Regulation of renal ion transport by the CUL3-WNK-SPAK pathway

项目摘要 肾脏在维持血浆[K+]中起关键作用，肾单位的远端节段微调K+ 保持在正常范围内。我们以前提出，肾远曲小管（DCT） 通过传感等离子体[K+]起关键作用。通过饮食K+限制降低血浆[K+]可激活WNK- SPAK/OSR 1-NCC途径，增加NaCl重吸收，但NCC减少钠的输送， 可能重塑远端K+分泌节段以降低K+分泌。家族性高钾血症 高血压（FHHt）是由WNK、Cullin 3（CUL 3）和Cullin 3（CUL 3）的突变引起的NCC活化增加引起的。 KLHL 3. Cullin环连接酶（CRL）复合物，由支架CUL 3、底物衔接子KLHL 3， 和连接酶RING降解WNK。突变体CUL 3的作用，产生跳跃的外显子9， 引起57个氨基酸（CUL 3-CUL 9）的内部缺失，存在争议。CUL 3-CUL 9引发自身降解 在体外，以及在CUL 3 FHHt小鼠模型中。因此，流行的模型是，CUL 3-CUL 9导致FHHt， 诱导CUL 3单倍不足。我们在CUL 3杂合子小鼠中的初步数据和一种新的小鼠模型， CUL 3 - 109 FHHt不支持这一点，我们假设CUL 3 - 109发挥主导作用，导致FHHt 使血浆[K+]传感器失调。我们认为，CUL 3-CUL 9通过以下因素的联合作用引起FHHt： 降低自身和KLHL 3的丰度。我们的数据表明，NKCC 2激活沿着厚的上升 肢体（TAL）也可能导致FHHt。最后，我们之前产生了肾脏特异性CUL 3敲除（KO） 小鼠，并发现他们显示出严重的表型（多尿和慢性肾病），与缺陷沿着 多个肾单位节段。我们的总体目标是确定CUL 3-BUL 9介导的细胞凋亡的机制。 FHHt，并深入了解CUL 3在肾脏中的功能。在目标1中，我们将确定CUL 3-CUL 9的作用。 表达和CUL 3 KO特异性沿着DCT，以确定沿沿着DCT的CRL破坏是否足够 导致FHHT我们将在小鼠中确定CRL破坏对KLHL 3的影响，因为我们发现CUL 3-p53 9 在培养的细胞中不适当地降解它。我们还将直接测试具有较低丰度的 CUL 3和KLHL 3发育FHHt。在目标2中，我们将确定K+分泌节段的重塑是否 发生在由CUL 3-CUL 9介导的FHHt中，并检查CRL破坏对NKCC 2活性的影响。一些 模型表明，CUL 3-CUL 9导致CRL活性显著降低，但数据表明这将是致命的。 我们认为，CUL 3-CUL 9可能发挥独特的作用，使其优先降解某些CRL 适配器。因此，在目标3中，我们将在我们的研究中检查CUL 3-CUL 9对其他CRL衔接子和底物的影响。 小鼠模型和原代细胞培养中。

Project Summary The kidney plays a key role in maintaining plasma [K+], with distal segments of the nephron fine-tuning K+ secretion to keep it in the normal range. We previously proposed that the renal distal convoluted tubule (DCT) plays a key role by sensing plasma [K+]. Decreasing plasma [K+] by dietary K+ restriction activates the WNK- SPAK/OSR1-NCC pathway, and increased NaCl reabsorption though NCC reduces delivery of sodium to, and possibly remodels, distal K+ secreting segments to lower K+ secretion. The disease Familial Hyperkalemic Hypertension (FHHt) is caused by increased NCC activation due to mutations in WNKs, Cullin 3 (CUL3), and KLHL3. The Cullin Ring Ligase (CRL) complex, composed of the scaffold CUL3, the substrate adaptor KLHL3, and the ligase RING, degrades WNKs. The effects of mutant CUL3, produced by skipping of exon 9 which causes internal deletion of 57 amino acids (CUL3-∆9), are controversial. CUL3-∆9 triggers its own degradation in vitro, and also in a mouse model of CUL3 FHHt. Thus, the prevailing model is that CUL3-∆9 causes FHHt by inducing CUL3 haploinsufficiency. Our preliminary data in CUL3 heterozygote mice and a new mouse model of CUL3-∆9 FHHt do not support this, and we hypothesize that CUL3-∆9 exerts dominant effects to cause FHHt and dysregulate the plasma [K+] sensor. We propose that CUL3-∆9 causes FHHt by a combined effect of lowering abundance of itself and of KLHL3. Our data suggest that NKCC2 activation along the thick ascending limb (TAL) may also contribute to FHHt. Finally, we previously generated kidney-specific CUL3 knockout (KO) mice, and found that they display a severe phenotype (polyuria and chronic kidney disease), with defects along multiple nephron segments. Our overall aim is to determine the mechanisms underlying CUL3-∆9-mediated FHHt, and gain insight into CUL3 function in the kidney. In Aim 1 we will determine the effects of CUL3-∆9 expression and CUL3 KO specifically along DCT to determine whether CRL disruption along DCT is sufficient to cause FHHt. We will determine the effects of CRL disruption on KLHL3 in mice, since we found CUL3-∆9 inappropriately degrades it in cultured cells. We will also directly test whether mice with lower abundance of CUL3 and KLHL3 develop FHHt. In Aim 2 we will determine whether remodeling of K+-secreting segments occurs in FHHt mediated by CUL3-∆9, and examine the effects of CRL disruption on NKCC2 activity. Some models suggest that CUL3-∆9 leads to dramatically lower CRL activity, but data suggest this would be lethal. We propose that CUL3-∆9 may exert unique effects that cause it to preferentially degrade certain CRL adaptors. Therefore, in Aim 3 we will examine effects of CUL3-∆9 on other CRL adaptors and substrates in our mouse models and in primary cell culture.