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【摘要】 Induction of heme oxygenase-1 (HO-1) in renal tubules occurs as an adaptive and beneficial response in acute renal failure (ARF) following ischemia and nephrotoxins. Using an in vitro model of polarized Madin-Darby canine kidney (MDCK) epithelial cells, we examined apical and basolateral cell surface sensitivity to HO-1 induction by heme. Basolateral exposure to 5 µM hemin (heme chloride) resulted in higher HO-1 induction than did apical exposure. The peak induction of HO-1 by basolateral application of hemin occurred between 12 and 18 h of exposure and was dose dependent. Similar cell surface sensitivity to hemin-induced HO-1 expression was observed using a mouse cortical collecting duct cell line (94D cells). Hepatocyte growth factor (HGF) is known to decrease cell polarity of MDCK cells. Following pretreatment with HGF, apically applied hemin gave greater stimulation of HO-1 expression, whereas HGF alone did not induce HO-1. We also examined the effect of hypoxia on hemin-mediated HO-1 induction. MDCK cells were subjected to hypoxia (1% O 2 ) for 24 h to simulate the effects of ischemic ARF. Under hypoxic conditions, both apical as well as basolateral surfaces of MDCK were more sensitive to HO-1 induction by hemin. Hypoxia alone did not induce HO-1 but appeared to potentiate both apical and basolateral sensitivity to hemin-mediated induction. These data demonstrate that the induction of HO-1 expression in polarized renal epithelia by heme is achieved primarily via basolateral exposure. However, under conditions of altered renal epithelial cell polarity and hypoxia, increased HO-1 induction occurs following apical exposure to heme.
【关键词】 acute renal failure kidney hepatocyte growth factor
HEME OXYGENASE - 1 (HO-1) is a 32-kDa microsomal enzyme that catalyzes the rate-limiting step in heme degradation, producing equimolar amounts of biliverdin, iron, and carbon monoxide ( 38 ). Two isoforms of HO have been identified: an inducible enzyme, HO-1, and a constitutive isoform, HO-2 ( 24 ). Several reviews have emphasized the importance of HO-1 as a potent cytoprotective enzyme as well as the biological effects of the HO-1 reaction product(s), which possess important antioxidant, anti-inflammatory, and antiapoptotic functions ( 9, 13, 14 ). HO-1 induction in the kidney provides an important adaptive and protective mechanism in response to acute renal injury secondary to ischemia-reperfusion, nephrotoxins (e.g., cisplatin), glomerulonephritis, renal transplant rejection, and rhabdomyolysis ( 18, 33 ). Therefore, the regulation of HO-1 in renal tubular epithelium is of critical importance.
HO-1 is dramatically induced after exposure to a wide variety of stimuli including heme ( 33 ). Heme serves as the prosthetic moiety for heme proteins such as hemoglobin, myoglobin, cytochromes, catalase, peroxidase, and respiratory burst oxidase ( 18, 29 ). Interestingly, heme exhibits a dual function by serving as a substrate for and inducer of HO-1 in vivo and in cultured cells ( 2, 3 ). In various models of renal disease, HO-1 expression is restricted to the renal epithelia lining the nephron ( 19, 26, 28 ). However, the relative cell surface (apical or basolateral) sensitivity of the renal tubular epithelium to heme-mediated induction of HO-1 is not understood.
The mammalian renal tubule is lined with a simple epithelium that is polarized with regard to cell surfaces and includes apical (facing the tubular lumen) and basolateral (facing the interstitium and vasa recta) surfaces. This renal epithelial cell polarity is important and necessary for renal physiology and host defense mechanisms.
Under normal, nonstressed conditions, levels of HO-1 in the renal tubule epithelium are low or undetectable ( 28 ). The epithelial cells lining the renal tubule are exposed to inducers of HO-1 such as heme from both the apical and basolateral surface during stressful conditions such as rhabdomyolysis. Because of water reabsorption, the concentration of heme becomes much higher in the renal tubule lumen especially in the distal nephron segments. Therefore, the apical surface would be exposed to relatively higher concentrations of heme. Because of these differences in concentrations of heme exposed to the apical and basolateral surface of the epithelium lining the renal tubule, we hypothesized that there would be differences in the sensitivity of HO-1 induction by heme on the apical and basolateral surfaces. To test this hypothesis, we have examined the role of epithelial cell polarity in the induction of HO-1 using an in vitro model of polarized renal epithelial cells. The data demonstrate that the apical surface of renal epithelial cells is relatively resistant to HO-1 induction compared with basolateral surface exposure. In addition, alteration of epithelial cell polarity with HGF or hypoxia increases apical sensitivity of HO-1 induction to heme.
MATERIALS AND METHODS
Cell culture. MDCK strain II cells were used between passages 3 and 10. Cells were cultured as previously described ( 5, 6 ) in modified Eagle's MEM containing Earl's balanced salt solution and glutamine supplemented with 5% fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B. The 94D mouse cortical collecting duct cell line was developed as previously described ( 42 ). 94D cells were cultured in DMEM/F-12, 5% fetal calf serum, 1.3 µg/l sodium selenite, 1.3 µg/l 3,3'5-triiodo-thyronine, 5 mg/l insulin, 5 mg/l transferrin, 2.5 mM glutamine, 5 µM dexamethasone, 100 U/ml penicillin, and 100 µg/ml streptomycin at 5% CO 2. All renal cell lines were plated at confluency on Transwell filter units (Costar, Cambridge, MA) with a pore size of 0.4 µm. R. Schwall generously provided recombinant human hepatocyte growth factor (HGF; Genentech, South San Francisco, CA). Because the c-met receptor is localized to the basolateral surface of MDCK cells ( 10 ), HGF was added to the basolateral compartment. Moreover, polarized MDCK cells do not respond to apically applied HGF ( 4 ). For hypoxia experiments, cells were placed in an incubator with 1% O 2 and at 37°C with 5% CO 2. Hemin (heme chloride, Sigma) was dissolved in DMSO. A 1 mM stock solution of hemin in DMSO was diluted in the MDCK cell medium containing serum to give the desired concentration of hemin. Equal amounts of DMSO were added to controls. All results are representative of at least two to three independent experiments.
Western blot analysis. Western blot analysis was performed as described previously with minor modifications ( 5 ). Cells were cultured on 24-mm Transwell filters. Briefly, equal amounts of protein in MDCK cell lysates were run on appropriate (12%) acrylamide gels at 160 V for 45 min. Proteins were transferred to Immobilon P membranes (Millipore, Bedford, MA). Membranes were blocked for 30 min with PBS- (PBS without Ca 2+ and Mg 2+ ), 5% milk, and 0.1% Tween 20 (block solution). For HO-1 and actin protein analysis, membranes were probed with primary antibodies of interest (rabbit anti-HO-1, 1:5,000 from Stressgen, SPA-896; rabbit anti- -actin, 1:2,000 from Sigma; mouse anti-E-cadherin, 1:500 from Transduction Laboratories) for 45 min and then washed with PBS-, 0.1% Tween 20 four times for 5 min each. Membranes were then probed with appropriate secondary horseradish peroxidase-labeled antibody (1:10,000 for both HO-1 and -actin and 1:25,000 for E-cadherin) for 60 min, washed with PBS-, 0.1% Tween 20 (4 x 5 min), and developed using the enhanced chemiluminescence kit (Amersham, Piscataway, NJ).
Immunofluorescent labeling of cells. MDCK strain II cells were grown on 6.5-mm-diameter Costar Transwell filters with 0.4-µm pores. Cells were washed with PBS+ (PBS containing Ca 2+ and Mg 2+ ) and then fixed with 4% paraformaldehyde for 20 min at 4°C. After filters were washed three times with PBS +, the cells were quenched with 75 mM NH 4 Cl and 20 mM glycine, pH 8.0, with KOH (quench solution) for 10 min at room temperature. Filters were washed one time with PBS + and permeabilized with PBS +, 0.7% fish skin gelatin, and 0.025% saponin (PFS) for 15 min at 37°C. Cells were exposed to primary antibody diluted in PFS [rabbit anti-HO-1 (1:500)] for 1 h at 37°C. Filters were then washed four times for 5 min each with PFS at room temperature and then labeled with the FITC-conjugated secondary antibody diluted 1:100 in PFS for 1 h at 37°C. Filters were rinsed four times for 5 min each with PFS, one time with PBS +, two times with PBS + containing 0.1% Triton X-100, and one time with PBS +. Cells were postfixed in 4% PFA for 15 min at room temperature. Filters were cut from the support with a scalpel and mounted in Vectashield Mounting Medium (Burlingame, CA). Immunofluorescent images were obtained with a laser-scanning confocal microscope (Leica LSCM, Heidelberg, Germany). The generated photomicrographs were captured and labeled using Adobe Photoshop. Representative data are presented in RESULTS.
Cytotoxicity assays. Cytotoxicity was determined by using the LIVE/DEAD viability/cytotoxicity assay (Molecular Probes, Eugene, OR) ( 41 ) and the clonogenic assay as previously described ( 11 ).
Measurement of transepithelial electrical resistance. For transepithelial electrical resistance (TER) experiments, MDCK strain II cell monolayers were grown on 12-mm-diameter, 0.4-µm pore-size filters, and electrical resistance was measured with the EVOM electrical resistance system (World Precision Instruments, New Haven, CT). All TER experiments were performed at least three times. The TER results are expressed as the measured resistance ( ) multiplied by the area of the filter (1 cm 2 ).
RESULTS
Effect of apical or basolateral surface exposure to hemin on HO-1 induction. We exposed the apical, basolateral, or both surfaces of filter-grown, polarized MDCK II cells to 5 µM hemin for 16 h. Following hemin exposure, cell lysates were prepared and analyzed for HO-1 expression by Western blot analysis. Figure 1 A demonstrates greater induction of HO-1 protein expression following basolateral surface exposure to hemin. To a lesser degree, HO-1 protein induction was also observed following apical surface exposure to hemin. Quantitation of HO-1 band densities following apical or basolateral exposure to hemin for 18 h was performed in six independent experiments. On average, basolateral exposure to hemin induced HO-1 expression 15-fold greater than that observed following apical exposure to hemin. Combined apical and basolateral exposure to hemin resulted in similar induction of HO-1 protein as observed in the cells exposed only to basolateral hemin. These results demonstrate that in a polarized renal epithelial cell line, the basolateral surface is more sensitive than the apical surface to hemin-induced HO-1 protein expression. We also examined apical and basolateral hemin exposure on HO-1 protein expression in a mouse cortical collecting duct cell line (94D) ( 42 ). In polarized 94D cells grown on filters, basolateral surface hemin exposure also induced higher levels of HO-1 protein expression than did apical surface exposure (data not shown).
Fig. 1. Madin-Darby canine kidney (MDCK) II cell basolateral surface is more sensitive to hemin-mediated induction of heme oxygenase-1 (HO-1) than the apical surface. A : apical (A), basolateral (B), or both (A+B) surfaces of polarized MDCK II cells were exposed to hemin (5 µM) for 16 h (C, control: DMSO, vehicle). Cell lysates were prepared in RIPA buffer and probed for HO-1 and actin (protein loading control) expression. B : time course of HO-1 induction by apical (A) or basolateral (B) exposure to hemin (5 µM) for indicated time points. C : dose response of hemin-induced HO-1 expression after apical (A) or basolateral (B) surface exposure for 16 h. In B and C, the samples were probed for E-cadherin expression as a protein-loading control.
The time course of HO-1 induction by apical or basolateral surface exposure to hemin was examined ( Fig. 1 B ). Again, basolateral exposure to hemin resulted in markedly higher levels of HO-1 protein induction than seen with apical exposure. Regardless of the cell surface exposed to hemin, induction of HO-1 protein was detected after 6 h of exposure, and the maximal stimulation of HO-1 protein was observed after exposure to hemin between 12 and 18 h and remained elevated for 24 h. This time course of HO-1 induction is similar to that observed in other model systems ( 32 ).
We next examined the hemin dose response of hemin-mediated HO-1 protein induction by either apical or basolateral exposure. Polarized MDCK II cells were apically or basolaterally exposed to increasing concentrations of hemin (0.005, 0.05, 0.5, or 10 µM resulted in cell toxicity. The results in Fig. 1 C confirm that the basolateral surface exposure to hemin gives greater induction of HO-1 protein expression than observed following apical surface exposure. However, at hemin concentrations below 5 µM, little to no HO-1 protein was observed in MDCK II cells regardless of the cell surface exposure. These data suggest that a threshold concentration of 5 µM is required to induce expression of HO-1 protein in MDCK II cells.
The induction of HO-1 may reflect the severity of cell injury. To assess whether the lesser induction of HO-1 with apical exposure, compared with basolateral exposure, was due to less cell injury that occurred with apical exposure, we examined the effect of cell surface exposure to hemin on cell viability using the LIVE/DEAD viability/cytotoxicity assay (Molecular Probes) and the clonogenic assay. At the concentrations used (5 µM), we did not detect an effect of hemin exposure on cell viability. In both assays, polarized MDCK strain II cells were exposed to vehicle or 5 µM hemin on the apical, the basolateral, or both surfaces for 16 h. With the LIVE/DEAD assay, the mean percentage of dead cells in the monolayers ± SD ( n = 5) was 0.195 ± 0.18, 0.15 ± 0.07, 0.28 ± 0.08, and 0.22 ± 0.10% for vehicle, apical, basolateral, and apical+basolateral hemin exposure, respectively. The percentages of dead cells in the all of the hemin exposed monolayers were not statistically different from the vehicle-exposed cell monolayers ( P 0.05). In the clonogenic assay, cell monolayers were exposed to vehicle or hemin on the apical, basolateral, or apical + basolateral surface for 16 h and trypsinized into a single-cell suspension. Cells were counted using a hemocytometer, equal numbers of cells were plated, and cell colonies were counted 5 days thereafter. The number of colonies ± SD ( n = 3) were 174.5 ± 19.7, 193.1 ± 12.1, 197.1 ± 13.0, and 187.7 ± 25.0 for vehicle, apical, basolateral, and apical+basolateral hemin exposure, respectively, and were not statistically different from the vehicle-exposed cells ( P 0.05).
Effect of HGF pretreatment on HO-1 induction by apical or basolateral exposure to hemin. Treatment of polarized MDCK II cells with HGF reduces epithelial cell polarity ( 5, 6, 8, 40, 41 ). Using this model system, we have demonstrated the importance of cell polarity in renal epithelial cell resistance to bacterial pathogens ( 41 ), apical surface susceptibility to adenoviral based gene delivery vectors ( 8 ), and production of antimicrobial factors ( 40 ). In addition, other investigators have provided evidence that HGF, by itself, is also an inducer of HO-1 in HepG2 cells ( 36 ). Therefore, we examined the effects of HGF treatment of MDCK II cells (100 ng/ml for 24 and 48 h) on cell surface sensitivity of hemin induction of HO-1 as well as on HO-1 induction. Figure 2 A shows that HGF treatment alone for periods extending to 48 h did not induce detectable levels of HO-1 as determined by Western blotting. However, HGF-treated MDCK II cells expressed higher levels of HO-1 following apical exposure to hemin by Western blotting ( Fig. 2 A ) and confocal microscopy ( Fig. 2 B ), which showed a perinuclear, endoplasmic reticulum staining pattern for HO-1 following induction. These data provide evidence that altering cell polarity with HGF increases the sensitivity of the apical (or luminal) surface to HO-1 induction by hemin.
Fig. 2. Hepatocyte growth factor (HGF) treatment increases apical sensitivity of MDCK II cells to hemin induction of HO-1. A : MDCK II cells were treated with HGF (100 ng/ml) for 24 and 48 h and then exposed to hemin (5 µM) on the apical (A), basolateral (B), or both (A+B) surfaces for 16 h (C, control: vehicle). Cell lysates were prepared in RIPA buffer and probed for HO-1 and actin (protein loading control) expression. B : control (no HGF) and HGF-treated (100 ng/ml) MDCK II cells were apically or basolaterally exposed to hemin (5 µM) for 16 h and HO-1 expression was visualized by laser-based confocal microscopy. Bars = 20 µm. C : HGF washout for 72 h restores relative apical resistance to HO-1 induction by hemin. MDCK II cells were treated with HGF (100 ng/ml) for 48 h and then exposed to hemin (5 µM) on the apical (A), basolateral (B), or both (A+B) surfaces for 16 h (C, control: vehicle). Another set of 48-h HGF-pretreated MDCK II cell monolayers was rinsed in fresh medium and cultured in fresh medium without HGF for 72 h. After the 72-h HGF washout, the cells were then exposed to hemin (5 µM) on the apical (A), basolateral (B), or both (A+B) surfaces for 16 h (C, control: vehicle). Cell lysates were prepared in RIPA buffer and probed for HO-1 and actin (protein-loading control) expression.
We have previously shown that HGF alters MDCK II cell polarity and that removal of HGF restores epithelial cell polarity ( 8 ). We examined whether removal of HGF from MDCK cells previously treated with HGF for 48 h restores the relatively low induction of HO-1 by apical exposure to hemin. These results are shown in Fig. 2 C. Following a 72-h washout period in medium not containing HGF, the MDCK cells express lower levels of HO-1 with apical exposure to hemin compared with MDCK cells treated with HGF for 48 h. These data correlate with our previously published morphological data showing that MDCK cell polarity is restored following removal of HGF stimulation ( 8 ).
Effect of hypoxia on HO-1 induction by apical and basolateral exposure to hemin. Hypoxia is a potent inducer of HO-1 in rat, bovine, mouse, and monkey cells but is a repressor in human cells ( 21, 22, 33 ). We examined the effects of hypoxia on HO-1 induction in general as well as cell surface sensitivity of induction by hemin in MDCK II cells. The results are shown in Fig. 3 and demonstrate that hypoxia leads to greater stimulation of HO-1 protein expression in MDCK II cells following either apical or basolateral stimulation with hemin. Hypoxia alone did not stimulate HO-1 protein expression in MDCK II cells. We next examined the dose response of hemin-mediated HO-1 induction under normoxic and hypoxic conditions. The apical or basolateral surface of polarized MDCK II cells was exposed to increasing concentrations of hemin for 24 h under normoxic and hypoxic conditions. Western blot analysis demonstrated that under hypoxic conditions, HO-1 induction was observed following exposure to hemin at lower concentrations on both the apical and basolateral surface of the renal epithelial cells ( Fig. 4 ). These data provide evidence that hypoxia increases both the apical and basolateral cell surface sensitivity to hemin-mediated HO-1 induction and that hypoxia alone does not stimulate HO-1 induction in MDCK II cells.
Fig. 3. Hypoxia increases HO-1 expression following apical (A), basolateral (B), or both (A+B) surface exposure to hemin. Polarized MDCK II cells were simultaneously exposed to hemin (5 µM) for 24 h on specific cell surfaces under normoxic and hypoxic (1% O 2 ) conditions. Cell lysates were prepared in RIPA buffer and probed for HO-1 and actin (protein-loading control) expression. C, control: vehicle.
Fig. 4. Dose-response of MDCK II cells to HO-1 induction by hemin [apical (A), basolateral (B), or both (A+B) surface exposure] under normoxic and hypoxic (1% O 2 ) conditions. The above surfaces of polarized MDCK II cells were exposed to increasing concentrations of hemin for 24 h under normoxic and hypoxic conditions. Cell lysates were prepared in RIPA buffer and probed for HO-1 and actin (protein loading control) expression. To allow for direct comparison of expression of HO-1 and actin, blots were exposed to film for identical times. C, control: vehicle.
TER is a surrogate marker for tight junction (TJ) function and paracellular permeability. We observed a 17% decrease in TER of MDCK II cells after 24 h of hypoxia ( P < 0.01; data not shown). Therefore, hypoxia may increase the apical surface sensitivity to hemin-mediated HO-1 induction by inhibition of TJ and allowing paracellular movement of apically applied hemin to the basolateral surface (see DISCUSSION ).
Cell surface sensitivity of HO-1 induction by cadmium. To examine the relative specificity of the apical and basolateral sensitivity of MDCK II cells to HO-1 induction by hemin, we examined the effects of apical, basolateral, and or both surface stimulation to cadmium chloride (another known inducer of HO-1). Figure 5 shows that cadmium exposure does result in the induction of HO-1. Both apical or basolateral surface exposure alone to cadmium resulted in similar induction of HO-1. However, exposure of both the apical and basolateral surface to cadmium resulted in greater HO-1 induction than seen with cadmium exposure to either surface alone. These data show that cell polarity does not influence cadmium-mediated HO-1 induction in MDCK II cells and that the differential cell surface sensitivity to hemin-mediated HO-1 induction is relatively specific.
Fig. 5. Cell surface polarity does not influence HO-1 induction by cadmium. Polarized MDCK II cells were exposed to cadmium (10 µM) for 16 h on the apical (A), basolateral (B), or both (A+B) surfaces. Cell lysates were prepared in RIPA buffer and probed for HO-1 and actin (protein-loading control) expression. C, control: vehicle.
DISCUSSION
The MDCK cell line, derived from the kidney tubules of a normal cocker spaniel in 1957 ( 23 ), is one of the best-characterized and most widely used cell lines in the study of epithelial cell polarity ( 34, 37 ) and represents an excellent model for the study of the role of renal epithelial cell polarity in HO-1 induction by hemin. The plasma membrane of epithelial cells in the renal tubule is divided into two major domains ( 27 ). The apical surface faces the lumen of the tubule, which is in contact with the glomerular ultrafiltrate. The basolateral surface faces adjoining tubular epithelial cells and the underlying interstitium and vasa recta. The apical and basolateral surfaces are separated by TJs. Apical and basolateral surfaces perform very different functions and therefore have distinct protein and lipid compositions. The apical surface is specialized for exchange of materials with the lumen, whereas the basolateral surface is specialized for interaction with other cells and exchange with the bloodstream. Our results demonstrate HO-1 induction is dramatically higher following hemin exposure to the basolateral surface compared with the apical surface in MDCK II cells as well as in a mouse cortical collecting duct cell line (94D). In addition, alteration of cell polarity changes this epithelial surface domain's responsiveness to hemin-mediated HO-1 induction. HGF treatment of well-polarized MDCK II cells leads to loss of polarity ( 5 ) and increases HO-1 induction following apical exposure to hemin. We also demonstrate that hypoxia increases both the apical and basolateral surface sensitivity to hemin-mediated HO-1 induction.
What are the potential mechanisms accounting for differences in apical and basolateral cell surface sensitivity to HO-1 induction by hemin? The results of this study suggest the presence of a hemin sensitivity factor that is relatively concentrated on the basolateral surface of polarized renal epithelial cells. Possible factors could include a receptor or transporter for hemin. Recently, the intestinal hemin transporter was cloned ( 30 ). This transporter has been identified as hemin carrier protein 1 (HCP-1) ( 30 ) and is localized to the apical surface of duodenal enterocytes. Northern blot analysis shows expression of HCP-1 mRNA in the duodenum and very little transcript in the kidney. In Caco-2 cells (an established model of intestinal epithelial cells) ( 17 ), hemin transport exhibits polarity with evidence for both hemin secretion and absorption, with hemin secretion greater than absorption ( 39 ). Interestingly, this same study also showed that cells exposed to hemin on the basolateral side demonstrated a higher HO-1 induction than cells exposed to hemin on the apical surface of Caco-2 cells ( 39 ). Thus intestinal epithelium shows a similar epithelial cell surface sensitivity to hemin-mediated HO-1 induction as seen in renal epithelial cells. These findings also suggest that the hemin transporter is not the major determinant of cell surface sensitivity of HO-1 induction by hemin in Caco-2 cells (if the heme transporter is also apically localized in this cell line).
Bacteria have developed outer membrane receptors to bind hemin. Hemin receptors have been characterized in numerous prokaryotic organisms including Vibrio fluvialis, Porphyromonas gingivalis, and Bordetella avium ( 1, 20, 35 ). With regard to eukaryotic organisms, evidence for a hemin receptor has been provided for on duodenal apical membranes and on the plasma membrane of erythroleukemia cells ( 15, 16 ). While the present work also provides evidence for the presence of a renal epithelial hemin receptor/transporter, further studies are required to define the hemin transporter and/or receptors in renal tubule epithelial cells.
Epithelial cell polarity plays a central role in physiology and host defense against pathogens. Using an in vitro model of renal epithelial tissue, this study demonstrates the importance of epithelial cell polarity in determining the sensitivity of the epithelium to hemin-mediated HO-1 induction. Alterations of epithelial cell polarity are thought to be the basis of several disease processes such as carcinogenesis, ischemia-reperfusion, polycystic kidney disease, and microvillus inclusion disease ( 25 ). These findings are also relevant to acute renal failure in rhabdomyolysis, wherein the kidney is challenged with an inordinate burden of filtered heme proteins. Vasoconstriction also occurs in this setting, and the resulting ischemia potentiates the heme protein-induced tubular injury ( 28 ). HO-1 is markedly induced in renal tubules in rhabdomyolysis, and increased filtered heme delivered to the lumen can cause HO-1 induction via apical stimulation in such pathological conditions.
The finding that under hypoxic conditions both the apical and basolateral surface of renal epithelial cells are sensitized to hemin-mediated HO-1 induction may be accounted for by different mechanisms. First, because hypoxia inhibits TJ function as demonstrated by the TJ surrogate marker TER, apically applied hemin may increase accessibility to the hemin-sensitive basolateral surface and greater HO-1 induction. However, the increased basolateral sensitivity to hemin-induced HO-1 by hypoxia cannot be accounted for by alterations in TJ permeability (if this were the case, basolaterally applied hemin would be diluted by leaking into the apical compartment and resulting in less HO-1 induction). Another possibility is the induction of HIF-1 by hypoxia and augmentation of HO-1 expression following basolateral exposure to hemin. Induction of HIF-1 has been shown to also upregulate HO-1 expression in other cell types but not in all human cells ( 31, 36 ). Consistent with our cell culture data, it has been recently shown that ischemia-reperfusion of the rat kidney increases the sensitivity to HO-1 induction by hemin ( 12 ). Because HO-1 is a protective enzyme during ischemic renal injury ( 33 ), the augmentation of luminal hemin-mediated HO-1 induction by alterations in polarity and hypoxia are likely to be very relevant in the pathogenesis and repair during acute renal injury following ischemia-reperfusion and rhabdomyolysis.
GRANTS
Support of this work was provided by the Department of Veterans Affairs (D. F. Balkovetz) and National Institutes of Health Grants RO1-DK-59600, HL-68157, and R21-67472 (A. Agarwal).
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作者单位：Veterans Administration Medical Center and Departments of Medicine, Surgery, and Cell Biology, Nephrology Research and Training Center, University of Alabama at Birmingham, Birmingham, Alabama