Effect of Varying Amino Acid Levels on Protein Metabolism in Nephrotic Rats During Total Parenteral Nutrition

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1 Effet of Varying Amino Aid Levels on Protein Metabolism in Nephroti Rats During Total Parenteral Nutrition AKIRA KAWASHIMA,* KAZO HIK,t MEGMI KAWASHIMA,* KOSAK NITTA,* WAKO YMRA,* NOBHIRO SGINO,* HIROSHI NIHEI,* and YASO NATORIt *Depart,ile,lt of Mediine, Kidney enter, Tokyo Women s Medial ollege, Tokyo, Japan; and Department of Nutritional hemistry, Shool of Mediine, Tokushima niversit,, Tokushima, Japan Abstrat In an attempt to determine appropriate diet in nephroti syndrome nephroti rats, indued by puromyin aminonuleoside, were nourished by total parenterab nutrition fluid ontaining the same energy, but three different levels (165, 33, and 66%) of amino aids for 7 d The frational rate of total protein synthesis in the liver was determined by injeting a flooding dose of [3Hjphenybabanine The proportion of newly synthesied proteins retained and exported by the liver was estimated by injeting a traer dose of [ 4]leuine and then measuring the protein radioativity remaining in the liver and present in the plasma after seretion was ompleted Nephroti animals synthesied more protein than ontrol animals Although the absolute synthesis rates of total protein in liver were inreased with inreasing amino aid administration, the absolute rates of synthesis of albumin were higher in the 33% group than in the other groups in nephroti rats However, kidney protein synthesis in nephroti rats was higher in the 165% group than in the 33% group Interestingly the 33% group revealed the smallest urinary exretion of total protein and albumin In addition, in the 33% group, plasma onentrations of total protein and albumin were higher, and plasma onentrations of total holesterol and triglyeride were lower than in other groups It was onluded that the 33% group, orresponding to a normal protein diet, has the greatest salutary effet on urinary protein exretion, followed by protein and lipid metabolism, in nephroti rats Not only protein intake but also the energy:protein ratio are important for diet therapy in nephroti animals The tehnique of total parenteral nutrition may be useful in defining the fators involved in gbomerular permeability or permseletivity and intraellular protein metabolism (J Am So Nephrob 8: , 1997) Patients with nephroti syndrome were one advised to inrease dietary protein intake as a means of offsetting urinary protein losses ( 1,2) However, it has been known sine the first observation reported by Farr (3) that when nephroti patients are on a high-protein diet, urinary protein exretion inreases, but serum protein onentration does not Reently, sine it has beome apparent that exess dietary protein may be responsible for inreases in GFR and renal blood flow, whih in turn aelerate the progression of glomerular slerosis (4-9), the effet of dietary protein intake on albumin metabolism in nephroti animals and humans has been investigated Kaysen et a! (1, 1 1) have demonstrated that a low-protein diet, ompared with a high-protein diet, resulted in dereased proteinuria and inreased plasma albumin onentration, despite dereased albumin synthesis However, few studies are available so far about protein synthesis other than albumin synthesis in nephroti animals and patients The present study was designed to investigate the influene Reeived August 9 l996 Aepted April 23 l997 orrespondene to Dr Akira Kawashima Department of Mediine Kidney enter, Tokyo Women s Medial ollege, 8-I Kawada-ho Shinjuku-ku, Tokyo 162, Japan I / $3OO/ Journal of the Amerian Soiety of Nephrology opyright 1997 by the Amerian Soiety of Nephrology of amino aid supply on protein metabolism in liver in nephroti rats while the animals were maintained with total parenteral nutrition (TPN) fluid ontaining different levels of amino aids, regardless of appetite loss and redued food intake due to any diet To determine the rate of protein synthesis, we used a flooding dose tehnique of [3H]phenylalanine (12) to minimie problems resulting from the identifiation of the amino aid preursor pool at the site of protein synthesis In addition, beause the flooding dose measures the rate of total protein synthesis, inluding proteins destined for seretion and export, the proportion of plasma proteins relative to other proteins synthesied in the liver was estimated by labeling them first with a trae dose of [ 4]leuine and then measuring the protein radioativity remaining in the liver and that present in the plasma after seretion was ompleted The results show that nephroti syndrome results in a seletive inrease in plasma protein synthesis and that TPN fluid ontaining 33% amino aid keeps plasma protein and albumin onentration at the highest level and urinary protein exretion of proteins and albumin, and plasma lipid onentration at the lowest level in nephroti animals Materials and Methods Experimental Animals and infusion Proedure Surgial proedure and subsequent experiments were performed after approval and aording to riteria established by the Animal

2 14 Journal of the Amerian Soiety of Nephrology are and se ommittee at Tokushima niversity, whih followed guidelines of National Institutes of Health on the humane use of laboratory animals Male Wistar rats (Japan lea, Tokyo, Japan), eah weighing approximately 2 g were housed in a ontrolled environment (temperature 23 ± 2#{176}; light from 7 am to 7 pm) and allowed free aess to a ommerial diet (MF#{174},Oriental, Tokyo, Japan) and water The animals were divided into six body weight-mathed groups of 1 animals eah and prepared for ontinuous infusion, using tehniques similar to that desribed by Steigner et a! (13) Rats were made nephroti by a single intravenous injetion of puromyin aminonuleoside (25 mg/loo g body wt in 9% Nal; Sigma, St Louis, MO) at the beginning of TPN All of the experimental animals were plaed in metaboli ages to ollet 24-h urine samples During the overnight surgial reovery period, a TPN solution was infused intravenously at a rate of 3 ml/d, and the rate was doubled on and after the next day, whih was designated as day I of the experiment The ompositions of the infusion solutions are shown in Table 1 In addition to gluose, eletrolytes, vitamins, and trae elements, the infusion solutions were adjusted to ontain various onentrations of amino aids by mixing with suitable proportions of MPR-F amino aid solution (Morishita Pharmaeutial, Osaka, Japan) The MPR-F solution has a onventional or standard amino aid omposition, whih was desribed in a previous artile from our laboratory (14) The energy ontent was maintained by replaing amino aids in the infusion solutions with gluose Infusion of experimental solutions (6 ml/d ontaining 646 kal) ontinued for 6 or 7 onseutive days Protein Turnover Rates On day 7 of the experiment, protein synthesis was measured in vivo after an intravenous injetion of a large dose of phenylalanine to flood the preursor pool (1 This injetion was administered to eah rat via the atheter used for delivery of TPN solution and ontained 15 mol of the amino aid, inluding 925 kbq of L-[4-3H]phenylalanine (74 GBq/mmol; Amersham, Bukinghamshire, nited Kingdom) in 1 ml of water/loo g body wt Anesthetied rats were killed by deapitation 2 or 1 mm after the injetion, blood was olleted in heparinied tubes, and tissues were rapidly exised Determination of the speifi radioativity of [3H]phenylalanine involved its enymi onversion into -phenethylamine The frational rate of protein synthesis (Ks: the perentage of the protein pool renewed eah day) was alulated from measurements of the speifi radioativity of phenylalanine in protein (5B) at 1 mm and the mean speifi radioativity of free phenylalanine in the liver (5A) between and 1 mm The value of A was obtained by killing animals at 2 and 1 mm, and the formula for alulating Ks was: Ks 5B X 5A X t, where is the inorporation time in days The absolute rate of protein synthesis was alulated from the frational rate of protein synthesis and tissue protein mass The liver synthesies proteins not only for intraorgan use but also for export into irulation as plasma proteins Newly synthesied plasma proteins are sereted by the liver after a lag of a few minutes The proportion of plasma proteins relative to other proteins synthesied in the liver an be studied by labeling them with a radioative preursor and then waiting for a suitable time until the seretion of plasma proteins is ompleted One an then measure the protein radioativity remaining in the liver and present in the plasma (16) The plasma proteins an be further frationated into albumin at room temperature ( I 7) In brief, the plasma was preipitated in saturated ammonium sulfate and to the supernatant was then added I N aeti aid, resulting in the preipitation of albumin We injeted intravenously a trae amount of L-[- 4]leuine (185 kbq) (127 GBq/ mmol; Amersham) 3 h before the animals were killed, and the total protein radioativity in the liver, plasma, and albumin fration of the plasma was determined The plasma volume was estimated by measuring the volume of distribution of Evans blue dye, whih was injeted intravenously, together with the flooding dose of [3H]phenylalanine 1 mm before the rats were killed Double-labeled samples were ounted in an Aboka liquid sintillation ounter (Tokyo, Japan) Proteins were measured by the biuret method, using bovine serum albumin as standard Table 1 omposition of intravenous infusates ategory Ami no Aid Level 165% 33% 66% Amino aid (g/dl) Gluose (g/dl) Na (meq/dl) K (meq/di) l (meq/dl) Mg (meq/dl) a (meq/dl) P (meq/dl) Vitaminsa Trae elementsb Water (mb/rat per d) Total energy (kal/rat per d) a Provided the following (mg/l): retinol, 3; thiamine, 5; riboflavin, 1; pyridoxine 15; niotinamine, 1; pantotheni aid, 25; asorbi aid, 5; ergoaliferol 25; all-a-toopheryl aetate, 5 h Providing the following (mgil): Fe, 5; Zn, 75; u, 75; I, 38 Analytial Methods On day 7 of the experiment, plasma was analyed for total protein (biinhonini protein assay reagent, Piere no G, Rokford, IL), albumin (Sigma kit no 631-2), total holesterol (Sigma kit no 352-2), and triglyerides (Sigma kit no 337-B) Twenty-four-hour urine samples were analyed for total protein and albumin Statistial Analyses All results are expressed as means ± SEM Signifiant differenes between individual means were determined by one-way ANOVA followed by Dunan s new multiple-range test (18) When P < 5, the differenes were regarded as statistially signifiant Results Experimental Animals Rats injeted with puromyin aminonuleoside exhibited harateristi symptoms assoiated with the nephroti syndrome On day 7 of the experiment, ontrol and experimental rats were evaluated using several parameters, as summaried in Table 2 The experimental rats developed marked proteinuria, albuminuria, hypoproteinemia, hypoalbuminemia, and hyperlipidemia Additionally, they revealed edema and asites

3 TPN in Nephroti Rats 141 i o r oooo -- r -- Nutritional Status ofanimals During Infusion Solution ontaining Graded Levels ofamino Aids Rats were nourished for 7 d by intravenous infusion of isoalori TPN solutions ontaining 1 65, 33, or 66% amino aid As shown in Table 1, nephroti animals that reeived the solutions ontaining 33% amino aids revealed lighter pro- rnn-n-n Nrfirf o NN teinuria, albuminuria, hyperlipidemia, hypoproteinemia, and hypoalbuminemia than the 1 65 and 66% amino aid-reeiving groups The liver and kidney weight and protein ontent after 7 d of infusion were higher in eah nephroti animal than orresponding ontrol animals The total protein ontent of liver was inreased with inreasing amino aid administration However, the protein ontent of kidney was highest in the 1 65% amino aid-reeiving group than in the other groups I) t- j \ Orr- Proportion of Newly Synthesied Proteins Retained and Exported by Liver The liver synthesies not only proteins for domesti use but also proteins for export into irulation as plasma proteins The proportion of newly synthesied proteins retained and exported by the liver was estimated, as desribed in Materials and Methods a I- ) 4- ) - ) Ln r1 1- tt -:Q rl rn1 r-r o tr r or- or-qri Q ,r1 o or Noi r-i t -, - n 2 8 B e e )! ;- S - J - 4- ) ), 1 - B l) +1 = o # r Ir Ir tr -VVV a - As shown in Table 3, the liver synthesied approximately equal amounts of domesti and plasma proteins at all levels of amino aid intake in both ontrol and nephroti rats Total Liver and Kidney Protein Synthesis Rates sing Flooding Dose of [3H]phenylalanine The frational rates of liver and kidney protein synthesis were determined by the flooding dose of [3H]phenylalanine (Table 4) These values, representing the total protein synthesis in the liver, were pro-rated among domesti liver proteins, albumin, and nonalbumin plasma proteins aording to the data given in Table 3, and then the absolute synthesis rate of eah fration was alulated by taking into aount the protein mass in the liver As shown in Table 4, the absolute synthesis rates of total protein in liver in nephroti animals were higher than in ontrol animals, and lower in the 165% amino aid-reeiving group than in the other groups The absolute rates of synthesis of albumin were also lower in the 1 65% group than in the other groups in nephroti animals (Table 4) On the other hand, in kidney, the 1 65% group revealed higher protein syntheti rates than the 33% group (Table 4) Disussion Earlier attempts to determine the rate of protein synthesis in vivo were hampered by a bak of aurate and reliable tehniques Although a number of methods involving administration of labeled amino aids with subsequent measurement of inorporation of label into protein have been used, the main diffiulty is the assessment of the speifi radioativity of the preursor amino aid at the site of protein synthesis An additional diffiulty arises from the possibility that the low- or high-protein diet is assoiated with loss of appetite and redued food intake, espeially in experimental animals The use of a

4 142 Journal of the Amerian Soiety of Nephrology Table 3 Distribution of protein radioativity in liver and plasma 3 h after injetion of radioative leuine Amino Aid Level Group 165% 33% 66% ontrol Nephrosis ontrol Nephrosis ontrol Nephrosis Liver (%) 56 ± ± ± ± ± ± 452 Plasma (%) 494 ± ± ± ± ± ± 451 albumin (%) 22 ± ± ± ± ± ± 34 nonalbumin (%) 292 ± ± ± ± ± ± 269 a Values denote perentage of total radioativity and are means ± SEM of at least six rats per group flooding dose of [3H]phenybalanine ( 12) ould overome this problem If removal of protein from the diet is assoiated with loss of appetite and redued food intake, the distintion of the sequelae of protein deprivation from those experiening starvation poses a diffiult problem An additional diffiulty arises from the possibility that the rate of protein turnover varies from time to time and from individual to individual (19) The tehnique of TPN offers a unique opportunity to rigorously modulate the quality and quantity of nutrient influx into animals, abolishing individual and diurnal variations in quantity and timing of food intake In the present study, we investigated the effet of parenteral infusion of three different amino aid onentrations, ie, 165, 33, and 66%, orresponding to 48-, 92-, and l83-g amino aids/kg per d, on protein metabolism in nephroti rats Judging from the growth of the animals in eah group, these amino aid levels may orrespond to low-protein, normal-protein, and high-protein diets, respetively, in the feeding experiments (2) Nephroti animals synthesied more proteins than ontrol animals Although the absolute synthesis rates of total protein in liver were inreased with inreasing amino aid administration, the absolute rates of synthesis of albumin were lower in the 1 65% group than in the other groups in nephroti rats On the other hand, kidney protein synthesis in nephroti rats was higher in the 1 65% group than the 33% group A hange in amino aid onentration led to a hange in urinary exretion of total protein and albumin in nephroti rats Interestingly, the 33% group revealed the smallest urinary exretion of total protein and albumin In addition, in the 33% group, plasma onentrations of total protein and albumin were higher, and plasma onentrations of total holesterol and triglyeride were lower than in the other groups The finding of augmented albumin synthesis in nephroti syndrome is in agreement with several previous reports (21-24) High-protein diets were one reommended for nephroti patients to inrease protein synthesis Although high-protein diets inrease albumin synthesis, inreased dietary protein also auses greater urinary albumin losses and inreased albumin atabolism, whih negate the effets of inreased synthesis (12 onversely, redued dietary protein in nephroti patients and rats redues albumin synthesis, but plasma albumin onentration is signifiantly inreased beause of a onomitant redution in urinary albumin loss that ours in the low-protein diet, offsetting the redued albumin synthesis (1, 1 1) However, our results demonstrated that the 33% group revealed the highest synthesis of albumin than the other groups These may be attributed to the problem of energy: protein ratio A high ratio of energy to protein leads to redued albumin synthesis and depletion of body albumin pool (26,27), and indues lipid synthesis We also demonstrated that the 33% group showed the lowest urinary protein exretion and maintained plasma levels of proteins and lipids among nephroti rats Kaysen et al reported that an inrease in the albumin synthesis is not linked to the derangement in lipid metabolism, but that a lose orrelation exists between the urinary exretion of albumin and the onentration of plasma lipids in nephroti patients (28) Inreased proteinuria leads to redued lipoprotein learane as a result of the urinary loss of a liporegulatory substane (29) Our data are onsistent with their study It is urious that the 33% group revealed lower urinary protein exretion even ompared with the 1 65% group This finding may be attributable to the abnormality of protein metabolism in the kidney itself Indeed, the 1 65% group revealed higher protein synthesis than the 33% group by the proteinuri kidney These findings suggest that the lower plasma protein synthesis and onentration or the inappropriate ratio of energy to amino aids may inrease kidney protein synthesis, probably supplying a defiieny of protein synthesis in liver and produing toxi substanes in quality or in quantity to hange the permeability or permseletivity of the gbomerular basement membrane Further studies to larify fators affeting the glomerular permeability or permseletivity should provide new insights into diet therapy for nephroti patients Exess dietary protein an inrease GFR and renal blood flow, whih in turn aelerate the progression of glomerular slerosis (4-9) Several investigators have reported inreased rates of atheroslerosis in nephroti patients (3,3 1) Redution of urinary protein exretion and improvement of abnormal metabolism is extremely important for any therapy of nephroti syndrome Our results indiate that the 33% group, orresponding to a normal protein diet, has the greatest salutary effet on urinary protein exretion, and then on protein and lipid metabolism, in nephroti rats This effet may be responsible for appropriate protein intake or energy:protein ratio

5 TPN in Nephroti Rats 143 Our urrent studies demonstrate that not only protein intake 1 N r s1 QQr oor1 r- rr, OON\IN x,r e- but also energy:protein ratio are important for diet therapy in nephroti animals Reently, Kaysen et a! reported that branhed-hain amino aids (32), arginine proline, glutamate, aspartate, or total nitrogen (33) augment neither albuminuria nor albumin synthesis in nephroti rats D Amio et a! reported that a soy protein diet redued urinary protein exretion signifiantly in nephroti patients (34) These findings suggest that it is neessary to study in detail the effets of speifi amino aids on protein metabolism and urinary protein exretion in nephroti animals The tehnique of TPN, desribed here, may be useful in defining the fators involved in gbomerular permeability or permseletivity and intraellular protein metabolism a ) ),,, I- 4- O < ), ) LI, B ooo trto, LI N r en N r (\N r rn 1 intravenous feeding in unrestrained rats Arh Surg 14: ri o- t- - #{231}I, I, - >- ; > LI,,, > B B B > -E5 ) ), ) > - VVVVV a Referenes I Blainey JD: High protein diets in the treatment of the nephroti syndrome /in Si (Lond) I 3: oggins H: Management of nephroti syndrome In: ontemporarv mssues in Nephro/ogv, edited by Brenner BM, Stein JH, Vol 9, New York, hurhill Livingstone, 1982, pp Fan LE: The assessment of protein by young hildren with the nephroti syndrome Am J Med Si 195: 7-83: Meyer TW, Lawrene WE, Brenner BM: Dietary protein and the progression of renal disease Kidney Int 24: , Jones MG, Lee K, Swaminathan R: The effet of dietary and the progression of renal disease /in Nephro/ 27: 71-75, Bosh JP, Saaggi A, Layer A, Rono, Belledone M, Glabman 5: Renal funtional reserve in humans: Effet of protein intake on gbomerular filtration rate Am J Med 75: , El Nahas AM, Masters-Thomas A, Brady SA, Farrington K, Wilkinson V Hilson AJ, Varghese Z, Moorhead JF: Seletive effet of low-protein diets in hroni renal disease Br J Med 289: , Mith WE, Waber M, Steinman THI, Hell 5, Zeger 5, Tungsanga K: The effet of a keto aid supplement to a restrited diet on the progression of hroni renal failure N Eng! J Med 311: 623, Young VR: Some metaboli and nutritional onsiderations of dietary protein restrition In: The Progressive Nature of Rena! Disease, Guest editor: Mith WE, edited by Brenner BM, Stein JH, New York, hurhill Livingstone, 1986, pp Kaysen GA, Gambeltoglio J, Jimine I, Jones H, Huthson FN: Effet of dietary protein-intake on albumin homeostasis in nephroti patients Kidney Int 29: , Kaysen GA, Jones H, Martin V Huthson FN: A low protein diet restrits albumin synthesis in nephroti rats J /in Invest 83: , Garlik PJ, Mnurlan MA, Preedy VR: A rapid and onvenient tehnique for measuring the rate of protein synthesis in tissues by injetion of [3H]phenylalanine Biohem J 192: , Steigner E, Vars HM, Dudrik SJ: A tehnique for long-term 332, Kikuhi T, Fukudome 5, Ikemoto H, Tsutsui I, Tanaka H, Kokuba Y, Orita Y, hiku K, Natori Y: Effet of enrihment of infusion solutions with branhed-hain amino aids in parenteral nutrition of rats J Nutr Si Vitaminol 33: 63-73, Garlik PJ, Millward DJ, James WPT, Waterbow J: The effet of protein deprivation and starvation on the rate of protein synthesis in tissues Biohi,n Biophvs Ata 414: 71-84, 1975

6 144 Journal of the Amerian Soiety of Nephrology 16 Sornik OA, Botbol V: Role ofhanges in protein degradation in the growth of regenerating liver J Biol hem 25 1 : , 1976 I 7 Debro JR, Tarver H, Korner A: The determination of serum albumin and globulin by a new method J Lab /in Med 5: , Steel RGD, Tone JH: Priniples and Proedures of Statistis, New York, MGraw-Hill, Haider M, Tarver H: Effet of diet on protein synthesis and nulei aid levels in rat liver J Nutr 99: , National Researh ounil: Nutrient requirements of the laboratory rat In: Nutrient Requirements of Laboratory Animals, Washington National Aademy of Siene, 1972, pp Jensen H Rossing N, Andersen SB, Jarnum 5: Albumin metabolism in the nephroti syndrome in adults /in Si 33: , I Kats J Bonorris G, Sellers AL: Albumin synthesis in perfused liver of normal and nephroti rats Am J Physiol 2 12: , I Marsh JB, Drabkin DL: Metaboli hanneling in experimental nephrosis IV Net synthesis of plasma albumin by liver slies from normal and nephroti rats J Biol hem 23: , Marsh JB Drabkin DL: Experimental reonstrution of metaboli pattern of lipid nephrosis: Key robe of hepati protein synthesis in hyperlipidemia Metabolism 9: , Kaysen GA, Kirkpatrik WG, ouser WG: Albumin homeostasis in the nephroti rat Am J Phvsio! 247: F192-F22, oward WA, Sawyer MB: Whole-body albumin mass and distribution in rats fed on low protein diets Br J Nutr 37: , ohen 5, Hansen JOL: Metabolism of albumin and gammaglobulin in kwashiorkor /in Si 23: , Kaysen GA, Gambertoglio J, Felts J, Huthson FN: Albumin synthesis, albuminuria and hyperlipidemia in nephroti patients Kidney hit 31: , Kaysen GA: Hyperlipidemia of the nephroti syndrome Kidney mt 39: , Berlyne GM, Mablik NP: Ishemi heart disease as a ompbiation of nephroti syndrome Lanet 2: 399-4, Mallik NP, Short D: The nephroti syndrome and ishemi heart disease Nephron 27: 54-57, Kaysen GA, Al-Bander H, Martin VI, Jones H Jr, Huthson FN: Branhed-hain amino aids augment neither albuminuria nor albumin synthesis in nephroti rats Am J Physio! 26: Fl77- Fl84, Kaysen GA, Martin VI, Jones H Jr: Arginine augments neither albuminuria nor albumin synthesis aused by high protein diets in nephrosis Am J Physiol 263: F97-F914, D Amio G, Gentile MG, Manna G, Fellin G, ieri R, ofano F, Petrini, Lavarda F, Perolini 5, Pomni M: Effet of vegetarian soy diet on hyperlipidemia in nephroti syndrome Lanet 339: , 1992