PEBP1/RAF/MEK/ERK信号通路在输尿管部分梗阻早期内质网应激中的调控机制及早期诊断肾损伤

Obstructive nephropathy is the main cause of renal failure in children and infants，and represents 16.1% of all pediatric kidney transplantation. However, there is no good method to treat these children and there are also no reliable invasive biomarkers for diagnosing renal injury at early stage at present. Our previous research revealed that podocyte-macrophage transformation initiated irreversible changes of renal injury in unilateral ureteral obstruction（UUO）. Taken phenotypic changes of podocyte as a pathological biomarker of early renal injury, we found that endoreticulum stress (ER stress) exist in the early period of renal injury in complete and partial ureteral obstruction model. In addition, PEBP1 was differently expressed in the renal tissue and urine in the early period of UUO. Some studies showed that PEBP1/Raf/MEK/ERK signal pathway was involved in the endoreticulum stress. So we inferred that PEBP1/Raf/MEK/ERK might participate in renal injury in the early period of UUO through regulating the endoreticulum stress. In this study, we will interfere in ER stress and PEBP1 expression by RANi or over expression techniques through in vivo and vitro study to reveal the role of PEBP1/Raf/MEK/ERK signal pathway in the early renal injury in obstructive nephropathy. In addition, the differentially expressed proteins related with PEBP1 pathway and ER stress in above studies will be chosen as the candidate proteins. The expression of these candidate proteins in the urine of infants with congenital hydronephrosis will be detected by multiple reaction monitoring (MRM) techniques. The value of these proteins as biomarkers for diagnosing early renal injury will be evaluated. This study would provide more theory base for the early invasive diagnosis and treatment of obstructive nephropathy.

梗阻性肾病是儿童肾脏功能衰竭的主要原因，占儿童肾移植的16.1%,目前尚无好的早期诊断和治疗方法。课题组前期以足细胞转化作为肾脏不可逆损伤病理标志，首次发现输尿管梗阻早期肾脏即处于异常的内质网应激状态，并且梗阻早期大鼠和儿童肾积水肾脏及尿液PEBP1表达异常，有研究表明PEBP1/Raf/MEK/ERK信号通路与内质网应激有关，因此推测PEBP1/Raf/MEK/ERK可能通过调控内质网应激参与输尿管梗阻早期肾损伤。本研究拟通过细胞和动物实验，构建干扰和表达载体，调控内质网应激及PEBP1信号通路关键环节，探讨PEBP1信号通路在输尿管梗阻早期肾脏损伤中的机制；同时，前瞻性大样本随访新生儿肾积水，采用MRM技术检测PEBP1信号通路中表达差异显著的蛋白质在新生儿肾积水尿液中的变化和肾积水转归的关系，评价这些蛋白质作为早期诊断梗阻肾病肾损伤的价值，为梗阻性肾病肾损伤的早期诊断和治疗奠定基础。

梗阻性肾病是儿童肾脏功能衰竭的主要原因，占儿童肾移植的16.1%,目前尚无好的早期诊断和治疗方法。课题组前期以足细胞转化作为肾脏不可逆损伤病理标志，首次发现输尿管梗阻早期肾脏即处于异常的内质网应激状态，并且梗阻早期大鼠和儿童肾积水肾脏及尿液PEBP1表达异常，有研究表明PEBP1/Raf/MEK/ERK信号通路与内质网应激有关，因此推测PEBP1/Raf/MEK/ERK可能通过调控内质网应激参与输尿管梗阻早期肾损伤。本研究通过细胞和动物实验，构建干扰载体，调控内质网应激及PEBP1信号通路关键环节，探讨PEBP1信号通路在输尿管梗阻早期肾脏损伤中的机制。结果我们发现PEBP1在大鼠肾小管上皮细胞上皮间质化过程以及输尿管部分梗阻所致早期肾损伤中起重要调控作用。ERK通路在大鼠肾小管上皮细胞间质化过程以及输尿管部分梗阻所致早期肾损伤中被激活。但PEBP1并不是单独通过PEBP1/Raf/ERK信号通路调控ERK活性。PUUO术后肾脏纤维化进展中IRE1α的上调，IRE1α特异性抑制剂STF-083010可抑制肾小管上皮细胞发生上皮间充质转化，坎地沙坦（CAN）可抑制肾小管上皮细胞发生上皮间充质转化但机制不同。通过双荧光素酶实验验证mir-615与PEBP1间不存在直接相互调控关系，通过双荧光素酶实验验证mir-142-5p与aRaf间不存在直接相互调控关系。PEBP1和IRE1α在大鼠肾小管上皮细胞上皮间质化过程以及输尿管部分梗阻所致早期肾损伤中起重要调控作用。ERK通路在大鼠肾小管上皮细胞间质化过程以及输尿管部分梗阻所致早期肾损伤中被激活。但PEBP1并不是单独通过PEBP1/Raf/ERK信号通路调控ERK活性。