The Role And Mechanism Of AMPK Regulating Neuropeptide Y In Diabetic Nephropathy

Diabetic nephropathy,in which albuminuria is an early manifestation,occurs in approximately one third of diabetic individuals and is the leading cause of end-stage renal failure worldwide.In patients with either T1 DM or T2 DM,the ability of insulin to elicit cellular responses is impaired,a concept termed ‘cellular insulin resistance',and is associated with altered renal cell glucose metabolism that may directly contribute to progressive renal damage independently of hyperglycemia.Interestingly,in addition to being associated with albuminuria and nephropathy in both type 1 and type 2 diabetes,insulin resistance is also associated with the development of albuminuria in non-diabetic individuals..Furthermore,in patients with either T1 DM or T2 DM,insulin resistance per se contributes to higher salt sensitivity,which closely associates with increases in blood pressure,albuminuria,and decline in renal function.Insulin is a metabolic hormone that not only regulates the metabolism of glucose and other substrates,but also directly modulates the biology of podcytes.Podocytes are highly specialized epithelial cells essential for the integrity of the glomerular filtration,and their ability of sensing insulin to prevent proteinuria,making them attractive early target cells in DN.In the glomeruli,approximately a decade ago it was discovered that human podocytes respond to insulin and express the hallmark components of insulin-sensitive cells,including the insulin receptor(IR),and key glucose transporters,including GLUT4 and GLUT1.Mice depleted of the insulin receptor specifically in podocytes developed albuminuria and podocyte loss,characteristic of DN pathology.Moreover,it has been shown that podocytes exposed to a diabetic environment become insulin resistant.This response is,at least in part,mediated by chronic exposure to elevated insulin levels promoting podocyte insulin receptor(IR)degradation.In a word,insulin signaling is a key regulator for normal kidney function of the glomerular podocyte,which is implicated in the pathogenesis of diabetic nephropathy.Hence potential molecular targets for modulating podocyte insulin responses thus treating DN are highly desirable.Neuropeptide Y(NPY)is a sympathetic neurotransmitter with wide-ranging effects in various organ systems,from the central nervous system to the cardiovascular(CV)system,the bone and the kidney.This highly conserved peptide containing 36 amino acids,is one of the most common peptides in the brain and is an abundant neurotransmitter in the peripheral sympathetic nervous system(SNS).NPY has been shown important in energy metabolism,appetite regulation,cardiac rhythm,blood pressure,smooth muscle contraction and relaxation.More and more evidence suggest that NPY acts as a metabolic signal that may contribute to obesity,hyperinsulinemia,and hyperglycemia.Insulin acts on NPY neurons and modulates the balance of energy regulation and the dysfunction of which may result in the development of metabolic diseases.Moreover,NPY acts as a potent therapeutic target for obesity and type 2 diabetes.It has been reported that NPY inhibits glucose-stimulated insulin secretion,and it regulates renal blood flow.More importantly,it has also been implied the therapeutic potential of neuropeptide Y in kidney disease specifically in DN.Both in vivo and in vitro studies have demonstrated that NPY not only protects kidney function from nephrotoxicity,but also associated with nutritional status and cardiovascular events in adults with chronic kidney disease.The possibility that this neuropeptide is involved in CKD progression is suggested by sparse observations in various experimental models such as kidney inflammation in mice with systemic lupus and kidney fibrosis and renal dysfunction in early postnatal hyperalimentation in the rat.Furthermore,circulating NPY levels have already been associated with left ventricular hypertrophy and incident CV events as well as with bone disease in advanced CKD.A cohort study unveiled that NPY associates with proteinuria and faster CKD progression as well as with a higher risk of kidney failure.Of note,a genetic polymorphism in the precursor molecule to NPY,prepro NPY,associates strongly with proteinuria and the risk of nephropathy in type 2 diabetics.Another clinical study indicates that Leu7 Pro polymorphism in NPY gene confers the susceptibility to the development of DN in Swedish female T1 D patients.Although these evidences imply that NPY may play a role in the pathogenesis of diabetes and CKD,the effect and specific mechanism of NPY in DN is still unknown.In this study,we hypothesized that NPY has an impact on podocyte insulin responses and protection from high glucose injury in DN.To test this hypothesis,we investigate the effect of NPY on the IR signaling cascade and podocyte damage both in mouse podocytes and diabetic db/db mice.The major pathway of insulin receptor signal transduction,phosphatidylinositol 3-kinase(PI3K)–Akt(also known as PKB)pathway and the positive regulator of insulin sensitivity AMP-activated protein kinase(AMPK),were assessed in our study.GSK3?was also detected since Akt acts as a central node in the regulation of the biological effects of insulin that it phosphorylates and activates essential downstream kinases including glycogen synthase kinase 3(GSK3).More importantly,there is evidence suggesting that GSK3 contributes to the induction by insulin resistance independently of insulin receptor signaling or PI3K-AKT activity.Based on this hypothesis,we aimed to establish the potential involvement of NPY in podocyte insulin sensitivity and its molecular mechanism in the pathogenesis of DN.Objective: To investigate whether NPY has a role in the amelioration of podocyte injury and insulin resistance,thus suppressing the progression of DN.Moreover,the molecular mechanisms responsible for the effects were examined.Methods: Glomerular podocytes were cultured in vitro.Podocytes were divided into normal control group,HG-stimulation group,HG+NPY group,HG+NPY+PI3K inhibitor LY294002 group,mannitol+ scambled si RNA group,mannitol+ NPY si RNA group,mannitol + NPY si RNA + GSK-3? inhibitor AZD1080 group.The treated cells were pre-treated with 100 nmol/L insulin for 15 min to extract total protein.The levels of PI3 K,p-PI3 K,AKT,p-AKT,IR-?,GSK3?,and p-GSK3?were detected by western blotting.The levels of IR-?and NPY m RNA were measured by q RT-PCR.The expression of IR-?in podocytes of each group was detected by immunofluorescence.The expression of NPY in the supernatant was detected by ELISA.Fluorescent D-glucose derivative 2NBDG was used for podocyte glucose uptake assay.CCK-8 was used to detect cell viability in each group.Scratch assay was used to determine the podocyte migration ability.Results:(1)NPY upregulated the insulin receptor signaling pathway in HG-stimulated podocytes.The level of IR-? protein and the phosphorylation of PI3 K and AKT were significantly increased.(2)NPY inhibited the phosphorylation of GSK3 ? protein.(3)NPY significantly increased the glucose uptake of HG-stimulated podocytes,and restored the viability of podocytes in the high glucose group from 55.56±2.29% to 75.49±3.19%,and inhibited the high cell migration induced by high glucose.(4)ELISA results and q RT-PCR results showed that activation of AMPK upregulated the expression of NPY in podocytes stimulated by HG.(5)Compared with the HG-stimulated control group,AMPK activation up-regulated the expression of IR-?protein and phosphorylation of AMPK,PI3 K and AKT,and down-regulated the phosphorylation of GSK3 ? level.(6)After HG-stimulation,treatment with AMPK activator AICAR significantly increased the uptake of 2-NBDG in the cells,which restored the viability of podocytes in the high glucose group from 53.95±3.43% to 70.22±1.38%,and inhibited HG-induced enhanced cell migration ability.Conclusions: The activation of AMPK promoted the increase of NPY expression in HG-stimulated podocytes.NPY can upregulate the insulin receptor signaling pathway,mediate PI3K/Akt/GSK3? signaling pathway to regulate podocyte glucose uptake,and improve podocyte activity,migration ability in HG-stimulated podocytes.Objective: To investigate the protective effects of NPY activated by AMPK and its molecular mechanism in DN mice.Methods: The experimental mice were grouped into: db/m group: db/m mice + normal saline injection,db/db group: db/db mice + normal saline injection,db/db+AICAR group: db/db mice + AICAR injection,db/db+NPY group:db/db mice + NPY injection,dbdb+AZD1080 group: db/db mice + AZD1080 injection.24 hours urine and blood urinary protein were collected,and blood biochemical tests were performed.Renal pathology and IR-? immunofluorescence were also tested.Glomeruli were isolated to obtain glomerular protein and western blotting was used to detect AMPK,p-AMPK,PI3 K,p-PI3 K,AKT,p-AKT,IR-?,GSK3?,p-GSK3? levels.q RT-PCR was used to detect IR-? and NPY m RNA levels.ELISA was used to detect the expression of NPY in serum and renal tissues of db/m group and db/db group.Linear regression analysis was used to analyze the correlation between NPY levels in serum and renal tissues and progression of DN.Results:(1)Activation of AMPK significantly increased NPY protein expression and its m RNA levels in serum and kidney tissues of db/db mice(p<0.01).(2)In db/db mice,activation of AMPK,up-regulation of NPY,or inhibition of GSK3? activity significantly reduced blood glucose and serum creatinine,cholesterol,triglyceride,and blood urea nitrogen levels(p<0.01).Serum albumin levels were significantly increased(p<0.01),and renal pathological changes in diabetic nephropathy mice were significantly improved.(3)Serum and renal NPY levels in DN mice were negatively correlated with blood glucose,serum creatinine,blood urea nitrogen,plasma triglyceride,cholesterol,and urinary protein(P<0.05),whereas serum and renal tissue NPY levels were positively correlated with serum albumin(P<0.05).(4)Activation of AMPK and up-regulation of NPY upregulate the insulin receptor signaling pathway in renal tissue of db/db mice.IR-? expression and PI3 K and AKT phosphorylation levels were significantly increased,and phosphorylation levels of GSK3? were significantly reduced.(5)Activation of AMPK,increase of NPY expression or inhibition of GSK3? can significantly improve the decrease of GLUT4 protein level in db / db mice.Conclusion: The activation of AMPK promoted the increase of NPY expression in serum and renal tissues of db/db mice,and then up-regulated the expression of GLUT4 protein through up-regulation of insulin receptor signaling pathway,mediated improvement of PI3K/Akt/GSK3? signaling pathway to ameliorate kidney damage in db/db mice,thus evoking a novel therapeutic concept for DN.