PDF(Pubmed)
Abstract：
The most common genetic causes of steroid-resistant nephrotic syndrome (SRNS) are mutations in the NPHS2 gene, which encodes the cholesterol-binding, lipid-raft associated protein podocin. Mass spectrometry and cDNA sequencing revealed the existence of a second shorter isoform in the human kidney in addition to the well-studied canonical full-length protein. Distinct subcellular localization of the shorter isoform that lacks part of the conserved PHB domain suggested a physiological role. Here, we analyzed whether this protein can substitute for the canonical full-length protein. The short isoform of podocin is not found in other organisms except humans. We therefore analysed a mouse line expressing the equivalent podocin isoform (podocinΔexon5) by CRISPR/Cas-mediated genome editing. We characterized the phenotype of these mice expressing podocinΔexon5 and used targeted mass spectrometry and qPCR to compare protein and mRNA levels of podocinwildtype and podocinΔexon5. After immunolabeling slit diaphragm components, STED microscopy was applied to visualize alterations of the podocytes\' foot process morphology.Mice homozygous for podocinΔexon5 were born heavily albuminuric and did not survive past the first 24 h after birth. Targeted mass spectrometry revealed massively decreased protein levels of podocinΔexon5, whereas mRNA abundance was not different from the canonical form of podocin. STED microscopy revealed the complete absence of podocin at the podocytes\' slit diaphragm and severe morphological alterations of podocyte foot processes. Mice heterozygous for podocinΔexon5 were phenotypically and morphologically unaffected despite decreased podocin and nephrin protein levels.The murine equivalent to the human short isoform of podocin cannot stabilize the lipid-protein complex at the podocyte slit diaphragm. Reduction of podocin levels at the site of the slit diaphragm complex has a detrimental effect on podocyte function and morphology. It is associated with decreased protein abundance of nephrin, the central component of the filtration-slit forming slit diaphragm protein complex.
摘要：
激素耐药型肾病综合征（SRNS）最常见的遗传原因是NPHS2基因的突变，编码胆固醇结合，脂筏相关蛋白podocin。质谱和cDNA测序显示，除了经过充分研究的经典全长蛋白外，人类肾脏中还存在第二种较短的同工型。缺乏部分保守PHB结构域的较短同种型的不同亚细胞定位表明了生理作用。这里,我们分析了这种蛋白质是否可以替代典型的全长蛋白质。在除人类以外的其他生物中未发现podocin的短同工型。因此，我们通过CRISPR/Cas介导的基因组编辑分析了表达等效podocin同工型（podocinΔexon5）的小鼠系。我们表征了这些表达podocinΔexon5的小鼠的表型，并使用靶向质谱法和qPCR来比较podocinwildtype和podocinΔexon5的蛋白质和mRNA水平。免疫标记狭缝隔膜组件后，应用STED显微镜观察足细胞足过程形态的变化。podocinΔexon5纯合子的小鼠出生时白蛋白含量很高，并且在出生后的前24小时内无法存活。靶向质谱显示podocinΔexon5的蛋白质水平大大降低，而mRNA丰度与podocin的规范形式没有差异。STED显微镜显示足细胞的狭缝隔膜完全不存在足细胞素，足细胞足过程的形态严重改变。尽管podocin和nephrin蛋白水平降低，但podocinΔexon5杂合子的小鼠在表型和形态学上均不受影响。与人短亚型podocin等效的鼠不能稳定足细胞狭缝隔膜处的脂质-蛋白质复合物。狭缝光阑复合物位点处的足细胞素水平的降低对足细胞功能和形态具有不利影响。它与nephrin的蛋白质丰度降低有关，过滤狭缝形成狭缝隔膜蛋白复合物的中心成分。
