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Renal Dysfunction in the Setting of Massive Hepatic Necrosis: A Network-based Analysis in a Mouse

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE24971 | GEO | 2012/01/01

SECONDARY ACCESSION(S): PRJNA131899

REPOSITORIES: GEO

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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.

Project description:Renal hypoxia is widespread in acute kidney injury (AKI) of various aetiologies. Hypoxia adaptation, conferred through the hypoxia-inducible factor (HIF), appears to be insufficient. Here we show that HIF activation in renal tubules through Pax8-rtTA-based inducible knockout of von Hippel-Lindau protein (VHL-KO) protects from rhabdomyolysis-induced AKI. In this model, histological observations indicate that injury mainly affects proximal convoluted tubules, with 5% necrosis at d1 and 40% necrosis at d2. HIF-1alpha up-regulation in distal tubules reflects renal hypoxia. However, lack of HIF in proximal tubules suggests insufficient adaptation by HIF. AKI in VHL-KO mice leads to prominent HIF activation in all nephron segments, as well as to reduced serum creatinine, serum urea, tubular necrosis, and apoptosis marker caspase-3 protein. At d1 after rhabdomyolysis, when tubular injury is potentially reversible, HIF mediated protection in AKI is associated with activated glycolysis, cellular glucose uptake and utilization, autophagy, vasodilation, and proton removal as demonstrated by qPCR, pathway enrichment analysis and immunohistochemistry. Together, our data provide evidence for a HIF-orchestrated multi-level shift towards glycolysis as a major mechanism for protection against acute tubular injury. All experiments were carried out in transgenic mice in which selective renal tubular VHL knockout (VHL-KO) was inducible by doxycycline (Reference: Mathia S, Paliege A, Koesters R, Peters H, Neumayer HH, Bachmann S, Rosenberger C. Action of hypoxia-inducible factor in liver and kidney from mice with Pax8-rtTA-based deletion of von Hippel-Lindau protein. Acta Physiol (Oxf). 2013; 207(3):565-76.). Four groups of animals were used: 1) controls: untreated mice; 2) VHL-KO: injected with doxycycline (0.1 mg per 10 g body weight SC), 4 days prior to sacrifice; 3) AKI: rhabdomyolysis; 4) VHL-KO/AKI: doxycycline plus rhabdomyolysis. To induce AKI, 50% glycerol (0.05 ml per 10 g body weight) was injected IM into the left hind limb under isoflurane narcosis. Drinking water was withdrawn between 20 h prior and 24 h after glycerol injection.

Project description:Microarray analysis of human kidneys with acute kidney injury (AKI) has been limited because such kidneys are seldom biopsied. However, all kidney transplants experience AKI, and early kidney transplants without rejection are an excellent model for human AKI: they are screened to exclude chronic kidney disease, frequently biopsied, and have extensive follow-up. We used histopathology and microarrays to compare indication biopsies from 28 transplants with AKI to 11 pristine protocol biopsies of stable transplants. Kidneys with AKI showed increased expression of 394 injury-repair response associated transcripts, including many known epithelial injury molecules (e.g. ITGB6, LCN2), tissue remodeling molecules (e.g. VCAN), and inflammation molecules (S100A8, ITGB3). Many other genes also predict the phenotype, depending on statistical filtering rules, including AKI biomarkers as HAVCR1 and IL18. Most mouse orthologs of the top injury-repair transcripts were increased in published mouse AKI models. Pathway analysis of the injury-repair transcripts revealed similarities to cancer, development, and cell movement. The injury-repair transcript score AKI kidneys correlated with reduced function, future recovery, brain death, and need for dialysis, but not future graft loss. In contrast, histologic features of "acute tubular injury" did not correlate with function or with the molecular changes. Thus the injury-repair associated transcripts represent a massive coordinate injury-repair response of kidney parenchyma to AKI, similar to mouse AKI models, and provide an objective measure for assessing the severity of AKI in kidney biopsies and validation for the use of many AKI biomarkers. AKI biopsies sample names and CEL files are from GSE21374. All consenting renal transplant patients undergoing biopsies for cause as standard of care between 09/2004 and 10/2007 at the university of Alberta or between 11/2006 and 02/2007 at the University of Illinois were included in the analysis. In addition to the cores required for standard histopathology, we collected one core for gene expression studies. the relationship between gene expression in the biopsy and subsequent graft loss was analyzed. This dataset is part of the TransQST collection.

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Renal Dysfunction in the Setting of Massive Hepatic Necrosis: A Network-based Analysis in a Mouse

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