The Expression of Intermediate Filament Protein Nestin and Its Association With Cyclin- dependent Kinase 5 in the Glomeruli of Rats With Diabetic Nephropathy

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The Expression of Intermediate Filament Protein Nestin and Its Association With Cyclin- dependent Kinase 5 in the Glomeruli of Rats With Diabetic Nephropathy Wei Liu, MD, Shuxia Liu, MD, Qingjuan Liu, MD, Jun Hao, MD, Yonghong Shi, MD, Song Zhao, MD, Huijun Duan, MD, PhD The American Journal of the Medical Sciences Volume 345, Issue 6, Pages 470-477 (June 2013) DOI: 10.1097/MAJ.0b013e3182648459 Copyright © 2013 Southern Society for Clinical Investigation Terms and Conditions

Figure 1 Histological examination of the glomeruli of rats. Renal tissue sections from the rats of the control, diabetes and diabetes+roscovitine groups at 16 weeks were stained with periodic acid-Schiff (A; ×400). Renal tissues from the rats of the control, diabetes and diabetes+roscovitine groups at 16 weeks were observed by transmission electron microscopy (B; white arrow: glomerular basement membranes, black arrow: foot process; ×20,000). Representative photographs of each group (n=6) are shown. The American Journal of the Medical Sciences 2013 345, 470-477DOI: (10.1097/MAJ.0b013e3182648459) Copyright © 2013 Southern Society for Clinical Investigation Terms and Conditions

Figure 2 Immunohistochemical staining of nestin protein. Renal tissue sections from the rats of the control and diabetes groups (4, 8, 12 and 16 weeks) were analyzed for the expression of nestin in the glomeruli by immunohistochemistry (×400). Representative photographs of each group (n=6) are shown (A). B, Semiquantitative analysis of nestin expression. Data are expressed as mean ± standard deviation. *P < 0.05 versus controls. The American Journal of the Medical Sciences 2013 345, 470-477DOI: (10.1097/MAJ.0b013e3182648459) Copyright © 2013 Southern Society for Clinical Investigation Terms and Conditions

Figure 3 Immunohistochemical staining of vimentin protein. Renal tissue sections from the rats of the control and diabetes groups (4, 8, 12 and 16 weeks) were analyzed for the expression of vimentin in the glomeruli by immunohistochemistry (×400). Representative photographs of each group (n=6) are shown (A). B, Semiquantitative analysis of vimentin expression. Data are expressed as mean ± standard deviation. *P < 0.05 versus controls. The American Journal of the Medical Sciences 2013 345, 470-477DOI: (10.1097/MAJ.0b013e3182648459) Copyright © 2013 Southern Society for Clinical Investigation Terms and Conditions

Figure 4 Expression of nestin in the glomeruli of diabetic rats. The renal glomeruli were isolated from the rats of the control and diabetes groups (4, 8, 12 and 16 weeks). The expression of nestin protein was examined by Western blot analysis. β-Actin was used as an internal control (A and B). The messenger RNA (mRNA) level of nestin was detected by real-time polymerase chain reaction. The 18S mRNA was used as an internal control (C). The levels of vimentin that assembled with nestin were detected by co-immunoprecipitation and Western blott analysis. The result was expressed as fold of control. The lysates were incubated with unspecific IgG as a negative control (D). Data are expressed as mean ± standard deviation (n=6). *P < 0.05 versus controls. The American Journal of the Medical Sciences 2013 345, 470-477DOI: (10.1097/MAJ.0b013e3182648459) Copyright © 2013 Southern Society for Clinical Investigation Terms and Conditions

Figure 5 Expression of Cdk5 in the glomeruli of diabetic rats. The renal glomeruli were isolated from the rats of the control and diabetes groups (4, 8, 12 and 16 weeks). The expression of Cdk5 protein was examined by Western blot analysis. β-Actin was used as an internal control (A and B). The messenger RNA (mRNA) level of Cdk5 was detected by real-time polymerase chain reaction. The 18S mRNA was used as an internal control (C). Data are expressed as mean ± standard deviation (n=6). *P < 0.05 versus controls. The American Journal of the Medical Sciences 2013 345, 470-477DOI: (10.1097/MAJ.0b013e3182648459) Copyright © 2013 Southern Society for Clinical Investigation Terms and Conditions

Figure 6 Effect of blockage of Cdk5 on nestin expression in the glomeruli of diabetic rats. The renal glomeruli were isolated from the rats of the control, diabetes, diabetes+DMSO and diabetes+roscovitine groups at 16 weeks. The expression of nestin protein was examined by Western blot analysis. β-Actin was used as an internal control (A and B). Data are expressed as mean ± standard deviation (n=6). *P < 0.05 versus controls, #P < 0.05 versus diabetes or diabetes+DMSO group. DMSO, dimethyl sulfoxide. The American Journal of the Medical Sciences 2013 345, 470-477DOI: (10.1097/MAJ.0b013e3182648459) Copyright © 2013 Southern Society for Clinical Investigation Terms and Conditions