Liposome-mediated transfer of nitric oxide synthase gene improves renal function in ureteral obstruction in rats.
BACKGROUND
The protective effect of nitric oxide has been demonstrated in several renal disease models. We augmented renal nitric oxide production by transfer of the inducible nitric oxide synthase (iNOS) gene into rat kidney in controls and in unilateral ureteral obstruction (UUO).


METHODS
The human iNOS gene was inserted into a pcDNA 3.1-backbone plasmid with the FLAG epitope (FLAG-iNOS). In vitro, transduction of FLAG-iNOS was confirmed by Western blot and Griess reaction. In vivo, we transfected either FLAG-iNOS or control plasmid (CMV-LacZ), using cationic liposomes. Urinary nitric oxide metabolites and immunohistochemistry confirmed iNOS transduction. Renal function was also assessed.


RESULTS
In vitro, increased iNOS expression was demonstrated in human embryonic kidney (HEK293) cells, along with increased release of nitric oxide metabolites, NO(2)/NO(3). In vivo, FLAG-iNOS was detected by polymerase chain reaction (PCR) up to 35 days after the transfection. Urine collection documented increased urinary NO(2)/NO(3). Immunohistochemistry localized iNOS to collecting ducts, distal tubules, and glomerulus of the injected kidney. Renal function measured up to 21 days after transfection in control animals was not significantly different between the two groups. In contrast, renal function after 24 hours of UUO was significantly improved in FLAG-iNOS-treated animals.


CONCLUSIONS
This study demonstrates the feasibility of liposome-mediated iNOS gene transfer into the kidney. Furthermore, the improvement of renal function in UUO demonstrates that the transfected iNOS gene is active and suggests that decreased iNOS activity contributes to the decreased renal function in UUO. This iNOS construct may have therapeutic utility in the pathophysiologic sequelae of UUO and other renal diseases.
Ito
Keiichi
K
Institute for Pediatric Urology, Department of Urology, Weill Cornell Medical Center, New York, New York 10021, USA.
Chen
Jie
J
Khodadadian
Jonathan J
JJ
Seshan
Surya V
SV
Eaton
Christian
C
Zhao
Xinyu
X
Vaughan
E Darracott
ED
Lipkowitz
Michael
M
Poppas
Dix P
DP
Felsen
Diane
D
D002478:Cells, Cultured; D000939:Epitopes / Q000235:genetics; D005629:Frozen Sections; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry; D007668:Kidney / Q000166:cytology / Q000502:physiology*; D008081:Liposomes; D009569:Nitric Oxide / Q000652:urine; D019001:Nitric Oxide Synthase / Q000235:genetics* / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D016612:Paraffin Embedding; D020127:Recovery of Function; D014161:Transduction, Genetic; D014517:Ureteral Obstruction / Q000628:therapy*; D001616:beta-Galactosidase / Q000235:genetics
D000939:Epitopes; D008081:Liposomes; D009569:Nitric Oxide; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D001616:beta-Galactosidase