Role of increased neutrophil extracellular trap formation on acute kidney injury in COVID-19 patients.

Background: A strong association between elevated neutrophil extracellular trap (NET) levels and poor clinical outcomes in patients with coronavirus infection 2019 (COVID-19) has been reported. However, while acute kidney injury (AKI) is a common complication of COVID-19, the role of NETs in COVID-19-associated AKI is unclear. We investigated the association between elevated NETs and AKI and the prognostic role of NETs in COVID-19 patients. Methods: Two representative markers of NETs, circulating nucleosomes and myeloperoxidase-DNA, were measured in 115 hospitalized patients. Serum levels of interleukin [IL]-6, monocyte chemotactic protein-1 [MCP-1], plasma von Willebrand factor (vWF) and urinary biomarkers of renal tubular damage (ß2-microglobulin [ß2M] and kidney injury molecule 1 [KIM-1]) were measured. Results: AKI was found in 43 patients (37.4%), and pre-existing chronic kidney disease (CKD) was a strong risk factor for AKI. Higher circulating NET levels were a significant predictor of increased risk of initial ICU admission, in-hospital mortality (adjusted HR 3.21, 95% CI 1.08-9.19) and AKI (OR 3.67, 95% CI 1.30-10.41), independent of age, diabetes, pre-existing CKD and IL-6 levels. There were strong correlations between circulating nucleosome levels and urinary KIM-1/creatinine (r=0.368, p=0.001) and ß2M (r=0.218, p=0.049) levels. NETs were also strongly closely associated with serum vWF (r = 0.356, p<0.001), but not with IL-6 or MCP-1 levels. Conclusions: Elevated NETs were closely associated with AKI, which was a strong predictor of mortality. The close association between NETs and vWF may suggest a role for NETs in COVID-19-associated vasculopathy leading to AKI.

Uremic serum damages endothelium by provoking excessive neutrophil extracellular trap formation.

Cardiovascular disease (CVD) is the leading cause of death in patients with chronic kidney disease (CKD). Endothelial cell (EC) dysfunction is a key CKD-specific risk factor; however, the mechanisms by which uremia harms the endothelium are still unclear. We report a role for excessive neutrophil extracellular trap (NET) formation induced by uremic serum on EC injury. Level of plasma nucleosome and myeloperoxidase-DNA, established in vivo markers of NETs, as well as intracellular adhesion molecule (ICAM)-1 were measured in hemodialysis (HD) patients and healthy volunteers (HV) and their prognostic role evaluated. For in vitro studies, HV-derived neutrophils and differentiated HL-60 cells by retinoic acid were used to determine the effect of uremic serum-induced NETs on human umbilical vein EC (HUVEC). The level of in vivo NETs was significantly higher in incident HD patients compared to HV, and these markers were strongly associated with ICAM-1. Specifically, nucleosome and ICAM-1 levels were independent predictors of a composite endpoint, all-cause mortality, or vascular access failure. In vitro, HD-derived uremic serum significantly increased NET formation both in dHL-60 and isolated neutrophils compared to control serum, and these NETs decreased EC viability and induced their apoptosis. In addition, the level of ICAM-1, E-selectin and von Willebrand factor in HUVEC supernatant was significantly increased by uremic serum-induced NETs compared to control serum-induced NETs. Dysregulated neutrophil activities in the uremic milieu may play a key role in vascular inflammatory responses. The high mortality and CVD rates in ESRD may be explained in part by excessive NET formation leading to EC damage and dysfunction.

Neutrophil extracellular traps and heparin-induced antibodies contribute to vascular access thrombosis in hemodialysis patients.

BACKGROUND: Anti-heparin/platelet factor 4 (PF4) antibodies may trigger severe thrombotic complications in hemodialysis (HD) patients. Tetrameric PF4 has a high affinity for extracellular DNA, which is a key component of neutrophil extracellular traps (NETs); therefore, the interactions between anti-heparin/PF4 antibodies and NETs can contribute to prothrombotic events. METHODS: Anti-heparin/PF4 antibody levels were measured by enzyme-linked immunosorbent assay and an optical density > 1.8 was regarded as clinically significant. We additionally measured serum nucleosome levels as representative markers of NETs, and the contributions of anti-heparin/PF4 and increased serum nucleosome levels to the primary functional patency loss of vascular access was assessed. RESULTS: The frequency of anti-heparin/PF4 antibodies was significantly higher in incident HD patients compared to prevalent HD patients (23.6% vs. 7.7%). Serum nucleosome levels, as well as the white blood cell counts, neutrophil counts, and high- sensitivity C-reactive protein levels, were significantly higher in anti-heparin/PF4 antibody-positive patients compared to the control. Platelet counts tended to be lower in the patients with anti-heparin/PF4 of >1.8 than in the controls. Relative risk calculations showed that the presence of anti-heparin/PF4 antibodies increased the risk of primary functional patency failure by 4.28-fold, and this risk increased further with higher nucleosome levels. Furthermore, in the anti-heparin/PF4 antibody-positive group, the time to first vascular intervention was much shorter, and the risk of repeated intervention was higher, compared to the controls. CONCLUSION: In incident HD patients, the presence of anti-heparin/PF4 antibodies was associated with increased NET formation; this could be a strong predictor of vascular access complications.

Prognostic role of circulating neutrophil extracellular traps levels for long-term mortality in new end-stage renal disease patients.

Dysregulation of innate immunity has been proposed as an important contributing factor for advanced atherosclerosis and resultant high mortality in hemodialysis (HD) patients. To evaluate the long-term prognostic role of in vivo neutrophil extracellular traps (NETs), we measured circulating serum nucleosome, myeloperoxidase (MPO), and DNase I levels in 281 incident HD patients. Circulating nucleosome level was significantly higher in HD patients compared to controls, and it was closely associated with MPO levels, suggesting increased in vivo NETs in uremia. Patients in the nucleosome Q4 group had significantly increased all-cause and adverse CV mortality compared to those in the Q1-3 group even after adjusting traditional risk factors Also, serum DNase I level was significantly higher in HD patients than controls (2.76 ±â€¯1.02 ng/ml and 1.93 ±â€¯0.85 ng/ml), but it had no correlation with NETs. Interestingly, it serves an additive biomarker for predicting poor CV outcomes. The two novel biomarkers might provide an importance independent prognostic significance in incident HD patients.

The relationship between autophagy, increased neutrophil extracellular traps formation and endothelial dysfunction in chronic kidney disease.

In chronic kidney disease (CKD), the number of circulating neutrophils are increased, and this is usually accompanied by an increased basal activation state. However, the possible association between neutrophil extracellular traps (NETs) with vascular complications has not been evaluated. We assessed the relationship between NETs, autophagy and endothelial dysfunction in maintenance hemodialysis (MHD) patients. NET formation, neutrophil elastase (NE) activities, and serum nucleosome levels were measured in MHD (n = 60) and controls (n = 20). Basal NET formation were markedly increased in MHD patient compared to controls. After PMA stimulation, MHD neutrophils showed significantly increased NETs formation response than controls. The degree of NETs was strongly associated with lower flow-mediated dilatation(%) of brachial artery even after adjustment for cardiovascular risk factors and uremic toxins. Moreover, MHD neutrophils showed increased basal autophagy activity. Interestingly, the levels of NETs were markedly augmented after autophagy inhibition, suggesting a protective role of autophagy in excessive NET formation.

Increased viability of endometrial cells by in vitro treatment with di-(2-ethylhexyl) phthalate.

Based on the findings of several reports that have shown an increased plasma level of di-(2-ethylhexyl) phthalate (DEHP) in women with endometriosis, the present study was designed to evaluate whether in vitro treatment with DEHP can increase viability of endometrial cells. Utilizing 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide assay, fluorescent activated cell sorter analysis, and microscopic evaluation after Hoechst staining, we revealed that in vitro treatment with DEHP leads to increased viability of Ishikawa cells as well as endometrial stromal cells in serum-free condition and following exposure to hydrogen peroxide, which suggests that exposure to phthalate might play a role in the establishment of endometriosis.

Increased expression of p21-activated kinase in adenomyosis.

The present study was designed to evaluate whether the expression of p21-activated kinase 1 (Pak1), which recently has been shown to be increased in the eutopic endometrium of women with endometriosis, is also increased in adenomyotic nodules as well as in the eutopic endometrium in women with adenomyosis. Comparative immunohistochemical study using a monoclonal antihuman Pak1 antibody revealed that the expression of Pak1 is increased in the eutopic endometrium of women with adenomyosis during the secretory phase, along with its increased expression in adenomyotic nodules, suggesting a possible role of Pak1 in the establishment of adenomyosis.

Increased expression of glutathione by estradiol, tumor necrosis factor-alpha, and interleukin 1-beta in endometrial stromal cells.

PROBLEM: The intracellular antioxidant system, based on glutathione (GSH), plays a key role in endometrial detoxification reactions and has been proposed to be involved in the pathogenesis endometriosis. This study was designed to evaluate whether estradiol (E(2)) and proinflammatory cytokines have any effects on expression of glutathione in endometrial stromal cells (ESCs). METHOD OF STUDY: Glutathione levels were measured utilizing high-performance liquid chromatography following in vitro culture and treatment of ESCs with estradiol, tumor necrosis factor-alpha (TNF-alpha) and interleukin 1-beta (IL-1beta). RESULTS: The GSH level in E(2) (10(-8) m) treatment group was significantly higher than in the control group at 48 h (P < 0.05). In vitro treatment of ESCs with TNF-alpha 10 ng/mL as well as E(2) (10(-8) m) plus TNF-alpha 10 ng/mL for 48 hr also led to a significant increase in GSH level (P < 0.05; P < 0.05, respectively). Both IL-1beta 10 ng/mL and E(2) (10(-8) m) plus IL-1beta 10 ng/mL for 48 hr increased GSH level significantly (P < 0.05; P < 0.05, respectively) as well. CONCLUSIONS: These findings might suggest that increased production of estradiol and proinflammatory cytokines in the peritoneal cavity possibly leads to the establishment of endometriosis through increased level of GSH.

Down-regulation of p21-activated kinase 1 by progestin and its increased expression in the eutopic endometrium of women with endometriosis.

BACKGROUND: P21-activated kinase 1 (Pak1) integrates various signaling pathways that are vital to cell survival and function. This study was performed to evaluate whether sex steroids may regulate the expression of Pak1 in endometrial cells as well as whether its expression is increased in the eutopic endometrium of women with endometriosis. METHODS: Following in vitro estradiol (E(2)) and/or medroxyprogesterone acetate (MPA) treatment of Ishikawa cells and endometrial stromal cells (ESCs), Pak1 protein was analyzed utilizing western blot analysis and immunocytochemistry. Immunohistochemistry was performed to evaluate Pak1 immunoreactivity semiquantitatively in women with endometriosis and in controls. To assess the role of Pak1 on endometrial cell viability, crystal violet assay was performed following transfection of Ishikawa cells with Pak1 small interfering RNA (siRNA). RESULTS: In vitro treatment with E(2) plus MPA or MPA alone led to a significant decrease of Pak1 protein in Ishikawa cells and ESCs (both P < 0.05 versus control). Immunohistochemistry also revealed that Pak1 protein is significantly decreased during the secretory phase in both epithelial and stromal cells in the control subjects (P < 0.001 and P < 0.01, respectively). The immunoreactivity of Pak1 in glandular cells was significantly increased in the eutopic endometrium of women with endometriosis compared with the controls during the secretory phase (P < 0.01). Crystal violet assay has shown that transfection of Ishikawa cells with Pak1 siRNA led to a significant decrease of cellular viability (P < 0.05). CONCLUSIONS: These findings suggest that Pak1 is down-regulated by progesterone during the secretory phase in normal endometrium and increased Pak1 activity during the secretory phase might lead to establishment of endometriosis.