An oxygen consumption assay using adenosine diphosphonucleotide (ADP) was performed on canine kidneys to measure the efficiency of oxidative phosphorylation. The purpose of the assay was to establish a respiratory control index (RCI) that could be used to evaluate kidneys preserved by different methods. Of the four methods tested, perfusion yielded the best results after 24-hour preservation. Additionally, four pairs of human cadaver kidneys were evaluated with the same assay. Some correlation existed between the RCI values of the nontransplanted kidney in each pair and the clinical viability of its transplanted mate

Dienst, Stanley G.

Samuel, Devprakash

Henry Ford Health System Scholarly Commons

Henry Ford Hospital Medical Journal Manuscript 2019 A Comparison of Kidney Preservation Methods by Oxidative Phosphorylation Studies Devprakash Samuel Stanley G. Dienst Follow this and additional works at: https://scholarlycommons.henryford.com/hfhmedjournal  Part of the Life Sciences Commons, Medical Specialties Commons, and the Public Health Commons Henry Ford Hosp Med Journal Vol 26, No 3, 1978 A Comparison of Kidney Preservation Methods by Oxidative Phosphorylation Studies Devprakash Samuel* and Stanley G. Dienst, MD* An oxygen consumption assay using adenosine di-phosphonucleotide (ADP) was performed on canine kid-neys to measure the efficiency of oxidative phosphorylation. The purpose of the assay was to establish a respiratory control index (RCI) that could be used to evaluate kidneys preserved by different methods. Of the four methods tested, perfusion yielded the best results after 24-hour preservation. Additionally, four pairs of human cadaver kidneys were evaluated with the same assay. Some correlation existed between the RCI values of the nontransplanted kidney in each pair and the clinical viability of its transplanted mate. OXIDATIVE phosphorylation is the process by which the energy released in the oxidation of organic foodstuffs is made available for cell metabolism, including protein syn-thesis. When cells are deprived of oxygen, molecular changes take place that f inal ly result in the loss of oxidative phosphorylation capacity. From these general observations, the ability of renal cells to carry on oxidative phosphoryla-tion has been suggested as a means of evaluating their viability after anoxia (and hypothermia).^'^ Methods currently used for determining the viability of kidneys harvested for transplantation include an assessment o f the vascular resistance from measurements of pressure and flow on preservation machines, measurements of lactic acid accumulation determined from the perfusate, deter-mination of the progressive release of intercellular enzymes, particularly lactic dehydrogenase, and measurements of tissue concentration of total adenine nucleotides.^"^ In the ischemically-damaged kidney the values indicated by these tests are abnormal. However, there is substantial overlap between nonviable kidneys and those which recover and have good function. The aim of this study is to measure tissue oxygen consump-tion wfth an oximeter during the oxidative metabolism of succinate. By adding adenosine diphosphonucleotide (ADP) to this system and determining the active phos-phorylating capacity of the tissue, ft is possible to estimate the immediately recoverable energy production and poten-tial function of the kidney. This work differs from previous studies '"^ in that this biochemical test is used to compare and evaluate several kidney preservation methods. Submitted for publication: July 7, 1978 Accepted for publication: September 15, 1978 * Departmentof Surgery, Section ofTransplantation and Surgical Research, Henry Ford Hospital Address reprint requests to Dr, Dienst, Departmentof Surgery, Henry Ford Hospital, 2799 W Grand Blvd, Detroit, Ml 48202 Materials and Methods Preservation method Kidneys were removed from anesthetized adult mongrel dogs and treated by one of the following procedures: 1. Warm Ischemia (N = 5 kidneys). After heparinization and clamping of the renal vasculature, an atraumatic nephrec-57 Samuel and Dienst tomy was performed. The kidney was immediately placed in a thin sterile plastic enclosure, without flushing and left in a 37°C water bath. 2. Collins (N = 4 kidneys) or Ringer's lactate flush (N = 5 kidneys). The nephrectomy was performed as described for the warm ischemia. Immediately after nephrectomy, the kidney was flushed with Collins or Ringer's lactate (approxi-mately 150 ml) at4°C until the effluent was clear. The kidney was stored in a sal ine ice slush bath for varying periods upto 24 hours, before cortical tissue samples were excised for the oxygen consumption assay. 3. Perfusion (N = 8 kidneys). Bilateral nephrectomy was performed, and the kidneys were placed en bloc on a Waters MOX-100 Renal Perfusion Console. Cryoprecipitated dog plasma with standard additives was used as the perfusate. Periodic flow, pressure, pH, PCO2, and PO2 measurements were obtained over a 24-hour period. Cortical biopsies were taken before the kidneys were placed on perfusion, at 24 hours, and in some cases at 48 hours. Oxygen consumption was determined immediately after taking the biopsies. Oxygen consumption assay Oxygen consumption was measured polarographically with a Clark-type oxygen electrode and a Yellow Springs Instru-ment Biological Oxygen Monitor, To prepare a 10% tissue homogenate, kidney biopsies of 300 to 400 mgm were placed in ice cold 0.25 M Sucrose and 0.01 M Tris-HCI pH 7.4. Sixteen p moles of sodium succinate was added to 3 ml of a solution containing potassium 115 mEq/L, sodium lOmEq/L , phosphate (HPO4) 100 mEq/L , ch lor ide 15 mEq/L, bicarbonate lOmEq/L, and magnesium 60 mEq/L. A cuvette containing 2.8 ml of this solution was placed in a water bath at 37°C. Tissue homogenate (0.2 ml) was added tothe reaction media. Oxygen consumption was monitored and expressed as p moles of O2 per milligram of wet tissue weight per hour. After a steady rate of oxygen consumption was obtained, 0.4 p moles of ADP was added. The tissue was then allowed to respire in the presence of ADP until oxygen consumption ceased. From these rates, the Respiratory Control Index (RCI) or ratio of the respiratory rate in the presence of ADP to that in the absence of ADP was computed (Figure 1). This index is an exact measurement of the phosphorylation capacity of the mitochondria.^ Four pairs of human cadaver kidneys were evaluated for their ability to phosphorylate ADP oxidatively while main-tained on continuous perfusion. One kidney from each pair was used for transplantation, and its function after one month was correlated wi th the RCI values dur ing preservation. Results The mean RCI values forthe different preservation methods are shown in Figure 2. Warm ischemia produced a rapid drop inthe phosphorylation capacity ofthe kidney homoge-nate, as reflected by the decreasing RCI values, which fell to 1.0, with no phosphorylation response after three hours. In contrast, the other preservation methods showed sustained phosphorylation capacity over at 24-hour period, with the following mean RCI values: perfused kidneys 1,66 ± .17, Collins flush 1.36 ± .11, and Ringer's lactate flush 1.13 ± .09. These results suggest that perfusion is the best method of preserving a kidney over a 24-hour period. CALCULATION OF OXYGEN CONSUMPTION SUSTAINED PHOSPHORYLATION IN KIDNEY PRESERVATION F I E S P I R A T O R Y C O N T R O L I N D E X I R C d R A T E O 5 C O N S U M P T I O N + A D P R A T E 0 , C O N S U M P T I O N - A D P ^88% SATURATION 79% ADDITION OF ADP 64% SATURATION 1.50- V 1 WARM ISCHEMIA \ NO PHOSPHORYLATION 1.00 - - - v N = 3 1 — I — I — I — r • PERFUSION 5 l l l l l l l l l l l l 10 12 14 16 18 20 TIME IMINUTESI Fig.1 Calculation of oxygen consumption rates and Respiratory Control Index (RCI). Fig. 2 Phosphorylation capacity expressed in terms of mean RCI (Respiratory Control Index) values during various kidney preservation methods. N = the number of kidney biopsies assayed. 58 Kidney Preservation by Oxidative Phosphorylation In addition, this oxygen consumption assay was performed on four pairs of marginally viable human kidneys evaluated for the Michigan Donor Program. One kidney from each pair was transplanted. The biopsies for the RCl were taken from the other kidney. As indicated on the table, the results show that some correlation may exist between the RCI values of the nontransplanted kidney and the clinical status of its transplanted mate. The kidney with an RCI of 1.48 is still functioningwell, while those with RCI values of 1.13-1.22 never established good renal function after transplantation. Discussion The respiratory control of kidney homogenates with and without normothermic ischemia has been previously stud-ied by polarographic measurement of oxygen consump-tion.^ However, this early study failed to quantitate the data and correlate the findings with actual kidney transplants. Renewed interest in the energy metabol ism of kidney tissue has led several investigators to use total adenine nucleotides (TAN), as postulated by Caiman, to predict v i a b i l i t y . T h e use of TAN, however, fails to yield a dynamic measurement of tissue metabolism, and the existence of a relationship between recoverable renal function and TAN levels has not been established. The theoretical basis of applying the respiratory control index can be explained by the two-fold process of oxidative phosphorylation as it occurs atthe cellular level in the inner mitochondrial membrane.^^"^^ Electronsoriginatingfrom the oxidation of high-energy reduced substrates are ultimately transferred to a low-energy terminal acceptor such as mo-lecular oxygen. This reaction provides sufficient chemical energy for the phosphorylation of ADP to yield adenosine triphosphonucleotide (ATP). This coupled relationship lends itself well to functional assessment for the quantitative measurement of RCI values. In the presence of oxidizable substrate (i.e., succinate), oxygen, ADP, and phosphate, respiration and phosphorylat ion rates are fast, and mitochondria are in the active state (State 3) ." In the controlled state (State 4), i.e., in the presence of substrate and oxygen but in the absence of excess ADP, there is minimal respiration in tightly coupled mitochondria. The RCI, orthe ratio of the rates in the active and the controlled states is a measure of the degree of control imposed on oxidation by phosphorylation. In oxidative phosphorylation hydrogen ions are conveyed by molecules of Flavin Adenine Dinucleotide (FAD) to the electron transport chain in the mitochondrial membrane (Figure 3). The transport of H+ ions across the inner mem-brane generates a H^ ion gradient, which results in an electric potential that forces H^ ions back through the inner membrane spheres and releases the energy for ATP syn-thesis. The ATP thus generated provides the chemical energy for many metabolic processes, such asthe transport of ions and small molecules, and the molecular synthesis needed for maintenance of intracellular structure. The application of oxidative phosphorylation measurement to the in vitro evaluation of preserved canine kidneys was successful even though the ultimate test of viability (i.e., allotransplantation) was not performed. Our results suggest thatthose kidneys with RCI values between 1.4-2.0 indicate MEAN RC I VALUES ON FOU R PA I RS OF HUMAN KIDNEYS HUMAN KIDNEY RC\ { p moles O /̂Hr x mgm) CLINICAL STATUS 1 1.19 Neither Kidney Transplanted 2 1.13 Mate Transplanted -NonFunctional 3 1.22 Mate Transplanted -Function doubtful for 3 wks., slow recovery time. 4 1.48 Mate Transplanted -Functional 59 Samuel and Dienst OXIDATIVE PHOSPHORYLATION Fig. 3 Oxidative phosphorylation in the mitochondria. tightly coupled mitochondria with controlled oxidation. Low RCI values below 1.4can be correlated witha low level of mitochondrial coupling, with an RCI of 1.0 indicating no active phosphorylation (Figure 2.) Our results show that at 24 hours several preservation methods will yield RCI values within the active range (1.4-2.0), and that perfusion has significantly higher RCI values than other methods. These find ings coincide with other reports in the d i n ical transplan-tation literature,^^"^* which suggestthat perfusion of cadaver kidneys for 24 hours is a more effective method of preserva-tion than ice storage. While the phosphorylation capacity of the perfused kidneys (RCI 1.66 ± .17) at 24 hours indicates that the tissue is metabol icaliy active, this may not be the only factor contributing to the metabolic requirements ofthe preserved kidney. Other factors, such as the TAN levels of the tissue preserved and ionic concentration gradients, may play an equally important role. These findings using oxidative phosphorylation to evaluate d ifferent preservation methods were extended to the cl in ical transplant setting, and the predictive ability of the RCI was analyzed in four pairs of marginally viable human kidneys (See Table). Although we are not able to present statistically significant evidence ofthe relationship between RCI values and the success of transplantation at this time, the results suggest that a correlation may exist between RCI and immediate posttransplant function. More studies are need-ed in this area to confirm this finding. In the interim, utilization of micro techniques with needle biopsies of 30-50 mgm of cortical tissue rather than invasive wedge biopsies of 300-400 mgm are currently being investigated. Modifications of the biopsy technique may encourage wider acceptance of this procedure in evaluating marginally via-ble human kidneys on preservation machines (i.e., long warm ischemic times or excessively long perfusion times with deteriorating flow characteristics). Conclusions 1. Three nonperfusion preservation methods and preserva-tion by perfusion have been evaluated in the canine model. When the Respiratory Control Index (RCI) was used to measure the phosphorylating capacity of kidney tissue homogenate, kidneys on perfusion yielded the best results after 24-hour preservation. 2. Four perfused human cadaver kidneys were shown to have RCI values which correlated with eventual clinical function. This finding suggests the possible value of this assay in determining v iabi l i ty and immediate post-transplant function of human cadaver kidneys. References 1. Bautista EM and Cohn HE: A prelimiary report: A single method of de termin ing k idney v iabi l i ty . Trans Amer Soc Ar t i f Int Organs 13:360-363, 1967, 2. Lannon SG, Tukaram KT, Oliver JA, et at: Preservation of kidneys assessed by a b iochemica l parameter. Surg Gyneco l Obstet 124:999-1004, 1967. 3. Belzer FO, Ashby BS, Huang JS, et at: Etiology of rising perfusion pressure in isolated organ perfusion. Ann Surg 168:382-391, 1968, 4. Liebau G, Klose HJ, Fischbach H, et a/; Simple tests for viability of the hypothermic pulsatile perfused dog kidney. Surgery 7:459-466, 1971, 5. Grundmann R, EichmannJ, Keckstein J,et a/: Relationship between the prolongation of warm ischemia and the maximum available preserva-tion period. Surgery 81:542-550, 1977. 6. Caiman KC: The prediction of organ viability — I. An hypothesis. Cryobiotogy 11:1-6, 1974. 7. Chance B and Will iams GR: Advances in Enzymotogy, ed Nord FF. 17:65-134, 1956, 8. Southard JH, Senzig KA, Hoffmann RM, and Belzer FO: Energy metabolism in kidneys stored by simple hypothermia. Transplant Proc 9:1535-1539, 1977, 9. Warnick CT and Lazarus H M : Adenine nucleotides during organ storage. Transplant Proc 9:1575-1577, 1977. 10. Bishop MC and Ross BD: Evaluation of hypertonic citrate flushing solution for kidney preservation using the isolated perfused rat kidney. Transplantation 25:235-239, 1978. 11. Lehninger AL: Biochemistry, ed 2. New York, Worth, 1975. 12. Slater EC: Mechanism of oxidative phosphorylation. Ann Rev Biochem 46:1015-1026, 1977. 13. Scott DF, Whiteside D, Redhead), eta/ : Ice storage versus perfusion for preservation of kidneys before transplantation. Br Med I 4:76-77,1974. 14. Toledo-Pereyra LH, Simmons RL, Moberg AW, et a l : Organ preserva-tion in successof cadaver transplants. Arch Surg 110:1031-1035, 1975. 15. Halasz NAand Col I ins G M : Forty-eight hour kidney preservation. Arch Surg 111:175-177, 1976. 60 61 

A Comparison of Kidney Preservation Methods by Oxidative Phosphorylation Studies

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