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 CYS). Protein lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with the indicated antibodies. G and H Western blot analyses (G) and quantitative data (H) showed that PRR siRNA blocked HG-induced DPP4 abundance and phosphorylation of JNK, and restored HG-suppressed SIRT3 and FGFR1 expression in HK-2 cells (n = 3). I and J Representative Western blot (I) and quantitative data (J) showed increased DPP4, upregulated phosphorylation of JNK, suppressed SIRT3 and reduced FGFR1 in PRR overexpressed HK-2 cells (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