nephrin and Disease-Models--Animal

Mycophenolic acid (MPA) was introduced into clinical practice as immunosuppressive drug therapy to prevent allograft rejection. Since then, its clinical application has widened. Our goal was to review the lessons learned from experimental nontransplant glomerular disease models on the mechanisms of MPA therapy. T and B lymphocytes are preferentially dependent on de novo purine synthesis. By inhibiting the rate-limiting enzyme of de novo purine synthesis, MPA depletes the pool of deoxyguanosine triphosphate (dGTP) and inhibits proliferation of these immune cells. Furthermore, MPA can also induce apoptosis of immune cells and is known to inhibit synthesis of fucose- and mannose-containing membrane glycoproteins altering the surface expression and binding ability of adhesion molecules. However, MPA exerts a direct effect also on nonimmune cells. Mesangial cells are partially dependent on de novo purine biosynthesis and are thus susceptible to MPA treatment. Additionally, MPA can inhibit apoptosis in podocytes and seems to be beneficial in preserving the expression of nephrin and podocin, and by attenuation of urokinase receptor expression leads to decreased foot-process effacement. In summary, our manuscript sheds light on the molecular mechanisms underlying the antiproteinuric effect of MPA. Overall, MPA is an excellent treatment option in many immunologic glomerulopathies because it possesses immunosuppressive properties, has a remarkable effect on nonimmune cells and counteracts the proliferation of mesangial cells, expansion of mesangial matrix, and foot-process effacement of podocytes combined with a low systemic toxicity.

Topics: Animals; Cell Proliferation; Disease Models, Animal; Glomerular Basement Membrane; Glomerulonephritis; Humans; Immunosuppressive Agents; IMP Dehydrogenase; Intracellular Signaling Peptides and Proteins; Lymphocytes; Membrane Proteins; Mesangial Cells; Monocytes; Mycophenolic Acid; Nephrotic Syndrome; Podocytes; Purines; Rats

Topics: Animals; Disease Models, Animal; Glomerulonephritis; Immune Sera; Immunoglobulin A; Immunotoxins; Interleukin-2 Receptor alpha Subunit; Japan; Membrane Proteins; Thy-1 Antigens

The glomerular visceral epithelial cell plays a central role in ultrafiltration of the blood and in a wide variety of inherited and acquired diseases of the kidney. The discovery of nephrin and other slit diaphragm proteins has led to an explosion of knowledge in the biology of this cell type. The most significant recent discoveries are reviewed in this paper.. Together with the glomerular endothelial cells and intervening glomerular basement membrane, the podocyte constitutes a major portion of the glomerular filtration barrier that separates blood from the urinary space. A number of proteins have been identified that are localized to the slit diaphragms that separate podocyte foot processes. Although it has been suggested that the slit diaphragm represents the ultimate filtration barrier, additional roles for this structure as a signaling centre and in endocytosis have been identified. Mutations in genes that reside in the slit pore or interact with the actin cytoskeleton have been linked to a variety of inherited diseases of the podocyte. Additional mutations in these genes have been linked to sporadic forms of nephrotic syndrome and proposed as modifiers of renal risk. The generation of podocyte-specific transgenic models and genomic tools for the murine podocyte provide important resources for the glomerular biologist.. Over the past year, studies using human genetics, conditional gene targeting and cell biological approaches have led to a rapid increase in our understanding of podocyte and glomerular biology, which should lead to the development of novel therapies for individuals with glomerular disease.

Topics: Animals; Animals, Genetically Modified; Disease Models, Animal; Gene Targeting; Humans; Kidney Diseases; Kidney Glomerulus; Membrane Proteins; Proteins

Proteinuria has been demonstrated to be not only a representative sign of renal lesion but also a risk factor for the progression to renal failure through its injurious effects on tubulointerstitium. The responsible gene for Finnish type congenital nephrotic syndrome was identified and its product was named 'nephrin' which is located on slit membrane between foot processes of glomerular epithelial cells and is considered to be concerned also in the induction of acquired renal lesions with proteinuria. The monoclonal antibody against rat nephrin can induce proteinuria. These facts suggest that the important role for final barrier against macromolecules is played by the slit membrane. Understanding the proteinuria mechanism at molecular level is expected to lead to the establishment of appropriate treatments. Nephrin is regarded as the most promising and attractive molecule for the development of new therapeutic strategy. Many nephrologists are now much interested in the intimate relationship between nephrin and angiotensin II.

Topics: Angiotensin II; Angiotensin-Converting Enzyme Inhibitors; Animals; Antibodies, Monoclonal; Disease Models, Animal; Humans; Membrane Proteins; Nephrotic Syndrome; Proteins; Proteinuria; Rats

The past 5 years have witnessed an exponential increase in our understanding of the structure and function of the glomerular slit diaphragm. The identification of nephrin as the first transmembrane slit diaphragm protein was a watershed event in slit diaphragm biology. This article correlates some of the observations of the prenephrin era with more recent studies, and elaborates on the individual characteristics of each slit diaphragm protein. Recent studies on protein-protein interactions related to slit diaphragm permeability and cell signaling are elaborated, along with observations on their expression in human disease and experimental models of proteinuria. Developmental expression of components of the slit diaphragm in normal and knockout mice also is discussed. Finally, some areas of future investigation are proposed.

Topics: Animals; Biopsy, Needle; Cadherins; Disease Models, Animal; Forecasting; Humans; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Kidney Diseases; Membrane Proteins; Mice; Mice, Knockout; Molecular Biology; Molecular Structure; Protein Binding; Proteins; Rodentia; Sensitivity and Specificity

There is increasing recognition of the importance of genetic factors in the development of focal segmental glomerulosclerosis and related proteinuric disorders. Recently, four genes have been identified which, when defective, cause focal segmental glomerulosclerosis or nephrosis. All of these genes appear to be important in the maintenance of glomerular podocyte function. However, not all cases of familial nephrosis or proteinuria are explained by defects in these genes.

Topics: Actinin; Adaptor Proteins, Signal Transducing; Animals; Cytoskeletal Proteins; Disease Models, Animal; Glomerulosclerosis, Focal Segmental; Humans; Membrane Proteins; Mice; Microfilament Proteins; Mutation; Proteins

Glomerular endothelial injury and effectiveness of glomerular endothelial repair play a crucial role in the progression of glomerulonephritis. Although the potent immune suppressive everolimus is increasingly used in renal transplant patients, adverse effects of its chronic use have been reported clinically in human glomerulonephritis and experimental renal disease. Recent studies suggest that progenitor stem cells could enhance glomerular endothelial repair with minimal adverse effects. Increasing evidence supports the notion that stem cell therapy and regenerative medicine can be effectively used in pathological conditions within the predictive, preventive and personalized medicine (PPPM) paradigm. In this study, using an experimental model of glomerulonephritis, we tested whether bone marrow-derived stem cells (BMDSCs) could provide better effect over everolimus in attenuating glomerular injury and improving the repair process in a rat model of glomerulonephritis. Anti-Thy1 glomerulonephritis was induced in male Sprague Dawley rats by injection of an antibody against Thy1, which is mainly expressed on glomerular mesangial cells. Additional groups of rats were treated with the immunosuppressant everolimus daily after the injection of anti-Thy1 or injected with single bolus dose of BMDSCs after one week of injection of anti-Thy1 (n = 6-8). Nine days after injection of anti-Thy1, glomerular albumin permeability and albuminuria were significantly increased when compared to control group (

Topics: Animals; Apoptosis; Bone Marrow Cells; Caspase 3; Disease Models, Animal; Everolimus; Kidney Glomerulus; Male; Membrane Proteins; Necrosis; Oxidative Stress; Rats, Sprague-Dawley; Stem Cell Transplantation; Stem Cells; Thiobarbituric Acid Reactive Substances

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

Crumbs2 is expressed at embryonic stages as well as in the retina, brain, and glomerular podocytes. Recent studies identified. To study the function of Crb2 at the renal filtration barrier, mice lacking Crb2 exclusively in podocytes were generated. Gene expression and histologic studies as well as transmission and scanning electron microscopy were used to analyze these. Crb2 is an essential component of the podocyte's slit diaphragm, interacting with Nephrin. Loss of slit diaphragm targeting and increasing ER stress are pivotal factors for onset and progression of CRB2-related SRNS.

Topics: Animals; Disease Models, Animal; Endoplasmic Reticulum; Female; Kidney Glomerulus; Male; Membrane Proteins; Mice; Nephrotic Syndrome; Podocytes; Proteinuria

We established a novel mouse model of chronic kidney disease (CKD) using acetic acid and compared it with the 5/6-nephrectomized mouse model. In our novel model, significant increases were observed in blood biochemical values and urinary parameters. Moreover, a decrease in creatinine clearance (Ccr) was observed. This model also demonstrated a higher survival rate than the 5/6-nephrectomized model. Observed histological changes in our model included cell infiltration in the renal interstitium, tubular dilation, regenerated tubules, and glomerulosclerosis. Inflammation of the renal interstitium was particularly remarkable. TNF-α, IL-1β, and ICAM-1 mRNA expression were up-regulated prior to elevation of mean blood pressure and prior to changes in blood biochemical values and urinary parameters. Up-regulation of TGF-β mRNA and down-regulation of nephrin mRNA were also observed at 12 weeks after acetic acid treatment. However, no correlation between the progression of CKD and the decrease in renal blood flow was observed. Finally, repeated losartan administration attenuated the effects of acetic acid-induced renal injury. Our findings suggest that chronic kidney conditions associated with this model may be triggered by interstitial inflammation. Moreover, we suggest that this model is useful for understanding the pathophysiological mechanisms of CKD, and for evaluating the effects of therapeutic agents.

Topics: Acetic Acid; Animals; Creatine; Disease Models, Animal; Gene Expression; Interleukin-1beta; Kidney; Losartan; Male; Membrane Proteins; Metabolic Clearance Rate; Mice, Inbred Strains; Nephrectomy; Renal Insufficiency, Chronic; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Under healthy conditions, foot processes of neighbouring podocytes are interdigitating and connected by an electron-dense slit diaphragm. Besides slit diaphragm proteins, typical adherens junction proteins are also found to be expressed at this cell-cell junction. It is therefore considered as a highly specialized type of adherens junction. During podocyte injury, podocyte foot processes lose their characteristic 3D structure and the filtration slits typical meandering structure gets linearized. It is still under debate how this change of structure leads to the phenomenon of proteinuria. Using super-resolution 3D-structured illumination microscopy, we observed a spatially restricted up-regulation of the tight junction protein claudin-5 (CLDN5) in areas where podocyte processes of patients suffering from minimal change disease (MCD), focal and segmental glomerulosclerosis (FSGS) as well as in murine nephrotoxic serum (NTS) nephritis and uninephrectomy DOCA-salt hypertension models, were locally injured. CLDN5/nephrin ratios in human glomerulopathies and NTS-treated mice were significantly higher compared to controls. In patients, the CLDN5/nephrin ratio is significantly correlated with the filtration slit density as a foot process effacement marker, confirming a direct association of local CLDN5 up-regulation in injured foot processes. Moreover, CLDN5 up-regulation was observed in some areas of high filtration slit density, suggesting that CLND5 up-regulation preceded the changes of foot processes. Therefore, CLDN5 could serve as a biomarker predicting early foot process effacement.

Topics: Adult; Aged; Aged, 80 and over; Animals; Claudin-5; Disease Models, Animal; Female; Glomerulosclerosis, Focal Segmental; Humans; Kidney Diseases; Kidney Glomerulus; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Middle Aged; Podocytes

Huangqi Guizhi Wuwu Decoction(HQGZWWD) is a Traditional Chinese Medicine formula from Synopsis of Golden Chamber used to treat blood arthralgia. According to the principle that the same treatment can be used for different diseases, HQGZWWD has proven effective for IgA nephropathy (IgAN) associated with spleen and kidney yang deficiency.. In this study, we investigated the mechanism by which HQGZWWD alleviates proteinuria and protects renal function in rats with IgAN by regulating the AT1R/Nephrin/c-Abl pathway.. Rats were randomly divided into six groups: control, IgAN model, IgAN model treated with low-dose HQGZWWD, IgAN model treated with medium-dose HQGZWWD, IgAN model treated with high-dose HQGZWWD, and IgAN model treated with valsartan. IgAN was induced using bovine γ-globulin. We evaluated the mediating effects of HQGZWWD on podocyte cytoskeletal proteins, the AT1R/Nephrin/c-Abl pathway, upstream tumor necrosis factor-α (TNF-α), and TNF-α receptor-1 (TNFR1).. The IgAN rats displayed proteinuria, IgA deposition in the mesangial region, and podocyte cytoskeletal protein damage. The expression of TNF-α, TNFR1, AT1R, and c-Abl was increased in the IgAN rat kidney, whereas the expression of nephrin, podocin, ACTN4, and phosphorylated nephrin (p-nephrin) was reduced. HQGZWWD treatment significantly alleviated podocyte cytoskeletal protein damage in the IgAN rats, upregulated the expression of nephrin, podocin, and ACTN4, and the colocalized expression of F-actin and nephrin. This study demonstrates that HQGZWWD attenuates podocyte cytoskeletal protein damage by regulating the AT1R-nephrin- c-Abl pathway, upregulating the expression of p-nephrin, and downregulating the expression of AT1R and c-Abl.. These results indicate that HQGZWWD attenuates podocyte cytoskeletal protein damage in IgAN rats by regulating the AT1R/Nephrin/c-Abl pathway, providing a potential therapeutic approach for IgAN.

Topics: Actinin; Actins; Animals; Cytoskeletal Proteins; Disease Models, Animal; Down-Regulation; Drugs, Chinese Herbal; Glomerulonephritis, IGA; Immunoglobulin A; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Podocytes; Protective Agents; Proteinuria; Proto-Oncogene Proteins c-abl; Rats, Sprague-Dawley; Receptor, Angiotensin, Type 1; Receptors, Tumor Necrosis Factor, Type I; Signal Transduction; Tumor Necrosis Factor-alpha

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

While chronic kidney disease is prevalent in adults, obstructive nephropathy (ON) has been reported in both young and old patients. In ON, tubulointerstitial lesions (TILs) have been widely investigated, but glomerular lesions (GLs) have been largely neglected. Here, we show a novel mechanism underlying GL development in ON in young and old mice. TILs develop earlier than GLs owing to infiltration of inflammatory cells in the tubulointerstitium, but GLs develop following the activation of

Topics: Age Factors; Animals; Cells, Cultured; Cytokines; Disease Models, Animal; Endothelial Cells; Kidney Diseases; Kidney Glomerulus; Ligands; Membrane Proteins; Mice, Inbred C57BL; Microfilament Proteins; MicroRNAs; Podocytes; Signal Transduction; Toll-Like Receptor 8

B-cell lymphoma/leukaemia 10 (Bcl10) is a member of the CARMA-Bcl10-MALT1 signalosome, linking angiotensin (Ang) II, and antigen-dependent immune-cell activation to nuclear factor kappa-B signalling. We showed earlier that Bcl10 plays a role in Ang II-induced cardiac fibrosis and remodelling, independent of blood pressure. We now investigated the role of Bcl10 in Ang II-induced renal damage.. Bcl10 knockout mice (Bcl10 KO) and wild-type (WT) controls were given 1% NaCl in the drinking water and Ang II (1.44 mg/kg/day) for 14 days. Additionally, Bcl10 KO or WT kidneys were transplanted onto WT mice that were challenged by the same protocol for 7 days. Kidneys of Ang II-treated Bcl10 KO mice developed less fibrosis and showed fewer infiltrating cells. Nevertheless, neutrophil gelatinase-associated lipocalin (Ngal) and kidney injury molecule (Kim)1 expression was higher in the kidneys of Ang II-treated Bcl10 KO mice, indicating exacerbated tubular damage. Furthermore, albuminuria was significantly higher in Ang II-treated Bcl10 KO mice accompanied by reduced glomerular nephrin expression and podocyte number. Ang II-treated WT mice transplanted with Bcl10 KO kidney showed more albuminuria and renal Ngal, compared to WT- > WT kidney-transplanted mice, as well as lower podocyte number but similar fibrosis and cell infiltration. Interestingly, mice lacking Bcl10 in the kidney exhibited less Ang II-induced cardiac hypertrophy than controls.. Bcl10 has multi-faceted actions in Ang II-induced renal damage. On the one hand, global Bcl10 deficiency ameliorates renal fibrosis and cell infiltration; on the other hand, lack of renal Bcl10 aggravates albuminuria and podocyte damage. These data suggest that Bcl10 maintains podocyte integrity and renal function.

Topics: Acute Kidney Injury; Albuminuria; Angiotensin II; Animals; B-Cell CLL-Lymphoma 10 Protein; Cell Movement; Disease Models, Animal; Fibrosis; Hepatitis A Virus Cellular Receptor 1; Kidney; Kidney Transplantation; Lipocalin-2; Macrophages; Membrane Proteins; Mice, Inbred C57BL; Mice, Knockout; Podocytes; T-Lymphocyte Subsets; Time Factors

We have found that calcium calmodulin kinase IV is increased in T cells, podocytes, and mesangial cells from patients with systemic lupus erythematosus, as well as in lupus-prone mice, podocytes of patients with focal segmental glomerulosclerosis, and in mice injected with doxorubicin. We showed that this accounts for aberrant T cell function and glomerular damage. Using nanoparticles (nlg) loaded with a small drug inhibitor of calcium calmodulin kinase IV and tagged with antibodies directed to CD4 we have been able to show inhibition of autoimmunity and lupus nephritis. Also, using nlg tagged with antibodies to nephrin, we showed suppression of nephritis in lupus-prone mice and of glomerular damage in mice exposed to doxorubicin. We propose the development of approaches to deliver drugs to cells in a targeted and precise manner.

Topics: Animals; Antibiotics, Antineoplastic; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinase Type 4; CD4 Antigens; Disease Models, Animal; DNA Methylation; Doxorubicin; Drug Delivery Systems; Glomerulosclerosis, Focal Segmental; Humans; Lupus Erythematosus, Systemic; Lupus Nephritis; Membrane Proteins; Mice; Mice, Inbred MRL lpr; Molecular Targeted Therapy; Nanoparticles; Protein Kinase Inhibitors; Sulfonamides; T-Lymphocytes; T-Lymphocytes, Regulatory; Th17 Cells

This study aimed to investigate the effect of the Phellinus linteus (Mesima) decoction on podocyte injury in a rat model of focal and segmental glomerulosclerosis (FSGS) and evaluate the potential mechanisms.. FSGS resembling primary FSGS in humans was established in rats by uninephrectomy and the repeated injection of doxorubicin. The FSGS rats were randomly divided into the model group, low-dose group of P. linteus decoction (PLD-LD), medium-dose group of P. linteus decoction (PLD-MD), and high-dose group of P. linteus decoction (PLD-HD). Blood and urine analysis were performed after 12 weeks and the molecular indicators of renal function and the renal pathological changes were examined.. FSGS developed within 12 weeks in the test group and showed progressive proteinuria and segmental glomerular scarring. Urinary protein, serum creatinine, urea nitrogen, triglycerides and cholesterol were significantly reduced following the 12-week intervention with P.linteus decoction, especially in the PLD-LD group. Renal nephrin and podocin were markedly increased. Moreover, the pathological damage in the renal tissue was alleviated by the PLD-LD intervention.. The P. linteus decoction alleviated the podocyte injury in the FSGS rat model, thus minimizing the progression of glomerular sclerosis and improving renal function.

Topics: Animals; Disease Models, Animal; Glomerulosclerosis, Focal Segmental; Humans; Intracellular Signaling Peptides and Proteins; Kidney; Male; Membrane Proteins; Phellinus; Plant Extracts; Podocytes; Rats; Rats, Sprague-Dawley

Proteinuric chronic kidney disease (CKD) remains a major health problem worldwide. While it is well established that the progression of primary glomerular disease induces tubulointerstitial lesions, how tubular injury triggers glomerular damage is poorly understood. We hypothesized that injured tubules secrete mediators that adversely affect glomerular health. To test this, we used conditional knockout mice with tubule-specific ablation of β-catenin (Ksp-β-cat-/-) and subjected them to chronic angiotensin II (Ang II) infusion or Adriamycin. Compared with control mice, Ksp-β-cat-/- mice were dramatically protected from proteinuria and glomerular damage. MMP-7, a downstream target of β-catenin, was upregulated in treated control mice, but this induction was blunted in the Ksp-β-cat-/- littermates. Incubation of isolated glomeruli with MMP-7 ex vivo led to nephrin depletion and impaired glomerular permeability. Furthermore, MMP-7 specifically and directly degraded nephrin in cultured glomeruli or cell-free systems, and this effect was dependent on its proteolytic activity. In vivo, expression or infusion of exogenous MMP-7 caused proteinuria, and genetic ablation of MMP-7 protected mice from Ang II-induced proteinuria and glomerular injury. Collectively, these results demonstrate that β-catenin-driven MMP-7 release from renal tubules promotes glomerular injury via direct degradation of the key slit diaphragm protein nephrin.

Topics: Angiotensin II; Animals; beta Catenin; Cells, Cultured; Disease Models, Animal; Doxorubicin; Humans; Kidney Tubules; Male; Matrix Metalloproteinase 7; Membrane Proteins; Mice; Mice, Knockout; Microscopy, Electron, Transmission; Podocytes; Primary Cell Culture; Proteolysis; Rats; Renal Insufficiency, Chronic

Podocyte injury is a key event for progressive renal failure. We have previously established a mouse model of inducible podocyte injury (NEP25) that progressively develops glomerulosclerosis after immunotoxin injection. We performed polysome analysis of intact and injured podocytes utilizing the NEP25 and RiboTag transgenic mice, in which a hemagglutinin tag is attached to ribosomal protein L22 selectively in podocytes. Podocyte-specific polysomes were successfully obtained by immunoprecipitation with an antihemagglutinin antibody from glomerular homogenate and analyzed using a microarray. Compared with glomerular cells, 353 genes were highly expressed and enriched in podocytes; these included important podocyte genes and also heretofore uncharacterized genes, such as Dach1 and Foxd2. Podocyte injury by immunotoxin induced many genes to be upregulated, including inflammation-related genes despite no infiltration of inflammatory cells in the glomeruli. MafF and Egr-1, which structurally have the potential to antagonize MafB and WT1, respectively, were rapidly and markedly increased in injured podocytes before MafB and WT1 were decreased. We demonstrated that Maff and Egr1 knockdown increased the MafB targets Nphs2 and Ptpro and the WT1 targets Ptpro, Nxph3, and Sulf1, respectively. This indicates that upregulated MafF and Egr-1 may promote deterioration of podocytes by antagonizing MafB and WT1. Our systematic microarray study of the heretofore undescribed behavior of podocyte genes may open new insights into the understanding of podocyte pathophysiology.

Topics: Animals; Cells, Cultured; Disease Models, Animal; Gene Expression Profiling; Genetic Predisposition to Disease; Glomerulosclerosis, Focal Segmental; Interleukin-2 Receptor alpha Subunit; Membrane Proteins; Mice, Transgenic; Oligonucleotide Array Sequence Analysis; Phenotype; Podocytes; Polyribosomes; Renal Insufficiency, Chronic; Transcriptome

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

Focal and Segmental Glomerulosclerosis (FSGS) is a severe glomerulopathy that frequently leads to end stage renal disease. Only a subset of patients responds to current therapies, making it important to identify alternative therapeutic options. The interleukin (IL)-1 receptor antagonist anakinra is beneficial in several diseases with renal involvement. Here, we evaluated the potential of anakinra for FSGS treatment. Molecular process models obtained from scientific literature data were used to build FSGS pathology and anakinra mechanism of action models by exploiting information on protein interactions. These molecular models were compared by statistical interference analysis and expert based molecular signature matching. Experimental validation was performed in Adriamycin- and lipopolysaccharide (LPS)-induced nephropathy mouse models. Interference analysis (containing 225 protein coding genes and 8 molecular process segments) of the FSGS molecular pathophysiology model with the drug mechanism of action of anakinra identified a statistically significant overlap with 43 shared molecular features that were enriched in pathways relevant in FSGS, such as plasminogen activating cascade, inflammation and apoptosis. Expert adjudication of molecular signature matching, focusing on molecular process segments did not suggest a high therapeutic potential of anakinra in FSGS. In line with this, experimental validation did not result in altered proteinuria or significant changes in expression of the FSGS-relevant genes COL1A1 and NPHS1. In summary, an integrated bioinformatic and experimental workflow showed that FSGS relevant molecular processes can be significantly affected by anakinra beyond the direct drug target IL-1 receptor type 1 (IL1R1) context but might not counteract central pathophysiology processes in FSGS. Anakinra is therefore not suggested for extended preclinical trials.

Topics: Animals; Collagen Type I; Disease Models, Animal; Doxorubicin; Drug Repositioning; Gene Expression; Glomerulosclerosis, Focal Segmental; Humans; Interleukin 1 Receptor Antagonist Protein; Membrane Proteins; Metabolic Networks and Pathways; Mice; Mice, Inbred BALB C; Podocytes

Pre-eclampsia (PE) is closely associated with perinatal morbidity and mortality and we want to investigate tetramethylpyrazine (TMP)'s effects on PE. Pregnant Sprague-Dawley rats were randomly divided into five groups: normal pregnant (PC), PE, PE+TMP 20 mg/kg, PE+TMP 40 mg/kg, and PE+TMP 60 mg/kg group. The PE rat model was established via L-NAME treatment. Systolic blood pressures (SBP) and urinary protein concentration were detected via the tail-cuff method and CBB kit, respectively. mRNA levels of key genes were analyzed via quantitative PCR and protein levels of key genes were measured by ELISA or western blot. TMP decreased SBP and urinary protein concentration of PE rats. TMP inhibited L-NAME-induced decrease in pups alive ratio, pups weight, and the ratio of pups/placenta weight and reversed L-NAME induced changes in placental histology, whereas it had little effect on placental weight. Urinary nephrin and podocin expressions were enhanced and serum placental growth factor level was decreased in PE rats, whereas TMP inhibited the above phenomena. TMP suppressed L-NAME-induced sFlt-1 upregulation in serums and kidneys of PE rats, whereas it downregulated IL-6 and MCP-1 expression in PE rats' serums, placentas and kidneys. TMP also suppressed the increase in placental sFlt-1 and vascular endothelial growth factor level caused by L-NAME. In addition, TMP inhibited CHOP and GRP78 expressions and decreased the ratio of p-elF2α/elF2α in PE rats. TMP attenuated the consequences of NO inhibition in pregnant rats.

Topics: Animals; Blood Pressure; Disease Models, Animal; Endoplasmic Reticulum Chaperone BiP; Female; Gene Expression Regulation; Heat-Shock Proteins; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; NG-Nitroarginine Methyl Ester; Nitric Oxide; Placenta; Pre-Eclampsia; Pregnancy; Pyrazines; Rats; Transcription Factor CHOP; Vascular Endothelial Growth Factor Receptor-1

We previously reported that humanized sickle cell (HbSS) mice develop spontaneous nephropathy, a major cause of morbidity and mortality in sickle cell disease (SCD). Because sex-dependent protective mechanisms in SCD have been reported, we examined the course of nephropathy in male and female HbSS mice to determine contributors and/or predictors of disease severity. In male HbSS mice, glomerular filtration rate was characterized by a rapid onset of hyperfiltration and subsequent progressive decline of renal function over 20 weeks. Early tubular injury presented with increased excretion of kidney injury marker 1 (KIM-1), progressive loss of tubular brush border, and interstitial fibrosis that preceded the onset of glomerular damage, suggesting a tubuloglomerular mechanism of kidney injury in these mice. Additionally, we observed a strong association between the magnitude of hyperfiltration and the degree of long-term kidney injury in male HbSS mice. Unlike males, female HbSS mice did not demonstrate a significant loss of renal function or severe kidney damage during the time course of the study. These results suggest that magnitude of hyperfiltration predicts the onset of chronic kidney damage in male HbSS mice, whereas protective mechanisms in female HbSS mice delay the onset of SCD nephropathy.

Topics: Anemia, Sickle Cell; Animals; Disease Models, Animal; Female; Glomerular Filtration Rate; Hemoglobin, Sickle; Hepatitis A Virus Cellular Receptor 1; Humans; Kidney; Kidney Diseases; Kidney Tubules, Proximal; Longitudinal Studies; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic

Glomerulonephritis (GN) is a common cause of end-stage kidney disease and is characterized by glomerular inflammation, hematuria, proteinuria, and progressive renal dysfunction. Transforming growth factor (TGF)-β is involved in glomerulosclerosis and interstitial fibrosis. TGF-β activates multiple signaling pathways, including the canonical SMAD pathway. We evaluated the role of SMAD signaling in renal injury and proteinuria in a murine model of GN. SMAD3

Topics: Animals; Anti-Glomerular Basement Membrane Disease; Autoantibodies; Cell Line; Disease Models, Animal; Endothelial Cells; Fibrosis; Kidney Glomerulus; Membrane Proteins; Mice, Inbred C57BL; Mice, Knockout; Microfilament Proteins; Paracrine Communication; Podocytes; Proteinuria; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta

Podocytes are differentiated post-mitotic cells that cannot replace themselves after injury. Glomerular parietal epithelial cells are proposed to be podocyte progenitors. To test whether a subset of parietal epithelial cells transdifferentiate to a podocyte fate, dual reporter PEC-rtTA|LC1|tdTomato|Nphs1-FLPo|FRT-EGFP mice, named PEC-PODO, were generated. Doxycycline administration permanently labeled parietal epithelial cells with tdTomato reporter (red), and upon doxycycline removal, the parietal epithelial cells (PECs) cannot label further. Despite the presence or absence of doxycycline, podocytes cannot label with tdTomato, but are constitutively labeled with an enhanced green fluorescent protein (EGFP) reporter (green). Only activation of the Nphs1-FLPo transgene by labeled parietal epithelial cells can generate a yellow color. At day 28 of experimental focal segmental glomerulosclerosis, podocyte density was 20% lower in 20% of glomeruli. At day 56 of experimental focal segmental glomerulosclerosis, podocyte density was 18% lower in 17% of glomeruli. TdTomato

Topics: Animals; Cell Transdifferentiation; Disease Models, Animal; Epithelial Cells; Genes, Reporter; Glomerulosclerosis, Focal Segmental; Humans; Intravital Microscopy; Luminescent Proteins; Membrane Proteins; Mice; Mice, Transgenic; Microscopy, Fluorescence; Podocytes; Red Fluorescent Protein

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

The glomerular filtration barrier, has historically only been spatially resolved using electron microscopy due to the nanometer-scale dimensions of these structures. Recently, it was shown that the nanoscale distribution of proteins in the slit diaphragm can be resolved by fluorescence based stimulated emission depletion microscopy, in combination with optical clearing. Fluorescence microscopy has advantages over electron microscopy in terms of multiplex imaging of different epitopes, and also the amount of volumetric data that can be extracted from thicker samples. However, stimulated emission depletion microscopy is still a costly technique commonly not available to most life science researchers. An imaging technique with which the glomerular filtration barrier can be visualized using more standard fluorescence imaging techniques is thus desirable. Recent studies have shown that biological tissue samples can be isotropically expanded, revealing nanoscale localizations of multiple epitopes using confocal microscopy. Here we show that kidney samples can be expanded sufficiently to study the finest elements of the filtration barrier using confocal microscopy. Thus, our result opens up the possibility to study protein distributions and foot process morphology on the effective nanometer-scale.

Topics: Animals; Autoantibodies; Biomarkers; Collagen Type IV; Disease Models, Animal; Fluorescent Antibody Technique, Indirect; Glomerular Filtration Barrier; Glomerulonephritis; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mice, Transgenic; Microscopy, Confocal; Microscopy, Fluorescence; Rats; Tissue Expansion

It is accepted that alteration of connexin43 (Cx43) expression in glomeruli is a common pathological response in several forms of kidney diseases. To date, however the change of the Cx43 expression in obesity-related glomerulopathy (ORG) has not been reported. In this study, the alteration of Cx43 expression in the glomeruli of rat with ORG was defined. Five-week-old rats were fed with high-fat diet for 18weeks to establish the ORG model, then the histological change of glomeruli, the foot process effacement of podocyte, the markers for podocyte injury (nephrin,podocin and WT1) and Cx43 expression in glomeruli were examined respectively. The results demonstrated metabolic disorder, hyperinsulinemia, systemic inflammation and microalbuminuria in ORG rats. There was significant hypertrophy, glomerular expansion and inflammatory cell infiltration in the kidney of ORG rats compared to the control group. Significant foot process effacement of the podocyte in the glomeruli, nephrin loss and density reduction were shown in the ORG rats, and Cx43 expression was significant upregulated in glomeruli of ORG rats compared to the control group. The results indicate the correlation of overexpressed Cx43 with the obesity related renal inflammation and suggest that Cx43 might be a potential target in the development of obesity related glomerulopathy.

Topics: Animals; Connexin 43; Diet, High-Fat; Disease Models, Animal; Glomerulonephritis; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Male; Membrane Proteins; Obesity; Podocytes; Rats; Rats, Sprague-Dawley; WT1 Proteins

Obesity-related glomerulopathy (ORG) is morphologically characterized by glomerulomegaly with or without observable focal segmental glomerulosclerosis under light microscope, with decreased podocyte density and number, and with increased foot‑process width observed under electron microscope. The severity of podocyte injury is correlated with the degree of proteinuria and renal dysfunction. However, the pathogenesis of ORG is not well understood. The aim of the present study was to explore the possible pathogenic role of aldosterone (ALDO) in ORG. In the in vivo animal experiments, body weight, Lee's obesity index, abdominal fat index, urinary protein excretion, average glomerular diameter were significantly increased, the mRNA and protein expression of podocyte‑associated molecules including nephrin, podocin, podoplanin and podocalyxin were significantly reduced, and the Wnt/β‑catenin signaling pathway was activated in ORG model mice compared with the Control mice, whereas the administration of spironolactone significantly ameliorated these effects. In the in vitro experiments on cultured podocytes, the mRNA and protein expression levels of the aforementioned podocyte‑associated molecules were significantly downregulated and the Wnt/β‑catenin signaling pathway was activated following ALDO stimulation, whereas eplerenone significantly attenuated all the above effects. Dickkopf‑related protein 1 (DKK1), an inhibitor of Wnt/β‑catenin signaling pathway, also reduced the effects of ALDO exposure on the expression of podocyte‑associated molecules. The present study hypothesized that ALDO may be involved in the pathogenesis of ORG through the activation of Wnt/β‑catenin signaling pathway in podocytes.

Topics: Aldosterone; Animals; beta Catenin; Disease Models, Animal; Down-Regulation; Eplerenone; Glomerulonephritis; Intercellular Signaling Peptides and Proteins; Intracellular Signaling Peptides and Proteins; Kidney Cortex; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Obesity; Podocytes; Sialoglycoproteins; Spironolactone; Wnt Proteins; Wnt Signaling Pathway

Lupus nephritis (LN) is one of the most common and severe complications in Systemic lupus erythematosus patients, and the mechanism underlining the pathogenesis of LN is still unknown. Autophagy plays vital roles in maintaining cell homeostasis and is involved in the pathogenesis of many diseases. In this study, we investigated the role of autophagy in the progression of LN.. Autophagic activities in podocytes of both LN patients (Class IV and V) and mice were evaluated. Podocytes were observed by electron microscopy, and autophagic activity was evaluated by immunofluorescence staining and western blot analysis. Apoptotic activity was evaluated by immunohistochemistry, TUNEL assays and flow cytometric analysis.. Significantly greater podocyte injury and discrepant autophagic levels were observed in LN patients. Differentiated mouse podocytes in the LN group showed reduced nephrin expression and increased apoptosis, as well as significantly higher levels of apoptosis-related proteins (cleaved caspase-3 and Bax). In the mice LN group, the increased number of autophagosomes was accompanied by increased LC3-II/LC3-I ratios and decreased p62, suggesting increased autophagic and apoptotic activity in podocytes. Blockade of autophagic activity by 3-MA or siRNA-mediated silencing of Atg5 resulted in decreases in LC3-II/LC3-I ratios, podocyte apoptosis and damage in the mice LN group. Futhermore, Rapamycin treatment increased LC3-II/LC3-I ratios, and enhanced LN-induced apoptosis in podocyte from modal animal.. This study demonstrates that autophagic activity of podocytes is a crucial factor in renal injury by directly affecting the function of podocyte; thus, inhibiting this activity during the early stages of LN is implicated as a potential therapeutic strategy for delaying the progression of LN. Also, clinical application in LN needs to consider patients' pathological type and drugs' comprehensive effectiveness.

Topics: Adenine; Adolescent; Adult; Animals; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Autophagy-Related Proteins; Case-Control Studies; Cells, Cultured; Disease Models, Animal; Disease Progression; Female; Humans; Lupus Nephritis; Male; Membrane Proteins; Mice, Inbred C57BL; Mice, Inbred MRL lpr; Podocytes; Sirolimus; Young Adult

Neonatal nephron loss may follow hypoxic-ischemic events or nephrotoxic medications. Its long-term effects on the kidney are still unclear. Unlike term infants, preterm neonates less than 36 weeks gestational age show ongoing nephrogenesis. We hypothesized that nephron loss during nephrogenesis leads to more severe renal sequelae than nephron loss shortly after the completion of nephrogenesis. Rats show nephrogenesis until day 10 of life resembling the situation of preterm infants. Animals were uninephrectomized at day 1 (UNX d1) resulting in nephron reduction during nephrogenesis and at day 14 of life (UNX d14) inducing nephron loss after the completion of nephrogenesis. 28 days after uninephrectomy the compensatory renal growth was higher in UNX d1 compared to UNX d14. Nephrin was reduced and collagen deposition increased in UNX d1. At 1 year of age, glomerulosclerosis and markers of tubulointerstitial damage were most prevalent in UNX d1. Moreover, the number of desmin-positive podocytes was higher and nephrin was reduced in UNX d1 indicating podocyte damage. Infiltration of inflammatory cells was heightened after UNX d1. Uninephrectomized animals showed no arterial hypertension. We conclude that neonatal nephron loss during active nephrogenesis leads to more severe glomerular and tubulointerstitial damage, which is not a consequence of compensatory arterial hypertension.

Topics: Animals; Animals, Newborn; Collagen; Desmin; Disease Models, Animal; Kidney Diseases; Kidney Glomerulus; Kidney Tubules; Membrane Proteins; Nephrectomy; Nephrons; Organogenesis; Rats; Rats, Wistar

Increased endoplasmic reticulum (ER) stress contributes to development of cardiorenal syndrome (CRS), and Silent Information Regulator 1 (SIRT1), a class III histone deacetylase, may have protective effects on heart and renal disease, by reducing ER stress. We aimed to determine if SIRT1 alleviates CRS through ER stress reduction.. Wild type mice (n=37), mice with cardiac-specific SIRT1 knockout (n=29), or overexpression (n=29), and corresponding controls, were randomized into four groups: sham MI (myocardial infarction) +sham STNx (subtotal nephrectomy); MI+sham STNx; sham MI+STNx; and MI+STNx. To establish the CRS model, subtotal nephrectomy (5/6 nephrectomy, SNTx) and myocardial infarction (MI) (induced by ligation of the left anterior descending (LAD) coronary artery) were performed successively to establish CRS model. At week 8, the mice were sacrificed after sequential echocardiographic and hemodynamic studies, and then pathology and Western-blot analysis were performed.. Neither MI nor STNx alone significantly influenced the other healthy organ. However, in MI groups, STNx led to more severe cardiac structural and functional deterioration, with increased remodeling, increased BNP levels, and decreased EF, Max +dp/dt, and Max -dp/dt values than in sham MI +STNx groups. Conversely, in STNx groups, MI led to renal structural and functional deterioration, with more severe morphologic changes, augmented desmin and decreased nephrin expression, and increased BUN, SCr and UCAR levels. In MI+STNx groups, SIRT1 knockout led to more severe cardiac structural and functional deterioration, with higher Masson-staining score and BNP levels, and lower EF, FS, Max +dp/dt, and Max -dp/dt values; while SIRT1 overexpression had the opposite attenuating effects. In kidney, SIRT1 knockout resulted in greater structural and functional deterioration, as evidenced by more severe morphologic changes, higher levels of UACR, BUN and SCr, and increased desmin and TGF-β expression, while SIRT1 overexpression resulted in less severe morphologic changes and increased nephrin expression without significant influence on BUN or SCr levels. The SIRT1 knockout but not overexpression resulted in increased myocardial expression of CHOP and GRP78. Cardiac-specific SIRT1 knockout or overexpression resulted in increased or decreased renal expression of CHOP, Bax, and p53 respectively.. Myocardial SIRT1 activation appears protective to both heart and kidney in CRS models, probably through modulation of ER stress.

Topics: Animals; Cardio-Renal Syndrome; Creatinine; Desmin; Disease Models, Animal; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Heart; Kidney; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocardial Infarction; Myocardium; Nephrectomy; Sirtuin 1; Transcription Factor CHOP; Transforming Growth Factor beta

Astragali Radix (AR) has been used in the traditional Chinese medicine (TCM) in the treatment of various renal diseases for many years. In this study, a NMR based metabolomic approach coupled with biochemical assay and histopathological inspection had been employed to study the protective effect of total flavonoids (TFA) in AR against adriamycin-induced nephropathy using rats model. Multivariate analysis revealed that 11 of perturbed metabolites could be reversed by TFA, and the MetaboAnalyst analysis revealed that the anti-nephrotic syndrome effect of TFA was probably related with regulation of alanine, aspartate and glutamate metabolism, citrate cycle, pyruvate metabolism, cysteine and methionine metabolism and glyoxylate and dicarboxylate metabolism. The regulatory effects on the gene expression (ACE, nephrin, podocin) suggested that the anti-nephrotic syndrome effect of TFA was also related with the protection of renal filtration function and regulation of blood pressure. The system pharmacology analysis revealed 43 potential targets for TFA, and suggested that the protective effect of TFA on the nephrotic syndrome was probably related with the regulation of immune and renin-angiotensin system. These metabolic changes and the associated pathways, as well as the compound-target-disease network provide insights into the mechanisms of TFA for the treatment of nephrotic syndrome, and further studies are needed to validate the bioactive compounds responsible for the anti-nephrotic syndrome effect of TFA.

Topics: Animals; Astragalus propinquus; Disease Models, Animal; Doxorubicin; Drug Evaluation, Preclinical; Drugs, Chinese Herbal; Flavonoids; Humans; Intracellular Signaling Peptides and Proteins; Kidney; Kidney Diseases; Male; Medicine, Chinese Traditional; Membrane Proteins; Metabolomics; Rats; Rats, Sprague-Dawley; Renin-Angiotensin System

BACKGROUND The aim of this study was to determine whether senescence in renal glomeruli is involved in lupus nephritis (LN); the expression of senescence-associated β-galactosidase (SA-β-Gal) and its association with glomerular lesions were investigated in a mouse model of LN. MATERIAL AND METHODS Eighteen MRL/lpr mice with severe proteinuria were randomly divided into 2 equal groups and intraperitoneally injected with dexamethasone (DEX) or saline; 4 age-matched mice with mild proteinuria served as controls. Serum creatinine and urinary protein levels were analyzed, and kidney histological changes were observed by periodic acid-Schiff and Sirius Red staining. SA-β-Gal was detected via histochemistry. Glomerular expression of collagen IV, α-SMA, and nephrin was analyzed by immunohistochemistry, and glomerular complement C3 deposition was tested by immunofluorescence. The relationships between SA-β-Gal expression and renal function or glomerular lesion markers were determined by Spearman's correlation analysis. RESULTS Mice with severe proteinuria exhibited glomerular segmental sclerosis and endothelial cell proliferation. DEX administration suppressed these lesions but had no significant effect on 24-hour urinary protein levels. The elevated glomerular expression of SA-β-Gal in proteinuric mice was attenuated by DEX treatment. In addition, DEX treatment markedly downregulated glomerular C3 deposition and collagen IV and α-SMA expression, while significantly increasing nephrin expression. Furthermore, SA-β-Gal expression was positively correlated with urinary protein levels and expression of α-SMA. CONCLUSIONS Accelerated senescence of glomerular cells may contribute to glomerular injury in LN.

Topics: Actins; Animals; beta-Galactosidase; Cellular Senescence; Collagen Type IV; Creatinine; Dexamethasone; Disease Models, Animal; Female; Immunohistochemistry; Kidney Glomerulus; Lupus Nephritis; Membrane Proteins; Mice; Mice, Inbred MRL lpr; Proteinuria

Topics: Acute Kidney Injury; Animals; Apoptosis; Disease Models, Animal; Doxorubicin; Humans; Inflammation; Kidney Diseases; Male; Membrane Proteins; Mice; Oxidative Stress; Piperidones; Pyrimidines; Repressor Proteins; WT1 Proteins

Podocyte depletion plays a major role in focal segmental glomerular sclerosis (FSGS). Because cells of the renin lineage (CoRL) serve as adult podocyte and parietal epithelial cell (PEC) progenitor candidates, we generated Ren1cCre/R26R-ConfettiTG/WT and Ren1dCre/R26R-ConfettiTG/WT mice to determine CoRL clonality during podocyte replacement. Four CoRL reporters (GFP, YFP, RFP, CFP) were restricted to cells in the juxtaglomerular compartment (JGC) at baseline. Following abrupt podocyte depletion in experimental FSGS, all four CoRL reporters were detected in a subset of glomeruli at day 28, where they co-expressed de novo four podocyte proteins (podocin, nephrin, WT-1 and p57) and two glomerular parietal epithelial cell (PEC) proteins (claudin-1, PAX8). To monitor the precise migration of a subset of CoRL over a 2w period following podocyte depletion, intravital multiphoton microscopy was used. Our findings demonstrate direct visual support for the migration of single CoRL from the JGC to the parietal Bowman's capsule, early proximal tubule, mesangium and glomerular tuft. In summary, these results suggest that following podocyte depletion, multi-clonal CoRL migrate to the glomerulus and replace podocyte and PECs in experimental FSGS.

Topics: Adult Stem Cells; Animals; Cell Lineage; Cell Movement; Claudin-1; Cyclin-Dependent Kinase Inhibitor p57; Disease Models, Animal; Epithelial Cells; Female; Glomerulosclerosis, Focal Segmental; Intracellular Signaling Peptides and Proteins; Intravital Microscopy; Kidney Glomerulus; Membrane Proteins; Mice; Mice, Transgenic; Microscopy, Fluorescence, Multiphoton; PAX8 Transcription Factor; Podocytes; Renin; Repressor Proteins; Stochastic Processes; WT1 Proteins

Injury of the glomerular filter causes proteinuria by disrupting the sensitive interplay of the glomerular protein network. To date, studies of the expression and trafficking of glomerular proteins have been mostly limited to in vitro or histologic studies. Here, we report a novel in vivo biotinylation assay that allows the quantification of surface expression of glomerular proteins in mice. Kidneys were perfused in situ with biotin before harvest. Afterwards glomeruli were isolated and lyzed. The protein of interest was separated by immunoprecipitation and the amount of surface-expressed protein was quantified by Western blot analysis with streptavidin staining. As proof-of-concept, we examined the presence of nephrin in the slit diaphragm in two well-established murine models of proteinuric kidney disease: nephrotoxic nephritis and adriamycin nephropathy. In proteinuric animals, significantly less nephrin was detected in the slit diaphragm. When proteinuria decreased once again during the course of disease, the amount of surface nephrin returned to the baseline. Our present results suggest that our assay is a valuable tool to study the glomerular filter in proteinuric kidney diseases. Note that the assay is not limited to proteins expressed in the slit diaphragm, and all surface proteins that are accessible to biotin perfusion and immunoprecipitation qualify for this analysis.

Topics: Albuminuria; Animals; Disease Models, Animal; Gene Expression; Kidney Diseases; Kidney Glomerulus; Male; Membrane Proteins; Mice; Nephritis; Proteinuria; Time Factors

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

Icariin has previously been demonstrated to attenuate hyperglycemia‑induced renal injury, however the renoprotective effects of icariin in a rat model of pregnancy‑induced hypertension (PIH) remain to be elucidated. The present study aimed to investigate the effect of icariin on PIH‑induced acute kidney injury (AKI) and proteinuria. Following 18 days of icariin treatment between day 1 and day 18 of gestation, which was combined with NG‑nitro‑L‑arginine methyl ester (L‑NAME) treatment between day 12 and day 18 of gestation to induce PIH, the 24 h urine protein level, blood urea nitrogen and serum creatinine were measured by using the Coomassie Brilliant Blue method, a commercial enzymatic kit and the picric acid method, respectively. Renal tissues were collected at day 18 of gestation for hematoxylin and eosin staining and immunohistochemistry. The mRNA expression of AGT and protein expression of angiotensin II (Ang II) in the kidneys of control and PIH rats was investigated by reverse transcription‑quantitative polymerase chain reaction and western blot analysis, respectively, to determine the effect of icariin on components of the renin‑angiotensin system. The results demonstrated that L‑NAME treatment in pregnant rats resulted in significant increases in systolic blood pressure (SBP) and diastolic blood pressure, in addition to the induction of severe proteinuria. The significant increase in SBP and proteinuria in PIH rats was prevented by icariin. L‑NAME‑induced AKI resulted in profound renal histological alterations, including mesangial expansion and glomerular lesions. L‑NAME administration exerted a marked decrease in the mRNA and protein expression levels of nephrin in the kidneys from PIH rats compared with control group. Furthermore, upregulation of circulating and renal Ang II levels in PIH rats was observed. However, icariin treatment significantly reversed the L‑NAME‑induced downregulation of nephrin and upregulation of circulating and renal Ang II levels in PIH rats. These results demonstrated that icariin administration improved urinary protein excretion levels and renal tissue damage in PIH rats, and the underlying mechanism was mediated in part, via upregulation of nephrin expression and downregulation of Ang II.

Topics: Acute Kidney Injury; Angiotensin II; Animals; Blood Pressure; Blood Urea Nitrogen; Creatinine; Disease Models, Animal; Down-Regulation; Female; Flavonoids; Gestational Age; Hypertension, Pregnancy-Induced; Kidney; Membrane Proteins; NG-Nitroarginine Methyl Ester; Pregnancy; Proteinuria; Rats; Rats, Wistar; Renin-Angiotensin System; Reproduction; Up-Regulation

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

Many cellular structures directly imply specific biological functions. For example, normal slit diaphragm structures that extend from podocyte foot processes ensure the filtering function of renal glomeruli. These slits are covered by a number of surface proteins, such as nephrin, podocin, podocalyxin and CD2AP. Here we report a human patient presenting with congenital nephrotic syndrome, omphalocele and microcoria due to two loss-of-function mutations in PODXL, which encodes podocalyxin, inherited from each parent. This set of symptoms strikingly mimics previously reported mouse Podxl

Topics: Adaptor Proteins, Signal Transducing; Animals; Cytoskeletal Proteins; Disease Models, Animal; Humans; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Membrane Proteins; Mice; Mutation; Nephrotic Syndrome; Podocytes; Sialoglycoproteins

Podocytes play a central role in the formation of the glomerular filtration barrier in the kidney, and their dysfunction has been shown to result in proteinuria. In the present study, we sought to determine the cell-autonomous role of NF-κB, a proinflammatory signaling, within podocytes in proteinuric kidney disease.. Podocyte-specific IκBΔN transgenic (Pod-IκBΔN) mice, in which NF-κB was inhibited specifically in podocytes, were generated by the Cre-loxP technology, and their phenotype was compared with control mice in adriamycin-induced nephropathy.. Pod-IκBΔN mice were phenotypically normal and did not exhibit proteinuria at the physiological condition. By the intravenous administration of adriamycin, overt proteinuria appeared in Pod-IκBΔN mice, as well as in control mice. However, of interest, the amount of proteinuria was significantly lower in adriamycin-injected Pod-IκBΔN mice (373 ± 122 mg albumin/g creatinine), compared with adriamycin-injected control mice (992 ± 395 mg albumin/g creatinine). Expression of podocyte-selective slit diaphragm-associated proteins, such as nephrin and synaptopodin, was markedly decreased by adriamycin injection in control mice, whereas the reduction was attenuated in Pod-IκBΔN mice. Adriamycin-induced reduction in synaptopodin expression was also seen in cultured podocytes derived from control mice, but not in those from Pod-IκBΔN mice.. Because nephrin and synaptopodin are essential for the maintenance of the slit diaphragm in podocytes, these results suggest that proteinuria in adriamycin-induced nephropathy is caused by the reduction in expression of these proteins. The results also suggest that the NF-κB signalling in podocytes cell-autonomously contributes to proteinuria through the regulation of these proteins.

Topics: Albuminuria; Animals; Cells, Cultured; Disease Models, Animal; Doxorubicin; Gene Expression Regulation; Genotype; Humans; I-kappa B Proteins; Integrases; Kidney Diseases; Male; Membrane Proteins; Mice, Inbred C57BL; Mice, Transgenic; Microfilament Proteins; NF-kappa B; Phenotype; Podocytes; Signal Transduction

Renal fibrosis is characterized by glomerulosclerosis and tubulointerstitial fibrosis. Transforming growth factor-β (TGF-β) is postulated to play a central role in the development of both fibrotic processes. Extracellular matrix proteins, particularly type I collagen and fibronectin, accumulate in the tissue during renal fibrogenesis. CCN2, also known as connective tissue growth factor (CTGF), is increased in the setting of fibrosis and modulates a number of downstream signaling pathways involved in the fibrogenic properties of TGF-β. Unilateral ureteral obstruction is one of the most widely used models of renal tubulointerstitial fibrosis. Herein, we describe unilateral ureteral obstruction in mice as an animal model of renal fibrosis and methods for immunohistochemical analyses of extracellular matrix proteins and CCN2. In addition, we describe the construction of podocyte-specific CCN2-transgenic mice for analyzing mesangial matrix expansion and glomerulosclerosis.

Topics: Animals; CCN Intercellular Signaling Proteins; Disease Models, Animal; Extracellular Matrix Proteins; Fibrosis; Genetic Vectors; Humans; Immunohistochemistry; Kidney Diseases; Membrane Proteins; Mice; Mice, Transgenic; Promoter Regions, Genetic; Receptor, Platelet-Derived Growth Factor beta; Transforming Growth Factor beta

Topics: Animals; Disease Models, Animal; Female; Gene Expression Regulation; Humans; Intracellular Signaling Peptides and Proteins; Kidney; Membrane Proteins; NG-Nitroarginine Methyl Ester; Pre-Eclampsia; Pregnancy; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sildenafil Citrate

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

Altered expression of nephrin underlies the pathophysiology of proteinuria in both congenital and acquired nephrotic syndrome. However, the epigenetic mechanisms of nephrin gene regulation remain elusive. Here, we show that Wolf-Hirschhorn syndrome candidate 1-like 1 long form (WHSC1L1-L) is a novel epigenetic modifier of nephrin gene regulation. WHSC1L1-L was associated with histone H3K4 and H3K36 in human embryonic kidney cells. WHSC1L1-L gene was expressed in the podocytes, and functional protein product was detected in these cells. WHSC1L1-L was found to bind nephrin but not other podocyte-specific gene promoters, leading to its inhibition/suppression, abrogating the stimulatory effect of WT1 and NF-κB. Gene knockdown of WHSC1L1-L in primary cultured podocytes accelerated the transcription of nephrin but not CD2AP. An in vivo zebrafish study involving the injection of Whsc1l1 mRNA into embryos demonstrated an apparent reduction of nephrin mRNA but not podocin and CD2AP mRNA. Immunohistochemistry showed that both WHSC1L1-L and nephrin emerged at the S-shaped body stage in glomeruli. Immunofluorescence and confocal microscopy displayed WHSC1L1 to colocalize with trimethylated H3K4 in the glomerular podocytes. Chromatin immunoprecipitation assay revealed the reduction of the association of trimethylated H3K4 at the nephrin promoter regions. Finally, nephrin mRNA was upregulated in the glomerulus at the early proteinuric stage of mouse nephrosis, which was associated with the reduction of WHSC1L1. In conclusion, our results demonstrate that WHSC1L1-L acts as a histone methyltransferase in podocytes and regulates nephrin gene expression, which may in turn contribute to the integrity of the slit diaphragm of the glomerular filtration barrier.

Topics: Adaptor Proteins, Signal Transducing; Animals; Binding Sites; Cytoskeletal Proteins; Disease Models, Animal; Doxorubicin; Epigenesis, Genetic; Gene Expression Regulation; Gene Expression Regulation, Enzymologic; HEK293 Cells; Histone-Lysine N-Methyltransferase; Histones; Humans; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Methylation; Mice, Inbred BALB C; Mice, Inbred C57BL; Nephrotic Syndrome; Nuclear Proteins; Podocytes; Promoter Regions, Genetic; RNA Interference; Transfection; Zebrafish; Zebrafish Proteins

In most forms of glomerular diseases, loss of size selectivity by the kidney filtration barrier is associated with changes in the morphology of podocytes. The kidney filtration barrier is comprised of the endothelial lining, the glomerular basement membrane, and the podocyte intercellular junction, or slit diaphragm. The cell adhesion proteins nephrin and neph1 localize to the slit diaphragm and transduce signals in a Src family kinase Fyn-mediated tyrosine phosphorylation-dependent manner. Studies in cell culture suggest nephrin phosphorylation-dependent signaling events are primarily involved in regulation of actin dynamics and lamellipodium formation. Nephrin phosphorylation is a proximal event that occurs both during development and following podocyte injury. We hypothesized that abrogation of nephrin phosphorylation following injury would prevent nephrin-dependent actin remodeling and foot process morphological changes. Utilizing a biased screening approach, we found nonreceptor Src homology 2 (sh2) domain-containing phosphatase Shp2 to be associated with phosphorylated nephrin. We observed an increase in nephrin tyrosine phosphorylation in the presence of Shp2 in cell culture studies. In the human glomerulopathies minimal-change nephrosis and membranous nephropathy, there is an increase in Shp2 phosphorylation, a marker of increased Shp2 activity. Mouse podocytes lacking Shp2 do not develop foot process spreading when subjected to podocyte injury in vivo using protamine sulfate or nephrotoxic serum (NTS). In the NTS model, we observed a lack of foot process spreading in mouse podocytes with Shp2 deleted and smaller amounts of proteinuria. Taken together, these results suggest that Shp2-dependent signaling events are necessary for changes in foot process structure and function following injury.

Topics: Amino Acid Sequence; Animals; Disease Models, Animal; Gene Deletion; Humans; Kidney; Membrane Proteins; Mice, Inbred C57BL; Molecular Sequence Data; Nephritis; Phosphorylation; Podocytes; Protamines; Protein Interaction Maps; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Tyrosine

Activation of small GTPase Rac1 in podocytes is associated with rodent models of kidney injury and familial nephrotic syndrome. Induced Rac1 activation in podocytes in transgenic mice results in rapid transient proteinuria and foot process effacement, but not glomerular sclerosis. Thus it remains an open question whether abnormal activation of Rac1 in podocytes is sufficient to cause permanent podocyte damage. Using a number of transgenic zebrafish models, we showed that moderate elevation of Rac1 activity in podocytes did not impair the glomerular filtration barrier but aggravated metronidazole-induced podocyte injury, while inhibition of Rac1 activity ameliorated metronidazole-induced podocyte injury. Furthermore, a further increase in Rac1 activity in podocytes was sufficient to cause proteinuria and foot process effacement, which resulted in edema and lethality in juvenile zebrafish. We also found that activation of Rac1 in podocytes significantly downregulated the expression of

Topics: Animals; Animals, Genetically Modified; Disease Models, Animal; Down-Regulation; Glomerular Filtration Barrier; Intracellular Signaling Peptides and Proteins; Kidney Diseases; Membrane Proteins; Metronidazole; Podocytes; rac1 GTP-Binding Protein; Zebrafish

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

Although calcineurin (CN) is distributed in many cell types and functions in regulating cell functions, the precise roles ofCNremained in each type of the cells are not well understood yet. ACNinhibitor (CNI) has been used for steroid-resistant nephrotic syndrome. ACNIis assumed to ameliorate proteinuria by preventing the overproduction of T-cell cytokines. However, recent reports suggest thatCNIhas a direct effect on podocyte. It is accepted that a slit diaphragm (SD), a unique cell-cell junction of podocytes, is a critical barrier preventing a leak of plasma protein into urine. Therefore, we hypothesized thatCNIhas an effect on theSD In this study, we analyzed the expression ofCNin physiological and in the nephrotic model caused by the antibody against nephrin, a critical component of theSD We observed thatCNis expressed at theSDin normal rat and human kidney sections and has an interaction with nephrin. The staining ofCNat theSDwas reduced in the nephrotic model, whileCNactivity in glomeruli was increased. We also observed that the treatment with tacrolimus, aCNI, in this nephrotic model suppressed the redistribution ofCN, nephrin, and otherSDcomponents and ameliorated proteinuria. These observations suggested that the redistribution and the activation ofCNmay participate in the development of theSDinjury.

Topics: Animals; Antibodies, Monoclonal; Calcineurin; Calcineurin Inhibitors; Cell Line; Child; Disease Models, Animal; Female; Humans; Intercellular Junctions; Male; Membrane Proteins; Mice; Nephrotic Syndrome; Podocytes; Protein Transport; Proteinuria; Rats, Wistar; Tacrolimus; Time Factors

Preclinical Research Vanillic acid (VA) is a dihydroxybenzoic acid derivative widely used as a flavoring agent. It has chemopreventive effects on experimentally-induced carcinogenesis and in ulcerative colitis. The object of the present study was to investigate the effects of VA, alone and in combination with methylprednisolone (MP), on cationic bovine serum albumin (cBSA induced immune-complex glomerulonephritis in female BALB/c mice. Pre-immunization was carried out with cBSA in BALB/c mice and repeated (cBSA, 13 mg/kg, 3 times/week, i.v.) for 6 weeks to induce glomerulonephritis which was confirmed by the presence of severe proteinuria. The effect of VA (50, 100, and 200 mg/kg, p.o.) and its combination with MP (12.5 mg/kg, p.o.) was assessed in the nephrotic disease model. Treatment with VA decreased inflammatory nephrotic injury as evidenced by decreased proteinuria, serum creatinine, blood urea nitrogen, serum IgG1 and TNF-α levels. Co-administration of VA with MP showed an improvement in the immunohistochemistry of glomerular nephrin and podocin. The present results indicate that VA has a nephroprotective effect in the management of autoimmune nephritis. Drug Dev Res 77 : 171-179, 2016.   © 2016 Wiley Periodicals, Inc.

Topics: Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Therapy, Combination; Female; Glomerulonephritis; Immune Complex Diseases; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Methylprednisolone; Mice; Mice, Inbred BALB C; Serum Albumin, Bovine; Treatment Outcome; Vanillic Acid

Minimal change disease (MCD) is a common pathological type of nephrotic syndrome. Its main histology is the fusion of podocyte foot process. The pathogenesis of MCD is not clear, but previously it was thought to be related to immune mechanism. In recent years more studies show that podocyte injury is the key link in the pathogenesis of MCD. In MCD mouse model and human kidney tissues, the expressions of podocyte slit membrane protein-nephrin and podocin, skeleton protein-synaptopodin are decreased, and the expression of synaptopodin is correlated with the response to hormone therapy. In addition, newest studies focused on another two potocyte associated proteins, CD80 and Angiopoietin-like-4. CD80, a T cell stimulating molecule, is expressed in potocyte. Kappa B gene sequences can be activated by external microbes, antigens through acting potocytes, which can induce the upregulation of CD80 expression, cytoskeletal protein damage and the glomerular filtration rate changes, resulting in proteinuria. Angiopoietin-like-4 can be expressed in normal potocytes, but over-expression of angiopoietin-like-4 may injure the GBM charge barrier and induce the foot process fusion, leading to MCD. However, further studies on the factors inducing CD80 and Angiopoietin-like-4 expression, and the interaction between glomerular basement membrane and the two proteins are needed. Based on the mechanism of MCD, NF-kappa B inhibitors and sialylation therapy would be a novel non-immune therapy for MCD.

Topics: Angiopoietin-Like Protein 4; Angiopoietins; Animals; B7-1 Antigen; Disease Models, Animal; Humans; Intracellular Signaling Peptides and Proteins; Kidney; Membrane Proteins; Mice; Microfilament Proteins; Nephrosis, Lipoid; NF-kappa B; Podocytes

Mutations in the INF2 (inverted formin 2) gene, encoding a diaphanous formin family protein that regulates actin cytoskeleton dynamics, cause human focal segmental glomerulosclerosis (FSGS). INF2 interacts directly with certain other mammalian diaphanous formin proteins (mDia) that function as RhoA effector molecules. FSGS-causing INF2 mutations impair these interactions and disrupt the ability of INF2 to regulate Rho/Dia-mediated actin dynamics in vitro. However, the precise mechanisms by which INF2 regulates and INF2 mutations impair glomerular structure and function remain unknown. Here, we characterize an Inf2 R218Q point-mutant (knockin) mouse to help answer these questions. Knockin mice have no significant renal pathology or proteinuria at baseline despite diminished INF2 protein levels. INF2 mutant podocytes do show impaired reversal of protamine sulfate-induced foot process effacement by heparin sulfate perfusion. This is associated with persistent podocyte cytoplasmic aggregation, nephrin phosphorylation, and nephrin and podocin mislocalization, as well as impaired recovery of mDia membrane localization. These changes were partially mimicked in podocyte outgrowth cultures, in which podocytes from knockin mice show altered cellular protrusions compared to those from wild-type mice. Thus, in mice, normal INF2 function is not required for glomerular development but normal INF2 is required for regulation of the actin-based behaviors necessary for response to and/or recovery from injury.

Topics: Actins; Acute Kidney Injury; Animals; Cells, Cultured; Disease Models, Animal; Formins; Glomerulosclerosis, Focal Segmental; Heparin; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mice; Microfilament Proteins; Microscopy, Electron, Transmission; Phenotype; Phosphorylation; Podocytes; Point Mutation; Protamines; rho GTP-Binding Proteins; rhoA GTP-Binding Protein; Signal Transduction

The concentration of adenosine in the normal kidney increases markedly during renal hypoxia, ischemia, and inflammation. A recent study reported that an A3 adenosine receptor (A3AR) antagonist attenuated the progression of renal fibrosis. The adriamycin (ADX)-induced nephropathy model induces podocyte injury, which results in severe proteinuria and progressive glomerulosclerosis. In this study, we investigated the preventive effect of a highly selective A3AR antagonist (LJ1888) in ADX-induced nephropathy. Three groups of six-week-old Balb/c mice were treated with ADX (11 mg/kg) for four weeks and LJ1888 (10 mg/kg) for two weeks as following: 1) control; 2) ADX; and 3) ADX + LJ1888. ADX treatment decreased body weight without a change in water and food intake, but this was ameliorated by LJ1888 treatment. Interestingly, LJ1888 lowered plasma creatinine level, proteinuria, and albuminuria, which had increased during ADX treatment. Furthermore, LJ1888 inhibited urinary nephrin excretion as a podocyte injury marker, and urine 8-isoprostane and kidney lipid peroxide concentration, which are markers of oxidative stress, increased after injection of ADX. ADX also induced the activation of proinflammatory and profibrotic molecules such as TGF-β1, MCP-1, PAI-1, type IV collagen, NF-κB, NOX4, TLR4, TNFα, IL-1β, and IFN-γ, but they were remarkably suppressed after LJ1888 treatment. In conclusion, our results suggest that LJ1888 has a renoprotective effect in ADX-induced nephropathy, which might be associated with podocyte injury through oxidative stress. Therefore, LJ1888, a selective A3AR antagonist, could be considered as a potential therapeutic agent in renal glomerular diseases which include podocyte injury and proteinuria.

Topics: Actins; Adenosine; Adenosine A3 Receptor Antagonists; Albuminuria; Animals; Body Weight; Creatinine; Dinoprost; Disease Models, Animal; Doxorubicin; Immunohistochemistry; Kidney; Kidney Diseases; Lipid Peroxidation; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; NF-kappa B; Oxidative Stress; Plasminogen Activator Inhibitor 1; Proteinuria; Transforming Growth Factor beta1

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

Renin-angiotensin system (RAS) inhibitors reduce glomerular injury and proteinuria, indicating that angiotensin II (Ang II) is involved in glomerular diseases. Although the local RAS is reported to play an essential role in maintaining local tissue functions, the role of the local RAS in regulating glomerular function is not well evaluated. In this study, we analyzed the glomerular expression of RAS components in nephrotic models and the effect of Ang II receptor blockers (ARB) on the expression of angiotensinogen (AGT).. The levels of glomerular expression of RAS components were analyzed in two nephrotic models: anti-nephrin antibody-induced nephropathy and PAN nephropathy, a mimic of human minimal change nephrotic syndrome. The effect of the ARB irbesartan on the expression of AGT in the nephrotic model was analyzed.. Glomerular expression of AGT and the receptors for Ang II was clearly increased in the nephrotic models, while the expression levels of renin, ACE and ACE2 were decreased. ARB treatment suppressed the increase of glomerular expression of AGT in the nephrotic model.. It is conceivable that the promoted local RAS action participated in the glomerular dysfunction, and that ARB treatment ameliorated slit diaphragm injury by inhibiting the positive feedback loop of the activated local Ang II action.

Topics: Angiotensin II Type 1 Receptor Blockers; Angiotensinogen; Animals; Cells, Cultured; Disease Models, Animal; Female; Kidney Glomerulus; Membrane Proteins; Nephrotic Syndrome; Podocytes; Rats, Wistar; Renin-Angiotensin System; RNA, Messenger

Podocytes are critically involved in the maintenance of the glomerular filtration barrier and are key targets of injury in many glomerular diseases. Chronic injury leads to progressive loss of podocytes, glomerulosclerosis, and renal failure. Thus, it is essential to maintain podocyte survival and avoid apoptosis after acute glomerular injury. In normal glomeruli, podocyte survival is mediated via nephrin-dependent Akt signaling. In several glomerular diseases, nephrin expression decreases and podocyte survival correlates with increased vascular endothelial growth factor (VEGF) signaling. How VEGF signaling contributes to podocyte survival and prevents apoptosis remains unknown. We show here that Gα-interacting, vesicle-associated protein (GIV)/girdin mediates VEGF receptor 2 (VEGFR2) signaling and compensates for nephrin loss. In puromycin aminonucleoside nephrosis (PAN), GIV expression increased, GIV was phosphorylated by VEGFR2, and p-GIV bound and activated Gαi3 and enhanced downstream Akt2, mammalian target of rapamycin complex 1 (mTORC1), and mammalian target of rapamycin complex-2 (mTORC2) signaling. In GIV-depleted podocytes, VEGF-induced Akt activation was abolished, apoptosis was triggered, and cell migration was impaired. These effects were reversed by introducing GIV but not a GIV mutant that cannot activate Gαi3. Our data indicate that after PAN injury, VEGF promotes podocyte survival by triggering assembly of an activated VEGFR2/GIV/Gαi3 signaling complex and enhancing downstream PI3K/Akt survival signaling. Because of its important role in promoting podocyte survival, GIV may represent a novel target for therapeutic intervention in the nephrotic syndrome and other proteinuric diseases.

Topics: Animals; Apoptosis; Cell Line; Cell Survival; Cells, Cultured; Disease Models, Animal; GTP-Binding Protein alpha Subunits, Gi-Go; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Mice; Microfilament Proteins; Nephrosis; Phosphatidylinositol 3-Kinases; Podocytes; Proto-Oncogene Proteins c-akt; Puromycin Aminonucleoside; Rats; Rats, Sprague-Dawley; Signal Transduction; TOR Serine-Threonine Kinases; Vascular Endothelial Growth Factor A

We investigated the relationship between proteinuria in L-NAME induced preeclampsia and the expression of nephrin and podocin, and the effect of low-molecular-weight-heparin (LMWH) on proteinuria in rats.. We detected nephrin and podocin expression of kidneys of pregnant rats after L-NAME and after LMWH intervening pregnant rats.. Glomerular nephrin expression in L-NAME induced preeclampsia significantly decreased, but not podocin. Nephrin was relatively increased after LMWH intervention and this was accompanied by a decrease in proteinuria.. We demonstrate that down-regulation of nephrin is involved in L-NAME induced proteinuria, and that LMWH reduces proteinuria by up-regulation of neprhin.

Topics: Animals; Disease Models, Animal; Drug Evaluation, Preclinical; Female; Heparin, Low-Molecular-Weight; Intracellular Signaling Peptides and Proteins; Kidney; Male; Membrane Proteins; NG-Nitroarginine Methyl Ester; Pre-Eclampsia; Pregnancy; Proteinuria; Random Allocation; Rats, Wistar

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

Nephrin, a cell surface signaling receptor, regulates podocyte function in health and disease. We study the role of nephrin in β-cell survival signaling. We report that in mouse islet β-cells and the mouse pancreatic beta-cell line (βTC-6 cells) nephrin is associated and partly co-localized with PI3-kinase. Incubation of cells with functional anti-nephrin antibodies induced nephrin clustering at the plasma membrane, nephrin phosphorylation and recruitment of PI3-kinase to nephrin thus resulting in increased PI3K-dependent Akt phosphorylation and augmented phosphorylation/inhibition of pro-apoptotic Bad and FoxO. Nephrin silencing abolished Akt activation and increased susceptibility of cells to apoptosis. High glucose impaired nephrin signaling, increased nephrin internalization and up-regulated PKCα expression. Interestingly, a marked decrease in nephrin expression and phosphorylated Akt was observed in pancreatic islets of db/db lepr-/- diabetic mice. Our findings revealed that nephrin is involved in β-cell survival and suggest that glucose-induced changes in nephrin signaling may contribute to gradual pancreatic β-cell loss in type 2 diabetes.

Topics: Animals; Antibodies, Monoclonal; Apoptosis; bcl-Associated Death Protein; Cell Line, Transformed; Cell Membrane; Cell Survival; Diabetes Mellitus, Type 2; Disease Models, Animal; Forkhead Box Protein O1; Forkhead Transcription Factors; Gene Expression Regulation; Glucose; Insulin; Insulin-Secreting Cells; Membrane Proteins; Mice; Mice, Knockout; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Kinase C-alpha; Proto-Oncogene Proteins c-akt; Receptors, Leptin; 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

Proteinuria is a central component of chronic kidney disease and an independent risk factor for cardiovascular disease. Kidney podocytes have an essential role as a filtration barrier against proteinuria. Kruppel-like Factor 4 (KLF4) is expressed in podocytes and decreased in glomerular diseases leading to methylation of the nephrin promoter, decreased nephrin expression and proteinuria. Treatment with an angiotensin receptor blocker (ARB) reduced methylation of the nephrin promoter in murine glomeruli of an adriamycin nephropathy model with recovery of KLF4 expression and a decrease in albuminuria. In podocyte-specific KLF4 knockout mice, the effect of ARB on albuminuria and the nephrin promoter methylation was attenuated. In cultured human podocytes, angiotensin II reduced KLF4 expression and caused methylation of the nephrin promoter with decreased nephrin expression. In patients, nephrin promoter methylation was increased in proteinuric kidney diseases with decreased KLF4 and nephrin expression. KLF4 expression in ARB-treated patients was higher in patients with than without ARB treatment. Thus, angiotensin II can modulate epigenetic regulation in podocytes and ARB inhibits these actions in part via KLF4 in proteinuric kidney diseases. This study provides a new concept that renin-angiotensin system blockade can exert therapeutic effects through epigenetic modulation of the kidney gene expression.

Topics: Albuminuria; Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Animals; Benzimidazoles; Biphenyl Compounds; Cell Line; Disease Models, Animal; DNA Methylation; Dose-Response Relationship, Drug; Doxorubicin; Epigenesis, Genetic; Irbesartan; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors; Male; Membrane Proteins; Mice, Inbred C57BL; Mice, Knockout; Podocytes; Promoter Regions, Genetic; Renal Insufficiency, Chronic; Renin-Angiotensin System; Signal Transduction; Tetrazoles; Time Factors; Transfection

Intrarenal angiotensin II is increased in kidney diseases independently of plasma angiotensin II and is thought to promote progressive deterioration of renal architecture. Here we investigated the mechanism of enhanced renal angiotensin II generation in kidney glomerular diseases. For this, kidney- or liver-specific angiotensinogen gene (Agt) knockout was superimposed on the mouse model of inducible podocyte injury (NEP25). Seven days after induction of podocyte injury, renal angiotensin II was increased ninefold in NEP25 mice with intact Agt, accompanied by increases in urinary albumin and angiotensinogen excretion, renal angiotensinogen protein, and its mRNA. Kidney Agt knockout attenuated renal Agt mRNA but not renal angiotensin II, renal, or urinary angiotensinogen protein. In contrast, liver Agt knockout markedly reduced renal angiotensin II to 18.7% of that of control NEP25 mice, renal and urinary angiotensinogen protein, but not renal Agt mRNA. Renal angiotensin II had no relationship with renal Agt mRNA, or with renal renin mRNA, which was elevated in liver Agt knockouts. Kidney and liver dual Agt knockout mice showed phenotypes comparable to those of liver Agt knockout mice. Thus, increased renal angiotensin II generation upon severe podocyte injury is attributed to increased filtered angiotensinogen of liver origin resulting from loss of macromolecular barrier function of the glomerular capillary wall that occurs upon severe podocyte injury.

Topics: Albuminuria; Angiotensin II; Angiotensinogen; Animals; Antibodies, Monoclonal; Disease Models, Animal; Exotoxins; Genotype; Kidney Diseases; Kidney Tubules, Proximal; Liver; Membrane Proteins; Mice, Knockout; Phenotype; Podocytes; RNA, Messenger; Sodium; Time Factors; Up-Regulation

Nephrotic syndrome (NS) is a clinical syndrome with a variety of causes, mainly characterized by heavy proteinuria. Podocyte injury plays a key role in proteinuria, one of the principal means for the control of NS is to prevent podocyte injury. Qi-Dan Fang consists of two of the most extensively applied herbal remedies among Traditional Chinese Medicine (TCM) (Radix Astragali Mongolici and Radix Salviae Miltiorrhizae, with a weight ratio of 5:1) which are specifically used for the treatment of various kidney diseases. In previous studies, we found that Qi-Dan Fang provides improvement to patients with adriamycin-induced nephrotic syndrome by alleviating proteinuria and serum lipid. The aim of this study is to study the efficiency of Qi-Dan Fang on NS model rat with renal dysfunction and podocyte injury, something which has not been carried out yet.. The rats were divided into Normal, Model, Jin Gui Shen Qi Pill (4.12 g/kg), Qi-Dan Fang (3.09, 6.17 and 12.34 g/kg/d) groups, they were each given a single tail intravenous injection of Adriamycin (6.0 mg/kg) except for the Normal group and were orally administered dosages of Qi-Dian Fang and Jin Gui Shen Qi pills once daily for 7 weeks. Following the treatment, the content of cystation C (CysC), blood urea nitrogen (BUN), serum creatinine (Scr) were measured with an autobiochemical analyser. The pathomorphological changes to the glomeruli, the mRNA expressions of nephrin, podocin, CD2AP genes and p53, bax, bcl-2 proteins expressions were also carried out to probe the effects of Qi-Dan Fang.. (1) Qi-Dan Fang treatment raised the level of CysC in blood serum while lowering the content of BUN and Scr in the adriamycin-induced nephrotic syndrome rat model; (2) Long-term administration of Qi-Dan Fang was able to ameliorate pathomorphological change of glomeruli and repair the organization structure of Glomerulus; (3) Qi-Dan Fang could increase the mRNA expression of nephrin, podocin and CD2AP genes, down-regulate the expression of p53, bax proteins, while increased bcl-2 protein to protect the podocyte and restore Glomerular selective filtration function.. Results of our present studies reveal that Qi-Dan Fang is able to enhance renal function, inhibit podocyte injury to provide improvements to the Adriamycin-induced nephrotic syndrome.

Topics: Adaptor Proteins, Signal Transducing; Animals; Antibiotics, Antineoplastic; bcl-2-Associated X Protein; Cytoskeletal Proteins; Disease Models, Animal; Doxorubicin; Drugs, Chinese Herbal; Gene Expression Regulation; Intracellular Signaling Peptides and Proteins; Kidney; Male; Membrane Proteins; Microscopy, Electron, Transmission; Nephrotic Syndrome; Phytotherapy; Podocytes; Protective Agents; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; RNA, Messenger; Tumor Suppressor Protein p53

ANG (1-7) contributes to the blood pressure (BP)-lowering effect of angiotensin receptor blockers (ARBs) in male experimental animals. Females have greater ANG (1-7) concentrations than males; however, the contribution of ANG (1-7) to ARB-mediated decreases in BP in females is unknown. The current study tested the hypothesis that female spontaneously hypertensive rats (SHR) have a larger ANG (1-7) contribution to the BP-lowering effects of the ARB candesartan than male SHR. Twelve-week-old male and female SHR were randomized to receive candesartan (0.5 mg·kg(-1)·day(-1); 7 days), candesartan plus ANG II (200 ng·kg(-1)·min(-1); 7 days), the ANG (1-7) antagonist A-779 (48 μg·kg(-1)·h(-1)) plus candesartan and ANG II. Candesartan decreased basal BP in males and females (baseline vs. candesartan: 142 ± 2 vs. 122 ± 3 and 129 ± 1 vs. 115 ± 1 mmHg, respectively; P < 0.05); however, the decrease was greater in males. ANG II increased BP in males in the presence of candesartan (149 ± 2 mmHg; P < 0.05); candesartan blocked ANG II-induced increases in BP in females (116 ± 1 mmHg). Pretreatment with A-779 abolished candesartan-mediated decreases in BP in females, but not males. A-779 also exacerbated ANG II-induced proteinuria (26 ± 6 vs. 77 ± 11 μg·kg(-1)·day(-1), respectively; P < 0.05) and nephrinuria (20 ± 5 vs. 202 ± 58 μg·kg(-1)·day(-1), respectively; P < 0.05) in candesartan-treated female SHR, with no effect in males. In conclusion, females are more sensitive to the BP-lowering effect of ARBs during ANG II infusion, whereas males are more sensitive under basal conditions. In addition, ANG (1-7) has a greater contribution to ARB-mediated decreases in BP, protein, and nephrin excretion in females relative to males.

Topics: Angiotensin I; Angiotensin II; Angiotensin Receptor Antagonists; Animals; Benzimidazoles; Biphenyl Compounds; Blood Pressure; Cell Adhesion Molecules; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Hypertension; Male; Membrane Proteins; Peptide Fragments; Rats; Rats, Inbred SHR; Receptor, Angiotensin, Type 1; Sex Factors; Tetrazoles

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

Membrane-associated guanylate kinase inverted 2 (MAGI-2) is a tight junction protein in epithelial tissues. We previously reported the detailed expression patterns of MAGI-2 in mouse tissues, including kidney podocytes, based on results obtained from Venus knock-in mice for Magi2 locus. In the present study, homozygous deletion of the Magi2 gene in mice caused neonatal lethality, which was explained by podocyte morphological abnormalities and anuria. Immunohistological analysis showed that loss of MAGI-2 function induced a significant decrease in nephrin and dendrin at the slit diaphragm of the kidney, although other components of the slit diaphragm were unchanged. Furthermore, nuclear translocation of dendrin was observed in the podocytes of the MAGI-2-null mutants, along with enhanced expression of cathepsin L, which is reported to be critical for rearrangement of the actin cytoskeleton in podocytes. Expression analysis of the null mutants showed that loss of MAGI-2 function induces abnormal expression of various types of adhesion-related molecules. The present study is the first to demonstrate that MAGI-2 has a critical role in maintaining the functional structure of the slit diaphragm and that this molecule has an essential role in the functioning of the kidney filtration barrier.

Topics: Adaptor Proteins, Signal Transducing; Animals; Creatine; Disease Models, Animal; Female; Gene Expression Regulation; Genotype; Glomerular Filtration Barrier; Guanylate Kinases; Humans; Intercellular Junctions; Kidney; Male; Membrane Proteins; Mice; Mice, Knockout; Nerve Tissue Proteins; Phenotype; Podocytes

Experiments determined whether the combination of endothelin A (ETA) receptor antagonist [ABT-627, atrasentan; (2R,3R,4S)-4-(1,3-benzodioxol-5-yl)-1-[2-(dibutylamino)-2-oxoethyl]-2-(4-methoxyphenyl)pyrrolidine-3-carboxylic acid] and a thiazide diuretic (chlorthalidone) would be more effective at lowering blood pressure and reducing renal injury in a rodent model of metabolic syndrome compared with either treatment alone. Male Dahl salt-sensitive rats were fed a high-fat (36% fat), high-salt (4% NaCl) diet for 4 weeks. Separate groups of rats were then treated with vehicle (control), ABT-627 (ABT; 5 mg/kg per day, in drinking water), chlorthalidone (CLTD; 5 mg/kg per day, in drinking water), or both ABT plus CLTD. Mean arterial pressure (MAP) was recorded continuously by telemetry. After 4 weeks, both ABT and CLTD severely attenuated the development of hypertension, whereas the combination further reduced MAP compared with ABT alone. All treatments prevented proteinuria. CLTD and ABT plus CLTD significantly reduced nephrin (a podocyte injury marker) and kidney injury molecule-1 (a tubulointerstitial injury marker) excretion. ABT, with or without CLTD, significantly reduced plasma 8-oxo-2'-deoxyguanosine, a measure of DNA oxidation, whereas CLTD alone had no effect. All treatments suppressed the number of ED1(+) cells (macrophages) in the kidney. Plasma tumor necrosis factor receptors 1 and 2 were reduced only in the combined ABT and CLTD group. These results suggest that ABT and CLTD have antihypertensive and renal-protective effects in a model of metabolic syndrome that are maximally effective when both drugs are administered together. The findings support the hypothesis that combined ETA antagonist and diuretic treatment may provide therapeutic benefit for individuals with metabolic syndrome consuming a Western diet.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Antihypertensive Agents; Arterial Pressure; Atrasentan; Cell Adhesion Molecules; Chlorthalidone; Deoxyguanosine; Disease Models, Animal; Diuretics; Drug Combinations; Endothelin A Receptor Antagonists; Endothelins; Hypertension; Inflammation; Kidney Diseases; Male; Membrane Proteins; Metabolic Syndrome; Oxidative Stress; Proteinuria; Pyrrolidines; Rats; Rats, Inbred Dahl; Rats, Sprague-Dawley; Receptor, Endothelin A; Sodium Chloride, Dietary

The precise pathogenic mechanism and role of angiotensin II (Ang II) action in the development of proteinuria in minimal change nephrotic syndrome (MCNS) is uncertain.. The glomerular expressions of the slit diaphragm (SD) molecules nephrin, podocin and NEPH1 in rat puromycin aminonucleoside (PAN) nephropathy, a mimic of MCNS, were analyzed. The effects of Ang II receptor blockade (ARB) (irbesartan 15 mg/kg body weight/day) on proteinuria and on the expression of the SD molecules were analyzed.. mRNA expressions of nephrin, podocin and NEPH1 were decreased to an undetectable level at 1 h. The staining of these SD molecules shifted to a discontinuous pattern, and their intensity was reduced. NEPH1 staining was reduced to an undetectable level on day 10. ARB treatment ameliorated the peak value of proteinuria (237.6 ± 97.0 vs. 359.0 ± 63.3 mg/day, p < 0.05), and prevented the decrease in the mRNA expression of the SD molecules (nephrin 66.96 %, podocin 60.40 %, NEPH1 77.87 % of normal level). The immunofluorescence staining of NEPH1 was restored by ARB. ARB treatment enhanced the expression of NEPH1 of normal rats.. Dysfunction of the SD molecules including NEPH1 is a crucial initiation event of PAN nephropathy. ARB treatment ameliorates proteinuria in PAN nephropathy by inhibiting the reduction of NEPH1 and nephrin. Ang II action regulates the expression of NEPH1 and nephrin in not only the pathological but also physiological state.

Topics: Angiotensin II Type 1 Receptor Blockers; Animals; Biphenyl Compounds; Disease Models, Animal; Disease Progression; Female; Gene Expression Regulation; Intracellular Signaling Peptides and Proteins; Irbesartan; Kidney Glomerulus; Membrane Proteins; Nephrosis, Lipoid; Proteinuria; Puromycin Aminonucleoside; Rats, Wistar; Receptor, Angiotensin, Type 1; Tetrazoles; Time Factors

Western blot is the gold standard method to determine individual protein expression levels. However, western blot is technically difficult to perform in large sample sizes because it is a time consuming and labor intensive process. Dot blot is often used instead when dealing with large sample sizes, but the main disadvantage of the existing dot blot techniques, is the absence of signal normalization to a housekeeping protein.. In this study we established a one dot two development signals (ODTDS) dot blot method employing two different signal development systems. The first signal from the protein of interest was detected by horseradish peroxidase (HRP). The second signal, detecting the housekeeping protein, was obtained by using alkaline phosphatase (AP).. Inter-assay results variations within ODTDS dot blot and western blot and intra-assay variations between both methods were low (1.04-5.71%) as assessed by coefficient of variation.. ODTDS dot blot technique can be used instead of western blot when dealing with large sample sizes without a reduction in results accuracy.

Topics: Alkaline Phosphatase; Animals; Biomarkers; Blotting, Western; Collagen Type I; Disease Models, Animal; Glyceraldehyde-3-Phosphate Dehydrogenases; Horseradish Peroxidase; Humans; Immunoblotting; Kidney; Kidney Diseases; Male; Membrane Proteins; Nephrectomy; Rats, Wistar; Reproducibility of Results; Smad2 Protein; Smad3 Protein; Specimen Handling

Collapsing focal segmental glomerulosclerosis (cFSGS) is a progressive kidney disease characterized by glomerular collapse with epithelial hyperplasia. Here we used a transgenic mouse model of cFSGS with immunotoxin-induced podocyte-specific injury to determine the role for Notch signaling in its pathogenesis. The mice exhibited progressive loss of podocytes and severe proteinuria concomitant with histological features of cFSGS. Hyperplastic epithelium was negative for genetic podocyte tags, but positive for the parietal epithelial cell marker claudin-1, and expressed Notch1, Jagged1, and Hes1 mRNA and protein. Enhanced Notch mRNA expression induced by transforming growth factor-β1 in cultured parietal epithelial cells was associated with mesenchymal markers (α-smooth muscle actin, vimentin, and Snail1). Notch inhibition in vitro suppressed these phenotypic transcripts and Notch-dependent cell migration. Moreover, Notch inhibition in vivo significantly decreased parietal epithelial cell lesions but worsened proteinuria and histopathology in our cFSGS model. Thus, aberrant Notch1-mediated parietal epithelial cell migration with phenotypic changes appears to underlie the pathogenesis of cFSGS. Parietal epithelial cell hyperplasia may also represent an adaptive response to compensate for a disrupted filtration barrier with progressive podocyte loss.

Topics: Amyloid Precursor Protein Secretases; Animals; Antibodies, Monoclonal; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Biomarkers; Calcium-Binding Proteins; Cell Line; Cell Movement; Cell Proliferation; Claudin-1; Dibenzazepines; Disease Models, Animal; Enzyme Inhibitors; Epithelial Cells; Exotoxins; Glomerulosclerosis, Focal Segmental; Homeodomain Proteins; Humans; Hyperplasia; Integrases; Intercellular Signaling Peptides and Proteins; Interleukin-2 Receptor alpha Subunit; Jagged-1 Protein; Kidney Glomerulus; Membrane Proteins; Mice; Mice, Transgenic; Podocytes; Proteinuria; Receptor, Notch1; RNA, Untranslated; Serrate-Jagged Proteins; Time Factors; Transcription Factor HES-1; Transforming Growth Factor beta1

The role of parietal epithelial cells (PECs) in glomerular disease is unclear because they also express podocyte proteins under pathophysiological conditions. To help resolve this, we established a novel PEC isolation technique in rats and mice to investigate which regulatory mechanisms lead to podocyte protein expression in PECs. This pure pool of naive PECs was then compared with PECs in primary culture and immortalized PECs in permanent culture. The naive PECs expressed low levels of podocyte-specific mRNA. Accordingly, in crescentic glomerulonephritis, single PECs activated the podocin promoter in vivo. In primary culture, PECs expressed a distinct morphology from podocytes but with high transcript and protein levels of PEC markers. In contrast to naive PECs, cultured PECs also expressed podocyte proteins, and this correlated with reduced proteolytic activity but not with increased transcript levels. Activation of autophagy or proteasomal degradation decreased the levels of podocyte proteins in PECs, whereas inhibition of proteasomal degradation led to the stabilization of podocyte proteins in PECs. Thus, naive PECs express podocyte transcripts physiologically and these podocyte proteins are stable under pathological conditions through decreased proteolysis.

Topics: Animals; Autophagy; Cells, Cultured; Desmin; Disease Models, Animal; Female; Glomerulonephritis; In Vitro Techniques; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Podocytes; Proteolysis; Rats; Rats, Sprague-Dawley; Rats, Wistar; RNA, Messenger; Sialoglycoproteins; Transcriptome

Alterations within the renal renin angiotensin system play a pivotal role in the development and progression of cardiovascular and renal disease. Angiotensin converting enzyme 2 (ACE2) is highly expressed in renal tubules and has been shown to be renoprotective in diabetes. The protease, a disintegrin and metalloprotease (ADAM) 17, is involved in the ectodomain shedding of several transmembrane proteins including ACE2. Renal ACE2 and ADAM17 were significantly increased in db/db mice compared to controls. We investigated the effect of the insulin sensitizer, rosiglitazone, on albuminuria, renal ADAM17 protein expression and ACE2 shedding in db/db diabetic mice. Rosiglitazone treatment of db/db mice normalized hyperglycemia, attenuated renal injury and decreased urinary ACE2 and renal ADAM17 protein expression. Urinary excreted ACE2 is enzymatically active. Western blot analysis of urinary ACE2 demonstrated two prominent immunoreactive bands at approximately 70 & 90 kDa. The predominant immunoreactive band is approximately 20 kDa shorter than the one demonstrated for kidney lysate, indicating possible ectodomain shedding of active renal ACE2 in the urine. Therefore, it is tempting to speculate that renoprotection of rosiglitazone could be partially mediated via downregulation of renal ADAM17 and ACE2 shedding. In addition, there was a positive correlation between blood glucose, urinary albumin, plasma glucagon, and triglyceride levels with urinary ACE2 excretion. In conclusion, urinary ACE2 could be used as a sensitive biomarker of diabetic nephropathy and for monitoring the effectiveness of renoprotective medication.

Topics: ADAM Proteins; ADAM17 Protein; Albuminuria; Angiotensin-Converting Enzyme 2; Animals; Blood Glucose; Diabetes Mellitus, Type 2; Disease Models, Animal; Glucagon; Glucose Tolerance Test; Glycosuria; Hyperglycemia; Hypoglycemic Agents; Kidney Diseases; Male; Membrane Proteins; Mice; Peptidyl-Dipeptidase A; Rosiglitazone; Thiazolidinediones; Tissue Inhibitor of Metalloproteinase-3; Triglycerides

Podocytes are highly specialized epithelial cells with complex actin cytoskeletal architecture crucial for maintenance of the glomerular filtration barrier. The mammalian Rho GTPases Rac1 and Cdc42 are molecular switches that control many cellular processes, but are best known for their roles in the regulation of actin cytoskeleton dynamics. Here, we employed podocyte-specific Cre-lox technology and found that mice with deletion of Rac1 display normal podocyte morphology without glomerular dysfunction well into adulthood. Using the protamine sulfate model of acute podocyte injury, podocyte-specific deletion of Rac1 prevented foot process effacement. In a long-term model of chronic hypertensive glomerular damage, however, loss of Rac1 led to an exacerbation of albuminuria and glomerulosclerosis. In contrast, mice with podocyte-specific deletion of Cdc42 had severe proteinuria, podocyte foot process effacement, and glomerulosclerosis beginning as early as 10 days of age. In addition, slit diaphragm proteins nephrin and podocin were redistributed, and cofilin was dephosphorylated. Cdc42 is necessary for the maintenance of podocyte structure and function, but Rac1 is entirely dispensable in physiological steady state. However, Rac1 has either beneficial or deleterious effects depending on the context of podocyte impairment. Thus, our study highlights the divergent roles of Rac1 and Cdc42 function in podocyte maintenance and injury.

Topics: Actin Depolymerizing Factors; Acute Kidney Injury; Albuminuria; Animals; cdc42 GTP-Binding Protein; Cell Shape; Desoxycorticosterone Acetate; Disease Models, Animal; Genotype; Hypertension; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Knockout; Nephrectomy; Neuropeptides; Phenotype; Phosphorylation; Podocytes; Protamines; rac1 GTP-Binding Protein; Renal Insufficiency; Signal Transduction; Time Factors

Membranous proliferative glomerulonephritis (MPGN) is a major primary cause of chronic kidney disease (CKD). Podocyte injury is crucial in the pathogenesis of glomerular disease with proteinuria, leading to CKD. To assess podocyte injuries in MPGN, the pathological features of spontaneous murine models were analyzed.. The autoimmune-prone mice strains BXSB/MpJ-Yaa and B6.MRL-(D1Mit202-D1Mit403) were used as the MPGN models, and BXSB/MpJ-Yaa(+) and C57BL/6 were used as the respective controls. In addition to clinical parameters and glomerular histopathology, the protein and mRNA levels of podocyte functional markers were evaluated as indices for podocyte injuries. The relation between MPGN pathology and podocyte injuries was analyzed by statistical correlation.. Both models developed MPGN with albuminuria and elevated serum anti-double-strand DNA (dsDNA) antibody levels. BXSB/MpJ-Yaa and B6.MRL showed severe proliferative lesions with T and B cell infiltrations and membranous lesions with T cell infiltrations, respectively. Foot process effacement and microvillus-like structure formation were observed ultrastructurally in the podocytes of both MPGN models. Furthermore, both MPGN models showed a decrease in immune-positive areas of nephrin, podocin and synaptopodin in the glomerulus, and in the mRNA expression of Nphs1, Nphs2, Synpo, Actn4, Cd2ap, and Podxl in the isolated glomerulus. Significant negative correlations were detected between serum anti-dsDNA antibody levels and glomerular Nphs1 expression, and between urinary albumin-to-creatinine ratio and glomerular expression of Nphs1, Synpo, Actn4, Cd2ap, or Podxl.. MPGN models clearly developed podocyte injuries characterized by the decreased expression of podocyte functional markers with altered morphology. These data emphasized the importance of regulation of podocyte injuries in MPGN.

Topics: Adaptor Proteins, Signal Transducing; Animals; CD3 Complex; Cytoskeletal Proteins; Disease Models, Animal; Female; Glomerulonephritis, Membranoproliferative; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Leukocyte Common Antigens; Male; Membrane Proteins; Mice; Microfilament Proteins; Microscopy, Electron; Myosin Heavy Chains; Nonmuscle Myosin Type IIA; Podocytes; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sialoglycoproteins

Familial amyloid polyneuropathy is characterized by transthyretin (TTR) deposition in various tissues, including the kidneys. While deposition induces organ dysfunction, renal involvement in TTR-related amyloidosis could manifest from proteinuria to end-stage kidney failure. As proteinuria is considered result of glomerular filtration barrier injury we investigated whether TTR deposition affects either glomerular basement membrane (GBM) or podocytes.. Immunohistochemistry, immunoblot and gene expression studies for nephrin, podocin and WT1 were run on renal tissue from human-TTRV30M transgenic mice hemizygous or homozygous for heat shock factor one (Hsf-1). Transmission electron microscopy was used for evaluation of podocyte foot process width (PFW) and GBM thickness in Hsf-1 hemizygous mice with or without TTRV30M or amyloid deposition.. Glomeruli of hsf-1 hemizygous transgenic mice showed lower nephrin and podocin protein levels but an increased podocyte number when compared to Hsf-1 homozygous transgenic mice. Nephrin, podocin and WT1 gene expression levels were unaffected by the Hsf-1 carrier status. TTRV30M deposition was associated with increased PFW and GBM thickness.. Under the effect of Hsf-1 hemizygosity, TTRV30M deposition has deleterious effects on GBM thickness, PFW and slit diaphragm composition, without affecting nephrin and podocin gene expression.

Topics: Amyloid; Amyloid Neuropathies, Familial; Animals; Biomarkers; Disease Models, Animal; DNA-Binding Proteins; Gene Expression; Glomerular Basement Membrane; Heat Shock Transcription Factors; Hemizygote; Homozygote; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mice; Mice, Transgenic; Podocytes; Prealbumin; Transcription Factors; WT1 Proteins

Recent studies have shown a beneficial effect of rapamycin in passive and active Heymann Nephritis (HN). However, the mechanisms underlying this beneficial effect have not been elucidated.. Passive Heymann Nephritis (PHN) was induced by a single intravenous infusion of anti-Fx1 in 12 Sprague-Dawley male rats. One week later, six of these rats were commenced on daily treatment with subcutaneous rapamycin 0.5 mgr/kg (PHN-Rapa). The remaining six rats were used as the proteinuric control group (PHN) while six more rats without PHN were given the rapamycin solvent and served as the healthy control group (HC). All rats were sacrificed at the end of the 7th week.. Rapamycin significantly reduced proteinuria during the autologous phase of PHN. Histological lesions were markedly improved by rapamycin. Immunofluorescence revealed attenuated deposits of autologous alloantibodies in treated rats. Untreated rats showed decreased glomerular content of both nephrin and podocin whereas rapamycin restored their expression.. Rapamycin monotherapy significantly improves proteinuria and histological lesions in experimental membranous nephropathy. This beneficial effect may be mediated by inhibition of the alloimmune response during the autologous phase of PHN and by restoration of the normal expression of the podocyte proteins nephrin and podocin.

Topics: Animals; Disease Models, Animal; Gene Expression Regulation; Glomerulonephritis, Membranous; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Male; Membrane Proteins; Proteinuria; Rats; Sirolimus

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

The present study was conducted to investigate the effects of probucol on the progression of diabetic nephropathy and the underlying mechanism in type 2 diabetic db/db mice. Eight weeks db/db mice were treated with regular diet or probucol-containing diet (1%) for 12 weeks. Non-diabetic db/m mice were used as controls. We examined body weight, blood glucose, and urinary albumin. At 20 weeks, experimental mice were sacrificed and their blood and kidneys were extracted for the analysis of blood chemistry, kidney histology, oxidative stress marker, and podocyte marker. As a result, 24 h urinary albumin excretions were reduced after probucol treatment. There were improvements of extracellular matrix accumulation and fibronectin and collagen IV deposition in glomeruli in the probucol-treated db/db mice. The reduction of nephrin and the loss of podocytes were effectively prevented by probucol in db/db mice. Furthermore, probucol significantly decreased the production of thiobarbituric acid-reactive substances (TBARS), an index of reactive oxygen species (ROS) generation and down-regulated the expression of Nox2. Taken together, our findings support that probucol may have the potential to protect against type 2 diabetic nephropathy via amelioration of podocyte injury and reduction of oxidative stress.

Topics: Albuminuria; Animals; Antioxidants; Collagen Type IV; Diabetes Mellitus, Type 2; Disease Models, Animal; Fibronectins; Kidney Diseases; Male; Membrane Glycoproteins; Membrane Proteins; Mice; Mice, Mutant Strains; NADPH Oxidase 2; NADPH Oxidases; Podocytes; Probucol; WT1 Proteins

We used a rat model to assess the role of nephrin, podocin, and desmin in the pathogenesis of IgA nephropathy (IgAN). A rat IgAN model was established by administration of BSA, CCl(4), and lipopolysaccharide (LPS) and compared with healthy control rats. Urinary protein, urine red blood cells, and biochemical parameters were measured for 12 weeks. Renal morphology and ultrastructure were examined by light and electron microscopy. Immunofluorescence was used to assess IgA deposition in the glomeruli and to measure expression of nephrin, podocin, and desmin. Real-time quantitative PCR was used to measure expression of nephrin, podocin, and desmin mRNAs. IgAN rats developed proteinuria at week-6 and this worsened over time. Pathological changes were evident under light microscopy at week-8 and under electron microscopy at week-4. Immunofluorescence analysis showed deposition of IgA in the kidneys of IgAN rats, but not control rats. IgAN rats had increased expression of glomerular podocin, nephrin, and desmin mRNAs and proteins at week-4. The expression of nephrin, podocin and desmin proteins and the expression of podocin and desmin mRNAs preceded the increase in urinary protein. Taken together, our study of a rat model of IgAN indicates that changes in the expression and distribution of nephrin, podocin, and desmin precede and may cause foot process fusion and proteinuria.

Topics: Animals; Desmin; Disease Models, Animal; Disease Progression; Fluorescent Antibody Technique; Glomerulonephritis, IGA; Hematuria; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Microscopy, Electron, Transmission; Podocytes; Proteinuria; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors

Acute kidney injury (AKI) is a major complication for infants following an asphyxic insult at birth. We aimed to determine if kidney structure and function were affected in an animal model of birth asphyxia and if maternal dietary creatine supplementation could provide an energy reserve to the fetal kidney, maintaining cellular respiration during asphyxia and preventing AKI.. Pregnant spiny mice were maintained on normal chow or chow supplemented with creatine from day 20 gestation. On day 38 (term ~39 d), pups were delivered by cesarean section (c-section) or subjected to intrauterine asphyxia. Twenty-four hours after insult, kidneys were collected for histological or molecular analysis. Urine and plasma were also collected for biochemical analysis.. AKI was evident at 24 h after birth asphyxia, with a higher incidence of shrunken glomeruli (P < 0.02), disturbance to tubular arrangement, tubular dilatation, a twofold increase (P < 0.02) in expression of Ngal (early marker of kidney injury), and decreased expression of the podocyte differentiation marker nephrin. Maternal creatine supplementation prevented the glomerular and tubular abnormalities observed in the kidney at 24 h and the increased expression of Ngal.. Maternal creatine supplementation may prove useful in ameliorating kidney injury associated with birth asphyxia.

Topics: Acute Kidney Injury; Animals; Asphyxia Neonatorum; Biomarkers; Creatine; Cytoprotection; Dietary Supplements; Disease Models, Animal; Female; Gene Expression Regulation, Developmental; Gestational Age; Humans; Infant, Newborn; Kidney; Lipocalins; Membrane Proteins; Murinae; Pregnancy; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Time Factors

Podocyte depletion is a major mechanism driving glomerulosclerosis. Progression is the process by which progressive glomerulosclerosis leads to end stage kidney disease (ESKD). In order to determine mechanisms contributing to persistent podocyte loss, we used a human diphtheria toxin transgenic rat model. After initial diphtheria toxin-induced podocyte injury (over 30% loss in 4 weeks), glomeruli became destabilized, resulting in continued autonomous podocyte loss causing global podocyte depletion (ESKD) by 13 weeks. This was monitored by urine mRNA analysis and by quantitating podocytes in glomeruli. Similar patterns of podocyte depletion were found in the puromycin aminonucleoside and 5/6 nephrectomy rat models of progressive end-stage disease. Angiotensin II blockade (combined enalapril and losartan) restabilized the glomeruli, and prevented continuous podocyte loss and progression to ESKD. Discontinuing angiotensin II blockade resulted in recurrent glomerular destabilization, podocyte loss, and progression to ESKD. Reduction in blood pressure alone did not reduce proteinuria or prevent podocyte loss from destabilized glomeruli. The protective effect of angiotensin II blockade was entirely accounted for by reduced podocyte loss. Thus, an initiating event resulting in a critical degree of podocyte depletion can destabilize glomeruli and initiate a superimposed angiotensin II-dependent podocyte loss process that accelerates progression resulting in eventual global podocyte depletion and ESKD. These events can be monitored noninvasively in real-time through urine mRNA assays.

Topics: Angiotensin II; Animals; Antihypertensive Agents; Disease Models, Animal; Humans; Intracellular Signaling Peptides and Proteins; Kidney Failure, Chronic; Kidney Glomerulus; Male; Membrane Proteins; Podocytes; Rats; Rats, Inbred F344; Rats, Sprague-Dawley; Rats, Transgenic; RNA, Messenger

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

Studies were performed to determine if early treatment with an angiotensin II (Ang II) receptor blocker (ARB), olmesartan, prevents the onset of microalbuminuria by attenuating glomerular podocyte injury in Otsuka Long-Evans Tokushima Fatty (OLETF) rats with type 2 diabetes mellitus.. OLETF rats were treated with either a vehicle, olmesartan (10 mg/kg/day) or a combination of nonspecific vasodilators (hydralazine 15 mg/kg/day, hydrochlorothiazide 6 mg/kg/day, and reserpine 0.3 mg/kg/day; HHR) from the age of 7-25 weeks.. OLETF rats were hypertensive and had microalbuminuria from 9 weeks of age. At 15 weeks, OLETF rats had higher Ang II levels in the kidney, larger glomerular desmin-staining areas (an index of podocyte injury), and lower gene expression of nephrin in juxtamedullary glomeruli, than nondiabetic Long-Evans Tokushima Otsuka (LETO) rats. At 25 weeks, OLETF rats showed overt albuminuria, and higher levels of Ang II in the kidney and larger glomerular desmin-staining areas in superficial and juxtamedullary glomeruli compared to LETO rats. Reductions in mRNA levels of nephrin were also observed in superficial and juxtamedullary glomeruli. Although olmesartan did not affect glucose metabolism, it decreased blood pressure and prevented the renal changes in OLETF rats. HHR treatment also reduced blood pressure, but did not affect the renal parameters.. This study demonstrated that podocyte injury occurs in juxtamedullary glomeruli prior to superficial glomeruli in type 2 diabetic rats with microalbuminuria. Early treatment with an ARB may prevent the onset of albuminuria through its protective effects on juxtamedullary glomerular podocytes.

Topics: Albuminuria; Angiotensin II Type 1 Receptor Blockers; Animals; Antihypertensive Agents; Blood Glucose; Blood Pressure; Body Weight; Diabetes Mellitus, Type 2; Disease Models, Animal; Hydralazine; Hydrochlorothiazide; Imidazoles; Juxtaglomerular Apparatus; Male; Membrane Proteins; Podocytes; Rats; Rats, Inbred OLETF; Reserpine; Tetrazoles

Pathological glomerular hyposialylation has been implicated in certain unexplained glomerulopathies, including minimal change nephrosis, membranous glomerulonephritis, and IgA nephropathy. We studied our previously established mouse model carrying a homozygous mutation in the key enzyme of sialic acid biosynthesis, N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase. Mutant mice died before postnatal day 3 (P3) from severe glomerulopathy with podocyte effacement and segmental glomerular basement membrane splitting due to hyposialylation. Administration of the sialic acid precursor N-acetylmannosamine (ManNAc) led to improved sialylation and survival of mutant pups beyond P3. We determined the onset of the glomerulopathy in the embryonic stage. A lectin panel, distinguishing normally sialylated from hyposialylated glycans, used WGA, SNA, PNA, Jacalin, HPA, and VVA, indicating glomerular hyposialylation of predominantly O-linked glycoproteins in mutant mice. The glomerular glycoproteins nephrin and podocalyxin were hyposialylated in this unique murine model. ManNAc treatment appeared to ameliorate the hyposialylation status of mutant mice, indicated by a lectin histochemistry pattern similar to that of wild-type mice, with improved sialylation of both nephrin and podocalyxin, as well as reduced albuminuria compared with untreated mutant mice. These findings suggest application of our lectin panel for categorizing human kidney specimens based on glomerular sialylation status. Moreover, the partial restoration of glomerular architecture in ManNAc-treated mice highlights ManNAc as a potential treatment for humans affected with disorders of glomerular hyposialylation.

Topics: Animals; Biomarkers; Carbohydrate Epimerases; Carrier Proteins; Dietary Supplements; Disease Models, Animal; Drug Evaluation, Preclinical; Hexosamines; Humans; Kidney Diseases; Kidney Glomerulus; Membrane Proteins; Mice; Mice, Mutant Strains; Microscopy, Electron; Mutation; N-Acetylneuraminic Acid; Podocytes; Real-Time Polymerase Chain Reaction; Sialoglycoproteins

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

Rho family GTPases are molecular switches best known for their pivotal role in dynamic regulation of the actin cytoskeleton. The prototypic members of this family are Cdc42, Rac1, and RhoA; these GTPases contribute to the breakdown of glomerular filtration and the resultant proteinuria, but their functions in normal podocyte physiology remain poorly understood. Here, mice lacking Cdc42 in podocytes developed congenital nephropathy and died as a result of renal failure within 2 weeks after birth. In contrast, mice lacking Rac1 or RhoA in podocytes were overtly normal and lived to adulthood. Kidneys from Cdc42-mutant mice exhibited protein-filled microcysts with hallmarks of collapsing glomerulopathy, as well as extensive effacement of podocyte foot processes with abnormal junctional complexes. Furthermore, we observed aberrant expression of several podocyte markers and cell polarity proteins in the absence of Cdc42, indicating a disruption of the slit diaphragm. Kidneys from Rac1- and RhoA-mutant mice, however, had normal glomerular morphology and intact foot processes. A nephrin clustering assay suggested that Cdc42 deficiency, but not Rac1 or RhoA deficiency, impairs the polymerization of actin at sites of nephrin aggregates. Taken together, these data highlight the physiological importance of Cdc42, but not Rac1 or RhoA, in establishing podocyte architecture and glomerular function.

Topics: Animals; cdc42 GTP-Binding Protein; Disease Models, Animal; Female; Glomerular Filtration Barrier; Kidney Diseases; Membrane Proteins; Mice; Mice, Knockout; Mice, Transgenic; Podocytes; Pregnancy; rac1 GTP-Binding Protein; rho GTP-Binding Proteins; rhoA GTP-Binding Protein

Multiple transforming growth factor (TGF)-β-induced fibrogenic signals have been described in vitro. To evaluate mechanisms in vivo, we used an adriamycin nephropathy model in 129x1/Svj mice that display massive proteinuria by days 5 to 7 and pathological findings similar to human focal segmental glomerulosclerosis by day 14. TGF-β mRNA expression increased after day 7 along with nuclear translocation of the TGF-β receptor-specific transcription factor Smad3. Inhibiting TGF-β prevented both pathological changes and type-I collagen and fibronectin mRNA expression, but proteinuria persisted. Renal Akt was phosphorylated in adriamycin-treated mice, suggesting PI3-kinase activation. Expression of mRNA for the p110γ isozyme of PI3-kinase was specifically increased and p110γ colocalized with nephrin by immunohistochemistry early in disease. Nephrin levels subsequently decreased. Inhibition of p110γ by AS605240 preserved nephrin expression and prevented proteinuria. In cultured podocytes, adriamycin stimulated p110γ expression. AS605240, but not a TGF-β receptor kinase inhibitor, prevented adriamycin-induced cytoskeletal disorganization and apoptosis, supporting a role for p110γ in podocyte injury. AS605240, at a dose that decreased proteinuria, prevented renal collagen mRNA expression in vivo but did not affect TGF-β-stimulated collagen induction in vitro. Thus, PI3-kinase p110γ mediates initial podocyte injury and proteinuria, both of which precede TGF-β-mediated glomerular scarring.

Topics: Animals; Apoptosis; Cells, Cultured; Class I Phosphatidylinositol 3-Kinases; Collagen Type I; Disease Models, Animal; Doxorubicin; Fibronectins; Fibrosis; Glomerulosclerosis, Focal Segmental; Immunohistochemistry; Kidney; Male; Membrane Proteins; Mice; Mice, 129 Strain; Phosphorylation; Podocytes; Protein Kinase Inhibitors; Proteinuria; Proto-Oncogene Proteins c-akt; Receptors, Transforming Growth Factor beta; RNA, Messenger; Signal Transduction; Smad3 Protein; Time Factors; Transforming Growth Factor beta; Up-Regulation

The slit diaphragm (SD) is an intercellular junction between renal glomerular epithelial cells (podocytes) that is essential for permselectivity in glomerular ultrafiltration. The SD components, nephrin and Neph1, assemble a signaling complex in a tyrosine phosphorylation dependent manner, and regulate the unique actin cytoskeleton of podocytes. Mutations in the NPHS1 gene that encodes nephrin cause congenital nephrotic syndrome (CNS), which is characterized by the loss of the SD and massive proteinuria. Recently, we have identified the expression of the transmembrane glycoprotein signal regulatory protein α (SIRPα) at the SD. In the present study, we analyzed the expression of SIRPα in developing kidneys, in kidneys from CNS patients and in proteinuric rat models. The possibility that SIRPα interacts with known SD proteins was also investigated. SIRPα was concentrated at the SD junction during the maturation of intercellular junctions. In the glomeruli of CNS patients carrying mutations in NPHS1, where SD formation is disrupted, the expression of SIRPα as well as Neph1 and nephrin was significantly decreased, indicating that SIRPα is closely associated with the nephrin complex. Indeed, SIRPα formed hetero-oligomers with nephrin in cultured cells and in glomeruli. Furthermore, the cytoplasmic domain of SIRPα was highly phosphorylated in normal glomeruli, and its phosphorylation was dramatically decreased upon podocyte injury in vivo. Thus, SIRPα interacts with nephrin at the SD, and its phosphorylation is dynamically regulated in proteinuric states. Our data provide new molecular insights into the phosphorylation events triggered by podocyte injury.

Topics: Animals; Antigens, Differentiation; Disease Models, Animal; Humans; Kidney Glomerulus; Membrane Proteins; Mutation; Nephrotic Syndrome; Phosphorylation; Podocytes; Protein Binding; Proteinuria; Rats; Receptors, Immunologic; Tyrosine

Renal podocytes and their slit diaphragms ensure the integrity of renal basement membrane and prevent urinary protein loss. We have previously reported that decreases of the podocyte slit diaphragm proteins nephrin and podocin represent early events in the podocytopathy of lupus nephritis (LN). We asked whether immunosuppressive agents such as glucocorticoids and cyclophosphamide may have direct effects on podocytes. We assessed in New Zealand Black/New Zealand White (NZB/W) F1 LN mice glomerular nephrin and podocin expression and localization by the use of Western blot and immunofluorescence; mRNA levels were measured by real-time polymerase chain reaction (PCR) and renal histology by light and electron microscopy. Early treatment with glucocorticoids and cyclophosphamide halted the histologic alterations associated with LN, preserving podocyte foot processes. Nephrin and podocin protein expression significantly increased in both glucocorticoid and cyclophosphamide groups as early as after three months of therapy. Real-time PCR revealed similar enhancement in nephrin and podocin mRNA levels after three to six months of treatment. This study documents that early treatment in experimental LN with glucocorticoids or cyclophosphamide preserves slit diaphragm proteins in podocytes and halts histological changes of the glomeruli, thus raising the possibility of a direct protective effect of these drugs on podocytes.

Topics: Animals; Blotting, Western; Cyclophosphamide; Disease Models, Animal; Female; Glucocorticoids; Immunosuppressive Agents; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Lupus Nephritis; Membrane Proteins; Mice; Mice, Inbred NZB; Podocytes; Real-Time Polymerase Chain Reaction; RNA, Messenger; Time Factors

We previously reported that in a model of spontaneously progressive glomerular injury with early podocyte loss, abnormal migration, and proliferation of glomerular parietal epithelial progenitor cells contributed to the formation of synechiae and crescentic lesions. Here we first investigated whether a similar sequence of events could be extended to rats with adriamycin (ADR)-induced nephropathy. As a second aim, the regenerative potential of therapy with bone marrow-derived mesenchymal stem cells (MSCs) on glomerular resident cells was evaluated. In ADR-treated rats, decrease of WT1(+) podocyte number due to apoptosis was associated with reduced glomerular expression of nephrin and CD2AP. As a consequence of podocyte injury, glomerular adhesions of the capillary tuft to the Bowman's capsule were observed, followed by crescent-like lesions and glomerulosclerosis. Cellular components of synechiae were either NCAM(+) parietal progenitor cells or nestin(+) podocytes. In ADR rats, repeated injections of MSCs limited podocyte loss and apoptosis and partially preserved nephrin and CD2AP. MSCs attenuated the formation of glomerular podocyte-parietal epithelial cell bridges and normalized the distribution of NCAM(+) progenitor cells along the Bowman's capsule, thereby reducing glomerulosclerosis. Finding that MSCs increased glomerular VEGF expression and limited microvascular rarefaction may explain the prosurvival effect by stem cell therapy. MSCs also displayed anti-inflammatory activity. Coculture of MSCs with ADR-damaged podocytes showed a functional role of stem cell-derived VEGF on prosurvival pathways. These data suggest that MSCs by virtue of their tropism for damaged kidney and ability to provide a local prosurvival environment may represent a useful strategy to preserve podocyte viability and reduce glomerular inflammation and sclerosis.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Cell Count; Cell Movement; Coculture Techniques; Cytoskeletal Proteins; Disease Models, Animal; Doxorubicin; Kidney; Kidney Diseases; Male; Membrane Proteins; Mesenchymal Stem Cell Transplantation; Podocytes; Rats; Rats, Inbred Lew; Regeneration; Stem Cells; Treatment Outcome; Vascular Endothelial Growth Factor A

Diphtheria toxin (DTx) receptor (DTR)-mediated conditional cell ablation in transgenic mice is a powerful tool to analyze cell function in vivo. Transgenic mice with cell-specific expression of the human DTR have been developed that allow conditional depletion of these cells in vivo through administration of the toxin. We have performed a careful analysis of mice after DTx injection and found an unexpected side effect. Treatment of wild-type C57BL/6 mice with DTx leads to a marked transient and completely reversible proteinuria, as a consequence of podocyte dysfunction that is morphologically characterized by foot process fusion and detachment from the glomerular basal membrane. In vitro analysis displayed that DTx-treated podocytes show diminished attachment to basal membrane proteins. Five to 9 days after DTx application the mice recover completely. Glomerular proteinuria is a hallmark of glomerular disease due to dysfunction of the filtration barrier. Rodents have been extensively used experimentally to better define the mechanisms of disease induction and progression. However, nongenetic mouse models of proteinuric glomerular damage are limited and display various shortcomings. We suggest DTx-induced transient kidney dysfunction as a new reversible model of experimental podocyte injury, which could be used as an additional approach to complement studies in human.

Topics: Analysis of Variance; Animals; Body Weight; Cell Adhesion; Cell Survival; Diphtheria Toxin; Disease Models, Animal; Female; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, SCID; Mice, Transgenic; Podocytes; Proteinuria

In addition to skeletal muscle and the nervous system, α-dystroglycan is found in the podocyte basal membrane, stabilizing these cells on the glomerular basement membrane. Fukutin, named after the gene responsible for Fukuyama-type congenital muscular dystrophy, is a putative glycosyltransferase required for the post-translational modification of α-dystroglycan. Chimeric mice targeted for both alleles of fukutin develop severe muscular dystrophy; however, these mice do not have proteinuria. Despite the lack of a functional renal defect, we evaluated glomerular structure and found minor abnormalities in the chimeric mice by light microscopy. Electron microscopy revealed flattening of podocyte foot processes, the number of which was significantly lower in the chimeric compared to wild-type mice. A monoclonal antibody against the laminin-binding carbohydrate residues of α-dystroglycan did not detect α-dystroglycan glycosylation in the glomeruli by immunoblotting or immunohistochemistry. In contrast, expression of the core α-dystroglycan protein was preserved. There was no statistical difference in dystroglycan mRNA expression or in the amount of nephrin and α3-integrin protein in the chimeric compared to the wild-type mice as judged by immunohistochemistry and real-time RT-PCR. Thus, our results indicate that appropriate glycosylation of α-dystroglycan has an important role in the maintenance of podocyte architecture.

Topics: Animals; Blotting, Western; Cell Shape; Disease Models, Animal; Dystroglycans; Glycosylation; Immunohistochemistry; Integrin alpha3; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Electron, Transmission; Podocytes; Polymerase Chain Reaction; Protein Processing, Post-Translational; Proteins; RNA, Messenger; Transferases; Walker-Warburg Syndrome

Activation of endothelin receptor-A (ET(A)) increases glomerular permeability to albumin (P(alb)) and elevates pro-inflammatory markers in hyperglycaemic rats.. Male Sprague-Dawley rats were given streptozotocin (n = 32) or saline (sham; n = 32). Half of the animals in each group received the ET(A)-selective antagonist, ABT-627 (atrasentan; orally), beginning immediately after hyperglycaemia was confirmed. Glomeruli were isolated by sieving techniques and P(alb) determined from the change in glomerular volume induced by oncotic gradients of albumin. Glomerular nephrin levels were assessed by immunofluorescence, whereas urinary nephrin was measured by immunoassay.. At 3 and 6 weeks after streptozotocin injection, proteinuria was significantly increased compared with sham controls and significantly reduced by ABT-627 treatment. P(alb) was also increased at 3 and 6 weeks post-streptozotocin. ABT-627 had no effect on P(alb) or protein excretion in sham control rats. In glomeruli isolated from hyperglycaemic rats, incubation with BQ-123, a selective ET(A) antagonist, reduced P(alb), whereas BQ-788, a selective endothelin receptor-B antagonist had no effect (n = 6 rats per group, 5-8 glomeruli per rat). Glomerular and plasma content of soluble intercellular adhesion molecule-1 and monocyte chemoattractant protein-1 were significantly increased 6 weeks after streptozotocin (ELISA). ABT-627 attenuated these increases. After 6 weeks of hyperglycaemia, glomerular nephrin content was decreased with a concurrent increase in urinary nephrin excretion. ABT-627 prevented glomerular nephrin loss in hyperglycaemic rats (n = 5-8 rats per group; eight groups).. These observations support the hypothesis that endothelin-1, via the ET(A) receptor, directly increases P(alb), possibly via nephrin loss, as well as early inflammation in the hyperglycaemic rat.

Topics: Animals; Atrasentan; Diabetes Mellitus, Experimental; Disease Models, Animal; Endothelin A Receptor Antagonists; Immunoassay; Kidney Glomerulus; Male; Membrane Proteins; Oligopeptides; Piperidines; Pyrrolidines; Rats; Rats, Sprague-Dawley; Receptor, Endothelin A

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

Podocytes provide a slit diaphragm to inhibit proteinuria, and nephrin between podocytes functions as a barrier during glomerular filtration. Hepatocyte growth factor (HGF) can improve proteinuria in rodents with various renal injuries, but little is known about the role of HGF in podocyte-based events during glomerulonephritis. In the present study, we examined whether and how nephrin expression is sustained by podocytes during the HGF-mediated attenuation of albuminuria.. Lipopolysaccharide (LPS)-treated mice were used as an animal model of albuminuria. We evaluated the effect of HGF on slit proteins using immunohistochemistry, western blotting and real-time polymerase chain reaction.. Albuminuria occurred 36 h after LPS treatment in mice. This albuminuria did not involve podocyte loss, but was associated with a decrease in nephrin and its key anchor, synaptopodin. In these processes, c-Met tyrosine phosphorylation, which represented HGF signal activation, occurred in glomerular cells including podocytes. When recombinant HGF was administrated to nephritic mice, c-Met tyrosine phosphorylation became evident in podocytes. The enhancement of the HGF-c-Met signal was associated with increases in nephrin and synaptopodin. An electron microscopic examination revealed that LPS induced the foot process effacement of podocytes, while HGF injections suppressed the foot process injury. Overall, albuminuria was attenuated in the LPS-treated mice after HGF administration.. HGF protects podocytes from a loss of nephrin, at least in part, through maintaining synaptopodin. As a result, HGF was shown to sustain foot process structure, and albuminuria was attenuated under inflammation.

Topics: Albuminuria; Animals; Blotting, Western; Disease Models, Animal; Female; Glomerulonephritis; Hepatocyte Growth Factor; Humans; Immunohistochemistry; Lipopolysaccharides; Membrane Proteins; Mice; Mice, Inbred C57BL; Microfilament Proteins; Phosphorylation; Podocytes; Polymerase Chain Reaction; Proto-Oncogene Proteins c-met; Recombinant Proteins; Time Factors

Doxorubicin (DOX) is an anthracycline antibiotic utilized in antitumor therapy; however, its clinical use is frequently impeded by renal toxic effects. As peroxisome proliferator-activated receptor-α (PPAR-α) has renoprotective effects in drug-related kidney injuries, we tested its ability to inhibit DOX-induced renal injury. Although both male PPAR-α knockout mice and their wild-type littermates (pure 129/SvJ background) had significant proteinuria 4 weeks after DOX treatment, those with deletion of PPAR-α had more severe proteinuria. This was associated with more serious podocyte foot process effacement compared with wild-type mice. In contrast, the PPAR-α agonist fenofibrate effectively reduced proteinuria and attenuated DOX-induced podocyte foot process effacement. Consistently, glomerular nephrin expression was significantly lower in the knockout compared with wild-type mice following DOX treatment. Fenofibrate therapy significantly blunted the reduction in glomerular nephrin levels in DOX-treated wild-type mice. In cultured podocytes, DOX induced apoptosis, increased cleaved caspase-3 levels, and decreased Bcl-2 expression, all attenuated by pretreatment with fenofibrate. Thus, PPAR-α deficiency exacerbates DOX-related renal injury, in part, due to increased podocyte apoptosis.

Topics: Animals; Apoptosis; Caspase 3; Cell Line; Cytoprotection; Disease Models, Animal; Doxorubicin; Fenofibrate; Kidney Diseases; Kidney Glomerulus; Male; Membrane Proteins; Mice; Mice, 129 Strain; Mice, Inbred BALB C; Mice, Knockout; Podocytes; PPAR alpha; Proteinuria; Proto-Oncogene Proteins c-bcl-2; Species Specificity; Time Factors; Transcription, Genetic

Focal and segmental glomerulosclerosis (FSGS) is one of the most important causes of end-stage renal failure. The bradykinin B1 receptor has been associated with tissue inflammation and renal fibrosis. To test for a role of the bradykinin B1 receptor in podocyte injury, we pharmacologically modulated its activity at different time points in an adriamycin-induced mouse model of FSGS. Estimated albuminuria and urinary protein to creatinine ratios correlated with podocytopathy. Adriamycin injection led to loss of body weight, proteinuria, and upregulation of B1 receptor mRNA. Early treatment with a B1 antagonist reduced albuminuria and glomerulosclerosis, and inhibited the adriamycin-induced downregulation of podocin, nephrin, and α-actinin-4 expression. Moreover, delayed treatment with antagonist also induced podocyte protection. Conversely, a B1 agonist aggravated renal dysfunction and even further suppressed the levels of podocyte-related molecules. Thus, we propose that kinin has a crucial role in the pathogenesis of FSGS operating through bradykinin B1 receptor signaling.

Topics: Actinin; Albuminuria; Animals; Bradykinin; Bradykinin B1 Receptor Antagonists; Disease Models, Animal; Doxorubicin; Gene Expression Regulation; Glomerulosclerosis, Focal Segmental; Heme Oxygenase-1; Interleukin-1beta; Intracellular Signaling Peptides and Proteins; Macrophages; Male; Matrix Metalloproteinase 9; Membrane Proteins; Mice; Mice, Inbred BALB C; Podocytes; Receptor, Bradykinin B1; RNA, Messenger; Signal Transduction; Time Factors; Tissue Inhibitor of Metalloproteinase-1

Our previous studies using puromycin aminonucleoside (PAN) established that podocyte damage leads to glomerular growth arrest during development and glomerulosclerosis later in life. This study examined the potential benefit of maintaining podocyte-derived VEGF in podocyte defense and survival after PAN injury using conditional transgenic podocytes and mice, in which human VEGF-A (hVEGF) transgene expression is controlled by tetracycline responsive element (TRE) promoter and reverse tetracycline transactivator (rtTA) in podocytes. In vitro experiments used primary cultured podocytes harvested from mice carrying podocin-rtTA and TRE-hVEGF transgenes, in which hVEGF can be induced selectively. Induction of VEGF in PAN-exposed podocytes resulted in preservation of intrinsic VEGF, α-actinin-4 and synaptopodin, antiapoptotic marker Bcl-xL/Bax, as well as attenuation in apoptotic marker cleaved/total caspase-3. In vivo, compared with genotype controls, PAN-sensitive neonatal mice with physiologically relevant levels of podocyte-derived VEGF showed significantly larger glomeruli. Furthermore, PAN-induced up-regulation of desmin, down-regulation of synaptopodin and nephrin, and disruption of glomerular morphology were significantly attenuated in VEGF-induced transgenic mice. Our data indicate that podocyte-derived VEGF provides self-preservation functions, which can rescue the cell after injury and preempt subsequent deterioration of the glomerulus in developing mice.

Topics: Actinin; Animals; Animals, Newborn; Apoptosis; Autocrine Communication; bcl-2-Associated X Protein; bcl-X Protein; Caspase 3; Cells, Cultured; Desmin; Disease Models, Animal; Doxycycline; Glomerulonephritis; Humans; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microfilament Proteins; Podocytes; Puromycin Aminonucleoside; Response Elements; Vascular Endothelial Growth Factor A

Multi-glycoside of Tripterygium wilfordii Hook. f. (GTW) has been proved clinically effective in reducing proteinuria in chronic kidney disease in China. However, the mechanisms involved are still unclear. In this study we examined the effects of GTW at the different dosages on proteinuria and podocyte slit diaphragm (SD) dysfunction in anti-Thy1.1 glomerulonephritis (GN).. Rats with anti-Thy1.1 GN were divided into 2 groups, a GTW group and a vehicle group, and sacrificed at 30 min, on day 7, and on day 14 in Experiments 1, 2 and 3, respectively. The administration of GTW at the moderate and high doses was started 3 days before or at the same time of antibody injection till sacrifice. Proteinuria was determined in Experiments 1, 2, and 3. After sacrifice, the staining intensity of SD-associated key functional molecules including nephrin and podocin, podocyte structure, mesangial change, macrophage infiltration, and blood biochemical parameters were examined, respectively. Protein and mRNA expressions of nephrin and podocin in glomeruli were also investigated. Besides, liver histological characteristics were analyzed.. In Experiment 1, GTW pretreatment at the medium dose (75 mg/kg body weight) caused no influence on the induction of anti-Thy1.1 GN and the basal nephrin expression. In Experiment 2, the high dosage (100mg/kg body weight) of GTW ameliorated proteinuria, the distribution of nephrin and podocin, mesangial proliferation, and the activated macrophage accumulation, as compared with vehicle group (P<0.05). Additionally, it increased mRNA and protein expressions of nephrin and podocin in glomeruli on day 7, but had no influence on podocyte structure. In Experiment 3, the medium dosage (75 mg/kg body weight) of GTW improved proteinuria, the partial matrix expansion, and the distribution of nephrin and podocin on day 14, as compared with anti-Thy1.1 GN rats (P<0.05). GTW at the high or moderate dose did not affect hepatic function on day 7 and on day 14.. Podocyte SD dysfunction, such as the disordered distribution and down-regulation of nephrin and podocin expression, is critically involved in the pathogenesis of anti-Thy1.1 GN induced by mAb 1-22-3. The restoration of the distribution and expression of nephrin and podocin by GTW could be an important mechanism by which GTW ameliorates proteinuria and podocyte SD dysfunction.

Topics: Animals; Disease Models, Animal; Female; Glomerular Mesangium; Glomerulonephritis, Membranoproliferative; Intracellular Signaling Peptides and Proteins; Macrophage Activation; Membrane Proteins; Phytotherapy; Plant Extracts; Podocytes; Proteinuria; Rats; Rats, Wistar; RNA, Messenger; Thy-1 Antigens; Tripterygium

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

Immunosuppressants and inhibitors of the renin angiotensin system are major reagents to treat nephrotic syndrome but their clinical effects are not necessarily satisfactory. Injection of doxorubicin in several strains of mice causes nephrotic syndrome-like disorder. Zhou et al. report that PPAR-α expression is downregulated in murine doxorubicin nephropathy and a PPAR-α agonist, fenofibrate, partially ameliorates the disorder induced likely through stabilization of nephrin expression and suppression of apoptosis in podocytes, providing a new preventive strategy.

Topics: Animals; Apoptosis; Caspase 3; Cytoprotection; Disease Models, Animal; Doxorubicin; Fenofibrate; Kidney Diseases; Kidney Glomerulus; Membrane Proteins; Mice; Mice, Knockout; Podocytes; PPAR alpha; Proteinuria; Proto-Oncogene Proteins c-bcl-2; Time Factors; Transcription, Genetic

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

Acute Tubular Necrosis (ATN) causes severe damage to the kidney epithelial tubular cells and is often associated with severe renal dysfunction. Stem-cell based therapies may provide alternative approaches to treating of ATN. We have previously shown that clonal c-kit(pos) stem cells, derived from human amniotic fluid (hAFSC) can be induced to a renal fate in an ex-vivo system. Herein, we show for the first time the successful therapeutic application of hAFSC in a mouse model with glycerol-induced rhabdomyolysis and ATN. When injected into the damaged kidney, luciferase-labeled hAFSC can be tracked using bioluminescence. Moreover, we show that hAFSC provide a protective effect, ameliorating ATN in the acute injury phase as reflected by decreased creatinine and BUN blood levels and by a decrease in the number of damaged tubules and apoptosis therein, as well as by promoting proliferation of tubular epithelial cells. We show significant immunomodulatory effects of hAFSC, over the course of ATN. We therefore speculate that AFSC could represent a novel source of stem cells that may function to modulate the kidney immune milieu in renal failure caused by ATN.

Topics: Amniotic Fluid; Animals; Apoptosis; Blood Urea Nitrogen; Cell Proliferation; Creatinine; Cytokines; Disease Models, Animal; Embryonic Stem Cells; Gene Expression; Glycerol; Humans; Karyotyping; Kidney; Kidney Tubular Necrosis, Acute; Luciferases; Luminescent Measurements; Membrane Proteins; Mice; Mice, Nude; PAX2 Transcription Factor; Reverse Transcriptase Polymerase Chain Reaction; Rhabdomyolysis; Stem Cell Transplantation; Transplantation, Heterologous

Glomerular slit diaphragm (SD) represents a modified adherens junction composed of molecules belonging to both immunoglobulin and cadherin superfamilies. Cadherins associate with the cytosolic scaffolding protein beta-catenin, but the precise role of beta-catenin in mature or injured podocytes is not known.. The conditional podocyte-specific beta-catenin-deficient mouse line was generated using the doxycycline-inducible Cre-loxP system. Expression of the beta-catenin-deficient gene was turned off at the age of 8 weeks by doxycycline treatment and the kidney phenotype was analysed. In addition, beta-catenin-deficient and control mice were treated with adriamycin (ADR) and analysed for albuminuria and morphological alterations.. Deletion of beta-catenin in mature podocytes did not change the morphology of podocytes nor did it lead to albuminuria. However, lack of beta-catenin attenuated albuminuria after ADR treatment. Electron microscopic examination showed increased podocyte foot process effacement associated with SD abnormalities in ADR-treated control mice compared to beta-catenin-deficient mice.. These results show that beta-catenin in podocytes is dispensable for adult mice, but appears to be important in modulating the SD during ADR-induced perturbation of the filtration barrier.

Topics: Albuminuria; Animals; Antibiotics, Antineoplastic; beta Catenin; Cadherins; Disease Models, Animal; Doxorubicin; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Membrane Proteins; Mice; Mice, Transgenic; Phenotype; Podocytes

Hepatocyte growth factor (HGF) is a potent antifibrotic protein that inhibits kidney fibrosis through several mechanisms. To study its role in podocyte homeostasis, injury, and repair in vivo, we generated conditional knockout mice in which the HGF receptor, c-met, was specifically deleted in podocytes using the Cre-LoxP system. Mice with podocyte-specific ablation of c-met (podo-met(-/-)) developed normally. No albuminuria or overt pathologic lesions were detected up to 6 months of age, suggesting that HGF signaling is dispensable for podocyte maturation, survival, and function under normal physiologic conditions. However, after adriamycin treatment, podo-met(-/-) mice developed more severe podocyte injury and albuminuria than their control littermates. Ablation of c-met also resulted in more profound suppression of Wilms tumor 1 (WT1) and nephrin expression, and podocyte apoptosis after injury. When HGF was expressed ectopically in vivo, it ameliorated adriamycin-induced albuminuria, preserved WT1 and nephrin expression, and inhibited podocyte apoptosis. However, exogenous HGF failed to significantly reduce albuminuria in podo-met(-/-) mice, suggesting that podocyte-specific c-met activation by HGF confers renal protection. In vitro, HGF was able to preserve WT1 and nephrin expression in cultured podocytes after adriamycin treatment. HGF also protected podocytes from apoptosis induced by a lethal dose of adriamycin primarily through a phosphoinositide 3-kinase (PI3K)/Akt-dependent pathway. Collectively, these results indicate that HGF/c-met signaling has an important role in protecting podocytes from injury, thereby reducing proteinuria.

Topics: Age Factors; Albuminuria; Animals; Apoptosis; Cells, Cultured; Cytoprotection; Disease Models, Animal; Doxorubicin; Hepatocyte Growth Factor; Kidney Diseases; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Phosphatidylinositol 3-Kinases; Podocytes; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-met; Signal Transduction; Time Factors; WT1 Proteins

Several proteins constituting the slit diaphragm are considered important for maintaining capillary wall permselectivity. Early intervention with blockers of angiotensin II receptors (AR) and mineralocorticoid receptors (MR) is effective against proteinuria in models of chronic hypertensive and protein-induced renal damage. However, the effects of AR and/or MR blockers in a model of acute nephrotic syndrome remain unknown. The effects of AR and MR blockers were examined in puromycin aminonucleoside (PAN)-treated rats.. Six week old male Sprague-Dawley (SD) rats were injected with PAN or vehicle and assigned to groups as follows: vehicle (group C); PAN (group P); PAN followed 3 days later by administration of the MR blocker, eplerenone (group MR), and by the AR blocker, losartan (group AR). Blood pressure and urinary protein excretion were measured and all rats were killed for immunohistochemical investigation on day 14 after PAN administration.. Blood pressure did not change throughout the study period. Proteinuria was decreased in groups MR and AR compared with group P (on day 14 after PAN administration, respectively; group P vs AR, P < 0.01; group P vs MR, P < 0.05). Nephrin, podocin and podocalyxin staining was preserved in the glomeruli of groups MR and AR compared with group P.. The MR and AR blockers decreased proteinuria in the acute model of nephrotic syndrome with preserved expression of glomerular podocyte protein independently of blood pressure.

Topics: Angiotensin II Type 1 Receptor Blockers; Animals; Blood Pressure; Disease Models, Animal; Eplerenone; Fluorescent Antibody Technique; Intracellular Signaling Peptides and Proteins; Losartan; Male; Membrane Proteins; Mineralocorticoid Receptor Antagonists; Nephrosis, Lipoid; Podocytes; Proteinuria; Puromycin Aminonucleoside; Rats; Rats, Sprague-Dawley; Sialoglycoproteins; Spironolactone; Time Factors

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

Membranous nephropathy is a disease that affects the filtering units of the kidney, the glomeruli, and results in proteinuria accompanied by loss of kidney function. Passive Heymann nephritis is an experimental model that mimics membranous nephropathy in humans, wherein the glomerular epithelial cell (GEC) injury induced by complement C5b-9 leads to proteinuria. We examined the role of cytochrome P450 2B1 (CYP2B1) in this complement-mediated sublytic injury. Overexpression of CYP2B1 in GECs significantly increased the formation of reactive oxygen species, cytotoxicity, and collapse of the actin cytoskeleton following treatment with anti-tubular brush-border antiserum (anti-Fx1A). In contrast, silencing of CYP2B1 markedly attenuated anti-Fx1A-induced reactive oxygen species generation and cytotoxicity with preservation of the actin cytoskeleton. Gelsolin, which maintains an organized actin cytoskeleton, was significantly decreased by complement C5b-9-mediated injury but was preserved in CYP2B1-silenced cells. In rats injected with anti-Fx1A, the cytochrome P450 inhibitor cimetidine blocked an increase in catalytic iron and ROS generation, reduced the formation of malondialdehyde adducts, maintained a normal distribution of nephrin in the glomeruli, and provided significant protection at the onset of proteinuria. Thus, GEC CYP2B1 contributes to complement C5b-9-mediated injury and plays an important role in the pathogenesis of passive Heymann nephritis.

Topics: Animals; Antibodies; Cimetidine; Complement Membrane Attack Complex; Cytochrome P-450 CYP2B1; Disease Models, Animal; Enzyme Inhibitors; Gene Silencing; Glomerulonephritis, Membranous; Heymann Nephritis Antigenic Complex; Kidney Glomerulus; Kidney Tubules; Membrane Proteins; Microvilli; Rats; Reactive Oxygen Species

Sepsis is induced by infectious challenges, and septic organ failure often occurs under local and systemic inflammation. Albuminuria is also evident during sepsis, but little is known about the molecular basis of septic albuminuria. Using lipopolysaccharide (LPS)-treated mice as a sepsis model, we found that the loss of nephrin, a key component for maintaining podocyte slit diaphragm, became evident in accordance with the onset of albuminuria, especially 36 h post-LPS challenge (i.e., albumiuric stage). Likewise, nephrin mRNA levels were decreased to 13% of saline-treated mice. Such a transcriptional suppression of nephrin was associated with the loss of nucleus-localized Wilms tumor-1 (WT1), a transcriptional factor for up-regulating nephrin gene. Thereafter, urinary albumin levels were decreased in mice between 72 and 96 h post-LPS challenge (i.e., recovery-stage). Notably, nuclear localization of WT1 seemed to be normalized, and nephrin mRNA and protein levels returned near the basal level 72 h post-LPS challenge. During LPS-mediated sepsis, there was a transient increase in blood interleukin-1β, a suppressor of nephrin production in podocytes. Therefore, down-regulation of nephrin by the loss in nuclear WT1, along with hyper-cytokinemia, may underlie the mechanisms by which albuminuria is induced by infectious stresses.

Topics: Albuminuria; Animals; Disease Models, Animal; Down-Regulation; Female; Interleukin-1beta; Lipopolysaccharides; Membrane Proteins; Mice; Mice, Inbred C57BL; Podocytes; RNA, Messenger; Sepsis; Transcription Factors; WT1 Proteins

Pigment epithelium-derived factor (PEDF) is a glycoprotein with potent neuronal differentiating activity. We, along with others, have recently found that PEDF inhibits retinal hyperpermeability by counteracting the biological effects of vascular endothelial growth factor (VEGF). However, the protective role of PEDF against nephrotic syndrome (NS), a condition of hyperpermeability in the glomerular capillaries, remains to be elucidated. In this study, we investigated whether and how PEDF reduced proteinuria in rats with adriamycin (ADR)-induced nephropathy (ADN), an experimental model of NS.. ADN was induced by a single intravenous injection of doxorubicin hydrochloride (n = 12). Half the ADN rats were intravenously administrated human recombinant PEDF; the other half were given vehicle everyday for up to 14 days. Control rats (n = 6) received vehicle only.. In ADN, expression levels of PEDF in isolated glomeruli were significantly decreased, which were associated with a marked proteinuria and increased urinary excretion of nephrin, an index of podocyte damage. Loss of nephrin and decreased podocyte cell number and fusion of foot processes of podocytes with nuclear factor-kappa B (NF-kappaB) activation and VEGF overexpression were also observed in the glomeruli of rats with ADN. Intravenous administration of PEDF ameliorated all of these changes in ADN rats.. The present findings suggest that PEDF could reduce proteinuria by suppressing podocyte damage and decreased nephrin as well as increased VEGF expression in the glomeruli of ADN rats. Pharmacological up-regulation or substitution of PEDF may offer a promising therapeutic strategy for the treatment of nephrotic syndrome.

Topics: Animals; Disease Models, Animal; Doxorubicin; Eye Proteins; Humans; Injections, Intravenous; Kidney Glomerulus; Male; Membrane Proteins; Nephrotic Syndrome; Nerve Growth Factors; NF-kappa B; Podocytes; Proteinuria; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Recombinant Proteins; Serpins; Vascular Endothelial Growth Factor A

Progressive proteinuria and glomerulosclerosis characterize chronic allograft nephropathy. However, the causes are not fully elucidated. Podocytes function to prevent proteinuria; injury to this glomerular cell leads to glomerulosclerosis. The potential role of podocytes in the failing transplanted kidney is unknown. A rat model of kidney transplantation, characterized by proteinuria and glomerulosclerosis, was utilized to examine the potential role of podocytes.. Archival tissue was examined from allografts (Dark Agouti kidneys transplanted into operationally tolerant Albino Surgery rats), isografts (Dark Agouti) and controls (Dark Agouti: age-matched or after unilateral nephrectomy). The number of podocytes (by WT-1 staining) as well as the podocyte proteins podocin, nephrin and synaptopodin (by immunostaining) were measured at days 0, 2, 6 and at 6 months after transplantation. Changes in these parameters were compared between groups and correlated with urinary protein excretion.. At 6 months, podocyte number was reduced in allografted kidneys, accompanied by a decrease in nephrin and synaptopodin, but not podocin staining. Remnant kidneys in the uninephrectomized rats also showed a decreased podocyte number but no change in podocyte protein staining. Podocyte loss in allografts was established on day 6, whereas a decrease in nephrin and synaptopodin was not evident until 6 months. In contrast, podocyte number and protein staining was decreased but not significantly so in remnant and isografted kidneys.. A decrease in the slit diaphragm proteins, nephrin and synaptopodin, is a component of chronic allograft pathology.

Topics: Animals; Cell Count; Chronic Disease; Creatinine; Disease Models, Animal; Glomerulonephritis; Kidney Transplantation; Male; Membrane Proteins; Microfilament Proteins; Podocytes; Proteinuria; Rats; Rats, Inbred Strains; Transplantation, Homologous

Nephrin is an essential structural component of the glomerular slit diaphragm (SD), a highly organized intercellular junction that constitutes the ultrafiltration barrier of the kidney. Recent studies have identified two additional nephrin-interacting SD proteins (NEPH1 and NEPH2), suggesting that the zipper-like pattern of the SD is formed through complex intra- and intermolecular interactions of these proteins. However, the complexity of the SD structure suggests that additional SD components remain to be discovered. In this study, we identified galectin-1 (Gal-1) as a new component of the SD, binding to the ectodomain of nephrin. Using dual-immunofluorescence and confocal microscopy and dual-immunoelectron microscopy, we found Gal-1 co-localizing with the ectodomain of nephrin at the SD in normal human kidney. By immunoprecipitation and surface plasmon resonance, we confirmed a direct molecular interaction between Gal-1 and nephrin. Moreover, recombinant Gal-1 induced tyrosine phosphorylation of the cytoplasmic domain of nephrin and activation of the extracellular signal-regulated kinase 1/2 in podocytes. We also showed that podocytes are a major site of biosynthesis of Gal-1 in the glomerulus and that the normal expression patterns and levels of Gal-1 are altered in patients with minimal change nephrotic syndrome. Finally, in puromycin aminonucleoside-induced rat nephrosis, an apparent reduction in the levels of Gal-1 and nephrin around the onset of heavy proteinuria was also revealed. Our data present Gal-1 as a new extracellular ligand of nephrin localized at the glomerular SD, and provide further insight into the complex molecular organization, interaction, and structure of the SD, which is an active site of intracellular signaling necessary for podocyte function.

Topics: Animals; Cell Line, Transformed; Disease Models, Animal; Female; Fluorescent Antibody Technique, Indirect; Galectin 1; Humans; Kidney; Membrane Proteins; Microscopy, Confocal; Microscopy, Fluorescence; Microscopy, Immunoelectron; Mitogen-Activated Protein Kinase 3; Nephrosis, Lipoid; Phosphorylation; Podocytes; Rats; Recombinant Proteins; Tyrosine

Activation of protein kinase C (PKC) has been implicated in the pathogenesis of diabetic nephropathy where therapy targeting the beta isoform of this enzyme is in advanced clinical development. However, PKC-beta is also increased in various forms of human glomerulonephritis with several potentially nephrotoxic factors, other than high glucose, resulting in PKC-beta activation. Accordingly, we sought to examine the effects of PKC-beta inhibition in a non-diabetic model of progressive kidney disease.. Subtotally nephrectomized (STNx) rats were randomly assigned to receive either the selective PKC-beta inhibitor, ruboxistaurin or vehicle. In addition to functional and structural parameters, gene expression of the podocyte slit-pore diaphragm protein, nephrin, was also assessed.. STNx animals developed hypertension, proteinuria and reduced glomerular filtration rate (GFR) in association with marked glomerulosclerosis and tubulointerstitial fibrosis. Glomerular nephrin expression was also reduced. Without affecting blood pressure, ruboxistaurin treatment attenuated the impairment in GFR and reduced the extent of both glomerulosclerosis and tubulointerstitial fibrosis in STNx rats. In contrast, neither proteinuria nor the reduction in nephrin expression was improved by ruboxistaurin.. These findings indicate firstly that PKC-beta inhibition may provide a new therapeutic strategy in non-diabetic kidney disease and secondly that improvement in GFR is not inextricably linked to reduction in proteinuria.

Topics: Animals; Disease Models, Animal; Enzyme Inhibitors; Glomerular Filtration Rate; Humans; Indoles; Kidney; Kidney Diseases; Male; Maleimides; Membrane Proteins; Mesangial Cells; Protein Kinase C; Protein Kinase C beta; Rats; Rats, Sprague-Dawley; RNA, Messenger; Transforming Growth Factor beta

Recent studies have reported that statins have renoprotective effects, independent from lowering plasma cholesterol. In this study, we examined whether statins were beneficial in a murine model of HIV-associated nephropathy (HIVAN).. We used conditional transgenic mice that express one of the HIV-1 accessory genes, vpr, selectively in podocytes using podocin promoter and the Tet-on system. These mice develop aggressive collapsing focal segmental glomerular sclerosis with massive proteinuria and deterioration of renal function within 4 weeks following heminephrectomy and doxycycline administration. Fluvastatin was administrated simultaneously with doxycycline, and the effect was compared with untreated controls after 4 weeks.. Fluvastatin at 10 mg/kg/day significantly decreased urinary albumin excretion (87 versus 11 mg/day, P < 0.01) and glomerular sclerosis (2.4 versus 1.0, P < 0.01, assessed by semi-quantitative scoring: 0-4). Fluvastatin also decreased serum creatinine and total cholesterol, but these differences were not statistically significant (0.36 versus 0.32 mg/dl, P = 0.35; 492 versus 378 mg/dl, P = 0.11, respectively). Phenotypic changes in podocytes, as indicated by the downregulation of nephrin, Wilms' tumour 1 and synaptopodin, along with upregulation of proliferating cell nuclear antigen, were attenuated by fluvastatin, suggesting its protective effects against podocyte injuries. In cultured podocytes, angiotensin II treatment decreased nephrin expression to 13% of basal levels, which was reversed to 58% by adding fluvastatin.. In conclusion, fluvastatin was effective in treating experimental HIVAN. The beneficial effect of this drug might be caused, in part, by preserving nephrin expression in podocytes against angiotensin II-mediated injury.

Topics: AIDS-Associated Nephropathy; Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Animals; Blotting, Western; Cells, Cultured; Disease Models, Animal; Fatty Acids, Monounsaturated; Fluorescent Antibody Technique; Fluvastatin; Genes, Wilms Tumor; Glomerulosclerosis, Focal Segmental; Immunoenzyme Techniques; Indoles; Kidney; Male; Membrane Proteins; Mice; Mice, Transgenic; Microfilament Proteins; Nephrectomy; Phenotype; Podocytes; Proteinuria

Podocytopenia characterizes many forms of glomerular disease, preceding the development of glomerulosclerosis. While detachment of viable podocytes from the underlying glomerular basement membrane is an important mechanism of podocyte loss, the underlying factors involved remain unclear. Secreted protein acidic and rich in cysteine (SPARC), a matricellular protein with counteradhesive properties, is normally expressed at low levels by the podocyte but is markedly increased following podocyte injury. Accordingly, we elucidate the role of SPARC in mediating experimental crescentic glomerulonephritis by inducing passive nephrotoxic nephritis in SPARC(+/+) and SPARC(-/-) mice. By days 4, 7, and 21 following disease induction, podocyte number is better preserved, glomerulosclerosis is ameliorated, and proteinuria is reduced in SPARC(-/-) mice as compared with SPARC(+/+) littermates. Moreover, the preserved podocyte number in SPARC(-/-) mice correlates with reduced urinary levels of both nephrin and podocin. To establish a causal role for SPARC in mediating detachment, cultured SPARC(+/+) and SPARC(-/-) podocytes were subjected to mechanical strain as well as trypsin digestion, and detachment assays were performed. While podocytes lacking SPARC were more resistant to stretch-induced detachment, stable re-expression of SPARC restored detachment rates to levels comparable with SPARC(+/+) podocytes. Taken together, this study proves that SPARC plays a causal role in mediating podocyte detachment and accelerating glomerulosclerosis in experimental crescentic glomerulonephritis.

Topics: Animals; Cells, Cultured; Disease Models, Animal; Disease Progression; Glomerulonephritis; Immunoblotting; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mice; Mice, Knockout; Osteonectin; Podocytes

Focal segmental glomerulosclerosis (FSGS) is a progressive renal disease, and the glomerular visceral cell hyperplasia typically observed in cellular/collapsing FSGS is an important pathological factor in disease progression. However, the cellular features that promote FSGS currently remain obscure. To determine both the origin and phenotypic alterations in hyperplastic cells in cellular/collapsing FSGS, the present study used a previously described FSGS model in p21-deficient mice with visceral cell hyperplasia and identified the podocyte lineage by genetic tagging. The p21-deficient mice with nephropathy showed significantly higher urinary protein levels, extracapillary hyperplastic indices on day 5, and glomerular sclerosis indices on day 14 than wild-type controls. X-gal staining and immunohistochemistry for podocyte and parietal epithelial cell (PEC) markers revealed progressive podocytopenia with capillary collapse accompanied by PEC hyperplasia leading to FSGS. In our investigation, non-tagged cells expressed neither WT1 nor nestin. Ki-67, a proliferation marker, was rarely associated with podocytes but was expressed at high levels in PECs. Both terminal deoxynucleotidyl transferase dUTP nick-end labeling staining and electron microscopy failed to show evidence of significant podocyte apoptosis on days 5 and 14. These findings suggest that extensive podocyte loss and simultaneous PEC hyperplasia is an actual pathology that may contribute to the progression of cellular/collapsing FSGS in this mouse model. Additionally, this is the first study to demonstrate the regulatory role of p21 in the PEC cell cycle.

Topics: Animals; Apoptosis; Cell Lineage; Cyclin-Dependent Kinase Inhibitor p21; Disease Models, Animal; Glomerular Filtration Rate; Glomerulosclerosis, Focal Segmental; Hyperplasia; In Situ Nick-End Labeling; Integrases; Ki-67 Antigen; Kidney Glomerulus; Male; Membrane Proteins; Mice; Mice, Knockout; Podocytes; Proteinuria; WT1 Proteins

Within the glomerulus, the scaffolding protein nephrin bridges the actin-rich foot processes that extend from adjacent podocytes to form the slit diaphragm. Mutations affecting a number of slit diaphragm proteins, including nephrin, cause glomerular disease through rearrangement of the actin cytoskeleton and disruption of the filtration barrier. We recently established that the Nck family of Src homology 2 (SH2)/SH3 cytoskeletal adaptor proteins can mediate nephrin-dependent actin reorganization. Formation of foot processes requires expression of Nck in developing podocytes, but it is unknown whether Nck maintains podocyte structure and function throughout life. Here, we used an inducible transgenic strategy to delete Nck expression in adult mouse podocytes and found that loss of Nck expression rapidly led to proteinuria, glomerulosclerosis, and altered morphology of foot processes. We also found that podocyte injury reduced phosphorylation of nephrin in adult kidneys. These data suggest that Nck is required to maintain adult podocytes and that phosphotyrosine-based interactions with nephrin may occur in foot processes of resting, mature podocytes.

Topics: Adaptor Proteins, Signal Transducing; Animals; Anti-Bacterial Agents; Cell Line; Disease Models, Animal; Doxycycline; Glomerular Filtration Rate; Glomerulonephritis; Kidney Glomerulus; Male; Membrane Proteins; Mice; Mice, Transgenic; Oncogene Proteins; Phosphorylation; Podocytes; Proteinuria; Puromycin Aminonucleoside; Rats; Rats, Sprague-Dawley; Tight Junctions

Elevated level of homocysteine (Hcy), known as hyperhomocysteinemia (HHcy), is associated with end-stage renal diseases. Hcy metabolizes in the body to produce hydrogen sulfide (H(2)S), and studies have demonstrated a protective role of H(2)S in end-stage organ failure. However, the role of H(2)S in HHcy-associated renal diseases is unclear. The present study was aimed to determine the role of H(2)S in HHcy-associated renal damage. Cystathionine-beta-synthase heterozygous (CBS+/-) and wild-type (WT, C57BL/6J) mice with two kidney (2-K) were used in this study and supplemented with or without NaHS (30 micromol/l, H(2)S donor) in the drinking water. To expedite the HHcy-associated glomerular damage, uninephrectomized (1-K) CBS(+/-) and 1-K WT mice were also used with or without NaHS supplementation. Plasma Hcy levels were elevated in CBS(+/-) 2-K and 1-K and WT 1-K mice along with increased proteinuria, whereas, plasma levels of H(2)S were attenuated in these groups compared with WT 2-K mice. Interestingly, H(2)S supplementation increased plasma H(2)S level and normalized the urinary protein secretion in the similar groups of animals as above. Increased activity of matrix metalloproteinase (MMP)-2 and -9 and apoptotic cells were observed in the renal cortical tissues of CBS(+/-) 2-K and 1-K and WT 1-K mice; however, H(2)S prevented apoptotic cell death and normalized increased MMP activities. Increased expression of desmin and downregulation of nephrin in the cortical tissue of CBS(+/-) 2-K and 1-K and WT 1-K mice were ameliorated with H(2)S supplementation. Additionally, in the kidney tissues of CBS(+/-) 2-K and 1-K and WT 1-K mice, increased superoxide (O(2)(*-)) production and reduced glutathione (GSH)-to-oxidized glutathione (GSSG) ratio were normalized with exogenous H(2)S supplementation. These results demonstrate that HHcy-associated renal damage is related to decreased endogenous H(2)S generation in the body. Additionally, here we demonstrate with evidence that H(2)S supplementation prevents HHcy-associated renal damage, in part, through its antioxidant properties.

Topics: Animals; Antioxidants; Apoptosis; Cystathionine beta-Synthase; Desmin; Disease Models, Animal; Glutathione; Glutathione Disulfide; Homocysteine; Hydrogen Sulfide; Hyperhomocysteinemia; Kidney; Kidney Failure, Chronic; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Nephrectomy; Oxidative Stress; Proteinuria; Sulfides; Superoxides

Inhibition of soluble epoxide hydrolase (sEH) has been shown to be renal protective in rat models of salt-sensitive hypertension. Here, we hypothesize that targeted disruption of the sEH gene (Ephx2) prevents both renal inflammation and injury in deoxycorticosterone acetate plus high salt (DOCA-salt) hypertensive mice. Mean arterial blood pressure (MAP) increased significantly in the DOCA-salt groups, and MAP was lower in Ephx2-/- DOCA-salt (129 +/- 3 mmHg) compared with wild-type (WT) DOCA-salt (145 +/- 2 mmHg) mice. Following 21 days of treatment, WT DOCA-salt urinary MCP-1 excretion increased from control and was attenuated in the Ephx2-/- DOCA-salt group. Macrophage infiltration was reduced in Ephx2-/- DOCA-salt compared with WT DOCA-salt mice. Albuminuria increased in WT DOCA-salt (278 +/- 55 microg/day) compared with control (17 +/- 1 microg/day) and was blunted in the Ephx2-/- DOCA-salt mice (97 +/- 23 microg/day). Glomerular nephrin expression demonstrated an inverse relationship with albuminuria. Nephrin immunofluorescence was greater in the Ephx2-/- DOCA-salt group (3.4 +/- 0.3 RFU) compared with WT DOCA-salt group (1.1 +/- 0.07 RFU). Reduction in renal inflammation and injury was also seen in WT DOCA-salt mice treated with a sEH inhibitor {trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid; tAUCB}, demonstrating that the C-terminal hydrolase domain of the sEH enzyme is responsible for renal protection with DOCA-salt hypertension. These data demonstrate that Ephx2 gene deletion decreases blood pressure, attenuates renal inflammation, and ameliorates glomerular injury in DOCA-salt hypertension.

Topics: Albuminuria; Animals; Benzoates; Blood Pressure; Chemokine CCL2; Desoxycorticosterone; Disease Models, Animal; Enzyme Inhibitors; Epoxide Hydrolases; Gene Deletion; Hypertension; Kidney Glomerulus; Macrophages; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Nephritis; Protein Structure, Tertiary; Sodium Chloride, Dietary; Time Factors; Urea

Induction of heme oxygenase (HO)-1 is a key defense mechanism against oxidative stress. Compared with tubules, glomeruli are refractory to HO-1 upregulation in response to injury. This can be a disadvantage as it may be associated with insufficient production of cytoprotective heme-degradation metabolites. We, therefore, explored whether 1) targeted HO-1 expression can be achieved in glomeruli without altering their physiological integrity and 2) this expression reduces proteinuria in immune injury induced by an anti-glomerular basement membrane (GBM) antibody (Ab). We employed a 4.125-kb fragment of a mouse nephrin promoter downstream to which a FLAG-tagged hHO-1 cDNA sequence was inserted and subsequently generated transgenic mice from the FVB/N parental strain. There was a 16-fold higher transgene expression in the kidney than nonspecific background (liver) while the transprotein immunolocalized in glomerular epithelial cells (GEC). There was no change in urinary protein excretion, indicating that GEC-targeted HO-1 expression had no effect on glomerular protein permeability. Urinary protein excretion in transgenic mice with anti-GBM Ab injury (days 3 and 6) was significantly lower compared with wild-type controls. There was no significant change in renal expression levels of profibrotic (TGF-beta1) or anti-inflammatory (IL-10) cytokines in transgenic mice with anti-GBM Ab injury. These observations indicate that GEC-targeted HO-1 expression does not alter glomerular physiological integrity and reduces proteinuria in glomerular immune injury.

Topics: Animals; Autoantibodies; Disease Models, Animal; Epithelial Cells; Gene Expression Regulation, Enzymologic; Genetic Vectors; Glomerulonephritis; Heme Oxygenase-1; Interleukin-10; Kidney Glomerulus; Macrophages; Membrane Proteins; Mice; Mice, Transgenic; Permeability; Promoter Regions, Genetic; Proteinuria; Transforming Growth Factor beta1

Impaired glomerular endothelial integrity is pivotal in various renal diseases and depends on both the degree of glomerular endothelial injury and the effectiveness of glomerular endothelial repair. Glomerular endothelial repair is, in part, mediated by bone marrow-derived endothelial progenitor cells. Peroxisome proliferator activated receptor-gamma (PPAR-gamma) agonists have therapeutic actions independent of their insulin-sensitizing effects, including enhancement of endothelial progenitor cell function and differentiation. We evaluated the effect of PPAR-gamma agonist rosiglitazone (4 mg.kg(-1).day(-1)) on the course of anti-Thy1-glomerulonephritis in rats. Rosiglitazone limited the development of proteinuria and prevented plasma urea elevation (8.1 +/- 0.4 vs. 12.5 +/- 1.1 mmol/l, P = 0.002). Histologically, inflammatory cell influx was not affected, but rosiglitazone-treated rats did show fewer microaneurysmatic glomeruli on day 7 (26 +/- 3 vs. 41 +/- 5%, P = 0.01) and reduced activation of matrix production with reduced renal cortical transforming growth factor-beta, plasminogen activator inhibitor type 1, and fibronectin-1 mRNA expression. However, bone marrow-derived endothelial cell glomerular incorporation was not enhanced (3.1 +/- 0.4 vs. 3.6 +/- 0.3 cells/glomerular cross section; P = 0.31). Rosiglitazone treatment in nonnephritic rats did not influence proteinuria, urea, or renal histology. In conclusion, treatment with PPAR-gamma agonist rosiglitazone ameliorates the course of experimental glomerulonephritis in a nondiabetic model, but not through enhancing incorporation of bone marrow-derived endothelial cells in the glomerulus.

Topics: Aneurysm; Animals; Blood Pressure; Bone Marrow Transplantation; Cell Movement; Disease Models, Animal; Endothelial Cells; Extracellular Matrix; Fibronectins; Gene Expression; Glomerulonephritis, Membranous; Hypoglycemic Agents; Isoantibodies; Kidney Cortex; Kidney Glomerulus; Male; Membrane Proteins; Plasminogen Activator Inhibitor 1; PPAR gamma; Proteinuria; Rats; Rats, Inbred BN; Rosiglitazone; Stem Cells; Thiazolidinediones; Transforming Growth Factor beta; Urea

There are several lines of evidence that the podocyte slit diaphragm (SD), which serves as a structural framework for the filtration barrier in kidney glomerulus, also plays an essential role as a signaling platform. Several SD components including nephrin and TRPC6 are known to be phosphorylated by a Src family tyrosine kinase (SFK), Fyn. Here we have characterized Neph1, another SD component, as a novel substrate of SFK. Fyn interacts with and phosphorylates the cytoplasmic domain of Neph1 in vitro and in intact cells. Peptide mass fingerprinting and site-directed mutagenesis identified several tyrosine phosphorylation sites. In pull-down assays using rat glomerular lysates, Neph1 but not nephrin specifically binds to adaptor protein Grb2 and tyrosine kinase Csk in a phosphorylation-dependent manner. Both tyrosine 637 and 638 of Neph1 are crucial for Neph1-Grb2 binding. Phosphorylation of tyrosine 637 is significantly up-regulated in in vivo models of podocyte injury. Furthermore, Neph1 attenuates ERK activation elicited by Fyn, and this inhibitory effect requires the intact binding motif for the Grb2 SH2 domain. Our results shown here demonstrate that Neph1 is a novel in vivo substrate of SFK and suggest that Neph1 modulates ERK signaling through phosphorylation-dependent interaction with Grb2. Thus, SFK orchestrates a wide spectrum of protein-protein interactions and intracellular signaling networks at SD through tyrosine phosphorylation.

Topics: Amino Acid Motifs; Animals; Cell Line; CSK Tyrosine-Protein Kinase; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; GRB2 Adaptor Protein; Humans; Kidney Diseases; Male; MAP Kinase Signaling System; Membrane Proteins; Peptide Mapping; Peptides; Phosphorylation; Podocytes; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-fyn; Rats; Rats, Wistar; src Homology Domains; src-Family Kinases; TRPC Cation Channels; TRPC6 Cation Channel

Mutations in the NPHS1 gene cause congenital nephrotic syndrome of the Finnish type. The gene product nephrin is a structural component of the glomerular slit diaphragm formed by neighboring podocytes. Nephrin has also been suggested to be involved in signaling processes that are important for podocyte survival and differentiation. A new study by Doné et al. reports that the absence of nephrin leads to the lack of slit diaphragms but does not affect podocyte apoptosis and gene expression patterns.

Topics: Animals; Apoptosis; Disease Models, Animal; Gene Expression; Humans; Kidney Glomerulus; Membrane Proteins; Mice; Mice, Knockout; Nephrotic Syndrome; Podocytes

Rho kinase (ROCK) inhibition reduces systemic blood pressure (BP) and decreases renal damage in animal models of kidney disease. The aim of this study was to determine if ROCK inhibition might have beneficial effects in glomerular disease processes that are independent of systemic BP.. We investigated the effects of the ROCK inhibitor Y27632 and hydralazine in murine puromycin aminonucleoside (PAN) nephrosis.. Treatment with either Y27632 or hydralazine similarly reduced systolic BP compared to vehicle-treated controls. Seven days after treatment with PAN, albuminuria, proteinuria and effacement of podocyte foot processes were significantly reduced in Y27632- and hydralazine-treated mice compared to vehicle-treated animals. Treatment with PAN significantly reduced expression of the podocyte proteins nephrin and Neph1, and the loss of glomerular nephrin was attenuated by treatment with Y27632 but not by treatment with hydralazine. In cultured podocytes, PAN potently activated both Rho and ROCK, and PAN-induced ROCK activation was prevented by Y27632.. The ROCK inhibitor Y27632 attenuated glomerular nephrin loss in murine PAN nephrosis independent of its effects on systemic BP.

Topics: Amides; Animals; Antihypertensive Agents; Cells, Cultured; Disease Models, Animal; Epithelial Cells; Kidney Glomerulus; Membrane Proteins; Mice; Nephrosis; Podocytes; Puromycin Aminonucleoside; Pyridines; rho-Associated Kinases

Preeclampsia is a pregnancy-specific disorder characterized by hypertension and proteinuria. In other disease states, proteinuria has been linked to altered expressions of podocyte foot-process proteins, but this has not been studied in women with preeclampsia. We sought to test the hypothesis that proteinuria in preeclampsia is associated with dysregulated expression of the podocyte cytoskeleton and/or tight junction proteins.. Renal tissue was obtained from autopsy material from seven women who had severe preeclampsia during the second half of their pregnancies up to 48 h after delivery, and who subsequently died. As controls, we used autopsy material from two women who died accidentally during the second half of their otherwise normal pregnancies. Immunohistochemical stains for nephrin, synaptopodin and podocin were performed on representative sections prepared from paraffin-embedded material.. Expression of both nephrin and synaptopodin was markedly decreased in preeclamptic compared with control kidney sections. By contrast, both cases and controls demonstrated strong staining for podocin.. We conclude that down-regulation of nephrin and synaptopodin is associated with proteinuria in women with preeclampsia. Recent studies have demonstrated that soluble vascular endothelial growth factor receptor 1 (sFlt-1) levels are elevated in preeclampsia compared with normal pregnancy. Studies in mice have shown that sFlt-1 may play a role in inducing proteinuria by neutralizing vascular endothelial growth factor (VEGF) and suppressing nephrin. Proteinuria and elevations of sFlt-1 in preeclampsia are temporally related, further supporting a possible role of sFlt-1 in the dysregulation of podocyte foot-process proteins.

Topics: Adolescent; Adult; Animals; Antibodies; Biopsy; Case-Control Studies; Disease Models, Animal; Female; Gene Expression Regulation; Humans; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Membrane Proteins; Mice; Mice, Inbred Strains; Microfilament Proteins; Pre-Eclampsia; Pregnancy; Proteinuria; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-1

Podocyte injury is a significant contributor to proteinuria and glomerulosclerosis. Recent studies have shown a renoprotective effect of erythropoietin (EPO) during ischemic kidney disease. In this study, we examine mechanisms by which a long acting recombinant EPO analog, darbepoetin, may confer renoprotection in the puromycin aminonucleoside-induced model of nephrotic syndrome. Darbepoetin decreased the proteinuria of rats treated with puromycin. This protective effect was correlated with the immunohistochemical disappearance of the podocyte injury markers desmin and the immune costimulator molecule B7.1 with the reappearance of nephrin expression in the slit diaphragm. Podocyte foot process retraction and effacement along with actin filament rearrangement, determined by electron microscopy, were all reversed by darbepoetin treatment. The protective effects were confirmed in puromycin-induced nephrotic rats that had been hemodiluted to normal hematocrit levels. Furthermore, puromycin treatment of rat podocytes in culture caused actin cytoskeletal reorganization along with deranged nephrin distribution. All these effects in vitro were reversed by darbepoetin. Our study demonstrates that darbepoetin treatment ameliorates podocyte injury and decreases proteinuria by a direct effect on podocytes. This may be accomplished by maintenance of the actin cytoskeleton and nephrin expression.

Topics: Actins; Animals; Apoptosis; B7-1 Antigen; Cells, Cultured; Cytoskeleton; Darbepoetin alfa; Desmin; Disease Models, Animal; Dose-Response Relationship, Drug; Erythropoietin; In Situ Nick-End Labeling; Male; Membrane Proteins; Nephrotic Syndrome; Podocytes; Protective Agents; Proteinuria; Puromycin Aminonucleoside; Rats; Rats, Wistar; Receptor Protein-Tyrosine Kinases; Receptors, Erythropoietin; RNA, Messenger; Time Factors

Our previous comprehensive analysis of the genes expressed in kidneys with anti-glomerular basement membrane (GBM) nephritis using DNA microarrays showed that SM22alpha was one of the highly expressed genes. SM22alpha is a 22-kDa cytoskeletal protein that is exclusively expressed in smooth muscle cells. We investigated the localization of SM22alpha at mRNA and protein levels, and its pathological significance in anti-GBM nephritis kidneys.. Northern blot analysis, in situ hybridization, immunohistochemistry and double immunofluorescence studies were performed. The specific antibody (Ab) against SM22alpha was obtained by immunization of rabbits with recombinant rat SM22alpha protein.. SM22alpha mRNA expression was upregulated in kidneys and inducibly expressed in the parietal and visceral glomerular epithelial cells in anti-GBM nephritis kidneys. Immunohistochemistry with anti-SM22alpha Ab showed that SM22alpha protein was localized in the same series of cells. Double immunofluorescence with anti-SM22alpha and anti-glomerular cell markers demonstrated that SM22alpha might be expressed in epithelial cells of injured glomeruli. In visceral epithelial cells, SM22alpha might be expressed in cells in which podocyte specific markers, podocalyxin and nephrin were lost.. The injured glomerular epithelial cells in anti-GBM nephritis might undergo structural and functional alterations, including the expression of a smooth muscle marker, SM22alpha.

Topics: Animals; Basement Membrane; Biomarkers; Disease Models, Animal; Epithelial Cells; Gene Expression Regulation; Glomerulonephritis; Immunoglobulin G; Kidney Glomerulus; Male; Membrane Proteins; Microfilament Proteins; Muscle Proteins; Oligonucleotide Array Sequence Analysis; Phenotype; Random Allocation; Rats; Rats, Inbred WKY; RNA, Messenger; Sialoglycoproteins

Studies in experimental animals and younger women suggest a protective role for estrogen; however, clinical trials may not substantiate this effect in older females. Therefore, the present study assessed the outcome of ovariectomy in older mRen2. Lewis rats subjected to a high-salt diet for 4 wk. Intact or ovariectomized (OVX, 15 wk of age) mRen2. Lewis rats were aged to 60 wk and then placed on a high-salt (HS, 8% sodium chloride) diet for 4 wk. Systolic blood pressures were similar between groups [OVX 169 +/- 6 vs. Intact 182 +/- 7 mmHg; P = 0.22] after the 4-wk diet; however, proteinuria [OVX 0.8 +/- 0.2 vs. Intact 11.5 +/- 2.6 mg/mg creatinine; P < 0.002, n = 6], renal interstitial fibrosis, glomerular sclerosis, and tubular casts were lower in OVX vs. Intact rats. Kidney injury molecule-1 mRNA, a marker of tubular damage, was 53% lower in the OVX HS group. Independent from blood pressure, OVX HS rats exhibited significantly lower cardiac (24%) and renal (32%) hypertrophy as well as lower C-reactive protein (28%). Circulating insulin-like growth factor-I (IGF-I) levels were not different between the Intact and OVX groups; however, renal cortical IGF-I mRNA and protein were attenuated in OVX rats [P < 0.05, n = 6]. We conclude that ovariectomy in the older female mRen2. Lewis rat conveys protection against salt-dependent increase in renal injury.

Topics: Aging; Angiotensin I; Angiotensin II; Animals; Animals, Congenic; Blood Pressure; C-Reactive Protein; Cell Adhesion Molecules; Disease Models, Animal; Female; Fibrosis; Hypertension; Hypertrophy; Insulin-Like Growth Factor I; Intracellular Signaling Peptides and Proteins; Kidney; Kidney Diseases; Membrane Proteins; Ovariectomy; Peptide Fragments; Proteinuria; Rats; Rats, Inbred Lew; Renin; Renin-Angiotensin System; RNA, Messenger; Sodium Chloride, Dietary

IFN-inducible protein-10 (IP-10/CXCL10) is a potent chemoattractant for activated T lymphocytes and was reported recently to have several additional biologic activities. In this study, the pathophysiologic role of IP-10 in the glomerular visceral epithelial cell (podocyte) was investigated. In cultured podocytes subjected to recombinant IP-10 treatment, the expression of slit-diaphragm (SD) components nephrin and podocin clearly was heightened. Rats that had puromycin aminonucleoside nephropathy and anti-nephrin antibody-induced nephropathy and were subjected to anti-IP-10 function-blocking antibody (anti-IP-10 mAb) treatment displayed a decrease in the protein level of SD components, as well as exacerbated proteinuria. For exploration of the mechanisms of this process, the interaction between IP-10 and the cell-cycle regulatory proteins was investigated. Cultured podocytes subjected to recombinant IP-10 treatment displayed an increase in the protein level of p27(Kip1), whereas the levels of cyclins E and A decreased. The expression of IP-10 and SD components was heightened by the treatment of siRNA of cyclin A, whereas these expressions were lowered by the treatment of siRNA of p27(Kip1). Proteinuric rats subjected to anti-IP-10 mAb treatment displayed a heightened expression of cyclin A from the early phase of the disease, which indicates that the anti-IP-10 mAb treatment exacerbates podocyte injury by disturbing the cell-cycle balance. These results raise the possibility that IP-10 could become a novel therapeutic target in nephrotic syndrome and several diseases with altered cell-cycle balance.

Topics: Animals; Cell Culture Techniques; Cell Cycle; Chemokine CXCL10; Chemokines, CXC; Disease Models, Animal; Female; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Nephrotic Syndrome; Podocytes; Rats; Rats, Wistar; Receptors, Chemokine; Receptors, CXCR3

Changes in podocyte number or density have been suggested to play an important role in renal disease progression. Here, we investigated the temporal relationship between glomerular podocyte number and development of proteinuria and glomerulosclerosis in the male Munich Wistar Fromter (MWF) rat. We also assessed whether changes in podocyte number affect podocyte function and focused specifically on the slit diaphragm-associated protein nephrin. Age-matched Wistar rats were used as controls. Estimation of podocyte number per glomerulus was determined by digital morphometry of WT1-positive cells. MWF rats developed moderate hypertension, massive proteinuria, and glomerulosclerosis with age. Glomerular hypertrophy was already observed at 10 weeks of age and progressively increased thereafter. By contrast, mean podocyte number per glomerulus was lower than normal in young animals and further decreased with time. As a consequence, the capillary tuft volume per podocyte was more than threefold increased in older rats. Electron microscopy showed important changes in podocyte structure of MWF rats, with expansion of podocyte bodies surrounding glomerular filtration membrane. Glomerular nephrin expression was markedly altered in MWF rats and inversely correlated with both podocyte loss and proteinuria. Our findings suggest that reduction in podocyte number is an important determinant of podocyte dysfunction and progressive impairment of the glomerular permselectivity that lead to the development of massive proteinuria and ultimately to renal scarring.

Topics: Animals; Blotting, Western; Disease Models, Animal; Glomerulosclerosis, Focal Segmental; Hypertension; Immunohistochemistry; Male; Membrane Proteins; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Podocytes; Proteinuria; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Pax2 is a transcription factor with important functions during kidney development . Ectopic expression of Pax2 in podocytes has been reported in various glomerular diseases , but the functional relevance remains unknown. We developed an inducible mouse model that allows activation of Pax2 specifically in podocytes. Persistent expression of Pax2 did not interfere with the initial differentiation of podocytes, but mice ectopically expressing PAX2 developed end-stage renal failure soon after birth. Similarly, activation of PAX2 in healthy adult animals resulted in renal disease within 3 weeks after podocyte-specific induction of a deleter Cre. PAX2 activation caused repression of the podocyte key regulator molecule Wt1 and consequently a dramatic reduction of nephrin expression. Recruitment of the groucho-related protein TLE4 may be involved in converting Pax2 into a transcriptional repressor of Wt1. Finally, treatment of mice with an angiotensin-converting enzyme (ACE) inhibitor normalized renal function and induced upregulation of the important structural molecule nephrin via a Wt1-independent pathway. Our data demonstrate the functional significance of PAX2 reexpression in mature podocytes for the development of glomerular diseases and suggest that reactivation of PAX genes in terminally differentiated cells leads to a more dedifferentiated phenotype.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Disease Models, Animal; Enalapril; Gene Expression Regulation; Kidney Failure, Chronic; Male; Membrane Proteins; Mice; PAX2 Transcription Factor; Podocytes; Proteinuria; WT1 Proteins

Nephrin, a product of the NPHS1 gene, is a component of the slit diaphragms that are found between glomerular foot processes and is a crucial element for glomerular filtration barrier. Recently, nephrin has been focused in a number of studies of proteinuria development including various types of acquired glomerular diseases including minimal change nephrotic syndrome and membranous nephropathy. However, the precise role of nephrin in such acquired glomerular diseases is still unknown. To analyse the role of nephrin further, two kinds of anti-nephrin antibodies were raised in the rabbits and applied to an experimental mouse model of chronic graft-versus-host disease, in which (C57BL/10 x DBA/2) F1 mice developed clinically apparent severe proteinuria with significant glomerular lesions 7 weeks after parental DBA/2 cell transfer. Antibody-sandwich ELISA detected anti-nephrin antibodies during week 2 to week 6, with the peak at week 2 or week 4. Colocalization of nephrin and IgG on week 4, week 6, and week 8 was revealed by confocal microscopic analysis, suggesting that in situ immune complex formation with nephrin in glomerular lesion. Taken together, it seems to be suggested nephrin and its autoantibody have a certain role in the development of glomerular lesion in our model mice.

Topics: Animals; Autoantibodies; Chronic Disease; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Glomerulonephritis; Graft vs Host Disease; Immune Complex Diseases; Lupus Nephritis; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Proteinuria; Rabbits

Nephrin, podocin and alpha-actinin are all involved in proteinuria, but it is unclear which molecular event plays a crucial role during the development of proteinuria. Immunofluorescence staining and real-time quantitative polymerase chain reaction were used to study the glomerular expression of these molecules in puromycin aminonucleoside (PAN) nephrosis. Morphometric methods were applied to evaluate the podocyte foot process (FP) morphology. Two days after PAN injection, nephrin and podocin staining became discontinuous, podocin intensity decreased and FP swelled. Nephrin protein and mRNA decreased at day 5. Both podocin and nephrin intensity decreased dramatically when heavy proteinuria occurred, but nephrin mRNA was regained. When proteinuria disappeared, podocin recovered whereas nephrin did not (P = 0.02); alpha-actinin intensity increased (P = 0.009) and the distribution changed. The podocyte FP volume density correlated negatively with nephrin (r = -0.78, P = 0.0001) and podocin immunofluorescence intensity (r = -0.76, P = 0.0001). We conclude that, before the onset of proteinuria, the first response was the nephrin and podocin distribution change, podocin protein decrease and swollen FP; the podocin quantitative change was earlier than nephrin. Podocin and nephrin distribution and the protein level was associated with proteinuria more closely than their mRNA level. The delayed alpha-actinin induction might be a reparative response.

Topics: Actinin; Animals; Antibiotics, Antineoplastic; Disease Models, Animal; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Male; Membrane Proteins; Nephrosis; Proteins; Proteinuria; Puromycin Aminonucleoside; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Nephrin is a recently identified protein that is a key component of the slit diaphragm. This protein may play a crucial role in maintaining the glomerular filtration barrier, and mutations in the gene for nephrin reportedly lead to congenital nephrosis. However, the expression of nephrin in acquired glomerular disease has not yet been fully clarified. To address this issue, we analysed the expression and localization of nephrin by morphological analysis based on immunoelectron microscopy in normal glomeruli and in glomeruli from proteinuric experimental models.. Twenty rats were divided into three experimental groups (n = 16 total) and a control group (n = 4). Rats in the experimental groups received a single intravenous injection of puromycin aminonucleoside (PAN), and were sacrificed at 1 (n = 4), 2 (n = 6) and 3 weeks (n = 6) post-injection. Nephrin expression was assessed by immunoelectron microscopy using a polyclonal antibody against nephrin and gold particles. It was quantified by counting the gold particles and the slit diaphragms and by measuring the average foot process width in microphotographs.. The average foot process width in the 1 week group (5924.5 +/- 1523.9 nm) was far greater than that of controls (1112.9 +/- 79.8 nm), but decreased thereafter. The average number of total gold particles per unit length (10 000 nm) of the glomerular basement membrane (GBM) underlying the foot processes was reduced at 1 week (26.0 +/- 9.5), compared with controls (335.3 +/- 125.5), but increased thereafter. Also, the average number of junctional gold particles per unit length of the GBM was lower than controls (208.4 +/- 1.7) at 1 week (10.1 +/- 3.5), but increased thereafter. There were no significant differences between the numbers of junctional gold particles per slit diaphragm among the groups, but significant differences were observed in the distributions of gold particles among the groups. Gold particles were more frequently seen in cytoplasm at 1 week.. The present ultrastructural studies showed that nephrin expression and its distribution were altered in PAN-treated rats, and this occurred in parallel with foot process effacement. Nephrin expression returned to normal with improved resolution of the effacement. Nephrin expression was found to be rather preserved in areas without foot process effacement, even in PAN-treated rats. The significance of the above findings in terms of proteinuria and foot process effacement needs further clarification.

Topics: Animals; Disease Models, Animal; Kidney; Kidney Glomerulus; Male; Membrane Proteins; Microscopy, Immunoelectron; Nephrosis; Proteins; Puromycin Aminonucleoside; Rats; Rats, Sprague-Dawley

Renal failure is a frequent and costly complication of many chronic diseases, including diabetes and hypertension. One common feature of renal failure is glomerulosclerosis, the pathobiology of which is unclear. To help elucidate this, we generated a mouse strain carrying the missense mutation Wt1 R394W, which predisposes humans to glomerulosclerosis and early-onset renal failure (Denys-Drash syndrome [DDS]). Kidney development was normal in Wt1(+/R394W) heterozygotes. However, by 4 months of age 100% of male heterozygotes displayed proteinuria and glomerulosclerosis characteristic of DDS patients. This phenotype was observed in an MF1 background but not in a mixed B6/129 background, suggestive of the action of a strain-specific modifying gene(s). WT1 encodes a nuclear transcription factor, and the R394W mutation is known to impair this function. Therefore, to investigate the mechanism of Wt1 R394W-induced renal failure, the expression of genes whose deletion leads to glomerulosclerosis (NPHS1, NPHS2, and CD2AP) was quantitated. In mutant kidneys, NPHS1 and NPHS2 were only moderately downregulated (25 to 30%) at birth but not at 2 or 4 months. Expression of CD2AP was not changed at birth but was significantly upregulated at 2 and 4 months. Podocalyxin was downregulated by 20% in newborn kidneys but not in kidneys at later ages. Two other genes implicated in glomerulosclerosis, TGFB1 and IGF1, were upregulated at 2 months and at 2 and 4 months, respectively. It is not clear whether the significant alterations in gene expression are a cause or a consequence of the disease process. However, the data do suggest that Wt1 R394W-induced glomerulosclerosis may be independent of downregulation of the genes for NPHS1, NPHS2, CD2AP, and podocalyxin and may involve other genes yet to be implicated in renal failure. The Wt1(R394W) mouse recapitulates the pathology and disease progression observed in patients carrying the same mutation, and the mutation is completely penetrant in male animals. Thus, it will be a powerful and biologically relevant model for investigating the pathobiology of the earliest events in glomerulosclerosis.

Topics: Adaptor Proteins, Signal Transducing; Animals; Base Sequence; Cell Division; Cytoskeletal Proteins; Denys-Drash Syndrome; Disease Models, Animal; DNA; Female; Gene Expression; Genes, Wilms Tumor; Glomerulosclerosis, Focal Segmental; Humans; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Microscopy, Electron; Phenotype; Point Mutation; Proteins; Renal Insufficiency; Species Specificity

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

Mutations in the gene encoding alpha-actinin-4 (ACTN4), an actin crosslinking protein, are associated with a form of autosomal dominant focal segmental glomerulosclerosis (FSGS). To better study its progression, a transgenic mouse model was developed by expressing murine alpha-actinin-4 containing a mutation analogous to that affecting a human FSGS family in a podocyte-specific manner using the murine nephrin promoter. Consistent with human ACTN4-associated FSGS, which shows incomplete penetrance, a proportion of the transgenic mice exhibited significant albuminuria (8 of 18), while the overall average systolic BP was elevated in both proteinuric and non-proteinuric ACTN4-mutant mice. Immunofluorescence confirmed podocyte-specific expression of mutant alpha-actinin-4, and real-time RT-PCR revealed that HA-ACTN4 mRNA levels were higher in proteinuric versus non-proteinuric ACTN4-mutant mice. Only proteinuric mice exhibited histologic features consistent with human ACTN4-associated FSGS, including segmental sclerosis and tuft adhesion of some glomeruli, tubular dilatation, mesangial matrix expansion, as well as regions of podocyte vacuolization and foot process fusion. Consistent with such podocyte damage, proteinuric ACTN4-mutant kidneys exhibited significantly reduced mRNA and protein levels of the slit diaphragm component, nephrin. This newly developed mouse model of human ACTN4-associated FSGS suggests a cause-and-effect relationship between actin cytoskeleton dysregulation by mutant alpha-actinin-4 and the deterioration of the nephrin-supported slit diaphragm complex.

Topics: Actin Cytoskeleton; Actinin; Actins; Animals; Blood Pressure; Cloning, Molecular; COS Cells; Disease Models, Animal; DNA, Complementary; Gene Expression; Glomerulosclerosis, Focal Segmental; Humans; In Vitro Techniques; Kidney Glomerulus; Membrane Proteins; Mice; Mice, Transgenic; Microfilament Proteins; Mutagenesis; Proteins; Proteinuria; Transgenes

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

The onset of proteinuria in passive Heymann nephritis, (PHN), a rat model of human membranous nephropathy (MN), is complement-dependent and is associated with altered podocyte slit diaphragm integrity and dissociation of nephrin from the actin cytoskeleton. These studies examined if complement is responsible for these podocyte changes.. PHN was induced with sheep anti-Fx1A. Controls were injected with normal sheep globulin. A third group was injected with anti-Fx1A and depleted of complement with cobra venom factor. Four days later, proteinuria was measured, slit diaphragm integrity was examined by electron microscopy, nephrin distribution was studied by immunofluorescence, and the glomerular content of nephrin and its association with actin were assessed by sequential extraction of isolated glomeruli and Western blotting.. Four days after immunization, seven out of eight PHN rats were proteinuric, whereas none of the complement depleted group had proteinuria despite similar levels of antibody deposition. Complement depletion preserved slit diaphragm morphology. Immunofluorescence microscopy with an antibody to the extracellular domain of nephrin showed a normal staining pattern in the rats depleted of complement and a shift to a more dispersed and clustered pattern in the PHN group. Western blot analysis of the glomerular extracts showed a significant reduction in the total amount of nephrin and in the fraction of actin-associated nephrin in the PHN group, whereas the amounts in the complement-depleted rats were similar to normal controls.. The onset of proteinuria in the PHN model of MN is coincident with complement-dependent alterations in the association of nephrin with the actin cytoskeleton and loss of podocyte slit diaphragm integrity.

Topics: Actins; Animals; Blotting, Western; Complement System Proteins; Disease Models, Animal; Fluorescent Antibody Technique; Glomerulonephritis; Glomerulonephritis, Membranous; Kidney; Membrane Proteins; Microscopy, Electron; Proteins; Proteinuria; Rats; Tissue Distribution

Topics: Animals; Disease Models, Animal; Glomerulonephritis; Glomerulonephritis, Membranous; Membrane Proteins; Proteins; Rats

Molecules of central functional significance for the glomerular podocytes are rapidly emerging and have been shown to be distinctly involved in diseases with altered glomerular filtration barrier. Here we used the puromycin aminonucleoside (PA) nephrosis model in the rat to study some key proteins associated with the maintenance of the functional glomerular filtration barrier in vivo. The molecules studied included the filtration slit component nephrin, the hairpin-like membrane protein podocin, the basolateral adhesion molecules beta1 integrin and alpha-dystroglycan, and the cytoskeleton-linking intermediary beta-catenin and the actin-associated alpha-actinin-4. The results showed diminished protein levels of podocin and nephrin in the PA-treated group. beta-catenin showed distinct down-regulation at 3 days of induction, and the control level was reached at 10 days. beta1 integrin was markedly up-regulated during induction. alpha-actinin-4 was not changed at the studied time points. The results show distinct differences in the different domains of podocytes during PA-induced proteinuria.

Topics: Actinin; Albuminuria; Animals; beta Catenin; Cytoskeletal Proteins; Disease Models, Animal; DNA Primers; Dystroglycans; Female; Immunohistochemistry; Integrin beta1; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Membrane Glycoproteins; Membrane Proteins; Microfilament Proteins; Nephrotic Syndrome; Proteins; Puromycin Aminonucleoside; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Trans-Activators

The location of nephrin has been identified as the slit-diaphragm of the glomerular podocyte. Recent evidence suggests that nephrin could play a key role in the function of the glomerular filtration barrier and the development of proteinuria but its status in long-term diabetes is still not understood. We studied the expression of nephrin in a hypertensive model of diabetic nephropathy and investigated the potential influence of angiotensin II blockade on nephrin gene and protein expression.. Streptozotocin-diabetic spontaneously hypertensive rats were given either no treatment or the angiotensin II antagonist, irbesartan, at a dose of 15 mg/kg per day by gavage for 32 weeks. Non-diabetic spontaneously hypertensive rats were used as a control group. Real time RT-PCR and immunohistochemistry were used to assess and quantify gene and protein expression of nephrin.. Diabetic spontaneously hypertensive rats developed albuminuria and had a reduction in both gene and protein expression of nephrin when compared with control rats. Irbesartan treatment prevented the development of albuminuria and completely abrogated the down regulation of nephrin in diabetic rats.. Long-term diabetes in spontaneously hypertensive rats is associated with a reduction in both gene and protein expression of nephrin within the kidney. These changes in nephrin levels were completely prevented by angiotensin II antagonist treatment, suggesting a potential novel mechanism to explain the antiproteinuric effect of agents which interrupt the renin-angiotensin system.

Topics: Albuminuria; Analysis of Variance; Angiotensin Receptor Antagonists; Animals; Biphenyl Compounds; Diabetes Mellitus, Experimental; Disease Models, Animal; Gene Expression Regulation; Hypertension; Irbesartan; Male; Membrane Proteins; Oligonucleotide Probes; Proteins; Rats; Rats, Inbred SHR; Reverse Transcriptase Polymerase Chain Reaction; Tetrazoles

The recently identified gene NPHS1 with its mutations causing congenital nephrotic syndrome of the Finnish type (CNF) is highly promising in providing new understanding of pathophysiology of proteinuria. Earlier we cloned a rat NPHS1 homologue, as well as characterized and raised antibodies to the respective protein product nephrin.. Changes in the expression levels of nephrin-specific mRNA in commonly used experimental models of proteinuria were examined using semiquantitative reverse transcription-polymerase chain reaction, immunofluorescence, and immunoelectron microscopy (IEM) of nephrin.. Notably, a 40% down-regulation of the nephrin-specific mRNA of cortical kidney was seen already at day 3 after induction of the puromycin aminonucleoside nephrosis (PAN), while no major elevation of urinary protein secretion was seen at this stage. A further decrease of 80% of nephrin message was seen at the peak of proteinuria at day 10. A similar decrease of up to 70% from the basal levels was seen in mercuric chloride-treated rats. Changes in the protein expression paralleled those of the mRNA in indirect immunofluorescence. Interestingly, a remarkable plasmalemmal dislocation from the normal expression site at the interpodocyte filtration slits could be observed in IEM.. Nephrin appears to be an important causative molecule of proteinuria and shows a remarkable redistribution from the filtration slits to the podocyte plasma membrane, especially in PAN.

Topics: Albuminuria; Animals; Antimetabolites, Antineoplastic; Disease Models, Animal; Disinfectants; Gene Expression; Glomerulonephritis; Kidney Glomerulus; Lipid Peroxidation; Male; Membrane Proteins; Mercuric Chloride; Microscopy, Immunoelectron; Proteins; Puromycin Aminonucleoside; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic