肾小管钠氯共转运子和BK通道介导的非醛固酮依赖的快速排钾机制研究

Urinary potassium (K+) excretion by distal renal tubule, which is regulated by aldosterone, is crucial to maintain body potassium balance. However, substantial evidence of recent research showed that kaliuresis after high K+ diet occurred much faster than aldosterone's effect could happen. Our previous study discovered that increased extracellular K+ concentration after high K+ diet caused membrane depolarization through basolateral kir4.1 channel and subsequent inhibited chloride efflux in distal convoluted tubule (DCT) cells, and then increased intracellular chloride concentration dephosporylated WNK4, which in turn dephosporylating SPAK/OSR1 and NCC. The quick NCC dephosphorylation is considered to facilitate luminal electrogenic Na+ absorption and K+ excretion in downstream connecting tubule (CNT) and collecting duct (CD) where epithelial sodium channel (ENaC) and renal outer medullary potassium channel (ROMK) are expressed. We also found different cullin3 (Cul3) neddylation level, accompanied with varying WNK4 abundance under different K+ diets. Cul3 neddylation level is associated with its degradation activity and can finally cause modified WNK4 abundance and NCC activity according to our previous study. Both abundance and phosporylation of WNK4 are demonstrated to be sufficient to regulate NCC function. Thus, our hypothesis is, the membrane depolarization induced by high K+ diet through kir4.1 happens not only in DCT, causing NCC dephosporylation, but also happens in downstream CNT and CD where another K+ excretion channel, BK is expressed in intercalated cells. BK should be activated quickly by the membrane depolarization and increased urine flow resulting from NCC inactivation. It is of great importance to avoid hyperkalemia in early period after high K+ diet. This is a direct effect, so it could happen faster than ROMK activation stimulated by aldosterone in principle cells. We also hypothesize that extracellular K+ can influence the neddylation level of Cul3, which through affected WNK4 abundance, finally magnifies NCC functional change caused by WNK4 phosporylation after high K+ or low K+ diet. Our preliminary data, especially of kidney–specific slo1 (BK) knockout mice we generated fully support the hypothesis we proposed. As shown here, we plan to verify the role of NCC and BK on rapid aldosterone-independent kaliuresis in multiple systems including MDCK cells, kidney slice, and mice, and seek for the mechanism of NCC regulation by Cul3 neddylation change under different extracellular K+ concentrations as well. This study will be important to clarify the subtle mechanism of electrolyte reabsorption and excretion in distal tubule, and shed light on the pathogenesis of essential hypertension.

远端肾小管的钾排泄对维持机体钾平衡至关重要，这一过程受醛固酮调节。然而高钾饮食后早在醛固酮激活之前，尿钾排泄已经显著增加。我们既往发现，高血钾可以通过细胞膜去极化导致肾远曲小管的钠氯共转运子（NCC）迅速去磷酸化失活，以促进下游肾小管排钾；不同钾饮食喂养小鼠呈现出不同的泛素化支架蛋白Cul3的类泛素化修饰程度，并出现WNK激酶丰度的变化，后者可调节NCC功能。据此我们提出假说：高钾饮食后远端肾小管上皮细胞膜去极化和NCC失活迅速激活BK通道排钾；血钾通过影响Cul3的类泛素化修饰，进一步维持NCC活性以利于下游排钾或保钾。我们的前期研究，包括建立的肾特异BK敲除小鼠完全支持该假说。本项目拟从分子、细胞、组织以及动物水平多层次明确NCC和BK介导高钾饮食后早期的尿钾排泄，探索血钾影响Cul3类泛素化修饰以调控NCC的机制，为最终阐明远端肾小管的离子重吸收机制，乃至高血压的发生机制提供新证据。

远端肾小管的钾排泄对维持机体钾平衡至关重要。高血钾可以通过细胞膜去极化导致肾远曲小管的WNK激酶失活，钠氯共转运子（NCC）活性下调以促进下游肾小管排钾。我们在稳定表达BK的细胞株和离体肾切片研究了BK通道在高钾时的活性，结果提示NCC活性下调的同时BK的表达并无变化。.糖尿病患者具有明显的高血压倾向，我们建立了1型和2型糖尿病小鼠模型，发现其肾脏WNK4-SPAK/OSR1-NCC活性亢进，负责降解WNK激酶的Cul3的neddylation显著增加，造成其连接蛋白KLHL3的泛素化降解增加。糖尿病小鼠主动脉表达CUL3以及其neddylation也明显增加，而连接蛋白KLHL2、RhoBTB1表达下降，造成其血管底物WNK3、RhoA/ROCK降解受阻，从而促进血管收缩。提示高糖，或同时存在高胰岛素的情况下，Cul3的neddylation增强，导致自身连接蛋白的降解，从而WNK激酶、RhoA/ROCK等底物堆积，导致肾小管NCC活性增强，大血管阻力增加，导致糖尿病时的高血压倾向。.我们还发现在先兆子痫患者的脐动脉和胎盘中，CRL3的关键成员Cullin3与它的Adaptor表达均显著下降。通过应用L-NAME干预小鼠模拟妊高症，发现L-NAME组孕鼠大动脉组织、肾脏小血管以及肾小管中Cullin3的丰度及neddylation均显著下降，其相应的Adaptors表达也下降。我们的研究提示，先兆子痫时机体处于缺血缺氧的状态导致PPARγ的表达下调和去泛素化水平增加，进而影响了Cullin3的丰度及活性。CRL3的功能不全导致了包括WNK激酶堆积，促进了血管的增殖及收缩，并导致钠离子的潴留、及远端小管的重塑。