Osteopontin modulates angiotensin II–induced inflammation, oxidative stress, and fibrosis of the kidney

Osteopontin modulates angiotensin II–inducedinflammation, oxidative stress, and fibrosis ofthe kidneyTalya Wolak1,2, HyunJu Kim3, Yuelan Ren1, Jason Kim1, Nosratola D. Vaziri3 and Susanne B. Nicholas1,2

1Division of Nephrology, Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA; 2Division ofEndocrinology, Diabetes, and Hypertension, Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California,USA and 3Division of Nephrology, Department of Medicine, University of California, Irvine, California, USA

Osteopontin, a secreted glycoprotein has been implicated

in several renal pathological conditions such as those due

to ureteral obstruction, ischemia, and cyclosporine toxicity.

We studied its possible role in angiotensin II–mediated

renal injury by infusing wild-type and osteopontin knockout

mice with angiotensin II and found that it raised blood

pressure and increased urinary albumin/creatinine ratios

in both strains of mice. However, while wild-type mice

responded to the infusion by macrophage infiltration and

increased expression of a-smooth muscle actin, fibronectin,

and transforming growth factor-b; the osteopontin knockout

mice developed none of these. Further, the knockout mice

had increased expression of monocyte chemoattractant

protein-1; NADPH oxidase subunits such as NOX2,

gp47phox, and NOX4; and plasminogen activator inhibitor-1

compared to the wild type animals. Proximal tubule epithelial

cells in culture treated with recombinant osteopontin and

angiotensin II had increased a-smooth muscle actin and

transforming growth factor-b expression. The effect of

angiotensin II was blocked by an antibody to osteopontin.

In addition, osteopontin attenuated angiotensin II-induced

plasminogen activator inhibitor-1 expression. These studies

show that osteopontin is a promoter and an inhibitor of

inflammation, oxidative stress, and fibrosis that is capable

of modulating angiotensin II–induced renal damage.

Kidney International (2009) 76, 32–43; doi:10.1038/ki.2009.90;

published online 8 April 2009

KEYWORDS: angiotensin II; fibrosis; inflammation; osteopontin; oxidative

stress

Angiotensin II (AngII) is a main player in the pathogenesis ofhypertensive nephropathy, which is a leading cause of chronickidney disease and its progression to end stage renal disease.1

Some of the deleterious effects of AngII on the kidney includerecruitment of inflammatory cells to the glomerulus,2,3

induction of collagen type IV synthesis in the glomerularbasement membrane,4 enhancement of an inflammatoryresponse in the proximal tubular cells,5 and fibrosis in therenal tubular interstitium. One of the mediators expressed inAngII-injured tissue is osteopontin (OPN).6,7 OPN is amatrix glycoprotein containing an arginine glycine aspartate(RGD) motif that binds to the integrin family of receptorsand a soluble cytokine. It is highly expressed in damagedtissues and plays a role in wound healing possibly byregulating inflammation and fibrosis.8,9 The link betweenOPN and AngII in the kidney is evident from studies inwhich blockade of one or more of the renin–angiotensin–aldosterone system components downregulated the OPNexpression.10,11 The role of OPN in renal damage has beeninvestigated in various animal models such as post-ischemicnephropathy,12 obstructive nephropathy,13 and chronicallograft damage.14 We and others have examined the effectof AngII on the cardiovascular structure and have shown thatthe lack of OPN attenuates both cardiac fibrosis15,16 andaortic aneurysm formation.17 This implied that OPN mightplay a role in AngII-mediated renal injury.

OPN may participate in the host response to tissueinjury by regulating inflammation, epithelial–mesenchymaltransition, and fibrosis.18,19 The balance between the pro-and anti-inflammatory actions of OPN results in modulationof processes involved in tissue damage and fibrosis.18 Follow-ing tissue damage, OPN facilitates macrophage and T-cellrecruitment20,21 and simultaneously attenuates secondaryinjury by decreasing production of peroxynitrite, a highlycytotoxic nitrogen species.22,23 By the induction of inducibleNO synthase and activation of NADPH oxidase, inflam-mation increases production of peroxynitrite, which is thebyproduct of NO interaction with superoxide anion(O2

�).24,25 By inhibiting inducible NO synthase, OPN reducesperoxynitrite production22,23 and thereby decreases oxidative/

o r i g i n a l a r t i c l e http://www.kidney-international.org

& 2009 International Society of Nephrology

Received 19 April 2008; revised 2 February 2009; accepted 10 February

2009; published online 8 April 2009

Correspondence: Susanne B. Nicholas, 900 Veteran Avenue Suite 24-130,

Los Angeles, California 90095, USA. E-mail: [email protected]

32 Kidney International (2009) 76, 32–43

http://dx.doi.org/10.1038/ki.2009.90

http://www.kidney-international.org

mailto:[email protected]

nitrosative stress in the inflammatory milieu. In proximaltubular cells, inflammation and oxidative stress are followedby mesenchymal transformation,26,27 in which epithelial cellslose their epithelial phenotype and acquire mesenchymalmarkers.28 This leads to deposition of collagen and othermatrix molecules, interstitial fibrosis, renal dysfunction,29,30

and potential progression towards end stage renal disease.Several studies have been performed in different diseasemodels using OPN knockout mice to elucidate the role ofOPN in fibrogenesis. For instance, lack of OPN has beenshown to attenuate mesenchymal transformation and renalfibrotic markers in neonatal chronic unilateral ureteralobstruction.13 In addition, OPN deficiency has been foundto impair wound healing19 and decrease collagen depositionafter myocardial infarction.31

In this study, we investigated the role of OPN in renalinflammation, oxidative stress, and interstitial fibrosis usingOPN-null (OPN�/�) and wild-type (OPNþ /þ ) mice infusedwith AngII for 4 weeks. The study revealed that OPNmodulates these processes to retard progression of renaldamage in AngII-induced hypertension.

RESULTSCharacteristics of study animals

The genotype of the study animals was confirmed byquantitative RT-PCR. As expected, OPN expression wasessentially absent in AngII- and vehicle-infused OPN�/� mice(Figure 1). AngII-infusion resulted in a threefold increase inOPN expression in the OPNþ /þ mice (Po0.05). Thebaseline body weight, blood pressure, and urine volume didnot differ significantly between OPNþ /þ and OPN�/� mice.AngII-infusion � 4 weeks led to a significant reductionof body weight and a significant increase in blood pressureand urine volume of equal magnitude in both OPNþ /þ andOPN�/� mice compared with their vehicle-infused counter-parts (Po0.05). Kidney weight compared with tibia length(to accommodate changes in body weight) was significantlylower in AngII-infused OPNþ /þ and OPN�/� than invehicle-infused mice (Table 1).

Effect of OPN on albuminuria

Baseline albumin-to-creatinine ratio (ACR) was minimal andof similar magnitude among the study groups. AngII infusionfor 4 weeks markedly increased ACR in both OPNþ /þ

(Po0.05) and OPN�/� (Po0.01). As in other models ofrenal injury in OPN-null animals,12 we found no significantdifference in ACR among AngII-infused OPN�/� comparedwith AngII-infused OPNþ /þ mice (338.4 vs 218.2 mg/mg),P¼ 0.10 determined by two-way ANOVA and non-parametric Mann–Whitney U-test (Figure 2).

Pro- and anti-inflammatory effects of OPN in the kidney

AngII-infusion resulted in substantial interstitial macrophageinfiltration in both OPNþ /þ and OPN�/� mice compared withvehicle-infused OPNþ /þ and OPN�/�mice. However, the mag-nitude of interstitial macrophage infiltration was significantly lessin AngII-infused OPN�/� than in the AngII-infused OPNþ /þ

mice. These findings were evident on immunohistochemical(IHC) staining for MOMA-2 (1.64±0.2/hfp compared with2.8±0.2/high powered field, Figure 3a and b) and CD68immunofluorescence (0.8±0.2/hfp compared with 2.18±0.25/high powered field, Figure 4a and b) in AngII-infused OPN�/�

compared with AngII-infused OPNþ /þ mice, respectively,(Po0.01). In addition, CD68 protein expression was 1.5-foldlower in AngII-infused OPN�/� compared with AngII-infused

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Figure 1 | Quantification of OPN and GAPDH mRNA expressionin kidney cortex RT-PCR. Animals were infused with AngII orvehicle (PBS) for 4 weeks and RNA was isolated from kidney cortexfor quantitative RT-PCR. The graph shows relative expressionof OPN/GAPDH mRNA. OPN expression was negligible invehicle- and AngII-infused OPN�/� mice. Values are expressedas mean±s.e.m. #Po0.01, *Po0.05, n¼ 4.

Table 1 | Physiologic characteristics of mice

OPN+/+ OPN�/�

Vehicle (n = 12) Ang II–infused (n = 10) Vehicle (n = 12) Ang II–infused (n = 12)

Body weight, initial (g) 27.9±0.7 29.0±0.3 31.0±0.4 28.7±0.3Body weight, final (g) 29.7±0.2 26.4±0.9a 29.0±0.0 24.2±0.3b

Systolic BP, initial (mm/Hg) 110.3±1.4 108.5±1.4 113.2±2.0 112.0±2.7Systolic BP, final (mm/Hg) 109.1±2.4 163.4±4.1a 112.6±1.6 165.0±5.4b

Urine volume, initial (ml) 1.4±0.1 1.3±0.1 2.6±0.2 2.0±0.2Urine volume, final (ml) 1.7±0.1 7.5±0.7a 1.3±0.2 5.0±0.8b

Kidney weight/tibia length � 10�3 15.8±0.5 14.8±0.6a 13.5±0.4 11.8±0.3b

AngII, Angiotensin II; BP, blood pressure; OPN, osteopontin; OPN�/�, OPN knockout mice; OPN+/+, wild-type mice. Values are mean±s.e.m.aPo0.05 compared with vehicle-infused OPN+/+. bPo0.05 compared with vehicle-infused OPN�/�.

Kidney International (2009) 76, 32–43 33

T Wolak et al.: Osteopontin, inflammation, oxidative stress, fibrosis o r i g i n a l a r t i c l e

OPNþ /þ mice, Po0.05 (Figure 4c and d). No difference wasfound between vehicle-infused OPNþ /þ and OPN�/� animals.These data indicate that OPN deficiency attenuated AngII-induced macrophage recruitment to the kidney.

Renal cortical MCP-1 expression was significantly greaterin the vehicle-treated OPN�/� mice than in their OPNþ /þ

counterparts. AngII infusion resulted in significant upregu-lation of MCP-1 mRNA and protein expression in bothOPN�/� and OPNþ /þ mice. However, MCP-1 mRNA andprotein expression was greater in AngII-infused OPN�/�

mice compared with AngII-infused OPNþ /þ mice. MCP-1mRNA expression was 2-fold higher (Figure 5a) and MCP-1protein expression was 3.5-fold higher (Figure 5b and c) inAngII-infused OPN�/� than in AngII-infused OPNþ /þ

mice, Po0.05. This observation is consistent with ourprevious findings which demonstrated that MCP-1, a potentmacrophage recruiter,32 is more intensely expressed inthe aorta of AngII-infused double knockout ApoE–/–/OPN–/–

than single knockout ApoE–/–/OPNþ /þ mice.17 Our resultssuggest that OPN significantly modulates renal AngII-induced inflammation by attenuating MCP-1 expression.

Effect of OPN on NAD(P)H oxidase

To determine whether the absence of OPN significantly altersAngII-induced upregulation of NADPH oxidase, we mea-sured the expression of NOX4 (a kidney-specific NADPHoxidase subtype33,34), NOX2, gp47phox and nitrotyrosine.AngII increased NOX4 mRNA 420-fold and protein 3.5-fold, Po0.01 (Figure 6a–c), NOX2 protein 1.7-fold, Po0.01and gp47phox 1.8-fold, Po0.05 in OPN�/� vs OPNþ /þ mice(Figure 6d–g). There was also a significant increase in NOX4(4-fold, Po0.01 and 7-fold, Po0.05) and NOX2 protein(1.3-fold Po0.01 and 3.8-fold, Po0.01) in OPN�/� andOPNþ /þ compared with vehicle-infused OPN�/� andOPNþ /þ mice, respectively. gp47phox increased sixfoldbetween AngII-infused OPNþ /þ and vehicle-infusedOPNþ /þ mice; however, the 1.2-fold increase in gp47phox

between AngII-infused OPN�/� and vehicle-infused OPN�/�

mice was not statistically different. Nitrotyrosine expressionincreased 1.5-fold, Po0.05 in AngII-infused OPNþ /þ and1.4-fold, Po0.05 in vehicle-infused OPN�/� mice vs vehicle-infused OPNþ /þ mice. The 1.2-fold increase in nitrotyrosineexpression in AngII-infused OPN�/� was not significant(Figure 6h and i). These data indicate that OPN deletion maypromote oxidative stress by upregulating NADPH oxidasesubtypes.

Effect of OPN on tubulointerstitial fibrosis

Both OPN and TGF-b1 have significant roles in woundhealing and fibrogenesis.35,36 Expression of TGF-b1, which is

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Figure 2 | Albumin-to-creatinine ratio (ACR) in OPNþ /þ vsOPN�/� animals. Following AngII or vehicle infusion for 4 weeks,animals were placed in metabolic cages for collection of urine foralbumin and creatinine measurements and albumin-to-creatininedetermination. The initial ACR (mg/mg) was comparable in all thestudy groups. Values are expressed as mean±s.d. #Po0.01,*Po0.05, n¼ 10–12.

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Figure 3 | Analysis of macrophage recruitment to thetubulointerstitial area after 4 weeks of AngII infusion. (a)Immunohistochemistry (IHC) for MOMA-2 staining. Macrophagesare stained brown. (b) Quantification of MOMA-2 IHC. Fifteen non-overlapping digitized images per slide at � 400 magnificationwere analyzed and expressed as number of MOMA-2 positive-stained cells per high-power field in the tubulointerstitialcross-section area. Values are expressed as mean±s.e.m.#Po0.01, n ¼ 5.


o r i g i n a l a r t i c l e T Wolak et al.: Osteopontin, inflammation, oxidative stress, fibrosis

also an early fibrosis regulator37 was examined in vivo.Quantitative RT-PCR demonstrated a 2-fold higher expres-sion of TGF-b1 mRNA in AngII-infused OPNþ /þ comparedwith AngII-infused OPN�/� animals (Figure 7a). TGF-b1

protein expression was similarly increased (Po0.01; Figure7b and c). To further investigate the role of OPN on

TGF-b1 expression, we incubated confluent porcine proximaltubular epithelial (PTE) cells with rmOPN or AngII.Incubation with either rmOPN (10 nM) or AngII (10�7 M)resulted in a 42-fold rise in TGF-b1 expression in a time-dependent manner up to 24 h, Po0.05 (Figure 8a and b)and a dose-dependent manner up to 2.3-fold with rmOPN

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Figure 4 | Immunofluorescence and quantification of kidney sections of CD68-positive staining and western blot analysis of renalcortex from OPNþ /þ and OPN�/� mice. (a) Arrows indicate regions of positive staining. (b) Fifteen non-overlapping digitized images perslide at �400 magnification were analyzed. Data are expressed as the total number of CD68 positive–stained cells per high-power field inthe tubulointerstitial cross-section area. Values are expressed as mean±s.e.m. #Po0.01, *Po0.05, n¼ 5. (c) Representative western blot ofCD68 and b-actin protein. (d) Quantification of signals from western blots. Values are expressed as mean±s.e.m., *Po0.05, n¼ 10–12.

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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 b-actin protein. (c) Quantification of signals from western blots. Values are expressedas mean±s.e.m. #Po0.01, *Po0.05, n¼ 8.



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Figure 6 | NOX4 mRNA and NOX4, NOX2, gp47phox, and nitrotyrosine protein expression in kidney cortex. (a) Quantification of NOX4and GAPDH by RT-PCR. (b) Representative western blot analysis of NOX4 and b-actin protein. (c) Quantification of signals from westernblots. Values are expressed as mean±s.e.m. #Po0.01, *Po0.01, n¼ 4. (d) Representative western blot analysis of NOX2 and b-actin protein.(e) Quantification of western blot signals for NOX2/b-actin protein ratio. (f) Representative western blot analysis of gp47phox and b-actinprotein. (g) Quantification of western blot signals for gp47phox/b-actin protein ratio. (h) Representative western blot analysis of nitrotyrosineand b-actin protein. (i) Quantification of nitrotyrosine/b-actin protein ratio. Values are expressed as mean±s.e.m.; #Po0.01, *Po0.05,n¼ 2–5.

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Figure 7 | TGF-b1 mRNA and protein expression in OPNþ /þ and OPN�/� mice. (a) Quantitative RT-PCR of TGF-b1 and GAPDH, n¼ 4.(b) Representative western blot analysis of TGF-b1 and b-actin protein. (c) Quantification of signals from western blots showing fold changein TGF-b1/b-actin protein expression. Values are expressed as mean±s.e.m. *Po0.05, n ¼ 10–12.



(1–10 nM) and AngII (10�9–10�4 M) at 24 h, Po0.05 (Figure8c and d).

Effects of OPN on fibrotic markers in vivo and in vitro

We examined the role of OPN on expression of a-SMA, as amarker of early interstitial fibrosis38 in AngII-mediated renalinjury by IHC (Figure 9a). Quantification showed thatcompared with vehicle, AngII significantly increased a-SMAexpression in both OPNþ /þ and OPN�/� mice (9.1±0.7 vs1.3±0.14, and 4.9±0.5 vs 0.8±0.09, respectively, Po0.01).The expression of a-SMA in AngII-infused OPNþ /þ wassignificantly higher compared with AngII-infused OPN�/�

mice, Po0.01 (Figure 9b). Incubation of PTE cells witheither AngII (10�7 M) or rmOPN (10 nM) resulted in a 2–3fold increase in a-SMA expression up to 24 h (Figure 9cand d), and a dose-dependent increase up to 1.5-fold withAngII (10�12–10�4 M) and rmOPN (1–10 nM), Po0.05(Figure 9e and f).

Effects of OPN on AngII-induced interstitial fibrosis

The expression of fibronectin and collagen IV was examined.Fibronectin expression by IHC was significantly higher inAngII-infused OPNþ /þ vs AngII-infused OPN�/� (4.6±0.8vs 1.2±0.3, Po0.01 (Figure 10a and b) and similarly increased5-fold in AngII-infused OPNþ /þ vs AngII-infused OPN�/�

mice by western blot, Po0.05 (Figure 10c and d). On theother hand, collagen IV IHC staining was increased in bothAngII-infused OPNþ /þ and OPN�/� (6.3±0.8 and 6.1±0.6)compared with vehicle-infused OPNþ /þ and OPN�/� (1.6±0.2 and 3.1±0.5) mice, Po0.01 (Figure 10e and f). Thissuggests that OPN may more directly regulate fibronectin thancollagen IV expression in the early stages of fibrosis.

Effects of OPN on expression of AngII-induced fibroticmarkers in vivo and in vitro

Our group has described the upregulation of renal corticalTGF-b1 by PAI-1.39 In this study, we showed that theexpression of PAI-1 mRNA and protein was B3-fold higherin AngII-infused OPNþ /þ compared with vehicle-infusedOPNþ /þ mice, Po0.05 and significantly increased in thevehicle-infused OPN�/� mice, but did not rise further withAngII infusion (Figure 11a–c). To investigate the dual role ofOPN to modulate fibrosis, we examined the expression ofa-SMA, TGF-b1 and PAI-1 in vitro. PTE cells were untreatedor treated for 24 h with rmOPN (10 nM) or AngII (10�6 M)or pre-treated � 2 h with anti-OPN antibody. OPN antibodydid not alter a-SMA or TGF-b1, but increased PAI-1expression 7-fold. OPN and AngII independently increasedthe expression of a-SMA (B4-fold, Po0.01), TGF-b1

(B3–4-fold, Po0.01) and PAI-1 (B10-fold, Po0.01).

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Figure 8 | TGF-b1 protein expression is upregulated in proximal tubular epithelial (PTE) cells by recombinant OPN (rmOPN) andAngII. (a) PTE cells were incubated with and without rmOPN (10 nM) or AngII (10�7) for up to 24 h. Representative western blot analysisof TGF-b1 and b-actin protein. (b) Quantification of signals from western blot showed a significant B2-fold higher TGF-b1 proteinexpression after 24 h incubation. *Po0.05 vs control, n¼ 6. (c) PTE cells were incubated with and without varying concentrations of rmOPN(1–10 nM) or AngII (10�4–10�9) for 24 h. Representative western blot analysis of TGF-b1 and b-actin protein. (d) Quantification of signalsfrom western blot shows a 2–2.5 fold higher TGF-b1 protein expression after 24 h incubation with rmOPN (5 and 10 nM) and AngII (10�4 and10�9 M) vs control. *Po0.05, n¼ 4.



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AngII 24 h

α-SMA50 kDa

Vehicle-OPN+/+

Vehicle-OPN−/−

AngII-OPN+/+

AngII-OPN−/−

#

#

##

*

***

*

*

#

β-actin43 kDa

rmOPN 10nMAngII 10−7M

α-SMA50 kDa

β-actin43 kDa

Figure 9 | Alpha-smooth muscle actin (a-SMA) expression in the renal tubulointerstitial area and in vitro. (a) Immunohistochemistry(IHC) staining of a-SMA in sections of renal cortex shows brown staining of a-SMA. (b) Quantification of a-SMA expression from IHC. Fifteennon-overlapping digitized images at � 200 magnification per slide were analyzed and expressed as percent of total tubulointerstitialcross-section area. Values are expressed as mean±s.e.m. #Po0.01, n¼ 5. (c) PTE cells were incubated with and without rmOPN (10 nM) orAngII (10�7) for up to 24 h. Representative western blot analysis of a-SMA and b-actin protein. (d) PTE cells were incubated with andwithout varying concentrations of rmOPN (1–10 nM) or AngII (10�4–10�12) for 24 h. Quantification of signals from western blots *Po0.05 vscontrol, n¼ 4. (e) Representative western blot analysis of a-SMA and b-actin protein. (f) Quantification of signals from western blots*Po0.05. Values are expressed as mean±s.e.m., n¼ 4.

Fib

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Fibronectin200 kDa

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7.5

5.0

2.5

0.0

Vehicle-OPN+/+


AngII-OPN+/+Vehicle-OPN+/+


AngII-OPN+/+

Vehicle-OPN+/+

Vehicle-OPN−/−

AngII-OPN+/+

AngII-OPN−/−

OPN+/+

OPN−/−

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Vehicle-OPN−/−

AngII-OPN+/+

AngII-OPN−/−

Vehicle-OPN+/+

Vehicle-OPN−/−

AngII-OPN+/+

AngII-OPN−/−

Vehicle Vehicle AngIIAngII

Figure 10 | OPN deficiency attenuates fibronectin, but not collagen IV. (a) IHC staining for fibronectin (brown color) in sections of renalcortex. (b) Quantification of fibronectin staining of 15 non-overlapping digitized �200 magnification images per slide and expressed aspercent total tubulointerstitial cross-section area. Values are expressed as mean±s.e.m. #Po0.01, n¼ 5. (c) Representative western blotanalysis of fibronectin and b-actin protein. (d) Quantification of signals from western blots. Values are expressed as mean±s.e.m. *Po0.05,n¼ 6. (e) IHC staining of a-SMA staining (brown color). (f) Quantification of collagen IV staining was done in a manner similar to that offibronectin staining. Values are expressed as mean±s.e.m. #Po0.01, n¼ 5.



However, the effect of AngII on a-SMA and TGF-b1 wasabolished with OPN antibody. There was a greater increase ofTGF-b1 compared with a-SMA or PAI-1 in the presence ofrmOPNþAngII (Figure 11d–i). This suggests that OPN alsoenhances a-SMA, TGF-b1 through AngII and attenuates theexpression of PAI-1.

DISCUSSION

The present study demonstrated that by modulatingprocesses involved in inflammation, oxidative stress andfibrosis, OPN may influence kidney damage in AngII-induced hypertension. We found that AngII infusionincreases OPN expression in OPNþ /þ animals. OPN-

PAI-147 kDa

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Vehicle VehicleAngll Angll

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+ +

+ ++++

−−

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Angll 10−6

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Anti-OPN −

−

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−−−− − −

−

+

+

+ +

+ ++++

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α-SMA TGF-β112.5 kDa

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#

#

§

§

§

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0Anti-OPN −

−

− − −

−−−

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++

+ +

+++

mOPN 10 nM

Angll 10−6 M

Anti-OPN −

−

− − −

−−−

− − −

++

+ +

+++

rmOPN 10 nM

Angll 10−6

M

Rel

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8

6

4

2

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*

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*

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*

Vehicle-OPN+/+

Vehicle-OPN−/−

Angll-OPN+/+

Angll-OPN−/−

Vehicle-OPN+/+

Vehicle-OPN−/−

Angll-OPN+/+

Angll-OPN−/−

Anti-OPN −−

− − − −

−−−− − −

−

++

+ ++ +

+++−−

mOPN 10 nM

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#

#*

Anti-OPN −−− − −

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+++

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M

PAI-147 kDa

PAI-

1/β-

actin

pro

tein

,fo

ld c

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e

4

3

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1

0

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Figure 11 | OPN modulates the expression of PAI-1 and fibrotic markers. mRNA and protein from renal cortex was used for quantitativeRT-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,n¼ 8. (b) Representative western blot analysis of PAI–1 and b-actin protein. (c) Quantification of signals from western blots. Data arefold change of PAI-1/b-actin protein. Values are mean±s.e.m. *Po0.05, n¼ 6. Cells were untreated or treated with rmOPN (10 nM)and AngII (10�6 M) for 24 h or pretreated with anti-OPN antibody for 1 h. (d) Representative western blot of a-SMA and b-actin protein.(e) Quantification of signals from western blots showing fold change of a-SMA/b-actin protein. (f) Representative western blot analysisof TGF-b1 and b-actin protein. (g) Quantification of signals from western blots showing fold change of TGF-b1/b-actin protein.(h) Representative western blot analysis of PAI-1 and b-actin protein. (i) Quantification of signals from western blots showing foldchange of PAI-1/b-actin protein. Values are expressed as mean±s.e.m. #Po0.01, *Po0.05, yPo0.001, n¼ 2–4.



deficient mice had attenuated tubulointerstitial macro-phage infiltration, despite enhanced MCP-1 expression andupregulation of NOX 4, NOX2, gp47phox, and nitrotyrosineexpression. In vivo analysis indicated that OPN functionedsimilarly to AngII by increasing TGF-b1, a-SMA andfibronectin expression, but unlike AngII, OPN reducedPAI-1. Similarly, in vitro studies revealed that both AngIIand OPN increased TGF-b1 and a-SMA but decreased PAI-1expression. These data suggest that OPN may exert bothpro- and anti-inflammatory effects to modulate the deleter-ious effects of AngII on inflammation, oxidative stress, andrenal fibrosis.

The baseline and AngII-induced increase in systemicblood pressure did not differ between OPNþ /þ and OPN�/�

mice. Albuminuria is a known feature of AngII-mediatedrenal injury.12,40 Urinary albumin excretion was similarin our AngII-infused OPN�/� and OPNþ /þ mice, suggest-ing that OPN may not directly contribute to the severity ofproteinuria and that in this model; proteinuria may besecondary to AngII-induced hyperfiltration.41

AngII-induced interstitial macrophage infiltration wassignificantly attenuated in our OPN�/� mice. This phenom-enon can be explained by the lack of OPN, which itself is amacrophage chemoattractant.42,43 Paradoxically, AngII infu-sion resulted in an exaggerated upregulation of MCP-1(which is an important mediator of macrophage recruit-ment44,45) in the OPN�/� mice. This finding is consistentwith our earlier studies.17 Antus et al. have demonstrated thatinfusion of AngII into animals facilitates macrophagerecruitment, perhaps either directly or through upregulationof chemotactic molecules such as RANTES and possiblyOPN.46,47 The current results in the kidney are comparable toour earlier findings in the aorta of ApoE–/–OPN–/– mice whichshowed reduced expression of macrophage CD68 andincreased MCP-1 expression in AngII-infused ApoE–/–OPN–/–

mice compared with AngII-infused ApoE–/–OPNþ /þ mice.17

It thus appears that OPN may mitigate the AngII-mediatedinflammatory process by limiting the exuberant expression ofMCP-1. It is of note that certain conditions such as diabeticnephropathy48,49 and autosomal-dominant polycystic kidneydisease50 are associated with concomitant upregulation ofMCP-1 and OPN. Conversely, in a rat model of anti-glomerular basement membrane glomerulonephritis, expres-sion of OPN and MCP-1 track inversely in differentcompartments of the kidney. For instance in the glomerulus,MCP-1 was highly expressed with lower OPN expression,whereas in the tubulo-interstitial area, MCP-1 expression wasonly slightly enhanced whereas OPN was maximally expres-sed and paralleled tubulo-interstitial macrophage infiltra-tion.51 Taken together, these results suggest that OPNmodulates inflammation through its pro- and anti-inflam-matory actions to enhance macrophage recruitment andsimultaneously inhibit MCP-1 expression.

The most frequently described anti-inflammatory actionof OPN is inhibition of inducible NO synthase,18,52 whichtogether with activation/upregulation of NAD(P)H oxidase

can result in increased production of ROS and reactivenitrogen species such as peroxynitrite. NOX2 is a homolog ofthe gp91phox catalytic subunit of NADPH oxidase and amajor source of ROS formation in the kidney.53–55 Here, wedemonstrated that the AngII-induced upregulation of NOX2,NOX4, and gp47phox was significantly amplified in theabsence of OPN. This was associated with a mild butinsignificant increase in nitrotyrosine abundance pointing toslight increase in AngII-induced superoxide production inOPN�/� mice. In contrast to the AngII-induced renal injury,Lai et al. reported that OPN deficiency downregulatesNADPH oxidase subunits NOX1, NOX2, p67phox, andp47phox as well as ROS production in diabetes-inducedvascular disease.56 This suggests that the effect of OPN onoxidative stress may be both tissue- and disease-specific.

The process of fibrosis is typically preceded by inflamma-tion57 and oxidative stress,27,58 and the role of OPN in earlyfibrosis has been extensively described. Van Timmeren et al.59

showed that the expression of OPN is limited to areas ofthe kidney exhibiting enhanced kidney injury molecule-1and a-SMA expression and macrophage accumulation. Persyet al. found that OPN deficiency attenuates macrophageinfiltration and interstitial fibrosis following ischemic injuryin mice.12 Yoo et al. reported lower interstitial fibrosisfollowing neonatal unilateral ureteral obstruction in OPN�/�

mice compared with OPNþ /þ mice.13 Bedke et al. showedthat the beneficial effect of CCR1 (chemokine receptor)antagonist in reducing fibrosis in the transplanted kidney isassociated with reductions of OPN, PAI-1, TGF-b1 anda-SMA.14 In our study, AngII-induced upregulation of TGF-b1 and a-SMA was significantly attenuated by OPNdeficiency pointing to the role of OPN in promoting TGF-b1 and a-SMA expression. This was confirmed by our in vitrostudies which showed that as with AngII, rmOPN inducesTGF-b1 and a-SMA protein expression and the AngII-induced increase in TGF-b1 and a-SMA was abolished bypretreatment with OPN antibody. We also showed that therise in tissue fibronectin abundance after four weeks of AngIIinfusion was attenuated by OPN deficiency. In contrast, therewas no attenuation of collagen IV expression.

PAI-1 mRNA and protein were significantly increased inour OPN�/� mice (compared with OPNþ /þ mice) suggest-ing that OPN can constrain PAI-1 production. Ma et al.60

have shown that PAI-1 may upregulate OPN, thus theincrease in PAI-1 in our OPN�/� mice may be, in part, due toloss of the inhibitory action of OPN. On the other hand, theincrease in PAI-1 abundance in our OPN�/� mice might bedue to the demonstrated increase in NOX4, as observedearlier by Djordjevic et al.61 In vitro, PAI-1 expression wasincreased by both rmOPN and AngII, but the AngII-inducedincrease in PAI-1 was not altered by pretreatment with OPNantibody. These data suggest that OPN may modulate fibrosisby activating mediators such as TGF-b1 and a-SMA (possiblythrough AngII) whereas concurrently inhibiting othermediators such as PAI-1, to maintain controlled fibrosisand perhaps promote tissue remodeling.



In conclusion, OPN modulates inflammation in theAngII-injured kidney by enhancing macrophage recruitmentto the tubular interstitium whereas simultaneously inhibitingthe expression of NOX2, gp47phox NOX4, and MCP-1.Similarly, OPN modulates renal tissue fibrosis by enhancingfibronectin, TGF-b1 and a-SMA expression and simulta-neously inhibiting PAI-1 expression. These observationsillustrate two faces of OPN as promoter and inhibitor ofinflammation, oxidation and fibrosis in the course of AngII-induced renal injury.

MATERIALS AND METHODSAnimalsBreeding pairs of osteopontin-knockout (OPN�/�) mice on a BlackSwiss background were the generous gift of R Johnson andC Giachelli with the permission of L Liaw.19 Wild-type OPN mice(OPNþ /þ ) were controls. Eight-week old mice (27.9–31 g) wereanesthetized with isoflurane and osmotic mini-pumps (Alza Corp.,Palo Alto, CA, USA) with AngII (2.5 mg/kg per min) in phosphate-buffered saline (PBS), or PBS (vehicle) were implanted subcuta-neously. Animals were habituated to BP measurements by indirecttail-cuff plethysmography (Visitech Systems, Apex, NC, USA) priorto initiation of the study until a plateau was reached (X10 times perday � 4 days) then weekly � 4 weeks for daily mean values. Animalswere placed in metabolic cages for 24-h urine collection at thebeginning and end of the study. Urinary albumin and creatininewere measured by Albuwell M Assay and Creatinine Companion(Exocel, Philadelphia, PA, USA) for ACR. After killing bypentobarbital overdose, the animals for body weight/tibia lengthwere weighed; tibia length was measured, which remains fairlyconstant with body weight changes. The kidneys were removed,weighed and either snap-frozen in liquid nitrogen and maintained at�801C or prepared for histologic analysis.

ImmunohistochemistryFibronectin, collagen IV and a-SMA expressed by myofibroblastsand macrophage MOMA-2 were detected by IHC by theavidin–biotin immunoperoxidase method (ZYMED detectionCarlsbad, CA, USA). Paraffin-embedded 4 mm sections weredeparaffinized with xylene, rehydrated in descending series ofethanol and boiled in 10 mM citrate buffer. Endogenous peroxidasewas applied in 10% hydrogen peroxide in PBS � 10 min followed byan avidin/biotin blocking reagent. Primary antibodies for mouseanti-a-SMA (1:200; Lab Vision Co., Fremont, CA, USA) and ratanti-MOMA-2 (1:100; Accurate Chemical & Scientific Co Westbury,NY, USA) mouse anti-fibronectin (1:50; Lab Vision Co.), rabbitanti-collagen IV (1:300; Santa Cruz Bio, Santa Cruz, CA, USA) orPBS negative control were incubated overnight at 41C. The sectionswere washed twice for 5 min in PBS, incubated � 30 min withsecondary antibodies, washed in PBS, and incubated � 30 min withVectastain ABC reagent. Bound antibodies were detected usingsubstrate-chromagen mixture for a brown color. The sections werestained with hematoxylin, dehydrated, and evaluated by lightmicroscopy and cortical tubular interstitial area. Myofibroblastsstained positive for a-SMA and tubulointerstitial zone stainedpositive for fibronectin and collagen IV. Both were quantified byscanning 15 non-overlapping randomly selected fields on eachsection at � 200 magnification (excluding areas with blood vessels)by a blinded observer. Immunohistochemical signal percent was

measured by computerized image analysis (Image Pro-Plus, MediaCybernetics, Silver Spring, MD, USA).

ImmunofluorescenceParaffin-embedded 4 mm sections were deparaffinized with xylene,rehydrated in descending series of ethanol and boiled in 10 mMcitrate buffer. The sections were incubated with 10% goat serum in3% BSA for 1 h at room temperature. Primary rabbit CD68 antibody(1:50; Santa-Cruz BIO) was added. As negative controls, PBS wassubstituted for a primary antibody. The sections were incubated at41C overnight, washed with PBS and incubated with goat anti-rabbitIgG-TRITC at 1:200 dilution for 1 h. The sections were mountedwith DAPI and observed under fluorescent microscope/TRITC forred color. Macrophages were counted manually in 15 non-overlapping randomly selected fields/section at � 400 magnificationby a blinded observer.

Proximal tubular epithelial cell cultureA vial of 2� 105 LLC-PK1 porcine proximal tubular epithelial cells(PTE: CL-101, ATTC, Manassas, VA, USA) was thawed and grown ingrowth media containing 1.5 g/l sodium bicarbonate, 3% fetalbovine serum and antibiotics. At 80% confluence, the cells werestarved for 16–20 h, and treated with rmOPN (10 nM) or AngII(10�7 M) for 0, 4, 8, 16 and 24 h or varying concentrations ofrmOPN (0–10 nM) or AngII (0–10�4 M) for 24 h. Protein fromwhole cell lysate was used for protein isolation in western blotanalysis (nX4 different cell preparations).

Western blot analysisTGF-b1, NADPH oxidase subunits NOX4, NOX2, and gp47phox andnitrotyrosine, PAI-1, CD68, MCP-1, and a-SMA protein (40–60mgrenal cortex) was measured by western blot analysis using standardprocedures.39 Antibodies against TGF-b1 (1:100), PAI-1 (1:1000),NOX2 (1:500), and gp47phox (1:1000; BD Biosciences/Pharmingen,San Diego, CA, USA), anti-a-SMA (1:400; Lab Vision Co., Fermont,CA), anti-MCP-1 (1:500), anti-CD68 (1:200) and anti-NOX4 (1:250) anti-fibronectin (1:100; Santa-Cruz BIO), and nitrotyrosine(1:500; Millipore Temecula, CA, USA) were used. Membranes wereprobed with primary antibody and corresponding secondaryantibodies, signals were scanned and quantified by Image J version1.28 U and NIH Image 1.60 scan software or by laser densitometry(Molecular Dynamics, Sunnyvale, CA, USA). Protein from PTE cellsas above or pretreated with either anti-OPN antibody (1:100;Santa-Cruz BIO) � 2 h were used for TGF-b1 or a-SMA expression.Following enhanced chemiluminescence (ECL) detection of pro-teins, the membranes were stripped and rehybridized with b-actinantibody as a loading control. At least four independent cellpreparations were used.

Reverse transcription real-time polymerase chain reaction(qRT-PCR)OPN, MCP-1, TGF-b1, NOX4, and PAI-1 mRNA expression inmouse renal cortex was measured by quantitative RT-PCR (n¼ 4–8animals/group). Total RNA (30–45 mg) was isolated by the Trizolmethod (Invitrogen Life Technologies, Carlsbad, CA, USA) andDNase-treated. Total RNA (1000 ng) was reverse-transcribed usingTaqMan Reverse Transcription Reagent Kit (Applied Biosystems,Foster City, CA, USA). The amount of contaminating DNA wasnegligible in the absence of reverse transcriptase. PCR amplification



of cDNA (100 ng) was performed with the ABI-PRISM 7700 system(total volume: 25 ml), using a TaqMan PCR Core Reagent Kit. Eachsample was analyzed in triplicate and normalized to GAPDH mRNAusing Taqman Rodent GAPDH Control Reagent. The forward andreverse primers (50 mm) and probes (50–100mm) labeled withcarboxyfluorescein dye used are listed in Table 2.

Statistical analysisData are expressed as mean±s.e.m. or mean±s.d., where indicated.Data were tested for normality and when non-normal distributionwas present, differences between groups were detected by Kruskal–-Wallis and Mann–Whitney U-tests; otherwise, analysis of variancewith Tukey post hoc tests was used. GraphPad Prizm 4 software (SanDiego, CA, USA) was used. Po0.05 was considered statisticallysignificant.

DISCLOSUREAll the authors declared no competing interests.

ACKNOWLEDGMENTSTW received funding from American Physicians Fellowship forMedicine in Israel. This work was supported by the WilsonFoundation, P01HD048721 and NIH/NHLBI RO1 HL071792-04. Someof these results were presented in poster form at the AmericanSociety of Nephrology, San Francisco, CA 2007 and ExperimentalBiology, San Diego, CA 2008. We thank Dr Willa A. Hsueh for hersupport.

REFERENCES1. Nissenson AR, Pereira BJ, Collins AJ et al. Prevalence and characteristics of

individuals with chronic kidney disease in a large health maintenance

organization. Am J Kidney Dis 2001; 37: 1177–1183.2. Wolf G, Bohlender J, Bondeva T et al. Angiotensin II upregulates toll-like

receptor 4 on mesangial cells. J Am Soc Nephrol 2006; 17: 1585–1593.3. Wolf G, Wenzel U, Burns KD et al. Angiotensin II activates nuclear

transcription factor-kappaB through AT1 and AT2 receptors. Kidney Int

2002; 61: 1986–1995.4. Chen S, Lee JS, Iglesias-de la Cruz MC et al. Angiotensin II stimulates

alpha3(IV) collagen production in mouse podocytes via TGF-beta and

VEGF signaling: implications for diabetic glomerulopathy. Nephrol Dial

Transplant 2005; 20: 1320–1328.5. Ruiz-Ortega M, Esteban V, Ruperez M et al. Renal and vascular

hypertension-induced inflammation: role of angiotensin II. Curr Opin

Nephrol Hypertens 2006; 15: 159–166.6. Rocha R, Martin-Berger CL, Yang P et al. Selective aldosterone blockade

prevents angiotensin II/salt-induced vascular inflammation in the rat

heart. Endocrinology 2002; 143: 4828–4836.7. de Borst MH, van Timmeren MM, Vaidya VS et al. Induction of kidney

injury molecule-1 in homozygous Ren2 rats is attenuated by blockade of

the renin-angiotensin system or p38 MAP kinase. Am J Physiol Renal

Physiol 2007; 292: F313–F320.8. Butler WT. The nature and significance of osteopontin. Connect Tissue Res

1989; 23: 123–136.9. Denhardt DT, Noda M. Osteopontin expression and function: role in bone

remodeling. J Cell Biochem Suppl 1998; 30–31: 92–102.

10. Yu XQ, Wu LL, Huang XR et al. Osteopontin expression in progressiverenal injury in remnant kidney: role of angiotensin II. Kidney Int 2000; 58:1469–1480.

11. Kramer AB, van der Meulen EF, Hamming I et al. Effect of combiningACE inhibition with aldosterone blockade on proteinuria and renaldamage in experimental nephrosis. Kidney Int 2007; 71: 417–424.

12. Persy VP, Verhulst A, Ysebaert DK et al. Reduced postischemicmacrophage infiltration and interstitial fibrosis in osteopontin knockoutmice. Kidney Int 2003; 63: 543–553.

13. Yoo KH, Thornhill BA, Forbes MS et al. Osteopontin regulates renalapoptosis and interstitial fibrosis in neonatal chronic unilateral ureteralobstruction. Kidney Int 2006; 70: 1735–1741.

14. Bedke J, Kiss E, Schaefer L et al. Beneficial effects of CCR1 blockade on theprogression of chronic renal allograft damage. Am J Transplant 2007; 7:527–537.

15. Matsui Y, Jia N, Okamoto H et al. Role of osteopontin in cardiac fibrosisand remodeling in angiotensin II-induced cardiac hypertrophy.Hypertension 2004; 43: 1195–1201.

16. Collins AR, Schnee J, Wang W et al. Osteopontin modulates angiotensinII-induced fibrosis in the intact murine heart. J Am Coll Cardiol 2004; 43:1698–1705.

17. Bruemmer D, Collins AR, Noh G et al. Angiotensin II-acceleratedatherosclerosis and aneurysm formation is attenuated in osteopontin-deficient mice. J Clin Invest 2003; 112: 1318–1331.

18. Denhardt DT, Noda M, O’Regan AW et al. Osteopontin as a means to copewith environmental insults: regulation of inflammation, tissueremodeling, and cell survival. J Clin Invest 2001; 107: 1055–1061.

19. Liaw L, Birk DE, Ballas CB et al. Altered wound healing in mice lackinga functional osteopontin gene (spp1). J Clin Invest 1998; 101:1468–1478.

20. Giachelli CM, Lombardi D, Johnson RJ et al. Evidence for a role ofosteopontin in macrophage infiltration in response to pathologicalstimuli in vivo. Am J Pathol 1998; 152: 353–358.

21. O’Regan AW, Chupp GL, Lowry JA et al. Osteopontin is associated withT cells in sarcoid granulomas and has T cell adhesive and cytokine-likeproperties in vitro. J Immunol 1999; 162: 1024–1031.

22. Hwang SM, Lopez CA, Heck DE et al. Osteopontin inhibits induction ofnitric oxide synthase gene expression by inflammatory mediators inmouse kidney epithelial cells. J Biol Chem 1994; 269: 711–715.

23. Rollo EE, Laskin DL, Denhardt DT. Osteopontin inhibits nitric oxideproduction and cytotoxicity by activated RAW264.7 macrophages.J Leukoc Biol 1996; 60: 397–404.

24. Beckman JS, Koppenol WH. Nitric oxide, superoxide, and peroxynitrite:the good, the bad, and ugly. Am J Physiol 1996; 271: C1424–C1437.

25. Park JB. Phagocytosis induces superoxide formation and apoptosis inmacrophages. Exp Mol Med 2003; 35: 325–335.

26. Strutz F, Zeisberg M, Ziyadeh FN et al. Role of basic fibroblast growthfactor-2 in epithelial-mesenchymal transformation. Kidney Int 2002; 61:1714–1728.

27. Zhang A, Jia Z, Guo X et al. Aldosterone induces epithelial-mesenchymaltransition via ROS of mitochondrial origin. Am J Physiol Renal Physiol2007; 293: F723–F731.

28. Li Y, Dai C, Wu C et al. PINCH-1 promotes tubular epithelial-to-mesenchymal transition by interacting with integrin-linked kinase.J Am Soc Nephrol 2007; 18: 2534–2543.

29. Iwano M, Plieth D, Danoff TM et al. Evidence that fibroblasts derive fromepithelium during tissue fibrosis. J Clin Invest 2002; 110: 341–350.

30. Chilosi M, Poletti V, Murer B et al. Abnormal re-epithelialization and lungremodeling in idiopathic pulmonary fibrosis: the role of deltaN-p63.Lab Invest 2002; 82: 1335–1345.

31. Trueblood NA, Xie Z, Communal C et al. Exaggerated left ventriculardilation and reduced collagen deposition after myocardial infarction inmice lacking osteopontin. Circ Res 2001; 88: 1080–1087.

Table 2 | Primers and probes

Gene Forward Reverse Probe

OPN 50-TCCCTCGATGTCATCCCTGT-30 50-CCCTTTCCGTTGTTGTCCTG-30 50-CCCAGCTTCTGAGCATGCCCTCTG-30

MCP-1 50-CAGCCAGATGCAGTTAACGC-30 50-GCCTACTCATTGGGATCATCTTG-30 50-CCACTCACCTGCTGCTACTCATTCACCA-30

TGF-b1 50-GAAACGGAAGCGCATCGA-30 50-GGGACTGGCGAGCCTTAGTT-30 50-56-FAM/CCATCCGTGGCCAGATCCTGTCC-30

PAI-1 50-TCAGCCCTTGCTTGCCTCAT-30 50-GCATAGCCAGCACCGAGGA-30 50-56-FAM/CAGGCCACCGACTTCGGAGTCAAAAG-30

NOX4 50-TCTCAGGTGTGCATGTAGCC-30 50-TTCTGGGATCCTCATTCTGG-30



32. Kanda H, Tateya S, Tamori Y et al. MCP-1 contributes to macrophageinfiltration into adipose tissue, insulin resistance, and hepatic steatosis inobesity. J Clin Invest 2006; 116: 1494–1505.

33. Geiszt M, Kopp JB, Varnai P et al. Identification of renox, an NAD(P)Hoxidase in kidney. Proc Natl Acad Sci USA 2000; 97: 8010–8014.

34. Gill PS, Wilcox CS. NADPH oxidases in the kidney. Antioxid Redox Signal2006; 8: 1597–1607.

35. Bennett MR, Czech KA, Arend LJ et al. Laser capture microdissection-microarray analysis of focal segmental glomerulosclerosis glomeruli.Nephron Exp Nephrol 2007; 107: e30–e40.

36. Kim JY, Lim SW, Li C et al. Effect of FTY720 on chronic cyclosporinenephropathy in rats. Transplantation 2005; 80: 1323–1330.

37. Fan JM, Ng YY, Hill PA et al. Transforming growth factor-beta regulatestubular epithelial-myofibroblast transdifferentiation in vitro. Kidney Int1999; 56: 1455–1467.

38. Yang J, Liu Y. Dissection of key events in tubular epithelial tomyofibroblast transition and its implications in renal interstitial fibrosis.Am J Pathol 2001; 159: 1465–1475.

39. Nicholas SB, Aguiniga E, Ren Y et al. Plasminogen activator inhibitor-1deficiency retards diabetic nephropathy. Kidney Int 2005; 67: 1297–1307.

40. Ophascharoensuk V, Giachelli CM, Gordon K et al. Obstructive uropathy inthe mouse: role of osteopontin in interstitial fibrosis and apoptosis.Kidney Int 1999; 56: 571–580.

41. de Seigneux S, Nielsen J, Olesen ET et al. Long-term aldosteronetreatment induces decreased apical but increased basolateral expressionof AQP2 in CCD of rat kidney. Am J Physiol Renal Physiol 2007; 293:F87–F99.

42. Tokuyama H, Kelly DJ, Zhang Y et al. Macrophage infiltration andcellular proliferation in the non-ischemic kidney and heart followingprolonged unilateral renal ischemia. Nephron Physiol 2007; 106:p54–p62.

43. Duvall CL, Weiss D, Robinson ST et al. The role of osteopontin in recoveryfrom hind limb ischemia. Arterioscler Thromb Vasc Biol 2007.

44. Tang WW, Qi M, Warren JS. Monocyte chemoattractant protein 1mediates glomerular macrophage infiltration in anti-GBM Ab GN.Kidney Int 1996; 50: 665–671.

45. Wenzel U, Schneider A, Valente AJ et al. Monocyte chemoattractantprotein-1 mediates monocyte/macrophage influx in anti-thymocyteantibody-induced glomerulonephritis. Kidney Int 1997; 51: 770–776.

46. Antus B, Exton MS, Rosivall L. Angiotensin II: a regulator of inflammationduring renal disease? Int J Immunopathol Pharmacol 2001; 14: 25–30.

47. Vaziri ND, Bai Y, Ni Z et al. Intra-renal angiotensin II/AT1 receptor,oxidative stress, inflammation, and progressive injury in renal massreduction. J Pharmacol Exp Ther 2007; 323: 85–93.

48. Kosugi T, Yuzawa Y, Sato W et al. Midkine is involved in tubulointerstitialinflammation associated with diabetic nephropathy. Lab Invest 2007; 87:903–913.

49. Chow F, Ozols E, Nikolic-Paterson DJ et al. Macrophages in mouse type 2diabetic nephropathy: correlation with diabetic state and progressiverenal injury. Kidney Int 2004; 65: 116–128.

50. Cowley Jr BD, Ricardo SD, Nagao S et al. Increased renal expression ofmonocyte chemoattractant protein-1 and osteopontin in ADPKD in rats.Kidney Int 2001; 60: 2087–2096.

51. Panzer U, Thaiss F, Zahner G et al. Monocyte chemoattractant protein-1and osteopontin differentially regulate monocytes recruitment inexperimental glomerulonephritis. Kidney Int 2001; 59: 1762–1769.

52. Katakam AK, Chipitsyna G, Gong Q et al. Streptozotocin (STZ) mediatesacute upregulation of serum and pancreatic osteopontin (OPN): a novelislet-protective effect of OPN through inhibition of STZ-induced nitricoxide production. J Endocrinol 2005; 187: 237–247.

53. Fujii M, Inoguchi T, Maeda Y et al. Pitavastatin ameliorates albuminuriaand renal mesangial expansion by downregulating NOX4 in db/db mice.Kidney Int 2007; 72: 473–480.

54. Gorin Y, Block K, Hernandez J et al. Nox4 NAD(P)H oxidase mediateshypertrophy and fibronectin expression in the diabetic kidney. J BiolChem 2005; 280: 39616–39626.

55. Etoh T, Inoguchi T, Kakimoto M et al. Increased expression of NAD(P)Hoxidase subunits, NOX4 and p22phox, in the kidney of streptozotocin-induced diabetic rats and its reversibity by interventive insulin treatment.Diabetologia 2003; 46: 1428–1437.

56. Lai CF, Seshadri V, Huang K et al. An osteopontin-NADPH oxidasesignaling cascade promotes pro-matrix metalloproteinase 9 activation inaortic mesenchymal cells. Circ Res 2006; 98: 1479–1489.

57. Aroeira LS, Aguilera A, Sanchez-Tomero JA et al. Epithelial tomesenchymal transition and peritoneal membrane failure in peritonealdialysis patients: pathologic significance and potential therapeuticinterventions. J Am Soc Nephrol 2007; 18: 2004–2013.

58. Kobayashi M, Sugiyama H, Wang DH et al. Catalase deficiency rendersremnant kidneys more susceptible to oxidant tissue injury and renalfibrosis in mice. Kidney Int 2005; 68: 1018–1031.

59. van Timmeren MM, Bakker SJ, Vaidya VS et al. Tubular kidney injurymolecule-1 in protein-overload nephropathy. Am J Physiol Renal Physiol2006; 291: F456–F464.

60. Ma J, Weisberg A, Griffin JP et al. Plasminogen activator inhibitor-1deficiency protects against aldosterone-induced glomerular injury. KidneyInt 2006; 69: 1064–1072.

61. Djordjevic T, BelAiba RS, Bonello S et al. Human urotensin II is a novelactivator of NADPH oxidase in human pulmonary artery smooth musclecells. Arterioscler Thromb Vasc Biol 2005; 25: 519–525.



Osteopontin modulates angiotensin II–induced inflammation, oxidative stress, and fibrosis of the kidney

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