Fig. 6: BCAAs induce podocyte apoptosis by promoting PKM2-DDIT3 cotransport into the nucleus to promote Chac1 and Trib3 transcription.

a PKM2 expression (green) and nuclei (blue) were detected by fluorescence microscope in LG-, HG- or HG/BCAA-treated differentiated MPC-5 podocytes. b Quantification of the protein expression of PKM2 in the cytoplasm and nucleus of differentiated MPC-5 podocytes. n = 4 total samples per group. c, d Coimmunoprecipitation (IP) and immunoblotting (IB) analysis of PKM2 with ATF5, ATF4, and DDIT3 in whole-cell or nucleus lysates of differentiated MPC-5 podocytes. e Chromatin immunoprecipitation (ChIP) assay of PKM2 with th.e Trib3 promoter in MPC-5 podocytes treated with DDIT3 siRNA (si-DDIT3) or scramble RNA (si-con). n = 6. f ChIP assay of DDIT3 with the Trib3 promoter in MPC-5 podocytes treated with Ad-PKM2 or Ad-empty. n = 3. g Representative Western blots of PKM2, Chac1, and Trib3 expression in primary podocytes isolated from PKM2 knockout mice (KO) or wild-type mice and transfected with adenovirus overexpressing PKM2 (OE) or empty vector (-). h Representative Western blots of PKM2, Chac1, and Trib3 expression in primary podocytes isolated from PKM2 KO mice that were exposed to 3 mM BCAAs for 24 h. i, j Representative confocal immunofluorescence images (i) and Western blots (j) of differentiated MPC-5 podocytes that were exposed to LG, HG, and/or 3 mM BCAA or 20 nM TEPP46 for 24 h. Both cytoplasmic and nuclear extracts were evaluated. n = 4 total samples per group. k, l Cell viability (k, n = 10 for LG and HG; n = 12 for HG/BCAA, n = 11 for HG/BCAA + TEPP46) and protein expression of Chac1 and Trib3 (l, n = 4 total samples per group) in differentiated MPC-5 podocytes were determined. The data were presented as mean ± SD, and analyzed by unpaired two-tailed Student’s t test (e, f) or one-way ANOVA (b, i, and j).