Kidney fibrosis is mediated by GARP-restricted TGF-β activation in fibroblasts
Yintong Chen, Weiwei Xu, Jieli Yu, Pei Deng, Nianping Liu, Yinyin Li, Hui Zhou, Hong Zhou, Jianchuan Wang, Bo Zhao, Florian Winau, Fan Fan Hou, Yu Hu
Yintong Chen, Weiwei Xu, Jieli Yu, Pei Deng, Nianping Liu, Yinyin Li, Hui Zhou, Hong Zhou, Jianchuan Wang, Bo Zhao, Florian Winau, Fan Fan Hou, Yu Hu
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Research Article Nephrology Public Health

Kidney fibrosis is mediated by GARP-restricted TGF-β activation in fibroblasts

Abstract

TGF-β is a central driver of kidney fibrosis, a common pathological hallmark of chronic kidney disease (CKD). Initiation of TGF-β signaling requires not only its synthesis but also the conversion of latent TGF-β to its bioactive form. However, the mechanisms governing TGF-β activation in the kidney and their contribution to kidney fibrosis remain poorly understood. Glycoprotein A repetitions predominant (GARP) anchors latent TGF-β on the cell surface and facilitates its bioactive release. Here, we show that GARP-mediated TGF-β activation promotes kidney fibrosis. GARP was upregulated in both human and mouse CKD kidneys, predominantly in fibroblasts, and was induced by TNF in an NF-κB–dependent fashion. In multiple mouse models of kidney fibrosis, either global or fibroblast-specific deletion of GARP markedly reduced fibrosis. Mechanistically, GARP enables sustained production of active TGF-β, thereby amplifying fibroblast stimulation. Deletion of GARP in kidney fibroblasts lowered active TGF-β levels and attenuated fibroblast activation, whereas GARP overexpression enhanced TGF-β signaling. Notably, tamoxifen-induced deletion of GARP after fibrosis onset attenuated kidney fibrosis. Together, our findings identify GARP-mediated release of active TGF-β as a critical step in sustaining fibroblast activation during kidney fibrosis and highlight GARP as a promising therapeutic target for CKD.

Authors

Yintong Chen, Weiwei Xu, Jieli Yu, Pei Deng, Nianping Liu, Yinyin Li, Hui Zhou, Hong Zhou, Jianchuan Wang, Bo Zhao, Florian Winau, Fan Fan Hou, Yu Hu

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Figure 5

GARP-dependent TGF-β activation sustains Smad signaling and induces fibroblast stimulation.

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GARP-dependent TGF-β activation sustains Smad signaling and induces fibr...
(A and B) Representative flow cytometry plots (A) and quantification (B) of GARP and LAP (TGF-β1) expression in kidney fibroblasts from sham or UUO-treated WT and GARP-KO mice (n = 3–4). MFI, mean fluorescence intensity. (C and D) Representative (C) and quantification (D) of immunoblot for p-Smad2/3 in sham or UUO-treated WT and GARP-KO mice (n = 5). (E) TGF-β reporter assay detecting active TGF-β released by PKFs with or without TGF-β1 priming. (F and G) Representative (F) and quantification (G) of immunoblot for p-Smad2/3 in medium only or TGF-β1–primed PKFs from WT and GARP-KO mice. (H) Flow cytometry of GARP expression in NRK-49F cells transduced with empty lentivirus (LV-vector) or GARP-overexpression lentivirus (LV-GARP). (I) Representative immunoblots showing TGF-β1 levels in cell lysates from medium (control) or TGF-β1–primed NRK-49F cells transduced with LV-vector or LV-GARP. (J) TGF-β reporter cell assay detecting active TGF-β release in NRK49F cells following TGF-β1 treatment in the absence or presence of CWHM-12 (an integrin αv inhibitor). (K and L) Representative (K) and quantification (L) of immunoblot for p-Smad2/3 in medium only or TGF-β1–primed NRK-49F cells. (M and N) Representative (M) and quantification (N) of immunoblot for GARP, FN, COL1A1, and Vimentin in medium only or TGF-β1–primed PKF cells from WT and GARP-KO mice. (O and P) Representative (O) and quantification (P) of immunoblot for GARP, FN, and α-SMA in medium only or TGF-β1–primed NRK-49F cells transduced with LV-vector or LV-GARP. Each lane in C, F, I, K, M, and O represents 1 well of cells. All in vitro experiments were independently repeated 3 times with similar results. Data represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Statistical significance was determined by 2-way ANOVA followed by Tukey’s multiple-comparison test (B, D, E, G, J, L, N, and P).