Department of Internal Medicine II, Martin Luther University Halle-Wittenberg, Halle,
Department of Anaesthesia and Intensive Care, Diakonie Hospital, Halle, Germany.
Nephron. 2019;141(1):61-74. doi: 10.1159/000493805. Epub 2018 Oct 16.
Decreased levels of leucocytic angiotensin converting enzyme 2 (ACE2) relate to atherosclerosis in patients with chronic kidney disease (CKD). Recently, micro RNA 421 (miR-421) was found to target and down-regulate ACE2 in human cardiac myofibroblasts. In this study, we investigated the correlation between serum levels of miR-421 and ACE2 transcripts in circulating leukocytes of healthy individuals (NP), CKD (3-5) and haemodialysis (HD) patients. Furthermore, we tested the possible interaction between miR-421 and 3'-UTR of ACE2 under normal and uremic milieu.
The levels of circulating miR-421, serum Ang1-7 and expression of leucocytic ACE2, ACE, MASR, AT1R and AT2R were investigated in 16 CKD3-5 (76 ± 10 years), 32 HD patients (65 ± 13 years) and 23 NP (51 ± 5 years) by employment of specific primers, TaqMan and competitive enzyme-linked immunosorbent assay assays. Interaction between miR-421 and ACE2-3'-UTR was investigated on THP-1 cells by employment of normal and uremic sera, reporter vectors and miR-421 inhibitor. Effects of uremic toxins indoxyl sulphate, p-cresol and p-cresyl sulphate on ACE2 and miR-421 levels were investigated in THP-1 monocytes.
The levels of serum miR-421 were significantly elevated, while Ang1-7 was significantly decreased in CKD3-5 and HD patients as compared with NP. Serum Ang1-7 correlated positively with leucocytic ACE2 (r2 = 0.213, p < 0.001). We found a significant and inverse correlation between the levels of circulating miR-421 and the expression of leucocytic ACE2 (r2 = 0.223, p < 0.0001). Further significant and positive correlations could be demonstrated between miR-421 and the transcripts of leucocytic AT1R (r2 = 0.094, p < 0.05) or eGFR (r2 = 0.231, p < 0.0001) or CRP (r2 = 0.092, p < 0.01). We found no correlations between miR-421 and ACE or AT2R or MASR expression. Treatment with miR-421 or uremic serum led to noticeable decrease of reporter 3'UTR-ACE2. Anti-miR-421 treatment resulted in the up-regulation of ACE2 protein. All uremic toxins tested were able to significantly elevate miR-421 levels and decrease the monocytic ACE2 transcripts.
Uremic patients show an enhanced expression of serum miR-421 as compared to healthy individuals. A strong association of circulating miR-421 with decreased transcripts of ACE2 may contribute to the low expression of the enzyme in leukocytes of CKD patients, further supporting the development of atherosclerotic events.
白细胞血管紧张素转换酶 2（ACE2）水平降低与慢性肾脏病（CKD）患者的动脉粥样硬化有关。最近，发现 micro RNA 421（miR-421）可靶向并下调人心肌成纤维细胞中的 ACE2。在这项研究中，我们研究了健康个体（NP）、CKD（3-5 期）和血液透析（HD）患者循环白细胞中血清 miR-421 水平与 ACE2 转录本之间的相关性。此外，我们还在正常和尿毒症环境下检测了 miR-421 与 ACE2 3'-UTR 之间可能的相互作用。
采用特定引物、TaqMan 和竞争性酶联免疫吸附试验检测 16 名 CKD3-5 期（76±10 岁）、32 名 HD 患者（65±13 岁）和 23 名 NP（51±5 岁）的循环 miR-421、血清 Ang1-7 以及白细胞 ACE2、ACE、MASR、AT1R 和 AT2R 的表达。采用正常和尿毒症血清、报告载体和 miR-421 抑制剂研究 miR-421 与 ACE2-3'-UTR 之间的相互作用。在 THP-1 单核细胞中研究尿毒症毒素吲哚硫酸酯、对甲酚和对甲酚硫酸盐对 ACE2 和 miR-421 水平的影响。
与 NP 相比，CKD3-5 和 HD 患者的血清 miR-421 水平显著升高，而 Ang1-7 水平显著降低。血清 Ang1-7 与白细胞 ACE2 呈正相关（r2=0.213，p<0.001）。我们发现循环 miR-421 水平与白细胞 ACE2 表达之间存在显著的负相关（r2=0.223，p<0.0001）。进一步显著的正相关可以在 miR-421 与白细胞 AT1R 的转录物之间得到证明（r2=0.094，p<0.05）或 eGFR（r2=0.231，p<0.0001）或 CRP（r2=0.092，p<0.01）。我们没有发现 miR-421 与 ACE 或 AT2R 或 MASR 表达之间的相关性。miR-421 或尿毒症血清的处理导致报告 3'UTR-ACE2 的明显减少。抗 miR-421 处理导致 ACE2 蛋白的上调。所有测试的尿毒症毒素都能显著升高 miR-421 水平并降低单核细胞 ACE2 转录物。
与健康个体相比，尿毒症患者表现出血清 miR-421 的增强表达。循环 miR-421 与 ACE2 转录本降低的强烈相关性可能有助于 CKD 患者白细胞中酶的低表达，进一步支持动脉粥样硬化事件的发生。
