Cytoskeleton disruption and apical redistribution of proximal tubule Na⁺- K⁺-ATPase during ischemia

The polar distribution of Na⁺-K⁺-ATPase to the basolateral membrane of proximal tubule cells is essential for the efficient and vectorial reabsorption of Na⁺ and may be dependent on the formation of a metabolically stable, detergent-insoluble complex of Na⁺-K⁺-ATPase with the actin membrane cytoskeleton. The present studies utilized immunocytochemical techniques to demonstrate and quantify the apical redistribution of Na⁺-K⁺- ATPase during mild ischemia (15 min) that occurred in proximal (1.3 ± 0.9 vs. 4.5 ± 1.1 particles/100 μm surface membrane, P < 0.01) but not distal tubule cells. Treatment of control apical membranes with 2-(2- methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanoate (A₂C), a fluidizing agent, markedly increased membrane fluidity without any effect on Na⁺-K⁺-ATPase activity. In brush-border membrane vesicles isolated after ischemia, however, A₂C further increased an already elevated Na⁺-K⁺-ATPase activity. During ischemia, total cellular Na⁺-K⁺-ATPase activity remained unaltered, but the Triton X-100-soluble (noncytoskeleton associated) fraction of Na⁺-K⁺-ATPase increased significantly following 15 and 30 min. There was a corresponding decrease in the Triton X-100-insoluble fraction of Na⁺-K⁺- ATPase, with the ratio of detergent-soluble to -insoluble Na⁺-K⁺-ATPase increasing from 13 ± 2 to 32 ± 5% (P < 0.01) during 30 min of ischemia. Western blot analysis of the Triton X-100-soluble fraction, following 30 min of ischemic injury, revealed the presence of Na⁺-K⁺-ATPase, actin, fodrin, and uvomorulin. However, in a fraction highly enriched for Na⁺-K⁺-ATPase, neither actin, fodrin, nor uvomorulin was detected. Taken together, these data suggest that in proximal tubule cells, as in Madin-Darby canine kidney cells, Na⁺-K⁺-ATPase exists primarily in a cytoskeletal-associated form. During ischemic injury, the actin cytoskeleton is disrupted, and Na⁺-K⁺- ATPase dissociates from the actin cortical cytoskeleton and is then free to redistribute to the apical membrane in proximal but not distal tubule cells.