Substrate Metabolism and Renal Function

Symposium on Pediatric Nephrology

Substrate Metabolism and Renal Function Takashi Yoshida, M.D. *

Sodium reabsorption and acidification of urine are major physiological functions of the kidney and are closely related to substrate metabolism in the kidney. Renal sodium reabsorption is finely controlled by physical mechanisms (glomerulotubular balance), hormonal influence (aldosterone, antidiuretic hormone) and substrate metabolism. Metabolism of fatty acids, glucose, lactate, amino acids, and Krebs cycle intermediates together provides more than sufficient energy for renal sodium reabsorption. Since the discovery of Na,K-ATPase by Skou,46 the relevance and importance of this enzyme have been explored extensively by countless investigators. While a link between sodium transport across the membrane, and activity of N a,K-ATPase has been well established in a variety of situations, it is still uncertain whether the so-called N a,K-ATPaselinked mechanism is solely responsible for total energy supply in gross sodium reabsorption as well as in fine adjustment of sodium reabsorption. It seems unlikely that processes of sodium reabsorption, which are located at different levels in the nephron, subserve different physiological requirements for the organisms, and are impaired by different inhibitors that act at different loci, could be regulated by a single enzyme-catalyzed step.33 Whittenbury50 proposed a "two pumps theory" to encompass the disparity which is caused by a single Na,K-ATPase theory. It is possible that indeed not only two, but many more "pumps" are linked to internal control which turns energy production on and off according to the need of sodium reabsorption and availability of energy. The network of these pumps must be so integrated that the information from proximal and distal nephrons is transmitted back and forth to compensate each other's function in order to adjust for continually changing internal and external milieu. The regulatory mechanism of glycolysis may serve one of these functions. Glucose metabolism also seems involved in homeostasis of intracellular acid-base balance. "Research ASSOciate, Department of Pediatrics, Stanford University School of Medicine, Stanford, California

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SPECIFIC SUBSTRATE METABOLISM AND SODIUM REABSORPTION Not all substrates taken up by renal tubular cells are completely oxidized. To what extent these substrates generate energy accountable for sodium reabsorption is not certain. Fatty Acids Earlier study indicated that oxidation of fatty acids appears to be an important energy source for the intact kidney35 and in vitro, particularly in renal cortex. 31. 48 N ieth and Schollmeyer35 studied renal metabolite uptake by 41 patients with various kidney diseases. The rate of uptake of the substrates was related to oxygen consumption; free fatty acids accounted for 59 per cent of the total oxygen uptake, followed by lactate 35 per cent. They concluded that the major portion of chemical energy is derived from the oxidation of free fatty acids and lactate. Lee et al. 32 observed in rabbit kidney cortex slices that the rate of uptake of albumin-bound palmitate1_14C was 4.6 ILmoles/100 gm per minute, whereas oxidation of palmitate occurred at a rate ofl. 7 ILmoles/100 gm per minute, accounting for 37per cent of total palmitate disappearance from the incubation media. Trimble and Bowman47 demonstrated that in the perfused rat kidney the maximal sodium reabsorption occurs only when glucose is present in the perfusate. When palmitate alone is present in the perfusate, as a metabolizable substrate, sodium reabsorption is greater than with no exogenous substrate present, but less than with glucose alone present. Addition of a-bromopalmitate, a known inhibitor of fatty acid oxidation, to the perfusate produced a significant decrease in sodium reabsorption both in the presence and absence of glucose with no change in tissue adenosine triphosphate levels. This indicates that at least a portion ofthe energy for sodium reabsorption is provided by oxidation of endogenous fatty acids. Ross et al. 42 demonstrated in the perfused rat kidney that endogenous fuels support 24.7 per cent of sodium reabsorption; fatty acid (oleate) is capable of 34.7 per cent when added as the only substrate. However, Cohen and Barac-Nietol l later demonstrated that the major fraction of palmitate-14 C extracted in vivo by the dog kidney was incorporated into either ketone bodies or triglycerides and only 5 to 10 per cent was completely oxidized to 14C0 2 • Park et al. 38 demonstrated that plasma fatty acids (palmitate, stearate, and oleate), contributed only about 6 per cent of total renal carbon dioxide production, and concluded that palmitate is not the sole free fatty acid of plasma which is metabolized by the kidney, nor are plasma free acids the major source of energy of the kidney. They state that plasma fatty acids are not used preferentially or exclusively to energize tubular reabsorption of sodium. Thus controversy remains as to the extent of fatty acid contribution in renal sodium reabsorption.

Lactic Acid Lactate is utilized by both cortical and medullary regions. Its use in cortex is influenced by other substrates, particularly free fatty acids. Krebs et al. 30 and Weidenmann and Krebs 48 have reported the metabolic fate oflactate in kidney cortical slices quantitatively. When lactate alone

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is present in the incubation media, approximately 50 per cent of it is oxidized to carbon dioxide and the rest metabolized to glucose, pyruvate, and glutamate. On the other hand, in the presence of fatty acids (acetoacetate), all carbon dioxide produced is accounted for by only acetoacetate, and lactate is converted to glucose quantitatively. The metabolism of lactate is more complicated in vivo. Lactate is utilized by cortex and medulla simultaneously. Unless the metabolic fate oflactate in each region in situ is known, the exact contribution of lactate metabolism to sodium reabsorption is not clearly delineated. Dies et al. 14 observed an inverse relationship between renal uptake of free fatty acids and lactate, although the relation was not proportional. Renal uptake of lactate is directly proportional to the arterial plasma concentration of lactate and there is a significant correlation between renal lactate uptake and tubular sodium reabsorption. 14 They suggested that the energy provision of lactate to sodium reabsorption may not be solely by the energy derived from oxidative phosphorylation in the mitochondria, but also by energy derived from the direct lactic dehydrogenase reaction in which NADH + H+ is generated stoichiometrically in the cytoplasmic milieu. Brand et al. 10 suggested that lactate oxidation supports that moiety of tubular sodium reabsorption which persists at high ureteral pressure. Cohen 12 suggested that lactate oxidation is supporting primarily proximal tubular sodium reabsorption. Glucose In a classic study, Gamble et al. 16 demonstrated loss of urinary sodium and potassium during fasting, and retention of sodium and potassium with carbohydrate refeeding. Ross et al. 42 demonstrated that glucose infusion to the isolated rat kidney increased the percentage sodium reabsorption from 77 to 98 per cent of the filtered load, and concluded that glucose is a better oxidizable substrate than fatty acid in the perfused rat kidney as compared with kidney cortex slices. In their calculation, they estimated that 75 per cent of sodium reabsorption can be supported by glucose when glucose is only an added substrate to the perfusate. On the other hand Garza-Quintero et alP observed in the dog experiment that: (1) at normal or at slightly elevated blood glucose concentrations there is a relatively low but constant rate of glucose oxidation by the kidney; the amount of glucose oxidized accounts for approximately 13 per cent ofthe total renal carbon dioxide production; and (2) there is a lack of correlation between glucose oxidation and decreases in sodium reabsorption which are produced by increased ureteral pressure. In vitro, the rate of uptake of glucose by medullary slices is greater than by cortical slices, although oxidation of glucose to carbon dioxide is much greater in cortical slices. ~o Both aerobic and anaerobic metabolism of glucose by outer medulla and inner medulla also can contribute energy to support renal sodium reabsorption. Kean et al. 27 considered anaerobic glycolysis as the major source of energy for medullary sodium reabsorption. Bernanke and Epstein 8 observed that the rate of glucose oxidation in medulla is the same irrespective of paz used in the incubation media. They estimated that about two thirds of the energy for medullary work is provided by aerobic glycolysis.

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Effect of Metabolic Inhibitor on Sodium Reabsorption Inhibition of renal glycolysis by strong natriuretic agents such as ethacrynic acid and furosemide 28 , 51 seems to indicate that the portion of tubular sodium reabsorption supported by glycolysis is labile and subjected to various metabolic influences. Yoshida and Metcoff51 reported that in the rat kidney there is a marked increase in the content of glycolytic intermediary metabolites above triosephosphate and a sharp decline below triosephosphate, two hours after a single intravenous injection of furosemide. This sharp cross-over indicates an inhibition of the reaction catalyzed by glyceroaldehyde-3-phosphate dehydrogenase (GAPDH). Indeed, the GAPDH activity ofthe rat kidney homogenate was inhibited by furosemide noncompetitively with respect to NAD+. Klahr et al. 28 also observed similar effects in a cell-free preparation of rat and rabbit by furosemide and ethacrynic acid. Bowman et al. 9 claimed that inhibition of glycolysis is not the cause of natriuresis by furosemide and ethacrynic acid since iodoacetic acid, which is a specific inhibitorofGAPDH, does not produce significant natriuresis. However, a recent report by Ross et al. 43 indicates that iodoacetamide inhibits residual sodium reabsorption which is not touched by excess ouabain in the perfusing media to the rat. They support the existence of a second pump for tubular sodium reabsorption, which requires metabolic energy but is not dependent on Na,K-ATPase. Earlier, Fujimoto et al.1 5 studied the effect of three metabolic inhibitors, cyanide, coenzyme Q, and dinitrophenol, on renal sodium reabsorption and oxygen consumption in an anesthetized dog. Cyanide decreased both sodium reabsorption and oxygen consumption. Coenzyme Q reduced sodium reabsorption but had no consistent effect on oxygen uptake. Dinitrophenol increased oxygen uptake (uncoupling of oxidative phosphorylation) without affecting the percentage of sodium reabsorption. From these results they concluded that energy for sodium movement may come directly from oxidative metabolism bypassing synthesis or breakdown of adenosine triphosphate. When oxygen uptake is increased by uncoupling agents, there should be no Pasteur effect because the requirement for inorganic phosphate to generate adenosine triphosphate from adenosine diphosphate is dissociated. Increased oxygen uptake under the influence of uncouplers creates a sink to oxidizable substrates particularly to lactate and pyruvate, and in turn whole glycolytic intermediates. Thus dinitrophenol can stimulate glycolysis greatly. Under this condition increased glycolysis may provide sufficient energy either by substrate level phosphorylation and/or generation ofreductive power (NADH) sufficient to maintain normal sodium reabsorption. Jones et al. 24 observed that microsomal membrane preparation of the rat kidney stimulates glycolytic activity more than 50 per cent. The stimulation seems to be due to adenosine triphosphatase activity of the membrane interacting with adenosine triphosphate associated with 3-phosphoglycerate kinase. The membrane preparation is shown to oxidize NADH generated by glycolysis. They interpret such interaction of cytoplasmic metabolism (glycolysis) and endoplasmic membrane-related enzymes to be a channeling of metabolic energy to the membrane for active transport. In the author's experience, the activity of NADH oxidase in the microsomal preparation of the rat kidney cortex is 41 JLmoles/gm protein/min.

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This is equivalent to 60 fLmole oxygen/gm wet weight/hour, constituting approximately 10 per cent of total oxygen uptake. Furosemide 10- 4 M inhibited about 32 per cent ofNADH oxidase activity (~ 19 fLmoles oxygen equivalents), whereas ouabain 10- 4 M did not affect it. In a rat, furosemide injection (2 mg/1 00 gm) caused a net sodium excretion of 500 fLEq/gm wet weight kidney in 2 hours, which is equivalent to a decrease of 17 fLmoles oxygen uptake by the kidney. Thus, although the energy derived from direct oxidation of NADH may share only a small portion of energy required for total renal sodium reabsorption, inhibition ofthe activity of this enzyme may be sufficiently accountable for the net change of sodium reabsorption caused by furosemide. It is conceivable that the major energy required for sodium reabsorption is supplied by hydrolysis of adenosine triphosphate which is generated in mitochondria via oxidative phosphorylation; a minor portion of energy may be supplied directly through a mechanism(s) which bypasses synthesis or hydrolysis of adenosine triphosphate. Total source of energy should be more than enough for sodium reabsorption, if they all are utilized for the work of sodium transport. Apparently some of the excess energy is used for other renal functions such as transport of other substances, detoxication, synthesis of glucose, protein, nucleic acids, and cell constituents. In renal tissue, the activity ofN a,K-ATPase may be so vastly excessive that unless a large portion of the enzyme activity or enzyme itself is decreased, no effect on hydrolysis of adenosine triphosphate and sodium reabsorption may be discerned. A common effect of glycolysis, fatty acid oxidation, and lactate oxidation is the generation of N ADH in cytoplasmic milieu and in mitochondrial compartment. Under normal conditions, mitochondrial oxidation ofNADH is coupled with phosphorylation of adenosine diphosphate to adenosine triphosphate along the electron transport chain. Oxidation of fatty acids generates in the mitochondria the large excess ofNADH, some equivalents of which are transferred out to the cytoplasmic compartment. This NADH, together with that generated by oxidation of lactate, can be utilized for gluconeogenesis. When and where glycolysis is prevailing, this NADH is not utilized for gluconeogenesis and may be channeled away for energizing sodium reabsorption.

AMMONIAGENESIS AND ACID-BASE BALANCE Formation and excretion of ammonia in maintaining normal acidbase homeostasis in body is one of the major metabolic functions of the kidney. Urinary ammonia is derived from deamidation of glutamine. Ammonia excretion into urine is facilitated by "nonionic diffusion" mechanism. 39 Deamidation of Glutamine Deamidation of glutamine is catalyzed by glutaminase I, glutamineketoacid-transaminase-w-lmlidase (so-called glutaminase 11)39 and glutamine-y-glutamyltransferase. 49 Glutamine I is distributed in the renal mitochondria and the other two in the cytoplasmic compartment. Of these, glutaminase I is the most important enzyme in relation to acidosis-

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induced ammoniagenesis. Glutaminase I has two isoenzymes: phosphate-dependent glutaminase (PDG) and phosphate-independent glutaminase (PIG). Under normal acid-base conditions, activity ofPDG is low in the proximal tubules and high in the distal tubules;13 the reverse is true with PID. However in the acidotic rat kidney, there is a marked elevation of PDG activity limited only to the proximal convolution. The activity of PDG in distal nephrons is not elevated, nor is any increase of PID activity seen in the entire length of the nephron. 13 This is in accord with the observation by Glabman et al. that secretion of ammonia in the proximal nephron is increased in acidosis. IS Adaptive increase of glutaminase I was first demonstrated by Davies and Yudkin. 13a The observation was repeatedly confirmed by a number of investigators. 39 However, the other investigators demonstrated that increased ammonia secretion occurred without increased glutaminase activity in acidotic animals. 39 Goldstein 20 showed that in adult rats administration of actinomycin D blocked the acidosis-induced rise of renal glutaminase activity without significantly altering the response of renal ammonia excretion to acidosis. They also observed that PDG level in the suckling rat kidney is about one third that of the adult kidneY,20 and the response to acidosis is significantly low. However, repeated administrations of the acid load eventually increased actinomycin D to such young suckling rats blocks not only glutaminase I activity but also urinary ammonia excretion. Krebs 3I states that the equilibrium of glutaminase I system favors complete hydrolysis of glutamine. If glutamine is relatively stable in the living cells, the glutaminase must be essentially inhibited under the physiological condition. He postulates that there must be some inhibitor of glutaminase in the renal tissue which becomes less effective in acidosis. Glutamate has been considered the best candidate as glutaminase I inhibitor. 20 Goldstein demonstrated that glutamate inhibits both PDG and PIG. Glutamate concentration in the kidney cells is about 7 mM. Inhibition constant (K i ) for PDG is 1 mM, and for PIG is 20 mM with respect to glutamine concentration. In the presence of 7 mM glutamate, PDG is 90 per cent inactive; PIG is 50 per cent inactive. 41 In metabolic acidosis glutamate content in the kidney decreases sharply. 23 Glutamate is de aminated oxidatively by glutamic dehydrogenase (GDH). glutamate + NAD+ + H 2 0

~

a-ketoglutarate + NADH + H+ + NH4+

In the physiological condition, the GDH system is in equilibrium, and GDH is practically inactive. 31 Therefore in order to move the reaction, a s'tate of disequilibrium must be created by removing either alpha-ketoglurate, NADH, H+, or NH4+.3I Preuss 40 demonstrated an increase in the ratio of NAD+ to NADH + H+, obtained either from the equilibrium constant of GDH system or from direct measurement of NAD+ and NADH + H+ in the rat kidney in acute acidosis. He argues that the ratio of N AD+ to N ADH in intramitochondria plays a regulatory role for cellular glutamate level. 91 However, Hems and Brosnan,23 and Gold-

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stein et al. 19 found that the NAD+ to NADH ratio was unchanged in the kidneys of acidotic rats. It was observed that in rats renal concentration of alpha-ketoglutarate is decreased in acidosis. 1 Balagura et al. 6 have shown that alphaketoglutarate infusion into an acidotic dog suppressed ammonia excretion significantly although the dog became more acidotic. The effect of alpha-ketoglutarate may be twofold: utilization of ammonia for synthesis of glutamate and feedback inhibition of glutaminase I by glutamate synthesized from alpha-ketoglutarate. Accumulating evidence indicates that increased glutaminase I activity and ammonia production in the kidney of the acidotic animal is not due to direct effect oflow pH.39 Lower pH inhibits glutaminase I activity. Because increased ammonia genesis can occur without changing glutaminase activity, some other factor must be operating to enhance ammoniagenesis in the kidney of the acidotic animal. Glutaminase I is localized in the inner membrane or matrix of kidney mitochondria. 13 Availability of glutamine to the site of glutaminase reaction is an important controlling factor for ammonia production. Intact mitochondria of the animals in normal acid-base balance shows poor permeability to glutamine. The permeability of glutamine is markedly increased in kidney mitochondria obtained from acidotic animals. Welbourne 49 proposed that during normal conditions, glutamine is distributed almost exclusively in the cytoplasmic compartment where it is deamidated by an enzyme glutamine glutamyltransferase (EC 2,3,2,1) by the following reaction: 2

GT

glutamine~2HN3

+ a-glutamylglutamate

Glutaminase I in the mitochondrial compartment is practically latent owing to the lack of glutamine. Therefore ammonia produced under physiological condition is attributed to glutamyltransferase. In acidosis, permeability of mitochondrial membrane to glutamine is altered and glutamine readily gets into inner mitochondrial compartments to stimulate glutaminase 1. In this theory, intramitochondrial concentration of glutamate should be far lower than the expected Ki • Therefore low production of ammonia is simply due to a lack of glutamine to stimulate glutaminase I activity rather than to inhibition of high glutamate concentration as claimed by Goldstein20 and Krebs. 31 Simpson45 proposed a specific carrier for glutamine transport in the kidney mitochondria. This carrier transfers glutamine to glutaminase I located in the inner membrane or matrix space and provides a site for regulation of glutamine metabolism and ammonia production. He suggests that increased renal formation from glutamine during metabolic acidosis results from an adaptive increase in transport of glutamine by the inner membrane carrier. In acidosis, alteration of mitochondrial glutamine transport seems to be an important initial factor that leads to subsequent changes in the number of metabolic pathways related to ammoniagenesis. However, the very cause that induces such alterations of permeability or transportcarrier of the mitochondria in acidosis is not known. Certainly low pH itself is not the cause as discussed earlier. Alleyene3 observed that when

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kidney slices from normal rat were incubated at an acid pH, there is a decrease rather than increase in ammonia production. When normal kidney slices are pretreated with plasma from acidotic rats, there is an increase in ammonia production and enhanced glutamine uptake. Thus he argues that there could be a factor which the rat produces in response to acidosis and which can stimulate ammonia production and renal gluconeogenesis. The chemical nature of this factor is not known; it is stable after neutralization and not dialyzable. Gluconeogenesis and Ammoniagenesis Kidney shares with liver in synthesizing glucose from lower carbon precursors. 7 The site of glucose synthesis in the kidney is the cortex, whereas the medulla carries on glycolysis only. In vitro, cortical slices can produce glucose comparable to that of liver per unit weight basis. However there is no significant contribution of kidney for blood glucose level under the normal condition because of the relatively small size of the kidney. On the other hand, after a prolonged fasting, the kidney can provide more than 40 per cent of blood glucose. 37 In the gluconeogenic pathway there are a few enzymatic steps considered to be rate limiting. For individual metabolic steps of the gluconeogenic pathway, readers are referred elsewhere. 33 The most important step is phosphenolpyruvate carboxykinase (PEPCK), whose distribution is mainly in the intramitochondrial compartment in the majority of species. In rats only proximal tubules contain appreciable amounts ofPEPCK activity. Moreover the proximal convolution contained twice the activity of the straight portion of proximal tubules. 21 Schmidt et al. 44 observed that acidosis induced with ammonium chloride in rats resulted in a threefold increase in PEPCK activity which is restricted to proximal tubules only. They suggested that renal gluconeogenesis takes place only in proximal tubules but not in distal tubules. Goodman et al. 21 observed that in acute acidosis induced in rats with ammonium chloride and in potassium deprivation, there was a concomitant increase in glucose production from alpha-ketoglutarate, glutamate, and glutamine, and increased ammonia production from glutamine. No increased glucose production was observed from glycerol or fructose by kidney cortical slices in vitro. They advanced an hypothesis that increased glucose production facilitates removal of intermediary metabolites such as alpha-ketoglutarate and in tum glutamate. Removal of both alphaketoglutarate and glutamate deactivates glutaminase I and thus results in enhanced ammonia production. Alleyene et al. 2 also reported similar observations, and suggested that the fall in HC0 3 - rather than change in pH is primarily an important factor in increased PEPCK activity. Goomo et al. 22 also confirmed the stimulatory effect of ammonium chloride induced acidosis on both gluconeogenesis and ammoniagenesis but failed to demonstrate increased gluconeogenic capacity in kidney cortical slices obtained from the dog with chronic respiratory acidosis who showed increased urinary excretion of ammonia. They suggested that the decrease in intracellular pH stimulated PEPCK activity during metabolic acidosis. Kamm and Strope26 have shown a similar response of PEPCK in acidotic and potassium-depleted rats. Recent evidence indicates that the apparent increase in PEPCK activity in the kidney of acidotic rats is not

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due to activation or new synthesis of the enzyme, but to the result of increased half-life of the enzyme in situ. The mechanism of enzyme protection in acidosis is unknown. PEPCK activity also is stimulated by its substrate oxalacetate. Most of the gluconeogenic substrates are metabolized through oxalacetate. Glucose synthesis from these substrates are all stimulated by calcium ions. 2, 34 The basic mechanism of calcium stimulation of gluconeogenesis is not well understood. Recently Alleyene 2 has shown that calcium stimulates glucose production by renal cortical slices without affecting ammonia production. Under the experimental condition utilization of glutamine and the formation of glutamate are unaffected by calcium. The observation clearly separates gluconeogenesis from ammonia production. Change in intracellular concentration of calcium with alteration of acid-base balance has not been delineated. Calcium is an inhibitor of pyru vic kinase. 25 If appreciable, this inhibition would result in stimulation of gluconeogenesis. Pyruvate kinase has been pointed to as an important modulator of gluconeogenesis by Krebs and Eggleston. 29 They observed an inverse relationship between gluconeogenesis and pyruvic kinase activity. Since potassium is an essential activator for pyruvate kinase,25 when the intracellular level of pot assium is sufficiently decreased, deactivation of pyruvic kinase can be expected. Increased renal gluconeogenesis observed by Goodman et al. 21 in the potassium-depleted animal can thus be construed in terms of deactivation of pyruvic kinase. Under the condition of dietary potassium depletion, plasma pH is alkalotic, but intracellular pH could be lower. Kamm and Strope26 have shown that both in potassium depletion and in acidosis there are increased renal but not hepatic PEPCK activity and decreased potassium content in muscle and kidney. Only diffusible potassium in renal cortex was significantly decreased in both conditions, but diffusible potassium content was not altered in alkalosis. Cohen and Barac-Nietoll attribute the loss of potassium from the kidney to the change in renal glucose metabolism in acidosis. They consider gluconeogenesis to be a regulatory mechanism of intracellular pH which may or may not be related to extracellular pH. Gluconeogenesis as a Mechanism of Energy Conservation It has been demonstrated that urinary excretion of organic acids in man is increased in metabolic acidosis and decreased in metabolic acidosis. 36 On the other hand, Balagura-Baruch and Pitts 4 observed that chronic metabolic acidosis and acute respiratory acidosis increased TNa for alpha-keto glutamic acid. Increased renal uptake and utilization of alpha-glutamic acid in acidosis also have been demonstrated. 5 Increased utilization of alpha-glutamic acid leads to increased titratable acid formation33 ,41 and enhanced gluconeogenesis. Glucose is an uncharged, "non acidic substance,"7 thus glucose synthesis from organic acids (lactic acids, alpha-keto glutamic acid) not only has a direct effect on cellular pH by converting acidic substance to "non acidic substance," but also saves organic acids which would otherwise be wasted by total oxidation or by excretion into urine. In this view increased renal gluconeogenesis in acidosis has a dual role: a regulatory role for intracellular pH and the role of salvaging energy.ll

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SUMMARY Glucose metabolism is related to two major renal functions. Glycolysis may be related to an energy requiring function-fine adjustment of renal sodium absorption. Gluconeogenesis may be related to controlling intracellular pH and salvation of energy otherwise wasted in acidosis. Ammoniagenesis is related to gluconeogenesis in the metabolic acidosis, but the individual phenomena are not necessarily dependent on each other.

REFERENCES 1. Alleyene, G. A. 0.: Concentrations of metabolic intermediates in kidney of rats with metabolic acidosis. Nature, 217:847,1968. 2. Alleyene, G. A. 0., Flores, H., and Roobol, A.: The interrelationship of the concentration of hydrogen ions, bicarbonate ions, carbon dioxide and calcium ions in the regulation of gluconeogenesis in the rat. Biochem. J., 136:445, 1973. 3. Alleyene, G. A. 0., and Roobol, A.: Regulation of renal cortex ammoniagenesis. I. Stimulation of renal cortex ammoniagenesis in vitro by plasma isolated from acutely acidotic rats. J. Clin. Invest., 53: 117, 1974. 4. Balagura-Baruch, S., and Pitts, R. F.: Renal handling of a-ketoglutarate by the dog. Am. J. PhysioL, 207:483, 1964. 5. Balagura-Baruch, S., and Stone, W. J.: Renal tubular secretion of a-ketoglutarate in dog. Am. J. PhysioL, 212:1319, 1967. 6. Balagura-Baruch, S., Shurland, L. M., and Welbourne, T. C.: Effect of a-ketoglutarate on renal ammonia release in the intact dog. Am. J. PhysioL, 218:1070, 1970. 7. Benoy, M. P., and Elliott, K. A.: Metabolism of lactic and pyruvic acids in normal and tumor tissues: Synthesis of carbohydrate. Biochem. J., 31 :1268, 1937. 8. Bernanke, D., and Epstein, F. H.: Metabolism of the renal medulla. Am. J. PhysioL, 208:541, 1965. 9. Bowman, R. H., Dolgin, J., and Coulson, R.: Furosemide, ethacrymic acid, and iodoacetate on function and metabolism in perfused rat kidney. Am. J. PhysioL, 224 :416, 1973. 10. Brand, P. H.o Cohen, J. J., and Bignal, M. C.: Independence oflactate oxidation from net Na+ reabsorption in the dog kidney, in vivo. Am. J. PhysioL, 227:1255, 1974. 11. Cohen, J. J., and Barac-Nieto, M.: Renal metabolism of substrate in relation of renal function. In Orloff, J.o and R. W. Berliner (eds.l: Renal Physiology. Washington, D.C., Am. Physiol. Soc., 1973, pp. 909-1001. , 12. Cohen, J. J.: Metabolic support for renal sodium reabsorption. Med. Clin. North Am., . 59:523, 1975. 13. Curthoys, N. P., and Lowry, O. H.: The distribution of glutaminase isoenzymes in the various structures of the nephron in normal, acidotic and alkalotic kidney. J. BioI. Chern., 248:162, 1973. 13a. Davies, B. M. A., and Yudkin, J.: Studies in biochemical adaptation. The origin of some urinary ammonia as indicated by the effects of chronic acidosis and alkalosis on some renal enzymes in the rat. Biochem. J., 25:407,1952. 14. Dies, F., Ramos, G., Avelar, E., et al.: Relationship between renal substrate uptake and tubular sodium reabsorption in dog. Am. J. PhysioL, 218:411, 1970. 15. Fujimoto, M., Nash, F. D., and Kessler, R. H.: Effects of cyanide, QQ, and dinitrophenol on renal sodium reabsorption and oxygen consumption. Am. J. PhysioL, 206:1327, 1964. 16. Gamble, J. L., Ross, G. S., and Tisdall, F. F.: The metabolism of fixed base during fasting. J. BioI. Chern., 57:633, 1923. 17. Garza-Quintero, R., Cohen, J. J., Brand, P. H., et al.: Steady state glucose oxidation by dog kidney in vivo: Relation to Na+ reabsorption. Am. J. PhysioL, 288:549, 1975. 18. Glabman, S. R., Klose, R. M., and Giebisch, G.: Micropuncture study of ammonia excretion in the rats. Am. J. PhysioL, 205: 127, 1963. 19. Goldstein, L., and Harley-DeWitt, S.: Renal gluconeogenesis and mitochondrial NAD+j NADH ratios in nursing and adult rats. Am. J. PhysioL, 224:752, 1973. 20. Goldstein, L.: Regulation of renal glutamine deamination. Med. Clin. North Am., 59:667, 1975. 21. Goodman, A. D., Fuisz, R. E., and Cahill, G. F., Jr.: Renal gluconeogenesis in acidosis, alkalosis, and potassium deficiency: Its possible role in regulation of renal ammonia production. J. Clin. Invest., 45 :612, 1966. 22. Goorno, W. E., Rector, F. C., and Seldin, D. W.: Relation of renal gluconeogenesis to ammonia production in dog and rat. Am. J. PhysioL, 213:969, 1967.

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