Stewart Gavin S, King Sarah L, Potter Elizabeth A, Smith Craig P
Faculty of Life Sciences, 2nd Floor, Core Technology Facility, Univ. of Manchester, Grafton St., Manchester M13 9NT, UK.
Am J Physiol Renal Physiol. 2007 Apr;292(4):F1157-63. doi: 10.1152/ajprenal.00183.2006. Epub 2006 Dec 5.
Renal facilitative urea transporters play a vital role in the urinary concentrating mechanism. UT-A3 is a phloretin-sensitive urea transporter that in the mouse is expressed on the basolateral membrane of renal inner medullary collecting duct (IMCD) cells. In this study, we engineered a Madin-Darby canine kidney (MDCK) I cell line that stably expresses mouse UT-A3 (MDCK-mUT-A3). Immunoblotting using the UT-A-targeted antibody ML446 detected a approximately 40-kDa signal in MDCK-mUT-A3 protein that corresponds to mUT-A3. Using cultured epithelial monolayers, radioactive (14)C-urea flux experiments determined that basolateral urea transport was no different between MDCK-mUT-A3 and control MDCK-FLZ cells under basal conditions [not significant (NS), ANOVA]. However, exposure to arginine vasopressin (AVP) significantly stimulated basolateral urea flux in MDCK-mUT-A3 monolayers (P < 0.05, ANOVA), while it had no effect in control MDCK-FLZ monolayers (NS, ANOVA). The AVP-stimulated basolateral urea transport in MDCK-mUT-A3 was inhibited by 1,3 dimethyl urea (P < 0.05, ANOVA) or phloretin (P < 0.05, ANOVA), both known inhibitors of facilitative urea transporters. MDCK-mUT-A3 basolateral urea flux was also stimulated by increasing intracellular levels of cAMP, via forskolin (P < 0.05, ANOVA), or intracellular calcium, via ATP (P < 0.05, ANOVA). Finally, 1-h preincubation with a specific PKA inhibitor, H89, significantly inhibited the increase in urea transport produced by AVP (P < 0.05, ANOVA). In conclusion, we have produced the first renal cell line to stably express the mUT-A3 urea transporter. Our results indicate that mUT-A3 is acutely regulated by AVP, via a PKA-dependent pathway. These findings have important implications for the regulation of urea transport in the renal IMCD and the urinary concentrating mechanism.
肾脏易化尿素转运蛋白在尿液浓缩机制中起关键作用。UT - A3是一种对根皮素敏感的尿素转运蛋白，在小鼠中表达于肾内髓集合管（IMCD）细胞的基底外侧膜上。在本研究中，我们构建了稳定表达小鼠UT - A3的Madin - Darby犬肾（MDCK）I细胞系（MDCK - mUT - A3）。使用针对UT - A的抗体ML446进行免疫印迹检测到MDCK - mUT - A3蛋白中有一条约40 kDa的条带，与mUT - A3相对应。利用培养的上皮单层细胞，通过放射性（14）C - 尿素通量实验确定，在基础条件下，MDCK - mUT - A3和对照MDCK - FLZ细胞之间的基底外侧尿素转运没有差异[无显著性差异（NS），方差分析]。然而，暴露于精氨酸加压素（AVP）显著刺激了MDCK - mUT - A3单层细胞的基底外侧尿素通量（P < 0.05，方差分析），而对对照MDCK - FLZ单层细胞没有影响（NS，方差分析）。MDCK - mUT - A3中AVP刺激的基底外侧尿素转运受到1,3 - 二甲基尿素（P < 0.05，方差分析）或根皮素（P < 0.05，方差分析）的抑制，这两种都是已知的易化尿素转运蛋白抑制剂。通过福斯可林增加细胞内cAMP水平（P < 0.05，方差分析）或通过ATP增加细胞内钙水平（P < 0.05，方差分析）也能刺激MDCK - mUT - A3的基底外侧尿素通量。最后，用特异性PKA抑制剂H89预孵育1小时可显著抑制AVP引起的尿素转运增加（P < 0.05，方差分析）。总之，我们构建了首个稳定表达mUT - A3尿素转运蛋白的肾细胞系。我们的结果表明，mUT - A3通过PKA依赖的途径受到AVP的急性调节。这些发现对肾内髓中尿素转运的调节和尿液浓缩机制具有重要意义。
