Identification of Functions of Peroxisome Proliferator- Activated Receptor in Proximal Tubules-猫眼课题宝

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2002

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Y. Kamijo;K. Hora;N. Tanaka;N. Usuda;K. Kiyosawa;T. Nakajima;F. Gonzalez;T. Aoyama

过氧化物酶体增殖物激活受体（PPAR）是类固醇/核受体超家族的成员，在肾脏中大量表达，但其生理功能尚不清楚。在这项研究中，使用 PPAR 缺失小鼠来帮助阐明其功能。研究发现，饥饿的 PPAR 缺失小鼠比饥饿的野生型小鼠分泌更多的尿白蛋白。此外，仅在饥饿的PPAR缺失小鼠的近端肾小管中发现巨大溶酶体的出现、白蛋白的显着积累以及白蛋白消化能力受损。这些异常可能是由于饥饿引起的碳水化合物代谢下降和 PPAR 依赖性脂肪酸代谢缺乏而导致 ATP 不足。有趣的是，当给予葡萄糖时，这些异常现象消失了。总而言之，这些发现证明了 PPAR 在近曲小管中的重要功能，通过维持 ATP 稳态来动态调节蛋白质降解系统，并强调了脂肪酸代谢在肾脏生理学中的重要性。最近，过氧化物酶体增殖物激活受体 (PPAR) 受到了相当多的关注，它被称为类固醇/核受体超家族的成员 (1)。根据一些研究(2,3)，肾脏中发现高水平的PPAR，并且主要位于近曲小管，尽管其生理功能尚未阐明。近端肾小管上皮细胞是高度分化的细胞，它们从肾小球滤液中重吸收许多对身体必需的物质，并分泌多种生理活性化合物。有人提出，近端肾小管上皮细胞含有较高密度的线粒体，产生的 ATP 对于支持其特定功能和维持基本细胞功能是必需的 (4)。为了阐明 PPAR 在肾脏中的生理作用，我们使用 PPAR 缺失小鼠检查了近端肾小管的重吸收过程。我们重点研究白蛋白重吸收，这是过滤蛋白质处理的典型组成部分，通过有效的受体介导的内吞作用来完成，其中巨蛋白充当主要受体 (5-8)。白蛋白重吸收与能量产生之间的关系也已确定，因为已知 PPAR 作为肝脏和心脏中线粒体能量产生的有效调节剂发挥着重要作用 (9,10)。此外，我们使用饥饿的小鼠来增加对脂肪酸作为能量燃料来源的依赖，并减少碳水化合物代谢物的影响。材料和方法材料抗小鼠白蛋白IgG 购自Beth Laboratories (Montgomery, TX)。抗大鼠组织蛋白酶 D IgG、ATP、乙酰辅酶 A 和三棕榈酸来自 Wako（日本大阪）。抗 Rab5a IgG、抗 Rab7 IgG 和抗小鼠组织蛋白酶 L IgG 来自 Santa Cruz Biotechnology (Santa Cruz, CA)。抗 LAMP-1 IgG 来自 American Research Products（马萨诸塞州贝尔蒙特）。抗巨蛋白多克隆抗血清 (11) 由 Marilyn G. Farquhar 博士（加利福尼亚大学）提供，抗大鼠葡萄糖醛酸酶 IgG (12) 由 K. Sukekawa 博士（日本岐阜大学医学院）提供。动物和饥饿治疗如前所述产生具有 Sv/129 遗传背景的 PPAR 缺失小鼠 (13)。野生型Sv/129在所有实验中用作对照。两组小鼠，野生型小鼠和PPAR缺失小鼠，饥饿48小时，随意提供水。再次喂食48小时，然后进行第二次饥饿48小时。第二次饥饿后，分别给(/)小鼠灌胃5％和50％葡萄糖溶液150μl以及2.2％和22％三棕榈酸甘油酯混悬液。每3小时重复给药3次。接收日期：2001 年 10 月 30 日。接收日期：2002 年 3 月 15 日。通讯作者：Kazuhiko Hora 博士，信州大学医学院第二内科系，地址：3-1-1 Asahi, Matsumoto, 3908621, Japan。电话：81-263-37-2634；传真：81-263-32-9412；电子邮件：yujibeat@hsp.md.shinshu-u.ac.jp

Peroxisome proliferator-activated receptor (PPAR ) is a member of the steroid/nuclear receptor superfamily that is intensively expressed in the kidney, but its physiologic function is unknown. In this study, PPAR -null mice were used to help clarify the function. Starved PPAR null mice were found to secrete significantly more quantities of urine albumin than starved wild-type mice. Furthermore, the appearance of giant lysosomes, marked accumulation of albumin, and an impaired ability concerning albumin digestion were found only in proximal tubules of the starved PPAR null mice. These abnormalities were probably derived from ATP insufficiency as a result of the starvation-induced decline of carbohydrate metabolism and a lack of PPAR -dependent fatty acid metabolism. It is interesting that these abnormalities disappeared when glucose was administered. Taken together, these findings demonstrate important functions of PPAR in the proximal tubules, the dynamic regulation of the proteindegradation system through maintenance of ATP homeostasis, and emphasize the importance of the fatty acid metabolism in renal physiology. Recently, considerable attention has been paid to the peroxisome proliferator-activated receptor (PPAR ), which is known as a member of the steroid/nuclear receptor superfamily (1). According to some studies (2,3), a high level of PPAR is found in the kidney and mainly localizes in the proximal tubules, although its physiologic function has not yet been clarified. Proximal tubular epithelial cells are highly differentiated cells that reabsorb many substances that are essential to the body from glomerular filtrate and secrete several physiologically active compounds. It was suggested that ATP produced in the proximal tubular epithelial cells, which contain a greater density of mitochondria, is necessary for supporting its specific functions and maintaining basic cell functions (4). To elucidate the physiologic role of PPAR in the kidney, we examined the reabsorption process in the proximal tubules, using PPAR -null mice. We focused on albumin reabsorption, a typical component of the filtered protein handling, done through efficient receptor-mediated endocytosis in which megalin acts as the main receptor (5–8). The relationship between albumin reabsorption and energy production was also determined, because PPAR is known to play an important role as a potent regulator of mitochondrial energy production in the liver and heart (9,10). In addition, we used starved mice to increase the dependence on fatty acids as an energy fuel source and to reduce the effect of carbohydrate metabolites. Materials and Methods Materials Anti-mouse albumin IgG was purchased from Bethyl Laboratories (Montgomery, TX). Anti-rat cathepsin D IgG, ATP, acetyl-CoA, and tripalmitin were from Wako (Osaka, Japan). Anti-Rab5a IgG, antiRab7 IgG, and anti-mouse cathepsin L IgG were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti–LAMP-1 IgG was from American Research Products (Belmont, MA). Anti-megalin polyclonal antiserum (11) was provided by Dr. Marilyn G. Farquhar (University of California, CA), and anti-rat -glucuronidase IgG (12) was from Dr. K. Sukegawa (Gifu University School of Medicine, Gifu, Japan). Animals and Starvation Treatment PPAR -null mice with an Sv/129 genetic background were produced as described previously (13). Wild-type Sv/129 were used as controls in all experiments. Two groups of mice, wild-type and PPAR -null mice, were starved for 48 h with water provided ad libitum. They were fed again for 48 h and then subjected to a second period of starvation for 48 h. After the second starvation, 150 l of 5% and 50% glucose solution and that of 2.2% and 22% tripalmitin suspension were respectively administered to the ( / ) mice by gavage. The administration was repeated three times every 3 h. Received October 30, 2001. Accepted March 15, 2002. Correspondence to Dr. Kazuhiko Hora, Second Department of Internal Medicine, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, 3908621, Japan. Phone: 81-263-37-2634; Fax: 81-263-32-9412; E-mail: yujibeat@hsp.md.shinshu-u.ac.jp

Identification of Functions of Peroxisome Proliferator- Activated Receptor in Proximal Tubules

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