JOURNALOF APPLIED PHYSIOLOGY Vol. 39, No. 3, September 1975.
Printed in U.S.A.
Renal blood flow distribution exercise
and exhaustion
during
in conscious
R. DELGADO, T. M. SANDERS, AND Department of Pathology, School of Medicine,
C. M. BLOOR Gu’uersity of California,
flow; microspheres; circulation; radiolabeling
cardiac
output;
dogs
San Diego,
La Jolla,
California
92037
clearance, nor does it require the prolonged steady-state conditions that limit the degree of exertion when PAH clearance is employed. Using the advantages of the radiolabeled tracer microsphere method our study assessed renal blood flow and its intrarenal distribution during rest and during steady state and exhaustive exercise in conscious dogs.
DELGADO, R., T. M. SANDERS, AND C. M. BLOOR. Renal blood flow distribution during steady-state exercise and exhaustion in conscious dogs. J. Appl. Physiol. 39(3) : 475478. 1975.-To determine whether renal blood flow is reduced or redistributed during exercise, we measured total renal flow (TRF) and intrarenal flow distribution (IRFD) in nine dogs. They ran on a motor-driven treadmill at 3-8 mph at grades of 8-15 y0 for an average of 35 min. We measured aortic pressure, heart rate, stroke volume, and cardiac output (CO) via chronically implanted catheters and an We injected 15-pm radiolabeled electromagnetic flow probe. microspheres ( 85Sr, 14Ce, and 51Cr) via a left atria1 catheter during resting control, steady state (SS) and exhaustive (EE) exercise; measured their distribution by gamma spectrometry; and determined TRF as % CO and as ml/l00 g per min. We determined IRFD for the outer and inner cortex and the outer medulla. TRF as yOCO dropped (P < 0.05) during both levels of exercise: from 10.2 s+: 0.7% to 3.9 h 0.4% (SS) and 3.4 + 0.6% (EE). TRF in ml/l 00 g per min did not change significantly from control (228 + 30 ml/l 00 g per min). IRFD was unchanged with exercise, remaining at about 80, 20, and 39i0 of TRF for the outer and inner cortex and outer medulla, respectively. We conclude that blood flow is not diverted from the kidneys during severe exercise in the dog. intrarenal shunting;
steady-state
METHODS
arteriovenous
INVESTIGATORS have measured sodium para -aminohippurate (PAH) clearance from the kidneys under various conditions of exercise in humans, and have reported that physical exertion reduced plasma flow to the kidneys to as low as 20 % of the resting value (7, 18, 28) Vatner et al. (27) and Van Citters and Franklin (26) used ultrasonic flowmeters to measure renal blood flow in severely exercising dogs and found that it did not change from resting values. The differing results from such studies may be due to differences in species, degrees of exertion, or methodology. For example, there has been some question whether PAH clearance can accurately estimate renal blood flow during exercise, since it may be redistributed through regions which do not extract PAH (3, 7). Recently several investigators have used the radiolabeled microsphere method (21) to evaluate renal and intrarenal blood flow under a variety of conditions (1, 3, 4, 15, 20, 22). Radiolabeled microspheres have not been used to assess renal blood flow and its intrarenal distribution during various degrees of exercise stress. This method is not affected by blood flow through nonextracting tissue as is PAH SEVERAL
Animal preparation. Nine healthy adult Labrador retrievers (18-25 kg) were trained to run on a motor-driven treadmill. We then performed a left thoracotomy using sodium pentobarbital (30 mg/kg) anesthesia and implanted polyvinyl catheters in the aortic arch and left atrium and a Biotronex electromagnetic flow probe on the aortic root (5). In five other dogs a catheter was also implanted in the right ventricle to determine the degree of recirculation of the microspheres. Antibiotics were administered before and for 7 days after surgery. The catheters were flushed daily with saline and filled with a solution of heparin and saline (10 mg/ml). Ten to twelve days after surgery cardiac output, aortic pressure, stroke volume, and heart rate were monitored. Cardiac output was measured using Biotronex BL-3 10 electromagnetic flowmeter amplifiers. Aortic pressure was measured using an Elema-Schonander transducer. Data were continuously recorded on a Q-channel highfrequency response Elema-Schonander Mingograf ink-jet recorder. The flowmeters were calibrated by passing measured flows of whole blood through the flow probe before implantation, and the procedure was repeated after removing from the dog. Calibration factors for all probes remained a constant =t5 % throughout the study. Control (resting) hemodynamics were recorded with the animals standing quietly on the treadmill. During these control observations cardiac output, aortic pressure and heart rate were monitored and one dose of radiolabeled microspheres was injected via the left atria1 catheter. Exercise. All animals were excised at least 12 h postprandially by running them on a treadmill at 3-8 mph and at grades of 8-15 % in an air-conditioned room maintained at 17 & 1 “C. We measured rectal temperature before and after exercise. Hemodynamic parameters were recorded routinely during all exercise episodes. Steady-state exercise was defined as an exercise workload which elicited a heart rate of 75 % of the previously determined maximal heart rate and at which the heart rate, mean aortic pressure, and stroke volume remained constant for at
475
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DELGADO,
476 least 5-8 min. Maximal heart rate, determined at least twice before on each animal by running each to exhaustion on the treadmill, was defined as that heart rate at which the dog could no longer maintain the work load imposed on it. During steady-state exercise a second dose of microspheres was injected after the animal had run at steady state for at least 5 min. Exhaustion (exhaustive exercise) was defined as the point at which the animal could no longer maintain the work load imposed on it. The length of time to exhaustion varied with each animal ranging from 25 to 55 min. At exhaustion a third dose of microspheres was injected. Microsphere method. We used tracer microspheres (15 & 5 pm, 3M Company) labeled with 85Sr, 141Ce, or 51Cr suspended in 20 % dextran and Tween 80 (polyoxyethylene). After vigorous mixing the mean activity per ~1 was determined. Activity for each isotope was expressed as counts/ min per ml. This method provided our standards for subsequent calculations. Immediately before injection of the microspheres the total activity of the solution injected was calculated. The solution was then further diluted with dextran and mixed continuously until injected into the animal. Light microscopy revealed less than 0.2 % of the microspheres clumped when mixed in this manner. Each injection contained 1.5-2.5 X 10” microspheres. The residual counts/min in the syringe and in the left atria1 catheter were also determined. Immediately after injecting the solution, the catheter was flushed. The activity of the standard and of the tissue samples was determined in a well-type Packard Autogamma spectrometer, counting only the photo peak for each isotope. A constant was obtained to extrapolate the entire spectrum for each isotope. The principles and methods we used are similar to the techniques used by Bud&erg et al. (6), Hales (12), Kjekshus (14), and Neutze et al. (16). In five animals blood was withdrawn from a venous or right ventricular catheter with a Harvard withdrawal pump at 15 ml/min for 2 min during and after injection of each batch of microspheres to determine the magnitude of arteriovenous shunting. In one dog under anesthesia, the right renal vein was catheterized and blood was withdrawn during the injection of an isotope into the left atria1 catheter to determine the magnitude of arteriovenous shunting of microspheres across the renal vascular bed. Postmortem studies. After the last isotope injection, the animals were killed with potassium citrate after being anesthetized with sodium pentobarbital. The kidneys were removed and placed in isotonic formalin for at least 24 h. They were subsequently decapsulated, blotted, weighed, and cut into coronal sections about 5 mm thick. At least five sections from various regions of the kidney were then divided the into the following zones: the outer section containing outer cortical zone, the next innermost section containing the inner cortical zone, and the medulla which was divided into outer and inner medullary zones. The inner medullary zone also included the pyramids and extended through the renal pelvis. Thus, all portions of the kidney were represented. The respective zones were weighed and expressed as a percentage of the total coronal section weight. We determined that the outer cortical zone made up 52 =t 3 %
SANDERS,
AND BLOOR
(SEM) of the weight of each kidney, inner cortical zone 23 rt 2 %, the outer medullary zone 15 & 1 %, and the inner medullary zone 11 & 1 %. We used these percentages as constants and multiplied the total kidney weight by them to obtain total weights of each zone for each kidney. After the representative zones were counted for radioactivity, the total counts of each section were determined by summation. The inner medulla and pelvis always contained less than 0.1 % of the total renal cpm and was therefore ignored in all subsequent calculations. The total counts/min per renal zone (RZ) was determined as follows RZ
= (counts/min
per g) X (total
wt of zone)
The total weight of each zone was determined as described in the preceding paragraph. Renal blood flow was calculated using the cardiac output determinations from the flow probe. Cardiac output (CO) represents absolute cardiac output less coronary blood flow (5). Total renal blood flow (Table 2) was expressed as a percentage of cardiac output ( %CO) as follows
%CO = 100 [(T counts/min)/(I
counts/min)]
where T countsjmin is the total counts/min per g in both kidneys and I counts/min is the total counts/min injected. Total renal flow (Table 2) expressed as ml/l00 g per min (TRF) was determined as follows TRF
= [(T counts/min)/(I
counts/min)] x (lOO/wt)
x co
where wt is total renal weight. The percentage of TRF (%TRF) (Table 3) to each renal zone was determined as follows %TRF
= 100 [RZ/(T
counts/min)]
All means were subjected to paired t-tests (23) to determine the significance of the difference between control flows and flow during exercise. A result of P < 0.05 was regarded as significant. RESULTS
Hemodynamics. Resting heart rate, mean aortic pressure, cardiac output, and stroke volume were comparable to those observed previously in the conscious dog (5, 10). Resting heart rate was 101 & 6 (SEM) beats/min, mean aortic pressure was 97 zt 3 mmHg, cardiac output was 2.8 + 0.2 l/min, and stroke volume was 27 zt 2 ml. Table 1 shows the hemodynamic changes observed. During steady TABLE
1. Hemodynamics during rest and exercise Heart Rate, beats/min
Resting control Steady-state exercise Exhaustive exercise
with
Values are control.
Mean Aortic Pressure, -Hg
Cardiac Output, l/min
Stroke Volume, ml
101 I+6 235&6*
97*3 12kt5*
2.8ztO.2 7.8ztO.5”
272t2 32&Z*
313*9t
124zt4”
10.3ztO.6t
31&Z*
means & SEM; iV = 9. t P < 0.05 compared with
* P < 0.05 compared steady-state exercise.
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RENAL
FLOW
DISTRIBUTION
IN EXERCISE
477
state exercise heart rate and mean aortic pressure rose to 233 and 125 % of control, respectively. Cardiac output and stroke volume rose to 279 and 119 % of control, respectively. At exhaustive exercise the heart rate and cardiac output had further increased to 133 and 132 % of steady state, or to 310 and 368 % of control. The rise in cardiac output was due primarily to cardioacceleration and to the rise in stroke volume to about 115 % of control. During these exercise bouts rectal temperature increased an average of 2.0 It 0.5”C. Renal and intrarenal blood Jaw. Table 2 displays total renal blood flow in two forms: flow expressed as percent of cardiac output ( %CO) and flow expressed as ml/ 100 g of tissue per min. Expressed as %CO renal blood flow declined to 38 % of control (P < 0.05) during steady-state exercise with no further decrease at exhaustion. However, expressed as ml/ 100 g per min the renal blood flow did not change significantly during steady-state exercise or at exhaustion. Intrarenal distribution of renal blood flow was expressed as a percentage of total renal blood flow (Table 3). The outer cortex, inner cortex and outer medulla received about 78, 20, and 2 % of the total renal blood flow, respectively. Thus, the cortical blood flow as measured with microspheres accounted for about 98 % of the total renal blood flow. During both levels of exercise the relative intrarenal distribution of renal blood flow remained essentially unchanged. Microsphere method. In the five dogs in which blood samples were obtained from the right heart during microsphere injection, 0.57 & 0.26% of the total radioactivity of the microspheres injected was collected in these samples under resting control conditions. During steady-state exercise and exhaustion similar sampling showed 0.54 + 0.27 % and 0.67 =t 0.24 %, respectively, of the radioactivity of the microspheres injected. Thus no significant degree of arteriovenous shunting occurred during the two stages of exercise as compared with controls. The withdrawal of blood from the renal vein during isotope administration into an anesthetized animal revealed no detectable radioactivity. This indicates that all the microspheres were trapped in the renal tissue. TABLE
2. Renal bloodjow
during exercise % co
Resting control Steady state Exhaustion Values output.
TABLE
10.2 3.9 3.4
ml/l00
It 0.7 zt 0.4* & 0.6*
228 & 30 266 & 39 267 zt 53
are means A SEM; N = 9. y0 CO * P < 0.05 compared with control.
3. Intrarenal
blood flow distribution Outer
Resting control Steady state Exhaustion
78.1 77.1 80.0
Cortex
zt 1.1 zk 1.6 rt 1 .O
Inner
19.7 20.1 18.2
g per min
= percent
of cardiac
( % total renal Jaw) Cortex
Outer
Medulla
rf: 0.9 & 1.3 zt 0.9
2.3 2.8 1.8
zt 0.3 zt 0.5 I+ 0.3
Values are means r+ SEM, representing y0 of total renal blood flow; iV = 9. No values were statistically different from control.
DISCUSSION
Our results show two basic findings. First, that exercise does not cause a redistribution of renal blood flow in dogs, and second, that the radiolabeled microsphere method can be applied to the study of blood flow distribution since it gives results similar to other, less flexible methods. Renal blood $0~. Our measurements of intrarenal flow distribution as a percentage of total renal blood flow perfusing various zones of the kidney agree with the results obtained by Rector et al. (20) who also used the tracer microsphere technique. The weight of the outer cortical zone represented 52 % of total kidney weight. This value is comparable to the 49 % that Rector obtained for the two outer zones. McNay and Abe (15) estimated that 78 % of total renal volume was cortical, which agrees with our findings. The sum of the outer cortical zone and inner cortical zones for each of the kidneys we evaluated was about 75 % of total renal weight. This also corresponds with other estimates of total cortical volume of 70-75 % (24, 29). The literature generally agrees that subcortical nephrons account for between 15 and 20 % of the total renal nephrons (19, 17). Kallskog et al. (13) have shown that juxtamedullary and cortical nephrons are perfused at the same rate. Accordingly, our subcortical zone (equivalent to juxtamedullary zone) should account for between 15 and 20 % of total nephron flow (i.e., outer cortical and juxtamedullary zones). Our result of 20 % is in good agreement. Significant redistribution of renal cortical blood flow has been shown with elevated perfusion pressure after vasopressor infusion (20). However, Abe (l), using microspheres, found that an increase in perfusion pressure from 102 to 126 mmHg results in no intrarenal blood flow distribution shift. Our results are in agreement with the findings of Abe, since we observed no redistribution of intrarenal blood flow during the raised mean aortic blood pressure induced by steady state and exhaustive exercise when mean aortic pressure was in the range of 120 mmHg (Table 1). Our findings that renal blood flow remains unchanged during exercise agrees with results presented by some investigators (26, 27), but not with others (7, 18, 28). Although the percentage of cardiac output which the kidney receives does drop during steady state and exhaustive exercise to 38 and 33 % / of control, respectively, the decrease is completely offset by the large increases in cardiac output. Although our data and others (26, 27) on the response of renal blood flow in exercising dogs differ markedly from measurements using PAH clearance in humans (7, 18, 28), the differences may be due to species. PAH clearance depends on the glomerular filtration of plasma and tubular secretion in the cortex; hence changes in intrarenal distribution of blood flow can alter this filtration and secretion, thereby affecting flow measurements. The filtration rate may change with exercise. During exercise, as water loss is accelerated by increased sweating and respiration, the blood colloidal osmotic pressure (COP) tends to increase (8, 19). Increasing the colloidal osmotic pressure affects renal function by decreasing the glomerular filtration rate (11). These renal regulatory mechanisms may
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478 change in concert without a change in total renal blood flow, but would affect the glomerular filtration rate and PAH clearance measurements. However, during heavy exercise when water loss through perspiration is great, the PAH method requires the maintenance of water diuresis to ensure a steady formation of urine for accurate volume determinations. Adequate amounts of water (about 6001,000 ml/h) were consumed by the subjects in the studies reviewed (7, 18, 28). Th us, it is unlikely that the sweat loss during moderate exercise would increase the COP. Also we did not observe any shift in blood flow from the cortical to medullary zone, which would have affected any measurements made with PAH. Since the two methods, i.e., PAH clearance and radiolabeled microspheres, correlate highly ( r = 0.94) in dogs at rest (Z), it is unlikely that a major redistribution of renal blood flow occurs during exercise in contrast to other forms of stress (3, 20). Our results support the view that there are major differences in the species’ responses to exercise in the regulation of renal blood flow. In our animals no significant arteriovenous shunting occurred during exercise, since samples obtained from the right heart showed less than 1 % of the total radioactivity of the microspheres injected, which was similar to values
DELGADO,
SANDERS,
AND
BLOOR
observed under resting control conditions. The fact that no significant shunting of radioactive microspheres occurred across the renal vascular bed into the renal vein of the kidney in the anesthetized animal indicated that particles were trapped in the renal tissue. These results agree well with those of Slotkoff et al. (22) who found less than 0.2 % of the total renal microsphere (15 & 5 r,cm) radioactivity in renal venous blood under similar experimental conditions. We conclude that our radiolabeled microsphere technique yields reproducible measurements of renal blood flow and intrarenal flow distribution and that only an insignificant quantity of microspheres are shunted past the renal circulation by arteriovenous shunts. The greatest portion of renal blood flow, about 98 %, goes to the renal cortex, and there is no significant change from resting values of renal flow with either steady state or exhaustive exercise. During exercise stress there is no redistribution of blood flow from the cortical zone. Finally, we conclude that there may be species differences between humans and dogs in the response of the renal circulation to exercise stress. This Institute Received
study was funded in part by National Program Project Grant HL- 12373. for publication 2 December 1974.
Heart
and
Lung
REFERENCES 1. ABE, Y. Intrarenal blood flow distribution and autoregulation of renal blood flow and glomerular filtration rate. Japan. Circulation J. 35: 1163-1173, 1971. 2. ARRUDA, J. A., S. BOONJAREN, C. WESTENFELDER, AND N. A. KURTZMAN. Measurement of renal blood flow with radioactive 1974. microspheres. Proc. Sot. ExptZ. Biol. Med. 146 : 263264, 3. BAY, W. H., J. H. STEIN, J. B. RECTOR, R. W. OSGOOD, AND T. F. FERRIS. Redistribution of renal cortical blood flow during elevated ureteral pressure. Am. J. Physiol. 222 : 33-37, 1972. 4. BISHOP, S. P., AND C. M. BLOOR. Regional myocardial blood flow following coronary occlusion in unanesthetized normal and hypoxemic dogs. Am. J. Cardiol. 33 : 127, 1974. 5. BLOOR, C. M., F. C. WHITE, AND B. E. SOBEL. Coronary and systemic hemodynamic effects of prostaglandins in the unanesthetized dogs. Cardiovascular Res. 7 : 156-l 66, 1973. 6. BUCKBERG, G. D., J. C. LUCK, D. B. PAYNE, J. E. HOFFMAN, J. P. ARCHIE, AND D. E. FIXLER. Some sources of error in measuring regional blood flow with radioactive microspheres. J. ApPl. Physiol. 3 1 : 598-604, 1971. 7. CHAPMAN, C. B., A. HENSCHEL, J. MINCKLER, A. FORSGREN, AND A. KEYS. The effect of exercise on renal plasma flow in normal male subjects. J. Clin. Invest. 27 : 639-644, 1948. 8. DELANNE, R. Variations Provoquets dans le Sang Veineux par I’Activiti Musculaire. Brussels : Impr. des Sciences, 1957, p. 174. 9. GILMORE, J. P. Renal PhysioZog>. Baltimore, Md.: Williams & Wilkins, 1972. 10. GREGG, D. E., E. M. KHOURI, AND C. R. RAYFORD. Systemic and coronary energetics in the resting unanesthetized dog. Circulation Res. 16 : 102- 113, 1965. 11. GUYTON, A. C. Textbook of Medical Physiology. Philadelphia, Pa.: Saunders, 1966. 12. HALES, J. R. S. Radioactive microsphere measurements of cardiac output and regional tissue blood flow in the sheep. Pfluegers Arch. 344: 119-l 32, 1973. 13. KALLSKOG, O., H. R. ULFENDAHL, AND M. WOLGAST. Single glomerular blood flow as measured with carbonized 141Celabelled microspheres. Acta Physiol. Stand. 85 : 408-413, 1972. 14. KJEKSHUS, J. K. Mechanisms for flow distribution in normal and ischemic myocardium during increased ventricular preload in the dog. Circulation Res. 33 : 489-499, 1973. 15. MCNAY, J. L., AND Y. ABE. Pressure dependent heterogenity of renal cortical blood flow in dogs. Circulation Res. 27 : 57 l-587, 1970.
16. NEUTZE, J. M., J. WYLER, AND ,4. M. RUDOLPH. Use of radioactive microspheres to assess distribution of cardiac output in Am. J. Physiol. 215 : 486-495, 1968. rabbits. of the Kidney and Body Fluids. Chicago, Ill. : 17. PITTS, R. F. Physiology Year Book, 1968. L. R., AND S. D. ROBINSON. Effects of environmental 18. RADIGAN, heat stress and exercise on renal blood flow and filtration rate. J. A#. Physiol. 2: 185-191, 1949. 19. RAISZ, 1~. G., W. Au, AND R. L. SCHEER. Studies on the renal concentration mechanism. III. Effect of heavy exercise. J. Clin. Invest. 38 : 8-l 3, 1959. 20. RECTOR, J. B., J. H. STEIN, W. H. BAY, R. W. OSGOOD, AND T. F. FERRIS. Effect of hemorrhage and vasopressor agents on distribution of renal blood flow. Am. J. Physiol. 222: 1125-l 132, 1972. A. M., AND M. A. HEYMANN. Circulation of the fetus 21. RUDOLPH, in utero: methods for studying distribution of blood flow, cardiac output and organ blood flow. Circulation Res. 21: 163-184, 1967. L. M., A. LOGAN, P. JOSE, J. D’AVELLA, AND G. M. 22. SLOTKOFF, EISNER. Microsphere measurement of the intrarenal circulation Res. 28 : 158-- 166, 197 1. of the dog. Circulation G. W. Statistical Methods. Ames, Iowa: Iowa State 23. SNEDECOR, Univ. Press, 1956. G. D., H. H. KOPALD, J. A. HERD, M. HOLLEN24. THORNBURN, BERG, C. C. C. O’MORCHOE, AND A. C. BARGER. Intrarenal distribution of nutrient blood flow determined with krypton in the unanesthetized dog. Circulation Res. 13 : 290-307, 1963. J., A. E. BARCKY, P. N. DANIEL, K. J. FRANKLIN, 25. TRUETA, Studies of the Renal Circulation. AND M. M. L. PRICHARD. Springfield, Ill. : Thomas, 1947. Cardiovascular per26. VAN CITTERS, R. L., AND D. L. FRANKLIN. formance of Alaska sled dogs during exercise. Circulation Res. 24: 33-42, 1969. S. F., C. B. HIGGINS, S. WHITE, T. PATRICK, AND D. 27. VATNER, FRANKLIN. The peripheral vascular response to severe exercise in untethered dogs before and after complete heart block. J. Clin. Invest. 50: 1950-1960, 1971. 28. WHITE, H. L., AND D. ROLF. Effects of exercise and of some other influence on the renal circulation in man. Am. J. Physiol. 152: 505-516, 1948. M. Studies on the regional blood flow with P32 29. WOLGAST, labeled red cells and small beta-sensitive semiconductor detectors. Acta Physiol. &and. Suppl. 3 13 : l- 109, 1968.
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Printed in U.S.A.
Renal blood flow distribution exercise
and exhaustion
during
in conscious
R. DELGADO, T. M. SANDERS, AND Department of Pathology, School of Medicine,
C. M. BLOOR Gu’uersity of California,
flow; microspheres; circulation; radiolabeling
cardiac
output;
dogs
San Diego,
La Jolla,
California
92037
clearance, nor does it require the prolonged steady-state conditions that limit the degree of exertion when PAH clearance is employed. Using the advantages of the radiolabeled tracer microsphere method our study assessed renal blood flow and its intrarenal distribution during rest and during steady state and exhaustive exercise in conscious dogs.
DELGADO, R., T. M. SANDERS, AND C. M. BLOOR. Renal blood flow distribution during steady-state exercise and exhaustion in conscious dogs. J. Appl. Physiol. 39(3) : 475478. 1975.-To determine whether renal blood flow is reduced or redistributed during exercise, we measured total renal flow (TRF) and intrarenal flow distribution (IRFD) in nine dogs. They ran on a motor-driven treadmill at 3-8 mph at grades of 8-15 y0 for an average of 35 min. We measured aortic pressure, heart rate, stroke volume, and cardiac output (CO) via chronically implanted catheters and an We injected 15-pm radiolabeled electromagnetic flow probe. microspheres ( 85Sr, 14Ce, and 51Cr) via a left atria1 catheter during resting control, steady state (SS) and exhaustive (EE) exercise; measured their distribution by gamma spectrometry; and determined TRF as % CO and as ml/l00 g per min. We determined IRFD for the outer and inner cortex and the outer medulla. TRF as yOCO dropped (P < 0.05) during both levels of exercise: from 10.2 s+: 0.7% to 3.9 h 0.4% (SS) and 3.4 + 0.6% (EE). TRF in ml/l 00 g per min did not change significantly from control (228 + 30 ml/l 00 g per min). IRFD was unchanged with exercise, remaining at about 80, 20, and 39i0 of TRF for the outer and inner cortex and outer medulla, respectively. We conclude that blood flow is not diverted from the kidneys during severe exercise in the dog. intrarenal shunting;
steady-state
METHODS
arteriovenous
INVESTIGATORS have measured sodium para -aminohippurate (PAH) clearance from the kidneys under various conditions of exercise in humans, and have reported that physical exertion reduced plasma flow to the kidneys to as low as 20 % of the resting value (7, 18, 28) Vatner et al. (27) and Van Citters and Franklin (26) used ultrasonic flowmeters to measure renal blood flow in severely exercising dogs and found that it did not change from resting values. The differing results from such studies may be due to differences in species, degrees of exertion, or methodology. For example, there has been some question whether PAH clearance can accurately estimate renal blood flow during exercise, since it may be redistributed through regions which do not extract PAH (3, 7). Recently several investigators have used the radiolabeled microsphere method (21) to evaluate renal and intrarenal blood flow under a variety of conditions (1, 3, 4, 15, 20, 22). Radiolabeled microspheres have not been used to assess renal blood flow and its intrarenal distribution during various degrees of exercise stress. This method is not affected by blood flow through nonextracting tissue as is PAH SEVERAL
Animal preparation. Nine healthy adult Labrador retrievers (18-25 kg) were trained to run on a motor-driven treadmill. We then performed a left thoracotomy using sodium pentobarbital (30 mg/kg) anesthesia and implanted polyvinyl catheters in the aortic arch and left atrium and a Biotronex electromagnetic flow probe on the aortic root (5). In five other dogs a catheter was also implanted in the right ventricle to determine the degree of recirculation of the microspheres. Antibiotics were administered before and for 7 days after surgery. The catheters were flushed daily with saline and filled with a solution of heparin and saline (10 mg/ml). Ten to twelve days after surgery cardiac output, aortic pressure, stroke volume, and heart rate were monitored. Cardiac output was measured using Biotronex BL-3 10 electromagnetic flowmeter amplifiers. Aortic pressure was measured using an Elema-Schonander transducer. Data were continuously recorded on a Q-channel highfrequency response Elema-Schonander Mingograf ink-jet recorder. The flowmeters were calibrated by passing measured flows of whole blood through the flow probe before implantation, and the procedure was repeated after removing from the dog. Calibration factors for all probes remained a constant =t5 % throughout the study. Control (resting) hemodynamics were recorded with the animals standing quietly on the treadmill. During these control observations cardiac output, aortic pressure and heart rate were monitored and one dose of radiolabeled microspheres was injected via the left atria1 catheter. Exercise. All animals were excised at least 12 h postprandially by running them on a treadmill at 3-8 mph and at grades of 8-15 % in an air-conditioned room maintained at 17 & 1 “C. We measured rectal temperature before and after exercise. Hemodynamic parameters were recorded routinely during all exercise episodes. Steady-state exercise was defined as an exercise workload which elicited a heart rate of 75 % of the previously determined maximal heart rate and at which the heart rate, mean aortic pressure, and stroke volume remained constant for at
475
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DELGADO,
476 least 5-8 min. Maximal heart rate, determined at least twice before on each animal by running each to exhaustion on the treadmill, was defined as that heart rate at which the dog could no longer maintain the work load imposed on it. During steady-state exercise a second dose of microspheres was injected after the animal had run at steady state for at least 5 min. Exhaustion (exhaustive exercise) was defined as the point at which the animal could no longer maintain the work load imposed on it. The length of time to exhaustion varied with each animal ranging from 25 to 55 min. At exhaustion a third dose of microspheres was injected. Microsphere method. We used tracer microspheres (15 & 5 pm, 3M Company) labeled with 85Sr, 141Ce, or 51Cr suspended in 20 % dextran and Tween 80 (polyoxyethylene). After vigorous mixing the mean activity per ~1 was determined. Activity for each isotope was expressed as counts/ min per ml. This method provided our standards for subsequent calculations. Immediately before injection of the microspheres the total activity of the solution injected was calculated. The solution was then further diluted with dextran and mixed continuously until injected into the animal. Light microscopy revealed less than 0.2 % of the microspheres clumped when mixed in this manner. Each injection contained 1.5-2.5 X 10” microspheres. The residual counts/min in the syringe and in the left atria1 catheter were also determined. Immediately after injecting the solution, the catheter was flushed. The activity of the standard and of the tissue samples was determined in a well-type Packard Autogamma spectrometer, counting only the photo peak for each isotope. A constant was obtained to extrapolate the entire spectrum for each isotope. The principles and methods we used are similar to the techniques used by Bud&erg et al. (6), Hales (12), Kjekshus (14), and Neutze et al. (16). In five animals blood was withdrawn from a venous or right ventricular catheter with a Harvard withdrawal pump at 15 ml/min for 2 min during and after injection of each batch of microspheres to determine the magnitude of arteriovenous shunting. In one dog under anesthesia, the right renal vein was catheterized and blood was withdrawn during the injection of an isotope into the left atria1 catheter to determine the magnitude of arteriovenous shunting of microspheres across the renal vascular bed. Postmortem studies. After the last isotope injection, the animals were killed with potassium citrate after being anesthetized with sodium pentobarbital. The kidneys were removed and placed in isotonic formalin for at least 24 h. They were subsequently decapsulated, blotted, weighed, and cut into coronal sections about 5 mm thick. At least five sections from various regions of the kidney were then divided the into the following zones: the outer section containing outer cortical zone, the next innermost section containing the inner cortical zone, and the medulla which was divided into outer and inner medullary zones. The inner medullary zone also included the pyramids and extended through the renal pelvis. Thus, all portions of the kidney were represented. The respective zones were weighed and expressed as a percentage of the total coronal section weight. We determined that the outer cortical zone made up 52 =t 3 %
SANDERS,
AND BLOOR
(SEM) of the weight of each kidney, inner cortical zone 23 rt 2 %, the outer medullary zone 15 & 1 %, and the inner medullary zone 11 & 1 %. We used these percentages as constants and multiplied the total kidney weight by them to obtain total weights of each zone for each kidney. After the representative zones were counted for radioactivity, the total counts of each section were determined by summation. The inner medulla and pelvis always contained less than 0.1 % of the total renal cpm and was therefore ignored in all subsequent calculations. The total counts/min per renal zone (RZ) was determined as follows RZ
= (counts/min
per g) X (total
wt of zone)
The total weight of each zone was determined as described in the preceding paragraph. Renal blood flow was calculated using the cardiac output determinations from the flow probe. Cardiac output (CO) represents absolute cardiac output less coronary blood flow (5). Total renal blood flow (Table 2) was expressed as a percentage of cardiac output ( %CO) as follows
%CO = 100 [(T counts/min)/(I
counts/min)]
where T countsjmin is the total counts/min per g in both kidneys and I counts/min is the total counts/min injected. Total renal flow (Table 2) expressed as ml/l00 g per min (TRF) was determined as follows TRF
= [(T counts/min)/(I
counts/min)] x (lOO/wt)
x co
where wt is total renal weight. The percentage of TRF (%TRF) (Table 3) to each renal zone was determined as follows %TRF
= 100 [RZ/(T
counts/min)]
All means were subjected to paired t-tests (23) to determine the significance of the difference between control flows and flow during exercise. A result of P < 0.05 was regarded as significant. RESULTS
Hemodynamics. Resting heart rate, mean aortic pressure, cardiac output, and stroke volume were comparable to those observed previously in the conscious dog (5, 10). Resting heart rate was 101 & 6 (SEM) beats/min, mean aortic pressure was 97 zt 3 mmHg, cardiac output was 2.8 + 0.2 l/min, and stroke volume was 27 zt 2 ml. Table 1 shows the hemodynamic changes observed. During steady TABLE
1. Hemodynamics during rest and exercise Heart Rate, beats/min
Resting control Steady-state exercise Exhaustive exercise
with
Values are control.
Mean Aortic Pressure, -Hg
Cardiac Output, l/min
Stroke Volume, ml
101 I+6 235&6*
97*3 12kt5*
2.8ztO.2 7.8ztO.5”
272t2 32&Z*
313*9t
124zt4”
10.3ztO.6t
31&Z*
means & SEM; iV = 9. t P < 0.05 compared with
* P < 0.05 compared steady-state exercise.
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RENAL
FLOW
DISTRIBUTION
IN EXERCISE
477
state exercise heart rate and mean aortic pressure rose to 233 and 125 % of control, respectively. Cardiac output and stroke volume rose to 279 and 119 % of control, respectively. At exhaustive exercise the heart rate and cardiac output had further increased to 133 and 132 % of steady state, or to 310 and 368 % of control. The rise in cardiac output was due primarily to cardioacceleration and to the rise in stroke volume to about 115 % of control. During these exercise bouts rectal temperature increased an average of 2.0 It 0.5”C. Renal and intrarenal blood Jaw. Table 2 displays total renal blood flow in two forms: flow expressed as percent of cardiac output ( %CO) and flow expressed as ml/ 100 g of tissue per min. Expressed as %CO renal blood flow declined to 38 % of control (P < 0.05) during steady-state exercise with no further decrease at exhaustion. However, expressed as ml/ 100 g per min the renal blood flow did not change significantly during steady-state exercise or at exhaustion. Intrarenal distribution of renal blood flow was expressed as a percentage of total renal blood flow (Table 3). The outer cortex, inner cortex and outer medulla received about 78, 20, and 2 % of the total renal blood flow, respectively. Thus, the cortical blood flow as measured with microspheres accounted for about 98 % of the total renal blood flow. During both levels of exercise the relative intrarenal distribution of renal blood flow remained essentially unchanged. Microsphere method. In the five dogs in which blood samples were obtained from the right heart during microsphere injection, 0.57 & 0.26% of the total radioactivity of the microspheres injected was collected in these samples under resting control conditions. During steady-state exercise and exhaustion similar sampling showed 0.54 + 0.27 % and 0.67 =t 0.24 %, respectively, of the radioactivity of the microspheres injected. Thus no significant degree of arteriovenous shunting occurred during the two stages of exercise as compared with controls. The withdrawal of blood from the renal vein during isotope administration into an anesthetized animal revealed no detectable radioactivity. This indicates that all the microspheres were trapped in the renal tissue. TABLE
2. Renal bloodjow
during exercise % co
Resting control Steady state Exhaustion Values output.
TABLE
10.2 3.9 3.4
ml/l00
It 0.7 zt 0.4* & 0.6*
228 & 30 266 & 39 267 zt 53
are means A SEM; N = 9. y0 CO * P < 0.05 compared with control.
3. Intrarenal
blood flow distribution Outer
Resting control Steady state Exhaustion
78.1 77.1 80.0
Cortex
zt 1.1 zk 1.6 rt 1 .O
Inner
19.7 20.1 18.2
g per min
= percent
of cardiac
( % total renal Jaw) Cortex
Outer
Medulla
rf: 0.9 & 1.3 zt 0.9
2.3 2.8 1.8
zt 0.3 zt 0.5 I+ 0.3
Values are means r+ SEM, representing y0 of total renal blood flow; iV = 9. No values were statistically different from control.
DISCUSSION
Our results show two basic findings. First, that exercise does not cause a redistribution of renal blood flow in dogs, and second, that the radiolabeled microsphere method can be applied to the study of blood flow distribution since it gives results similar to other, less flexible methods. Renal blood $0~. Our measurements of intrarenal flow distribution as a percentage of total renal blood flow perfusing various zones of the kidney agree with the results obtained by Rector et al. (20) who also used the tracer microsphere technique. The weight of the outer cortical zone represented 52 % of total kidney weight. This value is comparable to the 49 % that Rector obtained for the two outer zones. McNay and Abe (15) estimated that 78 % of total renal volume was cortical, which agrees with our findings. The sum of the outer cortical zone and inner cortical zones for each of the kidneys we evaluated was about 75 % of total renal weight. This also corresponds with other estimates of total cortical volume of 70-75 % (24, 29). The literature generally agrees that subcortical nephrons account for between 15 and 20 % of the total renal nephrons (19, 17). Kallskog et al. (13) have shown that juxtamedullary and cortical nephrons are perfused at the same rate. Accordingly, our subcortical zone (equivalent to juxtamedullary zone) should account for between 15 and 20 % of total nephron flow (i.e., outer cortical and juxtamedullary zones). Our result of 20 % is in good agreement. Significant redistribution of renal cortical blood flow has been shown with elevated perfusion pressure after vasopressor infusion (20). However, Abe (l), using microspheres, found that an increase in perfusion pressure from 102 to 126 mmHg results in no intrarenal blood flow distribution shift. Our results are in agreement with the findings of Abe, since we observed no redistribution of intrarenal blood flow during the raised mean aortic blood pressure induced by steady state and exhaustive exercise when mean aortic pressure was in the range of 120 mmHg (Table 1). Our findings that renal blood flow remains unchanged during exercise agrees with results presented by some investigators (26, 27), but not with others (7, 18, 28). Although the percentage of cardiac output which the kidney receives does drop during steady state and exhaustive exercise to 38 and 33 % / of control, respectively, the decrease is completely offset by the large increases in cardiac output. Although our data and others (26, 27) on the response of renal blood flow in exercising dogs differ markedly from measurements using PAH clearance in humans (7, 18, 28), the differences may be due to species. PAH clearance depends on the glomerular filtration of plasma and tubular secretion in the cortex; hence changes in intrarenal distribution of blood flow can alter this filtration and secretion, thereby affecting flow measurements. The filtration rate may change with exercise. During exercise, as water loss is accelerated by increased sweating and respiration, the blood colloidal osmotic pressure (COP) tends to increase (8, 19). Increasing the colloidal osmotic pressure affects renal function by decreasing the glomerular filtration rate (11). These renal regulatory mechanisms may
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478 change in concert without a change in total renal blood flow, but would affect the glomerular filtration rate and PAH clearance measurements. However, during heavy exercise when water loss through perspiration is great, the PAH method requires the maintenance of water diuresis to ensure a steady formation of urine for accurate volume determinations. Adequate amounts of water (about 6001,000 ml/h) were consumed by the subjects in the studies reviewed (7, 18, 28). Th us, it is unlikely that the sweat loss during moderate exercise would increase the COP. Also we did not observe any shift in blood flow from the cortical to medullary zone, which would have affected any measurements made with PAH. Since the two methods, i.e., PAH clearance and radiolabeled microspheres, correlate highly ( r = 0.94) in dogs at rest (Z), it is unlikely that a major redistribution of renal blood flow occurs during exercise in contrast to other forms of stress (3, 20). Our results support the view that there are major differences in the species’ responses to exercise in the regulation of renal blood flow. In our animals no significant arteriovenous shunting occurred during exercise, since samples obtained from the right heart showed less than 1 % of the total radioactivity of the microspheres injected, which was similar to values
DELGADO,
SANDERS,
AND
BLOOR
observed under resting control conditions. The fact that no significant shunting of radioactive microspheres occurred across the renal vascular bed into the renal vein of the kidney in the anesthetized animal indicated that particles were trapped in the renal tissue. These results agree well with those of Slotkoff et al. (22) who found less than 0.2 % of the total renal microsphere (15 & 5 r,cm) radioactivity in renal venous blood under similar experimental conditions. We conclude that our radiolabeled microsphere technique yields reproducible measurements of renal blood flow and intrarenal flow distribution and that only an insignificant quantity of microspheres are shunted past the renal circulation by arteriovenous shunts. The greatest portion of renal blood flow, about 98 %, goes to the renal cortex, and there is no significant change from resting values of renal flow with either steady state or exhaustive exercise. During exercise stress there is no redistribution of blood flow from the cortical zone. Finally, we conclude that there may be species differences between humans and dogs in the response of the renal circulation to exercise stress. This Institute Received
study was funded in part by National Program Project Grant HL- 12373. for publication 2 December 1974.
Heart
and
Lung
REFERENCES 1. ABE, Y. Intrarenal blood flow distribution and autoregulation of renal blood flow and glomerular filtration rate. Japan. Circulation J. 35: 1163-1173, 1971. 2. ARRUDA, J. A., S. BOONJAREN, C. WESTENFELDER, AND N. A. KURTZMAN. Measurement of renal blood flow with radioactive 1974. microspheres. Proc. Sot. ExptZ. Biol. Med. 146 : 263264, 3. BAY, W. H., J. H. STEIN, J. B. RECTOR, R. W. OSGOOD, AND T. F. FERRIS. Redistribution of renal cortical blood flow during elevated ureteral pressure. Am. J. Physiol. 222 : 33-37, 1972. 4. BISHOP, S. P., AND C. M. BLOOR. Regional myocardial blood flow following coronary occlusion in unanesthetized normal and hypoxemic dogs. Am. J. Cardiol. 33 : 127, 1974. 5. BLOOR, C. M., F. C. WHITE, AND B. E. SOBEL. Coronary and systemic hemodynamic effects of prostaglandins in the unanesthetized dogs. Cardiovascular Res. 7 : 156-l 66, 1973. 6. BUCKBERG, G. D., J. C. LUCK, D. B. PAYNE, J. E. HOFFMAN, J. P. ARCHIE, AND D. E. FIXLER. Some sources of error in measuring regional blood flow with radioactive microspheres. J. ApPl. Physiol. 3 1 : 598-604, 1971. 7. CHAPMAN, C. B., A. HENSCHEL, J. MINCKLER, A. FORSGREN, AND A. KEYS. The effect of exercise on renal plasma flow in normal male subjects. J. Clin. Invest. 27 : 639-644, 1948. 8. DELANNE, R. Variations Provoquets dans le Sang Veineux par I’Activiti Musculaire. Brussels : Impr. des Sciences, 1957, p. 174. 9. GILMORE, J. P. Renal PhysioZog>. Baltimore, Md.: Williams & Wilkins, 1972. 10. GREGG, D. E., E. M. KHOURI, AND C. R. RAYFORD. Systemic and coronary energetics in the resting unanesthetized dog. Circulation Res. 16 : 102- 113, 1965. 11. GUYTON, A. C. Textbook of Medical Physiology. Philadelphia, Pa.: Saunders, 1966. 12. HALES, J. R. S. Radioactive microsphere measurements of cardiac output and regional tissue blood flow in the sheep. Pfluegers Arch. 344: 119-l 32, 1973. 13. KALLSKOG, O., H. R. ULFENDAHL, AND M. WOLGAST. Single glomerular blood flow as measured with carbonized 141Celabelled microspheres. Acta Physiol. Stand. 85 : 408-413, 1972. 14. KJEKSHUS, J. K. Mechanisms for flow distribution in normal and ischemic myocardium during increased ventricular preload in the dog. Circulation Res. 33 : 489-499, 1973. 15. MCNAY, J. L., AND Y. ABE. Pressure dependent heterogenity of renal cortical blood flow in dogs. Circulation Res. 27 : 57 l-587, 1970.
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