鞘氨醇激酶1在肾间质纤维化中的作用及机制研究

Role of SphK1 in renal tubulointerstitial fibrosis and its mechanism

目的观察鞘氨醇激酶1(SphK1)在单侧输尿管结扎导致的小鼠肾间质纤维化模型中的作用及分子机制。方法 CD-1小鼠随机分为假手术组(Sham)、假手术加药物干预组(Sham+PF-543)、单侧输尿管结扎组(unilateral ureteral obstruction,UUO)和输尿管结扎加药物干预组(UUO+PF-543)。分别于手术后1、3、7、14 d取材,采用Western blot方法检测SphK1、纤维化相关指标mature TGF-β1、FN和ColⅠ及自噬相关因子LC3、Beclin1、Atg5和Atg12的蛋白表达水平;免疫组织化学染色方法观察SphK1、FN和ColⅠ的肾组织定位表达情况;Masson染色光镜下观察肾小管间质纤维化的病变面积,电镜下观察肾小管细胞内自噬体形成情况。结果与Sham组相比,UUO组小鼠肾组织SphK1及自噬相关的蛋白表达水平明显升高,呈现时间依赖性;单侧输尿管结扎导致肾小管细胞内自噬体增多;纤维化相关因子的表达明显升高并伴随肾间质纤维化的病变面积增加;与UUO组相比,PF-543干预明显降低肾组织SphK1及自噬相关蛋白的表达,同时肾小管细胞内自噬体形成减少;但PF-543干预导致UUO肾组织纤维化相关因子的表达进一步升高,间质纤维化的病变进一步加重。结论 SphK1在肾间质纤维化过程中发挥肾保护作用,这一作用可能主要是通过诱导自噬实现的。

Aim To investigate the effect of sphingo-sine kinase 1 （SphK1 ）on unilateral ureteral obstruc-tion（UUO）-induced tubulointerstitial fibrosis and ex-plore the possible mechanism.Methods The CD-1 mice were randomly divided into four groups：sham-op-eration group（Sham）,PF-543 treatment control group （Sham ＋PF-543）,model group（UUO）and PF-543 treatment group（UUO ＋PF-543）.On 1 ,3,7 and 1 4 d after operation,eight mice were selected randomly from each group and sacrificed.The protein expressions of SphK1 ,mature TGF-β1 ,FN,ColⅠ,LC3,Beclin1 ,Atg5 and Atg1 2 were observed by Western blot.The histo-logical changes were examined by Masson′s trichrome stain.Immunhistochemistry was performed to measure the levels of expression of SphK1 ,FN and Col Ⅰ. Transmission electron microscope was used to observe the autophagic body.Results SphK1 expression and autophagy were both upregulated in a mouse model of kidney fibrosis induced by UUO. Meanwhile, in-creased mature TGF-β1 and deposition of extracellular matrix（ECM）were observed in tubulointerstitial areas compared with sham-operated mice.After intraperito-neal injection with the SphK1 specific inhibitor PF-543 in UUO mice,enhanced expression of SphK1 and acti-vated autophagy were significantly abrogated.Howev-er,aggravation of renal fibrosis was detected when SphK1 inhibitor PF-543 was applied to suppress SphK1 expression in UUO mice.Conclusion SphK1 activa-tion is renoprotective through the induction of autoph-agy in the pathogenesis of kidney fibrosis.