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Radioisotope studies in isolated perfused baboon kidneys
CRYOBIOLOGI,
8,
134-137 (1971)
RADIOISOTOPE
STUDIES IN ISOLATED PERFUSED BABOON KIDNEYS’
R. SCHOONEES,za 3 G. R. JOHNSTON,4 J. H. GI~OENEWALD,2z HEEItDEN,3 AND G. P. MURPHY2
3 P. D. 1~. VAN
Baboon kidneys were studied during isolated bloodless perfusion as part of an experimental renal transplantation project. These studies were conducted upon freshly excised as well as preserved kidneys. Data was obtained on the basic function and preservability of baboon kidneys. This paper considers the use of various radioactive isotopic methods in the characterization of these kidneys. MATERIALS
AND
METHODS
Kidneys from both male and female adult Cape Chacma baboons (Papio ursinus) were used. The animals were sedated with phencyclidine hydrochloride (Sernylan, Parke, Davis Co., Detroit, Mich.) 1 mg/kg, atropine 0.6 mg, and Valium (Roche, Basle, Switzerland) 10 mg before operation. The kidneys were removed under sterile conditions and then gently irrigated with chilled, heparinized normal saline (2000 U of heparin to each 150 ml of normal saline) until the venous effluent was clear. At least 150 ml was employed in each instance. On an average 6 min elapsed from the time of removal of the kidney to the institution of successful isolated perfusion. The perfusion apparatus used has been described previously (4). It incorporates a warming coil, a water bath at 37”C, and a roller-type pump to produce a pulsating flow. The flow rate was maintained at 2 ml/ min/g of kidney, but in some experiments the rate was varied in order to study the effect on renal function. The perfusion solution employed was Travert 5% replacement solution (Travenol Inc., Morton Grove, Ill.) with 50 ml of 6% clinical dextran added to each liter. The perfusate was equilibrated with either 100 y0 oxygen or helium gas. The perfusion system was maintained at’ its capacity of 1000 ml throughout the 3-hr duration of the experiment. Measurements were made of the in-flow line pressure (mmHg), pOZ, pCOZ, pH, and urine output (ml/min and ml/min/g kidney). Samples were obtained of urine and of in-flow and out-flow perfusion fluid for the determination of osmolar clearance, free water excretion, effective renal cortical perfusion, percentage sodium reabsorption, PAH extraction, creatinine clearance, and radioactivity. These results have been described elsewhere (4). Mercury-203 chlormerodrin. Thirty kidneys were studied with *03Hg chlormerodrin, 3kCi added to the in-flow (arterial) perfusate. In 14 instances, 5 ml of plasma was mixed with the 3 PCi of *03Hg chlormerodrin prior to injection. Timed samples were collected from the in-flow, out-flow, and ureteral lines over a 50-min period. Tissue samples were obtained from each kidney at the end of the experiment for measurement of radioactivity of the cortex and medulla. The radioactivity of all samples was expressed as counts per minute per milliliter of fluid, or per gram of tissue. The additional effect of a 30-min ureteral occlusion was studied in 10 instances. Xenon-l%. The perfusion flow in 20 baboon kidneys was studied with iasXe. Following the perfusion period, a bolus of 200-400 &i of lasXe dissolved in saline was injected into the in-flow line while the venous line was diverted to prevent recirculation of the radioactive gas. The clearance rate of the 133Xe was monitored externally by a scintillation de1 Presented at the Fifth Cryopreservation Conference convened Meeting of the Society for Cryobiology, Buffalo, New York, August in part by the Office of Naval Research Contract ONR 3700. 2 Roswell Park Memorial Institute, Buffalo, New York 14203. 3 University of Stellenbosch Medical School, Bellville, South Africa.
4 Walter Reed Hospital,
Washington, D.C., 20012. 134
during the Annual 3-7, 1969. Supported
I:A1)IOISOTOPE
STUDIES
IN BABOON
KII)NEYS
tector probe plared 6 in. above the kidney. The curve obtained by direct writer was then plotted against t,irne on semilogarithmic paper and analyzed in its various coml)onents in thr standard manner (1, 2). Iodine-131 hippuran. Ten rnicrocuries of 1311hippuran was injected into the ill-flow line of 12 kidneys. L\ scintillation detector probe with direct writer print-out was positioned above the kidney at a distance determined by the isotope dose. A hippuran renogram was then obtained. The perfusion flow rate varied from 0.5 t’o 2.0 ml/min/g of kidney. .~1 renogram was obtained on a living baboon for comparison. One additional kidney was l)erfused with pl:tsma instead of the usual perfusate. Cc&on microspheres. StrontiumWabeled microspheres (15 + 5 p) and scandium46labeled mirrosl)heres (35 f 5 p) were used to determine renal blood flow distribution in intact baboons versus perfusion flow distribution in isolated kidneys. Fourteen baboons and 22 vervet monkeys received microspheres containing 20 &‘i of radioactivity by injection into either the left ventricle or descending aorta (above the renal arteries). Tht> kidney:: of these animals were then excised and assayed for radioactivity. Other kidneys were perfused for 3 hr and at the end of this period microspheres containing a similar amount of radioactivity were introduced into the in-flow line. Some of these kidneys had been in storage for 24 hr prior to perfusion. These kidneys were similarly assayed for rndinactivi@.
RESULTS Jfercury-2020J chlormerodrin. All kidneys perfused with ahelium-equilibrated perfusat,e showed rapid excretion of m3Hg chlormerodrin. Ureteral occlusion delayed *03Hg chlormerodrin removal from the perfusate until the occlusion was released. Addition of protein (plasma) to the z03Hg chlormerodrin prior to injection resulted in reduction in clearance of radioactivity from the perfusate. Oxygen equilibration of the perfusate did not alter these findings (Table 1). Renal tissue concentration of z03Hg was predominantly cortical in every experiment with no differential between outer and inner cortex. The presence of protein in the perfusate tended to reduce the concentration of *03Hg in the renal tissue in helium-exposed kidneys while enhancing it in oxygen-exposed kidneys. A reduction of flow rate u-as accompanied by a decrease in tissue radioactivity. xenon-1%. Flow rates through the cortex, outer medulla, and inner medulla were calculated from the curves obtained following injection of 133Xeinto the in-flow line of perfused kidneys. A fourth component was not observed due to the absence of perirenal fat. from the perfused specimens. In the nine kidneys exposed to helium 42 f 14% (mean f 1 SD) of the renal mass was supplied by component I of the curve (cortical flow). The 11 kidneys exposed to oxygen had 22 f 18% (mean f 1 SD) of the renal mass supplied b) component I. These findings would indicate that oxygen exposure of the perfusate reduces cortical perfusion to a level approaching that of the outer medulla. This redistribution of intrarenal perfusion flow is presumably related to vasoconstriction of the cortical vessels. Iodine-131 hippuran. Clearance of 1311hippuran by the perfused kidney was increasingly prolonged as the perfusion rate was reduced. Oxygen-exposed kidneys tend to clear Ia11hippuran slower than helium-exposed kidneys at similar perfusion rates (Fig. 1). At a perfusion rate of 2.0 ml/min/g a sharper isotopic peak is noted with helium than with oxygen exposure. At 1.5 ml/min/g this is not the case (Fig. 1). *it lower perfusion rates the average values for helium-exposed kidneys generally manifest higher peaks than the oxygell-exposed kidneys. This is a result of better diffusion of the isotopic tag and may be attributed to differences in vascular perfusion. Carbon microspheres, Helium versus oxygen exposure of the perfusate had no effect on the intrarenal distribution of labeled microspheres. The smaller (15 CL)a5Sr-labeled microspheres tended to wash out of the perfused kidneys and prevented in vitro versus in viva perfusion flow comparisons in the same kidney. The normal corticomedullary ratio
135
136
SCHOONEES
ET AL.
TABLE 1 ISOTOPE STUDIES DURING ISOLATEII BLOODLESS PERFUSION %&b&d microspheres (35f 5~):Corticomedullary ratios
l=Xe clearance:Renal Masssuppliedby component I
203Hg chlormerodrin corticomedullary ratios
Exposed to helium Number animals Mean =k 1 SD
studied
9 42 f
Exposed to oxygen Number animals studied Mean f 1 SD p Value
11 22 f
6
5 4.68 f
149&
1.67
3.62 f
l.G5
5 3.59 f 2.62
5 5.79 f
18%
Not significant
Not significant
0.01
1.78
of microsphere concentration in the in vivo baboon kidney was 46.4. Perfused kidneys had a 4.68 f 1.67 (mean f 1 SD) ratio when exposed to helium and a 5.79 i 1.65 (mean f 1 SD) ratio with oxygen exposure (Table 1). DISCUSSION These studies of isolated perfused kidneys were done during evaluation of a system for preservation of kidneys intended for allotransplantation. By isolated perfusion techniques, harvested donor kidneys could be evaluated, then stored if necessary and reevaluated prior to implantation in a recipient. A successful perfusion evaluation system would eliminate faulty or damaged kidneys from the preservation pool and allow for functional evaluation of preserved kidneys prior to implantation. Radioisotopic methods of evaluation are simple and atraumatic. The results of these studies indicate that the in vitro kidney performs better when oxyIO
10 8” 7t
2Dml/gm/min .I\ \ I \.
6t
9 7 6
b
1.5ml/gm/min
‘\
5-
5‘\
I
4-
“* ‘\
3-
‘\
4-
‘\
I
‘\
‘.
‘\
‘.
3. 2-
2-
P I
‘0 IO 9-
3
5
7
9
II
13
IL
loo
;6-
: 7t 6
5-
5t
3
5
7
‘.\
*-..
*. --.. . *. ‘. ‘.
Legend
1
‘0
TIME
or oxygen.
More
rapid
I3
-.
4
FIG. 1. The equilibration
II
O.Jml/gm/min
4.
T\
helium
9
of I311 hippuran equilibration
---
oxygen Helium
1---d3 5
7
Perfusions Perfusions LA 9
II
I3
IN MINUTES
in isolated bloodless renal perfusion and run-off
were noted with
helium
with
exposure.
RAI)IOISOTOPE
STUDIES
IN BABOON
KII)NEYS
gen is excluded from the perfusion system. Oxygen exposure mainly affects the distribut.iorr of the perfusate resulting in reduced cortical flow. No difference was found to exist in the corticomedullary concentration ratios of zo3Hg chlormerodrin in helium- and oxygenexposed kidneys. Xenon-133 studies, however, showed a reduction in cortical perfusion with oxygen as compared with helium equilibration. The i3iI hippuran renogram reflected more rapid clearance of hippuran in helium-exposed than in oxygen-exposed kidneys. The distribution of labeled microspheres did not reflect the decrease in cortical perfusion flow during oxygen exposure. This may represent particle distribution as au ent)ity which differs from fluid distribution in the perfused kidney. SUMMARY Radioisotopic studies were performed on baboon kidneys during isolated bloodless perfusion. The perfusion was equilibrated with either 100% oxygen or 100~0 helium gas. The clearance of 13aXe was found to be a very sensitive method in assessing renal circulation. Oxygen had a vasoconstrictive effect on the cortical vessels resulting in a decreased renal cortical perfusion flow. The distribution of labeled carbon microspheres did not reflect a difference in cortical flow during oxygen and helium exposure. Particles of the size employed did not remain fixed in the vascular tree. In some instances free or unbound isotope was detected. zoaHg chlormerodrin was poorly concentrated both during oxygen and helium exposure, presumably as a result of the absence of plasma proteins in the perfusate. Helium-exposed kidneys showed a more normal distribution and excretion of @lI hippuran than oxygen-exposed kidneys. REFERENCES 1. Carriere, S., Thorburn, G. D., O’Morchoe, C. C., and Berger, A. C. Intrarenal distribution of blood flow in dogs during hemorrhagic hypotension. Circ. Res., 19: 167-179, 1966. 2. Corm, H. L., Jr. Equilibrium distribution of radioxenon in tissue: Xenon-hemoglobin association curve. J. Appl. Physiol., 16: 1065-1070,1961. 3. Groenewald, J. H., van Zyl, J. J. W., Weber, H. W., and Murphy, G. P. Comparison of perfusion and non-perfusion of preserved baboon kidneys with and without oxygen. Trans. Amer. Sot. Artif. Inter. Organs, 15: 219-223, 1969. 4. Ward, M. J., Van Zyl, J. A., van Zyl, J. J. W., Retief, C. P., and Murphy, G. P. Functional characterization of isolated bloodless perfusion in baboon kidneys with oxygen and helium gases. J. Surg. Res., 9: 173-182, 1969.
137
8,
134-137 (1971)
RADIOISOTOPE
STUDIES IN ISOLATED PERFUSED BABOON KIDNEYS’
R. SCHOONEES,za 3 G. R. JOHNSTON,4 J. H. GI~OENEWALD,2z HEEItDEN,3 AND G. P. MURPHY2
3 P. D. 1~. VAN
Baboon kidneys were studied during isolated bloodless perfusion as part of an experimental renal transplantation project. These studies were conducted upon freshly excised as well as preserved kidneys. Data was obtained on the basic function and preservability of baboon kidneys. This paper considers the use of various radioactive isotopic methods in the characterization of these kidneys. MATERIALS
AND
METHODS
Kidneys from both male and female adult Cape Chacma baboons (Papio ursinus) were used. The animals were sedated with phencyclidine hydrochloride (Sernylan, Parke, Davis Co., Detroit, Mich.) 1 mg/kg, atropine 0.6 mg, and Valium (Roche, Basle, Switzerland) 10 mg before operation. The kidneys were removed under sterile conditions and then gently irrigated with chilled, heparinized normal saline (2000 U of heparin to each 150 ml of normal saline) until the venous effluent was clear. At least 150 ml was employed in each instance. On an average 6 min elapsed from the time of removal of the kidney to the institution of successful isolated perfusion. The perfusion apparatus used has been described previously (4). It incorporates a warming coil, a water bath at 37”C, and a roller-type pump to produce a pulsating flow. The flow rate was maintained at 2 ml/ min/g of kidney, but in some experiments the rate was varied in order to study the effect on renal function. The perfusion solution employed was Travert 5% replacement solution (Travenol Inc., Morton Grove, Ill.) with 50 ml of 6% clinical dextran added to each liter. The perfusate was equilibrated with either 100 y0 oxygen or helium gas. The perfusion system was maintained at’ its capacity of 1000 ml throughout the 3-hr duration of the experiment. Measurements were made of the in-flow line pressure (mmHg), pOZ, pCOZ, pH, and urine output (ml/min and ml/min/g kidney). Samples were obtained of urine and of in-flow and out-flow perfusion fluid for the determination of osmolar clearance, free water excretion, effective renal cortical perfusion, percentage sodium reabsorption, PAH extraction, creatinine clearance, and radioactivity. These results have been described elsewhere (4). Mercury-203 chlormerodrin. Thirty kidneys were studied with *03Hg chlormerodrin, 3kCi added to the in-flow (arterial) perfusate. In 14 instances, 5 ml of plasma was mixed with the 3 PCi of *03Hg chlormerodrin prior to injection. Timed samples were collected from the in-flow, out-flow, and ureteral lines over a 50-min period. Tissue samples were obtained from each kidney at the end of the experiment for measurement of radioactivity of the cortex and medulla. The radioactivity of all samples was expressed as counts per minute per milliliter of fluid, or per gram of tissue. The additional effect of a 30-min ureteral occlusion was studied in 10 instances. Xenon-l%. The perfusion flow in 20 baboon kidneys was studied with iasXe. Following the perfusion period, a bolus of 200-400 &i of lasXe dissolved in saline was injected into the in-flow line while the venous line was diverted to prevent recirculation of the radioactive gas. The clearance rate of the 133Xe was monitored externally by a scintillation de1 Presented at the Fifth Cryopreservation Conference convened Meeting of the Society for Cryobiology, Buffalo, New York, August in part by the Office of Naval Research Contract ONR 3700. 2 Roswell Park Memorial Institute, Buffalo, New York 14203. 3 University of Stellenbosch Medical School, Bellville, South Africa.
4 Walter Reed Hospital,
Washington, D.C., 20012. 134
during the Annual 3-7, 1969. Supported
I:A1)IOISOTOPE
STUDIES
IN BABOON
KII)NEYS
tector probe plared 6 in. above the kidney. The curve obtained by direct writer was then plotted against t,irne on semilogarithmic paper and analyzed in its various coml)onents in thr standard manner (1, 2). Iodine-131 hippuran. Ten rnicrocuries of 1311hippuran was injected into the ill-flow line of 12 kidneys. L\ scintillation detector probe with direct writer print-out was positioned above the kidney at a distance determined by the isotope dose. A hippuran renogram was then obtained. The perfusion flow rate varied from 0.5 t’o 2.0 ml/min/g of kidney. .~1 renogram was obtained on a living baboon for comparison. One additional kidney was l)erfused with pl:tsma instead of the usual perfusate. Cc&on microspheres. StrontiumWabeled microspheres (15 + 5 p) and scandium46labeled mirrosl)heres (35 f 5 p) were used to determine renal blood flow distribution in intact baboons versus perfusion flow distribution in isolated kidneys. Fourteen baboons and 22 vervet monkeys received microspheres containing 20 &‘i of radioactivity by injection into either the left ventricle or descending aorta (above the renal arteries). Tht> kidney:: of these animals were then excised and assayed for radioactivity. Other kidneys were perfused for 3 hr and at the end of this period microspheres containing a similar amount of radioactivity were introduced into the in-flow line. Some of these kidneys had been in storage for 24 hr prior to perfusion. These kidneys were similarly assayed for rndinactivi@.
RESULTS Jfercury-2020J chlormerodrin. All kidneys perfused with ahelium-equilibrated perfusat,e showed rapid excretion of m3Hg chlormerodrin. Ureteral occlusion delayed *03Hg chlormerodrin removal from the perfusate until the occlusion was released. Addition of protein (plasma) to the z03Hg chlormerodrin prior to injection resulted in reduction in clearance of radioactivity from the perfusate. Oxygen equilibration of the perfusate did not alter these findings (Table 1). Renal tissue concentration of z03Hg was predominantly cortical in every experiment with no differential between outer and inner cortex. The presence of protein in the perfusate tended to reduce the concentration of *03Hg in the renal tissue in helium-exposed kidneys while enhancing it in oxygen-exposed kidneys. A reduction of flow rate u-as accompanied by a decrease in tissue radioactivity. xenon-1%. Flow rates through the cortex, outer medulla, and inner medulla were calculated from the curves obtained following injection of 133Xeinto the in-flow line of perfused kidneys. A fourth component was not observed due to the absence of perirenal fat. from the perfused specimens. In the nine kidneys exposed to helium 42 f 14% (mean f 1 SD) of the renal mass was supplied by component I of the curve (cortical flow). The 11 kidneys exposed to oxygen had 22 f 18% (mean f 1 SD) of the renal mass supplied b) component I. These findings would indicate that oxygen exposure of the perfusate reduces cortical perfusion to a level approaching that of the outer medulla. This redistribution of intrarenal perfusion flow is presumably related to vasoconstriction of the cortical vessels. Iodine-131 hippuran. Clearance of 1311hippuran by the perfused kidney was increasingly prolonged as the perfusion rate was reduced. Oxygen-exposed kidneys tend to clear Ia11hippuran slower than helium-exposed kidneys at similar perfusion rates (Fig. 1). At a perfusion rate of 2.0 ml/min/g a sharper isotopic peak is noted with helium than with oxygen exposure. At 1.5 ml/min/g this is not the case (Fig. 1). *it lower perfusion rates the average values for helium-exposed kidneys generally manifest higher peaks than the oxygell-exposed kidneys. This is a result of better diffusion of the isotopic tag and may be attributed to differences in vascular perfusion. Carbon microspheres, Helium versus oxygen exposure of the perfusate had no effect on the intrarenal distribution of labeled microspheres. The smaller (15 CL)a5Sr-labeled microspheres tended to wash out of the perfused kidneys and prevented in vitro versus in viva perfusion flow comparisons in the same kidney. The normal corticomedullary ratio
135
136
SCHOONEES
ET AL.
TABLE 1 ISOTOPE STUDIES DURING ISOLATEII BLOODLESS PERFUSION %&b&d microspheres (35f 5~):Corticomedullary ratios
l=Xe clearance:Renal Masssuppliedby component I
203Hg chlormerodrin corticomedullary ratios
Exposed to helium Number animals Mean =k 1 SD
studied
9 42 f
Exposed to oxygen Number animals studied Mean f 1 SD p Value
11 22 f
6
5 4.68 f
149&
1.67
3.62 f
l.G5
5 3.59 f 2.62
5 5.79 f
18%
Not significant
Not significant
0.01
1.78
of microsphere concentration in the in vivo baboon kidney was 46.4. Perfused kidneys had a 4.68 f 1.67 (mean f 1 SD) ratio when exposed to helium and a 5.79 i 1.65 (mean f 1 SD) ratio with oxygen exposure (Table 1). DISCUSSION These studies of isolated perfused kidneys were done during evaluation of a system for preservation of kidneys intended for allotransplantation. By isolated perfusion techniques, harvested donor kidneys could be evaluated, then stored if necessary and reevaluated prior to implantation in a recipient. A successful perfusion evaluation system would eliminate faulty or damaged kidneys from the preservation pool and allow for functional evaluation of preserved kidneys prior to implantation. Radioisotopic methods of evaluation are simple and atraumatic. The results of these studies indicate that the in vitro kidney performs better when oxyIO
10 8” 7t
2Dml/gm/min .I\ \ I \.
6t
9 7 6
b
1.5ml/gm/min
‘\
5-
5‘\
I
4-
“* ‘\
3-
‘\
4-
‘\
I
‘\
‘.
‘\
‘.
3. 2-
2-
P I
‘0 IO 9-
3
5
7
9
II
13
IL
loo
;6-
: 7t 6
5-
5t
3
5
7
‘.\
*-..
*. --.. . *. ‘. ‘.
Legend
1
‘0
TIME
or oxygen.
More
rapid
I3
-.
4
FIG. 1. The equilibration
II
O.Jml/gm/min
4.
T\
helium
9
of I311 hippuran equilibration
---
oxygen Helium
1---d3 5
7
Perfusions Perfusions LA 9
II
I3
IN MINUTES
in isolated bloodless renal perfusion and run-off
were noted with
helium
with
exposure.
RAI)IOISOTOPE
STUDIES
IN BABOON
KII)NEYS
gen is excluded from the perfusion system. Oxygen exposure mainly affects the distribut.iorr of the perfusate resulting in reduced cortical flow. No difference was found to exist in the corticomedullary concentration ratios of zo3Hg chlormerodrin in helium- and oxygenexposed kidneys. Xenon-133 studies, however, showed a reduction in cortical perfusion with oxygen as compared with helium equilibration. The i3iI hippuran renogram reflected more rapid clearance of hippuran in helium-exposed than in oxygen-exposed kidneys. The distribution of labeled microspheres did not reflect the decrease in cortical perfusion flow during oxygen exposure. This may represent particle distribution as au ent)ity which differs from fluid distribution in the perfused kidney. SUMMARY Radioisotopic studies were performed on baboon kidneys during isolated bloodless perfusion. The perfusion was equilibrated with either 100% oxygen or 100~0 helium gas. The clearance of 13aXe was found to be a very sensitive method in assessing renal circulation. Oxygen had a vasoconstrictive effect on the cortical vessels resulting in a decreased renal cortical perfusion flow. The distribution of labeled carbon microspheres did not reflect a difference in cortical flow during oxygen and helium exposure. Particles of the size employed did not remain fixed in the vascular tree. In some instances free or unbound isotope was detected. zoaHg chlormerodrin was poorly concentrated both during oxygen and helium exposure, presumably as a result of the absence of plasma proteins in the perfusate. Helium-exposed kidneys showed a more normal distribution and excretion of @lI hippuran than oxygen-exposed kidneys. REFERENCES 1. Carriere, S., Thorburn, G. D., O’Morchoe, C. C., and Berger, A. C. Intrarenal distribution of blood flow in dogs during hemorrhagic hypotension. Circ. Res., 19: 167-179, 1966. 2. Corm, H. L., Jr. Equilibrium distribution of radioxenon in tissue: Xenon-hemoglobin association curve. J. Appl. Physiol., 16: 1065-1070,1961. 3. Groenewald, J. H., van Zyl, J. J. W., Weber, H. W., and Murphy, G. P. Comparison of perfusion and non-perfusion of preserved baboon kidneys with and without oxygen. Trans. Amer. Sot. Artif. Inter. Organs, 15: 219-223, 1969. 4. Ward, M. J., Van Zyl, J. A., van Zyl, J. J. W., Retief, C. P., and Murphy, G. P. Functional characterization of isolated bloodless perfusion in baboon kidneys with oxygen and helium gases. J. Surg. Res., 9: 173-182, 1969.
137