Klotho抑制尿毒症心肌肥厚的作用及机制研究

Left ventricular hypertrophy is the most important characteristic and pathological change of uremic cardiomyopathy. Oxidative stress induced by massive accumulation of uremic toxins is likely to play a crucial role in the development of uremic cardic hypertrophy. Secreted Klotho protein,which is mainly produced and secrected from the kidneys and has strong ability to resist oxidative stress, can act as a hormonelike factor in the circulation to protect cardiomyocytes from oxidative stress. Our previous study supported by NFSC indicated that the expression of Klotho in the kidneys and the level of secreted Klotho protein in serum were decreased markedly under uremic conditions, while Klotho gene transfection mediated by adenoassociated virus could significantly alleviate left ventricular hypertrophy in uremic mice. Therefore, we speculate that the development of cardic hypertrophy under uremic conditions might be related closely to the decrease of Klotho,which is probably due to the fact that the decrease of Klotho protein in the circulation could result in reduced anti-oxidiative response of cardiomyocytes to uremic toxins. To further investigate the role of Klotho in uremic cardic hypertrophy, we plan to carry out a study on neonatal rat cardiomyocytes and uremic rats. Firstly, the correlation analysis of Klotho protein level and the development of cardic hypertrophy during the progress of uremia will be studied in uremic rats. Then the role and mechanism of Klotho protein in the inhibition of cardiomyocyte hypertrophy induced by uremic toxins will be investigated. Finally, the prevention and treatment of cardic hypertrophy with Klotho gene transfection in uremic rats will be evaluated. The purpose of this study is to further reveal the mechanism of uremic cardiomyopathy through deep understanding of cardio-renal relationship,and to provide new insight into the prevention and treatment of uremic cardiomyopathy.

左室肥厚是尿毒症心肌病最主要的病变特征，大量尿毒症毒素蓄积引起的高氧化应激是其发生的一个突出诱因。Klotho是主要由肾脏产生的内源性抗氧化应激蛋白，可进入血循环对心肌细胞发挥保护作用。前一个基金研究发现，尿毒症时Klotho蛋白水平显著降低，而转染了该基因的尿毒症小鼠左室肥厚明显减轻，推测Klotho水平与尿毒症心肌肥厚发生有着密切联系，其机制可能与Klotho水平降低后不能有效保护心肌细胞对抗尿毒症毒素所诱导的氧化应激损伤有关。为进一步明确Klotho在尿毒症心肌肥厚中的作用，本项目以新生大鼠心肌细胞和尿毒症大鼠为研究对象，分析尿毒症进展过程中Klotho水平与心肌肥厚的相关性；探讨Klotho蛋白抑制尿毒症毒素诱导心肌细胞肥大的作用及机制；评价Klotho基因转染对尿毒症大鼠心肌肥厚的干预作用。旨在从心、肾内在关联角度揭示尿毒症心肌病的发病机制，为确立有效防治措施提供实验依据。

尿毒症心肌病是慢性肾脏病（CKD）最常见的心血管并发症和首位死亡原因，而左室肥厚是尿毒症心肌病最主要的心脏结构改变，是心力衰竭的重要病理基础。大量毒素蓄积引起的高氧化应激可能是尿毒症心肌肥厚的一个突出诱因。Klotho是主要由肾脏产生的内源性抗氧化应激蛋白，可进入血液循环对心肌细胞发挥保护作用。我们既往研究发现，尿毒症时Klotho水平显著降低，而转染了Klotho的尿毒症小鼠左室肥厚明显减轻，推测Klotho水平与尿毒症心肌肥厚密切相关。为进一步明确Klotho在尿毒症心肌肥厚中的作用，本项目首先在CKD患者人群中分析了血清尿毒症蛋白结合毒素硫酸吲哚酚（IS）、Klotho水平与eGFR和左室肥厚的相关性，然后以新生大鼠心肌细胞、Klotho基因缺陷（kl/kl）小鼠、尿毒症小鼠为研究对象，探讨Klotho抑制尿毒症毒素诱导心肌细胞肥大的作用及机制以及Klotho对CKD左室肥厚的干预作用。本项目的研究发现：1） CKD患者血清Klotho水平与eGFR呈正相关，与IS水平和左室肥厚呈负相关；并且Klotho水平低于均值的患者与左室肥厚呈现更显著的负相关。2）C57BL/6J小鼠腹腔注射IS 8周后可以诱导左室肥厚并下调肾脏Klotho表达；与野生型小鼠相比，kl+/-小鼠腹腔注射IS后左室肥厚更加明显，提示蛋白结合毒素IS是左室肥厚的重要诱因，而Klotho蛋白的缺失将加重左室肥厚的发生和发展。3）IS可以通过活化Nox2/Nox4促进新生大鼠心肌细胞ROS生成，并通过激活MAPKs信号诱导心肌细胞肥大，而Klotho和Nox 抑制剂DPI可以显著抑制IS诱导的心肌细胞肥大，说明Klotho可以通过抑制IS诱导的Nox/ROS/MAPKs信号通路活化抑制心肌细胞肥大。4）Klotho腹腔注射显著抑制尿毒症小鼠心肌组织Nox2/Nox4表达和ROS水平，并明显改善CKD左室肥厚。本项目研究结果表明，尿毒症毒素导致的氧化应激增强和Klotho缺失导致的内源性抗氧化应激能力减弱可能是左室肥厚发生、发展的关键机制之一，上调机体Klotho水平，即内源性抗氧化应激能力则是干预尿毒症心肌病的有效措施。本项目研究不仅从心、肾内在关联角度诠释了尿毒症心肌病的发病原因，同时也为临床转化医学提供了可能的防治靶点。