Materials and Methods

The experimental protocol was approved by the Animal Experiments Committee of the Radboud University Nijmegen Medical Centre and was performed in accordance to the European guidelines on animal experiments. Forty female virgin Wistar Hannover rats (Harlan Netherlands BV, Horst, The Netherlands) were studied in 2 groups at an age of 8-10 weeks, followed by 1-week acclimatization. Group 1 (mid-pregnant rats, 10; nonpregnant, 10) was used for studies in the Mulvany Halpern myograph; group 2 (mid-pregnant rats, 10; nonpregnant rats, 10) was used for perfusion-pressure myograph experiments. We used the isolated perfused rat kidney model for experiments on both groups (n = 40). We housed all rats by 2 in filter-top cages on a 12/12-hour light/dark cycle and provided them with standard diet (ssniff R/M-H; Ssniff Spezialdieten GmbH, Soest, Germany) and water ad libitum. Pregnancy was accomplished by mating with an experienced male of similar age. The presence of a semen plug at the bottom of the cage was considered successful mating and day 1 of pregnancy. Mid-pregnant (mid-gestation, day 11 of the pregnancy) and nonpregnant animals were anesthetized with an intraperitoneal injection of 6 mg/100 g pentobarbital (Apharmo, Arnhem, The Netherlands). Furosemide (1 mg/100 g; Sigma-Aldrich Chemie BV, Zwijndrecht, The Netherlands) was injected intraperitoneally to achieve maximal urethral distention for optimal catheter placement. We weighed all animals before surgery. During surgery, blood was withdrawn and plasma was stored at –80°C to measure relaxin concentration with a homologous radioimmunoassay. The assay was performed according to the method of Sherwood and Crnekovic. In our hands, the analytic sensitivity of the assay was 0.51 ng/mL. Dilution experiments showed good parallelism in the range of 25–100 μL.

Flow-mediated vasodilation, myogenic reactivity to pressure, and compliance were analyzed in a pressure-perfusion myograph (Pressure Myograph System-Model P100; J. P. Trading, Aarhus, Denmark). The responses were determined in phosphate-buffered saline solution (119 mmol/L NaCl, 4.69 mmol/L KCl, 25 mmol/L NaHCO ₃ , 1.17 mmol/L MgSO ₄ , 1.18 KH ₂ PO ₄ , 5.5 mmol/L glucose, and 10 mmol/L HEPES), to which were added 2 mmol/L ethylene glycol tetraacetic acid and 0.01 mmol/L Na-Nitroprusside (for calcium-free phosphate-buffered saline solution) to measure compliance or 2.5 mmol/L CaCl ₂ , 0.027 mmol/L Na ₂ ethylene diamine tetraacetic acid (for calcium phosphate-buffered saline solution) to assess flow-mediated vasodilation and myogenic reactivity. The buffers were oxygenated with 95% O ₂ and 5% CO ₂ at a temperature of 37°C. Second-order mesenteric arteries (150-400 μm) were mounted on 2 opposing glass cannulas (125 μm). Vascular inner diameter was measured through video recording (Vessel View; J. P. Trading). Arteries were equilibrated at an intraluminal pressure of 60 mm Hg for 20 minutes. Subsequently, preconstriction was achieved by the addition of the thromboxane A2 agonist, U46619 (9,11-Dideoxy-11α, 9α-epoxymethanoprostaglandin F _2α ; Sigma-Aldrich Chemie BV, Zwijndrecht, The Netherlands; bath concentration of 10 ^–7 −10 ^–6 mol/L), thereby reducing the vessel diameter by 30-40% of their basal diameter.

Flow-mediated vasodilation was defined as the vasodilator response to a certain blood flow increase. After reaching a stable contraction, the blood flow in the vessel was increased by 16.7-μL/min steps (2-minute interval) from 0-100 μL/min (comparable with a shear-stress range of 0-10 dyne/cm ² ), in the absence or presence of 100 μmol/L of the NO antagonist l -NAME ( l -Nitro-Arginine Methyl Ester; Sigma-Aldrich Chemie BV). Meanwhile, the intraluminal pressure was maintained at 60 mm Hg. Vessels were excluded from the study if they attained <20% preconstriction, <10% vasodilation over the completed flow-range, or instable pressure because of fluid leakage. Flow-mediated vasodilation was expressed as a percentage of the preconstriction status.

Myogenic reactivity was defined as the vasoconstrictive response to a certain pressure increase in the presence of calcium. Compliance was defined as the vasodilator response to a certain pressure increase in the absence calcium, to avoid smooth muscle cell contraction. To withdraw interference of previous assessments, a new mesenteric vessel was isolated and prepared as described earlier. Intraluminal pressure was increased every 2 minutes by 10-mm Hg steps, from 20 mm Hg up to 110 mm Hg, to successively determine myogenic reactivity and compliance (expressed in micrometer, μm).

We studied the vasoconstrictor response to phenylephrine (Sigma-Aldrich Chemie BV) with a Mulvany Halpern myograph (Dual Wire Myograph-Model 400A; J. P. Trading). The bath was filled with physiologic salt solution (119 mmol/L NaCl, 4.69 mmol/L KCl, 2.5 mmol/L CaCl ₂ , 25 mmol/L NaHCO ₃ , 1.17 mmol/L MgSO ₄ , 1.18 KH ₂ PO ₄ , 5.5 mmol/L glucose and 0.027 mmol/L ethylene diamine tetraacetic acid) and oxygenated with 95% O ₂ and 5% CO ₂ at a temperature of 37°C. Four second-order mesenteric arteries (150-400 μm) were mounted in 2 wire myographs, which were stabilized as previously described, and set at a tension equivalent to that generated at 90% of the inner circumference at 100 mm Hg. The response to 124 mmol/L KCl was measured to allow normalization of the phenylephrine response. The response to phenylephrine was determined in 8 steps over a range from 10 ^–7 -10 ^–5 mol/L, in the absence or presence of 100 μmol/L l -NAME. The data for the 2 arteries in each myograph were averaged when available.

The intrinsic adaptation of the kidney was assessed in the isolated perfused rat kidney model. Within the time frame of our protocol, this model was known to represent stable renal function. Briefly, the right renal artery was cannulated through the left renal artery and aorta. The right ureter was cannulated for urine collection. Perfusion was started in situ, and the kidney was removed and placed in a perfused bath at a temperature of 37.5°C. The kidney was perfused at a constant pressure of 90 mm Hg with oxygenated cell-free Krebs-Ringer-Henseheit (which contained 113 mmol/L NaCl, 4.8 mmol/L KCL, 25 mmol/L NaHCO ₃ , 1.4 mmol/L KH ₂ PO ₄ , 2.2 mmol/L CaCl ₂ , 1.4 mmol/L MgCl ₂ , 5 mmol/L glucose), 1.7 mmol/L Pluronic F-108 (oxygen carrier and oncotic agent; BASF, Arnhem, The Netherlands). Renal perfusion flow (RPFF) was recorded real-time with the use of a computer system (Midac testorganizer, W95 [version 3.0]; Radboud University Nijmegen Medical Centre, The Netherlands).

Stabilization of the RPFF was accomplished during a 40-minute period. From 40-60 minutes, the basal RPFF (RPFF _baseline ) was determined. At 60 minutes l -NAME was added to the perfusate (achieving a concentration of 100 μmol/L) to investigate the contribution of NO to the relaxin- (or placebo-) evoked vasodilator response up to 160 minutes (RPFF ₁₆₀ ). RPFF was normalized for body weight (milliliters per minute per 100 grams of body weight).

The data were expressed as means ± standard error of the mean (SEM); n indicates the number of animals. Flow-mediated vasodilation, response to phenylephrine, and RPFF were analyzed by nonlinear regression curve fitting (GraphPad Prism 4.0; Institute for Scientific Information, San Diego, CA). Subsequent curve fit estimates were (1) maximal vascular diameter after flow change (Diam _max ), (2) flow rate inducing 50% dilation, (3) flow-sensitivity (Flow _50% ), (4) maximum response to phenylephrine, and (5) phenylephrine concentration that induced a 50% response (C _50% ), RPFF _baseline , and RPFF _baseline minus RPFF ₁₆₀ (RPFF _delta ). Overall myogenic reactivity (corrected for percentage preconstriction) and overall vascular compliance were analyzed by analysis of variance for repeated measures (Greenhouse-Geisser correction; version 16.0.2; SPSS Inc, Chicago, IL). Selective straight-line curve fitting was performed over the range of 60–110 mm Hg, for both myogenic reactivity and compliance to estimate the hill slope. Baseline characteristics were analyzed with the Student t test. A probability value of < .05 was considered to represent statistical significance.