Abstract:
Most kidney stones are composed of calcium oxalate, and small increases in urine oxalate enhance the stone risk. The mammalian intestine plays a crucial role in oxalate homeostasis, and we had recently reported that Oxalobacter-derived factors stimulate oxalate transport by human intestinal Caco2-BBE (C2) cells through PKA activation. We therefore evaluated whether intestinal oxalate transport is directly regulated by activation of the PKA signaling pathway. To this end, PKA was activated with forskolin and IBMX (F/I). F/I significantly stimulated (3.7-fold) [14C]oxalate transport by C2 cells [≥49% of which is mediated by the oxalate transporter SLC26A6 (A6)], an effect completely blocked by the PKA inhibitor H89, indicating that it is PKA dependent. PKA stimulation of intestinal oxalate transport is not cell line specific, since F/I similarly stimulated oxalate transport by the human intestinal T84 cells. F/I significantly increased (2.5-fold) A6 surface protein expression by use of immunocytochemistry. Assessing [14C]oxalate transport as a function of increasing [14C]oxalate concentration in the flux medium showed that the observed stimulation is due to a F/I-induced increase (1.8-fold) in Vmax and reduction (2-fold) in Km. siRNA knockdown studies showed that significant components of the observed stimulation are mediated by A6 and SLC26A2 (A2). Besides enhancing A6 surface protein expression, it is also possible that the observed stimulation is due to PKA-induced enhanced A6 and/or A2 transport activity in view of the reduced Km. We conclude that PKA activation positively regulates oxalate transport by intestinal epithelial cells and that PKA agonists might therapeutically impact hyperoxalemia, hyperoxaluria, and related kidney stones.
摘要:
大多数肾结石由草酸钙组成,尿液中草酸盐的少量增加会增加结石的风险。哺乳动物肠道在草酸盐稳态中起着至关重要的作用,我们最近报道了草酸杆菌衍生因子通过PKA激活刺激人肠道Caco2-BBE(C2)细胞的草酸盐转运。因此,我们评估了肠草酸盐转运是否直接受PKA信号通路激活的调节。为此,PKA用毛喉素和IBMX(F/I)激活。F/I显着刺激(3.7倍)C2细胞的[14C]草酸盐转运[其中≥49%由草酸盐转运蛋白SLC26A6(A6)介导],该效应被PKA抑制剂H89完全阻断,表明它是PKA依赖性的。PKA刺激肠草酸盐转运不是细胞系特异性的,因为F/I类似地刺激人肠T84细胞的草酸盐转运。通过使用免疫细胞化学,F/I显著增加(2.5倍)A6表面蛋白表达。评估[14C]草酸盐转运作为通量培养基中[14C]草酸盐浓度增加的函数表明,观察到的刺激是由于F/I诱导的Vmax增加(1.8倍)和Km降低(2倍)。siRNA敲低研究表明,所观察到的刺激的显著组分是由A6和SLC26A2(A2)介导的。除了增强A6表面蛋白表达外,还可能的是,观察到的刺激是由于PKA诱导的增强的A6和/或A2转运活性(鉴于降低的Km)。我们得出结论,PKA激活正调节肠上皮细胞的草酸盐转运,PKA激动剂可能在治疗上影响高草酸血症。高草酸尿症,和相关的肾结石。