Molecular Mechanism Studies On The Alleviation Of Fructose-Induced Chronic Renal Injury By Polyphenols In Traditional Chinese Herb

Metabolic syndrome is a group of metabolic disorders with insulin resistance as the central pathological feature.Epidemiological studies have proved the positive correlation between the increase in the incidence of metabolic syndrome with long-term excessive fructose intake.High fructose intake causes metabolic syndrome,accompanied with insulin resistance,oxidative stress,inflammatory response,renal fibrosis with collagen deposition,proteinuria and other kidney damage.The molecular pathological mechanism of renal injury caused by high fructose intake needs to be further clarified.Mature interleukin-1?(IL-1?)is the cleaved product from its precursor by activating the nuclear transcription factor kappa B(NF-?B)signaling pathway and NLRP3 inflammasome.IL-1? interferes insulin signaling to decrease insulin sensitivity.1-phosphate sphingosine(SlP)is produced by the phosphorylation of sphingosine,catalyzed by sphingosine kinase 1(SphK1),which,SlP regulates multiple cellular functions through binding to SlP receptor 1 and 3(S1PR1/3).S1P upregulates NF-?B signaling and NLRP3 inflammasome to induce IL-1? secretion.The MicroRNAs(miRNAs)are a class of non-coding RNAs with a length of 18-24 nucleotides.MiR-330 is predicted to bind to the 3'noncoding region of SphK1 mRNA according to the Targetscan database.Our previous studies confirmed that high fructose feeding caused renal inflammation by activating NF-?B signaling and NLRP3 inflammasome,with dysregulation of insulin signaling and miR-330 decrease.Meanwhile,the activation of SphK1/S11P/S1PR1/3 axis,NF-?B signaling pathway and IL-1? over-express were observed in the liver of high fructose-fed rat.Therefore,we are wondering whether fructose activates SphK1/S1P/S1PR1/3 axis to upregulate the NF-?B signaling and NLRP3 inflammasome,leading to IL-1? release and insulin signaling disorder in the kidney?And whether the process has relationship with the change of miR-330 expression?Transform of proximal renal tubular epithelial cells(PTECs)into mesenchymal cells(EMT)is a major event in the development of renal fibrosis,regulated by transforming growth factor(TGF-?1).TGF-?1 binds to the ligand of type II transforming growth factor receptor(TGFBR2)and subsequently aggregates with type I transforming growth factor receptor to activate the transforming growth factor receptors(TGFBRs).Therefore Smad2 and Smad3 are simultaneously phosphorylated and aggregated with Smad4 to form a dimer,and subsequently be transferred into the nucleus to act as a transcriptional factor in regulating the expression of fibrosis related genes and proteins.Insulin resistance is considered to be an crucial pathological character in the development of fibrosis.The advanced glycation end products can activate TGF-?1/Smads signaling to promote tubulointerstitial fibrosis.Our previous studies confirmed that high fructose intake caused rat glomerular insulin resistance and increased serum TGF-?1 levels.We are wondering whether fructose could induce EMT of PTECs to promote renal fibrosis?And,whether activation of renal cortex TGF-?1/TGFBR/Smads signaling gives rise to renal fibrosis under high fructose consumption.Podocyte is the terminal differentiation cell,the structural or functional impairment of which causes proteinuria.Podocyte exhibits a high level of basal autophagic activity.The rapamycin target protein(mTOR)is a serine/threonine protein kinase which can regulate podocyte autophagy.Adenosine monophosphate activated protein kinase(AMPK)and mTOR can interact with each other to regulate autophagy,maintaining the cellular homeostasis.ROS,a second messenger molecule,can affect autophagy by modulating AMPK,mTOR or p38 MAPK.In addition,nuclear factor E2-related factor(Nrf2).a key factor in response to oxidative stress is regulated by Kelch-like ECH-associated protein 1(Keap1)and interacted with antioxidant elements(ARE)to regulate cellular ROS level.In our previous study,fructose induced ROS overproduction in kidney and impairment of the structure and function in podocytes of rat.Meanwhile,increased p38 MAPK phosphorylation in the podocytes were detected.We are wondering whether AMPKa is activated by ATP over-depletion under high fructose feeding in the podocytes.And whether the mTOR downregulation promotes autophagy to induce proteinuria?Does fructose active Nrf2 signaling simultaneously to trigger ROS accumulation?As a result,whether activation of AMPKa and p38 MAPK,leads to upregulation of mTOR-mediated autophagy?In clinical and basic research,the active ingredients of traditional Chinese medicine polyphenols have been confirmed to exhibit multiple pharmacological activities,including anti-inflammation,anti-oxidation and anti-fibrosis.In our previous study,morin improved kidney function in hyperuricemic mice,and ameliorated insulin signaling and inflammatory response in the liver of fructose-fed rats.But the molecular mechanism is still unknown.We are wondering whether pterostilbene improves insulin signaling and fibrosis in renal tubular epithelial cells?Whether polydatin relieves proteinuria by reducing oxidative stress and high level of autophagy levels in the podocytes of fructose-fed rats.In this study,male Sprague-Dawley rats were fed with 10%fructose water for 8 weeks.At the end of 5th week,rats were given morin(30 and 60 mg·kg-1)and allopurinol(5 mg·kg-1,positive control drug)for 4 weeks.Oral glucose tolerance test(OGTT)and insulin tolerance test(ITT)test confirmed that the decreased model animal insulin and glucose tolerance compared with the normal group,while serum insulin,uric acid,creatinine and IL-1? levels were increased significantly.Morin and allopurinol significantly improved the insulin sensitivity in the kidney of fructose-fed rats and decreased the levels of insulin,uric acid,creatinine and IL-1? in the serum of fructose-fed rats.On the basis of the above research work,we further investigate whether high fructose can down-regulate miR-330 level and affect the expression of SphKl,NF-?B signaling or NLRP3 inflammasome to affect insulin resistance in the kidney of fructose-fed rats and fructose-exposed HK-2 cells.,Activation of NF-?B signaling and NLRP3 inflammasome,as well as insulin resistance were observed in the renal cortex of fructose-fed rats compared with normal group,with elevated expressions of nuclear NF-?B,phosphorylated NF-KB-inhibiting kinase alpha subunit(p-I?B?),and phosphorylated I?B kinase beta subunit(p-IKK?),pro-IL-1?,NLRP3,apoptosis-associated speck-like protein(ASC),cysteine protease-1(caspase-1),IL-1? and p-insulin receptor substrate-1(IRS-1)(S318)protein levels,decreased expressions of p-insulin receptor(IR)(Y1345),p-IRS-1(Y895)and p-extracellular signal-regulated kinase 1/2(ERK1/2)(T202/Y204)levels.Consistently,activations of NF-?B signaling and NLRP3 inflammasome activation,as well as insulin resistance were observed in the human proximal tubular epithelial cells(HK-2)with 5 mM fructose treatment These results suggested that high fructose may induce activation of NF-?B signaling pathway,NLRP3 inflammasome and insulin resistance in the kidney,leading to renal injury.Morin restored above changes induced by fructose in the kidney of rats and HK-2 cells.SphK1 activity,SlP level and protein levels of SphK1,S1PR1/3 protein were significantly increased in the kidney of fructose-fed rats and fructose-exposed HK-2 cells,which were restored by morin and allopurinol.HK-2 cells were incubated with SphKl inhibitor,SlPR1/3 siRNA respectively,with or without 5 mM fructose,50 ?M morin,and 100 ?M allopurinol.SphK1 inhibitor and S1PR1/3 siRNA significantly inhibited NF-?B signaling and NLRP3 inflammasome to alleviate insulin resistance induced by high fructose treatment,remaining the SphK1 protein levels unchanged Morin and allopurinol further restored fructose-induced alternation of nuclear NF-?B,NLRP3,pro-IL-1?,IL-1? and insulin signaling related protein levels in HK-2 cells co-incubated with fructose and SphK1 inhibitor or S1PR1/3 siRNA,suggesting that morin and allopurinol restored fructose-induced activation of NF-?B signaling and NLRP3 inflammasome by inhibiting SphK1/S1P/S1PR1/3 axis,resulting in improvement of insulin signaling.The expression of miR-330 in the kidney cortex of fructose-fed rats was significantly decreased compared with the control group demonstrated by gene chip analysis.The results were verified by qRT-PCR assay.Consistently,high fructose down-regulated miR-330 expression in HK-2 cells compared with the normal group Morin and allopurinol significantly increased miR-330 expression in renal cortex of fructose-fed rats and fructose-exposed HK-2 cells.In the present study,miR-330 mimic and inhibitor were transfected into HK-2 cells for 24 h,and then treated with 5 mM fructose,morin(50 ?M)and allopurinol(100 ?M)for 48 h.MiR-330 mimic significantly up-regulated the miR-330 expression and down-regulated SphK1 protein levels in HK-2 cells.Meanwhile,miR-330 inhibitor significantly reduced the expression of miR-330 in HK-2 cells,and increased SphK1 protein levels.These data suggested that miR-330 may function by targeting SphKl.MiR-330 mimic significantly decreased fructose-induced SphK1/S1P over-expression,activation of NF-?B signaling and NLRP3 inflammasome,as well as insulin resistance in HK-2 cells.miR-330 inhibitor further upregulated SphK1/S1P levels in fructose-exposed HK-2 cells,activated NF-?B signaling and NLRP3 inflammasome,enhanced insulin resistance.In miR-330 mimic or miR-330 inhibitor transfected HK-2 cells with high fructose co-incubation,morin and allopurinol significantly reversed the abnormal expression of the above parameters.However,morin and allopurinol failed to restore fructose-induced alterations of these proteins in HK-2 cells with negative transfection.These results suggested that morin may suppress the activation of SphK1/S1P/S1PR1/3 axis by down-regulating miR-330.As a result,NF-?B/NLRP3 signaling and the insulin resistance is improved.Next,we focuses on whether high fructose consumption causes Epithelial-mesenchymal transition(EMT)of proximal renal tubular epithelial cells and renal fibrosis based on renal insulin resistance?And whether pterostilbene functions to protect kidney against fibrosis.Male Sprague-Dawley rats were fed with 10%fructose water for 12 weeks.After 6 weeks fructose feeding,the rats were randomly divided into four groups receiving physiological saline(fructose-vehicle group),pterostilbene(10,20,40 mg·kg-1)(n=7)and pirfenidone(200 mg·kg-1,positive control)(n=7)for an additional 6 weeks.Compared with the control group,OGTT test confirmed the decreased glucose tolerance and systemic insulin resistance induced by fructose in this model.The levels of insulin,uric acid,hyaluronic acid,TGF-?1,TC and TG levels in serum were significantly increased in fructose-fed rats.Pterostilbene restored these alterations under high fructose consumption in rats.Fructose-induced insulin resistance in the renal cortex of fructose-fed rats,accompanied with tubulointerstitial collagen deposition and EMT in the proximal tubules.EMT is characterized with the decreased expression of E-cadherin and increased expression of a smooth muscle actin(?-SMA)in the proximal tubules.Meanwhile,the expressions of renal cortex collagen at protein and mRNA levels were increased by fructose.Insulin resistance,EMT and collagen overexpression were also observed in high fructose(5 mM)exposed HK-2 cells.These results suggested that high fructose promoted fibrosis in the kidney with insulin resistance.Pterostilbene relieved insulin resistance in the renal cortex of fructose-fed rats and fructose-exposed HK-2 cells.Pterostilbene and pirfenidone alleviated renal fibrosis by reducing EMT and collagen accumulation in vivo and in vitro model under high fructose.The protein levels of TGF-?1,TGFBR1,p-Smad2,p-Smad3 and Smad4 were significantly increased in the renal cortex of fructose-fed rats and fructose-exposed HK-2 cells.The mRNA levels of TGFBR1 and Smad4 were significantly increased by fructose as well.These changes were restored by pterostilbene and pirfenidone.To investigate the possibly role of TGF-?1/TGFBR1/Smads signaling in kidney fibrosis induced by fructose,HK-2 cells were treated with TGFBR1/2 inhibitor and co-incubated with fructose,pterostilbene(5 ?M)and pirfenidone(5 ?M)respectively.TGFBR1/2 inhibitor significantly down-regulated the activation of Smads signaling and over-expressions of collagen ? and ?-SMA,while the E-cadherin protein levels were upregulated in fructose-exposed HK-2 cells.Pterostilbene and pirfenidone failed to restore these alterations fructose exposed HK-2 cells co-incubated with TGFBR1/2 inhibitor.In the last section of this thesis,we focuses on exploring the adverse effect of ROS produced by ATP depletion during the fructose consumption on renal glomerular podocytes.And the mechanism of podocytes injury resulted from redox state unbalance under high fructose,as well as the amelioration of polydatin were further investigated.Male Sprague-Dawley rats were fed with 10%fructose water for 12 weeks.After 7 weeks feeding,the rats were fed with polydatin(10,20,40 mg·kg-1)(n=7)and chloroquine(50 mg·kg-1,positive control)(n=7)for 6 weeks.Serum uric acid and urinary albumin levels were significantly increased in rats with high fructose consumption,which were decreased by polydatin.Fossa fusion was observed in the podocytes of fructose-fed rats,with decreases in the protein levels of Nephrin and podocin in the glomeruli.In vitro,fructose(5 mM)induced fusion of foot process,decreased expressions of split-hole diaphragm membrane protein Nephrin and podocin protein in human podocytes HPCs.These results suggested that high fructose may cause proteinuria by impairing the microstructure and function of podocyte.Polydatin and chloroquine alleviated the fusion of podocyte process in high fructose-fed rats and up-regulated the levels of Nephrin and podocin in glomeruli.Same results were observed in fructose-exposed HPCs incubated with polydatin and chloroquine.Moreover,increased LC3 ? protein levels and decreased levels of LC3 ? protein and p-mTOR were detected in the glomeruli of fructose-fed rats.Fructose decreased LC3 ? and p-mTOR protein levels in HPCs,with LC3 ? expression increased.These results suggested that high fructose may impair podocytes by inhibiting mTOR-mediated autophagy in rats.More importantly,polydatin and chloroquine up-regulated the LC3 I and p-mTOR protein levels and down-regulated LC3 II protein levels in the glomeruli of fructose-fed rats and fructose-exposed HPCs.Fructose-exposed podocytes were treated with polydatin(10 ?M)and chloroquine(5 ?M)with mTOR inhibitor rapamycin(1 ?M)co-incubation to investigate the effect of fructose on autophagy in podocyte.It was proved that rapamycin combined with fructose can not cause further changes in autophagy levels in podocytes.Polydatin restored the changes in autophagy induced by fructose with rapamycin co-treatment,while chloroquine had no effect.In order to investigate the effect of ATP depletion under fructose overconsumption in podocytes,the levels of ATP were assayed in fructose-exposed podocytes at different time points.The results showed that ATP levels continuously decreased from 8 h to 24 h in fructose-exposed HPCs,and reached the lowest level at 24 h.Meanwhile,the uric acid level in podocytes increased significantly at 24 h under high fructose.Moreover,the lysosomal levels were significantly decreased,with the increases in the production of oxidative stress(including total ROS,O2·-and H2O2).These results suggest that t fructose-induced ATP metabolic abnormalities in podocytes induced oxidative stress.Polydatin and chloroquine could significantly down-regulate the levels of uric acid and oxidative stress in HPCs(24 h),and up-regulate the lysosomal level.In addition,it was found that the levels of oxidative stress(including total ROS,O2·-and H2O2)were increased in fructose-fed rats,which were restored by polydatin and chloroquine.The protein level of Nrf2 and the total antioxidant capacity of the glomeruli were significantly decreased in the high fructose-fed rats.In vitro experiments also confirmed that high fructose decreased Nrf2 protein levels(48 h)and total antioxidant capacity(72 h),indicating that high fructose may attenuate Nrf2-mediated antioxidant balance and increase oxidative stress in podocytes.Polydatin and chloroquine significantly up-regulated Nrf2 expression in the nucleus and increased total antioxidant capacity in the glomeruli of fructose-fed rats and fructose-exposed HPCs.Furthermore,the protein levels of p-AMPK? and p-p38 MAPK were increased in the glomeruli of fructose-fed rats.In vitro experiments,we also found that high fructose exposure upregulated p-AMPKa and p-p38 MAPK protein levels in HPCs(24 h).Therefore,it was speculated that fructose may upregulate oxidative stress levels by ATP depletion and inhibiting Nrf2-mediated antioxidant balance.As a result.activation of AMPKa and p38 MAPK signaling increases the mTOR-mediated autophagy levels in podocytes,leading to proteinuria.In summary,the present study further investigated the pathological mechanism of kidney injury under high fructose consumption,including renal insulin resistance,inflammatory response,fibrosis and oxidative stress.Moreover,the new molecular pharmacological activities of morin,pterostilbene and polydatin provides the theoretical and experimental basis for the clinical applications in prevention and treatment of kidney injury in metabolic syndrome.