title: Dynamics of absolute amount of nephrin in a single podocyte in puromycin aminonucleoside nephrosis rats calculated by quantitative glomerular proteomics approach with selected reaction monitoring mode.
abstract: 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.
authors.affiliation: Laboratory of Veterinary Pathology, Faculty of Veterinary Medicine, Azabu University, Sagamihara, Japan.
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Kawakami
authors.forename: Junichi
authors.initials: J
authors.lastname: Kamiie
authors.forename: Kyohei
authors.initials: K
authors.lastname: Yasuno
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Kobayashi
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Aihara
authors.forename: Kinji
authors.initials: K
authors.lastname: Shirota
mesh_terms: D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000633:toxicity*; D007678:Kidney Glomerulus / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000652:urine*; D009401:Nephrosis / Q000139:chemically induced / Q000652:urine*; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D050199:Podocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D040901:Proteomics; D011692:Puromycin Aminonucleoside / Q000633:toxicity*; D051381:Rats; D011916:Rats, Inbred F344; D053719:Tandem Mass Spectrometry; D025721:WT1 Proteins / Q000652:urine
chemical_list: D000903:Antibiotics, Antineoplastic; D008565:Membrane Proteins; D010446:Peptide Fragments; D025721:WT1 Proteins; C113267:nephrin; D011692:Puromycin Aminonucleoside