nephrin and Diabetes-Mellitus--Type-1

The present study aimed to investigate the interaction between early diabetes and renal IR-induced AKI and to clarify the mechanisms involved. C57BL/6J mice were assigned to the following groups: (1) sham-operated; (2) renal IR; (3) streptozotocin (STZ-55 mg/kg/day) and sham operation; and (4) STZ and renal IR. On the 12th day after treatments, the animals were subjected to bilateral IR for 30 min followed by reperfusion for 48 h, at which time the animals were euthanized. Renal function was assessed by plasma creatinine and urea levels, as well urinary protein contents. Kidney morphology and gene and protein expression were also evaluated. Compared to the sham group, renal IR increased plasma creatinine, urea and albuminuria levels and decreased Nphs1 mRNA expression and nephrin and WT1 protein staining. Tubular injury was observed with increased Havcr1 and Mki67 mRNA expression accompanied by reduced megalin staining. Renal IR also resulted in increased SQSTM1 protein expression and increased proinflammatory and profibrotic factors mRNA expression. Although STZ treatment resulted in hyperglycemia, it did not induce significant changes in renal function. On the other hand, STZ treatment aggravated renal IR-induced AKI by exacerbating renal dysfunction, glomerular and tubular injury, inflammation, and profibrotic responses. Thus, early diabetes constitutes a relevant risk factor for renal IR-induced AKI.

Topics: Acute Kidney Injury; Albuminuria; Animals; Biomarkers; Creatinine; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Disease Progression; Gene Expression; Ischemia; Kidney; Kidney Tubules; Membrane Proteins; Mice, Inbred C57BL; Reperfusion Injury; Risk Factors; Sequestosome-1 Protein

The transient receptor potential canonical 6 (TRPC6) channel and podocin are colocalized in the glomerular slit diaphragm as an important complex to maintain podocyte function. Gain of TRPC6 function and loss of podocin function induce podocyte injury. We have previously shown that high glucose induces apoptosis of podocytes by activating TRPC6; however, whether the activated TRPC6 can alter podocin expression remains unknown. Western blot analysis and confocal microscopy were used to examine both expression levels of TRPC6, podocin, and nephrin and morphological changes of podocytes in response to high glucose. High glucose increased the expression of TRPC6 but reduced the expression of podocin and nephrin, in both cultured human podocytes and type 1 diabetic rat kidneys. The decreased podocin was diminished in TRPC6 knockdown podocytes. High glucose elevated intracellular Ca

Topics: Animals; Calcium; Cell Line; Cells, Cultured; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Gene Knockdown Techniques; Glucose; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Podocytes; Rats; TRPC6 Cation Channel; Zonula Occludens-1 Protein

Assessment of urinary extracellular vesicles including exosomes and microparticles (MPs) is an emerging approach for non-invasive detection of renal injury. We have previously reported that podocyte-derived MPs are increased in diabetic mice in advance of albuminuria. Here, we hypothesised that type 1 diabetes and acute hyperglycaemia would increase urinary podocyte MP levels in uncomplicated diabetes.. In this post hoc exploratory analysis, we examined archived urine samples from normoalbuminuric patients with uncomplicated type 1 diabetes studied under clamped euglycaemia and hyperglycaemia and compared with healthy controls. Urinary vesicles were assessed by electron microscopy and nanoparticle tracking while podocyte MPs were assessed by flow cytometry.. Neither vesicle size nor total number were significantly altered in type 1 diabetes or acute hyperglycaemia. By contrast, urinary podocyte MP levels were higher in type 1 diabetes (0.47 [0.00-3.42] MPs/μmol creatinine [Cr]) compared with healthy controls (0.00 [0.00-0.00] MPs/μmol Cr, p < 0.05) and increased under hyperglycaemic clamp (0.36 [0.00-4.15] MPs/μmol Cr during euglycaemia vs 2.70 [0.00-15.91] MPs/μmol Cr during hyperglycaemia, p < 0.05). Levels of urinary albumin to creatinine ratio and nephrin (surrogates of podocyte injury) were unchanged by type 1 diabetes or acute hyperglycaemia.. Taken together, our data show that urinary podocyte MP levels are higher in patients with type 1 diabetes in advance of changes in other biomarkers (albuminuria, nephrin). Examination of podocyte MPs may serve as an early biomarker of glomerular injury in uncomplicated type 1 diabetes.

Topics: Adult; Albuminuria; Biomarkers; Blood Pressure; Creatinine; Diabetes Mellitus, Type 1; Flow Cytometry; Humans; Hyperglycemia; Male; Membrane Proteins; Microscopy, Electron; Microscopy, Electron, Transmission; Nanoparticles; Podocytes; Young Adult

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

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

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

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

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

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

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

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

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

Nephrin plays a key role in maintaining the structure of the slit diaphragm in the glomerular filtration barrier. Our previous studies have demonstrated potent renoprotective activity for 1,25-dihydroxyvitamin D (1,25(OH)(2)D(3)). Here we showed that in podocytes 1,25(OH)(2)D(3) markedly stimulated nephrin mRNA and protein expression. ChIP scan of the 6-kb 5' upstream region of the mouse nephrin gene identified several putative vitamin D response elements (VDREs), and EMSA confirmed that the VDRE at -312 (a DR4-type VDRE) could be bound by vitamin D receptor (VDR)/retinoid X receptor. Luciferase reporter assays of the proximal nephrin promoter fragment (-427 to +173) showed strong induction of luciferase activity upon 1,25(OH)(2)D(3) treatment, and the induction was abolished by mutations within -312VDRE. ChIP assays showed that, upon 1,25(OH)(2)D(3) activation, VDR bound to this VDRE leading to recruitment of DRIP205 and RNA polymerase II and histone 4 acetylation. Treatment of mice with a vitamin D analog induced nephrin mRNA and protein in the kidney, accompanied by increased VDR binding to the -312VDRE and histone 4 acetylation. 1,25(OH)(2)D(3) reversed high glucose-induced nephrin reduction in podocytes, and vitamin D analogs prevented nephrin decline in both type 1 and 2 diabetic mice. Together these data demonstrate that 1,25(OH)(2)D(3) stimulates nephrin expression in podocytes by acting on a VDRE in the proximal nephrin promoter. Nephrin up-regulation likely accounts for part of the renoprotective activity of vitamin D.

Topics: Acetylation; Animals; Bone Density Conservation Agents; Calcitriol; Cell Line, Transformed; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Gene Expression Regulation; HEK293 Cells; Histones; Humans; Mediator Complex Subunit 1; Membrane Proteins; Mice; Mutation; Podocytes; Receptors, Calcitriol; Retinoid X Receptors; RNA Polymerase II; RNA, Messenger; Vitamin D Response Element

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

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

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

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

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

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

Patients with type 1 diabetes typically develop autoantibodies to antigens of the pancreatic islet cells including insulin, glutamic acid decarboxylase and the protein tyrosine phosphatase-related islet antigen 2 protein. Nephrin is a protein shared by the kidney glomeruli, pancreatic beta-cells and islet microendothelia. Since circulating antibodies to nephrin have been shown to cause proteinuria, we wanted to test whether such autoantibodies can be detected in diabetic patients.. We developed a radioimmunoprecipitation assay and analysed samples in a follow-up series of 66 patients with type 1 diabetes.. A total of 24% of the patients tested positive for nephrin autoantibodies at diagnosis, whereas 23, 14 and 18% had these antibodies at 2, 5 and 10 years, respectively. During the follow-up at 16-19 years after diagnosis, 14 patients had signs of renal injury and 29% of them tested positive for nephrin autoantibodies in at least one sample.. We conclude that a subset of patients with type 1 diabetes present with circulating autoantibodies to nephrin. However, the present data do not allow conclusions of a causative role for these antibodies in the pathogenesis of proteinuria in diabetes.

Topics: Adolescent; Autoantibodies; Child; Child, Preschool; Cohort Studies; Diabetes Mellitus, Type 1; Female; Humans; Immunoprecipitation; Infant; Kidney; Male; Membrane Proteins; Microscopy, Fluorescence; Time Factors

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

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

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

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

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

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

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

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