The glomeruli are unique filtration units. The glomerular endothelium has a high hydraulic conductivity in the context of a high reflection co-efficient. Intact isolated human glomeruli are amenable to both molecular (Whittle et al. 1999) and functional (Savin et al. 1992) study ex vivo. Although gene transfer into rodent glomeruli has been accomplished (Ye et al. 2002), gene transfer ex vivo in human glomeruli has not been published. We therefore studied gene transfer techniques on isolated human glomeruli with the aim of establishing a method for the manipulation of glomerular gene products for dynamic studies.

Glomeruli were isolated from the normal pole of human nephrectomy specimens removed for renal cell carcinoma. All patients gave informed consent and the study was approved by the North Bristol NHS Trust Ethical committee. Glomeruli were collected by standard sieving techniques. The glomeruli were washed in phosphate-buffered saline and cultured in RPMI medium supplemented with L-glutamine, 10 % (v/v) fetal calf serum (FCS), penicillin/streptomycin and Insulin Transferrin Sodium Selenite (Sigma). Fifty glomeruli were transferred to a well of a 24-well cell culture plate as a droplet. 1 X 10⁷ plaque-forming units (PFU) (multiplicity of infection, 100) of Adenovirus expressing red fluorescent protein (Ad-RFP) were added to the droplet and incubated for 60 min at 37°C, 5 % CO₂. After incubation the glomeruli were washed three times with RPMI/10 % (v/v) FCS, allowing 5 min in between washes for the glomeruli to settle. RPMI/10 % (v/v) FCS was added to the glomeruli, which were incubated overnight at 37°C, 5 % CO₂. Areas of infection of the glomeruli were visualised using confocal microscopy, 21 sections of 5 µm thick were imaged.

Glomerular cells infected with Ad-RFP fluoresced red. All cell types within the glomerulus appeared to have been infected and expressed the RFP.

Despite the difficulty of gene transfer into particular glomerular cells this protocol does indicate that exogenous proteins may be expressed in the glomeruli and the local micro-environment of the glomerular filtration barrier. We suggest that this simple and reliable protocol for gene transfer into human glomeruli ex vivo allows functional studies to be performed on the same glomeruli (normal or diseased) over time after specific manipulations.

The authors are grateful to Alan Leard for help with confocal microscopy and to Professor David Murphy for the gift of the Ad-RFP. This work was supported by The Wellcome Trust (grant number 57936).