The Role Of KATP On Glomerular Mesangial Cells Proliferation And Excessive Extracellular Matrix Synthesis Induced By High Glucose

With the increasing epidemic of diabetes mellitus,the incidence of diabetic kidney disease is increasing.Diabetes mellitus has become one of the main causes of end-stage renal disease.As intensive researches in diabetic nephropathy continued to deepen,Hemodynamic changes,metabolic pathway,inflammation / oxidative stress,and others(autophagy)were recognized as the current pathogenesis of diabetic nephropathy.But there is no improvement in the prevalence of diabetic kidney disease.It suggests there may be an underlying mechanism.ATP sensitive potassium channel(KATP)is a type of potassium channel sensitive to intra-or extra-cellular ATP,formed by heteromultimers.KATP participate in a variety of important biological processes,expressed in various tissues and cells,such as heart,pancreas,kidney,neuron and so on,mediating metabolic status and membrane excitability.KATP dysfunction plays a crucial role in the development of diabetes,while KATP regulating the secretion of insulin and glucagon.The studies on the role of KATP on the diabetic chronic complications have been less reported domestically and abroad so far.Research suggests that KATP channel openers protect the kidneys by reducing inflammatory cell infiltration and oxidative stress in chronic kidney disease,avoiding further injury as well.KATP opener nicorandil could improve renal pathological changes in eNOS-defective diabetic rats.Moreover,Clinical studies show that renal function and urinary albumin levels of patients in early diabetic nephropathy were significantly improved by using KATP channel opener nicorandil.It suggests that KATP channels may play an important role in the occurrence and development of diabetic nephropathy.KATP channels are an important mediator of cellular energy status and cell excitability.AMP activated protein kinase(AMPK)is a vital component of cellular energy metabolism pathway.There are increasing evidences that activation of AMPK inhibited the occurrence and development of diabetic nephropathy.Researchers have reported AMPK activators or increased AMPK 172 threonine phosphorylation raised the activation of KATP channels in a number of tissues such as pancreatic beta cells and ventricular muscle cells.Coimmunoprecipitation and mass spectrometry analysis showed that the AMPK alpha subunit combined with KATP channel subunits.It had shown that AMPK might be an important regulatory factor of KATP activity,which played an significant role in the pathogenesis of diabetic nephropathy possibly.In view of the regulation of AMPK and KATP channels playing an important role in the development of diabetic nephropathy,we propose that the suppressed expression of AMPK in renal cells decreased the activity of KATP channels under high glucose condition,followed by a series of pathological reactions.All of these maybe promote occurrence and development of diabetic nephropathy.Object: Two of the main pathological characters of early diabetic nephropathy are the increased extracellular matrix secretion and the excessive proliferation of mesangial cells.Glomerular mesangial cells(GMC)as the research objects,we want to identify the existence and location of KATP channels in mesangial cells firstly.And then we want to confirm the role of KATP channels activation in increased extracellular matrix secretion and the excessive proliferation of mesangial cells with high glucose treatment and by using KATP channels opener.At last we want to verify KATP channels can be activated by AMPK through the use of AMPK activator.Methods: 1.Glomerular mesangial cells were isolated from male sprague-dawley rat for primary culture.Real-time PCR was performed to measure the mRNA levels of KATP channels subunits(Kir6.1,Kir6.2,SUR1,SUR2 A,SUR2B).The distribution and localization of KATP were observed by fluorescence double staining and confocal microscopy.Primary mesangial cells were treated with 0,10,20,30,40mmol/L glucose for 12 and 24 hours.Cell proliferation was measured using a Cell Counting Kit-8.Cell apoptosis was observed by Annexin V-FITC/PI method.Cell cycle was counted by flow cytometric analysis after stained by Propidium PI.The level of fibronectin(FN)was assayed by using ELISA kit.2.(1)Primary mesangial cells were treated with or without 30mmol/L glucose for 24 hours to be high glucose group(HG group)or control group(CN group).Cell proliferation,Cell apoptosis,Cell cycle were observed.The level of FN and type IV collagen(IV-C)were assayed by using ELISA kits.The level of pAMPK/AMPK were measured by using western-blotting to detect the expression and activation of AMPK.The whole-cell configuration of the patch-clamp technique was performed to evaluate the activity of KATP channels.The mRNA levels of KATP channels subunits(Kir6.1,Kir6.2,SUR1,SUR2 A,SUR2B)were assessed by Real-time PCR as above.(2)Primary mesangial cells were treated with high glucose(HG group),high glucose and DMSO(vehicle of KATP channels opener)(HG+DMSO group),high glucose and DZX(KATP channels opener)(DZX+HG group),high glucose+ DZX+5-HD(KATP channels blocker)(DZX+5-HD+HG group)for 24 hours.Cell proliferation,the level of FN and IV-C,activity and the mRNA expression of KATP channels were measured as above.3.Primary mesangial cells were treated with high glucose(HG group),high glucose and DMSO(vehicle of AMPK activator)(HG+DMSO group),high glucose and 5-amino-4-imidazolecarboxamide riboside(AICAR,AMPK channels activator)(AICAR+HG group)for 24 hours.The expression of p AMPK/AMPK and activity of KATP channels were evaluated as above.Results:1.Double fluorescence staining showed KATP channels were expressed on the cytomembrane and mitochondria of GMC.Realtime-PCR results displayed that the KATP channel subunits in glomerular mesangial cells mainly existed in the form of Kir6.1/SUR2 A and / or Kir6.1/SUR2 B.It was shown by gradient glucose culture that GMC in 30mmol/L glucose for 24 hours had great proliferation rate(p<0.01)and highest FN level(p<0.01).2.(1)High glucose increased primary mesangial cells proliferation(p<0.01).The level of FN and IV-C in HG group was significantly higher than in CN group(p<0.01).Compared with CN group,activity of KATP channels and the expression of KATP channels subunits decreased significantly treated with high glucose,the level of pAMPK/AMPK as well(p<0.01).(2)DZX significantly decreased primary mesangial cells proliferation(p<0.01).The level of FN and IV-C decreased remarkably in DZX+HG group compared with HG and HG+DMSO groups(p<0.01).Activity of KATP channels rose significantly(p<0.05).The m RNA level of SUR2A/SUR2 B decreased in DZX+HG group(p<0.01)compared with HG and HG+DMSO group,while Kir6.1 had no difference among these groups.Compared with DZX+HG group,DZX+5-HD+HG group had higher primary mesangial cells proliferation(p<0.01)and higher level of FN and IV-C(p<0.01).And activity of KATP channels declined(p<0.05).The m RNA level of SUR2A/SUR2 B in DZX+5-HD+HG group were higher than in DZX+HG group(p<0.01 and p<0.05).3.Compared with HG and HG+DMSO group,AICAR+HG group enhanced the activity of KATP channels(p<0.01),while pAMPK/AMPK as well(p<0.01).Conclusion: KATP channels located in mitochondria and plasma membranes of primary mesangial cells in the form of Kir6.1/SUR2 A and/or Kir6.1/SUR2 B.High glucose promoted primary mesangial cells proliferation and the synthesis of extracellular matrix proteins.High glucose decreased activity of KATP channels and the mRNA level of KATP channels subunits,the same to the activity of AMPK.Treated with DZX,the activity of KATP channels raised.Meanwhile,Cell proliferation decreased significantly,so did the level of FN and IV-C.5-HD could reverse the above effects.All of above indicated KATP channels opener could improve mesangial cells proliferation and excessive extracellular matrix secretion induced by high glucose.The activity of KATP channels were raised by AMPK activator,revealing that AMPK enhanced the role of KATP channels in mesangial cells proliferation and excessive extracellular matrix synthesis induced by high glucose.KATP may play an important role in the the occurrence and development of diabetic nephropathy.There is a regulatory relationship between KATP and AMPK.