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：血压调节激素醛固酮在体外对肾脏细胞具有纳摩尔浓度的遗传毒性，并引起醛固酮处理的动物肾脏的DNA损伤。流行病学研究发现，高血压患者患肾癌的风险增加。在这些患者中，多达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对其主要靶细胞--远端小管细胞--DNA造成损伤，而且对导致大多数肾脏肿瘤的近端小管细胞也有损伤。检测到抗凋亡蛋白的诱导和促凋亡蛋白的减少，这支持了我们关于醛固酮具有致瘤潜力的假设。醛固酮还激活抗氧化性细胞防御的主要调节器--转录因子Nrf2。由异硫氰酸盐萝卜硫酸酯对Nrf2的更明显的激活保护了接受醛固酮治疗的动物的肾脏免受损害，并降低了他们的血压。项目内容：使用肾脏细胞中Nrf2的抑制物Keap1的特定缺失的小鼠，这将在上游先导实验中确定，问题将被解决，如果局部定义的Nrf2的激活足以保护肾脏细胞免受醛固酮诱导的损伤。这只小鼠还将显示这种激活是否有助于降低血压，或者是否需要全身激活Nrf2来实现这一效果。有了磷酸蛋白阵列，小鼠肾脏组织将被筛选出受醛固酮调节的信号转导通路。将在小鼠和大鼠的肾组织中检测到实质上受调控的候选蛋白的激活/抑制和肾脏定位。此外，如果已知的支持生存的信号和阵列识别的信号的激活发生在同时显示DNA损伤的细胞中，将对其进行测试。重点将在低剂量范围内研究上述效应，即高血压患者血浆中可预期的剂量。目的：本项目的预期结果将有助于阐明肾脏抗氧化防御的作用。目前，NRF2激活剂作为治疗急、慢性肾功能衰竭的可能药物引起了人们的极大兴趣。在这里，我们可以通过机制数据来帮助我们理解Nrf2在肾脏中的作用。通过分析信号通路获得的发现可能定义下一步澄清内源性物质是否可以作为致癌启动因素，在自发发展的恶性肿瘤中发挥作用的问题。

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.