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

CUL3-WNK-SPAK 通路对肾离子转运的调节

• 批准号: 10544339
• 负责人: JAMES A MCCORMICK
• 金额: $ 33.88万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10544339
• 关键词: Address; Affect; Amino Acids; Animals; Autophagocytosis; Binding; Blood Pressure; Cardiac Myocytes; Cells; Chronic Kidney Failure; Complex; Cullin Proteins; Cultured Cells; Data; Defect; Development; Dietary Potassium; Disease; Disputes; Distal; Distal convoluted renal tubule structure; Equilibrium; Exons; Familial disease; Functional disorder; Gitelman syndrome; Goals; Heart failure; Heterozygote; Homeostasis; Human; Hypertension; Hypokalemia; Immunofluorescence Immunologic; In Vitro; Ion Transport; Kidney; Knock-out; Knockout Mice; Knowledge; Ligase; Limb structure; Maintenance; Mediating; Messenger RNA; Modeling; Molecular; Mus; Mutation; Nephrons; Neurons; Normal Range; Pathway interactions; Phenotype; Phosphorylation; Phosphotransferases; Physiology; Plasma; Play; Polyuria; Potassium; Primary Cell Cultures; Progress Reports; Proteins; Rare Diseases; Regulation; Role; SLC11A2 gene; Scaffolding Protein; Skeletal Muscle; Sodium; System; Testing; Thick; Ubiquitination; Work; absorption; blood pressure regulation; cullin-3; driving force; exon skipping; extracellular; familial hyperkalemic hypertension; gain of function; hyperkalemia; in vivo; inhibitor; insight; loss of function; meter; mouse model; mutant; novel therapeutics; renal epithelium; scaffold; sensor; symporter; ubiquitin ligase

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.

项目摘要 肾脏在维持血浆[K+]方面起着关键作用，肾单位的远端节段微调K+ 分泌，使其保持在正常范围内。我们先前提出了肾脏远端曲管(DCT) 通过感知血浆[K+]起关键作用。膳食K+限制降低血浆[K+]激活WNK- Spak/OSR1-NCC途径，并增加盐的重吸收，尽管NCC减少了钠向 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WNKS、cullin 3(CUL3)和 KLHL3.该CRL复合体由支架CUL3、底物适配器KLHL3、 而连接酶环则降解WNKS。跳过外显子9产生的突变体CUL3的作用 导致57个氨基酸(CUL3-∆9)的内部缺失，是有争议的。CUL3-∆9引发自身降解 在体外，也在CUL3FHHt小鼠模型中。因此，流行的模型是CUL3-∆9通过以下方式引起FHHT 导致CUL3单倍体不足。我们在CUL3杂合子小鼠和一种新的CUL3杂合子小鼠模型中的初步数据 CUL3-∆9 FHHT不支持这一点，我们假设CUL3-∆9发挥显性效应导致FHHT 并使血浆[K+]感受器失调。我们认为CUL3-∆9通过共同作用引起FHHT。 降低自身和KLHL3的丰度。我们的数据表明，NKCC2沿着厚厚的上升方向被激活 肢体(TAL)也可能参与FHHt。最后，我们之前生成了肾脏特异的CUL3基因敲除(KO) 小鼠，发现它们表现出严重的表型(多尿和慢性肾脏疾病)，并伴随着缺陷 多个肾单位节段。我们的总体目标是确定CUL3-∆9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Cl3-∆9的效果 在DCT上特异性地表达和CUL3KO，以确定沿着DCT的CRL中断是否足够 导致了FHHT。我们将确定CRL中断对小鼠KLHL3的影响，因为我们发现了CUL3-∆9 在培养的细胞中不适当地降解它。我们还将直接测试低丰度的小鼠 CUL3和KLHL3形成FHHt。在目标2中，我们将确定K+分泌节段的重塑 发生在CUL3-∆9介导的FHHt中，并检测CRL中断对NKCC2活性的影响。一些人 模型表明，CUL3-∆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CUL3-∆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，我们将检查Cl3-∆9对我们的 小鼠模型和原代细胞培养。

项目成果

Regulation of the water channel aquaporin-2 by cullin E3 ubiquitin ligases.

cullin E3 泛素连接酶对水通道 aquaporin-2 的调节。

• DOI: 10.1152/ajprenal.00049.2024
• 发表时间: 2024
• 期刊: American journal of physiology. Renal physiology
• 作者: Murali,SathishK;McCormick,JamesA;Fenton,RobertA
• 通讯作者: Fenton,RobertA

Cullin 3 mutant causing familial hyperkalemic hypertension lacks normal activity in the kidney.

Cullin 3 突变体导致家族性高钾性高血压，肾脏缺乏正常活动。

• DOI: 10.1152/ajprenal.00153.2022
• 发表时间: 2022
• 期刊: American journal of physiology. Renal physiology
• 作者: Maeoka,Yujiro;Cornelius,RyanJ;Ferdaus,MohammedZubaerul;Sharma,Avika;Nguyen,LuanT;McCormick,JamesA
• 通讯作者: McCormick,JamesA

Potassium Effects on NCC Are Attenuated during Inhibition of Cullin E3-Ubiquitin Ligases.

• DOI: 10.3390/cells11010095
• 发表时间: 2021-12-29
• 期刊: Cells
• 影响因子: 6
• 作者: Murali SK;Little R;Poulsen SB;Ferdaus MZ;Ellison DH;McCormick JA;Fenton RA
• 通讯作者: Fenton RA

The CUL3/KLHL3-WNK-SPAK/OSR1 pathway as a target for antihypertensive therapy.

• DOI: 10.1152/ajprenal.00132.2016
• 发表时间: 2016-06
• 期刊: American journal of physiology. Renal physiology
• 作者: Mohammed Z. Ferdaus;J. McCormick

A five amino acids deletion in NKCC2 of C57BL/6 mice affects analysis of NKCC2 phosphorylation but does not impact kidney function.

• DOI: 10.1111/apha.13705
• 发表时间: 2021-09
• 期刊: Acta physiologica (Oxford, England)
• 作者: Moser S;Sugano Y;Wengi A;Fisi V;Lindtoft Rosenbaek L;Mariniello M;Loffing-Cueni D;McCormick JA;Fenton RA;Loffing J
• 通讯作者: Loffing J