Protease-activated receptor-1 contributes to renal injury and interstitial fibrosis during chronic obstructive nephropathy

Abstract

End-stage renal disease, the final stage of all chronic kidney disorders, is associated with renal fibrosis and inevitably leads to renal failure and death. Transition of tubular epithelial cells (TECs) into mesenchymal fibroblasts constitutes a proposed mechanism underlying the progression of renal fibrosis and here we assessed whether protease-activated receptor (PAR)-1, which recently emerged as an inducer of epithelial-to-mesenchymal transition (EMT), aggravates renal fibrosis. We show that PAR-1 activation on TECs reduces the expression of epithelial markers and simultaneously induces mesenchymal marker expression reminiscent of EMT. We next show that kidney damage was reduced in PAR-1-deficient mice during unilateral ureter obstruction (UUO) and that PAR-1-deficient mice develop a diminished fibrotic response. Importantly, however, we did hardly observe any signs of mesenchymal transition in both wild-type and PAR-1-deficient mice suggesting that diminished fibrosis in PAR-1-deficient mice is not due to reduced EMT. Instead, the accumulation of macrophages and fibroblasts was significantly reduced in PAR-1-deficient animals which were accompanied by diminished production of MCP-1 and TGF-β. Overall, we thus show that PAR-1 drives EMT of TECs in vitro and aggravates UUO-induced renal fibrosis although this is likely due to PAR-1-dependent pro-fibrotic cytokine production rather than EMT.

Keywords: epithelial-to-mesenchymal transition; obstructive nephropathy; protease-activated receptor-1; renal fibrosis; unilateral ureter obstruction.

Figure 1

PAR‐1 activation induces mesenchymal transition of imPTECs. A, Relative mRNA expression levels of AQP‐1, ZO‐1, vimentin, α‐SMA, fibronectin, and collagen I in imPTECs 24 hours after stimulation with thrombin (1 U/mL) or TGF‐Β (5 ng/mL). Indicated is the average of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.005 (one‐way ANOVA followed by Bonferroni multiple comparisons test). B, Protein expression levels of AQP‐1, ZO‐1, vimentin, α‐SMA, fibronectin, and collagen I in imPTEC whole cell lysates 72 h after stimulation with thrombin (1 U/mL), TRAP‐6 (100 μM), or TGF‐Β (5 ng/mL). GAPDH or β‐actin expression served as a loading control. C: Representative images of imPTECs 72 h after stimulation with thrombin (1 U/ml), TRAP‐6 (100 μM), or TGF‐Β (0.5 ng/mL). Blue: DAPI, red: phalloidin, and green: α‐SMA (upper panels) and ZO‐1 (lower panels)

Figure 2

General evaluation of renal damage after UUO. A, PAR‐1 mRNA expression in kidney lysates of contralateral and obstructed (UUO) kidneys from wild‐type and PAR‐1‐deficient mice 1, 3, 7, and 10 d after UUO. B, Tubular injury score of PAS‐D‐stained kidney sections of mice killed at the indicated time points after the induction of UUO. *P < 0.05; **P < 0.01; (one‐way ANOVA followed by Bonferroni multiple comparisons test)

Figure 3

PAR‐1 deficiency limits renal fibrosis. A, α‐SMA and vimentin mRNA expression in whole kidney lysates of unobstructed (sham) and obstructed (UUO) kidneys of wild‐type (WT) and PAR‐1‐deficient (PAR‐1^−/−) mice, 7 and 10 d after UUO. B, Western blot analysis of α‐SMA and vimentin in whole kidney lysates of unobstructed (sham) and obstructed (UUO) kidneys of wild‐type (WT) and PAR‐1‐deficient (PAR‐1^−/−) mice, 7 and 10 d after UUO. GAPDH expression served as loading control. C, Quantification of Western blots depicted in panel B. D, mRNA expression of collagen I and fibronectin in whole kidney lysates of unobstructed (sham) and obstructed (UUO) kidneys of wild‐type (WT) and PAR‐1‐deficient (PAR‐1^−/−) mice, 7 and 10 d after UUO. E, Representative pictures of picrosirius red staining. F‐G, Western blot analysis (left: representative picture, right: quantification) of collagen I (F) and fibronectin (G) in whole kidney lysates of unobstructed (sham) and obstructed (UUO) kidneys of wild‐type (WT) and PAR‐1‐deficient (PAR‐1^−/−) mice, 7 and 10 d after UUO. GAPDH expression served as loading control. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001 (one‐way ANOVA followed by Bonferroni multiple comparisons test)

Figure 4

Interstitial expression of mesenchymal markers, in wild‐type and PAR‐1‐deficient mice. Representative images of α‐SMA (A)‐ and vimentin (B)‐stained kidney slides of wild‐type and PAR‐1‐deficient mice 7 and 10 d after UUO and in unobstructed (Sham) control kidneys; scale bars represent 50 μm. C, Western blot analysis of E‐cadherin, SGLT2 and AQP1 in whole kidney lysates of unobstructed (sham) and obstructed (UUO) kidneys of wild‐type (WT) and PAR‐1‐deficient (PAR‐1^−/−) mice, 7 and 10 d after UUO. GAPDH expression served as loading control. D, Quantification of Western blots depicted in panel C. E, SNAI1 mRNA expression in whole kidney lysates of unobstructed (sham) and obstructed (UUO) kidneys of wild‐type (WT) and PAR‐1‐deficient (PAR‐1^−/−) mice, 7 and 10 d after UUO. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001 (one‐way ANOVA followed by Bonferroni multiple comparisons test)

Figure 5

PAR‐1 activation induces pro‐fibrotic cytokine expression and potentiates macrophage influx during renal fibrosis. A, imPTEC mRNA expression of cytokines 24 h after PAR‐1 stimulation with thrombin (1 U/mL). Indicated is the average of three independent experiments. B‐C, Protein expression of MCP‐1 (B), total TGF‐β and active TGF‐β (C) measured by ELISA in whole kidney lysates of wild‐type and PAR‐1‐deficient (PAR‐1^−/−) mice 7 and 10 d after UUO and in unobstructed control kidneys. D, F4/80 (ie, macrophage marker) mRNA expression in kidney lysates of wild‐type and PAR‐1‐deficient (PAR‐1^−/−) mice 1, 3, 7, and 10 d after UUO and in unobstructed (Sham) control kidneys. E: Representative images of F4/80‐stained kidney slides of wild‐type and PAR‐1‐deficient (PAR‐1^−/−) mice 1, 3, 7, and 10 d after UUO and in unobstructed (Sham) control kidneys; scale bars represent 100 μm. *P < 0.05; ***P < 0.005; ****(t test [A] and one‐way ANOVA followed by Bonferroni multiple comparisons test [B‐D])