基于Klotho基因的生物活性探讨中医“心肾相交”理论

"心肾相交"是指肾水上济于心,心火下降于肾。Klotho作为肾精的物质基础,在生理功能上与肾精作用相同,二者均秉承于父母,具有抗衰老、主生殖和生长发育、保护骨骼调节钙磷代谢等功用。Klotho衍生物可溶性Klotho(soluble Klotho,sKlotho)延续了Klotho的生理功能,并参与调节心肌细胞和血管平滑肌细胞的钙离子代谢,抑制钙离子沉积,调节脉道,保护心血管,具有交通心藏系统的功能。Klotho基因的这种微观调节作用,使得心藏系统和肾藏系统之间的关系更加紧密,加强了中医心肾之间的交互关系,也丰富了中医"心肾相交"理论。     

Potentielle application de l’axe fibroblast growth factor 23-Klotho dans la maladie rénale chronique

Membrane Alpha Klotho (α-klotho) is expressed in the kidney and functions as a co-receptor of FGF receptors (FGFRs) to activate specific fibroblast growth factor 23 (FGF23) signal pathway. FGF23 is produced in bones and participates in mineral homeostasis. The extracellular domain of transmembrane αklotho can be cleaved by proteases and released into the circulation as soluble α-klotho. Klotho deficiency is a pathogenic factor for chronic kidney disease progression and cardiovascular diseases. The FGF23 excess may also contribute to cardiovascular diseases where its pathogenic effect acts via the FGFR4 and independently of α-klotho. The decline in serum α-klotho followed by a rise in serum FGF23 at an early stage of chronic kidney disease can serve as a robust predictor for risk of cardiovascular diseases and mortality in both CKD patients and the general population. The first randomized trials suggest the possibility to reduce FGF23 excess in chronic kidney disease by controlling the phosphate serum using phosphate binders and reducing PTH levels with calcimimetic drug. New strategies emerge, including the administration of α-klotho recombinant and the use of epidrugs in order to correct the klotho deficiency. The FGR4 inhibitors are promising to limit the development of left ventricular hypertrophy linked to FGF23 excess. Finally, a better understanding of the molecular mechanisms of FGF23/α-klotho axis will allow us to find new strategic approaches and improve the CKD patient's management and their outcomes.     

帕立骨化醇抑制糖尿病肾病大鼠肾小管间质纤维化

目的:探讨帕立骨化醇(paricalcitol,P)对糖尿病肾病(DN)肾小管间质纤维化的干预作用及可能机制。方法:大鼠禁食后,采用单次无菌腹腔注射链脲佐菌素建立DN模型。将DN大鼠随机分为:(1)帕立骨化醇干预组(P组):帕立骨化醇溶于丙二醇中,于造模成功后第2天以0.4μg/kg的剂量腹腔注射,每周3次;(2)糖尿病肾病组(D组):给予等体积的丙二醇腹腔注射。设置正常对照组(C组)。帕立骨化醇连续干预12周后,测血、尿生化指标;进行肾脏病理学检查;利用免疫组化及Western blotting检测肾组织TGF-β1、Wnt-4、β-catenin及Klotho蛋白的表达;并进行指标间的相关分析。结果:(1)与C组比较,D组大鼠SCr、BUN及24 h尿蛋白水平均升高,而P组均较D组降低(P0.05)。(2)与C组比较,D组大鼠肾小管间质纤维化面积增加,而P组较D组减小(P0.05)。(3)D组大鼠肾组织Klotho蛋白表达低于C组,而P组高于D组(P0.05);与C组比较,D组大鼠肾组织TGF-β1、Wnt-4及β-catenin蛋白表达增加,而P组表达均较D组减少(P0.05)。(4)Klotho与纤维化面积、TGF-β1、Wnt-4及β-catenin均呈负相关(P0.05)。结论:帕立骨化醇可抑制DN大鼠肾小管间质纤维化,其作用可能与增加肾组织Klotho表达,抑制Wnt/β-catenin信号通路激活,同时减少TGF-β1合成相关。     

抗衰老Klotho蛋白减轻大鼠乳鼠心肌细胞缺氧/复氧损伤

目的:观察抗衰老Klotho蛋白对大鼠乳鼠原代心肌细胞缺氧/复氧(H/R)损伤的作用并探讨其作用机制。方法:建立大鼠乳鼠心肌细胞H/R模型,并将心肌细胞分为正常对照组、H/R模型组和不同浓度(0.1μmol/L、1μmol/L和10μmol/L)Klotho作用H/R组。观察各组心肌细胞H/R前后搏动频率变化,利用MTT方法检测细胞存活率;测定各组心肌细胞H/R后LDH、CK、AST的漏出量及MDA含量、SOD活性;流式细胞术检测各组心肌细胞的凋亡率;real-time PCR检测各组心肌细胞中内质网应激标记及凋亡相关分子GRP78、CRT和CHOP和caspase-12 mRNA的表达情况;Western blot法检测心肌细胞内内质网应激凋亡蛋白CHOP和caspase-12蛋白表达及Akt磷酸化水平。结果:与正常对照组相比,H/R模型组中心肌细胞搏动频率和细胞存活率显著下降,细胞凋亡率显著升高(P0.05);LDH、CK、AST和MDA含量升高而SOD活性显著降低(P0.05);GRP78、CRT、CHOP和caspase-12 mRNA表达显著增高(P0.05);CHOP和caspase-12蛋白表达随之增高而Akt的磷酸化水平显著降低(P0.05)。与H/R模型组相比,抗衰老Klotho蛋白作用H/R心肌细胞后,心肌细胞搏动频率和细胞存活率显著升高,细胞凋亡率逐渐降低(P0.05),LDH、CK、AST和MDA含量下降而SOD活性显著增高(P0.05),GRP78、CRT、CHOP和caspase-12 mRNA的表达逐渐降低(P0.05),CHOP和caspase-12蛋白表达也随之降低,而Akt磷酸化水平显著增加(P0.05)。结论:抗衰老Klotho蛋白能够提升H/R损伤后心肌细胞的存活率,抑制细胞凋亡,通过抵抗氧化应激和过度内质网应激反应发挥作用,并与激活Akt磷酸化有关。     

Melatonin Attenuates Memory Impairment Induced by Klotho Gene Deficiency Via Interactive Signaling Between MT2 Receptor,ERK, and Nrf2-Related Antioxidant Potential

Background:

We demonstrated that oxidative stress plays a crucial role in cognitive impairment in klotho mutant mice, a genetic model of aging. Since down-regulation of melatonin due to aging is well documented, we used this genetic model to determine whether the antioxidant property of melatonin affects memory impairment.

Methods:

First, we examined the effects of melatonin on hippocampal oxidative parameters and the glutathione/oxidized glutathione (GSH/GSSG) ratio and memory dysfunction of klotho mutant mice. Second, we investigated whether a specific melatonin receptor is involved in the melatonin-mediated pharmacological response by application with melatonin receptor antagonists. Third, we examined phospho-extracellular-signal-regulated kinase (ERK) expression, nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation, Nrf2 DNA binding activity, and glutamate-cysteine ligase (GCL) mRNA expression. Finally, we examined effects of the ERK inhibitor SL327 in response to antioxidant efficacy and memory enhancement mediated by melatonin.

Results:

Treatment with melatonin resulted in significant attenuations of oxidative damage, a decrease in the GSH/GSSG ratio, and a significant amelioration of memory impairment in this aging model. These effects of melatonin were significantly counteracted by the selective MT2 receptor antagonist 4-P-PDOT. Importantly, 4-P-PDOT or SL327 also counteracted melatonin-mediated attenuation in response to the decreases in phospho-ERK expression, Nrf2 nuclear translocation, Nrf2 DNA-binding activity, and GCL mRNA expression in the hippocampi of klotho mutant mice. SL327 also counteracted the up-regulation of the GSH/GSSG ratio and the memory enhancement mediated by melatonin in klotho mutant mice.

Conclusions:

Melatonin attenuates oxidative stress and the associated memory impairment induced by klotho deficiency via signaling interaction between the MT2 receptor and ERK- and Nrf2-related antioxidant potential.     

基于妊娠期糖尿病血糖控制效果对Klotho基因表达影响的研究

目的 研究妊娠期糖尿病血糖控制不达标状态下和体外高糖环境下对Klotho基因表达的影响。 方法 选择妊娠期糖尿病患者103例,其中血糖控制不达标56例,血糖控制达标47例,采用ELISA检测血清Klotho蛋白水平并分析各组妊娠结局,同时在体外高糖环境下培养HEK293细胞,采用RT-PCR方法检测细胞Klotho基因表达水平。结果 血清α-Klotho 和β-klotho蛋白在妊娠期糖尿病血糖控制不达标组表达水平明显低于血糖控制达标组(p<0.05),此外,血糖不达标组剖宫产率、巨大儿发生率高于达标组(p<0.05)。RT-PCR结果表明高糖可诱导HEK293细胞α-Klotho 和β-klotho mRNA表达水平下降。结论 体内外高糖是导致α-Klotho 和β-klotho表达下降重要因素之一,妊娠期糖尿病治疗目标应以血糖达标最为获益。     

Impaired function of fibroblast growth factor 23 / Klotho protein axis in prediabetes and diabetes mellitus: Promising predictor of cardiovascular risk

The discovery of clear molecular mechanisms of early cardiac and vascular complications in patients with prediabetes and known diabetes mellitus are core element of stratification at risk with predictive model creation further. Previous clinical studies have shown a pivotal role of impaired signaling axis of fibroblast growth factor 23 (FGF23), FGF23 receptor isoforms and its co-factor Klotho protein in cardiovascular (CV) complications in prediabetes and diabetes. Although there were data received in clinical studies, which confirmed a causative role of altered function of FGF-23/Klotho protein axis in manifestation of CV disease in prediabetes and type 2 diabetes mellitus (T2DM), the target therapy of these diseases directing on improvement of metabolic profiles, systemic and adipokine-relating inflammation by beneficial restoring of dysregulation in FGF-23/Klotho protein axis remain to be not fully clear. The aim of the review was to summarize findings regarding the role of impaired FGF-23/Klotho protein axis in developing CV complications in patients with prediabetes and type 2 diabetes mellitus. It has been elucidated that elevated levels of FGF-23 and deficiency of Klotho protein in peripheral blood are predictors of CV disease and CV outcomes in patients with (pre) diabetes, while predictive values of dynamic changes of the concentrations of these biomarkers require to be elucidated in detail in the future.     

Renoprotective effect of N-acetylcysteine on chronic cyclosporine A nephrotoxicity

Objective To explore the renoprotective effect of N-acetylcysteine (NAC) on chronic cyclosporine A (CsA) nephrotoxicity in mice model. Methods ICR mice were randomly divided into 4 groups: normal control group [olive oil, 1 ml/kg subcutaneous injection (s.c)], NAC control group (olive oil, 1 ml/kg s.c plus NAC 150 mg•kg-1•d-1 in drinking water), model group [ICR mice maintained on a salt-depleted diet (0.05% sodium) plus CsA (30 mg•kg-1•d-1) for four weeks]; Treatment group [ICR mice maintained on a salt-depleted diet (0.05% sodium) plus CsA (30 mg•kg-1•d-1 s.c) and NAC (150 mg•kg-1•d-1 in drinking water). Basic parameters, histopathology, 8-hydroxydeoxyguanosine (8-OHdG), the expression of Klotho and AKT-FoxO1 were studied. Results Compared with the normal control group, mice in model group showed deterioration in renal function [Scr (27.8±1.2) μmol/L vs (19.0±2.2) μmol/L, P＜0.01], development of tubulointerstitial fibrosis, increased urinary 8-OHdG output [(41.2±16.8) ng/d vs (28.7±7.4) ng/d, P＜0.05], and decreased Klotho expression [(17.8±4.5)% vs (100.0±4.0)%, P＜0.01]. Concomitant administration of NAC significantly improved all above parameters (all P＜0.05). Correlation analysis revealed that Klotho expression was negatively correlated with urinary 8-OHdG excretion (r＝-0.934, P＜0.01) and AKT-FoxO1 expression （AKT: r＝-0.939, P＜0.01; FoxO1: r＝-0.919, P＜0.01). Conclusion NAC can attenuate tubulointerstitial fibrosis in chronic CsA nephrotoxicity mice, which may be associated with the up-regulation of Klotho expression and the inhibition of AKT-FoxO1 signaling pathway.     

Efecto regulador de paricalcitol sobre parámetros inflamatorios,fibróticos y anticalcificantes en el paciente con enfermedad renal crónica. Más allá de la regulación de la enfermedad óseo-mineral

BackwardCardiovascular events are the major cause of morbidity and mortality in patients with chronic kidney disease (CKD). Inflammation and mineral-bone disorder are pathological conditions that have been associated with an increased cardiovascular risk.ObjectiveShow paricalcitol regulation overinflammatory, fibrotic and mineral disorder parameters in CKD.Material and methodsProspective Study in 46 CKD stages III-V patients without dialysis patients whith elevated parathormone in which we introduced paricalcitol. We evaluated classic and newest mineral and bone metabolism serum parameters (calcium, phosphorus, parathormone, fibroblast growth factor-23 [FGF-23], Klotho, calcidiol), inflammatory-fibrosis and anticalcifying parameters (interleukin-6 and 10, tumor necrosis factor-a [TNF- α], transforming growth factor-b [TGF-β],bone morphogenic protein-7 [BMP-7] and fetuin-A) for four months.ResultsAt the end of study soluble Klotho increased (p = .001), FGF-23 remained stable, calcium and phosphorus levels were not increased, calcidiol increased (p = .010) and PTH decreased (p = .002). Inflammation-fibrosis and calcification parameters showed positive regulation after paricalcitol treatment: interleukin-6 decreased significantly (p = .001) and also TNF-α did (p = .005), on the contrary, interleukin-10 and fetuin-A increased (p = .001 for both). Anti-fibrosis marker BMP-7 increased (p = .001) and TGF-b decreased (p = .001). We did not find significant changes in renal function.ConclusionsParicalcitol treatment might be profitable in regulating inflammatory and anticalcificant parameters, unmodified calcium or phosphorus seric levels and preserving kidney function in renal patients with no dialysis. Our selected parameters could indicate paricalcitol effects in mineral and endothelial disorder related to renal disease.