The Study On Effects And Mechanisms Of The Vascular Calcification Due To Chronic Inflammation In End Stage Renal Diseases Rats

Objective: To study on effects and mechanisms of the vascular calcification due to chronic inflammation in end stage renal diseases(ESRD) rats and provide a practically theoretical basis for hunting the efficacious methods to prevent and cure it. Methods: (1) To set up animal model of chronic renal failure: Thirty SD males rats had been randomly divided into the control group (Ctr, n=6), chronic renal failure group (CRF, n=12), CRF plus inflammation group (CRF+I, n=12). 72 hours later, the rats in Ctr group were dealed with normal saline (10ml/Kg) intragastrically daily. The rats in CRF group and CRF+I group were dealed with adenine (300mg/Kg) intragastrically daily. At the same time, the CRF+I group rats were also dealed with 10% casein subcutaneous injection (1.2g/Kg) every other day. General mental condition, diet, mortality and other conditions were observed during the experiment. (2) The treatment of tissues samples and the detection of indicators: Six weeks later, the rats were anaesthetized by 3% napental (30mg/Kg) lumbar injection, and, to sample blood from abdominal artery and the artery sample from thoracic aorta, then, the concentration of Calcium, Phosphorous, Creatinine and Urea nitrogen in Serum were biochemical analysised and the level of parathyroid hormone (PTH) in serum was assayed in Elisa. The 5mm of thoracic aorta was fixed with 4% paraformaldehyde for Von Kossa Staining and the detection of protein expression ofα-SMA in Immunohistochemistry(IHC), and the other parts of aorta were stored in -70℃freezer for RT-PCR detections(IL-6,BMP-2,Cbfα-1,Wnt3a & ALP mRNA). Results: (1) Biochemical Indicators in Serum: Six weeks later, chronic renal failure was induced in the CRF and (CRF+I) group rats. The levels of serum creatinine (μmol/L) and urea nitrogen (mmol/L) in the rats of CRF (270.93±29, 27.9±4.1) and (CRF+I) group (270.63±24.54, 33.65±8.26) were significantly higher than those in the Ctr group rats (66.85±1.5, 6.62±0.28 ) (P<0.05), however, there was no significant difference between which in CRF and (CRF+I) group rats (P>0.05). Compared with Ctr group (2.57±0.14) the concentration of blood calcium (mmol/L) in the rats, the lowest was CRF group (2.23±0.09) , and the(CRF+I)group (2.37±0.1) was lower. There were significant differences among three groups (P<0.05). The concentration of serum phosphrous (mmol/L) in the rats of CRF (5.23±0.27) and (CRF+I) group (5.37±0.42) were significantly higher than the Ctr group (2.1±0.2) (P<0.05), but, there was no significant difference between CRF and (CRF+I) group (P>0.05). (2) The concentration of PTH: The concentration of serum PTH in the rats (ng/L) of CRF (42.70±3.29) and (CRF+I) group (43.98±2.49) were remarkably higher than in Ctr group (29.60±2.85) (P<0.05). There was no significant difference between CRF and (CRF+I) group (P>0.05). (3) Morphological Detection:①Von Kossa Staining: The result of Von Kossa staining showed there was no significant calcification in the aorta of Ctr and CRF group rats, however, the distinct positive calcification staining was observed in tunica media in the aorta of the (CRF+I) group rats.②The protein expression ofα-SMA in IHC: the relative protein expression quantity ofα-SMA in the aorta of Ctr group rats (0.16±0.01) was the most, and that in CRF group rats (0.13±0.01) was more, and which in the (CRF+I) group rats (0.08±0.04) was the least. There were significant differences among each group (P<0.05). (4) The relative mRNA expression of IL-6, BMP-2, Wnt3a, Cbfα-1, and ALP: The relative expression of IL-6 mRNA in the aorta of (CRF+I) group rats (0.85±0.35) was higher than that in Ctr (0.51±0.18) and CRF group (0.58±0.13) (P<0.05), while there wasn't significant difference between that in the Ctr and CRF group (P>0.05). The relative expression of BMP-2, Wnt3a, and Cbfα-1 mRNA in the aorta of CRF (1.01±0.28, 0.81±0.23 & 1.38±0.56) and (CRF+I) group rats (1.30±0.43, 0.94±0.40 & 1.47±0.55) were significantly higher than those in Ctr group (0.54±0.17, 0.43±0.09 & 0.72±0.25) (P<0.05), while there were not significant differences between those in CRF and (CRF+I) group (P>0.05). The relative expression of ALP mRNA in the aorta of (CRF+I) group rats (0.62±0.22) was remarkably higher than that in Ctr (0.08±0.16) and CRF group (0.13±0.25) (P<0.05),while there wasn't significant difference between that in Ctr and CRF group (P>0.05). Conclusions: (1) The hyperparathyroidism and mineral imbalance result from chronic renal failure would promote the osteoblast/chondrocyte-like cells differentiation of vascular smooth muscle cells and start the vascular calcification. (2) Chronic inflammation could accelerate the process of vascular calcification in ESRD. (3) The possible mechanisms of vascular calcification in ESRD participated by chronic inflammation were: in ESRD, the expression of calcification promoters ( IL-6, BMP-2, Wnt3a, Cbfα-1, ALP and so on ) were upregulated by the chronic inflammation which would promote the transdifferentiation of vascular smooth muscle cells and mineralization. In this conditions, calcification started.