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目的探讨组织型转谷氨酰胺酶(tTG)在局灶节段性肾小球硬化(FSGS)模型大鼠肾脏中的表达及其意义。方法FSGS大鼠模型采用左颈静脉插管单次注射嘌呤霉素氨基核苷(PAN)方法建立,对照组大鼠注射等量生理盐水,20周后处死各组大鼠。肾组织切片用PAS染色,光镜下观察病理变化;用免疫组织化学、底物掺入免疫荧光法、Western印迹法分别观察肾内tTG蛋白分布、原位活性和蛋白水平变化;用免疫组化半定量方法分析细胞外基质纤连蛋白(FN)表达。结果模型组肾组织病理呈典型局灶节段肾小球硬化病变,伴大量蛋白尿。对照组肾组织tTG蛋白表达较弱,分布于肾小球内;模型组肾组织tTG蛋白分布于肾小球硬化部位。Western印迹结果显示模型组肾组织tTG蛋白水平比对照组增加2.61倍(P<0.05)。对照组肾组织tTG原位活性微弱;模型组肾小球tTG活性明显增强。模型组FN主要分布在肾小球,表达水平明显高于对照组(3.73±0.57比2.50±1.00,P<0.05):并且LTG蛋白水平与FN水平呈显著正相关(r=0.73,P<0.05)。结论tTG可能通过交联细胞外基质蛋白,抵抗降解,参与FSGS发生发展。
Objective To investigate the expression and significance of tissue transglutaminase (tTG) in the kidney of focal segmental glomerulosclerosis (FSGS) model rats. Methods The rat model of FSGS was established by single injection of puromycin aminonucleoside (PAN) into the left jugular vein, and the rats in the control group were injected with the same amount of normal saline. After 20 weeks, the rats in each group were sacrificed. Kidney sections were stained with PAS and pathological changes were observed under light microscope. The distribution of tTG protein, in situ activity and protein level in kidney were observed by immunohistochemistry, substrate incorporation of immunofluorescence and Western blotting. Immunohistochemistry Semiquantitative analysis of extracellular matrix fibronectin (FN) expression. Results The pathological changes of renal tissue in model group were typical focal segmental glomerulosclerosis with a large amount of proteinuria. In the control group, the expression of tTG protein in the kidney tissue was weak and distributed in the glomerulus. The tTG protein in the model group was distributed in the glomerular sclerosis site. Western blotting showed that the level of tTG protein in model group was 2.61 times more than that in control group (P <0.05). In the control group, the tTG activity in renal tissue was weak; the activity of glomerular tTG in the model group was significantly increased. The expression of FN in glomeruli was significantly higher in model group than in control group (3.73 ± 0.57 vs 2.50 ± 1.00, P <0.05), and there was a significant positive correlation between LTG protein and FN (r = 0.73, P <0.05 ). Conclusion tTG may be involved in the development of FSGS by cross-linking extracellular matrix proteins and resistance to degradation.