Carbon flux through tricarboxylic acid cycle in rat renal tubules

194

Biochimica et Biophysica Acta, 1033 (1990) 194-200 Elsevier

BBAGEN 23259

Carbon flux through tricarboxylic acid cycle in rat renal tubules Itzhak Nissim, Ilana Nissim and Marc Y u d k o f f Division of Biochemical Development and Molecular Diseases, The Children's Hospital of Philadelphia and the Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, PA (U.S.A.) (Received 6 June 1989) (Revised manuscript received 6 November 1989)

Key words: Ammoniagenesis; Chronic acidosis; NMR, 13C-; GC-MS; Tricarboxylic acid cycle; (Renal tubule).

Our aim was to delineate the effect(s) of chronic metabolic acidosis on renal TCA-cycle metabolism. Renal tubules isolated from control and chronically acidotic rats were incubated at pH 7.4 with either 2 mM [2,3-13C]pyruvate or [2-13C]acetate. GC-MS a n d / o r 13C-NMR were utilized to monitor the flux of 13C through pyruvate dehydrogenase, pyruvate carboxylase and the TCA-cycle. With either, precursor acidosis was associated with significantly decreased formation of 13C-labelled citrate, malate, aspartate and alanine and increased formation of glucose, lactate and acetyl-CoA as compared with the control. The results indicate that adaptation of renal metabolism to chronic metabolic acidosis is associated with diminished flux through citrate synthetase and concomitantly increased flux through pyruvate carboxylase. The data suggest that depletion of TCA-cycle intermediates and enhanced ammoniagenesis in the kidney of chronically acidotic rats may be regulated at the site of mitochondrial citrate-condensing enzyme.

Introduction

Critical questions yet remain regarding the mechanisms by which changes of renal tricarboxylic acid cycle (TCA-cycle) metabolism abet ammoniagenesis in response to acidosis [1-9]. An often stated, but still unproved, hypothesis is that acidosis is associated with reduced flux through the citrate synthetase pathway, thereby resulting in diminished formation of 2-oxoglutarate and heightened flux through the glutamate dehydrogenase reaction in order to maintain the partially depleted pool of this TCA-cycle intermediate. The overall result is increased ammonia formation through the oxidative deamination of glutamate and increased flux of glutamate carbon into the TCA-cycle [1,9]. Our purpose in the current investigation was to learn whether the diminished formation of citrate previously noted in the renal tubules of chronically acidotic rats [9] is due to reduced flux through citrate synthetase (CS) or to inhibition of flux through pyruvate carboxylase (PC)

Abbreviations: CS, citrate synthetase; PC, pyruvate carboxylase; NMR, nuclear magnetic resonance; GC-MS, gas-liquid chromatography linked to mass spectometry; t-BDMS, t-butyldimethyl-silyl derivatives. Correspondence: I. Nissim, Division of Biochemical Development and Molecular Diseases, The Children's Hospital of Philadelphia, 34th and Civic Center Boulevard, Philadelphia, PA 19104, U.S.A.

and, presumably, a decreased availability of oxaloacetate. We therefore utilized NMR and GC-MS to estimate flux through CS and PC by monitoring the metabolism of both [2-13C]acetate and [2,3-~3C]pyruvate in rat renal tubules. The acetate is metabolized to [2-13C]acetyl-CoA in the thiokinase reaction and then to [1-13C]citrate via citrate synthetase. The pyruvate is metabolized to [1,2,3,4-13C]citrate after formation of [1,2-~3C]acetyl-CoA in the pyruvate dehydrogenase reaction and condensation of this species with the [2,3~3C]oxaloacetate generated in the pyruvate carboxylase pathway [10]. These species can be readily discriminated with GC-MS and NMR. The data demonstrate that flux through CS is reduced in the kidney of chronically acidotic rats and that this adaptation, rather than an inhibition of PC, explains the lower rate of citrate production in acidosis. Materials and Methods

Preparation and incubation of renal cortical tubules Male Sprague-Dawley rats weighing between 250-300 g were obtained from Charles River Breeding Laboratories. Induction of chronic metabolic acidosis by ammonium chloride administration and preparation of tubules was carried out as described previously [6-9]. Tubules (25-30 mg wet weight/ml) were incubated at 37 °C for 60 min in 4 ml of Krebs-Henseleit buffer (pH 7.4) containing 2.5% albumin (dialyzed bovine fraction

0304-4165/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

195 V) in stoppered 25 ml Erlenmeyer flasks. The buffer was pregassed with 95% 02/5% CO 2. Incubations were initiated by the addition of 250 /tl of either 32 mM [2,3-13C]pyruvate or [2-13C]acetate (99 atom% excess; MSD Ltd.) to 4 ml of cell suspension and terminated by the addition of 400 /tl of 40% perchloric acid to each flask. After neutralization with KOH, the supernatants were utilized for the determination of metabolites as well as measurement of isotopic abundance with either GC-MS or NMR.

13C-NMR techniques The neutralized tissue HC104 extracts from four flasks of approx. 400 mg of tubules (wet weight) were pooled. This was lyophilized and reconstituted in 2 ml of 33% D20. The tissue suspension (2 ml) was placed in a 10 mm N M R tube with 10/~1 of 10% (v/v) dioxane in 33% D20 as internal standard (66.5 ppm). ~3C-NMR spectra were recorded at 2 5 ° C and 125.77 MHz for ~3C nuclei using a Bruker AM 500 spectrometer equipped with the ASPECT-3000 Data System. For most analyses 2000-3000 transients were accumulated by using radiofrequency 90 ° pulses (25 /~S) and a 25 KHz spectral window. Proton decoupling was achieved by composite pulse proton decoupling.

Expression of results and calculation of flux rates In each of the studies described above we measured metabolite concentration and 13C isotopic enrichments. The concentrations were normalized to wet weight tubules. The time course of [2,3-~3C]pyruvate utilization rates were best fitted to a single exponential equation, I t = I o e x p ( - k , t) were I t and I o are the concentrations of labelled pyruvate at time t and time 0, respectively, and k is the disappearance rate constant (min 1) [6]. The rate of appearance of 13C-labelled metabolite was calculated by either linear or non-linear regression, in the latter analysis using the expression: y = A(1 - e x p - k t ) where y is the concentration (nmol 13C wet weight) isotopically labelled metabolite at any given time, t; A is the amount of isotopically labelled metabolites at plateau (steady-state) and k is the turnover rate constant (min-1). The flux through a pathway in question is derived from the product of A times k.

Statistical analysis The Student's t-test or the method of Dunnett [15] for multiple comparisons with a control population was used for the statistical analysis. Results

GC-MS techniques GC-MS measurements of 13C isotopic enrichment were performed on a Hewlett-Packard 5990A GC-MS system. Glucose was derivatized as the 6-acetylbis1,2,3,5-butaneboronyl 2-D-glucofuranose by the method of Wiecko and Sherman [11]. Amino acids and organic acids were measured as their t-butyldimethylsilyl derivatives (t-BDMS) as previously described [12]. The isotopic enrichments of ~3C in jglucose, amino acid and organic acid derivatives were determined with selected monitoring of ions obtained by electron impact. The m / z 303/297 ratio, corresponding to glucose labelled at all six carbons, was utilized. ~3C enrichments in the citrate t-BDMS derivative were measured using m / z 595/591 for citrate labelled at four carbons in experiments with [2,3-13C]pyruvate as a substrate and using m / z 592/591 for singly labelled citrate in experiments with [2-a3C]acetate as a substrate. Aspartate, alanine, lactate, pyruvate and malate t-BDMS derivatives ( M + 2 / M ) were monitored using the m / z 420/418, 262/260, 263/261, 261/259 and 421/419 ratio, respectively, for labelling at two carbons in experiments with [2,3-13C]pyruvate.

Analytical measurements Amino acids, lactate, pyruvate and glucose were measured as previously described [6-9]. Citrate was determined by the method of Dagley [13] and malate by method of Goldberg and Passonneau [14].

13C-NMR spectroscopy Fig. 1 demonstrates the ~3C-NMR spectra obtained from renal tubules following a 60 min incubation with 2 mM [2,3-~3C]pyruvate. In the control, the spectra exhibit a significantly higher production of 13C-labelled alanine, aspartate, glutamate, citrate and malate compared with acidosis. However, in chronic acidosis 13CN M R spectra illustrate a significantly higher production of ~3C-labelled glucose. In the control, the peak at 18.81 ppm corresponding to C-2 of acetyl-CoA is hardly distinguished from background, whereas in acidosis this resonance demonstrates a significant accumulation of acetyl-CoA. In both control and acidosis the 13C-13C splitting are due to adjacent 13C atoms derived from [2,3-13 C]pyruvate.

GC-MS analysis In Fig. 2 the utilization of isotopically-labelled pyruvate during the course of the incubation is shown. The rate of [2,3-13C]pyruvate metabolism shows little differences between tubules obtained from control chronically acidotic rats, (Table I). The time course of 13C-labelled metabolite formation following incubation of renal tubules with [2,3-~3C] pyruvate is illustrated in Fig. 3. The formation of ~3Clabelled citrate and malate reached a steady-state at approx. 40 min after the start of the incubation, demonstrating an equilibrium between the formation and the utilization of these intermediates in the incubation sys-

196 o

CONTROL

_=o

,~

o o

o

0

=o

,4

o

o

'

d

o

an I "I-

o

r

o

4

O :~

II1

o

m~ 2 , _ll

'

~t'~l

/

I0

(~,

I

0~

e~ll

m

o.

I

~

,~-,~

I

0 f'~

o

,m

ql~ .

0

1

°

.J

o

ACIDOSIS

. ,

.

.

.

.

,

.

.

.

.

100

,

.

.

.

.

90

,

.

.

.

.

,

.

80

.

.

.

,

.

.

.

,

.

70

.

.

.

,

.

.

.

.

,

.

.

.

.

,

.

.

.

60

.

,

.

.

.

.

,

.

50

.

.

.

,

.

.

.

,

.

.

40

.

.

,

.

30

.

.

.

,

.

.

.

.

,

.

.

.

.

,

20

.

.

.

.

10

PPM

Fig. 1. ]3C-NMR spectra of neutralized perchloric acid extracts of renal tubules incubated for 60 min with 2 m M [2,3-13 C]pyruvate.

tern. However, formation of ]3C-labelled aspartate, alanine, glucose and lactate increased linearly throughout the course of the incubation (Fig. 3). Calculations based on the data points of Fig. 3 demonstrate a greater flux through the citrate synthetase, malate dehydrogenase and pyruvate alanine transaminase pathways in the control compared with acidosis (Table I). However, the rates of n C glucose and lactate formation were significantly'higher in acidosis compared with the control (Table I). These observations, which were obtained by GC-MS measurements, are in agreement with the data obtained by 13C-NMR spectroscopy (Fig. 1), which exhibit a higher resonance of ~3C malate, citrate, alanine and aspartate and a concomitantly smaller resonance of

13C glucose and lactate in the control compared with acidosis. The data clearly indicate an increased rate of gluconeogenesis from pyruvate during acidosis. In order to delineate the mechanism by which pyruvate carbon is exported from the mitochondria for the purpose of glucose production in the cytosol, experiments were performed in the presence of 1 mM amino-oxyacetate

TABLE I (a) Rate of [2,3-13 C]pyruuate utilization and (b) [ l SC]metabolite production in renal cortical tubules" isolated from chronically acidotic rats and rats in normal acid-base balance The initial rates of pyruvate utilization were determined by fitting the data points in Fig. 2 to a single exponential function and the rates of [13 C]metabolite production were determined by fitting the data points in Fig. 3 to either linear or non-linear regression analysis as described under Material and Methods. Results expressed in n m o l / g wet weight per min (mean _+S.D., n = 3 experiments). Significance: "P > 0.05 (not significant) and bp < 0.05 (compared with control).

7O 60

.~ 4o $ 3o :m.

20

,b z'o 3'0

s'o do

TIME(min.)

Fig. 2. Time course of [2,3J3C]pyruvate utilization by renal tubules obtained from control (e) or chronically acidotic rats (&). Values are the concentration (p, mol 13C/g wet weight) determined from the product of concentration and isotopic enrichment (atom% excess/100). Each point is the mean of three experiments.

Labeled Metabolite

Control

Acidosis

(a) [2,3-13 C]Pyruvate Utilization

765.7 + 88.5

906.3 -+ 80.8 ~

(b) 13C Metabolite Production: Citrate 16.9_+ Malate 15.9-+ Aspartate 13.6-+ Alanine 17.2_+ Lactate 106.4_+ Glucose 17.4_+

0.7 1.6 1.8 0.8 5.2 2.1

3.5_+ 5.8_+ 6.8_+ 3.1_+ 177.4_+ 54.4_+

1.2 b 0.1 b 0.8 b 0.2 b 9.9 b 3.1 b

197 4OO -~ 350

8OO 7OO Citrate 600 = 5O0

3oo

250

-

®8

~ o ~

zoo

300

L50

~ 200

i

2O I0 O' 7OO = 600

50

~

I0 2 O 5 0 4 O 5 0 6 O

900 _ 800

9OO

~ -

Asparta/

500

700 --- 600 o ~ 500

400

m ~ 400

700 600

E

¢ 3oo

Pyruvote

I0 20 50 40 50 60

I0 20 50 40 50 60 3.0

12 II Lactate I0

2.5 2.0

~

~

i.o

/-

/ 0.5 ~ , ~ J , ,

;

~ ="

~ " ,

,

8 7

4 3

2 I

I0 20 30 40 50 60 TIME (rain)

I0 20 30 40 50 60 TIME (mm)

p,c0.001

20C I00 0

~200 I00

: 200 I00

• 0.0001

® ~ 50C =400

3OO

-6 3OO

~0.0001 _T__

3o

~ I00

I00

8OO

80 70 60 50 40

Fig. 3. Time course of a3C-labelled metabolites formation from [2,313C]pyruvate by control (@) or chronically acidotic rat renal tubules (A). The data points are the product of isotopic enrichment (atom% excess/100) and concentration ( n m o l / g wet weight). Each point represents the mean + S.D. of three experiments.

Acetote

Fig. 4. Formation of 13C-labelled citrate following 60 min incubation of renal tubules with either 2 mM [2,3-13C]pyruvate or [2J3C]acetate. Results are m e a n _ S.D. of 3-4 experiments with control (open bar) or chronic acidosis (shaded bar). P values refer to control vs. acidosis.

(AOA), an inhibitor of transaminase reactions [18]. As demonstrated in Table II, AOA remarkably inhibited the formation of t3C-labelled aspartate, but it had little effect on t3C-malate (Table II) or 13C glucose and lactate formation (data not shown). The 13C isotopic enrichments represented in Table II indicate that approx. 70% of C-2 and C-3 of malate were derived from pyruvate, regardless of acid-base status. Furthermore, there was little difference between isotopic enrichment in aspartate and malate, indicating isotopic equilibrium between oxaloacetate and malate. However, the absolute production of 13C malate and aspartate were markedly lower in acidosis compared with the control (Table II). These observations presumably reflect a

TABLE II

Effect of amino-oxyacetate on the production of l 3C.labelled aspartate and malate from [2, 3 - 1.~C]pyruvate Renal tubules obtained from control and chronically acidotic rats were incubated for 60 min with 2 mM [2,3J3C]pyruvate in the presence and absence of 1 mM AOA. GC-MS was utilized to monitor 13C isotopic enrichment as described under Material and Methods. Values are mean _+S.D. of three experiments. Experiments

Control Acidosis Control + AOA Acidosis + AOA

13C-Enrichment (atom% excess)

13C-Formation(nmol/60 min per g wet wt. tubules) "

Malate

Aspartate

Malate

Aspartate

60.3 + 75.6 + 63.4 + 65.6 +

72.6 ___5.4 63.7 + 8.7 b 15.3 + 2.3 r 14.2 + 3.8 r

355.8 198.8 384.3 227.6

778.6 + 417.9 + 74.2 + 50.4 ±

8.6 6.4 b 4.2 ~ 9.4 ~

+ 28.7 ___18.3 c + 25.2 e + 23.4 ¢

a Values are the product of metabolite concentration ( n m o l / 6 0 min per g wet wt.) and (atom% excess/100). b p > 0.05 compared with control. c p < 0.05 and; d p < 0.01 compared with control. e p > 0.05 and; f P < 0.001 compared with incubation in the absence of AOA.

58.3 41.4 d 13.6 f 12.3 f

198 higher utilization of malate for the process of enhanced gluconeogenesis in tubules obtained from acidotic rat [8,9,16,17]. To further explore the effect of chronic metabolic acidosis on citrate synthesis, experiments were carried out with [2-13C]acetate as a sole substrate in the incubation medium. Fig. 4 shows the 13C enrichment (upper panel) and concentrations (lower panel) in citrate following incubation of renal tubules with either [2,3~3C]pyruvate or [2-13C]acetate. In the control 13C enrichment in citrate was approx. 2-fold higher compared with that in acidosis regardless of the precursor. In agreement with the isotopic enrichment data the formation of 13C citrate (nmol/g wet weight per 60 min) was approx. 4-fold higher in the control compared with chronic acidosis (Fig. 4, bottom panel). In addition, the data of Fig. 4 indicate that regardless of acid-base state the incorporation of pyruvate carbons into the TCA-cycle was approx. 3-fold higher than that of acetate. The availability of ATP may regulate the conversion of acetate to acetyl-CoA. However, prior observations have shown little change in ATP contents in renal tubules of chronically acidotic compared with control rats [6,7]. Thus, the availability of ATP was not a limiting factor for acetate metabolism in the current investigation. Presumably, in renal proximal tubules there is no difference in the uptake of pyruvate and acetate. Hence, the difference in the formation of ~3C citrate with either pyruvate or acetate as sole substrate may reflect a higher flux through pyruvate dehydrogenase than through acetate thiokinase. Discussion

The current investigation substantiates that flux through citrate synthetase is significantly diminished in renal tubules obtained from chronically acidotic rats (Figs. 3 and 4). This conclusion is consistent with the hypothesis that augmented renal ammoniagenesis in chronic acidosis is regulated at the site of citrate synthetase [1,9]. According to this formulation, diminished feeding of carbon skeletons into the TCA-cycle via the citrate-condensing enzyme leads ultimately to reduced production of mitochondrial 2-oxoglutarate and consequent enhancement of the oxidative deamination of glutamate via the G L D H reaction. The reductive amination of 2-oxoglutarate also is decreased (Fig. 1), thereby favoring augmented flux through the glutaminase pathway as the glutamate pool is diminished [3]. This scheme is consistent with our previous studies involving the metabolism of [3-13C]glutamate [8] and [3-13C]alanine [9]. These conclusions also are compatible with previous observations demonstrating that: (a) renal cortical concentrations of citrate of 2-oxoglutarate are elevated in metabolic alkalosis [19] and depleted in acidosis [20,21]; (b) that either 2-oxo-glutarate or pyru-

LAC

\

ACETATE

PYRUVATE ALA

MAL

/

I

) \

~ Acetote -

/

I,, Pyruvate [ |

AcCoA,,-,--,m-AcAc- ~ / ' ~ - Hb

Fig. 5. Schematic representation of the pathways involved in pyruvate and acetate metabolism. Bold arrows indicate more prominent pathways of carbons flux in chronic metabolic acidosis. The bold rectangle indicates the site of TCA-cycle inhibition following adaptation to chronic acidosis. AOA, indicates the site of inhibition by aminooxyacetate.

vate inhibits ammoniagenesis from glutamine by reducing flux through the glutamate dehydrogenase reaction [2-4,6,22,23]; and (c) that citrate synthetase exhibits maximal activity at pH 7.8-8.0. Beyond these limits the activity fall rapidly, particularly at acidic pH [24,25]. The TCA-cycle provides reducing equivalents to the electron transport chain and also, via ancillary reactions, substrates for biosynthetic reactions in the cytosol [10]. Fig. 5 illustrates schematically the adaptations occurring in the flux of carbon in response to chronic metabolic acidosis in rat renal tissue. The proposed carbon flows are consistent with the pathways shown by Lardy et al. [27], Krebs et al. [28] and Rognstad and Katz [18]. As proposed in Fig. 5, in acidosis the metabolism of pyruvate to oxaloacetate via PC is enhanced, but the entry of pyruvate carbon into the TCA-cycle via the sequential actions of pyruvate dehydrogenase and CS is decreased. The latter is inhibited by the accumulation of acetyl-CoA (Fig. 1) presumably consequent to reduced consumption of this thioester in the CS reaction [10]. Some pyruvate also may be carboxylated through the cytosolic NAD-linked malic enzyme [29]. The flow of carbon from mitochondria to cytosol is primarily through malate rather than aspartate efflux because amino-oxyacetate almost completely abolished formation of aspartate (Table II) with little effect on 13C glucose formation. Thus, the enhanced conversion of oxaloacetate to malate and malate efflux from mitochondria provides both the carbon and reducing potential for augmented gluconeogenesis in chronic acidosis, as demonstrated in Figs. 1 and 3. However, 13C-NMR spectra (Fig. 1) and GC-MS analysis (Figs. 3 and 4) suggest that in the control flux through both pyruvate carboxylase and citrate synthetase are equally important pathways feeding carbon into the TCA-cycle. In both control and acidotic conditions there was a linear formation of 13C-labelled lactate and glucose

199 (Fig. 3). According to Baverel et al. [30] and Saggerson [31] a linear production of lactate and glucose from pyruvate implies a constant flow of reducing equivalents from the mitochondria to the cytosol. Hence, as proposed in Fig. 5, the N A D H necessary for glucose and lactate formation is likely derived by the transport of the mitochondrial malate and its metabolism to oxaloacetate in the cytosol as previously suggested [18,27,28,31]. The net cytosolic reducing equivalent provision (CREP) is equal to 2 mol of N A D H / m o l of glucose + 1 mol of N A D H per mol of lactate formed [31]. Based on the rates of glucose and lactate production (Table I) the calculated C R E P is approx. 141 and 285 n m o l / m i n per g wet tissue in control and acidosis conditions, respectively. These calculations indicate that in acidosis there was a 2-fold higher production of mitochondrial malate and metabolism of the latter to OAA in the cytosol. Hence the current studies suggest that the flux through both NAD-linked and N A D H - l i n ked malate dehydrogenase are elevated in renal tubules obtained from chronically acidotic rats. It should be stressed that analysis of the m/z 595/591 ratio to determine labelling of 13C-citrate reflects the fact that with [2,3-13C]pyruvate as sole substrate [1,2,3,4-13C]citrate will be produced. This occurs following formation of [2,3-13C]oxaloacetate in the pyruvate carboxylase reaction and condensation of this species with [1,2-13C]acetyl-CoA derived from the pyruvate dehydrogenase reaction. Thus, we detected no appreciable formations of ' M + 1' or ' M + 2' species, denoting labelling to citrate at either 1 or 2 carbons. Similarly, in experiments involving [2-~3C]acetate as sole precursor, citrate is expected to be mainly singly labelled, this being derived from the methyl carbon of acetyl-CoA. We therefore employed the m/z 592/591 ratio to monitor ~3C-citrate synthesis in studies with [2-13C]acetate. Metabolism in the TCA-cycle will transfer 13C from citrate to 2-oxoglutarate, which is converted to 13Clabelled glutamate following reductive amination or transamination. In agreement with our previous observations [6,8,9], ~3C-NMR spectra demonstrate that the reductive amination of 2-oxoglutarate is much greater in control compared with acidotic tissue. The metabolic fate of the glutamate so derived is complex. The data indicate that some glutamate carbon is converted to aspartate. Fig. 1 shows that in control tissue labelling of C-2 aspartate/C-3 aspartate reaches a ratio of approx. 1:1. In contrast, in acidosis this ratio is approx. 1 : 2. The cause for this observation, which has been noted by others [32], is not immediately apparent. Cohen et al. [32] suggested that it is due to a compartmentalization of intramitochondrial oxaloacetate such that a significant fraction of the pool accessible to aspartate aminotransferase has not been randomized in the sequential reactions of fumarase and malate dehydrogenase [33]. The possibility of compartmentalization

in renal cortex is suggested by Schoolwerth and LaNoue [34]. The fact that the disparity in C-2 and C-3 labelling is evident during acidosis implies that endogenous carbon sources, presumably glutamine and glutamate, are directed into the T C A cycle (Fig. 5) because of the higher flux through glutaminase and glutamate dehydrogenase in acidotic tissue [3,4,6,8,34]. Another important observation is that the tubules produced acetoacetate and fl-hydroxybutyrate (Fig. 1), a finding which supports the suggestion of Krebs et al. [35] that kidney cortical slices produce ketone bodies, perhaps thereby supplementing hepatic production. The formation of [13C]acetoacetate is higher in acidosis, whereas [fl-13C]hydroxybutyrate is greater in control tissue. The finding presumably reflects a higher NAD+/NADH ratio in the renal cortical mitochondria of chronically acidotic rats. In summary, the current investigation indicates that acidosis is associated with multi-focal changes of carbon flux through the various steps of the TCA-Cycle, including citrate synthetase, pyruvate carboxylase and malate dehydrogenase. It should be emphasized that the extent to which the current conclusions can be extrapolated to the in vivo situation remains to be established.

Acknowledgements Supported by N I H grants RO1-DK34771 and RO1DK39348. The authors are grateful to Mr. Ziphing Lin and Mr. Marc Melincoff for expert technical assistance and Ms. Isabella Fisher for secretarial assistance.

References 1 2 3 4

Pitts, R.F. (1966) Physiologist 9, 97-109. Tannen, R.L. (1978) Am. J. Physiol. 235, F265-F277. Tannen, R.L. and Sastrasina, S. (1984) Kidney Int. 25, 1-10. Schoolwerth, A.C. and LaNoue, K.F. (1983) Am. J. Physiol. 244, F399-F408. 5 Vinay, P., Limieux, G., Gougoux, A. and Halperin, M. (1986) Kidney Int. 29, 68-79. 6 Nissim, I., Yudkoff, M. and Segal, S. (1985) J. Biol. Chem. 260, 13955-13967. 7 Nissim, I., Yudkoff, M. and Segal, S. (1986) J. Biol. Chem. 261, 6509-6514. 8 Nissim, I., Yudkoff, M. and Segal, S. (1987) Biochem. J. 241, 361-370. 9 Nissim, I., Yudkoff, M. and Segal, S. (1988) Contrib. Nephrol. 63, 60-70. 10 Williamson, J.R. and Cooper, R.H. (1980) FEBS Lett. 117, K73-K84. 11 Wiecko, J. and Sherman, W.R. (1976) J. Am. Chem. Soc. 98, 7631-7637. 12 Schwenk, W.F., Berg, P.J., Beaufrere, B., Miles, M. and Haymond, M.W. (1984) Anal. Biochem. 141, 101-109. 13 Dagley, S. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H.U., ed.), pp. 1562-1569, Academic Press, New York. 14 Goldberg, N.D. and Passonneau, J.V. (1974) in Methods in Enzymatic Analysis (Bergmeyer,H.U., ed.), pp. 1600-1603, Academic Press, New York.

200 15 Dunnett, C.W. (1964) Biometrika 42, 482-491. 16 Schoolwerth, A.C., Schmidt, B.C. and Culpepper, R.M. (1988) Miner. Electrolyte Metab. 14, 347-361. 17 Wirthenson, G. and Guder, W.G. (1986) Physiol. Rev. 66, 469-497. 18 Rognstad, R. and Katz, J. (1970) Biochem. J. 116, 483-491. 19 Bulagura-Baruch, S., Shurland, L.M. and Welbourne, T.C. (1970) Am. J. Physiol. 218, 1070-1075. 20 Vinay, P., Allignet, E., Pichette, C., Watford, M., Lemieux, G. and Gougoux A. (1980) Kidney Int. 17, 213-315. 21 Alleyne, G.A.O. (1986) Nature 217, 847-848. 22 Baurke, E., Delaney, V., Risquez, A. and Preuss, H.G. (1985) Contrib. Nephrol. 47, 28-35. 23 Tannen, R.L. and Kunin, A.S. (1982) Kidney Int. 22, 280-285. 24 Stern, J.R. and Ochoa, S. (1952) J. Biol. Chem. 198, 313-321. 25 Kosicki, G.W. and Srere, P.A. (1961) J. Biol. Chem. 236, 2566-2570. 26 Wiegand, G. and Remington, S.J. (1986) Annu. Rev. Biophys. Chem. 15, 97-117.

27 Lardy, H.A., Paetkau, V. and Walter, P. (1985) Proc. Natl. Acad. Sci USA 53, 1410-1415. 28 Krebs, H.A., Cascoyne, T. and Notton, B.M. (1967) Biochem. J. 102, 275-281. 29 Scaduto, R. and Davis, E.J. (1986) Biochem. J. 237, 691-698. 30 Baverel, G., Bonnard, M., Castanet, E.A. and Pellet, M. (1978) Kidney Int. 14, 567-575. 31 Saggerson, E.D. (1978) Biochem. J. 174, 131-142. 32 Cohen, S.M., Glynn, P. and Shulman, R.G. (1981) Proc. Natl. Acad. Sci. USA 78, 60-64.33. 33 Rognstad, R. and Katz, J. (1972) J. Biol. Chem. 247, 6047-6054. 34 Schoolwerth, A.C. and LaNoue, D.A. (1980) J. Biol. Chem. 255, 34003-3411. 35 Krebs, H.A., Hems, R., Weidemann, M.J. and Speake, R.N. (1966) Biochem. J. 101, 242-249.