Targeting VDR-sirt5-PHB2-mediated mitophagy to reverse vascular endMT and prevent chronic renal allograft dysfunction

Background: Chronic allograft dysfunction (CAD) with interstitial fibrosis/tubular atrophy (IF/TA) remains the leading barrier to long-term renal allograft survival, with no specific targeted therapies available. Endothelial-to-mesenchymal transition (EndMT) is the initiating event of allograft fibrosis, while mitochondrial homeostasis imbalance is a core trigger of EndMT. This study aimed to delineate the role and mechanism of the Vitamin D Receptor (VDR)-Sirt5-PHB2 axis in renal allograft fibrosis.
Methods: We validated target gene expression in clinical renal allograft biopsy tissues from CAD patients and normal controls. Murine allogeneic kidney transplantation models were established using wild-type and global VDR-knockout donors, with intervention of calcitriol (VDR agonist) combined with endothelial-specific Sirt5 knockdown via adeno-associated virus. Single-cell and spatial transcriptome sequencing, ChIP, Co-IP, and in vitro TNF-α-induced EndMT models were used to clarify molecular mechanisms.
Results: VDR, Sirt5 and PHB2 were significantly downregulated in CAD allografts, closely correlated with EndMT and pathological injury severity (P<0.001). Mechanistically, inflammatory stress (TNF-α) did not alter VDR transcription levels but transcriptionally upregulated the E3 ubiquitin ligase RBCK1. RBCK1 subsequently targeted VDR for proteasomal degradation via K48-linked polyubiquitination at the K302 site. Preserved VDR directly bound to the Sirt5 promoter and transcriptionally activated its expression. Sirt5 mediated desuccinylation of PHB2 at K236, which dually activated mitophagy and blocked Trim21-mediated K48-linked polyubiquitination of PHB2 at K142 to stabilize its expression. Calcitriol significantly improved allograft function, reduced interstitial fibrosis area and inhibited EndMT in wild-type mice (P<0.01), while these protective effects were completely abrogated by endothelial-specific Sirt5 knockdown.
Conclusions: This study identifies the VDR-Sirt5-PHB2 axis as a critical novel regulator of endothelial mitochondrial homeostasis and EndMT in renal allograft fibrosis. Our findings provide a rigorous mechanistic rationale for repurposing the widely used clinical agent calcitriol to prevent CAD, offering a highly translatable therapeutic strategy for improving long-term renal allograft survival.