Expression and molecular regulation of Na(+)-K(+)-ATPase after renal ischemia

Abstract

Renal ischemia causes redistribution of Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) to the apical membrane of proximal tubules. We determined the time course of regeneration of Na(+)-K(+)-ATPase polarity and sought evidence of increased enzyme production during recovery as a means to restore polarity. Anesthetized rats underwent 45 min renal ischemia and reflow of 15 min, 2 h, 6 h, and 24 h. Immunofluorescent and electron microscopy showed loss of strict basolateral localization of Na(+)-K(+)-ATPase at 15 min reflow with repolarization by 24 h in sublethally injured cells. Both alpha 1- and beta-subunits were only in microsomal fractions at all reflow intervals. Immunodetectable levels of both subunits declined to 60-70% of control by 24 h reflow. Levels of mRNA for each subunit declined in parallel through 24 h to 55% of control. Overall transcription was profoundly depressed through 6 h but had recovered to near control by 24 h. Specific transcription of alpha 1- and beta-subunit mRNA was markedly decreased after ischemia and only partially recovered by 24 h. These results suggest that recycling of misplaced units rather than new Na(+)-K(+)-ATPase production is the means by which renal epithelia initially repolarize after ischemic injury.

Ouabain-sensitive H,K-ATPase Functions as Na,K-ATPase in Apical Membranes of Rat Distal Colon

“…4). Similar to the earlier observations, both HKc␣ and NaK␣1 antibodies identify 100-kDa proteins (7,25). HKc␣ protein was predominantly expressed in apical membrane with modest expression in basolateral membranes (Fig.…”

“…In brief, to avoid aggregation, the protein samples of colonic apical and basolateral membranes were incubated at 37°C for 20 min before loading. Proteins were electrophoretically transferred from SDS-polyacrylamide gel electrophoresis to nitrocellulose membrane (Biotrace; Gelman Science Inc., Ann Arbor, MI) in 192 mM glycine, 25 mM Tris (pH 8.5), 20% methanol for 5 h at 60 V. After blocking nonspecific sites with 20 mM Tris, 137 mM NaCl, 0.1% Tween-20 buffer (pH 7.5) containing 5% nonfat dry-milk, immunostaining was performed with polyclonal antibodies to the ␣-subunits of the colonic HKc␣ (M1) at 1:1000 dilution (10) and Na,K-ATPase (NaK␣1) at 1:2000 dilution (25). Anti-rabbit IgG horseradish peroxidase conjugate (1:5000 dilution) was used as the secondary antibody for M1 antibody-stained blots, whereas anti-mouse IgG horseradish peroxidase conjugate (1:5000 dilution) was used as the secondary antibody for NaK␣1 antibody-stained blots.…”

Sex differences in heat shock protein 72 expression and localization in rats following renal ischemia-reperfusion injury

“…Recent studies revealed that ischemia-induced expression of HSP72 has beneficial effects on the reintegration of cytoskeletal structure and protection of renal tubular epithelial cell polarity (1), which is essential for the postischemic recovery and efficient enzyme function of Na-K-ATPase (NKA) (20). Our previous study reported that female rats have better survival rates and postischemic renal function than males (14).…”

Energetic Determinants of Tyrosine Phosphorylation of Focal Adhesion Proteins during Hypoxia/Reoxygenation of Kidney Proximal Tubules

“…Although the process is frequently also termed acute tubular necrosis, much of the tubule cell damage in both animal models and human acute renal failure is sublethal and reversible within affected cells. [1][2][3][4][5][6] Effects on multiple subcellular structures have been described including loss of brush border microvilli and simplification of the basolateral membrane, 1-3 disruption of the normal polar distribution of major membrane-associated proteins including Na ϩ ,K ϩ -ATPase and its associated cytoskeletal proteins, 7,8 disruption of tight junctions and adherens junctions, 5,9 -13 and abnormalities of integrin distribution and function, 14 -19 all of which can contribute to impaired barrier function and vectorial transport by the epithelium. ATP depletion and the resulting protein dephosphorylation 10 -13,20,21 are the primary processes initiating these events.…”