nephrin and Fibrosis

Topics: Albuminuria; C-Reactive Protein; Creatinine; Diabetes Mellitus, Type 2; Fibrosis; Humans; Inflammation; Interleukin-6; Kidney Diseases; Membrane Proteins; Sodium-Glucose Transporter 2 Inhibitors; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

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

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

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

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

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

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

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

Chronic renal fibrosis is the final common pathway of end stage renal disease caused by glomerular or tubular pathologies. Genetic background has a strong influence on the progression of chronic renal fibrosis. We recently found that Rowett black hooded rats were resistant to renal fibrosis. We aimed to investigate the role of sustained inflammation and oxidative/nitrative stress in renal fibrosis progression using this new model. Our previous data suggested the involvement of podocytes, thus we investigated renal fibrosis initiated by doxorubicin-induced (5 mg/kg) podocyte damage. Doxorubicin induced progressive glomerular sclerosis followed by increasing proteinuria and reduced bodyweight gain in fibrosis-sensitive, Charles Dawley rats during an 8-week long observation period. In comparison, the fibrosis-resistant, Rowett black hooded rats had longer survival, milder proteinuria and reduced tubular damage as assessed by neutrophil gelatinase-associated lipocalin (NGAL) excretion, reduced loss of the slit diaphragm protein, nephrin, less glomerulosclerosis, tubulointerstitial fibrosis and matrix deposition assessed by periodic acid-Schiff, Picro-Sirius-red staining and fibronectin immunostaining. Less fibrosis was associated with reduced profibrotic transforming growth factor-beta, (TGF-β1) connective tissue growth factor (CTGF), and collagen type I alpha 1 (COL-1a1) mRNA levels. Milder inflammation demonstrated by histology was confirmed by less monocyte chemotactic protein 1 (MCP-1) mRNA. As a consequence of less inflammation, less oxidative and nitrative stress was obvious by less neutrophil cytosolic factor 1 (p47phox) and NADPH oxidase-2 (p91phox) mRNA. Reduced oxidative enzyme expression was accompanied by less lipid peroxidation as demonstrated by 4-hydroxynonenal (HNE) and less protein nitrosylation demonstrated by nitrotyrosine (NT) immunohistochemistry and quantified by Western blot. Our results demonstrate that mediators of fibrosis, inflammation and oxidative/nitrative stress were suppressed in doxorubicin nephropathy in fibrosis-resistant Rowett black hooded rats underlying the importance of these pathomechanisms in the progression of renal fibrosis initiated by glomerular podocyte damage.

Topics: Aldehydes; Animals; Body Weight; Chemokine CCL2; Connective Tissue Growth Factor; Disease Progression; Disease Resistance; Dose-Response Relationship, Drug; Doxorubicin; Fibrosis; Kidney; Male; Membrane Proteins; Oxidative Stress; Proteinuria; Rats; Reactive Nitrogen Species; Species Specificity; Transforming Growth Factor beta1; Tyrosine

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

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

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

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

Although angiotensin (Ang) II blockade has become a standard antifibrotic therapy in kidney disease, the therapeutic efficacy of Ang II blockade is yet to be optimized. Considering the prognostic impact of proteinuria reduction, we hypothesized that titration of Ang II blockade for optimal anti-proteinuric effect would improve renoprotection. One day after induction of Thy 1.1 glomeruonephritis, rats were treated with increasing doses of the Ang II receptor blocker valsartan in drinking water. Six days after disease induction, the therapeutic effect on proteinuria, podocyte injury and glomerular fibrosis was evaluated. Increasing doses of valsartan resulted in increasing reduction of proteinuria. The maximally effective dose of valsartan was determined to be 1000 mg/l, which reduced proteinuria by 80% and maximally reduced glomerular matrix expansion, fibronectin, collagen I and collagen III staining and glomerular mRNAs for TGFß1, PAI-1, FN and collagen I. Notably, valsartan given at this dose prevented podocyte dysfunction by preserving expression of podocin and nephrin and the counter-regulating molecule B7-1 that is involved in podocyte injury. These results support the hypothesis that higher doses of valsartan are required to optimize proteinuria reduction and glomerulosclerosis amelioration. Further, the optimal dose of valsartan also provides an additional therapeutic effect by preventing podocyte dysfunction.

Topics: Angiotensin II Type 1 Receptor Blockers; Animals; Dose-Response Relationship, Drug; Fibrosis; Glomerulonephritis; Intracellular Signaling Peptides and Proteins; Kidney; Kidney Function Tests; Male; Membrane Proteins; Podocytes; Proteinuria; Rats; Rats, Sprague-Dawley; Tetrazoles; Valine; Valsartan

The present study employed a zinc-finger nuclease strategy to create heterozygous knockout (KO) rats for the transforming growth factor-β1 (Tgfb1) gene on the Dahl SS/Jr genetic background (TGF-β1(+/-) Dahl S). Intercrossing TGF-β1(+/-) rats did not produce any homozygous KO rats (66.4% +/-, 33.6% +/+), indicating that the mutation is embryonic lethal. Six-week-old wild-type (WT) littermates and TGF-β1(+/-) Dahl S rats were fed a 0.4% (low salt, LS) or 8% NaCl (high salt, HS) diet for 5 wk. Renal cortical expression of TGF-β1, urinary TGF-β1 excretion, proteinuria, glomerular injury and tubulointerstitial fibrosis, and systolic blood pressure were similar in WT and TGF-β1(+/-) Dahl S rats maintained on the LS diet. The expression and urinary excretion of TGF-β1 increased to a greater extent in WT than in TGF-β1(+/-)Dahl S rats fed an HS diet for 1 wk. Systolic blood pressure rose by the same extent to 235 ± 2 mmHg in WT and 239 ± 4 mmHg in TGF-β1(+/-) Dahl S rats fed a HS diet for 5 wk. However, urinary protein excretion was significantly lower in TGF-β1(+/-) Dahl S than in the WT animals. The degree of glomerular injury and renal cortical and outer medullary fibrosis was markedly less in TGF-β1(+/-) than in WT rats. These findings suggest that the loss of one copy of the TGF-β1 gene blunts the increase in renal TGF-β1 protein expression and slows the progression of proteinuria, glomerulosclerosis, and renal interstitial fibrosis in Dahl S rats fed an HS diet independently of changes in blood pressure.

Topics: Animals; Base Sequence; Fibrosis; Gene Expression Regulation; Gene Knockout Techniques; Hypertension; Intracellular Signaling Peptides and Proteins; Kidney; Kidney Diseases; Membrane Proteins; Molecular Sequence Data; Proteinuria; Rats; Rats, Inbred Dahl; Transforming Growth Factor beta1; Transforming Growth Factor beta2; Transforming Growth Factor beta3

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

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

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

Angiotensin (Ang) II induces vascular injury in part by activating innate and adaptive immunity; however, the mechanisms are unclear. We investigated the role of interferon (IFN)-γ and interleukin (IL)-23 signaling. We infused Ang II into IFN-γ receptor (IFN-γR) knockout mice and wild-type controls, as well as into mice treated with neutralizing antibodies against IL-23 receptor and IL-17A. Ang II-treated IFN-γR knockout mice exhibited reduced cardiac hypertrophy, reduced cardiac macrophage and T-cell infiltration, less fibrosis, and less arrhythmogenic electric remodeling independent of blood pressure changes. In contrast, IL-23 receptor antibody treatment did not reduce cardiac hypertrophy, fibrosis, or electric remodeling despite mildly reduced inflammation. IL-17A antibody treatment behaved similarly. In the kidney, IFN-γR deficiency reduced inflammation and tubulointerstitial damage and improved glomerular filtration rate. Nonetheless, albuminuria was increased compared with Ang II-treated wild-type controls. The glomeruli of Ang II-treated IFN-γR knockout mice exhibited fewer podocytes, less nephrin and synaptopodin staining, and impaired podocyte autophagy. Thus, IFN-γ blockade, but not IL-23 receptor antibody treatment, protects from Ang II-induced cardiac damage and electric remodeling. In the kidney, IFN-γ signaling acts in a cell type-specific manner. Glomerular filtration rate is preserved in the absence of the IFN-γR, whereas podocytes may require the IFN-γR in the presence of Ang II for normal integrity and function.

Topics: Angiotensin II; Animals; Blood Pressure; Cardiomegaly; Fibrosis; Heart; Inflammation; Interferon-gamma; Interleukin-17; Interleukin-23; Kidney; Membrane Proteins; Mice; Mice, Knockout; Microfilament Proteins; Myocardium; Podocytes; Signal Transduction

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

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

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

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

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

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

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

Sustained proteinuria is a major factor leading to kidney fibrosis and end-stage renal failure. Tubular epithelial cells are believed to play a crucial role in this process by producing mediators leading to fibrosis and inflammation. Congenital nephrotic syndrome of the Finnish type (NPHS1) is a genetic disease caused by mutations in a podocyte protein nephrin, which leads to constant heavy proteinuria from birth. In this work we studied the tubulointerstitial changes that occur in NPHS1 kidneys during infancy.. The pathologic lesions and expression of profibrotic and proinflammatory factors in nephrectomized NPHS1 kidneys were studied by immunohistochemistry, Western blotting, and cytokine antibody array. Oxidative stress in kidneys was assessed by measurement of gluthatione redox state.. The results indicated that (1) severe tubulointerstitial lesions developed in NPHS1 kidneys during infancy; (2) tubular epithelial cells did not show transition into myofibroblasts as studied by the expression of vimentin, alpha-smooth muscle actin (alpha-SMA), collagen, and matrix metalloproteinases 2 and 9 (MMP-2 and -9); (3) the most abundant chemokines in NPHS1 tissue were neutrophil activating protein-2 (NAP-2), macrophage inhibiting factor (MIF), and monocyte chemoattractant protein-1 (MCP-1); (4) monocyte/macrophage cells expressing CD14 antigen were the major inflammatory cells invading the interstitium; (5) the arteries and arterioles showed intimal hypertrophy, but the microvasculature in NPHS1 kidneys remained quite normal; and (6) excessive oxidative stress was evident in NPHS1 kidneys.. Heavy proteinuria in NPHS1 kidneys was associated with interstitial fibrosis, inflammation, and oxidative stress. The tubular epithelial cells, however, were resistant to proteinuria and did not show epithelial-mesenchymal transition.

Topics: Capillaries; Chemokines; Child, Preschool; Cytokines; Fibrosis; Humans; Infant; Kidney Tubules; Macrophages; Membrane Proteins; Monocytes; Nephritis; Nephrotic Syndrome; Oxidative Stress; Phenotype; Proteinuria

Blockade of the renin-angiotensin system has been established as a treatment for heart failure with hypertension and left ventricular hypertrophy, and for progressive kidney diseases. The present study was conducted to examine whether spironolactone, a mineralocorticoid receptor antagonist, alone or in combination with cilazapril, an angiotensin converting enzyme (ACE) inhibitor, ameliorates proteinuria and renal lesions in an immune-initiated progressive nephritis model. Wistar rats were uninephrectomized 7 days before injection of anti-Thy-1 monoclonal antibody 1-22-3 to induce progressive glomerulonephritis. The nephritic rats were untreated or treated with spironolactone (400 mg/kg body weight/day), cilazapril (1 mg/kg body weight/day), or both for 10 weeks. Proteinuria was increased in the untreated rats 1 week after nephritis induction and was maintained throughout the experiment. Compared with the untreated animals (212.9+/-49.2 mg/day), proteinuria was significantly reduced in the spironolactone-treated group (62.0+/-4.0 mg/day, p=0.0046) and the cilazapril-treated group (71.8+/-26.0 mg/day, p=0.0048) on day 70 after antibody injection. Further reduction of proteinuria (42.4+/-4.5 mg/day, p=0.0019 vs. the untreated group) and less renal cortex interstitial fibrotic change (fibrosis score: 142.0+/-18.4 vs. 80.3+/-18.5 in the untreated group, p=0.0123) were detected in the spironolactone plus cilazapril-treated group. Blood pressure did not differ among the three treatment groups. In conclusion, spironolactone ameliorates proteinuria to the same degree as cilazapril, and concomitant use of spironolactone and an ACE inhibitor further suppresses renal disease progression. These data suggest that concomitant treatment with spironolactone and an ACE inhibitor has beneficial effects on immune-initiated progressive kidney disease.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Antibodies, Monoclonal; Blood Pressure; Cilazapril; Drug Therapy, Combination; Fibrosis; Glomerulonephritis, Membranoproliferative; Kidney; Male; Membrane Proteins; Mineralocorticoid Receptor Antagonists; Proteinuria; Rats; Rats, Wistar; Spironolactone; Thy-1 Antigens

The molecular mechanisms maintaining glomerular filtration barrier are under intensive study. This study describes a mutant Nphs1 mouse line generated by gene-trapping. Nephrin, encoded by Nphs1, is a structural protein of interpodocyte filtration slits crucial for formation of primary urine. Nephrin(trap/trap) mutants show characteristic features of proteinuric disease and die soon after birth. Morphologically, fibrotic glomeruli with distorted structures and cystic tubular lesions were observed, but no prominent changes in the branching morphogenesis of the developing collecting ducts could be found. Western blotting and immunohistochemical analyses confirmed the absence of nephrin in nephrin(trap/trap) glomeruli. The immunohistochemical staining showed also that the interaction partner of nephrin, CD2-associated protein (CD2AP), and the slit-diaphragm-associated protein, ZO-1alpha (-), appeared unchanged, whereas the major anionic apical membrane protein of podocytes, podocalyxin, somewhat punctate as compared with the wild-type (wt) and nephrin(wt/trap) stainings. Electron microscopy revealed that >90% of the podocyte foot processes were fused. The remaining interpodocyte junctions lacked slit diaphragms and, instead, showed tight adhering areas. In the heterozygote glomeruli, approximately one third of the foot processes were fused and real-time RT-PCR showed >60% decrease of nephrin-specific transcripts. These results show an effective nephrin gene elimination, resulting in a phenotype that resembles human congenital nephrotic syndrome. Although the nephrin(trap/trap) mice can be used to study the pathophysiology of the disease, the heterozygous mice may provide a useful model to study the gene dose effect of this crucial protein of the glomerular filtration barrier.

Topics: Animals; Fibrosis; Genotype; Immunohistochemistry; Kidney; Kidney Glomerulus; Kidney Tubules; Membrane Proteins; Mice; Proteins; RNA, Messenger