Introduction: Optimization of organ utilization is critical to overcome the continually increasing organ shortage. Both high quality and marginal organs are often discarded due to prolonged cold ischemic times during storage. The aim of this study is to investigate the functional impacts of static cold storage (SCS) on kidneys using a normothermic machine perfusion model (NMP).  
Methods:Five porcine kidney pairs were either subjected to 24 hours of SCS followed by 48 hours of NMP (SCS group) or continual NMP for 48 hours (control group) using a novel prototype device. An autologous, leukocyte filtered, whole blood-based perfusate was used. Cortex biopsies were taken before, during, and after perfusion to assess mitochondrial function using high-resolution respirometry. Histological assessment was carried out using the Martius Scarlet Blue (MSB) staining. Perfusate and urine were sampled and analyzed at different timepoints during the perfusion.
Results: In both groups, 48 hours stable perfusion was achieved. Median flow rates were lower (244 mL/min vs. 277 mL/min, p < 0.0001) and corresponding renal resistances higher (0.39 min/mL/mmHg vs. 0.33 min/mL/mmHg, p < 0.0001) in the SCS group compared to the control. Median injury marker levels including ASAT (SCS vs. control group: 1341 U/L vs. 184 U/L, p = 0.031), LDH (1598 U/L vs. 909 U/L, p = 0.031) and NGAL (319 ng/mL vs. 143 ng/mL, p = 0.002) were significantly higher in the SCS group. While respiratory capacity declined in all kidneys, the efficiency of oxidative phosphorylation remained significantly higher in the control group (p = 0.016). In the SCS kidneys, pronounced suburothelial bleedings were found in the kidney pelvis and proximal ureter that were absent in control kidneys. Prolonged SCS resulted in a marked reduction of coagulation Factors II, VII and X (median SCS vs. control group: 14% vs. 22%, p = 0.031; 10% vs. 24%, p = 0.016; 24% vs. 35%, p = 0.031), increasing serum levels of tissue factor, a reduced maximal clot firmness early after reperfusion, and prolonged clotting times after 24 hours perfusion time.
Conclusion: Extended SCS exerts adverse effects on hemodynamics, metabolism and tissue integrity upon reperfusion. We have identified significant suburothelial bleedings resulting from extended SCS as a previously unrecognized manifestation of ischemia reperfusion injury.