Regulation of the renal epithelial sodium channel (ENaC) by the vascular endothelial growth factor (VEGF) and the pathogenetic role of renal ENaC for VEGF receptor inhibitor-induced hypertension.

血管内皮生长因子受体(VEGFR)抑制剂被用于肿瘤治疗。一种限制治疗的副作用是动脉高血压。肾脏机制在VEGFR抑制剂诱导的高血压中的作用目前尚不清楚。在对大鼠的实验研究中，我们发现VEGFR抑制剂舒尼替尼显著减少部分Na+的排泄，而对血管功能几乎没有影响。作为近端Na+重吸收标志的部分Li+排泄没有变化，表明Na+转运在更远的部位受到影响。在小鼠大脑皮质CD(M1)细胞上的实验进一步表明，血管内皮生长因子(VEGF-A)显著降低上皮细胞Na+通道(ENaC)亚单位mRNA的丰度，这一作用可被舒尼替尼抑制。血管内皮生长因子也显著降低了M1细胞中α-ENaC亚单位蛋白的丰度。我们的研究结果表明，血管内皮生长因子可能是ENaC在肾脏中的负生理调节因子，ENaC依赖的Na+重吸收增加可能参与了舒尼替尼诱导的高血压。我们提议的项目的第一个目标是检查血管内皮生长因子是否下调M1细胞中的ENaC。我们将验证这样的假说，即血管内皮生长因子降低ENaC蛋白丰度和伽马-ENaC的蛋白水解性切割，激活VEGFR-2，诱导ENaC内化和降低ENaC活性(膜片钳)。此外，我们还将研究细胞外信号调节激酶(ERK1/2)和一氧化氮合酶(NOS)依赖的通路是否参与了血管内皮生长因子诱导的ENaC下调。第二个目标是探讨VEGFR抑制剂孙尼替尼是否增加大鼠肾脏ENaC的活性。我们将测试假说，即舒尼替尼治疗4天增加了肾脏ENaC的表达，增强了伽马-ENaC亚基的蛋白水解性切割，减少了β-和伽马-ENaC亚基的磷酸化，或者刺激了ENaC亚基从细胞质插入顶端质膜，导致ENaC活性增强。第三个目标是测试舒尼替尼是否能减少大鼠肾脏一氧化氮的生成。我们将验证舒尼替尼治疗4天可减少肾脏一氧化氮合酶表达、降低肾脏一氧化氮合酶活性及改变肾脏一氧化氮合酶同工酶定位的假说。此外，我们将用舒尼替尼和NO供体硝酸盐联合治疗大鼠，以检验NO降低舒尼替尼诱导的动脉血压和肾脏钠重吸收增加以及减少舒尼替尼诱导的肾脏ENaC表达增加的假设。最后，我们将在CD-特异性NOS1基因敲除小鼠身上进行实验，以验证这样的假设，即舒尼替尼在基因敲除小鼠中导致动脉压和肾脏钠重吸收比对照小鼠更大的升高。

Vascular endothelial growth factor receptor (VEGFR) inhibitors are used in tumor therapy. One therapy-limiting side effect is arterial hypertension. The role of renal mechanisms in VEGFR inhibitor-induced hypertension is currently unclear. In experimental studies in rats, we showed that the VEGFR inhibitor, sunitinib, significantly decreases fractional Na+ excretion while having little effects on vascular function. Fractional Li+ excretion, as a marker for proximal Na+ reabsorption, was unchanged suggesting that Na+ transport was affected at more distal sites. Experiments in murine cortical CD (M1) cells further revealed that VEGF (VEGF-A) significantly decreases epithelial Na+ channel (ENaC) subunit mRNA abundances and that this effect is inhibited by sunitinib. VEGF also significantly reduced alpha-ENaC subunit protein abundance in M1 cells. Again, sunitinib diminished this effect.Our findings suggest that VEGF may be a negative physiological regulator of ENaC in the kidney and that increased ENaC-dependent Na+ reabsorption may contribute to sunitinib-induced hypertension. The first objective of our proposed project is to examine whether VEGF down-regulates ENaC in M1 cells. We will test the hypotheses that VEGF reduces ENaC protein abundances and proteolytic cleavage of gamma-ENaC, activates the VEGFR-2, induces internalization of ENaC and decreases ENaC activity (patch clamp). In addition, we will investigate if the extracellular signal-regulated kinase (ERK1/2)- and the nitric oxide synthase (NOS)-dependent pathways are involved in the VEGF-induced ENaC down-regulation.The second objective is to investigate if the VEGFR inhibitor, sunitinib, increases renal ENaC activity in rats. We will test the hypotheses that sunitinib treatment for 4 days increases renal ENaC expression, enhances proteolytic cleavage of the gamma-ENaC subunit, decreases phosphorylation of beta- and gamma-ENaC subunits or stimulates the insertion of ENaC subunits from the cytoplasm into the apical plasma membrane, leading to enhanced ENaC activity. The third objective is to test if sunitinib reduces renal NO formation in rats. We will test the hypotheses that sunitinib treatment for 4 days decreases renal NOS expression, decreases renal NOS activity and alters the localization of renal NOS isoenzymes. Furthermore, we will treat rats with a combination of sunitinib and the NO donor, nitrate, to test the hypotheses that NO reduces the sunitinib-induced increases in arterial blood pressure and renal sodium reabsorption and reduces the sunitinib-induced increases in renal ENaC expression. Finally, we will perform experiments in CD-specific NOS1 knockout mice to test the hypothesis that sunitinib causes greater elevations in arterial pressure and renal sodium reabsorption in knockout mice than in controls.