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. 2015 Jan;26(1):107-20.
doi: 10.1681/ASN.2014010085. Epub 2014 Jul 10.

Multiple genes of the renin-angiotensin system are novel targets of Wnt/β-catenin signaling

Lili Zhou  1 , Yingjian Li  2 , Sha Hao  2 , Dong Zhou  2 , Roderick J Tan  3 , Jing Nie  4 , Fan Fan Hou  4 , Michael Kahn  5 , Youhua Liu  6
Affiliations

Multiple genes of the renin-angiotensin system are novel targets of Wnt/β-catenin signaling

Lili Zhou et al. J Am Soc Nephrol. 2015 Jan.

Abstract

Activation of the renin-angiotensin system (RAS) plays an essential role in the pathogenesis of CKD and cardiovascular disease. However, current anti-RAS therapy only has limited efficacy, partly because of compensatory upregulation of renin expression. Therefore, a treatment strategy to simultaneously target multiple RAS genes is necessary to achieve greater efficacy. By bioinformatics analyses, we discovered that the promoter regions of all RAS genes contained putative T-cell factor (TCF)/lymphoid enhancer factor (LEF)-binding sites, and β-catenin induced the binding of LEF-1 to these sites in kidney tubular cells. Overexpression of either β-catenin or different Wnt ligands induced the expression of all RAS genes. Conversely, a small-molecule β-catenin inhibitor ICG-001 abolished RAS induction. In a mouse model of nephropathy induced by adriamycin, either transient therapy or late administration of ICG-001 abolished established proteinuria and kidney lesions. ICG-001 inhibited renal expression of multiple RAS genes in vivo and abolished the expression of other Wnt/β-catenin target genes. Moreover, ICG-001 therapy restored expression of nephrin, podocin, and Wilms' tumor 1, attenuated interstitial myofibroblast activation, repressed matrix expression, and inhibited renal inflammation and fibrosis. Collectively, these studies identify all RAS genes as novel downstream targets of Wnt/β-catenin. Our results indicate that blockade of Wnt/β-catenin signaling can simultaneously repress multiple RAS genes, thereby leading to the reversal of established proteinuria and kidney injury.

Keywords: CKD; Wnt; renal fibrosis; renin angiotensin system; β-catenin.

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Figures

Figure 1.
Figure 1.
Multiple genes of the RAS are direct targets of Wnt/β-catenin signaling. (A) Schematic presentation of the genes in the RAS. (B) Activation of intrarenal RAS in ADR nephropathy. The expression of RAS components, such as AGT, renin, ACE, and AT1, was assessed in the kidneys at 5 weeks after ADR injection. Frozen or paraffin kidney sections were used for immunostaining for RAS components. Arrows indicate positive staining. Scale bar, 50 µm. (C) Bioinformatics analyses revealed the presence of putative TBSs in the promoter regions of all human RAS genes. The sequences and positions of the putative TBS in multiple RAS genes are highlighted in red, whereas the TBS consensus sequence is given at the bottom of this panel. (D) Overexpression of β-catenin induced the expression of multiple RAS genes in kidney proximal tubular cells. HKC-8 cells were transfected with N-terminally truncated, FLAG-tagged, constitutively activated β-catenin expression vector (pDel-β-cat) or pcDNA3 empty vector for 24 hours. Cells were then analyzed for mRNA expression of multiple RAS genes. (E) Exogenous β-catenin induced protein expression of multiple RAS components in a dose-dependent fashion. HKC-8 cells were transfected with different amounts of pDel-β-cat plasmid as indicated (micrograms per well) or pcDNA3 empty vector (4 µg/well). Cell lysates were analyzed for protein expression of various RAS components by Western blotting. (F and G) ChIP assay showed that ectopic expression of β-catenin promoted the binding of LEF-1 to putative TBS in the promoter of RAS genes. HKC-8 cells transfected with either pcDNA3 or pDel-β-cat plasmids were subjected to ChIP analyses using specific anti–LEF-1 antibody. Increased binding of LEF-1 to putative TBS in (F) AGT and (G) renin gene promoters after β-catenin expression is shown. Representative ChIP assay (upper panels) and quantitative ChIP data (lower panels) are presented. *P<0.05 (n=3). (H) Colocalization of β-catenin and various RAS components in ADR nephropathy. Kidney cryosections were coimmunostained for AGT or renin (red) and β-catenin (green). Arrows indicate AGT (or renin) and β-catenin colocalization in the same tubule. Scale bar, 50 µm.
Figure 2.
Figure 2.
Wnts induce RAS expression, and small-molecule β-catenin inhibitor abolishes RAS induction in vitro. (A) Representative RT-PCR analyses showed that various Wnts induced the expression of RAS genes in tubular epithelial cells. HKC-8 cells were transfected with either pcDNA3 or the expression vectors for various Wnts as indicated for 24 hours. The mRNA expression of various RAS genes was analyzed by RT-PCR. (B–E) Small-molecule inhibitor ICG-001 abolished β-catenin–mediated RAS induction in vitro. HKC-8 cells were transfected with either pcDNA3 or N-terminally truncated, FLAG-tagged, constitutively activated β-catenin expression vector (pDel-β-cat) plasmid for 24 hours in the absence or presence of ICG-001 (10 µM). Quantitative real-time RT-PCR analyses showed the relative mRNA abundances of (B) AGT, (C) renin, (D) ACE, and (E) AT1 and AT2 after various treatments as indicated. Data are expressed as means±SEMs of three independent experiments. *P<0.05 versus pcDNA3 alone; †P<0.05 versus pDel-β-cat alone. (F) Representative Western blot analyses showed that ICG-001 abolished β-catenin–mediated RAS induction in tubular epithelial cells in vitro. HKC-8 cells were treated as indicated. Whole-cell lysates were immunoblotted for the protein expression of AGT, renin, ACE, and AT1, respectively.
Figure 3.
Figure 3.
Small-molecule inhibitor ICG-001 ameliorates an established proteinuria and kidney injury in ADR nephropathy. (A) Diagram shows the experimental design. Arrows indicate the starting point of ADR injection. Green bars indicate ICG-001 treatment. (B) Urinary albumin levels in mice at 5 weeks after ADR injection. Urinary albumin was expressed as milligrams per milligram creatinine. (C) Representative micrographs show kidney injury at 5 weeks after ADR injection in different groups of mice as indicated. Images of periodic acid–Schiff staining with different magnifications are shown. Scale bar, 50 µm. (D) Quantitative determination of kidney fibrotic lesions in different groups. Ctrl, control. *P<0.05 versus normal controls; †P<0.05 versus ADR alone (n=5–6).
Figure 4.
Figure 4.
Inhibition of β-catenin signaling by ICG-001 abolished RAS induction in ADR nephropathy. (A) Representative Western blot analyses revealed that ICG-001 abolished RAS induction in ADR nephropathy. Kidney lysates from different groups as indicated were immunoblotted with antibodies against AGT, renin, ACE, AT1, and actin. Numbers 1 and 2 represent different animals in a given group. (B–E) Graphic presentations of the relative abundances of (B) AGT, (C) renin, (D) ACE, and (E) AT1 in different groups as indicated. *P<0.05 versus normal controls; †P<0.05 versus ADR (n=5–6). (F) Representative micrographs showed RAS components in different groups as indicated. Kidney sections were stained with different antibodies against AGT, renin, ACE, and AT1. Arrows indicate positive tubules. Ctrl, control. Scale bar, 50 µm.
Figure 5.
Figure 5.
ICG-001 restores podocyte integrity in ADR nephropathy. (A) ICG-001 restores podocyte-specific proteins and inhibits desmin expression. Kidney lysates were immunoblotted with specific antibodies against nephrin, podocin, WT1, desmin, and actin. Numbers 1 and 2 represent different animals in a given group as indicated. (B–E) Graphic presentations of (B) nephrin, (C) podocin, (D) WT1, and (E) desmin expressions in different groups as indicated. *P<0.05 versus normal controls; †P<0.05 versus ADR alone (n=5–6). (F) Representative micrographs showing glomerular nephrin and WT1 expression. Frozen kidney tissue sections were stained with nephrin (red) and WT1 (green). (G) Graphic presentation shows the numbers of WT1-positive podocytes per glomerular cross-section. *P<0.05 versus normal controls; †P<0.05 versus ADR alone (n=5–6). (H–J) Expression of active β-catenin induces mRNA expression of RAS genes in vitro. Mouse podocytes were transiently transfected with N-terminally truncated, FLAG-tagged, constitutively activated β-catenin expression vector (pDel-β-cat) or control pcDNA3. The mRNA expression of (H) AGT, (I) renin, and (J) AT1 was assessed by quantitative RT-PCR. *P<0.05 (n=3). (K) Expression of active β-catenin induces AT1 protein expression in cultured podocytes. Mouse podocytes after transfection were immunostained for AT1 protein (green). Cell nuclei were visualized with DAPI staining (blue). Ctrl, control; DAPI, 4′,6-diamidino-2-phenylindole; Veh, vehicle.
Figure 6.
Figure 6.
ICG-001 inhibits renal expression of the Wnt/β-catenin target genes in ADR nephropathy. (A) Western blot analyses of renal expressions of the Wnt/β-catenin target genes in different groups. (B–E) Graphic representations of (B) Snail1, (C) PAI-1, (D) MMP-7, and (E) MMP-9 expressions in different groups as indicated. *P<0.05 versus normal controls; †P<0.05 versus ADR alone (n=5–6). (F) Representative micrographs show FSP-1 expression in different groups. Paraffin sections were stained with anti–FSP-1 antibody. Arrows indicate FSP-1–positive tubules. (G) Graphic presentation shows FSP-1 staining in different groups. Ctrl, control; Veh, vehicle. *P<0.05 versus normal controls; †P<0.05 versus ADR alone (n=5–6).
Figure 7.
Figure 7.
ICG-001 inhibits matrix gene expression and reduces renal fibrosis in ADR nephropathy. (A) Western blot analyses show the expression of multiple fibrosis-related genes. Kidney lysates were immunoblotted with specific antibodies against fibronectin, types I and III collagen, α-SMA, and actin. (B–E) Graphic representations of (B) fibronectin, (C) collagen I, (D) collagen III, and (E) α-SMA expressions in different groups as indicated. *P<0.05 versus normal controls; †P<0.05 versus ADR alone (n=5–6). (F) Representative micrographs show fibronectin and α-SMA protein expression and collagen deposition in different groups. Frozen kidney tissue sections were stained with antifibronectin antibody (green), whereas paraffin sections were used for α-SMA and Masson's Trichrome staining. Scale bar, 50 µm. (G and H) Graphic presentation shows fibronectin and α-SMA expression in different groups. Ctrl, control; MTS, Masson's Trichrome Staining; Veh, vehicle. *P<0.05 versus normal controls; †P<0.05 versus ADR alone (n=5–6).
Figure 8.
Figure 8.
ICG-001 inhibits inflammatory cytokines expression and reduces renal infiltration of macrophages in ADR nephropathy. (A–C) Quantitative real-time RT-PCR shows renal mRNA levels of (A) RANTES, (B) MCP-1, and (C) TNF-α in different groups. *P<0.05 versus normal controls; †P<0.05 versus ADR alone (n=5–6). (D) Representative micrographs show renal expression and localization of RANTES and F4/80 antigen in different groups. Paraffin-embedded kidney sections were stained with RANTES and F4/80 antibodies. Arrows indicate positive staining. Scale bar, 50 µm. (E) Graphic presentation of F4/80+ macrophages and CD3+ T cells in the kidney sections. Data are presented as the numbers of positive cells per high-power field (hpf). *P<0.05 versus normal controls; †P<0.05 versus ADR alone (n=5–6). (F) Western blot analyses show the active phosphorylated p65 (p-p65) and total p65 expressions in vivo. (G and H) Graphic presentations show the (G) p-p65 and (H) p65 protein abundances in different groups. Relative protein levels over the controls (fold induction) are reported. Ctrl, control; Veh, vehicle. *P<0.05 versus normal controls; †P<0.05 versus ADR alone (n=5–6).
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