Silencing of Acid Sphingomyelinase Gene Prevented Glomerular Oxidative Stress and Sclerosis in Hyperhomocysteinemic Mice

Hyperhomocysteinemia (hHcys) has been reported to enhance ceramide production, thereby leading to activation of NADPH oxidase and consequent glomerular oxidative stress and sclerosis. The present study was performed to determine whether acid sphingomyelinase (ASM), a ceramide producing enzyme, is implicated in the development of hHcys)‐induced glomerular oxidative stress and injury. After uninephrectomy and intra‐renal cortical transfection of vectors expressing shRNA against ASM, mice were fed a folate free (FF) diet for 8 weeks, which significantly elevated the plasma Hcys level compared to mice on normal show (ND). By in vivo molecular imaging, it was found that transfected shRNAs were expressed in the renal cortex starting on day 3 and continuing for 24 days. In isolated mouse glomeruli after 8 weeks, real‐time PCR analysis showed that ASM gene silencing significantly decreased ASM mRNA level by 46% and by 75%, respectively, when mice were on the ND and FF diet. Correspondingly, increases in glomerular NOX4 mRNA level and superoxide production measured by ESR in mice on the FF diet were substantially inhibited when ASM gene was silenced. Glomerular damages in mice with hHcys as shown by elevated urine protein excretion (0.024±0.006 mg/24h BW vs. 0.010±0.002 mg/24h BW of control) and glomerular damage index (2.95±0.05 vs. 0.87±0.03 of control) were also significantly attenuated by ASM gene silencing. Confocal microscopy demonstrated that hHcys‐induced podocytes injury as shown by reduction of podocin expression and increase of desmin production was improved by ASM gene silencing. These observations reveal that ASM importantly mediates podocyte injury and glomerular injury associated with NADPH oxidase activation and local oxidase stress during hHcys (supported by NIH grants HL‐091464, HL‐75316 and DK54927).