Sirtuin-3 Deletion in the Distal Nephron Protects Against Ischemic AKI

Background: The kidney is highly enriched in mitochondria, and mitochondrial dysfunction is a central contributor to AKI pathogenesis. Sirtuin-3 (SIRT3), a mitochondrial deacetylase, plays a critical role in preserving mitochondrial integrity. SIRT3 has been shown to protect against toxic AKI. However, the segment-specific functions of SIRT3 in the nephron following AKI remain poorly understood. We hypothesized that deletion of SIRT3 in both proximal and distal tubules would exacerbate ischemic AKI. Methods: Transgenic mice: whole nephron SIRT3 deletion (Pax8-rtTA/Tet-O-Cre/SIRT3fl/fl – SIRT3 iKKO), proximal tubule deletion (PepCK-Cre/SIRT3fl/fl – SIRT3 PTKO), and distal tubule deletion (Ksp-Cre/SIRT3fl/fl – SIRT3 DTKO) and with mitochondrial reporter (Ksp-Cre/SIRT3fl/fl/MITO-Tag - SIRT3 DTKOMITO). Model: unilateral ischemia/reperfusion (I/R) with contralateral nephrectomy harvested at 24 hours. Primary outcome: serum creatinine. Secondary outcomes: serum BUN, kidney histologic injury scoring, and kidney injury markers. Exploratory analysis: mRNA and protein levels of SIRT3 in whole kidneys of transgenic mice, single-cell RNA sequencing (scRNA-seq) of kidneys from wildtype mice following I/R. Statistics: 2 groups – unpaired T test; 2 groups with independent variables - two-way ANOVA with Bonferroni post-hoc. Results: Neither SIRT3 iKKO nor SIRT3 PTKO displayed worsened acute ischemic AKI compared to littermate counterparts. However, we noted a significant decrease in SIRT3 expression in the kidneys of mice following ischemic AKI. scRNA-seq revealed that SIRT3 expression is highest at baseline in proximal tubule segments but decreases following I/R. In contrast, in medullary distal tubular segments chronically exposed to hypoxic environment, SIRT3 expression is lower at baseline and increases after I/R injury. Interestingly, compared to littermates, SIRT3 DTKO mice displayed improved ischemic AKI. TUNEL staining revealed fewer TUNEL-positive cells in SIRT3 DTKO kidneys. Sorted distal tubule cells from SIRT3 DTKOMITO mice showed reduced expression of ferroptosis-related genes compared to littermates. Conclusion: Unexpectedly, SIRT3 deletion in kidney distal tubule cells reduces ferroptosis-mediated cell death and attenuates ischemic AKI, findings that could have implications for ischemic preconditioning. Funding: NIDDK Support - NIDDK Support, NIDDK Support