Erythrocytes are the sole oxygen (O2) transporters and highly sensitive hypoxia responders, yet their active role in tumor development remains unclear. Here, we identify a compensatory erythrocyte adaptation to hypoxia that enhances O2 release in head and neck squamous cell carcinoma (HNSCC). Untargeted metabolomics revealed a 13-metabolite erythrocyte signature with diagnostic potential, with sphingosine emerging as a central metabolic node altered across erythrocytes, plasma, and tumors. Erythrocytes from patients with HNSCC exhibited metabolic reprogramming characterized by increased sphingosine and sphingosine kinase 1 (SPHK1)-dependent sphingosine-1-phosphate (S1P) production, together with loss of the S1P transporter major facilitator superfamily domain-containing protein 2B (MFSD2B), resulting in systemic sphingolipid imbalance. Genetic ablation of erythrocyte-specific SPHK1 disrupted O2 and S1P release, impaired angiogenesis, and promoted an immunosuppressive tumor microenvironment, thereby accelerating tumor growth. These findings establish erythrocytes as active metabolic regulators of tumor progression via SPHK1/S1P signaling.

Glaucoma, long considered an ocular-limited, age-dependent and hypoxia-driven neurodegeneration, is here reframed as a systemic erythroid-inosine axis failure that originates in the bone marrow yet culminates in retinal ganglion cell (RGC) death. By mining UK Biobank datasets (n = 127,028) and validating our findings in an independent clinical cohort (n = 178), we reveal that glaucoma is preceded by dyserythropoiesis and a compensatory, AMPK-driven metabolic rewiring of mature erythrocytes that hypercatabolizes inosine to enhance oxygen unloading. This adaptation collapses when accelerated erythrocyte inosine metabolism drains systemic pools, starving high-energy demand hematopoietic progenitors, driving retinal microenvironment hypoxia and accelerating RGC loss. Genetic ablation of murine erythroid equilibrative nucleoside transporter 1 (ENT1) recapitulates the hallmark features of patients with glaucoma, including impaired erythropoiesis, reduced oxygen delivery, retinal hypoxia and RGC apoptosis in both age and intraocular pressure-induced glaucoma models. Conversely, inosine repletion reconstitutes erythroid output, restores oxygen delivery from mature erythrocytes and halts neurodegeneration in inducible glaucoma models. A ten-metabolite erythrocyte signature centered on inosine metabolism offers diagnostic potential. Altogether, our work redefines glaucoma as the first treatable systemic erythroid-driven hypoxic syndrome, positioning inosine as a pleiotropic metabolic rescue factor for neurodegeneration and a powerful biomarker for intercepting hypoxia-driven pathologies across organs.

ABSTRACT Background Rapid ascent to high altitude causes acute mountain sickness (AMS) and life-threatening pulmonary/cerebral edema, yet no prophylaxis enables immediate acclimatization. Intermittent hypoxia training (IHT) establishes a “hypoxic memory” that accelerates adaptation, to high altitude, but its cellular and molecular basis remains undefined, precluding effective pharmacological strategies. Methods A human cohort of 18 sea-level inhibitants was equally divided into two groups, one group received IHT prior to ascent to 3,500 meters, the other group did not. Multi-omics profiling of erythrocytes and plasma, along with isotopic glucose tracing, was employed to examine the metabolic effects of IHT upon high altitude acclimatization. Preclinical studies with genetically engineered mice were used to further define the molecular and metabolic basis of IHT-induced hypoxic memory allowing rapid acclimatization to high altitude. Results Metabolomics revealed glucocorticoids as previously unrecognized endogenous erythroid hypoxic memory orchestrators induced by IHT that negatively correlated with AMS severity. Lipidomics and isotopic glucose tracing demonstrated that glucocorticoid signaling via the glucocorticoid receptor (GR) coordinately enhanced glucose metabolism and activated sphingosine kinase-1 (SPHK1)-driven sphingosine-1-phosphate (S1P) synthesis, pre-conditioning erythrocyte oxygen unloading and antioxidant capacity. Glucocorticoid supplementation enhanced erythrocyte SPHK1 activation and oxygen delivery, counteracting multi-tissue hypoxia and pulmonary and renal neutrophil infiltration. Conversely, erythrocyte-specific Sphk1 ablation abolished glucocorticoid-induced S1P production causing severe tissue hypoxia and exaggerated pulmonary neutrophil infiltration. Conclusions We establish a new function of glucocorticoids in erythrocyte metabolic plasticity to enhance oxygen delivery as a hypoxic memory mechanism for rapid adaptation to high altitude. This previously unrecognized GR-mediated reprograming of glucose and sphingolipid metabolism offers a transformative precision pharmacologic strategy for high altitude preconditioning, high altitude emergencies and hypoxia-driven diseases.

Introduction A fundamental challenge in treating chronic kidney disease (CKD) is the lack of therapies to reverse established fibrosis. While systemic hypoxia sensors like erythrocyte sphingosine kinase 1 (eSPHK1) can initiate fibrotic signaling, the mechanisms driving self-perpetuating and progressive fibrosis remain unknown. Methods eSphk1 specific deficient mice underwent four-week angiotensin-II infusion, unilateral ureteral obstruction or ischemia reperfusion injury. Untargeted metabolomics profiled purified kidney macrophages, and [13C615N4]-arginine fluxomic tracked arginine-creatine metabolism in hypoxia treated macrophages. Multi-color immunofluorescent images of kidney tissues were scanned and scored. Preclinical studies by hypoxia inducible factor-1α (HIF-1α) inhibitor or knockdown macrophage creatine kinase B (Ckb) were performed. Clinical relevance was evaluated by measuring CKB mRNA level in peripheral blood mononuclear cells obtained from 131 patients with CKD and determining its correlation with disease severity. Results We identify a HIF-1α-CKB feedforward loop within profibrotic macrophages that functions as an autonomous engine of kidney fibrosis. This circuit, which can be triggered by established pathways such as eSphk1 dysfunction, is characterized by its capacity for self-renewal. Specifically, HIF-1α drives Ckb expression, reprogramming arginine metabolism toward creatine phosphate shunt (CPS) to generate an ATP surge that synergizes with an S1PR3-PKC signaling cascade to phosphorylate and stabilize HIF-1α, effectively bypassing the need for continued hypoxic input. This metabolic rewiring drives macrophage profibrotic polarization and leads to kidney fibrosis. Preclinically, pharmacologic HIF 1α inhibition or knockdown macrophage Ckb collapses this autonomous loop and halts fibrosis. Translationally, CKB mRNA in peripheral blood mononuclear cells rises in parallel with estimated glomerular filtration rate decline and histologic fibrosis score in patients with CKD. Conclusions Our work identifies a novel HIF-1α-CKB feedforward circuit in macrophages that sustains HIF-1α induction, channeling arginine metabolism toward CPS and thus promoting kidney fibrosis. These findings highlight that fibrosis is conceptualized from a passive end-stage outcome to an actively maintained process driven by the macrophage metabolic-polarization circuit, suggesting that breaking this malicious loop, in addition to initiating triggers, is critical to halt kidney fibrosis.

BACKGROUND AND AIMS:Obstructive sleep apnoea-hypopnoea syndrome (OSAHS) has emerged as a global epidemic with profound cardiovascular and renal consequences, yet its early pathogenic mechanisms remain poorly understood. Whether red blood cells (RBCs) act as the primary hypoxia sensor that transduces intermittent apnoea into irreversible outcomes remains enigmatic. This study aims to define the pathogenic nature of RBCs during the progression of OSAHS with a goal of identifying early biomarkers and targeted treatments to prevent detrimental outcomes. METHODS:A large OSAHS cohort and matched controls underwent quantification of RBC O2 off-loading capability and nitric oxide (NO) bioactivity. Untargeted metabolomics and [13C6, 15N4] arginine flux mapping identified specific metabolic pathway bottlenecks. The effect of OSAHS erythrocytes on endothelial function was evaluated by measuring acetylcholine-induced vasodilation in rat aortic rings incubated with the erythrocytes and perfused in a microfluidic system. Erythrocyte-specific sphingosine kinase-1 knockout mice (eSphK1-/-) and controls were exposed to chronic intermittent hypoxia (CIH). Therapeutic studies include a preclinical manipulation with the arginase inhibitor nor-NOHA, and a pilot continuous positive airway pressure (CPAP) observational study. RESULTS:OSAHS patients display dysfunctional RBCs with reduced O2 delivery and NO bioactivity alongside excessive oxidative stress, driven by impaired glucose and arginine metabolism. Moreover, arginine metabolism is preferentially channelled into ornithine and urea rather than NO production due to reduced endothelial nitric oxide synthase (eNOS) activity. Dysfunctional RBC-mediated blunted endothelium-dependent vasodilation is rescued by co-infusion of sodium nitroprusside (SNP) and pretreatment with S1P or nor-NOHA. These RBC anomalies correlate with peripheral hypoxia, hypertension, and metabolic disorders in patients and precede measurable hypertension and tissue damage in a CIH-exposed OSAHS murine model. Preclinically, nor-NOHA restores RBC-NO bioactivity and O2 delivery, normalizes blood pressure, and prevents tissue fibrosis. A three-circulating-metabolite fingerprint, including sphingosine, S1P, and arginine, is validated as an early and sensitive biomarker for its diagnosis and stratifies OSAHS severity. Genetically, CIH-challenged eSphK1-/- mice exhibit decreased eNOS activity and O2 offload capacity, severe tissue hypoxia, hypertension, and fibrosis. Mechanistically, this study revealed that decreased intracellular S1P and AMPK activity underlie reduced eNOS activation in RBCs of OSAHS by blocking its trafficking from the membrane to the cytosol and phosphorylation. In contrast, CPAP-treated patients exhibited lower erythrocyte dysfunction and arginine and sphingolipid metabolic impairment compared to untreated OSAHS patients. CONCLUSIONS:Altogether, this study demonstrates that OSAHS is a systemic RBC disease in which S1P-mediated O2 delivery and eNOS trafficking act as the master toggle between physiological O2 delivery and hypoxic vasculopathy. Circulating S1P, sphingosine, and arginine configuration constitute a sensitive metabolic signature enabling early diagnoses, while pharmacological or CPAP-mediated repair of the RBC S1P-eNOS axis offers precision cardiovascular and renal protection upstream of irreversible vascular injury.

This study employs Barnes maze behavioral assessments, untargeted liquid chromatography-mass spectrometry metabolomics, and 13C6-glucose isotopic tracing to systematically investigate cognitive function and metabolic profiles in hippocampal and cortical tissues of male and female mice across five distinct age-ranges. Behavioral analyses reveal significant cognitive decline in both sexes by 16-months-of-age, with females exhibiting more severe impairment by 23-months, demonstrating a sex-related variation. 13C6-glucose tracing analyses reveals that glucose is rapidly and preferentially metabolized toward the Tricarboxylic acid cycle over glycolysis and the pentose phosphate pathway (PPP), with metabolism rates increasing from juvenility to meet developmental demands and maintaining homeostasis into pre-elderly. Surprisingly, glucose metabolism continues to rise in elderly males but declines in females. Developmental shifts from purine biosynthesis to degradation display sex-related variation, highlighting sustained synthesis in elderly males versus degradation in aging females. Finally, age and sex- related differences in amino acids, neurotransmitters, histidine-derived antioxidants, and the arginine-urea cycle further underscore complex metabolic reprogramming in the CNS. Overall, our study elucidates from a metabolic perspective the molecular basis of sex-related variation in age-related cognitive decline by characterizing sex-related variation in reprogramming of glucose, purine, and amino acid metabolic networks. Integrated behavioural metabolomic profiling across the mouse lifespan reveals sex-specific brain metabolic reprogramming underlying divergent trajectories of age-related cognitive decline.

Aging-related diseases are aggravated by tissue hypoxia; however, the underlying mechanism remains unknown. Here, we report that the oxygen (O2) release capacity of red blood cells (RBCs) gradually decreases with age and is closely associated with aging-related tissue dysfunction. Metabolomic profiling of human and mouse RBCs and genetic studies in mice revealed that the reduction in 2,3-bisphosphoglyceric acid (2,3-BPG) content mediated by a decrease in bisphosphoglycerate mutase (BPGM) activity is a metabolic checkpoint underlying decreased RBC O2 release capability and dysfunction with advancing age. When glucose metabolism is impaired, erythroid inosine, transported by equilibrative nucleoside transporter 1 and converted to ribose 1-phosphate by increased purine nucleoside phosphorylase (PNP) activity, is an important compensatory fuel for RBCs during aging. In a preclinical study, inosine supplementation successfully alleviated the age-dependent reduction in BPGM activity that mediates glucose metabolic impairment, decreased O2 delivery, and tissue dysfunction. Finally, we unexpectedly discovered that 2,3-BPG acts as an inhibitor of PNP in RBCs by competing with the phosphate (Pi)-binding domain and interacting with residues serine 33 and alanine 116. Our studies revealed that impaired glucose metabolic reprogramming resulting from decreased BPGM activity underlies RBC bioenergetic decline and is a novel hallmark of aging. As 2,3-BPG levels decrease during aging, its inhibitory effect on PNP is reduced, resulting in increased PNP activity and inosine catabolism as an alternative fuel, suggesting that inosine is a potential rejuvenating therapy.

Metabolic adaptations that fuel metastatic dissemination are increasingly mapped, yet the existence of intrinsic metabolic "brakes" that actively restrain metastatic progression remains enigmatic. Here, we unveil bisphosphoglycerate mutase (BPGM) as a previously unrecognized metastasis suppressor that orchestrates a phospho-epigenetic relay linking glycolytic flux to carnitine-dependent fatty acid oxidation. Through high-resolution metabolomics, we discover that BPGM and its catalytic product 2,3-bisphosphoglycerate (2,3-BPG) constitute a metabolic checkpoint whose disruption predicts metastatic virulence in multiple cancers. Mechanistically, BPGM suppresses metastasis by triggering CDK1-T14 phosphorylation-dependent assembly of an EZH2-H3K27me3 repressor complex that silences γ-butyrobetaine hydroxylase (BBOX1), the rate-limiting enzyme in carnitine biosynthesis. This phospho-switch mechanism converts glycolytic 2,3-BPG levels into epigenetic orchestrator, thereby starving metastatic cells of carnitine-required fatty acid oxidation. Hypoxia-mediated KDM4A-H3K9me3 cascade emerges as the upstream inactivator of this metabolic-epigenetic checkpoint, explaining how tumor microenvironmental stress liberates metastatic potential. Therapeutically, pharmacological BBOX1 inhibition with Meldonium recapitulates BPGM-mediated metastasis suppression in orthotopic models, reducing metastatic burden. These findings reveal BPGM as a metabolic gatekeeper that integrates bioenergetic sensing with chromatin remodeling to constrain metastatic competence, while hypoxia-mediated checkpoint failure unleashes carnitine-fueled metastatic progression. Targeting the hypoxia-BPGM-BBOX1 axis represents an innovative approach for metastasis-preventive therapy.

Age-related hearing loss (ARHL) is a major public health concern, driven by the interplay of multiple factors. Here, we reveal spatiotemporal metabolic shifts in murine inner ears mirroring erythrocytes and plasma. Isotope-labeled glucose tracing demonstrates a metabolic rerouting favoring glycolysis over the pentose phosphate pathway, alongside downregulation of the tricarboxylic acid cycle, indicating impaired energy production and redox homeostasis. Accumulation of medium- and long-chain acylcarnitines further exacerbates lipotoxicity. Notably, age-dependent depletion of arginine, lysine, proline, and glycine disrupts the arginine-polyamine-urea cycle. Translationally, UK Biobank plasma metabolomics links omega-6 fatty acids, linoleic acid, glycine, and albumin to ARHL resilience, while branched-chain amino acids, tyrosine, creatinine, glycoprotein acetyls and urea confer risk. Sex differences in ARHL were linked to fatty acid metabolism divergence. These bioenergetic disruptions in the inner ear are mirrored in erythrocytes and plasma, highlighting potential biomarkers for early ARHL diagnosis and treatment.

Longevity individuals have lower susceptibility to chronic hypoxia, inflammation, oxidative stress, and aging-related diseases. It has long been speculated that "rejuvenation molecules" exist in their blood to promote extended lifespan. We unexpectedly discovered that longevity individuals exhibit erythrocyte oxygen release function similar to young individuals, whereas most elderly show reduced oxygen release capacity. Untargeted erythrocyte metabolomics profiling revealed that longevity individuals are characterized by youth-like metabolic reprogramming and these metabolites effectively differentiate the longevity from the elderly. Quantification analyses led us to identify multiple novel longevity-related metabolites within erythrocytes including adenosine, sphingosine-1-phosphate (S1P), and glutathione (GSH) related amino acids. Mechanistically, we revealed that increased bisphosphoglycerate mutase (BPGM) and reduced MFSD2B protein levels in the erythrocytes of longevity individuals collaboratively work together to induce elevation of intracellular S1P, promote the release of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from membrane to the cytosol, and thereby orchestrate glucose metabolic reprogramming toward Rapoport-Luebering Shunt to induce the 2,3-BPG production and trigger oxygen delivery. Furthermore, increased glutamine and glutamate transporter expression coupled with the enhanced intracellular metabolism underlie the elevated GSH production and the higher anti-oxidative stress capacity in the erythrocytes of longevity individuals. As such, longevity individuals displayed less systemic hypoxia-related metabolites and more antioxidative and anti-inflammatory metabolites in the plasma, thereby healthier clinical outcomes including lower inflammation parameters as well as better glucose-lipid metabolism, and liver and kidney function. Overall, we identified that youthful erythrocyte function and metabolism enable longevity individuals to better counteract peripheral tissue hypoxia, inflammation, and oxidative stress, thus maintaining healthspan.

Abnormal adipogenesis is a major contributor to fetal growth restriction (FGR) and its associated complications. However, the underlying etiology remains unclear. Here, it is reported that the placentas of women with pregnancies complicated with FGR exhibit peroxisome proliferator-activated receptor γ (PPARγ) inactivation. In mice, trophoblast-specific ablation of murine PPARγ reproduces the phenotype of human fetuses with FGR and defective adipogenesis. Coculture of trophoblasts with preadipocytes significantly improves preadipocyte commitment and differentiation and increases the transcription of a series of adipogenic genes via intercellular transfer of exosomal PPARγ proteins. Moreover, nanoparticle-mediated placenta-specific delivery of rosiglitazone (RGZ) significantly rescues adipogenesis defects in an FGR-induced mouse model. In summary, the placenta is a major reservoir of PPARγ. An insufficient supply of placental PPARγ to fetal preadipocytes via exosomes during late gestation is a major mechanism underlying FGR. Preclinically, placenta-targeted RGZ administration can be a promising interventional therapy for FGR and/or defective intrauterine fat development.

Introduction: The renin-angiotensin system (RAS) is an important regulator of blood pressure and fluid balance through the activation of AT receptors (ATRs) by angiotensin II (Ang II). Dysregulation of RAS leads to hypertension, inflammation, organ damage, fibrosis and progression of multiple chronic diseases. It is known that Ang II induces erythropoiesis. However, as the most abundant and only cells responsible for oxygen delivery within our body, the function of RAS and its underlying mechanism in mature erythrocytes remains unknown. Aim: We sought to define the specific expression of ATRs in mature erythrocytes, its function of and underlying mechanisms in multiple chronic disease conditions. Methods: Using gene expression profiling and Western blot analyses, we probed the expression of ATRs in both human and mouse erythroblasts and mature erythrocytes. Using genetic approaches, we generated the specific ablation of AT1aR in erythrocytes (eAT1aR-/-) by crossbreeding AT1aRloxP/loxP mice with EpoR-Cre+mice. Using two well accepted chronic hypertension and chronic kidney disease experimental models including Ang II infusion and unilateral ureteral obstruction (UUO), we compared the erythrocyte function and its life span as well as peripheral tissue hypoxia, function and fibrosis among eAT1aR-/- mice and controls with or without Ang II infusion and UUO challenge. Using comprehensive metabolomics and U-13C6 isotopically labeled glucose flux analysis coupled with multidisciplinary cellular and molecular approaches, we determined its molecular and metabolic bases. Results: We precisely defined that only AT1aRs but not AT1bRs or AT2Rs were expressed in both human and murine erythroblasts and mature erythrocytes. Genetically, eAT1aR-/- mice werefertile and morphologically indistinguishable from littermates with the similar systolic blood pressure and erythrocyte lifespan as control mice. Ang II infusion induced hypertension, severe cardiorenal hypoxia, myocardial hypertrophy, cardiorenal damage and fibrosis but less oxygen delivery from erythrocytes in eAT1aR-/- mice comparing to the Ang II-infused controls. Metabolically, we revealed that Ang II failed to induce glucose metabolic reprogramming channeled toward glycolysis over pentose phosphate pathway, resulting in less erythroid-specific Rapoport-Luebering Shunt and reduced 2,3-bisphosphoglycerate (2,3-BPG) production and O2 delivery from erythrocytes of Ang II infused eAT1aR-/- mice comparing to the controls. Similar as Ang II challenge, UUO murine models exhibited severe renal hypoxia, inflammation and fibrotic changes without effects on blood pressure in eAT1aR-/- mice comparing to the controls, indicating the common beneficial role of eAT1-mediated oxygen delivery in two independent animal models of hypertension and obstructive induced cardiorenal damages and progression. Molecularly, we defined that Ang II directly induced oxygen offload by activating AT1R-mediated SphK1-S1P-dependent BPGM activation in both primary cultured human and murine erythrocytes. Conclusion: We revealed a previously unrecognized protective role of eAT1 to combat tissue hypoxia, damage and fibrosis in two distinct experimental models via SphK1-S1P signaling cascade-mediated BPGM activation and enhanced O2 delivery from erythrocytes. These findings add a significant new chapter to the importance of erythrocyte RAS system in counteracting tissue hypoxia, cardiorenal damage and disease progression and suggest new therapeutic avenues.

Introduction: Erythrocytes, the major reservoir and supplier for plasma sphingosine 1 phosphate (S1P), is a potent biolipid signaling via its specific receptors responsible for immune cell trafficking and function. Current evidence has revealed that erythrocyte intracellular S1P has its own function to promote oxygen (O 2) delivery to counteract tissue hypoxia. Aim: To understand whether O 2 delivery mediated by erythrocyte S1P is involved in tissue macrophage polarization and fibrosis. Methods and Results: Genetic ablation of erythrocyte SphK1 (the only enzyme to generate S1P in erythrocyte, eSphK1 -/-) resulted in less O 2 delivery from erythrocytes, thus leading to severe renal hypoxia, damage and fibrosis in angiotensin II induced hypertensive kidney disease mouse model. Surprisingly, although plasma S1P level was reduced to 50% in Ang II-infused eSphK1 -/- mice, these mice displayed preferentially elevated pro-fibrosis M2 macrophages surrounding the hypoxic renal tubules. Surprisingly, there was no significant difference of S1P level in either kidney or renal macrophages between Ang II-infused controls and eSphK1 -/- mice, ruling out the possibility of difference of kidney or macrophage S1P levels responsible for renal M2 macrophage polarization. Metabolically, untargeted high-throughput metabolic profiling in macrophages purified from mouse kidney revealed a substantial reduction of amino acids (AAs) and fatty acids (FAs) but increased creatine. We further validate this in vitro by 13C 6, 15N 4-labeled arginine tracer. Hypoxia (1~2% O 2) promoted increased 13C 2-labeled creatine (arginine downstream metabolite) in murine bone marrow derived macrophages (BMDMs) time dependently. At cellular and molecular level, we further demonstrated that hypoxia signaling via HIF-1a directly enhanced IL-4 induced M2 polarization of BMDMs as well as expression of Ckb (a gene encoding creatine shuttle), which can be reversed by Chrysin (an HIF-1α inhibitor). Preclinical studies revealed that chrysin rescued the severe renal damage in eSphK1 -/-mice manipulated with unilateral ureteral obstruction (UUO) and prevented development of chronic kidney disease (CKD) in the control mice. Translational research validated mouse findings in kidney tissue of CKD patients. Conclusion: Our work has revealed that erythrocyte S1P combats HIF1α-dependent creatine shuttle, renal macrophage polarization and fibrosis and highlighted potential diagnostic and therapeutic possibilities for macrophage polarization and renal fibrosis.

Hypoxia is considered a common cause of myriad diseases and drives tissue damage and progression. Erythrocytes are vital under hypoxia since they can sense and act rapidly to hypoxia by promoting metabolomic reprogramming and oxygen (O 2) release. Adenosine monophosphate deaminase 3 (AMPD3) is highly enriched in erythrocytes and plays an important role in maintaining erythrocyte intracellular purine homeostasis. However, how it senses and regulates oxygen release remains poorly understood. Here we report that genetic ablation of AMPD3 led to more O 2 release from erythrocytes, resulting in less tissue hypoxia, damage, inflammation and fibrosis in two independent pathological hypoxia models including Angiotensin II infusion and unilateral ureteric obstruction (UUO) models. Metabolically, untargeted high-throughput metabolomic profiling in erythrocytes revealed that nucleotides including AMP, ADP and ATP levels were significantly increased in WT mice and further elevated in Ampd3 -/-mice. Isotopically adenosine flux experiment demonstrated that adenosine was rapidly and largely converted to AMP. At molecular level, we further demonstrated that hypoxia-induced AMPD3 activity as a compensatory mechanism led to accumulation of AMP, subsequently inducing AMP kinase (AMPK)-dependent metabolic reprogramming by inducing bisphosphoglycerate mutase (BPGM), which shifts glycolysis toward erythroid unique Rapoport-Leubering Shunt (RSL) to promote 2,3-BPG production, O 2 delivery and anti-ROS capacity to mitigate tissue hypoxia, dysfunction, inflammation and fibrosis. At cellular level, we revealed that hypoxia directly inhibited AMPD3, leading to activation of AMPK by lowering ROS in both cultured primary human and murine erythrocytes. Finally, we conducted human translational studies demonstrating that the production of 2,3-BPG, BPGM activity, p-AMPK level, and P50 were increased, while AMPD3 activity was decreased in patients with chronic kidney disease (a common pathological condition) compared with normal controls. Altogether, eAMPD3is a previously unrecognized master intracellular purinergic componentsensing hypoxia and promoting adaptative metabolic reprogramming tomitigate tissue hypoxia, insufficient energy, tissue damage and fibrosis by enhancing O 2 delivery and antioxidative stress capacity in a positive feedforward manner.

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 O2 delivery, which combat renal hypoxia and progression of CKD. ENT1-AMPD3-AMPK-BPGM comprise a group of circulating erythroid-specific biomarkers, providing early diagnostic and novel therapeutic targets for CKD. Background 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. Methods Mice with an erythrocyte-specific deficiency in equilibrative nucleoside transporter 1 (eEnt1 −/− ) and a global deficiency in AMP deaminase 3 (Ampd3 −/− ) were generated to define their function in two independent CKD models, including angiotensin II (Ang II) infusion and unilateral ureteral obstruction (UUO). Unbiased metabolomics, isotopic adenosine flux, and various biochemical and cell culture analyses coupled with genetic studies were performed. Translational studies in patients with CKD and cultured human erythrocytes examined the role of ENT1 and AMPD3 in erythrocyte function and metabolism. Results eEnt1 −/− mice display severe renal hypoxia, kidney damage, and fibrosis in both CKD models. The loss of eENT1-mediated adenosine uptake reduces intracellular AMP and thus abolishes the activation of AMPKα and bisphosphoglycerate mutase (BPGM). This results in reduced 2,3-bisphosphoglycerate and glutathione, leading to overwhelming oxidative stress in eEnt1 −/− mice. Excess reactive oxygen species (ROS) activates AMPD3, resulting in metabolic reprogramming and reduced O2 delivery, leading to severe renal hypoxia in eEnt1 −/− mice. By contrast, genetic ablation of AMPD3 preserves the erythrocyte adenine nucleotide pool, inducing AMPK-BPGM activation, O2 delivery, and antioxidative stress capacity, which protect against Ang II-induced renal hypoxia, damage, and CKD progression. Translational studies recapitulated the findings in mice. Conclusion eENT1-AMPD3, two highly enriched erythrocyte purinergic components that sense hypoxia, promote eAMPK-BPGM–dependent metabolic reprogramming, O2 delivery, energy supply, and antioxidative stress capacity, which mitigates renal hypoxia and CKD progression.

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. The PDI function catalyzes the trimerization and activation of heat shock factor-1 in the nucleus and regulates the oxidation state of several mitochondrial complexes. Cytosolic TG2 modifies proteins by the addition of serotonin or other primary amines and in this way affects cell signaling. Modification of protein-bound glutamines reduces ubiquitin-dependent proteasomal degradation. At the cell membrane, TG2 is associated with G protein-coupled receptors (GPCRs), where it functions in transmembrane signaling. TG2 is also found in the extracellular space, where it functions in protein cross-linking and extracellular matrix stabilization. Of particular importance in transglutaminase research are recent findings concerning the role of TG2 in gene expression, protein homeostasis, cell signaling, autoimmunity, inflammation, and hypoxia. Thus, TG2 performs a multitude of functions in multiple cellular compartments, making it one of the most versatile cellular proteins. Additional evidence links TG2 with multiple human diseases including preeclampsia, hypertension, cardiovascular disease, organ fibrosis, cancer, neurodegenerative diseases, and celiac disease. In conclusion, TG2 provides a multifunctional and multisite response to physiological stress.

Due to lack of nuclei and de novo protein synthesis, post-translational modification (PTM) is imperative for erythrocytes to regulate oxygen (O2) delivery and combat tissue hypoxia. Here, we report that erythrocyte transglutminase-2 (eTG2)-mediated PTM is essential to trigger O2 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. Carnitine supplementation is an effective and safe therapeutic approach to counteract hypertension and progression of CKD by enhancing erythrocyte O2 delivery. Altogether, we reveal eTG2 as an erythrocyte protein stabilizer orchestrating O2 delivery and tissue adaptive metabolic reprogramming and identify carnitine-based therapy to mitigate hypoxia and CKD progression.

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. Elevated levels of polyubiquitinated membrane-associated proteins in human sRBCs are reported here. High throughput and untargeted proteomic analyses of membrane proteins immunoprecipitated by ubiquitin antibodies detected elevated levels of ubiquitination of a series of proteins including cytoskeletal proteins, transporters, ROS-related proteins, and UPS machinery components in sRBCs. Polyubiquitination of membrane-associated catalase was increased in sRBCs, associated with decreased catalase activity and elevated ROS. Surprisingly, shuttling of p97 (ATP-dependent valosin-containing chaperone protein), a key component of the UPS to shuttle polyubiquitinated proteins from the membrane to cytosol for proteasomal degradation, was significantly impaired, resulting in significant accumulation of p97 along with polyubiquitinated proteins in the membrane of human sRBCs. Functionally, inhibition of p97 directly promoted accumulation of polyubiquitinated membrane-associated proteins, excessive ROS levels, and sickling in response to hypoxia. Overall, we revealed that p97 dysfunction underlies impaired UPS and contributes to oxidative stress in sRBCs.

As the only cells that carry and deliver oxygen (O2), erythrocytes play a vital role in maintaining the normal energy metabolism, function, and survival of every cell and organ within our body. Erythrocytes senses and acts rapidly to hypoxia by promoting metabolic reprogramming and O2 delivery. However, the erythroid specific hypoxic sensor and its metabolic role in tissue hypoxia and fibrosis remain poorly understood. Here we report that erythrocyte enriched equilibrative nucleotide transporter 1 (eENT1)-mediated rapid uptake of extracellular adenosine coordinating with the inhibition of erythrocyte specific AMPD3 (eAMPD3) is an imperative "erythrocyte hypoxia sensory machinery" boosting intracellular AMP and promoting AMP kinase (AMPK)-dependent metabolic reprogramming by inducing bisphosphoglycerate mutase (BPGM), which wires glucose metabolism toward specific erythroid glycolytic-Rapoport-Leubering Shunt (RSL) to trigger 2,3-BPG production, O2 delivery and anti-ROS capacity to mitigate renal hypoxia and progression in both patients and mice. Mechanistically, we revealed that hypoxia directly inhibits eAMPD3, leading to activation of eAMPK by lowering ROS in both cultured primary human and murine erythrocytes. Altogether, eENT1-eAMPD3 are previously unrecognized master intracellular purinergic components sensing hypoxia and promoting adaptative metabolic reprogramming to mitigate renal hypoxia, insufficient energy, kidney damage and progression by enhancing O2 delivery and antioxidative stress capacity in a positive feedforward manner. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal