Influence of triiodothyronine and dexamethasone on renal amino acid handling in rats loaded with various amino acid mixtures

Summary In adult female rats, the influence of dexamethasone or triiodothyronine on renal amino acid handling was investigated in amino acid loaded animals. Amino acids were administered intravenously as two mixtures, each containing four amino acids to overload amino acid reabsorption capacity. Bolus injections of both mixtures were followed by temporary increase in fractional excretion of the administered amino acids as well of the amino acids which were not covered in the mixtures. The administration of the two mixtures was followed by different interactions between various amino acid carriers.After dexamethasone pretreatment (60µg/100g b.wt. for 3 days, once daily) a stimulation of the renal amino acid handling could be shown. Triiodothyronine (20µg/100g b.wt. for 3 days, once daily) did not increase tubular reabsorption capacity for amino acids. It even increased fractional amino acid excretion in amino acid loaded rats as a sign of enhanced amino acid metabolism in the kidney and/or increased amino acid uptake into the tubular cells from the luminal site.     

Renal handling of amino acids in 5/6-nephrectomized rats: Stimulation of renal amino acid reabsorption after treatment with triiodothyronine or dexamethasone under amino acid load

Summary In anaesthetized adult female rats, the renal amino acid handling was measured six days after 5/6 nephrectomy (5/6NX). The distinct rise in blood urea nitrogen as well as the significant reduction in urine flow and GFR indicate an impairment of kidney function. In principle, in 5/6NX rats amino acid plasma concentrations were comparable to those of control animals with two intact kidneys, whereas the fractional excretions (FE_AA) of most endogenous amino acids measured were significantly enhanced. After bolus injection of leucine or taurine (each 20 mg/100 g b.wt.) or glutamine (90 mg/ 100 g b.wt.), dissolved in 2m1 normal saline per 100 g b.wt., the FE_AA of both the amino acids administered and the endogenous amino acids increased as a sign of overloaded amino acid reabsorption capacity. This effect was more pronounced in 5/6NX rats than in controls. As early as one hour after amino acid load, plasma concentrations and FE_AA returned to baseline values of 5/6NX rats. A pretreatment with triiodothyronine (20,µg/100 g b.wt.) or dexamethasone (60 µg/100 g b.wt.), both given intraperitoneally once daily for 3 days, stimulated the renal amino acid transport capacity in 5/6NX rats: the increase in FE_AA after amino acid load was significantly lower compared to non-pretreatred animals. This stimulation could be shown for the bolus amino acids and the endogenous amino acids and was more distinct in 5/6NX rats than in controls with two intact kidneys.     

Epidermal growth factor (EGF) increases the renal amino acid transport capacity in amino acid loaded rats

Summary In anaesthetized adult female rats, the influence of epidermal growth factor (EGF) on renal amino acid handling was investigated in glutamine, arginine (both 50 mg/100 g b. wt. per hour), or alanine (90 mg/ 100 g b. wt. per hour) loaded animals. Continuous infusions of the three amino acids were followed by an increase in the fractional excretion (FE) of the administered amino acids as well as of the other endogenous amino acids. Under load conditions (alanine, arginine or glutamine), EGF pretreatment (8g/100g b. wt. subcutaneously for 8 days, twice daily 8 a.m. and 4 p.m.) was followed by a stimulation of renal amino acid reabsorption. The increase in the fractional excretion of the administered amino acids was significantly lower than in non-EGF-treated rats. These changes in amino acid transport were connected with a significant reduction of GFR after EGF pretreatment (0.96 ± 0.10 vs. 0.62 ± 0.07 ml/min X 100 g b. wt.) and a distinct increase in sodium excretion (2.98 ± 0.55 vs. 4.97 ± 0.71val/100 g b. wt. X 20 min). After loading with p-aminohippurate (PAH; 200mg/100g b. wt.), PAH excretion in EGF rats was increased by about 20%, whereas urinary protein excretion was lower in EGF pretreated rats (control: 0.45 ± 0.04 vs. EGF: 0.18 ± 0.03 mg/ 100 g b. wt. X 20 min). The PAH load reduced amino acid reabsorption as a sign of overloading of renal tubular transport capacity, but in EGF pretreated animals the amino acid excretion was only slightly increased under these conditions. Furthermore, EGF pretreatment depressed normal kidney weight gain significantly (874 ± 18 vs. 775 ± 32mg/100g b. wt.). EGF can improve the renal tubular transport capacity, but, compared to well-known stimulators of renal transport like dexamethasone or tri-iodothyronine, its effect is only of a moderate degree.     

Renal amino acid transport in immature and adult rats during chromate and cisplatinum-induced nephrotoxicity

Summary. The effects of sodium dichromate (chromate; 1 mg/100 g b. wt. s.c.) and cisdiamminedichloroplatinum(II) (CP; 0.6 mg/100 g b. wt. i.p.) on renal amino acid excretion and plasma amino acid composition were investigated in 10- and 55-day-old anaesthetised rats. On the basis of diuresis experiments on conscious rats the mentioned doses and times (1^st day after chromate in both age groups and in 10-day-old rats after CP and 3^rd day after CP in adult rats) were found out to be optimal for the characterisation of amino acid transport after heavy metal poisoning. Interestingly, in conscious 10-day-old rats chromate nephrotoxicity is not detectable after 1 mg/100 g b. wt. whereas all of the other experimental groups showed nephrotoxic effects of chromate and CP in conscious rats. Urine volumes are lower, but not significantly, in anaesthetised immature rats, independently of the administered nephrotoxin. But GFR is significantly lower in 10-day-old rats, both in controls and after CP, whereas after chromate GFR is significantly reduced only in adult rats and age differences disappeared. In principle the renal fractional excretion (FE) of amino acids was distinctly higher in immature rats as a sign of lower amino acid reabsorption capacity. Nevertheless, the amino acid plasma concentrations were relatively high in immature rats. However, both chromate and CP did not distinctly influence amino acid plasma concentrations. But in both age groups the administration of chromate and CP significantly decreased amino acid reabsorption capacity (increase in FE) as a sign of nephrotoxicity, most pronounced in adult rats after CP. The investigation of renal amino acid handling confirms (1) that both CP and chromate are nephrotoxins, (2) that CP was more nephrotoxic in 55-day-old animals compared to immature rats as could be demonstrated before using other parameters for nephrotoxicity testing and showed (3) that determination of renal amino acid handling is a highly sensitive marker for nephrotoxicity testing, especially in immature rats. Received March 3, 2000 Accepted October 11, 2000     

Regulation of L-leucine transport in rat kidney by dexamethasone and triiodothyronine

We have investigated the transport mechanisms involved in the stimulation of renal tubular reabsorption of large amino acids by glucocorticoids in vivo through the examination of activity and expression of specific transport systems L and y(+)L for L-leucine in membrane preparations of rat kidneys. Kidneys were removed from adult female Wistar rats treated with dexamethasone or triiodothyronine, and the fractions of brush-border and basolateral membranes were isolated by density gradient centrifugation. Functional analysis of L-leucine uptake using rapid filtration technique revealed induction of a sodium-dependent, arginine-inhibitable system y(+)L transport component in the basolateral membrane in the dexamethasone-treated group. A minor sodium-independent, BCH-inhibitable, system L transport component was unaffected by glucocorticoids. L-leucine uptake remained unaffected in the triiodothyronine-treated group. Expression of both subunits of the system y(+)L transporter was increased in dexamethasone-treated rat kidneys: Western blot analysis showed a significant (46%) increase of 4F2hc protein abundance in the basolateral membrane fraction and competitive RT-PCR revealed an almost 4-times induced expression of y(+)LAT1 mRNA. Our results indicate that system y(+)L in rat kidney is regulated by glucocorticoids. We suggest that enhancement of both 4F2 heavy chain and y(+)LAT1 light chain is necessary for induction of this transport system in the kidney.     

The effect on amino acid transport of trypsin treatment of rat renal brush border membranes

Trypsin treatment of isolated rat renal brush border membrane vesicles which preferentially releases l-leucine aminopeptides (EC 3.4.11.2) decreases their ability to take up a variety of amino acids under Na⁺-gradient conditions. Such treatment did not alter the osmotic properties of the vesicles nor affect their fragility. A linear correlation could be demonstrated between the l-leucine aminopeptidase activity of the membranes and the initial rate of uptake of l-leucine and l-proline. Velocity of uptake-concentration dependence studies with these substrates indicate that the major effect of trypsinization is to decrease the maximum velocity (V_max1) of the low-K_m high-affinity system with little effect on the V_max2 of the high-K_m low-affinity transport process and no effect on the apparent Michaelis constants of either. Although the data indicate that l-leucine aminopeptidase activity and uptake of l-leucine and l-proline are affected in parallel, they should not be construed to imply a role of the enzyme in the transport process, especially in view of the global decrease in the uptake of various amino acids and sugars.     

The effect of diamide on amino acid transport by rat renal cortex slices

Diamide directly added to renal cortical slices inhibits the uptake of amino acids. Steady-state kinetic analysis indicates an inhibition of α-amino acid influx without effect on efflux. The effect could be reversed by addition of pyruvate to the incubation medium. Although there was a good correlation of the transport effect of diamide with its ability to decrease cellular reduced glutathione concentration, there did not appear to be a necessary connection between them. This was shown by the fact that renal cortical slices stored at 4°C have no alteration in amino acid uptake despite the fact that GSH concentration is as low as that seen with diamide. Diamide was shown to have a direct effect on the uptake of glycine by isolated renal brush border membrane vesicles.     

Comparative analysis of amino acid metabolism and transport in CHO variants with different levels of productivity

Chinese hamster ovary (CHO) cells are widely used for the production of biopharmaceuticals; however, our understanding of several physiological elements that contribute to productivity is limited. One of these is amino acid transport and how its limitation and/or regulation might affect productivity. To further our understanding, we have examined the expression of 40 mammalian amino acid transporter genes during batch cultures of three CHO cell lines: a non-producer and two antibody-producing cell lines with different levels of productivity. In parallel, extracellular and intracellular levels of amino acids were quantified. The aim was to identify differences in gene regulation between cell lines and within culture. Our results show that three transporters associated with transport of taurine and β-alanine, acidic amino acids and branched chain amino acids, are highly upregulated in both antibody-producing cell lines but not in the non-producer. Additionally, genes associated with the transport of amino acids related to the glutathione pathway (alanine, cysteine, cystine, glycine, glutamate) were found to be highly upregulated during the stationary phase of cell culture, correlating well with literature data on the importance of the pathway. Our analysis highlights potential markers for cell line selection and targets for process optimization.     

Regulation of amino acid transport in the renal epithelial cell line NBL-1

Summary The activities of the transport systems A, B° and X_AG- are induced by various forms of stress in renal epithelial cells. Amino acid deprivation induces System A and X_AG- in a protein-synthesis dependent process. In the case of System X_AG- evidence is presented that induction of transport does not involve an increase in the amount of mRNA for the transporter or of the amount of transport protein. Preliminary evidence for the existence of a novel glycoprotein which is induced in parallel to the induction of these transport systems is presented. It is suggested that the induction of amino acid transport proteins and of some of the so-called stress proteins may be triggered by a common molecular mechanism.     

Regulation of amino acid transport in growing cells of Streptomyces hydrogenans

(1) The active uptake of different amino acids by growing cells of Streptomyces hydrogenans was shown to be correlated with the physiological age of the cells. During the lag phase of growth the transport capacity increased and attained its highest level when the growth rate was maximum. During further growth the transport capacity declined progressively. The lowest transport activity was observed when the culture shifted into the stationary growth phase. (2) Such modulation of transport capacity was independent on the presence or absence of amino acids in the growth medium of the cells. (3) The size and the composition of the pool of free intracellular amino acids was also undergoing substantial variations during the growth cycle of the culture. In the lag phase, the levels of all amino acids decreased markedly and attained their lowest values at the end of this phase. During further growth the pool size was slowly replenished. (4) Removal of the pool resulted in a considerable gain of transport capacity. Therefore, it was concluded that active amino acid transport in growing Streptomyces hydrogenans is under feedback control by intracellular amino acids. (5) Quantitatively, the modulation of the pool size could not fully account for the variation of the transport capacity. Since a pool-independent stimulation of transport was found to be correlated with the increase of the growth rate of the cells, the possibility is discussed that the stimulation of transport is either due to increased levels of distinct RNA species, which might provide positive feedback signals for transport, or by increased rates of de novo synthesis of transport limiting proteins.List of Abbreviations AIB 2-aminoisobutyric acid - CM complete medium - MM mineral medium     

Effects of a new amino acid supplement on blood AA pools in patients with chronic renal failure

Summary We designed a new formula for AA supplement in order to correct blood pools of AA in chronic renal failure (CRF). This supplement was given to 5 patients with CRF and its effectiveness was tested during long term (12–24 weeks) administration. The patients had previously been on a diet providing 0.6 g of protein and 34–36 kcal/kg/day. The diet was then modified to one providing the same caloric content but only 0.3 g/kg high biological value protein per day with the addition of the AA supplement (0.3 g/kg). The new diet corrected most of the abnormalities in blood AA pools. After 1 month of treatment Val, Leu, Thr, Ser and Tyr levels rose and became normal throughout the study. Ratios Tyr/Phe, Ser/Gly and Val/Gly also improved. During the treatment no side effect or toxicity was observed, and serum albumin, transferrin and nutritional anthropometric parameters persisted to be normal. It is concluded that this specially designed AA supplement added to a hypoproteic diet is an acceptable regimen which can quite completely correct the imbalance in blood AA pools in CRF.     

Driving forces and current-voltage characteristics of amino acid transport in Riccia fluitans

The complete steady-state I–V relationship of α-aminoisobutyric acid transport across the plasmalemma of rhizoid cells from Riccia fluitans has been measured and analysed with special emphasis on α-aminoisobutyric acid equilibrium and saturation conditions. (A) The electrical data show that: (1) the amino acid-induced electrical current saturates after the addition of the amino acid, regardless of the concentration; (2) a steady state is reached 1–2 h after incubation in α-aminoisobutyric acid, but after less that 5 min in the presence of 1 mM CN⁻; (3) the steady-state I–V characteristic of α-aminoisobutyric acid transport is a sigmoid curve and fairly symmetric in current with respect to the voltage axis; and (4) the equilibrium potential is clearly a function of the amino acid accumulation ratio. It is suggested that the sigmoid curve represents the characteristic of carrier-mediated α-aminoisobutyric acid transport with a voltage-insensitive step, possibly the translocation of the unloaded carrier, rate-limiting. Since under normal conditions the voltage-sensitive rate constant k_oi is much greater than k_io, it is further suggested that the energy to drive this system is put into the transfer of positive charge from outside to the cytoplasm. (B) Accumulation ratios have been determined by inspection of current-voltage data, and additionally by compartmental analysis on green thalli from Riccia fluitans. Both methods give ratios far too low compared with the thermodynamically possible accumulation of about 10⁴. It is suggested that substantial leakages via different non-electrical pathways prevent equilibrium at steady state, and it is concluded that in such leaky systems the thermodynamic equilibrium condition is not suitable for estimating stoichiometries.     

Dietary deficiencies of single amino acids: Whole-body amino acid composition of adult rats

Abstract

To ascertain whether chronic amino acid deficiency alters the amino acid composition of the body, 44 adult female rats were randomly allocated to one of 11 treatments which included one control group, ingesting an adequate diet with balanced protein, and ten deficient groups in which one group received protein-deficient diets and the other groups consumed diets each deficient in a single essential amino acid. The degree of deficiency was adjusted to achieve a gradual decline in body weight to 85% of the initial weight and was then adjusted so that this weight was maintained until the end of the experiment at 93 days, when the rats were killed. Deficient rats had lower absolute weights of liver, gastrointestinal tract and muscle than animals given the adequate diet but greater relative weights (% of body weight) of heart and kidneys. There were no significant differences amongst groups in percentages of lipid, nitrogen, protein plus lipid or dry matter. Chronic marginal amino acid deficiencies did not selectively alter amino acid composition.     

Sodium dependence of neutral amino acid uptake into rabbit ileum

Alanine uses two mediated pathways to enter the rabbit ileal mucosa. Present results suggest that one of them (K_m = 4.1 mM) is fully dependent on sodium in the mucosal medium, while the other (K_m = 91 mM) is sodium-independent. Similar results are obtained for methionine and serine. Reinterpretation of previous alanine/sodium coupling coefficients suggests that two sodium ions per alanine molecule are transported via the high affinity system.     

Isolation and function of the amino acid transporter PAT1 (slc36a1) from rabbit and discrimination between transport via PAT1 and system IMINO in renal brush-border membrane vesicles

Reabsorption of amino acids is an important function of the renal proximal tubule. pH-dependent amino acid transport has been measured previously using rabbit renal brush-border membrane vesicles (BBMV). The purpose of this investigation was to determine whether this pH-dependent uptake represents H⁺/amino acid cotransport via a PAT1-like transport system. The rabbit PAT1 cDNA was isolated (2296bp including both 5′ and 3′ untranslated regions and poly(A) tail) and the open reading frame codes for a protein of 475 amino acids (92% identity to human PAT1). Rabbit PAT1 mRNA was found in all tissues investigated including kidney. When expressed heterologously in a mammalian cell line, rabbit PAT1 mediates pH-dependent, Na⁺-independent uptake of proline, glycine, l-alanine and α-(methylamino)isobutyric acid. Proline uptake was maximal at pH?5.0 (K_m?2.2±0.7?mM). A transport system with identical characteristics (ion dependency, substrate specificity) was detected in rabbit renal BBMV where an overshoot was observed in the absence of Na⁺ but in the presence of an inwardly directed H⁺ gradient. In the presence of Na⁺ and under conditions in which PAT1 transport function was suppressed, a second proline uptake system was detected that exhibited functional characteristics similar to those of the IMINO system. The functional characteristics of rabbit PAT1 in either mammalian cells or renal BBMV suggest that PAT1 is the low-affinity transporter of proline, glycine and hydroxyproline believed to be defective in patients with iminoglycinuria.     

Different diets and amino acid supplementation do not affect the voluntary consumption of ethanol by rats

The effects of four different diets (control diet: 19.5% protein, 60.5% carbohydrate, 10% fat; diet I: 65% protein, 10% carbohydrate, 10% fat; diet II: 5% protein, 76% carbohydrate, 10% fat; diet III: 20% protein, 69% carbohydrate, 1% fat; diet IV: 69% protein, 15% carbohydrate, 1% fat) and supplementation with 3 amino acids (tryptophan: 150 mg/kg/d; arginine: 400 mg/kg/d; taurine: 380 mg/kg/d) on the voluntary consumption of ethanol were investigated in rats using the 2 bottle method. First, rats received the control diet and diets I, II, III and IV for 20 days with a choice of ethanol for the last 6 days only. Ethanol consumption was similar in all dietary groups. Second, rats received the control diet for 8 days followed by diets I, II and IV for another 8 days. Ethanol was offered throughout both periods. The switch to the special diets did not affect ethanol consumption. Third, rats received a control diet with arginine, tryptophan or taurine added to the drinking fluids for 16 days with a choice of ethanol for the last 5 days; thereafter supplementation stopped but the ethanol choice remained. No difference in the voluntary intake of ethanol was noted but ethanol consumption fell after cessation of arginine supplementation. In conclusion, diets differing greatly in their composition or supplementation with these 3 amino acids did not affect the voluntary choice of ethanol by rats in a significant manner.     

Chloroquine,a novel inhibitor of amino acid transport by rat renal brush border membrane vesicles

Summary Chloroquine is an antimalarial and antirheumatic lysosomotropic drug which inhibits taurine uptake into and increases efflux from cultured human lymphoblastoid cells. It inhibits taurine uptake by rat lung slices and affects the uptake and release of cystine from cystinotic fibroblasts. Speculations on its mode of action include a proton gradient effect, a non-specific alteration in membrane integrity, and membrane stabilization. In this study, the effect of chloroquine on the uptake of several amino acids by rat renal brush border membrane vesicles (BBMV) was examined. Chloroquine significantly inhibited the secondary active, NaCl-dependent component of 10µM taurine uptake at all concentrations tested, but did not change equilibrium values. Analysis of these data indicated that the inhibition was non-competitive. Taurine uptake was reduced at all osmolarities tested, but inhibition was greatest at the lowest osmolarity. Taurine efflux was not affected by chloroquine, nor was the NaCl-independent diffusional component of taurine transport. Chloroquine (1 mM) inhibited uptake of the imino acids L-proline and glycine, and the dibasic amino acid L-lysine. It inhibited the uptake of D-glucose, but not the neutral-amino acids L-alanine or L-methionine. Uptake of the dicarboxylic amino acids, L-glutamic acid and L-aspartic acid, was slightly enhanced. With regard to amino acid uptake by BBMV, these findings may support some of the currently proposed mechanisms of the action of chloroquine but further studies are indicated to determine why it affects the initial rate of active amino acid transport.     

Feeding and arginine deficient diets differentially alter free amino acid concentrations of hindlimb muscle in young rats

Summary The objective of these experiments was to examine short- and long-term (7 d) effects of arginine-deficient diets on free amino acid concentrations in hindlimb muscle of rats. In rats fed the control diet containing arginine (+Arg), muscle alanine and methionine concentrations were higher 1 and 2h after feeding compared to food-deprived rats, whereas branched-chain amino acids, arginine and asparagine concentrations were lower postprandially. In Experiment 1, rats were fed an arginine-deficient (–Arg) diet with glutamate (+Glu) substituted for arginine; alanine (+Ala), ornithine (+Orn) or citrulline (+Cit) were substituted for arginine in Experiment 2. In Experiment 1, arginine concentrations decreased in blood but not in muscle. This contrasts with rats fed –Arg/+Ala or –Arg/+Orn diets which had muscle arginine concentrations less than half the concentrations in controls or in rats fed the –Arg/+Cit diet. Muscle essential amino acids in Experiment 2 did not differ by diet, but muscle branched-chain amino acids were elevated relative to controls in the rats fed –Arg/+Ala or –Arg/+Orn diets; however, rats fed the –Arg/+Cit diet had levels similar to the controls. Also, muscle branched-chain amino acids were correlated with glutamine concentrations in both blood and muscle. The measurements in the post-meal period suggest that muscle amino acid concentrations may more closely reflect dietary amino acid patterns than do blood amino concentrations.Abbreviations BCAA branched-chain amino acids - BCKADH branched-chain ketoacid dehydrogenase - EAA essential amino acids - LNAA large neutral amino acids - NEAA nonessential amino acids - PDV portal-drained viscera - SELSM standard error of least squares means - SSA 5-sulfosalicylic acid - TAA total amino acids Mention of a trade name, proprietary product or specific equipment does not constitute a guarantee by the US Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.     

Expression of atrial natriuretic peptide in thymic macrophages after dexamethasone treatment of rats

Summary The rat thymus represents a site of synthesis of atrial natriuretic peptide (ANP); the immunosuppressor dexamethasone strikingly increases ANP-expression in this immune organ. The presented data suggest that this increase can be attributed to macrophages. By means of immunohistochemistry and Northern blot analysis these immune cells were found to express ANP-immunoreactivity as well as mRNA coding for ANP. In contrast, macrophages of control thymi displayed only weak ANP-immunoreactivity. Thus, ANP appears to be a constituent of rat thymic macrophages, and its synthesis in the thymus is strongly elevated by acute exposure of the animals to glucocorticoids.     

The effect of heat shock on amino acid transport and cell volume in 3T3 cells

Summary. In 3T3 cells temperatures higher than physiological stimulated amino acid transport activity in a dose-dependent manner up to 44°C. However, the temperature increase did not induce widespread transport increase of all other nutrients tested. The activities of both amino acid transport systems A and ASC were enhanced within a few minutes following cell exposure to increased temperature. The maintenance of this effect required continuous exposure of the cells to hyperthermia. Kinetic analysis indicated that the stimulation of the activity of transport System A occurred through a mechanism affecting Vmax rather than Km. The continuous presence of cycloheximide did not prevent the transport changes induced by hyperthermia. These results suggest that the increased amino acid uptake reflects an activation or relocation of existing amino acid transport proteins. During the hyperthermic treatment, the content of ninhydrin-positive substances (NPS), mostly amino acids, increased within the cells and the accumulation of these compatible osmolytes was parallelled by an increase in cell volume. The withdrawal of amino acids from the culture medium immediately before and during the shock phase counteracted the increase and reduced the NPS content but did not prevent the increase in amino acid transport, the cell swelling and the induction of the heat shock response. Received June 30, 1999 Accepted July 27, 2000