DHHC6-Mediated SDHB Palmitoylation Protects against Tubular Cell Death and AKI

Background: Acute kidney injury (AKI), a syndrome characterized by an abrupt of loss of kidney function, is associated with considerable morbidity and mortality. The tubular cell injury and death contributes to the initiation and progression of AKI. Palmitoylation, a reversible post-translational modification, is involved in the development of renal fibrosis. However, the role of palmitoylation in tubular cell survival and AKI remains unclear. We aimed to investigate the role and mechanism of palmitoylation in tubular cell survival and AKI. Methods: 1.Acyl–biotin exchange，ABE; 2.Blue native polyacrylamide gel electrophoresis，BN-PAGE. Results: 1. The level of protein palmitoylation was markedly downregulated in the kidney of mice after UNx+IRI surgery. Analysis of the transcription levels of DHHCx in the GSE98622 database revealed significant downregulation of Zdhhc6 in the murine renal tissue of AKI. The expression of DHHC6 was reduced in tubular cells of AKI mice and patients. 2. To elucidate the role of DHHC6 in tubular cell survival and AKI, we generated DHHC6-deleted tubular cells mice by Cre/Loxp system and DHHC6 overexpressing mice by renal in situ injection of adeno-associated virus. We found that DHHC6 deficiency could exacerbate IRI induced renal injury, while overexpression of DHHC6 exerted renal protective effects. DHHC6 deficiency in vitro promoted tubular cell mitochondrial dysfunction and cell death, and significantly downregulated SDHB abundance. SDHB knockdown also promoted tubular cell death. 3. ABE assays showed that SDHB could be palmitoylated, and DHHC6 deficiency reduced, while DHHC6 overexpression elevated the palmitoylation level of SDHB. Immunoprecipitation and immunofluorescent analyses revealed the interaction of DHHC6 and SDHB. Furthermore, DHHC6 deficiency promoted the degradation of SDHB and reduced the activity of mitochondrial complex II. Based on the proteomics of palmitoylation modification and CSS/GPS-palm prediction results, the Cys115 site of SDHB was identified as its palmitoylation modification site. The palmitoylation of Cys115 was also associated with SDHB degradation and tubular cell death. Conclusion: In summary, this study revealed that the palmitoyltransferase DHHC6 protects against UNx+IRI induced AKI and tubular cell death by catalyzing SDHB palmitoylation. Funding: Government Support – Non-U.S.