**2. Materials and Methods**

#### *2.1. Induction of Experimental Uraemic Model*

All procedure and animals in this study were in accordance with the UK Animals (Scientific Procedure) Act 1986 and were approved by the University of Hull Ethical Review Process (No. PPL 70/7966). Experimental uraemia was induced in male Sprague-Dawley rats (obtained from Charles River Laboratories, Kent, UK) via a one-stage subtotal nephrectomy, as described previously [17].

Briefly, animals (250 g) were anaesthetised with 3% isofluorane in 3 L/min O2 and subsequently maintained on 2.5% isofluorane in 1 L/min oxygen. Depth of anaesthesia was confirmed using the pedal withdrawal reflex. Rimadyl was administered (4 mg/Kg body weight) pre-operatively via s/c injection for post-operative pain relief. A midline abdominal incision was made and the left kidney was exposed and decapsulated. Following clamping of the renal vasculature, approximately half of the kidney was excised comprised principally of cortical tissue. Haemorrhaging was controlled using Surgicel ® (Johnson & Johnson, Maidenhead, Berkshire, UK) before the remnant kidney was replaced. The right kidney was then exposed and decapsulated. Renal vasculature was ligated using a non-absorbable suture (Mersilk ® Johnson & Johnson, Maidenhead, Berkshire, UK) prior to excision of the kidney. Sterile isotonic saline (0.9% w/v) was administered into the abdominal cavity prior to closure to compensate for intraoperative fluid losses. The abdominal muscular layer was closed using an absorbable suture (Ethicon 3-0 Vicryl braided, Johnson & Johnson, Maidenhead, Berkshire, UK.) The dermal layer was closed with non-absorbable sutures (Ethicon 3-0 blue monofilament, Johnson & Johnson, Maidenhead, Berkshire, UK.) Sham animals were subjected to a sham procedure comprised of exposure and decapsulation of both kidneys.

Iron therapy was initiated 6 weeks post-surgery by a single intravenous (iv) injection of ferumoxytol (supplied by Takaeda UK Ltd., Holborn, London, UK) at a dose of 10 mg/Kg body weight. All animals were maintained in individual cages for a total of 12 weeks post-surgery (six weeks after the iv iron) and pair fed (sham and uraemic) with a standard chow diet. Water was available ad libitum.
