AMERICAN JOURNAL OF PHYSIOLOGY Vol. 231, No. 6, December 1976. Printed

Glucose proximal

in U.S.A.

transport tubules

in isolated perfused of snake kidney

DELON W. BARFUSS AND WILLIAM H. DANTZLER Department of Physiology, College of Medicine, Uniuersity BARFUSSJIELON W., AND WILLIAM H. DANTZLER,GZUCOS~ transport in isolated perfused proximal tubules of snake kidney. Am. J. Physiol. 231(6): 17164728. 1976. - Glucose tram+ port was studied in isolated, perfused snake (Thamnophis spp.) renal tubules. When 14C-labeled and unlabeled glucose concentrations for bath and perfusate were identical, net transepithelial glucose transport occurred from lumen to bath. Maximum rates of transport were 1.24 x lOAl2 and 2.17 x XV2 mol min-I mm+ in proximal-proximal and distal-proximal segments, respectively. Glucose concentration in cells of perfused tubules of both segments was less than that of bath or lumen during maximum glucose absorption. This cellular glucose concentration increased to that of bath and lumen when tubules spontaneously stopped transporting glucose. Transepithelial glucose permeability (bath + lumen) was about 0.25 x lo+ cm set-’ for both segments. Peritubular membrane permeability (bath -+ cell) was about 0.50 x lop5 cm set-* for both segments. Luminal membrane permeabilities (cell -+ lumen) were 0.29 x lo-” and 0.65 x 10s5 cm set-l for proximalproximal and distal-proximal segments, respectively. Luminal membrane permeability in opposite direction (lumen + cell) was about 10.0 x lop5 cm set-l for both segments. These results indicate that, during maximum glucose absorption, glucose enters cells down concentration gradient across luminal membrane by a mediated process and is transported out of the cells against concentration gradient at peritubular membrane.

isolated

renal

tubules;

comparative

renal physiology

GLUCOSE ABSORPTION from the luminal fluid of proximal tubules in frog, Necturus, rat, guinea pig, oppossum, rabbit, and dog kidneys has been well documented (13, 18, 23, 28-31). This glucose absorption process is generally considered to involve active transport and to show a maximum transport rate (Tm) as first described by Shannon and his colleagues (19,20) from clearance studies in dogs. Such a tubular maximum (Tm) for glucose absorption has been demonstrated more directly by perfusion of rabbit tubules in vitro (23) and by micropuncture of rat tubules in vivo (2, 27). However, the concept of an absolute Tm for glucose absorption in rats has also been brought into question by some results from in vivo microperfusion studies (12). The cellular mechanism involved in the glucose transport process is not yet well defined. However, the main active step is generally considered to be located at the luminal side of the cells. Major evidence for this site has come from studies with isolated, perfused rabbit proximal tubules (23) and from studies with luminal

of Arizona,

Tucson,

Arizona

85724

membrane fragments (3) and luminal membrane vesicles (15) from rat proximal tubules. Although the active step for glucose transport is considered to be located on the luminal membrane, it has also been suggested that the peritubular membrane may be more than passively involved (16, 22, 23). Sodium has also been shown to be requ ired for maximum glucose absorption by the proximal renal tubules of frogs (26), Necturus (14), and rats (25), This observation has suggested that glucose absorption by the renal tubules may be coupled to the diffusion of sodium into the cells as in the case of glucose absorption in the intestine (9). This is supported by work with isolated vesicles of luminal membranes from rat proximal tubules (15). Since the steps in the renal tubular transport of glucose were not clearly understood for any species, we decided to study glucose absorption in isolated, perfused snake proximal renal tubules where the environment on both sides of the epithelium could be controlled. The saturation kinetics were evaluated by varying the perfusion rate and the glucose concentration in the perfusate. We measured the luminal, cellular, and peritubular concentrations of glucose simultaneously in order to determine the possible cellular site of the active transport step. We also estimated the permeabilities of the whole epithelium and of the luminal and peritubular membranes from the glucose fluxes and the concentration differences. The results indicate that: 1) a maximum rate for glucose absorption exists in both the proximal and distal portions of the proximal tubule but that it is nearly twice as great in the distal portion as in the proximal portion; 2) when the glucose absorptive mechanism is saturated, the major active step appears to be located at the peritubular membrane and glucose appears to enter the cells from the lumen down a concentration gradient by a mediated process; and 3) sodium appears to be involved in glucose transport at the luminal membrane and possibly at the peritubular membrane. METHODS

Animals and dissection of tubules. Garter snakes (Thamnophis spp.) were obtained from commercial suppliers in Wisconsin and maintained as described previously (10, 11). The snakes (25-75 g) were killed by decapitation. Their kidneys were removed quickly and placed in cold snake-Ringer solution (see below for com-

1716

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GLUCOSE

TRANSPORT

BY

SNAKE

RENAL

TUBULES

position of Ringer solution). Proximal tubules were dissected free without the aid of enzymatic agents as described previously (IO, 11). The proximal tubule has been divided into two distinct anatomical regions based on differences in ability to transport p-aminohippurate (PAH) (11) The region of the proximal tubule from the neck to the first 180” bend has been designated the proximal-proximal tubule. No significant PAH transport from bath to lumen occurs in this segment. The remainder of the proximal tubule from the first 180” bend to the thin segment has been termed the distal-proximal tubule. Significant PAH transport occurs from bath to lumen in this segment. Both segments of the snake proximal renal tubule were used in the present study. Ringer composition. The composition of the basic Ringer medium used for dissecting, perfusing, and bathing the tissue was the same as that used previously (lo), It contained, in millimoles per liter: NaCl, 126; KCl, 3.0; NaHCO,, 24.0; NaH,PO,, 0.71; MgSO+ 1.2; CaCl,, 1.8. In addition, dextran (40,000 mol wt) was added to the outside bathing medium in a concentration of 4 g/l00 ml to approximate the plasma protein concentration of these snakes (10). In some experiments, a sodium-free Ringer was used for the perfusion fluid. This solution contained, in millimoles per liter: TRIS-HCl, 24.0; choline chloride, 128.0; KCl, 1.0; K2HP04, 1.0; MgS04, 1.2; CaCl,, 1.8. It had the same osmolality, measured by freezing-point depression, as the control solution. The bathing medium was bubbled with a 95% O&5% CO, gas mixture before and during all experiments to provide adequate mixing and to maintain the bicarbonate buffered Ringer at pH 7.4. The TRIS-HCl buffer maintained the pH at 7.4 in the sodium-free solution. Appropriate amounts of glucose were added to these various solutions just prior to the experiments. Uniformly labeled [14C]glucose (New England Nuclear, 150250 mCi/mmol) was used as a tracer for glucose an alysis. In some experiments with nonperfused tubules, aPpropriate amounts of 3-0-methylglucose labeled with 14C (New England Nuclear, 20-25 mCi/mmol) were added to the bathing medium. [Metho&H]inulin (New England Nuclear, 50-150 mCi/g) was added to the perfusion fluid as a marker for fluid absorption in the perfused tubules. It was also used as an extracellular volume marker in studies with nonperfused tubules, Prior to use, the [“H]inulin was dialyzed for 24 h at room temperature to remove any low-molecular-weight fragments. This dialysis tubing (Arthur H. Thomas Co.) had a molecular weight cut-off point of 3,500. Perfusion of tub&s. After a tubule was dissected, it was transferred to a special perfusion chamber (25-~1 volume) on the stage of a combined stereomicroscope and inverted compound microscope. The tubule was then perfused in a manner similar to that originally described by Burg et al., (6) and modified for use with snake renal tubules (10). Briefly, the two ends of the tubule were held between glass micropipets, and the tubule was perfused through an inner micropipet. The perfusion flui d tha .t accumulated in the co1letting pipet was collected and the volume determin .ed as described previously (10). The tubule was sealed into the collect-

1717 ing pipet with Sylgard 184 (Dow Corning Corp.). All perfusion experiments were performed at 25 t 2OC. Uptake of glucose and 3-O-methylglucose by nonperfused tubules. Segments of proximal-proximal and distal-proximal tubules were teased from fresh tissue as described above. For most studies, five to six such segments were incubated together for 60 min in a sealed bath of Ringer which was continuous1.y gassed with 95% O,-5% CO,. The temperature was maintained at 25 t 2OC. Glucose or 3-0-methylglucose was present in the bathing medium in a concentration of 0.14 mM. In some experiments on the uptake of glucose, phlorizin (0.1 mM) was present in the bath or the bath was sodium free. Also, in a few experiments on glucose uptake, the incubation period was extended for 24 h. The concentration of glucose or 3-0-methylglucose in the cell water at the end of the incubation period was determined as described below. The concentration in the bathing medium was also determined at the end of the incubation period. Cellular glucose concentration. The glucose concentration in the tubule cell water was determined for many of the perfused tubules and all the nonperfused tubules. For this purpose, each tubule was placed in 10 ~1 of 3% trichloroacetic acid (TCA) to extract 14C-labeled glucose and “H-labeled inulin. At the end of a perfusion experiment, the tubule was pulled free of the perfusion system with fine forceps and transferred into the TCA which was maintained under mineral oil. Under these circumstances, the transfer to the TCA took place in less than 2 s from the time the perfusion ended. Each nonperfused tubule was similarly transferred to TCA under oil. After 1 h of extraction, each tubule was removed and the TCA containing the extracted glucose and inulin was transferred to a scintillation vial for radioactive counting. Preliminary studies showed that this extraction period was long enough to remove all the labeled glucose and inulin from the tubules. Following the TCA extraction, each tubule was placed in chloroform for 10 min to extract the oil. The tubule was dried and weughed on a quartz-fiber ultramicrobalance (4). The final dry weight was multiplied by 1.249 to correct for weight lost during the TCA and chloroform extractions (10). This corrected dry weight was then multiplied by 3.52, as determined previously (lo), to obtain a value for the cell water. The total amount of 14C-labeled glucose extracted from the tubules came from the lumen and any contaminating bathing medium, as well as from the tubule cells. Therefore, it was necessary to subtract the glucose in the lumen and contaminating bathing medium from the total amount of glucose extracted from the tubule in order to obtain the glucose concentration in the cell water only. In the case of perfused tubules, the amount of glucose in the lumen was determined by measuring the amount of “H-labeled inulin extracted from the tubule. Since there was no inulin in the bathing medium, all of this had to come from the tubule lumen. The ratio of “H-labeled inulin to 14C-labeled glucose in the perfusate was also measured. The total amount of 3H-labeled inulin extracted from the tubule was divided by this ratio to determine how much 14C-labeled glucose was in

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1718 the lumen at the time of extraction. This amount was subtracted from the total amount of glucose extracted from the tubule. The amount of 14C-labeled glucose in the lumen of each perfused tubule was usually about 310% of the total 14C-labeled glucose extracted from the tubule. Since no lumen was visible in the nonperfused tubules at the end of incubation, it was assumed that the lumen was completely collapsed and no correction was made for glucose in the lumen of these tubules. The amount of extracellular bathing medium contaminating the extracted tubules was determined in the nonperfused tubules by adding “H-labeled inulin to the bathing medium. In these nonperfused tubules the average extracellular bathing medium transferred was 31.82 t 2.68% (mean t SE for 25 tubules) of the cell water. The amount of 14C-labeled glucose transferred with this extracellular bathing medium was determined by multiplying the concentration in the bathing medium by the volume transferred with each tubule. This amount was then subtracted from the total 14C-labeled glucose extracted. [Methoxy-“H]inulin was not present in the bathing medium during perfusion experiments. However, the tubules were removed from the bathing medium in a manner identical to that for the nonperfused tubules. Therefore, the same amount of extracellular bathing medium contamination was assumed to occur in these tubules and the total 14C-labeled glucose extracted was corrected for this degree of contamination as described above. The concentration of 14C-labeled 3-O-methylglucase in the cells of nonperfused tubules was determined as described for glucose. Chromatography. Thin-layer chromatography was used to determine if the intracellular 14C was still incorporated in glucose or in some metabolite of glucose. The chromatographic technique was the same as that used by Tune and Burg (25). The solvent system, n-butanolacetone-water (4050: IO), discriminates glucose from glucose 6-phosphate, fructose 6-phosphate, fructose 1,6diphosphate, lactate, and glycogen (23). Using this sytern, we found that 92% (91.66 -+ 1.42; mean & SE) of the total 14C extracted from four perfused tubules was in the form of glucose. For all other perfused tubules, the glucose concentration in the cell water was calculated on the assumption that 92% of the extracted 14C represented glucose. Extracts from four nonperfused tubules incubated for 60 min were found to have 88% (87.87 t 2.64; mean t SE) of the total 14C extracted still in the form of glucose. For all other nonperfused tubules incubated for 60 min, the glucose concentration in the cell water was calculated on the assumption that 88% of the extracted 14C represented glucose. Extracts from four nonperfused tubules incubated for 24 h had only 45% (45.08 t 9.17; mean -t- SE) of the total 14C extracted remaining in the form of glucose. For other nonperfused tubules incubated for 24 h, the glucose concentration in the cell water was calculated on the assumption that 45% of the extracted 14C represented glucose. Since 3-Omethylglucose is not normally metabolized by cells, extracts from tubules incubated with this sugar were not chromatographed. AnaZyticaZ methods. The activities of 14C and 3H were

D. W.

BARFUSS

AND

W. He DANTZLER

determined simultaneously in a liquid scintillation spectrometer (Nuclear Chicago Mark I, Unilux II, or Isocap/300). The scintillation solution used for all samples consisted of 2,5 ml of Aquasol (New England Nuclear) and 0.75 of ml of water in each vial. The net rate of glucose absorption from the lumen of a perfused tubule, expressed in moles per minute per millimeter tubule length (moles min-l mm+), was determined from the following relationship: net glucose

absorption

rate =

v,c - VOG ’ i TxL

(1)

In this equation Ci and C, are the concentrations (mol/ nl) of glucose in the initial perfusate and collected fluid, respectively. V~ is the volume (nl) of fluid accumulated in the collec tion pipet during perfusion period, It was mea sured directly as described previously (10). Vi is the volume (nl) of initial perfusate that entered the tubule from the perfusion pipet during the perfusion period. It was determined as described below. L is the length (mm) of the tubule segment perfused, exclusive of the portion held in the perfusion and collecting pipets. T is the time (min) of the perfusion period. Vi was determined from the fol lowing relationship: V i = V-0

I Ii 0

In this equation, Ii and I,, are the concentrations (cpml nl) of “H-labeled inulin in the nitial perfusate and collected fluid, respectively. Vi and V, are as defined above. Values were generally expressed as means t SE. Levels of statistical significance were determined with the Student t test. RESULTS

Maximum rate of glucose absorption. Net glucose absorption from lumen to bath occurred in both the proximal and distal portions of the proximal tubules when the concentrations of labeled and unlabeled glucose in both the perfusate and bathing medium were identical (Figs. 1 and 2). Since the concept of a maximum rate for glucose absorption had been challenged by the results of microperfusion studies in rats (12), it was of considerable interest to determine if there was a maximum rate for glucose absorption in snake proximal tubules. This was studied by varying the load of glucose delivered to the absorptive sites in the tubule lumen while glucose absorption was being measured. The glucose delivery rate was varied by changing the glucose concentration in the initial perfusate and by changing the perfusion rate. However, the concentrations of labeled and unlabeled glucose in the initial perfusate were always maintained identical to those in the bathing medium. The results for 16 proximal-proximal tubules are shown in Fig. 1. In these experiments, the perfusion rate was varied from about 2.5 to about 8,O nl minP and the glucose concentration was 0.14 mM at the lower glucose delivery rates or 1.14 mM at the higher glucose delivery rates. Net glucose absorption increased

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GLUCOSE

TRANSPORT

BY

SNAKE

RENAL

1719

TUBULES

2.17 t 0.16 x IO-l2 mol minl

1.8r

0.6

v

3.0

GI ucose

6.0

Delivery

9.0

12.0

Ra te(moles

I 5.0

I 8.0

min? x 10-12)

FIG. 1. Absorption of glucose by 16 proximal-proximal snake tubules as a function of glucose delivery rate. Perfusion rate ranged from about 2.5 to about 8.0 nl min?. Unlabeled glucose and 14Clabeled glucose concentrations for bath and perfusate were identical. Total glucose concentration was 0.14 or 1.14 mM. Data were grouped within intervals of glucose delivery rates and means were calculated for delivery rates and glucose absorption. Solid points indicate mean values. Horizontal and vertical lines indicate SE. Figures in parentheses indicate number of determinations at each point. Curve was fitted by eye.

with increasing glucose delivery rates until this rate reached about 4.0 x lo-l2 mol min+, at which point the transport mechanism appeared to saturate. The maximum net transport rate for glucose in these proximalproximal segments was about 1.2 X lo-l2 mol min-l mm-l. The convoluted portion of the rabbit proximal tubules has a much greater capacity to absorb glucose than the straight portion (23). In terms of p-aminohippurate transport, the proximal portion of the snake proximal tubule is analogous to the convoluted portion of the rabbit proximal tubule and the distal portion of the snake proximal tubule is analogous to the straight portion of the rabbit proximal tubule (11, 24). Therefore, the transport capacity for glucose in the distal-proximal tubule was of great interest. The results of varying the glucose delivery rate in eight distal-proximal tubules are shown in Fig. 2. In these experiments, the perfusion rate was varied from about 4.0 to about lo,0 nl min-’ and the glucose concentrations in perfusate and bathing medium were 0.15, 1.14, or 5.14 mM, In contrast to the proximal-proximal segment, glucose absorption in the distal-proximal segment increased with increasing glucose delivery rates until this rate reached about 12.0 x lo-l2 mol min+, at which time the transport mechanism appeared to saturate. The maximum net transport rate for glucose in these segments was about 2.3 x lo-l2 mol min-l mm+ or nearly twice that observed in the proximal-proximal segment. If the mean value for net glucose absorption is determined for all delivery rates greater than that necessary for saturation for each tubule segment, the value for proximal-proximal tubules is 1.24 t 0.14 x IOAf2 mol minP mm+ (mean -+ SE for 12 determinations) and that for distal-proximal tubules is

mm-’ (mean ? SE for 18 determinations). These values are significantly different from one another (P < 0.001). As noted above, in these experiments to determine the maximum rate of glucose absorption, the concentrations of 14C-labeled and unlabeled glucose were identical in both the perfusate and the bathing medium. This eliminated any chemical or isotopic gradients that could have produced incorrect values for glucose absorption. The importance of not having a gradient from lumen to bath for 14C-labeled glucose when glucose absorption was being measured is illustrated in Figs. 3 and 4. These figures summarize data on the relationship between the glucose delivery rate and net glucose absorption for proximal-proximal and distal-proximal tubules that were perfused with 14C-labeled glucose in the lumen only. However, the unlabeled glucose concentrations in the perfusate and bathing medium were identical. The isotopic gradient from lumen to bath led to a movement of 14C-labeled glucose into the bath out of proportion to the net movement of unlabeled glucose. Since the calculations of net glucose absorption had to be made on the assumption that the labeled and unlabeled glucose moved proportionately, the failure of this to occur led to the determination of a falsely high rate for glucose absorption. Although the transport system for glucose in the proximal-proximal tubules tended to saturate under these circumstances, this is not very clear (Fig, 3). Moreover, the glucose absorption rates (Fig 3) were more than 10 times the maximum transport rate seen when no isotopic gradient was present (Fig. 1). In segments of distal-proximal tubules, glucose absorption continued to increase even with very high glucose delivery rates (Fig. 4). No maximum rate for glucose absorption was seen in the presence of an isotopic gradient (Fig. 4), in contrast to the situation when l

.

N

T --a -

2.4

+

(4)

I

6

Glucose

I

I

12

Delivery

I1

I

I

I

18

Rate(moles

I 24

I

I

I

Ii

30

min-lx

I

36

10-12)

2. Absorption of glucose by eight distal-proximal snake tubules as a function of glucose delivery rate. Perfusion rates range from about 4.0 to about 10.0 nl min? Unlabeled glucose and 14Clabeled glucose concentration for bath and perfusate were identical. Total glucose concentration was 0.15, 1.14, or 5.14 mM. Data were grouped within intervals of glucose delivery rates and means were calculated for delivery rates and glucose absorption, Solid points indicate mean values. Horizontal and vertical lines indicate SE. Figures in parentheses indicate number of determinations at each point. Curve was fitted by eye, FIG.

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1720

D. W.

1

t

(7)

18-

6-

(II)

-

I

t(241

i

I

(7)

I (20)

+

(17)

U2) WlL

(1

Illllllllllltl~l~ltllllJ

O

l

45

Glucose

90

I35

180

225

Delivery Rate (moles

270

min”

315

360

x IO-‘*)

FIG. 3. Absorption of glucose by proximal-proximal tubules as a function of glucose delivery rate. Perfusion rate ranged from about 1.0 to about 10.0 nl min-‘. W-labeled glucose was present only in luminal fluid. Unlabeled glucose concentrations in bath and perfusate were identical at 1.16, 5.43, or 25.0 mM. Data were grouped within intervals of glucose delivery rates and means were calculated for delivery rates and glucose absorption. Solid points indicate mean values. Horizontal and vertical lines indicate SE. Figures in parentheses indicate number of determinations at each point. Curve was fitted by eye.

the tubules were perfused without an isotopic gradient (Fig. 2). Glucose concentration in the cell water. Whether an active step for glucose absorption is located on the luminal or peritubular membrane might be indicated if the transepithelial profile for the glucose concentrations during the absorptive process were known. With this in mind, we measured the concentrations of glucose in the collected perfusate, cell water, and bathing medium of tubules that were absorbing glucose. In these studies, the glucose delivery rate was always more than sufficient to saturate the glucose absorptive mechanism. This insured that the concentration of glucose in the lumen would not be reduced much below that in the bath as the perfusate moved from one end of the tubule to the other. This was important because, in order for the transepithelial profile to give meaningful information about the transport system, all the epithelial cells along the length of the tubule had to be functioning under nearly identical conditions. The glucose concentration in the lumen and cell water of each perfused tubule was expressed as a percent of the concentration in the bathing medium in the same study (Figs. 5 and 6). The average glucose concentration in the lumen was considered to be the arithmetic mean of the concentrations in the initial perfusate and in the collected fluid. The transepithelial glucose concentration profile for proximal-proximal tubules under control conditions is shown in Fig. 5. The average concentration of glucose in the lumen was significantly less (P < 0.005) than that in the bath, in accord with the observation that net glucose absorption was occurring during these studies. However, since the glucose delivery rate was well above that necessary to saturate the transport system, the concentration in the lumen was still about 85% of that in the bath. 0f greater importance was the finding that the glucose concentration in the cell water at this time was significantly less than that in either the

BARFUSS

AND

W.

H.

DANTZLER

bath (P < 0.02) or the lumen (P < 0.05). It averaged about 65% of the bath concentration. These findings indicate that glucose can enter the cells from the lumen down a concentration gradient but that it must be transported out of the cells against a concentration gradient on the peritubular side. This is compatible with the presence of an active step for glucose absorption at the peritubular membrane. Similar data were obtained for distal-proximal tubules under control conditions (Fig. 6). The average concentration of glucose in the lumen was significantly less (P < 0.05) than that in the bath, again in accord with the fact that glucose was being transported out of the lumen during these studies. As in the proximalproximal tubules, since the glucose delivery rate was greater than that necessary to saturate the transport system, the concentration in the lumen was still about 90% that in the bath. The concentration of glucose in the cell water was significantly less (P < 0.01) than that in the bath or lumen and averaged about 65% of the bath concentration. These findings indicate that glucose can enter the cells in this tubule segment down a concentration gradient, but that it must be transported out of the cells against a concentration gradient on the peritubular side. Again, this is compatible with an active step for glucose transport located at the peritubular membrane. If an active step for glucose transport is located at the peritubular membrane, the glucose in the cells would be expected to equilibrate with that in the lumen and bath if this transport step stopped functioning. This would be reflected in an increase in the cell-to-bath glucose concentration ratio from its normal value of less than unity to a value equal to unity. In order to examine this possibility, perfused distal-proximal and proximal-proxT

(4) -++

a

80

t

[I

l

180

Glucose

111

240

1

III1

300

Delivery Rate (moles

IllI

360

I]

420

480

min-’ x IO-'*)

FIG. 4. Absorption of glucose by distal-proximal tubules as a function of glucose delivery rate. Perfusion rate ranges from about 1.0 to about 10.0 nl min? W-labeled glucose was present only in luminal fluid. Unlabeled glucose concentrations in bath and perfusate were identical at 1.16, 5.43, or 25.0 mM. Data were grouped within intervals of glucose delivery rates and means were calculated for delivery rates and glucose absorption. Solid points indicate mean values. Horizontal and vertical lines indicate SE. Figures in parentheses indicate number of determinations at each point. Curve was fitted by eye.

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GLUCOSE

TRANSPORT 120-

BY

Proxinwl-

SNAKE

Proximal

RENAL

1721

TUBULES

Tubde

Proximal-

120 I

L

Proximd

0 mM No+

Tubule in Lumen

120 -

II

Proximal-Proximal

Tubule

0.1 mM Phloririn

in Lumen

loo-(51

8060-

6)

4020-

0

Both

Cdl

Lumen

Both

Ruth

PL = 0.29 x l@ cm se&

l

Pp= 0.50 x lo-s - cm se

- _ F Glucosi4~

-5

PL= 4.22 X IO-? cm set-’ Con t rot Absorption

Absorption

FIG. 5. Relative glucose concentrations in lumen fluid, cell water, and bathing medium under control conditions and in presence of 0 mM sodium and 0.1 mM phlorizin for proximal-proximal tubule. Bars indicate mean values as percent of bath concentration. Bath concentration is considered 100%. Vertical lines indicate SE. Numbers in parentheses indicate number of tubules. Model for each Distal-Proximal

P,=O.O8 Xl0 cm secmt = 19.5%

of Control

= 0.8”k

of Control

condition is shown in lower part of figure. Circles and solid arrows indicate active transport. Size of circles indicates relative effectiveness of transport step. Broken arrows indicate passive fluxes. Apparent permeabilities of luminal (PI,) and peritubular (PJ membranes with arrows indicating directions are also shown.

Distal- Proximal Tubule 0 mM No+ in Lumen

Tubule

Absorption

IV

Distol- Proximal 0. I mM Phloririn

Tubule in Lumen

--L,

6) 6)

Lumen

Ccl I

Bath

Bath

Lumen

Lumen

PL = 0.65 X IO-“, cm set-1

Absorption

Absorption

Both

t 0.46 X lo-5 cm see-

*

pL = 0.86 x locm stc”

PL = 5.98 X IO cm SIC-’

Control

Gel I

= 23.8%

of Control

Absorption

= I .2% of Control

6. Relative glucose concentrations in lumen fluid, cell water, and bathing medium under control conditions and in presence of 0 mM sodium and 0.1 mM phlorizin for distal-proximal tubule. Bars indicate mean values as percent of bath concentrations. Bath concentration is considered 100%. Vertical lines indicate SE. Numbers in parentheses indicate number of tubules. Model for each condition

is shown in lower part of figure. Circles and solid arrows indicate active transport. Size of circles indicates relative effectiveness of transport step, Broken arrows indicate passive fluxes. Apparent permeabilities of luminal (P,) and peritubular (PI,) membranes with arrows indicating directions are also shown.

imal tubules that had been absorbing glucose, but had then spontaneously stopped transporting glucose, were analyzed for their glucose content. The mean cell-tobath glucose concentration ratios were 1.02 t 0.03 (mean t SE; IL = 4) for distal-proximal tubules and 1.01 -t 0,03 (mean -+ SE; n = 9) for proximal-proximal tubules that had spontaneously stopped transporting glucose. These values are not significantly different from unity and further support the idea of an active step for glucose transport located at the peritubular membrane. Effects of sodium on glucose transport. Sodium has been implicated in the absorption of glucose by proximal renal tubules (14, 25, 26). According to the “sodiumgradient hypothesis” for the coupled transport of sodium and glucose, sodium movement from the lumen into the cells is required to provide energy for the transport of

glucose (9) Although the movement of glucose from the lumen into the cells in snake renal tubules appeared to be down a concentration gradient, it still appeared important to determine whether the concentration of sodium in the tubule lumen had any effect on glucose absorption. In order to examine this, we studied the effect of removing sodium from the perfusate on glucose absorption in four proximal-proximal and six distalproximal tubules. The tubules were perfused initially with a control Ringer solution with concentrations of labeled and unlabeled glucose equal to those in the bathing medium. Two or three lo-min control collections were made to determine the glucose absorption rate. Then the perfusion fluid was changed to a sodiumfree one containing concentrations of labeled and unlabeled glucose equal to those in the bath. After two or

FIG.

l

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3722 three additional collection peri ods, the tubules were collected and analyzed for their glucose content. During these experiments, the glucose delivery rates were kept below the level required to saturate the glucose transport system. This was necessary in order to see any changes in the glucose concentration in the collected fluid when the tubules were perfused with the sodium-free solution. However, perfusing at these low delivery rates made it impossible to compare the absolute absorption of one tubule to that of another. For this reason, the glucose absorption rates are expressed as percents of control values with each tubule serving as its own control. When proximal-proximal tubules were perfused with the sodium-free solution, the net glucose absorption rate decreased to a minimum of 19.5 t 545% (mean t SE for four tubules) of the control value by the time the tubules were collected. This decrease was highly significant (P < 0.001). The concentration of glucose in the lumen in these tubules was 93.0 -+ 4.0% (mean 2 SE) of that in the bath during the period of reduced glucose absorption that resulted from perfusion with the sodium-free solution (Fig. 5). The simultaneously determined cellular glucose concentration was 77.0 t 6.0% (mean t SE) of the bath concentration. Although this concentration of glucose in the cell water was somewhat greater than that in the cell water of the control tubules (Fig. 5), the two values were not significantly different (P < 0,40). Moreover, the concentration in the cells was still significantly below (P < 0.05) that in the bath (Fig. 5). Similar data were obtained for distal-proximal tubules. When sodium was removed from the perfusate (Fig. 6), the net glucose absorption rate was reduced to 23.8 * X3% (mean t SE for six tubules) of the control value, The glucose concentration in the lumen in these tubules was 91.0 k 4.0% (mean t SE) of that in the bath during the period when glucose absorption was reduced by perfusion with sodium-free fluid (Fig. 6). The glucose concentration in the cells of these tubules, determined at this time, was 78.0 t 10% (mean * SE) of that in the bath. In contrast to the proximal-proximal tubules, this concentration in the cells was not significantly less than the concentration in the bath (0.05 < P < 0.10). However, although this value for the cellular glucose concentration was closer to the bath concentration than the corresponding value in the control tubules, it was still not significantly greater (P < O-40) than the control value. These data for both the proximal-proximal and distal-proximal tubules indicate that the control concentration of sodium in the initial perfusate is necessary for optimum glucose absorption, but that a portion of that absorption can occur when sodium is absent from the initial perfusate. The tendency for the glucose concentration in the cells to be higher during sodium-free perfusion than in the control situation -also suggests that sodium may have some effect on a transport step at the peritubular membrane. To determine if the tubular glucose absorption mechanism could recover after exposure to sodium-free perfusion fluid, four proximal-proximal tubules were perfused with control solution after being perfused with sodium-free solution. The perfusion fluid was then

D.

W. BARFUSS

AND

W.

H.

DANTZLER

changed to a sodium-free one. Net glucose absorption decreased significantly (P < 0.001) in 10 min and was maximally depressed in 20 min (Fig. 7). After 30 min of perfusion with sodium-free medium, the perfusate was changed to control Ringer. Glucose absorption increased, but did not completely reach the control level even after 40 min. Effect of phlorizin on glucose absorption. Phlorizin appears to inhibit competitively renal tubular glucose absorption in those species studied (7). It is believed that this inhibition comes from the interaction of phlorizin with the luminal membrane (21). Although an active step for glucose absorption appeared to be present at the peritubular membrane of snake renal tubules and glucose appeared to enter the cells from the lumen d.own a concentration gradient, the nature of this entry step was not clear. Therefore, it seemed important to determine whether phlorizin in the lum en could block glucase absorption in these tubules. The effect of phlorizin was studied in four proximalproximal tubules and five distal-proximal tubules. As in the studies on the effects of sodium-free perfusion medium, the glucose delivery rates were kept below the level required to saturate the glucose transport system. This was done to make certain that any changes in the glucose concentration of the collected perfusate could be observed easily. Since this made it impossible to compare absolute absorption rates among tubules, the absorption rates were again expressed as percents of control values with each tubule serving as its own control. The tubules were first perfused with a control Ringer solution with concentrations of labeled and unlabeled glucose equal to those in the bath. After two or three lomin control collections, the perfusion fluid was changed 100 2 6 o

I

80

0

,f \\ \ \\ \

A Perfusion

Fluid

(-J---c +--•

to 0 mM Na+

0 mM Na+ 150 mM No”

T

\ \I

r -

T N=4

\\ 1\ \\i 0\ \\ I \\\\

TI ‘0-N’

QJ 20

: 3 c3 -

t 01

A Perfusion 150 mM

1

I

I 30

1

I 50

1

1 70

Fluid Nat

to

I

I 90

Time (mid 7. Effect of restoring sodium to perfusion fluid on net glucose absorption by proximal-proximal tubules. Unlabeled glucose concentrations were identical in perfusate and bathing medium, but 14Clabeled glucose was present in perfusate only. Values are shown as percents of control in each individual tubule. Vertical lines indicate FIG.

SE.

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GLUCOSE

TRANSPORT

BY

SNAKE

RENAL

1723

TUBULES

~0.1 mM Phloririn Added to an identical one except that it contained 0.1 mM phlorizin. After two or three additional collection periods, the tubules were collected and analyzed for their a-- - * Phloririn glucose content. When proximal-proximal tubules were perfused with a solution containing phlorizin, net glucose absorption almost ceased entirely within the first collection period. At the time the tubules were collected for determination of their glucose content, the net absorption rate was only 0 80 -+ 0.8% (mean 2 SE for four tubules) of the control level. Such a decrease was clearly statistically significant (P < 0.001). At this point, the concentration of glucose in the lumen was lOLO t 3.0% (mean & SE) of that in the bath (Fig. 5). This is a reflection of the almost complete inhibition of glucose absorption. The concentration of glucose in the cells was 81.0 -f- 15.0% 1% Phloririn Removed (mean k SE) of that in the bath. This was somewhat higher than the concentration in the cells of the control tubules (Fig. 5), but the difference was not statistically I I I 1 I I 1 1 significant (P -C 0.50). 30 50 70 90 A somewhat similar response to phlorizin was obTime (mid served with distal-proximal tubules. Net glucose abFIG. 8. Effect of removing phlorizin from perfusion fluid on net sorption almost ceased entirely within the first collecglucose absorption by proximal-proximal tubules. Unlabeled glucose tion period after the tubules were perfused with phloriconcentrations were identical in perfusate and bathing medium, but zin, At the time the tubules were collected for determi“C-labeled glucose was present in perfusate only. Values are shown nation of their glucose content, the net glucose absorp- as percents of control in each individual tubule. Vertical lines indition rate was reduced to 1.2 -+ 1.2% (mean -t- SE for five cate SE. tubules) of the control level. Again, this decrease was clearly statistically significant (P < 0.001). The concen- level in about 30 min. These data indicate that the tration of glucose in the lumen was 103 t 1.4% (mean k phlorizin inhibition of glucose transport is reversible. Membrane permeabilities. Glucose is actively abSE) of that in the bath at this time (Fig. 6). This again sorbed from the lumen of the tubule, but it also moves reflects the almost complete inhibition of glucose abpassively through the cell membranes. In order to detersorption. The concentration of glucose in the cell water was 52.0 t 5.0% (mean ,t SE) of that in the bath. This mine the importance of such passive movements, the was somewhat, but not significantly (0.20 < P -C 0.25), apparent permeabilities of the epithelium and of the lower than the glucose concentration in the cells of luminal and peritubular membranes to glucose were determined. To measure these passive permeabilities in control tubules (Fig. 6). These data for both the proxithe inward (bath -+ lumen) direction, the proximalmal-proximal and distal-proximal tubules clearly indiproxi .mal and distal-proximal tubules were perfused cate that phlorizin blocks net glucose absorption when it with ution that contai .ned no glucose. In addiRinger sol is present in the lumen. The tendency for the glucose concentration in the cells of distal-proximal tubules to tion this perfusion solution contained 0 .l mM phlorizin to prevent absorption of glucose that entered the lumen be lower in the presence of phlorizin than in the control situation further suggests that an active step for glucose from the bath. The bathing medium contained labeled and unlabeled glucose in a total concentration of 1.1 transport exists at the peritubular membrane and that mM. Both proximal-proximal and distal-proximaltuthis is not blocked by phlorizin. To determine if the tubular transport system for glu- bules were perfused long enough to obtain two or three cose could recover after inhibition with phlorizin, five collections for flux measuremen is. The tubules were then analyzed for their glucose content to determine the proximal-proximal tubules were perfused with control glucose gradients across the individual cell membranes, solution after being perfused with phlorizin-containing In estimating the various membrane permeabilities, solution (Fig. 8). In these studies, the unlabeled glucose we assumed that no significant amount of glucose concentration was the same in both the perfusate and bathing medium, but labeled glucose was present in the moved across the epithelium through extracellular per&sate only. Therefore, the absolute transport rates channels. If a significant amount of glucose moved bewere higher than the true rates should have been. How- tween the cells, then the individual membrane permeaever, the general pattern should have been indicative of bilities in the luminal direction would be lower than the the true response, These tubules were first perfused for present estimates+ The apparent passive transepithelial two or three control periods. The perfusion fluid was permeability to glucose from bath to lumen (I’$+,, cm then changed to one containing 0.1 mM phlorizin. The s-l) was calculated with the following equation: net glucose absorption was maximally depressed within FR+L PL 10 min. After 30 min of perfusion with phlorizin, the ‘+L = A,(& - CIA) perfusate was changed to the control solution Glucose absorption increased rapidly and approached the control In this equation, F BeL is the unidirectional glucose 1 I

I I

Tg---&---o

1 ’

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1724

D.

transfer rate (mol s-l) from bath to lumen; AL is the surface area (cm2) of the luminal membrane (for a mean luminal diameter of 27.5 pm and a tubule length of 3.0 mm; AL is 86.4 x lO+ cm”); Ce is the glucose concentration tmol cm+) in the bathing medium; and CL is the mean glucose concentration (mol cm-“) in the luminal fluid. This last value was calculated as the arithmetic mean of the concentration in the initial perfusate and the collected fluid. As can be seen in Table 1, the transepithelial permeabilities from bath to lumen for the proximal-proximal tubule (about 0.26 x 10+ cm s-l) and distal-proximal tubule (about 0.23 x lo+ cm s-l) were not significantly different (P < 0.70) from one another. Since the concentration of glucose in the cell water was measured in these flux studies, the apparent permeabilities of the peritubular and luminal membranes to glucose also could be determined. The apparent passive permeability of the peritubular membrane from bath to cell (PE+C, cm s-l) was calculated with the following equation:

W. BARFUSS

AND

W.

H.

DANTZLER

ity from cell to lumen for glucose appeared to be greater for the distal-proximal tubule (about 0.65 x 10B5 cm s-l) than for the proximal-proximal tubule (about 0.29 x lop5 cm s-l), these values were not statistically different from one another (Table 1; Figs. 5 and 6). These values are similar in magnitude to those for the peritubular membrane in the same direction. Since there was a glucose concentration gradient from the tubule lumen into the cells during the studies of glucose absorption with perfused tubules, it was possible to calculate the apparent permeability of the luminal membrane to glucose in the lumen-to-cell direction. This lumen-to-cell permeability for the luminal membrane (P[:+, cm s-l) was calculated with the following equation: P’

=

i-c:

FL+B A& - c,>

(6)

In this equation FL+B is the net absorption rate (mol s-l) for glucose obtained during the absorption studies. The other terms have been defined above. The luminal FB-1, membrane permeability from lumen to cell was estiP’ (4) mated under control conditions, during perfusion with B4C = A&, - cc) sodium-free solution, and during perfusion with soluIn this equation, AI, is the surface area (cm”) of the tion containing 0.1 mM phlorizin. The values are peritubular membrane (for a mean outside diameter of shown in Table 1 and Figs. 5 and 6. For both proximal50 pm and a tubule length of 1.0 mm, AP is 157.08 x proximal and distal-proximal tubule segments under lo-” cm”); and & is the mean concentration of glucose control conditions the apparent permeability of the in the cell water. The other terms have been defined luminal membrane to glucose in the lumen-to-cell dipreviously. The apparent permeabilities of the periturection was more than 10 times that in the cell-tobular membrane to glucose for the proximal-proximal lumen direction. These data suggest that the movetubule (about 0.50 x lo-” cm s-l) and the distal-proximent of glucose from the lumen into the cells is not a mal tubule (about 0.46 x lo+ cm s-l) are not signifiprocess of simple diffusion, but is mediated in some cantly different (P > 0.90) from one another and are fashion. about twice the corresponding transepithelial permeaWhen the tubules were perfused with a sodium-free bility (Table 1; Figs. 5 and 6). solution, the apparent Pt:4c for both segments of the The apparent passive permeability of the luminal proximal tubule decreased to about half the control membrane from cell to lumen (Pk+L, cm s-l), deter- value (Table 1; Figs. 5 and 6). This suggests that somined from these flux studies, was calculated with the dium is required for the normal movement of glucose following equation: across the luminal membrane into the cells and further supports the idea that this movement is mediated in F B-+1, PL = (51 some fashion. AL(C,: - GA) When phlorizin was present in the perfusion soluAll the terms in this equation have been defined above. tion, the apparent Pk+ for both the proximal-proximal Although the apparent luminal membrane permeabiland distal-proximal tubules decreased to a value that c--,1,

TABLE

1. Apparent permeabilities

of luminal

and peritubular

membranes and

of epithelium

to glucose

cm s-l X 10-j -

Proximal-proximal Control 0 mM Na+ 0.1 mM phlorizin

0.23

k

0.03

(4)

0.50 k 0.18 (3)

0.29

k 0.09 (4)

0.50 + 0.18 (3)

0.29

It

0,65

k 0.21

Distal-proximal Control 0 mM Na+ 0.1 mM phlorizin Values peritubular

are means permeability

0.26

st 0.05

(4)

tubules

0.46

0.09

(4)

10.62 4.22 0.08

k k

3.85 3.06 2 0.08

(4) (4) (4)

-+ 2.60

(5) (5) (5)

tubules 5 0.09

(5)

(6)

10.0

5.98 + 3.50 0.46

k Sk Numbers in parentheses (bath * cell). P& luminal

2 0.09

(5)

indicate number of tubules. permeability (cell + lumen).

0.65

* 0.21

PgdL, epithelial Pi;--tC, Iuminal

(6)

permeability permeability

0.86

(bath (lumen

Ik 0.86

-+ lumen). --, cell).

P&

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GLUCOSE

TRANSPORT

BY

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1725

TUBULES

was not, statistically different from zero (P > 0.40) (Table 1; Fi gs. 5 and 6). These data indicate that phlorizin can markedly inhibit the entrance of glucose into the cells across the luminal membrane. This is additional evidence that this entry is mediated in some fashion. Uptake of glucose and 3-0-methylglucose by nonperfused tubules. The possibility of an active step for glucose transport at the peritubular membrane was also examined by incubating nonperfused tubules in a bath containing 14C-labeled and unlabeled glucose in a concentration of 0.14 mM. In these tubules the lumens are collapsed so that glucose does not, enter the lumen. If an active step to move glucose out of the cells existed at the peritubular membrane, then the cells would be expected to maintain a glucose concentration below that in the bathing medium. If no such transport step were functioning, then the glucose concentration in the cell water would be expected to equilibrate with that in the bath. Nonperfused tubules were generally incubated for 60 min. This was long enough for a steady state to be attained. The control cell-to-bath concentration ratios (C/B) were 0.21 k 0.03 (mean t SE; n = 8) for the proximal-proximal tubules and 0.28 * 0.02 (mean -t SE; n = 5) for the distal-proximal tubules. These values are significantly (P < 0.01) less than 1.0. They are also significantly (P < 0.01) lower than the control ratios in perfused tubules (Figs. 5 and 6). These observations further support the concept, of an active transport step for glucose at the peritubular membrane. A C/B ratio lower in the nonperfused than in the perfused tubules may indicate the maximum gradient, that can be established by the pump when no glucose is being delivered to the cells across the luminal membrane. Incubating distal-proximal tubules in sodium-free medium or medium containing 0.1 mM phlorizin did not alter the C/B ratio for glucose from the control value obtained simultaneously (control value: 0.28 k 0.024; value for sodium-free medium: 0.28 t 0.050; value for phlorizin: 0.28 * 0.035; mean t SE, n = 6, for each situation). These fmdings suggest that any active transport step at the peritubular membrane is not inhibited by phlorizin and is not, dependent, on sodium in the outside bathing medium. However, when distal-proximal tubules were incubated in control medium for 24 h, the C/B ratio for glucose increased to about 1.0 (1.10 * 0.05; mean t SE, n = 4). This observation suggests that the transport system deteriorates with time. It agrees with the observation in perfused tubules that had stopped absorbing glucose and further supports the idea of an active transport system at the peritubular side of the cells. The chromatography data suggested that glucose was not so rapidly metabolized by the cells that this could occur during the transport process. This was further examined by studying the transport of 3-O-methylglucaseby nonperfused tubules. This sugar is reported to be transported by the renal tubules in the same manner as glucose, but it is not metabolized (25). Proximal-proximal and distal-proximal tubules were incubated in control medium containing 0.1 mM 3Gmethylglucose labeled with 14C.The cell-to-bath concentration ratios for

this sugar (0.27 5 0.02; mean t SE, n = 7 for proximalproximal tubules; 0.20 * 0.03; mean t SE, n = 4 for distal-proximal tubules) were significantly less (P < 0.001) than 1.0 and were not significantly different from the control ratios for glucose. These data further support the concept, of a transport step for glucose and similar sugars at the peritubular side of the renal tubule cells. DISCUSSION

In the present study, net transport of glucose from lumen to bath occurred in isolated, perfused proximal renal tubules from snakes of the genus Thamnophis. This absorptive process had a maximum rate at which it could transport, glucose. These findings directly confirm Shannon and Fisher’s (20) early clearance studies that indicated that the renal tubules were limited in their ability to absorb filtered glucose. Maximum rates for glucose transport were well defined in both proximal-proximal and distal-proximal tubules when the concentrations of unlabeled glucose and 14C-labeled glucose were identical for bath and perfusate. However, when the tubules were perfused with 14C-labeled glucose in the lumen only, glucose absorption increased with increasing delivery rates. No clearly defined maximum rate of transport was observed. These data illustrate the importance of matching the concentrations of an isotopically labeled compound on both sides of an epithelium when determining transepithelial transport rates by net, movement, of the isotope only. The presence of a gradient fur labeled glucose from tubule lumen to peritubular blood may have contributed to the apparent absen.ce of an absolute maximum rate of transport for gl.ucose in earlier in vivo microperfusion studies of rat proximal tubules (12). The finding of a maximum rate for glucose absorption in isolated, perfused proximal renal tubules from snakes is in agreement with the finding of a maximum rate for glucose transport in isolated proximal tubules from rabbits (23). However, the maximum rate for glucose transport in snake proximal tubules (2.2 x lo-l2 mol min-l mm+) is only about l/36 that (78.5 x 1O-12 mol min+) in rabbit proximal tubules. This large difference may reflect differences in the need for glucose absorption in the proximal tubules of these two species. In rabbits, the glomerular filtration rate for single nephrons is about 21 nl min+ (8) and the concentration of glucose in the plasma is 7.1 mM (23). Thus the filtered load of glucose per nephron is about 150 x lo-l2 mol min? In snakes, the glomerular filtration rate for single nephrons averaged only 1.8 nl minP (range, 0.5-5.0 for six nephrons) in an early study (5). The concentration of glucose in the plasma is about 2.8 mM (1). If this average glomerular filtration for single nephrons is appropriate for the snakes in the present study, then the filtered load of glucose per nephron is about 5 x lo-l2 mol min? This is about l/30 of that of rabbits. Thus, the relative magnitude of the filtered loads of glucose per nephron for these two species is about the same as the relative magnitude of the maximum transport rates. If the absorptive process were always operating at the

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1726 maximum rate measured in the present study, the minimum tubular length over which complete absorption of the filtered load could occur would be about 2 mm. Although the transport system will not continue to operate at a maximum rate as the glucose concentration in the tubule lumen decreases, it appears that the absorptive capacity measured in vitro is more than adequate to account for complete absorption along a proximal tubule about 4.5 mm in length (10). The maximum rate of glucose absorption in the snake distal-proximal tubule (2.2 x lo-l2 mol min+ mm-9 was about twice that in the proximal-proximal tubule (1.2 x lo-l2 mol min-l mm-l). This is in direct contrast withthe observations of Tune and Burg (23) on the proximal tubule of the rabbit. They found that the proximal portion of the proximal tubule (pars convoluta) transported glucose at a rate about ten times greater than that of the distal portion of the p&ma1 $ubule (pars recta). The reason for this apparent difference between the snake and the rabbit is not clear, but it may be related to the involvement of the tubule with available blood supply. During the dissection of the tubular segments, we observed that the microcirculation is ‘more closely associated with the distal-proximal tubules than with the proximal-proximal tubules of the snake. This anatomic arrangement may be of functional importance for the absorptive processes by rapidly carrying away absorbed glucose and thereby minimizing the gradient against which it must be transported. In the present study, the glucose concentration in the cells of isolated perfused proximal tubules was always less than that in the lumen or bathing medium when glucose was being absorbed at .maximum rates under control conditions. These findings indicate that glucose enters the cells from the lumen down ,a ,concentration gradient and is then transported out of the cells against a concentration gradient by a mechanism Ioctited at the peritubular membrane. This model. for ,glucose absorption under control conditions is shown for both proximal-proximal and distal-proximal +tubule segments in Figs. 5 and 6. The concept of a possible active transport step for glucose at the peritubular membrane is also supported by the observation that the glucose concentration in the cells of nonperfused tubules incubated in glucose-containing medium was always less than that in the bathing medium under steady-state conditions. When net transepithelial glucose absorption by perfused tubules ceased, the concentration of glucose in the cell water was found to be equal to that in the bathing medium rather than less. Similarly, nonperfused tubules incubated in glucose-containing medium for 24 h were no longer able to maintain an intracellular glucose concentration less than th.at in the bathing medium. These data support the concept of a transport step for glucose at the peritubular membrane that is responsible for maintaining the low intracellular glucose, concentration and is of fundamental importance to the transepithelial transport process. In determining the intracellular glucose concentration, we have assumed that, the glucose is, uniformly distributed throughout the cell water. It is possible, of

Il.

W.

BARFUSS

AND

W. H.

DANTZLER

course, that all the glucose in the cells is restricted to some very small compartment. The concentration in this compartment could then be higher than that in the lumen or bath, indicating an active step for glucose transport on the luminal membrane and passive movement into the bath. If the glucose were restricted to such a small intracellular. compartment and a high concentration were maintained by a transport step on the luminal me,mbrane only, th.en the cells would be expected. to lose glucose when the transport system stopped,’ However, when transepithelial transport ceased, the cells gained glucose and the intracellular concentration, calculated on the assumption of uniform distribution, rose to equal that of the bath and lumen Although some compartmentalization may occur, the present observation supports the idea that the low intracellular concentration is maintained by _a peritubular transport step and not simply by compartmentalization. Rapid metabolism of glucose could not have accounted for the low intracellular glucose concentrations in the present study since about 92% of the 14Cin extracts from perfused tubules and about 88% of. the 14Cin extracts from unperfused tubules was still in the form of glucose. Tune and Burg (23) also found that 92% of the radioactive label extracted from perfused rabbit tubules was in the form of glucose. Work by. others has also suggested that an active transport step,for glucose might exist at the peritubular membrane. Studies of glucose uptake by mammalian kidney slices have generally shown a steady-state tissue-to-bath glucose concentration ratio less than 1.0 (16, 17). A ratio greater than LO was achieved only when the glucose concentration in .the bathing medium was low and gluconeogenesis was stimulated (16). Similarly, the cell-to-bath glucose concentration ratio achieved by isolated, nonperfused rabbit tubules incubated in a glucose medium was only 0.68 (23). In contrast to these data with slices and nonperfused tubules, Tune and Burg (23) found a cell-to-bath glucose concentration ratio of 2.1 in perfused rabbit tubules during maximum glucose absorption. This suggests an active transport step for glucose at the luminal membrane. Snakes and rabbits may differ in their tubular transport mechanisms for glucose. However, it should be noted that the ratio of 2.1 in the rabbit tubules resulted from the sum of the cellular concentrations of 14C-labeled glucose from the perfusate and 3H-labeled glucose from the bathing medium. The concentration of 14C-labeled glucose in the cellsdid not exceed that in the perfusate as might have been expected if the main step for transepithelial transport were an uphill step at the luminal membrane. The transepithelial absorption of glucose by renal tubules is limited not only by the capacity of the transport mechanism but also by the extent to which glucose can diffuse from the peritubular fluid back into the tubule lumen. The measured transepithelial permeability of snake tubules to glucose was essentially the same for both proximal tubule segments (0.23 x lop5 cm s-l for the proximal-proximal segment and 0.26 x lop5 cm s-l for the distal-proximal segments). If the plasma

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GLUCOSE

TRANSPORT

BY

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RENAL

TUBULES

glucose level in these animals averages about 23 mM, then the maximum backflux of glucose from peritubular fluid to lumen that could occur when all the luminal glucose had been absorbed would be about 0.3 x lo-l2 mol min-1 mm? This is only about 15% of the maximum net rate of glucose absorption in the distal-proximal tubule or about 25% of that in the proximal-proximal tubules. Tune and Burg (23) found a transepithelial permeability to glucose of 7.2 x IO-” cm s-l in isolated, perfused rabbit proximal tubules. This is more than 25 times greater than that for the snake tubules. If the plasma glucose level is 7.1 mM in these animals, then the maximum backflux from peritubular fluid to lumen that could occur when all the luminal glucose had been absorbed would be about 19.5 x 10-12 mol mini mm+ This is many times what could occur in the snake tubules, but it is still only 25% of the net maximum reabsorptive rate (78.5 x 1O-12 mol min-l mm-l) +in rabbit tubules. Therefore, the transepithelial permeabilities appear reasonably appropriate for the plasma glucose levels and maximum rates of glucose absorption in each species. The apparent permeabilities of the individual peritubular and luminal membranes, estimated in these snake renal tubules from passive flux in the luminal direction, were quite low in both tubule segments. These reflect the low transepithelial permeability in the same direction. Using the estimates of the peritubular membrane permeabilities (0.46 x 10B5 cm s-l in the distal-proximal segment. and 0.50 x IO-” cm s-l in the proximal-proximal segment), we can calculate the glucaseconcentration difference between the cell water and the bath .ing m.ediurn that would have to be established if glucose were to cross the peritubu lar membrane by purely passive means during maximum absorption. Under these circumstances, the glucose concentration in the cell water would have had to be about 6 times that of the bath in the distal-proximal tubules and about 3 times that of the bath in the proximal-proximal tubules to account for the observed maximum rates of transport. If the present estimates of the peritubular membrane permeabilities are too high because of glucose movement between cells, then the intracellular glucose concentration would have to be even higher. However, the glucose concentration in the cell water was only about 0.65 times that in the bath. These estimates of the gradient required for passive glucose movement across the peritubular membrane further support the idea of an active step for glucose transport at the peritubular membrane shown in the models in Figs. 5 and 6. For both tubule segments, the apparent permeability of the luminal membrane when determined in the lumen-to-cell direction (about 10 x lOL5 cm s-9 was more than 15 times the apparent permeability determined in the opposite direction. This observation suggests that glucose entry into the cells is mediated in some fashion and is not simple diffusion. This idea is supported by the fact that when phlorizin was present in the lumen, the apparent perm eability of the luminal membrane in the lumen-to-cell direction was extremely low and not sig-

1727 nificantly different from zero. This suggests that phlorizin binds to some type of carrier in the luminal membrane to prevent the entry of glucose into the cell. In addition, sodium appears to play a role in the entry of glucose into the cells across the luminal membrane. When the tubules were perfused with a sodium-free solution, the apparent luminal membrane permeability of both tubule segments was reduced to about one-half the control value. This reduction in the apparent permeability of the luminal membrane was accompanied by a reduction in net glucose absorption. These findings also suggest that sodium is necessary for the optimum operation of some mediated step for glucose transport. The sodium requirement in the present study is in agreement with studies that have shown that sodium is required for maximum glucose absorption by rat proximal tubules (25). It was found that sodium must be removed from both luminal and peritubular sides ofthe tubule to produce a decrease in glucose absorption. This probably reflected the fact that when sodium was present in the peritubular fluid, it could diffuse into the lumen to play a role in the glucose transport mechanism. Since sodium was present in the bathing medium in the .present study, it should have diffused into the lumen. Even if sodium were an absolute requirement for glucose transport, enough may have been present in the lumen to prevent complete inhibition of glucose absorption. This also may have been reflected in the fact that the apparent permeability of the luminal membrane was reduced less in sodium-free medium than in the presence of phlorizin. On the other hand, it is possible that there is another path for glucose movement across the luminal membrane which can be inhibited by phlorizin but is independent of sodium. It should be mentioned, moreover, that the increase in the glucose concentration in the cell water when the tubules were perfused with sodium-free medium suggests that sodium in the lumen and, possibly, the sodium absorptive process may be required for maximum function of the apparent transport step at the peritubular membrane. Since sodium-free bathing medium had no effect on the glucose concentration in nonperfused tubules, any effect on a peritubtilar transport system may be related only to the absorption of sodium from the lumen and not to the intracellular sodium concentration. The effects of phlorizin and sodium-free perfusate on the luminal membrane permeability together with the very high control value for this permeability in the lumen-to-cell direction suggests that a phlorizin-sensitive, sodium-dependent system may facilitate glucose movement down a concentration gradient from the lumen into the cells when the glucose concentration in the lumen is high. It is also possible that, as glucose absorption goes to completion and the concentration in the lumen is markedly reduced, an active transport process functions at the luminal membrane to more glucose into the cells against a concentration gradient. However, this cannot be determined from the data in the present study, and it is not possible to study the transepithelial profile during glucose absorption with isolated perfused

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1728 tubules when the glucose falls very low.

D. W.

concentration

in the lumen

This investigation was supported in part by National Science Foundation Research Grants GB 38033 and BMS 75-09918, National Institutes of Health Research Grant AM-19294, and National Institutes of Health Training Grant HL-05884. A portion of this work is contained in a dissertation submitted to the Graduate College, University of Arizona by D. W. Barfuss in

BARFUSS

AND

W. H.

DANTZLER

partial fulfillment of the requirements for the PhD in Physiology. A preliminary report of a portion of this work was presented at the Fall Meeting of the American Physiological Society, San Francisco, Calif., 6-8 October, 1975. The present address of D. W. Barfuss is Departments of Physiology and Biophysics and Medicine, University of Alabama Medical Center, Birmingham, Ala, 35294. Received

for publication

26 December

1975.

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227, 1924. 31. WHITE, H. L,, AND 0. SCHMITT. kidney

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Am.

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