Introduction: Ischemia reperfusion injury (IRI) causes inflammation and cell death in kidneys obtained from donation after circulatory death (DCD) donors. This leads to poor organ function posttransplant. Therapeutics that prevent such damage, caused by hyperactive Toll-like receptors (TLR) systems, should be helpful in preserving organ quality pretransplant. We have previously reported that perfusion of porcine DCD kidneys with an oxygen carrier at 22°C significantly reduces TLR-induced inflammation and injury. To further improve this method, and therefore facilitate a high-throughput screening of a drug library, we have developed a simulated ischemia reperfusion injury (sIRI) model in human kidney tubular HK-2 cells.

Method: To mimic clinical IRI conditions, HK-2 cells are subjected to hypoxia for 1h at 37°C in a hypoxia chamber, sealed in a bag for 24h at 4°C (anoxia), and reoxygenated at 37°C for 24h. Cell supernatants are tested for injury markers (KIM1, NGAL), TLR ligands (damage associated molecular patterns; DAMPs) and inflammation markers (IL-6, TNF-α etc.) by ELISA. Cell death and viability are also assessed. For drug repurposing, THP1-Blue™ cells are incubated with DAMPs+drug simultaneously for 24h. NF-κB-inducible alkaline phosphatase reporter gene products are measured by Quanti-Blue. Optimization of discarded human kidney perfusion methods are currently underway.

Preservation of human kidneys with subnormothermic machine perfusion: Human DCD kidneys were obtained in pairs with matching blood (~ 1L) from the same donor or Canadian Blood Services. Organs were flushed with cold HTK solution and subjected to cold storage at 4°C or pulsatile blood perfusion at 22°C for 12h with hemoglobin-based oxygen carrier solution. Thereafter, kidneys are reperfused with normothermic (37°C) oxygenated blood for 4h. Blood and urine were collected every hour for biochemical analysis. Total urine output, urinary protein, albumin/creatinine ratio, and flow rate are measured. The kidneys were examined for acute tubular necrosis (H&E), apoptosis (TUNEL) and inflammation.

Result: Our results show sIRI condition increases cell death by both necrosis and apoptosis. DAMPs such as HMGB1, which activates the innate immune system through TLRs, and other predominant cytokines, are present in sIRI cell culture supernatants, as indicated by bead-based high throughput Multiplex ELISA assays. Kidneys preserved at 22°C had greater creatinine clearance and urine production compared to 4°C cold storage. Also, ischemia reperfusion injury-induced inflammatory markers were markedly reduced at 22°C compared to 4°C preservation. However, H&E and TUNEL scores may require further modification of evaluation.

Significance: Our model provides the foundation for future research aimed at improving organ preservation. The identification of a drug candidate that can ameliorate the damage from IRI will help optimize current organ preservation conditions.