*2.2. Model Characterisation*

Urine samples collected over 24 h was filtered through Millex syringe-driven Filter unit (Merck KGaA, Darmstadt, Germany) and serum samples collected at week 12 were analysed using the RX Monza analyser (Randox, Antrim, UK) for creatinine, urea and total protein according to manufacturer's protocol. Renal function was assessed by glomerular filtration rate (GFR) or creatinine clearance calculated using equation 1. Cardiac hypertrophy was evaluated by wet heart weight to tibia length ratio (HW/TL). Haematocrit was measured on an ABL77 Radiometer (Battery Universe Inc., USA) to confirm anaemia and packed cell volume (PCV) was subsequently measured to assess the impact of iv iron on anaemia. Briefly, heparinised blood samples were centrifuged and the ratio of packed red cell volume to whole blood volume calculated to give PCV. Iron status was determined from serum and urine biochemistry. Markers of iron status including serum iron, transferrin and total iron binding capacity were measured on the RX Monza analyser using Randox kits (Randox Laboratory Ltd., Crumlin, UK) as were urine samples.

Serum ferritin was analysed using the Enzyme linked immunosorbent assay ELISA ferritin commercial Kit (Abcam, Cambridge UK). Hepatic, renal and cardiac tissue contents of non-bound and total iron were measured. Briefly, 200 mg of tissue or faecal excrete was extracted with 1 mL 7.35 mM sodium acetate trihydrate bu ffer (4.65 pH) for 10 min and centrifuged at 12,000× *g*, 4 ◦C in a microfuge (Scientific Laboratory Supplies, UK) for 10 min and the supernatant filtered through Millex syringe-driven Filter unit (Merck KGaA, Germany), the resultant filtrate and urine filtrate were analysed for non-bound iron on the RX Monza.

Hepatic, renal and cardiac iron contents were evaluated by total elemental iron analysis on Perkin Elmer Optima 5300DV emission ICP-OES instrument (PerkinElmer, Inc, Waltham, MA, USA). Briefly, Tissue or serum samples were extracted with concentrated HNO3 (Romil SpA trace metals, Cambridge UK) and digested in Teflon microwave vessels (MARS Xpress, CEM Ltd., Buckingham

UK). The samples were allowed to cool, diluted with ultra-pure water and analysed on the Perkin Elmer Optima 5300 DV emission ICP instrument.

$$\frac{\text{Creatinine Clearance}}{\text{(mL/min/kg body weight)}} = \frac{\left[\frac{\text{Creatinine}\_{\text{wire}}}{\text{Time} \left(\text{h}\right) \times 60}\right]}{\text{Body Weight (kg)}}$$
