HSP70 binding modulates detachment of Na-K-ATPase following energy deprivation in renal epithelial cells

Abstract

The molecular mechanisms associated with reestablishment of renal epithelial polarity after injury remain incompletely delineated. Stress proteins may act as molecular chaperones, potentially modulating injury or enhancing recovery. We tested whether overexpression of heat shock protein 70 (HSP70) would stabilize Na-K-ATPase attachment to the cytoskeleton, under conditions of ATP depletion, and whether a direct association existed between Na-K-ATPase and HSP70 in cultured renal epithelial cells. LLC-PK1 cells were transfected with a tagged HSP70 (70FLAG) or vector alone (VA). Detachment of Na-K-ATPase was detected in Triton soluble lysate after ATP depletion. 70FLAG cells demonstrated a significant (P < 0.01) decrease in detachment of Na-K-ATPase after either 2 or 4 h of ATP depletion. Interactions between HSP70 and Na-K-ATPase were determined by coimmunoprecipitation of 70FLAG and Na-K-ATPase, by direct and competitive binding assays and by immunocytochemical localization. Binding of HSP70 and Na-K-ATPase increased dramatically following injury. Interactions were: 1) reversible; 2) reciprocal to changes in the HSP70 binding protein clathrin; and 3) present only when ATP turnover was inhibited in cell lysate, an established characteristic of HSP binding. These studies indicate that 1) overexpression of HSP70 is associated with decreased detachment of Na-K-ATPase from the cytoskeleton following injury; 2) HSP70 binds to Na-K-ATPase; and 3) binding of HSP70 to Na-K-ATPase is dynamic and specific, increasing in response to injury and decreasing during recovery. Interaction between the molecular chaperone HSP70 and damaged or displaced Na-K-ATPase may represent a fundamental cellular mechanism underlying maintenance and recovery of renal tubule polarity following energy deprivation.

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

“…In vitro studies also revealed that the overexpression of HSP72 is associated with decreased detachment of NKA from the cytoskeleton following ischemic injury (17). Based on our and others' previous results, here we hypothesized that the different expression and localization of HSP72 might also be responsible for gender disparity in NKA localization after the ischemic insult.…”

“…Ischemic preconditioning with HSP72 overexpression prevented NKA ␣ 1 -subunit dissociation after repeated ischemic insult (1). Very recently, a direct interaction has been approved between HSP72 and damaged or displaced NKA ␣ 1 in an in vitro model of renal ischemic injury (17). Although these studies further support the concept that HSP72 and NKA have important interactions, no one has as yet investigated the gender differences in colocalization of these proteins after renal I/R injury.…”

“…Regarding the direct interaction between HSP72 and NKA (17) and the gender differences in the expression of HSP72, one can postulate that the higher levels of HSP72 in F rats contribute to the postischemic restoration of membrane and protein structure of proximal tubular cells. The costaining of HSP72 and NKA on the basolateral membrane of F postischemic kidneys suggests that HSP72 might directly help to prevent postischemic damage to NKA and to preserve enzyme function.…”

Pseudomonas syringae hijacks plant stress chaperone machinery for virulence

“…J proteins bind Hsp70 through the J domain and stimulate Hsp70's ATP hydrolysis activity as well as other activities such as de novo folding of client proteins, intervening when proteins are improperly folded-often during stress conditions, protein degradation, and the disassembly of complexes, protein translocation, and trafficking (8)(9)(10). A conserved HPD loop of J domains is essential for interaction with Hsp70 and modulating Hsp70 activities (8).…”

Targeting Molecular Chaperones in Diabetic Peripheral Neuropathy

“…HSP70 directly interacts with the Na + /K + -ATPase and stabilizes its cytoskeletal anchorage in renal outer medulla (Ruete et al, 2008) and epithelial cells (Riordan et al, 2005) after ATP depletion. Whether this interaction may represent a fundamental mechanism underlying cellular protection is unknown, but warrants investigating if stabilization of Na + /K + -ATPase by HSP70 contributes to restoring NCV in diabetic nerves.…”