Male Wistar rats were fed a potassium-free diet with or without 4-phenylbutyric acid (4-PBA) or the selective HDAC3 inhibitor RGFP966 for 4 days. Primary renal inner medullary collecting duct (IMCD) cells and immortalized mouse cortical collecting duct (mpkCCD) cells were cultured in potassium-deprivation medium with or without HDAC inhibitors.
4-PBA increased the levels of AQP2 mRNA and protein in the kidney inner medullae in hypokalaemic (HK) rats, which was associated with decreased urine output and increased urinary osmolality. The level of acetylated H3K27 (H3K27ac) protein was decreased in the inner medullae of HK rat kidneys; this decrease was mitigated by 4-PBA. The H3K27ac levels were decreased in IMCD and mpkCCD cells cultured in potassium-deprivation medium. Decreased H3K27ac in the Aqp2 promoter region was associated with reduced Aqp2 mRNA levels. HDAC3 protein expression was upregulated in mpkCCD and IMCD cells in response to potassium deprivation, and the binding of HDAC3 to the Aqp2 promoter was also increased. RGFP966 increased the levels of H3K27ac and AQP2 proteins and enhanced binding between H3K27ac and AQP2 in mpkCCD cells. Furthermore, RGFP966 reversed the hypokalaemia-induced downregulation of AQP2 and H3K27ac and alleviated polyuria in rats. RGFP966 increased interstitial osmolality in the kidney inner medullae of HK rats but did not affect urinary cAMP levels.
HDAC inhibitors prevented the downregulation of AQP2 induced by potassium deprivation, probably by enhancing H3K27 acetylation.
雄性Wistar大鼠饲喂含或不含4-苯基丁酸(4-PBA)或选择性HDAC3抑制剂RGPP966的无钾饮食4天。将原代肾髓内收集管(IMCD)细胞和永生化小鼠皮质收集管(mpkCCD)细胞在含或不含HDAC抑制剂的钾剥夺培养基中培养。
4-PBA增加低钾血症(HK)大鼠肾脏髓质中AQP2mRNA和蛋白的水平,这与尿量减少和尿渗透压增加有关。HK大鼠肾脏内部髓质中乙酰化H3K27(H3K27ac)蛋白的水平降低;4-PBA减轻了这种降低。在缺钾培养基中培养的IMCD和mpkCCD细胞中H3K27ac水平降低。Aqp2启动子区域中降低的H3K27ac与降低的Aqp2mRNA水平相关。HDAC3蛋白表达在mpkCCD和IMCD细胞中上调,以响应缺钾,HDAC3与Aqp2启动子的结合也增加。RGFP966增加了MPKCCD细胞中H3K27ac和AQP2蛋白的水平,并增强了H3K27ac和AQP2之间的结合。此外,RGFP966逆转低钾血症诱导的大鼠AQP2和H3K27ac下调并减轻多尿。RGFP966增加了HK大鼠肾脏髓质的间质渗透压,但不影响尿cAMP水平。
HDAC抑制剂阻止了钾剥夺引起的AQP2下调,可能通过增强H3K27乙酰化。