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Abstract: Ingestion of aristolochic acid (AA) is associated with the development of aristolochic acid nephropathy (AAN), which is characterized by progressive tubulointerstitial fibrosis, chronic renal failure and urothelial cancer. Our previous study showed that bone morphogenetic protein-7 (BMP-7) could attenuate AA-induced epithelial-to-mesenchymal transition (EMT) in human proximal tubule epithelial cells (PTEC). However, how gremlin (a BMP-7 antagonist) antagonizes the BMP-7 action in PTEC remained unsolved. The aim of the current study was to investigate the role of gremlin in AA-induced EMT in PTEC (HK-2 cells). HK-2 cells were treated with AA (10μmol/L) for periods up to 72h. Cell viability was determined by tetrazolium dye (MTT) assay. Morphological changes were assessed by phase-contrast microscopy. Markers of EMT, including E-cadherin and α-smooth muscle actin (α-SMA) were detected by indirect immunofluorescence stains. The BMP-7 and gremlin mRNA and protein expression in HK-2 cells were analyzed by quantitative real-time PCR (real-time RT-PCR) and western blotting after exposure to AA. The level of phosphorylated Smad1/5/8, a marker of BMP-7 activity, was also determined by western blot analysis. Cells were transfected with gremlin siRNA to determine the effects of gremlin knockdown on markers of EMT following treatment with AA. Our results indicated that AA-induced EMT was associated with acquisition of fibroblast-like cell shape, loss of E-cadherin, and increases of alpha-SMA and collagen type I. Interestingly, exposure of HK-2 cells to 10μmol/L AA increased the mRNA and protein expression of gremlin in HK-2 cells. This increase was in parallel with a decrease in BMP-7 expression and a down-regulation of phosphorylated Smad1/5/8 protein levels. Moreover, transfection with siRNA to gremlin was able to recover BMP-7 signaling activity, and attenuate EMT-associated phenotypic changes induced by AA. Together, these observations strongly suggest that gremlin plays a critical role in the modulation of reno-protective action of BMP, and that inhibition of gremlin will be a promising means of developmenting novel treatments for AAN. [Copyright &y& Elsevier]

Abstract: Aristolochic acid nephropathy (AAN) is regarded as a kind of rapidly progressive renal fibrosis caused by the ingestion of herbal remedies containing aristolochic acid (AA). Recent studies showed that bone morphogenetic protein-7 (BMP-7) exerts beneficial effects on acute and chronic kidney injuries induced by different pathological conditions. We examined whether BMP-7 protects human renal tubular epithelial cells (HK-2) against AA-induced injury in vitro. HK-2 cells were cultured with different concentrations of AA and BMP-7 for 48h. Cell viability was determined by Cell Counting Kit-8 assay and lactate dehydrogenase (LDH) release. The apoptosis rate and the activity of caspase 3 protease were also examined. Epithelial-to-mesenchymal transition (EMT) was determined by cell morphology, E-cadherin and α-smooth muscle actin (α-SMA) protein expression, and TGF-β1 and collagen III secretion. Additionally, the effect of anti-TGF-β1 antibody on AA-induced EMT was assessed. Our results indicated that BMP-7 significantly increased cell proliferation, decreased apoptosis rate and attenuated activation of caspase-3, resulting in the protection of HK-2 cells from AA-induced cytotoxicity. In addition, studies on EMT revealed that BMP-7 could inhibit AA-induced myofibroblast phenotype and restored the epithelial morphology in a dose-dependent manner. It was partially through reducing the activation of a myofibroblast phenotype and production TGF-β1. Treatment with neutralizing anti-TGF-β1 antibody also blocked AA-induced EMT 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. [Copyright &y& Elsevier]