Targeting RNF145 in Renal Proximal Tubular Cells Protects against Lipotoxicity in Diabetes

Background: In diabetic patients, dyslipidemia is associated with both excessive deposition of triacylglycerol (TAG) in lipid droplets (LDs) and lipotoxicity. Yet, it is unclear how these two effects correlate with each other in the kidney and how they are influenced by dietary patterns. By using a diabetes mouse model, we previously found that a high fat diet enriched in the monounsaturated oleic acid (OA) caused more lipid storage in LDs in renal proximal tubular cells (PTCs), but less tubular damage than a corresponding butter diet with the saturated palmitic acid (PA). Mechanistically, endoplasmic reticulum (ER) stress was caused by elevated levels of saturated TAG precursors, reduced LD formation, and, consequently, higher membrane order in the ER (Perez-Marti et al, eLife 2022). Methods: Rnf145 knockout induced renal epithelial cells (iRECs) were generated using adenoviral CRISPR/Cas9. Cells were treated for 16 - 18 hrs with PA and subsequently analyzed for protein and mRNA expression, cell viability (crystal violet) and apoptosis (IncuCyte). Proteomic and lipidomic analyses were performed, and mitochondrial respiration and glycolysis were measured with Seahorse. Results: To identify pathways mediating PA-induced lipotoxicity, we applied transcriptomics to PA- and OA-treated induced renal epithelial cells (iRECs) and uncovered a set of genes differentially regulated by PA and OA, including the ER resident E3-ubiquitin ligase RNF145. Interestingly, RNF145 knockout protected iRECs against PA-mediated cytotoxicity and reduced ER stress, while its overexpression had opposite effects. Moreover, inhibition of desaturases in conditions of high lipid saturation rendered the Rnf145 depleted cells more sensitive than control cells, indicating that desaturases, such as SCD1, play a crucial role in Rnf145 deficient cells. Since proteomic analysis revealed changes in mitochondrial proteins and proteins involved in glycolysis and lipid metabolism, we confirmed that Rnf145-knockout cells undergo a shift from glycolysis towards mitochondrial respiration which might potentially contribute to the higher PA resistance. Conclusion: Altogether, our findings suggest RNF145 as a major regulator of lipid homeostasis and cellular metabolism, providing an interesting target for counteracting lipotoxicity in renal PTCs. Funding: Government Support – Non-U.S.