Interspecies differences in renal localization of cyclooxygenase isoforms: implications in nonsteroidal antiinflammatory drug-related nephrotoxicity.

Cyclooxygenase (COX) exists in 2 related but unique isoforms: one is constitutive (COX-1) and functions in normal cell physiology, and the other is inducible (COX-2) and is expressed in response to inflammatory stimuli. Nonsteroidal antiinflammatory drugs (NSAIDs) cause renal toxicity following inhibition of renal cyclooxygenases. Humans and animals exhibit differences in susceptibility to NSAID-related renal toxicity, which may be associated with differences in expression of 1 or both isoforms of COX in the kidney. In this study, we evaluated COX-1 and COX-2 expression in the kidneys of mixed-breed dogs, Sprague-Dawley rats, cynomolgus monkeys, and humans. In addition, the effect of volume depletion on renal COX expression was investigated in rats, dogs, and monkeys. COX expression was evaluated using 1 or more of the following procedures: reverse transcriptase polymerase chain reaction, in situ hybridization, and immunohistochemistry. We demonstrated that both COX isoforms are expressed in the kidneys of all species examined, with differences in the localization and level of basal expression. COX-1 is expressed at high levels in the collecting ducts and renal vasculature of all species and in a small number of papillary interstitial cells in rats, monkeys, and humans. Basal levels of COX-2 are present in the maculae densa, thick ascending limbs, and papillary interstitial cells in rats and dogs and in glomerular podocytes and small blood vessels in monkeys and humans. COX-2 expression is markedly increased in volume-depleted rats and dogs but not monkeys. These results indicate that significant interspecies differences exist in the presence and distribution of COX isoforms, which may help explain the difference in species susceptibility to NSAID-related renal toxicity.

Temporal variation and factors affecting measurement of canine von Willebrand factor.

OBJECTIVE: To determine whether canine plasma von Willebrand factor (vWf) varies between and within individuals over time and with different blood sample collection and processing procedures. ANIMALS: 26 adult dogs and 6 pups. PROCEDURE: Blood was obtained from the jugular or cephalic vein daily for 8 to 19 days and weekly for 9 to 23 weeks in adult dogs and periodically up to 180 days of age in pups. Temporal variation in vWf concentration and the effect of vascular occlusion, venipuncture site, lipemia, hemolysis, anticoagulant, storage time, freeze-thawing, and centrifugation speed on plasma vWf concentration, measured by ELISA, were determined. RESULTS: Plasma vWf concentration varied over time. In dogs with mean vWf concentration > or = 79 U/dl, the largest intraindividual range in vWf spanned 64 U/dl with daily and 53 U/dl with weekly sample collection. In dogs with mean vWf concentration < or = 24 U/dl, the largest individual variation was 12 U/dl with daily and weekly sample collection. In dogs with mean vWf concentration > or = 53 and < or = 74 U/dl, the largest intraindividual range spanned 35 U/ dl. Mean vWf concentration of pups from 3 to 180 days of age did not change. Sample hemolysis decreased mean vWf by 37%. Mean vWf concentration was 9% higher in cephalic than jugular vein samples (P = 0.056). Other sample collection/preparation procedures did not affect vWf concentration. CONCLUSION: There was substantial temporal variation in vWf concentration within individual dogs. CLINICAL RELEVANCE: Multiple tests may be necessary to obtain a reliable estimate of vWf concentration in dogs.

Two antipeptide monoclonal antibodies that recognize adhesive sequences in fibrinogen: identification of antigenic determinants and unrelated sequences using synthetic combinatorial libraries.

The fine specificity of two different monoclonal antibodies raised against synthetic peptides, each representing one of the two Arg-Gly-Asp (RGD) sequences in fibrinogen, was examined using synthetic combinatorial libraries (SCLs). The monoclonal antibodies (mAb), mAb LJ-134B/29 and mAb LJ-155B/16, recognize both the immunogenic peptide and native fibrinogen. The specificity of mAb LJ-134B29 was mapped using hexa- and decapeptide positional scanning SCLs (PS-SCLs) and competitive ELISA. The most active amino acids at each position of the two libraries were identified from a single screening. Individual hexa- and decapeptides were synthesized and assayed to determine their binding affinities. The 16 individual hexapeptides represented single and multiple substitutions of the antigenic determinant sequence, -GDSTFE-, eight of which had affinities less than 10nM. Four of the twelve individual decapeptides were found to have binding affinities of approximately 300nM, or nearly three-fold less than the peptide immunogen. A dual-defined hexapeptide library was screened against mAb LJ-155B/16, and individual peptides were obtained through an iterative selection and synthesis process. Surprisingly, one of the most active sequences was Ac-WWYESW-NH2 (IC50 = 40nM), which showed no similarity to the sequence of the immunizing peptide. Further mapping of the specificity of this antibody revealed that the antigenic determinant within the peptide immunogen was not completely linear. Recognition of this unrelated sequence by mAb LJ-155B/16 was confirmed in a direct binding assay using biotinylated peptide. The use of SCLs for the elucidation of high affinity peptides recognized by these two antibodies may provide additional information on the molecular mechanisms of fibrinogen binding to different integrin receptors.

Experimentally induced renal failure in the dog as an animal model of uremic bleeding.

Experimental canine renal failure was studied as a potential animal model for human uremic bleeding. Renal failure accompanied by hemostatic alterations was induced in eight dogs by means of two surgical techniques of renal mass reduction. The hemostatic deficits consisted of immediate and marked reduction of the platelet glass bead retention (PR) to less than 10% of normal and gradual prolongation of the buccal mucosal bleeding time (BMBT) to approximately four times the normal value. Platelet count, volume, aggregation responses, and coagulation were normal. A packed cell volume (PCV) of less than 30% was observed in three dogs. Elevation of the PCV normalized the BMBT in two dogs, but because the PR was unchanged and the BMBT effect was temporary, anemia was not considered the primary cause of the prolonged bleeding time. There was a significant, positive correlation between BMBT and BUN, suggesting that the altered hemostasis may be related to the accumulation of urea or other uremic toxins of protein origin. The finding of a defect in PR and BMBT--tests that require normal platelet adhesion and aggregation--in azotemic dogs were platelet numbers and aggregation are normal indirectly implicates platelet adhesion as the primary hemostatic defect.

von Willebrand factor is not altered in azotemic dogs with prolonged bleeding time.

The effects of azotemia on von Willebrand factor (vWf) plasma concentration, structure, and function were studied by utilizing canine models for both uremic bleeding and type I vWf deficiency (vWd). Seventy-five percent to 80% renal mass reduction in eight mixed-breed dogs induced marked azotemia (blood urea nitrogen [BUN] 103 +/- 7 mg/dl [mean +/- SEM]; creatinine 5.8 +/- 1 mg/dl) and prolonged mean buccal mucosal bleeding time (BMBT) from 1.8 +/- 0.2 minutes to 7.0 +/- 0.4 minutes. The mean vWf plasma concentration increased from 0.88 +/- 0.11 U/ml to 1.26 +/- 0.14 U/ml. The pre- and postsurgical sodium dodecyl sulfate-agarose gel electrophoresis multimeric patterns were similar in all dogs. Administration of cryoprecipitate from pooled azotemic mixed-breed dog plasma to five Doberman pinschers with type I vWd increased the mean plasma vWf from 0.14 +/- 0.01 U/ml to 0.48 +/- 0.04 U/ml and decreased the BMBT from 7.1 +/- 0.6 minutes to 3.14 +/- 0.09 minutes. After renal mass reduction, five type I vWd Dobermans developed marked azotemia (BUN 79 +/- 8.6 mg/dl; creatinine 3.7 +/- 0.6 mg/dl) and prolonged BMBT (16.1 +/- 3.6 minutes). Findings in the eight azotemic mixed-breed dogs indicated that (1) vWf plasma levels were normal to increased in azotemic dogs; (2) vWf structure and multimeric distribution were not altered in canine azotemia; and (3) vWf was functional when placed in a non-azotemic environment. The prolongation of the BMBT in azotemic vWd dogs indicated that factors other than alteration of vWf function were responsible for the prolonged BMBT in canine azotemia.

Effect of cryoprecipitate and plasma on plasma von Willebrand factor multimeters and bleeding time in Doberman Pinschers with type-I von Willebrand's disease.

We determined whether administration of cryoprecipitate or fresh-frozen plasma (FFP) would enhance glass bead platelet retention and shorten the bleeding time in von Willebrand factor (vWf)-deficient Doberman Pinschers. Plasma concentration of vWf was < 15% of the reference value in these dogs and, on the basis of multimeric analysis of vWf, these dogs had type-I von Willebrand's disease (vWd). Concentration of vWf in cryoprecipitate (prepared from FFP of clinically normal dogs) was enriched almost 20 times, and the preparation was a concentrate of the largest and most physiologically active multimers. Administration of a dose of cryoprecipitate calculated to increase plasma vWf concentration of recipient dogs to 50 U/dl increased plasma vWf concentration in recipient dogs to about 40 U/dl. Mean buccal mucosal bleeding time (BMBT) shortened from 6.7 minutes before treatment to 3.8 minutes at 2 hours after treatment. Cryoprecipitate from donor dogs treated with deamino-8-D-arginine vasopressin (1 micrograms/kg of body weight) effectively shortened mean BMBT from 6.4 minutes to 3.1 minutes. Administration of cryoprecipitate from vWf-deficient dogs prolonged, rather than shortened, the BMBT. After FFP (450 ml) infusion, plasma vWf concentration increased in recipient dogs, but the BMBT did not shorten. Glass bead platelet retention did not change after administration of cryoprecipitate or FFP.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of the buccal bleeding time and platelet glass bead retention as assays of hemostasis in the dog: the effects of acetylsalicylic acid, warfarin and von Willebrand factor deficiency.

The study evaluated two hemostatic assays in the dog, a modified version of the buccal mucosal bleeding time (BMBT) and the platelet glass bead retention (PR), to describe the aspects of hemostasis measured by these assays. Von Willebrand factor (vWf)-deficient Doberman pinscher dogs were used in evaluating the effects of altered platelet adhesion. Normal dogs were treated with either acetylsalicylic acid (ASA) or warfarin to evaluate the effects of altered platelet aggregation and coagulation. There was significant prolongation of the BMBT and reduction of the PR in vWf-deficient dogs as compared to normal dogs. In ASA treated dogs the BMBT was slightly prolonged; the PR was significantly reduced. The change in ASA-induced BMBT did not correlate with the sensitivity of the dog platelets to arachidonic acid. In warfarin treated dogs there was no change in the BMBT; however, the PR was significantly reduced. The BMBT is a test of hemostasis that is sensitive to platelet adhesion and aggregation deficits. The PR is useful in detecting general abnormalities in hemostasis including platelet adhesion defects due to reduced vWf.

Comparison of the healing of prescrotal urethrotomy incisions in the dog: sutured versus nonsutured.

Comparison was made of the healing of sutured prescrotal urethral incisions (12 dogs) with that of nonsutured incisions (6 dogs). Comparison was also made of the healing of 5-0 polyglactin 910-sutured urethral incisions (6 dogs) with that of 5-0 polydioxanone-sutured incisions (6 dogs). Three dogs from each treatment group were euthanatized 3 weeks and 6 weeks after the surgical procedure. Surgical sites were examined grossly, and urethral circumference measurements were taken at 3 locations (surgical site, 1 cm cranially, and 1 cm caudally). Transverse sections of the surgical sites were prepared and examined by light microscopy. Hemorrhage occurred postoperatively in dogs in which the incisions were not sutured. The surgical sites from the 6 dogs in which incisions healed by second intention had more fibrosis and less inflammation than did those that were sutured. There was little difference between incisions sutured with polyglactin 910 and those sutured with polydioxanone suture material. Postoperative urethral stricture formation did not occur in any of the dogs.