Novel Role for Sphingomyelin Phosphodiesterase Acid-like 3b in Radiation Nephropathy

Abstract

Background: Radiation nephropathy remains a challenging complication for cancer patients who receive abdominal or genitourinary radiation therapy (RT). The kidneys are highly radiosensitive organs that impose an ablative dose limitation. Sphingolipids are key bioactive lipids that cross-talk with DNA damage response (DDR) effectors to determine cell fate after genotoxic injuries. The specific expression of sphingomyelin phosphodiesterase acid-like 3b (SMPDL3b) in podocytes modulates stress signaling and ceramide-1-phosphate (C1P) levels. Prior work suggested that radiation-induced loss of SMPDL3b mediates podocyte injury through cytoskeletal remodeling, filopodia effacement, and altering sphingolipids homeostasis. However, the molecular mechanisms involved in radiation podocytopathy remain to be further explored. Aim: Our study investigates the role of SMPDL3b in regulating the DDR of renal podocytes after radiation injury. Methods: Wild-type (WT), SMPDL3b overexpressors (OE), and SMPDL3b knock-down (KD) human podocytes were used as in vitro models for this study and were exposed to a single radiation dose of 2 Gy. We assessed the kinetics of DNA double-strand breaks (DSBs) recognition and repair along with ATM pathway activation post-irradiation. We also assessed the extent of DNA damage repair and apoptosis in an in vivo model using C57BL6 WT and podocyte-specific SMPDL3b-knock out (KO) mice at 24 hours after a single 14 Gy dose of focal renal irradiation. Additionally, we examined the effect of SMPDL3b expression on nuclear sphingolipids and nuclear membrane fluidity along with their impact on the DDR. Results: SMPDL3b overexpression enhanced DSBs recognition and repair through the modulation of ATM nuclear shuttling and pathway activation. On the other hand, a significant delay in DSBs repair was observed when SMPDL3b was knocked down in vitro and in vivo. Ionizing Radiation (IR) altered the expression and subcellular localization of SMPDL3b from the lipid rafts of plasma membranes to the perinuclear and nuclear regions. Moreover, the expression of SMPDL3b regulated the nuclear membrane fluidity by altering its sphingomyelin content. Furthermore, SMPDL3b overexpression prevented radiation-induced alterations in the nuclear levels of C1P and ceramide. Exogenous administration of C1P radiosensitized OE podocytes by delaying ATM foci formation and activity, and subsequent DSBs repair. Conversely, pretreatment with ceramide kinase inhibitor (CERKI) radioprotected WT podocytes by enhancing ATM foci formation and activity, DSBs repair, and cell survival. These results suggest potential roles for the SMPDL3b and CERK/C1P axes in modulating radiation-induced podocyte injury. Conclusion: We suggest that SMPDL3b regulates nuclear membrane fluidity, nuclear sphingolipids, ATM nuclear shuttling and pathway activation, DSBs repair, and consequently podocytes survival. The current work unmasks novel roles for SMPDL3b and C1P in radiation-induced DDR and paves the way towards further investigations on promising, novel therapeutic targets that may prevent radiation nephropathy.