Eur. surg. Res. 9: 206-216 (1977)
Metabolism in the Hypothermically Perfused Dog Kidney Incorporation Rate of Leucine and Threonine into Proteins
Sven L undstam , R udolf J agenburg , O lof J onsson , Kent L undholm , J acob N aucler, Silas P ettersson and T ore Schersten Surgical Metabolic Research Laboratory, Departments of Clinical Chemistry, Surgery and Urology, Sahlgrenska sjukhuset, University of Göteborg, Göteborg
Key Words. Dog kidney • Hypothermic perfusion • Protein metabolism • [u C]Ieucine • [,4C]threonine Abstract. The incorporation of [u C]leucine and [14C]threonine into kidney cor tex proteins was studied during 6 days’ hypothermic perfusion of dog kidneys at 8-10 °C and during in vitro incubation of dog kidney cortex slices at 37 °C. Leucine carbon was incorporated into proteins at a higher rate than threonine carbon both during in vitro incubation of kidney cortex slices and during hypo thermic kidney perfusion. The incorporation of leucine and threonine during hypo thermic perfusion was linear for 6 days but 50-100 times lower than the incorpora tion of leucine and threonine in kidney cortex slices at 37 °C. During hypothermic perfusion there was a decrease in specific activity of leucine and threonine in the perfusate corresponding to a degradation of proteins which was greater than protein synthesis as calculated from the incorporation of label into pro teins. Leucine carbon was recovered in C 0 2 during hypothermic perfusion and in vitro incubation of kidney cortex slices at 37 °C. The incorporation of threonine carbon into C 0 2 was about 10°/o of the corresponding value for leucine both during hypo thermic kidney perfusion and during in vitro incubation of kidney cortex slices at 37 °C. It is concluded that there is a turnover of kidney proteins during hypothermic perfusion with a perfusate containing amino acids.
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Received: October 20, 1976; accepted: January 31, 1977.
Lundstam /J agenburg/J onsson/L undholm /N auclér/P ettersson/S cherstén
207
Amino acids are metabolized in the hypothermically perfused dog kid ney as indicated by amino acid uptake from the perfusate and release to the perfusate [13]. Glutamine, proline and glycine dominated the uptake and alanine and ammonia the release of nitrogen in accordance with the condition in vivo [18]. Uptake and release of amino acids are not neces sarily associated with an active turnover of proteins. The a-amino group of the amino acids may be utilized predominantly in the formation of alanine and ammonia [4, 20] and the carbon skeleton may be used for ox idation and gluconeogenesis [3]. The present study was designed to evaluate protein synthesis and deg radation during hypothermic perfusion of dog kidneys. As marker of pro tein synthesis, the incorporation rate of leucine and threonine carbon into proteins was determined during hypothermic perfusion and for compari son during in vitro incubation of kidney cortex slices at 37 °C.
Material and Methods 24 kidneys from adult mongrel dogs were used. The dogs were fasted but had free access to water for 24 h before the operation. Nephrectomy and treatment of the kidney before connection to the perfusion machine were performed according to previous descriptions [12,19]. The mean kidney weight was 84 + (SD) 16 g. Perfusion Procedure The perfusion was performed at 8-10 °C in a Gambro perfusion machine (Gambro, PF 2D, Lund, Sweden). The perfusion was run according to our previous de scription [13]. The perfusate was based on human albumin with addition of electrolytes of ex tracellular concentration, glucose, 17 L-amino acids, insulin, hydrocortisone, and benzyl-penicillin in concentrations as described previously [13]. [U-,4C]i.-Ieucine or [U-14C]L-threonine was added to the perfusate as presented in figures 2 and 3. In two experiments, puromycin hydrochloride was added to the perfusate to a final concentration of 210 «m ol'L 1. Before perfusion was started, and at various time intervals during perfusion, samples were taken from the perfusate to determine the concentration of amino acids and the incorporation of radioactive carbon into amino acids and C 02. A biopsy from cortex was taken after 1 h of perfusion and then after 1, 2, 4, and 6 days to determine the incorporation of label into tissue proteins.
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Tissue Incubation Procedure Cortical biopsies were taken for in vitro determination of incorporation rate of amino acid carbon into C 0 2 and proteins. This tissue was sliced in a Mcllwain tissue chopper (Mickle Laboratory Engineering Company, England). The kidney slices
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(0.4 mm) were washed in ice-chilled Krebs-Ringer bicarbonate (KRB) buffer solu tion, blotted and rapidly weighed on a torsion balance. They were then placed in Hagedorn tubes containing 4 ml of the medium. The flasks were gassed with 0 2:C02 (95:5), sealed with rubber stoppers, and incubated at 37 °C in a metabolic shaker with constant agitation, 80 strokes per minute. The incubation medium consisted of KRB buffer, pH 7.4, and a mixture of 19 L-amino acids at a concentration corre sponding to that of normal human plasma [22] or 10 times that of human plasma. [U-14C]L-leucine or [U-,4C]L-threonine, about 0.5 /iCi, was added to the incubation medium. The incorporation of label into proteins was studied at plasma concentra tion of amino acids with and without addition of insulin (25 UT"1) and at 10 times plasma concentration without insulin. The incubation time was 4 h. Kidney slices were incubated for 1 h in a medium with plasma concentration of amino acids for determination of the incorporation rate of label into C 02. After incubation, the kidney slices were homogenized with an Ultra-Turrax homogenizer (Jankel & Kunkel, FRG). The incorporation rate of amino acid carbon into proteins and CO, was calculated by dividing the radioactivity in proteins and C 0 2, respectively, with the specific activity in the incubation medium. Protein Preparation After homogenization of the kidney biopsies and slices, 10 ml 10% trichloroacet ic acid (TCA) were added and the mixture was heated to 80 °C for 40 min. After centrifugation the sediment was resuspended in 5 ml 5% TCA. The insoluble materi al was extracted for lipids with ethanol acetone (1:1) at 40 °C for 60 min. This ex traction was repeated twice at 20 °C and finally with 5 ml ether at 20 °C. The pre cipitate after lipid extraction was dissolved in 5 ml of 1 M NaOH at 40 °C for 60 min. Protein precipitation was then performed with 10 ml of 10% TCA. The pre cipitate was redissolved in Soluene (100 TM, Packard Instrument Company, USA) before radioactivity counting. Protein was quantified according to L owry et al. [9]. In two experiments, purified protein precipitate was hydrolyzed in 6 m HC1 at 110 °C for 24 h to determine the content of leucine and threonine in kidney cortex proteins. The subcellular distribution of the radioactivity was determined in one kidney perfused with [,4C]leucine: a cortical biopsy was homogenized in a 0.25 M sucrose solution with 0.001 m EDTA (w/v 1/10). The homogenate was then subfractioned by centrifugation in a nuclear fraction; (850g-10min)-2; heavy and light mitochondrial fraction: (20.000 g • 10 min) • 2; microsomal fraction: 100 000 g • 60 min; and a crude supernatant. All the fractions were precipitated with 10 ml 10% TCA. Protein was then prepared from the precipitate of the fraction as described above.
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Analytical Procedures The incorporation of the radioactive carbon into C 0 2 in the perfusate or incuba tion medium was determined as described previously [17, 19]. The amino acids in the perfusate were separated and quantified by ion exchange chromatography with an automatic amino acid analyzer (Jeol-ILC-5AH) equipped with a sample splitter. With the aid of the splitter aliquots of the separated amino acids were taken for de termination of the radioactivity.
Protein Metabolism in Hypothermically Perfused Dog Kidney
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Table I. Distribution of amino acids in dog kidney cortex proteins in molar percentage Compound
Kidney 1
Kidney 2
Mean
Aspartate Threonine Serine Glutamate Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine
8.2 3.8 3.7 12.0 5.6 8.0 9.8 8.4 2.4 6.1 10.4 3.3 4.3 6.7 2.4 5.3
8.4 4.3 3.9 13.2 5.0 7.0 9.7 8.0 1.5 5.0 11.0 3.2 4.9 7.4 2.6 4.9
8.3 4.0 3.8 12.6 5.3 7.5 9.8 8.2 2.0 5.6 10.7 3.3 4.6 7.0 2.5 5.1
Analyses from two kidneys are presented. Cystine and tryptophan were destroyed during hydrolysis and were not determined.
The radioactivity in aqueous samples was determined in Insta-Gel® (Packard In strument Company, USA) and in non-aqueous samples in PPO, POPOP, and tol uene by the aid of a Packard Tri-Carb liquid scintillation spectrometer (3320). Correction for quenching was performed by the external standard method.
Results The amino acid composition of kidney cortex proteins is given in table I. Leucine and threonine which were used in the incorporation studies amounted to 10.7 and 4.0 molar percent, respectively.
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In vitro Incubations Table II illustrates the incorporation rate of leucine and threonine into kidney cortex proteins. Amino acids but not insulin in the incubation me dium increased the incorporation rate of leucine. Thus, an increase from 1 to 10 times normal human plasma level of amino acids stimulated the
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Table II. Incorporation rate of leucine carbon (n=8) and threonine carbon (n = 7) into kidney cortex proteins Amino acid concentration
Leucine (mean ± SE) mmol • h-1 • kg protein-*
Threonine (mean ± SE) mmol • h-* • kg protein-1
Plasma concentration
0.22 ±0.03
0.15 ±0.03
10 times plasma concentration
0.31 ±0.04*
0.16±0.03
Plasma concentration + insulin (25 U • l ' 1)
0.22 ±0.02
0.17±0.05
Dog kidney cortex slices were incubated at 37 °C in a complete amino acid solution of human plasma concentration with and without insulin and at 10 times human plasma concentration (see Material and Methods). * ptoholm/N auciJ2 i /P ettersson /S cherstf.n
Days Fig. 3. Incorporation rate of leucine (A ; mean + SE; n = 5) and threonine ( • , ■ ) into kidney cortex proteins during hypothermic perfusion of dog kidneys with a medium containing amino acids at a concentration corresponding to that of normal human plasma. A: y = 27 4- 105 x, r = 0.988; • : y = 13 + 31 x, r = 0.999; ■: y = 20 + 13 x, r = 0.990.
The recovery of label in CO, during kidney perfusion with [14C]leucine was 10 times higher than during perfusion with [,4C]threonine. This ratio was almost the same from the 1st to the 6th day of perfusion. Puromycin, added to the perfusate, extinguished completely the incor poration of leucine carbon into proteins. However, the incorporation of label into CO, was unaffected by the presence of puromycin in the perfus ate. In one of the puromycin experiments the specific activity of leucine in the perfusate was determined. The specific activity decay curve was consonant with those obtained in experiments without puromycin added to the perfusate. Discussion
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The present results indicate a protein synthesis and degradation in the hypothermically perfused dog kidney. Leucine and threonine were incorporated into kidney cortex proteins both during in vitro incubations of tissue slices at 37 °C and during hy pothermic perfusion of kidneys with a medium containing 17 amino acids at approximately normal human plasma concentration.
Protein Metabolism in Hypothermically Perfused Dog Kidney
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The concentration of leucine in the perfusate decreased while that of threonine increased during perfusion. However, the specific radioactivity in the perfusate of both leucine and threonine decreased indicating a re lease of these amino acids and thus a protein degradation throughout the experiment. Leucine was oxidized during in vitro incubation of kidney cortex slices at a higher rate than in other in vitro preparations [2, 22], The oxidation rate of threonine in tissue slices was about 10°/o of that of leucine. The same relationship between leucine and threonine oxidation (10:1) was found during hypothermic perfusion. The incorporation rate of threonine into proteins was lower than that of leucine both during in vitro incubation of slices and during kidney per fusion. This difference is in accordance with the different mass fraction of these two amino acids in the kidney cortex proteins (table I). Amino acids stimulated the incorporation rate of leucine into proteins in kidney cortex slices, which is in accordance with findings in other preparations [11, 14, 22]. Insulin, on the other hand, did not stimulate the incorporation rate into proteins. This lack of stimulation may suggest that the initiation of peptide synthesis was not rate-limiting under these experimental condi tions [15]. The incorporation rate of leucine and threonine carbon into kidney cortex proteins in slices incubated at 37 °C was 50-100 times higher than during kidney perfusion at 8-10 °C. This difference may be explained by the temperature difference only [8]. A greater isotope dilution by amino acids from degraded proteins in the immediate precursor pool for protein synthesis in the perfusion experiments may be another contributing factor to this difference. In these studies the incorporation rate of amino acid into proteins was calculated from the specific activity in the perfusate. It is not known if the amino acids in the perfusate are in direct continuity with the immediate precursor pool for protein synthesis. However, it has been shown for kidney cortex slices [21] and for many other in vitro preparations [1, 6, 7, 10, 22] that the specific activity of amino acids in the incubation medium rapidly equilibrates with the immediate precursor pool for protein biosynthesis. Thus, as long as the immediate precursor pool of amino acids for protein synthesis is not clearly defined, it seems reasonable to use the specific activity in the perfusate for calculation of the incorporation rate of amino acid into proteins. The degradation rate of proteins, as estimated from the decrease in leucine and threonine specific activity in the perfusate, was higher than
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the protein synthesis rate as estimated from the incorporation rate of these amino acids. In a recent study [13] we obtained evidence for a ni trogen balance close to unity during hypothermic perfusion of dog kid neys. The discrepancy in the present study between synthesis and degra dation is seemingly discordant with that finding. However, the reutiliza tion of amino acids from degraded proteins [5, 10, 16] may cause an iso tope dilution in the precursor pool of amino acids for protein synthesis which in part may be responsible for this discrepancy between estimated synthesis and degradation rate. The present determinations of amino acid incorporation into proteins during kidney perfusion do not qualify for absolute quantitation of prote in synthesis rate. On the other hand, the extinguished incorporation of amino acids in the presence of puromycin in the perfusate and the distri bution of incorporated label in proteins of subcellular fractions support the conclusion that synthesis of polypeptides occurred under these conditions. If so, determination of the incorporation rate of amino acid into proteins during kidney perfusion can be used for qualitative comparisons for eval uation of various perfusates.
Acknowledgements The kind help with the sterility controls by Dr. P er WAhl£n, Department of Bacteriology, and the skilful technical assistance by Mrs. Berit J ohansson are gratefully acknowledged. This work was supported by grants from the Swedish Re search Council (No. 3X-652), Göteborgs Läkresällskap, the University of Göteborg, Svenska Sällskapet för Medicinsk Forskning, AB Gambro, and Harald Jeanssons Stiftelse och Harald och Greta Jeanssons Stiftelse.
References
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1 Adamson, L. F.; H erington, A. C., and Bornstein, J.: Evidence for the selec tion by the membrane transport system of intracellular or extracellular amino acids for protein synthesis. Biochim. biophys. Acta 282: 352-365 (1972). 2 Bylund, A.-C.: Skeletal muscle metabolism in man. Studies with special refer ence to methodology and effects of physical training; thesis Göteborg (1976). 3 C ahill, G. F., jr. and A oki, T. T.: Renal gluconeogenesis and amino acid me tabolism in man. Med. Clins N. Am. 59: 751-761 (1975). 4 F elig , P.: Amino acid metabolism in man. A. Rev. Biochem. 44: 933-955 (1975).
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5 G an, J. C. and J ei-tay , H.: Origins and metabolism of the intracellular amino acid pools in rat liver and muscle. Biochim. biophys. Acta 148: 448-459 (1967). 6 H ider, R. C.; F ern, E. B., and London, D. R.: Identification in skeletal muscle of a distinct extracellular pool of amino acids, and its role in protein synthesis. Biochem. J. 121: 817-827 (1971). 7 K ipnis , D. M.; R eiss, E., and H elmreich, E.: Functional heterogeneity of the in tracellular amino acid pool in mammalian cells. Biochim. biophys. Acta 51: 519-524 (1961). 8 L evy, M. N.: Oxygen consumption and blood flow in the hypothermic, perfused kidney. Am. J. Physiol. 197: 1111-1114 (1959). 9 Lowry, O. H.; Rosebrough, N. J.; F arr, A. L., and Randall, R. J.: Protein measurement with the Folin phenol reagent. J. biol. Chem. 193: 265-275 (1951). 10 L undholm, K. and Schersten, T.: Incorporation of leucine into human skeletal muscle proteins. A study of tissue amino acid pools and their role in protein bio synthesis. Acta physiol, scand. 93: 433-441 (1975). 11 L undholm, K. and Schersten, T.: Determination in vitro of the rate of protein synthesis and degradation in human-skeletal-muscle tissue. Eur. J. Biochem. 60: 181-186 (1975). 12 L undstam, S.; C laes, G.; J onsson, O.; Pettersson, S., and Schersten, T.: Me tabolism in the hypothermically perfused kidney. Production and utilization of lactate and utilization of acetate in the dog kidney. Eur. surg. Res. 8: 300-310 (1976). 13 L undstam, S.; J agenburg, R.; J onsson, O.; L undholm, K.; N aucler, J.; Pet tersson, S., and Schersten , T.: Metabolism in the hypothermically perfused dog kidney. Utilization and production of amino acids. Eur. surg. Res. 9: 191— 205 (1977). 14 M organ, H. E.; E arl, D. C. N.; Broadus, A.; Wolpert, E. B.; G iger, K. E., and J efferson, L. S.: Regulation of protein synthesis in heart muscle. I. Effect of amino acid levels on protein synthesis. J. biol. Chem. 246: 2152-2162 (1971). 15 Morgan, H. E.; J efferson, L. S.; W olpert, E. B., and Rannels, D. E.: Regula tion of protein synthesis in heart muscle. II. Effect of amino acid levels and in sulin on ribosomal aggregation. J. biol. Chem. 246: 2163-2170 (1971). 16 M ortimore, G. E.; W oodside, K. H., and H enry, J. E.: Compartmentation of free valine and its relation to protein turnover in perfused rat liver. J. biol. Chem. 247: 2776-2784 (1972). 17 N ilsson, S. and ScherstLn , T.: Synthesis of phospholipids and triglycerides in human liver slices. I. Experimental conditions and the synthesis rate in normal liver tissue. Scand. J. clin. Lab. Invest. 24: 237-249 (1969). 18 O wen , E. E. and Roblnson, R. R.: Amino acid extraction and ammonia metabo lism by the human kidney during the prolonged administration of ammonium chloride. J. clin. Invest. 42: 263-276 (1963). 19 P ettersson, S.; C laes, G., and Schersten, T.: Fatty acid and glucose utilization during continuous hypothermic perfusion of dog kidney. Eur. surg. Res. 6: 79-94 (1974). 20 P itts, R. F.; P ilkington, L. A.; Mac L eod, M. B., and Leal-P into, E.: Metabo
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lism of glutamine by the intact functioning kidney of the dog. J. clin. Invest. 51: 557-565 (1972). 21 R osenberg, L. E.; Berman, M., and Segal, S.: Studies of the kinetics of amino acid transport, incorporation into protein and oxidation in kidney-cortex slices. Biochim. biophys. Acta 71: 664-675 (1963). 22 Stakeberg, H.; G ustafson, A., and Scherstén , T.: Incorporation rate of leu cine into proteins in human liver slices. Eur. J. clin. Invest. 4: 393-398 (1974).
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Sven L undstam , Department of Surgery, Sahlgrenska sjukhuset, University of Göteborg, Göteborg (Sweden)
Metabolism in the Hypothermically Perfused Dog Kidney Incorporation Rate of Leucine and Threonine into Proteins
Sven L undstam , R udolf J agenburg , O lof J onsson , Kent L undholm , J acob N aucler, Silas P ettersson and T ore Schersten Surgical Metabolic Research Laboratory, Departments of Clinical Chemistry, Surgery and Urology, Sahlgrenska sjukhuset, University of Göteborg, Göteborg
Key Words. Dog kidney • Hypothermic perfusion • Protein metabolism • [u C]Ieucine • [,4C]threonine Abstract. The incorporation of [u C]leucine and [14C]threonine into kidney cor tex proteins was studied during 6 days’ hypothermic perfusion of dog kidneys at 8-10 °C and during in vitro incubation of dog kidney cortex slices at 37 °C. Leucine carbon was incorporated into proteins at a higher rate than threonine carbon both during in vitro incubation of kidney cortex slices and during hypo thermic kidney perfusion. The incorporation of leucine and threonine during hypo thermic perfusion was linear for 6 days but 50-100 times lower than the incorpora tion of leucine and threonine in kidney cortex slices at 37 °C. During hypothermic perfusion there was a decrease in specific activity of leucine and threonine in the perfusate corresponding to a degradation of proteins which was greater than protein synthesis as calculated from the incorporation of label into pro teins. Leucine carbon was recovered in C 0 2 during hypothermic perfusion and in vitro incubation of kidney cortex slices at 37 °C. The incorporation of threonine carbon into C 0 2 was about 10°/o of the corresponding value for leucine both during hypo thermic kidney perfusion and during in vitro incubation of kidney cortex slices at 37 °C. It is concluded that there is a turnover of kidney proteins during hypothermic perfusion with a perfusate containing amino acids.
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Received: October 20, 1976; accepted: January 31, 1977.
Lundstam /J agenburg/J onsson/L undholm /N auclér/P ettersson/S cherstén
207
Amino acids are metabolized in the hypothermically perfused dog kid ney as indicated by amino acid uptake from the perfusate and release to the perfusate [13]. Glutamine, proline and glycine dominated the uptake and alanine and ammonia the release of nitrogen in accordance with the condition in vivo [18]. Uptake and release of amino acids are not neces sarily associated with an active turnover of proteins. The a-amino group of the amino acids may be utilized predominantly in the formation of alanine and ammonia [4, 20] and the carbon skeleton may be used for ox idation and gluconeogenesis [3]. The present study was designed to evaluate protein synthesis and deg radation during hypothermic perfusion of dog kidneys. As marker of pro tein synthesis, the incorporation rate of leucine and threonine carbon into proteins was determined during hypothermic perfusion and for compari son during in vitro incubation of kidney cortex slices at 37 °C.
Material and Methods 24 kidneys from adult mongrel dogs were used. The dogs were fasted but had free access to water for 24 h before the operation. Nephrectomy and treatment of the kidney before connection to the perfusion machine were performed according to previous descriptions [12,19]. The mean kidney weight was 84 + (SD) 16 g. Perfusion Procedure The perfusion was performed at 8-10 °C in a Gambro perfusion machine (Gambro, PF 2D, Lund, Sweden). The perfusion was run according to our previous de scription [13]. The perfusate was based on human albumin with addition of electrolytes of ex tracellular concentration, glucose, 17 L-amino acids, insulin, hydrocortisone, and benzyl-penicillin in concentrations as described previously [13]. [U-,4C]i.-Ieucine or [U-14C]L-threonine was added to the perfusate as presented in figures 2 and 3. In two experiments, puromycin hydrochloride was added to the perfusate to a final concentration of 210 «m ol'L 1. Before perfusion was started, and at various time intervals during perfusion, samples were taken from the perfusate to determine the concentration of amino acids and the incorporation of radioactive carbon into amino acids and C 02. A biopsy from cortex was taken after 1 h of perfusion and then after 1, 2, 4, and 6 days to determine the incorporation of label into tissue proteins.
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Tissue Incubation Procedure Cortical biopsies were taken for in vitro determination of incorporation rate of amino acid carbon into C 0 2 and proteins. This tissue was sliced in a Mcllwain tissue chopper (Mickle Laboratory Engineering Company, England). The kidney slices
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(0.4 mm) were washed in ice-chilled Krebs-Ringer bicarbonate (KRB) buffer solu tion, blotted and rapidly weighed on a torsion balance. They were then placed in Hagedorn tubes containing 4 ml of the medium. The flasks were gassed with 0 2:C02 (95:5), sealed with rubber stoppers, and incubated at 37 °C in a metabolic shaker with constant agitation, 80 strokes per minute. The incubation medium consisted of KRB buffer, pH 7.4, and a mixture of 19 L-amino acids at a concentration corre sponding to that of normal human plasma [22] or 10 times that of human plasma. [U-14C]L-leucine or [U-,4C]L-threonine, about 0.5 /iCi, was added to the incubation medium. The incorporation of label into proteins was studied at plasma concentra tion of amino acids with and without addition of insulin (25 UT"1) and at 10 times plasma concentration without insulin. The incubation time was 4 h. Kidney slices were incubated for 1 h in a medium with plasma concentration of amino acids for determination of the incorporation rate of label into C 02. After incubation, the kidney slices were homogenized with an Ultra-Turrax homogenizer (Jankel & Kunkel, FRG). The incorporation rate of amino acid carbon into proteins and CO, was calculated by dividing the radioactivity in proteins and C 0 2, respectively, with the specific activity in the incubation medium. Protein Preparation After homogenization of the kidney biopsies and slices, 10 ml 10% trichloroacet ic acid (TCA) were added and the mixture was heated to 80 °C for 40 min. After centrifugation the sediment was resuspended in 5 ml 5% TCA. The insoluble materi al was extracted for lipids with ethanol acetone (1:1) at 40 °C for 60 min. This ex traction was repeated twice at 20 °C and finally with 5 ml ether at 20 °C. The pre cipitate after lipid extraction was dissolved in 5 ml of 1 M NaOH at 40 °C for 60 min. Protein precipitation was then performed with 10 ml of 10% TCA. The pre cipitate was redissolved in Soluene (100 TM, Packard Instrument Company, USA) before radioactivity counting. Protein was quantified according to L owry et al. [9]. In two experiments, purified protein precipitate was hydrolyzed in 6 m HC1 at 110 °C for 24 h to determine the content of leucine and threonine in kidney cortex proteins. The subcellular distribution of the radioactivity was determined in one kidney perfused with [,4C]leucine: a cortical biopsy was homogenized in a 0.25 M sucrose solution with 0.001 m EDTA (w/v 1/10). The homogenate was then subfractioned by centrifugation in a nuclear fraction; (850g-10min)-2; heavy and light mitochondrial fraction: (20.000 g • 10 min) • 2; microsomal fraction: 100 000 g • 60 min; and a crude supernatant. All the fractions were precipitated with 10 ml 10% TCA. Protein was then prepared from the precipitate of the fraction as described above.
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Analytical Procedures The incorporation of the radioactive carbon into C 0 2 in the perfusate or incuba tion medium was determined as described previously [17, 19]. The amino acids in the perfusate were separated and quantified by ion exchange chromatography with an automatic amino acid analyzer (Jeol-ILC-5AH) equipped with a sample splitter. With the aid of the splitter aliquots of the separated amino acids were taken for de termination of the radioactivity.
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Table I. Distribution of amino acids in dog kidney cortex proteins in molar percentage Compound
Kidney 1
Kidney 2
Mean
Aspartate Threonine Serine Glutamate Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine
8.2 3.8 3.7 12.0 5.6 8.0 9.8 8.4 2.4 6.1 10.4 3.3 4.3 6.7 2.4 5.3
8.4 4.3 3.9 13.2 5.0 7.0 9.7 8.0 1.5 5.0 11.0 3.2 4.9 7.4 2.6 4.9
8.3 4.0 3.8 12.6 5.3 7.5 9.8 8.2 2.0 5.6 10.7 3.3 4.6 7.0 2.5 5.1
Analyses from two kidneys are presented. Cystine and tryptophan were destroyed during hydrolysis and were not determined.
The radioactivity in aqueous samples was determined in Insta-Gel® (Packard In strument Company, USA) and in non-aqueous samples in PPO, POPOP, and tol uene by the aid of a Packard Tri-Carb liquid scintillation spectrometer (3320). Correction for quenching was performed by the external standard method.
Results The amino acid composition of kidney cortex proteins is given in table I. Leucine and threonine which were used in the incorporation studies amounted to 10.7 and 4.0 molar percent, respectively.
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In vitro Incubations Table II illustrates the incorporation rate of leucine and threonine into kidney cortex proteins. Amino acids but not insulin in the incubation me dium increased the incorporation rate of leucine. Thus, an increase from 1 to 10 times normal human plasma level of amino acids stimulated the
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Table II. Incorporation rate of leucine carbon (n=8) and threonine carbon (n = 7) into kidney cortex proteins Amino acid concentration
Leucine (mean ± SE) mmol • h-1 • kg protein-*
Threonine (mean ± SE) mmol • h-* • kg protein-1
Plasma concentration
0.22 ±0.03
0.15 ±0.03
10 times plasma concentration
0.31 ±0.04*
0.16±0.03
Plasma concentration + insulin (25 U • l ' 1)
0.22 ±0.02
0.17±0.05
Dog kidney cortex slices were incubated at 37 °C in a complete amino acid solution of human plasma concentration with and without insulin and at 10 times human plasma concentration (see Material and Methods). * ptoholm/N auciJ2 i /P ettersson /S cherstf.n
Days Fig. 3. Incorporation rate of leucine (A ; mean + SE; n = 5) and threonine ( • , ■ ) into kidney cortex proteins during hypothermic perfusion of dog kidneys with a medium containing amino acids at a concentration corresponding to that of normal human plasma. A: y = 27 4- 105 x, r = 0.988; • : y = 13 + 31 x, r = 0.999; ■: y = 20 + 13 x, r = 0.990.
The recovery of label in CO, during kidney perfusion with [14C]leucine was 10 times higher than during perfusion with [,4C]threonine. This ratio was almost the same from the 1st to the 6th day of perfusion. Puromycin, added to the perfusate, extinguished completely the incor poration of leucine carbon into proteins. However, the incorporation of label into CO, was unaffected by the presence of puromycin in the perfus ate. In one of the puromycin experiments the specific activity of leucine in the perfusate was determined. The specific activity decay curve was consonant with those obtained in experiments without puromycin added to the perfusate. Discussion
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The present results indicate a protein synthesis and degradation in the hypothermically perfused dog kidney. Leucine and threonine were incorporated into kidney cortex proteins both during in vitro incubations of tissue slices at 37 °C and during hy pothermic perfusion of kidneys with a medium containing 17 amino acids at approximately normal human plasma concentration.
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The concentration of leucine in the perfusate decreased while that of threonine increased during perfusion. However, the specific radioactivity in the perfusate of both leucine and threonine decreased indicating a re lease of these amino acids and thus a protein degradation throughout the experiment. Leucine was oxidized during in vitro incubation of kidney cortex slices at a higher rate than in other in vitro preparations [2, 22], The oxidation rate of threonine in tissue slices was about 10°/o of that of leucine. The same relationship between leucine and threonine oxidation (10:1) was found during hypothermic perfusion. The incorporation rate of threonine into proteins was lower than that of leucine both during in vitro incubation of slices and during kidney per fusion. This difference is in accordance with the different mass fraction of these two amino acids in the kidney cortex proteins (table I). Amino acids stimulated the incorporation rate of leucine into proteins in kidney cortex slices, which is in accordance with findings in other preparations [11, 14, 22]. Insulin, on the other hand, did not stimulate the incorporation rate into proteins. This lack of stimulation may suggest that the initiation of peptide synthesis was not rate-limiting under these experimental condi tions [15]. The incorporation rate of leucine and threonine carbon into kidney cortex proteins in slices incubated at 37 °C was 50-100 times higher than during kidney perfusion at 8-10 °C. This difference may be explained by the temperature difference only [8]. A greater isotope dilution by amino acids from degraded proteins in the immediate precursor pool for protein synthesis in the perfusion experiments may be another contributing factor to this difference. In these studies the incorporation rate of amino acid into proteins was calculated from the specific activity in the perfusate. It is not known if the amino acids in the perfusate are in direct continuity with the immediate precursor pool for protein synthesis. However, it has been shown for kidney cortex slices [21] and for many other in vitro preparations [1, 6, 7, 10, 22] that the specific activity of amino acids in the incubation medium rapidly equilibrates with the immediate precursor pool for protein biosynthesis. Thus, as long as the immediate precursor pool of amino acids for protein synthesis is not clearly defined, it seems reasonable to use the specific activity in the perfusate for calculation of the incorporation rate of amino acid into proteins. The degradation rate of proteins, as estimated from the decrease in leucine and threonine specific activity in the perfusate, was higher than
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the protein synthesis rate as estimated from the incorporation rate of these amino acids. In a recent study [13] we obtained evidence for a ni trogen balance close to unity during hypothermic perfusion of dog kid neys. The discrepancy in the present study between synthesis and degra dation is seemingly discordant with that finding. However, the reutiliza tion of amino acids from degraded proteins [5, 10, 16] may cause an iso tope dilution in the precursor pool of amino acids for protein synthesis which in part may be responsible for this discrepancy between estimated synthesis and degradation rate. The present determinations of amino acid incorporation into proteins during kidney perfusion do not qualify for absolute quantitation of prote in synthesis rate. On the other hand, the extinguished incorporation of amino acids in the presence of puromycin in the perfusate and the distri bution of incorporated label in proteins of subcellular fractions support the conclusion that synthesis of polypeptides occurred under these conditions. If so, determination of the incorporation rate of amino acid into proteins during kidney perfusion can be used for qualitative comparisons for eval uation of various perfusates.
Acknowledgements The kind help with the sterility controls by Dr. P er WAhl£n, Department of Bacteriology, and the skilful technical assistance by Mrs. Berit J ohansson are gratefully acknowledged. This work was supported by grants from the Swedish Re search Council (No. 3X-652), Göteborgs Läkresällskap, the University of Göteborg, Svenska Sällskapet för Medicinsk Forskning, AB Gambro, and Harald Jeanssons Stiftelse och Harald och Greta Jeanssons Stiftelse.
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Sven L undstam , Department of Surgery, Sahlgrenska sjukhuset, University of Göteborg, Göteborg (Sweden)
