Department of Molecular Pharmacology and Physiology, University of South Florida, Tampa, Florida.
Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Houston, Texas.
Am J Physiol Renal Physiol. 2020 Sep 1;319(3):F541-F551. doi: 10.1152/ajprenal.00146.2020. Epub 2020 Aug 3.
Plasma phosphate (P) levels are tightly controlled, and elevated plasma P levels are associated with an increased risk of cardiovascular complications and death. Two renal transport proteins mediate the majority of P reabsorption: Na-phosphate cotransporters Npt2a and Npt2c, with Npt2a accounting for 70-80% of P reabsorption. The aim of the present study was to determine the in vitro effects of a novel Npt2a inhibitor (PF-06869206) in opossum kidney (OK) cells as well as determine its selectivity in vivo in Npt2a knockout (Npt2a) mice. In OK cells, Npt2a inhibitor caused dose-dependent reductions of Na-dependent P uptake (IC: ~1.4 μmol/L), whereas the unselective Npt2 inhibitor phosphonoformic acid (PFA) resulted in an ~20% stronger inhibition of P uptake. The dose-dependent inhibitory effects were present after 24 h of incubation with both low- and high-P media. Michaelis-Menten kinetics in OK cells identified an ~2.4-fold higher for P in response to Npt2a inhibition with no significant change in apparent . Higher parathyroid hormone concentrations decreased P uptake equivalent to the maximal inhibitory effect of Npt2a inhibitor. In vivo, the Npt2a inhibitor induced a dose-dependent increase in urinary P excretion in wild-type mice (ED: ~23 mg/kg), which was completely absent in Npt2a mice, alongside a lack of decrease in plasma P. Of note, the Npt2a inhibitor-induced dose-dependent increase in urinary Na excretion was still present in Npt2a mice, a response possibly mediated by an off-target acute inhibitory effect of the Npt2a inhibitor on open probability of the epithelial Na channel in the cortical collecting duct.
血浆磷酸盐（P）水平受到严格控制，而血浆 P 水平升高与心血管并发症和死亡风险增加有关。两种肾脏转运蛋白介导了大部分 P 的重吸收：Na-磷酸盐共转运蛋白 Npt2a 和 Npt2c，其中 Npt2a 占 P 重吸收的 70-80%。本研究的目的是确定新型 Npt2a 抑制剂（PF-06869206）在负鼠肾（OK）细胞中的体外作用，并确定其在 Npt2a 敲除（Npt2a）小鼠体内的选择性。在 OK 细胞中，Npt2a 抑制剂引起 Na 依赖性 P 摄取的剂量依赖性降低（IC：1.4 μmol/L），而无选择性的 Npt2 抑制剂膦甲酸（PFA）导致 P 摄取的抑制作用强约 20%。在低 P 和高 P 培养基孵育 24 小时后，存在剂量依赖性的抑制作用。OK 细胞中的米氏动力学表明，Npt2a 抑制后 P 的 值升高了约 2.4 倍，但表观 Km 值没有显著变化。甲状旁腺激素浓度升高导致 P 摄取减少，相当于 Npt2a 抑制剂的最大抑制作用。在体内，Npt2a 抑制剂在野生型小鼠中诱导了剂量依赖性的尿磷排泄增加（ED：23 mg/kg），而在 Npt2a 小鼠中完全不存在，同时血浆 P 没有降低。值得注意的是，Npt2a 抑制剂诱导的尿钠排泄的剂量依赖性增加在 Npt2a 小鼠中仍然存在，这一反应可能是由 Npt2a 抑制剂对皮质集合管上皮钠通道的开放概率的急性非靶标抑制作用介导的。
