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

利用分子生物学技术阐明急性肾衰竭的机制

基本信息

批准号：
04454234
负责人：
TOMITA Kimio
金额：
$ 4.29万
依托单位：
Kumamoto University (1994)Tokyo Medical and Dental University (1992-1993)
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04454234/
关键词：
Acute renal failure Endothelin Endothelin receptor glomerulus tubule collecting duct vessel cGMP 水,Na アンギオテンシンII 腎糸球体腎尿細管腎血管微小尿細管分離法

项目摘要

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.

我们以前已经证明，ET-1主要在肾小球（GLM）和内部髓质收集管（IMCD）中合成。在这项研究中，使用逆转录和聚合酶链反应（RT-PCR）分析对A型和B型ET受体编码的MRNA进行了微定位，该肾小管沿肾小管沿肾小管，glomeruli，glomeruli，vasa recta recta bundle and arcuite arcuite arcuite arcuite arcuite arcuite arcuite arcuye arcuyeies a in early肾小管链接进行了逆转录和聚合酶链反应（RT-PCR）测定。在初始和末端的髓质收集导管和肾小球中检测到B型受体PCR产物的大信号，而在皮质收集导管和髓外收集管，Vasa Recta束和弧形动脉中发现了小信号。相比之下，仅在肾小球，VASA Recta束和弧形动脉中检测到A型受体mRNA。因此，两个ET受体亚型沿肾单位分布不同。尚不清楚如何在急性肾衰竭中调节它们。因此，我们利用rt-PCR使用点突变的竞争模板来对IMCD分离的GLM的ET-1，ET-AR和ET-BR mRNA进行定量分析。在SD大鼠中将左肾动脉夹住45分钟。然后将左肾脏重新解释为3,6,12,24,36，或再灌注后48小时。血浆ET-1，肌酐和BUN水平从缺血后的3-48h增加。 ET-1 mRNA的水平从3H急剧增加，在第12小时，ESCD的最大水平达到14.4倍，IMCD的水平为7.8倍。 ET-BR mRNA的水平也从6H升高，并且在GLM和IMCD中保持高水平，另一方面，ET-AR mRNA在GLM缺血后12H下降到30％。这些结果表明，在肾脏缺血期间，ET-1产生增加了GLM和IMCD，ET-AR和ET-BR mRNA受不同的调节。