Study On The New Targets And Mechanisms Of Podocytosis

Glomerular disease is the third leading cause of end-stage kidney disease(ESRD)in the United States and the cause of 25%of chronic kidney disease(CKD)worldwide.Podocytes are visceral epithelial cells and are the core component of the glomerular filtration barrier.Glomerular disease is a recognized risk factor for cardiovascular,metabolic,infection and other diseases.Podocyte injury(podocyte disease)is considered to be the initial feature of glomerular disease.The treatment of glomerular disease depends on its etiology and type,but currently it is mainly limited to the use of blockers and immunosuppressants of the renin-angiotensin-aldosterone system,including glucocorticoids,alkylating agents,and calcineurin inhibitors,antimetabolites,etc.However,the progress in the treatment of glomerular diseases is very limited,and there is an urgent need to find new therapeutic targets or treatment methods.Melanocortin hormone system plays a key role in maintaining homeostasis in human body through its neuro immune endocrine activity,and regulates a variety of physiological functions,including melanogenesis,inflammation,immune regulation,adrenal corticosteroid production,hemodynamics,diuresis,energy homeostasis,sexual function and secretion.The pathobiological effects of all melanocortins are transmitted by melanocortin receptors.A large amount of evidence has shown that melanocortin has renal protective effects in rodents and human kidney diseases,especially after the recent recognition of ACTH.ACTH monotherapy can effectively alleviate steroid resistant nephrotic syndrome,which indicates that ACTH has another mechanism to protect the kidney in addition to adrenal corticosteroid production.In view of the biological effects of ACTH binding to the melanocortin receptor and the expression of a large number of melanocortin receptors in the kidney,this not fully explored mechanism probably belongs to the melanocortin system.Five melanocortin receptors are known,but their signal transduction and regulation are poorly understood.In particular,the exact effect of melanocortin signaling mediated by melanocortin receptors remains uncertain.This was the first study that report the role of receptor 5 in the regulation of podocytes.Part 1 The role of melanocortin receptors under normal physiological conditionsObjectiveTo detect the related morphology and function of the kidney and podocytes in normal mice(WT),and to test the changes in the morphology and function of the kidneys and podocytes in mice with mutations of melanocortin 5 receptor,Detect if the mutation of the melanocortin 5 receptor affect the morphology and function of the kidney and podocytes under physiological conditions.Methods1.Detect the appearance,body weight,kidney weight and body weight ratio of mice,and compare the difference between MC5R-/-and WT mice.2.Through general laboratory tests(hematuria creatinine and urine protein),compare the kidney function of MC5R-/-and WT mice.3.Through PAS staining,electron microscope,immunohistochemical staining,and western blot,compare the structure of kidney and podocyte in MC5R-/-and WT mice under physiological conditions.Results1.Under physiological conditions,MC5R gene deletion does not affect the expression of Podocin,a marker of podocytes.2.Under physiological conditions,MC5R gene deletion does not affect the appearance and weight of mice.3.Under physiological conditions,there is no significant difference between MC5R-/-and WT mice in serum creatinine,urine albumin excretion rate,and kidney weight-to-weight ratio.4.Under physiological conditions,there is no significant difference in the structure and morphology of kidney and podocyte tissue between MC5R-/-and WT two groups.ConclusionsThere was no significant difference in the physiological and biochemical indexes of the kidney between the MC5R-/-and WT two groups of mice under physiological conditionsPart 2 Activation of melanocortin receptor on podocyte alleviates podocyte injuryObjectiveDetect the related morphological and functional changes of MC 5R-/-mouse podocytes in the adriamycin environment,test the effect of melanocortin agonists on podocytes,and then use plasmids to reconstruct the melanocortin 5 in MC5R-/mouse podocytes,to explore the effect of melanocortin on podocytes.Methods1.Through PAS staining,electron microscopy,immunohistochemistry,immunofluorescence staining,general laboratory tests(creatinine,urea and urine protein)and western blot,compare the kidney structure and function of MC5R-/-and WT mice under physiological and pathological(ADR)conditions.2.Compare the inhibition of GSK3β activity with the normal control podocyte morphology and the expression of downstream molecules,and explore the role of GSK3-/-phosphorylation in the melanocortin signaling system.3.Reconstruct the melanocortical receptors of MC5R-/-mice with MC5R plasmids and detect their response to ADR injury.Results1.After ADR injury,urinary albumin and glomerulosclerosis scores of MC5R-/group were significantly higher than those of WT group,and the difference was statistically significant(P<0.05).2.After ADR injury,the expression levels of WT-1 and podocin protein in podocytes in the MC5R-/-group were significantly lower than those in the WT group,and the difference was statistically significant(P<0.05).3.After ADR injury,the activities of GSK3β and NFκB are enhanced,the cytoskeleton is disordered,and the podocytes are reduced.4.After ADR injury,the activation of MC5R can reduce the activity of GSK3βand NFκB,and correct the disorder of actin cytoskeleton and podcytopenia.When the MC5R gene is deleted,the effect of the melanocortin pan-agonist NDP-MSH is greatly weakened,while the effect of the melanocortin-specific receptor agonist MC5A is cancelled.5.After ADR injury,urinary albumin after MC5R reconstruction was significantly lower than that of MC5R-/-group,the difference was statistically significant(P<0.05).The expression levels of WT-1 and Fibronectin protein in podocytes after MC5R reconstruction were significantly higher than those in the MC5R-/-group,and the difference was statistically significant(P<0.05).Conclusions1.Under pathological(ADR)injury,after MC5R is missing,podocyte damage is aggravated and GSK3β is overactivated.2.Melanocortin agonists can alleviate ADR-induced podocyte damage.3.Melanocortin agonists increase GSK3β phosphorylation and inhibit GSK3βactivity.4.Melanocortin agonists protect podocytes through MC5R.Part 3 Melanocorticoid relieves podocyte damage by down-regulating Gsk3βObjectiveBy transfecting the plasmid S9A to continuously activate GSK3β in the immortalized mouse podocytes,to explore whether it affects the protective effect of melanocortin agonists on podocytes.MethodsIn podocytes,use plasmid transfection to block the phosphorylation site of GSK3β to inhibit the phosphorylation of GSK3β,and explore the role of phosphorylation of GSK3β in the melanocortin signaling system.ResultsAfter ADR injury,EV+ADR,EV+ADR+MC5A and S9A+ADR+MC5A were treated with mouse podocytes cultured in vitro to prepare cell lysates.In EV+ADR+MC5A podocytes,p-RelA/p65 S467,The protein levels of p-paxillin,desmin,caspase-3,b7-1,and MCP-1 were significantly lower than those in the S9A+ADR+MC5A group,while phosphorylated GSK3β was significantly higher than that in the S9A+ADR+MC5A group.The difference was statistically significant(P<0.05).ConclusionsIn podocytes,after overactivation of GSK3β,the beneficial effects of MC5R agonists on podocytes disappear.