Euthatal

All animal procedures were approved by the Alfred Medical Research and Education Precinct (AMREP) Animal Ethics Committee in accordance with the National Health and Medical Research Council of Australia guidelines. The animals were maintained on a 12-hour light/dark cycle with unrestricted access to water and standard chow (Specialty Feeds, Glen Forrest, WA, Australia) under pathogen-free conditions in the Precinct Animal Centre of the Baker IDI Heart and Diabetes Institute. Nox4^−/− [21 (link)] mice were backcrossed with Apoe^−/− mice on the C57BL/6J background (B6.129P2-Apoe^tm1Unc /J, Jax Labs, Bar Harbor, ME, USA) for 10 generations to generate Nox4^−/−Apoe^−/− double knockout animals (Transnetyx, USA); as previously described in [9 ]. Diabetes was induced in six-week-old Nox4^−/−Apoe^−/− and Nox4^−/−Apoe^−/− wildtype male mice by five daily i.p. injections of streptozotocin [55 mg/kg dissolved in citrate buffer (Sigma-Aldrich, St Louis, MO, USA)] [22 (link)]. Mice with a blood glucose level ≥15 mmol/L were deemed diabetic and included in the study. After 20 weeks, the mice were killed by an i.p. injection of sodium pentobarbital (100 mg/kg; Euthatal, Sigma-Aldrich, Castle Hill, NSW, Australia).

Diabetes was induced in 6-week-old male floxedNox4, podNox4KO and podCre mice by five daily i.p. injections of streptozotocin (Sigma-Aldrich, St Louis, MO, USA), at a dose of 55 mg/kg body weight in citrate buffer, with control mice receiving citrate buffer alone. Only mice with blood glucose ≥15 mmol/l after injection of streptozotocin have been included in experiments; mice with blood glucose <15 mmol/l were excluded from the study (<10% of the total number of mice). After 10 and 20 weeks, the animals were anaesthetised using sodium pentobarbitone i.p. (100 mg/kg body weight; Euthatal, Sigma-Aldrich, Castle Hill, NSW, Australia). The kidneys were rapidly dissected, weighed and snap-frozen or processed in paraffin for subsequent analysis.