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J Am Soc Nephrol 13:2094-2101, 2002
© 2002 American Society of Nephrology

Functional Activation of Heat Shock Factor and Hypoxia-Inducible Factor in the Kidney

Oliver Eickelberg*, Frank Seebach*, Michael Riordan{dagger}, Gunilla Thulin{dagger}, Andrea Mann*, Kimberly H. Reidy{dagger}, Scott K. Van Why{dagger}, Michael Kashgarian* and Norman Siegel{dagger}

Departments of *Pathology and {dagger}Pediatrics, Yale University School of Medicine, New Haven, Connecticut.

Correspondence to Dr. Oliver Eickelberg, Giessen University School of Medicine, Department of Medicine II, Aulweg 123, D-35392 Giessen, Germany. Phone: +49-641-99-42300; Fax: +49-641-99-42309; E-mail: oliver.eickelberg{at}innere.med.uni-giessen.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. Renal ischemia is the result of a complex series of events, including decreases in oxygen supply (hypoxia) and the availability of cellular energy (ATP depletion). In this study, the functional activation of two stress-responsive transcription factors, i.e., heat shock factor-1 (HSF-1) and hypoxia-inducible factor-1 (HIF-1), in the kidney was assessed. When rats were subjected to 45 min of renal ischemia, electrophoretic mobility shift assays of kidney nuclear extracts revealed rapid activation of both HIF-1 and HSF. Western blot analyses further demonstrated that this activation resulted in increased expression of the HSF and HIF-1 target genes heat shock protein-72 and heme oxygenase-1, respectively. Whether hypoxia or ATP depletion alone could produce similar activation patterns in vitro was then investigated. Renal epithelial LLC-PK1 cells were subjected to either ATP depletion (0.1 µM antimycin A and glucose deprivation) or hypoxia (1% O2). After ATP depletion, HSF was rapidly activated (within 30 min), whereas HIF-1 was unaffected. In contrast, hypoxia led to the activation of HIF-1 but not HSF. Hypoxic activation of HIF-1 was observed within 30 min and persisted for 4 h, whereas no HSF activation was detected even with prolonged periods of hypoxia. HIF-1 was transcriptionally active in LLC-PK1 cells, as demonstrated by luciferase reporter gene assays using the vascular endothelial growth factor promoter or a synthetic promoter construct containing three hypoxia-inducible elements. Interestingly, intracellular ATP levels were not affected by hypoxia but were significantly reduced by ATP depletion. These findings suggest that HIF-1 is activated specifically by decreased O2 concentrations and not by reduced ATP levels alone. In contrast, HSF is activated primarily by metabolic stresses associated with ATP depletion and not by isolated O2 deprivation. In vivo, the two transcription factors are simultaneously activated during renal ischemia, which might account for observed differences between in vivo and in vitro epithelial cell injury and repair. Selective modulation of either pathway might therefore be of potential interest for modification of the response of the kidney to ischemia, as well as the processes involved in recovery from ischemia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tissue ischemia is a major underlying cause of death in the Western world and is observed in many disease states, including stroke, myocardial infarction, cancer, and acute renal failure (1). In the kidney, ischemia may cause severe epithelial cell injury and result in acute renal failure (2–4). The basic components of any ischemic insult include (1) a decrease in local oxygen supplies, (2) decreases in the levels of metabolic substrates (e.g., ATP or glucose), and (3) local accumulation of toxic metabolic end products (5–8). This cascade of events triggers multiple reactive metabolic and genetic mechanisms, of adaptive or maladaptive nature, in the respective tissue compartment. Depending on the severity of the insult and the inherent susceptibility of the tissue, ischemia can result in a permanent loss of function attributable to cell death or a transient compromise of function attributable to sublethal injury, with subsequent recovery (2,7,9).

Activation of transcription factors is one of the most rapid cellular events that occur in response to ischemia (10). Heat shock factor-1 (HSF-1) and hypoxia-inducible factor-1 (HIF-1) represent two separate classes of transcription factors that are specifically and rapidly activated by cellular stress (11–13). HSF-1 is important in the induction of heat shock proteins (HSP), which in turn act as chaperones, preventing the aggregation and inactivation of essential cellular proteins (12). In unstressed cells, HSF-1 remains inactive in the cytosol via its binding to HSP-90. During cellular stress, these monomers rapidly dissociate from HSP-90, trimerize, and are translocated to the nucleus, where HSF activates the transcription of genes that contain the characteristic heat shock element (14–16).

HIF-1 is specifically activated by decreases in tissue oxygen supplies, although some reports have suggested that HIF-1 activation can also be observed in response to stresses associated with energy deprivation (17). HIF-1 is a heterodimer composed of a highly regulated HIF-1{alpha} subunit and a constitutively expressed HIF-1{beta}/aryl hydrocarbon nuclear translocator subunit. HIF-1{alpha} is the DNA binding partner, and its translation is tightly controlled by tissue O2 levels. With physiologic O2 concentrations (>10% O2), HIF-1{alpha} mRNA is generated and translated but HIF-1{alpha} protein is immediately ubiquitinated, targeted to proteasomes, and degraded. Decreases in O2 levels inhibit the ubiquitination and degradation of HIF-1{alpha}, leading to exponentially increased HIF-1{alpha} protein levels and DNA binding activity (11,18–21). Once it is transcriptionally active, HIF-1 activates a family of genes containing a characteristic hypoxia-inducible element, including angiogenic factors [vascular endothelial growth factor VEGF-receptor (FLT)–1, and transforming growth factor-{beta}], glycolytic enzymes (hexokinase-1 and -2 and lactate dehydrogenase), glucose transporters, and cytoprotective genes [heme oxygenase-1 (HO-1) and -2] (22).

In this study, we investigated HSF-1 and HIF-1 activation during renal ischemia in vivo and in vitro, in an effort to characterize transcription factors that might be involved in the renal response to ischemia. We also analyzed the expression of known target genes for these transcription factors after varying reflow periods. In an attempt to further delineate the precise triggers for these events, we then subjected LLC-PK1 renal epithelial cells to hypoxia or ATP depletion and assessed the activation of HIF-1 and HSF-1 in vitro.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal Model
Studies were conducted with anesthetized male Sprague-Dawley rats (age, 12 to 14 wk; weight, 225 to 280 g), as described previously (23). Rats were housed in metal cages with access to regular chow and tap water ad libitum. Animals were maintained at ambient room temperature (25°C) with a 12-h/12-h light/dark cycle. Unilateral renal ischemia was produced by selective occlusion of one renal artery. After 45 min of ischemia, control and ischemic kidneys were removed from the animals, immediately placed on ice, and processed for tissue homogenization and nuclear extraction. Sham-operated animals underwent the same anesthesia, laparotomy, renal vessel isolation, and harvesting procedures as ischemic rats (24).

Cell Culture
LLC-PK1 cells (clonal line Cl4 3-B, a gift from C. Slayman, Yale University), derived from porcine renal proximal tubules, were cultured in {alpha}-minimal essential medium supplemented with 10% fetal bovine serum, 8 mM L-glutamine, and 20 mM Hepes (25). For all experiments, cells were grown until confluent and subjected to treatments as indicated. ATP depletion was achieved by placing cells in substrate-free {alpha}-minimal essential medium (containing no D-glucose or amino acids) supplemented with 100 mg/dl L-glucose and 0.1 µM antimycin A (a mitochondrial respiratory chain inhibitor) (13). Hypoxia was achieved by placing identical cultures in incubators equilibrated to 0.5% O2, as indicated. No antibiotics or antimycotics were added to the cultures at any time.

Western Blot Analyses
Kidneys were immediately removed from the retroperitoneum and frozen in a dry ice/ethanol bath. For Western blot analyses, tissues were homogenized, with a tissue grinder, in lysis buffer consisting of 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM ethylenediaminetetraacetate (EDTA), 1 mM ethylene glycol bis({beta}-aminoethyl ether)-N,N, N', N'-tetraacetate (EGTA), 1% Triton X-100, 2.5 mM sodium pyrophosphate, and 1 mM {beta}-glycerophosphate. A proteinase inhibitor set (Complete;Roche Molecular Biochemicals, Indianapolis, IN) and Na3VO4 (2 mM) were added to the lysis buffer immediately before homogenization. Homogenates were sheared by multiple aspirations through a 24-gauge needle, agitated at 4°C for 30 min, and then centrifuged at 15,000 rpm (at 4°C) for 10 min. Supernatants were used as whole-tissue lysates, and protein concentrations were measured by using the Bradford assay (BioRad Laboratories, Richmond, CA), according to the instructions provided by the manufacturer. Equal amounts of protein (10 µg) were separated on 7.5% sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose membranes, as described previously (25). Western blotting was performed with antibodies against HO-1, constitutive HSP-70, or HSP-72 (StressGen, Victoria, British Columbia, Canada). Specific bands were observed, after incubation with the respective secondary antibodies, via autoradiography using enhanced chemiluminescence analysis, according to the instructions provided by the manufacturer (SuperSignal; Pierce Chemicals, Rockford, IL).

Preparation of Nuclear Extracts
For analysis of DNA binding activities, kidneys were homogenized in buffer containing 10 mM Hepes (pH 7.5), 10 mM KCl, 0.5 mM spermidine, 5 mM EDTA, 1 mM EGTA, and 0.5 M sucrose, supplemented with a set of proteinase inhibitors (Complete). Homogenates were centrifuged for 10 min at 6000 x g (at 4°C), and the resulting pellets were washed twice with ice-cold phosphate-buffered saline (PBS) and processed for preparation of nuclear and cytosolic extracts, as described previously (26). In brief, cell pellets were resuspended in 200 µl of low-salt buffer (20 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM NaVO4, 1 mM EDTA, 1 mM EGTA, 0.2% Nonidet P-40, 10% glycerol, with Complete proteinase inhibitors). After 10 min of incubation on ice, the samples were centrifuged at 8000 x g for 1 min (at 4°C), and the supernatants (cytosolic extracts) were immediately frozen in a dry ice/ethanol bath. Pelleted nuclei were resuspended in 120 µl of high-salt buffer (20 mM Hepes, pH 7.9, 420 mM NaCl, 10 mM KCl, 0.1 mM NaVO4, 1 mM EDTA, 1 mM EGTA, 20% glycerol, supplemented with Complete), and nuclear proteins were extracted with shaking on ice for 30 min. Samples were centrifuged at 13,000 x g for 10 min (at 4°C), and the supernatants were used as nuclear extracts. Protein concentrations of all samples were determined by using the standard Bradford assay.

Electrophoretic Mobility Shift Assays
DNA mobility shift assays were performed with oligonucleotides comprising the consensus sequences for HSF-1 (5'-GCCGCGAACCTTCCCGAAACC-3') or HIF-1 (5'-GCCCTACGTGCTGTCTCA-3'). Oligonucleotides were end-labeled with [{gamma}-32P]ATP by using T4 polynucleotide kinase. Aliquots of nuclear extracts (4 µg) were incubated with labeled oligonucleotides (5 ng) under binding conditions [4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol, 50 mM NaCl, 10 mM Tris-HCl, pH 7.5, 50 µg/ml poly(dI-dC)], in a total volume of 10 µl. Incubations were performed at room temperatures for 30 min. Protein-DNA complexes were applied to 4% polyacrylamide gels, subjected to electrophoresis, and analyzed by autoradiography, as described previously (23,26).

Luciferase Reporter Gene Assays
For luciferase assays, LLC-PK1 cells were seeded onto 48-well plates (1 x 104 cells/well) until 50% confluent. Cells were transfected with one of the following plasmids: pVEGF-luc (comprising 700 bp of the rat VEGF promoter, kindly provided by A. Levy, Harvard University) (27), pHIF3wt-luc [containing a triple repeat of the hypoxia response element (HRE)], or pHIF3mut-luc (containing three inactivated HRE, achieved via site-directed mutagenesis) (both kindly provided by R. Wenger, University of Leipzig) (28). Transfections were performed by using the cationic lipid Tfx-50 (Promega, Madison, WI), at a DNA/lipid ratio of 1:3 (using 0.3 µg plasmid/well) (25). Transfections were performed in the absence of fetal bovine serum for 2 h at 37°C, in a humidified atmosphere. Cells were then subjected to hypoxia for 16 h, washed twice with ice-cold PBS, and lysed; equal amounts of lysates were analyzed for firefly luciferase expression. In brief, 10-µl aliquots of cell lysates were mixed with 50 µl of luciferase reagent buffer, and the luminescence of the samples was integrated for a period of 10 s, in a luminometer. Nonspecific effects and transfection efficiencies were monitored with coexpression of Renilla luciferase (dual luciferase assay; Promega).

Analyses of Intracellular ATP Concentrations
Intracellular ATP concentrations were measured by using a luciferase-based method, as described previously (13). In brief, control cells or cells subjected to ATP depletion or hypoxia for the indicated times were washed twice with ice-cold PBS and lysed with the addition of equal volumes of 3.6% perchloric acid. Samples were centrifuged, and ATP concentrations in the supernatants were determined by using an ATP bioluminescence assay (Sigma Chemical Co., St. Louis, MO), according to the instructions provided by the manufacturer.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In Vivo Renal Ischemia
When rats were subjected to unilateral renal ischemia, rapid activation of HSF was observed in ischemic but not control kidneys (Figure 1A). When kidney nuclear extracts were incubated with labeled oligonucleotides containing the heat shock element, a DNA binding complex appeared in ischemic kidneys (Figure 1A, lanes 2 and 4); the complex was absent in control kidney extracts (Figure 1A, lanes 1 and 3). Analysis of the ischemic extracts for the presence of HSF revealed that this complex could be supershifted with the addition of antibodies against HSF-1 or HSF-2 (Figure 1C, compare lanes 2 and 3 with lane 1). The addition of excess amounts of unlabeled oligonucleotides led to the disappearance of the observed DNA-binding HSF complex, as demonstrated previously (23). Therefore, HSF was clearly activated after 45 min of renal ischemia and was identified as a hetero-oligomeric complex consisting of HSF-1 and HSF-2 isoforms.



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  		Figure 1. Activation of heat shock factor (HSF) and hypoxia-inducible factor-1 (HIF-1) in ischemic rat kidneys in vivo. (A and B) Nuclear extracts from control and ischemic rat kidneys were incubated with labeled oligonucleotides containing specific binding sequences for HSF [termed the heat shock element (HSE)] (A) or HIF-1 [termed the hypoxia response element (HRE)] (B) and were separated on 5% polyacrylamide gels. Transcription factor complexes were then observed autoradiographically. (A) A representative gel-shift assay with control (lanes 1 and 3) and ischemic (45 min of unilateral ischemia) (lanes 2 and 4) rat kidneys demonstrates activation of HSF, as confirmed by the appearance of specific HSF-heat shock element complexes in ischemic extracts. (B) A representative gel-shift assay with control (lanes 5 and 7) and ischemic (lanes 6 and 8) rat kidneys demonstrates HIF-1 activation after 45 min of unilateral ischemia, as confirmed by the appearance of specific HIF-HRE complexes in ischemic extracts. (C) The HSF-heat shock element complexes in ischemic rat kidneys (lane 1, HSF-HSE) could be supershifted (HSF-supershift) by preincubation of the samples with antibodies against HSF-1 (lane 2) or HSF-2 (lane 3). (D) The HIF-HRE complexes (lane 4, HIF-HRE) could be supershifted (HIF-supershift) by preincubation of the samples with antibodies against HIF-1{alpha} (lane 5) or HIF-1{beta} (lane 6). All gels are representative of data obtained with six different animals subjected to 45 min of unilateral ischemia.

 
We then evaluated whether renal ischemia would also activate transcription factors that bind to the HRE (Figure 1B). A protein complex binding to labeled oligonucleotides containing the HRE was present in nuclear extracts derived from ischemic rat kidneys (Figure 1B, lanes 6 and 8) but not control kidneys (Figure 1B, lanes 5 and 7). Analysis of these ischemic extracts for the presence of HIF proteins revealed that this complex could be supershifted with the addition of antibodies specific for HIF-1{alpha} or HIF-1{beta}(Figure 1D, compare lanes 5 and 6 with lane 4). Therefore, HIF-1, like HSF, is activated in rat kidneys subjected to 45 min of ischemia.

Expression of HSF and HIF Target Genes
To assess whether the observed DNA binding activity of HSF and HIF resulted in increased expression of known target genes for either factor, we subjected rats to 45 min of ischemia, followed by varying reflow periods of 15 min to 24 h. After the indicated reflow periods, whole-kidney extracts were prepared and analyzed for expression of HSP-72 and HO-1 (Figure 2), i.e., genes that are known to be activated by HSF and HIF, respectively. As depicted in Figure 2, the expression of HSP-72 and HO-1 was significantly upregulated during the reflow periods, with different kinetics. HSP-72 expression increased after 2 h of reflow, progressively increased with time, and reached a maximum after 24 h of reflow. HO-1 expression, in contrast, was already maximal after 2 h of reflow (Figure 2). The expression of constitutive HSP-70, which served as a loading control, remained unchanged during and after ischemia. Therefore, the HSF and HIF DNA binding activities observed during renal ischemia were functionally active and led to increased expression of downstream target genes. Of note, we observed some interindividual differences in the time courses of HSP-72 and HO-1 induction. Although all animals exhibited increased expression of both target genes, some animals demonstrated faster induction than others, probably because of inherent variations in the stress response system.



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  		Figure 2. Effects of reflow periods on the expression of heat shock protein-72 (HSP-72), heme oxygenase-1 (HO-1), and constitutive HSP-70 (HSC-70) gene expression in rat kidneys subjected to 45 min of ischemia. Rats were subjected to unilateral renal ischemia for 45 min, followed by reflow periods of 15 min, 2 h, 6 h, or 24 h, as indicated. After the reflow periods, whole-kidney extracts were prepared, and equal aliquots (10 µg) were applied to 7.5% sodium dodecyl sulfate-polyacrylamide gels. Proteins were separated by gel electrophoresis and blotted onto nitrocellulose membranes. Blots were hybridized with antibodies specific for inducible HSP-72, HO-1, or constitutive HSP-70 (all from StressGen).

 
In Vitro Injuries
Because both HSF and HIF-1 were activated by renal ischemia in vivo, we sought to delineate the functional activation of each factor by either ATP depletion or hypoxia in vitro. Renal epithelial LLC-PK1 cells were exposed to either isolated ATP depletion (Figure 3, A and C) or hypoxia (1% O2) (Figure 3, B and D) for various time periods, and identical nuclear extracts were assayed for either HSF or HIF-1 activation in gel-shift analyses.



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  		Figure 3. Activation of HSF and HIF in renal epithelial LLC-PK1 cells in vitro. Nuclear extracts from LLC-PK1 cells exposed to 30 to 240 min of ATP depletion or hypoxia (1% O2) were investigated for HSF or HIF activation, as described in the legend to Figure 1. (A and B) Representative gel-shift assays demonstrating activation of HSF during ATP depletion [indicated by the appearance of specific HSF-heat shock element (HSF-HSE) complexes] (A) but not during hypoxia (B). (C and D) Representative gel-shift assays demonstrating HIF-1 activation during hypoxia (indicated by the appearance of specific HIF-HRE complexes) (D) but not during ATP depletion (C). Data shown are representative of three independent experiments with different LLC-PK1 cell passages.

 
HSF was inactive in control LLC-PK1 cells (Figure 3, A and B, 0 min) (23). When cells were subjected to ATP depletion, HSF was rapidly activated as early as 30 min after the injury, with a progressive increase after 240 min of ATP depletion (Figure 3A). In contrast, HSF was not activated by isolated hypoxia, even with prolonged exposure to 1% O2 (Figure 3B).

Similar to HSF, HIF was inactive in control LLC-PK1 cells (Figure 3, C and D, 0 min). In contrast to HSF, however, HIF-1 was not activated in the nuclear extracts generated from LLC-PK1 cells subjected to ATP depletion (Figure 3C). Rapid activation of HIF-1 could be observed under hypoxic conditions (Figure 3D). A specific HIF-1 complex was noted as early as 30 min after exposure of the cells to 1% O2, with maximal activation after 240 min (Figure 3D). This complex could be supershifted with the addition of antibodies against HIF-1{alpha} or HIF-1{beta}, and the complex disappeared after the addition of unlabeled competitor oligonucleotides (data not shown; similar to the data presented in Figure 1D). Exposure of LLC-PK1 cells to ATP depletion thus results in rapid activation of HSF but fails to activate HIF-1, whereas exposure of the same cells to hypoxia activates HIF-1 but not HSF.

HIF-1{alpha} Function in LLC-PK1 Cells
We previously reported that HSF activation results in a functional heat shock response in the kidney in vivo (23) and in LLC-PK1 cells in vitro (13). Relatively little is known, however, regarding the function of HIF in LLC-PK1 cells. To investigate whether the observed activation of HIF-1 in LLC-PK1 cells would result in transcriptional activation of target genes, we analyzed the transcriptional response to HIF-1 activation by performing luciferase assays with two different reporter genes, i.e., a reporter construct containing 651 bp of the VEGF promoter (pVEGF-luc) and a synthetic HIF-1-responsive reporter containing a triple repeat of the HRE cloned in front of a minimal promoter (pHIF3wt-luc). As depicted in Figure 4, the expression of both reporter constructs was significantly increased after 24 h of hypoxia in LLC-PK1 cells; pVEGF-luc was induced 5.9-fold and pHIF3wt-luc was induced 4.3-fold after 24 h of hypoxia (Figure 4, A and B). These inductions were specific, because hypoxic exposure of LLC-PK1 cells did not alter the expression of pHIF3mut-luc (a construct identical to pHIF3wt-luc but with the HRE rendered inactive via site-directed mutagenesis) (Figure 4C).



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  		Figure 4. Effects of hypoxia on HIF-1-dependent promoter activity. The plasmids pVEGF-luc [containing 850 bp of the vascular endothelial growth factor (VEGF) promoter] (A), pHIF3wt-luc (containing a triple repeat of the HRE) (B), and pHIF3mut-luc (identical to pHIF3wt-luc but with the HRE rendered inactive via site-directed mutagenesis) (C) were transfected into LLC-PK1 cells via lipofection. Cells were then exposed to hypoxia for 24 h and lysed, and luciferase activity was measured. Transfection efficiency was controlled for with the concomitant expression of Renilla luciferase (dual luciferase assay). Values are means ± SEM of quadruplicate determinations. *P < 0.001. Data are representative of three independent sets of experiments. RLU, relative luminescence units.

 
Cellular ATP Levels during Hypoxia
To determine whether the activation of HIF-1 coincided with changes in cellular ATP levels, ATP levels in LLC-PK1 cells were determined after 2 or 4 h of substrate deprivation (Figure 5A) or 4 or 24 h of hypoxia (Figure 5B). As described previously (13), exposure of LLC-PK1 cells to substrate deprivation for 2 h resulted in a dramatic decrease in cellular ATP levels, to <25% of control levels (9.4 ± 0.4 nmol/mg protein versus 38.7 ± 1.7 nmol/mg protein in control cells) (Figure 5A). However, exposure of LLC-PK1 cells to hypoxia did not alter cellular ATP levels (41.8 ± 0.6 nmol/mg protein versus 38.7 ± 1.7 nmol/mg protein in control cells) (Figure 5B), indicating that HIF-1 activation occurred independently of changes in cellular ATP levels.



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  		Figure 5. Effects of substrate deprivation or hypoxia on intracellular ATP levels. ATP levels in LLC-PK1 cells were determined after 2 or 4 h of ATP depletion (A) or 4 or 24 h of hypoxia (B), with a luciferase bioluminescence method (23), and were referred to total cellular protein contents. ATP levels were measured in triplicate, and data from two independent experiments were pooled. *P <0.001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our understanding of the molecular events that occur during renal ischemia is incomplete and is the subject of intense investigation (29,30). Renal ischemia leads to epithelial cell injury predominantly in the S3 segment of the proximal tubules, and the outcomes after injury depend largely on the regeneration of this specific cell population. In general, the activation of transcription factors is one of the earliest events in the cellular response to ischemia (10). Therefore, the delineation of such molecular events might provide valuable insights into the regulatory pathways that determine the changes in gene expression patterns during and after renal ischemia. The complexity of the ischemic insult suggests coactivation of multiple transcription factor families, inducing the expression of entire gene families, some of which might lead to adaptive responses and others to maladaptive responses. This study focused on the functional activation of two transcription factor systems, namely the HSF and HIF systems, in kidneys in vivo and in LLC-PK1 cells in vitro. Each of these transcription factors can activate a large number of genes that have been associated with cellular and molecular alterations in ischemic kidneys.

One of the most intriguing findings of this study was the observation that, although both HSF and HIF-1 were activated during ischemia in vivo, distinct mechanisms of activation could be elicited for each transcription factor in vitro. It was previously reported that HSF is activated by renal ischemia or ATP depletion (13,23); this study compared the HSF and HIF systems during renal ischemia in vivo and ATP depletion in vitro. HSF was activated by ATP depletion but not by a decrease in oxygen levels. Exposure of LLC-PK1 cells to hypoxia did not result in decreased levels of ATP, whereas substrate deprivation caused a profound decrease in cellular ATP levels. Therefore, activation of HSF might be primarily induced by cellular events that are initially triggered during ATP depletion. Previous studies linked HSF activation with decreased cellular ATP levels in the kidney and heart (23,31). As confirmed in this study, activation of HSF by ATP depletion is known to result in functional upregulation of inducible HSP (e.g., HSP-72) during reflow after ischemia (32–34).

In contrast to HSF activation, HIF-1 activation in LLC-PK1 cells occurred independently of a decrease in cellular ATP levels but was dependent on a reduction in tissue oxygenation. HIF-1 activation in response to hypoxia in vivo has been observed in multiple organs, such as heart, muscle, and brain (35–39). Those studies described rapid activation of HIF-1 after exposure to hypoxia; however, the net effects of HIF-1 activation on outcomes after cellular injury remain to be elucidated. Many in vitro studies have identified adaptive genes whose expression requires HIF-1 activation, including VEGF, VEGF receptors, erythropoietin, HO-1, and enzymes of the glycolytic pathway (11,27,35,40,41). However, HIF-1 also mediates the induction of multiple maladaptive genes, such as the proapoptotic genes Nip3 and caspase-3 (42–45). It remains to be determined whether HIF-1 activation, with subsequent induction of target genes, propagates an adaptive or maladaptive response to hypoxia during renal ischemia.

In cells lacking HIF-1{alpha}, the induction of hypoxia-responsive genes is absent, indicating that HIF-1 activity is absolutely required for the induction of this family of genes (40). Mice with a homozygous deletion of HIF-1{alpha} die in utero as a result of multiple developmental abnormalities (21,46). Heterozygous mice lacking one HIF-1{alpha} allele demonstrate reduced hypoxia-induced activation of HIF-1 and develop less severe hypoxic pulmonary hypertension and right ventricular hypertrophy, compared with wild-type mice (47), suggesting that HIF-1{alpha} activity might indeed contribute to some pathophysiologic changes observed during chronic hypoxia.

However, erythropoietin expression is rapidly induced by HIF-1 and has been demonstrated to accelerate functional recovery from ischemic renal injury (48) or cisplatin-induced acute renal failure (49). Similar observations have been made for HO in ischemic acute renal failure (50) and for VEGF in experimental thrombotic microangiopathy (51); both genes are also target genes induced by HIF-1 activation. These observations indicate that, at least in the kidney, HIF-1 activation might result in an adaptive response to renal ischemia. In this study, we demonstrated that ischemia in vivo and hypoxia in vitro (Figures 2 and 4) functionally activated HIF-1, resulting in the induction of gene products that might affect the outcomes of ischemic renal injuries. Therefore, manipulation of HIF-1 activation in the kidney might have profound effects on injury severity or the processes of recovery and repair.

Our observations clearly indicate that in vitro cellular injuries might not completely recapitulate the complexity of in vivo ischemia and that specific insults induce distinct transcription factor subsets in vitro. These findings suggest that similar discrepancies might exist for other transcription factors or entire gene families, indicating that interactions between different groups of genes might be fundamental to the critical processes of injury or repair. Moreover, the modulation of one group of genes might substantially affect either the severity of epithelial cell insults or the fate of injured cells (4).

In summary, our study demonstrates the existence of two independent transcription factor pathways that are coactivated during renal ischemia. Because many of the downstream effects of these pathways are still unknown, it would be interesting to selectively modify (via inactivation or overexpression) either pathway in the kidney. Modulation of HSF and/or HIF-1, either together or separately, might have profound effects on injury severity or the process of recovery from renal ischemia. An understanding of the complex cellular and molecular events that lead to ischemic injury or promote recovery will require the elucidation of synergistic and/or antagonistic interactions of entire families of transcription factors such as HSF and HIF-1. This study is a first step in that direction.


   Acknowledgments
 
Dr. Eickelberg was the recipient of an Advanced Postdoctoral Fellowship from the Juvenile Diabetes Foundation International (Grant 10-2000-71) and of the Sofja Kovalevskaja Award from the Alexander von Humboldt Foundation. Support for this work was provided by the National Institutes of Health (Grants DK44336, DK38979, and HD32573). We thank Andrew P. Levy (Technion-Israel Institute of Technology) for providing the VEGF-luciferase construct and Roland H. Wenger (University of Leipzig, Germany) for providing the HIF-luciferase construct.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication January 4, 2002. Accepted for publication April 3, 2002.




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