Branched-chain amino acid metabolism disorder promotes the progression of diabetic kidney disease by inducing podocyte apoptosis in glomeruli via activating the mTOR-ATF4-Chop/Trib3 signaling pathway

To investigate the molecular mechanisms by which branched-chain amino acids (BCAAs) catabolic disorder induces glomerular podocyte injury, and to elucidate its role and mechanism in promoting the pathogenesis of diabetic nephropathy (DN) in a high-fat diet (HFD)-induced mouse model of obesity.

(1) Cellular Experiments: ① Primary glomerular podocytes were isolated from 6- to 8-week-old male C57 BL/6J mice (weight 20 to 25 g) and cultured, and then were randomly divided into 3 groups (n=4): low-glucose (LG, 5.5 mmol/L) group, high-glucose (HG, 25 mmol/L) group, and HG+3 mmol/L BCAA group. Western blotting was used to detect the expression of BCAA metabolism-related molecules (PPM1K, BCKD, and BCKD pSer293), podocyte functional markers (Nephrin, Synaptopodin, and WT1), and the apoptotic molecule Cleaved Caspase-3. Immunohistochemistry staining and Western blotting were performed to detect the expression levels of mTOR signaling pathway molecules (p70s6K pThr389, p70s6K, ATF4 pSer166, and ATF4) and apoptotic pathway molecules (Chop and Trib3). ② ATF4-knockout fibroblast cells were randomly divided into control (n=4) and BCAA (n=4) groups. Western blotting was used to detect the expression levels of apoptosis pathway molecules. ③ Primary glomerular podocytes isolated from 6- to 8-week-old C57 BL/6J mice were randomly divided into 3 groups (n=4): HG (25 mmol/L) group, HG + 3 mmol/L BCAA group, and HG + 3 mmol/L BCAA + rapamycin (HG + BCAA + Rap) group. Western blotting was performed to assess the expression levels of mTOR signaling pathway and apoptotic pathway molecules. (2) Animal Experiments: An HFD-induced mouse model of obesity was established. Male C57 BL/6J mice aged 6 to 8 weeks were randomly divided into an HFD group, an HFD/Paired group (food intake matched to the HFD/BCAA group), an HFD/BCAA group (4% BCAAs supplemented in drinking water), and an HFD/BCAA + Rap intervention group. Periodic acid-Schiff (PAS) staining, Oil Red O staining, and detection of renal triglyceride content were performed to assess renal pathological morphology and lipid deposition. Biochemical assays were applied to detect the urinary microalbumin and creatinine levels, as well as to calculate the urinary albumin-to-creatinine ratio. Glomerular podocyte functional markers (Nephrin and Synaptopodin) and fibrosis-related molecules (Collagen Ⅳ) were detected by immunofluorescence assay and RT-PCR. The expression levels of renal lipid metabolism-related molecules (FABP4, CD36, PGC1α, and PPARα), as well as the mTOR signaling pathway and apoptotic pathway molecules were detected by immunohistochemistry and Western blotting.

(1) Cellular Experiments: ① High-glucose treatment decreased PPM1K expression and increased BCKD pSer293 expression in the podocytes (P<0.0001). BCAAs downregulated the expression of Nephrin (P<0.0001) and Synaptopodin (P<0.01) and upregulated Cleaved Caspase-3 expression (P<0.0001) in the podocytes under high glucose condition. BCAAs significantly upregulated the protein levels of p70s6K pThr389, p70s6K, ATF4 pSer166, ATF4, Chop, and Trib3 in the podocytes (P<0.0001). ② Knockout of ATF4 significantly blocked BCAA-induced Chop and Trib3 expression activated by mTOR (P<0.0001). ③ The mTOR inhibitor Rap significantly suppressed the expression of ATF4 pSer166, ATF4, Chop, and Trib3 (P<0.0001). (2) Animal Experiments: ① Exogenous BCAA supplementation increased the urinary albumin-to-creatinine ratio (P<0.0001), aggravated the damage to glomerular filtration barrier, downregulated the expression of WT1, Nephrin, and Synaptopodin (P<0.0001), and upregulated glomerular p70s6K pThr389 (P<0.01) and Chop (P<0.0001) expression. Additionally, BCAA supplementation promoted renal tubular cast formation and renal lipid accumulation (P<0.0001), and upregulated the expression of FABP4 and CD36 in renal tubules of HFD-fed mice. ② The mTOR inhibitor Rap blocked the mTOR-ATF4-Chop/Trib3 pathway, downregulated the expression of ATF4 pSer166, ATF4, Chop, and Trib3 (P<0.0001), and ameliorated renal lipid deposition (P<0.0001), renal tubular cast formation, and proteinuria (P<0.0001).

BCAA metabolism disorder induces glomerular podocyte dysfunction and apoptosis by activating the mTOR-ATF4-Chop/Trib3 signaling pathway, leading to renal lipid deposition, renal tubular cast formation, and progression of renal fibrosis, thereby promoting the development and progression of DN.