Volume 69, Issue 5, Pages 806-814 (March 2006) Glucose and diabetes: Effects on podocyte and glomerular p38MAPK, heat shock protein 25, and actin cytoskeleton  T. Dai, R. Natarajan, C.C. Nast, J. LaPage, P. Chuang, J. Sim, L. Tong, M. Chamberlin, S. Wang, S.G. Adler  Kidney International  Volume 69, Issue 5, Pages 806-814 (March 2006) DOI: 10.1038/sj.ki.5000033 Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 1 Dose-dependent activation of p38MAPK, phosphorylation of small HSP25, and maintenance of normal Pod cytoskeleton after short-term exposure of Pods to HG in culture. Differentiated Pods placed for 24 h in 0.4% fetal bovine serum were treated with indicated concentrations of glucose and/or mannitol (in triplicate) for 10 min before lysates were prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis, IEF, and subsequent Western blot analysis with anti-phospho p38MAPK antibody, p38MAPK antibody, and anti-HSP25 antibody. (a) Representative Western blots of phospho-specific p38MAPK (pp38MAPK) and quantitative evaluation of relative pp38MAPK to total p38MAPK (p38) ratios by densitometry analysis. (b, c) Representative IEF, Western blot analysis of HSP25, and quantitative evaluation of relative phosphorylated HSP25 isoforms to total HSP25 ratios by densitometry analysis. By using IEF, HSP25 can be separated into its nonphosphorylated isoform (P0), mono- (P1), and bi-phosphorylated (P2) isoforms. With incremental glucose concentrations, there is a relative increase in the phosphorylated HSP25 isoforms ((b) (P1+P2)/total HSP25), especially (c) the biphosphorylated form (P2/total HSP25). pp38MAPK and P2HSP25 did not change when mannitol was used as a control for osmolarity. (d, e) Differentiated Pods seeded on coverslips were exposed to HG and followed by fixation for staining. The cytoskeleton in HG-treated cells was similar to NG-treated cells. A representative photomicrograph stained with Texas red phalloidin showing normal F-actin filaments (d) and nuclear DNA staining, with very few cytoplasmic GA monomers stained with Oregon green DNase I (e). *P<0.05, **P<0.01, versus 5.5 mM glucose control. Kidney International 2006 69, 806-814DOI: (10.1038/sj.ki.5000033) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 2 Effect of p38MAPK inhibitor SB on p38MAPK activation, HSP25 phosphorylation, F/G-actin ratio, and Pod cytoskeleton in Pods cultured in NG and HG. Differentiated Pods were exposed to HG (25 mM) for 10 min without pre-exposure (HG) or with 4 h of pre-exposure (HG+I) to the p38MAPK inhibitor SB. SB202474 was used as an inhibitor control (HG+IC). Mannitol was added to NG (5.5 mM, NG) media as an osmolarity control (NG+M). (a) Representative Western blots of phospho-specific p38MAPK with or without SB and quantitative evaluation of relative pp38MAPK to total p38MAPK ratios by densitometry analysis. *P<0.05, HG versus NG, HG+I versus NG. (b) Representative IEF gel, Western blots of HSP25 with or without SB, and quantitative evaluation of relative phosphorylated HSP25 isoforms to total HSP25 ratios ((P1+P2)/(P0+P1+P2)) by densitometry analysis. The same samples used in (a) were centrifuged and resuspended in urea buffer; 30 μg protein of each sample was subjected to Bio-Rad Model 111 mini-IEF cell cast gel and subsequent Western blotting to detect the different phosphorylated HSP25 isoforms using the antibody against HSP25. The right two lanes are controls, in which Pod lysates were prepared before (Podo) and after (Podo+HS) heat shock stress. *P<0.05: NG versus HG; NG+M versus HG; NG versus HG+IC; NG+M versus HG+IC. **P<0.01: NG versus HG+I; NG+M versus HG+I; HG versus HG+I; HG+IC versus HG+I. (c) GA and total cellular actin were measured on the same samples as those in (a), using a DNase I assay to determine F/G actin ratios. *P<0.05: NG versus HG; NG+M versus HG; NG versus HG+I; HG+IC versus HG+I; **P<0.01: HG versus HG+I. (d) Differentiated Pods seeded on coverslips were exposed to HG or HG+I, fixed, and stained. As shown in Figure 1d and e, Pods incubated in HG without inhibitor maintain normal staining of speculated F-actin fibrils and only very few cytoplasmic GA staining. In Pods incubated with HG+I, a representative photomicrograph stained with Texas red phalloidin shows disrupted F-actin filaments and representative photomicrograph stained with Oregon green DNase I shows diffuse cytoplasmic GA monomers. The expected binding to nuclear DNA is also observed. Kidney International 2006 69, 806-814DOI: (10.1038/sj.ki.5000033) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 3 Glomerular expression of pp38MAPK, each of the two phosphorylated HSP25 isoforms, and F/GA ratios in 1-week, 1-month, and 4-month C and SZ-DM rats. (a) Representative Western blots of glomerular pp38MAPK from SZ-DM and C rats and quantitative evaluation of relative pp38MAPK to total p38MAPK ratios by densitometry analysis. *P<0.05: DM versus control. (b) The upper panel consists of representative Western blots of a mini-IEF from sieved glomeruli at the indicated time points from SZ-DM and C rats. The right two lanes are positive controls using samples with (Podo+HS) or without (Podo) heat shock stress. The bottom figure and (c) are quantitative densitometry analyses of relative phosphorylated HSP25 (P1+P2) and biphosphorylated HSP25 isoform (P2) to total HSP25 (P0+P1+P2) ratios, respectively. *P<0.05. (d) GA and total cellular actin were measured on the same samples as those in (a), using a DNase I assay to determine F/G-actin ratios. *P<0.05. Kidney International 2006 69, 806-814DOI: (10.1038/sj.ki.5000033) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 4 The number of slit membranes per 100 μm of GBM, and relationship analysis between Ualb/cr ratio and glomerular p2HSP25 levels, Ualb/cr ratio and glomerular slit pore frequency, the p2HSP25 level and the slit membrane frequency in 1-week, 1-month, and 4-month control and SZ-DM rat glomeruli. (a) A sequential decline in slit membrane number occurs with increasing duration of DM (*P<0.05: DM 4 months vs DM 1 week, DM 4 months vs DM 1 month; #P<0.01: DM 4 months vs Control, DM 1 month vs control). (b) A strong inverse correlation was found between the ln Ualb/cr ratios of SZ-DM rats and glomerular biphosphorylated HSP25 (p2HSP25) levels. r=-0.43, P=0.03. (c) A strong inverse correlation was noted between the ln Ualb/cr ratio and the number of slit membranes per 100 μm of GBM in control and DM rats. r=-0.72, P=0.0003. (d) A strong positive correlation was noted between the glomerular p2HSP25 level measured on a pooled sample of glomeruli from four rats (eight kidneys) for each data point and the slit membrane frequency measured on the tissue of a single rat from each of the four pooled cohorts whose glucose approximated the average of the group. (Each data point represents n=4 P2HSP25 measurements from eight kidneys and slit membrane density measurements derived from one kidney from that group.) r=0.93, P=0.0004. Kidney International 2006 69, 806-814DOI: (10.1038/sj.ki.5000033) Copyright © 2006 International Society of Nephrology Terms and Conditions