| Objective:Diabetic nephropathy (DN) is one of the major "microvascular" complications of diabetes, which is the leading cause of end-stage renal disease in many countries. Diabetic nephropathy is characterized by excessive amassing of extracellular matrix (ECM) with thickening of glomerular and tubular basement membranes and increased amount of mesangial matrix, which ultimately progress to glomerulosclersis and tubulo-interstitial fibrosis. Recent studies have shown that podocyte injury plays an important role in the progression of DN.Podocytes are terminally differentiated and highly specialized cells. They line the urinary side of the glomerular basement membrane (GBM) and function as a fine filter contributing ultimate size-selectivity, permitting permeability to molecules smaller than albumin. Among the characteristic findings of diabetic nephropathy, podocytes are involved in the development of glomerular hypertrophy, podocytopenia, glomerulosclerosis, and foot process effacement. At present, little is known about the mechanisms of podocyte injury in DN, but some studies have shown that the cytoskeleton changes are involved in this process.Nestin is a type VI intermediate filament (IF) protein mainly expressed in rapidly dividing progenitor cells of developing and regenerating tissues. In adult kidney, nestin was only expressed in podocytes, and plays an important role in maintaining the morphology and function of podocyte. The downregulation of nestin is associated with proteinuria in IgA nephropathy, membranous nephropathy and focal segmental glomerular sclerosis. However, little is known about whether the cytoskeleton protein is involved in the diabetic glomerular damage and the changes of nestin during the development of DN have not yet been established. Nestin was recently reported to exhibit cytoprotective functions in some studies. The depletion of nestin by siRNA increased the apoptosis of neural progenitor cells and vascular smooth muscle cells induced by H2O2, and the presence of nestin protects cells from cell death mediated by oxidative stress. Podocyte apoptosis is a major reason of podocyte loss, renal injury, proteinuria and the decreased renal function. However, the underlying mechanisms of podocyte apoptosis under high glucose environment remain poorly understood, and whether nestin involved in this process is unknown. In the present study, the expression of nestin, vimentin and Cdk5/p35and the interrelation among these factors were observed in diabetic models and conditionally immortalized mouse podocytes exposed to high glucose. In addition, the role of nestin on podocyte apoptosis induced by high glucose was also investigated, in order to provide theoretical basis for the prevention and treatment of DN.Methods:1Induction of rat diabetic models and detection of nestin, p-nestin, vimentin and Cdk5/p35in glomeruli.Male Wistar rats were randomly divided into two groups:control group and diabetic group (DM). Diabetes were induced by intraperitoneal injection of streptozotocin (STZ,65mg/kg) dissolved in0.1M citrate buffer (pH4.5). Individual animals with blood glucose concentrations more than16.7mM and the glucose in urine positive for2consecutive days after injection were confirmed as diabetes. The control rats were received0.1M citrate buffer solution only. At4,8,12and16weeks after treatment,5rats from each group were weighed and housed individually for24h in metabolic cages for collecting urine, to measure24-hour urinary albumin or proteins. Their sera were prepared for the measurement of serum blood glucose and blood urea nitrogen (BUN) concentrations. Then rats were sacrificed and their kidneys were removed. One portion of the renal tissues was fixed in4%paraformaldehyde for histological and immunohistochemical examinations, another portion of the renal tissues was fixed immediately in2.5%glutaraldehyde for transmission electron microscopy. The remaining renal tissues were prepared for isolating glomeruli. Immunohistochemistry and western blot were used to detected the expression of nestin, p-nestin, vimentin and Cdk5/p35proteins in renal tissues; immunofluorescence was used to detected the co-expression of nestin with vimentin, Cdk5, WT1, α-SMA, synaptopodin and CD31; immunoprecipitation was used to detected the co-assembled of nestin with vimentin; the levels of nestin, vimentin and Cdk5mRNA in glomeruli were detected by Real-time PCR.2The effects of high glucose on nestin expression in conditionally immortalized mouse podocytes.To induce proliferation, the conditionally immortalized mouse podocytes were cultured at33℃with y-IFN under growth permissive conditions, and then cells were cultured at37℃without y-IFN under growth restrictive conditions for10-14days to induce quiescence and the differentiated phenotype. Synaptopodin was detected by immunofluorescence to validate the differentiation of podocytes. The podocytes cultured at37℃for10days were randomly divided into3groups:normal glucose group (NG), normal glucose+mannitol group (NG+M), high glucose group (HG). The three groups were cultured for6,12,24,48and72h respectively, and then podocytes were harvested to abstract total RNA and protein. The expression of nestin, vimentin, p-nestin and Cdk5/p35were detected by immunocytochemistry and Western blot. The co-expression of nestin with vimentin was detected by immunofluoresence and immunoprecipitation. The mRNA levels of nestin, vimentin and Cdk5were detected by real-time PCR.3The effect of inhibition of Cdk5on nestin expression of high glucose stimulating podocytes.①Male Wistar rats were used to induce diabetes by injection of STZ. Rats were randomly divided into4groups:control group, diabetic group (DM), diabetes treated with DMSO group (DM+D) and diabetes treated with roscovitine group (DM+R). The method to make diabetic model is the same as that of part1. After the diabetic model was affirmed to be successful, the rats of DM+R group were daily given by intraperitoneal injection of roscovitine at a concentration of2.8mg/kg body weight dissolved in a total volume of400μl DMSO. While rats in diabetes with DMSO treatment group were received by intraperitoneal injection of400μl DMSO as control. Blood,24h urine and kidney samples were collected at week16after diabetes induced. One portion of the renal tissues was fixed in4%paraformaldehyde in for histological and immunohistochemical examinations, another portion of the renal tissues was fixed immediately in2.5%glutaraldehyde for transmission electron microscopy. The remaining renal tissues were prepared for isolating glomeruli to abstract total RNA and protein. The expression of nestin and p-nestin was detected by immunohistochemisry and Western blot.②Podocytes which cultured under nonpermissive conditions (at37℃) for10days were divided into4groups:Normal control group (NG), high glucose group (HG), high glucose+Cdk5siRNA group (HG+C-siRNA) and high glucose+negative siRNA group (HG+N-siRNA). Total RNA and protein were extracted from podocytes at48h after incubation. The expression of nestin and p-nestin was measured by immunohistochemistry and Western blot.4The effect of nestin inhibition on podocyte apoptosis induced by high glucose.The nestin miRNA plasmid was designed and constructed to inhibit the expression of nestin in podocytes. The scrambled plasmid was used as a negative control. For RNAi research, the podocytes were divided into5groups: normal glucose control group (NG), mannitol group (M), high glucose group (HG), high glucose+nestin miRNA transfection group (HG+miRNA) and high glucose+scrambled miRNA transfection group (HG+Scrambled). All groups were incubated for24h and48h. Immunocytochemistry and immunofluorescence were used to detecte the effect of miRNA inhibition. The survival rate of podocytes after high glucose simulation was detected by MTT. The podocyte apoptosis induced by high glucose was detected by TUNEL and flow cytometry. The protein levels of Bcl-2, Bax and cleaved caspase-3were measured by Western blot.Result: 1Induction of rat diabetic models and detection of nestin, p-nestin, vimentin and Cdk5/p35in glomeruli.①Compared with control group, histological sections of kidneys from the diabetic rats revealed significant hypertrophy of glomerular and expansion of the mesangial matrix of glomeruli. Uneven increase in thickness of glomerular basement membrane, and the fusion of podocyte foot processes in the glomeruli of diabetic rats were observed by transmission electron microscopy.②Immunofluorescence staining showed that the positive staining for nestin was only observed in podocytes of normal kidney, which co-expressed with WT1and synaptopodin.③Compared with control group, the expression of nestin was stronger from4weeks to8weeks (p<0.05), the expression of vimentin was higher at week4(p<0.05). The positive staining of nestin and vimentin was weaker in diabetic renal tissues at week16compared with control group (p<0.05). In DM group, some tubular epithelial cells were expressed nestin and vimentin from week4. Immunofluorescence staining showed that nestin co-expressed with vimentin or a-SMA in some tubular epithelial cells in diabetic renal tissues.④Western blot indicated that the expression of nestin was significantly increased from week4to week8after induction of disease in DM group (p<0.05). The expression of vimentin was increased at week4, and then gradually decreased. At week16, the expression levels of nestin and vimentin in DM group were remarkably declined compared with control group (p<0.05). The expression levels of Cdk5/p35and p-nestin (Thr-316) proteins were significantly increased in DM group (p<0.05). Co-immunoprecipitation indicated that nestin assembled with vimentin was significantly increased at week4, and then gradually decreased (p<0.05).⑤In parallel with the results of Western blotting analysis, significant increases in nestin and vimentin transcript were also observed in the mRNA from the glomeruli of diabetic rats at week4and week8(p<0.05), and then, the expression levels were gradually reduced. At week16of diabetes, the expression levels of nestin and vimentin mRNA were markedly declined (p<0.05). The mRNA level of Cdk5in the glomeruli of diabetic rats was also upregulated in a time-dependent manner, from week4to week16compared with controls (p<0.05).2The effects of high glucose on nestin expression in conditionally immortalized mouse podocytes.①Podocytes can be cultured in two different phenotypes. These cells proliferate and maintain an epithelial phenotype with cobblestone-like morphology under permissive conditions (at33℃). In contrast, culturing under nonpermissive conditions (at37℃) stops proliferation and induces conversion into arborized cells. The differentiation results in sprouting of differently shaped long processes from cell bodies and spindle-like projections arise from these primary processes. Synaptopodin, a differentiation specific actin cytoskeleton associated protein, is only expressed in differentiating podocytes.②Immunocytochemical staining showed that positive staining for nestin, vimentin and Cdk5was observed mainly in cytoplasm of podocytes. Compared with control, the expression of nestin and vimentin were significantly decreased in podocytes after high glucose stimulation, whereas the expression of Cdk5was increased.③The result of western blot showed that, compare with control, the expression levels of nestin and vimentin proteins were significantly decreased in HG group (p<0.05). After48hours, the expression of nestin almost disappeared. The levels of Cdk5/p35proteins were significantly increased by high glucose stimulating (p<0.05). The protein level of p-nestin (Thr-316) was increased in HG group, and reached its peak at24h.④In normal podocytes, the nestin co-expressed with vimentin, and formed a filamentous network extending from the nucleus to the plasma membrane. After simulation by high glucose, nestin disassembled with vimentin (p<0.05).⑤The levels of nestin, vimentin and Cdk5mRNA were detected by real-time PCR. Compared with control, the expression of nestin and vimentin mRNA were significantly decreased in HG group (p<0.05). The levels reached0.19and0.16folds to control group at72h. The Cdk5mRNA of HG group was significantly increased to3.75folds to control at72h.3The effect of inhibition of Cdk5on nestin expression of high glucose stimulating podocytes.①Compared with the diabetic group, the rats of diabetes with roscovitine treatment group showed significant reduction of blood urea nitrogen or albuminuria. Whereas there was no difference between these two groups on the levels of body weight, kidney weight, the radio of kidney to body weight, even glucose. Histopathological examination showed that the hypertrophy of glomerular, expansion of the mesangial matrix, the thickness of GBM and the fusion of foot processes were attenuated by treatment with roscovitine.②The results of immunohistochemistry and Western blot indicated that the expression level of nestin was stronger in DM+R group than in DM and DM+D groups (p<0.05).③Immunocytochemical staining showed that the expression of nestin in podocytes with Cdk5siRNA treatment was stronger than HG and HG+N-siRNA group.④Western blot indicated that the protein level of nestin was higher in HG+C-siRNA than that in HG and HG+N-siRNA group.4The effect of nestin inhibition on podocyte apoptosis induced by high glucose.①Nestin miRNA markedly reduced the expression of nestin in podocytes.②Compared with normal control group, the survival rate of HG group was significantly decreased at72h (p<0.05).③TUNEL and flow cytometry indicated that the podocyte apoptosis was markedly increased after high glucose stimulation. Downregulation of nestin clearly sensitized the cells to high glucose induced cell death. Compared with untransfected podocytes and podocytes trasfected with the scrambled vector, the survival rate of nestin-depleted podocytes was significantly reduced (p<0.05). The results of TUNEL and flow cytometry showed that the apoptotic podocytes among transfected with nestin miRNA were markedly higher than that in untransfected podocytes and podocytes trasfected with the scrambled vector (p<0.05).④The protein ratio of Bcl-2/Bax was decreased in HG group. Compared with untransfected podocytes and podocytes trasfected with the scrambled vector, the protein level of cleaved caspase-3significantly increased in nestin miRNA group.Conclusions:1In normal kidney, nestin expression is restricted to differentiated podocytes. The protein and mRNA expression of nestin and vimentin were up-regulated in renal glumeruli at the early stage of diabetes, and then decreased. The level of nestin assembled with vimentin in diabetic rats was decreased in the progression of diabetes.2In vitro, high glucose could reduce the protein and mRNA levels of nestin and vimentin in cultured conditionally immortalized mouse podocytes, and decrease the levels of nestin which assembled with vimentin.3Diabetic rats treated with roscovitine could reduce the injury of glumeruli and renal function. The decreased expression of nestin in diabetic glumeruli was also attenuated by roscovitine treatment. In cultured podocytes, inhibition of Cdk5by siRNA could also attenuate the decrease of nestin induced by high glucose.4High glucose could induce the podocyte apoptosis. Downregulation of nestin by miRNA clearly sensitized the podocytes to high glucose induced cell death. The apoptosis of nestin-depleted podocytes was significantly increased after high glucose stimulation. These results suggested that nestin serves as a prosurvival determinant in podocyte. High glucose maybe induce podocyte apoptosis by reduce the expression of nestin. |
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