(Received 23 January 1961) Crawford & Kennedy (1959) found the prolonged saluretic and diuretic

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1 454 J. Phyeiol. (1961), 157, pp With 3 text-figure Printed in Great Britain THE ACTION OF CHLOROTHIAZIDE IN THE PERFUSED CAT KIDNEY BY T. DE LIMA AND MARY F. LOCKETT From the Department of Physiology and Pharmacology, Chelsea College of Science and Technology, London, S. W. 3 (Received 23 January 1961) Crawford & Kennedy (1959) found the prolonged saluretic and diuretic effects which characterize the action of chlorothiazide in man (Laragh, 1958; Bayliss, Marrack, Perkis, Rees & Silva, 1958; and others) and in dogs (Beyer, 1958) were absent both in clinical and experimental diabetes insipidus. The first dose of chlorothiazide caused loss of salt without change in urine volume in these diabetics, so that the osmolarity of the urine rose. Thereafter the saluresis abated but the urine remained concentrated so long as the drug was given, and the daily volume of urine remained about halved. Kennedy & Crawford (1959) attributed this adaptation to the initial saluretic effects of chlorothiazide to change in the effects of the drug on tubular function. Then de los Reyes, Gomez & Bradford (1960) found that chlorothiazide decreased the filtration rate in diabetic patients; this they considered sufficient to explain the decrease in urine volume. They were confirmed in their observation by DAvid, Lazlo & Kovacs (1960), who also showed that the antidiuretic action of chlorothiazide in patients with diabetes insipidus was absent in patients suffering from psychic polydypsia, although both groups of patients lack circulating anti-diuretic hormone (ADH). Davey & Lockett (1960) found differences in the actions of aldosterone monacetate on the isolated cat kidney related to the presence or absence of a circulation through the head during the collection of blood for the perfusion circuit and the making of the heart-lung-kidney preparation. They attributed this difference to the absence of an oxytocin-like substance from blood collected in the absence of a head circulation. We were therefore interested to compare the actions of chlorothiazide on isolated kidneys perfused with these two types of blood, and to discover whether oxytocin modified the effects of chlorothiazide in these preparations. METHODS Male, female or neuter cats were used. Anaesthesia was induced with ether and was maintained by the intravenous injection of 1.0 % chloralose in 0.9 % NaCl, 8-0 ml./kg. The larger cats were used as blood donors. Heparinized blood (heparin 1500 u.f100 ml. blood)

2 CHLOROTHIAZIDE AND ISOLATED KIDNEYS 455 was withdrawn by femoral arterial cannula, either without further interference with the circulation (intact donors) or after ligature of the brachiocephalic and left subelavian arteries and section of the vagi (headless donors). The blood was usually stored overnight at 70C and next day until the heart-lung-kidney preparation was being made. Then mg NaHCO3 in 2-5 ml. 0-9 % NaCl was added. To each 100 ml. blood was added 3 0 ml. of a mixture of 5 % creatinine and 2-5 % p-aminohippuric acid (PAH) in 0.9 % NaCl, and an infusion of PAH was made into the venous reservoir to maintain a constant concentration of PAH in the blood. Heart-lung-kidney preparations were made in cats weighing kg as described by Davey & Lockett (1960), without exclusion of the head from the circulation until approximately 4-5 min before the perfusion of the kidney began. The temperature of the blood and the kidney chamber varied from 36-0 to 37-0 C and the arterial pressure from 114 to 148 mm Hg on different occasions, but these remained constant throughout each experiment. The methods used for the measurement of renal blood flow (R.B.F.), the collection of urine and blood samples, the estimation of sodium, potassium and creatinine in blood and urine, have been reported (Davey & Lockett, 1960). The carbon dioxide combining power of plasma was estimated by the method of Hawk, Oser & Summerson (1947), and p-aminohippuric acid in plasma and urine by the method of Bratton & Marshall as modified by Smith, Finkelstein, Aliminosa, Crawford & Graber (1945). Chlorothiazide (Merck, Sharp and Dohme Ltd), Pitocin and Pitressin (Parke Davis and Co. Ltd), creatinine, and inulin (British Drug Houses Ltd), p-aminohippuric acid (L. Light and Co. Ltd) and mannitol (Hopkin and Williams Ltd) were obtained directly from the manufacturers. RESULTS The effect of chlorothiazide on cat kidneys perfused with blood drawn from animals in which the head had not been excluded from the circulation (intact donors) The influence of chlorothiazide on the excretion of sodium and potassium ions and water was examined at spontaneous rates of urine flow in four heart-lung-kidney preparations perfused with blood collected from intact donors. Figure 1 shows results from one experiment typical of this series. When the urine and blood flows had remained nearly constant for half an hour, 10 mg chlorothiazide was injected into the venous reservoir to mix with 145 ml. of circulating blood. Within 10 min the rates of urine flow and of excretion of Na and K had begun to increase and the Tm ofp-amino hippuric acid (PAH) to decrease. The maximum effects of chlorothiazide on urine flow, excretion of these ions and Tm PAH were reached in min, were well maintained, and were unaccompanied by changes in R.B.F. or creatinine clearance. The CO2 combining power of the plasma fell steadily throughout all experiments, sometimes reaching values as low as 29 ml./100 ml. Neither this, nor the addition of 40 m-u. vasopressin/ 150 ml. circulating blood influenced the recorded actions of chlorothiazide.

3 456 T. DE LIMA AND MARY F. LOCKETT The effect of chiorothiazide on cat kidneys perfused with blood drawn from animalk in which the head had been excluded from the circulation (headless donors) Kidneys perfused with blood taken from headless donors excreted a greater proportion of the filtered load of Na than those perfused with blood 230 Na -E Na K E 170 4a0 Na E Na 34- z (ml./min) 0-5 Urine 01 Urine (ml./min) 0-O - G.F.R. 1-0 _ ~~~~~~~~~~~G.F.R. R.B.F. c h o t o o N K a w (ml./min) 9_ * 115 T,, PAH 95 =- (% control) _ Minutes Fig. 1.- The effect ofchlorothiazide on the excretion ofna+, K+ and water by a cat's kidney, 7*4 g, perfused at 142 mm Hg with blood taken from an intact donor. Ordinates, from above downward: urinary concentration of Na, K; urinary excretion of Na, K; Na excreted (% filtered load); G.F.lR.; urine volume; R.B.F.; Tm PAH. Abscissae, time. Chlorothiazide, 10 mg/145 ml. blood given at arrow. R.B.it

4 CHLOROTHIAZIDE AND ISOLATED KIDNEYS 457 taken from intact animals (Table 1). The effect of chlorothiazide in blood from headless donors was also characteristic and is described by reference to a typical result in Fig. 2. A small increase in urine flow, accompanied by an increase in the rate of excretion of Na and K ions and decrease in Tm PAH, became evident in the 10 min immediately following the addition of 10 mg of the drug to the venous reservoir. The concentrations of Na and K ions in the urine remained unchanged (Fig. 2) or rose slightly to reach a maximum in the next 10 min period. Thereafter these concentrations remained unaltered, but the creatinine clearance and urine volume decreased progressively and in parallel. The proportion of the filtered load of Na excreted was raised by chlorothiazide, then remained constant; it did not decline as the rates of filtration and excretion of Na and K decreased (Fig. 2). TABLE 1. The effect of chlorothiazide (10 mg/ ml.) on cat kidneys perfused with blood taken from intact animals is contrasted with the action of the drug in kidneys perfused with blood from headless donors In blood from intact donors In blood from headless donors At maximum At maximum Effect measured Before drug effect of drug Before drug effect of drug Urine flow (ml./min) (4) (4) (4) (4) Na excretion rate (4) (4) 34L1 ±+41 (4) (4) (t,-equivlmin) Na excreted (% (4) 9* (4) (4) (4) filtered load) Creatinine clearance (4) (4) (4) *19 (4) Weight of kidneys (g) 7* (4) (4) The values given are means+ standard error of the mean, followed by the number of preparations within brackets. Each kidney was perfused with one type of donor blood only. Oxytocin, 20 m-u./ ml. blood, produced only short-lasting antagonism of reductions in filtration rate caused by chlorothiazide in these preparations, and did not alter the proportions of the filtered load of sodium reabsorbed (Fig. 3). DISCUSSION The first point of interest arising out of these experiments is the very great similarity of the effect of chlorothiazide in man (Laragh 1958; Bayliss et al. 1958) and in intact animals (Beyer, 1958) on the excretion of sodium, potassium and water, and the effect of this drug on isolated cat kidneys perfused with blood drawn from intact animals (Fig. 1). In these perfused preparations chlorothiazide decreased the reabsorption of sodium andwater, enhanced the secretion ofpotassium (Kessler, Hierholzer, Gurd & Pitts, 1959) decreased Tm PAH and was without effect on total renal blood flow (Fig. 1).

5 458 T. DE LIMA AND MARY F. LOCKETT Secondly, there was remarkable similarity between the action of chlorothiazide on the excretion of salt and water in the patient with diabetes insipidus (Crawford & Kennedy, 1959; de los Reyes et al. 1960) and in the isolated cat kidney perfused with blood collected from headless donors (Figs. 2, 3). The only difference in the effect of chlorothiazide on the kidneys of normal man and of the patient with diabetes insipidus was the E 220 _ Na Na _ E 210 K20_ 10 _ K ' Na >- go _Na Er I I- 70_l _ K 10 2S0- a G.F.R. (ml./min) Urine Urine 04_ GFR (ml./min) Minutes Fig. 2. The effect of chlorothiazide on the excretion ofna+, K+ and water by a cat's kidney, 9-2 g, perfused at 148 mm Hg with blood taken from a headless donor under chioralose anaesthesia. Ordinates and abscissae as in Fig. 1. C:hiorothiazide, 10 mg/140 ml. blood given at arrow.

6 CHEOROTHIAZIDE AND ISOLATED KIDNEYS 459 reduction in filtration rate that accompanied the effect of the drug in the diabetic (de los Reyes et al. 1960). Reduction in ifitration rate was also a constant finding when kidneys perfused with blood from headless donors were treated with chlorothiazide (Figs. 2, 3); again it accounted for all the differences in the action of the drug on sodium, potassium and water excretion by isolated kidneys when perfused, on the one hand, with blood from intact donors (Fig. 1), and on the other, with blood from headless donors (Figs. 2, 3). The effect of chlorothiazide on fitration rate in the diabetic patient and isolated kidnley perfused with blood from headless Na (m-equiv/min) Chlorothiazide Oxytocin 10 mg 20 m-u. Na excreted (% filtered load) G.F.R. (ml./min) 1-5 5_. 0-5 _ GFR Urine (ml./min) R.B.F. 20- R.B.F _- (ml./min) Minutes Fig. 3. Transient antagonism by oxytocin of the reduction in G.F.R. and urine flow caused by chlorothiazide in a cat kidney, 12-2 g, perfused at 146 mm Hg with blood taken from headless donors under chloralose anaesthesia. Ordinates, from above downward: Urinary excretion of Na; Na excreted (% filtered load); G.R.F.; urine volume; R.B.F. Abscissae, time. Chlorothiazide, 10 mg and oxytocin 20 m-u. added to 140 ml. perfusing blood at first and second arrows respectively. donors cannot be attributed to lack of ADH for two reasons. First, David et at. (1960) found that chlorothiazide failed to produce antidiuresis in psychic polydypsics, in whom the circulating concentration of ADH is very low; secondly, the cat kidney perfused under chloralose anaesthesia is remarkably insensitive to ADH (Davey & Lockett, 1960), yet the differences between the diabetic and non-diabetic response to chlorothiazide were apparent in these preparations. Neither can the reduction of filtration rate characterizing the action of chlorothiazide in diabetes

7 460 T. DE LIMA AND MARY F. LOCKETT insipidus be attributed to lack of oxytocin, because 20 m-u./150 ml. blood produced only transient antagonism of this reduction (Fig. 3). It is therefore concluded that the patient with diabetes insipidus and blood drawn from headless animals both lack some other substance or substances, stable during the night in blood at 70 C. and surviving 5 hr of perfusion through a heart-lung-kidney preparation without excretion or destruction, which conditions some part of the renal vascular bed to withstand the vascular action of chlorothiazide. This same substance or substances may be concerned with the maintenance of normal sodium reabsorption, since kidneys perfused with blood from headless donors leak sodium at more than the normal rate (Table 1). Neither lack of oxytocin or of vasopressin influenced this steady sodium leak from kidneys perfused with blood from headless donors (Table 1). SUMMARY 1. Chlorothiazide 10 mg/ ml. blood, increased the excretion of Na, K and water, decreased Tm PAH, and caused no change in total R.B.F. of isolated perfused cat kidneys. These changes were evident in 5 min and reached a sustained maximum in min. 2. The drug was without effect on glomerular filtration rate (G.F.R.) in preparations perfused with blood from intact animals, but caused progressive decline in G.F.R. and the minute volume of urine in those perfused with blood drawn after exclusion of the head from the circulation. 3. Oxytocin caused only short-lived antagonism of this reduction in G.F.R. and no change in the proportion of the filtered Na load which was reabsorbed. This work was undertaken while one of us (T. de L.) was receiving a grant for personal maintenance during training in research from Cipla Laboratories, Bombay. REFERENCES BAYLISs, R. I. S., MARRACK, D., PE1xIs, J., REES, J. R. & SIVA, J. F. (1958). The use of chlorothiazide in the treatment of edema: a comparison with other diuretic agents. Ann. N.Y. Acad. Sci. 71, BEYER, K. H. (1958). The mechanism of action of chlorothiazide. Ann. N.Y. Acad. Sci. 71, CRAWFORD, J. D. & KENNEDY, G. C. (1959). Chlorothiazide in diabetes insipidus. Nature, Lond., 183, DAVEY, M. J. & LocsTTr, M. F. (1960). Actions and interactions of aldosterone monacetate and neurohypophysial hormones on the isolated cat kidney. J. Physiol. 152, Divm, M. A., Lkxo, F. A. & Kovkcs, K. (1960). Contrasting effect of chlorothiazide in diabetes insipidus and psychic polydypsia. Lancet, 279, DE LOs REYES, M. P., G6mEz, R. & BDFORD, I. (1960). Hydrochlorothiazide and diabetes insipidus. Lancet, 278, 650. HAWK, P. B., OsEz, B. L. & SummERsoN, W. H. (1947). Practical Physiological Chemistry, London: Churchill.

8 CHLOROTHIAZIDE AND ISOLATED KIDNEYS 461 KENNEDY, G. C. & CRAWFORD, J. D. (1959). Treatment of diabetes insipidus with hydrochlorothiazide. Lancet, 276, KEssIER, R. H., HEmRHOTL.ZR, K., GURD, R. S. & PiTrs, R. F. (1959). Localisation of the action of chlorothiazide in the nephron of the dog. Amer. J. Physiol 196, LARAGHE, J. H. (1958). Some effects of chlorothiazide on electrolyte metabolism and its use in edematous states. Ann. N.Y. Acad. Sci. 71, SMITH, H. W., FINKErsLE1N, N., ALIMINoSA, L., CRAWFORD, B. & GRABER, M. (1945). The renal clearances of substituted hippuric acid derivatives and other aromatic acids in dogs and man. J. clin. Invest. 24,