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. 2012 Sep;82(5):537-47.
doi: 10.1038/ki.2012.173. Epub 2012 May 23.

Tubule-specific ablation of endogenous β-catenin aggravates acute kidney injury in mice

Dong Zhou  1 , Yingjian Li, Lin Lin, Lili Zhou, Peter Igarashi, Youhua Liu
Affiliations

Tubule-specific ablation of endogenous β-catenin aggravates acute kidney injury in mice

Dong Zhou et al. Kidney Int. 2012 Sep.

Erratum in

  • Kidney Int. 2013 Sep;84(3):626

Abstract

β-Catenin is a unique intracellular protein functioning as an integral component of the cell-cell adherens complex and a principal signaling protein mediating canonical Wnt signaling. Little is known about its function in adult kidneys in the normal physiologic state or after acute kidney injury (AKI). To study this, we generated conditional knockout mice in which the β-catenin gene was specifically disrupted in renal tubules (Ksp-β-cat-/-). These mice were phenotypically normal with no appreciable defects in kidney morphology and function. In the absence of β-catenin, γ-catenin functionally substituted for it in E-cadherin binding, thereby sustaining the integrity of epithelial adherens junctions in the kidneys. In AKI induced by ischemia reperfusion or folic acid, the loss of tubular β-catenin substantially aggravated renal lesions. Compared with controls, Ksp-β-cat-/- mice displayed higher mortality, elevated serum creatinine, and more severe morphologic injury. Consistently, apoptosis was more prevalent in kidneys of the knockout mice, which was accompanied by increased expression of p53 and Bax, and decreased phosphorylated Akt and survivin. In vitro activation of β-catenin by Wnt1 or stabilization of β-catenin protected tubular epithelial cells from apoptosis, activated Akt, induced survivin, and repressed p53 and Bax expression. Hence, endogenous β-catenin is pivotal for renal tubular protection after AKI by promoting cell survival through multiple mechanisms.

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Figures

Figure 1
Figure 1. Up-regulation of renal β-catenin in mouse model of acute kidney injury induced by folic acid
(a, b) Immunohistochemical staining shows the expression and localization of β-catenin in the kidneys at 2 days after folic acid injection. Arrowheads indicate positive staining. Boxed area was enlarged. Scale bar, 50 μm. (c, d) Western blots demonstrate renal β-catenin protein levels at 2 days after folic acid injection. Representative Western blot (c) and quantitative data (d) are presented. Numbers (1, 2 and 3) indicate each individual animal in a given group. **P < 0.01 versus vehicle controls (n = 4). (e) Co-staining for β-catenin and tubular segment-specific markers in the injured kidneys after folic acid injection. Immunofluorescence staining demonstrated the co-staining of β-catenin (green) and various tubular markers (red) in the kidneys at 2 days after folic acid injection. Segment-specific tubular markers used are as follows: proximal tubule, aquaporin-1 (AQP1); cortical thick ascending limb, Tamm-Horsfall glycoprotein (THP); distal tubule, thiazide-sensitive NaCl cotransporter (TSC)/NCC; and collecting duct, aquaporin-3 (AQP3). Arrowheads indicate β-catenin-positive tubules.
Figure 2
Figure 2. Generation of conditional knockout mice with tubule-specific ablation of endogenous β-catenin
(a) Experimental design shows the strategy of cross-breeding of the β-catenin floxed mice (β-catfl/fl) with Cre transgenic mice under the control of Ksp-cadherin promoter (Ksp-Cre). Black boxes indicate the exons of β-catenin gene. Orange boxes denote LoxP site. (b) Genotyping of the mice by PCR analysis of genomic DNA. Lane 1 shows the genotyping of the control mice used in this study (genotype: β-catfl/fl), whereas lane 2 denotes the genotyping of the tubule-specific β-catenin knockout mice (genotype: β-cateninfl/fl,Cre), designated as Ksp-β-cat−/−. (c) Western blot analyses demonstrated a substantial reduction of renal β-catenin protein in Ksp-β-cat−/− mice. Kidney lysates were made from control and Ksp-β-cat−/− mice at 2 days after folic acid injection, and immunoblotted with specific antibodies against β-catenin and GAPDH, respectively. Numbers (1, 2 and 3) indicate each individual animal in a given group. (d, e) Representative micrographs show renal β-catenin staining in the control (d) and Ksp-β-cat−/− (e) mice at 2 days after folic acid injection. Boxed areas in cortex and medulla regions are enlarged. Arrowheads indicate renal tubules. Scale bar, 50 μm.
Figure 3
Figure 3. Mice with tubule-specific ablation of endogenous β-catenin are phenotypically normal
(a) Representative micrographs show the morphology of control and Ksp-β-cat−/− kidneys. Cortical and medullar regions were shown. Scale bar, 50 μm. (b–d) There was no difference in body weight (b), serum creatinine (c) and urinary albumin level (d) between control and Ksp-β-cat−/− mice (n = 3). (e) γ-catenin functionally compensates for the lost β-catenin at adherens junctions by augmenting interaction with E-cadherin in the kidneys of Ksp-β-cat−/− mice. Kidney lysates from control and Ksp-β-cat−/− mice were immunoprecipitated with anti-E-cadherin antibody, followed by immunoblotting with antibodies against β-catenin, γ-catenin and E-cadherin, respectively. (f) Loss of tubular β-catenin did not affect the expression of γ-catenin and E-cadherin in the kidneys. Kidney lysates were immunoblotted with antibodies against β-catenin, γ-catenin, E-cadherin and actin, respectively.
Figure 4
Figure 4. Tubule-specific ablation of β-catenin aggravates acute kidney injury induced by folic acid in mice
(a) Serum creatinine level in control and Ksp-β-cat−/− mice at 2 days after folic acid injection. *P < 0.05 (n = 9–10). (b, c) Morphological injury assessed in the PAS-stained kidney sections in control and Ksp-β-cat−/− mice. Representative micrographs of the kidneys at 2 days after folic acid injection (b) and quantitative assessment of injury (c) are presented. Yellow asterisks in the enlarged boxed areas indicate injured tubules. **P < 0.01 (n = 4).
Figure 5
Figure 5. Tubule-specific ablation of β-catenin promotes apoptosis and Bax expression after acute kidney injury
(a, b) Tubule-specific loss of β-catenin aggravates apoptosis in acute kidney injury. (a) Representative micrographs show apoptotic cell death detected by TUNEL staining. Arrows in the enlarged boxed areas indicate apoptotic cells. Scale bar, 50 μm. (e) Quantitative determination of apoptotic cells in renal cortex and medulla regions at 2 days after folic acid injection. Data are presented as numbers of apoptotic cells per high power field (HPF). **P < 0.01 (n = 4). (c, d) Loss of β-catenin promoted renal Bax protein expression. Representative Western blot data (c) and quantitative analysis (d) are presented. Numbers (1, 2, 3 and 4) indicate each individual animal in a given group. *P < 0.05 (n = 4). (e) Representative micrographs showed immunohistochemical staining for Bax in the kidneys at 2 days after folic acid injection. Arrows indicate Bax-positive tubules in renal cortex and medulla. Scale bar, 50 μm.
Figure 6
Figure 6. Tubule-specific ablation of β-catenin induced p53 and inhibited Akt phosphorylation and surviving expression in vivo
(a, b) Western blot analyses demonstrated that tubule-specific ablation of endogenous β-catenin induced renal expression of p53 protein. Numbers (1, 2, 3 and 4) indicate each individual animal in a given group. Quantitative data are presented in Panel (b). **P < 0.01 (n = 4). (c, d) Tubule-specific ablation of endogenous β-catenin suppressed renal Akt phosphorylation. Kidney lysates at 2 days after folic acid injection were immunoblotted with specific antibodies against phosphorylated Akt (Ser473) and total Akt, respectively. Numbers (1, 2, 3 and 4) indicate each individual animal in a given group. Quantitative data are presented in Panel (d). *P < 0.05 (n = 4). (e) qRT-PCR demonstrated a decreased expression of survivin mRNA in Ksp-β-cat−/− kidneys. *P < 0.05 (n = 5–6). (f) Diagram shows the potential pathways leading to apoptosis induced by the tubule-specific ablation of endogenous β-catenin.
Figure 7
Figure 7. Loss of tubular β-catenin aggravates AKI in renal ischemia/reperfusion injury model
(a) Serum creatinine level in control and Ksp-β-cat−/− mice at 1 day after renal ischemia/reperfusion injury. *P < 0.05 (n=3–4). (b) Representative micrographs of the kidneys in control and Ksp-β-cat−/− mice at 1 day after renal ischemia/reperfusion injury. Yellow asterisks in the enlarged boxed areas indicate injured tubules. Scale bar, 50 μm. (c) Quantitative assessment of renal injury. Injury score (% of injured tubules) are presented. **P < 0.01 (n=3). (d, e) Tubule-specific ablation of β-catenin aggravates apoptosis after renal ischemia/reperfusion injury. (d) Representative micrographs show apoptotic cell death detected by TUNEL staining. White arrows indicate apoptotic cells. (e) Quantitative determination of apoptotic cells in renal cortical and medullar regions at 1 day after renal ischemia/reperfusion injury. Data are presented as numbers of apoptotic cells per high power field (HPF). **P < 0.01 (n = 3). (f, g) Loss of β-catenin promoted renal Bax protein expression in ischemic AKI. Western blot (f) and quantitative analysis (g) are presented. Numbers (1, 2 and 3) indicate each individual animal in a given group. *P < 0.05 (n= 3).
Figure 8
Figure 8. Activation of endogenous β-catenin promotes kidney tubular cell survival in vitro.
(a, b) Ectopic expression of exogenous Wnt1 protects tubular cells against apoptosis induced by staurosporine (STS). HKC-8 cells were transfected with Wnt1 expression vector (pHA-Wnt1) or empty vector (pcDNA3), followed by treatment with STS (1 μM) for various periods of time as indicated. Apoptosis was assessed by TUNEL staining. Representative micrographs of TUNEL staining (a) and quantitative data of apoptotic cells per high power field (HPF) (b) are presented. **P < 0.01 versus vehicle control (n = 3); †P < 0.05 versus pcDNA3 control (n = 3). (c) qRT-PCR showed that activation of endogenous β-catenin by Wnt1 induced survivin mRNA expression in tubular epithelial cells. *P < 0.05 (n = 3). (d, e) Ectopic expression of Wnt1 prevented Bax (d) and p53 (e) expression induced by STS in tubular epithelial cells. Wnt1 also promoted Akt phosphorylation after STS incubation (e). (f–h) Ectopic expression of stabilized β-catenin also prevented tubular cell apoptosis (f), induced survivin expression (g) and blocked STS-induced Bax expression (h). HKC-8 cells were transfected with N-terminally truncated, stabilized β-catenin (pDel-cat) or empty vector (pcDNA3), respectively. Apoptosis was assessed by TUNEL staining after incubation with STS (1 μM) for 6 hours (f). Survivin mRNA expression was detected by qRT-PCR (g). Bax protein expression was assessed by Western blot and quantified (h). *P < 0.05 (n = 3).
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