硫化氢在单侧输尿管梗阻大鼠肾小管间质纤维化中的水平变化及其干预影响

Change in plasma H2S level and therapeutic effect of H2S supplementation in tubulointerstitial fibrosis among rats with unilateral ureteral obstruction

摘要:

目的：本研究通过构建单侧输尿管梗阻（UUO）诱导肾小管间质纤维化（TIF）大鼠模型，观察硫化氢（H2S）在血浆中的水平变化及两种关键合成酶胱硫醚β-合酶（CBS）、胱硫醚γ-裂解酶（CSE）在梗阻肾中的表达，并探讨H2S在TIF中的作用。方法：构建UUO致TIF大鼠模型，96只Sprague Dawley大鼠随机分为假手术组、模型组、NaHS低剂量治疗组（低剂量组）和NaHS高剂量治疗组（高剂量组），每组24只。治疗组分别于术后立即腹腔注射NaHS 1.4 μmol/kg和7 μmol/kg，每日两次；假手术组和模型组同时腹腔注射等量生理盐水。各组分别于术后7 d、14 d及21 d随机处死8只大鼠，采用去蛋白法测定血浆H2S含量；梗阻肾组织行苏木精-伊红及Masson染色，观察肾脏病理学变化；免疫组化方法检测梗阻肾组织CBS、CSE蛋白表达；RT-PCR法检测梗阻肾组织CBS mRNA、CSE mRNA的表达。结果：UUO大鼠肾小管间质损伤程度与血浆H2S浓度呈负相关（r=-0.891，P<0.01）；外源性给予NaHS能显著减轻肾小管间质损伤（P<0.01），上调肾组织CBS、CSE蛋白及其mRNA的表达和血浆H2S水平（P<0.01），肾小管间质纤维化程度明显减轻（P<0.01）；低、高NaHS剂量组间血浆H2S水平、CBS、CSE蛋白及其mRNA表达比较差异均无统计学意义（P>0.05）。结论：H2S 参与UUO致肾小管间质纤维化的发展过程，而且CBS/H2S体系和CSE/H2S体系在TIF中发挥关键作用。外源性补充NaHS可以延缓TIF的进展。

Abstract:

OBJECTIVE: To observe the level in plasma hydrogen sulfide (H2S) and the expression of cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE) (two key synthetases for endogenous H2S generation in the kidney) in obstructed kidney tissue among rats with tubulointerstitial fibrosis (TIF) induced by unilateral ureteral obstruction (UUO), and to explore the role of H2S in TIF. METHODS: Ninety-six male Sprague-Dawley rats were randomly divided into sham-operated, model, low-dose NaHS and high-dose NaHS groups (n=24 each). TIF was induced by UUO in the model, low-dose NaHS and high-dose NaHS groups. The low-dose and high-dose NaHS groups were intraperitoneally injected with NaHS (1.4 and 7.0 μmol/kg respectively) twice daily immediately after operation, and the sham-operated and model groups were intraperitoneally injected with an identical volume of normal saline. In each group, 8 rats were randomly selected and sacrificed at 7, 14 or 21 days after operation. Plasma H2S concentration was measured by deproteinization. The obstructed kidney tissue was subjected to hematoxylin and eosin staining and Masson staining, and the renal tubulointerstitial injury was evaluated under a microscope. mRNA and protein expression of CBS and CSE in the obstructed kidney tissue was measured by RT-PCR and immunohistochemistry respectively. RESULTS: The degree of UUO-induced renal tubulointerstitial injury was negatively correlated with plasma H2S concentration in (r=-0.891, P<0.01). With H2S supplementation, renal tubulointerstitial injury was reduced (P<0.01), the expression of mRNA and protein of CBS and CSE in the kidney tissue and plasma H2S level were upregulated (P<0.01), and the degree of TIF was reduced (P<0.01). There were no significant differences in plasma H2S level and mRNA and protein expression of CBS and CSE between the low-dose and high-dose NaHS groups (P>0.05). CONCLUSIONS: H2S is involved in the development of UUO-induced TIF, and the CBS/H2S and CSE/H2S systems play key roles in this process. H2S supplementation can delay the progression of TIF.