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George O’Brien Center for Kidney and Urologic Diseases, and Departments of Cell and Developmental Biology (M.-Z.Z., J.A.M.) and Medicine (P.S.L., R.C.H.), Vanderbilt University School of Medicine, Nashville, Tennessee 37232
Address all correspondence and requests for reprints to: Raymond C. Harris, M.D., C-3121 Medical Center North, Departments of Medicine, Vanderbilt University, Nashville, Tennessee 37232-4794. E-mail: ray.harris{at}vanderbilt.edu.
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Abstract |
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Introduction |
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Vasopressin acts through two distinct types of G protein-coupled receptors, classified as V1 and V2 (13). V1 receptors, which are comprised of V1a and V1b subtypes, are coupled to heterotrimeric G protein subtype Gq, and receptor activation leads to increases in intracellular calcium and constriction of smooth muscles (14, 15, 16, 17, 18, 19). The V2 receptor is coupled to Gs, and its antidiuretic actions are mediated through generation of cAMP (14). In the kidney, V2 receptors are localized to epithelial cells of collecting ducts and TALs, and V1a receptors reside in the vasculature. V1b has been detected in the medulla, but has not been localized to specific structures (18, 20).
The current investigation explored the regulation of COX isoforms in renal medulla by vasopressin receptor subtypes acutely and chronically. The results suggest that COX-2 expression is significantly down-regulated with vasopressin deficiency, and acute or chronic activation of either V1 or V2 stimulates renal medullary COX-2 expression secondary to increasing medullary tonicity; furthermore, COX-1 is also moderately down-regulated under conditions of chronic vasopressin deficiency.
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Materials and Methods |
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Animals
Experiments conformed to ethical guidelines for animal research established by the Department of Animal Care in Vanderbilt University and received prior approval before the initiation of the experiments. Male Long-Evans (LE) rats with or without hypophysectomy (HPX) and male Brattleboro (BB) rats were purchased from Harlan (Indianapolis, IN) and used at 200–300 g. Diabetes insipidus was diagnosed on the basis of daily water intake, urine output, and urinary osmolality. Both HPX and BB rats are characterized by vasopressin deficiency, although it is due to removal of the pituitary gland in the former and it is genetic in the latter. The rats were caged individually at least 1 wk before the experiments began. All rats had free access to tap water and were fed a commercial dry diet. Long-term administration of vasopressin receptor agonists was achieved via osmotic minipumps (Alzet model 2002, Palo Alto, CA) implanted sc under sterile conditions and ether anesthesia. Short-term administration of receptor agonists or antagonists was achieved by im or ip injection. Combinations of different protocols are explained in Results. Vasopressin, dDAVP, and other vasopressin receptor agonists and antagonists were all dissolved in distilled water.
Immunohistochemistry
In general, at the termination of an experiment, one kidney was removed from each rat for Western blot analysis, and the other was perfused with fixative in situ for histology. Under deep anesthesia with nembutal (70 mg/kg, ip), rats were exsanguinated with 50 ml/100 g heparinized saline (0.9% NaCl, 2 U/ml heparin, 0.02% sodium nitrite) through a transcardial aortic cannula and fixed with 3.7% formaldehyde in an acidic solution (pH 4.5) containing phosphate, periodate, acetate, and sodium chloride as described (22). The acidified aldehyde fixatives were crucial for reliable preservation of inner medulla/papillary structure and COX-2 antigenicity. The fixed kidney was dehydrated through a graded series of ethanols, embedded in paraffin, sectioned (4 µm), and mounted on glass slides. Internal controls and comparisons were facilitated by creating compound blocks with multiple specimens that were sectioned and stained together.
COX-1-immunoreactivity (COX-1-ir) was immunolocalized with affinity purified goat polyclonal antihuman COX-1 carboxyl terminus antibody no. C-20 (Santa Cruz Biotechnology, Santa Cruz, CA) at a 0.1-µg IgG/ml dilution. COX-2-ir was immunolocalized with affinity purified rabbit polyclonal antimurine COX-2 peptide (residues 570–598) antibody no. 160126 (Cayman Chemicals, Ann Arbor, MI) at a 0.1-µg IgG/ml dilution. The primary antibodies were localized by using Vectastain ABC-Elite (Vector Laboratories, Burlingame, CA) with diaminobenzidine as chromogen, followed by a light counterstain with toluidine blue. The specificity of our COX-2 immunolocalization was confirmed by two fundamental tests (23). Staining was eliminated by preabsorption of the primary serum with COX-2 protein purified from the rat distal vas deferens epithelium (24); COX-2-ir was colocalized with COX-2 mRNA detected by in situ hybridization (25).
Immunoblotting
Homogenates of papillae (4 ml per papilla) were prepared in 20 mM Tris-HCl (pH 8.0) with proteinase inhibitor mixture (Roche Molecular Biochemicals, Basel, Switzerland). After 10-min centrifugation at 10,000 x g, the supernatant was centrifuged for 60 min at 100,000 x g to sediment microsomes as described (11). The microsomes were resuspended in homogenizing buffer, mixed with equal volumes of 2x sodium dodecyl sulfate sample buffer, and boiled for 5 min. When Western blot analysis was performed, each lane was loaded with 5 µg of microsomes. The proteins were separated on 10% SDS-PAGE under reducing conditions and transferred to Immobilon-P transfer membranes (Millipore, Bedford, MA). After blocking overnight with 20 mM Tris-HCl (pH 7.4)/500 mM NaCl/5% nonfat milk/0.5% Tween 20, the blots were incubated for 3 h at room temperature with rabbit polyclonal antimurine COX-2 (Cayman Chemicals) at a 0.05 µg/ml dilution or goat polyclonal antihuman COX-1 at a 0.05 µg/ml dilution. The primary antibodies were detected with peroxidase-labeled goat antirabbit IgG or rabbit antigoat IgG (Santa Cruz Biotechnology) and exposed on film by using enhanced chemiluminescence (Amersham International, Arlington Heights, IL). Western blot was quantitated with an IS-1000 digital imaging system; (Alpha Innotech Corp., San Leandro, CA) the COX-2-ir band density from the control animal was designated as one, and that from the experiment animal was expressed as fold of control.
Cell culture
Renomedullary interstitial cells (RMICs) were prepared as previously described (27, 28). Cells were used after the fourth passage. After the cells were treated with vasopressin at different concentrations for 6, 12, or 24 h, the cells were collected for determination of COX-2 expression. We have recently reported that COX-2 expression in RMICs was stimulated by increased tonicity in the medium with NaCl, and suppressed by corticosterone (28). For positive controls, the RMICs were also treated with increased tonicity with NaCl and different concentrations of corticosterone in the medium for 24 h.
Prostaglandin E2 (PGE2) assay
Twenty-four-hour urine samples were collected and stored at -80 C and were assayed within 3 months. Urinary PGE2 was directly determined by enzyme immunoassay according to the manufacturer’s instruction (catalog no. 514010, Cayman Chemicals). Both intra- and interassay variations were less than 10%.
Micrography
Bright-field images from a Leitz Orthoplan microscope with DVC digital RGB video camera were digitized by the BIOQUANT image analysis system and saved as computer files (R & M Biometrics, Nashville, TN). Contrast and color level adjustment (Adobe Photoshop, Adobe Systems, Inc., San Jose, CA) were performed for the entire image; i.e. no region- or object-specific editing or enhancements were performed.
Statistical analysis
All values are presented as means ± SE ANOVA. The Bonferroni t test was used for statistical analysis, and differences were considered significant for P < 0.05.
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Results |
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). In contrast, COX-2-ir was confined to medullary interstitial cells (Fig. 1B). In age-matched HPX rats, COX-1-ir was moderately reduced in papillary collecting ducts and interstitial cells (Fig. 1C), whereas COX-2-ir was undetectable (Fig. 1D). Administration of dDAVP (10 ng/h) for 6 d to HPX rats led to increases in both medullary COX-1 and COX-2 expression (illustrated in the upper third of Fig. 1, C and D), suggesting a possible role for vasopressin in regulation of medullary COX expression.
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, left panel) and a significant decrease in papillary COX-2 expression (9.4 ± 1.4% of control; P < 0.01; Fig. 2, right panel). Immunohistochemistry showed diminished COX-1-ir and undetectable COX-2-ir in the papillae of BB rats (Fig. 1
, E and F).
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, vasopressin reduced 24-h urine volume and stimulated 24-h urine PGE2 excretion in a dose-dependent pattern [urine volume (ml/d): vehicle, 169 ± 10; 250 ng/d vasopressin: 170 ± 13; 500 ng/d vasopressin: 36 ± 3; 1000 ng/d vasopressin: 13 ± 1 ml. Urinary PGE2 excretion (ng/d): vehicle: 54 ± 5; 250 ng/d vasopressin: 52 ± 19; 500 ng/d vasopressin: 145 ± 28; 1000 ng/d vasopressin: 504 ± 70. n = 5]. Immunohistochemistry showed that COX-1 expression in the papilla of rats receiving 1000 ng/d vasopressin was slightly more intense in collecting ducts and detectably more intense and widespread in interstitial cells than in BB rats receiving vehicle (Fig. 1, E and G). The vasopressin effects on COX-2 expression were more pronounced. Similar to controls, no COX-2-positive cells were found in the papillae of BB rats receiving AVP at the dose of 250 ng/d (Fig. 1F). At 500 ng/d, some papillary interstitial cells were COX-2-ir positive (not illustrated); at 1000 ng/d, many COX-2-ir-positive interstitial cells were observed in a pattern resembling LE controls (Fig. 1H).
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, 24-h urine volume decreased 2-fold in PV-OT- or D3PVP-treated animals, but 10-fold in dDAVP-treated animals (vehicle, 200 ± 25 ml/d; dDAVP, 20 ± 5 ml/d; PV-OT, 80 ± 12 ml/d; D3PVP, 90 ± 9 ml/d). Immunoblotting (Fig. 3B) demonstrated that medullary expression of both COX-1 and COX-2 was increased after treatment with each of these three receptor agonists. COX-1 levels increased 2-fold after treatment with each receptor agonist, whereas COX-2 levels increased about 8-fold in dDAVP or PV-OT-treated animals and 2.5-fold in D3PVP-treated animals.
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), medullary COX-2 demonstrated significantly increased expression by 3 h after dDAVP administration and continued to increase at 6 and 9 h. In contrast, papillary COX-1 expression did not change over this time period. Immunohistochemical staining of papillary COX-2 after 9-h dDAVP treatment is shown in Fig. 6C. Acute effects of the other agonists were tested at a single time point of 8 h. Stimulation of medullary COX-2 expression by single im injection of 40 µg PV-OT was similar to dDAVP (Fig. 5). Administration of 40 µg D3PVP with a single im injection had no significant effect on medullary COX-2 expression (Fig. 5). Immunohistochemical analysis confirmed the immunoblotting results (Fig. 6, A and B). In a confirmation of a prior report (26), depriving BB rats of water for 12 h led to detectable levels of COX-2 expression (Figs. 4B and 6D). No additional detectable increases in COX-2 expression were seen in BB rats with 24-h water deprivation.
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, the V2 receptor antagonist significantly blunted the 8-h stimulating effects of dDAVP on papillary COX-2 expression, but the V1 receptor antagonist had little effect. In contrast, the stimulating effects of PV-OT on renal papillary COX-2 expression were significantly blunted by the V1 receptor antagonist, but not by theV2 receptor antagonist. Thus, dDAVP stimulates papillary COX-2 predominantly by activation of V2 receptors, whereas PV-OT stimulates medullary COX-2 predominantly by activation of V1 receptors.
Medullary osmolality and COX-2 up-regulation
We and other groups have reported that renal medullary COX-2 expression is regulated by medullary osmolality (26, 27, 28). To investigate whether increased medullary osmolality might also be a factor responsible for the increases in medullary COX-2 expression induced by different vasopressin receptor agonists, BB rats were first treated with furosemide (2 mg/kg, ip) 45 min before administration of 250 ng dDAVP, 40 µg PV-OT, or 40 µg D3PVP via a single im injection. Supplemental furosemide injections were given at 3- and 6-h time points. As indicated in Fig. 5, furosemide significantly blunted the increases in papillary COX-2 expression induced by dDAVP and PV-OT at 8 h, suggesting that the increases in papillary COX-2 expression induced by acute treatment of dDAVP or PV-OT appeared to be secondary to increased medullary osmolality.
RMIC in vitro
It has been reported that vasopressin and dDAVP stimulate PGE2 production in cultured RMICs through activation of PLA2 (6). Recently, we and other investigators reported that COX-2, but not COX-1, expression in the cultured mouse RMIC is stimulated by high osmolality by addition of NaCl to the medium (28). To investigate whether vasopressin stimulates COX-2 expression in the cultured RMIC, the cells were treated with different doses of vasopressin for 6, 12, and 24 h. As shown in Fig. 7C, COX-2 expression in RMICs did not change after 24 h of treatment with vasopressin at different doses (from 1–10,000 ng/ml). Similar results were achieved when RMICs were treated with different doses of vasopressin for 6 or 12 h (data not shown). As a positive control, RMIC COX-2 expression was stimulated by treatment with increased electrolyte tonicity with NaCl, but not urea, for 24 h (Fig. 7A). In another positive control, RMIC COX-2 expression was suppressed by 24-h treatment with different doses of corticosterone (Fig. 7B). These results indicate that vasopressin has no direct effect on RMIC COX-2 expression.
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Discussion |
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Given that increased vasopressin secretion is a physiological response to water deprivation, previous studies demonstrating that medullary COX-2 expression increased in water-deprived rats (26, 27, 28) suggest a possible role of vasopressin in mediation of papillary COX expression. Plasma vasopressin levels are very low in the HPX rat. Medullary COX-1 and COX-2 levels in HPX rats were much lower compared with age-matched LE controls, and these reduced COX-1 and COX-2 levels were stimulated by the V2 receptor-selective agonist dDAVP (Fig. 1, C and D). These data support a possible role of vasopressin in regulation of medullary COX-1 and COX-2 expression. BB rats represent a genetic model of vasopressin deficiency due to impaired vasopressin synthesis. Immunohistochemistry and Western blot analysis demonstrated that both papillary COX-1 and COX-2 expression were lower in BB rats compared with LE controls (Figs. 1, E and F, and 2). Increases in papillary COX-1 and COX-2 expression and urinary PGE2 excretion after vasopressin treatment indicate that vasopressin stimulates papillary COX-1 and COX-2 expression and that increased COX-1 and COX-2 are enzymatically active (Fig. 1, G and H, and Table 1).
The use of specific receptor-subtype agonists confirmed that medullary COX-1 and COX-2 expression increased in response to either V1 or V2 activation. The use of vasopressin receptor-selective antagonists confirmed that dDAVP stimulates medullary COX-2 via activation of V2 receptors, whereas PV-OT stimulates medullary COX-2 through V1 receptor activation (Fig. 5B).
The TAL plays a key role in the urinary concentrating mechanism (29, 30). Net NaCl reabsorption in this segment leads to dilute luminal fluid and increased medullary tonicity; and vasopressin, acting through V2 receptors, acutely increases the rate of NaCl absorption in the medullary TAL by stimulating the activity of the furosemide-sensitive Na-K-2Cl cotransporter and apical potassium channel (4, 31, 32, 33, 34, 35). Activation of V2 receptors also increases the activity of the vasopressin facilitated urea transport in the rat terminal inner medullary collecting duct, and the integrated effect of increased reabsorption by these two transport processes is an increase in medullary osmolality (36). Cowley et al. (37) found that papillary sodium and other osmolyte concentrations in BB rats increased significantly after treatment with vasopressin for 5 d. Administration of the vasopressin V1a receptor agonist PV-OT at a dose of 2 ng/kg·min over a period of 5 d led to a chronic reduction of medullary blood flow without cortical blood flow change (38). In the present study, PV-OT was used at a dose of 7 ng/kg·min, so we expect that renal medullary blood flow would be reduced. Reduced medullary blood flow prevents medullary washout and promotes increases in medullary osmolality (13). Therefore, although these receptors are located on different cell types and in different regions of the kidney and act through different mechanisms, the net effect of activation of either receptor is to increase interstitial osmolality in the papilla.
Furosemide is an inhibitor of Na-K-2Cl cotransporter in the TALs. Treatment with furosemide leads to a washout of the corticopapillary osmolarity gradient. Prevention of vasopressin induction of COX-2 by furosemide and lack of COX-2 induction in the cultured RMIC by vasopressin further support the hypothesis that vasopressin induces medullary COX-2 expression secondary to increased medullary osmolality. Although furosemide significantly blocked vasopressin-induced COX-2 expression, some residual increase was noted. However, full dose responses with furosemide were not performed in the present studies, so it is possible that TAL NaCl reabsorption was not completely inhibited with the furosemide concentration used.
In animal modes of the syndrome of inappropriate antidiuretic hormone secretion, sustained administration of vasopressin and water results in water retention and a secondary natriuresis, leading to progressive hyponatremia. However, after several days, increased free water excretion occurs despite sustained administration of vasopressin. This phenomenon is known as vasopressin escape from antidiuresis (39, 40, 41, 42). Although investigated extensively, the mechanisms of vasopressin escape are unclear. It has been reported that urinary PGE2 excretion increased concomitant with the onset of escape and that inhibition of prostaglandin production by indomethacin delays the onset of escape (39, 43). Kidney prostaglandins antagonize the antidiuretic effect of vasopressin. Murase et al. (43) recently reported that the combination of a nonspecific cyclooxygenase inhibitor, diclofenac, and the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester produced a marked inhibitory effect on vasopressin escape. All these data suggest that prostaglandins might be involved in the mechanism of vasopressin escape. Our result that medullary COX-2 expression increased shortly after treatment with vasopressin agonists suggests a possible role of COX-2 expression in vasopressin escape.
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Footnotes |
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Abbreviations: BB, Brattleboro; COX, cyclooxygenase; COX-1-ir, COX-1-immunoreactivity; dDAVP, [deamino-Cys1,D-Arg8]-vasopressin; D3PVP, [deamino-Cys1,D-3-(pyridyl)-Ala2,Arg8]-vasopressin; HPX, hypophysectomy/hyphosectomized; LE, Long-Evans; PgE2, prostaglandin E2; PLA2, phospholipase A2; PV-OT, [Phe2, Ile3, Orn8]-vasopressin; RMIC, renomedullary interstitial cell; TAL, thick ascending limb.
Received July 22, 2003.
Accepted for publication December 4, 2003.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in diabetes insipidus rats. Am J Physiol Endocrinol Metab Gastrointest Physiol 235:E624–E627
B-driven, COX2-dependent survival mechanism in renal medullary interstitial cells. J Clin Invest 106:973–982[Medline]
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