Zhang Mingzhuo, Liu Mingda, Ren Zhiyun, Wang Weiwan, Upadhyay Kiran, Asico Laureano Asico, Armando Ines, Jia Yutao, Wang Ping, Xue Ying, Wang Xiaoyan
bioRxiv. 2024 Feb 16:2024.02.14.580405. doi: 10.1101/2024.02.14.580405.
The thiazide-sensitive sodium chloride cotransporter (NCC) is the major apical sodium transporter located in the mammalian renal distal convoluted tubule (DCT). The amount of sodium reabsorbed in the DCT through NCC plays an important role in the regulation of extracellular fluid volume and blood pressure. Dopamine and its receptors constitute a renal antihypertensive system in mammals. The disruption of Drd4 in mice causes kidney-related hypertension. However, the pathogenesis of D4R-deficiency associated hypertension is not well documented.
We assessed the effects of D4R on NCC protein abundances and activities of DCT in mice with renal or global Drd4-deficiencies and expressing human D4.7 variant and in cultured mouse DCT cells, and explored the molecular mechanism.
NCC inhibitor hydrochlorothiazide enhanced the natriuresis in Drd4-/- mice. Renal NCC protein was greater while ubiquitination of NCC was less in Drd4-/- than Drd4+/+ mice. Silencing of D4R in cultured mouse DCT cells increased NCC protein but decreased NCC ubiquitination. D4R agonist had opposite effects that were blocked by the antagonist. In mouse kidneys and DCT cells D4R and NCC colocalized and co-immunoprecipitated. Moreover, D4R-agonist promoted the binding between the two proteins demonstrated by fluorescence resonance energy transfer. D4R agonism internalized NCC, decreased NCC in the plasma membrane, increased NCC in lysosomes and reduced NCC-dependent-intracellular-sodium transport. The lysosomal inhibitor chloroquine prevented the D4R-induced NCC-reduction. A shortened NCC half-life was suggested by its decay under cycloheximide-chase. Ubiquitin-specific-protease 48 (USP48, a deubiquitinating enzyme) was increased in the kidneys and cells with Drd4-deficiency while D4R stimulation decreased it in vitro and reduction of USP48 with siRNA decreased NCC expression. The mice carrying human D4.7 variant or with renal supcapsular-Drd4-siRNA-delivery developed hypertension with increased NCC.
Our data demonstrates that D4R downregulates NCC by promoting USP48-associated deubiquitination and subsequent internalization, lysosome relocation and degradation.
噻嗪类敏感的氯化钠协同转运蛋白（NCC）是位于哺乳动物肾远曲小管（DCT）的主要顶端钠转运蛋白。通过NCC在DCT中重吸收的钠量在细胞外液容量和血压调节中起重要作用。多巴胺及其受体构成哺乳动物的肾性抗高血压系统。小鼠中Drd4的破坏会导致与肾脏相关的高血压。然而，D4R缺乏相关高血压的发病机制尚未得到充分记录。
我们评估了D4R对肾或全身Drd4缺乏且表达人D4.7变体的小鼠以及培养的小鼠DCT细胞中NCC蛋白丰度和DCT活性的影响，并探索了分子机制。
NCC抑制剂氢氯噻嗪增强了Drd4-/-小鼠的利钠作用。与Drd4+/+小鼠相比，Drd4-/-小鼠的肾脏NCC蛋白含量更高，而NCC的泛素化程度更低。在培养的小鼠DCT细胞中沉默D4R会增加NCC蛋白，但会降低NCC泛素化。D4R激动剂具有相反的作用，且被拮抗剂阻断。在小鼠肾脏和DCT细胞中，D4R与NCC共定位并共免疫沉淀。此外，D4R激动剂通过荧光共振能量转移促进了这两种蛋白质之间的结合。D4R激动作用使NCC内化，降低了质膜中的NCC，增加了溶酶体中的NCC，并减少了NCC依赖性细胞内钠转运。溶酶体抑制剂氯喹可防止D4R诱导的NCC减少。在放线菌酮追踪下NCC的衰减提示其半衰期缩短。泛素特异性蛋白酶48（USP48，一种去泛素化酶）在Drd4缺乏的肾脏和细胞中增加，而D4R刺激在体外使其减少，用siRNA降低USP48会降低NCC表达。携带人D4.7变体或肾包膜下递送Drd4-siRNA的小鼠出现高血压，且NCC增加。
我们的数据表明，D4R通过促进USP48相关的去泛素化以及随后的内化、溶酶体重新定位和降解来下调NCC。
