Urinary Angiotensin Excretion During Sodium Restriction and Diuretics

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1 Nephrol Dial Transplant (1989)4: European Dialysis and Transplant Association-European Renal Association Nephrology Dialysis Transplantation Original Article Urinary Angiotensin Excretion During Sodium Restriction and Diuretics P. Boer, H. A. Koomans, W. H. Boer and E. J. Dorhout Mees Department of Nephrology and Hypertension, University Hospital, Utrecht, The Netherlands Abstract. The urinary excretion rate and plasma concentration of angiotensin I (, PAI) and angiotensin II (I, ), and plasma renin activity () were measured in seven healthy volunteers in the supine and upright position both on free and restricted sodium intakes. The same variables were measured before and after intravenous injection of furosemide, as well as before and during intravenous infusion of chlorothiazide. Assumption of the upright posture as well as sodium restriction increased PAI,, and, but and I excretion were not altered by these manoeuvres. Furosemide injection resulted in increases of all variables, albeit that PAI and were elevated to a lesser extent then. Chlorothiazide infusion caused a reduction in PAI,, and, whereas and I excretion increased modestly. The discrepancies between changes in plasma and urinary AI and All may indicate that urinary angiotensin excretion provides information which is supplementary to that of plasma angiotensins. Key words: Angiotensin; Diuretics; Kidney; Renin; Sodium; Urine Abbreviations: PAI, plasma angiotensin I;, plasma angiotensin II;, urinary angiotensin I; I, urinary angiotensin II;, plasma renin activity; EDTA, ethylenediaminetetraacetate; PMSF, phenylmethylsulphonylfluoride Correspondence and offprint requests to: P. Boer, Department of Nephrology and Hypertension, University Hospital, P.O. Box 85500, 3508 G. A. Utrecht, The Netherlands. Tel: Introduction Intrarenally produced All plays an important role in the regulation of renal haemodynamics and salt handling [1]., PAI, and in peripheral or renal venous blood are generally employed as indicators of the intrarenal activity of the renin-angiotensin system. Some observations suggest that this is not invariably justified. For instance, and do not always change in the same direction as directly measured kidney concentrations [2,3]. Renal tissue and lymph concentrations of renin and All are much greater than might be expected on the basis of renal vein concentrations [4-6], indicating nonvascular renal release or generation. Renal perfusion studies with AH show that only a small fraction of the infused All appears in the renal vein blood [7], indicating renal uptake or degradation. In man, studies providing direct information on intrarenal angiotensin production cannot be done. To examine the value of the urinary angiotensin excretion rate as an indicator of the activity of the intrarenal renin-angiotensin system, and to investigate whether changes in urinary and plasma angiotensins are parallel, we performed studies involving sodium intake, change of posture, and the use of diuretics acting at different sites of the tubule in man. Methods Subjects Four studies as detailed below were performed in seven healthy normotensive subjects (4 male, 3 female, age 22 ±1 years, systolic and diastolic blood pressure and 74±3mmHg, respectively). The protocol was approved by the Hospital Committee for Research in

2 714 P. Boeretal Humans, and informed consent was obtained from all participants. The subjects were studied both on a free sodium intake and during sodium restriction, achieved by intravenous administration of 20 mg furosemide followed by four days on a lommol/day sodium diet. Sodium restriction resulted in a decrease in body weight from to kg. The mean sodium excretion on the days preceding the studies were and 11+2 mmol/day on the free and sodium restricted diet, respectively. Study Protocols All investigations were done after an overnight fast. was ensured by giving an oral water load of ml/kg body weight before the studies were started, and subsequent substitution of urinary water loss throughout the experiments. Under these conditions urinary flow rates of > 10 ml/min are obtained. Study 1: assumption of upright posture. The subjects consumed the free sodium diet. They remained recumbent for 90 min; during the last 30 min, a urine portion and a mid-point blood sample were collected. A similar study was done while the subjects remained in the upright position for 90 min. Study 2: sodium restriction. The protocol of study 1 was repeated while the subjects consumed the sodium restricted diet. Study 3: furosemide administration. The subjects were studied on the free sodium intake in the supine position before and after intravenous injection of 20 mg furosemide (which was administered to initiate the period of sodium restriction). One 30-min urine specimen before and four subsequent 20-min specimens after injection as well as corresponding mid-point blood samples were obtained. Study 4: chlorothiazide infusion. The subjects were investigated on the sodium-restricted diet in the recumbent position before and during intravenous infusion of chlorothiazide (5 mg/min, preceded by an intravenous bolus of 250 mg). Other parts of this study have been published elsewhere [8]. Three 20-min and five 15-min urine specimens were collected before and during the infusion, respectively. Mid-point blood specimens were obtained in the third control period and in the second and fifth infusion periods. Preliminary studies had shown that sodium losses due to chlorothiazide infusion amount to 9 mmol/collection period. To maintain sodium balance during the experiment, these losses were supplemented by slow intravenous infusion of a 0.9% NaCl solution at a rate of 4 ml/min during the last four infusion periods. The volume of saline infused was subtracted from the volume of water given orally. During the study, a sustained intravenous infusion of inulin was administered for assessment of the glomerular filtration rate (GFR). Urine specimens were analysed for AI, All, sodium, creatinine, and inulin (study 4 only); plasma samples for the same variables and. Analytical Methods Angiotensin Determination., I, PAI, and PAH were measured by a modification of a previously reported radioimmunoassay [9]. Blood was collected on ice with EDTA (15mmol/l), pepstatin (10 umol/1), captopril (100 umol/1), and PMSF (6 umol/1) to inhibit the activity of renin, converting enzyme, and angiotensinases (the final concentrations in plasma are given in parentheses). Urines were collected on ice with PMSF (final concentration 3 umol/1). Collected in this way, plasma and urinary angiotensins are stable for 3 and 20 h, respectively. The samples were stored at 20 C, at which temperature they were stable for at least 9 months. Concentrating and cleaning up of the samples was performed by reversed-phase chromatography using a Baker SPE-10 vacuum extraction device and butylsilane bonded silica gel columns (Baker Chemical Co., Deventer, Netherlands), prewashed with methanol and equilibrated with deionised water. Urine (20 ml), acidified with glacial acetic acid to ph 3, or plasma (2 ml), 1:5 diluted and acidified with 8 ml mol/1 HC1 to ph 3, were applied on the columns. The effluents were discarded, and the columns were washed with 0.01 mol/1 HC1 and eluted with methanol/trifluoro-acetic acid 99:1 v/v. The eluate was dried under nitrogen at 37 C and dissolved in assay buffer. Dilution curves of urine or plasma extracts, as well as standard curves containing aliquots of extract, were parallel to the standard curves used in the radioimmunoassay. Characteristics of the AI and All assays are, respectively: sensitivity, 2 and 4 fmol; recovery of added standard material, in urine 72% and 90%, in plasma 73% and 89%; within-assay variation coefficient, for urine ll%and 10%,forplasma9%and9%;betweenassay variation coefficient, for urine 19% and 17%, for plasma 17% and 16%. Corrections were made for procedural losses. Cross reactions of the All antibody with AI and the C-terminal hepta-, hexa-, and penta-peptide are 1.2, 100, 100, and 80%, respectively. All concentrations were corrected for cross reaction with AI, but not with peptide fragments. Cross reactions of the AI antibody with All or All-fragments are negligible (< 0.001%). Other Determinations. was measured by radioimmunoassay [10]. Sodium and creatinine in urine and plasma were measured by standard laboratory techniques. Inulin was determined by a colorimetric method [11].

3 Urinary Angiotensin Excretion Table 1. Studies 1 and 2: response of urinary angiotensins and other variables to assumption of upright posture and sodium restriction 715 Condition C-Cr 1 PAI Supine, free Na Upright, free Na Supine, low Na Upright, low Na 9±2 11± * 18±5f 21±2t ± * f *t 26±2 35 ±2* 48±4f *t ±3* 24±3t 38±5*t, urinary sodium excretion; C-Cr, creatinine clearance; and UAH, urinary excretion of angiotensin 1 and II;, plasma renin activity; PAI and, plasma concentration of angiotensin I and II; * = P<0.05 with respect to corresponding supine value; f = P<0.05 with respect to corresponding value on free Na intake Table 2. Study 3: response of urinary angiotensins and other variables to intravenous injection of furosemide Baseline Furosemide 1 Furosemide 2 Furosemide 3 Furosemide 4 9± * * * * ±300* 2461 ±210* 1536 ±120* 860 ±100* C-Cr 131 ± ± ±12 118±11 78 ±25 151±44* 148 ±22* 143 ±30* I 67± * I12±30* ±21 190± * 400±100* 310±60* * Furosemide 1-4, collection periods after intravenous injection of furosemide; * = / > <0.05 with respect to baseline value Statistics. Values of, I, PAI, PAH, and are given as geometric mean + standard error (SEM). According to Bartlett's chi-square test [12], the variances of these variables were homogeneous. The studies were evaluated by analysis of variance, using a two-way randomised block design for studies 1 and 2, and oneway randomised block designs for studies 3 and 4 [12]. If a variance ratio (F) reached statistical significance (P<0.05), the differences between the means were analysed at the 5% level by the least significant difference test [12], using the treatment mean square error or the interaction mean square error (when appropriate) to calculate the least significant difference. Results Studies 1 and 2 The results are shown in Table 1. Irrespective of the sodium intake, and I excretion did not change significantly with assumption of the upright posture, whereas PAI,, and increased (P<0.05). In the supine as well as in the upright position, and I excretion was not significantly altered by sodium restriction, whereas PAI,, and increased (P<0.05). The creatinine clearance did not change; the sodium excretion decreased with assumption of the upright posture and sodium restriction (P<0.05). Study 3 The results are presented in Table 2. Intravenous injection of furosemide gave a significant increase in and I PAI 29±4 34± ±3 11 ±3 I3± ±4 12±3 excretion, as well as in (P<0.05). The increases in PAI and were smaller and did not reach statistical significance (/ > <0.10), because the time between baseline and peak value differed from subject to subject. In every subject, however, the peak value was greater than the baseline value: AI increased from to 40 ± 4 pmol/1, All from to 16 ± 3 pmol/1 (P < 0.05). The creatinine clearance showed a non-significant transient increase during the first period. The sodium excretion increased 25-fold, and had not yet returned to the baseline value in the last collection period (P<0.05). Study 4 The results are given in Table 3. During chlorothiazide infusion, and I excretion tended to increase (P<0.05). In contrast, PAI,, and decreased (P<0.05). The inulin clearance diminished by 12% (/ > <0.05); the sodium excretion increased 25-fold Discussion A notablefindingin our study is the absence of parallelism between changes in plasma and urinary angiotensins. Plasma angiotensins are generated in the systemic circulation after stimulation of renin release, but they may also originate from the kidney. However, the major part of intrarenally formed All is degraded in the kidney before

4 716 Table 3. Study 4: response of urinary angiotensins and other variables to intravenous infusion of chlorothiazide P. Boer et al C-Inulin UAH PA1 Baseline 1 Baseline 2 Baseline 3 Chlorothiazide 1 Chlorothiazide 2 Chlorothiazide 3 Chlorothiazide 4 Chlorothiazide 5 10±l ±I* 15±1* 15±1* I4+I* 14+1* ± * 550 ±50* 540 ±30* 530 ±30* 520 ±30* * 93+9* 90±9* 91+8* 94 ±7* ±14 92±12* 92±I8* ±12 56 ±12 59± ±11* 64±12 60±10 59± ±190* 790 ±180* ±4 35±2* 20±7 18±6 15±6* C-Inulin. inulin clearance: Chlorothiazide 1-5, collection periods during intravenous infusion of chlorothiazide;' -P<0.05 with respect to baseline values reaching the renal vein, as demonstrated in the dog by in vivo kidney perfusion studies with physiological concentrations of All [7], or by injection of radiolabelled All into the renal artery [14]. Urinary angiotensins may arise from glomerular filtration, local production by a tubular reninangiotensin system, or transtubular diffusion or secretion from the renal interstitium. The behaviour of the urinary excretion of AI and All was similar in the four studies (Tables 1-3), implying that the information which can be obtained from the excretion of both peptides is equivalent for the protocols used in our studies. From the angiotensin data and glomerular filtration rates presented in the tables it can be calculated that the urinary agiotensin excretion rate amounts to 1 %-5% of the filtered load. Small peptides like angiotensins are freely filtered. Subsequently, the major part is rapidly hydrolysed and reabsorbed in the proximal tubule, as shown in in vivo microinfusion studies with radiolabelled All in the rat [15]. We found no parallelism in changes of plasma and urinary angiotensins, which suggests that filtration from plasma is not the major source of and I. However, we can not exclude that alterations in tubular degradation or reabsorption of filtered angiotensins also contribute to the observed changes in angiotensin excretion. Local production by a tubular renin-angiotensin system requires the presence of a complete reninangiotensin system within or at the surface of renal tubule cells. Although literature on this subject is scarce, renin and angiotensinogen m-rna were found to be present in mesangial, proximal, and collecting duct cells [16]. In addition, AI may be converted into All intraiuminally, since converting enzyme activity has been demonstrated in the brush border zone of the proximal tubular epithelial cells [17]. Renin is not only secreted from the juxtaglomerular granular cells into the blood, but also - and even predominantly - into the renal interstitium, in which all the components of a renin-angiotensin system are present [1,7,13]. It is conceivable that the urinary angiotensins originate partly from transtubular diffusion or transport in regions where the interstitial concentrations are great. In vivo microperfusion studies in the rat have shown that peptides introduced in the distal parts of the tubule are recovered largely intact in the urine [15,18]. Thus, peptides entering the tubule at this site may escape from hydrolysation by brush border peptidases and tubular reuptake, which mainly occurs in the proximal tubule [15,18]. During chlorothiazide infusion, plasma All and decreased, whereas I increased. Intrarenal All is probably involved in the adaptation of the GFR to an increase in sodium load at the macula densa by increasing the pre- and postglomerular resistance, the net effect being a decrease in GFR [19]. If I is a marker of intrarenal All production, our finding of an increase in I and a decrease in inulin clearance is compatible with a role of All in the tubuloglomerular feedback mediated decrease in GFR. The increase in urinary angiotensin excretion in studies 3 and 4 corresponds with an increase in urine flow and may thus be flow-related. To obtain flow-independent values we calculated the urinary angiotensin:creatinine ratio, which increased in the same way as and I (data not shown). This makes it unlikely that the increased excretion represents a wash-out phenomenon. On the other hand, tubular degradation, if present, may be less complete when the urineflowis greater. The modest increase in PAI and after furosemide despite a large increase in was unexpected, although not unprecedented [20]. In each subject, the peak was greater than the baseline value (/><0.05), but due to scatter in the time-to-peak values the mean increase did not reach statistical significance (0.05 < P< 0.10). Literature reporting on urinary All excretion is scanty. Fukuchi [21] found normal values in patients with essential hypertension or chronic glomerulonephritis, increased values in renovascular hypertension, and decreased values in primary aldosteronism. The reported excretion rates are about 50 times lower than ours. This

5 Urinary Angiotensin Excretion may be due to differences in extraction technique, to intravesical degradation caused by prolonged collection periods (2 h), or to incomplete voiding of the bladder due to low urinary flow rates (< 0.5 ml/min). We did not confirm by high performance liquid chromatography that the All-like immunoreactive material in urine actually was All. If All-like peptide fragments cross-reacting with the All antibody are present in urine, they most probably originate from All, and they will provide the same kind of information as All. In conclusion, during various manoeuvres in healthy humans we found a discrepant behaviour of plasma and urinary AI and All. Our study gave no definitive information on the exact origin of urinary AI and All. Local production by a tubular renin-angiotensin system and transtubular diffusion or secretion from the interstitium are among the possibilities. More studies, both in humans (e.g. during converting enzyme inhibition and angiotensin infusion) and animals (comparison of urine and lymph angiotensin excretion) are needed to define the source and possible meaning of urinary angiotensin excretion. References 1. Navar LG, Rosivall L. Contribution of the renin-angiotensin system to the control of intrarenal hemodynamics. Kidney lm 1984; 25: Mendelsohn FAO. Angiotensin II: evidence for its role as an intrarenal hormone. Kidney lm 1982; 22: suppl. 12, S78-S81 3. Thurau KWC, Dahlheim H, Griiner A, Mason J, Granger P. Activation of renin in the single juxtaglomerutar apparatus by sodium chloride in the tubular fluid at the macula densa. Circ Res 1972; suppl. 2: Bailey MD. Rector FC, Seldin DW. Angiotensin II in arterial and renal venous plasma and renal lymph in the dog. J Clin Invest 1971; 50: Mendelsohn FAO. Evidence for the local occurrence of angiotensin II in rat kidney and its modulation by dietary sodium intake and converting enzyme blockade. Clin Sci 1979; 57: OMorchoe CC, O'Morchoe PJ, Albertine K.H, Jarosz HM. Concentration of renin in the renal interstitium, as reflected in lymph. Renal Physiot 1981; 4: Rosivall L, Narkates AJ, Oparil S, Navar LG. De novo intrarenal formation of angiotensin II during control and enhanced renin secretion. Am J Physiot 1987; 252: Fl 118-F Boer WH. Koomans HA, Dorhout Mees EJ. Acute effect of thiazides, with or without carbonic anhydrase activity, on lithium and free water clearance in man. Clin Sci 1989; 76: Boer P, Geyskes GG. Apparently high angiotensin II levels in patients with essential hypertension treated by converting enzyme inhibition. ScandJ Clin Lab Invest 1985; 45: Boer P, Sleumer JH, Spriensma M. Confirmation of the optimal ph for measuring renin activityin plasma. Clin Chem 1985; 31: Heyrowski A. A new method for the determination of inulin in plasma and urine. Clin Chim Ada 1956; 1: Snedecor GW, Cochran WG. Statistical Methods. Iowa State University Press, Ames, 1979; Ch. 10, Hackenthal E, Metz R, Buhrle CP, Taugner R. Intrarenal and intracellular distribution of renin and angiotensin. Kidney lm 1987; 31, suppl.20:s4-s Bailey MD, Oparil S. Relation of renal hemodynamics to metabolism of angiotensin II by the canine kidney. Circ Res 1977; 41: Carone F, Peterson DR, Oparil S, Pullman TN. Renal tubular transport and catabolism of proteins and peptides. Kidney Int 1979; 16: Rajamaran S, Graves K, Kunapuli P. Intrarenal renin-angiotensin system: in situ hybridization study. FedAbs Soc Exp BiolJ 1988; 2: A Taugner R, Hackenthal E, Helmchen U et al. The intrarenal reninangiotensin system. An immunocytological study on the localization of renin, angiotensin, converting enzyme and the angiotensins in the kidney of mouse and rat. Klin Wochenschr 1982; 60: Ganapathy V, Leibach FH. Carrier-mediated reabsorption of small peptides in renal proximal tubule. Am J Physiol 1986; 251: F945-F Navar LG, Rosivall L, Carmines PK, Oparil S. Effects of locally formed angiotensin II on renal hemodynamics. Fed Proc 1986; 45: Walker WG, Moore MA. Horvath JS, Whelton PK. Arterial and venous angiotensin II in normal subjects. Relation to plasma renin activity and plasma aldosterone concentration, and response to posture and volume changes. Circ Res 1976; 38: Fukuchi S. Estimation of urinary angiotensin II by radioimmunoassay. TohokuJ Exp Med 1974; 114: Receivedfor publication Accepted in revised form