Novel Role for Sphingolipid Metabolism in Radiation Nephropathy

Abstract

The molecular mechanisms of radiation nephropathy remain largely unidentified. Sphingomyelin phosphodiesterase acid like 3b (SMPDL3b) is a lipid modulating enzyme that plays a major role in podocyte injury, and in the pathogenesis of proteinuria in glomerular diseases. The current study investigates the role of bioactive sphingolipids and SMPDL3b in radiation-induced podocytopathy, and identifies a monoclonal antibody targeting CD20, rituximab, as a radioprotector. Immortalized human podocytes were X-irradiated with 8 Gy ± rituximab (100 μg/mL) 30 minutes prior to radiation. Metabolic activity and viability were assessed. Transcriptional and translational levels of SMPDL3b were measured by RT-PCR and Western blot respectively. Cytoskeletal remodeling and morphological changes were detected using immunofluorescence microscopy. C57BL6 mice, 12-14 week-old, were subjected to bilateral kidney irradiation (14 Gy) using a CT-image guided X-irradiator and functional parameters of kidney damage measured every 10 weeks for 40 weeks (BUN, hematocrit, serum creatinine, urine albumin and creatinine ratio). Histological assessments (H&E, Picro-sirius red, and Periodic Acid Schiff), podocyte number, and SMPDL3B expression were measured in harvested kidneys post-RT. In culture, podocytes viability and metabolic activity decreased after X-irradiation. Cytoskeletal remodeling, manifesting as loss of lamellipodia and remodeling of cortical actin, was detected. Ezrin, an actin binding protein, relocated from the plasma membrane to the cytosol as early as 2h post radiation. Ceramide levels increased while sphingosine-1 phosphate (S1P) and sphingosine levels decreased. In contrast, overexpression of SMPDL3b and exogenous S1P rescued podocytes from radiation-induced cytoskeletal changes. Treatment with rituximab protected podocytes from SMPDL3b loss and caspase-3 cleavage. In irradiated animals, kidney weight and volume were reduced significantly, SMPDL3b expression and podocyte number decreased, changes that were not evident after rituximab administration. Loss of SMPDL3b expression and podocyte number were associated with reduction in functional and histological parameters. Radiation-injury induces downregulation of SMPDL3b, and elevation of ceramide levels as well as early cytoskeletal remodeling. Overexpression of SMPDL3b or pretreatment with rituximab is radioprotective in cultured podocytes and animals. These findings suggest a potential role for SMPDL3b in radiation-induced podocytopathy.