COL4A3 Mutation Induced Podocyte Apoptosis by Dysregulation of NADPH Oxidase 4 and MMP-2

INTRODUCTION: Podocyte apoptosis is a common mechanism driving progression in Alport syndrome (AS). This study aimed to investigate the mechanism of podocyte apoptosis caused by COL4A3 mutations. METHODS: We recruited patients with autosomal dominant AS (ADAS). Patients with minimal change disease (MCD) were recruited as controls. Microarray analysis was carried out on isolated glomeruli from the patients and validated. Then, corresponding mutant human podocytes (p.C1616Y) and 129 mice (p.C1615Y, the murine homolog to the human p.C1616Y) were constructed. The highest differentially expressed genes (DEGs) from microarray analysis were validated in transgenic mice and podocytes before and after administration of MMP-2 inhibitor (SB-3CT) and NOX4 inhibitor (GKT137831). We further validated NOX4/MMP-2/apoptosis pathway by real-time polymerase chain reaction (PCR), immunohistochemistry, and western blot in renal tissues from the ADAS patients. RESULTS: Using microarray analysis, we observed that DEGs, including NOX4/H(2)O(2,) MMP-2, and podocyte apoptosis-related genes were significantly upregulated. These genes were validated by real-time PCR, histologic analysis, and western blot in corresponding mutant human podocyte (p.C1616Y) and/or mice models (p.C1615Y). Moreover, we found podocyte apoptosis was abrogated and MMP-2 expression was down-regulated both in vivo and in vitro by NOX4 inhibition, urinary albumin-to-creatinine ratio, 24-hour proteinuria; and renal pathologic lesion was attenuated by NOX4 inhibition in vivo. Furthermore, podocyte apoptosis was attenuated whereas NOX4 expression remained the same by inhibition of MMP-2 both in vivo and in vitro. CONCLUSION: These results indicated that NOX4 might induce podocyte apoptosis through the regulation of MMP-2 in patients with COL4A3 mutations. Our findings provided new insights into the mechanism of ADAS.