Cinacalcet ameliorates cardiac fibrosis in uremic hearts through suppression of endothelial-to-mesenchymal transition

Abstract

Cardiac fibrosis is a common feature in patients with chronic kidney disease (CKD). Increasing evidence has shown that elevated parathyroid hormone (PTH) is causally involved in cardiac fibrosis [1–4]. Cinacalcet (CINA), a calcimimetic agent, reduces PTH levels without increasing circulating levels of calcium and phosphorus [5,6]. Recent studies have shown that CINA treatment markedly attenuated myocardial fibrosis and vascular remodeling [6,7]. However, the underlying mechanisms of this action are still unknown. Endothelial-to-mesenchymal transition (EndMT) plays an important role in the development of cardiac fibrosis [8,9]. The purpose of this study was to investigate whether CINA ameliorated cardiac fibrosis in rats with uremia was mediated by suppression of EndMT. Study protocols were approved by the Ethical Committee of Southeast University. The rat model of uremia and secondary hyperparathyroidism was established as previously described [10]. Briefly, 8-week-old male Wistar rats were randomly divided into three groups: the control group (CTL, n = 6), the uremia group (U, n = 8) and the CINA-treated uremia group (U + CINA, n = 8). Uremia was induced by feeding rats a 0.75% adenine diet for 4 weeks. After adenine withdrawal, all animals were maintained on a 1.03% phosphorus diet for next 8 weeks. At initiation of the adenine diet, rats in the U + CINA group were orally treated with CINA (Santa Cruz Biotechnology, USA) once daily (10 mg/kg) until the end of experiment. The effect of elevated PTH on EndMT was also studied in primary aortic endothelial cells (ECs). It was demonstrated that the area of cardiac fibrosis in the U group was significantly increased compared to controls, and it was significantly attenuated by the administration of CINA (Fig. 1A–B). And CINA also inhibited the expression of the extracellular matrix elements type I collagen and fibronectin in uremic hearts (Fig. 1C–E). Furthermore, there was significant up-regulation of mesenchymal markers FSP1 and α-SMA, and down-regulation of the endothelial marker CD31, while CINA markedly abrogated expression of EndMT-associated markers in uremic hearts (Fig. 2). Compared with the U group, serum PTH concentrations were significantly lower in uremic rats treated with CINA (440.64 ± 43.25 vs 326.54 ± 47.86, p < 0.05). To further confirm the causal effect of elevated PTH on induction of EndMT, an in vitro experiment with primary aortic ECs was performed. The results showed that PTH incubation increased expression of FSP1 and α-SMA and decreased expression of CD31 at mRNA and protein levels in a concentration and time dependent manner (p < 0.05). Fig. 1 Effect of CINA on fibrotic lesions in uremic hearts. (A) Cardiac fibrosis was assessed by Masson’s trichrome staining. Images with different magnifications are shown. (B) Quantitative determination of cardiac fibrosis in the different groups. ... Fig. 2 Effect of CINA on the expression of EndMT-related markers in uremic hearts. (A) Representative micrographs showing immunohistochemical staining for cardiac expression of CD31 and FSP1 (original magnification is ×200). (B) Representative micrographs ... In conclusion, our data demonstrated that elevation of PTH induces EndMT and contributes to cardiac fibrosis in uremic rats, which could be prevented by CINA treatment. These results suggest that strategies aiming at lowering PTH levels might exert cardiac protective effects through an anti-EndMT mechanism.