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Highlights • AAІ inhibits Slit2-induced migration and tube formation of HUVECs; • Impaired angiogenesis by AA is involved in inactivated Slit2/Robo-NCK signaling. • Overexpression of NCK1 can rescue AAІ-impaired angiogenesis. Abstract Robo1/Robo2-NCK1/NCK2 signaling pathway controls endothelial cell sprouting and migration induced by Slit2 or VEGF, but whether it is involved in peritubular capillary (PTC) rarefaction of Aristolochic acid nephropathy (AAN) is unclear. In the present study, we evaluated whether AA exerts antiangiogenic effects by targeting this signaling pathways in HUVECs. HUVECs or lentivirus-mediated NCK1-overexpressing HUVECs were stimulated with AA (1, 2 or 3 μg/ml) in the absence or presence of 6 nM Slit2. Our results showed that AAІ (1–3 μg/ml) dose-dependently inhibited the migration and tube formation of HUVECs. This inhibition was in parallel with down-regulated mRNA and protein expression of Slit2/Robo1/Robo2-NCK1/NCK2 signaling pathway. Importantly, overexpression of NCK1 rescued AAІ-impaired angiogenesis, as evidenced by the increase of cell migration and tube formation of HUVECs in response to Slit2. The down-regulation of NCK2 and decreased activation of Rac1 was also restored by overexpression of NCK1. Taken together, our findings show that AA inhibits Slit2-induced migration and tube formation via inactivation of Robo1/Robo2-NCK1/NCK2 signaling pathway in HUVECs, and NCK1 might be a potential agent for vascular remodeling in AAN and diseases associated with impaired angiogenesis. [ABSTRACT FROM AUTHOR]

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]