| Background:Ig A Nephropathy(Ig AN)is the most common primary glomerular disease worldwide.The main pathological changes are the deposition of Galactose deficient Ig A1(Gd-Ig A1)in the mesangial region of the glomerulus,accompanied by the deposition of complement C3,Ig G/Ig M.Although environmental factors such as immunity are involved in the development of Ig AN,the exact factors are not fully understood.Previous studies on glomerular diseases have focused more on mesangial cells and podocytes,and endothelial cell damage is also involved in the progression of many glomerular diseases.Studies have shown that glomerular endothelial cell(GEC)damage is closely related to the progression of Ig A nephropathy.Endothelial cells form a semi-permeable membrane barrier between them through adherens junctions(AJs),tight junction(TJs)and a series of adhesion molecules.Vascular endothelial-cadherin(VE-cad)is a key transmembrane component of endothelial adhesion and is only expressed in vascular endothelial cells.Many inflammatory mediators such as Vascular Endothelial growth factor(VEGF),lipopolysaccharide(LPS),tumor necrosis factorα(TNF-α),regulates endothelial permeability by altering the activity of the Rho family of small GTP enzymes.Ig A nephropathy often develops secondary to mucosal infection,which leads to elevated interleukin 6(IL-6)levels locally or throughout the body.The inflammatory cytokine IL-6 is involved in the occurrence and development of various diseases,as well as the pathogenesis of Ig AN.In Ig AN patients,urinary IL-6 concentration correlates with renal function and predicts long-term renal outcome.However,whether IL-6 is related to glomerular endothelial injury in Ig AN,whether it affects the expression of VE-cad in GEC,whether it increases the permeability of GEC,and its possible molecular mechanism need to be further studied.Objective: To investigate whether there was an increase in glomerular endothelial cell permeability in Ig AN rats,and to observe the effect of IL-6 on the expression level of VE-cad in HRGECs.To explore the possible mechanism of IL-6 affecting GEC permeability,and to provide a theoretical basis for the treatment of Ig A nephropathy.Methods:1.Ig AN rat model was established,and IL-6 was used to stimulate HRGEC to observe its effect on cell permeability.(1)Establishment of Ig AN rat model.Twenty SPF 6-week-old male SD rats were divided into two groups: normal group(N,N = 10)Ig AN group(M,N = 10).Ig AN model was established by “BSA + CCl4 + LPS” method.At the 1st,3rd,6th and 8th week,24 h urine was collected by metabolic cage method under fasting and free drinking water condition,and urine erythrocyte count and urine protein were determined.The rats in both groups were sacrificed at the end of 8 weeks.(2)3ml of blood was taken from abdominal aorta,and the serum was separated and preserved.Serum IL-6 levels were determined by enzyme-Linked immunosorbent assay(ELISA).(3)The kidneys were divided in the direction perpendicular to the long axis,and the amount of Ig A deposition in the glomerulus was detected by direct immunofluorescence method to confirm the successful preparation of Ig AN model.The morphology of GEC was determined by electron microscopy after fixation with 2% glutaraldehyde.The expression of VE-cad in glomerulus was detected by immunofluorescence(IF).The protein expressions of VE-cad,and IL-6 in renal tissues were determined by Western blotting(Wb).(4)Cell proliferation and toxicity detection.The experimental concentrations of IL-6,IL-6R and tocilizumab(TCZ)were determined by cell Counting Kit-8(CCK-8).(5)HRGEC cells were cultured in the Transwell culture plate for 14 days,and the time to establish the monolayer cell model was determined by detecting transepithelial electrical Resistance(TEER).Cells were stimulated as follows: A: 90%DMEM-H drug solvent and 10% FBS medium were used as negative control;B: IL-6 stimulation alone;C: TCZ stimulation;D: IL-6 combined with 15-fold IL-6R stimulation;E:TCZ combined with IL-6 and IL-6R stimulation.TEER value was measured after treated for 24 h and48h.(6)Monolayer cell models were prepared on Transwell culture plates and stimulated with the above-mentioned drugs A,B,C,D and E for 24 and 48 h,respectively.The permeability of fluorescein isothiocyanate-dextran(FITC-dextran)in the single layer model was determined.2.HRGEC cells were stimulated with IL-6 in vitro to observe the effects of IL-6 on HRGEC cell adhesion molecules and related proteins.(1)The expressions of IL-6R and gp130 in HRGECs were determined by Western blotting(Wb).(2)Wb IF method was used to detect the expression changes of HRGEC cell adhesion protein VE-cad,claudin-5,occludin after different drug treatments,and to observe the influence of TCZ pretreatment on the expression of these proteins after IL-6stimulation.(3)The changes of β-catenin phosphorylation level in Ig A nephropathy renal tissues and HRGEC cells treated with different drugs and the effect of TCZ pretreatment were detected by Wb method.(4)Effect of sgp130 Fc blocking IL-6 trans signaling pathway on VE-cad expression.3.Pathway validation of IL-6 affecting VE-cad expression in HRGECs.(1)HRGECs were blocked by Rac1 inhibitor(NSC23766),Src inhibitor(PP2)and IL-6 trans-signaling pathway blocker(sgp130Fc),and the changes of upstream and downstream proteins were detected.(2)Sh RNA transfected with lentivirus silenced Vav2 and Tiam-1 respectively,and the changes of IL-6 stimulated HRGECs were observed.(3)Wb method was used to detect the expression changes of HRGECs adhesive proteins VE-cad,claudin-5,occludin,Src,Rac1,Tiam-1,Vav2 and other indicators after different drug treatments.(4)The level of active Rac1 in renal tissue was assessed using a pull-down assay kit.Results:1.In the Ig AN rat model,glomerular endothelial cells were damaged and IL-6levels increased,and IL-6/IL-6R could increase the permeability of HRGECs and reduce the cell TEER.(1)Electron microscopy and IF analysis showed that GEC damage was found in Ig AN rats.Wb analysis showed that the level of VE-cad protein in Ig AN group was significantly lower than that in normal group(p < 0.01).(2)Wb results showed that IL-6 level in renal tissue of Ig AN group was significantly higher than that in normal group(p < 0.01).Changes in serum IL-6 levels detected by ELISA were consistent with changes in renal tissue(p < 0.05).(3)CCK-8 showed that TCZ had no effect on the proliferation of HRGECs,and IL-6 had a weak effect on the proliferation of HRGEC cells.When IL-6/IL-6R(1:15)was combined to stimulate HRGECs for 48 hours,IL-6 concentration was greater than 75ng/m L and the cell proliferation was significantly inhibited(p < 0.01).IL-6 100 ng/m L,IL-6R 1500 ng/m L and TCZ 2000 g/m L were selected to stimulate HRGECs according to A,B,C,D and E in subsequent experiments.(4)IL-6 or IL-6/IL-6R stimulation significantly reduced TEER compared to the control group,with no significant difference between the two groups(p > 0.05),TCZ pretreatment can alleviate the decrease of IL-6/IL-6R on cell TEER,playing a therapeutic role.FITC-dextran transendothelial permeability test showed that IL-6 or IL-6/IL-6R stimulation significantly increased HRGECs permeability,and the increase was more significant in the IL-6/IL-6R group than in the IL-6 group(p < 0.05),TCZ treatment significantly reduced the increase of IL-6/IL-6R permeability to HRGECs.2.Il-6 /IL-6R decreased the expression of VE-cad,occludin,and claudin-5 in HRGECs,and increased the phosphorylation of β-catenin at Y654 and Y142.(1)Wb results showed that IL-6 or IL-6/IL-6R significantly reduced the expression of VE-cad,claudin-5 and occludin between HRGEC(p < 0.05),the above effects of IL-6/IL-6R were significantly inhibited in TCZ-pretreated HRGECs(p <0.05).IF showed that IL-6/IL-6R stimulated HRGECs significantly reduced the expression of VE-cad,claudin-5 and occludin,and TCZ reversed the decline of adhesion proteins induced by IL-6/IL-6R to a certain extent.(2)Wb detection showed that there was no significant difference in the expression of β-cateinin protein in kidneys between normal and Ig AN groups.The phosphorylation of β-cateinin at Y654 Y142 in Ig AN group was significantly increased(p < 0.01).(3)Phosphorylation of β-catenin at Y654 and Y142 in HRGECs was significantly increased after IL-6 or IL-6/IL-6R stimulation compared to the control group(p < 0.01).The phosphorylation of β-catenin at Y654 and Y142 was significantly decreased in the IL-6/IL-6R/TCZ group compared with the IL-6/IL-6R group(p < 0.01).(4)After sgp130 Fc blocked the IL-6 trans-signaling pathway,IL-6/IL-6R stimulated HRGECs to induce VE-cad decline was significantly reduced(p < 0.01).3.IL-6 exerts a pro-inflammatory effect on HRGECs via the trans-signaling pathway,and reduces the expression of VE-cad in HRGECs through the Src/Vav2/Rac1 signaling pathway.(1)Il-6 affects HRGECs adhesion molecule expression by activating Rac1.Rac1 blocker NSC23766 significantly reduced IL-6/IL-6R-induced HRGECs VE-cad decline,and the phosphorylation level of beta-catenin at Y654 and Y142 was significantly decreased(p < 0.01).Rac1-GTP levels were also significantly decreased after the application of the blocker NSC23766(p < 0.01).(2)Vav2 is the main target requried by IL-6 to stimulate Rac1 activation and down-regulate VE-cad.After Vav2 silencing,IL-6/IL-6R was used to stimulate HRGECs,and the decrease of VE-cad expression was significantly alleviated.Pull-down experiment showed that the increase of Rac1-GTP was significantly alleviated.(3)Src is the upstream target of IL-6 down-regulation of VE-cad expression via Vav2 and Tiam-1.After PP2 blocked Src activity,the decrease in VE-cad expression induced by IL-6/IL-6R stimulation was significantly alleviated(p < 0.01).Meanwhile,PP2 can block IL-6 /IL-6R induced p-Vav2,Tiam-1 elevation(p < 0.01).(4)IL-6 induces Src phosphorylation through the trans-signaling pathway.The phosphorylation of Src induced by IL-6/IL-6R and the expression of VE-cad were significantly reduced by sgp130 Fc blocking the trans-signal pathway(p < 0.01).At the same time,PP2 can block the elevation of p-vav2 and Tiam-1 induced by IL-6/IL-6R stimulation of HRGEC(p < 0.01).Conclusions:1.IL-6 was elevated in Ig AN rat glomerular endothelial cell injury model.IL-6/IL-6R can significantly increase the permeability of HRGECs and reduce cell TEER.TCZ can significantly reduce the effect of IL-6/IL-6R on HRGECs’ permeability.2.IL-6/IL-6R can reduce the expression of VE-cad,occludin,and claudin-5 in HRGECs,which may change the permeability of HRGECs by increasing the phosphorylation of β-catenin or the expression of adhesive proteins.3.TCZ significantly reduced the effect of IL-6/IL-6R on increasing HRGECs’ permeability,reduced the expression of VE-cad,occludin and claudin-5,and reduced the phosphorylation level of β-catenin.4.IL-6 plays a pro-inflammatory role in HRGECs via the trans-signaling pathway,leading to the decreased expression of VE-cad.IL-6 decreased the expression of VE-cad in HRGECs through the Src/Vav2/Rac1 signaling pathway.This finding reveals the molecular mechanism by which IL-6 induces increased permeability of GEC and provides a theoretical basis for the development of new therapies for kidney disease. |
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