Background: The slit diaphragm (SD) is a complex of podocyte-specific proteins and plays a significant role in glomerular filtration. To understand podocyte biology, it is important to determine the expression amount of the SD complex proteins. This study aimed to quantify the absolute amount of nephrin, which is believed to be a major component of SD, in podocytes and to apply that method to normal and puromycin aminonucleoside (PAN) nephrosis rats.
Methods: The counting method for podocyte number in a glomerulus was developed by three-dimensional reconstruction imaging of Wilms tumor (WT-1) immunofluorescence on isolated glomeruli. Absolute amount of nephrin was quantified by mass spectrometry using the selected reaction monitoring (SRM) mode with a stable isotope-labeled peptide.
Results: The number of podocytes per glomerulus was 95.5±17.6 in the control rats, 90.7±19.2 on Day 4 and 90.7±26.2 on Day 7 in PAN nephrosis rats. The amount of nephrin per glomerulus in control rats was 1.02±0.11 fmol and those in PAN nephrosis rats were reduced to 0.46±0.06 fmol and 0.35±0.04 fmol on Day 4 and Day 7. The nephrin amount per podocyte was significantly decreased association with the development of proteinuria in PAN nephrosis rats.
Conclusions: This study established the absolute quantification of nephrin and determined the amount of nephrin in a podocyte of normal and PAN nephrosis rat kidneys. This highly sensitive and selective quantification method for protein is a useful tool for the analysis of SD protein in a podocyte.