PRDM16 Alleviates Tubular Injury in Diabetic Nephropathy by Promoting Lipophagy via the Activation of UCP1 Gene Transcription

Background: Abnormal lipid deposition and dysregulated lipophagy are important characteristics of diabetic nephropathy (DN). And PR domain containing protein 16 (PRDM16) plays a crucial role in lipid metabolism. However, the role of PRDM16 associated with lipid in DN is unclear. Methods: Bioinformatics analyzing the PRDM16 expression in DN and its biological function. Mice with tubule epithelial cell-specific overexpression of PRDM16-tdTomato (PRDM16Tg/+) mice were generated. And we assessed the injury of kidneys via histopathological staining, Oil red O staining and Western blot (WB) analyses etc. Lipophagy in the human proximal tubular cell line (HK-2 cells) was detected by boron-dipyrromethene, monodansylcadaverine and lysosomes trichrome staining. The dual-luciferase reporter assays and chromatin immunoprecipitation binding polymerase chain reaction (ChIP-PCR) were used to identify the effect and binding region of PRDM16 to the UCP1 promoter. Results: Bioinformatics analysis showed that PRDM16 was mainly expressed in proximal tubular epithelial cells, and was descreased in DN groups. Its molecular function was primarily concentrated in lipid metabolism, lipid oxidation, and autophagy pathways. PRDM16Tg/+ mice exhibited alleviated renal pathological injury, and accompanied by increased lipophagy, decreased oxidative stress, apoptosis and fibrosis in renal tubular cells. Expression of fibronectin (FN), Kim-1, and c-Caspase3 was downregulated, while LC3 and Beclin1 expression was upregulated. These similar results were found in cells with transfection of PRDM16 overexpression plasmids. Furthermore，the protein levels of autophagy-related indicators LC3 and Beclin1 were increased in lipid droplet proteins, and enhanced the fluorescence colocalization regions of lipid droplets and autophagy indicators, indicating that PRDM16 promotes lipophagy in HK-2 cells. However, these protective effects of PRDM16 were diminished by UCP1 siRNA intervention. Dual-luciferase reporter assays showed that PRDM16 transcriptionally activated UCP1 promoter activity, and ChIP-PCR indicated that PRDM16 may bind to the UCP1 promoter R2 motif (-1250 bp to -1001 bp). Conclusion: PRDM16 facilitates lipophagy by transcriptionally activating UCP1 gene expression, thereby ameliorating lipid deposition and lipid-related kidney injury in DN. Funding: Other NIH Support - NSFC Support