小鼠腎臟損傷下之尿中 D-乳酸濃度與腎臟中乳酸轉運蛋白表現之探
討
The Study of the Urinary D-lactate Concentration and Lactate Transporters Expression in Mice Renal Injury
中文摘要
D-乳酸和人類許多疾病有關，例如腦病、酸中毒、糖尿病等；尿中 D-乳酸含量 與糖尿病大鼠腎病病程具有高度的相關性，可能可作為腎病變的指標。另一方 面，許多臨床及體外試驗證實，馬兜鈴酸可引起腎小管細胞受損，為了研究尿中 D-乳酸與腎損傷的關係，本研究選擇誘導馬兜鈴酸腎病變來探討尿中 D-乳酸含 量的變化，並研究腎臟中 D-乳酸轉運蛋白的表現，以了解 D-乳酸在腎臟中再吸 收機制與腎病的關連。
實驗動物為 C3H/He 雌鼠，以尾靜脈注射方式每天給予每公斤體重 10 毫克的馬 兜鈴酸、共給予 5 天藉以誘導腎損傷，並從尿蛋白、尿酶
N-acetyl-beta-D-glucosaminidase（NAG）的觀察判斷腎功能。另外並利用西方轉 漬法分析小鼠腎均質液中的腎臟乳酸轉運蛋白 MCT 2、slc5a8 的表現量。結果發 現，給予馬兜鈴酸後，尿蛋白與 NAG 皆大量增加，顯示腎小管已受到破壞；而 尿中 D-乳酸含量是對照組的將近 40 倍（與尿中肌胺酸酐的比例分別是 311.31 ± 71.7 及 8.60 ± 1.80 μM/mM）；小鼠腎臟乳酸轉運蛋白 MCT 2 及 slc5a8 的表現量 在給予馬兜鈴酸後則減少。
本研究證實尿中 D-乳酸含量與馬兜鈴酸腎損傷具高度相關性，造成這種情形的 可能原因之ㄧ是因為腎臟乳酸轉運蛋白的表現改變，尿中 D-乳酸或許可為臨床 上判斷腎病病程的指標之ㄧ。
英文摘要
D-lactate is associated with some human diseases such as diabetes mellitus and encephalopathy, and urinary D-lactate may be used as an indicator to determine the level of kidney damage in diabetic rats. Clinical and in vitro findings have previously suggested that the proximal tubule was the target of aristolochic acid (AA). In this study, the mice model of AA-induced nephropathy was used to investigate the relation between urinary D-lactate and renal injury.
The proximal tubular lesions were induced by intravenous injections of AA at a high dose of 10 mg/kg body weight/day for 5 days and were characterized biochemically.
Urinary excretion of proteins, urinary N-acetyl-beta-D-glucosaminidase (NAG), and plasma creatinine was determined. Urinary D-lactate was separated by our proposed column-switching high-performance liquid chromatography (HPLC) method with
fluorescence detection. The expressions of lactate transporters, slc5a8 and MCT 2, in the proximal tubule of normal and AA treated mice kidney were studied by western blot analysis.
The results showed that a remarkable increase of urinary markers including urinary proteins and urinary NAG, and the histological examination (PAS stain) confirmed the proximal tubule injury. The ratio of D-lactate (μM) to creatinine (mM) in the urine of AA treated mice were approximately 40-fold greater than that in control groups, which were 311.31 ± 71.7 and 8.60 ± 1.80, respectively. By Western blot analysis of lactate transporters, which have an affinity for D-lactate, we found that the expression of transporter MCT 2 and slc5a8 were decreased in AA treated mice. These data confirmed in vivo that urinary D-lactate reflected the renal injury conditions in AA-treated mice and may be a marker for the assessment of nephropathy.
