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. 2019 Apr;95(4):830-845.
doi: 10.1016/j.kint.2018.10.032. Epub 2019 Feb 12.

Wnt/β-catenin links oxidative stress to podocyte injury and proteinuria

Lili Zhou  1 , Xiaowen Chen  2 , Meizhi Lu  2 , Qinyu Wu  2 , Qian Yuan  2 , Chengxiao Hu  2 , Jinhua Miao  2 , Yunfang Zhang  3 , Hongyan Li  3 , Fan Fan Hou  2 , Jing Nie  2 , Youhua Liu  4
Affiliations

Wnt/β-catenin links oxidative stress to podocyte injury and proteinuria

Lili Zhou et al. Kidney Int. 2019 Apr.

Abstract

Podocyte injury is the major cause of proteinuria in primary glomerular diseases. Oxidative stress has long been thought to play a role in triggering podocyte damage; however, the underlying mechanism remains poorly understood. Here we show that the Wnt/β-catenin pathway is involved in mediating oxidative stress-induced podocyte dysfunction. Advanced oxidation protein products, a marker and trigger of oxidative stress, were increased in the serum of patients with chronic kidney disease and correlated with impaired glomerular filtration, proteinuria, and circulating level of Wnt1. Both serum from patients with chronic kidney disease and exogenous advanced oxidation protein products induced Wnt1 and Wnt7a expression, activated β-catenin, and reduced expression of podocyte-specific markers in vitro and in vivo. Blockade of Wnt signaling by Klotho or knockdown of β-catenin by shRNA in podocytes abolished β-catenin activation and the upregulation of fibronectin, desmin, matrix metalloproteinase-9, and Snail1 triggered by advanced oxidation protein products. Furthermore, conditional knockout mice with podocyte-specific ablation of β-catenin were protected against podocyte injury and albuminuria after treatment with advanced oxidation protein products. The action of Wnt/β-catenin was dependent on the receptor of advanced glycation end products (RAGE)-mediated NADPH oxidase induction, reactive oxygen species generation, and nuclear factor-κB activation. These studies uncover a novel mechanistic linkage of oxidative stress, Wnt/β-catenin activation, and podocyte dysfunction.

Keywords: AOPPs; Wnt; oxidative stress; podocyte; proteinuria; β-catenin.

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Figures

Figure 1.
Figure 1.. Circulating AOPPs are increased in CKD patients and induce Wnt/β-catenin activation in podocytes.
(a) Serum AOPPs levels inversely correlate with kidney function. Significant difference in serum AOPPs levels was observed in patients with different stages of CKD. Patients were grouped according to their eGFR (ml/min per 1.73m2) as indicated. Healthy controls: n=22, CKD patients: n=94. (b) Linear regression shows a correlation between serum AOPPs and urine albumin-to-creatinine ratio (UACR). (c) Linear regression shows a correlation between serum AOPPs and serum Wnt1 protein. The Spearman correlation coefficient (rs) and P value are shown. (d) Western blot analyses show that CKD serum induced active β-catenin and repressed ZO-1 and podocalyxin. Ctrl, control serum from normal subjects. (e, f) Graphic presentation of the relative abundances of active β-catenin (e), ZO-1 and podocalyxin (f) in different groups as indicated. *P< 0.05 versus controls (n = 3). (g) Western blot analyses show that CKD serum induced Wnt/β-catenin activation in podocytes, which was blocked by αRAGE. Podocyte lysates were immunoblotted with antibodies against Wnt1, Wnt7a, active β-catenin, podocalyxin, ZO-1 and α-tubulin, respectively. (h-k) Graphic presentations show the relative abundances of Wnt1 and Wnt7 (h), active β-catenin (i), podocalyxin (j) and ZO-1 (k) in different groups as indicated. *P< 0.05 versus controls; †P< 0.05 versus CKD serum alone (n = 3). (I) Representative micrographs show AOPPs and β-catenin staining in human focal and segmental glomerulosclerosis (FSGS). Human kidney biopsies from the patient with FSGS were stained with specific antibodies against AOPPs (red) and β-catenin (green), respectively. Arrows indicate positive staining in podocytes. Scale bar, 10 μm.
Figure 2.
Figure 2.. AOPPs induce Wnt/β-catenin activation and cause podocyte injury in vivo.
(a) Representative micrographs show renal Wnt1, Wnt7a and β-catenin expressions in mice after injections of AOPPs. Paraffin sections were stained with different antibodies. Arrows indicate Wnt1, Wnt7a and β-catenin expression specifically in glomerular podocytes. Scale bar, 25 μm.(b-f) Western blot analyses show that AOPPs induced Wnt1 and Wnt7a expression, and activated β-catenin and its downstream Snaill. Numbers (1 to 3) in (b) represent different animals in a given group. Graphic presentations show the relative abundances of Wnt1 (c), Wnt7a (d), active β-catenin (e) and Snail1 (f) proteins in different groups as indicated. *P < 0.05 versus controls. (g) Representative micrographs of desmin and nephrin staining in normal control and AOPPs groups. Scale bar, 20 μm. Arrows indicate positive staining. (h-l) Western blot analyses show that AOPPs induced α-SMA and fibronectin and repressed podocalyxin and nephrin. Numbers (1 to 3) in (h) represent different animals in a given group. Graphic presentations show the relative abundances of α-SMA (i), fibronectin (j), podocalyxin (k) and nephrin (l) proteins in different groups as indicated. *P < 0.05 versus controls.
Figure 3.
Figure 3.. Wnt ligands blocade attenuates AOPPs-induced podocyte injury and proteinuria.
(a) Experimental design. Red arrows indicate intravenous injection of either emty vector pcDNA43 or pV5-sKlotho at 1, 2 and 3 weeks after vehicle or AOPPs administration. (b) Western blot analyses show that both full-length and secreted Klotho were induced in isolated glomeruli from mice injected with pV5-sKlotho plasmid, compared to pcDNA3. (c) Delivery of sKlotho reduced AOPPs-induced albuminuria. Urinary albumin was expressed as μg/mg creatinine. *P < 0.05 versus control group; †P< 0.05 versus AOPPs group (n = 6). (d-h) Western blot analyses show that sKlotho restored AOPPs-induced loss of full-length Klotho and inhibited active β-catenin and its downstream fibronectin and α-SMA. Numbers (1 to 3) in (d) represent different animals in a given group. Graphic presentations show the relative abundances of full-length Klotho (e), active β-catenin (f), fibronectin (g) and α-SMA (h) proteins in different groups as indicated. (i) Antagonism of Wnt signaling by Klotho preserves podocyte integrity after AOPPs treatment. PAS staining and immunofluorescence staining for podocalyxin and nephrin revealed that sKlotho attenuated glomerular matrix accumulation and restored podocalyxin and nephrin. Arrows indicate positive staining. Scale bar, 25 μm. Representative transmission electron microscopy (TEM) microgarphs show podocyte ultrastructure after various treatments. Arrow indicates foot process effacement. Scale bar, 1 μm. (j-l) Western blot analyses show that sKlotho largely prevented AOPPs-induced loss of WT-1 and podocalyxin. Numbers (1 to 3) in (j) represent different animals in a given group. Graphic presentations show the relative abundances of WT-1 (k) and podocalyxin (l) proteins in different groups as indicated. *P < 0.05 versus controls; †P< 0.05 versus AOPPs (n = 6).
Figure 4.
Figure 4.. Podocyte-specific ablation of β-catenin protects against proteinuria induced by AOPPs in vivo.
(a) Scheme depicts experimental design. Conditional knockout mice with podocyte-specific deletion of β-catenin (podo-βcat−/−) and control littermates (podo-β-cat+/+) were described previously. (b) Podocyte-specific deletion of β-catenin protects mice from developing proteinuria after AOPPs administration in vivo. Urinary albumin was expressed as μg/mg creatinine. *P < 0.05 versus vehicle group; †P< 0.05 versus AOPPs group (n = 5 to 6). (c-f) Western blot analyses show that podocyte-specific deletion of β-catenin prevented β-catenin activation and inhibited fibronectin and α-SMA induction by AOPPs. Numbers (1 to 2) in (c) represent different animals in a given group. Graphic presentations show the relative abundances of active β-catenin (d), fibronectin (e) and α-SMA (f) expressions in different groups as indicated. *P< 0.05 versus vehicle group; †P < 0.05 versus AOPPs alone (n = 6). (g) Immunohistochemical and immunofluorescence staining shows β-catenin and fibronectin expression in different groups as indicated. AOPPs promoted active β-catenin and fibronectin expression in podocytes of podo-β-cat+/+ mice, but not in podo-β-cat−/− mice. Arrows indicate positive staining. Scale bar, 25 μm.
Figure 5.
Figure 5.. Mice with podocyte-specific ablation of β-catenin are protected against podocyte injury by AOPPs.
(a) Representative micrographs show that administration of AOPPs repressed nephrin and podocalyxin, and upregulated vimentin and MMP-9. However, podocyte-specific deletion of β-catenin largely restored nephrin and podocalyxin and inhibited vimentin and MMP-9. Arrows indicate positive staining. Scale bar, 25 μm. (b-d) Western blot analyses show that podocyte-specific deletion of β-catenin abolished desmin and MMP-9 induction by AOPPs. Numbers (1 to 2) in (b) represent different animals in a given group. Graphic presentations show the relative abundances of desmin (c) and MMP-9 (d) expressions in different groups as indicated. *P< 0.05 versus vehicle group; †P < 0.05 versus AOPPs (n = 5).
Figure 6.
Figure 6.. Wnt/β-catenin signaling is activated by AOPPs in cultured podocytes.
(a) RT-PCR analyses show that AOPPs induced multiple Wnt ligands expression in cultured podocytes in a time-dependent manner. Unmodified mouse serum albumin (MSA) incubated for 24 hours was used as a negative control. (b-d) Western blot analyses show the induction of Wnt1 and Wnt7a proteins in podocytes after AOPPs treatment. Graphic presentation of Wnt1 (c) and Wnt7a (d) proteins in different groups as indicated. *P < 0.05 versus controls (n = 3). (e) Immunofluorescence staining shows that AOPPs induced β-catenin activation and its nuclear translocation. Podocytes were treated with AOPPs for 12 hours. Cells were then immunostained for β-catenin and DAPI. Arrows indicate nuclear staining of β-catenin. Scale bar, 5 μm. (f-h) Western blot analyses of cell lysates show that AOPPs time-dependently induced active β-catenin and its downstream Snail1 protein expression. Graphic presentations of active β-catenin (g) and Snail1 (h) protein expressions in different groups as indicated. *P < 0.05 versus controls (n = 3).
Figure 7.
Figure 7.. Wnt/β-catenin activation is required for AOPPs-mediated podocyte injury.
(a-f) Western blot analyses show that recombinant Klotho (100 ng/ml) blocked AOPPs-mediated β-catenin activation and Snail1 induction, and restored the expressions of podocalyxin and ZO-1. Representative western blots (a, d) and graphic presentation of active β-catenin (b), Snail1 (c), podocalyxin (e) and ZO-1 (f) proteins in different groups are shown. *P < 0.05 versus controls; †P < 0.05 versus AOPPs (n = 3). (g) Western blot analyses show that overexpression of Flag-tagged, constitutively activated β-catenin repressed ZO-1, podocalyxin and nephrin expression. (h) Immunofluorescence staining shows that AOPPs induced reorganization of the F-actin cytoskeleton in podocytes. Arrow indicates stress-fiber in control podocytes, whereas arrowhead denotes peripheral localization of actin cytoskeleton in cells with over-expression of active β-catenin. Scale bar, 10 μm. (i-m) Western blot analyses show that knockdown of β-catenin by lentivirus-mediated β-catenin shRNA significantly inhibited AOPPs-mediated fibronectin, desmin, MMP-9 and Snail1 induction in podocytes. Graphic presentations show the relative abundances of fibronectin (j), desmin (k), MMP-9 (l) and Snail1 (m) proteins in different groups as indicated. *P < 0.05 versus controls; † P< 0.05 versus AOPPs plus Ctrl-shRNA (n = 3).
Figure 8.
Figure 8.. Wnt/β-catenin activation is dependent on ROS generation.
(a) ROS production is increased following AOPPs treatment in cultured podocytes. ROS was assessed by detection of DCFH fluorescence. Representative micrographs show that AOPPs induced ROS production in a time-dependent manner. Scale bar, 50 μm. (b) Quantitative data show the relative DCFH fluorescence intensity in different groups. The intensity of DCFH fluorescence in cells was assessed using a flow cytometer, and expressed as relative fluorescence intensity/100μg cell protein. (c-e) Western blot analyses show that AOPPs induced Nox2 and p47phox expression in cultured podocytes. Graphic presentations show the relative abundances of Nox2 (d) and p47phox (e) proteins in different groups as indicated. *P < 0.05 versus controls (n = 3). (f-k) Western blotting shows that NAC abolished AOPPs-induced Wnt1, Wnt7a, active β-catenin, Snail1 and restored nephrin expression in cultured podocytes. Podocytes were pre-incubated with NAC (2 mM), followed by incubation with AOPPs (10 μg/ml) for 24 hours. Graphic presentations of the relative abundances of Wnt1 (g), Wnt7a (h), active β-catenin (i), Snail1 (j) and nephrin (k) in different groups as indicated. *P< 0.05 versus controls; †P< 0.05 versus AOPPs alone (n = 3). (l) Representative micrographs show that NAC restored ZO-1 expression in cultured podocytes after AOPPs treatment. Arrows indicate positive staining. Scale bar, 20 μm.
Figure 9.
Figure 9.. RAGE/ROS/p65 NF-κB mediates AOPPs-triggered Wnt/β-catenin activation in podocytes.
(a) Western blotting shows that overexpression of RAGE induced Wnt1 and active β-catenin. (b) Blockade of RAGE by neutralizing antibody abolished AOPPs-induced Wnt1 and active β-catenin expression. (c) AOPPs induced phosphorylation of NF-κB p65 subunit in a time-dependent manner. (d) RT-PCR analyses show that NF-κB inhibitor SN50 repressed Wnt1 and Wnt7a mRNA expression induced by AOPPs. (e-h) Western blotting shows that SN50 inhibited AOPPs-mediated Wnt1, Wnt7a and active β-catenin induction in cultured podocytes. Graphic presentations of the relative abundances of Wnt1 (f), Wnt7a (g) and active β-catenin (h) in different groups are shown. *P< 0.05 versus controls; †P< 0.05 versus AOPPs alone (n = 3). (i) Representative micrographs show that SN50 blocked AOPPs-induced β-catenin nuclear translocation. Arrowheads indicate β-catenin in cell-cell junction, whereas arrows denote its nuclear staining. Scale bar, 20 μm. (j) Schematic presentation depicts the potential mechanism by which oxidative stress induces podocyte injury. AOPPs bind to plasma membrane receptor RAGE and trigger the activation of NADPH oxidase, thereby leading to an increased generation of ROS. This causes activation of p65 NF-κB, which in turn induces Wnt ligands such as Wnt1 and Wnt7a, and subsequently activates β-catenin. The activation of β-catenin triggers podocyte injury through inducing their dedifferentiation and mesenchymal transition. Multiple approaches such as blockade RAGE by neutralizing antibody, reduction of ROS by NAC, and genetic or pharmacologic ablation of β-catenin protect podocytes from injury.
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