Enhanced metanephric specification to functional proximal tubule enables toxicity screening and infectious disease modelling in kidney organoids.

While pluripotent stem cell-derived kidney organoids are now being used to model renal disease, the proximal nephron remains immature with limited evidence for key functional solute channels. This may reflect early mispatterning of the nephrogenic mesenchyme and/or insufficient maturation. Here we show that enhanced specification to metanephric nephron progenitors results in elongated and radially aligned proximalised nephrons with distinct S1 - S3 proximal tubule cell types. Such PT-enhanced organoids possess improved albumin and organic cation uptake, appropriate KIM-1 upregulation in response to cisplatin, and improved expression of SARS-CoV-2 entry factors resulting in increased viral replication. The striking proximo-distal orientation of nephrons resulted from localized WNT antagonism originating from the organoid stromal core. PT-enhanced organoids represent an improved model to study inherited and acquired proximal tubular disease as well as drug and viral responses.

An albumin-angiotensin converting enzyme 2-based SARS-CoV-2 decoy with FcRn-driven half-life extension.

The emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutants and breakthrough infections despite available coronavirus disease 2019 (COVID-19) vaccines calls for antiviral therapeutics. The application of soluble angiotensin converting enzyme 2 (ACE2) as a SARS-CoV-2 decoy that reduces cell bound ACE2-mediated virus entry is limited by a short plasma half-life. This work presents a recombinant human albumin ACE2 genetic fusion (rHA-ACE2) to increase the plasma half-life by an FcRn-driven cellular recycling mechanism, investigated using a wild type (WT) albumin sequence and sequence engineered with null FcRn binding (NB). Binding of rHA-ACE2 fusions to SARS-CoV-2 spike protein subdomain 1 (S1) was demonstrated (WT-ACE2 KD = 32.8 nM and NB-ACE2 KD = 31.7 nM) using Bio-Layer Interferometry and dose-dependent in vitro inhibition of host cell infection of pseudotyped viruses displaying surface SARS-CoV-2 spike (S) protein. FcRn-mediated in vitro recycling was translated to a five times greater plasma half-life of WT-ACE2 (t½ ß = 13.5 h) than soluble ACE2 (t½ ß = 2.8 h) in humanised FcRn/albumin double transgenic mice. The rHA-ACE2-based SARS-CoV-2 decoy system exhibiting FcRn-driven circulatory half-life extension introduced in this work offers the potential to expand and improve the anti-COVID-19 anti-viral drug armoury. STATEMENT OF SIGNIFICANCE: The COVID-19 pandemic has highlighted the need for rapid development of efficient antiviral therapeutics to combat SARS-CoV-2 and new mutants to lower morbidity and mortality in severe cases, and for people that are unable to receive a vaccine. Here we report a therapeutic albumin ACE2 fusion protein (rHA-ACE2), that can bind SARS-CoV-2 S protein decorated virus-like particles to inhibit viral infection, and exhibits extended in vivo half-life compared to ACE2 alone. Employing ACE2 as a binding decoy for the virus is expected to efficiently inhibit all SARS-CoV-2 mutants as they all rely on binding with endogenous ACE2 for viral cell entry and, therefore, rHA-ACE2 constitutes a versatile addition to the therapeutic arsenal for combatting COVID-19.

Study of Albumin Oxidation in COVID-19 Pneumonia Patients: Possible Mechanisms and Consequences.

Oxidative stress induced by neutrophils and hypoxia in COVID-19 pneumonia leads to albumin modification. This may result in elevated levels of advanced oxidation protein products (AOPPs) and advanced lipoxidation end-products (ALEs) that trigger oxidative bursts of neutrophils and thus participate in cytokine storms, accelerating endothelial lung cell injury, leading to respiratory distress. In this study, sixty-six hospitalized COVID-19 patients with respiratory symptoms were studied. AOPPs-HSA was produced in vitro by treating human serum albumin (HSA) with chloramine T. The interaction of malondialdehyde with HSA was studied using time-resolved fluorescence spectroscopy. The findings revealed a significantly elevated level of AOPPs in COVID-19 pneumonia patients on admission to the hospital and one week later as long as they were in the acute phase of infection when compared with values recorded for the same patients 6- and 12-months post-infection. Significant negative correlations of albumin and positive correlations of AOPPs with, e.g., procalcitonin, D-dimers, lactate dehydrogenase, aspartate transaminase, and radiological scores of computed tomography (HRCT), were observed. The AOPPs/albumin ratio was found to be strongly correlated with D-dimers. We suggest that oxidized albumin could be involved in COVID-19 pathophysiology. Some possible clinical consequences of the modification of albumin are also discussed.

SARS-CoV-2 spike protein inhibits megalin-mediated albumin endocytosis in proximal tubule epithelial cells.

Patients with COVID-19 have high prevalence of albuminuria which is used as a marker of progression of renal disease and is associated with severe COVID-19. We hypothesized that SARS-CoV-2 spike protein (S protein) could modulate albumin handling in proximal tubule epithelial cells (PTECs) and, consequently contribute to the albuminuria observed in patients with COVID-19. In this context, the possible effect of S protein on albumin endocytosis in PTECs was investigated. Two PTEC lines were used: HEK-293A and LLC-PK1. Incubation of both cell types with S protein for 16 h inhibited albumin uptake at the same magnitude. This effect was associated with canonical megalin-mediated albumin endocytosis because: (1) DQ-albumin uptake, a marker of the lysosomal degradation pathway, was reduced at a similar level compared with fluorescein isothiocyanate (FITC)-albumin uptake; (2) dextran-FITC uptake, a marker of fluid-phase endocytosis, was not changed; (3) cell viability and proliferation were not changed. The inhibitory effect of S protein on albumin uptake was only observed when it was added at the luminal membrane, and it did not involve the ACE2/Ang II/AT1R axis. Although both cells uptake S protein, it does not seem to be required for modulation of albumin endocytosis. The mechanism underlying the inhibition of albumin uptake by S protein encompasses a decrease in megalin expression without changes in megalin trafficking and stability. These results reveal a possible mechanism to explain the albuminuria observed in patients with COVID-19.

Vitamin D, vitamin D-binding protein, free vitamin D and COVID-19 mortality in hospitalized patients.

BACKGROUND: Vitamin D deficiency has been associated with worse coronavirus disease 2019 (COVID-19) outcomes, but circulating 25-hydroxyvitamin D [25(OH)D] is largely bound to vitamin D-binding protein (DBP) or albumin, both of which tend to fall in illness, making the 25(OH)D status hard to interpret. Because of this, measurements of unbound ("free") and albumin-bound ("bioavailable") 25(OH)D have been proposed. OBJECTIVES: We aimed to examine the relationship between vitamin D status and mortality from COVID-19. METHODS: In this observational study conducted in Liverpool, UK, hospitalized COVID-19 patients with surplus sera available for 25(OH)D analysis were studied. Clinical data, including age, ethnicity, and comorbidities, were extracted from case notes. Serum 25(OH)D, DBP, and albumin concentrations were measured. Free and bioavailable 25(OH)D were calculated. Relationships between total, free, and bioavailable 25(OH)D and 28-day mortality were analyzed by logistic regression. RESULTS: There were 472 patients with COVID-19 included, of whom 112 (23.7%) died within 28 days. Nonsurvivors were older (mean age, 73 years; range, 34-98 years) than survivors (mean age, 65 years; range, 19-95 years; P = 0.003) and were more likely to be male (67%; P = 0.02). The frequency of vitamin D deficiency [25(OH)D < 50 nmol/L] was similar between nonsurvivors (71/112; 63.4%) and survivors (204/360; 56.7%; P = 0.15) but, after adjustments for age, sex, and comorbidities, increased odds for mortality were present in those with severe deficiency [25(OH)D < 25 nmol/L: OR, 2.37; 95% CI, 1.17-4.78] or a high 25(OH)D (≥100 nmol/L; OR, 4.65; 95% CI, 1.51-14.34) compared with a 25(OH)D value of 50-74 nmol/L (reference). Serum DBP levels were not associated with mortality after adjustments for 25(OH)D, age, sex, and comorbidities. Neither free nor bioavailable 25(OH)D values were associated with mortality. CONCLUSIONS: Vitamin D deficiency, as commonly defined by serum 25(OH)D levels (<50 nmol/L), is not associated with increased mortality from COVID-19, but extremely low (<25 nmol/L) and high (>100 nmol/L) levels may be associated with mortality risks. Neither free nor bioavailable 25(OH)D values are associated with mortality risk. The study protocol was approved by the London-Surrey Research Ethics Committee (20/HRA/2282).

Usefulness of C-reactive protein/albumin ratio as a predictor of new-onset atrial fibrillation in SARS-COV-2.

Aim: To investigate whether C-reactive protein/albumin ratio (CAR) has an association with new onset atrial fibrillation (NOAF) in SARS-CoV-2. Materials & methods: This study included 782 patients with SARS-CoV-2 infection, who were hospitalized in Turkey. The end point of the study was an occurrence of NOAF. Results: NOAF was identified in 41 patients (5.2%). Subjects who developed NOAF had a higher CAR compared with those who did not develop NOAF (p < 0.001). In the multivariate logistic regression analysis the CAR (odds ratio = 2.879; 95% CI: 1.063-7.793; p = 0.037) was an independent predictor of NOAF. Conclusion: A high level of CAR in blood samples is associated with an increased risk of developing NOAF in SARS-CoV-2.

National Early Warning Score 2 and laboratory predictors correlate with clinical deterioration in hospitalized patients with COVID-19.

Aim: We aimed to determine the prognostic values of the National Early Warning Score 2 (NEWS2) and laboratory parameters during the first week of COVID-19. Materials & methods: All adult patients who were hospitalized for confirmed COVID-19 between 11 March and 11 May 2020 were retrospectively included. Results: Overall, 611 patients were included. Our results showed that NEWS2, procalcitonin, neutrophil/lymphocyte ratio and albumin at D0, D3, D5 and D7 were the best predictors for clinical deterioration defined as a composite of ICU admission during hospitalization or in-hospital death. Procalcitonin had the highest odds ratio for clinical deterioration on all days. Conclusion: This study provides a list of several laboratory parameters correlated with NEWS2 and potential predictors for clinical deterioration in patients with COVID-19.

Lay abstract The COVID-19 pandemic is a grueling problem worldwide. There is a lack of knowledge about the predictive value of National Early Warning Score 2 (NEWS2) for severe COVID-19 illness. We analyzed the prognostic value of NEWS2 and laboratory parameters during the clinical course of COVID-19. This study provides a list of several laboratory parameters correlated with NEWS2 and potential predictors for intensive care unit admission during hospitalization or in-hospital death.

Influence of nutritional status on clinical outcomes among hospitalized patients with COVID-19.

BACKGROUND AND AIMS: Several factors that worsen the prognosis of the new coronavirus SARS-CoV-2 have been identified, such as obesity or diabetes. However, despite that nutrition may change in a lockdown situation, little is known about the influence of malnutrition among subjects hospitalized due to COVID-19. Our study aimed to assess whether the presence of malnutrition among patients admitted due to COVID-19 had any impact on clinical outcomes compared with patients with the same condition but well nourished. METHODS: 75 patients admitted to hospital due to COVID-19 were analyzed cross-sectionally. Subjective Global Assessment (SGA) was completed by phone interview. Clinical parameters included were extracted from the electronic medical record. RESULTS: According to the SGA, 27 admitted due to a COVID-19 infection had malnutrition. Patients not well nourished were older than patients with a SGA grade A (65 ± 14.1 vs 49 ± 15.1 years; p < 0.0001). Length of hospital stay among poorly nourished patients was significantly higher (18.4 ± 15.6 vs 8.5 ± 7.7 days; p = 0.001). Mortality rates and admission to ICU were greater among subjects with any degree of malnutrition compared with well-nourished patients (7.4% vs 0%; p = 0.05 and 44.4% vs 6.3%; p < 0.0001). CRP (120.9 ± 106.2 vs 60.8 ± 62.9 mg/l; p = 0.03), D-dimer (1516.9 ± 1466.9 vs 461.1 ± 353.7 ng/mL; p < 0.0001) and ferritin (847.8 ± 741.1 vs 617.8 ± 598.7mcg/l; p = 0.03) were higher in the group with malnutrition. Haemoglobin (11.6 ± 2.1 vs 13.6 ± 1.5 g/dl; p < 0.0001) and albumin 3.2 ± 0.7 vs 4.1 ± 0.5 g/dl; p < 0.0001) were lower in patients with any degree of malnutrition. CONCLUSIONS: The presence of a poor nutritional status is related to a longer stay in hospital, a greater admission in the ICU and a higher mortality.

Neglecting Plasma Protein Binding in COVID-19 Patients Leads to a Wrong Interpretation of Lopinavir Overexposure.

Boffito et al. recalled the critical importance to correctly interpret protein binding. Changes of lopinavir pharmacokinetics in coronavirus disease 2019 (COVID-19) are a perfect illustration. Indeed, several studies described that total lopinavir plasma concentrations were considerably higher in patients with severe COVID-19 than those reported in patients with HIV. These findings have led to a reduction of the dose of lopinavir in some patients, hypothesizing an inhibitory effect of inflammation on lopinavir metabolism. Unfortunately, changes in plasma protein binding were never investigated. We performed a retrospective cohort study. Data were collected from the medical records of patients hospitalized for COVID-19 treated with lopinavir/ritonavir in intensive care units or infectious disease departments of Toulouse University Hospital (France). Total and unbound concentrations of lopinavir, C reactive protein, albumin, and alpha-1-acid glycoprotein (AAG) levels were measured during routine care on the same samples. In patients with COVID-19, increased total lopinavir concentration is the result of an increased AAG-bound lopinavir concentration, whereas the unbound concentration remains constant, and insufficient to reduce the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) viral load. Although international guidelines have recently recommended against using lopinavir/ritonavir to treat severe COVID-19, the description of lopinavir pharmacokinetics changes in COVID-19 is a textbook case of the high risk of misinterpretation of a total drug exposure when changes in protein binding are not taken into consideration.

Diagnostic utility of C-reactive protein to albumin ratio as an early warning sign in hospitalized severe COVID-19 patients.

C-reactive protein-to-albumin ratio (CAR) has been used as an indicator of prognosis in various diseases. Here, we intended to assess the CAR's diagnostic power in early differentiation of hospitalized severe COVID-19 cases. In this retrospectively designed study, we evaluated 197 patients in total. They were divided into two groups based on their severity of COVID-19 as non-severe (n = 113) and severe (n = 84). The comparison of groups' demographic data, comorbidities, clinical symptoms, and laboratory test results were done. Laboratory data of the patients within the first 24 h after admission to the hospital were evaluated. The calculation of receiver operating characteristic (ROC) curve was used to determine the diagnostic power of CAR in differentiating severity of COVID-19. Independent risk factors predictive of COVID-19 severity were determined by using logistic regression analysis. Although lymphocyte count levels were lower, severe COVID-19 patients had higher mean age, higher levels of neutrophil count, CRP, aspartate aminotransferase (AST), ferritin, and prothrombin time (P < 0.05). Compared with non-severe patients (median, 0.23 [IQR = 0.07-1.56]), patients with severe COVID-19 had higher CAR levels (median, 1.66 [IQR = 0.50-3.35]; P < 0.001). Age (OR = 1.046, P = 0.003), CAR (OR = 1.264, P = 0.037), and AST (OR = 1.029, P = 0.037) were independent risk factors for severe COVID-19 based on the multivariate logistic regression analysis. ROC curve analysis assigned 0.9 as the cut-off value for CAR for differentiation of severe COVID-19 (area under the curve = 0.718, 69.1% sensitivity, 70.8% specificity, P < 0.001). CAR is a useful marker in early differentiation of severity in patients hospitalized due to COVID-19 that have longer hospital stay and higher mortality.

Laboratory parameters in patients with COVID-19 on first emergency admission is different in non-survivors: albumin and lactate dehydrogenase as risk factors.

Prompt identification of the clinical status and severity of COVID-19 can be a challenge in the emergency department (ED), as the clinical severity of the disease is variable, real-time reverse-transcription PCR (RT-PCR) results may not be immediately available, and imaging findings appear approximately 10 days after the onset of symptoms. There is currently no set of simple, readily available and fast battery of tests that can be used in the ED as prognostic factors. The purpose was to study laboratory test results in patients with COVID-19 at hospital emergency admission and to evaluate the results in non-survivors and their potential prognostic value. A profile of laboratory markers was agreed with the ED providers based on the International Federation of Clinical Chemistry and Laboratory Medicine recommendation of its usefulness, which was made in 218 patients with COVID-19. Non-survivors were significantly older, and the percentage of patients with pathological values of creatinine, albumin, lactate dehydrogenase (LDH), C reactive protein, prothrombin time, D-dimer, and arterial blood gas, PaO2/FIO2 and satO2/FIO2 indices were significantly higher among the patients with COVID-19 who died than those who survived. Patients who died also presented higher neutrophil counts. Among all studied tests, albumin and LDH were independent prognostic factors for death. The results of the study show pathology in nine laboratory markers in patients with COVID-19 admitted in the ED, valuable findings to take into consideration for its prompt identification when there is no immediate availability of RT-PCR results.

C-reactive protein and albumin association with mortality of hospitalised SARS-CoV-2 patients: A tertiary hospital experience.

OBJECTIVE: The objective was to study hospitalised COVID-19 patients' mortality and intensive care unit (ICU) admission with covariates of interest (age, gender, ethnicity, clinical presentation, comorbidities and admission laboratory findings). METHODS: Logistic regression analyses were performed for patients admitted to University Hospital, University Hospitals Coventry and Warwickshire NHS Trust, between 24 January 2020 - 13 April 2020. RESULTS: There were 321 patients hospitalised. Median age was 73 years and 189 (59%) were male. Ethnicity was divided between Caucasian (77%), and black, Asian, and minority ethnic (BAME) groups (23%). Commonest symptoms were dyspnoea (62.9%), fever (59.1%) and cough (56%). Gastrointestinal symptoms amounted to 11.8%.Forty-four patients (13.7%) received ICU care. ICU male to female ratio was 3:1 (p=0.027; odds ratio (OR) 2.3; 95% confidence interval (CI) 1.1-4.9), BAME (p=0.008; OR 2.5; 95% CI 1.3-4.9), age >65 years (p=0.026; OR 0.28; 95% CI 0.09-0.93), heart disease (p=0.009; OR 0.2; 95% CI 0.1-0.6) and elevated C-reactive protein (CRP; p<0.001; OR 1.004; 95% CI 1.002-1.008) were associated with ICU admission.One-hundred and four patients (32.4%) died. Age >65 years (p=0.011; OR 5; 95% CI 1.6-21.9), neutrophils (p=0.047), neutrophil:lymphocyte ratio (NLR; p=0.028), CRP (p<0.001) and albumin (p=0.002) were associated with mortality. When analysis adjusted for age, CRP (p<0.001; OR 1.006; 95% CI 1.004-1.008) and albumin (p=0.005; OR 0.94; 95% CI 0.90-0.98) remained associated with mortality. CONCLUSIONS: COVID-19 has high mortality. BAME and male patients were associated with ICU admission. High CRP and low albumin (after correcting for age) were associated with mortality.