Role Of Wnt/?-catenin Signaling On AKI To CKD Progression And Blood Pressure Elevation

BackgroundThe incidence of acute kidney injury(AKI)is increasingly worldwide and causes 2 million people death every year.It is showed that AKI incidence in the inpatient department is 3.19%and increase to 30%in the Intensive Care Unit(ICU)by China clinical studies.AKI is increasingly recognized as a major risk factor leading to subsequent progression to chronic kidney disease(CKD).Therefore,it is really urgent to take measures to prevent AKI from developing CKD.It was thought that AKI is a kind of reversible disease and injured kidneys could be recovered.However,recently clinical studies showed AKI patients after hospital discharge,those with damaged renal function could be more easily progress to CKD,end stage renal disease(ESRD)and death compared with these with normal renal function.Even those AKI patients with normal renal function after hospital discharge,they will more probably develop to CKD compared with patients without AKI.All these clinical studies show that AKI is an independent risky factor leading to CKD progression.However,what factors govern AKI to CKD progression remains poorly understood.The renin-angiotensin system(RAS)plays a fundamental role in controlling blood pressure,body fluid balance and tissue homeostasis.RAS consists of several key components including angiotensinogen(AGT),renin,angiotensin converting enzyme(ACE),angiotensin type ? and type ?receptors(AT1 and AT2).In normal physiological conditions,RAS components are tightly regulated and separately produced by distinct organs such as liver,kidney and lung,respectively,which ensures a delicate control of their activities in the circulation.After kidney injury,however,intra-renal RAS is markedly activated,as a result of the simultaneous up-regulation of many RAS components in diseased kidneys.Intra-renal RAS components exercise numerous biological actions,such as hypertension,renal inflammation and fibrosis.Therefore,using angiotensin converting enzyme inhibitors(ACEI)or/and angiotensin receptor blockers(ARB)as first-line treatment for CKD.However,current anti-RAS therapy only displays limited efficacy,partly because of compensatory up-regulation of renin expression.As a result,in addition to ACEI and ARB drugs,we need new therapeutic strategies to slow the progression of CKD and Blood pressure(BP)elevation.The Wnt/?-catenin is a development pathway,plays an essential role in organogenesis,tissue homeostasis and tumor formation.The Wnt family as secreted signaling proteins with 19 different Wnts.The canonical intracellular signaling route is ?-catenin-dependent.Wnt proteins transmit their signal across the plasma membrane through interacting with serpentine receptors,the Frizzled(Fzd)family of proteins,and co-receptors,members of the LDL receptor-related protein(LRP5/6).Upon binding to their receptors,Wnt proteins induce a series of downstream signaling events resulting in dephosphorylation of ?-catenin.This leads to the stabilization of P-catenin,rendering it to translocate into the nuclei,where it binds to T cell factor/lymphoid enhancer-binding factor(LEF)to stimulate the transcription of Wnt target genes.In adult kidney,Wnt signaling seems to be silenced.However,our studies show that Wnt/?-catenin signaling is re-activated in a wide variety of AKI,CKD animal models and human kidney diseases.What's more,we recently demonstrated that ?-catenin,the principal intracellular mediator of canonical Wnt signaling,is a master transcriptional regulator controlling the expression of all RAS genes in diseased kidneys,including AGT,renin,ACE,ATl and AT2.We speculate that sustained activation of Wnt/?-catenin signaling may plays important roles in accelerating AKI to CKD,and promoting BP elevation by the virtue of its ability to promote RAS activation.Since Wnt/p-catenin signaling has been shown to play a role in renal fibrosis,block this pathway could prevent or relief the AKI progression and BP elevation.However,many Wnt were of activation in diseased kidneys,it is inefficient to target single Wnt to slow kidney disease progression.As all the canonical Wnt signaling is P-catenin-dependent,block P-catenin could be an effective and specific target to ameliorate renal fibrosis and CKD progression.Upon activation,p-catenin binds to T-cell factor(TCF)/lymphoid enhancer-binding factor(LEF)family of transcription factors in the nucleus,and then recruiting the transcriptional co-activators,cyclic AMP response-element binding protein(CREB)binding protein(CBP),to trans-activate its target genes.Therefor,disruption of the p-catenin binding to CBP could be an effective strategy to block trans-activate its target genes.In that regard,recent studies have identified ICG-001,a peptidomimetic small molecule that selectively inhibits ?-catenin signaling in a CBP-dependent fashion.ICG-001 is shown to repress the(3-catenin-driven gene transcription and inhibit bleomycin-induced lung fibrosis in mice.However,whether targeted inhibition of?-catenin/CBP signaling by ICG-001 prevents AKI progression and BP elevation remains unknown.In this study,we will examine the effects of activation of Wnt/?-catenin on AKI to CKD progression,and BP elevation.And present a novel and effective approach of a small-molecule inhibitor ICG-001 target ?-catenin signaling for the treatment of AKI to CKD progression,and BP elevation.Chapter ISustained activation of Wnt/?-catenin signaling drives AKI to CKD progressionObjectiveTo investigate the role of sustained activation of Wnt/?-catenin signaling on AKl to CKD progression,and evaluate the effective and feasible treatment of inhibiting ?-Catenin signaling to prevent AKI from developing to CKD.Methods1.Animal experiments1)Male C57BL/c mice were randomly divided into three groups:Sham controls,moderate bilateral renal ischemia/reperfusion injury(BIRI)and server BIRI.For this model,AKI happens at 1 to 3 days post-surgery and gradually develops to CKD at 10 days.Sham controls only exposed renal pedicle without clamping.For BIRI model,bilateral renal pedicles were clamped for 20 or 30 minutes using non-traumatic vascular clamps for moderate or severe kidney injury.Mice were sacrificed at 1,3 and 10 days post-surgery,blood and kidney tissues were harvested for renal function assay,RT-PCR and Western blot respectively.2)For the unilateral renal ischemia/reperfusion injury(UIRI)model,male BALB/c mice were undergo unilateral clamping of the renal pedicle for 30 minutes followed by contralateral nephrectomy at 10 days post-surgery.At 5 days post-surgery,mice were subjected to either a single intravenous injection of Wntl expression plasmid or daily intraperitoneal injections of ICG-001 for 6 days.Mice were sacrificed at 11 days post-surgery;blood and kidney tissues were harvested for renal function assay,immunohistochemistry,immunofluorescence,Real-time PCR and Western blot respectively.3)Serum creatinine levels were measured by using QuantiChrom Creatinine Assay Kit(DICT-500).And blood urea nitrogen levels were measured by using QuantiChrom Urea Assay Kit(DIUR-500).4)The induction of Wnt mRNA induced by moderate and server BIRI at 1,3 and 10 days post-surgery were determined by RT-PCR.5)The ?-catenin protein expression induced by moderate and server BIRI at 1,3 and 10 days post-surgery were analyzed by Western blot analysis.6)Collagen deposition and fibrotic lesions was examined by Masson-Trichromatic staining.The location and expression of Wntl,(?-catenin,a-SMA,MMP-7,ACE and renin were determined by immunohistochemistry.7)The induction of fibronectin was analyzed by immunofluorescence.8)The mRNA level of Wntl,?-SMA,fibronectin,Collagen ?,Collagen ?,Snail1,MMP-7 and FSPlwere determined by Real-time PCR?9)The expression of Wntl,?-catenin,fibronectin,?-SMA and PAI-1 protein level was examined by western blot.2.Cell experiments1)Preparation of Wnt condition medium Human proximal tubular epithelial cells(HKC,clone-8)were provided by provided by Dr.L.Racusen(Johns Hopkins University,Baltimore,Maryland).Cells were maintained in DMEM/F-12 containing 10%Fetal Bovine Serum.HKC-8 cells were transfected with various expression vectors,such as pHA-Wnt1 pV5-Wnt2,pV5-Wnt3a,pHA-Wnt4 and pV5-Wnt16.After 48 hours transfection,the cell supernatant of Wnt condition medium was harvested for further use.2)Normal rat kidney interstitial fibroblast(NRK-49F)cells were obtained from the American Type Culture Collection(Manassas,VA).Cells were maintained in DMEM/F12 medium supplemented with 10%fetal bovine serum.Serum starved NRK-49F Cells were treated with different concentration of Wnt condition medium as indicated.In some experiments,cells were incubated with ICG-001 at 10?M.And then cells were collected and subjected to various analyses.3)The induction of Fibronectin,Vimentin and Collagen ? were measured by immunofluorescence.4)The protein expression of Fibronectin was examined by western blot.5)The mRNA level of Fibronectin,FSP1,Collagen?and Collagen ?were determined by Real-time PCR?ResultsAnimal experiments1.Effect of moderate and server BIRI on activation of Wnt/?-catenin signaling1)Renal function assay displayed that the level of Scr(F=29.292,P=0.000)in mice of moderate BIRI were significant increased at 1 day post-surgery compared with sham controls,and reduced to normal level at 3 and 10 days post-surgery.While the level of Scr(F=114.061,P=0.000)induced by server BIRI was significant increased at 1 day post-surgery,and still higher than sham controls at 3,10 days post-surgery.2)RT-PCR results demonstrated that compared with sham controls,the expression of many Wnt mRNA in mice of moderate BIRI were dramatically upregulated at 1 day post-surgery,such as Wntl,2,3,3a,7a,8a,8b and 16.And the mRNA level of these Wnts were decreased at 3 days and backed to normal level at 10 days post-surgery.However,the expression of Wnt mRNA induced by server BIRI were dramatically upregulated at 1 and 10 days post-surgery,such as Wntl,3a,4,5b,7a,7b,9b,10a,10b and 16.3)Western blot analysis indicated that the ?-catenin(F=67.095,P=0.000)protein expression induced by moderate BIRI at 3 days post-surgery was significant increased compared with sham controls.While ?-catenin(F=16.212,P=0.000)protein expression in mice of sever BIRI was greatly increased at 1 day post-surgery,and with the peak induction at 3 days post-surgery.4)RT-PCR results demonstrated that,compared with moderate BIRI,the expression of many Wnt mRNA in mice of sever BIRI were dramatically upregulated at 1 day post-surgery,such as Wntl,2,3,3a,7a,7b,8a,8b,10a,10b and 16.5)Western blot analysis indicated that the ?-catenin(F=41.267,P=0.000)protein expression induced by severe BIRI at 3 days post-surgery was significant increased compared with moderate BIRI.2.Effect of over expression of Wnt on AKI to CKD progression1)Renal function assay displayed that the level of Scr(F=28.760,P=O.OOO)and BUN(F=31.719,P=0.000)induced by UIRI was significant increased compared with sham controls.Of note,the level of Scr and BUN in UIRI mice received Wntl plasmid injection was higher than mice with pcDNA3 plasmid injection.2)Masson staining results demonstrated that collagen deposition(F=56.824,P=0.000)in the renal interstitial in mice of UIRI was much more than sham controls.And collagen deposition in UIRI mice received Wntl plasmid injection was much more than mice with pcDNA3 plasmid injection.3)Immunohistochemistry data revealed that the distribution of Wntl,?-catenin,MMP-7 and renin were induced in cytoplasm,and a-SMA located in interstitial.The expression of Wntl,?-catenin,MMP-7,a-SMA and renin proteins in mice of UIRI were dramatically increased compared with sham controls.Of note,these proteins in UIRI mice with Wntl plasmid injection were much more.4)Immunofluorescence analysis indicated that fibronectin located in interstitial.Compared with sham controls,the expression of fibronectin protein induced by UIRI was significant increased,which was much more in UIRI mice with Wntl plasmid injection.5)Western blot results showed that compared with sham controls,the expression of?-catenin(F= 48.522,P=O.OOO),Fibronectin(F=77.352,P=0.000),a-SMA(F=78.627,P=O.OOO)and PAI-1(F=38.037,P=0.000)proteins induced by UIRI were significant increased,which were much more in UIRI mice with Wntl plasmid injection.6)Real-time PCR results displayed that compared with sham controls,the expression of a-SMA(F=12.302,P=0.001),MMP-7(F=24.334,P=0.000),Fibronectin(F=15.223,P=O.001),Collagen I(F=8.064,P=0.006),Collagen III(F=8.655,P=0.005)and FSPl(F=26.984,P=0.000)mRNA in mice of UIRI was significant increased,which were much more in UIRI mice with Wntl plasmid injection.3.Effects of block Wnt/?-catenin signaling on AKI to CKD progression1)Renal function assay displayed that the levels of Scr and BUN induced by UIRI were significant increased compared with sham controls.While ICG-001 treatment could obviously attenuated the levels of Scr(F=43.154,P=0.000)and BUN(F=25.984,P=0.000)compared with vehicle controls.2)Masson staining results demonstrated that compared with sham controls,collagen deposition in the renal interstitial induced by UIRI was dramatically up regulated.However,ICG-001 therapy significantly reduced collagen deposition(F=116.347,P=0.000)compared with vehicle controls.3)Immunohistochemistry data revealed that the distribution of ?-catenin,?-SMA and renin proteins in mice of UIRI were dramatically increased compared with sham controls.While ICG-001 treatment could attenuated these proteins expression.4)Immunofluorescence analysis indicated that compared with sham controls,the expression of Fibronectin protein in vehicle controls was significant increased,While ICG-001 treatment could inhibit Fibronectin protein expression.5)Western blot results showed that compared with sham controls,the expression of?-catenin(F=17.416,P=0.000),fibronectin(F=13.978,P=0.000),a-SMA(F=12.615,P=0.000)and PAI-1(F=24.711,P=0.001)proteins in vehicle controls was significant increased.However,ICG-001 therapy significantly reduced these proteins deposition.6)Real-time PCR results displayed that compared with sham controls,the expression of MMP-7(F=28.216,P=0.000),Fibronectin(F=17.416,P=0.000),Collagen ?(F=13.978,P=0.000),Collagen ?(F=12.615,P=0.000),FSP1(F=12.615,P=0.000)Snail1(F=6.158,P=0.000)and a-SMA(F=24.711,P=0.001)mRNA were significant increased in vehicle controls.And all mRNA was attenuated after ICG-001 therapy.Cell experiment1)Cell counting and MTT results showed that cells numbers(F=43.448,P=0.000)and OD value(F=18.487,P=0.000)were significant increased in NRK-49F cells treated with 10%or 40%Wnt condition medium for 24 hours.And these results could be ameliorated by ICG-001.2)BrdU results indicated that Wnt condition medium could obviously promoted NRK-49F cells proliferation,which could be abolished by ICG-001.3)Western blot results displayed that compared with controls,the expression of Fibronectin(F=208.122,P=0.000)protein was significant increased in NRK-49F cells treated with 10%or 40%Wnt condition medium for 24 or 48 hours,which could be blocked by ICG-001.And the expression of Fibronectin protein was dramatically decreased in NRK-49F cells after withdrawing Wnt condition medium.4)Immunofluorescence analysis indicated that Fibronectin,Collagen ? and Vimentin were significantly induced in NRK-49F cells treated with 10%or 40%Wnt condition medium for 24 hours.And ICG-001 treatment could reduce Fibronectin,Collagen ? and Vimentin proteins expression in NRK-49F cells.5)Real time-PCR results displayed that the expression of Fibronectin(F=55.445,P=0.000),Collagen?(F=23.098,P=0.000),Collagen ?(F=92.855,P=0.000)and FSP1(F=9.006,P=0.002)mRNA were significantly increased in NRK-49F cells induced by 40%Wnt condition medium for 48 hours.And all these mRNA were reduced after ICG-001 therapy.Conclusions1.Moderate IRI accounted for transient activation of Wnt/?-catenin signaling,while server IRI leaded to sustained activation of Wnt/?-catenin signaling and promoted AKI to CKD.2.Over expression of Wntl promoted activation of Wnt/?-catenin signaling,which accelerated AKI to CKD progression in vivo.3.Using ICG-001 target ?-catenin signaling could prevent AKI from developing CKD in vivo.4.Wnts condition medium promoted proliferation and activation of renal fibroblast cells to myofibroblast cells,which accelerate renal fibrosis.Chapter ?Wnt/?-catenin signaling participates in blood pressure regulationObjectiveTo investigate the role of Wnt/p-catenin signaling on blood pressure regulation,and evaluate the effective and feasible treatment of inhibiting ?-Catenin signaling to prevent hypertension and renal injury.MethodsAnimal experiments1.Angiotensin ?(Ang ?)infusion experiments1)Male SD Rats were divided into three groups:sham control,Ang II infusion rats treated with vehicle,and Ang II infusion rats injected with ICG-001.Ang II infusion model using osmotic mini-pump implanted subcutaneously.The blood pressure of rats was measured by the tail-cuff technique at 1,3 and 7 days post-surgery.At 7 days after Ang II infusion,all rats were sacrificed.Urine,blood and kidney tissue were collected for various analyses.2)Urinary albumin level was determined using rat Albuminuria ELISA Quantization kit.3)The mRNA level of Wnts were determined by RT-PCR.4)The protein expression of ?-catenin,ACE and AT1 were examined by western blot.2.5/6NX experiments1)For the 5/6NX model,male SD rats,weighting 200-220g,were subjected to 5/6NX using two-step protocol.Briefly,under general anesthesia,rats were subjected to surgical resection of two thirds of the left kidney.One week later,rats underwent right nephrectomy.Sham-operated rats had their kidneys exposed without nephrectomy.Six weeks post-operation,the 5/6NX rats were assigned into three groups:sham control,5/6NX rats treated with vehicle,and 5/6NX rats injected with ICG-001.ICG-001 was administered by intraperitoneal injection three times per week.The blood pressure of rats was measured by the tail-cuff technique at 6,8,10 and 12 weeks post-surgery.At 12 weeks after 5/6NX,all animals were sacrificed.Urine,blood and kidney tissues were collected for analysis.2)Serum and urine creatinine as well as blood urea nitrogen levels were measured by using automatic biochemical analyzer.3)Urinary albumin level was determined using rat Albuminuria ELISA Quantization kit.4)The expression of Fibronectin and Collagen I were examined by immunofluorescence.5)Renal morphological changes were measured by PAS staining.Collagen deposition was examined by Masson-Trichromatic staining.The location and expression of Wntl,?-catenin,a-SMA,AGT,ACE,AT1,renin,CD3 and CD68 were determined by immunohistochemistry.6)The mRNA level of Fibronectin,Collagen I,Collagen III,PAI-1 Rantes and TNF-a were determined by Real-time PCR.7)The protein expression of ?-catenin,Fibronectin and Collagen I were examined by western blot.Cell experiments1)The NRK 49F cells were seeded on six-well culture plates to 60 to 70%confluence in the complete medium containing 10%fetal bovine serum for 16 hours,and then changed to serum-free medium after washing twice with medium.Angiotensin II was added to the culture at the concentrations as indicated and incubated for 24 h.Cells were then analyzed for Wnt mRNA expression.2)HKC-8 cells were transfected with Wntl expressing vectors(pHA-Wntl),N-terminally truncated,constitutively activated P-catenin expression vector(pDel-?-cat)or empty vector(pcDNA3)by lipofectamine 2000(Invitrogen,Carlsbad,CA),according to the manufacturer's protocol.In some experiments,cells were incubated with ICG-001 at 1O?M or losartan at 10-6 M.Whole-cell lysates were prepared and subjected to Western blot analyses.Results1.Angiotensin II(Ang II)infusion experiments1)Blood pressure results of ail-cuff technique indicated that compared with sham controls,rats of Ang II infusion caused significant SBP(F=30.329,P=0.000),MBP(F=31.880,JP=0.000)elevation at 3 days post-surgery.And with peak infuction of SBP(F=30.572,P=O.OO1),MBP(F=25.819,P=0.000)at 7 days post-surgery.However,rats with injections of ICG-001 completely alleviated the elevation in BP induced by Ang ?.2)Urinary albumin results showed that rats of Ang II infusion significantly elevated albuminuria(F=14.359,P=0.000)at 7 days post-surgery.While injections of ICG-001 obviously attenuated albuminuria in rats induced by Ang II.3)Western blot results showed that compared with sham controls,the expressions of?-catenin(F=13.265,P=0.001),ACE(F=11.539,P=0.002)and AT1(F=26.597,P=0.000)proteins in rats of Ang II infusion were significant increased.However,ICG-001 therapy significantly abolised these proteins deposition.4)RT-PCR results displayed that compared with sham controls,the expression of Wnt mRNA was significant increased in rats of Ang II infusion.There were no changes of Wnt mRNA expression after ICG-001 therapy.2.5/6NX experiments1)The results of blood pressure revealed that compared with sham controls,rats of 5/6NX caused significant SBP(F=8.077,P=0.004)and MBP(F=9.105,P=0.003)elevation at 12 weeks post-surgery.However,rats with injections of ICG-001 obviously attenuated the elevation in BP induced by 5/6NX.2)Renal function assay showed that the level of Scr(F=69.692,P?0.000)and BUN(F=63.449,P=0.001)induced by 5/6NX was significantly increased compared with sham controls.However,rats with injections of ICG-001 alleviated the elevation in Scr and BUN induced by 5/6NX.3)ELISA results showed that albuminuria was significantly elevated(F=60.011,P=0.000)in rats of 5/6NX at 12 weeks post-surgery.While injections of ICG-001 obviously attenuated albuminuria in rats induced by 5/6NX.4)Masson staining results demonstrated that collagen deposition(F= 102.093,P=0.000)in the renal interstitial in rats of 5/6NX was much more than sham controls.And ICG-001 treatment attenuated the collagen deposition in rats with 5/6NX.5)PAS staining demonstrated that varying degrees of glomerular hypertrophy and sclerosis in the rats of 5/6NX compared with sham controls.While ICG-001 therapy improved these symptoms.6)Immunohistochemistry data revealed that the distribution of ?-catenin,AGT and renin were induced in cytoplasm;a-SMA,CD3 and CD68 located in interstitial;AT1 located in cytoplasm and interstitial and ACE lied in brush border membrane.The expression of P-catenin,a-SMA,AGT,ACE AT1,renin,CD3 and CD68 proteins in rats of 5/6NX were dramatically increased compared with sham controls.While ICG-001 treatment could obviously reduced these proteins expression.7)Immunofluorescence analysis indicated that compared with sham controls,the expression of Fibronectin and Collagen I proteins in rats of 5/6NX were significantly increased.While ICG-001 treatment could abolised these proteins expression.8)Western blot results showed that compared with sham controls,the expression of Fibronectin(F=11.485,P=0.001)and Collagen I(F=6.959,P=0.007)proteins in rats induced by 5/6NX were significantly increased.However,ICG-001 therapy significantly attenuated these proteins deposition.9)Real time-PCR results displayed that compared with sham controls,the expression of Fibronectin(F=6.288,P=0.000),Collagen I(F=15.806,P=0.000),Collagen III(F=17.767,P=0.001),PAI-1(F=14.135,P=0.001),Rantes(F=4.951,P=0.022)and TNF-a(F=10.926,P=0.001)mRNA were significant increased in rats of 5/6NX.And all mRNA expressions were decreased after ICG-001 therapy.3.Cell experiments(1)Compared-with controls,incubation of NRK-49F cells with Ang II induced the mRNA expression of numerous Wnt genes in a dose-dependent manner.(2)HKC-8 cells were transfected with constitutively activated p-catenin expression vector(pDel-?-cat).Over-expression of ?-catenin induced the expression of Fibronectin,a-SMA and Snaill proteins in HKC-8 cells.However,incubation with ICG-001 completely abolished the induction of these proteins.Interestingly,blockade of RAS signaling by losartan also abolished the ?-catenin-triggered induction of Fibronectin,a-SMA and Snail l in HKC-8 cells.Similarly,losartan also inhibited the expression of Fibronectin,a-SMA and Snaill induced by over-expression of Wntl.Conclusion1.Wnt/?-catenin is activated in two rat models of experimental hypertension:chronic Ang II infusion and remnant kidney after subtotal renal ablation.2.Wnt?-catenin activation is accompanied by intra-renal RAS activation and development of hypertension and kidney injury.3.Inhibition of ?-catenin with a small-molecule inhibitor ICG-001 lowers BP,represses RAS expression,reduces proteinuria,inhibits renal inflammation and fibrosis,and preserves kidney function.4.RAS activation is required for the Wnt/?-catenin-mediated fibrogenic response.