BK通道在平滑肌细胞转型和基质小泡分泌介导的慢性肾脏病血管钙化中的作用和机制研究

Chronic kidney disease(CKD), which impacts millions of people, is a complex health problem with a rising incidence and poor outcomes, including hypertension, hyperkalemia, mineral bone disorder, and cardiovascular disease, and faces an increasing risk of mortality. Vascular calcification happens with age and is obviously observed in CKD, the combination of vascular calcification and impaired bone metabolism in CKD has been identified as the chronic kidney disease mineral bone disorder (CKD-MBD). Remarkably, vascular calcification is frequently accompanied by decreased bone mineral density or disturbed bone turnover. This contradictory association, observed mainly in osteoporosis and CKD, is called the “calcification paradox”. During vascular calcification, high concentrations of phosphate and calcium induce a rise of the deposition of hydroxyapatite in the vessel wall and it was once considered a passive process. Now, vascular calcification is regarded as an active cell-regulated process, but the definite mechanism remains elusive. Vascular smooth muscle cells(VSMCs) play a pivotal role in calcification by undergoing osteoblast-like cells differentiation, during which the osteoblast-like cells express alkaline phosphatase(ALP) and other bone-related proteins, such as oteopontin, collagen type I and osteoprotegrin, and by releasing matrix vesicles(MVs), which serve as a nidus for deposition of calcium and phosphate ...The large conductance calcium- and voltage-activated K+ channel (BK, KCa1.1, Maxi K) channel is encoded by the KCNMA1 gene. It can be activated by Ca2+ alone, membrane depolarization alone, or synergistically by both. BK channels exist in many different tissues in the human body, including nerve, smooth and skeletal muscle and endocrine cells. The ability of integrating changes in intracellular Ca2+ and membrane potential makes BK channel play important roles in regulating neurotransmitter release, neuronal excitability and smooth muscle tone. But the function of BK channel in nonexcitable tissues is not well defined. Our preliminary study found that the BK channel controls osteoblast differentiation, and that BK opener NS1619 plays an important role in inhibiting the process of vascular calcification. For that vascular calcification and impaired bone metabolism are two common complications in CKD patients, and both vascular calcification and osteopenia share the same metabolic disturbance of calcium , inorganic phosphate, and calcified adjustment factors, we ypothesize that BK channel may take an important part in regulating CKD induced vascular calcification...In this study, a myriad of techniques will be performed, such as molecular biology, patch clamp, transmission electron microscope, MV-collagen calcification assay, In vitro biomineralization by MVs, immunohistochemical and immunostaining etc , to investigate the pathological propertyrole of BK in vascular calcification based on the in vitro (rats primary vascular smooth muscle cells and human VSMCs) , in vivo (CKD induced vascular calcification and BK knockout mice ), and ex vivo (vascular rings) calcification model. The aim of the study: (1) Whether the expression and activity of BK channels changes in the process of CKD induced vascular calcification; (2) How BK channels affects the progression and severity of vascular calcification; (3) What’s the mechanism on BK channels influencing the differentiation of VSMCs to osteoblast-like cells; (4) How BK channels affects the formation, secretion and functioin of MVs . This study will provide deep insight into understanding the pathogenesis of vascular calcification and explore the new therapeutic target.

慢性肾脏病（CKD）在国内外呈逐年上升趋势，血管钙化是CKD患者死亡的独立危险因素，但缺少有效治疗方法。目前研究认为其主要是平滑肌细胞转型为成骨细胞及转型细胞分泌基质小泡介导的主动调控过程，但分子机制不明确。血管钙化与骨代谢异常是CKD常见的并发症，二者负向关联。我们前期研究发现大电导钙激活钾通道（BK）在成骨细胞分化中发挥重要作用，由此我们假设BK通过调节该主动过程影响CKD血管钙化。我们拟采用原代血管平滑肌细胞钙化模型、CKD血管钙化动物模型、体外血管环钙化模型、BK基因敲除小鼠模型，结合免疫组化，膜片钳，免疫共沉淀，透射电镜、胶原钙化和体外矿化诱导实验等，验证CKD血管钙化模型中BK表达和活性变化，BK对血管钙化的调节作用，BK在平滑肌向成骨细胞样转变中的作用机制及BK在钙化平滑肌细胞分泌基质小泡中的作用。本项目不仅丰富人们对血管钙化分子机制的认识，而且为探索治疗靶点提供新的思路。

【目的】慢性肾脏病（CKD）血管钙化是体内钙磷代谢失调，导致钙盐在细胞和组织间异常沉积，VSMC向成骨样细胞转化，其发生机制十分复杂，目前关于BK通道是否参与CKD 血管钙化调控尚未有报道，本课题主要研究了BK通道在血管钙化平滑肌细胞表型转换中的作用及机制，旨在深入探究血管钙化的分子机制，寻求血管钙化的潜在治疗靶点。.【方法】 通过建立原代VSMC钙化模型，CKD血管钙化动物模型及血管环钙化模型，观察在血管钙化中BK通道表达量的变化，及其对VSMC向成骨样细胞转化的作用。采用茜素红染色，Western Blot，实时定量PCR，免疫组化等方法检测钙化模型建立是否成功，并检测钙化过程中BK通道表达量的变化。通过BK通道的开放剂或抑制剂的干预检测BK通道对血管钙化进程的影响，探索Akt的变化。通过Akt抑制剂干预检测血管钙化过程中Akt是否参与了BK通道调节血管钙化。同时，联合应用BK通道开放剂和BK沉默质粒，探究BK通道对细胞分泌的MV粒径、数量及其钙化程度的影响。通过自噬抑制剂3-MA联合BK通道开放剂NS1619，检测BK是否通过自噬调节钙化过程。.【结果】结果发现细胞、血管环及动物钙化模型BK通道的表达量较正常组显著降低。BK通道开放剂NS1619干预可延缓VSMC钙化进程，p-Akt 表达较钙化组显著升高，BK通道抑制剂Paxiline 干预促进VSMC钙化进程，p-Akt 表达较钙化组显著降低，Akt 抑制剂MK2206和NS1619共同干预后和NS1619单独联合钙化培养基诱导相比，NS1619延缓VSMC钙化进程的作用减弱，表明BK调控CKD血管钙化是通过Akt通路介导的。此外，纳米颗粒跟踪分析检测显示，BK通道开放剂和BK过表达质粒同样可以抑制MV的分泌并且延缓钙化过程，同时激活了自噬活动，BK shRNA对此有反作用。自噬抑制剂3-MA的干预，减弱了BK通道开放剂对钙化程度的改善。这表明BK通道通过调控自噬过程，抑制MV的分泌，从而改善血管钙化。.【结论】BK通道通过上调Akt通路抑制CKD血管钙化和平滑肌细胞转型，且可通过调控自噬过程，抑制MV的分泌，从而改善血管钙化。这提示我们BK通道可能作为治疗血管钙化的潜在靶点。