EPO激活PI3K/Akt信号通路调控梗阻性肾病AQP2表达机制的实验研究

Congenital hydronephrosis due to partial ureteral obstruction （PUO）is common and one reason of terminal kidney disease in children.It is reported that downregulation of the renal waterchannel aquaporin 2 (AQP2) play a role in the water transport in renal collecting ducts principal cells.The downregulation of renal AQP2 remains after release of obstruction, and it may lead to severe water-electrolyte imbalance in the cases with bilateral ureteral obstruction. Accumulating evidence suggests that EPO has organ protective effects，such as protecting the brain and kidneys from injury. Recently, we found that administration of EPO attenuates, at least partially, the downregulation of AQP2 in the obstructive kidney and promote recovery of downregulated AQP2 in the kidney immediately post releasing the ureteral obstruction. However, the underlying mechanisms for the downregulation of AQP2 is unknown. It has been reported that PI3K/Akt pathways mediate the AQP2 expression in the plasma membranemouse in the mouse kidney cortical collecting duct cell line (M-1 cells and mpkCCDc14 cells) in the literature.We hypothesized that EPO prevents the downregulation of renal AQP2 in rat with ureteral obstruction induced neonatelly by activation of the PI3K/Akt pathway which increase transcription or/and trafficking of AQP2 to the plasma membrane. Therefore, the purpose of the present study is to study whether EPO has protective effect on the obstructed kidney in newborn rat and investigate the underlying mechanisms involved in prevention of AQP2 downregulation in both the obstructed kidney and in the kidney post releasing of the obstruction. Immunochemistry, radioimmunoassay, western blot, RT-PCR, electron microscope and laser confocal microscope will be used in this study (Either in animal model and renal collecting cell line)to detect the change in AQP2 expression and trafficking and the underlying mechanisms for the regulation of AQP2. This study might open a new research area for finding new treatment strategies to prevent AQP2 downregulation and protect renal function in children with congenital hydronephrosis in the future.

先天性输尿管不全梗阻性肾积水是儿童终末肾病原因之一。研究显示梗阻肾脏水通道蛋白2（AQP2）表达下调与梗阻肾脏尿液浓缩功能受损有关。梗阻解除后肾脏已经下调的AQP2不能马上恢复。如何预防梗阻肾脏AQP2下调和促使解除梗阻肾脏下调的AQP2恢复是临床有待解决的问题。本课题组前期研究发现促红细胞生成素（EPO）可以有效抑制幼鼠梗阻肾脏AQP2下调和促使解除梗阻的肾脏AQP2恢复。文献报道三磷酸酰肌醇蛋白激酶/蛋白激酶B（PI3K/Akt）信号通路能调控鼠集合管细胞系AQP2合成和向细胞膜转运。EPO是否通过该信号通路调节梗阻肾脏AQP2表达有待进一步研究。本研究拟采用免疫组化、RT-PCR、激光共聚焦显微镜和电镜等技术，在新生鼠和幼鼠梗阻肾脏模型研究EPO调控AQP2的转运过程，并在体外细胞中进一步验证EPO调控AQP2表达的机制。为进一步研究梗阻性肾病EPO干预AQP2调控机制提供参考。

尿路梗阻是终末型肾衰竭的一个重要原因。本研究探讨了大鼠单双侧双尿管梗阻及解除梗阻后4W内大鼠肾脏的功能及肾脏水通道蛋白和Na/K-ATPase的变化。结果显示单侧输尿管梗阻48h及双侧输尿管梗阻24h显著影响肾脏水通道蛋白（AQPs）和Na/K-ATPase的表达，在梗阻解除后的4W内肾脏浓缩功能和钠的重吸收功能并不能完全恢复。促红细胞生成素（EPO）临床已用于新生儿缺血缺氧性脑病的研究，对肾性贫血患者也有较好的疗效。大量的动物研究发现EPO除了促造血功能，还有抗炎抗纤维化等作用。在本研究中我们在输尿管梗阻解除时用EPO对其进行不同时间的干预，用药时间分别为解除梗阻后1W、2W、3W和4W，发现EPO能够促进梗阻解除后肾脏功能和肾脏水通道蛋白和Na/K-ATPase表达的恢复。肾脏水通道蛋白在肾脏浓缩功能方面发挥重要的作用，Na/K-ATPase可以为肾脏水的重吸收提供动力，所以EPO对AQPs和Na/K-ATPase的保护作用也是其发挥肾脏保护作用的机制之一。体外实验，我们用EPO对肾脏集合管细胞进行干预，发现EPO能够通过PI3K、AKT信号通路上调AQP2的表达。肾脏纤维化也是许多肾脏疾病发展过程中一个常见的病理变化，在本研究中我们也对梗阻解除后肾脏纤维化的变化进行了相关检测，发现梗阻解除后短时间内（3d）肾脏纤维化并不明显，随着时间的延长，梗阻解除后2W内纤维化逐渐加重。我们用EPO对梗阻大鼠进行干预，发现EPO能够促进梗阻解除后肾脏纤维化的恢复。我们对人胎儿发育时期肾脏水通道蛋白的表达规律进行了研究，发现随孕周的增加，胎儿肾脏AQP1-4的表达也逐渐增加。同时我们收集不同孕期的羊水，并对其中的AQP2进行检测，发现羊水中的AQP2的表达与胎儿肾脏中AQP2的表达具有良好的相关性。在该基金的资助下，完成和在国际国内杂志发表相关论文和著作18篇。