Alterations of slit diaphragm proteins and the pathogenesis of albuminuria and FSGS

大多数儿童和青少年类固醇耐药肾病综合征（SRNS）和局灶节段性肾小球硬化（FSGS）病例是由单基因突变引起的。虽然FSGS的单基因原因的概率随着年龄的增长而下降，但遗传易感性在成年期散发形式的FSGS中也有明显的作用。虽然在过去的二十年里，在对SRNS/FSGS的遗传基础的理解方面取得了惊人的突破，但在描述了编码狭缝隔膜（SD）蛋白nephrin和podocin的基因中的基因缺陷作为SRNS/FSGS的原因之后的二十年里，SRNS/FSGS发展的分子基础远未被理解。在第一个资助期内，我们使用CRISPR/Cas基因组工程来开发新的小鼠模型，这些模型在遗传和表型上模仿人类SRNS。这些模型进行了细致的研究，超分辨率（STED）显微镜和定量形态分析。数学模型揭示了令人惊讶的发现，这些发现将改变我们对蛋白尿的看法。我们发现裂孔隔膜缩短先于白蛋白尿的发生，裂孔隔膜长度与白蛋白丢失的程度呈负相关，这是一个非常意外的发现。此外，疾病中肾小球滤过屏障的形态学改变似乎损害了抵消滤过压的压缩力，这导致肾小球基底膜压缩减少，最终导致蛋白尿。这些数据产生了第一个实验验证的肾小球超滤模型。该后续研究项目的总体目标是阐明将基因突变与这些初始形态学改变联系起来的致病机制，并应用深度学习算法来更深入地了解这些变化。因此，该项目继续致力于最新的突破性技术成果。具体而言，我们将（1）阐明决定SD和足细胞足突形态学特性定量变化的机制，（2）使用深度学习算法和计算建模破译肾滤过的生物物理机制，以及（3）表征SD和足突形态学改变对FSGS遗传易感性的影响。我们预计，该项目中应用的创新技术和模型的独特组合将导致延迟发病遗传形式的FSGS的潜在治疗策略。

The majority of cases of steroid-resistant nephrotic syndrome (SRNS) and focal segmental glomerulosclerosis (FSGS) in childhood and adolescence is caused by mutations in single genes. While the probability of single-gene causes of FSGS declines with age there is a clear role for genetic susceptibility also in sporadic forms of FSGS in adulthood. Although the past two decades have witnessed spectacular breakthroughs in the understanding of the genetic basis of SRNS/FSGS, two decades after the description of gene defects in the genes encoding for the slit diaphragm (SD) proteins nephrin and podocin as a cause of SRNS/FSGS, the molecular basis of SRNS/FSGS development is far from being understood. In the first funding period we used CRISPR/Cas genome engineering to develop new mouse models that genetically and phenotypically mimick human SRNS. These models were meticulously studied by super-resolution (STED) microscopy and quantitative morphological analyses. Mathematical modeling revealed surprising discoveries that will change the way how we think about albuminuria. We showed that shortening of the slit diaphragm precedes albuminuria development and that slit diaphragm length inversely correlated with the magnitude of albumin loss, a very unexpected finding. Moreover, morphological alterations of the glomerular filtration barrier in disease appeared to impair compressive forces that counteract filtration pressure which resulted in reduced compression of the glomerular basement membrane and ultimately in albuminuria. These data resulted in the first experimentally validated model of glomerular ultrafiltration. The overall aim of this follow-up research project is to elucidate the pathogenic mechanisms that link the genetic mutation with these initial morphologic alterations and to apply deep learning algorithms to gain a deeper understanding of those changes. As such, this project continues to work on most recent breakthrough technical achievements. Specifically, we will (1) elucidate the mechanisms that determine the quantitative changes in the morphological properties of the SD and of podocytes’ foot processes, (2) decipher the biophysical mechanisms of renal filtration using deep learning algorithms and computational modelling, and (3) characterize the impact of altered SD and foot process morphology on genetic susceptibility for FSGS. We anticipate that this unique combination of innovative technologies and models applied in this project will result in potential treatment strategies in delayed-onset genetic forms of FSGS.