Acta Physiol Scand 1990, 139, 9-13
Amino acid stimulation of Na,K-ATPase activity in rat proximal tubule after high-protein diet B. JAKOBSSON, S. H . L A R S S O N , " A. W I E S L A N D E R " and A. A P E R I A " Department of Pediatrics, Huddinge Hospital, and * Department of Pediatrics, St Goran's Children's Hospital, Karolinska Institute, Stockholm, Sweden
B., LARSSON, s. H., WIESLANDER, A. & APERIA,A. 1990. Amino acid stimulation of Na, K-ATPase activity in rat proximal tubule after high-protein diet. Acta Physzol Scand 139, 9-13. Received 20 March 1989,accepted 18 December 1989.ISSN 0001-6772.Department of Pediatrics, Huddinge Hospital, and Department of Pediatrics, St Goran's Children's Hospital, Karolinska Institute, Stockholm, Sweden.
JAKOBSSON,
Ouabain-sensitive (0s)0, consumption was determined in proximal tubular cells from weanling rats fed 21 yo (normal-protein, NP) or 50% (high-protein, HP) protein diet for 4 days. Butyric acid I O - ~M was added as a substrate for mitochondria1 respiration and the ionophore amphotericin B (10 ,ug m1-l) was used to sodium-load the cells. 0s respiration was higher in HP than in NP cells in both DME and amino acid-free electrolyte solution (ES). Amphotericin B significantly increased 0s respiration in both NP and HP cells, implying that the Na-K pump was activated by increased intracellular Na. In cells incubated in ES, addition of amino acids stimulated 0s respiration f I .4us 2 1.zf I . I nmol min-l mg-l protein) and in NP significantly in HP cells (16.9 cells (13.9ko.3 vs 14.9fo.6nmol min-' mg-l protein). Stimulation was significantly higher in HP cells (26&4%) than in NP cells (7f4%) ( P < 0.001). The amino acids did not stimulate ouabain-insensitive respiration. The results indicate that an HP diet to weanling rats will increase proximal tubule cell Na, K-ATPase-dependent respiration by enhancing Na entry via the Na-amino acid symports. Key words ; development, high-protein intake, Na-amino acid symports, Na-H .antiporter, Na,K-ATPase, oxygen consumption, proximal tubule cells.
High-protein diet stimulates kidney growth. Growth stimulation is prominent in the proximal parts of the nephron (Jakobsson et al. 1987, 1988). T h e mechanisms behind growth stimulation have not been clarified, but several factors have been proposed. These include the release of growth factors and an increase in the uptake of substrate, i.e. amino acids (Leffert & Koch 1979, Morgan & Naismith 1980, Paller & Hostetter 1986). T h e capacity of the proximal tubule cells to reabsorb amino acids is high. T h e purpose of this study has been to determine whether HP intake increases the amino
Correspondence : Birgir Jakobsson, Department of Pediatrics, Huddinge Hospital, S-141 86 Huddinge, Sweden.
acid uptake by the proximal tubule cell. I n this cell type the amino acids are reabsorbed with Na, and the electrochemical gradient created by active Na,K-ATPase-mediated extrusion of Na from the cells facilitates this transport (Ullrich 1979, Schafer & Williams 1985). I n a previous communication (Jakobsson & Aperia 1990) it was shown that Na,K-ATPase activity in proximal tubule segments from weanling rats is increased after 4 days on high-protein diet. I n that study Na, K-ATPase was measured as the rate of phosphate hydrolysis under V,,, conditions for Na, K and ATP. T h e turnover of Na, K-ATPase is limited in vivo because normally the cell Na concentration is less than Ki for this enzyme. T h e activity of the Na-K pump will therefore be modulated by the rate of N a entry into the cell (9). I n this
9
10
B.Jakobsson et al.
study we have assessed the relationship between the Na-amino acid symport and the Na-K p u m p by determining N a , K-ATPase-dependent respiration in proximal tubule cells suspended in media with and without amino acids. The renal proximal tubule cells were obtained from weanling rats given 21 yo ( N P ) or 50% ( H P ) protein diets.
M A T E R I A L S A ND M E T H O D S The studies were performed on male Sprague-Dawley rats. They were weaned at 16 days of age. At that age the body weight (body wt) ranged between 36 and 40 g. Rats from the same litter were then randomly allocated to two experimental groups that were given N P or HP diet (Ewos, Sodertalje, Sweden). T h e diets were isocaloric ; the difference in protein caloric value was compensated for by variations in carbohydrate content. The source of protein was pure casein. Since casein contains phosphorus, the total amount of phosphorus was higher in the HP diet than in the N P diet (0.72 yo and 0.45yo respectively). The contents of sodium, potassium and calcium were identical. Five rats were housed in each cage and they had free access to food and water. T h e food consumption was measured in each cage and was found not to differ significantly between the two experimental groups. At 20 days the body weight in both experimental groups ranged between 45 and 5 2 g, and there was no significant difference between the groups. Prepuration of cortical cell suspension. After anaesthesia with Inactin-Byk (Byk Gulden, Constanz, FRG) 80 mg kg-' i.p. the aorta was cut and the rats were bled, whereupon the kidneys were removed. I n each experiment kidneys from 6 9 animals were used. The cortex was separated from the medulla and thoroughly minced on ice with a razor-blade to a paste-like consistency. It was then incubated in one of two different solutions containing collagenase (0.065 g IOO ml-') at 37 "C in a shaking water bath for 90 min. T h e incubation solutions were Dulbecco's modified Eagle's medium (DME) (Gibco Laboratories) or a glucose- and amino acid-free electrolyte solution (ES) containing (mM) NaCl 105, KCI 5, NaHCO, 25, CaCI, 1.8, MgSO, I, sodium acetate 10 and Hepes 10.During incubation the solutions were continuously exposed to 95y00, and 5 % CO,. The cell suspension was cooled on ice and poured through graded filters (pore size 180pm, 75 pm, 53 p m and 38 pm). It was kept on ice for 15 min to allow the remains of tubule fragments to form a sediment. The remaining suspension, which contained a population of 75-80% proximal tubule cells (Kaissling & Criz 1979)was washed 2-3 times in 20 ml of the incubation solution without collagenase and the cells were collected after a slow spin (100 g for 4 min). The
remaining pellet was then diluted with 4-6 ml of the incubation solution. Oxygen consumption measurements. Cells suspended in D M E or in ES were transferred to a continuously stirred 3-ml chamber maintained at 37 "C with a circulating water bath. Aliquots containing 500 of the cell suspension were diluted with 2-5 ml of the incubation solution. The chamber was sealed by inserting a Clark-type polarographic oxygen probe (Scientific Division, Yellow Springs Instruments Co., Inc., Yellow Springs, Ohio, USA). Four minutes were allowed for temperature equilibration before measurements of the oxygen tension were started. T o optimi7e mitochondria1 respiration, butyric acid 10 ' M was added just before the cell suspension was added to the chamber. T o determine Na,K-ATPase-independent respiration, ouabain 2 x I O - ~M was added. This concentration of ouabain completely inhibits the Na-K pump in proximal tubule cells in primary culture (Larsson et al. 1986). In some of the protocols amphotericin B or a mixture of amino acids" (Kabi Vitrum, Sweden) was added just before determination of Qo,.
Ouabain was added to the chamber in 30pl of distilled water and amphotericin B in 30 p1 of dimethyl sulphoxide (DMSO). Fifteen microlitres of amino acid mixture was added. The osmolality in all solutions was approximately 300 mosmol kg-' and the pH was adjusted to 7.4. All studies were run in duplicate or triplicate. I n each study period, a parallel experiment using only vehicle was performed. The protein content of each sample was determined by the Bio-Rad method (Bio-Rad Laboratories 1977). Statistical analysis. The unpaired Student t-test was used for comparison of the N P and H P groups and the paired Student t-test for comparison within the N P and H P groups. A P-value less than 0.05 was considered significant.
RESULTS Basal 0, consumption was determined in cells incubated with a DME solution and in a glucoseand amino acid-free ES. T o t a l as well as ouabainsensitive oxygen consumption (OSQo0,was significantly higher in HP cells than in NP cells in all conditions of incubation (Table I ) . T h e difference in OSQoo,between HP and NP cells "g 1000 ml-': glycine, 7.9; L-aspartic, 3.4; Lglutamine, 5.6; L-alanine, 16.0; L-arginine, 1 1 . 3 ; Lcysteine and L-cystine, 0.56; L-histidine, 6.8; Lisoleucine, 5.6; L-leucine, 7.9; L-lysine, 9.0; Lmethionine, 5.6 ; L-phenylalanine, 7.9 ; L-proline, 6.8 ; L-serine, 4.5 ; L-threonine, 5.6; L-tryptophan, 1.9, Ltyrosine, 0.23; I.-valine, 7.3.
Na-amino acid symports in high protein intake
11
Table I . Total and ouabain-sensitive oxygen consumption (QoJ in proximal tubule cells from zo-day-old rats fed NP and HP diets for 4 days. The cells were incubated either in Dulbecco's modified Eagle's medium (DME) or in a amino acid- and glucose-free electrolyte solution (ES). Butyric acid I O - ~M has been added to both solutions Total Qo, (nmol min-' mg-' protein)
Ouabain-sensitive Qo, (nmol min-' mg-' protein) __
NP
DME ES
HP
__
49.1 k 10.6 (6) 37.7k0.9 (6)
*63.3*3.9
(5) *45.7 f 3 . 2 (4)
NP
HP
'7.9 f3.5 (6) 13.9kO.3 (6)
"24.7k 1 . 5 (5) *16.9+ 1.4 (4)
Values are means f.I SD, number of experiments is given in parenthesis. * Significantly different from NP cells in the same medium.
was less pronounced if the cells were incubated in ES (20%) than if they were incubated in DME (37%) (Table I ) . I n the studies presented in Table I , butyric acid was added to the cell suspension to provide the mitochondria with an optimal substrate (Harris et al. 1981). I n DME-incubated cells, removal of butyric acid decreased Qo, by approximately 20% in both NP cells (49.1f 10.6 us 4 1 . 4 f 4 . 8 , n = 4-6) and HP cells (63.3 f3.8 us 50.3 f5.9, n = 4-6). To determine Na, K-ATPase-dependent respiration under V,,, conditions for Na, cells incubated in DME were Na-loaded by the addition of the ionophore amphotericin B 0
.
-* I
C
b
ml-l) (Fig. I a and b). Amphotericin B significantly increased OSQo, in NP cells (17.9f3.5 us 42.8f6.1 nmol min-' mg-' protein) and in HP cells (24.7 1 . 5 us 60.3 & 6. I nmol min-' mg-' protein). Amphotericin B also significantly increased total Qo, in NP cells and HP cells. T h e amphotericin B stimulation of OSQo, was significantly higher in HP cells than in N P cells (P< 0.001) (Fig. I a). Amphotericin B did not increase Qo, in the presence of ouabain (data not shown), which implies that the increase in Qo, caused by amphotericin B is due to increased Na-K pump activity. I n cells incubated in ES, the addition of amino acids (4-5 mM) significantly stimulated OSQo, in both NP and HP cells (Fig. 2). I n N P cells OSQO, increased from 13.9fo.3 to
( 1 0p g
-***-
I -
ambh
C
arnph
Fig. I . Ouabain-sensitive (a) and total (b) Qo, during control (c) conditions and after addition of amphotericin B 10 ,ug ml-' (amph) in proximal tubule cells from zo-day-old rats fed NP (closed circles) or HP (open circles) diets for 4 days. Values are meansf I SD, n = 5 4 experiments in each group, * P < 0.05,
** P < 0.01, *** P < 0.001.
c'
ad
'c
aa'
Fig. 2. Ouabain-sensitive Qo, during control (c) , , conditions and after addition-of amino acids 4-5 mM (aa) in proximal tubule cells from 20-day-old rats fed NP (open bars) or HP (hatched bars) diets for 4 days. Values are meansf I SD, n = 4 4 experiments in each group, * P < 0.05, *** P < 0.001.
12
B . Jakobsson et al.
Oxygen consumption (Qo,) in the of mitochondrial uncoupler (FCCP 3 x I O - ~M) without or with amino acids 4-5 mM (aa) in proximal tubule cells from ao-day-old rats fed NP or HP diets for 4 days. The cells were incubated in an amino acid- and glucose-free solution in the presence of butyric acid I O - ~M Table
2.
presence
Total Qo, (nmol min-' mg-' protein)
NP
HP
FCCP
FCCP + aa
P
63.5f0.6 *72.3f5.0
63.23 fz.5 +69.3 f2.3
NS NS
Values are meansf I SD, n = 3-5 experiments in each group. * Significantly different from NP cells.
14.9& 0.6 nmol min-' mg-' protein ( P < 0.05). In HP cells OSQO, increased from 16.9f1.4 to 2 1 . 2 f 1 . 1 nmol min-' mg-' protein (P < 0.001). Amino acids did not increase Qo, in the presence of ouabain. I n N P cells ouabain-insensitive Qo, was 23.7 f0.6 nmol min-' mg-' protein in the absence and 22.7 f0.7 nmol min-' mg-' protein in the presence of amino acids. I n H P cells the corresponding values were 28.8 f 1.9 and 27.2 2.4 nmol min-' mg-' protein respectively. Amino acids stimulated OSQO, significantly more in H P cells than in N P cells. OSQo, was increased by 7 4 yoin N P cells and by 26 4 yo (mean f I SD) in HP cells ( P < 0.001). I n the presence of the. mitochondrial uncoupler FCCP ( 3 x I O - ~M), Qo, was the same with and without amino acids in both N P and HP cells (Table 2 ) . In the presence of FCCP, Qo, was significantly higher in H P cells than in NP cells (P < 0.05) (Table 2). DISCUSSION In proximal tubule cells from H P rats OSQO, was increased both when intracellular Na concentration was normal and when it was increased by an ionophore. Since ouabain is a specific inhibitor of Na, K-ATPase, it is assumed that OSQo, represents Na,K-ATPase-dependent respiration. The observations confirm results from a previous study (Jakobsson & Aperia 1990) in which Na, K-ATPase activity in isolated proximal tubules from H P rats was measured as the rate of phosphorate hydrolysis at Y,,, for Na, K and ATP. Na, K-ATPase was then found to be 42 yo higher in tubules from weanling rats
on an H P diet than in tubules from weanling rats on an N P diet. When the intracellular Na was normal, OSQO, ranged between 14 and 18 nmol min-' mg-' protein depending on the type of solution used for the incubation. In Na-loaded cells OSQO, was 43 nmol min-' mg-' protein. These results are similar to those found by Harris et al. (1982) in proximal tubules from adult rabbits (12 and 33 nmol min-' mg-' protein respectively). Amphotericin B was used to increase the intracellular Na in both NP and HP cells. This stimulated Na, K-ATPase-dependent respiration approximately 1 5 0 % in both NP and HP cells. The finding is in accordance with the concept that under in-vivo conditions the Na-K pump is unsaturated with respect to intracellular Na, and that the entry of Na into the proximal tubule cell modulates the Na-K pump activity. Studies of Na, K-ATPase-dependent respiration can therefore be used to evaluate the Na entry into the cell. Almost all amino acids that are filtered will be reabsorbed in the proximal tubule, where they are co-transported with sodium (Ullrich 1979, Schafer & Williams 1985). In the isolated perfused tubule, the removal of amino acids from the perfusate reduces Na reabsorption by approximately 20 yo(Kokko 1973). The influence of Na-amino acid symports on Na, K-ATPasedependent respiration was studied by adding a mixture of amino acids to cells incubated in an amino acid-free medium. T o provide substrate for mitochondrial respiration, butyric acid was added to the incubation medium. The addition of amino acids significantly increased OSQo, in both N P and HP cells. The effect was much more pronounced in the H P cells. There are at least two possible explanations for this enhanced effect in HP cells. The amino acids may increase mitochondrial respiration by increasing substrate availability. It is unlikely that this is the main cause of the increase in Qo,. The amino acids increased OSQo,, but not ouabain-insensitive Qo,. Moreover, in the uncoupler studies the mitochondrial respiration was found to be the same with and without amino acids in both NP and H P cells. Amino acids, with the exception of glutamine, are not oxidized to any extent by the proximal tubules (Mandel 1985). The concentration of glutamine in the amino acid mixture was less than 0.2 mM, which, in the presence of butyric acid, should have little influence on
Na-amino acid symports in high protein intake
APERIA, A. 1990.High protein intake accelerates the maturation of Na, K-ATPase in rat renal tubules. Actu Physiol Scund 139, 1-7. JAKOBSSON, B., CELSI,G., LINDBALD, B.S. & APERIA, A. 1987. Influence of different protein intake on renal growth in young rats. Actu Puediulr Scund 76, 293-299. JAKOBSSON, B., BOHMAN,S.O., SUNDELIN, B. & APERIA,A. 1988. Mitotic response to high protein intake in different renal cell types in weanling rats. Kidney Int 33, 662-666. KAISSLING, B. & CRIZ,W. 1979. Structural analysis of the rabbit kidney. A d v Anut Embryo1 Cell Biol 56, 1-123. KOKKO,J.P. 1973. Proximal tubule potential difference. Dependence on glucose, IICO,, and amino acids. 3 Clin Invest 52, 1362-1367. LARSSON, L., APERIA,A. & LECHENE, C. 1986. Ionic transport in individual renal epithelial cells from adult and young rats. Actu Ph,ysiol Scund 126, 321-332. LEFFERT,H.L. & KOCH,K.S. 1979. Regulation of growth of hepatocytes by sodium ions. In: , H . Popper & F. Schaffner (eds.) Progress in Licer Diseuses, pp. 123-134. Grune & Stratton, New This work was supported by grants from the Swedish York. Medical Research Council (projects nos. 0364402049). Expert technical assistance was provided by MANDEL,L.J. 1985. Metabolic substrates, cellular energy production, and the regulation of proximal Ms Ingrid Muldin and Ms Ann-Christine Eklof. tubular transport. Ann Rev Physinl47, 85-101. MORGAN, B.L.G. & NAISMITH, D.J. 1980. Value of REFERENCES dietary protein for hyperplastic growth at restricted BIO-RADLABORATORIES 1977. Bio-Rad protein assay. energy intakes. 3 Nutr 110, 618-626. Technical Bulletin 10sI . PALLER,M.S. & HOSTETTER, T.H. 1986. Dietary HARRIS, S.I., BALABAN, R.S., BARRET, L. & MANDEL, protein increases plasma renin and reduces pressor L.J. 1981. Mitochondria1 respiration capacity and reactivity to angiotensin 11. A m 3 Ph,ysiol 251, Na+- and K+-dependent adenosine triphosphataseF34-F39. mediated ion transport in the intact renal cell. 3 SCHAFER, J.A. & WILLIAMS JR, J.C. 1985. Transport Biol Chem 256, 10319-10328. of metabolic substrates by the proximal nephron. HARRIS, S.I., PATTON, L., BARRET, L. & MANDEL, L.J. Ann Rev Physiol47, 103-125. 1982. (Na+, K+)-ATPase kinetics within the intact ULLRICH,K.J. 1979. Sugar, amino acid, and Na+ cell. The role of oxidative metabolism. 3 Bid Chem cotransport in the proximal tubule. Ann Rev Physiol 41, 181-195. 257, 6996-7002.
mitochondrial respiration. Therefore, a more likely explanation of the amino acid-induced increase in OSQo, is that the Na-K pump has been activated by increased Na entry via the Na-amino acid symports. As indicated in Table 2, uncoupled mitochondrial respiration was higher in HP cells than in N P cells. Since this respiration is independent of pump activity, HP intake also seems to have some direct effect on mitochondrial activity. In conclusion, Na, K-ATPase-dependent respiration is higher in PCT cells from HP rats than from NP rats both in cells with normal intracellular Na levels and in Na-loaded cells. Stimulation of the Na-amino acid symport causes disproportionate increase in the Na, KATPase-dependent respiration in HP cells. T h i s observation suggests that, in proximal tubule cells from weanling rats, HP intake increases cellular Na uptake, mainly via the Na-amino acid symports.
JAKOBSSON, B. &
13
Amino acid stimulation of Na,K-ATPase activity in rat proximal tubule after high-protein diet B. JAKOBSSON, S. H . L A R S S O N , " A. W I E S L A N D E R " and A. A P E R I A " Department of Pediatrics, Huddinge Hospital, and * Department of Pediatrics, St Goran's Children's Hospital, Karolinska Institute, Stockholm, Sweden
B., LARSSON, s. H., WIESLANDER, A. & APERIA,A. 1990. Amino acid stimulation of Na, K-ATPase activity in rat proximal tubule after high-protein diet. Acta Physzol Scand 139, 9-13. Received 20 March 1989,accepted 18 December 1989.ISSN 0001-6772.Department of Pediatrics, Huddinge Hospital, and Department of Pediatrics, St Goran's Children's Hospital, Karolinska Institute, Stockholm, Sweden.
JAKOBSSON,
Ouabain-sensitive (0s)0, consumption was determined in proximal tubular cells from weanling rats fed 21 yo (normal-protein, NP) or 50% (high-protein, HP) protein diet for 4 days. Butyric acid I O - ~M was added as a substrate for mitochondria1 respiration and the ionophore amphotericin B (10 ,ug m1-l) was used to sodium-load the cells. 0s respiration was higher in HP than in NP cells in both DME and amino acid-free electrolyte solution (ES). Amphotericin B significantly increased 0s respiration in both NP and HP cells, implying that the Na-K pump was activated by increased intracellular Na. In cells incubated in ES, addition of amino acids stimulated 0s respiration f I .4us 2 1.zf I . I nmol min-l mg-l protein) and in NP significantly in HP cells (16.9 cells (13.9ko.3 vs 14.9fo.6nmol min-' mg-l protein). Stimulation was significantly higher in HP cells (26&4%) than in NP cells (7f4%) ( P < 0.001). The amino acids did not stimulate ouabain-insensitive respiration. The results indicate that an HP diet to weanling rats will increase proximal tubule cell Na, K-ATPase-dependent respiration by enhancing Na entry via the Na-amino acid symports. Key words ; development, high-protein intake, Na-amino acid symports, Na-H .antiporter, Na,K-ATPase, oxygen consumption, proximal tubule cells.
High-protein diet stimulates kidney growth. Growth stimulation is prominent in the proximal parts of the nephron (Jakobsson et al. 1987, 1988). T h e mechanisms behind growth stimulation have not been clarified, but several factors have been proposed. These include the release of growth factors and an increase in the uptake of substrate, i.e. amino acids (Leffert & Koch 1979, Morgan & Naismith 1980, Paller & Hostetter 1986). T h e capacity of the proximal tubule cells to reabsorb amino acids is high. T h e purpose of this study has been to determine whether HP intake increases the amino
Correspondence : Birgir Jakobsson, Department of Pediatrics, Huddinge Hospital, S-141 86 Huddinge, Sweden.
acid uptake by the proximal tubule cell. I n this cell type the amino acids are reabsorbed with Na, and the electrochemical gradient created by active Na,K-ATPase-mediated extrusion of Na from the cells facilitates this transport (Ullrich 1979, Schafer & Williams 1985). I n a previous communication (Jakobsson & Aperia 1990) it was shown that Na,K-ATPase activity in proximal tubule segments from weanling rats is increased after 4 days on high-protein diet. I n that study Na, K-ATPase was measured as the rate of phosphate hydrolysis under V,,, conditions for Na, K and ATP. T h e turnover of Na, K-ATPase is limited in vivo because normally the cell Na concentration is less than Ki for this enzyme. T h e activity of the Na-K pump will therefore be modulated by the rate of N a entry into the cell (9). I n this
9
10
B.Jakobsson et al.
study we have assessed the relationship between the Na-amino acid symport and the Na-K p u m p by determining N a , K-ATPase-dependent respiration in proximal tubule cells suspended in media with and without amino acids. The renal proximal tubule cells were obtained from weanling rats given 21 yo ( N P ) or 50% ( H P ) protein diets.
M A T E R I A L S A ND M E T H O D S The studies were performed on male Sprague-Dawley rats. They were weaned at 16 days of age. At that age the body weight (body wt) ranged between 36 and 40 g. Rats from the same litter were then randomly allocated to two experimental groups that were given N P or HP diet (Ewos, Sodertalje, Sweden). T h e diets were isocaloric ; the difference in protein caloric value was compensated for by variations in carbohydrate content. The source of protein was pure casein. Since casein contains phosphorus, the total amount of phosphorus was higher in the HP diet than in the N P diet (0.72 yo and 0.45yo respectively). The contents of sodium, potassium and calcium were identical. Five rats were housed in each cage and they had free access to food and water. T h e food consumption was measured in each cage and was found not to differ significantly between the two experimental groups. At 20 days the body weight in both experimental groups ranged between 45 and 5 2 g, and there was no significant difference between the groups. Prepuration of cortical cell suspension. After anaesthesia with Inactin-Byk (Byk Gulden, Constanz, FRG) 80 mg kg-' i.p. the aorta was cut and the rats were bled, whereupon the kidneys were removed. I n each experiment kidneys from 6 9 animals were used. The cortex was separated from the medulla and thoroughly minced on ice with a razor-blade to a paste-like consistency. It was then incubated in one of two different solutions containing collagenase (0.065 g IOO ml-') at 37 "C in a shaking water bath for 90 min. T h e incubation solutions were Dulbecco's modified Eagle's medium (DME) (Gibco Laboratories) or a glucose- and amino acid-free electrolyte solution (ES) containing (mM) NaCl 105, KCI 5, NaHCO, 25, CaCI, 1.8, MgSO, I, sodium acetate 10 and Hepes 10.During incubation the solutions were continuously exposed to 95y00, and 5 % CO,. The cell suspension was cooled on ice and poured through graded filters (pore size 180pm, 75 pm, 53 p m and 38 pm). It was kept on ice for 15 min to allow the remains of tubule fragments to form a sediment. The remaining suspension, which contained a population of 75-80% proximal tubule cells (Kaissling & Criz 1979)was washed 2-3 times in 20 ml of the incubation solution without collagenase and the cells were collected after a slow spin (100 g for 4 min). The
remaining pellet was then diluted with 4-6 ml of the incubation solution. Oxygen consumption measurements. Cells suspended in D M E or in ES were transferred to a continuously stirred 3-ml chamber maintained at 37 "C with a circulating water bath. Aliquots containing 500 of the cell suspension were diluted with 2-5 ml of the incubation solution. The chamber was sealed by inserting a Clark-type polarographic oxygen probe (Scientific Division, Yellow Springs Instruments Co., Inc., Yellow Springs, Ohio, USA). Four minutes were allowed for temperature equilibration before measurements of the oxygen tension were started. T o optimi7e mitochondria1 respiration, butyric acid 10 ' M was added just before the cell suspension was added to the chamber. T o determine Na,K-ATPase-independent respiration, ouabain 2 x I O - ~M was added. This concentration of ouabain completely inhibits the Na-K pump in proximal tubule cells in primary culture (Larsson et al. 1986). In some of the protocols amphotericin B or a mixture of amino acids" (Kabi Vitrum, Sweden) was added just before determination of Qo,.
Ouabain was added to the chamber in 30pl of distilled water and amphotericin B in 30 p1 of dimethyl sulphoxide (DMSO). Fifteen microlitres of amino acid mixture was added. The osmolality in all solutions was approximately 300 mosmol kg-' and the pH was adjusted to 7.4. All studies were run in duplicate or triplicate. I n each study period, a parallel experiment using only vehicle was performed. The protein content of each sample was determined by the Bio-Rad method (Bio-Rad Laboratories 1977). Statistical analysis. The unpaired Student t-test was used for comparison of the N P and H P groups and the paired Student t-test for comparison within the N P and H P groups. A P-value less than 0.05 was considered significant.
RESULTS Basal 0, consumption was determined in cells incubated with a DME solution and in a glucoseand amino acid-free ES. T o t a l as well as ouabainsensitive oxygen consumption (OSQo0,was significantly higher in HP cells than in NP cells in all conditions of incubation (Table I ) . T h e difference in OSQoo,between HP and NP cells "g 1000 ml-': glycine, 7.9; L-aspartic, 3.4; Lglutamine, 5.6; L-alanine, 16.0; L-arginine, 1 1 . 3 ; Lcysteine and L-cystine, 0.56; L-histidine, 6.8; Lisoleucine, 5.6; L-leucine, 7.9; L-lysine, 9.0; Lmethionine, 5.6 ; L-phenylalanine, 7.9 ; L-proline, 6.8 ; L-serine, 4.5 ; L-threonine, 5.6; L-tryptophan, 1.9, Ltyrosine, 0.23; I.-valine, 7.3.
Na-amino acid symports in high protein intake
11
Table I . Total and ouabain-sensitive oxygen consumption (QoJ in proximal tubule cells from zo-day-old rats fed NP and HP diets for 4 days. The cells were incubated either in Dulbecco's modified Eagle's medium (DME) or in a amino acid- and glucose-free electrolyte solution (ES). Butyric acid I O - ~M has been added to both solutions Total Qo, (nmol min-' mg-' protein)
Ouabain-sensitive Qo, (nmol min-' mg-' protein) __
NP
DME ES
HP
__
49.1 k 10.6 (6) 37.7k0.9 (6)
*63.3*3.9
(5) *45.7 f 3 . 2 (4)
NP
HP
'7.9 f3.5 (6) 13.9kO.3 (6)
"24.7k 1 . 5 (5) *16.9+ 1.4 (4)
Values are means f.I SD, number of experiments is given in parenthesis. * Significantly different from NP cells in the same medium.
was less pronounced if the cells were incubated in ES (20%) than if they were incubated in DME (37%) (Table I ) . I n the studies presented in Table I , butyric acid was added to the cell suspension to provide the mitochondria with an optimal substrate (Harris et al. 1981). I n DME-incubated cells, removal of butyric acid decreased Qo, by approximately 20% in both NP cells (49.1f 10.6 us 4 1 . 4 f 4 . 8 , n = 4-6) and HP cells (63.3 f3.8 us 50.3 f5.9, n = 4-6). To determine Na, K-ATPase-dependent respiration under V,,, conditions for Na, cells incubated in DME were Na-loaded by the addition of the ionophore amphotericin B 0
.
-* I
C
b
ml-l) (Fig. I a and b). Amphotericin B significantly increased OSQo, in NP cells (17.9f3.5 us 42.8f6.1 nmol min-' mg-' protein) and in HP cells (24.7 1 . 5 us 60.3 & 6. I nmol min-' mg-' protein). Amphotericin B also significantly increased total Qo, in NP cells and HP cells. T h e amphotericin B stimulation of OSQo, was significantly higher in HP cells than in N P cells (P< 0.001) (Fig. I a). Amphotericin B did not increase Qo, in the presence of ouabain (data not shown), which implies that the increase in Qo, caused by amphotericin B is due to increased Na-K pump activity. I n cells incubated in ES, the addition of amino acids (4-5 mM) significantly stimulated OSQo, in both NP and HP cells (Fig. 2). I n N P cells OSQO, increased from 13.9fo.3 to
( 1 0p g
-***-
I -
ambh
C
arnph
Fig. I . Ouabain-sensitive (a) and total (b) Qo, during control (c) conditions and after addition of amphotericin B 10 ,ug ml-' (amph) in proximal tubule cells from zo-day-old rats fed NP (closed circles) or HP (open circles) diets for 4 days. Values are meansf I SD, n = 5 4 experiments in each group, * P < 0.05,
** P < 0.01, *** P < 0.001.
c'
ad
'c
aa'
Fig. 2. Ouabain-sensitive Qo, during control (c) , , conditions and after addition-of amino acids 4-5 mM (aa) in proximal tubule cells from 20-day-old rats fed NP (open bars) or HP (hatched bars) diets for 4 days. Values are meansf I SD, n = 4 4 experiments in each group, * P < 0.05, *** P < 0.001.
12
B . Jakobsson et al.
Oxygen consumption (Qo,) in the of mitochondrial uncoupler (FCCP 3 x I O - ~M) without or with amino acids 4-5 mM (aa) in proximal tubule cells from ao-day-old rats fed NP or HP diets for 4 days. The cells were incubated in an amino acid- and glucose-free solution in the presence of butyric acid I O - ~M Table
2.
presence
Total Qo, (nmol min-' mg-' protein)
NP
HP
FCCP
FCCP + aa
P
63.5f0.6 *72.3f5.0
63.23 fz.5 +69.3 f2.3
NS NS
Values are meansf I SD, n = 3-5 experiments in each group. * Significantly different from NP cells.
14.9& 0.6 nmol min-' mg-' protein ( P < 0.05). In HP cells OSQO, increased from 16.9f1.4 to 2 1 . 2 f 1 . 1 nmol min-' mg-' protein (P < 0.001). Amino acids did not increase Qo, in the presence of ouabain. I n N P cells ouabain-insensitive Qo, was 23.7 f0.6 nmol min-' mg-' protein in the absence and 22.7 f0.7 nmol min-' mg-' protein in the presence of amino acids. I n H P cells the corresponding values were 28.8 f 1.9 and 27.2 2.4 nmol min-' mg-' protein respectively. Amino acids stimulated OSQO, significantly more in H P cells than in N P cells. OSQo, was increased by 7 4 yoin N P cells and by 26 4 yo (mean f I SD) in HP cells ( P < 0.001). I n the presence of the. mitochondrial uncoupler FCCP ( 3 x I O - ~M), Qo, was the same with and without amino acids in both N P and HP cells (Table 2 ) . In the presence of FCCP, Qo, was significantly higher in H P cells than in NP cells (P < 0.05) (Table 2). DISCUSSION In proximal tubule cells from H P rats OSQO, was increased both when intracellular Na concentration was normal and when it was increased by an ionophore. Since ouabain is a specific inhibitor of Na, K-ATPase, it is assumed that OSQo, represents Na,K-ATPase-dependent respiration. The observations confirm results from a previous study (Jakobsson & Aperia 1990) in which Na, K-ATPase activity in isolated proximal tubules from H P rats was measured as the rate of phosphorate hydrolysis at Y,,, for Na, K and ATP. Na, K-ATPase was then found to be 42 yo higher in tubules from weanling rats
on an H P diet than in tubules from weanling rats on an N P diet. When the intracellular Na was normal, OSQO, ranged between 14 and 18 nmol min-' mg-' protein depending on the type of solution used for the incubation. In Na-loaded cells OSQO, was 43 nmol min-' mg-' protein. These results are similar to those found by Harris et al. (1982) in proximal tubules from adult rabbits (12 and 33 nmol min-' mg-' protein respectively). Amphotericin B was used to increase the intracellular Na in both NP and HP cells. This stimulated Na, K-ATPase-dependent respiration approximately 1 5 0 % in both NP and HP cells. The finding is in accordance with the concept that under in-vivo conditions the Na-K pump is unsaturated with respect to intracellular Na, and that the entry of Na into the proximal tubule cell modulates the Na-K pump activity. Studies of Na, K-ATPase-dependent respiration can therefore be used to evaluate the Na entry into the cell. Almost all amino acids that are filtered will be reabsorbed in the proximal tubule, where they are co-transported with sodium (Ullrich 1979, Schafer & Williams 1985). In the isolated perfused tubule, the removal of amino acids from the perfusate reduces Na reabsorption by approximately 20 yo(Kokko 1973). The influence of Na-amino acid symports on Na, K-ATPasedependent respiration was studied by adding a mixture of amino acids to cells incubated in an amino acid-free medium. T o provide substrate for mitochondrial respiration, butyric acid was added to the incubation medium. The addition of amino acids significantly increased OSQo, in both N P and HP cells. The effect was much more pronounced in the H P cells. There are at least two possible explanations for this enhanced effect in HP cells. The amino acids may increase mitochondrial respiration by increasing substrate availability. It is unlikely that this is the main cause of the increase in Qo,. The amino acids increased OSQo,, but not ouabain-insensitive Qo,. Moreover, in the uncoupler studies the mitochondrial respiration was found to be the same with and without amino acids in both NP and H P cells. Amino acids, with the exception of glutamine, are not oxidized to any extent by the proximal tubules (Mandel 1985). The concentration of glutamine in the amino acid mixture was less than 0.2 mM, which, in the presence of butyric acid, should have little influence on
Na-amino acid symports in high protein intake
APERIA, A. 1990.High protein intake accelerates the maturation of Na, K-ATPase in rat renal tubules. Actu Physiol Scund 139, 1-7. JAKOBSSON, B., CELSI,G., LINDBALD, B.S. & APERIA, A. 1987. Influence of different protein intake on renal growth in young rats. Actu Puediulr Scund 76, 293-299. JAKOBSSON, B., BOHMAN,S.O., SUNDELIN, B. & APERIA,A. 1988. Mitotic response to high protein intake in different renal cell types in weanling rats. Kidney Int 33, 662-666. KAISSLING, B. & CRIZ,W. 1979. Structural analysis of the rabbit kidney. A d v Anut Embryo1 Cell Biol 56, 1-123. KOKKO,J.P. 1973. Proximal tubule potential difference. Dependence on glucose, IICO,, and amino acids. 3 Clin Invest 52, 1362-1367. LARSSON, L., APERIA,A. & LECHENE, C. 1986. Ionic transport in individual renal epithelial cells from adult and young rats. Actu Ph,ysiol Scund 126, 321-332. LEFFERT,H.L. & KOCH,K.S. 1979. Regulation of growth of hepatocytes by sodium ions. In: , H . Popper & F. Schaffner (eds.) Progress in Licer Diseuses, pp. 123-134. Grune & Stratton, New This work was supported by grants from the Swedish York. Medical Research Council (projects nos. 0364402049). Expert technical assistance was provided by MANDEL,L.J. 1985. Metabolic substrates, cellular energy production, and the regulation of proximal Ms Ingrid Muldin and Ms Ann-Christine Eklof. tubular transport. Ann Rev Physinl47, 85-101. MORGAN, B.L.G. & NAISMITH, D.J. 1980. Value of REFERENCES dietary protein for hyperplastic growth at restricted BIO-RADLABORATORIES 1977. Bio-Rad protein assay. energy intakes. 3 Nutr 110, 618-626. Technical Bulletin 10sI . PALLER,M.S. & HOSTETTER, T.H. 1986. Dietary HARRIS, S.I., BALABAN, R.S., BARRET, L. & MANDEL, protein increases plasma renin and reduces pressor L.J. 1981. Mitochondria1 respiration capacity and reactivity to angiotensin 11. A m 3 Ph,ysiol 251, Na+- and K+-dependent adenosine triphosphataseF34-F39. mediated ion transport in the intact renal cell. 3 SCHAFER, J.A. & WILLIAMS JR, J.C. 1985. Transport Biol Chem 256, 10319-10328. of metabolic substrates by the proximal nephron. HARRIS, S.I., PATTON, L., BARRET, L. & MANDEL, L.J. Ann Rev Physiol47, 103-125. 1982. (Na+, K+)-ATPase kinetics within the intact ULLRICH,K.J. 1979. Sugar, amino acid, and Na+ cell. The role of oxidative metabolism. 3 Bid Chem cotransport in the proximal tubule. Ann Rev Physiol 41, 181-195. 257, 6996-7002.
mitochondrial respiration. Therefore, a more likely explanation of the amino acid-induced increase in OSQo, is that the Na-K pump has been activated by increased Na entry via the Na-amino acid symports. As indicated in Table 2, uncoupled mitochondrial respiration was higher in HP cells than in N P cells. Since this respiration is independent of pump activity, HP intake also seems to have some direct effect on mitochondrial activity. In conclusion, Na, K-ATPase-dependent respiration is higher in PCT cells from HP rats than from NP rats both in cells with normal intracellular Na levels and in Na-loaded cells. Stimulation of the Na-amino acid symport causes disproportionate increase in the Na, KATPase-dependent respiration in HP cells. T h i s observation suggests that, in proximal tubule cells from weanling rats, HP intake increases cellular Na uptake, mainly via the Na-amino acid symports.
JAKOBSSON, B. &
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