**4. Discussion**

#### *4.1. Induction of Uraemia and Impact of Iron Therapy*

A significant deterioration of renal function was observed 12 weeks post-surgical induction of uraemia as indicated by the 124.6% and 92.7% increments in serum creatinine and urea, respectively. This is consistent with previous findings [29]. Progressive renal damage causes increased retention of creatinine and urea [30] resulting in decreased urinary levels as observed in Table 1. Impairment of kidney function as a result of persistent and progressive renal damage increases glomerular permeability and decreases tubular protein and fluid reabsorption. This in turn explains the 100% and 94% increments in urinary protein and volume, respectively. Weight gain 12 weeks post-surgery was similar between sham and uraemic groups, showing that malnutrition or loss of muscle mass reported in patients with CKD [31] was less likely to be a confounding factor. The elevated protein loss in urine correlated with a reduction in serum total protein.

The remnant left kidney of uraemic animals underwent compensatory hypertrophy indicated by increased kidney weight without affecting renal function (measured by GFR, Table 1). This change in the remnant kidney could be an adaptive response in an attempt to "normalise" or improve renal function, albeit with limited success. Previous work from this group has demonstrated progressive compensatory hypertrophy of the remnant kidney at week 6 (18.8%; *p* < 0.05), with diminishing function [32].

Parenteral iron treatment did not affect renal function, but increased serum total protein and transferrin concentrations. There are several potential safety issues concerning iv iron therapy including increased oxidative stress, infection and proteinuria as biomarkers [33–35]. Nephrotoxicity of iv iron therapy is dependent on the iron formulation as reported by Agarwal et al. [36]. These authors reported that unlike ferric gluconate, iron sucrose produced a 78% increased proteinuric response that was unaltered following repeated doses of iv iron. This was consistent with other reports of worsening proteinuria in response to iron sucrose therapy but not ferric gluconate [37]. The lack of significant change in urinary proteins levels by 6 weeks post therapy indicated that ferumoxytol did not elicit a chronic proteinuria effect in uraemic animals in this study. This is not unsurprising given that there are known physiochemical differences between iron preparations; hence, this confounder cannot be excluded [38].

#### *4.2. Anaemia in Uraemia and the E*ff*ect of Iron*

Anaemia significantly increases the risk of morbidity and mortality in CKD [39]. Iron deficiency anaemia in CKD is associated with diminished cytochrome c oxidase activity, decreased mitochondrial oxidative capacity and reduced total anti-oxidant capacity resulting in enhanced mitochondrial oxidative stress [40]. Therefore, anaemia correction by erythropoietin and/or iron replenishing therapy is an integral component in the managemen<sup>t</sup> of anaemia of CKD. The observation of reduced haematocrit as an indicator of anaemia is consistent with previous findings, which was improved following erythropoietin treatment [29]. Previous data indicated an inverse correlation between serum creatinine and haematocrit, suggesting the degree of anaemia is related to the severity of renal dysfunction [32]. Iron replenishment therapy via iv administration of iron complexes such as the third generation preparations (ferric carboxymaltose, iron isomaltoside and ferumoxytol) and the older formulations (low molecular weight iron dextran, iron sucrose and ferric gluconate) has proven to be effective in correcting iron deficiency anaemia in CKD [41,42].

Experimental uraemia resulted in a biomarker profile comparable to the clinical scenario of anaemia of inflammatory/chronic disease characterised by decreased serum iron with reduced serum TIBC [43]. This, together with maintenance of ferritin levels and liver iron content in sham and uraemic groups, indicates the inability to access stored iron in uraemia, similar to the classical setting of a pro-inflammatory state [44]. In absolute iron deficiency, reduction of serum iron would indicate a more readily available transferrin for iron binding (giving rise to increased TIBC). The reduction of TIBC

found here can partly be explained by the decreased circulating transferrin (perhaps partly related to the reduction in protein concentrations). This observation was in agreemen<sup>t</sup> with the report of Alfrey and Hammond [45] where serum iron and transferrin decreased rapidly following the induction of nephrotoxic syndrome. The investigators also observed increased urinary iron and transferrin loss similar to this study's observations. Urinary transferrin loss at week 12 of uraemia was 241% greater in uraemic animals compared to sham. Urinary iron loss increased by 66%. The present study has shown an inverse relationship between serum and faecal iron. No sign of blood in the faecal excreta of uraemic animals is indicative of little or no intestinal bleeding. Hence, the increase in faecal iron excretion is suggestive of malabsorption of iron or impaired absorption in the gut. The evidence also highlights increased urinary iron loss in relation to the severity of renal dysfunction. These two factors may be critical in determining the mechanism of iron deficiency anaemia in this model of CKD.

Studies have shown the elevation of inflammatory markers and concomitant upregulated expression of hepcidin [46,47] can lead to functional iron deficiency (ID) anaemia in CKD [48,49]. Given the central role of hepcidin in iron metabolism, assessment of serum, urinary and hepatic hepcidin levels and inflammatory markers such as interleukins (IL-1 and IL-6) could provide insight into the aetiology of iron deficiency. Nonetheless, uraemia may mediate hepcidin over-secretion resulting in enhanced destruction of ferroportin [50] and accumulation of dietary absorbed iron in the enterocytes. Subsequent loss of the iron via enterocyte shedding could explain the 52.7% increment in faecal iron in uraemic animals and the ensuing ID. However, hepcidin over-secretion may also prevent the release of iron from hepatocyte stores, causing an increase in hepatic iron, a phenomenon not observed in the present study. ID impairs red blood cells (RBCs) production, which could explain the presence of anaemia in this study. Persistent anaemia leads to significant compensatory left ventricular hypertrophy (as observed in this model of CKD in Figure 2), which eventually results in congestive heart failure in later stages of uraemia [51].

Iron therapy in the present study resulted in a significantly increased PCV (8%) in the uraemic group. Two prospective randomised studies found that a greater increase in haemoglobin above baseline was observed in CKD patients on ferumoxytol therapy than those on oral iron [52,53]. Parenteral ferumoxutol therapy in a randomised study was effective in raising the mean haemoglobin level and tolerable in patients in whom oral iron was ineffective [53]. Iron treatment restored serum transferrin levels in uraemic animals without changing urinary transferrin loss, suggesting increased synthesis. Serum iron remained low in the iron treated group with significant reduction in the sham group which could in part be due to the enhanced urinary loss. Iron therapy results in a rapid increase in circulating iron levels, which might trigger a homeostatic response [54]. This includes increased expression of transferrin and the transferrin receptor, which mediates cellular iron uptake. It is expressed at low levels in hepatocytes and is down-regulated in response to iron. Liver overload occurs when transferrin is completely saturated and hepatocytes internalise non-transferrin-bound iron present in the bloodstream explaining the observation of increased liver iron. Consequently, over time, total iron binding capacity increases as serum iron falls, as observed here.

Despite maintenance in total cardiac iron content, iron therapy led to a trend in reduced non-bound iron in cardiac and hepatic tissues that accounts for the cytotoxicity of iv iron. Intravenous iron therapy was associated with increased accumulation of iron in the liver. This is similar to the clinical evidence where iv iron therapy in haemodialysis patients was associated with iron accumulation in the spleen and liver but not the heart [55]. The accumulated iron in the spleen and liver serves as reserves that maintained erythropoiesis and could explain the amelioration of anaemia in the present study 6 weeks after bolus iv iron injection. The urinary iron loss alongside the transferrin loss observed in uraemic animals was exacerbated following therapy. There were no mortalities or observable side effects associated with ferumoxytol therapy, though adverse effects such as dizziness, pruritus, headache, fatigue and nausea have been reported in clinical studies [56]. These could not be reliably assessed in this rat model of CKD.
