Experimental design Beginning at 20 weeks of age, disease progression was mon itored weekly by assessing proteinuria. A cohort of mice, selected at 20 weeks of age, served as the asymptomatic group. Once fixed proteinuria of 30 to 100 mgdL had appeared on two consecutive occasions. the diseased mice were randomly assigned to either the sirolimus treated group or the untreated group. Sirolimus, dissolved in car boxymethylcellulose, was subcutaneously adminis tered three times weekly in single doses of 1 mgkg or 5 mg kg for eight weeks. Mice were monitored weekly until 52 weeks of age. Assessment of proteinuria Urine was manually expressed from each mouse on a weekly basis, collected into a sterile container and assayed for the presence of protein using a colorimetric method.
Proteinuria evaluations were scored as follows grade 0. 5trace proteinuria. grade 1about 30 mgdL. grade 2about 100 mgdL. grade 3about 300 mgdL. and grade 4more than 2000 mgdL. If mice achieved a grade 4 reading on two consecutive JAK inhibitor days they were euthanised. Assessment of renal pathology Kidneys were harvested from mice one to four months after an eight week course of treatment. Three to five mice were exam ined in each group. One half of a kidney was fixed by overnight immersion in 10% formaldehyde and paraffin embedded. The other half was snap frozen for RNA preparation. To determine the extent of renal damage, sections were stained with H E and periodic acid Schiff and scored for pathological changes. In addition, glomerulopathy was scored on a 0 to 5 scale. Severity grades were as follows 0normal or within normal limits.
1minimal or slight. 2mild. 3moderate. 4marked. 5severe. RNA purification and microarray hybridisation Snap frozen murine kidney tissue was homogenised in RLT buffer containing 1% beta mercaptoethanol selleck inhibitor using the polytron and RNA purified by Qiagen RNeasy columns. Eluted RNA was quantified using a Spectramax 96 well plate UV reader monitoring A260280 OD values. The quality of each RNA sample was assessed by capillary electrophoresis alongside an RNA molecular weight ladder on the Agilent 2100 bioana lyser. Microarray processing Five micrograms of total kidney RNA was prepared from indi vidual NZBW F1 mice of the following groups. untreated mice at 12 weeks. untreated F1 mice at 36 and 42 weeks combined. and sirolimus treated mice at 36 and 42 weeks combined. The animals selected for expres sion analysis reported here were representative of multiple studies performed that confirmed the data on proteinuria, mor tality rates and histopathology here. Biotin labelled cRNA was prepared using an oligo T7 primed reverse transcription reaction followed by in vitro transcription reaction with biotin labelled UTP and CTP.