Project description:To date, no published reports (human or animal) have examined the impact of acute liver failure on global gene expression profiles in remote organ systems like the kidney. In this study, we have characterized a model of acute kidney injury (AKI) using two highly-accurate techniques for assessing renal function in a mouse. In this model, mice developed massive hepatocyte necrosis, disordered hepatosplanchnic hemodynamics, and alterations consistent with ALF. Simultaneously, acute renal insufficiency developed, manifesting as oliguria, azotemia, and decreased glomerular filtration. In this paper, renal function is corroborated using two independent methodologies. These techniques are used in addition to hemodynamic, biochemical, and histologic analyses to demonstrate that acute hepatic injury promulgates renal dysfunction in a mouse. Similar to network-based analyses conducted in other models of human disease, we present a comprehensive, genome-wide assessment of the differentially-regulated, renal transcriptome in the setting of massive hepatic necrosis. Using this approach, mice receiving the select hepatotoxin D-(+)-Galactosamine HCl (GalN) were found to have significant perturbations in renal pathways related to lipid metabolism, small molecule biochemistry, the cell cycle, molecular transport, and amino acid metabolism, despite normal renal histology. By combining data obtained from clinical, physiologic, and molecular investigations, our findings have direct implications for exploring potential pharmacological approaches to the prevention of AKI in this setting. Male C57BL6 mice weighing 20 grams were given i.p. injections of the select hepatotoxin D-(+)-Galactosamine HCl (GalN). Animals were divided into 3 groups (n =3 per group): 1) Saline control injection (necropsy at 24 hours), 2) 4.5 g / kg GalN injection (necropsy at 24 hours), and 3) 4.5 g / kg i.p. injection (necropsy at 48 hours). At necropsy, liver and kidney were harvested after in situ perfusion with 10 ml of ribonuclease (RNAse)-free PBS and 10 ml of RNA Later reagent (Qiagen, Valencia, CA). Total cellular RNA was isolated from morcellated whole kidneys and liver using a commercial kit (RNA Easy, Qiagen). To extract RNA, 30 mg of tissue was processed according to the manufacturer’s instructions. RNA integrity was monitored using an Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA). One microgram of denatured RNA was used for cDNA synthesis, with reverse transcription carried out using random hexamer primers (Invitrogen, Carlsbad, CA). For quantitative real-time PCR (RT-PCR), 100 ng of cDNA template, 20 μL of SYBR green master mix and pre-optimized and validated mouse primer sets (Qiagen, Valencia, CA) were used and analyzed in an ABI Prism 7000 Thermocycler or a Roche LightCycler. Relative expression of gene targets was determined by the ΔΔCt method first normalizing to expression of the housekeeping gene, GAPDH, and then to expression in the sham treated control. Expression of RPLP0 was included in the analysis to verify the “housekeeping” status of GAPDH. Efficiencies for each primer pair were determined using the REST program and verified to be comparable to efficiencies for housekeeping gene primer pairs Equal amounts of total RNA from the three individual mice for each treatment condition were prepared for Affymetrix array hybridization according to the Affymetrix GeneChip Expression Analysis Technical Manual (www.affymetrix.com). A total of 18 array measurements were made (sham, 24 hr., and 48 hr. for each animal, liver and kidney, respectively). These correspond to the various CEL files and are labeled 1-18. All samples were hybridized to the GeneChip Mouse Genome 430 2.0 Array (Affymetrix, Santa Clara, CA) and preprocessed using R packages affy and gcRMA from the Bioconducter project. Greater than 45,000 transcripts and variants on the mouse genome 430 2.0 gene chip array were analyzed. Probe sets significantly perturbed after GalN administration were identified using rank product analysis. False discovery rate was used to control for multiple comparisons using the method of Benjamini and Hochberg. Adjusted p-values <0.05 were considered significant.
2012-01-01 | E-GEOD-24971 | biostudies-arrayexpress
