1 Section of Nephrology, Department of Medicine, University of Illinois at Chicago, Chicago 60612 - 7315; 3 Department of Physiology and Biophysics, College of Medicine, University of Illinois at Chicago, Chicago 60612 - 7342; and 2 West Side Division, Veterans Administration Chicago Health Care System, Chicago, Illinois 60612
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ABSTRACT |
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Regulation of renal
Na-HCO
carbachol; angiotensin II; acute acidosis; autophosphorylation; sodium-bicarbonate cotransporter; proline-rich tyrosine kinase 2
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INTRODUCTION |
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WE HAVE
PREVIOUSLY CHARACTERIZED the regulation of
Na-HCO
The nonreceptor tyrosine kinase (NRTK) proline-rich tyrosine kinase 2 (Pyk2) (2, 10), also referred to as related adhesion focal
tyrosine kinase (1), cellular adhesion kinase-
(26), and calcium-dependent tyrosine kinase
(34), is known to interact with SFKs, an association that
requires autophosphorylation and thereby plays an important role in
coupling both G protein-coupled receptors (GPCR) and growth factor
receptors to classic MAPK pathway activation in a number of cell types.
Calcium-dependent Pyk2 activation and Src interaction have been
demonstrated after GPCR stimulation by carbachol and ANG II
(8). We therefore examined whether Pyk2 could play a
similar role in the calcium-dependent carbachol and ANG II stimulation
of NBC1 activity in renal epithelial cells.
The role of Pyk2 in epithelial cells has been incompletely explored
(9, 11) and, to our knowledge, has not been examined in
the kidney. Pyk2 exhibits renal expression (10, 12, 30) and has been immunohistochemically localized to the renal proximal tubule (12). We have recently reported, in preliminary
form, the presence of endogenous Pyk2 in the opossum kidney (OK)
proximal tubule cell line (6). In the present study, we
examined both Pyk2 phosphorylation and Pyk2-SFK interaction for roles
in the regulation of basolateral NBC activity in these cells. This
cotransporter is a major determinant of HCO
We have previously demonstrated a general role for G proteins in the regulation of proximal tubule cell NBC activity (19). More recently, we have shown that activation of specific GPCRs for both muscarinic cholinergic agonists (14) and ANG II (15) increase NBC activity by means of the sequential activation of Src family tyrosine kinases (SFKs) and the classic MAPK pathway. Although a general requirement for calcium/calmodulin has been demonstrated for cholinergic stimulation of NBC activity (16) and calcium is a known effector of many of angiotensin's cellular actions, the specific mechanisms coupling GPCR activation to SFK activation, and ultimately to increased NBC activity, have not been defined. We therefore sought to examine whether Pyk2 might play a role in these processes.
In addition to the GPCR-mediated processes above, acute acidosis has also been shown to activate both SFKs (22, 32, 33) and the classic MAPK pathway (22) in this cell type, thereby increasing NBC activity (22). We therefore also examined the role of Pyk2 in this unrelated, non-GPCR-mediated process.
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MATERIALS AND METHODS |
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Reagents.
The pH-sensitive fluorophore BCECF was obtained from Molecular Probes
(Eugene, OR) as the membrane-permeable AM (BCECF-AM). Amiloride was
purchased from Research Biochemicals (Natick, MA). Src-specific
antibodies (GD11) were obtained from Upstate Biotechnology (Lake
Placid, NY), as were the SFK and Src assay kits and ERK kinase kits
employed herein. Phosphotyrosine-specific antibodies were obtained from
Zymed (PY Plus; South San Francisco, CA) or BD Transduction
Laboratories (RC20, Lexington, KY). Pyk2-specific monoclonal antibodies
were also obtained from BD Transduction Laboratories. Rabbit polyclonal
anti-Pyk2 phosphospecific antibodies (-Pyk2-pTyr402)
were obtained from Biosource (Camarillo, CA).
Agarose-immobilized Src-SH2 domain peptides were purchased from
Calbiochem (La Jolla, CA). Immunoblots were analyzed with a
commercially available chemiluminescence detection system (Phototope)
from New England Biolabs (Beverly, MA). All other immunoblotting
reagents, including nitrocellulose membranes, were routinely obtained
from Bio-Rad (Hercules, CA). Cell culture reagents, including serum and
additives, were purchased from GIBCO BRL (Grand Island, NY). All other
reagents, including carbachol, ANG II, the calmodulin antagonist
N-(4-aminobutyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-13), and murine monoclonal antibodies directed against
the c-myc epitope (MEQKLISEEDL, clone 9E10) (7)
were obtained from Sigma (St. Louis, MO).
Cell culture. Mycoplasma-free American OK cells were obtained from the American Type Culture Collection (Rockville, MD) at passage 37 and were maintained in Eagle's minimum essential medium containing Earle's balanced salts and supplemented with 10% fetal bovine serum as described previously (14). Cells were routinely serum deprived for 24 h before and during testing and were typically preincubated with inhibitors for at least 30 min where appropriate.
Fluorometric assays of pHi and NBC activity.
Confluent cell monolayers cultured on uncoated plastic coverslips were
made quiescent, loaded with BCECF, and continuously monitored for
pH-dependent changes in fluorescence at 37°C as described previously
(14, 23). In brief, cells were perfused at 37°C with a
Cl-free physiological solution [(in mM) 25 NaHCO
Src phosphorylation and kinase activity assays.
Src phosphorylation was assessed by quantitative immunoblot analysis of
whole cell lysates by using both Src-specific polyclonal antisera and
recombinant monoclonal anti-phosphotyrosine antibodies (RC20) as
described previously (14, 22). Src kinase activity was
evaluated in parallel by using a commercially available
immunoprecipitated kinase activity assay (Upstate Biotechnology, Lake
Placid, NY) according to the manufacturer's recommendations. In brief,
we tested the in vitro ability of Src immunoprecipiates to
phosphorylate a synthetic oligopeptide substrate (KVEKIGEGTYGVVYK)
corresponding to residues 6-20 of
p34cdc2 (3). Samples were incubated
in (in mM) 25 Tris · HCl (pH 7.2), 31.3 MgCl2, 25 MnCl2, 0.5 EGTA, and 0.5 DTT, as well as 62.5 µM Na3VO4 and 112.5 µM ATP containing 10 µCi
[-32P]ATP at 30°C for 10 min before the reaction was
stopped by the addition of trichloroacetic acid. After application to
P81 phosphocellulose paper, unincorporated radionuclides were eluted
with 7.5% (vol/vol) phosphoric acid. Phosphotranferase activity was
then assayed as specific 32P incorporation into the
substrate by liquid scintillation counting.
ERK1/2 activity assay.
Total ERK1/2 kinase activity was measured by using a commercially
available nonradioactive in vitro immunoprecipitated kinase activity
assay (Upstate Biotechnology) according to the manufacturer's recommendations. In brief, ERK1/2 immunoprecipitates prepared from whole cell lysates were tested for the ability to phosphorylate myelin basic protein in the presence of inhibitors of PKA, PKC (PKC
inhibitor peptide), and calmodulin kinase II (R24571). The total
concentration mixture consisted of (in mM) 15 MOPS, pH 7.2, 18.75 -glycerolphosphate, 18.75 MgCl2, 3.75 EGTA, 0.75 sodium
orthovanadate, 0.75 DTT, and 112.5 ATP, and (in µM) 5 PKC inhibitor,
0.5 PKA inhibitor, and 0.5 compound R24571, as well as 20 mg of
dephosphorylated myelin basic protein. Reactions were carried out at
30°C for 20 min. Five microliters of the reaction were resolved in
12% SDS-PAGE and probed with anti-pMBP. Carbachol and ANG II findings
were also confirmed with a radioactive immunoprecipitated ERK1/2
activity assay as described previously (14).
Ectopic Pyk2 expression. Mammalian expression vectors encoding wild-type Pyk2 (Pyk2-WT) mutant (pCMV-Pyk2-WT) and a K457A-kinase-dead Pyk2 (Pyk2-KD) mutant (pCMV-Pyk2-KD) were the generous gift of Dr. J. Thomas Parsons (University of Virginia) and have been described previously (31). These vectors express full-length Pyk2-WT or Pyk2-KD fused to an NH2-terminal c-myc epitope tag (MEQKLISEEDL) (7). Transient gene transfer was achieved in subconfluent OK cells by using LipofectAMINE lipofection reagent (Life Technologies, Rockville, MD) according to the manufacturer's recommendations and as described previously (14). Transiently transfected cells were routinely tested within 48 h of gene transfer. Ectopic Pyk2-WT or Pyk2-KD (K457A) expression was routinely confirmed by immunoblot analysis with the 9E10 murine monoclonal antibody directed against the c-myc epitope tag (7). For fluorometric assay, stable transfection of OK cells with Pyk2-KD was performed by using the same method and was selected with 400 µg/ml G418.
Phoshorylation and immunoprecipitation. Whole cell lysates (500 µg) were incubated with antibodies specific for Pyk2, phosphotyrosine, or Src for 6 h at 4°C. The resulting immunocomplexes were precipitated with immobilized protein G/protein A (Calbiochem). Constituent proteins were eluted from these immunocomplexes by boiling for 10 min in 2× SDS sample buffer and were resolved by denaturing PAGE before transfer to nitrocellulose for immunoblotting. To examine the effect of W-13 on Pyk2 phosphorylation, quiescent cells transiently transfected with Pyk2-WT were treated with 100 µM of W-13 for 30 min and stimulated with carbachol. The cells were lysed and 500 µg of lysates were immunoprecipitated with anti-Pyk2 or anti-phosphotyrosine and immunoblotted with anti-phosphotyrosine and anti-Pyk2, respectively.
Pyk2 autophosphorylation was determined by probing an equal amount of carbachol stimulated and paired control cell lysates withImmunoblot analysis. Whole cell lysates were routinely prepared in radioimmunoprecipitation assay buffer (50 mM Tris · Cl, 1% Nonidet P-40, 0.25% Na-deoxycholate, 150 mM NaCl, and 1 mM EDTA) supplemented with both protease inhibitor and phosphatase inhibitor cocktails (Sigma). Lysate proteins were electrophoretically resolved and transferred to nitrocellulose for immunoblotting as described previously (14). After incubation in Tris-buffered saline with 0.1% Tween 20, 20 mM Tris · Cl, and 500 mM NaCl containing 10% (wt/vol) immunoblot grade nonfat dry milk for 1 h at room temperature to block nonspecific binding, primary antibodies were routinely incubated.
Pyk2-Src coprecipitation assays.
Pyk2-Src interaction was assayed by precipitating 500 µg of
stimulated and unstimulated Pyk2-WT-overexpressing cell lysates with
-Src or
-Pyk2 antibodies. The eluted proteins were analyzed by
immunoblotting with
-Pyk2 or anti-Src, respectively. Coprecipitation with SH2 domain linked to protein A-agarose was also performed.
Statistical analysis. Densitometric analysis of Western blots was carried out by using NIH Image software. All data are expressed as means ± SE for a series of n experiments. Student's t-tests or ANOVA were used. P values <0.05 were considered to represent significant differences.
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RESULTS |
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Cholinergic stimulation of Src kinase activity is
calcium/calmodulin dependent.
We have previously demonstrated a general requirement for increased
intracellular calcium in the stimulation of NBC activity by both
carbachol and ANG II. We have also shown that carbachol- and ANG
II-stimulated NBC activity share a common requirement for sequential
SFK and classic MAPK pathway activation (14, 21). The
mechanism whereby intracellular calcium is coupled to these other
signaling intermediates, however, has not been defined. The ubiquitous
calcium-binding protein calmodulin is known to couple intracellular
calcium to the activation of a number of signaling intermediates,
including SFKs. Thus, to better define the relationship between
intracellular calcium and other signaling intermediates implicated in
NBC regulation, we examined the calcium chelator BAPTA and the
calmodulin antagonist W-13 for the ability to prevent activation of
both SFKs and the classic MAPK pathway. As shown in Table
1, both carbachol and ANG II increased
the activities of SFK and Src, as well as ERK, the distal element of
the classic MAPK pathway. We have examined the effect of ANG II,
carbachol, or 10% CO2 on MAPK activity by using ERK1/2
immunoprecipitates for their ability to phosphorylate myelin basic
protein. As can be seen in Fig. 1, ANG
II, carbachol, or 10% CO2 all increased ERK1/2
activity. These effects were inhibited by intracellular calcium
chelation (+BAPTA), as well as by calmodulin antagonism (+W-13).
Neither BAPTA nor W-13 had a significant effect on the basal activities
of these signaling intermediates, suggesting that neither agent
directly inhibits the measured kinase activities.
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Cholinergic stimulation increases endogenous Pyk2 tyrosine
phosphorylation.
Cholinergic stimulation of SFK activation is coupled to
calcium/calmodulin and Pyk2 activation in PC12 cells. Similar coupling has been observed for ANG II stimulation in myocardial cells. We
therefore studied a possible role for Pyk2 in coupling intracellular calcium to the regulation of NBC activity in renal epithelial cells. As
depicted in Fig. 2A, Pyk2 is
strongly expressed in OK cells. Cholinergic stimulation increased
tyrosine phosphorylation of a protein that comigrates with Pyk2 (Fig.
2B) but not total Pyk2 abundance in quiescent OK cells
stimulated with 100 µM carbachol for 5 min.
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Ectopic expression of both Pyk2-WT and Pyk2-KD in OK cells and Pyk2
phosphorylation.
To determine whether a Pyk2 catalytic site plays a role in
phosphorylation and association, we transiently transfected OK cells
with Pyk2-WT and Pyk2-KD. After 48 h of transfection, cells were
lysed and Pyk2 were analyzed by using Pyk2 and c-myc
antibodies. An intense band of ~110 kDa for c-myc was
noted in Pyk2-WT and Pyk2-KD transfectants, confirming successful
overexpression because Pyk2-WT and Pyk2-KD were both targeted with
c-myc epitope (Fig. 3A). Phosphorylation assays
were also performed on cells overexpressing Pyk2-WT and Pyk2-KD with
and without carbachol stimulation. Figure 3B shows that Pyk2
phosphorylation of cells transfected with Pyk2-WT was increased by
carbachol compared with control, and transfection of Pyk2-KD prevented
phosphorylation. Quantitative analysis of Pyk2 phosphorylation showed a
significant increase in phosphorylation by carbachol (Fig.
3C).
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Cholinergic stimulation increases Pyk2 autophosphorylation.
Increased phosphorylation was shown in Fig. 3. To examine whether
autophosphorylation is involved, we used Pyk2-pTyr402
antibodies to detect phosphorylation of Tyr402 residue.
Figure 4A shows that
endogenous Pyk2 is phosphorylated by cholinegic stimulation. The same
result was obtained in cells overexpressing Pyk2-WT, and
autophosphorylation was completely abolished in Pyk2-KD-overexpressing
cells (Fig. 4B).
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Pyk2 autophosphorylation requires calcium/calmodulin.
To examine the calcium/calmodulin dependence of Pyk2
autophosphorylation, we preincubated cells overexpressing Pyk2-WT with W-13 (100 µM × 30 min) before carbachol stimulation. At 5 min, cells were harvested and lysed. Whole cell lysates were precipitated with anti-Pyk2 or anti-phosphotyrosine and probed with
anti-phosphotyrosine and anti-Pyk2, respectively. There was an increase
in Pyk2 phosphorylation with carbachol, but this effect was totally
blocked with W-13 (Fig. 5A),
consistent with a proximal effector function for calcium/calmodulin in
Pyk2 phosphorylation. Identical results were observed for endogenous Pyk2 (Fig. 5B).
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Autophosphorylated Pyk2 physically associates with Src.
In previous experiments, Pyk2 autophosphorylation
(Pyk2-Tyr402) in response to carbachol stimulation was
shown, and the result was consistent with increased total tyrosine
phosphorylation of Pyk2 in response to carbachol. Thus we sought to
determine whether autophosphorylation plays a role in Pyk2 and Src
association as previously shown in PC12 cells. Cells overexpressing
Pyk2-WT were stimulated with carbachol, and Src-Pyk2
association was analyzed by coprecipitation assays. Figure
6A shows that in the presence of carbachol, there was an increased Src-Pyk2 association. This association was shown to be mediated by Src-SH2 domains. Pyk2 was found
to physically interact with Src under conditions associated with Pyk2
autophosphorylation. The ability of immobilized Src-SH2 domains to
mimic Src in coprecipitating Pyk2 suggests specific contributions by
SH2-phosphotyrosine interactions. Figure 6B shows that
carbachol increases SH2-phospho-Pyk2 interaction compared with
unstimulated cells. This association was enhanced in cells overexpressing Pyk2-WT and was prevented in cells expressing Pyk2-KD.
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Disruption of Pyk2 function abrogates stimulated, but not basal,
NBC activity.
We have previously shown that OK cells express the proximal tubule NBC1
isoform (15), and NBC activity is increased by ANG II,
carbachol, and 10% CO2 (14, 15, 20). We have
shown here that Pyk2-KD overexpression decreased both Pyk2
autophosphorylation and Src-Pyk2 association in carbachol-stimulated
cells. As an indirect test of the hypothesis that Pyk2 activation and
Pyk2-Src association play a central role in the transduction of diverse extracellular signals that serve to increase NBC activity, we examined
ectopic Pyk2-KD expression for the ability to attenuate the stimulatory
effects of carbachol (10 µM), ANG II (0.1 nM), and acute acidosis
(10% CO2). Table 2 shows
that Pyk2-KD overexpression prevented the effect of carbachol, ANG II,
or 10% CO2 to stimulate NBC activity without altering
baseline activity.
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DISCUSSION |
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Pyk2 is a recently described NRTK with structural similarities to focal adhesion kinase (FAK) that associates with SFK (2, 5, 8) through phosphorylation of Tyr402 and creates a binding site for SH2 domains of SFKs (29). Despite close structural homology to FAK, which also interacts with Src, Pyk2 is activated by various stimuli and has been shown to couple both GPCRs and growth factor receptors to classic MAPK pathway activation (8). Pyk2 has been shown to mediate different functions from those associated with FAK in a number of cell types (31). Pyk2 is also distinguished from FAK by means of its association with stimuli capable of increasing free intracellular calcium, activation of PKC, and exposure to stress factors (28, 29).
Although Pyk2 is predominantly expressed in the central nervous system and best characterized in PC12 cells, it is expressed in the kidney (10, 12, 30) and has been immunohistochemically localized to the proximal tubule (12). In the present work, we have established the renal epithelial cell expression of Pyk2, as well as its function in coupling diverse stimuli to NBC activation and signal transduction mechanisms associated with NBC activation in an OK renal epithelial cell line. We demonstrated that calcium/calmodulin and the classic MAPK pathway serve as important signaling effectors proximal and distal to Pyk2-SFK interaction, respectively. This also showed a common requirement of calcium/calmodulin and Pyk2-SFK interaction in the cholinergic and ANG II stimulation of epithelial cell NBC activity.
The presence of endogenous Pyk2 and its activation by acute acidosis in the OK renal epithelial cell line have also previously been reported in preliminary form (27), but Pyk2 activation in this cell type has not been characterized and its functional consequences are not known. In the present work, we confirmed the endogenous expression of Pyk2 in OK cells and have shown that this NRTK plays an important role in coupling the effect of carbachol, ANG II, and acute acidosis to SFK activation and ultimately to increased NBC activity. To our knowledge, this represents the first description of a functional interaction between Pyk2 and Src in renal epithelial cells.
Pyk2 autophosphorylation plays an important role in the transduction of diverse GPCR signals (5, 8) and was recently implicated in the translocation of GLUT4 (25). Both carbachol (4, 8-10) and ANG II (13, 24) have been shown to stimulate Pyk2 activation in a variety of cells types. In cardiac fibroblasts, this activation has been shown to be calcium/calmodulin sensitive (13) and the first description of Pyk2 activation by acute acidosis. Our findings suggest a specific mechanism whereby SFK activation may be coupled to diverse GPCR and non-GPCR stimuli through Pyk2 and show a central role of Pyk2 in transducing a number of physiologically important signals that regulate pHi by means of coincident effects on NBC.
Ectopic expression of the kinase-inactive K457A-Pyk2 mutant (Pyk2-KD) completely abrogated Pyk2 tyrosine phosphorylation in both stimulated and unstimulated cells. This mutant contains a single Lys for Ala mutation in the ATP binding site of the kinase domain of Pyk2 (31). The absence of detectable tyrosine phosphorylation for both unstimulated and stimulated cells ectopically expressing Pyk2-KD is compatible with the known requirement for an intact kinase domain and the absence of Pyk2 Tyr402 phosophorylation with Pyk2-KD is consistent with this result and emphasizes the importance of autophosphorylation. Overexpression of Pyk2-KD totally prevented Pyk2-Src association by means of the SH2 domain. Taken together, these findings suggest that Pyk2 autophosphorylation is required for both activity and interaction with Src. These findings are compatible with those reported previously in other cell types (31).
We have previously shown that the glucocorticoid stimulation of NBC activity was not inhibited by overexpression of the negative regulator of Src, Csk (23), indicating that the effect of ANG II, carbachol, and CO2 on the Src/MAPK pathway is specific. The exact mechanism whereby acute changes in pHi activate NRTKs, such as Pyk2 and Src, is not known, but acute acidosis has been variably associated with both increased and decreased free intracellular calcium content in a variety of cell types. A previous study has suggested that proximal tubule cell Src activation by acute intracellular acidosis is calcium independent (33). In our laboratory, CO2 stimulation of NBC1 activity appears to be calcium/calmodulin dependent (Ruiz OS and Arruda JAL, unpublished observations), suggesting that this may serve as a common mechanism of initiating the signaling cascade examined herein and culminating in increased NBC activity. The present studies show that the ability of both ANG II and CO2 to stimulate ERK1/2 activity was also inhibited by BAPTA and W-13 (Fig. 1). The ability of CO2 to mimic the effects of ANG II and carbachol in activating the calcium-dependent NRTK Pyk2 is compatible with this hypothesis.
In the present work, we have attempted to define the molecular basis for SFK activation and signaling in the stimulation of NBC activity by acute acidosis, cholineric agonists, and ANG II. These stimuli have been individually shown to signal by means of both SFK-dependent and SFK-independent mechanisms in a variety of cell types. It is therefore of considerable interest that these diverse stimuli share a common mechanism of NBC activation involving both SFK activation and SFK-MAPK coupling in renal epithelial cells (6, 21). We have clearly demonstrated that Pyk2-SFK interaction serves as a unifying coupling mechanism for these diverse stimuli and thereby plays an important role in the integration of a wide range of signals known to increase epithelial cell NBC activity. In addition to providing important mechanistic insights into the regulation of epithelial cell NBC activity, our findings provide new regulatory and functional insights into the role of Pyk2 in these cells. We have demonstrated the novel activation of Pyk2 by acute acidosis, and our findings also suggest a novel functional role for this NRTK in the regulation of pHi. A common requirement for Pyk2 in both the activation of SFKs and the subsequent stimulation of NBC activity is compatible with a pivotal role for Pyk2-SFK coupling in the regulation of vectorial ion transport in this cell type.
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ACKNOWLEDGEMENTS |
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The authors acknowledge the technical assistance of Yi-Yong Qiu, Fely Kear, and Dolores L. Mahmud.
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FOOTNOTES |
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This work was supported in part by grants-in-aid from local affiliates of the National Kidney Foundation and the American Heart Association, as well as by the Merit Review Program Awards from the US Department of Veterans Affairs. Portions of this work were presented in preliminary form at the 33rd Annual Meeting of the American Society of Nephrology on October 14, 2000 in Toronto, Ontario, Canada (6).
Address for reprint requests and other correspondence: J. A. L. Arruda, Rm. 418W CSN, Section of Nephrology (M/C 793), Univ. of Illinois at Chicago, 820 South Wood St., Chicago, IL 60612-73155 (E-mail JAArruda{at}uic.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
May 7, 2002;10.1152/ajprenal.00338.2001
Received 9 November 2001; accepted in final form 30 April 2002.
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