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Kidney International,Vol. 47 (1995), pp. 1558—1568

Effects of urodilatin in the rat : Comparison with ANFand interaction with vasoactive substances

KARLHANS ENDLICH, WOLF-GEORG FORSSMANN, and MICHAEL STEINHAUSEN, with the technical assistance of RUDOLF DUSSEL

Institute of Physiology, University of Heidelberg, Heidelberg, and Lower Saxony Institute for Research, Hannover, Germany

EWects of urodilatin in the rat kidney: Comparison with ANFand humans as well as in animals [6—9], and the physiological role of interaction with vasoactive substances. We compared the effects of urodilatin (URO) and atrial natriuretic factor (ANF) in normal andboth and the adequate stimuli for their release are still hydronephrotic kidneys (HNK) of rats. Furthermore, the impact ofdebated [2, 10—12]. Nonetheless, URO has been successfully used blocking different vasoactive on the action of natriureticin the clinical treatment of acute renal failure after organ trans- peptides on vessels of cortical (C) and juxtamedullary (JM) glomeruli was plantation [13]. studied in HNK by using URO. In normal kidneys, effects of URO and ANF (1.2, 2.4, 4.8, 12, and 19 10molkg1 min1 i.v.) were not Up to now the question is unsolved as to whether the natriuretic significantly different. At 12 lO molkg1 min1, URO and ANFeffect of ANF is solely due to inhibition of tubular sodium increased flow 5.4 1.7 and 3.00.8-fold, increased urinaly sodium reabsorption or whether vascular effects of ANF are either excretion 20.7 8.8 and 10.34.0-fold, and decreased blood pressure by predominantly or at least partially involved in natriuresis [101. 13 2% and 12 1%, respectively (mean SCM). In HNK, URO and ANF (0.4, 0.9, and 2.0 10_11 mol kg1 min1 i.v. and local application ANF increases GFR primarily by raising glomerular capillary of 0.5, 1.0, and 2.0 i0 M) dose-dependently dilated preglomerularhydraulic pressure [14]. An altered ratio of pre- to postglomerular vessels (max 20%), constricted efferent arterioles (max 15%), and resistance is responsible for the increase in glomerular pressure increased glomerular blood flow of C glomeruli in an identical fashion. [14—16]. Preglomerular and efferent vasoconstriction Comparing URO effects on C and JM arterioles (0.4 and 0.9 10 mol .kg mini.v.), JM responses were about one third of C responses. of renal vessels by ANF was directly visualized by us [17, 18] and Angiotensin converting inhibition (ACEI, 2 10 mol kg'others [19, 20]. In contrast, ANF dilated only preglomerular quinapril i.v.), combined ACEI and cyclooxygenase inhibition (CYOI, vessels in the juxtamedullary nephron preparation [21] and had no 2.8 iO M indomethacin), and (ET) receptor blockade (10—6 effect on isolated renal vessels of the rabbit [22]. M BQ 123 and IRL 1038) diminished preglomerular vasodilation (C and JM) caused by URO infusion. Efferent vasoconstriction (C and JM) The biological effects of URO, ANF and brain natriuretic caused by URO was exaggerated by blockade of nitric oxide synthesis peptide (BNP) are thought to be mediated by the natriuretic (10—i M L-NAME) and abolished by combined ACEI and CYOI, by peptide receptor (NPR) A subtype which uses guanosine receptor blockade (4 i0 M Hoe 140), and by ET blockade. CYOI attenuated only JM efferent constriction. Our results show that 3',5'-cyclic monophosphate (cGMP) as its second messenger URO and ANF possess equipotent vascular and similar natriuretic effects [23, 24]. These peptides have similar NPR binding properties in in the rat kidney. The magnitude of preglomerular vasodilation, which is the rat and human kidney [25]. Binding of URO or ANF to directly mediated by these peptides, depends on the basal level ofvascular smooth muscle results in vasodilation as demonstrated endogenous vasoconstrictors, while efferent vasoconstriction may be me- in preconstricted arcuate arteries [26] and afferent arterioles diated by the secondary release of ET. [20]. Efferent vasoconstriction, however, is poorly understood. It was hypothesized that ANF may constrict the efferent arteriole via secondary release of a constrictor [14, 20]. How- ever, blockade of angiotensin II receptors and formation, and In 1988 urodilatin (URO) was isolated from human urine andof thromboxane and a-adrenergic receptors failed to inhibit was claimed to be a produced by the kidney efferent constriction [14, 20]. Vasoconstrictor effects of ANF itself [1, 2]. More recently, transcripts of the gene for atrialare not restricted to the renal vasculature; they were also natriuretic factor (ANF) have indeed been found in the kidney [3]. URO has four additional amino acids in comparison to ANFobserved in the coronary and gastrointestinal circulation [27, which has been known since 1981 [I• In contrast to ANF, URO28]. could not be detected in the circulating blood of humans [5]. The In one part of our present study we compared the natriuretic natriuretic potency of both peptides showed some differences ineffects of URO and ANF in the normal rat kidney and the vascular effects in the hydronephrotic kidney model, which per- mits visualization of the vasculature of cortical (C) and juxtamed- ullary (JM) glomeruli in vivo [29, 30]. In the other part of the study Received for publication July 8, 1994 we explored the interaction of URO with several vasoactive and in revised form December 15, 1994 hormones at C and JM vessels to gain insight into the mechanisms Accepted for publication January 12, 1995 by which natriuretic peptides induce preglomerular vasodilation © 1995 by the International Society of Nephrology and efferent constriction.

1558 Endlich et al: Renal effects of urodilatin 1559

Table1. Control values of vessel lumen diameters, GBF, blood pressure, and body weight

Series 1 Series 2 Series 3 Series 4 (N=6) (N=6) (N=5) (N=5) Arcuate artery proximal pm 80.4 10.9 71.5 3.6 77.84.5 80.09.3 distal p.m 50.0 6.4 46.7 2.7 56.3 2.9 44.1 3.1 Interlobular artery proximal pm 32.1 1.9 26.4 1.0 30.2 1.7 26.01.8 distal pin 13.40.7 13.0 1.1 11.4 1.3 12.0 1.1 C afferent arteriole proximal pin 10.40.6 9.8 0.6 9.70.9 11.2 1.1 distal pin 8.20.5 8.1 0.9 8.20.6 7.00.5 C efferent arteriole proximal pin 14.90.7 15.1 1.2 10.00.8 12.6 0.8 distal p.m 18.6 1.1 19.3 1.6 14.9 1.3 15.5 1.7 JM afferent arteriole proximal pm 23.2 0.9 19.7 2.2 distal pin 21.9 1.0 17.4 1.5 JM efferent arteriole proximal p.m 41.9 1.7 31.7 3.1 distal pm 44.4 1.8 35.8 4.1 C GBF ni min ' 48.1 10.6 30.4 8.0 29.74.3 32.7 5.9 JMGBF ni min1 397.5 39.7 312.5 102.2 Blood pressure mm Hg 115.8 2.4 105.8 2.4 104.05.1 109.0 2.9 Body weightg 242 16 257 8 240 11 232 8 Data are mean SEM.

Methods arcuate artery (near the interlobular artery), (3) proximal inter- lobular artery (near the arcuate artery), (4) distal interlobular Experiments on hydronephrotic kidneys artery (near the afferent arteriole), (5) proximal afferent arteriole Preparation of the hydronephrotic kidney. Experiments were(near the interlobular artery), (6) distal afferent arteriole (at the performed on 58 female Wistar rats weighing 237 23 g (mean narrowest segment before entering the glomerulus), (7) proximal SD). The technique of splitting the rat kidney has been previouslyefferent arteriole (within 50 j.m of the glomerulus), (8) distal described in detail [29, 301. In brief, ligation of the ureter via a efferent arteriole (near the welling point). To identify JM glomer- flank incision was performed during pentobarbital sodium anes-uli we used the length of the unbranched portion of the efferent thesia (Nembutal' 60 mg kg' i.p., Ceva, Bad Segeberg, Ger-arteriole (cf. [30]). C and JM glomeruli fed from the same arcuate many). The final experiments were performed under thiobutabar-artery were selected for the purpose of comparison. bital anesthesia (InactinR 100 mg kg' i.p., Byk Gulden, Glomerular blood flow (GBF). Red blood cell velocity was Konstanz, Germany) two to three months after the induction ofdetermined in the efferent arteriole by a velocity tracking correla- hydronephrosis. Body temperature was maintained at 37.0 to tor (Model 102B; 1PM Inc., San Diego, CA, USA) [31]. To obtain 37.5°C via a heating table, systemic blood pressure was monitoredGBF, the measured red cell velocity was multiplied by the luminal via a cannula in the left femoral artery, and isotonic saline (50diameter of the efferent arteriole and corrected for the Fahraeus min) was continuously infused via a cannula placed in the effect [32]. jugular vein. After exposure of the left hydronephrotic kidney by a flank incision, the kidney was split along the greater curvature with a thermal cautely. In the first two series of experiments the Experimental protocol ventral half and in the other series the dorsal half of the kidney was sutured to a semicircular frame that was attached to the in the following, specified concentrations for locally applied bottom of a plexiglas bath. in the first two series intravitaldrugs refer to the final concentration of the drug in the tissue microscopy was confined to the outside of the kidney, whereas inbath. Each protocol started with two control periods separated by the other series intravital microscopy of the inside of the kidney10 minutes. Each protocol was made up of several periods during allowed us to visualize JM glorneruli [30]. The entry of the renalwhich systemic blood pressure, GBF, and vascular diameters were hilus into the bath was sealed with silicone grease, and the bathmeasured. was filled with an isotonic, isocolloidal solution (HaemaccelR, Effects of i.v. or locally applied URO and ANFwerecompared Behringwerke, Marburg/Lahn, Germany) maintained at 37°C. Ain two series. Leitz Ultropac water immersion objective (UO-55) was combined Series I (N =6).After two control periods URO (produced by with a television system and video recording for intravital micros-W. G. Forssmann) was intravenously infused in three periods of copy. Kidney preparations were allowed to equilibrate in the12 minutes each in the following doses: 0.4, 0.9, and 2.0 tissue bath for at least one hour after the surgical procedure. Themol kg1 min' dissolved in isotonic saline at a rate of 5, 14, microcirculatory parameters of this preparation have been dem-and 29 1id min ,respectively.Measurements were done 10 onstrated to be stable for more than three hours [18]. minutes after the start of each infusion. Forty minutes after stop Renal vascular segments. Lumen diameter measurements of theof the third URO infusion a control measurement was performed. following vessel segments were carried out, the vessels beingThereafter URO was added to the tissue bath in increasing identified according to the branching pattern of the vessels fromconcentrations: 0.5, 1.0, and 2.0 iO M. The kidney was exposed the selected cortical (C) or juxtamedullary (JM) glomerulus: (1)to each concentration for 12 minutes. Measurements were per- proximal arcuate artery (near the interlobar artery), (2) distalformed 10 minutes after the addition of each concentration. In 1560 Endlich et a!: Renal effects of urodilatin

Table 1. Continued Series 5 Series 6 Series 7 Series 8 Series 9 (N =6) (N =6) (N =7) (N =6) (N =7) Arcuate artery proximal pm 66.27.2 75.5 3.9 65.7 2.2 72.8 5.0 57.3 4.1 distal pin 41.8 1.6 46.5 2.0 45.5 1.4 45.8 2.6 42.2 3.2 Interlobular artery proximal pm 23.8 1.1 30.0 1.0 29.70.9 25.70.5 26.1 1.2 distal pm 13.90.8 14.60.7 15.3 1.0 15.21.6 12.6 0.6 C afferent arteriole proximal pm 10.20.6 10.90.8 10.90.8 9.90.6 11.0 0.5 distal pm 8.40.6 8.00.5 8.40.5 7.1 0.4 8.5 0.6 C efferent arteriole proximal p.m 14.3 1.1 12.20.6 10.80.8 14.41.4 15.8 1.5 distal pm 17.2 1.2 14.70.9 14.70.8 17.41.1 19.4 0.8 JM afferent arteriole proximal p.m 19.7 1.1 17.70.6 19.9 1.0 16.31.0 20.5 1.0 distal pm 15.50.9 15.00.5 17.4 1.5 13.40.8 17.5 1.0 JM efferent arteriole proximal pm 32.02.1 28.1 2.3 31.8 3.9 29.32.5 31.3 2.6 distal pm 34.42.4 29.3 1.3 35.2 4.7 28.6 1.6 33.4 3.3 C GBF ni min' 25.64.3 32.33.0 33.6 10.3 41.2 4.2 36.9 8.5 JM GBF ni min1 315.9 56.7 244.4 24.2 281.1 46.5 211.5 18.7 238.3 50.6 Blood pressure mm Hg 107.5 1.7 113.32.8 106.42.6 104.2 1.5 121.40.9 Body weight g 249 7 227 8 224 5 240 6 231 7 Data are mean SEM.

two additional experiments only saline (without URO) was in-4 to 8 was followed by the identical protocol of URO application fused at the same rates to exclude unspecific effects of theas in series 3. infusion. Series 9 (N =7).Endothelin (ET) ETA and ETB receptor Series 2 (N =6).Equimolar amounts of ANF (produced byblockade by local application of 10_6 M BQ 123 and 106 M IRL W. G. Forssmann) were used with the identical protocol as in1038 (California Peptide Research, Napa, CA, USA), measure- series 1. ments after 15, 30, 45, and 60 minutes. The last period was Senes 3 (N 5). The effects of i.v. infused URO (0.4 andfollowed by the identical protocol of URO infusion (0.4, 0.9, and 0.9 10_11 mol kg min1) on C and JM vessels and GBFwere2.0• 10—11 mol kg .min')as in series 1. After stop of the examined. Each infusion period lasted 12 minutes. MeasurementsURO infusion the tissue bath solution was exchanged three times were done 10 minutes after the start of each infusion. In twoat intervals of 10 minutes to wash out the ET antagonists. additional experiments URO was i.v. infused with the identicalThereafter, a new control measurement was done (30 mm after protocol as in the clearance experiments (see below). In theseinfusion stop), which was followed by i.v. infusion of URO with experiments measurements were done 10 and 20 minutes after thethe same protocol as before; 10_6 M BQ 123 as well as 106 M IRL beginning of each infusion period. 1038 antagonized the ET-1-induced decrease in GBF in the In the following six series the impact of blocking severalhydronephrotic kidney (Endlich et al, manuscript submitted). vasoactive hormones on C and JM effects of URO was studied. After two control periods the rats were pretreated with: Clearance experiments Series 4 (N =5).Angiotensin converting enzyme inhibition Animal preparation. In 18 female Wistar rats weighing 206 9 (ACEI) by i.v. bolus injection of 2 10—6 mol kg' quinaprilg (mean SD) effects of i.v. infusion of URO and ANF on urine (provided by Goedecke-Parke-Davis, Freiburg, Germany) [33],volume flow (V), urinary sodium excretion (UaV), GFR (esti- measurements after 20 and 40 minutes. mated by clearance), and blood pressure were mea- Series 5 (N 6). Cyclooxygenase inhibition (CYOI) by localsured. Animals were anesthetized with thiobutabarbital (100 application of 2.8 iO M indomethacin (Sigma, Deisenhofen,mg kg1 i.p.), placed on a heating table to maintain body Germany) into the tissue bath [301, measurements after 15, 30, 45,temperature at 37.0 to 37.5°C, and tracheotomized. The jugular and 60 minutes. vein was cannulated for intravenous infusion and the femoral Series 6 (N =6).Combined ACEI and CYOI by local applica-artery was cannulated for registration of systemic blood pressure tion of 2.8 10 M indomethacin 20 minutes after i.v. bolusand for removal of blood samples. A 0.5 ml bolus was followed by injection of 2 10_6 mol kg1 quinapril, measurements beforecontinuous i.v. infusion of 17 p.1 .min1isotonic saline containing indomethacin application and 15, 30, 45, and 60 minutes thereaf-1.5 g dl 1creatinineto reach a plasma creatinine concentration ter. of about 15 mg dl* The ureters were exposed by a median Series 7 (N =7).Blockade of nitric oxide (NO) synthesis byabdominal incision and cannulated without touching the kidneys. local application of i0— M L-nitroarginine-methylester (L-The ureter catheters were externalized and the abdominal incision NAME, Sigma) [341, measurements after 15, 30, and 60 minutes.was closed with clips. Series 8 (N =6).Bradykinin B2 receptor blockade by local Measurements. Urine from each ureter was collected in 20 application of 4 10—8 M Hoe 140 (provided by Hoechst, Frank-minute periods. Urine volume was determined gravimetrically. furt, Germany) [35], measurements after 15, 30, 60, 90, and 120Urinary sodium concentration was measured by flame photometry minutes. (FMD 3, Zeiss, Oberkochen, Germany). Creatinine concentration After a stable state was reached, the last measurement in seriesin urine and plasma was determined with a creatinine analyzer Endlich et al: Renal effects of urodilatin 1561

(Model 2, Beckman Instruments, Munich, Germany). Creatinineafferent arteriole near the glomerulus (Fig. 3). C and JM vessels clearance was calculated from urine volume flow and creatininewere equally sensitive to CYOI and GBF was reduced by the same concentrations of plasma and urine. amount (—44 3% and —42 5%, respectively). After CYOI, Experimental protocol. Animals were divided into three groupsURO induced preglomerular dilation, which was shifted to larger (each N =6).After one hour of equilibration urine was collectedvessels, and a significant constriction only at C efferent arterioles for 200 minutes divided into ten periods. The protocol started(Fig. 2). Efferent constriction by URO after CYOI appeared to be with three control periods (Cl, C2, C3). Then, i.v. infusion ofenhanced at C glomeruli and to be blunted at JM glomeruli URO was started in the first group and i.v. infusion of ANF in thecompared with URO only (series 3). However, no statistical second group. The third group received only saline. During thesignificance was achieved. While C GBF increased, JM GBF five experimental periods (El, E2, E3, E4, E5) the infusedremained unchanged. amounts of URO and ANF were as follows: 1.2, 2.4, 4.8, 12, and Series 6. CYOI after ACEI did not fundamentally change the 19 10_11 mol kg1 min1. The total infusion rate was keptpattern of vasodilation which was obtained by ACEI alone, except constant at 17 d -min'.The experimental periods were followedfor a pronounced vasoconstriction at distal JM efferent arterioles by two recovery periods (Ri, R2). (Fig. 3). C GBF increased by 32 8%, while JM GBF decreased by —29 3%. After this combined ACE! and CYOI, URO Data analysis almost completely lost its characteristic pre- and postglomerular All values are presented as mean SEM. Changes in vascularvascular effects (Fig. 2). Only a few preglomerular segments were diameters and GBF are expressed as percentage changes from theslightly dilated. GBF was increased to a similar extent by URO as first control values unless otherwise stated. Clearance data arewithout pretreatment. expressed as relative changes to the third control period (C3). Series 7. Blockade of NO synthesis (60 mm after local applica- Analysis of variance and the Bonferroni method for multipletion of io— M L-NAME) reduced pre- and postglomerular comparisons were used to test for statistical significance. Thediameters of C and JM vessels and decreased C GBF by —51 overall significance level was set to P < 0.05. 4% and JM GBF by —54 6% (Fig. 3). After NO synthase inhibition, URO again dilated the preglomerular vasculature (Fig. Results 2). However, the constriction of C and JM efferent arterioles caused by URO was significantly exaggerated in the absence of Experiments on hydronephrotic kidneys NO production. The URO-induced GBF increase was unchanged The control values of vessel diameters, GBF, blood pressure,by pretreatment with L-NAME. and body weight are summarized in Table 1. Blood pressure did Series 8. Bradykinin B2 receptor blockade (local application of not significantly change in any series except for series 6 where it4 10_s M Hoe 140) diminished C and JM GBF in a time- fell from a control value of 113.3 2.8 mm Hg to 109.22.1 mm dependent manner, reaching a stable state after about 90 minutes. Hg after ACEI and CYOI. C and JM GBF were lowered by —46 2% and —42 5%, Series 1 and 2. Intravenous infusion of URO or ANF inducedrespectively, after 120 minutes as a result of a diameter reduction preglomerular vasodilation, efferent vasoconstriction, and an in-of nearly all pre- and postglomerular vessels (Fig. 3). Although crease in GBF in a dose-dependent fashion (Fig. 1). The afferentpreglomerular vasodilation by URO was preserved after admin- arteriole near the glomerulus dilated only slightly. Forty minutesistration of Hoe 140, efferent vasoconstriction was significantly after stopping the infusion all diameters returned towards theirblocked (Fig. 2). C and JM GBF were massively increased by control values. GBF decreased from 53 9% to 14 13% andURO under this condition. from 47 10% to —7 7% after stopping the infusion of URO Series 9.ETreceptor blockade (60 mm after local application of and ANF, respectively. Subsequent local application of URO or106 M BQ 123 and 106 M IRL 1038) dilated pre- and postglo- ANF elicited identical vascular effects compared with i.v. infusionmerular vessels and increased C and JM GBF by 15 1% and 11 of these peptides. On the whole, the effects of URO and ANF 2%, respectively (Fig. 4). Subsequent infusion of URO dilated were not significantly different from each other. In two additionalpreglomerular vessels and increased C and JM GBF dose-depen- experiments, the URO infusion was replaced by saline. In bothdently. However, efferent vasoconstriction was absent. After wash experiments fluctuations of GBF were less than 5%. out of the ET antagonists vascular diameters and GBF returned to In the following series, vascular segments and GBF of C and JM baseline. In the absence of ET antagonists, the second infusion of glomeruli were studied. URO constricted efferent arterioles. Furthermore, the preglo- Series 3. Intravenous infusion of URO dose-dependently dilatedmerular vasodilation by URO was enhanced. preglomerular vessels, constricted efferent arterioles, and in- creased GBF of JM as well as of C glomeruli (Fig. 2). Vascular effects of URO were more pronounced in C arterioles. Clearance experiments Series 4. ACEI by quinapril (20 nun after i.v. bolus injection of 2 10—6 mol kg1) resulted in preglomerular vasodilation of C The control values of the three treatment groups (saline, ANF, and JM vessels (Fig. 3). Efferent arterioles were only significantlyand URO) are summarized in Table 2. Intravenous infusion of dilated at the distal JM site. C GBF rose by 48 13% and JMsaline did not result in any significant change of the measured GBF by 23 7%. After ACEI, URO was unable to further dilateparameters. Parameters were also not altered during the three preglomerular vessels (Fig. 2). ACEI did not affect efferentcontrol periods (Cl to C3) and at the three lower rates (1.2, 2.4, vasoconstriction by URO resulting in a decrease of GBF. and 4.8 iO mol -kg'min'; El to E3) of infused ANF or Series 5. CYOI by indomethacin (60 mm after local applicationURO (Fig. 5). of 2.8- i0 M) induced constriction of all vessels except for the C ANF and URO infusion significantly increased urine flow 3.0± 1562 Endlichet al: Renal effects of urodilatin

0.4 x 10_il rnol • mm1. kg i.V. GBF, .\% 0.5 x 10 mol • t in bath GBF, .X! +20 +20 +10 78 j +10 7 8 —10 123456 —10 123456

** 0.9 x 10- mol • mirn1 • k iv. +40 t +30 +30 +20 * 1 x109moI•I1 in bath I ______78 ] 12:4s6J** g—1o Ct * -c(0 * C, U I— * I— a, a, +50 . . -f60I ÷40 2 x 10" mol • min . kg iv. . I

+30 I - * ÷40 2 x iO mol• I in bath

78 ** 1 23 4 5 6 [ILL! 40 mm after infusion stop ¾* +20 +10 0 4 56 Fig.1. Percentchanges of renal vascular diameters and GBF of Cglomeruli in response to iv. infusion (0.4, 0.9, and 2.0 101Jmolkg' min') and local application (0.5, 1.0, and 2.0 i0° M) of Urodilatin (LI)andANF (U).N=6each, data are mean SEM. < 0.05 vs. control, °P < 0.05 vs. previous value, P < 0.05 Urodilatin vs. ANF. Preglomerular: (1) arcuate arteries-proximal; (2) arcuate arteries-distal; (3) iriterlobular arteries-proximal; (4) interlobular arteries-distal; (5) C afferent arterioles-near interlobular arteries; (6) C afferent arterioles-near glomeruli. Postglomerular: (7) C efferent arterioles-near glomeruli; (8) C efferent arterioles-near welling point.

0.8-fold and 5.4 1.7-fold, respectively, at an infusion rate of Discussion 12 i0 mol kg1 min (E4). At this rate, ANF and URO raised urinary sodium excretion 10.3 4.0-fold and 20.7 In the clearance experiments, i.v. infused URO and ANF 8.5-fold, respectively. Also, blood pressure declined significantlyincreased urine volume flow and sodium excretion to a similar by 12.2 0.8% (ANF) and 13.21.6% (URO). GFR tended todegree with a concomitant fall in blood pressure (Fig. 5). URO increase by about 20%, but did not change significantly through-tended to be the more potent natriuretic substance than ANF, but out the experiments. After stopping the infusion (R1-R2) allthe effects of both peptides were not significantly different from values returned to baseline. Though URO effects on urine floweach other. At similar high doses of URO and ANF, a comparable and on sodium excretion tended to be greater than ANF effects,increase in natriuresis was also observed by Abassi et al [81 and URO and ANF effects on all measured parameters were notSaxenhofer, Fitzgibbon and Paul in rats [36]. URO and ANF significantly different from each other. effects were almost identical in one study [36], while URO was In two experiments on hydronephrotic kidneys using the sameabout two times more potent than ANF in the other study [81. infusion protocol for URO as in the clearance experiments, effects In humans and dogs, however, URO was consistently found to of URO on GBF and vessels were similar to those in series 3. Atbe the more potent natriuretic substance compared with ANF [6, 4.8 10_li mol kg1 -min'(E3) GBF increased by 14 and 24% 7,9]. In contrast to ANF, URO is not inactivated by peptidases in JM glomeruli and by 21 and 41% in C glomeruli, and wasfrom dog kidney cortex membranes [37]. It was therefore argued further increased by the two higher infusion rates. that intravenously applied URO may reach intraluminal receptors Endlich et al: Renal effects of urodilatin 1563

OBE

Urodilalin Lv. vs. control

+30 +75 +20 +50 - * +10 T8'+25 0 Ii.1I _____ 2 3 4 5 6 cu-I;. 5' 6' "911JI p —10 * —25 ** 4 B Urodilatin after 2 x 106 mol• kg' Quinapill iv.

0 01

—10 cii* — fl25jI Urodilatin after 2.8 x 10 mol • r' indomethacin in bath

+20 +50

+10 +25 •1 0) —10 -25

C)

a) Urodilatin after Quinapril + indomethacin —10

* E * Urodilatin* after — 1x 10 'mol • r' L-NAME in bath o +50 * +10 [11 +9! + Fig.2. Percent changes of renal vascular —20 * diameters and GBF of C (El, ) and JM (LI, 1111) glomeruli in response to URO only [0.4 El, ) and 0.9 (l)JOmolkg'. min' iv.] 4 F Urodilatin aftor 4 x 10 niol • I HOE 140 in bath and to URO after different pretreatments. Data +30 +75 * are mean SEM.*P< 0.05vs. pretreatment, * ° P<0.05vs. URO without pretreatment. Preglomerular: (1) to (6) cf. Fig. 1; (5') JM afferent arterioles-near interlobular arteries; +10 iiiit1 78 +: [I (6') JM afferent arterioles-near glomeruli. Postglomerular: (7)to(8) cf. Fig. 1; (7') JM 5' —10 - 1 2 3 4 5 6 6' 78' efferent arterioles-near glomeruli; (8') JM —25 efferent arterioles-near first branching point.

in the collecting duct in sufficiently high concentration to inhibitmode of application (i.v. or local). This is in agreement with our sodium reabsorption [6, 7]. While URO was as potent as ANF inprevious findings [17, 181 and those of others [19, 201. binding to and activating guanylate cyclase of inner medullary Furthermore, URO was also able to dilate JM afferent arte- collecting duct cells of the rat kidney in one study [25], it wasrioles and to constrict JM efferent arterioles (Fig. 2). However, about 10-fold less potent in another study using the same prepa-JM diameter changes were only about one third of C diameter ration [36]. changes. URO changed the diameter of JM afferent and efferent In the experiments on hydronephrotic kidneys, we observedarterioles by about 5%. Since control diameters of JM efferent preglomerular vasodilation and efferent vasoconstriction in Carterioles in our preparation are about twice those in the JM vessels during URO or ANFapplication(Fig. 1). The vascularnephron preparation (32 vs. 16 gm), such small changes could efi'ects were identical for URO and ANF and independent of thehave been below the optical resolution in the latter preparation. 1564 Endlich et a!: Renal effects of urodilatin

GBF A 2 x 10 mol • kg-1 Quinapril iv. +30 * +75 - * +20 +50 -

+10 +25 - n 0 1nhLson 0 1234567 8 5'6'7' 8 B 2.8x105mo1'L1 indomethacin in bath

0 —10 * * * * —20 *uyuu* * flfl25* —50- * UI a2

1) +50 - Ca+20 Ca C) +10 * +25 a) a) E 0 aCa —10

* D 1 x 10 mol• L' L-NAME in bath

0 VA 0

—10 * yy fl25 p* * —20 * * * —50 E4 x 10-8 mol • M HOE 140 in bath U] 123456 78 56' 7181 0- 0 * —10-IHHEU u* n"* * —25 Fig. 3. Percent changes of renal vascular yy* * * * diameters and (JBF of C (U) and JM () —20 - —50 glomemli after different treatments prior to URO infusion. Data are mean 5EM. < 0.05 vs. control.

This could account for the reported lack of efferent constriction byact via the same receptors with similar binding affinity and signal ANFat3- io° M in the JM nephron preparation [21]. At thetransduction efficacy in vivo. same concentration, however, afferent arterioles of the JM Interestingly, i.v. infusion of URO or ANFhadalready signif- nephron preparation were dilated by about 20%. In addition, theicant effects on vascular diameters and GBF in the hydronephrotic reported lack of efferent constriction could be caused by alteredkidney at 0.9 10_li mol kg min. In contrast, sodium ex- basal levels of endogenous vasoactive hormones interfering withcretion as well as blood pressure were not affected at 1.2W lO URO. In spite of the small effects of URO on JM vessels, increasemol kg1 min' URO or ANF, and they did not significantly in GBF of C and JM glomeruli was very similar. A participation ofchange until a tenfold higher dose was infused in the clearance medullary blood flow in mediating natriuresis caused by UROexperiments. In the study of Saxenhofer et al on rats, bolus cannot be excluded from our experiments, while the results of twoinjection of 10 mol kg URO already increased sodium other studies suggest that medullary blood flow is not primarilyexcretion about fourfold [36]. These results suggest that the renal involved [38, 39]. Since URO and ANFpossessequipotentvasculature is sensitive enough to natriuretic peptides to partici- vascular effects in our preparation, both peptides most probablypate in elevating sodium excretion. While an increase in GFR was Endlich et al. Renal effects of urodilatin 1565

GBF, .% A lx 1OmoI'r' BQ123+I x 10moI•r' IRLIO38inbath

++ + + + + + +251+ 0 n i_i_i_itt— o1 n 1 2 3 4 5 67 8 5' 6' 7'8'

B Urodilatin Lv. after BQ 123 + IRL 1038 £ +20 +501 * I +10 ** **i * +25 0 00 0; 0 o11• C, •rIrItàLL C tt C Washout a +10 +251

0 T Fig.4. Percent changes of renal vascular diame- a ters and GBF of C (LI, U) and JM (P4 [II) glomendi in response to URO [0.9 (LI, )and2.0 ( D Urodilatin iv. after washout FEE) 10-11molkg min iv.] afterETreceptor ** blockade (local application of 106 M BQ 123 and * +50 10-6 M IRL 1038) and after washout of ETantag- +25 onists. Diameter and GBF changes after ET receptor blockade and after washout are shown 11111178 S7'B' as percent changes from the first control values. N = data are mean SEM.+P <0.05vs. 1 2 3 4 5 6 5' 6' qi1j* 7; * control, *D< 0.05vs. ET receptor blockade or I wash out, °P <0.05vs. URO after washout.

Table 2. Control values of urine volume flow (V), urinary sodium sufficiently high to affect the renal vasculature. Presently, how- excretion (UNaV), GFR, blood pressure and body weight (during ever, the paracrine route by which URO increases sodium excre- collection period C3, mean SEM,per kidney) tion is a matter of speculation [12]. Nonetheless, vascular effects Treatment evoked by i.v. infusion of URO are representative for those Saline ANF URO natriuretic peptides that bind to NPR-A and NPR-C receptors, (N=6) (N=6) (N=6) like ANF and BNP. V pJmin' 6.1 1.7 4.4 1.2 6.53.2 In the other part of our study we examined the vascular effects UNaV pmolmin' 0.56 0.16 0.31 0.16 0.52 0.36 of URO after blockade of several vasoactive hormones that could GFR ml min1 0.88 0.15 1.06 0.15 0.83 0.08 possibly interfere with preglomerular vasodilation and efferent Blood pressure mm Hg 115.0 1.3 116.7 1.1 113.3 2.5 constriction. ACEI and ET receptor blockade revealed a basal 203 3 2084 2084 Body weightg tone of vasoconstriction by angiotensin II (Ang II) and ET (Figs. 3 and 4). However, ACE inhibitors not only block the formation of Ang II, they also induce an endothelium-dependent vasodila- frequently measured at higher doses, sodium excretion was re-tion by the release and the diminished degradation of bradykinin ported to increase about threefold in spite of unchanged GFR atand by a direct interaction at the bradykinin B2 receptor level or lower doses [6, 7, 36]. However, since basal sodium excretion istransduction process [42, 43]. Further, ACE inhibitors increase less than 1% of the filtered load, an increase of GFR by a fewangiotensin I levels which could be enzymatically degraded to percent may be sufficient to increase sodium excretion measur-angiotensin-(1-7) [441. This peptide may also induce endothelium- ably. Presently, measurement of GFR with such a high accuracy isdependent vasodilation [45]. In conclusion, it remains unclear in technically impossible. By micropuncture of end-proximal tubulesthe present study to what extent the observed vasodilation after Cogan found that ANF increased GFR and solute delivery out ofACEI is due to inhibition of Ang II formation. A basal constrictor proximal tubules by the same percentage [40]. Further, if GFRtone caused by ET in our preparation has also been shown by was kept at control level during ANF application by an aorticusing anti-ET-1/3-antibodies [46]. clamp, natriuresis was substantially reduced [40, 41]. CYOI, blockade of NO synthase, and blockade of bradykinin B2 It is generally accepted that ANF and perhaps BNP, which arereceptors revealed basal vasodilation by dilating prostaglandins, secreted into the blood, are capable of modulating renal hemo-by NO release, and bradykinin (Fig. 3). A basal predominance of dynamics. URO most likely acts as a paracrine , since itdilating prostaglandins are in agreement with our previous obser- could not be detected in human plasma (detection limit aboutvation of local CYOI [30], whereas systemic CYOI in female 0.1 10— 12 M) [5]. URO is synthesized in cells of the connectingWistar rats only decreased medullary blood flow [47]. One reason tubule and secreted into the lumen. If URO is also basolaterallymight be the surgical trauma inherent in the preparation of the secreted, concentrations of URO in the interstitium might behydronephrotic kidney. CYOI after ACEI only constricted JM 1566 Endlichet al: Renal effects of urodilatin

10 20

Sc,)C) —0 0) a)— cC) 0 liz 20 mm 5 10.5. .a)za) 5 (U2. a)

C1 C2 C3 E1 E2 E3 C4 E5 R1R2 C1 C2C3 E E2 E3 E4 E5 R1 Ft2 '1 a) E ÷40 E 120 Fig. 5. Relative changes of urine volume flow C +20 (V), urinaty sodium excretion (UNJ/), GFR, and 0 ir a) blood pressure during iv. infusion of saline (—— — Ca 0 a) ), URO(—•), orANF (0—0) vs. C3. N =6 —20 0. 90 each, data are mean SCM. *P<0.05 vs. C3 (U 1. C1 C2 C3 E C2 C3 E4 E5 Ft1R2 ., and saline. C1-C3 control, E1-E5 experimental a, C1 C2 C3 E E2 E3 C4 E5 A1 A2 (i.v. infusion of 1.2, 2.4, 4.8, 12, and 19 10 0E 0 mol kg1 .min1URO or ANF), R1-R2 recovery periods.

efferent arterioles (Fig. 3). Thus, ACE! seems to reduce the basalsuggest that the vascular endothelium of the efferent arteriole level of dilating prostaglandins at all C vessels and at JM afferentis crucial in mediating constriction in response to URO. arterioles. After ACEI, vascular effects of CYOI are confined toEfferent vasoconstriction by URO is most probably mediated JM vessels as observed in the normal kidney of female rats [47]. Aby the secondary release of ET. The release of a constrictor, basal release of NO has recently been shown in our preparationthrough which URO acts on efferent arterioles, was assumed [34] in line with studies in the normal rat kidney [48]. However, aon the basis of a greater latency in the onset of efferent bradykinin B2 receptor-mediated basal vasodilation, which isconstriction compared with preglomerular dilation [10]. This present in the hydronephrotic kidney, seems to be absent inrelease of ET by URO could be modulated by , prostag- conscious rats [49]. landins, and NO. Blockade of endothelial bradykinin B2 recep- Preglomerular vasodilation by URO was diminished aftertors might interfere with URO by stimulation of endothelin ACEI, combined ACEI and CYOI, and ET receptor blockaderelease, since GBF did not reach a stable state until 90 minutes (Figs. 2 and 4). A dependence of the ANF-mediated preglomeru-after the addition of the bradykinin antagonist. Many of the lar vasodilation on the presence of vasoconstrictors was alsobiological effects of bradykinin are brought about by the observed in other studies [15, 20]. As efferent constriction bysecondary release of other substances [53]. Since blockade of URO persisted after ACEI, in agreement with others [14, 20],NO synthesis enhances efferent constriction by URO, endothe- URO reduced GBF under this condition. A decrease of renalhal release of NO may serve as a negative feedback. This could blood flow by ANF after ACEI was also demonstrated in abe accomplished either by a shear stress-dependent release of clearance study on humans [50]. In another study on humans,NO in the efferent arteriole or by stimulation of NO production ACEI reduced the increase in GFR by ANF [51]. ANF was shownvia endothelial ET receptors [54] or by a direct effect of URO to elicit an endothelium-independent vasodilation [52]. Afteron NO production. blockade of prostaglandins and NO synthesis URO was still able In summary, URO and ANF possess similar natriuretic and to dilate preglomerular vessels (Fig. 2). However, the pattern ofidentical vascular effects in normal and hydronephrotic rat kid- vasodilation was shifted to larger vessels, which are the preferen-neys, suggesting similar receptor binding and signal transduction tial site of endothelium-controlled vasodilation in our preparationefficacy. Both peptides identically induce preglomerular vasodila- [46, 34]. tion and efferent constriction in the hydronephrotic kidney. URO effects on efferent arterioles after different pretreatmentsPreglomerular vasodilation, which is caused by receptor-mediated were similar in the C and JM circulation except for URO effectsactivation of guanylate cyclase in vascular smooth muscle cells, after CYOI (Figs. 2 and 4). After CYOI, URO significantlydepends on the presence of endogenous vasoconstrictors. Efferent constricted only C efferent arterioles, which could reflect thevasoconstriction by natriuretic peptides might be the result of the specific role of prostaglandins in the female renal medullarysecondary release of endothelin, which could be modulated by circulation. Blockade of NO synthesis amplified efferent vasocon-kinins, prostaglandins, and NO. striction at C and JM vessels. Efferent vasoconstriction by URO was abolished after com- Acknowledgments bined ACE! and CYOI, after blockade of bradykinin B2 This work was supported by the German Research Foundation (DFG, receptors, and after ET receptor blockade (Figs. 2 and 4).Ste 107/12-2). We gratefully acknowledge the technical assistance of Helga These results, together with the observation that Ang II, Adler, Martina Seyfert, and Nicole Schneider. thromboxane, and a-adrenergic agonists are not involved in Reprint requests to Dr. M Steinhausen, I. Physiologisches Institut der efferent constriction mediated by URO [14, 20], strongly Universität, Im Neuenheimer Feld 326, D-69120 Heidelberg, Germany. Endlich et al: Renal effects of urodilatin 1567

References 24. McK T: Receptors of atrial natriuretic factor. Annu Rev Physiol 54:11—27, 1992 25. VALENTIN J-P, SECHI LA, SCI-IAMBELAN M, HUMPHREYS MH: Urodi- 1. SCHULZ-KNAPPE P, FORSSMANN K, HERBST F, HOCK D, PIPKORN R, latin binds and activates renal receptors for atrial natriuretic peptide. FORSSMANN WG: Isolation and structural analysis of "urodilatin", a Hypertension 21:432—438, 1993 new peptide of the cardiodilatin-(ANP)-family, extracted from human 26. AALKJAER C, MULVANY MJ, NYBORG NCB: Atrial natriuretic factor urine. Kim Wochenschr 66:752-759, 1988 causes specific relaxation of rat renal arcuate arteries. Br J Pharmacol 2. GOETZ KL: Renal natriuretic peptide (urodilatin?) and atriopeptin: 86:447—453,1985 Evolving concepts. Am J Physiol 261:F921—F932, 1991 27.WANGLER RD, BREUHAUS BA, OTERO HO, HASTINGS DA, HOLZMAN 3. GREENWALDJE,RITTER D, TETENS E, R0TwEIN PS: Renal expression MD, SANEII HH, SPARKS HV JR,CHIMOSKEYJE: Coronary vasocon- of the gene for atrial natriuretic factor. Am J Physiol 263:F974—F978, strictor effects of atriopeptin II. Science 230:558—561,1985 1992 28.WooDS RL, SMOLICH JJ: Regional blood flow effects of ANP in 4. DE BOLD A.J, BORENSTEIN HB, VERRES AT, SONNENBERG H: A rapid conscious dogs: Preferential gastrointestinal vasoconstriction. Am J and potent natriuretic response to intravenous injection of atrial Physiol 261:H1961—H1969, 1991 myocardial extract in rats. Life Sci 28:89—94, 1981 29. STEINHAUSEN M, SN0EL H, PAREKH N, BAKER R, JOHNSON PC: 5. DRUMMER C, FIEDLER F, BUB A, KLEEFELD D, DIMITRIADES E, Hydronephrosis: A new method to visualize vas afferens, efferens, and GERZER R, FORSSMANN WG: Development and application of a glomerutar network. Kidney mt 23:794—806,1983 urodilatin (CDD/ANP-95-126)-speciflc radioimmunoassay. Pfiugers 30.STEINHAUSEN M, BALLANTYNE D, FRETSCHNER M, PAREKH N: Sex Arch 423:372—377, 1993 differences in autoregulation of juxtamedullary glomerular blood flow 6. BESTLE MH, BIE P: Renal effects of urodilatin and atrial natriuretic peptide in volume expanded conscious dogs. Acta Physiol Scand in hydronephrotic rats. Am J Physiol 258:F863—F869, 1990 31. WAYLAND H, JOHNSON PC: Erythrocyte velocity measurement in 149:77—83, 1993 7. HILDEBRANDT DA, MIZELLE HL, BRANDS MW, HALL JE: Comparison microvessels by a two slit-photometric method. JAppi Physiol 22:333— of renal actions of urodilatin and atrial natriuretic peptide. Am J 337, 1967 32.GAEHTGENS P, BENNER KU, SCHICKENDANTZ 5, ALBRECHT KH: Physiol 262:R395—R399, 1992 8. ABASSI ZA, POWELL JR, GOLOMB E, KEISER HR: Renal and systemic Method for simultaneous determination of red cell and plasma flow effects of urodilatin in rats with high-output heart fai!ure.Am JPhysiol velocity in vitro and in vivo. Pfidgers Arch 361:191—195, 1976 262:F615—F621, 1992 33. DIETRICH MS, STEINHAUSEN M: Differential reactivity of cortical and 9. SAXENHOFER H, RASELLI A, WEIDMANN P, FORSSMANN WG, BUB A, juxtamedullary glomeruli to adenosine-1 and adenosine-2 receptor FERRARI P, SHAW SG: Urodilatin, a natriuretic factor from kidneys, stimulation and angiotensin-converting enzyme inhibition. Microvasc can modify renal and cardiovascular function in men. Am J Physiol Res 45:122—133,1993 259:F832—F838, 1990 34. HOFFEND J, CAVARAPE A, ENDLICH K, STEINHAUSEN M: Influence of 10. ATLAS SA, MAACK T: Atrial natriuretic factor, in Handbook of endothelium-derived relaxing factor on renal microvessels and pres- Physiology, edited by EE WINDHAGER, New York, Oxford University sure-dependent vasodilation. Am J Physiol 265:F285—F292,1993 Press, 1992 35.HOCK FJ, WIRTH K, ALBUS U, LINZ W, GERHARDS HJ, WIEMER G, 11. BALLERMANN BJ, ZEIDEL ML: Atrial natriuretic hormone, in The HENKE ST, BREIPOHL G, KONIG W, KNOLLE J, SCHOLKENS BA: Hoe Kidney: Physiology and Pathophysiology, edited by DW SELDIN, G 140 a new potent and long acting bradykinin-antagonist: In vitro GIEBISCH, New York, Raven Press, 1992 studies. Br J Pharmacol 102:769—773,1991 12. VALENTIN J-P, HUMPHREYS MH: Urodilatin: A paracrine renal natri- 36. SAXENHOFER H, FITZGIBBON WR, PAUL RV: Urodilatin: Binding uretic peptide. Sem Nephrol 13:61—70, 1993 properties and stimulation of cGMP generation in rat kidney cells. Am 13. CEDIDI C, KUSE E-R, MEYER M, OLDHAFER K, RINGE B, WAHLERS T, J Physioi 264:F267—F273,1993 CREMER J, FREI U, PICHLMAYR R, FORSSMANN WG: Treatment of 37. GAGELMANN M, HOCK D, FORSSMANN WG: Urodilatin (CDD/ANP- acute postoperative renal failure after liver and heart transplantation 95-126) is not biologically inactivated by a peptidase from dog kidney by urodilatin. Clin Invest 71:435—436, 1993 cortex membranes in contrast to atrial natriuretic peptide/cardiodila- 14. DUNN BR, ICHIKAWA I, PFEFFER JM, TROY JL, BRENNER BM: Renal tin (a-hANP/CDD-99-126). FEBS Lett 233:249—254, 1988 and systemic hemodynamic effects of synthetic atrial natriuretic 38. KIBERD BA, LARSON TS, ROBERTSON CR, JAMISON RL: Effect of atrial peptide in the anesthetized rat. Circ Res 59:237—246, 1986 natriuretic peptide on vasa recta blood flow in the rat. Am J Physiol 15. CAMARGO MJF, KLEINERT HD, ATLAS SA, SEALEY JE, LARAGH JH, 252:F1112—F1117,1987 MAACK T: Ca-dependent henodynamic and natriuretic effects of 39.TAKEZAWA K, COWLEY AW JR, SKELTON M,ROMAN RJ: Atriopeptin atrial extract in isolated rat kidney. Am J Physiol 246:F447—F456, 1984 IIIalters renal medullary hemodynamics and the pressure-diuresis 16. FRIED TA, MCCOY RN, OSGOOD RW, STEIN JH: Effect of atriopeptin response in rats. Am J Physiol 252:F992—F1002, 1987 II on determinants of glomerular filtration rate in the in vitro perfused 40. COGAN MG: Atrial natriuretic factor can increase renal solute excre- dog glomerulus.Am JPhysiol 250:F1119—F1122, 1986 17. MARIN-GREZ M, FLEMING iT, STEINI-IAUSEN M: Atrial natriuretic tion primarily by raising glomerular filtration. Am JPhysiol 250:F710— F714,1986 peptide causes pre-glomerular vasodilatation and post-glomerular 41. DAVIS CL, BRIGGS JP: Effect of reduction in renal artery pressure on vasoconstriction in rat kidney. Nature 324:473—476, 1986 atrial natriuretic peptide-induced natriuresis. Am J Physiol 252:F146— 18. STEINI-IAUSEN M, BLUM M, FLEMING JT, HOLZ FG, PAREKH N, WJEGMANDL: Visualization of renal autoregulation in the split F153,1987 42. KON V, FoGo A, ICHIKAwA I: Bradykinin causes selective efferent hydronephrotic kidney of rats. Kidney mt 35:1151—1160, 1989 19. LOUTZENHISER R, HAYASHI K, EPSTEIN M: Atrial natriuretic peptide arteriolar dilation during angiotensin I converting enzyme inhibition. reverses afferent arteriolar vasoconstriction and potentiates efferent Kidney hit 44:545—550,1993 arteriolar vasoconstriction in the isolated perfused rat kidney. J 43.HECKER M, PORSTI I, Bu AT, BUSSE R: Potentiation by ACE Pharmacol Exp Ther 246:522—528, 1988 inhibitors of the dilator response to bradykinin in the coronary 20. LANESE DM, YUAN BH, FALK SA, CONGER JD: Effects of atriopeptin microcirculation: Interaction at the receptor level. Br J Pharmacoi III on isolated rat afferent and efferent arterioles. Am J Physiol 111:238—244,1994 261:F1102—F1109, 1991 44. FERRARIOCM,BROSHINANKB,DIz DI, JAISWALN, KI-ioSLA MC, 21. VELDKAMP PJ, CARMINES PK, INSCI-lo EW, NAVAR LG: Direct MILSTED A, TALLANT EA: Angiotensin-(1-7): A new hormone of the evaluation of the microvascular actions of ANP in juxtamedullary angiotensinsystem. Hypertension18:S-III-126-—S-III-133, 1991 nephrons. Am J Physiol 254:F440—F444, 1988 45. PORSTI I, BARA AT, BUSSE R, HECKER M: Release of nitric oxide by 22. EDWARDS RM, WEIDLEY EF: Lack of effect of atriopeptin II on rabbit angiotensin-(1-7) from porcine coronary endothelium: Implications glomerular arterioles in vitro. Am J Physiol 252:F317—F321, 1987 for a novel angiotensin receptor. Br J Pharmacol 1111:652—654,1994 23. KOLLER KJ, GOEDDEL DV: Molecular biology of the natriuretic 46.GULBINS E, HOFFEND J, Z0U AP, DIETRICI-I MS, SCHLOTTMANN K, peptides and their receptors. Circulation 86:1081—1088, 1992 CAVARAPE A, STEINHAUSEN M: Endothelin and endothelium-derived 1568 Endlich et al: Renal effects of urodilatin

relaxing factor control of basal renovascular tone in hydronephrotic Opposite effects of enalapril and nitrendipine on natriuretic response rat kidneys. J Physiol 469:571—582, 1993 to atrial natriuretic factor. Hypertension 13:173—180, 1989 47. PAREKFI N, Zou AP, JONGLING I, ENDLICH K, SADOWSKI J, STEIN- 51. WAMBACH G, SCHITrENHELM U, STIMPEL M, BONNER G, KAUFMANN HAUSEN M: Sex differences in control of renal outer medullary W: Natriuretic action of ANP is blunted by ACE inhibition in humans. circulation in rats: Role of prostaglandins. Am J Physiol 264:F629— J Cardiovasc Pharmacol 13:748—753, 1989 F636, 1993 52. WINQUIST RJ, FAIs0N EP, WALDMAN SA, SCHWARTZ K, MURAD F, 48. BAYLIS C, HARTON P, ENGELS K: Endothelial derived relaxing factor RAPOPORT RM: Atrial natriuretic factor elicits an endothelium- controls renal hemodynamics in the normal rat kidney. JASN 1:875— independent relaxation and activates particulate guanylate cyclase in 881, 1990 vascular smooth muscle. Proc Natl Acad Sci USA 81:7661—7664, 1984 49. MADEDDU P, ANANIA V, PARPAGLIA PP, DEMONTIS MP, VARONI MV, 53. REGOLI D, RHALEB N-E, DPAPEAU G, DI0N S: receptor PisANu 0, TROFFA C, ToNoLo G, GLoRloso N: Effects of Hoe 140, a subtypes. J Cardiovasc Pharmacol 15:S30—S38, 1990 bradykinin B2-receptor antagonist, on renal function in conscious 54. HIRATA Y, EM0RI T, EGUCHI 5, KANNO K, 1ati T, OHTA K, MARUMO normotensive rats. Br J Pharmacol 106:380—386, 1992 F: subtype B mediates synthesis of nitric oxide by 50. GAILLARD CA, KOOMANS HA, RABELINK TJ, BOER P, MEES EJD: cultured bovine endothelial cells. I Clin Invest 91:1367—1373, 1993