The Differentiation Of Mice Macrophages ANA-1and Their Diverse Roles In Folic Acid Nephropathy

Background:The various roles of different state of macrophages in the process of renal interstitial fibrosis have been studied more in recent years. Macrophages infiltrate from circulation to the kidneys when kidney damage occurred due to various pathogens. Depending on the tissue microenvironment, classically activated macrophages(M1) promoted the development of kidney inflammation while the alternatively activated macrophages(M2) relieved tissue inflammation through releasing regulatory factors like IL-4,IL-13, IL-10. A lot of molecules induced the phenotype differentiation of macrophage, but less were reported about the roles of costimulatory molecules involved in the process, especially B7-H3which plays an important role in the inflammatory reaction. The actions of different phenotypes of macrophages in renal tubulointerstitial fibrosis were also less studied.Objective:In this study, different methods were used to stimulate murine macrophage (ANA-1) and induced different phenotypes of macrophages. By comparing different cytokine production with various stimulation, phenotypic differentiation methods of ANA-1were established. This will provide theoretical basis for the clinical application of B7-H3. In addition, macrophages stimulated by lipopolysaccharide(LPS) or IL-4were injected into mouse with folic acid nephropathy and followed effects on tubulointerstitial fibrosis were explored.Methods:1.When cultured by1640and fetal bovine serum(FBS), ANA-1with LPS, IL-4, B7-H3monoclonal antibody (B7-H3mAb), B7-H3mAb+LPS, B7-H3mAb+IL-4, blank and mice IgG control group were established. The cells and supernatant were collected after a24hours’culture. Inducible nitric oxide synthase (iNOS) and macrophage mannose receptor (CD206) were detected by flow cytometry. IL-10, tumor necrosis factor a(TNF-a) level were tested by enzyme-linked immunosorbent assay (ELISA).2.CD1mouse were randomly assigned to four groups:normal control group, folic acid group(FA group), LPS+FA group, IL-4+FA group. The model of interstitial fibrosis was induced with240mg/kg of folic acid (FA) by intraperitoneal injection. Five mouse in each group were sacrificed on the14th and21th day. Blood was collected to detect blood urea nitrogen(UN), creatinine(Cr), uric acid (UA) respectively. Samples of kidneys were processed for hematoxylineosin stain(HE), Masson trichrome stain and F4/80macrophages, α-smooth muscle actin (a-SMA), collagen I (COLI) detected by immunohistochemical stain(IHC).Results:The first part:1. The expression of B7-H3on ANA-1stimulated with LPS was up-regulated while decreased with IL-4(P<0.05);2. Compared with the blank control group, LPS group had a high protein expression of iNOS, and decreased significantly when cocultured with B7-H3mAb (P<0.05);3. The high expression of CD206was seen in each group without obvious change;4. Compared with the blank control group, the production of IL-10in IL-4group and IL-4+B7-H3mAb group significantly increased (P<0.05), while no significant was found in LPS group, B7-H3mAb group and LPS+B7-H3mAb group; Compared with the blank control group, although the level of TNF-a in the IL-4+B7-H3mAb group had statistical difference, the change was not obvious and the other five groups had no significant difference. The second part:1. After two times of240mg/kg FA administration by intraperitoneal injection, serum UN, Cr, UA level significantly increased in FA group. HE staining and Masson staining showed LPS+FA group and IL-4+FA group had different renal tubulointerstitial fibrosis. The severity of fibrosis in LPS+FA folic acid group was the worst, followed by FA group and last by IL-4+FA group.2. Immunohistochemistry results:the FA group, LPS+FA group, IL-4+FA group showed a high level of F4/80+macrophage infiltration at two weeks, the expression of a-SMA and COLI were significantly higher than the normal control group, there was no significant difference among three model groups. At the third week, the expression of a-SMA in IL-4+FA group was lower than which in FA group and LPS+FA group (P<0.05), the expression of COL I in LPS+FA group was dominant, followed by FA group and IL-4+FA group weakest (P<0.05).Conclusion:The first part:1.ANA-1stimulated by1μg/ml LPS up-regulated the level of iNOS, polarized to M1macrophages and up-regulated expression of B7-H3. B7-H3mAb down-regulated the increased iNOS induced by LPS.2. ANA-1with lOng/ml IL-4increased the secretion of IL-10, polarized macrophages to M2phenotypic with lower expression of B7-H3. The second part:1. After two times of240mg/kg FA administration by intraperitoneal injection, serum UN, Cr, UA level significantly increased. HE and Masson staining showed a large number of inflammatory cell infiltration in the tubulointerstitial fibrosis area. Immunohistochemistry showed a significant increase of the number of F4/80+macrophages and the expression of COL I and a-SMA. Tubulointerstitial fibrosis model was successfully established by FA.2. LPS stimulated macrophages exacerbated significantly the severity of tubulointerstitial fibrosis in folic acid nephropathy, manifested by the enlarged area of interstitial fibrosis and increased COLI expression; whereas macrophages activated by IL-4inhibited the development of tubulointerstitial fibrosis with the decreased COL I and a-SMA expression in the folic acid nephropathy.