key: cord-1039582-0m78kfux
authors: Henry, Brandon Michael; de Oliveira, Maria Helena Santos; Cheruiyot, Isaac; Benoit, Justin L.; Cooper, David S.; Lippi, Giuseppe; Le Cras, Timothy D.; Benoit, Stefanie W.
title: Circulating level of Angiopoietin-2 is associated with acute kidney injury in coronavirus disease 2019 (COVID-19)
date: 2021-03-23
journal: Angiogenesis
DOI: 10.1007/s10456-021-09782-w
sha: 427efd4f029cb38352795c4b617793a2a22b93f0
doc_id: 1039582
cord_uid: 0m78kfux

nan

Emerging evidence suggests that endothelial dysfunction plays a central role in the pathophysiology of coronavirus disease 2019 . Recent post-mortem studies have documented extensive endothelial damage and inflammatory infiltrates in pulmonary and extra-pulmonary capillary beds of COVID-19 patients [1, 2] . This results in loss of endothelial integrity, activation of pro-coagulant pathways, disruption of the alveolar-capillary barrier, and vascular hyperpermeability [2] . Endothelial damage is a common denominator of thrombosis (micro-and macrovascular), acute respiratory distress syndrome (ARDS), acute kidney injury (AKI), and multiorgan failure (MOF), which are major drivers of morbidity and mortality in COVID-19 patients [3] . AKI is a common feature of COVID-19, impacting nearly half of all hospitalized patients, and is associated with high mortality, especially among those requiring renal replacement therapy [4] [5] [6] . We have recently shown that AKI may be driven in COVID-19 by a secondary thrombotic microangiopathy (TMA) phenomenon, as evidenced by low ADAMTS13 activity to von Willebrand factor (VWF:Ag) ratio [7] . However, the mechanism by which AKI occurs in COVID-19 has yet to be fully elucidated.

With the high frequency of AKI and thromboses in patients with COVID-19, biomarkers of endothelial damage/activation-related biomarkers have become of interest. Angiopoietin-1 (Ang-1) is an angiogenic growth factor that promotes vessel maturation and survival by activation of the Tie2 receptor (Tie2) on endothelial cells [8] . Ang-1 is expressed by pericytes and vascular smooth muscle cells and can stabilize endothelial functions by reducing inflammation and apoptosis of endothelial cells [9] . On the contrary, Angiopoietin-2 (Ang-2) enhances endothelial inflammation and hyperpermeability as it can act as an antagonist to Ang-1 and Tie2 signaling [9, 10] . We hypothesized that elevated Ang-2 would be associated with an increased risk for developing severe COVID-19-related AKI during the course of infection.

In this prospective observational study, adults (≥ 18 years old) presenting to the University of Cincinnati Medical Center Emergency Department (ED) with respiratory symptoms at triage suggestive of COVID-19 and with positive reverse transcription-polymerase chain reaction (RT-PCR) test for COVID-19 via nasopharyngeal swab were enrolled. This study was approved by the University of Cincinnati institutional review board (IRB) and performed under a waiver of informed consent. Blood samples were collected via routine draws for clinical indications in the ED. Circulating levels of Ang-1 and Ang-2 were determined in EDTA plasma using an enzyme-linked immunosorbent assay following the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA) using a DS2 ELISA processing system (Dynex Technologies, Inc, Chantilly, Virginia, USA). Serum creatinine was measured using a kinetic alkaline picrate (modified Jaffe) method using either a Beckman Coulter AU480 Chemistry Analyzer (Brea, California, USA) or a Beckman Coulter AU5822 Chemistry Analyzer (Brea, California, USA). Patients were monitored through hospitalization until discharge/death if admitted from the ED or for 30 days if discharged from the ED. The primary outcome of interest was the development of severe AKI, defined as Kidney Disease: Improving Global Outcomes (KDIGO) Stage 2 + 3 according to serum creatinine (SCr) criteria [11] . The secondary outcome was the need for renal replacement therapy (RRT). Ang-2 levels were correlated with white blood cell count (WBC), C-reactive protein (CRP), interleukin (IL) 6, 8, 10, tumor necrosis factor-alpha (TNF-α), plasminogen, fibrinogen, D-Dimer, ADAMTS13 activity, VWF:ag, myoglobin, plasma neutrophil gelatinase-associated lipocalin (NGAL), and serum cystatin C.

Analysis of data was carried out using R software (version 4.0.2, R Foundation for Statistical Computing, Vienna, Austria). Categorical data were reported as frequencies (%), while continuous data were reported as the median and interquartile range (IQR). Comparison of baseline Ang-1 and Ang-2 levels, as well as other laboratory values between COVID-19 patients with and without severe AKI, was carried out using the Mann-Whitney U-test. Proportions were compared between groups using Fisher's exact test. Logistic regression analysis was performed to estimate the effect of changes in Ang-1 and Ang-2 levels when adjusted for the presence of comorbidities, and variable selection was performed using the stepwise algorithm.

A total of 51 COVID-19 patients were included. The median age was 50.5 (IQR: 39.3-66.0) years, and 57.7% were males. Their comorbidities are shown in Table 1 (Fig. 1b) . In multivariate logistic regression, both pre-existing chronic kidney disease and hypertension were significantly associated with increased odds of severe AKI, with adjusted odds ratios (ORs) of 31.8 (95%CI 1.18-854.88) and 22.0 (95%CI 1.15-420.32), respectively. An increase in 1000 pg/ mL of Ang-2 was associated with a 39% increase in odds of severe AKI (OR 1.39 [95%CI 1.05-1.86]). Full results are presented in Supplemental Table 1 .

In this prospective study, we observed that Ang-2 levels measured at ED presentation are significantly increased in patients at risk of developing severe AKI. Moreover, we observed that elevated Ang-2 is an independent predictor of severe AKI and RRT. Our findings are in agreement with Smadja et al. [12] , who reported significantly higher levels of Ang-2 in intensive care unit-admitted COVID-19 patients. They observed that patients with Ang-2 levels greater than 5000 pg/mL had ninefold higher odds of ICU admission. Our findings are also in agreement with Araujo et al. [13] who observed that elevated Ang-2 levels were significantly associated with increased odds of severe AKI and need for RRT in ICU-admitted non-COVID-19 acute respiratory distress syndrome (ARDS) patients.

Overall, our results are consistent with a picture of endothelial injury and a thrombotic microangiopathy phenomenon in COVID-19-associated AKI, further supported by the negative correlation with ADAMTS13 activity and positive correlations with fibrinogen and D-dimer. These results are consistent with elevations of Ang-2 observed in other forms of TMA [14] [15] [16] . Ang-2 was also correlated with several pro-inflammatory biomarkers, consistent with a hyperinflammatory response that can produce endothelial injury. Endothelium activation can lead to the release of Ang-2 from Weibel-Palade (WP) bodies [17] . Interestingly, however, we did not observe significant correlation between Ang-2 and VWF:ag. Philippe et al. [18] reported observing two distinct biomarker profiles, with VWF:ag increased in accordance with disease severity, while Ang-2 was elevated only in the critically ill. Taken together, this suggests that endothelial VWF secretion in COVID-19 may in part occur via pathways different than that of Ang-2. Indeed, while VWF is also secreted via WP bodies in the basal and regulated secretory pathways, the endothelium may also directly secrete VWF via a constitutive secretory pathway using small anterograde carriers [19] . Moreover, COVID-19 is associated with platelet hyperactivity, which occurs via multiple mechanisms, including spike protein binding to platelet angiotensin-converting enzyme 2 (ACE2) receptors, resulting in platelet activation and alpha granule release, which contains VWF in high molecular weight forms [20] .

Ang-2 inhibits the protective anti-inflammatory Ang-1/ Tie2 signaling cascade [17] . The Tie2 receptor is a central regulator in protecting the vasculature against thrombus formation in the setting of systemic inflammation, such as that seen in sepsis [21] . In a pilot study of critically ill patients with TMA and anti-glomerular basement membrane disease, plasma exchange was shown to be an effective method to remove excess circulating Ang-2, returning to almost normal values with ≤ 4 treatments [17] . As such, the investigation of the pathophysiologic role of Ang-2 in COVID-19 should be prioritized as targeting Ang-2 via plasma exchange or other inhibitory approaches are potential therapies in patients with severe COVID-19. Future longitudinal studies are needed to fully elucidate the role of Ang-2 in COVID-19 endothelial dysfunction and multiorgan injury and the specificity of Ang-2 for COVID-19 AKI.

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