Albumin is a major serum survival factor for renal tubular cells and macrophages through scavenging of ROS

José Iglesias, Vivian Elizabeth Abernethy, Zhiyong Wang, Wilfred Lieberthal, Jason S. Koh, and Jerrold S. Levine

Renal Section, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

We have previously shown that lysophosphatidic acid (LPA), an abundant serum lipid that binds with high affinity to albumin, is a potent survival factor for mouse proximal tubular cells and peritoneal macrophages. We show here that BSA also has potent survival activity independent of bound lipids. Delipidated BSA (dBSA) protected cells from apoptosis induced by FCS withdrawal at concentrations as low as 1% of that in FCS. dBSA did not activate phosphatidylinositol 3-kinase, implying that its survival activity occurs via a mechanism distinct from that for most cytokines. On the basis of the following evidence, we propose that dBSA inhibits apoptosis by scavenging reactive oxygen species (ROS): 1) FCS withdrawal leads to ROS accumulation that is inhibitable by dBSA; 2) during protection from apoptosis, sulfhydryl and hydroxyl groups of dBSA are oxidized; and 3) chemical blockage of free sulfhydryl groups or preoxidation of dBSA with H2O2 removes its survival activity. Moreover, dBSA confers almost complete protection from cell death in a well-established model of oxidative injury (xanthine/xanthine oxidase). These results implicate albumin as a major serum survival factor. Inhibition of apoptosis by albumin occurs through at least two distinct mechanisms: carriage of LPA and scavenging of ROS.

apoptosis; reactive oxygen species; renal epithelial cells


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ABSTRACT
INTRODUCTION
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ALBUMIN IS A 69-kDa plasma protein with a variety of physiological functions, including maintenance of plasma oncotic pressure, buffering of acid-base changes, and transport to and from tissues of multiple substances within the bloodstream (7, 40, 41). Molecules carried by albumin include free fatty acids, phospholipids such as lysophosphatidic acid (LPA), steroid-based hormones, prostaglandins, and heavy metals (25, 27, 40, 41). Albumin is unique among plasma proteins in having a free sulfhydryl group at Cys34 as well as being devoid of any carbohydrate side chains (22, 41). The redox state of the free sulfhydryl group at Cys34 has been shown to be important in regulating the ligand binding capacity of albumin (22).

Albumin is frequently used as a carrier protein in cell culture systems. However, the potential role of albumin, or its multiple ligands, on the regulation of cell survival and apoptosis has not been fully explored. Recently, we have shown that albumin-bound LPA is a major serum noncytokine survival factor for primary cultures of murine peritoneal macrophages (mphi ) and renal tubular epithelial cells (25, 27). Because LPA was able to promote survival at concentrations as low as 50 nM, it is clear that even trace amounts of BSA can have a substantial effect on cell survival. During these studies, we observed that 99.95% delipidated BSA alone, which is devoid of LPA, was itself able to inhibit apoptosis of murine peritoneal mphi subjected to serum withdrawal (25).

We show here a novel role for albumin as a potent survival factor for primary cultures of murine mphi and renal tubular epithelial cells. This effect of albumin is independent of both bound lipids, such as LPA or free fatty acids, and the oncotic properties of albumin. The survival activity of albumin appears to depend on its ability to act as an antioxidant, most likely through scavenging of reactive oxidative species (ROS) by the free hydroxyl groups of albumin plus its free sulfhydryl group at Cys34.


    METHODS
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INTRODUCTION
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Reagents. Neutral dextran-70 was obtained from Pharmacia (Uppsala, Sweden). alpha 1-Glycoprotein (orosomucoid; alpha 1GP) and beta 2-microglobulin (beta 2MG) were both generous gifts of Dr. Michael Shia (Boston Univ. School of Medicine, Boston, MA). All other reagents, unless otherwise stated, were obtained from Sigma Chemical (St. Louis, MO).

Primary culture of mouse peritoneal mphi . Peritoneal exudate cells were harvested by lavage from C57Bl/6 mice (The Jackson Laboratory, Bar Harbor, ME) 3 days after ip injection of 1.5 ml of 4.05% thioglycollate broth (28). Cells were washed twice in RPMI 1640 and plated in 24-well tissue culture plates at 2 × 105 cells/well in R.10 culture medium (RPMI 1640 plus 10% FCS, with 2 mM L-glutamine, 5 mM HEPES, 100 U/ml penicillin, and 100 mg/ml streptomycin). After a 4-h incubation at 37°C, nonadherent cells were removed by washing with RPMI 1640. The remaining adherent cells, >98% mphi , as determined by morphological examination and nonspecific esterase staining (28), were cultured in R.10, R.0 medium (R.10 minus FCS), or R.0 plus various concentrations of BSA or 99.95% delipidated fraction V BSA (dBSA).

Primary culture of mouse proximal tubular cells. Mouse proximal tubular (MPT) cells were cultured from collagenase-digested fragments of proximal tubules isolated from the cortices of kidneys of C57Bl/6 mice by a modification of previously described methods (30). Cortical tubules were plated in FCS-free, defined culture medium (1:1 mixture of DMEM and Ham's F-12, with 2 mM L-glutamine, 1 mM HEPES, 5 µg/ml transferrin, 5 µg/ml insulin, 50 nM hydrocortisone, 50 U/ml penicillin, and 50 µg/ml streptomycin), denoted as "full medium". Growth factor-free medium is defined as full medium minus insulin and hydrocortisone. MPT cells grew to confluence from tubules over 5-7 days and were studied within 2 wk of achieving confluence. Cell monolayers were previously shown to be of proximal tubular origin by a combination of morphological, biochemical, and transport characteristics (30). After confluence was achieved, cells were washed twice in Ham's F-12 and subsequently grown in full medium, growth factor-free medium, or growth factor-free medium plus various concentrations of BSA or dBSA.

Phase contrast and immunofluorescent microscopy. Nuclear morphology was assessed by staining with H33342 (Calbiochem, San Diego, CA), a supravital DNA dye with excitation and emission wavelengths of 348 and 479 nm, respectively. H33342 enters live cells and so stains the nuclei of viable as well as apoptotic and necrotic cells. The nuclei of apoptotic cells are readily distinguishable from those of viable and necrotic cells on the basis of chromatin condensation, nuclear fragmentation, and increased brightness of H33342 fluorescence (25, 27, 30, 31). Adherent cells and cells that had detached spontaneously from culture wells were washed separately in PBS before being stained with H33342 (1.0 µg/ml) for 10 min at 37°C. Wet preparations were made on glass slides and examined under phase contrast and epifluorescence microscopy for visualizing cell morphology and H33342 nuclear staining in the same cells.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Mphi and MPT cells were cultured for 72 h and 7-10 days, respectively, in R.0 and growth factor-free medium alone or with FCS, high-dose insulin, and various concentrations of BSA or dBSA. The number of viable cells remaining was determined with the use of a modification of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (37). This assay is based on the ability of mitochondria from viable cells to cleave the tetrazolium rings of the pale yellow MTT and form a dark blue formazan product. The number of surviving cells is directly proportional to the level of the formazan product created. After removal of the growth medium, 165 µl of MTT dissolved in R.0 (mphi ) or growth factor-free medium (MPT cells) at 1 mg/ml were added to each well. After incubation at 37°C for 4 h, the MTT formazan was dissolved by addition of 165 µl of 10% SDS in 0.01 N HCl. Aliquots from each well were read with the use of a Dynatech microELISA plate reader with a test wavelength of 570 nm and a reference wavelength of 650 nm.

Data are presented as percent increased viability above R.0 (mphi ) or growth factor-free medium (MPT cells) and are normalized so that culture in R.10 (mphi ) or full medium (MPT cells) represents 100%. For example, the following formula was used for mphi cultured with dBSA
% Viability = <FR><NU>viability (dBSA) − viability (R.0)</NU><DE>viability (R.10) − viability (R.0)</DE></FR>

Mphi survival assay for N-acetylcysteine. N-acetylcysteine alone, in the absence of cells, promotes the conversion of MTT to its blue formazan product, so that an MTT assay could not be used to assess the effect of N-acetylcysteine on mphi survival. Instead, viability was determined by counting the number of cells that remained adherent to the culture well and excluded trypan blue. Percent increased viability above R.0 was then calculated identically as for data obtained by MTT assay.

Thymidine incorporation. Mphi and MPT cells were cultured for 24 h in survival factor-free medium alone or with FCS, high-dose insulin, and various concentrations of BSA or dBSA. A quantity of 2 µCi of [3H]thymidine (2 Ci/mmol; NEN, Boston, MA) was added for the final 12 h. Cells were washed three times with RPMI 1640, then incubated with 2.0 ml of ice-cold 5% TCA for 1 h at 4°C. The TCA was removed, and cells were washed once with fresh TCA. A quantity of 2.0 ml of ice-cold ethanol containing 200 µM potassium acetate was added to each well for 5 min, after which cells were incubated twice in 2.0 ml of a 3:1 mixture of ethanol and ether for 15 min per incubation. After air drying, cells were solubilized in 1.0 ml of 0.1 N NaOH. We measured [3H]thymidine counts per minute by adding samples to scintillation fluid and counting with the use of a model no. 1600TR Tri-Carb liquid scintillation analyzer beta -counter (Packard Instrument, Meriden, CT).

Oncotic pressure. Oncotic pressure (in mmHg) was measured with the use of a Wescor oncometer (model no. 4400; Logan, UT).

ROS accumulation. Mphi were cultured in R.10, R.0, or R.0 plus dBSA (10 mg/ml). MPT cells were cultured in full medium (high-dose insulin), growth factor-free medium, or growth factor-free medium plus dBSA (5 mg/ml). Assays were conducted at the onset of visible apoptosis: after 72 h for mphi and after 7-10 days for MPT cells. Cells were loaded with dichlorodihydrofluorescein diacetate (DCF) (3, 12, 38) for 45 min, after which point the dye was removed and the cells were washed and incubated in PBS for 60 min at 37°C. After collection of supernatants, cells were lysed in 1% Triton X-100 in PBS. DCF fluorescence of supernatants and lysates was measured with a Perkin-Elmer 650-105 fluorescence spectrophotometer (Norwalk, CT) with the use of excitation and emission wavelengths of 488 and 530 nm, respectively. Values were normalized for differences in cell number by quantitation of cell protein in parallel identically treated wells with the use of a microtiter bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL), according to manufacturer's instructions.

Phosphatidylinositol 3-kinase assay. Cell lysates were normalized for protein content and then incubated for 24 h with polyclonal anti-phosphatidylinositol 3-kinase (PI3K) antibodies directed against the 85-kDa regulatory subunit of the p85/p110 isoform (Upstate Biotechnology, Lake Placid, NY). Immune complexes were adsorbed onto protein A (p85/p110)-Sepharose; washed twice with PBS containing 1% Nonidet P-40 and 1 mM Na3VO4; washed three times with 100 mM Tris, pH 7.4, containing 5 mM LiCl and 1 mM Na3VO4; and finally washed twice with 10 mM Tris, pH 7.4, containing 160 mM NaCl, 5 mM EDTA, and 1 mM Na3VO4. PI3K assays were carried out for 10 min in a buffer containing 10 mM HEPES, pH 7.2, 1 mM EGTA, 20 mM MgCl2, 100 µM ATP, 10 µg phosphatidylinositol (Avanti Polar Lipids, Alabaster, AL), and 20 µCi [gamma -32P]ATP (20,000 Ci/mmol; NEN). Reactions were run for 10 min and then stopped by addition of 2 N HCl. Lipids were extracted with a 1:1 mixture of CHCl3 and CH3OH and then analyzed by TLC.

Preparation of BSA and dBSA. BSA and dBSA were dissolved at a concentration of 20 mg/ml in growth factor-free medium or R.0 for experiments involving MPT cells or mphi , respectively. Before use, solutions were filtered with a 0.2-µm filter. Protein content was determined with the use of a BCA protein assay (Pierce).

Modifications of dBSA. Carboxyamidation of the free sulfhydryl groups of dBSA was performed by treating dBSA with equimolar iodoacetamide at pH 8.0 for 10 h in the dark at 37°C (22). Peroxidation of dBSA was performed by treating dBSA with a 700 µM solution of H2O2 for 2 h at room temperature (44). Modified dBSA was then dialyzed extensively against growth factor-free medium or R.0 to remove excess iodoacetamide or H2O2.

Determination of protein free sulfhydryl content. The reaction of 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) with free sulfhydryl groups was used to determine the free sulfhydryl content of dBSA, carboxyamidated dBSA, peroxidized dBSA, or dBSA that had been exposed to cells in culture for 72 h (6). A quantity of 3 ml of protein solution was mixed with 2 ml of PBS, pH 8.0, and 5 ml of H2O. Then 20 µl of DTNB was added to 3 ml of this mixture, and absorbance was measured at 412 nm with the use of a Spectronic 1201 spectrophotometer (Milton Roy, Rochester, NY). All values were zeroed by subtracting the absorbance at 412 nm for 20 µl of DTNB in 3 ml of either RPMI 1640 or growth factor-free medium. Free sulfhydryl concentration (M) was then calculated with the use of the following formula
Free sulfhydryl concn = <FR><NU>(AD)</NU><DE>&egr;</DE></FR>
where A is the absorbance at 412 nm, D is the dilution factor (equal to 10/3), and epsilon  is the molar extinction coefficient, or 13,600 M-1 · cm-1. Protein content was determined with the use of a BCA protein assay (Pierce).

Determination of protein carbonyl content. The reaction of 2,4-dinitrophenylhydrazine (DNPH) with protein carbonyl groups was used to determine the carbonyl content of freshly prepared dBSA or dBSA that been exposed to cells in culture for 72 h (29). A quantity of 500 µl of protein solution was precipitated in 10% TCA at 4°C. After microcentrifugation at 11,000 g for 15 min, supernatants were discarded and precipitates were resuspended in 500 µl of 10 mM DNPH in 2 N HCl. Samples were incubated at room temperature for 60 min, with vortexing every 15 min. After addition of 500 µl of 20% TCA, samples were microcentrifuged at 11,000 g for 3 min and the supernatants were discarded. Precipitates were washed three times in ethanol-ethyl acetate (1:1, vol/vol), then redissolved at 37°C in 6 M guanidine in 20 mM potassium phosphate adjusted to pH 2.3 with 0.05% trifluoracetic acid. After removal of any remaining insoluble material by microcentrifugation, the absorbance at 374 nm was measured with the use of a Spectronic 1201 spectrophotometer (Milton Roy). All values were zeroed by subtracting the absorbance at 374 nm for a control dBSA sample treated identically except that no DNPH was added. Protein carbonyl concentration (M) was calculated by using the same formula as for sulfhydryl content except that A is the absorbance at 374 nm, the dilution factor D is 1, and the molar extinction coefficient epsilon  is 22,000 M-1 · cm-1. Protein content was determined with the use of a BCA protein assay (Pierce).

Xanthine/xanthine oxidase model. Xanthine was prepared as a 287 mM stock solution in 1 N NaOH. This stock solution was further diluted to 0.8 mM in R.0 or growth factor-free medium, and the pH was adjusted to 7.4. Xanthine oxidase was reconstituted in H2O at 5 U/ml. Before use, all reagents were filtered through a 0.2-µm filter.

Measurement of H2O2. Concentrations of H2O2 were determined with the use of the PeroXOQuant quantitative peroxide assay (Pierce), according to manufacturer's instructions.

Statistics. Quadruplicate wells for mphi and duplicate wells for MPT cells were examined in each experiment, and the results were averaged. A minimum of three experiments was performed for all data points. Data are expressed as means ± SE of the averaged values obtained from each experiment. Statistical significance was determined by a two-tailed Student's t-test.


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dBSA acts as a survival factor for primary cultures of murine peritoneal mphi and MPT cells. We have previously shown that primary cultures of murine peritoneal mphi and MPT cells undergo apoptosis on withdrawal of serum or growth factors (25, 27, 30). Loss of viability occurs asynchronously over several days, leading to gradual loss of the monolayer. On the basis of previous observations suggesting that BSA has survival activity for mphi independent of bound lipids (25), we assessed the ability of dBSA (99.95% delipidated) to inhibit apoptosis of mphi and MPT cells.

After 72 h in FCS-free medium (R.0), the majority of mphi exhibited typical features of apoptosis such as decreased cell size and rounding, followed by detachment from the monolayer (Fig. 1A). Hoechst staining of the remaining adherent mphi revealed many cells with homogeneous brightly staining nuclei, indicative of chromatin condensation, plus nuclear fragmentation, both unique features of cells dying by apoptosis (Fig. 1A). Detached mphi were uniformly apoptotic on Hoechst staining (data not shown), indicating that the decrease in cell number seen with R.0 reflects apoptotic cell death and not poor adherence of viable cells (25). In marked contrast, mphi cultured in R.0 plus FCS (R.10) or dBSA remained fully viable, with extensive spreading and dendritic-like processes (Fig. 1A). Hoechst staining of these mphi showed the faint nuclear staining, prominent nucleoli, and normal chromatin pattern of viable cells (Fig. 1A). No detached cells were found for mphi cultured with FCS or dBSA.



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Fig. 1.   A: 99.95% delipidated fraction V BSA (dBSA) maintains viability of macrophages (mphi ). Mphi were cultured for 72 h in serum-free medium alone (R.0) or R.0 supplemented with 10% FCS (R.10) or dBSA (5 mg/ml). After being washed, remaining adherent cells were photographed under phase microscopy. Cells were then gently scraped, stained with Hoechst dye, and photographed as a wet prep under phase and epifluorescence microscopy for visualization of cell morphology and Hoechst nuclear staining in same cells. B: dBSA maintains viability of mouse proximal tubular (MPT) cells. MPT cells were cultured for 7-10 days in full medium (high-dose insulin) and growth factor-free medium alone (GF-free) or supplemented with dBSA (5 mg/ml). Photography was then performed as described in A.

Similar results were obtained with MPT cells. After 7-10 days in growth factor-free medium, the majority of MPT cells underwent apoptosis and detached from the monolayer (Fig. 1B). Addition of high-dose insulin (which stimulates insulin-like growth factor I receptors) or dBSA inhibited apoptosis and maintained confluence of the monolayer (Fig. 1B). Hoechst staining of MPT cells cultured in growth factor-free medium showed nuclear condensation and fragmentation characteristic of apoptosis (Fig. 1B). In contrast, MPT cells cultured in the presence of high-dose insulin or dBSA appeared fully viable on Hoechst staining (Fig. 1B).

We next used the MTT assay to assess more quantitatively the role of BSA as a survival factor (Fig. 2). In these studies, we compared the effects of nondelipidated BSA vs. dBSA. Nondelipidated BSA contains high amounts of bound lipids, including LPA, a potent survival factor for both mphi and MPT cells (25, 27). Data are presented as percent increased viability above survival factor-free medium and normalized so that culture in 10% FCS (mphi ) or full medium (MPT cells) represents 100% (compare with METHODS). MTT assays were performed after 72 h of culture for mphi and after 7-10 days for MPT cells.



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Fig. 2.   dBSA is a survival factor for mphi and MPT cells. Viability of mphi and MPT cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. A: mphi were cultured for 72 h in R.0, R.10, or R.0 containing various concentrations of BSA or dBSA. P < 0.01, BSA (0.025 mg/ml) vs. R.0; P < 0.02, dBSA (0.5 mg/ml) vs. R.0. B: MPT cells were cultured for 7-10 days in full medium and GF-free medium alone or supplemented with various concentrations of BSA or dBSA. P < 0.05, BSA (0.0125 mg/ml) vs. GF-free medium; P < 0.02, dBSA (0.5 mg/ml) vs. GF-free medium.

Addition of BSA or dBSA led to enhanced survival of mphi (Fig. 2A) and MPT cells (Fig. 2B), with a clear dose-response relationship. Nondelipidated BSA had a significant effect on survival at concentrations as low as 0.025 mg/ml (~362 nM) for both mphi and MPT cells. This value, which is <0.01% of the concentration of BSA in FCS, is consistent with the known high potency of albumin-bound LPA as a survival factor for these cells (25, 27). Although dBSA had less survival activity than BSA at nearly all concentrations, a significant effect on survival by dBSA was seen at concentrations as low as 0.5 mg/ml (7.25 µM; ~1% of concentration in FCS). These results suggest that BSA promotes survival of mphi and MPT cells through two independent mechanisms, the first dependent on bound lipids such as LPA and a second unknown mechanism independent of lipids.

dBSA is not a mitogen for mphi and MPT cells. The increased numbers of viable cells seen with addition of dBSA could potentially be the result of two processes: inhibition of apoptosis and/or stimulation of proliferation. We assessed the contribution of dBSA-induced proliferation to increased mphi and MPT cell viability by measuring [3H]thymidine incorporation as an index of DNA synthesis. Cells were cultured for 24 h in growth factor-free medium alone or growth factor-free medium supplemented with FCS, high-dose insulin, BSA, or dBSA. [3H]thymidine was added for the final 12 h.

[3H]thymidine incorporation by mphi in the presence of dBSA and BSA was near background and did not differ from that seen with R.0 (data not shown). These results are consistent with our previous findings that peritoneal mphi , being terminally differentiated, are nonproliferative, and that the increased number of viable mphi seen in the presence of other mphi survival factors, such as FCS, LPA, and macrophage colony-stimulating factor, is almost entirely the result of inhibition of apoptosis rather than stimulation of proliferation (25).

Unlike mphi , MPT cells proliferate in response to mitogens such as high-dose insulin or LPA (27, 30). Data for MPT cells are presented as percent increased [3H]thymidine incorporation above growth factor-free medium and normalized so that culture with high-dose insulin represents 100%. As shown in Fig. 3, [3H]thymidine incorporation by MPT cells in the presence of BSA or dBSA was not significantly different from that in growth factor-free medium. We conclude that BSA is not a mitogen for mphi or MPT cells and hence that BSA maintains viability solely through inhibition of apoptosis.


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Fig. 3.   dBSA is not a mitogen for MPT cells. MPT cells were cultured for 24 h in GF-free medium alone or supplemented with high-dose insulin (5 µg/ml), BSA (5 mg/ml), or dBSA (5 mg/ml). [3H]thymidine incorporation was determined during final 12 h. Data are percent change in [3H]thymidine incorporation compared with high-dose insulin. P = not significant (NS), BSA or dBSA vs. GF-free medium.

Survival activity of dBSA is not due to trace contamination with lipopolysaccharide. The remainder of our studies were directed toward elucidating the lipid-independent mechanism by which BSA inhibits apoptosis. We first sought to exclude the possibility that contamination with lipopolysaccharide (LPS) might account for the survival activity of BSA. Although delipidation of BSA should remove most contaminating LPS, LPS is an extremely potent mphi survival factor, acting at concentrations as low as 100 pg/ml (34), so that even trace contamination with LPS could profoundly affect cell survival. We therefore determined the effect of BSA on survival in the presence and absence of polymyxin B, a cationic polypeptide antibiotic that neutralizes the effects of LPS by binding avidly to its lipid A core moiety (36). As shown in Fig. 4, the addition of polymyxin B (10 µg/ml) had no effect on the survival of mphi and MPT cells cultured in the presence of BSA, dBSA, FCS, or high-dose insulin. As a positive control, polymyxin inhibited LPS-induced release of interleukin-1 by mphi (not shown). These studies rule out any contribution from LPS contamination.



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Fig. 4.   Survival activity of dBSA is not due to lipopolysaccharide contamination. Viability of mphi and MPT cells was determined by MTT assay. A: mphi were cultured for 72 h with (+) and without (-) polymyxin B (10 µg/ml) in R.0 alone or R.0 supplemented with 10% FCS (R.10), BSA (10 mg/ml), or dBSA (10 mg/ml). P = NS, all comparisons of polymyxin B vs. no polymyxin B. B: MPT cells were cultured for 7-10 days in full medium and GF-free medium alone or supplemented with BSA (5 mg/ml) or dBSA (5 mg/ml). P = NS, all comparisons of polymyxin B vs. no polymyxin B.

Survival activity of dBSA is independent of its oncotic properties. Recent data have established the importance of adhesion-mediated changes in cell shape for the survival of endothelial cells (5). Because albumin is the major protein responsible for maintaining the colloid oncotic pressure of blood, we examined the possibility that the survival activity of BSA may be due to its oncotic properties and consequent changes in cell volume or shape. We compared the survival activity of dBSA (5 mg/ml or 725 µM) to that of an equimolar concentration of neutral dextran-70. The colloid oncotic pressures of these two solutions were measured independently and found to be 1.99 and 2.30 mmHg, respectively. Despite the slightly higher oncotic pressure for neutral dextran-70, the viability of both mphi and MPT cells in the presence of dBSA was significantly greater than that in the presence of dextran-70 (Fig. 5). We conclude that the survival activity of dBSA cannot be attributed to its oncotic properties.


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Fig. 5.   Survival activity of dBSA is independent of its oncotic properties. Mphi and MPT cells were cultured in survival factor-free media supplemented with dBSA or neutral dextran-70 of equimolar concentration (72.5 µM). Viability was determined after 72 h for mphi and after 7 days for MPT cells. P < 0.05, dBSA vs. neutral dextran-70 for both mphi and MPT cells.

Inhibition of apoptosis by dBSA is independent of PI3K activation. Activation of PI3K plays a critical role in survival factor signaling by a variety of cytokines. We have previously shown that LPA, a major serum survival factor tightly bound to albumin, prevents apoptosis of mphi and MPT cells subjected to survival factor withdrawal via activation of PI3K (25, 27). We therefore determined whether dBSA might also be activating PI3K. Mphi were FCS-starved for 6 h, then stimulated with dBSA or BSA. Consistent with its known carriage of LPA, nondelipidated BSA activated the p85/p110 isoform of PI3K within 2 min of stimulation, with peak activity occurring at 5 min (Fig. 6). These kinetics are consistent with our previously published data for LPA (25). In contrast, dBSA did not activate PI3K above baseline levels. We have previously shown that baseline PI3K activity is dependent on mphi adhesion and likely accounts for the ~25% survival seen after 72 h of FCS-free culture (25). We conclude that dBSA does not activate PI3K and that inhibition of apoptosis by dBSA occurs through a mechanism distinct from that for most cytokines.


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Fig. 6.   dBSA does not activate phosphatidylinositol 3-kinase (PI3K) in mphi . Mphi were precultured for 6 h in R.0. After preculture, mphi were stimulated with dBSA or BSA at 5 mg/ml for indicated times (in min). p85/p110 PI3K activity was determined as described in METHODS.

dBSA inhibits the accumulation of ROS after withdrawal of survival factors. Unlike the majority of circulating plasma proteins, in which cysteine residues form intermolecular or intramolecular disulfide bonds, albumin possesses a free sulfhydryl group at Cys34 (22, 41). By virtue of its reduced state, the sulfhydryl group at Cys34 may enable albumin to function as a circulating antioxidant. This property of albumin may be important to the regulation of apoptosis, since we have previously shown that apoptosis of MPT cells induced by growth factor withdrawal can be inhibited by a number of antioxidants and scavengers of ROS (30). Similarly, N-acetylcysteine (10 mM), which uses a free sulfhydryl group to scavenge ROS, promoted the survival of mphi cultured in R.0 (44 ± 17% survival, P < 0.05).

To test the hypothesis that the lipid-independent survival activity of BSA is attributable to scavenging of ROS, we first determined the effect of dBSA on the accumulation of ROS after withdrawal of survival factors. DCF is a cell-permeant fluorogenic dye that is trapped inside cells on cleavage of its lipophilic ester groups by intracellular nonspecific esterases (3, 12, 38). Reaction of DCF with ROS, in particular H2O2, leads to fluorescence of the fluorescein moiety within the DCF dye. As shown in Fig. 7, withdrawal of survival factors from mphi and MPT cells leads to ROS accumulation that is inhibitable by coincubation with dBSA. Accumulation of ROS was greater in supernatants than in cell lysates, most likely because intracellular oxidation of DCF is accompanied by significant leakage of the dye from cells (3) as well as because cells in the later stages of apoptosis lose cell membrane integrity. When the values for released and cell-associated ROS were combined, the reduction in ROS accumulation induced by dBSA was >10× for MPT cells and >100× for mphi .



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Fig. 7.   dBSA inhibits accumulation of reactive oxygen species (ROS) after withdrawal of survival factors. Mphi (A) and MPT cells (B) were cultured under indicated conditions until onset of visible apoptosis (3 days for mphi and 7-10 days for MPT cells). Cells were then loaded with dichlorodihydrofluorescein diacetate dye, washed, and incubated in PBS for 60 min. ROS accumulation was measured separately in supernatants and cell lysates by fluorometry, and values were normalized for differences in cell number by quantitation of cell protein. It should be noted that data are plotted on a log scale.

BSA is oxidized after withdrawal of survival factors. If dBSA inhibits apoptosis by scavenging of ROS, then dBSA should itself undergo progressive oxidation during coculture with mphi and MPT cells. Such oxidation would manifest as a decrease in free sulfhydryl content via oxidation of Cys34 and as an increase in carbonyl content via oxidation of free hydroxyl groups.

We used DTNB to measure the free sulfhydryl content of dBSA that had been coincubated with mphi and MPT cells in growth factor-free medium. DTNB reacts with protein sulfhydryl groups at pH 8.0 to produce a highly colored anion that can be measured by spectrophotometry (6). The free sulfhydryl content of freshly prepared dBSA (72.5 µM) was 40.5 ± 0.5 µM, or ~0.56 sulfhydryl groups/dBSA molecule, in accordance with the published value of ~0.60 sulfhydryls/dBSA molecule (20, 23, 44). After coincubation with MPT cells for 72 h in growth factor-free medium, the free sulfhydryl content of dBSA decreased to 22.3 ± 3.7 µM (Fig. 8A). To confirm that the decrease in sulfhydryl content was from oxidation and not from a decrease in dBSA concentration due to uptake by MPT cells, we determined the protein concentration of dBSA remaining in the supernatant at 72 h. This was 70 µM, or >95% of that at 0 h. Thus the sulfhydryl content of dBSA was reduced by almost 50% to ~0.32 sulfhydryls/molecule. Similarly, for mphi , the free sulfhydryl content of dBSA decreased from 43.5 ± 3.0 to 27.4 ± 2.8 µM after coincubation for 72 h in R.0 (Fig. 8A). The concentration of dBSA at 72 h was unchanged from that at 0 h.



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Fig. 8.   dBSA is oxidized after withdrawal of survival factors. Mphi and MPT cells were incubated in survival factor-free medium plus dBSA (5 mg/ml) for 72 h. Free sulfhydryl content (A) and carbonyl content (B) of cocultured dBSA were determined as described in METHODS and compared with those of freshly prepared dBSA. P < 0.02, sulfhydryl content of freshly prepared vs. 72-h coculture for both mphi and MPT cells; P < 0.002, carbonyl content of freshly prepared vs. 72-h coculture for both mphi and MPT cells.

We next measured the carbonyl content of dBSA that had been coincubated with mphi and MPT cells. The reaction of DNPH with protein carbonyls forms stable protein hydrazones, which can be quantitated spectrophotometrically (29). The carbonyl content of freshly prepared dBSA (72.5 µM) was 1.6 ± 0.2 nmol/mg. After coincubation with MPT cells or mphi for 72 h in growth factor-free medium, the carbonyl content of dBSA increased to 5.5 ± 1.2 and 7.9 ± 2.8 nmol/mg, respectively (Fig. 8B). Taken together, these data for sulfhydryl and carbonyl content show that dBSA undergoes oxidation during coincubation with MPT cells and mphi subjected to withdrawal of growth factors.

Oxidation and/or chemical blockade of the free sulfhydryl group in dBSA inhibits survival activity. Because coincubation of dBSA with MPT cells in growth factor-free medium led to a decrease in the free sulfhydryl content and an increase in the carbonyl content of dBSA, we next determined whether chemical blockade of the free sulfhydryl group and/or oxidation of dBSA would attenuate its survival activity during survival factor withdrawal. Modification of the free sulfhydryl group at Cys34 was accomplished in one of two ways. Oxidation by H2O2 reduced the free sulfhydryl content of dBSA (72.5 µM) from 41 ± 1 to 28 ± 3 µM, whereas irreversible carboxyamidation with iodoacetamide reduced the free sulfhydryl content to 17 ± 2 µM. As shown in Fig. 9, modification of dBSA by either or both of these two methods abolished most of the survival activity of dBSA for mphi and MPT cells undergoing survival factor withdrawal. We cannot be sure in these studies whether modification of the free sulfhydryl group at Cys34 and/or oxidation of hydroxyl groups are the only chemical changes induced in dBSA by H2O2 or iodoacetamide. Nonetheless, by showing that prior oxidation of dBSA and/or blockade of free sulfhydryl and hydroxyl groups profoundly diminish the survival activity of dBSA, these data further suggest that dBSA functions as a scavenger of ROS.


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Fig. 9.   Oxidation and/or chemical blockade of free sulfhydryl groups in dBSA attenuate survival activity. MPT cells and mphi were incubated in survival factor-free medium plus dBSA (5 mg/ml) that was either untreated, oxidized by treatment with H2O2, carboxyamidated by treatment with iodoacetamide to block free sulfhydryl groups, or both oxidized and carboxyamidated. Survival was assessed by MTT assay after 3 days for mphi and after 7-10 days for MPT cells. P < 0.005, untreated dBSA vs. all modified dBSA for both MPT cells and mphi .

BSA is a potent antioxidant, protecting in a well-established model of oxidant injury. As a final test of our hypothesis, we determined whether BSA would protect cells from ROS generated by the reaction between xanthine and xanthine oxidase, a well-established model of oxidant injury (39, 44). Xanthine oxidase catalyzes the oxidation of both xanthine and hypoxanthine to yield a number of ROS, including O-2, H2O2, and · OH (22). Mphi and MPT cells were incubated for 24 h in the presence of a nonlimiting concentration of xanthine. As shown in Fig. 10, A and B, the addition of xanthine oxidase led to the dose-dependent death of both cell types. At the highest concentration of xanthine oxidase (12.5 mU/ml), cell death occurred predominantly by necrosis (as assessed by increased cell size, loss of membrane integrity, and normal Hoechst nuclear staining), with only ~40% of cells still alive at 24 h. The addition of dBSA or BSA at 5 mg/ml led to nearly 100% protection from cell death at all concentrations of xanthine oxidase (Fig. 10, A and B). The limited cell death in the presence of dBSA or BSA was predominantly apoptotic (as assessed by decreased cell size, maintenance of membrane integrity, and nuclear condensation). Conversion from necrosis to apoptosis further implies a protective role for dBSA and is consistent with previous work showing that the severity of cellular injury determines the mechanism of cell death (31). Preoxidation of dBSA with H2O2 abolished most of the protective effect of dBSA for MPT cells (Fig. 10C).





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Fig. 10.   dBSA is a potent antioxidant, protecting cells from oxidant injury generated by reaction of xanthine and xanthine oxidase. Mphi (A) and MPT cells (B) were treated with excess xanthine (0.8 mM) and indicated concentrations of xanthine oxidase alone or plus either BSA (5 mg/ml) or dBSA (5 mg/ml). Survival was determined by MTT assay after 24 h. P < 0.05, BSA and dBSA vs. control for mphi at 12.5 and 1.25 mU/ml of xanthine oxidase; P < 0.05, BSA and dBSA vs. control for MPT cells at 12.5 and 2.5 mU/ml of xanthine oxidase. C: MPT cells were treated identically as in B except that cells were incubated with either dBSA (5 mg/ml) or H2O2-treated dBSA (oxidized). P < 0.005, dBSA vs. control for all concentrations of xanthine oxidase; P = NS, dBSA (oxidized) vs. control for all concentrations of xanthine oxidase. Xanthine alone, in absence of xanthine oxidase, had no effect on cell survival. D: xanthine oxidase (25 mU/ml) was added to excess xanthine (0.8 mM) in presence or absence of dBSA (5 mg/ml) in a cell-free system. Reduction in H2O2 concentration produced by dBSA was determined at indicated times. P < 0.05, dBSA vs. control for all times.

Finally, to show directly that dBSA can scavenge ROS, we determined the effect of dBSA on H2O2 accumulation generated by the reaction of xanthine and xanthine oxidase in a cell-free system (Fig. 10D). dBSA (5 mg/ml) reduced H2O2 accumulation by up to 35 µM. The effect of dBSA was maximal at 1 min. No additional effect of dBSA was seen up to 30 min, suggesting that the ability of dBSA to scavenge H2O2 was rapidly saturated. On average, each dBSA molecule scavenged ~0.48 molecules of H2O2. This value is close to the number of free sulfhydryls per dBSA molecule (~0.60) (20, 23, 44) and suggests that scavenging of H2O2 may occur predominantly via free sulfhydryl groups. Taken together, these data are consistent with the hypothesis that BSA promotes survival of cells treated with xanthine/xanthine oxidase by scavenging ROS.

Scavenging of ROS may be a general property of proteins. Proteins and amino acid acids vary in their susceptibility to oxidative attack (see DISCUSSION). Although albumin is unique among plasma proteins in having a free sulfhydryl group, we hypothesized that the presence of free hydroxyl groups and other sites capable of scavenging ROS should confer survival activity on a variety of proteins. We therefore determined the survival activity for mphi and MPT cells of three unrelated proteins, each having minimal free sulfhydryl content. These were ovalbumin, alpha 1GP, and beta 2MG. The free sulfhydryl content of 72.5 µM solutions of these proteins was 7.1, 5.4, and 6.7 µM, respectively, compared with a free sulfhydryl content of 40 µM for 72.5 µM dBSA. For mphi , only beta 2MG showed significant survival activity. In contrast, for MPT cells, all three proteins showed significant survival activity, with alpha 1GP and beta 2MG actually possessing survival activity equal to that of dBSA. Given probable differences in the ROS scavenged by these three proteins, our findings are consistent with at least two interpretations. MPT cells and mphi might differ in the type and quantity of ROS generated as a result of survival factor withdrawal, or, alternatively, the signaling pathways leading to induction of apoptosis in response to ROS might differ between these two cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Albumin is a major circulating plasma protein with a variety of functions, including maintenance of oncotic pressure and transport of multiple bioactive substances (7, 41, 42). Recently, we have shown that albumin-bound LPA is a major serum noncytokine survival factor for primary cultures of mphi and MPT cells (25, 27). In the course of these studies, we found that dBSA (99.95% delipidated) alone, in the absence of any other survival factors, was able to inhibit apoptosis of mphi subjected to FCS withdrawal (25).

Our objective here was to examine the lipid-independent role of BSA as a survival factor for mphi and MPT cells. We report several novel findings. First, dBSA alone is a highly potent survival factor. A significant effect on viability was seen at concentrations of dBSA as low as 1% of those normally found in plasma. At concentrations equivalent to those of plasma, the survival activity of dBSA was >= 50% of that achieved with FCS. Second, dBSA does not activate PI3K, which implies that its survival activity is attributable to a mechanism different from that for most cytokines. Third, the survival activity of dBSA seems to be attributable, at least in part, to its antioxidant properties through scavenging of ROS. Biochemical features of dBSA that are important in this regard include free hydroxyl groups and a free sulfhydryl group at Cys34, a unique feature of albumin in comparison with other plasma proteins (22, 41). Fourth, the antioxidant effect of dBSA is not limited to apoptosis induced by survival factor withdrawal. dBSA alone conferred almost complete protection from cell death in a well-established model of oxidative injury (xanthine/xanthine oxidase). Finally, protection against apoptosis induced by survival factor withdrawal may be a general feature of proteins, as alpha 1GP and beta 2MG inhibited apoptosis of MPT cells to a degree similar to dBSA. Protection, however, did not extend to all proteins (see ovalbumin in Fig. 11), nor did protection in one cell type generalize to protection in another cell type (compare data for mphi and MPT cells).


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Fig. 11.   Scavenging of ROS may be a general property of plasma proteins. MPT cells were cultured in full medium or GF-free medium alone or supplemented with indicated proteins at a concentration of 72.5 µM. Mphi were cultured in R.10, R.0, or R.0 supplemented with same proteins. Survival was determined by MTT assay after 7-10 days for MPT cells and after 3 days for mphi . P < 0.001, all proteins vs. GF-free medium for MPT cells; P < 0.001, beta 2-microglobulin vs. R.0 for mphi ; P = NS, ovalbumin and alpha 1-glycoprotein vs. R.0 for mphi .

In establishing the role of dBSA as an antioxidant, we ruled out several other mechanisms by which dBSA might promote cell survival. First, dBSA was not a mitogen for mphi or MPT cells, indicating that increased viability was the result of inhibition of apoptosis rather than stimulation of proliferation. Second, survival was not the result of trace contamination with LPS, as the addition of polymyxin B had no effect on dBSA-mediated survival. Finally, in light of a recent report indicating the importance of cell shape in promoting survival (5), we showed that oncotic effects did not play a major role, as the survival activity of neutral dextran-70 was significantly less than that of dBSA, despite equal oncotic pressures.

Although the precise role of ROS in apoptosis remains undefined (2, 21), antioxidants have been shown to be protective in several models of apoptosis induced by survival factor withdrawal, for example, sympathetic neurons deprived of nerve growth factor (13) or FL5.12 cells (a murine pro-B lymphocyte cell line) deprived of interleukin-3 (15). ROS most likely promote apoptosis by signaling its initiation, either by mediating cell damage or by acting as second messengers in the complex signal-transduction pathways involved in switching on the death program (2, 21). The role of ROS in the actual execution of apoptosis is far more uncertain. In accordance with these ideas, we have shown that apoptosis of MPT cells induced by survival factor withdrawal can be inhibited by a number of antioxidants and ROS scavengers (30). In this model, ROS appeared to act upstream of caspases in the apoptotic pathway (30). Here, we show that antioxidants also protect mphi , as N-acetylcysteine, which like albumin possesses a free sulfhydryl group, inhibited apoptosis of mphi subjected to FCS withdrawal.

The mechanism by which albumin and other plasma proteins exert their antioxidant effects is most likely multifactorial. Proteins such as transferrin, ceruloplasm, and albumin bind the transition metals Fe2+ and Cu2+, thereby preventing the generation of · OH via the Fenton reaction (15, 18, 32, 45). In addition, albumin and other proteins can act as sacrificial sinks for attack by ROS, either directly through oxidation of amino acid side chains or indirectly through reaction with lipid species and radicals arising from the peroxidation of cell membrane lipids (8, 9, 16). The amino acids cysteine, histidine, methionine, tyrosine, and tryptophan are particularly susceptible to direct oxidative attack, whereas lysine is most susceptible to attack by malondialdehyde, one of the principal products of lipid peroxidation (8, 9, 16). Albumin is unique among plasma proteins in possessing a free sulfhydryl group, making it a particularly effective scavenger of ROS (22, 41). In contrast, most sulfhydryl residues in other proteins form either inter- or intramolecular disulfide bonds (22). The role of albumin as an ROS scavenger has been confirmed in cell-free systems with the use of a wide variety of oxidative species, including HOCl, H2O2, · OH, carbon radicals, and peroxynitrite (8-11, 16, 17, 42-44, 46, 49-51). Finally, it is possible that the free sulfhydryl group of albumin enables it to act not only as an antioxidant but also as a reducing agent, affecting a wide variety of cell proteins, the function of which is dependent on redox state, including the N-methyl-D-aspartic acid receptor and the DNA binding proteins activator protein-1 and nuclear factor-kappa B (2, 21, 26, 35).

We explored the antioxidant role of dBSA by focusing on two potential oxidative reactions, namely, loss of the free sulfhydryl group at Cys34 and oxidation of hydroxyl groups to carbonyls. As assessed by oxidation of the fluorogenic dye DCF, dBSA inhibited the accumulation of ROS after withdrawal of survival factors by >10× in MPT cells and >100× in mphi . During protection of MPT cells and mphi from apoptosis, dBSA underwent progressive oxidation. Coincubation of dBSA with MPT cells and mphi subjected to 72 h of survival factor withdrawal led to an ~50% decrease in the sulfhydryl content of dBSA and an ~5× increase in carbonyl content. Importantly, peroxidation of dBSA by treatment with H2O2 or chemical blockade of the free sulfhydryl group at Cys34 by carboxyamidation almost completely eliminated the survival activity of dBSA for both MPT cells and mphi . In addition, peroxidation of dBSA also inhibited its ability to prevent the oxidative death of MPT cells and mphi exposed to xanthine and xanthine oxidase. These results strongly implicate free sulfhydryl and hydroxyl groups in the survival activity of dBSA. Nevertheless, it should be noted that our data do not rule out protective effects from these groups independent of oxidative changes, nor do they rule out the contribution of other potentially reactive sites in dBSA.

It is interesting to note that albumin has been previously reported to have survival activity in a number of systems. Lornage et al. (33) found that BSA was a survival factor for spermatozoa, and Twigg et al. (48) extended these findings by showing that BSA protected spermatozoa from oxidant injury. Burleson et al. (1) found that perfusion of transplantable kidneys with albumin produced viability of the organ for up to 50 h without structural damage. Guilbert and Iscove (14) reported that BSA was necessary for the survival of bone marrow-derived hematopoietic cell colonies. In two studies, dBSA also prevented injury to hepatocytes subjected to ischemia-reperfusion damage (4, 47). Finally, Zoellner et al. (52) showed that BSA, human serum albumin, and recombinant human albumin all inhibited apoptosis of adherent, but not nonadherent, endothelial cells.

Our studies provide a framework for interpreting these protective effects of albumin. Thus BSA may protect cells from apoptosis and/or oxidant injury by several distinct mechanisms. First, as shown here, dBSA is a potent antioxidant through scavenging of ROS. Second, nondelipidated BSA contains high concentrations of bioactive phospholipids. We have recently shown that LPA is a highly potent survival factor for mphi and MPT cells, acting at concentrations as low as 50 nM (25, 27). Finally, free unsaturated fatty acids, which also bind with high affinity to albumin, are potent survival factors for mphi (but not MPT cells) via activation of PI3K and nuclear factor-kappa B (24).

In conclusion, we have shown that BSA is a major survival factor in FCS. BSA inhibits apoptosis of primary cultures of mphi and MPT cells subjected to survival factor withdrawal by both lipid-dependent and -independent mechanisms. The lipid-dependent mechanism depends on activation of PI3K and relates to carriage of LPA and other bioactive lipids. The lipid-independent mechanism is independent of PI3K and is attributable, at least in part, to the ability of dBSA to act as a potent antioxidant through scavenging of ROS.


    ACKNOWLEDGEMENTS

We are grateful to Dr. Ronald McCaffrey for use of the microELISA plate reader.


    FOOTNOTES

This work was supported by National Institutes of Health Grants AR/AI-42732 (to J. S. Levine), DK-375105, and HL-53031 (both to W. Lieberthal) and a Clinical Scientist Award from the National Kidney Foundation (to J. S. Levine).

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J. S. Levine, Renal Section, E428, Boston Medical Center, One Boston Medical Center Place, Boston, MA 02118 (E-mail: jlevine{at}bu.edu).

Received 4 March 1999; accepted in final form 15 June 1999.


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Am J Physiol Renal Physiol 277(5):F711-F722
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