Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
National Center of Structural Biology and Bioimaging/CENABIO, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
Cell Physiol Biochem. 2020 Nov 18;54(6):1143-1162. doi: 10.33594/000000306.
BACKGROUND/AIMS: Chronic malnutrition (M) affects >1 billion people worldwide. Epidemiological data point to long-term renal and cardiovascular outcomes (e.g. arterial hypertension, cardiorenal syndromes). The renin-angiotensin-aldosterone system (RAAS) has been implicated in the physiopathology of these disturbances, but M-induced alterations in RAAS-modulated renal Na handling and their cardiovascular repercussions are not known. Moreover, altered tissue-specific histone deacetylases (HDAC) results in arterial hypertension and the use of sodium Valproate (Val; a HDAC inhibitor) reduces blood pressure. However, there are no reports regarding the renal and cardiovascular effects of HDAC inhibition in M, or on the signaling pathways involved. The central aim of our study has been to investigate whether alterations in the HDAC/RAAS axis underpin alterations in active Na transport in the kidney and heart, and affects blood pressure.
Male rats aged 28 days were given either a control (C) or a multideficient diet (Regional Basic Diet, RBD), which mimics alimentary habits from developing countries. Subgroups received Losartan (Los), a blocker of type 1 Angiotensin II receptors. When the rats reached 70 days, new subgroups received Val until they were 90 days of age. Homogenates and enriched plasma membrane fractions from renal cortex corticis and cardiomyocytes were obtained by differential centrifugation of the tissues. The activity of renal and cardiac deacetylases was assayed by measuring - after incubation with the membranes - the amount of deacetylated lysines in a substrate containing an acetylated lysine side chain. Protein kinases activities were measured following the incorporation of the γ-phosphoryl group of [γ-P]ATP into Ser/Thr residues of histone type III-S. The activity of Na-transporting ATPases (kidney and heart) was quantified by measuring the release of P from ATP that was sensitive to ouabain ((Na+K)ATPase), or sensitive to furosemide (Na-ATPase). Tail-cuff plethysmography was used to measure systolic blood pressure and heart rate.
M provoked HDAC downregulation, which was reversed by Los and Val, either alone or in combination, with selective upregulation of protein kinases C and A (PKC, PKA) in renal cortex corticis, but not in left ventricle cardiomyocytes. The 2 kinases were strongly inhibited by Los and Val in both organs. Malnourished rats developed elevated systolic arterial pressure (SAP) and heart rate (HR) at 70 days of age; Los and Val restored the control SAP, but not HR. Functional and the above biochemical alterations were associated with the deregulation of renal and cardiac Na-transporting ATPases. (Na+K)ATPase activities were downregulated in M rats in both organs, and were further inhibited by the pharmacological treatments in the renal cortex corticis (C and M groups) and the left ventricle (only in C rats). No additional effect was found in cardiac (Na+K)ATPase from M rats. Ouabain-resistant Na-ATPase was upregulated in renal cortex corticis and downregulated in cardiomyocytes, returning to C values after administration of Los and Val.
The HDAC/RAAS axis appears to be a key regulator of Na-transporting ATPases in renal cortex corticis and cardiomyocytes via an appropriate balance of PKC and PKA activities. Modifications within the HDAC/RAAS axis provoked by chronic M - with repercussions in renal and cardiac Na transport - underpin alterations in bodily Na homeostasis that culminate with the onset of arterial hypertension and potential cardiorenal syndrome.
背景/目的：慢性营养不良（M）影响全球超过 10 亿人。流行病学数据表明存在长期的肾脏和心血管结局（例如动脉高血压、心肾综合征）。肾素-血管紧张素-醛固酮系统（RAAS）与这些紊乱的病理生理学有关，但 M 引起的 RAAS 调节的肾脏钠处理改变及其心血管影响尚不清楚。此外，组织特异性组蛋白去乙酰化酶（HDAC）的改变会导致动脉高血压，而使用丙戊酸钠（Val；一种 HDAC 抑制剂）可以降低血压。然而，目前尚无关于 HDAC 抑制在 M 中的肾和心血管作用或涉及的信号通路的报道。我们研究的主要目的是研究 HDAC/RAAS 轴的改变是否是肾脏和心脏中主动钠转运改变的基础，并影响血压。
28 天大的雄性大鼠给予对照（C）或多缺陷饮食（区域基础饮食，RBD），这模仿了来自发展中国家的饮食习惯。亚组接受洛沙坦（Los），一种 1 型血管紧张素 II 受体阻滞剂。当大鼠达到 70 天时，新亚组接受 Val，直到它们 90 天大。通过组织的差速离心从肾皮质皮质和心肌细胞中获得肾和心肌细胞的质膜富集部分。通过测量在孵育后含乙酰化赖氨酸侧链的底物中去乙酰化赖氨酸的量来测定肾和心肌细胞的脱乙酰酶活性。蛋白激酶活性通过测量 [γ-P]ATP 的 γ-磷酸基团掺入组蛋白 III-S 的 Ser/Thr 残基来测量。通过测量对哇巴因（（Na+K）ATPase）敏感或对呋塞米（Na-ATPase）敏感的 ATP 释放来定量钠转运 ATP 酶（肾和心脏）的活性。尾套体积描记法用于测量收缩压和心率。
M 引起 HDAC 下调，洛沙坦和 Val 单独或联合使用均可逆转，同时选择性地上调肾皮质皮质中的蛋白激酶 C 和 A（PKC、PKA），但左心室心肌细胞中没有。这 2 种激酶在这两种器官中均被洛沙坦和 Val 强烈抑制。营养不良的大鼠在 70 天大时出现收缩压（SAP）和心率（HR）升高；洛沙坦和 Val 恢复了对照 SAP，但没有恢复 HR。功能和上述生化改变与肾脏和心脏钠转运 ATP 酶的失调有关。（Na+K）ATP 酶活性在 M 大鼠的两个器官中均下调，并且在肾皮质皮质（C 和 M 组）和左心室（仅在 C 大鼠中）的药理学治疗中进一步受到抑制。在 M 大鼠的心脏（Na+K）ATP 酶中未发现其他作用。肾皮质皮质中的钠泵被上调，心肌细胞中的钠泵被下调，在用洛沙坦和 Val 处理后恢复到 C 值。
HDAC/RAAS 轴似乎是通过 PKC 和 PKA 活性的适当平衡来调节肾皮质皮质和心肌细胞中钠转运 ATP 酶的关键调节剂。慢性 M 引起的 HDAC/RAAS 轴改变——对肾脏和心脏钠转运的影响——导致体内钠稳态的改变，最终导致动脉高血压和潜在的心肾综合征的发生。
