85

Biochimica et Biophysica Acta, 499 (1977) 85--98 © Elsevier/North-Holland Biomedical Press

BBA 28288

C O N T R I B U T I O N OF pH-SENSITIVE METABOLIC PROCESSES TO pH HOMEOSTASIS IN ISOLATED R A T KIDNEY TUBULES

ANTHONY G. DAWSON

Department of Biochemistry, University of Sydney, Sydney, N.S.W. 2006 (Australia) (Received December 6th, 1976)

Summary The metabolism of isolated rat kidney tubules suspended in calcium-free physiological saline buffered with phosphate was found to be sensitive to changes in the pH of the suspending medium. Lowering the pH from 7.8 to 6.4 brought a b o u t increases in the rates of oxidation of added succinate, glutamate or glutamine as well as in the production of glucose from lactate, glutamine, succinate and fructose. The cellular ATP level was also higher in tubules incubated at pH 6.4. In contrast, the utilization of added glucose was greater at pH 7.8 than at pH 6.4, a substantial a m o u n t of lactate being produced at the higher pH. When glucose and either lactate or glutamine were provided as co-substrates glucose was the preferred fuel at pH 7.8 but the alternative substrate was the more readily utilized at pH 6.4. As a consequence of the metabolic activities of the tubules the pH of the suspending medium changed, utilization of lactate, glutamate or glutamine causing a rise in pH while conversion of glucose to lactate caused a fall in pH. In cases where t w o substrates were metabolized concurrently over a period of 3 h the extracellular pH tended towards a plateau level of approximately pH 7.4. It is proposed that pH-sensitive metabolism in isolated kidney tubules contributes to pH homeostasis in the cellular environment.

Introduction Two kinds of change in mammalian kidney metabolism can occur in response to variations in the extracellular pH. One of these is an adaptive change which is first observed in the intact animal several hours after the induction of chronic or acute acidosis. The main feature of this adaptation is an increase in the rate at which extracellular glutamine is taken up by the kidney cells and converted to ammonia, CO2 and glucose [ 1 ] ; this is thought to be due to increases in the activities of the mitochondrial glutamine transport system [2,3], glutaminase [4] and phosphoenolpyruvate carboxykinase [5--9].

86 The second type of pH-dependent change is one in which certain metabolic activities of in vitro preparations of kidney taken from animals in normal acidbase balance respond directly and immediately to alterations in the pH of the extracellular medium. Processes affected include glycolysis [10], gluconeogenesis [11--17], ammoniagenesesis [17--20] and oxidation of substrates through the tricarboxylic acid cycle [14,15,21]. The increases in substrate oxidation, ammonia production and gluconeogenesis that accompany a lowering of the extracellular pH are similar to those that occur during adaptation to experimental acidosis but it has been pointed out that the two can not necessarily be related in any simple manner [22]. The rapidity of the in vitro response suggests that it might be mediated differently from the longer-term in vivo adaptive response and it has been proposed that H ÷ exerts direct regulatory influences on phosphofructokinase [23--25], some enzymes of the tricarboxylic acid cycle [13--15] and, possibly, phosphoenolpyruvate carboxykinase [16]. However, one problem encountered in some of the previous work has arisen through the use of suspending media buffered by the carbonic acid/bicarbonate system. This had led to difficulties in distinguishing between those effects caused by pH changes and those caused by the changes in bicarbonate concentration by which pH variations were engineered. Citrate oxidation, gluconeogenesis and ammoniagenesis are all affected by changes in bicarbonate concentration even when the pH remains unaltered [12,15,16,20,26]. In order to avoid this problem in the current investigation the suspending medium was buffered with phosphate instead of bicarbonate, and CO2 was continuously removed from the atmosphere in contact with the medium. The replacement of bicarbonate by phosphate was thought to be acceptable because phosphate, at concentrations of 1--10 mM, which accommodates the normal physiological levels, appears to have little effect on the gluconeogenic and respiratory activities of kidney preparations [27,28]. Only when the phosphate concentration is lowered to below 1 mM is there a significant effect on these activities, gluconeogenesis being stimulated and substrate oxidation inhibited [28]. However, because of problems associated with the precipitation of calcium phosphate, particularly at high pH, the not uncommon practice of omitting calcium from the suspending medium was also adopted. This has the additional benefit of minimising the interpretive difficulties posed by the interrelated effects of pH and calcium on renal metabolism [13,14,16]. It should be borne in mind that the omission of calcium leads to decreases in both gluconeogenesis and respiration in kidney preparations [13,14,16,27,29] but this loss of activity was considered acceptable when weighed against the problems likely to be set by the inclusion of this ion. In the work reported below an attempt was made to determine the interrelationships between the extracellular pH and integrated patterns of metabolism in isolated rat kidney tubules. Several substrates were used either alone or in combinations; included among them were glutamine, lactate and glucose which, according to Pitts [30], are major renal fuels. On the basis of the results it is proposed that the pH sensitivity of certain metabolic processes is important to the attainment and maintenance of pH homeostasis of the cellular environment.

87

Experimental Materials. Hyaluronidase (type I), collagenase (type I), glucose oxidase (type II), peroxidase (type II), hexokinase (type III), glucose-6-phosphate dehydrogenase (type V), lactate dehydrogenase (type II), NAD*, NADH, NADP ÷, ATP and L-lactic acid were obtained from Sigma Chemical Co., St. Louis, Mo., U.S.A.; L-glutamic acid, D-glucose and sodium succinate were from B.D.H. Ltd., Poole, Dorset, U.K.; L-glutamine was from E. Merck AG, Darmstadt, Germany; D-fructose was from Ajax Chemicals Ltd., Sydney, Australia; D-[U-~4C]glucose, D-[U-14C]fructose, L-[U-14C]glutamic acid, L-[U-14C]glutamine, [1-14C]lactic acid and [2,3-'4C2]succinic acid were from The Radiochemical Centre, Amersham, Bucks., U.K.; bovine serum albumin powder (fraction V) was from Armour Pharmaceutical Co. Ltd., Eastbourne, Sussex, U.K. All other reagents were A.R. grade. Tubule preparations. Tubules were isolated by the enzymatic disruption of kidney slices from adult albino rats (Wistar strain) as described by Dawson [29] except that calcium-free medium was used for washing and suspending the isolated tubules. Incubations. Tubules (5--11 mg of cell protein) were incubated in 2 ml of calcium-free medium containing the following ions (mequiv./1), Na ÷, 144; K÷, 4.2; Mg2÷, 1.1; SO~, 1.1; CI-, 134 or 142; HPO~ plus H2PO:,, 5 or 10, with an initial pH of 6.4--7.8. Substrates were added, either singly or jointly at the concentrations indicated in the text; 14C-labelled substrates were used only as single exogenous substrates because of the problems of interpreting data on the fate of the isotopic carbon when labelled and unlabelled substrates are added jointly [31]. Incubations were for periods of 0.5--3 h at 37°C in conventional Warburg manometry vessels fitted with stoppers and shaken at 120 oscillations/ min. The gas phase was air and the centre well of each flask contained 0.1 ml of 2 M NaOH to absorb metabolically produced CO2. At the end of the incubation period 0.2 ml of 3 M HC104 was added immediately to each flask except in experiments where the final extracellular pH was to be determined. In those cases the suspensions were cooled, centrifuged at 300 × g for 2 min and the pH of the supernatants determined before acidification. Each acidified mixture was centrifuged to remove the precipitate. The supernatant fluid was neutralized with 3 M KOH, made up to 3 ml with distilled H20 and the KC104 precipitate was sedimented by centrifugation. Samples of the neutral, perchlorate-free supernatant fluid were used for analyses. Measurements. Determinations of pH were made using a Titron pH electrode connected to a Horiba M-5 pH meter. The protein content of the tubule preparation was measured by the method of Lowry et al. [32] with a solution of bovine serum albumin as the reference standard. Glucose was determined by the glucose oxidase method [27]. Enzymatic methods were used for the measurement of lactate [33], pyrurate [34] and ATP [35], the formation or utilization of NAD(P)H being followed spectrophotometrically at 340 nm using an Aminco DW-2 UV/VIS spectrophotometer. Ammonia was estimated by the method of Kaplan [36].

88 14CO2 produced from 14C-labelled substrates and trapped in the centre well of the reaction vessel was determined by transferring a sample of fluid from the centre well on to a dry Whatman glass fibre disc previously impregnated with fresh 10% (w/v) barium acetate. After drying in air the disc was placed in a scintillation vial containing 5 ml of naphthalene/PPO/dioxane scintillation fluid [37] and was counted (minimum of 104 accumulated counts) in a Nuclear Chicago Isocap/300 Liquid Scintillation System. Oxygen consumption was measured manometrically [38]. Statistical analysis. All results are given as the mean + the standard error of the mean. Significance of difference was estimated by the Student's t-test. Results

Effect of pH on glucose formation in kidney tubules The formation of glucose from fructose, lactate, glutamine or succinate in tubules suspended in medium buffered with phosphate at pH 7.8, 7.3, 6.8 or 6.4 is shown in Fig. 1. Glucose production from all four substrates increased markedly between pH 7.8 and pH 6.8 but underwent little change between pH 6.8 and pH 6.4. Although the amount of glucose formed from fructose was much greater than that formed from the other substrates the similarities

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Fig. I . E f f e c t o f p H o n p r o d u c t i o n of glucose b y k i d n e y tubules. Tubules (8--11 mg of protein) w e r e i n c u b a t e d f o r 3 0 m i n a t 3 7 ° C in 2 m l o f t h e Ca2+-free m e d i u m b u f f e r e d w i t h p h o s p h a t e ( 1 0 m M ) and c o n t a i n i n g o n e o f t h e f o l l o w i n g substxates; o, D - f r u c t o s e (5 r a M ) ; I , L-lactate (5 m M ) ; ~, L - g l u t a m i n e (5 m M ) ; o, s u c c i n a t e ( 5 m M ) . E a c h point represents the m e a n v a l u e -+ S.E. f o r t h r e e s e p a r a t e e x p e r i m e n t s . Fig. 2. E f f e c t of p H on t h e o x i d a t i o n o f s u c c i n a t e b y k i d n e y t u b u l e s . E x p e r i m e n t a l c o n d i t i o n s as f o r Fig. 1, w i t h 0.1 #Ci [ 2 , 3 - 1 4 C 2 ] s u c c i n a t e (5 m M ) as s u b s t r a t e . E a c h p o i n t r e p r e s e n t s t h e m e a n v a l u e -+ S.E. for t h r e e s e p a r a t e e x p e r i m e n t s .

89 between the four curves suggested the probable involvement of at least one c o m m o n pH-sensitive control point.

Effect of pH on the oxidation of 14C-labelled substrates The results presented in Fig. 2 indicate that the oxidation of [2,3-~4C:]succi nate to ~4CO2 was strikingly stimulated as the pH of the suspending medium was lowered from pH 7.8 to pH 6.4. In parallel experiments the consumption of oxygen by the tubules rose from 1.30 + 0.04 to 1.64 + 0.04/~mol/mg protein per h over the same pH range (P < 0.002). The data in Table I show that a similar, though less marked, increase in ~4CO: production occurred with L-[U-~4C] glutamine and L-[U-14C]glutamate but n o t with L-[1-14C]lactate nor D-[U-~4C] glucose both of which were oxidized to a similar extent at pH 7.8 and pH 6.4. The strong stimulation of succinate, glutamate and glutamine oxidation by H ÷ suggested an effect exerted directly on the tricarboxylic acid cycle activity.

Effect of pH on ATP levels in tubules The ATP in tubules after a 30 min incubation with 5 mM succinate at different pH levels is shown in Fig. 3. The pre-incubation level of ATP is marked as a broken horizontal line in the figure. Incubation at pH 7.8 resulted in a significant fall in ATP from the pre-incubation level (P < 0.01) while incubation at pH 6.4 led to a significant rise (P < 0.05). The ATP level after incubation appeared to be influenced b y the pH of the medium in a fashion similar to that seen with succinate oxidation and glucose formation, possibly implying that the three parameters are related in some way. In three experiments with 5 mM glucose as substrate, under the same conditions as those given in Fig. 3, the ATP level after incubation at pH 7.8 was 5.8 + 0.4 nmol/mg cell protein whereas that after incubation at pH 6.4 was significantly higher at 8.9 + 0.4 nmol/mg cell protein ( P < 0.01) even though glucose oxidation was the same at the t w o pH levels (cf. Table I).

Relationship between extracellular pH and metabolism of glutamine, glutamate, lactate and glucose With 3 mM L-[U-14C]glutamine as sole exogenous substrate during a 3 h TABLE I E F F E C T O F p H O N T H E O X I D A T I O N O F 1 4 C - L A B E L L E D S U B S T R A T E S BY K I D N E Y T U B U L E S T u b u l e s ( 7 - - 1 1 m g o f p r o t e i n ) w e r e s u s p e n d e d in 2 m l o f t h e Ca2+-free m e d i u m b u f f e r e d w i t h p h o s p h a t e ( 5 rnM) at either p H 7.3 o r p H 6 . 4 a n d c o n t a i n i n g 0.1 /~Ci o f 14C-labelled substrate a t t h e c o n c e n t r a t i o n s s h o w n . I n c u b a t i o n s w e r e f o r 3 0 rain at 3 7 a C . T h e r e s u l t s are g i v e n as m e a n v a l u e s -+ S.E. f o r t h r e e s e p a r a t e experiments. Substrate

.

14CO2 (/~rnol/mg p r o t e i n per 3 0 rain)

p H 7.8 3 3 5 5

mM mM mM mM

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0.39 0.34 0.17 0.26

+ 0.04 * : + 0.03 * -+ 0 . 0 3 + 0.02

* Values a t p H 7.8 a n d p H 6 . 4 are significantly different. P