RENIN PROCESSING IN RENAL DEVELOPMENT

作为肾素-血管紧张素系统的第一和限速酶， 肾素在心血管稳态的控制中具有重要作用。 调节肾素基因表达的分子机制的定义 以及随后的细胞内合成，分选， 活性肾素的运输、加工、储存和最终释放 对了解肾素-血管紧张素系统至关重要。 大部分 调节细胞内肾素近端处理的分子机制 其分泌部位仍有待确定。 在正常成人肾脏中， 肾素由肾小球（JG）细胞合成。 但在 在发育早期，肾素在弓状和小叶间表达， 动脉 如果一只成年动物被转化酶 抑制时，存在肾素基因表达和释放的募集 细胞沿着肾小球前血管，类似于 未成熟的动物 我们假设平滑肌细胞 从肾动脉树的平滑肌细胞（SMC）打开和关闭肾素基因是 由结合到5'端的组织特异性横断因子介导 肾素基因的侧翼区。 我们建议隔离和 表征这些因子并鉴定顺式调节序列 参与了回应。 天然和突变的细胞的共转染研究 具有横切因子的肾素启动子表达构建体将允许 我们评估所涉及序列的功能意义。 的 负责细胞内处理的分子机制 对肾素尚未进行详细研究。 突变的前，或 将产生肾素基因的原和肾素序列， 引入AT-T20细胞，并使用EM显微镜， 贩运或肾素将受到调查。 这些研究将决定 肾素基因中的特定序列是否负责 在细胞内对肾素分子进行分选。 类似地，使用抗体 针对分子的特定结构域（前，原，肾素），我们 将决定处理肾素的特定细胞器。 进一步的研究将探讨肾素基因与 转录（通过插入序列探针测量）至肾素释放 来自通过反向溶血空斑试验检测的单个细胞。 导致肾素释放的细胞膜和胞浆事件将是 血管紧张素II、β受体激动剂和 拮抗剂和钙浓度的变化。 拟议 实验将增加我们对分子过程的理解 调节肾素的生物合成、运输和释放。

As the first and rate limiting enzyme of the renin-angiotensin system, renin has an important role in the control of cardiovascular homeostasis. Definition of the molecular mechanisms regulating renin gene expression and the subsequent intracellular events of synthesis, sorting, trafficking, processing, storage and finally release of the active renin is essential for understanding the renin-angiotensin system. Most of the molecular mechanisms regulating intracellular handling of renin proximal to its secretion site remain to be defined. In the normal adult kidney, renin is synthesized by the juxtaglomerular (JG) cells. However, during early development, renin is expressed in arcuate and interlobular arteries. If an adult animal is subjected to converting enzyme inhibition, there is a recruitment of renin gene expressing and releasing cells along the preglomerular vessels, resembling the situation in the immature animal. We hypothesize that the ability of smooth muscle cells (SMCs) from the renal arterial tree to turn on and off the renin gene is mediated by tissue specific transecting factors binding to the 5' flanking region of the renin gene. We propose to isolate and characterize those factors and to identify the Cis regulatory sequences involved in the response. Co-transfection studies of native and mutated renin promoter expression constructs with transecting factors will allow us to assess the functional significance of sequences involved. The molecular mechanisms responsible for the intracellular processing of renin have not been investigated in detail. Mutations of the pre-, or pro- and renin sequences of the renin gene will be produced and introduced into AT-T20 cells and, using EM microscopy, the intracellular trafficking or renin will be investigated. These studies will determine whether specific sequences within the renin gene are responsible for intracellular sorting of the renin molecule. Similarly, using antibodies directed against specific domains (pre, pro, renin) of the molecule, we will determine the specific organelles where renin is processed. Additional studies will examine the relationship of renin gene transcription (measured by intervening sequence probes) to renin release from individual cells detected by the reverse hemolytic plaque assay. The cell membrane and cytosolic events leading to renin release will be investigated in response to angiotensin II, beta-receptor agonists and antagonists and changes in the calcium concentration. The proposed experiments will increase our understanding of the molecular processes regulating renin biosynthesis, trafficking and release.