Renal Effects of a Urodilatin Infusion in Patients with Liver Cirrhosis, with and Without Ascites

J Am Soc Nephrol 9: 1489-1498, 1998

Renal Effects of a Urodilatin Infusion in Patients with Liver Cirrhosis, with and without Ascites

JAN CARSTENS,* JACOB GREISEN,t KAARE T. JENSEN,* HENDRIK VILSTRUP,t and ERLING B. PEDERSEN* *Research Laboratory of Nephrologv and Hypertension and Department of Medicine V, Aarhus University Hospital, Aarhus, Denmark.

Abstract. This study reports the effects of a short-term (60 mm) creased significantly, indicating a tubular effect of URO on low-dose (20 ng . kg . min ‘) infusion of synthetic urodilatin sodium handling. Filtration fraction, lithium clearance (a (URO) in patients with liver cirrhosis. URO is a natriuretic marker of end-proximal fluid delivery), and fractional excre- peptide. A total of 15 cirrhotic patients with ascites and nine tion of lithium increased, fractional proximal tubular sodium without ascites participated in a randomized, double-blind, reabsorption decreased, and absolute proximal tubular sodium placebo-controlled study in a crossover design. Renal hemo- reabsorption remained unchanged, suggesting increased deliv- dynamics were estimated by a clearance technique using ra- ery of isotonic fluid from the proximal tubule during URO dioactive tracers, and tubular handling of sodium was evalu- infusion. In addition, a significant decrease in fractional distal ated by the lithium clearance method. The renal effects of URO tubular sodium reabsorption contributed to the natriuresis. In were characterized by a significant increase in urine sodium conclusion, URO improved sodium and urine output in cir- excretion rate (UNa) and urine flow rate (V) in the cirrhotic rhotic patients with and without aseites by enhancing fluid patients without ascites (UNa: 173%; V: 94%) and with ascites delivery from the proximal tubules in addition to inhibiting

(UNa 219%, P < 0.01; V: 42%, P < 0.01) when compared fractional sodium reabsorption in the distal nephron. with placebo infusions. Fractional excretion of sodium in-

The human atrial natriuretic peptide (ANP) has been investi- onstrated that short-term infusion of synthetic URO caused a gated as an alternative agent in treatment of aseites because of significant natriuresis and diuresis with a threshold at a dose of its unique effects of increasing GFR, inhibiting sodium reab- 20 ng . kg ‘ . min (14). Whether infusion of URO has any sorption in the inner medullary collecting duct (IMCD), and beneficial renal effect in cirrhotic patients with sodium-water suppressing the renin-aldosterone system (1,2). Thus far, how- retention is unknown. It is hypothesized that URO might be a ever, the benefits from synthetic ANP in cirrhotic patients with good candidate to cause selective natriuresis in cirrhotic pa- ascites have been variable and relatively unimpressive, often tients because URO may stimulate GFR and reduce tubular limited by an intolerable drug-induced hypotension (3-6). sodium reabsorption without compromising the BP. This study Another natriuretic peptide named urodilatin (URO) has been was undertaken to investigate the natriuretic and diuretic ef- shown to be a stronger natriuretic and diuretic agent than ANP fects, to delineate the tubular sites of action of URO using the in healthy humans (7) and in experimental animal models of lithium clearance method (I 5), and to determine the effects on congestive heart failure (8). The mechanism underlying the renal hemodynamics, BP, and salt-water regulating hormones. greater potency of synthetic URO has not been clarified, but its resistance to the neutral endopeptidase degradation might be an Materials and Methods important factor (9). Endogenous URO is synthesized in the Patients kidney and secreted into the tubular lumen where it inhibits The inclusion criteria were: (1) presence of cirrhosis in a liver sodium reabsorption in the distal nephron (10,1 1). Specific biopsy; (2) in the absence of a liver biopsy, anamnestic, clinical, and guanylate cyclase-coupled receptors in the IMCD are the phys- laboratory evidence of cirrhosis including ascites (verified by ultra- iologie target sites for URO, and cGMP is the second messen- sound), esophagogastric varices (verified by gastroseopy), portal hy- ger for URO’s biological actions (12,13). pertension (verified by liver catheterization), hypoalbuminemia, and In a recent dose-response study with healthy men, we dem- reduced prothrombine index; and (3) written informed consent to participate in the study. The exclusion criteria were: clinical and laboratory evidence of (1) severe renal disease (serum ereatinine > Received May 29, 1997. Accepted January 30. 1998. 200 p.mob/L); (2) congestive heart failure; (3) arterial hypertension; Correspondence to Dr. Jan Carstens, Research Laboratory of Nephrology and (4) diabetes melbitus; (5) anemia (hemoglobin < 6.5 mmolIL); and (6) Hypertension, Aarhus University Hospital, Section Aarhus Amtssygehus. Tage history of bladder dysfunction. The withdrawal criteria during or after Hansens Gade 2, 8000 Aarhus, Denmark. the study were: (1) development of severe adverse events during or

1046-6673/0908- 1489$03.00/0 after infusion; (2) incomplete emptying of the urinary bladder dem- Journal of the American Society of Nephrology onstrated by a difference of 20% or more in GFR during the two Copyright © 1998 by the American Society of Nephrobogy preinfusion clearance periods. 1490 Journal of the American Society of Nephrology J Am Soc Nephrol 9: 1489-1498, 1998

Table 1. Clinical and biochemical characteristics of the patients’

Characteristic Cirrhosis with Aseites Cirrhosis without Ascites

tl 15 9 Age (yr) 50.6 ± 5.6 50.9 ± 10.4 Weight (kg) 72.6 ± 14.0 71.5 ± 18.2 Systolic BP (mmHg) 1 16 ± 19 1 17 ± 12 Diastolic BP (mmHg) 70 ± 8 69 ± 8

Pulse rate (min ‘ ) 64 ± I2 57 ± 1 1 24-h urinary sodium (mmol/d) 1 25 ± 60 1 32 ± 63 Prothrombin index 0.69 ± 17 0.77 ± 0.18 p-bilirubin (jmolIL) 19 ± 9 22 ± 10 p-abanine aminotransferase (UIL) 26 ± 15 48 ± 40 p-alkaline phosphatase (UIL) 22 1 ± 86 388 ± 260 p-albumin (gIL) 37 ± 4 35 ± 3

p-creatinine (jtmoblL) 72 ± 10 79 ± 18 p-sodium (mmol/L) I 37 ± 2 139 ± 2 p-potassium (mmollL) 4.0 ± 0.4 3.8 ± 0.3

b-hemoglobin (mmolIL) 8.2 ± 0.8 7.8 ± 0.4 b-thrombocytes(109/L) 121 ±69 144± 111

a Results are mean ± SD.

Table 2. GFR, ERPF, and FF before (periods 1 and 2), during (periods 3 and 4), and after (periods 5 and 6) infusion with URO and placebo in I 5 cirrhotic patients with ascites and nine patients without ascitesa

Preinfusion Infusion Infusion Postinfusion Postinfusion Parameter Periods 1 and 2 Period 3 Period 4 Period S Period 6 (0 mm) (30 mm) (60 mm) (90 mm) (120 mm)

GFR (m1’ . min’ 1.73 m2) ascites URO 90±23 98±31 96±21 98±49 81±25 placebo 91 ± 23 84 ± 24 93 ± 30 91 ± 27 90 ± 23 no ascites URO 77±19 86±25 81±18 74±19

placebo 81 ± 25 73 ± 27 81 ± 19 72 ± 22 72 ± 22

ERPF (m1’ . min’ 1.73 m2) ascites URO 429 ± 85 430 ± 78 396 ± 67 390 ± 146 359 ± 93C

placebo 427 ± 80 392 ± 90 426 ± 1 12 413 ± 91 416 ± 99 no ascites

URO 395 ± 120 410 ± 139 354 ± 93b 332 ± 72b 320 ± l22C placebo 383 ± 131 365 ± 134 389 ± 107 342 ± 118 344 ± 119 FF (%) ascites

URO 21 ± 3 23 ± 4C 24 ± 4C 25 ± 4 22 ± 3C placebo 21±3 21±3 22±3 22±3 22±2 no ascites

URO 20 ± 4 22 ± 5C 24 ± 4’ 22 ± 3C 21 ± 3b placebo 22 ± 4 20 ± 2 21 ± 4 22 ± 4 21 ± 4

a Results are given as means of the absolute values ± SD. ERPF, effective renal plasma flow; FF, filtration fraction; URO, urodilatin. b p < 0.05, significant deviation from preinfusion bevel (periods 1 and 2) within group.

C p < 0.01, significant deviation from preinfusion bevel (periods 1 and 2) within group.

Twenty-eight patients with cirrhosis were enrolled and investi- esophagogastrie varices and died 3 wk later. Another patient was gated. Twenty-five patients completed the study. One patient was withdrawn on the first study day (before infusion) because of diabetes hospitalized 1 d after the first study day because of bleeding from meblitus. A third patient simply did not want to attend the second J Am Soc Nephrol 9: 1489-1498, 1998 Renal Effects of Urodilatin in Liver Cirrhosis 1491

800 thiamin. B-combin vitamin, phytomenadione. codeine. panodil, and 750 -0- Placebo - no ascites ciprofloxacin. Medication was discontinued for 1 d before each study 700 -.- URO -no ascites 650 day. The cirrhotic patients in treatment with diuretics were in a state - Placebo -ascites 600 .-.- URO -ascites of normohydration to overhydration with mild-to-moderate beg edema ,r 550 and ascites. The patients had a normal electrocardiogram (one had a .E 500 pacemaker implanted), and none had proteinuria (albustix) or glucos- . 450 400 uria. Clinical data of the patients are given Table I . The study was .2. 350 approved by the local ethics committee and the Danish National 300 D 250 Board of Health, and it was carried out in accordance with the 200 Declaration of Helsinki.

150 -

100 - 50 - Design 0 . C I C C C Time(min) The study was double-blind and placebo-controlled. Each patient 0 30 60 90 120 was studied on two different days with an interval of 2 to 3 wk. The

16 subject received in randomized order intravenous infusion of URO and placebo during 60 mm. The dose of URO was 20 ng . kg body 14. weight . min’. The body weight was corrected for the amount of

12 abdominal fluid, estimated by a clinical examination on the study day.

,c_.10 - C Procedure ! 8- E The patients collected a 24-h urine sample before each study day,

> 6- and at 10 p.m. before the study day a lithium carbonate tablet of 300 mg was taken. None of the patients was hospitalized at the time of the 4- study. The subjects fasted from midnight. On the study day, 200 ml of

2. water was given orally every 30 mm from 7 am. until 1 p.m. The study day consisted of an equilibration phase of 120 mm (9 am. to 0. 1 1 am.), the infusion phase of 60 mm ( 1 1 am. to noon), and a 0 30 60 90 120 postinfusion phase of 60 mm (noon to 1 p.m.). Urine was collected in Time (mm) six consecutive 30-mm clearance periods from 10 am. to I p.m. After the second period at 1 1 a.m., synthetic URO (Boehringer Mannheim, INFUSI0NJ Mannheim, Germany) or placebo was infused by infusion pump with

Figure 1. Urine sodium excretion rate (UNa) and urine flow rate (V) an infusion rate of 0.2 ml kg ‘ . h . The patients were in the supine before (0 mm), during (30 and 60 mm), and after (90 and 120 mm) position, except during urination, which took place in upright position. infusion with urodilatin (URO) and placebo in 15 cirrhotic patients Urine from the clearance periods was analyzed for 5tCr-ethylenedi- with ascites and nine patients without ascites. Results are given as aminetetra-acetie acid (EDTA) and ‘25lhippuran activity, URO, so- means ± SD. * < 0.05; ** < 0.01, significant deviation from dium, potassium, lithium, osmolality, and eGMP. At the beginning preinfusion bevel (0 mm) within group. and end of each clearance period, venous blood samples were drawn for determination of 51Cr-EDTA activity, ‘25l-hippuran activity, sodium, potassium, lithium, osmolality, and hematocrit. Plasma renin concentration, angiotensin II (Angll), aldosterone (Aldo), arginine study day due to the blood samplings. One patient with cirrhosis vasopressin (AVP), brain natriuretic peptide (BNP), and cGMP were without ascites was withdrawn after the study because of incomplete determined before the start of infusion and at end of the clearance emptying of the bladder. Twenty-four patients were included in the periods 4, 5, and 6. All blood samples were taken in the supine statistical analyses. position before urination. After the drawing of a blood sample, an The mean age of the 24 patients was 50. 1 ± 5.3 yr and the average equal volume of isotonic saline was given intravenously. BP and heart body weight was 72.0 ± 15 kg. Fifteen men and nine women partie- rate were measured every 30 mm from 8:30 am. to I 1 am. During ipated in the study. Fifteen patients had liver cirrhosis with aseites and and after the infusion, the measurements were performed every IS nine patients had cirrhosis without aseites. In 21 subjects, the liver mm. The routine parameters were determined before each study day. disease was associated with excessive alcohol abuse. One patient had Sodium, potassium, and creatinine were measured in the 24-h urine. cirrhosis due to Wilson’s disease, one had primary biliary cirrhosis, and one had macronodulary cirrhosis of unknown etiology. Eighteen patients had cirrhosis verified by a liver biopsy. The seven patients in Methods whom a liver biopsy was not taken had years of excessive alcohol GFR and effective renal plasma flow (ERPF) were measured by a abuse; six of them had aseites, and six had portal hypertension. constant infusion clearance technique (16) using 5tCr-EDTA and Thirteen patients had esophagogastric varices (seven asetic patients), ‘251-hippuran as reference substances, respectively. A priming dose of and in 16 patients portal hypertension (12 ascitie patients) had been 5tCr-EDTA and ‘25I-hippuran at 9 am. was followed by continuous verified by liver catheterization (n = 10) or ultrasound (n = 6). The intravenous infusions of 5tCr-EDTA and ‘25I-hippuran until the end patients with aseites received diuretics. The diuretic treatment eon- of the postinfusion phase. The serum concentration of the radioactive sisted of either hexalactone (10 patients) or a combination therapy of tracers was kept stable by an infusion pump (VIAL Medical, Brezins, hexalactone and furosemide (five patients). The cirrhotic patients France). Serum and urinary concentrations of lithium were measured without aseites did not receive diuretic treatment. Other medication by atomic absorption spectrophotometry (Perkin Elmer 3 100). The

included propranobol, isosorbide mononitrate, omeprazole, disulfiram, routine parameters including sodium (N,)’ potassium, ereatinine, 1492 Journal of the American Society of Nephrology J Am Soc Nephrob 9: 1489-1498. 1998

800 1100 750 1000. 700 650 900. 600 800.

‘a550 C

. 500 :j 700.

-.. 450 i 600.

. 400 .2. 350 1 500. I 300 400. 250 D 300. 200 150 200. I 00 100. 50 0 0. 0 60 0 60

Time (mm) Time (mm)

Figure 2. UNa at baseline (0 mm) and after 60 mm of URO infusion in cirrhotic patients with aseites (U) and without ascites (#{149}).

hemoglobin, leukoeytes, thrombocytes, and albumin were determined method described by Pedersen et al. (19). Before the RIA, AVP was by autoanalyzer technique by the Department of Clinical Biochemis- extracted from plasma by Sep-Pak Cl8 cartridges. The minimum try (Skejby Hospital. Aarhus, Denmark). detection level was 0.5 pmol/L plasma. The CV were 13% (interassay) The clearance calculations are based on the formula: Clearance of and 9% (intra-assay). a substance X (Cs) = C X V/Ce. where C is the concentration of X BNP in plasma was measured by RIA. Immunoreactive BNP was in urine, V is the urinary output, and C is the mean value of the two extracted from plasma by use of Sep-Pak Cl8 cartridges eluted by plasma concentrations of X at the start and end of each clearance 80% ethanol in a 4% acetic acid solution. RIA was performed using period. Accepting that lithium is solely absorbed in the proximal a rabbit anti-BNP antibody without cross-reactivity with aANP and tububi and to the same degree as sodium and water, lithium clearance URO. The minimum detection level was 0.49 fmol per tube. The CV is a measure of end proximal flow of isotonic fluid into the loop of were 6% (intra-assay) and 1 1% (interassay) (20). Henle (15). GFR, ERPF, lithium clearance (CLI), and sodium clear- eGMP in plasma and urine was measured by RIA, using a com- anee (CNa) were all standardized to a body surface area of 1.73 m2. mercial kit (Amersham). The CV were 9% (interassay) and 6% (in- The tubular handling of sodium was calculated using the following traassay); the lower detectable limit for plasma eGMP was 0.3 formulas: proximal absolute reabsorption of sodium: (PARNa) nmol/L.

(GFR - Ci,) X ‘Na’ proximal fractional reabsorption of sodium and The osmolar concentration of serum and urine was determined by water: (I CLI/GFR) X 100%, distal absolute an osmometer, using freezing point depression (Advanced Cryomatie

reabsorption of sodium: (DARNa) (CL, CNa) X ‘3Na’ and distal osmometer model 3C2). BP was determined by Hawksley Random fractional reabsorption of sodium: (DFRNa) (1 CNa/CLI) X Zero Sphygmomanometer and a semiautomatic device (Takeda). 100%. Angll in plasma was measured by RIA using a modification of the method described by Kappelgaard et al. (17). RIA was performed Statistical Analyses after previous extraction from plasma by Sep-Pak Cl8 cartridges Data from 24 subjects were included in the statistical analyses. A (Waters Associates, Milford, MA). which were washed with 20% back of normality in the raw data was overcome by taking natural methanol and water. Elution was performed with 100% methanol. The logarithms of the variables. All of the statistical analyses were per- minimum detection level was 2 pmol/L plasma. The coefficients of formed on natural log-transformed data. A parametric repeated-mea- variation (CV) were 12% (interassay) and 8% (intra-assay). sures ANOVA, in conjunction with a paired samples t test, was used Aldosterone in plasma was measured using a modification of the to evaluate changes in the parameters over time both within the URO method of Rask-Madsen et al. (18). RIA was performed after extrac- group and the placebo group. For paired comparison within groups, tion from plasma by Sep-Pak Cb8 cartridges. The minimum detection data from the clearance periods 3, 4, 5, and 6 were compared with level was 42 pmollL. The CV were 13% (interassay) and 9% (intra- preinfusion values (average values of the measurements in clearance assay). periods 1 and 2). For comparisons between the URO group and the The quantitative determination of active renin in plasma was mea- placebo group, a paired samples t test was used, and comparisons sured by a commercial immunoradiometric assay (Nichols Institute between the two cirrhotic groups were carried out by an unpaired Diagnostics, Geneva, Switzerland). The CV were 7.4% (interassay) samples t test. The differences between groups were based on the and 2.5% (intra-assay). relative changes () from preinfusion level. A P value of 0.05 was the AVP in plasma was measured by a slight modification of the limit of significance. The mean values of the relative changes () J Am Soc Nephrol 9: 1489-1498, 1998 Renal Effects of Urodibatin in Liver Cirrhosis 1493

7,0 given in Table 2. ERPFURO was significant compared with 6,5 ERPFpIacebo in the group without aseites (period 4: 6,0- -0---- Placebo -no ascites 5,5 - -.- URO -no ascites URO, -9%; PL, 4%; P < 0.05) and in those with ascites 5,0 -U--- Placebo -ascites (period 6: URO, - 17%; PL, -3%; P < 0.05). 4,5 - -.-- URO -ascites Filtration fraction FF (GFR/ERPF) values during and after e 3,5 URO and placebo infusion are shown in Table 2. FFURO was 3,0. U. 25 significant compared with ZFFPICIceh() in the cirrhotic patients 2,0 without ascites (period 4: URO, 19%; PL, - 1%; P < 0.01) and 1,5 to in the group with ascites (period 4: URO, 16%; PL, 3%; P < 0,5 0.01). 0,0 C C C CC Time (mm) 0 30 60 90 120 44. Urine Sodium Excretion Rate (UN,) and Fractional 42 40 Excretion of Sodium (FEN(,) 38 UNa values during and after URO and placebo infusion are 36 34. shown in Figure 1. The cirrhotic patients with ascites had a 32 30 significant lower UNa (P 0.048) fl periods 1 and 2 (prein- 28 :i 26 fusion) than those without ascites. zUNaURO was significant 24 22 compared with LUNapIaceb() in the group with ascites (period 4: 20 18 URO, 219%; PL, 25%; P < 0.01) and in the group without 16 14 ascites (period 4: URO, 173%; PL, 6%; P < 0.01). UNaURO 12 was about the same size in the two cirrhotic groups (P > 0.05). 10 100 All nine cirrhotic patients without ascites and 12 of the 15 with 98 ascites had a natriuretic response to URO (Figure 2).

96 Na values during and after URO and placebo infusion are 94 shown in Figure 3. FENaURO was significant compared with 92 IFENapIacebo in the group with aseites (period 4: URO, 177%; 90 PL, 20%; P < 0.01) and in the group without ascites (period 4:

URO, 153%; PL, 6%; P < 0.01). 86 84 Urine Flow Rate (V) 82 V values during and after URO and placebo infusion are 80 Time (mm) depicted in Figure 1. VURO was significant in the group 0 30 60 90 120 without ascites (period 4: URO, 94%; PL, -4%; P < 0.01) and

INFUSION in the group with ascites (period 4: URO, 42%; PL, - 19%;

P < 0.01). VURo in the cirrhotic patients without ascites was Figure 3. Fractional excretion of sodium (FENa). fractional excretion larger (P < 0.05) than the change in the ascites group. of lithium (FELl), and distal fractional reabsorption of sodium (DFRNU) before (0 mm), during (30 and 60 mm), and after (90 and 120 Lithium Clearance (CL,) and Fractional Excretion of mm) infusion with URO and placebo in 15 cirrhotic patients with aseites and nine patients without aseites. Results are given as means ± Lithium (FEL,)

SD. * < 0.05; **D < 0.01, significant deviation from preinfusion CLI values during and after URO and placebo infusion are level (0 mm) within group. given in Table 3. C[1JRo was significant compared with CLiplaceb() in the group without ascites (period 4; URO, 25%;

PL, -2%; P < 0.05). FEL values are given in Figure 3. from preinfusion bevel are selected for presentation in the Results ZFELIURO was significant in the cirrhotic patients without section below. aseites (period 4: URO, 19%; PL, -6%; P < 0.01). In the acites group, CLiURO and FELIURO were not significant Results compared with the changes during placebo. Renal Hemodynamics GFR values during and after URO and placebo infusion are Proximal Absolute (PARN(,) and Fractional shown in Table 2. LGFR during URO infusion (LGFRURo) (PFRN,JHO) Reabsorption of Sodium was significant compared with the change during placebo PARNa did not change significantly within groups during URO (LGFRpIaceb) in the cirrhotic patients without ascites (period infusion or compared with placebo (data not shown). PFRNO

3: URO, 13%; Placebo (PL), - 10%; P < 0.05). However, this values are shown in Table 3. ZPFRNO during URO infusion significant difference disappeared in clearance period 4. was significant compared with the change during placebo in those

LGFRURO was not significant compared with ZGFRplacebo in without ascites (period 4: URO, -7%; PL, 2%; P < 0.05). There the aseites group. was no significant difference between APFRN*), URO and ERPF values during and after URO and placebo infusion are L1PFRN,,IO, placebo in the aseites group. 1494 Journal of the American Society of Nephrology J Am Soc Nephrol 9: 1489-1498, 1998

Distal Absolute (DARN(Z) and Fractional(DFRNa) bevels of renin, Angli, and aldosterone were higher (P < 0.05) Reabsorption of Sodium in the patients with ascites than in those without ascites. DARNavalues during and after URO and placebo infusion are &enin, zAngII, and L\Aldo induced by URO were not signif- given in Table 3. LDARNaURO was significant in the cirrhotic icant compared with the hormone changes during placebo. patients without ascites (period 3: URO, 12%; PL, - 10%; P < Preinfusion levels of AVP were identical in the two cirrhotic 0.05), but not in those with ascites. DFRNa values during groups. LAVP induced by URO was significant compared with infusion of URO and placebo are illustrated in Figure 3. placebo in the aseites group (period 4: URO, 97%; PL, -6%; DFRNaURO was significant both in the group without ascites P < 0.01) and in the group without ascites (period 4: URO, (period 4: URO, -7%; PL, -0.9%; P < 0.05) and with aseites I 14%; PL, 4%; P < 0.01). Plasma ANP and BNP levels were (period 4: URO, -5%; PL, -0.1%; P < 0.05). LDFRNaURO in elevated in both cirrhotic groups before infusion (with ascites, the cirrhotic patients without ascites was larger (P < 0.01) than ANP: 12.4 ± 7.6 pmol . L’, BNP: 6.2 ± 6.1 pmol . L) the change in the ascites group. (without ascites, ANP: 14.1 ± 8.7 pmol . L’, BNP: 11.3 ±

1 1. 1 pmol . L ) In our laboratory, the normal range for Second Messenger cGMP healthy subjects was ANP: 2.4 (0.6 to 4.0) pmol . L ‘ and Plasma concentrations of eGMP and urine excretion rates of BNP: 1 .6 (0.8 to 3. 1) pmol . L ‘ . The hormones were not cGMP are shown in Figure 4. The cirrhotic patients have measured during the infusions. preserved the ability to generate cGMP in response to URO. There was no significant difference in the second-messenger response between the two cirrhotic groups. Hematocrit Plasma Concentrations of Renin, Angil, Aldosterone, The hematoerit values are shown in Figure 5. zHemato- AVP, ANP, and BNP critURO was significant compared with the change during pla- The plasma concentrations of renin, AngII, and aldosterone cebo in the aseites group (period 4: URO, 5%; PL, -2%; during URO and placebo are shown in Table 4. Preinfusion P < 0.01), but not in the cirrhotic patients without ascites.

Table 3. Lithium clearance (CLI), proximal fractional reabsorption of sodium (PFRN,JO), and distal absolute reabsorption of sodium (DARNa) before (periods 1 and 2), during (periods 3 and 4), and after (periods 5 and 6) infusion with URO and placebo in 15 cirrhotic patients with ascites and nine patients without ascitesC

Preinfusion Infusion Infusion Postinfusion Postinfusion Parameter Periods I and 2 Period 3 (30 mm) Period 4 (60 mm) Period S (90 mm) Period 6 (120 mm) (0 mm)

CLI (ml . min’ 1.73 m2) ascites URO 22 ± 8 23 ± 11 26 ± 13 25 ± 11 22 ± 14 placebo 22±7 20±9 22±8 20±7 20±7 no aseites

URO 23±5 27±8b 29±7c 25±9 20±6 placebo 20±5 18±7 19±5 17±6 17±5

PFRNJH,O (%) aseites URO 74 ± 1 1 75 ± 12 72 ± 14 72 ± 12 76 ± 10

placebo 75 ± 9 75 ± 11 75 ± 10 77 ± 7 77 ± 9 no ascites URO 69±7 69±5 64±7” 67±7 70±4 placebo 75±4 76±5 77±6 77±5 77±3 DARNa (tmol/min) aseites URO 2.9 ± 1.1 2.9 ± 1.5 3.1 ± 1.9 3.0 ± 1.7 2.9 ± 1.7 placebo 2.9 ± 0.9 2.5 ± 1.4 2.9 ± 1.1 2.6 ± 0.9 2.6 ± 1.0 no ascites

URO 2.9 ± 0.7 3.4 ± 10b 0.9” 3.1 ± 1.1 2.5 ± 0.8 placebo 2.5 ± 0.7 2.3 ± 0.9 2.4 ± 0.7 2.2 ± 0.8 2.1 ± 0.6

a Results are given as means of the absolute values ± SD. b p < 0.05, significant deviation from preinfusion bevel (periods 1 and 2) within group.

C p < 0.01, significant deviation from preinfusion level (periods 1 and 2) within group. J Am Soc Nephrol 9: 1489-1498, 1998 Renal Effects of Urodilatin in Liver Cirrhosis 1495

Mean Arterial BP and Pulse Rate -0- Placebo - no ascites -.-. URO-noascites Mean arterial BP (MAP) and pulse rate (PR) values during 60 - -0-- Placebo - ascites 55 URO and placebo are depicted in Figure 5. L.MAPURO values -4- URO-ascites were not significant compared with the changes during pla- 50 - cebo. ZPRURO values were significant in the ascites group -J 45.

(period 4: URO, 4%; PL, -3%; P < 0.01, and period 5: URO, 40.

10%; PL, -0.1%; P < 0.01), but not in those without aseites. .E.. 35 a. 30. 0 24-Hour Urine Sodium Excretion and Body Weight c 25. The sodium content in the 24-h urine samples collected the E U) day before the URO and placebo infusions is indicated in Table . 15. a. 1. The body weight decreased 0.8 ± 0.5 kg during URO and 10

0.5 ± 0.6 kg during placebo in the group without ascites. In 5. those with ascites, the weight decreased 0.5 ± 0.5 kg during 0

URO and 0.2 ± 0.6 kg during placebo. 6

Adverse Effects 5 Two cirrhotic patients with ascites (nos. 108 and 1 15) de- veloped symptoms of dizziness 30 to 40 mm after diseontinu- ation of URO infusion. The symptoms appeared as the subjects were in the upright position, urinating. Systemic BP dropped moderately in one of them. They recovered in Trendelenburg position without any treatment.

Discussion 1. The purpose of the present study was to examine the renal effects of infusing a single dose of URO into cirrhotic patients 0 Time (mm) with and without aseites. Renal clearance of radioactive tracers 0 30 60 90 120 and lithium was used to study the renal mechanism(s) of action of URO, taking into account the effect of URO on the systemic INFUSION_] BP and the salt-water regulating hormones. This study is the first to demonstrate the renal effects of URO in cirrhotic Figure 4. Plasma and urine cGMP before (0 mm), during (30 and 60 mm), and after infusion (90 and 120 mm) of URO and placebo in 15 patients, and the truly new information is that the study pro- cirrhotic patients with aseites and nine patients without ascites. Re- vides convincing evidence for a natriuretic and diuretic effect sults are given as means ± SD. of a low-dose infusion of URO in liver patients with cirrhosis without causing intolerable hypotension. URO markedly improved sodium excretion rate and urine flow rate in the cirrhotic patients with and without ascites, and plasma levels of renin and aldosterone and a higher sympa- the responses were nearly the same size in the two groups. An thetic nerve activity than responders (22-24). It has been increase in filtration fraction and in isotonic fluid delivery from suggested that diminished distal tubular sodium delivery may the proximal tubule and a decrease in distal tubular fractional be involved in ANP resistance (25,26). In the present study, the sodium reabsorption contributed to the natriuretie and diuretic two cirrhotic groups had almost the same natriuretic response effects of URO. Second-messenger cGMP markedly increased, to URO, although the cirrhotic patients with ascites had bio- supporting the active biological responsiveness of the peptide chemical parameters indicating a more advanced degree of receptors in cirrhotic patients. Plasma concentrations of renin, cirrhosis, including a lower prothrombin index and higher Angli, and aldosterone were unaltered during URO infusion, preinfusion levels of plasma renin, AngIl, and aldosterone. The whereas the hematocrit values increased and mean BP gradu- decompensated cirrhotic patients had similar sodium excre- ally declined. The pharmacodynamic effects were relatively tions in the 24-h urine as those with early cirrhosis. This may short-lived after infusion, probably because of the short elim- be explained by some influence of diuretic treatment in the ination half-life (2 to 5 mm) of URO (7,21) and activation of ascites group. Spironolactone and its metabobites probably counter-regulatory mechanisms. circulated in plasma on the infusion day, antagonizing the The patients were divided into two groups depending on the tubular action of abdosterone and increasing the delivery of clinical finding of aseites. As shown in ANP studies, the sodium to the distal nephron to the site of action of URO. natriuretie responses to ANP might differ markedly depending Thereby, spironolactone may have contributed to the URO- on the stage of cirrhosis (22,23). Nonresponders, who failed to induced natriuresis, whereas the furosemide effect likely had show a natriuretie response to ANP or head-out-water immer- disappeared before URO infusion due to the short elimination sion, were found to have a severe sodium retention and higher half-life of the loop agent. The present findings suggest that 1496 Journal of the American Society of Nephrology J Am Soc Nephrol 9: 1489-1498. 1998

Table 4. Plasma renin, angiotensin II (Angil), and abdosterone (Aldo) before (periods 1 and 2), during (periods 3 and 4), and after (periods 5 and 6) infusion with URO and placebo in 15 cirrhotic patients with ascites and nine patients without ascites

Preinfusion Infusion Postinfusion Postinfusion P aramet er Periods 1 and 2 (0 mm) Period 4 (60 mm) Period 5 (90 mm) Period 6 (120 mm)

Renin (mIU . L) ascites URO 61 ± 59 52 ± 50 54 ± 50 61 ± 59

placebo 45 ± 41 44 ± 39 41 ± 36 43 ± 39 no ascites URO 21±14 21±16 21±17 23±17 placebo 23 ± 20 23 ± 20 23 ± 20 26 ± 19

Angli (pmob . L 1) ascites URO 11.7 ± 10.0 11.3 ± 9.8 10.5 ± 9.4 11.3 ± 10.0 placebo 9.0 ± 7.6 8.7 ± 7.8 8.5 ± 8.1 8.7 ± 8.3 no ascites URO 3.9 ± 2.1 4.3 ± 3.1 3.6 ± 1.9 4.8 ± 4.6

placebo 4.0 ± 2.6 4.0 ± 2.3 3.8 ± 2.1 4.3 ± 2.7

Aldo (pmol . L) ascites URO 305 ± 280 216 ± 140b 187 ± l26C 242 ± 176

placebo 292 ± 280 200 ± 170b 21 1 ± 188” 237 ± 220 no ascites URO 62±20 67±20 56±13 53±13 placebo 68±33 64±21 54±15 64±28

a Results are given as means of the absolute values ± SD. L p < o.os, significant deviation from preinfusion bevel (periods 1 and 2) within group. C p < 0.01, significant deviation from preinfusion level (periods 1 and 2) within group.

URO may have a beneficial effect as a therapeutic agent in creases were not as significant as seen in healthy subjects decompensated cirrhotic patients with moderate sodium reten- (7, 1 4). ERPF values were unchanged or decreased in the cir- tion and treated with an aldosterone antagonist. It is still rhotic patients, consistent with the findings in healthy subjects uncertain whether URO can induce natriuresis in decompen- (14). The natriuretic peptide, i.e., ANP, is not known as a sated cirrhotic patients with severe sodium retention and re- classic vasodilator but rather as a vasoconstrictor antagonist fractory ascites. If URO really has a natriuretic potency in (28). In the present study, a renal vasodilator effect of URO cirrhotic patients with diuretic-resistant ascites, it could have may have been counteracted by a BP-bowering effect, natriure- important clinical significance in the future treatment of these sis and diuresis, and an egression of fluid from the intravas- patients. cular compartment to the interstitium. A tubular effect on The natriuretic potency of URO in the cirrhotic patients sodium reabsorption in the cirrhotic patients is demonstrated without ascites is obvious. UNa was quite similar to the UNa by a significant increase in FENU. To determine whether URO observed in healthy subjects (60 mm of infusion of 20 ng had a proximal and a distal tubular site of action, the lithium

URO . kg I . min ‘; UNa: 562 p.mol . min ‘ and UNa: 110%) clearance (CLI) method was used (29). In the cirrhotic patients (14). The cirrhotic patients without ascites had normal plasma without ascites, CLI and FELl increased and PFRNH,O de- levels of renin, AngIl, and aldosterone, whereas plasma ANP creased, suggesting an inhibitory effect of URO on isotonic and BNP were increased. Impaired natriuretic response to ANP fluid reabsorption in the proximal tubule. In the decompen- has been demonstrated in cirrhotic patients without clinically sated cirrhotic patients and healthy volunteers, the lithium apparent volume retention (3), and a difference between the excretion increased as well, but not significantly compared responses to URO and ANP in early cirrhosis might be ex- with placebo (14). With a significant increase in filtration plained by a low BP after ANP but not after URO administra- fraction, one might expect an associated increase in proximal tion. absolute sodium reabsorption. The unchanged PFRN,H,O dur- Filtration fraction significantly increased, induced by an ing URO infusion supports the idea of an inhibitory proximal increase in GFR and a decrease in ERPF. The gbomerular tubular effect of URO. The mechanism of action of URO in the hydrostatic pressure probably increased due to a dilation of vas proximal nephron is not clarified. Peptide receptors are not afferent and a constriction of vas efferent (27).The GFR in- found in these segments, and the proximal tubule cells do not J Am Soc Nephrol 9: 1489-1498, 1998 Renal Effects of Urodilatin in Liver Cirrhosis 1497

50 - attributed to the aldosterone antagonist treatment. The inhibi- 48 - -0-- Placebo-no ascites tion of DFRNa was not blunted compared with the action in

46 - -.--- URO-no ascites healthy men (20 ng URO . kg I . min ‘; LDFRNa, -5%; . 44 - -0- Placebo-ascites PL, - 1 %) ( I 4). The cirrhotic patients had preserved the ability to generate high concentrations of cGMP as seen in healthy

subjects (60 mm infusion of 20 ng URO . kg I . min ; 32 nmol/L cGMP in plasma), unlike what was found in nephrotie

42-Ig0 o 3836. - * -.--- URO-ascites patients after ANP administration (32). 34. 32 URO did not have a suppressive effect on renin secretion, as 30 . CCI C C TIme(min) shown previously in healthy men (14). A suppressive effect 0 30 60 90 120 might have been antagonized by the BP-reducing effect of URO. The gradual decline in BP could be due to a fall in 100 - preload (33). A decrease in venous return may be explained by

90 a direct effect of URO on the vascular tone in the venous system (although this has not been investigated in any greater : 80. detail) or by a fall in plasma volume caused by the natriuretic . and diuretic effects of URO and a shift of fluid from the a. 70. intravascular compartment into the interstitium. A marked increase in hematocrit values is a consistent finding during 60. administration of natriuretic peptides (28).

50. In conclusion, this short-term, low-dose infusion of URO markedly improved sodium and urine output without severe 90 influence on mean BP in the cirrhotic patients with and without ascites. A marked increase in fractional excretion of sodium I 80 E indicated a tubular effect of URO on sodium handling. In-

: ‘#{176} creased isotonic fluid delivery from the proximal tubule and 60. inhibition of sodium reabsorption in the distal nephron con- 0 tributed to the natriuresis. A clear cGMP response emphasized ;50, ! 40. the action of synthetic URO on the guanylate cyclase-coupled .U) 30 receptors, whereas the renin-angiotensin-aldosterone axis was a. unaffected. With minor exceptions, the URO infusions caused

20 IIICCIIII Time(min) similar renal, hemodynamic, and endocrine effects in the cir- 0 15 30 45 60 75 90105120 rhotic patients as in a previously studied group of healthy men.

_INFUSION Acknowledgments Figure 5. Mean arterial BP (MAP), pulse rate, and hematocrit values This study was supported by the Danish Medical Research Council, before (0 mm), during (30 and 60 mm), and after (90 and 120 mm) Aarhus University Research Foundation, and Boehringer Mannheim infusion with URO and placebo in 15 cirrhotic patients with aseites (Mannheim, Germany). Boehringer Mannheim kindly supplied the and nine patients without ascites. Results are given as means ± SD. synthetic urodibatin and placebo. We thank Lisbeth Mik.kelsen,

*P < 0.05; ** < 0.01, significant deviation from preinfusion level Kirsten T#{216}nder,Dorte R#{248}nde,Jane Knudsen, Elsebeth Fibiger, Gitte (0 mm) within group. Paulsen, and Rikke Andersen for their skillful technical assistance.

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