- Top of page
- Materials and Methods
Research involving metabolically active and functioning organs, maintained ex vivo in culture-like conditions, could provide numerous opportunities for medical innovations and research. We report successful perfusion of isolated canine and human kidneys ex vivo at near physiologic temperature for 48 h. During the perfusions parameters of metabolism and function remained stable. Nitric oxide synthase (NOS) was identified as the underlying mechanism preserving vascular integrity. Most importantly, when the canine kidneys were reimplanted there was immediate normal renal function. This report highlights the potential significance of whole organ culture using a warm temperature ex vivo perfusion and discusses medical applications that could be developed.
Materials and Methods
- Top of page
- Materials and Methods
Exsanguineous metabolic support (EMS) technology (Breonics Inc., Schenectady, NY) was used for ex vivo warm perfusion (32 °C); a temperature range previously shown to adequately support oxidative metabolism and cellular reparative processes (18–24). Both human and canine kidneys were placed on a perfusion system including an oxygenator and a pulsatile pump, retrofitted with controllers to maintain PAO2, PACO2, pH and temperature. The renal artery was cannulated and perfusion was initiated with a pulsatile perfusion pressure of 50/30 mmHg. The volume of the circulating medium was 500 mL for the canine and 750 mL for the human kidneys. The EMS medium consisted of an enriched tissue culture-like solution (Table 1). The acellular EMS medium was supplemented with pyridoxylated bovine hemoglobin (6-g percentage) (Ezon Inc., Piscataway, NJ) to obtain a target PAO2 of 200 mmHg to support continued oxidative metabolism in the range previously shown to correlate with adequate (nondamaging) oxygen delivery (20). In order to minimize the risk of microbial infection and to avoid changes in osmolarity, the urine produced during the period of warm perfusion was recirculated. The circulating medium was exchanged (10%) every 4 h. PAO2 analysis of prerenal and postrenal samples was performed using an ABL5 blood gas analyzer (Radiometer Medical A/S, Copenhagen, Denmark). O2-consumption (mL/min/g) was calculated using the following formula:
Table 1. Composition of basal exsanguineous metabolic support medium
|DL-Alanine||0.12 g/L||Menadione (Na Bisulfate)||0.00003 g/L|
|L-Arginine HCl||0.14 g/L||Myo-Inositol||0.0001 g/L|
|DL-Aspartic Acid||0.12 g/L||Niacinamide||0.00005 g/L|
|L-Cysteine HCl H2O||0.00022 g/L||Nicotinic Acid||0.00005 g/L|
|L-Cystine 2HCl||0.52 g/L||Para-Aminobenzoic Acid||0.0001 g/L|
|DL-Glutamic Acid||0.2672 g/L||D-Pantothenic Acid Ca||0.00002 g/L|
|L-Glutamine||0.20 g/L||Polyoxyethylenesorbitan Monoolate||0.04 g/L|
|Glycine||0.10 g/L||Pyridoxal HCl||0.00005 g/L|
|L-Histidine HCl H2O||0.04376 g/L||Pyridoxine HCl||0.00005 g/L|
|L-Hydroxyproline||0.02 g/L||Retinol Acetate||0.00028 g/L|
|DL-Isoleucine||0.08 g/L||Riboflavin||0.00002 g/L|
|DL-Leucine||0.24 g/L||Ribose||0.001 g/L|
|L-Lysine HCl||0.14 g/L||Thiamine HCL||0.00002 g/L|
|DL-Methionine||0.06 g/L||Thymine||0.0006 g/L|
|DL-Phenylalanine||0.10 g/L||Uracil||0.0006 g/L|
|L-Proline||0.08 g/L||Xanthine HCl||0.00069 g/L|
|DL-Serine||0.10 g/L||Calcium Chloride 2H2O||0.265 g/L|
|DL-Theonine||0.12 g/L||Ferric Nitrate 9H2O||0.00144 g/L|
|DL-Tryptophan||0.04 g/L||Magnesium Sulfate (anhydrous)||1.2 g/L|
|L-Tyrosine 2Na||0.11532 g/L||Potasium Chloride||0.40 g/L|
|DL-Valine||0.10 g/L||Sodium Acetate (anhydrous)||0.10 g/L|
|Adenine Hemisulfate||0.02 g/L||Sodium Chloride||6.8 g/L|
|Adenosine Triphosphate 2Na 2Na||0.002 g/L||Sodium Phosphate Monobasic (anh)||0.224 g/L|
|Adenylic Acid||0.0004 g/L||D-Glucose||2.0 g/L|
|Alpha Tocopherol Phosphate 2Na||0.00002 g/L||Insulin||0.01 g/L|
|Ascorbic Acid||0.001 g/L||Bovine Serum Albumin||30 g/L|
|D-Biotin||0.00002 g/L||Sodium Bicarbonate||4.4 g/L|
|Calciferol||0.0002 g/L||Pyruvate||0.22 g/L|
|Cholesterol||0.0024 g/L||Transferin||0.10 g/L|
|Choline Chloride||0.001 g/L||Serum||10 mL|
|Deoxyribose||0.001 g/L||B-cyclodextrin||0.50 g/L|
|Folic Acid||0.00002 g/L||Chondroitin sulfate B||0.004 g/L|
|Glutathione (reduced)||0.0001 g/L||Fibroblast growth factor||0.02 g/L|
|Guanine HCL||0.0006 g/L||Heparin||0.18 g/L|
|Hypoxanthine||0.0006 g/L|| || |
Biopsies were not taken before initiating perfusion because small specimens would not be representative of the whole kidney and large biopsies would alter the subsequent perfusion characteristics. Instead postperfusion biopsies were taken from the human kidneys for histologic evaluation.
Hypothermic perfusion and cold storage
Control kidneys in groups 3 and 4 were flushed and placed into a MOX-100 perfusion machine (Water's instruments, MN) with 500 mL of Belzer's Machine Perfusate used as a circulating solution. After the kidneys were connected to the perfusion system, perfusion was set to a systolic pressure of 50 mmHg. After 1 h of machine perfusion, if needed, the systolic pressure was readjusted to 50 mmHg. Flow and pressure were continuously monitored.
Control kidneys in groups 5 and 6 were flushed and submerged in cold ViaSpanTM. The kidneys were then triple-bagged and packed in ice until reimplantation.
Human kidneys were procured from heartbeating donors with negligible warm ischemic time for the purpose of transplantation, and were later determined to be nontransplantable by institutional criteria that included the presence of a tumor on the paired kidney, atherosclerotic plaque, age of the donor, and the period of cold ischemia. The kidneys were stored in ViaSpan at 4 °C (mean cold storage time of 38 h) before their release for research use. The kidneys were weighed and the renal artery of each kidney was cannulated. The kidneys were flushed with approximately 250 cc of organ culture medium warmed to 32 °C to remove the hypothermic solution from within the vasculature. During the organ culture at 32 °C, the kidneys were evaluated for oxidative metabolism, vascular dynamics and organ function. The organ parameters were tested hourly. Human kidneys were perfused for 12 h (n = 2), 24 h (n = 2) or 48 h (n = 2).
Animals and surgical protocol
The autotransplantation experiments were performed on foxhounds weighing 20–30 kg. The animals demonstrated normal renal function before the start of the study. All experiments were performed following the principles of laboratory animal care according to the NIH standards. Kidneys were exposed through a midline incision and the left renal artery, vein and ureter were mobilized. Kidneys were then divided into six groups:
Group 1 (n = 6): 24-h organ culture (32 °C) and reimplantation
Group 2 (n = 3): 48-h organ culture (32 °C) and reimplantation
Group 3 (n = 3): 24-h hypothermic perfusion (4 °C) and reimplantation
Group 4 (n = 3): 48-h hypothermic perfusion (4 °C) and reimplantation
Group 5 (n = 3): 24-h cold storage (4 °C) and reimplantation
Group 6 (n = 3): 48-h cold storage (4 °C) and reimplantation
The mean anastomosis time was 28 min and ranged from 22 to 32 min. Contralateral nephrectomy was performed before reperfusion of the preserved kidney. The contralateral kidneys were used in the nitric oxide synthase (NOS) blocking studies. NOS was not inhibited (control; n = 3), inhibited by L-NAME (100 µM; n = 3); inhibited by iNOS (inducible NOS) specific with dexamethasone (15 µM; n = 3), or inhibited by iNOS specific L-NIL (100 µM; n = 3). Values are expressed as the mean with standard deviation for each experimental group. The concentration of nitrate in the EMS medium was determined by analysis on a Dionex DX-500 ion chromatograph using an AS-15 column and KOH eluant that varied in concentration by a programmed gradient. All samples were analyzed in duplicate sets diluted by a factor of 11. The concentrations of nitrate in the various EMS media specimens were within the standard calibration range.
Post-transplant graft function
Blood samples for BUN and serum creatinine were taken each morning and analyzed using an ACE analyzer (Schiapparelli Biosystems Inc., Fairfield, NJ). Serum creatinine was considered normal with values below 2.0 mg/dL.
- Top of page
- Materials and Methods
These results demonstrate that it is feasible to maintain intact human kidneys during an acellular perfusion with a cell culture-like medium administered via the vascular bed for 48 h. While the kidneys were maintained ex vivo at 32 °C, stable metabolism, vascular perfusion and organ function were observed. Moreover, when canine kidneys that were similarly warm perfused for 48 h with comparable metabolic rates, perfusion characteristics and function were reimplanted, the kidneys provided immediate life-sustaining function.
The canine kidney model represents a long-established methodology for developing and testing organ preservation technologies. The canine kidney was effectively utilized for the development of all the perfusate solutions used clinically, including ViaSpanTM, Belzer Machine Perfusate, Collins, Eurocollins, HSA-based, cryoprecipitated plasma, and Sacks solutions (26–36). Likewise, the canine kidney model has also been instrumental in the development of supporting preservation technologies such as perfusion preservation, static storage and in establishing their relative efficacy (37–39). The canine kidney model has also been employed to assess immunologic complications and injury following preservation, potential pharmacologic interventions and substrate requirements (40–48).
Most importantly, the various organ preservation technologies developed using the canine kidney model were, in every case, successfully transferred to clinical use without the necessity of reformulation or substantial optimization.
Although these human kidneys could not be transplanted, the results of the canine kidney transplant studies demonstrated the viability and functional status of kidneys following 48 h of near-normothermic perfusion. To our knowledge this is the first report of long-term ex vivo perfusion of kidneys at near normothermic temperature following reimplantation in which normal life-sustaining function is demonstrated.
Protective mechanisms involved in the organ culture appear to include the continued flux of NO by constitutive NOS, as well as activation of the inducible isoform, iNOS. Blockade of NOS by L-NAME, dexamethasone or L-NIL resulted in profound edema after several hours of organ culture. The literature is replete with reports of the versatility of this critical signaling messenger with demonstrations of both protective and adverse effects. It appears likely that NO generated by both the constitutive isoform found in endothelial cells and inducible isoform of NOS provide important signaling pathways during organ culture. The continued function of the constitutive NOS isoform during organ culture is suggested by the increased dysfunction observed with L-NAME blockade that targets multiple NOS isoforms in comparison with specific blockade of iNOS. L-NIL is approximately 28-fold more selective for iNOS in comparison with the constitutive endothelial NOS isoform (49). Similarly, dexamethasone inhibits the induction of iNOS without affecting the constitutive isoforms (50,51). While the edema was more severe in the kidneys perfused with the addition of L-NAME, blockade of iNOS also leads to profound edema supporting the function of iNOS during organ culture.
The induction of iNOS may be attributable a generalized modulatory mechanism when organs are removed from their physiologic setting and adapt to an altered state of equilibrium (52). However, the NO produced during the period of organ culture was not cytotoxic, as the canine kidneys following ex vivo organ culture supported normal serum chemistries with immediate resumption of urine flow upon reimplantation.
The ability to study the heterogeneous cell populations that constitute complex tissues with preservation of normal cell-cell and cell-extracellular matrice integrity holds the potential to better understand cellular interactions. Among the potential medical applications that may be developed, one of the most intriguing would be the possibility of repairing damaged organs. The application of cell-culture principles to an isolated organ in combination with adequate growth factor signaling could provide the basis for cellular repair mechanisms; as an optimized cell culture system can support a population doubling in <24h. The repair during ex vivo perfusion would be distinct from the physiologic processes involved in wound repair, because wound repair consists of the primary steps of coagulation and inflammation, along with a migratory/adhesion phase. As organ culture is acellular and ex vivo, any repair represents the cellular recovery that occurs with restoration of metabolism during cell culture.
Other potential medical applications would include using ex vivo metabolism to modulate allograft immunogenecity preventing allosensitization by rendering grafts nonimmunogenic. In the near term, the ability to perform prognostic testing during organ culture may well form the basis for expanding the organ donor pool with organs from marginal and nonheartbeating donors.