Modeling Hypoxia/Reoxygenation Injury in Proximal Tubular Epithelial Cells

Abstract

Kidney transplantation accounts for approximately 60%-65% of all transplanted solid organs. Most donor kidneys are obtained from deceased individuals, requiring extended cold preservation, which is a known contributor to poor transplant outcomes. Despite current preservation strategies, ischemia-reperfusion injury (IRI) remains an unavoidable consequence of transient blood flow interruption, leading to oxygen and nutrient deprivation. Within the nephron, proximal tubular epithelial cells (PTECs) of the S3 segment are particularly susceptible to IRI due to their high metabolic demand and dependence on mitochondrial oxidative phosphorylation. At the molecular level, IRI disrupts mitochondrial metabolism and reduces ATP production, compromising the energy requirements of proximal tubular epithelial cells (PTECs) and promoting apoptosis and necrosis. To investigate these mechanisms and evaluate potential therapeutic strategies, robust and reproducible in vitro models of renal IRI that accurately recapitulate the metabolic vulnerability of PTECs are essential. Here, we describe a protocol for the induction and assessment of hypoxia/reoxygenation (H/R) injury in murine immortalized PTECs (IM-PTECs). The protocol includes detailed information on the medium composition and culture conditions required to maintain these cells, followed by the induction of H/R injury through controlled hypoxia and reoxygenation phases that closely mimic the ischemia and reperfusion events in transplanted kidneys. This model provides a valuable platform for evaluating the effects of different interventions on renal epithelial cells exposed to H/R injury. The impact of these treatments can be assessed through the analysis of the expression of markers associated with PT damage, as well as through the assessment of the mitochondrial respiratory function. Together, these readouts offer mechanistic insights into compound efficacy and cellular recovery processes, supporting the development of targeted therapies for renal IRI.