肾远曲小管(DCT)中的噻嗪敏感性氯化钠共转运蛋白(NCC)在调节血压(BP)和K稳态中起着至关重要的作用。在高钾血症期间,减少的NCC磷酸化和总NCC丰度促进下游电K分泌和BP减少。然而,K+依赖性降低总NCC水平的机制尚不清楚。这里,我们显示,NCC水平在体外肾小管中降低,在高K+培养基中孵育24-48小时。这种降低与NCC转录无关,但使用蛋白酶体(MG132)或溶酶体(氯喹)的抑制剂被阻止。离体,高K+增加NCC泛素化,但是抑制泛素偶联途径阻止了高K+介导的NCC蛋白的减少。在体外在高K培养基中孵育的小管中或在高K饮食喂养4天的小鼠的肾皮质中,热休克蛋白70(Hsp70)的丰度和磷酸化,泛素依赖性蛋白质降解和蛋白质折叠的关键调节因子,减少了。相反,在类似的样本中,PP1α的表达,已知去磷酸化Hsp70,也增加。NCC与Hsp70和PP1α共免疫沉淀,并抑制其作用可防止高K介导的总NCC水平降低。总之,我们表明高钾血症通过Hsp70促进的PP1α依赖性过程驱动NCC泛素化和降解。该机制促进NCC的K依赖性降低以保护血浆K稳态并潜在地降低BP。
The thiazide-sensitive sodium-chloride cotransporter (NCC) in the renal distal convoluted tubule (DCT) plays a critical role in regulating blood pressure (BP) and K+ homeostasis. During hyperkalemia, reduced NCC phosphorylation and total NCC abundance facilitate downstream electrogenic K+ secretion and BP reduction. However, the mechanism for the K+-dependent reduction in total NCC levels is unknown. Here, we show that NCC levels were reduced in ex vivo renal tubules incubated in a high-K+ medium for 24-48 h. This reduction was independent of NCC transcription, but was prevented using inhibitors of the proteasome (MG132) or lysosome (chloroquine). Ex vivo, high K+ increased NCC ubiquitylation, but inhibition of the ubiquitin conjugation pathway prevented the high K+-mediated reduction in NCC protein. In tubules incubated in high K+ media ex vivo or in the renal cortex of mice fed a high K+ diet for 4 days, the abundance and phosphorylation of heat shock protein 70 (Hsp70), a key regulator of ubiquitin-dependent protein degradation and protein folding, were decreased. Conversely, in similar samples the expression of PP1α, known to dephosphorylate Hsp70, was also increased. NCC coimmunoprecipitated with Hsp70 and PP1α, and inhibiting their actions prevented the high K+-mediated reduction in total NCC levels. In conclusion, we show that hyperkalemia drives NCC ubiquitylation and degradation via a PP1α-dependent process facilitated by Hsp70. This mechanism facilitates K+-dependent reductions in NCC to protect plasma K+ homeostasis and potentially reduces BP.