Determination of the Preferred Conditions for the Isolated Perfusion of Porcine Kidneys

Abstract

Background: The isolated perfused porcine kidney (IPPK) model has been the method of choice for the early preclinical evaluation of kidney graft preservation techniques. The preferred reperfusion conditions have not yet been determined. Here, we examined the effects of pressure- or flow-controlled perfusion and oxygenation by pure oxygen or carbogen (95% O₂/5% CO₂) on normothermic reperfusion in the IPPK model. Methods: Porcine kidneys were cold-stored for 24 h in histidine-tryptophan-ketoglutarate solution and reperfused for 1 h with normothermic whole blood/Krebs-Henseleit buffer medium (20/80%). Kidneys (n = 5/group) were flow-controlled reperfused with pure oxygen (1 ml/min/g; Flow-O₂) or pressure-controlled reperfused (85 mm Hg mean arterial pressure) and oxygenated with either pure oxygen (Pressure-O₂) or carbogen (Pressure-O₂/CO₂). Renal function and damage were assessed during reperfusion and NGAL and HIF-1α levels were analyzed using an ELISA. Results: Pressure-O₂ and Pressure-O₂/CO₂ were associated with significantly better renal hemodynamics and acid-base homeostasis compared to Flow-O₂. Urine protein concentrations and the fractional excretion of sodium were lower with both Pressure-O₂ and Pressure-O₂/CO₂ than with Flow-O₂. NGAL and HIF-1α levels were also lower with Pressure-O₂ and Pressure-O₂/CO₂ than with Flow-O₂. Only Pressure-O₂/CO₂ could demonstrate a significantly increased urine production compared to Flow-O₂. The structural integrity was well preserved in the Pressure-O₂ and Pressure-O₂/CO₂ groups, whereas diffuse and global glomerular destruction was observed in the Flow-O₂ group. Conclusion: In the IPPK model, the application of pressure-controlled reperfusion with carbogen oxygenation, and to a lesser extent with pure oxygen, maintained physiological renal function for 1 h, thus providing a reliable and reproducible ex vivo evaluation of kidney preservation quality.

Extracorporeal perfusion of isolated organs of large animals – Bridging the gap between in vitro and in vivo studies

“…Flow controlled set-ups have been used most often (Chapman et al, 1961;Villeneuve et al, 1996;Grosse-Siestrup et al, 2002b;Zeitlin and Eshraghi, 2002;Dragu et al, 2011), but newer evidence suggests that pressure controlled perfusion improves organ preservation (Mancina et al, 2015). Two strategies may be applied in flow controlled experiments: a low flow or a high flow approach.…”

“…Carbogen may be chosen when the buffer capacity of commonly used cell-free perfusion solutions depends on the presence of CO 2 (Mancina et al, 2015) or when using solutions without oxygen carriers, as these require a higher O 2 content than in room air to meet the tissues' oxygen demand. Pure oxygen is rarely used in perfusion experiments as unphysiologically high oxygen tension may lead to the production of free radicals (Fuller and Lee, 2007) and a lack of buffer capacity of CO 2 may cause a pH drop.…”

“…Whole blood also contains erythrocytes which represent an optimal oxygen carrier and therefore enable sufficient oxygen transport to perfused tissues (Mancina et al, 2015).…”

“…So, despite the reduced concentration of oxygen carriers due to its dilution, the improved flow rate leads to better tissue supply and consequently to superior organ function (Dittrich et al, 2000). For dilution purposes, plasma (Patan et al, 2009) or artificial solutions such as Tyrode solution (Friebe et al, 2013b), modified Krebs-Henseleit buffer (Mancina et al, 2015) or dialysate solution (acetate hemodialysis concentrate HD 22, Fresenius Medical Care, Bad Homburg, Germany) (Wagner et al, 2003) have been used successfully.…”

“…This is reflected in relatively low weight gain from edema formation in experiments using whole blood as perfusate (Patan et al, 2009;Constantinescu et al, 2011;Mancina et al, 2015). However, one report shows a higher weight gain in limbs perfused over five hours with diluted autologous blood (blood: Tyrode solution 4:1) compared to limbs perfused over eight hours with adapted Tyrode solution containing sodium…”