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Kidney International, Vol. 29 (1986), pp. 54—67

Glucose metabolism in renal tubular function

BRIAN D. Ross, JOSEPH ESPINAL, and PATRIcI0 SILVA

Departments of Medicine and Clinical Biochemistry, University of Oxford, Oxford, United Kingdom, and Charles A. Dana Research Institute, Harvard-Thorndike Laboratory of Beth Israel Hospital and Department of Medicine, Harvard Medical School and Beth Israel Hospital, Boston, Massachusetts, USA

Much can be learned of what glucose does in renal tubular Glycogen synthesis (and degradation) have been noted in function from considering the major fuels of respiration in thestructures of the medulla [12, 13]; indirect evidence suggests kidney. Respiration provides most of the ATP required forthat the synthesis of glycogen occurs from blood glucose [14]. energy-dependent transport processes; the small residual pro-However, the rates of such reactions are low and unlikely to portion arises from glycolysis and substrate-level oxidations. Incontribute to metabolic balance [151. vitro studies reveal that, contrary to expectation, glucose is a By far the most important biosynthetic pathway in the kidney rather poor fuel of respiration in kidney cortex; preferred fuelsis that of glucose synthesis from noncarbohydrate precursors, are short- and long-chain fatty acids, endogenous lipids, ketonegluconeogenesis. It is this process, exceeding the capacity of bodies, lactate, and some amino-acids [1, 2]. Each of thesethe apparent enzyme activities of either glycolysis or glucose substrates successfully competes with glucose when substratesoxidation, which obscures the contribution of glucose to renal are offered in combination. In the outer medulla, glucose is arespiration and exaggerates the contribution of lactate. The fuel for respiration [3] but once again other potential fuels, suchdemonstration proving this follows and is from recent experi- as succinate 14] or lactate [5, 6], appear to be preferred. In thements using specific isotopic techniques considered in detail. inner medulla oxygen consumption is much less than in theThe role of renal gluconeogenesis in renal function is also cortex, and glucose is metabolized primarily to lactatetreated in detail. (glycolysis). The preferred fuel of respiration in deep medullary structures has not been identified but may be endogenous lipids, as in the remainder of the kidney [7]. Heterogeneity and compartmentation In the intact in vivo kidney, arterio-venous difference mea- surements again point to fuels other than glucose as the prime Maps of enzyme activity indicate that many enzymes are energy source. In humans, Neith and Schollmeyer [8] assesseddistributed unevenly along the nephron; for example, some lactate > free fatty acids > citrate> pyruvate in the order ofsegments showed high specific activity while in others, little or renal extraction without a significant arterio-venous differenceno activity is found [14, 16]. Relevant to the present discussion established for glucose. are the enzymes of glycolysis, hexokinase, phosphofructo- In marked contrast to this evidence is the now often repeatedkinase, and pyruvate kinase. All of these predominate in late demonstration that renal tubular sodium transport is betterstructures of the nephron, that is, medullary ascending limb, supported by glucose than by other respiratory fuels providedcortical ascending limb, distal convoluted tubules, and in the to the intact perfused kidney [9, 10]. This review discussesentire collecting duct. Significant activity of these three evidence that glucose is not only crucial in electrolyte trans-glycolytic enzymes is present, however, in all nephron struc- port, but, indeed is, a key respiratory fuel in selected structurestures, so that a low rate of glycolysis, or some degree of along the nephron. substrate cycling is feasible in many nephron structures. Mito- chondrial density and the enzymes of the TCA cycle are Biosynthetic processes in the kidney particularly high in the medullary thick ascending limb, and In part, the anomalies which arise from interpreting arterio-particularly low in the thin descending limb of Henle's loop as venous difference measurements in the kidney are resolved bywell as the medullary collecting tubule [17]. Hence, mitochon- considering key biosynthetic processes which also occur indna! respiration is exceptionally low in the inner medulla. A key kidney tubule cells. Triglyceride synthesis occurs fromenzyme in this discussion, pyruvate dehydrogenase, which exogenous fatty-acids and from glycerol, itself derived fromprovides the link between glycolysis and glucose oxidation has exogenous glucose or lactate [11]. Arterio-venous differencesnot been mapped. may therefore exaggerate the contribution of fatty acids to Gluconeogenesis, earlier recognized as a renal cortical func- respiration. tion [18], is shown to be confined even more precisely to the proximal tubule, because glucose 6-phosphatase, fructose 1,6- diphosphatase, and phosphoenolpyruvate carboxykinase (PEPCK) are virtually limited to these structures. The remain- Received for publication June 12, 1985 ing important cortical segments, the glomeruli [19], and the © 1986 by the International Society of Nephrology distal convoluted tubules [20] are predominantly glycolytic.

54 Glucose metabolism in renal tubular function 55 2Glucose i Glucose-6-phosphate 1 V Fructose-6-phosphate 5 Fructose-i ,6biphosphate

3-Phosphoglyceraldehyde . Dihydroxyacetone phosphate 8\ 1 3-Diphosphoglycerate

1 V 3-Phosphoglycerate I Phosphoenolpyruvate

Oxaloacetate Pyruvate/_Lactate

Mitochondria

TCA Cycle 2-Oxoglutarate / Fig. 1. Pathway of glycolysis.

Finally, and probably of overriding importance, the measuredContributionby the kidney to glucose homeostasis in the whole activity of enzymes greatly exceeds any physiologically deter- organism mined rate of metabolism in the kidney. This indicates an important modifying influence of metabolic regulation of en- It has been considered that renal glucose synthesis must zymes by allosteric and other modulators in the intact nephron.somehow supplement hepatic gluconeogenesis in maintaining Therefore, this review concentrates where possible on resultsblood glucose [27, 28]. However, direct evidence for this has obtained in the intact, functioning kidney. been difficult to obtain. When more information about the bi-directional flux of glucose metabolism in the kidney becomes available, it should be possible to assess more accurately what Renal tubular function and carbohydrate metabolism the net contribution of glucose to homeostasis may be. Therefore, this review deals with glycolysis, lactate oxida- Glucose, sodium cotransport has been well documented intion, glucose oxidation, futile cycles in glucose utilization, the kidney [211, but appears to be quite independent of renalgluconeogenesis, and analysis of the role of each process in metabolism of glucose [9, 211. Available data suggest thatrenal tubular transport function. glucose metabolism is involved intimately in potassium secre- tion [22], bicarbonate transport [23], hydrogen ion secretion, Glycolysis [24] and in active sodium transport [9] and maintenance of The utilization of glucose can be divided into three pathways: glomerular filtration rate [25]andfree-water clearance [26].glycolysis, oxidation, and the pentose phosphate shunt. Nevertheless, from the heterogeneity of transport functionsGlycolysis is defined as the formation of lactate from glucose or along the nephron, the details of any relationship betweenglycogen. The pathway (Fig. 1) consists of 11 enzymes, all of transport events and glucose metabolism will be difficult towhich are located in the cytoplasm of the cell. In the kidney, establish by direct examination of the whole or fragmentedglycolysis occurs primarily from glucose because glycogen kidney. stores are minimal [12]. Glycolysis could thus occur in all renal 56 Ross ci a!

COOR COOH COOH

"CH3 CH3 CH2 CH2 CH I I I HC-OH__C=O__C=O__HC-OH__CH

*COOH COOH COOH COOH COOH

Lactate Pyruvate Oxaloacetate Malate Fumarase randomization

COOH COOH COOH

CH3 CH3 CH2 CH2 CH

HC-OH i C=O H'C-OH 4 *CH

I I I I COOH COOH COOH COOH COOH Fig. 2. Pathway for lactate production via Phosphoenolpyruvate oxaloacet ate. cells, whether or not they contain mitochondria. Those cellsthelial transport is no longer present. Only an intact kidney which contain mitochondria may then further convert lactate orpreparation can offer such an answer. pyruvate to C02, completing the oxidation of glucose (see The use of the isolated perfused kidney preparation [34] below). allows delivery of the products of metabolism of one renal area Sites. Mapping glycolytic enzymes has been reviewed into any other by the recirculation of perfusion medium. Meta- detail [14, 16]. Briefly, the activities of the key regulatorybolic glucose rates determined in this preparation are therefore enzymes, hexokinase, phosphofructokinase, and pyruvatethe result of simultaneous and synchronous gluconeogenesis kinase, suggest that the main site of lactate production fromand glycolysis. The rates of both pathways will be underesti- glucose is the inner medulla and papilla. Extra lactate has beenmated seriously if only measurements of the concentrations of detected in the blood draining from the papilla in vivo [29]. Thesubstrates and products are made. Improved methods to study role of lactate produced by the medulla is unclear because,glucose metabolism in the functioning kidney involve radioiso- given the anatomical distribution of blood vessels in the kidneytope dilution. These methods exploit the recirculation of sub- [30], it cannot directly provide a substrate for gluconeogenesis.strates and products in the perfusion system so that changes in This question will be addressed below. the specific radioactivities of lactate or glucose can reflect the Pentose phosphate shunt. Location of the pentose phosphaterates of glycolysis, lactate consumption, and gluconeogenesis shunt can be defined by the distribution of the first enzyme inindependently, Using [U-14C]-lactate and [6-3H]-glucose in sep- the pathway, glucose 6-phosphate dehydrogenase (G6PD). Thisarate experiments, lactate production from glucose, that is, enzyme is universal throughout the nephron with relatively highglycolysis, can be calculated from the dilution of [U-'4C]- activity in the glomeruli and the proximal straight tubule. In thelactate. The method has been applied to cortical tubules [15] latter, where G6PD is maximal, phosphofructokinase (PFK)and the perfused rat kidney [35]. The use of [6-3H]-glucose activity is twice that of G6PD. This may indicate the relativeallows calculation of the rate of gluconeogenesis and/or glucose proportion of glucose carbon that traverses this particularrecycling [36, 37] (see below). At this point, it should be segment. In the glomeruli, G6PD and PFK activities are similar.emphasized that lactate may arise by processes other than Using the standard assay for pentose phosphate shunt activity,glycolysis, including recycling of carbon as shown in Figure 2. viz difference between C1—C6 labeled glucose consumption, To determine the rate of lactate production, a novel method two groups have established the existence of pentose phosphatehas been applied, originally presented by Janssens, Hems, and shunt activity in tissue slices [31] and nephron segments [21.Ross [151 for estimating lactate recycling in kidney tubules. The The rates obtained by these two groups are similar and consti-method involves incubation or perfusion with 2 m lactate plus tute only 10% of glucose utilization. In the absence of renal[U-'4C] lactate in the presence or absence of 5 m glucose. fatty acid synthesis, the role of the pentose phosphate shuntIsolated kidneys were perfused with 100 ml modified Krebs- (primarily in producing NADPH) in the kidney is unclear. Henseleit buffer pH 7.4, containing 6.7% (w/v) of bovine serum Measurement of glycolysis. A variety of in vitro techniquesalbumin, 5 m glucose, 2 m lactate, and [U-'4C] lactate to have been used that exclude the heterogeneity of the cortex andgive an initial specific radioactivity of approximately 100,000 medulla by using slices or tubules of either site. Moreover,dpm/tmole. The specific radioactivity of lactate will fall in techniques have been devised that allow separation of tubularproportion to the amount of unlabelled lactate that is being fragments along the nephron and within a single area [32, 33].produced from glucose, endogenous sources, or by lactate However, when considering the relationship between the met-"cycling" during incubation or perfusion. Since both labelled abolic and the transport function of the kidney, isolated celland unlabelled lactate will be removed continuously at the same preparations can offer only limited information since transepi-time as they are being formed (the result of glucose synthesis Glucose metabolism in renal tubular function 57

Table1. Rates of lactate productiona in kidney

Calculated glycolytic rate Cortical Perfused Diet Substrate tubules kidney Cortical Medullary Total Fed 2 msi Lactate + 5 146 26 176 24 mglucose 25 139 164 2 m Lactate 121 22 12 3

Starved (48 hr) 2 m Lactate + 5mtt — 172 39 glucose 0 167 167 2 m Lactate 15 15 <5 a Lactateproduction was calculated by the isotopic dilution method using [U-"C]-lactate, as described in the text. The data are compiled from [15,35].The rates are presented in tmoles/hr/g dry wt.

— and lactate oxidation to C02), a mathematical treatment isat time 0, Yo =c1and c2 = (8) requiredto calculate the true rate of production. Janssens, A Hems, and Ross [15]showedthat, in the absence of glucose, Yt =yoe_AY (9) lactate continued to be formed, probably owing to lactate recycling. z =.(1—e_At) (10) Therate of lactate production (that is, the rate of entry of A unlabelled lactate into the labelled lactate pool) may be calcu-Making t, the unit of time, =1: lated from Eqs. (1)and(2)below,knowing the initial (Yo) and final (Yt) amount of radioactive lactate present and the final y =yoe_A;z = (1—e) (11) concentration of unlabelled lactate (Zt): A Substituting: A = Yo in— (1) Yo Yt A =in— (1) Yt — Zt = 1 (2) — Zt = 1 (2) A Yo A Yo whereB is the rate of formation of unlabelled lactate, A is the Corticalglycolysis. Inisolated cortical tubules, lactate is rate constant for the removal of lactate from the pool, Yo 15produced at a rate of 121 22 moles/hr/g dry weight in the lactate (micromole) at initial specific activity, Yt is lactateabsence of added glucose, but surprisingly this rate does not (micromole) at the same specific radioactivity at time t, and Zt increase with the addition of glucose [15].Thus,in fed rats at is the concentration of unlabelled lactate present at the end ofany rate, glycolysis is effectively absent in the cortex, When the incubation. Thus: fasted the rate of lactate production in the rats in the absence of glucose falls near zero. It is suggested that such lactate produc- Zt =Xt—Yt ('3) tionobserved is due to recycling (see Futile cycles involved in where Xt =measuredlactate at time t (Xo =measuredlactaterenal glucose utilization below). With application of these data at time 0). Eqs. (1)and(2)havebeen derived as follows: Let c1to the perfused kidney, therefore, the technique may be used to and c2 be constants and d and d decrements in the amount ofdetermine the rate of medullary glycolysis. labelled and unlabelled lactate respectively in a unit of time, d. Medullaryglycolysis. Lactateproduction was almost zero in Assuming that all newly formed lactate mixes completely andthe absence of glucose, rising to 176 moles/hr/g wet weight rapidly with the pool, and that the rate of lactate removal (A) Swhen physiological concentrations of glucose were present. proportional to its concentration: This value permits calculation of cortical glycolysis and is between 12 3 (the value obtained in glucose-free perfusion) and =— zero (the difference observed in cortical tubules in the d A (4) presence and absence of glucose). On the other hand, a high rate of medullary glycolysis of 139 tmoles/hr/g total kidney can and: be calculated (Table 1) (that is, lactate produced in the presence of glucose, less that in its absence (—12), together with the = — B A (5) maximumvalue for glycolysis observed in cortical tubules [38]). d1 Expressed as the rate/g of medulla (one-third of the total kidney then: weight [17]), lactate production is 417 moles/hr/g dry wt. This -At is remarkably close to the glycolytic rate determined in medul- =c1e (6) lary tissues incubated directly (380 imoles/hr/g dry weight; see B [36] for an identical result obtained by a different isotopic =— +c2e_At (7)technique in the perfused kidney). A 58 Ross et a!

Table 2.Effects of variations in sodium transport on glycolysis and They suggested that '4CO, formed in one turn of the TCA cycle glucoseutilizationa derives from oxaloacetate, therefore, there is a "crossingover" Lactate Glucose atoxaloacetate, between glucose oxidation and glucose forma- Nafiltered load production utilization tion. Consequently, a considerable dilution of label may occur, uEqlmin/g p.rnoleslhrlgpmo!esIhrIg permitting unreliable estimates of the rate of glucose utilization. Diet wet wt dry wt dry wt Another problem with measurement of glucose oxidation by Fed 91±8 176±24 56±4 this technique in the perfused kidney is an underestimate of Starved 172 39 — radiolabel released as a result of poor trapping methods [41]. Fed and furosemide 77 8 98 14* 37 ÷4* These problems inspired Espinal, Bartlett, and Ross [36] to use Starved and [6-3H]-glucose. Tritium from [6-3H]-glucose is retained in furosemide 108 18* Fed, nonfiltering 0 — — pyruvate and enters the TCA cycle. Detritiation occurs at Starved, nonfiltering 0 87 10* pyruvate carboxylase and a-ketoglutarate transaminase. Thus, Glycolytic rate is presented as the rate of lactate production3H20 release from [6-3H]-glucose provides a reliable estimate of obtained by the isotopic dilution method as described in the text.the rate of glucose oxidation [42, 43]. Morever, use of this label Glucose utilization is obtained by the release of 3H20 from 6-3H- can also provide a method of measurement of glucose recycling glucose. The data are compiled from [351.Furosemidewas present at a when used in the intact perfused kidney [36]. concentration of 0.1 m. Statistical significance is indicated by *< Glucoseoxidation rates in relation torenal function. To our 0.01. knowledge, identification of the major sites of glucose oxidation in the kidney is largely the work of two laboratories. Using isolated single nephron segments, Klein et al [20] showed that Relationship between glycolysis and renal function. Whilesignificant rates of glucose oxidation occurred in both the the relationship between electrolyte transport functions andcortical and medullary thick ascending limbs, but little occurred gluconeogenesis is the subject of considerable attention (seein the proximal convoluted tubule. Similarly, Vinay, Gougoux, below), little is known of the relationship with glycolysis. Thisand Lemieux [20] showed that distal convoluted tubules oxi- study has been attempted by Bartlett et al [35]. In theirdized glucose at a rate three times greater than the proximal experiments, urine flow, GFR, and sodium reabsorption weretubules. It can be calculated from the work of Vinay, Gougoux, measured in parallel with lactate production in the functioningand Lemieux [20] that cortical tubules as a whole oxidized kidney. A significant correlation between lactate productionglucose at a rate of approximately 28 moles/hr/g dry weight. In and urine flow rate and GFR was found, There was also acontrast, Espinal, Bartlett, and Ross used [37] isolated cortical significant and inverse relationship between lactate productiontubules and demonstrated a rate of 3H20 production of 74.5 and fractional sodium excretion and a direct relationship be-1moles/hr/g dry weight. However, their studies with inhibitors tween lactate production and total sodium reabsorption (TNa)led them to suggest that only 35% of this rate can actually be [35].In the nonfiltering kidney, lactate production in the pres-ascribed to oxidation of glucose (as opposed to recycling), that ence of 5 m glucose and 2 m lactate was only half that in theis, approximately 26 imoles/hr/g dry wt—a remarkable agree- filtering kidney. Furosemide (l0- M) inhibited sodium reab-ment with the work of Vinay, Gougoux, and Lemieux [20]. sorption and lactate production (Table 2). On the other hand, The rate of glucose oxidation in medullary tubules has also they found no correlation of these two indices of renal functionbeen studied. Klein et al [2] obtained a figure of approximately with lactate consumption. These results suggest that glycolysis100 moles/hr/g dry wt (calculated from their data assuming 100 rather than lactate oxidation [39] is required for some aspect ofng dry wt/mm of nephron segment [14]), while Espinal, Bartlett, renal tubular function, probably for sodium reabsorption. and Ross [37] calculate a rate of 180 moles/hr/g dry weight. In marked contrast is the earlier work in medullary slices from Glucose oxidation rabbit kidney which obtained rates of 10 moles/hr/g dry wt [4, Definition and sites.Glucoseoxidation refers to the produc-44]. tion of CO2 from glucose by the TCA cycle. The link with Fromthe data of Espinal, Bartlett, and Ross [37] and assum- glycolysis is provided by the mitochondrial enzyme, pyruvateing a ratio of cortex to medulla of 3:1 [17], it can be calculated dehydrogenase. Therefore, glucose oxidation in the kidney canthat the expected rate of glucose oxidation by the intact kidney only occur in those areas rich in mitochondria and with anwould be around 80 to 90 /Lmoles/hr/g dry wt. This value is very active pyruvate dehydrogenase. Glucose oxidation is known toclose to their experimental result in the perfused kidney of 74 occur in the glomeruli, outer medulla, and the distal convoluted/Lmoles/hr/g dry wt [361. tubule. Unfortunately, pyruvate dehydrogenase has not been Starvationresults in decreased rates of glucose oxidation in mapped, and therefore, it is not known whether there is bothcortical and medullary tubules [37]. This reduction could capacity for oxidation in the other parts of the nephron. be due to a decreased glycolytic flux [15] or a decreased activity Release of CO2 from [U-'4C]-glucose is thought to provide aof pyruvate dehydrogenase [1, 45, 46]. reliable estimate of the rate of glucose oxidation in any tissue. Espinal, Bartlett, and Ross [36, 37] tested the effect of two This method has been used in isolated nephron segments [2],classes of diuretics on the rates of glucose oxidation on both tissue slices [4], and tubules [201. Despite the alleged reliabilitycortical and medullary tubules and in the perfused kidney of this method, the use of 14C02 measurements has been(Table2). Furosemide had no effect on glucose oxidation in the criticized by Krebs et al [40] as long ago as 1966. The work of cortexbut significantly inhibited it in the medulla. Amiloride Krebs et al [40] is worth noting here because they showed thehad no effect on either cortex or medulla. When the diuretics limitations involved in the use of single label measurements.wereusedtogether, inhibition was in both areas. In perfused rat Glucose metabolism in renal tubular function 59 kidneys, furosemide inhibited 3H20 production by 30% and Effects of modifying electrolyte transport on lactate oxida- amiloride by about 50%, an effect which was significantlytion. Cohen and Barac-Nieto [50] summarize the early evidence greater than that of furosemide. The data clearly indicate a rolethat lactate oxidation parallels net sodium reabsorption by the of glucose oxidation in some transport process which is dis-intact kidney and might therefore be a major fuel of renal rupted by slicing the kidney. Potassium transport is a likelyrespiration in vivo. Neith and Schollmeyer [8] drew the same candidate [221. conclusion from their arteriovenous difference measurements in Control of glucose oxidation: pyruvate dehydrogenase. Anyhumans; however, in neither case is proof of lactate oxidation discussion on the control of glucose oxidation in the kidneyavailable. In the perfused kidney, lactate appears to stimulate must center on the regulation of pyruvate dehydrogenase,respiration [27] and to substitute in part for glucose in support- because this enzyme controls the rate of entry of glucoseing sodium transport [25, 39, 51]. Lactate (and pyruvate) carbon (as pyruvate) into the TCA cycle. Pyruvate dehydrog-support Pi reabsorption by the kidney [51]. A role of cytoplas- enase is a mitochondrial multi-enzyme complex whichmic NADH in the regulation of phosphate transport remains catalyzes the oxidative decarboxylation of pyruvate. The com-controversial [52]. In the presence of physiological concentra- plex is regulated by reversible phosphorylation which results intions of glucose, lactate consumption measured by isotopic active (dephosphorylated) and inactive (phosphorylated) forms.methods which fail to distinguish between oxidation and con- These interconversions are catalyzed by specific kinase andversion to glucose [31] was not related to GFR nor affected by phosphatase reactions [47]. Although much is known about thefurosemide or amiloride. Overall, it seems improbable that regulation of pyruvate dehydrogenase in the heart, limitedlactate oxidations per se play any specific part in the tubular information is available on the activities in the kidney. Laceyevents. and Randle [461 showed 50% of the enzyme is in its active form. Even less is known of any relationship between pyruvate Futile cycles involved in renal glucose utilization dehydrogenase and renal function. Also, an inverse relationship It is probable that in the kidney, as in other tissues, futile has been shown between sodium reabsorption and pyruvatecycles occur, and that they are involved in the regulation of dehydrogenase activity (Ross, Stukowski, and Guder, unpub-metabolism [53]. A futile cycle [43] is defined as: 'If two lished observations). If this observation is coupled to theopposite nonequilibrium reactions operated simultaneously and relationship existing between glycolysis and sodium reabsorp-at the same rate, there is no flux of metabolites, but a 'futile' tion (see above), it may be suggested that glycolysis appearsrecycling, the net balance of which appears to be wasteful relevant to medullary sodium transport and possibly free-waterexpenditure of energy.' clearance. On the other hand, glucose oxidation may play a part Newsholme and Underwood [53] obtained evidence for the in distal convoluted tubule sodium transport, as well as inexistence of the fructose 6-phosphate/fructose 1 ,6-diphosphate supporting potassium excretion in the collecting duct [481. cycle in kidney cortex slices. Rognstad [54] and Janssens, Hems, and Ross [15] suggested that pyruvate was recycled in Lactate oxidation by the kidney cortical tubules, by a cycle between pyruvate/oxaloacetate, Definition and cell site(s) of lactate oxidation. Lactate oxi-rather than the conventional pyruvate/phosphoenolpyruvate dation, by conversion to pyruvate in the cytosol, and the furthercycle. Other cycles are known, and reviewed by Hue [43]. In metabolism within the mitochondria of pyruvate to CO2 andthis section we discussed the evidence for their existence in the water is analogous to the complete oxidation of glucose. How-pathways of glucose metabolism in the kidney and assess their ever, it differs in some details: Cytoplasmic NADH is generatedquantitative significance. in large amounts by lactic dehydrogenase (LDH), requiring Theoretically, the role of futile cycles in regulation may be either gluconeogenesis or the intervention of the malate:aspar-thermogenesis in amplification of allosteric control of metabolic tate shuttle for its disposal. Because of the almost universalflow, determining the orientation of flux at a metabolic cross occurrence of LDH, lactate can compete successfully withroads, or controlling the concentration of some key metabolites glucose as a respiratory fuel. High rates of lactate oxidation by(to review futile cycling, see [55]). Finally, compartmentation of kidney indeed have been reported, and cells of the proximalenzymes, or their division between adjacent cells may either tubule, in which glucose oxidation is poor, are capable of highprevent cycling, or be the object of the cycle. For example, the rates of lactate oxidation in parallel with equivalent rates oftransfer of reducing equivalents across the mitochondrial mem- glucose synthesis. In this simple case it is likely that all of thebrane, or of metabolites across a cell membrane may be energy for gluconeogenesis is derived from lactate oxidationfacilitated by a cycle. [49]. In the outer medulla, lactate is a respiratory fuel [4, 6, 44]. Substrate cycles in renal metabolism. In an earlier review, In the papilla, where lactate is produced in high rate, oxidationCohen and Barac-Nieto [50] considered the possibility that of lactate appears less likely but this has not been testedrenal sodium transport was in some way controlled by the then directly. recently described 'futile cycle' at fructose 6-phosphate/fruc- Methods of measurement. The usual pitfalls attend studies intose 1,6-diphosphate. The renal physiologist may even be which 14C02 production from '4C-lactate has been used in thestruck by the similarity to regulation of sodium transport by measurement of lactate oxidation (see above). Similarly, sup-passive back-flux; fine-control might be exerted over the net pression of 3H20 production from [6-3H]-glucose, by unlabelledsodium transport without altering the rate of the 'pump,' if lactate may indicate "dilution" of [3H]-pyruvate pools ratherpassive back-flux varied under different conditions. In the than competition between lactate and glucose as fuels. Directkidney, substrate cycling might regulate gluconeogenesis or assay of 02 consumption, lactate removal, and glucose synthe-glycolysis or might be involved in renal transport, to the extent sis advocated by Krebs et al [40] has stood the test of time. that ATP-supply is controlled by the rate of such cycling. In 60 Rosset a!

glucose or glucose precursors can support free-water clearance. This is a function of the distal convoluted tubule which seems to uniquely depend on glucose. Substrate provided for this reac- tion could be generated in the anatomically conveniently situ- ated proximal tubule cells: Baines and Ross [26] considered this to be glucose, the end product; it is also feasible that PEP traverses the intercellular space, to be returned to pyruvate and the TCA cycle by the high activity of pyruvate kinase in the C distal convoluted tubule. Fig. 3. Hypothetical models of substrate cycling in the kidney. A) (2) Pyru vate/oxaloacetate. The pyruvate/oxaloacetate cycle Cycling via pyruvate carboxylase, phosphoenolpyruvate carboxykinase provides an alternative route for the observed recycling of and pyruvate kinase, or via pyruvate carboxylase and oxaloacetatepyruvate and/or lactate. The two reactions catalyzed by pyruv- decarboxylase (or malic enzyme) could occur within a single cell in the ate carboxylase and oxaloacetate decarboxylase occur in the proximal convoluted tubule. B) Cycling between adjacent proximal and distal convoluted tubule cells could occur if both cells are permeable to mitochondria. The existence of this cycle in the kidney and liver PEP. C) A form of Con-cycle of the end-products of cortical andwas proposed simultaneously by Rognstad [54] and Janssens, medullary metabolism could occur during 'single-pass' of blood, ifHems, and Ross [15] who observed that inhibition of PEPCK connection, indicated by ?, can be established between cortex andfailed to limit the cycling. The reversal of pyruvate carboxylase medulla or vice-versa. Alternatively, this cycle can readily be demon- could also be achieved by malic enzyme, but its activity is strated during second-pass or recirculation of blood as occurs in vivo. Abbreviations: P, pyruvate; PEP, phosphoenolypyruvate; OAA,considered to be too low. The role of this cycle may be twofold: oxaloacetate; G, glucose; L, lactate. It could provide the means for the complete oxidation of oxaloacetate and the other 4-carbon skeletons arising in the TCA cycle (in the absence of the PEP/pyruvate path). Alterna- particular, cycling pyruvate/oxaloacetate/phosphoenolpyruvatetively, or in addition, the cycle could provide a fine control at (PEP) and back to pyruvate (via pyruvate carboxylase, PEPCKboth enzymes, controlling both the rate of oxidation of pyruvate and pyruvate kinase) or between fructose 6-phosphate/fructose(by limiting substrate supply) and the rate of gluconeogenesis. 1 ,6-diphosphate (via PFK and FDPase) are possible in a tissue (3) Fructose 6-phosphate/fructose diphosphate. The two cy- with the capacity for both gluconeogenesis and glycolysis. Ascles involving the hexose monophosphate pools are classic discussed elsewhere, the enzymes of glycolysis and glucofutile cycles, because they apparently waste ATP. The more neogenesis are separated spatially in the kidney between distalimportant F6P/FDP includes the major regulatory enzyme of and proximal structures, possibly limiting the extent of sub-glycolysis, PFK. In the liver this cycle is controlled by the strate cycling or be viewed as the physiological 'reason' foreffector fructose 2,6-diphosphate, a powerful stimulator of PFK separation. Nevertheless, intracellular cycling could occur inand inhibitor of FDPase. The existence of fructose 2,6-diphos- the kidney because even in the predominantly gluconeogenicphate has been confirmed in the kidney [43]; whether this cells of the proximal tubule the activity of pyruvate kinase andindicates an important role for a futile F6P/FDP cycle in the PFK actually exceeds the activity of PEPCK (see Ross andkidney remains to be established. Guder [14] for calculation). Furthermore, the peculiar folding of (4) Glucose/glucose 6-phosphate. This cycle could regulate nephron structures results in very close proximity within thethe rates of glucose phosphorylation, the production of glucose, cortex of structures with gluconeogenic and glycolytic capacity,or entry of substrate into the hexose monophosphate shunt. so that the concept of substrate cycling might extend beyondWhile the necessary enzymes are present in the kidney, they that generally implied to include cycling of metabolites betweenare spatially separate [16] and no evidence, to our knowledge, neighboring cells (Fig. 3B). Extending the model further tofor the cycle has been obtained. include whole tissues, cycling might be visualized between (5) Glucose/lactate. Recognized for many years as the Con cortex and medulla (Fig. 3C). cycle, the recycling of lactate carbon between separate tissues Individual substrate cycles in the kidney. (1) Phosphoenol-of the same animal is a vital part of glucose homeostasis. pyruvate/pyruvate. This cycle, shown in the liver [541,occurs Underlying the discussion of vascular inter-relationships be- across two cellular compartments and involves pyruvate kinasetween cortex and medulla is the possibility of an internal 'Con (PEP-pyr), pyruvate carboxylase (pyr/OxAc), and phospho-cycle' at work within the kidney. This is illustrated in Figure enolpyruvate carboxykinase (OxAc/PEP), In the cortical seg-3C. At the present state of knowledge, it seems extremely ments recycling of pyruvate was observed by Rognstad andunlikely that such a vascular connection exists so that a cycle Katz [56]. In cortical tubules, lactate cycling occurs in fed ratsduring a single 'pass' of the renal circulation is improbable [301. and was abolished in fasted rats [15]. However, inhibition ofThus, the attractive idea that glucose synthesized in the cortex PEPCK with mercaptopicolinate failed to suppress cycling anddirectly contributes to energy provision for transport processes lends doubt to the existence of this particular cycle in kidneyin the medulla has no experimental basis. Nor is it clear that the cortex. Subsequent experiments by Bartlett et al [35] andhigh rate of lactate released into the vasa recta draining the Espinal, Bartlett, and Ross [36] also support this conclusion andrenal medulla (demonstrated in hamster kidney [291) can con- suggest instead that pyruvate recycling could follow the alter-tribute to gluconeogenesis or respiration in the renal cortex. native route described below. Current suggestions say this mayBartlett, Lowry, and Ross [5] found that a cycle within the be the route of cycling in the liver as well. On the other hand,medulla appears more probable: Glucose delivered to the an inter-cellular substrate cycle (Type B in Fig. 3, pyr/PEP/pyr)papilla is converted only to lactate. This lactate is delivered to might explain the results of Baines and Ross [261 wherebythe outer medulla, where, as the preferred fuel for respiration Glucose metabolism in renal tubular function 61

1/ Glucose

/1 Glucose 6-phosphate

Fructose 6-phosphate

A Fructose 1 ,6-diphosphate V/i Mitochondrial membrane Glyceraldehyde 3-phosphate

NADH-4 3-Phosphoglycerol phosphate 01/ Pyruvate I ATP - IA 3-Phosphoglycerate CO2 ATP V/I NADH Oxaloacetate——' Malate-4--I-—1. Malate 2-Phosphoglycerate

i/ii OxaloacetateZ''°" ,.— Phosphoenolpyruvate '/12 f GTP V/i I Aspartate /1 ' Aspartate Fig. 4. The pathways for gluconeogenesis.

[4—6] it spares glucose. Glucose is thereby made available to thepathway is not the simple reversal like that of glycolysis deeper medullary structures, which, in turn, depend entirely onbecause of three energy barriers that exist: (1) pyruvate and glycolysis to generate ATP. Support for this type of internalphosphoenolpyruvate, (2) fructose 1 ,6-diphosphate and fruc- medullary economy of fuels by cycling comes from studying thetose 6-phosphate, and (3) glucose 6-phosphate and glucose. perfused kidney, in which furosemide inhibited both lactateThese barriers are side-stepped by reactions catalyzed by production [35] and glucose oxidation [36]. Finally, it is obviousenzymes specific for the gluconeogenic pathway. These en- that a cycle of glucose/lactate can and does occur between thezymes are pyruvate carboxylase and phosphoenolpyruvate cortex and medulla when second-pass is permitted. This is thecarboxykinase (PEPCK) that bypass the pyruvate phosphoenol- case in the kidney in vivo and has now been documented andpyruvate barrier; fructose 1 ,6-diphosphatase that reverses the quantified in the model of intact perfused rat kidney. fructose 6-phosphate fructose 1 ,6-diphosphate step; and glu- Physiological implications of futile cycles in the kidney. Incose 6-phosphatase that releases glucose (Fig. 4). The the absence of detailed evidence of modulating substrate cyclesgluconeogenic pathway has additional particular characteris- by diet, by hormone action or altered electrolyte transport intics. Pyruvate carboxylase is an intramitochondrial enzyme that the kidney, any conclusion regarding their physiological roleconverts pyruvate to oxaloacetate in the presence of ATP, must be tentative. biotin, and CO2. Oxaloacetate, its product, cannot cross the (1) Control of gluconeogenesis, glycolytic flux, and oxidativemitochondrial membrane [57]. The remaining enzymes of the metabolism. Inhibition of pyruvate/oxaloacetate cycling bygluconeogenic pathway are found in the cytosol. To cross the fasting results in accelerated gluconeogenesis. This mechanismmitochondrial membrane, oxaloacetate is converted to malate may apply to other conditions in which renal gluconeogenesis isby malate dehydrogenase with the expenditure of NADH, and modified, such as acidosis or steroid therapy. then leaves the mitochondrion via the malate-aspartate shuttle. The absence of pyruvate/oxaloacetate cycling when the intactOnce in the cytosol malate is reoxidized to oxaloacetate by the kidney is compared with isolated tubules suggests that somecytosolic form of malate dehydrogenase with the formation of mutual control exists between 'futile' cycling and the renalNADH. Malate dehydrogenase is thus an important enzyme in transport work of sodium reabsorption. On the other hand,the gluconeogenic pathway. Another way in which oxaloacetate direct control of sodium transport by the induction of PEPCK iscan leave the mitochondrion is by transamination to aspartate. made less likely by the finding (at least in the normal kidney)Aspartate also leaves the mitochondrion by the malate- that the cycle pyruvate/PEP does not occur in the kidneyaspartate shuttle and is deaminated back to oxaloacetate. cortex. Aspartate transaminase is present in both the mitochondrion (2) Whole-body glucose homeostasis. Sufficient evidence hasand the cytosol and is thus another important enzyme for been accumulated that the overall glucose/lactate cycle isgluconeogenesis. The importance of the malate-aspartate shut- important to maintain blood glucose in a variety of dietarytle goes beyond gluconeogenesis. It also regulates the oxidation conditions, although as discussed below, the net contribution ofof substrates that sustain sodium reabsorption by the kidney renal glucose synthesis to the process is less than has been[58]. Oxaloacetate is then converted into phosphoenolpyruvate suggested heretofore. by PEPCK with the expenditure of GTP. Gluconeogenesis requires NADH for the reduction of 3- Gluconeogenesis phosphoglycerol phosphate to glyceraldehyde 3-phosphate. Pathway of gluconeogenesis. Gluconeogenesis is the de novoThe conversion of oxaloacetate to malate in the mitochondria synthesis of glucose from noncarbohydrate precursors. Itsprovides means of exporting from the mitochondrion not only 62 Ross et a! oxaloacetate but also reducing equivalentsthat, asfor gluconeogenesis have not been identified clearly. PEPCK is oxaloacetate, cannot readily cross the mitochondrial mem-the enzyme most frequently perceived as the rate-limiting step brane. This step is important in the de novo synthesis of glucosein the gluconeogenic pathway because its activity is increased from precursors other than lactate, the main physiologicalin physiological and pathological conditions that stimulate precursor, that do not generate reducing equivalents such asgluconeogenesis. However, the nephron site with highest activ- pyruvate and aminoacids that yield pyruvate. ity of the enzyme does not coincide exactly with the nephron Gluconeogenesis is an energy-requiring process. When ATPportion that exhibits the highest rate of gluconeogenesis. The is decreased, gluconeogenesis is reduced [52]. For each mole-highest rates of PEPCK specific activity are found in the cule of glucose formed from pynivate six high energy phosphateconvoluted portion of the proximal tubule while the pars recta bonds are required: two ATP at the step catalyzed by pyruvatehas 50% of that [61]. However, the rate of glucose synthesis is carboxylase, two GTP at the generation of phosphoenolpyruv-higher in the pars recta than in the convoluted portion [77]. It is ate, and two ATP at the step of the conversion of 3-possible that the gluconeogenic pathway is regulated at the level phosphoglycerate to 3-phosphoglycerol phosphate. In addition,of pyruvate carboxylase, but the paucity of measurements of two NADH are required for the reduction of 3-phosphoglycerolthe activity of this enzyme in different conditions that modulate phosphate to glyceraldehyde 3-phosphate. The cost ofgluconeogenesis does not permit adequate evaluation of its role. gluconeogenesis should be compared with the net production of That gluconeogenesis and glycolysis occur in different neph- eight high energy bonds from glucose. ron segments in the kidney has been ascertained from the Sites of gluconeogenesis in the kidney. The portions of thedistribution of the enzymes that mediate both pathways. There- nephron where gluconeogenesis can occur are defined by thefore, gluconeogenesis is not reciprocally regulated by glycolysis presence of the enzymes that mediate this pathway. Thein the kidney as it is in the liver. The enzymes of gluconeogen- proximal tubule is the only site where gluconeogenesis canesis are, however, regulated by cellular modulators. Pyruvate occur [32, 59—63]; nephron segments beyond the proximalcarboxylase is regulated positively by acetyl CoA [78—80]. The tubule cannot synthesize glucose. PEPCK, fructose 1,6-enzyme is almost inactive in the absence of this compound. diphosphatase,glucose6-phosphatase, and aspartateMoreover, acetyl CoA reduces the activity of pyruvate dehy- transaminase are present almost exclusively in the proximaldrogenase, thus, its presence favors the gluconeogenic path- tubule. Pyruvate carboxylase distribution along the nephronway. Fructose 1,6-diphosphatase is inhibited by its own sub- has not been measured because of the particular lability of thisstrate at concentrations greater than 0.5 m [81] and also by enzyme that loses activity during sample preparation [32].low concentrations of AMP [82]. The inhibition by fructose Although malate dehydrogenase has not been mapped along the1 ,6-diphosphate and AMP is removed by precursors of nephron, it is a key enzyme of the citric acid cycle, present bothgluconeogenesis that supply reducing equivalents and are in the mitochondrion and cytosol, widely distributed in manyreadily oxidized [81]. Therefore, an increase in the supply of tissues and probably present in all nephron segments. readily oxidized metabolic substrates or a decrease in the Role of renal gluconeogenesis in glucose homeostasis. Theutilization of ATP should stimulate the gluconeogenic pathway capacity of the kidney to produce glucose was first demon-in the proximal tubule. strated by Benoy and Elliot [64]. Since then many studies have Pathophysiological conditions that alter renal gluconeogen- shown that the kidney contributes to blood glucose [65—67] andesis.Alterationsinacid-base balance modify renal this contribution has been variously estimated to be 5 to 25%gluconeogenesis. Metabolic acidosis stimulates it in a variety of [28, 68] under normal conditions and as much as 50% duringpreparations [83—88], but respiratory acidosis does not alter it starvation or diabetes [28]. Of interest is the observation that[84], while alkalosis inhibits it [86, 87]. Acidosis stimulates the transplantation of a normal kidney to a patient with Type 1activity of PEPCK [32, 88—92] associated with an increase in glycogenosis failed to improve fasting hypoglycemia despitePEPCK mRNA [93, 94]. Neither glucose 6-phosphatase nor glucocorticoid therapy [69]. The problems in defining the con-pyruvate carboxylase have been shown to change during aci- tribution of renal gluconeogenesis to blood glucose arise fromdosis [32]. Fructose 1 ,6-diphosphatase has been shown both to the fact that in vivo the high circulating levels of glucosebe increased [59] and remain unchanged [32]. It was initially minimize the magnitude of the changes in glucose concentra-suggested by Goodman, Fuisz, and Cahill [83] that tion. The rapid rate of renal blood flow that results in a smallgluconeogenesis could have a role in the regulation of ammonia venous-arterial difference in glucose concentration [68, 70, 71]production by the kidney. For ammonia production to contrib- further compounds this problem. The venous-arterial differenceute to the net disposal of hydrogen ion by the kidney, the two can even be negative [72—74]. Another source of error is theprotons that are released when glutamine is deaminated to utilization of glucose by the kidney that variably affects itsammonia and a-ketoglutarate must be neutralized. This occurs concentration in the renal vein [75, 76]. Recently, Kida et al [281when a-ketoglutarate is converted to glucose or metabolized to introduced an isotope dilution technique validated by unilateralCO2 and H2O. The fact that glucose is the major end product of nephrectomy but not without interpretation difficulties. It doesglutamine metabolism [87] and that acidosis stimulates PEPCK not take glycolysis by the kidney into account, which wouldand gluconeogenesis lends support to the hypothesis that falsely elevate the contribution of the kidney to blood glucose.gluconeogenesis regulates a-ketoglutarate levels and, therefore, It also assumes that the rate of glycolysis by the medulla of theammonia metabolism. Although acidosis and gluconeogenesis kidney is constant. In the absence of knowledge on the rate ofhave been shown to change in parallel in the rat, this does not glucose utilization by the kidney and factors that may modify it,occur in the rabbit, guinea pig, or dog [95—98]. Inhibitors of the method provides only limited information. gluconeogenesis do not invariably suppress ammonia produc- Regulation of renal gluconeogenesis. The rate-limiting stepstion or glutamine utilization [98—101] suggesting that the Glucose metabolism in renal tubular function 63

gluconeogenic pathway is not the only pathway for ammoniafact that in the isolated perfused rat kidney norepinephrine also production [98, 101]. Indeed, the gluconeogenic pathway needstimulates sodium reabsorption has led to the suggestion of a not be the only pathway for the neutralization of the protonslink between electrolyte transport and glucose metabolism [26]. released during the production of ammonia from glutamine. The Parathyroid hormone stimulates gluconeogenesis in the kid- oxidation or a-ketoglutarate would accomplish the same thing.ney [134—136]. The hormone stimulates gluconeogenesis in the However, the gluconeogenic pathway offers a convenient wayconvoluted portions of superficial but not juxtamedullary neph- of doing it, as described in a quote by Krebs [1021: "We nowrons in a way independent of its capacity to stimulate adenylate look upon renal gluconeogenesis as a salvage reaction for thecyclase in either type of nephron [136]. The effect is mediated glutamine carbon which is set free when ammonia has beenby cyclic AMP [134] and calcium ions [134, 135]. It appears that provided from glutamine. The carbon skeleton that remains isthe effect of the hormone is to increase the levels of intracellular not necessarily needed as a source of energy; it can be salvagedcalcium since the latter is necessary for parathyroid hormone in the form of glucose, a material which can either be stored asstimulation of gluconeogenesis. Parathyroid hormone has no glycogen or utilized in other tissues which depend upon carbo-effect on renal gluconeogenesis at calcium concentrations hydrate as a source of energy." greater than 3 m. Calcium itself can stimulate gluconeogene- Diabetes causes an increase in renal gluconeogenesis and insis; its effect appears to be maximal at physiological concentra- the key enzymes of the gluconeogenic pathway [103—110]. Thetions [18, 129, 134, 137, 138]. In addition, calcium ionophore effect of diabetes to increase gluconeogenesis may be related toA23187 can also stimulate gluconeogenesis [139]. Vitamin D3 the associated metabolic acidosis because alkalinization pre-and 1-alpha-OH D3 have been shown to stimulate gluconeo- vents both the increase in gluconeogenesis and in enzymegenesis in the kidney [140, 141]. The effect is inhibited by activity [105,106].However, others have failed to show such acalcitonin and is thought to be due to an increase in calcium relationship [28]. concentration [141]. Since the experiments by Krebs et a! [18], it has been known Miscellaneousconditions that affect renal gluconeogenesis. that a decrease in the intake of carbohydrates stimulatesAdditionalfactors that modify gluconeogenesis by the kidney gluconeogenesis in the kidney. The effect is seen with fasting orhave been identified recently. Somatostatin has been shown to starvation of variable duration [105,111—113].Fasting increasesincrease gluconeogenesis [142]. On the other hand, phosphate the activity of all key enzymes of the gluconeogenic pathway:depletion results in a decrease in gluconeogenesis at a time pyruvate carboxylase, PEPCK, fructose 1,6-diphosphatase,when renal inorganic phosphate and ATP were still within and glucose 6-phosphatase [32, 110, 113—115]. Both the in-normal limits [143]. The administration of branched chained creased gluconeogenesis and the increase in enzyme activity isalpha ketoacids was shown to inhibit gluconeogenesis although reversed rapidly by refeeding [113]. The increase in gluconeo-the actual mechanism has not been identified [144]. This last genesis induced by fasting appears to depend on the accompa-observation may have some clinical significance in patients with nying metabolic acidosis because it can be reversed by thechronic renal failure where these acids are used to reduce the administration of bicarbonate [105]. Similarly, the increase inrate of decline of the disease. Finally, clofibrate can decrease gluconeogenesis and PEPCK activity produced by a high pro-gluconeogenesis [145, 146]. tein diet can be reversed by alkalinization [88]. Gluconeogenesis and sodium transport. Thereare many Gluconeogenesis is stimulated by exercise [116]. This stimu-indications that gluconeogenesis and the reabsorption of so- lation is presumably the result of the increase in circulatingdium are reciprocally related. Inhibition of Na-K-ATPase and lactate, itself the consequence of anaerobic muscle metabolism,thereby sodium reabsorption by the kidney stimulates gluco- and of the accompanying acidosis because it can be reduced byneogenesis [38, 147, 148]. Stimulation of Na-K-ATPase by the administration of bicarbonate [117]. either nystatin [149] or monensin [150] inhibits gluconeogene- Effectsof hormones on gluconeo genesis. Glucocorticoids sis. Angiotensin stimulates gluconeogenesis while it inhibits stimulate gluconeogenesis [118]. Glucocorticoids exert a tonicproximal tubulereabsorption [151—153]. Furosemide, effect on gluconeogenesis; adrenalectomy decreases the rate ofethacrynic acid, and chlorothiazide stimulate gluconeogenesis gluconeogenesis by the kidney [119]. The effect of glucocorti-in rat kidney cortex slices [154].Inisolated kidneys of steroid- coids to stimulate gluconeogenesis can be demonstrated intreated rats, perfused solely with glucose, gluconeogenesis is isolated perfused kidneys [120], slices [121], and isolated tu-stimulated while sodium reabsorption is depressed [120]. Fur- bules [122]. Treatment with glucocorticoids increases the activ-thermore, sodium reabsorption and gluconeogenesis change ity of pyruvate carboxylase, PEPCK, and fructose I ,6-diphos-reciprocally when sodium reabsorption is increased abruptly phatase [108—110, 115, 123—125]. The increase in phosphoenol-[155].Thesedata suggest that two energy-requiring processes, pyruvate carboxykinase is mediated by an increase in mRNAsodium reabsorption and gluconeogenesis, may compete for [93]. Aldosterone can stimulate gluconeogenesis in the rat, butenergy availability in the proximal tubule. However, sodium the effect is seen only with supramaximal doses of the hormonereabsorption and gluconeogenesis are not always reciprocally suggesting that the effect is due to glucorticoid receptor occu-related. Inhibition of Na-K-ATPase with ouabain does not pation [126]. always result in stimulation of gluconeogenesis [38, 151, 156]. Norepinephrine stimulates gluconeogenesis in slices, isolatedIndeed, in isolated perfused kidneys of fed or starved rats, cortical tubules, the isolated perfused rat kidney and in vivoouabain inhibited gluconeogenesis from lactate [58].Theinter- [26, 38, 70, 127—131]. The effect of norepinephrine appears to bepretation of these data is difficult because of the varying nature mediated by an alpha1 receptor [132, 133], although a betaof the preparations and experimental conditions used. The receptor has also been proposed [128]. The effect is inhibited bysituation is further complicated by the heterogeneity of the methoxyverapamil, a calcium channel blocking agent [1321. Thenephron. Certain agents used to inhibit sodium reabsorption 64 Ross et a! exert their action at tubular levels where gluconeogenesis is not Kidney mt 20:29—35, 1981 thought to occur. It is likely that under normal conditions 3.EVELOFFJ,HAASE W, KINNE R:Separation ofrenal medullary sufficient energy is available in the kidney to sustain both cells: isolation of cells from the thick ascending limb of Henle's loop. JCell Biol87:672—680, 1980 gluconeogenesis and sodium reabsorption. 4. LEE JB, VANCE VK, CAHILL GF JR: Metabolism of '4C labelled substrates by rabbit kidney cortex and medulla. Am J Physiol Integration of carbohydrate metabolism within the kidney 203:27—36, 1962 5. BARTLETT S, LOWRY M, Ross BD: Competition between lactate Despite the many studies of renal glucose metabolism, there and glucose in the renal medulla. Proc 8th mt Cong Nephrol, is no proof that carbohydrate metabolism within any one Athens, 1981, RM.054,p66 segment of the kidney is integrated, either with metabolism or 6. BAVAREL G, FORISSTER M, PELLET M: Lactate and pyruvate other nephron segments, or with any specific transport func- metabolism in dog renal outer medulla: effects of oleate and tion. However, the most striking feature is the very narrow ketone bodies. In!J Biochem 12:163—168,1980 7. HOHENEGGER M, WITTMAN G, DAHLHEIMH:Oxidation of fatty limits within which renal arteriovenous glucose difference ap- acids by different zones of the rat kidney. PflugersArch pears to be controlled. This suggests that the function of one 341:105—I 12,1973 nephron segment does in some manner respond to differences in 8. NEITH H, SCI-IOLLMEYER P: Substrate utilization of the human the metabolic demand on another. kidney. Nature 209:1244—1245, 1966 9. Ross BD,EPSTEIN The most important function of 'futile' cycles of renal glucose FH, LEAF A: Sodium reabsorption in the perfused rat kidney. AmJ Physiol225:1165—1 171, 1973 metabolism may be considered as that of acting as a buffer to 10. SCHUREK Hi, BREZHT JP, LOHFERT H, HJERHOLZER K: The basic glucose and lactate levels in the blood during major metabolic requirements for the function of the isolated cell-free perfused rat variations. Thus, with futile cycles, intracellular cycles, and kidney. PflugersArch 354:349—365, 1975 11.WIRTHENSOHN GB, GUDER WG: Triacylglycerol metabolism in medullary recycling of glucose and lactate, the many-fold isolated rat kidney cortex tubules. Biochem3 186:317—324,1980 variation in filtration and reabsorptive capacity of the kidney 12. SCHLENDER KK: Regulation of renal glycogen synthetase imposed by eating, drinking or by acid-base, sodium and interconversion of two forms in vitro. Biochem Biophys Acta potassium balance might be achieved by simply reorganizing 297:384—394, 1973 intrarenal carbohydrate metabolism. No sudden changes in l3. KHANDELWAL RL,ZINMANSM, KNULL HR: The effect of streptozotocin-induced diabetes on glycogen metabolism in rat glucose demand by the kidney need be envisaged. kidney and its relationship to the liver system. Arch Biochem In longer-term dietary adaptations to starvation in animals Biophys 197:310—316,1979 and in humans, this mechanism breaks down, so that fasting 14. Ross BD,GUDERWG: Heterogeneity and compartmentation in natriuresis can only be reversed by the provision of exogenous kidney, in Metabolic compartmentation, edited by SIES H Aca- demic Press, 1982, pp 363—409 glucose [1581. 15. JANSSENS P, HEMS R, Ross B: The metabolic fate of lactate in renal cortical tubules. Biochem 3 190:27—37, 1980 Summarypoints 16. GUDER WG, Ross BD: Enzyme distribution along the nephron. • Kidney Jut 26:101—ill, 1984 Heterogeneity of metabolic activity along the nephron 17. PFALLER W: Structure function correlation on rat kidney. Quan- points to a very varied relationship between glucose metabo- titative correlation of structure and function in the normal and lism and ion transport. injured rat kidney. Adv Anat Embryol Cell Biol 70: 1—106, 1982 • Glycolysis is linked closely to free-water clearance and 18. KREBS HA, BENNETT DAH, DE GASQUET P, GASCOYNE T, possibly to sodium, potassium, and hydrogen ion transport. YOSHIDA T: Renal gluconeogenesis. The effect of diet on the • Glucose oxidation, while not the major source of renal gluconeogenic capacity of rat-kidney-cortex slices. Biochem J 86:22—27, 1963 energy, is crucial in sodium, potassium, and phosphate 19. GREGOIRE F: Oxidative metabolism of the normal rat glomerulus. reabsorption. Kidney mt7:86—93,1975 • Gluconeogenesis recovers carbon compounds generated 20. VINAY P, GouGoux A, LEMIEUX G: Isolation of a pure Suspen- during the process of renal ammoniagenesis. Glucose synthesis sion of rat proximal tubules. Am J Physiol 241:F403—F411, 1981 and active sodium transport appear to compete for renal ATP, 21.KLEINZELLERA: Active sugar transport in renal cortex cells; the electrolyte requirement. Biochim Biophys Acta 211:277—292, 1970 although no regulatory function for this competition has been 22. SILVA P, Ross BD, CHARNEY AN, BESARAB A, EPSTEIN FH: identified. Glucose formed in the proximal tubule may support Potassium transport by the isolated perfused kidney. JClin Invest free-water clearance in adjacent distal tubule, but is not thought 56:862—869,1975 to contribute to any medullary function. 23. BESARAB A, SILVA P, Ross B, EPSTEIN FH: Bicarbonate and sodium reabsorption by the isolated perfused kidney. Am J • The complex network of biosynthetic and catabolic path- Physiol 228:1525—1530, 1975 ways of glucose metabolism may have evolved in the kidney to 24. Ross BD, TANNEN RL: Effect of decrease in bicarbonate concen- protect the organism against wide variations in glucose demand tration on metabolism of the isolated perfused rat kidney. C/in Sci which would otherwise be unavoidable during the course of 57: 103—Ill, 1979 25. FREGA NS,WEINBERGJM, Ross BD, LEAF A: Stimulation of rapidly fluctuating renal electrolyte loads. sodium transport by glucose in the perfused rat kidney. AmJ Physiol 233:F235—F240, 1977 Reprint requests to Dr. P. Silva, Department of Medicine, Beth Israel 26. BAINESAD, Ross BD: Nonoxidative glucose metabolism a pre- Hospital, 330 Brookline Avenue, Boston, Massachusetts 02215, USA requisite for formation of dilute urine. Am J Physiol 242:F49l—F498, 1982 References 27. NisHlITsuTsuji-Uwo JM, Ross BD, KREBS HA: Metabolic activ- ities of the isolated perfused rat kidney. BiochemJ 103:825—862, 1.WEIDEMANMi,KREBSHA:The fuel of respiration of rat kidney 1967 cortex. Biochemf 112:149—166, 1969 28. KIDA K, NAKAJO 5, KAMIYA F, TOYAMA Y, NISI-Ho T, 2. KLEIN KL, WANG MS, TORIKAI S, DAVIDSON WD, KUROKAWA NAKAGAWA H: Renal net glucose release in vivo and its contri- K: Substrate oxidation by defined nephron segments of rat kidney. bution to blood glucose in rats. J Clin Invest 62:721—726, 1978 Glucose metabolism in renal tubular function 65

29. Rulz-GuINAzu A, PEHLING G,RUMMRICHG, ULLRICH KJ: 55. NEWSHOLME EA, CRABTREE B: Substrate cycle in metabolic Glukose undmilchsaurekonzentrationenan der spitze des regulation and heat generation. BiochemnSoc Symp 41:61—I10, vaskularen gegenstromsystem in nierenmark. PflugersArch 1976 274:311—317,1961 56. ROGNSTAD R,KATZJ:Gluconeogenesis in the kidney cortex. J 30.KRIz W: Der architektonischeundfunktionalleauthauder ratten- Biol Che,n 247:6047—6054,1972 niere. Z Ze!lforschMikrosk Anat 82:495—535,1967 57. LARDY HA,PAETKAUV, WALTER P:Paths of carbon in 31. SOCHOR M, BAQUERNZ,MCLEAN P: Glucose overutilization in gluconeogenesisand lipogenesis: The role of mitochondria in diabetes: evidence from studies on the changes in hexokinase, the supplying precursors of phosphoenolpyruvate. ProcNat Acad Sci pentose phosphate pathway and glucuronate-xylulose pathway in 53:1410—1415,1965 rat kidney cortex in diabetes. Biochem Biophys Res Commun 58. Ross B, SILvA P,BULLOCK5: Role of the malate—aspartate 86:32—39, 1979 shuttle in renal sodium transport in the rat. ClinSc! 60:419—426, 32.BURCHHB, NARINS RG, CHU C, FAGIOLI S, Ci-ioi S, MCCARTHY 1981 W, LOWRY OH: Distribution along the rat nephron of three 59. SCHMID H,SCHOLZM, MALL A, SCHMIDT U, GUDER WG, enzymes of gluconeogenesis in acidosis and starvation. Am J DUBACHUC: Carbohydrate metabolism in rat kidney: heteroge- Physiol 235:F246—F253, 1978 neous distribution of glycolytic and gluconeogenic key enzymes. 33. MOREL F: Sites of hormone action in the mammalian nephron. Am CurrProbl Clin Biochem 8:282—289,1977 JPhysiol 240:F159—F164,1981 60.GUDERWG,SCHMIDTU: The localization of gluconeogenesis in 34. Ross BD: The isolated perfused kidney. ClinSc! Mo! Med rat nephron: Determination of phosphoenolpyruvate carboxyki- 55:513—521,1978 nase (GTP) in microdissected tubules. HoppeSeylers ZPhysiol 35. 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