Li Zhe, Li Jing, Li Lin, Wang Qian, Zhang Qian, Tian Ling, Li Chenchen
Division of Nephrology, Affiliated Hospital of Hebei University, Baoding, China.
Key Laboratory of Bone Metabolism and Physiology in Chronic Kidney Disease of Hebei Province, Baoding, China.
Sci Rep. 2024 Dec 30;14(1):32091. doi: 10.1038/s41598-024-83596-w.
Klotho has been importantly linked to atherosclerosis, but little is known about its specific role. This study investigates the mechanism by which Klotho enhances the stability of atherosclerotic plaques in chronic kidney disease. apoE-/- knockout mice and C57BL/6 mice underwent 5/6 nephrectomy and then klotho-NC and klotho-mimic groups were set up to be fed a high-fat chow diet and a dummy group was created to be fed a normal chow diet. qPCR detected relative mRNA expression of klotho. Oil Red O and HE staining assessed lipid proportion in the aorta. Masson staining evaluated renal failure pathology in mice. Immunohistochemistry measured MAC-2 and α-SMA expression in the aorta. ELISA quantified urea, cholesterol, calcium ions, and triglycerides in mouse plasma. Western blotting detected associated protein expression, followed by cell-based experiments for validation. Compared with the Klotho-NC group, the plaque area and aortic lipid and renal fibrosis area were reduced in the Klotho-mimic group. Klotho-mimic reduced macrophage area, plasma urea, cholesterol, calcium ions, and triglyceride levels, and decreased the expression of p-PERK, NOX2, NOX4, Caspase-3, Caspase-9, Bax, p-GRK2, p-PLCβ, p-Src, and p-IP3R. Without ox-LDL stimulation, Klotho expression increased in the Klotho-mimic group, with no significant differences in NOX2, p-SHP1, p-Src, p-PERK, p-GRK2, and p-PLCβ. With ox-LDL in high-calcium medium, Klotho and p-SHP1 increased, while NOX2, p-Src, p-PERK, p-GRK2, and p-PLCβ decreased in the Klotho-mimic group. After ox-LDL and TPI-1 treatment, Klotho increased, NOX2 decreased, and other proteins showed no significant changes. Adding shRNA-GRK2 reduced NOX2, p-Src, and p-PERK, increased p-SHP1, with no changes in p-GRK2 and p-PLCβ. Differences in NOX2, p-GRK2, p-PLCβ, and p-PERK between groups were reduced in high-calcium medium, while p-SHP1 differences increased. Klotho enhances chronic kidney disease atherosclerotic plaque stability by inhibiting GRK2/PLC-β-mediated endoplasmic reticulum stress in macrophages via the ROS/SHP1 pathway.
α-klotho与动脉粥样硬化密切相关，但其具体作用尚不清楚。本研究探讨α-klotho增强慢性肾脏病动脉粥样硬化斑块稳定性的机制。将载脂蛋白E基因敲除小鼠和C57BL/6小鼠行5/6肾切除，然后分为α-klotho-NC组和α-klotho模拟组，给予高脂饲料喂养，设立假手术组给予普通饲料喂养。qPCR检测α-klotho的相对mRNA表达。油红O染色和苏木精-伊红染色评估主动脉脂质比例。Masson染色评估小鼠肾衰竭病理情况。免疫组织化学检测主动脉中巨噬细胞半乳糖凝集素-2（MAC-2）和α-平滑肌肌动蛋白（α-SMA）表达。酶联免疫吸附测定（ELISA）定量小鼠血浆中尿素、胆固醇、钙离子和甘油三酯。蛋白质免疫印迹法检测相关蛋白表达，随后进行细胞实验验证。与α-klotho-NC组相比，α-klotho模拟组的斑块面积、主动脉脂质和肾纤维化面积减小。α-klotho模拟物减少了巨噬细胞面积、血浆尿素、胆固醇、钙离子和甘油三酯水平，并降低了磷酸化蛋白激酶R样内质网激酶（p-PERK）、烟酰胺腺嘌呤二核苷酸磷酸氧化酶2（NOX2）、NOX4、半胱天冬酶-3（Caspase-3）、半胱天冬酶-9（Caspase-9）、促凋亡蛋白Bax、磷酸化G蛋白偶联受体激酶2（p-GRK2）、磷酸化磷脂酶Cβ（p-PLCβ）、磷酸化原癌基因酪氨酸蛋白激酶Src（p-Src）和磷酸化三磷酸肌醇受体（p-IP3R）的表达。在无氧化型低密度脂蛋白（ox-LDL）刺激下，α-klotho模拟组中α-klotho表达增加，NOX2、磷酸化含Src同源2结构域蛋白酪氨酸磷酸酶1（p-SHP1）、p-Src、p-PERK、p-GRK2和p-PLCβ无显著差异。在高钙培养基中加入ox-LDL后，α-klotho模拟组中α-klotho和p-SHP1增加，而NOX2、p-Src、p-PERK、p-GRK2和p-PLCβ减少。ox-LDL和TPI-1处理后，α-klotho增加，NOX2减少，其他蛋白无显著变化。加入shRNA-GRK2可降低NOX2、p-Src和p-PERK，增加p-SHP1，p-GRK2和p-PLCβ无变化。高钙培养基中各组间NOX2、p-GRK2、p-PLCβ和p-PERK的差异减小，而p-SHP1的差异增加。α-klotho通过ROS/SHP1途径抑制巨噬细胞中GRK2/PLC-β介导的内质网应激，从而增强慢性肾脏病动脉粥样硬化斑块的稳定性。
