Department of Pharmacology, New York Medical College, Valhalla, New York 10595
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ABSTRACT |
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Primary cultures of medullary thick ascending limb (mTAL)
cells retain the capacity to express calcium-sensing receptor (CaR) mRNA and protein. Increases in cyclooxygenase-2 (COX-2) mRNA
accumulation, protein expression, and PGE2 synthesis were
observed in a dose- and time-dependent manner after exposure of these
cells to extracellular calcium (Ca
cyclooxygense-2; calcium-sensing receptor; medullary thick ascending limb
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INTRODUCTION |
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EXPRESSION OF
CYCLOOXYGENSE-2 (COX-2)
APPEARS to be differentially regulated in the kidney. For
instance, low salt increases COX-2 expression in the cortex, whereas
high salt increases expression in the medulla (36). These
differences are consistent with the ability of renal prostaglandins to
affect vascular or tubular events in the cortex and medulla,
respectively. The medullary thick ascending limb (mTAL), which is
impermeable to water but actively reabsorbs salt from tubular fluid,
helps to establish the osmolarity gradient along the loop of Henle. The
Na+ pump located on the basolateral membrane of the mTAL
provides the energy for this process. Na+, K+,
and Cl are reabsorbed from the tubular fluid via the
Na+-K+-2Cl
cotransporter on the
apical membrane, and K+ is recycled via apical
K+ channels back to the tubular fluid. PGE2,
the major prostaglandin produced in the kidney, has been reported to
inhibit the Na+-K+-2Cl
cotransporter and apical K+ channel (6, 15,
16). Thus PGE2 derived from COX-2 in the mTAL may be
part of a mechanism that contributes to the regulation of renal
function because tumor necrosis factor-
mediated decreases in
rubidium uptake (an in vitro correlate of natriuresis) were COX-2
dependent (10).
Disturbances in renal concentrating ability, water excretion, and loop
of Henle function have been associated with hypercalcemia in humans and
experimental animals (5, 18, 22). Moreover, increased
prostaglandin levels in the kidney have been linked to inhibition of
NaCl reabsorption by the TAL in hypercalcemia (22). The
locus and mechanisms of these effects are not fully understood.
However, immunohistochemical data indicated an increase in COX-2
expression in the outer medulla (18), suggesting that COX-2 expression in the mTAL may increase after challenge with extracellular calcium (Ca
Calcium-sensing receptor (CaR), originally cloned from the bovine
parathyroid gland (2), has also been found in several nephron segments including the rat mTAL (25, 26). CaR are G protein-coupled receptors that transduce Ca
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METHODS |
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Animals. Male Sprague-Dawley rats (Charles River Lab, Wilmington, MA), weighing 100-110 g, were maintained on standard rat chow (Ralston-Purina, Chicago, IL) and given tap water ad libitum.
Reagents. Tissue culture media, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and COX-2 primers (sense: TACAAGCAGTGGCAAAGGC, antisense: CAGTATTGAGGAGAACAGATGGG) were obtained from Life Technologies (Grand Island, NY). Reagent-grade chemicals and collagenase (type 1A) were from Sigma (St. Louis, MO). COX-2 antisera were from Cayman (Ann Arbor, MI). NS-398 and nimesulide were from Biomol (Ann Arbor, MI). Polyvinylidene difluoride (PVDF) membranes were obtained from Amersham (Arlington Heights, IL).
Isolation of mTAL cells. mTAL cells (~95% purity) were isolated and characterized as previously described (9, 17). Briefly, male Sprague-Dawley rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (0.65 mg/100 g body wt). The kidneys were perfused with sterile 0.9% saline, via retrograde perfusion of the aorta and cut along the corticopapillary axis. The inner stripe of the outer medulla was excised, minced with a sterile blade, and incubated for 10 min at 37°C in a 0.1% collagenase solution gassed with 95% O2. The suspension was sedimented on ice, mixed with Hanks' balanced salt solution (HBSS) containing 2% BSA, and the supernatant containing the crude suspension of tubules was collected. The collagenase digestion was repeated three times with the remaining undigested tissue. The combined tubule suspensions were spun, resuspended in HBSS, and filtered through 52-µm nylon mesh (Fisher Scientific, Springfield, NJ). The filtrated solution was discarded, and the tubules retained on the mesh were resuspended in HBSS. Then, the solution was centrifuged at 500 rpm for 5 min, the supernatant was aspirated away, and the cells were cultured in DMEM-Ham's F-12 medium (1:1), 10% fetal bovine serum (FBS), epidermal growth factor (20 ng/ml), 1% glutamine, streptomycin-penicillin (100 U/ml), and Fungizone (1 µg/ml; Gemini, Woodland, CA). After 3 days, monolayers of cells were 80-90% confluent. The cells were kept quiescent in RPMI containing 0.42 mM Ca2+ and 0.5% FBS for 18-24 h before use.
Isolation of total RNA/RT-PCR analysis. Total RNA was isolated by lysing cells or outer medullary tissue in TriZol reagent (GIBCO BRL, Life Technologies, Grand Island, NY) and precipitated with isopropyl alcohol. A 3-µg aliquot of total RNA isolated from unstimulated or stimulated mTAL cells was used for cDNA synthesis using the Superscript Preamplification system (GIBCO BRL, Life Technologies) in a 20-µl reaction mixture containing Superscript II RT (200 U) and random hexamers (50 ng). The reaction was performed at room temperature for 10 min to allow extension of the primers by RT, then at 42°C for 50 min, 70°C for 15 min, and 4°C for 5 min. An aliquot of the cDNA was then amplified using Taq DNA polymerase (2.5 U) in the presence of sense and antisense primers (1 µM) for murine COX-2, CaR, or GAPDH. For COX-2 and GAPDH, the samples were first denatured for 4 min at 94°C, and the amplification (30 cycles) was then initiated by 0.5 min of denaturation at 94°C, 1 min of annealing at 53°C, and polymerization for 0.5 min at 72°C followed by autoextension at 72°C for 8 min. For CaR, the samples were first denatured for 3 min at 94°C, and the amplification (35 cycles) was then initiated by 0.5 min of denaturation at 94°C, 0.5 min of annealing at 47°C, and polymerization for 1 min at 72°C followed by autoextension at 72°C for 10 min. PCR products were quantified by normalizing mRNA accumulation for COX-2 with GAPDH.
Western blot analysis. The media were removed, and cells were washed three times with PBS (1×). Cells were lysed using 10 mM Tris · HCl, pH 7.5, 1 mM EDTA, and 1% SDS for 5 min on ice. Outer medulla and heart were lysed in the same buffer after homogenization on ice. Protein concentrations were determined using a detergent-compatible Bio-Rad protein assay kit. Thirty micrograms of cell lysate were mixed with an equal volume of 2× SDS-PAGE loading buffer (100 mM Tris · Cl, pH 6.8, 200 mM mercaptoethanol, 4% SDS, 0.2% bromophenol blue, and 20% glycerol) and boiled for 3 min. The proteins in the cell lysate were separated on a 10% SDS-PAGE gel and transferred to PVDF membranes. The membranes were blocked with a solution containing 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) at room temperature for 30 min. Membranes were immunoblotted with a rabbit anti-mouse COX-2 polyclonal antibody or anti-CaR primary antibody (a kind gift from Dr. Steven Hebert, Yale University) for 1 h at room temperature. Membranes were washed 3 times with TBST and incubated with alkaline phosphatase-conjugated secondary antibody (Santa Cruz, CA) for 30 min at RT. Membranes were washed, and enhanced chemifluorescence and phosphorimaging were used for analysis of COX-2 or CaR protein expression.
PGE2 ELISA. Quiescent mTAL cells were incubated with calcium chloride (1-2 mM) or poly-L-arginine (PLA; 10-100 nM) in media containing 0.5% serum for varying times, after which the cell-free supernatants were assayed for PGE2 by ELISA (Neogen, Lexington, KY). Briefly, 50 µl of the sample and 50 µl of horseradish peroxidase (HRP)-conjugated PGE2 were added to wells of a 96-well plate that had previously been coated with anti-PGE2 antibody for 1 h. After incubation, substrate for HRP was added to each well for 30 min, and the reaction was terminated by the addition of 50 µl/well 1 N HCl. Quantitation was achieved by measuring absorbance at 450 nm.
Gene transfection. mTAL cells were cultured to 70-80% confluence. The medium was removed, and cells were placed in 1 ml of serum-free OPTI-MEM medium containing 3 µg/ml of either the plasmid DNA expressing CaR (a generous gift from Dr. Karin Rodland, University of Oregon) or an empty plasmid vector (pcDNA3.1) and 10 µl lipofectamine reagent (Life Technologies) for 4 h at 37°C/5% CO2. After the transfection period, 1 ml of DMEM-F-12 containing 20% FBS was added, and the cells were incubated overnight at 37°C/5% CO2. The medium was then removed, and cells were cultured for an additional 12 h in DMEM-F-12 containing 10% FBS. Then cells were kept quiescent overnight in RPMI medium containing 0.5% FBS. Cells were treated with the appropriate reagents, then washed three times with PBS; supernatants were collected for determination of PGE2 levels, and cellular protein was determined to normalize the ELISA data.
Statistical analysis.
The responses were compared by unpaired Student's t-test or
by one-way ANOVA when multiple comparisons were made. Data are presented as means ± SE; P 0.05 was considered
statistically significant.
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RESULTS |
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Expression of CaR in cultured mTAL cells.
Expression of CaR has been reported for TAL tissues. However, the
ability of primary cultures of mTAL cells to express CaR has not been
studied. Thus CaR mRNA accumulation was assessed in mTAL cells after 3 days of culture. The inner stripe of the outer medulla (OM) was used as
a positive control because this region was used to prepare tubule
suspensions from which primary cultures of mTAL cells were then
established. RT-PCR analysis of an equal amount of total RNA isolated
from OM and primary cultures of mTAL cells revealed a 400-bp fragment
predicted by primers designed to detect the presence of CaR mRNA (Fig.
1). The identity of the 400-bp fragment
was confirmed by DNA sequencing analysis, which demonstrated that the
sequence was identical to that reported for rat CaR (data not shown).
CaR protein was detected by Western blot analysis of equal amounts of
protein samples indicating that cultured mTAL cells retained the
ability to express this receptor (Fig. 1).
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Ca
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Ca
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Ca ion,
stimulates PGE2 synthesis in mTAL cells, PGE2
levels in supernatants were measured by ELISA after treatment with 1.7 mM CaCl2 or 3.4 mM NaCl for 1, 6, and 22 h (Fig.
6). We found that CaCl2
strongly enhanced PGE2 production in primary cultured mTAL
cells. In contrast, PGE2 levels were similar to those
observed for unstimulated cells after exposure to NaCl (with the same
Cl
concentration as the CaCl2 treatment)
(Fig. 6). These data demonstrate that the enhanced PGE2
production by CaCl2 is caused by the Ca2+ ion
but not by Cl
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Effects of COX-2-selective inhibitors on
Ca
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Overexpression of CaR in mTAL cells.
We recently showed that addition of Ca
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DISCUSSION |
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We demonstrated that mTAL cells in primary culture express CaR.
COX-2 mRNA accumulation and protein expression were enhanced after
being challenged with Ca
The COX-1 and COX-2 proteins are encoded by separate genes located on different chromosomes. Multiple signaling pathways have been linked to stimulation of COX-2 gene expression including the protein kinase A pathway, the PKC pathway, viral transformation, and tyrosine kinase (28). We previously demonstrated that PMA and tumor necrosis factor, direct and indirect activators of PKC activity, respectively, enhance COX-2 protein expression and PGE2 production in cultured mTAL cells (10), suggesting that PKC activation may be a signaling pathway for upregulating the COX-2 gene and enhancing local PGE2 production in the mTAL.
The extracellular domain of CaR has several regions rich in negatively
charged amino acids, which may bind calcium and other cationic ligands
(4, 5). Thus polyvalent cations such as PLA can act as CaR
agonists and mimic effects of calcium initiated by CaR activation.
(3). Expression of a functional CaR has been demonstrated
on the basolateral side of the TAL (24, 25). Some cell
types may not retain this receptor when cultured (20). We
recently showed that exposure of cultured mTAL cells to increasing concentrations of Ca
The free calcium concentration ranges from 1.0 to 1.2 mM in serum and
is tightly regulated by several mechanisms including the CaR (1,
14). Mutations in this receptor have been shown to cause
disorders of calcium homeostasis such as familial hypocalciuric hypercalcemia. Heterozygous and homozygous CaR knockout mice exhibit mild and severe alterations in Ca
Ca2+ has been reported to inhibit K+ recycling
in TAL (35) and to disrupt both NaCl and divalent cation
reabsorption by the TAL (27). Thus modulation of TAL NaCl
and divalent cation reabsorption by CaR activation provides a mechanism
to regulate both monovalent and divalent mineral ion homeostasis
(1). For instance, raising the serum ionized
Ca2+ level by 25% increased the urinary excretion of
Na+ by 150% (8, 23). CaR modulation of TAL
function also may be linked to alterations in the urinary concentrating
capacity of the kidney via alterations in medullary tonicity. Thus
hypercalcemia patients have diminished urinary concentrating ability.
Hypercalcemia stimulated the expression of intrarenal phospholipase
A2 and COX-2 in rats (18), and endogenous
PGE2 mediated the inhibition of rat TAL Cl
reabsorption in chronic hypercalcemia (22). Thus the
extracellular calcium concentration gradient along the loop of Henle
(30) may provide the necessary concentration range to
alter local COX-2 protein expression and PGE2 production in
the mTAL.
Renal cortical COX-2 mRNA levels decreased 2.9-fold in rats on a
high-salt diet and increased 3.3-fold in rats on a low-salt diet
(13). In contrast, medullary COX-2 level was increased in
rats on a high-salt diet (36). Divergent regulation of
COX-2 in cortex and medulla by dietary salt suggests that
prostaglandins in different kidney regions serve different functions,
with medullary production playing a role in promoting the excretion of
salt and water in volume overload, whereas cortical prostaglandins may protect glomerular circulation in volume depletion (13,
36). Recent studies have shown that adrenalectomy (ADX) caused
higher COX-2 protein expression in rat TAL segments (32,
37). Previous studies showed that ADX caused inhibition of
sodium reabsorption by 33% in the loop of Henle, an effect mediated by
PGE2 in the mTAL and reversed by aldosterone (6, 15,
29, 37). Thus aldosterone and other steroid hormones produced by
the adrenal gland may be one group of physiological factors that
inhibit COX-2 protein expression in the mTAL in vivo to promote salt
reabsorption. Immunohistochemical data showed that the mTAL appears to
express COX-2 protein constitutively in a subpopulaton of cells
(33). Given the effects of PGE2 on mTAL ion
transport (15, 16), Ca
Ca
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-56423 and PPG HL-34300 and American Heart Association Grant 9740001N.
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FOOTNOTES |
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N. R. Ferreri is an Established Investigator of the American Heart Association.
Address for reprint requests and other correspondence: N. R. Ferreri, Dept. of Pharmacology, New York Medical College, Valhalla, NY 10595 (E-mail: nick_ferreri{at}nymc.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.
Received 29 March 2001; accepted in final form 15 June 2001.
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