1Department of Pediatrics, 2Magnetic Resonance Research Center, 3Department of Pathology, Yale University, New Haven, Connecticut
Submitted 16 August 2004 ; accepted in final form 28 September 2004
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ABSTRACT |
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newborn; renal proximal tubule; heat shock proteins; 70-kDa heat shock protein; ischemia
To establish the role of HSF-1 in the response of the immature tubule, developmental and injury-induced changes in nuclear protein expression were examined. The effect of specific inhibition of HSF-1 on tolerance of the immature nephron was explored using circular, ethylene glycol bridged, decoy oligodeoxynucleotides.
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METHODS |
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The effect of bilateral renal ischemia on HSF-1 expression was studied in adult male Sprague-Dawley rats, as previously described (20). After 45-min ischemia, the kidneys were rapidly removed, decapsulated, and harvested as above. Kidneys harvested from sham-operated animals served as controls.
Nuclear protein extraction. Nuclear protein extraction was performed using hypotonic lysis followed by high-salt extraction of nuclei (1). Homogenate was centrifuged for 10 s at 18,000 g using a Microfuge (Beckman Coultier) and the supernatant was discarded. The pellet was resuspended in 400 µl of a buffer containing 10 mM HEPES-KOH (pH 7.9) at 4°C, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.2 mM PMSF, 1 mM sodium vanadate, and Complete Protease Inhibitor Cocktail (Roche), incubated on ice for 10 min, and centrifuged at 18,000 g for 10 s. The supernatant was discarded and the remaining pellet was suspended in a volume of buffer equal to the volume of the pellet and containing 20 mM HEPES-KOH (pH 7.9) at 4°C, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM PMSF, 1 mM sodium vanadate, and Complete Protease Inhibitor Cocktail (Roche). Samples were incubated on ice for 20 min, centrifuged at 18,000 g for 2 min at 4°C, and the supernatant was removed and stored at 80°C.
Preparation of suspensions enriched in tubule segments. For each experiment, tubule segments were obtained from the kidneys of 12 immature (P10) rat pups and 1 mature (810 wk) male Sprague-Dawley rat. Animals were anesthetized with pentobarbital sodium; the kidneys were removed and bisected longitudinally. Kidneys were incubated for 45 min at 37°C with 21 FALGPA units of collagenase (Sigma, St. Louis, MO) in 7.5 ml of freshly prepared buffer (buffer C) containing 4 mM sodium lactate, 2 mM alanine, 3 mM butyrate, 2 mM glutamine, 5 mM malate, 5.5 mM glucose, 112 mM NaCl, 3 mM KCl, 1.2 mM MgSO4, 1 mM KH2PO4, 1.5 mM CaCl2, and 25 mM NaHCO3, equilibrated with 95% O2-5% CO2, and having a final pH of 7.4 (10). After digestion, tubules were separated by gentle scraping with a razor blade and resuspended in buffer C. To eliminate glomeruli, the tubules were filtered through a fabric mesh (Tetko, Briarcliff Manor, NY), 115 µm for the mature suspension, 50 µm for the immature suspension. The suspension was washed in buffer C by spinning at 500 g three times for 2 min. The final pellet was resuspended in buffer C and kept on ice until studied.
Induction of anoxia and subsequent recovery. Tubules from both the immature and mature rats were suspended in 6 ml of buffer C and studied in 500-µl aliquots. Before the induction of anoxia, tubule suspensions were bubbled with 95% O2-5% CO2 at 37°C for 10 min to allow for steady-state equilibration. Samples of tubular suspension obtained following equilibration were used to provide baseline measurements (mature and immature controls). Anoxia was produced by bubbling each aliquot for 2 min with 95% N2-5% CO2. Anoxia was confirmed by measurement of PO2 using a Clarke oxygen electrode. Each Eppendorf tube was capped and incubated at 37°C in a shaking water bath for 20 min, rebubbled with 95% N2-5% CO2, and incubated for a further 25 min, giving a total anoxic period of 45 min. Tubes were opened at the end of the anoxic period, bubbled with 95% O2-5% CO2, and incubated at 37°C in a shaking water bath for 1 h. Aliquots of tubular suspension for Western blotting (150 µl) were removed, sonicated, and stored at 80°C. Aliquots for additional studies were obtained and stored as described below.
Decoy treatment. Inhibition of HSF-1 was achieved using a transcription factor decoy as previously described (17). Briefly, multiple copies of a 23-bp oligodeoxynucleotide, of a sequence corresponding to HSE, were added to suspensions of immature tubules. After absorption, the decoy provides an alternative, and ineffective, target for binding of activated HSF-1. The decoy consisted of complementary strands of DNA joined at either end by ethylene glycol bridges 3'-cgaaacctctggaatattcctag-5'. Decoy specificity was assessed using a scrambled decoy, sharing the same base composition as the HSF-1 decoy but with a random base order 3'-ctaatgaaccgatactcgcgatt-5'.
LDH assay. To assess the integrity of the suspension of tubules, LDH release was determined as an index of plasma membrane damage. The tubular suspension (250 µl) was removed and centrifuged at 12,000 g for 1 min. The supernatant was removed and retained; the pellet was resuspended in an equal volume of buffer C and sonicated on ice for 10 s. Samples were stored at 4°C and processed within 24 h. Enzyme activity was measured as previously described (10). Released LDH from cells was expressed as a percentage of total LDH (released LDH plus nonreleased LDH in the tubules).
HSP70 assay. To assess the effect of decoy treatment on HSF-1-controlled proteins, levels of HSP70, the principle synthesis product of the heat shock response, were measured by Western blot analysis. The tubular suspension (100 µl) was mixed with an equal volume of chilled extraction buffer (PHEM) containing 0.1% Triton X-100, 60 mM piperazine-N,N'-bis(2-ethanesulfonic acid; pH 6.8), 25 mM HEPES, 10 mM EGTA, 2 mM magnesium chloride, 1 mM benzamidine, 2 mM sodium vanadate, and Complete Protease Inhibitor Cocktail (Roche). Samples were stored at 80°C before protein determination and Western blot analysis.
Protein assay and Western blot analysis. Protein concentrations of samples were determined, following protein precipitation using the Compat-Able Protein Assay Preparation Reagent Set (Pierce, Rockford, IL), by a bicinchoninic acid method using BSA as a protein standard (BCA Protein Assay Kit, Pierce).
Equal amounts of protein (10 µg) were mixed with an equal volume of 2x loading buffer containing 100 mM Tris (pH 6.8), 4% SDS, 0.2% bromophenol blue, 20% glycerol, and 200 mM DTT and separated by SDS-PAGE electrophoresis on 420% gradient gels (Criterion, Bio-Rad). Proteins were transferred onto nitrocellulose membranes (Biotrace NT, Pall, FL) and verification of comparable total protein load was established by means of Ponceau staining. Nonspecific binding sites were blocked with 5% skim milk in 10 mM Tris (pH 7.5), 37.5 mM sodium chloride, and 0.5% Tween 20, and membranes were then incubated for 1 h with monoclonal antibodies directed against HSF-1 (RT-629-P, Neo Markers, Fremont, CA) or inducible HSP-72 (SPA-810 Stressgen, B.C., Canada). After repeated washings, the membranes were incubated with an appropriate species-specific secondary antibody for 1 h. After being washed further, immunoreactive antigen was detected with enhanced chemiluminescence. Western blot analysis reagents and protocols were supplied by the manufacturer (Pierce). Chemiluminescence was detected by exposure of photographic film (Kodak X-OMAT AR, Rochester, NY) and quantified by means of densitometry. Films were scanned using a Linoscan 14000 and the resulting images were analyzed using Scion Image software (Scion, Frederick, MD).
Sample size and statistical analysis. Experimental groups, comprising immature and mature anoxic tubules, both treated and untreated with the decoy, were studied on a minimum of five occasions for each condition (n = 5). Values were expressed as means ± SE. Comparisons between groups were made by analysis of variance and Student's t-test. Values are considered significantly different if P < 0.05.
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RESULTS |
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The effect of HSF-1 inhibition on cellular viability after an anoxic insult was studied in tubular suspensions incubated with a cyclic oligonucleotide decoy sharing sequence homology with the HSE (i.e., the HSF-1 binding site). To confirm the anticipated molecular effect of decoy treatment, immature tubules were injured with or without the HSF-1 decoy and HSP70 levels were assessed by Western blotting (n = 3). Tubules treated with decoy demonstrated a consistent and substantial reduction in HSP70 abundance (Fig. 2).
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To determine whether the magnitude of injury could be modulated in proportion to the dose of decoy administered, immature tubules were treated with 40, 80, and 160 µg of decoy. The degree of anoxic injury was proportional to the dose of decoy administered (Fig. 4).
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DISCUSSION |
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Tolerance to injury has also been demonstrated within individual organ systems. Lambs exposed to hypoxemia demonstrate increasing preservation of glomerular filtration rate and sodium reabsorption with decreasing postnatal age (22). Proximal convoluted tubules, removed from immature rabbits, show greater preservation of active transport and cellular integrity following anoxia compared with tubules from mature animals (4).
Increased glycolytic capacity and preservation of high-energy phosphates have been proposed as potential mechanisms of resistance to injury in neonatal animals. However, although glycolysis is increased in immature proximal renal tubules exposed to hypoxia, inhibition of glycolysis does not alter immature tolerance (12).
Heat shock response has been proposed as a potential mediator of immature tolerance to hypoxia-anoxia because: 1) overexpression of HSP induces tolerance to hypoxia-ischemia in mature animals (8, 24); 2) constitutive levels of inducible HSP increase during postnatal development (21); 3) immature animals demonstrate a robust and amplified heat shock response following a variety of insults compared with that seen in mature animals of the same species (11, 18, 21); and 4) immature renal tubules exposed to anoxia demonstrated increased activation and binding of trimerized HSF-1 to the HSE (11).
The current study demonstrates a maturation of abundance of HSF-1 in immature rat renal tubules. Increased expression of HSF-1 during development parallels that of HSP70, one of the many heat shock proteins under the control of this promoter (21). The relative total abundance of this potent transcriptional activator in P10 rat pups is considerably greater than that seen in uninjured mature rats. Renal ischemia increases the abundance of HSF-1 in mature animals. However, the level of HSF-1 after 45 min of ischemic injury in mature rats remains lower than that seen in uninjured P10 pups. The process of development appears analogous to injury-induced preconditioning in the mature animal, which is associated with tolerance to a subsequent insult (16).
Previous studies demonstrated that the activation of HSF-1 in immature renal tubules is markedly more robust than that seen in tubules from mature rats following a variety of injuries including anoxia, hyperoxia, and hyperthermia (11). Results of the current study suggest that constitutive upregulation of the heat shock response in the immature kidney is primed by high levels of the primary transcriptional regulator of this stress-induced system, HSF-1.
When immature tubules were incubated with the HSF-1 decoy, expression of the principle synthesis product of the heat shock response, HSP70, was substantially reduced. This observation is in keeping with the predicted molecular mechanism of action of the oligonucleotide decoy to dampen the molecular sequence of events following the activation of HSF-1.
In the present study, vulnerability to anoxia was increased in immature tubules treated with the HSF-1 decoy. The severity of injury following targeted inhibition of HSF-1 was similar to that seen in mature tubules. The loss of tolerance to anoxia was specific to decoy-sharing sequence homology with the HSE and occurred in a dose-dependent manner. Increased injury following decoy treatment of immature tubules may be a reflection of direct inhibition of heat shock protein synthesis. Previous work in our laboratory indicated that treatment of cultured proximal tubule cells with an HSF-1 decoy produces a significant downregulation of both HSP70 and HSP25 following cellular ATP depletion. Decoy-induced dampening of the heat shock response was associated with a substantial loss of cellular polarity and more severe injury following energy depletion (17). The HSF-1 decoy increases both the severity of injury in cultured renal cells and the vulnerability of immature tubules to anoxia, suggesting the possibility of a common mechanism that modulates renal cell injury.
Although it is tempting to assume that the increased abundance of HSF-1 and HSP70 in P10 tubules is responsible for the tolerance of the immature kidney to anoxia, it is important to recognize that HSF-1 governs transcription and regulation of a myriad of genes that affect essential cellular functions. Homozygous HSF-1-deficient mice exhibit multiple phenotypes including prenatal lethality and abnormalities during extraembryonic development. Of note, basal heat shock protein expression is not altered appreciably in HSF-1 knockout mice, suggesting that HSF-1 might be involved in regulating other genes or signaling pathways important to critical pathobiological processes including cellular resistance to injury (23). In fact, HSF-1, although traditionally regarded as a transcription promoter, can act to repress transcription, which might suppress induction of pathways that propagate cell death (19).
The present series of studies indicates that there is a progressive increase in expression of HSF-1 in renal tubules during early postnatal development. In essence, the immature nephron responds to stress as if the process of development is analogous to a preconditioning insult in the mature kidney. Treatment with an HSF-1 decoy is associated with a loss of tolerance to anoxia in immature renal tubules. The precise cellular mechanism of this increased vulnerability to injury may be related to the modulation of the heat shock response or other critical cellular functions influenced by HSF-1. The immature nephron may serve as a paradigm of augmented heat shock response in the absence of prior injury.
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GRANTS |
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FOOTNOTES |
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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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REFERENCES |
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