Suzuki Hitoe, Uchida Keiko, Nitta Kosaku, Nihei Hiroshi
Department of Medicine, Kidney Center, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan.
Clin Exp Nephrol. 2004 Sep;8(3):188-95. doi: 10.1007/s10157-004-0297-8.
In diabetic nephropathy, tubulointerstitial fibrosis is an important component of renal injury. Transforming growth factor (TGF)-beta is a key cytokine that is involved in the pathogenesis of tubulointerstitial fibrosis. However, signal transduction cascades of TGF-beta under high-glucose conditions remain to be clarified. We undertook this study to elucidate whether mitogen-activated protein (MAP) kinase and Smad proteins were involved in TGF-beta-induced fibronectin (FN) production under high glucose in NRK fibroblasts.
After serum restriction, NRK cells were exposed to either normal glucose (5.5 mM d-glucose), high glucose (30 mM d-glucose), or 30 mM l-glucose in the presence or absence of TGF-beta for 24 h. MAP kinase inhibitors (SB 203580 and PD 98059) were added to the cultured NRK fibroblasts 2 h before TGF-beta1, and the incubations continued for 8 h. The phosphorylation of p38 MAP kinase, extracellular signal-regulated kinase (ERK)1/ERK2, and c-Jun N-amino terminal kinase (JNK) was assessed by immunoblotting. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were performed to determine FN mRNA and protein expression, respectively.
High glucose significantly increased the expression of FN mRNA, by 2.4 +/- 1.4-fold. In the presence of either SB 203580 or PD 98059, the high glucose-induced FN mRNA increase was completely inhibited. Incubation of NRK fibroblasts for 48 h in 30 mM d-glucose did not alter p38 MAP kinase, ERK1/ERK2, or JNK phosphorylation. The addition of exogenous TGF-beta1 (1 ng/ml) for 8 h increased FN mRNA by 2.7 +/- 1.1-fold. Both the TGF-beta1- and high glucose-induced FN mRNA increases were inhibited by SB 203580 and PD 98059. Dominant-negative Smad4 did not affect the FN mRNA increase induced by TGF-beta1 and high glucose. Exogenous TGF-beta1 under both normal and high glucose, enhanced the phosphorylation of both p38 MAP kinase and ERK1/ERK2, but not that of JNK.
NRK fibroblasts exposed to high glucose demonstrated increased TGF-beta1-induced p38 MAP kinase activation. The FN synthesis induced by high glucose and TGF-beta1 was not affected by the Smads pathway and was not due to increased osmolarity. The enhanced activation of p38 MAP kinase may contribute to the altered fibroblast phenotype that leads to progressive diabetic nephropathy.
在糖尿病肾病中，肾小管间质纤维化是肾损伤的重要组成部分。转化生长因子（TGF）-β是参与肾小管间质纤维化发病机制的关键细胞因子。然而，高糖条件下TGF-β的信号转导级联仍有待阐明。我们进行这项研究以阐明丝裂原活化蛋白（MAP）激酶和Smad蛋白是否参与高糖环境下TGF-β诱导的NRK成纤维细胞中纤连蛋白（FN）的产生。
血清饥饿后，NRK细胞在存在或不存在TGF-β的情况下，分别暴露于正常葡萄糖（5.5 mM d-葡萄糖）、高糖（30 mM d-葡萄糖）或30 mM l-葡萄糖中24小时。在加入TGF-β1前2小时，将MAP激酶抑制剂（SB 203580和PD 98059）添加到培养的NRK成纤维细胞中，并继续孵育8小时。通过免疫印迹评估p38 MAP激酶、细胞外信号调节激酶（ERK）1/ERK2和c-Jun N-末端激酶（JNK）的磷酸化。分别进行逆转录-聚合酶链反应（RT-PCR）和蛋白质免疫印迹法以测定FN mRNA和蛋白质表达。
高糖显著增加FN mRNA的表达，增加了2.4±1.4倍。在存在SB 203580或PD 98059的情况下，高糖诱导的FN mRNA增加被完全抑制。NRK成纤维细胞在30 mM d-葡萄糖中孵育48小时未改变p38 MAP激酶、ERK1/ERK2或JNK的磷酸化。加入外源性TGF-β1（1 ng/ml）8小时使FN mRNA增加2.7±1.1倍。SB 203580和PD 98059抑制了TGF-β1和高糖诱导的FN mRNA增加。显性负性Smad4不影响TGF-β1和高糖诱导的FN mRNA增加。正常和高糖条件下的外源性TGF-β1均增强了p38 MAP激酶和ERK1/ERK2的磷酸化，但不影响JNK的磷酸化。
暴露于高糖的NRK成纤维细胞显示TGF-β1诱导的p38 MAP激酶活化增加。高糖和TGF-β1诱导的FN合成不受Smads途径影响，也不是由于渗透压增加所致。p38 MAP激酶的活化增强可能导致成纤维细胞表型改变，进而导致糖尿病肾病进展。
