Illumina HiSeq 2000 sequencing; RNAseq analysis of human peritoneal dialysis effluent cells treated with or without alanyl-glutamine supplemented peritoneal dialysis fluid in a randomized controlled cross-over pilot study
RNAseq analysis of human peritoneal dialysis effluent cells treated with or without alanyl-glutamine supplemented peritoneal dialysis fluid in a randomized controlled cross-over pilot study
Peritoneal cells were isolated from overnight and PET-effluents by centrifugation 200 x g for 30 minutes. The randomized cross-over pilot study (EudraCT 2012-004004-36) was conducted at the Medical University of Vienna. Patients on peritoneal dialysis aged â‰¥ 19 years were included if they were stable for at least 2 months (absence of peritonitis or severe concomitant disease). Exclusion criteria included known hypersensitivity to or treatment with another investigational drug, presumed non-compliance, limited efficacy of peritoneal dialysis, clinically significant inflammation, body weight < 50 kg, or immune-suppressive therapy. One patient among 7 screened subjects was not enrolled in the trial due to kidney transplantation before initiation of the trial. Six clinically stable PD patients (4 male, 2 female) aged 55.3 (45-66) years, treated with PD for 17.8 (4.9-48.1) months, with total Kt/V of 2.4 (1.85-2.87), were included in the trial following written informed consent. Two patients had a history of peritonitis > 2 months before enrolment. 4 patients had residual renal function (Tab. 1). Patients were randomly allocated to cross-over treatment sequences. Treatments were separated by a wash-out period (> 28 days).  For AlaGln treatment, 17.4 ml or 34.8 ml DipeptivenÂ® (200 mg AlaGln/ml, Fresenius-Kabi, Bad Homburg, Germany) was added to 2.0 L of PDF (PhysionealÂ® 40 with 3.86% glucose, Baxter) immediately before instillation, resulting in a final AlaGln concentration of 8 or 16 mM AlaGln (= 0.17% or 0.34%). The increase in total osmolality of the modified PDF was less than 1%. The control treatment was carried out with unmodified PDF (PhysionealÂ® 40 with 3.86% glucose, Baxter). RNA was isolated from cell pellets using Qiazol buffer and the miRNeasy Mini Kit (Qiagen) per manufacturer's protocol. RNA was checked for integrity with the Agilent Bioanalyzer 2100.  RNA-seq libraries were prepared with TruSeq Stranded mRNA LT sample preparation kit (Illumina) using Sciclone and Zephyr liquid handling robotics (PerkinElmer).