Different effects of global osteopontin and macrophage osteopontin in glomerular injury

Abstract

Osteopontin (OPN) is a pro-and anti-inflammatory molecule that simultaneously attenuates oxidative stress. Both inflammation and oxidative stress play a role in the pathogenesis of glomerulonephritis and in the progression of kidney injury. Importantly, OPN is highly induced in nephritic kidneys. To characterize further the role of OPN in kidney injury we used OPN^−/− mice in antiglomerular basement membrane reactive serum-induced immune (NTS) nephritis, an inflammatory and progressive model of kidney disease. Normal wild-type (WT) and OPN^−/− mice did not show histological differences. However, nephritic kidneys from OPN^−/− mice showed severe damage compared with WT mice. Glomerular proliferation, necrotizing lesions, crescent formation, and tubulointerstitial injury were significantly higher in OPN^−/− mice. Macrophage infiltration was increased in the glomeruli and interstitium in OPN^−/− mice, with higher expression of IL-6, CCL2, and chemokine CXCL1. In addition, collagen (Col) I, Col III, and Col IV deposition were increased in kidneys from OPN^−/− mice. Elevated expression of the reactive oxygen species-generating enzyme Nox4 and blunted expression of Nrf2, a molecule that inhibits reactive oxygen species and inflammatory pathways, was observed in nephritic kidneys from OPN^−/− mice. Notably, CD11b diphteria toxin receptor mice with NTS nephritis selectively depleted of macrophages and reconstituted with OPN^−/− macrophages showed less kidney injury compared with mice receiving WT macrophages. These findings suggest that in global OPN^−/− mice there is increased inflammation and redox imbalance that mediate kidney damage. However, absence of macrophage OPN is protective, indicating that macrophage OPN plays a role in the induction and progression of kidney injury in NTS nephritis.

INTRODUCTION

Osteopontin (OPN) is a multifunctional cytokine and adhesion protein that is produced in several tissues, including kidney. OPN is expressed by several cell types, including epithelial and mesenchymal cells and cells of hematopoietic origin like T cells and macrophages. Cultured kidney tubular epithelial cells, mesangial cells, and podocytes produce OPN, and its expression can be increased by growth factors, cytokines, 1, 25(OH)₂-vitamin D, and mechanical stress. In the normal kidney, OPN is constitutively expressed by segments of the loop of Henle and distal nephrons (44).

OPN has been implicated in several physiological and pathological events, including maintenance of tissue integrity during inflammation. OPN is a pro-and anti-inflammatory molecule that simultaneously attenuates oxidative stress in the inflammatory milieu. Although the precise role of OPN in immune responses is not clear, it has been suggested that as a proinflammatory molecule, OPN recruits and modulates the function of macrophages and T cells in addition to enhancing Th1 cytokine expression (1). Once the infection or tissue injury is restrained, OPN may act as an anti-inflammatory molecule via inhibition of inducible NO synthase and NO production and by preventing synthesis of prostaglandin E₂ to limit fibrosis and to maintain tissue integrity. OPN also inhibits oxidative stress by preventing the production of peroxynitrite, a selective oxidant (2, 7). However, increased OPN expression also plays a role in fibrosis by modulating myofibroblast differentiation, increasing collagen (Col) I expression and decreasing matrix metalloproteinases expression (38).

Inflammation is the physiological response to pathogen invasion and tissue damage that is normally resolved in a timely manner by the action of proresolving mediators. However, excessive and nonresolved inflammation results in fibrosis that ultimately leads to organ failure and death. Many chronic inflammatory diseases are also associated with increased production of reactive oxygen species, which results in oxidative stress and causes tissue damage and dysfunction by attacking, denaturing, and modifying structural and functional molecules and by activating redox-sensitive factors. These events led to necrosis, apoptosis, inflammation, and fibrosis. Evidence suggests that both inflammation and oxidative stress play a significant role in the development and progression of chronic kidney diseases (36). Moreover, oxidative stress and inflammation are connected because inflammation induces oxidative stress and vice versa. In addition, elevation of biomarkers of increased oxidative stress and markers of inflammation (C-reactive protein, IL-1, IL-6, TNF-α) has been demonstrated in patients with chronic kidney disease (36).

Because OPN can act as both a pro- and anti-inflammatory molecule as well as modulate oxidative stress, we investigated the effect of absence of OPN in a progressive model of kidney injury. We found that global OPN-deficient mice showed increased inflammation and fibrosis with a loss of redox homeostasis in the kidney, consistent with an overall protective role of OPN in disease. However, an absence of macrophage OPN in the same model protected mice from kidney injury.

MATERIALS AND METHODS

Induction of nephrotoxic serum nephritis.

Male OPN-deficient mice (8 wk old, The Jackson Laboratory B6.129S6(Cg)-Spp1^tm1Blh/J stock no. 004936) and age-and-sex matched littermate wild-type (WT) controls were used. Mice were preimmunized with rabbit IgG 5 days before intravenous injection of 30 µl of anti-GBM antibody (Ab). Preparation of anti-GBM Ab has been described (6, 42). Mice were euthanized at day 8 after the injection of nephrotoxic serum (NTS) to collect kidney tissue and blood. At this time, pronounced glomerular injury and marked tubulointerstital abnormalities were observed (23, 42). Serum creatinine (Cr) was determined by liquid chromatography tandem mass spectrometry and urine microalbuminuria by using mouse specific ELISA kit (Exocell Inc., Philadelphia, PA) and normalized with urine Cr (albumin/creatinine ratio µg/mg) (41). All experiments were performed under protocols approved by the Institutional Animal Care and Use Committee at The University of Colorado Denver.

mRNA expression of cytokines/chemokines.

Kidney total RNA was isolated using TRIzol (Thermo Fischer Scientific, Grand Island, NY). Five micrograms of total RNA from each sample was used for RNase protection assay. mCK1 and mCK5c multi-probe template sets (BD Biosciences, San Jose, CA) were used to investigate cytokine and chemokine expression. RNase protection assay was performed using the Torrey Pines Biolabs kit (Secaucus, NJ) following a described protocol (10, 13). Phosphoimage quantitation of blots was performed using the PhosphorImager SI scanning instrument and ImageQuaNT software (Molecular Dynamics, Sunnyvale, CA). L-32 was used as a housekeeping gene. Final values were expressed as a ratio of counts per minute for the specific mRNA/L-32 mRNA to ensure a constant quantity of RNA in each sample.

Histopathology.

Kidney samples fixed in 10% neutralized buffered formalin and methanol-Carnoy fixative solution were embedded in paraffin. For light microscopy examination, 3- to 4-µm sections were stained with periodic acid-Schiff reagent. For staining of macrophages and T cells, sections of periodate-lysine-paraformaldehyde-fixed tissue were reacted with monoclonal Ab anti-CD68 (Bio-Rad AbD Serotec Ltd., Raleigh, NC) and the pan-T cell marker CD3 (BD Biosciences), respectively, using frozen sections (6, 15). We selected CD68 Ab to detect macrophages, because CD68 is expressed by all macrophages that accumulate in nephritic glomeruli. In contrast, CD11b and F4/80 appear to be expressed by a subset of glomerular macrophages (26). Ab binding was detected using a horseradish peroxidase-based detection system. Positively stained cells per 60 glomeruli were counted and expressed per glomerular section. All quantitative morphological analyses were performed in a blinded fashion.

Immunohistochemistry of Col.

Paraffin sections of methanol-Carnoy fixed tissue were stained for Cols I, III, and IV with specific Abs (SouthernBiotech, Birmingham, AL). The secondary Abs consisted of peroxidase-coupled rabbit anti-goat IgG (Dako North America Inc., Carpinteria, CA). Histological morphometry for Cols was performed using a ScanScope digital scanner (Aperio Technologies Inc., Vista, CA) and results were expressed as mean ± SE % area.

Western blot analysis.

Kidney total protein was obtained using the protein extraction reagent (T-PER, Thermo Fisher Scientific Inc., Waltham, MA) containing protease inhibitors. Nuclear extracts were prepared as described (12). The protein levels of NADPH oxidase (Nox) 4 and Nrf2 were analyzed by Western blot using anti-NOX4 Ab (Novus Cell Signaling Technology, Danvers, MA) and Nrf2 Ab (Abcam, Cambridge, MA). Anti-β-actin Ab (BD Biosciences) or histone H1 (Santa Cruz Biotechnology, Dallas, TX) were used as a loading control.

Bone marrow-derived macrophages isolation.

Bone marrow-derived macrophages (BMDM) were isolated and cultured as described (20, 29). In brief, bone marrow cells were harvested from femurs and tibias of OPN-deficient mice or WT mice. Cells were incubated in DMEM medium containing 10% FBS, 1% penicillin/streptomycin, and 15% L929 cell conditioned medium as a source of macrophage colony-stimulating factor at 37°C. After being cultured overnight, nonadherent cells (containing BMDM) were transferred to a new dish and incubated for an additional 7 days. At the end of the culture period, the purity of isolated macrophages was determined by flow cytometric analysis using Abs against CD68 and Cd11b and gating live cells by forward scatter/side scatter. In addition, to investigate if there was a difference in the expression of cytokines between BMDM from OPN-deficient mice and WT mice at basal level, mRNA expression of several cytokines was analyzed by RNase protection assay.

Conditional ablation of macrophages and reconstitution with WT or OPN-deficient macrophages.

For these studies, we used CD11b-DTR transgenic mice (The Jackson Laboratory, stock no. 006000), these mice are transgenic for human diphtheria toxin (DT) receptor (DTR) under the control of the CD11b promoter. Administration of nanogram doses of DT results in rapid and marked macrophage ablation in the different tissues, including kidney. These mice are on the same background that OPN^−/− mice. CD11b-DTR transgenic mice were preimmunized with rabbit IgG 5 days before intravenous injection with anti-GBM Ab, and at day 5 macrophages were depleted with DT (20 ng/g, i.p.) and reconstituted 24 h later (day 6 after the induction of NTS nephritis), when there is >80% reduction of glomeruli macrophages as described (9, 21, 22, 43). Mice received 1,000,000 of WT or OPN-deficient macrophages (the number of monocytes in one blood volume (21, 39) as described by several groups, including ours (8, 9, 43). Mice received a second dose of DT at day 7 after induction of the NTS nephritis to maintain a depleted endogenous macrophage and were euthanized at day 8. We tracked glomerular accumulation of transferred BMDM isolated from WT and OPN^−/− mice by labeling them with PKH-26GL (Sigma, St. Louis, MO) according the manufacturer’s instructions, and harvested into serum-free medium immediately before injection. One million macrophages were injected on day 6 after induction of NTS nephritis in CD11b-DTR mice ablated of macrophages by injecting DT at days 5 and 7 as described above. Mice were euthanized at day 8 after induction of the disease, and frozen sections from the kidney were stained with Alexa Fluor 488 phalloidin (Life Technologies, Grand Island, NY) and visualized under laser scanning confocal fluorescence microscopy to detect macrophages recruited in the kidney as described (19, 43).

Antigen-specific humoral immune response and glomerular IgG deposition.

Total mouse anti-rabbit IgG titers were measured by enzyme-linked immunosorbent assay using sera collected 8 days after the induction of the nephritis as described (33, 42). Bound mouse IgG was detected using peroxidase-conjugated anti-mouse IgG (Dako) at 1:1,000 dilution, tetramethylbenzidine peroxidase substrate, and absorbance reading at 450 nm. Normal sera served as a negative control. IgG1, IgG2a, and IgG2b isotypes were measured as described above. The bound mouse Ig isotypes were detected using peroxidase-conjugated goat anti-mouse IgG1, IgG2a, and IgG2b Abs (Thermo Fisher) at a dilution of 1:500. IgG deposition was determined in kidney frozen sections using FITC-labeled anti-rabbit IgG (Dako) or anti-mouse IgG (Dako) as described (11, 42). Immunofluorescence images were analyzed by ImageJ software (National Institutes of Health, Bethesda, MD) as described (42).

Statistics.

Mice sample size was determined using SD values and power analyses of our previous studies on the NTS nephritis (42). Statistical analyses were performed using one-way ANOVA with multiple pairwise comparisons with the Bonferroni adjustment for multiple hypothesis testing. Student’s t-test (Mann-Whitney U-test) was used to compare mean values between two experimental groups. Data are reported as mean values ± SE. Values of P < 0.05 were considered statistically significant.

RESULTS

Absence of OPN increases kidney injury in NTS nephritis.

OPN-deficient mice showed unaltered fertility, developed normally, and showed no significant pathological manifestations as described (30, 35). In addition, we also examined kidney histology in OPN-deficient mice under normal conditions. Two-month-old OPN-deficient mice and WT mice did not show differences in the glomeruli and the tubulointerstitium (Fig. 1A). However, at day 8 after the induction of glomerulonephritis (GN) severe kidney injury was observed in OPN-deficient mice. The index of glomerular proliferation (2.06 ± 0.08 vs. 1.04 ± 0.10 P < 0.0001), necrotizing lesions (80.3 ± 9.6% vs. 17.6 ± 6.6%, P < 0.02), and crescentic glomeruli (55.4 ± 7.2 vs. 2.43 ± 2.9. P < 0.01) were markedly increased in OPN-deficient mice compared with WT mice. The absence of OPN also led to prominent tubulointerstitial injury (3.8± 0.54 vs. 0.33 ± −0.08, P < 0.0001) (Fig. 1, A and B). As a result of kidney damage in OPN-deficient mice, kidney function was severely reduced compared with WT mice with both an increase in Cr and microalbuminuria (Fig. 2, A and B).

Fig. 1.

Severe kidney injury is observed in mice deficient in OPN in NTS nephritis. A: periodic acid-Schiff (PAS) staining of kidney sections of WT and OPN-deficient mice with and without GN. Nephritic kidneys from mice lacking OPN had more severe lesions compared with WT mice. B: quantitation of kidney injury in WT and OPN^−/− mice with NTS nephritis. Each data point represents sections sampled from 6 WT (10 wk old) and 9 OPN^−/− (10 wk old) mice and is expressed as mean ± SE. *P < 0.05, **P < 0.01, ****P < 0.0001. PAS staining, original magnification ×400. GN, glomerulonephritis; KO, knockout; Nl, normal; OPN, osteopontin; WT, wild type.

Fig. 2.

Kidney function is deteriorated in mice lacking OPN. A: serum creatinine (mg/dl) was highly increased in OPN^−/− mice. B: OPN^−/− mice showed a significant increase in albumin/creatinine ratio (ACR). Results were sampled from 3 normal WT mice (10 wk old) and 6 WT (10 wk old) and 9 OPN^−/− (10 wk old) mice with NTS nephritis. Results are expressed as mean ± SE. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. KO, knockout; Nl, NL, normal; OPN, osteopontin; WT, wild type.

Severity of NTS nephritis in OPN^−/− mice is associated with increased infiltration of macrophages.

Because macrophages and T cells are implicated in kidney injury in NTS nephritis, we examined infiltration of these cells into the kidney in OPN^−/− mice and WT mice. There was higher accumulation of macrophages in both the glomeruli and in the interstitium in OPN^−/− mice compared with WT mice (P < 0.0001, Fig. 3, A and C). The induction of nephritis also resulted in significant increase of T cells in the interstitium in WT and OPN^−/− mice but there was not significant difference between both groups (Fig. 3, B and C).

Fig. 3.

Glomerular and kidney interstitium macrophage infiltration is increased in OPN^−/− mice but no difference is observed in CD3 cells infiltration. Immunohistochemistry stained for CD68+ monocytes/macrophages (A) and CD3+ cells of kidney sections of WT and OPN-deficient mice (B). Spleen was used as positive control (C). Quantitation of CD68⁺ and CD3⁺ cells infiltration from WT and OPN^−/− mice with NTS nephritis. Each data point represents sections sampled from 3 normal (WT, 10 wk old) mice, 6 WT (10 weeks old), and 9 OPN^−/− (10 weeks old) mice with NTS nephritis and is expressed as mean ± SEM **P > 0.01, ****P > 0.0001. CD68 and CD3 staining, original magnification ×200. hpf, high-power field; KO, knockout; Nl, normal; NTS, nephrotoxic serum; OPN, osteopontin; WT, wild type.

Differential expression of cytokines/chemokines in kidneys from OPN^−/− mice and WT mice with NTS nephritis.

In view of the pro- and anti-inflammatory effects of OPN, the effect of its deletion on the level of cytokine expression was examined. In nephritic kidneys from OPN^−/− mice, higher expression of the proinflammatory cytokines IL-6 mRNA was observed compared with WT mice. Interestingly, the levels of IL-15 mRNA were significantly lower in OPN^−/− mice (Fig. 4A). In addition, CCL2/MCP-1 and CXCL1/MIP-2 were higher in OPN^−/− mice although the induction of RANTES/CCL5 expression was greater in WT mice (Fig. 4B).

Fig. 4.

Analysis of cytokines/chemokines expression in NTS nephritis. A and B: higher expression of cytokines/chemokines is induced in kidneys from OPN-deficient mice compared with WT mice. Densitometric analysis of blots from RNase protection assay of cytokines and chemokines expressed in the kidneys of OPN^−/− mice and WT mice with NTS nephritis. The data are presented as a ratio of the counts per minute (cpm) for the specific mRNA/L-32 mRNA to ensure a constant quantity of RNA in each sample. Results were sampled from 3 normal (WT, 10 wk old) mice and 6 WT (10 wk old) and 9 OPN^−/− (10 weeks old) mice with NTS nephritis and expressed as mean ± SE. *P > 0.05, **P > 0.01, ***P > 0.001, ****P > 0.0001. KO, knockout; MCP, monocyte chemoattractant protein; MIP, macrophage inflammatory protein; Nl, NL, normal; NTS, nephrotoxic serum; OPN, osteopontin; WT, wild type.

Enhanced Col deposition in kidneys from OPN^−/− mice with NTS nephritis.

In OPN^−/− mice increased deposition of Col I and Col III was observed compared with WT mice. In addition, the expression of Col IV, an important component of glomerular extracellular matrix, was also enhanced in the nephritic glomeruli of OPN^−/− mice (Fig. 5).

Fig. 5.

Col deposition is increased in kidneys from OPN^−/− mice. Col I and Col III expression was increased in the interstitium and Col IV deposition was enhanced in the glomeruli from nephritic kidneys in OPN^−/− mice (A). Morphometric analysis of Col I (B), Col III (C), and Col IV (D). Each data point represents sections sampled from 3 normal (WT, 10 wk old) mice and 6 WT (10 wk old) and 9 OPN^−/− (10 wk old) mice with NTS nephritis. Results are expressed as mean ± SE. **P > 0.01, ***P > 0.001, ****P < 0.0001. Cols staining I and III, original magnification ×200 and Col IV original magnification ×400. Col, collagen; KO, knockout; Nl, normal; OPN, osteopontin; WT, wild type.

Nox4-Nrf-2 imbalance in nephritic kidneys from OPN^−/− mice.

Because oxidative stress plays an important role in the pathogenesis of NTS nephritis and OPN is an antioxidant molecule, we investigated if there was redox imbalance in nephritic kidneys in the absence of OPN. Elevated expression of the reactive oxygen species-generating enzyme Nox 4 was observed in nephritic kidneys from OPN^−/− mice. In contrast, the expression of Nrf2, the transcription factor that regulates genes encoding antioxidant and detoxifying molecules was suppressed in OPN^−/− mice (Fig. 6).

Fig. 6.

Redox imbalance in nephritic kidneys from OPN^−/− mice: Western blot analysis of Nox4 (A and B) and Nrf2 (C) shows increased expression of Nox4 and blunted expression of Nrf2. Representative Western blots are shown. Densitometry analyses for Nox 4 (B) was performed from 3 normal WT and 3 normal OPN^−/−-deficient mice and 6 WT and 6 OPN^−/− mice with NTS nephritis, all mice were 10 wk old. *P > 0.05, **P > 0.01. GN, glomerulonephritis; KO, knockout; Nl, normal; OPN, osteopontin; WT, wild type.

Systemic humoral immune response, but not the binding of rabbit IgG and mouse IgG, was affected in OPN^−/− mice.

To analyze the systemic humoral immune response in WT and OPN^−/− mice with NTS nephritis, we assessed antigen-specific mouse anti-rabbit IgG in serum by specific ELISA. Total mouse anti-rabbit globulin IgG levels were similar in both groups (Fig. 7A). Although IgG1 and IgG2b titers were comparable in both groups, the levels of IgG2a were significantly higher in OPN^−/− mice (Fig. 7A). In addition, the binding of rabbit and mouse IgG was not different between WT and OPN^−/− nephritic kidneys (Fig. 7, B and C).

Fig. 7.

Absence of OPN affects systemic humoral immune response but not IgG deposition. A: circulating titers of total mouse anti-rabbit IgG at serum dilution of 1:100 (left) or 1:200 (right) were not different between the nephritic groups *P < 0.05 vs. normal. However, although IgG1 and IgG2b titers were comparable in both groups, there were significantly higher titers of IgG2a in OPN^−/− mice (*P < 0.05). B: immunofluorescence analysis of mouse (left) and rabbit IgG (right) deposition in WT mice and OPN^−/− mice by ImageJ software. Immunofluorescence is expressed as arbitrary units. C: immunofluorescence staining of rabbit IgG and mouse IgG. Original magnification ×200. KO, knockout; Nl, NL, normal; OD450, optical density 450 nm; OPN, osteopontin; Rb, rabbit; WT, wild type.

Macrophage purity and in vivo tracking of transferred macrophages.

Macrophages were isolated from bone marrow as described in material and methods. The fraction of CD68/CD11b-positive cells from bone marrows from WT and OPN^−/− mice was >92% (Fig. 8, A and B). In the negative controls (no adding Ab or using purified IgG) 0.2% cells were positive for CD68 or CD11b. Importantly, there was no difference in the mRNA expression of proinflammatory cytokines and chemokines between unstimulated BMDM from WT and OPN-deficient mice (Fig. 8C). In addition, when we tracked glomerular accumulation of transferred BMDM isolated from OPN-deficient mice and WT mice, a similar number of macrophages was observed into nephritic glomeruli in both groups (5.23 ± 0.4254, 5.86 ± 0.358, respectively, Fig. 8, D and E).

Fig. 8.

WT macrophages and OPN-deficient macrophages isolated from bone marrow show similar production of cytokines/chemokines at a basal level and migrate similarly to nephritic kidneys. Representative FACS plots showing CD68 and CD11b-positive cell populations isolated from WT (A) and OPN^−/− (B) mice. Determinations were performed at the end of the culture period. (C) RNase protection assay of cytokines and chemokines expressed in BMDM. Probes contain polylinker regions and are longer than the protected bands. GAPDH and L-32 were used as housekeeping genes. Densitometric analysis of blots showed from RNase protection assay are presented as a ratio of the counts per minute (cpm) for the specific mRNA/L-32 mRNA to ensure a constant quantity of RNA in each sample. Representative figure from BMDM isolated from three different WT mice and OPN^−/− mice. (D and E) Representative fluorescence micrographs of PKH-26GL-labeled BMDM. Similar number of WT (D) and OPN^−/− (E) macrophages migrate to nephritic glomeruli. Original magnification ×400. BMDM; bone marrow-derived macrophages; Chemok, chemokine; Cytok, cytokine; LT, lymphotoxin; MIP, macrophage inflammatory protein; OPN, osteopontin; TGF, transforming growth factor; WT, wild type.

Absence of macrophage OPN attenuates kidney injury.

Because macrophages play critical roles in the initiation and progression of NTS nephritis we determined the role of OPN expressing macrophages in kidney injury in GN. Endogenous macrophages were eliminated with DT in CD11b-DTR Tg mice with NTS nephritis at day 5 after induction of the diseases. Mice received a second dose of DT at day 7 after induction of the NTS nephritis to maintain a depleted endogenous macrophages as described above. Adoptive transfer of macrophages from OPN^−/− mice or WT mice was performed at day 6, and mice were euthanized at day 8. At the time mice were euthanized, glomerular hypercellularity, necrotizing lesions, crescent formation and tubulointerstitial damage were attenuated in mice reconstituted with OPN^−/− macrophages compared with mice receiving WT macrophages (Fig. 9, A and C). In addition, the number of tubular casts, a good indicator for chronic tubulointerstitial injury, was significantly reduced in mice receiving macrophages deficient in OPN (36.4 ± 14 vs. 5.0 ± 1.9, P < 0.0001). As a consequence of attenuated kidney damage in mice receiving OPN^−/− macrophages, normal kidney function was maintained (Fig. 9B).

Fig. 9.

Lack of macrophage OPN attenuates kidney injury in CD11b-DTR mice with NTS nephritis. A: periodic acid-Schiff (PAS) staining of kidney sections of nephritic kidneys from mice that were depleted of macrophages and received adoptive transfer of WT macrophages (AT WT Mac) or adoptive transfer of macrophages deficient in OPN (AT OPN^−/− Mac). Each data point represents sections sampled from six AT WT Mac (10 wk old) and six AT OPN^−/− Mac (10 wk old) mice with NTS nephritis. B: serum creatinine (mg/dl) creatinine values, showing preserved kidney function in mice receiving AT OPN^−/− Mac. C: quantitation of kidney injury. Data are expressed as mean ± SE *P < 0.05, ***P < 0.001, ****P < 0.0001. PAS staining, original magnification ×400. glom, glomeruli; KO, knockout; Mac, macrophage; Nl, normal; NTS, nephrotoxic serum; OPN, osteopontin; WT, wild type.

DISCUSSION

Our results indicate that absence of global OPN increases susceptibility to kidney injury in NTS nephritis by increasing inflammation, systemic humoral immune response, and oxidative stress. Because inflammation and oxidative stress are major mediators of progression of kidney damage, renal fibrosis was also increased in mice lacking OPN. These results suggest an overall protective response of OPN on kidney injury. In contrast, an absence of macrophage OPN attenuates kidney damage in NTS nephritis, suggesting that macrophage OPN is proinflammatory and plays a role in the induction and progression of kidney injury in NTS nephritis.

OPN is highly expressed in chronic inflammatory and autoimmune diseases, and it is specifically localized in and around inflammatory cells. Although OPN is not expressed in circulating monocytes, it is highly upregulated during macrophage differentiation and is one of the crucial products of macrophage in several diseases (34). Macrophages are a source and a target of OPN. OPN regulates macrophage migration, survival, phagocytosis, and proinflammatory cytokine production (31). We have found that OPN is expressed in the kidney in GN and macrophages infiltrating the nephritic glomeruli express OPN. In addition, inactivating macrophages using an agonist for the adenosine receptor A_2A reduced OPN expression that was associated with reduction in kidney injury, supporting a role of macrophage OPN in mediating kidney damage in GN (11).

Interestingly, global OPN^−/− mice showed severe kidney damage compared with WT mice, indicating that global/kidney OPN has a protective role against tissue damage. Although studies have demonstrated that OPN promotes accumulation of macrophages and may play a role in macrophage-mediated kidney injury, OPN also has renoprotective effects in kidney damage, such as inhibiting inducible NO synthase and suppressing NO synthesis, reducing cell peroxide formation, decreasing cell apoptosis, and playing a role in the regeneration of cells (27, 30, 44). Moreover, OPN has been identified as a strong and independent predictor for survival in critically ill patients with acute kidney injury (24). In addition, in animal models, OPN global deficient mice showed more severe kidney injury in acute renal ischemia and marked glomerulosclerosis after uninephrectomy (30, 37).

Previously, there was a report that kidney injury was reduced in accelerated crescentic GN in rats by using a neutralizing anti-OPN Ab (48). In addition, in a passive model of crescentic GN, blocking OPN with a neutralizing Ab in the early fibrotic stage attenuates glomerular fibrosis. However, using OPN-deficient mice, there was no difference in kidney injury and macrophage infiltration compared with WT mice in NTS nephritis (3, 48, 49). The variation in the results could be related to preferential inhibition of macrophage OPN with the neutralizing Abs. It may also relate to differences in the species, strain, sex, and use of neutralizing Ab versus global deficient mice. In this study we use male OPN knockout mice on a C57BL/6 background compared with female mice in a mixed 129 Sv/C57BL6 background (crossed once with C57BL6) in the previous study inducing NTS nephritis in OPN-deficient mice (3).

It has been suggested that through its proinflammatory effects, OPN contains tissue injury whereas its anti-inflammatory function can limit fibrosis and help to repair the tissue damage. In contrast, an absence of OPN causes defective macrophage recruitment and differentiation with enhanced production of toxic metabolites and oxidative stress resulting in extensive fibrosis (7). The severity in kidney damage in global OPN-deficient mice with NTS nephritis could be explained, at least in part, by difference in cytokines/chemokine expression between WT and OPN-deficient mice. Interestingly, the levels of IgG2a were significantly higher in OPN^−/− mice, suggesting a dysregulation of production of IgG in the absence of OPN. This is important because IgG2a binds all of the γ chain-containing activating Fcγ receptors that are expressed in the majority of innate immune effector cells. Some of these cells also play an important role in the pathogenesis of NTS nephritis, such as dendritic cells and this could in part explain the increased damage observed in OPN^−/− mice (4, 25). In addition, Nrf2 was blunted and Nox4 was increased in OPN-deficient mice. Since oxidative stress and inflammation are inseparably associated and each one amplifies the other, increased oxidative stress could promote recruitment and activation of leukocytes and resident cells intensifying inflammation. Conversely, by generating reactive oxygen, chlorine, and nitrogen species, activated inflammatory cells and resident cells could increase oxidative stress. Moreover, the protective effects of Nrf2 are demonstrated by a decrease of oxidative stress, inflammation, and kidney disease in response to Nrf2 activators, whereas Nrf2 deletion amplifies oxidative stress and inflammatory pathways and leads to autoimmune nephritis (36, 47).

Previously, Nrf2 has been reported to be reduced in kidney diseases such as 5/6 nephrectomy, focal segmental glomerulosclerosis, and crescentic glomerulonephritis, and in our study OPN-deficient mice with GN also lacked expression of Nrf2 (17, 18, 36, 46). Whether OPN regulates Nrf2 expression will require further studies.

Interestingly, in global deficient OPN mice there was an increase in macrophage infiltration in NTS nephritis. Although it has been reported that OPN can downregulate M1 macrophages, an absence of OPN shifts macrophage polarization toward a proregenerative phenotype by reducing M1 and M2a and increasing M2c subsets (5, 14). Consequently, it is likely that the infiltrating macrophages in NTS nephritis observed in the OPN global deficient mice are protective.

In contrast to increased kidney damage in NTS nephritis in global OPN-deficient mice, an absence of macrophage OPN attenuated kidney injury. Glomerular macrophage accumulation was similar in CD11b-DTR mice receiving WT macrophages and macrophages deficient in OPN, suggesting that absence of OPN does not affect migration of these cells in NTS nephritis. Notably, macrophages deficient in OPN did not show any difference in the basal expression of cytokines compared with WT macrophages. However, after BMDM migrate to the nephritic kidney and become exposed to an inflammatory milieu, OPN-deficient macrophages and WT macrophages may respond differently because OPN stimulates production of proinflammatory cytokines by macrophages (1).

In addition to its protective effects, studies have shown a deleterious role for OPN in kidney diseases. Studies reducing OPN or using OPN deficient mice have shown attenuation of diabetic nephropathy, aldosterone-mediated renal fibrosis, chronic allograft nephropathy, and obstructive uropathy (16, 28, 32, 40, 45). These data support that OPN is a molecule with complex function and raises the possibility that it is a “wound healing molecule” that, depending on the condition and cell type producing it, may function to aid healing or, conversely, trigger excess inflammation, oxidative stress and fibrosis. In addition, proteolytically cleaved OPN fragments possess higher activity than the full-length form and several forms of OPN may have distinct effect in different tissues. Tissue-specific deletion of OPN in different kidney diseases will be necessary to determine its role in kidney injury and protection.

GRANTS

This work was supported in part by National Institutes of Health Grants DK-082509 (to G. Garcia), R01-DK-096030 (to M. Miyazaki), R01-HL-117062 (to M. Miyazaki), R01-HL-133545 (to M. Miyazaki), and R01-HL-132318 (to M. Miyazaki).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

J.T. and G.E.G. conceived and designed research; J.T., C.R.-J., M.M., S.M.-A., R.M., and G.E.G. performed experiments; J.T., L.D.T., C.R.-J., M.M., S.M.-A., M.K., R.M., A.A.-H., Y.S., T.J., M.A.L., R.J.J., and G.E.G. analyzed data; J.T., L.D.T., C.R.-J., M.M., S.M.-A., M.K., R.M., A.A.-H., Y.S., T.J., M.A.L., R.J.J., and G.E.G. interpreted results of experiments; J.T. and G.E.G. prepared figures; J.T. and G.E.G. drafted manuscript; J.T., L.D.T., R.J.J., and G.E.G. edited and revised manuscript; J.T., L.D.T., C.R.-J., M.M., S.M.-A., M.K., A.A.-H., Y.S., T.J., M.A.L., R.J.J., and G.E.G. approved final version of manuscript.