A self-powered constant infusion device for use in unrestrained rats.

We developed a device that delivers fluid through a catheter at a constant rate and can be used in conscious animals to solve a variety of problems. For example, this device can be used for delivering drugs and maintaining intravascular catheter patency. The device provides infusions at low flows (1.0-1.5 ml/day), so that experimental agents may be administered with minimal volume loading of the rat. Arterial and venous catheter patency is maintained by infusion of heparinized saline through indwelling catheters attached to the device. The catheters exit from the rat in the intrascapular area and are routed through a protective spring to the device, which is suspended above the cage. The catheters may be attached to pressure transducers, blood may be sampled, and injections or infusions may be made without disturbing the rat. Because the device is self-contained, it can be suspended by a fluid-free swivel that rotates through 360 degrees, providing minimal restraint. The device has been used successfully to measure arterial and central venous blood pressures in two studies using rats.

State-dependent expression of pressure diuresis in conscious rats.

In 1967, Guyton and Coleman modeled pressure diuresis as the underlying, essential, long-term mechanism that regulates arterial pressure when sodium intake changes. Other mechanisms that influence renal function interact with pressure diuresis to achieve sodium balance and determine the blood pressure. Increases in sodium intake suppress sodium conserving mechanisms and activate natriuretic mechanisms; decreases in sodium intake have the opposite effect. If the Guyton-Coleman model is correct, then pressure diuresis should be more readily detected in animals on a high-salt diet than in animals on a low-salt diet. We measured spontaneous changes in arterial pressure and urine flow in conscious rats fed low-salt (0. 4% NaCl) and high-salt (8.0% NaCl) chow. For 10 rats fed a high-salt diet, arterial pressure and urine flow were positively correlated in 19 of 32 (59%) trials. In 10 rats fed a low-salt diet, a positive correlation was observed in 10 of 33 (30%) trials. Chi-square analysis revealed that differences in Na+ content of the diet were significantly associated with the probability of a positive relationship between blood pressure and urine flow. These results support the hypothesis that the expression of pressure diuresis across time is dependent on the state of sodium balance.

Body fluid expansion is not essential for salt-induced hypertension in SS/Jr rats.

To evaluate the importance of volume in the development of hypertension in inbred Dahl salt-sensitive rats (SS/Jr), we measured the changes in blood pressure (BP) that occurred with oral intake of food (salt) and water in rats whose body weight was permitted to increase versus those in which body weight was maintained constant with a servo-control system. We hypothesized that if volume expansion is essential in the development of hypertension, then BP would not increase if body weight was held constant. We found that oral presentation of chow containing 4% salt to SS/Jr rats caused BP to increase 32.2 +/- 2.9 mmHg over 4 days when body weight was controlled at its initial value. Plasma sodium increased from 142.0 to 145.2 meq/l during 4 days of high salt. Neither plasma volume, hematocrit, nor central venous pressure changed significantly on the high-salt diet. In contrast, the inbred Dahl salt-resistant rats (SR/Jr) did not increase their BP during body weight control when given 4% salt. This demonstrates that volume expansion is not an obligatory step in the pressure response to increased salt in SS/Jr rats. Our results obtained with oral presentation of salt, in contrast to intravenous, represent a physiological evaluation of the significance of volume changes in response to dietary salt because no potential regulatory reflexes have been bypassed.

A gravimetric method for the measurement of total spontaneous activity in rats.

Currently available methods for the measurement of spontaneous activity of laboratory animals require expensive, specialized equipment and may not be suitable for use in low light conditions with nocturnal species. We developed a gravimetric method that uses common laboratory equipment to quantify the total spontaneous activity of rats and is suitable for use in the dark. The rat in its home cage is placed on a top-loading electronic balance interfaced to a computer. Movements are recorded by the balance as changes in weight and transmitted to the computer at 10 Hz. Data are analyzed on-line to derive the absolute value of the difference in weight between consecutive samples, and the one-second average of the absolute values is calculated. The averages are written to file for off-line analysis and summed over the desired observation period to provide a measure of total spontaneous activity. The results of in vitro experiments demonstrated that: 1) recorded weight changes were not influenced by position of the weight on the bottom of the cage, 2) values recorded from a series of weight changes were not significantly different from the calculated values, 3) the constantly decreasing force exerted by a swinging pendulum placed on the balance was accurately recorded, 4) the measurement of activity was not influenced by the evaporation of a fluid such as urine, and 5) the method can detect differences in the activity of sleeping and waking rats over a 10-min period, as well as during 4-hr intervals recorded during active (night-time) and inactive (daytime) periods. These results demonstrate that this method provides an inexpensive, accurate, and noninvasive method to quantitate the spontaneous activity of small animals.

Phenotypic variation in sensorimotor performance among eleven inbred rat strains.

As a first step toward identifying the genes that determine sensorimotor ability (motor coordination) we subjected 11 inbred strains of rats to three different tests for this trait. Rats were tested at 13 wk of age to determine how long they could remain on 1) a rotating cylinder as the velocity of rotation increased every 5 s (1-direction rotation test), 2) a rotating cylinder that reversed direction every 5 s and increased velocity every 10 s (2-direction rotation test), and 3) a platform that was tilted 2 degrees every 5 s from 22 to 47 degrees (tilt test). On all three tests, rats of the PVG strain demonstrated the greatest sensorimotor ability. In contrast, rats of the MNS strain were most often represented among the group of strains that demonstrated the lowest performance on all tests. Considering all three tests, there was a 3- to 13-fold range in sensorimotor performance between the highest and lowest strains. This large divergence between the highest and lowest strains provides a genetic model that can be used to identify intermediate phenotypes and quantitative trait loci that contribute to sensorimotor ability.

Phenotypic variation in strength among eleven inbred strains of rats.

As a first step toward the long-range goal of identifying the genes that determine strength, we subjected 11 inbred strains of rats to three tests of muscular strength. The tests consisted of measuring (1) the force exerted by the rat as it was pulled by the base of the tail off a grid on the pan of a top-loading electronic balance (scale test); (2) the length of time the rat hung from a 2.5-mm-diameter U-shaped wire (wire-hanging test); and (3) the length of time the rat hung from a vertically oriented grid consisting of 4-mm-diameter rods (grid-hanging test). Six rats of each gender from each strain were tested at 12 weeks of age, once/day for 5 consecutive days. For the two tests that required use of all four limbs (the scale and grid-hanging tests), one strain performed best (DA). In contrast, on the test that required primarily the use of the front limbs (wire-hanging test), the DA was the lowest performing strain and the F344 rats the best. This differential ranking suggests that the tests selected for variance in the morphological distribution of strength among the strains. There was a 1.5- to 5.2-fold divergence observed between the males of the highest and lowest strains on the scale test and grid hanging tests. This large divergence provides the opportunity to search for intermediate phenotypes and quantitative trait loci that contribute to the different performances of the strains on these strength tests.

Evaluation of the renin-angiotensin system in a congenic renin Dahl salt-sensitive rat.

When an approximately 30 centiMorgan (cM) region of chromosome 13 containing the renin gene from the Dahl salt-resistant rat (R) was introgressed into the Dahl salt-sensitive rat (S), the resulting congenic rat (designated S.R-Ren) had a systolic blood pressure on a 2% (w/w) salt diet that was 24 mmHg lower than that of its S counterpart. Due to the large size of the transferred segment (over 30 million bp), the question remained as to whether or not the renin gene was the cause of the blood-pressure difference between the strains. We evaluated the role of the renin-angiotensin system in S.R-Ren and S rats fed a 0.05% salt diet by examining differences between strains in (1) expression of renin in three tissue types, (2) the blood-pressure response to blockade of both angiotensin-converting enzyme and angiotensin II receptors, and (3) pressure natriuresis. No differences were found in renin levels in plasma, kidney or adrenal gland between strains. The blood-pressure responses to the angiotensin-converting-enzyme inhibitor captopril and to the angiotensin II-receptor blocker saralasin in conscious S and S.R-Ren rats were similar. Furthermore, renal function, evaluated by a pressure-natriuresis index that took into account both the time and the arterial pressure needed to excrete an acute salt load, did not differ between strains. Our findings therefore fail to demonstrate a role for the renin gene in conferring lower blood pressure in the congenic rat and suggest that there is an unknown arterial-pressure-regulating locus in this 30 cM region of chromosome 13.

Hepatorenal reflex in the rat.

The possible existence of a hepatorenal reflex was evaluated in male Sprague-Dawley rats. Sodium excretion was measured in two groups of six rats each, during the first 4 h following acute ingestion of a known amount of high salt chow (2.0-2.5 mequiv. NaCl). Hourly excretion rates for sodium before surgery were compared with results following 7 days of recovery from either hepatic denervation (n = 6) or sham denervation (n = 6). Before denervation, hourly sodium excretion between the groups was not different. Following surgery for hepatic denervation, sodium excretion was 91% lower than presurgery values for the 1st h (p < 0.02) and 44% lower in the 2nd h (p < 0.04). Sham denervation caused no significant change in sodium excretion when compared with presurgery results. A test for completeness of denervation showed that norepinephrine concentration in liver tissue taken from denervated rats was 5.1 +/- 8 ng/g and that taken from sham rats was 22.8 +/- 1 ng/g (p < 0.001). These data demonstrate that the liver is essential for the normal postprandial excretion of sodium following ingestion of a high salt meal in rats.

Spontaneous changes in arterial blood pressure and renal interstitial hydrostatic pressure in conscious rats.

1. Previous work has demonstrated a positive relationship between experimentally induced changes in arterial pressure (AP) and renal interstitial hydrostatic pressure (RIHP). The purpose of the present study was to test the hypothesis that RIHP is positively correlated with the normal changes in AP that occur spontaneously in conscious rats. 2. Rats were chronically instrumented for the recording of AP (via an aortic catheter) and RIHP. RIHP was measured by implanting a Millar microtransducer, whose tip had been encapsulated in a 35 microns pore polyethylene matrix (5 mm long, 2 mm o.d.), approximately 5 mm below the renal cortical surface. 3. A total of 56 h of simultaneous analog recording of AP and RIHP was obtained from ten rats. Each 1 h segment was digitized and evaluated at frequencies of 1, 0.1, 0.02 and 0.01 Hz. 4. In forty-nine out of fifty-six of these 1 h recordings taken at 1 Hz, there were significant positive linear correlations between AP and RIHP (mean r = 0.32) with a mean slope of 0.11 mmHg RIHP/1 mmHg AP. Low-pass filtering to 0.01 Hz significantly increased the r value to 0.48. 5. These results demonstrate that spontaneous changes in AP and RIHP are positively correlated. The spontaneous coupling of AP and RIHP may be of importance in the regulation of salt and water excretion by the pressure diuresis mechanism.

Dynamic, short-term coupling between changes in arterial pressure and urine flow.

Pressure diuresis refers to the direct effect of arterial pressure (AP) on the rate of urine flow (UF). On the basis of computer modeling, pressure diuresis has been viewed as a long-term mechanism that acts to set the level of the blood volume and, thus, the steady-state AP. There are no systematic studies, however, on the rapidity with which changes in AP induce changes in UF in vivo. Therefore, we measured the delay between induced changes in AP and the subsequent change in UF. Nine anesthetized rats were instrumented with arterial, venous, and ureteral catheters. AP and UF were measured every 2 s, while acute changes in AP were induced by 1) occlusion of the aorta above or below the renal vessels; 2) brief tail pinch; or 3) intravenous administration of acetylcholine (1 microgram), phenylephrine (1 microgram), or angiotensin II (0.1 microgram). The rapidity of the urinary response to induced changes in AP was determined by calculating the delay between a significant change in AP (+/- 2 SD from baseline) and a significant change in UF. The delay averaged 6.0 +/- 0.5 s for all conditions. Also, examining the relationship between the magnitude of the induced changes in AP and the magnitude of the responses in UF revealed an exponential influence of AP on UF. That is, there were proportionately larger changes in UF compared with AP (< or = 10 times greater magnitude) in response to the experimental interventions.(ABSTRACT TRUNCATED AT 250 WORDS)

Gravimetric method for the dynamic measurement of urine flow.

The rate of urine formation is a primary index of renal function, but no techniques are currently available to accurately measure low rates of urine flow on a continuous basis, such as are normally found in rats. We developed a gravimetric method for the dynamic measurement of urine flow in anesthetized rats. Catheters were inserted directly into the ureters close to the renal pelves, and a siphon was created to collect all of the urine formed as rapidly as it was produced. Urine flow was determined by measuring the weight of the urine using a direct-reading analytical balance interfaced to a computer. Basal urine flow was measured at 2-sec intervals for 30 to 60 min. The dynamic response of urine flow to a rapid decrease in arterial pressure produced by a bolus intravenous injection of acetylcholine (0.5 micrograms) was also measured. Intrinsic drift, evaporative losses, and the responsiveness of the system to several fixed pump flows in the low physiologic range were evaluated in vitro. The gravimetric method described was able to continuously measure basal urine flows that averaged 37.3 +/- 12.4 microliters/min. Error due to drift and evaporation was negligible, totaling less than 1% of the measured urine flow. Acetylcholine-induced declines in arterial pressure were followed within 8 sec by a decline in urine flow. These data demonstrate that this new gravimetric method provides a simple, inexpensive, dynamic measurement of urine flow in the microliter/min range.

Spontaneous pressure-flow relationships in renal circulation of conscious dogs.

Renal pressure-flow (P-F) relationships are usually evaluated by measuring effects of mechanically induced changes in renal arterial pressure (AP) on renal blood flow (RBF). We devised a method allowing evaluation of renal P-F relationships during normal changes in AP occurring spontaneously in a conscious animal rather than during artificially induced changes in AP. In 18 trials in 6 dogs standing at rest, we measured average AP and RBF for each cardiac cycle over periods of approximately 35 min (approximately 3,100 cardiac cycles/trial). AP and RBF values for each cardiac cycle were expressed as percent change (%delta) from the 35-min average (beat-to-beat changes). Slope and angle of each consecutive beat-to-beat P-F change were calculated and collated into one of eight zones representing the possible physiological mechanisms responsible for concurrent, spontaneous changes in RBF and AP. In a predominance of the cardiac cycles (approximately 43%), the spontaneous AP-RBF relationship was consistent with being mediated by arterial baroreflexes (i.e., increases in AP were accompanied by proportionately greater increases in RBF during 44.4% of cardiac cycles in which AP increased, and decreases in AP were accompanied by proportionately greater decreases in RBF during 41.4% of cardiac cycles in which AP decreased). Blockade of autonomic ganglionic transmission with hexamethonium markedly attenuated this pattern. Our results indicate that renal circulation participates in moment-to-moment control of AP via a predominant baroreflex-like pattern.

Pressure diuresis and autonomic function in conscious dogs.

Pressure diuresis is thought to be a major long-term regulator of arterial blood pressure (AP). Previously, pressure diuresis has been characterized using pharmacological or surgical blockade of other mechanisms known to affect renal function. This study evaluated pressure diuresis in conscious dogs with minimal experimental interference. Dogs were chronically instrumented under pentobarbital anesthesia with aortic and urinary bladder catheters. AP was increased by 10% in resting dogs by exposure to increased light and sound intensity (arousal) for 90 min. During arousal, urine flow (UV) and Na+ excretion (UNa+ V) correlated with AP (UV vs. AP, r = 0.12, P less than 0.05; UNa+ V vs. AP, r = 0.19, P less than 0.005; 17 trials in 7 dogs). Arousal did not affect the plasma concentration of atrial natriuretic factor, suggesting that this hormone did not contribute to the correlations between UV or UNa+ V and AP. Because arousal may induce an autonomically mediated antidiuresis, studies were repeated during autonomic ganglionic blockade with hexamethonium. During autonomic blockade, the correlations between UV or UNa+ V and AP were increased (UV vs. AP, r = 0.72; UNa+ V vs. AP, r = 0.72, P less than 0.001; 6 trials in 4 dogs). We conclude that the effect of pressure diuresis on UV and UNa+ V can be detected in the intact animal, during normal operation of all the mechanisms that control renal function. Furthermore, when autonomic reflexes are blocked, the pressure-diuresis mechanism is a major determinant of UV and UNa+ V.

Support of arterial blood pressure by major pressor systems in conscious dogs.

The roles of the autonomic nervous system, vasopressin, and angiotensin II in support of blood pressure were evaluated in seven conscious, resting dogs while hydrated or dehydrated. Mean arterial blood pressure (MAP) was monitored, and the dogs were given hexamethonium to block autonomic ganglia. Thirty minutes later, they were given captopril, and after another 30 min, a vasopressin V1 antagonist, d(CH2)5TyrMeAVP, was given. The order okf administration of captopril and d(CH2)5TyrMeAVP was alternated in different experiments. Hexamethonium had no effect on steady-state MAP in either hydrated or dehydrated dogs. In hydrated dogs, the average MAP was 100 mmHg; d(CH2)5TyrMeAVP decreased MAP by approximately 12 mmHg, and captopril decreased MAP by 24 mmHg. The magnitude of the effect of these two inhibitors was independent of the order of their administration. Dehydration doubled the effect of d(CH2)5TyrMeAVP on MAP but had no effect on the response to captopril. The results suggest that 1) autonomic function is not essential for maintenance of arterial blood pressure in resting dogs; 2) during autonomic ganglionic blockade, arterial blood pressure is supported by both angiotensin II and vasopressin; and 3) dehydration increases the role of vasopressin in control of blood pressure.

Lactate oxidation by three segments of the rabbit proximal tubule.

Oxidation of [U14C]lactate to 14CO2 was measured in vitro, in nonperfused anatomically defined segments of rabbit proximal tubule (S1, proximal convoluted, and S2 and S3, proximal straight tubules). The rate of lactate oxidation was similar in S2 and S3 segments, and within the range of lactate oxidation rates measured in vivo. In contrast, the oxidation rate of S1 segments was significantly lower than that of S2 or S3. In proximal straight tubules, lactate oxidation was inhibited by incubation at 0 degrees C, or by application of 1 mM ouabain. To determine if the rate of transepithelial transport affected the rate of lactate oxidation, lactate oxidation was measured in proximal straight tubules after the lumen had been opened by perfusion with Ringer's containing 10 mM polyethylene glycol. No difference in lactate oxidation rate was observed between tubules with patent lumina and nonperfused tubules. These results suggest that the various segments of the renal proximal tubule have different metabolic characteristics, and that the rate of substrate oxidation is related to the activity of the Na+, K+-ATPase.

Active tetraethylammonium uptake across the basolateral membrane of rabbit proximal tubule.

Tetraethylammonium (TEA) uptake was measured in isolated, nonperfused rabbit S2 proximal tubule segments. The TEA cell-to-bath concentration ratio in bicarbonate-Ringer bathing medium was 10-fold higher than that predicted by passive equilibration according to the basolateral electrochemical potential, indicating that TEA uptake is an active process as reported by previous investigators. Removing bicarbonate and CO2 reduced TEA uptake to 22% of control. When bicarbonate-CO2 was replaced by HEPES-O2 or butyrate-O2, TEA uptake was unaltered, but uptake was inhibited when the major buffer anion was omitted. In the presence of 10(-4) M ouabain and bicarbonate-CO2, the TEA cell-to-bath concentration ratio was reduced to 20% of control. TEA uptake in the absence of bicarbonate and CO2 was unaltered by the addition of 10(-4) M ouabain. TEA uptake was inhibited when the bathing medium contained 0 mM K+ or 2 mM Ba2+. These data 1) demonstrate that active basolateral TEA uptake is dependent on medium buffer capacity and 2) support the concept that a portion of TEA uptake occurs via a passive equilibration pathway.

Lactate transport by Thamnophis proximal tubule: sodium dependence.

We examined the effects of experimental conditions on unidirectional lactate fluxes in isolated perfused Thamnophis proximal tubule. Fluxes were determined by adding L(+)-[U-14C]lactate to perfusate or bath as appropriate (lactate concentration = 1 mM). The lumen-to-bath lactate flux (Jlb lact) was not affected by the manipulations required to exchange perfusate or bath or by substitution of phosphate for bicarbonate buffer. During measurement of Jlb lact, lactate was ordinarily added to the perfusate only. However, addition of 1 mM lactate to the bath had no effect on Jlb lact, indicating no role for exchange diffusion in renal lactate absorption. In contrast, substitution of tetramethylammonium (TMA+) or choline for Na+ decreased Jlb lact by about 75%. The bath-to-lumen flux (Jbl lact) was also decreased by TMA+ substitution for Na+, although by only about 25%. From these and previous results we suggest that in Thamnophis proximal tubule, lactate is absorbed by an active Na+-dependent transport process that probably derives its energy from the lumen-to-cell electrochemical gradient for Na+.

Peritubular uptake of lactate by Thamnophis proximal tubule.

Lactate is absorbed in the proximal tubule and also enters tubular cells at the peritubular membrane. To characterize peritubular lactate entry, lactate uptake was measured in isolated nonperfused proximal tubules. Tubules were dissected and incubated in Ringer solution with L(+)-[U-14C]lactate and 3H2O. After incubation, the tubules were extracted, and the extracts were assayed for 14C and 3H or were chromatographed to determine the percentage of tubule 14C identifiable as lactate. Maximal steady-state tubular fluid-to-bath lactate concentration ratios (TF/B lactate) occurred by 30-60 min incubation at 25 degrees C. In 30 min, one-third of the tubules established a TF/B lactate ratio greater than 1.00, and 61.4 +/- 18.6% of tubule 14C was lactate. There was no difference in TF/B lactate ratio in proximal and distal proximal segments. Uptake was depressed at 5 degrees C. Mersalyl at 10(-4) M increased the TF/B lactate ratio and tubule water content. Probenecid at 7.5-30 x 10(-4) M also increased the TF/B lactate ratio. Distal proximal tubules incubated with [3H]PAH showed a control TF/B para-aminohippurate (PAH) ratio of approximately 30, but with 10(-4) M mersalyl the TF/B PAH ratio was approximately 1.00. Lactate uptake at the peritubular membrane occurs against an electrochemical gradient, independently from the PAH transport mechanism.

Lactate absorption in Thamnophis proximal tubule: transport versus metabolism.

Proximal tubules from the kidney of Thamnophis (garter snake) were perfused in vitro and unidirectional fluxes of lactate measured using L(+)-[U-14C]lactate, (lactate concentration, 1 mM). The lumen-to-bath (absorptive) flux (Jlb lact) significantly exceeded the bath-to-lumen flux (backflux) (Jbl lact) in each of 12 tubules (seven distal proximal and five proximal proximal). The flux ratio (Jlb lact/Jbl lact) was approximately 3.00. At flow rates of 13-16 nl/min and lactate concentration of 1 mM the net flux was about 1.60 pmol . min-1 . mm-1 in both proximal proximal and distal proximal segments. Both fluxes were decreased by perfusion at 5 degrees C. To determin e the contribution of metabolism of lactate to its absorption, Jlb lact was measured at 25 degrees C in 10 distal proximal tubules during perfusion with [14C]lactate, lactate concentration, 1 mM, and with [methoxy-3H]inulin. In these experiments, the amount of 14C found in the bath was 93% of the amount of 14C absorbed from the lumen. Chromatography showed that all of the 14C found in the bath was [14C]lactate. These data establish that in Thamnophis proximal tubule lactate absorption occurs against an electro chemical gradient by transport of the intact lactate molecule without significant metabolism.