Circulating miR-421 Targeting Leucocytic Angiotensin Converting Enzyme 2 Is Elevated in Patients with Chronic Kidney Disease.

BACKGROUND: 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.

METHODS: 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.

RESULTS: 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.

CONCLUSIONS: 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.