Proteomic analysis of urinary extracellular vesicles highlights specific signatures for patients with primary aldosteronism.

Background: Urinary extracellular vesicles (uEVs) can be released by different cell types facing the urogenital tract and are involved in cellular trafficking, differentiation and survival. UEVs can be easily detected in urine and provide pathophysiological information "in vivo" without the need of a biopsy. Based on these premises, we hypothesized that uEVs proteomic profile may serve as a valuable tool in the differential characterization between Essential Hypertension (EH) and primary aldosteronism (PA). Methods: Patients with essential hypertension (EH) and PA were enrolled in the study (EH= 12, PA=24: 11 Bilateral Primary Aldosteronism subtype (BPA) and 13 Aldosterone Producing Adenoma (APA)). Clinical and biochemical parameters were available for all the subjects. UEVs were isolated from urine by ultracentrifugation and analysed by Transmission Electron Microscopy (TEM) and nanotrack particle analysis (NTA). UEVs protein content was investigated through an untargeted MS-based approach. Statistical and network analysis was performed to identify potential candidates for the identification and classification of PA. Results: MS analysis provided more than 300 protein identifications. Exosomal markers CD9 and CD63 were detected in all samples. Several molecules characterizing EH vs PA patients as well as BPA and APA subtypes were identified after statistical elaboration and filtering of the results. In particular, some key proteins involved in water reabsorption mechanisms, such as AQP1 and AQP2, were among the best candidates for discriminating EH vs PA, as well as A1AG1 (AGP1). Conclusion: Through this proteomic approach, we identified uEVs molecular indicators that can improve PA characterization and help in the gain of insights of the pathophysiological features of this disease. In particular, PA was characterized by a reduction of AQP1 and AQP2 expression as compared with EH.

Urinary extracellular vesicle mRNA analysis of sodium chloride cotransporter in hypertensive patients under different conditions.

Urinary extracellular vesicles (UEV) mainly derive from cells of the urogenital tract and their cargo (proteins, nucleic acids, lipids, etc.) reflects their cells of origin. Na chloride cotransporter (NCC) is expressed at the kidney level in the distal convoluted tubule, is involved in salt reabsorption, and is the target of the diuretic thiazides. NCC protein has been recognized and quantified in UEV in previous studies; however, UEV NCC mRNA has never been studied. This study aimed to identify and analyze NCC mRNA levels in primary aldosteronism (PA). The rationale for this investigation stems from previous observations regarding NCC (protein) as a possible biomarker for the diagnosis of PA. To evaluate modulations in the expression of NCC, we analyzed NCC mRNA levels in UEV in PA and essential hypertensive (EH) patients under different conditions, that is, before and after saline infusion, anti-aldosterone pharmacological treatment, and adrenal surgery. NCC mRNA was measured by RT-qPCR in all the samples and was regulated by volume expansion. Its response to mineralocorticoid receptor antagonist was correlated with renin, and it was increased in PA patients after adrenalectomy. NCC mRNA is evaluable in UEV and it can provide insights into the pathophysiology of distal convolute tubule in different clinical conditions including PA.

Red Blood Cell Morphologic Abnormalities in Patients Hospitalized for COVID-19.

Peripheral blood smear is a simple laboratory tool, which remains of invaluable help for diagnosing primary and secondary abnormalities of blood cells despite advances in automated and molecular techniques. Red blood cells (RBCs) abnormalities are known to occur in many viral infections, typically in the form of mild normo-microcytic anemia. While several hematological alterations at automated complete blood count (including neutrophilia, lymphopenia, and increased red cell distribution width-RDW) have been consistently associated with severity of COVID-19, there is scarce information on RBCs morphological abnormalities, mainly as case-reports or small series of patients, which are hardly comparable due to heterogeneity in sampling times and definition of illness severity. We report here a systematic evaluation of RBCs morphology at peripheral blood smear in COVID-19 patients within the first 72 h from hospital admission. One hundred and fifteen patients were included, with detailed collection of other clinical variables and follow-up. A certain degree of abnormalities in RBCs morphology was observed in 75 (65%) patients. Heterogenous alterations were noted, with spiculated cells being the more frequent morphology. The group with >10% RBCs abnormalities had more consistent lymphopenia and thrombocytopenia compared to those without abnormalities or <10% RBCs abnormalities (p < 0.018, and p < 0.021, respectively), thus underpinning a possible association with an overall more sustained immune-inflammatory "stress" hematopoiesis. Follow-up analysis showed a different mortality rate across groups, with the highest rate in those with more frequent RBCs morphological alterations compared to those with <10% or no abnormalities (41.9%, vs. 20.5%, vs. 12.5%, respectively, p = 0.012). Despite the inherent limitations of such simple association, our results point out towards further studies on erythropoiesis alterations in the pathophysiology of COVID-19.

ZOOMICS: Comparative Metabolomics of Red Blood Cells From Guinea Pigs, Humans, and Non-human Primates During Refrigerated Storage for Up to 42 Days.

Unlike other rodents, guinea pigs (Cavia porcellus) have evolutionarily lost their capacity to synthesize vitamin C (ascorbate) de novo and, like several non-human primates and humans, rely on dietary intake and glutathione-dependent recycling to cope with oxidant stress. This is particularly relevant in red blood cell physiology, and especially when modeling blood storage, which exacerbates erythrocyte oxidant stress. Herein we provide a comprehensive metabolomics analysis of fresh and stored guinea pig red blood cell concentrates (n = 20), with weekly sampling from storage day 0 through 42. Results were compared to previously published ZOOMICS studies on red blood cells from three additional species with genetic loss of L-gulonolactone oxidase function, including humans (n = 21), olive baboons (n = 20), and rhesus macaques (n = 20). While metabolic trends were comparable across all species, guinea pig red blood cells demonstrated accelerated alterations of the metabolic markers of the storage lesion that are consistent with oxidative stress. Compared to the other species, guinea pig red blood cells showed aberrant glycolysis, pentose phosphate pathway end product metabolites, purine breakdown products, methylation, glutaminolysis, and markers of membrane lipid remodeling. Consistently, guinea pig red blood cells demonstrated higher end storage hemolysis, and scanning electron microscopy confirmed a higher degree of morphological alterations of their red blood cells, as compared to the other species. Despite a genetic inability to produce ascorbate that is common to the species evaluated, guinea pig red blood cells demonstrate accelerated oxidant stress under standard storage conditions. These data may offer relevant insights into the basal and cold storage metabolism of red blood cells from species that cannot synthesize endogenous ascorbate.

Beta thalassemia minor is a beneficial determinant of red blood cell storage lesion.

Blood donor genetics and lifestyle affect the quality of red blood cell (RBC) storage. Heterozygotes for beta thalassemia (bThal+) constitute a non-negligible proportion of blood donors in the Mediterranean and other geographical areas. The unique hematological profile of bThal+ could affect the capacity of enduring storage stress, however, the storability of bThal+ RBC is largely unknown. In this study, RBC from 18 bThal+ donors were stored in the cold and profiled for primary (hemolysis) and secondary (phosphatidylserine exposure, potassium leakage, oxidative stress) quality measures, and metabolomics, versus sex- and age-matched controls. The bThal+ units exhibited better levels of storage hemolysis and susceptibility to lysis following osmotic, oxidative and mechanical insults. Moreover, bThal+ RBC had a lower percentage of surface removal signaling, reactive oxygen species and oxidative defects to membrane components at late stages of storage. Lower potassium accumulation and higher uratedependent antioxidant capacity were noted in the bThal+ supernatant. Full metabolomics analyses revealed alterations in purine and arginine pathways at baseline, along with activation of the pentose phosphate pathway and glycolysis upstream to pyruvate kinase in bThal+ RBC. Upon storage, substantial changes were observed in arginine, purine and vitamin B6 metabolism, as well as in the hexosamine pathway. A high degree of glutamate generation in bThal+ RBC was accompanied by low levels of purine oxidation products (IMP, hypoxanthine, allantoin). The bThal mutations impact the metabolism and the susceptibility to hemolysis of stored RBC, suggesting good post-transfusion recovery. However, hemoglobin increment and other clinical outcomes of bThal+ RBC transfusion deserve elucidation by future studies.

Detection of Urinary Exosomal HSD11B2 mRNA Expression: A Useful Novel Tool for the Diagnostic Approach of Dysfunctional 11ß-HSD2-Related Hypertension.

Objective: Apparent mineralocorticoid excess (AME) is an autosomal recessive disorder caused by the 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) enzyme deficiency, traditionally assessed by measuring either the urinary cortisol metabolites ratio (tetrahydrocortisol+allotetrahydrocortisol/tetrahydrocortisone, THF+5αTHF/THE) or the urinary cortisol/cortisone (F/E) ratio. Exosomal mRNA is an emerging diagnostic tool due to its stability in body fluids and its biological regulatory function. It is unknown whether urinary exosomal HSD11B2 mRNA is related to steroid ratio or the HSD11B2 662 C>G genotype (corresponding to a 221 A>G substitution) in patients with AME and essential hypertension (EH). Aim of the Study: To detect and quantify HSD11B2 mRNA from urinary exosomes in samples from family members affected by AME and EH, and to evaluate the relationship between exosomal HSD11B2 mRNA, steroid ratio, 662C>G genotype, and hypertension. Methods: In this observational case-control study, urinary steroid ratios and biochemical parameters were measured. Urinary exosomes were extracted from urine and exosomal HSD11B2 mRNA was quantified by Droplet Digital PCR (ddPCR). B2M (ß-2 microglobulin) gene was selected as the reference housekeeping gene. Results: Among family members affected by AME, exosomal urinary HSD11B2 mRNA expression was strictly related to genotypes. The two homozygous mutant probands showed the highest HSD11B2 mRNA levels (median 169, range 118-220 copies/µl) that progressively decreased in 221 AG heterozygous with hypertension (108, range 92-124 copies/µl), 221 AG heterozygous normotensives (23.35, range 8-38.7 copies/µl), and wild-type 221 AA subjects (5.5, range 4.5-14 copies/µl). Heterozygous hypertensive subjects had more HSD11B2 mRNA than heterozygous normotensive subjects. The F/E urinary ratio correlated with HSD11B2 mRNA copy number (p < 0.05); HSD11B2 mRNA strongly decreased while THF+5αTHF/THE increased in the two probands after therapy. In the AME family, HSD11B2 copy number correlated with both F/E and THF+5αTHF/THE ratios, whereas in EH patients, a high F/E ratio reflected a reduced HSD11B2 mRNA expression. Conclusions: HSD11B2 mRNA is detectable and quantifiable in urinary exosomes; its expression varies according to the 662 C>G genotype with the highest levels in homozygous mutant subjects. The HSD11B2 mRNA overexpression in AME could be due to a compensatory mechanism of the enzyme impairment. Exosomal mRNA is a useful tool to investigate HSD11B2 dysregulation in hypertension.

Blood donor obesity is associated with changes in red blood cell metabolism and susceptibility to hemolysis in cold storage and in response to osmotic and oxidative stress.

BACKGROUND: Obesity is a global pandemic characterized by multiple comorbidities, including cardiovascular and metabolic diseases. The aim of this study was to define the associations between blood donor body mass index (BMI) and RBC measurements of metabolic stress and hemolysis. STUDY DESIGN AND METHODS: The associations between donor BMI (<25 kg/m2 , normal weight; 25-29.9 kg/m2 , overweight; and ≥30 kg/m2 , obese) and hemolysis (storage, osmotic, and oxidative; n = 18 donors) or posttransfusion recovery (n = 14 donors) in immunodeficient mice were determined in stored leukocyte-reduced RBC units. Further evaluations were conducted using the National Heart, Lung, and Blood Institute RBC-Omics blood donor databases of hemolysis (n = 13 317) and metabolomics (n = 203). RESULTS: Evaluations in 18 donors revealed that BMI was significantly (P < 0.05) and positively associated with storage and osmotic hemolysis. A BMI of 30 kg/m2 or greater was also associated with lower posttransfusion recovery in mice 10 minutes after transfusion (P = 0.026). Multivariable linear regression analyses in RBC-Omics revealed that BMI was a significant modifier for all hemolysis measurements, explaining 4.5%, 4.2%, and 0.2% of the variance in osmotic, oxidative, and storage hemolysis, respectively. In this cohort, obesity was positively associated (P < 0.001) with plasma ferritin (inflammation marker). Metabolomic analyses on RBCs from obese donors (44.1 ± 5.1 kg/m2 ) had altered membrane lipid composition, dysregulation of antioxidant pathways (eg, increased oxidized lipids, methionine sulfoxide, and xanthine), and dysregulation of nitric oxide metabolism, as compared to RBCs from nonobese (20.5 ± 1.0 kg/m2 ) donors. CONCLUSIONS: Obesity is associated with significant changes in RBC metabolism and increased susceptibility to hemolysis under routine storage of RBC units. The impact on transfusion efficacy warrants further evaluation.

ZOOMICS: Comparative Metabolomics of Red Blood Cells From Old World Monkeys and Humans.

As part of the ZOOMICS project, we set out to investigate common and diverging metabolic traits in the blood metabolome across various species by taking advantage of recent developments in high-throughput metabolomics. Here we provide the first comparative metabolomics analysis of fresh and stored human (n = 21, 10 males, 11 females), olive baboon (n = 20), and rhesus macaque (n = 20) red blood cells at baseline and upon 42 days of storage under blood bank conditions. The results indicated similarities and differences across species, which ultimately resulted in a differential propensity to undergo morphological alterations and lyse as a function of the duration of refrigerated storage. Focusing on purine oxidation, carboxylic acid, fatty acid, and arginine metabolism further highlighted species-specific metabolic wiring. For example, through a combination of steady state measurements and 13C6 15N4-arginine tracing experiments, we report an increase in arginine catabolism into ornithine in humans, suggestive of species-specific arginase 1 activity and nitric oxide synthesis-an observation that may impact the translatability of cardiovascular disease studies carried out in non-human primates (NHPs). Finally, we correlated metabolic measurements to storage-induced morphological alterations via scanning electron microscopy and hemolysis, which were significantly lower in human red cells compared to both NHPs.

COVID-19 infection results in alterations of the kynurenine pathway and fatty acid metabolism that correlate with IL-6 levels and renal status.

Previous studies suggest a role for systemic reprogramming of host metabolism during viral pathogenesis to fuel rapidly expanding viral proliferation, for example by providing free amino acids and fatty acids as building blocks. In addition, general alterations in metabolism can provide key understanding of pathogenesis. However, little is known about the specific metabolic effects of SARS-COV-2 infection. The present study evaluated the serum metabolism of COVID-19 patients (n=33), identified by a positive nucleic acid test of a nasopharyngeal swab, as compared to COVID-19-negative control patients (n=16). Targeted and untargeted metabolomics analyses specifically identified alterations in the metabolism of tryptophan into the kynurenine pathway, which is well-known to be involved in regulating inflammation and immunity. Indeed, the observed changes in tryptophan metabolism correlated with serum interleukin-6 (IL-6) levels. Metabolomics analysis also confirmed widespread dysregulation of nitrogen metabolism in infected patients, with decreased circulating levels of most amino acids, except for tryptophan metabolites in the kynurenine pathway, and increased markers of oxidant stress (e.g., methionine sulfoxide, cystine), proteolysis, and kidney dysfunction (e.g., creatine, creatinine, polyamines). Increased circulating levels of glucose and free fatty acids were also observed, consistent with altered carbon homeostasis in COVID-19 patients. Metabolite levels in these pathways correlated with clinical laboratory markers of inflammation and disease severity (i.e., IL-6 and C-reactive protein) and renal function (i.e., blood urea nitrogen). In conclusion, this initial observational study of the metabolic consequences of COVID-19 infection in a clinical cohort identified amino acid metabolism (especially kynurenine and cysteine/taurine) and fatty acid metabolism as correlates of COVID-19, providing mechanistic insights, potential markers of clinical severity, and potential therapeutic targets.

COVID-19 infection alters kynurenine and fatty acid metabolism, correlating with IL-6 levels and renal status.

BACKGROUNDReprogramming of host metabolism supports viral pathogenesis by fueling viral proliferation, by providing, for example, free amino acids and fatty acids as building blocks.METHODSTo investigate metabolic effects of SARS-CoV-2 infection, we evaluated serum metabolites of patients with COVID-19 (n = 33; diagnosed by nucleic acid testing), as compared with COVID-19-negative controls (n = 16).RESULTSTargeted and untargeted metabolomics analyses identified altered tryptophan metabolism into the kynurenine pathway, which regulates inflammation and immunity. Indeed, these changes in tryptophan metabolism correlated with interleukin-6 (IL-6) levels. Widespread dysregulation of nitrogen metabolism was also seen in infected patients, with altered levels of most amino acids, along with increased markers of oxidant stress (e.g., methionine sulfoxide, cystine), proteolysis, and renal dysfunction (e.g., creatine, creatinine, polyamines). Increased circulating levels of glucose and free fatty acids were also observed, consistent with altered carbon homeostasis. Interestingly, metabolite levels in these pathways correlated with clinical laboratory markers of inflammation (i.e., IL-6 and C-reactive protein) and renal function (i.e., blood urea nitrogen).CONCLUSIONIn conclusion, this initial observational study identified amino acid and fatty acid metabolism as correlates of COVID-19, providing mechanistic insights, potential markers of clinical severity, and potential therapeutic targets.FUNDINGBoettcher Foundation Webb-Waring Biomedical Research Award; National Institute of General and Medical Sciences, NIH; and National Heart, Lung, and Blood Institute, NIH.

Impact of taurine on red blood cell metabolism and implications for blood storage.

BACKGROUND: Taurine is an antioxidant that is abundant in some common energy drinks. Here we hypothesized that the antioxidant activity of taurine in red blood cells (RBCs) could be leveraged to counteract storage-induced oxidant stress. STUDY DESIGN AND METHODS: Metabolomics analyses were performed on plasma and RBCs from healthy volunteers (n = 4) at baseline and after consumption of a whole can of a common, taurine-rich (1000 mg/serving) energy drink. Reductionistic studies were also performed by incubating human RBCs with taurine ex vivo (unlabeled or 13 C15 N-labeled) at increasing doses (0, 100, 500, and 1000 µmol/L) at 37°C for up to 16 hours, with and without oxidant stress challenge with hydrogen peroxide (0.1% or 0.5%). Finally, we stored human and murine RBCs under blood bank conditions in additives supplemented with 500 µmol/L taurine, before metabolomics and posttransfusion recovery studies. RESULTS: Consumption of energy drinks increased plasma and RBC levels of taurine, which was paralleled by increases in glycolysis and glutathione (GSH) metabolism in the RBC. These observations were recapitulated ex vivo after incubation with taurine and hydrogen peroxide. Taurine levels in the RBCs from the REDS-III RBC-Omics donor biobank were directly proportional to the total levels of GSH and glutathionylated metabolites and inversely correlated to oxidative hemolysis measurements. Storage of human RBCs in the presence of taurine improved energy and redox markers of storage quality and increased posttransfusion recoveries in FVB mice. CONCLUSION: Taurine modulates RBC antioxidant metabolism in vivo and ex vivo, an observation of potential relevance to transfusion medicine.