CD36 restricts lipid-associated macrophages accumulation in white adipose tissues during atherogenesis.

Visceral white adipose tissues (WAT) regulate systemic lipid metabolism and inflammation. Dysfunctional WAT drive chronic inflammation and facilitate atherosclerosis. Adipose tissue-associated macrophages (ATM) are the predominant immune cells in WAT, but their heterogeneity and phenotypes are poorly defined during atherogenesis. The scavenger receptor CD36 mediates ATM crosstalk with other adipose tissue cells, driving chronic inflammation. Here, we combined the single-cell RNA sequencing technique with cell metabolic and functional assays on major WAT ATM subpopulations using a diet-induced atherosclerosis mouse model (Apoe-null). We also examined the role of CD36 using Apoe/Cd36 double-null mice. Based on transcriptomics data and differential gene expression analysis, we identified a previously undefined group of ATM displaying low viability and high lipid metabolism and labeled them as "unhealthy macrophages". Their phenotypes suggest a subpopulation of ATM under lipid stress. We also identified lipid-associated macrophages (LAM), which were previously described in obesity. Interestingly, LAM increased 8.4-fold in Apoe/Cd36 double-null mice on an atherogenic diet, but not in Apoe-null mice. The increase in LAM was accompanied by more ATM lipid uptake, reduced adipocyte hypertrophy, and less inflammation. In conclusion, CD36 mediates a delicate balance between lipid metabolism and inflammation in visceral adipose tissues. Under atherogenic conditions, CD36 deficiency reduces inflammation and increases lipid metabolism in WAT by promoting LAM accumulation.

Machine learning predicts which rivers, streams, and wetlands the Clean Water Act regulates.

We assess which waters the Clean Water Act protects and how Supreme Court and White House rules change this regulation. We train a deep learning model using aerial imagery and geophysical data to predict 150,000 jurisdictional determinations from the Army Corps of Engineers, each deciding regulation for one water resource. Under a 2006 Supreme Court ruling, the Clean Water Act protects two-thirds of US streams and more than half of wetlands; under a 2020 White House rule, it protects less than half of streams and a fourth of wetlands, implying deregulation of 690,000 stream miles, 35 million wetland acres, and 30% of waters around drinking-water sources. Our framework can support permitting, policy design, and use of machine learning in regulatory implementation problems.

Disparities in Child Welfare Referrals for Patients Seen in a Pediatric Emergency Department for Unintentional Ingestions.

OBJECTIVE: To examine the characteristics of patients visiting the pediatric emergency department (PED) for unintentional ingestions and associations between patient race and ethnicity in referrals to Child Protective Services (CPS) for supervisory neglect. METHODS: We conducted a cross-sectional analysis of children <12 years old who presented to the PED between October 2015 and December 2020 for an unintentional ingestion. Patients were identified by searching the electronic health record for diagnosis codes corresponding to unintentional ingestions. Patient demographics, ingestion type, disposition, and referrals to CPS were abstracted by manual chart review. Logistic regression models were used to evaluate associations between patient demographics and visit characteristics with referral to CPS. RESULTS: We identified 129 PED encounters for unintentional ingestions that were included for analysis. Overall, 22 patients (17.1%) were referred to CPS for neglect. In the univariate analysis, both ingestion of an illicit drug and arrival to the PED by ambulance were associated with a higher odds of referral to CPS. In the multivariable model adjusted for parent language, ingestion type, and mode of arrival to the PED, Hispanic patients had higher odds of referral to CPS than White patients (adjusted odds ratio (aOR) = 17.2, 95% confidence intervals [1.8-162.3], P = .03). There was not a statistically significant association between Black race and referral to CPS. CONCLUSIONS: Referrals to CPS from the PED after unintentional ingestions are common and disproportionally involve Hispanic patients. More research is needed to promote equitable child maltreatment reporting for children presenting to the PED following unintentional ingestions.

Erratum: Role of adipocyte Na,K-ATPase oxidant amplification loop in cognitive decline and neurodegeneration.

[This corrects the article DOI: 10.1016/j.isci.2021.103262.].

Adipocyte Na, K-ATPase Signaling Attenuates Experimental Uremic Cardiomyopathy.

Oxidative stress has been shown to cause an alteration of intracellular signaling in adipocytes that may lead to various comorbidities of obesity and cardiovascular complications. Evidence suggests that dysregulation of Na, K-ATPase signaling can contribute to systemic inflammation and redox signaling that leads to various metabolic disturbances. Hence the present study aims to explore the specific role of adipocyte Na, K-ATPase signaling in the amelioration of pathophysiological alterations of experimental uremic cardiomyopathy. Experimental uremic cardiomyopathy was induced by partial nephrectomy (PNx), and adipocyte-specific expression of NaKtide, a peptide that inhibits Na, K-ATPase signaling, was achieved using a lentivirus construct with NaKtide expression driven by an adiponectin promoter. Cardiomyopathy and anemia induced in partial nephrectomy mice were accompanied by an altered molecular phenotype of adipocytes, increased systemic inflammatory cytokines and oxidant stress within 4 weeks. These changes were significantly worsened by the addition of a Western diet (enriched in fat and fructose contents) but were prevented with specific expression of NaKtide in adipocytes. The skeletal muscle-specific expression of NaKtide did not ameliorate the disease phenotype. Adipocyte dysfunction and uremic cardiomyopathy developed in PNx mice, both were significantly ameliorated by the adipocyte-specific expression of NaKtide. These findings suggest that oxidative milieu in the adipocyte has a pivotal role in the development and progression of uremic cardiomyopathy in mice subjected to partial nephrectomy. If confirmed in humans, this may be a lead for future research to explore novel therapeutic targets in chronic renal failure.

Substrate-dependent metabolomic signatures of myeloperoxidase activity in airway epithelial cells: Implications for early cystic fibrosis lung disease.

Myeloperoxidase (MPO) is released by neutrophils in inflamed tissues. MPO oxidizes chloride, bromide, and thiocyanate to produce hypochlorous acid (HOCl), hypobromous acid (HOBr), and hypothiocyanous acid (HOSCN), respectively. These oxidants are toxic to pathogens, but may also react with host cells to elicit biological activity and potential toxicity. In cystic fibrosis (CF) and related diseases, increased neutrophil inflammation leads to increased airway MPO and airway epithelial cell (AEC) exposure to its oxidants. In this study, we investigated how equal dose-rate exposures of MPO-derived oxidants differentially impact the metabolome of human AECs (BEAS-2B cells). We utilized enzymatic oxidant production with rate-limiting glucose oxidase (GOX) coupled to MPO, and chloride, bromide (Br-), or thiocyanate (SCN-) as substrates. AECs exposed to GOX/MPO/SCN- (favoring HOSCN) were viable after 24 h, while exposure to GOX/MPO (favoring HOCl) or GOX/MPO/Br- (favoring HOBr) developed cytotoxicity after 6 h. Cell glutathione and peroxiredoxin-3 oxidation were insufficient to explain these differences. However, untargeted metabolomics revealed GOX/MPO and GOX/MPO/Br- diverged significantly from GOX/MPO/SCN- for dozens of metabolites. We noted methionine sulfoxide and dehydromethionine were significantly increased in GOX/MPO- or GOX/MPO/Br--treated cells, and analyzed them as potential biomarkers of lung damage in bronchoalveolar lavage fluid from 5-year-olds with CF (n = 27). Both metabolites were associated with increasing bronchiectasis, neutrophils, and MPO activity. This suggests MPO production of HOCl and/or HOBr may contribute to inflammatory lung damage in early CF. In summary, our in vitro model enabled unbiased identification of exposure-specific metabolite products which may serve as biomarkers of lung damage in vivo. Continued research with this exposure model may yield additional oxidant-specific biomarkers and reveal explicit mechanisms of oxidant byproduct formation and cellular redox signaling.

Contribution of adipocyte Na/K-ATPase α1/CD36 signaling induced exosome secretion in response to oxidized LDL.

Introduction: Adipose tissue constantly secretes adipokines and extracellular vesicles including exosomes to crosstalk with distinct tissues and organs for whole-body homeostasis. However, dysfunctional adipose tissue under chronic inflammatory conditions such as obesity, atherosclerosis, and diabetes shows pro-inflammatory phenotypes accompanied by oxidative stress and abnormal secretion. Nevertheless, molecular mechanisms of how adipocytes are stimulated to secrete exosomes under those conditions remain poorly understood. Methods: Mouse and human in vitro cell culture models were used for performing various cellular and molecular studies on adipocytes and macrophages. Statistical analysis was performed using Student's t-test (two-tailed, unpaired, and equal variance) for comparisons between two groups or ANOVA followed by Bonferroni's multiple comparison test for comparison among more than two groups. Results and discussion: In this work, we report that CD36, a scavenger receptor for oxidized LDL, formed a signaling complex with another membrane signal transducer Na/K-ATPase in adipocytes. The atherogenic oxidized LDL induced a pro-inflammatory response in in vitro differentiated mouse and human adipocytes and also stimulated the cells to secrete more exosomes. This was largely blocked by either CD36 knockdown using siRNA or pNaKtide, a peptide inhibitor of Na/K-ATPase signaling. These results showed a critical role of the CD36/Na/K-ATPase signaling complex in oxidized LDL-induced adipocyte exosome secretion. Moreover, by co-incubation of adipocyte-derived exosomes with macrophages, we demonstrated that oxidized LDL-induced adipocyte-derived exosomes promoted pro-atherogenic phenotypes in macrophages, including CD36 upregulation, IL-6 secretion, metabolic switch to glycolysis, and mitochondrial ROS production. Altogether, we show here a novel mechanism through which adipocytes increase exosome secretion in response to oxidized LDL and that the secreted exosomes can crosstalk with macrophages, which may contribute to atherogenesis.

Inhibition of Na/K-ATPase signaling Attenuates Steatohepatitis and Atherosclerosis in Mice Fed a Western Diet.

We have previously reported that the α1 subunit of sodium-potassium adenosine triphosphatase (Na/K-ATPase), acts as a receptor and an amplifier for reactive oxygen species, in addition to its distinct pumping function. On this background, we speculated that the blockade of Na/K-ATPase-induced ROS amplification with a specific peptide, pNaKtide, might attenuate the development of steatohepatitis. To test this hypothesis, pNaKtide was administered to a murine model of NASH: the C57Bl6 mouse fed a "western" diet containing high amounts of fat and fructose. The administration of pNaKtide reduced obesity as well as hepatic steatosis, inflammation and fibrosis. Of interest, we also noted a marked improvement in mitochondrial fatty acid oxidation, insulin sensitivity, dyslipidemia and aortic streaking in this mouse model. To further elucidate the effects of pNaKtide on atherosclerosis, similar studies were performed in ApoE knockout mice also exposed to the western diet. In these mice, pNaKtide not only improved steatohepatitis, dyslipidemia, and insulin sensitivity but also ameliorated significant aortic atherosclerosis. Collectively, this study demonstrates that the Na/K-ATPase/ROS amplification loop contributes significantly to the development and progression of steatohepatitis and atherosclerosis. Furthermore, this study presents a potential treatment, the pNaKtide, for the metabolic syndrome phenotype.

Na/K-ATPase signaling tonically inhibits sodium reabsorption in the renal proximal tubule.

Through its classic ATP-dependent ion-pumping function, basolateral Na/K-ATPase (NKA) generates the Na+ gradient that drives apical Na+ reabsorption in the renal proximal tubule (RPT), primarily through the Na+ /H+ exchanger (NHE3). Accordingly, activation of NKA-mediated ion transport decreases natriuresis through activation of basolateral (NKA) and apical (NHE3) Na+ reabsorption. In contrast, activation of the more recently discovered NKA signaling function triggers cellular redistribution of RPT NKA and NHE3 and decreases Na+ reabsorption. We used gene targeting to test the respective contributions of NKA signaling and ion pumping to the overall regulation of RPT Na+ reabsorption. Knockdown of RPT NKA in cells and mice increased membrane NHE3 and Na+ /HCO3 - cotransporter (NBCe1A). Urine output and absolute Na+ excretion decreased by 65%, driven by increased RPT Na+ reabsorption (as indicated by decreased lithium clearance and unchanged glomerular filtration rate), and accompanied by elevated blood pressure. This hyper reabsorptive phenotype was rescued upon crossing with RPT NHE3-/- mice, confirming the importance of NKA/NHE3 coupling. Hence, NKA signaling exerts a tonic inhibition on Na+ reabsorption by regulating key apical and basolateral Na+ transporters. This action, lifted upon NKA genetic suppression, tonically counteracts NKA's ATP-driven function of basolateral Na+ reabsorption. Strikingly, NKA signaling is not only physiologically relevant but it also appears to be functionally dominant over NKA ion pumping in the control of RPT reabsorption.

Hypertonic Saline Infusion for Hyponatremia: Limitations of the Adrogué-Madias and Other Formulas.

Hypertonic saline infusion is used to correct hyponatremia with severe symptoms. The selection of the volume of infused hypertonic saline ( VInf ) should address prevention of overcorrection or undercorrection. Several formulas computing this VInf have been proposed. The limitations common to these formulas consist of (1) failure to include potential determinants of change in serum sodium concentration ([ Na ]) including exchanges between osmotically active and inactive sodium compartments, changes in hydrogen binding of body water to hydrophilic compounds, and genetic influences and (2) inaccurate estimates of baseline body water entered in any formula and of gains or losses of water, sodium, and potassium during treatment entered in formulas that account for such gains or losses. In addition, computing VInf from the Adrogué-Madias formula by a calculation assuming a linear relation between VInf and increase in [ Na ] is a source of errors because the relation between these two variables was proven to be curvilinear. However, these errors were shown to be negligible by a comparison of estimates of VInf by the Adrogué-Madias formula and by a formula using the same determinants of the change in [ Na ] and the curvilinear relation between this change and VInf . Regardless of the method used to correct hyponatremia, monitoring [ Na ] and changes in external balances of water, sodium, and potassium during treatment remain imperative.

pNaKtide Inhibits Na/K-ATPase Signaling and Attenuates Obesity.

Obesity is a growing public health crisis across the world and has been recognized as an underlying risk factor for metabolic syndrome. Growing evidence demonstrates the critical role of oxidative stress in the pathophysiological mechanisms of obesity and related metabolic dysfunction. As we have established previously that Na/K-ATPase can amplify oxidative stress signaling, we aimed to explore the effect of inhibition of this pathway on obesity phenotype using the peptide antagonist, pNaKtide. The experiments performed in murine preadipocytes showed the dose-dependent effect of pNaKtide in attenuating oxidant stress and lipid accumulation. Furthermore, these in vitro findings were confirmed in C57Bl6 mice fed a high-fat diet. Interestingly, pNaKtide could significantly reduce body weight, ameliorate systemic oxidative and inflammatory milieu and improve insulin sensitivity in obese mice. Hence the study demonstrates the therapeutic utility of pNaKtide as an inhibitor of Na/K-ATPase oxidant amplification signaling to alleviate obesity and associated comorbidities.

Na/K-ATPase suppresses LPS-induced pro-inflammatory signaling through Lyn.

Na/K-ATPase (NKA), besides its ion transporter function, is a signal transducer by regulating Src family kinases (SFK). The signaling NKA contributes to oxidized LDL-induced macrophage foam cell formation and interacts with TLR4. However, its role in lipopolysaccharides (LPS)-induced signaling and glycolytic switch in macrophages remains unclear. Using peritoneal macrophages from NKA α1 haploinsufficient mice (NKA α1+/-), we found that NKA α1 haploinsufficiency led to enhanced LPS-stimulated NF-κB pathway, ROS signaling, and pro-inflammatory cytokines. Intraperitoneal injection of LPS resulted in more severe lung inflammation and injury with lower survival rate in NKA α1+/- mice. Additionally, LPS induced a higher extent of the metabolic switch from oxidative phosphorylation to glycolysis. Mechanistically, NKA α1 interacted with TLR4 and Lyn. The presence of NKA α1 in this complex attenuated Lyn activation by LPS, which subsequently restricted the downstream ROS and NF-κB signaling. In conclusion, we demonstrated that NKA α1 suppresses LPS-induced macrophage pro-inflammatory signaling through Lyn.

Disposition Decisions in Cases of Medical Complexity and Health Inequity.

The question of optimal disposition for children with complex medical and social circumstances has long challenged the well-intentioned clinician. The coronavirus disease 2019 pandemic created unique difficulties for patients, families, and health care providers, in addition to highlighting long-standing racial and socioeconomic inequities in health care. In pediatric hospitals, necessary public health measures such as visitor restrictions shifted many shared decision-making processes such as discharge planning from complicated to impossible. Here, we present the case of a medically complex adult (with a long-standing pediatric condition) whose surrogate decision-maker objected to discharge to a long-term care facility because of restrictions and risks associated with the coronavirus disease 2019 pandemic. We offer the commentary of experts in clinical ethics, intensive care, inpatient subacute care, and palliative care. Our discussion includes analysis of the ethical considerations involved in the case, concrete guidance on steps toward an ethically permissible discharge, and suggestions for how a health equity lens can improve communication and decision-making for families who are victims of systemic racism and economic discrimination.

Gene module regulation in dilated cardiomyopathy and the role of Na/K-ATPase.

Dilated cardiomyopathy (DCM) is a major cause of cardiac death and heart transplantation. It has been known that black people have a higher incidence of heart failure and related diseases compared to white people. To identify the relationship between gene expression and cardiac function in DCM patients, we performed pathway analysis and weighted gene co-expression network analysis (WGCNA) using RNA-sequencing data (GSE141910) from the NCBI Gene Expression Omnibus (GEO) database and identified several gene modules that were significantly associated with the left ventricle ejection fraction (LVEF) and DCM phenotype. Genes included in these modules are enriched in three major categories of signaling pathways: fibrosis-related, small molecule transporting-related, and immune response-related. Through consensus analysis, we found that gene modules associated with LVEF in African Americans are almost identical as in Caucasians, suggesting that the two groups may have more common rather than disparate genetic regulations in the etiology of DCM. In addition to the identified modules, we found that the gene expression level of Na/K-ATPase, an important membrane ion transporter, has a strong correlation with the LVEF. These clinical results are consistent with our previous findings and suggest the clinical significance of Na/K-ATPase regulation in DCM.

Tumor-Suppressor Role of the α1-Na/K-ATPase Signalosome in NASH Related Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality worldwide, with an estimate of 0.84 million cases every year. In Western countries, because of the obesity epidemic, non-alcoholic steatohepatitis (NASH) has become the major cause of HCC. Intriguingly, the molecular mechanisms underlying tumorigenesis of HCC from NASH are largely unknown. We hypothesized that the growing uncoupled metabolism during NASH progression to HCC, manifested by lower cell redox status and an apoptotic 'switch' activity, follows a dysregulation of α1-Na/K-ATPase (NKA)/Src signalosome. Our results suggested that in NASH-related malignancy, α1-NKA signaling causes upregulation of the anti-apoptotic protein survivin and downregulation of the pro-apoptotic protein Smac/DIABLO via the activation of the PI3K → Akt pro-survival pathway with concomitant inhibition of the FoxO3 circuit, favoring cell division and primary liver carcinogenesis. Signalosome normalization using an inhibitory peptide resets apoptotic activity in malignant cells, with a significant decrease in tumor burden in vivo. Therefore, α1-NKA signalosome exercises in HCC the characteristic of a tumor suppressor, suggesting α1-NKA as a putative target for clinical therapy.