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Abstract：
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: Using microarray analysis, we observed that DEGs, including NOX4/H2O2, 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.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: Using microarray analysis, we observed that DEGs, including NOX4/H2O2, 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.
UNASSIGNED: 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.
摘要：
■足细胞凋亡是驱动Alport综合征（AS）进展的常见机制。本研究旨在探讨COL4A3突变引起足细胞凋亡的机制。
■我们招募了常染色体显性遗传AS（ADAS）患者。招募具有微小改变疾病(MCD)的患者作为对照。对来自患者的分离的肾小球进行微阵列分析并验证。然后，相应的突变人足细胞(p.C1616Y）和129只小鼠（p。C1615Y,构建了与人类p.C1616Y）的鼠同源物。在施用MMP-2抑制剂（SB-3CT）和NOX4抑制剂（GKT137831）之前和之后，在转基因小鼠和足细胞中验证了来自微阵列分析的最高差异表达基因（DEGs）。我们通过实时聚合酶链反应（PCR）进一步验证了NOX4/MMP-2/凋亡途径，免疫组织化学，和来自ADAS患者的肾组织的蛋白质印迹。
■使用微阵列分析，我们观察到DEG,包括NOX4/H2O2、MMP-2和足细胞凋亡相关基因均显著上调。这些基因通过实时PCR验证，组织学分析,和相应突变人足细胞的蛋白质印迹（p。C1616Y）和/或小鼠模型（p。C1615Y)。此外，我们发现，在体内和体外，通过抑制NOX4，足细胞凋亡被消除，MMP-2表达下调，尿白蛋白与肌酐比值，24小时蛋白尿；体内NOX4抑制减轻了肾脏病理损害。此外，通过体内和体外抑制MMP-2，足细胞凋亡减弱，而NOX4表达保持不变。
■这些结果表明，NOX4可能通过调节MMP-2诱导COL4A3突变患者足细胞凋亡。我们的发现为ADAS的机制提供了新的见解。
■我们招募了常染色体显性遗传AS（ADAS）患者。招募具有微小改变疾病(MCD)的患者作为对照。对来自患者的分离的肾小球进行微阵列分析并验证。然后，相应的突变人足细胞(p.C1616Y）和129只小鼠（p。C1615Y,构建了与人类p.C1616Y）的鼠同源物。在施用MMP-2抑制剂（SB-3CT）和NOX4抑制剂（GKT137831）之前和之后，在转基因小鼠和足细胞中验证了来自微阵列分析的最高差异表达基因（DEGs）。我们通过实时聚合酶链反应（PCR）进一步验证了NOX4/MMP-2/凋亡途径，免疫组织化学，和来自ADAS患者的肾组织的蛋白质印迹。
■使用微阵列分析，我们观察到DEG,包括NOX4/H2O2、MMP-2和足细胞凋亡相关基因均显著上调。这些基因通过实时PCR验证，组织学分析,和相应突变人足细胞的蛋白质印迹（p。C1616Y）和/或小鼠模型（p。C1615Y)。此外，我们发现，在体内和体外，通过抑制NOX4，足细胞凋亡被消除，MMP-2表达下调，尿白蛋白与肌酐比值，24小时蛋白尿；体内NOX4抑制减轻了肾脏病理损害。此外，通过体内和体外抑制MMP-2，足细胞凋亡减弱，而NOX4表达保持不变。
■这些结果表明，NOX4可能通过调节MMP-2诱导COL4A3突变患者足细胞凋亡。我们的发现为ADAS的机制提供了新的见解。
