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1Department of Pathology, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131; and 2Departments of Obstetrics, Gynecology, and Reproductive Sciences, and Cell Biology and Physiology, University of Pittsburgh School of Medicine and Magee-Womens Research Institute, Pittsburgh, Pennsylvania 15213
Submitted 22 May 2003 ; accepted in final form 18 June 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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6 ng/ml). The peak response was observed during the 0.4 µg/h infusion rate (serum rhRLX of 11 ng/ml), which was comparable to our previous work using a 4.0 µg/h (serum rhRLX of 20 ng/ml). In contrast, a dose of 40 µg/h was ineffective (serum rhRLX of 80 ng/ml). When 4.0 µg/h rhRLX was administered by osmotic minipump for shorter periods (
24 h), renal circulatory and osmoregulatory changes were observed by 6 h. After removal of the osmotic minipump, these changes persisted for at least 12 h, but they were fully restored by 24 h. Even briefer administration of 4.0 µg/h rhRLX by intravenous infusion showed an onset of action in the kidney by 1-2 h. In contrast, the 40 µg/h dose of rhRLX elicited minimal effects, and comparable to our earlier report, 4.0 µg/h purified porcine relaxin was also relatively ineffective during short-term intravenous administration. In conclusion, the effect of relaxin on the renal circulation and osmoregulation is biphasic, insofar as high doses are relatively inactive, and the onset of action is more rapid than previously believed. These findings may be important to consider when evaluating relaxin in the treatment of renal disease. glomerular filtration; renal circulation; pregnancy
2 days) to conscious, nonpregnant intact or ovariectomized female (6) and male (5) rats. In fact, by neutralizing endogenous circulating relaxin with specific antibodies or eliminating relaxin from the circulation by ovariectomy, the renal circulatory and osmoregulatory changes of pregnancy are completely abrogated (14). An important mechanism for pregnancy (relaxin)-induced renal vasodilation, hyperfiltration, and reduced myogenic reactivity of small renal arteries involves upregulation of vascular matrix metalloproteinase-2 activity, which, in turn, processes big endothelin (ET) to ET1-32, thereby activating the endothelial ET-B receptor and nitric oxide (3-7, 11, 15).
Previous work demonstrating the renal vasodilatory action of relaxin employed an infusion rate of 4 µg/h for as long as 5 days that produced serum concentrations of 20 ng/ml, i.e., more or less equivalent to gestational day 11 of rat pregnancy when renal vasodilation is virtually maximal (2, 5, 6, 11, 16). However, both glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) return toward nonpregnant levels on gestational days 18-20, when serum relaxin levels are 80-120 ng/ml (2, 16). Therefore, one objective here was to determine the threshold dose of relaxin that induces renal vasodilation and hyperfiltration in conscious rats, and another was to test whether the renal effects of relaxin are biphasic, i.e., capable of producing renal vasodilation at lower doses, whereas being relatively inactive at higher doses.
An earlier report (6) showed that intravenous infusion of porcine relaxin (pRLX) for 4 h failed to significantly alter GFR or ERPF. In contrast, 3-h incubation of small renal arteries with recombinant human relaxin (rhRLX) in vitro produced a reduction in myogenic reactivity (Novak and Conrad, unpublished observations). Therefore, another objective of the present investigation was to revisit the time course of relaxin-induced renal vasodilation and hyperfiltration in conscious rats by using both pRLX and rhRLX with particular emphasis on shorter infusion times (1-6 h). Finally, the time required for restoration of GFR and ERPF after cessation of relaxin administration is presently unknown, and thus our last goal was to examine this issue.
In short, the objective of the present investigation was threefold: to determine the dose response and time course of relaxin-induced renal vasodilation, hyperfiltration, and osmoregulatory changes, as well as to determine the time required for the recovery of these variables after cessation of relaxin administration.
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METHODS |
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Before surgical procedures, rats were habituated to a Plexiglas experimental cage (Braintree Scientific, Braintree, MA) from 1 to 5 h/day for a minimum of 5 days. The rats were then retrained 7-10 days after surgery and before the onset of the experimental protocol.
The surgical preparation has been previously described in detail (2). Briefly, under halothane anesthesia and with the use of the aseptic technique, catheters were implanted in the abdominal aorta and inferior vena cava via the femoral artery and vein, respectively, and externalized subcutaneously between the scapulae. These catheters were filled with a 1:1 mixture of 50% dextrose and heparin (1,000 U/ml) to keep them patent. A catheter made of Silastic-covered stainless steel tubing was secured in the bladder with a purse-string suture and exteriorized through the ventral abdominal wall. The stainless steel cannula was then stoppered with a Silastic-covered pin (18-gauge blunted needle), thus allowing the animals to urinate through the urethra while in their home cages. After surgery, the rats were returned to their home cages, provided with a 5% dextrose solution for hydration and additional nourishment for 48 h, and allowed 7-10 days of recovery before experimentation. All catheters were assembled in house and gas sterilized before surgery.
Measurement of renal function. After 7-10 days of recovery from surgery, renal function and mean arterial blood pressure (MAP) were measured in age-matched, chronically instrumented conscious rats. The rats were placed in the experimental cage, and after the obturator was removed from the bladder catheter, the bladder was rinsed with a small amount of sterile water and the cannula was extended with a short polyethylene tube that led to a collection container. Approximately 100 µl of blood were collected from the femoral arterial catheter for measurement of plasma osmolality (Posmol), sodium, and hematocrit. The arterial catheter was then attached to a Statham pressure transducer (Gould P23 ID) and a Gilson Universal amplifier and chart recorder (model ICT-2H Duograph, Gilson Medical Electronics, Middleton, WI). MAP was continuously recorded. The arterial catheter was also used for collection of blood samples at the midpoint of each timed urine collection. The femoral venous catheter was attached to a Sage infusion pump (model 355, Orion Research, Cambridge, MA) for delivery of inulin (In; Sigma Products) and p-aminohippurate (pAH; p-AH sodium, Merck, Sharpe, and Dohme, West Point, PA) diluted with sterile Ringer solution. Timed urine collections were made in small preweighed tubes. Urine volume was measured gravimetrically. This technique of urine collection proved reliable: after reaching steady-state conditions in the present study, the excretion rates of In and pAH were 95 ± 3 and 98 ± 2%, respectively, of their infusion rates (n = 60 rats).
To begin, a bolus of In (0.2 ml of 10% In/100 g body weight) and pAH (0.1 ml of 2% pAH/100 g body weight) was slowly given over 5 min, followed by constant infusion (0.4 and 0.1 mg · min-1 · 100 g body weight-1, respectively; flow rate of 14 µl/min). After a 60-min equilibration period, three baseline urine collections of 30 min each with midpoint blood collections of 200 µl each were obtained. After centrifugation of blood and removal of plasma, the red blood cells were suspended in an equal amount of Ringer solution and returned to the rat to avoid volume depletion.
Experimental protocols. After baseline measurements of renal function and MAP, the rats were implanted with an Alzet minipump 1003D (Durect, Cupertino, CA), which delivered various doses of rhRLX (0.04, 0.15, 0.40, or 40 µg/h). The Alzet osmotic minipump model 1003D (3-day delivery) containing rhRLX or vehicle (20 mM sodium acetate, pH 5.0) was implanted subcutaneously by using halothane anesthesia and aseptic technique. Renal function was again assessed at 48 h. A 1-ml blood sample was collected at the end to measure rhRLX levels.
For the time course studies, baseline renal function was measured as described above. The rats were then randomly divided into pairs; one received an osmotic minipump delivering a known vasoactive dose of rhRLX (4 µg/h; Refs. 5, 6, 11, 15), and the other received a minipump filled with vehicle. Renal function was measured again at 6, 12, and 24 h in each pair of rats. These pumps were not primed in vitro before implantation. Therefore, infusion durations may have actually varied from 2-6, 8-12, and 20-24 h. After the 20- to 24-h time point, the osmotic minipumps were removed by using halothane anesthesia, a procedure that required <5 min. Renal function was then measured 12, 24, and 48 h after the removal of the minipumps.
In other experiments, relaxin was administered intravenously into the femoral venous catheter concurrently with the In or pAH infusion by using a Tygon manifold. To begin, after a 60-min equilibration period, three 30-min urine collections with midpoint blood samples were made to establish baseline renal function. Then, a bolus (pRLX and rhRLX: 2.0 µg/0.5 ml Ringer solution or 0.5 ml of Ringer solution alone) was given over 5 min, followed by a constant intravenous infusion of pRLX (4 µg/h), rhRLX (4 or 40 µg/h), or Ringer solution by Sage infusion pump. The RLX bolus was calculated to produce circulating levels ranging from 10 to 30 ng/ml, depending on the theoretical volume of distribution. Six 60-min urine collections and midpoint blood samples were obtained during the infusion of relaxin or vehicle.
Analytical techniques. Plasma and urine In and pAH were assayed by standard techniques, as reported previously (2). Posmol was measured by a freezing-point depression instrumentation osmometer (model 3MO, Advanced Instruments, Needham Heights, MA). The levels of rhRLX in serum were measured by a quantitative ELISA immunoassay (11, 17). Briefly, a 96-well microtiter plate (Falcon polystyrene U bottom, Becton Dickinson, Franklin Lakes, NJ) was coated overnight with affinity-purified anti-rhRLX rabbit polyclonal antibody. Standards and rat sera were diluted in PBS containing Tween-20, BSA, and goat IgG (Sigma Chemical, St. Louis, MO), and 100 µl were added to each well in duplicate or triplicate, respectively. After overnight incubation at 4°C, the wells were washed, and 100 µl of affinity-purified, peroxidase-conjugated anti-rhRLX rabbit polyclonal antibody were added to each well, followed by incubation at room temperature while shaking for 3 h. After appropriate washing, tetramethylbenzidine solution was added, and color development was stopped with phosphoric acid. Plates were read on a microtiter plate spectrophotometer (Labsystems Multiskan RC, Fisher Scientific) at a wavelength of 450 nm with a reference wavelength of 630 nm. After correction for the blank, sample concentrations were determined by entering data into a four-parameter logistic curve-fitting program. Concentrations were corrected for the dilution factor and reported. Validation of the assay included spiking rat plasma at three levels: 50, 200, and 500 pg/ml, which yielded recoveries of 98, 95, and 102%, respectively. Low-, medium-, and high-level controls (n = 10 each) in the range of the standard curve (11.72-750 pg/ml) were then used to validate the intra-assay coefficient of variation. These values were 5.1, 1.9, and 3.5%, respectively. The interassay variation was 6.9, 12.1, and 13.1%, respectively, when the low, medium, and high controls were evaluated over 10 plates. The detection limit of the assay was 5 pg/ml. This assay does not detect either rat or pRLX.
Preparation of drugs. pAH (Merck & Co., West Point, PA) and IN (Sigma Chemical) were freshly prepared on the morning of the experiment by using Ringer solution as diluent. In, characteristically insoluble at ambient temperature, was prepared for infusion by heating a 15-ml aliquot in a boiling water bath for 10 min. When diluted in Ringer solution and mixed with pAH, it remained in solution throughout the experiment. A model 1003D, 3-day osmotic minipump (Durect, Cupertino, CA) was used to deliver the rhRLX or vehicle (20 mM sodium acetate, pH 5.0). The rhRLX (from Elaine Unemori, Connectics, Palo Alto, CA) was supplied as a 5.0 mg/ml solution in 20 mM sodium acetate, pH 5.0. For intravenous infusion, both rhRLX and pRLX (the latter provided by O. David Sherwood as a lyophilized preparation) were diluted in Ringer solution.
Statistical analysis. Data are presented as means ± SE. Baseline renal clearances and clearances during the various doses of rhRLX infusion were averaged. The data was analyzed by using two-factor repeated-measures multivariate ANOVA. If significant main effects or interactions were observed, then group means were compared by using Tukey's test (18). P < 0.05 was considered statistically significant.
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RESULTS |
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20 ng/ml, a level found on gestational day 11, when pregnancy-induced renal vasodilation is virtually maximal in this species (2, 5, 6, 11, 16). Of interest, the influence of rhRLX dosage on renal function was biphasic, because after 48 h of rhRLX administration at 40 µg/h, GFR, ERPF, and effective renal vascular resistance were comparable to baseline (Fig. 1, A-C). The Posmol was significantly decreased during the 0.15 and 0.4 µg/h but not during the 40 µg/h infusion rates compared with baseline (Fig. 1D).
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GFR and ERPF were significantly increased as early as 6 h after the infusion of rhRLX by subcutaneous osmotic minipump at 4 µg/h was begun (Fig. 2, A and B). By 12 h, GFR and ERPF were at their peak and comparable to our earlier studies in which we measured renal function after 2 and 5 days of rhRLX administration (5, 6, 11). Serum rhRLX concentrations at 6, 12, and 24 h were 10.7 ± 1.1, 14.0 ± 2.4, and 16.8 ± 2.7 ng/ml, respectively. Posmol was decreased at all time points compared with baseline (Fig. 2C). After the 24-h time point, the osmotic minipumps were removed. Renal function and Posmol returned to levels observed in vehicle-infused rats by 24 h (Fig. 3).
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To investigate even shorter durations of infusion, rhRLX or pRLX was administered through the femoral venous catheter. rhRLX produced a significant increase in GFR and ERPF by as early as 1-2 h after the intravenous infusion was begun compared with vehicle administration (Fig. 4, A and B). Here, serum rhRLX concentration averaged 6.8 ± 1.0 ng/ml after6hof infusion. pRLX was considerably less effective than rhRLX when administered at the same infusion rate (4 µg/h).
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Finally, renal function was measured at baseline and during intravenous infusion of vehicle or 40 µg/h rhRLX. This high dose of rhRLX modestly increased GFR relative to vehicle and baseline at 1, 3, and 4 h, but then returned to baseline values by 5 and 6 h (Fig. 5A). ERPF was significantly increased relative to vehicle infusion only at the 2-h time point (Fig. 5B). Serum rhRLX averaged 47.9 ± 12.1 ng/ml after6hof infusion.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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0.15 µg/h, which produced an average serum concentration of 6 ng/ml, whereas the peak response was observed during an infusion rate of 0.4 µg/h (serum rhRLX of 11 ng/ml). The latter was comparable to the renal circulatory effects of the 4.0 µg/h infusion rate previously reported (5, 6, 11). Higher infusion rates of 40 µg/h, which achieved a mean serum concentration of 80 ng/ml did not increase renal function. This serum level is comparable to that observed during late pregnancy in the rat, when renal function is returning to prepregnant values (2, 16). One possible explanation (among many) for the biphasic effect of relaxin is that high serum concentrations preferentially vasodilate other organ beds such as the uterus (13). Thus the kidneys may be effectively deprived of blood flow during late gestation in the rat.
We previously reported that renal function was unaffected during a 4-h period after a 2.0-µg intravenous bolus and 4 µg/h infusion of pRLX (6). This result, however, conflicted with our more recent work showing that incubation of small renal arteries with rhRLX in vitro for 3 h reduced myogenic reactivity (Novak and Conrad, unpublished observations). Therefore, we revisited the issue of time course. When rhRLX was administered by subcutaneous osmotic minipump at 4.0 µg/h, the onset of action in the renal circulation was 6 h. Of additional interest, when the osmotic minipumps were subsequently removed, renal vasodilation and hyperfiltration persisted for at least 12 h with complete restoration of renal function observed by 24 h.
After a 2.0-µg intravenous bolus and 4 µg/h infusion of rhRLX, significant renal vasodilation and hyperfiltration were observed by as early as 1 to 2 h, which then persisted throughout the 6-h period of study. However, at the same dose, pRLX was relatively inactive between 1 and 6 h of infusion, comparable to our laboratory's earlier report (6). Nevertheless, pRLX is equally effective as rhRLX on the renal circulation when administered for 2 or 5 days by osmotic minipump (6). Thus pRLX is considerably less potent than rhRLX when administered acutely but is comparable when administered chronically. Presently, we do not have a ready explanation for these discrepant results between the two relaxin preparations during acute administration.
Although the infusion of rhRLX for 6 h at 40 µg/h significantly increased GFR at the 1-, 3-, and 4-h time points, and ERPF at the 2-h time point relative to vehicle, these increases were at best modest compared with the 4 µg/h infusion rate. Indeed, ERPF was significantly lower at the 5-h time point during the 40 µg/h infusion of rhRLX relative to vehicle. By and large, these data on short-term infusion of rhRLX at 40 µg/h are consistent with those obtained after 2 days of infusion, i.e., a minimal effect on renal function (if any) compared with infusion rates of 0.4 or 4.0 µg/h.
In addition to mediating the renal circulatory adaptations to pregnancy (14), relaxin also initiates the osmoregulatory adjustments (14). After 2 days of rhRLX administration, the threshold dose for a decline in Posmol was 0.15 µg/h, which was identical to the threshold dose for the renal circulatory changes. Further reduction was observed during the 0.4 µg/h infusion that produced levels of Posmol comparable to the 4 µg/h infusion rate, as previously reported (5, 6, 11). Of note, the 40 µg/h dose failed to significantly reduce Posmol. This result was unexpected, because Posmol remains decreased during late gestation in rats in the face of high serum concentrations of relaxin that are comparable to those reached with the 40 µg/h infusion (2, 12). One potential explanation for this apparent inconsistency is that the moderate levels of serum relaxin observed during early to midgestation in the rat initiate the decline in Posmol (14), whereas the maintenance of hypoosmolality with advancing gestation is independent of the hormone. During short-term administration of rhRLX by subcutaneous osmotic minipump at 4.0 µg/h, the Posmol was significantly decreased after as little as6hof infusion. On cessation of infusion, Posmol remained significantly decreased at 12 h but was restored by 24 h.
In view of both the renal vasodilatory (6) and matrix-degrading (17) attributes of relaxin, it has been suggested that the hormone may be efficacious in the treatment of various renal diseases (1). So far, the hormone has been tested in several rat models of renal disease: the bromoethylamine model of renal interstitial fibrosis (9), renal mass reduction by infarction or surgical excision of both poles (8), and cyclosporin nephrotoxicity (10). In all cases, amelioration of renal injury was observed. The findings of the present study related to the biphasic dose response of relaxin and relatively rapid onset of action may facilitate further evaluation of the hormone in the treatment of both chronic and acute renal diseases.
In summary, the present results provide further insight into the action of relaxin on the renal circulation and osmoregulation in conscious rats. Surprisingly, the onset of action is considerably more rapid than previously believed (6), i.e., within hours, and the dose response is biphasic, with high doses being relatively inactive. Overall, both the dose response and time course effects of rhRLX on renal function and Posmol were comparable.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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