Modulation of renal cell injury by heat shock proteins: lessons learned from the immature kidney.

The mechanisms that underlie tolerance to injury in immature animals and tissues have been a subject of interest since 1670. Observations in neonatal units that premature infants are less prone to develop acute renal failure than adults in critical care units have prompted a series of investigations. Although initially attributed to metabolic adaptation such as increased glycolytic capacity and preservation of high energy phosphate, more recent studies have indicated a prominent role for the heat shock response. Observed modulations of injury by heat shock proteins in the immature kidney have significant implications for advancement of our understanding of renal cell injury in both adults and children.

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

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.

Reduced tolerance of immature renal tubules to anoxia by HSF-1 decoy.

Immature animals demonstrate an amplified heat shock response following a variety of insults compared with that seen in mature animals (M). The potential role of the heat shock response in modulating immature tolerance to injury was compared between rat pups, 10 postnatal days of age (P10), and M. Baseline levels of the heat shock transcription factor (HSF-1) were substantially elevated in P10 compared with M animals. In uninjured P10 pups, HSF-1 level was comparable to that of M animals subjected to 45 min of ischemia. As anticipated, the integrity of suspensions of tubules exposed to anoxia was preserved in P10 animals (23% LDH release) compared with M (40%), P < 0.01. The effect of targeted inhibition of HSF-1 on tubular integrity was studied using a cyclic oligonucleotide decoy. The HSF-1 decoy increased the severity of anoxic injury in P10 pups to a level comparable with M animals. LDH release was 33% in decoy-treated P10 tubules compared with 40% in M. When P10 tubules were treated with scrambled decoy, resistance to anoxia remained intact (24%). The increased vulnerability of the tubular suspension to injury was specific to the HSF-1 decoy and proportional to the dose of decoy applied. This study demonstrates maturation in the abundance of HSF-1 in the immature rat kidney. The loss of resistance of immature tubules to anoxia with specific inhibition of HSF-1 may be due to its effect on the heat shock response or other signaling pathways of critical pathobiological importance in renal cell injury.

Differential inhibition of HSP72 and HSP25 produces profound impairment of cellular integrity.

To test a putative cause and effect relationship between heat-shock protein (HSP) expression and response to renal cell injury, HSP72 and HSP25 were differentially inhibited in LLC-PK1 cells by means of transcription factor decoy and short interference RNA (siRNA). Cellular injury was assessed by solubilization of NaK ATPase (S-NaK). An exonuclease-resistant, ethylene glycol-bridged, circular oligonucleotide decoy for heat-shock transcription factor (HSF)-1, based on the sequence of the porcine heat-shock element, was constructed and validated. It was found that under all experimental conditions, cells had comparable ATP levels; that decoy of unligated or scrambled sequence was ineffective; that HSP72 mRNA and HSP72/HSP25 proteins were significantly reduced in decoy-treated cells; and that the dampened response to HSF activation in decoy-treated, injured cells was accompanied by a substantially amplified loss of cellular integrity (S-NaK was 85% compared with baseline levels). Specific inhibition of HSP72 that used siRNA directed against an inducible porcine HSP72 gene resulted in complete ablation of injury-induced HSP72. Isolated inhibition of HSP72 was also associated with marked NaK ATPase detachment from the cytoskeleton (S-NaK was 135% compared with baseline levels). These studies suggest that an HSF-1 decoy effectively dampens the HSP72/HSP25 response to injury in renal cells; that HSP72 siRNA ablates injury-induced induction of HSP72; and that dampening of the HSP72/HSP25 response and ablation of the HSP72 response are both associated with impaired restitution of cellular polarity.

Developmental expression of HSP-72 and ischemic tolerance of the immature kidney.

The resistance of the immature kidney to ischemic injury is well documented, but the mechanisms involved in this tolerance have been elusive. Previous studies have demonstrated that tubules obtained from immature rats exhibit a bigger stress response than mature tubules. Consequently, we evaluated the developmental expression of HSP-72 in the postnatal kidney and determined whether or not that pattern of expression was correlated with the previously known tolerance of the immature kidney to injury. A distinct pattern of HSP-72 expression with a peak abundance at postnatal day 10 (P10), with a subsequent decline toward values seen in mature rats, was found. Moreover, this stress protein is located predominantly in tubular segments, the site of ischemic injury. To determine if this constitutive, non-induced expression of HSP-72 in the immature rat could be protective of cellular integrity and renal function, both immature (P10) and mature (8 weeks) rats were subjected to 45 min of bilateral renal artery ischemia. The postischemic induction of HSP-72 in the P10 animals was robust and the peak expression 2 h after ischemia was even greater than that detected in mature animals. Thus, the constitutive enhanced expression of HSP-72 did not prohibit or mute the inducible response of this stress protein in the immature animals. Immature animals, when compared with mature rats, also experienced cytoprotection, demonstrated by decreased detachment of Na-/K-ATPase from the cytoskeleton and substantial protection of renal function determined by serum creatinine level. These findings suggest that the developmental expression of heat shock proteins may play a critical and fundamental role in the well-observed tolerance of immature tubules to ischemic or anoxic injury.

Hsp27 associates with actin and limits injury in energy depleted renal epithelia.

The purpose of the study was to determine whether Hsp27 interacts with actin and could protect against selected manifestations of injury from energy depletion in renal epithelia. LLC-PK1 cells were stably transfected to overexpress human Hsp27 tagged with green fluorescence protein (GFP). Transfected expression of the labeled Hsp27 did not reduce endogenous Hsp25 levels in the cells compared with either nontransfected cells or cells transfected with GFP alone used as the transfectant control (G). By fluorescence energy transfer (FRET) between GFP-tagged Hsp27 and rhodamine phalloidin-decorated actin, minimal interaction was found in uninjured control cells. In ATP-depleted cells, Hsp27 was associated closely with F-actin at lateral cell boundaries and with aggregated actin within the cell body. Less Hsp27 interaction with actin was found during recovery; but when adjusted for total phalloidin fluorescence, FRET between Hsp27 and F-actin did not change between 2-h ATP depletion and 4-h recovery. Where Hsp27 association with actin persisted during recovery, it was principally with the residual aggregates of actin in the cell body. Detachment of Na,K-ATPase from the cytoskeleton at 2-h ATP depletion was significantly less in Hsp27 cells compared with transfectant control G cells but not at 4-h ATP depletion. Detachment of ezrin from the cytoskeleton during ATP depletion was nearly complete and was not prevented in the Hsp27 cells. Protection of the Hsp27 cells was not attributable to preservation of cellular ATP levels. Hsp27 appears to have specific actions in renal epithelia subjected to energy depletion, including interacting with actin to preserve architecture in specific intracellular domains.

Ischemic conditioning prevents Na,K-ATPase dissociation from the cytoskeletal cellular fraction after repeat renal ischemia in rats.

Recent studies have suggested that heat shock proteins (HSPs) are involved in the restoration of the cytoskeletal anchorage of Na,K-ATPase after renal ischemia. To determine their role in ischemic conditioning, we investigated whether cytoskeletal Na,K-ATPase was stabilized during repeat ischemia concurrent with 25-kD and 70-kD HSPs induction. Anesthetized rats either underwent single unilateral renal ischemia or were conditioned with bilateral renal ischemia and, after 18 h of reflow, were then subjected to repeat unilateral renal ischemia. Renal cortex was harvested, and effects of single versus repeat ischemia were compared by Triton X-100 extraction, by immunohistochemistry, and by an in vitro assay of Na,K-ATPase association with isolated cytoskeletal fractions. In contrast to single ischemia, repeat ischemia did not result in increased Triton X-100 extractability of Na,K-ATPase. Levels of 25-kD and 70-kD HSPs were significantly induced by ischemic conditioning and redistributed into the cytoskeletal fraction after single and repeat ischemia. Immunohistochemistry also showed significant disruption of Na,K-ATPase within proximal tubules only after a single episode of ischemia, whereas repeat ischemia did not alter the pattern of restored Na,K-ATPase localization in conditioned renal cortex. The preserved association of Na,K-ATPase with the cytoskeletal fraction of conditioned renal cortex was effectively abolished in vitro by addition of antibodies against 25-kD or 70-kD HSP. These results suggest that 25-kD and 70-kD HSPs induced by ischemic conditioning stabilize the cytoskeletal anchorage of Na,K-ATPase during repeat renal ischemia.