以動物模式探討小鼠骨髓細胞對巴拉刈造成急性腎損傷的再生潛能

Abstract

巴拉刈（Paraquat, PQ）又稱百草枯，其化學名為 1,1’-dimethyl-4,4’-bipyridylium dichloride，分子式為 C 12 H 14 C l2 N 2 ，屬於聯嘧啶（bypyridiniums）類的化合物，目前已超過一百三十個國家正式登記並使用，為全球使用最廣泛的除草劑之一。巴拉刈中毒大多由口服途徑引起，進入體內後，最早達到最高組織濃度的器官為腎臟，同時它也是最主要的排泄器官。若腎臟功能正常，在口服後6小時內約有百分之八十至九十的巴拉刈以原型型態從尿液中排出體外，在24小時內幾乎為百分之百會由腎臟排出。若腎功能不正常，則組織中(尤其是肺)的巴拉刈濃度將增高，且其濃度頂點將延到 15 至 20 小時之後。當巴拉刈進到腎臟細胞內後，隨即進行氧化還原循環(redox cycling)，消耗NADPH，產生大量的超氧化物 (superoxide anion, O 2.-)等自由基，導致腎臟的傷害。根據 2001 到 2002 年的統計資料指出，在台灣所有農藥中毒事件中，巴拉刈中毒佔了 18.9 %，造成的死亡率為 72.1 %。 過去文獻顯示，高劑量巴拉刈中毒後，會引發多重器官衰竭，其中包括急性腎損傷(acute kidney injury, AKI)，在 24 至 96 小時內出現的蛋白尿(proteinuria)、膿尿(pyuria)、氮血(azotemia)、血尿(hematuria)等較輕微症狀會出現，一旦出現寡尿或無尿，則代表更嚴重的急性腎小管壞死，也會發生腎絲球腎炎的情況。 傳統的治療方法包括洗胃(gastric lavage)、給予口服吸附劑如活性碳（active charcoal）、血液灌流(charcoal haemoperfusion)及糖質皮質固醇類(glucocorticoid)藥物治療，但這些治療往往無法有效提高存活率。急性腎損傷（AKI）目前仍然為高死亡率的疾病之一，主要是因為在臨床治療上有抗藥性的問題而造成治療無效的結果。因此在血管內移植骨髓間葉幹細胞（bone marrow mesenchymal stem cells,BM-MSCs）對於治療AKI是一種新穎且有希望的方法，過去已有研究利用BM-MSCs治療 AKI 模式小鼠，觀察到BM-MSCs會在受傷的腎臟上，修復腎小管及改善腎臟功能，且會分化成腎小管上皮細胞、腎小球系膜細胞（mesangial cells）及腎絲球上皮細胞。因此，本實驗利用雄鼠骨髓細胞對於巴拉刈中毒的雌鼠進行細胞處理，觀察是否能減少傷害並提高其存活率，接著探討其中的調節機制。 為了建立巴拉刈所造成的急性腎臟損傷，首先利用八週大雌性 C57BL/6小鼠以腹腔注射給予 55 mg/kg BW劑量的巴拉刈，使C57BL/6 小鼠腎功能指數與對照組相比有顯著上升的趨勢，隨著暴露巴拉刈後的天數增加，腎功能指數也有增加的趨勢。由組織切片中觀察到腎小管刷狀緣失去、細胞排列不規則且細胞因為毒性死亡而掉落，足細胞結構不完整及核掉落等現象。而在給予三次小鼠骨髓細胞 進行細胞治療後，可將C57BL/6小鼠存活率由20%提高至60%，但利用 dexamethasone (DEX)進行治療無法有效提升小鼠存活率。實驗結果也顯示細胞治療可改善巴拉刈對小鼠引起的腎臟組織傷害，並減少腎功能的破壞，且在處理後第六天可使腎功能恢復到與控制組相似，同時，可減少細胞凋亡及嗜中性白血球浸潤的現象。 綜合以上結論，實驗結果顯示在高劑量 55 mg/kg BW 巴拉刈處理後的 C57BL/6小鼠，以骨髓細胞進行細胞治療，恢復小鼠腎臟功能，減少腎臟傷害並且提高其存活率。而這樣的恢復的現象，或許是藉由骨髓細胞分泌出的細胞激素及生長因子作為免疫抑制的調節以改善巴拉刈對腎臟造成的傷害。本實驗是首先探討巴拉刈對腎臟足細胞造成傷害的研究。

Paraquat(1,1’-dimethyl-4,4’-bipyridylium dichloride, PQ),a potent nephrotoxicant, belongs to the class of bipyridylium quaternary ammonium herbicides. Paraquat mainly excrete by kidney and undergoes a process of redox-cycling and finally leads to reactive oxygen species (ROS) production at the expense of NADPH. This alters the normal cell functions and causes kidney toxicity. Paraquat poisoning is the most common cause of fulminated and fatal herbicide intoxication. From 2001 to 2002, paraquat intoxication contributed to 18.9 % of all pesticide poisonings in Taiwan. Studies showed that high doses of paraquat lead to multiple organ failure such as lung, kidney, liver and so on. Kidney has the most highest concentration compare to the other organs at any detection time. Paraquat-induced acute kidney injury (AKI) caused proteinuria, pyuria, azotemia, uremia and other minor symptoms within 24-96 hours and this can be used as a predict marker of AKI. Despite severe tubular necrosis, paraquat may also cause glomerular damage and eventually lead to glomerulonephritis. The conventional clinical therapies for the patients of fulminant paraquat poisoning are ineffective. On the other hand, AKI patients remain high mortality after clinical treatment due to drug-resistance. Recently, bone marrow mesenchymal stem cells(BM-MSCs) have been reported to have the ability to repair injured kidney and improve survival rate of mice. Therefore, this study aim to evaluate the treatment efficacy of mouse bone marrow cells（mBMCs）on mice with acute kidney paraquat poisoning. In animal model,to mimic AKI, 8 weeks female C57BL/6 mice were intraperitoneally administered with 55 mg/kg BW paraquat. Histopathologically, kidneys showed tubular brush border lost, irregular cell arrangement and inflammatory cells infiltration. Furthermore, kidney functions evaluation also revealed significant differences in BUN, SCr, urine total protein and urine Cr compared to the control group. After three times co-treatment with mBMCs, paraquat-induced AKI mice showed an increased survival rate from 20% to 60%. One time treatment with mBMCs or dexamethasone, however, did not elevate the survival rate of mice. Histopathologically, three times mBMCs co-treatment repaired the injury caused by paraquat and the structure of renal tubular and podocytes seemed to be similar as control group. Renal functions study also suggested that three times mBMCs co-treatment would restore the physiological functions of kidney and results showed that there was no differences between treatment group and control group on Day 6. As a conclusion, the results of this study demonstrated that mBMCs co-treatment could decrease kidney damage,repair kidney functions and increase the survivalrate of mice with acute kidney paraquat poisoning. Besides, this is also the first study to evaluate the damage of podocytes that caused by paraquat.