Urodilatin, a -Derived Natriuretic Factor, Is Excreted with a Circadian Rhythm and Is Stimulated by Saline Infusion in Man1

Christian Drummer, M.D., Fritz Fiedler, M.D., August KOnig, M.D., and Rupert Gerzer, M.D.2

kidney either by a neural reflex or by a humoral Medizinische Klinik, Klinikum lnnenstadt der Universit#{228}t, factor (1). Since the discovery of atnial natri- MUnchen, Federal Pepublic of Germany uretic (ANP) in 1 98 1 (2), many investigations were conducted to find out whether the ANP is the (J. Am. Soc. Nephrol. 1991; 1:1109-1113) missing that increases renal sodium and water excretion after a hypervolemic stimulus. How- ever, recent dog experiments showed that plasma ABSTRACT ANP is not, or Is only partially. responsible for the natniuretic response after expansion of a cardiac bal- Urodilatin is a recently described, presumably kid- loon (3)-a model that simulates hypervobemia. In ney-derived member of the atrial man, saline Infusion is a commonly accepted stimu- family. The first data on a physiological role for uro- bus that also induces hypervobemia. In almost all dilatin in the regulation of sodium in saline infusion studies that were described in recent humans is presented in this work. Urinary urodilatin years. only the renal responses during 2 to 3 h pos- excretion during a 9-day study in healthy volunteers tinfusion were determined. One study followed uri- paralleled the circadian rhythm in urinary sodium nary excretion for 5 h postinfusion and showed that excretion. Furthermore, urodilatin and sodium excre- urinary sodium excretion was still elevated at the end tion were slightly increased during the first 3 h after of this observation period. whereas plasma ANP was an acute isotonic saline infusion and about three- only transiently elevated at 1 5 mm postinfusion (4). fold elevated during 14 h postinfusion; both param- We have recently extended this observation (acute saline Infusion of about 1 .6 L in healthy man). show- eters remained closely correlated up to 28 h postin- ing that only 1 5% of the infused sodium was excreted fusion. These data suggest that urodilatin is involved within the first 3 h and that sodium excretion was in the circadian regulation of sodium excretion and increased for 1 to 2 days. whereas plasma ANP was is a physiological regulator of long-term sodium ex- only marginally affected shortly after the infusion cretion after an acute saline infusion. (Drummer et at. , submitted for publication). Urodi- Key Words: Urodilatin excretion. atrial natriuretic peptide an- latin, a new member of the ANP family, was recently alog. natriuresis isolated from human and identified as a 32- amino-acid peptide containing the se- quence 95 through 1 26 of the cardiac ANP pnopeptide (5). Compared with the circulating plasma form of T he mechanisms of water and sodium homeosta- ANP-(99-l26), urodilatin is N terminally extended, is sis are a major field of Investigation In human probably originated from kidney tubules (6). and is physiology, but our knowledge on circulatory blood only slowly degraded and inactivated by kidney cor- volume control is still far from being complete. In tex membrane that rapidly inactivate ANP- 1 956, Henry et at. stated that there must be a vol- (99- 1 26) (7). Urodilatln binding properties to recep- ume-sensing mechanism in the thoracic circulatory tors and the activation of receptor coupled guanylate organs that influences the excretory function of the cyclase by urodilatin are similar as for ANP-(99- 126) (8). The new peptide has vasonelaxant properties (5) and stimulates natriuresis in dogs (9). Recently, we I Received October 2. 1990. Accepted December 13, 1990. have reported about urinary urodilatin excretion In a 2 correspondence to PD Dr. med. Rupert Gerzer, Labor f#{252}rklinische Pharmakol- ogie. Medizlnische Kilnik, Kilnikum Innenstadt der Universit#{248}t. Ziemssenstralt,e I. series of physiological experiments in the dog (10). In 8000 MUnchen 2, Federal Republic ot Germany. the study presented here, we have investigated six 1046-6673/0109-1 109$03.00/0 healthy men over a 9-day period. to determine the Journal of the American society of Nephrology copyght © 1991 by the American society of Nephrology cmrcadian rhythm in urodilatin excretion and the par-

Journal of the Amermcan Society of Nephrology 1109 Urodilatin in Healthy Subjects

ticipation of urodilatin in the renal response to an the tracer. Separation of bound and free antigen was acute saline infusion compared with the participa- achieved by a double-antibody method. The assay tion of the cardiac member, ANP-(99-126). detection limit was 7. 1 ± 4. 1 pM. and the 50% inter- cept was 67 pM. Intraassay and Interassay variations METHODS were 8.3 and 12.2%, respectively. Recovery of syn- thetic urodilatin added to urine before ethanol ex- Subjects and Study Design traction was greater than 90%. The urodibatin anti- Six healthy male volunteers (age. 23 to 3 1 years) body did not cross-react with ANP-(99- 1 26), brain gave their written informed consent and participated natniuretic peptide, 13-ANP, atniopeptmn I. atniopeptmn in the study which was conducted at the Microgravity III, or with several other ANP analogs with shortened User Support Center (MUSC). Cologne. Federal Re- amino acid sequences, even in micromolar concen- public of Germany. The study was approved by the trations (Drummer et at. , manuscript In preparation). Ethical Committee of the University of Munich. All urodilatin determinations were performed in trip- During the whole 9-day study period, the subjects licate. remained In a recumbent body position except for Plasma ANP immunoreactivity was determined eliminating body wastes or taking a shower. Any from EDTA plasma samples after extraction with C18- exhaustion or physical exercise was prohibited for cartridges by a commercial RIA (Amersham-Buchler, the volunteers. The subjects received a strictly con- Hannover, (Federal Republic of Germany). trolled diet consisting of 1 ,940 kcal/day. 45 mL/kg body wt of water, and 2.6 mmol/kg body wt of sodium each day. Statistics Throughout the whole study period. urine and The circadlan rhythm in the excretion of water, blood samples were collected to determine those pa- sodium, and urodllatin was evaluated by an analysis rameters and that are involved in body of variance (BMDP 2V; Statistical Software Inc., Los water and sodium homeostasis. Urine samples were Angeles, CA) by using the daily sampling time and distributed to four sampling periods/day (10 p.m. to the different study days as two “within” factors. 6 a.m., 6 a.m. to noon, noon to 6 p.m.. and 6 p.m. to The urinary excretion rates during the control and 10 p.m.) and blood samples were drawn at 6 a.m., 2 infusion experiments were evaluated by analysis of p.m., and 10 p.m. each study day. On the fifth study variance (BMDP 2V) by using sampling time as within day, each subject additionally received an intrave- factor and infusion yes/no as grouping factor. nous isotonic saline infusion (about 2 liters within For comparison between sodium and urodilatin ex- 25 mm) and the renal and hormonal responses were cretlon, the linear regression was calculated by using compared with those of a control experiment which the BMDP 6D program (Statistical Software Inc.). was conducted 2 days before the infusion. During the infusion and control experiments, respectively, additional blood samples (30 mm and 1 . 2, and 3 h RESULTS postinfusion) and urine samples (1 , 2, and 3 h postin- Figure 1 shows urine flow (UV), urinary sodium fusion) were collected. excretion (UNaV) and urinary urodilatin excretion (UUrocillatlnV) in healthy volunteers during 9 days with Determinations four sampling periods/day. The analysis of variance Urinary sodium and potassium concentrations showed a significant circadlan 24-h rhythm (P < were determined by flame photometry. Urinary uro- 0.0003) for all excretion rates (urine flow, sodium, dilatin was measured with a newly developed radio- and urodilatin), with minimal excretion rates during immunoassay (RIA) (Drummer et at. , manuscript in the night periods and maximal excretion rates during preparation). Briefly, a specific unodilatin antibody the day periods. A comparable rhythm In potassium (kindly provided by Professor Forssmann, Hannoven) excretion with peak excretion rates in the afternoon was developed in rabbits. A small peptide composed (99 ± 5 Eq/mIn, mean ± SE) and minimum excretion of amino acids identical to the six N-terminal residues rates during the night periods (28 ± 1 zEq/mln) was of urodibatin was coupled to hemocyanin and was observed. Furthermore, these circadian rhythms used as the antigenic stimulus. This amino acid does were significantly Influenced (P < 0.0001) by study not occur In human ANP or any of the commercially days (impact of within factor “study day” on within available ANP analogues. For the RIA, the antibody factor “daily sampling time”), obviously caused by the was used In a final dilution of 1:2750. increased excretion rates during the infusion day. Synthetic urodilatin (Bissendorf Blochemicals, Table 1 shows that, during the 9-day study period, Hannover, Federal Republic of Germany) was used both urodilatin and sodium excretion in the Individ- to prepare the standard curve (range, 3 to 400 pM); ual subjects. as well as for all subjects (r = 0.706), ‘25I-labeled urodilatin (2.000 /LC1/nmob) was used as were closely correlated. The respective correlation

1110 Volume I ‘ Number 9 ‘ 1991 Drummer et al

circadian urinary excretion TABLE I . Correlation between urinary sodium and urodilatin excretion#{176} meoniSEM n=6

Excretion Data Subject r PN 6.0

C 4.5 - C Four sampling pe- A 0.856 <0.001 33 E 0 .9 I0 riods/dayover9 B 0.777 <0.001 33 C E 3 days C 0.784 <0.001 33

> V V D 0.506 <0.002 33 E 0.827 <0.001 33 F 0.741 <0.001 33 All 0.706 <0.001 198 Saline infusion and A 0.885 0.001 26 control experi- B 0.873 0.001 26 C ments C 0.898 0.001 26 E - C 0 .2 D 0.621 0.001 26 C C E 0.854 0.001 26 S ‘ F 0.572 0.002 26 V V All 0.756 0.001 156

0 The table shows correlation coefficients (r). error probability (I. and the respective number of observations (Al) for linear regression be- C tween urodilatin and sodium excretion. The data from four sampling E periods per day show individual results (subjects A to F) on circadian

0 - C urine samples over the whole study period and the evaluation for all 0 subjects (corresponding to Figure 1). The data from the infusion and E C C control experiments show the respective results only considering the S ‘ > sampling periods during these particular experiments (corresponding C V V to Figure 2). 0 V 0

D 0 1 2 3 4 5 6 7 8 pling periods, no Infusion). The data show a slight increase in urine flow and sodium excretion during Duration of study (days) the 3 h that are usually investigated In such studies,

Figure 1. Urine flow (UV), urinary sodium excretion (UNOV) as well as a small increase in urodilatin excretion and urinary urodilatin excretion (Ura,nV) in six healthy during the second and third hour postinfuslon. The volunteers (mean ± SE) during 9 days with four sampling comparison of excretion rates during the Infusion periods per day. The x-axis labels indicate ‘midnight’ each and control experiments during this second and third study day. Triangles show the time of control and infusion hour postinfusion showed that an absolute amount experiments during the 9-day study period. All excretion of 53 and 42 mL of water, 9.2 and 8. 1 mEq of sodium, rates (urine flow, sodium, and urodilatmn) showed a signifi- 5.2 and 4.6 mEq of potassium. and also 499 and 416 cant circadian 24-h rhythm (P < 0.0003), with minimal ex- fmol of urodilatin, respectively, was excreted more cretion rates during the night periods. during the Infusion sequence in comparison with the control sequence (data are averages from six volun- between urine flow and urodilatin excretion was r = teens). However, a pronounced diuresis and natri- 0.588 (all subjects). uresis occurred during the long-term period. Urodi- Plasma ANP immunoreactivity was 1 7.2 ± 0.9 pg/ latin excretion was also stimulated and closely pan- mL (mean ± SE) In the morning, 15.8 ± 0.8 pg/mL at albeled the natniuretic response for more than 24 h. 2 p.m., and 16.2 ± 0.7 pg/mL before sleep. The All excretion rates (urine flow, sodium, potassium. analysis of variance did not show a circadian rhythm and urodilatin) significantly Increased (P < 0.0001) in plasma ANP concentrations, and the regression after infusion. The most pronounced Increases In analysis denied any relation between plasma ANP sodium and urodilatin excretion were detected be- and the urinary sodium excretion rhythm (R = tween 3 and 14 h postinfusion. During the sampling -0.078). periods 3 to 10 h and 10 to 14 h postinfusion, re- Figure 2 shows urine flow, and sodium and urodi- spectiveby, absolute amounts of 1 , 1 80 mL and 122 latin excretion after an acute isotonic saline infusion mL of water, 136.5 and 65.8 mEq of sodium, 36.7 (30 mL/kg body wt, within 25 mm). Regarding the and 0.3 mEq of potassium, and 25,500 and 11,600 circadlan rhythms in urinary excretion of water, so- fmol of urodilatin were excreted more during the dium, and urodilatin, the Infusion effects were com- infusion sequence in comparison with the control pared with the control experiment (comparable sam- sequence.

Journal of the American Society of Nephrology 1111 Urodilatin in Healthy Subjects

F 1 control [ 1 infusion DISCUSSION

mean±SEM n=6 The data presented here for healthy subjects show that the urinary excretion of the recently discovered 6.0 peptide urodilatin significantly increases after an C 0 acute saline infusion and closely parallels the pro- 4.5 U) -- nounced sodium excretion that occurs during 3 and E C 14 h postinfusion. This long-term period, where the , 3.0 main part of sodium excretion after an acute saline

> infusion is found, has not been investigated in recent 1.5 D years. In addition, this study shows a circadian

0.0 rhythm in urinary urodilatin excretion under strictly controlled conditions at rest. This circadian rhythm 800 parallels the day/night variations in renal water and C C 0 sodium excretion which was already described in the E 600 U) - last century (1 1). C In contrast to urinary urodilatin excretion, plasma 400 ANP immunoreactivity neither paralleled the circa- 200 dian natniuretic rhythm nor was increased after an

D acute saline infusion. Thus, it appears unlikely that 0 the cardiac member of the ANP family is really in- volved in the regulation of sodium excretion under 125 physiological conditions, which is consistent with E C 0 - 100 previous reports (3. 1 0). Because urodilatin, as a phys- 0 U) E iobogically occurring ANP analog. stimulates sodium .- 75 > excretion in dogs (9) and an intravenous urodilatin .c 50 bobus also induces sodium excretion in healthy hu- 0 25 mans (Kentsch et at. , manuscript in preparation), 1” ‘ -‘-- ‘L4 the present data suggest that urodilatin is involved D D 0 In the regulation of natniuresis after an acute saline -2 01233 10 20 30 40 infusion. Time after infusion (hours) Because we directly observed urinary urodibatin excretion rates, but not intrarenal urodilatin release, Figure 2. Urine flow (UV), urinary sodium excretion (UNOV) and urinary urodilatin excretion (Uu,OdOtflV) in six healthy the possibility of a passive flow-dependent “wash- volunteers during a control experiment without infusion out” phenomenon for urodilatin remains. However, (dotted area) or with infusion (superimposed white area). several observations suggest that this is unlikely. The The left panel shows a 2-h baseline period before the individual correlations between urodilatin excretion intervention, followed by three 1-h periods after the infusion and urine flow were weaker than the respective con- of 0.9% saline solution (30 mL/kg body wt, within 25 mm). relations between unodilatin and sodium excretion The right panel shows the long-term excretory responses for all volunteers, during the infusion experiment as during the control and infusion experiment, starting with the well as during the resting condition. In one volunteer sampling period 3 to 10 h postinfusion up to 40 h. The (subject D), there was no relation between urodilatin infusion significantly (P< 0.0001) affected the time course in urine flow and sodium and urodilatin excretion. The most and urine flow (circadian. r = 0. 1 63: infusIon, r = obvious increases in sodium and urodilatin excretion are 0.222). whereas a less-than-perfect but still signifi- detectable between 3 and 14 h postinfusion. cant correlation was found between urodilatin and sodium excretion (Table 1 ). During the sampling period 1 0 to 1 4 h postinfusion (Figure 2), urodIbatin Table 1 also shows the close correlations between (and sodium) excretion was still considerably In- sodium and unodilatin excretion only considering the creased despite urine flow that approached the con- sampling periods during the saline infusion and the trob values. Furthermore, on the second day postin- control experiment. High individual correlation coef- fusion (Figures 2, sampling periods from 28 to 38 h ficients were found as well as a close correlation for postinfusion). urine flow again increased whereas all subjects (r = 0.756). The respective correlation urodilatin excretion did not. Finally. a wash-out ef- between urine flow and urodibatin excretion was r = fect for urodilatin should be transient. Because tre- 0.635 (all subjects). Plasma ANP immunoreactivity mendous increases in urodilatin excretion were still was almost unaffected after saline infusion, and the detectable after the long period of 1 4 h postinfusion regression analysis did not promote any relation with and on the day after the infusion, urodilatin excretion urinary sodium excretion (r = -0.192). was not exhausted (illustrated in Figure 1 ): the data

1112 Volume I ‘ Number 9 ‘ 1991 Drummer et al

suggest that Intrarenal urodilatin release, on more possible and to Professor K. Goetz, Kansas City. MO. for helpful likely synthesis. must have been induced by the sa- suggestions and discussions. R. Gerzer is a recipient of a Heisenberg Fellowship from the Deutsche Forschungsgemeinschaft. line infusion. A comparable conclusion has recently been drawn from urodilatin excretion data in a series of experiments In dogs (10). REFERENCES Considering the present data, one could speculate 1 . Henry JP, Gauer OH, Reeves JL: Evidence of that the infusion-induced hypervolemia stimulates the atrial location of receptors influencing urine urodilatin secretion or even synthesis in kidney tu- flow. Cinc Res 1956:4:85-90. 2. de Bold AJ, Borenstein HB, Veress AT, Son- bular cells either by a neural reflex or directly via an nenberg H: A rapid and potent natriuretic re- intrarenal pressure- or osmolanity-medlated mecha- sponse to intravenous injection of atnial myocar- nism. Feller et at. provided evidence that urodilatin dial extract in rat. Life Sci 1981:28:89-94. is of renal origin, because they found that most of 3. Goetz KL, Wang BC, Geer PG. Leadley RJ, Reinhardt HW: Atriab stretch increases sodium the ANP immunoreactivity from porcine kidney tis- excretion independently of release of atnial pep- sue elutes on a cation exchange high-performance tides. Am J Physiol 1 986:250:R946-950. liquid chromatography with synthetic urodilatin. 4. Sagnella GA, Singer DRJ, Markandu ND, et at.: Furthermore, by using the specific urodilatin RIA, Atrial natiuretic peptide-cyclic GMP coupling we did not detect any urodilatin in extracted plasma and urinary sodium excretion during acute vol- ume expansion in man. Can J PhysiofPharmacol from healthy volunteers, even when the samples 1 990;68:535-538. were concentrated 1 0-fold. Therefore, we do not as- 5. Schulz-Knappe P. Forssmann K, Herbst F, sume that urodibatin is a circulating plasma hormone Hock D, Pipkorn R, Forssmann WG: Isolation and favor the hypothesis of a renal origin. Once and structural analysis of “urodilatin”, a new peptide of the cardlodilatin-(ANP)-famlly. ex- secreted, this degradation-resistant peptide might tracted from human urine. Klin Wochenschr bind to receptors in the tubular system. Specific 1988:66:752-759. receptors for ANP have been detected in distal tu- 6. Feller SM, Gagelmann M, Forssmann WG: Uro- bules and collecting ducts of dog kidney, whereas no dibatin: A newly described member of the ANP family. Trends Pharmacol Sd 1989:10:93-94. ANP receptors were found in the proximal tubules 7. Gagelmann M, Hock D, Forssmann WG: Urodi- (1 2). This distribution of binding properties in the latin (CDD/ANP-95- 1 26) is not biologically mac- dog tubular system corresponds with the distribution tivated by a peptidase from dog kidney cortex of ANP-sensitive particulate guanylate cyclase (13), membranes in contrast to atnial natniuretic pep- suggesting that the intrarenal effects of ANP or uro- tide/cardiodilatin (-hANP/CDD-99- 1 26). FEBS Lett 1988;233:249-254. dibatin are mediated by cGMP as a second messenger. 8. Heim J-M, Kiefersauer 5, Fulle H-J, Gerzer R: Furthermore, ANP increases sodium delivery and re- Urodilatin and fl-ANF: Binding properties and duces sodium reabsorption in the collecting ducts In activation of particulate guanylate cyclase. a rat model (14), beading to a natniuretic response. Biochem Biophys Res Commun 1989:163:37- Considering that plasma ANP-(99- 1 26), but not uro- 41. 9. Riegger GAJ, Elsner D, Schulz-Knappe P. dilatin, is rapidly degraded and inactivated in the Forssmann WG: The new peptide urodilatin kidney (7) by a kidney cortex membrane-derived me- ANP-95-126) In dogs with and without heart talboendoprotease (15), it appears likely that the in- failure. Circulation 1 988:78(suppb II):II-429. trarenal ANP-receptor/guanylate cyclase system Is 1 0. Goetz KL, Drummer C, Zhu JL, Leadley R, Fiedler F, Gerzer R: Evidence that urodilatin, the physiological target for the new peptide urodilatin rather than ANP, regulates renal sodium excre- rather than for ANP-(99- 126). tion. J Am Soc Nephrol 1990:1:867-874. In conclusion, although the study presented here 1 1 . Smith E: Health and Disease as Influenced by does not establish any cause-and-effect relationship the Daily. Seasonal, and Other Cyclical Changes between urodilatin and sodium excretion, the results in the Human System. London: Walton and Marbely; 1861. at least suggest that urodilatin is a physiologically 12. de Lean A, Vinay P. Cantin M: Distribution of regulated hormone or paracnine factor that is in- atrial natniunetic factor receptors in dog kidney volved in sodium homeostasis and In the regulation fractions. FEBS Lett 1985:193:239-242. of urinary sodium excretion. 1 3. Tremblay J, Gerzer R, Vinay P. Pang SC, B#{233}li- veau R, Hamet P: The Increase of cGMP by atriab natriuretic factor correlates with the distribution ACKNOWLEDGMENTS of particulate guanylate cyclase. FEBS Lett The work presented here was part of an international collaborative 1985; 181:17-22. study in preparation for the European Spacelab Mission D-2 and 14. Sonnenberg H, Honrath U. Chong CK, Wilson was supported by the German Bundesministerium f#{252}rForschung DR: Atrial natriuretic factor inhibits sodium und Technologie. We thank Professor W.G. Forssmann. Hannover, transport in medullany collecting duct. Am J Federal Republic of Germany. for kindly providing the urodilatin Physiol 1 986:250:F963-F966. antibody. We also thank B. Endres for technical assistance and H. 15. Koehn A, Norman JA, Jones BN, Le Sueur L, Beranek for statistical support. We are especially thankful to Dr. D. Sakane Y, Ghai RD: Degradation of atnial natni- Padeken and his team from the Microgravity User Support Center uretic factor by kidney cortex membranes. J Blob (MUSC, Cologne, Federal Republic of Germany) for making this study Chem 1987:262:11623-11627.

Journal of the American Society of Nephrology 1113