Segmental localization of mRNAs encoding Na+-K+-ATPase α-and β-subunit isoforms in rat kidney using RT-PCR

Abstract

To characterize the expression of genes encoding the α- and β-subunit isoforms of the Na+-K+-ATPase in rat kidney, we used reverse transcription (RT)-PCR of microdissected renal structures combined with quantitation of subunit isoform mRNAs in the major renal parenchymal zones. Transcripts for α1, α2, α3, β1, and β2 subunit isoforms were detected by RT-PCR in microdissected glomeruli, proximal convoluted tubules, medullary thick ascending limbs of Henle, cortical and inner medullary collecting ducts. The truncated α1 (α1-T) isoform was also amplified from cortex, outer and inner medulla and isolated glomeruli, but it was not detected in these nephron segments. The DNA sequence of the renal α1-T PCR product was identical to that of the cDNA previously cloned from aortic smooth muscle cells. RNA dot-blot analysis indicated that the α1, α2, and α3 isoforms contributed ∼70%, ∼20%, and ∼10%, respectively, of the total α isoform mRNA in each parenchymal zone. RNase protection assays determined that the β1 and β2 isoforms accounted for ∼95% and ∼5%, respectively, of the βisoform mRNA in each zone. These data provide definitive evidence for the differential expression of mRNAs encoding all the α and β isoforms in the renal parenchyma, and for the coexpression of these isoforms in the nephron segments examined. The results suggest the potential expression of up to eight different Na+-K+-ATPase isoenzymes in the kidney, and for multiple molecular levels of regulation of renal Na+-K+-ATPase expression.