| Objective: To investigate the expression change of adhesion molecules(ICAM-1 and VCAM-1), C3, IL-17(the marker of Th17 cell) and ED1(the marker of mononuclear macrophage) in the renal transplantation tissue and the effect of hyperbaric oxygenation(HBO) therapy on their expressions by establishing a syngenic rat model of renal transplantation ischemia reperfusion injury(IRI), and to further investigate the molecular mechanism of the therapeutic action of HBO on the occurrence and development of the renal IRI.Methods: Sprague-Dawley(SD) rats were randomized divided into renal transplantation group, renal transplantation group + HBO group(hereafter referred to as HBO-treated group) and sham group, and each group was subdivided into 1h, 3h and 5h group(n = 6 in each group). The renal transplantation group was gived renal transplantation only; for HBO-treated group, renal transplantation rats received HBO in the oxygen chamber for animal experiment for 1 h at 0, 2 and 4 h after reperfusion, respectively, while the sham group received right nephrectomy only, not renal transplantation. Rats in both renal transplantation group and HBO-treated group were sacrificed at 1, 3 and 5 h after renal transplantation and their renal transplantation tissues and blood samples were taken; rats in the sham group were also sacrificed at the corresponding time points and their left kidney tissues and blood samples were taken. Pathological change in renal tissues was observed by HE staining. Expression of ICAM-1, VCAM-1, C3, ED1 and IL-17 was detected by immunohistochemistry(IHC), expression of ICAM-1 and C3 m RNAs was observed by real-time PCR, and Serum creatinine(SCr) was detected by fully automatic biochemical analyser.Results: 1. Changes in renal function: At 1, 3 and 5 h after reperfusion, SCr values increased distinctly in both renal transplantation group and HBO-treated group compared with the sham group(P<0.05); compared with the renal transplantation group, no significantdifference was noted in the HBO-treated group at 1 and 3 h, but SCr values decreased distinctly at 5 h(P<0.05). Both renal transplantation group and HBO-treated group, SCr values increased over time(P<0.05). In the sham group, there was no statistical difference in SCr value at each time point(P>0.05). 2. Changes in renal pathology: HE staining found that in the sham group the renal tissue structure was normal and no distinct change was noted at each time point. In the renal transplantation group, tissue injury was deteriorated over reperfusion time, and dilated lumen of renal tubule, swollen epithelial cells, and enlarged glomerular volume were noted; protein-like casts, red cell casts, granular degeneration and neutrophil infiltration appeared gradually. 3. Changes in expression of ICAM-1, VCAM-1, C3, ED1 and IL-17 in renal tissues by IHC:(1) ICAM-1: ICAM-1 was mainly expressed in nuclei of renal tubular epithelial and stromal cells. Relative quantification results showed distinctly higher expression of ICAM-1 in the renal transplantation group than in both sham group and HBO-treated group at 1, 3 and 5 h after reperfusion(P<0.05) and no significant difference between HBO-treated group and sham group(P>0.05). In both renal transplantation group and HBO-treated group, expression of ICAM-1 distinctly rose over time(P<0.05).(2) VCAM-1: VCAM-1 was expressed mainly in the cytoplasm of renal tubular epithelial cell, and slightly in the cytoplasm of glomerular epithelial cell. Relative quantification results showed that expression of VCAM-1 was distinctly higher in the renal transplantation group than in the sham group at 1, 3 and 5 h after reperfusion(P<0.05), and that expression of VCAM-1 markedly decreased after HBO(P<0.05) but was still higher than that of sham group. Expression of VCAM-1 distinctly rose over time in both renal transplantation group and HBO-treated group(P<0.05).(3) C3: C3 was mainly expressed in the cytoplasm of renal tubular epithelial cell. Relative quantification results showed that expression of C3 was distinctly higher in the renal transplantation group than in the sham group at 1, 3 and 5 h after reperfusion(P<0.05), and that expression of C3 markedly decreased after HBO(P<0.05) but was still higher than that of sham group. Expression of C3 distinctly rose over time in both renal transplantation group and HBO-treated group(P<0.05).(4) ED1(the surface marker of mononuclear macrophage): ED1 was mainly expressed in the cytoplasm of renal tubular epithelial cell. Relative quantification results showed that: at 1 h after reperfusion, expression of ED1 was distinctly higher in the renal transplantation group than in both HBO-treated group and sham group(P<0.05), and there was no significant difference between HBO-treated group and sham group; at 3 and 5 h after reperfusion, expression of ED1 was distinctly higher in the renal transplantation group than in the sham group(P<0.05), and expression of ED1 markedly decreased after HBO(P<0.05) but was still higher than that of sham group. Expression of ED1 distinctly rose over time in both renal transplantation group and HBO-treated group(P<0.05).(5) IL-17(the marker of Th17 cell): IL-17 was expressed mainly in the cytoplasm of renal tubular epithelial cell and very slightly in the cytoplasm of glomerular epithelial cell in both renal transplantation group and HBO-treated group, whereas it was expressed only in the cytoplasm of renal tubular epithelial cell and none in the glomerulus in the sham group. Relative quantification results showed that expression of IL-17 was distinctly higher in the renal transplantation group than in the sham group at 1, 3 and 5 h after reperfusion(P<0.05), and that expression of IL-17 markedly decreased after HBO(P<0.05) but was still higher than that of sham group. Expression of IL-17 distinctly rose over time in both renal transplantation group and HBO-treated group(P<0.05). 4. Changes in expression of ICAM-1 and C3 m RNAs in renal tissues by real-time PCR:(1) ICAM-1 m RNA: Relative expression analysis results showed that: at 1 h after reperfusion, no significant difference in expression of ICAM-1 m RNA was noted among three groups; at 3 h after reperfusion, expression of ICAM-1 m RNA was higher in the renal transplantation group than in both sham group and HBO-treated group(P<0.05), and expression of ICAM-1 m RNA markedly decreased after HBO(P<0.05) but was still higher than that of sham group. In both renal transplantation group and HBO-treated group, there was no significant difference in expression of ICAM-1 m RNA at 1 and 3 h after reperfusion(P>0.05), and expression of ICAM-1 m RNA was markedly higher at 5 h than at 1 and 3 after reperfusion(P<0.05).(2) C3 m RNA: Relative expression analysis results showed that expression of C3 m RNA was higher in the renal transplantation group than in the sham group at 1, 3 and 5 h after reperfusion(P<0.05), and that expression of C3 m RNA markedly decreased after HBO(P<0.05) but was still higher than that of sham group. Expression of C3 m RNA distinctly rose over time in both renal transplantation group and HBO-treated group(P<0.05). 5. Correlation analysis revealed that the rat Scr values and the P values of expression of ICAM-1, VCAM-1, C3, ED1 and IL-17 in renal tissues were at a level of less than 5%, and that there were positive correlations.Conclusions: 1. Renal dysfunction and damaged renal tissues could appear immediately at the early stage of reperfusion after renal transplantation; adhesion molecules, C3, Th17 cells and mononuclear macrophages may be involved in and promote the renal IRI, and tissue injury may deteriorate gradually over reperfusion time. 2. HBO in the treatment of early renal IRI could play a renoprotective role through the suppression of overexpression of adhesion molecules, complement system activation, Th17 cell aggregation and mononuclear macrophage infiltration, and the role become more distinct over reperfusion time. |
PDF Full Text Request