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Dimethyl sulfoxide distribution in rabbit kidneys and function following hypertonic washout
ABSTRACTS,
17th ANNUAL
on a simple ex viva shunt circuit consisting of silastic tubing with Teflon vessel tips connecting the perfusor aorta and vena cava to the vessels of the preserved kidney. Side arms on the shunt provide a mechanism for monitoring pressure and flow. Three groups of kidneys were studied; (a) normal in situ controls, (b) immediate plug-in controls, and (c) preserved kidneys following 24-hr machine perfusion at 7°C. Endogenous creatinine clearance was measured on urine collected from 30 to 90 min after connection to the circuit. The mean creatinine clearance values for Groups 1 to 3, respectively, were 316 SD 138 mUhr, 291 SD 145 ml/hr, and 140 SD 45 mVhr. Renal blood flows ranged between 3.92 and 5.26 ml/g kidney weight. These are within the normal physiologic range for this species. The blood flow and function of unpreserved kidneys on this perfusion circuit were indistinguishable from the in situ controls. The model proved to be at least as sensitive as the standard transplant model for the detection of preservational renal injury as evidenced by a significantly lower level of creatinine clearance in the perfused compared with the control kidneys (P < 0.05).
603
MEETING
phrectomized animals. There were five to seven animals in each group. The controls, group 1, as well as the others received 100 ml/kg Ringer’s lactate over the course of the experiment. Groups 2-4 received mannitol 1 g/kg 15 min prior to ischemia. Group 3 received allopurinol 100 mg/kg and group 4 allopurinol 100 mg/kg plus inosine 100 mg/kg by iv infusion 1 hr before pedicle clamping. The maximum serum creatinine levels and TAN levels are shown in the table. Renal function was significantly improved by the use of mannitol alone without any improvement in AN regeneration. Allopurinol and inosine administration did not enhance TAN levels at any time, and, when used in combination with mannitol, abolished the beneficial effects of the latter. 33. Dimethyl Sulfoxide Distribution in Rabbit Kidneys and Function Following Hypertonic Washout. N. B. SEGAL, H. GALLEY, AND F. M. GUTTMAN (Department of Surgery, Lady Davis Institute, Sir Mortimer B. Davis Jewish General Hospital, Montreal, Quebec).
An in vitro perfusion system has been described to allow assessment of rabbit kidney function after 32. Adenine Nucleotide Levels and Recovery oj cryobiological manipulation. Rabbit kidneys were Function After Renal Ischemic Injury. GEOFperfused at 10°C (P = 60 mm Hg) with a high K+, low FREY M. COLLINS, RICHARD D. GREEN, AND Na+ medium containing 14C inulin and [3H]Me,S0. NICHOLAS A. HALASZ (Veterans AdministraMe,SO concentration was increased gradually to 3.OM tion Medical Center, and University of Califorover 45 min. Kidneys were then perfused with 3.0 M nia School of Medicine, San Diego, California). Me,SO for an additional 15, 30, or 45 min. Total Marked change in adenine nucleotide (AN) levels is H,O/Me,SO space, Me,SO space, and inulin space in a conspicuous feature of renal ischemic injury and subcortical + medullary sections were calculated. Comsequent recovery. However, it remains uncertain as to plete intracellular Me,SO equilibration was achieved whether these changes are primary or secondary in the in both cortical and medullary sections 30 min after pathophysiology of organ damage. To help resolve this reaching 3.0 M concentration. Inulin space was increased significantly when Me,SO perfusion was carquestion, we have undertaken experiments on ischemically injured rabbit kidneys to determine (a) ried out (mean rise of 19%). Kidneys were perfused whether pharmacologic protection from ischemia is with 3.0 M Me,SO added gradually at two different dependent upon enhanced regeneration of adenine nurates equilibrated for 30 min and removed by subcleotide and (b) whether the administration of agents sequent perfusion with Me,SO-free medium made reported to enhance AN regeneration would result in hypertonic with mannitol: (A) Me,SO added over 45 improved renal function. Rabbit kidneys were exposed minutes, stepwise washout with 600, 500, 400 to 1 hr in situ normothermic ischemia. TAN (AMP + mOsm/kg media, 20 min at each step; (B) Me,SO ADP + ATP) measurements were made on cortical added as in (A), stepwise washout with 800, 700, 600, biopsies obtained before and after the ischemic period 500, 400 mOsm/kg media, 15 min at each step; (C) as well as 1 hr after restoration of blood flow. Function Me,SO added over 67 min, same washout as (A). Kidwas evaluated in parallel series of unilaterally neney function was assayed at 37°C for 1 hr. Kidneys ABSTRACT Max. Group 1 2 3 4 t Mean + SD.
Serum Creatinine Cm%) 8.9 3.8 7.9 9.9
k zt + +
3.9t 1.9 3.8 4.7
32-TABLE
Before W.I. 17.7 20.3 16.0 18.0
2 2 r +
3.2 4.2 1.6 0.9
1 TAN mM/kg dry wt After W.I. I-hr recovery 5.7 6.1 5.9 4.2
-+ 1.3 t 0.8 t 1.8 r+ 1.6
8.3 9.3 8.9 8.7
r+_3.1 k 1.2 + 2.7 t 2.1
604
ABSTRACTS,
17th ANNUAL
MEETING
ABSTRACT 33-TABLE
1
Control
Me,SO
Cortex
Medulla
Cortex
Medulla
Total H,O/ Me,SO space Me,SO space
83.17 + 0.186 -
83.07 + 1.30 -
Inulin space
36.09 k 2.03
32.60 5 2.46
79.90 2 0.62 78.23 + 1.370 65.45 f 3.40
78.86 k 0.90 79.05 + 2.01 59.92 k 2.95
were able to function after equilibration with 3.0 M Me,SO followed by hypertonic washout. Increased osmolarity (800 mOsm/kg) has some deleterious effects; gradual addition of Me,SO at different rates did not affect function. 34. Problems of In Vivo Heterotopic Rabbit Kidney implants. J. BORZONE, N. B. SEGAL, AND
F. M. GUTTMAN (Department of Surgery, Lady Davis Institute, Sir Mortimer B. Davis, Jewish General Hospital, Montreal, Quebec, Canada). The use of rabbit kidneys implanted through the artifice of cannulation of the aorta and vena cava for short term studies of cryobiological manipulation is described. New Zealand white rabbits weighing 2.3-3.4 kg received 200 mg aspirin in 3 gal of drinking water for 1 week. A tracheotomy to reduce dead space was found to be beneficial (Collins) and a CVP catheter into the right jugular vein. The iv fluid was Ringer’s lactate with 2.5% of BSA at 30 mYkg/hr. Heparin, 100 U/kg wt, was injected. Laparotomy through a long midline incision and placement of the intestines in a plastic bag. A heating lamp was used. Donor kidneys were dissected out after infiltration of the renal pedicle with 2% xylocaine and papavarine. The artery and vein were catheterized with etched (extra-corporeal) vessel tips No. 17-20 and the kidney was immediately flushed with 200 ml cold (4°C) oxygenated media and placed on ice. Mannitol, 1 g/kg, and furosemide,’ 0.2 mg/kg, were given 5-10 min prior to attaching test kidneys. In group 1 the kidney was placed into the recipient animal immediately after flushing: group 2, after 2 hr of cold ischemia; group 3 after 48 hr of cold storage. In the recipient animal the aorta and vena
cava were isolated with meticulous care and cannulated with Teflon vessel tips No. 13-14attached to prepared silastic tubing in T form allowing for BP monitoring on the arterial line and sampling on the venous line. The ends of these catheters were fitted such that the donor kidney could be plugged in. GFR, as measured by exogenous creatinine clearance, Na reabsorption, and glucose reabsorption were measured as parameters of kidney function. There were no significant differences in GFR, Na reabsorption, and glucose reabsorption, between groups 1 and 2, but 48-hr storage resulted in significant reduction of all parameters. Further work to eliminate some of the technical problems in this model remains to be done. 35. Effect of Perfusate Composition on Canine Renal Ultrastructure During Long-Term Preservation.
LEO MENZ, JOHN CODD, MAX JELLINEK, AND PAUL GARVIN (Saint Louis University Medical Center, Department of Surgery, and Cochran Veterans Administration Hospital, Saint Louis, Missouri). We previously demonstrated that redox control of a cryoprecipitated plasma (CPP) perfusate was beneticial to ischemically injured canine kidneys (J. Surg. Res. 22, 585 (1977)). Ultrastructural and biochemical data demonstrated functional integrity when reducing agents, ascorbic acid, glutathione-, and potentiostatic control were included in hypothermic perfusion. We now report on 7-day storage of optimally harvested kidneys perfused under identical conditions for a comparison of three perfusates: (i) CPP; (ii) human plasma protein fraction (PPF), and (iii) canine serum (CS). Ultrastructure was studied in three nephric compo-
ABSTRACT 3ATABLE
1
Group
GFR (ml/mm)
Na reabsorption (So)
Glucose reabsorption (%)
1 2 3
2.74 2 0.67 2.72 + 0.04 1.10 + 0.12
86.90 2 3.21 80.62 -c 0.66 25.05 2 1.91
97.07 2 2.67 94.96 k 0.15 33.08 ” 0.85
17th ANNUAL
on a simple ex viva shunt circuit consisting of silastic tubing with Teflon vessel tips connecting the perfusor aorta and vena cava to the vessels of the preserved kidney. Side arms on the shunt provide a mechanism for monitoring pressure and flow. Three groups of kidneys were studied; (a) normal in situ controls, (b) immediate plug-in controls, and (c) preserved kidneys following 24-hr machine perfusion at 7°C. Endogenous creatinine clearance was measured on urine collected from 30 to 90 min after connection to the circuit. The mean creatinine clearance values for Groups 1 to 3, respectively, were 316 SD 138 mUhr, 291 SD 145 ml/hr, and 140 SD 45 mVhr. Renal blood flows ranged between 3.92 and 5.26 ml/g kidney weight. These are within the normal physiologic range for this species. The blood flow and function of unpreserved kidneys on this perfusion circuit were indistinguishable from the in situ controls. The model proved to be at least as sensitive as the standard transplant model for the detection of preservational renal injury as evidenced by a significantly lower level of creatinine clearance in the perfused compared with the control kidneys (P < 0.05).
603
MEETING
phrectomized animals. There were five to seven animals in each group. The controls, group 1, as well as the others received 100 ml/kg Ringer’s lactate over the course of the experiment. Groups 2-4 received mannitol 1 g/kg 15 min prior to ischemia. Group 3 received allopurinol 100 mg/kg and group 4 allopurinol 100 mg/kg plus inosine 100 mg/kg by iv infusion 1 hr before pedicle clamping. The maximum serum creatinine levels and TAN levels are shown in the table. Renal function was significantly improved by the use of mannitol alone without any improvement in AN regeneration. Allopurinol and inosine administration did not enhance TAN levels at any time, and, when used in combination with mannitol, abolished the beneficial effects of the latter. 33. Dimethyl Sulfoxide Distribution in Rabbit Kidneys and Function Following Hypertonic Washout. N. B. SEGAL, H. GALLEY, AND F. M. GUTTMAN (Department of Surgery, Lady Davis Institute, Sir Mortimer B. Davis Jewish General Hospital, Montreal, Quebec).
An in vitro perfusion system has been described to allow assessment of rabbit kidney function after 32. Adenine Nucleotide Levels and Recovery oj cryobiological manipulation. Rabbit kidneys were Function After Renal Ischemic Injury. GEOFperfused at 10°C (P = 60 mm Hg) with a high K+, low FREY M. COLLINS, RICHARD D. GREEN, AND Na+ medium containing 14C inulin and [3H]Me,S0. NICHOLAS A. HALASZ (Veterans AdministraMe,SO concentration was increased gradually to 3.OM tion Medical Center, and University of Califorover 45 min. Kidneys were then perfused with 3.0 M nia School of Medicine, San Diego, California). Me,SO for an additional 15, 30, or 45 min. Total Marked change in adenine nucleotide (AN) levels is H,O/Me,SO space, Me,SO space, and inulin space in a conspicuous feature of renal ischemic injury and subcortical + medullary sections were calculated. Comsequent recovery. However, it remains uncertain as to plete intracellular Me,SO equilibration was achieved whether these changes are primary or secondary in the in both cortical and medullary sections 30 min after pathophysiology of organ damage. To help resolve this reaching 3.0 M concentration. Inulin space was increased significantly when Me,SO perfusion was carquestion, we have undertaken experiments on ischemically injured rabbit kidneys to determine (a) ried out (mean rise of 19%). Kidneys were perfused whether pharmacologic protection from ischemia is with 3.0 M Me,SO added gradually at two different dependent upon enhanced regeneration of adenine nurates equilibrated for 30 min and removed by subcleotide and (b) whether the administration of agents sequent perfusion with Me,SO-free medium made reported to enhance AN regeneration would result in hypertonic with mannitol: (A) Me,SO added over 45 improved renal function. Rabbit kidneys were exposed minutes, stepwise washout with 600, 500, 400 to 1 hr in situ normothermic ischemia. TAN (AMP + mOsm/kg media, 20 min at each step; (B) Me,SO ADP + ATP) measurements were made on cortical added as in (A), stepwise washout with 800, 700, 600, biopsies obtained before and after the ischemic period 500, 400 mOsm/kg media, 15 min at each step; (C) as well as 1 hr after restoration of blood flow. Function Me,SO added over 67 min, same washout as (A). Kidwas evaluated in parallel series of unilaterally neney function was assayed at 37°C for 1 hr. Kidneys ABSTRACT Max. Group 1 2 3 4 t Mean + SD.
Serum Creatinine Cm%) 8.9 3.8 7.9 9.9
k zt + +
3.9t 1.9 3.8 4.7
32-TABLE
Before W.I. 17.7 20.3 16.0 18.0
2 2 r +
3.2 4.2 1.6 0.9
1 TAN mM/kg dry wt After W.I. I-hr recovery 5.7 6.1 5.9 4.2
-+ 1.3 t 0.8 t 1.8 r+ 1.6
8.3 9.3 8.9 8.7
r+_3.1 k 1.2 + 2.7 t 2.1
604
ABSTRACTS,
17th ANNUAL
MEETING
ABSTRACT 33-TABLE
1
Control
Me,SO
Cortex
Medulla
Cortex
Medulla
Total H,O/ Me,SO space Me,SO space
83.17 + 0.186 -
83.07 + 1.30 -
Inulin space
36.09 k 2.03
32.60 5 2.46
79.90 2 0.62 78.23 + 1.370 65.45 f 3.40
78.86 k 0.90 79.05 + 2.01 59.92 k 2.95
were able to function after equilibration with 3.0 M Me,SO followed by hypertonic washout. Increased osmolarity (800 mOsm/kg) has some deleterious effects; gradual addition of Me,SO at different rates did not affect function. 34. Problems of In Vivo Heterotopic Rabbit Kidney implants. J. BORZONE, N. B. SEGAL, AND
F. M. GUTTMAN (Department of Surgery, Lady Davis Institute, Sir Mortimer B. Davis, Jewish General Hospital, Montreal, Quebec, Canada). The use of rabbit kidneys implanted through the artifice of cannulation of the aorta and vena cava for short term studies of cryobiological manipulation is described. New Zealand white rabbits weighing 2.3-3.4 kg received 200 mg aspirin in 3 gal of drinking water for 1 week. A tracheotomy to reduce dead space was found to be beneficial (Collins) and a CVP catheter into the right jugular vein. The iv fluid was Ringer’s lactate with 2.5% of BSA at 30 mYkg/hr. Heparin, 100 U/kg wt, was injected. Laparotomy through a long midline incision and placement of the intestines in a plastic bag. A heating lamp was used. Donor kidneys were dissected out after infiltration of the renal pedicle with 2% xylocaine and papavarine. The artery and vein were catheterized with etched (extra-corporeal) vessel tips No. 17-20 and the kidney was immediately flushed with 200 ml cold (4°C) oxygenated media and placed on ice. Mannitol, 1 g/kg, and furosemide,’ 0.2 mg/kg, were given 5-10 min prior to attaching test kidneys. In group 1 the kidney was placed into the recipient animal immediately after flushing: group 2, after 2 hr of cold ischemia; group 3 after 48 hr of cold storage. In the recipient animal the aorta and vena
cava were isolated with meticulous care and cannulated with Teflon vessel tips No. 13-14attached to prepared silastic tubing in T form allowing for BP monitoring on the arterial line and sampling on the venous line. The ends of these catheters were fitted such that the donor kidney could be plugged in. GFR, as measured by exogenous creatinine clearance, Na reabsorption, and glucose reabsorption were measured as parameters of kidney function. There were no significant differences in GFR, Na reabsorption, and glucose reabsorption, between groups 1 and 2, but 48-hr storage resulted in significant reduction of all parameters. Further work to eliminate some of the technical problems in this model remains to be done. 35. Effect of Perfusate Composition on Canine Renal Ultrastructure During Long-Term Preservation.
LEO MENZ, JOHN CODD, MAX JELLINEK, AND PAUL GARVIN (Saint Louis University Medical Center, Department of Surgery, and Cochran Veterans Administration Hospital, Saint Louis, Missouri). We previously demonstrated that redox control of a cryoprecipitated plasma (CPP) perfusate was beneticial to ischemically injured canine kidneys (J. Surg. Res. 22, 585 (1977)). Ultrastructural and biochemical data demonstrated functional integrity when reducing agents, ascorbic acid, glutathione-, and potentiostatic control were included in hypothermic perfusion. We now report on 7-day storage of optimally harvested kidneys perfused under identical conditions for a comparison of three perfusates: (i) CPP; (ii) human plasma protein fraction (PPF), and (iii) canine serum (CS). Ultrastructure was studied in three nephric compo-
ABSTRACT 3ATABLE
1
Group
GFR (ml/mm)
Na reabsorption (So)
Glucose reabsorption (%)
1 2 3
2.74 2 0.67 2.72 + 0.04 1.10 + 0.12
86.90 2 3.21 80.62 -c 0.66 25.05 2 1.91
97.07 2 2.67 94.96 k 0.15 33.08 ” 0.85