PROXIMAL REABSORPTION WITH CHANGING TUBULAR FLUID INFLOW IN RAT NEPHRONS

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1 Experimental Physiology (1998), 83, Printed in Great Britain PROXIMAL REABSORPTION WITH CHANGING TUBULAR FLUID INFLOW IN RAT NEPHRONS G. ROMANO, G. FAVRET, R. DAMATO AND E. BARTOLI* Department of Internal Medicine, University of Udine, Medical School, Udine, Italy (MANUSCRIPT RECEIVED 8 MAY 1997, ACCEPTED 10 SEPTEMBER 1997) SUMMARY The relative contribution of intraluminal versus peritubular factors in mediating glomerulo-tubular balance (GTB) is still controversial. We modulated the load of tubular fluid to the proximal tubule of single nephrons of rats by injecting oil into the efferent arteries (EAO). In fifty nephrons the changes in reabsorption induced by obstruction occurred in the same direction as, and were significantly correlated with, the simultaneous changes in single nephron glomerular filtration rate (SNGFR) (y = x, R = 0-91, P < ). In an additional set of thirty-nine nephrons the load of tubular fluid was changed, during EAO, by partial collection from Bowman's space or from the early proximal convolution. Thus, the rate of tubular fluid delivery along the proximal tubule was changed in an experimental situation that prevented any modification in the oncotic pressure of peritubular capillaries. The changes in proximal deliveries during this experimental condition were significantly correlated with those during reabsorption (y = lx, R = 0-82, P < ). These data demonstrate that GTB is fully expressed even when the native peritubular environment is kept constant while the rate of perfusion of proximal tubular segments with native tubular fluid is changed. INTRODUCTION Proximal reabsorption depends upon diffusion of Na+ from lumen to cell, followed by active transport from cell to blood across the basolateral membrane. A slight fall in luminal Na+ concentration will drag solvent flow from lumen to blood (Green, Giebisch, Unwin & Weinstein, 1991). It seems difficult to reconcile this observation with the known ability of proximal tubular reabsorption to adapt quickly to the changes in single nephron glomerular filtration rate (SNGFR). This is the phenomenon of glomerulo-tubular balance (GTB). Several studies suggested a peritubular control of reabsorption, mediated by capillary oncotic and hydrostatic pressure (Brenner & Troy, 1971; Brenner, Troy, Daugharty & Maclnnes 1973). Other studies reported a proportionality between perfusion rates of artificial fluids and tubular reabsorption (Andreoli, Schafer & Troutman, 1978; Green, Moriarty & Giebisch, 1981; Schafer, 1984). Flow-related reabsorption has been shown during perfusion of in vitro isolated tubular segments only at low perfusion rates (Imai, Seldin & Kokko, 1977; Schafer, 1984). However, the transepithelial flow in these structures is so low that the detection of changes may become indistinguishable from errors inherent in the technique at high flow rates (Du Bois, Vernoiry & Abramow, 1976). Therefore, the mechanisms mediating GTB are controversial and as yet unsettled. We attempted to separate luminal from peritubular factors in a number of studies, which disclosed a strong relationship between intraluminal flow rate of native tubular fluid and reabsorption (Bartoli, Conger & Earley, 1978; Bartoli, 1981), even in circumstances where the peritubular environment could not have been altered (Bartoli, Conger & Earley, 1973). * Corresponding author 1647

2 36 G. ROMANO, G. FAVRET, R. DAMATO AND E. BARTOLI The aim of the present experiments was to modulate the rate of flow of native tubular fluid along the full length of the proximal tubule, in conditions where the peritubular environment was kept either constant or this was changed in a direction presumably opposite to that required by the theory of peritubular mediation of GTB. METHODS The experiments were performed on thirty-five Munich-Wistar rats with superficial glomeruli, weighing g, bought commercially (Charles River Italia, Calco, Lecco, Italy). The animals were fed a standard pellet diet and then fasted for 2 days before the experiment, with water allowed ad libitum. All details on anaesthesia, surgical procedures, maintenance fluids, the urine reinfusion system, clearance and micropuncture procedures, determinations and analytical measurements have been reported previously (Romano, Favret, Bartoli, 1995; Bartoli, Romano & Favret, 1996a,b). Anaesthesia was induced by the intraperitoneal injection of Trapanal (Byk Gulden, Konstanz, Germany; 100 mg kg-'). The animals were placed on a heated operating table throughout the experiment, and their body temperature maintained at 37 'C. An endotracheal tube was inserted, and polyethylene catheters were inserted into the jugular vein for fluid infusion and for administration of the glomerular marker, and in the femoral artery for blood pressure recording and blood sampling. The left kidney was exposed through a flank incision, carefully dissected free of surrounding fat and tissues, and immobilized in a lucite cup that was firmly attached to the operating table. Both ureters were cannulated with plastic tubing near the renal pelvis. Urine was allowed to drip from both kidneys into a reservoir, connected by a Silastic tube (Dow Corning) and a peristaltic micropump to the femoral vein catheter. The pump rate was adjusted continuously to match the urine flow rate. The urine level in the reservoir was therefore kept constant. When the jugular catheter was inserted, the animal received a priming infusion followed by a constant maintenance infusion to replace insensible losses and those due to sampling, amounting to 50,1 for blood and to 10,ul for urine. On average, six blood and six urine samples were withdrawn during each experiment. Sixty minutes before tubular sampling, 135,uCi kg-1 of [i4c]carboxy-inulin (Amersham International plc) were injected though the jugular vein. Micropuncture techniques Individual nephrons were identified on the surface of the kidney by direct observation through a Zeiss double-headed microscope at x 120 magnification. Two trained micropuncturists were therefore able to work simultaneously. A thin (<5,um outer diameter tip) pipette filled with standard mammalian Ringer solution containing 50 g F1 Lissamine Green was inserted into a proximal tubule surface loop. By observing the progression of two consecutive boluses of the dye, the proximal tubules of individual nephrons were mapped and the last proximal convolutions accessible to micropuncture on the surface (LP) were identified and carefully drawn. The procedure was repeated for an average of seven nephrons on each animal. The study was carried out with the technique of free-flow total collection of tubular fluid. This was accomplished by injecting an oil block into the tubular lumen, approximately eight tubular diameters long. The flow displaced the oil block downstream, beyond the collecting pipette, allowing the beginning of manual tubular fluid aspiration. This continued at a rate that kept the oil block stationary. Blood samples and timed urine collections were obtained immediately before and after sampling from each nephron studied. The urine samples were taken from the left kidney only, which had been prepared for micropuncture. The blood samples were immediately centrifuged, the haematocrit measured, the plasma separated and precisely measured aliquots were saved for counting and for Na+ and osmolality determinations. These same measurements were performed on the urine samples. The collections of tubular fluid (TF) lasted 1-5 min. At the end of the collections of tubular fluid, the tip of the collecting pipette was sealed with oil aspirated either from the tubular oil block or from that bathing the surface of the kidney. The pipette was immediately removed from the micromanipulator holder, and the sample transferred to a precalibrated constant-bore glass capillary tube. The length of the tubular fluid sample was carefully measured with a suitable apparatus, and the volume calculated. The sample was then transferred into 1 ml of a scintillation counting solution that was then put into a 5 ml vial (Ultima Gold, Packard, Groningen, The Netherlands). A similar procedure was used for plasma and urine samples. Counting was performed for 30 min or up to at least 1000 counts min-' with a B 1900 TR Tricarb Liquid Scintillation Analyser (Packard Canberra Company, Groningen, The Netherlands).

3 INTRALUMINAL CONTROL OF GTB 37 Using methods already described in detail (Romano et al. 1995; Bartoli et al. 1996a, b) we calculated the following: (1) collection rate (CR; ni min-'), the rate of tubular fluid collected per minute, given by the tubular volume collected divided by the time of collection; (2) TFIn/Pin, the tubular fluid-to-plasma inulin concentration ratio, given by the ratio of counts per minute per unit volume of TF over those of plasma; (3) percentage reabsorption, given by1 - Pin/TFin multiplied by 100; (4) SNGFR (nl min-1), the rate of filtration of individual nephrons, given by CR times TFin/Pin; (5) absolute reabsorption, (AR; nl min-'), given by SNGFR times1 - Pin/TFin. These calculations were preferred to simpler ones (such as AR= SNGFR - CR) to be consistent with calculations used for the double-collection experiments. The priming (20 ml kg-') and maintenance (10 ml kg-' h-1) infusions were given as Ringer solution. These were slightly larger than usual, to avoid low baseline SNGFR levels. Besides mapping the glomerulus and the proximal convoluted tubule we also identified the efferent arteriole (EA) stemming from that glomerulus. Two protocols were followed. Protocol1 We studied each tubule twice, before and during occlusion of the efferent arteriole (EAO). (a) For the accessible control measurement, we performed a free flow total collection of tubular fluid from the last convolution of the proximal tubule of each nephron. (b) After completing this collection, a pipette was inserted into the EA of the same glomerulus, and black stained castor oil was injected to block the EA and its vascular territory. During injection the oil was continuously displaced peripherally, filling most of the superficial capillaries located at the periphery of the nephron under study. (c) Recollection during oil block of the EA was accomplished by repeating the collection from the last proximal convolution. Protocol 2 We studied each tubule twice, before and during partial aspiration of tubular fluid either from Bowman's space or from an early convolution of the proximal tubule, during blockade of the efferent artery (EAO). (a) The experiment began with the EAO procedure, as previously described. (b) For the control collection, we performed a free flow total collection of tubular fluid from the last proximal convolution. (c) Immediately after the control collection, we performed a simultaneous double collection of tubular fluid by aspirating all tubular fluid at the last proximal sampling site using the technique of total collection with distal oil blockade, and by partially collecting tubular fluid with a thin-tipped pipette inserted more proximally, without distal oil blockade. This proximal pipette was inserted when possible in Bowman's space, or the first proximal convolution leaving the glomerulus. When this was not technically possible, it was inserted into the earliest accessible convolution of the same nephron. The collection rate was kept lower than the inflowing tubular fluid rate by aspirating approximately at the same rate as that of the more distal collecting pipette. This required manipulating the aspiration of the collecting system by changing the hydrostatic level between the collecting pipette and the syringe connected to the line used for manual suctioning of tubular fluid. (d) For the proximal collection, we then performed a free flow total collection of tubular fluid from the earliest convolution of the proximal tubule, the same position in which the proximal pipette had been inserted during the double collection. This required inserting a new sampling pipette of larger tip size. This technique is similar to that already described in previous studies (Bartoli et al. 1973), with the difference that it was performed during EAO, and that the more proximal pipette was inserted near the glomerulus. Therefore, the proximal tubular segment along which delivery and, presumably, reabsorption were altered during double collection was then longer than the segments where microperfusion and double collection had been performed previously (Bartoli & Earley, 1971; Buentig & Earley, 1971; Bartoli et al. 1973). In each sample of tubular fluid we measured the volume, the sampling time, and the inulin concentration. We calculated the collection rate (CR; nl min-1), nephron filtration rate (SNGFR; nl min-'), tubular fluid to plasma inulin concentration ratio (TFin/Pin), percentage reabsorption (PR) and absolute reabsorption (AR; nl min-'). The rates of delivery and reabsorption of tubular fluid between the two pipettes, as required by Protocol 2, were calculated as follows. (1) During single collections (Protocol 2), delivery was given by CREP, the collection rate measured at the early proximal (EP) sampling site during the final single collection (step (d) of Protocol 2): Baseline delivery between pipettes = CREP, which is the fluid flow rate delivered into the nephron segment between the early and last proximal tubular puncture site.

4 38 G. ROMANO, G. FAVRET, R. DAMATO AND E. BARTOLI Reabsorption was given by last proximal SNGFR (SNGFRLP) multiplied by the difference between last proximal and early proximal percentage reabsorptions: Baseline AR between pipettes = SNGFRLP x (PRLP -PREP). (2) During the double collections (Protocol 2), the delivery along the same segments was calculated as SNGFR - CREP - AREP, where SNGFR was the sum of the rates calculated during double collection from the early proximal and the last proximal sample. CREP was the collection rate at the early proximal pipette during double collection. AREP was the absolute reabsorption between the glomerulus and the early proximal pipette (SNGFR x PREP): Double collection delivery between pipettes = SNGFR - CREP -AREP. Reabsorption by the segment between the two pipettes during the double collection was given by a formula identical to that used for single collections, although we used the data obtained from double collections only: Double collection AR between pipettes = Delivery x (PRLP -PREP). This is the fluid reabsorption rate between the early and late proximal tubular collecting pipettes. The PR was calculated as: PR = (1 - P/TF) x 100. The plasma value was measured as the average of blood samples withdrawn before and after the samplings from each nephron. Other methods of calculation were available. For instance, we could have used the same SNGFR value for single and double collection (by taking the average), and the same early proximal reabsorptions. To avoid early TFin/Pin values lower than unity due to random errors in TFi. and Pin measurements, we could have introduced corrections for plasma water content. We elected to use raw data, as we did in all previous studies, even though this implies the occurrence of errors. These errors remained within the limits already measured by us (Romano, Favret, Federico & Bartoli, 1997) and others (Oken, Wolfert, Laveri & Choi, 1985). The measurements were analysed statistically. Means, standard errors of the mean, differences between means by Student's paired t tests, regressions, correlations, differences between regressions by covariance analysis were computed with statistical packages adapted for personal computers. RESULTS We attempted to separate the effect of intraluminal factors from that of the efferent peritubular oncotic pressure by keeping the latter constant and at a high value by partial oil blockade of the efferent arteriole. The blood reaching the capillaries through collaterals has a higher protein concentration than during baseline, and, presumably, a lower hydrostatic pressure. If we assume that peritubular mediation of reabsorption occurs, this should cause the reclamation of more reabsorbate for any given value of SNGFR. Table 1 shows that the mean values of SNGFR and reabsorption before and during partial obstruction of the efferent glomerular artery (EAO) were not significantly different. However, the distribution of the present data was significantly different from the Gaussian distribution (probit test, P < 0.004). In contrast, the distribution observed in 147 previously published (Romano et al. 1997) recollection pairs in collection-recollection experiments was not different from the Gaussian distribution (probit test, P > 0-277). These data suggest that oil injection into the efferent artery can result, in several instances, in a slight increase in efferent arteriolar resistance. In turn, this can increase the glomerular hydrostatic pressure and, consequently, SNGFR through a significant rise in filtration fraction. A major increase in

5 INTRALUMINAL CONTROL OF GTB 39 Table 1. Efferent artery obstruction (EAO) SNGFR PR AR (nl min-1) (ni min-') Control PairedP >0.191 >0.595 >0-348 n EAO ± ± 1-5 Data are the means ± S.E.M. measured in fifty proximal tubules during control conditions, and remeasured, from the same sites in a paired fashion during efferent artery obstruction (EAO). The paired P value shows the significance of the difference between control and EAO data. SNGFR, single nephron glomerular filtration rate; PR, percentage reabsorption; AR, absolute reabsorption. resistance of the efferent glomerular artery should reduce glomerular blood flow markedly, and, notwithstanding a rise in filtration fraction, cause an attendant reduction of SNGFR. Thus, we examined the relationship between the changes in absolute reabsorption and the paired changes in SNGFR which occurred on switching from control conditions to EAO in order to obtain more information than could be obtained by inspection of mean values only. Figure 1 shows the relationship between SNGFR and rate of reabsorption before (top) and during EAO (bottom). The regression slopes between SNGFR and absolute reabsorption during control conditions (AR = x SNGFR, R = 0-90, P < ) and during EAO (AR = x SNGFR, R = 0*84, P < ) are both significantly different from zero but are not different from each other by covariance analysis (P > 0.88). Figure 2 portrays the changes (A) in AR and SNGFR on switching from control conditions to EAO. The paired changes in AR, shown on the ordinate, closely follow those in filtration, shown on the abscissa. The regression slopes and correlation coefficients were significantly different from zero (y = x, R = 0.91, P < ). In two nephrons extreme and opposite changes in filtration were measured, indicating that the technique can result in unpredictable and profound effects in resetting total and efferent glomerular resistances. Experimental error might have contributed to these rather large changes. During EAO we measured reabsorption in the same thirty-nine proximal tubular segments before and during an induced change in the load of tubular fluid. This was obtained by aspirating part of the filtered tubular fluid through a proximal pipette at the beginning of the proximal segment. A similar study was performed previously by us (Bartoli et al. 1973), and reproduced subsequently by others (Haberle, Shiigai, Maier, Schiffl & Davis, 1981). The results are shown in Table 2. They show that SNGFR was not different when measured at the last and early proximal sampling sites. During double collection from both early and last proximal sites of the same nephrons, SNGFR was not significantly different from the mean value computed from the isolated single collections. The mean percentage reabsorptions (PR) were not different in the samples taken from the early proximal sites in control conditions and those obtained during double collection. In contrast, the PR values measured at the last proximal site were significantly higher during double collection. These data are in agreement with other observations demonstrating the well known incompleteness of GTB (Bartoli & Earley, 1972).

6 40 G. ROMANO, G. FAVRET, R. DAMATO AND E. BARTOLI 60 Control measurements. 0.. y Efferent artery obstruction cc 0 / A. A SNGFR (nl min-1) Fig. 1. Least-squares regressions between nephron filtrations (abscissa) and reabsorptions (ordinate). During control conditions (top) the regression equation is AR = x SNGFR, R = 0-90, P < During EAO (bottom) the regression equation is AR = x SNGFR, R = 0-84, P < The two slopes are not different by covariance analysis (P > 0-88). Table 2 also reports the data of flow and reabsorption rates along the proximal segment between the two pipettes. The inflow into the experimental segment was significantly reduced during double collection with respect to that occurring in control conditions. Reabsorption by these same segments changed in the same direction and in near proportion to the changes in inflow. This is also portrayed in Figs 3 and 4. Figure 3 shows on the abscissa the inflow to the proximal segment between early and last proximal pipettes. This value of fluid delivery is equivalent to SNGFR and it is plotted against reabsorption by the same segment on the ordinate. The regression slopes obtained in control conditions and observed during double

7 INTRALUMINAL CONTROL OF GTB , 0 7E 20- -_ 00 S w -20- / ASNGFR (ni min-) Fig. 2. Changes in filtration between control conditions and EAO (abscissa), plotted against the paired changes in reabsorption (ordinate). The regression equation is: AAR = x ASNGFR, R = 0.91, P < Table 2. Reabsorptions and deliveries in thirty-nine proximal tubular segments during control conditions and double collections SNGFR (nl min-) PR Delivery * Reabsorption* (nl min-') (nl min-') EP P LP EP P LP Control 24.1 ± 2-2 > ± < ± ± ± 1-5 Paired P > < < < 0.02 Double collection 29-5 ± < ± ± ± 0-8 Data are paired values measured during control conditions, and remeasured during double collection of tubular fluid. During control, SNGFR was separately measured at the early proximal (EP) and last proximal (LP) sampling site. The paired P (>0.292) is not significant and the values are significantly correlated. Their mean (23 3 ± 1-9 nl min-') is not different by paired t test from that of double collection. The paired P values between EP and LP measurements are reported. The calculated deliveries and reabsorptions in the proximal tubular segments between EP and LP collecting pipettes are shown at the right-hand side, marked by the asterisks. The ratios of absorptions to deliveries between pipettes are diffierent from the PR reported, which refer to the entire proximal tubule, from the glomerulus to the last proximal collecting site. For definitions of abbreviations, see Table 1. collection are significantly different from zero, but are not significantly different from each other by covariance analysis (P > 0.09). Due to the proximity to the glomerulus, the experimental error and the use of plasma inulin values uncorrected for plasma water content, there are a few calculated negative deliveries. The major factors causing the computation of negative deliveries were the random changes in SNGFR and early PR on switching from single

8 42 G. ROMANO, G. FAVRET, R. DAMATO AND E. BARTOLI 35 - Control conditions 25- c Double collections 25 - C E 15-5 < Delivery (nl min-') Fig. 3. Least-squares regressions between delivery of tubular fluid (abscissa) and reabsorption (ordinate) along the proximal tubular segments between early proximal and last proximal pipettes. During control conditions the regression equation (top) is AR = x Delivery, R = 0-77, P < During double collection (bottom) the regression equation is AR= x Delivery, R = 0.64, P < The two regression slopes are not significantly different by covariance analysis (P > 0.09). to double collections. By using the mean SNGFR, PR and corrected Pin, no negative values were computed. However, we considered it more accurate to perform our calculation with raw uncorrected data, without resorting to complex recalculations based on assumptions which might be different among animals and tubules. The reader can evaluate the experimental error, which is consistent with that specifically measured and analysed by us (Romano et al. 1997). Figure 4 reports the changes (A) in inflow on switching from control conditions to the experimental period of double collection, on the abscissa, plotted against the paired changes in reabsorption, on the ordinate. The regression slopes and correlation coefficients are significantly different from zero.

9 INTRALUMINAL CONTROL OF GTB '.EC ce / ADelivery (ni min-1) Fig. 4. Changes in delivery from control to double collection (abscissa), plotted against the paired changes in reabsorption (ordinate). The regression equation is AAR = ( x ADelivery, R = 0.82, P < DISCUSSION Glomerulo-tubular balance (GTB) is the term used to describe the ability of proximal tubular reabsorption to adapt proportionately to changes in filtered load (Bartoli & Earley, 1971). The perfusion of proximal tubular segments in vivo at different rates in some laboratories indicated a lack of GTB (Wiederholt, Hierholzer, Windhager & Giebish, 1967; Buentig & Earley, 1971), suggesting that peritubular mediation of the adaptive changes occurred during spontaneous alterations of nephron filtration rate (SNGFR). Several studies have demonstrated a correlation and a lack of dissociation between the initial peritubular protein concentration and tubular reabsorption (Brenner, Falchuk, Keimowitz & Berliner 1969; Ichikawa & Brenner, 1979). There have also been experiments on peritubular capillary perfusion using different protein concentrations which demonstrated a proportionality between capillary oncotic pressure and tubular reabsorption (Brenner & Troy, 1971). Thus, these data indicate a peritubular mediation of GTB. In contrast, the perfusion of proximal tubular segments in vivo with plasma ultrafiltrate (Bartoli & Earley, 1973) and with native tubular fluid (Haberle et al. 1981; Haberle & Von Baeyer, 1983; Tucker & Blantz, 1978) suggested an additional mechanism for modulating proximal reabsorption by intraluminal flow rate per se. These studies were considered necessary after it was demonstrated that in vivo perfusion with artificial perfusates may depress reabsorption (Bartoli & Earley, 1973). Moreover, perfusion of peritubular capillaries with ultrafiltrates of plasma of different oncotic pressures did not always confirm the dependence of proximal reabsorption on peritubular oncotic pressure (Agerup, 1975; Conger, Bartoli & Earley, 1976). Perfusion with colloid solutions artificially prepared with bovine albumin gave controversial results (Brenner & Troy, 1971; Holzgreve & Schrier, 1975). Experiments designed to measure the effects of

10 44 G. ROMANO, G. FAVRET, R. DAMATO AND E. BARTOLI hydrostatic pressure gradients between lumen and capillary were also controversial (Brenner & Troy, 1971; Bank, Aynedjian & Wada, 1972; Agerup, 1975). Thus, the data on the mechanisms that mediate GTB are conflicting. The major difficulty with these experiments is to produce changes in native flow of tubular fluid without modification of the peritubular environment, and vice versa. In order to dissociate these factors we devised appropriate experiments by inducing hydronephrosis (Bartoli et al. 1978) or partial obstruction of the lumen of single tubules (Romano, Favret, Federico & Bartoli, 1996). Finally, we resorted to double-collecting proximal tubular fluid to reduce its delivery to the last proximal convolution (Bartoli et al. 1973). These studies indicated a high degree of tubular balance when the intraluminal flow rate was decreased while the peritubular factors were presumably left unchanged. We performed the opposite experiments, manipulating the peritubular factors while leaving the intraluminal flow rate unchanged. This was achieved by obstructing with oil all the tubules surrounding the experimental one. Reabsorption of this experimental tubule was measured before and during the reduction of peritubular oncotic pressure in the efferent arteries stemming from all adjacent nephrons and was not found to be changed by these manoeuvres (Bartoli, 1981). Thus, the bulk of our own evidence indicates that GTB is mainly mediated by factors acting inside the tubular lumen. We thought it important to design experiments in which the changes in intraluminal flow are associated with changes in peritubular factors opposite to those required by the peritubular theory for mediation of GTB. This was the aim of the present experiments. In Protocol 1, we obstructed the efferent artery by continuous oil injection. This produces two consequences. If the rise in efferent resistance was slight, filtration pressure and SNGFR increased. Alternatively, SNGFR will fall during EAO if the rise in resistance causes a significant rise in total resistance to flow in the vasculature of the glomerulus. In either circumstance the filtration fraction of that glomerulus will increase, and the initial peritubular oncotic pressure in the capillaries supplied by it will be maximal. Since one cannot know in advance which effect will prevail, the data should be analysed by observing the relationships between the changes in SNGFR and the concomitant changes in reabsorption. According to the peritubular theory of mediation of GTB, reabsorption should remain relatively constant, and more or less independent from the changes in tubular flow rate (SNGFR) per se. Reabsorption should be reset at a high level because of the high post-glomerular oncotic pressure. Figure 2 shows that the changes in reabsorption occurred in the same direction as, and were significantly correlated with, those in filtration during EAO. We examined by covariance analysis the relationships between filtration and reabsorption in control conditions and during EAO. This analysis showed that the slopes were not different (P > 0.88), although fractional reabsorption rose slightly, on average, in spite of the slight mean fall in SNGFR. This is consistent with the known fact that GTB is not perfect, and there is a slight resetting of reabsorption (Bartoli & Earley, 1972). This resetting causes reabsorption to increase and to fall less than SNGFR when filtration is changed by experimental manoeuvres (Romano et al. 1996). Therefore, the relationship between reabsorption and filtration should be different, in conditions where the GTB is expressed, from that observed in conditions where these changes are random and attributable to experimental errors in measurements, as during collection-recollection sampling. Therefore we compared, by covariance analysis, the present data with the relationship between changes in filtration and

11 INTRALUMINAL CONTROL OF GTB 45 reabsorption measured in 147 collection-recollection pairs previously obtained in the proximal tubule (Romano et al. 1997). The covariance analysis demonstrates that the slopes are not different (P > 0.08). Only the recollection pairs follow a normal distribution. Clearly, the rise in efferent oncotic pressure during EAO cannot be demonstrated, since it is not possible to sample from the artery during continuous oil injection. However, it is indisputable that the oil clogs part of the surrounding capillaries towards which it is displaced by the blood flowing from the efferent artery. This suggests the establishment of frictional forces larger than those of blood, indicating increased efferent outflow resistance. Thus, the data obtained using Protocol 1 suggest that reabsorption follows filtration, even when the oncotic pressure of the EA is reset at a maximum level, and that GTB is then largely mediated by intraluminal events. The peritubular factor determining reabsorption may not therefore be the initial EA oncotic pressure, but, rather, the mean effective oncotic pressure bathing the peritubular capillaries (Buentig & Earley, 1971). In other words, as the oncotic pressure rises along the glomerular capillaries because of the filtration of a protein-free ultrafiltrate, the oncotic pressure falls along peritubular capillaries because of the progressive reclamation of a protein-free reabsorbate. In either circumstance, the net force is given by the integral of the change in protein concentration along the length of the capillary. Hence, even though the initial oncotic pressure may be high, the post-glomerular plasma flow may represent a factor more important in determining reabsorption than the initial post-glomerular protein concentration. This peritubular flow may cause the observed relationship between reabsorption and filtration, which could then be mainly attributed to peritubular factors. This explanation would, however, exclude any effect of the initial peritubular protein concentration as a determing factor in GTB and would indicate the product between oncotic pressure and post-glomerular plasma flow as the main determinant. To investigate this possibility we resorted to Protocol 2. When we double-collected the proximal tubular fluid, we reduced the delivery to more distal sites of the proximal tubule while diluting to a minor extent the peritubular protein concentration from its initial postglomerular value. This initial value was already maximal during efferent obstruction. Therefore we had induced an important discrepancy between the putative peritubular reabsorbing force and the bulk of reabsorbate. The oncotic pressure was maximal and unchanged with respect to baseline conditions, while the volume of absorbable tubular fluid was lower. This should have resulted in increased reabsorption according to the peritubular theory. Instead, our data showed that even during this situation reabsorption fell significantly. Moreover, the relationships depicted in Fig. 2 could be reproduced, as shown in Figs 3 and 4. The regression slopes between filtration and reabsorption were significantly different from zero and similar before and during double collection. The changes in proximal tubular fluid delivery between baseline and double collection were significantly correlated with those in reabsorption. The data obtained using Protocol 2 demonstrate that the rate of flow of native filtrate along the proximal tubule is the major determinant of proximal reabsorption during rapid variations of filtration. Hence, GTB is a phenomenon rapidly mediated by events acting at the luminal side of the proximal tubular membrane. It could be argued that the peritubular environment is a milieu where significant mixing of blood occurs between adjacent nephrons. In this case the experiments involving double collection of tubular fluid, originally performed by us (Bartoli et al. 1973) and reproduced by

12 46 G. ROMANO, G. FAVRET, R. DAMATO AND E. BARTOLI others (Haberle et al. 1981) without EAO, should already represent conclusive evidence in favour of the intraluminal theory. Furthermore, this would allow us to consider as fully valid the experiments involving manipulation of intraluminal flow rate per se, such as those using single nephron hydronephrosis (Bartoli et al. 1978) and single nephron external compression (Romano et al. 1996), which invariably demonstrated a strong correlation between the fall in reabsorption and that in SNGFR. The single nephron function hypothesis states that each proximal convoluted tubule is perfused by the parent efferent arteriolar blood. Although this is not the case in certain rat species (Beeuwkes, Moffat & Fourman, 1963), we do not know if the hypothesis holds in the Munich-Wistar strain. If it does, it would account for GTB in single nephron hydronephrosis and compression by the peritubular hypothesis. However, even if the single nephron function hypothesis does apply to Munich-Wistar rats, Protocol 2 produced a complete dissociation between the change in intraluminal flow delivery and that in peritubular factors. Experimental manoeuvres devised in other studies only depressed SNGFR, limiting the impact of the experiment to one direction of change only. In contrast, the present experiments caused, in several instances, a rise in SNGFR, which allowed us to explore the whole range of changes in intraluminal flow rate. Changes in hydrostatic pressure, which should be considered among the peritubular factors, could have had an important influence on the results obtained. However, during EAO peritubular hydrostatic pressure is expected to fall, leading to a further rise in the overall transtubular gradient. This effect may have been buffered by a fall in intraluminal hydrostatic pressure when SNGFR fell during EAO or during double collection. The effect of the hydrostatic pressure gradient was not supported by the evidence from most (Brenner et al. 1973; Brenner & Troy, 1971; Ichikawa & Brenner, 1979), although not all (Agerup, 1975; Bank et al. 1972), authors favouring the peritubular theory. The changes which occur in the present experiments were expected to enhance the effect of the peritubular factors which act in a direction opposite to that anticipated by intraluminal control of GTB. Therefore, they do not support the hypothesis that the effects which seem to be determined mainly by intraluminal factors are caused by peritubular events. Finally, it is important to recognize that the modulations of tubular fluid flow rate and reabsorption could be measured, in the present experiments, along almost the full length of the proximal tubule. The changes measured in former studies, where shorter proximal segments were available, were close to the experimental error of the methods. In conclusion, we manipulated the load of filtrate to the proximal tubule, and measured reabsorption during experimental conditions in which the initial peritubular protein concentration was kept constant, presumably at a maximum level. Both in the situation where the loads of filtrate and peritubular protein delivery may have remained proportional (Protocol 1), and in the situation where protein delivery was experimentally increased above tubular fluid flow rate (Protocol 2), reabsorption closely followed intraluminal flow. These data demonstrate that the filtered load is the principal determinant of proximal reabsorption by intraluminally mediated events. This paper was supported by grants from Consiglio Nazionale delle Ricerche and Ministero della Universita Ricerca Scientifica, Rome, Italy.

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