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Renal physiology of the pig: Application of stop-flow
0300-9629/90$3.00+ 0.00 Pergmon Pressplc
Camp. Biochem. Physiol. Vol. 96A, No. 1, pp. 41-43, 1990
Printedin GreatBritain
RENAL PHYSIOLOGY OF THE PIG: APPLICATION OF STOP-FLOW JAMB M. TERRIS*and CARMELE. BIXBY~ *Department of Physiology and TDepartment of Laboratory Animal Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 208144799, USA
(Receiued 10 October 1989) Abstract-l. The stop-flow method in the pig, with modified kidneys, produced patterns for sodium, chloride, glucose and PAH that were similar to those obtained previously in other animals with simple kidneys. 2. Ethacrynic acid, under the conditions of the experiment, abolished distal sodium and chloride reabsorption in the pig. 3. We conclude that this method, viewed in perspective, could provide useful in situ information about the physiology of modified kidneys where the more difficult micropuncture and in oitro perfusion techniques are impractical.
method is also applicable for demonstrating secretion when secretory rates are so low that they cannot be observed in free flow clearance experiments. Although there are limitations with this technique (e.g. potential modification of proximal samples as they pass through more distal nephron segments), as there are with all other techniques for assessing renal function, there are several advantages over micropuncture, in vitro perfusion and clearance methods. For example, many nephron segments are inaccessible to micropuncture, and information concerning these segments is not obtainable by clearance studies. Clearance studies provide only information which is the sum of the reabsorption and secretory processes. No special equipment or training is necessary, as is true with micropuncture and perfusion techniques (microscopes, micropipettes, holding devices and dissection of nephron fragments). Finally, in some species (e.g. the pig), the immobility of the kidneys makes micropuncture studies very difficult, if not impossible, and in vitro perfusion requires collagenase during the dissection procedure. Stop-flow has been applied to simple kidneys. This study was designed to investigate the possibility of evaluating renal physiology, by the stop-flow method, in a multipapillary kidney similar to that found in the human (preliminary report, Bixby and Terris, 1988). The pig was selected because it is the only mammal shown to have such a kidney.
INTRODUCTION
The stop-flow technique was introduced in the late 1950s by Malvin and co-workers (Malvin et al., 1958a, b; Wilde and Malvin, 1958) as a convenient method, in a single experiment, to characterize the simultaneous transport of many solutes within segments along the entire nephron, in situ, by the concentration pattern developed during stopped flow. The technique consists of clamping the ureter for a short period (7-15 min) during the steady state that arises from the constant infusion of an osmotic diuretic that is neither reabsorbed, secreted nor metabolized (mannitol). Test substances of interest are included in the mannitol infusion solution. The intratubular fluid at the end of this period differs from fluid during free flow because it has had longer contact with tubular epithelium and its composition reflects reabsorptive and secretory activities to a greater extent. Immediately following the period of stopped flow, serial samples of urine are collected and analysed. The first samples represent urine trapped in the renal pelvis and most distal parts of the nephron (collecting duct and distal tubule). Later samples are composed of urine trapped in successively more proximal areas. Solutes reabsorbed show decreasing concentrations; and solutes secreted exhibit increasing concentrations. Inulin is employed to correct for water movement. Results obtained during stopped flow for solutes related to simultaneously obtained determinations for PAH, inulin, glucose and sodium make it possible to determine the site and direction of transport of these solutes across the tubule. The simultaneous secretion and reabsorption of a substance in different segments of the nephron can be determined by this method, regardless of the direction of net transport (e.g. proximal reabsorption and distal reabsorption or secretion of potassium). Also, the capacity of a single tubule area to secrete and/or reabsorb a solute under different conditions can be demonstrated. This
MATERIALS
AND METHODS
Domestic pigs (six animals, 14.5-25.9 kg) were preanesthetized with ketamine (25-30mg/kg body wt, IM) and acepromazine (1.0 mg/kg IM). Atropine (0.04 mg/kg IM) was administered to decrease salivation. Anesthesia during the surgical procedure was maintained with pentobarbital to effect (25-30 mg/kg IV). Following anesthesia, a midline abdominal incision was performed. One ureter was exposed and catheterized with PE200 tubing (which delivered 38 drops/ml of urine). The tube was advanced into the ureter, placed close to the beginning of the pelvic space, and secured with ligatures to prevent urine from leaking between the 41
42
JAMES
M. TERRISand CARMELE. Brxnv
catheter and ureteral wall. The catheter was connected via a 3-way stopcock to a Statham transducer and Gould recorder for renal pelvic pressure monitoring during the occlusion period. A catheter was placed in the contralateral ureter (or bladder) to collect urine delivered by the contralateral kidney (this urine was discarded). Tygon catheters (0.4 x 0.07”, 18 gauge) were placed in a carotid artery for blood pressure measurement and blood sampling and femoral vein for adminsitration of fluids. A priming infusion of inulin (780 mg), PAH (44 mg), and glucose (5-10 g) was followed by a constant infusion of 0.9% saline containing 10% mannitol (to induce a brisk diuresis) and 0.5-1.5% glucose (to saturate the glucose reabsorption mechanism in the proximal tubule), delivered at a rate of approximately 0.75 ml/kg/min with a constant flow infusion pump. Blood and urine samples were taken at 10 min intervals until the occlusion period. The mannitol infusion also contained inulin and PAH in a suitable concentration to deliver approximately 1.O and 0.04 mg/kg/min, respectively. After urine flow had stabilized (0.18-0.43 ml/kg/min, approximately 45 min after beginning the mannitol infusion), the ureter was occluded and ureteral pressure recorded. Total occlusion times ranged from 12 to 15 min. Stable pressures were reached 7-10 min following occlusion and ranged from 23 to 56 mm Hg. Following approximately 6 min of stable ureteral pressure, the occlusion was released and sufficient 0.5 ml urine samples (75) obtained to ensure adequate collection of urine (determined by preliminary studies to be approximately 50 samples). Blood samples were taken at the beginning, midpoint and end of the occlusion period. A blood sample was also obtained at the end of the urine collection period (approximately 45 min after release of the occlusion). Ethacrynic acid was studied in two animals. Following the first occlusion period, the mannitol infusion was continued for an additional 45 min and a second occlusion performed. In one animal (pig number 26B), 87mg of ethacrynic acid were injected into the venous infusion line at the start of the 45 min period. Ethacrynic acid was included in the mannitol infusion at this point and delivered at a rate of 0.03 mg/kg/min. In the second animal (pig number 996), 87mg of ethacrynic acid were injected into the venous infusion line 10min before occlusion and the ethacrynic acid added to the mannitol infusion at this point. No differences were noted in the results between the two animals. With ethacrynic acid the ureteral pressure stabilized within I-2min following the occlusion in both
animals. The remainder of the study was conducted as described above. Urinary and serum sodium and potassium were determined with a Beckman Klina Flame lithium internal standard flame photometer, urine and serum chloride with a Radiometer CMTlO chloride titrator and glucose with a Beckman Glucose analyzer 2. PAH was determined according to the method of Bratton et al. (1939). All samples were read at 545 nm in a Beckman Model 26 Dual Beam Spectrophotometer. Inulin was determined by the method of Schreiner (1950) and read at a wavelength of 490 nm. RESULTS
Patterns not unlike those reported for simpler (unipapillary, crest) kidneys were obtained (Fig. 1). Markers identified samples with predominantly distal urine (sodium, cumulative urine volume approximately 12-15% kidney weight) and predominantly proximal urine (PAH and glucose, cumulative urine volume approximately 30% kidney weight). An apparent proximal secretion of potassium in the proximal tubule of this species (Fig. 1) will require further investigation. Additionally, complete inhibition of sodium and chloride reabsorption in the thick ascending limb of Henle’s loop was demonstrated in the two animals studied with the diuretic ethacrynic acid (Fig. 2, Pig 996). Reproducibility of the distal reabsorption of sodium is illustrated with five studies (Fig. 3). Stop-flow occlusion times varied from 12 to lSmin, with periods of stable ureteral pressures averaging approximately 6 min. Stable urine flow rates ranged from 5 to lOml/min with infusions of 0.75 ml/kg/min. DISCUSSION
The stop-flow technique has been applied to a number of species with simple kidneys, such as the unipapillary and crest. These include the rat (Young and Edwards, 1964), dog (Malvin et al., 1958a; Wilde and Malvin, 1958), monkey (Vander and Cafruny, 1962), and rabbit (Beechwood et al., 1964). A clinical appraisal of the stop-flow method was published
Pig 3QA
CUMULATIVE
URINE VOLUME
(% Kidney WeIghtI
Fig. 1. Stop-flow patterns for sodium, chloride, glucose, para-aminohippuric acid (PAH) and potassium. Distal minimum for sodium and chloride reabsorption occur at a cumulative urine volume equivalent to approximately 12 and 15% kidney weight, respectively. The proximal minimum for glucose reabsorption occurs at approximately 32% kidney weight. % Free flow (FF) is determined as: %FF = ((SAMPLE UX/PX/SAMPLE UIN/PIN) - (FFUX/PX)/(FFUIN/FFPIN)) x 100 ((FFUX/FFPX)/(FFUIN/FFPIN)) where UX = urine concentration of substance, PX = plasma concentration of substance, UIN = urine concentration of inulin and PIN = plasma concentration of inulin.
43
Stop-flow in the pig Ethacrynic Acid (EA) Pig 996 godlum
ttw
Chloride 4tt.r
-1ool
I
10
0
CUMULATIVE
Fig.
2.
20
EA EA EA
I 30
URINE VOLUME (% Kidney Wclght)
Effect of ethacrynic acid on distal sodium and chloride reabsorption.
Distal Reabsorption of Sodium
found to be dependent on sodium reabsorption and unrelated to potassium secretion. Ureteral occlusion was applied for 6-8 min. Differences in stop-flow sodium and chloride patterns observed between the pig in these studies and those of others in the dog were thought to be due to differences in nephron population (pig 34% long looped nephrons, dog 100%). Nielsen and Buus Lassen studied the effects of the diuretic triamterene in small pigs (10-15 kg) with stop-flow. Only potassium and ammonium ions were reported. Occlusion periods lasted 3 min and a 3% sodium chloride solution was infused to induce a diuresis. Results suggested a distal inhibition of potassium secretion. Effects of triamterene on ammonium ions in these studies were inconclusive. The present studies clearly demonstrate the applicability of the stop-flow method to modified kidneys with a complex renal pelvis. Acknowledgements-The
experiments reported herein were conducted according to the principles set forth in the Guide .&or the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council, NIH Pub. No. 85-23. This work was supported by Uniformed Services University Grant R076CV. .... ,..-. --
PI‘, Pig Pig Pig Pig
76A a33 986 23A 268
30 CUMULATIVE
REFERENCES
40
URINE VOLUME (K Kidney Wslght)
Fig. 3. Distal reabsorption of sodium in five animals. Sodium minimum occurs in a range of approximately 5.7-8.9ml (9.5-13.5% kidney weight).
by Imamura (1963). The author indicated that the method produced vague results when compared to the dog, suggesting that it was because man possesses twice as many nephrons as the dog and that the renal pelvis in man is larger, resulting in greater mixing of urine. He concluded that there was a need for a non-human subject with a human-like kidney to better evaluate the method as it applies to humans. We are aware of only two studies utilizing the stop-flow method in animals with more complicated kidneys, such as the pig, which have multipapillary kidneys similar to the human. In the first, with Landrace/Duroc/Hampshire swine, Vogh and Cassin (1966) found it was impossible to maintain adequate urine flow rates in the newborn (less than 14 days), compared to older animals (2-3 months), utilizing 20% mannitol. The maximum flow rate obtained in a newborn was reported to be 1.6 ml/kg/min. In animals with flows between 1.3 and 1.5 ml/kg/min patterns became less distinct and at 0.8 ml/kg/min or less localization of solute movement was impossible. However, it was possible to localize the PAH secretory system to the proximal tubule, as has been shown for other species including the human. In these experiments potassium secretion was observed to occur in the distal tubule. Chloride reabsorption was
Beechwood E. C., Bemdt W. 0. and Mudge G. H. (1964) Stop-flow analysis of tubular transport of uric acid in rabbits. Am. J. Physiol. 207, 1265-1272. Bixby C. and Terris J. M. (1988) Distal Na and Cl reabsorption in the pig kidney: Application of stop flow. FASEB Journal 2, 5883.
Bratton C. A. and Marshall E. K. Jr (1939) A new coupling component for sulfanilamide determination. J. biol. Chem. 126, 537-550. Imamura A. (1963) Clinical appraisal of stop flow method. Jpn J. Ural. 54, 1200-I 206.
Malvin R. L., Wilde W. S. and Sullivan L. P. (1958a) Localization of nephron transport by stop flow analysis. Am. J. Physiol. 164, 135-142. Malvin R. L., Wilde W. S., Vander A. J. and Sullivan L. P. (1958b) Localization and characterization of sodium transport along the renal tubule. Am. J. Physiol. 105, 549-557. Nielsen 0. E. and Lassen J. B. (1963) Triamterene activity investigated by the stop-flow technique and in vitro studies on carbonic anhydrase. Acta Pharmacol. Toxicol. 20, 351-356.
Schreiner G. E. (1950) Determination of inulin by means of resorcinol. Proc. Sbc. exp. biol. Med. 14, 117~121. Vander A. J. and Cafrunv E. J. (1962) Stoo flow analvsis of renal function in the monkey. hrn. j. Ph&iol. 202, lIO5-8. Vogh B. and Cassin S. (1966) Stop flow analysis of renal function in newborn and maturing swine. Biol. Neonate 10, 153-165. Wilde W. S. and Malvin R. L. (1958) Graphical placement of transport segments along the nephron from urine concentration pattern developed with stop flow technique. Am. J. Physiol. 105, 153-160. Young J. A. and Edwards K. D. G. (1964) Stop-flow analysis of phenosulphonpthalein excretion and visual localization of the site of maximal urine acidification in the rat nephron. Ausr. J. exp. biol. med. Sci. 42, 539-542.
Camp. Biochem. Physiol. Vol. 96A, No. 1, pp. 41-43, 1990
Printedin GreatBritain
RENAL PHYSIOLOGY OF THE PIG: APPLICATION OF STOP-FLOW JAMB M. TERRIS*and CARMELE. BIXBY~ *Department of Physiology and TDepartment of Laboratory Animal Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 208144799, USA
(Receiued 10 October 1989) Abstract-l. The stop-flow method in the pig, with modified kidneys, produced patterns for sodium, chloride, glucose and PAH that were similar to those obtained previously in other animals with simple kidneys. 2. Ethacrynic acid, under the conditions of the experiment, abolished distal sodium and chloride reabsorption in the pig. 3. We conclude that this method, viewed in perspective, could provide useful in situ information about the physiology of modified kidneys where the more difficult micropuncture and in oitro perfusion techniques are impractical.
method is also applicable for demonstrating secretion when secretory rates are so low that they cannot be observed in free flow clearance experiments. Although there are limitations with this technique (e.g. potential modification of proximal samples as they pass through more distal nephron segments), as there are with all other techniques for assessing renal function, there are several advantages over micropuncture, in vitro perfusion and clearance methods. For example, many nephron segments are inaccessible to micropuncture, and information concerning these segments is not obtainable by clearance studies. Clearance studies provide only information which is the sum of the reabsorption and secretory processes. No special equipment or training is necessary, as is true with micropuncture and perfusion techniques (microscopes, micropipettes, holding devices and dissection of nephron fragments). Finally, in some species (e.g. the pig), the immobility of the kidneys makes micropuncture studies very difficult, if not impossible, and in vitro perfusion requires collagenase during the dissection procedure. Stop-flow has been applied to simple kidneys. This study was designed to investigate the possibility of evaluating renal physiology, by the stop-flow method, in a multipapillary kidney similar to that found in the human (preliminary report, Bixby and Terris, 1988). The pig was selected because it is the only mammal shown to have such a kidney.
INTRODUCTION
The stop-flow technique was introduced in the late 1950s by Malvin and co-workers (Malvin et al., 1958a, b; Wilde and Malvin, 1958) as a convenient method, in a single experiment, to characterize the simultaneous transport of many solutes within segments along the entire nephron, in situ, by the concentration pattern developed during stopped flow. The technique consists of clamping the ureter for a short period (7-15 min) during the steady state that arises from the constant infusion of an osmotic diuretic that is neither reabsorbed, secreted nor metabolized (mannitol). Test substances of interest are included in the mannitol infusion solution. The intratubular fluid at the end of this period differs from fluid during free flow because it has had longer contact with tubular epithelium and its composition reflects reabsorptive and secretory activities to a greater extent. Immediately following the period of stopped flow, serial samples of urine are collected and analysed. The first samples represent urine trapped in the renal pelvis and most distal parts of the nephron (collecting duct and distal tubule). Later samples are composed of urine trapped in successively more proximal areas. Solutes reabsorbed show decreasing concentrations; and solutes secreted exhibit increasing concentrations. Inulin is employed to correct for water movement. Results obtained during stopped flow for solutes related to simultaneously obtained determinations for PAH, inulin, glucose and sodium make it possible to determine the site and direction of transport of these solutes across the tubule. The simultaneous secretion and reabsorption of a substance in different segments of the nephron can be determined by this method, regardless of the direction of net transport (e.g. proximal reabsorption and distal reabsorption or secretion of potassium). Also, the capacity of a single tubule area to secrete and/or reabsorb a solute under different conditions can be demonstrated. This
MATERIALS
AND METHODS
Domestic pigs (six animals, 14.5-25.9 kg) were preanesthetized with ketamine (25-30mg/kg body wt, IM) and acepromazine (1.0 mg/kg IM). Atropine (0.04 mg/kg IM) was administered to decrease salivation. Anesthesia during the surgical procedure was maintained with pentobarbital to effect (25-30 mg/kg IV). Following anesthesia, a midline abdominal incision was performed. One ureter was exposed and catheterized with PE200 tubing (which delivered 38 drops/ml of urine). The tube was advanced into the ureter, placed close to the beginning of the pelvic space, and secured with ligatures to prevent urine from leaking between the 41
42
JAMES
M. TERRISand CARMELE. Brxnv
catheter and ureteral wall. The catheter was connected via a 3-way stopcock to a Statham transducer and Gould recorder for renal pelvic pressure monitoring during the occlusion period. A catheter was placed in the contralateral ureter (or bladder) to collect urine delivered by the contralateral kidney (this urine was discarded). Tygon catheters (0.4 x 0.07”, 18 gauge) were placed in a carotid artery for blood pressure measurement and blood sampling and femoral vein for adminsitration of fluids. A priming infusion of inulin (780 mg), PAH (44 mg), and glucose (5-10 g) was followed by a constant infusion of 0.9% saline containing 10% mannitol (to induce a brisk diuresis) and 0.5-1.5% glucose (to saturate the glucose reabsorption mechanism in the proximal tubule), delivered at a rate of approximately 0.75 ml/kg/min with a constant flow infusion pump. Blood and urine samples were taken at 10 min intervals until the occlusion period. The mannitol infusion also contained inulin and PAH in a suitable concentration to deliver approximately 1.O and 0.04 mg/kg/min, respectively. After urine flow had stabilized (0.18-0.43 ml/kg/min, approximately 45 min after beginning the mannitol infusion), the ureter was occluded and ureteral pressure recorded. Total occlusion times ranged from 12 to 15 min. Stable pressures were reached 7-10 min following occlusion and ranged from 23 to 56 mm Hg. Following approximately 6 min of stable ureteral pressure, the occlusion was released and sufficient 0.5 ml urine samples (75) obtained to ensure adequate collection of urine (determined by preliminary studies to be approximately 50 samples). Blood samples were taken at the beginning, midpoint and end of the occlusion period. A blood sample was also obtained at the end of the urine collection period (approximately 45 min after release of the occlusion). Ethacrynic acid was studied in two animals. Following the first occlusion period, the mannitol infusion was continued for an additional 45 min and a second occlusion performed. In one animal (pig number 26B), 87mg of ethacrynic acid were injected into the venous infusion line at the start of the 45 min period. Ethacrynic acid was included in the mannitol infusion at this point and delivered at a rate of 0.03 mg/kg/min. In the second animal (pig number 996), 87mg of ethacrynic acid were injected into the venous infusion line 10min before occlusion and the ethacrynic acid added to the mannitol infusion at this point. No differences were noted in the results between the two animals. With ethacrynic acid the ureteral pressure stabilized within I-2min following the occlusion in both
animals. The remainder of the study was conducted as described above. Urinary and serum sodium and potassium were determined with a Beckman Klina Flame lithium internal standard flame photometer, urine and serum chloride with a Radiometer CMTlO chloride titrator and glucose with a Beckman Glucose analyzer 2. PAH was determined according to the method of Bratton et al. (1939). All samples were read at 545 nm in a Beckman Model 26 Dual Beam Spectrophotometer. Inulin was determined by the method of Schreiner (1950) and read at a wavelength of 490 nm. RESULTS
Patterns not unlike those reported for simpler (unipapillary, crest) kidneys were obtained (Fig. 1). Markers identified samples with predominantly distal urine (sodium, cumulative urine volume approximately 12-15% kidney weight) and predominantly proximal urine (PAH and glucose, cumulative urine volume approximately 30% kidney weight). An apparent proximal secretion of potassium in the proximal tubule of this species (Fig. 1) will require further investigation. Additionally, complete inhibition of sodium and chloride reabsorption in the thick ascending limb of Henle’s loop was demonstrated in the two animals studied with the diuretic ethacrynic acid (Fig. 2, Pig 996). Reproducibility of the distal reabsorption of sodium is illustrated with five studies (Fig. 3). Stop-flow occlusion times varied from 12 to lSmin, with periods of stable ureteral pressures averaging approximately 6 min. Stable urine flow rates ranged from 5 to lOml/min with infusions of 0.75 ml/kg/min. DISCUSSION
The stop-flow technique has been applied to a number of species with simple kidneys, such as the unipapillary and crest. These include the rat (Young and Edwards, 1964), dog (Malvin et al., 1958a; Wilde and Malvin, 1958), monkey (Vander and Cafruny, 1962), and rabbit (Beechwood et al., 1964). A clinical appraisal of the stop-flow method was published
Pig 3QA
CUMULATIVE
URINE VOLUME
(% Kidney WeIghtI
Fig. 1. Stop-flow patterns for sodium, chloride, glucose, para-aminohippuric acid (PAH) and potassium. Distal minimum for sodium and chloride reabsorption occur at a cumulative urine volume equivalent to approximately 12 and 15% kidney weight, respectively. The proximal minimum for glucose reabsorption occurs at approximately 32% kidney weight. % Free flow (FF) is determined as: %FF = ((SAMPLE UX/PX/SAMPLE UIN/PIN) - (FFUX/PX)/(FFUIN/FFPIN)) x 100 ((FFUX/FFPX)/(FFUIN/FFPIN)) where UX = urine concentration of substance, PX = plasma concentration of substance, UIN = urine concentration of inulin and PIN = plasma concentration of inulin.
43
Stop-flow in the pig Ethacrynic Acid (EA) Pig 996 godlum
ttw
Chloride 4tt.r
-1ool
I
10
0
CUMULATIVE
Fig.
2.
20
EA EA EA
I 30
URINE VOLUME (% Kidney Wclght)
Effect of ethacrynic acid on distal sodium and chloride reabsorption.
Distal Reabsorption of Sodium
found to be dependent on sodium reabsorption and unrelated to potassium secretion. Ureteral occlusion was applied for 6-8 min. Differences in stop-flow sodium and chloride patterns observed between the pig in these studies and those of others in the dog were thought to be due to differences in nephron population (pig 34% long looped nephrons, dog 100%). Nielsen and Buus Lassen studied the effects of the diuretic triamterene in small pigs (10-15 kg) with stop-flow. Only potassium and ammonium ions were reported. Occlusion periods lasted 3 min and a 3% sodium chloride solution was infused to induce a diuresis. Results suggested a distal inhibition of potassium secretion. Effects of triamterene on ammonium ions in these studies were inconclusive. The present studies clearly demonstrate the applicability of the stop-flow method to modified kidneys with a complex renal pelvis. Acknowledgements-The
experiments reported herein were conducted according to the principles set forth in the Guide .&or the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council, NIH Pub. No. 85-23. This work was supported by Uniformed Services University Grant R076CV. .... ,..-. --
PI‘, Pig Pig Pig Pig
76A a33 986 23A 268
30 CUMULATIVE
REFERENCES
40
URINE VOLUME (K Kidney Wslght)
Fig. 3. Distal reabsorption of sodium in five animals. Sodium minimum occurs in a range of approximately 5.7-8.9ml (9.5-13.5% kidney weight).
by Imamura (1963). The author indicated that the method produced vague results when compared to the dog, suggesting that it was because man possesses twice as many nephrons as the dog and that the renal pelvis in man is larger, resulting in greater mixing of urine. He concluded that there was a need for a non-human subject with a human-like kidney to better evaluate the method as it applies to humans. We are aware of only two studies utilizing the stop-flow method in animals with more complicated kidneys, such as the pig, which have multipapillary kidneys similar to the human. In the first, with Landrace/Duroc/Hampshire swine, Vogh and Cassin (1966) found it was impossible to maintain adequate urine flow rates in the newborn (less than 14 days), compared to older animals (2-3 months), utilizing 20% mannitol. The maximum flow rate obtained in a newborn was reported to be 1.6 ml/kg/min. In animals with flows between 1.3 and 1.5 ml/kg/min patterns became less distinct and at 0.8 ml/kg/min or less localization of solute movement was impossible. However, it was possible to localize the PAH secretory system to the proximal tubule, as has been shown for other species including the human. In these experiments potassium secretion was observed to occur in the distal tubule. Chloride reabsorption was
Beechwood E. C., Bemdt W. 0. and Mudge G. H. (1964) Stop-flow analysis of tubular transport of uric acid in rabbits. Am. J. Physiol. 207, 1265-1272. Bixby C. and Terris J. M. (1988) Distal Na and Cl reabsorption in the pig kidney: Application of stop flow. FASEB Journal 2, 5883.
Bratton C. A. and Marshall E. K. Jr (1939) A new coupling component for sulfanilamide determination. J. biol. Chem. 126, 537-550. Imamura A. (1963) Clinical appraisal of stop flow method. Jpn J. Ural. 54, 1200-I 206.
Malvin R. L., Wilde W. S. and Sullivan L. P. (1958a) Localization of nephron transport by stop flow analysis. Am. J. Physiol. 164, 135-142. Malvin R. L., Wilde W. S., Vander A. J. and Sullivan L. P. (1958b) Localization and characterization of sodium transport along the renal tubule. Am. J. Physiol. 105, 549-557. Nielsen 0. E. and Lassen J. B. (1963) Triamterene activity investigated by the stop-flow technique and in vitro studies on carbonic anhydrase. Acta Pharmacol. Toxicol. 20, 351-356.
Schreiner G. E. (1950) Determination of inulin by means of resorcinol. Proc. Sbc. exp. biol. Med. 14, 117~121. Vander A. J. and Cafrunv E. J. (1962) Stoo flow analvsis of renal function in the monkey. hrn. j. Ph&iol. 202, lIO5-8. Vogh B. and Cassin S. (1966) Stop flow analysis of renal function in newborn and maturing swine. Biol. Neonate 10, 153-165. Wilde W. S. and Malvin R. L. (1958) Graphical placement of transport segments along the nephron from urine concentration pattern developed with stop flow technique. Am. J. Physiol. 105, 153-160. Young J. A. and Edwards K. D. G. (1964) Stop-flow analysis of phenosulphonpthalein excretion and visual localization of the site of maximal urine acidification in the rat nephron. Ausr. J. exp. biol. med. Sci. 42, 539-542.