Locau/ation and quantil ai ion of P4504A mRNA in microdissfcted nephron segments by RT-PCR

K. A. Hopp, Osamu Ito, R. J. Roman

Research output: Contribution to journalArticle

Abstract

CYP4A en/ymes catalyze the formation of 20-HKTE which plays a critical role in the regulation of renal tubular and vascular function. '1 he high degree of homology between isoforms (especially between 4A2 and 4A3 ) has limited the ability to develop specific probes to study expression of this system. In the present study, primers were designed at a unique 9 basepair region in exon 3 of the 4A3 isoform, and PC'R wa.s performed using stepdown conditions (10 cycles 70 C, 25 cycles 60 C) to distinguish the 4A3 and 4A2 products. We aiso developed a competitive RT-PCR assay tor measurement of 4A2 mRNA using an RNA mimic. 4A2 and 4Ai mRNA was detected in 5 mm lengths of proximal tubule (PT). cortical and medullary thick ascending loop of Henle (cTAL and m'FAL). cortical and medullary collecting duet (CCD and MCD) and glomeruli ((i) of male rats. 4A8 mRNA was detected in cortical ((i. PT, CCI) and cTAl.) but not in medullary nephron segments. In contrast. 4A1 mRNA wa.s not detecled in any nephron segment. I 'sing the competitive assay. 4A2 mRNA levels averaged 20.5±8.25, 3.62.78. 2.1 ±0.89. and 0.4±0.07 attomoles/ng RNA in the PT, mTAL, G and MCD. respectively. These results indicate that 4A2 and 4A3 are the major isoforms expressed in nearly every nephron segment of the kidney of male rats. Supported by Mil grants IIL36279 and HE 29587.

Original languageEnglish
JournalFASEB Journal
Volume11
Issue number9
Publication statusPublished - 1997 Dec 1

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

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