1. Volume 76, Issue 1, Pages 32-43 (July 2009)
Osteopontin modulates angiotensin II–induced inflammation, oxidative stress, and fibrosis of the kidney 
Talya Wolak, HyunJu Kim, Yuelan Ren, Jason Kim, Nosratola D. Vaziri, Susanne B. Nicholas 
Kidney International 
Volume 76, Issue 1, Pages (July 2009)
DOI: /ki
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2. Figure 1
Quantification of OPN and GAPDH mRNA expression in kidney cortex RT-PCR. Animals were infused with AngII or vehicle (PBS) for 4 weeks and RNA was isolated from kidney cortex for quantitative RT-PCR. The graph shows relative expression of OPN/GAPDH mRNA. OPN expression was negligible in vehicle- and AngII-infused OPN−/− mice. Values are expressed as mean±s.e.m. #P<0.01, *P<0.05, n=4.
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3. Figure 2
Albumin-to-creatinine ratio (ACR) in OPN+/+ vs OPN−/− animals. Following AngII or vehicle infusion for 4 weeks, animals were placed in metabolic cages for collection of urine for albumin and creatinine measurements and albumin-to-creatinine determination. The initial ACR (μg/mg) was comparable in all the study groups. Values are expressed as mean±s.d. #P<0.01, *P<0.05, n=10–12.
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4. Figure 3
Analysis of macrophage recruitment to the tubulointerstitial area after 4 weeks of AngII infusion. (a) Immunohistochemistry (IHC) for MOMA-2 staining. Macrophages are stained brown. (b) Quantification of MOMA-2 IHC. Fifteen non-overlapping digitized images per slide at × 400 magnification were analyzed and expressed as number of MOMA-2 positive-stained cells per high-power field in the tubulointerstitial cross-section area. Values are expressed as mean±s.e.m. #P<0.01, n=5.
Kidney International  , 32-43DOI: ( /ki )
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5. Figure 4
Immunofluorescence and quantification of kidney sections of CD68-positive staining and western blot analysis of renal cortex from OPN+/+ and OPN−/− mice. (a) Arrows indicate regions of positive staining. (b) Fifteen non-overlapping digitized images per slide at × 400 magnification were analyzed. Data are expressed as the total number of CD68 positive–stained cells per high-power field in the tubulointerstitial cross-section area. Values are expressed as mean±s.e.m. #P<0.01, *P<0.05, n=5. (c) Representative western blot of CD68 and β-actin protein. (d) Quantification of signals from western blots. Values are expressed as mean±s.e.m., *P<0.05, n=10–12.
Kidney International  , 32-43DOI: ( /ki )
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6. Figure 5
MCP-1 mRNA and protein expression in the kidney cortex. (a) Quantification of MCP-1 and GAPDH by quantitative RT-PCR. (b) Representative western blot analysis of MCP–1 and β-actin protein. (c) Quantification of signals from western blots. Values are expressed as mean±s.e.m. #P<0.01, *P<0.05, n=8.
Kidney International  , 32-43DOI: ( /ki )
Copyright © 2009 International Society of Nephrology Terms and Conditions

7. Figure 6
NOX4 mRNA and NOX4, NOX2, gp47phox, and nitrotyrosine protein expression in kidney cortex. (a) Quantification of NOX4 and GAPDH by RT-PCR. (b) Representative western blot analysis of NOX4 and β-actin protein. (c) Quantification of signals from western blots. Values are expressed as mean±s.e.m. #P<0.01, *P<0.01, n=4. (d) Representative western blot analysis of NOX2 and β-actin protein. (e) Quantification of western blot signals for NOX2/β-actin protein ratio. (f) Representative western blot analysis of gp47phox and β-actin protein. (g) Quantification of western blot signals for gp47phox/β-actin protein ratio. (h) Representative western blot analysis of nitrotyrosine and β-actin protein. (i) Quantification of nitrotyrosine/β-actin protein ratio. Values are expressed as mean±s.e.m.; #P<0.01, *P<0.05, n=2–5.
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8. Figure 7
TGF-β1 mRNA and protein expression in OPN+/+ and OPN−/− mice. (a) Quantitative RT-PCR of TGF-β1 and GAPDH, n=4. (b) Representative western blot analysis of TGF-β1 and β-actin protein. (c) Quantification of signals from western blots showing fold change in TGF-β1/β-actin protein expression. Values are expressed as mean±s.e.m. *P<0.05, n=10–12.
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9. Figure 8
TGF-β1 protein expression is upregulated in proximal tubular epithelial (PTE) cells by recombinant OPN (rmOPN) and AngII. (a) PTE cells were incubated with and without rmOPN (10nM) or AngII (10−7) for up to 24h. Representative western blot analysis of TGF-β1 and β-actin protein. (b) Quantification of signals from western blot showed a significant ~2-fold higher TGF-β1 protein expression after 24h incubation. *P<0.05 vs control, n=6. (c) PTE cells were incubated with and without varying concentrations of rmOPN (1–10nM) or AngII (10−4–10−9) for 24h. Representative western blot analysis of TGF-β1 and β-actin protein. (d) Quantification of signals from western blot shows a 2–2.5 fold higher TGF-β1 protein expression after 24h incubation with rmOPN (5 and 10nM) and AngII (10−4 and 10−9M) vs control. *P<0.05, n=4.
Kidney International  , 32-43DOI: ( /ki )
Copyright © 2009 International Society of Nephrology Terms and Conditions

10. Figure 9
Alpha-smooth muscle actin (α-SMA) expression in the renal tubulointerstitial area and in vitro. (a) Immunohistochemistry (IHC) staining of α-SMA in sections of renal cortex shows brown staining of α-SMA. (b) Quantification of α-SMA expression from IHC. Fifteen non-overlapping digitized images at × 200 magnification per slide were analyzed and expressed as percent of total tubulointerstitial cross-section area. Values are expressed as mean±s.e.m. #P<0.01, n=5. (c) PTE cells were incubated with and without rmOPN (10nM) or AngII (10−7) for up to 24h. Representative western blot analysis of α-SMA and β-actin protein. (d) PTE cells were incubated with and without varying concentrations of rmOPN (1–10nM) or AngII (10−4–10−12) for 24h. Quantification of signals from western blots *P<0.05 vs control, n=4. (e) Representative western blot analysis of α-SMA and β-actin protein. (f) Quantification of signals from western blots *P<0.05. Values are expressed as mean±s.e.m., n=4.
Kidney International  , 32-43DOI: ( /ki )
Copyright © 2009 International Society of Nephrology Terms and Conditions

11. Figure 10
OPN deficiency attenuates fibronectin, but not collagen IV. (a) IHC staining for fibronectin (brown color) in sections of renal cortex. (b) Quantification of fibronectin staining of 15 non-overlapping digitized × 200 magnification images per slide and expressed as percent total tubulointerstitial cross-section area. Values are expressed as mean±s.e.m. #P<0.01, n=5. (c) Representative western blot analysis of fibronectin and β-actin protein. (d) Quantification of signals from western blots. Values are expressed as mean±s.e.m. *P<0.05, n=6. (e) IHC staining of α-SMA staining (brown color). (f) Quantification of collagen IV staining was done in a manner similar to that of fibronectin staining. Values are expressed as mean±s.e.m. #P<0.01, n=5.
Kidney International  , 32-43DOI: ( /ki )
Copyright © 2009 International Society of Nephrology Terms and Conditions

12. Figure 11
OPN modulates the expression of PAI-1 and fibrotic markers. mRNA and protein from renal cortex was used for quantitative RT-PCR and western blot analyses. (a) Quantitative RT-PCR of PAI-1 and GAPDH. Data are expressed as relative ratio of PAI-1/GAPDH mRNA, n=8. (b) Representative western blot analysis of PAI–1 and β-actin protein. (c) Quantification of signals from western blots. Data are fold change of PAI-1/β-actin protein. Values are mean±s.e.m. *P<0.05, n=6. Cells were untreated or treated with rmOPN (10nM) and AngII (10−6M) for 24h or pretreated with anti-OPN antibody for 1h. (d) Representative western blot of α-SMA and β-actin protein. (e) Quantification of signals from western blots showing fold change of α-SMA/β-actin protein. (f) Representative western blot analysis of TGF-β1 and β-actin protein. (g) Quantification of signals from western blots showing fold change of TGF-β1/β-actin protein. (h) Representative western blot analysis of PAI-1 and β-actin protein. (i) Quantification of signals from western blots showing fold change of PAI-1/β-actin protein. Values are expressed as mean±s.e.m. #P<0.01, *P<0.05, §P<0.001, n=2–4.
Kidney International  , 32-43DOI: ( /ki )
Copyright © 2009 International Society of Nephrology Terms and Conditions