FC 110PTH INDCUED ENDMT VIA MIR-29A-5P/GSAP/NOTCH1 PATHWAY CONTRIBUTED TO VALVULAR CALCIFICATION IN RATS WITH CKD

Abstract

Abstract Background and Aims Endothelial-to-mesenchymal transition (EndMT) of valvular endothelial cells is the common pathophysiology of valvular calcification (VC) among the patients of non-chronic kidney disease (CKD). However, there are few studies of valvular calcification in CKD. In our previous work, parathyroid hormone (PTH) was considered as an important player of EndMT in vascular diseases. Meanwhile, the increasing serum level of PTH in CKD patients is closely related to VC, so whether PTH could induce valvular EndMT and the mechanism involved are worthy of further study. Method 5/6 nephroectomy (5/6Nx) with high phosphorus diet was used to construct a VC model in rats with CKD. miRNA sequencing was used to find the changes of miRNA in human umbilical vein endothelial cells (HUVECs) intervened by PTH. The expression levels of miR-29a-5p, markers of EndMT and Notch1 pathway were determined by PCR, western blot and immunofluorescence. The activity of γ-secretase was determined by ELISA. VC was observed by VonKossa staining and scanning electron microscope. Results Firstly, we found PTH could induce valvular EndMT in VC among the rat with CKD, and VC could be alleviated by cinacalcet. As for the contribution of PTH on EndMT, in vitro, global microRNA(miRNA) expression profiling of HUVECs was examined in PTH versus control and miR-29a-5p was identified as the miRNA that was most notably decreased under PTH stimulation, which could be resumed by PTHrP(7-34) (PTHR1 inhibitor). Overexpressing miR-29a-5p could inhibit PTH-induced EndMT in vitro and valvular EndMT in vivo. The dual luciferase assay was verified that γ-secretase activating protein (GASP) is the target of miR-29a-5p. miR-29a-5p-mimics, si-GSAP and DAPT (γ-secretase inhibitor) could inhibit PTH-induced γ-secretase activation thus blocking Notch1 pathway activation to inhibit PTH-induced EndMT in vitro. In vivo, Notch1 pathway activation was observed in VC among the rats with CKD. Blocking Notch1 pathway activation by AAV-miR-29a and DAPT could inhibit vavular EndMT in rats with CKD. In addition, blocking Notch1 pathway activation could also alleviate VC among the rats with CKD. Conclusion PTH could activate valvular EndMT via miR-29a-5p/GSAP./Notch1 pathway, and that could contributed to VC in rats with CKD.