Metabolic activities of the isolated perfused rat kidney

1. A technique for perfusing the isolated rat kidney is described. It is primarily designed for the study of renal metabolism but is also suitable for studying some aspects of the secretory function; this was normal with respect to minimal glucosuria. The glomerular filtration rate as measured by creatinine clearance was lower than in vivo and slowly decreased with time. 2. Gluconeogenesis from a variety of precursors was rapid and similar to that in kidney-cortex slices, in contrast with liver where the perfused organ is more effective than slices. Whereas the maximal rates of gluconeogenesis from glycerol and pyruvate were similar in liver and kidney, the rates from succinate, malate and fumarate were 14–20 times, and those from glutamate and aspartate about three times, as high in the kidney. 3. The oxygen consumption of the perfused organ was about twice that of cortex slices, presumably because of the secretory work done in the perfused organ but not in slices. 4. The rate of acetoacetate oxidation was about the same in the perfused organ and in slices but, because of the higher rate of oxygen consumption, the percentage contribution of acetoacetate to the fuel of respiration was lower in the perfused organ. The results suggest that acetoacetate can supply energy for the basal requirements and for gluconeogenesis but not for the secretory work. 5. Glutamine was formed at a high rate from glutamate and at a lower rate from aspartate. The high rates indicate that, in the rat, the kidney is a major source of body glutamine.     

Dopamine production by the isolated perfused rat kidney

We used isolated perfused rat kidneys to examine dopamine (DA) production and its relation to renal function. Both innervated and chronically surgically denervated kidneys perfused with a solution containing neither albumin nor tyrosine, excreted 0.2 +/- 0.1 ng DA X min-1 X g wet weight-1 during the 10-min collection period between 30 and 40 min after starting perfusion. When perfused with 6.7% albumin, without tyrosine, innervated kidneys excreted 1.0 +/- 0.06 ng DA X min-1 X g-1 and denervated kidneys excreted 1.0 +/- 0.07 DA X min-1 X g-1. When 0.03 mM tyrosine was included in the albumin perfusate, innervated kidneys excreted 1.2 +/- 0.1 ng DA X min-1 X g-1 (p less than 0.1). Under these conditions DA excretion continued for at least 100 min at which time it was 0.6 ng X min-1 X g-1 and 86 ng/g kidney weight had been excreted. Denervated kidneys perfused with albumin + tyrosine excreted 0.9 +/- 0.13 ng DA X min-1 X g-1. Renal stores of free DA, conjugated DA, and dihydroxyphenylalanine (DOPA) could have provided at the most 30 ng/g of DA. Carbidopa inhibited DA excretion completely. DA excretion did not correlate with renal vascular resistance, inulin clearance, or fractional sodium excretion. In summary, nonneural tissue in isolated perfused kidneys produced DA at the same rate as denervated kidneys in vivo. Less than one-third of the DA produced by isolated kidneys could have come from intrarenal stores of DOPA, free DA, and conjugated DA; the rest was synthesized from unknown precursors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alanine transport by the isolated perfused rat kidney

L-alanine transport kinetics were examined in the isolated perfused rat kidney (1) using different perfusate concentrations of alanine (PAla) to obtain different filtered loads and (2) under conditions of osmotic diuresis. The transport maximum for alanine (TmAla) was found to be very high relative to $\text{in}$$\text{vivo}$ filtered loads of alanine. The apparent TmAla was dependent on glomerular filtration rate (GFR) and it could be modified by osmotic diuresis. It is suggested that the variation of TmAla with changes in GFR may be the consequence of variations in fractional volume flow through the proximal tubule.     

Carbohydrate metabolism in the isolated perfused rat kidney

1. Anaerobic formation of lactate from glucose by isolated perfused rat kidney (411mumol/h per g dry wt.) was three times as fast as in aerobic conditions (138mumol/h per g). 2. In aerobic or in anaerobic conditions, the ratio of lactate production to glucose utilization was about 2. 3. Starvation or acidosis caused a decline of about 30% in the rate of aerobic glycolysis. 4. The rate of formation of glucose from lactate by perfused kidney from a well-fed rat, in the presence of 5mm-acetoacetate (83mumol/h per g dry wt.), was of the same order as the rate of aerobic glycolysis. 5. During perfusion with physiological concentrations of glucose (5mm) and lactate (2mm) there were negligible changes in the concentration of either substrate. 6. Comparison of kidneys perfused with lactate, from well-fed or starved rats, showed no major differences in contents of intermediates of gluconeogenesis. 7. The tissue concentrations of hexose monophosphates and C(3) phosphorylated glycolytic intermediates (except triose phosphate) were decreased in anaerobic conditions. 8. Aerobic metabolism of fructose by perfused kidney was rapid: the rate of glucose formation was 726mumol/h per g dry wt. and of lactate formation 168mumol/h per g (dry wt.). Glycerol and d-glyceraldehyde were also released into the medium. 9. Aerobically, fructose generated high concentrations of glycolytic intermediates. 10. Anaerobic production of lactate from fructose (74mumol/h per g dry wt.) was slower than the aerobic rate. 11. In both anaerobic and aerobic conditions the ratio [lactate]/[pyruvate] in kidney or medium was lower during perfusion with fructose than with glucose. 12. These results are discussed in terms of the regulation of renal carbohydrate metabolism.     

Dipeptide metabolism in the isolated perfused rat kidney

Renal handling of glycyl-proline was studied in the isolated perfused rat kidney. Glycyl-proline disappeared from the perfusate as a function of time. The dipeptide was freely filtered at the glomerulus but only 6% of the filtered load was excreted in the urine as the intact peptide. More than 90% of the filtered dipeptide was reabsorbed as the intact peptide and/or its hydrolytic products. Non-filtration mechanisms were also involved to a significant extent in the clearance of the peptide. Hydrolysis at intratubular, intracellular and peritubular sites all contribute to the disappearance of the dipeptide from the perfusate, though the relative contributions of each mechanism are not known. Significant metabolic conversions, especially the conversion of glycine to serine, were also observed during perfusion.     

Basal prostaglandin synthesis by the isolated perfused rat kidney

In order to assess the main characteristics of the prostaglandin (PG) biosynthesis by the isolated perfused rat kidney, the urinary and venous outputs of PGE2, PGF2alpha, 6-keto-PGF1alpha and of thromboxane (Tx)B2 were followed during 120 min after an equilibration period of 30 min. Single pass kidneys were perfused with a Krebs-Henseleit solution added with Polygeline at a constant flow rate providing a perfusion pressure about 90 mm Hg. From the beginning of the study, major differences could be observed in the renal biosynthetic rate of the 4 PG studied which were mainly excreted into the venous effluent. During the perfusion, urinary and venous outputs of PGE2, PGF2alpha and of TxB2 remained stable whereas those of 6-keto-PGF1alpha sharply increased and were found inversely related to the glomerular filtration rate (r = -0.95; p n 0.001). Finally, the urinary and venous outputs of each of the four PGs studied were found positively related. It is concluded that the isolated perfused rat kidney is a valuable preparation for studying the biosynthesis of PGs and that, at least in thi model, the urinary excretion of PGs is a good index of their renal synthesis.     

The shunt pathway of mevalonate metabolism in the isolated perfused rat kidney

The shunt pathway of mevalonate metabolism (Edmond, J., and Popják, G. (1974) J. Biol. Chem. 249, 66-71) has been studied in isolated kidneys from rats perfused with physiological concentrations of variously labeled [14C]- and [3H]mevalonates. The rate of operation of the shunt pathway was quantified by the production of either 14CO2 or 3H2O from the tracers. The measured rates of 14CO2 production from [14C] mevalonate were converted to rates of mitochondrial acetyl-CoA production by methods which take into account underestimations of metabolic rates derived from 14CO2 production. We have shown that the sex difference in renal shunting of mevalonate (Wiley, M. H., Howton, M. M., and Siperstein, M. D. (1979) J. Biol. Chem. 254, 837-842) occurs at physiological levels of substrate. The shunt pathway diverts up to 17% of the flux of mevalonate entering the cholesterol synthesis pathway in the kidney. It may, therefore, play a role in the long term regulation of cholesterol synthesis in this organ, as had been hypothesized by Edmond and Popják.     

Volume-regulatory potassium release from isolated perfused rat kidney

The present study has been performed to test for cell volume regulatory potassium release from the isolated perfused rat kidney exposed to hypotonic perfusate and for its sensitivity to potassium channel blocker barium and calcium channel blocker verapamil. Replacement of 25 mmol/l NaCl with 50 mmol/l mannitol has little effect on effluent potassium activity, whereas subsequent omission of mannitol from the perfusate leads to a transient increase of effluent potassium activity, reflecting volume regulatory potassium release. Barium (1 mmol/l) leads to a marked transient decrease of effluent potassium activity, pointing to net cellular uptake of potassium. Verapamil (1 mumol/l) leads to a slight decrease of effluent potassium activity. Both barium and verapamil virtually abolish the rapid, transient increase of effluent potassium activity upon exposure to hypotonic perfusates. Thus, the substances either block or markedly retard volume regulatory potassium release. The apparent renal vascular resistance is transiently increased by exposure to hypotonic perfusates and by barium, but is reduced by verapamil. Cell volume regulation of isolated perfused mouse straight proximal tubules is retarded but not abolished by verapamil (0.1 mmol/l). In conclusion, cellular potassium release from rat kidney can be determined by continuous measurement of effluent potassium activity. The volume regulatory potassium release and cell volume regulation are impaired by both barium and verapamil. The persisting cell volume regulation could be due either to slow potassium release and/or some mechanism independent of potassium.     

Cortisol metabolism and excretion in the isolated perfused rat kidney

The isolated perfused rat kidney allows a simultaneous kinetic study of both the renal metabolism and the urinary excretion of cortisol and its metabolites in the rat. In this system, cortisol was completely metabolized within 120 minutes. The main renal metabolites of cortisol (cortisone, 20 reduced cortisol and 20 reduced cortisone) were found in the recirculating perfusate and in urine. The formation of these metabolites was quantitatively evaluated and compared to a theoretical model.     

Nitrendipine-induced stimulation of renin release by the isolated perfused rat kidney

The direct effects of the organic calcium antagonist nitrendipine upon renin release were assessed using the isolated rat kidney perfused at constant pressure. This model circumvents the indirect actions of vasodilating agents by artificially maintaining perfusion pressure constant, thereby avoiding the hypotensive effects associated with the systemic administration of such agents. Renin release as assessed by radioimmunoassay was stimulated 2.6-fold upon the administration of 10(-6) M nitrendipine. Since this stimulation of renin release occurred in the absence of any alteration in perfusion pressure, we conclude that it represents a direct action of nitrendipine. This finding is in support of the current hypothesis concerning the inverse relationship between cytosolic Ca2+ and renin secretory rate, and suggests that Ca entry into the juxtaglomerular cells of the juxtaglomerular apparatus is sensitive to blockade by organic calcium antagonists such as nitrendipine.     

Metabolism of cysteinyl leukotrienes by the isolated perfused rat kidney.

The metabolism of cysteinyl leukotrienes by the isolated perfused rat kidney was investigated. For this purpose LTC4, LTD4 or LTE4 were studied in separate experiments. The isolated perfused rat kidney metabolized all cysteinyl leukotrienes to the final metabolite N-acetyl-LTE4. In the presence of 5% albumin 50% of LTC4 was metabolized to LTD4 (22%), LTE4 (15%) and N-acetyl-LTE4 (13%) within 60 min. Excretion of radioactivity into urine was less than 1%. In contrast, in the absence of albumin, LTC4 was completely metabolized within 45 min to N-acetyl-LTE4, the sole and final metabolite of LTC4 found in the perfusion medium as well as in urine. After 60 min 19% and 42% of total radioactivity were found in the perfusion medium and in urine, respectively. Isolated glomeruli metabolized LTC4 to LTD4 and to LTE4 but not to N-acetyl-LTE4 at a rate comparable to the rate observed by the isolated perfused kidney in the absence of albumin. In contrast to isolated glomeruli isolated tubuli metabolized LTE4 to N-acetyl-LTE4 at a rate comparable to that observed by the isolated perfused kidney in the absence of albumin. The present study shows that the isolated perfused rat kidney metabolizes cysteinyl leukotrienes to the sole and final metabolite N-acetyl-LTE4. In the presence of albumin metabolism is slowed down and excretion of N-acetyl-LTE4 into urine is prevented.     

Carboplatin-induced alteration of the thiol homeostasis in the isolated perfused rat kidney

Carboplatin elicits minor side effects with respect to its first generation analogue cisplatin. Nevertheless, a dose-dependent nephrotoxicity of the drug has been reported to occur both in patients and in rats and a possible pathogenic role have been attributed to oxidative stress. We have studied the effect of carboplatin administration on the thiol/disulfide balance, on other biomarkers of oxidative stress and on antioxidant enzymes in the isolated perfused rat kidney. A 5-500 μM carboplatin dose range did not alter renal function but significantly decreased levels of cysteine, glutathione and exposed protein sulfhydryl groups. Only a minimal increment in disulfides was observed, whereas malonyldialdehyde and protein carbonyls did not increase significantly. Among the antioxidant enzymes studied, only thioltransferase was inhibited by the treatment. Our data suggest that a minimal oxidative stress is present under our experimental conditions, thus indicating that platinum-based drugs do not produce significant amount of reactive oxygen species.     

Serine synthesis by an isolated perfused rat kidney preparation.

The isolated perfused rat kidney was shown to synthesize serine from aspartate or glutamate, both of which are also precursors of glucose. The major products of aspartate metabolism were ammonia, serine, glutamate, glucose, glutamine and CO2. Perfusion of kidneys with aspartate in the presence of amino-oxyacetate resulted in a near-complete inhibition of aspartate metabolism, illustrating the essential role of aspartate aminotransferase in the metabolism of this substrate. Radioactivity from 14C-labelled aspartate and from 14C-labelled glycerol was incorporated into serine and glucose. Production of both glucose and serine from aspartate was suppressed in the presence of 3-mercaptopicolinic acid. These data provide evidence for the operation of the phosphorylated and/or non-phosphorylated pathway for serine production to the presence of 3-mercaptopicolinic acid. This is explained by simultaneous glycolysis. The rate of glucose production, but not that of serine, was greater in kidneys perfused with glutamate or with aspartate plus glycerol than the rates obtained by perfusion with aspartate alone. These data are taken to suggest that serine synthesis occurred at a near-maximal rate, and that the capacity of the kidney for serine synthesis from glucose precursors is lower than that for glucose synthesis.     

Melatonin prevents cyclosporine-induced nephrotoxicity in isolated and perfused rat kidney

Cyclosporine A (CsA) is a potent and effective immunosuppressive agent, but its action is frequently accompanied by severe renal toxicity. The precise mechanism by which CsA causes renal injury is not known. Reactive oxygen species (ROS) have been shown to play a role, since CsA-induced renal lipid peroxidation is attenuated in vivo and in vitro by the concomitant administration of antioxidants such as vitamin E. We show here the effect of the antioxidant melatonin (MLT), a hormone produced by the pineal gland during the dark phase of the circadian cycle, in a model of CsA nephrotoxicity in the isolated and perfused rat kidney. Kidneys isolated from rats were divided into seven groups. At the end of perfusion, malondialdehyde and 4-hydroxyalkenals (MDA+4-HDA), metabolites of nitric oxide N O 2 - +N O 3 - were measured and histopathological examination was performed. CsA treatment induced a significant increase in MDA+4-HDA while not affecting the nitric oxide metabolite level. MLT remarkably prevented glomerular collapse and tubular damage as revealed by morphometric analysis. Our study suggests that lipid peroxidation is an early important event in the pathogenesis of CsA nephrotoxicity and that MLT is able to protect kidneys from CsA at a relatively low concentration.     

Synthesis of bile acid monosulphates by the isolated perfused rat kidney.

Perfusion of an isolated rat kidney with labelled bile acids, in a protein-free medium, resulted in the urinary excretion of the labelled bile acid, 3% being converted into polar metabolities in 1h. These metabolities were neither glycine nor taurine conjugates, nor bile acid glucuronides, and on solovolysis yielded the free bile acid. On t.l.c. the metabolite of [24-14C]lithocholic acid had the mobility of lithocholate 3-sulphate. The principal metabolite of [24-14C]chenodeoxycholic acid had the mobility of chenodeoxycholate 7-sulphate; trace amounts appeared as chenodeoxycholate 3-sulphate. [35S]sulphate was incorporated in chenodeoxycholic acid by the kidney, resulting in a similar pattern of sulphation. No disulphate salt of chenodeoxycholic acid was detected. These findings lend support to the hypothesis that renal synthesis may account for some of the bile acid sulphates present in urine in the cholestatic syndrome in man.     

Influence of cyclosporin A on the respiration of isolated rat kidney mitochondria

In vitro exposure of isolated rat kidney mitochondria to cyclosporin A, a new immunosuppressive agent with serious nephrotoxic side-effects, leads to alterations of both succinate- and glutamate plus malate-supported respiration in a dose-related manner. ADP- and 2,4-dinitrophenol-stimulated respiration, respiratory control indices, and ADP/O ratios are decreased. The mitochondrial alterations are discussed as possible pathogenetic reasons of cyclosporin A nephrotoxicity.     

The localization of renal glutathione oxidase activity studied in the isolated, perfused rat kidney

The metabolism of extracellular glutathione was studied in the isolated, perfused rat kidney. Both recirculating and single-pass perfusions were associated with rapid conversion of reduced glutathione to glutathione disulfide in the perfusate. Only a minor fraction of perfusate glutathione was recovered in urine; however, this fraction was markedly increased in the presence of the inhibitor of γ-glutamyltransferase, serine·borate. In contrast, serine·borate had no effect on either oxidation or disappearance of perfusate glutathione. The results indicate that renal glutathione oxidase activity is restricted to glutathione present in plasma, while γ-glutamyltransferase acts on glutathione in the glomerular filtrate.