| ObjectivesDiabetes Mellitus(DM) and its complications are diseases which severely jeopardize health and quality of life. Diabetic nephropathy(DN) is one of the most severe complications of DM and is also an important cause of death. The structural remodeling and excessive deposition of extracellular matrix(ECM) in the glomeruli eventually leads to the renal failure. At present, it is believed that synthesis and degradation of ECM by various cytokines (such as TGF-β1, c-fos, inflammatory factors,ect) and several signaling pathways (such as the smad and MAPK pathway,ect), is a complex pathological process.Collagen IV is the main component of ECM, and a major indicator of the ECM accumulation. TGF-β1is a member of TGF-β1super family. Smads family are delineated as the classic signal transducers of the downstream of TGF-β1. C-fos is a important proto-oncogene in nucleus, and mediate many cytokines gene transcription and activation in immune responses, also enhance the immune damage. In addition, DN is a inflammatory disease. Being important inflammatory mediators, interleukin-6(IL-6) and intercellular adhesion molecule-1(ICAM-1) participate in many pathophysiologic processes and have significant function in the development of DN.G protein coupled receptor(GPCR) is a transmembrane receptor protein, which is involved in α、β1、AT1receptor. A lot of study have demonstrated that renal damage associated with GPCR autoantibody, and using the corresponding receptor antagonist can have kidney protection effect in a certain degree.Most research on the relationship between renal damage in hypertension and GPCR autoantibodies, while study on the relationship between DN leads to renal damage and GPCR autoantibodies were relatively less.Doxazosin is α1receptor blockers, to be able to highly selective irreversible binds to α1receptors, which is widely used in the treatment of hypertension in clinic. Our team has been confirmed in previous animal experiments that Doxazosin and α1-receptor autoantibody (autoantibodies against α1-adrenergic receptor, andα1-AA) competitive combination on the α1-receptor, blocking the high expression of TGF-β1and related signal transduction pathways caused by α1-AA injected into mice, and eventually block the renal matrix reconstruction.But its role in the DN mechanism are still not fully elucidated.In the present study DN rats model was established, adopt the method of injected α1-AA for mediating in rats, and continue to delve into the α1-AA mediated kidney tissues of DN rats of collagen Ⅳ, Smad2/3, c-fos protein expression and the influence of the level of serum IL-6, ICAM-1,and application of Doxazos in renal protective effects and mechanisms. Hope to lay the foundation for further research of the pathogenesis of DN.Methods Part one:α1-AA mediated in diabetic rat kidney tissues of collagen IV, Smad2/3, c-fos expression and the expression of inflammatory cytokinesRats were divided into normal group(N group), DM rats without α1-AA mediated group (DM group), DM rats α1-AA mediated group (DM+α1-AA group).STZ(30mg/kg) was injected by intraperitoneal injection. After the success of making model, the DM rats α1-AA mediated group were syringe α1-AA by rats’tails vein at0,4,8,12,16week, l00μg/100g by weight. At week16, the rats were sacrificed, the24hours urinary protein count, serum creatinine were measured. Renal and ultrasturctural pathological changs were examined. Renal tissue collagen IV, Smad2/3and c-fos expression were measured by immunohistochemical assay. Serum α1-AA, IL-6and ICAM-1were measured by ELISA.Part two:The effects of α1-AA on the expression of collagen IV, Smad2/3and c-fos inhibitted by Doxazosin in renal cortex of DM ratsThe established of DM animal model and α1-AA mediated method were the same with Part one. After the success of making model, rats were delivered into the group of DM rats non-mediated group, DM rats α1-AA mediated group, DM rats both α1-AA mediated and Doxazosin intervention group and DM rats Doxazosin intervention group. The DM rats both α1-AA mediated and the Doxazosin intervention group and DM rats Doxazosin intervention group were giving0.36mg/kg dosage,10ml/kg per rat capacity through intragastric administration.1/d, successive16weeks. After the rats were sacrificed, the24hours urinary protein count, serum creatinine were measured. Renal and ultrasturctural pathological changs were examined. Renal tissue collagen Ⅳ, Smad2/3and c-fos expression were measured by immunohistochemical assay.Part three:The effects of α1-AA on the expression of IL-6and ICAM-1inhibitted by Doxazosin in serum of DM rats IL-6and ICAM-1were measured by ELISA.The rest were same with Part two.ResultsPart one:α1-AA mediated in diabetic rat kidney tissues of collagen IV, Smad2/3, c-fos expression and the expression of inflammatory cytokines1、Comparison of Weight,FBG in three groupsAfter mediated in16weeks, healthy rats’ weight is increased gradually than before, DM rats’ weight is significantly less than before. The FBG of DM rat were above16.7mmol/L, and had been maintained at a high level, compared with the N group had significant difference; there was no difference in DM+α1-AA group and DM group.2、Comparison of Scr and24hUpro in three groupsAfter mediated in16weeks, the level of Scr and24hUpro were increased gradually in DM rats, and there is no significant changes in healthy rats. DM+α1-AA group compared with DM group, the index increased more significantly.3、Related changes of renal cortex in three groups in electron microscopeResults in electron microscope:Anormaly had not been seen in the normal group. Anormaly had been seen in the DM rats group. Serious destruction had been seen in the DM rats group mediated by α1-AA.4、Expression of collagen IV, Smad2/3and c-fos in renal cortex in three groupsThe expression level of collagen IV, Smad2/3and c-fos were evaluated by average luminosity. The collagen IV, Smad2/3and c-fos were exanmined in renal cortex in both healthy rats group and DM rats. DM rats group was significantly higher than healthy rats group, and the DM rats group mediated by α1-AA was the highest.5、Comparison of α1-AA, IL-6and ICAM-1in three groupsDM+α1-AA group serum IL-6and ICAM-1levels higher than that of DM group.Part two:The effects of α1-AA on the expression of collagen IV, Smad2/3and c-fos inhibitted by Doxazosin in renal cortex of DM rats1、Comparison of weight before and after treatment in every group ratsAfter mediating in16weeks, healthy rats’ weight is increased gradually than before. DM rats’ weight is significantly less than before.2、Comparison of Scr and24hUpro in every groupsAfter mediating in16weeks, the level of Scr and24hUpro in α1-AA mediated group were higher than non-mediated DM rats. The level of Scr and24hUpro in the DM rats both α1-AA mediated and Doxazosin intervention group were obviously lower than the DM rats α1-AA mediated group. The level of Scr and24hUpro in DM rats Doxazosin intervention group were lower than the non-mediated DM rats.3、Related changes of renal cortex in every groups in electron microscopeAnormaly slightly had been seen in the non-mediated DM rats. Serious destruction had been seen in the DM rats α1-AA mediated group, and it had been taken a turn for the better in the DM rats both α1-AA mediated and Doxazosin intervention group. Anormaly had been seen in the DM rats intervented by Doxazosin.4、Expression of collagen IV, Smad2/3and c-fos in renal cortex in three groupsThe collagen IV, Smad2/3and c-fos were exanmined in renal cortex in both α1-AA mediated group and α1-AA non mediated group. The expression of collagen Ⅳ, Smad2/3and c-fos in α1-AA mediated group was significantly higher than α1-AA non mediated groups. α1-AA mediated and Doxazosin intervention group was significantly lower than α1-AA mediated group. Doxazosin intervention group was significantly lower thanα1-AA non mediated group.Part three:The effects of α1-AA on the expression of IL-6and ICAM-1 inhibitted by Doxazosin in serum of DM rats1、Comparison of24h Upro、KW/BW in every groupsAfter mediating in16weeks, the level of24hUpro and KW/BW in α1-AA mediated group were higher than non-mediated DM rats. The level of24hUpro and KW/BW in the DM rats both α1-AA mediated and Doxazosin intervention group were obviously lower than the DM rats α1-AA mediated group. The level of24hUpro and KW/BW in DM rats Doxazosin intervention group were lower than the non-mediated DM rats.2、Comparison of IL-6、ICAM-1in every groupsThe IL-6、ICAM-1were exanmined in both α1-AA mediated group and α1AA non mediated group. The IL-6and ICAM-1in α1-AA mediated group was significantly higher than α1-AA non mediated groups. α1-AA mediated and Doxazosin intervention group was significantly lower than α1-AA mediated group. Doxazosin intervention group was significantly lower thanα1-AA non mediated group.3、Correlations between IL-6and ICAM-1level and24hUpro excretion, kidney/body weightThe results of correlation analysis showed that:24hUpro、KW/BW and IL-6, ICAM-1levels were positively correlated.4、Related changes of renal cortex in every groups in electron microscope The same with Part two.Conclusions1、DN is a kind of immune response, also is a kind of inflammation, which are closely connected and positive correlation. 2、α1-AA may through MAPK, TGF-β1/smad pathway and GPCR signal transduction, increased expression of collagen IV, Smad2/3and c-fos, eventually leading to renal extracellular matrix remodeling, worsening renal function; DM rat by α1-AA mediated, serum IL-6, ICAM-1levels were significantly increased, suggesting that the occurrence, development of inflammatory mechanisms involved in DN.3、Doxazosin as α1-AA antagonists may be competitive with α1-receptor, blocking the signal transduction pathways involved in α1-AA injected mice, thereby, improving renal extracellular matrix remodeling, to protect renal function. |
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