INTRODUCTION

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RENAL ISCHEMIA INDUCES the rapid, duration-dependent relocation of apical and basolateral membrane proteins into the alternate domain (8, 16-19). For Na⁺-K⁺-ATPase to be translocated to the apical domain, it must first be detached from its cytoskeletal tether, which has been defined functionally by detergent extractability (17, 18, 27). Reestablishment of the membrane-cytoskeletal complex, and thus cell polarity, appears to occur by recycling of misplaced Na⁺-K⁺-ATPase subunits rather than by increased biosynthesis (3, 29). The heat-shock proteins (HSP) are ideal candidates for participating in such a posttranslational repair mechanism (21). Renal ischemia rapidly induces the elaboration of 70-kDa HSPs (HSP-70), and the subcellular distribution of the inducible HSP-72 within proximal tubules during recovery from ischemic injury parallels the distribution of Na⁺-K⁺-ATPase and cytoskeletal elements (12, 28). Therefore the pattern of induction and elaboration of HSP-72 suggests a role in the reassembly of disrupted or denatured proteins during postischemic cellular reorganization.

Specific functions attributed to HSP-70 proteins include actions as a chaperone, which allows for proper protein folding by preventing deleterious peptide interactions or aggregations (24). HSP-70 binds to hydrophobic, normally hidden, domains of denatured proteins, thereby preventing aggregation or, in some instances, assisting aggregated proteins to become soluble (10, 24). The ability of this family of proteins to reactivate denatured proteins has been demonstrated (7, 23, 25, 30). Hydrolysis of ATP is required for this chaperone function. HSP-70 stress proteins readily bind to denatured proteins in the absence of ATP but do not process and release the substrate without hydrolysis of ATP (21).

To investigate putative functional interactions between heat-shock proteins and cellular proteins disrupted by renal ischemia, a subfraction of aggregated proteins was purified from rat renal cortex and examined for the presence and abundance of HSP-72, Na⁺-K⁺-ATPase, and actin. ATP hydrolysis was enhanced or inhibited in renal cortex homogenates to determine the effect on the subsequent composition of the protein aggregates with respect to each protein. ATP hydrolysis was also altered in resuspended, purified aggregates from injured kidneys to alter HSP-72 activity and to assess the effect on the release of each of these proteins from the aggregates.

	MATERIALS AND METHODS

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Animal Preparation

Cell Protein Fractionation

Isolation of aggregated proteins. Aggregates were isolated by further differential centrifugation by modifying a protocol reported by Oberg et al. (22) for purification of mutant and overexpressed recombinant proteins with similar physicochemical properties. The Triton-insoluble subfraction was twice resuspended in extraction buffer, sonicated, and pelleted at 17,000 g for 30 min at 4°C. The resultant pellet was again twice resuspended in extraction buffer, sonicated, and centrifuged at 5,000 g for 30 min at 4°C, and the final purified pellet (aggregates) was stored in extraction buffer at 70°C.

Enhancement and inhibition of ATP hydrolysis. The effect of enhancing versus inhibiting HSP-72 activity was examined by adding excess Mg-ATP to promote ATP hydrolysis or by adding CDTA to inhibit ATP hydrolysis in parallel aliquots from the same extract. In one series of experiments ATP hydrolysis was altered in homogenates of renal cortex before isolation of aggregates and in a separate series after isolation of aggregates.

Alteration of ATP hydrolysis before isolation of aggregates. CDTA was omitted from the initial extraction buffer. Mg-ATP (5 mM) was then added to half of the homogenate while endogenous ATP hydrolysis was inhibited in the other half of the homogenate by the addition of 2 mM CDTA. Both homogenates were incubated for 60 min at room temperature before the isolation of aggregates by the fractionation procedure as described above.

ATP levels were determined by luciferase assay (9) in homogenates from 2-h reflow cortex. There was a 30% decline in homogenate ATP over the 1-h incubation in the samples that contained CDTA. Parallel addition of Mg-ATP to a separate aliquot of the same homogenates resulted in a greater than 300-fold increase in homogenate ATP levels compared with the CDTA group. Subsequent to the Mg-ATP addition, levels fell by 98% over the 1-h incubation interval, indicating enhanced ATP hydrolysis in the Mg-ATP augmented homogenates compared with the CDTA homogenates in which ATP hydrolysis was inhibited.

Alteration of ATP hydrolysis after isolation of aggregates. After completion of the fractionation procedure, CDTA was omitted from the final extraction buffer used to resuspend the aggregates. Mg-ATP (5 mM) was then added to one-half of the suspension while endogenous ATP hydrolysis was blocked in the other half via the addition of 2 mM CDTA. The resuspended aggregates were incubated for 60 min at room temperature before the aggregates were recovered by a repeat of the final centrifugation at 5,000 g at 4°C. Both repelleted aggregates and the corresponding supernatants containing proteins released from the aggregates during the incubations were studied. In this group of experiments, the intermediate washes at 17,000 g were omitted to achieve equivalent total Triton X-100 incubation time and comparable protein content of the aggregates with the additional Mg-ATP or CDTA incubation periods. Coomassie blue staining of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the different protein fractions and Western analysis for Na⁺-K⁺-ATPase, HSP-72, and actin showed no differences in protein abundance or pattern in aggregates isolated with this modified procedure. All samples were stored at 70°C until further analysis.

To evaluate the ability of this protocol to isolate a subfraction that is enriched in denatured proteins, bovine serum albumin (BSA) was denatured by reductive carboxymethylation (rcm-BSA) (1). Protein suspensions of native and denatured rcm-BSA in extraction buffer were fractionated in parallel as described above. As shown in Fig. 1, minimal native BSA but 90% of denatured rcm-BSA was recovered in the aggregates.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Percentage recovery of native versus denatured bovine serum albumin (BSA) in aggregate subfraction. BSA was denatured by reductive carboxymethylation (rcm-BSA). Native and denatured BSA were suspended in extraction buffer, and the aggregate subfraction was isolated. Minimal native BSA, but 90% of denatured rcm-BSA, was recovered in aggregate subfraction.

Western Analysis

Statistics

	RESULTS

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Na⁺-K⁺-ATPase Extractability and HSP-72 Induction

View larger version (44K): [in this window] [in a new window]   Fig. 2.   Na+-K+-ATPase and 72-kDa heat-shock protein (HSP-72) after Triton extraction of control and postischemic rat renal cortex during reflow: immunoblot of representative results from n = 5 samples from controls and at each reflow interval. Postischemic dissociation of Na+-K+-ATPase from cytoskeleton is demonstrated by increased Na+-K+-ATPase in Triton-soluble fraction at 15 min reflow and to a lesser extent at 2 h reflow. Restoration of normal Na+-K+-ATPase distribution coincides with progressive increase in expression of HSP-72 in both cellular fractions.

Na⁺-K⁺-ATPase and HSP-72 in Protein Aggregates During Reflow

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Na+-K+-ATPase and HSP-72 in protein aggregates isolated from control and postischemic rat renal cortex during reflow: immunoblot of protein aggregates representing results of n = 5-7 at each interval. After an initial decrease at 15 min reflow, Na+-K+-ATPase abundance in aggregate fraction returned to control levels at 2-6 h reflow. HSP-72 first appeared in aggregates at 15 min and progressively increased through 2 and 6 h of reflow.

Effects of Enhancement or Inhibition of ATP Hydrolysis

View larger version (111K): [in this window] [in a new window]   Fig. 4.   Coomassie stain of Triton-soluble (TS) and aggregate subfractions (paired lanes A, B, and C) of postischemic rat renal cortex after enhancing (+) or inhibiting () ATP hydrolysis. ATP incubation (+) did not result in nonspecific degradation of proteins in any of the subfractions. Trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) incubation () resulted in an identical protein pattern in aggregates isolated before (A) or after incubation (B). Changes in protein abundance from altered ATP hydrolysis in isolated aggregate subfraction (B) are mirrored in supernatant (C). Molecular mass markers in far right lane were (from top) 105, 71, 43, and 28 kDa. Arrow, location of the high-molecular-mass protein that moves from aggregate to soluble fraction with ATP incubation.

Alteration of ATP Hydrolysis in Homogenates Before Isolation of Aggregates

View larger version (59K): [in this window] [in a new window]   Fig. 5.   Alteration of ATP hydrolysis in homogenates before isolation of aggregates. Na+-K+-ATPase, HSP-72, and actin in aggregates and Triton-soluble fraction obtained from rat renal cortex that had been incubated with Mg-ATP (+ATP) or CDTA (ATP): immunoblots demonstrating results from n = 3 determinations of each condition represented. Na+-K+-ATPase, actin, and HSP-72 all decrease in the aggregate subfraction with ATP incubation (+ATP). This effect was observed not only in postischemic kidney cortex when HSP-72 was increased but also in control kidneys with minimal HSP-72 expression. Altering ATP hydrolysis had no effect on Na+-K+-ATPase, HSP-72, or actin in Triton-soluble fraction.

Alteration of ATP Hydrolysis After Isolation of Aggregates

View larger version (54K): [in this window] [in a new window]   Fig. 6.   Alteration of ATP hydrolysis after isolation of aggregates: purified aggregates were incubated with ATP (+ATP) or CDTA (ATP), and proportion of Na+-K+-ATPase, HSP-72, and actin remaining in aggregates or release into supernatant was assayed by Western analysis. CDTA incubation (ATP) resulted in decreased Na+-K+-ATPase in aggregates obtained from 2 h reflow samples compared with control with a concomitant increased release of Na+-K+-ATPase into supernatant from the same aggregates. ATP incubation (+ATP) had no effect on controls but resulted in stabilization of Na+-K+-ATPase in aggregates obtained at 2 h reflow with less dissociation into the supernatant. At 2 h reflow, ATP released HSP-72 from the aggregate into the supernatant. There were no significant changes in actin from control or reflow samples.

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Densitometric analysis of changes in Na+-K+-ATPase and HSP-72 abundance resulting from ATP incubation of isolated aggregates. Changes are expressed as arbitrary densitometric units (AU) normalized for the signal from the aggregates incubated with CDTA (ATP) as 100 AU (n = 5 controls and 5 at each reflow). Following incubation with Mg-ATP, Na+-K+-ATPase association with aggregated proteins was markedly increased at 2 h reflow but was not significantly affected at 6 h reflow or in controls. ATP released a significant portion of HSP-72 from aggregated proteins at both reflow intervals. Altering ATP hydrolysis caused no change in actin abundance in isolated aggregates in controls or at any reflow interval (not shown).

	DISCUSSION

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Our results confirm that nonlethal renal ischemia causes transient dissociation of Na⁺-K⁺-ATPase from the membrane-cytoskeleton complex associated with increased expression of the inducible cytosolic heat-shock protein, HSP-72. Moreover, these results directly demonstrate the temporal relationship between the Triton X-100 extractability of Na⁺-K⁺-ATPase and HSP-72 elaboration (12, 28, 29). During recovery from ischemia, HSP-72 expression increases while detergent-soluble Na⁺-K⁺-ATPase decreases, suggesting that reestablishment of Na⁺-K⁺-ATPase anchorage to the cytoskeleton may be facilitated by the action of HSP-72.

Increasing evidence suggests that denatured proteins are not only the substrate but also the trigger for the heat-shock response (2, 10, 14, 15, 20, 26). The common denominator appears to be some feature of the denatured protein recognized by HSP-70 proteins, such as exposure of normally hidden hydrophobic domains. In support of this hypothesis microinjection of denatured BSA, but not of the respective native protein, into Xenopus laevis oocytes caused the activation of a coinjected HSP-70 hybrid gene (1). More recent studies have shown that the best predictor of stress response intensity was the extent of denatured protein aggregation (14, 15). Therefore, alterations of physicochemical properties of proteins, such as aggregation and solubility, seem to parallel the ability to elicit a stress response.

To determine whether a similar dynamic interaction occurs in the injured kidney, a protocol was adapted to purify aggregated proteins from renal cortex to examine potential interactions between HSP-72 and cell proteins known to be disrupted by renal ischemia (22). We also sought to evaluate whether the cell proteins isolated in this aggregate fraction might have relevance to the previously established link between denatured proteins and the HSP-70 chaperones. Two groups have used reductive carboxymethylation to denature and aggregate BSA in vitro (rcm-BSA) to examine the relationship between denatured and aggregated proteins with the constitutively expressed cognate of HSP-70 (HSC-70) and with stress response induction in vivo (1, 14, 15). They demonstrated that rcm-BSA, in distinction to native BSA, both is preferentially recognized by HSC-70 and induces the stress response in oocytes. Furthermore, progressive aggregation of the denatured BSA correlated with the degree of stress response activation in oocytes; coinjection of HSC-70 blocked the stress response elicited by the aggregated BSA (14, 15). Using the same technique to denature BSA, we found nearly complete recovery of the rcm-BSA, compared with essentially no recovery of native BSA using our aggregate isolation procedure. This indicates that the isolated aggregates may contain denatured proteins that are substrates for HSP-70 chaperone action, which is further supported by the presence of HSP-72 in the aggregates early in the reflow period after renal ischemia. These findings are consistent with our previous in situ immunolocalization of HSP-72 to a distinct granular pattern at 2 and 6 h of reflow, which is suggestive of aggregated proteins (28).

It may seem surprising that we purified aggregated proteins from uninjured, control kidney cortex as well as from postischemic cortex. However, the aggregate fraction is operationally defined as highly aggregable proteins containing exposed hydrophobic domains, which would be expected to include nascent, partially folded proteins as well as denatured or other nonnative proteins. Thus we would expect to find Na⁺-K⁺-ATPase in the nascent form within the aggregates from control tissue. The contribution of nascent forms to the total aggregated enzyme may explain the initial decline in Na⁺-K⁺-ATPase present in the aggregates isolated at 15 min of reflow, as we previously have demonstrated a marked decrease in transcription for new Na⁺-K⁺-ATPase units at this reflow interval (29). At 2 h reflow, the aggregates likely contain significantly more disrupted Na⁺-K⁺-ATPase, since at this interval aggregated Na⁺-K⁺-ATPase had risen above levels at 15 min even though transcription for new enzyme remains severely depressed (29). In addition, the aggregated Na⁺-K⁺-ATPase in the injured samples behaves differently from both the control and later recovery samples upon addition of ATP. The present studies, then, indicate that the aggregates contain Na⁺-K⁺-ATPase in different structural forms, nascent proteins under control conditions and disrupted forms after ischemia.

These results may be further interpreted by considering the essence of Na⁺-K⁺-ATPase interaction with the cytoskeleton. Classically, increased solubilization of Na⁺-K⁺-ATPase by Triton X-100 extraction is regarded as the marker for dissociation of the membrane-cytoskeleton complex (8, 16-19). However, the cytoskeletal anchorage of Na⁺-K⁺-ATPase may represent a continuum ranging from complete assembly to a partly assembled but unstable complex (both Triton insoluble) to complete dissociation (Triton soluble). Renal ischemia may result in a shift of this continuum toward higher degrees of instability and dissociation. In this study, the decreased content of Na⁺-K⁺-ATPase in the aggregates early after the injury (at 15 min reflow, Fig. 3), when Triton-soluble enzyme is maximally increased (Fig. 2), suggests the aggregates contain partially denatured, unstable, or incompletely assembled membrane protein-cytoskeleton complexes.

Support for functional interaction of HSP-72 with specific proteins can be provided by taking advantage of a cardinal feature of stress protein activity. Molecular chaperones such as HSP-72 readily bind to other proteins in the absence of ATP hydrolysis but do not act and release the attached protein without hydrolysis of ATP (4-6, 21, 25, 30). These HSPs use the energy of ATP hydrolysis to undergo a conformational change, which may result in 1) refolding or partial stabilization of denatured proteins and 2) release of reconformed proteins (24). Repeated cycles of this kind would result in the repair of more complex structures, with the released substrates then reassembling into native form. In a similar manner, cellular proteins disrupted by renal ischemia may be restored by ATP-dependent action of stress proteins. Such HSP-mediated repair should be evident in a cellular subfraction, which contains denatured proteins and HSP-72, such as the aggregates that were isolated in this study.

Enhancement of ATP hydrolysis in homogenates of renal cortex before the isolation of aggregates reduced HSP-72 content in aggregates obtained during reflow, concomitant with decreases of Na⁺-K⁺-ATPase and actin. This effect is consistent with ATP-dependent functional interactions between these proteins in this aggregated cell protein subfraction. Although the postischemic studies suggest that HSP-72 was released from its substrate (4, 6), similar effects of ATP hydrolysis on actin and Na⁺-K⁺-ATPase were seen in aggregates obtained under control conditions with low expression of inducible HSP-72. Thus, in this series of experiments, HSP-72-mediated effects during reflow were not separable from constitutive effects of ATP hydrolysis, raising the possibility that other protein chaperones are involved as well.

However, the effects of altering ATP hydrolysis after isolation of the aggregates were unique to those purified after ischemia. Inhibition and enhancement of ATP hydrolysis effected no change in the aggregated Na⁺-K⁺-ATPase or actin in controls, indicating that effects observed in the reflow samples were specific for postischemic recovery and that nonspecific degradation or redistribution of the proteins of interest by the separate incubations did not occur. Inhibition of ATP hydrolysis prevented and enhancement of ATP hydrolysis increased release of HSP-72 from the aggregate into the supernatant at each reflow interval, as would be expected if there were specific, functional interaction between HSP-72 and denatured substrates in the aggregates (4, 6). At 2 h reflow, inhibiting ATP hydrolysis allowed increased dissociation of Na⁺-K⁺-ATPase from the aggregates into the supernatant compared with nonischemic controls, but enhancing ATP hydrolysis preserved the association of Na⁺-K⁺-ATPase with the aggregates. These effects of altering ATP hydrolysis on Na⁺-K⁺-ATPase in the aggregates were less prominent at 6 h reflow.

Together these results suggest that, following renal ischemia, HSP-72 interacts either directly with Na⁺-K⁺-ATPase or with other elements of the membrane cytoskeleton-complex in a manner dependent upon ATP hydrolysis. Enhanced HSP-72 function may have assisted refolding and stabilization of Na⁺-K⁺-ATPase or other nonnative elements of the membrane-cytoskeleton complex, allowing increased binding and preserved association of Na⁺-K⁺-ATPase with proteins in the isolated aggregates and thereby resulting in reassembly into a more organized state. This interpretation is further supported by the maximal effect of altering ATP hydrolysis in the aggregates found at 2 h reflow compared with the diminished effect at 6 h reflow. On the basis of the known pattern of recovery of cell polarity where at 2 h the cell is highly disorganized but by 6 h polarity is largely restored (12, 29), one would expect that at later reflow (6 h) the membrane-cytoskeleton complex in aggregates would be more intact and organized, thus diminishing the reparative effects of ATP hydrolysis. The continued release of HSP-72 at 6 h reflow, when the ATP effect on Na⁺-K⁺-ATPase is diminished, suggests that this stress protein may be complexed with other denatured proteins in the aggregates at this time. However, it cannot be excluded that ATP incubation resulted in release of HSP-72 from its substrate before it could complete its chaperone functions. In this case, the removal of other adjunctive cytosolic factors by the aggregate isolation procedures and the subsequent release of HSP-72 by ATP might have resulted in the formation of more aggregable complexes of nonnative Na⁺-K⁺-ATPase from the injured renal epithelia. Further studies are needed to characterize the pattern and sequence of postischemic disruption of the injured membrane-cytoskeletal complex and to identify other specific aggregated proteins that are substrates for HSP-72-mediated repair processes.

In conclusion, this study confirms the temporal associations between induction of HSP-72 and changes in Na⁺-K⁺-ATPase in renal cortex after ischemic injury, demonstrating that restoration of Na⁺-K⁺-ATPase anchorage to the cytoskeleton coincides with increased expression of HSP-72. It further demonstrates that a cellular subfraction of aggregated proteins can be isolated after renal ischemia. Moreover, functional interaction between HSP-72 and Na⁺-K⁺-ATPase during the recovery process is suggested by the effects of manipulating ATP hydrolysis on the release of HSP-72 from and the association of Na⁺-K⁺-ATPase with this aggregate subfraction. HSP-mediated and ATP-dependent repair processes may thus play an integral role in the restoration of cellular integrity during recovery from renal ischemia.