J. Phy8iOl. (1978), 275, pp. 481-493 Printed in Great Britain

481

EFFECTS OF GLUCOSE ON WATER AND SODIUM REABSORPTION IN THE PROXIMAL CONVOLUTED TUBULE OF RAT KIDNEY

By J. H. V. BISHOP, R. GREEN AND S. THOMAS From the Department of Physiology, University of Manchester, Manchester M13 9PT

(Received 12 September 1977) SUMMARY

1. The effects of glucose on sodium and water reabsorption by rat renal proximal tubules was investigated by in situ microperfusion of segments of proximal tubules with solutions containing glucose or no glucose, with and without phlorizin. 2. Absence of glucose did not significantly alter net water flux. Sodium flux was reduced by about 10 % but this was not statistically significant. 3. In the absence of glucose in the perfusion fluid net secretion of glucose occurred. 4. Phlorizin reduced either net reabsorption or net secretion of glucose; and net water flux. 5. The data suggest that in later parts of the proximal convoluted tubule some sodium may be co-transported with glucose, but that this represents only a small

fraction of the total sodium reabsorption. 6. It is suggested that the glucose carrier is reversible and in appropriate circumstances could cause glucose secretion. 7. Althoughphlorizin alters net water flux the underlying mechanisms are not clear. 8. The calculated osmolality of the reabsorbate was significantly greater than the perfusate osmolality and greater than plasma osmolality although this was not quite significant statistically. INTRODUCTION

In the mammalian kidney, filtered glucose is almost completely reabsorbed in the proximal convoluted tubule (Walker, Bott, Oliver & MacDowell, 1941; Rohde & Deetjen, 1968; Fr6hnert, H6hmann, Zwiebel & Baumann, 1970) by an active mechanism with a maximum transport rate (TmG). There is now extensive evidence (see review by Morel & de Rouffignac, 1973) that TmG is influenced by changes in glomerular filtration rate (GFR) and in extracellular fluid volume; and that renal reabsorption of glucose is influenced by concurrent reabsorption of sodium. The suggestion (Ullrich, Rumrich & Kl6ss, 1974) that this latter effect involves glucose-sodium transport, in the manner proposed by Crane (1962) for the intestine, is supported by direct studies on the rat proximal tubule (Ullrich, Rumrich & Kloss, 1974) and on brush-border vesicles prepared from proximal tubules (Kinne, Murer, Kinne-Saffran, Thees & Sachs, 1975). The evidence as to whether the converse applies, that primary changes in proximal tubular reabsorption of glucose influence sodium (and hence isotonically equivalent water) transport, is more equivocal. The increase in transepithelial potential difference (lumen more negative) induced by glucose in the isolated, perfused proximal I6

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J. H. V. BISHOP AND OTHERS tubule of rabbit (Kokko, 1973; Burg, Patlak, Green & Villey, 1976) and in the rat proximal tubule, in vivo (Fr6mter & Gessner, 1974) may be interpreted in terms of glucose-induced electrogenic cotransport of sodium; similar electrical effects are induced by glucose in brush-border vesicle preparations (Kinne et al. 1975). But this evidence is indirect; the quantitative relation between changes in electrical potential and in sodium transport is unknown, and changes in potential are not directly related to changes in fluid transport (Cardinal, Lutz, Burg & Orloff, 1975). Furthermore, Stolte, Hare & Boylan (1972) reported that fractional proximal tubular reabsorption of fluid was independent of intraluminal glucose concentration. Similarly, in a study primarily designed to examine quantitative relations between ionic and water fluxes in rat proximal tubules, Green & Giebisch (1975) found that the presence of glucose in the perfused lumen and peritubular capillaries did not significantly affect fluid or sodium fluxes. In contrast, in recent studies on isolated perfused proximal tubule of rabbit (Burg et al. 1976) and with in situ perfusions of rat proximal tubule (Weinman, Suki & Eknoyan, 1976), glucose appeared to augment water reabsorption significantly. However, in these two latter studies, neither glucose nor sodium fluxes were measured. It seems clear that possible resolution of these apparent inconsistencies and a more complete description of the quantitative effects of changes in glucose transport on sodium and water reabsorption in the proximal tubule (with, in consequence, a better assessment of the mechanisms involved) requires direct, simultaneous measurements of the pertinent fluxes. To this end, proximal tubules have been perfused in situ in the rat with solutions designed to induce changes in glucose transport (presence and absence of glucose and of phlorizin) and the fluxes of glucose, sodium, potassium and water were measured. The results of a study on the whole kidney (Bishop, Elegbe, Green & Thomas, 1978) were used in planning an appropriate protocol. 482

METHODS All experiments were performed on male Sprague-Dawley rats weighing 140-190 g which had free access to a rat pellet diet until 16 hr before the experiment and to water until the experiment began. The rats were anaesthetized by an i.P. injection of Inactin (Promonta Corp., Hamburg), 120 mg kg body weight-', and placed on a thermostatically heated operating table, set to maintain body temperature at 37 'C. A tracheostomy was performed and two indwelling catheters (PP10, Portex Plastics Ltd., Hythe, Kent) were inserted into the left jugular vein, one for the administration of priming and sustaining infusions and one for injection of lissamine green. The right carotid artbry was cannulated (PP50) and blood pressure monitored (Statham strain gauge P23DC connected to a Grass recorder model 79). Experiments were terminated if the blood pressure fell below 95 mmHg. The left kidney was exposed through a flank incision and freed from the adrenal gland and the perirenal fat. The capsule was left intact and the kidney placed in a Perspex cup which was securely clamped to the operating table. The surface of the kidney was bathed in liquid paraffin B.P. at 37 'C and the ureter cannulated with polyethylene tubing (PP10). Each animal received 1 ml. physiological saline to replace any loss of fluid due to surgery and thereafter was infused with physiological saline at a rate of 20 1l. min-. Transit time of lissamine green (Steinhausen, 1963) was measured before any tubules were punctured, and any animal with a proximal transit time of greater than 13 sec was discarded. After this, perfusion of individual proximal tubules was performed. Tubular perfusion was carried out using a 12 ,zm diameter pipette ground on three sides to form a point, connected by thickwalled tubing (PP20) to an oil-filled 10t jl.

PROXIMAL TUBULAR GLUCOSE AND FLUID

483

Hamilton syringe (Micromeasure, The Hague, Holland) mounted on an infusion pump (Special slow infuser, Scientific and Research Instruments, Edenbridge, Kent). The pump was set to deliver approximately 30 nl. min-' and allowed to run continuously throughout the experiment. The method of perfusion is a development of that described by Bank & Aynedjian (1972), in turn modified from Sonnenburg & Deetjen (1964). Randomly selected proximal tubules were punctured by a pipette (12-14 #am diameter) filled with castor oil stained with Sudan black. Two or three drops of oil were allowed to flow down the tubule to determine its course and to ascertain that it had at least two more surface loops. The perfusion pipette was inserted into the next most distal surface loop and oil was injected from the first pipette to fill the intervening tubule. The length of the oil-filled loop was always more than eight times the tubular diameter, to allow adequate sealing of the perfused segment. The first pipette was withdrawn and thrust repeatedly into the same segment to allow easy egress of glomerular filtrate, then reinserted into a surface loop distal to that which received the perfusion pipette. A column of oil was rapidly injected and collection of fluid initiated by applying a small negative pressure to the pipette. Care was taken to maintain the position of the oil blocks constant and to avoid distending or collapsing the tubule. Fluid was collected over 3-7 min and then the pipette was rapidly withdrawn into the surface oil, where a small amount of oil was sucked into the tip of the pipette to prevent evaporation of the collected perfusate. The tubule was drawn for later reference. Using the above techniques, four different solutions (detailed composition in Table 1) were used to perfuse tubules: (a) glucose perfusate containing 5 mM-glucose; (b) glucose-phlorizin perfusate, as (a) but with 0-1 mM-phlorizin (K and K Rare and Fine Chemicals, Plainview, New York); (c) saline perfusate with saline substituted for glucose; (d) saline-phlorizin perfusate, as (c) but with 0-1 mM-phlorizin. The composition of the control glucose perfusate (a) was chosen to provide an approximation to a plasma ultrafiltrate in respect of glucose and ionic concentrations, osmolality, pH and buffer capacity. TABLE 1. Composition of perfusates (m-mole 1.-i)

NaCl

NaHCO3 KCl Na2HPO4 KH2PO4 CaCl2

Glu- Phlorcose izin 0 5-0 5-0 0-1

Glucose perfusate 122-3 25-0 40 2-0 0-5 2-0 Glucose perfusate + 40 122*3 25-0 2-0 05 2-0 phlorizin Saline perfusate 125-0 25-0 4-0 2-0 05 2-0 0 0 Saline perfusate + 125-0 25-0 4-0 2-0 2-0 0-1 05 0 phlorizin To 1 ml. of each solution was added 50 ,uc [3H]inulin and 10 1d. 10% lissamine green. Solutions were gassed with 5% CO2 and 95% 02 to give a final pH of 7 40. The measured osmolality was 300 m-osmole kg-1 water. At the end of the experiments a blood sample was obtained from a tail vein, collected into heparinized tubes, centrifuged and analysed. Tubules were reidentified and filled with Microfil (Canton Biomedical Products Inc., Boulder, Colorado). The kidney was removed and stored overnight in deionised water at 4 'C. Next day the kidney was partially digested in 20% NaOH for 25-30 min and the silicone rubber casts dissected out. Using a camera lucida attachment on a Leitz TS stereomicroscope (E. Leitz GMBH D6330, Wetzlar, W. Germany) both the cast and an object micrometer were drawn and the original tubule measured by comparing the lengths of the two with a curvilinear map measurer. The volume of fluid collected was measured in a calibrated constant bore capillary of about 0-3 mm internal diameter, and a measured aliquot was taken so that [3H]inulin (Radiochemical Centre, Amersham) could be counted in a liquid scintillation counter (Intertechnique SL.30), with PCS (Radiochemical Centre, Amersham) as the scintillant. The rate of delivery of the perfusion pump was measured in vitro after each experiment by counting the radioactivity delivered into a counting vial during a period of time the same as that over which the tubule was perfused. The total amounts of inulin delivered and collected during each perfusion were calculated. 10-15% of experiments in each series were discarded because the rate of inulin I6-2

484

J. H. V. BISHOP AND OTHERS

recovery did not exceed 80 % of the perfusion rate. Of the remainder, the mean recovery rate for all series was 99 2%. Table 2 shows the separate data for each series. For plasma, perfusates and collected fluids, sodium and potassium were measured on an Aminco Helium Glow Photometer (Aminco Inc., Silver Spring, Maryland) and osmolality on a nanolitre osmometer (Clifton Technical Physics, Hartford, N.Y.). Glucose was measured using a micromodification of the hexokinase, glucose-6-phosphate dehydrogenase reaction (Bergermeyer, 1963). Nine ml. 0.1 M-Tris buffer (pH 7-6), 0-2 ml. triphosphopyridine nucleotide (17 mg ml.-'), 0*2 ml. ATP (25 mg ml.-'), 0 2 ml. MgCl2 (1 5 M), 0-2 ml. glucose-6-phosphate dehydrogenase (70 units dissolved in 1 ml. 2 m-ammonium acetate), 0X2 ml. hexokinase (95 units TABLE 2. Perfusion and collection rates (mean + S.E.)

Glucose perfusate Glucose-phlorizin perfusate Saline perfusate

No. of animals 12 8

No. of tubules 61 46

Perfusion rate (nl. min-) 32-81 ± 0-42 32-34 + 0-63

Collection (% of perfusion) 99 4+ 1*4 99.3 + 1.4

13 57 32-16 + 0-72 98-1± 1-4 11 43 Saline-phlorizin 33-88 + 0.88 100.5 + 1.5 perfusate Collection rate was calculated from amounts of [3H]inulin delivered and collected.

dissolved in 1 ml. 2M-ammonium acetate) and 0-2 ml. albumen (10 g l.-) were mixed together and 1 PI. dispensed into cuvettes made from 50 Ill. Microcaps (Drummond Scientific Co., U.S.A.). To each cuvette was added 10-30 nl. of sample, the whole was mixed, incubated at 37 °C for 30 min and read on a fluoromicrophotometer (Aminco Inc., Silver Spring, Maryland), with filters to give an excitation wave-length of 360 nm and emission wave-length of 440 nm. With care, it was possible to read solutions of 0.1-10 m-mole 1.-i glucose with a coefficient of variation of 4%. All the chemicals and enzymes for the assay were obtained from Sigma Chemical Co., London. Fluxes of sodium, potassium, osmoles, glucose and water were calculated: water flux, DW = VO (1-Ino/InL) where V0 is the perfusion rate and Ino and InL the concentration of inulin in the perfusate and collected fluid respectively; other fluxes, (A = AO VO-AL (VO-DOW) where (DA is the flux of any substance and AO and AL are the concentration in the perfusate and collected fluid respectively. Statistical comparisons for mean fluxes between series were performed by t test. For each experiment, estimation of any constituent of plasma, perfusate and collected fluid were always performed with standards in the same analytical run. Accordingly, within any one series, comparisons between concentrations of any constituent were assessed by paired t test. RESULTS

The numbers of animals and of perfusions are shown in Table 2. The pooled plasma data for all four series are summarized in Table 3, from which it is evident that with respect to these constituents, the perfusates (Table 1) represent satisfactory approximations to a plasma ultrafiltrate. The estimated perfusion rates were closely similar for all four series and the inulin data show that fluid collections were essentially complete in all the experiments providing the data presented here (Table 2). From the increase in inulin concen-

485 PROXIMAL TUBULAR GLUCOSE AND FLUID trations in the collected fluid, approximately 7-9 % of each perfusate was reabsorbed

per mm length of tubule. For each series, mean paired differences in concentrations of sodium, potassium, glucose and osmoles between perfusate and corresponding collected fluid are given in Table 4, and the calculated fluxes of glucose, sodium, potassium and water are presented in Table 5. Any changes in concentration for a solute must result from the relative water and solute fluxes. TABLE 3. Concentrations in plasma (mean + S.E.; n = 44) K Glucose Na Osmolality (m-osmole kg-1 water) (m-mole 1.-i) (m-mole 1.-i) (m-mole 1.-i) 145-5 + 1-2 4-82 + 0-21 4-58 + 0-23 302-8 ± 1-9 TABLE 4. Changes in concentration (collected fluid perfusate) during perfusion (mean ± S.E.) Na K P p (m-mole 1.-i) (m-mole 1. -1) -7-1 Glucose perfusate +0-05 < 0-6 < 0-001 + 1-0 +0-08 > 0-5 (n= 61) -8-7 < 0-001 -0-38 Glucose-phlorizin < 0-01 +0-8 perfusate (n = 46) + 0-13 Saline perfusate -5-0 + 0-07 < 0-001 < 0-4 + 1-1 (n = 57) + 0-07 > 0-3 -0-45 -5-6 Saline-phlorizin < 0-001 < 0-005 + 1-5 perfusate (n 43) + 0-14 Osmole Glucose (m-osmole P m-mole 1.-i kg-1 water) p Glucose perfusate -0-45 -4-00 < 0-001 < 0-1 + 0-10 > 0-05 ± 2-03 (n= 61) Glucose-phlorizin +0-71 + 1-86 < 0-05 < 0-02 + 0-35 perfusate (n = 46) > 0-1 + 1-34 Saline perfusate +2-04 + 0-70 < 0-001 < 04 + 0-06 (n = 57) > 0-3 +2-15 Saline-phlorizin + 0-36 < 0-001 -2-73 > 0-1 perfusate (n = 43) + 0-05 + 1-45 > 0-05 P represents probability that the change (paired difference) is different from zero. =

Sodium For each series, sodium concentration in the collected fluid was significantly lower than that in the corresponding perfusate (Table 4). For the glucose perfusate only, the sodium concentration in the collected fluid was also lower than that in the corresponding plasma (mean paired difference 2-7 + 1-18 m-mole 1.-i; P < 0-05). As compared with the values in glucose perfusions, small (but non-significant) reductions in mean net sodium flux (Table 5) occurred with glucose-phlorizin (15 %; 0-1 > P > 0-05) and with saline (12%; 0-2 > P > 0-1) perfusions. With the combination of absent glucose and added phlorizin, the mean sodium flux in the saline-phlorizin series was substantially reduced (27 %) compared with the glucose -

J. H. V. BISHOP AND OTHERS 486 series (P < 0.01). As with the comparison between the glucose and glucose-phlorizin series, addition of phlorizin to a saline perfusate was associated with a non-significant (0.2 > P > 0-1) reduction (17 %) in net sodium flux. Although the mean reductions in net sodium flux with phlorizin addition to glucose and saline were non-significant for both, the fact that the direction of change was similar in both and also paralleled significant changes in water flux suggests that these were real effects. TABLE 5. Net flux measurement (mean S.E.)

Glucose perfusate (n = 61)

Glucose-phlorizin perfusate (n = 46) Saline perfusate (n = 57) Saline-phlorizin

perfusate (n

=

43)

Na K (p-mole (p-mole min-' min1 lnr1) mm-1) 595 97 ±38 ±1D6 503 19-0 + 34 +3-1 521 11.6 + 42 + 1-8 434 20-9

+41

+3.9

Glucose (p-mole min-' mm-,)

-16*9

Water (nl. min-' mm-1) 2.70 ±011 2*18 +0-13 2-71

+1-7

+0.20

24.4 +2-7 0-14 + 7-6

- 8-1

2-10

1-4

+0.15

+

Potassium For glucose and saline perfusions, no significant changes in potassium concentrations were evident in collected fluid as compared with perfusate (Table 4). In contrast, addition of phlorizin to glucose and saline perfusates was associated with significant decreases in potassium concentrations in collected fluid in both cases (glucose-phlorizin, P < 0-01; saline-phlorizin P < 0-005). Potassium fluxes were influenced by the presence of phlorizin rather than of glucose (Table 5). Thus, there were no significant differences between the glucose and saline series nor between the glucose-phlorizin and saline-phlorizin series; whereas the presence of phlorizin caused an approximate twofold increase for both glucose and saline perfusions (glucose-phlorizin, P < 0-01; saline-phlorizin, P < 0-05).

Glucose With glucose perfusate, glucose concentrations in the collected perfusate (Table 4) were consistently reduced (mean - 0-45 m-mole 1.-i; P < 0-001). The change in concentration was essentially similar irrespective of the length of tubule perfused; the mean concentration of glucose in collected fluid for perfused segments 0-8-1-2 mm in length was 4-48 + 0-26 m-mole 1.-i; and for perfused segments greater than 2 mm long 4-50 + 0-31 m-mole l.-'. Thus the reduction in concentration must have occurred in the first 0-8 mm of tubule perfused and thereafter concentration changed little. In contrast, addition of phlorizin to the glucose perfusate was associated with an increase in glucose concentration in the collected fluid (mean + 0-71 mmole L-'7; P < 0-05) due to abolition of significant net glucose movement together with continued net water reabsorption (Table 5). In both the saline and saline-phlorizin series, where there was no glucose in the

487 PROXIMAL TUBULAR GLUCOSE AND FLUID perfusate, glucose appeared in the collected fluid (Table 4). With saline perfusate, the mean glucose concentration in collected fluid was 0 70 m-mole 1.-i; and with saline-phlorizin perfusate was 0-36 m-mole 1.-i, a value significantly lower than with saline alone (P < 0.001). As with the glucose series, the concentrations of glucose in the saline and saline-phlorizin series did not appear to alter with the length of tubule. With the glucose perfusate, there was a net reabsorption of glucose of 24-4 p-mole min- mm-1. Addition of phlorizin completely abolished glucose reabsorption (Table 5). With the two glucose-free solutions, however, net flux into the tubule occurred; with saline perfusate there was a net secretion of 16-9 and with salinephlorizin perfusate of 8 1 p-mole min- mm-1. The latter was significantly lower than with saline alone (P < 0'001).

Water Mean net water fluxes (Table 5) were not significantly different between the glucose and saline perfusate series (P > 0.95), despite the differences in glucose fluxes; nor between the glucose-phlorizin and saline-phlorizin series (P < 0.7). Addition of phlorizin caused similar reductions (approximately 20 %) in net water flux for both glucose and saline perfusions (glucose-phlorizin, P < 0 005; saline-phlorizin P < 0.025).

Osmolality The mean perfusate osmolality for all four series (300 + 1 9 m-osmole kg-1 water) was very similar to that of the plasma (303 + 1-9 m-osmole kg-1 water) (Table 3). In none of the four series was there a significant difference between the osmolality of the perfusate and that of the corresponding collected fluid (Table 4), although in the glucose and saline-phlorizin series, the lower values for collected fluid approached significance (0.1 > P > 0.05). In order to examine this further, osmolal fluxes were calculated for each experiment, and factored bv the corresponding net water flux so as to provide a calculated osmolality for the reabsorbate. The mean value for the pooled data from the four series was 322 + 9 7 m-osmole kg-1 water (n = 207). Using the paired t test on the data from individual experiments, the difference between reabsorbate and perfusate (+ 20*0 + 9*0 m-osmole kg-1 water) was statistically significant (P < 0.05) and that between reabsorbate and plasma (+ 18-7 + 10-2) was almost so (0.10 > P > 0.05). DISCUSSION

The main findings in the present study are first, that absence of glucose from the perfusate failed to produce quantitatively large or statistically significant effects on net sodium, potassium or water fluxes, either in the presence or absence of phlorizin; secondly, that in the absence of glucose a significant net flux of glucose into the luminal fluid occurred, which was reduced (but remained significant) when phlorizin was added to the glucose-free perfusate; thirdly, that addition of phlorizin to both glucose and saline perfusates was associated with significant reductions in net glucose flux (to zero with the glucose-phlorizin perfusate) and in net water flux, with significant increases in net potassium flux and with relatively small (statistically not

J. H. V. BISHOP AND OTHERS significant) reductions in mean sodium flux. In addition, the collected data from all the series provide tentative evidence for hypertonicity of the reabsorbate. 488

Relations between glucose, sodium and water fluxes The present data showing that absence of glucose from the perfusate produced no significant change in net water flux are in agreement with the results of Stolte et al. (1972) and Green & Giebisch (1975). In the present study this also applies in the presence of phlorizin. Furthermore, absence of glucose was associated with only relatively small, statistically not quite significant, reductions in mean sodium flux (again both in the presence and absence of phlorizin). In view of the abundant evidence that the presence of sodium is mandatory for adequate renal reabsorption of glucose, this may appear surprising; although the evidence as to whether, conversely, proximal tubular reabsorption of sodium and fluid is significantly influenced by the presence of luminal glucose is less consistent, particularly with respect to the quantitative effect. The enhancement of glucose of the negative electrical potential difference in isolated perfused rabbit proximal tubule (Kokko, 1973; Burg et al. 1976) and in the earliest portions of proximal tubules of Munich-Wistar rats (Fr6mter & Luer, 1973; Fr6mter & Gessner, 1974) has been discussed (e.g. Fromter & Gessner, 1974) in terms of glucose-stimulated enhancement of electrogenic cotransport of sodium. However, we emphasize that such electrical data provide no evidence as to the quantitative significance of glucose-sodium cotransport relative to total proximal tubular reabsorption of sodium and fluid; and in another study, the electrical potential difference was found not to be directly related to water reabsorption (Cardinal et al. 1975). However, in two recent reports, significant effects of glucose on proximal tubular reabsorption of fluid have been described (Burg et at. 1976; Weinman et al. 1976). In the study of Burg et al. (1976) on isolated perfused rabbit tubules, removal of glucose from both perfusate and bath caused a mean reduction in fluid transport of 25 %, from the perfusate alone of some 10 % (but non-significant) and from bath alone caused no effect; addition of glucose to previously glucose-free perfusate increased fluid reabsorption by some 25 %. These complex effects were interpreted as arising from a combination of glucose-sodium cotransport with a direct osmotic effect of the transported glucose, although neither sodium nor glucose fluxes were directly measured. We consider that any quantitative differences from the present study may arise from differences in (1) species, (2) experimental preparation and protocol. and (3) the proximal convoluted segments utilized for perfusion. The experimental situation of Weinman et al. ( 1976) was more similar to that of the present study in that perfusion of individual superficial convoluted tubules was performed in situ in Sprague-Dawley rats. However, they found that perfusion with isotonic saline alone caused a 37 % reduction in fluid reabsorption compared with that from a balanced artificial perfusate containing glucose; and that addition of either 2 or 5 mm-glucose caused restoration to the higher values. Again, sodium and glucose fluxes were not directly measured; and again, there are sufficient differences in protocol from the present study (in composition of perfusate with respect to potassium, calcium, chloride and bicarbonate concentrations and buffer capacity;

489 PROXIMAL TUBULAR GLUCOSE AND FLUID and in perfusion rates) to render the differences less discrepant than might appear. Thus, the lower fluid reabsorption rates in the present work are more similar to those reported by others (Neumann & Rector, 1976). Because Weinman et al. (1976) found that solutions with different glucose concentrations (which would have been expected to alter glucose reabsorption and hence fluid reabsorptive rates, in the event of tight glucose-sodium coupling) produced no significant differences in fluid reabsorption, they attributed their results to an effect of glucose on ionic permeability characteristics of the luminal membrane. In view of the differences in perfusate composition mentioned above, this suggestion might be particularly pertinent in explaining quantitative differences between their results and those of the present study. Thus, it has been shown (Suki, Hebert, Stinebaugh, Martinez-Maldano & Eknoyan, 1974; Wen, 1976) that glucose and anion transports may be interrelated in the proximal tubule. An additional explanation of differences between these studies may arise from the suggestion originally proposed by Walker et al. (1941), and subsequently confirmed (Seely, 1973; Lingard, Rumrich & Young, 1973; Fr6mter & Gessner, 1974; Jacobson & Kokko, 1976), that different parts of the proximal tubule possess different transport characteristics. The data of Frdmter & Gessner (1974) relate to both the first 25 % and later portions of the proximal tubule in Munich-Wistar rate; those of Burg et al. (1976) to perfused, isolated segments of unspecified parts of the rabbit proximal tubule. The present data concern the later parts, those segments most accessible for the protocol used here, of proximal tubule of Sprague-Dawley rats; it is possible that similar segments were used by Weinman et al. (1976). We are not able to comment on the characteristics of the most proximal 25 o/o of the rat tubule, that portion where most glucose is reabsorbed at normal plasma con-

centrations.

Collecting all the available information from the present study and from the literature, we consider it probable that the reductions in mean sodium flux in the glucose-phlorizin and saline series are real effects, albeit not statistically significant, and that there does exist a linkage between glucose and sodium reabsorption. Whether this represents a tight 1:1 coupling in all segments of the proximal tubule and whether glucose may influence sodium via factors other than direct coupling (as discussed by Burg et al. (1976) and Weinman et al. (1976)) is not clear. In the later parts of the proximal convoluted tubule, the present results suggest that the more limited capacity to reabsorb glucose is associated with a quantitatively limited effect of glucose on sodium reabsorption. From the data in Table 5, it would be expected that even with a tight 1:1 coupling, reabsorption of 24 p-mole min- mm-' glucose could directly influence only a small proportion of the total proximal reabsorption of sodium (up to 600 p-moles min- mm-'), and that such a direct effect would be difficult to detect, experimentally, relative to the large total sodium reabsorption. The present data also show that the conventional use of changes in fluid reabsorption as an index of equivalent changes in sodium reabsorption may be questionable in some circumstances.

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Glucose secretion When glucose-free perfusate was used, glucose was secreted to a mean concentration of 0 7 m-mole L.-i in the collected fluid. This confirms the pioneer study of Walker & Hudson (1937) on Necturus. Similar equilibrium concentrations have been reported by others (van Liew, Deetjen & Boylan, 1967; Stolte et al. 1972; von Baeyer, von Conta, Haeberle & Deetjen, 1973). Two main explanations might be proposed. First, the passive permeability to glucose might be sufficiently high in these circumstances to permit substantial diffusive back-leak of glucose into the initially glucose-free perfusates. From such data as are available on rat proximal tubule (Loeschke, Baumann, Renschler & Ullrich, 1969; Stolte et al. 1972, von Baeyer et al. 1973), the permeability is much too low for this to be an adequate explanation. In the study of Tune & Burg (1971), a higher permeability was inferred; but this was on isolated, perfused rabbit tubule in the absence of phlorizin. Thus an alternative explanation is required permitting glucose inflow into the lumen in quantities greater than can be explained by diffusion. We consider that this is most simply explained if the glucose carrier is reversible, permitting glucose transport in either direction. If this is so, net glucose secretion might be expected under appropriate circumstances, particularly with a favourable transmembrane glucose concentration gradient. Furthermore, Ullrich et al. (1974) suggested that the carrier affinity for glucose is determined by ambient sodium concentrations. Thus for a reversible glucose carrier, net movement would be determined by relative, sodium-influenced intraluminal and intracellular affinities for glucose as well as by the transmembrane glucose concentration gradients. It would be predicted that a steady-state intraluminal concentration of glucose would arise in appropriate circumstances, as in the present study and in some previous reports (Rohde & Deetjen, 1968; Frohnert et al. 1970; Hare & Stolte, 1972; von Baeyer et al. 1973). Furthermore, inhibition of such a reversible carrier would be expected to reduce net secretion of glucose, as was found when phlorizin was added to the glucose-free saline perfusate (Table 5). We conclude that the present data are most readily explained by a reversible glucose carrier, with relative intraluminal and intracellular affinities influenced by the respective sodium concentrations. This conclusion is similar to that proposed for L-glucose transport (Huang & Woosley, 1968; Baumann & Huang, 1969).

Effects of phlorizin With the dosage used here, addition of phlorizin to the glucose perfusate completely abolished net proximal tubular glucose reabsorption (Table 5) as has been shown previously (von Baeyer et al. 1973) and as would be expected from the extensive biochemical evidence concerning the properties of phlorizin as a competitive inhibitor of glucose transport (Silverman, 1976). As discussed above, the reduction in net glucose secretion into a glucose-free perfusate induced by phlorizin is also explicable in terms of inhibition of a reversible glucose-carrier. However, there is also evidence that, at least in high doses, phlorizin may inhibit renal metabolic reaction (Lotspeich, 1960); and our study on the whole rat kidney

491 PROXIMAL TUBULAR GLUCOSE AND FLUID (Bishop et al. 1978) shows effects of phlorizin additional to those on glucose transport. In the present study, addition of phlorizin to glucose and saline perfusates reduced net water flux, significantly, and net sodium flux, not quite significantly, in both. Considering all four series of perfusion together, differences between net water fluxes are not simply related to those between glucose fluxes. The present experiments provide no direct evidence concerning the mechanisms by which such effects of phlorizin on sodium and water were achieved, but it may be permissible to speculate on the basis of other evidence. Thus, as has been shown for red cell membranes (Gerlach, Deuticke & Duhm, 1964; Schnell, 1972; Passow & Wood, 1974), phlorizin might inhibit anion transport in the proximal tubule, and so secondarily reduce sodium (and water) flux apart from its action on glucose transport. Alternatively, the small amount of glucose secreted into the tubule might reduce net osmotic efflux of fluid, both directly and via any effect on sodium cotransport; or any effects of phlorizin on the availability of glucose as on intracellular metabolic substrate might interfere with fluid reabsorption.

Reabsorbate osmolality The apparent demonstration of significant hypertonicity of the reabsorbate involves calculation from the data of five separate ultramicro determinations (perfusate volume; inulin and osmolal concentrations of both perfusate and collected fluid), the individual errors of which contribute to the large standard error of the pooled mean. It should be noted that the relative volumes of perfusate and reabsorbate are such that the calculated hypertonic value for the reabsorbate would arise from a very small difference (less than 1 m-osmole kg-1 water) between the osmolalities of perfusate and collected fluid. This is much too small to be detected with consistency in individual experiments; and the statistical significance of the difference arises from the very large number ot perfusions (over 200). Appropriate reservations have to be applied concerning the functional significance to be attached to the data. However, in view of the paucity of direct experimental evidence supporting the various theoretical models of proximal tubular reabsorption of fluid (e.g. Diamond & Bossert, 1967; Sackin & Boulpaep, 1975), we consider that presentation of the collected data from the large number of tubular perfusions is justified. Diamond &T Bossert (1967) constrained their model with values selected to produce an isotonic reabsorbate. Recently, Sackin & Boulpaep (1975) produced a theroetical analysis showing that the reabsorbate must be hypertonic, the extent of which is uncertain. With the reservations expressed above, we consider that the present evidence of a significant reabsorbate hypertonicity adds to the similar, statistically non-significant tendency previously noted in a few experiments (Green & Giebisch, 1975); and provides some support to the conclusions of Sackin & Boulpaep. In conclusion, in the later parts of the proximal tubule in rat, glucose may influence sodium reabsorption but to an extent of only 10-15 % of total proximal reabsorption of sodium and by uncertain mechanisms. The proximal tubular glucosecarrier can operate reversibly; net movement of glucose depends on transmembrane concentration gradients and probably on variable carrier affinities influenced by intraluminal and intracellular sodium concentrations. Phlorizin inhibits the reversible

J. H. V. BISHOP AND OTHERS glucose carrier; but may also inhibit fluid reabsorption via independent mechanisms. Finally, we have provided tentative evidence that proximal tubular reabsorbate is hypertonic. 492

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