Franny Jongbloed

38 CHAPTER 2 by orbital puncture while the animals were anesthetized as well as on postoperative day 1, 2, 3, 7 and 28 if applicable. Serum urea and creatinine levels were measured using the QuantiChrom™ assay kits (DIUR-500 and DICt-500, Gentaur Europe, Brussels, Belgium). Statistical analysis was performed with IBM SPSS statistics version 20.0, GraphPad Prism version 5.0, R software and/or Microsoft Excel. Statistical significance was defined as P< 0.05 unless noted otherwise. For assessment of the kidney function, the one-way ANOVA was used including the Bonferroni test for multiple comparisons. Means were compared using either the non-parametric Mann-Whitney U test or the independent t-test for parametric data. The survival curves were compared using the Log-rank (Mantel-Cox) test and visualized by the Kaplan-Meier curve. Histology After euthanasia, both kidneys were harvested. Half of the left and the right kidney of each mouse were fixed in formalin for histological analysis in Hematoxylin and Eosin (HE) stained sections. Kidneys were analyzed in a blinded manner. Semi-quantitative scoring of the severity of the lesions was performed for acute tubular necrosis (0-5) and tubular epithelial regeneration (0-4). Scores were defined as: 0= none; 1=minimal; 2=mild; 3=moderate; 4=marked and, if applicable, 5=severe necrosis or regeneration. Pathology scores were analyzed with the Kruskal-Wallis test for K independent samples and the Dunn’s test for multiple comparisons. Microarray analysis Of the 10 male mice used for microarray analysis, half of both the left and the right kidney (taken in longitudinal direction) were snap frozen in liquid nitrogen until further analysis directly after fasting. For gene expression analysis, total RNA was extracted from frozen kidney tissue using the QIAzol lysis Reagent and miRNAeasy Mini Kit (QIAGEN, Hilden, Germany), as described in the Qiagen protocol (males: fasted n=5, ad libitumn=5). Addition of wash buffers RPE and RWT (QIAGEN) was done mechanically by using the QIAcube (QIAGEN, Hilden, Germany) via the miRNeasy program. RNA was eluted in RNase free water and stored at -80°C. The concentration of RNA was measured by Nanodrop (Thermo Scientific). Quality assessment of the RNA was done using the 2100 Bio-Analyzer (Agilent Technologies, Amstelveen, the Netherlands). The quality of the RNA is expressed as the RNA integrity number (RIN, range 0-10). Samples with a RIN below 8 were excluded from analysis. RNA was further processed for hybridization to Affymetrix HTMG-430 PMArray Plates at the Microarray Department of the University of Amsterdam, the Netherlands according to Affymetrix protocols. Four to six biological replicates were used for each group. Quality control and normalization were performed using the pipeline at the www. arrayanalysis.org website (Maastricht University, the Netherlands). Normalization was done