2790 - Novel Role for SMPDL3b in Radiation Nephropathy

M. S. Francis^1,2, A. Ahmad³, L. Bodgi⁴, A. Fornoni⁵, B. Marples⁶, and Y. Zeidan^1,7; ¹American University of Beirut, Beirut, Lebanon, ²Department of Radiation Oncology, Stanford University, School of Medicine, Stanford, CA, ³Department of Radiation Oncology, University of Miami/Sylvester Comprehensive Cancer Center, Miami, FL, Miami, FL, ⁴American University of Beirut Medical Center, Beirut, Lebanon, ⁵University of Miami, Miami, FL, ⁶Department of Radiation Oncology, University of Rochester, Rochester, NY, ⁷Lynn Cancer Institute, Baptist Health South Florida, Boca Raton, FL

Purpose/Objective(s): Radiation nephropathy (RN) remains a challenging complication for patients who receive radiation therapy (RT) targeting abdominal or pelvic tumors. Prior work showed that RT-induced loss of sphingomyelin phosphodiesterase acid-like 3b (SMPDL3b) mediates RN by delaying DNA damage repair in podocytes. The current study investigates the interplay between SMPDL3b and ceramide kinase (CERK)/ceramide-1-phosphate (C1P) in the DNA damage response of renal podocytes. Materials/

Methods: RT-induced changes in nuclear sphingolipids and nuclear membrane fluidity were assessed in wild-type (WT) and SMPDL3b overexpressing (OE) human podocytes after an X-ray dose of 2 Gy. The kinetics of DNA double-strand breaks (DSBs) recognition and repair were assessed in WT cells by quantifying ?-H2AX and p-ATM nuclear foci. Pharmacological inhibition of CERK was performed by administering a series of concentrations of the CERK inhibitor (CERKI), NVP-231, 6 hrs pre-RT. Alternatively, CERK silencing was done using 30 nM siRNA for 48 hrs pre-RT. These parameters were also assessed in radioresistant OE cells with and without exogenous C1P treatment (10 µM) 1 hr pre-RT. The effect of C1P on the enzymatic activity of ATM kinase was further explored using purified recombinant human ATM protein kinase and p53 protein substrate at various C1P concentrations.
Results: SMPDL3b overexpression abrogated RT-induced alterations in the nuclear levels of C1P and ceramide, downregulated the basal levels of nuclear sphingomyelin, and increased nuclear membrane fluidity. CERKI-treated WT cells (50 nM) scored significantly higher numbers of g-H2AX (93±2 vs. 83±3, p<0.001) and p-ATM (87±2 vs. 75±3, p<0.0001) nuclear foci at 10 min, and lower numbers of residual g-H2AX foci (7±0.6 vs. 12.0±0.3, p<0.0001) at 24 hrs post-RT. Moreover, CERKI treatment significantly mitigated RT-induced cytotoxicity to 8±1% compared to non-treated cells (42±2%, p<0.0001). Notably, CERK silencing yielded comparable results to CERKI treatment. On the other hand, the exogenous administration of C1P radiosensitized OE cells by increasing cytotoxicity to 38±2% post-RT compared to non-treated cells (8±1%, p<0.0001). C1P treatment delayed DSBs recognition (75±1 vs. 94±4, p=0.0001) and repair (10±0 vs. 6.0±1.7, p<0.05) through delayed ATM foci formation (57±2 vs. 88±1, p<0.0001). Moreover, a 10 µM concentration of C1P induced around 40% decrease in ATM kinase activity, noted by a significant reduction in p53 phosphorylation.
Conclusion: Our results suggest that the SMPDL3b/ C1P signaling modulates radiation podocytopathy by regulating nuclear sphingolipids and membrane fluidity. Such changes alter ATM nuclear shuttling, ATM activation, and DSBs repair. The current work unmasks a novel role for SMPDL3b/ C1P in radioprotecting normal renal tissues.