钙化性纳米微粒致肾结石大鼠尿液中性粒细胞明胶酶相关脂质运载蛋白、骨桥蛋白及单核细胞趋化蛋白-1的变化及其意义

Changes in urine neutrophil gelatinase-associated lipocalin,osteopontin and monocyte chemoattractant protein-1 in rats with renal calculi induced by calcified nanoparticles and their significance

目的在建立钙化性纳米微粒(CNPs)致大鼠肾结石模型的过程中,检测大鼠不同时间尿液中的中性粒细胞明胶酶相关脂质运载蛋白(NGAL)、骨桥蛋白(OPN)、单核细胞趋化蛋白-1(MCP-1)的含量,初步分析CNPs致肾结石的形成机制。方法利用肾结石患者术中所得结石培养出CNPs,制成CNPs混悬液。20只SD雄性大鼠随机分为CNPs诱石组(A组)和空白对照组(B组)。通过鼠尾静脉注射CNPs感染大鼠,分别于注射后4 h、12 h、24 h、1周、2周及8周用代谢笼收集大鼠尿液,酶联免疫分析法(ELISA)检测大鼠各时间尿液中NGAL、OPN、MCP-1的含量。于注射CNPs后1、2和8周分批次处死大鼠,肾脏组织HE染色后于光镜下观察其结晶的形成及病理改变,对照分析结晶的形成与大鼠尿液中NGAL、OPN、MCP-1含量变化的关系。结果在注射CNPs后4 h,与B组比较,A组的NGAL水平显著增高(P<0.05),2周时A组的OPN、MCP-1水平开始大幅度升高(P<0.05),8周时A组的NGAL、OPN、MCP-1的含量均明显升高(P<0.05)。1周时,A组肾脏组织可见少量肾小管上皮细胞颗粒样变性,肾小管内未见钙盐晶体沉积;2周时,A组肾小管内可见少量钙盐晶体沉积,伴有少数肾小管上皮细胞空泡样变性和颗粒样变性;2周后肾小管内钙盐晶体的沉积量与NGAL、OPN、MCP-1的含量呈正相关;8周时,A组肾小管内可见大量钙盐晶体沉积,伴有广泛肾小管上皮细胞空泡样变性,肾小管上皮细胞的病理改变程度与NGAL的含量呈正相关;B组肾脏组织未见钙盐沉积和病理改变。结论大鼠感染CNPs 4 h后即肾小管上皮细胞开始受到损伤,并且损伤程度与NGAL水平呈正相关。2周后肾小管内开始出现钙盐晶体的黏附和聚集,单核-巨噬细胞和OPN参与其黏附和聚集过程,并且钙盐晶体的沉积量随损伤程度加重也相应增加。

[Objective] To detect the content of neutrophil gelatinase-associated lipocalin （NGAL）, osteopontin （OPN） and monoeyte chemoattractant protein-1 （MCP-1） in rat urine in different periods during the process of establishment of renal calculus rat model induced by calcified nanopartieles （CNPs） so as to analyze the mechanism of kidney stone formation induced by CNPs. [Methods] Calcified nanoparticles were extracted and cultivated from clinically diagnosed patients with nephrolithiasis. Twenty adult male SD rats were ran- domized into 2 groups （10 in each group）： group A was given an intravenous injection of calcified nanoparti- cles while group B was used as normal control. Urine of rats was collected by using metabolic cages in dif- ferent periods after injection of CNPs. The content of NGAL, OPN and MCP-1 in rat urine was detected using ELISA. Rats were sacrificed in different periods. The kidneys were examined for pathology. [Results] In the group A, NGAL began to increase 4 hours after injection of CNPs （P 〈 0.05）; OPN and MCP-1 began to largely increase 2 weeks after injection of CNPs （P〈 0.05）; NGAL, OPN and MCP-1 rose more significantly 8 weeks after injection of CNPs （P〈 0.05） compared to those in the group B. At the 1st week, histopathological studies of the renal tissue of the group A showed granular degeneration of a small amount of renal tubular epithelial cells without renal tubular crystallization. At the 2nd week, histopathological studies revealed small quantities of calcium crystals in the renal tubules accompanied with vacuolar degeneration and granular degeneration of a few renal tubular epithelial cells. After 2 weeks, the deposition of calcium crystals in the re- nal tubules was positively related to the levels of NGAL, OPN and MCP-1. At the 8th week, histopathological changes included a large number of calcium crystals in the renal tubules and extensive vacuolar degeneration of renal tubular epithelial cells which were positively correlated to the content of NGAL. No calcium crystal deposition or pathological changes could be seen in the renal tissue of group B. [Conclusions] Calcified nanoparticles begin to injure renal tubular epithelial cells 4 hours after injection of CNPs, and NGAL level is positively correlated with the degree of injury. The adhesion and aggregation of calcium crystals begin to ap- pear in the renal tubules 2 weeks after injection of CNPs, and the crystals increase with the increased severity of renal tubular injury. Mononuclear macrophage and OPN are involved in the adhesion and aggregation pro- cess.