核心结合因子和Ⅱ型胶原在高磷诱导的残肾大鼠血管钙化中的表达

Expression of core-binding factor and collagen type 2 in hyperphosphatemia-induced vascular calcification in rats with remnant kidneys

目的观察核心结合因子(Cbfα1)和Ⅱ型胶原在高磷诱导的残肾大鼠血管钙化中的表达,探讨慢性肾脏病5期血管钙化发生的可能机制。方法 40只SD雄性大鼠随即分为4组:残肾+高磷组(n=10),残肾+正常磷组(n=10),假手术+正常磷组(n=10),假手术+高磷组(n=10)。分别行5/6肾切除或假手术,术后开始给予高磷饮食或正常磷饮食16周。收集尿量检测24h尿蛋白,取血检测血清磷、肌酐、尿素氮,取胸主动组织,von kossa染色检测血管钙化,RT-PCR检测Cbfα1和Ⅱ型胶原mRNA表达,免疫组化或Western-Blot检测Cbfα1和Ⅱ型胶原蛋白表达。结果与其他3组比较,残肾+高磷组大鼠尿蛋白、血磷、尿素氮和肌酐明显增高(P<0.05)。von kossa染色显示,残肾+高磷组大鼠主动脉呈现明显钙化,而残肾+正常磷组仅个别出现钙化,假手术+高磷组和假手术+正常磷组未见钙化。免疫组化显示,与其他3组比较,残肾+高磷组大鼠主动脉Cbfα1、Ⅱ型胶原的表达明显增多(P<0.05)。相关性分析表明,血磷水平与Cbfα1、Ⅱ型胶原阳性积分呈正相关(r=0.525,0.640,P<0.05)。RT-PCR和Western-Blot显示,与其他3组比较,残肾+高磷组主动脉Cbfα1mRNA和蛋白的表达明显上调(P<0.05)。残肾+高磷组、残肾+正常磷组与另外2组相比,动脉Ⅱ型胶原mRNA表达明显上调(P<0.05)。结论高血磷是残肾大鼠血管钙化的重要影响因素,血磷水平与Cbfα1和Ⅱ型胶原的表达呈正相关。Cbfα1、Ⅱ型胶原的表达增多可能是高磷诱导的残肾大鼠血管钙化的重要机制之一。

Objective To observe the expression of core-binding factor （Cbfa） and collagen type 2（ColⅡ） in hyperphosphatemia-induced vascular calcification in rats with remnant kidneys, and to explore the possible mechanisms of vascular calcification of chronic renal disease （CKD） stage 5. Methods Forty sprague dawley rats were divided into 4 groups as follows： remnant kidney models receiving high phosphorous （HP） diet （n= 10）; remnant kidney models receiving normal phosphorous （NP） diet （n= 10）; sham operation models receiving NP diet （n= 10）; sham operation models receiving HP diet （n= 10）. After 5/6 nephrectomy or sham operation, the rats were fed with HP diet Ediet formula： phosphate （P} 1.2%, calcium （Ca） 1.6%] or NP diet [diet formula： P （0.9%）, Ca （1.2%] for 16 weeks respectively. At the 16th week, urine samples were collected for 24 h urine protein, bloods were collected for serum phosphorus, serum creatinine （Scr）, and urea nitrogen （UN）, and thoracic aorta was removed. Calcification was confirmed by von kossa staining. Cbfa and Col Ⅱ mRNA expressions were determined by RT-PCR, and Cbfal and Col II protein expressions were determined By immunohistochemistry or Western blot. Results After16 weeks, urine protein, serum phosphorus, urea nitrogen, and creatinine levels were significantly higher in remnant kidney models receiving HP diet than those of the other three groups （P〈0.05）. Von kossa staining showed that significant vascular calcification was found in remnant kidney models receiving HP diet while the remnant kidney ones receiving NP diet and sham operation models receiving HP diet occasionally had vascular calcification, and sham operation models receiving NP diet had no vascular calcification. The expression of Cbfa and Col increased significantly in remnant kidney models receiving HP diet, compared with the other three groups （P〈0.05）. Serum phosphorus was positively correlated with Cbfal and Col Ⅱ staining scores （r=0. 525, 0. 640, P〈 0.05, respectively）. RT-PCR and Western-Blot showed the expressions of Cbfal mRNA increased significantly in remnant kidney modeiss receiving HP diet compared with the other three groups （P〈0.05）, whiie the expression of Colli mRNA increased significantly in remnant kidney models receiving HP and NP diet compared with the other two groups （P〈0.05}. Conclusion Hyperphosphatemia is an important influencing factor of vascular calcification in rats with remnant kidneys, and serum phosphorus level is positively correlated with Cbfi, and Col Ⅱexpressions. The increase of Cbfal and Col Ⅱ expressions may be one of the important mechanisms of hyperphosphatemia-indeced vascular calcification in rats with remnant kidneys.