Osmolality of distal tubular fluid in the dog. J R Clapp, R R Robinson J Clin Invest. 1966;45(12):1847-1853. https://doi.org/10.1172/JCI105488. Research Article Find the latest version: https://jci.me/105488/pdf Journal of Clinical Investigation Vol. 45, No. 12, 1966 Osmolality of Distal Tubular Fluid in the Dog * JAMES R. CLAPP t AND ROSCOE R. ROBINSON t (From the Department of Medicine, Duke University Medical Center, Durham, N. C.) Renal micropuncture observations in rodents suggested that dog distal fluid is normally hypo- have demonstrated that the tubular fluid from early tonic rather than isotonic at the end of the distal portions of the distal convoluted tubule is always convolution. If so, at high rates of urine flow dur- hypotonic to plasma (1, 2). A limited number of ing osmotic diuresis, the ability of the collecting observations have suggested that its hypotonicity ducts to abstract solute-free water might be so is maintained along the entire distal tubule dur- exceeded that hypotonic urine could be excreted ing water diuresis (1). In contrast, during hy- despite the presence of antidiuretic hormone. dropenia with or without a superimposed osmotic Possible relationships among rates of solute excre- diuresis, the distal fluid achieves osmotic equi- tion, vasopressin dosage, and solute-free water librium with plasma in later portions of the distal reabsorption by the collecting ducts have been tubule (1, 2). Consequently, in the presence of discussed fully by Orloff, Wagner, and Davidson antidiuretic hormone, it has been widely accepted (11). that distal tubular fluid is always isosmotic to Heretofore, direct measurements of distal fluid plasma as it enters the cortical collecting ducts. osmolality utilizing micropuncture techniques have These findings in rodents have precluded a satis- not been feasible in the dog because the distal con- factory explanation for the observation that man voluted tubules could not be recognized on the and dog can excrete hypotonic final urine during surface of the kidney (15). Renal micropuncture certain experimental and pathological conditions experiments in this species have therefore been when antidiuretic hormone activity is presumably confined to studies of proximal tubular function. adequate (3-11). These observations have been However, in the present study, segments of the most apparent at high rates of urine flow and sol- distal convoluted tubules have been identified on ute excretion. If, as in the rat (2, 12), the tubu- the surface of the kidney, thus permitting the ap- lar fluid of the dog is isosmotic as it enters the col- plication of renal micropuncture techniques to an lecting ducts under these conditions, the subse- evaluation of the contribution of distal nephron quent excretion of hypotonic urine would require segments to urinary concentration and dilution in that the collecting ducts reabsorb solute in excess normal dogs. In contrast to earlier observations in of water. Since this explanation is inconsistent rodents (1, 2) measurements of osmolality have with current concepts of solute and water reab- demonstrated that the tubular fluid is markedly sorption by the collecting ducts in the presence of hypotonic along the entire length of the distal tu- maximal amounts of vasopressin (water in ex- bule during hydropenia as well as water diuresis. cess of solute), some investigators (9, 13, 14) have Methods * Submitted for publication May 31, 1966; accepted Au- Renal micropuncture experiments were performed on gust 19, 1966. nine healthy mongrel dogs weighing from 8.2 to 14.5 kg. Supported by grants from the U. S. Public Health Five dogs were examined during hydropenia and antidiu- Service (AM 05930 and AM 07420), the American resis; four animals were evaluated during water diure- Heart Association, the Life Insurance Medical Research sis. Anesthesia was induced by the iv administration of Fund, and the North Carolina Heart Association. Pentothal sodium (25 mg per kg); thereafter small sup- t Established Investigator of the American Heart plemental doses were administered as necessary. Respira- Association. tion was maintained through a cuffed endotracheal tube Address requests for reprints to Dr. James R. Clapp, via a Harvard respirator. The ventilatory rate was ad- Dept. of -Medicine, Duke University Medical Center, justed to maintain the blood Pco2 between 35 and 45 mm Durham, N. C. 27706. Hg. The left kidney was exposed through a flank in- : Senior Investigator of the North Carolina Heart cision, and indwelling polyethylene catheters were in- Association. serted into both foreleg veins and the ureter of the 1847 1848 JAMES R. CLAPP AND ROSCOE R. ROBINSON experimental kidney. An additional polyethylene cathe- an interval of at least 7 to 10 minutes. In general, no ter was introduced into the abdominal aorta via a fe- more than a short segment of a single distal tubule ex- moral artery to the approximate level of the renal tended to the kidney surface. The number of distal tu- arteries. bules on the surface of the kidney varied greatly from Dogs studied during antidiuresis were deprived of wa- one animal to another, and none at all were observed on ter after receiving an im injection of Pitressin tannate occasion. The diameter of the distal tubular lumen was in oil (1 U) about 16 hours before the experiment. After usually smaller than that of the proximal tubule; oc- the induction of anesthesia these animals received a single casionally, they were equal in size. The location of a iv injection of aqueous vasopressin (33 mU per kg). A single distal tubule had to be noted with great care dur- maintenance infusion of aqueous vasopressin in 5% dex- ing the passage of dye because the transparency of their trose in water (50 mU per kg per hour, 0.25 ml per min- epithelial lining made them difficult to see once the dye ute) was administered via a foreleg vein throughout had cleared the distal tubular lumen. However, once a each experiment. distal tubule had been identified and punctured, samples Animals examined during water diuresis were allowed could be collected with relative ease because of a brisk free access to water before each study. After anes- flow of tubular fluid even during hydropenia. Sites of thesia, water diuresis was induced by the rapid iv ad- distal tubular puncture were identified by the latex in- ministration of 2.5% dextrose in water (40 to 80 ml per jection and dissection technique (16). As in the rat, the kg). The initial water load was followed by a constant distal convoluted tubule is defined as that segment of the iv infusion of 2.5%o dextrose in water at a rate equal to distal nephron between the macula densa and the point the urine flow. Water diuresis occurred within 3 to 7 at which it joins with another distal tubule to form a hours. Qualitative tests for urinary glucose were nega- cortical collecting duct. tive during the entire period of water diuresis. Before the collection of distal fluid samples, the di- In each group, preparatory to micropuncture, the left rection of flow along the tubule was first ascertained by kidney was immobilized on a Lucite holder as described the injection of a droplet of colored mineral oil. Samples previously (15). The administration of an initial pri- of tubular fluid (approximately 0.01 Al) were then col- ming dose of inulin was followed by a constant infu- lected at a rate sufficient to maintain the distal position sion (0.3 ml per minute) of 2.5 to 5%o inulin in either of an injected oil droplet. Only a brief period of time was 5%o dextrose in water (antidiuresis experiments) or required for the collection of these small samples. 0.85%o saline (water diuresis experiments). After equili- Immediately after collection, the samples were emptied bration of the priming dose of inulin, sequential renal from the collection pipettes into a siliconized petri dish clearance periods of 15 to 20 minutes' duration were ob- filled with hydrated mineral oil. Duplicate aliquots tained throughout each study. Heparinized samples of (< 0.001 A0) of the sample were transferred into separate arterial blood were obtained at the midpoint of each mineral oil-filled quartz capillaries (i.d. 25 AL), and their urine collection period for the determination of the plasma osmolality was measured cryoscopically in an Advanced inulin concentration and osmolality. Similar measure- nanoliter osmometer. This apparatus permitted simul- ments were also performed on timed collections of urine taneous measurements on the unknown sample and two from the experimental kidney. standard solutions of known osmolality (500, 100, or 50 A single sample of proximal fluid and two to nine mOsm per kg of H20) . In this fashion, each of the samples of distal tubular fluid were obtained from the duplicate readings was bracketed by simultaneous read- surface convolutions during each experiment. The tech- ings on two standard solutions of known osmolality. nique for collection of proximal fluid samples and the Ultramicro osmolality measurements on plasma samples means of identifying the sites of tubular puncture in this from each experiment averaged 98.2% of the macro segment of the nephron have been described previously measurements on the same sample. Replicate ultramicro (15). The identification of surface convolutions of the analyses on different aliquots of the same sample always distal tubule was facilitated by the intra-aortic injection agreed within ± 2%. of 1 or 2 ml of 5% lissamine green before each distal Plasma and urinary inulin concentrations were deter- puncture. After its injection the dye appeared rapidly mined by the anthrone method of Fuhr, Kaczmarczyk, and in the peritubular circulation and proximal tubular fluid. Kruttgen. Macro measurements of the osmolality of Its passage through the lumina of the proximal convo- plasma and urine were made on an Advanced osmometer luted tubules was usually complete within 20 to 25 sec- (model 64-31).