fluid, *7 in peritubular capillary plasma and in

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1 J. Phy8iol. (1977), 273, pp With 2 text-ftgurem Printed in Great Britain COMPARISON OF CHLORIDE CONCENTRATION AND OSMOLALITY IN PROXIMAL TUBULAR FLUID, PERITUBULAR CAPILLARY PLASMA AND SYSTEMIC PLASMA IN THE RAT BY JOHN C. ATHERTON From the Department of Physiology, University of Manchester, Manchester M13 9PT (Received 25 May 1977) SUMMARY 1. Chloride concentration and osmolalities were compared in consecutively collected samples of proximal tubular fluid, peritubular capillary plasma and systemic plasma. 2. Mean chloride concentrations (m-mole/l) were 141' in tubular fluid, *7 in peritubular capillary plasma and in systemic plasma. 3. Mean osmolalities (m-osmole/kg H2) were in tubular fluid, in peritubular capillary plasma and in systemic plasma. 4. These differences are discussed in relation to the anatomical and functional organization of the peritubular capillaries and renal tubules. INTRODUCTION In recent years the mechanisms of fluid reabsorption by the renal proximal tubule have been extensively investigated. To account for passive reabsorption of the fraction of the filtrate normally reabsorbed along the tubule it would be necessary to postulate the existence of an osmotic driving force of approximately 2 m-osmole/l (Giebisch, 1972). The assumption that such a gradient does not exist has necessitated the development of complicated models to explain fluid reabsorption (reviewed by Sackin & Boulpaep, 1975). Evidence has also accumulated to suggest that the establishment of a transtubular concentration gradient for chloride in the early part of the proximal tubule is important for fluid reabsorption in the remainder of this nephron segment (Fr6mter, Rumrich & Ullrich, 1973; Barratt, Rector, Kokko & Seldin, 1974; Neumann & Rector, 1976). The actual

2 766 J. C. ATHERTON contribution of this gradient to total fluid reabsorption is still questioned (Green & Giebisch, 1975; Bank, Aynedjian & Weinstein, 1976; Burg & Green, 1976; Green, 1977). Despite the obvious importance of transtubular ionic gradients for fluid reabsorption, evaluation of the mechanisms responsible has usually occurred in the absence of accurate knowledge of their magnitude. The gradients have been calculated as the difference between tubular fluid and systemic plasma rather than the true comparison of tubular fluid and the peritubular environment; the inherent assumption being equality of composition between systemic and peritubular capillary plasmas. The present study was undertaken to test this assumption for chloride concentration and osmolality. Furthermore, by the consecutive sampling of proximal tubular fluid and peritubular capillary blood from vascular stars immediately adjacent to the site of tubular fluid collection, it was possible to obtain a more accurate assessment of the magnitude of the transtubular gradients. METHODS Experiments were performed on male Sprague Dawley rats (weighing 15-2 g) allowed free access to water but fasted for 18 h before experimentation. Animals were anaesthetized by an i.p. injection of Inactin (Promonta, Hamburg) 12 mg/kg body wt. Catheters were placed in the left jugular vein for infusion of -9 % saline (3H inulin containing 1 #sc/ml.) at 2 #I/min; in the right carotid artery to record blood pressure (Statham pressure transducer connected to a Grass polygraph, model 7B), and in the left ureter to drain pelvic urine. Body temperature was monitored throughout the experiment and maintained at 37 C. The left kidney was exposed by a mid-flank incision, cleared of perirenal fat and placed in a perspex cup. The surface of the kidney was bathed with oil heated to 37C. The internal surfaces of all micropipettes, tip o.d. of jum and sharpened on three sides, were coated with a dilute silicone solution (Sigmacote) before use. Three fluid samples were obtained as quickly as possible, the interval of time between each collection being less than 3 min. Sample 1. Peritubular capillary blood from a surface vascular star. Collection was entirely spontaneous. The blood was transferred immediately to an oil-containing siliconized constant bore glass capillary which was then sealed at one end and centrifuged. The haematocrit was determined. Sample 2. A timed collection of tubular fluid from a surface convolution of a proximal tubule immediately adjacent to the site of peritubular capillary blood collection. Sample 3. Systemic blood collected from the tail was centrifuged and the haematocrit determined. Only the results of samples when all three were collected and subsequently analysed are included. Chloride concentrations, determined by the method described by Ramsay, Brown & Croghan (1955) for nanolitre samples and osmolalities by freezing point depression (Clifton Technical Physics, Hartford, N.Y.) were obtained for peritubular capillary plasma, systemic plasma and tubular fluid. 3H Inulin was measured (Intertechniques

3 RENAL TRANSTUBULAR GRADIENTS Liquid Scintillation Spectrometer) in tubular fluid and systemic plasma. Since the primary use of 3H inulin in this study was to calculate water reabsorption by the proximal tubules and not to determine the transtubular inulin concentration gradient, its concentration in peritubular capillary plasma was not measured. Caculions Nephron filtration fraction was calculated from peritubular capillary blood and systemic blood haematocrits as described by Brenner &; Galla (1971): FFpTCB = 1- Htc,8 (1- Htcp-) H4Tm (1-Htc.B)' 1 where FFprCB = peritubular capillary nephron filtration fraction; HtcsB = systemic blood haematocrit; HtcpTcB = peritubular capillary blood haematocrit. Measured plasma chloride concentrations were corrected for plasma water using factors of 1-6 for systemic plasma (Pitts, 1963; Fromter et al. 1973) and 1- FFPTCBI (1- (FFPTCB +.6)) for peritubular capillary plasma. Single nephron glomerular filtration rate (SNGFR) was calculated as TF Vn - In. (2) P Fractional water reabsorption by the proximal tubule was calculated as (1- ( TF PIn) ) x 1. (3) Fractional reabsorption of chloride and osmoles by the proximal tubule were calculated as ( P TF )() 767 In eqns. (2)-(4) the following abbreviations have been used; Vn = volume of tubular fluid collected per minute (nl./min); TF/P = proximal tubular fluid to systemic plasma concentration ratio where In = inulin; a = chloride or osmolality. Data are presented as means ± s.e. of mean. Statistical analyses used were the Student's t test for paired observations and calculation of linear regression lines by least-squares analysis. RESULTS Only those data from animals whose mean blood pressure exceeded 9 mmhg and which had a proximal tubule transit time (Steinhausen, 1963) of less than 12 sec are presented. Mean SNGFR was nl./min for all tubules punctured. Mean haematocrits obtained for peritubular capillary blood and systemic blood were 53% ±.8 (range 46-61) and 42 / + -4 (range 37-45) respectively. Table 1 includes data for chloride concentrations and osmolality in tubular fluid, peritubular capillary plasma and systemic plasma. The individual comparisons for osmolality and chloride concentrations in peritubular capillary plasma and systemic plasma are included in Fig. 1. The majority of points fall below the line of identity, indicating

4 768 J. C. ATHERTON that peritubular capillary plasma chloride concentration and osmolality are lower than the corresponding systemic plasma values (paired t test, P <.1 for chloride, P <.5 for osmolality). TABLx 1. Mean + S.E. of mean of chloride concentrations and osmolalities in tubular fluid, peritubular capillary plasma and systemic plasma. The values in parentheses represent the number of comparisons between tubular fluid, peritubular capillary plasma and systemic plasma. The values for peritubular capillary plasma and systemic plasma chloride concentrations have been corrected for plasma water - see Methods Peritubular Tubular capillary Systemic fluid plasma plasma *6 114*8± ± 1*8 297 ± ± ± 1*8 Chloride (m-molefl.) (3) Osmolality (m-osmole/ kg Hs (25) 14 i Chloride (m-mole/l.) W E co a co J 14. M I- 33 r * Osmolality (m-osmole/kg H2) Systemic plasma Fig. 1. Relation between peritubular capillary plasma and systemic plasma for chloride concentration and osmolality. Continuous lines represent the lines of identity. 33

5 RENAL TRANSTUBULAR GRADIENTS 769 Thus for both chloride and osmolality the assumption of equality in composition between peritubular capillary plasma and systemic plasma leads to an underestimate of the magnitude of the transtubular concentration gradient. When calculated as the tubular fluid to plasma concentration ratio the mean transtubular gradient for chloride is c U- C I- a. CA,. M - C c) U- U. 8 r- B a) ECn 41- l l l ' 4 8 Fractional water reabsorption (%) A Fig. 2. Relation between fractional reabsorption by the proximal tubule for chloride (A) and osmoles (B) and fractional water reabsorption. Equations for the regression lines are y = 18-6 x , and y = 93-8 x for chloride and osmoles respectively and the correlation coefficients -91 and -98. and using peritubular capillary plasma and systemic plasma respectively (paired t test, P < -1) and for osmolality is and using peritubular capillary plasma and systemic plasma respectively (paired t test, P <.5). How such differences might arise is suggested at least for chloride in Fig. 2 where fractional reabsorption of chloride (A) and osmoles (B) are

6 77 J. C. ATHERTON plotted against fractional water reabsorption. Extrapolation of line B goes through the origin. Extrapolation of line A cuts the X-axis at a point significantly above zero (P < x1), indicating that 15-2% fractional water reabsorption has occurred before chloride reabsorption becomes significant. DISCUSSION The present data, obtained during infusion of -9 % saline at 2 csl./min, demonstrate that there are differences in composition between systemic and peritubular capillary plasmas; namely the chloride concentration and osmolality of peritubular capillary plasma are significantly lower. The data add, therefore, to the recent observations of Weinstein & Szyjewicz (1976) that during infusion of Ringer solution at 5,ul./min in the rat, peritubular capillary plasma chloride concentration is approximately 6 % lower than in systemic plasma. In addition, since peritubular capillary blood and fluid from proximal tubules immediately adjacent to the site of blood collection were collected as nearly as possible simultaneously the present data enable an accurate assessment of the true magnitude of the transtubular gradients not only for chloride but also for osmolality. It is considered that the observed differences in composition between peritubular capillary plasma and systemic plasma are real since it is unlikely that they resulted from contamination of the peritubular capillary blood sample with either tubular fluid or renal surface fluid during collection since (a) both tubular fluid chloride concentration and osmolality were always higher and (b) the peritubular capillary blood haematocrit was higher than the systemic blood haematocrit. The anatomical organization of the efferent arterioles and the first portion of the proximal tubules derived from glomeruli of superficial cortical nephrons is such that they probably ascend in close proximity to each other to the subcapsular surface of the kidney (Beeuwkes, 1971). At the surface of the kidney the efferent arterioles form vascular stars from which the peritubular capillary network is derived (Steinhausen, Eisenbach & Galasky, 197; Brenner & Galla, 1971). It is likely, therefore, that the reabsorptive characteristics of the early part of the proximal tubule (that part inaccessible to micropuncture) would determine the composition of the blood collected from the surface vascular stars. Measurement of the transtubular potential profile along the proximal tubule (Fromter et al. 1973; Barratt et al. 1974; Fromter & Gessner, 1974; Seely & Chirito, 1975) suggests that the reabsorptive characteristics of the early part of the proximal tubule differ from those found

7 RENAL TRANSTUBULAR GRADIENTS 771 in later segments. Of particular relevance to the present work is the finding that in the early part of the proximal tubule there is only negligible reabsorption of chloride (Le Grimellec, 1975). In addition, associated with the reabsorption of solutes from the proximal tubule is iso-osmotic water reabsorption. Support for these suggestions is provided by the relationship between percentage water reabsorption and percentage chloride and osmolal reabsorption (Fig. 2). The data are consistent with the view that there is a linear and direct relationship between osmolal reabsorption and water reabsorption. However, chloride is not reabsorbed in significant amounts until water reabsorption is 15-2 % of the filtered load. Since the reabsorbate from the early part of the tubule is characterized by being relatively free of chloride (Le Grimellec, 1975) and rich in bicarbonate (Rector, Carter & Seldin, 1965), the chloride in the plasma escaping filtration will be diluted as it passes directly to the vascular stars on the subcapsular surface of the kidney. The small yet significant difference between the osmolality of peritubular capillary and systemic plasmas is incompatible with the generally accepted view that solute reabsorption in the proximal tubule is accompanied by iso-osmotic water reabsorption. To account for this observation water reabsorption in excess of solute is required. One possible explanation is 'active' water reabsorption as suggested by Fr6mter et al. (1973). Alternatively, since the effective osmotic pressure need not necessarily be the same as that found by direct measurement (Staverman, 1951), solutions with different ionic composition and different reflection coefficients could give rise to a situation whereby the directly measured osmotic gradient did not reflect the effective osmotic gradient. For example, the effective osmotic gradient between solutions with measured osmolalities of 3 m-osmole/kg H2 composed of sodium chloride, which has a reflection coefficient of 7, and sodium bicarbonate, which has a reflection coefficient of 1, would be 9 m-osmole/kg H2. Therefore, measured osmolalities do not give true measurements of effective osmotic pressures. Finally, it is possible that since the tubular and capillary organisation on the surface of the kidney is such that the distal tubules come into contact with the same peritubular capillary network as the proximal tubules (Beeuwkes & Bonventre, 1975) the possibility exists that ADH-induced water reabsorption would dilute the peritubular capillary plasma. In summary, the present data demonstrate the existence of differences in chloride concentration and osmolality between peritubular capillary and systemic plasmas. These differences appear to be the resultant of the structural organization on the surface of the kidney and the transport

8 772 J. C. ATHERTON 772 J.AHRO characteristics of the different nephron segments, in particular the first portion of the proximal tubule. The significance of transtubular concentration gradients to fluid reabsorption is such that these differences may be of importance in proximal tubular function. It is pertinent, therefore, to suggest that data interpreted with the assumption of equality in composition between systemic and peritubular capillary plasmas should be re-evaluated. REFERENCES BANx, N., AYNXDJIAN, H. S. & WEINSTEIN, S. W. (1976). Effect of intraluminal bicarbonate and chloride on fluid absorption by the rat renal proximal tubule. Kidney int. 9, BARRATT, L. J., RECTOR, F. C., KoKxo, J. P., & SELDIN, D. W. (1974). Factors governing the transepithelial potential difference across the proximal tubule of the rat kidney. J. clin. Invest. 53, BEEuWKES, R. (1971). Efferent vascular patterns and early vascular tubular relations in the dog kidney. Am. J. Physiol. 221, BEEVIWKES, R. & BONVENTRE, J. V. (1975). Tubular organisation and vascular relations in the dog kidney. Am. J. Physiol. 229, BRENNER, B. M. & GALLA, J. H. (1971). Influence of postglomerular haematocrit and protein concentration on rat nephron fluid transfer.' Am. J. Physiol. 22, BURG, M. & GREEN, N. (1976). Role of monovalent ions in the reabsorption of fluid by isolated perfused proximal renal tubules of the rabbit. Kidney int. 1, FROMTER, E. & GESSNER, K. (1974). Free-flow potential profile along rat kidney proximal tubule. Pflugers Arch. ges. Physiol. 351, FROMTER, E., RUMRICH, G. & ULLRIcH, K. J. (1973). Phenomenologic description of Na+, Cl- and HC3- absorption from proximal tubules of the rat kidney. Pfluigers Arch. ges. Physiol. 343, GrEBISH, G. (1972). Coupled ion and fluid transport in the kidney. New Engl. J. Med. 287, GREEN, R. (1977). Ionic requirements of proximal tubular fluid reabsorption in the rat in vivo. In Macy conference on Renal Function, ed. GIEBIH, G. Charleston: Macy Foundation. (in the Press). GREEN, R. & GIEBISCE, G. (1975). Ionic requirements of proximal tubular sodium transport. I. Bicarbonate and chloride. Am. J. Physiol. 229, LE GRIMELLEC, C. (1975). Micropuncture study along the proximal convoluted tubule. Electrolyte reabsorption in first convolutions. Pflugers Arch. yes. Physiol. 354, NEUMANN, K. H. & RECTOR, F. C. (1976). Mechanism of Na Cl and water reabsorption in the proximal convoluted tubule of rat kidney. J. clin. Invest. 58, PITrs, R. F. (1963). Physiology of the Kidney and Body Fluids, 2nd edn., p. 29. Chicago: Year book medical publishers Inc. RAMSEY, J. A., BROWN, R. M. J. & CROGHAN, P. C. (1955). Electrometric titration of chloride in small volumes. J. exp. Biol. 32, RECTOR, F. C., CARTER, N. W. & SELDIN, D. W. (1965). The mechanism of bicarbonate reabsorption in the proximal and distal tubules of the kidney. J. cdin. Inve8t. 44, SARKIN, H. & BouLPAEP, E. L. (1975). Models for coupling salt and water transport: proximal tubular reabsorption in Necturus kidney. J. gen. Physiol. 66,

9 RENAL TRANSTUBULAR GRADIENTS 773 SEELY, J. F. & CHIRITO, E. (1975). Studies of the electrical potential difference in rat proximal tubule. Am. J. Physiol. 229, STAVEBMA, A. J. (1951). The theory of measurement of osmotic pressure Reel. Trav. chir. Pay8-Ba8. Beig. 7, STENHAusEN, M. (1963). Eine Methode zur Differenzierung proximaler und distaler Tubuli der Nierenrinde von Ratten in vivo und ihre Anwendung zur Bestimmung tubularer Stromungsgeschwindigkeiten. Pfluger8 Arch. gem. Phy&iol. 227, STEnX;AusEN, M., EISENBACH, G. M. & GALASKY, R. (197). A countercurrent system of the surface of the renal cortex of rats. Pfluger8 Arch. ge8. Phy8iol. 318, WEINsTEIN, S. W. & SZYJEWIcZ, J. (1976). Early postglomerular plasma concentrations of chloride, sodium, and inulin in the rat kidney. Am. J. Physiol. 231,