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:In temperate and sub-tropical regions of Latin America, complicated malaria manifested as hepatic dysfunction or renal dysfunction is seen in all age groups. There has been a concerted focus on understanding the patho-physiological and molecular basis of complicated malaria in children, much less is known about it in adults. This can be attributed to many factors: one of which is the expression of clonally variant families of adhesion proteins: PfEMP1 on the iRBC surface. We report here, the analysis of data from a custom, cross strain microarray (Agilent Platform) using material from patient samples, showing hepatic dysfunction or renal failure. These are the most common manifestations seen in adults along with cerebral malaria. The data has been analyzed with reference to variable surface antigens, encoded by the var, rifin and stevor gene families. The differential regulation profiles of key genes (comparison of PFC and PFU) have been observed. The exportome has been analyzed using similar parameters. GO term based functional enrichment of differentially regulated genes identified, up-regulated genes stastically enriched (p<0.5) to critical biological processes. Systems network based functional enrichment of overall differentially regulated genes yielded a similar result. We are reporting here, up-regulation of var group A and B genes whose proteins are predicted to interact with CD36 receptor in the host as also the up-regulation of group A rifins and many of the stevors. This is contrary to most other reports from pediatric patients, with cerebral malaria where the up-regulation of var A group genes have been seen. A protein-protein interaction based network has been created and analysis performed. This co-expression and text mining based network has shown overall connectivity between the variable surface antigens and the exportome. The up-regulation of var group B and C genes encoding PfEMP1 with different domain architecture would be important for deciding strategies for disease prevention. Plasmodium falciparum isolates were collected from patients (n=12) with differing clinical conditions. The patients exhibited symptoms categorized as uncomplicated (n=5) or complicated malaria (n=7). Criteria for determination of complicated disease were based on World Health Organization year 2000 guidelines. Microarray array based transcriptional profiling was carried out to find out differentailly expressed genes in complicated malaria isolates (non-cerebral malaria) compared to un-complicated malaria isolates.

Project description:In temperate and sub-tropical regions of Latin America, complicated malaria manifested as hepatic dysfunction or renal dysfunction is seen in all age groups. There has been a concerted focus on understanding the patho-physiological and molecular basis of complicated malaria in children, much less is known about it in adults. This can be attributed to many factors: one of which is the expression of clonally variant families of adhesion proteins: PfEMP1 on the iRBC surface. We report here, the analysis of data from a custom, cross strain microarray (Agilent Platform) using material from patient samples, showing hepatic dysfunction or renal failure. These are the most common manifestations seen in adults along with cerebral malaria. The data has been analyzed with reference to variable surface antigens, encoded by the var, rifin and stevor gene families. The differential regulation profiles of key genes (comparison of PFC and PFU) have been observed. The exportome has been analyzed using similar parameters. GO term based functional enrichment of differentially regulated genes identified, up-regulated genes stastically enriched (p<0.5) to critical biological processes. Systems network based functional enrichment of overall differentially regulated genes yielded a similar result. We are reporting here, up-regulation of var group A and B genes whose proteins are predicted to interact with CD36 receptor in the host as also the up-regulation of group A rifins and many of the stevors. This is contrary to most other reports from pediatric patients, with cerebral malaria where the up-regulation of var A group genes have been seen. A protein-protein interaction based network has been created and analysis performed. This co-expression and text mining based network has shown overall connectivity between the variable surface antigens and the exportome. The up-regulation of var group B and C genes encoding PfEMP1 with different domain architecture would be important for deciding strategies for disease prevention.

Project description:The liver has inherent regenerative capacity via mitotic division of mature hepatocytes. However, if the hepatic loss is massive or mature hepatocyte proliferation is impaired by chronic liver injury, HSPC are activated to support liver regeneration. Access to liver tissue from 4 patients who underwent liver transplantation for hepatitis B virus (HBV)- associated acute liver failure (ALF) provided us with the opportunity to investigate the molecular mechanisms of liver regeneration in humans by means of gene expression profiling and immunohistochemistry (IHC). Gene expression profiling of 17 liver specimens from the 4 ALF cases and individual liver specimens from 10 liver donors documented a distinct gene signature for ALF. However, unsupervised multidimensional scaling and hierarchical clustering identified two-well defined clusters that segregated according to the histopathological severity, i.e. massive hepatic necrosis (MHN; 2 patients) and submassive hepatic necrosis (SHN; 2 patients). We found that ALF is characterized by a strong hepatic stem/progenitor cell (HSPC) gene signature, as also confirmed by IHC, along with ductular reaction, both of which are more prominent in MHN. Interestingly, no evidence of further lineage differentiation was seen in MHN, whereas in SHN we detected cells with hepatocyte-like morphology. Strikingly, ALF was associated with a strong tumorigenesis gene signature. MHN had the greatest upregulation of cancer stem cell genes (EpCAM, CK19 and CK7), whereas the most upregulated genes in SHN were related to cellular growth and proliferation (AKR1B10, NQO1, RRM2, SFN, TOP2A, CCNB1, CDC20, ANLN and KI67). The extent of liver necrosis correlated with an overriding fibrogenesis gene signature, reflecting the wound healing process. Conclusion: Our data provide evidence of marked HSPC cell activation and fibrogenesis in HBV-associated ALF, which positively correlate with the extent of liver necrosis. Moreover, we detected a strong tumorigenesis gene signature in ALF, which underlines the relationship between liver regeneration and liver cancer. Liver samples were obtained from explanted liver of four patients with HBV-associated acute liver failure (4-5 samples per liver) and 10 individual normal liver donors and gene expression profiling was used to establish a molecular definition of this disease. This dataset is part of the TransQST collection.

Project description:The liver has inherent regenerative capacity via mitotic division of mature hepatocytes. However, if the hepatic loss is massive or mature hepatocyte proliferation is impaired by chronic liver injury, HSPC are activated to support liver regeneration. Access to liver tissue from 4 patients who underwent liver transplantation for hepatitis B virus (HBV)- associated acute liver failure (ALF) provided us with the opportunity to investigate the molecular mechanisms of liver regeneration in humans by means of gene expression profiling and immunohistochemistry (IHC). Gene expression profiling of 17 liver specimens from the 4 ALF cases and individual liver specimens from 10 liver donors documented a distinct gene signature for ALF. However, unsupervised multidimensional scaling and hierarchical clustering identified two-well defined clusters that segregated according to the histopathological severity, i.e. massive hepatic necrosis (MHN; 2 patients) and submassive hepatic necrosis (SHN; 2 patients). We found that ALF is characterized by a strong hepatic stem/progenitor cell (HSPC) gene signature, as also confirmed by IHC, along with ductular reaction, both of which are more prominent in MHN. Interestingly, no evidence of further lineage differentiation was seen in MHN, whereas in SHN we detected cells with hepatocyte-like morphology. Strikingly, ALF was associated with a strong tumorigenesis gene signature. MHN had the greatest upregulation of cancer stem cell genes (EpCAM, CK19 and CK7), whereas the most upregulated genes in SHN were related to cellular growth and proliferation (AKR1B10, NQO1, RRM2, SFN, TOP2A, CCNB1, CDC20, ANLN and KI67). The extent of liver necrosis correlated with an overriding fibrogenesis gene signature, reflecting the wound healing process. Conclusion: Our data provide evidence of marked HSPC cell activation and fibrogenesis in HBV-associated ALF, which positively correlate with the extent of liver necrosis. Moreover, we detected a strong tumorigenesis gene signature in ALF, which underlines the relationship between liver regeneration and liver cancer.

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