Department of Nephrology, Affiliated Hospital of Xuzhou Medical University, Xuzhou, 221002, PR China; Department of Internal Medicine and Diagnostics, Xuzhou Medical University, Xuzhou, 221002, PR China.
Department of Nephrology, Affiliated Hospital of Xuzhou Medical University, Xuzhou, 221002, PR China.
Biomed Pharmacother. 2020 Dec;132:110798. doi: 10.1016/j.biopha.2020.110798. Epub 2020 Oct 1.
Calcium dobesilate (Cad), a protective agent, protects against microvascular damage, and diseases such as diabetic retinopathy and diabetic nephropathy. However, these vascular protective effects have not been demonstrated in chronic kidney disease (CKD). In this study, we aimed to determine the ability of Cad to protect against renal interstitial fibrosis induced by unilateral ureteral obstruction (UUO) and identify the underlying therapeutic mechanisms of Cad during hypoxia/serum deprivation (H/SD) in human umbilical vein endothelial cells (HUVECs). A total of 36 male mice were randomly assigned into 3 groups (12 mice in each group): the Sham-operated group (Sham), the saline solution-treated UUO mice group (UUO), and the Cad administration (intragastrically) group (Cad). The mice in Cad group were administered Cad (100 mg/kg) daily by oral gavage and slaughtered on the 7th and 14th days post-surgery. Six mice from each group were sacrificed by sodium pentobarbital injection on the 7th and 14th day after surgery. Tissue hypoxia, cell apoptosis and fibrotic lesions were detected by Immunostaining and Western blot. Peritubular capillaries (PTCs) injury was measured by a novel technique of fluorescent microangiography (FMA). Endothelial cell-to-mesenchymal transition (EndMT) were identified by immunofluorescence and Western blot. HUVECs proliferation was measured via Cell Counting Kit‑8 assays and Edu staining. Sirt1 and its downstream gene in Cad regulation of endothelial were detected. Hematoxylin-eosin (HE), Masson-trichrome stains and Histological findings showed that Cad administration markedly reduced hypoxia and renal interstitial fibrosis at each time point in UUO. Meanwhile, Cad protect against EndMT process of PTCs by increasing CD31 expression and decreasing α-smooth muscle actin and fibronectin expression. in vitro studies showed that there was a proliferative response of the HUVECs incubated with Cad (10 μM) in H/SD. Sirt1 was suppressed after small interfering RNA (siRNA) was transfected in HUVECs. Mechanistically, Cad enhanced Sirt1 signaling, which was accompanied by increased levels of p53 acetylation (ac-p53). Meanwhile, protein expression of Bcl-2, and VE-cadherin were downregulated, Bax, and α-SMA were upregulated. In summary, the therapeutic effect of Cad in obstructive nephropathy were likely through suppressing EndMT progression and promoting anti-apoptotic effects after via activating the Sirt1/p53 signaling pathway.
羟苯磺酸钙（Cad）是一种保护剂，可预防微血管损伤和糖尿病视网膜病变、糖尿病肾病等疾病。然而，在慢性肾脏病（CKD）中尚未证明这些血管保护作用。在这项研究中，我们旨在确定 Cad 在单侧输尿管梗阻（UUO）诱导的肾间质纤维化中的保护作用，并确定 Cad 在缺氧/血清剥夺（H/SD）下人脐静脉内皮细胞（HUVEC）中的潜在治疗机制。共 36 只雄性小鼠随机分为 3 组（每组 12 只）：假手术组（Sham）、盐水处理 UUO 小鼠组（UUO）和 Cad 给药（灌胃）组（Cad）。Cad 组小鼠每日经口灌胃给予 Cad（100mg/kg），术后第 7、14 天处死。每组各有 6 只小鼠于术后第 7、14 天经戊巴比妥钠注射处死。免疫染色和 Western blot 检测组织缺氧、细胞凋亡和纤维病变。荧光微血管造影术（FMA）测量肾小管道损伤。免疫荧光和 Western blot 鉴定内皮细胞-间充质转化（EndMT）。通过细胞计数试剂盒-8 测定和 Edu 染色测量 HUVEC 增殖。检测 Cad 调节内皮中 Sirt1 及其下游基因。苏木精-伊红（HE）、Masson 三色染色和组织学发现，Cad 给药在 UUO 的每个时间点均明显减轻缺氧和肾间质纤维化。同时，Cad 通过增加 CD31 表达和减少α-平滑肌肌动蛋白和纤维连接蛋白表达来防止 PTCs 的 EndMT 过程。体外研究表明，在 H/SD 中孵育 Cad（10μM）的 HUVECs 有增殖反应。转染小干扰 RNA（siRNA）后，HUVECs 中的 Sirt1 被抑制。机制上，Cad 增强了 Sirt1 信号，伴随着 p53 乙酰化（ac-p53）水平的升高。同时，Bcl-2 和 VE-cadherin 的蛋白表达下调，Bax 和α-SMA 的蛋白表达上调。总之，Cad 在梗阻性肾病中的治疗效果可能是通过抑制 EndMT 进展和通过激活 Sirt1/p53 信号通路促进抗凋亡作用来实现的。
