KLF4-dependent epigenetic remodeling modulates podocyte phenotypes and attenuates proteinuria
Kaori Hayashi, … , Yusuke Sakamaki, Hiroshi Itoh
Kaori Hayashi, … , Yusuke Sakamaki, Hiroshi Itoh
Published May 8, 2014
Citation Information: J Clin Invest. 2014;124(6):2523-2537. https://doi.org/10.1172/JCI69557.
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Research Article Nephrology

KLF4-dependent epigenetic remodeling modulates podocyte phenotypes and attenuates proteinuria

Abstract

The transcription factor Kruppel-like factor 4 (KLF4) has the ability, along with other factors, to reprogram somatic cells into induced pluripotent stem (iPS) cells. Here, we determined that KLF4 is expressed in kidney glomerular podocytes and is decreased in both animal models and humans exhibiting a proteinuric. Transient restoration of KLF4 expression in podocytes of diseased glomeruli in vivo, either by gene transfer or transgenic expression, resulted in a sustained increase in nephrin expression and a decrease in albuminuria. In mice harboring podocyte-specific deletion of Klf4, adriamycin-induced proteinuria was substantially exacerbated, although these animals displayed minimal phenotypical changes prior to adriamycin administration. KLF4 overexpression in cultured human podocytes increased expression of nephrin and other epithelial markers and reduced mesenchymal gene expression. DNA methylation profiling and bisulfite genomic sequencing revealed that KLF4 expression reduced methylation at the nephrin promoter and the promoters of other epithelial markers; however, methylation was increased at the promoters of genes encoding mesenchymal markers, suggesting selective epigenetic regulation of podocyte gene expression. Together, these results suggest that KLF4 epigenetically modulates podocyte phenotype and function and that the podocyte epigenome can be targeted for direct intervention and reduction of proteinuria.

Authors

Kaori Hayashi, Hiroyuki Sasamura, Mari Nakamura, Tatsuhiko Azegami, Hideyo Oguchi, Yusuke Sakamaki, Hiroshi Itoh

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Figure 8

KLF4 expression increases nephrin promoter activity in podocytes.

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KLF4 expression increases nephrin promoter activity in podocytes. (A) Ma...
(A) Map of nephrin promoter regions included in the luciferase constructs. CpG 1–5 correspond to the sites shown in Supplemental Figure 8. (B) Luciferase activity 48 hours after transfection of KLF4-overexpressing podocytes (KLF4) or control podocytes (empty) with constructs containing regions a–c (n = 6). (C) ChIP assay for the presence of KLF4 in the promoter region of nephrin in KLF4-overexpressing podocytes (KLF4) or control podocytes (empty). The amplified fragment corresponds to region B in Figure 8A. The bar graph shows the quantification of KLF4/input intensity (n = 4). (D) Effect of KRE deletion on nephrin promoter activity. Luciferase activity was assayed 48 hours after transfection of luciferase constructs containing region B with (KRE[–]) or without (KRE[+]) specific deletion of the KRE (shown boxed in the upper panel of Supplemental Figure 8) into KLF4-overexpressing podocytes or control podocytes (n = 6). (E) Effect of KRE deletion on DNA binding to podocyte nuclear extracts analyzed by EMSA. Nuclear extracts from KLF4-overexpressing podocytes were pretreated with or without unlabeled competitor DNA or KLF4 antibody before incubation with labeled probes for EMSA to confirm specificity of the complexes. KRE (–) and (+) refer to probes with or without a deletion of the KRE sequence, respectively. **P < 0.01 vs. controls. †P < 0.05, ††P < 0.01 vs. the respective groups.