Micropuncture study of the effect of various diuretics on sodium reabsorption by the proximal tubules of the dog. J H Dirks, … , W J Cirksena, R W Berliner J Clin Invest. 1966;45(12):1875-1885. https://doi.org/10.1172/JCI105492. Research Article Find the latest version: https://jci.me/105492/pdf Journal of Clinical Investigation Vol. 45, No. 12, 1966 Micropuncture Study of the Effect of Various Diuretics on Sodium Reabsorption by the Proximal Tubules of the Dog * JOHN H. DIRKS,t WILLIAM J. CIRKSENAt AND ROBERT W. BERLINER § WITH THE TECHNICAL ASSISTANCE OF NORDICA GREEN (From the Laboratory of Kidney and Electrolyte Metabolism, National Heart Institute, Bethesda, Md.) The precise sites of action of various diuretics ulin ratios are compared for each tubule. Re- within the nephron have been the subject of nu- peated collections from the same tubules do not merous investigations and considerable debate for appreciably alter tubule fluid reabsorption during several decades. Inhibition of sodium transport in continued hydropenia or continued saline infusion. the proximal tubule has, at one time or another, Acute saline infusion, however, markedly de- been considered to play a major role in the ac- pressed sodium reabsorption by the proximal tu- tion of virtually every group of diuretics. How- bule, an effect that was independent of changes in ever, such actions on the proximal tubules have glomerular filtration rate and occurred in the pres- been inferred only from indirect evidence. The ence of high circulating levels of mineralocorticoid technique of micropuncture allows a more direct hormones and vasopressin. This effect of acute evaluation of the effects of pharmacologic agents salt loading on reabsorption in the proximal tu- on sodium reabsorption by the proximal tubules. bule is thought to be mediated by some unknown The exploration by micropuncture technique of humoral agent (5-7) and has also been shown to the effects of diuretics on various portions of the occur in the rat (8). The reabsorption of fluid nephron is as yet very limited. Carbonic anhy- undergoes a considerable redistribution from prox- drase inhibitors have been shown to impair acidi- imal to distal segments after saline infusion. This fication in the proximal convoluted tubules in the makes it difficult to assess the localization of the rat (1, 2). Recently Deetjen (3) and Malnic, action of a diuretic from the magnitude of the diu- Vieira, and Enokibara (4) have examined the resis produced, since the fraction of the filtered effect of the new sulfonamide diuretic furosemide sodium that is reabsorbed in the various nephron on fluid reabsorption in the proximal tubule. segments may vary so extensively. It furthermore For the detection of the effects of interventions makes necessary careful attention to the state of on reabsorption by the tubules, a considerable ad- hydration in evaluating any changes detected in vantage accrues from repeated collections from reabsorption in the proximal tubule, since differ- the same site in the tubules, a procedure used in ences in hydration can in themselves produce recently reported studies (5). Identical convolu- marked changes in reabsorption. tions of proximal tubules are sampled during each In the studies to be reported here we have used experimental phase, and changes in sodium reab- the technique of collections from the same tubule sorption as measured by tubule fluid to plasma in- segments in dogs before and after administration of diuretics. In most of the studies, the dogs were * Submitted for publication May 24, 1966; accepted hydropenic throughout, but a few were infused August 30, 1966. Presented in part at the Eastern Section of the Ameri- with saline before the control periods, and in can Federation for Clinical Research, New York, N. Y., others sufficient isotonic saline solution was ad- December 3, 1965. ministered to replace losses due to diuresis. Al- t Present address: Department of Medicine, Royal Vic- though the methods were sensitive enough to de- toria Hospital, Montreal 2, Canada. tect the effects of small doses of mannitol, none t Special Fellow, Office of the Surgeon General, De- partment of the Army, Washington, D. C. of the other diuretics was found to yield signifi- § Address requests for reprints to Dr. Robert W. Ber- cant depression of reabsorption in the proximal liner, National Heart Institute, Bethesda, Md. 20014. convoluted tubule. 1875 1876 J. H. DIRKS, W. J. CIRKSENA, AND R. W. BERLINER Methods The following experiments with diuretics were con- ducted. Studies were performed on mongrel dogs weighing Mannitol. Graded intravenous infusions of mannitol 10 to 30 kg. The anesthesia, inulin infusion, and surgi- were used to assess the sensitivity of the technique for cal preparation of the dogs for clearances and micropunc- the detection of depressed fluid reabsorption by the ture were identical to those previously reported (5, 9, proximal convoluted tubules. Mannitol was infused into 10). Collections from convolutions of proximal tubules a leg vein at various flow rates (2 to 10.7 ml per min- were first made during a control period, and the precise ute) and at varying concentrations (5, 10, and 20%) in site of micropuncture was carefully marked in each case eight hydropenic dogs. This resulted in the excretion of with nigrosine dye layered on adjacent tubules. A dose 2 to 46% of the volume of glomerular filtrate (V/GFR) of a diuretic agent sufficient to produce a maximal 1 during resampling of previously punctured tubules. In effect was then administered intravenously. This initial four hydropenic dogs the repeat collections were done dose was followed by an infusion delivering the same in periods when V/GFR fell in the range of 10 to 46%. amount of drug per hour. When the urine flow had in- In four hydropenic dogs, repeat collections were made creased to several times the control rate, samples were when urine flow was 2 to 9% of glomerular filtration obtained from the same proximal tubule convolutions. rate. Collections of fluid from convolutions of proximal tu- Ethacrynic acid. Initial collections were performed in bules were made as described previously (5). The mi- six hydropenic dogs. Ethacrynic acid2 was then ad- cropipette, filled to the tip with colored mineral oil, was ministered intravenously in doses of 1 to 5 mg per kg inserted into a tubule, and a small amount of oil was in- with an equal hourly infusion rate. In five dogs, initial jected into the lumen. The oil drop was permitted to flow collections were performed after initial saline loading as distally, and tubule fluid was allowed to flow spontane- described for the saline controls. Ethacrynic acid was ously into the pipette. After the administration of diu- then administered as above, and the fluid loss resulting retics, the intratubular pressure was increased; conse- from the diuresis was replaced with intravenous infusion quently, the samples were analyzed only if the oil drop of an equal volume of isotonic saline solution. A single remained stationary or moved distally while in view. experiment in which the initial collections were carried In this way, the possibility of retrograde collections was out in hydropenia but in which the fluid losses were re- averted. Inulin in plasma and urine was determined by placed with isotonic saline was combined with this the anthrone method of Fiuhr, Kaczmarczyk, and Kriitt- latter group. gen (12), and filtration rates were calculated for 15- Chlormerodrin. Three dogs were loaded with 10 g minute periods during the micropuncture procedure. ammonium chloride 18 hours before the experiment. Con- Inulin in tubule fluid was determined by a modification of trol collections were performed in hydropenia, and then the microanthrone method (13, 14) as previously de- chlormerodrin was administered intravenously as a prim- scribed in detail (5) ; the method yielded reproducibility ing dose of 2 mg Hg per kg with an equal hourly infu- with a standard deviation of 4.7%o. Tubule fluid to sion rate. Collections were repeated when diuresis had plasma (TF/P) inulin ratios were calculated with the increased the flow rate severalfold over control values. plasma inulin interpolated to the midpoint of the sample In three dogs initial collections were obtained during sa- collection when there were changes in plasma inulin line loading as described and then chlormerodrin was concentration. administered as above. The losses due to diuresis were Controls. The results of control re-collection during replaced by the intravenous infusion of roughly equal continued hydropenia (thirsted 18 hours) or continued volumes of isotonic saline solution. In one dog, the saline infusion have' been reported (5). Additional con- diuretic-induced fluid losses were similarly replaced af- trols obtained during continued saline infusions from ter initial collections were performed in hydropenia. seven dogs are included in this report. Dogs were again Furosemide. Furosemide 3 was studied in four hydro- infused with 0.4 ml isotonic saline per kg per minute for penic dogs. In two animals furosemide was administered 30 minutes and then 0.2 ml per kg per minute to the as an initial dose of 5 mg per kg followed by an infu- conclusion of the experiment. sion of the same dose each hour. The other two ani- Reduced filtration rate. The results of reduced filtra- mals received five times this dose. No attempt was tion rate on proximal TF/P inulin ratios during continued made in the furosemide experiments to preserve the ani- saline infusion have also been reported (5). Similarly, mal's sodium balance with isotonic saline infusion. filtration rate was reduced in seven hydropenic dogs by Hydrochlorothiazide. In three dogs, initial collections clamping the left renal artery with a screw clamp. were performed in hydropenia. Hydrochlorothiazide was Urine flow was reduced and the previously punctured administered at a dose of 2 mg per kg initially, followed tubules were resampled.