Wnt/β-catenin signaling mediates both heart and kidney injury in type 2 cardiorenal syndrome

Abstract

In type 2 cardiorenal syndrome, chronic heart failure is thought to cause or promote chronic kidney disease; however, the underlying mechanisms remain poorly understood. We investigated the role of Wnt signaling in heart and kidney injury in a mouse model of cardiac hypertrophy and heart failure induced by transverse aortic constriction (TAC). At 8 weeks after TAC, cardiac hypertrophy, inflammation, and fibrosis were prominent, and echocardiography confirmed impaired cardiac function. The cardiac lesions were accompanied by upregulation of multiple Wnt ligands and activation of β-catenin, as well as activation of the renin-angiotensin system (RAS). Wnt3a induced multiple components of the RAS in primary cardiomyocytes and cardiac fibroblasts in vitro. TAC also caused proteinuria and kidney fibrosis, accompanied by klotho depletion and β-catenin activation in the kidney. Pharmacologic blockade of β-catenin with a small molecule inhibitor or the RAS with losartan ameliorated cardiac injury, restored heart function, and mitigated the renal lesions. Serum from TAC mice was sufficient to activate β-catenin and trigger tubular cell injury in vitro, indicating a role for circulating factors. Multiple inflammatory cytokines were upregulated in the circulation of TAC mice, and tumor necrosis factor-α was able to inhibit klotho, induce β-catenin activation, and cause tubular cell injury in vitro. These studies identify Wnt/β-catenin signaling as a common pathogenic mediator of heart and kidney injury in type 2 cardiorenal syndrome after TAC. Targeting this pathway could be a promising therapeutic strategy to protect both organs in cardiorenal syndrome.

Keywords: TAC; Wnt; cardiac hypertrophy; chronic kidney disease; fibrosis; β-catenin.

Figure 1.. Transverse aortic constriction (TAC) causes heart disorder and kidney injury in mice.

(a) Western blot analyses show protein expression of β-MHC, fibronectin, TNF-α in the heart of mice subjected to TAC for 8 weeks. (b-d) Quantitative data on β-MHC, fibronectin, TNF-α protein abundances in different groups as indicated. Relative levels (fold induction over the controls) of proteins were presented. *P < 0.05. (e) Representative micrographs show the histology (H.E staining) of cardiac sections of control and TAC mice. Heart cross-sections show overt cardiac hypertrophy in TAC mice. Scale bar, 1 mm. (f) Western blot analyses show protein expression of podocalyxin, fibronectin, Snail1 in the kidney of mice subjected to TAC for 8 weeks. (g-i) Quantitative data on podocalyxin, fibronectin, Snail1 proteins in different groups as indicated. Relative levels (fold induction over the controls) of proteins were presented. *P < 0.05. (j) Representative micrographs show Masson’s trichrome staining of kidney sections of control and TAC mice. Yellow arrow indicates collagen deposition. Scale bar, 20 μm.

Figure 2.. Wnt/β-catenin is activated in the heart of TAC mice.

(a) qRT-PCR shows that a battery of Wnt genes was induced in the heart of mice at 8 weeks after TAC. *P<0.05 vs controls (n=6). (b-f) Western blot analyses confirm the induction of Wnt1, Wnt3a, active β-catenin and total β-catenin protein in the heart of mice at 8 weeks after TAC. Representative Western blots (b) and quantitative data (c-f) were presented. *P<0.05 vs controls (n=4). (g) Representative micrographs show that Wnt3a was induced in cardiomyocytes in mice at 8 weeks after TAC. Black arrow indicates positive staining. (h) Representative micrographs show the immunohistochemical staining for β-catenin in heart. The β-catenin protein was induced and predominantly localized in the cytoplasma of cardiomyocytes in mice after TAC (black arrow), whereas β-catenin in sham control mice was mainly localized in the site of cell-cell junction (empty arrow). Scale bar, 20 μm.

Figure 3.. Blockade of Wnt/β-catenin signaling by ICG-001 prevented cardiac injury in mice after TAC.

(a) Experimental design. Red arrow indicates the timing of TAC surgery, while black arrow show the timing of sacrifice. (b) Representative Western blots show cardiac protein levels of β-MHC, fibronectin, collagen I, TNF-α, active β-catenin, total β-catenin in various groups as indicated. (c-h) Quantitative data on protein levels of β-MHC, fibronectin, collagen I, TNF-α, active β-catenin and total β-catenin are presented in given groups as indicated. *P<0.05 vs sham controls; †P<0.05 vs TAC alone (n=6). (i) Representative micrographs show histology and β-MHC and fibronectin protein expression in the heart of mice at 8 weeks after TAC. Upper panel, H.E. staining of heart sections in various groups as indicated; Middle panel, immunostaining for cardiac β-MHC; Bottom panel, immunostaining for fibronectin in the heart of mice as indicated. Yellow arrows indicate positive staining. Scale bar, 20 μm.

Figure 4.. Wnt/β-catenin activates renin-angiotensin system in the heart after TAC.

(a) Representative Western blots show cardiac expression of ACE, renin, AT1 in various groups as indicated. (b-d) Quantitative data on cardiac expression of ACE (b), renin (c), AT1 (d) proteins are presented in various groups as indicated. *P<0.05 vs sham controls; †P<0.05 vs TAC alone (n=6). (e) Representative Western blots show that Wnt3a induced the expression of active β-catenin, β-catenin, ACE, renin, AT1 in primary cardiomyocytes. (f-j) Quantitative data show the induction of active β-catenin, β-catenin, ACE, renin, AT1 by Wnt3a in cardiomyocytes. *P<0.05 vs controls; †P<0.05 vs Wnt3a alone (n=3). (k) Representative Western blots show protein expression of active β-catenin, β-catenin, ACE, angiotensinogen, AT1 in primary cardiac fibroblasts after various treatments as indicated. (i-p) Quantitative data show the protein levels of β-catenin, active β-catenin, ACE, angiotensinogen, AT1 in primary cardiac fibroblasts after various treatments as indicated. *P<0.05 vs controls; †P<0.05 vs Wnt3a alone (n=3). Ctrl, control; TAC, transverse aortic constriction; ICG, ICG-001; Los, losartan.

Figure 5.. Blockade of Wnt/β-catenin by ICG-001 also abolishes renal β-catenin signaling and ameliorates kidney injury after TAC.

(a) Representative Western blots show protein expression of fibronectin, podocalyxin, Snail1, Kim-1, active β-catenin and total β-catenin in the kidney of mice as indicated. (b-g) Quantitative data on renal expression of fibronectin, podocalyxin, Snail1, Kim-1, active β-catenin and total β-catenin proteins are presented in various groups as indicated. *P<0.05 vs sham controls; †P<0.05 vs TAC alone (n=6). (h) Representative micrographs show renal collagen deposition and the expression of podocalyxin and β-catenin proteins in various groups as indicated. Upper panel, Masson’s trichrome staining of kidney sections; Middle panel, immunohistochemical staining for podocalyxin; Lower panel, immunohistochemical staining for β-catenin. Arrows denote positive staining. Scale bar, 20 μm. (i) Urinary albumin levels in various groups as indicated. Albuminuria was expressed after normalization with urinary creatinine. *P<0.05 vs sham controls; †P<0.05 vs TAC alone (n=6). (j) Graph shows a linear correlation between urinary albumin level and BNP in serum. Ctrl, control; TAC, transverse aortic constriction; ICG, ICG-001.

Figure 6.. Circulating factors in mouse serum after TAC mediate renal β-catenin activation and kidney injury in vitro.

Human kidney proximal tubular epithelial cells (HKC-8) were incubated with mouse serum (2.5%) from different groups as indicated. (a) Representative Western blots show the expression of active β-catenin, total β-catenin, Snail1 and Kim-1 in HKC-8 cells after incubation for 24 hours. (b-e) Quantitative data on protein levels of active β-catenin, total β-catenin, Snail1 and Kim-1 are presented. *P<0.05 vs sham controls; †P<0.05 vs TAC alone (n=6). (f-n) Knockdown of β-catenin abolishes kidney injury and renin angiotensin system activation in vitro. HKC-8 cells were infected with lentiviral β-catenin shRNA vector (β-cat-shR) or control vector (ctrl-shR), and followed by incubation with mouse serum (2.5%) from different groups as indicated. Representative Western blots (f) show the expression of active β-catenin, total β-catenin, fibronectin, Kim-1, Snail1, angiotensinogen, renin and AT1 in HKC-8 cells after incubation for 24 hours. Quantitative data on protein levels of β-catenin (g), active β-catenin (h), fibronectin (i), Snail1 (j), Kim-1 (k), angiotensinogen (l), renin (m) and AT1 (n) are presented. *P<0.05 vs control (ctrl) serum; †P<0.05 vs ctrl-shR (n=3). Ctrl, control; TAC, transverse aortic constriction; ICG, ICG-001; Lentiviral Ctrl shR, lentiviral control shRNA; Lentiviral β-catenin-shR, lentiviral β-catenin shRNA.

Figure 7.. Circulating levels of pro-inflammatory cytokines after TAC correlate with heart and kidney injury.

(a) ELISA shows the circulating levels of TNF-α in various groups at 8 weeks after TAC. *P<0.05 vs sham controls; †P<0.05 vs TAC alone (n=6). (b) Circulating MCP-1 level in various groups at 8 weeks after TAC. *P<0.05 vs sham controls; †P<0.05 vs TAC alone (n=6). (c) Circulating IL-1β level in various groups at 8 weeks after TAC. *P<0.05 vs sham controls; †P<0.05 vs TAC alone (n=6). (d) Circulating BNP level in various groups at 8 weeks after TAC. *P<0.05 vs sham controls; †P<0.05 vs TAC alone (n=6). (e) Circulating level of TNF-α correlates with cardiac injury. Diagram shows a linear correlation between TNF-α (pg/ml) and BNP (pg/ml) in serum. (f) Circulating level of TNF-α correlates with kidney injury. Diagram shows a linear correlation between albuminuria (mg/mg Ucr.) and TNF-α (pg/ml) in serum. Ctrl, control; TAC, transverse aortic constriction; ICG, ICG-001; Los, Losartan.

Figure 8.. TNF-α activates β-catenin and triggers injurious response in kidney tubular cells in vitro.

(a) Representative micrographs show immunostaining for β-catenin in HKC-8 cells after various treatments as indicated. HKC-8 cells were pretreated with ICG-001 (5 μM) or losartan (10 μM) for 1 hour before incubating with TNF-α for 12 hours. White arrows denote positive nuclear staining. (b) Representative Western blot analyses revealed protein level of β-catenin, active β-catenin, Snail1 and Kim-1 in HKC-8 cells after various treatments for 24 hours as indicated. (c-f) Quantitative data for β-catenin, active β-catenin, Snail1 and KIM1 proteins in various groups are shown. *P<0.05 vs sham controls; †P<0.05 vs TNF-α alone (n=6).

Figure 9.. Depletion of Klotho aggravates cardiomyocyte hypertrophy and cardiac fibroblast activation induced by Wnt/β-catenin.

(a, b) Western blot analyses show that TAC caused renal depletion of Klotho in mice, which was abrogated by ICG-001 or losartan. Representative Western blotting for Klotho in the kidney in various groups (a) and quantitative data on Klotho protein levels were presented (b). *P<0.05 vs sham controls; †P<0.05 vs TAC alone (n=6). (c) ELISA assay shows serum levels of Klotho protein in mice after various treatments as indicated. *P<0.05 vs sham controls; †P<0.05 vs TAC alone (n=6). (d) Representative micrographs show renal expression of Klotho protein in mice after various treatments as indicated. Black arrows indicate positive staining. (e) Representative Western blots show the protein levels of β-MHC, α-actin, active β-catenin in primary cardiomyocytes after various treatments as indicated. Rat primary cardiomyocytes were incubated with Wnt3a (100 ng/ml) in the absence or presence of Klotho (100 ng/ml) for 24 hours. (f-h) Quantitative data on the protein levels of β-MHC (f), α-actin (g), active β-catenin (h) in various groups as indicated. (i) Representative Western blot show the protein levels of fibronectin, α-SMA, active β-catenin, β-catenin in rat primary cardiac fibroblasts after various treatments as indicated. Rat primary cardiac fibroblasts were incubated with Wnt3a (100 ng/ml) in the absence or presence of Klotho (100 ng/ml) for 24 hours. (j-m) Quantitative data on the protein levels of fibronectin (j), α-SMA (k), active β-catenin (l), β-catenin (m) in various groups as indicated. (n) Schematic diagram depicts cardiorenal inter-organ crosstalk after TAC. Heart injury following TAC activates Wnt/β-catenin and RAS, leads to systemic inflammation with up-regulation of TNF-α, IL-1β, MCP-1 in the circulation. Pro-inflammatory cytokines in the circulation subsequently suppresses renal Klotho expression, which leads to activation of Wnt/β-catenin and RAS in the kidney and cause podocyte and tubular injury, and fibroblast activation. As the main source of systemic Klotho, depressed renal production of Klotho in the damaged kidney directly causes Klotho deficiency in the circulation, which further aggravates Wnt-mediated cardiomyocyte hypertrophy and cardiac fibroblast activation after TAC, forming a vicious cycle. Ctrl, control; TAC, transverse aortic constriction; ICG, ICG-001; Los, losartan.