Normothermic human kidney preservation drives iron accumulation and ferroptosis

Introduction

Machine perfusion technologies have emerged as an important new modality in the field of organ preservation and transplantation. To address the growing disparity between the available donor organs and the number of patients on the transplant waiting list¹, significant efforts have been devoted to developing advanced perfusion platforms. These platforms aim to create a window of opportunity to facilitate the assessment, reconditioning, repair, and regeneration of deceased donor organs².

While normothermic machine perfusion (NMP) techniques for liver transplantation have become routine clinical practice^3,4, successful translation into the field of kidney transplantation, which constitutes the majority of transplant organs, remains elusive. This raises the question how ex vivo NMP of deceased donor livers has been extended up to 12 days⁵, with successful transplantation after 3 days of perfusion⁶, whereas studies on normothermic kidney perfusion typically range from 1 to 6 h, and even then, the first randomized controlled trial was discontinued in the absence of a discernible positive effect⁷. This is concerning given the increasing clinical application of normothermic kidney perfusion by organ procurement organizations and highlights the need to critically evaluate whether all organs are equally suited for each preservation method.

In this study, we sought to elucidate the molecular changes that occur in kidney grafts during NMP, encompassing a detailed analysis of the multi-day perfusion of 19 deceased human donor kidneys. Of these, seven kidneys were perfused up to 48-h. We observed that hemolysis provokes iron accumulation in perfusate and tissue. Through untargeted lipidomic profiling we could confirm the accumulation of oxidized phospholipid species (oxPL), pointing towards iron-dependent cell death known as ferroptosis. Next, we demonstrate that dialysis-based free hemoglobin (fHb) removal reduces lipid peroxidation, but a ferroptosis gene signature persists. Finally, we show that cell-free perfusion at subnormothermia, an approach we recently demonstrated to support four-day human kidney preservation⁸, negates iron accumulation, the ferroptosis gene signature, phospholipid peroxidation, and acute kidney injury (AKI).

Results

Study design and donor characteristics

In total, 19 human donor kidneys deemed unsuitable for transplantation were perfused. During the first phase, seven kidneys were perfused up to 48-h at normothermia with an RBC-based perfusate. In the second phase, an additional twelve human kidneys were included...