Tubulointerstitial injury is exacerbated in streptozotocin-induced diabetic matrix metalloproteinase-2 knockout mice

背景与目的：基质金属蛋白酶-2 （Matrix metalloproteinase -2, MMP-2）是一类能降解多种细胞外基质（extracellular Matrix， ECM）的金属蛋白酶。有研究表明，糖尿病诱导的ECM积累至少部分是由MMP-2的下调引起的。然而，体内MMP-2的活性和表达是否影响糖尿病引起的肾损害尚无数据。因此，我们研究了MMP-2缺乏对糖尿病动物小管间质损伤的影响。材料与方法：用链脲佐菌素（STZ）（50mg/kg）诱导MMP-2敲除雄性小鼠（MMP-2 KO）和C57BL/6J小鼠（Ctrl）发生糖尿病。16周后，分别用酶谱法和实时PCR法检测皮质MMP-2的表达和活性。采用酶联免疫吸附试验（ELISA）检测尿白蛋白排泄量（UAE）和n -乙酰-β- d -氨基葡萄糖苷酶（NAG）。western blot和免疫组织检测α-平滑肌肌动蛋白（α-SMA）含量。分别采用苏木精和伊红染色（HE）和马松三色染色评价大鼠小管间质损伤和纤维化程度。结果：16周糖尿病小鼠血浆葡萄糖和HbA1c水平比非糖尿病对照组小鼠升高约2-3倍（16周糖尿病小鼠血浆葡萄糖；490.3+/-7.5mg/dl, HbA1c; 9.91+/-0.22%）。酶谱分析和RT-PCR结果显示，糖尿病小鼠肾皮质总组织中MMP-2的表达和活性升高。与对照组相比，MMP-2 KO DM小鼠血清BUN、肌酐（Cr）和UAE水平显著升高（BUN: 23.2+/-1.7 vs 39.8+/-6.0mg/dl, p<0.01, Cr; 0.07+/-0.01 vs 0.17+/-0.04mg/dl, p<0.01， UAE; 0.08+/-0.01 vs 0.16+/-0.03mg/mgCr, p<0.05）。此外，DM小鼠的小管间质损伤和皮质α-SMA表达增强，在MMP-2 KO DM小鼠中进一步增加。结论：我们首次证明糖尿病MMP-2基因敲除小鼠的小管间质损伤和纤维化加剧，尽管糖尿病小鼠的MMP-2水平升高。本研究提示其他基质降解酶的减少可能参与糖尿病肾病的小管间质损伤和纤维化。少

Background and Aims : Matrix metalloproteinnase-2 (MMP-2) is one of the potent metalloproteinases which can degrade various types of extracellular matrix (ECM) such as collagen IV, V and laminin. It has been suggested that diabetes-induced accumulation of ECM are, at least in part, by down-regulation of MMP-2. However there is no data whether MMP-2 activity and expression affect diabetes-induced renal damage in vivo. Therefore, we investigated the effects of MMP-2 deficiency on tubulointerstitial injury in diabetic animals.Materials and Methods : Diabetes was induced by streptozotocin (STZ)(50mg/kg) in male MMP-2 knockout mice (MMP-2 KO) and C57BL/6J mice (Ctrl). After 16 weeks, cortical MMP-2 expression and activity were measured by zymography and real-time PCR, respectively. Urinary albumin excretion (UAE) and N-acetyl-β-D-glucosaminidase (NAG) were measured by enzyme-linked immunosorbent assay (ELISA). Alfa-smooth muscle actin (α-SMA) was evaluated by western blots and immnohistoche … More mistry. Tubulointerstitial injury and fibrosis were evaluated by hematoxylin and eosin staining (HE) and Masson-trichrome staining, respectively.Results : Plasma levels of glucose and HbA1c were increased by about 2-3-folds in 16-week diabetic mice compared with non-diabetic Ctrl mice (plasma glucose ; 490.3+/-7.5mg/dl, HbA1c ; 9.91+/-0.22% in 16-week diabetic mice). MMP-2 expression and activity in the total kidney cortex of diabetic mice were increased in zymography and RT-PCR analysis. Serum levels of BUN, creatinine (Cr) and UAE were significantly increased in MMP-2 KO DM mice compared with Ctrl DM mice (BUN ; 23.2+/-1.7 vs 39.8+/-6.0mg/dl, p<0.01, Cr ; 0.07+/-0.01 vs 0.17+/-0.04mg/dl, p<0.01, UAE ; 0.08+/-0.01 vs 0.16+/-0.03mg/mgCr, p<0.05). Further, tubulointerstitial injury and cortical α-SMA expression were enhanced in DM mice, which were further increased in MMP-2 KO DM mice.Conclusions : We demonstrate for the first time that tubulointerstitial injury and fibrosis were exacerbated in diabetic MMP-2 knockout mice, even though MMP-2 levels were increased in diabetic mice. The present study suggests that the decrease in other matrix-degrading enzymes may be involved in the tubulointerstitial injury and fibrosis in diabetic nephropathy. Less