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

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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.