Urodilatin, a natriuretic factor from kidneys, can modify renal and cardiovascular function

in men

HERMANN SAXENHOFER, ADOLF0 RASELLI, PETER WEIDMANN, WOLF-GEORG FORSSMANN, ACHIM BUB, PAOLO FERRARI, AND SIDNEY G. SHAW Medizinische Poliklinik, University of Berne, 3010 Berne, Switzerland; and Anatomisches Institut, University of Heidelberg, 6900 Heidelberg, Federal Republic of Germany

SAXENHOFER, HERMANN, ADOLFO RASELLI, PETER WEIDMANN, WOLF-GEORG FORSSMANN, ACHIM BUB, PAOLO FERRARI, AND SIDNEY G. SHAW. Urodilatin, a natriuretic factor

from kidneys, can modify renal and cardiovascular function in men. Am. J. Physiol. 259 (Renal Fluid Electrolyte Physiol. 28): F832-F838, 199O.-Urodilatin is a newly identified analogue of human atria1 natriuretic factor-(99-126) [ANF-(99-126)], which has recently been isolated from human urine and has 32 amino acid residues [ANF-(95-126)]. To investigate renal and cardiovascular effects in men, eight healthy subjects received injections of 25, 50, and 100 ,ug urodilatin iv compared with 50 pug ANF-(99-126) and placebo. Blood pressure decreased (P < 0.05) after 50 pug ANF-(99-126), whereas urodilatin lowered diastolic blood pressure only at the highest dose (P < 0.01). Heart rate increased (P < 0.05-0.01) dose dependently after urodilatin injections. Glomerular filtration rate rose after 100 pug (from 120 t 3 to 156 t 7 mlomin-l* 1.73 m-“, P < 0.001) and 50 pg urodilatin (from 116 t 7 to 149 t 13 mLrnir-?= 1.73 mV2, P < 0.01) but not after 25 pg urodilatin, ANF-(99-126), or placebo. Effective renal plasma flow was not significantly modified. Diuresis and excretion of sodium, chloride, and guanosine 3’,5’-cyclic monophosphate increased (P < 0.001) dose dependently; effects of 25 I_cgurodilatin equaled those of 50 lug ANF-(99-126). Plasma renin, aldosterone, and catecholamines were unchanged. We conclude that urodilatin can acutely modify renal and cardiovascular function in men and seems to exert more potent renal effects than ANF-(99-126). atria1 natriuretic factor; glomerular filtration renal plasma flow; diuresis; natriuresis; urinary cyclic monophosphate; blood pressure

rate; effective guanosine 3 ’ ,5’ -

THE DISCOVERY of atria1 natriuretic factor (ANF) (6), several molecular forms have been recognized. The majority of these peptides appear to be products of a single gene, transcribed in heart atria and ventricles, lung, adrenal medulla, some vessels, and various parts of the nervous system (3, 13, 15, 16, 22, 28). In mammalian atria1 myocytes, a prohormone containing 126 amino acids [ANF-(l-126)] is stored. The biologically active ANF-(99-126), which has 28 amino acid residues of the COOH terminus of the prohormone, is secreted into the bloodstream (12, 37, 40). Synthetic ANF-(99-126) can modulate hemodynamics, produce an extravascular shift of fluid volume (40), enhance diuresis and natriuresis (5, 9, 4O), inhibit renin and aldosterone production (8, 20, 41), and lower blood pressure (BP) (9, 39, 41). There is increasing evidence that endogenous ANF-(99-126) may exert similar actions (2, 5). SINCE

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It has been suggested that ANF is excreted in the urine, as immunoreactive (ir) material could be detected in normal and diseased individuals (26). However, ANF immunoreactivity in urine did not correlate with levels in blood (26). Recently a new 32-amino acid ANF peptide was isolated from human urine and termed urodilatin (31). Urine and blood ANF immunoreactivity were identified as two different molecular forms. Compared with the circulating ANF-(99-126), the NH, terminus of urodilatin is extended by four amino acid residues (ThrAla-Pro-Arg). Urodilatin accounts largely for the ANF immunoreactivity in human urine but could not be detected in plasma (C. Drummer, F. Fiedler, R. Gerzer, A. Bub, and W. G. Forssmann, unpublished observations). Moreover, inactivation of bioactive circulating ANF(99-126) occurs by proteolysis and phosphorylation, whereas urodilatin is, in contrast, strongly inert to proteolytic cleavage by endopeptidase (EC 3.4.24.11) (14), the major ANF-(99-126)-degradating activity in the kidneys (36). On the basis of immunohistological studies (10, II), urodilatin originates probably in the kidney, although no gene expression of the ANF or urodilatin gene could so far be detected (H. J. Magert, unpublished data). Bioactivity of urodilatin was demonstrated in a vascular smooth muscle relaxation assay (14). Moreover, initial observations revealed a vasorelaxant as well as a strong natriuretic action in the rat (27). To investigate whether urodilatin may modify cardiovascular and renal function in men, we assessed the effects of different intravenous doses of synthetic urodilatin compared with a standard dose of 50 pugANF-(99-126) and placebo on plasma urodilatin or ANF; BP; indexes of hemoconcentration; effective renal plasma flow (ERPF); glomerular filtration rate (GFR); urinary excretion of water, electromonophosphate lytes, and guanosine 3’ ,5’ -cyclic (cGMP); and blood levels of renin, aldosterone, and catecholamines. SUBJECTS

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Subjects. Eight healthy men aged 20-30 yr were investigated. All had a BP consistently less than 140/90 mmHg, and none was taking drugs. They were instructed to follow a diet containing 130-150 mmol sodium and 60-80 mmol potassium daily for at least 3 days before each study day. The study was approved by the Ethical Committee of the University of Bern, and all subjects

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gave written informed consent to undergo the protocol described below. Synthetic urodilatin and ANF-(99-126). Urodilatin was derived by Merrifield solid phase synthesis (27) by Bissendorf Peptide, Wedemark, FRG. It was provided as urodilatin acetate of >98% purity in the form of a lyophilized white powder. Sterile ampules containing 50 pug were used, and the urodilatin was dissolved in 1 ml of 0.9% saline. Synthetic ANF-(99-126) (Bissendorf) was also provided in sterile ampules containing 50 pg and was prepared similarly. Study procedure. The subjects were investigated on 5 different days. On each study day, they received a singleblind and in randomized order an intravenous bolus injection of 2550, or 100 pg urodilatin, 50 ,ugANF-(99126), or placebo. The interval between study days was at least 2 wk. During the day preceding each test, 24 h of urine was collected. After an overnight fast, the subjects drank, between 7 and 8 A.M., 20 ml/kg of water. Thereafter, they remained supine until the completion of the test except for voiding. At 8 A.M., plastic cannulas were inserted into an antecubital vein on each arm. GFR and ERPF were determined by constant infusion clearance techniques, using Yr-labeled EDTA and p-aminohippuric acid (PAH), respectively (29). An intravenous priming dose of 15 &i ‘ICr-EDTA and 0.5 g PAH in 50 ml of 0.9% NaCl was administered, followed by a maintenance infusion of a solution containing 13 &i “‘CrEDTA and 0.5 g PAH/l. Carrier solution consisted of one-third 0.9% NaCl and two-thirds 5% glucose and was administered at an infusion rate of 0.2 ml. min. kg-’ by a calibrated pump (Perfusor V, Braun, Melsungen, FRG). Urine produced during an initial 60-min equilibration period was discarded. Starting at 9 A.M., two clearance periods (CP) of 30 min each served as baseline (CPl and CP2). Thereafter, a bolus injection of either 25, 50, or 100 pg synthetic urodilatin, 50 ,ug ANF-(99-126), or placebo was administered, and effects were monitored during 120 min (CP3-6). Body weight was recorded before and at the completion of each test. Plasma irANF and ir-urodilatin levels were obtained before and 1, 2, 3, 5, 10, 15, 20, 25, 30, 45, 60, 75,105, and 135 min after bolus injections. BP and heart rate (HR) were obtained at -120, -60, and -1 min and, after the bolus injection, at 1-min intervals during the initial 10 min and every 10 min thereafter. Urine volume, plasma and urinary sodium, chloride, potassium, osmolality, “lCr-EDTA and PAH values, and urinary cGMP were measured in each CP. Hematocrit and plasma protein concentrations were measured before and 15, 30, 45, 75, and 105 min after bolus injections. Plasma renin activity (PRA), aldosterone, norepinephrine, epinephrine, and dopamine levels were measured in CP2-6. Blood samples were obtained at the midpoint of each CP Analytical methods. BP was determined with a standard cuff and the semiautomatic recorder Sphygmo-Digital (Asulab, Neuchatel, Switzerland). Hematocrit was measured by the microcrit method; plasma and urinary “‘Cr-EDTA activity were measured in a gamma-counter (Tri-Carb-Scintillation Spectrometer, Packard Instruments, Downers Grove, IL); PAH was measured by

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FIG. 1. Plasma levels of urodilatin, measured specific radioimmunoassay (RIA) after urodilatin injections. Data are means t SE.

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standard photometric method; and sodium, potassium, and chloride were measured by autoanalyzer (Greiner, Langenthal, Switzerland). Osmolality was determined by freezing point depression, using a cryoscope with a Peltier element. Plasma urodilatin concentrations were measured with a urodilatin-specific radioimmunoassay (RIA) developed from a synthetic peptide containing the Thr-Ala-Pro-Arg epitope as an immunogen. This assay does not cross-react with circulating ANF-(99-126) (Drummer et al., unpublished observations). The coefficient of intra-assay averaged ~5% (n = 3), and the coefficient of interassay variation was 1000 c, ..-> z 800

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2. Plasma levels of urodilatin and atria1 natriuretic factor(99-126) [ANF-(99-126)] measured as immunoreactive ANF by RIA after urodilatin, ANF-(99-126), or placebo bolus injections. Data are means t SE.

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t 20, 407 t 126, and 1,123 t 270 pmol/l, respectively. Peak plasma concentrations were reached l-2 min after injection and decreased rapidly with a half-life of 2-3 min (Fig. 1). Thirty to 45 min after injection, urodilatin had completely disappeared from the circulating blood. Analyzed with the ANF RIA, basal plasma irANF values did not differ significantly among the 5 study days (Table 1, Fig. 2) and were unchanged after placebo administration. After urodilatin bolus injections of 25, 50, and 100 pg, plasma ANF immunoreactivity increased to peak levels of 184 t 56,365 t 94, and 1,545 t 352 pmol/ 1 (Fig. 2). Injection of 50 pg ANF-(99-126) increased plasma irANF to 678 & 112 pmol/l with similar kinetics as urodilatin (Fig. 2). Blood pressure and heart rate. Basal values did not differ significantly among the 5 study days and were

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FIG. 3. Effects of urodilatin, ANF-(99-126), or placebo bolus injections on blood pressure and heart rate in 8 normal subjects. Data are means * SE. bpm, beats/min.

stable after the injection of placebo (Fig. 3). Systolic BP was lowered significantly (P < 0.05) after ANF-(99126) but not after urodilatin injections. Diastolic BP decreased acutely within 1 min after 50 pg ANF-(99126) (P < 0.05) or 100 pg urodilatin (P < 0.01); a similar tendency after the lower urodilatin doses did not reach statistical significance. HR was unchanged after placebo or ANF-(99-126) but increased after 25, 50, or 100 pug

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of urodilatin (P < 0.05 and P < 0.01, respectively); effects of urodilatin appeared to be dose dependent. Moreover, cardiovascular effects of both urodilatin or ANF-(99126) dissipated 20 min after bolus injections. Fluid electrolyte state and renal function. The basal fluid electrolyte state and renal function did not differ significantly among the 5 study days (Table 1). ERPF was stable after placebo and not significantly modified after ANF- (99-126) or urodilatin injections (Fig. 4). GFR rose after urodilatin injection, with marked increases after 100 pg (from 120 t 3 to 156 t 7 ml. min-’ . 1.73 rnR2, P < O.OOl), and 50 ,ug (from 116 t 7 to.149 t 13 mlomin-l* 1.73 mB2, P < 0.01) (Fig. 4). Effects of urodilatin bolus injection on GFR lasted 30 min. In contrast, variations in GFR after 25 pg urodilatin or 50 pg ANF-(99-126) did not differ from placebo (Fig. 4). FF was stable after placebo but increased during the initial 30 min after urodilatin injections (P < 0.05 to P < 0.01). A tendency for a rise after 50 lug ANF-(99-126) did not reach statistical significance (Fig. 4). Diuresis and the fractional excretion rates of sodium and chloride were unchanged by placebo but increased very markedly after 50 or 100 pg of urodilatin (P < 0.001) (Fig. 5). Fifty micrograms of ANF-(99-126) had less pronounced effects (about one-half)(P < 0.01 to P < O.OOl), the magnitude of which was similar to that of 25

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FIG. 4. Effects of urodilatin, ANF-(99-126), tions on renal function in 8 normal subjects. CP, clearance period.

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