**1. Introduction**

Iron-deficiency anaemia (IDA) is a major health problem worldwide. It is commonly associated with the progression of chronic kidney disease (CKD), affecting both quality of life and mortality [1–4]. Iron deficiency can exacerbate mitochondrial dysfunction and enhance oxidative stress in this patient population [5]. Mitochondrial proteins involved in oxidative phosphorylation (OXPHOS) pathway (respiratory complexes I-III) are iron complexes. Other mitochondrial enzymes, including aconitase of the Krebs cycle, require iron-sulphur (Fe-S) clusters (ISC) for their function [6,7]. Thus, derailment of mitochondrial iron homeostasis can result in human diseases associated with mitochondrial dysfunction such as Friedreich's ataxia [8]. Iron deficiency distorts the tightly regulated biosynthesis of haem and ISC which are needed for mitochondrial function and aggravate mitochondrial oxidative stress [9]. Mitochondria are also increasingly recognised as major contributors to reactive oxygen species (ROS) production [10]. In dysfunctional mitochondria, the proton or potentially electron leak at complexes I and III causes excessive wasting of molecular oxygen into superoxide radical (O2•<sup>−</sup>). This can lead to the generation of more radicals (hydoxy (OH−) and peroxynitrite (ONOO−) radicals) [10]. Therefore, parenteral iron therapy employed in clinical practice as an integral component of managing anaemia of CKD may enhance mitochondrial function, and reduce overall oxidative stress (pro-oxidant versus anti-oxidant activity) without compromising renal function.

The mechanisms of progression of CKD are complex and multi-factorial and not completely elucidated. However, evidence implicating mitochondrial dysfunction in the initiation and progression of kidney disease comes from mitochondrial cytopathies [11]. Mitochondrial cytopathies (inherited or sporadic mtDNA mutations in mitochondrial genes) in kidneys lead to glomerular diseases, tubular defects and cystic kidney disease. Focal segmental glomerular sclerosis (FSGS), an example of glomerular disease emanating from mtDNA mutations, is a frequent cause of end stage kidney disease [12,13]. Other evidence of mitochondrial dysfunction in renal disease has come from in vivo and in vitro studies [14,15]. Mitochondrial dysfunction with reduced complex I, II and IV expression potentiated podocytes injury, impaired nephrin synthesis and increased ROS production [14]. Patients with iron deficiency anaemia from causes such as gastro-intestinal loss or menstruation do not appear to develop overt renal dysfunction. This suggests compensatory upregulation of mitochondrial function even in uraemic scenarios [16]. Although there is evidence of iron deficiency in CKD, tissue iron levels may vary between organs and may be preserved in the kidney minimising any detrimental renal effect.

Studies to date have provided evidence that the 5/6 nephrectomy used in this study is a suitable animal model of CKD showing enhanced vulnerability to oxidative stress and mitochondrial dysfunction [17]. However, limited information is available on the iron status of the model and response to therapy with intravenous (iv) iron. Most studies on the impact of iv iron are limited to acute systemic oxidative effects, disregarding their longer-term impact and benefits. Despite the central role of mitochondria (within the kidney) in the possible initiation and progression of CKD [18–20] and the integral role of iron in key mitochondrial proteins (aconitase, complex I, II or III), the impact of parenteral iv iron therapy as employed in clinical practice on mitochondrial function, oxidative stress and CKD progression has not been sufficiently characterised.

The aim of this study was to determine the longer-term impact of iv iron administration on these parameters in an experimental animal model of CKD. The Ferumoxytol therapy (one form of available parenteral iron) protocol used in this study mimics that of clinical practice using an equivalent weight adjusted bolus injection at a dose of 510 mg in adults (i.e., 8–10 mg/Kg body weight) [21]. Ferumoxytol is a third-generation iron complex whose slow dissociation from the carbohydrate complex leads to the release of less "labile" iron, thus allowing a rapid bolus infusion of high doses clinically with favourable outcomes [22,23].

Initially, the aim was to ensure that the phenotype of the uraemic model exhibited IDA and further that iv iron therapy would ameliorate this deficiency. It was hypothesised that compromised renal mitochondrial function in this model would increase susceptibility to oxidative stress. Parenteral iron therapy could improve mitochondrial function of the remnant kidney, lessening the deleterious effect of oxidative stress. Therefore, iron status (systemic and tissue) and iron deficiency (ID) anaemia in the 5/6 nephrectomy model of CKD were studied and the impact of iv iron investigated. Hence, renal mitochondrial function in uraemia and renal and systemic oxidative stress before and following iron treatment were studied in detail.
