Key points
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Exosomes are vesicles that are released from the kidney into the urine. They contain RNA and protein from the cell of origin and can track changes in renal physiology non‐invasively.
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...Current methods for the identification and quantification of urinary exosomes are time consuming and only semi‐quantitative.
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In this study, we applied nanoparticle tracking analysis to human urine and identified particles with a range of sizes, including a subpopulation of characteristic exosomal size that labelled positively with antibodies to exosome proteins.
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Nanoparticle tracking analysis was able to track an increase in exosomal aquaporin 2 concentration following desmopressin treatment of a kidney cell line, a rodent model and a patient with central diabetes insipidus.
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With appropriate sample storage, nanoparticle tracking analysis has potential as a tool for the rapid characterization and quantification of exosomes in human urine. This new method can be used to develop urinary extracellular vesicles further as a non‐invasive tool for investigating human renal physiology.
Exosomes are vesicles that are released from the kidney into urine. They contain protein and RNA from the glomerulus and all sections of the nephron and represent a reservoir for biomarker discovery. Current methods for the identification and quantification of urinary exosomes are time consuming and only semi‐quantitative. Nanoparticle tracking analysis (NTA) counts and sizes particles by measuring their Brownian motion in solution. In this study, we applied NTA to human urine and identified particles with a range of sizes. Using antibodies against the exosomal proteins CD24 and aquaporin 2 (AQP2), conjugated to a fluorophore, we could identify a subpopulation of CD24‐ and AQP2‐positive particles of characteristic exosomal size. Extensive pre‐NTA processing of urine was not necessary. However, the intra‐assay variability in the measurement of exosome concentration was significantly reduced when an ultracentrifugation step preceded NTA. Without any sample processing, NTA tracked exosomal AQP2 upregulation induced by desmopressin stimulation of kidney collecting duct cells. Nanoparticle tracking analysis was also able to track changes in exosomal AQP2 concentration that followed desmopressin treatment of mice and a patient with central diabetes insipidus. When urine was stored at room temperature, 4°C or frozen, nanoparticle concentration was reduced; freezing at −80°C with the addition of protease inhibitors produced the least reduction. In conclusion, with appropriate sample storage, NTA has potential as a tool for the characterization and quantification of extracellular vesicles in human urine.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Blood pressure (BP) normally dips during sleep, and nondipping increases cardiovascular risk. Hydrochlorothiazide restores the dipping BP profile in nondipping patients, suggesting that the NaCl ...cotransporter, NCC, is an important determinant of daily BP variation. NCC activity in cells is regulated by the circadian transcription factor per1. In vivo, circadian genes are entrained via the hypothalamic–pituitary–adrenal axis. Here, we test whether abnormalities in the day:night variation of circulating glucocorticoid influence NCC activity and BP control. C57BL6/J mice were culled at the peak (1:00 AM) and trough (1:00 PM) of BP. We found no day:night variation in NCC mRNA or protein but NCC phosphorylation on threonine (pNCC), required for NCC activation, was higher when mice were awake, as was excretion of NCC in urinary exosomes. Peak NCC activity correlated with peak expression of per2 and bmal1 (clock genes) and sgk1 and tsc22d3 (glucocorticoid-responsive kinases). Adrenalectomy reduced NCC abundance and blunted the daily variation in pNCC levels without affecting variation in clock gene transcription. Chronic corticosterone infusion increased bmal1, per1, sgk1, and tsc22d3 expression during the inactive phase. Inactive phase pNCC was also elevated by corticosterone, and a nondipping BP profile was induced. Hydrochlorothiazide restored rhythmicity of BP in corticosterone-treated mice without affecting BP in controls. Glucocorticoids influence the day:night variation in NCC activity via kinases that control phosphorylation. Abnormal glucocorticoid rhythms impair NCC and induce nondipping. Night-time dosing of thiazides may be particularly beneficial in patients with modest glucocorticoid excess.
Extracellular vesicles (ECVs) facilitate intercellular communication along the nephron, with the potential to change the function of the recipient cell. However, it is not known whether this is a ...regulated process analogous to other signaling systems. We investigated the potential hormonal regulation of ECV transfer and report that desmopressin, a vasopressin analogue, stimulated the uptake of fluorescently loaded ECVs into a kidney collecting duct cell line (mCCD
) and into primary cells. Exposure of mCCD
cells to ECVs isolated from cells overexpressing microRNA-503 led to downregulated expression of microRNA-503 target genes, but only in the presence of desmopressin. Mechanistically, ECV entry into mCCD
cells required cAMP production, was reduced by inhibiting dynamin, and was selective for ECVs from kidney tubular cells. In vivo, we measured the urinary excretion and tissue uptake of fluorescently loaded ECVs delivered systemically to mice before and after administration of the vasopressin V2 receptor antagonist tolvaptan. In control-treated mice, we recovered 2.5% of administered ECVs in the urine; tolvaptan increased recovery five-fold and reduced ECV deposition in kidney tissue. Furthermore, in a patient with central diabetes insipidus, desmopressin reduced the excretion of ECVs derived from glomerular and proximal tubular cells. These data are consistent with vasopressin-regulated uptake of ECVs in vivo We conclude that ECV uptake is a specific and regulated process. Physiologically, ECVs are a new mechanism of intercellular communication; therapeutically, ECVs may be a vehicle by which RNA therapy could be targeted to specific cells for the treatment of kidney disease.
Drug-induced liver injury (DILI) results in over 100 000 hospital attendances per year in the UK alone and is a leading cause for the post-marketing withdrawal of new drugs, leading to significant ...financial losses. MicroRNA-122 (miR-122) has been proposed as a sensitive DILI marker although no commercial applications are available yet. Extracellular blood microRNAs (miRNAs) are promising clinical biomarkers but their measurement at point of care remains time-consuming, technically challenging, and expensive. For circulating miRNA to have an impact on healthcare, a key challenge to overcome is the development of rapid and reliable low-cost sample preparation. There is an acknowledged issue with miRNA stability in the presence of hemolysis and platelet activation, and no solution has been demonstrated for fast and robust extraction at the site of blood draw. Here, we report a novel microfluidic platform for the extraction of circulating miR-122 from blood enabled by a vertical approach and gravity-based bubble mixing. The performance of this disposable cartridge was verified by standard quantitative polymerase chain reaction analysis on extracted miR-122. The cartridge performed equivalently or better than standard bench extraction kits. The extraction cartridge was combined with electrochemical impedance spectroscopy to detect miR-122 as an initial proof-of-concept toward an application in point-of-care detection. This platform enables the standardization of sample preparation and the detection of miRNAs at the point of blood draw and in resource limited settings and could aid the introduction of miRNA-based assays into routine clinical practice.
Vascular and kidney dysfunction commonly co-exist. There is a need for biomarkers of vascular health. Circulating microRNAs are biomarkers; miR-126 is endothelial cell-enriched. We measured ...circulating miR-126 in rats with nephrotoxic nephritis (NTN) and humans with acute endothelial and renal injury (vasculitis associated with autoantibodies to neutrophil cytoplasm antigens (ANCAs)). We compared these findings to those from patients with chronic kidney disease (CKD) and end-stage renal disease (ESRD) and explored the relationship between miR-126 and vascular dysfunction. In NTN, miR-126 was reduced. In ANCA vasculitis (N = 70), pre-treatment miR-126 was reduced compared to health (N = 60) (88-fold). miR-126 increased 3.4-fold post-treatment but remained lower than in health (∼26-fold). Argonaute 2-bound miR-126 increased with ANCA vasculitis treatment. miR-126 did not differ between CKD (N = 30) and health but its concentration correlated with endothelial dysfunction. miR-126 was reduced in ESRD (N = 15) (∼350 fold). miR-126 may be a marker of vascular inflammation and could aid decision-making.
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•New biomarkers are needed that report vascular function in patients with kidney disease•The endothelial microRNA, miR-126, was reduced with vascular inflammation (vasculitis)•miR-126 correlated with vascular health in chronic kidney disease•Circulating miR-126 may be a marker of vascular dysfunction
Molecular Physiology; Molecular Genetics; Molecular Biology; Immunology
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
It is unclear what effect the pattern of health-care use before admission to hospital with COVID-19 (index admission) has on the long-term outcomes for patients. We sought to describe mortality and ...emergency readmission to hospital after discharge following the index admission (index discharge), and to assess associations between these outcomes and patterns of health-care use before such admissions.
We did a national, retrospective, complete cohort study by extracting data from several national databases and linking the databases for all adult patients admitted to hospital in Scotland with COVID-19. We used latent class trajectory modelling to identify distinct clusters of patients on the basis of their emergency admissions to hospital in the 2 years before the index admission. The primary outcomes were mortality and emergency readmission up to 1 year after index admission. We used multivariable regression models to explore associations between these outcomes and patient demographics, vaccination status, level of care received in hospital, and previous emergency hospital use.
Between March 1, 2020, and Oct 25, 2021, 33 580 patients were admitted to hospital with COVID-19 in Scotland. Overall, the Kaplan-Meier estimate of mortality within 1 year of index admission was 29·6% (95% CI 29·1–30·2). The cumulative incidence of emergency hospital readmission within 30 days of index discharge was 14·4% (95% CI 14·0–14·8), with the number increasing to 35·6% (34·9–36·3) patients at 1 year. Among the 33 580 patients, we identified four distinct patterns of previous emergency hospital use: no admissions (n=18 772 55·9%); minimal admissions (n=12 057 35·9%); recently high admissions (n=1931 5·8%), and persistently high admissions (n=820 2·4%). Patients with recently or persistently high admissions were older, more multimorbid, and more likely to have hospital-acquired COVID-19 than patients with no or minimal admissions. People in the minimal, recently high, and persistently high admissions groups had an increased risk of mortality and hospital readmission compared with those in the no admissions group. Compared with the no admissions group, mortality was highest in the recently high admissions group (post-hospital mortality HR 2·70 95% CI 2·35–2·81; p<0·0001) and the risk of readmission was highest in the persistently high admissions group (3·23 2·89–3·61; p<0·0001).
Long-term mortality and readmission rates for patients hospitalised with COVID-19 were high; within 1 year, one in three patients had died and a third had been readmitted as an emergency. Patterns of hospital use before index admission were strongly predictive of mortality and readmission risk, independent of age, pre-existing comorbidities, and COVID-19 vaccination status. This increasingly precise identification of individuals at high risk of poor outcomes from COVID-19 will enable targeted support.
Chief Scientist Office Scotland, UK National Institute for Health Research, and UK Research and Innovation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Thiazide diuretics are among the most widely used antihypertensive medications worldwide. Thiazide-induced hyponatremia (TIH) is 1 of their most clinically significant adverse effects.
TIH must ...result from excessive saliuresis and/or water reabsorption. We hypothesized that pathways regulating the thiazide-sensitive sodium-chloride cotransporter NCC and the water channel aquaporin-2 (AQP
) may be involved. Our aim was to assess whether patients with TIH would show evidence of altered NCC and AQP
expression in urinary extracellular vesicles (UEVs), and also whether abnormalities of renal sodium reabsorption would be evident using endogenous lithium clearance (ELC).
Blood and urine samples were donated by patients admitted to hospital with acute symptomatic TIH, after recovery to normonatremia, and also from normonatremic controls on and off thiazides. Urinary extracellular vesicles were isolated and target proteins evaluated by western blotting and by nanoparticle tracking analysis. Endogenous lithium clearance was assessed by inductively coupled plasma mass spectrometry.
Analysis of UEVs by western blotting showed that patients with acute TIH displayed reduced total NCC and increased phospho-NCC and AQP
relative to appropriate control groups; smaller differences in NCC and AQP
expression persisted after recovery from TIH. These findings were confirmed by nanoparticle tracking analysis. Renal ELC was lower in acute TIH compared to that in controls and convalescent case patients.
Reduced NCC expression and increased AQP
expression would be expected to result in saliuresis and water reabsorption in TIH patients. This study raises the possibility that UEV analysis may be of diagnostic utility in less clear-cut cases of thiazide-associated hyponatremia, and may help to identify patients at risk for TIH before thiazide initiation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Patients with cancer are at greater risk of dying from COVID-19 than many other patient groups. However, how this risk evolved during the pandemic remains unclear. We aimed to determine, on the basis ...of the UK national pandemic protocol, how factors influencing hospital mortality from COVID-19 could differentially affect patients undergoing cancer treatment. We also examined changes in hospital mortality and escalation of care in patients on cancer treatment during the first 2 years of the COVID-19 pandemic in the UK.
We conducted a prospective cohort study of patients aged older than 19 years and admitted to 306 health-care facilities in the UK with confirmed SARS-CoV-2 infection, who were enrolled in the International Severe Acute Respiratory and emerging Infections Consortium (ISARIC) WHO Clinical Characterisation Protocol (CCP) across the UK from April 23, 2020, to Feb 28, 2022; this analysis included all patients in the complete dataset when the study closed. The primary outcome was 30-day in-hospital mortality, comparing patients on cancer treatment and those without cancer. The study was approved by the South Central–Oxford C Research Ethics Committee in England (Ref: 13/SC/0149) and the Scotland A Research Ethics Committee (Ref 20/SS/0028), and is registered on the ISRCTN Registry (ISRCTN66726260).
177 871 eligible adult patients either with no history of cancer (n=171 303) or on cancer treatment (n=6568) were enrolled; 93 205 (52·4%) were male, 84 418 (47·5%) were female, and in 248 (13·9%) sex or gender details were not specified or data were missing. Patients were followed up for a median of 13 (IQR 6–21) days. Of the 6568 patients receiving cancer treatment, 2080 (31·7%) died at 30 days, compared with 30 901 (18·0%) of 171 303 patients without cancer. Patients aged younger than 50 years on cancer treatment had the highest age-adjusted relative risk (hazard ratio HR 5·2 95% CI 4·0–6·6, p<0·0001; vs 50–69 years 2·4 2·2–2·6, p<0·0001; 70–79 years 1·8 1·6–2·0, p<0·0001; and >80 years 1·5 1·3–1·6, p<0·0001) but a lower absolute risk (51 6·7% of 763 patients <50 years died compared with 459 30·2% of 1522 patients aged >80 years). In-hospital mortality decreased for all patients during the pandemic but was higher for patients on cancer treatment than for those without cancer throughout the study period.
People with cancer have a higher risk of mortality from COVID-19 than those without cancer. Patients younger than 50 years with cancer treatment have the highest relative risk of death. Continued action is needed to mitigate the poor outcomes in patients with cancer, such as through optimising vaccination, long-acting passive immunisation, and early access to therapeutics. These findings underscore the importance of the ISARIC-WHO pandemic preparedness initiative.
National Institute for Health Research and the Medical Research Council.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Aims
Patients on antituberculosis (anti‐TB) therapy are at risk of drug‐induced liver injury (DILI). MicroRNA‐122 (miR‐122) and cytokeratin‐18 (K18) are DILI biomarkers. To explore their utility in ...this global context, circulating miR‐122 and K18 were measured in UK and Ugandan populations on anti‐TB therapy for mycobacterial infection.
Methods
Healthy subjects and patients receiving anti‐TB therapy were recruited at the Royal Infirmary of Edinburgh, UK (ALISTER—ClinicalTrials.gov Identifier: NCT03211208). African patients with human immunodeficiency virus–TB coinfection were recruited at the Infectious Diseases Institute, Kampala, Uganda (SAEFRIF—NCT03982277). Serial blood samples, demographic and clinical data were collected. In ALISTER samples, MiR‐122 was quantified using polymerase chain reaction. In ALISTER and SAEFRIF samples, K18 was quantified by enzyme‐linked immunosorbent assay.
Results
The study had 235 participants (healthy volunteers n = 28; ALISTER: active TB n = 30, latent TB n = 88, nontuberculous mycobacterial infection n = 25; SAEFRIF: human immunodeficiency virus‐TB coinfection n = 64). In the absence of DILI, there was no difference in miR‐122 and K18 across the groups. Both miR‐122 and K18 correlated with alanine transaminase (ALT) activity (miR‐122: R = .52, 95%CI = 0.42–0.61, P < .0001. K18: R =0.42, 95%CI = 0.34–0.49, P < .0001). miR‐122 distinguished those patients with ALT>50 U/L with higher sensitivity/specificity than K18. There were 2 DILI cases: baseline ALT, 18 and 28 IU/L, peak ALT 431 and 194 IU/L; baseline K18, 58 and 219 U/L, peak K18 1247 and 3490 U/L; baseline miR‐122 4 and 17 fM, peak miR‐122 60 and 336 fM, respectively.
Conclusion
In patients treated with anti‐TB therapy, miR‐122 and K18 correlated with ALT and increased with DILI. Further work should determine their diagnostic and prognostic utility in this global context‐of‐use.
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ABSTRACT—Hypertension and arterial stiffness are important independent cardiovascular risk factors in chronic kidney disease (CKD) to which endothelin-1 (ET-1) contributes. Loss of nocturnal blood ...pressure (BP) dipping is associated with CKD progression, but there are no data on 24-hour arterial stiffness variation. We examined the 24-hour variation of BP, arterial stiffness, and the ET system in healthy volunteers and patients with CKD and the effects on these of ET receptor type A receptor antagonism (sitaxentan). There were nocturnal dips in systolic BP and diastolic BP and pulse wave velocity, our measure of arterial stiffness, in 15 controls (systolic BP, −3.2±4.8%, P<0.05; diastolic BP, −6.4±6.2%, P=0.001; pulse wave velocity, −5.8±5.2%, P<0.01) but not in 15 patients with CKD. In CKD, plasma ET-1 increased by 1.2±1.4 pg/mL from midday to midnight compared with healthy volunteers (P<0.05). Urinary ET-1 did not change. In a randomized, double-blind, 3-way crossover study in 27 patients with CKD, 6-week treatment with placebo and nifedipine did not affect nocturnal dips in systolic BP or diastolic BP between baseline and week 6, whereas dipping was increased after 6-week sitaxentan treatment (baseline versus week 6, systolic BP−7.0±6.2 versus −11.0±7.8 mm Hg, P<0.05; diastolic BP−6.0±3.6 versus −8.3±5.1 mm Hg, P<0.05). There was no nocturnal dip in pulse pressure at baseline in the 3 phases of the study, whereas sitaxentan was linked to the development of a nocturnal dip in pulse pressure. In CKD, activation of the ET system seems to contribute not only to raised BP but also the loss of BP dipping. The clinical significance of these findings should be explored in future clinical trials.
CLINICAL TRIAL REGISTRATION—URLhttp://www.clinicaltrials.gov. Unique identifiersNCT01770847 and NCT00810732.
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