Water-loaded Dogs Research Articles

Antidiuretic Effects of a Novel Nonpeptide Vasopressin V2-Receptor Agonist, OPC-51803, Administered Orally to Dogs

We elucidated the pharmacological properties of a novel nonpeptide vasopressin V2-receptor agonist, OPC-51803 ((5R)-2-[1-(2-chloro-4-(1-pyrrolidinyl)benzoyl-2,3,4,5-tetra-hydro-1H-1-benzazepine-5-yl]-N-isopropylacetamide), via both in vitro binding experiments incorporating canine kidney and platelet membrane fractions and in vivo experiments that would determine the compound’s antidiuretic effects after oral administration to water-loaded dogs. OPC-51803 displaced [3H]arginine vasopressin (AVP) binding to canine V2 and V1a receptors, as determined by resulting Ki values of 15.2 ± 0.6 nM (n = 4) and 653 ± 146 nM (n = 4), respectively. These data indicate that OPC-51803 was about 43 times more selective for V2 receptors than for V1a receptors. Antidiuretic studies showed that orally administered doses of OPC-51803 (0.03 to 0.3 mg · kg−1) decreased urine volume and increased urinary osmolality in a dose-dependent manner in water-loaded dogs. Intravenous OPC-51803 infusions (0.3 and 3 μg · kg−1 · min−1) did not affect renal or systemic hemodynamics in anesthetized dogs. Since these results confirm that OPC-51803 shows antidiuretic action in dogs, the compound may be useful for treating AVP-deficient pathophysiological states.

Body sodium and volume homeostasis.

maintenance of a “milieu interieur,” which allows our cells to function away from the ancient sea, is one of the most important functions of the body. Maintenance of constant extracellular osmolarity and sodium concentration depends on a precise balance between intake and excretion of sodium and

Renal effects of nitric oxide synthase inhibition in conscious water-loaded dogs.

The renal effects of the nitric oxide (NO) synthase inhibitor nitro-L-arginine methyl ester (L-NAME) were investigated in conscious dogs undergoing sustained water diuresis and replacement of urinary sodium losses. Experiments were performed with and without additional extracellular volume expansion (isotonic saline, 2% body wt). L-NAME (10 microg. kg(-1). min(-1)) infused during water diuresis decreased urine flow (2.5 +/- 0.2 to 1.5 +/- 0.3 ml/min), free water clearance (1.9 +/- 0.2 to 1.0 +/- 0.2 ml/min), and sodium excretion (4.0 +/- 1.7 to 2.1 +/- 0.6 micromol/min). Arterial blood pressure increased from 112 +/- 2 to 126 +/- 3 mmHg, but creatinine clearance did not measurably change. Plasma endothelin and vasopressin concentrations and plasma renin activity (PRA) were unchanged. Urinary endothelin concentration increased (3.4 +/- 0.8 to 6.2 +/- 1.7 pg/ml), but the excretion rate remained constant. L-Arginine infusion (0.6 mg. kg(-1). min(-1)) along with L-NAME abolished the renal effects but not the blood pressure increase. Volume expansion increased urine flow (2.5 +/- 0.4 to 5.7 +/- 0.5 ml/min) and sodium excretion (3.8 +/- 1.6 to 76.5 +/- 14.5 micromol/min). L-NAME attenuated the renal effects of volume expansion: urine flow increased to 2.8 +/- 0.7 ml/min and sodium excretion to 34 +/- 17 micromol/min. PRA decreased with control volume expansion but not during L-NAME. Urinary endothelin levels were elevated by L-NAME, decreased with volume expansion in all series, but excretion rate remained constant. Infusion of L-arginine partially reversed these effects of L-NAME. The results demonstrate that NO synthase inhibition increases blood pressure and blunts the renal responses to water and saline loading.

Evidence for cholinergic innervation in dog renal tissue.

Denervation procedures that affect the sympathetic system of the kidney, as demonstrated by norepinephrine depletion of renal tissue, increased urine volume, fractional sodium excretion, and free water clearance in anesthetized water-loaded dogs. These increases were reduced by atropine, which also blocked the increase above those basal functional levels produced by acetylcholine in both innervated and denervated kidneys. An in vitro tubular cell preparation of innervated kidneys corresponding to the outer cortex showed [3H]quinuclidinyl benzilate (QNB) binding parameters characteristic of muscarinic receptors. Denervation did not change either [3H]QNB binding parameters or the ability of inner and outer cortex cells to perform the hemicholinium-3-inhibitable, sodium-dependent choline uptake and conversion of [3H]choline to [3H]acetylcholine. This cell membrane behavior is similar to that observed in tissues where cholinergic neuronal structures are present, suggesting the existence of a cholinergic innervation of the kidney, independent of the integrity of vessel-traveling nerves. Similarly, the finding of choline acetyltransferase in renal tissue, unaffected by sympathetic denervation, seems to confirm the presence of cholinergic nerve terminals. The cholinergic system may thus contribute to the regulation of tubular reabsorption of sodium and water in some conditions.

Renal responses to atrial natriuretic factor in hydrated and hydropenic dogs

Atrial natriuretic factor (ANF 101–126) was compared to the standard diuretics, furosemide and hydrochlorothiazide, and to the vasodilator, acetylcholine in hydrated and dehydrated anesthetized dogs. ANF 101–126 (20 pmole/kg/min, ira) modestly reduced solute-free water clearance in water-loaded dogs and slightly lowered free water reabsorption in dehydrated animals. This pattern of responses most closely resembled those produce by 10 mg/kg, ira of the distally-acting diuretic, hydrochlorothiazide and a natriuretic dose of acetylcholine (2.5 μg/kg/min, ira). In contrast, the loop diuretic, furosemide (1 mg/kg, ira) drastically suppressed both free water clearance and reabsorption. ANF 101–126 produced changes in free water handling which were not readily distinguishable from those induced by either hydrochlorothiazide, a distally-acting diuretic, or acetylcholine, a vasodilator.

Role of antidiuretic activity in the inhibition of renin secretion by vasopressin in anesthetized dogs

The nature of the activity of vasopressin which is responsible for the inhibition of renin secretion was studied by comparing the effects of vasopressin (AVP) and analogs of AVP in anesthetized water-loaded dogs. Infusion of AVP (1.0 ng/kg/min) increased mean arterial pressure (MAP) and decreased heart rate (HR) and free water clearance (C H2O). Plasma renin activity (PRA) decreased from 11.9±4.7 to 3.8±1.7 ng/ml/3 hr ( p<0.05). A selective antidiuretic agonist, 1-deamino-8-D-arginine vasopressin (1.0 ng/kg/min), which had no effect on MAP or HR but was effective as AVP in decreasing C H2O, decreased PRA from 13.5±4.6 to 7.0±2.9 ng/ml/3 hr ( p<0.05). Infusion of a selective vasoconstrictor agonist, 2-phenylalanine-8-ornithine oxytocin (1.0 ng/kg/min), increased MAP and decreased HR but did not decrease C H2O or PRA. A vasoconstrictor antagonist, d(CH 2) 5Tyr(Me)AVP (10 μg/kg), completely blocked the MAP and HR responses to AVP but did not block the decrease in C H2O or PRA ( 5.9±1.8 to 2.9±1.6 ng/ml/3 hr ) ( p<0.001). Infusion of the 0.45% saline vehicle had no significant effect on MAP, HR, C H2O or PRA. These results indicate that the inhibition of renin secretion by vasopressin in anesthetized water-loaded dogs is due to its antidiuretic activity.

Failure of delta-aminolaevulinic acid and porphobilinogen to alter renal salt and water excretion in the dog.

We tested the hypothesis that the hyponatremia commonly observed in patients with acute hepatic porphyria is mediated by the presence of aminolaevulinic acid (ALA) and (or) porphobilinogen (PBG) at the level of the renal tubule to alter water handling, or at the level of the nervous system to release antidiuretic hormone (ADH). When these substances were infused intravenously into hydropenic dogs, there was no effect on systemic hemodynamics or renal function. Neither ALA or PBG, when infused directly into the renal circulation of water-loaded dogs, could effect a decrease in urinary flow, osmolar clearance, or renal perfusion. Similar results were obtained when these substances were infused directly into the renal circulation of hydropenic dogs. When ALA was administered directly into the carotid artery of nine water-loaded dogs and eight hydropenic dogs, there was again no effect on systemic hemodynamics or renal function. These data suggest that ALA (and PBG) have no effect on the renal excretion of sodium or water, and ALA does not cause the release of ADH. The hyponatremia of porphyria may be related to factors other than the elevated plasma levels of ALA and PBG.

Effect of somatostatin on renal water handling in the dog.

When somatostatin was infused into the left renal artery of anaesthetized, hydropenic dogs in doses ranging from 1 to 10 micrograms/min, it produced an increased flow of a more dilute urine from the ipsilateral kidney. Similar infusions in dogs undergoing a maximal water diuresis had no effect. If aqueous antidiuretic hormone (ADH) was administered intravenously into water-loaded dogs prior to the intraarterial infusion of somatostatin, this latter peptide was able to produce an augmented flow of a more dilute urine from the ipsilateral kidney. If the left kidney was made to excrete a concentrated urine in the face of maximal water loading by restricting arterial perfusion, then the infusion of somatostatin had no effect on urinary dilution, though this peptide could increase water excretion in hydropenic dogs when the left kidney was similarly restricted as to arterial inflow. In dogs undergoing a water diuresis that were given cyclic AMP (4 mg/min) into the left renal artery, a decrease in ipsilateral water excretion was observed. The subsequent infusion of somatostatin produced no urinary dilution. We conclude that somatostatin increases renal water excretion by antagonizing the ADH effect on the renal tubule, and that this event probably occurs at a pre-cAMP site within the cell.

(3-aryl-1,2-benzisoxazol-6-yl)oxy]acetic acids. A new diuretic series.

A series of [(3-aryl-1,2-benzisoxazol-6-yl)oxy]acetic acids was synthesized and tested for diuretic activity in saline-loaded mice and in conscious, water-loaded dogs. The structural requirements for good diuretic activity in both mice and dogs were found to be very specific. In summary, the compounds with the best diuretic activity (13i, 13q, and 13ff) were substituted with a 2-fluorophenyl group at the 3 position and chlorine or bromine at the 7 position. Compound 13ff, [(7-bromo-3-(2-fluorophenyl)-1,2-benzisoxazol-6-yl]oxy]acetic acid (HP 522), was found to be moderately uricosuric in chimpanzees and was selected for further development.

Urinary concentrating ability during dehydration in the absence of vasopressin.

Despite the apparent absence of vasopressin (ADH), Brattleboro homozygotes [diabetes insipidus (DI) rats] can concentrate their urine when deprived of drinking water. Since other investigators have shown that reducing glomerular filtration rate (GFR) improves the concentrating ability of water-loaded dogs, the present studies were undertaken to quantify the magnitude and time course of changes in GFR during dehydration. Clearance experiments were performed in 10 conscious DI rats before and following 3, 6, 9, 12, 15, and 24 h of dehydration. Urine osmolality increased from 155.0 +/- 12.6 (SE) to 696.7 +/- 8.4 mosmol/kg H2O after 24 h. GFR averaged 984.3 +/- 79.6 microliters . min-1 . 100 g body wt-1 in the control phase, fell to about 80% of this value over the first 12 h of dehydration, and then declined to 27% at 24 h. The rats lost 20% of their body weight over the 24 h. The osmolality of the papillary tip averaged 896 +/- 44 mosmol/kg H2O at 24 h compared to a control value of 493 +/- 28. The lack of osmotic equilibration between urine and papillary interstitium suggests that dehydration did not appreciably increase the water permeability of the distal nephron. These experiments clearly show a progressive decline in GFR as urine becomes concentrated during dehydration in the absence of ADH; these events may or may not be causally related.

Arginine vasopressin metabolism in dogs. I. Evidence for a receptor-mediated mechanism.

The plasma clearance rates (PCR) of arginine vasopressin (AVP), and iodinated AVP (125I-AVP) were determined after pulse injection in conscious water-loaded dogs. Both the PCR and the apparent initial volume of distribution were significantly greater for AVP than for the biologically inactive iodinated AVP 37.4 +/- 4.8 ml/kg per min vs. 6.7 +/- 0.8 ml/kg per min (P less than 0.001) and 12.7 +/- 0.9% body wt vs. 7.1 +/- 0.4% body wt (P less than 0.001). AVP clearance was then determined by the constant-infusion technique at doses that produced equilibrium AVP concentrations within and above the physiological range. AVP-PCR was 37.4 +/- 7.1 ml/kg per min at 34 microU/kg per min, which was comparable to that after pulse injection (P less than 0.9). AVP clearance fell progressively, and urine osmolality progressively increased with increasing AVP infusion rates to plateau values at 136 microU/kg per min; a strong negative correlation was observed between mean AVP-PCR and urine osmolality (r = -0.993). The data suggest a relationship between the biological activity of AVP and its clearance. It is proposed that plasma membrane receptors may mediate a portion of the metabolic clearance of AVP.

A bilateral antidiuresis to renal artery infusion of prostaglandin E1 in dogs treated with phenylbutazone.

1. In acute experiments, high levels of endogenous prostaglandins, provoked by operative stress, could obscure or alter the actions of infused prostaglandins on the kidney. For this reason we decided to compare the effects of infusing prostaglandin E(1) into the renal artery of the dog before and after the administration of phenylbutazone, a prostaglandin synthetase inhibitor.2. Infusion of prostaglandin E(1) into the left renal artery of the pre-phenylbutazone treated dog undergoing a mannitol diuresis increased renal plasma flow, glomerular filtration rate and the excretion of salt and water. The findings are in general agreement with those reported by others.3. Following phenylbutazone administration the vascular and saluretic actions of prostaglandin E(1) were unchanged but a reduced diuretic effect was observed. The response to a low dose of prostaglandin E(1) (0.05 mug/min) was reduced from 1.46 +/- 0.15 to 0.96 +/- 0.16 ml./min (P < 0.001) and the response to a high dose (0.5 mug/min) from 1.82 +/- 0.19 to 0.99 +/- 0.31 ml./min (P < 0.002).4. A significantly less dilute urine was excreted during prostaglandin infusion in the dog after phenylbutazone treatment than before. The reduction in the diuretic response was of the same order as the decrease in the free water clearance response, while the increase in osmolar clearance was unchanged.5. In water-loaded dogs treated with phenylbutazone, infusion of prostaglandin E(1) into the left renal artery had a biphasic effect on urine output from the left kidney. An initial diuretic response to a low dose of prostaglandin E(1) disappeared with the infusion of higher doses, and antidiuresis developed in the immediate post-infusion period.6. As prostaglandin was infused into the left kidney progressive antidiuresis was seen in the non-infused right kidney.7. It is concluded that endogenous prostaglandins do not obscure or alter the vascular and saluretic actions of intrarenal prostaglandin E(1). The findings question the proposed link between the vascular and saluretic actions of this compound.8. It is suggested that the reduced diuretic effect of prostaglandin E(1) in series no. 1, and the antidiuresis in the water-loaded dogs, are caused by the release of endogenous ADH. It is further suggested that phenylbutazone unmasks this release by removing the endogenous prostaglandins. If these deductions are correct, the findings support the anti-ADH role assigned to endogenous prostaglandins by Anderson, Berl, McDonald & Schrier (1975).

Renal response to vasopressin after inhibition of prostaglandin synthesis.

The renal effects of vasopressin (VP) in water-loaded and hydropenic conscious dogs were examined with and without the previous administration of prostaglandin (PG) synthesis inhibitors (indomethacin and meclophenamate). The parameters studied were: urinary output, sodium and potassium excretion, plasma and urinary osmotic concentration, total renal blood flow, mean arterial pressure, heart rate, and the clearance and extraction ratio of p-aminohippuric acid and inulin. The infusion of VP caused antidiuresis and marked saluresis during water diuresis, whereas it was found to be diuretic and saluretic in hydropenic animals. Inhibition of PG synthesis greatly enhanced the antidiuretic activity of VP and abolished its saluretic and diuretic actions. Changes in renal water and solute excretion and changes in the hemodynamic parameters are uncorrelated. It is concluded that intrarenal PGs may play an important role in modulating the renal action of VP.

Left atrial receptors and the antidiuretic hormone.

1. Stimulation of left atrial receptors by distending balloons located at the junction between the pulmonary veins and the left atrium and in the body of the left atrium resulted in a diuresis in anaesthetized dogs.2. In the same dogs the diuresis was not consistently associated with a reduction in the antidiuretic activity of plasma as compared with that during the initial control period. The antidiuretic activity of dog plasma was assayed in water-loaded rats anaesthetized with ethanol.3. Intravenous injections of vasopressin suppressed a water diuresis in water-loaded dogs anaesthetized with chloralose. The changes in antidiuretic activity of dog plasma which resulted from these injections were reliably detected by the bio-assay in the rat.4. It is concluded that the diuretic response to the stimulation of left atrial receptors is not accompanied by a reduction in the plasma antidiuretic hormone level.

Neuroendocrine and behavioral correlates of constitutional differences.

Consistent and stable individual differences were observed in the renal responses of water-loaded dogs during the development of Pavlovian conditional motor defense reflexes by electrocutaneous reinforcement. Some dogs developed persistent and intense conditional antidiuretic responses (with a high urine osmolality) to the entire Pavlovian conditioning room complex, whereas other dogs failed to exhibit conditional antidiuresis. In contradistinction to the conditional motor defense reflexes, the conditional antidiuretic responses showed poor differentiation, thus demonstrating the phenomenon of schizokinesis discovered by Gantt. In addition to conditional water retention, the antidiuretic dogs also exhibited persistent hyperpnea, copious salivation and tachycardia. The physiologic responses of the antidiuretic dogs to the Pavlovian conditioning room resemble those described by Walter B. Cannon for a “fight or flight” response,i.e., physiologic reactions of animals engaged in intense muscular effort. The antidiuresis serves the function of conserving body water so that it may be available for evaporative cooling in order to maintain thermal homeostasis. Since our experimental dogs cannot engage in a consummatory “fight or flight” response, their physiologic reaction to a stressful psychologic environment can be considered maladaptive. Pharmacologic analysis suggested that the antidiuretic responses may involve cholinergic transmitters.

EFFECT OF UREA OSMOTIC DIURESIS ON POTASSIUM EXCRETION.

An increased solute load per remaining functioning nephron may enhance K excretion and aid in maintaining K balance in chronic renal failure. An analogous model was sought by inducing progressive osmotic diuresis with urea in a) water-loaded dogs with minimal K excretion, b) dogs receiving K infusions at intermediate rates of 207–280 µEq/min, and c) dogs receiving K infusions at maximal tolerated rates of 400–600 µEq/min. Urea diuresis significantly augmented K excretion in experiments in which basal K excreted-to-K filtered ratios (KE:KF) were minimal (.02–.03) or intermediate (.20–.73), but when control ratios approached 1.0, urea loading became progressively less effective. The slight increment in K excretion with low basal KE:KF ratios (19 µEq/min) suggested partial inhibition of proximal reabsorption; the more marked increment in K excretion at intermediate KE:KF levels (130 µEq/min) is consistent with enhancement of K secretion following increased delivery of Na to the distal exchange sites. Once distal K secretion was maximally stimulated by K loading, however, it was not further affected by solute diuresis or augmented Na excretion.

OSMOLALITY, BICARBONATE CONCENTRATION, AND WATER REABSORPTION IN PROXIMAL TUBULE OF THE DOG NEPHRON.

Micropuncture and microanalytical techniques were used to study the effect of antidiuresis and water diuresis on osmolality, bicarbonate concentration, and water reabsorption in the proximal tubule of the dog nephron. Samples collected during antidiuresis and water diuresis remained isotonic to plasma throughout the first 50% of the proximal convoluted tubule. Mean bicarbonate concentrations of 16 mEq/liter and 17 mEq/liter were found in the middle third of the tubule during antidiuresis and water diuresis, respectively. These values were slightly less than the plasma concentration of 22 mEq/liter. Proximal tubular fluid samples for inulin concentration were collected during antidiuresis, water diuresis, and during vasopressin infusion in water-loaded dogs. A mean tubular fluid to plasma (TF/P) inulin ratio of 2.3 was found in the middle third of the proximal tubule during antidiuresis. This value is significantly different ( P &lt; 0.01) from a mean of 1.6 in the same portion of the tubule during water diuresis. Vasopressin administration to hydrated dogs returned the TF/P inulin ratio in the middle third of the proximal tubule to 2.0. These results suggest that vasopressin stimulated Na reabsorption in the proximal tubule of the dog nephron.