Uremic conditions lead to decreased vascular expression of lncRNA CARMN in vitro and in vivo

Background: Chronic kidney disease (CKD) is closely associated with cardiovascular disease. This association is largely driven by vascular pathologies that are induced and aggravated by CKD. Advanced CKD leads to uremia, which is characterized as a complex and heterogeneous condition with numerous effectors on the cardiovascular system. Mineral dysregulation, especially the retention of inorganic phosphate, is a key player in this context. In the process of uremia-induced vascular pathologies, a change in vascular smooth muscle cell phenotype is observed, which is accompanied by a loss of myofibroblast markers. Ultimately, this leads to the loss of the myofibroblast phenotype and progresses to vascular calcification. Recent research has identified that an SMC-specific long non-coding RNA, cardiac mesoderm enhancer-associated noncoding RNA (CARMN), plays an important role in maintaining the contractile phenotype of VSMCs. Vascular disease is associated with dramatically decreased CARMN expression in VSMC. To date, there is no data investigating CARMN in the context of uremia-induced vascular disease.

Methods and Results: A murine model of uremia was established using adenine induced nephropathy. We investigated two different dose regimes of adenine-rich diet (high and low-dose) (A). Mice fed with a high-dose adenine diet for 10 weeks showed more prominent renal fibrosis as well as pronounced uremia in the plasma ("severe uremia") (B,C). The low-dose regime of adenine-diet showed less pronounced renal fibrosis marker and uremic plasma compared to control ("mild uremia"). Thoracic aortas were explanted and investigated by gene expression analysis. We observed statistical significant downregulation of CARMN in animals with mild and severe uremia compared to control (D). This effect was more pronounced in animals with severe uremia and accompanied by a down regulation of myofibroblast marker ACTA2. Echocardiography of control and uremic animals did not reveal significant differences between the groups (E). As uremia is a multifactorial and heterogeneous state, we performed cell culture experiments to identify underlying mechanisms. SMCs derived from human coronary arteries were stimulated with phosphate (NaH₂PO₄) and uremic toxin indoxyl sulfate (IS) in concentrations from 50-250μmol/l to mimic uremic plasma. VSMCs were stimulated for 24, 48 and 72 h and gene expression analysis was performed. HCASMC treated with phosphate-containing medium showed a decreased expression of ACTA2 and CARMN (F). This was observed after 48h of treatment and maintained over 72h. These findings indicate loss of myofibroblastic and contractile phenotype of VSMCs, which is observed in various diseases such as uremia-induced vascular calcification. Furthermore, we observed that this effect was phosphate dependent as increasing doses of IS (50-250μmol) showed no influence on ACTA2 and CARMN expression after 24, 42, and 72h. 

Conclusion: Uremic conditions lead to a downregulation of CARMN in the aortas of mice. This effect is associated with decreased expression of myofibroblastic marker ACTA2 and is dose-dependent under more pronounced uremia. In vitro, we identified hyperphosphatemia as a cause of these effects in HCASMCs. These findings indicate that the hyperphosphatemic conditions observed in uremic plasma lead to a decreased expression of the vasculoprotective CARMN and the subsequent loss of myofibroblast and contractile properties of HCASMCs.