Identification of target cells for aldosterone-induced genotoxicity and characterisation of aldosterone effects with regard to the triggering of pro-survival pathways in kidney cells

背景：调节血压的激素醛固酮在纳摩尔浓度的体外肾细胞中具有遗传毒性，并引起醛固酮治疗动物肾脏的DNA损伤。流行病学研究发现高血压患者患肾癌的风险增加。其中13-20%的患者血浆醛固酮水平升高。在上一个项目期间，我们发现醛固酮不仅在其主要靶细胞远端小管细胞中引起DNA损伤，而且在近端小管细胞中也引起DNA损伤，这是大多数肾脏肿瘤的发生原因。检测到抗凋亡诱导和促凋亡蛋白的减少，支持我们的假设，醛固酮的致瘤潜能。醛固酮还能激活抗氧化细胞防御的主要调节因子Nrf2。异硫氰酸萝卜硫素更明显地激活了Nrf2，保护了醛固酮治疗动物的肾脏免受损害，并降低了它们的血压。项目内容：在上游试点实验中，使用具有Nrf2抑制剂Keap1特异性缺失的小鼠肾细胞，将解决Nrf2局部激活是否足以保护肾细胞免受醛固酮诱导的损伤的问题。这只小鼠也将显示这种激活是否有助于降低血压，或者是否需要Nrf2的全身激活才能达到这种效果。利用磷酸蛋白阵列，小鼠肾组织将筛选醛固酮调节的信号转导途径。将在小鼠和大鼠的肾组织中检测到大量调节的候选蛋白的激活/抑制和肾定位。此外，它将被测试，如果已知的促生存信号的激活，以及由阵列识别的信号，发生在同时显示DNA损伤的细胞中。重点将放在低剂量范围内的上述效应的研究，这些剂量可以在高血压个体的血浆中预期。目的：本项目的预期结果将有助于阐明抗氧化防御在肾脏中的作用。目前，人们对Nrf2激活剂作为治疗急性和慢性肾衰竭的可能药物有很大的兴趣。在这里，我们可以通过力学数据来帮助理解Nrf2在肾脏中的作用。通过分析信号通路获得的发现可能会明确下一步的问题，即内源性物质是否可以作为致癌启动因子，在自发发展的恶性肿瘤中发挥作用。

Background: The blood pressure-regulating hormone aldosterone is genotoxic in vitro in kidney cells in nanomolar concentrations, and provoked DNA damage in kidneys of aldosterone-treated animals. Epidemiological studies have found an increased kidney cancer risk in hypertensive patients. In up to 13-20% of these patients plasma aldosterone levels are elevated. During the last project period, we found DNA damage caused by aldosterone not only in its primary target cells, cells of the distal tubulus, but also in cells of the proximal tubulus, which give rise to the majority of kidney tumors. An induction of anti-apoptotic and a decrease of pro-apoptotic proteins was detected, supporting our hypothesis of a tumorigenic potential of aldosterone. Aldosterone also activates the main regulator of the antioxidative cellular defense, the transcription factor Nrf2. An even more pronounced activation of Nrf2 by the isothiocyanate sulforaphane protected kidneys of aldosterone-treated animals from damage and lowered their blood pressure.Project content: Using mice with a specific deletion of the inhibitor of Nrf2, Keap1, in kidney cells to be determined in an upstream pilot experiment, the question will be addressed if a locally defined activation of Nrf2 is sufficient to protect kidney cells from aldosterone-induced damage. This mouse will also show if this activation contributes to the blood pressure lowering, or if a systemic activation of Nrf2 is needed for this effect. With a phospho-protein array, mouse kidney tissue will be screened for signal transduction pathways modulated by aldosterone. The activation/inhibition and renal localisation of substantially regulated candidate proteins will be detected on kidney tissue of mice and rats. Further, it will be tested, if the activation of the already known pro-survival signals, and of signals identified by the array, takes place in cells which simultaneously show DNA damage. Emphasis will be placed on the study of the mentioned effects in low dose ranges, doses which can be expected in the plasma of hypertensive individuals.Aims: The anticipated results of the present project will help to elucidate the role of the antioxidative defense in the kidney. Currently, there is a great interest in activators of Nrf2 as possible drugs to treat acute and chronic kidney failure. Here, we can contribute to the understanding of Nrf2 effects in the kidney with mechanistical data. Findings gained from analyzing the signaling pathways might define the next steps in clarifying the question if endogenous substances can act as carcinogenesis initiating factors, playing a role in spontaneous developing malignancies.