目的 探究不同造模周期對(duì)單側(cè)輸尿管結(jié)扎（UUO）誘導(dǎo)大鼠腎纖維化模型表型的影響及可能的代謝物質(zhì)基礎(chǔ)。方法 54只SD雄性大鼠隨機(jī)分為9組，除對(duì)照組外，其余各組均采用UUO的方法誘導(dǎo)大鼠腎臟纖維化模型，各模型組分別于7、14、21、28、35、42、49、56 d取材。檢測(cè)大鼠24 h尿量，尿蛋白及血清肌酐、尿素氮、白蛋白、總蛋白、三酰甘油、總膽固醇水平，測(cè)定腎比重（術(shù)側(cè)與對(duì)側(cè)腎臟質(zhì)量比）及腎臟、心臟、脾臟、膀胱組織臟器系數(shù)，蘇木素-伊紅（HE）染色和馬松（Masson）染色檢測(cè)術(shù)側(cè)腎臟病理變化。采用液相色譜串聯(lián)質(zhì)譜技術(shù)（LC-MS/MS）對(duì)血清代謝物進(jìn)行非靶向檢測(cè)分析，結(jié)合模式識(shí)別技術(shù)篩選血清差異代謝物，初步揭示UUO模型的代謝物質(zhì)基礎(chǔ)。為進(jìn)一步評(píng)價(jià)差異代謝物用于腎纖維化疾病診斷和療效監(jiān)測(cè)的可行性，采用馬來酸依那普利治療UUO大鼠28 d（術(shù)后第2天開始給藥），檢測(cè)血清中差異代謝物相對(duì)含量。結(jié)果 UUO術(shù)后1周，大鼠24 h尿量、尿蛋白含量、腎比重、血肌酐和尿素氮等生化指標(biāo)含量顯著上升，病理檢測(cè)可見腎小球萎縮、早期硬化，皮質(zhì)區(qū)可見少量炎癥細(xì)胞浸潤，血清代謝指紋譜與對(duì)照組相比明顯偏移，血清中內(nèi)源性代謝物硫酸吲哚酚、2-氨基馬尿酸、黃嘌呤核苷的含量急劇上升。術(shù)后2周，機(jī)體代償功能發(fā)揮作用，心臟、脾臟及右腎臟器指數(shù)明顯上升，尿蛋白、血肌酐、白蛋白、三酰甘油及差異代謝物含量較第1周有所下降，血清中正相代謝物二十二碳六烯酸（DHA）含量上升。術(shù)后3～4周，大鼠尿液、血生化指標(biāo)以及溶血磷脂酰乙醇胺（20∶ 4）、黃嘌呤核苷等血清代謝物呈波動(dòng)性變化，病理可見腎臟組織充血、結(jié)締組織增生等病變，血清代謝指紋譜相對(duì)穩(wěn)定，形成局灶型腎臟纖維化模型。術(shù)后5～8周，除腎臟組織病理持續(xù)惡化外，血清代謝指紋譜及各項(xiàng)血液、尿液指標(biāo)處于相對(duì)穩(wěn)定狀態(tài)。術(shù)后5～6周時(shí)2-氨基馬尿酸、馬尿酸、2-苯乙酰胺、DHA、硫酸吲哚酚等代謝物含量存在波動(dòng)。術(shù)后7～8周代謝物含量呈現(xiàn)穩(wěn)定狀態(tài)，形成彌散型腎臟纖維化模型。體內(nèi)驗(yàn)證實(shí)驗(yàn)顯示，與模型組相比，馬來酸依那普利組大鼠血清中硫酸吲哚酚、黃嘌呤核苷、馬尿酸、2-氨基馬尿酸、2-苯乙酰胺等差異代謝物含量呈現(xiàn)顯著回調(diào)（P＜0.05、0.01、0.001）。結(jié)論 UUO造模4周可形成局灶型腎臟纖維化模型，造模8周可形成穩(wěn)定的彌散型腎臟纖維化模型。硫酸吲哚酚、黃嘌呤核苷、馬尿酸、2-氨基馬尿酸、2-苯乙酰胺、DHA等內(nèi)源性血清代謝物是評(píng)價(jià)UUO致腎臟纖維化疾病進(jìn)程的靈敏度高的潛在指標(biāo)。

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

國家自然科學(xué)基金資助項(xiàng)目（82304849）；廣東省自然科學(xué)基金資助項(xiàng)目（2021A1515110784）；廣州市基礎(chǔ)研究計(jì)劃市校（院）聯(lián)合資助項(xiàng)目（202201020329）。