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Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
ABSTRACT
Ischemic acute renal failure is a frequent clinical problem in hospitalized patients and is associated with significant mortality. Hypoxia-inducible factor 1 (HIF-1) mediates cellular adaptation to hypoxia by regulating biological processes important for cell survival, which include glycolysis, angiogenesis, erythropoiesis, apoptosis, and proliferation. To investigate the role of HIF-1 in hypoxia-induced renal epithelial cell death, we generated mice that allow inactivation of HIF-1 by tetracycline-inducible Cre-loxP-mediated recombination in primary renal proximal tubule cells (PRPTC), resulting in a suppression of HIF-1-mediated gene transcription during oxygen deprivation. In the absence of glucose, the onset and the degree of hypoxia-induced cell death in HIF-1-deficient PRPTC were comparable to wild-type cells. However, when glucose availability was limited, the onset of cell death was delayed in either PRPTC that were HIF-1 deficient or in wild-type PRPTC when glycolysis or glucose uptake was partially inhibited. Our findings suggest in an in vitro genetic model that 1) the generation of adequate energy levels for the maintenance of PRPTC viability under hypoxia does not require HIF-1 and 2) that HIF-1 regulates the timing of hypoxia-induced cell death and apoptosis onset through its effects on glucose consumption.
apoptosis; glycolysis and glucose uptake; renal proximal tubule; Cre-loxP-mediated recombination
THE RENAL PROXIMAL TUBULE plays a central role in ischemic acute tubular necrosis (ATN) (37, 38, 47), a common cause of acute renal failure and major clinical problem in hospitalized patients (48, 72). Despite the availability of renal replacement therapy, ATN, especially in the intensive care setting, is associated with a very high mortality rate of 4080% and little progress has been made in improving this outcome (19). On a cellular level, the development of ischemic ATN is associated with inadequate oxygen and nutrient delivery resulting in gene expression changes orchestrated by hypoxia-responsive transcription factors. A key mediator of hypoxia-induced gene expression is hypoxia-inducible factor 1 (HIF-1). HIF-1 is a member of the Per-ARNT-Sim (PAS) family of basic helix-loop-helix transcription factors. As a global regulator of oxygen homeostasis, this heterodimeric transcription factor facilitates both oxygen delivery and adaptation to oxygen deprivation by regulating biological processes that include glucose uptake and metabolism, angiogenesis, erythropoiesis and iron homeostasis, cellular proliferation, and apoptosis (for comprehensive reviews, see Refs. 60, 63). HIF activation is dependent on stabilization of the oxygen-sensitive -subunit and subsequent nuclear translocation to form a functional complex with HIF- and cofactors such as CBP/p300. In conditions of adequate oxygen supply, iron- and oxygen-dependent prolyl-hydoxylation of the oxygen-dependent degradation domain within HIF- is necessary for binding to the von Hippel-Lindau (VHL) E3-ubiquitin ligase complex, which targets HIF- for proteasomal degradation (recently reviewed in Ref. 60). A second hypoxic switch operates in the COOH-terminal transactivation domain of HIF- with the hydroxylation of an asparagine residue; in hypoxia, asparagine hydroxylation is blocked and CBP/p300 recruitment is facilitated (35).
Cell death by oxygen deprivation has been shown to involve mitochondrial signaling as ablation of cytochrome c, Apaf-1, and caspase 9 confers resistance to hypoxia-induced cell death in murine fibroblasts and in transformed cells (45, 65). This notion is furthermore supported by the observation that increased expression of anti-apoptotic BCL-2 family members (BCL-2, BCL-XL) or the inactivation of proapoptotic BCL-2 family members (BAX, BAK) protects cells from hypoxia-induced apoptosis (8, 45, 64). In addition, protection from hypoxia-induced programmed cell death in immortalized rat kidney proximal tubular epithelial cells has been shown to involve HIF-independent activation of IAP-2 (17). However, severe ATP depletion in conjunction with hypoxia results in necrotic cell death that becomes insensitive to mitochondrial signaling (45). Although ATP depletion can result in both apoptosis and necrosis, a reduction of ATP levels by 80% or more was associated with necrosis in mouse proximal tubular cells (36).
The role of HIF-1 in the regulation of hypoxic cell death remains controversial (for recent reviews on this topic, see Refs. 22, 25, 51) and may be context dependent (3). HIF-1 has a key regulatory function in cellular energy metabolism and thus cell survival by increasing glucose uptake and glycolysis under conditions when generation of ATP through oxidative phosphorylation is not possible due to a lack of oxygen (30, 62). On the other hand, it has also been suggested that HIF-1 promotes cell death through an increase in the expression of proapoptotic factors. BNIP-3 and BNIP-3L are BH3-only proapoptotic BCL-2 family members that are induced by hypoxia in a HIF-1-dependent manner (7). Overexpression of BNIP-3 in particular has been found to induce cell death in tumor cell lines and cardiomyocytes (13, 34, 44, 54, 55, 74). In addition, a HIF-1-dependent decrease in antiapoptotic BCL-2 (12), its involvement in p53-dependent apoptosis (66), and HIF-1-dependent activation of caspase 8 (6) are in support of a proapoptotic role for HIF-1, which appears to be cell type independent.
To investigate the role of HIF-1 in hypoxia-induced cell death in the renal epithelium, we examined primary renal proximal tubule cells (PRPTC) isolated from mice that allow conditional inactivation of HIF-1 by tetracycline-inducible Cre-loxP-mediated recombination. Here, we report that the onset of hypoxia-induced cell death in PRPTC cultures is HIF-1 dependent when glucose availability is limited and also demonstrate that HIF-1 is not required for growth and viability of PRPTC, which are known to be highly glycolytic in culture. Based on our findings, we propose that, once activated, the signaling pathways that are responsible for the execution of hypoxia-induced cell death are largely HIF-1 independent.
MATERIALS AND METHODS
Culture of PRPTC. Primary renal proximal tubular epithelial cells were isolated from the kidney cortex of 3- to 4-wk-old mice that allow tetracycline-inducible inactivation of HIF-1 (Hif1a2lox/2lox;R26-rtTA2;Lc-1; for details, see Ref. 27); Hif1a is the gene symbol for murine HIF-1. Genotyping of mice was carried out as described previously (27). After being minced, renal cortex was digested with type IV collagenase (Worthington Biochemical, Lakewood, NJ, cat. no. S9S3564) in the presence of soybean trypsin inhibitor (Invitrogen, Grand Island, NY) as described previously (70, 71). An equal number of tubule fragments were plated in 24-well plates or larger dishes as needed. Primary cells were expanded in DMEM (low glucose)/Ham’s F-12 (Invitrogen, Carlsbad, CA), supplemented with 50 ng/ml insulin, 200 ng/ml hydrocortisone, 5 μg/ml apotransferrin, 1% penicillin, and 1% streptomycin (Sigma, St. Louis, MO). Primary cells were characterized by immunocytochemistry with antibodies directed against E-cadherin (Transduction Laboratories, Lexington, KY), cytokeratin (Sigma), and -smooth muscle actin (Neomarkers, Lab Vision, Fremont, CA) following standard procedures. For the inactivation of Hif1a, primary cells were treated with 4 μg/ml doxycycline at day 1 after isolation for a total of 3 days; doxycycline was removed at least 24 h before the initiation of hypoxia experiments. For experiments under hypoxia, cells were either exposed to 0.2% O2-5% CO2 in an Invivo2 200 hypoxia chamber (Ruskinn Technologies, Leeds, UK) or cultured under normoxic conditions (20.9% O2-5% CO2) in DMEM (Invitrogen) containing different glucose concentrations, in the absence or presence of growth factors. Glucose concentration in the culture medium was determined with the glucoseoxidase-peroxidase method (Sigma). Cell viability experiments were repeated at least three times.
All procedures involving mice were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Live Animals and were approved by the University of Pennsylvania Institutional Animal Care and Use Committee.
MTS assay. Viability of PRPTC grown under hypoxia was assessed initially with an MTS assay (CellTiter96, Promega, Madison, WI). In this assay, the tetrazolium compound 3-(4,5 dimethylthiazol-2-yl)-53-carboxymethoxyphenyl-2-(4-sulfophenyl)-2H-tetrazolium (MTS) is chemically reduced by metabolically active cells to a water-soluble formazan compound in the presence of phenazine methosulfate. The production of formazan is proportional to the number of living cells. Near-confluent PRPTC (>80% confluency) were grown under hypoxia (0.2% O2) for a duration of 12, 24, 36, and 48 h. After removal of cells from the hypoxia chamber, MTS assay solution was added to the medium according to the manufacturer’s instructions. Absorbance at 490 nm was measured with a Bio-Rad ELISA reader. Cell viability was expressed as viability index: absorbance measured over absorbance of normoxic controls.
7-AAD viability assay. For assessing the percentage of viable cells after exposure to hypoxia, single-cell suspension renal epithelial cells were stained with vital dye 7-aminoactinomycin D (7-AAD; Viaprobe, Pharmingen) and analyzed by fluorescence-activated cell sorting (FACS). Cells with intact cell membrane will not take up 7-AAD. Acquisition was performed on a FACS Calibur (BD Biosciences). FACS staining results with 7-AAD were confirmed manually by Trypan blue exclusion using a hemocytometer.
BrdU incorporation assay. 5-Bromo-2'-deoxy-uridine (BrdU) incorporation in wild-type and Hif-1a/ cells was determined with the BrdU incorporation assay kit II by immunodetection of labeled cells with peroxydase-conjugated antibodies following the manufacturer’s instructions (Roche Diagnostics, Indianapolis, IN).
TUNEL staining. For the analysis of apoptosis, terminal-deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) was used (In Situ Cell Death Detection kit, Roche Applied Science). Briefly, PRPTC were grown on chamber slides and after exposure to hypoxia fixed in 4% paraformaldehyde for 1 h at room temperature. Cells were then permeabilized in 0.1% Triton X-100 and 0.1% sodium citrate for 2 min on ice and incubated with the TUNEL reaction mixture in a wet chamber for 1 h at 37°C. The TUNEL reaction was terminated by washing the slides with phosphate-buffered saline. Omission of TUNEL enzyme was used as negative control, and DNase I-treated cells were used as a positive control. TUNEL-positive nuclei were visualized using a Nikon E600 fluorescent microscope equipped with IP Lab image-analysis software (Scanalytics).
RNA isolation and semiquantitative RT-PCR. Total RNA from PRPTC was prepared with TRIzol reagent according to the manufacturer’s guidelines (Invitrogen). cDNA was synthesized from 1 μg total RNA with SuperScript first-strand synthesis system for RT-PCR (Invitrogen); 2 μl of cDNA were subjected to PCR amplification with gene-specific primers. The following primers were used for PCR: Bcl-XL forward 5'-ttcgggatggagtaaactgg-3', reverse 5'-gccaagagaactgagatgtgg-3'; Fadd forward 5'-cgcgtgagcaaacgaaag-3', reverse 5'-tccatcttggcctcagacac-3'; Glut-1 forward 5'-gtcctatctgagcatcgtggc-3', reverse 5'-taatacgactcactatagggct-3'; Ldh-A forward 5'-cgtctccctgaagtctcttaacc-3', reverse 5'-cccacaccatctcaacacc-3'. Primer sequences and reaction conditions for Hif1a exon-2, Bax, Bnip3, Pgk, and -actin were used as described before (6).
Chemicals. 2-Deoxyglucose (2-DOG) and phloretin were purchased from Sigma.
Statistical analysis. A two-tailed Student’s t-test was used for statistical analysis. P values of <0.05 were considered statistically significant.
RESULTS
Efficient inactivation of Hif1a in PRPTC. To examine the role of HIF-1 in hypoxia-induced death of renal epithelial cells, we established a mouse strain that allows conditional inactivation of Hif1a (gene symbol for murine HIF-1) in primary cells. In this mouse strain, a tetracycline-inducible Cre-recombinase, Lc-1 Cre (61), is regulated by a reverse transactivator (rtTA), which is under transcriptional control of the ubiquitously expressed ROSA26 promoter (ROSA26 knock-in) (79). PRPTC can be successfully isolated and cultured from these mice and addition of 4 μg/ml of doxycycline to the culture medium is sufficient to recombine the Hif1a allele with high efficiency (>90%) and without any cytotoxic effects. In the Hif1a conditional 2-lox allele (59), Cre-mediated recombination results in an out-of-frame deletion of Hif1a exon 2, which encodes the basic-helix-loop-helix domain (Fig. 1A). We previously showed that the nonrecombined 2-lox allele is not detectable by genomic PCR in doxycycline-treated cells and that Hif1a protein is absent at 1% O2, whereas it is upregulated in nontreated cells (27). Furthermore, the absence of functional Hif1a protein was indicated by the lack of hypoxic upregulation of Vegf or Pgk (27). Immunohistochemical staining for epithelial cell markers was used to confirm the epithelial origin of PRPTC (Fig. 1B). For the generation of Hif1a-deficient PRPTC, cells were treated with 4 μg/μl of doxycycline for 3 days and hypoxia experiments were carried out 1 day after the removal of doxycycline from the culture medium.
Early onset of hypoxia-induced cell death and apoptosis in HIF-1-competent PRPTC. To mimic ischemic conditions in vitro, near-confluent wild-type (wt) or Hif1a-deficient (Hif1a/) PRPTC were exposed to 0.2% O2 in DMEM containing low glucose (100 mg/dl) without growth factors and compared with normoxic wt and Hif1a/ PRPTC cultured in the same medium. Before we examined cell viability under hypoxia, we assessed for possible differences in cellular growth between wt and Hif1a/ PRPTC and used a BrdU incorporation assay to determine proliferative activity. Compared with normoxia, we found an overall 8- to 10-fold decrease in BrdU incorporation and no significant difference in the number of BrdU-positive cells between wt and Hif1a/ cells after 15 h of hypoxia treatment, where significant changes in cell viability were not yet apparent (data not shown). On average, five to seven BrdU-positive cells per x200 high-power field were found in both wt and Hif1a/ PRPTC. This is not a surprising result, as ATP generation through oxidative phosphorylation is severely limited in hypoxic cells, resulting in reduced proliferation.
To assess PRPTC viability under hypoxia, an MTS assay was used first. In this colorimetric assay, the formation of a MTS formazan compound in the culture medium correlates directly with the number of metabolically active/viable cells and was not affected by hypoxia itself (data not shown). MTS assays were performed on wt and Hif1a/ PRPTC treated with 0.2% O2 for 12, 24, 36, and 48 h and on PRPTC treated with normoxia; all were cultured in low glucose without growth factors for the entire duration of treatment. After completion of hypoxia and normoxia treatments, absorbance was measured and cell viability was expressed as the ratio of absorbance obtained from treated PRPTC over absorbance from untreated genotype-matched normoxic controls cultured in the presence of growth factors.
Under normoxia, we found that the viability of Hif1a/ PRPTC was similar to wt, although slightly improved; absorbance ratio of 0.99 ± 0.03 in wt vs. 1.1 ± 0.05 in Hif1a/ cells, P = 0.0034 (Fig. 2A). We cannot exclude that this small difference was due to low levels of HIF-1 activity in normoxic PRPTC (27), inhibiting cell cycle progression through functional interaction with c-MYC, as has been shown in HCT116 cells (33).
Hypoxia treatment for 12 h resulted in no significant change in absorbance in wt and Hif1a/ cultures, whereas after 24 h of hypoxia treatment the viability of wt PRPTC was dramatically decreased compared with Hif1a/ PRPTC; absorbance ratio for wt was 0.05 ± 0.03 and 0.81 ± 0.09 for Hif1a/ cells, P < 0.001 (Fig. 2B). A comparable decrease in Hif1a/ cell viability was observed only after 36 h of hypoxia (absorbance ratio of 0.14 ± 0.10; Fig. 2B). Significant differences in cell viability between Hif1a/ and wt PRPTC were also obtained in a separate experiment with 7-AAD staining by FACS. After 24 h of hypoxia, 55.67 ± 2.61% of Hif1a/ PRPTC were found to be viable by 7-AAD exclusion in contrast to 33.63 ± 3.13% of wt cells. To exclude the possibility that the early decrease in viability of wt cells resulted from the absence of growth factors, insulin, apotransferrin, and hydrocortisone (IHT) were added during hypoxia treatment. Hypoxic treatment in the presence of IHT and pyruvate in low glucose did not result in improved survival of wt compared with Hif1a/ PRPTC (Fig. 2C). These data suggest that the hypoxia-induced decrease in PRPTC viability is growth factor independent and that the timing of cell death onset is HIF-1 dependent.
Because HIF-1 is involved in the regulation of apoptosis, we next used TUNEL staining to examine whether there was a difference between wt and Hif1a/ cells and whether the degree of apoptosis in hypoxic PRPTC correlated with cell viability. Because 24 h of hypoxia treatment resulted in a high percentage of dead wt cells that had already detached from the culture plate, a treatment duration of 18 h was chosen as morphological changes associated with apoptosis precede actual cell death. The decrease in viability found in wt cells after 18 h of hypoxia was associated with a fivefold increase in the percentage of TUNEL-positive cells; the percentage of TUNEL-positive cells increased from 5.67 ± 2.42% in normoxia to 28.5 ± 4.58% in hypoxia (P < 0.001). In contrast, the percentage of TUNEL-positive Hif1a/ PRPTC did not differ between normoxia and hypoxia after 18 h; 3.66 ± 2.5 vs. 5.68 ± 2.42% in hypoxia, P = 0.29 (Fig. 3, A and B). However, when treated with hypoxia for 28 h, the percentage increased 5.5-fold, similar to the increase seen in wt cells after 18 h (4.0 ± 1.79% in normoxia and 22.0 ± 3.57% in hypoxia, P < 0.001). We conclude that the degree of apoptosis in hypoxic PRPTC directly correlates with cell viability determined by MTS assay and that the onset of hypoxia-induced apoptosis is delayed in Hif1a/ PRPTC.
We next analyzed whether altered expression of apoptosis genes could account for the differences in the timing of apoptosis onset. We focused our analysis primarily on factors that had previously been shown to be directly or indirectly regulated by HIF-1, such as BNIP3, BCL-2, BCL-XL, BAX, and caspase8/FADD signaling. We only found that transcript levels for proapoptotic Bnip-3 were significantly different between wt and Hif1a/ cells after 18 h of hypoxia (Fig. 3C). Bnip-3 is a direct transcriptional target of HIF-1 and was found to be significantly upregulated under hypoxia in wt cells.
In summary, our data suggest that the execution of hypoxia-induced cell death and apoptosis does not require HIF-1 but that HIF-1 regulates the timing of hypoxia-induced cell death, which may or may not involve BNIP-3.
Effect of glucose on hypoxia-induced cell death in wt and HIF-1-deficient PRPTC. Because nutrient supply in ischemic tissues is limited, we examined the effect of differences in glucose availability on hypoxia-induced cell death in wt and Hif1a-deficient PRPTC. Near-confluent wt and Hif1a/ PRPTC (>80% confluency) were cultured in DMEM in the absence of growth factors and glucose while being subjected to severe hypoxia (0.2% O2) for 2, 4, 6, 8, and 16 h. Surprisingly, differences in the onset and the degree of hypoxia-induced cell death between wt and Hif1a/ PRPTC (Fig. 4A) were not found by MTS assay or Trypan blue exclusion. After 8 h of hypoxia, viability of both wt and Hif1a/ PRPTC was equally decreased by 50% (no statistically significant difference, P = 0.56); absorbance ratios for wt PRPTC decreased from 1.02 ± 0.05 to 0.47 ± 0.06 and for Hif1a/ cells from 1.03 ± 0.06 to 0.45 ± 0.04. Viable cells were not found after 16 h of hypoxia in either group.
In contrast to hypoxia treatment in the absence of glucose, even prolonged hypoxia treatment for up to 48 h in the presence of high glucose (450 mg/dl) did not affect PRPTC cell viability in either group despite an increase in Bnip-3 and a significant decrease in pH (Fig. 4B). We found that the acidity of wt PRPTC culture medium was significantly increased (pH <6.5) compared with Hif1a/ PRPTC (pH 7), which most likely was the result of a decrease in HIF-1-dependent lactic acid production. Similar observations were made in Hif1a-deficient fibroblasts, indicating that HIF-1 is a critical mediator of the Pasteur effect (62). Taken together, our analysis of hypoxia-treated PRPTC in absent, low, or high glucose suggest that glucose availability and the rate of glucose consumption determine the timing of cell death and apoptosis onset and that in the absence of glucose hypoxia-induced death is HIF-1 independent.
We next investigated whether differences in the expression of genes involved in glucose uptake and anaerobic metabolism could account for the delayed onset of cell death in Hif1a-deficient PRPTC. It is well established that hypoxic induction of genes involved in glucose uptake and glycolysis such as glucose transporter 1 (GLUT-1), phosphoglycerate kinase 1 (PGK), and lactate dehydrogenase A (LDH-A) is directly regulated by HIF-1. Analysis of mRNA levels in hypoxic PRPTC showed that Glut-1, Pgk, and Ldh-A were induced in wt PRPTC, whereas hypoxic induction in Hif1a/ PRPTC was suppressed (Fig. 5A), suggesting decreased glucose utilization and uptake in hypoxic Hif1a/ PRPTC. We then measured glucose levels in the culture media of wt and Hif1a/ cells. We found that glucose in the culture medium of wt PRPTC was not detectable after 18 h of hypoxia, whereas glucose in the medium of Hif1a/ cells was detectable at a concentration of 30 mg/dl (Fig. 5B), indicating that increased expression of genes involved in glucose uptake and utilization resulted in a faster depletion of glucose from the media of Hif1a-competent PRPTC cultures. In contrast, under normoxia differences in glucose levels between wt and Hif1a/ PRPTC cultures were not detectable (Fig. 5B). We predicted that maintenance of adequate extracellular glucose levels in the medium of wt PRPTC (DMEM with 100 mg/dl glucose) would reverse this phenotype and extend the lifespan of wt cells beyond that of Hif1a/ cells. We therefore replenished glucose in the medium of wt PRPTC every 9 h to a final concentration of 100 mg/dl and performed MTS assays after 24 and 36 h of hypoxia treatment. Indeed, we found that the absorbance ratios for wt and Hif1a/ cells were now reversed at both time points. While viability of Hif-1a/ cells was severely decreased after 36 h of hypoxia (absorbance ratio of 0.32 ± 0.03), viability of wt PRPTC was not affected by treatment with severe hypoxia when glucose was replenished (absorbance ratio of 1.07 ± 0.06 after 36 h of hypoxia). No significant differences in viability between wt and Hif1a/ cells were found when glucose was replenished in both wt and Hif1a/ PRPTC cultures; absorbance ratio after 36 h of hypoxia was 1.07 ± 0.06 for wt and 1.11 ± 0.05 for Hif1a/ cells. These findings cement our hypothesis that HIF-1 regulates the onset of hypoxia-induced cell death in PRPTC through its effects on glucose consumption and thus glucose availability.
In summary, our data suggest that increased glucose uptake and metabolism in Hif1a-competent PRPTC lead to enhanced glucose consumption and faster depletion of glucose from the culture medium and thus to earlier onset of hypoxia-induced cell death, whereas glucose consumption by Hif1a/ PRPTC was slower compared with wt, resulting in prolonged survival under severe hypoxia. Our findings also indicate that in the absence of HIF-1, cultured PRPTC are able to generate energy levels adequate for growth and viability under normoxia and that a HIF-1-mediated increase in glucose uptake and metabolism is not required for cell survival of hypoxic PRPTC in vitro.
Inhibition of glucose transport and glycolysis improves cell survival of severely hypoxic wt PRPTC. Because we found that the delayed onset of cell death in hypoxic Hif1a/ PRPTC cultured in low glucose was associated with slower glucose consumption and suppression of mRNAs encoding Glut-1 and glycolytic genes, we hypothesized that either partial inhibition of Glut-1 and/or glycolysis in wt cells would lead to a similar timing of cell death onset in wt and Hif1a/ cells. We found that cell viability between wt and Hif1a/ cells after 24 h of hypoxia treatment was comparable after the addition of glycolysis inhibitor 2-DOG at a concentration of 3 mM (Fig. 6A). The absorbance ratios for wt were 0.48 ± 0.03 and 0.42 ± 0.04 for Hif1a/ PRPTC (P = 0.03). Three millimolar deoxyglucose only partially inhibits glycolysis, as higher concentration doses of 2-DOG (10 mM) in the presence of respiratory chain inhibition were shown to decrease glycolytic lactate production by 50% in rabbit renal proximal tubule isolates, which was, however, also associated with an additional decrease in cell viability (15). We therefore conclude that partial inhibition of glycolysis is sufficient to increase survival of wt cells (Fig. 6A).
Without 2-DOG treatment, 24 h of hypoxia resulted in a severe decrease in viable wt cells. This was associated with a dramatic increase in the percentage of TUNEL-positive cells; 57.5 ± 8.80% of wt cells that were still attached to the culture plate stained positive for TUNEL in hypoxia, vs. 4.5 ± 2.07% in normoxia (Fig. 6B). Treatment with 2-DOG reduced the percentage of TUNEL-positive wt cells to 18.17 ± 2.14% (P < 0.001). Although the use of 2-DOG resulted in a statistically significant decrease in viability of Hif1a/ cells compared with wt [TUNEL: 18.17 ± 2.14% for wt and 24.33 ± 3.33% for Hif1a/, P = 0.02; absorbance ratio: 0.48 ± 0.03 and 0.42 ± 0.04 for Hif1a/ PRPTC (P = 0.03)], the degree of hypoxia-induced cell death and apoptosis in both groups was in a comparable range. This minor difference in viability is compatible with the notion that the glycolytic capacity of hypoxic wt cells is increased compared with hypoxic Hif1a/ cells and therefore is less susceptible to inhibition with a given concentration of 2-DOG; however, we cannot exclude that this observation is secondary to unspecific toxic effects of 2-DOG.
Similarly, we tested whether inhibition of enhanced glucose uptake through a hypoxia-induced increase in Glut-1 would delay the onset of cell death in wt PRPCT exposed to 0.2% O2 and low glucose. We found that after treatment with 25 μM of phloretin, absorbance ratios in wt cells were similar to Hif-1a/ cells after 18, 24, and 30 h of hypoxia (Fig. 6C, shown is the 24-h result), indicating that partial inhibition of sodium-independent glucose transport via facilitative glucose transporters (GLUT) delayed the onset of hypoxia-induced cell death in wt cells (absorbance ratio for wt was 48.41 ± 4.48 and 32.33 ± 2.97 for Hif1a/ cells, P = 0.006). Higher concentrations of phloretin, and thus more efficient inhibition of glucose uptake, resulted in severely decreased viability of both wt and Hif1a/ PRPTC compared with nonphloretin-treated hypoxic cells (data not shown). As was found for treatment with 2-DOG, the statistically significant increase in viability of phloretin-treated wt cells compared with Hif1a/ cells could reflect enhanced expression of GLUT-1 on the cell surface and thus diminished susceptibility to GLUT inhibition or could be the result of unspecific effects on cell viability.
Taken together, our data support the notion that a HIF-1-mediated increase in glucose consumption through a hypoxia-induced upregulation of glycolytic genes and increase in glucose uptake results in early depletion of available glucose resources, and thus earlier onset of hypoxia-induced cell death.
DISCUSSION
ATN as a result of renal ischemia represents a common clinical problem in hospitalized patients and is associated with high mortality. Identification of key molecular events that lead to the development of and govern the recovery phase of ATN is critical for the design of effective therapies to improve its clinical outcome. Ischemia-reperfusion injury in renal epithelial cells typically results in loss of polarity and brush border followed by cell death and sloughing of dead cells, dedifferentiation and proliferation of remaining viable cells, eventually leading to reestablishment of a functional nephron during the recovery phase of ATN (72). Identification of HIF-1 and the characterization of the molecular machinery that mediates its rapid proteolysis under conditions of normal oxygen supply (60) represented a significant advance in understanding the global hypoxic response of cells. During renal ischemia when HIF proteolysis is inhibited, HIF-1 can be detected in the nucleus of renal tubular epithelial cells, where it dimerizes with HIF-1 to form transcriptionally active HIF-1, whereas the HIF-1 homolog EPAS-1 (HIF-2) is not detectable in this cell type, suggesting that HIF-1 is the key mediator of hypoxic HIF signaling in renal epithelial cells (56, 58, 78). As a global regulator of cellular adaptation to hypoxia, HIF-1 controls glycolytic ATP production, enhances oxygen delivery through the stimulation of angiogenic growth factor production and erythropoiesis, and is involved in regulating cell survival and proliferation in hypoxic tissues (60, 63); the role of renal epithelial HIF-1 during kidney ischemia, however, remains to be defined. Because of its central function in cellular adaptation to hypoxia, HIF-1 signaling and the oxygen sensing mechanisms, which control HIF-1 proteolysis, represent potential pharmacological targets in the design of therapeutic strategies aimed at improving the outcome of ischemic tissue injuries in general (20). An important prerequisite for the design of such therapies, however, is the precise understanding of HIF-1’s role in ischemic tissue injury.
Because the role of HIF-1 in the regulation of cell survival is controversial and its role in hypoxia-induced renal epithelial cell death has yet to be defined, we used a tetracycline-inducible system of Cre-loxP-mediated recombination that allows highly efficient inactivation of Hif-1 in PRPTC in vitro. We have chosen a primary cell culture system over the use of established renal epithelial cell lines or immortalized cells to avoid unwanted effects on apoptosis signaling resulting from genetic alterations during long-term passage of cells or from the expression of viral or cellular oncogenes commonly used for the establishment of cell lines.
When we investigated the effects of HIF-1 inactivation on cell survival, differences between severely hypoxic wt and Hif1a-deficient PRPTC were found only in cells that were exposed to hypoxia in the presence of low glucose. This difference in cell survival was a consequence of a more rapid depletion of glucose from the culture medium of wt PRPTC, resulting in an earlier onset of hypoxia-induced cell death. Under hypoxia, the generation of ATP through oxidative phosphorylation is not possible and mammalian cells switch to glycolysis, a much less efficient way of generating ATP (2 mol of ATP/mol of glucose vs. 36 mol of ATP/mol glucose as per traditional estimate). This metabolic switch, also known as the Pasteur effect, is dependent on HIF-1, which is necessary for the hypoxic induction of genes involved in glucose uptake and glycolysis to compensate for the decreased energy yield per mole of metabolized glucose (62). Inactivation of Hif1a in PRPTC suppressed hypoxic induction of glycolytic genes and glucose transporter Glut-1, thus resulting in less glucose uptake and consumption by hypoxic Hif1a/ PRPTC compared with wt PRPTC. However, inactivation of Hif1a did not negatively impact on PRPTC viability when cells were treated with severe hypoxia in the presence of glucose, indicating that a HIF-1-mediated increase in glucose uptake and metabolism during severe hypoxia is not required for cell survival of cultured PRPTC.
In vivo, proximal tubule cells are normally highly gluconeogenic and thus lactate production as the result of glycolysis is low. When cultured under normoxia in vitro or during ischemia in vivo, however, renal proximal tubule cells shift their metabolism from gluconeogenesis to glycolysis (2, 4, 49, 68, 75). Consistent with our findings, previous studies have suggested that this metabolic switch in vitro is a consequence of proliferation and does not depend on the Pasteur effect, which is mediated by HIF-1 (62, 68, 69), although oxygen dependence has been established in "shake-cultures" (49). While trace levels of HIF-1 are detectable in normoxic wt PRPTC (27), suggesting relative hypoxia despite normoxic incubator conditions, we found that absence of HIF-1 does not negatively affect ATP generation in PRPTC, as ADP/ATP ratios between wt and Hif1a/ cells were identical (data not shown). However, the impact of HIF-1 absence on the ability of the proximal tubule to switch to glycolysis in vivo under ischemic conditions is difficult to predict from our in vitro analysis. Studies in Hif1a-deficient fibroblasts have shown that the Pasteur effect in these cells is HIF-1 dependent (62), whether this is the case in renal proximal tubule cells in vivo is currently being determined in our laboratory. Nevertheless, our in vitro findings demonstrate that ATP generation in cultured HIF-1-deficient PRPTC is adequate for normal growth and viability in normoxia.
Differences in cell viability between Hif1a/ and wt PRPTC were not observed in the absence of glucose indicating that molecular pathways, which execute hypoxia-induced cell death in renal epithelial cells, are largely HIF-1 independent. Both apoptotic and necrotic forms of cell death exist in ischemic renal tissues (73) and it has been suggested that necrosis occurs when intracellular ATP levels fall below 20% (36). HIF-1 has been implicated in the regulation of apoptosis by altering the balance of proapoptotic vs. anti-apoptotic factors, which may occur in a cell type- and context-specific manner. Its exact role in this process remains controversial. Studies from our group, for example, have suggested that HIF-1 in a VHL-deficient background induces cell death in thymocytes through a caspase 8-dependent mechanism (6). Others have suggested a proapoptotic role for HIF-1 through downregulation of BCL-2 in ES cell-derived teratocarcinomas (12), through increased BNIP-3 in cardiomyocytes (7, 34, 74), or through increased BAX in renal epithelial cells immortalized with temperature-sensitive SV40 large T at nonpermissive temperatures (67). In contrast to these reports, which predict that HIF-1 would potentiate apoptosis in renal epithelial cells, we found that when medium glucose levels fell below the detection limit, the degree of apoptosis in hypoxic wt and Hif1a/ PRPTC was comparable, suggesting that HIF-1 is involved in the regulation of hypoxia-induced cell death predominantly through its effects on glycolytic substrate utilization and not through effects on mitochondrial signaling per se, including signaling through BNIP-3.
The importance of glucose, glycolytic substrate uptake, and metabolism in the regulation of apoptosis and cell viability is well recognized. In vitro studies of hypoxic and nonhypoxic cell death have shown that the availability of glycolytic substrate (42), an increase in glycolytic metabolism, and the augmentation of glucose uptake through increased expression of GLUT-1 under conditions where glycolytic substrate was not depleted (16, 39, 52) resulted in improved cell survival in neonatal cardiomyocytes and other cell types. A potential cytoprotective role for increased glycolytic metabolism in the setting of nonhypoxic HIF-1 stabilization remains to be investigated. Recent studies have provided more direct and mechanistic links between glucose metabolism and apoptosis. Hexokinase isozymes, which are rate-limiting glycolytic enzymes, associate with the outer mitochondrial membrane and prevent cytochrome c release and thus apoptosis (9, 14, 21, 41, 50). This interaction appears to be mediated by serine/threonine kinase AKT/PKB (40), which has a strong anti-apoptotic role in response to growth factors and stimulates glucose uptake and glycolysis in nontransformed and transformed cells. In some cases, AKT-transformed cells lose their ability to utilize nonglycolytic bioenergetic substrates and become "glucose and glycolysis dependent" under normoxia, resulting in cell death on withdrawal of glycolytic substrate (10, 18). Activation of fatty acid -oxidation through supraphysiological levels of AMP-activated protein kinase (AMPK) has been shown to reverse this phenotype (10). AMPK in nontransformed cells acts as a critical intracellular energy sensor and is activated by increased AMP levels. To conserve intracellular ATP stores, AMPK inhibits anabolic pathways, such as cap-dependent protein translation via phosphorylation of the tuberous sclerosis tumor suppressor TSC2 (29), and stimulates glycolysis and glucose uptake via GLUT-1 and GLUT-4, as well as fatty acid -oxidation (24). Hypoxic cells, however, are completely dependent on the availability of glycolytic substrates and cannot utilize bioenergetic pathways that involve oxidative phosphorylation, as is the case in severely hypoxic PRPTC.
Although our data suggest that hypoxia-induced death of nonpreconditioned renal epithelial cells in the acute phase of renal ischemia may not depend on functional HIF-1, HIF-1’s role in ischemia-reperfusion injury of renal epithelial cells and in the recovery from ATN remains unclear. HIF-1’s involvement in the regulation of a multitude of cellular functions such as migration, the maintenance of matrix and barrier function, cellular transport, vasomotor regulation and angiogenic signaling, and direct and indirect evidence from rodent models of acute ischemia support the notion that HIF-1 has a critical role in the pathogenesis of renal ischemic injury (5, 11, 26, 57, 60). It is unclear, however, where, when, and how in the pathogenesis of ATN signaling through HIF-1 intersects with key cellular functions that affect the clinical outcome of this disease. Increased HIF-1 activity upregulates factors that have been shown to be "cytoprotective" in the setting of different renal diseases, including ischemia. These, for example, include VEGF (31, 32), heme oxygenase-1 (1, 28, 46, 77), and EPO (76). In recent reports, pretreatment of rats with a HIF-specific prolyl-hydroxylase inhibitor (23) or cobalt chloride (43), a chelating agent known to inhibit HIF- proteolysis, resulted in improved renal clearance after clamping of the renal pedicle. Histologically, pretreatment with cobalt chloride was also associated with amelioration of tubulointerstitial damage and a decrease in macrophage infiltration, providing evidence that HIF-1 signaling is involved in ischemic preconditioning of the kidney, as has been suggested for other organ systems (11, 53). Efforts in our laboratory are currently under way to clarify the role of HIF-1 in renal ischemia-reperfusion injury and renal preconditioning in vivo.
In summary, in this report we provided genetic evidence that HIF-1 signaling is not required for growth and viability of cultured renal proximal tubule cells and for the execution of hypoxia-induced renal epithelial cell death and apoptosis in vitro. However, we propose that a HIF-1-dependent increase in glucose metabolism and consumption may result in early cell death under conditions when renal epithelial cells are exposed to severe hypoxia after or during entering a proliferative phase and glucose availability is limited, for example in the setting of repeated and prolonged hypoxic insults during the recovery phase of ATN. Our findings should stimulate further investigations into the role of HIF-1 in renal ischemia-reperfusion injury.
GRANTS
This work was supported by Grant 0365342U from the American Heart Association (to V. H. Haase).
ACKNOWLEDGMENTS
We are grateful to the members of the Morphology Core, Penn Center for Molecular Studies in Digestive and Liver Disease (P30-DK50306) for assistance with the image analysis and we thank all members of the Haase lab for helpful discussions and critical review of the manuscript.
Present address of Y. Akai: Nara Medical University, First Department of Internal Medicine, 840 Shijo-cho, Kashihara, Nara 634-8522, Japan.
FOOTNOTES
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. Section 1734 solely to indicate this fact.
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