We used heterozygous SERCA2 C674S gene mutation knock-in (SKI) mice, where one copy of C674 was substituted by serine to represent partial C674 oxidation. In renal proximal tubule (RPT) cells, the substitution of C674 by serine decreased mitochondrial Ca2+ content, increased mitochondrial membrane potential, ATP content, and reactive oxygen species (ROS), which could be reversed by ERS inhibitor 4-phenylbutyric acid or SERCA2 agonist CDN1163. In SKI RPT cells, the redox modulator Tempol alleviated oxidative stress, downregulated the protein expression of ERS markers and soluble epoxide hydrolase, upregulated the protein expression of dopamine D1 receptor, and reduced Na+/K+- ATPase activity. In SKI mice, SERCA2 agonists CDN1163 and [6]-Gingerol, or the redox modulator Tempol increased urine output and lowered blood pressure.
The irreversible oxidation of C674 is not only an indicator of increased ROS, but also further inducing oxidative stress to cause hypertension. Activation of SERCA2 or inhibition of oxidative stress is beneficial to alleviate hypertension caused by SERCA2 dysfunction.
结果:我们使用了SERCA2C674S基因突变敲入(SKI)小鼠,其中C674的一个拷贝被丝氨酸取代以代表C674的部分氧化。在肾近曲小管(RPT)细胞中,丝氨酸取代C674降低了线粒体Ca2+含量,线粒体膜电位增加,ATP含量,和活性氧(ROS),可以通过ERS抑制剂4-苯基丁酸或SERCA2激动剂CDN1163逆转。在SKIRPT细胞中,氧化还原调节剂Tempol减轻了氧化应激,下调ERS标记和可溶性环氧化物水解酶的蛋白表达,上调多巴胺D1受体的蛋白表达,并降低Na+/K+-ATP酶活性。在SKI小鼠中,SERCA2激动剂CDN1163和[6]-姜辣素,或氧化还原调节剂Tempol增加尿量和降低血压。
结论:C674的不可逆氧化不仅是ROS增加的指标,还会进一步诱导氧化应激导致高血压。激活SERCA2或抑制氧化应激有利于缓解SERCA2功能障碍引起的高血压。