| Background: Mesangial proliferative glomerulonephritis(Mes PGN)is the most common chronic kidney disease(CKD)in China,and it is also the leading cause of end stage renal disease(ESRD).Nowadays,Mes PGN is considered to be a kind of immune & inflammation related diseases.Mesangial cells are not only the target cells in immuno inflammatory milieu,but also can be activated and secrete chemokine(such as CXCL10,CCL2),cytokines(such as TNF-? and IL-6)and mesangial matrix components,which exacerbate the renal injury.Renal fibrosis is a hallmark pathological change in patients with Ig A nephropathy progressing to ESRD.It has been reported that the prognosis of Ig A nephropathy is closely related to renal interstitial fibrosis(RIF).CXCL10/IP10 is one of the IFN-gamma induced proteins,belongs to CXC chemokine family.It is involved in the development of many glomeruli diseases through chemotaxis,cell growth regulation and angiogenesis inhibition.It has been reported that the CXCL10 is highly expressed in human glomerulonephritis and is involved in the pathogenesis of fibrosis in a variety of human tissues.However,its effects on Mes PGN and renal fibrosis still need further study.Objective: 1.Based on the classic animal model of mesangial proliferative glomerulonephritis,Habu nephritis model,which is established by Cxcl10-deficient(Cxcl10-/-)mice and wildtype(Cxcl10+/+)mice,we compared the pathological phenotypes of different genotypes of model mice to explore the specific role of CXCL10 in Mes PGN.2.Through in vitro and in vivo experiments,we studied the effects of CXCL10 on mouse mesangial cells,which may provide a new theoretical basis to reveal the pathogenesis of Mes PGN.3.The unilateral ureteral obstruction(UUO)model was established using Cxcl10-/-and Cxcl10+/+ mice to study the effect of CXCL10 on renal fibrosis and provide a new therapeutic target for the treatment of disease.Method: 1.Wildtype(Cxcl10+/+)mice and Cxcl10-deficient(Cxcl10-/-)mice were used to generate a murine model of Mes PGN.The mice were sacrificed at 1,3,7 and 14 days after injection.The histological changes in glomeruli were examined by PAS staining(Periodic Acid-Schiff staining),and cell proliferation was detected by PCNA immunohistochemistry staining.The levels of the expression of cell cycle regulatory proteins and ERK phosphorylation was analyzed by Western blot.2.Primary mouse renal mesangial cells(MRMCs)was stimulated by recombinant CXCL10 protein.MRMCs proliferation were detected using the EDU assay and immunofluorescence of Ki67,while MRMCs migration was detected by the wound healing assay.3.MRMCs were treated by ERK inhibitor U0126.The regulation of ERK1/2 signaling pathway on the effects of CXCL10 was evaluated by EDU assay and Western blot.4.The unilateral ureteral obstruction(UUO)model was established and the mice were sacrificed at 3,5,7 and 14 days after surgery.The histological changes and collagen deposition levels in kidney tissue was examined by PAS,Masson and Sirius red staining.The expression of fibrosis related proteins were examined by Western blot.Results: 1.A marked increase expression of Cxcl10 m RNA and protein were observed in whole kidney tissue in both Habu nephritis model and UUO model.2.Compared with Cxcl10+/+ mice,Cxcl10-/-mice expressed mitigation of extracellular matrix accumulation and cell proliferation in the mesangial proliferation stage of Habu nephritis(7 day).PCNA immunohistochemistry staining results showed that Cxcl10-/-mice expressed a lower percentage of PCNA positive cells at 7d.3.The western blot results showed that Cxcl10-/-mice exhibited a lower expression of cell cycle regulatory proteins(such as Cyclin D1,Cyclin D3,CDK2,CDK4 and CDK6)and phosphorylated ERK compared to the Cxcl10+/+ mice at 7d.4.Treatment primary mouse renal mesangial cells with different concentration of recombinant CXCL10 protein.The Edu assay and Ki67 immunofluorescence staining results indicated that CXCL10 could promote the proliferation of MRMCs.The wound healing assay results showed that CXCL10 could stimulate MRMCs migration.5.Western blot results showed that recombinant CXCL10 protein stimulation could significantly increase the level of ERK1/2 phosphorylation in MRMCs and ERK1/2 inhibitor U0126 could prevented CXCL10 induced MRMC proliferation and the activation of phosphorylated ERK.6.A mouse model of UUO based on Cxcl10+/+ mice and Cxcl10-/-mice was established successfully.The levels of serum creatinine and urea nitrogen were significantly lower in Cxcl10-/-mice than in Cxcl10+/+ mice.Pathological staining results showed that the injury degree of renal tissue and the collagen deposition levels were lighter in Cxcl10-/-mice.Western blot results showed that the expression of ?-SMA?FN and Col ? in Cxcl10-/-mice was significantly reduced compared with Cxcl10+/+ mice.Conclusion: 1.In the mesangial proliferative glomerulonephritis model,Cxcl10 knockout alleviated glomerular cell proliferation in mesangial proliferative phase by downregulating the expression of cell cycle regulatory proteins.2.CXCL10 could promote the proliferation and migration of mouse mesangial cells via ERK1/2 signaling pathway.3.In the unilateral ureteral obstruction model,Cxcl10 knockout could reduce renal injury,mitigate the deposition of collagen and inhibit renal fibrosis. |
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