The Effect And Mechanism Of Acidification On VSMCs Calcification In Uremic Rats

Objective: Patients with ESRD has a vastly increased risk of cardiovascular disease.Among hemodialysis patients,nearly 50% of the mortality is a consequence of cardiovascular disease.Vascular calcification has emerged as a risk for cardiovascular morbidty and mortality.Also,the presence of vascular calcification is an independent predictor of cardiovascular events in ESRD patients.The pathogenesis of vascular calcification is a complex mechanism,as in bone formation and apoptosis play a chiefly role in the progression of vascular calcification.Methods: 1 Animals and Experimental Design 1.1 Animals Sprague-Dawley rats were purchased from Animal of the Hebei Medical University?China?.All the rats were maintain in an air condition house with a controlled 12/12-h light/dark cycle and free access to normal food and water.Since the uremic rats lose weight,food was restricted in the control rats so that aggregate weights were similar.To produce a wider range of calcification,calcitriol?600ng/kg every other day?was gavaged to enhance calcification.Renal failure was induced by?5/6?nephrectomy with a standard two-step surgical procedure.1.2 Experimental Design After the second surgery,the rats were randomly assigned?on the basis of the normal distribution of baseline body weights?into four experimental groups: Sham-operated?n=6,used as a control?,5/6Nx+vehicle+distilled water?n=5?,5/6 Nx+calcitriol 600 ng/kg every other day?n=5?,5/6 Nx+calcitriol 600 ng/kg every other day +acidosis?n=6,0.28mmol/L NH4 CL in drinking water everyday?,0.28mmol/L solution of NH4 Cl was added to the drinking water in order to induce metabolic acidosis in 5/6Nx.After 5 weeks,rats of each Sham-operated and treatment group were sacrificed.The aortas were divided into three parts.The cranial part of the aorta was studied by histology?von Kossa?and Immunohistochemistry for LTCC ?3 subunits and runx2.The last part was used for the assessment of calcification.Aortic speciments were rinsed in normal saline,minced,and decalcified in 1 ml of 0.6 mmol/L HCL for 72 h with gentle agitation at room temperature.Then,the calcium content of HCL supernatants was determined colorimetrically and by spectroph-otometry normalized by tissue weight.Creatinine and BUN concentrations were measured using by spectrophotometry.pH and bicarbonate measured a selective electrode.Expression of LTCC ?3 subunits and runx2 were studied by Immun-ohistochemistry staining.2 Rat VSMCs were obtained from the tunica media of an adult male Sprague Dawley rat thoracic aorta.After verified using morphologic analysis and alpha smooth muscle actin immunohistochemical staining.To induce calcification,the VSMCs 10 mmol/L ?-glycerophosphate,10mol/l insulin,and 50mg/ml ascorbic acid in the presence of 15% serum.Control VSMCs were cultured in identical conditions,but without the ?-glycerophosphate.The media was changed every day,which was acidified by HCL to adjust at pH 7.1?according to the pH of CKD+VC+AC group?.These changes in pH did not decrease VSMC viability.In some experiments,VSMCs were also treated with calcium channel blockers: verapamil,20mmol/L.Culture medium was changed every day.Calcification assays After incubation period?14 days?,cells were washed with phosphate buffered saline and calcification was quantified:?a?by spectrophotometry,using a calcification Kit and?b?by histology,using 1% Alizarin red stain.3 Intracellular Ca²⁺ measurement cells were loaded with 5?mol/L fluo-3AM,a fluorescent calcium indicator,for 1¹.5h in darkness at room temperature.VSMCs were depolarized by 80 mmol/l K+ solution to increase in [Ca2+]i..Intracellular calcium changes were examined by microplate reader.The ratio of emissions with excitations at a certain wavelength,which was used as a measure of [Ca²⁺]i.4 RT-PCR The target genes LTCC ?3 subunit and runx2 were determined by RT-PCR,following incubation for 4 days.The GAPDH gene was used as an endogenous control.Western Blotting Total protein was extracted from the VSMCs,and the concentrations were measured with the BCA protein assay kit following incubation for 4 days.5 Alkaline phosphatase activity Cellular alkaline phosphatase activity was measured by p-nitrophenyl substrate supplied in an alkaline phosphatase assay kit.Protein concentrations were determined with a Bio-Rad protein assay kit?Bio-Rad?,which was used for normalizing to alkaline phosphatase activity.6 Statistical analysis Data analyses were conducted using SPSS 13.0 software.All results were expressed as mean ± standard deviation.Differences among groups were determined by analysis of variance?ANOVA?,and the Student-Newman-Keuls method was determined by post-hoc testing.P<0.05 denoted a statistically significant difference.Results:1 Effect of acidosis on aortic calcification in vivo.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 nonacidotic groups,pH and bicarbonate were significantly decreased in CKD+VC+AC group?P<0.05?.Ca content was higher in CKD+VC than sham and CKD?Ca content in CKD+VC: 9.66±1.62,vs 2.33±0.42,P<0.05 vs 5/6 sham,vs 2.41±0.41,p<0.05 vs CKD?.Received with NH4 CL effectively decreased Ca content?Ca content in CKD+VC+AC: 4.83±0.73,vs 9.66±1.62,P<0.05 vs CKD+VC?.Von Kossa-stained tissue sections of the aorta showed in Figure 1,CKD+VC showed significantly calcium deposits in the sections.Rats treated with NH4 CL did not have calcium deposits.2 Effect of acidosis on VSMCs calcification in vitro.Ca content was shown in Table 2.Ca content and Alizarin red stain were obviously shown that at pH 7.1 with ?-glycerophosphate had low calcium level and slight Alizarin red stain?P<0.05?with incubated period?14 days?.Verapamil could significantly decreased calcium deposition when compared with group at pH 7.4 with the present of ?-glycerophosphate.3 Effect of acidosis on subunits of LTCC expression in vitro and intracellular calcium [Ca]i changes in VSMCsRT-PCR and western blot were performed to quantify mRNA and protein levels,which were shown in Figure 4.mRNA and protein expression of ?3 subunit were induced decrease by acidosis?P<0.05?.There was a decrease in [Ca]i in pH7.1 with ?-glycerophosphate in response to acidosis?P<0.05??Table 2?.Interestingly,there was a significant difference in expression of ?3 subunit and intracellular calcium [Ca]i between VSMCs with and without ?-glycerophosphate at pH 7.4?P<0.05?.Verapamil blocked the increase in [Ca]i as expected?Table 2?.4 Effect of acidosis on runx2 expression and ALP activity in vitroIt has been performed that vascular mineralization in vitro;therefore,we examined whether acidosis suppresses the phenotype transition of VSMCs into osteoblast-like cells.The effect of calcifying medium on runx2,a vital marker of osteoblast differentation,was markedly enhanced following ?-GP treatment when the pH was 7.4?Figure 4?,while inhibited by acidosis and in the presence of verapamil as well?Figure 4?.ALP activity had the same trend,acidosis provided a significant inhibition on ALP activity,compared with cells grown in calcification medium with pH 7.4.Addition of verapamil into the calcification medium inhibited the increase of ALP activity?Table 2?.5 Effect of acidosis on LTCC ?3 subunit and runx2 expression in vivoWe next investigated whether the observed effect of acidosis on LTCC ?3 subunit and runx2 expression on in vitro VSMCs could be reproduced in vivo.Immunohistochemistry performed to determine the changes of LTCC ?3 subunit and runx2 in vivo.5/6Nx rats were treated with or without NH4 CL and calcitriol.As shown in Figure 5 and 6,LTCC ?3 subunit and runx2 expression were upregulated in CKD+VC group.There was a gradual decrease in LTCC ?3 subunit and runx2 expression in CKD +VC+AC group.Immunoreactive score showed that LTCC ?3 subunit and runx2 IRS in CKD +VC group was higher than that in sham and CKD group?runx2 IRS: 6.20±1.64,vs 2.16±0.75,P<0.05 vs sham;vs 3.00±1.00,P<0.05 vs CKD;LTCC ?3 subunit IRS:6.60±2.51,vs 1.83±0.98,P<0.05 vs sham;vs 2.60±0.55,P<0.05 vs CKD?.Acidosis marked decreased LTCC ?3 subunit and runx2 expression?LTCC ?3 subunit,vs 3.00±1.45,P<0.05 vs CKD+VC;runx2,vs 3.83±1.33,P<0.05 vs CKD+VC?Conclusion:One of the possible mechanisms of the inhibitory effect of acidification on VSMCs calcification is that acidification prevented LTCC ?3 subunit expression and calcium influx,leading to the degradation of VSMCS phenotype transforming.