Evaluation of Temporal Changes in Urine-based Metabolomic and Kidney Injury Markers to Detect Compound Induced Acute Kidney Tubular Toxicity in Beagle Dogs.

Urinary protein biomarkers and metabolomic markers have been leveraged to detect acute Drug Induced Kidney Injury (DIKI) in rats; however, the utility of these indicators to enable early detection of DIKI in canine models has not been well documented. Therefore, we evaluated temporal changes in biomarkers and metabolites in urine from male and female beagle dogs. Gentamicin- induced kidney lesions in male dogs were characterized by moderate to severe tubular epithelial cell degeneration/necrosis, epithelial cell regeneration and dilation; and a unique urinebased metabolomic fingerprint. These metabolite changes included time and treatment-dependent increases in lactate, taurine, glucose, lactate, alanine, and citrate as well as 9 other known metabolites. As early as 3 days post dose, gentamicin induced increases in urinary albumin, clusterin, neutrophil gelatinase associated protein (NGAL) and total protein concentrations. Urinary albumin, clusterin, and NGAL showed earlier and more robust elevations than traditional kidney safety biomarkers, blood urea nitrogen and serum creatinine. Elevations in urinary kidney injury molecule 1 (KIM-1) were less reliable for detection of gentamicin nephrotoxicity in dogs based on values generated utilizing multiple first-generation, canine-specific KIM-1 immunoassays. The metabolic fingerprint was further evaluated in male and female dogs that received Compound A which induced slightly reversible renal tubular alterations characterized as degeneration/necrosis and concurrent significant increases in urinary taurine amongst other markers. These data support further investigations to demonstrate the value of urinary metabolites, albumin, clusterin, NGAL and taurine as promising markers to enable early detection of DIKI in dogs.

Polymeric gadolinium chelate magnetic resonance imaging contrast agents: design, synthesis, and properties.

We have synthesized and evaluated five series of polymeric gadolinium chelates which are of interest as potential MRI blood pool contrast agents. The polymers were designed so that important physical properties including molecular weight, relaxivity, metal content, viscosity, and chelate stability could be varied. We have shown that, by selecting polymers of the appropriate MW, extended blood pool retention can be achieved. In addition, relaxivity can be manipulated by changing the polymer rigidity, metal content affected by monomer selection, viscosity by polymer shape, and chelate stability by chelator selection.

Etiologic significance of arginine vasopressin in motion sickness.

There is abundant evidence implicating the role of arginine vasopressin in motion sickness. The effects of AVP analogs on motion sickness were investigated in squirrel monkeys. Two specific V1 antagonists (SK&F 100273 and SK&F 103561) and three mixed V1/V2 antagonists (SK&F 101926, SK&F 105494, and SK&F 104146-D) were tested on six highly susceptible monkeys. Intravenous injections of 200 ug of a V1 antagonist abolished emesis in all six monkeys, and few prodromal symptoms remained (latency to emesis > 120 minutes, P < .001). Mixed V1/V2 antagonists failed to abolish emesis in all monkeys. However, there was a slight increase in the latency to the first bout of emesis/retching with the mixed antagonists when compared with the baseline. The dose-response relationship and rate of onset of action of the V1 antagonists (SK&F 100273) were explored. Latency to the first bout of emesis/retching increased to about twice that of the baseline when half of the effective antiemetic dose was used. The efficacy demonstrated by the specific V1 antagonists indicates that V1 receptors may modulate emesis.

The application of toxicokinetic data to dosage selection in toxicology studies.

Appropriate dosage selection is a key element in the design of toxicology studies and, hence, is the first step in the process of evaluating the safety of a new chemical or pharmaceutical agent. This demands careful consideration of exposure to the drug or chemical under investigation in relation to the pharmacological or toxicological effects it evokes in an experimental animal. Toxicokinetic data provide this perspective, but they should not be considered exclusively of other data which reflect the specific activity, potency, or metabolism of the drug or chemical in each individual test species. It is equally inappropriate to base dosage selection in toxicology studies exclusively on functional or morphological endpoints that cause effects outside the range which can be accommodated by homeostatic mechanisms and repair processes. Finally, extrapolation of toxicokinetic data across species lines can lead to serious miscalculations with respect to both dosage selection and the process of risk assessment. In each case, decisions should be based on the integration of toxicokinetic data with other measures and endpoints of biological and toxicological effect.

Effects of catechol ring fluorination on cardiovascular and renal activities of fenoldopam enantiomers.

SK&F 87516 is a potent DA1 receptor agonist with demonstrated renal vasodilator activity. SK&F 87516 is the 6-fluoro analog of another DA1 agonist/renal vasodilator agent, fenoldopam. SK&F 87516 is a racemic mixture of two enantiomers, SK&F(R)-87516 and SK&F(S)-87516, and like fenoldopam, the (R)-enantiomer is responsible for the biological activities of the racemate. SK&F(R)-87516 is diuretic in spontaneously hypertensive rats and in dogs, whereas its enantiomer, SK&F(S)-87516 is inactive. SK&F(R)-87516 increases glomerular filtration rate, an effect which may account, in part, for its diuretic activity. Unlike fenoldopam, SK&F(R)-87516 is not associated with acute hypotensive activity, tachycardia, or stimulation of the renin-angiotensin-aldosterone system. The activity differences between SK&F(R)-87516 and fenoldopam are not related to differences in DA1 agonist potency. The activity differences may be due to the differing effects of fluorine and chlorine on the electron distribution in the catechol ring, resulting in an enhanced effect of SK&F(R)-87516 at alpha 2-adrenoceptors.

Urea transport in toad skin (Bufo marinus)

Urea is transported from mucosa to serosa across the skin of the stenohaline toad, Bufo marinus, studied under short circuit current (SCC) conditions. Mucosal to serosal transepithelial urea transport (Jm-->s(urea)) was markedly and asymmetrically enhanced in toads adapted to hypertonic (150 mM) NaCl and showed saturation kinetics with an estimated Kd for urea in the bathing solution of approximately 1 mM and a maximal rate of Jm-->s(urea) = 9.4 nmol.cm-2 x hr-1, consistent with a carrier-mediated transport mechanism. Jm-->s(urea) in the skin of 150 mM NaCl-adapted toads was characterized with drugs known to affect transepithelial urea transport (J(urea)) in the urinary bladder of this species. Amiloride (10(-8)-10(-3) M) inhibited Jm-->s(urea) in a dose-dependent fashion, but with a potency only 1/1000th of that for inhibition of SCC in the same skins. Phloretin (< or = 5 x 10(-4) M) had no effect on Jm-->s(urea) or SCC; ouabain (5 x 10(-4) M) and NaCN (10(-3) M) had no effect on Jm-->s(urea) but inhibited SCC (indicating inhibition of active sodium transport) by 70 and 67%, respectively and vasopressin (10(-8) M) had no effect on Jm-->s(urea), but stimulated SCC 179% above base line. The pyrazinoyl amiloride analog, 2-pyrazinoylguanidine (10(-4) M), reported to inhibit urea transport in mammals, also had no effect on Jm-->s(urea), but inhibited SCC approximately 30%. A 1.5 unit pH gradient (m-->s or s-->m) had no effect on Jm-->s(urea).(ABSTRACT TRUNCATED AT 250 WORDS)

Antagonism of antidiuretic hormone in domestic pigs (Sus scrofa).

1. Morphological and physiological aspects of renal function are shared by humans and swine. SK&F 101926 is a potent antagonist of vasopressin binding to V2 receptors and vasopressin stimulation of adenylate cyclase activity in renomedullary membranes from both species. 2. Unexpectedly, SK&F 101926 proved to be an antidiuretic agonist in humans. Hence, we evaluated SK&F 101926 for antidiuretic agonist and antagonist activities in conscious domestic pigs. 3. During water diuresis (Uosm < 230 mOsm/kg H2O), administration of SK&F 101926 (100 micrograms/kg, i.v.) produced a maximal Uosm of 192 +/- 18 mOsm/kg H2O, a concentration not significantly different from that in vehicle-treated pigs. 4. In hydropenia, SK&F 101926 produced a modest decrease in Uosm, from 945 to 629 mOsm/kg H2O (P < 0.05). 5. In in vitro studies subsequently performed using renomedullary tissue from the same pigs, SK&F 101926 displayed high affinity for V2 receptors (Kbind = 11.8 nM) and high potency to inhibit vasopressin-stimulation of adenylate cyclase (Ki = 3.9 nM). 6. No activity of SK&F 101926 to stimulate adenylate cyclase activity was detected. 7. We conclude that, in spite of its activity in in vitro assays, SK&F 101926 is a weak antidiuretic antagonist in domestic pigs. 8. These results underscore the limited utility of assessments of vasopressin receptor binding and vasopressin-stimulated adenylate cyclase activities in vitro to predict functional antidiuretic activities in vivo.

Characterization of maximal intravenous dose volumes in the dog (Canis familiaris).

1. Pharmacological evaluation of intravenously administered drugs is frequently limited by the dose volume. Commonly used osmolytes in dosing solutions are isotonic sodium chloride (0.9%) and dextrose (5%). 2. The objective of this study was to determine the maximum volume of an isotonic intravenous (i.v.) solution to be given to conscious dogs. 3. Four dogs (2 male, 2 female), prepared for urine collections and for blood pressure and heart rate determinations, were given saline or dextrose intravenously at infusion rates of 0.3 ml(kg min)-1, 1.0 ml(kg min)-1 or 3.0 ml(kg min)-1 for 60 min; a sham-infusion was also performed. 4. Hematocrit and serum osmolality, BUN, sodium, potassium, chloride, creatinine and glucose, urinary excretion of sodium, potassium, chloride, and urine flow rate, urine osmolality, and renal creatinine and glucose clearances were determined. 5. Intravenous infusion of greater than 1.0 ml(kg min)-1 (60 ml/kg) 0.9% saline was associated with significant hemodilution and tachycardia. 6. Intravenous infusion of greater than 0.3 ml(kg min)-1 (18 ml/kg) 5% dextrose was associated with significant hemodilution, hyponatremia, tachycardia and delayed CNS disturbances associated with hyponatremia. 7. Hence, the limiting volumes of isotonic vehicles that can be administered intravenously to conscious dogs without contributing substantial effects on systemic or renal function are approx. 60 ml/kg 0.9% saline (or an equivalent non-metabolizable osmolyte) and approx. 18 ml/kg 5.0% dextrose (or an equivalent metabolizable osmolyte), administered over 60 min.

Characterization of the renal handling of p-aminohippurate (PAH) in the beagle dog (Canis familiaris).

1. The renal clearance of p-aminohippurate (PAH) is the most commonly used method for estimation of renal blood flow in laboratory animals and in humans. 2. This study characterizes the renal handling of PAH in 11 beagle dogs and describes for this species critical parameters for the valid use of the renal PAH clearance technique to estimate renal blood flow. 3. PAH was given as a priming dose plus infusion rates of 0.01-6 mg/kg per min. 4. Steady-state plasma concentrations were achieved within 60 min. 5. Urinary excretion of PAH accounted for > or = 90% of the PAH infused. 6. Plasma PAH concentrations increased linearly with dose. 7. Renal secretion of PAH exceeded filtered PAH at plasma PAH concentrations less than approx. 6 mg%. 8. The TmPAH for renal PAH secretion was approx. 0.4 mg/kg per min. 9. At PAH infusion rates above TmPAH the clearance of PAH decreased and asymptotically approached the value of the GFR.

Vasopressin antagonist inhibition of clotting factor release in the rhesus monkey (Macaca mulatta).

The antidiuretic (V2) agonist, dDAVP, stimulates release of the clotting factors von Willebrand factor (vWF) and factor VIIIc (FVIIIc) in humans. The objective of these studies was to identify and characterize dDAVP stimulation of clotting factor release in pentobarbital-anesthetized rhesus monkeys using V2 receptor agonists (dDAVP, SK&F 101926, SK&F 104146, SK&F 104244) and a V2 receptor antagonist (SK&F 105494) given i.v. dDAVP (3.0 micrograms/kg) stimulated release of vWF, FVIIIc and renin and was associated with tachycardia and blood pressure-lowering activity. The V2 receptor antagonist, SK&F 105494 (30 micrograms/kg, i.v.), had no effect on clotting factor release, heart rate or blood pressure, but prevented dDAVP stimulation of clotting factor release and tachycardia. The V2 agonists SK&F 101926, SK&F 104146 and SK&F 104244 did not stimulate clotting factor release, blood pressure, heart rate or plasma renin activity in this species at the doses tested. Thus, the rhesus monkey is a nonhuman primate species in which dDAVP stimulation of FVIIIc and vWF occurs. The results support the hypothesis that dDAVP stimulation of clotting factor release is mediated by a low-affinity, V2-like receptor mechanism. The tachycardia and renin release associated with dDAVP administration are most likely secondary to vasodilation, also mediated by a V2-like mechanism.

Investigation of anti-motion sickness drugs in the squirrel monkey.

Early attempts to develop an animal model for anti-motion sickness drugs, using dogs and cats; were unsuccessful. Dogs did not show a beneficial effect of scopolamine (probably the best single anti-motion sickness drug for humans thus far) and the findings in cats were not definitive. The authors have developed an animal model using the squirrel monkey (Saimiri sciureus) of the Bolivian phenotype. Unrestrained monkeys in a small lucite cage were tested in an apparatus that induces motion sickness by combining vertical oscillation and horizontal rotation in a visually unrestricted laboratory environment. Signs of motion sickness were scored using a rating scale. Ten susceptible monkeys (weighing 800-1000 g) were given a total of five tests each, to establish the baseline susceptibility level. Based on the anticholinergic activity of scopolamine, the sensitivity of squirrel monkey to scopolamine was investigated, and the appropriate dose of scopolamine for this species was determined. Then various anti-motion sickness preparations were administered in subsequent tests: 100 ug scopolamine per monkey; 140 ug dexedrine; 50 ug scopolamine plus 70 ug dexedrine; 100 ug scopolamine plus 140 ug dexedrine; 3 mg promethazine; 3 mg promethazine plus 3 mg ephedrine. All these preparations were significantly effective in preventing motion sickness in the monkeys. Ephedrine, by itself, which is marginally effective in humans, was ineffective in the monkeys at the doses tried (0.3-6.0 mg). The squirrel monkey appears to be a good animal model for antimotion sickness drugs. Peripherally acting antihistamines such as astemizole and terfenadine were found to be ineffective, whereas flunarizine, and an arginine vasopressin V1 antagonist, showed significant activity in preventing motion sickness.

Characterization of the renal effects and renal elimination of sulotroban in the dog.

The renal effects and renal handling of the nonprostanoid thromboxane receptor antagonist, sulotroban (4-[2-(phenylsulfonylamino)ethyl]phenoxyacetic acid), were characterized in dogs. Sulotroban was infused i.v. at 0.06, 0.2, 0.6 and 1.0 mg kg-1 min-1 (plus prime) for 180 min. Arterial blood pressure was reduced significantly during infusion of the 1.0 mg kg-1 min-1 dosage only. Diuresis, characterized by increases in both fractional and absolute urinary excretion of sodium, potassium, chloride and calcium, and decreases in urine osmolality occurred at each of the sulotroban dosages tested. The renal clearance of sulotroban exceeded the glomerular filtration rate, suggesting renal secretion of sulotroban. The transport maximum for sulotroban secretion was approximately 160 micrograms kg-1 min-1. Renal cortical slices from naive dogs accumulated [14C]sulotroban against a concentration gradient. Sulotroban accumulation was blocked by metabolic inhibitors (dinitrophenol and sodium azide) and inhibitors of organic anion transport (probenecid and p-aminohippurate), but not by inhibitors of organic cation transport (cyanine and tetraethylammonium), suggesting that tubular secretion of sulotroban is mediated by an organic anion transport system. It was concluded that: 1) decreases in blood pressure occurred only after high dosages and were associated with high plasma sulotroban concentrations; 2) diuresis occurred at all dosages and may represent a separate pharmacological action unrelated to thromboxane receptor antagonism; and 3) renal excretion of sulotroban in the dog occurs by both filtration and tubular secretion with secretion occurring via an organic acid transporter.

Up-regulation of renal adenylate cyclase-coupled vasopressin receptors after chronic administration of vasopressin antagonists to rats.

Chronic administration of vasopressin [antidiuretic hormone (ADH)] antagonists has been shown to produce a paradoxical antidiuresis in both ADH-replete and ADH-deplete (diabetes insipidus) rats. The antidiuretic effect is progressive, reaching maximal levels in 4 to 5 days, and sustained, persisting for at least 24 hr after cessation of treatment. The antidiuretic profiles associated with these antagonists do not coincide with the profiles of potent ADH agonists, arginine vasopressin and 1-deamino-8-D-arginine vasopressin. To investigate the mechanism of the antidiuretic effect of ADH antagonists, male diabetes insipidus rats were administered antagonists selective for the renal [adenylate cyclase-coupled (V2)] or pressor (phosphytidyl inositol-coupled) vasopressin receptor and urine output (volume and osmolality) and renal vasopressin receptor properties (concentration and affinity) were determined and compared to rats treated with arginine vasopressin or 1-deamino-8-D-arginine vasopressin. After acute administration, only the V2-acting antagonists were antidiuretic, but were 3 orders of magnitude less potent than 1-deamino-8-D-arginine vasopressin. Following chronic administration, all of the antagonists were antidiuretic, but the level of antidiuresis achieved with the phosphytidyl inositol-coupled vasopressin receptor-selective antagonist was 2- to 3-fold lower than for analogs with V2 activity. Maximal antidiuretic effects were realized in 5 days and were apparent at 24 hr after cessation of treatment. The antidiuretic activities and potencies of the ADH antagonists appeared to be increased following chronic antagonist administration. Finally, renal vasopressin receptor concentration was significantly elevated 24 hr after cessation of antagonist treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Yb-DTPA, a novel contrast agent in magnetic resonance imaging: application to rat kidney.

We have developed a novel contrast agent, ytterbium-diethylenetriamine pentaacetic acid (Yb-DTPA), for magnetic resonance imaging (MRI) and its mechanism of action is predominantly by the susceptibility mechanism. We have applied this contrast agent for renal MRI studies. We also demonstrate that Yb-DTPA has a renal clearance similar to that of inulin in rats and may therefore be useful for the estimation of glomerular filtration rate.

Effect of V1/V2-receptor antagonism on renal function and response to vasopressin in conscious dogs.

To characterize effects of V1- and V2-receptor stimulation on renal function, eight conscious mongrel female dogs were studied in four separate studies greater than or equal to 2 wk apart during the following six consecutive 20-min periods: 1) intrarenal administration of the full V1/V2-receptor antagonist SKF 105494 (100 ng.kg-1.min-1) during basal circulating vasopressin (VP) levels (n = 3), 2) elevation of renal arterial plasma VP concentrations by intrarenal administration of exogenous arginine VP (0.05 mU.kg-1.min-1, n = 5), 3) simultaneous administration of SKF 105494 at (100 ng.kg-1.min-1) with intrarenal administration of exogenous VP (0.05 mU.kg-1.min-1; n = 5), and 4) intrarenal vehicle alone (n = 5). When administered during conditions of basal circulating endogenous VP, the full receptor antagonist effects were limited to opposition of hydrosmotic effects of VP. Elevation of renal arterial plasma VP levels through infusion of exogenous VP resulted in decreased renal plasma flow, glomerular filtration rate, osmolar clearance, urinary flow rate, and free water clearance and increased urine osmolality. These effects were all abolished by simultaneous administration of V1/V2-receptor antagonist. These data suggest that, under basal low levels of circulating VP, VP only influences renal water excretion. However, when plasma VP concentrations are elevated, VP may contribute to renal vasoconstriction and secondarily to reduced solute excretion, in addition to its effects on free water clearance.

Vasopressin antidiuretic agonist and antagonist activity in dogs: structural and stereochemical relationship between bridge and carboxyl terminus.

A series of vasopressin analogs with various amino acid tail modifications were tested for antidiuretic agonist and antagonist (water diuretic) activity in the water-loaded indomethacin-treated and hydropenic dogs, respectively. Changing the carboxy terminus from Cys6-Pro7-Arg8-NH2 in SK&F 101926 to Cys6-Arg7-NH2 or to D-Cys6-Pro7-Arg8-NH2 or to D- or L-Cys6-Arg7-D-Arg8-NH2 reduced antidiuretic agonist and increased water diuretic activity. Replacement of the sulfur atoms in the cysteine residues with methylene groups further reduced the antidiuretic agonist activity of all carboxy terminus-modified compounds which possessed full agonist activity. It also increased the water diuretic activity of those disulfide analogs with both weak agonist and antagonist activity. These results indicate that alterations in the geometry of the hexapeptide ring and in the stereochemical relationship between the ring and the carboxy terminus of the molecule substantially modify the in vivo agonist and antagonist activity of vasopressin analogs.

Effect of cyclo-oxygenase blockade on the renal actions of vasopressin and SK&F 105494 in the rhesus monkey.

1. Vasopressin administration to pentobarbitone-anaesthetized, hydrated female rhesus monkeys resulted in dose-dependent increases in urine osmolality and decreases in urine flow. Treatment with indomethacin at a dose (5 mg kg-1, i.v.) that reduced urinary prostaglandin E2 (PGE2) excretion by at least 70% did not alter these responses. 2. The vasopressin antagonist, SK&F 105494 (Pas1,6D-Tyr(Et)2Val4Arg7D-Arg8desGly9 arginine vasopressin; 10 micrograms kg-1), caused significant rightward shifts (P less than 0.05) of both the vasopressin-urine osmolality and the vasopressin-urine flow dose-response curves. Treatment with indomethacin did not alter these responses. 3. SK&F 105494 alone or after indomethacin treatment had minimal effects on urine osmolality and urine flow. 4. The data indicate that indomethacin does not alter the antidiuretic activity of vasopressin in the rhesus monkey and that SK&F 105494 is a potent antagonist of exogenous vasopressin with minimal agonist activity.

Physiological regulation of the renal vasopressin receptor-effector pathway in dogs.

Physiological regulation of receptor-effector pathways is recognized as a significant factor determining target organ selectivity and sensitivity in several hormonal systems. Whether or not physiological regulation of the renal vasopressin (V2) receptor-effector pathway participates in the control of body fluid homeostasis is unknown. We evaluated four states likely to be associated with altered sensitivities of the renal V2 receptor-effector pathway as follows: dehydration (18-h hydropenia), volume expansion, exogenous arginine vasopressin (AVP) infusion (10 ng/kg + 0.25 ng.kg-1.h-1), and cyclooxygenase blockade (indomethacin, 2 mg/kg + 2 mg.kg-1.h-1) for effects on the antidiuretic efficacies and potencies of putative V2-receptor antagonists in conscious dogs. The antidiuretic efficacies of desGly9[Pmp1-D-Tyr(Et)2Val4]AVP [Smith Kline & French (SK&F) 101926; 0.01-1,000 micrograms/kg] ranged from that of a full agonist to that of an antagonist, depending on the physiological state studied. The vasopressin antagonist potency of SK&F 101926 was increased 150-fold in association with extracellular volume expansion and decreased by blockade of renal cyclooxygenase activity. This spectrum of activities is that anticipated for a partial agonist under conditions where receptor number and/or sensitivity of receptor-effector coupling is increased or decreased, respectively. Thus volume expansion and increased circulating vasopressin concentration are associated with effective decreases, whereas hydropenia and cyclooxygenase blockade are associated with effective increases in sensitivity of the renal V2 receptor-effector pathway in the dog kidney. We conclude that the V2 receptor-effector pathway is a site of integration of physiological mechanisms participating in the control of body fluid homeostasis in conscious dogs.

Characterization of the hemodynamic activities of fenoldopam and its enantiomers in the dog.

Fenoldopam (SK&F 82526) is a potent and selective dopamine DA-1 agonist with demonstrated renal vasodilator and antihypertensive activities in experimental animals and humans. Fenoldopam is a racemic mixture of two enantiomers, SK&F R-82526 and SK&F S-82526. The R-enantiomer is uniformly reported to be more potent than the racemate; in contrast, there is controversy regarding potency of the S-enantiomer. In these studies, the renal and systemic hemodynamic activities of fenoldopam and its enantiomers are characterized in anesthetized, phenoxybenzamine-treated dogs. The results show that the renal and systemic vasodilator activities of fenoldopam are properties of the R-enantiomer; the S-enantiomer is essentially inactive. The renal and systemic vasodilator properties of SK&F R-82526 are antagonized in a competitive fashion by the DA-1 antagonist, SK&F R-83566, but not the DA-2 antagonist, domperidone. Ganglionic blockade did not attenuate renal vasodilation associated with SK&F R-82526. Thus, the mechanism of SK&F R-82526-associated vasodilation, like that previously established for fenoldopam, is via stimulation of postganglionic DA-1 receptors.