目的 探究不同造模周期对单侧输尿管结扎（UUO）诱导大鼠肾纤维化模型表型的影响及可能的代谢物质基础。方法 54只SD雄性大鼠随机分为9组，除对照组外，其余各组均采用UUO的方法诱导大鼠肾脏纤维化模型，各模型组分别于7、14、21、28、35、42、49、56 d取材。检测大鼠24 h尿量，尿蛋白及血清肌酐、尿素氮、白蛋白、总蛋白、三酰甘油、总胆固醇水平，测定肾比重（术侧与对侧肾脏质量比）及肾脏、心脏、脾脏、膀胱组织脏器系数，苏木素-伊红（HE）染色和马松（Masson）染色检测术侧肾脏病理变化。采用液相色谱串联质谱技术（LC-MS/MS）对血清代谢物进行非靶向检测分析，结合模式识别技术筛选血清差异代谢物，初步揭示UUO模型的代谢物质基础。为进一步评价差异代谢物用于肾纤维化疾病诊断和疗效监测的可行性，采用马来酸依那普利治疗UUO大鼠28 d（术后第2天开始给药），检测血清中差异代谢物相对含量。结果 UUO术后1周，大鼠24 h尿量、尿蛋白含量、肾比重、血肌酐和尿素氮等生化指标含量显著上升，病理检测可见肾小球萎缩、早期硬化，皮质区可见少量炎症细胞浸润，血清代谢指纹谱与对照组相比明显偏移，血清中内源性代谢物硫酸吲哚酚、2-氨基马尿酸、黄嘌呤核苷的含量急剧上升。术后2周，机体代偿功能发挥作用，心脏、脾脏及右肾脏器指数明显上升，尿蛋白、血肌酐、白蛋白、三酰甘油及差异代谢物含量较第1周有所下降，血清中正相代谢物二十二碳六烯酸（DHA）含量上升。术后3～4周，大鼠尿液、血生化指标以及溶血磷脂酰乙醇胺（20∶ 4）、黄嘌呤核苷等血清代谢物呈波动性变化，病理可见肾脏组织充血、结缔组织增生等病变，血清代谢指纹谱相对稳定，形成局灶型肾脏纤维化模型。术后5～8周，除肾脏组织病理持续恶化外，血清代谢指纹谱及各项血液、尿液指标处于相对稳定状态。术后5～6周时2-氨基马尿酸、马尿酸、2-苯乙酰胺、DHA、硫酸吲哚酚等代谢物含量存在波动。术后7～8周代谢物含量呈现稳定状态，形成弥散型肾脏纤维化模型。体内验证实验显示，与模型组相比，马来酸依那普利组大鼠血清中硫酸吲哚酚、黄嘌呤核苷、马尿酸、2-氨基马尿酸、2-苯乙酰胺等差异代谢物含量呈现显著回调（P＜0.05、0.01、0.001）。结论 UUO造模4周可形成局灶型肾脏纤维化模型，造模8周可形成稳定的弥散型肾脏纤维化模型。硫酸吲哚酚、黄嘌呤核苷、马尿酸、2-氨基马尿酸、2-苯乙酰胺、DHA等内源性血清代谢物是评价UUO致肾脏纤维化疾病进程的灵敏度高的潜在指标。

Objective To systematically evaluate the impact of modeling duration on the progression of renal fibrosis induced by unilateral ureteral obstruction (UUO) in rats, utilizing pathophysiological indices and serum metabolite analysis. Methods A total of 54 male Sprague-Dawley (SD) rats were randomly assigned to nine groups. With the exception of the control group, all other groups underwent unilateral ureteral ligation. Each experimental group was assessed at 7, 14, 21, 28, 35, 42, 49, 56 d post-induction. The 24 h urine volume of rats was detected. The urine protein, serum creatinine, urea nitrogen, albumin, total protein, triglyceride and total cholesterol levels were detected by the kit method. The renal specific gravity (the mass ratio of the surgical side to the contralateral kidney) and the organ coefficients of the kidney, heart, spleen and bladder tissues were also detected. Hematoxylin-eosin (HE) staining and Masson staining were used to detect the pathological changes of the operated kidney. Differential serum metabolites were identified using high-resolution liquid chromatography-mass spectrometry in conjunction with pattern recognition techniques. More, UUO rats were treated with ACEI drugs (enalapril maleate) for 28 d, and the relative contents of differential metabolites in serum were detected to evaluate the feasibility of differential metabolites in the diagnosis and curative effect monitoring of renal fibrosis diseases. Results One week following the UUO procedure, significant increases were observed in the 24 h urine volume, urinary protein concentration, renal specific gravity, serum creatinine, and blood urea nitrogen levels in the rats. Histological examination via HE staining revealed glomerular atrophy and presclerosis, and a few inflammatory cell infiltration in cortex. In comparison to the control group, the serum metabolic profile exhibited significant deviations, characterized by a marked increase in the levels of endogenous metabolites such as indoxyl sulfate, 2-aminohippuric acid, and xanthosine. Two weeks post-surgery, compensatory physiological mechanisms appeared to be activated, as evidenced by notable increases in the organ indices of the heart, spleen, and right kidney. Concurrently, there was a significant reduction in the levels of urinary protein, serum creatinine, albumin, triglycerides, and other differential metabolites when compared to the first week, alongside an increase in the serum concentration of DHA. Between three to four weeks following the operation, fluctuations were observed in biochemical markers and serum metabolites, such as lysoPE (20:4) and xanthosine. Additionally, pathological assessments revealed renal congestion and hyperplasia of connective tissue, while the serum metabolic profile stabilized. Focal renal fibrosis model was established in this point. By the fifth to eighth week post-operation, despite a continued deterioration in renal histopathology, both the serum metabolic profile and various hematological and urinary parameters remained relatively stable. By the fifth to eighth week post-operation, despite a persistent decline in renal histopathology, both the serum metabolic profile and various hematological and urinary parameters exhibited relative stability. Metabolites, including 2- aminohippuric acid, hippuric acid, 2-phenylacetamide, DHA and indoxyl sulfate, demonstrated fluctuations during the fifth to sixth week period post-operation. And the metabolite levels stabilized by the seventh to eighth week period. Model of diffuse renal fibrosis was established in this point. In vivo verification experiments demonstrated that the serum levels of indoxyl sulfate, xanthosine, hippuric acid, 2-aminohippuric acid, and 2-phenylacetamide in renal fibrosis rats were significantly decreased, showing statistically significant differences compared with the model group (P < 0.05, 0.01, and 0.001). Conclusion UUO induces a focal renal fibrosis model within four weeks and a stable diffuse renal fibrosis model by eight weeks. Endogenous serum metabolites, such as indoxyl sulfate, xanthosine, hippuric acid, 2-aminohippuric acid, 2-phenylacetamide and DHA, are potential indicators with heightened sensitivity to evaluate the progression of renal fibrosis induced by UUO.

R965

国家自然科学基金资助项目（82304849）；广东省自然科学基金资助项目（2021A1515110784）；广州市基础研究计划市校（院）联合资助项目（202201020329）。