Contains reports on one research project.National Institutes of Health (Grant 2 TO1 GM01555-06

Conrad, Chester H.

Mark, R. G.

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X. COMMUNICATIONS BIOPHYSICSProf.Prof.Prof.Prof.Prof.Prof.Prof.Prof.Prof.Prof.Academic and Research StaffProf. T. F. Weisst, ttProf. T. R. WillemainDr. J. S. BarlowjfDr. H. Duifhuis***N. I. DurlachDr. R. D. HallDr. A. J. M. IloutsmaDr. N. Y. S. KiangtDr. L. U. E. KbhlloffelltttDr. E. C. MoxontDr. M. J. Mulroy?. D. Braida. K. Burns[. S. Colburnt,. S. Frishkopf. L. GoldsteintJ. Guinan, JrL. G. Mark**i. M. OmanV. T. PeakeTi. M. SiebertO. Bayer, Jr.E. BerlinerL. BonnR. BourkII. ConradM. CroninIH. DomnitzE. GorelickIlasanL. flicksD. W. AltmanntD. J. CallahanA. HI. CristtB. GaimanP. W. Herman, Jr.W. F. KelleyElizabeth M. MarrtJ. M. Onorato, Jr.tL. H. SeifelJ. B. Walters, Jr.M. RabinowitzL. RhyneA. ScudderT. ShepardSingerL. SmithM. Stern, Jr.L. SulmanTomTungA. NORMOTHERMIC PERFUSION OF CANINE KIDNEYS- THE EFFECT OF PERFUSATESNational Institutes of Health (Grant 2 TO1 GM01555-06)C. H. Conrad, R. G. Mark[With B. P. Kekis, C. D. Derry, and NI. Slapak, Sears Surgical Laboratory,Boston City Hospital.]tAlso at the Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary,Cambridge, Massachusetts.tOn leave - Department of Biophysics, University College, London.Also Instructor in Medicine, Harvard Medical School, Boston, Massachusetts.ttAlso Instructor in Preventive and Social Medicine, Harvard Medical School,Boston, Massachusetts.TResearch Affiliate in Communication Sciences from the Neurophysiological Labo-ratory of the Neurology Service of the Massachusetts General Hospital, Boston, Massa-chusetts.Visiting Scientist from Institute for Perception Research, Eindhoven, TheNetherlands.Visiting Scientist from University of Stuttgart, Germany.ITDepartment of Physiology, Harvard University, Cambridge, Massachusetts.QPR No. 109Graduate StudentsP. HochfeldD. II. JohnsonM. S. KeshnerM. C. LibermanjTLynette L. LindenR. P. LippmannN. D. MegillS. L. MoshierJ. A. MyersV. NedzelnitskyS. R. Purks179(X. COMMUNICATIONS BIOPHYSICS)1. IntroductionAlthough hypothermic perfusion is now used as the basis of all long-term methods oforgan preservation, a period of seven days is the longest for which preservation hasbeen successfully achieved.I By using intermediate living hosts, perfusion at 37°C withwhole blood has allowed kidney preservation for periods of more than three weeks.2 Atpresent, however, limitations imposed by the materials used for artificial preservationsystems prohibit the perfusion of organs with whole blood for periods exceeding morethan a few days, because of phenomena such as cell aggregation and hemolysis.In a recent study 3 it was shown that a 25°C 24-hour kidney perfusion could be suc-cessfully achieved with life supporting function after transplantation by the use of eitherplatelet-poor or platelet-rich plasma. The advantages of plasma as a perfusate in anormothermic system are obvious. It contains many of the substrates required by thekidney and its proteins confer appropriate buffering and osmotic properties. The useof plasma also eliminates problems of hemolysis and sludging. Although protein dena-turation might still be damaging, the use of a membrane oxygenator and the minimiza-tion of the air-plasma interface area might minimize this problem. Inherent in the useof plasma as a perfusate at 37CC, however, is the question of whether it is capable ofcarrying enough oxygen to support a normothermic kidney. Theoretical considerationof the oxygen consumption and blood flow of normal kidneys in vivo would suggest thatit is. At the normal flow rate of 3-4 ml/gram of kidney tissue/minute, a very small arte-riovenous oxygen difference is found,4 of the order of 2 ml/100 ml as compared with4 ml/100 ml for systemic venous blood. Since it has been shown 5 that renal oxygenconsumption during normal sodium filtration is of the order of 0. 08 ml/gm/min, the2 ml/100 ml of oxygen dissolved in oxygen-saturated plasma might be enough to sup-port the kidney, provided that sufficiently high flows could be maintained. Thus, byusing a 97% oxygen 3% CO 2 mixture and flow rates of 3-4 ml/gm/min, an adequate sup-ply of oxygen should theoretically be available.The purpose of the present study, then, was to ascertain whether or not a normo-thermic kidney could be supported by using plasma as the perfusate. The methods andresults have been described in detail elsewhere. 72. MethodThe experiment involved perfusion of kidneys from 19 apparently healthy 10-20 kg mongrel dogs. Three groups of 3-4 perfusions were done. Group A (9 kid-neys) was perfused with whole blood, Group B (4 kidneys) with low hematocritblood (4-7%), Group C (6 kidneys) with plasma. Anesthesia was induced withsodium thiamylal at an approximate dosage of 20 mg/kg, and additional thiam-ylal was given intravenously as needed. After intubation, a midline incision wasQPR No. 109 180(X. COMMUNICATIONS BIOPHYSICS)made, and the left kidney was dissected free after infiltration of the surrounding tissuewith 5% Cyclaine (topical hexylcaine hydrochloride, 50 mg/ml). The artery, vein andureter were freed and the ureter divided and cannulated with polyethylene tubing.The dog was maintained in a diuretic state with a fluid load of 5% dextrose in normalsaline or 25% mannitol in 5% dextrose in normal saline. The renal artery and vein werethen clamped and divided and both cannulated with siliconized glass cannulae. The kid-ney was then weighed and transferred to the perfusion apparatus while the donor animalwas maintained under light thiamylal anesthesia until reimplantation.The perfusion circuit is shown schematically in Fig. X-1. It included a pulsatileRECORDERTHERMOMETERPRESSURETRANSDUCERBUBBLE 96%O0TRAP 24% CO2H2038' 40o CURINERESERVOIR(VENT)H2 038' CMEMBRANEOXYGENATORPUMPPUMPDRIVERRESERVOIR /HEAT EXCHANGERFig. X-1. The perfusion system. The silastic pump is driven by a pneumaticoscillator which has been described previously. 8QPR No. 109 181(X. COMMUNICATIONS BIOPHYSICS)pump, membrane oxygenator (silicone rubber), bubble trap, organ chamber, and venousreservoir/heat exchanger with an incorporated filter. The pump was a silicone rubberventricle with one-way valves, driven by a pneumatic oscillator with adjustable systolicand diastolic intervals and pressures similar to the one described by Slapak et al.8The oxygenator was a Travenol 5M0321 membrane oxygenator ventilated by 97% oxygenwith 3% CO 2 or 96% oxygen with 4% CO 2 . There was a bubble trap in the arterial line,and a graduated tube in the venous line which made possible direct measurement of flowby occlusion of the venous line between the graduated tube and the reservoir. The venousreturn was filtered by a blood administration set filter as it entered the reservoir. Theorgan rested in a glass organ chamber covered by a moist sponge and supported by asilicone rubber coil through which water at 38°C was circulated. Urine was collectedin a graduated reservoir and could be returned to the venous line if desired. The venousreservoir contained a glass coil through which 38 0 C water was circulated, to serve asa heat exchanger. The priming volume of the apparatus was approximately 200 ml.The arterial perfusion pressure and organ surface temperature were recorded con-tinuously. Flow, arterial and venous pH, PCO 2 and P0O2 were monitored periodically,as were hematocrit, plasma glucose, and urine flow. Samples of perfusate and urine werealso taken periodically for determination of osmolarity, sodium, potassium and creati-nine. After termination of perfusion, the kidney was flushed with cold lactated Ringer' ssolution and reweighed. It was then reimplanted by end-to-end anastomosis of the renalartery to the external iliac artery and end-to-side anastomosis of the renal vein to theexternal iliac vein. Total ischemic times were from 25 to 35 minutes. The ureter wasimplanted directly in the bladder and contralateral nephrectomy was performed imme-diately following the implantation. Postoperatively, blood samples were drawn every fewdays for measurement of BUN, serum creatinine, sodium, potassium, and osmolarity.Since in all groups venous PO was above 150 mm Hg, a value at which hemoglobinremains fully saturated, oxygen consumption was calculated from the dissolved oxygenalone by using the formulaAO2 (ml/100 ml)0 CONSUMPTION (ml/gm/min) = FLOW (ml/gm/min) X 100where FLOW is the perfusate flow, and AO2A V is the arteriovenous oxygen difference,given by(PO artPO 2ven)AO2A V (ml/100 ml)= 760 X a (ml/100 ml/atm)6where a is the solubility of oxygen in plasma as given by Sendroy et al. The solubilitywas corrected for temperature and for hematocrit, since acells is greater than aplasma.QPR No. 109 182Table X-1. Viability after perfusion, and oxygen consumption, creatinine clearance,and sodium reabsorption during perfusion of the perfused kidneys. Note inGroups A,planted orB, and C that 6, 2, and 2 kidneys, respectively, were not reim-function was not evaluated because of unrelated technical failure.FunctionafterReim -plantationOxygenConsumption(ml/gm/min)CreatinineClearance(ml/100 gm/min)I I.024SodiumReabsorption( LEq/gm/min)6711 - .0206890 U .0356924 U .038 2.7 3.76946 U .021 3. 6 4.76760 U .041MEAN ± S. D. (n) .030 ±. 009(6) 3. 1 ±. 7(2) 4. 2 ±. 7(2)6977 - .039 20. 2 29. 0LOW 6206 . 049 44. 3 63. 5HE MATOCRIT6978 S .043 17. 1 24.4(Group B) 6990 U .041MIEAN S. D. (n) .041 i. 008(4) 27. 2 ±(3) 39. 0 ±21.4(3)6856 - .0506860 - .0426875 - .0386872 - .039WHOLEBLOOD 6903 - . 028(Group A) 6925 - . 037 19. 0 28. 36885 S .025 5. 5 7. 96945 S .040 16. 8 25. 06966 S .034 12.3 17.3MIEAN S. D. (n) .037 ±. 007(9) 13.4 ±6. 0(4) 19. 6 ±9. 1(4)Notes: - No reimplant, or function not evaluated.S SurvivalU Failure (uremic).QPR No. 109DogNo.6873PLAS MA(Group C)183(X. COMMUNICATIONS BIOPHYSICS)2. ResultsResults are summarized in Table X-1.a. ViabilityNone of theFuthermore, at4 kidneys perfused by plasma were functional after reimplantation.reimplantation, these kidneys were dark and sometimes mottled. TheyS6760X6946--- x ---- x- PLASMA6924---- LOW HEMATOCRITx - -- WHOLE BLOOD6945990 6978- 696610 20 30DAYS POSTOPERATIVEFig. X-2.Postoperative renal function after reim-plantation for dog numbers indicated,measured by blood urea nitrogen values.Serum creatinine values not shown weresimilar in their behavior.produced little, if any, urine. In contrast, all of the three kidneys perfused by wholeblood and reimplanted regained normal color and were immediately life-sustaining; theanimals were sacrificed some months later with normal renal function, as shown inFig. X-2. One of the two kidneys perfused with low hematocrit blood was life-sustaining.b. Oxygen Consumption and Other ResultsThere were no statistically significant differences in the oxygen consumption of thethree groups. Figure X-3 shows the relationship between Na reabsorption and 02consumption in the three groups. There was, however, a statistically significantdifference (P<. 05) in favor of the whole blood and low hematocrit groups for sodiumreabsorption and creatinine clearance, although all groups showed impaired filtration.It was found in all three groups that renal venous PO 2 during perfusion was greater than150 mm Hg, arterial PO 2 averaging approximately 560 mm Hg.3. DiscussionThe data indicate that inadequate oxygen supply or consumption does not seem toprovide a convincing explanation of the striking difference in the viability between theplasma- and whole blood-perfused kidneys, although there is a possibility that failure ofQPR No. 109 184(X. COMMUNICATIONS BIOPHYSICS)0.140.12 -0.10 -Z (9)0.08 -(5) x PLASMAzO 0.06 6978 o LOW HEMATOCRIT0 - 69780( 6924 6945 o 6206 * WHOLE BLOOD,69770.04 6925x 6925696668850.02 694650 100 150Na REABSORPTION (pEq/gm/min)Fig. X-3. Oxygen consumption and sodium reabsorption of perfused kidneys.Note that.although oxygen consumption is not significantly differentin the three groups, Na reabsorption values in the plasma group(shown by X's) are much lower. The straight lines indicate therelations obtained by other researchers.5, 9the plasma-perfused kidneys is related to oxygen supply or consumption. Renal corticalPO 2 is generally10 below renal venous PO 2 and localized areas of hypoxia might existeven with a high venous PO 2 . The presence of intrarenal "shunt diffusion" has beenproposed to explain this effect, although limited oxygen diffusion at the capillary levelwould produce the same effect. These effects would allow oxygen to bypass respiringtissue, and make the organ incapable of extracting all seemingly available oxygen. Ashunt diffusion effect would be most significant with high arterial PO 2 because of thelarge gradient for diffusion that is thus created.Thus, since the venous PO 2 was 150 mm Hg in all groups, the possibilities are(i) there was more than adequate oxygen; (ii) the oxygen could not reach the renal paren-chyma because of a shunting effect; and (iii) oxygen diffusion at the capillary level wasinadequate, or the cells were unable to utilize the available oxygen. Even if the plasma-perfused kidneys in these experiments did have adequate oxygen, the low creatinineclearances leave open the possibility that a normally filtering kidney perfused withplasma would become hypoxic as a result of increased sodium load and the consequentneed for oxygen to provide energy for sodium reabsorption.QPR No. 109 185(X. COMMUNICATIONS BIOPHYSICS)Work done by Gimbrone et al.12 suggests that platelets might play a role in themaintenance of vascular endothelium. In their studies, organs perfused at 370C showeddiminished function and increased endothelial damage in the absence of platelets. Inour experiment, platelets were present in Group A (whole blood) and Group B (lowhematocrit), and the lack of platelets in Group C (plasma) might be responsible for theobserved failure in this group. Although kidneys perfused with platelet-rich and platelet-poor plasma at 25 0 C did not demonstrate advantages of platelet-rich plasma,3 resultsmight differ at 37 0 C. Further experiments including a platelet-rich plasma groupwould be necessary to test this possibility.4. ConclusionThese data show that perfusion with plasma at 37 0C does not produce life-supportingfunction, whereas perfusion with whole or diluted blood can. The cause for the failureof the plasma-perfused group seems to be due to a fact or factors other than oxygen.The absence of platelets is one of the possible factors.References1. J. E. Woods, "Successful Three-to-Seven Day Preservation of Canine Kidneys,"Arch. Surg. (Chicago) 102, 614 (1971).2. A. R. Lavender, M. Forland, J. J. Rams, J. S. Thompson, H. P. Russe, andB. H. Spargo, "Extracorporeal Renal Transplantation in Man," J. Am. Med.Assoc. 203, 265 (1968).3. M. J. Wexler, A. D. Ginsburg, A. Latsena, R. A. Aster, and M. Slapak, "Twenty-four Hour Renal Preservation and Perfusion Utilizing Platelet-rich Plasma," Ann.Surg. 174, 811 (1971).4. L. G. Wesson, Jr., Physiology of the Human Kidney (Grune and Stratton, New York,1969).5. N. A. Lassen, O. Munck, and J. H. Thaysen, "Oxygen Consumption and SodiumReabsorption in the Kidney," Acta Physiol. Scand. 51, 371 (1961).6. J. Sendroy, Jr. . R. T. Dillon, and D. D. van Slyke, "Studies of Gas and Elec-trolyte Equilibria in Blood. XIX. Solubility and Physical State of Uncombined Oxy-gen in Blood," J. Biol. Chem. 105, 597 (1934).7. C. IH. Conrad, "A Study of the Oxygen Consumption of Isolated Perfused Normo-thermic Kidneys," S. M. Thesis, Department of Electrical Engineering, M. I. T.,1972.8. M. Slapak, R. A. Wigmore, and R. Demers, et al., "Preservation Using a SimplePortable Apparatus Suitable for Controlled Hyperbarbic Conditions," in J. C.Norman (Ed.), Organ Perfusion and Preservation (Appleton-Century-Crofts,New York, 1968), Chap. 25, pp. 317-335.9. P. Deetjen and K. Kramer, "Die Abhangigkeit des 02-Verbrauchs der Niere vonder Na-Ruckresorption," Pflueger Arch. Ges. Physiol. 273, 636 (1961).10. IH. P. Leichtweiss, D. W. Libbers, and C. Weiss, et al., "The Oxygen Supplyof the Rat Kidney. Measurements of Intrarenal PO2," Pflueger Arch. Ges.Physiol. 309, 328 (1969).QPR No. 109 186(X. COMMUNICATIONS BIOPHYSICS)11. M. N. Levy and G. Sauceda, "Diffusion of Oxygen from Arterial to Venous Seg-ments of Renal Capillaries," Amer. J. Physiol. 196, 1336 (1959).12. MI. A. Gimbrone, Jr., R. H. Aster, and R. S. Cotran, et al., "Preservation ofVascular Integrity in Organs Perfused in vitro with a Platelet-rich Medium,"Nature 222, 33 (1969).QPR No. 109 187

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