*3.1. Renal Function*

Uraemic animals demonstrated reduced renal clearance (estimated by glomerular filtration rate (GFR)) as evidenced by increased serum creatinine and urea concentrations (Table 1). Increased urinary and decreased serum protein (indicators of proteinuria) supported the renal insu fficiency observed in this model. The remnant kidney underwent significant remodelling indicated by an increase of 37% and 48% kidney mass in untreated and iron treated uraemic groups, respectively (Figure 1).

**Table 1.** Markers of renal function in sham and uraemic animals with and without iv iron. Data are presented as mean ± SEM. GFR (glomerular filtration rate) \* *p* < 0.05; sham versus uraemic; }*p* < 0.05; uraemic untreated versus treated.


**Figure 1.** Renal hypertrophy in uraemic and sham animals (*n* = 22) and in animals exposed to intravenous (iv) iron therapy at six weeks was assessed by measuring left kidney mass. Data are presented as mean ± SEM, (\* *p* < 0.05 and \*\* *p* < 0.01).

Uraemic animals exposed to iv iron therapy had a significantly lower serum creatinine (*p* < 0.05) and increased serum protein than those without iron. There was no change in the degree of proteinuria or renal dysfunction as a result of iron therapy in the uraemic group (Table 1).

## *3.2. Left Ventricular (LV) Hypertrophy*

Induction of uraemia resulted in significant cardiac hypertrophy evidenced by an increased heart weight to tibia length ratio (HW/TL) (Figure 2). This is in agreemen<sup>t</sup> with previous observations [17]. Administration of iron did not impact on the extent of LV hypertrophy.

**Figure 2.** Cardiac hypertrophy 12 weeks post-surgical induction of uraemia was evaluated by measurement of heart weight to tibia length ratio in uraemia and sham animals with and without iv iron. Data are presented as mean ± SEM (\* *p* < 0.05).

#### *3.3. Anaemia and Iron Status*

The iron profile and packed cell volume in this model is given in Table 2. Uraemia was associated with anaemia characterised by a reduced haematocrit (Figure 3A) and a decreased serum iron. There was also increased faecal iron loss which may reflect reduced absorption or possible gastrointestinal bleeding and also increased urinary loss (Figure 3C). Serum transferrin was reduced alongside enhanced urinary loss (Figure 4) and lowered total iron binding capacity (TIBC). Liver iron stores and cardiac total iron concentrations were unchanged.

**Table 2.** Markers of iron status in sham and uraemic animals with and without iron therapy. Data are presented as mean ± SEM. \* *p* < 0.05; sham versus uraemic. }*p* < 0.05; uraemic untreated versus treated. TIBC = total iron bonding capacity.


**Figure 3.** Iron analysis. (**A**) Haematocrit was measured to confirm anaemia; (**B**) Faecal Iron loss as a measure of iron malabsorption; (**C**) Urinary iron excretion was evaluated to study the cause of iron deficiency. Data are presented as mean ± SEM (\* *p* < 0.05, \*\* *p* < 0.01).

**Figure 4.** Transferrin analysis (**A**): Serum transferrin level and (**B**): Urinary transferrin loss at various stages of uraemia. Data are presented as mean ± SEM (\* *p* < 0.05, \*\* *p* < 0.01).

## *3.4. Impact of Iron Therapy*

Intravenous iron therapy had a modest impact on iron deficiency anaemia in uraemic animals. There was restoration of serum transferrin and TIBC to a level similar to that observed in the sham group with an 8% increased PCV in the uraemic group without any significant change in the sham animals (Table 2). Increased faecal iron content was observed in the iron treated sham group (Figure 3B). Urinary transferrin and iron excretion in uraemic and sham operated groups were unchanged by week 12 in treated animals, in contrast to measurements 3 weeks after the iron bolus (Figure 4). Total iron measured by the elemental iron analysis in the uraemic remnant kidney was 46% higher in the treated group relative to the baseline data; this did not reach statistical significance. In cardiac tissue, there was a non-significant 22% reduction. Liver iron was increased significantly by 33% in the iron treated uraemic group relative to the iron treated sham group. This reflected a 45% increase relative to the untreated uraemic group (Table 2).

#### *3.5. Systemic and Renal Oxidative Stress*

This experimental model of CKD was associated with a 24% reduction of systemic GPx antioxidant activity (Figure 5) without evidence of systemic lipid peroxidation (Figure 6). This may reflect a possibly generalised reduction in protein synthesis or a marker of oxidative stress. There was an increased concentration of oxidised glutathione (GSSG) (Figure 7A) in the remnant kidney without any change in the reduced form (GSH) (Figure 7B). Treatment with iv iron was associated with reduced TBARS (*p* < 0.01) and upregulation of systemic GPx activity by 35% and 32% in sham and uraemic groups, respectively, relative to untreated but lower levels when comparing sham versus uraemic exposed to iv iron (Figures 5 and 6).

**Figure 5.** Serum glutathione peroxidase activity. Systemic anti-oxidant capacity was investigated through the measurement of glutathione peroxidase activity in the serum of uraemic and sham animals. Results are presented as mean ± SEM (\* *p* < 0.05).

**Figure 6.** Serum TBARS. TBARS (thiobarbuturic acid reactive substances) were measured to access lipid peroxidation in uraemic and sham animals. Results are presented as mean ± SEM. (\* *p* < 0.01).

**Figure 7.** Endogenous antioxidant glutathione level in kidney tissue. (**A**) Renal oxidised glutathione in sham (*n* = 10) and uraemic animals (*n* = 10) with and without i.v. iron therapy; (**B**) renal reduced glutathione in sham (*n* = 10) and uraemic animals (*n* = 10) with and without i.v. iron therapy. Results are presented as mean ± SEM. (\* *p* < 0.05).
