Introduction: Ex vivo normothermic machine perfusion (NMP) of the kidney may aid in reducing the organ shortage. However, during prolonged preservation periods, red blood cells (RBC) in the perfusion solution are damaged and free hemoglobin and hemin are released. Oxidative stress and mitochondrial damage are induced and may trigger acute and chronic kidney dysfunction. Herein, we investigated the effects of free heme on renal proximal tubular epithelial cells (RPTEC) to assess the effects of hemolysis on these cells. 
Method: RPTEC cells were seeded in 6-well plates (1×10⁶/well) and were allowed to attach for 3h. Cells were then treated with hemin (50µM dissolved in 1% DMSO) or control (1% DMSO) and incubated overnight. The next day, ATP was quantified to assess cell viability and MTS assay was performed to evaluate metabolic activity. The mitochondrial function was assessed by high resolution respirometry including oxidative phosphorylation (OXPHOS), efficiency of ATP production and integrity of the outer mitochondrial membrane. 
Results: The OXPHOS capacity was decreased three fold in the presence of hemin compared to the control (64.86±22.39 vs 24.07±5.25 pmol/s×m, p-value <0.0002, paired t test). The efficiency of ATP production was also diminished (0.83±0.02 vs 0.73±0.05, p-value <0.0003, paired t test) and lower ATP levels were found (p-value <0.0001, one sample t test). Similarly, a decreased metabolic activity was revealed by MTS assay (p-value <0.0001, one sample t test). Outer mitochondrial membrane integrity stayed intact reflected by cytochrome c control efficiencies <0.1 in both groups. 
Conclusion: Free heme leads to a compromised bioenergetic state in RPTEC cells. Thus, hemolysis during kidney NMP may contribute to mitochondrial damage and impaired kidney function.