Mechanisms of the development of mesangial proliferative glomerulonephritis via constitutive-active expression of IKK2 in juxtaglomerular renin precursor cells

肾小球外/肾小球内肾素系细胞（Extra-/acetaglomerular renin lineage cells，RLC）是肾小球损伤后迁移到肾小球内替代系膜细胞的系膜前体细胞。因此，RLC必须接收来自肾小球内过程（损伤、细胞死亡、炎症）的信号，并通过失去肾素产生、迁移、增殖和系膜分化/肌成纤维细胞活化来反应。目前还不清楚哪些细胞外和细胞内信号对RLC的这种表型变化是必不可少的，以及这种主要的再生机制是否也会导致系膜增生性肾小球肾炎。为了解决这个问题的实验，在一个公正的方法，我们差异化的RLC之前和期间，我们的小鼠系膜溶解性肾小球肾炎模型在体内之前和期间的肾小球修复反应的转录组分析。在迁移过程中显示早期TNF-α和持续的核因子κB（NF-κB）刺激。在使用肾素谱系细胞系As 4.1的体外研究中，已经证明TNF-α/NF-κB系统关闭肾素产生并导致向活化的系膜细胞/肌成纤维细胞的表型变化。通过建立一种新的转基因小鼠系，诱导NF-κB激活剂IKK 2特异性地在RLC中组成型活性表达，不仅观察到肾小球装置的变化，而且还观察到致密斑细胞的激活和系膜增生性肾小球肾炎。NF-κB是一个广泛分布的转录因子家族，它作为一个“传感器系统”被细胞外的炎症信号激活，特别是改变细胞表型，使其分别向存活、增殖、侵袭/迁移和肌成纤维细胞分化和激活的方向发展。通过本申请，我们想检验主要假设，即RLC中IKK 2的组成型活性表达（通过NF-κB活化）足以通过诱导表型变化引起系膜增生性肾小球肾炎，所述表型变化从肾小球肾素产生细胞变为肾素阴性、迁移、增殖、成肌纤维细胞活化和系膜分化的肾小球内细胞。通过流式细胞仪分选、组织学和活体显微镜细胞示踪方法，我们将研究选择性激活的RLC是否以及如何主动迁移到肾小球。机制和因素将分别进行评估和细胞特征的RLC，肾小球以及与相邻的致密斑细胞的相互作用。同时，使用鼠肾素谱系细胞系As4.1的体外研究将分析转染后不同IKK 2构建体的表型影响。此外，我们想探讨的问题是否TNF-α/NF-κB激活肾小球生态位是不同的人类肾脏疾病的验证。

Extra-/juxtaglomerular renin lineage cells (RLC) are mesangial precursor cells migrating into the glomerulus to replace mesangial cells after glomerular injury. Hereby, the RLC must receive signals from the intraglomerular processes (injury, cell death, inflammation) and react by loss of renin production, immigration, proliferation and mesangial differentiation / myofibroblast activation. It is unclear which extracellular and intracellular signals are essential for this phenotypical change of the RLC and whether this primarily regenerative mechanism can also lead pathogenetically to a mesangial proliferative glomerulonephritis. To solve this question experimentally, in an unbiased approach we differentially characterized the RLC before and during our murine mesangiolytic glomerulonephritis model in vivo before and during the glomerular repair reaction by transcriptome analysis. An early TNF-α and continuing nuclear factor κB (NF-κB) stimulation were shown during immigration. In in vitro studies using the renin lineage cell line As4.1, it was already demonstrated that the TNF-α/NF-κB system shuts down renin production and causes a phenotypical change towards activated mesangial cells/myofibroblasts. By creating a novel transgene mouse line with induction of the constitutive-active expression of the NF-κB activator IKK2 specifically in RLC, not only changes of the juxtaglomerular apparatus, but also activation of macula densa cells and a mesangial proliferative glomerulonephritis were observed. NF-κB represents a ubiquitously distributed family of transcription factors that are activated especially by inflammatory signals from extracellular area in terms of a “sensor system” and particularly change the cell phenotype toward survival, proliferation, invasion/migration and myofibroblast differentiation and activation respectively. With this application, we would like to test the main hypothesis that solely the constitutive-active expression of IKK2 (via NF-κB activation) in RLC is sufficient to cause a mesangial proliferative glomerulonephritis via induction of a phenotypical change from a juxtaglomerular renin producing cell to a renin-negative, migrating, proliferative, myofibroblast-activated and mesangial differentiated intraglomerular cell. By FACS sorting, histological and intravital microscopic cell tracing methods, we will examine whether and how the selectively activated RLC actively immigrate into the glomerulus. Mechanisms and factors will be separately evaluated and cellularly characterized concerning the RLC, the glomerulus as well as the interaction with the neighboring macula densa cells. In parallel, in vitro studies using the murine renin lineage cell line As4.1 will analyze the phenotypical impact of different IKK2 constructs after transfection. In addition, we would like to explore the question whether a TNF-α/NF-κB activation in the juxtaglomerular niche is verifiable with different human renal diseases.