| Background and objective:Aristolochic acid nephropathy (AAN), a rapidly progressive tubulointerstitial renalfibrosis was reported in patients after ingestion of Chinese herb containing aristolochic acid(AA) in1993by Belgian nephrologists. AAN is characterized by progressivetubulointerstitial fibrosis, chronic renal failure and urothelial cancer. However, the precisemechanism of the renal injury caused by AA is unexplored. In our previous study, wedemonstrated that BMP-7was a potent inhibitor of AA-induced renal tubular epithelial cellinjury，but the mechanism of BMP-7in AAN is unclear. Gremlin，a BMP-7antagonist，whether it could antagonize the BMP-7action in PTEC remained unsolved. So weinvestigated the role of gremlin in AA induced EMT in PTEC (HK-2cells), deploying themechanism of AAN and design the method in prevention and cuteIn the present study, we developed a cellular model of AAN and investigated theexpression of gremlin, BMP-7and P-Smad1/5/8in AA induced EMT. Moreover, the studyexplored the mechanism of gremlin, regulating the renoprotective actions of BMP-7inAAN. It will help to illustrate the mechanism of aristolochic acid nephropathy and providea new therapy strategie.Method：The study was followed as two parts.Part one: The expression of gremlin and activation of BMP-7after aristolochic acidinduced epithelial-to-mesenchymal transition (EMT) in human renal tubular epithelial cellsin vitro.1. The best concentration of AA which induced epithelial-to-mesenchymal transition(EMT) in human proximal tubule epithelial cells was chosen by MTT.2. Using the best concentration of AA (10μmol/L) induced HK-2cells for0h,24h,48h, 72h, the morphological changes were observed by inverted phase contract microscope.TheBMP-7and gremlin mRNA and protein expression in HK-2cells were analyzed byquantitative real-time PCR (RT-PCR) and western blotting after being cultured for0h,24h,48h,72h exposure to AA. The level of phosphorylated Smad1/5/8, a marker of BMP-7activity, was also determined by western blotting analysis. E-cadherin, α-smooth muscleactin (α-SMA) and collagen type I expression were observed by immunostaining.Part two: The effect of gremlin silencing on the expression of gremlin and activationof BMP-7under the condition of AA induced HK-2epithelial-to-mesenchymal transition(EMT).1. According to gremlin mRNA sequence in Genebank of NCBI and using softwaredesign and reference literature, we designed3siRNA oligonucleotides sequences targetingto Gremlin mRNA (named siRNA-Gremlin-1，siRNA-Gremlin-2，siRNA-Gremlin-3) and1non-sense sequence (siRNA-Gremlin-neg).The shRNA templates were cloned into shRNAexpression vector pcDNA6.2-GW/EmGFPsiR, which construct the sepecificity RNAplasmid targeting inhibited the expression of Gremlin(named pshRNA-Gremlin-1,pshRNA-Gremlin-2, pshRNA-Gremlin-3), and negative control vector(namedpshRNA-Gremlin-neg) contains the non-sense sequence.2. The HK-2cells cultured in the condition of AA were transfected with combinedplasimid (pshRNA-Gremlin-3) by liposome Lipofectamine2000.Use the empty plasmidsas blank control. Cultured HK-2was divided into four groups:1) Normal control group:cells were cultured in DMEM/F12medium.2) AA group: cells were cultured inDMEM/F12medium containing AA((10μmol/L).3) AA+negative control group: cellswere transfected with pshRNA-Gremlin–neg and cultured in DMEM/F12mediumcontaining AA((10μmol/L).4) AA+Gremlin siRNA group: cells were transfected withpshRNA-Gremlin-3and cultured in DMEM/F12medium containing AA((10μmol/L).3. The morphological changes were observed by inverted phase contract microscope.after collectting of cells of every group. The BMP-7and gremlin mRNA and proteinexpression in HK-2cells were analyzed by quantitative real-time PCR (real-time RT-PCR)and western blotting. The level of phosphorylated Smad1/5/8, E-cadherin, α-smooth muscle actin (α-SMA) and collagen type I were also determined by western blot analysis.E-cadherin and α-smooth muscle actin (α-SMA) expression were observed byimmunostaining.Results:Part one:1. We have found out that10umol/L was the vulnerate concentration. The HK-2cellsresulted in morphological change to a fibroblast-like shape identifiable by the presence ofelongated lamellipodia and a spindle shape in the time-dependent.2. Incubation of cultured HK-2cells under AA (10umol/L) conditions for differenttimes revealed a gradual decrease of E-cadherin expression and increase of α-SMAexpression.3. Incubation of cultured HK-2cells under AA conditions for different times revealeda remarkable increase in gremlin mRNA and protein expression as early as24h, and then agradual increase for48h and72h as demonstrated by real-time RT-PCR and Westernblotting, respectively. The BMP-7expression was gradually decreased at both the mRNAlevel and protein level, especially for72h. At the same time, the level of phosphorylatedSmad1/5/8, a marker of BMP-7activity, significantly and gradually went down when thecells were treated with AA.Part two:1. Gremlin siRNA significantly inhibited AA-induced HK-2cells gremlin mRNA aswell as protein expression, while had no effect on BMP-7expression. Transfection withgremlin siRNA reverted the AA-induced decreased levels of phosphorylated Smad1/5/8inHK-2cells2. Gremlin siRNA significantly inhibited AA-induced HK-2cells E-cadherindecreasing, α-SMA and collagen type I protein increasing. Phase-contrast photomicroscopeof the cultures revealed that knockdown of gremlin significantly reversed thespindle-shaped morphology to a cobblestone appearance.Conclusion：1. Aristolochic acid (AA) increased expression of gremlin and decreased the level of BMP-7and phosphorylated Smad1/5/8in HK-2Cells.The balance of gremlin/bmp-7wasdisturbed in EMT.2. Gremlin siRNA significantly reverted the AA-induced decreased levels ofphosphorylated Smad1/5/8, while had no effect on BMP-7expression.The mechanism ofEMT might be that the increasing of gremlin regulated the activity of BMP-7.3. Transfection with siRNA to gremlin will attenuate EMT induced by AA in HK-2cells. Inhibition of gremlin might be a promising agent for aristolochic acid-induced kidneydamage. |
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