Tubule-Derived Wnts Are Required for Fibroblast Activation and Kidney Fibrosis

Abstract

Cell-cell communication via Wnt ligands is necessary in regulating embryonic development and has been implicated in CKD. Because Wnt ligands are ubiquitously expressed, the exact cellular source of the Wnts involved in CKD remains undefined. To address this issue, we generated two conditional knockout mouse lines in which Wntless (Wls), a dedicated cargo receptor that is obligatory for Wnt secretion, was selectively ablated in tubular epithelial cells or interstitial fibroblasts. Blockade of Wnt secretion by genetic deletion of Wls in renal tubules markedly inhibited myofibroblast activation and reduced renal fibrosis after unilateral ureteral obstruction. This effect associated with decreased activation of β-catenin and downstream gene expression and preserved tubular epithelial integrity. In contrast, fibroblast-specific deletion of Wls exhibited little effect on the severity of renal fibrosis after obstructive or ischemia-reperfusion injury. In vitro, incubation of normal rat kidney fibroblasts with tubule-derived Wnts promoted fibroblast proliferation and activation. Furthermore, compared with kidney specimens from patients without CKD, biopsy specimens from patients with CKD also displayed increased expression of multiple Wnt proteins, predominantly in renal tubular epithelium. These results illustrate that tubule-derived Wnts have an essential role in promoting fibroblast activation and kidney fibrosis via epithelial-mesenchymal communication.

Keywords: Wnt signaling; Wntless; chronic kidney disease; fibroblast; renal fibrosis.

Figure 1.

Tubule-specific ablation of endogenous Wls suppresses activation of Wnt/β-catenin signaling after UUO. (A) Schematic diagram shows the strategy of crossbreeding of Wls floxed mice (Wls^fl/fl) with Ksp-Cre transgenic mice. The Wls floxed mutant mice possess a loxP site before the ATG start site in the 5′ untranslated region and another in the upstream of exon 2 of Wls gene. (B) Mouse genotyping analyzed by PCR. Lane 1 shows the genotyping of the control mice used in this study (genotype: Wls^fl/fl), and lane 2 denotes the genotyping of the tubule-specific Wls knockout mice (genotype: Wls^fl/fl_, Cre), designated as Ksp-Wls−/−. (C) Western blot analyses demonstrate a substantial reduction of renal Wls protein in Ksp-Wls−/− mice, compared to the controls. Kidney lysates were prepared from control and Ksp-Wls−/− mice at 7 days after UUO. Numbers (1–5) indicate each individual animal in a given group. (D) Costaining for Wls and tubule segment–specific markers in control and Ksp-Wls−/− kidneys. Immunofluorescence staining demonstrated the costaining of Wls (red) and various tubular markers (green) in the kidneys. Segment-specific tubular markers used are as follows: proximal tubule, aquaporin-1 (AQP1); distal tubule, thiazide-sensitive Na-Cl cotransporter (TSC)/NCC; and collecting duct, aquaporin-3 (AQP3). Arrows indicate Wls-positive tubules. Scale bar, 50 µm. (E) Tubule-specific deletion of Wls does not cause renal abnormality. Representative micrographs of PAS staining show kidney cortex and medulla in control and Ksp-Wls−/− mice. Scale bar, 50 µm. (F and G) Western blot analyses of renal expression of β-catenin and PAI-1 proteins in the obstructive kidneys of control and Ksp-Wls−/− mice at 7 days after UUO. Representative western blot (F) and quantitative data (G) are presented. Numbers (1–3) indicate each individual animal in a given group. *P<0.05 versus controls (n=6–9). (H) Representative immunofluorescence micrographs show β-catenin expression and localization in the control and Ksp-Wls−/− mice kidneys at 7 days after UUO. Arrows indicate positive nuclear β-catenin staining in kidney tubular epithelial cells. Scale bar, 50 µm. (I) qRT-PCR demonstrated renal MMP-7 mRNA levels at 7 days after UUO. *P<0.05 versus controls (n=6–9). Ctrl, control.

Figure 2.

Tubule-specific deletion of Wls ameliorates kidney fibrosis after UUO. (A and B) qRT-PCR analyses show significant downregulation of collagen type I and type III mRNA in the obstructed kidneys of Ksp-Wls−/− mice at 7 days after UUO, compared with the controls. *P<0.05 versus controls (n=3–5). (C and D) Western blot analyses demonstrate a decreased fibronectin protein in Ksp-Wls−/− kidney after UUO, compared with the controls. Representative western blot (C) and quantitative data (D) are presented. *P<0.05 versus controls (n=6–9). (E) Representative micrographs of MTS, Picrosirius Red staining, and immunofluorescence staining for fibronectin and collagen type III are presented. Arrows indicate positive staining. (F–H) Graphical presentation of quantitative data of fibrosis score (F), fibronectin (G), and collagen type III (H) in control and Ksp-Wls−/− mice at 7 days after UUO. *P<0.05 versus controls (n=3). Scale bar, 50 µm. Fn, fibronectin; Ctrl, control.

Figure 3.

Tubule-specific deletion of Wls reduces partial EMT after UUO. (A and B) Western blot analyses show E-cadherin protein levels in the obstructive kidneys of the control and Ksp-Wls−/− mice at 7 days after UUO. Representative western blot (A) and quantitative data (B) are presented. *P<0.05 versus controls (n=6–9). Numbers (1–5) denote each individual animal in a given group. (C) Representative micrographs show E-cadherin and vimentin expression in control and Ksp-Wls−/− kidneys at 7 days after UUO. Arrows indicate positive staining in renal tubules. Scale bar, 50 μm. (D) Quantitative data show vimentin-positive tubular cells per high power field (hpf). *P<0.05 versus controls (n=3). Ctrl, control; Vim+, vimentin positive.

Figure 4.

Tubule-specific deletion of Wls represses myofibroblast activation and renal inflammation after UUO. (A and B) Quantitative determination of renal α-SMA and Fsp-1 mRNA levels of the control and Ksp-Wls−/− mice at 7 days after UUO by qRT-PCR. *P<0.05, **P<0.01 versus controls (n=3–5). (C) Western blot analyses of renal α-SMA, PDGFR-β, PCNA, and Cyclin D1 in the control and Ksp-Wls−/− mice at 7 days after UUO. (D–G) Quantitative data on the protein expression of α-SMA (D), PDGFR-β (E), PCNA (F), and cyclin D1 (G) in the obstructed kidney after UUO. *P<0.05 versus controls (n=6–9). Numbers (1–5) indicate each individual animal in a given group. (H) Representative micrographs show α-SMA, PDGFR-β, and PCNA expression in the control and Ksp-Wls−/− kidneys at 7 days after UUO. Arrows indicate positive staining. Scale bar, 50 µm. (I) Quantitative data on renal α-SMA staining are shown. (J) Knockout of Wls in renal tubules decreases PCNA-positive cells in renal interstitium. PCNA-positive cells per high power field (hpf) are counted and shown. (K) qRT-PCR demonstrates renal mRNA levels of FasL in the control and Ksp-Wls−/− mice at 7 days after UUO. (L) qRT-PCR demonstrates renal mRNA levels of MCP-1 in the control and Ksp-Wls−/− mice at 7 days after UUO. *P<0.05 versus controls. Ctrl, control.

Figure 5.

Fibroblast-specific deletion of Wls exhibits little effect on kidney fibrosis after obstructive injury. (A) Schematic diagram depicting generation of fibroblast-specific deletion of Wls in mice by using Cre-LoxP system. Wls floxed mice (Wls^fl/fl) were crossbred with tamoxifen-inducible Cre transgenic mice under the control of endogenous Gli1 promoter/enhancer elements. Tamoxifen-inducible, Cre-mediated recombination resulted in deletion of the flanked sequences in Gli1-expressing fibroblasts. (B) Mouse genotyping analyzed by PCR. Lanes 1 and 2 show the genotyping of the control mice used in this study (genotype: Wls^fl/fl), lanes 3 and 4 denote the genotyping of fibroblast-specific Wls knockout mice (genotype: Wls^fl/fl Cre), designated as FC-Wls−/−. (C) Coimmunostaining with antibodies against Cre recombinase (red) and various cell type–specific markers (green). Arrows indicate Cre-positive cells. Scale bar, 20 µm. (D) Representative immunofluorescent micrographs show colocalization of Wls and α-SMA expression in control and FC-Wls−/− kidneys at 7 days after UUO. Arrows indicate α-SMA–positive, active fibroblasts in kidney interstitial space. Scale bar, 50 μm. (E) Quantitative data show the percentage of Wls⁺ cells in the α-SMA⁺ fibroblast population in control and FC-Wls−/− kidneys at 7 days after UUO. **P<0.01 versus controls (n=3). (F–H) qRT-PCR analyses show the relative mRNA levels of α-SMA, fibronectin, and collagen type III in the obstructive kidneys of control and FC-Wls−/− mice at 7 days after UUO (n=5). (I) Western blot analyses of renal fibronectin and α-SMA levels in control and FC-Wls−/− mice at 7 days after UUO. (J) Representative micrographs of immunohistochemical staining show α-SMA expression in control and FC-Wls−/− kidneys at 7 days after UUO. (K) Representative micrographs of MTS show collagen deposition in control and FC-Wls−/− kidneys at 7 days after UUO. Arrows indicate positive staining. Scale bar, 50 µm. Ctrl, control.

Figure 6.

Wnt ligands promote fibroblast proliferation and matrix production in vitro. (A) Representative micrographs show the phase-contrast images of fibroblasts after incubation with Wnts-CM in the absence or presence of ICG-001 for 48 hours. (B) Wnts promotes fibroblast proliferation. NRK-49F cells were incubated with Wnts-CM for 48 hours. Cell numbers were counted and presented. *P<0.05, **P<0.01 versus controls. ^†P<0.05, versus 10% or 40% Wnts-CM alone (n=3). (C) Representative micrographs show that Wnts-CM promoted fibroblasts DNA synthesis as demonstrated by BrdU incorporation. NRK-49F cells were incubated with 10% and 40% Wnts-CM for 48 hours, respectively. Cells were immunostained with mouse anti-BrdU antibody (red). SYTO-Green (green) was used to visualize the nuclei. Arrows indicate BrdU-positive cells. (D) Quantitative determination of the percentage of BrdU-positive cells after the treatment. *P<0.05, **P<0.01 versus controls. ^†P<0.05, versus 10% or 40% Wnt-CM (n=3). (E) Colorimetric MTT assay shows that Wnts-CM promoted fibroblast proliferation. *P<0.05, **P<0.01 versus controls. ^†P<0.05, versus 10% Wnts-CM (n=3). OD, optical density. (F) Western blot analyses show that Wnts-CM induced PCNA protein expression in fibroblasts. NRK-49F cells were incubated with varying concentrations of Wnts-CM for 48 hours as indicated.

Figure 7.

Multiple Wnt ligands are induced in human CKD. Human kidney biopsy samples were immunohistochemically stained with specific antibodies against Wnt1, Wnt2, Wnt4, Wnt5a, Wnt9b, and Wnt10b proteins. Representative micrographs show expression and localization of Wnts protein in a variety of human CKDs. Wnt1, Wnt9b, and Wnt10b were largely expressed in tubular epithelial cells (boxed area, solid arrows) but not in interstitial cells (open arrows). Wnt2, Wnt4, and Wnt 5a were detected in both tubular and interstitial cells in diseased kidneys (boxed area, black arrows and yellow arrows). Nontumor kidney tissue from the patients who had renal cell carcinoma and underwent nephrectomy was used as normal controls. IgAN, IgA nephropathy. Boxed areas are enlarged. Scale bar, 50 μm.

Figure 8.

Tubule-derived Wnts play a major role in the pathogenesis of kidney fibrosis. Diagram shows the cellular sources of Wnt ligands and their actions in renal fibrogenesis. Multiple Wnts are induced in renal tubular epithelium as well as in interstitial fibroblasts after kidney injury and secreted by a Wls-dependent mechanism. Pool of Wnts targets tubular cells and impairs epithelial integrity characterized by loss of E-cadherin and de novo expression of vimentin, and promotes fibroblast activation characterized by an enhanced cell proliferation and matrix production. ECM, extracellular matrix.