Identification and localization of renal Na(+)-Ca2+ exchanger by polymerase chain reaction

Abstract

The molecular identity of the renal Na(+)-Ca2+ exchanger was determined by a homology-based polymerase chain reaction (PCR) cloning strategy. Rat kidney RNA was amplified by PCR, using oligonucleotide primers based on regions of low degeneracy in the published canine cardiac Na(+)-Ca2+ exchanger cDNA sequence, and the products were subcloned and sequenced. A 452-bp clone (NCX1) was identified, which shares 89% nucleotide and 98% amino acid sequence identity with the canine cardiac exchanger, suggesting that they are products of the same gene. NCX1 was shown, by Northern analysis, to hybridize to an abundant major transcript of 7 kb and a minor one of approximately 14 kb both localized predominantly to kidney cortex. Microdissected tubule PCR analysis revealed that NCX1 was enriched in distal convoluted tubule compared with other cortical nephron segments. Such a location is consistent with a Na(+)-Ca2+ exchanger corresponding to NCX1 playing a major role in active Ca2+ reabsorption at this site.