| Objective: Cardiovascular complications are common among patients with chronic kidney disease(CKD). It is increasingly apparent that cardiovascular complications are more likely leading death to individuals with chronic kidney disease(CKD). Vascular calcification is regarded as an key point of cardiovascular risk relating to osteogenic/chondrogenic differentiation. In uremic patients, serum bicarbonate peaks following bicarbonate loading during hemodialysis. Present study has suggested that alkalinization increases vascular calcification in uremic rats. KCa3.1(Intermediate-conductance Ca2+-activated K+ channels) channel is sensitive to intracellular Ca2+ concentration and widely distributed throughout the vascular smooth muscle cells contributing to a variety of cell activation processes. In this study, we present evidence that the KCa3.1 channels are required for rat VSMCs calcification in response to Alkalinization, trying to find new therapy of vascular calcification.Methods:1 Six healthy male four-week-old SD rats were used for VSMCs culture. Six healthy male six-week-old SD rats were selected for Aortic rings cultured in vitro. All the rats were purchased from animal experiment center of Hebei Medical University.2 Vascular smooth muscle cells(VSMCs) and aortic rings were obtained from rat thoracic aorta, then randomly divided into control group(p H was provided into7.4, 8.0), high phosphorus groups(p H was provided into7.4, 7.7 and 8.0, VSMCs in three groups were treated with 10mmol/L β-glycerophosphate. 10mmol/L HCl and 10mmol/L Na HCO3 were used to adjust the p H) and TRAM-34 group(20nmol/L was added into p H8.0 high phosphorus dulbecco’s modified eagle’s medium).3 Calcium deposition was measured by Alizarin red staining, calcium content after cells simulated for 12 days. Calcium deposition was measured by von Kossa staining and calcium content after aortic rings were cultured for 12 days. Intracellular free Ca2+ was measured by probe Fluo 3-AM. The expression of KCa3.1, runt-related transcription factor 2(Runx2)were detected by RT-PCR and Western Blot after cells stimulated for 4 days. ALP activity was measured by enzyme linked immunosorbent assay after cells simulated for 12 days. The expression of KCa3.1, Runx2 were detected by Immunohistochemistry after aortic rings were cultured for 4 days.4 Results were presented as means ± SD, and SPSS 17.0 software(SPSS Company, Chicago, IL, USA) was used for the ANOVA analysis and Dunnett test. For all the statistical tests, significance was defined as P<0.05.Results: 1 The effect of calcification in alkalinization and β-glycerophosphate induced VSMCs and aortic rings 1.1 Compared with control group, calcification in VSMCs was significantly increased in high phosphorus group(P<0.05) and decreased in TRAM-34 group(P<0.05). 1.2 Compared with control group, calcification in aortic rings was significantly increased in high phosphorus group(P<0.05) and decreased in TRAM-34 group(P<0.05). 2 Compared with control group, the Ca2+ influx in VSMCs was significantly increased in high phosphorus group(P<0.05) and blocked in TRAM-34 group(P<0.05). 3 The expression of KCa3.1, Runx2 and the activity of ALP in alkalinization and β-glycerophosphate induced VSMCs and aortic rings 3.1 Compared with control group,the expression of KCa3.1, Runx2 and the activity of ALP in high phosphorus groups were increased(P<0.05) and decreased in TRAM-34 group(P<0.05). Besides, expression of Runx2 and KCa3.1 were augmented as the p H was higher(P<0.05). 3.2 The expression of KCa3.1 and Runx2 in aortic rings was higher in high phosphorus groups(P<0.05) and lower in TRAM-34 group(P<0.05).Conclusions:Up-regulation of KCa3.1 may contribute to alkalinization and β-glycerophosphate induced VSMCs calcification through osteogenic/ chondrogenic differentiation. |
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