Kidney and Liver Injuries After Major Burns in Rats Are Prevented by Resolvin D2 - PubMed (original) (raw)

. 2016 May;44(5):e241-52.

doi: 10.1097/CCM.0000000000001397.

Yong-Ming Yu, Tomohiro Kurihara, Aleksandr Vasilyev, Amir Ibrahim, Rahmi Oklu, Gaofeng Zhao, Anil V Nair, Dennis Brown, Alan J Fischman, Ronald G Tompkins, Daniel Irimia

Affiliations

Kidney and Liver Injuries After Major Burns in Rats Are Prevented by Resolvin D2

Yoshitaka Inoue et al. Crit Care Med. 2016 May.

Abstract

Objectives: Innate immune dysfunction after major burn injuries increases the susceptibility to organ failure. Lipid mediators of inflammation resolution, e.g., resolvin D2, have been shown recently to restore neutrophil functionality and reduce mortality rate in a rat model of major burn injury. However, the physiological mechanisms responsible for the benefic activity of resolvin D2 are not well understood.

Design: Prospective randomized animal investigation.

Setting: Academic research setting.

Subjects: Wistar male rats.

Interventions: Animals were subjected to a full-thickness burn of 30% total body surface area. Two hours after burn, 25 ng/kg resolvin D2 was administered IV and repeated every day, for 8 days. At day 10 post burn, 2 mg/kg of lipopolysaccharide was administered IV, and the presence of renal and hepatic injuries was evaluated at day 11 post burn by histology, immunohistochemistry, and relevant blood chemistry.

Measurements and main results: In untreated animals, we found significant tissue damage in the kidneys and liver, consistent with acute tubular necrosis and multifocal necrosis, and changes in blood chemistry, reflecting the deterioration of renal and hepatic functions. We detected less tissue damage and significantly lower values of blood urea nitrogen (26.4 ± 2.1 vs 36.0 ± 9.3 mg/dL; p ≤ 0.001), alanine aminotransferase (266.5 ± 295.2 vs 861.8 ± 813.7 U/L; p ≤ 0.01), and total bilirubin (0.13 ± 0.05 vs 0.30 ± 0.14 mg/dL; p ≤ 0.01) in resolvin D2-treated rats than in untreated animals. The mean blood pressure of all animals was above 65 mm Hg, indicating adequate tissue perfusion throughout the experiments. We measured significantly larger amounts of chromatin in the circulation of untreated than of resolvin D2-treated rats (575.1 ± 331.0 vs 264.1 ± 122.4 ng/mL; p ≤ 0.05) and identified neutrophil extracellular traps in kidney and liver tissues from untreated rats, consistent with the tissue damage.

Conclusions: Pathologic changes in kidney and liver tissues in a rat model of major burn and endotoxin insults are ameliorated by resolvin D2.

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Figures

Figure 1

Figure 1. Timeline of the burn injuries, treatment, and renal cortex injury

A. Blood samples were collected before the burn procedure (day 0), before injecting LPS (day 10 pb), and 24 hours after LPS (day 11 pb). Hemodynamic measurements were also performed at the same time points. One additional measurement was performed at 12 hours after LPS injection. For animals receiving treatment, 25 ng/kg of RvD2 were administered first at 2 hours after burn injury (day 0) and continued daily until day 7 after burn. B, C. Representative section of renal cortex at day 11 pb, in burn and LPS, untreated group. Tubular injuries were observed, including tubular cast formation (arrows), and ballooning degeneration/vacuolization of renal proximal tubular cells (H&E, ×400, scale bar 50 μm). D. Light microscopic image at day 11 pb in burn and LPS, RvD2 treated group, indicated that tubular injury was reduced by RvD2 treatment in renal cortex (H&E, ×400, scale bar 50 μm). E – G. Quantitative analysis of pathological changes in the renal cortex (N=3 rats total, N=2–3 experiments for each sham burn group; N=6 rats total, N=3–4 experiments for each burn group). We employed semi-quantitative scores (from 0 (normal) to 3 (severe)), as previously described (17)). We assessed the presence of tubular casts, tubular dilatation/flattening, and tubular degeneration/vacuolization in the renal cortex. In all comparisons, RvD2 treatment reduced the pathological changes of tubular cells in renal cortex (Burn/LPS vs. Burn/LPS/RvD2; **_p_≤0.01, ns, and *_p_≤0.05, respectively).

Figure 2

Figure 2. Pathology of the liver after burn injury and endotoxin injection with and without Resolvin D2 treatment

A. Spotty necrosis of the liver at day 11 pb, after burn and LPS, untreated group (24 hours after LPS injection). Several necrotic foci (N) were identified in the liver tissue sections, in the burn and LPS group. Large numbers of inflammatory cells, including neutrophils, macrophages, and lymphocytes infiltrated the portal area (H&E, ×200, scale bar 100 μm). B. Representative section of liver at day 11 pb, in the burn and LPS with RvD2 treatment groups. Spotty necrotic changes were significantly diminished in the RvD2 treatment group (H&E, ×200, scale bar 100 μm). C – E. Quantitative analysis of pathological changes in the liver (N=3 rats total, N=2–3 experiments for each sham burn group; N=6 rats total, N=3–4 experiments for each burn group). We quantified the spotty necrosis, degeneration or edematous changes of hepatocytes, and infiltration of inflammatory cells in the portal area. RvD2 treatment suppressed these impaired pathological changes in liver (Burn/LPS vs. Burn/LPS/RvD2; *_p_≤0.05). In particular, RvD2 treatment reduced spotty necrotic changes in hepatocytes (Burn/LPS vs. Burn/LPS/RvD2; ***_p_≤0.001).

Figure 3

Figure 3. Hemodynamic changes in rats after burns and endotoxin, in treated and untreated groups

A, B. Body weight change in sham burn (A) and burn groups (B). Rats in sham burn groups lost few % of total body weight in the first 1 or 2 days due to general anesthesia, and recovered quickly by day 4 pb. Burned rats (all) lost about 5 % of total body weight in the 3 or 4 days post burn injury, and recovered at 10 days after burn. The body weight loss was comparable in treated and untreated groups (orange and green lines). After LPS injection, both RvD2 treated (blue) and untreated (red) groups showed comparable body weight loss. C–F. Systolic and mean blood pressure in sham burn (C, E) and burn groups (D, F). We monitored hemodynamic changes using non-invasive tail cuff measurement at four points, day 0 (pre burn), day 10 pb (before LPS), and then at 12 (day10.5 pb) and 24 (day 11 pb) hours after the LPS injection. After LPS the systolic blood pressure (SBP) and the mean blood pressure (MBP) decreased slightly in untreated groups. Differences between untreated and treated rats were significant at 12 hours after LPS (Burn/LPS vs. Burn/LPS/RvD2; *_p_≤0.05). It is important to note that MBP was maintained above 65 mmHg for all animals throughout the study. G, H. Pulse rate (PR) increased after LPS, more in untreated than in RvD2 treated group (H). The difference between untreated and treated animals increased at 24 hours after endotoxin in burn and LPS groups (Burn/LPS vs. Burn/LPS/RvD2; *_p_≤0.05). Hemodynamic condition, including SBP, MBP, and PR, did not change significantly in all sham burn groups (A–H: N=3 rats total, N=2–3 experiments for each sham burn group; N=6 rats total, N=3–4 experiments for each burn group).

Figure 4

Figure 4. Blood chemistry changes for renal function and Megalin staining of renal proximal tubules

Blood chemistry tests were performed at day 0 (pre burn), day 10 pb (before LPS), and day 11 pb (24 hours after LPS) (A–F: N=3 rats total, N=2–3 experiments for each sham burn group; N=6 rats total, N=3–4 experiments for each burn group). A, B. The renal function marker, blood urea nitrogen (BUN), increased significantly after LPS injection, and restored by RvD2 pre treatment (A: Sham Burn/LPS vs. Sham Burn/LPS/RvD2; *_p_≤0.05). In burned rats, BUN increased more significantly after the second septic insult (Burn/LPS vs. Burn/LPS/RvD2; ***_p_≤0.001). C, D. Serum creatinine values did not change in any of the groups. E, F. In sham burn groups, serum albumin values were preserved during treatment period, and decreased after LPS, in both treated and untreated groups. The decrease in serum albumin after LPS was ameliorated by RvD2 treatment (E: Sham Burn/LPS vs. Sham Burn/LPS/RvD2; *_p_≤0.05). On the other hand, levels of serum albumin decreased after burn and even further after LPS, in both treated and untreated groups. The decrease in serum albumin after burn and burn and LPS was also ameliorated by RvD2 treatment (F: Burn vs. Burn/RvD2; †_p_≤0.05, Burn/LPS vs. Burn/LPS/RvD2; *_p_≤0.05). G, H. Representative images of renal proximal tubules at day 11 pb in burn only group (G) and burn + RvD2 treatment group (H). Green fluorescence shows megalin, and the blue fluorescence shows the nuclei. Megalin is localized in the apical brush border region of the proximal tubule. Staining intensity after burn injury is higher in the RvD2 treated group. Scale bars, 50 μm. I. Quantitative analysis of fluorescence intensity of megalin staining. Megalin intensity decreased after burn and LPS groups, and was restored in the burn and RvD2 treatment group, but not in the burn and LPS and RvD2 group (Sham Burn and Burn groups N=3 rats total; all other groups N=4 rats total).

Figure 5

Figure 5. Blood chemistry changes for hepatic function in rats after burns and endotoxin, in treated and untreated groups

A – C. After LPS, as second septic insult post burn, we measured significant increases in serum ALT, ALP, and total bilirubin at day 11 pb (Burn/LPS vs. Burn/LPS/RvD2; **_p_≤0.01, ***_p_≤0.001, and **_p_≤0.01, respectively). Changes were significantly reduced in the RvD2 treated group. D. Levels of blood glucose were significantly depressed after LPS injection in the untreated vs. treated group (Burn/LPS vs. Burn/LPS/RvD2; **_p_≤0.01) (A–D: N=6 rats total, N=3–4 experiments for each burn group).

Figure 6

Figure 6. Neutrophil Extracellular Traps and Plasma genomic DNA values 24 hours after endotoxin

A. Plasma genomic DNA values at day 11 pb (24 hours after injecting 2 mg/kg LPS) in burn groups increase after the second septic insult (N=6 rats total, N=3–4 experiments for each burn group). Administering RvD2 suppresses the increase in plasma genomic DNA (Burn/LPS vs. Burn/LPS/RvD2; *_p_≤0.05). B. Fluorescence microscopic detection of NETs in renal cortex at day 11 pb in burn and LPS group. NETs are identified by fluorescence from histone/DNA staining (red) and neutrophil elastase (green). Nuclei of intact parenchymal cells are identified by cell-permeant DAPI staining (blue). NETs appear to be located outside of proximal tubular cells in the renal cortex. Scale bar, 10 μm. C. Immunohistochemistry section of hepatic tissues at day 11 pb in burn and LPS groups. High fluorescence intensity, corresponding to the presence of NETs can be observed outside of the hepatocyte in burn and LPS without treatment groups. Scale bar, 10 μm. D. Representative section of hepatic focal necrosis at day 11 pb in burn and LPS groups (immunohistochemistry). High fluorescence intensity of histone/DNA and elastase, corresponding to large amounts of NETs can be observed at area of necrosis. Scale bar, 10 μm.

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