Abstract:

Objective　To investigate the effects of Klotho protein on renal injury and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome in spontaneous hypertensive rats (SHR). Methods　The experiment was conducted from January 10 to March 25, 2024. The 14-week-old SPF male spontaneously hypertensive rats with body mass of 220-260 g were selected, and the other SPF male WKY rats with the same body mass were selected. All rats were fed alone in a 12 h/12 h light-dark cycle at 25 ℃ and 60% humidity, and after 7 days of adaptive feeding, the rats were treated in groups at the same time in the morning during the following 21 days. The spontaneously hypertensive rats were divided into a model group (gavage with equal volume of normal saline), a positive control group (gavage with 0.67 mg/kg of enalapril), low and high dose Klotho groups (gavage with 50 and 100 mg/kg of Klotho), and a high dose Klotho+NLRP3 inflammasome activator group (gavage with 100 mg/kg of Klotho protein followed by 5 mg/kg of nigericin sodium), 12 rats in each group; another 12 WKY rats were selected as a control group (gavage with equal volume of normal saline). The rats in each group were given gavage once a day for 21 days, and other treatment methods were the same. The systolic and diastolic blood pressure of the tail artery, renal function (serum creatinine, blood urea nitrogen, and urinary microalbumin), pathological morphology of renal tissue, content of collagen fibers, levels of inflammatory factors [interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and IL-1β], Klotho protein, NLRP3, apoptosis-associated speck-like protein (ASC), Caspase-1, Collagen Ⅲ, and Collagen Ⅰ protein contents of each group were detected. One-way analysis of variance and LSD-t test were used for statistical analysis. Results　Compared with the control group, the model group had severe renal tissue damage, with increased tail artery systolic and diastolic blood pressure, serum creatinine, urea nitrogen, and urinary microalbumin contents, levels of inflammatory factors in renal tissue (IL-6, TNF-α, and IL-1β), Collagen volume fraction, and NLRP3, ASC, Caspase-1, Collagen Ⅲ, and Collagen Ⅰ protein expressions in renal tissue, and reduced Klotho protein expression level (0.24±0.01 vs. 0.60±0.02), with statistically significant differences (all P<0.05). Compared with the model group, the positive control group and low and high dose Klotho groups had reduced kidney tissue lesions, with reduced tail artery systolic and diastolic blood pressure, serum creatinine, urea nitrogen, and urinary microalbumin contents, levels of inflammatory factors in renal tissue (IL-6, TNF-α, and IL-1β), Collagen volume fraction, and NLRP3, ASC, Caspase-1, Collagen Ⅲ, and Collagen Ⅰ protein expressions in renal tissue, and increased Klotho protein expression level (0.53±0.03, 0.36±0.03, 0.51±0.04 vs. 0.24±0.01), with statistically significant differences (all P<0.05). Compared with the high dose Klotho group, the high-dose Klotho+NLRP3 inflammasome activator group had severe renal tissue damage, intensified renal fibrosis and inflammatory response, and decreased renal function (all P<0.05). Conclusion　Supplementing with Klotho protein can alleviate the renal tissue inflammation and fibrosis in spontaneously hypertensive rats by inhibiting the NLRP3 inflammasome pathway, thereby improving the renal injury and enhancing the renal function.

Key words:

Nucleotide-binding oligomerization domain-like receptor protein 3, Inflammasome, Klotho protein, Spontaneously hypertensive rats,  , Renal function, Renal injury

摘要：

目的　探讨Klotho蛋白对自发性高血压大鼠肾损伤及核苷酸结合结构域富含亮氨酸重复序列和含热蛋白结构域受体3（NLRP3）炎症小体的影响。方法　实验时间为2024年1月10日至3月25日。选择14周龄、体质量220～260 g的SPF级雄性自发性高血压大鼠，另取相同体质量范围的SPF级雄性WKY大鼠。所有大鼠单独饲养在温度25℃、相对湿度60%的光暗（12 h/12 h）循环中，经过7 d适应性喂养后，在随后21 d期间的上午同一时间对大鼠进行分组治疗。将自发性高血压大鼠分为模型组（给予等体积生理盐水灌胃）、阳性对照组（给予0.67 mg/kg依那普利灌胃治疗）和低、高剂量Klotho组（分别给予50、100 mg/kg Klotho灌胃治疗）、高剂量Klotho+NLRP3炎症小体激活剂组（给予100 mg/kg Klotho蛋白灌胃治疗后，再给予5 mg/kg尼日利亚菌素钠盐灌胃治疗），每组12只大鼠；另取12只WKY大鼠作为对照组（给予等体积生理盐水灌胃）。各组大鼠灌胃治疗1次/d，持续21 d，其他处理方式相同。检测各组大鼠尾动脉收缩压和舒张压、肾功能（血肌酐、尿素氮和尿微量白蛋白）、肾脏组织病理变化及纤维化程度、炎症因子［白细胞介素6（IL-6）、肿瘤坏死因子-α（TNF-α）和IL-1β］水平、Klotho蛋白、NLRP3、凋亡相关斑点样蛋白质（ASC）、半胱氨酸天冬氨酸酶-1（Caspase-1）、Collagen Ⅲ和Collagen Ⅰ蛋白含量。采用单因素方差分析、LSD-t检验进行统计分析。结果　与对照组相比，模型组大鼠肾脏组织病损程度严重，尾动脉收缩压及舒张压、血肌酐、尿素氮和尿微量白蛋白含量、IL-6、TNF-α、IL-1β、胶原容积分数、肾脏组织中NLRP3、ASC、Caspase-1、Collagen Ⅲ和Collagen Ⅰ蛋白表达量均升高，Klotho蛋白表达量降低（0.24±0.01比0.60±0.02），差异均有统计学意义（均P<0.05）；与模型组相比，阳性对照组以及低、高剂量Klotho组大鼠肾脏组织病损减轻，尾动脉收缩压及舒张压、血肌酐、尿素氮和尿微量白蛋白含量、IL-6、TNF-α、IL-1β、胶原容积分数、肾脏组织中NLRP3、ASC、Caspase-1、Collagen Ⅲ和Collagen Ⅰ蛋白表达量均降低，Klotho蛋白表达量均升高（0.53±0.03、0.36±0.03、0.51±0.04比0.24±0.01），差异均有统计学意义（均P<0.05）；与高剂量Klotho组相比，高剂量Klotho+NLRP3炎症小体激活剂组大鼠肾脏组织病损严重、肾脏纤维化和炎症反应加剧、肾功能下降（均P<0.05）。结论　补充Klotho蛋白通过抑制NLRP3炎症小体通路，可以减轻自发性高血压大鼠肾脏组织的炎症反应和纤维化，从而改善肾损伤，增强肾功能。

关键词:

核苷酸结合结构域富含亮氨酸重复序列和含热蛋白结构域受体3, 炎症小体, Klotho蛋白, 自发性高血压大鼠, 肾脏功能, 肾损伤