| Background and objective: Diabetic nephropathy(DN)is one of the main causes of end-stage renal disease(ESRD).It has been found that inflammatory immune response plays a vital role in the development of DN.Macrophages are a common type of immune inflammatory cells,whose infiltration and activation can be regarded as a crucial connection in the development of DN.Macrophages can release inflammatory mediators to produce cells and tissues injury after activation,but whether there are other mechanisms to aggravate renal injury has attracted great attention in recent years.The pathogenesis of diabetic nephropathy includes inflammation,fibrosis,and so on.The means how to induce inflammation,and which pathway involved in is the problem we need to be concentrated on.Bruton’s tyrosine kinase(BTK)is a member of the non receptor tyrosine kinase Tec family,which participated in mediating signaling pathways of human and animals.It plays an important role in the proliferation,differentiation and apoptosis of cells.Nucleoside binding oligomerization domain like receptor protein 3(NLRP3)inflammasome including NLRP3,Apoptosis-associated speck-like protein containing a caspase recruitment domain(ASC)and cysteine aspartic acid specific protease-1(caspase-1),NLRP3 recruited ASC and activated the precursor of caspase-1.Recent studies have shown that NLRP3 alone or in coordination with BTK can aggravate the inflammatory response.In this experiment,we will discuss how NLRP3 inflammasome mediated by BTK regulate DN inflammatory response and its mechanism from three perspectives: DN patients,BTK knockout mice and bone marrow-derived macrophages(BMMs),which provides a new direction and strategy for DN treatment.Methods: Part 1: Activation of macrophages and NLRP3 inflammasomes in kidney of patients with type 2 diabetic nephropathy.We collected a total of 16 patients with diabetic nephropathy from January 2017 to December 2019,who were from the Department of Nephrology of the First Affiliated Hospital of Anhui Medical University.The clinical data of the patients were analyzed.At the same time,we collected 10 cases of renal tissue over 2cm away from the pathological changes of renal tumors in urology department of our hospital as the control group,and the morning fasting blood of normal subjects in physical examination center of our hospital was collected as the control group.Serum samples from patients with diabetic nephropathy(DN group)and control group(control group)were collected.The levels of IL-1β,TNF-α and MCP-1 were measured by ELISA.The expression of CD68 and Btk in renal tissues of DN group and control group were detected by immunohistochemistry.CD68 was co-expressed with inducible nitric oxide synthase(i NOS),BTK,p-BTK and NLRP3 in DN and control groups by immunofluorescence.Part 2: The effect of Bruton tyrosine kinase knockout and inhibitor application on the activation of macrophage inflammasomes stimulated by high glucoseFlow cytometry results showed that F4/80 and CD11 b double-positive cells accounted for 93.88% of the total cells,which met the experimental requirements of purity and maturity.Optimization of experimental conditions: CCK-8 results showed that inhibitor concentration at 10-8 to 10-5 mmol/L had no effect on cell activity,Western blot results showed that the optimal inhibitor concentration was 10-6 mmol/L,and the optimal high glucose stimulation time was 12 h.Transwell results showed that the migration function of macrophages increased under high glucose stimulation,while the cell migration ability of HG+BTK-/-group and HG+PCI-32765 group was significantly weaker than that of HG group.ELISA results showed that the inflammatory factors MCP-1,TNF-α and IL-1β in the supernatant of cell culture medium of HG group were significantly higher than those of C group(P<0.05),there was no significant difference between C group and M group(P>0.05),and HG+BTK-/-group and HG+PCI-32765 group were significantly lower than those of HG group(P<0.05).The results of PCR showed that the expression of MCP-1,TNF-α and IL-1β in HG group was significantly higher than that in C group(P < 0.05),while the expression of MCP-1,TNF-α and IL-1β in HG + BTK-/-group and HG + PCI-32765 group was significantly lower than that in HG group(P < 0.05).Laser confocal microscope results showed that i NOS expression was significantly increased in HG group compared with C group(P<0.05),while it was significantly decreased in HG+BTK-/-group and HG+PCI-32765 group(P<0.05),and immunofluorescence results showed that NLRP3 inflammasome activation was significantly increased in HG group compared with C group(P < 0.05),while it was significantly decreased in HG+BTK-/-group and HG+PCI-32765 group(P < 0.05).Laser confocal microscope results showed that NLRP3 and ASC were expressed in the same location in cells,and the expression in HG group was higher than that in C group(P<0.05),while the expression in HG+BTK-/-group and HG+PCI-32765 group was lower than that in HG group(P<0.05).Co-immunoprecipitation results showed that NLRP3 could co-interact with ASC.Western blot showed that the expression of NLRP3,ASC,pro-caspase-1,caspase-1p20 protein in HG group was significantly higher than that in C group(P<0.05),while the expression in HG+BTK-/-group and HG+PCI-32765 group was lower than that in HG group(P<0.05).Part 3: The protective effect and mechanism of Bruton tyrosine kinase knockout on renal tissue injury in diabetic mice by reducing the activation of NLRP3 inflammasomeThe animals were divided into wild C57BL/6J mice group(WT),diabetes model group(STZ),BTK knockout control group(BTK-/-),and BTK knockout diabetes model group(BTK-/-+ STZ),with 8 animals in each group.The method of model establishment was to inject streptozocin(STZ)intraperitoneally into mice,the dose of each mouse was 50 mg/kg,the same volume of sodium citrate solution was injected into the control group,and the continuous injection was carried out within 5 days.One week later,the random blood glucose was detected.The blood glucose value ≥ 16.7 mmol/l was regarded as the success of model establishment.The blood,urine and kidney tissue samples were collected after 12 weeks of routine feeding.The ratio of kidney weight to body weight,blood glucose and 24-hour urinary albumin excretion rate were measured.PAS staining was used to observe the pathological changes of renal tissues of mice in each group;immunohistochemistry staining was used to observe the expression of macrophage markers F4/80,podocyte markers WT1 and nephrin in renal tissues of mice in each group;transmission electron microscopy was used to observe the ultrastructures of renal tissues of mice in each group,including the pathological changes of podocyte,foot process,mesangial area and basement membrane,and the thickness of basement membrane was measured;The expression of p-Btk,Btk,IL-1β,TNF-α and MCP-1 in renal tissue of mice in each group was detected by western blot;the expression of IL-1β,TNF-α and MCP-1 m RNA in renal tissue of mice in each group was detected by real-time quantitative PCR;the expression of NLRP3,ASC and caspase-1 in renal tissue of mice in each group was detected by Western blot.Results: Part 1: Activation of macrophages and NLRP3 inflammasomes in kidney of patients with type 2 diabetic nephropathy.The results of ELISA showed that the levels of IL-1β,TNF-α and MCP-1 in DN group were significantly higher than those in control group(P < 0.05);the expression of CD68 and BTK in DN group was significantly higher than that in control group(P < 0.05);the expression of i NOS,BTK,p-BTK and NLRP3 in DN group was significantly higher than that in control group(P < 0.05).The above results suggest that the level of inflammatory factors and macrophage expression in the serum of DN patients increased.The expression of BTK,p-BTK and NLRP3 in renal macrophages increased.Part 2: The effect of Bruton tyrosine kinase knockout and inhibitor application on the activation of macrophage inflammasomes stimulated by high glucoseFlow cytometry results showed that the double positive cells of F4/80 and CD11 b accounted for 93.88% of the total cell number,which was in line with the purity and maturity of the experimental requirements.Experimental conditions were optimized: CCK-8 results showed that the concentration of the inhibitor had no effect on cell activity at 10-8 to 10-5 mmol/L,while Western blot results showed that the optimal concentration of the inhibitor was 10-6 mmol/L and the optimal time for high-glucose stimulation was 12 h.Transwell results showed that the migration function of macrophages increased under high glucose stimulation,while the migration ability of cells in HG + BTK-/-group and HG + PCI-32765 group was significantly decreased compared with that in HG group.ELISA results showed that IL-1β,TNF-α and MCP-1 levels of inflammatory factors in the cell supernatant of HG group were significantly higher than those in group C(P<0.05),while there was no significant difference between group C and group M(P>0.05),while HG + BTK-/-group and HG + PCI-32765 group were significantly lower than those in group HG(P<0.05).PCR results showed that m RNA levels of IL-1β,TNF-α and MCP-1 patients in HG group were significantly higher than those in C group(P<0.05),while those in HG+BTK-/-group and HG + PCI-32765 group were significantly lower than those in HG group(P<0.05).The results showed that iNOS expression in HG group increased significantly compared with that in group C(P<0.05),and decreased significantly in group HG+BTK-/-and HG + PCI-32765(P<0.05).Meanwhile,immunofluorescence results indicated that the activation of NLRP3 inflammasomes increased significantly in group HG compared with group C(P<0.05),while decreased significantly in group HG + BTK-/-and HG + PCI-32765(P<0.05).The results of laser confocal microscope indicated that the expression of NLRP3 and ASC was consistent in cells,and the expression of HG group was increased compared with that of group C(P<0.05),while the expression of HG + BTK-/-group and HG + PCI-32765 group was decreased compared with that of group HG(P<0.05).The results of co-immunoprecipitation showed that NLRP3 co-acted with ASC.Western blot results showed that the protein expressions of NLRP3,ASC,pro-caspase-1 and caspase-1p20 in HG group were significantly higher than those in C group(P<0.05),while those in HG + BTK-/-group and HG + PCI-32765 group were significantly lower than those in HG group(P<0.05).Part 3: The protective effect and mechanism of Bruton tyrosine kinase knockout on renal tissue injury in diabetic mice by reducing the activation of NLRP3 inflammasomeThe blood glucose,kidney-body weight ratio and 24-hour urinary protein excretion rate of mice in STZ group were significantly higher than those in WT group(P<0.05);the related indexes of mice in BTK-/-+STZ group were decreased compared with those in STZ group(P<0.05).PAS staining results showed that mesangial expansion and tubulointerstitial injury were increased in STZ group compared with WT group,and the mesangial expansion index was increased(P <0.05),while the pathological changes in BTK-/-+STZ group were significantly alleviated(P<0.05).Immunohistochemical results showed that the expression of F4/80 was increased in STZ group compared with WT group,and the expression of podocyte markers WT1 and Nephrine was decreased with statistical difference(P<0.05),while the expression of F4/80 was decreased in BTK-/-+STZ group compared with STZ group,and the expression of WT1 and Nephrine was increased with statistical difference(P<0.05).The results of electron microscopy showed that the basement membrane thickness was widened(P < 0.05),mesangial cells and matrix proliferated,foot processes partially fused,and podocyte number decreased in STZ group compared with WT group,while the basement membrane thickness was thinner in BTK-/-+STZ group compared with STZ group(P < 0.05),mesangial cells and matrix proliferation decreased,podocyte number increased,and podocyte fusion alleviated.The protein expression of BTK,p-BTK,i NOS and inflammatory factors IL-1β,TNF-α,MCP-1 in STZ group was significantly increased compared with WT group(P<0.05),while the expression decreased after BTK knockout(P<0.05).Real-time quantitative PCR results showed that the expression of inflammatory factors TNF-α,MCP-1,IL-1β m RNA in renal tissue was significantly increased in STZ group compared with WT group(P<0.05),while the expression was significantly decreased after BTK knockout(P<0.05).Western blot showed that the relative expression of inflammasome-associated protein NLRP3,ASC and caspase-1 in STZ group was significantly higher than that in WT group(P < 0.05),while the expression was significantly decreased after BTK gene knockout(P < 0.05).Conclusion: 1.Type 2 diabetic nephropathy patients with elevated inflammatory markers in serum,macrophage infiltration was found in the kidney tissues and BTK expression increased,further study found within macrophages iNOS,BTK,P-BTK and NLRP3 expression increased,DN patients in microcirculation inflammatory state,macrophage infiltration was found in the kidney tissues increased,activation of inflammasomes increased.2.Under high glucose stimulation,macrophages can undergo activation,and the expressions of inflammatory cytokines TNF-α,MCP-1 and IL-1β are significantly increased.In terms of mechanism of action,high glucose can increase the protein expression of BTK,NLRP3,ASC and Caspase-1 in macrophages,while the inflammation is alleviated after BTK inhibition and knockout,and the protein expression level related to the inflammasome signaling pathway is reduced,suggesting that BTK can regulate the inflammatory effect of macrophages through the NLRP3-ASC-Caspase1 axis.3.Protein expressions of P-BTK,iNOS,NLRP3,ASC,Caspase1,TNF-α,MCP-1 and IL-1β in renal tissues of diabetic mice were significantly increased,and relative m RNA expression levels of inflammatory factors were increased.After BTK gene knockout,the urine protein of mice decreased,the macrophage infiltration and podocyte injury in renal tissue decreased,and the inflammation in renal tissue decreased,suggesting that BTK may be involved in the inflammatory effect of diabetic mouse kidney tissue.Further studies showed that protein expressions of NLRP3,ASC and caspase1 in renal tissues were decreased,suggesting that BTK could mediate renal inflammation in diabetic mice through the NLRP3-ASC-Caspase1 axis,and that BTK knockout could protect diabetic kidney injury in mice. |
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