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. 2019 Jun 1;1865(6):1313-1322.
doi: 10.1016/j.bbadis.2019.01.027. Epub 2019 Jan 30.

Wnt/β-catenin Regulates Blood Pressure and Kidney Injury in Rats

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Free PMC article

Wnt/β-catenin Regulates Blood Pressure and Kidney Injury in Rats

Liangxiang Xiao et al. Biochim Biophys Acta Mol Basis Dis. .
Free PMC article

Abstract

Activation of the renin-angiotensin system (RAS) plays a pivotal role in mediating hypertension, chronic kidney and cardiovascular diseases. As Wnt/β-catenin regulates multiple RAS genes, we speculated that this developmental signaling pathway might also participate in blood pressure (BP) regulation. To test this, we utilized two rat models of experimental hypertension: chronic angiotensin II infusion and remnant kidney after 5/6 nephrectomy. Inhibition of Wnt/β-catenin by ICG-001 blunted angiotensin II-induced hypertension. Interestingly, angiotensin II was able to induce the expression of multiple Wnt genes in vivo and in vitro, thereby creating a vicious cycle between Wnt/β-catenin and RAS activation. In the remnant kidney model, renal β-catenin was upregulated, and delayed administration of ICG-001 also blunted BP elevation and abolished the induction of angiotensinogen, renin, angiotensin-converting enzyme and angiotensin II type 1 receptor. ICG-001 also reduced albuminuria, serum creatinine and blood urea nitrogen, and inhibited renal expression of fibronectin, collagen I and plasminogen activator inhibitor-1, and suppressed the infiltration of CD3+ T cells and CD68+ monocytes/macrophages. In vitro, incubation with losartan prevented Wnt/β-catenin-mediated fibronectin, α-smooth muscle actin and Snail1 expression, suggesting that the fibrogenic action of Wnt/β-catenin is dependent on RAS activation. Taken together, these results suggest an intrinsic linkage of Wnt/β-catenin signaling with BP regulation. Our studies also demonstrate that hyperactive Wnt/β-catenin can drive hypertension and kidney damage via RAS activation.

Keywords: Blood pressure; Renal fibrosis; Renin-angiotensin system; Wnt; β-Catenin.

Conflict of interest statement

Conflict of interest

The authors declare that they have no competing interests.

Figures

Figure 1.
Figure 1.
Inhibition of Wnt/β-catenin signaling by ICG-001 blocks blood pressure elevation induced by chronic angiotensin II (Ang II) infusion in rats. (A) Diagram shows experimental design. Red bar denotes Ang II treatment. Green bar shows the treatment schedule of ICG-001. Arrows indicate the timing for BP assessment. Blood pressure was measured using the tail-cuff technique at 3 and 7 days after Ang II infusion. Both systolic blood pressure (B, D) and mean blood pressure (C, E) are shown, respectively. **P < 0.01 versus sham controls; †P < 0.05 versus vehicle (n=5).
Figure 2.
Figure 2.
Angiotensin II induces Wnt expression and RAS activation in vivo and in vitro. (A) RT-PCR analysis shows that multiple Wnt genes were induced in rat kidneys at 7 days after Ang II infusion. Numbers (1, 2, and 3) indicate each individual animal in a given group. (B) Quantitative data of the relative Wnt mRNA levels among different groups. *P < 0.05 versus control group (n=5). (C, D) Western blot analysis shows the abundance of Wnt proteins at 7 days after Ang II infusion in the absence or presence of ICG-001. Representative Western blot (C) and quantitative data (D) are presented. *P < 0.05 versus control group (n=5). (E, F) Western blot analysis shows a dramatic increase in renal β-catenin, ACE and AT1 abundance at 7 days after Ang II infusion, which could be blocked by ICG-001. Representative Western blot (E) and quantitative data (F) are presented. *P < 0.05 versus sham controls, †P < 0.05 versus vehicle controls (n=5). (G) ICG-001 ameliorated Ang II-mediated proteinuria in rats. Urinary albumin levels were assessed by a specific ELISA, and reported after correction with urinary creatinine. *P < 0.05 versus sham controls, †P < 0.05 versus vehicle controls (n=5). (H) Ang II induces multiple Wnt expression in rat kidney interstitial fibroblasts (NRK-49F) in vitro. NRK-49F cells were incubated with different doses of Ang II as indicated for 24 h, and Wnt mRNA expression was assessed by RT-PCR. (I, J) Ang II induces protein expression of β-catenin and RAS components in a dose-dependent manner. NRK-49F cells were treated with different doses of Ang II for 24 hours. Representative Western blot (I) and quantitative data (J) are presented. *P < 0.05 versus controls (n=3). (K) Representative Western blot shows that Ang II induced β-catenin, AT1 and ACE expression in a time-dependent manner. NRK-49F cells were treated with 100 nM Ang II for various periods of time as indicated.
Figure 3.
Figure 3.
Inhibition of Wnt/β-catenin signaling by ICG-001 normalizes blood pressure and activation of renin-angiotensin system in rat remnant kidney model. (A) Wnt/β-catenin is activated in rat remnant kidney model in vivo. Representative micrographs demonstrate the expression and localization of β-catenin in remnant kidney. Rat kidney sections from sham control and 5/6NX at 12 weeks were stained immunohistochemically for β-catenin protein. Arrows indicate β-catenin–positive staining in renal tubules. Scale bar, 50 μm. The images were representative from 6 rats per group. (B and C) Western blot analyses show a dramatic increase in renal β-catenin abundance after 5/6NX. Representative Western blot (B) and quantitative data (C) are presented. Data are means ± SEM of 6 animals per group. *P < 0.05 versus sham controls (n=6). (D) Diagram shows experimental design. Green bar shows the treatment schedule of ICG-001 or vehicle. (E, F) Blood pressure was measured using the tail-cuff technique at 12 weeks after 5/6NX. Both systolic blood pressure (E) and mean blood pressure (F) are shown, respectively. *P < 0.05 versus sham controls; †P < 0.05 versus vehicle (n=6). Scale bar, 50 μm. (G) Kidney paraffin sections from different groups were stained for angiotensinogen (AGT), renin, angiotensin-converting enzyme (ACE) and angiotensin II type 1 receptor (AT1), respectively. Arrows indicate positive staining in renal tubules, and arrowhead denotes AT1-positive interstitial cells. The images were representative from 6 animals per group.
Figure 4.
Figure 4.
Blockade of Wnt/β-catenin signaling by ICG-001 ameliorates kidney dysfunctions and renal interstitial fibrosis after 5/6NX. (A, B) ICG-001 treatment improves kidney function. Serum creatinine (A) and blood urea nitrogen level (B) were assessed at 12 weeks after 5/6NX. *P < 0.05 versus sham controls; †P < 0.05 versus vehicle (n=6). (C) SDS-PAGE analysis shows the abundance and composition of urinary proteins in rats from different groups. Urine samples after normalization to creatinine were analyzed on SDS-PAGE and stained with Comarossi Blue reagent. Numbers (1, 2 and 3) indicate each individual animal in a given group. A protein at the size of 65 kDa, presumably albumin, is indicated by asterisk. (D) Urinary albumin levels in rats at 12 weeks after 5/6NX. Urinary albumin was expressed as μg/mg creatinine. (E) Kidney sections were stained with periodic acid-Schiff (PAS) and Masson-trichrome (MTS) reagents, respectively. Representative micrographs from different groups as indicated are shown. Arrows indicate positive staining. Scale bar, 50 μm. (F) Kidney weights in different groups after ICG-001 treatment. The kidney weight to body weight ratio (KW/BW) at 12 weeks after 5/6NX was calculated and presented. *P < 0.05 versus sham controls, †P < 0.05 versus vehicle controls (n=6). (G) Quantitative determination of renal fibrotic lesions in different groups. *P < 0.05 versus sham controls; †P < 0.05 versus vehicle (n=6). (H) Immunohistochemical staining shows that ICG-001 inhibited myofibroblast activation in remnant kidney. Rat kidney sections at 12 weeks after 5/6NX were immunostained with specific antibody against α-SMA. Arrows indicate positive staining. Scale bar, 50 μm. The images were representative from 6 animals per group. Except for vessel, there was no α-SMA-positive interstitial cell in normal rat kidney (not shown).
Figure 5.
Figure 5.
ICG-001 inhibits matrix gene expression in remnant kidney. (A-D) Quantitative, real-time RT-PCR results reveal that ICG-001 inhibited renal expression of fibronectin (A), type I collagen (B), type III collagen (C), and PAI-1 (D) mRNA at 12 weeks after 5/6NX. Relative mRNA levels are reported after normalization with β-actin. Ctrl, control. *P < 0.05 versus sham controls; †P < 0.05 versus vehicle (n=6). (E-I) Western blot analyses show the expression of fibronectin, type I collagen, α-SMA and PAI-1 in different groups. Numbers (1, 2 and 3) indicate each individual animal in a given group. Representative Western blot (E, H) and quantitative data for fibronectin (F), type I collagen (G), α-SMA and PAI-1 (I) are shown. Relative protein levels (fold induction versus control group) are presented after normalization with actin. *P < 0.05 versus sham controls; †P < 0.05 versus vehicle (n=6). (J) Representative micrographs show that ICG-001 inhibited fibronectin and type I collagen deposition in rat remnant kidney at 12 weeks after 5/6NX. Frozen kidney sections were stained with specific antibodies against fibronectin and type I collagen, respectively. Arrows indicate positive staining. Scale bar, 50 μm. The images were representative from 6 animals per group.
Figure 6.
Figure 6.
ICG-001 attenuates inflammatory infiltration and represses proinflammatory cytokines expression. (A) Representative micrographs show renal infiltration of CD3+ T cells and CD68+ monocytes/macrophages. Kidney sections from different groups were immunohistochemically stained with specific antibodies against CD3 and CD68 antigens, respectively. Arrows indicate positive staining. Scale bar, 50 μm. (B, C) Quantitative determination of CD3− (B) and CD68− (C) positive cells in various groups as indicated. *P < 0.05 versus sham controls, †P < 0.05 versus vehicle (n=6). (D) Graphic presentation shows the relative mRNA levels of RANTES and TNF-α mRNA determined by qRT-PCR. Relative mRNA levels were determined after normalization with β-actin and expressed as fold induction over sham controls. Data are expressed as mean ± SEM. *P < 0.05 versus sham controls, †P < 0.05 versus vehicle (n=6).
Figure 7.
Figure 7.
RAS activation is required for Wnt/β-catenin-mediated fibrogenic response in vitro. (A, B) Western blot analyses and quantitative data showed that ICG-001 abolished β-catenin-mediated fibronectin, α-SMA and Snail1 expression in tubular epithelial cells. HKC-8 cells were transfected with empty vector (pcDNA3) or constitutively activated β-catenin expression plasmid (pDel-β-cat) for 24 hours, following by incubation with ICG-001 (10 μM). *P < 0.05 versus controls (n=3); †P < 0.05 versus pDel-β-cat alone (n=3). (C, D) Losartan abolished β-catenin-mediated fibrogenic action. HKC-8 cells were treated as indicated. Whole cell lysates were immunoblotted for the protein expression of fibronectin, α-SMA and Snail1, respectively. Representative Western blot analyses (C) and quantitative data (D) are presented. *P < 0.05 versus controls (n=3); †P < 0.05 versus pDel-β-cat alone (n=3). (E) Losartan also inhibited the expression of fibrosis-related genes induced by Wnt1. HKC-8 cells were transfected with pcDNA3 or pHA-Wnt1 plasmid for 24 hours, followed by incubation with losartan (10−6 M). Quantitative data on the relative abundance of fibronectin, α-SMA and Snail1 are presented. *P < 0.05 versus controls (n=3); †P < 0.05 versus pDel-β-cat alone (n=3). (F) Diagram denotes the potential role and mechanism of Wnt/β-catenin signaling in the pathogenesis of CKD induced by 5/6NX.

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