Xuehai Ye1, Marina Jerebtsova, Xue-Hui Liu, Zhuangwu Li, Patricio E Ray. 1. Centers for Genetic Medicine and Molecular Physiology, Children's Research Institute, Children's National Medical Center, and The George Washington University, Washington, DC 20010, USA.
Abstract
BACKGROUND: The expression of foreign genes into renal glomerular cells holds enormous potential to modulate the outcome of renal diseases. Recombinant adenoviruses (rAds) are promising gene transfer vectors because they have the ability to infect a wide range of nondividing cells. However, despite the fact that renal glomeruli are easily accessible via the renal circulation, adenovirus-mediated gene transfer into rodent glomeruli has been problematic. Here, we described our experience using rAd vectors to express foreign genes in rodent renal glomeruli in vivo and in cultured human renal glomerular cells. METHODS: We developed two techniques--the "portal clamping" and "prolonged renal infusion"--to infect mouse and rat renal glomeruli in vivo, respectively. We used E-1-deleted rAd vectors carrying the lacZ gene encoding beta-galactosidase (Ad. CBlacZ) under the control of the cytomegalovirus enhancer and chicken beta-actin promoter. Cultured human renal glomerular podocytes, endothelial and mesangial cells were grown following standard techniques. Transgene expression was evaluated by doing beta-galactosidase staining and reverse transcription-polymerase chain reaction studies. RESULTS: We found that both a prolonged exposure and a high concentration of circulating adenoviral vectors were required to achieve efficient gene transfer to renal glomerular cells in rodents. The virus-mediated transgene expression in renal glomeruli lasted for at least 42 days in mice and 21 days in rats without causing significant renal injury. CONCLUSIONS: These data demonstrate the feasibility of using rAd vectors as a tool to express foreign genes in rodent renal glomerular cells and suggest that all types of human renal glomerular cells are equally susceptible to rAd infection.
BACKGROUND: The expression of foreign genes into renal glomerular cells holds enormous potential to modulate the outcome of renal diseases. Recombinant adenoviruses (rAds) are promising gene transfer vectors because they have the ability to infect a wide range of nondividing cells. However, despite the fact that renal glomeruli are easily accessible via the renal circulation, adenovirus-mediated gene transfer into rodent glomeruli has been problematic. Here, we described our experience using rAd vectors to express foreign genes in rodent renal glomeruli in vivo and in cultured human renal glomerular cells. METHODS: We developed two techniques--the "portal clamping" and "prolonged renal infusion"--to infect mouse and rat renal glomeruli in vivo, respectively. We used E-1-deleted rAd vectors carrying the lacZ gene encoding beta-galactosidase (Ad. CBlacZ) under the control of the cytomegalovirus enhancer and chickenbeta-actin promoter. Cultured human renal glomerular podocytes, endothelial and mesangial cells were grown following standard techniques. Transgene expression was evaluated by doing beta-galactosidase staining and reverse transcription-polymerase chain reaction studies. RESULTS: We found that both a prolonged exposure and a high concentration of circulating adenoviral vectors were required to achieve efficient gene transfer to renal glomerular cells in rodents. The virus-mediated transgene expression in renal glomeruli lasted for at least 42 days in mice and 21 days in rats without causing significant renal injury. CONCLUSIONS: These data demonstrate the feasibility of using rAd vectors as a tool to express foreign genes in rodent renal glomerular cells and suggest that all types of human renal glomerular cells are equally susceptible to rAd infection.