Ultrastructural study on nephrin expression in experimental puromycin aminonucleoside nephrosis.
BACKGROUND
Nephrin is a recently identified protein that is a key component of the slit diaphragm. This protein may play a crucial role in maintaining the glomerular filtration barrier, and mutations in the gene for nephrin reportedly lead to congenital nephrosis. However, the expression of nephrin in acquired glomerular disease has not yet been fully clarified. To address this issue, we analysed the expression and localization of nephrin by morphological analysis based on immunoelectron microscopy in normal glomeruli and in glomeruli from proteinuric experimental models.


METHODS
Twenty rats were divided into three experimental groups (n = 16 total) and a control group (n = 4). Rats in the experimental groups received a single intravenous injection of puromycin aminonucleoside (PAN), and were sacrificed at 1 (n = 4), 2 (n = 6) and 3 weeks (n = 6) post-injection. Nephrin expression was assessed by immunoelectron microscopy using a polyclonal antibody against nephrin and gold particles. It was quantified by counting the gold particles and the slit diaphragms and by measuring the average foot process width in microphotographs.


RESULTS
The average foot process width in the 1 week group (5924.5 +/- 1523.9 nm) was far greater than that of controls (1112.9 +/- 79.8 nm), but decreased thereafter. The average number of total gold particles per unit length (10 000 nm) of the glomerular basement membrane (GBM) underlying the foot processes was reduced at 1 week (26.0 +/- 9.5), compared with controls (335.3 +/- 125.5), but increased thereafter. Also, the average number of junctional gold particles per unit length of the GBM was lower than controls (208.4 +/- 1.7) at 1 week (10.1 +/- 3.5), but increased thereafter. There were no significant differences between the numbers of junctional gold particles per slit diaphragm among the groups, but significant differences were observed in the distributions of gold particles among the groups. Gold particles were more frequently seen in cytoplasm at 1 week.


CONCLUSIONS
The present ultrastructural studies showed that nephrin expression and its distribution were altered in PAN-treated rats, and this occurred in parallel with foot process effacement. Nephrin expression returned to normal with improved resolution of the effacement. Nephrin expression was found to be rather preserved in areas without foot process effacement, even in PAN-treated rats. The significance of the above findings in terms of proteinuria and foot process effacement needs further clarification.
Lee
Young Ki
YK
Division of Nephrology, Department of Medicine, Samsung Medical Center, 50 Ilwondong, Kangnamgoo, Seoul, Korea 135-710.
Kwon
Taegun
T
Kim
Dae Joong
DJ
Huh
Wooseong
W
Kim
Yoon-Goo
YG
Oh
Ha-Young
HY
Kawachi
Hirashi
H
D000818:Animals; D004195:Disease Models, Animal; D007668:Kidney / Q000473:pathology / Q000648:ultrastructure; D007678:Kidney Glomerulus / Q000473:pathology / Q000648:ultrastructure; D008297:Male; D008565:Membrane Proteins; D016253:Microscopy, Immunoelectron; D009401:Nephrosis / Q000209:etiology / Q000473:pathology*; D011506:Proteins / Q000378:metabolism*; D011692:Puromycin Aminonucleoside / Q000633:toxicity; D051381:Rats; D017207:Rats, Sprague-Dawley
D008565:Membrane Proteins; D011506:Proteins; C113267:nephrin; D011692:Puromycin Aminonucleoside