[Metabolic alkalosis on the surface but metabolic acidosis in depth]. / Alcalose métabolique en surface, mais acidose métabolique en profondeur.

Our purpose in writing this article is to emphasize the acid-base consequences and total body imbalances which follow the selective depletion of HCl. The initial body balance is an equimolar deficit of chloride and gain of bicarbonate. Within a short period of time, body balance changes; the net deficits are closer to equimolar losses of potassium and chloride. Since the loss of potassium occurred without the simultaneous loss of existing body anions (chloride or phosphate), this negative balance of potassium is accompanied by an equimolar gain of hydrogen ions. Thus when the negative balance is that of KCl, acid-base balance is present but there is a surplus of bicarbonate in the extracellular fluid (ECF) together with an equal surplus of hydrogen ions in another compartment (the intra-cellular fluid (ICF)). Moreover, if the ECF volume is contracted, a more severe degree of acidosis of the ICF may occur due to a higher PCO2 in venous blood. Given the acid-base balance and a deficit of KCl, one should not view this disorder as being "corrected" by saline at any time other than in the acute phase before a large potassium deficit occurs. Sodium chloride should be restricted to repair a deficit of sodium chloride. The emphasis on therapy is obviously to replace the deficit of KCl.

Mechanisms of proximal proton secretion in BBM of herbivorous, omnivorous, and carnivorous species.

The mechanisms of proton secretion by the proximal brush-border membrane (BBM) were compared in carnivorous (dog), omnivorous (human, pig, rat), and herbivorous (rabbit, hamster) species. The activity of the proton pump (V-type bafilomycin-sensitive H(+)-adenosinetriphosphatase) and of the Na+/H+ exchanger (amiloride-sensitive quenching of acridine orange fluorescence), the two major proton secretion mechanisms, was measured. The enzymatic activity of the H(+)-adenosinetriphosphatase activity was measured in intact (endosomes) and solubilized (0.1% deoxycholate or Triton X-100) BBM vesicles isolated by conventional Mg2+ precipitation techniques. In all species, but not in humans, the fraction of the ATP turnover energizing the proton pump (bafilomycin-sensitive respiration) was also measured in isolated proximal tubules. Significant differences in acid transport mechanisms were noted between species, with the proton pump predominating in the BBM of carnivorous species and the Na+/H+ exchanger predominating in the BBM of herbivorous species. The fraction of respiration suppressible by bafilomycin in proximal tubules was also different in all the species considered. This may indicate a different organization of proximal H+ transport related to the species-specific menace to acid-base balance.

Could cytoplasmic concentration gradients for sodium and ATP exist in intact renal cells?

In renal cells, the Na+ pump maintains a transmembrane concentration gradient for sodium ensuring the net reabsorption of sodium with or without cotransported species. This process requires a significant fraction of the ATP turnover of proximal tubules and thick ascending limbs. To understand the potential regulatory influences of Na+ and ATP on the activity of the Na+ pump in these nephron segments, the apparent kinetics of the membrane-bound Na+-K+ ATPase and of the cellular Na+ pump were studied in different preparations of dog proximal tubules and thick ascending limbs (tubular suspensions, tissue homogenates, and basolateral membrane vesicles) obtained from dog kidney cortex and red medulla. Two determinant kinetic parameters, i.e., the apparent Michaelis constant (Km) and the saturating concentrations for sodium and ATP, were compared with the intracellular concentrations of Na+ and ATP measured under physiological conditions. In both types of tubules, the apparent Km value for Na+ (5-15 mM) is set well below the measured mean intracellular concentration of sodium (50-60 mM), suggesting that the Na+ pump should be saturated by sodium ions under normal conditions. Nevertheless, a modest increment of the Na concentration in the vicinity of the pump, obtained by equilibrating the intra- and extra-cellular sodium concentrations at various extracellular [Na+] with nystatin, increases the activity of the Na+ pump in intact cortical tubules and thick ascending limbs, even when the extracellular [Na+] is set at the estimated intracellular [Na+], demonstrating that the pump is not saturated by sodium in situ. Similarly, the kinetics of the renal Na+ pump as a function of the ATP concentration suggested that the pump should be saturated by ATP in physiological conditions, since in both tissues the cellular ATP level (3-6 mM) is higher than the concentration required to achieve saturation of this activity (< 2.5 mM). However, in renal cortical tubules, the steady-state intracellular [Na+] is affected by modest changes of ATP concentration, suggesting that the Na+ pump is not functionally saturated by ATP. Our data suggest that concentration gradients for Na+ and ATP may exist in the cytosol of renal cells. These gradients would be related to the polarity of sodium transport and of the ATP-consuming and ATP-regenerating processes in intact cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Glucose metabolism in dog inner medullary collecting ducts.

The adenosine triphosphate (ATP) generating pathways of dog inner medullary collecting ducts (IMCD) were examined in vitro using suspensions of dog IMCD tubules incubated under aerobic and anaerobic conditions. Glucose is always the preferred substrate for this tissue, even if lactate can be oxidized under aerobic conditions. The metabolism of glucose proceeds largely towards lactate accumulation in the presence or absence of oxygen. Glycogen is also consumed and more markedly so during anoxia. The pentose shunt represents a minor pathway for glucose metabolism in this tissue. Under aerobic conditions, the net oxidation of glucose to CO2 contributes significantly to the cell energetics, mitochondrial and cytoplasmic mechanisms sharing equally the ATP synthesis. In the absence of oxygen, only the cytoplasmic routes of ATP synthesis are used, but the apparent ATP turnover is markedly reduced. A marked inhibition of the activity of the Na-K-ATPase during anoxia explains this observation. The utilization of glucose for osmolyte synthesis is a minor process and appears to be suppressed under anaerobic conditions. It is concluded that the ATP turnover is low in dog IMCD cells as compared with that of other nephron segments and is largely dependent upon glucose availability under aerobic or anaerobic conditions.

A method to evaluate renal ammoniagenesis in vivo.

A reduced rate of excretion of ammonium (NH4+) can be due to either a low rate of production and/or a low transfer of NH4+ to the urine. At present, there is no way to obtain a measure of the rate of production of NH4+ in vivo without invasive techniques. Hence, our purpose was to develop a non-invasive test to reflect this rate in vivo. Conditions were selected so that there would be a wide range in the rate of production of NH4+ in the kidney. Initial experiments were performed in dogs because both the rate of production and excretion of NH4+ could be measured directly. The rate of excretion of NH4+ in normal dogs on their usual diet varied over a wide range and was not directly related to its rate of production. Nevertheless, 59% of the NH4+ produced was excreted when the pH of urine was < 6 or when the rate of flow of urine was high (after administering a loop diuretic). To produce a urine with a low pH and high flow rate in humans, a loop diuretic (20 mg of furosemide) and a mineralocorticoid (200 micrograms of fludrocortisone) were given. The pH of urine fell to 5.1 and the rate of urine flow rose to 8 ml/min; the rate of excretion of NH4+ rose from 21 to 33 mumol/min when the urine flow rate rose.(ABSTRACT TRUNCATED AT 250 WORDS)

Heterogeneous metabolism and toxicity of 4-pentenoate along the dog nephron.

4-Pentenoate (4P) is a short-chain fatty acid which causes a complete renal Fanconi syndrome. We have examined the mechanism of 4P toxicity along the nephron after a prolonged (30 min) exposition of isolated renal tubular segments to this agent. In proximal tubules, 4P inhibited the activity of alpha-ketoglutarate dehydrogenase, pyruvate dehydrogenase, and beta-oxidation, but not in thick ascending limb or inner medullary collecting duct tubules in suspension. These proximal effects were accompanied by a marked oxidation of the proximal redox state, with a fall in the tissue respiration and a low content of ATP. The acetyl-CoA content of proximal tubules was simultaneously reduced. Butyrate, acetate, hexanoate or octanoate did not exert these effects. In proximal tubules the metabolism of 4P led to the tissue accumulation of 3-keto-4-pentenoyl-CoA, a known unspecific inhibitor of metabolic oxidation. This metabolite was not detectable in thick ascending limbs which metabolized 4P rapidly. No metabolism of 4P was noted in collecting ducts. We conclude that beta-oxidation probably differs in proximal and thick ascending limb tubules, allowing 4P metabolism to exert a specific toxicity in proximal tubules. A selective proximal defect in energy metabolism probably explains the Fanconi syndrome observed with exposition to 4P.

Metabolic effects of 4-pentenoate on isolated dog kidney tubules.

The effects of 4-pentenoate (0.01 to 10 mM) were studied on suspensions of cortical tubules and of thick ascending limbs (TAL) prepared from dog kidneys. When cortical tubules were incubated with 1 mM glutamine, 4-pentenoate accelerated glutamine uptake, ammoniagenesis, and the production of alpha-ketoglutarate, lactate and pyruvate, but decreased gluconeogenesis. With 5 mM glutamine, the marked accumulation of alpha-ketoglutarate reversed the net fluxes through the alanine and aspartate aminotransferases. When cortical tubules or TAL were incubated with lactate, its utilization and gluconeogenesis (in cortical tubules) were markedly decreased by 4-pentenoate. The mitochondrial NAD+/NADH ratio was markedly increased by 4-pentenoate in cortical tubules but not in TAL. The production of 14CO2 from 14C[1]-pyruvate or 14C-[1]-alpha-ketoglutarate was decreased by approximately 60% by 4-pentenoate in cortical tubules but not in TAL. In cortical tubules, these findings are best explained by depletion of mitochondrial free CoA, inhibition of pyruvate and alpha-ketoglutarate dehydrogenases and decreased mitochondrial NADH. By contrast, in TAL, accumulation of reducing equivalents probably resulted from the metabolism of 4-pentenoate itself.

Brush border membrane proteins in experimental Fanconi's syndrome induced by 4-pentenoate and maleate.

Fanconi's syndrome was investigated using brush border membrane (BBM) vesicles isolated from dog kidney. Sodium-dependent uptake of glucose, phosphate, and amino acids and protein phosphorylation were studied in BBM isolated from normal and from 4-pentenoate- and maleate-treated animals. The time course of D-glucose and phosphate uptake, in BBM vesicles, remained unchanged, indicating that both treatments had no effect on carrier properties, and that permeabilities to these substrates and to sodium were not modified. Furthermore, sodium-dependent transport of alanine, phenylalanine, proline, glycine, and glutamate into vesicles remained unaltered by either treatment. 4-Pentenoate treatment caused modifications of the phosphorylation pattern of BBM proteins: the phosphorylation of two proteins (61 and 74 kDa) was increased and that of two others (48 and 53 kDa) was decreased. Maleate treatment caused an increase in the phosphorylation for the same 61-kDa protein, which was also affected by 4-pentenoate treatment, suggesting that phosphorylation of this protein could be related to a mechanism involved in both 4-pentenoate- and maleate-induced Fanconi's syndrome. These changes were also observed in the presence of sodium fluoride and L-bromotetramisole, indicating that the modification of phosphorylation was not due to a difference in phosphatase activities. These results suggest that Fanconi's syndrome induced by 4-pentenoate or maleate is not caused by an inhibition of BBM Na(+)-dependent transport systems. Our results also suggest that protein phosphorylation may play an important role in the molecular defect involved in Fanconi's syndrome.

Basolateral glucose transport in distal segments of the dog nephron.

The transport of glucose by canine thick ascending limbs (TAL) and inner medullary collecting ducts (IMCD) was studied using tubule suspensions and membrane vesicles. The uptake of D-[14C(U)]glucose by a suspension of intact TAL tubules was reduced largely by phloretin (Pt), moderately by phlorizin (Pz), and completely suppressed by a combination of both agents. A selective effect of Pz on the transport of [14C]alpha-methyl-D-glucoside, but not on 2-[3H]deoxyglucose, was also observed in TAL tubules. In contrast, glucose transport was unaffected by Pz but entirely suppressed by Pt alone in IMCD tubules. The metabolism of glucose was largely suppressed by Pt but unaffected by Pz in both types of tubules. Membrane vesicles were prepared from the red medulla and the white papilla or from TAL and IMCD tubules isolated from these tissues. Vesicle preparations from both tissues demonstrated a predominant carrier-mediated, sodium-independent, Pt- and cytochalasin B-sensitive glucose transport. Following purification of basolateral membrane on a Percoll gradient, the sodium-insensitive D-[14C(U)]glucose transport activity copurified with the activity of the basolateral marker Na(+)-K+ ATPase in both tissues. However, a small sodium-dependent and Pz-sensitive component of glucose transport was found in membrane vesicles prepared from the red medulla or from thick ascending limb tubules but not from the papilla nor collecting duct tubules. The kinetic analysis of the major sodium-independent processes showed that the affinity of the transporter for glucose was greater in collecting ducts (Km = 2.3 mM) than in thick ascending limbs (Km = 4.9 mM). We conclude that glucose gains access into the cells largely through a basolateral facilitated diffusion process in both segments. However a small sodium-glucose cotransport is also detected in membranes of TAL tubules. The transport of glucose presents an axial differentiation in the affinity of glucose transporters in the renal medulla, ensuring an adequate supply of glucose to the glycolytic inner medullary structures.

Metabolism of lactate by a suspension of dog thick ascending limbs: relations with transport.

The addition of substrate in the form of lactate (L), but not glucose (G), increases the respiration of canine thick ascending limb (TAL) segments in a saturable (above 2 mM) fashion. More than 60% of this stimulation is ouabain-sensitive (1 mM ouabain) even if L and G transport are both sodium-insensitive processes in TAL. Thus L, but not G, specifically stimulates Na+ entry in TAL cells and its subsequent transport by the Na+,K(+)-ATPase. If chloride is substituted for by gluconate, no significant substrate-induced stimulation of ouabain-sensitive respiration is observed. SITS (4-acetamino-4'-isothiocyanostilbene-2,2'-disulfonic acid) also interferes with the L-induced stimulation of respiration. Thus L entry in TAL appears to be directly or indirectly coupled to the transepithelial flux of Cl-. Furosemide (F), but not amiloride, also inhibits this stimulation suggesting that the accelerated Na+ entry triggered by the application of L occurs through the F-sensitive carrier or that lactate transport is F-sensitive in TAL cells. In accord, F specifically impairs the metabolism of L (as compared to G). These data suggest that in intact TAL tubules both lactate uptake and oxidation are directly or indirectly influenced by the transcellular flux of NaCl. This organization may participate to maintain a stoichiometry between the transport of NaCl and the availability of L to support the energetic needs of TAL cells.

Basolateral transport of lactate in dog thick ascending limbs.

Basolateral membrane vesicles (BLMV) isolated from both red outer medulla or from thick ascending limb segments isolated from the dog kidney were used to examine the process of lactate transport in this nephron segment. The BLMV preparation was enriched in Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) that represented 96% of the total ATPase activity of this preparation and the vesicles were largely under the right side-out orientation. On application of a OH- or HCO3- gradient (inside greater than outside), a secondary active lactate accumulation was observed, with characteristic transient overshoot. This phenomenon was shown to occur irrespective of the presence or absence of Na+, K+, or Cl-. The pH, but not the bicarbonate-driven, overshoot was abolished by nigericin (in presence of K+). Studies with valinomycin and K+ demonstrated that the generation of a membrane potential was not responsible for the acceleration of lactate transport, even if the amplitude of lactate accumulation was reduced in the presence of a bicarbonate gradient and valinomycin. A significant trans-stimulation of [14C]lactate transport by cold lactate was observed (under voltage-clamp condition). The transport was 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid insensitive but sensitive to furosemide (IC50 = 0.1 mM) and alpha-hydroxycyanocinnamate (IC50 = 1 mM). The kinetic parameters of the transporter revealed a single carrier with an apparent Michaelis constant of 1.7 mM and an apparent Vmax of 9.7 nmol.mg protein-1.30 s-1. The transporter was shown to be distinct from that of proximal tubule brush-border membrane or mitochondria (pyruvate). Thus thick ascending limbs possess a carrier-mediated lactate transport that can be used for lactate uptake (aerobic condition) or for lactate release (anaerobic glycolysis) according to metabolic processes imposed by the local oxygenation condition.

Experimental Fanconi's syndrome resulting from 4-pentenoate infusion in the dog.

Studies were performed in anesthetized dogs to evaluate the effects of 4-pentenoate on urinary electrolyte excretion and renal metabolism. The intravenous administration of 4-pentenoate (1 mumol.kg-1.min-1 during 180 min) markedly increased the urinary excretion of bicarbonate, phosphate, potassium, amino acids, glucose, and various organic anions, whereas that of sodium and chloride also rose but less strikingly. These results suggest that 4-pentenoate markedly inhibits the proximal reabsorption of various solutes and therefore reproduces an experimental Fanconi's syndrome. Despite the rise in renal cortical concentration of alpha-ketoglutarate, glutamine utilization and total ammonia production expressed per 100 ml glomerular filtration rate increased following 4-pentenoate infusion, the ammonia being diverted into the renal vein. This increment in glutamine utilization was equal to the combined rise in the renal production of glutamate and alpha-ketoglutarate. By contrast, renal lactate utilization was drastically reduced. A causal relationship between the decreased renal cortical ATP concentration and the inhibited reabsorption of various solutes is suggested but cannot be established unequivocally.

Renal and extrarenal use of glutamine in normal and acidotic dogs.

The total body clearance of glutamine in five normal and four acidotic dogs was estimated from the kinetics of disappearance from the blood of 14C-[U]-L-glutamine administered in a central vein as a single bolus. The disappearance curve was analyzed as reflecting a biexponential phenomenon with both a mixing and a metabolism component occurring, respectively, in the extracellular (mixing) and intracellular compartment (metabolism). The apparent total body metabolism of glutamine (total body clearance x arterial concentration) was compared with the renal use of this aminoacid as directly determined by renal A-V differences and blood flow. It was demonstrated that the renal use of glutamine represents between 13% and 25% of the total body use, and was increased by acidosis, which did not change significantly the overall rate of synthesis or use.

Enzymatic basis of renal adaptation to acidosis in the dog.

The effect of pH on the activities of various enzymes along the ammoniagenic pathway was tested on dog kidney cortex homogenates in an attempt to identify the metabolic steps that could be directly influenced by a low pH in this species. The activity of alphaketoglutarate dehydrogenase was markedly stimulated by acidification of the medium that decreased abruptly the km for alphaketoglutarate. By contrast, succinyl CoA synthetase and malate dehydrogenase remained relatively insensitive to pH changes. The apparent km of malic enzyme for malate was markedly decreased by an acid medium. Therefore these findings suggest that an acid pH regulates the ammoniagenic pathway at two critical sites: alphaketoglutarate dehydrogenase and malic enzyme. These stimulated enzymatic activities may account for the changes in renal cortical concentrations of metabolites observed during metabolic or respiratory acidosis.

Metabolic effects of acetate on the heart.

The effects of various substrates (15 mM glucose, 5 mM glucose, 20 mM acetate, or a combination of these substrates) on the coronary blood flow and on the energetic status of myocytes were studied in isolated perfused rat hearts. We demonstrate that low level glucose (5 mM) or high concentration of acetate (20 mM) leads to a simultaneous fall in tissue ATP, rise in tissue adenosine, and significant increment in coronary blood flow. The latter effect is especially marked with 20 mM acetate. Dipyridamole (10(-6) M) does not enhance the vasodilatation induced by acetate. The provision of 15 mM glucose together with 20 mM acetate fully prevents these changes, indicating that the vasodilatation induced by acetate is probably mediated by metabolic changes. The evidence supports the concept that a redistribution of blood flow together with a fall in tissue ATP may explain some of the adverse effects of acetate dialysis in man, and suggests that the provision of glucose may alleviate these changes.

Effect of valproate on lactate and glutamine metabolism by rat renal cortical tubules.

The metabolic effects of sodium valproate (VPA) on rat renal cortical tubules have been examined. When 1 or 5 mM lactate was used as substrate in the incubation medium, VPA decreased markedly the lactate uptake by the tubules. When 1 or 5 mM glutamine was used, the addition of VPA accelerated glutamine uptake, ammoniagenesis, but also stimulated markedly the accumulation of lactate and pyruvate produced from glutamine. VPA had a dose-dependent inhibitory effect on gluconeogenesis from both glutamine and lactate. With 5 mM glutamine, VPA also induced a significant accumulation of glutamate in the medium. The oxygen consumption by the tubules was diminished by 40% following VPA addition. It is concluded that VPA modifies the metabolism of rat cortical tubules by interfering with the oxidation of natural substrates and stimulates in this fashion the production of ammonia by kidney tubules.

Metabolic effects of valproate on dog renal cortical tubules.

The effect of valproate (0.01-10 mM), an antiepileptic drug inducing hyperammonemia in humans, was studied in vitro on a suspension of renal cortical tubules (greater than 85% proximal tubules) obtained from six normal dogs. When these tubules were incubated with 1 mM glutamine, the addition of valproate accelerated glutamine uptake, ammoniagenesis, and the production of alanine, lactate, and pyruvate. With 5 mM glutamine, a rise in glutamate accumulation, a much greater synthesis of alanine, an important aspartate production, and a striking accumulation of lactate and pyruvate were observed. With 1 or 5 mM lactate, lactate utilization and gluconeogenesis were markedly reduced with increasing concentrations of valproate. Oxygen consumption was reduced by only 15-20% by 10 mM valproate. The accelerated glutamine utilization resulting from valproate could not be prevented by aminooxyacetate, an inhibitor of transamination. Valproate also reduced various enzymatic activities, a finding that could not explain its metabolic effects. Four sites of action may explain these various metabolic changes: (i) a stimulation of mitochondrial glutamine transport, (ii) an increase in the flux of glutamate to malate, and (iii) a reduction in the net oxidation of pyruvate and (iv) in the flux through pyruvate carboxylase.

Effect of valproate on renal metabolism in the intact dog.

Valproate is an antiepileptic drug known to induce hyperammonemia in humans. This hyperammonemia might result from a reduced detoxification of ammonium in the liver and/or from an accelerated renal ammoniagenesis. Six dogs with normal acid-base equilibrium and eight dogs with chronic metabolic acidosis were infused with valproate directly into their left renal artery in order to obtain arterial concentrations around 3 to 4 mM. The arterial ammonium concentration rose only in chronically acidotic dogs, whereas the lactate concentration and the lactate/pyruvate ratio increased in both groups. The urinary excretion of lactate and pyruvate increased markedly but the urinary excretion of other relevant metabolites remained minimal. Renal glutamine utilization and ammonium production were not changed by valproate administration in normal dogs but increased modestly in acidotic dogs. However, renal lactate utilization was drastically reduced and in fact, changed into a net production of lactate. Valproate strikingly reduced the renal cortical concentrations of glutamine, glutamate, alphaketoglutarate and citrate, and more modestly those of malate, oxaloacetate, aspartate, alanine and ATP. By contrast, the tissue lactate concentration and the lactate/pyruvate ratio were markedly increased. In experiments with brush border membrane vesicles, valproate inhibited the lactate transporter. These results suggest that high concentrations of valproate drastically inhibited the proximal reabsorption and the proximal and distal oxidation of lactate and pyruvate. Valproate probably became itself a significant energetic substrate for the kidney.

Characterization and metabolism of canine proximal tubules, thick ascending limbs, and collecting ducts in suspension.

Preparations of distinct nephron segments were obtained from dog kidneys by collagenase treatment. Four morphologically different tissues were isolated: glomeruli, proximal tubules, thick ascending limbs, and papillary collecting ducts. Each segment possessed a characteristic assay of membrane-bound and cytoplasmic enzymes. Specific metabolic characteristics also were found: gluconeogenesis and ammoniagenesis in proximal tubules, glycolytic aerobic metabolism in thick ascending limbs, and glycolytic anaerobic metabolism in papillary collecting ducts. The assay of Na+ -K+ ATPase, H+ -ATPase, and Ca2+ -ATPase activities in these nephron segments demonstrated a specific enrichment of Na+ -K+ ATPase in thick ascending limbs, and of H+ -ATPase in proximal tubules and papillary collecting ducts. Tubular respiration in the absence or presence of ouabain, 1,3-dicyclohexylcarbodiimide, or furosemide demonstrated that the respiration of each segment could be correlated to the activity of specific ion motive ATPases. Furthermore, a tight coupling between ion transport, ATP turnover, and substrate oxidation was demonstrated. These isolated tubular structures are thus viable and capable of transepithelial transport. Our preparation provides large amounts of defined population of tubules and are thus useful for the study of biochemical and functional heterogeneity along the nephron.