Gene expression and molecular mechanism of Annexins on urolithiasis

目的：肾结石的形成是一个复杂的过程，许多分子参与了这一级联反应。新形成的晶体附着在肾上皮细胞上似乎是这一级联反应发展的关键步骤。膜联蛋白是一种钙结合分子，受草酸钙晶体的上调。Annexin V通常被用作探针来证明磷脂酰丝氨酸暴露在凋亡细胞的表面。为了更详细地了解膜联蛋白在尿石症和肾上皮损伤中的作用，我们检测了成石大鼠肾脏细胞凋亡的发生率和凋亡相关基因的表达。材料和方法：雄性Wistar大鼠饮水中给予乙二醇(EG)，强制喂饲1α-OH-D_3。通过末端脱氧核苷酸转移酶介导的dUTP-生物素缺口末端标记…定位凋亡细胞更多的是LING(TUNEL)方法。结果：对照组和给药后第1天TUNEL法未检测到细胞凋亡相关基因的表达，第3天开始在外髓质肾小管上皮细胞中检测到标记细胞，且标记细胞数在观察期内逐渐增多。给药2周后，大鼠肾脏出现DNA梯状条带。免疫组织化学染色显示Fas配体(Fas L)、bax和白介素1β转换酶(ICE)在Henle环降支和远曲小管肾小管上皮细胞中的表达。结论：在EG和1α-OH-D3诱导的高草酸血症大鼠肾结石模型中，肾上皮细胞发生了细胞凋亡，以Henle环降支和远端曲小管的凋亡最为明显。逆转录聚合酶链式反应和免疫组织化学分析显示Fas-L、ICE、bax、c-myc和P53蛋白表达上调。这些结果表明，EG和1α-OH-D3致结石大鼠肾上皮损伤可诱导上述细胞凋亡相关基因的表达，且不同途径可诱导细胞凋亡。较少

Purpose : Kidney stone formation is a complex process, and numerous molecules participate in this cascade. Attachment of newly formed crystals to renal epithelial cells appears to be a critical step in the development of this cascade. Annexins are one of the calcium binding molecules and up-regulated by calcium oxalate crystals. Annexin V is routinely used as a probe to demonstrate phosphatidylserine exposed on the surface of apoptotic cells. To obtain more detailed insight into annexins on urolithiasis and renal epithelial injury, we examined an incidence of apoptotic cells and expression of apoptosis related genes in kidneys of stone-forming rat.Materials and Methods : Male Wistar rats were administrated ethylene glycol (EG) in drinking water and force fed withl alpha-OH-D_3. Apoptosis was detected as a ladder of fragmented DNA in agarose gels of electrophoresed genomic DNA. Apoptotic cells were localized by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labe … More ling (TUNEL) method. Expression of apoptosis-related genes was analyzed by both reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry.Results : While no labeling was detected in the control or the first day of administration by the TUNEL method, labeling began to be detected in the renal tubular epithelium of the outer medulla at day 3, and the number of labeled cells increased progressively during the observation period. A ladder of DNA fragments was demonstrated in the kidneys of rats administrated after 2 weeks. Immunohistochemical studies revealed the expressions of Fas ligand (Fas L), bax and interleukin-1 beta converting enzyme (ICE) in the renal tubular epithelium of the descending limb of loop of Henle and distal convoluted tubules. mRNA of ICE, c-myc, p53 and Fas L genes were also up-regulated in the kidneys of stone forming-rat model.Conclusion : In the hyperoxaluric, subsequent stone-forming rat kidney caused by the administration of EG and 1 alpha-OH-D_3, apoptosis was detected in the renal epithelial cells, especially in those of the descending limb of the loop of Henle and distal convoluted tubules. RT-PCR and immunohistochemical analyses revealed up-regulations of Fas-L, ICE, Bax, c-myc and p53. These findings indicate that renal epithelial injury in the stone-forming rat model following EG and 1 alpha-OH-D_3 feeding may induce the expression of those apoptosis-related genes, and different pathways in the stone-forming rat model may induce apoptosis. Less