Gene expression analysis of diabetic kidney cortices obtained from ICAM-1 deficient mice and wild type mice

In the present study, we aimed to determine the genes involved in inflammatory process of diabetic nephropathy. ICAM-1+/+ and ICAM-1-/- mice aged 8 weeks were divided into four groups: 1) nondiabetic ICAM-1+/+ mice (ND-WT), 2) nondiabetic ICAM-1-/- mice (ND-KO), 3) streptozotocin (STZ)-induced diabetic ICAM-1+/+ mice (DM-WT), and 4) STZ-induced diabetic ICAM-1-/- mice (DM-KO). Three months after the induction of diabetes, total RNA was extracted from each specimen of renal cortex. We examined gene expression profiles of four groups. We identified 193 genes; the ratio of expression level of DM-WT was >2 or <0.5 of that of DM-KO. Of 193 genes, hierarchical clustering identified 33 genes that were significantly upregulated only in DM-WT but not remarkable in ND-WT and ND-KO. Functional annotation of these 33 genes revealed that the significant functions of them were related to the immune or inflammatory process: immune response, response to stimulus, defense response, immune effector process and antigen processing and presentation. These genes contained several inflammatory related genes, such as chemokine (C-X-C motif) ligand 10, chemokine (c-c motif) ligand 12, and chemokine (c-c motif) ligand 8. In this cluster, we focused on cholecystokinin (CCK) because CCK is one of the most up-regulated genes. Real-time RT-PCR revealed that CCK mRNA expression was significantly up-regulated in DM-WT compared with DM-KO. These results suggest that CCK may play a critical role in the progression of diabetic nephropathy by controlling inflammation in diabetic kidney. Male ICAM-1-/- mice (C57BL/6J background) were purchased from The Jackson Laboratory (Bar Harbor, ME). Male C57BL/6J mice (ICAM-1+/+) mice were used as controls. ICAM-1+/+ and ICAM-1-/- mice aged 8 weeks were divided into four groups (n = 5 each): 1) nondiabetic ICAM-1+/+ mice (ND-WT), 2) nondiabetic ICAM-1-/- mice (ND-KO), 3) streptozotocin (STZ)-induced diabetic ICAM-1+/+ mice (DM-WT), and 4) STZ-induced diabetic ICAM-1-/- mice (DM-KO). Mice in the diabetic groups received an intraperitoneal injection of STZ (Sigma Chemical, St.Louis, MO) at 200 mg/kg in citrate buffer (pH 4.5). Blood glucose levels were determined 7 days after STZ injection and only mice with blood glucose concentrations >16 mmol/L were used in the study. Nondiabetic ICAM-1+/+ and ICAM-1-/- mice received citrate buffer injections only. All mice had free access to standard diet and tap water. All animal procedures were performed according to the Guidelines for Animal Experiments at Okayama University Medical School, Japanese Government Animal Protection and Management Law (no. 105), and Japanese Government Notification on Feeding and Safekeeping of Animals (no. 6). Three months after the induction of diabetes, all mice were killed, and the kidneys were harvested. Total RNA was extracted from each specimen of renal cortex using standard protocol from RNeasy midi kit (Qiagen, Valencia, CA) at 3 months. The concentration and the quality of the total RNA sample were assessed by Agilent Bioanalyzer. Biotin-labeled target cRNA was prepared using First and Second strand cDNA Synthesis Kit (Amersham Biosciences, No. 320000) and In Vitro Transcription Kit (Amersham Biosciences, No.320001). Incubate total RNA (5 ÎŒg), diluted bacterial mRNA spike controls and T7 oligo (dT) primer for 10 min at 70 C. Add first-strand reaction components and incubate 1 hour at 42C. Add the first-strand cDNA product to the second-strand reaction components and incubate for 2 hours at 16 C. Double-strand cDNA was purified using QIAquick purification kit (Qiagen), eluted twice, each time with 30ÎŒl of nuclease-free water then dried in a SpeedVac concentrator. Prepare IVT mix of biotinylated UTP, ribonucleotides, and the 10x T7 enzyme mix and add to the resuspended cDNA. Incubate at 37C for 14 hours. Purify cRNA using RNeasy Mini Kit (Qiagen) and elute the cRNA twice, each time with 50 ÎŒl nuclease-free water. Measure the absorbance at 260 nm and 280 nm to determine the ratio (A260:A280=2). In 25 ÎŒl total volume, add 10 ÎŒg of cRNA to 5 ÎŒl of 5x fragmentation buffer and incubate at 94 C for 20 min. Bring 10 ÎŒg of fragmented cRNA, 78 ÎŒl of hybridization buffer component A, and 130 ÎŒl of hybridization buffer component B to a final volume of 260 ÎŒl with water. Incubate at 90C for 5 min and immediately chill on ice for 5 min. Slowly inject 250 ÎŒl of hybridization reaction mixture into array input port and seal ports with sealing strips. Set the shaker speed to 300 rpm and incubate slides for 18 hours at 37C in an Innove 4080 shaker. Remove the Flex Chamber using the hybridization removal tool. Then, place the bioarrays into the bioarray rack while it sits inside the medium reagent reservoir containing 0.75xTNT. Transfer the bioarray rack to the large reagent reservoir containing preheated 0.75xTNT, and incubate at 46 C for exactly 1 hour. Fill each slot of the small reagent reservoir with 3.4 ml of Cy5-Streptavidin working solution. Transfer the bioarray rack from the large reagent reservoir in to the small reagent reservoir and incubate bioarrays at RT for 30 min. Wash the bioarrays four times with 1xTNT at RT for 5 min. Rinse the bioarrays in 0.1xSSC/0.05%Tween by moving rack up and down 5 times in 5 seconds. Immediately follow the rinse with centrifugation to dry bioarrays, and store dried bioarrays in the dark. We scanned bioarrays with Axon GenePix 4000B and analyzed with CodeLink Expression Analysis software version 2.3.2. The 10,000 spot intensities on the scanned microarray image were normalized to a median value of 1 and data were exported for analysis with CodeLink Expression Analysis software version 2.3.2本研究旨在明确参与糖尿病肾病炎症过程的基因。选取8周龄的细胞间黏附分子1（ICAM-1）野生型（ICAM-1+/+）与基因敲除型（ICAM-1-/-）小鼠，分为4组：1）非糖尿病野生型ICAM-1+/+小鼠（ND-WT），2）非糖尿病基因敲除型ICAM-1-/-小鼠（ND-KO），3）链脲佐菌素（streptozotocin, STZ）诱导的糖尿病野生型ICAM-1+/+小鼠（DM-WT），4）链脲佐菌素诱导的糖尿病基因敲除型ICAM-1-/-小鼠（DM-KO）。糖尿病诱导3个月后，提取各小鼠肾皮质标本的总RNA，检测4组小鼠的基因表达谱。本研究筛选得到193个基因，其DM-WT组的表达水平与DM-KO组相比，比值大于2或小于0.5。在这193个基因中，经分层聚类分析鉴定出33个仅在DM-WT组中显著上调，而在ND-WT与ND-KO组中无明显变化的基因。对该33个基因进行功能注释后发现，其显著富集的功能与免疫或炎症过程相关：包括免疫应答、刺激应答、防御反应、免疫效应过程以及抗原加工与呈递。上述基因包含多种炎症相关基因，如趋化因子（C-X-C基序）配体10、趋化因子（C-C基序）配体12以及趋化因子（C-C基序）配体8。在该聚类基因集中，我们重点关注胆囊收缩素（cholecystokinin, CCK），因其为上调幅度最高的基因之一。实时逆转录聚合酶链反应（real-time RT-PCR）结果显示，与DM-KO组相比，DM-WT组的CCK mRNA表达水平显著上调。上述结果提示，CCK可能通过调控糖尿病肾脏的炎症反应，在糖尿病肾病的进展中发挥关键作用。雄性ICAM-1-/-小鼠（C57BL/6J背景）购自杰克逊实验室（美国缅因州巴尔港），雄性C57BL/6J小鼠（即ICAM-1+/+小鼠）作为对照。8周龄的ICAM-1+/+与ICAM-1-/-小鼠分为4组，每组n=5，分组同上。糖尿病模型组小鼠腹腔注射链脲佐菌素（Sigma Chemical公司，美国密苏里州圣路易斯），剂量为200 mg/kg，溶媒为pH 4.5的柠檬酸盐缓冲液。STZ注射7天后检测血糖水平，仅选取血糖浓度大于16 mmol/L的小鼠进入本研究。非糖尿病组小鼠仅注射柠檬酸盐缓冲液。所有小鼠自由采食标准饲料与自来水。所有动物实验操作均遵循冈山大学医学院动物实验指南、日本《动物保护与管理法》（第105号）以及日本《动物饲养与看护告示》（第6号）。糖尿病诱导3个月后，处死所有小鼠，摘取肾脏。采用RNeasy midi试剂盒（Qiagen公司，美国加利福尼亚州巴伦西亚）的标准操作流程提取各肾皮质标本的总RNA。采用安捷伦生物分析仪（Agilent Bioanalyzer）评估总RNA的浓度与质量。生物素标记的靶标互补RNA（cRNA）制备采用第一链与第二链cDNA合成试剂盒（Amersham Biosciences，货号320000）以及体外转录试剂盒（Amersham Biosciences，货号320001）。具体步骤如下：将5 μg总RNA、稀释的细菌mRNA spike对照与T7寡聚(dT)引物在70℃孵育10分钟；加入第一链反应组分，42℃孵育1小时；将第一链cDNA产物加入第二链反应组分，16℃孵育2小时。采用QIAquick纯化试剂盒（Qiagen）纯化双链cDNA，每次用30 μl无核酸酶水洗脱2次，随后经SpeedVac浓缩仪干燥。制备含生物素化UTP、核糖核苷酸与10× T7酶混合物的体外转录（IVT）混合液，加入重悬后的cDNA，37℃孵育14小时。采用RNeasy Mini试剂盒（Qiagen）纯化cRNA，每次用50 μl无核酸酶水洗脱2次。通过测定260 nm与280 nm处的吸光度值，确定吸光度比值（A260:A280=2）。取10 μg cRNA，加入5 μl 5×片段化缓冲液，总体积补至25 μl，94℃孵育20分钟进行片段化。取10 μg片段化cRNA、78 μl杂交缓冲液组分A与130 μl杂交缓冲液组分B，加水至终体积260 μl。90℃孵育5分钟后立即置于冰上冷却5分钟。缓慢将250 μl杂交反应混合液注入芯片进样口，并用密封条密封进样口。将摇床转速设为300 rpm，在Innove 4080摇床中37℃孵育芯片18小时。使用杂交拆卸工具移除Flex Chamber，随后将生物芯片置于生物芯片架中，该架置于含0.75×TNT的中型试剂槽内。将生物芯片架转移至含预热0.75×TNT的大型试剂槽中，46℃准确孵育1小时。向小型试剂槽的每个孔中加入3.4 ml Cy5-链霉亲和素工作液。将生物芯片架从大型试剂槽转移至小型试剂槽中，室温孵育30分钟。室温下用1×TNT洗涤生物芯片4次，每次5分钟。将生物芯片架在0.1×SSC/0.05%吐温溶液中上下移动5次，历时5秒进行漂洗。漂洗后立即离心干燥生物芯片，避光保存干燥后的芯片。采用Axon GenePix 4000B扫描仪扫描生物芯片，使用CodeLink Expression Analysis软件版本2.3.2进行数据分析。将扫描得到的微阵列图像上的10000个点信号强度归一化至中位数为1，导出数据后使用CodeLink Expression Analysis软件版本2.3.2进行后续分析。