Signaling Mechanisms Involved In Angiotensin II-induced Mesangial Cell Proliferation And Inflammatory Mediators Expression: Role Of ROS/EGFR/JNK/AP-1 Pathway

Chronic kidney disease(CKD)is an emerging top public health problem affecting 1,300,000,000 Chinese.CKD arises from a wide variety of etiologies and develops into a common manifestation of end-stage renal disease characterized by glomerulosclerosis and tubulointerstitial fibrosis and the complete loss of renal function,which require dialysis or kidney transplantation to maintain life.The use of inhibitors of the components of the renin-angiotensin axis,namely, angiotensin-converting enzyme inhibitors(ACEi)and angiotensin type 1(AT1) receptor antagonists(ARBs),has become a standard clinical practice for treatment of CKD.Despite intensive investigation,the mechanism of the renal beneficial effects of ACEi and ARBs is incompletely understood.It is clear,however,that these effects of ACEi and ARBs cannot be entirely attributed to their hemodynamic effects.Consistent with this notion,Angiotensinâ…¡(Angâ…¡)exerts a direct detrimental effect in the renal cells.In particular,Angâ…¡stimulates activation of mesangial cells(MCs),a hallmark of the pathological changes in CKD.Therefore,it is imperative to understand the signaling mechanism of the Angâ…¡-elicited activation-promoting effect in the MCs.The elucidation the signaling pathway involved in Angâ…¡-induced mesangial cells activation provides insights into the mechanisms of CKD and may also help identify new targets for treatment of CKD.The present study consists two parts:Partâ… :c-Jun NH₂-terminal kinase mediation of angiotensinâ…¡-induced proliferation and inflammatory mediators expression in human mesangial cells Objective:The octapeptide hormone Angâ…¡has been shown to activate c-Jun NH2-terminal kinase(JNK)in cultured mesangial cells,but the functional implication of this phenomenon remains to be determined,largely due to the lack of an effective approach to block JNK.Therefore,the present study was carried out to examine whether JNK is involved in Angâ…¡-induced cell proliferation and expression of inflammatory mediators,such as monocyte chemoattractant protein-1 (MCP-1),transforming growth factor-β(TGF-β)and fibronectin(FN),in cultured human mesangial cells(HMCs)with the use of a newly developed JNK-selective blocker,SP-600125.Methods:Normal-appearing portions of human kidneys that were surgically removed for renal carcinoma were used to culture MCs from outgrowths of collagenase-treated glomeruli.The incorporation of ³H-thymidine(³H-TdR)and cell count were used as the measure of mesangial cell proliferation.MCP-1 mRNA expression was determined by ribonuclease protection assay.MCP-1,TGF-β1,and FN excretion was examined by ELISA.JNK and c-Jun phosphorylation was detected by Western Blot.Transient luciferase reporter assay and electrophoretic mobility shift assay(EMSA)were used to detect the transcription activity of c-Jun and the activity of AP-1.Results:1.The increase in JNK activity and c-Jun phosphorylation in HMCs was detected at 15 minutes after the addition of Angâ…¡.Maximal activity occurred at 30 minutes(6.2-fold increase)and reducing thereafter.Angâ…¡activated JNK in a dose-dependent manner,with a maximal stimulation seen at 100 nmol/L.2. SP600125 countered the Angâ…¡-induced Ser63 phosphorylation of c-Jun in a dose-dependent manner.The reduction in JNK activity was 75%at 10μmol/L concentration and 90%at 20μmol/L concentration.SP600125 had no significant effect on the activation of ERK1/2 and p38 MAPK.3.Angâ…¡-induced transcriptional activity of the c-Jun was inhibited by SP600125.Nuclear extract from HMCs treated with Angâ…¡showed increased binding to the AP-1 motif in a time-dependent manner.SP600125 effectively countered the increased AP-1 binding in a dose-dependent manner.4.Angâ…¡strongly increased MCP-1 promoter activity and MCP-1 mRNA expression in a time-dependant manner.SP600125 had a concentration-dependent inhibitory effect on Angâ…¡-induced MCP-1 promoter activity and mRNA expression at a concentration range of 1 to 20μmol/L. SP600125 inhibited Angâ…¡-induced MCP-1,TGF-βand FN production in a concentration-dependent manner.5.SP600125(1 20μmol/L)inhibited Angâ…¡-induced mesangial cell proliferation as determined by ³H-thymidine incorporation and cell count in a concentration-dependent manner,the incorporation was decreased by 1,5,10,and 20μmol/L of SP600125,respectively.Conclusion:These results show that the JNK/AP-1 pathway is involved in the cell proliferation,MCP-1 and TGF-βexpression,and ECM production and JNK is an important therapeutic target for glomerulonephritis and glomerulosclerosis.Partâ…¡:NADPH oxidase-derived ROS mediated Angiotensinâ…¡-induced Human Mesangial Cells ProliferationObjective:We have previously shown that Angâ…¡induces mesangial cell proliferation via JNK/AP-1 pathway.The present study attempted to determine the upstream mediators of the JNK activation with emphasis on reactive oxygen species(ROS).Methods:The incorporation of ³H-thymidine(³H-TdR)and cell count were used as the measure of mesangial cell proliferation.ROS production was determined by DCFDA fluorescence.Nicotinamide adenine dinucleotide phosphate(NADPH) oxidase activity was examined by lucigenin chemiluminescence.JNK activation was assayed by Western Blot.Results:1.In cultured human mesangial cells,Angâ…¡time-dependently and dose-dependently increased intracellular ROS production as early as 3 min and peak at 60 min.Incubation with different dose of Angâ…¡(1,10,100 nmol/L Angâ…¡) for 60 min,ROS production increased for 1.82-,2.92-,and 4.08-fold,respectively. 2.Angâ…¡-induced ROS generation was sensitive to diphenyleneiodonium sulfate (DPI)(10μmol/L)and apocynin(500μmol/L),two structurally distinct NADPH oxidase inhibitors.In contrast,inhibitors of other oxidant-producing enzymes, including the mitochondrial complexâ… inhibitor rotenone,the xanthine oxidase inhibitor allopurinol,the cyclooxygenase inhibitor indomethacin,the lipoxygenase inhibitor nordihydroguiaretic acid,the cytochrome P450 oxygenase inhibitor ketoconazole and the nitric oxide synthase inhibitor G-nitro-L-arginine methyl ester were without an effect.3.Angâ…¡-induced ROS generation was inhibited by the AT1 antagonist losartan(10μmol/L)but not the AT2 antagonist PD 123319(10μmol/L).4.Angâ…¡treatment induced translocation of cytosolic of p47phox and p67phox to the membrane.The antioxidants almost abolished Angâ…¡-mitogenic response,associated with a remarkable blockade of the activation of JNK(83% inhibition).Conclusions:NADPH oxidase-derived ROS involved in Angâ…¡-induced JNK/AP-1 activation and mesangial cell proliferation.