Patrick Vinay

The increase of cGMP by atrial natriuretic factor correlates with the distribution of particulate guanylate cyclase

We have demonstrated previously that atrial natriuretic factor (ANF) augments urinary, plasma and kidney cGMP levels but has no significant effect upon cAMP. Using cGMP as a marker, we searched for specific target sites involved in the action of ANF in the dog kidney, and observed no changeof cGMP in the proximal tubules, a 2‐fold increase over basal levels in the thick loop of Henle and a 3‐fold elevation in the collecting duct. The most striking action on cGMP occurred in the glomeruli with a rise of up to 50‐fold being evident at 1–2 min. after the addition of ANF. The results obtained in the absence or presence of a phosphodiesterase inhibitor support the notion that the effects of ANF were exerted at the level of guanylate cyclase stimulation rather than cGMP phosphodiesterase inhibition. The action of sodium nitroprusside (SNP), a direct stimulator of soluble guanylate cyclase, differed from that of ANF. The ability of the factor to enhance cGMP levels was correlated with the distribution of particulate guanylate cyclase. This study identifies the glomeruli and the distal part of the nephron as specific targets of ANF and implicates particulate guanylate cyclase as the enzyme targetted for the expression of its action.

Hypoxemia During Hemodialysis: A Critical Review of the Facts

The literature describing the fall in PaO2 during dialysis is intensively and critically reviewed. This phenomenon is related to both the type of membrane used (cellulosic v noncellulosic membrane), and to the composition of the dialysate (acetate v bicarbonate). It appears that a ventilation/perfusion mismatch due to pulmonary leukostasis can, in part, explain hypoxemia in patients dialyzed with cellulosic membranes. This phenomenon is especially apparent in patients with preexisting pulmonary abnormalities. However, hypoventilation remains the major cause of hypoxemia. This hypoventilation is mainly due to CO2 consumption during acetate metabolism (acetate dialysis), or alkalinization of the blood (bicarbonate dialysis). The metabolic consequences of acetate metabolism, and of bicarbonate and CO2 losses through the dialyzer are critically analyzed. The cause for the increment in oxygen consumption during acetate dialysis is examined. Finally, the respective role of these combined factors are described and used to explain the changes in VCO2, VO2, respiratory quotient (RQ), and PaO2 reported in the literature during dialysis against acetate and/or bicarbonate.

Distribution of atrial natriuretic factor receptors in dog kidney fractions

Specific receptors for atrial natriuretic factor were studied in purified glomeruli, proximal tubules, thick ascending limbs of Henles loops and collecting ducts from dog kidney. Glomeruli contain the highest concentration of receptor sites (pK = 9.9, B max = 200 protein), followed by collecting ducts (pK = 9.4, B max = 150 ). Low levels of receptor sites were also detectable in thick ascending limbs of Henles loops (pK = 9.4, B max = 36 ) while proximal tubules were completely devoid of specific binding sites. These results indicate that the glomeruli appear to be the primary site of interaction of artrial natriuretic factor in kidney cortex but that it might also act in the medulla on lower nephron tubular function.

Impact of nitric oxide on blood pressure in hemodialysis patients

Nitric oxide (NO) is a powerful vasoactive agent that contributes to the regulation of blood pressure (BP). However, the role of NO in uremic patients and during the course of hemodialysis is still debated. Blood L-arginine concentrations and exhaled NO concentrations were measured in 22 healthy controls and in 22 hemodialysis patients before and after dialysis. On the basis of their BP response during hemodialysis, the patients were divided into three groups: 6 of the 22 patients presented with a decrease in BP during dialysis (group 1), eight presented with a stable BP (group 2), and eight with an increase in BP (group 3). The exhaled NO concentration was higher in dialysis patients than in healthy controls (22.7 +/- 2.6 ppb in dialysis patients v 16.7 +/- 0.9 ppb in controls, mean +/- SEM, P = 0.044). The predialysis and postdialysis exhaled NO concentrations were inversely correlated with the change in BP during hemodialysis (r = -0.47, P = 0.013). Patients with a decrease in BP (group 1) had the highest NO concentrations; patients with an increase in BP (group 3) had the lowest values; and patients with a stable BP during the course of dialysis (group 2) had intermediary values (trend test, P = 0.0291). In addition, both the exhaled NO concentration and the blood L-arginine concentration decreased during dialysis in all patients (P = 0.005 and P = 0.001, respectively). These results provide several novel insights into NO metabolism and BP regulation during hemodialysis: (1) maintenance hemodialysis is associated with a chronic increase in NO concentrations; (2) changes in BP during hemodialysis are inversely correlated with exhaled NO concentrations, higher NO levels being associated with a decrease in BP and lower NO levels with an increase in BP during dialysis; (3) blood L-arginine levels decrease during hemodialysis, and this reduction may in turn influence NO production.

Renal Hemodynamics and Ammoniagenesis: CHARACTERISTICS OF THE ANTILUMINAL SITE FOR GLUTAMINE EXTRACTION

Renal production of ammonia by the left kidney was studied in 31 acidotic dogs (NH(4)Cl) after acute constriction of the renal artery. Renal ammoniagenesis fell in direct proportion with the reduction in glomerular filtration rate and renal plasma flow. The renal extraction of glutamine by the experimental kidney fell in direct proportion with the reduction in renal hemodynamics. Extracted glutamine remained greater than filtered glutamine indicating that both the luminal and antiluminal transport sites were operative. The relationship between renal extraction of glutamine and ammoniagenesis observed during control was maintained after renal artery constriction (1.7 mumol NH(3) produced for each mumol of glutamine extracted). Systemic venous or renal intra-arterial infusion of glutamine during arterial constriction increased renal production of ammonia to or above control values. These observations indicate that the mechanisms responsible for glutamine extraction and ammonia production were operating normally despite reduced hemodynamics. When measured immediately after arterial clamping, the renal venous pNH(3) was found to rise significantly decreasing progressively thereafter towards control values. The extracted fraction of total glutamine delivered to the kidney (31%) did not change after acute reduction of the glutamine load. Thus, the antiluminal extraction site was incapable of lowering renal venous plasma glutamine concentration below 0.33 muM/ml. In a second series of experiments, the properties of the antiluminal site of transport for glutamine were studied after complete occlusion of the left ureter in acidotic and nonacidotic animals. Under these circumstances, it was demonstrated that the antiluminal site is capable of extracting sufficient glutamine to maintain total ammonia production at 60% or more of control. In acidotic animals, changes in cellular pNH(3) appeared to play a key role on the antiluminal extraction of glutamine since the significant rise in renal blood flow often observed after ureteral occlusion prevented the rise in pNH(3) noted when blood flow remained constant. Thus, when renal blood flow rose glutamine extraction and ammonia production were maintained at control values. In these acidotic animals, glutamine infusion failed to influence ammonia production until luminal transport was restored by release of ureteral clamp and resumption of glomerular filtration. The latter observation establishes that reabsorbed glutamine is utilized at least in part for ammonia production.

The effect of ketone bodies on renal ammoniogenesis

Infusion of ketone bodies to ammonium chloride-loaded acidotic dogs was found to induce significant reduction in urinary excretion of ammonia. This effect could not be attributed to urinary pH variations. Total ammonia production by the left kidney was measured in 25 animals infused during 90 min with the sodium salt of D,L-beta-hydroxybutyric acid adjusted to pH 6.0 or 4.2. Ketonemia averaged 4.5 mM/liter. In all experiments the ammonia content of both urine and renal venous blood fell markedly so that ammoniogenesis was depressed by 60% or more within 60 min after the onset of infusion. Administration of equimolar quantities of sodium acetoacetate adjusted to pH 6.0 resulted in a 50% decrease in renal ammonia production. Infusion of ketone bodies adjusted to pH 6.0 is usually accompanied by a small increase in extracellular bicarbonate (3.7 mM/liter). However infusion of D,L-sodium lactate or sodium bicarbonate in amounts sufficient to induce a similar rise in plasma bicarbonate resulted in only a slight decrement in ammonia production (15%). The continuous infusion of 5% mannitol alone during 90-150 min failed to influence renal ammoniogenesis. Infusion of pure sodium-free beta-hydroxybutyric acid prepared by ion exchange (pH 2.2) resulted in a 50% decrease in renal ammoniogenesis in spite of the fact that both urinary pH and plasma bicarbonate fell significantly. During all experiments where ketones were infused, the renal extraction of glutamine became negligible as the renal glutamine arteriovenous difference was abolished. Renal hemodynamics did not vary significantly. Infusion of beta-hydroxybutyrate into the left renal artery resulted in a rapid decrease in ammoniogenesis by the perfused kidney. The present study indicates that ketone bodies exert their inhibitory influence within the renal tubular cell. Since their effect is independent of urinary or systemic acid-base changes, it is suggested that they depress renal ammoniogenesis by preventing the transformation of glutamine and glutamate into alpha-ketoglutarate in the mitochondria of the renal tubular cell.

Metabolic changes in skeletal muscle during chronic metabolic acidosis.

1. 1. In an attempt to clarify the importance of skeletal muscle as a source of glutamine during chronic metabolic acidosis, in vivo and in vitro experiments were performed in normal and NH4Cl acidotic rats. 2. 2. In the acidotic animals, arterial glutamine, glutamate and alanine were significantly lower than in normal rats. The arterio-venous difference for glutamine across the hindlimb was not statistically different in acidotic and normal animals. 3. 3. No net release or uptake of glutamate was observed in either control or acidotic rats. However the uptake of alanine observed in normal animals was completely abolished in the acidotic rats. 4. 4. The metabolite profile of freeze-clamped hindlimb skeletal muscle from acidotic animals revealed a lower concentration of glutamine, glutamate, alanine, aspartate, alphaketoglutarate, lactate and pyruvate and increased ammonia concentration. 5. 5. Alanine aminotransferase activity of the hindlimb skeletal muscle was unchanged by acidosis. 6. 6. In vitro glutamine production by the isolated rat diaphragm was not different in acidotic and control animals. 7. 7. The addition of various substrates (valine, leucine and NH4Cl) stimulated glutamine production to similar extent in control and acidotic animals. 8. 8. While alanine was produced by the diaphragm of the normal animals, it did not occur in the acidotic rats. Furthermore the addition of substrates did not stimulate alanine production by the diaphragm of the acidotic animals. 9. 9. Addition of methionine sulfoximine (MSO) 5 mM to inhibit diaphragm glutamine synthetase activity resulted in a 55% decrease in glutamine synthesis in both normal and acidotic animals. 10. 10. The production of glutamine or alanine was not affected by acidification of the incubation medium from pH 7.4 to 7.0. 11. 11. Our findings suggest that the capacity for glutamine synthesis by the skeletal muscle is not affected by metabolic acidosis. 12. 12. The fall in glutamate and the rise in ammonia concentrations in the skeletal muscle in vivo during metabolic acidosis suggest that glutamate availability is rate-limiting for the production of glutamine. This could explain the marked variations in alanine metabolism. 13. 13. In view of the very small changes observed in glutamine production by the skeletal muscle during acidosis, the role of other tissues such as the liver, the brain and, in the rat, the kidney, should be envisaged. 14. 14. The possibility of a significant decrease in glutamine metabolism by the intestine during acidosis making more glutamine available for renal extraction should also be seriously considered.

Effect of Valproate on Lactate and Glutamine Metabolism by Rat Renal Cortical Tubules

Abstract 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.

Response of the rat and dog kidney to H+ concentration in vitro—A comparative study with slices and tubules

Abstract 1. 1. The present study reevaluates the metabolic response of rat and dog renal cortical tissue to changes in H + concentration of incubation medium and compares the response of kidney slices to (hat of isolated tubules. 2. 2. In the rat, acidification (pH 7.1) depresses glutamate accumulation and enhances gluconeogenesis with both slices and tubules, but glutamine extraction and ammonia production increase significantly only with tubules. 3. 3. Alkalinization (pH 7.7) depresses glucose and ammonia production and enhances glutamate accumulation with both slices and tubules but glutamine extraction is also slightly stimulated with tubules only. 4. 4. Thus, in the rat, variations of incubation medium pH modulates (a) glutamate utilization and (b) glutamine transport from the medium to the mitochondria, the second effect being apparent with tubules only. The different response observed with slices and tubules is attributed to a difference of permeability for glutamine between these two preparations. 5. 5. The intracellular metabolite profile of tubules rapidly separated from the incubation medium after 30 min suggests that in vitro acidosis increases and alkalosis decreases glutamate utilization and ammonia production probably by affecting the activity of the alpha-ketoglutarate dehydrogenase complex. 6. 6. In the dog, no effect of H + concentration on glutamine transport or utilization can be seen. However, H + concentration modifies glutamate accumulation and stimulates gluconeogenesis (from glutamate and endogenous substrates). 7. 7. Both effects are in accord with observations in the rat and may involve similar mechanisms.

A Brush Border Membrane-Bound H+ -ATPase from the Dog Proximal Tubule

We have characterized the H+-ATPase activity (ATP hydrolysis and proton transport) of brush border membranes (BBMs) isolated from the dog kidney cortex. In solubilized BBMs, two thirds of t