significant replacement of larger intracellular anions by extracellular chloride. The

Transcription

1 J. Physiol. (1979), 297, pp With 8 text-ftgurem Printed in Great Britain ANION EXCHANGE AND VOLUME REGULATION DURING METABOLIC BLOCKADE OF RENAL CORTICAL SLICES BY MICHAEL B. PINE, DAVID RHODES, KATHRYN THORP AND YVONNE TSAI From the Department of Medicine and Thorndike Laboratory, Harvard Medical School and Beth Israel Hospital, Boston, Massachusetts 2215 U.S.A. (Received 9 January 1979) SUMMARY 1. The development of swelling of rat and guinea-pig renal cortical slices was studied after metabolic blockade (hypoxia plus glycolytic blockade with iodo-acetic acid) and/or exposure to 'isotonic' high potassium, no sodium solution. 2. Swelling was greater after exposure to oxygenated high potassium solution than after metabolic blockade in physiologic Krebs-Henseleit solution. Swelling was reduced after metabolic blockade in high potassium solution compared to incubation in oxygenated high potassium solution. Increasing periods of transient metabolic blockade in Krebs-Henseleit solution progressively blunted swelling when slices were subsequently incubated in oxygenated high potassium solution. 3. Metabolic blockade in Krebs-Henseleit solution resulted in marked reductions in potassium and increases in sodium. Incubation in high potassium solution resulted in marked increases in potassium and similar low levels of sodium regardless of associated interventions. Metabolic blockade in both media resulted in significantly greater increases in renal cortical chloride than in monovalent cations (potassium plus sodium). Incubation in oxygenated high potassium solution was associated with similar increases in renal cortical chloride and total monovalent cations. 4. Renal cortical losses of solids and protein and increases in renal cortical inulin space were greater after metabolic blockade than after incubation under oxygenated conditions regardless of the incubation media. 5. These data support the conclusion that during metabolic blockade there is a significant replacement of larger intracellular anions by extracellular chloride. The loss of osmotically active intracellular anions limits the increase in renal cortical volume during metabolic inhibition and exposure to high potassium solution. INTRODUCTION The 'pump-leak' hypothesis of cell volume regulation has emphasized active extrusion of intracellular sodium to counterbalance the colloid osmotic force of intracellular anions to which the cell membrane is normally impermeable (Leaf, 1956; Tosteson & Hoffman, 196; Cook, 1965; Okamoto & Quastel, 197). According to this hypothesis, increases in cell membrane permeability to and inhibition of active removal of major extracellular cations can both contribute to cell swelling. Thus, metabolic blockade would be expected to produce cell swelling by preventing active /79/ $ The Physiological Society 13-2

2 388 M. B. PINE extrusion of sodium and possibly also increasing cell membrane sodium permeability, while replacement of sodium by potassium, ion for ion, in interstitial fluid should result in cell swelling since the cell membrane is more permeable to potassium than to sodium, and potassium is not actively pumped out of cells (Macknight & Leaf, 1977). In the present study, the development of renal cortical swelling during metabolic blockade and/or exposure to an 'isotonic' high potassium, no sodium solution was evaluated in the light of the 'pump-leak' hypothesis. Differences in swelling in response to these interventions were observed which support a significant role for anion exchange in cell volume regulation during metabolic blockade. METHODS Preparation. Adult male rats and guinea-pigs were decapitated. Kidneys were rapidly removed and slices of renal cortex approximately 5 mm thick were prepared using a Stadie-Riggs slicer (Stadie & Riggs, 1944). Slices were submerged in Krebs-Henseleit solution (Krebs & Henseleit, 1932) with 5.5 mm-glucose which was bubbled With 95 % oxygen-5 % carbon dioxide at 25 C (Po2> 6 mmhg, ph= 7 4). All tissues equilibrated for 1 hr before the study. Media. Krebs-Henseleit solution contained (mm): Na, ; K, 5-87; Cl-, ; Ca2, 2-52; Mg2, 1P16; HCO3, 25-88; S42-, 1-16; H2PO-, 1418; glucose, 595. Potassium substituted solution its which sodium was replaced by potassium, ion for ion, contained (mm): K, 15-25; Cl-, ; Ca2, 2-52; Mg2, 1-16; HCO3-, 25-88; SO42-, 1-16; H2P4-, 1-18; glucose, 5.5. The osmolarity of both solutions was 285m-osmole/1. Metabolic blockade consisted of hypoxia and glycolytic blockade with 1-4 M-iodo-acetic acid (Eastman Kodak Co., U.S.A.). Hypoxia (Po2 <25 mmhg) was produced by bubbling-with 95 % nitrogen-5 % carbon dioxide. Sodiumpotassium exchange pump inhibition was achieved in guinea-pig renal slices with 1-3 M-ouabain (Sigma Chemical Co., U.S.A.). [3H]inulin (New England Nuclear, U.S.A.) was added to the bath 2 hr before the termination of an experiment to determine the volume of distribution of inulin. All experiments were performed at 25 'C. Interventions. Rat renal cortical slices were studied after (a) equilibration and 2 and 4 hr of additional incubation in oxygenated Krebs-Henseleit solution; (b) 1, 2 and 4 hr in oxygenated potassium substituted solution; (c) 1, 2 and 4 hr of metabolic blockade in Krebs-Henseleit solution; and (d) 1 hr of metabolic blockade in Krebs-Henseleit solution followed by either1 or 3 additional hours of metabolic blockade in potassium substituted solution. Transient metabolic blockade was evaluated by incubating equilibrated rat renal cortical slices for: (a) 2, 4 and 6 hr in oxygenated Krebs-Henseleit solution; (b) 1, 2, 4 and 6 hr during metabolic blockade in Krebs- Henseleit solution; (c) 1, 2, 3 and 4 hr during metabolic blockade in Krebs-Henseleit solution followed by 2 additional hours in oxygenated Krebs-Henseleit solution; (d) 1, 2, 3 and 4 hr during metabolic blockade in Krebs-Henseleit solution followed by 2 additional hours in oxygenated potassium substituted solution; and (e) 2 hr in oxygenated potassium substituted solution immediately after equilibration and after equilibration plus 4 additional hours in oxygenated Krebs-Henseleit solution. Renal cortical slices from guinea-pigs were studied after: (a) equilibration and 2 hr in oxygenated Krebs-Henseleit solution; (b) 1 and 2 hr in oxygenated potassium substituted solution; (c) 1 and 2 hr of metabolic blockade in Krebs-Henseleit solution; (d) 1 hr of metabolic blockade in Krebs-Henseleit solution followed by 1 hr of metabolic blockade in potassium substituted solution; (e) 1 and 2 hr in oxygenated Krebs-Henseleit solution containing 1O-3 M-ouabain; and (f) 1 hr in oxygenated Krebs-Henseleit solution containing 1O-3 M-ouabain followed by1 hr in oxygenated potassium substituted solution containing 1-3 M-ouabain. Analy8i8. At the end of each experiment, tissues were blotted with bibulous paper for 3 sec and weighed on a Mettler H 54 balance. Tissues were dried to constant weight and reweighed to determine water contents. Tissue electrolytes were measured after dried slices were dissolved in IN-HNO,. Potassium and sodium were measured using a flame photometer with an internal lithium standard (Instrumentation Laboratory, U.S.A.). Chloride was measured by coulometricamperometric titration with silver using a Buchler-Cotlove chloridometer (Buchler Instruments, U.S.A.). Tissues dried after exposure to [3H]inulin were rehydrated and dissolved in 1 ml.

3 ANION EXCHANGE AND RENAL CORTICAL VOLUME 389 ProtosolR (New England Nuclear, U.S.A.). Ten milliliters of EconofluorTM (New England Nuclear, U.S.A.) were added and 3H was counted in a Tricarb Liquid Scintillation Counter (Packard Instrument, U.S.A.) using standards containing 5 and 1 ml. bathing solution. Loss of tissue solids in the course of an intervention was determined by removing tissues immediately after equilibration, blotting them for 3 sec, weighing them on a Mettler H-54 balance, and then rapidly submerging them in the appropriate solution. Water content at the end of equilibration was determined independently and used to calculate the dry weight of each tissue before the study. After each intervention tissues were again blotted and weighed. Tissue solids at the end of the intervention were measured directly after drying to constant weight. Tissue protein was analysed using the method of Lowry, Rosebrough, Farr & Randall (1951). Calculations. Water and electrolytes are reported as ml. and molee per gram dry tissue at the end of each intervention. Solid and protein loss are calculated based on tissue dry weight before each intervention. Results are reported as mean s.e. of the mean. Determinations after different periods of a single intervention were compared using Student's t test for unpaired data (two samples) (Snedecor & Cochran, 1967) or Duncan's test for multiple samples (Duncan, 1955). Determinations after similar periods of exposure to several interventions were also compared using one of these tests. Changes in tissue chloride were compared to changes in monovalent cations using Student's t test for paired data (Snedecor & Cochran, 1967). Calculated losses of solids after different interventions were used to reanalyse changes in rat renal cortical volume when these were related to initial rather than final dry weights. Changes in renal cortical volume were related to changes in cell volume (1) assuming interstitial volume was not altered by the interventions studied and (2) assuming interstitial volume was equal to the measured inulin space. RESULTS Water Renal cortical water at the end of equilibration was ml./g in rats and ml./g in guinea-pigs. There were no significant changes in water content during additional incubation in oxygenated Krebs-Henseleit solution in either rats (6 hr) or guinea-pigs (2 hr). In rats (Fig. 1), metabolic blockade in Krebs-Henseleit solution resulted in significant progressive increases in renal cortical water (P <.1 for each pair of determinations). Incubation in oxygenated potassium substituted solution for 1, 2 and E 4 / - * Time (hr) Fig. 1. Rat renal cortical water (as ml./g dry tissue wt.) after incubation in oxygenated Krebs-Henseleit solution (controls, e *), in oxygenated potassium substituted solution (o o), in Krebs-Henseleit solution with metabolic blockade (El - -D), and in potassium substituted solution with metabolic blockade (A- * - A). s.e. of means are not illustrated since they are all < 15 ml./g and would be obscured by symbols. Immediate post-equilibration determination (n = 18) is at hr. n= 8 for controls and 1 and 2 hr determinations. n = 18 for 4 hr interventions.

4 39 M. B. PINE 4 hr resulted in significantly greater swelling than after similar periods of metabolic blockade in Krebs-Henseleit solution (P < 1). Renal cortical volume in oxygenated potassium substituted solution was stable after 1 hr. After metabolic blockade for 1 hr in Krebs-Henseleit solution, 1 and 3 additional hours of metabolic blockade in potassium substituted solution resulted in less swelling than equilibration plus 1 and 3 hr of incubation in oxygenated potassium substituted solution (P < 1). In rat renal cortex, there was no significant difference in swelling after metabolic blockade in high and low potassium solutions. In rat renal cortical slices (Fig. 2), swelling after metabolic blockade in Krebs- Henseleit solution for 1 and 2 hr was partially reversed by subsequent incubation in oxygenated Krebs-Henseleit solution for 2 hr ( to and 4*11 4 to 3-89 '1 ml./g respectively, P < 1 for both comparisons). After 4 hr of metabolic blockade in Krebs-Henseleit solution, 2 hr of reoxygenation in Krebs- Henseleit solution resulted in continued swelling (P < 1) at a slower rate (P < 1) than swelling during 2 hr of continued metabolic blockade in Krebs-Henseleit 8 6. <x,e 4t- ; -;j Time (hr) Fig. 2. Rat renal cortical water (as ml./g dry tissue wt.) after incubation in oxygenated Krebs-Henseleit solution (@- ), in oxygenated potassium substituted solution for 2 hr after equilibration and after 4 hr of incubation in oxygenated Krebs-Henseleit solution (o o), in Krebs-Henseleit solution with metabolic blockade (L - ---l), in oxygenated potassium substituted solution for 2 hr after transient metabolic blockade in Krebs-Henseleit solution ( - *), - and in oxygenated Krebs-Henseleit solution - for 2 hr after transient metabolic blockade in Krebs-Henseleit solution ( -* -.). Two hours after equilibration, incubation in oxygenated Krebs-Henseleit solution(@) and zero hours of transient metabolic blockade in Krebs-Henseleit solution followed by 2 hr of incubation in oxygenated Krebs-Henseleit solution () are identical. Thereafter, control values remain constant, while increasing periods of metabolic blockade in Krebs-Henseleit solution followed by 2 hr of incubation in oxygenated Krebs-Henseleit solution result in increases in renal cortical water. Similarly, 2 hr after equilibration, incubation in oxygenated potassium substituted solution (o) and zero hours of transient metabolic blockade in Krebs-Henseleit solution followed by 2 hr of incubation in oxygenated potassium substituted solution ( ) are identical. Thereafter, oxygenation in Krebs-Henseleit solution preceding 2 hr incubation in oxygenated potassium substituted solution does not alter swelling from exposure to the high potassium medium, while increasing periods of metabolic blockade in Krebs-Henseleit solution followed by 2 hr of incubation in oxygenated potassium substituted solution result in reductions in the amount of renal cortical swelling associated with 2 hr of incubation in oxygenated potassium substituted solution. S.E. of means are not illustrated since they are all < -15 ml./g and would be obscured by symbols. Immediate post-equilibration determination is at hr. n = 16 for each determination.

5 ANION EXCHANGE AND RENAL CORTICAL VOLUME 391 solution. When transient metabolic blockade was followed by 2 hr of incubation in oxygenated potassium substituted solution, renal cortical swelling was progressively reduced as the preceding period of metabolic blockade was lengthened from to 2 hr (P < -1). Transient metabolic blockade for more than 2 hr did not result in any further significant decrease in swelling in renal cortical slices exposed to oxygenated potassium substituted solution for 2 hr. Four hours in oxygenated Krebs-Henseleit prior to 2 hr in oxygenated potassium substituted solution did not alter swelling induced by the high potassium medium. Renal cortical water was higher after 4 hr of metabolic blockade in Krebs-Henseleit solution followed by 2 hr in oxygenated potassium substituted solution than it was after 6 hr of metabolic blockade in Krebs-Henseleit solution ( vis ml./g, P < -1). 8- A 8 I - -4 ~ Time (hr) Fig. 3. Guinea-pig renal cortical water (as ml./g dry tissue wt.) after incubation in oxygenated Krebs-Henseleit solution oxygenated potassium substituted solution ( O), Krebs-Henseleit solution with metabolic blockade (C- --, Fig. 3A only), potassium substituted solution with metabolic blockade (A A, Fig. 3A only), oxygenated Krebs-Henseleit solution containing 1-3 M-ouabain (_- --U, Fig. 3 B only), and oxygenated potassium substituted solution containing 1-3 M-ouabain (A--- --A, Fig. 3 B only). S.E. of means are not illustrated since they are all < -2 ml./g and would be obscured by symbols. Immediate postequilibration determination is at hr. n = 8 for each determination. In renal cortical slices from guinea-pigs (Fig. 3) incubation in oxygenated potassium substituted solution resulted in more rapid swelling than metabolic blockade in Krebs-Henseleit solution (P < -1). Metabolic blockade for 1 hr in Krebs-Henseleit solution followed by metabolic blockade for a second hour in potassium substituted solution resulted in significantly more swelling than 2 hr of metabolic blockade in Krebs-Henseleit solution (P < -1), but significantly less swelling than 1 hr of incubation in oxygenated high potassium solution (P < - 1). Blockade of the sodium-potassium exchange pump with 1-3M-ouabain did not alter renal cortical volume in oxygenated Krebs-Henseleit solution, nor did it inhibit or slow swelling in oxygenated potassium substituted solution. Potassium and sodium Renal cortical potassium at the end of equilibration was /smole/g in rats and 281 4,umole/g in guinea-pigs. Sodium at the end of equilibration was #umole/g

6 392 Mf. B. PINE in rats and 222 5,umole/g in guinea-pigs. There were no significant changes in potassium or sodium during additional incubation in oxygenated Krebs-Henseleit solution in either rats (6 hr) or guinea-pigs (2 hr). In rats (Figs. 4 and 5), metabolic blockade resulted in a progressive fall in renal cortical potassium (P < 1 for each pair of determinations) and a progressive rise in sodium (P < -1 for each pair of determinations). Elevated renal cortical potassium contents were present in all tissues exposed to potassium substituted solution (P < 1), but significant differences in potassium were found between continuously oxygenated slices and slices subjected to metabolic blockade either preceding or during incubation in the high potassium medium (P < -1). Exposure to potassium substituted solution resulted in a fall in sodium so that similar low sodium contents were found in all tissues exposed to the high potassium solution regardless of preceding or coexisting interventions. 12 -e 1 Potassium Sodium z 6!Z4.// 2 I ' Time (hr) Fig. 4. Rat renal cortical potassium and sodium (as #smole/g dry tissue wt.) after incubation in oxygenated Krebs-Henseleit solution in oxygenated potassium substituted solution (o o), in Krebs-Henseleit solution with metabolic blockade (El- - -) and in potassium substituted solution with metabolic blockade (E- - A). S.E. of means are not illustrated since they are all < 2 /Zmole/g and - would be obscured by symbols. Immediate post-equilibration determination (n = 18) is at hr. n = 8 for controls and 1 and 2 hr determinations. n = 18 for 4 hr interventions. In rat renal cortex (Fig. 5), 2 hr in oxygenated Krebs-Henseleit solution after 1 hr of metabolic blockade in Krebs-Henseleit solution resulted in return of potassium to control values, but only a partial removal of excess renal cortical sodium (P < 1). After 2 hr of metabolic blockade in Krebs-Henseleit solution, 2 hr in oxygenated Krebs-Henseleit solution resulted in only partial return of potassium towards normal (P < 1), while sodium did not change significantly. After 4 hr of metabolic blockade in Krebs-Henseleit solution, 2 hr in oxygenated Krebs-Henseleit solution did not result in a significant rise in potassium, and sodium continued to rise (P < -1). However, after 4 hr of metabolic blockade and 2 hr of reoxygenation in Krebs- Henseleit solution, potassium was significantly higher (P <.1) and sodium significantly lower (P < 1) than after 6 hr of continuous metabolic blockade in Krebs-Henseleit solution.

7 ANION EXCHANGE AND RENAL CORTICAL VOLUME Potassium Sodium 6, v 4 / / 2 o Time (hr) Fig. 5. Rat renal cortical potassium and sodium (as #umole/g dry tissue wt.) after incubation in oxygenated Krebs-Henseleit solution (-O), in oxygenated potassium substituted solution for 2 hr after equilibration and after 4 hr incubation in oxygenated Krebs-Henseleit solution (o o), in Krebs-Henseleit solution with metabolic blockade ([]- - -[l), in oxygenated potassium substituted solution for 2 hr after transient metabolic blockade in Krebs-Henseleit solution ( - - -(), and in oxygenated Krebs-Henseleit solution for 2 hr after transient metabolic blockade in Krebs- Henseleit solution (I - * The interrelationship of these interventions is explained more fully in the legend to Fig. 2. Values are mean s.e. of mean unless the s.e. of mean is < 2,csmole/g and would be obscured by symbol. Immediate postequilibration determination is at hr. n= 8 for each determination. In guinea-pig renal cortex (Fig. 6), metabolic blockade and exposure to ouabain resulted in similar losses of potassium after 1 and 2 hr, but the increases in renal cortical sodium after 1 and after 2 hr were greater after metabolic blockade than after ouabain (P < 1). Incubation in potassium substituted solution resulted in similar low renal cortical sodium contents in the presence of oxygen, metabolic blockade, and oxygen plus ouabain. Elevated potassium contents were present in all tissues exposed to potassium substituted solution (P < 1), but potassium was significantly less in slices after metabolic blockade in the high potassium medium than after incubation in oxygenated potassium substituted solution with or without ouabain (P < -1). Chloride and monovalent cations Renal cortical chloride at the end of equilibration was Fsmole/g in rats and 17 1,umole/g in guinea-pigs. There were no significant changes in chloride during incubation in oxygenated Krebs-Henseleit solution in rats (4 hr) or guinea-pigs (2 hr). Changes in rat renal cortical chloride and monovalent cations (potassium plus sodium) are recorded in Table 1. There was a progressive increase in chloride (P < -1 for each pair of determinations) and monovalent cations (P < 1 for each pair of determinations except 2 hr vs. 4 hr) during 4 hr of metabolic blockade in Krebs- Henseleit solution. There was a marked rise in renal cortical chloride and total monovalent cations during the first hour of incubation in oxygenated potassium substituted solution (P <.1) after which chloride and monovalent cation contents remained stable during 3 additional hours of incubation in oxygenated high potas-

8 394 M. B. PINE 12. A Potassium 1 o 8 / Sodium 6-4 /-~ - 2 o E ~- //- z B o , Time (hr) Fig. 6. Guinea-pig renal cortical potassium and sodium (as,umole/g dry tissue wt.) after incubation in oxygenated Krebs-Henseleit solution (- *), oxygenated potassium substituted solution (o o), Krebs-Henseleit solution with metabolic blockade ([l - - -s, Fig. 6A only), potassium substituted solution with metabolic blockade (a- * - A, Fig. 6A only), oxygenated Krebs-Henseleit solution containing 1O-3 M-ouabain (M - --, Fig. 6 B only), and oxygenated potassium substituted solution containing 1O-3 M-ouabain (,- * M *, Fig. 6B only). s.e. of means are not illustrated since they are all < 25 gmole/g and would be obscured by symbols. Immediate postequilibration determination is at hr. n= 8 for each determination. sium medium. Metabolic blockade for 1 hr in Krebs-Henseleit solution followed by 1 or 3 additional hours of metabolic blockade in potassium substituted solution resulted in increases in renal cortical chloride and monovalent cations similar to increases found during metabolic blockade in Krebs-Henseleit solution and significantly less than increases observed after comparable periods of incubation in oxygenated potassium substituted solution (P < 1). When post-equilibration changes in renal cortical chloride were compared to changes in total monovalent cations, no significant differences were found after 1, 2 and 4 hr in oxygenated potassium substituted solution. However, the increase in tissue chloride exceeded the increase in total monovalent cations after 1, 2 and 4 hr of metabolic blockade both in Krebs-Henseleit solution and in potassium substituted solution (P < 1). Changes in guinea-pig renal cortical chloride and total monovalent cations are recorded in Table 2. Chloride and total monovalent cations increased each hour during 2 hr of metabolic blockade in Krebs-Henseleit solution (P < -1). Incubation in oxygenated potassium substituted solution for 1 hr resulted in greater increases in chloride and total monovalent cations than were found after 1 and 2 hr of metabolic

9 ANION EXCHANGE AND RENAL CORTICAL VOLUME 4 b4 1ō S z 1 l eq M 1 O 1 ro 1~ eq V4 V4 Ni 1 7) 395 ) ).4 O4Q eq 1- bo S-- w *1 - l 1 1 V- M C a _ 1 d V V bl ;) 1 O t4 1 1 O t- 1 1~ 1 eq - ec e o. C) ) ) ) 54 ) &4..- I-- -) rs, z _ 1 1 I t- eco C1V m V4 c _ T- ee fx CO _ O g 84 Q4 * II 5z ; w 54 Q b 1 (3 ) z w 5, Is *- =4 ).t.) C; 1 b *-5 V) OZZ

10 396 ( -4 S 41) 4) I 1') SN bd I 4) z w I - l M. B. PINE equ 1 1 r F-4 o z ' Q l I O e -P- 1 1 co 1 1t O~ 1 1 m 1 v 1 l l es 1 IF Q zq CO l CO o. v l CO 5- S t,4 X X *_ U zg * _ *(3!2 Ci CO 1 l o 1 I ~~~~~~~~~~I z S- ad 1 4 Ca t- 2 ~~~.D Xe1 c o o w = 1 o ~O

11 ANION EXCHANGE AND RENAL CORTICAL VOLUME 397 blockade in Krebs-Henseleit solution (P <.1). During a second hour in oxygenated high potassium solution, mean chloride and total monovalent cations both increased by about 1 % of the rise seen in the first hour, but this further increase was statistically significant only for chloride (P < 1). One hour of metabolic blockade in Krebs-Henseleit solution followed by 1 hr of metabolic blockade in potassium substituted solution resulted in greater accumulation of chloride and total monovalent cations than 2 hr of metabolic blockade in Krebs-Henseleit solution (P < '1), but less than after 1 hr of incubation in oxygenated potassium substituted solution (P < -1). Renal cortical chloride and monovalent cations were similar to controls after 1 and 2 hr in oxygenated Krebs-Henseleit solution containing 1-3 M-ouabain. Similar increases in chloride and monovalent cations were present after 1 hr in oxygenated potassium substituted solution and after 1 hr in oxygenated Krebs- Henseleit solution containing ouabain followed by 1 hr in oxygenated high potassium medium containing ouabain. When post-equilibration changes in renal cortical chloride were compared to changes in total monovalent cations, no significant differences were found when tissues were incubated in oxygenated Krebs-Henseleit solution and oxygenated potassium substituted solution, both with and without ouabain. Increases in renal cortical chloride were significantly greater than increases in total monovalent cations after metabolic blockade in both Krebs-Henseleit solution and high potassium medium (P < 5). Solids, protein, and inulin space Changes in rat renal cortical solids, protein, and inulin space are recorded in Table 3. There was no significant change in solids during 2 and 4 hr of post-equilibration incubation in oxygenated Krebs-Henseleit solution. Renal cortical protein was 54 4 g/g after equilibration and did not change significantly after 2 and 4 additional hours in oxygenated Krebs-Henseleit solution. Inulin space could not be assessed until 2 hr post-equilibration and was ml./g at that time. A similar value was obtained after equilibration plus 4 hr in oxygenated Krebs- Henseleit solution. The inulin space obtained 2 hr after equilibration was used as the equilibration value for purposes of analysis. During incubation in oxygenated potassium substituted solution, there was a loss of renal cortical solids and an increase in inulin space during the first 2 hr (P < -1) with no change in total solids and inulin space during the next 2 hr. Renal cortical protein content remained stable during 4 hr of incubation in oxygenated high potassium medium. Metabolic blockade in Krebs-Henseleit solution resulted in a decrease in total solids and an increase in inulin space during the first 2 hr (P < -1) with a continued loss of solids (P <.5) and increase in inulin space (P < -1) during the next 2 hr. A significant loss of protein was found only after 4 hr of metabolic blockade (P <.5). Losses of total solid after metabolic blockade in Krebs- Henseleit were greater than after incubation in oxygenated potassium substituted solution after 2 hr (P <.5) and after 4 hr (P < -1). Inulin space and protein loss were significantly greater after metabolic blockade in Krebs-Henseleit solution than after incubation in oxygenated potassium substituted solution only after 4 hr (P < *1 and P < 5, respectively). Changes in renal cortical solids, protein, and

12 398 M. B. PINE C) o C) OQ w " XJ C) C) 8 W _ -~-- 14 W.4 C) Co 44--l _; _ E P4 14 >, ~2 P., _ 6 1 9? l eq l >6 o 1 1 eq H o O * I eq m 1 * I * I X= l O 6 6 l C> ms 6 eq eq 1 CO co o o *- - o~ l l o 1 1 * - l l o l l o eq ' l D o o l l o * U, l UD C) 11 o 6 o C).. o = * " 1 1 o W C) " "2 C) f Q e 4ZO~ ~ ~ 144 C ) * - C)n W.- C) o bez # ;! x X ~,e, CA) C)- U u Q c *~ a I ~ _ V4 O t o~lv C)

13 ANION EXCHANGE AND RENAL CORTICAL VOLUME 399 inulin space after 1 hr of metabolic blockade in Krebs-Henseleit solution followed by either 1 or 3 hr of metabolic blockade in potassium substituted solution were all similar to changes after corresponding periods of metabolic blockade in Krebs-Henseleit solution and were significantly different from values obtained after 4 hr of incubation in oxygenated potassium substituted solution (P < O5 for solids and protein and P < 1 for inulin space). The use of dry weights immediately prior to each intervention did not substantially alter the relation among changes in water or electrolytes after metabolic blockade and/or exposure to potassium substituted solution compared to results obtained using dry weights at the end of each intervention. Furthermore, the relation among changes in cellular volume after metabolic blockade and/or exposure to potassium substituted solution (Fig. 7) was similar both when volumes were cal- 2h A B C 4 4h Fig. 7. Rat renal cortical cellular water calculated from (A) total tissue water per gram dry weight at the conclusion of each experiment and an extracellular space containing 114 ml. H2 per gram dry weight (inulin space in oxygenated Krebs- Henseleit solution); (B) total tissue water and inulin space per gram dry weight at the end of each experiment; and (C) total tissue water and inulin space per gram calculated dry weight at the end of equilibration before each intervention. Values are mean s.e. of mean. n= 8 for 2 hr interventions and 4 hr of oxygenation in Krebs-Henseleit solution. n = 18 for other 4 hr interventions. El, 2 in Krebs-Henseleit solution;., 2 in potassium substituted solution; %, metabolic blockade in Krebs-Henseleit solution; M, metabolic blockade in potassium substituted solution. culated assuming that interstital volume was unaltered by the interventions studied and when volumes were calculated assuming that inulin remained a valid extracellular marker and did not enter cells during metabolic blockade and/or exposure to potassium substituted solution. DISCUSSION Maintenance of normal cell volume in a physiologic high sodium, low potassium medium is generally attributed to the active extrusion of sodium (Macknight & Leaf,

14 4 M. B. PINE 1977). Data supporting this theory of cell volume regulation include (1) increases in cell volume often but not invariably observed after metabolic inhibition (Leaf, 1956; Heckman & Parsons, 1959; Okamoto & Quastel, 197; Grochowski, Ganote, Hill & Jennings, 1976; Hughes & Macknight, 1977; Pine, Bing, Weintraub & Abelmann, 1979); (2) increases in cell volume when sodium is replaced by a cation to which the cell membrane is permeable and which cannot be actively extruded (Boyle & Conway, 1941; Trump & Ginn, 1968; Okamoto & Quastel, 197; Mazet, Claret & Claret, 1974; Cooke, 1975; Hughes & Macknight, 1977); and (3) decreases in swelling during metabolic blockade when sodium is replaced by a cation to which the cell membrane is less permeable (Hughes & Macknight, 1977). In experiments in which cell volume increases in an 'isotonic' medium, increases in cell water are accompanied by increases in cell monovalent cations and chloride so that the calculated osmolarity of the fluid gained is approximately equal to the osmolarity of the bathing medium (Mudge, 1951; Leaf, 1956; Macknight & Leaf, 1977). Active removal of sodium by metabolizing cells is accomplished, at least in part, by a ouabain sensitive sodium-potassium exchange pump (Post & Jolly, 1957; Skou, 1965), although other sodium pumps may contribute significantly (Whittembury, 1968; Whittembury & Grantham, 1976). Blockade of the sodium-potassium exchange pump with ouabain results in significant increases in cell sodium along with marked losses of cell potassium, but these changes do not invariably result in either cell swelling or an increase in cell total monovalent cations or chloride (Maude, 1969; Macknight, Pilgrim & Robinson, 1974; Allison, 1975). The failure of some cells to swell after exposure to ouabain and the relative resistance of some tissues to swelling after metabolic blockade (Pine, Bing, Brooks & Abelmann, 1978) has raised significant questions about the relationship of active removal of cell sodium and maintenance of normal cell volume. The effectiveness of sodium pumps in maintaining a low intracellular sodium concentration is dependent upon the balance between their activity and cell membrane permeability to sodium (Tosteson & Hoffman, 196). If inhibition of active sodium extrusion occurs while sodium permeability remains low, sodium may enter cells in an ion for ion exchange for intracellular potassium without a net gain in either cell total monovalent cations, chloride, or water. Continued low membrane permeability to sodium may account for the absence of cell swelling in some tissues after ouabain or after metabolic blockade. Likewise, the more rapid swelling observed after complete ion for ion replacement of medium sodium by potassium than after metabolic blockade in a physiologic high sodium, low potassium medium (Cooke, 1975; Hughes & Macknight, 1977) may reflect a higher membrane permeability to the major extracellular cation in the former preparation. In the present experiments, renal cortical swelling after exposure to potassium substituted solution was significantly less during co-existent metabolic blockade than under oxygenated conditions in both rats and guinea-pigs. Furthermore, increasing periods of prior transient metabolic blockade resulted in progressive blunting of the increase in renal cortical volume associated with exposure to oxygenated high potassium medium. These data are consistent with and extend the observations of Hughes & Macknight (1977) concerning the interaction of metabolic blockade and exposure of renal cortical slices to rubidium and caesium. On the other hand, these

15 ANION EXCHANGE AND RENAL CORTICAL VOLUME 41 data are inconsistent with a theory in which volume regulation depends solely upon cation pumps and leaks. In the present study, the precise relation between changes in renal cortical cell volume and total renal cortical water content is uncertain. Renal cortical cell water may be determined accurately using inulin as an extracellular marker during incubation in oxygenated Krebs-Henseleit solution, but the loss of tissue solids and protein during interventions used in these experiments casts doubt upon the exclusively extracellular location of inulin at the conclusion of these studies. However, conclusions concerning both the relation of swelling in oxygenated potassium substituted solution to swelling during metabolic blockade in Krebs-Henseleit solution and the effect of metabolic blockade on swelling in potassium substituted solution are identical regardless of whether they are based on analyses assuming a constant interstitial volume with leakage of inulin into cells or analyses assuming an exclusively extracellular localization of inulin. It is, therefore, possible to extend conclusions regarding renal cortical volume regulation from tissue slices to renal cortical cells despite the lack of exact measurements of cell water content. In the present study, a 'cation gap' was observed when increases in electrolytes were analysed after metabolic blockade in either incubation medium. This excess of chloride gained over monovalent cations gained during metabolic blockade is not unique to this study. After metabolic blockade for 1 hr or less, the net gain in renal cortical chloride exceeded the net gain in monovalent cations in the studies of Leaf (1956; by 41 jtmole/g dry tissue) and Robinson (1961; by 23,umole/g dry tissue). Longer periods of metabolic blockade consisting of hypoxia and iodo-acetic acid resulted in the development of a striking 'cation gap' after 1 hr in renal cortical slices from rats, guinea-pigs, and rabbits (Fig. 8, calculated from data of Hughes & c, 15 )- E Time of metabolic blockade (min) Fig. 8. 'Cation gap' in fluid gained by renal cortical slices from rats (@), guinea-pigs (Ei), and rabbits (A) after metabolic blockade in a physiologically high sodium, low potassium medium. The difference between changes in renal cortical chloride and monovalent cations (potassium plus sodium) from values immediately post-equilibration was calculated from data of Hughes & Macknight (1977) and is recorded as,umole/g dry tissue wt. Immediate post-equilibration determination is at hr. Each point represents the mean of six to eight measurements.

16 42 M. B. PINE 42 M.PN Macknight, 1977). The presence of a 'cation gap' implies that either additional cations entered cells or cell anions were lost. Since neither Krebs-Henseleit solution nor potassium substituted solution contains substantial amounts of cations other than potassium and sodium, the first alternative appears highly improbable, while the loss of intracellular anions through a cell membrane damaged by metabolic inhibition is a perfectly reasonable possibility. The contention that extracellular chloride replaced intracellular anions is further strengthened by the greater loss of renal cortical solids and protein and the greater increase in inulin space observed during metabolic blockade compared to continued oxygenation, regardless of incubation medium. An increase in the permeability of renal cortical cell membranes resulting in the loss of intracellular anions during metabolic blockade can account for the smaller increases in renal cortical water observed during metabolic blockade in Krebs-Henseleit or potassium substituted solution than during incubation in oxygenated high potassium medium. It is also consistent with the failure of ouabain, which inhibits cation exchange pump function without substantially altering cell membrane permeability, to reduce the rate of swelling in oxygenated potassium substituted solution. The role of cell swelling in the progression from reversible injury to cell death after metabolic inhibition has been a topic of recent interest (Leaf, 197; Jennings, 1975). In the present study, metabolic blockade for 1 hr was associated with completely reversible potassium loss and at least partially reversible gains in sodium and water. Even after 4 hr of metabolic blockade, although reversal of potassium loss and sodium and water gains did not occur during 2 hr of reoxygenation in a physiologic medium, continued potassium loss and sodium and water gains were reduced compared to continued metabolic blockade, indicating some residual viability. Thus, the present studies, which include a range of increasing metabolic injuries approaching complete loss of cell function, suggest that reversible injury secondary to metabolic blockade may occur in association with cell swelling which is limited by the loss of intracellular constituents. It is also possible that these losses of intracellular constituents, rather than increases in cell volume, may be critical in defining the point at which injury is irreversible. This view of a non-critical role for cell swelling in irreversible cell injury is supported by the substantial return of cell function which has been observed in renal cortical slices after massive increases in volume were produced by 1 hr of incubation in oxygenated 'isotonic' high potassium, no sodium solution (M. B. Pine, unpublished observations). Future studies of cell volume regulation and metabolic injury should consider the role of anion exchange in preparations which more closely approximate in vivo conditions. Support for this work was provided by grants HL 1539, HL 599 and RR 132 from the National Institutes of Health and by grants from the Whitaker Health Sciences Fund and the Milton Fund. REFERENCES ALLISON, J. V. (1975). Effects of ouabain at different concentrations upon slices of rat renal cortex. Proc. Univ. Otago med. Sch. 53, BOYLE, P. J. & CONWAY, E. J. (1 94 1). Potassium accumulation in muscle and associated changes. J. Phy8iol. 1, 1-63.

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