Genome-wide analysis of renal gene expression during hyperoxaluria development

Previous studies have proposed that production of reactive oxygen species (ROS) is an important contributor to renal injury and inflammation following exposure to oxalate or calcium-oxalate crystals. The present study was conducted to determine, utilizing global transcriptome analyses, if the NADPH oxidase system is activated in kidneys of rats fed a diet leading to hyperoxaluria and crystal deposition. HLP was used to induce hyperoxaluria. Hyperoxaluria will lead to crystallization and up regulation of various NADPH oxidase subunits followed by increased expression of different specific genes. It is our hypothesis that crystallization induces inflammation of the kidneys via the activation of renin-angiotensin system (RAS) and production of reactive oxygen species (ROS) through NADPH oxidase complex. Apocynin was used to block hyperoxaluria and production of reactive oxygen species (ROS) with the inhibition of NADPH oxidase system as Apocynin inhibits membrane translocation of p47 subunit of NADPH oxidase. The present study was designed based on NADPH oxidase system and rats were divided into 4 groups (n= 6/group). Group 1 was fed regular rat chow diet, Group 2 received regular rat chow diet supplemented with 5% (Hydroxy-L-Proline) HLP, Group 3 received rat chow diet with 4 mmol Apocynin to drink, and Group 4 received regular rat chow diet with 5% HLP and 4 mmol Apocynin. After 28 days each rat was euthanized, their kidneys freshly explanted and dissected to obtain both cortex and medulla tissues. So the 4 groups were divided into cortex and medulla forming 8 groups. RNA was isolated from all the 8 specimens既往研究表明，活性氧簇（reactive oxygen species, ROS）的生成是草酸盐或草酸钙晶体暴露后引发肾损伤与炎症的重要诱因。本研究借助全局转录组分析，旨在探究喂食可诱导高草酸尿症与晶体沉积的饲料后，大鼠肾脏内NADPH氧化酶系统是否被激活。本研究采用羟脯氨酸（Hydroxy-L-Proline, HLP）构建高草酸尿症模型。高草酸尿症可引发晶体形成，并上调多种NADPH氧化酶亚基的表达，随后促使多种特异性基因的表达水平升高。本研究提出假说：晶体形成可通过激活肾素-血管紧张素系统（renin-angiotensin system, RAS），并经由NADPH氧化酶复合物产生活性氧簇，进而诱发肾脏炎症。夹竹桃麻素（Apocynin）可通过抑制NADPH氧化酶p47亚基的膜转位，阻断高草酸尿症的发生与活性氧簇的生成，从而抑制NADPH氧化酶系统。本研究围绕NADPH氧化酶系统设计实验，将大鼠随机分为4组（每组n=6）。第1组喂食普通大鼠饲料；第2组喂食添加5%羟脯氨酸的普通大鼠饲料；第3组喂食普通大鼠饲料，同时饮用含4 mmol夹竹桃麻素的溶液；第4组喂食添加5%羟脯氨酸的普通大鼠饲料，同时饮用含4 mmol夹竹桃麻素的溶液。造模28天后，对所有大鼠实施安乐死，迅速摘取肾脏并分离肾皮质与肾髓质组织。据此，4组大鼠分别按肾皮质与肾髓质进行细分，最终形成8个亚组。随后从全部8份标本中提取RNA。