Life Sciences Vol . 17, pp . 1799-1806 Printed in the U .S .A .
Pergamon Press
EFFECTS OF L-GL000SE ON SODIUM REABSORPTION IN THE ISOLATED PERFUSED RAT KIDNEY Mary Ellen Trimble Veterans Administration Hospital and Department of Physiology Upstate Medical Center, Syracuse, New York 13210 (Received in final form November 7, 1975) S omis ry In the isolated perfused rat kidney, sodium reabsorption is enhanced in the presence of 5 .5 mM D-glucose . However, it is unclear whether this effect is metabolic or whether it is due to a requirement for sodium transport in the process of glucose reabsorption . A third possibility is solvent drag . In an attempt to differentiate between these possibilities, kidneys were perfused with the D-glucose isomer, L-glucose (L-G), a nonmetabolizable hexose . At a perfusate concentration of 5 " 5 mM L-G, par cent L-G reabsorption was approximately 30 . Inhibition of L-G reabsorption by D-glucose suggests carriermediated transport . In the presence of 5 .5 mM L-G, sodium reabsorption approximated 92$ during the course of perfusion . When L-G was omitted from perfusate, sodium reabsorption ulti mately declined to 85$ . Since significant metabolism of L-G was not observed, .the results are compatible with the hypothesis that enhanced sodium reabsorption may be brought about by some still to be defined aspect of glucose transport . In a previous study, we reported that in the isolated perfused rat kidney, sodium reabsorption was enhanced in the presence of 5 .5 mM D-glucose (1) . Similar findings have been reported by others (2) . The mechanism for this enhanced sodium reabsorption is unclear . It could be due to an effect of glucose on renal metabolism or alternatively it could be associated with transport of the intact glucose molecule . In an attempt to distinguish between these possibilities, sodium reabsorption was studied to the presence of the nomietabolizable D-glucose isomer, L-glucose . Enhanced sodium reabsorption in the presence of a reabsorbed but nonmetabolizable hexose would have to be explained in terms of the transport process per se . Observations on the nature of L-glucose transport have been conflicting In the present experiments in the isolated perfused rat kidney, it (3-6) . was possible to study L-glucose transport in the absence of other substrates . When the initial perfusate L-glucose concentration was 5 .5 mM, fractional L-glucose reabsorption was approximately 30$ . Under these conditions, reabsorption of L-glucose was associated with significantly greater sodium reabsorption than observed in the absence of substrate .
1799
1800
L-Glucose and Renal Na Transport
Vol . 17, No . 12
Methods Perfusion rocedure : Kidneys were isolated from male Charles River rats weighing 350-F50~ere perfused according to a previously described technique (7) . All kidneys were perfused with a recirculatlon volume of 40 ml Krebs bicarbonate buffer containing 7 .5$ bovine serum albumin . To this was added 1 uCi (3H]polyethylene glycol (PEG), MW 4000, SA 12 .7 mCi/mmole (New England Nuclear), which was used to measure glomerular filtration rate (GFR) (8) . Following a 10 min equilibration period, five 10-min urine collections were obtained with perfusate samples taken at the midpoint of each collection . Chemicals : Other substances added to the perfusate in some experiments included 1 1 .5 mM, 5 .5 mM or 11 .0 mM L-glucose (Sigma) ; 2) 1 .1 mM or 5 " 5 ~ D-glucose (Matheson Coleman â Bell) ; 3) 2 .2 mM sodium stets mlamide (Dtamox) (Lederle) ; and 4) L-[1-14C]glucose (New England Nuclear, SA 50 mCi/mmole) . The purity of chemical L-glucose was assessed with paper chromatography in a butanol : alchohol : water (50 :32 :18) system . L-glucose migrated as a single homogeneous spot with an Rf, 0 .25, identical to D-glucose . in standard L-glucose solutions, no D-glucose could be detected when assayed by the glucose oxidase method (9) . The purity of the L-[1- 14 C]glucose was 99$ as determined by paper chromatograpby using the above solvent system . Standard solutions containing L-[1-l~+C]glucose were also reacted with glucose oxidase which is specific for D-glucose . Thin layer chromatography on SilicAR (Malilnkrodt) coated plates showed that at least 96$ of the radioactivity migrated with L-glucose . 14C0 roductton from L-[1 : 14C] lucose : To determine whether L-g lucose was metabolized to a signi Icant degree, CO2 production from L-[1-14C]glucose was determined as described previously (1) . Oxygenation In this closed, preoxygenated system was adequate since continuously monitored perfusate P0 2 levels were between 350-450 mm Hg throughout the course of the perfusion . One to 5 uCi L - [1 -1 C]glucose was added l ~o 40 ml perfusate in the presence and absence of unlabelled L-glucose . CO2 production was also determined In kidney cortical slices as described by others (10) . Approximately 200 mg of tissue was Incubated for 90 to at 32 C in 5 ml of Krebs phosphate buffer containing 2 .5-5 .0 uCi L-[1-I~C]glucose and 5 .5 mM unlabeled L-glucose . Total reducing Analyses : Sodium was determined by flame photometry . sugar was measured with the ferrtcyanide method using the Technicon AutoAnalyzer . With this method, standard L-glucose solutions give readings identical to standard D-glucose solutions with an error of 3$ . The isolated kidney releases small amounts of endogenous D-glucose Into the perfusate so D-glucose was routinely measured using the glucose oxidase method . When L-glucose was the only exogenous sugar added, L-glucose concentrations were determined from the difference between total reducing sugar and D-glucose concentrations . In some experiments, both L- and D-glucose were added to In addiperfusate . In these cases, luCi L-[1-14C]glucose was also added . tion to chemical measurements, per cent reabsorption of L-glucose was calculated from the clearance ratio, clearance of L-[1-14C]glucose divided by clearance of [3H]PEG . The Student t-test was used in statistical analyses and P values of 0 .05 or less were considered significant . Recul is Characteristics of L-glucose transport : Net reabsorption of L-glucose was consistently observed when the filtered load of L-glucose was greater
Vol . 17, No . 12
L-Glucose and Reaal Na Transport
1801
L-GLUCOSE
3A 2.5 c 2.0 ° LS â
T L-G
(~moln/minl
10 0.5
L0
2.0
3.0
4.0
S.0
6.0
FI G. 1 Effect of Variations In Filtered L-Glucose Load on L-Glucose Transport. Initial perfusate L-glucose concentrations ranged from 1 .0-11 .0 mM . Each point represents one 10 min clearance period . than 0 .6 umoles/min (Fig . 1) . Fractional L-glucose reabsorption was approximately 30~ when the initial perfusate L-glucose concentration was 5 " 5 mM (Fig . 2) . Raising the L-glucose concentration to 11 .0 mM resulted In somewhat decreased fractional L-glucose reabsorption . Absolute amounts of sugar reabsorbed were similar with both 5 "5 and 11 mM L-glucose (F1 g. 3) . For purposes of compar(son, experiments were also done with a concentration of D-glucose (l .l mM) which yielded the same absolute amount of sugar reabsorption as with $ .5 or 11 .0 mM L-glucose (Fig . 3) " L-GLUCOSE
Per Cant L-Glucose (L-G) Reabsorption During the Course of Perfusión . n ~ number of kidneys . 5 .5 mM L-G, n ~ 8 ; 11 .0 mM L-G, n ~ 5 . Mean ± SE .
L-Glucose and Renal Na Transport
1802
Vol. 17, No . 12
LI " mM D-GlucoseS .S mM L-Glucose ILOmM " L-Glucose
L0 To 0.8 (I+moles/min) 0.6 0.4 0.2 0~ 0
FI G . 3 Comparison of Absolute Amounts of D- and L-Glucose Transported (TG) During the Course of Perfusion. n as in Ftg . 2 and D-G, n ~ 6. Mean ± SE . To determine whether L-glucose reabsorption could be accounted for by simple diffusion alone, L-glucose transport was examined when D-glucose was added to the perfusate (Table 1) . Net L-glucose reabsorption was s(gnifl cantly decreased in the presence of D-glucose . Acetazolamide (2 .2 mM), added to perfusate to decrease the amount of fluid reabsorbed in the proxtTABLE 1 Inhibition of Net L-Glucose Reabsorption by Various Substances In addition to 5 .5 mM L-glucose :
N
$ L-glucose reabsorption
[3H]PEG Urine/Plasma
Method of L-glucose ânalysts
Control
8
31 .1 ± 2.0
14 .6 ± 1 .6
chemical
D-Glucose (5 " 5 mM)
6
13 .5 ± 0 .5
l4 C
Acetazolamide (2 .2 mM)
4
9.5 ± 2 .1* -3 .0 ± 5 .3*
Values are mean ±SE .
16 .8 ± 2.2*
chemical 3 .5 ± 0 .1*
N = number of animals.
*P
Pergamon Press
EFFECTS OF L-GL000SE ON SODIUM REABSORPTION IN THE ISOLATED PERFUSED RAT KIDNEY Mary Ellen Trimble Veterans Administration Hospital and Department of Physiology Upstate Medical Center, Syracuse, New York 13210 (Received in final form November 7, 1975) S omis ry In the isolated perfused rat kidney, sodium reabsorption is enhanced in the presence of 5 .5 mM D-glucose . However, it is unclear whether this effect is metabolic or whether it is due to a requirement for sodium transport in the process of glucose reabsorption . A third possibility is solvent drag . In an attempt to differentiate between these possibilities, kidneys were perfused with the D-glucose isomer, L-glucose (L-G), a nonmetabolizable hexose . At a perfusate concentration of 5 " 5 mM L-G, par cent L-G reabsorption was approximately 30 . Inhibition of L-G reabsorption by D-glucose suggests carriermediated transport . In the presence of 5 .5 mM L-G, sodium reabsorption approximated 92$ during the course of perfusion . When L-G was omitted from perfusate, sodium reabsorption ulti mately declined to 85$ . Since significant metabolism of L-G was not observed, .the results are compatible with the hypothesis that enhanced sodium reabsorption may be brought about by some still to be defined aspect of glucose transport . In a previous study, we reported that in the isolated perfused rat kidney, sodium reabsorption was enhanced in the presence of 5 .5 mM D-glucose (1) . Similar findings have been reported by others (2) . The mechanism for this enhanced sodium reabsorption is unclear . It could be due to an effect of glucose on renal metabolism or alternatively it could be associated with transport of the intact glucose molecule . In an attempt to distinguish between these possibilities, sodium reabsorption was studied to the presence of the nomietabolizable D-glucose isomer, L-glucose . Enhanced sodium reabsorption in the presence of a reabsorbed but nonmetabolizable hexose would have to be explained in terms of the transport process per se . Observations on the nature of L-glucose transport have been conflicting In the present experiments in the isolated perfused rat kidney, it (3-6) . was possible to study L-glucose transport in the absence of other substrates . When the initial perfusate L-glucose concentration was 5 .5 mM, fractional L-glucose reabsorption was approximately 30$ . Under these conditions, reabsorption of L-glucose was associated with significantly greater sodium reabsorption than observed in the absence of substrate .
1799
1800
L-Glucose and Renal Na Transport
Vol . 17, No . 12
Methods Perfusion rocedure : Kidneys were isolated from male Charles River rats weighing 350-F50~ere perfused according to a previously described technique (7) . All kidneys were perfused with a recirculatlon volume of 40 ml Krebs bicarbonate buffer containing 7 .5$ bovine serum albumin . To this was added 1 uCi (3H]polyethylene glycol (PEG), MW 4000, SA 12 .7 mCi/mmole (New England Nuclear), which was used to measure glomerular filtration rate (GFR) (8) . Following a 10 min equilibration period, five 10-min urine collections were obtained with perfusate samples taken at the midpoint of each collection . Chemicals : Other substances added to the perfusate in some experiments included 1 1 .5 mM, 5 .5 mM or 11 .0 mM L-glucose (Sigma) ; 2) 1 .1 mM or 5 " 5 ~ D-glucose (Matheson Coleman â Bell) ; 3) 2 .2 mM sodium stets mlamide (Dtamox) (Lederle) ; and 4) L-[1-14C]glucose (New England Nuclear, SA 50 mCi/mmole) . The purity of chemical L-glucose was assessed with paper chromatography in a butanol : alchohol : water (50 :32 :18) system . L-glucose migrated as a single homogeneous spot with an Rf, 0 .25, identical to D-glucose . in standard L-glucose solutions, no D-glucose could be detected when assayed by the glucose oxidase method (9) . The purity of the L-[1- 14 C]glucose was 99$ as determined by paper chromatograpby using the above solvent system . Standard solutions containing L-[1-l~+C]glucose were also reacted with glucose oxidase which is specific for D-glucose . Thin layer chromatography on SilicAR (Malilnkrodt) coated plates showed that at least 96$ of the radioactivity migrated with L-glucose . 14C0 roductton from L-[1 : 14C] lucose : To determine whether L-g lucose was metabolized to a signi Icant degree, CO2 production from L-[1-14C]glucose was determined as described previously (1) . Oxygenation In this closed, preoxygenated system was adequate since continuously monitored perfusate P0 2 levels were between 350-450 mm Hg throughout the course of the perfusion . One to 5 uCi L - [1 -1 C]glucose was added l ~o 40 ml perfusate in the presence and absence of unlabelled L-glucose . CO2 production was also determined In kidney cortical slices as described by others (10) . Approximately 200 mg of tissue was Incubated for 90 to at 32 C in 5 ml of Krebs phosphate buffer containing 2 .5-5 .0 uCi L-[1-I~C]glucose and 5 .5 mM unlabeled L-glucose . Total reducing Analyses : Sodium was determined by flame photometry . sugar was measured with the ferrtcyanide method using the Technicon AutoAnalyzer . With this method, standard L-glucose solutions give readings identical to standard D-glucose solutions with an error of 3$ . The isolated kidney releases small amounts of endogenous D-glucose Into the perfusate so D-glucose was routinely measured using the glucose oxidase method . When L-glucose was the only exogenous sugar added, L-glucose concentrations were determined from the difference between total reducing sugar and D-glucose concentrations . In some experiments, both L- and D-glucose were added to In addiperfusate . In these cases, luCi L-[1-14C]glucose was also added . tion to chemical measurements, per cent reabsorption of L-glucose was calculated from the clearance ratio, clearance of L-[1-14C]glucose divided by clearance of [3H]PEG . The Student t-test was used in statistical analyses and P values of 0 .05 or less were considered significant . Recul is Characteristics of L-glucose transport : Net reabsorption of L-glucose was consistently observed when the filtered load of L-glucose was greater
Vol . 17, No . 12
L-Glucose and Reaal Na Transport
1801
L-GLUCOSE
3A 2.5 c 2.0 ° LS â
T L-G
(~moln/minl
10 0.5
L0
2.0
3.0
4.0
S.0
6.0
FI G. 1 Effect of Variations In Filtered L-Glucose Load on L-Glucose Transport. Initial perfusate L-glucose concentrations ranged from 1 .0-11 .0 mM . Each point represents one 10 min clearance period . than 0 .6 umoles/min (Fig . 1) . Fractional L-glucose reabsorption was approximately 30~ when the initial perfusate L-glucose concentration was 5 " 5 mM (Fig . 2) . Raising the L-glucose concentration to 11 .0 mM resulted In somewhat decreased fractional L-glucose reabsorption . Absolute amounts of sugar reabsorbed were similar with both 5 "5 and 11 mM L-glucose (F1 g. 3) . For purposes of compar(son, experiments were also done with a concentration of D-glucose (l .l mM) which yielded the same absolute amount of sugar reabsorption as with $ .5 or 11 .0 mM L-glucose (Fig . 3) " L-GLUCOSE
Per Cant L-Glucose (L-G) Reabsorption During the Course of Perfusión . n ~ number of kidneys . 5 .5 mM L-G, n ~ 8 ; 11 .0 mM L-G, n ~ 5 . Mean ± SE .
L-Glucose and Renal Na Transport
1802
Vol. 17, No . 12
LI " mM D-GlucoseS .S mM L-Glucose ILOmM " L-Glucose
L0 To 0.8 (I+moles/min) 0.6 0.4 0.2 0~ 0
FI G . 3 Comparison of Absolute Amounts of D- and L-Glucose Transported (TG) During the Course of Perfusion. n as in Ftg . 2 and D-G, n ~ 6. Mean ± SE . To determine whether L-glucose reabsorption could be accounted for by simple diffusion alone, L-glucose transport was examined when D-glucose was added to the perfusate (Table 1) . Net L-glucose reabsorption was s(gnifl cantly decreased in the presence of D-glucose . Acetazolamide (2 .2 mM), added to perfusate to decrease the amount of fluid reabsorbed in the proxtTABLE 1 Inhibition of Net L-Glucose Reabsorption by Various Substances In addition to 5 .5 mM L-glucose :
N
$ L-glucose reabsorption
[3H]PEG Urine/Plasma
Method of L-glucose ânalysts
Control
8
31 .1 ± 2.0
14 .6 ± 1 .6
chemical
D-Glucose (5 " 5 mM)
6
13 .5 ± 0 .5
l4 C
Acetazolamide (2 .2 mM)
4
9.5 ± 2 .1* -3 .0 ± 5 .3*
Values are mean ±SE .
16 .8 ± 2.2*
chemical 3 .5 ± 0 .1*
N = number of animals.
*P
