Radiation-induced DNA damage in podocytes : role of SMPDL3b in ATM nuclear shuttling

Abstract

Radiotherapy (RT) is the primary, non-invasive treatment for over 50percent of cancer patients. Despite its curative potential, RT can induce damage in normal healthy tissues surrounding the tumor. The kidneys are radiosensitive organs, hence dose-limiting for RT targeting abdominal and paraspinal tumors. Excessive radiation doses to the kidneys ultimately lead to radiation nephropathy. In fact, podocytes are key players in the pathogenesis of radiation-induced proteinuria. Prior work points to a potential role for the lipid modifying enzyme, SMPDL3b, in regulating the response of podocytes to radiation injury. Aim: Our current study aims to determine the role of SMPDL3b in DNA double strand breaks (DSBs) repair after radiation injury. Methods: WT (wild-type) and OE (SMPDL3b overexpressors) immortalized human podocytes were used for this study. Differentiated cultured cells were irradiated and the treatment was stopped at selected time points. -H2AX and p-ATM nuclear foci were quantified by confocal microscopy after immunofluorescence (IF) staining. Further investigations on ATM nuclear shuttling were performed on isolated nuclear and cytoplasmic fractions through Western Blotting. Cell survival was assessed by quantifying the protein expression of p-p53 (ser15) and cleaved caspase 3. Furthermore, liquid chromatography – mass spectrometry (LC-MS) analysis was used for the assessment of radiation-induced changes in nuclear sphingolipids in both WT and OE cells. The impact of ceramide-1-phosphate (C1P) or ZOPRA pre-treatments on DSBs recognition and repair through ATM nuclear shuttling was also assessed by IF staining. Viability of irradiated WT and OE podocytes after treatments was assessed by MTT assay. Results: After assessing the kinetics of -H2AX and p-ATM nuclear foci appearance- disappearance in both cell lines, we established that SMPDL3b overexpression in podocytes enhances DSBs recognition and repair by modulating ATM nuclear shuttling. Western Blotting analysis on isolated nuclear and cy