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.

Novel insights into the pathobiology of pulmonary hypertension in heart failure with preserved ejection fraction.

Heart failure (HF) with preserved ejection fraction (HFpEF) is the most common cause of pulmonary hypertension (PH) worldwide and is strongly associated with adverse clinical outcomes. The American Heart Association recently highlighted a call to action regarding the distinct lack of evidence-based treatments for PH due to poorly understood pathophysiology of PH attributable to HFpEF (PH-HFpEF). Prior studies have described cardiophysiological mechanisms to explain the development of isolated postcapillary PH (ipc-PH); however, the consequent increase in pulmonary vascular (PV) resistance (PVR) may lead to the less understood and more fatal combined pre- and postcapillary PH (cpc-PH). Metabolic disease and inflammatory dysregulation have been suggested to predispose PH, yet the molecular mechanisms are unknown. Although PH-HFpEF has been studied to partly share vasoactive neurohormonal mediators with primary pulmonary arterial hypertension (PAH), clinical trials that have targeted these pathways have been unsuccessful. The increased mortality of patients with PH-HFpEF necessitates further study into viable mechanistic targets involved in disease progression. We aim to summarize the current pathophysiological and clinical understanding of PH-HFpEF, highlight the role of known molecular mechanisms in the progression of PV disease, and introduce a novel concept that lipid metabolism may be attenuating and propagating PH-HFpEF.NEW & NOTEWORTHY Our review addresses pulmonary hypertension (PH) attributable to heart failure (HF) with preserved ejection fraction (HFpEF; PH-HFpEF). Current knowledge gaps in PH-HFpEF pathophysiology have led to a lack of therapeutic targets. Thus, we address identified knowledge gaps in a comprehensive review, focusing on current clinical epidemiology, known pathophysiology, and previously studied molecular mechanisms. We also introduce a comprehensive review of polyunsaturated fatty acid (PUFA) lipid inflammatory mediators in PH-HFpEF.

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.

Paraoxonases at the Heart of Neurological Disorders.

Paraoxonase enzymes serve as an important physiological redox system that participates in the protection against cellular injury caused by oxidative stress. The PON enzymes family consists of three members (PON-1, PON-2, and PON-3) that share a similar structure and location as a cluster on human chromosome 7. These enzymes exhibit anti-inflammatory and antioxidant properties with well-described roles in preventing cardiovascular disease. Perturbations in PON enzyme levels and their activity have also been linked with the development and progression of many neurological disorders and neurodegenerative diseases. The current review summarizes the available evidence on the role of PONs in these diseases and their ability to modify risk factors for neurological disorders. We present the current findings on the role of PONs in Alzheimer's disease, Parkinson's disease, and other neurodegenerative and neurological diseases.

Eutherian-Specific Functions of BetaM Acquired through Atp1b4 Gene Co-Option in the Regulation of MyoD Expression.

Vertebrate ATP1B4 genes represent a rare instance of orthologous gene co-option, resulting in radically different functions of the encoded BetaM proteins. In lower vertebrates, BetaM is a Na, K-ATPase ß-subunit that is a component of ion pumps in the plasma membrane. In placental mammals, BetaM lost its ancestral role and, through structural alterations of the N-terminal domain, became a skeletal and cardiac muscle-specific protein of the inner nuclear membrane, highly expressed during late fetal and early postnatal development. We previously determined that BetaM directly interacts with the transcriptional co-regulator SKI-interacting protein (SKIP) and is implicated in the regulation of gene expression. This prompted us to investigate a potential role for BetaM in the regulation of muscle-specific gene expression in neonatal skeletal muscle and cultured C2C12 myoblasts. We found that BetaM can stimulate expression of the muscle regulatory factor (MRF), MyoD, independently of SKIP. BetaM binds to the distal regulatory region (DRR) of MyoD, promotes epigenetic changes associated with activation of transcription, and recruits the SWI/SNF chromatin remodeling subunit, BRG1. These results indicate that eutherian BetaM regulates muscle gene expression by promoting changes in chromatin structure. These evolutionarily acquired new functions of BetaM might be very essential and provide evolutionary advantages to placental mammals.

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.

Dirty Jobs: Macrophages at the Heart of Cardiovascular Disease.

Cardiovascular disease (CVD) is one of the greatest public health concerns and is the leading cause of morbidity and mortality in the United States and worldwide. CVD is a broad yet complex term referring to numerous heart and vascular conditions, all with varying pathologies. Macrophages are one of the key factors in the development of these conditions. Macrophages play diverse roles in the maintenance of cardiovascular homeostasis, and an imbalance of these mechanisms contributes to the development of CVD. In the current review, we provide an in-depth analysis of the diversity of macrophages, their roles in maintaining tissue homeostasis within the heart and vasculature, and the mechanisms through which imbalances in homeostasis may lead to CVD. Through this review, we aim to highlight the potential importance of macrophages in the identification of preventative, diagnostic, and therapeutic strategies for patients with CVD.

Evaluating the Roles of Different Types of Laser Therapy in Becker's Nevus Treatment.

Becker's nevus (BN) is a cutaneous hamartoma of benign nature that develops through adolescence and affects mostly young men. The nevus is usually located unilaterally and is characterized by hypertrichosis and hyperpigmentation. Despite recent advances in treatment modalities, no effective treatment has been established for BN hyperpigmentation. We sought to assess the efficacy and safety of fractional Erbium: YAG 2940 nm and Q-switched Nd: YAG 1064 nm lasers in the treatment of BN hyperpigmentation. Twenty-three patients with BN were included in a prospective, randomized-controlled, observer-blinded, split-lesion comparative technique trial. In each patient, two similar square test regions were randomized to either be treated with a fractional Erbium: YAG 2940 nm laser or with a Q-switched Nd: YAG 1064 nm laser. Each patient was treated with three sessions at six-week intervals. At the follow-up, clearance of hyperpigmentation was assessed by physician global assessment, visual analogue scale, grade of improvement, patient global assessment, and patient satisfaction. Regions treated with the fractional Erbium: YAG 2940 nm laser demonstrated significantly better improvement compared to ones treated with the Q-switched Nd: YAG 1064 nm (p-value = 0.001) laser. Adverse effects such as repigmentation and hypertrophic scarring were not reported during the follow-up period. The outcomes were cosmetically acceptable with overall high satisfaction among the included patients. Our data suggest a superior role for the fractional Erbium: YAG (2940 nm) laser in the treatment of BN hyperpigmentation compared to the Q-switched Nd: YAG (1064 nm) laser, along with being a safer method and having no reported side effects.

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.

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.

Vascular Calcification in Chronic Kidney Disease: Diversity in the Vessel Wall.

Vascular calcification (VC) is one of the major causes of cardiovascular morbidity and mortality in patients with chronic kidney disease (CKD). VC is a complex process expressing similarity to bone metabolism in onset and progression. VC in CKD is promoted by various factors not limited to hyperphosphatemia, Ca/Pi imbalance, uremic toxins, chronic inflammation, oxidative stress, and activation of multiple signaling pathways in different cell types, including vascular smooth muscle cells (VSMCs), macrophages, and endothelial cells. In the current review, we provide an in-depth analysis of the various kinds of VC, the clinical significance and available therapies, significant contributions from multiple cell types, and the associated cellular and molecular mechanisms for the VC process in the setting of CKD. Thus, we seek to highlight the key factors and cell types driving the pathology of VC in CKD in order to assist in the identification of preventative, diagnostic, and therapeutic strategies for patients burdened with this disease.

Regulation of Na/K-ATPase expression by cholesterol: isoform specificity and the molecular mechanism.

We have reported that the reduction in plasma membrane cholesterol could decrease cellular Na/K-ATPase α1-expression through a Src-dependent pathway. However, it is unclear whether cholesterol could regulate other Na/K-ATPase α-isoforms and the molecular mechanisms of this regulation are not fully understood. Here we used cells expressing different Na/K-ATPase α isoforms and found that membrane cholesterol reduction by U18666A decreased expression of the α1-isoform but not the α2- or α3-isoform. Imaging analyses showed the cellular redistribution of α1 and α3 but not α2. Moreover, U18666A led to redistribution of α1 to late endosomes/lysosomes, while the proteasome inhibitor blocked α1-reduction by U18666A. These results suggest that the regulation of the Na/K-ATPase α-subunit by cholesterol is isoform specific and α1 is unique in this regulation through the endocytosis-proteasome pathway. Mechanistically, loss-of-Src binding mutation of A425P in α1 lost its capacity for regulation by cholesterol. Meanwhile, gain-of-Src binding mutations in α2 partially restored the regulation. Furthermore, through studies in caveolin-1 knockdown cells, as well as subcellular distribution studies in cell lines with different α-isoforms, we found that Na/K-ATPase, Src, and caveolin-1 worked together for the cholesterol regulation. Taken together, these new findings reveal that the putative Src-binding domain and the intact Na/K-ATPase/Src/caveolin-1 complex are indispensable for the isoform-specific regulation of Na/K-ATPase by cholesterol.

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.

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.

Chronic Low Dose Oral Exposure to Microcystin-LR Exacerbates Hepatic Injury in a Murine Model of Non-Alcoholic Fatty Liver Disease.

Microcystins are potent hepatotoxins that have become a global health concern in recent years. Their actions in at-risk populations with pre-existing liver disease is unknown. We tested the hypothesis that the No Observed Adverse Effect Level (NOAEL) of Microcystin-LR (MC-LR) established in healthy mice would cause exacerbation of hepatic injury in a murine model (Leprdb/J) of Non-alcoholic Fatty Liver Disease (NAFLD). Ten-week-old male Leprdb/J mice were gavaged with 50 µg/kg, 100 µg/kg MC-LR or vehicle every 48 h for 4 weeks (n = 15-17 mice/group). Early mortality was observed in both the 50 µg/kg (1/17, 6%), and 100 µg/kg (3/17, 18%) MC-LR exposed mice. MC-LR exposure resulted in significant increases in circulating alkaline phosphatase levels, and histopathological markers of hepatic injury as well as significant upregulation of genes associated with hepatotoxicity, necrosis, nongenotoxic hepatocarcinogenicity and oxidative stress response. In addition, we observed exposure dependent changes in protein phosphorylation sites in pathways involved in inflammation, immune function, and response to oxidative stress. These results demonstrate that exposure to MC-LR at levels that are below the NOAEL established in healthy animals results in significant exacerbation of hepatic injury that is accompanied by genetic and phosphoproteomic dysregulation in key signaling pathways in the livers of NAFLD mice.

Circulating Lactonase Activity but Not Protein Level of PON-1 Predicts Adverse Outcomes in Subjects with Chronic Kidney Disease.

The burden of cardiovascular disease and death in chronic kidney disease (CKD) outpaces that of the other diseases and is not adequately described by traditional risk factors alone. Diminished activity of paraoxonase (PON)-1 is associated with increased oxidant stress, a common feature underlying the pathogenesis of CKD. We aimed to assess the prognostic value of circulating PON-1 protein and PON lactonase activity on adverse clinical outcomes across various stages and etiologies of CKD. Circulating PON-1 protein levels and PON lactonase activity were measured simultaneously in patients with CKD as well as a cohort of apparently healthy non-CKD subjects. Both circulating PON-1 protein levels and PON lactonase activity were significantly lower in CKD patients compared to the non-CKD subjects. Similarly, across all stages of CKD, circulating PON-1 protein and PON lactonase activity were significantly lower in patients with CKD compared to the non-CKD controls. Circulating PON lactonase activity, but not protein levels, predicted future adverse clinical outcomes, even after adjustment for traditional risk factors. The combination of lower circulating protein levels and higher activity within the CKD subjects were associated with the best survival outcomes. These findings demonstrate that diminished circulating PON lactonase activity, but not protein levels, predicts higher risk of future adverse clinical outcomes in patients with CKD.