Influence of retinol on kidney in 5/6 nephrectomy mice

The circadian clock has been found to be associated with various diseases. We showed that 5/6 nephrectomy (5/6Nx) Clk/Clk mice, which show mutation in the gene encoding circadian locomotor output cycles (Clock) do not show aggravation of renal fibrosis because transforming growth factor-1 (Tgf-1) expression is not increased. In wild-type 5/6Nx kidneys, we found that retinoid, a metabolite of retinol, led to alteration of the expresion 24-h rhythm of Clock expression. Renal Tgf- 1 expression is activated by Clock and further aggravates renal dysfunction by causing fibrosis. We also showed that, in 5/6Nx mice fed a retinol-free diet, renal fibrosis and apoptosis are reduced, leading to a marked improvement in serum creatinine levels. Moreover, our study identified hepatic Cyp3a11 and Cyp26a1 as key retinol metabolism-related genes whose expression decreased in 5/6Nx mice. Our data indicated that the negative chain reaction of metabolic clock alteration in between the kidney and liver aggravates renal dysfunction. Differential gene expression between retinol (-) feeding and clock mutant in 5/6 nephrectomized mouse was measured on the kidney at 8 weeks after operation. Four-week-old male ICR mice (Charles River Japan, Inc., Yokohama, Japan) were housed in a light-controlled room (lights on from Zeitgeber time [ZT] 0 to ZT12) at 24 ± 1°C and 60 ± 10% humidity, with food and water available ad libitum. Mice were synchronized to the lighting conditions for 2 weeks before surgery. Male ICR mice (5 weeks old) were purchased from Charles River Japan, Inc. (Kanagawa, Japan). Clock mutant mice (C57BL/6J-ClockmlJt/J) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). We placed them in the ICR genetic background to enhance breeding robustness and care of the young. These mice were backcrossed using a Jcl:ICR background for more than eight generations. We prepared mouse models of CRF by 5/6Nx operation (Ope) under sodium pentobarbital (40 mg/kg, i.p.) or diethyl ether anesthesia. 5/6Nx was performed in two stages. In the first surgical procedure (at 6 weeks of age), two-thirds of the left kidney was removed by cutting off both poles. Seven days later, the right kidney was completely removed. After the operation, mice were housed for 8 weeks (until they were 16 weeks old) in order to achieve CRF. Sham-operated (Sham) mice were subjected to laparotomy on the same days as the procedure in the 5/6Nx mice. This method was also used for treating Clk/Clk mice. Retinol-free food (A minus) was purchased form KBT ORIENTAL CO., LTD. To investigate the influences of retinol-free feeding on kidney, mice were fed from the fourth week to the eighth week after an operation.

昼夜节律时钟（circadian clock）已被证实与多种疾病相关。本研究发现，在编码昼夜节律运动输出周期蛋白（circadian locomotor output cycles, Clock）的基因存在突变的5/6肾切除（5/6 nephrectomy, 5/6Nx）Clk/Clk小鼠中，由于转化生长因子-1（transforming growth factor-1, Tgf-1）的表达未出现上调，肾脏纤维化并未加重。在野生型5/6Nx小鼠肾脏中，我们发现视黄醇的代谢产物类视色素（retinoid）会改变Clock基因表达的24小时节律。肾脏Tgf-1的表达可被Clock激活，并通过诱发纤维化进一步加重肾功能障碍。我们还证实，在喂食无视黄醇饮食的5/6Nx小鼠中，肾脏纤维化与细胞凋亡均有所减轻，血清肌酐水平得到显著改善。此外，本研究鉴定出肝脏Cyp3a11与Cyp26a1作为关键的视黄醇代谢相关基因，其在5/6Nx小鼠中的表达水平出现下调。我们的研究数据表明，肾脏与肝脏之间代谢节律紊乱的负向级联反应会加重肾功能障碍。本研究于术后8周采集肾脏样本，检测了5/6肾切除小鼠喂食无视黄醇饮食与Clock基因突变小鼠之间的差异基因表达情况。4周龄雄性ICR小鼠（购自日本Charles River公司，横滨，日本）饲养于光照可控环境中，光照周期为授时因子时间（Zeitgeber time, ZT）0时至ZT12时，环境温度控制在24±1℃，湿度为60±10%，自由进食饮水。小鼠在术前于该光照条件下同步饲养2周。另有5周龄雄性ICR小鼠购自日本Charles River公司（神奈川，日本）。Clock突变小鼠（C57BL/6J-Clock^mlJt/J）购自美国杰克逊实验室（Bar Harbor, ME, USA）。我们将其回交至Jcl:ICR遗传背景以提升繁殖稳定性与幼崽照料效率，该回交过程持续至少8代。我们通过戊巴比妥钠（40 mg/kg，腹腔注射）或乙醚麻醉，采用5/6Nx手术制备慢性肾功能衰竭（chronic renal failure, CRF）小鼠模型。5/6Nx手术分两阶段进行：第一阶段手术（小鼠6周龄时）切除左肾两极的三分之二组织；7天后完全切除右肾。术后小鼠饲养8周（直至16周龄）以构建慢性肾功能衰竭模型。假手术（Sham）小鼠在与5/6Nx小鼠相同的时间点接受剖腹手术，该处理方法同样应用于Clk/Clk小鼠。无视黄醇饲料（A minus）购自KBT ORIENTAL CO., LTD.。为探究无视黄醇饮食对肾脏的影响，小鼠于术后第4周至第8周喂食该饲料。