International Journal of
Molecular Sciences Article
Renalase Protects against Renal Fibrosis by Inhibiting the Activation of the ERK Signaling Pathways Yiru Wu, Liyan Wang, Dai Deng, Qidong Zhang and Wenhu Liu * Department of Nephrology, Affiliated Beijing Friendship Hospital, Faculty of Kidney Diseases, Capital Medical University, No. 95 Yong An Road, Xi Cheng District, Beijing 100050, China; [email protected] (Y.W.); [email protected] (L.W.); [email protected] (D.D.); [email protected] (Q.Z.) * Correspondence: [email protected]; Tel.: +86-10-6313-8579; Fax: +86-10-6313-9144 Academic Editor: Gregor Drummen Received: 5 February 2017; Accepted: 4 April 2017; Published: 27 April 2017
Abstract: Renal interstitial fibrosis is a common pathway for the progression of chronic kidney disease (CKD) to end-stage renal disease. Renalase, acting as a signaling molecule, has been reported to have cardiovascular and renal protective effects. However, its role in renal fibrosis remains unknown. In this study, we evaluated the therapeutic efficacy of renalase in rats with complete unilateral ureteral obstruction (UUO) and examined the inhibitory effects of renalase on transforming growth factor-β1 (TGF-β1)-induced epithelial–mesenchymal transition (EMT) in human proximal renal tubular epithelial (HK-2) cells. We found that in the UUO model, the expression of renalase was markedly downregulated and adenoviral-mediated expression of renalase significantly attenuated renal interstitial fibrosis, as evidenced by the maintenance of E-cadherin expression and suppressed expression of α-smooth muscle actin (α-SMA), fibronectin and collagen-I. In vitro, renalase inhibited TGF-β1-mediated upregulation of α-SMA and downregulation of E-cadherin. Increased levels of Phospho-extracellular regulated protein kinases (p-ERK1/2) in TGF-β1-stimulated cells were reversed by renalase cotreatment. When ERK1 was overexpressed, the inhibition of TGF-β1-induced EMT and fibrosis mediated by renalase was attenuated. Our study provides the first evidence that renalase can ameliorate renal interstitial fibrosis by suppression of tubular EMT through inhibition of the ERK pathway. These results suggest that renalase has potential renoprotective effects in renal interstitial fibrosis and may be an effective agent for slowing CKD progression. Keywords: renalase; chronic kidney disease; renal interstitial fibrosis; ERK signaling pathway; epithelial–mesenchymal transition
1. Introduction It is generally recognized that the incidence of chronic kidney disease (CKD) is increasing worldwide and is a public health problem posing a serious threat to human health. Renal interstitial fibrosis (RIF), characterized by excessive deposition of extracellular matrix, is recognized as a common pathological feature of CKD which leads to the development of end-stage renal disease (ESRD), accompanied by progression of renal dysfunction [1]. Accumulating evidence suggests that epithelial–mesenchymal transition (EMT) of tubular epithelial cells contributes significantly to the onset and pathogenesis of RIF [2]. EMT, a process whereby epithelial cells lose their epithelial phenotype and gain attributes of mesenchymal cells, has been implicated in the generation of myofibroblasts and fibroblasts in kidney disease [3]. Of the many factors that trigger EMT, transforming growth factor-β1 (TGF-β1) is considered to be the most potent inducer of EMT in various types of epithelial cells via both Smad-dependent and -independent mechanisms [4]. Therefore, targeting TGF-β1-mediated EMT may ameliorate RIF and the progressive loss of renal function. Int. J. Mol. Sci. 2017, 18, 855; doi:10.3390/ijms18050855
www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2017, 18, 855
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Renalase is a recently discovered flavoprotein with oxidoreductase activity, which is expressed in a variety of tissues including the kidney, heart and nervous system [5,6]. In addition to its enzymatic function, renalase may also act as a signaling molecule which can lighten acute kidney injury (AKI) by interacting with cell surface receptors, such as the plasma membrane calcium ATPase isoform PMCA4b [7,8]. The expression level of renalase is significantly diminished in sub-totally nephrectomized rats and patients with ESRD and CKD [9–11]. Clinical studies show that renalase deficiency and single-nucleotide polymorphisms in renalase are associated with essential hypertension, coronary heart disease, stroke and diabetes [10,12,13]. Administration of recombinant renalase protects against AKI, contrast-induced nephropathy and cardiac ischemia/reperfusion injury [14–17]. However, there is no research on whether renalase can reverse EMT and reduce renal interstitial fibrosis. In the present study, we examined the anti-fibrosis effects of renalase in vivo in a rat model of tubulointerstitial fibrosis induced by unilateral ureteral obstruction (UUO) and in vitro in EMT of human proximal tubular epithelial cells induced by TGF-β1. In addition, we explored whether the effects of renalase effects are mediated by interfering with canonical or non-canonical TGF-β1 signaling pathways through investigation of signaling molecules in vitro. 2. Results 2.1. Establishment of the UUO Model and Expression of Renalase in Rat Kidneys At 7, 10, 14, or 21 days after UUO, rats were killed and their kidneys harvested to analyze fibrosis. In the sham group, the structure of the kidney was normal; the renal tubules were arranged closely and orderly, the basement membrane was smooth and continuous, and there was no inflammatory cell infiltration of the interstitial area. After UUO, the kidney on the obstructed side was enlarged while renal parenchyma was thinned. Seven days after the operation, there was a large number of patchy inflammatory cell infiltrates in the renal interstitium, moderate to severe dilatation of the renal tubules, portions of distal tubular atrophy, luminal occlusion, renal tubule epithelial cell swelling and degeneration, and renal interstitial edema and broadening, showing mild fibrous tissue proliferation. Moreover, with prolonged obstruction, the above phenomena were further aggravated (Figure 1A). Masson’s trichrome staining (MTS) revealed that, in the sham group, the linear distribution of blue interstitial collagen fibers can only be seen in the renal tubular basement membrane. In the UUO group, as time passed, the blue interstitial collagen fibers gradually increased, suggesting progressive fibrosis (Figure 1A,B). Moreover, immunofluorescence demonstrated that the expression of E-cadherin decreased while α-SMA increased (Figure 1C). Immunohistochemistry revealed that the expression of fibronectin (FN) and collagen I (Col-I) increased with the time of obstruction (Figure 1E), consistent with western blot data (Figure 1D). However, there was no statistically significant difference in FN between the 14-day group and 21-day group (p = 0.242). Given that at 14 days post-obstruction renal fibrosis was already evident, we selected 14 days as the endpoint at which to analyze fibrosis in the subsequent experiments. To observe the relationship between fibrosis and renalase, we first investigated the expression level of renalase in the kidney. Immunohistochemistry showed that, in 8-week-old rats, renalase was mainly distributed in the renal tubules under normal conditions. There was also significant expression in glomerular mesangial areas (Figure 1F). After UUO, with the aggravation of fibrosis, the expression of renalase was gradually decreased, both in the kidney (Figure 1F,G) and in plasma (Figure 1H).
Int. J. Mol. Sci. 2017, 18, 855 Int. J. Mol. Sci. 2017, 18, 855
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Figure 1. Cont. Figure 1. Cont.
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Figure Figure1.1.Cont. Cont.
Int. J. Mol. Sci. 2017, 18, 855 Int. J. Mol. Sci. 2017, 18, 855
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Figure Figure 1. 1.Cont. Cont.
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Figure1. 1. Establishment of unilateral the unilateral ureteral obstruction model and expression Figure Establishment of the ureteral obstruction (UUO) (UUO) model and expression of renalaseof in rat (A) Histological changesin in kidneysureter in unilateral inrenalase rat kidneys. (A)kidneys. Histological changes in kidneys unilateral ligation.ureter Kidneyligation. sectionsKidney from sections from were various groupstowere subjectedeosin to Hematoxylin eosin (H&E) and Masson’s trichrome various groups subjected Hematoxylin (H&E) and Masson’s trichrome staining (MTS) at staining (MTS) at different times after UUO. With the prolongation of obstruction, renal tubular different times after UUO. With the prolongation of obstruction, renal tubular epithelial cell atrophy, epithelial cell atrophy, luminal expansion, inflammatory cell infiltration and tubule luminal expansion, interstitial inflammatory cell interstitial infiltration and tubule widening were aggravated and wideningfibrosis were increased aggravated and interstitial fibrosis gradually; magnification: 40×; interstitial gradually; magnification: 40×increased ; (B) Quantitative analysis of renal fibrotic (B) Quantitative analysisRenal of renal fibrotic lesions in various Renal lesions (defined lesions in various groups. fibrotic lesions (defined as thegroups. percentage of fibrotic the MTS-positive fibroticas the percentage of thebyMTS-positive fibrotic area) were quantified byImmunofluorescence computer-aided morphometric area) were quantified computer-aided morphometric analyses; (C) showing the expression E-cadherin and smooth muscle (α-SMA). Representative photomicrographs analyses; (C) of Immunofluorescence showing the actin expression of E-cadherin and smooth muscle actin are shown. With the extension of obstruction, fluorescence intensity E-cadherin became weak and (α-SMA). Representative photomicrographs are shown. Withofthe extension of obstruction, fluorescence intensity of of α-SMA strengthen gradually. The fluorescence difference wasintensity not statistically significant at fluorescence intensity E-cadherin became weak and of α-SMA strengthen 14gradually. and 21 days; 40×not ; (D,E) Western blotting andatimmunohistochemistry demonstrated Themagnification: difference was statistically significant 14 and 21 days; magnification: 40×; that, after UUO, blotting the expression of fibronectin and collagen-I increased a time-dependent manner.of (D,E) Western and immunohistochemistry demonstrated that,inafter UUO, the expression The differenceand wascollagen-I not statistically significant at 14 days and manner. 21 days. The brown/yellow indicates fibronectin increased in a time-dependent difference wascolor not statistically antibody binding; Arrows to positive results in Figure 1E.indicates Magnification: 40×binding; ; (F) Representative significant at 14 days andrefer 21 days. The brown/yellow color antibody Arrows refer photomicrographs of immunohistochemistry showing40×; the expression and localization of renalase inof to positive results in Figure 1E. Magnification: (F) Representative photomicrographs the ligated kidney. Renalase was mainly distributed the renal tubules underinnormal conditions; immunohistochemistry showing the expression andinlocalization of renalase the ligated kidney. After UUO,was with the aggravation of the fibrosis, the expression of renalase was gradually decreased. Renalase mainly distributed in renal tubules under normal conditions; After UUO, with the The differenceofwas not statistically significant in 14 days and 21decreased. days; Arrows refer to positive aggravation fibrosis, the expression of renalase was gradually The difference was not results in Figure 1E. Magnification: 40× ; (G)21Western blotting and analysisinofFigure renalase statistically significant in 14 days and days; Arrows referdensitometric to positive results 1E. protein levels in various groups. The results were consistent with immunohistochemistry; (H) The Magnification: 40×; (G) Western blotting and densitometric analysis of renalase protein levels in concentration of renalase in plasma. the prolongation of obstruction, the concentration of renalaseof various groups. The results were With consistent with immunohistochemistry; (H) The concentration inrenalase plasma in decreased Results are presented as percentages of controlofvalues andinare the plasma. gradually. With the prolongation of obstruction, the concentration renalase plasma means ± Standard deviation five animals per group. of * pcontrol < 0.05, compared the control decreased gradually. Results(SD) are of presented as percentages values andwith are the means ± # p < 0.05, compared with the 7-day group; & p < 0.05, compared with the 10-day group; group; Standard deviation (SD) of five animals per group. * p < 0.05, compared with the control group; # p < ˆ p0.05, < 0.05, compared withthe the7-day 14-daygroup; group;&NS: statistical difference; Col-I:^ collagen compared with p
Molecular Sciences Article
Renalase Protects against Renal Fibrosis by Inhibiting the Activation of the ERK Signaling Pathways Yiru Wu, Liyan Wang, Dai Deng, Qidong Zhang and Wenhu Liu * Department of Nephrology, Affiliated Beijing Friendship Hospital, Faculty of Kidney Diseases, Capital Medical University, No. 95 Yong An Road, Xi Cheng District, Beijing 100050, China; [email protected] (Y.W.); [email protected] (L.W.); [email protected] (D.D.); [email protected] (Q.Z.) * Correspondence: [email protected]; Tel.: +86-10-6313-8579; Fax: +86-10-6313-9144 Academic Editor: Gregor Drummen Received: 5 February 2017; Accepted: 4 April 2017; Published: 27 April 2017
Abstract: Renal interstitial fibrosis is a common pathway for the progression of chronic kidney disease (CKD) to end-stage renal disease. Renalase, acting as a signaling molecule, has been reported to have cardiovascular and renal protective effects. However, its role in renal fibrosis remains unknown. In this study, we evaluated the therapeutic efficacy of renalase in rats with complete unilateral ureteral obstruction (UUO) and examined the inhibitory effects of renalase on transforming growth factor-β1 (TGF-β1)-induced epithelial–mesenchymal transition (EMT) in human proximal renal tubular epithelial (HK-2) cells. We found that in the UUO model, the expression of renalase was markedly downregulated and adenoviral-mediated expression of renalase significantly attenuated renal interstitial fibrosis, as evidenced by the maintenance of E-cadherin expression and suppressed expression of α-smooth muscle actin (α-SMA), fibronectin and collagen-I. In vitro, renalase inhibited TGF-β1-mediated upregulation of α-SMA and downregulation of E-cadherin. Increased levels of Phospho-extracellular regulated protein kinases (p-ERK1/2) in TGF-β1-stimulated cells were reversed by renalase cotreatment. When ERK1 was overexpressed, the inhibition of TGF-β1-induced EMT and fibrosis mediated by renalase was attenuated. Our study provides the first evidence that renalase can ameliorate renal interstitial fibrosis by suppression of tubular EMT through inhibition of the ERK pathway. These results suggest that renalase has potential renoprotective effects in renal interstitial fibrosis and may be an effective agent for slowing CKD progression. Keywords: renalase; chronic kidney disease; renal interstitial fibrosis; ERK signaling pathway; epithelial–mesenchymal transition
1. Introduction It is generally recognized that the incidence of chronic kidney disease (CKD) is increasing worldwide and is a public health problem posing a serious threat to human health. Renal interstitial fibrosis (RIF), characterized by excessive deposition of extracellular matrix, is recognized as a common pathological feature of CKD which leads to the development of end-stage renal disease (ESRD), accompanied by progression of renal dysfunction [1]. Accumulating evidence suggests that epithelial–mesenchymal transition (EMT) of tubular epithelial cells contributes significantly to the onset and pathogenesis of RIF [2]. EMT, a process whereby epithelial cells lose their epithelial phenotype and gain attributes of mesenchymal cells, has been implicated in the generation of myofibroblasts and fibroblasts in kidney disease [3]. Of the many factors that trigger EMT, transforming growth factor-β1 (TGF-β1) is considered to be the most potent inducer of EMT in various types of epithelial cells via both Smad-dependent and -independent mechanisms [4]. Therefore, targeting TGF-β1-mediated EMT may ameliorate RIF and the progressive loss of renal function. Int. J. Mol. Sci. 2017, 18, 855; doi:10.3390/ijms18050855
www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2017, 18, 855
2 of 25
Renalase is a recently discovered flavoprotein with oxidoreductase activity, which is expressed in a variety of tissues including the kidney, heart and nervous system [5,6]. In addition to its enzymatic function, renalase may also act as a signaling molecule which can lighten acute kidney injury (AKI) by interacting with cell surface receptors, such as the plasma membrane calcium ATPase isoform PMCA4b [7,8]. The expression level of renalase is significantly diminished in sub-totally nephrectomized rats and patients with ESRD and CKD [9–11]. Clinical studies show that renalase deficiency and single-nucleotide polymorphisms in renalase are associated with essential hypertension, coronary heart disease, stroke and diabetes [10,12,13]. Administration of recombinant renalase protects against AKI, contrast-induced nephropathy and cardiac ischemia/reperfusion injury [14–17]. However, there is no research on whether renalase can reverse EMT and reduce renal interstitial fibrosis. In the present study, we examined the anti-fibrosis effects of renalase in vivo in a rat model of tubulointerstitial fibrosis induced by unilateral ureteral obstruction (UUO) and in vitro in EMT of human proximal tubular epithelial cells induced by TGF-β1. In addition, we explored whether the effects of renalase effects are mediated by interfering with canonical or non-canonical TGF-β1 signaling pathways through investigation of signaling molecules in vitro. 2. Results 2.1. Establishment of the UUO Model and Expression of Renalase in Rat Kidneys At 7, 10, 14, or 21 days after UUO, rats were killed and their kidneys harvested to analyze fibrosis. In the sham group, the structure of the kidney was normal; the renal tubules were arranged closely and orderly, the basement membrane was smooth and continuous, and there was no inflammatory cell infiltration of the interstitial area. After UUO, the kidney on the obstructed side was enlarged while renal parenchyma was thinned. Seven days after the operation, there was a large number of patchy inflammatory cell infiltrates in the renal interstitium, moderate to severe dilatation of the renal tubules, portions of distal tubular atrophy, luminal occlusion, renal tubule epithelial cell swelling and degeneration, and renal interstitial edema and broadening, showing mild fibrous tissue proliferation. Moreover, with prolonged obstruction, the above phenomena were further aggravated (Figure 1A). Masson’s trichrome staining (MTS) revealed that, in the sham group, the linear distribution of blue interstitial collagen fibers can only be seen in the renal tubular basement membrane. In the UUO group, as time passed, the blue interstitial collagen fibers gradually increased, suggesting progressive fibrosis (Figure 1A,B). Moreover, immunofluorescence demonstrated that the expression of E-cadherin decreased while α-SMA increased (Figure 1C). Immunohistochemistry revealed that the expression of fibronectin (FN) and collagen I (Col-I) increased with the time of obstruction (Figure 1E), consistent with western blot data (Figure 1D). However, there was no statistically significant difference in FN between the 14-day group and 21-day group (p = 0.242). Given that at 14 days post-obstruction renal fibrosis was already evident, we selected 14 days as the endpoint at which to analyze fibrosis in the subsequent experiments. To observe the relationship between fibrosis and renalase, we first investigated the expression level of renalase in the kidney. Immunohistochemistry showed that, in 8-week-old rats, renalase was mainly distributed in the renal tubules under normal conditions. There was also significant expression in glomerular mesangial areas (Figure 1F). After UUO, with the aggravation of fibrosis, the expression of renalase was gradually decreased, both in the kidney (Figure 1F,G) and in plasma (Figure 1H).
Int. J. Mol. Sci. 2017, 18, 855 Int. J. Mol. Sci. 2017, 18, 855
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Figure 1. Cont. Figure 1. Cont.
Int. J. Mol. Sci. 2017, 18, 855 Int. J. Mol. Sci. 2017, 18, 855
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Figure Figure1.1.Cont. Cont.
Int. J. Mol. Sci. 2017, 18, 855 Int. J. Mol. Sci. 2017, 18, 855
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Figure Figure 1. 1.Cont. Cont.
Int. J. Mol. Sci. 2017, 18, 855 Int. J. Mol. Sci. 2017, 18, 855
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Figure1. 1. Establishment of unilateral the unilateral ureteral obstruction model and expression Figure Establishment of the ureteral obstruction (UUO) (UUO) model and expression of renalaseof in rat (A) Histological changesin in kidneysureter in unilateral inrenalase rat kidneys. (A)kidneys. Histological changes in kidneys unilateral ligation.ureter Kidneyligation. sectionsKidney from sections from were various groupstowere subjectedeosin to Hematoxylin eosin (H&E) and Masson’s trichrome various groups subjected Hematoxylin (H&E) and Masson’s trichrome staining (MTS) at staining (MTS) at different times after UUO. With the prolongation of obstruction, renal tubular different times after UUO. With the prolongation of obstruction, renal tubular epithelial cell atrophy, epithelial cell atrophy, luminal expansion, inflammatory cell infiltration and tubule luminal expansion, interstitial inflammatory cell interstitial infiltration and tubule widening were aggravated and wideningfibrosis were increased aggravated and interstitial fibrosis gradually; magnification: 40×; interstitial gradually; magnification: 40×increased ; (B) Quantitative analysis of renal fibrotic (B) Quantitative analysisRenal of renal fibrotic lesions in various Renal lesions (defined lesions in various groups. fibrotic lesions (defined as thegroups. percentage of fibrotic the MTS-positive fibroticas the percentage of thebyMTS-positive fibrotic area) were quantified byImmunofluorescence computer-aided morphometric area) were quantified computer-aided morphometric analyses; (C) showing the expression E-cadherin and smooth muscle (α-SMA). Representative photomicrographs analyses; (C) of Immunofluorescence showing the actin expression of E-cadherin and smooth muscle actin are shown. With the extension of obstruction, fluorescence intensity E-cadherin became weak and (α-SMA). Representative photomicrographs are shown. Withofthe extension of obstruction, fluorescence intensity of of α-SMA strengthen gradually. The fluorescence difference wasintensity not statistically significant at fluorescence intensity E-cadherin became weak and of α-SMA strengthen 14gradually. and 21 days; 40×not ; (D,E) Western blotting andatimmunohistochemistry demonstrated Themagnification: difference was statistically significant 14 and 21 days; magnification: 40×; that, after UUO, blotting the expression of fibronectin and collagen-I increased a time-dependent manner.of (D,E) Western and immunohistochemistry demonstrated that,inafter UUO, the expression The differenceand wascollagen-I not statistically significant at 14 days and manner. 21 days. The brown/yellow indicates fibronectin increased in a time-dependent difference wascolor not statistically antibody binding; Arrows to positive results in Figure 1E.indicates Magnification: 40×binding; ; (F) Representative significant at 14 days andrefer 21 days. The brown/yellow color antibody Arrows refer photomicrographs of immunohistochemistry showing40×; the expression and localization of renalase inof to positive results in Figure 1E. Magnification: (F) Representative photomicrographs the ligated kidney. Renalase was mainly distributed the renal tubules underinnormal conditions; immunohistochemistry showing the expression andinlocalization of renalase the ligated kidney. After UUO,was with the aggravation of the fibrosis, the expression of renalase was gradually decreased. Renalase mainly distributed in renal tubules under normal conditions; After UUO, with the The differenceofwas not statistically significant in 14 days and 21decreased. days; Arrows refer to positive aggravation fibrosis, the expression of renalase was gradually The difference was not results in Figure 1E. Magnification: 40× ; (G)21Western blotting and analysisinofFigure renalase statistically significant in 14 days and days; Arrows referdensitometric to positive results 1E. protein levels in various groups. The results were consistent with immunohistochemistry; (H) The Magnification: 40×; (G) Western blotting and densitometric analysis of renalase protein levels in concentration of renalase in plasma. the prolongation of obstruction, the concentration of renalaseof various groups. The results were With consistent with immunohistochemistry; (H) The concentration inrenalase plasma in decreased Results are presented as percentages of controlofvalues andinare the plasma. gradually. With the prolongation of obstruction, the concentration renalase plasma means ± Standard deviation five animals per group. of * pcontrol < 0.05, compared the control decreased gradually. Results(SD) are of presented as percentages values andwith are the means ± # p < 0.05, compared with the 7-day group; & p < 0.05, compared with the 10-day group; group; Standard deviation (SD) of five animals per group. * p < 0.05, compared with the control group; # p < ˆ p0.05, < 0.05, compared withthe the7-day 14-daygroup; group;&NS: statistical difference; Col-I:^ collagen compared with p
