The Role of SMPDL3b in Regulating Radiation Nephropathy

Abstract

Radiation nephropathy (RN) is functionally characterized by an initial acute phase (proteinuria) followed by a chronic decline of glomerular filtration rate leading to renal failure. RN is irreversible and affects the quality of life of both short- and long-term cancer survivors. The molecular mechanisms responsible for RN remain largely unknown. The current study investigates the role of sphingolipids in radiation-induced podocytopathy and RN. Sphingomyelin phosphodiesterase acid-like 3B (SMPDL3b) expression was assessed in cultured podocytes after X-irradiation (1-8 Gy) by real-time PCR and Western blotting. Changes in podocyte morphology and radiation-induced DNA damage repair were assessed in wild-type, SMPDL3b-KO and SMPDL3b-over-expressing podocytes. Wild-type C57BL/6 male and female mice (10–14 weeks) were X-irradiated using CT image-guidance (SD 14 Gy, fractionated 24x2 Gy; and 4 Gy SD + 6 mg/kg cisplatin). 50 mg/kg Rituximab (or IgG) was administered IP 30 min before irradiation to protect SMPDL3b. Functional kidney parameters (proteinuria and albumin/creatinine urine ratio, along with serum BUN [blood urea nitrogen], creatinine and hematocrit) were analyzed 10–40 weeks post treatment. Kidney histology, podocyte number and SMPDL3b levels were assessed by immunohistochemistry and Western blotting. Following irradiation, SMPDL3b expression was significantly reduced in vitro and in vivo . In irradiated kidneys, podocyte number decreased significantly and H&E kidney sections demonstrated a multifocal increase in the number of pericytes, tubular atrophy and glomerular damage. Transmission electron microscopy indicated radiation-mediated changes to glomerular base membrane thickness and podocyte foot process effacement. Periodic Acid-Schiff (PAS) staining demonstrated increases in glomerular mesangial matrix accumulation post-irradiation, along with diffuse intertubular fibrosis as determined by Sirius red staining, especially in the renal cortex. Rituximab improved kidney functional parameters, vascular structure, normalization of pericyte coverage and suppressed the development of fibrosis and tubular damage post irradiation. SMPDL3b expression was identified as a molecular determinant of podocyte injury after single dose and fractionated X-irradiation. Rituximab pretreatment protected mice against radiation-induced nephrotoxicity, which may have therapeutic implications for radiation-induced injuries in cancer patients.