Study On The Mechanism Of Improving Chronic Renal Injury By The Damp-clearing Chinese Medicine Through Regulating RAS

Renal fibrosis is the common process and main pathological feature of all kinds of chronic renal disease developing to end-stage renal disease.Growth factors such as transforming growth factor-β1（TGF-β1）,chemotactic factors such as monocyte chemotactic proteins and renin-angiotensin system（RAS）,TGF-β1/Smad,Wnt/β-catenin pathways are involved in the occurrence and development of renal fibrosis.RAS is composed of renin,angiotensin,angiotensinⅠ,angiotensin-converting enzyme,angiotensinⅡ（AngⅡ）,and angiotensin receptor.Extensive studies have proved that activated renal RAS is involved in renal fibrosis.Damp-clearing drug such as the surface layer of Poria cocos（Schw.）Wolf,Grifola umbellate and Rhizoma alismatis have renoprotective and diuretic action.Based on our previous studies,we investigate the effects on RAS,TGF-β1/Smad and Wnt/β-catenin signaling pathways of poricoic acid ZA（PZA）,and poricoic acid B（PAB）isolated from the surface layer of Poria cocos（Schw.）Wolf（Fulingpi in Chinese）,ergone or ergosterone（ERG）from Grifola umbellate and alisol B 23-acetate（ABA）from Rhizoma alismatis to reveale their anti-renal fibrosis mechanisms.Objective:1.To investigate the mechanism of PZA in improving the injury of human proximal tubular epithelial cells（HK-2）and podocytes（MPC5）through mediating RAS and TGF-β/Smad signaling pathway activated by TGF-βor AngⅡ,and to reveal the mechanism of its renal protective effect.2.To investigate the mechanism of PAB,ERG and ABA in improving the injury of HK-2and MPC5 through mediating RAS and Wnt/β-catenin signaling pathway activated by AngⅡ,and to reveal the mechanism of their renal protective effects.Methods:1.Fulingpi,Rhizoma alismatis and Grifola umbellate was pulverized with methanol and ethanol,extracted with petroleum ether and ethyl acetate,then separated and purified by MCI colum chromatography and HPLC,finally,identified by high resolution mass spectrometry,HNMR and NMR carbon spectroscopy to get PZA,PAB,ERG and ABA.2.The effects of PZA on extracellular matrix components,RAS and TGF-β1/Smad pathway in TGF-β1 or AngⅡinduced HK-2 cells and MPC5 cells were studied by qRT-PCR,siRNA,immunofluorescence staining,immunoprecipitation and Western blots.3.The effects of PAB,ERG and ABA on RAS,Wnt/β-catenin,IκB/NF-κB and Keap1/Nrf2 signaling pathways in HK-2 cells and MPC5 cells induced by AngⅡwere studied by qRT-PCR,immunofluorescence staining and Western Blots.Results:1.PZA,PAB,ABA and ERG were identified by high resolution mass spectrometry,¹H NMR and ¹³C NMR.2.PZA inhibited the expression of AGT,Renin,ACE and AT1 protein in HK-2 cells and MPC5 cells induced by TGF-β1 or AngⅡ,indicating that PZA had an intervention effect on RAS activation.PZA inhibits the expression of fibrotic proteinα-smooth muscle actin（α-SMA）,collagenⅠand fiberectin,and down-regulates the expression of E-cadherin,indicating that PZA can improve the excessive deposition of extracellular matrix components.PZA inhibited the up-regulated expression of TGF-β1,p-Smad2,p-Smad3,and Smad4 and the down-regulated expression of Smad7 proteins,suggesting that PZA has a regulatory role in the activation of TGF-β1/Smad signaling pathway.3.AngⅡinduced the up-regulated protein expressions of AGT,Renin,ACE and AT1 in RAS components in HK-2 cells and MPC5 cells,and the up-regulated protein expressions of Wnt1,Activeβ-catenin andβ-catenin in Wnt/β-catenin signaling pathway.ICG-001,aβ-catenin inhibitor,inhibited the up-regulation of RAS,while Losartan（LOS）,an AngⅡtype 1 receptor blocker,inhibited the activation of Wnt/β-catenin signaling pathway,suggesting that RAS interacts with the Wnt/β-catenin signaling pathway.AngⅡinduced the expression ofα-smooth muscle actin（α-SMA）,collagenⅠand fiberectin in HK-2 cells were up-regulated,and the expression of E-cadherin was down-regulated,indicating that AngⅡinduced excessive deposition of extracellular matrix components.The expression of podocin,nephrin,podocalyxin,synaptopodin and other proteins in MPC5 cells induced by AngⅡwere significantly down-regulated,indicating that Ang can cause podocyte injury.The expression of Wnt1,Activeβ-catenin,β-catenin and their downstream target proteins Snail1,Twist,matrix metalloproteinase-7（MMP-7）,and fibroblast specific protein 1（FSP-1）in HK-2 cells and MPC5 cells induced by AngⅡwere up-regulated.The results showed that RAS activated the Wnt/β-catenin signaling pathway.The protein expressions of MCP-1 and COX-2 and Nrf-2 and HO-1 in HK-2 cells and MPC5 cells induced by AngⅡwere significantly up-regulated,indicating that RAS activated the IκB/NF-κB and Keap1/Nrf2 signaling pathways.PAB,ABA and ERG can regulate RAS,Wnt/β-catenin,IκB/NF-κB and Keap1/Nrf2 signaling pathways in HK-2 cells and MPC5 cells.Conclusion:The active components PZA,PAB,ABA and ERG in Fulingpi,Rhizoma alismatis and Grifola umbellate can improve renal injury,and play a role in renal protection by regulating RAS,TGF-β1/Smad,Wnt/β-catenin,IκB/NF-κB and Keap1/Nrf2 signaling pathways,respectively.The study revealed the mechanism of these damp-clearing Chinese medicine medicines to improve kidney injury.