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Am J Physiol Renal Physiol 275: F613-F622, 1998;
0363-6127/98 $5.00
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Vol. 275, Issue 4, F613-F622, October 1998

Selective increase of cyclooxygenase-2 expression in a model of renal ablation

Jun-Ling Wang1, Hui-Fang Cheng1, Ming-Zhi Zhang2, James. A. McKanna2, and Raymond C. Harris1

George M. O'Brien Kidney and Urologic Diseases Center and 1 Division of Nephrology, Department of Medicine, 2 Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Previous studies have suggested a possible role for prostaglandins (PGs) in mediating alterations in nephron structure and function ensuing after renal ablation. Two isoforms of cyclooxygenase (COX) have been described: constitutive (COX-1) and inducible (COX-2). We examined expression of these isoforms following subtotal renal ablation (5/6 ablation, RA) in rats. In renal cortex, COX-2 mRNA and immunoreactive protein (IP) increased progressively compared with sham-operated littermates. In contrast, there were no significant changes in COX-1 mRNA expression. In normal kidney, cortical COX-1 IP was immunolocalized predominantly to mesangial cells and collecting tubules, whereas COX-2 IP was found in a subset of cortical thick ascending limb of Henle's loop (CTAL) cells in the region of the macula densa (MD). Following RA, significantly increased COX-2 IP was detected in the MD and surrounding CTAL cells. In addition, fainter immunoreactive COX-2 was detected in scattered visceral epithelial cells and mesangial cells of the glomerulus. Immunoblotting of isolated glomeruli demonstrated a selective increase of glomerular immunoreactive COX-2 expression following RA. No change of COX-1 expression was seen. To determine COX activity, isolated glomeruli were incubated with arachidonic acid and PGE2 measured by enzyme immunoassay (EIA). Compared with sham, glomeruli from 2 wk RA produced significantly more PGs. SC-58560, a selective COX-1 inhibitor, did not inhibit PG production in the remnant glomeruli at concentrations up to 10-4 M, whereas SC-58236, a relatively selective COX-2 inhibitor, significantly inhibited PG production by RA glomeruli. In preliminary studies, to define mechanisms of altered expression of glomerular COX-2, rat mesangial cells were incubated with serum from sham or 2 wk RA. There were significant increases in COX-2 expression in response to 2 wk RA serum. In summary, these results indicate selective increases in renal cortical COX-2 expression following renal ablation.

prostaglandin G/H synthase; mesangial cell; renal failure; cortical thick ascending limb of Henle's loop; macula densa

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

PROSTANOIDS COMPRISE a diverse family of biologically active lipids derived from enzymatic metabolism of arachidonic acid by prostaglandin G2/H2 synthase (cyclooxygenase, COX) and further metabolism by specific synthetases. Although prostanoid synthesis occurs in all cells and tissues, the kidney is a particularly rich source for prostanoids. Prostaglandin E2 (PGE2) is the major renal metabolite, and urinary PGE2 concentrations are typically in the nanomolar range, well above circulating picomolar concentrations (3, 13) Cellular responses are mediated by specific membrane-associated receptors, which are members of the G protein-coupled receptor superfamily. Since receptor affinity for the prostanoids is in the nanomolar range, prostanoids act locally on the tissues in which they are synthesized or on tissues adjacent to those in which they are synthesized.

As would be predicted from their local action, the kidney is an important biological target for these intrarenally produced prostaglandins. Prostaglandins regulate both renal hemodynamics and epithelial water and solute transport. Each prostaglandin has distinct effects on these renal targets. Additionally, a single prostaglandin, such as PGE2, may also have multiple, and at times apparently opposing, functional effects on a given target tissue. There is now firm evidence that these diverse effects can be accounted for by multiple receptor subtypes for individual prostaglandins.

The gene for constitutive prostaglandin G2/H2 synthase (COX-1) encodes a 2.7- to 2.9-kb transcript (11). In the normal adult kidney, immunoreactive COX-1 has been localized to arteries and arterioles, glomeruli, and collecting ducts. No immunoreactive COX-1 has been found in the proximal or distal convoluted tubules, Henle's loop, or macula densa (MD) (56). Recent studies have demonstrated that, in addition to COX-1, certain cells express a gene encoding a 4.0- to 4.4-kb transcript, COX-2, the expression of which is activated by mitogenic stimuli (27). Although the translation products of both COX-1 and COX-2 are of similar size (~73 kDa) and possess similar cyclooxygenase activity, they share only ~66% homology in amino acid sequence (27, 41, 42). Eicosanoids produced by COX-1 and COX-2 are indistinguishable, except that after treatment with aspirin [but not other nonsteroidal antiinflammatory drugs (NSAIDs)], COX-2 metabolizes arachidonic acid to 15(R)-hydroxyeicosatetraenoic acid. The physiological significance of this reaction is unknown (34).

The message for COX-2 increases in cultured cells in response to mitogenic and inflammatory stimuli (15) and appears to mediate prostanoid production from arachidonic acid released by endotoxin or mitogens (50). Regulated COX-2 expression occurs during murine and rodent development (62, 69); COX-2 expression is highly regulated during metanephric development (62, 69), and targeted deletion of COX-2 results in abnormal renal development (10, 36), whereas targeted deletion of COX-1 produces no developmental renal abnormalities or renal pathology in adult animals (29). In adult animals, COX-2 expression occurs predominantly during cell growth or in inflammatory states (10, 33, 35, 42). However, normal adult mammalian kidney has low but measurable levels of COX-2 mRNA (12, 21, 43). Furthermore, in situ hybridization and immunohistochemical localization demonstrate that in rat kidney, cortical COX-2 expression is localized to scattered cortical thick ascending limb of Henle's loop (CTAL) cells in the region of the MD (65). In animals chronically salt depleted, COX-2 expression in the peri-MD region increases significantly (21). In addition, COX-2 is expressed in a subset of medullary interstitial cells in the papilla.

Cyclooxygenase metabolites have been implicated in functional and structural alterations in glomerular and tubulointerstitial inflammatory diseases (14, 25, 61). Studies have suggested that prostanoids may also mediate altered renal function and glomerular damage following subtotal renal ablation, and glomerular prostaglandin production may be altered in such conditions (11, 39, 45, 49, 53, 54, 57-59). The current studies were designed to investigate renal expression of COX-1 and COX-2 following subtotal renal ablation in the rat.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. COX-1 mouse cDNA probe and polyclonal anti-COX-1 antibodies were from Oxford Biomedical Research (Oxford, MI) and Santa Cruz Biotechnology (Santa Cruz, CA). Anti-COX-2 antibodies and PGE2 EIA were from Cayman (Ann Arbor, MI). The COX-1 inhibitor, SC-58560, and COX-2 inhibitor, SC-58236, were gifts from Searle Monsanto. [32P]CTP (3,000 Ci/mmol), and the enhanced chemiluminescence (ECL) kit and ECL hyperfilm were from Amersham (Arlington, Heights, IL). Bicinchoninic acid (BCA) protein assay reagent kit, Immunopure ABC peroxidase staining kit, and biotin-labeled mouse anti rabbit IgG(H+L) antibody were from Pierce (Rockford, IL). Other reagents were purchased from Sigma Chemical (St. Louis, MO).

Animals. Male Sprague-Dawley rats (Harlan, Indianapolis, IN), initially weighing 150-200 g, were utilized. For ablation of renal mass, animals were anesthetized with ketamine:xylene (9:1) and placed on a warming table, and their kidneys exposed under aseptic conditions via a ventral abdominal incision. The right kidneys were removed, and the posterior and anterior (if present) apical segmental branches of the left renal artery were individually ligated, as described (3). Sham-operated animals from the same litter were used as controls. In a subset of additional animals, subtotal renal ablation of the remaining kidney was produced by excision of both renal poles.

Glomeruli isolation and PGE2 measurement. Rat glomeruli were collected by differential sieving as previously described (23). Glomeruli from sham or 2 wk postrenal ablation were incubated in RPMI 1640 with or without 10-5 M arachidonic acid at 37°C for 1 h. Additional groups were incubated with selective COX-1 or COX-2 inhibitors. The glomeruli were then centrifuged, and protein was assayed. The supernatant was removed, and PGE2 was measured by EIA. Medium PGE2 content was normalized to glomerular protein content.

Cell culture. Rat glomerular mesangial cells were cultured as previously described (23). Cells of passages 5-6 were used in these experiment. The cells were grown to confluence in RPMI 1640 + 10% FCS, switched to RPMI 1640 without serum for 48 h, and then incubated for an additional 4 h prior to RNA extraction either with no addition (control) or with 1% (final volume) of serum from sham-operated or 14-day post renal ablation rats.

RNA extraction and Northern blotting. Renal cortex or mesangial cell RNA was extracted by the acid guanidium thiocyanate-phenol chloroform method, as described previously. (6). RNA samples were electrophoresed in denatured agarose gel and transferred to nitrocellulose membrane and hybridized with a 1.3-kb 32P-labeled cDNA Kpn I/Xho I fragment of 3'-untranslated region of rat COX-2 (21) or with 32P-labeled COX-1 cDNA probe. The membranes were then stripped and rehybridized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Immunoblotting. The cortex or glomeruli were homogenized in 30 mM Tris · HCl, pH 8.0, and 100 µM phenylmethylsulfonyl fluoride (PMSF; 1:9 wt/vol). Following a 10-min centrifugation at 10,000 g, the supernatant was centrifuged for 60 min at 110,000 g to prepare microsomes as described previously (22). The microsomes were resuspended in SDS-sample buffer, heated to 100°C for 5 min, and the protein was separated on 8% SDS gels under reducing conditions and transferred to Immobilon-P transfer membranes (Millipore, Bedford, MA). The blots were blocked overnight with 100 mM Tris · HCl, pH 7.4, containing 5% nonfat dry milk, 3% albumin, and 0.5% Tween 20, followed by incubation for 16 h with rabbit anti-murine polyclonal antiserum to COX-2 (Cayman) at 2.5 µg/ml dilution. The second reagent, biotinylated goat anti-rabbit antibody, was detected using avidin and biotinylated horseradish peroxidase (Pierce) and exposed on film using ECL (Amersham).

Immunohistochemistry. To obtain uniform and reproducible immunostaining, it was necessary to perfuse the kidney in situ prior to fixation (22). 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, and 0.02% sodium nitrite) through a transcardial aortic cannula and fixed with glutaraldehyde-periodate acid saline (GPAS) as previously described (22, 69). GPAS contains final concentrations of 2.5% glutaraldehyde, 0.011 M sodium metaperiodate, 0.04 M sodium phosphate, 1% acetic acid, and 0.1 M NaCl and provides excellent preservation of tissue structure and COX-2 antigenicity, as previously described (21). The fixed kidneys were dehydrated through a graded series of ethanols, embedded in paraffin, sectioned at 4 µm thickness, and mounted on glass slides. COX-1 and COX-2 immunoreactivity was localized with polyclonal goat anti-murine COX-1 (Santa Cruz) or polyclonal rabbit anti-murine COX-2 serum (Cayman Chemical), respectively, diluted to 2.5 µg/ml. The first antibody was localized using Vectastain ABC-Elite (Vector, Burlingame, CA) with diaminobenzidine as chromogen, followed by a light counterstain with toluidine blue.

Quantitative image analysis based on the distinctive density and color of irCOX-2 in video images and the number, size, and position of stained cells from kidney sections was quantified using BIOQUANT true-color windows system (R & M Biometrics, Nashville, TN) equipped with digital stage encoders that allow high-magnification images to be mapped to global coordinates throughout the whole kidney. Sections from at least four different rats were analyzed for each time point.

Blood pressure and renal function. Blood pressure was measured weekly by the tail-cuff method (5). Urine was collected for 24 h for determination of volume and total protein concentration using BCA protein assay reagent kit (Pierce). Blood urea nitrogen was determined by standard diacetyl monoxime technique (Sigma).

Statistical analysis. All values are presented as mean ± SE. ANOVA and Bonferroni t-tests were used for statistical analysis, and differences were considered significant when P < 0.05.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

To determine expression of cyclooxygenase subtypes following loss of renal mass, we used the model of subtotal ablation in rats (2). In this model, renal function is relatively well preserved during the first 3 wk after ablation of renal mass (2). In our studies, baseline blood urea nitrogen was 15 ± 1 mg/dl (n = 6) and was increased to 31 ± 2, 35 ± 3, and 34 ± 3 mg/dl in rats 1 wk, 2 wk, and 3 wk, respectively, after subtotal renal ablation (n = 7). Similarly, 24-h urine protein excretion was 154 ± 28 mg/24 h in control rats and was 281 ± 66, 300 ± 42, and 273 ± 23 mg/24 h in rats 1 wk, 2 wk, and 3 wk, respectively, after subtotal renal ablation (n = 6-7); and mean blood pressure was elevated from 80 ± 5 mmHg in sham-operated animals to 133 ± 7, 132 ± 8, and 134 ± 8 mmHg at 1, 2, and 3 wk, respectively, after subtotal renal ablation (n = 5-7). RNA from renal cortices of sham or experimental animals was probed with cDNAs for COX-1 or COX-2, and relative expression was normalized to GAPDH expression. COX-2 mRNA expression in renal cortex 1 wk after subtotal nephrectomy was 1.4 ± 0.08-fold of sham (n = 5); there were significant increases in COX-2 mRNA expression at 2 wk (2.0 ± 0.2 of sham, n = 5; P < 0.05) and 3 wk (1.5 ± 0.1 of sham, n = 5; P < 0.05) after renal ablation (Fig. 1A). Although there was some variability of expression among experiments in the 3 wk group, there were no statistically significant differences between the level of expression at 2 and 3 wk. Immunoblotting with COX-2-specific antisera demonstrated a similar time course of increases in expression of COX-2 immunoreactive protein in microsomes prepared from renal cortex. One week after renal ablation, COX-2 immunoreactivity was 1.5 ± 0.2 of sham (n = 5), whereas there were significant increases at 2 wk (1.9 ± 0.2 of sham; n = 5; P < 0.01) and 3 wk (1.6 ± 0.2 of sham; n = 5 P < 0.05) after renal ablation. (Fig. 1B).


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  		Fig. 1.   Cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) expression in renal cortex after subtotal ablation. COX-2 mRNA (A) (n = 5) and immunoreactive (ir) protein (B) (n = 5) increased after subtotal ablation, whereas COX-1 mRNA (C) (n = 4) and immunoreactive protein (D) (n = 3) were not altered. Relative expression of mRNA was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. * P < 0.05, compared with sham. Insets: representative experiments. Lane 1, sham; lane 2, 1 wk after ablation; lane 3, 2 wk after ablation; lane 4, 3 wk after ablation.

There were no significant changes in renal cortical COX-1 mRNA expression following renal ablation [1 wk, 1.2 ± 0.2 of sham; 2 wk, 1.0 ± 0.1 of sham; 3 wk, 0.9 ± 0.1 of sham; n = 4, not significant (NS)] (Fig. 1C). Renal cortical COX-1 immunoreactive protein expression was similarly unchanged (1 wk, 0.9 ± 0.1 of sham; 2 wk, 1.0 ± 0.1 of sham; 3 wk, 1.1 ± 0.1 of sham; n = 4, NS) (Fig. 1D).

Glomerular cyclooxygenase expression was further examined in isolated glomeruli from sham-operated and renal ablation animals. Immunoblotting indicated significant increases in glomerular immunoreactive COX-2 expression following renal ablation (1 wk, 1.7 ± 0.1 of sham, P < 0.05; 2 wk, 2.2 ± 0.2 of sham, P < 0.01; 3 wk, 2.1 ± 0.2 of sham, P < 0.05; n = 3) (Fig. 2A). No changes in glomerular COX-1 expression were detected (1 wk, 1.3 ± 0.1 of sham; 2 wk, 1.0 ± 0.1 of sham; 3 wk, 1.1 ± 0.2 of sham; NS; n = 3) (Fig. 2B).


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  		Fig. 2.   COX-1 and COX-2 immunoreactivity in glomeruli after subtotal ablation. COX-2 immunoreactive protein (A) (n = 3) increased after subtotal ablation, but COX-1 immunoreactive protein (B) (n = 3) was unchanged. * P < 0.05, compared with sham. Insets: representative experiments. Lane 1, sham; lane 2, 1 wk after ablation; lane 3, 2 wk after ablation; lane 4, 3 wk after ablation.

Immunohistochemical localization indicated that, in sham-operated kidneys, cortical COX-1 was immunolocalized predominantly to mesangial cells and collecting tubules, whereas COX-2 was found in a subset of CTAL cells in the region of MD, as we have previously described (Fig. 3, A and B) (22). No COX-2 immunoreactivity was detected in intrinsic glomerular cells. Increased COX-2 immunoreactive cells CTAL in the region of the MD were detected after renal ablation (Fig. 3C), and quantification confirmed significant increases after renal ablation (expressed as irCOX-2 area in µm2 vs. total cortical area in mm2: sham, 44 ± 4 ; n = 6; 1 wk, 42 ± 7, n = 4, NS; 2 wk, 103 ± 15, n = 6, P < 0.01; 3 wk, 117 ± 13, n = 6, P < 0.01). In addition, although no COX-2 immunoreactivity could be detected in intrinsic glomerular cells of sham-operated animals, there was detectable irCOX-2 in occasional visceral glomerular epithelial cells and in mesangial cells in the remnant nephrons (Fig. 3C).


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  		Fig. 3.   Immunohistochemical localization of COX-2 immunoreactivity in renal cortex after subtotal renal ablation. Adult rat kidney fixed with glutaraldehyde-periodate acid saline and stained to localize COX-1 (A) or COX-2 (B-D), either sham operated (A and B) or 2 wk after 5/6 nephrectomy (C and D). A: mesangial cells (m) exhibit irCOX-1 in perinuclear cisternae; magnification, ×220. B: control nephrons display sparse individual COX-2-positive cells in the epithelium of the cortical thick ascending limb of Henle's loop (CTAL, arrow) or near the macula densa (md, arrowhead); magnification, ×220. C: kidney 2 wk after 5/6 nephrectomy; low-magnification (×22) scanning the cortex from the outer medulla (om) to the adjacent necrotic zone (nz) reveals COX-2-positive cells (arrows) at least 10-fold more numerous than in controls. D: at higher (×220) magnification (boxed region in C), irCOX-2 is shown to be increased in some macula densa cells as well as in adjacent cells of the CTAL (arrows).

To determine cyclooxygenase activity, isolated glomeruli were incubated with exogenous arachidonic acid (10-5 M) for 1 h to provide substrate excess, and PGE2 released into the media was measured by EIA. Compared with glomeruli from sham-operated animals, in the presence of exogenous arachidonic acid, glomeruli from rats 2 wk after renal ablation produced significantly more PGE2 [18.8 ± 1.4 vs. 38.7 ± 3.4 ng immunoreactive PGE2 (irPG)/mg protein; n = 7, P < 0.01] (Fig. 4A). In glomeruli from sham-operated rats, the COX-1-selective inhibitor, SC-58560, inhibited arachidonic acid-stimulated PGE2 production by glomeruli (10-5 M, 10.1 ± 0.7 ng irPG/mg protein; n = 3, P < 0.05). In contrast, in glomeruli from 2 wk renal ablation rats, SC-58560, even concentrations of 10-3 M did not inhibit arachidonic acid-stimulated PGE2 production (39.5 ± 3.1 ng irPG/mg protein; n = 3, NS) (Fig. 4B). However, a relatively selective COX-2 inhibitor, SC-58236, inhibited remnant glomerular arachidonic acid-stimulated PGE2 production in a concentration-dependent manner, with maximum effects at 10-5 (14.1 ± 1.5 ng irPG/mg protein; n = 7, P < 0.01) (Fig. 4B).


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  		Fig. 4.   Increased prostaglandin production in isolated glomeruli from remnant kidneys. A: glomeruli were isolated from remnant and sham kidneys 2 wk after operation and were incubated with or without 10 µM arachidonic acid for 1 h, the supernatant was collected, and irPGE2 production was measured by EIA (n = 7; * P < 0.01). B: concentration response of irPGE2 production from arachidonic acid-stimulated remnant glomeruli in presence of the COX-1-selective inhibitor, SC-58560, or the COX-2-selective inhibitor, SC-58236.

To explore potential mechanisms of altered expression of glomerular COX-2, quiescent cultured rat mesangial cells were incubated for 4 h with or without 1% serum from sham-operated or 2 wk renal ablation rats. Serum from sham-operated animals led to numerical but nonsignificant increases in mesangial cell COX-2 mRNA expression compared with control; however, the addition of serum from renal ablation rats induced a significant increase in steady-state COX-2 mRNA expression (3.8 ± 0.3; n = 3, P < 0.05) (Fig. 5).


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  		Fig. 5.   Effect of remnant serum on COX-2 mRNA expression in cultured mesangial cells. Quiescent rat mesangial cells were either not stimulated (control) or stimulated for 4 h with 1% rat serum from sham-operated rats or 2 wk subtotal renal ablation rats. Relative COX-2 mRNA expression was normalized to GAPDH expression. * P < 0.05, compared with control (n = 3). Inset: representative experiment. Lane 1, control; lane 2, sham serum; lane 3, remnant serum.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The present studies determined that, following subtotal renal ablation, there were selective increases in renal cortical COX-2 mRNA and immunoreactive protein expression, without significant alterations in COX-1 expression. This increased COX-2 expression was most prominent in the CTAL in the region of the MD, the site of expression of cortical COX-2 in the normal rat kidney (21, 22, 65). In addition, there was detectable COX-2 immunoreactivity in some glomeruli from remnant kidneys, with increased expression in visceral epithelial cells and mesangial cells. Of interest, COX-2 expression has been reported in visceral epithelial cells of human kidney (26). There were no apparent differences in the pattern or extent of increased COX-2 immunoreactivity in the two remnant models (subtotal infarction vs. excision of the remaining kidney). Given that there are increased macrophages and other inflammatory cells in the remnant kidney, it is also possible that a component of increased cortical COX-2 immunostaining was secondary to infiltrating cells.

The present studies were designed to investigate specifically the expression of COX-1 and COX-2 in the renal cortex; however, in the rat, there is significant COX-1 expression in medullary collecting tubule and medullary interstitial cells. In addition, COX-2 immunoreactivity is found in a subset of medullary interstitial cells, near the tip of the papilla (23). Although quantitative analysis of medullary structures was not performed, immunohistochemical analysis did not reveal detectable alterations in medullary COX-1 or COX-2 expression.

Isolated glomeruli also demonstrated selective increases in COX-2 immunoreactivity and increased PGE2 production in response to exogenous arachidonic acid. These studies are consistent with previous studies indicating that glomeruli from remnant kidneys (46, 58), as well as from animals fed a high-protein diet (11, 57), demonstrated increased prostanoid production. In the present studies, PGE2 production was utilized as a marker of cyclooxygenase activity. Previous studies have examined the prostanoid profile in isolated glomeruli from remnant kidneys and have documented increases in both vasodilatory (PGE2) and vasoconstrictive (thromboxane A2 and PGE2alpha ) prostanoid production (58).

The increased PGE2 production seen in remnant glomeruli in the present studies was sensitive to selective COX-2 inhibitors but not selective COX-1 inhibitors, suggesting that the increased glomerular PGE2 production was secondary to increased COX-2 expression. Increases in prostanoid production were noted when excess exogenous arachidonic acid was added in the present studies, as well as previous studies in models of increased glomerular prostanoid production (11), suggesting an increase in cyclooxygenase enzyme activity per se rather than increased substrate availability. The lack of significant differences in the absence of arachidonic acid addition in the present studies is consistent with a lack of significant phospholipase A2 (PLA2) activation; however, whether there are alterations in PLA2 expression or activity in the remnant glomeruli was outside the scope of the present studies and was not examined.

The mechanism(s) regulating increased COX-2 expression in the remnant kidneys was not determined in the present studies. In previous studies, we determined that application of mechanical stress to cultured mesangial cells induced COX-2 mRNA and immunoreactive protein (1). Intrarenal ANG II production is increased in the remnant model (47), and angiotensin converting enzyme (ACE) inhibition normalizes the increased PGC and decreases the development of nephrosclerosis (3). Although the effect of ACE inhibitors on glomerular prostaglandin production has not been examined to date in the remnant model, similar studies in glomeruli from rats with bilateral ureteral obstruction demonstrated that the increased glomerular cyclooxygenase activity in this model was normalized if the animals were treated with ACE inhibitors (67). However, whether ANG II regulates expression of COX-2 in hyperfiltering states has not been addressed. The finding in the present studies that substances in uremic serum stimulated COX-2 mRNA expression in cultured mesangial cells is intriguing but must require further characterization to determine whether such factors are important in mediation of COX-2 expression in vivo.

NSAIDs that are currently commercially available are relatively nonselective, and at therapeutic concentrations these may inhibit both COX-1 and COX-2 (28). Pelayo et al. (47) found that when given 24 h after subtotal renal ablation, indomethacin reversed increases in renal blood flow and single-nephron glomerular filtration rate; similar decreases in hyperfiltration were noted by Nath et al. (39) when indomethacin was given acutely to rats 14 days after subtotal nephrectomy. After either subtotal ablation or excessive dietary protein, defective autoregulation of renal blood flow due to decreased myogenic tone of the afferent arteriole is present; some (37, 46) but not all (18) investigators have reported that inhibition of cyclooxygenase activity corrected the autoregulatory defect.

Recent studies have indicated a role for prostaglandins in MD-mediated renin release in normal kidneys (21). Our previous findings indicating localized COX-2 expression to the MD region and increased expression after salt restriction suggest a potentially important role in the MD-mediated regulation of renin release (22); in this regard, recent studies by Harding et al. (20) have indicated that COX-2 inhibitors prevented increased renal renin mRNA in response to dietary salt restriction. Although an important role for the renin-angiotensin system in the pathological changes occurring in the remnant kidney model has been strongly suggested by the numerous studies indicating amelioration by either ACE inhibitors or angiotensin receptor blockers, the evidence suggesting increased intrarenal renin expression in this model is still controversial. In general, previous investigators have not detected increases in plasma renin and renal renin expression (48, 52), although renin expression in the juxtaglomerular apparatus may be selectively increased in the glomeruli adjacent to the scarred area of the remnant kidney, suggesting that these glomeruli may be selectively hypoperfused (8). In addition, increased intraglomerular renin expression has been described in remnant glomeruli (53).

Given that there is not necessarily globally increased juxtaglomerular renin expression in the hyperfiltering remnant model, the importance of the increased COX-2 expression in the MD region of the CTAL is unclear. In our studies, there was no apparent localization of the increased MD COX-2 expression only to the "peri-scar" glomeruli. Previous studies using a nonselective cyclooxygenase inhibitor (indomethacin) found that treatment of rats with remnant kidneys decreased renal vasodilatation but did not affect autoregulation or renin release (18).

There is suggestive evidence that prostanoids may play a role in mediating or modulating the progressive fibrosis occurring in the glomerulus and tubulointerstitium following renal ablation. Four groups have reported that thromboxane synthase inhibitors decreased proteinuria and glomerulosclerosis in rats with remnant kidneys, in association with increased renal prostacyclin production and lower systolic blood pressure (49, 51, 59, 70). Schmitz et al. (54) confirmed increases in thromboxane B2 excretion in the remnant kidney and correlated decreased arachidonic and linoleic acid levels with increased thromboxane production, since the thromboxane synthase inhibitor U-63557A restored fatty acid levels and retarded progressive glomerular destruction (54). In contrast to the putative deleterious effects of thromboxane, the prostacyclin analog, cicaprost, retarded renal damage in uninephrectomized dogs fed a high-sodium and high-protein diet, an effect that was not mediated by amelioration of systemic hypertension (64). Indirect studies have also implicated cyclooxygenase metabolites in the genesis of renal hypertrophy following ablation of renal mass, as indomethacin administration prevented renal hypertrophy following contralateral nephrectomy (30).

Studies in cultured mesangial cells indicate that thromboxane A2 and PGF2alpha , which signal predominantly through Gq, with phospholipase C activation, stimulate mitogenesis (24). Most investigators have suggested that PGE2 and prostacyclin, both of which signal in mesangial cells predominantly through Gs, with adenylate cyclase activation, inhibit mitogenesis (33, 38), although Mahadevan et al. (31) reported that, with hyperglycemia, PGE2 stimulated mesangial cell proliferation. In general, although prostanoids that have vasoconstrictive properties may be promitogenic and vasodilatory prostaglandins may be antimitogenic, these effects appear to be distinct from hemodynamic effects since they are seen in vitro and thus may reflect differences in intracellular signaling pathways.

Prostanoids have also been shown to alter extracellular matrix production by mesangial cells in culture. Thromboxane A2 stimulates matrix production by both transforming growth factor-beta -dependent and -independent pathways (40, 60). PGE2 has been reported to decrease steady-state mRNA levels of alpha 1(I) and alpha 1(III) procollagens, but not alpha 1(IV) procollagen and fibronectin mRNA, and to reduce secretion of all studied collagen types into the cell culture supernatants. Of interest, this effect did not appear to be mediated by cAMP (68). PGE2 has also been reported to increase production of matrix metalloproteinase-2 (MMP-2) and to mediate ANG II-induced increases in MMP-2 (55). Whether vasodilatory prostaglandins mediate decreased fibrillar collagen production and increased matrix-degrading activity in glomeruli in vivo has not yet been studied; however, there is compelling evidence in nonrenal cells that prostanoids may either mediate or modulate matrix production (16, 17, 63). Significantly, recent studies have indicated that cultured lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis exhibit decreased ability to express COX-2 and to synthesize PGE2 (66). In addition to direct effects on fibroblast production of collagen, PGE2 may also inhibit actions of leukocytes and lymphocytes (7, 19, 44).

In summary, our results indicate that one of the consequences of subtotal renal ablation is an increase in cortical COX-2 expression. This increased COX-2 is localized predominantly to the CTAL in the region of the MD and to intrinsic glomerular cells and appears to be in large part responsible for the observed increases in glomerular cyclooxygenase activity. Whether this increased COX-2 expression is involved in modulating the functional and structural abnormalities that ensue from loss of functional renal mass must await further investigation.

    ACKNOWLEDGEMENTS

This work was supported by the Vanderbilt George O'Brien Kidney and Urologic Diseases Center (National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-39261), by a grant from Searle Monsanto (St. Louis, MO), and by funds from the Department of Veterans Affairs.

    FOOTNOTES

Address for reprint requests: R. C. Harris, Division of Nephrology, S-3322, MCN, Vanderbilt Univ. School of Medicine, Nashville, TN 37232.

Received 18 December 1997; accepted in final form 22 July 1998.

    REFERENCES
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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