ATP releases HSP-72 from protein aggregates after renal ischemia

Abstract

The pattern of 72-kDa heat-shock protein (HSP-72) induction after renal ischemia suggests a role in restoring cell structure. HSP-72 activity in the repair and release from denatured and aggregated proteins requires ATP. Protein aggregates were purified from normal and ischemic rat renal cortex. The addition of ATP to cortical homogenates reduced HSP-72, Na+-K+-ATPase, and actin in aggregates subsequently isolated, suggesting that their interaction is ATP dependent. Altering ATP hydrolysis in the purified aggregates, however, had different effects. ATP released HSP-72 during reflow and preserved Na+-K+-ATPase association with aggregates at 2 h but had no effect in controls or at 6 h reflow and caused no change in actin. These results indicate that HSP-72 complexes with aggregated cellular proteins in an ATP-dependent manner and suggests that enhancing HSP-72 function after ischemic renal injury assists refolding and stabilization of Na+-K+-ATPase or aggregated elements of the cytoskeleton, allowing reassembly into a more organized state.

Ischemic Conditioning Prevents Na,K-ATPase Dissociation from the Cytoskeletal Cellular Fraction after Repeat Renal Ischemia in Rats

“…The abdomen was opened by a median incision, and the kidneys were exposed and, if macroscopically normal, carefully dissected. After anticoagulation with 125 IU of heparin, renal ischemia was performed as previously described (4,5). After the protocol, anesthetized animals were killed by i.v.…”

“…Protein concentrations were assessed in duplicate by Bradford analysis. Protein samples were electrophoresed through SDS-polyacrylamide gels and electrophoretically transferred to nitrocellulose (4,5). Membranes were incubated with the primary antibodies against 70-kD HSP (Sigma Chemical Co., St. Louis, MO, U.S.A.), 25-kD HSP (SPA801; Stressgen Biotechnologies), and alpha-subunit of Na,K-ATPase (Upstate Biotechnology, Lake Placid, NY, U.S.A.).…”

“…The redistribution of Na,K-ATPase from pellet to supernatant has been used in several studies as a reproducible marker for ischemic injury of the proximal tubule cell. Using the same technique, the increased Triton X-100 extractability of Na,K-ATPase after renal ischemia was shown to be fully reversible during recovery at a rate dependent on the severity of the insult (3)(4)(5).…”

“…The 70-kD HSPs, a family of molecular chaperons, show a distinct temporal pattern of postischemic cellular localization and intracellular distribution that coincides with the restoration of disrupted cytoskeletal elements during recovery of the proximal tubule cell from ischemia (8). Using functional in vitro assays, we recently demonstrated that enhanced 70-kD HSP activity and abundance results in stabilization of Na,K-ATPase in cytoskeletal subfractions (4,5).…”

“…For Na,K-ATPase to be translocated, it must first dissociate from its cytoskeletal anchorage (2)(3)(4)(5). Triton X-100 extraction fractionates the cellular pool of Na,K-ATPase into an insoluble pellet (cytoskeletal-associated fraction) and a soluble supernatant by simple differential centrifugation.…”