This study aims to observe the changes and the function of p38MAPK-HSP27 signaling pathways in acute lung injury (ALI) induced by acute ischemic kidney injury in rats. Wistar rats were randomly divided into Group A (control group), Group B (acute kidney injury group), and Group C (acute kidney injury +SB203580). The concentration of protein in BALF, neutrophil counts, PI, W/D; the concentration of TNF-
AKI, one of the common but complicated clinical issues, is experienced in 30% of critical patients. About 5%-6% of the patients in the intensive care unit can suffer from AKI and need renal replacement [
ALI is generally aggravated by nephrogenic pulmonary edema, that is, increased capillary transmural hydrostatic pressure (similar to the increased volume load in heart failure) because of water-sodium retention caused by acute renal failure. Based on recent studies on animals, however, the changes in pulmonary vascular permeability vary at the very onset of AKI [
As shown by previous studies, the signal pathway of p38 MAPK-HSP27 regulates the assembly/disassembly of actin, reconstructs the cytoskeleton, and changes the pulmonary vascular permeability [
About 90 healthy male Wistar rats weighing 300 g–320 g from the animal center of the China Medical University with postintraperitoneal anesthesia (5% chloral hydrate) underwent tracheotomy and catheterization in carotid arterial and jugular vein. The respiration, heart rate, diastolic/systolic pressure, mean arterial pressure, and central venous pressure of the rats were monitored. The rats that were in stable condition for 30 min were randomly divided into three groups with 30 rats in each group. In Group A (control group), bilateral arteriovenous were separated, and bilateral renal arteriovenous were not ligated. Intraperitoneal injection was performed with 2 mL of a saline solution. In Group B (AKI group), bilateral arteriovenous were separated and bilateral renal arteriovenous were ligated. Intraperitoneal injection was then performed with 2 mL of a saline solution. In Group C (AKI +SB203580), bilateral arteriovenous were separated, and bilateral renal arteriovenous were ligated. Intraperitoneal injection was then performed with 2 mL of SB203580 (the inhibitors of p38MAPK, namely, pyridine imidazole medicament derivatives at 2 mg/kg). This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal use protocol has been reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the First Hospital of China Medical University.
After the establishment of the model, six animals in each group were killed at 0, 2, 4, 6, and 8 h. The cytokine in venous blood was tested. Lungs exposed after thoracotomy were observed for gross changes. After the right hilum was ligated, the upper-right lung was taken, fixed with 4% paraformaldehyde, embedded in paraffin, sliced, HE-stained, and observed for pathological changes by using a microscope. The lower-right lung was then observed. The blood was eliminated using a filter, weighed, and heated in an oven (80°C for 72 h) to obtain a stable weight. The blood was then weighed to determine its dry weight, and the (W/D) weight ratio was then calculated. Bronchoalveolar lavage was performed on the left lung in triplicate with 3 mL of a phosphate buffer solution per test. The collected BALFs in the three tests were mixed together and cryopreserved for cytokine analysis. Coomassie brilliant blue staining was performed to identify the protein in BALF. The rats received intravenous injections of human serum albumin (HSA) an hour before extermination. The HSP levels in the plasma and in BALF were determined by enzyme-linked immunosorbent assay (ELISA). PI was calculated as the BALF-to-plasma ratio of the HSA concentration. The sediments of BALF were used to determine the neutrophil count.
Lung tissue was normally fixed, dehydrated, embedded, and sliced to obtain hematoxylin- and eosin- (HE) stained slices. Multiple high-power fields were randomly selected for each HE-stained slice to compare the variations in lung tissues and cells.
Double antibody sandwich ELISA was used to determine the concentrations of IL-1
The middle lobe of the right lung that was taken after thoracotomy was immediately cryopreserved in liquid nitrogen and made into a homogenate. Thiobarbituric acid and nitrate reductase methods were used to determine the MDA and NO contents, respectively, according to the instruction in the assay kit (Beyotime).
From the obtained homogenate in Section 1.5, the protein was extracted and its concentration was measured. The homogenate was kept under −80°C. A 100
SPSS 16.0 was used in statistical calculations. Count data were expressed in
The degree of ALI was evaluated by the protein content and neutrophilic count in BALF. The protein content and neutrophilic count had no significant variations at each time point after the experiment in Group A. Both values in BALF began to increase 2 h after the experiment in Group B and maintained a gradual increasing trend with significant difference compared with Group A. Compared with Group B, both values in BALF were significantly decreased in Group C, which was treated with SB203580. The difference was statistically significant (
Change of protein content, neutrophil counts in rat’s Bronchoalveolar lavage (BALF) (
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The permeability of pulmonary vascular endothelial cells or alveolar epithelial cells was evaluated via PI and W/D. PI and W/D had no significant variations at each time point after the experiment in Group A. Both values began to increase 2 h after the experiment in Group B and maintained a gradually increasing trend with significant difference compared with Group A. When compared with group B, both values significantly decreased in Group C. The difference was statistically significant (
Comparison of W/D and PI.
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The concentrations of TNF-
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MDA and NO reflect the level of lipid peroxidation. The MDA and NO contents in the rat lung tissue were measured by thiobarbituric acid and nitrate reductase methods, respectively, to evaluate the level of lipid peroxidation in the rat lung tissue. Both values had no significant variation at each time point after the experiment in Group A. Both values began to increase 2 h after the experiment in Group B and maintained a gradual increasing trend with significant difference compared with Group A. Compared with Group B, both values significantly decreased in Group C. The difference was significantly significant (
The content of MDA, NO in the lung tissue (
Time (h) | MDA (nmol/mg) | NO ( |
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The changes in the morphology were observed by HE staining to evaluate the effect of acute renal injury and SB203580, which were the inhibitors of P38 on pulmonary morphology. The alveolar structure appeared intact without exudates in the alveoli or pulmonary interstitial edema in the normal control group at 0 and 8 h. In the kidney injury group (Group B), swollen epithelium was found with thickened alveoli wall, as well as telangiectasia, capillary hyperemia, evident edema in the stroma and cavity of alveoli, and inflammatory cells in the alveoli cavity. The exudates of red blood cells and protein increased because of the infiltration of numerous inflammatory cells. In other areas, injury in the small air tracts was observed with disorder in the alveoli structure, which indicates the pathological changes in ALI. In Group C, where the inhibitor of p38MAPK (SB203580) was used, exudates of inflammatory cells, red blood cells, and protein were observed in the alveoli. However, the edema in the stroma or cavity of alveoli was mild (Figure
Morphology of the lung tissues of different groups after HE staining. (a) and (b) The lung tissue of the control group (Group A) at 0 and 8 h, respectively, in which the alveolar structures are integral without effusion from inside the alveoli or pulmonary interstitial edema; (c) the lung tissue of the acute lung injury group, in which swollen alveolar epithelia, thickened alveolar walls, telangiectasia and capillary hyperemia, and pulmonary interstitial edema, noticeably increased effusion of inflammatory cells, red blood cells, and proteins from inside the alveoli, and disordered alveolar structures are observed, showing pathological changes of acute lung injury; and (d) the lung tissue of Group C after the administration of the p38MAPK inhibitor SB203580, in which effusion of inflammatory cells, red blood cells, and proteins from inside the alveoli is observed, but the interstitial and alveolar edemata are milder compared with Group B.
The concentrations of p-p38MAPK and p-HSP27 were measured by Western blot to evaluate the effect of AKI on the p38 MAPK-HSP27 signal pathway. As shown in Figures
p-p38MAPK expression in Groups A, B, and C at different time points using Western blot analysis. The strip concentration of p-p38MAPK in Group B begins to rise from 2 h after test, whereas that in Group A does not show any significant difference; compared with Group B, p-p38MAPK expression in Group C decreases at different time points after the administration of the p38MARPK inhibitor SB203580.
p-HSP27 expression in Groups A, B, and C at different time points using Western blot analysis. The strip concentration of p-HSP27 in Group B begins to rise from 2 h after test, whereas that in Group A does not show any significant difference; compared with Group B, p-HSP27 expression in Group C decreases at different time points after the administration of the p38MARPK inhibitor SB203580.
The concentrations of F-actin and G-actin were measured by Western blot. The ratios of F- to G-actin were calculated to evaluate indirectly the changes in the endothelial cytoskeleton after AKI. As shown in Figure
F-actin and G-actin expression in Groups A, B, and C at different time points using Western blot analysis. The concentration of Western blot of banding of F-Actin and G-Actin had no significant variation at each time point after the experiment in Group A. The concentration of Western blot of banding of F-Actin begin to increase at 2 hours after the experiment in Group B, appeared to maintain a gradually increasing trend, but the concentration of Western blot of banding of G-Actin began to decrease, which is a significant difference compared with Group A. Compared with Group B, the concentration of Western blot of banding of F-Actin was significantly decreased, and that of G-actin were significantly increased in group C which was treated by SB203580,
The ratios of F- to G-actin had no significant variations at each time point after the experiment in Group A. The ratios began to increase 2 h after the experiment in Group B, and maintained a gradually increasing trend with significant difference compared with Group A. Compared with Group B, the ratios significantly decreased in Group C. The difference was statistically significant (
Figure
Analyses of the correlations of TNF-
p38MAPK %
p38MAPK %
This research showed that the protein level and neutrophil counts in BALF significantly increased 2 h after AKI with higher (W/D) lung weight ratios and PI. Pathological changes in ALI, which include swollen alveoli epithelium, expanded alveoli wall, telangiectasia, capillary hyperemia, evident edema in the stroma and cavity of alveoli, and inflammatory cells in alveoli, significantly increased the exudates of red blood cells and protein, the infiltration of numerous inflammatory cells, the injury of small air tracts in several areas, and the disorderly arrangement of alveoli. Previous studies have proven that ALI occurred at the early stage of AKI, which was characterized by the infiltration of inflammatory cells, the hyperemia in alveoli cavity, and the changes in the alveoli structure [
TNF-
Anti-inflammatory cytokine (IL-10) content also increased 2 h after AKI, wherein the peak was reached within 4 h. IL-10 content gradually declined after the peak was reached. The balance between inflammation and anti-inflammation was affected, which caused an inflammation imbalance. In addition, numerous inflammatory mediators in the circulation can be deposited in the lungs because of the massive vascular system in the lungs; therefore, the pulmonary levels of TNF-
Another important feature of ALI is the undermined integrity and function of the pulmonary endothelial-epithelial barrier. The three-dimensional structure is maintained by a cytoskeleton, which is the vital structure that sustains the normal shape and function of cells. Microfilament is one of the vital cytoskeletons that consists of F-actin and actin-binding proteins, wherein the assembly/disassembly of actins involves multiple cellular activities [
In this study, p-p38MAPK correlated positively with p-HSP27 expression levels. The expression levels decreased when the inhibitor of p38MAPK (SB203580) was used, thereby proving that p38MAPK regulates the expression of p-HSP27. MAPK is a vital cellular signal transduction system that regulates cellular proliferation, differentiation, apoptosis, and gene expression. p38MAPK is a member of the MAPK family. Based on previous studies, p38MAPK can be activated by UV, proinflammatory cytokines (TNF-
Typical ALI is caused by the induction of AKI and bilateral occlusion of renal arteries and veins. The effects include increased protein content in BALF, increased lung W/D and PI with infiltration of inflammatory cells in the alveoli, increased MDA and NO contents, increased inflammatory mediators (TNF-
This study was supported by Science and Technology Project of Shenyang City (no. F11-264-1-52) and Fund for Scientific Research of the First Hospital Of China Medical University, (no. fsfh1103).