Nonsteroidal anti-inflammatory drug-activated gene-1 plays a role in the impairing effects of cyclooxygenase inhibitors on gastric ulcer healing

Nonsteroidal anti-inflammatory drugs (NSAIDs) can impair gastric ulcer healing. This study investigates the involvement of NSAID-activated gene-1 (NAG-1) in ulcer repair impairment by cyclooxygenase (COX) inhibitors. Gastric ulcers were induced in rats by acetic acid. Four days later, animals received daily intragastric indomethacin (nonselective COX-1/COX-2 inhibitor; 1 mg/kg), 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole (SC-560) (selective COX-1 inhibitor; 2.5 mg/kg), (5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl) phenyl-2(5H)-furanone (DFU) (selective COX-2 inhibitor; 5 mg/kg), celecoxib (selective COX-2 inhibitor; 1 mg/kg), and valdecoxib (selective COX-2 inhibitor; 1 mg/kg), for 1, 3, or 7 days. Ulcerated tissues were processed to assess: 1) COX-1, COX-2, NAG-1, proliferating cell nuclear antigen (PCNA), and activated caspase-3 expression; 2) ulcer area; and 3) prostaglandin E(2) (PGE(2)) levels. COX-1 expression in ulcerated tissues was decreased, whereas COX-2 expression was enhanced. Ulcer healing was delayed by indomethacin, DFU, and SC-560, but not by celecoxib and valdecoxib. Ulcer PGE(2) levels were decreased by SC-560, DFU, celecoxib, valdecoxib, and indomethacin. NAG-1 was overexpressed in ulcerated tissues and further enhanced by indomethacin, DFU, and SC-560, but not by celecoxib or valdecoxib. PCNA expression in ulcerated areas was reduced by indomethacin, but not by the other test drugs. The expression of activated caspase-3 in ulcers was increased and enhanced further by indomethacin, DFU, and SC-560, but not by celecoxib and valdecoxib. These findings indicate that: 1) COX inhibitors exert differential impairing effects on gastric ulcer healing, through mechanisms unrelated to the inhibition of COX isoforms and prostaglandin production; and 2) NAG-1 induction, followed by activation of proapoptotic pathways, can contribute to the impairing effects of COX inhibitors on ulcer healing.     

A new role for complement in experimental membranous nephropathy in rats.

The only established role for complement in mediating immunologic renal disease involves elaboration of leukochemotactic factors and neutrophil-dependent glomerular injury. In the passive Heymann nephritis (PHN) model of experimental membranous nephropathy, rats injected with sheep antibody to rat proximal tubular brush border antigen (Fx1A) form subepithelial deposits of sheep IgG and rat complement (C3), and develop heavy proteinuria after 5 d without glomerular inflammatory changes. To study the role of complement in mediating proteinuria in PHN, 16 rats were treated daily with cobra venom factor from before antibody injection to maintain C3 levels at < 10% of pretreatment values and compared to 16 untreated controls. Proteinuria at 5 d was abolished in C3-depleted rats (4 +/- 1, controls 70 +/- 15 mg/d, P < 0.001), although renal deposition of 125I-labeled antibody ws the same in both groups (188 +/- 35 vs. 191 +/- 22 microgram IgG/2 kidneys, P > 0.5). Nephritogenic doses of both the noncomplement-fixing F(ab')2 portion and the gamma 2 subclass of anti-Fx1A IgG produced subepithelial deposits of immunoglobulin without C3, but proteinuria did not occur despite glomerular deposition of up to 70 microgram/2 kidneys of gamma 2. However, glomerular deposition of as little as 60 microgram of gamma 1 produced C3 fixation in vivo and heavy proteinuria. No neutrophil exudate could be detected histologically in PHN from the time of antibody injection through development of proteinuria. Proteinuria in five PHN rats depleted of neutrophils to < 200/mm3 with antineutrophil serum was not reduced compared to six controls with normal neutrophil counts (34 +/- 9.6 vs. 25 +/- 10.4 mg/d, P > 0.5). These results demonstrate that proteinuria in the PHN model of membranous nephropathy is complement-dependent and strongly suggest a neutrophil-independent mechanism. Thus a new role for the complement system in mediating immunologic glomerular injury is identified.     

Leukotriene D4 is a mediator of proteinuria and glomerular hemodynamic abnormalities in passive Heymann nephritis.

We assessed the role of leukotrienes (LTs) in Munich-Wistar rats with passive Heymann nephritis (PHN), an animal model of human membranous nephropathy. 10 d after injection of anti-Fx1A antibody, urinary protein excretion rate (Upr) in PHN was significantly higher than that of control. Micropuncture studies demonstrated reduced single nephron plasma flow and glomerular filtration rates, increased transcapillary hydraulic pressure difference, pre- and postglomerular resistances, and decreased ultrafiltration coefficient in PHN rats. Glomerular LTB4 generation from PHN rats was increased. Administration of the 5-LO activating protein inhibitor MK886 for 10 d markedly blunted proteinuria and normalized glomerular hemodynamic abnormalities in PHN rats. An LTD4 receptor antagonist SK&F 104353 led to an immediate reduction in Upr and to reversal of glomerular hemodynamic impairment. Ia(+) cells/glomerulus were increased in PHN rats. In x-irradiated PHN rats, which developed glomerular macrophage depletion, augmented glomerular LT synthesis was abolished. Thus, in the autologous phase of PHN, LTD4 mediates glomerular hemodynamic abnormalities and a hemodynamic component of the accompanying proteinuria. The synthesis of LTD4 likely occurs directly from macrophages or from macrophage-derived LTA4, through LTC4 synthase in glomerular cells.     

Interactions of dimethyl sulfoxide and nonsteroidal anti-inflammatory agents in passive Heymann's nephritis

The effect of treatment with indomethacin on the ability of dimethyl sulfoxide (DMSO) to reduce proteinuria in rats with passive Heymann's nephritis (PHN) was studied. PHN rats treated with DMSO alone excreted significantly less protein by day 14 than PHN rats treated with buffer or with indomethacin alone. Rats treated with DMSO excreted 19 +/- 6.0 mg protein/24 hr, and those treated with DMSO and indomethacin excreted 161 +/- 27.4 mg protein/24 hr (P less than 0.001). Rats treated with DMSO alone had significantly higher serum albumin and significantly lower serum cholesterol and triglyceride concentrations than those given the two drugs together. Glomerular deposits of C3 were reduced in DMSO-treated rats, but serum C3 concentrations and rat antirabbit serum antibody titers were similar in the two groups. When a higher dose of indomethacin (5 mg/kg) plus DMSO was used, protein excretion was significantly reduced. Rats treated with DMSO and acetylsalicylic acid (ASA) (37 mg/kg/day) or DMSO and meclofenamate (5 mg/kg/day) did not have a significant reduction in protein excretion compared with untreated controls. High-dosage indomethacin alone did not reduce proteinuria. Low doses of nonsteroidal anti-inflammatory agents therefore appear to block the effect of DMSO on proteinuria. This was in marked contrast to the finding of reduction of proteinuria induced by larger doses of indomethacin (5 mg/kg) plus DMSO. DMSO did not reduce proteinuria in rats with nephrosis induced by puromycin of aminonucleoside.     

Basic fibroblast growth factor augments podocyte injury and induces glomerulosclerosis in rats with experimental membranous nephropathy.

Podocyte injury is believed to contribute to glomerulosclerosis in membranous nephropathy. To identify the factors involved, we investigated the effects of basic fibroblast growth factor (bFGF), a cytokine produced by podocytes, on rats with membranous nephropathy (passive Heymann nephritis [PHN]). All rats received a daily i.v. bolus of 10 microg bFGF or vehicle from days 3-8 after PHN induction. In proteinuric PHN rats on day 8, bFGF injections further increased proteinuria. Podocytes of bFGF-injected PHN rats showed dramatic increases in mitoses, pseudocyst formation, foot process retraction, focal detachment from the glomerular basement membrane, and desmin expression. bFGF injections in PHN rats did not alter antibody or complement deposition or glomerular leukocyte influx. bFGF-injected PHN rats developed increased glomerulosclerosis when compared with control PHN rats. Also, bFGF induced proteinuria and podocyte damage in rats injected with 10% of the regular PHN-serum dose. None of these changes occurred in bFGF-injected normal rats, complement-depleted PHN rats or rats injected with 5% of the regular PHN serum dose. These divergent bFGF effects were explained in part by upregulated glomerular bFGF receptor expression, induced by PHN serum. Thus, bFGF can augment podocyte damage, resulting in increased glomerular protein permeability and accelerated glomerulosclerosis. This bFGF action is confined to previously injured podocytes. Release of bFGF from glomerular sources (including podocytes themselves) during injury may represent an important mechanism by which podocyte damage is enhanced or becomes self sustained.     

Interaction of cyclooxygenase isoenzymes, nitric oxide, and afferent neurons in gastric mucosal defense in rats

The cyclooxygenase (COX)-2 inhibitors 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl-2(5II)-furanone (DFU) (0.02-2 mg/kg) and N-[2-(cyclohexyloxy)-4-nitrofenyl]-methanesulfonamide (NS-398) (0.01-1 mg/kg), the COX-1 inhibitor 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole (SC-560) (0.05-5 mg/kg), and dexamethasone (1 mg/kg) were studied in rats challenged with intragastric acid (300 mM HCl). All compounds induced severe gastric damage when rats were treated concurrently with the inhibitor of constitutive and inducible nitric-oxide (NO) synthase N(G)-monomethyl-L-arginine methyl ester (L-NAME) (3 or 40 mg/kg). DFU and NS-398 caused significantly less damage in rats receiving the selective inhibitor of inducible NO synthase N-(3-(aminomethyl)benzyl)acetamidine (1400W) (0.3 mg/kg). The COX-1 inhibitor SC-560 induced moderate damage in the acid-challenged stomach even without suppression of NO, but damage was aggravated by L-NAME. The COX-3 inhibitor phenacetin (400 mg/kg) did not injure the gastric mucosa despite suppression of NO. Furthermore, DFU, NS-398, SC-560, and dexamethasone caused severe injury in the acid-challenged stomach of rats pretreated with capsaicin to ablate afferent neurons. The mucosal damage induced by the COX-1 inhibitor, the COX-2 inhibitors, and dexamethasone in L-NAME- or capsaicin-treated rats was reversed by coadministration of 16,16-dimethyl-prostaglandin E2 (2 x 8 ng/kg). Gross mucosal damage was paralleled by histology. Our results support the concept that endogenous NO, prostaglandins, and afferent neurons act in concert in the regulation of gastric mucosal integrity. The prostaglandins necessary for mucosal defense in the face of NO suppression, and afferent nerve ablation can be derived either from COX-1 or COX-2. The data do not propose a protective role for a phenacetin-sensitive COX-3. Our findings suggest that not only COX-1 but also COX-2 has important functions in the maintenance of gastric integrity.     

Roles of endogenous prostaglandins and cyclooxygenase isozymes in healing of indomethacin-induced small intestinal lesions in rats

The role of prostaglandins (PGs)/cyclooxygenase (COX) in the healing of indomethacin-induced small intestinal ulcers was examined in rats. Animals were given indomethacin (10 mg/kg s.c.) and killed 1, 2, 3, 5, and 7 days later. Indomethacin (2 mg/kg), 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole (SC560; COX-1 inhibitor; 3 mg/kg), and rofecoxib (COX-2 inhibitor; 3 mg/kg) were given p.o. once daily for 6 days, during the first 3 days or last 3 days of the experimental period. All COX inhibitors given for 6 days significantly impaired the healing of these ulcers. Healing was also impaired by rofecoxib given for the first 3 days or by SC560 given for the last 3 days. The expression of COX-2 mRNA in the intestine was up-regulated after ulceration, persisting for 3 days and dissipating thereafter. Mucosal PGE2 contents decreased within 3 h after ulceration, recovered 24 h later, and increased above normal 1 approximately 3 days later. The PGE2 content at 4 days after ulceration was decreased by rofecoxib but not SC560, whereas that at 7 days was suppressed by SC560 but not rofecoxib. Vascular content in the ulcerated mucosa decreased when the healing was impaired by COX inhibitors. The deleterious effect of indomethacin on healing was mimicked by a prostacyclin E receptor (EP) 4 antagonist and reversed by coadministration of PGE2 as well as an EP4 agonist. In conclusion, endogenous PGs play a role in the healing of intestinal ulcers through EP4 receptors, yet the COX isozyme involved differs depending on the stage of healing; COX-2 in the early stage and COX-1 in the late stage.     

Effect of alterations in glomerular charge on deposition of cationic and anionic antibodies to fixed glomerular antigens in the rat

Reduction of the negative charge of the glomerular capillary wall alters its charge- and size-selective properties. To investigate the effect of alteration in glomerular charge properties on antibody localization, we prepared cationic and anionic fractions of antibodies to subepithelial and glomerular basement membrane (GBM) antigens, and compared their deposition in normal rats and rats treated with protamine sulfate or aminonucleoside of puromycin to reduce capillary wall charge. IgG antibodies were eluted from kidneys of rats with active Heymann's nephritis (AICN), passive Heymann's nephritis (PHN), or anti-GBM nephritis (NTN), separated into cationic and anionic fractions, and radiolabeled with iodine 125 or iodine 131. Relative antibody content of each fraction was determined by incubation with an excess of glomerular antigen. Varying amounts of cationic and anionic IgG eluted from kidneys of rats with AICN or PHN were injected into 24 normal or protamine sulfate-treated rats. Glomerular binding of all antibodies was highly correlated with IgG delivery to the kidney. The ratio of cationic to anionic antibody deposited in the glomeruli of normal rats after 4 hours was 1.08 +/- 0.07 for AICN eluate and 0.37 +/- 0.04 for PHN eluate. The ratios were not significantly different in animals pretreated with protamine sulfate (1.15 +/- 0.06 and 0.44 +/- 0.06, respectively; P greater than 0.05). Varying amounts of cationic and anionic IgG eluted from kidneys of rats with NTN were injected into 10 normal rats and four rats treated with aminonucleoside of puromycin. Glomerular binding of antibody was again highly correlated with IgG delivery to the kidney. The ratio of cationic to anionic antibody deposited in the glomeruli of normal rats after 1 hour was 1.03 +/- 0.06, and was not significantly altered in rats treated with aminonucleoside of puromycin (1.05 +/- 0.03, P greater than 0.5). Proteinuria in PHN rats was also unaffected by treatment with protamine sulfate for 5 days (controls: 68 +/- 21 mg/day; protamine sulfate-treated: 65 +/- 14 mg/day; n = 25, P greater than 0.08). These results demonstrate that treatment to reduce glomerular polyanion does not significantly alter the ratio of cationic to anionic antibodies to fixed glomerular antigens that deposit in the glomerulus, or reduce proteinuria caused by deposition of antibody to a fixed subepithelial antigen.     

Rofecoxib [Vioxx, MK-0966; 4-(4'-methylsulfonylphenyl)-3-phenyl-2-(5H)-furanone]: a potent and orally active cyclooxygenase-2 inhibitor. Pharmacological and biochemical profiles.

The discoveries that cyclooxygenase (COX)-2 is an inducible form of COX involved in inflammation and that COX-1 is the major isoform responsible for the production of prostaglandins (PGs) in the gastrointestinal tract have provided a rationale for the development of specific COX-2 inhibitors as a new class of anti-inflammatory agents with improved gastrointestinal tolerability. In the present study, the preclinical pharmacological and biochemical profiles of rofecoxib [Vioxx, also known as MK-0966, 4-(4'-methylsulfonylphenyl)-3-phenyl-2-(5H)-furanone], an orally active COX-2 inhibitor, are described. Rofecoxib is a potent inhibitor of the COX-2-dependent production of PGE(2) in human osteosarcoma cells (IC(50) = 26 +/- 10 nM) and Chinese hamster ovary cells expressing human COX-2 (IC(50) = 18 +/- 7 nM) with a 1000-fold selectivity for the inhibition of COX-2 compared with the inhibition of COX-1 activity (IC(50) > 50 microM in U937 cells and IC(50) > 15 microM in Chinese hamster ovary cells expressing human COX-1). Rofecoxib is a time-dependent inhibitor of purified human recombinant COX-2 (IC(50) = 0.34 microM) but caused inhibition of purified human COX-1 in a non-time-dependent manner that could only be observed at a very low substrate concentration (IC(50) = 26 microM at 0.1 microM arachidonic acid concentration). In an in vitro human whole blood assay, rofecoxib selectively inhibited lipopolysaccharide-induced, COX-2-derived PGE(2) synthesis with an IC(50) value of 0.53 +/- 0.02 microM compared with an IC(50) value of 18.8 +/- 0.9 microM for the inhibition of COX-1-derived thromboxane B(2) synthesis after blood coagulation. Using the ratio of the COX-1 IC(50) values over the COX-2 IC(50) values in the human whole blood assay, selectivity ratios for the inhibition of COX-2 of 36, 6.6, 2, 3, and 0.4 were obtained for rofecoxib, celecoxib, meloxicam, diclofenac, and indomethacin, respectively. In several in vivo rodent models, rofecoxib is a potent inhibitor of carrageenan-induced paw edema (ID(50) = 1.5 mg/kg), carrageenan-induced paw hyperalgesia (ID(50) = 1.0 mg/kg), lipopolysaccharide-induced pyresis (ID(50) = 0.24 mg/kg), and adjuvant-induced arthritis (ID(50) = 0.74 mg/kg/day). Rofecoxib also has a protective effect on adjuvant-induced destruction of cartilage and bone structures in rats. In a (51)Cr excretion assay for detection of gastrointestinal integrity in either rats or squirrel monkeys, rofecoxib has no effect at doses up to 200 mg/kg/day for 5 days. Rofecoxib is a novel COX-2 inhibitor with a biochemical and pharmacological profile clearly distinct from that of current nonsteroidal anti-inflammatory drugs and represents a new therapeutic class of anti-inflammatory agents for the treatment of the symptoms of osteoarthritis and rheumatoid arthritis with improved gastrointestinal tolerability.     

Proteinuria in passive Heymann nephritis is associated with lipid peroxidation and formation of adducts on type IV collagen.

Passive Heymann nephritis (PHN) is a model of human membranous nephropathy that is characterized by formation of granular subepithelial immune deposits in the glomerular capillary wall which results in complement activation. This is causally related to damage of the filtration barrier and subsequent proteinuria. The local accumulation of injurious reactive oxygen species (ROS) is a major effector mechanism in PHN. ROS may induce tissue damage by initiating lipid peroxidation (LPO). In turn, this leads to adduct formation between breakdown products of LPO with structural proteins, such as formation of malondialdehyde (MDA) or 4-hydroxynonenal-lysine adducts. To examine the role of LPO in the development of proteinuria we have localized MDA and 4-hydroxynonenal-lysine adducts in glomeruli of PHN rats by immunofluorescence microscopy, using specific monoclonal antibodies. By immunogold electron microscopy, MDA adducts were localized to cytoplasmic vesicles and cell membranes of glomerular epithelial cells, to the glomerular basement membrane (GBM), and also to immune deposits. Type IV collagen was specifically identified as being modified by MDA adducts, using a variety of techniques. Collagenase pretreatment of GBM extracts indicated that the NC-1 domain of type IV collagen was a site of adduct formation. When LPO was inhibited by pretreatment of PHN rats with the antioxidant probucol, proteinuria was reduced by approximately 85%, and glomerular immunostaining for dialdehyde adducts was markedly reduced, even though the formation of immune deposits was not affected. By contrast, lowering of the serum cholesterol levels had no influence on the development of proteinuria. These findings are consistent with the premise that ROS-induced glomerular injury in PHN involves LPO and that this results not only in damage of cell membranes but in modification of type IV collagen in the GBM as well. The close temporal correlation of the occurrence of LPO with proteinuria and the ability of probucol to inhibit proteinuria support a causal role for LPO in the the alteration of the glomerular permselectivity which results in proteinuria.     

Role of cyclooxygenase-1 and -2, phospholipase C,and protein kinase C in prostaglandin-mediated gastroprotection

Oral administration of the nonselective cyclooxygenase (COX) inhibitor indomethacin (20 mg/kg), the COX-1 inhibitor 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole (SC-560) (20 mg/kg), or the COX-2 inhibitor rofecoxib (1-20 mg/kg) antagonized the gastroprotective effects of 16,16-dimethyl-prostaglandin (PG) E2 (75 ng/kg p.o.) and 20% ethanol in rats. The effects of the COX inhibitors were reversed by the activator of ATP-sensitive potassium (KATP) channels cromakalim (0.3-0.5 mg/kg p.o.). The protective effects of 16,16-dimethyl-PGE2 and 20% ethanol were counteracted by the phospholipase C inhibitor 1-(6-((17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U-73122), but not its inactive analog 1-(6-((17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-2,5-pyrrolidine-dione (U-73343) (1 mg/kg each i.v.). Likewise, the protein kinase C inhibitors chelerythrine (0.7 mg/kg i.v.) and staurosporine (3 microg/kg i.v.) inhibited gastroprotection. Effects of these enzyme inhibitors were not reversed by cromakalim. Submaximally effective doses of SC-560 (0.2 mg/kg p.o.) and rofecoxib (0.02 mg/kg p.o.) were additive and abolished the protection induced by 20% ethanol. The findings show that inhibition of COX-1 or COX-2 antagonizes not only adaptive gastroprotection by 20% ethanol but also the protective effect of exogenous PG in a cromakalimsensitive manner. Endogenous PG obviously add to the protective activity of exogenous PG. Gastroprotection by PG involves phospholipase C, protein kinase C, and KATP channels. Activation of KATP channels does not exert protection when the activity of phospholipase C or protein kinase C is suppressed.     

Distinct actions of endothelin A-selective versus combined endothelin A/B receptor antagonists in early diabetic kidney disease

Selective endothelin A (ET(A)) and combined ET(A) and ET(B) receptor antagonists are being investigated for use in treating diabetic nephropathy. However, the receptor-specific mechanisms responsible for producing the potential benefits have not been discerned. Thus, we determined the actions of ET(A) and ET(B) receptors on measures of glomerular function and renal inflammation in the early stages of diabetic renal injury in rats through the use of selective and combined antagonists. Six weeks after streptozotocin (STZ)-induced hyperglycemia, rats were given 2R-(4-methoxyphenyl)-4S-(1,3-benzodioxol-5-yl)-1-(N,N-di(n-butyl)aminocarbonyl-methyl)-pyrrolidine-3R-carboxylic acid (ABT-627) (5 mg/kg/day), a selective ET(A) antagonist; (2R,3R,4S)-4-(benzo[d][1,3]dioxol-5-yl)-2-(3-fluoro-4-methoxyphenyl)-1-(2-(N-propylpentylsulfonamido)ethyl)pyrrolidine-3-carboxylic acid hydrochloride (A-182086) (10 mg/kg/day), a combined ET(A/B) antagonist; or vehicle for 1 week. Sham controls received STZ vehicle (saline). Hyperglycemia led to significant proteinuria, increased glomerular permeability to albumin (P(alb)), nephrinuria, and an increase in total matrix metalloprotease (MMP) and transforming growth factor-β1 (TGF-β1) activities in glomeruli. Plasma and glomerular soluble intercellular adhesion molecule-1 (sICAM-1) and monocyte chemoattractant protein-1 (MCP-1) were elevated after 7 weeks of hyperglycemia. Daily administration of both ABT-627 and A-182086 for 1 week significantly attenuated proteinuria, the increase in P(alb), nephrinuria, and total MMP and TGF-β1 activity. However, glomerular sICAM-1 and MCP-1 expression was attenuated with ABT-627, but not A-182086, treatment. In summary, both selective ET(A) and combined ET(A/B) antagonists reduced proteinuria and glomerular permeability and restored glomerular filtration barrier component integrity, but only ET(A)-selective blockade had anti-inflammatory and antifibrotic effects. We conclude that selective ET(A) antagonists are more likely to be preferred for the treatment of diabetic kidney disease.     

Immunohistochemical and functional correlations of renal cyclooxygenase-2 in experimental diabetes

Prostaglandins (PGs) generated by the enzyme cyclooxygenase (COX) have been implicated in the pathological renal hemodynamics and structural alterations in diabetes mellitus, but the role of individual COX isoenzymes in diabetic nephropathy remains unknown. We explored COX-1 and COX-2 expression and hemodynamic responses to the COX-1 inhibitor valeryl salicylate (VS) or the COX-2 inhibitor NS398 in moderately hyperglycemic, streptozotocin-diabetic (D) and control (C) rats. Immunoreactive COX-2 was increased in D rats compared with C rats and normalized by improved glycemic control. Acute systemic administration of NS398 induced no significant changes in mean arterial pressure and renal plasma flow in either C or D rats but reduced glomerular filtration rate in D rats, resulting in a decrease in filtration fraction. VS had no effect on renal hemodynamics in D rats. Both inhibitors decreased urinary excretion of PGE(2). However, only NS398 reduced excretion of thromboxane A(2). In conclusion, we documented an increase in renal cortical COX-2 protein expression associated with a different renal hemodynamic response to selective systemic COX-2 inhibition in D as compared with C animals, indicating a role of COX-2-derived PG in pathological renal hemodynamic changes in diabetes.     

Angiotensin inhibition reduces glomerular damage and renal chemokine expression in MRL/lpr mice

Beneficial effects of angiotensin II inhibition during inflammatory renal disease may involve both hemodynamic and nonhemodynamic mechanisms. To analyze whether angiotensin II inhibition has protective effects on lupus-like, autoimmune-mediated renal damage in MRL/lpr mice, four groups of mice were treated orally for 6 weeks with: 1) vehicle, 2) enalapril (3.0 mg/kg per day), 3) candesartan cilexetil (5.0 mg/kg), or 4) amlodipine (10 mg/kg) as a blood pressure control (n = 9-12/group). All antihypertensive treatments lowered blood pressure to a similar level compared with vehicle group (enalapril: 99.8 +/- 8.3 mm Hg; candesartan: 101 +/- 9 mm Hg; amlodipine: 103.8 +/- 6.7 mm Hg; vehicle: 113.5 +/- 4.6 mm Hg). Vehicle-treated mice developed a moderate glomerular injury with albuminuria (35.1 +/- 39.0 microg/mg of creatinine). Glomerular lesions consisted of immune complex deposition and mesangial expansion with increased mesangial cell proliferation. Amlodipine treatment had no significant protective effects. In contrast to vehicle- and amlodipine-treated mice, those subjected to angiotensin II blockade with enalapril or candesartan had reduced albuminuria, glomerular expansion, and mesangial proliferation. This was associated with significantly reduced renal chemokine mRNA expression compared with vehicle treatment. Our results show that inhibition of angiotensin II has protective effects on the glomerular damage of MRL/lpr mice that extend beyond hemodynamics and involve down-modulation of glomerular inflammation, reduction of mesangial cell proliferation, and decrease in chemokine expression.     

Characterization of rofecoxib as a cyclooxygenase-2 isoform inhibitor and demonstration of analgesia in the dental pain model

BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin, ibuprofen, and indomethacin (INN, indometacin) inhibit both the constitutive (COX-1) and inducible (COX-2) isoforms of cyclooxygenase. The induction of COX-2 after inflammatory stimuli has led to the hypothesis that COX-2 inhibition primarily accounts for the therapeutic properties of NSAIDs. METHODS: Chinese hamster ovary (CHO) cell lines that express each COX isoform were used to characterize the in vitro selectivity of rofecoxib. Single oral doses of rofecoxib and indomethacin were then assessed in subjects with use of ex vivo COX-isoform specific assays (serum thromboxane B2 [TXB2] and lipopolysaccharide [LPS]-stimulated whole blood prostaglandin E2 and assays of COX-1 and COX-2 activity, respectively). A double-blind, parallel-group study compared the analgesic efficacy of rofecoxib to placebo and ibuprofen in 102 patients with dental pain. RESULTS: Rofecoxib showed a >800-fold COX-2 selectivity with use of CHO cells that express human COX-1 and COX-2. In subjects, dose- and concentration-dependent inhibition of LPS-stimulated prostaglandin E2 was observed with both rofecoxib (IC50 [the concentration estimated to produce 50% inhibition], 0.77 micromol/L) and indomethacin (IC50, 0.33 micromol/L). Whereas indomethacin inhibited TXB2, (IC50, 0.14 micromol/L), no inhibition was observed with rofecoxib even at doses of up to 1000 mg. In the dental pain study, total pain relief (TOTPAR) over the 6 hours after dosing was similar between 50 mg and 500 mg rofecoxib and 400 mg ibuprofen (P > .20). All active treatments showed greater improvement than placebo (P < .001) CONCLUSIONS: Rofecoxib inhibited COX-2 without evidence of COX-1 inhibition, even at oral doses of up to 1000 mg. Nonetheless, rofecoxib showed analgesic activity indistinguishable from that observed with ibuprofen, a nonisoform-selective COX inhibitor. These results support the hypothesis that the analgesic effects of NSAIDs primarily derive from inhibition of COX-2.     

Cyclooxygenase-2 inhibition by rofecoxib reverses naturally occurring fever in humans.

Cyclooxygenase (COX) exists as constitutive (COX-1) and inducible (COX-2) isoforms. Nonsteroidal antiinflammatory drugs (NSAIDs) such as ibuprofen and diclofenac inhibit both COX-1 and COX-2. The role of COX-2 in the genesis of fever in monkeys and humans was examined with use of the specific COX-2 inhibitor rofecoxib. Rofecoxib was administered to monkeys made febrile by 6 microg/kg intravenous lipopolysaccharide. Induced pyrexia was followed by oral rofecoxib (1 or 3 mg/kg), diclofenac (3 mg/kg), or vehicle. Rofecoxib and diclofenac rapidly reversed the elevated temperature (P < .05 versus vehicle for 3 mg/kg rofecoxib and diclofenac at 70 to 90 minutes after dosing). A single-dose, parallel-group, double-blind randomized trial was conducted in 94 patients with fever caused by a viral-type illness. Mean baseline temperature was similar for all groups (-38.5 degrees C). Patients received oral doses of 12.5 mg rofecoxib, 25 mg rofecoxib, 400 mg ibuprofen, or placebo and the mean +/- SE change in oral temperature at 4 hours after dosing was -0.97 degrees C +/- 0.11 degrees C, -1.19 degrees C +/- 0.09 degrees C, -1.20 degrees C +/- 0.11 degrees C, and 0.01 C +/- 0.17 C, respectively (P < .001 for active treatments versus placebo). Specific inhibition of COX-2 by rofecoxib results in antipyretic activity in monkeys and humans comparable to dual COX-1/COX-2 inhibitors such as diclofenac or ibuprofen. The data support the hypothesis that it is the COX-2 isoform that is primarily involved in the genesis of fever in humans.     

Role of prostaglandins and thromboxane in the control of renal hemodynamics in experimental liver cirrhosis

Although there is considerable evidence that vasodilator prostaglandins such as prostaglandin E2 (PGE2) modulate renal hemodynamics in liver cirrhosis, the role of the vasoconstrictor thromboxane A2 (TXA2) is controversial. We measured renal hemodynamics and glomerular eicosanoid production in cirrhotic and control rats. Renal plasma flow, as estimated by para-aminohippurate clearance (CPAH) and glomerular filtration rate, as determined by inulin clearance (CIN), were comparable between groups; glomerular production of PGE2 and TXA2 (estimated by the metabolite thromboxane B2 [TXB2]) was slightly but not significantly higher in cirrhotic than in control rats (PGE2: 1060 +/- 142 pg/mg glomerular protein vs 854 +/- 288 pg/mg glomerular protein; TXB2: 782 +/- 103 pg/mg glomerular protein vs 468 +/- 104 pg/mg glomerular protein). Addition of serum from cirrhotic rats to the incubation media failed to increase eicosanoid production in glomeruli obtained from either cirrhotic or control rats. Cyclooxygenase inhibition with 5 mg/kg indomethacin, a dose sufficient to result in a 68% inhibition of glomerular PGE2 synthesis, decreased both CPAH (from 6.59 +/- 0.69 ml/min to 4.52 +/- 0.67 ml/min, p less than 0.05) and CIN (from 1.34 +/- 0.16 ml/min to 0.68 +/- 0.07 ml/min, p less than 0.01) in cirrhotic rats. Thromboxane synthesis inhibition with 1 mg/kg UK-38485, which resulted in an 84% decrease in glomerular TXB2, did not significantly affect either CPAH or CIN; however, there was a strong trend toward improvement in CIN (from 1.23 +/- 0.11 ml/min to 1.43 +/- 0.15 ml/min (0.05 less than p less than 0.1). Neither drug affected renal hemodynamics in control rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complement-induced glomerular epithelial cell injury. Role of the membrane attack complex in rat membranous nephropathy.

In passive Heymann nephritis (PHN) in rats, antibody (anti-Fx1A) reacts in situ with a glomerular epithelial antigen and induces complement (C)-mediated cell-independent proteinuria. To assess the role of the membrane attack complex (MAC), we determined the need for C8 in the pathogenesis of proteinuria in an autologous-phase model of PHN. Isolated rat kidneys, containing nonnephritogenic, non-C-fixing gamma 2 sheep anti-Fx1A (planted antigen), when perfused in vitro with C-fixing guinea pig anti-sheep IgG and a source of C (fresh human plasma 50% vol/vol in buffer containing bovine serum albumin), developed marked proteinuria after 20 min (0.58 +/- 0.08 mg/min X g, n = 8) that increased further to 3.20 +/- 0.93 mg/min X g after 80 min. In contrast, identical kidneys perfused with antibody and heat-inactivated or C8-deficient human plasma and normal kidneys perfused with antibody and fresh plasma excreted only 0.27 +/- 0.03 (n = 6), 0.27 +/- 0.04 (n = 5), and 0.40 +/- 0.05 mg/min X g (n = 6) after 20 min, and 0.13 +/- 0.02, 0.22 +/- 0.03, and 0.32 +/- 0.05 mg/min X g after 80 min, respectively. When C8-deficient plasma was reconstituted with sources of C8 (n = 3), proteinuria was restored to the level observed with fresh normal plasma. Differences in protein excretion could not be explained by quantitative differences in glomerular antigen or antibody content. Extensive ultrastructural damage to glomerular visceral epithelial cells was exclusively seen in antigen-containing kidneys perfused with antibody and C8-replete plasma. Thus, glomerular injury in this model results from an antigen-specific, antibody-directed, C8-dependent reaction involving assembly of the MAC. The ultrastructural findings argue in favor of MAC-induced cytotoxicity of the glomerular visceral epithelial cells.