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Department of Physiology, University of Munich, Munich, Germany.
Correspondence to Dr. Wolfgang Neuhofer, Department of Physiology, University of Munich, Pettenkoferstrasse 12, 80336 München, Germany. Phone: +49-89-5996-534; Fax: +49-89-5996-532; E-mail: W.Neuhofer{at}physiol.med.uni-muenchen.de
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Abstract |
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Introduction |
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In the kidney, the abundance of HSP72 (the major stress-inducible HSP) is correlated with the corticomedullary osmotic gradient and changes appropriately with the diuretic state (7,8). These observations suggest that this HSP plays an important role in the adaptation of medullary cells to high extracellular solute concentrations. Under both normal and stressful conditions, members of the HSP70 family participate in various cellular processes, such as folding and refolding of proteins, intracellular protein trafficking and degradation, and signal transduction (9).
Although renal cells tolerate high and fluctuating solute concentrations, there are limits to the concentrations of NaCl and urea that even renal medullary cells can withstand. Increases in the medium osmolality to 
600 mosmol/kg H2O, through either NaCl or urea addition, activate death programs, leading primarily to apoptosis (10,11). Studies with MDCK and mIMCD3 cells indicate that hypertonic stress confers resistance to the deleterious effects of high urea concentrations by limiting the proapoptotic effects of high urea concentrations (3,12,13). In those studies, urea-induced apoptosis could be ameliorated by prior or simultaneous exposure to hypertonic stress. The finding that inhibition of HSP72 expression during hypertonic stress with antisense transfection attenuated the beneficial effects of hypertonic pretreatment on subsequent urea exposure suggests a cytoprotective role for HSP72 (14).
Because hypertonic stress promotes a variety of intracellular events, it is conceivable that factors other than hypertonicity-induced upregulation of HSP72 production contribute to the enhanced resistance to high urea concentrations. The aim of this study was thus to examine the effects of forced overexpression of HSP72 alone on the resistance of MDCK cells to high urea concentrations, in the absence of hypertonic stress. Because constitutive HSP72 overexpression inhibits cell growth and disrupts signaling pathways (15,16), an inducible vector construct was used to generate MDCK cells with regulated HSP72 overexpression. To gain a better understanding of the HSP72-mediated protection against high urea concentrations, markers of necrosis and apoptosis were used to characterize in greater detail the nature of urea-induced cell damage.
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Materials and Methods |
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Expression Vectors and Transfection
For inducible overexpression of HSP72, a 2.3-kb fragment containing the entire coding region of the stress-inducible human HSP70 gene (pH2.3) (17) was subcloned into the inducible mammalian expression vector pLKneo as follows (18). pH2.3 was released from pAT153-pH2.3 (American Type Culture Collection, Manassas, VA) with HindIII and BamHI and was gel purified, and recessed 3'-termini were then filled in with Klenow enzyme (MBI Fermentas, St. Leon-Rot, Germany). The eukaryotic expression vector pLKneo, containing a DEX-inducible mouse mammary tumor virus (MMTV) promoter (18), was linearized with HindIII and EcoRI; the recessed 3'-termini were filled in with Klenow enzyme and the 5'-termini were dephosphorylated with calf intestinal alkaline phosphatase (MBI Fermentas). Subsequently, the 2.3-kb HSP72 fragment was blunt-end ligated into pLKneo. The orientation of the insert was verified by restriction mapping and sequencing of the junctions. In the resulting construct pLKneo-HSP72, HSP72 is expressed under the control of a DEX-inducible MMTV promoter. pLKneo-HSP72 also contains resistance genes for ampicillin and geneticin (G418).
MDCK cells were transfected by using liposome-mediated procedures. Briefly, 24 h before transfection, exponentially growing MDCK cells were seeded at a density of 0.5 x 106 cells/75-mm dish. On the day of transfection, 20 µg of plasmid DNA was dissolved in 400 µl of serum-free Dulbecco’s modified Eagle’s medium (DMEM); in parallel, 20 µg of liposomes (Clonfectin; Clontech, Palo Alto, CA) dissolved in Hepes-NaCl buffer (10 mM Hepes, pH 7.4, 140 mM NaCl) was added to another 400-µl aliquot of serum-free DMEM. The two samples were then combined and incubated at room temperature for 20 min. Subsequently, the cells were washed with phosphate-buffered saline (PBS), incubated with the DNA-liposome mixture for 4 h, washed with PBS, and incubated in complete medium for another 24 h. After that period, the medium was replaced with DMEM containing 600 µg/ml G418 (Sigma), which was refreshed every 2 d. After 2 to 3 wk, G418-resistant clones were subcloned and expanded for analysis of inducible HSP72 expression. For this purpose, the respective clones were exposed to 1 µM DEX (diluted from a 10 mM stock solution in ethanol) for 24 h, assayed for HSP72 expression by Western blot analysis, and matched to the same clone grown in the absence of DEX. Clones with inducible HSP72 overexpression (MDCK-LK72) were used for experiments at passages 3 to 15. Mock-transfected MDCK (MDCK-LKm) cells were established by introducing pLKneo without the insert.
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Western Blot Analysis
After the treatments, the cells were washed three times with chilled PBS and scraped into 8 M urea/PBS (50 µl/35-mm plate). Cells were lysed with three cycles of snap-freezing and thawing. The extracts were stored for 15 min at room temperature, vigorously vortexed, and centrifuged at 12,000 x g for 15 min at 4°C. Protein concentrations in the supernatant were determined in duplicate, using a commercially available kit (BioRad, Munich, Germany) (19). Aliquots (40 µg) of total cell protein were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and Western blot analysis, and HSP72 and heat-shock cognate–70 (HSC70) (HSP73) were immunodetected as reported elsewhere (6).
Northern Blot Analysis
After the treatments, the cells were washed three times with ice-cold PBS and lysed by the addition of 1 ml of TRI-Reagent (Sigma). RNA was recovered according to the recommendations of the manufacturer. Aliquots (20 µg) of total RNA were subjected to electrophoresis through 1% agarose/formaldehyde gels, blotted onto nylon membranes (Hybond-N+; Amersham, Freiburg, Germany), and immobilized by ultraviolet-crosslinking. For detection of HSP72 mRNA expression, a digoxigenin-labeled HSP72 cDNA probe was prepared. Gel-purified pH2.3 was subjected to random-primed extension by the large fragment of DNA polymerase Iexo- (MBI Fermentas), in the presence of dNTP and digoxigenin-dUTP. For Northern blot analyses, the blots were prehybridized for 2 h at 55°C in a solution containing 50% formamide, 5x SSC, 0.1% SDS, and 10% blocking reagent (Roche, Mannheim, Germany) and were hybridized overnight in the same solution containing 20 ng/ml digoxigenin-labeled probe. After hybridization, the membranes were washed twice for 15 min each with 2x SSC/0.1% SDS at room temperature and twice for 15 min with 0.1x SSC/0.1% SDS at 68°C. Nonradioactive detection procedures were performed according to a previously described method (20). To correct for differences in RNA loading, the membranes were stripped and rehybridized with digoxigenin-labeled cDNA specific for glyceraldehyde phosphate dehydrogenase (6). Signals were quantified by laser densitometry (Ultrascan XL; Amersham).
Immunofluorescence Staining
Cells were grown on eight-well slides (Lab Tech chamber slides; Nunc, Wiesbaden, Germany). After the treatments, the cells were washed three times with chilled PBS and fixed in methanol for 15 min at -20°C. The cells were permeabilized for 15 min with 0.1% Triton X-100 in PBS (pH 7.4). Nonspecific binding sites were blocked for 30 min in immunofluorescence buffer (1% bovine serum albumin, 0.1% Tween 20, and 0.01% NaN3 in PBS, pH 7.4). HSP72 was immunodetected by sequential incubation with monoclonal anti-HSP72 antibody (SPA810, 1:500 in immunofluorescence buffer; StressGen, Victoria, British Columbia, Canada), polyclonal rabbit anti-mouse Ig antibody (1:1000 in immunofluorescence buffer; Dianova, Wiesbaden, Germany), streptavidin-conjugated goat anti-rabbit Ig antibody (1:500 in immunofluorescence buffer; Dianova), and tetrarhodamine isothiocyanate-labeled biotin (1:400 in immunofluorescence buffer; Dianova), each for 1 h at room temperature with gentle agitation. After each antibody exposure, the slides were washed three times, for 15 min each, with PBS. After the final wash, the slides were mounted with Fluorosave (Calbiochem, La Jolla, CA) and examined by using a fluorescence microscope (Zeiss, Oberkochen, Germany) equipped with the appropriate filters.
Cell Survival Assays
After urea exposure, detached cells were aspirated, adherent cells were collected by trypsinization, and both fractions were counted in a hemocytometer. The surviving fraction was expressed as the adherent/total (adherent plus detached) cell ratio. To confirm that the detached cells were actually dead, in some experiments they were washed and replated in control medium. During the reincubation, less than one in one thousand cells reattached.
Measurement of LDH Activity
Cells were plated in 24-well plates (Costar), in 1 ml of medium. After 24 h, the medium was replaced with DMEM, containing 0.1 µM DEX or lacking DEX (control samples), for 48 h. The cells were then exposed to the respective media containing 600 mM urea. After urea exposure, 25 µl of medium was removed and centrifuged at 250 x g for 5 min at 4°C; the supernatant was removed and stored at 4°C. For measurement of total cellular LDH activity, the cells were disrupted by vigorous passage five times through a 26-gauge needle, and cellular debris was pelleted by centrifugation at 12,000 x g for 5 min at 4°C. The supernatant was stored at 4°C. LDH activity was assessed by using a commercially available LDH assay (Sigma), as described by the manufacturer. Relative LDH activity was expressed as the ratio between activity in the supernatant and total LDH activity with the respective experimental protocol.
Flow Cytometry
After urea exposure, apoptosis and necrosis were determined by labeling with annexin V-FITC and PI (21). For this purpose, the detached cells from 75-mm dishes were aspirated and stored on ice. The adherent cells were collected by trypsinization, combined with the detached cells, and pelleted by centrifugation at 250 x g for 5 min at 4°C. The cell pellet was washed twice with ice-cold PBS, and 105 cells were resuspended in 100 µl of binding buffer (10 mM Hepes, pH 7.5, 140 mM NaCl, 2.5 mM CaCl2). Subsequently, 1 µl of annexin V-FITC conjugate (50 µg/ml) and 2 µl of PI (100 µg/ml) were added and allowed to bind for 10 min at room temperature, with protection from light. Detection of viable cells (annexin V-FITC-negative, PI-negative), early apoptotic cells (annexin V-FITC-positive, PI-negative), apoptotic-lysed cells (annexin V-FITC-positive, PI-positive), and necrotic cells (annexin V-FITC-negative, PI-positive) was performed by using a Becton Dickinson FACScan instrument in combination with Cell Quest software (Becton Dickinson, Heidelberg, Germany). A total of 30,000 events were used for analysis. Results are presented as bivariate dot blots of log FL1 (annexin V-FITC) and log FL3 (PI), representing a reproducible cell population gated by forward and side scatter.
Statistical Analyses
The data are presented as means ± SEM. Differences between the means were tested for significance by t test for independent samples and by multivariate ANOVA for multiple samples. P < 0.05 was regarded as significant.
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Results |
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0.1 µM. In contrast, HSP72 expression in MDCK-LKm cells was not affected by any concentration of DEX after 24 h. On the basis of this observation, a DEX concentration of 0.1 µM was used for further experiments. The abundance of constitutively expressed HSC70 (HSP73) was not altered by any concentration of DEX (Figure 1). Northern blot analysis revealed that 0.1 µM DEX increased HSP72 mRNA abundance in MDCK-LK72 cells 20-fold after 24 h of incubation (Figure 2). Interestingly, the transgene could be discerned from the endogenous canine HSP72 message because it contains a portion of the MMTV promoter and thus appears as a band with higher molecular weight (Figure 2). Endogenous canine HSP72 mRNA expression was only minimally stimulated by DEX.
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Protection against High Urea Concentrations with HSP72 Overexpression
Cell Survival.
MDCK-LK72, MDCK-LKm, and MDCK wild-type cells were incubated for 48 h in medium containing 0.1 µM DEX or in normal DMEM (control samples). When pretreated MDCK cells were subsequently exposed for 24 h to medium containing 600 mM urea, the number of cells remaining attached to the culture dish was three- to fivefold higher for DEX-treated MDCK-LK72 cells than for any other group (Figure 5). This improved resistance to high urea concentrations coincided with strongly increased HSP72 abundance in MDCK-LK72 cells pretreated with DEX (Figures 3 and 4). In some experiments, the floating cells were collected and replated in normal DMEM. Less than one in one thousand cells reattached; in other words, >99.9% of the detached cells were dead (data not shown).
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After 24 h of incubation in medium containing 600 mM urea, the vast majority of MDCK-LKm and wild-type cells, either pretreated with 0.1 µM DEX or not, were in the apoptotic-lysed stage, as indicated by staining with both annexin V-FITC and PI (Figure 7). In MDCK-LK72 cells, DEX induction of HSP72 before urea exposure significantly reduced the fraction of apoptotic-lysed cells; the fractions of early apoptotic cells (annexin V-FITC-positive, PI-negative) and viable cells (annexin V-FITC-negative, PI-negative) were significantly increased (Figure 7). The great majority of MDCK-LK72 cells not pretreated with DEX were in the early apoptotic and apoptotic-lysed stages. Inspection of Figure 7 reveals that the fraction of early apoptotic cells is higher for MDCK-LK72 cells not treated with DEX, compared with mock-transfected or wild-type cells. This finding might be explained by the elevated basal HSP72 levels in noninduced MDCK-LK72 cells, which might already be sufficient to retard progression through the apoptotic pathway.
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Discussion |
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We previously demonstrated that pretreatment of MDCK cells with hypertonic NaCl, which strongly increases HSP72 expression, conferred substantial resistance to the deleterious effects of subsequent exposure to high urea concentrations (600 mM, 24-h exposure) (3). In those studies, cellular HSP72 contents were positively correlated with the degree of resistance to 600 mM urea. A cytoprotective role for HSP72 was further suggested by the observation that inhibition of NaCl-induced HSP72 expression by antisense transfection abrogated the beneficial effect of prior hypertonic stress (24). By demonstrating that regulated overexpression of HSP72 significantly retards the progression of cells through the apoptotic pathway (which is usually entered by unprotected cells after exposure to high urea concentrations), this study provides positive experimental evidence that it is indeed HSP72 that contributes decisively to the survival of renal epithelial cells in an environment characterized by greatly elevated urea concentrations.
Urea concentrations exceeding 400 to 500 mM induce apoptosis in mIMCD3 and MDCK cells, as demonstrated by activation of caspase-3 as an effector of programmed cell death and internucleosomal DNA fragmentation (10,14). The initial step in urea-induced apoptosis is unclear, however. Urea signaling exhibits the hallmarks of a peptide mitogen-like signaling pathway, including activation of receptor tyrosine kinases, extracellular signal kinase, c-Jun N-terminal kinase, Elk-1, Shc, Grb2, SOS, and Ras S6 kinase (25,26). The precise mechanism by which HSP72 retards activation and progression through the apoptotic death program is not known. Considering that high urea concentrations activate c-Jun amino-terminal kinase (25), it is of interest that suppression of this stress kinase by overexpression of HSP72 is involved in the protection of myogenic cells from energy deprivation (27,28). This mechanism may be relevant for the protection of renal medullary cells from high urea concentrations in the model used in this study.
Another mechanism providing protection from high urea concentrations may be more general and related to the chaperoning activities of HSP72. High concentrations of urea are known to compromise the structure and function of cellular proteins by destabilizing polypeptides (29). HSP72 is capable of stabilizing partly denatured polypeptides, or even promoting their correct refolding, by binding to hydrophobic residues that are normally restricted to the interior of the protein, thus preventing aggregation and irreversible loss of function (9,30). Because the accumulation of damaged proteins is a well established stimulus that results in the induction of apoptosis (9), it is conceivable that elevated expression of HSP72 prevents or retards the onset of apoptosis through stabilizing effects on cellular macromolecules. In summary, these experiments clearly demonstrate that HSP72 protects renal epithelial cells against the adverse effects of high urea concentrations, and they suggest that HSP72, which is expressed at high levels in the renal inner medulla, is an integral part of the defense mechanism of medullary cells, for survival in an environment characterized by extreme concentrations of urea.
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Acknowledgments |
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