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Relaxin is a potent renal vasodilator in conscious rats
Lee A. Danielson,1 O. David Sherwood,2 and Kirk P. Conrad3
1Department of Pathology, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131, USA2Department of Molecular and Integrative Physiology and College of Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA3Departments of Obstetrics, Gynecology, and Reproductive Sciences, and of Cell Biology and Physiology, University of Pittsburgh and Magee-Womens Research Institute, Pittsburgh, Pennsylvania 15213, USA
Address correspondence to: Kirk P. Conrad, Magee-Womens Research Institute, 204 Craft Avenue, Pittsburgh, Pennsylvania 15213, USA. Phone: (412) 641-6019; Fax: (412) 641-1503; E-mail: rsikpc@mail.magee.edu
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1Department of Pathology, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131, USA2Department of Molecular and Integrative Physiology and College of Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA3Departments of Obstetrics, Gynecology, and Reproductive Sciences, and of Cell Biology and Physiology, University of Pittsburgh and Magee-Womens Research Institute, Pittsburgh, Pennsylvania 15213, USA
Address correspondence to: Kirk P. Conrad, Magee-Womens Research Institute, 204 Craft Avenue, Pittsburgh, Pennsylvania 15213, USA. Phone: (412) 641-6019; Fax: (412) 641-1503; E-mail: rsikpc@mail.magee.edu
Find articles by Sherwood, O. in: JCI | PubMed | Google Scholar
1Department of Pathology, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131, USA2Department of Molecular and Integrative Physiology and College of Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA3Departments of Obstetrics, Gynecology, and Reproductive Sciences, and of Cell Biology and Physiology, University of Pittsburgh and Magee-Womens Research Institute, Pittsburgh, Pennsylvania 15213, USA
Address correspondence to: Kirk P. Conrad, Magee-Womens Research Institute, 204 Craft Avenue, Pittsburgh, Pennsylvania 15213, USA. Phone: (412) 641-6019; Fax: (412) 641-1503; E-mail: rsikpc@mail.magee.edu
Find articles by Conrad, K. in: JCI | PubMed | Google Scholar
Published February 15, 1999 - More info
J Clin Invest. 1999;103(4):525–533. https://doi.org/10.1172/JCI5630.
© 1999 The American Society for Clinical Investigation
Received: October 27, 1998; Accepted: January 4, 1999
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Methods
Animal preparation.
Long-Evans female rats 10–14 weeks old were purchased from Harlan Sprague-Dawley (Frederick, Maryland, USA). They were fed PROLAB RMH 2000 diet containing 0.48% sodium (PME Feeds Inc., St. Louis, Missouri, USA) and were provided water ad libitum. To prepare the rats for experimental procedures, they were trained for several hours in a Plexiglas restraining cage (Braintree Scientific Co., Braintree, Massachusetts, USA) on at least five different occasions before surgical intervention. These cages afforded sufficient space for grooming of the face and front paws while preventing the rat from turning around. Thus, accurate timed urine collections and blood samplings were made possible from the chronically implanted bladder and vascular catheters, respectively. Rats failing to habituate to the cage were eliminated from the study (< 1%). All animal procedures were approved by the Institutional Animal Care and Use Committee of the Magee-Womens Research Institute.
The details of the surgical procedures have been described previously (18, 22). Briefly, using ketamine (6 mg/100 g body weight) and pentobarbital sodium (2.1 mg/100 g body weight) anesthesia, Tygon catheters were implanted in the abdominal aorta and inferior vena cava via the femoral artery and vein, respectively. The bladder catheter, a silastic-covered stainless steel cannula, was sewn into the urinary bladder with a purse-string suture and exteriorized through the ventral abdominal wall. This catheter was then plugged, allowing the animal to urinate through the urethra while in her home cage. In the ovariectomized and sham-ovariectomized animals, the ovaries were first ligated and then excised, or briefly manipulated, respectively, via a small incision made in the lateral abdominal wall immediately after the insertion of vascular and bladder catheters. All surgeries were conducted using aseptic technique. At least a 7-day recovery period was allowed before experimentation.
For chronic infusion of RLX, an osmotic minipump was inserted subcutaneously in the back of the animal under ether anesthesia. After shaving and cleaning the skin with alcohol and betadine, a small incision was made and the minipump containing either rhRLX, purified porcine RLX, or vehicle was inserted. Then the incision was closed with surgical staples. The model 2001 and 2ML1 osmotic minipumps (Alza Co., Palo Alto, California, USA) were used for the purified porcine RLX and rhRLX, respectively.
Experimental protocols
Influence of chronic infusion of purified porcine RLX, rhRLX, or vehicle on renal function in intact female rats. At the beginning of each experiment, immediately after opening the arterial catheter and before infusion of fluids, 100 μl of blood was collected into a heparinized tube. After centrifugation to separate blood cells from plasma, the latter was frozen at –20°C until it was assayed for osmolality. Then three 30-min baseline urine collections with midpoint blood samples and continuous recording of mean arterial pressure (MAP) were made on one or two occasions, separated by at least 48 h, to accurately measure control MAP, GFR, ERPF, and effective renal vascular resistance (ERVR) (MAP/ERPF). The hematocrit was also routinely determined on these midpoint blood samples. The renal clearances of inulin (IN) and para-aminohippurate (PAH) were used to assess GFR and ERPF, respectively. The femoral arterial catheter was connected to a Statham pressure transducer (Gould P23 ID; Statham Instruments, Hato Rey, Puerto Rico) and a Gould Universal amplifier for measurement of MAP, which was displayed on a Gould 5900 Series Signal Conditioner Cage and TA11 chart recorder. Next, an osmotic minipump containing purified porcine RLX (4 μg/h; n = 7), rhRLX (4 μg/h; n = 5), or vehicle (n = 4) was implanted. MAP, GFR, ERPF, and ERVR were assessed on days 2 and 5 of the infusion, and 5–12 days after depletion of the 7-day minipump. Details of our methodologies for the assessment of GFR and ERPF in chronically instrumented, conscious rats, including the measurement of IN and PAH in both plasma and urine, have been published previously (18, 22). Circulating RLX concentrations of 20–40 ng/ml have been measured in rats of 12–14 gestational days (19). Based on previous work (14, 25), an infusion rate of 4 μg/h for purified porcine RLX or rhRLX would be expected to produce plasma levels of 20–40 ng/ml. In fact, we reached this target level; see Results.
Influence of chronic infusion of rhRLX on renal function in ovariectomized or sham-ovariectomized rats. Six female rats each were subjected either to ovariectomy or sham ovariectomy immediately after the implantation of vascular and bladder catheters. Seven days later, renal function and MAP were assessed before the insertion of osmotic minipumps containing rhRLX. On day 5 of the RLX infusion (4 μg/h), renal function and MAP were again determined.
Acute infusion of Nω-nitro-L-arginine methyl ester or angiotensin II during chronic infusion of purified porcine RLX. At least 7 days after the implantation of vascular and bladder catheters, osmotic minipumps containing either porcine RLX (4 μg/h) or vehicle (Ringer's solution) were implanted in intact female rats (n = 7 and 6, respectively). Renal function and MAP were measured on day 5 of RLX infusion (three 30-min urine and midpoint blood collections). Next, an infusion of Nω-nitro-L-arginine methyl ester (L-NAME) (2 μg/min; a substrate competitive inhibitor of NO synthase) was administered intravenously by infusion pump (Model 200; KD Scientific, Boston, Massachusetts). Four 1-h urine collections with midpoint blood samples were collected for the assessment of GFR, ERPF, and ERVR during the L-NAME administration. Identical procedures were performed on additional animals chronically administered porcine RLX (n = 5) or vehicle (n = 5), except that angiotensin II (ANG II) (3 ng/min) was infused rather than L-NAME. Finally, another four age-matched control rats were administered Ringer's solution instead of either L-NAME or ANG II on day 5 of porcine RLX infusion.
Acute administration of purified porcine RLX. Renal function and MAP were measured in four intact female rats during acute infusion of purified porcine RLX. Three 30-min baseline urine collections were obtained with midpoint blood samples to accurately determine control GFR, ERPF, and ERVR. Then a bolus of 2 μg purified porcine RLX in 0.5 ml total volume was given over 5 min into the femoral venous catheter to fill the extracellular water compartment and reach a target concentration of 20–40 ng/ml. Immediately thereafter, porcine RLX was administered at 4 μg/h by infusion pump, and four 1-h urine collections with midpoint blood samples were made during the RLX infusion.
Metabolic cage studies. Six rats were individually housed in Nalgene metabolism cages (Rodent Metabolism Cages for 150–300 g rats; VWR Scientific, Bridgeport, New Jersey, USA). Water and food were provided ad libitum. After 5–7 days of habituation, two baseline 24-h urine collections were obtained. Then an osmotic minipump containing purified porcine RLX was implanted (4 μg/h). Additional 24-h urine collections were made on days 2 and 5 of RLX infusion and on days 4, 12, and 25 after exhaustion of the 7-day minipump. Food and water intake, as well as urinary flow rate were measured by gravimetric technique. The urinary excretion of sodium, cGMP, and nitrate + nitrite (NOX) were also determined. The measurements made during the two baseline collections were averaged, as were the measurements made during the three post-RLX collections.
Analytic techniques. Plasma osmolality was measured using a freezing-point depression instrumentation osmometer (Model 3MO; Advanced Instruments, Needham Heights, Massachusetts, USA). Plasma and urine IN and PAH were assayed by standard techniques as reported previously (18, 22). Urinary sodium concentration was measured by ion selective electrode (Sodium-Potassium Chemistry Module; Beckman Instruments Inc., Brea, California, USA). Urinary cGMP was determined by specific radioimmunoassay as described previously (26). Urinary NOX was measured by reduction of nitrate to nitrite, the latter determined by the Griess reaction, which produces a colorimetric product measured at 540 nm (Nitrate/Nitrite Colorimetric Assay Kit; Cayman Chemical, Ann Arbor, Michigan, USA).
One milliliter of blood was obtained from the rats administered with rhRLX for 5 days at the end of the renal function measurements. The levels of rhRLX in serum were then measured in a quantitative sandwich immunoassay (27). Briefly, wells of a 96-well microtiter plate (Maxisorp Immunomodules; Nalge Nunc International, Naperville, Illinois, USA) were coated overnight with affinity-purified anti-rhRLX rabbit polyclonal antibody. Sera were diluted in PBS containing Tween-20, Thimerosal, BSA (Sigma Chemical Co., St. Louis, Missouri, USA), and normal goat IgG (Organon Teknika-Cappel, Durham, North Carolina, USA), and 100 μl was added to wells in duplicate. After an overnight incubation at 4°C, the wells were washed and 100 μl of affinity-purified, peroxidase-conjugated anti-rhRLX rabbit polyclonal antibody was added to each well. After an appropriate incubation period at room temperature, the wells were washed again, and 100 μl of a tetramethylbenzidine solution was added to each well. After color development, the reaction was stopped, absorbances at 450/630 nm were measured, and RLX concentrations in the sera were determined by entering data into a four-parameter logistic curve–fitting program. The assay has been validated for use with murine serum, shows no detectable cross-reactivity with natural murine RLX, and has a lower detection limit of 20 pg/ml.
Preparation of drugs. PAH and IN were freshly prepared on the morning of the experiment using Ringer's 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's solution and mixed with PAH, it remained in solution throughout the experiment. L-NAME (Sigma Chemical Co.) was also prepared in Ringer's solution within 1 h of use. ANG II (5-ILE All or Hypertensin II; Sigma Chemical Co.) was prepared from a stock solution in 5% dextrose (100 μg/ml) that was frozen in aliquots at –20°C. Final dilutions were made in Ringer's solution immediately before infusion. For chronic infusion by osmotic minipump, rhRLX (Connectics, Palo Alto, California, USA) was prepared from a stock 1.5 mg/ml solution in 20 mM sodium acetate (pH 5.0) and diluted accordingly in the same buffer, and the lyophilized, purified porcine RLX (28) was dissolved in Ringer's solution. For acute infusion, the lyophilized purified porcine RLX was prepared daily immediately before use in Ringer's solution containing 0.01% rat albumin (Cappel Research Products, Durham, North Carolina, USA).
Statistical analysis. Data are presented as mean ± SEM. Most data were analyzed using repeated-measures mixed models with treatment group and time as fixed effects. If significant main effects or interactions were observed, then Dunnett's test was used to compare least-squares means for baseline values with all subsequent time periods. Least-squares means for rats that had been administered vehicle and recombinant human and purified porcine RLX were compared using Scheffe's procedure for multiple comparisons. For Table 5, one-factor repeated-measures ANOVA was used, and group means were compared by Orthogonal Contrasts. P < 0.05 was considered statistically significant (29).
Table 524-h urinary excretion of cGMP and NOx in conscious rats administered porcine RLX for 5 daysA
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Results
Chronic infusion of rhRLX, purified porcine RLX, or vehicle. The time-control experiments (Fig. 1, a–d) using either the vehicle for rhRLX (20 mM sodium acetate, pH 5.0; n = 2) or the vehicle for porcine RLX (Ringer's solution; n = 2 rats) showed relative stability of MAP, GFR, ERPF, and ERVR over the approximately two-week study period (P = not significant [ns] by ANOVA). Because the results obtained with the two different vehicle preparations were comparable, they were combined. While chronic infusion of purified porcine RLX at 4 μg/h did not significantly change MAP, there was a marked increase in GFR and ERPF, as well as a reciprocal reduction in ERVR on both days 2 and 5 of administration (P < 0.05 vs. baseline and vehicle). Similar results were obtained for rhRLX, except that significance was not reached until day 5 of infusion.
Figure 1Effect of long-term infusion of purified porcine RLX, rhRLX, or vehicle on (a) mean arterial pressure (b) glomerular filtration rate (c) effective renal plasma flow, and (d) effective renal vascular resistance. The numbers in parentheses depict the number of rats tested. *P < 0.05 vs. baseline and vehicle. rhRLX, recombinant human RLX; RLX, relaxin.
Chronic infusion of rhRLX in ovariectomized or sham-ovariectomized rats. The profound rise in GFR and ERPF, as well as fall in ERVR, observed on day 5 of RLX infusion were comparable in ovariectomized and sham-ovariectomized rats (P = ns) (Fig. 2a–d).
Figure 2Effect of 5-day administration of rhRLX to sham-ovariectomized and ovariectomized rats on (a) mean arterial pressure (b) glomerular filtration rate (c) effective renal plasma flow, and (d) effective renal vascular resistance. The numbers in parentheses depict the number of rats investigated. *P < 0.05 vs. baseline.
Acute infusion of L-NAME or ANG II during chronic infusion of purified porcine RLX. The renal vasodilation and hyperfiltration in the RLX-treated rats observed on day 5 of infusion was completely abrogated by NO synthase inhibition (Table 1). To minimize the possibility that the convergence of renal function in the RLX and vehicle infused rats elicited by L-NAME was merely a consequence of nonspecific renal vasoconstriction, the experimental paradigm was repeated using another vasoconstrictor, ANG II. Rather than converging, ERPF and ERVR diverged even further during the acute infusion of ANG II (Table 2). Thus, these results were diametrically opposed to those obtained using the L-NAME. To emphasize the contrasting effects of L-NAME and ANG II on renal function and MAP in conscious rats chronically administered RLX, the percent change in MAP, GFR, ERPF, and ERVR from baseline is portrayed in Figs. 3 and 4. L-NAME produced a significantly greater rise in MAP and ERVR and reduction in GFR and ERPF in the RLX-treated compared with vehicle-infused rats (P < 0.05 by ANOVA). In contrast, the percent increase in MAP and reduction in GFR elicited by ANG II was comparable in the RLX-treated and vehicle-infused rats, whereas the percent increase in ERVR and reduction in ERPF was markedly attenuated in the rats administered chronic RLX (P < 0.001 vs. vehicle by ANOVA). Another group of rats chronically treated with porcine RLX was administered Ringer's solution instead of L-NAME or ANG II, and thus served as time controls. Neither MAP nor any of the renal parameters were consistently altered over the approximately six hours of vehicle infusion (Table 3).
Figure 3The effect of L-NAME infusion in rats administered purified porcine RLX (n = 7) or vehicle (n = 6) for 5 days. The means ± SEM represent the percent change from baseline values for (a) mean arterial pressure (b) glomerular filtration rate (c) effective renal plasma flow, and (d) effective renal vascular resistance. L-NAME, Nω-nitro-L-arginine methyl ester.
Figure 4The effect of ANG II infusion in rats administered purified porcine RLX or vehicle for 5 days. There were five rats in each treatment group. The means ± SEM represent the percent change from baseline values for (a) mean arterial pressure (b) glomerular filtration rate (c) effective renal plasma flow, and (d) effective renal vascular resistance. ANG II, angiotensin II.
Table 1Effect of L-NAME on mean arterial pressure and renal function in conscious rats administered porcine RLX or Ringer's solution (vehicle) for 5 daysA
Table 2Effect of ANG II on mean arterial pressure and renal function in conscious rats administered porcine RLX or Ringer's solution (vehicle) for 5 daysA
Table 3Effect of Ringer's solution on mean arterial pressure and renal function in conscious rats administered porcine RLX for 5 days (time control)A
Acute administration of purified porcine RLX. A 2-μg bolus of purified porcine RLX followed by a sustained infusion of 4 μg/h for four hours did not affect either GFR or ERPF (Fig. 5, a–d).
Figure 5Effect of short-term infusion of purified porcine RLX on (a) mean arterial pressure (b) glomerular filtration rate (c) effective renal plasma flow, and (d) effective renal vascular resistance. n = 4 rats.
Hematocrit and plasma osmolality. In the intact female rats chronically administered either purified porcine or rhRLX for the study of renal function, both the hematocrit and plasma osmolality showed significant decrements by day 2 (P < 0.05) (Table 4).
Table 4Hematocrit and plasma osmolality of conscious rats chronically administered porcine RLX, rhRLX, or vehicle (time control)A
Urinary excretion of cGMP and NOX. The 24-hour urinary excretion of cGMP and NOX were not significantly affected by the chronic infusion of porcine RLX at 4 μg/h (P = ns by ANOVA). In the same animals, urinary sodium excretion and water intake were significantly increased on days 2 and 5, respectively, of RLX infusion (P < 0.05 vs. baseline and post-RLX) (Table 5).
Serum RLX. In 17 of the rats, we measured the serum concentration of the rhRLX that was infused by osmotic minipump at 4 μg/h for five days. The mean ± SEM was 28.1 ± 4.8 ng/ml.
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