SIRT3 Deacetylates PDHE1α to Regulated Mitochondria Metabolism in Tubular Epithelial Cells During Renal Fibrosis

Background: Abnormal energy metabolism is considered to be related to renal interstitial fibrosis. Pyruvate dehydrogenase α (PDHE1α) is the main catalytic enzyme of pyruvate dehydrogenase complex (PDHC) linking glycolysis to the TCA cycle. N-lysine acetylation is an important post-translational modification involves in energy metabolism. SIRT3 is a mitochondrial deacetylase that mediates the activity many metabolic enzymes. Methods: Unilateral ureteral obstruction (UUO) or ischemia-reperfusion (I/R) were used to induce renal fibrosis in C57BL/6J mice or SIRT3 knockout mice. Primary tubular epithelial cells (PTCs) were stimulated by TGF-β1. Protein array and the acetylation array by LC-MS/MS were performed on tubules separated from sham or UUO-operated mice. K149R, K267R, K385R mutations in PDHE1a were transfected into PTCs. Results: Acetylome showed that the majority of proteins were hyper-acetylation after UUO. GO enrichment analysis revealed that PDH was the most obviously enriched GO term. Immunoprecipitation analysis confirmed that PDHE1α acetylation was enhanced after UUO or I/R operation. Activation of SIRT3 by HKL could block the hyperacetylation of PDHE1α, restored PDH enzyme activity, and inhibited the phosphorylation of PDHE1α in mice with UUO or I/R. On the contrary, Sirt3 KO mice had more acetylated PDHE1α, more phosphorylated PDHE1α and defective PDH enzyme activity. In vitro, increased PDHE1 acetylation was accompanied by reduced PDH enzyme activity and increased PDHE1α phosphorylation in PTCs after TGF-β1 stimulation. Activation of SIRT3 by HKL repressed the effect of TGF-β1. Inhibition SIRT3 activity by 3-TYP or SIRT3 siRNA transfection have the same effect as TGF-β1. K149, K267, K385 were identified as the main potentially lysine acetylated sites in PDHE1α. Acetylation of PDHE1α, the activity of PDH and PDHE1α phosphorylation remained unchanged in PTCs with the K385R mutation stimulated with TGF-β1 or SIRT3 siRNA transfection. Conclusions: In summary, our data showed that mitochondrial proteins involved in regulating energy metabolism were acetylated and targeted by SIRT3 in PTCs. The deacetylation of PDHE1α at lysine 385 by SIRT3 plays a key role in metabolic reprogramming in renal fibrosis. Funding: Government Support - Non-U.S.