The percentage of abnormal glomeruli was determined by examining a minimum of 50 glomeruli/mouse for abnormalities according to previously published protocols . Abnormalities included glomerular hypercellularity, crescent formation, fibrinoid necrosis, segmental proliferation, hyalinosis and capillary wall thickening. Urine was collected using metabolic cages for 24 h prior to the end of experiments. Proteinuria was determined using a modified
Bradford assay and expressed as mg/24 h . Serum creatinine measurements were recorded after termination of the experiment using an alkaline picric acid method and an auto-analyser. Urinary nitric oxide (NO) was measured as described previously, using a Griess assay . Urine samples (collected GSK-3 activation from mice for a 24-h period before killing) were centrifuged at 2000 g for 10 min. A total of 50-µl aliquots of urine were added to 50 µl of Griess reagent (1·5% sulphanilamide/0·15% naphthyl ethylene diamine) in a 96-well selleck microtitre plate. Samples were incubated for 10 min at room temperature
and absorbance read at 540 nm. Urinary nitrite concentration was determined from standards of sodium nitrite of known concentrations. Samples were tested in duplicate and measured as micromolars (µm) per 24 h. Kidneys were fixed in periodate lysine paraformaldehyde for 4 h, washed with 20% sucrose solution, and then frozen in liquid nitrogen. Tissue sections were cut and a three-layered immunoperoxidase technique was used to stain for CD4+ T cells and macrophages. The primary antibodies used were GK1·5 for CD4+ T cells [anti-mouse CD4; American Type Culture Collection (ATCC), Manassas, VA, USA] and FA/11 for macrophages (anti-mouse CD68, provided by Dr G. Koch, Cambridge, UK). The secondary antibody used was rabbit anti-rat biotin (BD Biosciences, San Jose, CA, USA). A minimum of 20 consecutively viewed glomeruli were
assessed per animal. Results are expressed as cells per glomerular cross section (c/gcs) described previously . For measurement of T-bet, GATA3 and RORγ by reverse transcription–polymerase chain reaction (RT–PCR), 500 ng of RNA was treated with 1 unit of amplification grade DNase I (Invitrogen, Melbourne, Australia), Etofibrate primed with random primers (Applied Biosystems, Foster City, CA, USA) and reverse-transcribed using a high-capacity cDNA reverse transcription kit (Applied Biosystems). Gene-specific oligonucleotide primers designed using the Primer 3 software (Whitehead Institute for Biomedical Research, Cambridge, MA, USA) were synthesized by Invitrogen, using a protocol described previously . A Rotor Gene RG-3000 (Corbett Research, Mortlake, Australia) using Power SYBR green PCR master mix (Applied Biosystems) was used to perform RT–PCR.