Wilmes et al. 2013 - Springer Static Content Server

Cell culture and treatment conditions.
RPTEC/TERT1 human proximal tubular epithelial cells (Wieser et al. 2008) were obtained from
Evercyte GmbH, Vienna. Cells were cultured in a 1 to 1 ratio mixture of DMEM and Ham's F-12
nutrient mix (Invitrogen) mix containing 2 mM glutamax, 5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml
sodium selenite, 100 U/ml penicillin, 100 μg/ml streptomycin, 10 ng/ml epithelial growth factor and
36 ng/ml hydrocortisone and routinely split into 10 cm dishes (Sarstedt). Cultures were maintained at
37 °C in a 5 % CO2 humidified atmosphere. For experimental treatments, cells were sub-cultured
onto 96 well plates or 0.2 μm pore size, 25 mm diameter aluminum oxide filters (Nunc) using a
trypsinisation protocol. For treatment, compounds were prepared in complete culture media at the
indicated treatment concentrations with controls containing the corresponding vehicle when used.
Adefovir Dipivoxil, Cisplatin and Ifosfamide were obtained from Sigma-Aldrich, while Cidofovir and
Zoledronate were from Gilead and Alexis respectively. Before treatment, cells were allowed to
stabilise and achieve a differentiated state through maintenance for 10 days (feeding every 2-3 days)
after confluence is reached. Cells were then treated every day for up to 14days and processed for
experimental endpoints as indicated.
Transcriptomic and Pathway analysis.
After treatment, RNA was prepared from RLT buffer (Qiagen) cellular lysates as previously reported
(Leonard et al. 2006).Gene expression analysis was performed using Illumina ® HT 12 v4 BeadChip
arrays (Illumina Inc.) allowing the analysis of ~47,000 individual probe sets. Synthesis of biotinlabelledcRNA was performed using a Theonyx Liquid Performer (Aviso) and MessageAmp™ II aRNA
amplification Kit (Ambion) with several modifications as described previously (Wilmes et al. 2013;
Zidek et al. 2007). cRNAquality was determined using the 2100 Agilent Bio-Analyzer. Amplified
biotinylated cRNA (750ng) was hybridised to the beadchips in a hybridisation cartridge under
humidified conditions for 20hrs at 58 °C. After staining fluorescence detection was carried out by
confocal laser scanning with the Illumina® BeadArray Reader (Illumina) at 532nm and 0.8µm
resolution. Illumina® BeadStudio software was used for condensing raw data and to further ensure
array quality based on different control bead parameters as described for a previous study (Boehme
et al. 2009).Quantile normalisation was also applied to account for non-biological variancebetween
arrays. Collection and warehousing of array data was performedusing BASE (Emergentec
Biodevelopment GmbH). Identification of differentially regulated probes was carried out using the Rpackage “limma” incorporating a moderated one-way ANOVA with a Benjamini–Hochbergcorrected pvalue below or equal to 0.05 as previously reported (Wilmes et al. 2013). Pathway and regulatory
factor enrichment was performed using ingenuity pathway analysis (IPA) (IngenuitySystems) as
previously described (Crean et al. 2013). This software uses an input of differentially regulated genes
that have associated directional fold change and statistical p values as described above. It then
compares this input against an in house database (Ingenuity Knowledge Base) of gene interactions
and functional associations created from millions of individually modelled biological relationships
identified from publically available sources such as Pubmed. The software calculates statistical
associations between the input gene list and previously defined lists of genes categorised into
functional groupings such as “canonical pathways” and “upstream regulators”.
Protein Quantification by ELISA
Changes in protein levels of IL-19, LCN2 and SerpinA3 in cell culture media and Interleukin-19 and
Lipocalin-2 in urine were determined by enzyme-linked immunosorbent assay (ELISA). IL-19 and
LCN2 were assayed using Duoset kits from RND systems and SerpinA3 using an ELISA kit from
Genway BioSciences according to manufacturers’ instructions and as previously described (O'Reilly et
al. 2006).
Patient population and clinical analysis
Matched serum and urine were collected from 9 healthy volunteers and 77 chronic kidney disease
(CKD) patients with diagnosed conditions including ANCA-associated vasculitis, lupus nephritis,
minimal change nephropathy, membranous nephropathy and IgA nephropathy and familial juvenile
hyperuricaemic nephropathy as previously reported (Prajczer et al. 2010). Urine and serum samples
were stored below−20°C until assay. Only excess samples from routine clinical investigations were
used and such use for research purposes was approved by the local institutional ethics board. Urinary
and serum creatinine was measured by the Jaffe reaction. Urinary protein was measured by the
turbidimetricbenzalkonium chloride assay (Roche, Modular Analyser). Estimated glomerular filtration
rate (eGFR) was calculated using the Modification of Diet in Renal Disease (MDRD) formula (Levey et
al. 1999). Urinary N-acetyl-β-D-glucosaminidase (NAG) was measured by a colorimetric assay (PPR
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