Overexpression of Na+/K+-ATPase Parallels the Increase in Sodium Transport and Potassium Recycling in an In Vitro Model of Proximal Tubule Cellular Ageing

Abstract

Na(+)/K(+)-ATPase plays a key role in the transport of Na(+) throughout the nephron, but ageing appears to be accompanied by changes in the regulation and localization of the pump. In the present study, we examined the effect of in vitro cell ageing on the transport of Na(+) and K(+) ions in opossum kidney (OK) cells in culture. Cells were aged by repeated passing, and Na(+)/K(+)-ATPase activity and K(+) conductance were evaluated using electrophysiological methods. Na(+)K(+)-ATPase alpha(1)- and beta(1)-subunit expression was quantified by Western blot techniques. Na(+)/H(+) exchanger activity, changes in membrane potential, cell viability, hydrogen peroxide production and cellular proliferation were determined using fluorimetric assays. In vitro cell ageing is accompanied by an increase in transepithelial Na(+) transport, which results from an increase in the number of Na(+)/K(+)-ATPase alpha(1)- and beta(1)-subunits, in the membrane. Increases in Na(+)/K(+)-ATPase activity were accompanied by increases in K(+) conductance as a result of functional coupling between Na(+)/K(+)-ATPase and basolateral K(+) channels. Cell depolarization induced by both KCl and ouabain was more pronounced in aged cells. No changes in Na(+)/H(+) exchanger activity were observed. H(2)O(2) production was increased in aged cells, but exposure for 5 days to 1 and 10 microM: of H(2)O(2) had no effect on Na(+)/K(+)-ATPase expression. Ouabain (100 nM: ) increased alpha(1)-subunit, but not beta(1)-subunit, Na(+)/K(+)-ATPase expression in aged cells only. These cells constitute an interesting model for the study of renal epithelial cell ageing.

Impaired Stimulatory Effect of ETB Receptor on D₃ Receptor in Immortalized Renal Proximal Tubule Cells of Spontaneously Hypertensive Rats

“…+ -K + -ATPase Activity Assay Na + -K + -ATPase activity was determined as the rate of inorganic phosphate released in the presence or absence of ouabain [31][32][33] . To prepare membranes for Na + -K + -ATPase activity assay, RPT cells cultured in 21-cm 2 For determination of ouabain-insensitive ATPase activity, NaCl and KCl were omitted from the reaction mixtures containing ouabain.…”

Age-dependent effect of ouabain on renal Na+,K+-ATPase

“…Moreover, previously published results using opossum kidney cells demonstrated that ouabain treatment induced an increase in Na + ,K + -ATPase activity only in cells with high number of passages in culture. These cells had increased levels of ROS, alterations in the cytoskeleton and increased basal activity and expression of Na + ,K + -ATPase (Silva, et al, 2006;Silva and Soares-da-Silva, 2007;Silva and Soares-da-Silva, 2009). Altogether, the discrepancies between our results and previously published in vivo results demonstrate that the mechanisms responsible for ouabain-mediated Na + ,K + -ATPase regulation are complex, cell specific and may be dependent not only on the dosage and duration of the treatment but also on the strain of the rats used in the study.…”

“…Juvenile Sprague-Dawley rats treated intraperitoneally with ouabain (Holthouser, et al, 2010;Zhang, et al, 2010) showed that the development of hypertension was accompanied by an increase in renal Na + ,K + -ATPase activity. Studies using cell culture models demonstrated that the interaction between Na + , K + -ATPase and other membrane proteins may differ between cell lines leading to the activation of specific ouabain-mediated cell signaling pathways and different outcomes regarding Na + ,K + -ATPase regulation (Holthouser, et al, 2010;Khundmiri, et al, 2007;Khundmiri, et al, 2006;Liu, et al, 2002;Silva, et al, 2006). Moreover, previously published results using opossum kidney cells demonstrated that ouabain treatment induced an increase in Na + ,K + -ATPase activity only in cells with high number of passages in culture.…”

Inhibition of D 4 Dopamine Receptors on Insulin Receptor Expression and Effect in Renal Proximal Tubule Cells

“…Na + ‐K + ‐ATPase activity was determined as the rate of inorganic phosphate released in the presence or absence of ouabain 28, 29. Rat RPT cells were treated with vehicle (dH 2 O) and D 4 receptor agonist (PD168077) at the indicated concentrations and durations of incubation.…”