醛固酮-WNK4途径参与盐负荷对上皮钠通道的调节

目的 通过建立生理条件下的盐负荷饮食大鼠模型,观察醛固酮和WNK4在水盐代谢调节中的作用。 方法 将SD大鼠分为5组：高盐组（H,4% NaCl）、正常盐组（N,0.4% NaCl）、低盐组（L,0.07% NaCl）、高盐加醛固酮组（H+A,4% NaCl+1 mg&#8226;kg-1&#8226;d-1醛固酮）、低盐加螺内酯（L+S,0.07% NaCl+0.1 g&#8226;kg-1&#8226;d-1螺内酯）,所有大鼠自由饮水,喂养2周。用放射免疫法检测血浆醛固酮的变化。应用实时定量PCR和Western印迹法检测大鼠肾脏上皮钠通道γ亚基（γENaC）、WNK4的mRNA和蛋白的变化。 结果 H组大鼠血浆醛固酮水平低于N组（P < 0.05）,H+A组高于H组（P < 0.05）;L组大鼠血浆醛固酮水平高于N组（P < 0.05）,显示SD大鼠造模成功。L组大鼠肾脏γENaC蛋白表达高于N组,但是L+S低于L组;同时H组低于N组,H+A组高于H组,差异均有统计学意义（P < 0.05）。mRNA变化趋势和蛋白变化趋势一致。H组肾脏WNK4的蛋白表达高于N组,但是H+A组低于H组;同时L组低于N组,L+S组高于L组,差异均有统计学意义（P < 0.05）。mRNA的变化趋势和蛋白的变化趋势一致。 结论 饮食中的盐可以调节γENaC在肾脏的蛋白表达,醛固酮和WNK4都参与了机体对盐的调节,WNK4受到醛固酮的负调节作用。     

原发性肝癌患者外周血中IL-32表达水平及意义研究

目的：探讨原发性肝癌患者外周血中IL-32表达水平及临床意义。 方法：选择40例原发性肝癌患者（肝癌组）与20例健康体检者（对照组）,收集两组的外周血,用TRIzol试剂提取外周血中淋巴细胞总RNA,分别用real-time PCR与ELISA检测IL-32 mRNA与蛋白的表达,分析肝癌患者IL-32表达水平与AFP表达水平的相关性。 结果：肝癌组患者外周血中IL-32表达在RNA及蛋白质水平均高于对照组（均P<0.05）;原发性肝癌患者外周血中IL-32表达水平与AFP表达水平呈正相关（r=0.583,P<0.05）。 结论：原发性肝癌患者外周血中IL-32表达增高,IL-32可能与原发性肝癌的发病密切相关。     

内毒素诱导内皮细胞粘附分子表达的意义

目的　研究内毒素 (LPS)刺激下内皮细胞粘附分子 (ICAM 1)表达的规律及信号调节机制。　方法　 (1)在不同剂量和时间 ,用LPS刺激培养的人脐静脉血管内皮细胞株ECV 30 4 ,从mRNA水平观察ICMA 1的诱导表达规律 ;(2 )以信号通路阻断剂预处理细胞 30min后再行LPS刺激 ,从mRNA及蛋白水平观察不同信号通路对ICAM 1诱导表达的调节作用。　结果　 (1) 10 0 pg/mlLPS刺激细胞 6h就可以诱导ICAM 1mRNA的表达 ,随刺激浓度增加 ,ICAM 1mRNA表达增强 ,10 0～ 10 0 0ng/mlLPS具有最大的诱导效应。 6～ 8h是mRNA表达高峰 ,12h以后表达仍处于较高水平。 (2 )核因子 κB(NF κB)抑制剂PSI能显著抑制ICAM 1mRNA及蛋白表达 ;细胞外信号调节激酶 1/ 2 (ERK1/ 2 )丝裂原活化蛋白激酶 (MAPK)抑制剂PD980 5 9及p38MAPK抑制剂SB2 0 35 80 ,均能在mRNA及蛋白水平部分抑制ICAM 1的表达。　结论　LPS以剂量 时间依赖方式诱导内皮细胞ICAM 1mRNA表达 ,NF κB是调节ICAM 1表达的主要信号通路 ,p38、ERK1/ 2是调节ICAM 1表达的次要信号通路     

高血压肾损伤患者外周血单个核细胞PPARγ和NF-κB的mRNA表达及其与心血管重构的相关性

目的探讨高血压肾损伤患者外周血单个核细胞过氧化物酶体增殖物激活受体γ(PPARγ)和核因子-κB(NF-κB)的mRNA表达及其与心血管重构的相关性。 方法选择2016年5月至2019年5月本院收治的高血压患者,65例为单纯高血压组,50例高血压肾损伤患者为高血压肾损伤组,健康者50例为对照组。测定各组受试者的外周血单个核细胞PPARγ mRNA、NF-κB mRNA表达;应用心脏和血管超声分别检测颈动脉及心脏的重构指标：颈动脉平均内膜中层厚度(IMT)、颈动脉斑块积分、左心室质量指数(LVMI)。应用Pearson方法分析高血压肾损伤患者外周血单个核细胞PPARγ mRNA、NF-κB mRNA表达水平与心血管重构指标的相关性,多元线性回归分析高血压肾损伤患者IMT的影响因素。 结果与对照组相比,单纯高血压组、高血压肾损伤组患者的IMT、斑块积分、LVMI和NF-κB mRNA表达水平明显升高,而PPARγ mRNA表达水平均明显降低(P<0.05)。高血压肾损伤组IMT、斑块积分、LVMI各指标和NF-κB mRNA表达水平均明显高于单纯高血压组,PPARγ mRNA表达水平明显低于单纯高血压组(P<0.05)。高血压肾损伤患者PPARγ mRNA表达水平均与IMT、斑块积分、LVMI呈负相关关系(均P<0.05);NF-κB mRNA表达水平均与IMT、斑块积分、LVMI均呈正相关关系(P均<0.05)。PPARγ mRNA降低、NF-κB mRNA升高是高血压肾损伤患者IMT的影响因素(均P<0.05)。 结论高血压肾损伤患者外周血PPARγ mRNA表达明显降低,NF-κB mRNA表达明显升高,二者均与心血管重构有相关性。     

内毒素诱导大鼠肝细胞白蛋白表达下降的分子机制

目的　探讨内毒素诱导肝细胞白蛋白表达下降的分子机制。方法　 1μg/ml内毒素刺激肝细胞后 ,分别在 0 ,2 ,8,12和 2 4h时留取肝细胞及上清检测白蛋白mRNA及其蛋白水平的变化。细胞内信号蛋白p38激酶和ERK激酶的特异性阻断剂SB2 0 35 80和PD980 5 9预处理肝细胞后检测上清中白蛋白的浓度。结果　内毒素刺激后 2 4h白蛋白mRNA下降约为 30 % ,与此同时白蛋白浓度下降约 5 0 %。SB2 0 35 80和PD980 5 9可以在体外抑制内毒素诱导肝细胞白蛋白表达的下降。结论　内毒素在转录水平抑制肝细胞白蛋白mRNA的表达来抑制白蛋白的合成。这一过程与细胞内信号传导通路p38和ERK激酶密切相关 ,进一步阐明了感染时低白蛋白血症的分子机制。     

WNK4激酶通过溶酶体途径抑制BK通道表达

目的 研究WNK4激酶对BK通道的调节作用及机制.方法 将BK和WNK4野生型(WNK4-WT)或CD4(对照)质粒DNA共同转染进Cos-7细胞中,采用免疫染色-共聚焦激光显微镜、化学发光法、Western印迹法检测BK在细胞上的分布、细胞膜表面蛋白和总蛋白的表达;并使用质子泵抑制剂bafilomycin A1( Baf A1)阻断溶酶体降解检测BK蛋白表达水平的减少是否由于其蛋白降解增多所致.结果 免疫染色-共聚焦激光显微镜发现,与对照组相比,WNK4-WT组BK在细胞膜表面的分布明显减少.化学发光法检测结果显示,对照组BK的细胞膜表面蛋白表达水平为299.9±18.6,WNK4-WT组中其细胞膜表面蛋白表达水平为148.4±13.7,比对照组显著下降(P＜0.01).Western印迹结果提示,WNK4-WT组BK的总蛋白表达水平比对照组明显减少.和对照组(100％)相比,WNK4-WT显著减少BK的总蛋白水平(42.3％±15.2％,P＜0.01),而Baf A1则逆转WNK4-WT对BK蛋白的抑制作用(82.2％±12.1％,P＜0.05).结论 WNK4激酶能同时抑制BK在Cos-7细胞膜表面蛋白和总蛋白的表达水平;WNK4激酶抑制BK通道蛋白的表达是通过增加其在溶酶体内的降解所致的.     

限钠对肾血管性高血压的作用

晚近提出对中度高血压患者适量限止钠摄入能有效降低血压。对严重高血压更应严格限钠。肾血管性高血压是肾素依赖型高血压的典型例子,可以想象,钠缺乏可刺激肾素,从而使血压升高,故低钠饮食对     

肾结石大鼠钠/二羧基转运蛋白1的表达及枸橼酸钾防治机制的研究

目的 研究肾组织钠／二羧基转运蛋白1（SDCT1）与低枸橼酸尿的关系以及枸橼酸钾的干预作用，探讨肾结石发病的分子机制和防治措施。方法 雄性Wistar大鼠分为对照组、肾结石组及枸橼酸钾干预组。血、尿枸橼酸和草酸采用酶法测定，Northern blot检测大鼠肾组织SDCT1mRNA水平的改变，免疫组织化学观察SDCT1在肾组织的分布及表达变化。结果 与对照组比较，肾结石组第3天尿草酸水平显著升高，枸橼酸水平显著降低，同时肾组织SDCT1mRNA及其蛋白水平上调。第7天SDCT1mRNA及其表达产物增加更为显著，同时尿枸橼酸水平进一步降低，尿钙排泄显著增加，87.5%大鼠有中－大量的草酸钙结石形成。第14天上述改变更为明显，结石形成率达100％。枸橼酸钾干预组各时间点尿草酸水平与肾结石组差异无显著性意义，但尿枸橼酸水平显著高于肾结石组及对照组，肾组织SDCT1mRNA及蛋白表达显著低于肾结石组，与对照组差异无显著性意义；结石形成率显著低于肾结石组；肾小管扩张、炎细胞浸润等病变也明显减轻。结论 肾组织SDCT1表达上调可能是低枸橼酸尿的重要原因，与肾结石的形成有密切关系。枸橼酸钾可下调肾结石大鼠肾组织SDCT1的表达，对肾结石的形成具有明显的干预作用。     

Liddle综合征1例及国内文献复习

目的 了解我国Liddle综合征的临床特点及探讨其早期诊断,提高临床认识水平.方法 回顾性分析本院1例Liddle综合征及国内杂志发表文献共23篇,总共58例的临床资料.结果 男37例,女21例,比例为1.8:1,年龄14～71岁,平均(31.4±15.2)岁,首发症状,高血压39.7％ (23/58),乏力、低钾临床表现8.6％(5/58),同时出现高血压、低血钾12.1％ (7/58),无症状体检发现39.7％ (23/58)；发病年龄10～59岁,平均(22.3±10.4)岁,发病到确诊时间0～30年,平均(8.2±9.6)年；血压130～290/70～240mmHg,平均(收缩压195.96 ±39.66,舒张压123.67±29.1 mmHg)；合并脑卒中20.7％ (12/58)；10.3％ (6/58)仅低钠饮食或补钾即维持正常血压和血钾,55.2％( 32/58)低钠饮食、氨苯蝶啶及补钾后可控制血压和血钾,13.8％ (8/58)通过低钠饮食、阿米洛利及补钾治疗,15.5％(9/58)患者需要加用其他降压药方能控制血压.结论 Liddle综合征是一种可以得到很好治疗和控制的疾病,早发现、早诊断、早治疗可避免严重并发症的发生.     

胃癌外周血TWA1 mRNA的表达与预后价值

目的检测胃癌（GC）患者外周血TWA1 mRNA表达水平，分析其与患者长期预后的关系。 方法选择2016年6月至2017年6月于西宁市第二人民医院诊治的97例GC患者，实时荧光定量PCR（RT-PCR）检测外周血TWA1表达量，随访并记录患者无进展生存期（PFS）。受试者工作特征（ROC）曲线判定TWA1 mRNA的阈值，分组比较TWA1 mRNA不同表达患者的PFS情况。Cox回归模型评估TWA1 mRNA水平对GC患者预后的预测价值。 结果随访（20.91±5.76）个月，随访率为94.79%（91/94），平均PFS（21.83±5.13）个月。TWA1 mRNA对GC患者PFS判定的曲线下面积为0.781（95% CI：0.687~0.875，P<0.001），截断值为1.27。TWA1 mRNA高表达患者的PFS显著短于低表达患者（χ²=14.415，P<0.01）。淋巴结转移（HR=3.328，95% CI：2.098~5.971，P=0.044）、TNM Ⅲ~Ⅳ期（HR=3.400，95% CI：1.114~4.795，P=0.011）及外周血TWA1 mRNA高表达（HR=7.429，95% CI：1.711~32.263，P=0.007）是影响患者PFS的独立危险因素。 结论外周血TWA1的表达与GC患者PFS显著相关，TWA1 mRNA水平对GC患者随访预后情况具有较好的预测价值。     

Cotransporters, WNKs and hypertension: an update

PURPOSE OF REVIEW: Studies of inherited conditions characterized by high or low blood pressure reveal the importance of a new signalling cascade, With no Lysine kinases (WNK) --> ste20/SPS1-related proline/alanine-rich kinase (SPAK)/oxidative stress-responsive kinase-1 (OSR1) --> Cation-Chloride Cotransporters (CCC), in regulating blood pressure and in the pathogenesis of essential hypertension. This review explores how these molecules interact to co-ordinate sodium homeostasis and how errors in these interactions may result in hypertension. RECENT FINDINGS: Studies using transgenic animals and gene knockins have clarified the role of mutant WNK4 in hypertension, by revealing its main action to be increasing the expression and activity of sodium-chloride cotransporter (NCC) in the kidney. Functional studies show how phosphorylation of WNK1 regulates both its activity and ability to interact with SPAK/OSR1, and clearly place it upstream of SPAK/OSR1 in the cascade. The structural basis for the interactions between SPAK/OSR1 and targets has been identified. SUMMARY: WNKs, activated by upstream kinases or autophosphorylation, bind and phosphorylate SPAK/OSR1, which in turn phosphorylate and activate NCCs and Na-K-Cl cotransporters (NKCCs). This increases sodium retention in the kidney (NKCC2, NCC) and vascular resistance (NKCC1), but decreases renin release (NKCC1). Hypertension-associated mutant WNKs increase surface expression and activation of renal tubular NKCC2 and NCC. Whether this adequately explains the hypertension awaits studies of these mutants in other tissues.     

WNK kinases and essential hypertension

PURPOSE OF REVIEW: The present review summarizes recent literature and discusses the potential roles of WNKs in the pathogenesis of essential hypertension. RECENT FINDINGS: WNKs (with-no-lysine [K]) are a recently discovered family of serine-threonine protein kinases with unusual protein kinase domains. The role of WNK kinases in the control of blood pressure was first revealed by the findings that mutations of two members, WNK1 and WNK4, cause Gordon's syndrome. Laboratory studies have revealed that WNK kinases play important roles in the regulation of sodium and potassium transport. Animal models have been created to unravel the pathophysiology of sodium transport disorders caused by mutations of the WNK4 gene. Potassium deficiency causes sodium retention and increases hypertension prevalence. The expression of WNK1 is upregulated by potassium deficiency, raising the possibility that WNK1 may contribute to salt-sensitive essential hypertension associated with potassium deficiency. Associations of polymorphisms of WNK genes with essential hypertension in the general population have been reported. SUMMARY: Mutations of WNK1 and WNK4 cause hypertension at least partly by increasing renal sodium retention. The role of WNK kinases in salt-sensitive hypertension within general hypertension is suggested, but future work is required to firmly establish the connection.     

Mechanism of regulation of renal ion transport by WNK kinases

PURPOSE OF REVIEW: This review summarizes recent advances in the understanding of the mechanism of regulation of renal ion transport by WNK kinases. RECENT FINDINGS: There are four mammalian WNK [with-no-lysine (K)] kinases: WNK1-WNK4. Mutations of WNK1 and WNK4 in humans cause hypertension and hyperkalemia at least partly by altering renal sodium and potassium transport. WNK1 and WNK4 stimulate endocytosis of ROMK1 by recruiting an endocytic scaffold protein, intersectin. The recruitment is independent of the kinase activity and occurs between the PXXP motif of WNKs and the SH3 domain of intersectin. Regulation of cation-chloride-coupled cotransporters, Na+-K+-2Cl(-) cotransporter (NKCC) 1 and NKCC2 [and the Na-Cl co-transporter (NCC), under some conditions] by WNKs requires kinase activity. WNK1 and WNK4 bind with and phosphorylate two Ste20-related protein kinases, OSR1 and SPAK, which in turn bind with and phosphorylate NKCCs and NCC to increase their activity. Binding of OSR1/SPAK to upstream activators (WNKs) and downstream substrates (NKCCs and NCC) are both mediated by a docking site in the C-terminus of OSR1/SPAK and RFX[V/I] motifs present in WNKs or in NKCCs and NCC. SUMMARY: WNKs regulate ion transport via both catalytic and noncatalytic mechanisms. We discuss hypotheses that WNKs, contrasting with aldosterone, play important roles in dissociating sodium reabsorption from potassium secretion.     

Mechanisms of disease: WNK-ing at the mechanism of salt-sensitive hypertension

Potassium deficiency is associated with an increased prevalence of hypertension. Increasing potassium intake lowers blood pressure via an unknown mechanism. WNK (with no lysine) kinases are a novel family of large serine/threonine protein kinases. A large deletion from the first intron of the WNK1 gene results in increased levels of expression of WNK1 and causes Gordon's syndrome, of which hypertension and hyperkalemia are features. WNK1 activates the Na(+)/Cl(-) cotransporter NCC and the epithelial Na(+) channel ENaC, and inhibits the renal K(+) channel ROMK. Enhanced Na(+) reabsorption and inhibition of K(+) secretion resulting from increased WNK1 expression probably contribute to hypertension and hyperkalemia in Gordon's syndrome. Here, we review the role of dietary K(+) deficiency in the pathogenesis of salt-sensitive hypertension and summarize recent findings indicating that WNK1 might mediate renal Na(+) retention and hypertension in K(+) deficiency.     

WNK kinases regulate sodium chloride and potassium transport by the aldosterone-sensitive distal nephron

With-No-Lysine [K] (WNKs) are a recently discovered family of serine/threonine protein kinases that contain a uniquely structured catalytic domain. Mutations in the genes encoding two family members, WNK1 and WNK4, cause a chloride-dependent, thiazide-sensitive inherited syndrome of hypertension and hyperkalemia. Over the past 5 years, physiologic studies have demonstrated that these proteins regulate transcellular and paracellular epithelial ion flux. In this mini review, we discuss WNK1 and WNK4 gene products and their regulatory effects on sodium chloride and potassium handling in the aldosterone-sensitive distal nephron. Experimental observations regarding the effects of these proteins on transport processes mediated by the thiazide-sensitive Na-Cl co-transporter, the epithelial sodium channel, the renal outer medullary potassium channel, and the paracellular pathway integrate into a model that suggests an essential role for WNKs in coordinating renal Na-Cl reabsorption and K(+) secretion.     

The WNK kinase network regulating sodium, potassium, and blood pressure

The relationship between renal salt handling and hypertension is intertwined historically. The discovery of WNK kinases (With No lysine = K) now offers new insight to this relationship because WNKs are a crucial molecular pathway connecting hormones such as angiotensin II and aldosterone to renal sodium and potassium transport. To fulfill this task, the WNKs also interact with other important kinases, including serum and glucocorticoid-regulated kinase 1, STE20/SPS1-related, proline alanine-rich kinase, and oxidative stress responsive protein type 1. Collectively, this kinase network regulates the activity of the major sodium and potassium transporters in the distal nephron, including thiazide-sensitive Na-Cl cotransporters and ROMK channels. Here we show how the WNKs modulate ion transport through two distinct regulatory pathways, trafficking and phosphorylation, and discuss the physiologic and clinical relevance of the WNKs in the kidney. This ranges from rare mutations in WNKs causing familial hyperkalemic hypertension to acquired forms of hypertension caused by salt sensitivity or diabetes mellitus. Although many questions remain unanswered, the WNKs hold promise for unraveling the link between salt and hypertension, potentially leading to more effective interventions to prevent cardiorenal damage.     

Pseudo-hypoaldosteronisms

TWO FORMS: Pseudohypoaldosteronisms (PHA) are characterized by end-organ resistance to aldosterone inducing hyperkalemia and hyperaldosteronism. There are two forms of PHA classified according to the level of blood pressure with either hypotension (Type 1 PHA or PHA 1) or hypertension (Type 2 PHA or PHA 2). PHA 1: The association with hypotension and high renin level (PHA 1) is responsible for type 4 tubular acidosis and should suggest congenital or acquired excessive salt loss. Acquired forms are associated with salt wasting of urinary (nephropathy) or digestive (colon resection + ileostomy) origin. Congenital neonatal forms are either sporadic or autosomal dominant or recessive. Sporadic or autosomal dominant forms are caused by mutations in the mineralocorticoid receptor gene and generally remit with age. Autosomal recessive forms are caused by mutations in the gene encoding the amiloride-sensitive sodium channel and are clinically more severe with pulmonary symptoms. PHA 2: The association of hyperkalemia/hyperaldosteronism with high blood pressure should suggest PHA 2 or Gordon's syndrome, still called familial hyperkalemic hypertension. This form of low-renin hypertension is caused by mutations in the WNK genes (WNK 1 for PHA 2C and WNK 4 for PHA 2B), but other genes located on different loci are also involved. These WNK kinases constitute a new signalisation pathway that would regulate blood pressure and homeostasy of Na+, K+, H+ and Cl- ions.     

Dietary electrolyte-driven responses in the renal WNK kinase pathway in vivo

WNK1 and WNK4 are unusual serine/threonine kinases with atypical positioning of the catalytic active-site lysine (WNK: With-No-K[lysine]). Mutations in these WNK kinase genes can cause familial hyperkalemic hypertension (FHHt), an autosomal dominant, hypertensive, hyperkalemic disorder, implicating this novel WNK pathway in normal regulation of BP and electrolyte balance. Full-length (WNK1-L) and short (WNK1-S) kinase-deficient WNK1 isoforms previously have been identified. Importantly, WNK1-S is overwhelmingly predominant in kidney. Recent Xenopus oocyte studies implicate WNK4 in inhibition of both thiazide-sensitive co-transporter-mediated Na+ reabsorption and K+ secretion via renal outer medullary K+ channel and now suggest that WNK4 is inhibited by WNK1-L, itself inhibited by WNK1-S. This study examined WNK pathway gene expression in mouse kidney and its regulation in vivo. Expression of WNK1-S and WNK4 is strongest in distal tubule, dropping sharply in collecting duct and with WNK4 also expressed in thick ascending limb and the macula densa. These nephron segments that express WNK1-S and WNK4 mRNA have major influence on long-term NaCl reabsorption, BP, K+, and acid-base balance, processes that all are disrupted in FHHt. In vivo, this novel WNK pathway responds with significant upregulation of WNK1-S and WNK4 with high K+ intake and reduction in WNK1-S on chronic lowering of K+ or Na+ intake. A two-compartment distal nephron model explains these in vivo findings and the pathophysiology of FHHt well, with WNK and classic aldosterone pathways responding to drivers from K+ balance, extracellular volume, and aldosterone and cross-talk through distal Na+ delivery regulating electrolyte balance and BP.