Determining the Role of Canonical Notch Signalling in the Regulation of the Glucocorticoid Receptor in Steroid Resistant Nephrotic Syndrome

肾小球的瘢痕形成可导致终末期肾病。糖皮质激素治疗有有益的效果，但耐药性（GR）随着时间的推移而发展。有其他治疗方法，但有明显的副作用，只有30-60%的患者有反应。因此，需要新的治疗方法。最近，我们发现一个关键信号通路（Notch）的激活参与了肾小球瘢痕形成的启动。通过敲除小鼠模型和阻断该通路的药物，我们和其他人已经证明肾小球瘢痕形成是可以预防的。对白血病患者的研究表明，Notch活性可抑制糖皮质激素反应，抑制Notch可逆转这一效应。我的初步数据显示，糖皮质激素受体NR3C1在体外和体内的对照人肾小球细胞中表达，在类固醇抵抗患者衍生的人肾小球细胞中表达下调。由于Notch效应基因HES-1的激活已被证明下调NR3C1并介导白血病中GR的发展，我观察到在肾病综合征患者肾活检组织中硬化肾小球足细胞中HES-1的暂时上调，这些患者最初对糖皮质激素敏感，随后发展为GR。这些数据支持Notch激活在类固醇抵抗性肾病综合征中的作用。我的研究目标将包括测试一些基本原理。首先，我想证明糖皮质激素反应性基因表达在接受糖皮质激素治疗时发生在肾小球上皮细胞中，然后确定来自类固醇抵抗性肾病综合征（SRNS）患者肾活检的肾小球细胞中缺乏相同基因的表达。接下来，我建议确定SRNS患者足细胞系中Notch信号通路组分的表达是否上调。如果是这种情况，我将耗尽Notch效应基因，以确定我是否可以挽救糖皮质激素反应基因的表达。其次，我将使用遗传形式肾小球硬化（肾小球瘢痕形成）的小鼠模型，以确定肾小球上皮细胞中糖皮质激素反应性基因表达的缺失是否与肾小球硬化的发展有关，以及这是否也与Notch通路成分的上调有关。接下来，我将专门探讨遗传性肾小球硬化小鼠模型中的继发性Notch激活是否与肾小球上皮细胞中糖皮质激素反应性基因表达的丧失有关——通过产生Notch激活的转基因小鼠，并检查Notch激活导致的肾小球瘢痕形成是否与肾小球上皮细胞中糖皮质激素反应性基因表达的丧失有关，将有助于这一点。如果这被证明是正确的，抑制Notch通路的化学物质将被用于肾小球硬化遗传形式的小鼠模型，以观察是否有可能在肾小球瘢痕愈合的同时挽救糖皮质激素反应。与此同时，为了进一步明确肾小球硬化的分子机制，我们将与合作者一起对一大批肾病综合征患者进行遗传分析，这可能为肾病综合征的治疗策略开辟新的研究途径。

Scarring of the glomerulus can cause terminal kidney disease. Glucocorticoid treatment has a beneficial effect but resistance (GR) develops over time. Alternative treatments are available with significant side effects and only 30-60% of patients respond. Consequently, new treatments are needed. Recently, we have shown that activation of a critical signalling pathway (Notch) is involved in initiating glomerular scarring. Using knockout mouse models and drugs which block the pathway, we and others, have shown that glomerular scarring can be prevented. Studies in leukaemic patients have shown that Notch activity can inhibit the glucocorticoid response, an effect which can be reversed by inhibiting Notch. My preliminary data shows that the glucocorticoid receptor, NR3C1, is expressed in control human glomerular cells in vitro and in vivo and is downregulated in human glomerular cells derived from patients with steroid-resistance. As activation of the Notch effector gene, HES-1, has been shown to downregulate NR3C1 and mediate the development of GR in leukaemia, I have observed that HES-1 is temporally upregulated in podocytes of sclerosed glomeruli in kidney biopsy tissue in patients with nephrotic syndrome who have been initially glucocorticoid sensitive and subsequently developed GR. These data support a role for Notch activation in steroid-resistant nephrotic syndrome.My research objectives will involve testing a few basic principles.Firstly, I would like to demonstrate that glucocorticoid-responsive gene expression occurs in glomerular epithelial cells when treated with glucocorticoid therapy and then determine, absence of expression of the same genes in glomerular cells derived from kidney biopsies of steroid resistant nephrotic syndrome (SRNS) patients. Next, I propose to determine whether expression of components of the Notch signalling pathway are upregulated in podocyte cell lines of SRNS patients. If this is the case, I will then deplete Notch effector genes to determine if I can rescue expression of glucocorticoid-responsive genes.Secondly, I will use mouse models of genetic forms of glomerulosclerosis (scarring of the glomerulus), to determine whether loss of glucocorticoid-responsive gene expression in glomerular epithelial cells is associated with the development of glomerulosclerosis and whether this is also associated with an up regulation of Notch pathway components.I will next specifically explore whether secondary Notch activation in mouse models of genetic forms of glomerulosclerosis is associated with loss of glucocorticoid-responsive gene expression in glomerular epithelial cells - this will be facilitated by generating transgenic mice of Notch activation and examining whether onset of glomerular scarring as a result of Notch activation is associated with loss of glucocorticoid-responsive gene expression in glomerular epithelial cells. If this proves true, chemicals which inhibit the Notch pathway will be used in mouse models of genetic forms of glomerulosclerosis to see whether it is possible to rescue glucocorticoid-responsiveness concommitant with resolution of glomerular scarring.In parallel, genetic analysis of a large cohort of patients with nephrotic syndrome will be undertaken with collaborators in an attempt to further define molecular mechanisms of glomerulosclerosis which may open up new avenues of investigation for therapeutic strategies in nephrotic syndrome.