Elucidation of mechanism of acute renal failure by use molecular biological techniques

我们以前已经证明，ET-1主要在肾小球（Glm）和内髓集合管（IMCD）合成。在这项研究中，微定位的mRNA编码的A型和B型ET受体进行了在大鼠肾脏中使用的逆转录和聚合酶链反应（RT-PCR）分析的个别microdisserted肾小管段沿着肾单位，肾小球，直血管束，弓形动脉。B型受体PCR产物在起始和终末的内髓集合管、肾小球中检测到大信号，而在皮质集合管、外髓集合管、直血管束和弓状动脉中检测到小信号。相反，A型受体mRNA仅在肾小球、直小血管束和弓状动脉中检测到。因此，两种ET受体亚型沿肾单位沿着分布不同。目前尚不清楚它们在急性肾衰竭中是如何调节的。因此，我们采用点突变竞争性模板RT-PCR技术，对SD大鼠IMCD的Glm中ET-1、ET-AR和ET-BR的mRNA进行了定量分析。分别于再灌注后3、6、12、24、36、48 h取左肾行显微解剖。血浆ET-1、肌酐和BUN水平在缺血后3- 48小时升高。缺血3 h后ET-1 mRNA水平明显升高，12 h时Glm和IMCD中ET-1 mRNA水平分别为对照组的14.4倍和7.8倍。Glm和IMCD组ET-BR mRNA表达也从缺血6 h开始升高，并持续到缺血48 h，而Glm组ET-AR mRNA表达在缺血12 h下降到30%。这些结果表明，在Glm和IMCD中ET-1的产生增加，并且ET-AR和ET-BR mRNA在肾缺血期间受到不同的调节。

We have previously demonstrated that ET-1 is synthesized mainly in the glomerulus (Glm) and inner medullary collecting duct (IMCD). In this study, micro-localization of mRNA coding for A-type and B-type ET receptors was carried out in the rat kidney using a reverse transcription and polymerase chain reaction (RT-PCR) assay of individual microdissected renal tubule segments along the nephron, glomeruli, vasa recta bundle, and arcuate arteries. Large signals for B-type receptor PCR product were detected in the initial and terminal inner medullary collecting duct, and glomerulus, while small signals were found in the cortical collecting duct and outer medullary collecting duct, vasa recta bundle, and arcuate artery. In contrast, A-type receptor mRNA was detected only in the glomerulus, vasa recta bundle, and arcuate artery. Thus, the two ET receptors subtypes are distributed differently along the nephron. It is not known how they are regulated in acute renal failure. Therefore, we employed RT-PCR with point-mutated competitive templates for quantitative analysis of ET-1, ET-AR,and ET-BR mRNAs from isolated Glm of IMCD.The left renal artery was clamped for 45 min in SD rats. The left kidney was then microdissected 3,6,12,24,36, or 48h after reperfusion. The plasma ET-1, creatinine, and BUN levels were increased from 3-48h post ischemia. The levels of ET-1 mRNA dramatically increased from 3h, reaching maximum levels of 14.4 fold in Glm and 7.8 fold in IMCD at 12h post-ischemia. The level of ET-BR mRNA also increased from 6h and was sustained a high level until 48h in Glm and IMCD.On the other hand, ET-AR mRNA decreased to 30% at 12h after the ischemia in Glm. These results suggest that ET-1 production increases in Glm and IMCD,and ET-AR and ET-BR mRNAs are regulated differently during renal ischemia.