(Pro)renin receptor mediates tubular epithelial cell pyroptosis in diabetic kidney disease via DPP4-JNK pathway

Figures

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  Fig. 1. PRR expression and cell pyroptosis were increased in renal tubules of DKD patients. A and B Representative fluorescent images of coimmunostaining PRR (red) with GSDMD (green) in kidney biopsies from DKD patients (n = 16), and the control subjects were from paracancerous tissue (n = 5) (A), and quantitative data showed the upregulation of PRR in renal tubule. Bar = 20 μm. (B). C–F Analysis of the association between relative tubular PRR expression and eGFR(C), BUN (D), urinary albumin-to-creatinine ratio (UACR) (E) and Hemoglobin A1c (HbA1c) (F) of patients with DKD. (G–I) GSEA analysis of pyroptosis (G), NOD-like receptor signaling pathway (H) and Interleukin-1 production (I) in renal tubules of DKD patients (n = 10) and healthy controls (n = 12) by using public GSE dataset (GSE30529). J Representative microscopy images of tubular cell morphology in renal sections from DKD patients and health controls. Bar = 1 μm. (K and L) Elisa detection of urinary IL-1β (K) and IL-18 (L) from DKD patients (n = 28) and health controls (n = 23). M Elisa detection of urinary IL-6 from DKD patients (n = 28) and health controls (n = 18). Data are presented as mean ± SEM of biologically independent…

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  Fig. 2. Knockdown of PRR effectively blocked high glucose (HG)-augmented HK-2 cell pyroptosis. A and B Western blot analyses (A) and quantitative data (B) showed that knocking down by siRNA diminished HG-induced PRR expression. (n = 3). C and D Flow cytometry analysis showed that HG induced active Caspase1+PI+ HK-2 cells were decreased by PRR siRNA (C) and quantitative data (D). (n = 3). E PRR was silenced in HG stimulated HK-2 cells, and the Caspase1 activity in cell lysis was determined by kits (n = 5). F and G Western blot analyses (F) and quantitative data (G) showed that PRR siRNA blocked HG-induced NLRP3, cleaved-Caspase1, GSDMD-N, IL-1β and IL-18 expression in HK-2 cells (n = 3). H and I PRR was ablated in HG treated HK-2 cells, and the IL-1β (H), IL-18 (I) or IL-6 (J) concentration in the culture medium was determined by ELISA, and then normalized by protein concentration in cell lysates (n = 3). Data are presented as mean ± SEM of biologically independent samples. ∗ P < 0.05, ∗∗ P < 0.01. One-way ANOVA was used to analyze the data among multiple groups, followed by Tukey’s post hoc test

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  Fig. 3. Overexpression of PRR promotes HK-2 cell pyroptosis. (A and B) Western blot analyses. A and quantitative data (B) showed the increased protein abundance of PRR in PRR overexpression plasmid transfected HK-2 cells. (n = 3). (C and D) Flow cytometry analysis showed that active Caspase1+PI+ HK-2 cells were increased by PRR overexpression (C) and quantitative data (D) (n = 3). E PRR was overexpressed in HK-2 cells, and the Caspase1 activity in cell lysis was determined by kits (n = 5). (F and G) Western blot analyses (F) and quantitative data (G) showed that PRR overexpression increased NLRP3, cleaved-Caspase1, GSDMD-N, IL-1β and IL-18 expression in HK-2 cells. (n = 3). H and I PRR was overexpressed in HK-2 cells, and the IL-1β (H), IL-18 (I) or IL-6 (J) concentration in the culture medium was determined by ELISA, and then normalized by protein concentration in cell lysis. (n = 3). Data are presented as mean ± SEM of biologically independent samples. ∗ P < 0.05, ∗∗ P < 0.01. P values were determined by Student’s t-test

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  Fig. 4. PRR interacted with DPP4 to activate JNK signaling and inhibit SIRT3 signaling and FGFR1 signaling in vitro. A BioGRID database was used to analyze the potential interaction between PRR and DPP4. B Co-immunoprecipitation (Co-IP) to detect the interaction between PRR and DPP4. The protein lysates isolated from HK-2 cells were immunoprecipitated with anti-PRR antibody and were immunoblotted with indicated antibodies. C Representative fluorescent images of coimmunostaining PRR (red) with DPP4 (green) in renal sections from DKD patients and the healthy control was from paracancerous tissue. Bar = 20 μm. D Schematic representation of the DPP4 domain. E Co-IP was used to detect the interaction between PRR and Flag-DPP4. HK-2 cells were transfected with Flag-tagged truncation of DPP4 C-terminal 325–766 amino acids (Flag-DPP4 C), or 1–324 amino acids (Flag-DPP4 N). Protein lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with the indicated antibodies. F Co-IP was used to detect the interaction between PRR and Flag-DPP4. HK-2 cells were transfected with Flag-tagged truncation of DPP4 C-terminal 325–766 amino acids (Flag-DPP4 C), or 325–551 amino acids (Flag-DPP4…

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  Fig. 5. DPP4 facilitated HG-induced HK-2 cell pyroptosis by activating JNK and suppressing SIRT3 and FGFR1. A and B Western blot analyses. A and quantitative data (B) showed that knocking down by siRNA diminished HG-induced DPP4 expression and phosphorylated JNK, and restored HG-reduced SIRT3 and FGFR1 abundance. (n = 3). C and D Flow cytometry analysis showed that knocking down DPP4 reduced HG increased active Caspase1+PI+ HK-2 cells (C) and quantitative data (D) (n = 3). E DPP4 was ablated in HG stimulated HK-2 cells, and the Caspase1 activity in cell lysis was determined by kits. F and G Western blot analyses (F) and quantitative data (G) showed that DPP4 siRNA attenuated HG-induced NLRP3, cleaved-Caspase1, GSDMD-N, IL-1β and IL-18 expression in HK-2 cells (n = 3). ( Hand I) DPP4 was knocked down in HG treated HK-2 cells, and the IL-1β (H), IL-18 (I) or IL-6 (J) concentration in the culture medium was determined by ELISA, and then normalized by protein concentration in cell lysates (n = 3). Data are presented as mean ± SEM of biologically independent samples. ∗ P < 0.05, ∗∗ P < 0.01. One-way ANOVA was used to analyze the data among multiple groups, followed by Tukey’s post hoc test

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  Fig. 6. PRR exerted pyroptotic effects through DPP4-mediated signaling. A and B Representative western blot analyses (A) and quantitative data (B) showed that knocking down DPP4 by siRNA in HK-2 cells diminished PRR overexpression induced phosphorylation of JNK, and restored PRR overexpression reduced SIRT3 and FGFR1 expression (n = 3). C and D Flow cytometry analysis showed that knocking down DPP4 reduced PRR overexpression induced frequency of active Caspase1+PI+ HK-2 cells (C) and quantitative data (D) (n = 3). E PRR was silenced in HG stimulated HK-2 cells, and the Caspase1 activity in cell lysis was determined by kits. f and g Western blot analyses (F) and quantitative data (G) showed that DPP4 siRNA blocked HG-induced NLRP3, cleaved-Caspase1, GSDMD-N, IL-1β and IL-18 expression in HK-2 cells (n = 3). H and I DPP4 was knocked down in PRR overexpressed HK-2 cells, and the IL-1β (H), IL-18 (I) or IL-6 (J) concentration in the culture medium was determined by ELISA, and then normalized by protein concentration in cell lysates (n = 3). Data are presented as mean ± SEM of biologically independent samples. ∗ P < 0.05, ∗∗ P < 0.01. One-way ANOVA was used to analyze the data among multiple…

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  Fig. 7. Silencing of PRR ameliorated kidney injury and inflammation in db/db mice. A Schematic diagram of the AAV injection surgery. B and C The protein level of PRR was assessed by Western blot. Representative Western blot (B) and quantitative data (C) are showed (n = 6). D Measured of urinary albumin concentration by using albumin detection kit, the quantitative data was normalized with urinary creatinine. E–L Representative images of Masson, Sirius Red, PAS, KIM-1, F4/80 and Ly6G staining of renal section of WT mice and db/db mice with or without PRR silence (E) and quantitative analysis (F–L) (n = 6). Bar = 20 μm. Data are presented as mean ± SEM of biologically independent samples. ∗ P < 0.05, ∗∗ P < 0.01. One-way ANOVA was used to analyze the data among multiple groups, followed by Tukey’s post hoc test

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  Fig. 8. PRR knockdown blunted pyroptotic cell death and fibrotic response in db/db mice via DPP4-mediated signaling. A–F The protein levels of NLRP3, cleaved-Caspase1, GSDMD-N, IL-1β, IL-18 were assessed by Western blot. Representative Western blot (A) and quantitative data (B–F) are showed (n = 6). C Representative immunofluorescence microscopy images of coimmunostaining PRR (red) with GSDMD (green) in renal sections from WT and db/db mice with or without PRR ablation. Bar = 20 μm. H and I Elisa detection of urinary IL-1β (H) and IL-18 (I) concentration, the quantitative data were normalized with urinary creatine (n = 6). J–K western blot analyses (J) and quantitative data (K) showed the abundance of DPP4, phosphorylation level of JNK, SIRT3 and FGFR1 (n = 6). L Representative fluorescent images of renal sections from mice with or without PRR silence and stained with PRR (red), DPP4 (green). Bar = 20 μm. Data are presented as mean ± SEM of biologically independent samples. ∗ P < 0.05, ∗∗ P < 0.01. One-way ANOVA was used to analyze the data among multiple groups, followed by Tukey’s post hoc test