Thresholds for cellular disruption and activation of the stress response in renal epithelia

Scott K. van Why, Sunmi Kim, John Geibel, Frank A. Seebach, Michael Kashgarian, and Norman J. Siegel

Departments of Pediatrics, Pathology and Surgery, Yale University School of Medicine, New Haven, Connecticut 06520-8064


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Renal ischemia causes a rapid fall in cellular ATP, increased intracellular calcium (Cai), and dissociation of Na+-K+-ATPase from the cytoskeleton along with initiation of a stress response. We examined changes in Cai, Na+-K+-ATPase detergent solubility, and activation of heat-shock transcription factor (HSF) in relation to graded reduction of ATP in LLC-PK1 cells to determine whether initiation of the stress response was related to any one of these perturbations alone. Cai increased first at 75% of control ATP. Triton X-100 solubility of Na+-K+-ATPase increased below 70% control ATP. Reducing cellular ATP below 50% control consistently activated HSF. Stepped decrements in cellular ATP below the respective thresholds caused incremental increases in Cai, Na+-K+-ATPase solubility, and HSF activation. ATP depletion activated both HSF1 and HSF2. Proteasome inhibition caused activation of HSF1 and HSF2 in a pattern similar to ATP depletion. Lactate dehydrogenase release remained at control levels irrespective of the degree of ATP depletion. Progressive accumulation of nonnative proteins may be the critical signal for the adaptive induction of the stress response in renal epithelia.

heat-shock proteins; kidney; heat-shock transcription factor; calcium; sodium-potassium-adenosinetriphosphatase


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

A SERIES OF CELLULAR perturbations in renal epithelia are caused by energy deprivation from hypoxia, ischemia, or metabolic inhibition. Early in the injury process, reversible alterations include increases in free intracellular calcium (Cai), disruption of the cytoskeleton, and loss of cell polarity (6, 16, 22, 24, 25, 43). These changes are rapid, occur before lethal injury, and are reversible. Both calcium-dependent and calcium-independent mechanisms are involved in disruption of the cytoskeleton and plasma membrane that underlie the loss of cell polarity (7, 24, 25, 29, 35). The precise mechanism by which the injured epithelium recovers normal architecture is not known; however, stress proteins likely play a significant role. The stress proteins HSP-72 and HSP-25 are both induced by renal ischemia (1, 9, 32, 38). Both of these heat-shock proteins have been shown to be protein chaperones and to be instrumental in resurrecting denatured proteins (8, 12, 17).

Induction of the stress response and subsequent heat-shock protein synthesis has been well characterized in cell injury from a variety of insults (26). The initial event in the stress response is activation of the heat-shock transcription factor (HSF), which then undergoes trimerization and binding to the heat-shock element (HSE; see Refs. 26 and 44). The activity of HSF is itself regulated by HSP-70, which in turn may be affected by the quantity of denatured protein within a cell (23). In addition, HSF activity and stress protein induction may be modulated by changes in free Cai (5, 14, 15, 30). We previously studied the relationship of stress response initiation to specific decrements in ATP in renal cortex in vivo. As indicated by activation of HSF, the stress response was initiated when renal cortical ATP was reduced below a threshold of 50% of control. Further reductions in renal ATP resulted in a more vigorous stress response (40). Cellular ATP, then, or the metabolic consequences associated with its depletion may be threshold factors for initiation of the stress response in the kidney.

In the present study, we examined whether stress response induction was related to ATP depletion alone or whether other cellular perturbations, which could induce a stress response, occur before or coordinate with HSF activation. We determined the relationship of specific decrements in cellular ATP to HSF activation, to changes in Cai, and to disruption of membrane protein-cytoskeletal interactions. We also determined the relationship between HSF activation and subsequent cell death to determine whether the stress response may be adaptive or merely a manifestation of lethal injury. Using cultured renal epithelial cells, we found, as in renal cortex in vivo, that HSF was first activated when cellular ATP was reduced below a threshold of 50% of control and that further decrements in cellular ATP were associated with progressive activation of HSF. ATP depletion activated both HSF1 and HSF2. Proteasome inhibition also activated both HSF1 and HSF2 in a pattern similar to activation caused by ATP depletion. In addition, HSF activation from ATP depletion was not associated with increased cell death, indicating that the stress response is not merely a manifestation of lethal injury. Finally, changes in free Cai and Na+-K+-ATPase solubilization were detectable before HSF activation. Alterations in cell architecture and Cai homeostasis may be instrumental in activating and modulating the stress response in cells injured by energy deprivation.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell preparations. LLC-PK1 cells (clonal line Cl4 3-B; gift from Carolyn Slayman) derived from proximal tubules of porcine kidney were grown in alpha -MEM (GIBCO) with 10% FBS at 37°C in 95% air and 5% CO2. Culture inserts (25 mm diameter) coated with collagen IV (Collaborative Biomedical) were plated with 2.8 × 105 cells. Twenty-four hours after reaching confluence, parallel groups of cells were rinsed with 37°C PBS and then incubated for 2 h in prewarmed substrate-free alpha -MEM (containing no glucose or amino acids) to which was added 0.1 µM antimycin A and graded concentrations of D-glucose from 100 to 0 mg/dl. Media osmolality for each experimental group was equilibrated by providing a complementary quantity of the nonmetabolizable isomer L-glucose to bring the total glucose concentration (D + L) to 100 mg/dl. Controls for each experimental group were cells grown in parallel that underwent equivalent washes in substrate-free alpha -MEM incubation media containing 100 mg/dl D-glucose without antimycin A.

Cellular ATP determination. At the end of the 2-h incubation, cells in each treatment group were rinsed and then harvested in cold PBS. Cellular ATP concentration was measured using a modification of the luciferase assay described by Uchida and Endou (37). Adenine nucleotides were extracted from the cells by addition of an equal volume of 3.6% perchloric acid. ATP content was measured using a luminometer (Lumat LB 9501; Berthold, Wallac, Gaithersburg, MD) and an ATP bioluminescent assay kit (Sigma Chemical, St. Louis, MO). The protein content of each sample was measured using the method of Lowry et al. (20) with BSA as the standard. ATP content was expressed relative to the protein content for each sample, and then cellular ATP levels in each treatment group were expressed as a percentage of the respective control grown in parallel. In a separate series of experiments, cells from each treatment group were rinsed in 37°C PBS, replaced with normal alpha -MEM for 3 h, and then harvested for ATP determination.

Triton X-100 extraction and Western analysis. At the end of the 2-h treatment, cells from each condition were washed with PBS and then harvested in 60 mM PIPES, 10 mM EGTA, and 2 mM MgCl2, pH 6.9, with 0.1% Triton X-100, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 0.1 mM DTT, and 1.0 µg/ml leupeptin. Cells from four filters of the same condition were collected into a microfuge tube and rocked for 20 min at 4°C. The mixture was then centrifuged at 35,000 g for 15 min at 4°C. The supernatant containing the Triton X-100-soluble proteins was removed and stored at -80°C. Protein determination, electrophoresis, and transfer to nitrocellulose of equivalent 50-µg samples from the Triton X-100 extracts were performed as previously described (39). Nonspecific binding sites were blocked with 5% dried milk and 20 mM Tris, pH 7.6, 137 mM NaCl, and 0.2% Tween 20. Membranes were incubated for 1 h with mouse monoclonal antibody to the alpha -subunit of Na+-K+-ATPase (39) diluted 1:10,000. Detection was with secondary antibodies, reagents, and protocols for enhanced chemiluminescence (Amersham, Arlington Heights, IL). Multiple exposures on XAR-5 film (Kodak) were obtained to ensure that signals for each band were within the linear range of the film. Computerized densitometry of the specific bands on all autoradiograms was performed as described previously (39).

Measurement of free Cai changes. Before initiation of metabolic inhibition, 10 mM green 1-AM or calcium orange-AM (Molecular Probes, Eugene, OR) was added to cells in PBS with 100 mg/dl D-glucose and incubated for 30 min at 37°C. After loading the fluorochrome, cells were washed three times with PBS and then treated with experimental media described above to achieve graded decrements in cellular ATP. At 2 h of metabolic inhibition, the membranes containing the cells were removed from the culture inserts and placed on glass slides with PBS to prevent dessication. Images of Cai fluorescence were obtained with an MRC-600 scanning laser microscope (Bio-Rad) equipped with a 488-nm filter for calcium green or a rhodamine filter (514 nm) for calcium orange. To achieve a consistent fluorescence sampling, the variable confocal aperture size was fixed at a setting predetermined to have acceptable signal-to-noise ratio for all samples. The same objective lens (Leitz Water Emersion, X-50), electronic zoom factor, and neutral density filter setting were used for each determination. Each image thus would possess an identical "volume" of fluorescence signal and could be analyzed using the MRC system software. In general, 10 images were accumulated in 5 s using the Kalman mode. In addition, the same image was recorded 1 min apart to evaluate photobleaching, which uniformly was <5%. Ten separate experiments were performed per level of cellular ATP depletion, and 10 images were gathered for each experiment. Relative fluorescence intensity of cellular calcium was determined using IM-4000 image analysis software from Georgia Instruments (Roswell, GA). Free Cai concentration was estimated from the relative fluorescent intensity by using the response calibration equation Ca2+i Kd (F - Fmin)/(Fmax - F) where Kd is the dissociation constant, F is fluorescence, min indicates minimum, and max indicates maximal and using a calcium calibration buffer kit (Molecular Probes). Calcium green measurements demonstrated greater precision and less variation than calcium orange.

Assay of HSF activation. At the completion of the 2 h of metabolic inhibition, cells were rinsed and then harvested in chilled PBS. After removal of the wash by brief centrifugation, the cells were lysed by freezing in liquid nitrogen for 2 min and then thawing at room temperature. Homogenization buffer (20% glycerol, 20 mM HEPES, pH 7.9, 400 mM sodium chloride, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, and 10 µg/ml of leupeptin) was added to the lysates, which were then centrifuged for 15 min at 18,000 g at 4°C. Protein concentration of the supernatants was determined by the Bio-Rad protein assay (Hercules, CA), and aliquots were stored at -80°C. Gel retardation assays using end-labeled HSE and 10 µg of the LLC-PK1 cell protein extracts were then performed as previously described for rat renal cortex protein extracts (40).

Cell integrity. Lactate dehydrogenase (LDH) release was determined as an index of plasma membrane damage to assess the integrity of the cells both at the end of the 2-h interval of metabolic inhibition and 16 h later after replacement with normal media. LDH was assayed as described by Takano et al. (36). Released LDH (supernatant LDH) was expressed as a percentage of total LDH (supernatant LDH + cellular LDH), which is determined after sonication of the cells.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Graded cellular ATP depletion. Figure 1 shows cellular ATP levels, expressed as percentage of parallel control, in cells treated for 2 h with substrate-free media modified with antimycin A and progressive decrements in D-glucose from 100 to 0 mg/dl. In the presence of 100 mg/dl D-glucose, inhibition of aerobic metabolism with antimycin A alone reduced ATP to 75-80% of control. In cells treated with antimycin A, reducing D-glucose concentration down to 20 mg/dl had no additional effect on decreasing cellular ATP. However, progressive reduction in media D-glucose below 20 mg/dl resulted in a graded decline in ATP levels. Antimycin A with 0 mg/dl D-glucose caused a fall in cellular ATP to <5% of control. The inhibitory media was removed, cells were washed, and normal media were replaced to assess recovery of cell ATP in each treatment group. As shown in Fig. 1, at 3 h of recovery, cellular ATP levels in all treatment groups had returned to 80% of control, equivalent to the effect of antimycin A alone. By 24 h of substrate repletion, ATP levels had returned to 100% of control in all groups.


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Fig. 1.   Graded ATP depletion in LLC-PK1 cells. ATP levels in LLC-PK1 cells were determined after 2 h of energy depletion with antimycin A and graded decrements in D-glucose (x-axis). ATP levels were determined both at the end of 2 h of metabolic inhibition (0 h recovery) and after repletion of substrate with normal media (3 h recovery). ATP levels for each experimental condition are expressed as a percentage of parallel cultures of uninhibited controls (C). Bars shown for the 0-h recovery samples represent means ± SE calculated from 10-15 experiments from each experimental condition.

Detergent-extractable Na+-K+-ATPase. Cytoskeletal dissociated Na+-K+-ATPase was detected by Western analysis of Triton X-100 extracts performed at the end of the interval of metabolic inhibition at each level of ATP depletion (Fig. 2). A representative immunoblot (Fig. 2A) shows that, once ATP levels were reduced to <75% of control, increased cytoskeletal-dissociated Na+-K+-ATPase was detected compared with control. There was an additional incremental increase in Triton X-100 solubility of Na+-K+-ATPase with further decrements in cellular ATP. Figure 2B is the summation of densitometry of Na+-K+-ATPase bands from the Triton X-100-soluble extracts at the separate levels of ATP depletion. Reduction in cell ATP down to 75% of control did not cause a significant change in detergent-extractable, cytoskeletal-dissociated Na+-K+-ATPase. Reduction in cell ATP down to <70% of control caused an increase in Na+-K+-ATPase solubility that progressed with additional decrements in cell ATP.



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Fig. 2.   Detergent-soluble Na+-K+-ATPase and graded ATP depletion. Representative immunoblot (A) and densitometry (B) of Na+-K+-ATPase in detergent-soluble extracts from LLC-PK1 cells after graded ATP depletion. ATP levels are expressed as percentage of parallel untreated controls. Quantity of detergent-soluble Na+-K+-ATPase determined by densitometry is expressed relative to the quantity of soluble enzyme in control cells. Results expressed are the means of 4-10 experiments at each separate level of ATP depletion as indicated on the x-axis. Bars indicate SE.

Changes in free Cai with graded ATP depletion. Figure 3 shows the results of steady-state Cai fluorescence in relation to degree of ATP depletion. The fluorescent intensity is expressed on the 0-254 arbitrary unit scale of pixel intensity from the image analysis software. Calcium fluorescence remained at baseline levels until ATP depletion had reached 75% of control. At that level, Cai fluorescence had begun to increase; there was some variability between samples at this level using calcium orange compared with calcium green. Cai fluorescence with both dyes was consistently increased when ATP depletion reached 60% of control. Further decrements in cell ATP resulted in a progressively increased Cai, as measured with both dyes. The steady-state calcium fluorescence intensities were converted into estimated values of Cai concentration using external standards. The mean estimated Cai concentration in control cells was 127 nM using calcium green and 160 nM using calcium orange. Cai initially rose to ~230 nM, as measured with both dyes when ATP levels were reduced to 65% of control. Once Cai fluorescence began to rise, the estimated calcium concentration increased in a linear fashion to ~600 nM.


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Fig. 3.   Free intracellular calcium (Cai) and graded ATP depletion. Cai was measured by fluorescence of the cells loaded with calcium green and calcium orange. Fluorescence intensity is expressed on a scale of arbitrary units from 0 (minimum) to 254 (maximum). Steady-state calcium fluorescence was measured after 2 h of substrate deprivation at each level of ATP depletion. Bars indicate SE.

HSF activation with graded ATP depletion. Figure 4 is a gel retardation assay using protein extracts from LLC-PK1 cells after 2 h of complete metabolic inhibition with antimycin A and 0% D-glucose in substrate-free alpha -MEM. The unbound labeled probe of double-stranded oligonucleotide containing the consensus sequences of the HSE migrates to the bottom of the gel. Activated HSF bound to the labeled HSE are retarded in the gel. Lane 1 contains extracts from cells after 2 h of complete metabolic inhibition, indicating that ATP depletion activates HSF in LLC-PK1 cells to bind the HSE. The binding specificity of activated HSF from LLC-PK1 cells to labeled HSE was confirmed by competition assays. In lane 2, preincubation with excess unlabeled HSE successfully competed for the activated HSF, therefore eliminating subsequent binding of HSF to labeled probe. In lane 3, prior incubation with excess nonspecific double-stranded DNA did not prevent activated HSF from binding to the labeled HSE.


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Fig. 4.   Representative gel retardation assay showing specificity of heat-shock transcription factor (HSF)-heat-shock element (HSE) interaction after ATP depletion in LLC-PK1 cells. Unbound labeled HSE probe migrates freely to the bottom of the gel (open arrow). Activated HSF bound to labeled HSE is indicated by the filled arrow. In all three lanes, the protein extracts were from the same ATP-depleted LLC-PK1 cells. Lane 1, extracts incubated with labeled HSE; lane 2, incubation of extracts with excess unlabeled HSE before addition of labeled HSE; lane 3, preincubation of extracts with nonspecific double-stranded DNA before addition of labeled HSE. ATP depletion activates HSF in LLC-PK1 cells to bind HSE specifically.

The relationship between cellular ATP and activation of HSF in LLC-PK1 cells is shown in Fig. 5. The band shown represents activated HSF bound to labeled HSE. When cellular ATP was maintained above 65% of control, no change in activated HSF was detected. However, below 50% ATP, there was consistent activation of HSF. HSF activation increased incrementally with progressive decrements in cellular ATP below 50% of control.


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Fig. 5.   HSF activation and graded ATP depletion in LLC-PK1 cells. Representative gel retardation assay of LLC-PK1 extracts obtained at each level of ATP depletion. Region at the bottom of the gel containing unbound labeled probe is not shown. Bands shown represent activated HSF bound to HSE. Cellular ATP as a percentage of untreated control levels is indicated at bottom.

Activity of HSF1 and HSF2 with ATP depletion and proteasome inhibition. To determine whether ATP depletion preferentially activates HSF1 or HSF2 in LLC-PK1 cells, extracts from antimycin A-treated cells were incubated with antibody against HSF1 and HSF2 before addition of the labeled HSE (Fig. 6, lanes 2-4). Antibody to HSF1 supershifted nearly all of the activated HSF-HSE complex, whereas antibody to HSF2 supershifted a portion but not all of the activated HSF-HSE complex in extracts from ATP-depleted cells. ATP depletion, then, activates both HSF1 and HSF2 in LLC-PK1 cells.


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Fig. 6.   Activation of HSF1 and HSF2 by ATP depletion and by proteasome inhibition. Cells were ATP depleted for 1 h or treated with the proteasome inhibitor MG132 (10 mM; Peptides International) for 2 h, and nuclear extracts were evaluated for HSF activation by gel retardation assay using the labeled HSE. NS, nonspecific binding to the labeled HSE; HSF, activated HSF. Lanes 1 and 5, extracts from control, vehicle-treated cells showing no HSF activation; lanes 2-4, ATP-depleted cells; lanes 6-8, MG132-treated cells; lanes 2 and 6, no antibody addition; lanes 3 and 7, incubation of extracts with antibody to HSF1 before gel retardation assay; lanes 4 and 8, preincubation with antibody to HSF2 (both antibodies from Chemicon, Temecula, CA). ATP depletion and proteasome inhibition cause a similar activation of both HSF1 and HSF2 in LLC-PK1 cells. Results represent 4 separate sets of experiments for each condition in the ATP-depleted cells and two separate sets for each condition in the MG132-treated cells.

HSF2 has been described previously to be activated only with proteasome inhibition (21) or with hemin-induced differentiation of erythroid cells (34). Because protein degradation by the proteasome is ATP dependent, decreased proteasome activity could contribute to HSF activation in energy-depleted LLC-PK1 cells. To evaluate this possibility, cells were treated with hemin or with the specific proteasome inhibitor MG132. Hemin did not activate HSF in the LLC-PK1 cells, consistent with the previous report of hemin-induced HSF activation being specific to erythroid cell lines (21). On the other hand, the proteasome inhibitor MG132 caused significant HSF activation (Fig. 6, lanes 6-8). Incubation of the extracts with anti-HSF1 or anti-HSF2 antibody indicated that proteasome inhibition activated both HSF1 and HSF2. Similar to the findings in ATP-depleted cells, antibody to HSF1 supershifted nearly all of the activated HSF-HSE complex, but antibody to HSF2 shifted only a portion of the complex induced by MG132 treatment. Therefore, isolated proteasome inhibition results in a pattern of HSF activation in LLC-PK1 cells similar to that induced by energy depletion.

Cell integrity. LDH release was determined as an index of cell integrity both at the end of the 2-h metabolic inhibition (0 h recovery) at each level of ATP depletion and 16 h after substrate repletion (16 h recovery). LDH release was similar and <10% under all experimental conditions, suggesting that, irrespective of the degree of ATP depletion or repletion, the insult inflicted was not lethal (Fig. 7). Subjection of the cells to 24 h of total metabolic inhibition with no repletion of substrate resulted in 100% LDH release, indicating that prolonged energy depletion resulted in uniform cell death.


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Fig. 7.   Lactate dehydrogenase (LDH) release from graded ATP depletion. LDH released into the supernatant is expressed as a percentage of total cellular LDH. Percent LDH release was measured at the end of 2 h of metabolic inhibition (0 h recovery) at each level of ATP depletion and at 16 h of recovery after restoration of substrate with normal media (n = 2 for each experimental condition). LDH release was measured in a parallel set of cells subjected to 24 h of total metabolic inhibition as shown on right (Total).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

To determine whether initiation of the stress response was related to decrements in cell energy alone or whether HSF activation was coordinated with other cellular alterations that result from energy deprivation, we adapted the model of graded ATP depletion in LLC-PK1 cells described by Venkatachalam et al. (41). As they previously described, a stepwise reduction in cellular ATP could be achieved reproducibly using antimycin A with graded glucose deprivation for 2 h. Replacement of the energy deprivation media with normal growth media resulted in progressive recovery of cellular ATP. Compared with control cells, we found no evidence of lethal injury either immediately at the end of 2 h of energy deprivation at any level of ATP or during recovery with substrate repletion. Therefore, the observed increases in detergent extractability of Na+-K+-ATPase, free Cai, and HSF activation all occurred in sublethally injured cells.

Several investigators have carefully defined early events in renal epithelia injured by ATP depletion. Energy deprivation results in a rapid and duration-dependent alteration in cytoskeletal proteins that disrupts membrane-cytoskeletal protein interactions and is manifested by membrane blebbing and loss of cell polarity (6, 24, 25). The previous studies that defined these cellular alterations did so by examining changes over time during total energy deprivation. In each case, the manifestations of injury progressed as the interval of ATP depletion was lengthened. Our finding that detergent-extractable Na+-K+-ATPase progressively increased when ATP is reduced below a threshold of 70% control shows that, in addition to the duration of the energy depletion, the severity of the ATP depletion affects the degree of cellular disruption.

The relationship of specific decrements in cellular ATP to lethal events such as alteration in membrane lipids or commitment to necrotic versus apoptotic pathways to cell death have been defined (19, 41). We employed a shorter duration of metabolic inhibition than used in those studies, which allowed us to examine the relationship of sublethal events to specific decrements in cellular ATP. We found specific thresholds in cellular ATP below which there is disruption of Cai homeostasis and dissociation of Na+-K+-ATPase from the cytoskeleton. Remarkably, the relationship between HSF activation and cellular ATP was the same in LLC-PK1 cells, as we previously found in rat kidney cortex in vivo (40). In both instances, activated HSF was first detected when cellular ATP was reduced to 50% of control; additional reductions in ATP were accompanied by greater HSF activation. These results indicate that graded ATP depletion in cultured cells replicates cellular events in vivo that lead to stress response induction, further supporting this model as representative of early sublethal alterations in cell structure and function resulting from renal ischemia. In addition, the degree to which the stress response is activated is tightly linked to the severity of the insult. In renal epithelia injured by ischemia, then, the stress response is not an all-or-nothing affair. It appears to be an adaptive response that is measured to the severity of the injury.

The threshold where we first could detect increases in Cai was at 75% of control ATP. However, at this level of ATP depletion, the changes in Cai were variable. The threshold for consistent change in Cai was 60% of control ATP. Additional decrements in ATP caused further increases in Cai, but none resulted in lethal injury. Sublethal increases in Cai, therefore, occur early during energy depletion, and the changes in Cai are tightly linked to specific decrements in cellular ATP.

The preponderance of studies have examined changes in Cai at intervals following severe ATP depletion. In one other study, Cai was measured at more moderate levels of ATP depletion (4). Although specific decrements in cellular ATP were not assessed, the findings were similar to ours. Initial, variable increases in Cai were detectable at moderate levels of ATP depletion. A more rapid and consistent increase in Cai accompanied a more profound fall in cellular ATP (4).

As has been previously described in isolated proximal tubules and cultured renal epithelia (4, 7, 11, 16, 18, 22, 42), the early prelethal increases in Cai that we found were in the nanomolar range. Similar fivefold early increases in Cai were previously reported both in MDCK cells (22) and opossum kidney cells (18), which were associated with the rapid fall in cellular ATP. In both cases, these changes in Cai were reversible. We also found that the early changes in Cai from graded ATP depletion of limited duration did not result in cell death. This supports previous work in isolated proximal tubules in which changes in Cai preceded membrane damage and were not necessarily associated with lethal cell injury (11, 16, 42, 43).

The present studies do not and were not designed to determine whether a single specific signal or whether a combination of intracellular events conspire to induce the stress response. They do identify several cellular events that may be involved in the initial activation of HSF or subsequent regulation of its activity. Because we found that with partial ATP depletion early changes in Cai and cell architecture are accompanied by HSF activation, each of these cellular perturbations may contribute to stress response induction in injured renal epithelia. It has been suggested that HSF itself could be a sensor for cellular stress, since nascent HSF undergoes oligomerization that is dependent on the cognate of HSP-70 (Hsc-70) and ATP (31). In addition, in cell-free extracts containing HSF activated by heat, HSF-HSE binding was decreased by the addition of ATP, indicating that the level of cellular ATP itself could modulate HSF activity (33). More likely, the other cellular perturbations that follow energy depletion may be the initial signals for the stress response.

A series of investigators have examined the relationship between changes in Cai and the stress response. Shifts of calcium within specific intracellular domains can affect nucleocytoplasmic trafficking (13). Calcium, therefore, could be required for the translocation of inactive cytoplasmic HSF into the nucleus. Addition of calcium to HeLa cell extracts activated HSF, an effect ablated with stabilization of protein structure by glycerol (27). In permeabilized NIH/3T3 cells, calcium alone did not activate HSF but was required for HSF activation by heat shock (30). In primary cultures of rat proximal tubule epithelia, Cai chelators prevented HSP-70 induction by heat (45). Both calcium-dependent and -independent systems modulate stress response induction in epidermoid cells (15). Taken together, these studies indicate that increases in Cai can enhance stress protein production and are necessary but not sufficient for HSF activation. Therefore, changes in Cai are not the efficient cause of stress response induction but appear to be necessary to modulate the response in a variety of cells.

The principal trigger for the stress response in energy-depleted renal epithelia is, in all probability, the accumulation of disrupted, nonnative, or denatured cellular proteins. HSF is activated upon release from the constitutively expressed protein chaperones when the demand for these chaperones increases as denatured and aggregated proteins accumulate under conditions of cell stress (23, 44). ATP depletion alone causes denaturation and aggregation of proteins in mammalian cells, as demonstrated by the reduced activity and solubility of luciferase as a reporter protein (28). Furthermore, ATP releases HSP-72 complexed to aggregated proteins isolated from renal cortex injured by ischemia (2), and proteasome inhibition (which results in accumulation of abnormal proteins) induces expression of several HSPs in renal epithelia (3). Because the proteasome plays a significant part in ATP-dependent proteolysis, reduced proteasome activity during energy deprivation could contribute to signals for stress protein induction. In a recent report, it was suggested that separate HSFs may be regulated by distinct signalling mechanisms, since HSF2 was preferentially activated in erythroid cells treated with the proteasome inhibitor MG132 (21).

To determine whether inhibition of proteasome activity might contribute to stress response induction in energy-depleted renal epithelia, the pattern of HSF activation in response to ATP depletion was compared with that which occurred with isolated proteasome inhibition. The pattern of HSF activation in response to proteasome inhibition was similar to that caused by ATP depletion; each treatment activated both HSF1 and HSF2, with HSF1 activation being predominant. The dual activation of HSF1 and HSF2 in ATP-depleted and proteasome-inhibited renal epithelia is similar to that found in heat-shocked MEF cells (21). That proteasome inhibition did not preferentially activate HSF2 in LLC-PK1 cells is in contrast to findings in other cell lines reported by Mathew et al. (21) but is consistent with the variable pattern of HSF1 and HSF2 activation that they found in cell lines of different origins. Because the profile of HSF activation did not differ significantly between ATP-depleted and proteasome-inhibited cells, our study does not define the relative contribution of decreased clearance of abnormal proteins compared with increased protein disruption to the overall initiation of the stress response. Nevertheless, the findings suggest that induction of the stress response in energy-deprived renal epithelia is multifactorial and that reduced proteasome activity may play a significant role.

Based on the sequence of events and pattern of HSF activation observed in the present study, we speculate that the critical signal for induction of the stress response in renal epithelia is the accumulation of nonnative proteins through progressive disruption of cellular proteins in the face of reduced proteasome activity. Depletion of cellular ATP could cause accumulation of abnormal cellular proteins either directly or via activation of calcium-dependent enzymes. Because calcium-mediated alterations in the cytoskeleton occur early after ATP depletion and lead to disruption of cell architecture and polarity (7, 25, 29, 35), the link between Cai and HSF activation may be through the changes in these structural proteins. The accumulation of nonnative cellular proteins would then demand the attention of HSP-70 chaperones, triggering the release and activation of HSF. Activated HSF then initiates the stress response, which may be further enhanced by the increased levels of Cai.

By examining cells at graded decrements in cellular ATP, we were able to establish the relationship of specific manifestations of injury to initiation of the stress response. Changes in Cai and disruption of cytoskeletal-membrane protein interactions were detectable at levels of cellular ATP that are higher than required for HSF activation. In renal epithelia subjected to sublethal and reversible injury, then, Cai is increased and Na+-K+-ATPase is dissociated from the cytoskeleton before activation of HSF. The profile of HSF activation from ATP depletion is similar to that caused by proteasome inhibition, so reduced proteasome activity in energy-deprived renal epithelia may contribute to signals for the stress response. Thus the final common pathway for HSF activation in renal epithelia injured by energy depletion may be the accumulation of nonnative proteins, contributed to by both increased disruption of native proteins and decreased clearance of denatured proteins.


    ACKNOWLEDGEMENTS

We are grateful for the excellent assistance of Tom Ardito and Andrea Mann with technical aspects of the studies and Marie Campbell with manuscript preparation.


    FOOTNOTES

This work was supported by National Institutes of Health Grants DK-44336, DK-38979, and HD-32573. This work was performed during the tenure of a Clinician-Scientist Award (to S. K. Van Why) from the American Heart Association.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: S. Van Why, Yale Univ. School of Medicine, Dept. of Pediatrics, 333 Cedar St., PO Box 208064, New Haven, CT 06520-8064.

Received 10 September 1998; accepted in final form 15 April 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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