血液透析患者紅血球及白血球之適應機轉

Abstract

血液透析(haemodialysis [HD])病人之紅血球與嗜中性白血球如何適應尿毒症及血液透析仍屬未知。在此，我們計畫研究血液透析病人紅血球與嗜中性白血球的病理生理學。文獻顯示：紅血球可藉由第一型葡萄糖轉運蛋白(GLUT 1)來攝入脫氫抗壞血酸(DHA)並將之還原為抗壞血酸(Ascorbate)。紅血球內的抗壞血酸已被證明可以減少紅血球外抗壞血酸被氧化(oxidation)。在初步的實驗中，我們研究經年齡及性別校正的正常控制組（CN group）和血液透析病人（HD group）血漿與紅血球內抗壞血酸及脫氫抗壞血酸濃度，紅血球細胞膜（EM）上GLUT 1的表現量，以及進行「紅血球抗壞血酸循環」(Erythrocyte Ascorbate Recycling)體外試驗。結果發現，HD後(post-HD)紅血球內抗壞血酸較HD前(pre-HD)減少，但兩日後其濃度便會回復與HD前相同；而血漿中的抗壞血酸濃度則不會回復。體外試驗的結果則顯示HD group的紅血球維持細胞內抗壞血酸濃度之能力較CN group強，而且此種能力可以被細胞鬆弛素B(cytochalasin-B)(GLUT 1抑制劑)所抑制。我們也發現到HD group EM上GLUT 1的表現量較CN group為多(P = 0.002)。綜上所述，可以推測：血液透析病人的紅血球在面對血液透析所造成的抗壞血酸大量流失，已經發展出較CN group好的維持細胞內抗壞血酸濃度之能力；而GLUT 1的表現增加可能在其中扮演一部分的角色。 然而在血液透析病人GLUT 1的表現增加之原因仍然不明。因此，在進一步的實驗中，我們試圖找出血液透析病人GLUT 1的表現增加之原因。我們研究68個HD病人、35個不同血紅素（Hb）含量的慢性貧血病人及44個腎臟功能不同但有相似Hb之慢性腎功能不全患者的EM上 GLUT 1的表現量與嗜中性白血球中缺氧誘導因子-1（HIF-1α）mRNA的表現量。結果發現Hb較低的血液透析病人EM GLUT 1表現量較大，嗜中性白血球HIF-1α的mRNA表現量也較高；反之亦然。類似的情形也發生在慢性貧血病人身上，而且其GLUT 1與HIF-1α mRNA表現量呈現正相關(R = 0.742, P<0.001)。然而此種相關性並未在血液透析病人及慢性腎臟疾病病人身上發現。就以上結果可以得知：血液透析病人EM GLUT 1的表現增加可能是因貧血而非尿毒，而此一不尋常的現象可能與嗜中性白血球HIF-1α的活化有關。 在部分血液透析病人(17%)仍然有持續性之輕微代謝性酸中毒的問題，這類持續性之輕微代謝性酸中毒所造成血液透析病人之病態生理已被廣泛的研究；但其對血液透析病人嗜中性白血球細胞內pH值（pHi）及功能的作用從未被清楚的驗證。因此，在第三部分的實驗中，我們將血液透析前(pre-HD)的嗜中性白血球以血漿中重碳酸鹽濃度（PHCO3）的不同分為三個組別：Groups A、B and C，PHCO3分別為≦26 mmol/L，連同CN group來測定在嗜中性白血球的pHi、細胞凋亡(apoptosis)、吞噬作用(phagocytosis)與氧爆(oxidative burst reaction)作用。我們也測試在Group A中代謝性酸中毒(藉由以重碳酸鹽來矯正代謝性酸中毒)與體外試驗細胞內酸化對嗜中性白血球功能的影響。實驗數據顯示血液透析病人的PHCO3若較低，其嗜中性白血球pHi也較低，並有較明顯細胞凋亡延遲、吞噬作用與氧爆作用增加現象。這些改變在矯正代謝性酸中毒之後減少。體外實驗亦證明嗜中性白血球的pHi與細胞凋亡呈現正相關，而與吞噬作用、氧爆作用則呈現負相關。 從上述之三部分的實驗可以得知血液透析病人的紅血球與嗜中性白血球在面對HD及尿毒症時，會由基因這個階段開始，發展自我保護或自我危害的病理生理學改變。為了解此一難題，我們仍需更進一步的研究。

How do erythrocytes and neutrophils adapt themselves to uremia is still unclear. We here, plan to investigate the patho-physiology of erythrocytes and neutrophils in hemodialysis (HD) patients. Erythrocytes can take up dehydroascorbate on the glucose transporter 1 (GLUT 1) and reduce it to ascorbate. Intra-erythrocyte ascorbate has been proven to be directly responsible for the decreased oxidation of extra-erythrocytic ascorbate. In the first experiment, plasma and intra-erythrocyte ascorbate, dehydroascorbate concentrations, the GLUT 1 expression levels on the erythrocyte membrane (EM), and in vitro studies of “erythrocyte ascorbate recycling” were investigated in age- and sex-matched healthy subjects (CN group) and in HD patients (HD group). We found that intra-erythrocyte ascorbate concentrations decreased after one session of HD, compared to those of pre-HD, and recovered to the values of pre-HD two days later, whereas plasma ascorbate concentrations did not recover. In vitro studies suggested that erythrocytes of HD patients possess a stronger ability to maintain intracellular ascorbate concentrations compared to those of healthy subjects. This ability could be inhibited by cytochalasin B (GLUT 1 inhibitor). We also found increased expression levels of GLUT 1 (P = 0.002) on EM in HD group compared to those for CN group. Our results suggest that uremic erythrocytes lost large amount of ascorbate during HD, but regained it to the pre-HD level two days later. Enhanced expression of GLUT 1 on EM in HD patients may contribute to better preservation of intracellular ascorbate compared to the healthy subjects. However, the mechanism responsible for the enhancement is not clear. In the second experiments, we investigated the expression of GLUT 1 on EM and of hypoxia-inducible factor (HIF) 1α mRNA in neutrophil in 68 HD, 35 chronic anemic patients with different hemoglobin (Hb) levels and in 44 chronic renal insufficiency (CRI) patients having different renal functions with similar Hb levels. We found that HD patients having lower Hb expressed higher GLUT 1 on EM and higher HIF 1α mRNA in neutrophils and vice versa. Similar scenario was also found in chronic anemic patients. Furthermore, expression of GLUT 1 on EM was positively correlated with expression of HIF 1α mRNA in neutrophils of chronic anemic patients (R = 0.742, P<0.001). However, this correlation was not found in HD and CKD patients. Our data suggest that anemia, not uremia, may contribute to the enhanced expression of GLUT 1 on EM in HD patients and this abnormality was associated with the activation of HIF 1α. Chronic, but mild, metabolic acidosis is still commonly found in a number of HD patients. However, the effect of intracellular pH (pHi) on uremic neutrophil has not been clearly defined. In the third experiments, we used pre-HD neutrophils from three groups of HD patients having different levels of pre-HD plasma bicarbonate concentrations (PHCO3) and pH values (pre-HD PHCO3 of Groups A, B, and C were consistently ≤21, 21-26 and ≥26 mmol/L, respectively.) and neutrophils from age-, sex-matched healthy controls to determine pHi, apoptosis, phagocytosis and oxidative burst reactions in vivo. We also studied, in Group A, the effect of metabolic acidosis correction on neutrophil function. Furthermore, we investigated the effect of intracellular acidification on neutrophil functioning in vitro. We found that neutrophils from the HD patients in Group A exhibited significantly lower pHi than those in Groups B and C. In addition, Group A neutrophils had significantly delayed apoptosis, enhanced phagocytosis and increased oxidative burst reactions compared to those in Groups B and C. These alterations in neutrophil function in Group A were reduced by correcting metabolic acidosis over a period of one month. Moreover, our in vitro studies demonstrated that the pHi of neutrophils is positively correlated with apoptosis, and inversely correlated with phagocytosis and oxidative burst reactions. Our data suggest that HD patients having low PHCO3 exhibited low neutrophil pHi. This intracellular acidification may contribute to the delayed apoptosis, enhanced phagocytosis and increased oxidative burst reactions observed in these neutrophils compared to neutrophils having normal or higher pHi. To sum up, uremic erythrocytes and neutrophils have evolved genetically to protect or to jeopardize themselves from the pathological alterations in HD patients. Further study is necessary to complete the puzzle.