THE EFFECT OF POTASSIUM ON INTRACELLULAR BICARBONATE IN SLICES OF KIDNEY CORTEX Helen M. Anderson, … , Gilbert H. Mudge, Theodora J. Lannon J Clin Invest. 1955;34(11):1691-1697. https://doi.org/10.1172/JCI103222. Research Article Find the latest version: https://jci.me/103222/pdf THE EFFECT OF POTASSIUM ON INTRACELLULAR BICARBON- ATE IN SLICES OF KIDNEY CORTEX1 By HELEN M. ANDERSON AND GILBERT H. MUDGE 2 WITH THE TECHNICAL ASSISTANCE OF THEODORA J. LANNON (From the Department of Medicine, College of Physicians and Surgeons, Columbia University, and the Presbyterian Hospital, New York City, N. Y.) (Submitted for publication June 21, 1955; accepted July 20, 1955) An increase in serum bicarbonate concentration concentrations in the kidney cortex can be varied has been observed in association with potassium over a wide range by the use of in vitro techniques, depletion in a variety of clinical and experimental studies of tissue slices were undertaken in order to conditions. Although the development of the al- define the relationships of intracellular potassium kalosis may be extra-renal in origin, its perpetua- and bicarbonate. tion must depend upon an alteration in renal func- tion which can be characterized as either an in- METHODS crease in bicarbonate reabsorption or an increase The preparation of tissues has been described previously in hydrogen ion excretion. Many instances of so- in detail (7) and is summarized here. Fresh slices were made from the renal cortex of rabbits killed by carotid called "paradoxical" aciduria have been described exsanguination and were depleted of potassium by leach- (cf. 1, 2). ing for one hour in 0.15 N NaCl at room temperature. Recent attempts to interpret this phenomenon Half the slices were then incubated in Warburg flasks have been predicated upon the concept that the in 3 ml. of a medium containing NaHCO, 18 mEq. per extracellular alkalosis of potassium depletion is L., CaCl2 1.3 mEq. per L., sodium phosphate buffer (pH 7.4) 3.7 mM. per L. and sodium acetate 0.01 mM. per L.3 accompanied by an intracellular acidosis. Sup- The remaining slices were incubated in a medium identi- porting evidence has been derived from a variety cal in composition except for the addition of 10 mEq. per of experiments. In the muscle of potassium de- L. of KCl. Sufficient NaCl was added to both media to pleted rats Gardner, MacLachlan, and Berman provide constant osmolar concentrations of 300 mOs. per (3) observed a decrease in the intracellular pH as L. The Warburg vessels were gassed with a mixture of 5 per cent CO2 and 95 per cent 02. The carbon dioxide- calculated from whole tissue analyses corrected bicarbonate buffer system provided a pH of 7.4 in the for extracellular space. Cooke and co-workers external medium (8). Following incubation for 35 min- (4), on the basis of balance experiments and utes at 250 C. the slices were promptly removed, blotted, muscle analyses, concluded that the loss of intra- and then analyzed for water and electrolyte. This entire cellular potassium in rats was accompanied by an procedure will be referred to hereafter as the standard system; modification of individual variables will be de- intracellular accumulation of both sodium and hy- scribed for each special circumstance. drogen ions. In studying experimental potassium Tissue sodium and potassium were determined by flame depletion in man, Black and Milne (5) interpreted photometry using lithium as an internal standard. In their balance data as indicating a shift of hydrogen early experiments tissue chloride was determined by a ions into cells. In an attempt to obtain direct evi- modified Volhard titration. In later experiments, through the courtesy of Dr. Paul Marks, chlorides were meas- dence relating the electrolyte composition of the ured potentiometrically using a silver-silver chloride cell renal parenchyma to the excretion of acid urine, (9). Darrow, Cooke, and Coville (6) analyzed the kid- "Acid-labile CO2" was determined by a modification of neys of potassium depleted rats, but with inconclu- the technique devised by Conway and Fearon (10). Conway dishes (inner chamber 40 mm. diameter, 5 mm. sive results. high) were prepared with 1.3 ml. of 0.025 N Ba(OH), Since the intracellular sodium and potassium in the center well, covered with ground glass cover-slips 1 and sealed with anhydrous lanolin. After incubation in This study was supported by a grant from the Rocke- the Warburg flasks, tissue slices (about 300 mg. per feller Foundation to Dr. John V. Taggart. 2 Present Address: Department of Pharmacology and 3 Although these amounts of calcium and phosphate Experimental Therapeutics, The Johns Hopkins Uni- were routinely added, their omission did not influence versity School of Medicine, Baltimore 5, Maryland. the results. 1691 1692 HELEN M. ANDERSON AND GILBERT H. MUDGE TABLE I RESULTS Effect of potassium on tissue bicarbonate concentration A typical experiment employing the standard Tissue composition system is illustrated in Table I. Before incubation, HCO3- K+ Na+ +K+ the potassium concentration of the leached slice mEq./Kg. wet weight was 35 mEq. per Kg. After incubation in a potas- Before incubation 35.0 137 sium-free medium there was no change in tissue After incubation No K+ added to potassium, but incubation in the medium contain- external medium 13.5 34.9 138 ing 10 mEq. per L. of potassium resulted in an ac- lOmEq./L. K+added to external medium 18.0 77.2 135 cumulation of potassium by the tissue to a final concentration of 77.2 mEq. per Kg. This value is similar to that found in fresh tissue (7). The vessel) were quickly transferred to the outer well of a was prepared Conway dish. About 5 ml. of 2 N H2S04 was uptake of potassium by the slices associated then added to the outer well on the side opposite the tis- with a loss of sodium, so that there was no signifi- sues. At all times the cover glass was slipped open only cant change in the sum of sodium and potassium.5 far enough to allow insertion of the sample or the pipette The bicarbonate concentration of the tissue incu- The dish was resealed and rotated tip. promptly gently bated without potassium was 13.5 mEq. per Kg.; to provide complete penetration of acid into the slices. slices with added potassium had a significantly The Conway dishes so loaded with Ba(OH)2, tissue, and H2S04 were allowed to stand at room temperature for one higher bicarbonate concentration (18.0 mEq. per hour. A one ml. aliquot of fluid from the center well Kg.). was then removed and its content of Ba(OH)2 deter- mined by titration with 0.005 N HCl, using thymol blue TABLE 1I Average values from 17 experiments employing as indicator. Calculation by difference revealed the the Standard System * quantity of Ba(OH)2 converted to BaCO, and hence the in the tissue. A amount of "acid-labile C02" present Final tissue composition Conway dish without tissue was run with each experi- K+ added to ment as a control. Standardization of this technique using incubation known concentrations of NaHCO3 solution in the outer medium well gave 95 to 105 per cent recoveries. Longer stand- 10 mEq./ 0 L. a± S.D. P ing, increased temperature, or gentle agitation of the Conway dish did not significantly alter the recovery. In HCO3-, mEq./Kg. 12.3 17.0 4.7± .52 <.001 one experiment, through the courtesy of Dr. Duncan K+, mEq./Kg. 32.1 71.7 39.6 1.2 <.001 Holaday, "acid-labile CO," of the slices was measured by Na++K+, mEq./Kg. 140 141 1.0±1.5 >.5 the gasometric method of Danielson and Hastings (11).4 H20,%wetweight 77.8 78.7 0.94± .24 <.001 The results were the same as those obtained by the modi- fied Conway technique. * Eight analyses of fresh kidney cortex, removed as quickly as possible after killing the rabbit, yielded an All expressions for the concentration of electrolyte in average bicarbonate concentration of 14.4 mEq. per Kg. tissues are given per kilogram wet weight. The q02 was Fresh tissue values previously reported (7) are: K+, 69.3 calculated as the cubic millimeters of 02 consumed per mEq. per Kg.; Na++K+, 138 mEq. per Kg.; H20, 77.1 hour per mg. initial wet weight of tissue. Tissue bi- per cent wet weight. carbonate determinations were done in duplicate on tis- A was in a sue incubated in separate flasks and treated identically. similar relationship observed total Tissue Na, K, and Cl were determined in duplicate on of 72 experiments. In Table II are summarized the specimen from a single flask. the results from 17 experiments in which tissue water con- 4 "Acid-labile C02" is considered to include dissolved bicarbonate, sodium, potassium and C02, H2CO,, and HCO,7. For simplicity, except as indi- tent were measured. The average difference in cated in Table VIII, the term bicarbonate is used as a tissue bicarbonate between the slices with and synonym for "acid-labile C02," since this is by far the without added potassium was 4.7 mEq. per Kg.; largest component of the C02-H2COs-HCO8- system. It this was associated with an average change of is recognized that Conway and Fearon (10) have pre- in level of tissue sented evidence to suggest that compounds other than 39.6 mEq. per Kg. the potassium. C0-H2CO,-HC0O, are measured as "acid-labile C02." 5 In this presentation the convention of correlating tis- However, since the nature of these compounds is not sue bicarbonate to potassium will be employed.