Klotho G-395A基因多态性与ESRD患者钙磷代谢紊乱的相关性分析

目的观察终末期肾病(ESRD)患者klotho G-395A基因多态性分布与其合并钙磷代谢紊乱的相关性。 方法选取2015年4月至2016年12月期间就诊于扬州市多个血液净化中心的ESRD患者共137例,体检中心正常体检者80名为对照组;运用FQ-PCR(TaqMan法)对各研究对象进行klotho基因G-395A多态性分型;检测klotho蛋白、成纤维细胞生长因子(FGF23)及钙磷代谢相关生化指标水平,比较不同基因型间各生化指标间的水平。卡方检验、t检验、单因素方差分析、Mann-Whitney U检验及Kruskal-Wallis H检验用于各组生化指标间差异性分析,多因素采用二元logistic回归分析。 结果①ESRD组和健康对照组的基因型分布均符合Hardy-Weinberg平衡(χ²＝0.512, χ²＝0.134; P<0.05)。②ESRD组和对照组基因亚型及基因频率分布差异有统计学意义(χ² ＝11.467, P＝0.003; χ²＝10.130, P＝0.001),且ESRD组患者A等位基因频率较对照组高。③ESRD组不同的基因型中,血钙、klotho蛋白及FGF23水平差异有统计学意义(P<0.05),血钙在GA型与AA型间表达水平差异有统计学意义(t＝－2.469,P＝0.015), FGF23、klotho蛋白在GG与AA型(Z＝－4.020, Z＝－5.461; P<0.001),GA与AA型(Z＝－4.303, Z＝－5.610; P<0.001)之间的表达水平差异均有统计学意义。④二元logistic回归分析示GA＋AA型为ESRD患者钙磷代谢紊乱的独立危险因素。 结论Klotho G-395A基因多态性A等位基因位点可能为ESRD患者的易感基因,并与ESRD患者合并钙磷代谢紊乱有关。     

血清Klotho、FGF23、ALP、25-（OH）D水平对持续非卧床腹膜透析患者心脏瓣膜钙化的影响*

目的 探讨血清Klotho、成纤维细胞生长因子23（FGF23）、碱性磷酸酶（ALP）、25羟维生素D[25-（OH）D]水平对持续非卧床腹膜透析（CAPD）患者心脏瓣膜钙化的影响。方法 选取2018年 1月—2019年1月内蒙古医科大学附属医院收治的132例CAPD患者,另选取同期医院体检中心接收的40例健康体检者,检测并对比CAPD患者与健康受试者血清Klotho、FGF23、ALP、25-（OH）D水平;根据心脏超声评估心脏瓣膜钙化情况将CAPD患者分为钙化组和非钙化组,对比两组血清Klotho、FGF23、ALP、25-（OH）D水平及其他可能导致心脏瓣膜钙化影响因素的差异,应用Logistic回归分析相关危险因素;绘制受试者工作特征（ROC）曲线,分析血清Klotho、FGF23、ALP、25-（OH）D水平对心脏瓣膜钙化的诊断效能。结果 CAPD患者血清Klotho、25-（OH）D水平均低于健康受试者,血清FGF23、ALP水平均高于健康受试者（P?<0.05）;钙化组血清Klotho、25-（OH）D水平均低于非钙化组,血清FGF23、ALP水平均高于非钙化组（P?<0.05）;钙化组残余肾功能（RRF）低于非钙化组,血磷水平高于非钙化组（P?<0.05）;Logistic回归分析显示,RRF、血磷、血清Klotho、FGF23、ALP、25-（OH）D水平均是导致心脏瓣膜钙化的危险因素（P?<0.05）;ROC结果显示,血清Klotho、FGF23、ALP、25-（OH）D诊断心脏瓣膜钙化的最佳截断点分别为401.10?ng/L、231.91?ng/L、 76.50?u/L、11.36?nmol/L,敏感性分别为88.68%、90.57%、71.70%及67.92%,特异性分别为88.61%、92.41%、77.22%及70.89%,AUC分别为0.903（95% CI：0.854,0.952）、0.927（95% CI：0.873,0.945）、0.714（95% CI：0.622,0.770）及0.698（95% CI：0.610,0.759）。结论 CAPD患者血清Klotho、25-（OH）D水平降低,FGF23、ALP水平升高,且均与CAPD心脏瓣膜钙化关系密切,所有指标对CAPD患者心脏瓣膜钙化有较高的诊断效能。     

Implications of Klotho in vascular health and disease

Cardiovascular disease (CVD) is a prevalent condition in general population and the first cause of death overall. Klotho, a pleiotropic protein related to longevity that acts as a co-receptor of the fibroblast growth factor 23, has been proposed as a key regulator of the development of CVD. In the few clinical studies made, it has been observed a relationship between low levels of soluble Klotho and the occurrence and severity of CVD, as well as a reduction of cardiovascular risk when they are high. Also, different polymorphisms of human Klotho gene have been related to the incidence of cardiovascular events. Moreover, several experimental studies indicate that this protein acts in the maintenance of vascular homeostasis. Klotho improves endothelial dysfunction through promotion of NO production and mediates anti-inflammatory and anti-aging effects such as suppression of adhesion molecules expression, attenuation of nuclear factor-kappa B or inhibition of Wnt signaling. Furthermore, this protein is related to the attenuation of vascular calcification as well as prevention of cardiac hypertrophy. The expression of this protein in the vascular wall implies a new scenario for the treatment of vascular disorders. The purpose of this review is to provide an overview of the relationship between the Klotho protein and CVD, in addition to its role in the maintenance of functional vascular integrity.     

山茱萸多糖通过上调Klotho表达和抑制PI3K/AKT通路对肝癌HepG2细胞增殖、凋亡的影响

目的探究山茱萸多糖通过上调Klotho表达和抑制PI3K/AKT通路对肝癌HepG2细胞增殖、凋亡的影响。方法分别以6.25、12.5、25 mg/mL山茱萸多糖作用于人肝癌细胞株HepG2,CCK8法检测细胞增殖能力,采用Hoechst 33258荧光染色以及流式AnnexinV-FITC/PI双染法检测细胞凋亡率;通过免疫印迹法检测增殖相关蛋白Ki67、凋亡相关蛋白Bax、Bcl-2、Caspase-3以及PI3K/Akt通路相关蛋白水平,并检测Klotho蛋白表达水平。结果与对照组相比,6.25、12.5、25 mg/mL山茱萸多糖组HepG2细胞克隆形成数显著下降(P0.05),且随山茱萸多糖浓度升高克隆形成数显著下降;与对照组相比,6.25、12.5、25mg/mL山茱萸多糖组HepG2细胞凋亡率显著升高(P0.05),且随山茱萸多糖浓度升高细胞凋亡率逐渐升高(P0.05);与对照组相比,6.25、12.5、25mg/mL山茱萸多糖组HepG2细胞中Bax、cleaved-caspase-3、Klotho蛋白表达升高(P0.05),Ki67、Bcl-2、p-PI3K/PI3K、p-Akt/Akt蛋白表达下降(P0.05),且呈剂量相关性。结论山茱萸多糖可能通过上调Klotho表达抑制PI3K/AKT通路活化,抑制HepG2细胞增殖,促进HepG2细胞凋亡。     

Association of polymorphisms of the androgen receptor and klotho genes with bone mineral density in Japanese women

Genetic variants of the androgen receptor and klotho protein may contribute to variation in bone mass as well as to predisposition to osteoporosis. The relationship of a CAG repeat polymorphism of the androgen receptor gene (AR) and of a –395GA polymorphism of the klotho gene (KL) to bone mineral density (BMD) in Japanese women was examined in a population-based study. The subjects (1,101 and 1,110 women for AR and KL polymorphisms, respectively) were aged 40–79 years and were randomly recruited to a population-based prospective cohort study of aging and age-related diseases. BMD for the total body, lumbar spine, right femoral neck, right trochanter, and right Wards triangle was measured by dual-energy X-ray absorptiometry. Genotypes for the AR and KL polymorphisms were determined by polymerase chain reaction based assays. The number of CAG repeats of AR was inversely correlated with BMD for the lumbar spine in premenopausal women but not in postmenopausal women. The (CAG)_n22 and (CAG)_n23 alleles were designated S and L, respectively. Among premenopausal women, BMD for the total body was significantly lower in subjects with the LL genotype than in those with the SS genotype or those in the combined group of SS and SL genotypes. In contrast, BMD was not associated with AR genotype in postmenopausal women. Among all women, BMD for the lumbar spine was significantly lower in subjects with the GG genotype of the –395GA polymorphism of KL than in those with the AA genotype. BMD was not associated with –395GA genotype among premenopausal women. In postmenopausal women, BMD for the total body or lumbar spine tended to be lower in subjects with the GG genotype than in those with the AA genotype or those in the combined group of GA and AA genotypes. These results suggest that AR is a susceptibility gene for reduced BMD in premenopausal Japanese women, and that KL is a susceptibility gene for reduced BMD in all women.     

Secreted klotho protein attenuates osteogenic differentiation of human bone marrow mesenchymal stem cells in vitro via inactivation of the FGFR1/ERK signaling pathway

Abstract

Increasing evidence indicates that the osteogenic differentiation of mesenchymal stem cells (MSCs) is related to bone formation, heterotopic ossification, and even vascular calcification. Therefore, it is essential to understand the microenvironment that regulates these processes. The Klotho gene plays an important role in tissue mineralization, and its secreted protein functions as a hormone. We investigated the effects of secreted Klotho protein on the osteogenesis of human bone marrow MSC (hBMSCs). To this end, the cells received osteogenic medium with or without Klotho protein. The results showed that osteoblast-specific gene expression and mineral deposition were decreased when MSCs were incubated with Klotho. Klotho reduced the expression of fibroblast growth factor receptor 1 (FGFR1) and phosphorylated extracellular signal-regulated kinase 1/2. However, both MEK and FGFR1 inhibitors delayed bone mineral formation more than Klotho. These data suggest that secreted Klotho protein attenuates the osteogenic differentiation of hBMSCs in vitro through FGFR1/ERK signaling.     

基于FGF23-Klotho轴探讨肾元颗粒对db/db糖尿病肾病小鼠骨骼的保护作用

目的 观察肾元颗粒对db/db糖尿病肾病小鼠FGF23-Klotho轴的影响，并探讨其对骨骼的保护的可能机制。方法 30只db/db小鼠随机分为模型组（20 mL分为模^-1）、肾元颗粒组（6.0 g粒组模^-1）和厄贝沙坦组（30 mg坦组模^-1），10只同窝野生（wild type, WT）小鼠作为空白组（20 mL为空白^-1），各组小鼠给药容量均为（20 mL给药容^-1）。连续灌胃12周后，采用生化检测仪检测血钙、血磷含量；酶联免疫吸附测定法（ELISA）检测各组血清成纤维化生长因子23 （Fibroblast growth factor 23，FGF23）含量，以及骨骼钙盐含量、骨骼碱性磷酸酶（ALP）活性；蛋白免疫印迹法（Western blot）检测骨骼骨保护素（osteoprotegerin，OPG）蛋白表达水平；免疫荧光双标检测肾脏Klotho和成纤维化生长因子受体1（Fibroblast growth factor receptor 1，FGFR1）蛋白，并于激光共聚焦下观察拍照。结果 与正常组比，模型组小鼠血钙减少，血磷含量升高（P <0.05），小鼠血FGF23含量显著升高（P < 0.05），小鼠骨骼中钙盐含量减少，ALP活性降低（P < 0.05），小鼠骨骼OPG蛋白表达减少（P < 0.05），小鼠肾脏Klotho和FGFR1免疫荧光双标阳性细胞数显著减少（P < 0.05）；与模型组比，肾元颗粒组小鼠血钙升高，血磷减低（P < 0.05），血FGF23含量降低（P < 0.05），骨骼钙盐含量升高，骨骼ALP活性升高（P < 0.05），骨骼OPG蛋白表达量增加（P < 0.05），肾脏免疫荧光双标阳性细胞数增多（P < 0.05）。结论 肾元颗粒能改善db/db糖尿病肾病小鼠骨质的流失，而其作用机制可能与FGF23-Klotho轴平衡恢复有关。     

Promising novel therapeutic targets for kidney disease: Emphasis on kidney-specific proteins

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Histamine deficiency inhibits lymphocyte infiltration in the submandibular gland of aged mice via increased anti-aging factor Klotho

ObjectivesHistidine decarboxylase (HDC), a histamine synthase, is expressed in various tissues and is induced by proinflammatory cytokines such as TNFα. As they age, C57BL/6 mice show auto-antibody deposition and lymphocyte infiltration into various tissues, including salivary glands. However, the mechanism underlying cell infiltration and the change in HDC expression in salivary glands with aging remain unclear. Thus, we aimed to elucidate the relationship between histamine and inflammaging.MethodsWe investigated the change in histology and HDC expression in the major salivary glands (parotid, submandibular, and sublingual) of 6-week- and 9-month-old wild-type mice. We also determined the histological changes, cytokine expression, and anti-aging factor Klotho in the salivary glands of 9-month-old wild-type and HDC-deficient (HDC-KO) mice.ResultsCell infiltration was observed in the submandibular gland of 9-month-old wild-type mice. Although most cells infiltrating the submandibular glands were CD3-positive and B220-positive lymphocytes, CD11c-positive and F4/80-positive monocyte lineages were also detected. HDC, TNFα, and IL-1β mRNA expression increased in the submandibular gland of 9-month-old wild-type mice. The expression of PPARγ, an anti-inflammatory protein, declined in 9-month-old wild-type mice, and Klotho expression increased in 9-month-old HDC-KO mice. Immunohistochemistry showed that Klotho-positive cells disappeared in the submandibular gland of 9-month-old wild-type mice, while Klotho was detected in all salivary glands in HDC-KO mice of the same age.ConclusionOur findings demonstrate the multifunctionality of histamine and can aid in the development of novel therapeutic methods for inflammatory diseases such as Sjogren's syndrome and age-related dysfunctions.