Quantitative RT-PCR analysis of mRNAs encoding a colonic putative H,K-ATPase α subunit along the rat nephron: Effect of K⁺ depletion

Abstract

The rat nephron displays two ouabain-sensitive K-ATPases: one, which is present in proximal tubules and thick ascending limbs of normal rats, is specifically activated by K⁺ and is down-regulated by K⁺ depletion, whereas the other one appears in collecting ducts of K⁺-depleted rats and is activated by either Na⁺ or K⁺. To determine which of these two ATPases is similar to colonic-type H,K-ATPase, we quantitated by reverse transcriptase-polymerase chain reaction (RT-PCR) the mRNAs encoding the colonic H,K-ATPase α subunit in microdissected nephron segments. In normal rats, statistically significant amounts of colonic H,K-ATPase mRNAs were detected exclusively in cortical thick ascending limbs and cortical collecting ducts (200–500 copies/mm). Because these levels of expression were low (1–1.2 copies/target cell), they probably have no physiological relevance. In rats fed a K⁺-depleted diet for 2 weeks, expression of colonic H,K-ATPase was markedly enhanced in cortical and medullary collecting ducts (5000–12,000 copies/mm or 30–40 copies per cell), whereas it remained low in all other nephron segments. Thus, colonic H,K-ATPase α subunit is specifically expressed in cortical and outer medullary collecting ducts of K⁺-depleted rats where it likely accounts for the ouabain-sensitive K-ATPase activity.