延胡索酸酶基因(Fh)突变在高盐饮食肾损伤及血压盐敏感性形成中的作用

Renal injury and arterial blood pressure of many essential hypertensive patients exhibits an increased sensitivity to dietary salt intake. The molecular mechanisms underlying the exacerbated renal injury and blood pressure salt sensitivity are not well understood. In our previous proteomic study, we found dramatic differences in fumarase, the gene located on the chromosome 13, in the renal medulla between Dahl salt-sensitive rats and salt-insensitive Consomic 13. Sequencing of fumarase cDNA indicated one base mutation had been happened in SS rats, this led to the total fumarase activity was significantly reduced in the renal medulla compared with consomic 13 rats. Intravenous infusion of a fumarate precursor resulted in exacerbation salt-induced hypertension in consomic 13 rats. In addition, the fumarate infusion increased renal medullary tissue levels of H2O2. Treatment of cultured human renal epithelial cells with the fumarate precursor also increased cellular levels of H2O2. We also found that Hnrnp K in the renal medulla was upregulated by the 4% NaCl diet in Consomic 13 rats but down-regulated in SS rats. The level of angiotensinogen in the renal medulla was regulated in an opposite manner. Silencing of hnrnp K resulted in an up-regulation of angiotensinogen in cultured human kidney cells. The further ChIP analysis confirmed that Hnrnp K protein bound to the angiotensinogen promoter in the renal medulla in vivo. Combined with the knowledge of superoxide and NO produced by mTAL diffuses to the pericytes of the vasa recta, and modulated the sodium reabsorption and long-term control of arterial pressure and hypertension. The current proposal give the following hypothesis:①More reactive oxygen species were produced by pentose phosphate pathway and mitochondrial respiratory chain when fumarase activity was reduced in medulla of Dahl SS rats. Fumarase is the key enzyme in the TCA cycle, which catalyzes the conversion between fumarate and L-malate. The reduction of Fh activity will slow down the velocity of carbohydrate oxidation and production of ATP, as the compensation, the bypass metabolism pentose phosphate pathway should be enhanced. Whether this alteration causes more reactive oxygen species produced by NADPH oxydase will be the main focus of the problem. ②The synthesis of L-Arg and NO were reduced after fumarase activity was reduced. Fumarase is also involved in the urea cycle by the conversion of fumarate to L-malate and supply the aspartate to argininosuccinate synthase. The reduction of synthesis of L-Arg will reduce the production of NO in mTAL. ③ NADPH oxydase was activated by Ang II that came from renal medulla angiotensinogen. ④The key enzymes involved in the production of ROS were evoked by high salt diet. If the hypothesis were confirmed the mutation of fumarase gene could be considered as the real reason of renal injury and blood pressure salt sensitivity developed in high salt diet.

高盐饮食引起的肾脏损伤和血压升高在临床上具有很高的发病率且趋于低龄化，严重影响我国居民的健康质量，但目前对于高盐引起的肾脏损伤和血压盐敏感性产生的机制并不十分清楚。申请者通过蛋白质组学对这一问题的研究发现位于盐敏感大鼠13号染色体上的Fh基因发生点碱基突变并导致Fh酶的活性显著下降，动脉灌注延胡索酸乙脂可以引起显著的血压升高和肾脏氧化损伤。同时，高盐饮食还引起肾脏髓质血管紧张素原的高表达和氧自由基产生关键酶的高表达。结合近年形成的氧自由基/NO血压调控理论提出：①探讨Fh酶活性降低是否导致PPP途径和线粒体呼吸产生了更多的氧自由基？是否导致精氨酸合成减少，使得mTAL中NO产生不足？②高盐饮食后肾髓质血管紧张素原的高表达是否通过AngⅡ-PKCα途径激活NADPH氧化酶活性，导致氧自由基增加？最终明确Fh基因突变是否是肾脏氧化损伤和血压盐敏感性形成的根源，明确盐因素对氧自由基产生的贡献。

高盐饮食可以促进盐敏感个体高血压的发生和发展，在临床上具有很高的发病率且趋于低龄化，严重影响我国居民的健康质量。目前对于高盐引起的肾脏损伤和血压盐敏感性产生的机制并不十分清楚。本项目在前期对盐敏感大鼠模型研究的基础上，提出Fh酶活性的不足可能是盐敏感高血压发生发展的根源。项目展开以下三方面的研究工作：（1）Fh酶活性降低对氧自由基代谢的影响；（2）Fh酶活性降低后的代谢改变与代谢规律；（3）Fh酶活性降低对NO代谢的影响；从动物模型分析----细胞分子机制验证----动物模型验证，我们取得以下重要结果：（1）富马酸水合酶活性的不足导致苹果酸和门冬氨酸合成不足，特别是门冬氨酸，它是精氨酸合成的前体化合物。通过敲低HK2 细胞Fh基因的表达，细胞代谢减少了苹果酸、门冬氨酸、精氨酸以及NO的产出。这一机制通过给予盐敏感大鼠饮食中补偿苹果酸和门冬氨酸的量可以极大地缓解高盐引起的血压升高。（2）Fh活性不足，SS大鼠糖代谢效率降低，脂肪合成增强，蛋白质分解代谢所产生的氨基酸成为能量产生的主要来源，由于能量代谢消耗掉大量氨基酸，使得食物来源的精氨酸含量减少，NO合成减少，血压升高。（3）Fh活性降低后，NADPH氧化酶，OGDH的活性会增加，同时PPP途径的活性增强，用于提供拮抗氧自由基的NADPH。尽管和GSH合成与代谢的酶活性均显著提高，但总体GSH/GSSG依然低于对照，证实Fh酶活性降低的确可以引起细胞氧化还原状态的失衡，进而引起SS大鼠血压升高和血压的盐敏感性。从本项目的研究结果我们可以获得一下启示：（1）临床上可以通过改变膳食结构增加氨基酸代谢均衡达到盐敏感高血压的预防和治疗。本项目研究给出了具体的干预机制，即增加苹果酸，门冬氨酸的饮食可以阻滞盐敏感高血压的发生和发展。（2）由于氧自由基和NO不足是盐敏感高血压发病的主要根源，针对拮抗氧自由基和补充NO产生是临床盐敏感高血压预防和治疗的主要靶点。