Effects Of Acidic And Alkaline Environment On Vascular Calcification In Rats With Chronic Renal Failure

Objective: Cardiovascular disease(CVD) is the major cause of death in patients with chronic kidney disease(CKD). Vascular calcification is an important risk factor for CVD. Vascular calcification in CKD patients is mainly manifested as medial calcification of blood vessels, where the key link is presumably that VSMCs osteogenesis/chondrogenesis-like phenotypic transformation. In the previous studies, it was found that the acidic and alkaline environment could regulate the function of osteoblasts and osteoclasts, which could affect the formation of bone. The vascular calcification in CKD patients is similar to that of bone development, and we hypothesized that acid and alkaline environments may affect vascular calcification by affecting VSMCs phenotype transformation. In order to explore the effects of the acidic and alkaline conditions on vascular calcification, the vascular calcification in chronic renal failure rat and in vitro culture VSMCs were induced by high phosphorus as a model, to observe the effect of acidic and alkaline environment on vascular calcification and the expression of Runt related transcription factor 2(Runx2).Methods:1 Animal experimentIn vivo experiment: Clean grade, healthy, male, 5-week-old SD rats, adaptive feeding for 1 week in a barrier environment.In vitro experiment: Primary culture to get vascular smooth muscle cells(VSMCs).2 Experimental groupingIn vivo experiment: 33 SD rats were randomly divided into 5 groups: control group(6 rats), chronic renal failure group(6 rats), chronic renal failure with vascular calcification group(7 rats), acid intervention group(7 rats) and alkaline intervention group(7 rats). Renal failure was induced by(5/6) nephrectomy. To produce a wider range of calcification, calcitriol was gavaged to enhance calcification. Establishment of chronic metabolic acidosis model in rats by feeding and drinking NH4 CL, and constructing a model of chronic metabolic acidosis in rats by intraperitoneal injection of Na HCO3.In vitro experiment: VSMCs were randomly divided into 4 groups: normal control group, p H7.4+ high phosphorus group, p H7.1+ high phosphorus group and p H7.7+ high phosphorus group. Normal control group used low glucose DMEM medium containing 10% fetal bovine serum,and then adjusted the p H value to 7.4, as normal culture medium. PH7.4+ high phosphorus group was cultured with high phosphorus medium, which was added 10 mmol/L of β glycerol phosphate in normal medium, and the p H value was adjusted to 7.4. The p H value of p H7.1+ high phosphorus group was adjusted to 7.1 on the basis of high phosphorus medium. The p H value of p H7.7+ high phosphorus group was adjusted to 7.7 on the basis of high phosphorus medium.3 Rat aortic blood biomarkers detection: extraction of rat aortic blood serum creatinine, urea nitrogen, p H value and the concentration of HCO3-,to tell whether the model of chronic renal failure, acidosis model, alkalosis is established successfully or not.4 Calcification assay: the calcification of rat thoracic aorta and VSMCs were detected by using von Kossa stain and Alizarin Red stain of calcification and o-cresolphthalein complexone method to determine the calcium content.5 Alkaline phosphatase(ALP) activity:the ALP activity of VSMCs cells was determined by enzyme linked immunosorbent assay(ELISA).6 The expression of Runx2: the Runx2 expression of thoracic aorta in rats was detected by chemical methods with the immunohistochemical. The Runx2 expression of VSMCs was detected by using RT-PCR and Western-blot method.7 Statistical methods: Statistical analysis of data using SPSS 17.0 software for analysis.In line with the normal distribution of the measurement data were described by mean±standard deviation ( (?)±s).Differences among multi-groups by one-way analysis of variance (ANOVA) and means of two subgroups comparison in multi-groups by Student-Newman-Keuls(SNK). With P<0.05 for the difference was statistically significant.Results:1 Establishment of rat model of chronic renal failure: Compared with control group, serum creatinine and urea nitrogen levels were significantly increased in chronic renal failure group, chronic renal failure with vascular calcification group, acid intervention group and alkaline intervention group(P<0.05). Compared with chronic renal failure with vascular calcification group, p H and bicarbonate of arterial blood were significantly decreased in acid intervention group(P<0.05) and significantly increased in alkaline intervention group(P<0.05).2 Effects of acidic and alkaline environment on vascular calcification of thoracic aorta in rats with chronic renal failure: Kossa von staining showed that there was a large amount of brown and black calcium deposits in the thoracic aorta of rats with chronic renal failure. But in the control group, chronic renal failure group and acid intervention group were not found the brown black calcium salt deposits in the thoracic aorta of rats with chronic renal failure. Compared with chronic renal failure with vascular calcification group, the brown black calcium salt deposits were significantly increased in alkaline intervention group.Results the calcium content of o-cresolphthalein complexone method display, compared with control group, calcium content were significantly decreased in acid intervention group(P<0.05) and significantly increased in alkaline intervention group(P<0.05).3 Effect of acidic and alkaline environment of thoracic aorta in Runx2 rats on expression of chronic renal failure: immunohistochemical results showed that compared with control group and chronic renal failure group, the Runx2 immunohistochemistry score( 6.31 ± 1.32vs2.08 ± 0.83, 6.31 ±1.32vs3.27±1.08,9.15±1.38vs2.08±0.83,9.15±1.38vs3.27±1.08) was significantly increased in chronic renal failure with vascular calcification group and alkaline intervention group(P<0.05). Compared with chronic renal failure with vascular calcification group, the Runx2 immunohistochemistry score was significantly decreased in acid intervention group(3.83±1.3vs6.31±1.32,P<0.05)and significantly increased in alkaline intervention group(9.15±1.38vs6.31±1.32,P<0.05).4 Effects of acidic and alkaline environment on high phosphorus induced rat VSMCs calcification: alizarin red staining showed that compared with the normal control group, calcification dyeing of the VSMCs were significantly increased in p H7.4+ high phosphorus group and p H7.7+ high phosphorus group. Compared with p H7.4+ high phosphorus group, calcification dyeing of the VSMCs were significantly decreased in p H7.1+ high phosphorus group and significantly increased in p H7.7+ high phosphorus group.Results the calcium content of o-cresolphthalein complexone method display, compared with the normal control group, the calcium content of the VSMCs were significantly increased in p H7.4+ high phosphorus group and p H7.7+ high phosphorus group(P<0.05). Compared with p H7.4+ high phosphorus group, the calcium content of the VSMCs were significantly decreased in p H7.1+ high phosphorus group and significantly increased in p H7.7+ high phosphorus group(P<0.05).5 Effects of acidic and alkaline environment on high phosphorus induced rat VSMCs ALP activity: compared with the normal control group, the ALP activity of the VSMCs were significantly increased in p H7.4+ high phosphorus group and p H7.7+ high phosphorus group(P<0.05). Compared with p H7.4+ high phosphorus group, ALP activity of the VSMCs were significantly decreased in p H7.1+ high phosphorus group and significantly increased in p H7.7+ high phosphorus group(P<0.05).6. Effects of acidic and alkaline environment on high phosphorus induced rat VSMCs Runxs expression:The expression of Runx2 m RNA detected by RT-PCR showed that compared with the normal control group, the expression of Runx2 m RNA of the VSMCs were significantly increased in p H7.4+ high phosphorus group and p H7.7+ high phosphorus group(P<0.05). Compared with p H7.4+ high phosphorus group, the expression of Runx2 m RNA of the VSMCs were significantly decreased in p H7.1+ high phosphorus group and significantly increased in p H7.7+ high phosphorus group(P<0.05).The expression of Runx2 protein detected by Western-blot showed that compared with the normal control group, the expression of Runx2 protein expression of the VSMCs were significantly increased in p H7.4+ high phosphorus group and p H7.7+ high phosphorus group(P<0.05). Compared with p H7.4+ high phosphorus group, the expression of Runx2 protein expression of the VSMCs were significantly decreased in p H7.1+ high phosphorus group and significantly increased in p H7.7+ high phosphorus group(P<0.05).Conclusion:The acidic environment can inhibit the occurrence of vascular calcification in chronic renal failure rats, and the alkaline environment can promote the occurrence of vascular calcification in chronic renal failure rats. The mechanism may be achieved by regulating the expression of Runx2, which may affect the transformation of vascular smooth muscle cells into osteoblast like phenotype.