The Addition of Transcriptomics to the Bead-Enabled Accelerated Monophasic Multi-Omics Method: A Step toward Universal Sample Preparation.

Omics-based measurements enable the study of biomolecules in a high-throughput fashion, leading to the characterization and quantification of biological systems. Multi-omics methods aim to incorporate several omics measurements for a more holistic approach, which is crucial for advancing our understanding of the diversity and redundancy of biological systems. Current multi-omics sample preparation methods have achieved proteomics, lipidomics, and metabolomics from individual samples; however, the bioinformatic tools currently available for interpreting data generated from these omics are limited. Alternately, transcriptomics has a wide arsenal of available bioinformatic tools offering intensive sample characterization but has yet to be incorporated into a unified, multi-omics sample preparation technique. Herein we describe the modified bead-enabled accelerated monophasic multi-omics (mBAMM) method, which incorporates RNA extraction for transcriptomics analysis. mBAMM was shown to enable RNA-seq without compromising the isolation of biomolecules for proteomics, lipidomics, and metabolomics. This methodology greatly improves sample characterization and represents a major innovation toward cohesive insights into biological systems.

Feature-agnostic metabolomics for determining effective subcytotoxic doses of common pesticides in human cells.

Although classical molecular biology assays can provide a measure of cellular response to chemical challenges, they rely on a single biological phenomenon to infer a broader measure of cellular metabolic response. These methods do not always afford the necessary sensitivity to answer questions of subcytotoxic effects, nor do they work for all cell types. Likewise, boutique assays such as cardiomyocyte beat rate may indirectly measure cellular metabolic response, but they too, are limited to measuring a specific biological phenomenon and are often limited to a single cell type. For these reasons, toxicological researchers need new approaches to determine metabolic changes across various doses in differing cell types, especially within the low-dose regime. The data collected herein demonstrate that LC-MS/MS-based untargeted metabolomics with a feature-agnostic view of the data, combined with a suite of statistical methods including an adapted environmental threshold analysis, provides a versatile, robust, and holistic approach to directly monitoring the overall cellular metabolomic response to pesticides. When employing this method in investigating two different cell types, human cardiomyocytes and neurons, this approach revealed separate subcytotoxic metabolomic responses at doses of 0.1 and 1 µM of chlorpyrifos and carbaryl. These findings suggest that this agnostic approach to untargeted metabolomics can provide a new tool for determining effective dose by metabolomics of chemical challenges, such as pesticides, in a direct measurement of metabolomic response that is not cell type-specific or observable using traditional assays.

Transcriptomic Analysis of Arachidonic Acid Pathway Genes Provides Mechanistic Insight into Multi-Organ Inflammatory and Vascular Diseases.

Arachidonic acid (AA) metabolites have been associated with several diseases across various organ systems, including the cardiovascular, pulmonary, and renal systems. Lipid mediators generated from AA oxidation have been studied to control macrophages, T-cells, cytokines, and fibroblasts, and regulate inflammatory mediators that induce vascular remodeling and dysfunction. AA is metabolized by cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP) to generate anti-inflammatory, pro-inflammatory, and pro-resolutory oxidized lipids. As comorbid states such as diabetes, hypertension, and obesity become more prevalent in cardiovascular disease, studying the expression of AA pathway genes and their association with these diseases can provide unique pathophysiological insights. In addition, the AA pathway of oxidized lipids exhibits diverse functions across different organ systems, where a lipid can be both anti-inflammatory and pro-inflammatory depending on the location of metabolic activity. Therefore, we aimed to characterize the gene expression of these lipid enzymes and receptors throughout multi-organ diseases via a transcriptomic meta-analysis using the Gene Expression Omnibus (GEO) Database. In our study, we found that distinct AA pathways were expressed in various comorbid conditions, especially those with prominent inflammatory risk factors. Comorbidities, such as hypertension, diabetes, and obesity appeared to contribute to elevated expression of pro-inflammatory lipid mediator genes. Our results demonstrate that expression of inflammatory AA pathway genes may potentiate and attenuate disease; therefore, we suggest further exploration of these pathways as therapeutic targets to improve outcomes.

A systematic scoping review of the neurological effects of COVID-19.

BACKGROUND: The global coronavirus 2019 (COVID-19) pandemic began in early 2020, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In mid-2020 the CIAO (Modelling the Pathogenesis of COVID-19 Using the Adverse Outcome Pathway Framework) project was established, bringing together over 75 interdisciplinary scientists worldwide to collaboratively investigate the underlying biological mechanisms of COVID-19 and consolidate the data using the Adverse Outcome Pathway (AOP) Framework. Neurological symptoms such as anosmia and encephalitis have been frequently reported to be associated with infection with SARS-CoV-2. OBJECTIVE: Within CIAO, a working group was formed to conduct a systematic scoping review of COVID-19 and its related neurological symptoms to determine which key events and modulating factors are most commonly reported and to identify knowledge gaps. DESIGN: LitCOVID was used to retrieve 86,075 papers of which 10,244 contained relevant keywords. After title and abstract screening, 2,328 remained and their full texts were reviewed based on predefined inclusion and exclusion criteria. 991 studies fulfilled the inclusion criteria and were retrieved to conduct knowledge synthesis. RESULTS: The majority of publications reported human observational studies. Early key events were less likely to be reported compared to middle and late key events/adverse outcomes. The majority of modulating factors described related to age or sex. Less recognised COVID-19 associated AO or neurological effects of COVID-19 were also identified including multiple sclerosis/demyelination, neurodegeneration/cognitive effects and peripheral neuronal effects. CONCLUSION: There were many methodological and reporting issues noted in the reviewed studies. In particular, publication abstracts would benefit from clearer reporting of the methods and endpoints used and the key findings, to ensure relevant papers are included when systematic reviews are conducted. The information extracted from the scoping review may be useful in understanding the mechanisms of neurological effects of COVID-19 and to further develop or support existing AOPs linking COVID-19 and its neurological key events and adverse outcomes. Further evaluation of the less recognised COVID-19 effects is needed.

Novel Model of Oxalate Diet-Induced Chronic Kidney Disease in Dahl-Salt-Sensitive Rats.

Diet-induced models of chronic kidney disease (CKD) offer several advantages, including clinical relevance and animal welfare, compared with surgical models. Oxalate is a plant-based, terminal toxic metabolite that is eliminated by the kidneys through glomerular filtration and tubular secretion. An increased load of dietary oxalate leads to supersaturation, calcium oxalate crystal formation, renal tubular obstruction, and eventually CKD. Dahl-Salt-Sensitive (SS) rats are a common strain used to study hypertensive renal disease; however, the characterization of other diet-induced models on this background would allow for comparative studies of CKD within the same strain. In the present study, we hypothesized that SS rats on a low-salt, oxalate rich diet would have increased renal injury and serve as novel, clinically relevant and reproducible CKD rat models. Ten-week-old male SS rats were fed either 0.2% salt normal chow (SS-NC) or a 0.2% salt diet containing 0.67% sodium oxalate (SS-OX) for five weeks.Real-time PCR demonstrated an increased expression of inflammatory marker interleukin-6 (IL-6) (p < 0.0001) and fibrotic marker Timp-1 metalloproteinase (p < 0.0001) in the renal cortex of SS-OX rat kidneys compared with SS-NC. The immunohistochemistry of kidney tissue demonstrated an increase in CD-68 levels, a marker of macrophage infiltration in SS-OX rats (p < 0.001). In addition, SS-OX rats displayed increased 24 h urinary protein excretion (UPE) (p < 0.01) as well as significant elevations in plasma Cystatin C (p < 0.01). Furthermore, the oxalate diet induced hypertension (p < 0.05). A renin-angiotensin-aldosterone system (RAAS) profiling (via liquid chromatography-mass spectrometry; LC-MS) in the SS-OX plasma showed significant (p < 0.05) increases in multiple RAAS metabolites including angiotensin (1-5), angiotensin (1-7), and aldosterone. The oxalate diet induces significant renal inflammation, fibrosis, and renal dysfunction as well as RAAS activation and hypertension in SS rats compared with a normal chow diet. This study introduces a novel diet-induced model to study hypertension and CKD that is more clinically translatable and reproducible than the currently available models.

Microcystin-LR aerosol induces inflammatory responses in healthy human primary airway epithelium.

Harmful algal blooms plague bodies of freshwater globally. These blooms are often composed of outgrowths of cyanobacteria capable of producing the heptapeptide Microcystin-LR (MC-LR) which is a well-known hepatotoxin. Recently, MC-LR has been detected in aerosols generated from lake water. However, the risk for human health effects due to MC-LR inhalation exposure have not been extensively investigated. In this study, we exposed a fully differentiated 3D human airway epithelium derived from 14 healthy donors to MC-LR-containing aerosol once a day for 3 days. Concentrations of MC-LR ranged from 100 pM to 1 µM. Although there were little to no detrimental alterations in measures of the airway epithelial function (i.e. cell survival, tissue integrity, mucociliary clearance, or cilia beating frequency), a distinct shift in the transcriptional activity was found. Genes related to inflammation were found to be upregulated such as C-C motif chemokine 5 (CCL5; log2FC = 0.57, p = 0.03) and C-C chemokine receptor type 7 (CCR7; log2FC = 0.84, p = 0.03). Functionally, conditioned media from MC-LR exposed airway epithelium was also found to have significant chemo-attractive properties for primary human neutrophils. Additionally, increases were found in the concentration of secreted chemokine proteins in the conditioned media such as CCL1 (log2FC = 5.07, p = 0.0001) and CCL5 (log2FC = 1.02, p = 0.046). These results suggest that MC-LR exposure to the human airway epithelium is capable of inducing an inflammatory response that may potentiate acute or chronic disease.

Cardioprotective Role for Paraoxonase-1 in Chronic Kidney Disease.

Paraoxonase-1 (PON-1) is a hydrolytic enzyme associated with HDL, contributing to its anti-inflammatory, antioxidant, and anti-atherogenic properties. Deficiencies in PON-1 activity result in oxidative stress and detrimental clinical outcomes in the context of chronic kidney disease (CKD). However, it is unclear if a decrease in PON-1 activity is mechanistically linked to adverse cardiovascular events in CKD. We investigated the hypothesis that PON-1 is cardioprotective in a Dahl salt-sensitive model of hypertensive renal disease. Experiments were performed on control Dahl salt-sensitive rats (SSMcwi, hereafter designated SS-WT rats) and mutant PON-1 rats (SS-Pon1em1Mcwi, hereafter designated SS-PON-1 KO rats) generated using CRISPR gene editing technology. Age-matched 10-week-old SS and SS-PON-1 KO male rats were maintained on high-salt diets (8% NaCl) for five weeks to induce hypertensive renal disease. Echocardiography showed that SS-PON-1 KO rats but not SS-WT rats developed compensated left ventricular hypertrophy after only 4 weeks on the high-salt diet. RT-PCR analysis demonstrated a significant increase in the expression of genes linked to cardiac hypertrophy, inflammation, and fibrosis, as well as a significant decrease in genes essential to left ventricular function in SS-PON-1 KO rats compared to SS-WT rats. A histological examination also revealed a significant increase in cardiac fibrosis and immune cell infiltration in SS-PON-1 KO rats, consistent with their cardiac hypertrophy phenotype. Our data suggest that a loss of PON-1 in the salt-sensitive hypertensive model of CKD leads to increased cardiac inflammation and fibrosis as well as a molecular and functional cardiac phenotype consistent with compensated left ventricular hypertrophy.

Antioxidant Therapy Significantly Attenuates Hepatotoxicity following Low Dose Exposure to Microcystin-LR in a Murine Model of Diet-Induced Non-Alcoholic Fatty Liver Disease.

We have previously shown in a murine model of Non-alcoholic Fatty Liver Disease (NAFLD) that chronic, low-dose exposure to the Harmful Algal Bloom cyanotoxin microcystin-LR (MC-LR), resulted in significant hepatotoxicity including micro-vesicular lipid accumulation, impaired toxin metabolism as well as dysregulation of the key signaling pathways involved in inflammation, immune response and oxidative stress. On this background we hypothesized that augmentation of hepatic drug metabolism pathways with targeted antioxidant therapies would improve MC-LR metabolism and reduce hepatic injury in NAFLD mice exposed to MC-LR. We chose N-acetylcysteine (NAC, 40 mM), a known antioxidant that augments the glutathione detoxification pathway and a novel peptide (pNaKtide, 25 mg/kg) which is targeted to interrupting a specific Src-kinase mediated pro-oxidant amplification mechanism. Histological analysis showed significant increase in hepatic inflammation in NAFLD mice exposed to MC-LR which was attenuated on treatment with both NAC and pNaKtide (both p ≤ 0.05). Oxidative stress, as measured by 8-OHDG levels in urine and protein carbonylation in liver sections, was also significantly downregulated upon treatment with both antioxidants after MC-LR exposure. Genetic analysis of key drug transporters including Abcb1a, Phase I enzyme-Cyp3a11 and Phase II metabolic enzymes-Pkm (Pyruvate kinase, muscle), Pklr (Pyruvate kinase, liver, and red blood cell) and Gad1 (Glutamic acid decarboxylase) was significantly altered by MC-LR exposure as compared to the non-exposed control group (all p ≤ 0.05). These changes were significantly attenuated with both pNaKtide and NAC treatment. These results suggest that MC-LR metabolism and detoxification is significantly impaired in the setting of NAFLD, and that these pathways can potentially be reversed with targeted antioxidant treatment.

GeneToList: A Web Application to Assist with Gene Identifiers for the Non-Bioinformatics-Savvy Scientist.

The increasing incorporation of omics technologies into biomedical research and translational medicine presents challenges to end users of the large and complex datasets that are generated by these methods. A particular challenge in genomics is that the nomenclature for genes is not uniform between large genomic databases or between commonly used genetic analysis tools. Furthermore, outdated genomic nomenclature can still be found amongst scientific communications, including peer-reviewed manuscripts. Therefore, a web application (GeneToList) was developed to assist in gene ID conversion and alias matching, with a specific focus on achieving a user-friendly interface for the non-bioinformatics-savvy scientist. It currently includes gene information for over 38,000 different taxa retrieved from the National Center for Biotechnology and Information (NCBI) Gene resource. Supported databases of gene IDs include NCBI Gene Symbols, NCBI Gene IDs (Entrez IDs), OMIM IDs, HGNC IDs, Ensembl IDs, and 28 other taxa-specific identifiers. GeneToList is available at genetolist.com. The tool is a web application that is compatible with many standard browsers. The gene ID conversion feature of this application was found to outcompete the common gene ID conversion tools. Specifically, it was able to successfully convert all tested IDs, whereas the others were not able to recognize the gene aliases. Therefore, the gene ID disambiguation provided by this application should be beneficial for many scientists dealing with gene data when the uniformity of gene nomenclature is important for downstream analysis.

Paraoxonase-1 Regulation of Renal Inflammation and Fibrosis in Chronic Kidney Disease.

Papraoxonase-1 (PON1) is a hydrolytic lactonase enzyme that is synthesized in the liver and circulates attached to high-density lipoproteins (HDL). Clinical studies have demonstrated an association between diminished PON-1 and the progression of chronic kidney disease (CKD). However, whether decreased PON-1 is mechanistically linked to renal injury is unknown. We tested the hypothesis that the absence of PON-1 is mechanistically linked to the progression of renal inflammation and injury in CKD. Experiments were performed on control Dahl salt-sensitive rats (SSMcwi, hereafter designated SS rats) and Pon1 knock-out rats (designated SS-Pon1em1Mcwi, hereafter designated SS-PON-1 KO rats) generated by injecting a CRISPR targeting the sequence into SSMcwi rat embryos. The resulting mutation is a 7 bp frameshift insertion in exon 4 of the PON-1 gene. First, to examine the renal protective role of PON-1 in settings of CKD, ten-week-old, age-matched male rats were maintained on a high-salt diet (8% NaCl) for up to 5 weeks to initiate the salt-sensitive hypertensive renal disease characteristic of this model. We found that SS-PON-1 KO rats demonstrated several hallmarks of increased renal injury vs. SS rats including increased renal fibrosis, sclerosis, and tubular injury. SS-PON-1 KO also demonstrated increased recruitment of immune cells in the renal interstitium, as well as increased expression of inflammatory genes compared to SS rats (all p < 0.05). SS-PON-1 KO rats also showed a significant (p < 0.05) decline in renal function and increased renal oxidative stress compared to SS rats, despite no differences in blood pressure between the two groups. These findings suggest a new role for PON-1 in regulating renal inflammation and fibrosis in the setting of chronic renal disease independent of blood pressure.

As We Drink and Breathe: Adverse Health Effects of Microcystins and Other Harmful Algal Bloom Toxins in the Liver, Gut, Lungs and Beyond.

Freshwater harmful algal blooms (HABs) are increasing in number and severity worldwide. These HABs are chiefly composed of one or more species of cyanobacteria, also known as blue-green algae, such as Microcystis and Anabaena. Numerous HAB cyanobacterial species produce toxins (e.g., microcystin and anatoxin-collectively referred to as HAB toxins) that disrupt ecosystems, impact water and air quality, and deter recreation because they are harmful to both human and animal health. Exposure to these toxins can occur through ingestion, inhalation, or skin contact. Acute health effects of HAB toxins have been well documented and include symptoms such as nausea, vomiting, abdominal pain and diarrhea, headache, fever, and skin rashes. While these adverse effects typically increase with amount, duration, and frequency of exposure, susceptibility to HAB toxins may also be increased by the presence of comorbidities. The emerging science on potential long-term or chronic effects of HAB toxins with a particular emphasis on microcystins, especially in vulnerable populations such as those with pre-existing liver or gastrointestinal disease, is summarized herein. This review suggests additional research is needed to define at-risk populations who may be helped by preventative measures. Furthermore, studies are required to develop a mechanistic understanding of chronic, low-dose exposure to HAB toxins so that appropriate preventative, diagnostic, and therapeutic strategies can be created in a targeted fashion.

Microcystin-LR (MC-LR) Triggers Inflammatory Responses in Macrophages.

We were the first to previously report that microcystin-LR (MC-LR) has limited effects within the colons of healthy mice but has toxic effects within colons of mice with pre-existing inflammatory bowel disease. In the current investigation, we aimed to elucidate the mechanism by which MC-LR exacerbates colitis and to identify effective therapeutic targets. Through our current investigation, we report that there is a significantly greater recruitment of macrophages into colonic tissue with pre-existing colitis in the presence of MC-LR than in the absence of MC-LR. This is seen quantitatively through IHC staining and the enumeration of F4/80-positive macrophages and through gene expression analysis for Cd68, Cd11b, and Cd163. Exposure of isolated macrophages to MC-LR was found to directly upregulate macrophage activation markers Tnf and Il1b. Through a high-throughput, unbiased kinase activity profiling strategy, MC-LR-induced phosphorylation events were compared with potential inhibitors, and doramapimod was found to effectively prevent MC-LR-induced inflammatory responses in macrophages.

CD40/CD40L Signaling as a Promising Therapeutic Target for the Treatment of Renal Disease.

The cluster of differentiation 40 (CD40) is activated by the CD40 ligand (CD40L) in a variety of diverse cells types and regulates important processes associated with kidney disease. The CD40/CD40L signaling cascade has been comprehensively studied for its roles in immune functions, whereas the signaling axis involved in local kidney injury has only drawn attention in recent years. Clinical studies have revealed that circulating levels of soluble CD40L (sCD40L) are associated with renal function in the setting of kidney disease. Levels of the circulating CD40 receptor (sCD40), sCD40L, and local CD40 expression are tightly related to renal injury in different types of kidney disease. Additionally, various kidney cell types have been identified as non-professional antigen-presenting cells (APCs) that express CD40 on the cell membrane, which contributes to the interactions between immune cells and local kidney cells during the development of kidney injury. Although the potential for adverse CD40 signaling in kidney cells has been reported in several studies, a summary of those studies focusing on the role of CD40 signaling in the development of kidney disease is lacking. In this review, we describe the outcomes of recent studies and summarize the potential therapeutic methods for kidney disease which target CD40.

Impact of Comorbidities on SARS-CoV-2 Viral Entry-Related Genes.

Viral entry mechanisms for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are an important aspect of virulence. Proposed mechanisms involve host cell membrane-bound angiotensin-converting enzyme 2 (ACE2), type II transmembrane serine proteases (TTSPs), such as transmembrane serine protease isoform 2 (TMPRSS2), lysosomal endopeptidase Cathepsin L (CTSL), subtilisin-like proprotein peptidase furin (FURIN), and even potentially membrane bound heparan sulfate proteoglycans. The distribution and expression of many of these genes across cell types representing multiple organ systems in healthy individuals has recently been demonstrated. However, comorbidities such as diabetes and cardiovascular disease are highly prevalent in patients with Coronavirus Disease 2019 (COVID-19) and are associated with worse outcomes. Whether these conditions contribute directly to SARS-CoV-2 virulence remains unclear. Here, we show that the expression levels of ACE2, TMPRSS2 and other viral entry-related genes, as well as potential downstream effector genes such as bradykinin receptors, are modulated in the target organs of select disease states. In tissues, such as the heart, which normally express ACE2 but minimal TMPRSS2, we found that TMPRSS2 as well as other TTSPs are elevated in individuals with comorbidities compared to healthy individuals. Additionally, we found the increased expression of viral entry-related genes in the settings of hypertension, cancer, or smoking across target organ systems. Our results demonstrate that common comorbidities may contribute directly to SARS-CoV-2 virulence and we suggest new therapeutic targets to improve outcomes in vulnerable patient populations.

CD40 Receptor Knockout Protects against Microcystin-LR (MC-LR) Prolongation and Exacerbation of Dextran Sulfate Sodium (DSS)-Induced Colitis.

Inflammatory Bowel Disease (IBD) is one of the most common gastrointestinal (GI) disorders around the world, and includes diagnoses such as Crohn's disease and ulcerative colitis. The etiology of IBD is influenced by genetic and environmental factors. One environmental perturbagen that is not well studied within the intestines is microcystin-leucine arginine (MC-LR), which is a toxin produced by cyanobacteria in freshwater environments around the world. We recently reported that MC-LR has limited effects within the intestines of healthy mice, yet interestingly has significant toxicity within the intestines of mice with pre-existing colitis induced by dextran sulfate sodium (DSS). MC-LR was found to prolong DSS-induced weight loss, prolong DSS-induced bloody stools, exacerbate DSS-induced colonic shortening, exacerbate DSS-induced colonic ulceration, and exacerbate DSS-induced inflammatory cytokine upregulation. In addition, we previously reported a significant increase in expression of the pro-inflammatory receptor CD40 in the colons of these mice, along with downstream products of CD40 activation, including plasminogen activator inhibitor-1 (PAI-1) and monocyte chemoattractant protein-1 (MCP-1). In the current study, we demonstrate that knocking out CD40 attenuates the effects of MC-LR in mice with pre-existing colitis by decreasing the severity of weight loss, allowing a full recovery in bloody stools, preventing the exacerbation of colonic shortening, preventing the exacerbation of colonic ulceration, and preventing the upregulation of the pro-inflammatory and pro-fibrotic cytokines IL-1ß, MCP-1, and PAI-1. We also demonstrate the promising efficacy of a CD40 receptor blocking peptide to ameliorate the effects of MC-LR exposure in a proof-of-concept study. Our findings suggest for the first time that MC-LR acts through a CD40-dependent mechanism to exacerbate colitis.

Harmful Algal Bloom Toxicity in Lithobates catesbeiana Tadpoles.

Harmful algal blooms (HAB) have become a major health concern worldwide, not just to humans that consume and recreate on contaminated waters, but also to the fauna that inhabit the environments surrounding affected areas. HABs contain heterotrophic bacteria, cyanobacterial lipopolysaccharide, and cyanobacterial toxins such as microcystins, that can cause severe toxicity in many aquatic species as well as bioaccumulation within various organs. Thus, the possibility of trophic transference of this toxin through the food chain has potentially important health implications for other organisms in the related food web. While some species have developed adaptions to attenuate the toxic effects of HAB toxins, there are still numerous species that remain vulnerable, including Lithobates catesbeiana (American bullfrog) tadpoles. In the current study we demonstrate that acute, short-term exposure of tadpoles to HAB toxins containing 1 µg/L (1 nmol/L) of total microcystins for only 7 days results in significant liver and intestinal toxicity within tadpoles. Exposed tadpoles had increased intestinal diameter, decreased intestinal fold heights, and a constant number of intestinal folds, indicating pathological intestinal distension, similar to what is seen in various disease processes, such as toxic megacolon. HAB-toxin-exposed tadpoles also demonstrated hepatocyte hypertrophy with increased hepatocyte binucleation consistent with carcinogenic and oxidative processes within the liver. Both livers and intestines of HAB-toxin-exposed tadpoles demonstrated significant increases in protein carbonylation consistent with oxidative stress and damage. These findings demonstrate that short-term exposure to HAB toxins, including microcystins, can have significant adverse effects in amphibian populations. This acute, short-term toxicity highlights the need to evaluate the influence HAB toxins may have on other vulnerable species within the food web and how those may ultimately also impact human health.

Assessment of diagnostic biomarkers of liver injury in the setting of microcystin-LR (MC-LR) hepatotoxicity.

Microcystin-leucine arginine (MC-LR) is a potent liver toxin produced by freshwater cyanobacteria, also known as blue-green algae. While harmful algal blooms are increasing in frequency and severity worldwide, there is still no established method for the diagnosis and assessment of MC-LR induced liver damage. The guidelines for MC-LR safe exposure limits have been previously established based on healthy animal studies, however we have previously demonstrated that pre-existing non-alcoholic fatty liver disease (NAFLD) increases susceptiblity to the hepatotoxic effects of MC-LR. In this study, we sought to investigate the suitability of clinically used biomarkers of liver injury, specifically alanine aminotransferase (ALT) and alkaline phosphatase (ALP), as potential diagnostic tools for liver damage induced by chronic low dose administration of MC-LR in the setting of pre-existing NAFLD. In our Leprdb/J mouse model of NAFLD, we found that while MC-LR induced significant histopathologic damage in the setting of NAFLD, gene expression of ALT and ALP failed to increase with MC-LR exposure. Serum ALT and ALP also failed to increase with MC-LR exposure, except for a moderate increase in ALP with the highest dose of MC-LR used (100 µg/kg). In HepG2 human liver epithelial cells, we observed that increasing MC-LR exposure levels do not lead to an increase in ALT or ALP gene expression, intracellular enzyme activity, or extracellular activity, despite a significant increase in MC-LR induced cytotoxicity. These findings demonstrate that ALT and ALP may be unsuitable as diagnostic biomarkers for MC-LR induced liver damage.

Renal Fibrosis Is Significantly Attenuated Following Targeted Disruption of Cd40 in Experimental Renal Ischemia.

Background Renal artery stenosis is a common cause of renal ischemia, contributing to the development of chronic kidney disease. To investigate the role of local CD40 expression in renal artery stenosis, Goldblatt 2-kidney 1-clip surgery was performed on hypertensive Dahl salt-sensitive rats (S rats) and genetically modified S rats in which CD40 function is abolished (Cd40mutant). Methods and Results Four weeks following the 2-kidney 1-clip procedure, Cd40mutant rats demonstrated significantly reduced blood pressure and renal fibrosis in the ischemic kidneys compared with S rat controls. Similarly, disruption of Cd40 resulted in reduced 24-hour urinary protein excretion in Cd40mutant rats versus S rat controls (46.2±1.9 versus 118.4±5.3 mg/24 h; P<0.01), as well as protection from oxidative stress, as indicated by increased paraoxonase activity in Cd40mutant rats versus S rat controls (P<0.01). Ischemic kidneys from Cd40mutant rats demonstrated a significant decrease in gene expression of the profibrotic mediator, plasminogen activator inhibitor-1 (P<0.05), and the proinflammatory mediators, C-C motif chemokine ligand 19 (P<0.01), C-X-C Motif Chemokine Ligand 9 (P<0.01), and interleukin-6 receptor (P<0.001), compared with S rat ischemic kidneys, as assessed by quantitative PCR assay. Reciprocal renal transplantation documented that CD40 exclusively expressed in the kidney contributes to ischemia-induced renal fibrosis. Furthermore, human CD40-knockout proximal tubule epithelial cells suggested that suppression of CD40 signaling significantly inhibited expression of proinflammatory and -fibrotic genes. Conclusions Taken together, our data suggest that activation of CD40 induces a significant proinflammatory and -fibrotic response and represents an attractive therapeutic target for treatment of ischemic renal disease.

Hyperglycemia induces key genetic and phenotypic changes in human liver epithelial HepG2 cells which parallel the Leprdb/J mouse model of non-alcoholic fatty liver disease (NAFLD).

Non-alcoholic fatty liver disease (NAFLD) is a growing global health concern. With a propensity to progress towards non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma, NAFLD is an important link amongst a multitude of comorbidities including obesity, diabetes, and cardiovascular and kidney disease. As several in vivo models of hyperglycemia and NAFLD are employed to investigate the pathophysiology of this disease process, we aimed to characterize an in vitro model of hyperglycemia that was amenable to address molecular mechanisms and therapeutic targets at the cellular level. Utilizing hyperglycemic cell culturing conditions, we induced steatosis within a human hepatocyte cell line (HepG2 cells), as confirmed by electron microscopy. The deposition and accumulation of lipids within hyperglycemic HepG2 cells is significantly greater than in normoglycemic cells, as visualized and quantified by Nile red staining. Alanine aminotransferase (ALT) and alkaline phosphatase (ALP), diagnostic biomarkers for liver damage and disease, were found to be upregulated in hyperglycemic HepG2 cells as compared with normoglycemic cells. Suppression of CEACAM1, GLUT2, and PON1, and elevation of CD36, PCK1, and G6PK were also found to be characteristic in hyperglycemic HepG2 cells compared with normoglycemic cells, suggesting insulin resistance and NAFLD. These in vitro findings mirror the characteristic genetic and phenotypic profile seen in Leprdb/J mice, a well-established in vivo model of NAFLD. In conclusion, we characterize an in vitro model displaying several key genetic and phenotypic characteristics in common with NAFLD that may assist future studies in addressing the molecular mechanisms and therapeutic targets to combat this disease.

Interleukin 21 (IL-21) regulates chronic allograft vasculopathy (CAV) in murine heart allograft rejection.

IL-21 is the most recently discovered common gamma-chain cytokine that promotes persistent T-cell responses in chronic infections, autoimmunity and cancer. However, the therapeutic potential of inhibiting the IL-21-BATF signaling axis, particularly in transplant rejection, remains unclear. We used heart transplant models to examine the effects of IL-21 blockade in prevention of chronic cardiac allograft vasculopathy (CAV) using genetic knock-out and therapeutic approaches. Both wild-type C57BL/6 and IL-21-/- strains acutely rejected Balb/c skin grafts and once immunized with this skin graft, rejected Balb/c heart allografts in an accelerated fashion. However, when transplanted with heart grafts from the class-II major histocompatibility complex mutant, B6bm12 mice; wild-type recipients developed CAV, while IL-21-/- recipients were protected, even at day 100 post-transplant. Similarly, BATF-/- recipients, lacking the transcription factor BATF responsible for IL-21 production, did not develop CAV in B6-bm12 heart allografts. Strikingly, in a transient treatment protocol, the development of CAV in wild-type recipients of B6-bm12 hearts allografts was blocked by the administration of IL-21 receptor fusion protein (R-Fc). Thus, we demonstrate that CAV is regulated at least in part by IL-21 signaling and its blockade by genetic approaches or therapy with IL-21R-Fc prevents CAV in mice.