nephrin and Diabetic-Nephropathies

Deregulation of protecting factor signaling actions in podocytes has emerged as an alternative pathway of podocyte injury mechanisms. Here, we review recent knowledge that highlighted how podocyte protecting factors are modulated by protein phosphatases.. Protein tyrosine kinases and phosphatases participate in many, if not all, aspects of cellular function by turning on or off multiple signaling cascades and podocytes are no exception. Modulation of tyrosine residue phosphorylation of podocyte factors such as nephrin, vascular endothelial growth factor, insulin receptors and substrates has been shown to promote podocyte damage and cell death that contributed to multiple glomerular diseases. Protein phosphatase activity can cause either an increase [Src homology 2 domain-containing phosphatase 2 (SHP-2)] or a decrease [Protein tyrosine phosphatase1B (PTP1B), SHP-1 and SH2 domain-containing 5'-inositol phosphatase 2 (SHIP2)] in nephrin tyrosine phosphorylation depending on which podocyte injury model was used. Insulin resistance is closely linked to the development and progression of renal disease. Expression of PTP1B, SHP-1, phosphatase and tensin homolog and SHIP2 are potential mechanisms of podocytes insulin resistance in diabetic kidney disease.. Tight regulation of protein phosphatases is critical to maintain cell homeostasis and may offer new perceptive targets to restore protecting factor actions in order to prevent podocyte dysfunction and glomerular diseases.

Topics: Animals; Diabetic Nephropathies; Humans; Insulin Resistance; Membrane Proteins; Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; Phosphoprotein Phosphatases; Phosphorylation; Podocytes; Protective Factors; Protein Tyrosine Phosphatase, Non-Receptor Type 1; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Receptor, Insulin; Signal Transduction

Urinary podocalyxin and nephrin are urine markers of podocyte dysfunction that may reflect the integrity of kidney's filtration barrier. Studies on their respective roles in glomerular diseases are still underway. However, the isolated and unsystematic manner in which they are being studied does not permit proper identification of their roles in each glomerular disease. As such, there is little or no appreciation of what research has already achieved and what remains to be achieved as the research direction is not clearly defined. We explored the recent studies and outlined the major findings regarding the value of both biomarkers in each of the three glomerular disease entities. Our review covered diabetic nephropathy, membranous nephropathy and IgA nephropathy.

Topics: Biomarkers; Diabetic Nephropathies; Glomerulonephritis, IGA; Humans; Membrane Proteins; Podocytes; Proteinuria; Sialoglycoproteins

Epithelial-mesenchymal transition (EMT) is a tightly regulated process by which epithelial cells lose their hallmark epithelial characteristics and gain the features of mesenchymal cells. For podocytes, expression of nephrin, podocin, P-cadherin, and ZO-1 is downregulated, the slit diaphragm (SD) will be altered, and the actin cytoskeleton will be rearranged. Diabetes, especially hyperglycemia, has been demonstrated to incite podocyte EMT through several molecular mechanisms such as TGF-β/Smad classic pathway, Wnt/β-catenin signaling pathway, Integrins/integrin-linked kinase (ILK) signaling pathway, MAPKs signaling pathway, Jagged/Notch signaling pathway, and NF-κB signaling pathway. As one of the most fundamental prerequisites to develop ground-breaking therapeutic options to prevent the development and progression of diabetic kidney disease (DKD), a comprehensive understanding of the molecular mechanisms involved in the pathogenesis of podocyte EMT is compulsory. Therefore, the purpose of this paper is to update the research progress of these underlying signaling pathways and expound the podocyte EMT-related DKDs.

Topics: Diabetic Nephropathies; Epithelial-Mesenchymal Transition; Humans; Hyperglycemia; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Podocytes; Signal Transduction; Tight Junction Proteins

Diabetes is the leading cause of microalbuminuria and end-stage renal failure in industrial countries. Disruption of the filtration barrier, seen in almost all nephrotic diseases and diabetes, is the result of the loss or effacement of the podocyte foot process, notably damage of proteins within the slit diaphragm such as nephrin. For many years, nephrin has been viewed as a structural component of the slit diaphragm. It is now well recognized that nephrin contains several tyrosine residues in its cytoplasmic domain, which influences the development of glomerular injury. In this review, we propose an overview of nephrin signaling pathways in kidney injury.

Topics: Animals; Diabetic Nephropathies; Humans; Kidney; Membrane Proteins; Phosphorylation; Protein Processing, Post-Translational; Renal Insufficiency, Chronic; Signal Transduction

The prevalence of diabetes mellitus increased during the last century and it is estimated that 45% of the patients are not diagnosed. In South America the prevalence of diabetes and chronic kidney disease (CKD) increased, with a great disparity among the countries with respect to access to dialysis. In Ecuador it is one of the main causes of mortality, principally in the provinces located on the coast of the Pacific Ocean. The greatest single cause of beginning dialysis is diabetic nephropathy (DN). Even using the best therapeutic options for DN, the residual risk of proteinuria and of terminal CKD remains high. In this review we indicate the importance of the problem globally and in our region. We analyse relevant cellular and molecular studies that illustrate the crucial significance of glomerular events in DN development and evolution and in insulin resistance. We include basic anatomical, pathophysiological and clinical concepts, with special attention to the role of angiogenic factors such as the vascular endothelial growth factor (VEGF-A) and their relationship to the insulin receptor, endothelial isoform of nitric oxide synthase (eNOS) and angiopoietins. We also propose various pathways that have therapeutic potential in our opinion. Greater in-depth study of VEGF-A and angiopoietins, the state of glomerular VEGF resistance, the relationship of VEGF receptor 2/nephrin, VEGF/insulin receptors/nephrin and the relationship of VEGF/eNOS-NO at glomerular level could provide solutions to the pressing world problem of DN and generate new treatment alternatives.

Topics: Angiopoietins; Diabetic Nephropathies; Global Health; Humans; Insulin Resistance; Kidney Glomerulus; Membrane Proteins; Nitric Oxide; Nitric Oxide Synthase Type III; Receptor, Insulin; Receptors, Vascular Endothelial Growth Factor; Signal Transduction; Vascular Endothelial Growth Factor A

Recent studies have demonstrated the role of insulin resistance in renal injury related to obesity, with hyperfiltration leading to glomerulomegaly in a pattern similar to that found in diabetic nephropathy. Similarities in the histologic patterns of damage from obesity and diabetes point to overlapping mechanisms of injury. In this review, we will examine the hormonal mechanisms, signaling pathways and injury patterns in renal injury resulting from obesity and attempt to draw conclusions on the reasons for these similarities.

Topics: Adiponectin; Diabetic Nephropathies; Female; Glomerular Filtration Rate; Hemodynamics; Humans; Insulin Resistance; Insulin-Secreting Cells; Kidney; Leptin; Male; Membrane Proteins; Obesity; Podocytes; Resistin; Signal Transduction; Sleep Apnea, Obstructive

In recent years there has been an explosion of interest in the glomerular podocyte, which plays a central role in control of glomerular filtration. A host of new molecules have been identified as playing essential roles in the maintenance of podocyte integrity in both humans and mouse models. Of all of these, arguably the most pivotal is nephrin, a transmembrane receptor molecule located at the specialized podocyte cell-cell junction, termed the slit diaphragm. Mutations in this gene cause the most severe form of congenital nephrotic syndrome, and many interacting proteins have now been described to form a large multiprotein complex with complex dynamics. There is little evidence of functional nephrin expression outside the glomerulus, and there are accumulating data that nephrin is essential for the unique properties of podocyte biology. Utilizing a powerful human cell culture model, comparing wild-type with nephrin-null podocytes, we can show that several crucial functional properties of podocytes depend on nephrin, including insulin responsiveness and cytoskeletal reorganization. Thus, it is reasoned that nephrin is a signature molecule required to define distinct podocyte characteristics.

Topics: Animals; Diabetic Nephropathies; Genotype; Humans; Kidney Glomerulus; Membrane Proteins; Mice; Mutation; Nephrotic Syndrome; Phenotype; Podocytes

Nephrin, the main structural protein of the slit diaphragm, is expressed on the surface of glomerular podocytes and is critical in maintaining permselectivity and preventing proteinuria. This review focuses on the fate of nephrin in the context of endothelial injury and gives an update on the recent progress in understanding the pathomechanisms that lead to proteinuria.. The following conditions of endothelial injury were found to induce loss of nephrin.(1) Preeclampsia, in which the associated proteinuria is induced by the soluble variant of vascular endothelial growth factor, which stimulates production of endothelin 1 (ET1) in endothelial cells. ET1 in turn triggers nephrin shedding from podocytes.(2) Hypertension, in which increased levels of angiotensin II induce podocyte apoptosis and reduce nephrin expression, leading to proteinuria in rats.(3) Diabetes and high fat diet, which lead to a significant increase in inflammatory molecules and cytokines, including MCP-1, which induces changes in podocyte cytoskeleton and nephrin loss.. Recent results showed that damage to endothelial cells may alter endothelial-podocyte interaction and induces nephrin loss, a main cause of proteinuria.

Topics: Animals; Diabetic Nephropathies; Dietary Fats; Endothelial Cells; Endothelium; Female; Humans; Hypertension; Kidney; Kidney Glomerulus; Membrane Proteins; Podocytes; Pre-Eclampsia; Pregnancy; Signal Transduction

Topics: Animals; Bone Morphogenetic Protein 2; Claudins; Diabetic Nephropathies; Diagnosis, Differential; Glomerulonephritis, IGA; Glomerulosclerosis, Focal Segmental; GTP-Binding Proteins; Humans; Kidney Diseases; Membrane Proteins; Nephrosis, Lipoid; Podocytes; Protein Glutamine gamma Glutamyltransferase 2; Transglutaminases

Podocyte injury plays a key role in the development of diabetic nephropathy. This review discusses recent advances in our understanding of mechanisms of podocyte injury in diabetes mellitus and the associated alterations in the function of the glomerular filtration barrier.. The effects of hyperglycemia on critical podocyte parameters including cell-cell interactions, attachment to the glomerular basement membrane, and podocyte apoptosis have been determined in both cell culture and in-vivo models of diabetes mellitus. The podocyte has also been identified as a target of action for insulin and growth hormone, hormones with significant roles in the altered homeostasis of diabetes mellitus.. Understanding the cellular and molecular basis for changes in podocyte structure and function in diabetes mellitus may lead to novel diagnostic tools and treatment strategies for diabetic nephropathy.

Topics: Animals; Diabetic Nephropathies; Growth Hormone; Humans; Insulin; Kidney Glomerulus; Membrane Proteins; Podocytes

Microalbuminuria is the earliest detectable clinical abnormality in diabetic glomerulopathy. On a molecular level, metabolic pathways activated by hyperglycemia, glycated proteins, hemodynamic factors, and oxidative stress are key players in the genesis of diabetic kidney disease. A variety of growth factors and cytokines are then induced through complex signal transduction pathways. Transforming growth factor-beta 1 (TGF-beta1) has emerged as an important downstream mediator for the development of renal hypertrophy and the accumulation of mesangial extracellular matrix components, but there is limited evidence to support its role in the development of albuminuria. The loss of proteoglycans in the glomerular basement membrane (GBM) has been recently questioned as causative of the albuminuria, and current research has focused on the podocyte as a central target for the effects of the metabolic milieu in the development and progression of diabetic albuminuria. Podocyte-derived vascular endothelial growth factor (VEGF), a permeability and angiogenic factor whose expression is increased in diabetic kidney disease, is perhaps a major mediator of the increased protein filtration. Decreased podocyte number and/or density as a result of apoptosis or detachment, GBM thickening with altered matrix composition, and a reduction in nephrin protein in the slit diaphragm with podocyte foot process effacement, all comprise the principal features of diabetic podocytopathy that clinically manifests as albuminuria and proteinuria. Many of these events are mediated by angiotensin II whose local concentration is stimulated by high glucose, mechanical stretch, and proteinuria itself. Angiotensin II in turn stimulates podocyte-derived VEGF, suppresses nephrin expression, and induces TGF-beta1 leading to podocyte apoptosis and fostering the development of glomerulosclerosis. Proteinuria can then induce in tubular cells a genetic program leading to tubulointerstitial inflammation, fibrosis and tubular atrophy. Besides direct effects of albuminuria on tubular cells, pathophysiological changes in the ultrafiltration barrier lead to an increased tubular filtration of various growth factors (TGF-beta1, insulin-like growth factor I) that may further alter the function of tubular cells. Moreover, angiotensin II also stimulates uptake of ultrafiltered proteins into tubular cells and enhances the production of proinflammatory and profibrotic cytokines within the cells. Migration of macro

Topics: Angiotensin II; Diabetic Nephropathies; Endothelial Cells; Endothelium, Vascular; Humans; Kidney Glomerulus; Membrane Proteins; Models, Biological; Podocytes; Proteinuria; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Nephrin, a newly described protein, has been localized to the slit membrane between adjacent podocytes of the glomerulus. Its discovery followed the demonstration of the gene NPHS1 and its mutation, resulting in the absence of the protein product, nephrin, in the congenital nephrotic syndrome of the Finnish type. The link between permutations in nephrin expression and proteinuria has been shown in animal models by using neutralizing antibodies or studying mice with inactivation of the nephrin gene. Moreover, the expression of nephrin has been shown to be reduced in various animal models of proteinuric renal disease. The relationship between changes in nephrin expression and proteinuric renal disease in humans is not fully elucidated, with a reduction in expression of this protein reported in a range of renal diseases. Diabetic nephropathy, one of the major causes of end-stage renal disease, is associated with substantial proteinuria and in experimental models with a reduction in slit pore density. In experimental models of diabetes, nephrin expression has been described as being transiently increased in the first 8 weeks of diabetes, followed in longer-term studies with reduced nephrin expression in association with increasing proteinuria. An angiotensin II-receptor blocker has been shown to prevent depletion in glomerular nephrin expression in the diabetic kidney. Human studies in both type 1 and type 2 diabetes suggest down-regulation of nephrin expression in the diabetic kidney and it has been postulated that these changes may play a role in the pathogenesis of diabetic nephropathy, specifically the development of proteinuria in this condition. Although there are other proteins involved in the structure of the epithelial podocyte and specifically the slit pore, nephrin seems to play a pivotal role in preventing passage of protein through the glomerular barrier. Furthermore, it is suggested that the antiproteinuric effects of inhibition of the renin-angiotensin system may partly relate to the effects of these agents on nephrin expression.

Topics: Animals; Diabetic Nephropathies; Humans; Kidney Diseases; Membrane Proteins; Proteins; Proteinuria

Diabetic nephropathy is a severe complication of Type 2 diabetes. Tubular lesions may play an important role in its early stages. The aim of our study was to determine if atorvastatin protects the podocytes and the proximal tubule in patients with Type 2 diabetes.. A total of 63 patients with Type 2 diabetes completed this 6-months prospective pilot study. They were randomized to continue rosuvastatin therapy (control group) or to be administered an equipotent dose of atorvastatin (intervention group), and were assessed regarding urinary podocytes, podocyte-associated molecules, and biomarkers of proximal tubule dysfunction.. In patients with Type 2 diabetes, atorvastatin exerts favorable effects on the kidney. There is a correlation between the evolution of the podocytes and of the proximal tubule biomarkers, supporting the hypothesis that the glomerular changes parallel proximal tubule dysfunction in the early stages of diabetic nephropathy.

Topics: Aged; Albuminuria; Atorvastatin; Biomarkers; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Female; Glomerular Filtration Rate; Glycation End Products, Advanced; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Kidney Tubules, Proximal; Male; Membrane Proteins; Middle Aged; Pilot Projects; Podocytes; Prospective Studies; Rosuvastatin Calcium; Vascular Endothelial Growth Factor A

Podocyte injury and its subsequent loss in urine play an important role in the pathogenesis of diabetic nephropathy; blockade of the renin-angiotensin system may ameliorate the damage.. In a non-randomized setting, we studied 71 patients with diabetic nephropathy on a stable dose of angiotensin-converting enzyme inhibitor (ACEI). In 37 patients, angiotensin receptor blocker (ARB) was added (the combination group); ACEI alone was continued in the other 34 (the control group). The mRNA expressions of nephrin, podocin, and synaptopodin in urinary sediment were measured at 0 and 12 weeks.. Baseline glomerular filtration rate (GFR) correlated with the urinary expression of nephrin (r=0.320, P=0.007), podocin (r=0.336, P=0.004), and synaptopodin (r=0.350, P=0.003). After adjusting for the baseline expression, the combination group had a significantly lower urinary synaptopodin expression (7.49 (95% confidence interval CI, 0.62-115.29) vs 14.83 (95% CI, 1.03-241.43), P=0.026) than the control group after 12 weeks of treatment. The percentage change in urinary podocin expression over 12 weeks of treatment had a modest correlation with the rate of GFR decline in 1 year (r=-0.243, P=0.041).. In patients with diabetic nephropathy, urinary mRNA expression of podocyte markers correlated with baseline renal function. Urinary expression of synaptopodin was lower after 12 weeks of ACEI and ARB combination therapy. Our result suggests that serial measurement of urinary podocyte markers may have a value for the monitoring of therapeutic response.

Topics: Aged; Angiotensin II Type 1 Receptor Blockers; Angiotensin-Converting Enzyme Inhibitors; Biphenyl Compounds; Diabetic Nephropathies; Drug Therapy, Combination; Female; Gene Expression; Humans; Intracellular Signaling Peptides and Proteins; Irbesartan; Male; Membrane Proteins; Microfilament Proteins; Middle Aged; Podocytes; RNA, Messenger; Tetrazoles; Treatment Outcome

Proteinuria, reflecting increased glomerular permeability to macromolecules is a characteristic feature of diabetic nephropathy. Nephrin, a 1241-residue transmembrane protein is a key component of the podocyte slit pore membrane and a major contributor of the glomerular filtration barrier. We investigated the expression of nephrin in human kidney tissue from patients with diabetic nephropathy to elucidate its relationship with proteinuria and the effects of anti-proteinuric therapy with angiotensin converting enzyme inhibition.. Renal biopsies were examined from 14 patients with Type II (non-insulin-dependent) diabetes mellitus and proteinuria who had been randomised to receive treatment with the ACE inhibitor, perindopril (4 mg/day) or placebo for the preceding 2 years. These specimens were compared with control human tissue sections, obtained from areas of normal renal cortex following nephrectomy for malignancy. Proteinuria was measured, specimens were examined histologically for injury and the expression of nephrin messenger RNA was assessed by quantitative in situ hybridisation.. Glomeruli from placebo-treated patients with diabetic nephropathy, showed a 62% reduction in nephrin expression compared with control subjects (p=0.0003). In contrast, nephrin RNA in glomeruli from perindopril treated patients was similar to that in the non-diabetic control group. In both placebo and perindopril treated patients, a close inverse correlation was noted between the magnitude of nephrin gene expression and the degree of proteinuria (placebo: r=0.86, p=0.013, perindopril: r=0.91, p=0.004).. Modulation in nephrin expression is related to the extent of proteinuria in diabetic nephropathy. These changes define, at a molecular level alterations in the glomerulus that occur in relation to proteinuria in diabetes and the effects of anti-proteinuric treatment with ACE inhibition.

Topics: Angiotensin-Converting Enzyme Inhibitors; Biopsy; Blood Pressure; Creatinine; Diabetic Nephropathies; Gene Expression Regulation; Glycated Hemoglobin; Humans; In Situ Hybridization; Membrane Proteins; Perindopril; Placebos; Proteins; Proteinuria

Diabetic nephropathy is the leading cause of end-stage renal failure, but the effectiveness of currently available strategies for preventing diabetic nephropathy remains unsatisfactory. This study was designed to evaluate the changes in adipokines levels caused by dipeptidyl peptidase-4 inhibitor sitagliptin therapy as one of the possible mechanisms of sitagliptin's amelioration of diabetic nephropathy. Twenty-four male Wistar rats weighing 180-200 g were taken and divided into three groups, that is, control, diseased, and treatment group. High-fat diet and streptozotocin-induced Type 2 diabetic rats were divided into diseased and treatment groups. The treatment group was given sitagliptin orally, 10 mg/kg per day for 6 weeks. Serum glucose, serum insulin, serum blood urea nitrogen, serum creatinine, and 24-h urinary protein levels were measured in serum and urine samples. mRNA expression levels of podocin, nephrin, and adipokines in renal tissues were determined. Results showed that sitagliptin treatment effectively reduced serum glucose, serum creatinine, serum blood urea nitrogen, and 24-h proteinuria, along with partial prevention of insulinopenia, in the treatment group as compared to the diseased group. The renal mRNA expression levels of podocin, nephrin, and adiponectin were significantly upregulated, while those of leptin and resistin were significantly downregulated in the diabetic rats receiving sitagliptin therapy compared to the non-treated diabetic rats. Based on these findings, it is suggested that sitagliptin, via mediating the modulation of adipokines levels, upregulates renal nephrin and podocin expression, which leads to the amelioration of diabetic nephropathy.

Topics: Adipokines; Animals; Creatinine; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Glucose; Male; Rats; Rats, Wistar; RNA, Messenger; Sitagliptin Phosphate

Extra-capillary hypercellularity is a common finding in crescentic glomerulonephritis (GN) and focal segmental glomerulosclerosis (FSGS). In diabetic nephropathy (DN), extra-capillary hypercellularity is often observed as a finding of complications such as IgA nephropathy or microscopic polyangiitis superimposed on DN. However, in rare cases, epithelial cell proliferation may accompany DN. We experienced a case of nodular diabetic glomerulosclerosis with marked extra-capillary hypercellularity and revealed the origin of this atypical lesion using immunostainings.. A man in his 50 s was admitted to the hospital with nephrotic syndrome, and a renal biopsy was performed. Diffuse nodular lesions and extra-capillary hypercellularity were observed, but the results of serological examination or immunofluorescent assays did not implicate any other crescentic GN. Immunostaining for claudin-1 and nephrin was performed to identify the origin of the extra-capillary lesions. Given the clinical course and pathological findings, a diagnosis of DN-associated extra-capillary cell proliferation was made.. Extra-capillary hypercellularity, which resembles FSGS or crescentic GN, is a rare finding in DN and should therefore be treated with caution. In such cases, co-staining for claudin-1 and nephrin may facilitate the diagnosis of DN.

Topics: Cell Proliferation; Claudin-1; Diabetes Mellitus; Diabetic Nephropathies; Glomerulonephritis, IGA; Glomerulonephritis, Membranoproliferative; Glomerulosclerosis, Focal Segmental; Humans; Male; Membrane Proteins; Microscopic Polyangiitis; Middle Aged

Shenkang injection has been used clinically to lower creatinine levels. This study explored the mechanism of Shenkang injection on protecting kidney function from hyperglycemia-mediated damage.. This study utilized a STreptoZotocin (STZ)-induced rat model of diabetes. In total, 60 rats were randomized into either the control group (n = 15) injected with vehicle or treatment group (n = 45) injected with STZ to induce hyperglycemia. Eight weeks after diabetes onset, diabetic rats were further randomized to receive different treatments for 4 consecutive weeks, including vehicle (diabetic nephropathy group, n = 15), Shenkang (n = 15), or Valsartan (n = 15). At 12 weeks, a series of urine and blood measures were examined and damage to the kidney tissue was examined using histology. Expression of nephrin and transforming growth factor-β1 (TGF-β1) were characterized using immunohistochemistry and Western blot.. Compared to the control group, rats in the diabetic nephropathy group showed significant kidney damage demonstrated by high kidneyindex, high levels of urinary albumin, albumin/creatinine ratio (ACR), blood urea nitrogen as well as histological evidence. Shenkang injection significantly improved kidney function in the diabetic rats by decreasing kidney index, ACR, and serum creatinine. Shenkang treatment also mitigated kidney damage, improved nephrin expression, and decreased TGF-β1 expression in the kidneys.. Shenkang treatment protected renal function in diabetic rats by increasing nephrin expression, which protects diabetic rats from hyperglycemia-mediated kidney damage.

Topics: Albumins; Animals; Creatinine; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Hyperglycemia; Kidney; Rats; Transforming Growth Factor beta1

Podocytes, the key component of the glomerular filtration barrier (GFB), are gradually lost during the progression of diabetic kidney disease (DKD), severely compromising kidney functionality. The molecular mechanisms regulating the survival of podocytes in DKD are incompletely understood. Here, we show that membrane-associated guanylate kinase inverted 2 (MAGI2) is specifically expressed in renal podocytes, and promotes podocyte survival in DKD. We found that MAGI2 expression was downregulated in podocytes cultured with high-glucose in vitro, and in kidneys of db/db mice as well as DKD patients. Conversely, we found enforced expression of MAGI2 via AAV transduction protected podocytes from apoptosis, with concomitant improvement of renal functions. Mechanistically, we found that MAGI2 deficiency induced by high glucose levels activates TGF-β signaling to decrease the expression of anti-apoptotic proteins. These results indicate that MAGI2 protects podocytes from cell death, and can be harnessed therapeutically to improve renal function in diabetic kidney disease.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Communication; Diabetes Mellitus; Diabetic Nephropathies; Glucose; Guanylate Kinases; Humans; Mice; Podocytes; Transforming Growth Factor beta

Phloretin is a dihydrochalcone flavonoid from natural plants, which has protective activities against oxidative stress and inflammation. To date, its effect on diabetic nephropathy (DN) has not been investigated. In this study, we examined the potential role of phloretin in diabetes-induced renal damage and associated mechanisms in a type 2 diabetes mellitus (T2DM) model induced by streptozotocin (STZ) and high-fat diet (HFD) in Apolipoprotein E knockout (

Topics: Animals; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mice; Phloretin; Podocytes

Advanced glycation end products (AGEs) play an important role in oxidative stress and inflammation, processes implicated in the development and progression of kidney dysfunction. In the present study, we investigated the participation of the pro-oxidant protein thioredoxin-interacting protein (TXNIP) and of epigenetic mechanisms on kidney tissue (in vivo, in non-diabetic rats) and on terminally differentiated glomerular podocytes (in vitro) chronically exposed to AGEs. AGEs induced total kidney and glomerular TXNIP expression and decreased H3K27me3 content. Concomitant treatment with the antioxidant N-acetyl-cysteine (NAC) reversed only the increased TXNIP expression. TXNIP expression positively correlated with proteinuria and negatively correlated with H3K27me3 content. In vitro studies in podocytes showed that 72 h exposure to AGEs decreased nephrin expression and increased Txnip, Nox4, Col4a1, and epithelial-to-mesenchymal transition (EMT) markers (Acta2, Snail1, and Tgfb1). Podocytes treatment with NAC reversed Nox4, Col4a1, Acta2, and Tgfb1 increased expression but did not abrogate the reduced expression of nephrin. MiR-29a expression was downregulated by AGEs in vivo, but not in vitro. In conclusion, treatment of non-diabetic rats with AGEs induced TXNIP expression and decreased the contents of the repressive epigenetic mark H3K27me3 and of miR-29a, potentially driving injury to glomerular filtration barrier and podocytes dysfunction.

Topics: Animals; Antioxidants; Cell Cycle Proteins; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Epigenesis, Genetic; Epithelial Cells; Gene Expression; Gene Expression Regulation; Glycation End Products, Advanced; Histones; Kidney; Kidney Glomerulus; Male; Membrane Proteins; Oxidative Stress; Podocytes; Rats; Rats, Wistar; Reactive Oxygen Species

To investigate the effects of Tongluo Digui decoction on renal injury and streptozotocin-induced podocyte autophagy in diabetic rats.. Male Sprague-Dawley rats were randomly divided into six groups: normal, model, Tongluo Digui decoction (high, medium, and low dose) and valsartan. Streptozotocin was injected intraperitoneally to replicate the diabetic animal model. After 8 weeks, proteinuria was evaluated to establish the diabetic nephropathy model. Treatments were administered daily via the intragastric route. At 16 weeks after gavage, we determined 24 h urine protein concentration, and blood glucose, serum creatinine, and urea nitrogen concentrations. Then, rats were sacrificed, and kidneys were harvested and stained with periodic acid-Schiff to evaluate the pathological changes in glomeruli, including glomerular podocytes by transmission electron microscopy. Western blot analysis was used to determine the expression of nephrin, podocin, p62, beclin-1, LC3Ⅱ/Ⅰ, and p-mTOR/mTOR protein in kidney tissues.. Compared with the model group, Tongluo Digui decoction was associated with decreases in 24 h urine protein concentration, and blood glucose, hemoglobin A1c, serum creatinine, urea nitrogen concentrations, total serum protein and albumin. Concurrently, mesangial mesenteric broadening and fusion of foot processes were reduced, the glomerular basement membrane was not significantly thickened, and the number of podocytes and the number of autophagosomes in the podocytes was increased. Further, expression of nephrin, podocin, LC3Ⅱ, and beclin-1 protein in kidney tissue was up-regulated, while expression of p62 protein was down-regulated and mTOR phosphorylation was inhibited.. Tongluo Digui decoction may inhibit the progression of diabetic nephropathy by inhibiting mTOR phosphorylation, thereby increasing autophagy to protect podocytes and reducing proteinuria.

Topics: Animals; Autophagy; Diabetic Nephropathies; Disease Models, Animal; Drugs, Chinese Herbal; Humans; Intracellular Signaling Peptides and Proteins; Kidney; Male; Membrane Proteins; Podocytes; Rats; Rats, Sprague-Dawley; TOR Serine-Threonine Kinases

The sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that is now appreciated as key regulatory factor for various cellular functions in the kidney, including matrix remodeling. It is generated by two sphingosine kinases (Sphk), Sphk1 and Sphk2, which are ubiquitously expressed, but have distinct enzymatic activities and subcellular localizations. In this study, we have investigated the role of Sphk2 in podocyte function and its contribution to diabetic nephropathy. We show that streptozotocin (STZ)-induced nephropathy and albuminuria in mice is prevented by genetic depletion of Sphk2. This protection correlated with an increased protein expression of the transcription factor Wilm's tumor suppressor gene 1 (WT1) and its target gene nephrin, and a reduced macrophage infiltration in immunohistochemical renal sections of STZ-treated Sphk2

Topics: Albuminuria; Animals; Diabetic Nephropathies; Genes, Tumor Suppressor; Membrane Proteins; Mice; Mice, Knockout; Phosphotransferases (Alcohol Group Acceptor); Podocytes; Streptozocin; WT1 Proteins

To observe the effects of saxagliptin on the expression of mitogen-activated protein kinase 38 (p38MAPK), nephrin and podocin in renal tissue in type 2 diabetic (T2DM) rats, and to explore the possible mechanism of its renal protection.. Forty-eight male Sprague-Dawley rats were used for the study and divided into four different groups: normal controls (Group NC), DM controls (Group DM), DM + glibenclamide (Group Su) and DM + saxagliptin (Group Sa). The day drug administration started was defined as week 0. After 12 weeks, hemoglobin A1c (HbA1c), total cholesterol (TC), triglyceride (TG), urea nitrogen (BUN) and creatinine (Cr) in serum were detected, simultaneously albumin and creatinine in urine were measured, respectively, and then urinary albumin/creatinine ratio (UACR) was calculated. The pathological morphology of kidney tissue in different groups was observed, and the expression of nephrin and podocin mRNA and protein in kidney tissue were detected.. (1) After 12 weeks, FBG and HbA1c in Group Su and Group Sa were significantly lower than those in Group DM (both P < 0.05), while there was no significant difference between Group Su and Group Sa. TC, TG and UACR in Group Sa were significantly decreased than those in Group DM. (2) When compared with Group DM, the kidney weight/body weight ratios, the average width of glomerular basement membrane and foot process fusion ratio were all improved in Group Sa after 12 weeks. (3) The expression of p38MAPK mRNA and protein was significantly decreased, while nephrin and podocin mRNA and protein were significantly higher in Group Sa than those in Group DM after 12 weeks. (4) A significant negative correlation was detected between p38MAPK mRNA and nephrin (r = - 0.421, P = 0.009) and podocin mRNA (r = - 0.570, P = 0.000), respectively.. Saxagliptin can reduce urinary albumin excretion and exert renal protective effect, especially on podocytes in T2DM rats. The mechanism may be related to its inhibition of renal p38MAPK signaling pathway and the increase in the expression of nephrin and podocin in renal tissue, which is independent of its hypoglycemic effect.

Topics: Adamantane; Animals; Creatinine; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Dipeptides; Glycated Hemoglobin; Humans; Intracellular Signaling Peptides and Proteins; Kidney; Male; Membrane Proteins; p38 Mitogen-Activated Protein Kinases; Podocytes; Rats; Rats, Sprague-Dawley

A growing body of evidence suggests a role of proteolytic enzymes in the development of diabetic nephropathy. Cathepsin C (CatC) is a well-known regulator of inflammatory responses, but its involvement in podocyte and renal injury remains obscure. We used Zucker rats, a genetic model of metabolic syndrome and insulin resistance, to determine the presence, quantity, and activity of CatC in the urine. In addition to the animal study, we used two cellular models, immortalized human podocytes and primary rat podocytes, to determine mRNA and protein expression levels via RT-PCR, Western blot, and confocal microscopy, and to evaluate CatC activity. The role of CatC was analyzed in CatC-depleted podocytes using siRNA and glycolytic flux parameters were obtained from extracellular acidification rate (ECAR) measurements. In functional analyses, podocyte and glomerular permeability to albumin was determined. We found that podocytes express and secrete CatC, and a hyperglycemic environment increases CatC levels and activity. Both high glucose and non-specific activator of CatC phorbol 12-myristate 13-acetate (PMA) diminished nephrin, cofilin, and GLUT4 levels and induced cytoskeletal rearrangements, increasing albumin permeability in podocytes. These negative effects were completely reversed in CatC-depleted podocytes. Moreover, PMA, but not high glucose, increased glycolytic flux in podocytes. Finally, we demonstrated that CatC expression and activity are increased in the urine of diabetic Zucker rats. We propose a novel mechanism of podocyte injury in diabetes, providing deeper insight into the role of CatC in podocyte biology.

Topics: Animals; Cathepsin C; Diabetic Nephropathies; Disease Models, Animal; Female; Gene Knockdown Techniques; Glucose; Humans; Hyperglycemia; Insulin Resistance; Kidney; Membrane Proteins; Metabolic Syndrome; Obesity; Permeability; Podocytes; Rats; Rats, Zucker; RNA, Messenger; Serum Albumin; Transcriptome

EDA2R is a member of the large family of tumor necrosis factor receptor (TNFR). Previous studies suggested that EDA2R expression might be increased in the kidneys of diabetic mice. However, its mRNA and protein expression in kidneys were not analyzed; moreover, its role in the development of diabetic kidney disease was not explored. Here we analyzed the mRNA and protein expressions of EDA2R in diabetic kidneys and examined its role in the podocyte injury in high glucose milieu. By analysis with real-time PCR, Western blotting, we found that both the mRNA and protein levels of EDA2R were increased in the kidneys of diabetic mice. Immunohistochemical studies revealed that EDA2R expression was enhanced in both glomerular and tubular cells of diabetic mice and humans. In vitro studies, high glucose increased EDA2R expression in cultured human podocytes. Overexpression of EDA2R in podocytes promoted podocyte apoptosis and decreased nephrin expression. Moreover, ED2AR increased ROS generation in podocytes, while inhibiting ROS generation attenuates EDA2R-mediated podocyte injury. In addition, EDA2R silencing partially suppressed high glucose-induced ROS generation, apoptosis, and nephrin decrease. Our study demonstrated that high glucose increases EDA2R expression in kidney cells and that EDA2R induces podocyte apoptosis and dedifferentiation in high glucose milieu partially through enhanced ROS generation.

Topics: Animals; Apoptosis; Cells, Cultured; Diabetes Mellitus; Diabetic Nephropathies; Female; Kidney; Membrane Proteins; Mice; Podocytes; Reactive Oxygen Species; RNA, Messenger; Xedar Receptor

Diabetic nephropathy (DN) is a major and severe complication of diabetes mellitus. Ferulic acid (FA), a phenolic compound widespread in fruits and plants, displays a variety of pharmacological activities including regulating blood glucose and lipids, anti-oxidation, anti-inflammation and anti-fibrosis. The study was aimed to investigate the renal protective effects of FA on diabetic rats and elucidate the underlying mechanisms. FA (100 mg kg

Topics: Animals; Coumaric Acids; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Fibrosis; Inflammation; Intracellular Signaling Peptides and Proteins; Kidney; Male; Membrane Proteins; Protective Agents; Rats; Transcription Factor RelA; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease, and sleeve gastrectomy (SG) is considered to be an effective strategy to improve pre-existing DN. However, the mechanism remains unknown.. Animal model of DN was induced by high-fat diet (HFD) and streptozotocin (STZ). SG or sham surgery was performed and rats were sacrificed at 4, 8, and 12 weeks after surgery. The basic parameters (blood glucose, body weight, kidney weight), indicators of renal function including serum creatinine (Scr), blood urea nitrogen (BUN), urine microalbumin, urine creatinine (Ucr), microalbumin creatinine ratio (UACR), ultrastructural changes of glomerulus, and the expression of nephrin gene and protein in glomerular podocytes were compared among groups.. Blood glucose and body weight of SG rats were significantly lower than those of the sham-operated rats, and renal function of SG groups were also significantly improved within the postoperative period of 12 weeks. The results of periodic acid-Schiff staining (PAS) and transmission electron microscopy (TEM) showed that glomerular hypertrophy and accumulation of extracellular matrix proteins were significantly alleviated after SG, and the thickness of basement membrane and the fusion or effacement of foot processes were also significantly improved. The mRNA and protein expression of nephrin in SG groups was significantly higher than that in the sham group.. These results suggest that SG attenuates DN by upregulating the expression of nephrin and improving the ultrastructure of glomerular filtration membrane. This study indicates that SG can be used as an available therapeutic intervention for DN.

Topics: Animals; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Gastrectomy; Membrane Proteins; Obesity, Morbid; Rats; Rats, Sprague-Dawley

Myeloid-derived growth factor (MYDGF), mainly secreted by bone marrow-derived cells, has been known to promote glucagon-like peptide-1 production and improve glucose/lipid metabolism in mouse models of diabetes, but little is known about the functions of MYDGF in diabetic kidney disease (DKD). Here, we investigated whether MYDGF can prevent the progression of DKD.. In vivo experiments, both loss- and gain-of-function strategies were used to evaluate the effect of MYDGF on albuminuria and pathological glomerular lesions. We used streptozotocin-treated Mydgf knockout and wild-type mice on high fat diets to induce a model of DKD. Then, albuminuria, glomerular lesions and podocyte injury were evaluated in Mydgf knockout and wild-type DKD mice treated with adeno-associated virus-mediated Mydgf gene transfer. In vitro and ex vivo experiments, the expression of slit diaphragm protein nephrin and podocyte apoptosis were evaluated in conditionally immortalised mouse podocytes and isolated glomeruli from non-diabetic wild-type mice treated with recombinant MYDGF.. MYDGF deficiency caused more severe podocyte injury in DKD mice, including the disruption of slit diaphragm proteins (nephrin and podocin) and an increase in desmin expression and podocyte apoptosis, and subsequently caused more severe glomerular injury and increased albuminuria by 39.6% compared with those of wild-type DKD mice (p < 0.01). Inversely, MYDGF replenishment attenuated podocyte and glomerular injury in both wild-type and Mydgf knockout DKD mice and then decreased albuminuria by 36.7% in wild-type DKD mice (p < 0.01) and 34.9% in Mydgf knockout DKD mice (p < 0.01). Moreover, recombinant MYDGF preserved nephrin expression and inhibited podocyte apoptosis in vitro and ex vivo. Mechanistically, the renoprotection of MYDGF was attributed to the activation of the Akt/Bcl-2-associated death promoter (BAD) pathway.. The study demonstrates that MYDGF protects podocytes from injury and prevents the progression of DKD, providing a novel strategy for the treatment of DKD. Graphical abstract.

Topics: Albuminuria; Animals; Apoptosis; bcl-Associated Death Protein; Desmin; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Diet, High-Fat; Gene Transfer Techniques; Interleukins; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mice; Mice, Knockout; Podocytes; Proto-Oncogene Proteins c-akt; Signal Transduction

Podocyte injury is the primary cause of glomerular injury in diabetic nephropathy (DN). Advanced oxidation protein products (AOPPs), the triggers and markers of oxidative stress in DN, have been linked to podocyte damage. However, the underlying mechanism is not yet clear. Here, we investigated the potential role of FOXO3a, a key transcription factor in the response to stress, in mediating AOPPs-induced podocyte injury. We found that FOXO3a expression was increased in the glomeruli of kidney biopsies from patients with DN and it was positively correlated with proteinuria. The serum from patients with DN significantly increased FOXO3a and its downstream genes FasL and Bim, thereby inducing the high level of cleaved caspase3 and the loss of nephrin and podocin expressions in podocytes. Blockade of AOPPs signaling by a neutralizing antibody against the receptor of advanced glycation end products (αRAGE) abolished the effect of DN serum on podocytes, confirming the pathogenic role of AOPPs in DN serum. Downregulation of FOXO3a decreased AOPPs-induced podocyte apoptosis and restored the levels of podocyte markers nephrin and podocin, and upregulation of FOXO3a exacerbated these changes in podocytes after AOPPs treatment. Furthermore, FOXO3a specifically activated proapoptotic genes in podocytes only in the presence of AOPPs. Mechanistically, AOPPs increased the FOXO3a protein levels by inhibiting their autophagic degradation in a ROS/mTOR-dependent manner. Moreover AOPPs activated the accumulated FOXO3a by maintaining FOXO3a in the nucleus, and this process was dependent on ROS-mediated AKT signaling deactivation. These studies suggest that FOXO3a plays a critical role in mediating AOPPs-induced podocyte injury and reveal a new mechanistic linkage of oxidative stress, FOXO3a activation and podocyte injury in DN.

Topics: Advanced Oxidation Protein Products; Animals; Apoptosis; Autophagy; Biomarkers; Diabetic Nephropathies; Forkhead Box Protein O3; Glycation End Products, Advanced; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mice; Mice, Inbred C57BL; Oxidative Stress; Podocytes; Receptor for Advanced Glycation End Products

Diabetic kidney disease (DKD) is one of the leading pathological causes of decreased renal function and progression to end-stage kidney failure. To explore and characterize age-related changes in DKD and associated glomerular damage, we used a rat model of type 2 diabetic nephropathy (T2DN) at 12 wk and older than 48 wk. We compared their disease progression with control nondiabetic Wistar and diabetic Goto-Kakizaki (GK) rats. During the early stages of DKD, T2DN and GK animals revealed significant increases in blood glucose and kidney-to-body weight ratio. Both diabetic groups had significantly altered renin-angiotensin-aldosterone system function. Thereafter, during the later stages of disease progression, T2DN rats demonstrated a remarkable increase in renal damage compared with GK and Wistar rats, as indicated by renal hypertrophy, polyuria accompanied by a decrease in urine osmolarity, high cholesterol, a significant prevalence of medullary protein casts, and severe forms of glomerular injury. Urinary nephrin shedding indicated loss of the glomerular slit diaphragm, which also correlates with the dramatic elevation in albuminuria and loss of podocin staining in aged T2DN rats. Furthermore, we used scanning ion microscopy topographical analyses to detect and quantify the pathological remodeling in podocyte foot projections of isolated glomeruli from T2DN animals. In summary, T2DN rats developed renal and physiological abnormalities similar to clinical observations in human patients with DKD, including progressive glomerular damage and a significant decrease in renin-angiotensin-aldosterone system plasma levels, indicating these rats are an excellent model for studying the progression of renal damage in type 2 DKD.

Topics: Aging; Albuminuria; Animals; Blood Glucose; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Disease Progression; Hypertrophy; Kidney Glomerulus; Male; Membrane Proteins; Organ Size; Polyuria; Rats; Rats, Wistar; Renin-Angiotensin System; Water-Electrolyte Imbalance

Altered DNA methylation plays an important role in the onset and progression of kidney disease. However, little is known about how the changes arise in disease states. Here, we report that KAT5-mediated DNA damage repair is essential for the maintenance of kidney podocytes and is associated with DNA methylation status. Podocyte-specific KAT5-knockout mice develop severe albuminuria with increased DNA double-strand breaks (DSBs), increased DNA methylation of the nephrin promoter region, and decreased nephrin expression. Podocyte KAT5 expression is decreased, whereas DNA DSBs and DNA methylation are increased in diabetic nephropathy; moreover, KAT5 restoration by gene transfer attenuates albuminuria. Furthermore, KAT5 decreases DNA DSBs and DNA methylation at the same nephrin promoter region, which indicates that KAT5-mediated DNA repair may be related to DNA methylation status. These results suggest a concept in which an environment of DNA damage repair, which occurs with decreased KAT5, may affect DNA methylation status.

Topics: Albuminuria; Animals; Cells, Cultured; Diabetes Mellitus, Experimental; Diabetic Nephropathies; DNA Breaks, Double-Stranded; DNA Damage; DNA Methylation; DNA Repair; Glomerulosclerosis, Focal Segmental; Glucose; Humans; Kidney; Kidney Glomerulus; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors; Lysine Acetyltransferase 5; Membrane Proteins; Mice, Inbred C57BL; Mice, Knockout; Podocytes; Promoter Regions, Genetic; Tamoxifen; Trans-Activators

Diabetic kidney disease (DKD) is a complication associated with diabetes and is a major public health problem in modern society. Podocyte injury is the central target of the development of DKD, and the loss or dysregulation of nephrin, a key structural and signalling molecule located in the podocyte slit diaphragm (SD), initiates potentially catastrophic downstream events within podocytes. IQGAP1, a scaffold protein containing multiple protein-binding domains that regulates endocytosis, can interact with nephrin in podocytes. It is hypothesized that IQGAP1 contributes to nephrin endocytosis and may participate in the pathogenesis of DKD. The dramatically increased histo-nephrin granularity score in DKD glomeruli showed a significant positive correlation with increased IQGAP1-nephrin interaction without changes in the total protein content of nephrin and IQGAP1. In cultured human podocytes, hyperglycaemia induced the intracellular translocation of IQGAP1 from the cytosol to the vicinity of the cytomembrane, reinforced the IQGAP1-nephrin interaction, and augmented nephrin endocytosis. Moreover, impaired podocyte function, such as migration, extensibility and permeability, were further aggravated by wild-type IQGAP1 plasmid transfection, and these effects were partially restored by siRNA-mediated IQGAP1 downregulation. Collectively, these findings show that IQGAP1, an intracellular partner of nephrin, is involved in nephrin endocytosis and the functional regulation of podocytes in DKD.

Topics: Animals; Cell Line; Diabetic Nephropathies; Endocytosis; Humans; Kidney Glomerulus; Membrane Proteins; Mice; Mice, Inbred C57BL; Podocytes; ras GTPase-Activating Proteins

Macrophage-mediated inflammation plays a significant role in the development and progression of diabetic nephropathy (DN). However, the underlying mechanisms remain unclear. Studies suggest that T cell immunoglobulin domain and mucin domain-3 (Tim-3) has complicated roles in regulating macrophage activation, but its roles in the progression of DN are still completely unknown.. We downregulated Tim-3 expression in kidney (intrarenal injection of Tim-3 shRNA expressing lentivirus or global Tim-3 knockout mice) and induced DN by streptozotocin (STZ). We analyzed the degree of renal injury, especially the podocyte injury induced by activated macrophages in vitro and in vivo. Then, we transferred different bone marrow derived macrophages (BMs) into STZ-induced Tim-3 knockdown mice to examine the effects of Tim-3 on macrophages in DN.. First, we found that Tim-3 expression on renal macrophages was increased in patients with DN and in two diabetic mouse models, i.e. STZ-induced diabetic mice and db/db mice, and positively correlated with renal dysfunction of DN patients. Tim-3 deficiency ameliorated renal damage in STZ-induced diabetes with concurrent increase in protein levels of Nephrin and WT-1. Similar effects were observed in mice with Tim-3 knockdown diabetic mice. Second, adoptive transfer of Tim-3-expressing macrophages, but not Tim-3 knockout macrophages, accelerated diabetic renal injury in DN mice, suggesting a key role for Tim-3 on macrophages in the development of DN. Furthermore, we found NF-κB activation and TNF-α excretion were upregulated by Tim-3 in diabetic kidneys, and podocyte injury was associated with the Tim-3-mediated activation of the NF-κB/TNF-α signaling pathway in DN macrophages both in vivo and in vitro.. These results suggest that Tim-3 functions as a key regulator in renal inflammatory processes and serves as a potential therapeutic target for renal injury in DN.

Topics: Adoptive Transfer; Animals; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Disease Models, Animal; Gene Knockout Techniques; Hepatitis A Virus Cellular Receptor 2; Humans; Macrophage Activation; Macrophages; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; NF-kappa B; Podocytes; Streptozocin; Tumor Necrosis Factor-alpha

Diabetic nephropathy is the leading cause of end-stage renal disease. Although dysfunction of podocytes, also termed glomerular visceral epithelial cells, is critically associated with diabetic nephropathy, the mechanism underlying podocyte dysfunction still remains obscure. Here, we identify that KDM6A, a histone lysine demethylase, reinforces diabetic podocyte dysfunction by creating a positive feedback loop through up-regulation of its downstream target KLF10. Overexpression of KLF10 in podocytes not only represses multiple podocyte-specific markers including nephrin, but also conversely increases KDM6A expression. We further show that KLF10 inhibits nephrin expression by directly binding to the gene promoter together with the recruitment of methyltransferase Dnmt1. Importantly, inactivation or knockout of either KDM6A or KLF10 in mice significantly suppresses diabetes-induced proteinuria and kidney injury. Consistent with the notion, we also show that levels of both

Topics: Animals; Base Sequence; Cell Line, Transformed; Diabetic Nephropathies; Down-Regulation; Early Growth Response Transcription Factors; Epigenesis, Genetic; Exosomes; Feedback, Physiological; Histone Demethylases; Humans; Kidney; Kruppel-Like Transcription Factors; Male; Membrane Proteins; Mice, Inbred C57BL; Mice, Knockout; Phenotype; Podocytes; Promoter Regions, Genetic; Protein Binding

Multiple studies indicate that microRNAs (miRNAs) are involved in diabetes. However, the roles of miRNA in the target organ damages in diabetes remain unclear. This study investigated the functions of miR-320a in diabetic nephropathy (DN). In this study, db/db mice were used to observe the changes in podocytes and their function in vivo, as well as in cultured mouse podocyte cells (MPC5) exposed to high glucose in vitro. To further explore the role of miR-320a in DN, recombinant adeno-associated viral particle was administered intravenously to manipulate the expression of miR-320a in db/db mice. Overexpression of miR-320a markedly promoted podocyte loss and dysfunction in DN, including mesangial expansion and increased levels of proteinuria, serum creatinine and urea nitrogen. Furthermore, MafB was identified as a direct target of miR-320a through AGO2 co-immunoprecipitation, luciferase reporter assay, and Western blotting. Moreover, re-expression of MafB rescued miR-320a-induced podocyte loss and dysfunction by upregulating the expressions of Nephrin and glutathione peroxidase 3 (Gpx3). Our data indicated that miR-320a aggravated renal disfunction in DN by targeting MafB and downregulating Nephrin and Gpx3 in podocytes, which suggested that miR-320a could be a potential therapeutic target of diabetic nephropathy.

Topics: Animals; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Glutathione Peroxidase; Kidney; MafB Transcription Factor; Membrane Proteins; Mice; MicroRNAs; Podocytes

Wenshen Jianpi recipe (WSJPR), a blended traditional Chinese medicine, is considered to have the possible beneficial effect on the progression of diabetic nephropathy (DN). This present study was designed to elucidate this protective activity in a rat model with streptozotocin (STZ)-induced DN and to explore the possible underlying mechanism.. Adult Sprague Dawley (SD) rats were induced to develop DN through intraperitoneal injection of STZ (60 mg/kg). Animals were orally administered saline, WSJPR at 7.5, 15, 30 g/kg, and valsartan (25 mg/kg) daily for 8 weeks. Blood and 24-h urine samples of each rat were collected for biochemical examination at 2-week intervals. Microcirculatory blood flow in the renal cortex and hemorheology index were also measured. At the end of 8 weeks, all rats were sacrificed to obtain the kidney tissues for histological examination and reverse transcription polymerase chain reaction (RT-PCR) was used to analyze the transcriptional levels of nephrin and podocin genes.. WSJPR could improve serum total protein (TP) and albumin (ALB), reduce the excretion rates of urine-TP (U-TP), urine-ALB (U-ALB) and urine urea nitrogen (UUN) (P < 0.05), although it did not significantly alter the hyperglycemia. In addition, treatment with WSJPR could strongly reduce blood flow, erythrocyte aggregation index, and ameliorate microcirculation. In histological measurement, WSJPR-treated rats showed a significant amelioration in glomerular hypertrophy and mesangial expansion. By RT-PCR, we found WSJPR up-regulated the nephrin and podocin expression at mRNA levels.. This study suggested that WSJPR could effectively relieve renal damage and improve renal function of DN rats by ameliorating metabolism disorder and increasing the gene expression of nephrin and podocin, which might be a useful approach for the treatment of DN.

Topics: Animals; Diabetic Nephropathies; Disease Models, Animal; Drugs, Chinese Herbal; Humans; Intracellular Signaling Peptides and Proteins; Kidney; Male; Medicine, Chinese Traditional; Membrane Proteins; Proteinuria; Rats; Rats, Sprague-Dawley

To explore the protective effect of liraglutide on renal damage in rats with diabetic kidney disease (DKD) through the protein kinase B-mammalian target of rapamycin (Akt-mTOR) pathway.. A total of 45 specific pathogen-free male Sprague-Dawley rats were divided into healthy group (no diabetes, n=15), diabetes group (diabetes, n=15), and liraglutide group (diabetes + liraglutide intervention, n=15). The differences in the biochemical indexes, lesion degree, glomerular Nephrin expression level, and mRNA and protein expressions of Akt-mTOR in renal tissues were detected in three groups via hematoxylin-eosin (HE) staining, immunohistochemistry, Western blotting, and quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR), respectively.. The albumin-to-creatinine ratio (ACR) and levels of serum creatinine (Scr), urine microalbumin (UmAlb), fasting blood glucose (FBG), glycated hemoglobin (HbA1c), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and total cholesterol (TC) in renal tissues were the lowest in healthy group and the highest in diabetes group, while they significantly declined in liraglutide group compared with those in diabetes group. Also, there were statistically significant differences (p<0.05). The level of high-density lipoprotein cholesterol (HDL-C) in renal tissues was the highest in healthy group and the lowest in diabetes group, while it was significantly increased in liraglutide group compared with that in diabetes group. Also, there were statistically significant differences (p<0.05). In healthy group, the mesangial structure and renal tubules were normal, the tubular basement membrane was smooth and intact, and there were no interstitial widening and inflammatory cell infiltration. Compared with diabetes group, the mesangial cell proliferation and vacuolar degeneration were alleviated, while the tubular dilatation or atrophy, fibrous tissues, and inflammatory cells were reduced in liraglutide group. Moreover, the results of immunohistochemical staining revealed that the glomerular Nephrin protein was arranged uniformly and showed the blue-black particles in healthy group. The glomerular Nephrin protein expressed was significantly decreased and arranged disorderly in diabetes group compared with that in healthy group, while it was increased in liraglutide group compared with that in diabetes group (p<0.05). The protein expression of Akt-mTOR in renal tissues was the lowest in healthy group and the highest in diabetes group, while it markedly declined in liraglutide group compared with that in diabetes group, displaying statistically significant differences (p<0.05). Similarly, the mRNA expression of Akt-mTOR in renal tissues was the lowest in healthy group and the highest in diabetes group, while it markedly declined in liraglutide group compared with that in diabetes group, displaying also statistically significant differences (p<0.05).. Liraglutide can significantly reduce the blood glucose and improve the renal function in rats by suppressing the protein expression of AKT-mTOR, thereby exerting a protective effect on renal damage in rats with DKD.

Topics: Animals; Blood Glucose; Cells, Cultured; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Gene Expression Regulation; Hypoglycemic Agents; Liraglutide; Male; Membrane Proteins; Mesangial Cells; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Signal Transduction; Specific Pathogen-Free Organisms; Streptozocin; TOR Serine-Threonine Kinases

To determine the specific role of podocyte-expressed cannabinoid-1 receptor (CB. We developed a mouse model with a podocyte-specific deletion of CB. High glucose exposure for 48 hours led to an increase in CB. Activation of CB

Topics: Animals; Arginase; Cell Hypoxia; Cells, Cultured; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Glucose; Intracellular Signaling Peptides and Proteins; Kidney; Kidney Glomerulus; Kidney Tubules, Proximal; Membrane Proteins; Mice; Microcirculation; Oxidative Stress; Podocytes; Receptor, Cannabinoid, CB1; Receptors, Cannabinoid

BACKGROUND Gremlin has been reported to be up-regulated in glomerular mesangial cells in diabetic nephropathy (DN). However, the regulation of gremlin in podocytes is still rarely reported. This study aimed to investigate the underlying mechanisms by which gremlin mediates the pathogenesis of DN via transforming growth factor-β (TGF-β) signaling pathways. MATERIAL AND METHODS Lentiviral and RNAi transfection were performed to increase and decrease gremlin expression in high-glucose conditions. Expression at the mRNA and protein level was detected by RT-qPCR and Western blotting. RESULTS The expression of gremlin was significantly higher in high-glucose (HG, 30mM) than normal-glucose (NG, 5.5 mM) conditions. The gremlin overexpression significantly suppressed the expression of nephrin and synaptopodin. The phosphorylation of canonical TGF-b signaling pathway components, including Smad2/3 and MKK, was increased in the gremlin-overexpressing group. In addition, the expression levels of Bax and cleaved caspase-3 were also higher in the gremlin-overexpressing group. TGF-β pathway inhibitor (SB505124) significantly inhibited TGF-β pathway activity and enhanced the expression of nephrin and synaptopodin. CONCLUSIONS These results indicate that gremlin can aggravate podocyte lesions through the TGF-β signaling pathway, providing a novel therapeutic target for DN.

Topics: Animals; Apoptosis; Benzodioxoles; Cytokines; Diabetic Nephropathies; Imidazoles; Intercellular Signaling Peptides and Proteins; Lentivirus; Membrane Proteins; Mice; Microfilament Proteins; Podocytes; Pyridines; Signal Transduction; Transforming Growth Factor beta; Up-Regulation

BACKGROUND β-arrestins have been shown to play a critical role in the progression of diabetic nephropathy. Nevertheless, the potential mechanism of β-arrestins on the regulation of podocyte apoptosis has rarely been discussed. This study aimed to elucidate the regulation of β-arrestin 1/2 on podocyte apoptosis through the Wnt/b-catenin pathway. MATERIAL AND METHODS This study structured β-arrestin 1/2 down-regulated and up-regulated expression by plasmid transfection. The protein levels were detected with Western blotting, and mRNA expression was detected with RT-qPCR. The apoptotic cells were measured by flow cytometry. RESULTS β-arrestin 1/2 expression levels of podocytes were up-regulated in high-glucose-induced podocytes. β-arrestin 1/2 overexpression inhibited the expression of nephrin and podocin protein. Up-regulated β-arrestin 1/2 promoted podocyte apoptosis and p53 pathway by increasing Bax, cleaved caspase-3, and p-p53 levels in high-glucose-induced podocytes. Flow cytometry showed that the apoptotic cells were markedly higher in the b-arrestin 1/2 up-regulated group compared with the scramble group. Expression of β-catenin was increased in the β-arrestin 1/2 up-regulated group, which indicated that the Wnt/b-catenin pathway was activated. Wnt/b-catenin pathway inhibitor (Dkk1) distinctly suppressed the apoptosis induced by β-arrestin 1/2 overexpression and high glucose. CONCLUSIONS These results provide a molecular pathomechanism of β-arrestin 1/2 and Wnt/β-catenin pathway on podocyte apoptosis and provide new ideas for the treatment of diabetic nephropathy, which paves the way for the future study of diabetic nephropathy and podocytes.

Topics: Animals; Apoptosis; beta Catenin; beta-Arrestin 1; beta-Arrestin 2; Cell Line; Diabetic Nephropathies; Down-Regulation; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mice; Mice, Transgenic; Podocytes; Transcriptional Activation; Up-Regulation; Wnt Signaling Pathway

To analyze the correlation between the expressions of vascular endothelial growth factor (VEGF) and transient receptor potential canonical 6 (TRPC6) and their role in podocyte injury in rats with diabetic nephropathy.. Forty SD rats with diabetic nephropathy induced by intraperitoneal injection of 65 mg/kg streptozotocin were randomized equally into 5 groups, including a diabetic nephropathy model group and 4 treatment groups, with 8 normal SD rats as the normal control group. In the 4 treatment groups, the rats received intraperitoneal injections with SU5416 at 5 mg/kg or 10 mg/kg twice a week or with LY294002 at 1 mg/kg or 2 mg/kg once daily for 8 weeks. Blood glucose, serum creatinine, blood urea nitrogen, and 24-h urinary protein levels of the rats were detected at different time points, and the pathologies in the renal tissue were observed using HE staining, PAS staining and immunohistochemistry. The expressions of VEGF, nephrin, and TRPC6 at mRNA and protein levels were detected using RT-PCR and Western blotting.. Compared with normal control rats, the diabetic rats showed significantly increased fasting blood glucose, serum creatinine, blood urea nitrogen and 24-h urinary protein levels with decreased expressions of nephrin mRNA and protein (P<0.05) and increased expressions of VEGF and TRPC6 (P<0.05). Compared with the untreated diabetic rats, the rats with SU5416 treatment showed increased 24-h urinary protein, urea nitrogen, and nephrin expression and decreased TRPC6 expression without significant changes in fasting blood glucose, serum creatinine, or VEGF expression. The rats treated with LY294002 showed decreased 24-h urinary protein and TRPC6 expression without significant changes in fasting blood glucose, serum creatinine, urea nitrogen, or expressions of nephrin and VEGF.. The regulatory effect of VEGF on TRPC6 can be blocked by inhibiting VEGFR-2 or blocking PI3K/Akt signaling pathway.

Topics: Animals; Chromones; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Indoles; Kidney; Membrane Proteins; Morpholines; Podocytes; Pyrroles; Random Allocation; Rats; Rats, Sprague-Dawley; TRPC Cation Channels; Vascular Endothelial Growth Factor A

The maintenance of interpodocyte slit diaphragm is critical in the sieving function of glomerular filtration barrier. Eucalyptol is a natural constituent in aromatic plants with antioxidant properties. This study investigates whether and how eucalyptol inhibits podocyte slit diaphragm malfunction in glucose-exposed podocytes and diabetic mouse kidneys.. Podocytes were incubated in media containing 33 mm glucose with 1-20 μm eucalyptol. The in vivo model employed db/db mice orally administrated with 10 mg kg. Eucalyptol may be a potent agent antagonizing diabetes-associated malformation of interpodocyte slit junction and podocyte actin cytoskeleton.

Topics: Animals; Cell Line; Diabetic Nephropathies; Eucalyptol; Glucose; Glycation End Products, Advanced; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Male; MAP Kinase Signaling System; Membrane Proteins; Mice, Inbred C57BL; Podocytes; Proto-Oncogene Proteins c-myc; Receptor for Advanced Glycation End Products; Signal Transduction

Previous studies have shown that podocyte autophagy is an important trigger for proteinuria and glomerulosclerosis. The mammalian rapamycin target protein (mTOR) occupies a pivotal position in the autophagy pathway. In this study, we planned to clarify the mechanism of mTOR regulation of podocyte autophagy and the effect of rapamycin (RAPA).. All rats were randomly divided into normal control group (n = 8), DN group (n = 8), and RAPA group (n = 8). Blood and urine samples were collected at the 4th, 8th, and 12th weeks of the experiment. The serum creatinine (Scr), urine volume levels, and the 24 h urine protein (UP) levels were examined. The nephrin, podocin, mTOR, ribosomal S6 kinase 1 (S6K1), and autophagy marker light chain 3 (LC3II) expression levels were evaluated by immunohistochemistry, quantitative PCR, and immunoblotting.. The urine volume, 24 h UP, and Scr of the DN and RAPA groups increased significantly compared with the NC group (p < .05). Nephrin and podocin expression was decreased in the kidney tissues of the DN and RAPA groups compared with the NC group (p < .05). The expression levels of mTOR and S6K1 increased and LC3II expression decreased in the renal tissues of the DN and RAPA groups compared with the NC group (p < .05). After RAPA treatment, all the above indexes were improved compared with the DN group (p < .05), but were significantly abnormal compared with the NC group (p < .05).. The proteinuria and kidney function had improved after RAPA treatment. These results confirmed that RAPA specifically binds to mTOR kinase, and inhibits mTOR activity, thereby regulating the pathological autophagic process.

Topics: Animals; Autophagy; Creatinine; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Podocytes; Proteinuria; Rats; Rats, Sprague-Dawley; Ribosomal Protein S6 Kinases; Signal Transduction; Sirolimus; Streptozocin; TOR Serine-Threonine Kinases

Nephrin is a core component of podocyte (glomerular epithelial cell) slit diaphragm and is required for kidney ultrafiltration. Down-regulation or mislocalization of nephrin has been observed in diabetic kidney disease (DKD), characterized by albuminuria. Here, we investigate the role of protein kinase C and casein kinase 2 substrate in neurons 2 (PACSIN2), a regulator of endocytosis and recycling, in the trafficking of nephrin and development of DKD. We observe that PACSIN2 is up-regulated and nephrin mislocalized in podocytes of obese Zucker diabetic fatty (ZDF) rats that have altered renal function. In cultured podocytes, PACSIN2 and nephrin colocalize and interact. We show that nephrin is endocytosed in PACSIN2-positive membrane regions and that PACSIN2 overexpression increases both nephrin endocytosis and recycling. We identify rabenosyn-5, which is involved in early endosome maturation and endosomal sorting, as a novel interaction partner of PACSIN2. Interestingly, rabenosyn-5 expression is increased in podocytes in obese ZDF rats, and,

Topics: Adaptor Proteins, Signal Transducing; Animals; Carrier Proteins; Cytoskeletal Proteins; Diabetes Mellitus; Diabetic Nephropathies; Gene Expression Regulation; Humans; Membrane Proteins; Mice; Obesity; Podocytes; Protein Transport; Proteins; Rats, Zucker; Up-Regulation; Vesicular Transport Proteins

Renal podocytes form the main filtration barrier possessing unique phenotype maintained by proteins including podocalyxin and nephrin, which are modulated in pathological conditions. In diabetic nephropathy (DN), podocytes become structurally and functionally compromised. Nephrin, a structural backbone protein of the slit diaphragm, acts as regulator of podocyte intracellular signalling with renoprotective role. Vitamin D

Topics: Animals; Bone Density Conservation Agents; Cell Survival; Cholecalciferol; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Ergocalciferols; Glucose; Kidney Glomerulus; Membrane Proteins; Podocytes; Rats, Wistar; Sialoglycoproteins; Signal Transduction; Tissue Culture Techniques

Podocyte injury and glomerular basement membrane thickening have been considered as essential pathophysiological events in diabetic nephropathy. The aim of this study was to investigate the possible beneficial effects of vinpocetine on diabetes-associated renal damage. Male Wistar rats were made diabetic by injection of streptozotocin (STZ). Diabetic rats were treated with vinpocetine in a dose of 20mg/kg/day for 6 weeks. Treatment with vinpocetine resulted in a marked decrease in the levels of blood glucose, glycosylated haemoglobin, creatinine, blood urea nitrogen, urinary albumin and albumin/creatinine ratio along with an elevation in creatinine clearance rate. The renal contents of advanced glycation end-products, interleukin-10, tissue growth factor-β, nuclear factor (NF)-κB and Ras-related C3 botulinum toxin substrate 1 (Rac 1) were decreased. Renal nephrin and podocin contents were increased and their mRNA expressions were replenished in vinpocetine-treated rats. Moreover, administration of vinpocetine showed improvements in oxidative status as well as renal glomerular and tubular structures. The current investigation revealed that vinpocetine ameliorated the STZ-induced renal damage. This beneficial effect could be attributed to its antioxidant and antihyperglycemic effects parallel to its ability to inhibit NF-κB which eventually modulated cytokines production as well as nephrin and podocin proteins expression.

Topics: Animals; Blood Glucose; Body Weight; Diabetic Nephropathies; Disease Models, Animal; Fasting; Gene Expression Regulation; Glycated Hemoglobin; Intracellular Signaling Peptides and Proteins; Kidney; Male; Membrane Proteins; NF-kappa B; Podocytes; Proteinuria; rac1 GTP-Binding Protein; Rats; Rats, Wistar; RNA, Messenger; Vinca Alkaloids

Diabetic nephropathy (DN) is a serious and one of the most common microvascular complications of diabetes. There is accumulating evidence to indicate that advanced glycation end products (AGEs), senescent macroprotein derivatives formed at an accelerated rate under conditions of diabetes, play a role in DN. In this study, we found that the serum and urine levels of C-X-C motif chemokine ligand 9 (CXCL9) were significantly elevated in patients with DN compared with healthy controls. Based on an in vitro model of mouse podocyte injury, AGEs decreased the proliferation of podocytes and increased the expression of CXCL9 and C-X-C motif chemokine receptor 3 (CXCR3), and promoted the activation of signal transducer and activator of transcription 3 (STAT3). The knockdown of CXCL9 by the transfection of mouse podoyctes with specific siRNA significantly increased the proliferation and decreased the apoptosis of the podoyctes. Moreover, the levels of inflammatory factors, such as tumor necrosis factor (TNF)‑α and interleukin (IL)‑6 were also decreased in the podoyctes transfected with siRNA-CXCL9, accompanied by the increased expression of nephrin and podocin, and decreased levels of Bax/Bcl-2 and activated caspase-3. The knockdown of CXCL9 also led to the inactivation of the Janus kinase 2 (JAK2)/STAT3 pathway. Importantly, the use of the JAK2 inhibitor, AG490, and valsartan (angiotensin II receptor antagonist) attenuated the injury induced to mouse podoyctes by AGEs. On the whole, and to the best of our knowledge, this study demonstrates for the first time that AGEs exert pro-apoptotic and pro-inflammatory effects in mouse podoyctes through the CXCL9-mediated activation of the JAK2/STAT3 pathway. Thus, our data provide a potential therapeutic target for DN.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Cell Line; Chemokine CXCL9; Diabetic Nephropathies; Gene Expression Regulation; Glycation End Products, Advanced; Humans; Interleukin-6; Intracellular Signaling Peptides and Proteins; Janus Kinase 2; Membrane Proteins; Mice; Podocytes; Proto-Oncogene Proteins c-bcl-2; Receptors, Angiotensin; Receptors, CXCR3; RNA, Small Interfering; Signal Transduction; STAT3 Transcription Factor; Tumor Necrosis Factor-alpha; Tyrphostins; Valsartan

Renal damage and dysfunction is attributed to sustained hyperglycaemia in overt diabetes. Subcutaneous insulin injections are beneficial in delaying the progression of renal dysfunction and damage in diabetics. However, the current mode of administration is associated with severe undesirable effects. In this study, we evaluated the ameliorative effects of pectin-insulin dermal patches on renal dysfunction in diabetes.. Pectin-insulin patches (20.0, 40.8 and 82.9 µg/kg) were applied on the skin of streptozotocin-induced diabetic rats, thrice daily for 5 weeks. Blood glucose concentration, blood pressure and urine output volume were recorded on week 5 after which the animals were sacrificed after which the kidneys and plasma were collected. Kidney nephrin expression and urinary nephrin concentration, albumin excretion rate (AER), creatinine clearance (CC) and albumin creatinine ratio (ACR) were assessed.. Patch application resulted in reduced blood glucose concentration and blood pressure. Furthermore, pectin-insulin patch treatment resulted in increased kidney nephrin expression and reduced urinary nephrin concentration. AER, CC ACR were also reduced post patch application.. The application of pectin-insulin patch limited diabetes associated kidney damaged and improved kidney function. These observations suggest that pectin-insulin patches may ameliorate kidney dysfunction that is associated with chronic subcutaneous insulin administration.

Topics: Animals; Blood Glucose; Blood Pressure; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Insulin; Membrane Proteins; Pectins; Rats; Streptozocin; Transdermal Patch

Podocytes serve a critical role in the development of many glomerular diseases, including diabetic nephropathy (DN). Epigallocatechin‑3‑gallate (EGCG), a predominant polyphenolic component of green tea, has indicated its therapeutic effects in diabetes. In the present study, mouse podocyte cells were cultured in vitro, cell injury was induced by high glucose, and the protective effect of EGCG on cell proliferation and apoptosis and the underlying mechanisms were investigated. The results demonstrated that high glucose significantly inhibited cell proliferation after 48 and 72 h compared with normal glucose and mannitol treatment. EGCG (20 µmol/l) markedly promoted podocyte proliferation after 24, 48 and 72 h incubation with high glucose. Furthermore, high glucose significantly reduced WT‑1 and nephrin expression in podocytes compared with the normal glucose and mannitol groups, while EGCG (20 µmol/l) treatment largely restored their expression. High glucose also significantly increased the apoptotic cell population compared with normal glucose and mannitol groups. However, EGCG combined with high glucose greatly decreased the apoptotic cell number compared with high glucose treatment alone. Furthermore, high glucose treatment was demonstrated to significantly increase glucose‑regulated protein 78 (GRP78), phosphorylated‑ PKR‑like ER kinase (p‑PERK) and caspase‑12 protein expression levels, which is representative of endoplasmic reticulum (ER) stress, compared with the normal glucose and mannitol groups. However, EGCG treatment significantly attenuated GRP78, p‑PERK and caspase‑12 protein expression induced by high glucose. These findings suggested that EGCG serves a protective role in glucose‑induced podocyte apoptosis via suppressing ER stress, and may provide a novel therapeutic strategy to ameliorate the process of DN.

Topics: Animals; Apoptosis; Caspase 12; Catechin; Cells, Cultured; Diabetes Mellitus, Experimental; Diabetic Nephropathies; eIF-2 Kinase; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Glucose; Heat-Shock Proteins; Membrane Proteins; Mice; Podocytes; Protective Agents; WT1 Proteins

Hypertrophy is a prominent feature of damaged podocytes in diabetic kidney disease (DKD). mTORC1 hyperactivation leads to podocyte hypertrophy, but the detailed mechanism of how mTORC1 activation occurs under pathological conditions is not completely known. Moreover, reduced nephrin tyrosine phosphorylation has been observed in podocytes under pathological conditions, but the molecular mechanism linking nephrin phosphorylation and pathology is unclear so far. In this study, we observed a significant increase in C1-Ten level in diabetic kidney and in high glucose-induced damaged podocytes. C1-Ten acts as a protein tyrosine phosphatase (PTPase) at the nephrin-PI3K binding site and renders PI3K for IRS-1, thereby activating mTORC1. Furthermore, C1-Ten causes podocyte hypertrophy and proteinuria by increasing mTORC1 activity in vitro and in vivo. These findings demonstrate the relationship between nephrin dephosphorylation and the mTORC1 pathway, mediated by C1-Ten PTPase activity. We suggest that C1-Ten contributes to the pathogenesis of DKD by inducing podocyte hypertrophy under high glucose conditions.

Topics: Animals; Diabetic Nephropathies; Glucose; HEK293 Cells; Humans; Hypertrophy; Insulin Receptor Substrate Proteins; Male; Mechanistic Target of Rapamycin Complex 1; Membrane Proteins; Mice; Phosphatidylinositol 3-Kinases; Phosphorylation; Podocytes; Protein Tyrosine Phosphatases; Proteinuria; Signal Transduction; Tensins

Diabetic nephropathy (DN) is one of the major causes of end‑stage renal failure. Grape seed proanthocyanidin extracts (GSPE) are known to act as antioxidants. The current study aimed to determine the effects of GSPE on the streptozotocin (STZ)‑induced diabetic rat model and to explore the underlying mechanism of its action. Wistar rats were induced into a diabetic state by injection of STZ and were treated with 250 mg·kg‑1·day‑1 GSPE for 24 weeks. Kidney samples were collected for observation of renal pathological changes by light microscope (periodic acid‑Schiff staining) and electron microscopy. Reverse transcription‑polymerase chain reaction, western blotting, and immunohistochemical staining were used to detect the mRNA and protein expression of the receptor for advanced glycation end‑products (RAGE), nephrin and podocin. The results indicated that diabetic rats treated with GSPE had markedly reduced Ccr, urinary albumin excretion, ratio of kidney weight to body weight, AGEs and ECM accumulation (P<0.01) compared with that in the diabetic rats. GSPE treatment can also reverse the renal pathological damage in diabetic rats. Further results indicated that GSPE treatment significantly decreased the RAGE expression level (P<0.01), and significantly increased the expression level of nephrin in the kidney and glomeruli of diabetic rats (P<0.01). However, no significant differences were identified in the expression of podocin following GSPE treatment (P>0.05). In conclusion, the results demonstrated that GSPE exerts a reno‑protective effect by decreasing urinary albumin excretion and reversing renal pathological damage in diabetic rats. The underlying mechanism of GSPE activity is associated with the decreased expression of the AGEs/RAGE axis and the increased expression of nephrin in diabetic rats.

Topics: Animals; Antioxidants; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Disease Models, Animal; Gene Expression Regulation; Glycation End Products, Advanced; Grape Seed Extract; Humans; Kidney; Membrane Proteins; Oxidative Stress; Proanthocyanidins; Rats; Receptor for Advanced Glycation End Products

To investigate the effects of ferulic acid (FA) on the streptozocin (STZ) -induced kidney injury in diabetic rats and its possible mechanisms.. Diabetes was induced in male SD rats by an injection of STZ (40 mg/kg,i.v.). After 72 hours, blood glucose levels were detected and blood glucose levels exceeded 16.7 mmol/L were diagnosed as diabetic model rats. Diabetic model rats were randomly divided into model group and FA group, ten animal in each group. Another 10 healthy male SD rats were treated as control group. The rats in FA group were treated with FA (100 mg/kg, i.g.,qd) from the 5th week since the diabetic rats model was successfully established and lasted for 8 weeks. The levels of blood glucose, body weight, organ coefficient of kidney, blood urea nitrogen and creatinine were tested. HE staining was employed to observe the pathological changes of the renal tissue. Immunohistochemistry was employed to determine the protein of nephrin and podocin.. Compared to control group, the levels of blood glucose, organ coefficient of kidney, blood urea nitrogen(BUN) and serum creatinine(sCr) were increased significantly. Renal cells from model group rats showed atrophied and disordered after HE staining and interstitial proliferation were also appeared in renal tissue of the model group. Meantime, the levels of nephrin and podocin protein were obviously decreased. These changes were significantly attenuated in the model group treated with FA.. FA can evidently ameliorate renal damage in rats with diabetic nephropathy induced by STZ, which might be related to increase the level of nephrin and podocin protein.

Topics: Animals; Coumaric Acids; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Intracellular Signaling Peptides and Proteins; Kidney; Male; Membrane Proteins; Podocytes; Rats; Rats, Sprague-Dawley

Podocyte damage is the initial hallmark of diabetic nephropathy (DN), leading to the increasing morbidity and mortality in diabetic patients. Recent researches have corroborated the critical roles of miRNAs in the pathological progression of DN. Here, elevation of miR-217 was verified in high glucose (HG)-stimulated podocytes. Moreover, blocking miR-217 expression antagonized HG-induced cell injury by attenuating the adverse role of HG on cell viability and inhibiting ROS levels and cell apoptosis. Simultaneously, miR-217 repression restored HG-disrupted insulin resistance by elevating glucose uptake and nephrin expression, an essential component for insulin-induced glucose uptake. Mechanism assay substantiated the defective autophagy in HG-treated podocytes, which was resumed by miR-217 cessation. Importantly, suppressing autophagy pathway with 3-MA alleviated the protective roles of miR-217 down-regulation in podocyte injury and insulin resistance. Luciferase reporter analysis confirmed that PTEN was a target of miR-217 in podocytes. Additionally, blocking PTEN expression restrained autophagy restoration in miR-217-decreased cells. Furthermore, PTEN down-regulation attenuated the beneficial role of miR-217 suppression in HG-induced injury and insulin resistance. Together, this study manifests that miR-217inhibition can protectively antagonize HG-induced podocyte damage and insulin resistance by restoring the defective autophagy pathway via targeting PTEN, representing a novel and promising therapeutic target against diabetic nephropathy.

Topics: Animals; Apoptosis; Autophagy; Cell Survival; Cells, Cultured; Diabetic Nephropathies; Down-Regulation; Gene Expression Regulation; Glucose; Insulin Resistance; Membrane Proteins; Mice; MicroRNAs; Oligonucleotides; Podocytes; PTEN Phosphohydrolase; Reactive Oxygen Species; RNA, Small Interfering

Transforming growth factor beta 1 (TGF-β1) plays a critical role in the pathogenesis of glomerulosclerosis. The purpose of this study was to examine the effects of inhibition of transient receptor potential cation channel C6 (TRPC6) on podocyte injury induced by TGF-β1 via nephrin and desmin mechanisms.. A rat model of nephropathy was first induced by intravenous injections of adriamycin to determine TRPC6 signal pathway engaged in glomerulosclerosis in vivo. Conditionally immortalized podocytes were cultured in vitro and they were divided into four groups: control; TGF-β1 treatment; TGF-β1 with TRPC6 knockdown and TGF-β1 without TRPC6 knockdown. Real time RT-PCR and Western blot analysis were employed to determine the mRNA and protein of expression of nephrin, desmin and caspase-9, respectively. Flow cytometry was used to examine the apoptotic rate of podocytes and DAPI fluorescent staining was used to determine apoptotic morphology.. In vivo experiment, adriamycin significantly upregulated the protein expression of TGF-β1, TRPC6, desmin and caspase-9, and decreased nephrin. Consistent with the latter results, in vitro experiment mRNA and protein expression of desmin and caspase-9 was increased in cultured TGF-β1-treated podocytes, whereas nephrin was declined as compared with the control group. Importantly, TRPC6 knockdown significantly attenuated the upregulated desmin and caspase-9, and alleviated impairment of nephrin induced by TGF-β1. Moreover, typical morphologic features were presented in apoptotic podocytes. The number of apoptotic podocytes was increased after exposure to TGF-β1 and this was alleviated after TRPC6 knockdown. TRPC6 knockdown also decreased an apoptosis rate of TGF-β1-treated podocytes. Note that negative TRPC6 transfection control failed to alter an increase of the apoptosis rate in TGF-β1-treated podocytes.. TGF-β1 induced by glomerulosclerosis impairs the protein expression of nephrin and amplifies the protein expression of desmin and caspase -9 via TRPC6 signal pathway. Inhibition of TRPC6 alleviates these changes in podocytes-treated with TGF-β1 and attenuated apoptosis of podocytes. Our data suggest that TRPC6 signal plays an important role in mediating TGF-β1-induced podocyte injury via nephrin, desmin and caspase-9. Results of the current study also indicate that blocking TRPC6 signal pathway has a protective effect on podocyte injury. Targeting one or more of these signaling molecules may present new opportunities for treatment and management of podocyte injury observed in glomerulosclerosis.

Topics: Animals; Caspase 9; Cells, Cultured; Desmin; Diabetic Nephropathies; Disease Models, Animal; Doxorubicin; Gene Knockdown Techniques; Male; Membrane Proteins; Mice; Plasmids; Podocytes; Rats; Rats, Sprague-Dawley; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta1; TRPC Cation Channels; Up-Regulation

The present study aimed to investigate the relationship between wnt/β-catenin signaling pathway and kidney impairment in diabetic nephropathy (DN) mice as well as the renoprotective effect of rhein (RH). Mice were randomly divided into four groups (n = 6): db/db mice treated with RH (DN + RH), db/db mice (DN), db/m mice treated with RH (NC + RH) and db/m mice (NC). RH-treated groups were administered orally at a daily dose 120 mg/kg. Mice were sacrificed after 12 weeks of treatments. In our study, increased albuminuria, together with weight gain and hyperglycemia was observed in the beginning of the study and continued to increase throughout the length of the study (12 weeks). Histopathologic changes were observed in the DN group. Expectedly, mice receiving the treatment with RH were protected from this injury. Meanwhile, the expression of nephrin, a podocyte-specific marker, was significantly reduced while wnt1, p-GSK-3β/tGSK-3β, p-β-catenin/tβ-catenin were higher in the DN group mice when analyzed by immunofluorescence and Western blotting. RH reversed these above changes. wnt/β-catenin signaling pathway participates in RH ameliorating kidney injury in DN mice. The manipulation of RH might act as a promising therapeutic intervention for DN.

Topics: Animals; Anthraquinones; beta Catenin; Blood Glucose; Diabetic Nephropathies; Male; Membrane Proteins; Mice; Signal Transduction; Wnt Proteins

Discovery of common pathways that mediate both pancreatic β-cell function and end-organ function offers the opportunity to develop therapies that modulate glucose homeostasis and separately slow the development of diabetes complications. Here, we investigated the in vitro and in vivo effects of pharmacological agonism of the prostaglandin I2 (IP) receptor in pancreatic β-cells and in glomerular podocytes. The IP receptor agonist MRE-269 increased intracellular 3',5'-cyclic adenosine monophosphate (cAMP), augmented glucose-stimulated insulin secretion (GSIS), and increased viability in MIN6 β-cells. Its prodrug form, selexipag, augmented GSIS and preserved islet β-cell mass in diabetic mice. Determining that this preservation of β-cell function is mediated through cAMP/protein kinase A (PKA)/nephrin-dependent pathways, we found that PKA inhibition, nephrin knockdown, or targeted mutation of phosphorylated nephrin tyrosine residues 1176 and 1193 abrogated the actions of MRE-269 in MIN6 cells. Because nephrin is important to glomerular permselectivity, we next set out to determine whether IP receptor agonism similarly affects nephrin phosphorylation in podocytes. Expression of the IP receptor in podocytes was confirmed in cultured cells by immunoblotting and quantitative real-time PCR and in mouse kidneys by immunogold electron microscopy, and its agonism 1) increased cAMP, 2) activated PKA, 3) phosphorylated nephrin, and 4) attenuated albumin transcytosis. Finally, treatment of diabetic endothelial nitric oxide synthase knockout mice with selexipag augmented renal nephrin phosphorylation and attenuated albuminuria development independently of glucose change. Collectively, these observations describe a pharmacological strategy that posttranslationally modifies nephrin and the effects of this strategy in the pancreas and in the kidney.

Topics: Acetamides; Acetates; Animals; Cell Line; Cell Survival; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Humans; Hypoglycemic Agents; Insulin; Insulin Secretion; Insulin-Secreting Cells; Membrane Proteins; Mice, Inbred C57BL; Mice, Knockout; Mutation; Phosphorylation; Podocytes; Prodrugs; Protein Processing, Post-Translational; Pyrazines; Receptors, Epoprostenol; Renal Insufficiency; RNA Interference

Emerging evidence has demonstrated that microRNAs (miRNAs) play a mediatory role in the pathogenesis of diabetic nephropathy. In this study, we found that miR-218 was upregulated in high glucose (HG) treated podocytes, which are essential components of the glomerular filtration barrier and a major prognostic determinant in diabetic nephropathy. Additionally, up-regulation of miR-218 was accompanied by an increased rate of podocyte death and down-regulation in the level of nephrin, a key marker of podocytes. However, inhibition of miR-218 exerted the opposite effect. In addition, the dual-luciferase reporter assay showed that miR-218 directly targeted the 3'-untranslated region of heme oxygenase-1 (HO-1), and further study confirmed an increase of HO-1 in HG-treated podocytes transfected with anti-miR-218. Knockdown of HO-1 blocked the anti-apoptotic effect of anti-miR-218. Furthermore, inhibition of miR-218 was associated with decreased expression of the known pro-apoptotic molecule p38-mitogen-activated protein kinase (p38-MAPK) activation. Following preconditioning with SB203580, an inhibitor of p38-MAPK, the stimulatory effect of HG on podocyte apoptosis was strikingly ameliorated. These findings suggested that miR-218 accelerated HG-induced podocyte apoptosis through directly down-regulating HO-1 and facilitating p38-MAPK activation.

Topics: 3' Untranslated Regions; Animals; Apoptosis; Diabetic Nephropathies; Down-Regulation; Enzyme Activation; Gene Knockdown Techniques; Glucose; HEK293 Cells; Heme Oxygenase-1; Humans; Membrane Proteins; Mice; MicroRNAs; p38 Mitogen-Activated Protein Kinases; Podocytes; Up-Regulation

Although dipeptidyl peptidase IV (DPPIV) inhibitors are known to have renoprotective effects, the mechanism underlying these effects has remained elusive. Here we investigated the effects of DA-1229, a novel DPPIV inhibitor, in two animal models of renal injury including db/db mice and the adriamycin nephropathy rodent model of chronic renal disease characterized by podocyte injury. For both models, DA-1229 was administered at 300 mg/kg/day. DPPIV activity in the kidney was significantly higher in diabetic mice compared with their nondiabetic controls. Although DA-1229 did not affect glycemic control or insulin resistance, DA-1229 did improve lipid profiles, albuminuria and renal fibrosis. Moreover, DA-1229 treatment resulted in decreased urinary excretion of nephrin, decreased circulating and kidney DPPIV activity, and decreased macrophage infiltration in the kidney. In adriamycin-treated mice, DPPIV activity in the kidney and urinary nephrin loss were both increased, whereas glucagon-like peptide-1 concentrations were unchanged. Moreover, DA-1229 treatment significantly improved proteinuria, renal fibrosis and inflammation associated with decreased urinary nephrin loss, and kidney DPP4 activity. In cultured podocytes, DA-1229 restored the high glucose/angiotensin II-induced increase of DPPIV activity and preserved the nephrin levels in podocytes. These findings suggest that activation of DPPIV in the kidney has a role in the progression of renal disease, and that DA-1229 may exert its renoprotective effects by preventing podocyte injury.

Topics: Animals; Chemokine CCL2; Diabetic Nephropathies; Dipeptidyl Peptidase 4; Dipeptidyl-Peptidase IV Inhibitors; Disease Models, Animal; Doxorubicin; Inflammation Mediators; Kidney; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Osteopontin; Piperazines; Podocytes; Protective Agents

Accumulating evidence has suggested that the epithelial-mesenchymal transition (EMT) is a pathway that potentially leads to podocyte depletion and proteinuria in diabetic nephropathy (DN). Therefore, this study was designed to investigate the protective effects of forkhead transcription factor O1 (FOXO1) on podocyte EMT, under high-glucose (HG) conditions in vitro and under diabetic conditions in vivo. The results showed that HG-induced podocyte EMT was associated with FOXO1 inactivation, which was accompanied by activation of the transforming growth factor (TGF)-β1/SMAD3/integrin-linked kinase (ILK) pathway. Accordingly, constitutive FOXO1 activation suppressed the TGF-β1/Smad3/ILK pathway and partially reversed EMT, similar to the effects observed after treatment with SIS3 or QLT0267, which are selective inhibitors of TGF-β1-dependent SMAD3 phosphorylation and ILK, respectively. In addition, lentiviral-mediated FOXO1 overexpression in the kidneys of diabetic mice considerably increased FOXO1 expression and activation, while decreasing proteinuria and renal pathological injury. These data suggested that forced FOXO1 activation inhibited HG-induced podocyte EMT and ameliorated proteinuria and renal injury in diabetic mice. Our findings further highlighted that FOXO1 played a protective role against diabetes in mice and may potentially be used as a novel therapeutic target for treating diabetic nephropathy.

Topics: Animals; Azo Compounds; Desmin; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Epithelial-Mesenchymal Transition; Forkhead Box Protein O1; Forkhead Transcription Factors; Gene Expression Regulation; Glucose; Isoquinolines; Male; Membrane Proteins; Mice; Podocytes; Protein Serine-Threonine Kinases; Pyrazoles; Pyridines; Pyrroles; Smad3 Protein; Transforming Growth Factor beta1

The 12-lipoxygenase (12-LO) and angiotensin II (Ang II) interaction plays an important role in diabetic nephropathy (DN). Proteinuria in DN is associated with decreased slit diaphragm proteins including nephrin and P-cadherin. Therefore, we investigated whether Ang II type 1 receptor (AT1) blocker (ARB) regulates 12-LO activity and slit diaphragm protein expression in diabetic rat glomeruli.. Glomeruli were isolated with the sieving method, and classified into small glomeruli (SG; 75-μm sieve) and large glomeruli (LG; 125-μm sieve).. 12(S)-HETE, a lipid product of 12-LO, was increased by Ang II in the glomeruli. Infusion of 12(S)-HETE and Ang II significantly decreased nephrin expression in LG, but increased it in SG compared to control. Glomerular P-cadherin expression was reduced after Ang II and 12(S)-HETE treatment without differences between LG and SG. ARB did not influence glycemic levels but completely abolished the increases in 12(S)-HETE, AT1 expression, and proteinuria in diabetic rats. Nephrin expression was significantly reduced in LG but increased in SG in diabetic rats compared to control. P-cadherin expression decreased in both diabetic LG and SG. The abnormalities of nephrin and P-cadherin were partially but significantly reversed by ARB.. ARB potentially ameliorates DN via the up-regulation of glomerular nephrin and P-cadherin expression through the inhibition of 12-LO activation in the glomeruli of rats with DN.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Animals; Arachidonate 12-Lipoxygenase; Cadherins; Cells, Cultured; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Diet, High-Fat; Kidney Glomerulus; Lipoxygenase Inhibitors; Losartan; Male; Membrane Proteins; Mice; Podocytes; Rats, Sprague-Dawley; Receptor, Angiotensin, Type 1; Renin-Angiotensin System; Streptozocin

The aim of this research was to investigate the effects of cyclopropanyldehydrocostunolide (also named LJ), a derivative of sesquiterpene lactones (SLs), on high glucose (HG)-induced podocyte injury and the associated molecular mechanisms.. Differentiated mouse podocytes were incubated in different treatments. The migration and albumin filtration of podocytes were examined by Transwell filters. The protein and mRNA levels of MCP-1 were measured using enzyme-linked immunosorbent assay (ELISA) and quantitative real-time PCR (q-PCR). Protein expression and phosphorylation were detected by western blot, and the nuclear translocation of NF-κB was performed with a confocal microscope. The gene expression of the receptor activator for NF-κB (RANK) was silenced by small interfering RNA (siRNA).. Our results showed that HG enhanced migration, albumin filtration and MCP-1 expression in podocytes. At the molecular level, HG promoted the phosphorylation of NF-κB/p65, IKKβ, IκBα, mitogen-activated protein kinase (MAPK) and the nuclear translocation of p65. LJ reversed the effects of HG in a dose-dependent manner. Furthermore, our data provided the first demonstration that the receptor activator for NF-κB ligand (RANKL) and its cognate receptor RANK were overexpressed in HG-induced podocytes and were downregulated by LJ. RANK siRNA also attenuated HG-induced podocyte injury and markedly inhibited the activation of NF-κB and MAPK signaling pathways.. LJ attenuates HG-induced podocyte injury by suppressing RANKL/RANK-mediated NF-κB and MAPK signaling pathways.

Topics: Active Transport, Cell Nucleus; Animals; Biomarkers; Cell Line, Transformed; Cell Movement; Chemokine CCL2; Diabetic Nephropathies; Gene Expression Regulation; Hyperglycemia; Hypoglycemic Agents; Lactones; MAP Kinase Signaling System; Membrane Proteins; Mice; Phosphorylation; Podocytes; Protein Processing, Post-Translational; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; RNA Interference; Sesquiterpenes; Transcription Factor RelA

Urinary nephrin is a potential non-invasive biomarker of disease. To date, however, most studies of urinary nephrin have been conducted in animal models of diabetic nephropathy, and correlations between urinary nephrin-to-creatinine ratio and other parameters have yet to be evaluated in animal models or patients of kidney disease with podocyte dysfunction. We hypothesized that urinary nephrin-to-creatinine ratio can be up-regulated and is negatively correlated with renal nephrin mRNA levels in animal models of kidney disease, and that increased urinary nephrin-to-creatinine ratio levels are attenuated following administration of glucocorticoids. In the present study, renal nephrin mRNA, urinary nephrin-to-creatinine ratio, urinary protein-to-creatinine ratio, and creatinine clearance ratio were measured in animal models of adriamycin nephropathy, puromycin aminonucleoside nephropathy, anti-glomerular basement membrane glomerulonephritis, and 5/6 nephrectomy. The effects of prednisolone on urinary nephrin-to-creatinine ratio and other parameters in puromycin aminonucleoside (single injection) nephropathy rats were also investigated. In all models tested, urinary nephrin-to-creatinine ratio and urinary protein-to-creatinine ratio increased, while renal nephrin mRNA and creatinine clearance ratio decreased. Urinary nephrin-to-creatinine ratio exhibited a significant negative correlation with renal nephrin mRNA in almost all models, as well as a significant positive correlation with urinary protein-to-creatinine ratio and a significant negative correlation with creatinine clearance ratio. Urinary protein-to-creatinine ratio exhibited a significant negative correlation with renal nephrin mRNA. Following the administration of prednisolone to puromycin aminonucleoside (single injection) nephropathy rats, urinary nephrin-to-creatinine ratio was significantly suppressed and exhibited a significant positive correlation with urinary protein-to-creatinine ratio. In addition, the decrease in number of glomerular Wilms tumor antigen-1-positive cells was attenuated, and urinary nephrin-to-creatinine ratio exhibited a significant negative correlation in these cells. In conclusion, these results suggest that urinary nephrin-to-creatinine ratio level is a useful and reliable biomarker for predicting the amelioration of podocyte dysfunction by candidate drugs in various kidney disease models with podocyte dysfunction. This suggestion will also be validated in a clinical set

Topics: Animals; Anti-Glomerular Basement Membrane Disease; Biomarkers; Creatinine; Diabetic Nephropathies; Doxorubicin; Kidney Diseases; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; Podocytes; Puromycin Aminonucleoside; Rats; Rats, Wistar

Diabetic nephropathy (DN) is one of the major microvascular complications in diabetes. Podocyte injury such as slit diaphragm effacement is regarded as a determinant in the occurrence and development of albuminuria in DN. In this study, we examined the effect of hyperoside, an active flavonoid glycoside, on proteinuria and renal damage in a streptozotocin-induced DN mouse model at the early stage. The results showed that oral administration of hyperoside (30 mg/kg/day for 4 weeks could significantly decrease urinary microalbumin excretion and glomerular hyperfiltration in DN mice, but did not affect the glucose and lipid metabolism. Periodic acid-Schiff staining and transmission electron microscopy showed that glomerular mesangial matrix expansion and podocyte process effacement in DN mice were significantly improved by hyperoside. Further investigations via immunofluorescence staining, real-time reverse transcription polymerase chain reaction and Western blot analysis showed that the decreased slit diaphragm protein nephrin and podocin mRNA expression and protein levels in DN mice were restored by hyperoside treatment. Collectively, these findings demonstrated that hyperoside could decrease albuminuria at the early stage of DN by ameliorating renal damage and podocyte injury.

Topics: Albuminuria; Animals; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Female; Glycosides; Intracellular Signaling Peptides and Proteins; Kidney; Membrane Proteins; Mice, Inbred C57BL; Phytotherapy; Plant Extracts; Podocytes; Quercetin

Diabetic nephropathy (DN) is the major cause of end-stage renal disease worldwide. Podocytes are important for glomerular filtration barrier function and maintenance of size selectivity in protein filtration in the kidney. Podocyte damage is the basis of many glomerular diseases characterized by loss of interdigitating foot processes and decreased expression of components of the slit diaphragm. Nephrin, a podocyte-specific protein, is the main component of the slit diaphragm. Loss of nephrin is observed in human and rodent models of diabetic kidney disease. The long isoform of CIN85 (RukL) is a binding partner of nephrin that mediates nephrin endocytosis via ubiquitination in podocytes. Here we demonstrate that the loss of nephrin expression and the onset of proteinuria in diabetic mice correlate with an increased accumulation of ubiquitinated proteins and expression of CIN85/RukL in podocytes. CIN85/RukL deficiency preserved nephrin surface expression on the slit diaphragm and reduced proteinuria in diabetic mice, whereas overexpression of CIN85 in zebrafish induced severe edema and disruption of the filtration barrier. Thus, CIN85/RukL is involved in endocytosis of nephrin in podocytes under diabetic conditions, causing podocyte depletion and promoting proteinuria. CIN85/RukL expression therefore shows potential to be a novel target for antiproteinuric therapy in diabetes.

Topics: Adaptor Proteins, Signal Transducing; Animals; Creatinine; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Endocytosis; Genotype; Humans; Membrane Proteins; Mice; Microscopy, Electron, Transmission; Neoplasm Proteins; Nerve Tissue Proteins; Podocytes; Proteinuria

The present study was designed to examine the protective effect of notoginsenoside R1 (NR1) on podocytes in a rat model of streptozotocin (STZ)‑induced diabetic nephropathy (DN), and to explore the mechanism responsible for NR1-induced renal protection. Diabetes was induced by a single injection of STZ, and NR1 was administered daily at a dose of 5 mg/kg (low dose), 10 mg/kg (medium) and 20 mg/kg (high) for 16 weeks in Sprague-Dawley rats. Blood glucose levels, body weight and proteinuria were measured every 4 weeks, starting on the day that the rats received NR1. Furthermore, on the day of sacrifice, blood, urine and kidneys were collected in order to assess renal function according to general parameters. Pathological staining was performed to evaluate the renal protective effect of NR1, and the expression of the key slit diaphragm proteins, namely neprhin, podocin and desmin, were evaluated. In addition, the serum levels of inflammatory cytokines [tumor necrosis factor-α (TNF-α), tumor growth factor-β1 (TGF-β1), interleukin (IL)-1 and IL-6] as well as an anti-inflammatory cytokine (IL-10) were assessed, and the apoptosis of podocytes was quantified. Finally, the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway and the involvement of nuclear factor-κB (NF-κB) inactivation was further analyzed. In this study, NR1 improved renal function by ameliorating histological alterations, increasing the expression of nephrin and podocin, decreasing the expression of desmin, and inhibiting both the inflammatory response as well as the apoptosis of podocytes. Furthermore, NR1 treatment increased the phosphorylation of both PI3K (p85) and Akt, indicating that activation of the PI3K/Akt signaling pathway was involved. Moreover, NR1 treatment decreased the phosphorylation of NF-κB (p65), suggesting the downregulation of NF-κB. This is the first study to the best of our knowledge, to clearly demonstrate that NR1 treatment ameliorates podocyte injury by inhibiting both inflammation and apoptosis through the PI3K/Akt signaling pathway.

Topics: Animals; Apoptosis; Blood Glucose; Cell Nucleus; Cytokines; Desmin; Diabetic Nephropathies; Ginsenosides; Inflammation; Intracellular Signaling Peptides and Proteins; Kidney; Male; Membrane Proteins; Phosphatidylinositol 3-Kinases; Phosphorylation; Podocytes; Protective Agents; Proteinuria; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Signal Transduction

Podocyte dysfunction is important in the onset and development of diabetic nephropathy (DN). Histone deacetylases (HDACs) have been recently proved to play critical roles in the pathogenesis of DN. As one subtype of the class IIa HDACs, HDAC9 is capable to repress/de-repress their target genes in tumor, inflammation, atherosclerosis and metabolic diseases. In the present study, we investigate whether HDAC9 is involved in the pathophysiologic process of DN, especially the podocyte injury. Firstly, we explored the expression patterns and localization of HDAC9 and found that HDAC9 expression was significantly up-regulated in high glucose (HG)-treated mouse podocytes, as well as kidney tissues from diabetic db/db mice and patients with DN. Secondly, knockdown of HDAC9 in mouse podocytes significantly suppressed HG-induced reactive oxygen species (ROS) generation, cell apoptosis and inflammation through JAK2/STAT3 pathway and reduced the podocytes injury by decreasing the expression levels of Nephrin and Podocin. Moreover, in diabetic db/db mice, silencing of HDAC9 attenuated the glomerulosclerosis, inflammatory cytokine release, podocyte apoptosis and renal injury. Collectively, these data indicate that HDAC9 may be involved in the process of DN, especially podocyte injury. Our study suggest that inhibition of HDAC9 may have a therapeutic potential in DN treatment.

Topics: Animals; Apoptosis; Diabetic Nephropathies; Gene Knockdown Techniques; Gene Silencing; Glucose; Histone Deacetylases; Inflammation; Intracellular Signaling Peptides and Proteins; Janus Kinase 2; Kidney; Male; Matrix Metalloproteinases; Membrane Proteins; Mice, Inbred C57BL; Phosphorylation; Podocytes; Protein Kinase Inhibitors; Reactive Oxygen Species; Repressor Proteins; RNA, Messenger; Signal Transduction; STAT3 Transcription Factor; Up-Regulation

Epithelial-mesenchymal transition (EMT) is important for diabetic nephropathy (DN). Podocytes are specialized epithelial cells, which form a major component of the glomerular filtration barrier. Podocyte damage has been suggested to be the primary mechanism behind the albuminuria associated with DN. The present study aimed to determine the function of glycogen synthase kinase (GSK)‑3β in EMT and barrier dysfunction of mouse podocytes exposed to high glucose (HG) conditions. Matured and differentiated podocytes were treated with normal glucose (NG), HG or NG + mannitol. Podocytes were also transfected with a small interfering RNA (siRNA) against GSK‑3β or a scrambled siRNA, or were treated with lithium chloride (LiCl), a GSK‑3β inhibitor, under NG or HG conditions. The expression levels of the epithelial cell markers, nephrin and podocin, and the myofibroblast cell markers, α‑smooth muscle actin (SMA) and fibronectin, in podocytes by western blot analysis and immunofluorescence staining, respectively. The monolayer barrier function was assessed by albumin inflow. The phosphorylation and activity levels of GSK‑3β were also quantified. It was observed that HG promotes EMT in podocytes, due to the increased levels of podocin and nephrin expression and the reduced α‑SMA and fibronectin expression levels. HG also induced barrier dysfunction and increased the expression level of total GSK‑3β, Try216‑phosphorylated‑GSK‑3β and the GSK‑3β activity in podocytes. Transfection of GSK‑3β siRNA or treatment with LiCl reversed the HG‑induced EMT and barrier dysfunction in podocytes. In conclusion, the present study determined that GSK‑3β is required for EMT and barrier dysfunction in podocytes under HG conditions; therefore, GSK‑3β may be a novel target for the treatment of DN.

Topics: Actins; Animals; Diabetic Nephropathies; Disease Models, Animal; Epithelial-Mesenchymal Transition; Gene Expression Regulation; Glomerular Filtration Barrier; Glucose; Glycogen Synthase Kinase 3 beta; Humans; Intracellular Signaling Peptides and Proteins; Lithium Chloride; Membrane Proteins; Mice; Podocytes; RNA, Small Interfering

MiR-155 has been reported to be involved in both innate and adaptive immune responses. But the role of miR-155 in hyperglycemia-induced nephropathy is still unknown. In our current study, 3-month-old male wild-type C57 mice and Mir-155(-/-) mice were used to establish hyperglycemia-induced nephropathy. In our hyperglycemia-induced nephropathy model, the expression of podocyte injury marker desmin was markedly increased in the diabetes group when compared with control. Diabetes also significantly decreased the levels of nephrin and acetylated nephrin, whereas the expression of miR-155 was markedly increased in diabetes group when compared with control. MiR-155(-/-) mice showed significantly increased expression of nephrin, acetylated nephrin, and Wilm's tumor-1 protein (WT-1) when compared with wild-type control. MiR-155 deficiency results in significantly decrease in IL-17A expression both in vivo and in vitro. And the increased expression of WT-1, nephrin, and ac-nephrin was reversed with additional treatment of rmIL-17. Furthermore, we found that the inhibited Th17 differentiation induced by miR-155 deficiency was dependent on increased expression of SOCS1. In conclusion, miR-155 deficiency promotes nephrin acetylation and attenuates renal damage in hyperglycemia-induced nephropathy. This was associated with inhibited IL-17 production through enhancement of SOCS1 expression.

Topics: Acetylation; Acute Kidney Injury; Animals; Cell Differentiation; Desmin; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Hyperglycemia; Interleukin-17; Kidney; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; MicroRNAs; Podocytes; Repressor Proteins; Streptozocin; Suppressor of Cytokine Signaling 1 Protein; Suppressor of Cytokine Signaling Proteins; Th17 Cells; WT1 Proteins

Nephrin, a critical podocyte membrane component that is reduced in diabetic nephropathy, has been shown to activate phosphotyrosine signaling pathways in human podocytes. Nephrin signaling is important to reduce cell death induced by apoptotic stimuli. We have shown previously that high glucose level exposure and diabetes increased the expression of SHP-1, causing podocyte apoptosis. SHP-1 possesses two Src homology 2 domains that serve as docking elements to dephosphorylate tyrosine residues of target proteins. However, it remains unknown whether SHP-1 interacts with nephrin and whether its elevated expression affects the nephrin phosphorylation state in diabetes. Here we show that human podocytes exposed to high glucose levels exhibited elevated expression of SHP-1, which was associated with nephrin. Coexpression of nephrin-CD16 and SHP-1 reduced nephrin tyrosine phosphorylation in transfected human embryonic kidney 293 cells. A single tyrosine-to-phenylalanine mutation revealed that rat nephrin Tyr(1127) and Tyr(1152) are required to allow SHP-1 interaction with nephrin. Overexpression of dominant negative SHP-1 in human podocytes prevented high glucose-induced reduction of nephrin phosphorylation. In vivo, immunoblot analysis demonstrated that nephrin expression and phosphorylation were decreased in glomeruli of type 1 diabetic Akita mice (Ins2(+/C96Y)) compared with control littermate mice (Ins2(+/+)), and this was associated with elevated SHP-1 and cleaved caspase-3 expression. Furthermore, immunofluorescence analysis indicated increased colocalization of SHP-1 with nephrin in diabetic mice compared with control littermates. In conclusion, our results demonstrate that high glucose exposure increases SHP-1 interaction with nephrin, causing decreased nephrin phosphorylation, which may, in turn, contribute to diabetic nephropathy.

Topics: Animals; Caspase 3; Cell Line; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Disease Models, Animal; Gene Expression Regulation; Glucose; GPI-Linked Proteins; HEK293 Cells; Humans; Insulin; Kidney Glomerulus; Membrane Proteins; Mice; Mice, Transgenic; Phosphorylation; Phosphotyrosine; Podocytes; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Rats; Receptors, IgG; Signal Transduction

The onset of diabetic nephropathy (DN) is highlighted by glomerular filtration barrier abnormalities. Identifying pathogenic factors and targetable pathways driving DN is crucial to developing novel therapies and improving the disease outcome. Semaphorin3a (sema3a) is a guidance protein secreted by podocytes. Excess sema3a disrupts the glomerular filtration barrier. Here, using immunohistochemistry, we show increased podocyte SEMA3A in renal biopsies from patients with advanced DN. Using inducible, podocyte-specific Sema3a gain-of-function (Sema3a(+)) mice made diabetic with streptozotocin, we demonstrate that sema3a is pathogenic in DN. Diabetic Sema3a(+) mice develop massive proteinuria, renal insufficiency, and extensive nodular glomerulosclerosis, mimicking advanced DN in humans. In diabetic mice, Sema3a(+) exacerbates laminin and collagen IV accumulation in Kimmelstiel-Wilson-like glomerular nodules and causes diffuse podocyte foot process effacement and F-actin collapse via nephrin, αvβ3 integrin, and MICAL1 interactions with plexinA1. MICAL1 knockdown and sema3a inhibition render podocytes not susceptible to sema3a-induced shape changes, indicating that MICAL1 mediates sema3a-induced podocyte F-actin collapse. Moreover, sema3a binding inhibition or podocyte-specific plexinA1 deletion markedly ameliorates albuminuria and abrogates renal insufficiency and the diabetic nodular glomerulosclerosis phenotype of diabetic Sema3a(+) mice. Collectively, these findings indicate that excess sema3a promotes severe diabetic nephropathy and identifies novel potential therapeutic targets for DN.

Topics: Actins; Animals; Chromones; Collagen Type IV; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Gene Knockdown Techniques; Humans; Integrin alphaVbeta3; Laminin; Membrane Proteins; Mice; Mice, Knockout; Microfilament Proteins; Microtubule-Associated Proteins; Mixed Function Oxygenases; Nerve Tissue Proteins; Podocytes; Proteinuria; Receptors, Cell Surface; Renal Insufficiency; Semaphorin-3A; WT1 Proteins; Xanthones

Expression of thioredoxin-interacting protein (TxNIP), an endogenous inhibitor of the thiol oxidoreductase thioredoxin, is augmented by high glucose (HG) and promotes oxidative stress. We previously reported that TxNIP-deficient mesangial cells showed protection from HG-induced reactive oxygen species, mitogen-activated protein kinase phosphorylation, and collagen expression. Here, we investigated the potential role of TxNIP in the pathogenesis of diabetic nephropathy (DN) in vivo. Wild-type (WT) control, TxNIP(-/-), and TxNIP(+/-) mice were rendered equally diabetic with low-dose streptozotocin. In contrast to effects in WT mice, diabetes did not increase albuminuria, proteinuria, serum cystatin C, or serum creatinine levels in TxNIP(-/-) mice. Whereas morphometric studies of kidneys revealed a thickened glomerular basement membrane and effaced podocytes in the diabetic WT mice, these changes were absent in the diabetic TxNIP(-/-) mice. Immunohistochemical analysis revealed significant increases in the levels of glomerular TGF-β1, collagen IV, and fibrosis only in WT diabetic mice. Additionally, only WT diabetic mice showed significant increases in oxidative stress (nitrotyrosine, urinary 8-hydroxy-2-deoxy-guanosine) and inflammation (IL-1β mRNA, F4/80 immunohistochemistry). Expression levels of Nox4-encoded mRNA and protein increased only in the diabetic WT animals. A significant loss of podocytes, assessed by Wilms' tumor 1 and nephrin staining and urinary nephrin concentration, was found in diabetic WT but not TxNIP(-/-) mice. Furthermore, in cultured human podocytes exposed to HG, TxNIP knockdown with siRNA abolished the increased mitochondrial O2 (-) generation and apoptosis. These data indicate that TxNIP has a critical role in the progression of DN and may be a promising therapeutic target.

Topics: Albuminuria; Animals; Antigens, Differentiation; Apoptosis; Carrier Proteins; Cells, Cultured; Collagen Type IV; Creatinine; Cystatin C; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Fibrosis; Glomerular Basement Membrane; Glucose; Interleukin-1beta; Kidney Glomerulus; Membrane Proteins; Mice; Mice, Knockout; Mitochondria; NADPH Oxidase 4; NADPH Oxidases; Oxidative Stress; Oxygen; Podocytes; Repressor Proteins; RNA, Messenger; Streptozocin; Thioredoxins; Transforming Growth Factor beta1; WT1 Proteins

Chinese medicine comprised of all natural herbs is widespread used in the treatment of diabetic nephropathy (DN). Podocyte contributes to the integrity of glomerular filtration barrier whose injury plays an important role in the initiation and progression of DN. Our study aimed to investigate the effect of Qiwei granules on podocyte lesion in diabetic KK-A(y) mice kidney and its underlying mechanism.. Twelve-week-old male KK-A(y) mice were randomly divided in vehicle group and Qiwei granules group, while C57BL/6J mice were used as normal control. The mice were gavage with 1.37 g/kg/day Qiwei granules or water for 10 weeks. We measured water, food intake and body weight (BW) and fasting blood glucose (FBG) every 2 weeks, and urine protein every 4 weeks. At the end of the experiment, all surviving mice were sacrificed. The kidney weight and serum renal parameters were measured, and the renal morphology was observed. To search the underlying mechanism, we examined the podocyte positive marker, slit diaphragm protein expression and some involved cell signal pathway.. Qiwei granules treatment significantly improved the metabolic parameters, alleviated the urinary protein, and protected renal function in KK-A(y) mice. In addition, the glomerular injuries and podocyte lesions were mitigated with Qiwei granules treatment. Furthermore, Qiwei granules increased expression of nephrin, CD2AP, and integrin alpha3beta1 in the podocytes of KK-A(y) mice. Qiwei granules improved the phosphoration of Akt and inhibited cleaved caspase-3 protein expression.. These finding suggest that Qiwei granules protects the podocyte from the development of DN via improving slit diaphragm (SD) molecules expression and likely activating Akt signaling pathway in KK-A(y) mice.

Topics: Adaptor Proteins, Signal Transducing; Animals; Biomarkers; Body Weight; Caspase 3; Cytoskeletal Proteins; Diabetic Nephropathies; Drugs, Chinese Herbal; Integrin alpha3beta1; Kidney; Magnoliopsida; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Phytotherapy; Podocytes; Protective Agents; Signal Transduction

The transgenic E1-DN mice express a kinase-negative epidermal growth factor receptor in their pancreatic islets and are diabetic from two weeks of age due to impaired postnatal growth of β-cell mass. Here, we characterize the development of hyperglycaemia-induced renal injury in the E1-DN mice. Homozygous mice showed increased albumin excretion rate (AER) at the age of 10 weeks; the albuminuria increased over time and correlated with blood glucose. Morphometric analysis of PAS-stained histological sections and electron microscopy images revealed mesangial expansion in homozygous E1-DN mice, and glomerular sclerosis was observed in the most hyperglycaemic mice. The albuminuric homozygous mice developed also other structural changes in the glomeruli, including thickening of the glomerular basement membrane and widening of podocyte foot processes that are typical for diabetic nephropathy. Increased apoptosis of podocytes was identified as one mechanism contributing to glomerular injury. In addition, nephrin expression was reduced in the podocytes of albuminuric homozygous E1-DN mice. Tubular changes included altered epithelial cell morphology and increased proliferation. In conclusion, hyperglycaemic E1-DN mice develop albuminuria and glomerular and tubular injury typical of human diabetic nephropathy and can serve as a new model to study the mechanisms leading to the development of diabetic nephropathy.

Topics: Albuminuria; Animals; Capsid Proteins; Diabetic Nephropathies; Disease Models, Animal; ErbB Receptors; Homozygote; Humans; Kidney Glomerulus; Male; Membrane Proteins; Mice; Mice, Transgenic; Podocytes

Diabetic nephropathy is a major cause of end-stage kidney disease. Characterized by progressive microvascular disease, most efforts have focused on injury to the glomerular endothelium. Recent work has suggested a role for the podocyte, a highly specialized component of the glomerular filtration barrier. Here, we demonstrate that the Drosophila nephrocyte, a cell analogous to the mammalian podocyte, displays defects that phenocopy aspects of diabetic nephropathy in animals fed chronic high dietary sucrose. Through functional studies, we identify an OGT-Polycomb-Knot-Sns pathway that links dietary sucrose to loss of the Nephrin ortholog Sns. Reducing OGT through genetic or drug means is sufficient to rescue loss of Sns, leading to overall extension of lifespan. We demonstrate upregulation of the Knot ortholog EBF2 in glomeruli of human diabetic nephropathy patients and a mouse ob/ob diabetes model. Furthermore, we demonstrate rescue of Nephrin expression and cell viability in ebf2(-/-) primary podocytes cultured in high glucose.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Cells, Cultured; Diabetic Nephropathies; Dietary Carbohydrates; Drosophila; Drosophila Proteins; Humans; Immunoglobulins; Membrane Proteins; Mice; N-Acetylglucosaminyltransferases; Podocytes; Sucrose

Our recent publication showed that a small bioactive pigment epithelium derived factor (PEDF) peptide (P78-PEDF) prevents the development of diabetic nephropathy (DN). However, its effects on the progression of established DN were not clear. Therefore, the purpose of this study was to determine the effect of P78-PEDF in the progression of DN and to compare the effects of P78-PEDF and an ACE inhibitor (ACEi), a standard of care in DN. Experiments were conducted in Ins2(Akita) mice treated with P78-PEDF or captopril starting at 6 wks of age for 12 wks (early treatment) or starting at 12 wks of age for 6 wks (late treatment). We first established the optimal dose of the P78-PEDF peptide to ameliorate DN in Ins2(Akita) mouse for a 6 wk study period and found that the peptide was effective at 0.1- 0.5 µg/g/day. We next showed that early or late treatment with P78-PEDF resulted in protection from DN as indicated by reduced albuminuria, kidney macrophage recruitment, histological changes, inflammatory cytokines and fibrotic markers (kidney TNF-α, fibronectin, VEGFA and EGFR), and restored nephrin expression compared with vehicle-treated Ins2(Akita) mice. Interestingly, only early but not late treatment with captopril was as effective as P78-PEDF in reducing most DN complications, despite its lack of effect on nephrin, VEGFA and EGFR expression. These findings highlight the importance of P78-PEDF peptide as a potential therapeutic modality in both the development and progression of diabetic renal injury.

Topics: Animals; Blood Pressure; Captopril; Cytokines; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Disease Progression; Eye Proteins; Kidney; Macrophages; Male; Membrane Proteins; Mice; Nerve Growth Factors; Serpins

APPL1, an intracellular adaptor protein, takes part in numerous metabolic reactions. Although APPL1 plays a key role in glucose metabolism via adiponectin pathway and has been proved associated with type 2 diabetes, little is known about its role in diabetic nephropathy. To explore the role of APPL1 in diabetic nephropathy, we upregulated the expression of APPL1 in cultured mouse podocytes by adenovirus infection and tested the effects of APPL1 overexpression in podocytes treated with high glucose. Here, a mouse podocyte cell line (generated from H-2Kb-tsA58 immortmouse) was cultured and divided into four groups: Group 1 (normal glucose, NG), Group 2 (high glucose, HG), Group 3 (HG and infected with control adenovirus) and Group 4 (HG and infected with Ad-APPL1). Cell vitality of Group 4 is significantly higher than Group 2, but notably lower than Group 1 (P<0.01). The apoptosis rate of Group 4 was much lower (P<0.01) than Group 2 and Group 3. A decrease in phase G0/G1 and an increase in phase S was observed in Group 4 compared with Group 2 (P<0.01). These data suggested the protective role of APPL1 overexpression in high glucose condition. Moreover, the levels of Nephrin, AMPK and p-AMPK were decreased by high-glucose treatment, but increased by APPL1 overexpression. In conclusion, in the experimental high glucose condition, APPL1 acts as a protective factor against podocytes injury through regulating AMPK signaling, and may be a new therapy target for diabetic nephropathy.

Topics: Adaptor Proteins, Signal Transducing; AMP-Activated Protein Kinases; Animals; Apoptosis; Cell Cycle Checkpoints; Cell Line; Cell Proliferation; Cellular Microenvironment; Diabetic Nephropathies; Enzyme Activation; Glucose; Membrane Proteins; Mice; Podocytes; Signal Transduction; Time Factors; Transfection

Podocyte injury plays a crucial role in the pathogenesis and progression of diabetic nephropathy (DN). We investigated whether patients with diabetes mellitus (DM) without overt DN present podocyte markers in urine suggestive of early podocyte injury.. We studied 71 patients with DM type 2 and normal urine albumin excretion (UAE) and 39 non-diabetic controls. The mRNA abundance of 3 podocyte-specific markers in urinary sediment (nephrin, podocin and synaptopodin) was measured with real-time quantitative PCR. All the subjects were categorized according to their urinary podocyte marker profile into 2 groups, those with only synaptopodin mRNA presence (synaptopodin only group) and those with nephrin and/or podocin mRNA presence in addition to synaptopodin in their urine (nephrin and/or podocin group).. Synaptopodin mRNA was detected in the urine of all the diabetics and controls. The presence of nephrin and/or podocin mRNA in urine was more frequent among DM patients compared to controls (53.5 vs. 30.8%, respectively; p = 0.022). Binary logistic regression analysis revealed that the only significant predictor of the presence of nephrin and/or podocin mRNA in urine was the presence of DM (OR 2.59, 95% CI 1.14-5.91, p = 0.024, adjusted for all risk factors). A strong correlation between nephrin and podocin urinary mRNA levels was noted (r = +0.796, p < 0.001).. This study demonstrated that urinary podocyte markers are more prevalent in diabetic patients with normal UAE compared to controls, and this may reflect early podocyte injury. DM is the only significant determinant of the presence of nephrin and/or podocin mRNA in urine in this population. Therefore, urinary podocyte markers may emerge as a valuable tool in the early diagnosis of DN.

Topics: Aged; Albuminuria; Biomarkers; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Female; Humans; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Microfilament Proteins; Middle Aged; Podocytes; Prevalence; Risk Factors; RNA, Messenger

Diabetic nephropathy (DN) is the main cause of morbidity and mortality in diabetes and is characterized by mesangial matrix deposition and podocytopathy, including podocyte loss. The risk factors and mechanisms involved in the pathogenesis of DN are still not completely defined. In the present study, we aimed to understand the cellular mechanisms through which activation of B2 kinin receptors contribute to the initiation and progression of DN. Stimulation of cultured rat podocytes with bradykinin (BK) resulted in a significant increase in ROS generation, and this was associated with a significant increase in NADPH oxidase (NOX)1 and NOX4 protein and mRNA levels. BK stimulation also resulted in a signicant increase in the phosphorylation of ERK1/2 and Akt, and this effect was inhibited in the presence of NOX1 and Nox4 small interfering (si)RNA. Furthermore, podocytes stimulated with BK resulted in a significant increase in protein and mRNA levels of connective tissue growth factor (CTGF) and, at the same time, a significant decrease in protein and mRNA levels of nephrin. siRNA targeted against NOX1 and NOX4 significantly inhibited the BK-induced increase in CTGF. Nephrin expression was increased in response to BK in the presence of NOX1 and NOX4 siRNA, thus implicating a role for NOXs in modulating the BK response in podocytes. Moreover, nephrin expression in response to BK was also significantly increased in the presence of siRNA targeted against CTGF. These findings provide novel aspects of BK signal transduction pathways in pathogenesis of DN and identify novel targets for interventional strategies.

Topics: Animals; Bradykinin; Cells, Cultured; Connective Tissue Growth Factor; Diabetic Nephropathies; Extracellular Signal-Regulated MAP Kinases; Membrane Proteins; NADH, NADPH Oxidoreductases; NADPH Oxidase 1; NADPH Oxidase 4; NADPH Oxidases; Oxidative Stress; Phosphorylation; Podocytes; Proto-Oncogene Proteins c-akt; Rats; Receptor, Bradykinin B2; RNA Interference; RNA, Messenger; Signal Transduction; Time Factors; Transfection; Up-Regulation

To explore the combined detection of urine UmAlb and urinary nephrin (Unephrin), podocalyxin (UPCX) in podocyte of MKR mice with diabetic nephropathy.. Thirty 8 weeks old MKR mice were randomly divided into two groups as follows: negative control group, DN model group, and another 15 wild C57 mice were used as normal control. Mice in DN model group were received unilateral nephrectomy and high-fat diet feed for 2 months. The morphological structure changes of the podocytes were observed by transmission electron microscopes. The levels of FBG were detected by electrochemical detection method, The nephrin and PCX protein expression were measured by western blotting. The levels of UmAlb, Unephrin and UPCX were detected by ELISA.. The podocyte damage in the mice of DN model group increased significantly when compared with normal control. As compared with normal control, FBG in the model group increased significantly (P<0. 01), the expression level of nephrin and PCX in Renal Tissue and Unephrin, UPCX, and urine UmAlb were also increased significantly (P<0. 01).. The level of Unephrin and UPCX were positive correlated with the level of urine UmAlb, the loss of podocyte strcture protein might be one of the mechanism in leading proteinuria in diabetic nephropathy.

Topics: Animals; Blotting, Western; Diabetic Nephropathies; Enzyme-Linked Immunosorbent Assay; Membrane Proteins; Mice; Podocytes; Proteinuria; Sialoglycoproteins

Diabetic nephropathy (DN) is the leading cause of end-stage renal failure, and podocyte injury plays a major role in the development of DN. In this study, we investigated whether tacrolimus (FK506), an immunosuppressor, can attenuate podocyte injury in a type 2 diabetic mellitus (T2DM) rat model with DN. Transmission electron microcopy was used to morphologically evaluate renal injury. The urinary albumin (UAL), creatinine clearance rate (Ccr) and major biochemical parameters, including glucose, insulin, serum creatinine (Scr), urea nitrogen, total cholesterol (CHO) and triglyceride (TG), were examined 12 weeks after the administration of FK506. The expressions of the canonical transient receptor potential 6 (TRPC6), nuclear factor of activated T-cells (NFAT) and nephrin were detected by Western blotting and qPCR. In the rat model of DN, the expressions of TRPC6 and NFAT were significantly elevated compared with the normal rat group; however, the treatment with FK506 normalized the increased expression of TRPC6 and NFAT and attenuated podocyte ultrastructure injury. UAL, Ccr and the biochemical parameters were also improved by the use of FK506. In cell experiments, FK506 improved the decreased expression of nephrin and suppressed the elevated expression of both TRPC6 and NFAT caused by high glucose in accordance with TRPC6 blocker U73122. Our results demonstrated that FK506 could ameliorate podocyte injury in T2DM, which may be related to suppressed expressions of TRPC6 and NFAT.

Topics: Albuminuria; Animals; Biomarkers; Blood Glucose; Blood Urea Nitrogen; Creatinine; Cytoprotection; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Down-Regulation; Immunosuppressive Agents; Male; Membrane Proteins; Microscopy, Electron, Transmission; NFATC Transcription Factors; Podocytes; Rats, Wistar; Tacrolimus; TRPC Cation Channels

The objective of the present study was to investigate the effects of β-conglycinin and soya isoflavones on diabetic nephropathy (DN). DN was induced by an intravenous injection of streptozotocin (25 mg/kg) in spontaneously hypertensive rats. DN rats were divided into a non-diabetic group (C, control group) and three DN groups (D, DN with control diet; B, DN+control diet with one-eighth of casein replaced by β-conglycinin as the protein source; and I, DN+control diet with 0·01 % soya isoflavones). After a 4-week experimental period, we found that fasting blood sugar and plasma and kidney advanced glycation end product levels and 24 h urinary protein excretion of the B group were significantly lower than those of the D group and insulin sensitivity and nephrin expression of the B group were significantly higher than those of the D group. In addition, systolic blood pressure, angiotensin-converting enzyme activity, angiotensin II level and plasma TAG level of the B group were significantly lower than those of the D group, whereas only the levels of plasma TAG and thiobarbituric acid-reactive substances of the I group were lower than those of the D group. In conclusion, β-conglycinin may be beneficial for retarding DN progression and this effect cannot be completely explained by its isoflavone content.

Topics: Angiotensin II; Animals; Antigens, Plant; Blood Glucose; Blood Pressure; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Dietary Proteins; Globulins; Glycation End Products, Advanced; Glycine max; Insulin Resistance; Isoflavones; Kidney; Male; Membrane Proteins; Peptidyl-Dipeptidase A; Phytotherapy; Plant Preparations; Rats; Rats, Inbred SHR; Seed Storage Proteins; Soybean Proteins; Thiobarbituric Acid Reactive Substances; Triglycerides

Higher doses of AngII (angiotensin II) blockers are intended to optimize albuminuria reduction rather than for blood pressure control in chronic kidney disease. However, the long-term renoprotection of high-dose AngII blockers has yet to be defined. The present study sought to determine whether doses of ARB (AngII receptor blocker) that maximally reduce proteinuria could slow the progression of glomerulosclerosis in the uninephrectomized db/db mouse, a model of Type 2 diabetes. Untreated uninephrectomized db/db mice had normal blood pressure, but developed progressive albuminuria and mesangial matrix expansion between 18 and 22 weeks of age, which was associated with increased renal expression of TGFβ1 (transforming growth factor β1), PAI-1 (plasminogen-activator inhibitor-1), type IV collagen and FN (fibronectin). Treatment with valsartan in the drinking water of db/db mice from 18 to 22 weeks of age, at a dose that was determined previously to maximally reduce proteinuria, prevented the increases in albuminuria and the markers of renal fibrosis seen in untreated db/db mice. In addition, WT-1 (Wilms tumour protein-1)-immunopositive podocyte numbers were found to be lower in the untreated glomeruli of mice with diabetes. The expression of podocin and nephrin were continually decreased in mice with diabetes between 18 and 22 weeks of age. These changes are indicative of podocyte injury and the administration of valsartan ameliorated them substantially. Renal expression of TNFα (tumour necrosis factor α), MCP-1 (monocyte chemoattractant protein-1), Nox2 (NADPH oxidase 2), p22phox and p47phox and urine TBARS (thiobarbituric acid-reacting substance) levels, the markers of renal inflammation and oxidative stress, were increased during disease progression in mice with diabetes. Valsartan treatment was shown to reduce these markers. Thus high doses of valsartan not only reduce albuminuria maximally, but also halt the progression of the glomerulosclerosis resulting from Type 2 diabetes via a reduction in podocyte injury and renal oxidative stress and inflammation.

Topics: Animals; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Disease Progression; Extracellular Matrix Proteins; Fibronectins; Gene Expression Regulation; Inflammation; Intracellular Signaling Peptides and Proteins; Kidney; Kidney Cortex; Kidney Glomerulus; Membrane Proteins; Mice; Oxidative Stress; Plasminogen Activator Inhibitor 1; Podocytes; RNA, Messenger; Tetrazoles; Transforming Growth Factor beta1; Treatment Outcome; Valine; Valsartan; WT1 Proteins

Radix puerariae, a traditional Chinese herbal medication, has been used successfully to treat patients with early stage of diabetic nephropathy. However, the underlined mechanism of this renal protective effect has not been determined. In the current study, we investigated the effects and the mechanism of puerarin in Streptozotocin (STZ)-induced diabetic rats. We treated STZ-rats with either puerarin or losartan, an angiotensin II receptor blocker, as compared to those treated with vehicle. We found that both puerarin and losartan attenuated kidney hypertrophy, mesangial expansion, proteinuria, and podocyte foot process effacement in STZ rats. In addition, both puerarin and losartan increased expression of podocyte slit diaphragm proteins such as nephrin and podocin. Interestingly, we found that puerarin treatment induced a more pronounced suppression of oxidative stress production and S-nitrosylation of proteins in the diabetic kidneys as compared to losartan treatment. Furthermore, we found that matrix metalloproteinase-9 (MMP-9), which is known to be activated by oxidative stress and S-nitrosylation of proteins, was also suppressed more extensively by puerarin than losartan. In conclusion, these data provide for the first time the potential mechanism to support the use of puerarin in the treatment of early diabetic nephropathy.

Topics: Animals; Collagen Type IV; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Drug Evaluation, Preclinical; Enzyme Repression; Intracellular Signaling Peptides and Proteins; Isoflavones; Kidney; Losartan; Male; Matrix Metalloproteinase 9; Membrane Proteins; Podocytes; Proteinuria; Rats; Rats, Wistar; Reactive Oxygen Species; Streptozocin

Cellular crescents are typically inflammatory and associated with rapidly progressive glomerulonephritis. Their pathogenesis involves glomerular basement membrane rupture due to circulating or intrinsic factors. Crescents associated with diabetic glomerulosclerosis are rarely reported. Furthermore, the nature of cells forming crescents in diabetes is unknown. To investigate the nature of crescents in diabetes, we examined renal biopsies from diabetic patients with nodular glomerulosclerosis and crescents (n = 2), diabetes without crescents (n = 5), nondiabetic renal biopsies (n = 3), and crescentic glomerulonephritis with inflammatory crescents (n = 5). Electron microscopy and confocal immunofluorescence analysis with antibodies against nephrin (a podocyte marker) and claudin 1 (parietal epithelial cell marker) were performed. Diabetic glomeruli with crescents contained a mixture of crescentic cells expressing either claudin 1 (11 ± 1.4 cells/glomerulus) or nephrin (5.5 ± 3.0 cells/glomerulus). Rare crescentic cells coexpressed nephrin and claudin 1 (2.5 ± 1.6 cells/glomerulus). In contrast, inflammatory crescents were almost exclusively composed of claudin 1-positive cells (25 ± 5.3 cells/glomerulus). Cells coexpressing claudin 1 and nephrin were absent in inflammatory crescents and all cases without crescents. Electron microscopy showed podocyte bridge formation between the glomerular basement membrane and parietal basement membrane but no glomerular basement membrane rupture as in inflammatory crescents. Crescents in diabetes may occur in diabetes in the absence of a secondary etiology and are composed of a mixture of parietal epithelial cells and visceral podocytes. Cells coexpressing parietal epithelial and podocyte markers suggest that parietal epithelial cells may transdifferentiate into podocytes in response to severe glomerular injury.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers; Claudin-1; Diabetic Nephropathies; Female; Glomerulonephritis; Humans; Kidney Glomerulus; Male; Membrane Proteins; Middle Aged; Podocytes

We previously showed that the transcription factor Mafb is essential for podocyte differentiation and foot process formation. Podocytes are susceptible to injury in diabetes, and this injury leads to progression of diabetic nephropathy. In this study, we generated transgenic mice that overexpress Mafb in podocytes using the nephrin promoter/enhancer. To examine a potential pathogenetic role for Mafb in diabetic nephropathy, Mafb transgenic mice were treated with either streptozotocin or saline solution. Diabetic nephropathy was assessed by renal histology and biochemical analyses of urine and serum. Podocyte-specific overexpression of Mafb had no effect on body weight or blood glucose levels in either diabetic or control mice. Notably, albuminuria and changes in BUN levels and renal histology observed in diabetic wild-type animals were ameliorated in diabetic Mafb transgenic mice. Moreover, hyperglycemia-induced downregulation of Nephrin was mitigated in diabetic Mafb transgenic mice, and reporter assay results suggested that Mafb regulates Nephrin directly. Mafb transgenic glomeruli also overexpressed glutathione peroxidase, an antioxidative stress enzyme, and levels of the oxidative stress marker 8-hydroxydeoxyguanosine decreased in the urine of diabetic Mafb transgenic mice. Finally, Notch2 expression increased in diabetic glomeruli, and this effect was enhanced in diabetic Mafb transgenic glomeruli. These data indicate Mafb has a protective role in diabetic nephropathy through regulation of slit diaphragm proteins, antioxidative enzymes, and Notch pathways in podocytes and suggest that Mafb could be a therapeutic target.

Topics: Animals; Apoptosis; Blood Glucose; Body Weight; Cell Line, Transformed; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Gene Expression; Glutathione Peroxidase; Hyperglycemia; Insulin; MafB Transcription Factor; Membrane Proteins; Mice, Inbred C57BL; Mice, Transgenic; Podocytes; Promoter Regions, Genetic; Receptor, Notch2; Signal Transduction

The transcription factor Kruppel-like factor 4 (KLF4) has the ability, along with other factors, to reprogram somatic cells into induced pluripotent stem (iPS) cells. Here, we determined that KLF4 is expressed in kidney glomerular podocytes and is decreased in both animal models and humans exhibiting a proteinuric. Transient restoration of KLF4 expression in podocytes of diseased glomeruli in vivo, either by gene transfer or transgenic expression, resulted in a sustained increase in nephrin expression and a decrease in albuminuria. In mice harboring podocyte-specific deletion of Klf4, adriamycin-induced proteinuria was substantially exacerbated, although these animals displayed minimal phenotypical changes prior to adriamycin administration. KLF4 overexpression in cultured human podocytes increased expression of nephrin and other epithelial markers and reduced mesenchymal gene expression. DNA methylation profiling and bisulfite genomic sequencing revealed that KLF4 expression reduced methylation at the nephrin promoter and the promoters of other epithelial markers; however, methylation was increased at the promoters of genes encoding mesenchymal markers, suggesting selective epigenetic regulation of podocyte gene expression. Together, these results suggest that KLF4 epigenetically modulates podocyte phenotype and function and that the podocyte epigenome can be targeted for direct intervention and reduction of proteinuria.

Topics: Albumins; Animals; Diabetic Nephropathies; Disease Models, Animal; DNA Methylation; Doxorubicin; Epigenesis, Genetic; Gene Expression; Humans; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Permeability; Phenotype; Podocytes; Promoter Regions, Genetic; Proteinuria; Vimentin

The aim was to explore the effects of rapamycin on autophagy and injury of podocytes in streptozocin (STZ)-induced type 1 diabetic mice, and its role in delaying progression of diabetic nephropathy. In this study, male Balb/c mice were divided into three groups: control (n = 12), STZ-induced diabetic (n = 12), and rapamycin-treated diabetic (DM + Rapa) (n = 12), which received intraperitoneal injection of rapamycin (2 mg/kg/48 h) after induction of DM. Levels of urinary albumin (UA), blood urea nitrogen, serum creatinine, and kidney weight/body weight were measured at week 12. Renal pathologic changes, number of podocytes autophagy, and organelles injury were investigated by PAS staining, transmission electron microscopy, and immunofluorescence staining, respectively. Western blot was performed to determine the expression of LC3 (a podocyte autophagy marker), phosphorylated mammalian target of rapamycin, p-p70S6K, bax, and caspase-3 protein. Podocytes count was evaluated by immunofluorescence staining and Wilms tumor 1 immunohistochemistry, and Western blot of nephrin and podocin. The results indicated that rapamycin could reduce the kidney weight/body weight and UA secretion. It could alleviate podocyte foot process fusion, glomerular basement membrane thickening, and matrix accumulation, and increase the number of autophagosomes, and LC3-expressing podocytes. Down-regulation of bax and caspase-3 protein, and up-regulation of nephrin and podocin protein were observed in the glomeruli of diabetic mice after administration of rapamycin. In conclusion, rapamycin can ameliorate renal injury in diabetic mice by increasing the autophagy activity and inhibition of apoptosis of podocytes.

Topics: Albuminuria; Animals; Autophagy; bcl-2-Associated X Protein; Biomarkers; Blood Urea Nitrogen; Caspase 3; Cytoprotection; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Mice, Inbred BALB C; Microtubule-Associated Proteins; Podocytes; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; Time Factors

Presumed genetic risk for diabetic and nondiabetic end stage renal disease is strong in African Americans.. Exome sequencing data from African Americans with type 2 diabetic end stage renal disease and nondiabetic, non-nephropathy controls in the T2D-GENES study (Discovery, n=529 patients and n=535 controls) were evaluated, focusing on missense variants in NPHS1. Associated variants were then evaluated in independent type 2 diabetic end stage renal disease (Replication, n=1305 patients and n=760 controls), nondiabetic end stage renal disease (n=1705), and type 2 diabetes-only, non-nephropathy samples (n=503). All participants were recruited from dialysis facilities and internal medicine clinics across the southeastern United States from 1991 to present. Additional NPHS1 missense variants were identified from exome sequencing resources, genotyped, and sequence kernel association testing was then performed.. Initial analysis identified rs35238405 (T233A; minor allele frequency=0.0096) as associated with type 2 diabetic end stage renal disease (adjustment for admixture P=0.042; adjustment for admixture+APOL1 P=0.080; odds ratio, 2.89 and 2.36, respectively); with replication in independent type 2 diabetic end stage renal disease samples (P=0.018; odds ratio, 4.30) and nondiabetic end stage renal disease samples (P=0.016; odds ratio, 4.48). In a combined analysis (all patients with end stage renal disease versus all controls), T233A was associated with all-cause end stage renal disease (P=0.0038; odds ratio, 2.82; n=3270 patients and n=1187 controls). A P-value of <0.001 was obtained after adjustment for admixture and APOL1 in sequence kernel association testing. Two additional variants (H800R and Y1174H) were nominally associated with protection from end stage renal disease (P=0.036; odds ratio, 0.44; P=0.0084; odds ratio, 0.040, respectively) in the locus-wide single-variant association tests.. Coding variants in NPHS1 are associated with both risk for and protection from common forms of nephropathy in African Americans.

Topics: Aged; Apolipoprotein L1; Apolipoproteins; Black or African American; Case-Control Studies; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Female; Gene Frequency; Genetic Predisposition to Disease; Humans; Kidney Failure, Chronic; Lipoproteins, HDL; Male; Membrane Proteins; Middle Aged; Odds Ratio; Phenotype; Polymorphism, Single Nucleotide; Protective Factors; Risk Assessment; Risk Factors

We investigated whether aliskiren, a direct renin inhibitor, provided protection in a model of diabetic nephropathy in mice and compared its protective effects to valsartan, an angiotensin II type 1 receptor blocker.. Hyperglycemia was induced with streptozotocin (STZ, 40 mg/kg/day × 5 days) injection in DBA/2J mice fed on a high fat diet. Mice were treated with either aliskiren (25 mg/kg/day) or valsartan (8 mg/kg/day) for 6 weeks.. Aliskiren and/or valsartan treatment significantly attenuated albuminuria, urinary nephrin excretion and glomerulosclerosis. Aliskiren and/or valsartan prevented reduction of podocin and WT1 protein abundance in diabetic mice. Aliskiren and/or valsartan significantly prevented increased expression of profibrotic growth factors (TGFβ, CTGF and PAI-1), proinflammatory cytokines (MCP-1, TNFα and IL-1β), endoplasmic reticulum (ER) stress markers (CHOP and XBP-1) and lipid accumulation in the kidney of diabetic animals. Aliskiren showed similar efficacy compared to valsartan therapy and dual treatment in some aspects has synergistic protective effects.. Our study indicates that aliskiren and/or valsartan protects against diabetic kidney disease through multiple mechanisms, including decreasing podocyte injury, activation of profibrotic growth factors and proinflammatory cytokines, ER stress and accumulation of lipids.

Topics: Albumins; Amides; Animals; Creatinine; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Endoplasmic Reticulum Stress; Fumarates; Inflammation; Lipid Metabolism; Male; Membrane Proteins; Mesangial Cells; Mice, Inbred DBA; Podocytes; Protective Agents; Proteinuria; Proto-Oncogene Mas; Proto-Oncogene Proteins; Receptors, G-Protein-Coupled; Renin-Angiotensin System; Tetrazoles; Valine; Valsartan

The present study aimed to investigate the relationship between PI3K/p-Akt signaling pathway and podocyte impairment in DN rats as well as the protective effect of calcitriol.. SD rats were randomly divided into four groups: normal control (NC), normal treated with calcitriol (NC+VD), diabetic nephropathy (DN) and DN treated with calcitriol (DN+VD); all VD rats were treated with 0.1 μg/kg/d calcitriol by gavage. DN model rats were established by intraperitoneal injections of streptozotocin (STZ). Rats were sacrificed after 18 weeks of treatments.. In the present study, increased albuminuria was observed as early as 3 weeks of diabetes and continued to increase more than six-fold throughout the length of the study (18 weeks). Expectedly, animals receiving the treatment with calcitriol was protected from this increase, lower about one third. Meanwhile, the expression of podocyte specific markers, including nephrin and podocin, together with PI3K/p-Akt was significantly decreased in DN rats, whereas calcitriol reversed these above changes accompanied by elevated the expression levels of VDR. Additionally, a positive correlation was observed between the expression levels of nephrin and VDR (r = 0.776, P < 0.05). Likewise, the expression of nephrin was positively correlated with both PI3K-p85 and p-Akt (r = 0.736, P < 0.05; r = 0.855, P < 0.05, respectively).. PI3K/p-Akt signaling pathway participates in calcitriol ameliorating podocyte injury in DN rats. The manipulation of calcitriol might act as a promising therapeutic intervention for diabetic nephropathy.

Topics: Albuminuria; Animals; Calcitriol; Desmin; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Male; Membrane Proteins; Phosphatidylinositol 3-Kinases; Podocytes; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Receptors, Calcitriol; Signal Transduction

The aim of this study was to evaluate the effect of compound 21 (C21), a selective AT2 receptor agonist, on diabetic nephropathy and the potential additive effect of C21, when associated with losartan treatment, on the development of albuminuria and renal fibrosis in Zucker diabetic fatty (ZDF) rats. The experiments lasted 15 wk (from 5 to 20 wk of age) and were performed in 40 ZDF rats and 12 control lean rats. ZDF rats were divided into 4 groups: 1) 9 rats were treated with losartan; 2) 10 rats were treated with C21; 3) 9 rats were treated with losartan plus C21; and 4) 12 rats were maintained without any treatment. ZDF rats showed an increase in blood glucose level, albuminuria, renal fibrosis, macrophage infiltration, and TNF-α expression and a reduction of glomerular nephrin expression compared with control lean rats. C21 treatment reduced renal glomerular, tubulointerstitial, and perivascular fibrosis, and macrophage infiltration and TNF-α expression in ZDF rats. C21 treatment caused a decrease in albuminuria in ZDF rats up to 11 wk of age. Losartan decreased macrophage infiltration, TNF-α expression, and renal glomerular and perivascular fibrosis, restored glomerular nephrin expression, but did not affect tubulointerstitial fibrosis. Losartan treatment caused a decrease in albuminuria in ZDF rats up to 15 wk of age. At the end of the protocol, only the combination of C21 plus losartan significantly reduced albuminuria in ZDF rats. These data demonstrate that C21 has beneficial effects on diabetic nephropathy, suggesting the combination of C21 and losartan as a novel pharmacological tool to slow the progression of nephropathy in type II diabetes.

Topics: Albuminuria; Animals; Blood Pressure; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Drug Evaluation, Preclinical; Fibrosis; Interleukin-10; Kidney; Losartan; Male; Membrane Proteins; Rats, Zucker; Receptor, Angiotensin, Type 2; Sulfonamides; Thiophenes; Tumor Necrosis Factor-alpha

Increasing evidence suggests the heterogeneity of macrophage phenotype and function ultimately determines the outcome of diabetic nephropathy (DN). This study aimed to investigate the effects of vitamin D on macrophage M1/M2 phenotype and its role in preventing podocyte impairment in streptozotocin-induced DN rats. Calcitriol, a bioactive 1,25-dihydroxyvitamin D3, ameliorated proteinuria and renal damage as well as reversed the decline of both nephrin and podocin, crucial structural proteins in podocytes. DN rats showed increased infiltrating macrophages with M1 phenotype characterized by elevated expression of inducible nitric oxide synthase and TNF-α in glomeruli and interstitium, which were inhibited after calcitriol treatment. Interestingly, calcitriol promoted M2 macrophage activation with enhanced expression of CD163, arginase-1, and mannose receptor at week 18 but not at week 8 or 14. The ratio of CD163 to CD68, considered as the proportion of M2 macrophages, was about 2.9-fold higher at week 18 after calcitriol treatment. Furthermore, the protein expression of inducible nitric oxide synthase, a crucial marker of M1 macrophages, was negatively correlated with the expression of either nephrin or podocin, whereas CD163, indicating M2 macrophages, was positively correlated. In vitro, 1,25-dihydroxyvitamin D3 switched high-glucose-induced M1 macrophages toward an M2 phenotype in either U937-derived macrophages or RAW264.7 cells. Our results suggest that vitamin D not only reduces macrophage infiltration and inhibits M1 macrophage activation but also enhances M2 macrophage phenotype to protect against podocyte injury.

Topics: Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Blood Glucose; Body Weight; Calcitriol; Diabetic Nephropathies; Drug Evaluation, Preclinical; Intracellular Signaling Peptides and Proteins; Kidney Function Tests; Kidney Glomerulus; Macrophages; Male; Membrane Proteins; Nitric Oxide Synthase Type II; Phenotype; Podocytes; Random Allocation; Rats; Rats, Sprague-Dawley; Receptors, Cell Surface; Vitamins

Non-enzymatic protein glycation and resultant accumulation of advanced glycation endproducts (AGE) are implicated in the pathogenesis of diabetic complications including diabetic nephropathy (DN). It is considered that antiglycating agents offer protection against AGE mediated pathologies including DN. Earlier we characterized procyanidin-B2 (PCB2) as the active component from cinnamon (Cinnamomum zeylanicum) that inhibits AGE formation in vitro. In this study, we have investigated the potential of PCB2-enriched fraction of cinnamon to prevent in vivo accumulation of AGE and to ameliorate renal changes in diabetic rats. Streptozotocin-induced diabetic rats were fed with either 3% cinnamon or 0.002% PCB2-fraction in diet for 12weeks. Biochemical analysis of blood and urine was performed at the end of experiment. Evaluation of glomerular markers that serve as indicators of renal function was done by immunohistochemistry, immunoblotting and qRT-PCR. Supplementation of diabetic rats with cinnamon and PCB2-fraction prevented glycation mediated RBC-IgG cross-links and HbA1c accumulation in diabetes rats. Cinnamon and PCB2-fraction also inhibited the accumulation of N-carboxy methyl lysine (CML), a prominent AGE in diabetic kidney. Interestingly, cinnamon and its PCB2-fraction prevented the AGE mediated loss of expression of glomerular podocyte proteins; nephrin and podocin. Inhibition of AGE by cinnamon and PCB2-fraction ameliorated the diabetes mediated renal malfunction in rats as evidenced by reduced urinary albumin and creatinine. In conclusion, PCB2 from cinnamon inhibited AGE accumulation in diabetic rat kidney and ameliorated AGE mediated pathogenesis of DN.

Topics: Animals; Biflavonoids; Catechin; Cell Adhesion Molecules; Cinnamomum zeylanicum; Diabetic Nephropathies; Glycated Hemoglobin; Glycation End Products, Advanced; Glycosylation; Inflammation Mediators; Intracellular Signaling Peptides and Proteins; Kidney; Male; Membrane Proteins; Proanthocyanidins; Rats; Rats, Wistar; RNA, Messenger

Our recent publication showed that pharmacological blockade of arginases confers kidney protection in diabetic nephropathy via a nitric oxide (NO) synthase (NOS)3-dependent mechanism. Arginase competes with endothelial NOS (eNOS) for the common substrate L-arginine. Lack of L-arginine results in reduced NO production and eNOS uncoupling, which lead to endothelial dysfunction. Therefore, we hypothesized that L-arginine or L-citrulline supplementation would ameliorate diabetic nephropathy. DBA mice injected with multiple low doses of vehicle or streptozotocin (50 mg/kg ip for 5 days) were provided drinking water with or without L-arginine (1.5%, 6.05 g·kg(-1)·day(-1)) or L-citrulline (1.66%, 5.73 g·kg(-1)·day(-1)) for 9 wk. Nonsupplemented diabetic mice showed significant increases in albuminuria, blood urea nitrogen, glomerular histopathological changes, kidney macrophage recruitment, kidney TNF-α and fibronectin mRNA expression, kidney arginase activity, kidney arginase-2 protein expression, and urinary oxidative stress along with a significant reduction of nephrin and eNOS protein expression and kidney nitrite + nitrate compared with normal mice after 9 wk of diabetes. Surprisingly, L-arginine or L-citrulline supplementation in diabetic mice did not affect any of these parameters despite greatly increasing kidney and plasma arginine levels. These findings demonstrate that chronic L-arginine or L-citrulline supplementation does not prevent or reduce renal injury in a model of type 1 diabetes.

Topics: Amino Acids; Animals; Arginase; Arginine; Blood Pressure; Citrulline; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Dietary Supplements; Fibronectins; Kidney; Male; Membrane Proteins; Mice; Mice, Inbred DBA; Nitrates; Nitric Oxide Synthase Type III; Nitrites; Tumor Necrosis Factor-alpha

This study determined if blocking ligand occupancy of the αVβ3 integrin could inhibit the pathophysiologic changes that occur in the early stages of diabetic nephropathy (DN). Diabetic rats were treated with either vehicle or a monoclonal antibody that binds the β3 subunit of the αVβ3 integrin. After 4 weeks of diabetes the urinary albumin to creatinine ratio (UACR) increased in both diabetic animals that subsequently received vehicle and in the animals that subsequently received the anti-β3 antibody compared with control nondiabetic rats. After 8 weeks of treatment the UACR continued to rise in the vehicle-treated rats; however it returned to levels comparable to control nondiabetic rats in rats treated with the anti-β3 antibody. Treatment with the antibody prevented the increase of several profibrotic proteins that have been implicated in the development of DN. Diabetes was associated with an increase in phosphorylation of the β3 subunit in kidney homogenates from diabetic animals, but this was prevented by the antibody treatment. This study demonstrates that, when administered after establishment of early pathophysiologic changes in renal function, the anti-β3 antibody reversed the effects of diabetes normalizing albuminuria and profibrotic proteins in the kidney to the levels observed in nondiabetic control animals.

Topics: Albuminuria; Animals; Antibodies, Monoclonal; Biomarkers; Collagen Type IV; Creatinine; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Fibrosis; Integrin alphaVbeta3; Kidney; Ligands; Male; Membrane Proteins; Phosphorylation; Protein Binding; Rats, Sprague-Dawley; Streptozocin; Transforming Growth Factor beta

An increasing number of clinical and animal model studies indicate that activation of the innate immune system and inflammatory mechanisms are important in the pathogenesis of diabetic nephropathy. Nucleotide-binding oligomerization domain containing 2 (NOD2), a member of the NOD-like receptor family, plays an important role in innate immune response. Here we explore the contribution of NOD2 to the pathogenesis of diabetic nephropathy and found that it was upregulated in kidney biopsies from diabetic patients and high-fat diet/streptozotocin-induced diabetic mice. Further, NOD2 deficiency ameliorated renal injury in diabetic mice. In vitro, NOD2 induced proinflammatory response and impaired insulin signaling and insulin-induced glucose uptake in podocytes. Moreover, podocytes treated with high glucose, advanced glycation end-products, tumor necrosis factor-α, or transforming growth factor-β (common detrimental factors in diabetic nephropathy) significantly increased NOD2 expression. NOD2 knockout diabetic mice were protected from the hyperglycemia-induced reduction in nephrin expression. Further, knockdown of NOD2 expression attenuated high glucose-induced nephrin downregulation in vitro, supporting an essential role of NOD2 in mediating hyperglycemia-induced podocyte dysfunction. Thus, NOD2 is one of the critical components of a signal transduction pathway that links renal injury to inflammation and podocyte insulin resistance in diabetic nephropathy.

Topics: Adult; Aged; Aged, 80 and over; Animals; Biomarkers; Blood Glucose; Cells, Cultured; Creatinine; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Diet, High-Fat; Female; Glucose Transporter Type 4; Humans; Inflammation Mediators; Insulin; Insulin Resistance; Lipids; Male; MAP Kinase Signaling System; Membrane Proteins; Mice; Mice, Knockout; Middle Aged; Nephritis; Nod2 Signaling Adaptor Protein; Podocytes; Time Factors; Up-Regulation

We have shown previously that inhibition of sEH (soluble epoxide hydrolase) increased EETs (epoxyeicosatrienoic acids) levels and reduced renal injury in diabetic mice and these changes were associated with induction of HO (haem oxygenase)-1. The present study determines whether the inhibition of HO negates the renoprotective effect of sEH inhibition in diabetic SHR (spontaneously hypertensive rats). After 6 weeks of induction of diabetes with streptozotocin, SHR were divided into the following groups: untreated, treated with the sEH inhibitor t-AUCB {trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid}, treated with the HO inhibitor SnMP (stannous mesoporphyrin), and treated with both inhibitors for 4 more weeks; non-diabetic SHR served as a control group. Induction of diabetes significantly increased renal sEH expression and decreased the renal EETs/DHETEs (dihydroxyeicosatrienoic acid) ratio without affecting HO-1 activity or expression in SHR. Inhibition of sEH with t-AUCB increased the renal EETs/DHETEs ratio and HO-1 activity in diabetic SHR; however, it did not significantly alter systolic blood pressure. Treatment of diabetic SHR with t-AUCB significantly reduced the elevation in urinary albumin and nephrin excretion, whereas co-administration of the HO inhibitor SnMP with t-AUCB prevented these changes. Immunohistochemical analysis revealed elevations in renal fibrosis as indicated by increased renal TGF-β (transforming growth factor β) levels and fibronectin expression in diabetic SHR and these changes were reduced with sEH inhibition. Co-administration of SnMP with t-AUCB prevented its ability to reduce renal fibrosis in diabetic SHR. In addition, SnMP treatment also prevented t-AUCB-induced decreases in renal macrophage infiltration, IL-17 expression and MCP-1 levels in diabetic SHR. These findings suggest that HO-1 induction is involved in the protective effect of sEH inhibition against diabetic renal injury.

Topics: Actins; Albuminuria; Animals; Benzoates; Blood Pressure; Creatinine; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Disease Progression; Drug Evaluation, Preclinical; Enzyme Inhibitors; Epoxide Hydrolases; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Kidney Cortex; Male; Membrane Proteins; Metalloporphyrins; Rats; Rats, Inbred SHR; Urea

In nondiabetic rat models of renal disease, angiotensin II (Ang II) perpetuates podocyte injury and promotes progression to end-stage kidney disease. Herein, we wanted to explore the role of Ang II in diabetic nephropathy by a translational approach spanning from in vitro to in vivo rat and human studies, and to dissect the intracellular pathways involved. In isolated perfused rat kidneys and in cultured human podocytes, Ang II down-regulated nephrin expression via Notch1 activation and nuclear translocation of Snail. Hairy enhancer of split-1 was a Notch1-downstream gene effector that activated Snail in cultured podocytes. In vitro changes of the Snail/nephrin axis were similar to those in renal biopsy specimens of Zucker diabetic fatty rats and patients with advanced diabetic nephropathy, and were normalized by pharmacological inhibition of the renin-angiotensin system. Collectively, the present studies provide evidence that Ang II plays a relevant role in perpetuating glomerular injury in experimental and human diabetic nephropathy via persistent activation of Notch1 and Snail signaling in podocytes, eventually resulting in down-regulation of nephrin expression, the integrity of which is crucial for the glomerular filtration barrier.

Topics: Aged; Angiotensin II; Animals; Case-Control Studies; Cells, Cultured; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Down-Regulation; Female; Humans; Immunohistochemistry; Kidney; Linear Models; Male; Membrane Proteins; Microscopy, Electron, Transmission; Middle Aged; Rats; Rats, Sprague-Dawley; Rats, Zucker; Real-Time Polymerase Chain Reaction; Receptor, Notch1; Snail Family Transcription Factors; Transcription Factors

Pigment epithelium-derived factor (PEDF) is a multifunctional protein with antiangiogenic, antioxidative, and anti-inflammatory properties. PEDF is involved in the pathogenesis of diabetic retinopathy, but its direct role in the kidneys remains unclear. We hypothesize that a PEDF fragment (P78-PEDF) confers kidney protection in diabetic nephropathy (DN). The localization of the full-length PEDF protein were determined in DBA mice following multiple low doses of streptozotocin. Using immunohistochemistry, PEDF was localized in the kidney vasculature, interstitial space, glomeruli, tubules, and renal medulla. Kidney PEDF protein and mRNA expression were significantly reduced in diabetic mice. Continuous infusion of P78-PEDF for 6 wk resulted in protection from diabetic neuropathy as indicated by reduced albuminuria and blood urea nitrogen, increased nephrin expression, decreased kidney macrophage recruitment and inflammatory cytokines, and reduced histological changes compared with vehicle-treated diabetic mice. In vitro, P78-PEDF blocked the increase in podocyte permeability to albumin and disruption of the actin cytoskeleton induced by puromycin aminonucleoside treatment. These findings highlight the importance of P78-PEDF peptide as a potential therapeutic modality in early phase diabetic renal injury.

Topics: Albuminuria; Animals; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Eye Proteins; Kidney; Male; Membrane Proteins; Mice; Mice, Inbred DBA; Nerve Growth Factors; Peptide Fragments; Podocytes; Serpins

Monocyte/macrophage recruitment correlates strongly with the progression of renal impairment in diabetic nephropathy (DN), yet their direct role is not clear. We hypothesized that macrophages contribute to direct podocyte injury and/or an abnormal podocyte niche leading to DN. Experiments were conducted in CD11b-DTR mice treated with diphtheria toxin (DT) to deplete macrophages after streptozotocin-induced diabetes. Additional experiments were conducted in bone marrow chimeric (CD11b-DTR→ C57BL6/J) mice. Diabetes was associated with an increase in the M1-to-M2 ratio by 6 wk after the induction of diabetes. Macrophage depletion in diabetic CD11b-DTR mice significantly attenuated albuminuria, kidney macrophage recruitment, and glomerular histological changes and preserved kidney nephrin and podocin expression compared with diabetic CD11b-DTR mice treated with mutant DT. These data were confirmed in chimeric mice indicating a direct role of bone marrow-derived macrophages in DN. In vitro, podocytes grown in high-glucose media significantly increased macrophage migration compared with podocytes grown in normal glucose media. In addition, classically activated M1 macrophages, but not M2 macrophages, induced podocyte permeability. These findings provide evidence showing that macrophages directly contribute to kidney injury in DN, perhaps by altering podocyte integrity through the proinflammatory M1 subset of macrophages. Attenuating the deleterious effects of macrophages on podocytes could provide a new therapeutic approach to the treatment of DN.

Topics: Animals; CD11b Antigen; Cell Count; Cell Movement; Cells, Cultured; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Disease Models, Animal; Glucose; In Vitro Techniques; Intracellular Signaling Peptides and Proteins; Macrophages; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Podocytes; Streptozocin

Diabetic kidney disease has been associated with the presence of lipid deposits. We assumed that curcumin, a polyphenol, would attenuate the tissue dyslipidemic condition through activation of 5' adenosine monophosphate (AMP)-activated protein kinase (AMPK) phosphorylation and suppression of sterol regulatory element-binding protein (SREBP)-1c in the kidney and would prevent renal progression in experimental type 1 diabetic rats. Diabetes was induced with streptozotocin (STZ) (55 mg/kg) by intraperitoneal injection in male Sprague-Dawley rats. Three weeks after STZ injection, rats were divided into three groups, namely, control, diabetic and diabetic treated with curcumin (100 mg/kg/day) by gavage for 8 weeks. We found that curcumin decreased plasma triglyceride and the amount of renal triglyceride significantly. Furthermore, treatment of diabetic rats with curcumin increased the phosphorylation of AMPK and prevented the increased renal expression of SREBP-1c and, as a result, decreased the expression of acetyl CoA carboxylase and fatty acid synthase as well as adipose differentiation-related protein, a marker of cytoplasmic droplets. We also demonstrate that curcumin significantly suppressed the increased expression of transforming growth factor β, vascular endothelial growth factor and extracellular matrix proteins such as type IV collagen and fibronectin. In addition, curcumin treatment increased nephrin expression to near-normal levels in diabetic rats. These results demonstrated that curcumin protects against the development of diabetic nephropathy through the AMPK-SREBP pathway and the reduction of renal triglyceride accumulation which could be a possible mechanism by which curcumin preserves renal function in diabetes.

Topics: Acetyl-CoA Carboxylase; AMP-Activated Protein Kinases; Animals; Curcumin; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Extracellular Matrix Proteins; Fatty Acid Synthases; Kidney; Male; Membrane Proteins; Perilipin-2; Phosphorylation; Rats; Rats, Sprague-Dawley; Signal Transduction; Sterol Regulatory Element Binding Protein 1; Streptozocin; Transforming Growth Factor beta; Triglycerides; Vascular Endothelial Growth Factors

Diabetic nephropathy (DN) is the major life-threatening complication of diabetes. Abnormal permeability of glomerular basement membrane plays an important role in DN pathogenesis. This study was performed to assess the effect of arctiin, the lignan constituent from Arctium lappa L., on metabolic profile and aggravation of renal lesions in a rat model of streptozotocin (STZ)-induced DN. STZ-induced diabetic rats were treated with arctiin at the dosage of 60 or 40 mg/kg/day via intraperitoneal injection for 8 weeks. Blood glucose and 24-h urinary albumin content were measured, and kidney histopathological changes were monitored. RT-PCR and immunohistochemistry were used to detect the mRNA and protein levels of nephrin, podocin and heparanase (HPSE) in the kidney cortex of rats, respectively. Treatment with arctiin significantly decreased the levels of 24-h urinary albumin, prevented the sclerosis of glomeruli and effectively restored the glomerular filtration barrier damage by up-regulating the expression of nephrin and podocin and down-regulating HPSE level. Our studies suggest that arctiin might be beneficial for DN. The effects of arctiin on attenuating albuminuria and glomerulosclerosis are possibly mediated by regulating the expression of nephrin and podocin and HPSE in STZ-induced diabetic rats.

Topics: Albuminuria; Animals; Blood Glucose; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Furans; Gene Expression Regulation; Glomerular Filtration Barrier; Glucosides; Glucuronidase; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Male; Membrane Proteins; Metabolome; Rats; Rats, Sprague-Dawley; RNA, Messenger; Streptozocin

Diabetic nephropathy (DN) is characterized by podocyte damage and increased phosphodiesterase-5 (PDE-5) activity-exacerbating nitric oxide (NO)-cyclic 3',5' guanosine monophosphate (cGMP) pathway dysfunction. It has been shown that PDE-5 inhibition ameliorates DN. The role of podocytes in this mechanism remains unclear. We investigated how selective PDE-5 inhibition influences podocyte damage in streptozotocin (STZ) diabetic rats.. Male Sprague-Dawley rats (250-300 g) were injected with STZ and divided into two groups: (i) STZ control (non-treated, STZ, n=6) and (ii) STZ+vardenafil treatment (10 mg/kg/day, STZ-Vard, n=8). Non-diabetic rats served as negative controls (Control, n=7). Following 8 weeks of treatment, immunohistochemical and molecular analysis of the kidneys were performed.. Diabetic rats had proteinuria, increased renal transforming growth factor (TGF)-β1 expression and podocyte damage when compared with controls. Vardenafil treatment resulted in preserved podocyte cGMP levels, less proteinuria, reduced renal TGF-β1 expression, desmin immunostaining in podocytes and restored both nephrin and podocin mRNA expression. Diabetes led to increased glomerular nitrotyrosine formation and renal neuronal nitric oxide synthase and endothelial nitric oxide synthase mRNA expression, but vardenafil did not influence these parameters.. Our data suggest that a dysfunctional NO-cGMP pathway exacerbates podocyte damage in diabetes. In conclusion, vardenafil treatment preserves podocyte function and reduces glomerular damage, which indicates therapeutic potential in patients with DN.

Topics: Animals; Blotting, Western; Cells, Cultured; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Imidazoles; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Nitric Oxide Synthase Type III; Oxidative Stress; Phosphodiesterase 5 Inhibitors; Piperazines; Podocytes; Proteinuria; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sulfones; Transforming Growth Factor beta1; Triazines; Vardenafil Dihydrochloride

Mesenchymal stem cells (MSC) attenuate albuminuria and preserve normal renal histology in diabetic mice. However, the effects of MSC on glomerular podocyte injury remain uncertain. The aim of this study was to evaluate the effects of MSC on podocyte injury in streptozotocin (STZ)-induced diabetic rats. Thirty days after diabetes induction by STZ injection (65 mg/kg, intraperitoneally) in Sprague-Dawley rats, the diabetic rats received medium or 2 × 10(6) enhanced green fluorescent protein-labeled MSC via the renal artery. In vivo tracking of MSC was followed by immunofluorescence analysis. Diabetes-related physical and biochemical parameters were measured on day 60 after the MSC infusion. The expression of podocyte markers (nephrin and podocin), podocyte survival factors (VEGF and BMP-7), and the ultrastructural pathology of podocytes were also assessed. MSC were only detected in the glomeruli from the left kidney receiving MSC infusion. Compared with medium-treated diabetic rats, rats treated with MSC showed a suppressed increase in kidney weight, kidney to body weight index, creatinine clearance rate, and urinary albumin to creatinine ratio; however, the treatment had no effect on blood glucose or body weight levels. Furthermore, the MSC treatment reduced the loss of podocytes, effacement of foot processes, widening of foot processes, thickening of glomerular basal membrane (GBM), and loss of glomerular nephrin and podocin. Most important, MSC-injected kidneys expressed higher levels of BMP-7 but not of VEGF. Our results clearly demonstrated that intra-arterial administration of MSC prevented the development of albuminuria as well as any damage to or loss of podocytes, though there was no improvement in blood sugar levels. The protective effects of MSC may be mediated in part by increasing BMP-7 secretion.

Topics: Animals; Biomarkers; Blood Glucose; Bone Morphogenetic Protein 7; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Gene Expression; Genes, Reporter; Green Fluorescent Proteins; Intracellular Signaling Peptides and Proteins; Kidney; Male; Membrane Proteins; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Podocytes; Proteinuria; Rats; Rats, Sprague-Dawley; Streptozocin; Vascular Endothelial Growth Factor A

Diabetic nephropathy is the main cause of end-stage renal disease. N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP), a physiological tetrapeptide hydrolyzed by the angiotensin-converting enzyme (ACE), has antifibrotic effects in the cardiovascular system and in the kidney in experimental models of hypertension, heart failure and renal disease. The aim of the study was to evaluate the effect of Ac-SDKP in diabetic nephropathy and the potential additive effect of Ac-SDKP, when compared to ACE inhibitors alone, on the development of renal fibrosis.. Diabetes was induced in 28 Sprague-Dawley rats by a single intraperitoneal injection of streptozotocin. Control rats (n = 10) received only buffer solution. An ACE inhibitor (ramipril, 3 mg/kg/day) was administered to 11 diabetic rats. After 2 months, Ac-SDKP (1 mg/kg/day) was administered by osmotic minipumps for 8 weeks to 7 diabetic rats and to 6 diabetic rats treated with ramipril. Osmotic minipumps delivered saline solution in the corresponding sham-treated rats (diabetic rats, n = 10, and ramipril-treated diabetic rats, n = 5).. Diabetic rats showed a significant increase in blood glucose level, urinary albumin excretion and renal fibrosis, and a reduction of glomerular nephrin expression with respect to control rats. Ac-SDKP administration significantly reduced renal fibrosis in diabetic rats, without significantly reducing urinary albumin excretion. Ramipril treatment caused a significant decrease in albuminuria and renal fibrosis and restored glomerular nephrin expression. Administration of Ac-SDKP, in addition to ramipril, further reduced renal fibrosis with respect to ramipril alone, while it did not improve the antiproteinuric effect of ramipril.. Ac-SDKP administration reduces renal fibrosis in diabetic nephropathy. Addition of Ac-SDKP to ACE inhibition therapy improves the reduction of renal fibrosis with respect to ACE inhibition alone, suggesting a beneficial effect of this pharmacological association in diabetic nephropathy.

Topics: Albuminuria; Angiotensin-Converting Enzyme Inhibitors; Animals; Diabetic Nephropathies; Drug Evaluation, Preclinical; Glomerular Filtration Rate; Growth Inhibitors; Kidney Glomerulus; Male; Membrane Proteins; Nephrosclerosis; Oligopeptides; Ramipril; Rats; Rats, Sprague-Dawley

Emerging evidence has suggested that podocytes undergo epithelial-mesenchymal transition (EMT) in diabetic nephropathy (DN). Connective tissue growth factor (CTGF) and integrin-linked kinase (ILK) are involved in the progression of DN. However, the underlying mechanisms of EMT are not well understood. The study aimed to investigate the roles of CTGF and ILK in high glucose-induced phenotypic alterations of podocytes and determine whether ILK signaling is downstream of CTGF. The epithelial marker of nephrin and the mesenchymal marker of desmin were investigated by real-time RT-PCR and Western blotting. The results demonstrated that podocytes displayed a spreading, arborized morphology in normal glucose, whereas they had a cobblestone morphology in high glucose conditions, accompanied by decreased nephrin expression and increased desmin expression, suggesting podocytes underwent EMT. In response to high glucose, CTGF and ILK expression in podocytes were increased in a dose- and time-dependent manner, whereas the increase did not occur in the osmotic control. Furthermore, the inhibition of CTGF with anti-CTGF antibody prevented the phenotypic transition, as demonstrated by the preservation of epithelial morphology, the suppression of high glucose-induced desmin overexpression and the restoration of nephrin. Of note, the upregulation of ILK induced by high glucose was partially blocked by the inhibition of CTGF. In summary, these findings suggested that CTGF and ILK were involved in high glucose-induced phenotypic alterations of podocytes. ILK acted as a downstream kinase of CTGF and high glucose-induced ILK expression might occur through CTGF-dependent and -independent pathways.

Topics: Animals; Antibodies, Monoclonal; Cell Line; Connective Tissue Growth Factor; Desmin; Diabetic Nephropathies; Epithelial-Mesenchymal Transition; Glucose; Intercellular Signaling Peptides and Proteins; Membrane Proteins; Mice; Podocytes; Protein Serine-Threonine Kinases; Signal Transduction

Diabetic nephropathy (DN) is a progressive kidney disease that is caused by injury to kidney glomeruli. Podocytes are glomerular epithelial cells and play critical roles in the glomerular filtration barrier. Recent studies have shown the importance of regulating the podocyte actin cytoskeleton in early DN. The phosphoinositide 3-kinase (PI3K) inhibitor, wortmannin, simultaneously regulates Rac1 and Cdc42, which destabilize the podocyte actin cytoskeleton during early DN. In this study, in order to evaluate the reno-protective effects of wortmannin in early DN by regulating Rac1 and Cdc42, streptozotocin (STZ)-induced proteinuric renal disease (SPRD) rats were treated with wortmannin. The albuminuria value of the SPRD group was 3.55 ± 0.56 mg/day, whereas wortmannin group was 1.77 ± 0.48 mg/day. Also, the albumin to creatinine ratio (ACR) value of the SPRD group was 53.08 ± 10.82 mg/g, whereas wortmannin group was 20.27 ± 6.41 mg/g. Changes in the expression level of nephrin, podocin and Rac1/Cdc42, which is related to actin cytoskeleton in podocytes, by wortmannin administration were confirmed by Western blotting. The expression levels of nephrin (79.66 ± 0.02), podocin (87.81 ± 0.03) and Rac1/Cdc42 (86.12 ± 0.02) in the wortmannin group were higher than the expression levels of nephrin (55.32 ± 0.03), podocin (53.40 ± 0.06) and Rac1/Cdc42 (54.05 ± 0.04) in the SPRD group. In addition, expression and localization of nephrin, podocin and desmin were confirmed by immunofluorescence. In summary, we found for the first time that wortmannin has a reno-protective effect on SPRD rats during the early DN. The beneficial effects of wortmannin in SPRD rats indicate that this compound could be used to delay the progression of the disease during the early DN stage.

Topics: Albumins; Androstadienes; Animals; cdc42 GTP-Binding Protein; Creatinine; Desmin; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Disease Models, Animal; Humans; Intracellular Signaling Peptides and Proteins; Kidney; Membrane Proteins; Phosphoinositide-3 Kinase Inhibitors; Podocytes; rac1 GTP-Binding Protein; Rats; Rats, Inbred Strains; Wortmannin

Diabetic glomerulosclerosis is the hardening of the renal glomeruli that can lead to kidney failure. In the early stage of glomerulosclerosis occur renal mesangial expansion and renal filtration dysfunction. Purple corn has been classified as a functional food and is rich in anthocyanins exerting potential disease-preventive activities. The in vitro study using human renal mesangial cells examined that anthocyanin-rich purple corn butanol fraction (PCB) can attenuate high glucose (HG)-promoted mesangial cell proliferation and matrix accumulation.. Cells were cultured for 3 days in media containing 33 mM glucose in the presence of 1-20 μg/mL PCB. In the in vivo animal study, db/db mice were treated with 10 mg/kg anthocyanin-rich polyphenolic extracts of purple corn (PCE) for 8 weeks.. HG enhanced mesangial production of the fibrosis biomarkers of collagen IV and connective tissue growth factor (CTGF), which was markedly attenuated by adding PCB. Such mesangial fibrosis entailed interleukin-8 activation via eliciting Tyk2-STAT signaling pathway. PCB dampened HG-promoted mesangial hyperplasia that appeared to be attributed to increased expression of platelet-derived growth factor. The 8-week administration of PCE lowered plasma glucose level of db/db mice and ameliorated severe albuminuria. Moreover, PCE lessened collagen fiber accumulation in kidney glomeruli and CTGF expression via retarding TGF-β signaling. Protein expressions of nephrin and podocin, key proteins for filtration barrier function of the glomerular capillary wall, were repressed by treating mice with PCE.. Purple corn may be a potent therapeutic agent for the treatment for diabetes-associated glomerulosclerosis accompanying proteinuria and kidney filtration dysfunction.

Topics: Albuminuria; Animals; Anthocyanins; Biomarkers; Blood Glucose; Cell Proliferation; Collagen Type IV; Connective Tissue Growth Factor; Diabetic Nephropathies; Fibrosis; Humans; Interleukin-8; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Mesangial Cells; Mice; Plant Extracts; Platelet-Derived Growth Factor; Proteinuria; Signal Transduction; STAT Transcription Factors; Transforming Growth Factor beta; TYK2 Kinase; Zea mays

Akita mice are a genetic model of type 1 diabetes. In the present studies, we investigated the phenotype of Akita mice on the FVB/NJ background and examined urinary nephrin excretion as a marker of kidney injury. Male Akita mice were compared with non-diabetic controls for functional and structural characteristics of renal and cardiac disease. Podocyte number and apoptosis as well as urinary nephrin excretion were determined in both groups. Male FVB/NJ Akita mice developed sustained hyperglycemia and albuminuria by 4 and 8 weeks of age, respectively. These abnormalities were accompanied by a significant increase in systolic blood pressure in 10-week old Akita mice, which was associated with functional, structural and molecular characteristics of cardiac hypertrophy. By 20 weeks of age, Akita mice developed a 10-fold increase in albuminuria, renal and glomerular hypertrophy and a decrease in the number of podocytes. Mild-to-moderate glomerular mesangial expansion was observed in Akita mice at 30 weeks of age. In 4-week old Akita mice, the onset of hyperglycemia was accompanied by increased podocyte apoptosis and enhanced excretion of nephrin in urine before the development of albuminuria. Urinary nephrin excretion was also significantly increased in albuminuric Akita mice at 16 and 20 weeks of age and correlated with the albumin excretion rate. These data suggest that: 1. FVB/NJ Akita mice have phenotypic characteristics that may be useful for studying the mechanisms of kidney and cardiac injury in diabetes, and 2. Enhanced urinary nephrin excretion is associated with kidney injury in FVB/NJ Akita mice and is detectable early in the disease process.

Topics: Albuminuria; Animals; Biomarkers; Blotting, Western; Diabetes Complications; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Disease Models, Animal; Hyperglycemia; Immunoenzyme Techniques; Kidney; Male; Membrane Proteins; Mice; Mice, Inbred Strains; Phenotype; Podocytes; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Podocyte specific proteins are dysregulated in diabetic nephropathy, though the extent of their expression loss is not identical and may be subject to different regulatory factors. Quantifying the degree of loss may help identify the most useful protein to use as an early biomarker of diabetic nephropathy.. Protein expression of synaptopodin, podocin and nephrin were quantified in 15 Type 2 diabetic renal biopsies and 12 control patients. We found statistically significant downregulation of synaptopodin (P<0.0001), podocin (P = 0.0002), and nephrin (P<0.0001) in kidney biopsies of diabetic nephropathy as compared with controls. Urinary nephrin levels (nephrinuria) were then measured in 66 patients with Type 2 diabetes and 10 healthy controls by an enzyme-linked immunosorbent assay (Exocell, Philadelphia, PA). When divided into groups according to normo-, micro-, and macroalbuminuria, nephrinuria was found to be present in 100% of diabetic patients with micro- and macroalbuminuria, as well as 54% of patients with normoalbuminuria. Nephrinuria also correlated significantly with albuminuria (rho = 0.89, p<0.001), systolic blood pressure (rho = 0.32, p = 0.007), and correlated negatively with serum albumin (rho = -0.48, p<0.0001) and eGFR (rho = -0.33, p = 0.005).. These data suggest that key podocyte-specific protein expressions are significantly and differentially downregulated in diabetic nephropathy. The finding that nephrinuria is observed in a majority of these normoalbuminuric patients demonstrates that it may precede microalbuminuria. If further research confirms nephrinuria to be a biomarker of pre-clinical diabetic nephropathy, it would shed light on podocyte metabolism in disease, and raise the possibility of new and earlier therapeutic targets.

Topics: Biomarkers; Blood Pressure; Case-Control Studies; Cross-Sectional Studies; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Enzyme-Linked Immunosorbent Assay; Female; Humans; Immunohistochemistry; Male; Membrane Proteins; Middle Aged

To investigate the dynamic changes of urinary nephrin, and the relationship between it and urinary albumin excretion rate (UAER) in a diabetic rat model, as well the effects of yiqiyangyinhuayutongluo recipe.. Diabetic model was induced by high fat diet combined with low-dose Streptozotocin (STZ) in rats. Normal group (NG), model group (MG), and yiqiyangyinhuayutongluo recipe treated group (YHTG) were set. Gastrointestinal Yiqiyangyi-nhuayutongluo recipe was administered once daily for 32 w. At the end of the 2nd w (2 w), 8 w, 16 w, and 32 w, fasting blood glucose (FBG), UAER and 24h urinary nephrin (U-nephrin) were detected.. Compared with NG, FBG in MG increased notably (P < 0.05). Compared with MG, FBG of YHTG (P < 0.05) since 16 w. U-nephrin and UAER in MG increased significantly from 2 w, peaked at 16 w, lessened in different degree at 32 w, but were still higher than NG. The correlation analysis showed that there was a significant positive correlation between U-nephrin and UAER at different time, the correlation coefficient as r > 0.9, and P < 0.05. Compared with MG, U-nephrin and UAER in YHTG decreased markedly (P < 0.05) except for U-nephrin at 8 w.. U-nephrin and UAER in diabetic rat model have a positive linear correlation. Yiqiyangyinhuayutongluo recipe can reduce UAER markedly, and preventing the lose of nephrin in urine maybe one of the mechanisms.

Topics: Albuminuria; Animals; Blood Glucose; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Drugs, Chinese Herbal; Male; Membrane Proteins; Rats; Rats, Sprague-Dawley; Streptozocin

Peroxisome proliferator-activated receptor (PPAR)-δ is a ligand-activated transcription factor in regulating gene expression and is believed to play an important role in various kidney diseases including diabetic nephropathy. This study investigated the efficacy of GW610742, a highly specific agonist for PPAR-δ, for the treatment of diabetic nephropathy.. Type 2 diabetic Otsuka Long-Evans Tokushima Fatty rats were randomized into an untreated diabetic group (n = 9) and a GW610742-treated diabetic group (n= 9). The GW610742 was administered (10 mg/kg/day) orally for 11 weeks. Long-Evans Tokushima Otsuka rats (n = 9) were used as a non-diabetic control.. Albuminuria was markedly increased and renal PPAR-δ expression was decreased in diabetes. Diabetic albuminuria and renal injury markers, such as glomerular basement membrane thickening, decreased number of slit pores between podocyte foot processes, decreased nephrin expression, increased desmin expression and increased CCL2 expression, were significantly reversed through the treatment with GW610742. PPAR-δ agonist GW610742 markedly increased nephrin expression in cultured podocytes. Nephrin mRNA expression was markedly decreased in response to high glucose in cultured podocytes and effectively prevented by GW610742.. PPAR-δ activation by GW610742 ameliorates albuminuria by preventing diabetes-induced nephrin loss and restoring podocyte integrity, implying that GW610742 may be a potential therapeutic agent for diabetic nephropathy.

Topics: Albuminuria; Animals; Blotting, Western; Cell Culture Techniques; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Immunohistochemistry; Membrane Proteins; Podocytes; PPAR gamma; Rats; Rats, Inbred OLETF; Real-Time Polymerase Chain Reaction; Thiazoles

Rho-associated kinases (ROCK) are activated in the kidney as well as in cultured cells of diabetic models and have been implicated in renal pathophysiology. To explore whether inhibition of ROCK is protective, we studied its role in a model of accelerated diabetic nephropathy where uninephrectomized rats were made diabetic by streptozotocin. After establishing diabetes, rats were treated with the ROCK inhibitor fasudil continuously or for the final 6 weeks of an 18-week experimental period. The results were compared to similar rats given losartan, an established treatment of clinical and experimental diabetic nephropathy, or a combination of both agents. Vehicle-treated diabetic and non-diabetic uninephrectomized rats served as controls. Diabetes resulted in a rapid development of albuminuria, higher glomerulosclerosis and interstitial fibrosis scores, lower glomerular filtration rates, and increased expression of several molecular markers of diabetic nephropathy. Eighteen weeks of fasudil treatment reduced renal ROCK activity, and ameliorated diabetes-induced structural changes in the kidney and expression of the molecular markers in association with a modest anti-proteinuric effect but no change in blood pressure. Late intervention with fasudil reduced glomerulosclerosis, but did not influence proteinuria. Most effects of fasudil were comparable to those of losartan, although losartan lowered blood pressure and further lowered proteinuria. The combination of both treatments was no different than losartan alone. Thus, ROCK inhibition protected the kidney from diabetic nephropathy even though it did not reduce the blood pressure.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Angiotensin II Type 1 Receptor Blockers; Animals; Blood Pressure; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Epithelial-Mesenchymal Transition; Gene Expression; Kidney; Losartan; Male; Membrane Proteins; Nephrectomy; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; rho-Associated Kinases; Vascular Endothelial Growth Factor A

The involvement of VEGF-A as well as the therapeutic efficacy of angiogenesis inhibitors in diabetic nephropathy have been reported. We recently reported the therapeutic effects of vasohibin-1 (VASH-1), an endogenous angiogenesis inhibitor, in a type 1 diabetic nephropathy model (Nasu T, Maeshima Y, Kinomura M, Hirokoshi-Kawahara K, Tanabe K, Sugiyama H, Sonoda H, Sato Y, Makino H. Diabetes 58: 2365-2375, 2009). In this study, we investigated the therapeutic efficacy of VASH-1 on renal alterations in obese mice with type 2 diabetes. Diabetic db/db mice received intravenous injections of adenoviral vectors encoding human VASH-1 (AdhVASH-1) and were euthanized 8 wk later. AdhVASH-1 treatment resulted in significant suppression of glomerular hypertrophy, glomerular hyperfiltration, albuminuria, increase in the CD31(+) glomerular endothelial area, F4/80(+) monocyte/macrophage infiltration, the accumulation of type IV collagen, and mesangial matrix. An increase in the renal levels of VEGF-A, VEGFR-2, transforming growth factor (TGF)-β1, and monocyte chemoattractant protein-1 in diabetic animals was significantly suppressed by AdhVASH-1 (immunoblotting). AdhVASH-1 treatment significantly recovered the loss and altered the distribution patterns of nephrin and zonula occludens (ZO)-1 and suppressed the increase in the number of fibroblast-specific protein-1 (FSP-1(+)) and desmin(+) podocytes in diabetic mice. In vitro, recombinant human VASH-1 (rhVASH-1) dose dependently suppressed the upregulation of VEGF induced by high ambient glucose (25 mM) in cultured mouse podocytes. In addition, rhVASH-1 significantly recovered the mRNA levels of nephrin and the protein levels of ZO-1 and P-cadherin and suppressed the increase in protein levels of desmin, FSP-1, Snail, and Slug in podocytes under high-glucose condition. Taken together, these results suggest the potential use of VASH-1 as a novel therapeutic agent in type 2 diabetic nephropathy mediated via antiangiogenic effects and maintenance of podocyte phenotype in association with antiproteinuric effects.

Topics: Angiogenesis Inhibitors; Animals; Blood Glucose; Blood Pressure; Blotting, Western; Cell Cycle Proteins; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Humans; Immunohistochemistry; Kidney; Male; Membrane Proteins; Mice; Mice, Obese

Diabetic nephropathy (DN) is the major cause of end-stage renal disease. The early changes in DN are characterized by an increased in kidney size, glomerular volume, and kidney function, followed by the accumulation of glomerular extracellular matrix, increased urinary albumin excretion (UAE), glomerular sclerosis, and tubular fibrosis. Resveratrol (RSV) has been shown to ameliorate hyperglycemia and hyperlipidemia in streptozotocin-induced diabetic rats. In the present study, we examined the beneficial effects of RSV on DN and explored the possible mechanism of RSV action. Male Sprague-Dawley rats were injected with streptozotocin at 65mg/kg body weight. The induction of diabetes mellitus (DM) was confirmed by a fasting plasma glucose level ≥300mg/dL and symptoms of polyphagia and polydipsia. The DM rats were treated with or without RSV at 0.75mg/kg body weight 3 times a day for 8 weeks. Animals were sacrificed and kidney histology was examined by microscopy. Urinary albumin excretion, glomerular hypertrophy and expressions of fibronectin, collagen IV, and TGF-β in the glomeruli were alleviated in RSV-treated DM rats, but not in untreated DM rats. In addition, RSV treatment reduced the thickness of the glomerular basement membrane (GBM) to the original thickness and increased nephrin expressions to normal levels in DM rats. Moreover, RSV inhibited phosphorylation of smad2, smad3 and ERK1/2 in diabetic rat kidneys. This is the first report showing that RSV alleviates early glomerulosclerosis in DN through TGF-β/smad and ERK1/2 inhibition. In addition, podocyte injuries of diabetic kidneys are lessened by RSV.

Topics: Animals; Collagen Type IV; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Disease Models, Animal; Fibronectins; Hypoglycemic Agents; Kidney; Male; Membrane Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphorylation; Rats; Rats, Sprague-Dawley; Resveratrol; Signal Transduction; Smad Proteins; Stilbenes; Transforming Growth Factor beta

Nephrin, the key molecule of the glomerular slit diaphragm, is expressed on the surface of podocytes and is critical in preventing albuminuria. In diabetes, hyperglycemia leads to the loss of surface expression of nephrin and causes albuminuria. Here, we report a mechanism that can explain this phenomenon: hyperglycemia directly enhances the rate of nephrin endocytosis via regulation of the β-arrestin2-nephrin interaction by PKCα. We identified PKCα and protein interacting with c kinase-1 (PICK1) as nephrin-binding proteins. Hyperglycemia induced up-regulation of PKCα and led to the formation of a complex of nephrin, PKCα, PICK1, and β-arrestin2 in vitro and in vivo. Binding of β-arrestin2 to the nephrin intracellular domain depended on phosphorylation of nephrin threonine residues 1120 and 1125 by PKCα. Further, cellular knockdown of PKCα and/or PICK1 attenuated the nephrin-β-arrestin2 interaction and abrogated the amplifying effect of high blood glucose on nephrin endocytosis. In C57BL/6 mice, hyperglycemia over 24 h caused a significant increase in urinary albumin excretion, supporting the concept of the rapid impact of hyperglycemia on glomerular permselectivity. In summary, we have provided a molecular model of hyperglycemia-induced nephrin endocytosis and subsequent proteinuria and highlighted PKCα and PICK1 as promising therapeutic targets for diabetic nephropathy.

Topics: Albuminuria; Animals; Arrestins; beta-Arrestins; Blood Glucose; Carrier Proteins; Cell Cycle Proteins; Diabetic Nephropathies; Endocytosis; Humans; Hyperglycemia; Membrane Proteins; Mice; Mice, Knockout; Models, Biological; Nuclear Proteins; Phosphorylation; Protein Kinase C-alpha

Emerging evidence suggests that podocyte injury is a crucial event in the stage of diabetic nephropathy (DN), a process in which angiotensin II is implicated. In this study, the authors investigated the influence of irbesartan, an angiotensin receptor blocker, on the phenotypic alterations of podocytes in experimental DN.. DN was induced by combination of high-sucrose, high-fat diet and intraperitoneal injection of low dose of streptozotocin (35 mg/kg) in spontaneously hypertensive rats. Diabetic rats were treated with irbesartan (50 mg/kg/d) by gavage for 8 weeks. Nondiabetic normotensive Wistar-Kyoto rats, which have the same genetic background as spontaneously hypertensive rat, were used as controls. The renal histological changes were investigated by light and electron microscopy. The epithelial marker of nephrin and mesenchymal marker of desmin were detected by real-time reverse transcriptase-polymerase chain reaction and Western blotting.. Compared with controls, diabetic rats were associated with mesangial matrix deposition, thickening of glomerular basement membrane, albuminuria, loss of podocytes and effacement of foot processes. Furthermore, the expression of nephrin was significantly reduced whereas desmin was increased. Irbesartan treatment not only lowered blood pressure and albuminuria but also attenuated podocyte loss, maintenance of nephrin expression and inhibition of desmin expression.. This study demonstrates that early irbesartan intervention attenuates the podocyte damage and ameliorates phenotypic alterations of podocytes, which provides a novel insight for the early application of angiotensin receptor blocker to prevent the development of DN.

Topics: Albuminuria; Angiotensin II Type 1 Receptor Blockers; Animals; Antihypertensive Agents; Biomarkers; Biphenyl Compounds; Blood Pressure; Blotting, Western; Desmin; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Immunohistochemistry; Irbesartan; Male; Membrane Proteins; Microscopy, Electron; Podocytes; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Reverse Transcriptase Polymerase Chain Reaction; Tetrazoles; Time Factors

Diabetic nephropathy is associated with dedifferentiation of podocytes, losing the specialized features required for efficient glomerular function and acquiring a number of profibrotic, proinflammatory, and proliferative features. These result from tight junction and cytoskeletal rearrangement, augmented proliferation, and apoptosis.. Experiments were performed in conditionally immortalized human podocytes developed by transfection with the temperature-sensitive SV40-T gene. Cells were then cultured in the presence of transforming growth factor (TGF)-β1 or angiotensin II in the presence or absence of a selective inhibitor of the TGF-β type I receptor kinase, SB-431542. Gene and protein expression were then examined by real-time RT-PCR and immunofluorescence, and correlated with changes observed in vivo in experimental diabetes.. Treatment of cells with TGF-β1 resulted in dynamic changes in their morphology, starting with retraction and shortening of foot processes and finishing with the formation of broad and complex tight junctions between adjacent podocytes. This dedifferentiation was also associated with dose- and time-dependent reduction in the expression of glomerular epithelial markers (nephrin, p-cadherin, zonnula occludens-1) and increased expression of mesenchymal markers (α-smooth muscle actin, vimentin, nestin), matrix components (fibronectin, collagen I, and collagen IV α3), cellular proliferation, and apoptosis. The induction of diabetes in mice was also associated with similar changes in morphology, protein expression, and proliferation in glomerular podocytes.. In response to TGF-β and other TGF-dependent stimuli, mature podocytes undergo dedifferentiation that leads to effacement of foot processes, morphologic flattening, and increased formation of intercellular tight junctions. This simplification of their phenotype to a more embryonic form is also associated with reentry of mature podocytes into the cell cycle, which results in enhanced proliferation and apoptosis. These "pathoadaptive" changes are seen early in the diabetic glomerulus and ultimately contribute to albuminuria, glomerulosclerosis, and podocytopenia.

Topics: Actins; Angiotensin II; Apoptosis; Blotting, Western; Cadherins; Cell Differentiation; Cell Proliferation; Cells, Cultured; Collagen; Diabetic Nephropathies; Fibronectins; Humans; Intermediate Filament Proteins; Membrane Proteins; Nerve Tissue Proteins; Nestin; Podocytes; Transforming Growth Factor beta; Vimentin

The predominant transcription factors regulating key genes in diabetic kidney disease have not been established. The transcription factor upstream stimulatory factor 1 (USF1) is an important regulator of glucose-mediated transforming growth factor (TGF)-β1 expression in mesangial cells; however, its role in the development of diabetic kidney disease has not been evaluated. In the present study, wild-type (WT; USF1 +/+), heterozygous (USF1 +/-), and homozygous (USF1 -/-) knockout mice were intercrossed with Akita mice (Ins2/Akita) to induce type 1 diabetes. Mice were studied up to 36 wk of age. The degree of hyperglycemia and kidney hypertrophy were similar in all groups of diabetic mice; however, the USF1 -/- diabetic mice had significantly less albuminuria and mesangial matrix expansion than the WT diabetic mice. TGF-β1 and renin gene expression and protein were substantially increased in the WT diabetic mice but not in USF1 -/- diabetic mice. The underlying pathway by which USF1 is regulated by high glucose was investigated in mesangial cell culture. High glucose inhibited AMP-activated protein kinase (AMPK) activity and increased USF1 nuclear translocation. Activation of AMPK with AICAR stimulated AMPK activity and reduced nuclear accumulation of USF1. We thus conclude that USF1 is a critical transcription factor regulating diabetic kidney disease and plays a critical role in albuminuria, mesangial matrix accumulation, and TGF-β1 and renin stimulation in diabetic kidney disease. AMPK activity may play a key role in high glucose-induced regulation of USF1.

Topics: Albuminuria; Alleles; AMP-Activated Protein Kinases; Animals; Cell Line; Cell Nucleus; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Disease Progression; Extracellular Matrix; Female; Hyperglycemia; Hypertrophy; Kidney; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Renin-Angiotensin System; RNA, Messenger; Transforming Growth Factor beta; Upstream Stimulatory Factors

Diabetic nephropathy (DN) is among the most lethal complications that occur in type 1 and type 2 diabetics. Podocyte dysfunction is postulated to be a critical event associated with proteinuria and glomerulosclerosis in glomerular diseases including DN. However, molecular mechanisms of podocyte dysfunction in the development of DN are not well understood. Here we have shown that activity of mTOR complex 1 (mTORC1), a kinase that senses nutrient availability, was enhanced in the podocytes of diabetic animals. Further, podocyte-specific mTORC1 activation induced by ablation of an upstream negative regulator (PcKOTsc1) recapitulated many DN features, including podocyte loss, glomerular basement membrane thickening, mesangial expansion, and proteinuria in nondiabetic young and adult mice. Abnormal mTORC1 activation caused mislocalization of slit diaphragm proteins and induced an epithelial-mesenchymal transition-like phenotypic switch with enhanced ER stress in podocytes. Conversely, reduction of ER stress with a chemical chaperone significantly protected against both the podocyte phenotypic switch and podocyte loss in PcKOTsc1 mice. Finally, genetic reduction of podocyte-specific mTORC1 in diabetic animals suppressed the development of DN. These results indicate that mTORC1 activation in podocytes is a critical event in inducing DN and suggest that reduction of podocyte mTORC1 activity is a potential therapeutic strategy to prevent DN.

Topics: Adaptor Proteins, Signal Transducing; Animals; Carrier Proteins; Cell Differentiation; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Disease Models, Animal; Endoplasmic Reticulum; Enzyme Activation; Glomerular Basement Membrane; Glomerular Mesangium; Male; Mechanistic Target of Rapamycin Complex 1; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Mutant Strains; Multiprotein Complexes; Phosphorylation; Podocytes; Protein Processing, Post-Translational; Proteins; Regulatory-Associated Protein of mTOR; Ribosomal Protein S6 Kinases; Sirolimus; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 1 Protein; Tumor Suppressor Proteins

Monocyte/ chemoattractant protein-1/chemokine ligand (CCL) 2 and stromal cell-derived factor-1/CXCL12 both contribute to glomerulosclerosis in mice with type 2 diabetes mellitus, through different mechanisms. CCL2 mediates macrophage-related inflammation, whereas CXCL12 contributes to podocyte loss. Therefore, we hypothesized that dual antagonism of these chemokines might have additive protective effects on the progression of diabetic nephropathy. We used chemokine antagonists based on structured l-enantiomeric RNA (so-called Spiegelmers) ie, the CCL2-specific mNOX-E36 and the CXCL12-specific NOX-A12. Male db/db mice, uninephrectomized at the age of 6 weeks, received injections of Spiegelmer, both Spiegelmers, nonfunctional control Spiegelmer, or vehicle from the age of 4 months for 8 weeks. Dual blockade was significantly more effective than monotherapy in preventing glomerulosclerosis. CCL2 blockade reduced glomerular leukocyte counts and renal-inducible nitric oxide synthase or IL-6 mRNA expression. CXCL12 blockade maintained podocyte numbers and renal nephrin and podocin mRNA expression. Consistently, CXCL12 blockade suppressed nephrin mRNA up-regulation in primary cultures of human glomerular progenitors induced to differentiate toward the podocyte lineage. All previously mentioned parameters were significantly improved in the dual-blockade group, which also suppressed proteinuria and was associated with the highest levels of glomerular filtration rate. Blood glucose levels and body weight were identical in all treatment groups. Dual chemokine blockade can have additive effects on the progression of diabetic kidney disease when the respective chemokine targets mediate different pathomechanisms of disease (ie, inflammation and progenitor differentiation toward the podocyte lineage).

Topics: Animals; Blotting, Western; Cells, Cultured; Chemokine CCL2; Chemokine CXCL12; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Glomerular Filtration Rate; Glomerulonephritis; Immunoenzyme Techniques; Interleukin-6; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Obese; Nitric Oxide Synthase Type II; Podocytes; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stem Cells

The cannabinoid receptor type 2 (CB2) has protective effects in chronic degenerative diseases. Our aim was to assess the potential relevance of the CB2 receptor in both human and experimental diabetic nephropathy (DN).. CB2 expression was studied in kidney biopsies from patients with advanced DN, in early experimental diabetes, and in cultured podocytes. Levels of endocannabinoids and related enzymes were measured in the renal cortex from diabetic mice. To assess the functional role of CB2, streptozotocin-induced diabetic mice were treated for 14 weeks with AM1241, a selective CB2 agonist. In these animals, we studied albuminuria, renal function, expression of podocyte proteins (nephrin and zonula occludens-1), and markers of both fibrosis (fibronectin and transforming growth factor-β1) and inflammation (monocyte chemoattractant protein-1 [MCP-1], CC chemokine receptor 2 [CCR2], and monocyte markers). CB2 signaling was assessed in cultured podocytes.. Podocytes express the CB2 receptor both in vitro and in vivo. CB2 was downregulated in kidney biopsies from patients with advanced DN, and renal levels of the CB2 ligand 2-arachidonoylglycerol were reduced in diabetic mice, suggesting impaired CB2 regulation. In experimental diabetes, AM1241 ameliorated albuminuria, podocyte protein downregulation, and glomerular monocyte infiltration, without affecting early markers of fibrosis. In addition, AM1241 reduced CCR2 expression in both renal cortex and cultured podocytes, suggesting that CB2 activation may interfere with the deleterious effects of MCP-1 signaling.. The CB2 receptor is expressed by podocytes, and in experimental diabetes, CB2 activation ameliorates both albuminuria and podocyte protein loss, suggesting a protective effect of signaling through CB2 in DN.

Topics: Albuminuria; Animals; Cannabinoids; Chemokine CCL2; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Disease Models, Animal; Fibronectins; Humans; Kidney Cortex; Membrane Proteins; Mice; Podocytes; Receptor, Cannabinoid, CB2; Transforming Growth Factor beta1

Studies suggest that soluble epoxide hydrolase (sEH) inhibition reduces end-organ damage in cardiovascular diseases. We hypothesize that sEH gene (Ephx2) knockout (KO) improves endothelial function and reduces renal injury in streptozotocin-induced diabetes. After 6 wk of diabetes, afferent arteriolar relaxation to acetylcholine was impaired in diabetic wild-type (WT) mice, as the maximum relaxation was 72% of baseline diameter in the WT but only 31% in the diabetic mice. Ephx2 KO improved afferent arteriolar relaxation to acetylcholine in diabetes as maximum relaxation was 58%. Urinary monocyte chemoattractant protein-1 (MCP-1) excretion significantly increased in diabetic WT mice compared with control (868 ± 195 vs. 31.5 ± 7 pg/day), and this increase was attenuated in diabetic Ephx2 KO mice (420 ± 98 pg/day). The renal phospho-IKK-to-IKK ratio and nuclear factor-κB were significantly decreased, and hemeoxygenase-1 (HO-1) expression increased in diabetic Ephx2 KO compared with diabetic WT mice. Renal NADPH oxidase and urinary thiobarbituric acid reactive substances excretion were reduced in diabetic Ephx2 KO compared with diabetic WT mice. Albuminuria was also elevated in diabetic WT mice compared with control (170 ± 43 vs. 37 ± 13 μg/day), and Ephx2 KO reduced this elevation (50 ± 15 μg/day). Inhibition of sEH using trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (tAUCB) also reduced renal inflammation and injury in diabetic WT mice. Furthermore, inhibition of HO with stannous mesoporphyrin negated the reno-protective effects of tAUCB or Ephx2 KO during diabetes. These data demonstrate that Ephx2 KO improves endothelial function and reduces renal injury during diabetes. Additionally, our data also suggest that activation of HO-1 contributes to improved renal injury in diabetic Ephx2 KO mice.

Topics: Albuminuria; Animals; Chemokine CCL2; Collagen; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Dose-Response Relationship, Drug; Endothelium, Vascular; Epoxide Hydrolases; Heme Oxygenase-1; I-kappa B Kinase; Inflammation Mediators; Kidney; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; NADPH Oxidases; Nephritis; Oxidative Stress; Phosphorylation; Thiobarbituric Acid Reactive Substances; Time Factors; Transcription Factor RelA; Vasodilation; Vasodilator Agents

To explore the effect of VEGF inhibitor SU5416 on podocytopathy of rats with type I diabetic nephropathy.. Thirty male SD rats were randomly divided into three groups: normal control group(NC), diabetic nephropathy group(DN) and diabetic nephropathy treated with SU5416 group(SU5416). Rats with DN were induced by STZ. At the end of 8 weeks after SU5416 treatment, body weight (BW), kidney weight (KW), 24 h urine albuminuria excretion rate(24 h UAER), plasma glucose and creatinine were detected respectively. Renal morphology were stained with periodic acid-Schiff (PAS). And the expression of podocyte-specific genes nephrin and podocin were detected by immunofluorescence. The mRNA levels of genes and VEGF were assessed by real time-PCR respectively.. Compared with NC group, DN rats'BW were decreased but the KW were increased, and the levels of blood glucose, creatinine, 24 h UAER and kidney cortex VEGF mRNA were significantly higher. The expression of nephrin and podocin were decreased(P<0.05), and GBM thickening and mesangial matrix expansion were developed. Treatment with SU5416 leads to a marked decrease of KW and the level of 24 h UAER. Concurrently, the expressions of nephrin and podocin were revert partly in response to SU5416(P<0.05), and pathological changes were successfully ameliorated. However, the KW, glucose, creatinine and the level of VEGF mRNA were not significantly affected by SU5416 treatment(P>0.05).. VEGF-R inhibitor SU5416 can obviously ameliorate albuminuria and histologic changes, and restore the expression of podocyte-specific genes nephrin and podocin in DN rats, suggesting that VEGF-R inhibitor is beneficial for the repair of podocytes in DN, which might be an important adjunct for podocytopathy therapy.

Topics: Albuminuria; Animals; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Gene Expression Regulation; Indoles; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Podocytes; Protein Kinase Inhibitors; Pyrroles; Rats; Rats, Sprague-Dawley; Receptors, Vascular Endothelial Growth Factor; RNA, Messenger; Vascular Endothelial Growth Factor A

Increased glomerular permeability to proteins is a characteristic feature of diabetic nephropathy (DN). The slit diaphragm is the major restriction site to protein filtration, and the loss of nephrin, a key component of the slit diaphragm, has been demonstrated in both human and experimental DN. Both systemic and glomerular hypertension are believed to be important in the pathogenesis of DN. Human immortalized podocytes were subjected to repeated stretch-relaxation cycles by mechanical deformation with the use of a stress unit (10% elongation, 60 cycles/min) in the presence or absence of candesartan (1 microM), PD-123319 (1 microM), and rosiglitazone (0.1 microM). Nephrin mRNA and protein expression were assessed using quantitative real-time PCR, immunoblotting, and immunofluorescence, and the protein expression of AT(1) receptor and angiotensin II secretion were evaluated. Exposure to stretch induced a significant approximately 50% decrease in both nephrin mRNA and protein expression. This effect was mediated by an angiotensin II-AT(1) mechanism. Indeed, podocyte stretching induced both angiotensin II secretion and AT(1) receptor overexpression, podocyte exposure to angiotensin II reduced nephrin protein expression, and both the AT-1 receptor antagonist candesartan and a specific anti-angiotensin II antibody completely abolished stretch-induced nephrin downregulation. Similar to candesartan, the peroxisome proliferator-activated receptor (PPAR)-gamma agonist, rosiglitazone, also inhibited stretch-induced nephrin downregulation, suggesting interference with stretch-induced activation of the angiotensin II-AT(1) receptor system. Accordingly, rosiglitazone did not alter stretch-induced angiotensin II secretion, but it prevented AT(1) upregulation in response to stretch. These results suggest a role for hemodynamic stress in loss of nephrin expression and allude to a role of PPAR-gamma agonists in the prevention of this loss.

Topics: Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Apoptosis; Cell Proliferation; Cells, Cultured; Diabetic Nephropathies; Down-Regulation; Humans; Membrane Proteins; Podocytes; PPAR gamma; Receptor, Angiotensin, Type 1; RNA, Messenger; Rosiglitazone; Stress, Mechanical; Thiazolidinediones; Up-Regulation

Cannabinoid receptor 1 (CB1) is localized in the central nervous system and in peripheral tissues involved in energy metabolism control. However, CB1 receptors are also expressed at low level within the glomeruli, and the aim of this study was to investigate their potential relevance in the pathogenesis of proteinuria in experimental type 1 diabetes.. Streptozotocin-induced diabetic mice were treated with N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,3-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251), a selective CB1-receptor antagonist, at the dosage of 1 mg x kg(-1) x day(-1) via intraperitoneal injection for 14 weeks. Urinary albumin excretion was measured by enzyme-linked immunosorbent assay. CB1 receptor expression was studied by immunohistochemistry, immunoblotting, and real-time PCR. Expression of nephrin, podocin, synaptopodin, and zonula occludens-1 (ZO-1) was assessed by immunofluorescence and real-time PCR. Fibronectin, transforming growth factor-beta1 (TGF-beta1), and connective tissue growth factor (CTGF) mRNA levels were quantitated by real-time PCR.. In diabetic mice, the CB1 receptor was overexpressed within the glomeruli, predominantly by glomerular podocytes. Blockade of the CB1 receptor did not affect body weight, blood glucose, and blood pressure levels in either diabetic or control mice. Albuminuria was increased in diabetic mice compared with control animals and was significantly ameliorated by treatment with AM251. Furthermore, CB1 blockade completely prevented diabetes-induced downregulation of nephrin, podocin, and ZO-1. By contrast overexpression of fibronectin, TGF-beta1, and CTGF in renal cortex of diabetic mice was unaltered by AM251 administration.. In experimental type 1 diabetes, the CB1 receptor is overexpressed by glomerular podocytes, and blockade of the CB1 receptor ameliorates albuminuria possibly via prevention of nephrin, podocin, and ZO-1 loss.

Topics: Albuminuria; Animals; Connective Tissue Growth Factor; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Fibronectins; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Phosphoproteins; Piperidines; Polymerase Chain Reaction; Pyrazoles; Receptor, Cannabinoid, CB1; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta1; Zonula Occludens-1 Protein

Podocyte loss plays an important role in the pathogenesis of diabetic nephropathy, but counting the number of glomerular podocyte in renal biopsy specimen is a labor-intensive task. We study whether intra-renal and urinary messenger RNA expression of podocyte-associated molecules could be used to estimate glomerular podocyte number in patients with diabetic nephropathy.. We studied 21 consecutive patients with biopsy-proven diabetic nephropathy. The intra-renal and urinary mRNA expression of nephrin, podocin, and synaptopodin were measured by real-time quantitative polymerase chain reaction. Podocyte number was determined in micro-dissected glomerulus. The degree of histological scarring was quantified by morphometric analysis.. Glomerular podocyte number correlated with intra-renal expression of nephrin (r=0.510, p=0.044), podocin (r=0.605, p=0.013), and synaptopodin (r=0.480, p=0.060). Glomerular podocyte number also significantly correlated with urinary expression of synaptopodin (r=0.595, p=0.019) but not other targets. Baseline renal function correlated with intra-renal expression of nephrin (r=0.617, p=0.005), synaptopodin (r=0.474, p=0.040), and podocin (r=0.443, p=0.057). The degree of tubulointerstitial scarring also inversely correlated with intra-renal expression of nephrin (r=-0.462, p=0.047), podocin (r=-0.458, p=0.049), and synaptopodin (r=-0.500, p=0.029) but not with urinary gene expression.. Intra-renal expression of podocyte-associated molecules correlated with glomerular podocyte number, renal function, and tubulointerstitial scarring. The results suggest that intra-renal, but not urinary expression of podocyte-associated molecules, might be used to assess the degree of podocyte loss in diabetic nephropathy.

Topics: Biopsy; Diabetic Nephropathies; Female; Glomerular Filtration Rate; Humans; Intracellular Signaling Peptides and Proteins; Kidney; Kidney Glomerulus; Male; Membrane Proteins; Microfilament Proteins; Middle Aged; Podocytes; RNA, Messenger

We sought to examine the pathogenic role of excessive VEGF-A expression in podocytes, since it has been reported that diabetic nephropathy and other glomerular diseases are associated with increased VEGF-A expression. The induction of podocyte-specific VEGF164 overexpression in adult transgenic mice led to proteinuria, glomerulomegaly, glomerular basement membrane thickening, mesangial expansion, loss of slit diaphragms, and podocyte effacement. When doxycycline-mediated VEGF164 was stopped, these abnormalities reversed. These findings were associated with reversible downregulation of metalloproteinase 9 and nephrin expression. Using transmission electron microscopy, we established that VEGF-A receptor-2 (VEGFR2) was expressed in podocytes and glomerular endothelial cells. We also found that VEGF164 induced VEGFR2 phosphorylation in podocytes. Further, we were able to co-immunoprecipitate VEGFR2 and nephrin using whole kidney lysates, confirming interaction in vivo. This implies that autocrine and paracrine VEGF-A signaling through VEGFR2 occurs in podocytes and may mediate the glomerular phenotype caused by VEGF164 overexpression. Thus, we suggest that podocyte VEGF164 overexpression in adult mice is sufficient to induce glomerular filtration barrier structural and functional abnormalities similar to those present in murine diabetic nephropathy.

Topics: Age Factors; Animals; Autocrine Communication; Diabetic Nephropathies; Genotype; Glomerular Basement Membrane; Kidney Diseases; Matrix Metalloproteinase 9; Membrane Proteins; Mice; Mice, Transgenic; Paracrine Communication; Phenotype; Phosphorylation; Podocytes; Protein Binding; Proteinuria; Signal Transduction; Up-Regulation; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

Microalbuminuria is an early lesion during the development of diabetic nephropathy. The loss of high molecular weight proteins in the urine is usually associated with decreased expression of slit diaphragm proteins. Nephrin, is the major component of the glomerular slit diaphragm and loss of nephrin has been well described in rodent models of experimental diabetes as well as in human diabetic nephropathy.. In this manuscript we analyzed the role of PKC-alpha (PKCalpha) on endocytosis of nephrin in podocytes. We found that treatment of diabetic mice with a PKCalpha-inhibitor (GO6976) leads to preserved nephrin expression and reduced proteinuria. In vitro, we found that high glucose stimulation would induce PKCalpha protein expression in murine and human podocytes. We can demonstrate that PKCalpha mediates nephrin endocytosis in podocytes and that overexpression of PKCalpha leads to an augmented endocytosis response. After PKC-activation, we demonstrate an inducible association of PKCalpha, PICK1 and nephrin in podocytes. Moreover, we can demonstrate a strong induction of PKCalpha in podocytes of patients with diabetic nephropathy.. We therefore conclude that activation of PKCalpha is a pathomechanistic key event during the development of diabetic nephropathy. PKCalpha is involved in reduction of nephrin surface expression and therefore PKCalpha inhibition might be a novel target molecule for anti-proteinuric therapy.

Topics: Animals; Diabetes Mellitus; Diabetic Nephropathies; Endocytosis; Gene Expression Regulation, Enzymologic; Glucose; Humans; Membrane Proteins; Mice; Podocytes; Protein Kinase C-alpha

Rosiglitazone (Ros) has been shown to attenuate CXCL8 and ICAM-1 overexpression in renal tubular cells exposed to glycated albumin. The present study explores whether this can be translated into renoprotection in vivo. Uninephrectomized (Unx) type 2 diabetic db/db mice were chosen as a model of accelerated diabetic nephropathy.. Uninephrectomy was performed in 10-week-old db/db mice. They were then treated with vehicle, metformin or Ros for 8 weeks.. Unx-db/db mice treated with Ros had lower serum creatinine and albuminuria, less severe glomerulosclerosis, tubulointerstitial injury, fewer infiltrating macrophages, and less proliferating nuclear antigen-positive tubular cells compared with mice treated with metformin that had a similar level of glycemic control and insulin resistance. In addition, Ros but not metformin attenuated renal cortical expression of CCL2, MIP-2, and ICAM-1 and inhibited p-STAT1 signal activation. Ros also increased glomerular nephrin expression.. Our results delineated the biochemical and histologic characteristics of Unx-db/db mice and demonstrated the in vivo glucose-independent anti-inflammatory mechanisms of Ros in nephropathy of accelerated murine type 2 diabetes.

Topics: Animals; Anti-Inflammatory Agents; Cytokines; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Disease Models, Animal; Down-Regulation; Kidney; Kidney Glomerulus; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Nephrectomy; Protective Agents; Rosiglitazone; STAT1 Transcription Factor; Thiazolidinediones

The capacity of renin-angiotensin system (RAS) inhibitors to delay progression of diabetic nephropathy depends on the time at which therapy is started. A multimodal intervention is required to afford renoprotection in overt diabetic nephropathy. Here we assessed the effects of maximal RAS inhibition by angiotensin-converting enzyme (ACE) inhibitor plus angiotensin II type 1 receptor blocker (ARB) in combination with statin in rats with overt diabetic nephropathy. Uninephrectomized rats made diabetic by streptozotocin were orally treated from 4 (when proteinuria and renal lesions had developed) to 8 mo with vehicle, lisinopril plus candesartan, lisinopril plus candesartan plus rosuvastatin, or rosuvastatin alone. Systolic blood pressure increased in diabetic rats and was significantly lowered by combined therapies. Dual RAS blockade significantly reduced proteinuria compared with vehicle. Addition of statin further lowered proteinuria to control levels. Glomerulosclerosis was ameliorated by RAS inhibitors or statin, and regression was achieved by the addition of statin. Loss of podocytes of diabetic rats was limited by ACE inhibitor plus ARB while normalized by the three drugs. Defective nephrin expression of diabetes was increased by dual RAS blockade or statin and restored by the triple therapy. Tubular damage, interstitial inflammation, and expression of the fibrotic markers transforming growth factor (TGF)-β1 and phosphorylated Smad 2/3 in tubuli were significantly reduced by the triple regimen. These data suggest a strategy to target proteinuria to try to achieve regression of renal disease in diabetic patients who do not fully benefit from RAS inhibition alone.

Topics: Angiotensin II Type 1 Receptor Blockers; Angiotensin-Converting Enzyme Inhibitors; Animals; Blood Pressure; Cholesterol; Cholesterol Esters; Diabetic Nephropathies; Fibrosis; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Immunohistochemistry; Kidney; Kidney Function Tests; Kidney Glomerulus; Lipid Metabolism; Male; Membrane Proteins; Nephritis, Interstitial; Podocytes; Proteinuria; Rats; Rats, Sprague-Dawley; Renin-Angiotensin System

Renal inflammation and nephrin downregulation contribute to albuminuria in diabetes. We studied, in streptozotocin-induced diabetic rats, the effect of rosiglitazone (RSG), a peroxisome proliferator-activated receptor-γ agonist, on renal macrophage infiltration, MCP1, and nephrin expression in relation to albuminuria.. We investigated control and diabetic rats treated or untreated with RSG. Animals were sacrificed at 1, 3, and 9 months. Renal MCP1 and nephrin expression were studied by immunoblotting, renal macrophage infiltration by immunohistochemistry, and albuminuria by ELISA. Electron microscopy was used to assess glomerular ultrastructural morphology. In vitro experiments were conducted in isolated cultured rat glomeruli.. Glycaemic control was similar in diabetic rats treated and untreated with RSG, and blood pressure was comparable in all groups. RSG prevented diabetes-induced albuminuria at 9 months, and renal macrophage infiltration and MCP1 upregulation at 3 and 9 months. Diabetes-mediated nephrin downregulation was abolished by RSG. Diabetes-induced glomerulosclerosis, glomerular basement membrane thickening, and foot process fusion were not affected by RSG. In isolated glomeruli, MCP1 directly induced nephrin downregulation and this was prevented by RSG. RSG had no effect on nephrin expression.. RSG prevents albuminuria and nephrin downregulation in experimental diabetes independently of glycaemic and blood pressure control. This effect likely occurs via correction of diabetes-induced inflammatory processes.

Topics: Albuminuria; Animals; Cells, Cultured; Chemokine CCL2; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Hypoglycemic Agents; Kidney; Macrophages; Male; Membrane Proteins; PPAR gamma; Rats; Rats, Sprague-Dawley; Rosiglitazone; Thiazolidinediones

Development of diabetic nephropathy is accompanied by changes in integrin-mediated cell-matrix interactions. The α8-integrin chain is specifically expressed in mesangial cells of the glomerulus. During experimental hypertension, α8-integrin plays a protective role in the glomerulus. We hypothesized that α8-integrin is involved in maintaining the integrity of the glomerulus in diabetic nephropathy. Experimental streptozotocin (STZ) diabetes led to an increased expression and glomerular deposition of α8-integrin. To test the functional role of α8-integrin, STZ diabetes was induced in mice with a homozygous (α8-/-) or heterozygous (α8+/-) deletion of the α8-integrin gene and in wild-type litters (α8+/+). Blood glucose and mean arterial blood pressure were not different in α8-/- and α8+/+ mice after 6 wk of diabetes. However, diabetic α8-/- mice developed significantly higher albuminuria and more glomerulosclerosis than diabetic α8+/+ mice. Moreover, in diabetic α8-/- mice, the number of glomerular cells staining positive for the podocyte markers WT-1 and vimentin were reduced more prominently than in diabetic α8+/+. The filtration barrier protein nephrin was downregulated in diabetic glomeruli with the strongest reduction observed in α8-/- mice. Taken together, α8-/- mice developed more severe glomerular lesions and podocyte damage after onset of STZ diabetes than α8+/+ mice, indicating that α8-integrin is protective for the structure and function of the glomerulus and maintains podocyte integrity during the development of diabetic nephropathy.

Topics: Albuminuria; Animals; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Immunohistochemistry; Integrin alpha Chains; Kidney; Kidney Function Tests; Kidney Glomerulus; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Microdissection; Nephrectomy; Podocytes; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

High glucose levels can change podocyte gene expression and subsequently induce podocyte damage through altered glucose metabolism. l-Carnitine is known to play a beneficial role in diabetes; however, there are no studies on the effects of l-carnitine on podocyte alteration under high glucose conditions. This study investigated whether l-carnitine can attenuate diabetic podocyte injury through the prevention of loss of slit diaphragm proteins. The l-carnitine treatment group showed increased glucose uptakes compared to the control group, suggesting that glucose utilization in the podocytes was increased by l-carnitine. l-Carnitine treatment also prevented decreased mRNA expressions of nephrin and podocin in the high glucose-stimulated podocytes. However, mRNA expressions of CD2AP and α-actinin-4 were not significantly changed by the high glucose conditions. When these data are taken together, l-carnitine can increase glucose uptake in podocytes under high glucose conditions, and its mechanism may be at least partly related to the up-regulation of nephrin and podocin. Our results help clarify the beneficial effects of l-carnitine in diabetic nephropathy.

Topics: Animals; Carnitine; Cell Line; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Gene Expression Regulation; Glucose; Hyperglycemia; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Male; Membrane Proteins; Mice; Osmolar Concentration; Podocytes; Rats; Rats, Sprague-Dawley; RNA, Messenger

C-jun N-terminal kinase (JNK) regulates both the development of insulin resistance and inflammation. Podocytes of the widely used db/db mouse model of diabetic nephropathy lose their ability to respond to insulin as albuminuria develops, in comparison to control db/+ mice. Here we tested whether JNK inhibition or its gene deletion would prevent albuminuria in experimental diabetes. Phosphorylated/total JNK was significantly increased in vivo in glomeruli of db/db compared to db/+ mice. Treatment of podocytes isolated from these two strains of mice with tumor necrosis factor-alpha caused greater phosphorylation of JNK in those obtained from diabetic animals. When db/db mice were treated with a cell-permeable TAT-JNK inhibitor peptide, their insulin sensitivity and glycemia significantly improved compared to controls. We induced diabetes in JNK1 knockout mice with streptozotocin and found that they had significantly better insulin sensitivity compared to diabetic wild-type or JNK2 knockout mice. Albuminuria was, however, worse in all mice treated with the JNK inhibitor and in diabetic JNK2 knockout mice compared to controls. Nephrin expression was also reduced in JNK inhibitor-treated mice compared to controls. A similar degree of mesangial expansion was found in all diabetic mice. Our study shows that targeting JNK to improve systemic insulin sensitivity does not necessarily prevent diabetic nephropathy.

Topics: Albuminuria; Animals; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Genotype; Hyperglycemia; Insulin; Insulin Resistance; JNK Mitogen-Activated Protein Kinases; Membrane Proteins; Mice; Mice, Knockout; Protein Kinase Inhibitors

Omega-3 polyunsaturated fatty acids (n-3 PUFA) show beneficial effects in cardiovascular disease, IgA, and diabetic nephropathy; however, the mechanisms underlying these benefits are unknown. The study was performed in male Sprague-Dawley rats randomly divided into four treatment groups: nondiabetic (ND), streptozotocin-induced diabetic (D), diabetic and fed a high n-3 PUFA diet (D+canola), and diabetic and fed a high n-6 (omega-6) PUFA diet (D+corn). Study treatments were carried out for 30 wk. D+canola significantly decreased diabetes-associated increases in urine albumin excretion (ND 17.8 +/- 6.4; D 97.3 +/- 9.4; D+canola 8.3 +/- 2.2 mg/day); systolic blood pressure (ND 153 +/- 9; D 198 +/- 7; D+canola 162 +/- 9 mmHg); glomerulosclerosis (ND 0.6 +/- 0.2; D 1.8 +/- 0.2; D+canola 0.8 +/- 0.1 AU); and tubulointerstitial fibrosis in the renal cortex (ND 1.2 +/- 0.2; D 2.0 +/- 0.2; D+canola 1.1 +/- 0.1) and the inner stripe of the outer medulla (ND 1.0 +/- 0.2; D 2.1 +/- 0.2; D+canola 1.1 +/- 0.2 AU). D+corn also exerted renoprotection, but not to the same degree as D+canola (urine albumin excretion, 33.8 +/- 6.1 mg/day; systolic blood pressure, D+corn 177 +/- 6 mmHg; glomerulosclerosis, D+corn 1.2 +/- 0.3 AU; cortical tubulointerstitial fibrosis, D+corn 1.6 +/- 0.1 AU; medullary tubulointerstitial fibrosis, D+corn 1.5 +/- 0.1 AU). In addition, D+canola attenuated D-associated increase in collagen type I and type IV, IL-6, MCP-1, transforming growth factor-beta, and CD68 expression. These observations indicate a beneficial effect of high dietary intake of n-3 PUFA in reducing diabetic renal disease.

Topics: Albuminuria; Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Blood Glucose; Blood Pressure; Body Weight; Chemokine CCL2; Collagen Type I; Collagen Type IV; Creatinine; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Fatty Acids, Monounsaturated; Fatty Acids, Omega-3; Fibrosis; Interleukin-6; Intermediate Filament Proteins; Kidney; Male; Membrane Proteins; Nerve Tissue Proteins; Nestin; Organ Size; Rapeseed Oil; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta

Diabetic nephropathy is characterized by decreased expression of bone morphogenetic protein-7 (BMP-7) and decreased podocyte number and differentiation. Extracellular antagonists such as connective tissue growth factor (CTGF; CCN-2) and sclerostin domain-containing-1 (SOSTDC1; USAG-1) are important determinants of BMP signaling activity in glomeruli. We studied BMP signaling activity in glomeruli from diabetic patients and non-diabetic individuals and from control and diabetic CTGF(+/+) and CTGF(+/-) mice. BMP signaling activity was visualized by phosphorylated Smad1, -5, and -8 (pSmad1/5/8) immunostaining, and related to expression of CTGF, SOSTDC1, and the podocyte differentiation markers WT1, synaptopodin, and nephrin. In control and diabetic glomeruli, pSmad1/5/8 was mainly localized in podocytes, but both number of positive cells and staining intensity were decreased in diabetes. Nephrin and synaptopodin were decreased in diabetic glomeruli. Decrease of pSmad1/5/8 was only partially explained by decrease in podocyte number. SOSTDC1 and CTGF were expressed exclusively in podocytes. In diabetic glomeruli, SOSTDC1 decreased in parallel with podocyte number, whereas CTGF was strongly increased. In diabetic CTGF(+/-) mice, pSmad1/5/8 was preserved, compared with diabetic CTGF(+/+) mice. In conclusion, in human diabetic nephropathy, BMP signaling activity is diminished, together with reduction of podocyte markers. This might relate to concomitant overexpression of CTGF but not SOSTDC1.

Topics: Adaptor Proteins, Signal Transducing; Aged; Animals; Antigens, Differentiation; Bone Morphogenetic Proteins; Cell Count; Cell Differentiation; Connective Tissue Growth Factor; Diabetic Nephropathies; Female; Humans; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Male; Membrane Proteins; Mice; Mice, Transgenic; Microfilament Proteins; Middle Aged; Podocytes; Proteins; Signal Transduction

Monocyte chemoattractant protein-1 (MCP-1), a chemokine binding to the CC chemokine receptor 2 (CCR2) and promoting monocyte infiltration, has been implicated in the pathogenesis of diabetic nephropathy. To assess the potential relevance of the MCP-1/CCR2 system in the pathogenesis of diabetic proteinuria, we studied in vitro if MCP-1 binding to the CCR2 receptor modulates nephrin expression in cultured podocytes. Moreover, we investigated in vivo if glomerular CCR2 expression is altered in kidney biopsies from patients with diabetic nephropathy and whether lack of MCP-1 affects proteinuria and expression of nephrin in experimental diabetes.. Expression of nephrin was assessed in human podocytes exposed to rh-MCP-1 by immunofluorescence and real-time PCR. Glomerular CCR2 expression was studied in 10 kidney sections from patients with overt nephropathy and eight control subjects by immunohistochemistry. Both wild-type and MCP-1 knockout mice were made diabetic with streptozotocin. Ten weeks after the onset of diabetes, albuminuria and expression of nephrin, synaptopodin, and zonula occludens-1 were examined by immunofluorescence and immunoblotting.. In human podocytes, MCP-1 binding to the CCR2 receptor induced a significant reduction in nephrin both mRNA and protein expression via a Rho-dependent mechanism. The MCP-1 receptor, CCR2, was overexpressed in the glomerular podocytes of patients with overt nephropathy. In experimental diabetes, MCP-1 was overexpressed within the glomeruli and the absence of MCP-1 reduced both albuminuria and downregulation of nephrin and synaptopodin.. These findings suggest that the MCP-1/CCR2 system may be relevant in the pathogenesis of proteinuria in diabetes.

Topics: Animals; Biopsy; Cells, Cultured; Chemokine CCL2; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Down-Regulation; Humans; In Vitro Techniques; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Microfilament Proteins; Phosphoproteins; Podocytes; Proteinuria; Recombinant Proteins; rho-Associated Kinases; RNA, Messenger; Zonula Occludens-1 Protein

Podocyte dysfunction, one of the major causes of proteinuria, leads to glomerulosclerosis and end stage renal disease, but its underlying mechanism remains poorly understood. Here we show that Wnt/beta-catenin signaling plays a critical role in podocyte injury and proteinuria. Treatment with adriamycin induced Wnt and activated beta-catenin in mouse podocytes. Overexpression of Wnt1 in vivo activated glomerular beta-catenin and aggravated albuminuria and adriamycin-induced suppression of nephrin expression, whereas blockade of Wnt signaling with Dickkopf-1 ameliorated podocyte lesions. Podocyte-specific knockout of beta-catenin protected against development of albuminuria after injury. Moreover, pharmacologic activation of beta-catenin induced albuminuria in wild-type mice but not in beta-catenin-knockout littermates. In human proteinuric kidney diseases such as diabetic nephropathy and focal segmental glomerulosclerosis, we observed upregulation of Wnt1 and active beta-catenin in podocytes. Ectopic expression of either Wnt1 or stabilized beta-catenin in vitro induced the transcription factor Snail and suppressed nephrin expression, leading to podocyte dysfunction. These results suggest that targeting hyperactive Wnt/beta-catenin signaling may represent a novel therapeutic strategy for proteinuric kidney diseases.

Topics: Albuminuria; Animals; beta Catenin; Biopsy; Cell Line, Transformed; Diabetic Nephropathies; Glomerulosclerosis, Focal Segmental; Humans; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Podocytes; Signal Transduction; Snail Family Transcription Factors; Transcription Factors; Up-Regulation; Wnt1 Protein

We present the analysis of an intronic polymorphism of the nephrin gene and its relationship to the development of diabetic nephropathy in a study of diabetes type 1 and type 2 patients. The frequency of the single nucleotide polymorphism rs#466452 in the nephrin gene was determined in 231 patients and control subjects. The C/T status of the polymorphism was assessed using restriction enzyme digestions and the nephrin transcript from a kidney biopsy was examined. Association between the polymorphism and clinical parameters was evaluated using multivariate correspondence analysis. A bioinformatics analysis of the single nucleotide polymorphism rs#466452 suggested the appearance of a splicing enhancer sequence in intron 24 of the nephrin gene and a modification of proteins that bind to this sequence. However, no change in the splicing of a nephrin transcript from a renal biopsy was found. No association was found between the polymorphism and diabetes or degree of renal damage in diabetes type 1 or 2 patients. The single nucleotide polymorphism rs#466452 of the nephrin gene seems to be neutral in relation to diabetes and the development of diabetic nephropathy, and does not affect the splicing of a nephrin transcript, in spite of a splicing enhancer site.

Topics: Adult; Aged; Biopsy; Case-Control Studies; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Female; Genotype; Humans; Introns; Male; Membrane Proteins; Middle Aged; Multivariate Analysis; Polymorphism, Single Nucleotide; Reverse Transcriptase Polymerase Chain Reaction; RNA Splicing; Transcription, Genetic

Podocyte-specific, doxycycline (DOX)-inducible overexpression of soluble vascular endothelial growth factor (VEGF) receptor-1 (sFlt-1) in adult mice was used to investigate the role of the VEGF-A/VEGF receptor (VEGFR) system in diabetic glomerulopathy.. We studied nondiabetic and diabetic transgenic mice and wild-type controls treated with vehicle (VEH) or DOX for 10 weeks. Glycemia was measured by a glucose-oxidase method and blood pressure by a noninvasive technique. sFlt-1, VEGF-A, VEGFR2, and nephrin protein expression in renal cortex were determined by Western immunoblotting; urine sFlt-1, urine free VEGF-A, and albuminuria by enzyme-linked immunosorbent assay; glomerular ultrastructure by electron microscopy; and VEGFR1 and VEGFR2 cellular localization with Immunogold techniques.. Nondiabetic DOX-treated transgenic mice showed a twofold increase in cortex sFlt-1 expression and a fourfold increase in sFlt-1 urine excretion (P < 0.001). Urine free VEGF-A was decreased by 50%, and cortex VEGF-A expression was upregulated by 30% (P < 0.04). VEGFR2 expression was unchanged, whereas its activation was reduced in DOX-treated transgenic mice (P < 0.02). Albuminuria and glomerular morphology were similar among groups. DOX-treated transgenic diabetic mice showed a 60% increase in 24-h urine sFlt-1 excretion and an approximately 70% decrease in urine free VEGF-A compared with VEH-treated diabetic mice (P < 0.04) and had lower urine albumin excretion at 10 weeks than VEH-treated diabetic (d) mice: d-VEH vs. d-DOX, geometric mean (95% CI), 117.5 (69-199) vs. 43 (26.8-69) mug/24 h (P = 0.003). Diabetes-induced mesangial expansion, glomerular basement membrane thickening, podocyte foot-process fusion, and transforming growth factor-beta1 expression were ameliorated in DOX-treated diabetic animals (P < 0.05). Diabetes-induced VEGF-A and nephrin expression were not affected in DOX-treated mice.. Podocyte-specific sFlt-1 overexpression ameliorates diabetic glomerular injury, implicating VEGF-A in the pathogenesis of this complication.

Topics: Animals; Blotting, Western; Diabetes Complications; Diabetes Mellitus; Diabetic Nephropathies; Doxycycline; Enzyme-Linked Immunosorbent Assay; Gene Expression; Humans; Kidney Glomerulus; Membrane Proteins; Mice; Mice, Transgenic; Microscopy, Electron; Podocytes; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factor Receptor-2

Several works in the setting of early experimental diabetic nephropathy using anti-inflammatory drugs, such as mycophenolate mofetil (MMF), have shown that prevention of the development or amelioration of renal injury including proteinuria. The exact mechanisms by which anti-inflammatory drugs lower the albuminuria have no still to clarify well. In this study, diabetes was induced by injection of streptozotocin after uninephrectomy. Rats were randomly divided into three groups: control group, diabetic group and diabetic group treated with MMF. Elevated 24h urinary albumin excretion rate was markedly attenuated by MMF treatment. In diabetic rats receiving no treatment, there were increase in ED-1+ cells in the glomeruli, which were effectively suppressed by MMF treatment. The expression of nephrin and podocin protein was reduced in the glomeruli from diabetic rats, and MMF treatment significantly increased the expression of nephrin and podocin. The expression of IL-1, TNF-alpha and 3-NT protein in the glomeruli were significantly increased in diabetic rats, which were all significantly inhibited by MMF treatment. Our results show that MMF could decrease urinary albumin excretion, which mechanism may be at least partly correlated with upregulated expression of nephrin and podocin in the glomeruli of diabetic rat.

Topics: Albuminuria; Animals; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Interleukin-1; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Male; Membrane Proteins; Mycophenolic Acid; Rats; Tumor Necrosis Factor-alpha

The protein kinase C (PKC)-beta isoform has been implicated to play a pivotal role in the development of diabetic kidney disease. We tested this hypothesis by inducing diabetic nephropathy in PKC-beta-deficient (PKC-beta(-/-)) mice. We studied nondiabetic and streptozotocin-induced diabetic PKC-beta(-/-) mice compared with appropriate 129/SV wild-type mice. After 8 weeks of diabetes, the high-glucose-induced renal and glomerular hypertrophy, as well as the increased expression of extracellular matrix proteins such as collagen and fibronectin, was reduced in PKC-beta(-/-) mice. Furthermore, the high-glucose-induced expression of the profibrotic cytokine transforming growth factor (TGF)-beta1 and connective tissue growth factor were significantly diminished in the diabetic PKC-beta(-/-) mice compared with diabetic wild-type mice, suggesting a role of the PKC-beta isoform in the regulation of renal hypertrophy. Notably, increased urinary albumin-to-creatinine ratio persisted in the diabetic PKC-beta(-/-) mice. The loss of the basement membrane proteoglycan perlecan and the podocyte protein nephrin in the diabetic state was not prevented in the PKC-beta(-/-) mice as previously demonstrated in the nonalbuminuric diabetic PKC-alpha(-/-) mice. In summary, the differential effects of PKC-beta deficiency on diabetes-induced renal hypertrophy and albuminuria suggest that PKC-beta contributes to high-glucose-induced TGF-beta1 expression and renal fibrosis, whereas perlecan, as well as nephrin, expression and albuminuria is regulated by other signaling pathways.

Topics: Albuminuria; Animals; Chromosome Deletion; Collagen Type IV; Creatinine; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Fibronectins; Fibrosis; Heparan Sulfate Proteoglycans; Hypertrophy; Kidney; Membrane Proteins; Mice; Mice, Knockout; Organ Size; Protein Isoforms; Protein Kinase C; RNA; Streptozocin; Transforming Growth Factor beta1; Vascular Endothelial Growth Factors

To investigate nephrin and desmin expression in rat podocytes in early diabetic nephropathy (DN) and the rale of angiotensin II receptor antagonist in renal protection.. Rat models of DN established by a injection of a single dose of streptozotocin (STZ) were randomized into model group and irbesartan group, with rats without STZ injection as the normal control group. The rats in irbesartan group were subjected to daily intragastric irbesartan administration for 8 consecutive weeks, while those in the model group received only saline in the same manner. Upon completion of the treatment, the rats were sacrificed and pathological changes of the kidney were examined with optical and transmission electron microscope. Nephrin and desmin expressions in the podocytes were detected by immunohistochemistry.. In rats with DN, irbesartan administration alleviated podocyte injury and significantly lowered the expression of nephrin and desmin (P<0.05).. Angiotensin II receptor antagonist may offer renal protection against DN by alleviating structural and functional podocyte damage through decreasing nephrin expression in the podocytes.

Topics: Angiotensin II Type 1 Receptor Blockers; Animals; Biphenyl Compounds; Desmin; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Irbesartan; Kidney; Membrane Proteins; Podocytes; Rats; Rats, Sprague-Dawley; Tetrazoles

Diabetic nephropathy (DN) is clinically characterized by proteinuria. Many studies tried to demonstrate a relationship between proteinuria and changes in nephrin in various forms of glomerular diseases including DN, but the results are not consistent. Glomerular hypertrophy occurs in DN, yet hypertrophy does not develop in all glomeruli concurrently. For investigation of the differences in nephrin expression according to glomerular size, glomeruli were isolated from 10 control and 10 streptozotocin-induced diabetic rats at 6 wk after the induction of diabetes by a sieving technique using sieves with pore sizes of 250, 150, 125, and 75 microm. Glomeruli then were classified into large glomeruli (LG; on the 125-microm sieve) and small glomeruli (SG; on the 75-microm sieve) groups. Glomerular volumes were determined using an image analyzer, and mRNA and protein expression was determined by real-time PCR and Western blot, respectively. The mean volumes of diabetic LG (1.51 +/- 0.06 x 10(6) microm(3)) and control LG (1.37 +/- 0.05 x 10(6) microm(3)) were significantly higher than those of diabetic SG (0.94 +/- 0.03 x 10(6) microm(3)) and control SG (0.87 +/- 0.03 x 10(6) microm(3); P < 0.01). Nephrin mRNA expression was significantly reduced in the diabetic LG group compared with the diabetic SG and control glomeruli groups (P < 0.05). In contrast, nephrin mRNA expression was significantly higher in the diabetic SG group compared with the diabetic LG and control glomeruli groups (P < 0.05). Even after correction for 18s rRNA and Wilms' tumor-1 mRNA expression, the differences in nephrin mRNA expression remained significant. The expression of nephrin protein showed a similar pattern to the mRNA expression. In conclusion, these data suggest that the nephrin gene is differentially expressed according to glomerular size. Furthermore, more hypertrophied glomeruli with lesser nephrin expression may be responsible for albuminuria in the early stage of DN.

Topics: Albuminuria; Animals; Diabetic Nephropathies; Disease Progression; Early Diagnosis; Gene Expression Regulation; Kidney Glomerulus; Male; Membrane Proteins; Microscopy, Electron; Podocytes; Rats; Rats, Sprague-Dawley; RNA, Messenger

Podocyte has inflammatory role in the development of diabetic nephropathy (DN). Mycophenolate mofetil (MMF), an anti-inflammatory agent, can suppress macrophage infiltration and reduce renal injury in streptozotocin-induced diabetic rats. Angiotensin II receptor blocker (ARB), another renal protecting agent, can decrease podocyte loss in DN. In this study, we detected the expression levels of monocyte chemoattractant protein-1 (MCP-1) and nephrin to evaluate podocyte's role in inflammatory reaction in DN, observe and compare the effect of MMF alone and in combination with valsartan, on preventing podocyte loss in streptozotocin (STZ) induced diabetic rats.. Diabetic model was constructed in uninephrectomized male Wistar rats by single peritoneal injection of STZ (65 mg/kg). The successfully induced diabetic rats were randomly divided into four groups: diabetes without treatment group (DM), valsartan treated group (DMV), MMF treated group (DMM), and combined therapy group (DMVM). Normal rats of the same sibling were chosen as control (NC). At the end of the 8th week, serum biochemistry, 24-hour urinary protein (UP) and the ratio of kidney weight/body weight (RWK/B) were measured. The rats were sacrificed for the observation of renal histomorphology through light and electron microscope. Nephrin, desmin and MCP-1 levels were detected by semi-quantitative immunohistochemical assays. Real-time quantitative PCR was used to detect the mRNA levels of nephrin and MCP-1.. Compared with group NC, serum glucose level, 24-hour UP and RWK/B in group DM were significantly higher (P < 0.01), and the nephrin mRNA level in DM group was significantly lower (P < 0.05). The nephrin mRNA expression levels in group DMV, DMM and DMVM were all higher than that of DM group (P < 0.05) and no significant differences were found among the three treatment groups (P > 0.05). Treatment with MMF, valsartan or their combination could significantly decrease the 24-hour UP and RWK/B, and suppress glomerulosclerosis and interstitial fibrotic lesions in diabetic rats. In diabetic rats, the high expressions of desmin and MCP-1 in kidney were suppressed by valsartan, MMF or their combination.. Podocytes are involved in the inflammatory reaction of diabetic rats. MMF could suppress MCP-1 and desmin expression, enhance nephrin expression, and attenuate proteinuria in diabetic rats. The combined therapy of valsartan and MMF did not show any superiority over monotherapies on renal protection. MMF may have renoprotective effect in early stages of diabetic nephropathy through preventing podocytes loss and anti-inflammatory activity.

Topics: Animals; Chemokine CCL2; Desmin; Diabetic Nephropathies; Drug Therapy, Combination; Immunohistochemistry; Male; Membrane Proteins; Mycophenolic Acid; Podocytes; Rats; Rats, Wistar; Tetrazoles; Valine; Valsartan

To investigate the effect of podocyte ultrastructure and changes of nephrin and the podocin expression on the pathogenesis of diabetic nephropathy (DN) in rats.. Twenty Wistar rats were divided into 2 groups: a normal control group and a DN model group.We determined the 24h proteinuria and other biochemical indexes, measured the ultrastructures of podocytes with electronic microscope, and detected the expression of nephrin and podocin with immunohistochemical technique and RT-PCR at the 4th and 8th weeks.. The 24h proteinuria increased in the DN group; the number of podocytes was significantly lower; and the foot process width (FPW) obviously increased in the DN group compared with the normal group (P<0.01). The protein and mRNA expressions of nephrin and podocin reduced in the DN group.There was a negative correlation between the proteinuria and the protein expression of nephrin and podocin (P<0.01).. The reduction in glomerular podocyte number,the increased FPW, and the down-regulated expression of nephrin and podocin appear at the early stage of DN and become more serious with the disease progression.The podocyte lesion not only is associated with the degree of proteinuria,but also correlates with the development of glomerulosclerosis and damage of renal function.

Topics: Animals; Diabetic Nephropathies; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Podocytes; Proteinuria; Rats; Rats, Wistar

Diabetic nephropathy (DN) is one of the major complications of type 2 diabetes and is associated with coronary disease. Nephrin, a protein mainly expressed in glomeruli, is decreased in DN and other kidney diseases. Since insulin levels are misregulated in type 2 diabetes, a possible connection between DN and its decreased nephrin expression could be the presence of regulatory elements responsive to insulin in the nephrin gene (NPHS1) promoter region. In this work, using bioinformatic tools, we identified a purine-rich GAGA element in the nephrin gene promoter and conducted a genomic study in search of the presence of polymorphisms in this element and its possible association with DN in type 2 diabetic patients. We amplified and sequenced a 514 bp promoter region of 100 individuals and found no genetic variants in the purine-rich GAGA-box of the nephrin gene promoter between groups of patients with diabetes type 2 with and without renal and coronary complications, control patients without diabetes and healthy controls.

Topics: Adult; Aged; Case-Control Studies; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Female; Genetic Markers; Genetic Predisposition to Disease; Genotype; Humans; Male; Membrane Proteins; Middle Aged; Pilot Projects; Polymorphism, Genetic; Promoter Regions, Genetic

Albuminuria in diabetic nephropathy is due to endothelial dysfunction, a loss of negative charges in the basement membrane, and changes a of the slit-membrane diaphragm composition. We have recently shown that protein kinase C alpha (PKCalpha)-deficient mice are protected against the development of albuminuria under diabetic conditions. We here tested the hypothesis that PKCalpha mediates the hyperglycemia-induced downregulation of the slit-diaphragm protein nephrin. After 8 weeks of streptozotocin (STZ)-induced hyperglycemia the expression of glomerular nephrin was significantly reduced. In contrast, other slit-diaphragm proteins such as podocin and CD2AP were unaltered in diabetic state. In PKCalpha-/- mice, hyperglycemia-induced downregulation of nephrin was prevented. Podocin and CD2AP remained unchanged. In addition, the nephrin messenger RNA expression was also reduced in hyperglycemic wild-type mice but remained unaltered in PKCalpha-/- mice. We postulate that the underlying mechanism of the hyperglycemia-induced regulation of various proteins of the glomerular filtration barrier is a PKCalpha-dependent regulation of the Wilms' Tumor Suppressor (WT1) which previously has been shown to act as a direct transcription factor on the nephrin promoter. Our data suggest that PKCalpha activation may be an important intracellular signaling pathway in the regulation of nephrin expression and glomerular albumin permeability in the diabetic state.

Topics: Adaptor Proteins, Signal Transducing; Albuminuria; Animals; Cytoskeletal Proteins; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Gene Expression Regulation; Humans; Hyperglycemia; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Male; Membrane Proteins; Mice; Mice, Knockout; Protein Kinase C-alpha; RNA, Messenger; Signal Transduction; WT1 Proteins

Podocyte injury is associated with many forms of human and experimental proteinuric glomerular disease. The aim of this study was to investigate the level of nephrin expression in a model of obesity and type II diabetes mellitus, the obese Zucker rat, as well as to investigate whether nephrin expression is influenced by treatment with quinapril or diltiazem, 2 drugs frequently used in type II diabetes mellitus.. Obese Zucker rats were treated with either quinapril or diltiazem at a dose of 10 mg/kg body weight per day and 100 mg/kg body weight per day, respectively, for 6 months. Real time reverse transcription-polymerase chain reaction (RT-PCR) and immunoperoxidase assays were used to assess and quantify nephrin gene expression and other markers of podocyte damage, such as desmin and synaptopodin protein.. Quinapril treatment prevented the reduction of nephrin levels compared with the control group, while diltiazem treatment did not prevent the reduction. Similar results were obtained when other phenotypic markers, such as desmin, were assessed. Similarly, synaptodin showed this tendency, although it did not achieve statistically significant differences.. The podocyte phenotypic changes assessed in a model of obesity and type II diabetes mellitus were corrected by an angiotensin-converting enzyme (ACE) inhibitor. These results could be associated with an improvement in the slit diaphragm, and therefore, in the maintenance of the filtration barrier. Diltiazem did not achieve similar results.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Blood Pressure; Calcium Channel Blockers; Desmin; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Diltiazem; DNA, Complementary; Immunoenzyme Techniques; Male; Membrane Proteins; Microfilament Proteins; Obesity; Proteins; Quinapril; Rats; Rats, Zucker; Reverse Transcriptase Polymerase Chain Reaction; RNA; Tetrahydroisoquinolines

Albumin modified by Amadori-glucose adducts has been linked to the development of diabetic nephropathy through its ability, independent of hyperglycemia, to activate protein kinase C-beta (PKC-beta), up-regulate the transforming growth factor-beta (TGF-beta) system, and stimulate expression of extracellular matrix proteins in glomerular cells, and by the demonstration that reducing the burden of glycated albumin ameliorates renal structural and functional abnormalities in the db/db mouse.. To probe whether the salutary effects consequent to lowering glycated albumin, which include reduction of albuminuria, relate to an influence of the Amadori-modified protein on nephrin, the podocyte protein critical to regulation of protein excretion, and on the angiogenic vascular endothelial growth factor (VEGF), which induces microvascular permeability, diabetic db/db mice were treated with a small molecule that inhibits the nonenzymatic glycation of albumin.. Compared to nondiabetic db/m mice, diabetic controls exhibited increased urinary excretion of albumin and type IV collagen, elevated renal TGF-beta1 protein levels, reduced glomerular nephrin immunofluorescence and nephrin protein by immunoblotting, and increased glomerular VEGF immunostaining and renal VEGF protein content. Diabetic animals receiving test compound showed significant lowering of proteinuria, normalization of renal TGF-beta1 protein, and significant restoration of altered glomerular nephrin and VEGF expression.. The findings causally implicate the increased glycated albumin associated with the diabetic state in the abnormal renal nephrin and VEGF expression found in diabetes, thereby promoting proteinuria and glomerulosclerosis.

Topics: Albuminuria; Animals; Collagen Type IV; Diabetic Nephropathies; Fluorescent Antibody Technique; Glycated Serum Albumin; Glycation End Products, Advanced; Kidney Glomerulus; Male; Membrane Proteins; Mice; Mice, Mutant Strains; Proteinuria; Serum Albumin; Transforming Growth Factor beta; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A

Diabetic nephropathy is one of the major microvascular complications in diabetes and is the leading cause of end-stage renal disease worldwide. Among various factors, angiogenesis-associated factors such as vascular endothelial growth factor (VEGF)-A and angiopoietin (Ang)-2 are involved in the development of diabetic nephropathy. We previously reported the therapeutic efficacy of antiangiogenic tumstatin peptide in the early diabetic nephropathy model. Here, we examine the effect of endostatin peptide, a potent inhibitor of angiogenesis derived from type XVIII collagen, in preventing progression in the type 1 diabetic nephropathy mouse model. Endostatin peptide did not affect hyperglycemia induced by streptozotocin (STZ). Glomerular hypertrophy, hyperfiltration, and albuminuria were significantly suppressed by endostatin peptide (5 mg/kg) in STZ-induced diabetic mice. Glomerular mesangial matrix expansion, the increase of glomerular type IV collagen, endothelial area (CD31(+)), and F4/80(+) monocyte/macrophage accumulation were significantly inhibited by endostatin peptide. Increase in the renal expression of VEGF-A, flk-1, Ang-2, an antagonist of angiopoietin-1, transforming growth factor-beta1, interleukin-6, and monocyte chemoattractant protein-1 was inhibited by endostatin peptide in diabetic mice. Decrease of nephrin mRNA and protein in diabetic mice was suppressed by treatment with endostatin peptide. The level of endostatin in the renal cortex and sera was increased in diabetic mice. Endogenous renal levels of endostatin were decreased in endostatin peptide-treated groups in parallel with VEGF-A. Although serum levels of endostatin were decreased in the low-dose endostatin-peptide group, high-dose administration resulted in elevated serum levels of endostatin. These results demonstrate the potential use of antiangiogenic endostatin peptide as a novel therapeutic agent in diabetic nephropathy.

Topics: Albuminuria; Amino Acid Sequence; Animals; Blood Glucose; Body Weight; Collagen Type IV; Creatinine; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Endostatins; Female; Hyperglycemia; Hypertrophy; Immunohistochemistry; Integrin alpha5beta1; Kidney; Kidney Glomerulus; Membrane Proteins; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Organ Size; Peptide Fragments; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

Diabetic nephropathy (DN) is associated with functional changes in the filtration barrier, and microalbuminuria is a strong predictor of the development of overt DN. Nephrin is a novel podocyte-specific protein which localizes at the slit diaphragm. This study examines the expression of nephrin mRNA in the kidneys of type 2 diabetics with DN.. Renal tissues were obtained from 13 type 2 diabetics with DN. We also examined samples from five patients with minimal change nephrotic syndrome (MCNS) and five normal kidneys (normals) as control. The severity of DN was classified into two grades based on histopathological findings. DN grade 1 (DN1 = seven patients) presented mild mesangial expansion, and DN grade 2 (DN2 = six patients) moderate mesangial expansion. Nephrin mRNA was quantitated and localized by in situ hybridization.. Cells positive for nephrin mRNA were detected exclusively in glomerular epithelial cells. The percentage of cells positive for nephrin mRNA in DN2 was significantly lower than in MCNS and normal kidneys. Furthermore, there was an inverse correlation between the percentage of cells positive for nephrin mRNA and extent of proteinuria.. The low expression of nephrin mRNA may be closely linked to development and/or progression of proteinuria in human diabetic nephropathy.

Topics: Adult; Biopsy, Needle; Case-Control Studies; Cohort Studies; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Disease Progression; Female; Gene Expression Regulation; Genetic Markers; Humans; Immunohistochemistry; In Situ Hybridization; Kidney Function Tests; Male; Membrane Proteins; Middle Aged; Probability; Prognosis; Proteins; RNA, Messenger; Sensitivity and Specificity; Severity of Illness Index; Statistics, Nonparametric

Diabetic nephropathy (DN) is a frequent complication in patients with diabetes mellitus. To find improved intervention strategies in this disease, it is necessary to investigate the molecular mechanisms involved. To obtain more insight into processes that lead to DN, messenger RNA expression profiles of diabetic glomeruli and glomeruli from healthy individuals were compared.. Two morphologically normal kidneys and 2 kidneys from patients with DN were used for the study. Glomerular RNA was hybridized in duplicate on Human Genome U95Av2 Arrays (Affymetrix, Santa Clara, CA). Several transcripts were tested further in independent patient groups and at the protein level by immunohistochemistry.. Ninety-six genes were upregulated in diabetic glomeruli, whereas 519 genes were downregulated. The list of overexpressed genes in DN includes aquaporin 1, calpain 3, hyaluronoglucosidase, and platelet/endothelial cell adhesion molecule. The list of downregulated genes includes bone morphogenetic protein 2, vascular endothelial growth factor (VEGF), fibroblast growth factor 1, insulin-like growth factor binding protein 2, and nephrin. A decrease in VEGF and nephrin could be validated at the protein level and also at the RNA level in renal biopsy specimens from 5 additional patients with diabetes.. Results of oligonucleotide microarray analyses on control and diabetic glomeruli are presented and discussed in their relation to vascular damage, mesangial matrix expansion, proliferation, and proteinuria. Our findings suggest that progression of DN might result from diminished tissue repair capability.

Topics: Adult; Aged; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Gene Expression Profiling; Humans; Immunohistochemistry; Kidney Glomerulus; Male; Membrane Proteins; Middle Aged; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; Proteins; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A

Recent disclosure of podocyte proteins has unraveled previously rather mysterious mechanisms that govern glomerular perm-selectivity in health and disease. Here we addressed the role of nephrin, CD2-associated protein (CD2AP), and podocin together with the integrity of the slit diaphragm in the pathogenesis of proteinuria of patients with diabetes and nephropathy.. Nephrin mRNA and protein expression were evaluated in parallel in adult diabetic patients by in situ hybridization and immunohistochemistry. For comparison, nondiabetic patients with minimal change nephrosis and normal control patients were evaluated. CD2AP and podocin expression by immunohistochemistry was also assessed. The filtration slit was analyzed by morphometry and transmission electron microscopy.. Extracellular nephrin mRNA and protein were markedly reduced in diabetic patients. No changes were found in patients with minimal change versus controls. CD2AP and podocin were comparable in all subjects. Ultrastructural analysis showed in diabetic patients a remarkable reduction in the percentage of electron dense slit diaphragms, despite a frequency of the filtration slits comparable to control patients.. Down-regulation of nephrin and loss of the electron dense structure of slit diaphragm indicate a novel mechanism accounting for proteinuria in diabetic nephropathy. To the extent that glomerular protein trafficking contributes to renal disease progression, our findings may have clinical relevance. Reduction of nephrin in the context of normal expression of CD2AP and podocin can be taken reasonably as a specific marker of renal disease in diabetes. Therapies targeted at correcting podocyte nephrin might be of value for diabetic medicine.

Topics: Adaptor Proteins, Signal Transducing; Adult; Case-Control Studies; Cytoskeletal Proteins; Diabetic Nephropathies; Female; Gene Expression; Humans; Immunohistochemistry; In Situ Hybridization; Intracellular Signaling Peptides and Proteins; Kidney; Male; Membrane Proteins; Middle Aged; Proteins; RNA, Messenger

To explore the effects of various antihypertensive regimes which achieve similar blood pressure control using a range of agents including the angiotensin II type 1 receptor antagonist, valsartan, as monotherapy or in combination with two subclasses of calcium channel blockers (CCBs) (the dihydropyridine, amlodipine and the phenylalkylamine, verapamil) on the progression of renal disease and the expression of the podocyte slit pore protein, nephrin in an accelerated model of diabetic nephropathy.. Valsartan treatment reduced systolic blood pressure as assessed by radiotelemetry (135 +/- 3 versus diabetic 153 +/- 6 mmHg) as well as retarding the increase in albumin excretion rate by approximately 50%. Combination therapy of valsartan with either amlodipine or verapamil was equally effective in reducing blood pressure to valsartan monotherapy (valsartan + amlodipine 129 +/- 4 valsartan + verapamil 133 +/- 6 mmHg;) but was not as effective at reducing albuminuria. A reduction in glomerulosclerosis was observed with valsartan monotherapy with less reduction in injury with the valsartan + amlodipine combination, despite a similar reduction in blood pressure. The decrease in nephrin, in diabetic rats was attenuated by valsartan monotherapy, but not by other treatments.. The results of this study demonstrate that despite a similar reduction in blood pressure, the addition of the CCB amlodipine to the AII antagonist failed to provide similar renoprotection to that observed with an equihypotensive regimen of valsartan as monotherapy. Furthermore, the depletion in glomerular nephrin expression in diabetic animals was only abrogated by valsartan treatment, the therapy which was most effective at retarding the development of albuminuria in this model.

Topics: Albuminuria; Amlodipine; Angiotensin Receptor Antagonists; Animals; Antihypertensive Agents; Blood Pressure; Calcium Channel Blockers; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Disease Models, Animal; Drug Therapy, Combination; Hypertension; Kidney Glomerulus; Male; Membrane Proteins; Proteins; Rats; Rats, Inbred SHR; Receptor, Angiotensin, Type 1; Sclerosis; Systole; Tetrazoles; Valine; Valsartan; Verapamil

Several mutations in the nephrin gene are responsible for the lack of slit membrane of the glomeruli leading to massive proteinuria present already in utero. Variations in the nephrin gene may also affect the degree of proteinuria in acquired kidney diseases. We tested the hypothesis of whether any of the polymorphisms identified in the coding region of the nephrin gene were associated with diabetic nephropathy.. In a case-control, cross-sectional study, 996 Finnish type 1 diabetic patients from the FinnDiane Study were genotyped by standard polymerase chain reaction protocol.. The frequencies of the rare alleles in the E117K, R408Q, and N1077S polymorphisms in the entire cohort were 34%, 8%, and 12%, respectively. When comparing patients with a mutant allele with the wild genotype there was no difference between the patients with end-stage renal disease, proteinuria, microalbuminuria, and those with a normal albumin excretion rate (df =3, chi2 =1.62, 1.31 and 0.77). Neither were the polymorphisms associated with the progression of kidney disease, nor with creatinine clearance and albumin excretion rate.. This study does not support an involvement of the coding region of the nephrin gene in the pathogenesis of diabetic nephropathy in type 1 diabetic patients.

Topics: Adult; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Female; Gene Frequency; Genotype; Humans; Male; Membrane Proteins; Polymorphism, Genetic; Proteins

We studied the distribution of nephrin in renal biopsies from 17 patients with diabetes and nephrotic syndrome (7 type 1 and 10 type 2 diabetes), 6 patients with diabetes and microalbuminuria (1 type 1 and 5 type 2 diabetes), and 10 normal subjects. Nephrin expression was semiquantitatively evaluated by measuring immunofluorescence intensity by digital image analysis. We found an extensive reduction of nephrin staining in both type 1 (67 +/- 9%; P < 0.001) and type 2 (65 +/- 10%; P < 0.001) diabetic patients with diabetes and nephrotic syndrome when compared with control subjects. The pattern of staining shifted from punctate/linear distribution to granular. In patients with microalbuminuria, the staining pattern of nephrin also showed granular distribution and reduction intensity of 69% in the patient with type 1 diabetes and of 62 +/- 4% (P < 0.001) in the patients with type 2 diabetes. In vitro studies on human cultured podocytes demonstrated that glycated albumin and angiotensin II reduced nephrin expression. Glycated albumin inhibited nephrin synthesis through the engagement of receptor for advanced glycation end products, whereas angiotensin II acted on cytoskeleton redistribution, inducing the shedding of nephrin. This study indicates that the alteration in nephrin expression is an early event in proteinuric patients with diabetes and suggests that glycated albumin and angiotensin II contribute to nephrin downregulation.

Topics: Adult; Aged; Albuminuria; Angiotensin II; Biopsy; Blotting, Western; Cells, Cultured; Cytochalasin B; Cytoskeleton; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Female; Fluorescent Antibody Technique, Indirect; Gene Expression; Glycated Serum Albumin; Glycation End Products, Advanced; Humans; Kidney; Male; Membrane Proteins; Microscopy, Fluorescence; Middle Aged; Nephrotic Syndrome; Proteins; Receptor for Advanced Glycation End Products; Receptors, Immunologic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Serum Albumin; Tissue Distribution

Although ACE inhibitors slow progression of diabetic renal disease, the mortality and morbidity is still high. As other hormonal factors are involved, inhibition of vasopeptidases could further reduce progression. We studied dual inhibition of angiotensin converting enzyme and neutral endopeptidase in a model of progressive diabetic renal injury. The major endpoints were reductions in systemic blood pressure, albuminuria and renal structural injury.. Diabetic spontaneously hypertensive rats were treated with the ACE inhibitor perindopril (mg.kg(-1).day(-1)) or the vasopeptidase inhibitor omapatrilat at doses of 10 (oma10) and 40 (oma40) mg.kg(-1).day(-1) for 32 weeks. In vivo ACE and NEP inhibition was quantitated by in vitro autoradiography. Renal structural injury was assessed by measurement of the glomerulosclerotic (GS) index and tubulointerstitial area (TI). The expression of transforming growth factor beta, beta-inducible gene-h3 and nephrin were also quantitated.. Despite a similar reduction in blood pressure by perindopril and oma10, greater attenuation of albuminuria was afforded by oma10, with a complete amelioration observed with oma40. Oma40 lead to a 33% reduction in renal NEP binding and this was associated with less albuminuria and prevention of GS, TI area and overexpression of TGFbeta and betaig-h3. Diabetes-associated reduction in nephrin expression was restored by both drugs.. These findings suggest that other vasoactive mechanisms in addition to angiotensin II are important in the prevention of diabetic nephropathy, and that vasopeptidase inhibition might confer an advantage over blockade of the RAS alone in the treatment of diabetic renal disease.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Blood Pressure; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Disease Models, Animal; Hypertension; Immunohistochemistry; Kidney; Male; Membrane Proteins; Peptidyl-Dipeptidase A; Perindopril; Proteins; Rats; Rats, Inbred SHR; Reference Values

Diabetic nephropathy is the leading cause of end-stage renal disease. Because early diagnosis and treatment may prevent the complication, new tools for an early detection are needed. One of the key components of the glomerular filtration slit spanning between neighboring podocytes is nephrin. Its expression is altered in experimental models of diabetes and also in various human proteinuric diseases, including diabetes. We studied whether type 1 diabetic patients with or without nephropathy exhibit immunoreactive nephrin in the urine, reflecting early damage of the filtration barrier. Diabetic patients with normoalbuminuria (n = 40), with microalbuminuria (n = 41), and with macroalbuminuria (n = 39) and patients previously normoalbuminuric but now testing positive for microalbuminuria (newMicro, n = 39) were screened for nephrinuria with Western blotting using two affinity-purified anti-nephrin antibodies. Nondiabetic healthy subjects (n = 29) were also studied. Nephrinuria was present in 30% of normoalbuminuric, 17% of microalbuminuric, 28% of macroalbuminuric, and 28% of newMicro patients. Of female patients, 35% were nephrinuric compared with only 19% of male patients (P = 0.02). None of the control subjects was nephrinuric. In conclusion, glomerular filtration barrier may be affected in one-third of diabetic patients manifesting as early nephrinuria. Nephrinuria may have prognostic value and become a marker of susceptibility for kidney complications in diabetes.

Topics: Adult; Blotting, Western; Case-Control Studies; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Female; Fluorescent Antibody Technique; Humans; Male; Membrane Proteins; Proteins

Mutations in the gene coding for the podocyte slit-pore membrane protein, nephrin, are responsible for the Finnish-type congenital nephrotic syndrome. The present study sought to examine whether nephrin expression may also be altered in experimental diabetes, and how such changes related to the development of proteinuria. In addition, the study also sought to examine nephrin expression in animals treated with different anti-proteinuric therapies.. Nephrin gene expression and localization were examined in rats with streptozotocin-induced diabetes at 6 months duration (proteinuric phase) and at 7 days (pre-proteinuric phase). In addition, the effects of anti-proteinuric drug therapies were also assessed in long-term diabetic rats, treated with either the angiotensin-converting enzyme inhibitor perindopril, or the blocker of advanced glycation end-product formation, aminoguanidine. Nephrin expression was determined using quantitative real-time PCR and in situ hybridization.. When compared with control animals, nephrin expression was reduced in the late proteinuric phase (45% reduction vs controls, P<0.01) but not in the early, pre-proteinuric phase of experimental diabetic nephropathy. While ACE inhibition and aminoguanidine both reduced proteinuria, only the former attenuated the diabetes-associated reduction in nephrin expression.. These findings suggests that reduction in nephrin may be a determinant of glomerular hyperpermeability in diabetic nephropathy. Attenuation of these changes with ACE inhibition suggest that this mechanism may contribute to the anti-proteinuric effects of this, but not all classes of drug which reduce urinary protein in diabetic nephropathy.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Base Sequence; Diabetes Mellitus, Experimental; Diabetic Nephropathies; DNA Primers; Guanidines; In Situ Hybridization; Male; Membrane Proteins; Mutation; Perindopril; Proteins; Proteinuria; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Diabetic nephropathy is a major complication of diabetes leading to thickening of the glomerular basement membrane, glomerular hypertrophy, mesangial expansion, and overt renal disease. The pathophysiologic mechanisms of diabetic nephropathy remain poorly understood. Nephrin is a recently found podocyte protein crucial for the interpodocyte slit membrane structure and maintenance of an intact filtration barrier. Here we have assessed the role of nephrin in two widely used animal models of diabetes, the streptozotocin model of the rat and the nonobese diabetic mouse. In both models, the expression levels of nephrin-specific mRNA as determined by real-time quantitative polymerase chain reaction increased up to two-fold during several weeks of follow-up. Immunohistochemical stainings revealed nephrin also more centrally within the glomerular tuft along with its preferential site in podocytes. Interestingly, as detected by immunoblotting, nephrin protein was also found in the urine of streptozotocin-induced rats. We conclude that nephrin is connected to the early changes of diabetic nephropathy and thus may contribute to the loss of glomerular filtration function.

Topics: Animals; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Gene Expression; Kidney Glomerulus; Male; Membrane Proteins; Proteins; Rats; Rats, Wistar; RNA, Messenger; Tissue Distribution