Fig 2: PROTAC attenuates renal fibrosis and ameliorates renal function and renal anemia of 5/6Nx mice. A Experimental design of evaluating the therapeutic effect of PROTAC on 5/6Nx animal models. B Western blot analyses of HIF-1α, HIF-2α, Smad3, collagen-I and α-SMA expression and C quantification of them in 5/6Nx mice (n = 6/per group). D Representative Masson’s trichrome staining of renal sections among indicated groups. Scale bar: 500 μm. E The positive-stained area of Masson’s trichrome was quantitatively measured (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\times$$\end{document}×400 magnification, n = 8/per group). F Trajectory of Cystatin C of indicated groups at indicated times (n \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\ge$$\end{document}≥ 6/per group) and renal function was evaluated with G serum creatinine and H serum BUN after 5 weeks of vehicle or PROTAC treatment (n = 8/per group). Trajectory of I HGB and J RBC counts of indicated groups at indicated times (n \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\ge$$\end{document}≥ 6/per group, **P < 0.01 compared between PROTAC-treated and vehicle-treated mice in 5/6Nx group. #P < 0.05 compared between PROTAC-treated and vehicle-treated mice in Sham group). K The serum level of EPO was determined in indicated groups. The situation of iron mobilization was indicated with serum level of L Hepcidin, (M) Fe2+ and N Ferritin of indicated groups after 5 weeks of treatment. Veh = vehicle, 5/6Nx = 5/6 nephrectomy model, HGB = hemoglobin, RBC = red blood cells. Data are means ± SD. NS = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig 3: Mechanism of VHL-recruiting, Smad3-targeting PROTAC. A Schematic of PROTAC-mediated Smad3 degradation and HIF-2α stabilization. B The impact of different amount of PROTAC on the degradation of Smad3 recombinat protein was investigated with ACHN whole cell lysate expressing high level of VHL. C PROTAC-mediated polyubiquitination and degradation of Smad3 protein via ubiquitin–proteasome pathway. The degradation and ubiquitination on Smad3 protein induced by PROTAC was compared in the absence and presence of MG132 (a specific proteasome inhibitor) with ACHN whole cell lysate expressing high level of VHL

Fig 4: VHL-based Smad3-targeting PROTAC protects from chronic kidney disease through simultaneously reducing renal fibrosis and promoting renal repair. ECM = excessive extracellular matrix, EMT = epithelial–mesenchymal transition

Fig 5: PROTAC degrades Smad3 and stabilizes HIF-2α but not HIF-1α in vitro. A Treatment of NRK-49F cells with PROTAC of two linkers: a PROTAC-PEG and b PROTAC-(CH2COOH)2. B Treatment of NRK-49F cells with 25 nM PROTAC of two charities: PROTAC-R and PROTAC-S and the plot represented three independent experiments (n = 3). C The expression levels of different E3 ligases in human, mouse and rat renal tubular cells (HK2, mTEC, NRK52E). D Time course treatment of NRK-49F cells with 25 nM of PROTAC. E Treatment of NRK-49F cells with PROTAC of different concentration and the plot represented three independent experiments (n = 3). F Treatment of HK2 cells with PROTAC of different concentration and the plot represented three independent experiments (n = 3). PEG = Polyethylene glycol; (CH2COOH)2 = Succinic acid. Data are means ± SD; NS = not significant, *P < 0.05, **P < 0.01