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

黃子娟(Tzu-Chuan Huang)

指導教授 ： 李仁愛

臺北醫學大學/藥學院/藥學研究所/碩士(2006年)

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。

關鍵字

D-乳酸 ； 馬兜鈴酸 ； 乳酸轉運蛋白

並列摘要

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.

並列關鍵字

D-lactate ； Aristolochic acid ； lactate transporters

參考文獻

1. Tobert, J.A., et al., Enhancement of uricosuric properties of indacrinone by manipulation of the enantiomer ratio. Clin Pharmacol Ther, 1981. 29(3): p. 344-50.

2. Drayer, D.E., Pharmacodynamic and pharmacokinetic differences between drug enantiomers in humans: an overview. Clin Pharmacol Ther, 1986. 40(2): p. 125-33.

3. Raffa, R.B., et al., Complementary and synergistic antinociceptive interaction between the enantiomers of tramadol. J Pharmacol Exp Ther, 1993. 267(1): p. 331-40.

5. Takigawa, Y., et al., D-aspartate uptake into cultured rat pinealocytes and the concomitant effect on L-aspartate levels and melatonin secretion. Biochem Biophys Res Commun, 1998. 248(3): p. 641-7.

6. Tsai, Y.C., et al., Stereoselective effects of 3-hydroxybutyrate on glucose utilization of rat cardiomyocytes. Life Sci, 2006. 78(12): p. 1385-91.