LPS-induced endotoxic shock leads to NETs deposition in kidney tissue.

<p>Endotoxic shock was induced by LPS injection (15 mg/kg, iv.). In situ immunofluorescence analysis of frozen tissue sections of kidney harvested 12 h after endotoxic shock induction was conducted. (<b>A-B</b>) DNA stained. (<b>C-D</b>) DNA/histones stained. (<b>E-F</b>) MPO stained. (<b>G-H</b>) Tissue structure was visualized by differential interference contrast (DIC) microscopy. NETs were identified by the co-localization of DNA, histone and MPO markers. (<b>I-J</b>) Co-localization of DNA (blue), DNA/histone (green) and MPO (red) indicates intraglomerular NETs formation. n = 5 per experimental group.</p

Serum concentrations of cf-DNA are positively correlated with organ failure in septic patients.

<p>(<b>A</b>) Cf-DNA was quantified in serum samples obtained from healthy control volunteers (n = 8) and septic patients (n = 31). * p < 0.05 compared with healthy control volunteers (Mann-Whitney U test). (<b>B</b>) Correlation of cf-DNA serum concentrations with SOFA scores. Serum cf-DNA concentration according to stages of (<b>C</b>) AKIN score and (<b>D</b>) ARDS, according to the Berlin Definition. *p < 0.05 compared with 0/1 stages (Mann-Whitney U test). (<b>E</b>) Patient serum samples incubated with rhDNase for 30 minutes. *p < 0.05 compared with serum+rhDNase (Paired t-test). AKIN—Acute Kidney Injury Network; ARDS—Acute Respiratory Distress Syndrome; no—no ARDS; rS–Spearman’s rho; SOFA–Sequential Organ Failure Assessment.</p

Morphological changes in heart, lungs and liver tissues.

<p>Animals were euthanized 12 h after endotoxic shock induction, and the heart, lungs, and liver were isolated, fixed by immersion in 10% paraformaldehyde, dehydrated and embedded in paraffin wax. Then, 5-μm-thick sections were stained with hematoxylin and eosin for histological examination. The images are representative of heart, lung and liver sections from the CTRL (<b>A, D, G</b>), LPS+Sal (<b>B, E, H</b>) and LPS+rhDNase (<b>C, F, I</b>) groups. Arrows indicate edema. Stars indicate leukocyte infiltration. Arrowheads indicate hyperplasia and hypertrophy of Kupffer cells. n = 5 per each experimental group.</p

Systemic degradation of NETs attenuated organ damage during LPS-induced endotoxic shock.

<p>Endotoxic shock was induced by LPS injection (15 mg/kg, iv.) Mice were pre-treated (10 min) and post-treated (8 h) with saline or rhDNase (10 mg/kg, sc.). Twelve hours after endotoxic shock induction, the serum concentrations of cf-DNA (<b>A</b>) and TNF-α (<b>B</b>), serum levels of serum CK-MB (<b>C</b>), BUN (<b>D</b>), AST (<b>E</b>) and MPO in lung tissue (<b>F</b>) were determined. The data are reported as the mean ± SEM. * p < 0.05 compared with the sham group; # p < 0.05 compared with the LPS+Sal group (ANOVA followed by Tukey’s test, n = 5 per experimental group). (<b>G</b>) Survival rates of endotoxemic mice pre-treated (10 min) and post-treated (8/8 h) with saline or rhDNase (10 mg/kg, sc.) until the 48^th hour. * p < 0.05 compared with the LPS+Sal group (Mantel-Cox log-rank test, n = 10 per experimental group).</p

Degradation of systemic cf-DNA/NET by rhDNase treatment did not prevent organ damage during polymicrobial sepsis.

<p>Mice were subjected to sham surgery or CLP-induced severe sepsis. (<b>A</b>) Blood samples were collected 3, 6, 12 and 24 hours after sepsis induction, and plasma concentrations of cf-DNA/MPO (NET) were determined (white horizontal bar represents the sham group at the indicated times). Blood samples were also collected 6 and 12 hours after sepsis induction in mice treated pre-sepsis (10 min) and post-sepsis (4 h) with rhDNase (10 mg/kg, sc.) * p < 0.05 compared with the sham group, white line; # p < 0.05 compared to the CLP- Sal 6 and 12 h groups (ANOVA followed by Tukey’s test, n = 5 per experimental group). (B) Animals were treated pre-sepsis (10 min) and post-sepsis (4 h) with Sal (control) or rhDNase (10 mg/kg, sc.). Six hours after sepsis induction, the serum levels of CK-MB, BUN (C) and AST (D) were quantified. Blood bacterial levels 6 h (E) were also quantified; # p < 0.05 compared with the CLP-Sal 6 h group (ANOVA followed by Tukey’s test, n = 5 per experimental group). The horizontal bars represent the median (Mann-Whitney U test, n = 5–7 per experimental group). The data are reported as the mean ± SEM. * p < 0.05 compared with the sham group; # p < 0.05 compared with the CLP+Sal group (ANOVA followed by Tukey’s test, n = 5 per experimental group).</p