| Objective: Cardial vessel disease(CVD) is a major complication of patients with chronic kidney disease,and CVD is one of the leading cause of death for patients with chronic kidney disease(CKD), and vascular calcification(VC) is an high risk factor of CVD. In the past,vascular calcification was considered as a passive and degenerative process of calcium phosphate depositing in vessel wall, now a large number of studies have shown that vascular calcification is a reversible, adjustable biological process,which involved in many kinds of factors, and associated with bone metabolic abnormalities. Besides traditional factors such as age, diabetes, high blood pressure, non-traditional factors such as calcium phosphorus metabolism, oxidative stress, secondary hyperparathyroidism,vascular calcification also have some influence on patients with chronic kidney disease(CKD).Vascular calcification of End-stage Renal Disease(ESRD) patients occurs mainly in the media of vascular, which of main components is vascular smooth muscle cells(VSMC). VSMC phenotype transformation,which is VSMC transforms into a bone/cartilage phenotype,accompanied by secreting Runx2?BMPs?Cbf?-1 that regulating the process of vascular calcification recuperatively,is one of the main mechanism of vascular calcification in the media membrane of vascular. Many factors can stimulate VSMC transdifferentiation into osteogenesis cells, VSMC transformed into osteogenesis can produce collagen and collagen matrix proteins, which combined with phosphate and calcium salt causes mineralization under the effect of alkaline phosphatase(ALP). Numerous studies reported that the mechanism of high phosphorus causing vascular calcification was associated with runt-related transcription factor(Runx2). Under normal circumstances, Runx2,which used as a vascular calcification early evaluation marker,is not expressed in vascular tissue, its abnormal expression is closely related to occurrence and progress of vascular calcification, and the more calcification, the more Runx2. Our previous study have found that high phosphorus can promote Runx2 expression in aortic VSMC, thus leading to phenotype transformation. In vitro,Byon showed that endogenous knockout Runx2 expression can inhibit the TRAP positive osteoblast formation, RANKL expression and macrophage migration, thus decrease the vascular calcification induced by high-fat diet. Gangahar also confirmed MGP involved in calcification inhibition by reducing the expression of Runx2 and its activity. These datas suggested regulating Runx2 can inhibit VSMC calcification.Apoptosis is also one of the main mechanism of vascular calcification occurred in the media membrane. Many studies have shown that calcification began in membrane matrix vesicles which was released by dying vascular smooth muscle cells, so called the apoptotic bodies. Mc Nair using electron microscope to observe dialysis and non-dialysis patients with arterial calcification found that damage/death vascular smooth muscle cells released matrix vesicles that containing hydroxyapatite nanoparticles, and speculated that the matrix vesicles was the starting point of calcification, further study have found that the dialysis could accelerate the matrix vesicles released to accelerate vascular calcification in dialysis patients. Schoppet found that the arteries membrane apoptosis existed in Monckeberg 's sclerosis patients in situ ligation analysis. Sachiko observed that rat thoracic aorta tissue cultured with high phosphorus showed increased apoptosis. Kozaki found high phosphorus can accelerate apoptosis and promote vascular smooth muscle cells calcification, and this effect could be inhibited by statin in dose dependent manner. Growth arrest- specific gene 6(Gas6) is a member of the vitamin K dependent protein family, and its ligand is Axl. Studies have shown that statins may activate Gas6-Axl signaling pathways by increasing the Gas6 m RNA stability, rather than its transcriptional activity, and inhibite vascular smooth muscle cell apoptosis, and thus reduce high phosphorus induced calcification. And RNA interference or eliminate Axl extracellular structure can abolish statins inhibitory effect of high phosphorus induced vascular calcification.Calcification inhibitors MGP(matrix gla protein, MGP) can inhibit calcium phosphate deposit on the blood vessel through. Further research had found that MGP overexpression in cultured vascular smooth muscle cells with high phosphorus had reduced the expression and activity of Caspase3 and cytokine, repressed apoptosis, and alleviated vascular smooth muscle cells calcification. Hyunsoo found that ?-lipoic acid,on the one hand,can stabilize mitochondrial function, inhibit cytochrome c release to cytoplasm activating Caspase3, decrease apoptosis and reduce vascular calcification, on the other hand, can also activate the Gas6 Axl/Akt signaling pathway, enhance cell proliferation ability and reduce vascular calcification. In summary,Vascular smooth muscle cell apoptosis is one of the important mechanisms of vascular calcification, Gas6/Axl/Akt signaling pathway regulates vascular smooth muscle cell apoptosis and plays an important role in the process of calcification.Research shows that traditional and non-traditional factors affect End-Stage Renal Disease(ESRD) patients with vascular calcification through different mechanisms. Kirschbaum observed a statistically significant increase in p H in post-dialysis blood. Total and ionized calcium increased significantly only in the patients with central venous catheters but not in those with fistulas or grafts. All patients experienced a decrease in phosphate concentrations. But 2h after termination of dialysis, the calculated risk of metastatic calcification would increase 2.8-fold compared to pre-dialysis conditions. This suggests that blood PH value, or dialysate PH may be closely related to the risk of vascular calcification in patients receiving hemodialysis. In an experimental model of uremic rats, Pineda found that CKD rats had an increased p H level(7.57 ± 0.03) compared to non-CKD rats(7.36±0.1), and the CKD rats showed a high degree of vascular calcification compared to non-CKD rats. Further study found that blood anion gap has a high degree of correlation with rat aortic vascular calcification and aortic phosphate content.At present, the study about the effection of PH and its fluctuation on vascular calcification in patients with chronic kidney disease is rare. Therefore, this topic proposed to explore the mechanisms of PH and its fluctuation in the progress of vascular calcification in chronic kidney disease(CKD) and intervention Gas6 / AXL- PI3K/AKT signaling pathway wethercan serve as therapeutic targets of vascular calcification in chronic kidney disease(CKD) throurh in vivo and in vitro experimental study which study the the effects of PH and its fluctuation on vascular calcification in chronic kidney disease(CKD).Methods:1 The risk factors for coronary artery calcification inpatients with end-stage renal diseaseFifty-three ESRD patients on regular hemodialysis(3 times a week) were enrolled from 2014 to 2015 in hebei medical university hospital blood purification center in the present study and divided into a negative control group and three positive groups(mild, moderate and severe calcification groups), based on coronary artery calcification score(CACs). Clinical data of all patients at study entry were collected. Arterial blood samples were also collected at the start of the first hemodialysis(HD) session of the week to measure the levels of serum albumin, uric acid,calcium(Ca),phosphorus(Pi),magnesium,high density lipoprotein(HDL),low density lipoprotein(LDL), C-reactive protein(CRP), beta2 microglobulin(?2MG), freeparathyroid hormone(i PTH), alkaline phosphatase, fibrinogen, hemoglobin(HGB) and ferritin. In the meantime, levels of blood p H were detected after collecting pre- and post-HD blood samples, to calculate?p H(post-HDp H subtract pre-HDp H). Logistic regression analysis was performed to analyze the correlation of CACs to clinical parameters and previously-reported blood biochemical indicators, followed by analysis of the incidence of CAC and influential factors in ESRD patients.2 The effect and mechanism of PH and its volatility on chronic renal failure rat aortic vascular calcificationThe chronic renal failure rats model was built by 5/6 nephrectomy, and vascular calcification model was built by feeding calcitriol, chronic metabolic acidosis, chronic metabolic alkalosis and acid-base volatility model were built by respectively injecting NH4 Cl, Na HCO3, and the next day alternating injection of NH4 Cl, Na HCO3 into abdominal cavity.34 healthy male SD rats were randomly divided into 6 groups by random number method, namely sham group(n=6), chronic renal failure group(CKD)(n=5), calcification group(CKD+VC)(n=5), acid intervention group(CKD+VC+AI)(n=6), base intervention group(CKD+VC+BI)(n=6) and volatility group(CKD+VC+AB)(n=6). After 30 days, arterial blood were obtain from rat abdominal aorta puncture, blood p H were determined by blood gas analysis, and after arterial blood with heparin anticoagulation, plasma were separated by centrifugation, and stored in- 80?. Take over the chest aorta of rats for histologic observation and determination of calcium content.3 The effects of extracellular PH and its fluctuation on rat vascular smooth muscle cell phenotype transformation and calcification induced by high phosphorusA total of 6 healthy male SD rats aged 8-10-weeks were selected in the study. VSMCs from rat thoracic aorta were cultured in vitro, and then identified by immunocytochemistry. The VSMCs were randomly divided into 5 groups by random sampling method: PH7.4 group, PH6.8+P group, PH7.4+P group, PH7.7+P group, and PH6.8/7.7+P group. Alkaline phosphatase activity were detected after stimulating 6 days., The calcium content were evaluated, calcium nodules were detected by alizarin red staining, and the expression of Runx2 were detected by RT-PCR after stimulating 10 days.4 Effects of extracellular PH and its fluctuation on high-phosphorus-induced apoptosis of rat vascular smooth muscle cells and its mechanismA total of 6 healthy male SD rats aged 8-10-weeks were selected in the study. VSMCs from rat thoracic aorta were cultured in vitro, and then identified by immunocytochemistry. The VSMCs were randomly divided into 5 groups by random sampling method: PH7.4 group, PH6.8+P group, PH7.4+P group, PH7.7+P group, and PH6.8/7.7+P group. Expression of Bcl2 m RNA, Bax m RNA and caspase3 m RNA were measured by RT-PCR; and Bcl2/Bax was calculated..Apoptosis rate were measured by flow cytommetry.5 Effects of Gas6/AXL or PI3K/AKT signaling pathway on inhibition of rat vascular smooth muscle cell calcification in response to extracellular acid stimulationA total of 6 healthy male SD rats aged 8-10-weeks were selected in the study. VSMCs from rat thoracic aorta were cultured in vitro, and then identified by immunocytochemistry. The VSMCs were randomly divided into 4 groups by random sampling method: PH7.4 group, PH6.8+P group, PH7.4+P group, PH6.8+P+LY294002 group. RT-PCR were peformed to determinate the expression of Gas6 m RNA?Axl m RNA?caspase3 m RNA and Akt m RNA, and Western Blot were used to detect the expression of Caspase3?cleaved-Caspase3?t-Akt and p-Akt. Apoptosis rate were measured by flow cytommetryResults:1 The risk factors for coronary artery calcification inpatients with end-stage renal diseaseSeverity of CAC was positively correlated with age, HD duration, serum calcium, serum phosphorus and pre-HD p H, but negatively correlated with serum albumin and ?p H. Logistic regression analysis revealed that age, HD duration, serum phosphoruslevels and ?p H were independent risk factors for CAC in ESRD patients(P<0.05).2 The effect and mechanism of PH and its volatility on chronic renal failure rat aortic vascular calcification(1) The influence of acidic environment on chronic renal failure rat aortic calcification:Plasma biochemistry could be seen in the Table 2-1.Renal functions levels got worse in all 5/6 Nx groups(P<0.05). Nephrectomy alone did not have an effect on the acid–base balance of the rats. Compared with nonacidotic groups, p H and bicarbonate were significantly decreased in CKD+VC+AI group(P<0.05). Von Kossa-stained tissue sections of the aorta showed in Fig. 2-1, CKD+VC showed significantly calcium deposits in the sections. Rats treated with NH4 Cl did not have calcium deposits.Calcium content was higher in CKD+VC than sham and CKD(9.73±1.83vs2.44±0.56,9.73±1.83vs2.46±1.23,P<0.05). Received with NH4 Cl effectively decreased calcium content compared with CKD+VC group(9.73±1.83vs5.27±2.12,P<0.05). Immunohistochemical results showed in Fig. 2-2 to 2-3. Immunohistochemical score of Runx 2 and Caspase3 between sham and CKD group has no obvious difference( 2.95±1.10vs2.25±0.82;2.72±0.86vs1.46±1.22,P>0.05). Immunohistochemical scoreof Runx2 and Caspase3 in CKD+VC group is significantly higher than sham and CKD group(6.16±1.59vs2.25±0.82,6.16±1.59vs2.95±1.10;6.62±1.31vs1.46±1.22,6.62±1.31vs2.72±0.86, P<0.05). Compared with CKD+VC group, immunohistochemical score of Runx2 and Caspase3 in CKD+VC+AI reduced significantly(6.16±1.59vs4.11±0.92;6.62±1.31vs3.60±1.43,P<0.05).(2)The influence of alkali environment on chronic renal failure rat aortic calcification : Plasma biochemistry could be seen in the Table 1.Renal functions levels got worse in all 5/6 Nx groups(P<0.05). Nephrectomy alone did not have an effect on the acid–base balance of the rats. Compared with nonalkali groups, p H and bicarbonate were significantly increased in CKD+VC+BI group(P<0.05). Von Kossa-stained tissue sections of the aorta showed in Fig. 2-4, CKD+VC and CKD+VC+BI showed significantly calcium deposits in the sections. Calcium content was higher in CKD+VC+BI than sham, CKD and CKD+CV(13.86±3.16vs2.44±0.56,13.86±3.16vs2.46±1.23, 13.86±3.16vs9.73±1.83, P<0.05). Immunohistochemical results showed in Fig. 2-5 to 2-6. Immunohistochemical scoreof Runx2 and Caspase3 in CKD+VC+BI group is significantly higher than sham, CKD and CKD+VC group(8.96±1.36vs2.25±0.82, 8.96±1.36vs2.95±1.10, 8.96±1.36vs6.16±1.59;8.01±0.74vs1.46±1.22, 8.01±0.74vs2.72±0.86, 8.01±0.74vs6.62±1.31, P<0.05)?(3)The influence of acid-base fluctuations on chronic renal failure rat aortic calcification:Plasma biochemistry could be seen in the Table 1.Renal functions levels got worse in all 5/6 Nx groups(P<0.05). Nephrectomy alone did not have an effect on the acid–base balance of the rats. Von Kossa-stained tissue sections of the aorta showed in Fig. 2-7, except for CKD+VC+AI, CKD+VC and CKD+VC+BI showed significantly calcium deposits in the sections. Calcium content was higher in CKD+VC+AB group than CKD+CV+AI(8.59±1.84vs5.27±2.12,P<0.05), but lower than CKD+VC+BI(8.59±1.84vs13.86±3.16,P<0.05). Immunohistochemical results showed in Fig. 2-8 to 2-9.Immunohistochemical scoreof Runx2 and Caspase3 in CKD+VC+AB group is significantly higher than CKD+VC+AI group(6.74±1.22vs4.11±0.92; 6.41±1.15vs3.60±1.43, P<0.05), but lower than CKD+VC+BI(6.74±1.22vs8.96±1.36;6.41±1.15vs8.01±0.74,P<0.05)?3 The effects of extracellular PH and its fluctuation on rat vascular smooth muscle cell phenotype transformation and calcification induced by high phosphorus(1) After 10 days extracellular stimuli, calcium content among 5 groups is that PH6.8+P lower than PH7.7+P and PH6.8/7.7+P(P<0.05), and PH6.8/7.7+P lower than PH7.7+P(P<0.05) ? Alizarin red staining results consistent with the calcium content determination results.(2) After 6 days extracellular stimuli, alkaline phosphatase among 5 groups is that PH6.8+P lower than PH7.7+P and PH6.8/7.7+P(P<0.05), and PH6.8/7.7+P lower than PH7.7+P(P<0.05).(3) After 6 days extracellular stimuli, the expression of Runx2 among 5 groups is that PH6.8+P lower than PH7.7+P and PH6.8/7.7+P(P<0.05), and PH6.8/7.7+P lower than PH7.7+P(P<0.05).4 Effects of extracellular PH and its fluctuation on high-phosphorus-induced apoptosis of rat vascular smooth muscle cells and its mechanismApoptosis rate and caspase3 m RNA expression in PH6.8+P group were significantly lower than those in PH7.7+P group(P<0.05) and PH6.8/7.7+P group(P<0. 05);Bcl2/Bax levels in PH6.8+P group were significantly higher than those in PH7.7+P group(P<0.05) and PH6.8/7.7+P group(P<0.05).5 Effects of Gas6/AXL or PI3K/AKT signaling pathway on inhibition of rat vascular smooth muscle cell calcification in response to extracellular acid stimulation(1) Compared with p H7.4 group(normal environmental acidity), the calcium levels in VSMCs significantly increased in p H7.4 with ?-glycerol phosphate group(P<0.05). Even in p H6.8 with phosphorous group, the calcium content is significantly higher than that in p H7.4 group. As p H decreased, the calcium content in p H6.8 with phosphorous group was significantly decreased than that in p H7.4 with phosphorous group. The result indicated that alizarin red staining was in consistent with calcium determination results.(2)The results showed that the cells were mainly concentrated in D3, D4, B3, B4, C3, C4. The apoptosis of VSMCs in p H7.4 with phosphorous group was significantly higher than that in cells of p H7.4 group(P<0.05). The apoptosis of p H6.8 with phosphate cells was statistically significantly lower than that of p H7.4 with phosphorous group(P<0.05).(3)There was no significant difference in Gas6 expression among three groups. The AXL m RNA levels in p H7.4 with phosphorous group and p H6.8 with phosphorous group were significantly lower than that in p H7.4 group. While compared to p H7.4 with phosphorous group, the AXL m RNA expression was significantly increased in p H6.8 with phosphorous group with p H reduction(P<0.05). Moreover, we measured the AXL protein expression by western blot in VSMCs among the three groups. Similar results were showed in the AXL protein expression.(4)The calcium content in VSMCs of p H6.8 with phosphorous and PI3 K inhibitor LY294002 group was significantly increased after addition of LY294002(P<0.05), though still significantly lower the calcium content in p H7.4 with phosphorous. As can be seen, apoptosis rate had the same profile with the calcium content in VSMCs of p H 6.8 with phosphorous and PI3 K inhibitor LY294002 group(P<0.05). Casp3 and AKT expression were also measured by western blot. There was a significant increase of Casp3 expression and a decrease of AKT expression in in VSMCs of p H 6.8 with phosphorous and PI3 K inhibitor LY294002 group(P<0.05).Conclusion:1 Coronary calcification of ESRD patients affected by multiple factors, as independent risk factors, like age, age of hemodialysis, serum phosphorus, ?PH, as well as the PH of pre-HD play a decisive role for coronary calcification.2 Inhibition of aorta calcification in CKD rats was produced by acid environment, while it has contrary effect during the alkaline and PH fluctuation environment. This response was associated with the expression of Runx2 and Caspase3, which was influence vascular calcification by interfere VSCMs phenotypic change and apoptosis.3 VSMCs calcification in rats induced by hyperphosphate was suppressed in an extracellular acid environment, while it was promoted under extracellular alkaline and PH fluctuation environment. This mechanism might be regulate the osteoblast and chondrocyte phenotypic change by increase or decrease the expression of Runx2 and ALP.4 Extracellular acidic environment can inhibit high-phosphorus-induced VSMCs apoptosis, whereas extracellular alkaline5 Inhibition of VSMCs calcification induced by hyperphosphate in the extracellular acid environment may attribute to the signal pathway Gas6/Axl-PI3K/Akt. |
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