Quantification of mRNA 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/paracrine peptide of putative pathophysiological importance in renal transplant medicine. The aim of the present study was to develop a method for analysis of gene expression of the renal endothelin system in humans. Only small amounts of tissue are available from renal cortical needle biopsies. Thus, in the present study we developed a quantitative assay based on the competitive reverse transcriptase polymerase chain reaction (RT-PCR) technology. We quantified endothelin A (ET(A)) and B (ET(B)) receptor subtype mRNAs and preproET-1 mRNA levels in renal cortex biopsies obtained before nephrectomy of healthy kidney donors. Mean (+/- SEM) mRNA levels of the ET(A) and ET(B) receptor subtypes in 26 living donors were 212 +/- 23 and 368 +/- 56 amol/microg total RNA, respectively. The preproET-1 mRNA level in 19 living donors was 213 +/- 28 amol/microg total RNA. The inter-assay coefficient of variation (CV) for the assay was 10%; the intra-assay CV was 6-13%. The competitive RT-PCR assay described provides an accurate tool for gene expression investigation of the human endothelin system in renal cortical needle biopsies.
Asberg
A
A
Institute of Surgical Research, Medical Department B, The National Hospital, University of Oslo, Norway. anderas@farmasi.uio.no
Hartmann
A
A
Attramadal
H
H
D000328:Adult; D000818:Animals; D001707:Biopsy, Needle; D019332:Endothelin-1; D016232:Endothelins / Q000235:genetics*; D005260:Female; D006801:Humans; D007668:Kidney / Q000737:chemistry* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D011498:Protein Precursors / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D044022:Receptor, Endothelin A; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D019332:Endothelin-1; D016232:Endothelins; D011498:Protein Precursors; D012333:RNA, Messenger; D044022:Receptor, Endothelin A; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin