QUANTIFICATION OF MESSENGER-RNA LEVELS OF ENDOTHELIN RECEPTOR SUBTYPES AND PREPROENDOTHELIN-1 IN RENAL NEEDLE BIOPSIES BY COMPETITIVE REVERSE-TRANSCRIPTASE POLYMERASE-CHAIN-REACTION

The vasoconstrictive peptide endothelin-1 (ET-1) is an autocrine/parac rine peptide of putative pathophysiological importance in renal transp lant medicine. The aim of the present study was to develop a method fo r analysis of gene expression of the renal endothelin system in humans . Only small amounts of tissue are available from renal cortical needl e biopsies. Thus, in the present study we developed a quantitative ass ay based on the competitive reverse transcriptase polymerase chain rea ction CRT-PCR) technology. We quantified endothelin A (ETA) and B (ETB ) receptor subtype mRNAs and preproET-1 mRNA levels in renal cortex bi opsies obtained before nephrectomy of healthy kidney donors. Mean (+/- SEM) mRNA. levels of the ETA and ETB receptor subtypes in 26 living d onors were 212+/-23 and 368+/-56 amol/mu g total RNA, respectively. Th e preproET-1 mRNA level in 19 living donors was 213+/-28 amol/mu g tot al RNA. The inter-assay coefficient of variation (CV) for the assay wa s 10%; the intra-assay CV was 6-13%. The competitive RT-PCR assay desc ribed provides an accurate tool for gene expression investigation of t he human endothelin system in renal cortical needle biopsies.