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. 2020 Sep 1;130(9):4845-4857.
doi: 10.1172/JCI135773.

Methylation in pericytes after acute injury promotes chronic kidney disease

Yu-Hsiang Chou  1   2   3 , Szu-Yu Pan  1   3   4 , Yu-Han Shao  3 , Hong-Mou Shih  3   5 , Shi-Yao Wei  3   6 , Chun-Fu Lai  1 , Wen-Chih Chiang  1 , Claudia Schrimpf  7 , Kai-Chien Yang  8   9 , Liang-Chuan Lai  3 , Yung-Ming Chen  1 , Tzong-Shinn Chu  1 , Shuei-Liong Lin  1   3   10   11
Affiliations

Methylation in pericytes after acute injury promotes chronic kidney disease

Yu-Hsiang Chou et al. J Clin Invest. .

Abstract

The origin and fate of renal myofibroblasts is not clear after acute kidney injury (AKI). Here, we demonstrate that myofibroblasts were activated from quiescent pericytes (qPericytes) and the cell numbers increased after ischemia/reperfusion injury-induced AKI (IRI-AKI). Myofibroblasts underwent apoptosis during renal recovery but one-fifth of them survived in the recovered kidneys on day 28 after IRI-AKI and their cell numbers increased again after day 56. Microarray data showed the distinctive gene expression patterns of qPericytes, activated pericytes (aPericytes, myofibroblasts), and inactivated pericytes (iPericytes) isolated from kidneys before, on day 7, and on day 28 after IRI-AKI. Hypermethylation of the Acta2 repressor Ybx2 during IRI-AKI resulted in epigenetic modification of iPericytes to promote the transition to chronic kidney disease (CKD) and aggravated fibrogenesis induced by a second AKI induced by adenine. Mechanistically, transforming growth factor-β1 decreased the binding of YBX2 to the promoter of Acta2 and induced Ybx2 hypermethylation, thereby increasing α-smooth muscle actin expression in aPericytes. Demethylation by 5-azacytidine recovered the microvascular stabilizing function of aPericytes, reversed the profibrotic property of iPericytes, prevented AKI-CKD transition, and attenuated fibrogenesis induced by a second adenine-AKI. In conclusion, intervention to erase hypermethylation of pericytes after AKI provides a strategy to stop the transition to CKD.

Keywords: Epigenetics; Nephrology; Pericytes.

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. Acute kidney injury induced a transient increase in renal myofibroblasts.
(A) Representative images showing Col1a1-GFP+α-smooth muscle actin (αSMA)+ myofibroblasts in the kidneys of Col1a1-GFPTg mice before (Ctrl) injury and at the indicated time points (day 2–56) after acute kidney injury (AKI) induced by right nephrectomy (Nx) followed by left ischemia/reperfusion injury (IRI). Arrows, arrowheads, and asterisks indicate Col1a1-GFP+ pericytes, Col1a1-GFP+αSMA+ myofibroblasts, and Col1a1-GFP–αSMA+ vascular smooth muscle cells, respectively. Scale bar: 25 μm. Original magnification, ×400. (B) Dot chart showing the cell numbers of Col1a1-GFP+αSMA+ myofibroblasts per high-powered field (HPF) at ×400 at the indicated time points. (C) Dot chart showing the proportion of αSMA+Col1a1-GFP+ myofibroblasts to Col1a1-GFP+ pericytes. Horizontal bars represent the mean, error bars represent the SEM. **P < 0.01, ***P < 0.001 vs. Ctrl by 1-way ANOVA with post hoc Dunnett’s correction. n = 5.
Figure 2
Figure 2. Renal myofibroblasts were derived from pericytes during acute kidney injury.
(A) Experimental scheme showing cohort labeling by tamoxifen and AKI-induced by Nx + IRI (IRI-AKI) in Col1a2-CreERTTg ROSA26fstdTomato/+ mice. Analyses were performed at the indicated time points. (B) Representative images showing the Col1a2-RFP+ pericyte lineage and αSMA+Col1a2-RFP+ myofibroblasts in the kidneys. Arrows and arrowheads indicate Col1a2-RFP+ pericytes and αSMA+Col1a2-RFP+ myofibroblasts, respectively. Scale bars: 25 μm. Original magnification, ×400. (C) Dot chart showing the cell numbers of the Col1a2-RFP+ pericyte lineage/HPF at the indicated time points. (D) Dot chart showing the cell numbers of αSMA+Col1a2-RFP+ myofibroblasts/HPF. (E) Dot chart showing the proportion of αSMA+Col1a2-RFP+ myofibroblasts to Col1a2-RFP+ pericytes. (F) AKI induced the activation of qPericytes into aPericytes. Based on the fate of Col1a2-RFP+ pericytes (Supplemental Figures 4–6), aPericytes might undergo apoptosis or inactivation (iPericytes) by day 28 after IRI-AKI. Horizontal bars represent the mean, error bars represent the SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Ctrl by 1-way ANOVA with post hoc Dunnett’s correction. n = 5.
Figure 3
Figure 3. Inactivated pericytes retained a higher potential for fibrosis and cell proliferation in vitro.
(A) Primary cultures of qPericytes and iPericytes from the kidneys before and on day 28 after IRI-AKI, respectively, were exposed to TGF-β1 or Ctrl culture medium for 16 hours. Dot charts showed the expression of Acta2, Col1a1, and Timp1 assessed by quantitative PCR. The expression levels were normalized by Gapdh. Horizontal bars represent the mean, error bars represent the SEM. *P < 0.05, **P < 0.01 by 1-way ANOVA with post hoc Tukey’s correction. n = 4 for each group. (B) Line chart showing the cell proliferation of qPericytes and iPericytes assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Data are expressed as the mean ± SEM. *P < 0.05 by t test at each time point. n = 4 for each group.
Figure 4
Figure 4. Inactivated pericytes retained a higher potential for cell proliferation induced by a second injury in vivo.
(A) Experimental scheme of a second AKI on the kidneys that recovered from the first AKI. IRI-AKI was induced on day 0 and allowed to recover until the second AKI induced by 1-week adenine diet from day 28. Sham operation on day 0 and regular diet from day 28 served as the Ctrl. The mice were allocated to group 1 (IRI+adenine, AKI on kidneys that recovered from the first AKI), group 2 (adenine, single AKI), group 3 (IRI, single AKI), and group 4 (Nx only, Ctrl). (B) Dot charts showing the plasma levels of BUN and Cre. (C) Representative images showing αSMA+ myofibroblasts in the kidneys. Scale bars: 25 μm. Original magnification, ×400. (D) Dot chart showing the cell numbers of αSMA+ myofibroblasts/HPF at ×400 in the renal sections. (E) Representative images showing Ki67+αSMA+ myofibroblasts in the kidneys. Scale bars: 25 μm. Original magnification, ×400. (F) Dot chart showing the cell numbers of Ki67+αSMA+ myofibroblasts/HPF in the renal sections. Horizontal bars represent the mean, error bars represent the SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by 1-way ANOVA with post hoc Tukey’s correction. n = 5 for each group.
Figure 5
Figure 5. Demethylation by 5-azacytidine during acute kidney injury attenuated the progression to chronic kidney disease.
(A) Experimental scheme for the IRI-AKI and 5-azacytidine (Aza) treatment. Nx followed by sham operation served as the Ctrl. Plasma BUN and Cre were analyzed on day 28 and day 180. Kidneys were analyzed on day 180. (B) Dot charts showing the plasma BUN and Cre in each group. (C) Representative images showing Picrosirius red staining in the renal sections on day 180. Scale bars: 50 μm. (D) Dot chart showing the quantification of Picrosirius red–stained fibrotic area in the renal sections. (E) Dot chart showing the cell numbers of αSMA+ myofibroblasts/HPF in the renal sections. (F) Dot chart showing the renal Acta2 expression normalized by Gapdh. Horizontal bars represent the mean, error bars represent the SEM. *P < 0.05, ***P < 0.001 by 1-way ANOVA with post hoc Tukey’s correction. n = 5 for each group.
Figure 6
Figure 6. Demethylation by 5-azacytidine during acute kidney injury attenuated the reactivation of pericytes and renal fibrosis induced by a second injury.
(A) Experimental scheme of adenine-AKI on the kidneys that recovered from the first IRI-AKI. The protocol for the administration of Aza or vehicle is shown in Figure 5A. Sham operation on day 0 and regular diet from day 28 served as the Ctrl. The mice were allocated to group 1 (IRI + adenine), group 2 (adenine only), group 3 (IRI only), group 4 (IRI + Aza + adenine), and group 5 (Nx only, Ctrl). Analyses were performed on day 35. (B) Dot charts showing the plasma BUN and Cre. n = 5. (C) Representative images showing αSMA+ myofibroblasts in the renal sections. Scale bars: 25 μm. Original magnification, ×400. n = 5. (D) Representative images showing Ki67+αSMA+ myofibroblasts in the kidneys. Scale bars: 25 μm. Original magnification, ×400. n = 5. (E) Dot chart showing the cell numbers of αSMA+ myofibroblasts/HPF in the renal sections. n = 5. (F) Dot chart showing the cell numbers of Ki67+αSMA+ myofibroblasts in the renal sections. n = 5. (G) Dot chart showing the quantification of Picrosirius red–stained fibrotic area in the renal sections. n = 5. (H–J) Dot chart showing the expression of renal Acta2, Col1a1, and Col3a1. Horizontal bars represent the mean, error bars represent the SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by 1-way ANOVA with post hoc Tukey’s correction. n = 4 for each group.
Figure 7
Figure 7. Demethylation by 5-azacytidine reversed the vascular stabilizing function of myofibroblasts.
(A) The scheme showing the model of 3D capillary formation by mixing pericytes (green) and HUVECs (red) in collagen gels. (B) A representative bright-field image demonstrating the cross sections of capillary tubes 24 hours after mixing pericytes and HUVECs in collagen gels. Scale bars: 250 μm. Original magnification, ×4. (C) A representative immunofluorescent image showing capillary formation (red) 24 hours after mixing the pericytes and CellTracker Red–stained HUVECs in collagen gels. Scale bars: 250 μm. Original magnification, ×4. (D) Dot chart showing the vascular density in the collagen gels in the presence of coculture with qPericytes (qPC), aPericytes (aPC, myofibroblasts), aPericytes after Aza treatment for 3 days (aPC + Aza), or in the absence of pericyte coculture (HUVEC alone) for 24 hours. n = 10. (E) Images showing the collapse of collagen gels induced by kallikrein (KLK) in gels with HUVECs in the presence or absence of pericyte coculture. Scale bars: 250 μm. Original magnification, ×4. (F) Line chart showing the percentage of collapsed area in gels with HUVECs in the presence or absence of pericyte coculture at the indicated time points after KLK administration. n = 10. (G) Dot charts showing the expression of Timp3, Angpt1, and Angpt2 assessed by quantitative PCR. The expression levels were normalized to Gapdh. Horizontal bars represent the mean, error bars represent the SEM. n = 4. *P < 0.05, **P < 0.01, ***P < 0.001 by 1-way ANOVA with post hoc Tukey’s correction.
Figure 8
Figure 8. YBX2 expression in pericytes decreased after acute kidney injury.
(A) Representative images showing the immunohistochemistry staining of YBX2 in the Ctrl kidneys. Arrows indicate YBX2+ interstitial cells. Scale bars: 25 μm. Original magnification, ×400. (B) Representative images showing Col1a1-GFP+ qPericytes with YBX2 expression in the Ctrl kidney of Col1a1-GFPTg mice. Scale bars: 25 μm. Original magnification, ×400. (C) Representative images showing the immunohistochemistry staining of YBX2 in kidneys on day 7 after IRI-AKI. Arrowheads indicate YBX2+ tubular epithelial cells while asterisks indicate interstitial cells without YBX2. Scale bars: 25 μm. Original magnification, ×400. (D) Dot chart showing the expression of Ybx2 in the Ctrl and day 7 IRI-AKI kidneys. n = 5. (E and F) Dot chart showing the expression of Ybx2 and Acta2 in purified pericytes from the kidneys at the indicated time points after IRI-AKI. Horizontal bars represent the mean, error bars represent the SEM. n = 4. *P < 0.05, **P < 0.01, ***P < 0.001 by 1-way ANOVA with post hoc Tukey’s correction.
Figure 9
Figure 9. YBX2 repressed the expression of Acta2.
(A) Line chart showing the expression of Ybx2 mRNA in the primary kidney pericytes after TGF-β1 exposure and withdrawal at the indicated time points. Data were expressed as the mean ± SEM. n = 3. *P < 0.05 by t test vs. before TGF-β1 withdrawal. (B) Representative images showing the electrophoresis of the PCR products of the Ybx2 gene using MeDIP or input DNA from primary kidney pericytes after TGF-β1 exposure for 24 or 120 hours. Pericytes without TGF-β1 exposure served as the Ctrl. (C) Scheme showing the primer design in the promoter regions of the Acta2 gene. (D) Representative images showing the electrophoresis of PCR products of the promoter of the Acta2 gene using DNA immunoprecipitated by anti-YBX2 antibody (ChIP) or input DNA from the primary kidney pericytes with or without TGF-β1 exposure for 120 hours. ChIP using isotype IgG served as the Ctrl. (E) Representative Western blot analyses for YBX2, αSMA, and β-actin in pericytes with or without lentiviral transduction for YBX2 expression. (F) Dot chart showing the relative expression of YBX2 and αSMA in pericytes with or without lentiviral transduction for YBX2 expression. Horizontal bars represent the mean, error bars represent the SEM. n = 3. **P < 0.01, ***P < 0.001 by 1-way ANOVA with post hoc Tukey’s correction.
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