Nephrin Protein Research Articles

The protective effect of the EP2 receptor on TGF-β1 induced podocyte injury via the PI3K / Akt signaling pathway.

Transforming growth factor β1 (TGF-β1) plays a central role in chronic kidney diseases. TGF-β1 induction causes podocyte injury, which results in proteinuria and renal failure. However, the effect of the prostaglandin E2 /E-prostanoid receptor (EP2) on TGF-β1-induced podocyte injury remains unknown. Previous studies have shown that phosphoinositide 3-OH kinase (PI3K)/Akt is widespread in cells, and is vital for the regulation of cell proliferation, differentiation, apoptosis and metabolism. In this study, we cultured immortalized mouse podocytes in vitro in different groups: control group; TGF-β1 (5ng/ml) group; EP2 agonist Butaprost treatment (10−7, 10−6, or 10-5mol/L) +TGF-β1 group; EP2 antagonist AH6809 treatment (10−7, 10−6, or 10-5mol / L) + TGF-β1 group. We found that compared with the control group, proliferation of podocytes in the TGF-β1 group significantly decreased and apoptosis increased. Expression of cAMP decreased, whereas PGE2 increased. Meanwhile, expressions of nephrin, podocin and CD2AP mRNA and protein were dramatically downregulated, activated caspase-3 was increased, and activated PI3K/Akt activity were depressed. Butaprost intervention promoted podocyte proliferation with reduced apoptosis. Conversely, AH6809 intervention led to opposite results (P<0.05). Our findings suggested that EP2 agonist protects podocytes by increasing expression of cAMP, which creates feedback of inhibiting PGE2 expression. This causes the interaction of nephrin, podocin and CD2AP resulting the inhibition of apoptosis induced by activation of the PI3K / Akt signaling pathway.

Protective effect of calcitriol on podocytes in spontaneously hypertensive rat

Protective effect of calcitriol on podocytes in spontaneously hypertensive rat

β-Arrestin 1/2 Aggravates Podocyte Apoptosis of Diabetic Nephropathy via Wnt/β-Catenin Pathway.

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

Correlation between expressions of VEGF and TRPC6 and their roles in podocyte injury in rats with diabetic nephropathy

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

Podocytes are new cellular targets of haemoglobin-mediated renal damage.

Recurrent and massive intravascular haemolysis induces proteinuria, glomerulosclerosis, and progressive impairment of renal function, suggesting podocyte injury. However, the effects of haemoglobin (Hb) on podocytes remain unexplored. Our results show that cultured human podocytes or podocytes isolated from murine glomeruli bound and endocytosed Hb through the megalin-cubilin receptor system, thus resulting in increased intracellular Hb catabolism, oxidative stress, activation of the intrinsic apoptosis pathway, and altered podocyte morphology, with decreased expression of the slit diaphragm proteins nephrin and synaptopodin. Hb uptake activated nuclear factor erythroid-2-related factor 2 (Nrf2) and induced expression of the Nrf2-related antioxidant proteins haem oxygenase-1 (HO-1) and ferritin. Nrf2 activation and Hb staining was observed in podocytes of mice with intravascular haemolysis. These mice developed proteinuria and showed podocyte injury, characterized by foot process effacement, decreased synaptopodin and nephrin expression, and podocyte apoptosis. These pathological effects were enhanced in Nrf2-deficient mice, whereas Nrf2 activation with sulphoraphane protected podocytes against Hb toxicity both in vivo and in vitro. Supporting the translational significance of our findings, we observed podocyte damage and podocytes stained for Hb, HO-1, ferritin and phosphorylated Nrf2 in renal sections and urinary sediments of patients with massive intravascular haemolysis, such as atypical haemolytic uraemic syndrome and paroxysmal nocturnal haemoglobinuria. In conclusion, podocytes take up Hb both in vitro and during intravascular haemolysis, promoting oxidative stress, podocyte dysfunction, and apoptosis. Nrf2 may be a potential therapeutic target to prevent loss of renal function in patients with intravascular haemolysis. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Angiotensin II Type I Receptor Agonistic Autoantibody Induces Podocyte Injury via Activation of the TRPC6- Calcium/Calcineurin Pathway in Pre-Eclampsia

Background/Aims: Angiotensin II type I receptor agonistic autoantibody (AT1-AA) is closely related to pre-eclampsia, which is characterized by proteinuria and hypertension. AT1-AA has been shown to enhance the effect of AngII in pre-eclampsia, such as production of endothelin-1, activation of ROS, and vasoconstriction, which are considered to be associated with hypertension; however, whether or not AT1-AA participates in podocyte damage leading to the generation of proteinuria has not been reported. In this study we investigated the role of pre-eclamptic serum AT1-AA on podocytes and the mechanism underlying the generation of proteinuria. Methods: The levels of AT1-AA isolated from pre-eclamptic sera were determined by an enzyme-linked immunosorbent assay. Human podocytes were cultured in vitro and treated with various concentrations of AT1-AA. Whether or not an ERK1/2 inhibitor and TRPC6 siRNA inhibit the effect of AT1-AA on podocytes was determined. Western blot was used to detect the expression of podocyte-specific proteins (nephrin, synaptopodin, and podocin) and the phosphorylation of ERK1/2 and TRPC6. The arrangement of F-actin was observed by immunofluorescence. A Calcineurin Cellular Activity Assay Kit was used to detect calcineurin activity. Changes in the intracellular Ca²⁺ concentration was determined by confocal laser. Results: AT1-AA induced a decrease in podocyte-specific protein expression and calcineurin activity and increased expression of p-ERK1/2 and TRPC6 protein and the intracellular Ca²⁺ concentration. Immunofluorescence revealed rearrangement of F-actin. PD98059, an inhibitor of ERK1/2, and TRPC6 siRNA attenuated the decreased expression of podocyte-specific proteins and decreased intracellular Ca²⁺ concentration. The expression of TRPC6 was reduced following the addition of ERK1/2 inhibitor. Conclusion: AT1-AA induced podocyte damage in a dose-dependent manner. The underlying mechanism might involve activation of the TRPC6 –calcium/calcineurin pathway. This study provides new details regarding podocyte injury and the mechanism underlying the generation of proteinuria in pre-eclampsia.

Angiotensin II Stimulates the NLRP3 Inflammasome to Induce Podocyte Injury and Mitochondrial Dysfunction

Background: We previously reported that the NLRP3 inflammasome played an important role in mediating the podocyte injury induced by aldosterone. However, more studies on the role of the NLRP3 inflammasome in the pathogenesis of podocytopathy are still required. The present study was undertaken to study the role of the NLRP3 inflammasome in angiotensin II (Ang II)-induced podocyte injury, as well as the potential mechanisms. Methods: In this study, we used an Ang II infusion model in NLRP3^–/– mice. In cultured podocytes, we used siRNA to silence NLRP3; then we treated the podocytes with Ang II. Results: Following Ang II treatment, we found that the NLRP3 inflammasome was significantly activated in line with mitochondrial dysfunction in a dose- and time-dependent manner. Silencing NLRP3 by siRNA transfection ameliorated podocyte apoptosis, attenuated the loss of the podocyte proteins nephrin and podocin, and protected mitochondrial function. Ang II infusion activated the NLRP3 inflammasome, caused albuminuria, and induced podocyte damage, which was all blocked in the NLRP3^–/– mice. At the same time, NLRP3 deletion also ameliorated the mitochondrial dysfunction induced by Ang II infusion. However, the deletion of NLRP3 did not affect the Ang II hypertension. Conclusion: Taken together, these results demonstrate an important role of the NLRP3 inflammasome in mediating Ang II-induced podocyte injury and mitochondrial dysfunction, suggesting that the NLRP3 inflammasome might be an effective therapeutic target against podocytopathy.

Abstract 139: Loss of Thymosin b4 Exacerbates Renal and Cardiac Damage in Angiotensin-II Hypertension

Angiotensin-II (Ang-II)-induced hypertension is associated with tissue damage and fibrosis in the kidney and heart. Thymosin β4 (Tβ4) regulates cell morphology, inflammation and fibrosis in several organs and administration of exogenous Tβ4 is protective in diabetic nephropathy and unilateral ureteral obstruction model. However, role of endogenous Tβ4 in hypertension-induced organ damage is unknown. We hypothesize that, loss of Tβ4 accelerates renal and cardiac fibrosis and damage in Ang-II hypertension. To test our hypothesis, Tβ4 knockout (Tβ4 -/- ) and wild-type (Tβ4 +/+ ) C57BL/6 mice (n=6-10) were infused continuously for six-weeks with either Ang-II (980 ng/kg/min) or vehicle via osmotic minipumps. All the results are presented in table 1. In Ang-II infusion, systolic blood-pressure were not different between both strains (Table 1). Interestingly, urinary albuminuria was significantly higher in Tβ4 -/- mice compared to Tβ4 +/+ mice by Ang-II. High expression of Tβ4 is found in the glomeruli along with high expression of Nephrin, an important protein in the filtration barrier of the kidney. In Ang-II infusion, nephrin protein expression was greatly reduced in mice deficient of Tβ4, suggesting that loss of nephrin is one of the mechanism for elevated urinary albumin in Tβ4 -/- mice. Additionally, renal fibrosis was higher in Tβ4 -/- mice. We also studied cardiac damage and observed that in Ang-II infusion, cardiac hypertrophy and cardiac fibrosis were much higher in Tβ4 -/- mice. These data indicate that loss of endogenous Tβ4 caused significant tissue damage in the kidney and heart in Ang-II hypertension, suggesting renal and cardiac protective role of this peptide.

Vinpocetine mitigates proteinuria and podocytes injury in a rat model of diabetic nephropathy

Vinpocetine mitigates proteinuria and podocytes injury in a rat model of diabetic nephropathy

The impact of α-Lipoic acid on cell viability and expression of nephrin and ZNF580 in normal human podocytes

The impact of α-Lipoic acid on cell viability and expression of nephrin and ZNF580 in normal human podocytes

Effects of ferulic acid on the expressions of nephrin and podocin in podocytes of diabetic rats

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

Effects of a spleen tyrosine kinase inhibitor on progression of the lupus nephritis in mice

Effects of a spleen tyrosine kinase inhibitor on progression of the lupus nephritis in mice

Bone marrow-derived mesenchymal stem cells ameliorate nephrosis through repair of impaired podocytes.

The purpose of this study was to investigate the effects of bone marrow-derived mesenchymal stem cells (BMSC) on podocytes of puromycin amino nuclear glucoside (PAN) -induced nephrosis in mice. Mice were randomly divided into Control, PAN and BMSC groups. Mice were injected with PAN (0.5 mg/g weight) via the tail vein. The 24-h urinary protein was obtained after modelling, and urinary protein excretion was determined. The blood and kidney specimens were isolated after the tenth day of modelling. Blood samples were collected for measuring serum creatinine (SCr) and blood urea nitrogen (BUN). A sample of kidney was taken for observing pathological changes through hematoxylin-eosin staining and electron microscopy, and the rest of the kidney was used for detecting the protein and mRNA expression of nephrin, CD2AP, synaptopodin, TRPC6 by real-time quantitative PCR, Western-blot and immunohistochemistry. After PAN injection, podocyte foot process fusion was detected by electron microscopy, and the 24 h urinary protein excretion increased compared with control mice on days 3, 7 and 10 post-PAN injection (P.

NLRP3 inflammasome activation contributes to aldosterone-induced podocyte injury.

Aldosterone (Aldo) has been shown as an important contributor of podocyte injury. However, the underlying molecular mechanisms are still elusive. Recently, the pathogenic role of NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome in mediating renal tubular damage was identified while its role in podocyte injury still needs evidence. Thus the present study was undertaken to investigate the role of NLRP3 inflammasome in Aldo-induced podocyte damage. In vitro, exposure of podocytes to Aldo enhanced NLRP3, caspase-1, and IL-18 expressions in dose- and time-dependent manners, indicating an activation of NLRP3 inflammasome, which was significantly blocked by the mineralocorticoid receptor antagonist eplerenone or the antioxidant N-acetylcysteine. Silencing NLRP3 by a siRNA approach strikingly attenuated Aldo-induced podocyte apoptosis and nephrin protein downregulation in line with the blockade of caspase-1 and IL-18. In vivo, since day 5 of Aldo infusion, NLRP3 inflammasome activation and podocyte injury evidenced by nephrin reduction occurred concurrently. More importantly, immunofluorescence analysis showed a significant induction of NLRP3 in podocytes of glomeruli following Aldo infusion. In the mice with NLRP3 gene deletion, Aldo-induced downregulation of nephrin and podocin, podocyte foot processes, and albuminuria was remarkably improved, indicating an amelioration of podocyte injury. Finally, we observed a striking induction of NLRP3 in glomeruli and renal tubules in line with an enhanced urinary IL-18 output in nephrotic syndrome patients with minimal change disease or focal segmental glomerular sclerosis. Together, these results demonstrated an important role of NLRP3 inflammasome in mediating the podocyte injury induced by Aldo.

Computational analysis of nsSNPs of NPHS1 gene and their effect on protein structural stability

Computational analysis of nsSNPs of NPHS1 gene and their effect on protein structural stability

Modeling Monogenic Human Nephrotic Syndrome in the Drosophila Garland Cell Nephrocyte.

Steroid-resistant nephrotic syndrome is characterized by podocyte dysfunction. Drosophila garland cell nephrocytes are podocyte-like cells and thus provide a potential in vivo model in which to study the pathogenesis of nephrotic syndrome. However, relevant pathomechanisms of nephrotic syndrome have not been studied in nephrocytes. Here, we discovered that two Drosophila slit diaphragm proteins, orthologs of the human genes encoding nephrin and nephrin-like protein 1, colocalize within a fingerprint-like staining pattern that correlates with ultrastructural morphology. Using RNAi and conditional CRISPR/Cas9 in nephrocytes, we found this pattern depends on the expression of both orthologs. Tracer endocytosis by nephrocytes required Cubilin and reflected size selectivity analogous to that of glomerular function. Using RNAi and tracer endocytosis as a functional read-out, we screened Drosophila orthologs of human monogenic causes of nephrotic syndrome and observed conservation of the central pathogenetic alterations. We focused on the coenzyme Q10 (CoQ10) biosynthesis gene Coq2, the silencing of which disrupted slit diaphragm morphology. Restoration of CoQ10 synthesis by vanillic acid partially rescued the phenotypic and functional alterations induced by Coq2-RNAi. Notably, Coq2 colocalized with mitochondria, and Coq2 silencing increased the formation of reactive oxygen species (ROS). Silencing of ND75, a subunit of the mitochondrial respiratory chain that controls ROS formation independently of CoQ10, phenocopied the effect of Coq2-RNAi. Moreover, the ROS scavenger glutathione partially rescued the effects of Coq2-RNAi. In conclusion, Drosophila garland cell nephrocytes provide a model with which to study the pathogenesis of nephrotic syndrome, and ROS formation may be a pathomechanism of COQ2-nephropathy.

Childhood nephrotic syndrome: genetic basis in studying and treatment

Childhood nephrotic syndrome is a serious disease with the high risk of mortality. The prevalence worldwide is approximately 2 to 7 cases per 100,000 children. Hitherto, the patients are treatmed with steroid or immunosuppressive therapy and renal transplantation. However, many patients are steroid resistant and have a high risk of recurrence. The mutations in seven genes (NHPS1, NPHS2, CD2AP, PLCE1, ACTN4, TRPC6, INF2) have been implicated in different forms of nephrotic syndrome. Among them, two genes, NPHS1 and NPHS2, encoding for nephrin and podocin protein of the glomerular filtration barrier, have a particularly serious influence in almost of cases and steroid resistance in patients. So that, in the world there were a lot of researches conducted to identify gene mutations that related to disease manifestations and the response to treatment in the patients who have different genetic background. Mutations in NPHS1 and NPHS2 are considered the main cause of 75% cases with nephrotic syndrome. And NPHS2 mutations related to 40% cases with steroid resistance in patients. These results will help the doctors to have an effective treatment for the patients. However, in Vietnam no study has yet been done to detect gene mutations in patients with nephrotic syndrome. In this article, we summarize the research which have been published to give an overview of the genetic basis of causing and treatment for childhood nephrotic syndrome.

Effect of forkhead transcription factor O1 on high glucose-induced epithelial-mesenchymal transition in mice podocytes and its mechanism

Objective To study the role and molecular mechanism of forkhead transcription factor O1(FoxO1)on epithelial-mesenchymal transition(EMT)in mice podocytes cultured under high glucose conditions through inhibiting transforming growth factor-β1(TGF-β1)/Smad3 signaling. Methods The podocytes were transfected with lentiviral vectors expressing constitutively active FoxO1(LV-CA-FoxO1), the lentiviral vectors expressing FoxO1 short hairpin RNA(LV-FoxO1-shRNA), and lentiviral vectors expressing empty vectors(LV-NC). The podocytes were divided into five groups of normal glucose(5.6 mmol/L, group A), high glucose(25 mmol/L, group B), high glucose plus LV-CA-FoxO1(group C), high glucose plus LV-FoxO1-shRNA(group D), and high glucose plus empty lentiviral vectors(group E). After treatment with different periods of time(24, 48, and 72 h), the mRNA levels of podocytes were measured through realtime PCR in each group, including FoxO1, TGF-β1, Smad3, nephrin, and desmin. The protein expressions of FoxO1, p-FoxO1, TGF-β1, Smad3, p-Smad3, nephrin, and desmin were assessed by Western blot. The distribution of FoxO1 in podocytes was detected by immunofluorescence. In addition, the TGF-β1 secretion in cell culture supernatants was analyzed by ELISA. Results Compared with group A, there was no significant difference in either mRNA or protein levels of FoxO1 in group B(both P>0.05), whereas the p-FoxO1/FoxO1 ratio markedly increased(P<0.05). The expressions of TGF-β1 and Smad3, as well as the ratio of p-Smad3/ Smad3 all increased(all P<0.05). The nephrin mRNA and protein levels decreased, with increased desmin levels in a time-dependent manner(all P<0.05). Compared with group B, the levels of FoxO1 mRNA and protein increased in group C but decreased in group D(all P<0.05). There was a significant decrease of p-FoxO1/FoxO1 ratio in group C(P<0.05). The expressions of TGF-β1, p-Smad3, and desmin decreased in group C but increased in group D, with the nephrin mRNA and protein levels elevated in group C, but reduced in group D(all P<0.05). Conclusion FoxO1 plays a crucial role in down-regulating the activation of TGF-β1 pathway and thereby ameliorating EMT of podocytes. (Chin J Endocrinol Metab, 2016, 32: 767-772) Key words: Forkhead transcription factors O1; Diabetic nephropathy; Podocyte; Epithelial-mesenchymal transition; Transforming growth factor-β1

Correlation study of podocyte injury and kidney function in patients with acute kidney injury

Correlation study of podocyte injury and kidney function in patients with acute kidney injury

Metformin ameliorates podocyte damage by restoring renal tissue nephrin expression in type 2 diabetic rats.

Previous studies found that metformin provided some renoprotection for diabetic renal damage. In the present study, we evaluated the effects of different doses of metformin on the expression of renal tissue nephrin in type 2 diabetes mellitus (T2DM) model rats and the possible mechanism underlying its protective effect in kidney podocytes. A high-fat diet combined with a low dose of streptozotocin was used to induce T2DM model rats. Diabetic rats were treated with 150, 300, or 500 mg/kg metformin for 8 weeks. At the end of the study, urine and blood samples were collected for measurement of different indices. Light microscopy and transmission electron microscopy were used to identify morphological changes. Renal expression of nephrin protein was assayed by immunohistochemical staining, whereas real-time polymerase chain reaction was used to detect renal nephrin (Nphs1) mRNA expression. Metformin treatment of T2DM rats produced dose-dependent significant reductions in urinary albumin and nephrin concentrations, glomerular basement membrane thickness (GBMT), and the foot process fusion rate (FPFR) compared with control T2DM model rats, whereas renal expression of nephrin protein and Nphs1 mRNA was dose-dependently increased by metformin treatment. Metformin protects kidney podocytes in T2DM model rats by dose-dependently adjusting renal nephrin expression.