*Purpose: Ischemia reperfusion injury (IRI) affects all solid organ transplants and impairs renal graft function. However, the mechanisms by which IRI affects grafts are incompletely understood and effective therapies for IRI are lacking. Emerging evidence points to mitochondrial dysfunction playing a central role in IRI. While studying normothermic ex vivo kidney perfusion, we found that mitochondrial proteins (e.g. CPT2, ETFB) were significantly increased in association with superior graft function (Mol & Cell Proteomics 2021). We aimed to determine the key regulator of these mitochondrial proteins and to elucidate their role in IRI.

*Methods: Computational prediction using ChEA3 and Catrin was applied on the list of proteins increased by normothermic ex vivo kidney perfusion. Primary human proximal tubular epithelial cells (PTECs) were treated with pharmacological inhibitor of HNF4α or vehicle and exposed to 0, 12 or 24 hours of hypoxia (1% O₂) or normoxia. LDH release and gene expression were examined. Mitochondrial function was studied using a Seahorse instrument.

*Results: Two computational analyses predicted HNF4α as a key transcriptional regulator of 63/70 of the mitochondrial proteins identified. Compared to vehicle-treated PTECs, HNF4α inhibitor significantly increased LDH release (p<0.05) in a time and dose-dependent fashion under hypoxia. In parallel, HNF4A transcript was significantly decreased (p=0.044) relative to vehicle at 24 hours of hypoxia. HNF4α inhibitor also increased LDH in PTECs under normoxia, and caused a significant decline (p=0.004 at 6h, p=0.048 at 24h) in PPARGC1A, a master-regulator of mitochondrial biogenesis. Preliminary studies indicated reduced mitochondrial function in PTECs treated with HNF4α inhibitor that mimicked the effect of hypoxia.

*Conclusions: HNF4α inhibition increases proximal tubular cell death and appears to decrease mitochondrial function and biogenesis. Thus, HNF4α may play an important role in kidney IRI and in maintaining proximal tubular health. Increasing the activity of HNF4α may thus drive a protective phenotype in proximal tubular epithelial cells and ameliorate kidney injury.

« Back to 2022 American Transplant Congress