Comparison of the Inhibitory Mechanisms of Diethyl Citrate, Sodium Citrate, and Phosphonoformic Acid on Calcification Induced by High Inorganic Phosphate Contents in Mouse Aortic Smooth Muscle Cells.

OBJECTIVE: This study aimed to investigate the differences and inhibitory effects of diethyl citrate (Et2Cit), sodium citrate (Na3Cit), and phosphonoformic acid (PFA) on calcification induced by high inorganic phosphate (Pi) contents in mouse aortic smooth muscle cells (MOVAS) and to develop drugs that can induce anticoagulation and inhibit vascular calcification (VC). METHODS: Alive and fixed MOVAS were assessed for 14 days in the presence of high Pi with increasing Et2Cit, Na3Cit, and PFA concentrations. Calcification on MOVAS was measured through Alizarin red staining and the deposited calcium amount; apoptosis was detected by annexin V staining; and cell transdifferentiation was examined by measuring smooth muscle lineage gene (α-SMA) expression and alkaline phosphatase activity. RESULTS: Coincubation of MOVAS with Et2Cit, Na3Cit, and PFA significantly decreased Pi-induced VC in live MOVAS, and the apoptotic rate was reduced by low inhibitor concentrations. The 3 inhibitors could prevent the alkaline phosphatase activity induced by high Pi contents and increased the expression of α-smooth muscle actin genes. Thus, the transdifferentiation of MOVAS into osteoblast-like cells was blocked. Their inhibitory effects exhibited concentration dependence. The inhibitory effect of each inhibitor at the same concentration showed the following trend: PFA > Na3Cit > Et2Cit. CONCLUSIONS: Et2Cit, Na3Cit, and PFA prevented the calcification of MOVAS and inhibited the osteochondrocytic conversion of vascular smooth muscle cells. Thus, Et2Cit and Na3Cit as anticoagulants may alleviate VC in clinical applications.

UA promotes epithelial­mesenchymal transition in peritoneal mesothelial cells.

Long­term peritoneal dialysis is often limited or interrupted due to the development and progression of peritoneal fibrosis. Accumulating evidence suggests that epithelial­mesenchymal transition (EMT) is a major component of peritoneal injury associated with peritoneal fibrosis in the end stage of renal disease; however, at present, the underlying mechanisms remain unclear. Thus, in the present study, uric acid (UA)­induced EMT of peritoneal mesothelial cells was investigated by western­blot and immunofluorescence staining. The results revealed that peritoneal mesothelial cells stimulated with UA underwent EMT, as demonstrated by the decreased expression of epithelial markers (E­cadherin) and an increased expression of mesenchymal markers (α­smooth muscle actin and vimentin). Additionally, it was reported that UA could facilitate the progression of EMT of peritoneal mesothelial cells via EMT transcription pathways, including transforming growth factor­ß1/mothers against decapentaplegic homolog 3 and P38/mitogen­activated protein kinase by western­blot and reverse transcription semi­quantitative polymerase chain reaction. The results of the present study suggest that UA could promote EMT and may contribute to peritoneal chronic disease. Furthermore, the data obtained suggest that the levels of blood UA may account for the development of EMT; thus, lowering the levels of blood UA may be beneficial to inhibit the occurrence and development of peritoneal fibrosis.

Changes in urinary nanocrystallites in calcium oxalate stone formers before and after potassium citrate intake.

The property changes of urinary nanocrystallites in 13 patients with calcium oxalate (CaOx) stones were studied before and after ingestion of potassium citrate (K3cit), a therapeutic drug for stones. The analytical techniques included nanoparticle size analysis, transmission electron microscopy, X-ray diffraction, and Fourier-transform infrared spectroscopy. The studied properties included the components, morphologies, zeta potentials, particle size distributions, light intensity autocorrelation curves, and polydispersity indices (PDIs) of the nanocrystallites. The main components of the urinary nanocrystallites before K3cit intake included uric acid, ß-calcium phosphate, and calcium oxalate monohydrate. After K3cit intake, the quantities, species, and percentages of aggregated crystals decreased, whereas the percentages of monosodium urate and calcium oxalate dehydrate increased, and some crystallites became blunt. Moreover, the urinary pH increased from 5.96 ± 0.43 to 6.46 ± 0.50, the crystallite size decreased from 524 ± 320 nm to 354 ± 173 nm, and the zeta potential decreased from -4.85 ± 2.87 mV to -8.77 ± 3.03 mV. The autocorrelation curves became smooth, the decay time decreased from 11.4 ± 3.2 ms to 4.3 ± 1.7 ms, and the PDI decreased from 0.67 ± 0.14 to 0.53 ± 0.19. These changes helped inhibit CaOx calculus formation.