Kidneys were preserved by continuous pulsatile perfusion and their metabolic rate monitored by O2 consumption. Experiments were carried out to determine if dialyzing the perfusate to supply metabolic substrates would aid in kidney preservation. A control group was compared against two experimental groups at 25°C. Average preservation time before fall off of oxygen consumption differed for each group: control - 8 hours, dialysis with amino acids - 6 hours, dialysis with amino acids and cofactors - 11 hours. Results indicated that, if suitable concentrations of metabolic substrates and cofactors were used, the preserved kidneys maintained higher levels of O2 consumption for longer periods of time

Dall\u27Olmo, Daniel

Dienst, Stanley G.

English

Henry Ford Health System Scholarly Commons

Henry Ford Hospital Medical JournalVolume 24 | Number 3 Article 29-1976Prolonging oxygen consumption duringpreservation of canine kidneys by the addition ofcontinuous dialysisDaniel Dall'OlmoStanley G. DienstFollow this and additional works at: https://scholarlycommons.henryford.com/hfhmedjournalPart of the Life Sciences Commons, Medical Specialties Commons, and the Public HealthCommonsThis Article is brought to you for free and open access by Henry Ford Health System Scholarly Commons. It has been accepted for inclusion in HenryFord Hospital Medical Journal by an authorized editor of Henry Ford Health System Scholarly Commons.Recommended CitationDall'Olmo, Daniel and Dienst, Stanley G. (1976) "Prolonging oxygen consumption during preservation of canine kidneys by theaddition of continuous dialysis," Henry Ford Hospital Medical Journal : Vol. 24 : No. 3 , 125-130.Available at: https://scholarlycommons.henryford.com/hfhmedjournal/vol24/iss3/2Henry Ford Hosp. M e d . Journal Vo l . 24 , No . 3, 1976 Prolonging oxygen consumption during preservation of canine kidneys by the addition of continuous dialysis Danie l D a l l ' O l m o * and Stanley G. Dienst , M D * * Kidneys were preserved by continuous pul sad le perfusion and their metabolic rate monitored by Oi consumption. Experiments were carried out to determine ifdialyzing the perfusate to supply metabolic substrates would aid in kidney preservation. A control group was compared against two experi-mental groups at 25°C. Average preservation dme before fall off of oxygen consumption differed for each group: control - 8 hours, dialysis with amino acids-6 hours, dialysis with amino acids and cofactors - 11 hours. Results Indicated that, If suitable concentra-tions of metabolic substrates and cofactors were used, the preserved kidneys main-tained higher levels of O2 consumption for longer periods of time. N. * Summer Research Fellow in Renal Preservation Laboratory ** Depar tment of Surgery, Section of Trans-plantation Address reprint requests to authors at Henry Ford Hospital, 2799 West Grand Boulevard,-Detroit M l 48202 UMEROUS experiments have been un-dertaken to extend the period of organ pres-ervation by continuous pulsatile perfusion. Variations in the temperature and composi-tion of the perfusate have been investi-g a t e d . T o da te , the pe r fusa te recommended by Belzer et al (from 5-7°C) yields the most consistent results.' Usingthe same perfusate, this study seeks to determine if continuously dialyzing the perfusate wi l l extend the period of organ preservation. Method Female mongrel dogs weighing between 14 and 24 kg were anesthetized with sodium pentobarbital (40 mgm/kg) and intubated prior to surgery. Through a midline abdomi-nal inc is ion, one kidney was removed, placed in an ice bath, and flushed with cold heparinized Ringer's lactate. After flushing, the kidney was immediately placed on the MOX-lOO Renal Preservation Console. The total ischemia time was always less than two minutes. The pressure, PO2, and pH of the arterial and venous perfusates, as well as the flow and temperature, were measured at intervals of 15-30 minutes. 125 Dall'Olmo and Dienst The renal ve in was cannu la ted w i t h a large cannu la (16.6 mm^) for the purpose o f mea-sur ing the partial pressure o f oxygen . The oxygenat ion of the p lasma was main ta ined by a Lande-Edwards M e m b r a n e Oxygena to r (1M^ s u r f a c e a rea ) . T h e p e r f u s a t e w a s c ryoprec ip i ta ted can ine plasma prepared by-the Belzer m e t h o d . ' A d iagram of the per fu -sion c i r cu i t is shown in Figure 1. Three types of tests were per fo rmed d u r i n g t h e course of these exper iments . A. DeterminaUon of oxygen consumpUon as a function of change in temperature. Startingthe perfusate at 5°C, oxygen consump-tion measurements of each kidney were made foreach 5° increment rise in the temperature of the perfusate until 37°C was reached. For every five degree increase in temperature, the ar-terial and venous PO2 levels were allowed to stabilize for ten minutes before readings were taken. The pressure ranged from 60 mm / Hg at 5°C to 100 m m / H g at 37°C. This was done to increase flow and provide the additional oxy-gen required at higher temperatures. 6. Determination of O2 consumption measured at a constant temperature -controls. Several perfusions were carried out at the selected temperatureof25°C while monitoring O2 consumption. This was plotted against the duration o f the perfusion period in hours. Dialyzing perfusate at 25°C. As in B, oxygen consumption was recorded throughoutthe procedure. In thiscasecontinu-ous counter current dialysis of the perfusate was carried out through a commercial d i -alyzer.* Two dialysates were prepared. The first was an electrolyte solution prepared from a clinical dialyzing concentrate** plus dex-trose 400 mgm%, sodium citrate 12 meq/L, and an amino acid hydrolysate*** 0.25% final concentration. The remaining volume was made up wi th sterile water. The pH of the dialysatewas brought to 7.4 at25°C by adding disodium phosphate. The second dialysate was a solution of tissue culture media**** (diluted 10:1 with sterile water) and sodium citrate added 6 meq/L. A dialysate pH of 7.4 was achieved by add ing 2.2 grams per liter of sodium bicarbonate. *Cordis Dow Artificial Kidney **Renalyte 45/Cobe — Final concentration: Na 140 m e q / L , C l 106 m e q / L , Ca 3.5 meq/L, K 3.0 meq/L, Mg 1.5 meq/L, and Acetate 42 meq / L ***Amigen — Baxter: 5% protein (Casien) hy-drolysate. Bisulfite as preservative. ****Eagle's 199—Grand Island Biological—20 amino acids, 18 vitamins, nucleotides, pu-rines, and pyrimidines, dextrose and ribose and inorganic salts in Ringer's bicarbonate and Ca 1.3 meq / L and iron.'' ' Cooling unit Dialyzing soln. Oxygenator Temp, gauge Figure 1 Kidney Perfusion Circuit 126 Prolonging oxygen consumption by dialysis Results Experimental groups: Croup A -A l l kidneys showed an increase in oxygen consumption with increasing tempera-ture. Results of oxygen consumption as a function of temperature are shown in Figure 2. Croup 6 (ControO-ln this group kidneys main-tained at 25°C had constant oxygen consumption for six to seven hours after which the flow and A-V oxygen difference and subsequently the oxygen consumption decreased. Group C-Dialysis ofthe perfusate with the elec-trolyte solution (plus amino acids) did not extend the period of oxygen consumption. Continuous consumption was present for only five hours. The perfusate flows in this experiment remained con-stant as A-V oxygen differences narrowed. On the other hand, dialysis ofthe perfusate with Eagle's solution gave longer sustained oxygen consump-tion as compared to controls. With the Eagle's, constant oxygen consumption and plasma flow was maintained for eleven hours. Tfie A-V, PO2 difference was sustained for a time even after flow decreased. It was only after the flow decreased (below 20 cc/min) that the A-V, O2 difference disappeared. Table 1 shows the effect of the two dialysates on the length of oxygen consumption. Discussion Since the acceptance of cryoprecipitated plasma as a perfusate for kidney preserva-tion, modifications of this solution have been studied in an effort to extend reliable preservation time. Tests have been made on the effect of substituting fresh plasma perfu-sate every 12 hours,^ using silica gel treated plasma,''or using the albumin fraction (pu-rified protein fraction) of plasma^ as the perfusate. The objective of our study is to determine the length of effective metabolic preservation as influenced by continuously dialyzingthecryoprecipitated plasma perfu-sate during kidney preservation. The the-oretical basis of this approach is to replenish and stabilize substrate and soluble cofactor concentrations forthe actively metabolizing kidney cells. There is evidence that a lack of essential energy substrates can lead to irre-versible tissue damage during organ preser-vation.'' ^ Among the substrates implicated are unesterified fatty acids^ (the preferred substrate for energy production inthe kidney 40 n 30-20-10-- I -10 - I -15 - I -20 25 1 30 O2 C O N S U M P T I O N Figure 2 Changes in oxygen consumption (m-moles/ hr/grm) as a function of increasing temperature (°C). 127 Dal l 'Olmo and Dienst TABLE I Dialysate Oxygen Consumption (average) Number of Experiments (m-moles/gm x hr) Hour 1 2 3 4 5 6 7 8 9 10 11 None 2 18.2 16.4 16.7 16.1 16.1 17.3 16.0 10.0 6.0 — — Electrolyte solution plus Dextrose, Amigen and Citrate 5 37.0 36.7 26.6 20.5 19.5 6.1 4.1 2.0 — — — Eagle's Tissue Culture Medium (199) plus Citrate 6 18.0 17.5 16.6 15.4 21.4 17.7 19.1 20.9 20.0 15.8 9.3 Changes in oxygen consumption over time comparing the two dialysates to the control group (without dialysis) cortex), glycerol, and glucose. Another the-oretical advantage favoring the incorpora-tion of dialysis is the removal of metabolic end products. The kidney produces metab-olites that accumulate during continuous perfusion.'' ' One of these, lactic acid, increases the acidity of the perfusate and also leads to an increase in the production of ammonia.^' ' The increase in the concentra-tion of metabolic products and an ultimate decrease in the oxidizable substrates may be l im i t ing factors in extending kidney preservation. Oxygen consumption was chosen as a measure of metabolic activity due to this event being the final step in cellular respira-tion. When oxygen is consumed, it indicates that the respiratory chain is intact. The ar-terial-venous oxygen difference is a direct measure of how rapidly respiration is being carried on. Therefore, oxygen consumption was used as a measure of kidney metabo-lism. When the temperature was increased, the oxygen consumption also increased. These results coincide with previously pub-lished data."* To measure accurately the effects of di-alysis, the temperature of 25°C was selected. At the usual 5-8°C, the activation energy of biochemical reactions is very high. Even in the presence of enzymes, they proceed very slowly. The activities of many of the glyco-lytic and tricarboxylic acid cycle enzymes particularly are reduced or arrested at lower temperatures.'""'^Therefore, a temperature was selected at which enzyme activities would be close to normal. Looking at the glycolytic and respiratory enzymes together, the largest change in their activity (noted by Q'°) is 20-30°C. Therfore, 25°C was chosen. This temperature would provide sufficient activity to use substrates quickly if the sub-strates were to be the limiting factor. Table I shows the differences in the length of the preservation period between controls and the two dialysis groups. This suggests thatthe enzymes were more active at this elevated temperature and substrate concentration was a determ in ing factor. By maintaining the substrate concentration, as the Eagle's solu-tion does, metabolic activity is prolonged and therefore oxygen consumption is ob-tained for longer periods of time. 128 Prolonging oxygen consumption by dialysis ELECTRON TRANSPORT CHAIN NAOH—»-NAD dehydrogenase—^Coenzyme Q—»-Cytxhrome b—»-Cytxhrome c—^Cytochrome 883—^02 Cytochrome Cj Nicotinic Riboflavin Acid Figure 3 This figure indicates positions of sustained cofactor concentrations along an electron transport chain. The effect of dialysis differed depending on the dialysate used. With the electrolyte solution, failure to achieve longer preserva-tion could be due to loss of components such as glucose, adenosine, vitamins, and other soluble compounds into the dialysate. On the other hand, longer preservation per-iods were realized by dialyzing with Eagle's 199 solution. This could be due to a sus-tained concentrat ion of compounds re-qui red for respirat ion. For example, the solution maintained concentrations of co-factors and substrate such as biotin (which functions in the decarboxylation of pyru-vate), nicotinic acid (which is a precursor of NAD), and riboflavin (the precursor of FAD and FMN). These are all necessary for cell respiration. Deficiencies in these would re-sult in lowered cell respiration. Another molecule whose concentration was main-tained in the perfusate by dialysis was ATP. Figure 3 shows the sequence from NADH to oxygen in the electron transport chain. Posi-tions where cofactors were sustained by dialysis are indicated by arrows. Maintain-ing the cofactor and substrate concentra-tions by dialysis with Eagle's 199 solution resulted in prolonged oxygen consumption. Therefore, it is suggested that maintenance of a relatively high concentration of cofac-tors as well as oxidizable substrate may be i m p o r t a n t in e x t e n d i n g safe o rgan preservation. References 1. Humphries AL: Problems of organ preserva-t ion: A review. Transplantation 5:1138-1153, 1967 2. Abouna GM, Lim F, CookTS, Grubb W, Craig SS, Seibel HR, and Hume D M : Three day c a n i n e k i d n e y p r e s e r v a t i o n . Surgery 71:436-444, 1972 3. Johnson RWC, Anderson M, FlearCTG, Mur-ray SGH, Taylor RMR, and SwinneyJ: Evalua-tion of a new perfusion solution for kidney preservation. Transplantation 13:270-75, 1972 4. Toledo-Pereyra LH, Condie RM, Simmons RL, and Najarian JS: Renal preservation with silica gel fraction (SGF) of plasma. Dialysis and Transplantation 4:20-22, 1975 5. Belzer FO, Ashby BS, HuangJS, and Dunphy jE: Etiology of rising perfusion pressure in i s o l a t e d o r g a n p e r f u s i o n . A n n Surg 168:382-391, 1968 6. MorganJF, Morton HJ, and Parker RC: Nutri-tion of animal cells in tissue culture (part 1). Proc Soc Exp Biol & Med 73:1-8,1950 129 Dall'Olmo and Dienst 7. Salk, Younger, and Ward: Use of color change of phenol red as the indicator in titrating poliomyelitis virus or its antibody in a tissue culture system. Am j Hyg/ene 60:214-230, 1954 8. Grundmann R, Landes T, and Pichlmaier H: Hypothermic pulsatile perfusion of dog kid-neys. Transplantation 14:742-47, 1972 9. Levy M N : Oxygen consumption of blood flow in the hypothermic perfused kidney. Am j Physiol 19:1111-14, 1959 10. HannonJP: Effect of prolonged cold exposure on oxidative phosphorylation and adenosine-triphosphatase activity of rat liver tissue. Am j Physiol 196:890-92, 1959 11 Hannon JP: Effect of prolonged cold exposure on components of the electron transport sys-tem. Am j Physiol 198:740-44, 1960 12. Hannon JP and Vaughan DA: Effect of pro-longed co ld exposure on the g lyco ly t ic enzymes of liver and muscle. Am j Physiol 198:375-380,1960 13. Belzer FO, Ashby SB, Dunphy JE: Twenty-four hour and seventy-two hour preservation of canine kidneys. Lancet 2:536-39,1967 14. LehningerAlbertL:6/ochem/stry, Second Edi-t ion. New York: Worth publishers. Inc., 1975 130 

Prolonging oxygen consumption during preservation of canine kidneys by the addition of continuous dialysis

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Prolonging oxygen consumption during preservation of canine kidneys by the addition of continuous dialysis

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