Erythrocyte ENT1-AMPD3 Axis is an Essential Purinergic Hypoxia Sensor and Energy Regulator Combating CKD in a Mouse Model.

Significance Statement: Hypoxia drives kidney damage and progression of CKD. Although erythrocytes respond rapidly to hypoxia, their role and the specific molecules sensing and responding to hypoxia in CKD remain unclear. In this study, we demonstrated in a mouse model that erythrocyte ENT1-AMPD3 is a master energy regulator of the intracellular purinergic hypoxic compensatory response that promotes rapid energy supply from extracellular adenosine, eAMPK-dependent metabolic reprogramming, and O 2 delivery, which combat renal hypoxia and progression of CKD.

Tissue transglutaminase: a multifunctional and multisite regulator in health and disease.

Tissue transglutaminase (TG2) is a widely distributed multifunctional protein involved in a broad range of cellular and metabolic functions carried out in a variety of cellular compartments. In addition to transamidation, TG2 also functions as a Gα signaling protein, a protein disulfide isomerase (PDI), a protein kinase, and a scaffolding protein. In the nucleus, TG2 modifies histones and transcription factors.

p97 dysfunction underlies a loss of quality control of damaged membrane proteins and promotes oxidative stress and sickling in sickle cell disease.

Sickling is the central pathogenic process of sickle cell disease (SCD), one of the most prevalent inherited hemolytic disorders. Having no easy access to antioxidants in the cytosol, elevated levels of reactive oxygen species (ROS) residing at the plasma membrane in sickle red blood cells (sRBCs) easily oxidize membrane proteins and thus contribute to sickling. Although the ubiquitin-proteasome system (UPS) is essential to rapidly clear ROS-damaged membrane proteins and maintain cellular homeostasis, the function and regulatory mechanism of the UPS for their clearance in sRBCs remains unidentified.

Erythrocyte transglutaminase-2 combats hypoxia and chronic kidney disease by promoting oxygen delivery and carnitine homeostasis.

Due to lack of nuclei and de novo protein synthesis, post-translational modification (PTM) is imperative for erythrocytes to regulate oxygen (O) delivery and combat tissue hypoxia. Here, we report that erythrocyte transglutminase-2 (eTG2)-mediated PTM is essential to trigger O delivery by promoting bisphosphoglycerate mutase proteostasis and the Rapoport-Luebering glycolytic shunt for adaptation to hypoxia, in healthy humans ascending to high altitude and in two distinct murine models of hypoxia. In a pathological hypoxia model with chronic kidney disease (CKD), eTG2 is critical to combat renal hypoxia-induced reduction of Slc22a5 transcription and OCNT2 protein levels via HIF-1α-PPARα signaling to maintain carnitine homeostasis.

Erythrocyte adenosine A2B receptor prevents cognitive and auditory dysfunction by promoting hypoxic and metabolic reprogramming.

Hypoxia drives aging and promotes age-related cognition and hearing functional decline. Despite the role of erythrocytes in oxygen (O2) transport, their role in the onset of aging and age-related cognitive decline and hearing loss (HL) remains undetermined. Recent studies revealed that signaling through the erythrocyte adenosine A2B receptor (ADORA2B) promotes O2 release to counteract hypoxia at high altitude.

Adenosine A2B receptor: A pathogenic factor and a therapeutic target for sensorineural hearing loss.

Over 466 million people worldwide are diagnosed with hearing loss (HL). About 90% of HL cases are sensorineural HL (SNHL) with treatments limited to hearing aids and cochlear implants with no FDA-approved drugs. Intriguingly, ADA-deficient patients have been reported to have bilateral SNHL, however, its underlying cellular and molecular basis remain unknown.

Maternal erythrocyte ENT1-mediated AMPK activation counteracts placental hypoxia and supports fetal growth.

Insufficient O2 supply is frequently associated with fetal growth restriction (FGR), a leading cause of perinatal mortality and morbidity. Although the erythrocyte is the most abundant and only cell type to deliver O2 in our body, its function and regulatory mechanism in FGR remain unknown. Here, we report that genetic ablation of mouse erythrocyte equilibrative nucleoside transporter 1 (eENT1) in dams, but not placentas or fetuses, results in FGR.

Erythrocyte Metabolic Reprogramming by Sphingosine 1-Phosphate in Chronic Kidney Disease and Therapies.

Rationale: Hypoxia promotes renal damage and progression of chronic kidney disease (CKD). The erythrocyte is the only cell type for oxygen (O) delivery. Sphingosine 1-phosphate (S1P)-a highly enriched biolipid in erythrocytes-is recently reported to be induced under high altitude in normal humans to enhance O delivery.

Reciprocal upregulation of hypoxia-inducible factor-1α and persistently enhanced placental adenosine signaling contribute to the pathogenesis of preeclampsia.

Recent evidence indicates that elevated placental adenosine signaling contributes to preeclampsia (PE). However, the molecular basis for the chronically enhanced placental adenosine signaling in PE remains unclear. Here, we report that hypoxia-inducible factor-1α (HIF-1α) is crucial for the enhancement of placental adenosine signaling.

Differential role of adenosine signaling cascade in acute and chronic pain.

Adenosine is a signaling molecule induced under stress such as energy insufficiency and ischemic/hypoxic conditions. Adenosine controls multiple physiological and pathological cellular and tissue function by activation of four G protein-coupled receptors (GPCR). Functional role of adenosine signaling in acute pain has been widely studied.

Increased LIGHT leading to sFlt-1 elevation underlies the pathogenic link between hydatidiform mole and preeclampsia.

Hydatidiform moles are known to pose an extremely high risk of severe early-onset preeclampsia if left untreated. TNF superfamily cytokine, LIGHT has recently been reported to contribute to pathophysiology of preeclampsia. The present study aimed to investigate the involvement of LIGHT in hydatidiform moles.

Erythrocyte Adenosine A2B Receptor-Mediated AMPK Activation: A Missing Component Counteracting CKD by Promoting Oxygen Delivery.

Background: Oxygen deprivation or hypoxia in the kidney drives CKD and contributes to end organ damage. The erythrocyte's role in delivery of oxygen (O) is regulated by hypoxia, but the effects of CKD are unknown.

Methods: We screened all of the metabolites in the whole blood of mice infused with angiotensin II (Ang II) at 140 ng/kg per minute up to 14 days to simulate CKD and compared their metabolites with those from untreated mice.

Circadian period 2: a missing beneficial factor in sickle cell disease by lowering pulmonary inflammation, iron overload, and mortality.

The circadian clock is important for cellular and organ function. However, its function in sickle cell disease (SCD), a life-threatening hemolytic disorder, remains unknown. Here, we performed an unbiased microarray screen, which revealed significantly altered expression of circadian rhythmic genes, inflammatory response genes, and iron metabolic genes in SCD Berkeley transgenic mouse lungs compared with controls.

Tissue Transglutaminase-Mediated AT1 Receptor Sensitization Underlies Pro-inflammatory Cytokine LIGHT-Induced Hypertension.

Background: Although numerous recent studies have shown a strong link between inflammation and hypertension, the underlying mechanisms by which inflammatory cytokines induce hypertension remain to be fully elucidated. Hypertensive disorders are also associated with elevated pressor sensitivity. Tissue transglutaminase (TG2), a potent cross-linking enzyme, is known to be transcriptionally activated by inflammatory cytokines and stabilize angiotensin II (Ang II) receptor AT1 (AT1R) via ubiquitination-preventing posttranslational modification.

Elevated ecto-5'-nucleotidase: a missing pathogenic factor and new therapeutic target for sickle cell disease.

Although excessive plasma adenosine is detrimental in sickle cell disease (SCD), the molecular mechanism underlying elevated circulating adenosine remains unclear. Here we report that the activity of soluble CD73, an ectonucleotidase producing extracellular adenosine, was significantly elevated in a murine model of SCD and correlated with increased plasma adenosine. Mouse genetic studies demonstrated that CD73 activity contributes to excessive induction of plasma adenosine and thereby promotes sickling, hemolysis, multiorgan damage, and disease progression.

Sphingosine-1-phosphate receptor 1 mediates elevated IL-6 signaling to promote chronic inflammation and multitissue damage in sickle cell disease.

Sphingosine-1-phosphate (S1P) is a biolipid involved in chronic inflammation in several inflammatory disorders. Recent studies revealed that elevated S1P contributes to sickling in sickle cell disease (SCD), a devastating hemolytic, genetic disorder associated with severe chronic inflammation and tissue damage. We evaluated the effect of elevated S1P in chronic inflammation and tissue damage in SCD and underlying mechanisms.

Structural and Functional Insight of Sphingosine 1-Phosphate-Mediated Pathogenic Metabolic Reprogramming in Sickle Cell Disease.

Elevated sphingosine 1-phosphate (S1P) is detrimental in Sickle Cell Disease (SCD), but the mechanistic basis remains obscure. Here, we report that increased erythrocyte S1P binds to deoxygenated sickle Hb (deoxyHbS), facilitates deoxyHbS anchoring to the membrane, induces release of membrane-bound glycolytic enzymes and in turn switches glucose flux towards glycolysis relative to the pentose phosphate pathway (PPP). Suppressed PPP causes compromised glutathione homeostasis and increased oxidative stress, while enhanced glycolysis induces production of 2,3-bisphosphoglycerate (2,3-BPG) and thus increases deoxyHbS polymerization, sickling, hemolysis and disease progression.

Comprehensive Characterization of Alternative Polyadenylation in Human Cancer.

Background: Alternative polyadenylation (APA) is emerging as a major post-transcriptional mechanism for gene regulation, and dysregulation of APA contributes to several human diseases. However, the functional consequences of APA in human cancer are not fully understood. Particularly, there is no large-scale analysis in cancer cell lines.

Erythrocyte purinergic signaling components underlie hypoxia adaptation.

Erythrocytes are vital to human adaptation under hypoxic conditions because of their abundance in number and irreplaceable function of delivering oxygen (O). However, although multiple large-scale altitude studies investigating the overall coordination of the human body for hypoxia adaptation have been conducted, detailed research with a focus on erythrocytes was missing due to lack of proper techniques. The recently maturing metabolomics profiling technology appears to be the answer to this limitation.

Elevated Adenosine Induces Placental DNA Hypomethylation Independent of A2B Receptor Signaling in Preeclampsia.

Preeclampsia is a prevalent pregnancy hypertensive disease with both maternal and fetal morbidity and mortality. Emerging evidence indicates that global placental DNA hypomethylation is observed in patients with preeclampsia and is linked to altered gene expression and disease development. However, the molecular basis underlying placental epigenetic changes in preeclampsia remains unclear.

Inflammation, Autoimmunity, and Hypertension: The Essential Role of Tissue Transglutaminase.

Inflammatory cytokines cause hypertension when introduced into animals. Additional evidence indicates that cytokines induce the production of autoantibodies that activate the AT1 angiotensin receptor (AT1R). Extensive evidence shows that these autoantibodies, termed AT1-AA, contribute to hypertension.

Erythrocytes retain hypoxic adenosine response for faster acclimatization upon re-ascent.

Faster acclimatization to high altitude upon re-ascent is seen in humans; however, the molecular basis for this enhanced adaptive response is unknown. We report that in healthy lowlanders, plasma adenosine levels are rapidly induced by initial ascent to high altitude and achieved even higher levels upon re-ascent, a feature that is positively associated with quicker acclimatization. Erythrocyte equilibrative nucleoside transporter 1 (eENT1) levels are reduced in humans at high altitude and in mice under hypoxia.

Beneficial Role of Erythrocyte Adenosine A2B Receptor-Mediated AMP-Activated Protein Kinase Activation in High-Altitude Hypoxia.

Background: High altitude is a challenging condition caused by insufficient oxygen supply. Inability to adjust to hypoxia may lead to pulmonary edema, stroke, cardiovascular dysfunction, and even death. Thus, understanding the molecular basis of adaptation to high altitude may reveal novel therapeutics to counteract the detrimental consequences of hypoxia.

Hypoxia-mediated impaired erythrocyte Lands' Cycle is pathogenic for sickle cell disease.

Although Lands' cycle was discovered in 1958, its function and cellular regulation in membrane homeostasis under physiological and pathological conditions remain largely unknown. Nonbiased high throughput metabolomic profiling revealed that Lands' cycle was impaired leading to significantly elevated erythrocyte membrane lysophosphatidylcholine (LysoPC) content and circulating and erythrocyte arachidonic acid (AA) in mice with sickle cell disease (SCD), a prevalent hemolytic genetic disorder. Correcting imbalanced Lands' cycle by knockdown of phospholipase 2 (cPLA2) or overexpression of lysophosphatidycholine acyltransferase 1 (LPCAT1), two key enzymes of Lands' cycle in hematopoietic stem cells, reduced elevated erythrocyte membrane LysoPC content and circulating AA levels and attenuated sickling, inflammation and tissue damage in SCD chimeras.

Sphingosine-1-phosphate promotes erythrocyte glycolysis and oxygen release for adaptation to high-altitude hypoxia.

Sphingosine-1-phosphate (S1P) is a bioactive signalling lipid highly enriched in mature erythrocytes, with unknown functions pertaining to erythrocyte physiology. Here by employing nonbiased high-throughput metabolomic profiling, we show that erythrocyte S1P levels rapidly increase in 21 healthy lowland volunteers at 5,260 m altitude on day 1 and continue increasing to 16 days with concurrently elevated erythrocyte sphingonisne kinase 1 (Sphk1) activity and haemoglobin (Hb) oxygen (O2) release capacity. Mouse genetic studies show that elevated erythrocyte Sphk1-induced S1P protects against tissue hypoxia by inducing O2 release.