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WATER AND ELECTROLYTE HOMEOSTASIS

Reactive oxygen species and the regulation of renal Na⁺-K⁺-ATPase in opossum kidney cells

Institute of Pharmacology and Therapeutics, Faculty of Medicine, Porto, Portugal

Submitted 16 June 2007 ; accepted in final form 31 July 2007

Culture of opossum kidney (OK) cells over 40 passages resulted in marked overexpression of ₁-subunit and ₁-subunit Na⁺-K⁺-ATPase, which paralleled with increases in Na⁺-K⁺-ATPase activity, and enhanced availability in hydrogen peroxide (H₂O₂) (J Membr Biol 212: 163–175, 2006). The present study evaluated the mechanisms involved in increased H₂O₂ production in OK cells cultured over 40 passages (up to passage 80) and whether H₂O₂ plays a role in the regulation of Na⁺-K⁺-ATPase expression and activity. The accumulation of H₂O₂ in the extracellular medium over 24 h (236.5 ± 2.7 vs. 319.3 ± 2.9 nM) and the rate of H₂O₂ production (5.21 ± 0.02 vs. 7.37 ± 0.02 nM/min) in cells with 40 passages was lower (P < 0.05) than in cells with 80 passages. The increase in H₂O₂ production in cells with 80 passages was accompanied by overexpression of NOX1, but not of NOX2, and overexpression of SOD1, SOD2, and SOD3. Apocynin, a NADPH inhibitor, markedly attenuated the increase in H₂O₂ production in cells with 80 passages and significantly decreased Na⁺-K⁺-ATPase activity (26% reduction) and ₁-subunit Na⁺-K⁺-ATPase overexpression. Increases in Na⁺-K⁺-ATPase ₁-subunit mRNA abundance were also observed in OK cells with increased number of cell passages, but apocynin did not affect transcript abundance. In conclusion, the increased availability of H₂O₂ in the intracellular milieu plays an important role in the long-term regulation of Na⁺-K⁺-ATPase expression and activity. These cells may constitute an interesting model to study events related to oxidative stress, in which adaptation to increases in H₂O₂ had progressively adapted in a more natural manner.

NADPH oxidase; superoxide dismutase