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

项目概要 肾脏在维持血浆 [K ] 方面发挥着关键作用，肾单位的远端部分可微调 K 使其分泌量保持在正常范围。我们之前提出肾远曲小管（DCT） 通过感测等离子体发挥关键作用[K]。通过饮食 K 限制来降低血浆 [K ] 会激活 WNK- SPAK/OSR1-NCC 途径，尽管 NCC 减少了钠的输送，但增加了 NaCl 重吸收，并且 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) 是由于 WNK、Cullin 3 (CUL3) 和 NCC 突变导致 NCC 激活增加所致。 KLHL3。 Cullin 环连接酶 (CRL) 复合物，由支架 CUL3、底物接头 KLHL3 组成， 连接酶 RING 会降解 WNK。突变体 CUL3 的影响，由外显子 9 的跳跃产生， 导致 57 个氨基酸（CUL3-Δ9）内部缺失，这是有争议的。 CUL3-Δ9 触发自身退化 体外实验以及 CUL3 FHHt 小鼠模型中的实验。因此，普遍的模型是 CUL3-Δ9 通过以下方式导致 FHHt： 诱导 CUL3 单倍体不足。我们在 CUL3 杂合子小鼠和新小鼠模型中的初步数据 CUL3-Δ9 FHHt 不支持这一点，我们假设 CUL3-Δ9 发挥主导作用导致 FHHt 并使血浆 [K ] 传感器失调。我们认为 CUL3-Δ9 通过以下因素的综合作用导致 FHHt： 降低其自身和 KLHL3 的丰度。我们的数据表明 NKCC2 沿着厚层上升激活 肢体 (TAL) 也可能导致 FHHt。最后，我们之前生成了肾脏特异性 CUL3 敲除 (KO) 小鼠，发现它们表现出严重的表型（多尿和慢性肾病），并且存在缺陷 多个肾单位段。我们的总体目标是确定 CUL3-Δ9 介导的潜在机制 FHHt，并深入了解 CUL3 在肾脏中的功能。在目标 1 中，我们将确定 CUL3-Δ9 的影响 表达和 CUL3 KO 特异性地沿着 DCT 以确定沿着 DCT 的 CRL 破坏是否足够 导致 FHHt。我们将确定 CRL 破坏对小鼠 KLHL3 的影响，因为我们发现了 CUL3-Δ9 在培养细胞中不恰当地降解它。我们还将直接测试是否具有较低丰度的小鼠 CUL3 和 KLHL3 开发了 FHHt。在目标 2 中，我们将确定是否重塑 K 分泌片段 发生在 CUL3-Δ9 介导的 FHHt 中，并检查 CRL 破坏对 NKCC2 活性的影响。一些 模型表明 CUL3-Δ9 会导致 CRL 活性显着降低，但数据表明这将是致命的。 我们认为 CUL3-Δ9 可能会发挥独特的作用，导致其优先降解某些 CRL 适配器。因此，在目标 3 中，我们将检查 CUL3-Δ9 对我们的其他 CRL 接头和底物的影响 小鼠模型和原代细胞培养物。

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.