A METHOD FOR ISOLATION OF RAT RENAL MICROVESSELS AND MESSENGER-RNA LOCALIZATION

The objective of this study was to develop a technique to identify and dissect segments of the rat renal microcirculation and to apply rever se transcription (RT) to specific mRNAs with subsequent amplification of the cDNA by polymerase chain reaction (PCR) to evaluate gene expres sion. To circumvent the difficulty associated with visualizing specifi c microvessels, we intrarenally infused blue latex microparticles, 1-5 mu m in diameter, with subsequent identification and microdissection of specific segments of the renal microvasculature under stereomicrosc opy. To demonstrate its utility, we assessed expression of mRNAs encod ing fibronectin and renin. As expected, mRNA encoding fibronectin was localized along the renal microcirculation, and mRNA encoding renin wa s primarily present in afferent arterioles and interlobular arteries. Identity of the amplified cDNA fragments was verified by sequencing. T his perfusion-microdissection technique coupled to RT-PCR should be us eful in the evaluation of gene expression along the renal microvascula ture. It may also allow bridging of the gap between analysis of gene e xpression of rare mRNA species by in situ hybridization and physiology of the renal microcirculation.