Project description:We compared gene expression profiles from six donor kidneys prior to surgical manipulation to six kidneys removed after laparoscopic donor nephrectomy (LDN) and several hours of CO2 pneumoperitoneum. Biopsies were obtained from renal cortex and hybridized to Affymetrix HG-U133A GeneChips. For control kidneys we identified 1380 genes present on all 6 samples that had a signal intensity greater than 1000. Functional classification of these revealed genes for cellular signaling (201; 15%), regulation of transcription (156; 11%), cellular transport (144; 10%) and cellular metabolism (111; 8%). A class comparison between the controls and LDN kidneys yielded 865 differentially expressed genes. Functional classification of the 502 genes differentially up-regulated in LDN kidneys identified associations with apoptosis, cell adhesion, cell signaling, regulation of cell growth/proliferation, immune/inflammation, ischemia/stress response and proteolysis/peptidolysis. These data demonstrate an altered renal transcriptome induced by several hours of CO2 pneumoperitoneum and laparoscopic surgery characterized by up-regulation of ischemia and injury associated genes. Keywords: Comparative Expression Twelve healthy adult kidney donors signed a consent form, and were approved for donation according to the usual evaluation and practice of the Cleveland Clinic Renal Transplant Program. To avoid potentially significant differences in gene expression based on race and/or ethnicity, only white recipients were selected. The patients were divided into two groups; six controls from open donor nephrectomy and six from LDN. Demographic information included age, gender, race, HLA type, and renal function data including serum creatinine (mg/dL), GFR measured by iothalamate clearance (cc/min.), and urine protein excretion (mg/24 hr). Each patient was given general anesthesia and had a diuresis induced with saline, mannitol, and furosemide. The controls had an extraperitoneal flank incision made, and with only minimal manipulation of the kidney two subcapsular core renal biopsies were obtained using a 15-gauge spring-loaded needle. For the LDN cases two core renal biopsies were obtained using the same needles after the kidneys underwent 2 to 3 hours of C02 pneumoperitoneum. Study biopsies went immediately into 1.5 mL of RNALater (Ambion, Austin, TX), and were stored at –80 0C until they were processed. Biopsy specimens were homogenized in 1 mL of Trizol (Invitrogen, Carlsbad, CA); total RNA was purified using an RNeasy column (Qiagen, Valencia, CA); and quality confirmed with an Agilent 2100 BioAnalyzer (Palo Alto, CA). Standard Affymetrix GeneChip (Santa Clara, CA) protocols were used, and labelled samples were hybridized to HG-U133A GeneChip arrays containing 22,283 probe sets representing over 14,500 human genes. Data was analyzed using Signal intensities generated using GeneChip Operating System version 1.0 and were subsequently analysed by Robust Multichip Average Express (RMA Express) using all Affymetrix .CEL files as a training set. BRB ArrayTools (http://linus.nci.nih.gov/BRB-ArrayTools.html) was used to perform class comparisons (significance level of p < 0.005), and create dendrograms. We used NetAffyx (www.affymetrix.com) to annotate the differentially expressed genes according to biological function based on the Gene Ontology database. A Heatmap was created using Cluster and TreeView software. This dataset is part of the TransQST collection.

Project description:Fabry disease, an X-linked lysosomal storage disorder caused by galactosidase alpha (GLA) gene mutations, exhibits diverse clinical manifestations, and poses significant diagnostic challenges. Early diagnosis and treatment are crucial for improved patient outcomes, pressing the need for reliable biomarkers. In this study, we aimed to identify miRNA candidates as potential biomarkers for Fabry disease using the KingFisher™ automated isolation method and NanoString nCounter® miRNA detection assay. Clinical serum samples were collected from both healthy subjects and Fabry disease patients. RNA extraction from the samples was performed using the KingFisher™ automated isolation method with the MagMAX mirVana™ kit or manually using the Qiagen miRNeasy kit. The subsequent NanoString nCounter® miRNA detection assay showed consistent performance and no correlation between RNA input concentration and raw count, ensuring reliable and reproducible results. Interestingly, the detection range and highly differential miRNA between the control and disease groups were found to be distinct depending on the isolation method employed. Nevertheless, enrichment analysis of miRNA-targeting genes consistently revealed significant associations with angiogenesis pathways in both isolation methods. Additionally, our investigation into the impact of enzyme replacement therapy on miRNA expression indicated that some differential miRNAs may be sensitive to treatment. Our study provides valuable insights to identify miRNA biomarkers for Fabry disease. While different isolation methods yielded various detection ranges and highly differential miRNAs, the consistent association with angiogenesis pathways suggests their significance in disease progression. These findings lay the groundwork for further investigations and validation studies, ultimately leading to the development of non-invasive and reliable biomarkers to aid in early diagnosis and treatment monitoring for Fabry disease.

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Homo sapiens

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