1. Volume 88, Issue 4, Pages 745-753 (October 2015)
Renin-angiotensin blockade resets podocyte epigenome through Kruppel-like Factor 4 and attenuates proteinuria 
Kaori Hayashi, Hiroyuki Sasamura, Mari Nakamura, Yusuke Sakamaki, Tatsuhiko Azegami, Hideyo Oguchi, Hirobumi Tokuyama, Shu Wakino, Koichi Hayashi, Hiroshi Itoh 
Kidney International 
Volume 88, Issue 4, Pages (October 2015)
DOI: /ki
Copyright © 2015 International Society of Nephrology Terms and Conditions

2. Figure 1
Angiotensin receptor blocker decreases DNA methylation of the nephrin promoter with recovery of Kruppel-like Factor 4 (KLF4) expression in vivo. (a) Experimental protocol. Four weeks after injection of mice with adriamycin (ADM), candesartan (Cand), irbesartan (Irb), or captopril (Capto) was administered at the indicated doses for 14 days. (b) Quantification of albuminuria in each group at the end of the study. (c) Representative confocal photomicrographs and (d) quantification of glomerular KLF4 (green) and nephrin (red) staining at the end of the study. Scale bars; 25 μm. (e) Quantitative reverse transcription PCR (RT-PCR) analysis of KLF4 and nephrin in laser-microdissected glomeruli of mice treated with or without candesartan for 14 days. (f) Western blot analysis of KLF4 and nephrin expression in the kidney cortex of mice treated with or without candesartan for 14 days. (g) (Left panel) Representative photomicrographs of light microscopy of glomeruli (WT1 staining) and (right panel) quantification of WT1-positive cells per glomerulus in mice treated with or without candesartan for 14 days. (h) (Upper panel) Results of methylation-specific PCR (MSP) using primers for unmethylated (UM, top) or methylated (M, bottom) nephrin promoter. (Lower panel) Relative level of unmethylated nephrin promoter region using real-time MSP analysis in laser-microdissected glomeruli of mice treated with or without candesartan for 14 days, adjusted by number of podocytes per glomerulus. n=5 in each group with duplicates. *P<0.05, **P<0.01 versus untreated (ADM (+), Cand (-)). Data are mean±s.e.m.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

3. Figure 2
The effect of angiotensin receptor blocker (ARB) on proteinuria and the nephrin promoter methylation was attenuated by Kruppel-like Factor 4 (KLF4) knockdown in podocytes in vivo. (a) Experimental protocol. Four weeks after injection of podocyte-specific KLF4 knockout (KO) mice or wild-type control mice (WT) with adriamycin (ADM), candesartan (Cand) was administered at the indicated doses for 14 days. (b) Quantification of albuminuria in each group at the end of the study. (c) Representative confocal photomicrographs and (d) quantification of glomerular KLF4 (green) and nephrin (red) staining at the end of the study. Scale bars: 25 μm. (e) Quantitative reverse transcription PCR (RT-PCR) analysis of KLF4 and nephrin in laser-microdissected glomeruli of KLF4 KO or WT mice treated with or without candesartan for 14 days. (f) (Upper panel) Representative photomicrographs of light microscopy of glomeruli (WT1 staining) and (lower panel) quantification of WT1-positive cells per glomerulus in each group. (g) (Upper panel) Results of methylation-specific PCR (MSP) using primers for unmethylated (UM, top) or methylated (M, bottom) nephrin promoter. (Lower panel) Relative level of unmethylated nephrin promoter region using real-time MSP analysis in laser-microdissected glomeruli of mice treated with or without candesartan for 14 days, adjusted by number of podocytes per glomerulus. *P<0.05 versus WT control mice (ADM (+), Cand (-)). n=5 in each group with duplicates. #P<0.05 versus the respective groups. Data are mean±s.e.m.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

4. Figure 3
Angiotensin II (Ang II) increases the nephrin promoter methylation with decreased Kruppel-like Factor 4 (KLF4) expression in vitro. (a, b) Time course and dose dependency of decreases in KLF4 expression in podocytes treated with Ang II. The time course was examined with 10−6 mol/l Ang II, and the dose dependency was examined with Ang II for 6 h by (a) quantitative reverse transcription PCR (RT-PCR) or (b) western blot analysis. (c) (Upper panel) Western blot analysis of KLF4 and nephrin expression and (Lower panel) real-time RT-PCR analysis of KLF4 and nephrin mRNA in podocytes treated with Ang II (10−6 mol/l) for 24 h. (d) Chromatin immunoprecipitation (ChIP) assay for the presence of KLF4 in the promoter region of nephrin in podocytes treated with or without Ang II (10−6 mol/l) for 24 h. KLF4-Tg; KLF4-overexpressing podocytes. The bar graph shows the quantification of KLF4/input intensity. (e) KLF4 expression in Ang II-treated podocytes with or without treatment of angiotensin receptor blocker (ARB) candesartan (10−6 mol/l) 1 hour before administration of Ang II (10−6 mol/l). (f) (Upper panel) Results of methylation-specific PCR (MSP) using primers for unmethylated (UM, top) or methylated (M, bottom) nephrin promoter. (Lower panel) Relative levels of unmethylated CpG using real-time MSP analysis in podocytes treated with or without Ang II (10−5 or 10−4 mol/l) for 14 days. (g) Real-time RT-PCR analysis of nephrin mRNA in podocytes treated with or without Ang II (10−5 or 10−4 mol/l) for 14 days. n=4 independent experiments with duplicates. *P<0.05 versus controls. #P<0.05 versus the respective groups. Data are mean±s.e.m.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

5. Figure 4
The nephrin promoter methylation is increased in human kidney diseases with decreased Kruppel-like Factor 4 (KLF4) and nephrin expression. (a) Representative confocal photomicrographs and (b) quantification of glomerular nephrin staining in kidney biopsies from patients with proteinuric kidney diseases. MCD, minimal change disease; FSGS, focal segmental glomerulosclerosis; DN, diabetic nephropathy. Clinical data are presented in Supplementary Tables 1 and 2. Scale bar: 50 μm. (c) Results of real-time methylation-specific PCR (MSP) analysis in laser-microdissected glomeruli of human biopsy samples without (upper panel) or with (lower panel) adjustment by podocyte number per glomerulus. (d) Correlation between nephrin expression by immunofluorescent staining and the nephrin promoter methylation by real-time MSP analysis without (upper panel) or with (lower panel) adjustment by podocyte number per glomerulus. (e) Expression of KLF4 by immunofluorescent staining with or without angiotensin receptor blocker (ARB) treatment at the time of biopsy. (f) Results of real-time MSP analysis in laser-microdissected glomeruli of human biopsy samples with or without ARB treatment at the time of biopsy without (upper panel) or with (lower panel) adjustment by podocyte number per glomerulus. Sample size in each group is indicated in Supplementary Tables 1 and 2. *P<0.05, **P<0.01 versus controls. Data are mean±s.e.m.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions