Study Of The Relationship Between Heparanase And Renal Inflammatory Cell Infiltration

As a common symptome in the development of various Chronic kidney diseases(CKD), renal inflammatory cell infiltration plays an important role in mediation of the occurrence and development of both chronic renal tubulointerstitial inflammation and renal fibrosis. Pathologically , there is an increase of expression and activity of inflammation mediators in peripheral blood, meanwhile, intrinsic renal cells are activated , express various chemotatic factors, and conduct inflammatory cells such as mononuclear-macrophage, polymorphonuclear leucocytes and T cells to nephridial tissue ,which interact with intrinsic renal cells ,then express a series of cytokines and growth factors, adhesion molecules and extracellular matrix (ECM), so renal interstitial injury is directly or indirectly magnified, meanwhile, synergistic effect of cytokines and chemotatic factors will lead to more paths of inflammatory reactions to form a water fall effect. This cascade effect results in irreversible injury of renal interstitium. However, with regard to the mechanism of recruitment and migration of inflammatory cell to inflammatory site , it is still unclear whether there are other factors contributing to the biological behavior of migration and infiltration of inflammatory cells beside the effects of known inflammatory chemotatic factors up to now.To migrate to inflammatory reaction site, inflammatory cells have to pass through the barrier composed of extracellular matrix and basement membrane .This barrier mainly contains structural protein and glycosaminoglycan, the main component of glycosaminoglycan is heparan sulfate proteoglycan (HSPGs). HSPGs are glycoproteins mainly consisting of a protein core and side chains of several linear heparan sulfate (HS) covalent linking to the protein core. At the beginning, destruction of structural protein is regarded as the decisive step for cell invasion and migration. But actually, as a component of ECM and basement membrane, the degradation of glycosaminoglycan is essensial for cell invasion.Heparanase(Hpa)is an endoglycosidase which takes HSPGs as substrate,then splits core molecule HS of HSPGs . Hpa plays an important role in inflammation, invasion and migration of tumor cells. In inflammation and immune reaction , once on stimulation , lymphocytes and mononuclear-macrophages immediately release Hpa and split HSPG, so integrity of vessel wall basement membrane is destroyed ,which enables the extravasation of immune competent cell and they migrate to the inflammation site. The body's defensive reaction leads to inflammatory reaction, but pathologically, if inflammatory chemotaxis and inflammatory reaction haven't been stopped, persistent excessive repair will contribute to irreversible fibrosis of the inflammatory site.According to recent knowledge of the role that Hpa plays in metachoresis of immunocompetence cells and migration to inflammatory site of immune competent cells, we guess that in progressive CKD, renal inflammatory cell infiltration and successive chronic progressive renal fibrosis may have relationship with abnormal expression of Hpa, inflammatory cells such as activated T lymphocytes secrete Hpa, and Hpa performs its activity in regional kidney, which promotes inflammatory cell infiltration into nephridial tissue, activates intrinsic renal cells, releases HS binding factors, pro-fibrotic cytokines and growth factors, which triggers renal fibrosis proceeding.Therefore, our study aimed at investigation of the relationship between Hpa and renal inflammatory cell infiltration. As adriamycin nephrosis model has a character of continuous quantitative proteinuria, progressive inflammatory injury and fibrosis of renal interstitium, so similarly, we observed Hpa expression in adriamycin nephrosis and discussed the anti-inflammatory effect of Hpa inhibitors to explore new way to prevent CKD and delay its fibrosis.Main contents of the experiment1. In the first part of our study, RT-PCR and immunochemical method were used to investigate the Hpa expression in the nephridial tissue and peripheral blood lymphocyte of patients with progressive chronic nephritis and healthy donor for kidney transplant. Meanwhile, we observed the inflammatory cell infiltration of the nephridial tissue.2. In the second part, after cell culture, we used RT-PCR to observe the effect of Hpa inhibitor laminaran on Hpa expression in cultured peripheral blood lymphocytes from CKD patients.3. In the third part, we first established an adriamycin nephrosis model, then investigated the Hpa expression in murine peripheral blood lymphocytes and nephridial tissue, and their relationship with nephridial tissue inflammatory cell with RT-PCR and immunochemical method, tested clinical indexes to see the therapeutical effect of laminaran on adriamycin nephrosis model, observed the influence of laminaran on proliferation of lymphocytes in adriamycin nephrosis rat with liquid scintillation method, and measured the expression variance of chemokines, adhesion molecules and pro-fibrotic cytokines that were crucial to nephridial tissue with immunochemical method.Main result1. We use RT-PCR and immunohistochemistry to investigate the Hpa expression of peripheral blood lymphocytes and nephridial tissues in CKD patients and healthy donors of kidney for transplant, as well as the infiltration of inflammatory cells2. Hpa mRNA expression is still higher in cultured peripheral blood lymphocytes than normal control group. Laminaran can decrease Hpa expression.3. The Hpa expressions, in peripheral blood lymphocytes and nephridial tissue from adriamycin nephrosis rat, both are higher than normal rat, which has a similar phase characteristic to the degree of aggravation of inflammatory cells infiltration in nephridial tissue.4. Compared with control group with renal disease, clinical indexes including Urine protein, serum creatinine, blood urea nitrogen, etc, show that the disease is relieved in laminaran treated group rat, extent of inflammatory cell infiltration and fibrosis in nephridial tissue are palliated.5. Both Hpa mRNA expression in peripheral blood lymphocytes and nephridial tissue of laminaran treated group rats are lower than these of control group with renal disease.6. Lymphocytes proliferation activitiesof laminaran treated group rats are lower than these of control group with renal disease.7. Expression of IL-6,IL-8,ICAM-1 and TGF-βin nephridial tissue of laminaran treated group is lower than that of control group with renal disease.Main Conclusion1.There is high Hpa expression in CKD, infiltration extent of inflammatory cells in nephridial tissue closely relates with high expression of Hpa.2. Laminaran can inhibit the expression of Hpa in CKD Lymphocytes. 3. Laminaran has curative effect on adriamycin nephrosis, probably by inhibiting Hpa and Lymphocytes proliferation to palliate inflammatory reaction in renal interstitium, which palliates the expression of important chemotactic factors, adhesion molecules, profibrotic cytokines in nephridial tissue, and delays CKD fibrosis.