Inhibition of Sphingosine-1-Phosphate Receptor 4 (S1PR4) Improves Kidney Outcomes in a Mouse Model of Alport Syndrome via Inducing Lipophagy

Background: Alport syndrome is a hereditary glomerulopathy caused by mutations in collagen 4, a key component of the glomerular basement membrane. An experimental mouse model of Alport syndrome (Col4a3 -/-) develops glomerular disease associated with renal neutral lipid accumulation and rapidly progress to renal failure which can be temporarily prevented by lipid-lowering agents. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that exerts its effects via its GPCRs (S1PR1-5). A group of mutations in S1P lyase have been identified in a group of patients with nephrotic syndrome. However, the role of S1P signaling in podocytes and its therapeutic potential in glomerular disease is not well understood. Methods: S1P levels were measured in kidney cortices of 8-week-old mice by LC-MS and normalized to protein concentration. In vitro S1PR4 antagonism was achieved by serum starvation for 24 hours followed by treatment with CYM50358 for 24 hours. Lipid droplets and lysosomes were stained by Nile Red and LysoTracker staining and imaged on Opera Phenix HCS system. Apoptosis was determined by ApoTox-Glo™ Assay (Promega). CYM50358 (10mg/kg) was administered to Col4a3 -/- by intraperitoneal injection starting at 4 weeks of age and until sacrifice at 8 weeks of age. Plasma and urine samples were collected at time of sacrifice and used to determine albumin to creatinine ratio, blood urea nitrogen, and plasma creatinine. Results: Kidney cortices from Col4a3 -/- mice have increased levels of S1P, S1PR4, and neutral lipid accumulation. Immortalized podocytes isolated from Col4a3 -/- mice have increased S1PR4, apoptosis, and neutral lipid accumulation in the form of lipid droplets. Antagonism of S1PR4 results in a reduction of apoptosis and a reduction of neutral lipids. Treatment with a S1PR4 antagonist is sufficient to reduce albumin to creatinine ratio, blood urea nitrogen, plasma creatinine, fibrosis, glomerulosclerosis and neutral lipid accumulation. We see an increase in lysosome number, increase in LC3II/I, and decrease in P62 suggesting that autophagy is being induced. Conclusion: Our results suggest that inhibition of S1PR4 may be beneficial in preventing glomerular disease progression in a mouse model of alport syndrome, possibly by reducing neutral lipid accumulation via lipophagy. Funding: NIDDK Support, Other U.S. Government Support