Stewart Gavin S, O'Brien Jennifer H, Smith Craig P
Faculty of Life Sciences, Core Technology Facility, University of Manchester, Manchester, UK.
Am J Physiol Cell Physiol. 2008 Jul;295(1):C121-9. doi: 10.1152/ajpcell.00444.2007. Epub 2008 Apr 30.
The renal UT-A urea transporters UT-A1, UT-A2, and UT-A3 are known to play an important role in the urinary concentrating mechanism. The control of the cellular localization of UT-A transporters is therefore vital to overall renal function. In the present study, we have investigated the effect of ubiquitination on UT-A plasma membrane expression in Madin-Darby canine kidney (MDCK) cell lines expressing each of the three renal UT-A transporters. Inhibition of the ubiquitin-proteasome pathway caused an increase in basal transepithelial urea flux across MDCK-rat (r)UT-A1 and MDCK-mouse (m)UT-A2 monolayers (P < 0.01, n = 3, ANOVA) and also increased dimethyl urea-sensitive, arginine vasopressin-stimulated urea flux (P < 0.05, n = 3, ANOVA). Inhibition of the ubiquitin-proteasome pathway also increased basolateral urea flux in MDCK-mUT-A3 monolayers (P < 0.01, n = 4, ANOVA) in a concentration-dependent manner. These increases in urea flux corresponded to a significant increase in UT-A transporter expression in the plasma membrane (P < 0.05, n = 3, ANOVA). Further analysis of the MDCK-mUT-A3 cell line confirmed that vasopressin specifically increased UT-A3 expression in the plasma membrane (P < 0.05, n = 3, ANOVA). However, preliminary data suggested that vasopressin produces this effect through an alternative route to that of the ubiquitin-proteasome pathway. In conclusion, our study suggests that ubiquitination regulates the plasma membrane expression of all three major UT-A urea transporters, but that this is not the mechanism primarily used by vasopressin to produce its physiological effects.
已知肾脏尿素转运蛋白UT - A1、UT - A2和UT - A3在尿液浓缩机制中起重要作用。因此，控制UT - A转运蛋白的细胞定位对整体肾功能至关重要。在本研究中，我们研究了泛素化对表达三种肾脏UT - A转运蛋白的Madin - Darby犬肾（MDCK）细胞系中UT - A质膜表达的影响。泛素 - 蛋白酶体途径的抑制导致跨MDCK - 大鼠（r）UT - A1和MDCK - 小鼠（m）UT - A2单层的基础跨上皮尿素通量增加（P < 0.01，n = 3，方差分析），并且还增加了对二甲基尿素敏感的、精氨酸加压素刺激的尿素通量（P < 0.05，n = 3，方差分析）。泛素 - 蛋白酶体途径的抑制还以浓度依赖性方式增加了MDCK - mUT - A3单层的基底外侧尿素通量（P < 0.01，n = 4，方差分析）。这些尿素通量的增加对应于质膜中UT - A转运蛋白表达的显著增加（P < 0.05，n = 3，方差分析）。对MDCK - mUT - A3细胞系的进一步分析证实，加压素特异性增加了质膜中UT - A3的表达（P < 0.05，n = 3，方差分析）。然而，初步数据表明，加压素通过与泛素 - 蛋白酶体途径不同的途径产生这种效应。总之，我们的研究表明泛素化调节所有三种主要UT - A尿素转运蛋白的质膜表达，但这不是加压素产生其生理效应的主要机制。
