Tissue transglutaminase is a calcium-dependent enzyme that catalyzes the cross-linking of polypeptide chains, including those of extracellular matrix (ECM) proteins, through the formation of epsilon-(gamma-glutamyl) lysine bonds. This crosslinking leads to the formation of protein polymers that are highly resistant to degradation. As a consequence, the enzyme has been implicated in the deposition of ECM protein in fibrotic diseases such as pulmonary fibrosis and atherosclerosis. In this study, we have investigated the involvement of tissue transglutaminase in the development of kidney fibrosis in adult male Wistar rats submitted to subtotal nephrectomy (SNx). Groups of six rats were killed on days 7, 30, 90, and 120 after SNx. As previously described, these rats developed progressive glomerulosclerosis and tubulo-interstitial fibrosis. The tissue level of epsilon-(gamma-glutamyl) lysine cross-link (as determined by exhaustive proteolytic digestion followed by cation exchange chromatography) increased from 3.47+/- 0.94 (mean+/-SEM) in controls to 13.24+/-1.43 nmol/g protein 90 d after SNx, P </= 0.01. Levels of epsilon-(gamma-glutamyl) lysine cross-link correlated well with the renal fibrosis score throughout the 120 observation days (r = 0.78, P </= 0.01). Tissue homogenates showed no significant change in overall transglutaminase activity (14C putrescine incorporation assay) unless adjusted for the loss of viable tubule cells, when an increase from 5.77+/-0.35 to 13.93+/-4.21 U/mg DNA in cytosolic tissue transglutaminase activity was seen. This increase was supported by Western blot analysis, showing a parallel increase in renal tissue transglutaminase content. Immunohistochemistry demonstrated that this large increase in epsilon-(gamma-glutamyl) lysine cross-link and tissue transglutaminase took place predominantly in the cytoplasm of tubular cells, while immunofluorescence also showed low levels of the epsilon-(gamma-glutamyl) lysine cross-link in the extracellular renal interstitial space. The number of cells showing increases in tissue transglutaminase and its cross-link product, epsilon-(gamma-glutamyl) lysine appeared greater than those showing signs of typical apoptosis as determined by in situ end-labeling. This observed association between tissue transglutaminase, epsilon-(gamma-glutamyl) lysine cross-link, and renal tubulointerstitial scarring in rats submitted to SNx suggests that tissue transglutaminase may play an important role in the development of experimental renal fibrosis and the associated loss of tubule integrity.
展开▼
机译:组织转谷氨酰胺酶是一种钙依赖性酶,通过形成ε-(γ-谷氨酰胺)赖氨酸键,催化包括细胞外基质(ECM)蛋白的多肽链的交联。这种交联导致高度抗降解的蛋白质聚合物的形成。结果,该酶已牵涉在诸如肺纤维化和动脉粥样硬化的纤维化疾病中ECM蛋白的沉积。在这项研究中,我们已经调查了组织转谷氨酰胺酶参与成年次全肾切除术(SNx)的成年雄性Wistar大鼠肾脏纤维化的发展。在SNx后第7、30、90和120天杀死六只大鼠。如前所述,这些大鼠发展为进行性肾小球硬化和肾小管间质纤维化。 ε-(γ-谷氨酰)赖氨酸交联的组织水平(通过彻底的蛋白水解消化后通过阳离子交换色谱法测定)从对照中的3.47 +/- 0.94(平均值+/- SEM)增加到13.24 +/- 1.43nmol SNx后90 d / g蛋白,P = 0.01。在整个120天的观察日中,ε-(γ-谷氨酰胺)赖氨酸交联水平与肾纤维化评分密切相关(r = 0.78,P = 0.01)。组织匀浆显示总的转谷氨酰胺酶活性没有明显变化(14C腐胺掺入试验),除非将存活小管细胞的损失进行了调整(当胞浆组织转谷氨酰胺酶活性从5.77 +/- 0.35 U / mg DNA增加到13.93 +/- 4.21 U / mg)被看见。 Western blot分析证实了这种增加,表明肾脏组织中谷氨酰胺转移酶含量平行增加。免疫组织化学表明,ε-(γ-谷氨酰)赖氨酸交联和组织转谷氨酰胺酶的这种大量增加主要发生在肾小管细胞的细胞质中,而免疫荧光法也显示出ε-(γ-谷氨酰)赖氨酸交联的水平较低。在细胞外肾间质间隙中。通过原位末端标记确定,显示组织转谷氨酰胺酶及其交联产物ε-(γ-谷氨酰胺)赖氨酸增加的细胞数量大于显示典型凋亡迹象的细胞数量。在提交给SNx的大鼠中观察到的组织转谷氨酰胺酶,ε-(γ-谷氨酰)赖氨酸交联和肾小管间质瘢痕形成之间的关联表明组织转谷氨酰胺酶可能在实验性肾纤维化的发展以及相关的肾小管丢失中起重要作用诚信。
展开▼