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. 2010 May;16(5):544-50.
doi: 10.1038/nm.2135. Epub 2010 Apr 25.

Methylation determines fibroblast activation and fibrogenesis in the kidney

Wibke Bechtel  1 , Scott McGoohan, Elisabeth M Zeisberg, Gerhard A Müller, Hubert Kalbacher, David J Salant, Claudia A Müller, Raghu Kalluri, Michael Zeisberg
Affiliations

Methylation determines fibroblast activation and fibrogenesis in the kidney

Wibke Bechtel et al. Nat Med. 2010 May.

Abstract

Fibrogenesis is a pathological wound repair process that fails to cease, even when the initial insult has been removed. Fibroblasts are principal mediators of fibrosis, and fibroblasts from fibrotic tissues fail to return to their quiescent stage, including when cultured in vitro. In a search for underlying molecular mechanisms, we hypothesized that this perpetuation of fibrogenesis is caused by epigenetic modifications. We demonstrate here that hypermethylation of RASAL1, encoding an inhibitor of the Ras oncoprotein, is associated with the perpetuation of fibroblast activation and fibrogenesis in the kidney. RASAL1 hypermethylation is mediated by the methyltransferase Dnmt1 in renal fibrogenesis, and kidney fibrosis is ameliorated in Dnmt1(+/-) heterozygous mice. These studies demonstrate that epigenetic modifications may provide a molecular basis for perpetuated fibroblast activation and fibrogenesis in the kidney.

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Conflict of interest statement

Competing Financial Interests: The authors declare no competing financial interests.

Figures

Figure 1
Figure 1
5′-azacytidine ameliorates experimental renal fibrosis. (a) Representative photomicrographs of Masson's trichrome–stained kidney sections from mice that received folic acid (FA) (control) or folic acid and 5′-azacytidine (5′-Aza). Mice were analyzed at 0 d, 3 d, 28 d or 147 d after folic acid injection. Scale bar, 200 μm. (b) Interstitial fibrotic area in Masson's trichrome–stained sections. The graph summarizes average values at the indicated time points of each group. (c) Average serum creatinine concentrations at the indicated time points. (d) Representative confocal photomicrographs of immunofluorescence labeling for FSP-1 (top), α-SMA (middle) or type I collagen (bottom) on kidney sections of control CD1 mice (left column), kidneys of mice after folic acid injection (middle column) and from kidneys of mice that had received both folic acid and 5′-azacytidine (right column). Scale bar, 20 μm. (e–g) Relative stained area in frozen sections that were labeled with antibodies to FSP-1 (e), α-SMA (f) or type I collagen (g). Data are presented as means ± s.e.m. *P < 0.05.
Figure 2
Figure 2
RASAL1 hypermethylation in fibrotic kidney fibroblasts. (a) Electrophoresis of PCR products using primers for methylated RASAL1 (top) and unmethylated RASAL1 (bottom). Primary fibroblasts TK188, TK261, TK270, TK274, TK110 and TK257 were isolated from fibrotic human kidneys; TK124, TK173 and TK210 were derived from nonfibrotic human kidneys. (b) BGS of the indicated cells. Each box is representative of the indicated cell culture; each row of dots in the boxes is representative of the RASAL1 CpG island; each dot is representative of a single CpG. Empty dots indicate unmethylated CpGs; black dots indicate methylated CpGs. Each row represents a single sequenced clone (five for each cell line). We methylated DNA with the bacterial methyltransferase SssI as positive control. (c) Relative activity of pGL4-luciferase plasmid containing unmethylated or methylated RASAL1 promoter. (d) Average RASAL1 expression (by qRT-PCR) of each fibroblast culture (normalized to TK173 nonfibrotic fibroblasts set arbitrarily to 1). AU, arbitrary units. (e) Representative photomicrographs of Masson's trichrome–stained kidneys of control CD1 mice and of CD1 mice 5 months after folic acid injection. Scale bars, 20 μm. (f) Average serum creatinine concentrations of control CD1 mice and of mice with renal fibrosis. (g) Relative Rasal1 expression (qRT-PCR) in kidneys of control CD1 mice and of mice that had received folic acid. Expression in control mice was set to an arbitrary value of 1. (h) Rasal1 methylation, as analyzed by MeDIP. The top picture shows a virtual gel of Rasal1 PCR products of captured (methylated) DNA; the bottom picture shows Rasal1 PCR products of input DNA (to control for equal loading in immunoprecipitation). (i) Immunofluorescence double-labeling with antibodies to Rasal1 (red) and FSP-1 (green). The arrow highlights an FSP-1+Rasal1+ fibroblast. Scale bars, 20 μm. (j) The average number of FSP-1+Rasal1+ cells among all FSP-1+ fibroblasts per group (top) and the number of FSP-1+ fibroblasts per visual field in each group (bottom). Data are presented as means ± s.e.m. ***P < 0.001.
Figure 3
Figure 3
Absence of Rasal1 hypermethylation in kidneys that do not become fibrotic upon injury. (a) Representative photomicrographs of periodic acid–Schiff–stained kidney before and 3, 10 and 150 d after initiation of ischemia-reperfusion injury (IRI). Scale bars, 20 μm. (b) Average histopathological degree of tubular injury over time. (c) Rasal1 methylation upon ischemia-reperfusion injury, as analyzed by MeDIP. Top, PCR analysis of immunoprecipitated methylated DNA with primers specific to Rasal1. Middle, leucine aminopeptidase (LAP) positive control. Bottom, input DNA control. (d) Rasal1 methylation, as analyzed in control kidneys, in fibrotic kidneys of mice that had been challenged with folic acid and in kidneys of mice that had received folic acid and had been also treated with 5′-azacytidine. The picture shows a virtual gel of methylated DNA immunoprecipitation analysis. Data are presented as means ± s.e.m.
Figure 4
Figure 4
Rasal1 silencing causes renal fibroblast activation via Ras hyperactivity. (a) Methylation of Rasal1 in primary fibroblasts isolated from fibrotic and nonfibrotic mouse kidneys, as analyzed by MeDIP. Top, sample analysis; bottom, input DNA control reaction. (b) Relative Rasal1 expression in control fibroblasts, fibrotic fibroblasts, control fibroblasts transfected with Rasal1-specific siRNA and fibrotic fibroblasts transfected with Rasal1-specific siRNA. (c) Ras activity of nonfibrotic control fibroblasts, nonfibrotic control fibroblasts transfected with Rasal1-specific siRNA, fibrotic fibroblasts and fibrotic fibroblasts transfected with Rasal1-specific siRNA after 3 d of culture in serum-free medium. (d) Average cell counts for nonfibrotic control fibroblasts, nonfibrotic control fibroblasts transfected with Rasal1-specific siRNA, fibrotic fibroblasts and fibrotic fibroblasts transfected with Rasal1-specific siRNA at the indicated time points. (e,f) Relative type I collagen and α-SMA expression, respectively, in each group listed. (g) Representative photomicrographs of Masson's trichrome–stained kidneys of healthy CD1 mice (control), mice who had received a single injection of folic acid (24 weeks after folic acid injection) and mice who had received treatment with the Ras inhibitor FTS in addition to receiving folic acid. Scale bars, 100 μm. The bar graph summarizes the relative fibrotic area in each group. (h) Average serum creatinine concentrations at the indicated time points in the indicated groups. (i) Kidneys of three representative mice in each group were analyzed by MeDIP; the top picture shows amplified methylated Rasal1 DNA, and the bottom picture shows the control input DNA. For b, e and f, control values were arbitrarily set to 1. Data are presented as means ± s.e.m. * P < 0.05; **P < 0.01; ***P < 0.001.
Figure 5
Figure 5
Dnmts in the mouse model of folic acid–induced nephropathy. (a) Relative expression of Dnmt1, DNmt3a and Dnmt3b in nonfibrotic and fibrotic mouse kidneys. Expression in FA-treated kidneys was normalized to kidneys from uninjected control mice, with each control value set arbitrarily to 1. (b) Representative photomicrographs of immunofluorescence labeling with antibodies specific to Dnmt1 (green) and FSP-1 (red). The arrows highlight nuclear DNMT1 staining in FSP-1+ fibroblasts in fibrotic kidneys (bottom). Scale bars, 20 μm. (c) Dnmt1 protein–Rasal1 promoter complexes were captured with Dnmt1-specific antibodies, detected by Rasal1 PCR and analyzed by electrophoresis. (d) Representative photomicrographs of Masson's trichrome–stained kidneys from wild-type control mice (top left), wild-type mice that had received folic acid (top right), Dnmt1+/− control mice (bottom left) and Dnmt1+/− mice that had received folic acid (bottom right). Scale bars, 200 μm. (e) Average fibrotic area in each of the indicated groups. (f) Average serum creatinine concentrations in each of the indicated groups. (g) MeDIP analysis for Rasal1 (top) and the input control DNA (bottom) of DNA isolated from kidneys of each indicated group. Data are presented as means ± s.e.m. **P < 0.01, ***P < 0.001.
Figure 6
Figure 6
TGF-β1–induced methylation of Rasal1 in kidney fibroblasts is mediated by Dnmt1. (a) Relative Rasal1 expression over time. (b) Rasal1 methylation in primary mouse kidney fibroblasts in response to TGF-β1 exposure for 24 h and 5 d, as indicated by the asterisks in panel a. We analyzed methylation by methylation-specific PCR. Top, a virtual gel of the PCR with primers specific for methylated Rasal1; bottom, the corresponding result when primers specific for nonmethylated Rasal1. (c) Immunoblot with antibodies specific for Dnmt1. (d) Representative photomicrographs of fibroblasts labeled with antibodies to Dnmt1 (green). Fibroblasts were maintained in serum-free medium (control) or exposed to TGF-β1 for 72 h. Arrowheads highlight Dnmt1− nuclei in control fibroblasts, and arrows point to Dnmt1+ nuclei in TGF-β1–treated fibroblasts. (e) Rasal1 methylation in response to exposure to TGF-β1 (for 5 d) in primary fibroblasts isolated from kidneys of wild-type (WT) C57BL/6 mice and from littermate Dnmt1+/− mice. Top, MeDIP sample analysis; bottom, input DNA control reaction. (f) Rasal1 methylation in control fibroblasts, fibroblasts transfected with Dnmt1-specific siRNA, TGF-β1–stimulated fibroblasts and fibroblasts transfected with Dnmt1-specific siRNA and stimulated with TGF-β1 for 5 d. (g) Double-immunofluorescence labeling on control mouse kidneys (top) and on kidneys that were made fibrotic by a single injection of folic acid (bottom) using antibodies to Dnmt1 (green) and phosphorylated Smad2 and Smad3 (pSmad2/3, red). Arrows highlight nuclear pSmad2/3 colocalized with nuclear Dnmt1. Scale bars, 200 μm.
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