C57BL/6小鼠長期服用鋰鹽之腎臟病程觀測

Abstract

鋰鹽已被用於躁鬱症治療多年，但其作用機制是到近幾年才逐步釐清。已有多篇研究證實，鋰鹽對於糖原合成酶激酶3(glycogen synthase kinase-3，GSK3)的抑制效果，是促成神經細胞穩定的重要因素。而鋰鹽多需長期服用，且治療濃度與中毒濃度接近，因此負責鋰鹽代謝的腎臟常是最直接受到影響的器官。除了鋰鹽中毒造成急性腎衰竭外，長期治療的病人有很高比例會引發腎性尿崩症(Nephrogenic diabetes insipidus，NDI)，並有部分病患演變成慢性腎病變(chronic kidney disease，CKD)。目前的研究多著重於NDI成因的探討，已知的主要途徑是因鋰鹽抑制血管加壓素訊息(vasopressin signaling)傳遞，使集尿管上負責進行水分再吸收的第二型水通道蛋白(aquaporin 2，AQP2)表現量降低，並阻擋其運輸至主細胞(principal cell)的頂膜上執行作用，導致水分無法再吸收，病患尿液濃縮能力失調。至於長期服用鋰鹽所導致的CKD，目前已有多篇臨床追蹤研究證實鋰鹽的使用會緩慢降低病人的肌酸酐廓清率(creatinine clearance，CCr)和腎絲球過濾率(glomerular filtration rate，GFR)，而逐漸走向CKD。並由病人的組織切片觀察到慢性腎小管間質腎病(chronic tubulointerstitial nephropathy，CTIN)和局部腎絲球硬化(focal segmental glomerulosclerosis，FSGS)的病徵，但相關機制的探究至今仍然缺乏。 近期的研究，終於在實驗中建立大鼠長期(6個月)服用鋰鹽產生慢性間質纖維化的模型。但隨著基因轉殖/基因剔除小鼠使用的廣泛，若要作疾病機轉的探究，勢必要用到此工具。因此，我們選用C57BL/6(簡稱B6)小鼠作為研究對象，餵食小鼠鋰鹽飼料半年，並在過程中監控其腎功能，檢測有無蛋白尿和腎臟纖維化的發生，目的是要建立鋰鹽引發慢性腎臟病的小鼠模式。 我們給予小鼠含有鋰鹽的飼料，於服用飼料後第2、4、8、12、24週分批將小鼠放至代謝籠，留24小時尿液後進行犧牲，抽血並作腎臟組織的採集。在小鼠血中的鋰濃度皆維持在臨床治療濃度範圍的前提下，我們觀察到鋰鹽組小鼠飲水量和排尿量顯著較控制組高，且尿液滲透壓的數值顯著較低，但卻未低於血漿滲透壓因而認定為溶質利尿。自由水廓清率未看出水分過度排出，腎臟AQP2的基因表現也與控制組無異。於是我們另外進行了限水試驗，確實有看到鋰鹽小鼠水分再吸收反應較差的情形。而從滲透壓廓清率、鈉、氯排出量較控制組高的結果，我們無法排除水利尿(water diuresis)和溶質利尿(solute diuresis)同時存在的可能。服用鋰鹽的過程中，小鼠的腎功能與控制組無異，由尿中白蛋白的檢測也未能看出蛋白尿的情形。至於是否有間質纖維化的產生？我們未能看到纖維化相關基因的表現增加，而從組織切片染色分析結果，同樣沒有看到腎臟纖維化的跡象。總結來說，本次實驗雖有看到B6小鼠長期服用鋰鹽產生尿液濃縮的失調，但卻未能區辨其成因為水利尿還是溶質利尿。由腎功能、白蛋白檢測和組織切片染色分析，確認B6小鼠在服用鋰鹽半年後依然無法建立慢性腎臟病的模式。是故後人若要進行鋰鹽相關研究，可選用B6小鼠作尿崩症成因的探討，但若是要研究鋰鹽慢性腎臟病，B6小鼠就不會是個好選擇。

Lithium has been used therapeutically for more than 100 years and remains a common drug for bipolar disorders. However, the mechanism of this agent was unclear until recently studies explored. More and more studies proved that lithium’s neuroprotective ability comes from the inhibition of glycogen synthase kinase 3 (GSK3), a ubiquitously expressed serine/threonine kinase. Although the effect of lithium therapy is remarkable, long-term use brings renal toxicity frequently. Lithium nephrotoxicity can be divided into three main categories: acute intoxication, nephrogenic diabetes insipidus (NDI), and chronic kidney disease (CKD). In addition to the overdose of lithium contributes to acute renal failure, the most common side effect is NDI. Defective in urinary concentrating ability make patients present with polyuria and polydipsia. Numerous studies revealed that this defect is due to the downregulation of aquaporin 2 (AQP2), a water channel regulated by vasopressin signaling, and decrease in AQP2 trafficking to the apical membrane of the principal cells in the collecting ducts. Prolonged lithium treated patients display CKD in a high proportion. Chronic tubulointerstitial nephropathy (CTIN) and focal segmental glomerulosclerosis (FSGS) are dominant CKD form in these biopsy findings. Nevertheless, the researches of lithium-induced CKD are still limited. Recently, a lithium-induced chronic interstitial fibrosis model has been established in rat kidney. It will be a useful tool to understand of chronic interstitial fibrosis and find out the intervention strategies to prevent injury. However, lithium is a systemic drug that is difficult to study the detail mechanism or effect on specific cells. To reveal the molecular mechanism of diseases, transgenic mice are the irreplaceable instrument. Whether the lithium-induced chronic interstitial fibrosis also occurs on mice must be verify before the transgenic mice use. Therefore, we built up a mice model to study the disease progression of lithium-induced nephropathy. We chose C57BL/6, the most widely used stain for transgenic mice production, as our study subject. These mice fed on lithium diet for 6 months, and monitored plasma lithium level in therapeutic range. The lithium-treated mice remained polyuria and polydipsia over the duration of the study, except 6 month. Although the urine osmolality was significantly lower than control group, it was still concentrating urine (urine osmolality > plasma osmolality) that defined as solute diuresis. Higher osmolality clearance and sodium, chloride excretion may also be the solute diuresis evidences. Free water clearance and gene expression both could not prove the appearance of NDI. We verified with water deprivation test that lithium decreased urine concentrating ability. These mice remained normal renal function that compared to control group. There was no proteinuria occurred in this study. Renal interstitial fibrosis examined with gene expression and histology staining, but there was no different between the two groups. In conclusion, C56BL/6 mice can be used for lithium-induced NDI study, but not a good choice for chronic interstitial fibrosis investigation.