🎗Mitochondrial injury in human and mouse kidneys with ischemia reperfusion injury

Kidney transplantation is the best treatment for end-stage renal disease. However, ischemia-reperfusion injury (IRI) harms all transplanted kidneys, limiting their survival, with living donor (LD) kidneys being the least affected. Normothermic ex vivo kidney perfusion (NEVKP) is a storage method resulting in superior graft outcomes compared to static cold storage (SCS). Previously, we showed that NEVKP improves kidney function/structure after IRI and is characterized by preserved mitochondrial protein expression (i.e ETFB and CPT2) in a porcine model of autotransplantation. We aimed to examine the impact of NEVKP, SCS, and LD grafts, on mitochondrial health after transplantation, and the impact of IRI severity on kidney mitochondrial gene and protein expression.
Human kidney grafts were from LD or deceased donors (DD) that were stored in NEVKP or SCS. Biopsies were taken prior to implantation (DD-SCS-1, DD-NEVKP-1, LD) and at the time of delayed graft function (DGF) (DD-SCS-2 or DD-NEVKP-2). Patient blood was collected shortly after transplant (<7 days; DD-SCS-1, DD-NEVKP-1, LD) and at the time of DGF (DD-SCS-2 or DD-NEVKP-2). Immunohistochemistry was used to determine ETFB expression, and circulating cell-free mitochondrial DNA (ccf-mtDNA) was measured in patient plasma. Plasma proteomics was done with the SomaScan assay. Mice underwent unilateral IRI (uIRI) for 35 min with contralateral nephrectomy on post-op day 14 and sacrificed one day later.
Human kidney allograft expression of ETFB was significantly reduced at the time of DGF in grafts stored by SCS compared to NEVKP, which had higher expression than LD grafts (fig 1A-G). Ccf-mtDNA genes (ND1, ND4) were elevated in SCS compared to NEVKP plasma, supporting enhanced mitochondrial injury (fig 1H). SCS at DGF and NEVKP shortly after transplant had a significant inverse correlation between ccf-mtDNA and kidney ETFB expression (fig 1I-L). Plasma proteomics revealed significantly increased expression of proteins mediating mitochondrial triggered apoptosis, NLRP3 inflammasome, mitochondrial fatty acid oxidation, and mitochondrial fission in SCS patients, compared to NEVKP and LD (fig. 1M-O). uIRI model showed increases in serum creatinine (fig 2A-B) and fibrosis on post-op day 15 (fig 2C). In the uIRI model, male and female mice had reduced ETFB protein expression (fig 2D) and gene expression of Etfb, Cpt2, and Ppargc1a, (fig 2E-F) in the injured kidney compared to sham.
Severe kidney IRI resulting in DGF was associated with decreased ETFB kidney expression and increased ccf-mtDNA, indicating mitochondrial injury, supported by the plasma proteome. The converse expression of mitochondrial associated proteins in plasma compared to kidney tissue suggests leakage of intracellular contents in IRI. Additionally, the uIRI model supports diminished mitochondrial integrity in mice. These findings support the idea that NEVKP preserves mitochondria integrity, potentially improving long-term kidney grafts.