The Role And Mechanism Of Hyperoxalemia In Vascular Calcification Of Uremia

BackgroundCardiovascular disease is the most common cause of morbidity and mortality in patients with chronic kidney disease and end stage renal disease.Vascular calcification is a powerful factor which can predict cardiovascular and all-cause mortality in CKD.It’s well known that vascular calcification of CKD is mainly caused by hyperphosphatemia which is followed by decreased renal function,subsequently causes calcium phosphate precipitation.However,using calcium-free phosphorus binder does not completely improve vascular calcification in ESRD patients.This suggests that some other factors in ESRD may be related to the occurrence of vascular calcification.Oxalate is one of the uremic toxins retained in ESRD patients.At present,studies in vivo and in vitro have proved that hyperoxalemia can affect cardiovascular function.Clinical studies have found calcium oxalate crystals in coronary atherosclerotic plaques of blood vessels in autopsy cases,indicating that calcium oxalate can be deposited in the vascular wall.What’s more,oxidative stress plays an important role in the pathophysiology of vascular calcification.As we all known,high oxalic acid causes oxidative stress in renal tubular epithelial cells and leads to secondary cell damage.We supposed that hyperoxalemia caused by renal failure is related to the oxidative stress state of CKD and thus affects vascular calcification.ObjectiveThe main purpose of this study was to confirm the role of hyperoxalemia in uremic vascular calcification and its correlation with oxidative stress of CKD.Furthermore,to observe whether OxF can improve oxidative stress and vascular calcification in uremia.MethodsWe collected 20 patients in the stage of CKD3-5 and matched 20 patients with normal renal function according to sex and age to measure their plasma oxalate and advanced oxidation protein product.Using ion chromatography to detect the content of oxalate and phosphate in blood vessels.We want to investigate the effects of different concentrations of oxalate on vascular endothelial cells and vascular smooth muscle cells in vitro.The supernatant of endothelial cells and smooth muscle cells stimulated by different concentrations of oxalate was collected and the expression of SOD,MDA,TNF-,IL-6,CRP and AOPP was detected by ELISA.The expression of osteogenic transcription factors such as RUNX2 and ALP was detected by RT-PCR and WB to determine whether smooth muscle cells tended to transform into osteoblast-like cells stimulated by oxalic acid and the specific mechanism.In order to investigate the role of oxalate in uremic vascular calcification,left nephrectomy and right renal electrocoagulation were performed in apoE^-/-mice to establish uremic model.The rats were divided into two groups:control group and uremic model group.The animals were killed after 12 weeks,and the blood samples of each group were collected.Blood urea,oxalate,calcium,phosphorus,low density lipoprotein,high density lipoprotein,total cholesterol,and triglyceride were detected by automatic biochemical analyzer.We detected blood malondialdehyde,superoxide dismutase,AOPP,TNF-а,and IL-6 by ELISA.Urine samples were collected every four weeks to detect 24-hour urine oxalate levels in the two groups.Besides,the isolated aorta was stained with Von Kossa to observe calcified plaques,and the area of calcified plaques in the two groups was compared by semi-quantitative method.What’s more,the contents of oxalate and phosphate in blood vessels were quantitatively detected by ion chromatography.Subsequently,oxalobacter formigenes were used to reduce the blood oxalic acid level of the model mice.After 12 weeks of intervention,the animals were sacrificed to observe the variety of blood urinary acid,serum oxidative stress index,vascular calcification and vascular oxalate content.ResultsIt was found that the serum oxalate concentration in patients with CKD3 stage was about 3 times higher than that in the control group,and it was about 5 times higher in patients with CKD4-5 stage than that in the control group.Serum oxalate concentration was positively correlated with creatinine and AOPP,and the oxalate content was 4.89±2.12ug/g in the collected renal artery.Besides,there was a certain correlation between serum oxalate concentration and vascular oxalate quantity and the coefficients r was 0.78,but P>0.05,which may be attributed to the small number of blood vessels.After stimulation with high concentration of oxalate（200μM and 500μM）,MDA,TNF-,IL-6,CRP and AOPP in the supernatant of endothelial cells increased significantly,while SOD in the supernatant decreased to a certain extent.Western-blot results showed that high concentration of oxalate could activate Jak2/Stat3 signaling pathway,which indicated that high oxalate could promote the oxidative stress state in endothelial cells and the secretion of inflammatory factors by activating the Jak2/Stat3 signaling pathway.Under the stimulation of high oxalate,the expression of osteogenic transcription factors such as RUNX2 and ALP have improved at RNA level or protein level in smooth muscle cells through Erk1/2 signaling pathway.In vivo,the levels of blood urea,oxalate,serum phosphorus,low density lipoprotein and total cholesterol in uremic group were significantly higher than those in control group.And the oxidative stress indexes AOPP and MDA in serum were significantly higher than those in sham operation group,SOD was significantly lower than those in control group.Besides,it was found that the calcified plaques in uremic group were significantly higher than those in the control group.What’s more,serum calcium,phosphorus,calcium and phosphorus product and serum oxalate concentration were correlated with calcified plaque area,and the coefficients r was 0.67,0.56,0.50,0.53,respectively,P<0.05.The quantitative results showed that oxalate was detected in both control group and uremic group,and the content of oxalate in uremic group was significantly higher than that in control group（The uric acid group had an oxalate content of 24.96±7.59ug/g,and the ratio of oxalate to phosphate was 1:282.The control group had an oxalate content of 2.64±0.54ug/g and the ratio of oxalate to phosphate was 1:415）.And there was a certain correlation between serum oxalate level and the quantity of phosphate and oxalate in blood vessels.The correlation coefficients r was 0.77,0.59,respectively,P<0.05.In addition,there was also a certain correlation between the concentration of oxalic acid and MDA,AOPP,IL-6and SOD.The correlation coefficients r was 0.70,0.52,0.53,-0.53,respectively,P<0.05.After 12 weeks of intervention,the expression of OxF in feces was detected by real-time PCR and the concentration was maintained at 10⁶cfu/g.The plasma oxalate,MDA,AOPP,TNF-in serum decreased to a certain extent,and SOD increased to a certain extent after the intervention of OxF.The semi-quantitative analysis of Von Kossa staining showed that the vascular calcification area of the CKD+OxF intervention group was lower than that of the CKD group,and the vascular oxalate content of the CKD+OxF intervention group was significantly lower than that of the CKD group,P<0.05.ConclusionsThe results showed that the level of serum oxalate increased with the decrease of renal function in patients with renal failure.In order to confirm the correlation between plasma oxalate and vascular oxalate content,it is necessary to further expand the number of blood vessel samples.Besides,calcium oxalate deposition could be detected on the vascular wall of uremic patients,and the concentration of oxalate in blood was positively correlated with the content of calcium oxalate.In vitro experiments,we also further confirmed that oxalate on vascular endothelial cells can promote oxidative stress,and play part of the role in promoting the transformation of vascular smooth muscle cells.In vivo,oxalic acid levels and vascular oxalate levels in the CKD group were significantly higher than those in the control group,and there was a correlation between the concentration of oxalic acid and oxidative stress products and the quantitative determination of oxalate.The intervention of OxF decreased vascular wall oxalate content significantly and improved the oxidative stress state of CKD,which also has a very significant clinical worth.