Protective Effect Of BMP-7 Against Aristolochic Acid-induced Renal Tubular Epithelial Cell Injury

Background and objectiveRecently, Chinese herbal medicine containing aristolochic acid (AA)-induced nephrotoxicity called Aristolochic acid nephropathy(AAN) has been caused widespread interests by many scholars. Animal experiments and clinical data have confirmed that the main pathological appearance of AAN is a progressive renal tubulointerstitial fibrosis.AAN makes a faster progress than any other chronic interstitial nephritis,which will develop into final stage renal failure in several months or one to two years. It has caused a poor prognosis and big harm to people's health.In order to find out new cognition and medicines to provide important help for improving the level of treating AAN, it is very important to deploy the mechanism of AAN and design the method in prevention and cute.Bone morphogenetic protein-7(BMP-7), also known as osteogenic protein-1(OP-1), is a member of the transforming growth factor-β(TGF-β) superfamily, which plays a crucial role in renal development and many animal models of renal disease, such as ureteral obstruction model, renal ischema model, diabetic nephropathy model, IgA nephropathy model . However, its role in AAN has not yet been explored.In the present study, we developed a cellular model of AAN and investigated the renalprotective effect of BMP-7 on AA-induced tubular epithelial cell injury. MethodThe study was followed as two parts.Part one: Aristolochic acid induced injury in human renal tubular epithelial cells in vitro. In order to find out the best vulnerate concentration, cultured HK-2 was divided into four groups: normal control,treatment with AA at the concentration of 30μmol/L, 60μmol/L and 120μmol/L.Part two: Protective effect of BMP-7 against aristolochic acid-induced renal tubular epithelial cell injury . In Part one,we have found out that 60μmol/L was the best vulnerate concentration. Cultured HK-2 was divided into four groups: normal control, treatment with AA at the concentration of 60μmol/L, 60μmol/L AA combined with 500ng/mL or 1000ng/mL BMP-7.After treatment for 48h, in order to observe the relationship between AA and nephrotoxicity and whether BMP-7 take the protective effect,the cells were harvested for following assays.The morphological changes were observed by inverted phase contract microscope. Cell viability was determined by Cell Counting Kit-8 assay and lactate dehydrogenase (LDH) release. The apoptosis rate was identified by flow cytometry. The expression of active caspase 3 protease was measured by Western blot analysis. E-cadherin andα-smooth muscle actin (α-SMA) protein expression were observed by immunostaining.TGF-β1 and collagen III secretion were determined by ELISA.ResultsPart one:1.Untreated, control culture of HK-2 cells produced a confluent monolayer with cobblestone morphology . After treatment with 30μmol/L AA, a few cells were spindle shaped. When treated with 60μmol/L AA, a significant number of cells showed an elongated, spindle shaped, fibroblastic. When exposed to 120μmol/L AA, more cells appeared shrunken, rounded and underwent necrosis.2.AA inhibited HK-2 cells proliferation,induced apoptosis and increased the LDH level.The apoptosis might be caused by the expression of active Caspase-3.3.AA could inhibited E-cadherin expression,increasedα-SMA expression and TGF-β1 and collagen III secretion especially at the lower concentration (30,60μmol/L).4.In this experiment, we found that AA at 60μmol/L caused apparent injury appearance and induced EMT obviously. Therefore we chose this concentration (60μmol/L) for the following experiments.Part two: When incubated with both AA 60μmol/L and BMP-7 (500 ng/ml, 1000 ng/ml), morphology changes were not obvious. BMP-7 significantly increased cell proliferation, decreased apoptosis rate and LDH secretion ,and attenuated expression of active caspase-3. BMP-7 also decreased TGF-β1 and collage III secretion ,inhibited AA-induced myofibroblast phenotype and restored the epithelial morphology in a dose-dependent manner.Conclusion1. AA inhibited HK-2 cells proliferation,induced apoptosis and increased the LDH level.The mechanism of apoptosis might be caused by the expression of active Caspase-3.2. The lower concentration of AA(30,60μmol/L) could induce EMT in HK-2.The EMT might be related to TGF-β1 secretion.3. BMP-7 significantly increased cell proliferation, decreased apoptosis rate in HK-2 treated with AA .The mechanism of decreased apoptosis might be that BMP-7 could attenuated the expression of active Caspase-3.4.BMP-7 significantly reversed AA-induced EMT in HK-2,which might be related to the reason that BMP-7 could decrease TGF-β1 and collagen III secretion.Together, these observations strongly suggest that BMP-7 is a potent inhibitor of AA-induced renal tubular epithelial cell injury and might be a promising agent for aristolochic acid-induced kidney damage.