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 alpha- and beta-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 alpha 1, alpha 2, alpha 3, beta 1, and beta 2 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 alpha 1 (alpha 1-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 alpha 1-T PCR product was identical to that of the cDNA previously cloned from aortic smooth muscle cells. RNA dot-blot analysis indicated that the alpha 1, alpha 2, and alpha 3 isoforms contributed approximately 70%, approximately 20%, and approximately 10%, respectively, of the total alpha isoform mRNA in each parenchymal zone. RNase protection assays determined that the beta 1 and beta 2 isoforms accounted for approximately 95% and approximately 5%, respectively, of the beta isoform mRNA in each zone. These data provide definitive evidence for the differential expression of mRNAs encoding all the alpha and beta 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.

Acute and Chronic Effects of Hyperosmolality on mRNA and Protein Expression and the Activity of Na-K-ATPase in the IMCD

“…Our results are compatible with those reported by Orlowski and Lingrel [10], although these authors did not study mRNA expression of the β 2 isoform. Our results do not completely agree with those of previous reports [7, 12, 13, 14, 16, 17]. The mRNA expressions of α 1 , β 1 , and β 2 isoforms of Na-K-ATPase in the IMCD were similar to the protein expressions of them in the inner medulla.…”

“…The distribution of these isoforms differs depending on tissues or organs, suggesting different roles of isoforms. Reports on the distribution of isoforms in the kidney are conflicting [7, 10, 12, 13, 14, 15, 16, 17]. Studies performed by Ahn el al.…”

“…Studies performed by Ahn el al. [13]and Clapp et al [16]showed the presence of α 1 , α 2 , α 3 , β 1 , and β 2 mRNA in the nephron including IMCD. Tumlin et al [18]reported (1) that α 1 and α 2 mRNA were detected in cortical collecting duct (CCD) and proximal tubule S2 subsegments and (2) that α 1 , β 1 , and β 2 protein isoforms were detected in CCD, S2, medullary thick ascending limb and OMCD.…”

Expression regulation of Na+-K+-ATPase α1-subunit subtypes in zebrafish gill ionocytes

“…In zebrafish, different ionocyte types express respective NKA ␣1 paralogues, which may subtly differ in ouabain affinity, implying different signaling potentials in these ionocytes. In mammalian kidneys, combinations of ␣1-3-and ␤1-2-subunits were found to be differentially expressed in various segments by RT-PCR analysis on microdissected samples (9). In overexpression experiments of mouse collecting duct mpkCCD cells, the human ␣1-but not the ␣2-isoform was stimulated by aldosterone, a steroid con- Fig.…”

Proximal Nephron

“…In the kidney tubules, Na + -K + -ATPase is expressed (96; 444) and localized primarily on the basolateral plasma membranes (Fig. 4) (20; 353), where it is more active in proximal convoluted tubules than in proximal straight tubules (129; 132; 263; 264; 434; 489).…”