Volume 66, Issue 4, Pages 1365-1375 (October 2004) Liposome-mediated transfer of nitric oxide synthase gene improves renal function in ureteral obstruction in rats1  Keiichi Ito, Jie Chen, Jonathan J. Khodadadian, Surya V. Seshan, Christian Eaton, Xinyu Zhao, E. Darracott Vaughan, Michael Lipkowitz, Dix P. Poppas, Diane Felsen  Kidney International  Volume 66, Issue 4, Pages 1365-1375 (October 2004) DOI: 10.1111/j.1523-1755.2004.00899.x Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 1 Structure of FLAG-inducible nitric oxide synthase (iNOS) (A) and CMV-LacZ (B) constructs. The human iNOS gene was inserted into pcDNA3.1-backbone plasmid with a FLAG epitope sequence. Commercially obtained control plasmid (CMV-LacZ, pcDNA3.1/myc-His(-)/LacZ) was used for the control in vivo experiments. Kidney International 2004 66, 1365-1375DOI: (10.1111/j.1523-1755.2004.00899.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 2 Expression of FLAG-inducible nitric oxide synthase (iNOS) protein in human embryonic kidney (HEK293) cells. HEK293 were incubated with either (A) FLAG-iNOS or (B) liposome, as described in the Methods section. Strong expression was observed 24 hours after FLAG-iNOS transfection using liposome. Kidney International 2004 66, 1365-1375DOI: (10.1111/j.1523-1755.2004.00899.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 3 Western blot analyses for inducible nitric oxide synthase (iNOS) in human embryonic kidney (HEK293)-transfected cells. iNOS was detected in FLAG-iNOS–transfected cells by Western blot using an antibody directed against (A) the FLAG epitope or (B) iNOS. In both cases, a band at 130 kD was detected. Kidney International 2004 66, 1365-1375DOI: (10.1111/j.1523-1755.2004.00899.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 4 Polymerase chain reaction (PCR) analysis for FLAG-inducible nitric oxide synthase (iNOS). FLAG-iNOS cDNA was detected using PCR specific for FLAG-iNOS plasmid. (A) FLAG-iNOS cDNA could be detected 21 days after the transfection in the injected kidney. (B) FLAG-iNOS cDNA was detected in contralateral kidney and (C) other organs, including lung, spleen, and bladder. Kidney International 2004 66, 1365-1375DOI: (10.1111/j.1523-1755.2004.00899.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 5 Immunolocalization of FLAG-inducible nitric oxide synthase (iNOS) protein in frozen sections of kidney. iNOS was visualized using an antibody to FLAG, and was detected by immunofluorescence as described in the Methods section. (A) Strong expression was seen in FLAG-iNOS–transfected kidneys at day 3. The expression was mainly in medullary collecting ducts (B) No expression was seen in medulla of the CMV-LacZ–transfected kidney. (C) Weak staining was noted in glomeruli of CMV-LacZ–injected kidney. (D) Stronger glomerular staining was noted in the FLAG-iNOS–injected kidney. In addition, cortical tubules had weak baseline fluorescence (C and D). Kidney International 2004 66, 1365-1375DOI: (10.1111/j.1523-1755.2004.00899.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 6 Immunolocalization of inducible nitric oxide synthase (iNOS) protein in paraffin-embedded sections of kidney. iNOS was visualized using an antibody to iNOS, and was detected by immunoperoxidase as described in the Methods section. Representative picture from rats 3 days after injection is shown. (A) Uniform staining was noted in CMV-LacZ–injected kidney. (B and C) In FLAG-iNOS–injected kidney, increased staining was noted in distal tubules and collecting ducts in both the cortex (B) and medulla (C). Abbreviations are: PT, proximal tubule; DT, distal tubule; CD, collecting duct; LH, loop of Henle (in C). Kidney International 2004 66, 1365-1375DOI: (10.1111/j.1523-1755.2004.00899.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 7 Immunolocalization of β-galactosidase (β-gal) in paraffin-embedded sections of kidney. β-gal was visualized by immunoperoxidase as described in the Methods section. A representative picture from rats 7 days after injection is shown. (A) In the injected kidney, β-gal was mainly expressed in medullary collecting ducts. (B) In the contralateral kidney, no staining was noted. Kidney International 2004 66, 1365-1375DOI: (10.1111/j.1523-1755.2004.00899.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 8 Comparison of urinary nitric oxide (NO2/3) excretion between CMV-LacZ and FLAG-inducible nitric oxide synthase (iNOS)–injected groups. Urinary nitric oxide metabolites were analyzed in a 24-hour urine sample as described in the Methods section. There was significantly more urinary NO2/3 excretion in the FLAG-iNOS–injected group as compared to the CMV-LacZ group. Increased NO2/3 excretion was sustained for 32 days. *P < 0.05 vs. day 0;#P < 0.05 vs. same day with CMV-LacZ treatment. Kidney International 2004 66, 1365-1375DOI: (10.1111/j.1523-1755.2004.00899.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 9 Renal function in normal rats after either CMV-LacZ or FLAG-inducible nitric oxide synthase (iNOS) injection. Renal blood flow (RBF) and glomerular filtration rate (GFR) were measured from 3 to 21 days after injection of either CMV-LacZ or FLAG-iNOS as described in the Methods section. (A) RBF measurements demonstrated no significant effects of FLAG-iNOS injection. A similar finding was noted when (B) glomerular filtration was assessed. Abbreviations are: UK, uninjected kidney; IK, injected kidney, *P < 0.05 compared to uninjected kidney. Kidney International 2004 66, 1365-1375DOI: (10.1111/j.1523-1755.2004.00899.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 10 Immunolocalization of FLAG-inducible nitric oxide synthase (iNOS) protein in transfected kidneys after 24 hours of UUO. FLAG-iNOS protein was localized by immunofluorescence at 8 days after injection (coinciding with 24 hours of UUO). (A) iNOS was clearly expressed in medulla of FLAG-iNOS injected kidney. (B) In contrast, no staining was seen in CMV-LacZ–injected kidney. Kidney International 2004 66, 1365-1375DOI: (10.1111/j.1523-1755.2004.00899.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 11 Renal function at 24 hours after UUO following either CMV-LacZ or FLAG-inducible nitric oxide synthase (iNOS) injection. CMV or LacZ was injected intraureterally. At 7 days after injection, the ureter was ligated and renal function measured at 24 hours. Both renal blood flow (RBF) and glomerular filtration rate (GFR) were significantly decreased in the obstructed kidney of CMV-LacZ rats as compared to the contralateral kidney. The decreased renal function of the obstructed kidney was significantly attenuated in FLAG-iNOS–injected rats compared to CMV-LacZ rats. Abbreviations are: CK, contralateral kidney (LacZ; iNOS); PTK, previously transfected, obstructed kidney (LacZ; iNOS). *P < 0.05 compared to contralateral kidney; ∞P < 0.05, compared to LacZ-treated obstructed kidney. Kidney International 2004 66, 1365-1375DOI: (10.1111/j.1523-1755.2004.00899.x) Copyright © 2004 International Society of Nephrology Terms and Conditions