| Objective:Vascular calcification is one of the main performance of diabetic cardiovascular complications,and is also a major risk factor for cardiovascular death in the patients with diabetes,While vascular dysfunctional is closely connected with the development of diabetic nephropathy(DN),the important complication of diabetes mellitus.Diabetic patients seem to have increased prevalence of vascular calcification when compared with non-diabetic patients and vascular calcification are common and severe in DN patients.Currently the study of vascular caicification are focus on either peripheral or coronary arterial calcification,but we know little about renal artery.So in order to explore the effects of renal artery calcification on the progression of diabetic nephropathy(DN),the activation and its role of BMP2 signal pathway in renal artery of rats,we observed the relationship between renal function and renal artery calcification,and observed BMP2 and its downstream factor Smad1,Runx2 and Osterix gene and protein changes On DN rats renal artery.Methods:64 Male SD rats were randomly divided into control group(CON group,n=18),diabetic nephropathy group(DN group,n=22)and diabetic nephropathy with vascular calcification group(DN+VDN group,n=24).DN group and DN+VDN group rats received intraperitoneal injection with 35 mg/kg streptozotocin(STZ)in citrate solution after 8 weeks of a high fat diet to induce diabetes.Following this(after 3 days),blood glucose levels were detected utilizing One-Touch strip.Rats with blood glucose concentrations above 16.7 mmol/L were considered as diabetic and were used in the experiments followed.24 hours urinary protein of modeling rats after 2 weeks was detected.If it were higher than 30mg,the DN modeling succeed,otherwise it failed and would be given up.Then DN+VDN group rats were continued to treat with vitamin D3 and nicotine.The CON group included age-matched rats that were only given a vehicle buffer and a standard rat chow diet.rats were sacrificed in each group at 8 w,12 w and 16 w respectively after the success of modeling and 24-hour urine was reserved to test 24 hours urinary albumin(24-h Upro);blood was collected from heart to test serum urea nitrogen(BUN),creatinine(Scr),blood glucose,cystatin C(CysC);the pathologic change to the renal artery were detected by von Kossa staining and the calcium content were detected by calcium assay kit;double immunofluorescence staining was applied to detect the BMP2/a-SMA and Runx2/a-SMA protein expression;immunohistochemistry was applied to detect the protein expression of BMP2/Smadl/Runx2/Osterix signal pathway in the renal artery;real-time polymerase chain reaction(RT-PCR)was applied to detect the gene expression levels of BMP2 and Runx2;histopathology of kidney were assessed by hematoxylin/eosin staining.Results:1.General index①body weight:compared with CON group,model group decreased obviously(P<0.05),But the DN group and DN+VDN had no significant statistical difference at each time point.②blood glucose:The level of blood glucose in CON group were at normal level fluctuation,model group were higher than CON group(P<0.05),there were no obvious difference between DN group and DN+VDN group ③BUN,Scr:compared with CON group,the level of BUN in DN and DN+VDN group were significantly increased in 8th week(P<0.05)while the level of Scr were increased in 12th week(P<0.05),there were no difference between DN group and DN+VDN group.④CysC:The DN group rat serum Cys C level was obviously higher than that of CON group,lower than DN + VDN group.⑤ 24-h Upro:The 24-h Upro in group DN and group DN+VC were gradually increased in 8th,12th and 16th weeks,and were higher than that in group CON(P<0.05),the 24-h Upro level in DN+VDN group increased at 16th week while compared with DN group(P<0.05).2.The characteristics of the rat renal artery calcification ①Von Kossa staining:The deposition of black granules in DN and DN+VDN group were gradually increased in 8th,12th and 16th weeks,and were significantly higher than that in group CON at each time points(P<0.05).②Determination of calcium content:Calcium content in the renal artery of three group rats were gradually increased since 8w.Moreover,the calcium content in DN group was significantly higher than that in group CON and lower than DN+VDN group at each time points(P<0.05).3.Immunofluorescence test the expression of BMP2/a-SMA and Runx2/a-SMA:As compared with control group,the expression of BMP2 and Runx2 evaluated by immunofluorescence were relatively high while the expression of a-SMA was relatively low in the DN and DN+VDN rats at each time point.Furthermore,the expression intensity of BMP2 and Runx2 in DN+VDN group were the most strong and the expression of α-SMA was the most weak among groups.4.The expression of BMP2/Smad1 Runx2/Osterix signal protein:BMP2,Smadl,Runx2 and Osterix appeared in the media.The expression of BMP2,Smadl,Runx2 and Osterix in the control group was relatively low,yet relatively high in DN and DN+VDN group(P<0.05)and showed a continual increasing trend.As compared with DN group,the expression of BMP2,Smad1,Runx2 and Osterix at each time point in the DN+VDN group increased significantly(P<0.05).5.BMP2 and Runx2 mRNA Expression:The RT-PCR analysis results showed that,since 8w,BMP2 and Runx2 mRNA expressions in the DN and DN+VDN rats were significantly up-regulated compared to the control group(P<0.05)and showed an increasing tendency,DN+VDN group showed significantly higher levels of BMP2 and Runx2 mRNA when compared with DN group(P<0.05).6.Kidney HE staining showed:there was no significant change in kidney tissues of the CON group.But the volume of glomerulus increased,the visible mesangial region widened and the granular or vacuoles degeneration appeared in epithelia of renal tubules in the DN group at 8w.With the extension of time,the thickening of basement membrane is more obvious,and the hyaline degeneration appeared in renal arteriolars at 12w.At the16w of the experiment,the damage of renal tissue was aggravating,and the tuberous sclerosis appeared in glomerulus.Moreover,the histopathology damages of the kidney in group DN+VDN were more serious than group DN at the same time point.Conclusion:1.There has existed the RAC in the early stage of DN,and the activation of BMP2/Smad1/Runx2/Osterix signal pathway may play an important role in vascular calcification;2.The occurrence and severity of RAC directly participate in and promote the progression of DN;3.The BMP2/Smad1/Runx2/Osterix signal pathway may be an important regulating factor in DN with renal artery calcification. |
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