RESUMO
Objective To observe the expression of Notch1 signaling pathway in the aorta of chronic kidney disease (CKD) rats with vascular calcification and to explore the role of this signaling pathway activation in aortic calcification of CKD.Methods A total of 40 male SD rats were randomly divided into normal group (Nor group) and CKD with vascular calcification group (CKD+VC group).Rats in each group were sacrificed at 4 weeks,6 weeks and 8 weeks respectively after the success of modeling.Their 24-hour urine was reserved to test 24 hour urine protein (24 h Upro);blood sample was collected from abdominal aorta to test blood urea nitrogen (BUN),serum creatinine (Scr),Ca and P.The histopathology of renal was detected by HE staining.The aortic calcification was detected by alizarin red S staining.Immunohistochemistry (IHC) was used to test the protein expressions of alpha-smooth muscle actin (or-SMA),Runt-related transcription factor 2 (Runx2),Notchl,recombination signal-binding protein for immunoglobulin kappa J region (RBP-Jκ),Msh homeobox 2 (Msx2),Jagged1 and Notch1 intracellular domain (N1-ICD) in the aorta.Real time PCR was applied to detect the mRNA expressions of α-SMA,smooth muscle 22 alpha (SM22α),Runx2,alkaline phosphatase (ALP),Notch1,RBP-Jκ,Msx2 and Jagged1.Results Compared with those in Nor group,24 h Upro,BUN and Scr increased in the CKD+VC group at 4th,6th and 8th weeks (all P ＜ 0.05).Numerous continuous calcified nodules were detected in the vascular wall of the CKD+VC group,but none in Nor group.As compared with Nor group,the expression of α-SMA was low,while the expression of Runx2 was relatively high in the CKD+VC rats at each time point (all P ＜ 0.05).The expressions of Notchl,RBP-Jκ,Msx2,Jaggedl and NI-ICD in the Nor group were slightly appeared in the aortic wall,while in CKD+VC group these signal protein expressions increased relatively during the experimental period (all P ＜ 0.05).As compared with Nor group,the expressions of α-SMA and SM22α mRNA were low,yet the expressions of Runx2 and ALP mRNA were high in the CKD+VC rats at each time point (all P ＜ 0.05).The mRNA levels of Notch1,RBP-Jκ,Msx2 and Jagged1 in the CKD+VC group at each time point were significantly up-regulated as compared with the Nor group (all P ＜ 0.05).Conclusions There exist phenotypic changes in smooth muscle cells and activations of Notch1/RBP-Jκ/Msx2 signaling pathway in CKD rats with vascular calcification.It may be one of the important signal transduction pathways.
RESUMO
Objective To explore the effects of renal artery calcification on the renal function in type 2 diabetic ne-phropathy rats .Methods Rats were randomly divided into control group ( CON group ) , diabetic nephropathy group ( DN group) and DN with vascular calcification group ( DN+VC group) .Rats of group DN and DN +VC were fed with high sugar and fat diet and injected with streptozotocin (STZ)into abdominal cavity to induce type 2 diabetes. After diabetic models were made , rats of group DN+VC were treated by vitamin D 3 plus nicotine .The rats were sacrificed at 8 , 12 and16 week respectively and the pathologic change to the renal artery were microscoped by von Kossa staining .The calcium content were detected by calcium assay kit and double immunofluorescence staining and real-time polymerase chain reaction ( RT-qPCR) were applied to detect the protein and gene expression levels of BMP2 in the renal artery.Measure the levels of blood urea nitrogen (BUN),serum creatinine (Scr),cystatin C (Cys C) and 24 hour urinary protein (24-h UA)respectively at the 8th,12th and 16th weeks.Histopathology of kidney was assessed by hematoxylin/eosin staining .Results The deposition of black granules , the calcium content and the protein and gene expression levels of BMP 2 in DN group were significantly higher than those in group CON and lower than DN+VC group at each time points(P<0.05).The BUN, Scr, Cys C and 24-h UA in group DN and group DN+VC were gradually increased in 8th,12th and 16th weeks, and were higher than those in group CON( P<0.05 ) .Compared with the DN group , only the level of Cys C at each time point and the level of 24-h UA in 16th week in DN+VC group were significantly higher ( P<0.05 ) .The pathological damages of the kidney in group DN showed a continual worsening trend and the pathological changes of the kidney in group DN +VC were more serious than group DN .Calcium content was positively correlated with the increased serum BUN , Scr, Cys C, 24-h UA and BMP2 mRNA ( all P<0.01 ) .Conclusions The occurrence and severity of renal artery calcification may participate in and promote the progression of DN .
RESUMO
Objeetive To explore the effects of renal artery calcification on the progression of diabetic nephropathy (DN),the activation and its role of bone morphogenetic protein 2(BMP2) signal pathway in renal artery of rats.Methods Sixty male SD rats were randomly divided into control group(CON group),DN group and DN with vascular calcification group (DN+VDN group).Rats of group DN and DN + VDN were fed with high sugar and fat diet and injected with streptozocin (STZ) into abdominal cavity to induce diabetes.After diabetic models were successfully made,rats of group DN+ VDN were treated by vitamin D3 plus nicotine.The rats were sacrificed at 8th,12th and 16th week respectively and the levels of renal function,blood glucose and 24 h urinary protein (24-h Upro) were measured.The pathologic changes to the renal artery were observed by yon-Kossa staining and the calcium content was detected by calcium assay kit.The pathologic changes to the kidney were observed by HE.Immunohistochemistry was applied to detect the protein expression of BMP2/Smad1/Runx2/ Osterix signal pathway in the renal artery and real-time PCR were applied to detect the mRNA expression levels of BMP2 and Runx2.Results The calcium content and the deposition of black granules in DN group were significantly higher than those in group CON and lower than DN + VDN group at each time point (P ＜ 0.05).The renal function indices in group DN and group DN+VDN were gradually increased in 8th,12th and 16th weeks,and were higher than those in group CON (P ＜ 0.05).Compared with that in DN group,although the level of BUN,Scr,Cys C and 24-h Upro in DN+VDN group rats were higher at different time point,the level of Cys C at each time point and the level of 24-h Upro in the 16th week showed significant differences (P ＜ 0.05).The pathological damages of the kidney in group DN and DN+VDN showed a continual worsening trend and the pathological changes of the kidney in group DN+VDN were more serious than those in group DN.Furthermore,the levels of BMP2/Smad1/Runx2/Osterix signal protein and BMP2,Runx2 mRNA in DN rats were higher than those in CON group,lower than DN+VDN group at each time point (P ＜ 0.05).Correlation analysis demonstrated that calcium content was positively correlated with serum BUN,Scr,Cys C,24-h Upro and the expression of BMP2,Runx2 mRNA (r=0.835,0.705,0.829,0.897,0.641,0.683,P ＜ 0.01,respectively).Conclusion Renal artery calcification may participate in and promote the progression of DN,and the BMP2 signal pathway may be an important regulating factor in DN with renal artery calcification.