Franny Jongbloed

219 8 BENEFICIAL EFFECTS OF A PREOPERATIVE DIET tubular necrosis (ATN) was suspected and recorded. Delayed graft function (DGF) was defined as the need for dialysis in the first postoperative week 39 . Slow graft function (SGF) was defined as an estimated glomerular filtration rate (eGFR) ≤10 ml/min per 1.73 m 2 at day 6 after transplantation 39 . Classification of surgical complications was done using the Clavien-Dindo classification of surgical complications 40 . mRNA-sequence analysis For gene expression analysis, 10 donors were included in the CCPR group and 10 donors in the control group. Biopsies were taken at the end of the cold preservation period (kidney off ice), directly before implantation. Biopsies were put in 2 mL Eppendorf tubes (Eppendorf Group, New York, USA) containing 1 mL of RNAlater RNA Stabilization Reagent (QIAGEN Benelux B.V., Venlo, the Netherlands), and were kept at 4°C for at least 48 hours. Total RNA extraction and measurement of RNA concentration was done as described previously 13,14 . The RNA quality was expressed as the RNA integrity number (RIN, range 0-10), and values ranged between 6.1 and 8.2. Handling, analysis and visualization of the data was performed in R. Principal component analysis (PCA) and density plots of the raw counts revealed an outlying sample that had a very low RNA concentration. This sample was omitted from further analysis. A filtering step was performed to remove low count transcripts: transcripts had to be sequenced at least three times per sample and two times per experimental group within gender. The filtering step resulted in 175,756 transcripts for analysis. Differentially expressed transcripts between diet type within gender were calculated using DESeq2. False Discovery Rate correction was performed as described by Storey and Tibshirani 41,42 . Complete raw and normalized microarray data and their MIAME compliant metadata have been deposited at the Gene expression Omnibus (GEO) database GSE103532 (www.ncbi. nlm.nih.gov/geo). End points The primary end point was the postoperative kidney function in the kidney donors as measured by serum creatinine and CKD-EPI eGFR 43 . The main secondary end point was the transcriptional changes induced by the CCPR diet as measured by gene expression analysis in kidney biopsies. Additional secondary end points of the donors included: compliance to the CCPR diet, metabolic changes induced by the CCPR diet, and perioperative outcome. Secondary end points of the recipients were postoperative kidney transplant function, perioperative outcome (including blood loss, urine production), and the incidence of DGF, SGF and ATN defined as described previously 39 .