[Effect and mechanism of intermittent alkaline stimulation on high phosphorus induced calcification in vascular smooth muscle cells of rats].

Objective: To explore the effect and possible mechanisms of intermittent alkaline on rat vascular smooth muscle cells (VSMCs) calcification induced by high phosphorus. Methods: VSMCs were isolated from rat thoracic aorta and cultured in vitro. The fourth generation VSMCs were randomly divided into control group, high phosphorus+ pH7.4, high phosphorus+ pH7.5, high phosphorus+ pH7.6 and high phosphorus+ pH7.7 group with random number table. The control group was cultured in DMEM with 10% fetal bovine serum. Other groups were cultured in DMEM with 10 mmol/L ß-glycerophosphate and alkalized by 7.4% NaHCO(3) to adjust the pH respectively. After the intervention of 4 hours, the control group was replaced with the normal medium containing 10% fetal bovine serum, the other 4 groups were replaced with high phosphorus based on the pH value of the culture medium, and then replaced the culture medium every other day. After 4 days intervention, the mRNA and protein expression of L type calcium channel ß(3) subunit(LTCC ß(3)) and Runt related transcription factor 2 (Runx2) were detected by RT-PCR and Western blot. After 4 days intervention, the level of VSMC calcium ion was detected by Fluo-3/AM. After 14 days intervention, alkaline phosphatase (ALP) activity was measured by enzyme linked immunosorbent assay (ELISA) and the calcification was observed by measuring calcium content. Results: (1) Compared with control group, the gene and protein expressions of LTCC ß(3) were higher in high phosphorus+ pH7.4 group (0.49±0.03 vs. 0.23±0.02 and 0.45±0.03 vs. 0.26±0.02 respectively, all P<0.05). Compared with high phosphorus+ pH7.4 group, the mRNA(0.86±0.05) and protein(0.62±0.04) expressions of LTCC ß(3) were higher in high phosphorus+ pH7.5 group (P<0.05). Compared with high phosphorus+ pH7.5 group, the mRNA(0.99±0.05) and protein(0.80±0.03) expressions of LTCC ß(3) were higher in high phosphorus+ pH7.5 group (all P<0.05). Compared with high phosphorus+ pH7.6 group, the mRNA(1.16±0.05) and protein(0.93±0.03) expressions of LTCC ß(3) were higher in high phosphorus+ pH7.7 group (all P<0.05). (2) Compared with control group, calcium ion influx were higher in high phosphorus+ pH7.4 group (124.61±6.06 vs. 75.68±7.82, P<0.05). Compared with high phosphorus+ pH7.4 group, calcium ion influx was higher in high phosphorus+ pH7.5 group(210.85±9.75, P<0.05). Compared with high phosphorus+ pH7.5 group, calcium ion influx was higher in high phosphorus+ pH7.6 group(298.44±11.42, P<0.05). Compared with high phosphorus+ pH7.6 group, calcium ion influx was higher in high phosphorus+ pH7.7 group(401.13±11.41, P<0.05). (3) Compared with control group, the mRNA and protein expressions of Runx2 and ALP were higher in high phosphorus+ pH7.4 group (0.60±0.04 vs. 0.34±0.03, 0.42±0.04 vs. 0.21±0.02, 67.2±4.3 vs. 23.2±2.3 respectively, all P<0.05). Compared with high phosphorus+ pH7.4 group, the mRNA(0.76±0.05) and protein(0.68±0.03) expressions of Runx2 and ALP(102.1±5.4) were higher in high phosphorus+ pH7.5 group (all P<0.05). Compared with high phosphorus+ pH7.5 group, the mRNA(0.90±0.05) and protein(0.90±0.05) expressions of Runx2 and ALP(139.3±4.9) were higher in high phosphorus+ pH7.6 group (all P<0.05). Compared with high phosphorus+ pH7.6 group, the mRNA(1.11±0.05) and protein(1.08±0.06) expressions of Runx2 and ALP(197.0±6.7) were higher in high phosphorus+ pH7.7 group (all P<0.05). (4) Compared with control group, the calcium content were higher in high phosphorus+ pH7.4 group ((75.4±4.3)mg/g pro vs.(25.2±2.1)mg/g pro, P<0.05). Compared with high phosphorus+ pH7.4 group, the calcium content were higher in high phosphorus+ pH7.5 group ((100.8±5.7) mg/g pro, P<0.05). Compared with high phosphorus+ pH7.5 group, the calcium content were higher in high phosphorus+ pH7.6 group ((143.5±6.1) mg/g pro, P<0.05). Compared with high phosphorus+ pH7.6 group, the calcium content were higher in high phosphorus+ pH7.7 group ((205.1±8.2) mg/g pro, P<0.05). Conclusion: Intermittent alkaline stimulation can promote high phosphorus induced rat VSMCs calcification possibly through upregulating LTCC ß(3) subunit gene and protein expression, increasing calcium ion influx and enhancing VSMCs phenotypic transformation.

[Role of nuclear factor of activated T-cells cytoplasmic 1 on vascular calcification in rats with chronic renal failure].

Objective: To explore the role of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) on vascular calcification in chronic renal failure rats. Methods: Nineteen male Sprague-Dawley (SD) rats were randomly divided into three groups: sham-operated group (n=6), 5/6 Nephrectomy (Nx) group (n=6), 5/6 Nx+ calcitriol group (n=7). Vascular calcification was determined by von Kossa staining and orthocresolphthalein complexone (OCPC) method. Protein expressions of NFATc1 and runt-related transcription factor 2 (Runx2) in aortas were measured by immunohistochemistry.In vitro, vascular smooth muscle cells (VSMCs) were primarily cultured and calcification was induced by ß-glycerophosphate (ß-GP). These cells were then randomly divided into control group, calcification group (10 mmol/L ß-GP) and cyclosporin A (CsA) intervention group (10 mmol/L ß-GP+ 1 µg/ml CsA). Calcium deposition was measured by Alizarin red staining and OCPC method; alkaline phosphatase (ALP) activity was measured by enzyme-linked immunosorbent assay. RT-PCR and Western blotting were used to observe the mRNA and protein expression of VSMCs NFATc1 and Runx2 respectively. Results: Compared to that in sham-operated and 5/6 Nx group, the expression of NFATc1 was obviously up-regulated in 5/6 Nx+ calcitriol group (7.20±0.46 vs 1.52±0.77, 2.04±1.31, P<0.05). In vitro, VSMCs calcification was successfully induced by high phosphorus environment, and RT-PCR and Western blotting showed that the expressions of NFATc1 and Runx2 were up-regulated (P<0.05). The calcification level in CsA intervention group was lower than that in calcification group [(60.86±7.95) vs (107.20±11.07) mg/g, P<0.05], and expression of Runx2 (mRNA and protein level) and ALP activity [(48.63±3.02) vs (98.75±3.46) U/g, P<0.05] decreased as well. Conclusion: NFATc1 contributes to accelerating vascular calcification in rat with chronic renal failure, the possible mechanism of which is that NFATc1 promotes VSMCs transformation to osteogenic phenotype.

[Effects of intermediate conductance calcium-activated potassium channel blocker TARAM-34 on ß-glycerophosphate induced vascular smooth muscle cells calcification].

OBJECTIVE: To observe the role of TRAM-34 (1-((2-chlorophenyl)diphenylmethyl)-1H-pyrazole), the blocker of intermediate conductance calcium-activated potassium channel (KCa3.1), on ß-glycerophosphate induced vascular calcification in vitro. METHODS: Vascular smooth muscle cells(VSMCs) were obtained from rat thoracic aorta, and VSMCs after the fourth passage and aortic rings were divided into control group (cultured in DMEM with 10% fetal bovine serum), high phosphorus group (cultured in DMEM with 10% fetal bovine serum and 10% ß-glycerophosphate) and TRAM-34 group(20 nmol/L TRAM-34 was added into high phosphorus DMEM). Calcium deposition of VSMCs and aortic rings were measured by o-cresolphthalein complexone method.Calcium influx of VSMCs was measured by immunofluorescence probe Fluo-3 AM.The expression of runt-related transcription factor 2(Runx2)was detected by RT-PCR and Western blot for cells and immunohistochemistry for aortic rings.ALP activity was measured by alkaline phosphatase activity detection kit. RESULTS: (1) Compared with control group, calcification was significantly increased in high phosphorus group ((121.67±6.17) mg/g vs. (84.38±8.17) mg/g, P<0.05) and this effect could be attenuated by TRAM-34 ((93.31±11.36) mg/g, P<0.05 vs. high phosphorus group) after 12 days culture. Similar results were found in aortic rings cultured for 12 days-high phosphorus group: (7.17±0.57) mg/g vs. CONTROL: (1.18±0.13) mg/g (P<0.05) and TRAM-34: (4.71±0.42) mg/g, P<0.05 vs. high phosphorus group.(2) Compared with control group, the calcium influx was higher in high phosphorus group (349.22±40.47 vs. 151.67±16.94, P<0.05) and reduced in TRAM-34 group (194.67±22.21, P<0.05 vs. high phosphorus group) in VSMCs simulated for 4 days. (3) Both mRNA and protein expressions of Runx2 in high phosphorus groups were higher than in control group (0.630±0.033 vs.0.340±0.058 and 0.865±0.031 vs.0.414±0.011, both P<0.05) and lower in TRAM-34 group (0.399±0.023 and 0.575±0.014, both P<0.05 vs. high phosphorus group) in VSMCs simulated for 4 days.Besides, compared with high phosphorus group, the expression of Runx2 was decreased in control group(0.113±0.010 vs.0.067±0.008, P<0.05) and TRAM-34 group (0.069±0.006, P<0.05) after aortic rings were cultured for 4 days. (4) Compared with control group, the activity of ALP was significantly increased in high phosphorus group (96.56±9.84 vs.46.92±4.60, P<0.05) and decreased in TRAM-34 group(70.20±8.41, P<0.05 vs. high phosphorus group) in VSMCs simulated for 12 days. CONCLUSION: KCa3.1 blocker TRAM-34 can inhibit ß-glycerophosphate induced VSMCs and aortic ring calcification through inhibiting calcium influx, downregulating Runx2 expression and attenuating osteogenic differentiation.

[Polymorphism at the miR-502 binding site in the 3' untranslated region of SET8 gene is associated with the risk of clear cell renal cell carcinoma].

OBJECTIVE: To investigate the relationship between single nucleotide polymorphism of SET8 gene and the risk of clear cell renal cell carcinoma (CCRCC). METHODS: We selected 140 CCRCC patients and 130 healthy controls in this case-control study.Genotype of single nucleotide polymorphism (rs16917496) at the miR-502 binding site in the 3'UTR of SET8 mRNA in the CCRCC patients and healthy controls was tested and the association between genotype and risk of cancer was assessed. The expression of SET8 was determined by immunohistochemistry and the relationship between expression of SET8 and genotype of rs16917496 was analyzed. RESULTS: In the control group, CC, CT and TT genotypes were found in 30, 32 and 68 persons, respectively, while in the CCRCC patients, CC, CT and TT genotypes were found in 14 , 47 and 79 cases, respectively.The frequencies of rs16917496 CT and TT genotypes in the CCRCC group were significantly higher than those in the control group (P<0.05). Compared with the CC genotype, patients with CT and TT genotypes were more susceptible to develop CCRCC (P<0.05). CT and TT genotypes of rs16917496 at the miR-502 binding site of the SET8 gene were associated with expression of SET8. CONCLUSIONS: Genotype of the SNP rs16917496 at the miR-502 binding site in the 3' untranslated region of the SET8 gene is associated with the expression of SET8 protein. Analysis of genetic polymorphisms in miRNA binding sites may help to identify the subgroups of population susceptible to CCRCC.