Gene delivery into rat glomerulus using a mesangial cell vector.
To develop an effective protocol of gene transfer into glomeruli, an ex vivo gene delivery system using rat mesangial cells (RMC) as a vector was examined. RMC genetically engineered with a retrovirus harboring the Escherichia coli beta-galactosidase gene was used to estimate the efficacy of gene delivery and the location of the cells within the kidney. The RMC expressing beta-galactosidase, RMCLZ1, was cultured in vitro and the cells were injected into the left kidney through the renal artery of a normal Sprague Dawley rat. At least 1 x 10(6) RMCLZ1 was required for effective gene delivery into glomeruli. One hour and 1, 4, and 14 d after injection, glomeruli were isolated from the left kidneys injected with the cells and the expression of beta-galactosidase in each glomeruli was evaluated. One hour and 1 d after injection, more than 90 and 80%, respectively, of glomeruli from the left kidney showed strong beta-galactosidase activity, while no activity of beta-galactosidase was found in the glomeruli from the right kidneys. The number of glomeruli stained by X-gal and the intensity decreased with time. Fourteen days after injection, about 35% of the glomeruli retained the RMCLZ1. X-gal and periodic acid-Schiff staining of frozen sections obtained 14 d after injection allowed the estimation of the site where the mesangial cells injected were located. The mesangial cells were found mainly in two different locations, the glomerular capillary and the mesangium. The majority (about 90%) of the mesangial cells were located in the glomerular capillary and about 9% of the cells were in the mesangial area. Occasionally, the positive staining was found in proximal tubules and the interlobular artery. Although additional methods are required for the site-specific targeting of the mesangial area, the ex vivo gene transfer to glomeruli is feasible and may be a useful tool for future investigations in the pathological mechanisms of glomerular injury.
Kim
H J
HJ
Hyonam Kidney Laboratory, Soon Chun Hyang University, Seoul, Korea.
Kim
S I
SI
Yun
I J
IJ
Kwak
J H
JH
Yu
S H
SH
D000818:Animals; D018014:Gene Transfer Techniques / Q000592:standards*; D017930:Genes, Reporter; D005822:Genetic Vectors; D005920:Glomerular Mesangium / Q000166:cytology* / Q000637:transplantation; D007678:Kidney Glomerulus / Q000033:anatomy & histology / Q000166:cytology / Q000378:metabolism*; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D012190:Retroviridae / Q000235:genetics; D013997:Time Factors; D014018:Tissue Distribution; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D001616:beta-Galactosidase