| Background and purpose: Ig A nephropathy(Ig AN)is currently considered to be the most common primary chronic glomerulonephritis worldwide and is characterized by deposition of Ig A and complement C3 immunocomplexes in the glomerular mesangial region.Ig AN has obvious geographic variability and is more easier to progress in East and Southeast Asia.Previous studies have shown that 20-40% of Ig A nephropathy patients develop end-stage kidney disease within 5-25 years of diagnosis.Ig AN is an immune disease,the specific pathogenesis is not clear.There have been studies suggesting that inflammatory response may be closely related to the onset and progression of Ig A nephropathy.Bruton’s tyrosine kinase(BTK)encodes a cytoplasmic protein that belongs to the Tec family of non-receptor tyrosine kinases.It is an important kinase in the B cell antigen receptor signaling pathway and expresses all B lymphocytes and innate immune cells except T lymphocytes and plasma cells.More and more evidences show that BTK has multiple functions in monocytesand is closely related to the development of many immune diseases.It may promote end-organ damage by promoting the production of autoantibodies and mediating the inflammatory response of myeloid cells.However,the role of BTK in Ig AN has not been clarified.Macrophage is a kind of immune cells that can recruit to sites of infection or tissue damage after the local release of cytokines,chemokines,and/or other mediators.Macrophages mediate phagocytosis of pathogens through a variety of surface receptors,such as Toll-like receptors and FcγR At the same time,it presents and produces various cytokines to T cell antigens,which leads to inflammation and activates a more specific antigen response.Fc R are important regulators of immune responses,which mediate monocyte-macrophage phagocytosis is not affected by BTK inhibition or Xid mutations,and there have been reports of impaired polarization of BTK-deficient macrophages.B-cell activation and immune complex-mediated Fc have long been recognized.The importance of receptor activation in the pathogenesis of immune-mediated glomerulonephritis,and the deposition of Ig A immune complexes captured by FcγRI in the glomerulus is considered to be the main pathogenic mechanism of Ig A nephropathy.In this study,human specimens and animal models were used to investigate the mechanism of macrophage bruton tyrosine kinase activation on Ig A nephropathy.Method:Part 1: Collecting a total of 63 patients with a diagnosis of primary Ig A nephropathy confirmed by renal biopsy pathology from the Department of Nephrology,the First Affiliated Hospital of Anhui Medical University from March 1,2019 to August 30,2019.Paraffin tissue and early morning fasting blood were used as Ig A nephropathy group.PBMCs were isolated by polysucrose-diatrizoate density gradient centrifugation.PBMCs were collected from our hospital during the same period.A total of 20 cases of2 cm renal tissue adjacent to the posterior tumor were used as pathology control group;early morning fasting blood from 20 healthy volunteers at the physical examination center was selected as the clinical control group.Single and double staining methods were used for IHC method.The expression levels and localization of CD68 and BTK protein in the Ig AN group and adjacent cancer tissues were detected by IHC and WB.The expression of PBMCs phosphorylated BTK and phosphorylated NF-κB was detected in the Ig A nephropathy group.The correlation between BTK in the glomerulus and the expression of complements C3 b and C4 d in kidney tissue and the Oxford classification,Katafuchi semi-quantitative analysis and the clinical indicators of patients were compared.QRT-PCR and ELISA test TNF-ɑ,IL-1β,MCP-1 expression levels.Part 2: A total of 14 BTK knockout mice and 14 wild-type mice of the same background were selected.The model of Ig A nephropathy was jointly established by bovine serum albumin,carbon tetrachloride CCL4 and lipopolysaccharide.The mice were divided into four groups: WT group(wild-type mouse,n = 7),Ig AN group(wild-type mice modeled as Ig A nephropathy,n = 7),BTK-/-group(BTK knockout mice without any treatment,n = 7)and BTK-/-Ig AN group(BTK knockout mice modeled as Ig A nephropathy,n = 7).After 10 weeks,enter the metabolic cage one day before sacrifice to collect 24-hour urine specimens from all mice,and collect and record mouse body weight,kidney weight,blood specimens,and kidney tissue specimens.The use of immunofluorescence to examine Ig A deposition in the mesangial region and the electron dense deposition of the mesangial region under the electron microscope were considered as successful modeling.Detection of urine protein,blood urea nitrogen and other general indicators of the mouse;light microscope is used to observed kidney pathological changes;IHC single and double staining detect CD68,BTK protein expression and localization;IHC and WB methods detect p BTK and p-NF-κB p65.Using IHC,QRT-PCR and ELISA detect inflammatory cytokines levels of TNF-ɑ,IL-1β,MCP-1.Result:Part 1: IHC results revealed a significant increase in renal macrophage infiltration in Ig AN patients,single staining showed no significant positive expression in the glomeruli and tubulointerstitium of normal kidney tissues of CD68 and BTK,and CD68 and BTK of kidneys in Ig AN patients The expression of BTK was significantly increased(p<0.0001);double staining showed that CD68-positive cells expressing BTK in the glomeruli and tubulointerstitium were significantly higher than those in the normal control group(p<0.0001);phosphorylated BTK in the kidneys of patients with Ig A nephropathy was also significantly higher than those in normal kidney tissue;In Oxford classification,the expressions of CD68 and BTK of M1,E1,S1,T1-2,and C1-2were higher than those of M0,E0,S0,T0 and C0 respectively(p<0.05);CD68 and BTK expression in glomeruli and tubulointerstitium were significantly positively correlated with Katafuchi semi-quantitative glomerular score and tubulointerstitial scores(p<0.05);In the Ig AN group,patients with positive expression of complement C3 b and C4 d in renal tissues had significantly increased glomerular BTK expression(p<0.05);glomerular BTK expression was positively correlated with 24-hour urine protein and serum creatinine in clinical indicators(p<0.05),negatively correlated with serum albumin(p<0.05);IHC,WB,QRT-PCR and ELISA results suggest that p-NF-κB p65,TNF-ɑ,IL-1β and MCP-1 were significantly increased(p<0.05)in Ig AN patients renal tisues and PBMCs than control group.Part 2: There was no significant difference in kidney weight / weight ratio of mice in each group;blood urea nitrogen and 24-hour urine protein levels were significantly higher in the Ig AN group than in the WT group,and in the BTK-/-Ig AN group was significantly higher compared to BTK-/-group and was significantly relived than the Ig AN group(p<0.05).The Katafuchi semi-quantitative scores of the WT group and the BTK-/-group were not significantly different,tubulointerstitial scores and vascular scores were significantly increased(p<0.05),and the above indicators were significantly higher in BTK-/-Ig AN mice than in the BTK-/-group under the same state and were significantly lower than in the Ig AN group(p<0.05).Immunohistochemical single staining showed that the expression of CD68 and BTK in the glomeruli and tubulointerstitium of the Ig AN group was significantly increased compared with the mice in the WT group(p<0.05),and the expression of CD68 in BTK-/-Ig AN was ignificantly increased compared with BTK-/-group(p<0.05);double staining showed that BTK-positive CD68-positive cells in the glomeruli and tubulointerstitium of the Ig AN group were significantly higher than those in the WT group(p<0.05).There have no BTK-positive CD68-positive cells in the BTK-/-and BTK-/-Ig AN group.The activation of phosphorylated BTK in the glomeruli and tubulointerstitium of mice in Ig AN group was more significant elevated than in WT group(p<0.05),there was no expression in the kidneys of mice in the BTK-/-and BTK-/-Ig AN group;the expression of glomerular BTK was positively correlated with 24-hour urine protein and blood urea nitrogen in clinical indicators(p<0.05);the results of IHC,WB,and QRT-PCR suggested inflammation indexes in the mice of the Ig AN group were significantly increased.The expression of p-NF-κB p65,TNF-ɑ,IL-1β and MCP-1 in the kidney tissues of mice in the Ig AN group was significantly higher than that in the WT group,while the above inflammatory factors in the kidneys of the BTK-/-Ig AN group was significantly increased in the BTK-/-group and decreased significantly in the Ig AN group(p<0.05).Conclusion:In patients with Ig A nephropathy,the expression of BTK in macrophages is significantly increased and phosphorylated BTK is activated;The BTK levels in macrophages is related to Oxford classification and Katafuchi semi-quantitative scores of pathological indicators and complement C3 b,C4d.Patients with severe pathological manifestation and complement-positive expression have more pronounced BTK expression;glomerular BTK in macrophage expression is correlated with urine protein,serum creatinine and albumin.BTK may mediate progression of inflammation in Ig A nephropathy by activating the NF-kB signaling pathway and lead to the secretion of inflammatory factors.In animal experiments,blood urea nitrogen and urine protein levels of Ig AN mice increased significantly,mesangial cells and mesangial matrix were significantly proliferated,renal tubularinterstitial developed atrophy and inflammatory cells infiltration and arterial hyaline degeneration.BTK knockout Ig AN mice showed significant improvements of above manifestations.Ig AN mice found that phosphorylated BTK was activated and activated downstream NF-κB signaling pathways,inflammatory factors of TNF-ɑ,IL-1β and MCP-1 were significantly increased.The levels of p-NF-κB p65 and inflammatory factors in the kidneys of BTK knockout Ig AN mice were significantly reduced,suggesting that macrophage BTK activation may be involved in the development of renal inflammation in Ig A nephropathy. |
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