親蛋白質尿毒素對血管內皮細胞與心肌細胞黏附接合的影響及其機制

Abstract

慢性腎臟病患的心臟血管疾病發生率與死亡率遠高於其他一般正常人。累積於體內的尿毒素可能會造成血管內皮細胞功能異常與心律不整的發生。硫酸吲哚酚(indoxyl sulfate)和對甲酚(p-cresol)都是隨著腎功能惡化而累積於體內的尿毒分子。本論文主要探討硫酸吲哚酚對血管內皮細胞與對甲酚對心肌細胞的黏附接合(adherens junction)的影響，並闡明其作用機轉。第一部分研究是以小牛肺動脈內皮細胞株(Bovine pulmonary artery endothelial cells, BPAECs)的細胞研究模式進行。首先利用螢光免疫法可觀察到硫酸吲哚酚處理後，小牛肺動脈內皮細胞的vascular endothelial cadherin (VE-cadherin)、p120-catenin、β-catenin染色強度均明顯減少，細胞間隙變大，張力絲(stress fibers)呈現平行排列的變化。流式細胞儀則證明硫酸吲哚酚處理後內皮細胞之內的自由基增加。若以抗氧化劑MnTMPyP或ERK pathway抑制劑U0126前處理，可顯著減少硫酸吲哚酚處理所造成的細胞間連接離散。西方點墨法發現硫酸吲哚酚處理會增加細胞內肌凝蛋白輕鏈激酶與肌凝蛋白輕鏈磷酸化及extracellular-signal-regulated protein kinase (ERK1/ERK2) 活化。抗氧化劑MnTMPyP前處理可減少ERK2磷酸化，U0126前處理也可阻止硫酸吲哚酚造成的肌凝蛋白輕鏈激酶與肌凝蛋白輕鏈的磷酸化。本實驗結果可證明硫酸吲哚酚藉由增加自由基造成MEK-ERK-MLCK-MLC訊息路徑活化，使細胞收縮而造成血管內皮細胞間隙變大。第二部分研究是以新生大鼠心肌細胞為模式。對甲酚處理後，心肌細胞收縮速率下降且有不規則跳動情形。利用螢光免疫法可觀察到心肌細胞間隙增加、N-cadherin與p120-catenin染色減少。後續流式細胞儀與西方點墨法實驗發現對甲酚處理造成心肌細胞內鈣離子增加與蛋白激酶Cα (protein kinase Cα)磷酸化增加。免疫沉澱實驗證實蛋白激酶Cα的活化造成p120-catenin絲胺酸去磷酸化，進而和N-cadherin分離，使黏附接合瓦解。蛋白激酶Cα的抑制劑Go 6976可阻止上述對甲酚處理造成心肌細胞的變化。因此，對甲酚藉由造成心肌細胞內鈣離子增加與蛋白激酶Cα 活化，使p120-catenin絲胺酸去磷酸化，瓦解黏附接合。此結果再度於H9c2細胞株實驗證實，以siRNA剔除蛋白激酶Cα後，對甲酚處理就不再能造成心肌細胞間隙增加、黏附接合瓦解及p120-catenin絲胺酸去磷酸化。本論文研究結果證實：硫酸吲哚酚和對甲酚確實分別造成血管內皮細胞與心肌細胞黏附接合瓦解，與臨床觀察到的慢性腎臟病人合併有心血管疾病發生現象吻合。未來需要更進一步深入研究，探究更詳細的機制與可能的治療對策。

Chronic kidney disease patients have a much higher risk of cardiovascular diseases and death than general population. Uremic toxins are probably involved in the development of vascular endothelial dysfunction and cardiac arrhythmia. Indoxyl sulfate (IS) and p-cresol are both uremic toxins that accumulate with deterioration of renal function. This study explored the effects and underlying mechanisms of IS and p-cresol on the adherens junctions (AJs) of vascular endothelial cells and cardiomyocytes, respectively. In the first section of study, bovine pulmonary artery endothelial cells (BPAECs) model was adopted. After treatment with IS, the immunofluorescence study showed significantly decreased staining of vascular endothelial cadherin (VE-cadherin), p120-catenin, and β-catenin. IS treatment resulted in disruption of intercellular contacts between BPAECs with prominent parallel-oriented intracellular stress fiber formation. Intracellular free radical levels which measured by flow cytometry increased after IS treatment. Pre-treatment with antioxidant, MnTMPyP, and an ERK pathway inhibitor, U0126, both significantly prevented IS-induced disruption of intercellular contacts. Western blotting analyses demonstrated that IS induced phosphorylation of myosin light chain kinase (MLCK) and myosin light chains (MLC) as well as activation of extracellular-signal-regulated protein kinase (ERK1/ERK2). Pretreatment with MnTMPyP prevented ERK2 phosphorylation. U0126 prevented the IS-induced MLCK and MLC phosphorylation. MEK-ERK acted as the upstream regulator of the MLCK-MLC pathway. These findings suggest that the superoxide anion-MEK-ERK-MLCK-MLC signaling mediates IS-induced cell contraction and junctional dispersal of BPAECs. The second section of study was carried in the model of neonatal rat cardiomyocytes. The p-cresol treatment decreased cardiomyocyte contraction rates and resulted in arrhythmic contraction. In immunofluorescence experiment, a loss of N-cadherin and p120-catenin immunostaining from cell-cell contact sites was noted after p-cresol treatment. P-cresol disrupted AJs and caused the formation of intercellular gap formation. Flow cytometry and Western blotting analyses revealed that p-cresol treatment increased intracellular calcium levels and protein kinase Cα (PKCα) phosphorylation. The immunoprecipitation experiment revealed that PKCα activation induced serine dephosphorylation of p120-catenin, dissociation of p120-catenin and N-cadherin, and AJs disassembly. Inhibition of PKCα by Go 6976 blocked the above effects. Thus, p-cresol caused AJs disassembly between cardiomyocytes, which was mediated by intracellular calcium influx-PKCα activation-p120catenin serine dephosphorylation. This cascade was further confirmed in the study of H9c2 cells by siRNA approach. siRNA knockdown of PKCα prevented p-cresol-induced intercellular gaps formation, AJs disassembly, and serine dephosphorylation of p120-catenin. Our study showed that uremic toxin indoxyl sulfate and p-cresol caused vascular endothelial cell and cardiomyocyte AJ disassembly, respectively. These findings agree with the previously reported findings that indoxyl sulfate and p-cresol are associated with cardiovascular disease in chronic kidney disease populations.Additional studies are needed to elucidate the detailed molecular pathways and possible treatment policy.