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. 2023 Sep 7;11(9):2475.
doi: 10.3390/biomedicines11092475.

The Role of Hypoxia on the Trimethylation of H3K27 in Podocytes

Johanna Barth  1 , Ivonne Loeffler  1 , Tzvetanka Bondeva  1 , Marita Liebisch  1 , Gunter Wolf  1
Affiliations

The Role of Hypoxia on the Trimethylation of H3K27 in Podocytes

Johanna Barth et al. Biomedicines. .

Abstract

Epigenetic alterations contribute to the pathogenesis of chronic diseases such as diabetes mellitus. Previous studies of our group showed that diabetic conditions reduce the trimethylation of H3K27 in podocytes in a NIPP1- (nuclear inhibitor of protein phosphatase 1) and EZH2- (enhancer of zeste homolog 2) dependent manner. It has been previously reported that in differentiated podocytes, hypoxia decreases the expression of slit diaphragm proteins and promotes foot process effacement, thereby contributing to the progression of renal disease. The exact mechanisms are, however, not completely understood. The aim of this study was to analyze the role of hypoxia and HIFs (hypoxia-inducible factor) on epigenetic changes in podocytes affecting NIPP1, EZH2 and H3K27me3, in vitro and in vivo. In vivo studies were performed with mice exposed to 10% systemic hypoxia for 3 days or injected with 3,4-DHB (dihydroxybenzoate), a PHD (prolyl hydroxylase) inhibitor, 24 h prior analyses. Immunodetection of H3K27me3, NIPP1 and EZH2 in glomerular podocytes revealed, to the best of our knowledge for the first time, that hypoxic conditions and pharmacological HIFs activation significantly reduce the expression of NIPP1 and EZH2 and diminish H3K27 trimethylation. These findings are also supported by in vitro studies using murine-differentiated podocytes.

Keywords: EZH2; H3K27me3; NIPP1; enhancer of zeste homolog 2; epigenetic; hypoxia; nuclear inhibitor of protein phosphatase 1; podocyte.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Detection of NIPP1 (nuclear inhibitor of protein phosphatase 1) expression in cultured podocytes under hypoxic conditions: (a) NIPP1 mRNA expression; N = 3, n = 12; *** p ≤ 0.001 compared with normoxia; (b) Western blot of NIPP1 protein expression and quantification; TBP-TATA box binding protein; N = 5, n = 4; * p ≤ 0.05 compared with normoxia; (c) NIPP1 immunofluorescence and quantification; red-NIPP1, blue-DAPI (4′,6-diamidino-2-phenylindole), scale bar: 20 μm; N = 2, n = 10; *** p ≤ 0.001 compared with normoxia.
Figure 2
Figure 2
Detection of EZH2 (enhancer of zeste homolog 2) expression in cultured podocytes under hypoxic conditions: (a) EZH2 mRNA expression; N = 3, n = 12; *** p ≤ 0.001 compared with normoxia; (b) Western blot of EZH2 protein expression and quantification; TBP-TATA box binding protein; N = 6, n = 4; (c) EZH2 immunofluorescence and quantification; red-EZH2, blue-DAPI; N = 2, n = 10, scale bar: 20 μm; *** p ≤ 0.001 compared with normoxia.
Figure 3
Figure 3
Detection of H3K27me3 in cultured podocytes under hypoxic conditions: (a) Western blot of H3K27me3 and quantification; TBP-TATA box binding protein; N = 4, n = 4; *** p ≤ 0.001 compared with normoxia; (b) H3K27me3 immunofluorescence and quantification; purple-H3K27me3, blue-DAPI; N = 2, n = 10, scale bar: 20 μm; *** p ≤ 0.001 compared with normoxia.
Figure 4
Figure 4
Role of systemic hypoxia (10% O2) on NIPP1 expression, EZH2 expression and trimethylation of H3K27 in mice: (a) double immunofluorescence staining of NIPP1 and Synaptopodin (SYN); red-NIPP1, green-SYN, blue-DAPI; N = 6, n = 20; representative podocytes are tagged with white asterisks; ** p ≤ 0.01 compared with normoxia; (b) double immunofluorescence staining of EZH2 and SYN; red-EZH2, green-SYN, blue-DAPI; N = 6, n = 20; representative podocytes are tagged with white asterisks; *** p ≤ 0.001 compared with normoxia; (c) double immunofluorescence staining of H3K27me3 and SYN; purple-H3K27me3, green-SYN, blue-DAPI; N = 6, n = 20; representative podocytes are labelled with white asterisks; * p ≤ 0.05 compared with normoxia.
Figure 5
Figure 5
Role of pharmacological activation of HIFs on NIPP1, EZH2 and H3K27me3 in mice: (a) double immunofluorescence staining of NIPP1 and SYN; red-NIPP1, green-SYN, blue-DAPI; N = 6, n = 15; representative podocytes are tagged with white asterisks; 3,4-DHB—3,4-dihydroxybenzoate; *** p ≤ 0.001 compared with control; (b) double immunofluorescence staining of EZH2 and SYN; red-EZH2, green-SYN, blue-DAPI; N = 6, n = 15; representative podocytes are tagged with white asterisks; *** p ≤ 0.001 compared with control; (c) double immunofluorescence staining of H3K27me3 and SYN; purple-H3K27me3, green-SYN, blue-DAPI; N = 6, n = 15; representative podocytes are tagged with white asterisks; *** p ≤ 0.001 compared with control.
Figure 5
Figure 5
Role of pharmacological activation of HIFs on NIPP1, EZH2 and H3K27me3 in mice: (a) double immunofluorescence staining of NIPP1 and SYN; red-NIPP1, green-SYN, blue-DAPI; N = 6, n = 15; representative podocytes are tagged with white asterisks; 3,4-DHB—3,4-dihydroxybenzoate; *** p ≤ 0.001 compared with control; (b) double immunofluorescence staining of EZH2 and SYN; red-EZH2, green-SYN, blue-DAPI; N = 6, n = 15; representative podocytes are tagged with white asterisks; *** p ≤ 0.001 compared with control; (c) double immunofluorescence staining of H3K27me3 and SYN; purple-H3K27me3, green-SYN, blue-DAPI; N = 6, n = 15; representative podocytes are tagged with white asterisks; *** p ≤ 0.001 compared with control.
Figure 6
Figure 6
Schematic illustration of the assumed mechanism that contributes to epigenetic changes of H3K27me3 and podocyte injury in CKD.
See this image and copyright information in PMC

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