Critical role for osteopontin in diabetic nephropathy
2010; Elsevier BV; Volume: 77; Issue: 7 Linguagem: Inglês
10.1038/ki.2009.518
ISSN1523-1755
AutoresSusanne B. Nicholas, Joey Liu, Jason Kim, Yuelan Ren, Alan R. Collins, Lam Nguyen, Willa A. Hsueh,
Tópico(s)Cancer, Hypoxia, and Metabolism
ResumoThe profibrotic adhesion molecule, osteopontin (OPN), is upregulated in kidneys of humans and mice with diabetes. The thiazolidinedione (TZD) insulin sensitizers decrease albuminuria in diabetic nephropathy (DN) and reduce OPN expression in vascular and cardiac tissue. To examine whether OPN is a critical mediator of DN we treated db/db mice with insulin, rosiglitazone, or pioglitazone to achieve similar fasting plasma glucose levels. The urine albumin-to-creatinine ratio and glomerular OPN expression were increased in diabetic mice, but both were reduced by the TZDs more than by insulin. We administered streptozotocin to OPN-null and OPN-wild-type mice, and OPN-null mice were bred into both type 1 (Ins2akita/+) and 2 (db/db) diabetic mice. In each case, OPN deletion decreased albuminuria, mesangial area, and glomerular collagen IV, fibronectin and transforming growth factor (TGF)-β in the diabetic mice compared with their respective controls. In cultured mouse mesangial cells, TZDs but not insulin decreased angiotensin II-induced OPN expression, while recombinant OPN upregulated TGF-β, ERK/MAPK, and JNK/MAPK signaling. These studies show that OPN expression in DN mouse models enhances glomerular damage, likely through the expression of TGF-β, while its deletion protects against disease progression, suggesting that OPN might serve as a therapeutic target. The profibrotic adhesion molecule, osteopontin (OPN), is upregulated in kidneys of humans and mice with diabetes. The thiazolidinedione (TZD) insulin sensitizers decrease albuminuria in diabetic nephropathy (DN) and reduce OPN expression in vascular and cardiac tissue. To examine whether OPN is a critical mediator of DN we treated db/db mice with insulin, rosiglitazone, or pioglitazone to achieve similar fasting plasma glucose levels. The urine albumin-to-creatinine ratio and glomerular OPN expression were increased in diabetic mice, but both were reduced by the TZDs more than by insulin. We administered streptozotocin to OPN-null and OPN-wild-type mice, and OPN-null mice were bred into both type 1 (Ins2akita/+) and 2 (db/db) diabetic mice. In each case, OPN deletion decreased albuminuria, mesangial area, and glomerular collagen IV, fibronectin and transforming growth factor (TGF)-β in the diabetic mice compared with their respective controls. In cultured mouse mesangial cells, TZDs but not insulin decreased angiotensin II-induced OPN expression, while recombinant OPN upregulated TGF-β, ERK/MAPK, and JNK/MAPK signaling. These studies show that OPN expression in DN mouse models enhances glomerular damage, likely through the expression of TGF-β, while its deletion protects against disease progression, suggesting that OPN might serve as a therapeutic target. Activation of the nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) in the kidney attenuates albuminuria in animal models of both type 1 and 2 diabetes and in humans with type 2 diabetes.1.Imano E. Kanda T. Nakatani Y. et al.Effect of troglitazone on microalbuminuria in patients with incipient diabetic nephropathy.Diabetes Care. 1998; 21: 2135-2139Crossref PubMed Scopus (179) Google Scholar, 2.Isshiki K. Haneda M. Koya D. et al.Thiazolidinedione compounds ameliorate glomerular dysfunction independent of their insulin-sensitizing action in diabetic rats.Diabetes. 2000; 49: 1022-1032Crossref PubMed Scopus (170) Google Scholar, 3.Nicholas S.B. Kawano Y. Wakino S. et al.Expression and function of peroxisome proliferator-activated receptor-gamma in mesangial cells.Hypertension. 2001; 37: 722-727Crossref PubMed Google Scholar, 4.Tanimoto M. Fan Q. Gohda T. et al.Effect of pioglitazone on the early stage of type 2 diabetic nephropathy in KK/Ta mice.Metabolism. 2004; 53: 1473-1479Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar However, the mechanisms mediating these antialbuminuric effects are not known, but appear to be independent of blood glucose and blood pressure.1.Imano E. Kanda T. Nakatani Y. et al.Effect of troglitazone on microalbuminuria in patients with incipient diabetic nephropathy.Diabetes Care. 1998; 21: 2135-2139Crossref PubMed Scopus (179) Google Scholar,3.Nicholas S.B. Kawano Y. Wakino S. et al.Expression and function of peroxisome proliferator-activated receptor-gamma in mesangial cells.Hypertension. 2001; 37: 722-727Crossref PubMed Google Scholar Recently, osteopontin (OPN), a large phosphoglycoprotein adhesion molecule, has emerged as a potentially key pathophysiologic contributor in diabetic nephropathy (DN). OPN is expressed in all glomerular cells: mesangial cells, podocytes, and endothelial cells.5.Endlich N. Sunohara M. Nietfeld W. et al.Analysis of differential gene expression in stretched podocytes: osteopontin enhances adaptation of podocytes to mechanical stress.FASEB J. 2002; 16: 1850-1852PubMed Google Scholar, 6.Giachelli C.M. Vascular calcification: in vitro evidence for the role of inorganic phosphate.J Am Soc Nephrol. 2003; 14: S300-S304Crossref PubMed Google Scholar, 7.Giachelli C.M. Liaw L. Murry C.E. et al.Osteopontin expression in cardiovascular diseases.Ann NY Acad Sci. 1995; 760: 109-126Crossref PubMed Scopus (171) Google Scholar, 8.Sodhi C.P. Batlle D. Sahai A. Osteopontin mediates hypoxia-induced proliferation of cultured mesangial cells: role of PKC and p38 MAPK.Kidney Int. 2000; 58: 691-700Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar Microarray analyses of diabetic versus normal kidneys identified OPN as one of the major genes upregulated in humans with DN and in mice with either type 1 diabetes induced by streptozotocin (STZ) or the type 2 db/db model of diabetes.9.Susztak K. Bottinger E. Novetsky A. et al.Molecular profiling of diabetic mouse kidney reveals novel genes linked to glomerular disease.Diabetes. 2004; 53: 784-794Crossref PubMed Scopus (119) Google Scholar Ligands to PPARγ, thiazolidinediones (TZDs), suppress OPN expression in cardiomyocytes, vascular smooth muscle cells, and macrophages to attenuate cardiac fibrosis and atherosclerosis.10.Oyama Y. Akuzawa N. Nagai R. et al.PPARgamma ligand inhibits osteopontin gene expression through interference with binding of nuclear factors to A/T-rich sequence in THP-1 cells.Circ Res. 2002; 90: 348-355Crossref PubMed Scopus (53) Google Scholar,11.Oyama Y. Kurabayashi M. Akuzawa N. et al.Troglitazone, a PPARgamma ligand, inhibits osteopontin gene expression in human monocytes/macrophage THP-1 cells.J Atheroscler Thromb. 2000; 7: 77-82Crossref PubMed Scopus (11) Google Scholar We, therefore, hypothesized that OPN is a critical mediator in DN and that the renal protective effect of TZDs in DN may involve suppression of OPN. To address this hypothesis, we (1) determined whether glomerular OPN expression correlated with attenuation of albuminuria in db/db mice treated with TZDs versus insulin, (2) defined the role of OPN in STZ-induced albuminuria using OPN knockout mice, (3) examined the effect of genetic knockout of OPN in mouse models of both type 1 and 2 diabetes, and (4) investigated OPN regulation by angiotensin II (AngII) and TZDs, and the effect of mouse recombinant OPN on transforming growth factor (TGF)-β expression and signal transduction in cultured mouse mesangial cells. Fasting plasma glucose was elevated in db/db mice versus db/m mice and remained unchanged after treatment with PPARγ ligands, rosiglitazone or pioglitazone, or with insulin for 8 weeks, similar to pretreatment levels, but progressively increased in db/db control mice (Table 1). Initial and final blood pressures did not change among all animal groups. Before killing, db/db mice weighed significantly more than db/m mice. The ratio of body weight/tibia length was comparable in control, rosiglitazone- and pioglitazone-treated db/m and higher in db/db mice (Table 1). The ratio of kidney weight/tibia length was not different among the animals (data not shown).Table 1Physiological features of treated and untreated non-diabetic db/m and diabetic db/db micedb/m Controldb/db DiabeticPioRosiControlPioRosiInsulinControlNumber of mice648128820Initial plasma glucose (mg/dl)92±474±481±4271±31aDiabetic db/db versus non-diabetic db/m control, P<0.05.279±27aDiabetic db/db versus non-diabetic db/m control, P<0.05.299±39aDiabetic db/db versus non-diabetic db/m control, P<0.05.283±23aDiabetic db/db versus non-diabetic db/m control, P<0.05.Final plasma glucose (mg/dl)134±16114±15115±11291±18aDiabetic db/db versus non-diabetic db/m control, P<0.05.249±22aDiabetic db/db versus non-diabetic db/m control, P<0.05.278±44aDiabetic db/db versus non-diabetic db/m control, P<0.05.649±17bFinal versus initial, P<0.05.Initial SBP (mm Hg)98±7100±4100±792±6104±3103±397±5Final SBP (mm Hg)109±6100±5111±3108±11100±498±393±3Body weight/tibia length (g/mm)1.4±0.011.5±0.031.4±0.033.8±0.09aDiabetic db/db versus non-diabetic db/m control, P<0.05.3.0±0.05aDiabetic db/db versus non-diabetic db/m control, P<0.05.3.0±0.07aDiabetic db/db versus non-diabetic db/m control, P<0.05.2.9±0.05aDiabetic db/db versus non-diabetic db/m control, P<0.05.Abbreviations: Pio, pioglitazone; Rosi, rosiglitazone; SBP, systolic blood pressure.a Diabetic db/db versus non-diabetic db/m control, P<0.05.b Final versus initial, P<0.05. Open table in a new tab Abbreviations: Pio, pioglitazone; Rosi, rosiglitazone; SBP, systolic blood pressure. Baseline albumin-to-creatinine ratio (ACR) was elevated in 8-week-old db/db mice (∼400–700 mg/dl) and progressively increased to 1207±72 μg/mg by 16 weeks, P<0.05, as previously reported in this model.12.Breyer M.D. Bottinger E. Brosius III, F.C. et al.Mouse models of diabetic nephropathy.J Am Soc Nephrol. 2005; 16: 27-45Crossref PubMed Scopus (412) Google Scholar ACR in db/m mice (∼100 mg/dl) was unchanged from initial levels (Figure 1). Insulin treatment decreased ACR in db/db mice to 458±107 μg/mg. However, the TZDs had a greater effect to lower ACR (rosiglitazone, 217±50 μg/mg; pioglitazone, 233±26 μg/mg), P<0.05. Thus, PPARγ ligands improved ACR more than did insulin, suggesting a PPARγ effect on albumin excretion beyond the decrease in glucose. Periodic acid Schiff (PAS) staining was performed to quantify the % mesangial area that was glomerular mesangial matrix (Figures 2a, b). There was no significant difference among the db/m group. However, % mesangial area was significantly higher in db/db control compared with insulin-treated mice, which was also significantly higher than in either rosiglitazone- or pioglitazone-treated mice. Immunohistochemistry (IHC) for glomerular fibronectin was performed to assess matrix protein accumulation. Glomerular fibronectin staining was similar to % mesangial area (Figures 2c, d) with ∼6-fold increase in db/db control mice, a modest reduction with insulin treatment and a greater reduction by TZDs. Glomerular cores were isolated from a subset of animals to determine TGF-β protein expression. TGF-β levels were higher in db/db control and insulin-treated db/db compared with either db/m control or rosiglitazone-treated db/db mice, P<0.05 (Figure 2e, f). Glomerular expression of OPN was substantially increased in db/db mice compared with db/m mice, similar to that in whole kidney of humans and animals.9.Susztak K. Bottinger E. Novetsky A. et al.Molecular profiling of diabetic mouse kidney reveals novel genes linked to glomerular disease.Diabetes. 2004; 53: 784-794Crossref PubMed Scopus (119) Google Scholar There was a trend for insulin to decrease glomerular OPN expression after 8 weeks. In contrast, OPN expression was reduced by 40–50% in rosiglitazone- and pioglitazone-treated db/db mice, P<0.05 (Figure 3a). These observations suggested that glomerular OPN may be a potential target for PPARγ ligands, with a more modest effect of insulin. We incubated stable mouse mesangial cells with AngII in the absence and presence of insulin, rosiglitazone, or pioglitazone to examine their direct effects on OPN expression. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated ∼2-fold stimulation of OPN by AngII and 1.5-fold upregulation by insulin + AngII compared with untreated control cells, P<0.05. However, both rosiglitazone and pioglitazone downregulated OPN in the presence of AngII, to control levels, P<0.05. Insulin alone did not alter OPN expression in AngII-treated cells (Figure 3b). To determine the role of OPN in DN, we induced diabetes in OPN−/− and OPN+/+ control mice, on a 129 × Black Swiss background, with STZ to achieve plasma glucose levels of ∼450 to 600 mg/dl compared with control mice (∼100 mg/dl), P<0.05 (Table 2). There were no differences in tail-cuff systolic blood pressures over the 12-week study. At killing, body weight/tibia length was significantly higher in non-diabetic compared with diabetic mice, P<0.05. Kidney weight/tibia length was slightly less in the non-diabetic versus the diabetic OPN+/+, P<0.05, but remained unchanged in OPN−/−mice.Table 2Physiological characteristics of OPN+/+ and OPN−/− miceOPN+/+OPN−/−ControlDiabeticControlDiabeticNumber of mice1013810Initial plasma glucose (mg/dl)63±468±473±768±3Final plasma glucose (mg/dl)93±5aFinal versus initial, P<0.05.601±24aFinal versus initial, P<0.05.102±8aFinal versus initial, P<0.05.462±56aFinal versus initial, P<0.05.Initial SBP (mm Hg)93±5100±594±688±4Final SBP (mm Hg)101±397±4101±5105±2Body weight/tibia length (g/mm)1.9±0.1bControl versus diabetic, P<0.05.1.4±0.11.9±0.0bControl versus diabetic, P<0.05.1.6±0.1Kidney weight/tibia length (g/mm)0.01±0.0bControl versus diabetic, P<0.05.0.02±0.00.01±0.00.01±0.0Abbreviation: SBP, systolic blood pressure.a Final versus initial, P<0.05.b Control versus diabetic, P<0.05. Open table in a new tab Abbreviation: SBP, systolic blood pressure. The initial ACR was higher in OPN+/+ compared with OPN−/−mice, P<0.05 (Figure 4a). After 4, 8, and 12 weeks of STZ administration, the diabetic OPN−/− had higher ACR compared with non-diabetic OPN+/+ mice. However, ACR in diabetic OPN+/+ was substantially higher than in diabetic OPN−/− mice throughout the study. Thus, the lack of OPN attenuated albuminuria after STZ administration, despite similar plasma glucose levels. To further investigate the effect of OPN on the pathology of DN, kidneys were sectioned for PAS and IHC staining, and glomerular cores were isolated for western blots. STZ-diabetic OPN+/+ had greater glomerular % mesangial area (1.9-fold), collagen IV (2.5-fold), TGF-β (1.6-fold), and fibronectin (1.2-fold) expression compared with non-diabetic OPN+/+ mice, P<0.05 (Figures 4b–i). Collagen, TGF-β, and fibronectin protein levels were not different in STZ-diabetic OPN−/− versus non-diabetic OPN−/− mice. Quantitative RT-PCR of glomerular core collagen IV, fibronectin, and TGF-β expression showed parallel results (data not shown). Thus, the lack of OPN is associated with less albuminuria as well as less glomerular matrix, collagen IV, TGF-β, and fibronectin mRNA and protein, suggesting that OPN may have a pathophysiologic role in glomerular injury in diabetes. To further test the role of OPN in genetic models of types 1 and 2 DN, OPN−/− mice were bred onto Ins2akita/+; Ins2+/+ and (Leprdb/db; Lepr+/+ mice) (Figures 5 and 6). Quantitative RT-PCR of glomerular cores confirmed the absence of OPN expression in OPN−/− mice (data not shown). In both groups, initial and final plasma glucose was significantly lower in non-diabetic (OPN+/+Ins2+/+; OPN−/−Ins2+/+ and OPN+/+Lepr+/+; OPN−/−Lepr+/+) compared with diabetic (OPN+/+Ins2akita/+; OPN−/−Ins2akita/+, and OPN+/+Leprdb/db; OPN−/−Leprdb/db) mice, P<0.05. There were no differences in initial or final blood pressures among the groups. However, there was a significant increase in body weight and kidney weight/tibia length between Leprdb/db and Lepr+/+ mice, P<0.05 (Tables 3 and 4).Figure 6Breeding protocol for OPN−/− Leprdb/db.View Large Image Figure ViewerDownload (PPT)Table 3Physiological features of diabetic Ins2akita/+ and non-diabetic Ins2+/+ miceOPN+/+OPN−/−Ins2+/+ Non-diabeticIns2akita/+ DiabeticIns2+/+ Non-diabeticIns2akita/+ DiabeticNumber of mice10778Initial plasma glucose (mg/dl)95±3480±34aP<0.05, diabetic versus control mice.73±12453±40aP<0.05, diabetic versus control mice.Final plasma glucose (mg/dl)117±3.5465±34aP<0.05, diabetic versus control mice.114±10468±41aP<0.05, diabetic versus control mice.Initial SBP (mm Hg)100±594±692±3104±3Final SBP (mm Hg)97±4108±496±595±5Body weight/tibia length (g/mm)1.8±0.061.24±0.042.02±0.071.34±0.03Kidney weight/tibia length (g/mm)0.012±0.0000.014±0.0010.012±0.0000.0129±0.000Abbreviation: SBP, systolic blood pressure.Initial: age 2 months.Final: age 4 months.a P<0.05, diabetic versus control mice. Open table in a new tab Table 4Physiological features of OPN−/−Lepr and OPN+/+Lepr non-diabetic and diabetic miceOPN+/+OPN−/−Lepr+/+ Non-diabeticLeprdb/db DiabeticLepr+/+ Non-diabeticLeprdb/db DiabeticNumber of mice97915Initial plasma glucose (mg/dl)97±7417±50aP<0.05, diabetic versus non-diabetic.85±5497±35aP<0.05, diabetic versus non-diabetic.Final plasma glucose (mg/dl)116±4555±29aP<0.05, diabetic versus non-diabetic.,bP<0.05, final versus initial.86±9cP<0.05, OPN−/− versus OPN+/+.499±22aP<0.05, diabetic versus non-diabetic.Initial SBP (mm Hg)106±4100±5107±4109±4Final SBP (mmHg)105±4101±4115±±4109±4Body weight/tibia length (g/mm)1.70±0.062.80±0.18aP<0.05, diabetic versus non-diabetic.1.78±0.092.60±0.17aP<0.05, diabetic versus non-diabetic.Kidney weight/tibia length (g/mm)0.012±0.0010.015±0.001aP<0.05, diabetic versus non-diabetic.0.011±0.0010.015±0.001aP<0.05, diabetic versus non-diabetic.Abbreviation: SBP, systolic blood pressure.Initial: age 2 months.Final: age 4 months.a P<0.05, diabetic versus non-diabetic.b P<0.05, final versus initial.c P<0.05, OPN−/− versus OPN+/+. Open table in a new tab Abbreviation: SBP, systolic blood pressure. Initial: age 2 months. Final: age 4 months. Abbreviation: SBP, systolic blood pressure. Initial: age 2 months. Final: age 4 months. The protective effect of OPN depletion against albuminuria was demonstrated in genetic type 1 and 2 OPN−/− mice. ACR was 5- to 7.7-fold higher in OPN+/+Ins2akita/+ versus OPN+/+Ins2+/+ but not different in OPN−/−Ins2akita/+ versus OPN−/−Ins2+/+ mice at 2, 3, and 4 months, P<0.05. Similarly, ACR was ∼9-fold higher in OPN+/+Leprdb/db versus OPN+/+Lepr+/+ and three- to fourfold higher in OPN−/−Leprdb/db versus OPN−/−Lepr+/+ mice, P<0.05. OPN deletion attenuated ACR two- to threefold in both genetic OPN−/−-diabetic mice, P<0.05 (Figures 7a, b). OPN deletion markedly attenuated the diabetes-induced increases in % mesangial area (∼1.3-fold), collagen IV (∼1.5-fold), and fibronectin (four to fivefold) in both diabetic models by PAS and IHC, P<0.05 (Figures 8a–l; P<0.001). Glomerular TGF-β by quantitative RT-PCR showed ∼2-fold lower expression in both OPN−/−-diabetic models versus diabetic OPN+/+ mice, P<0.01 and there was no increase in TGF-β expression in diabetic versus non-diabetic OPN−/− mice (Figures 8m, n). To investigate the cellular effect of OPN, cultured mesangial cells were incubated with recombinant mouse OPN (rmOPN) for up to 24 h. There was a dose-dependent increase in TGF-β expression with OPN up to 10 nM (Figure 9a, b). At 10 nM, there was a time-dependent upregulation of OPN by TGF-β (Figure 9c, d), which peaked at 12 h. In contrast, pretreatment of mesangial cells grown in high glucose (30 mM) with antibody against OPN resulted in a dose-dependent inhibition of TGF-β expression (Figure 9e, f). These data suggested that OPN enhances TGF-β expression, and that OPN may promote glomerular matrix accumulation by upregulation of TGF-β. Several studies have shown that mitogen-activated protein kinase (MAPK) signaling pathways are activated in mesangial cells in the diabetic milieu. To understand the potential mechanisms by which OPN may regulate extracellular matrix expression and thus identify a potential link to promote albuminuria in the glomerulus, mesangial cells were preincubated with inhibitors of ERK/MAPK (PD98059) and JNK/MAPK (SP600125) for 30 min followed by incubation with rmOPN for 30 and 45 min, respectively. Phosphorylated ERK and phosphorylated JNK were upregulated two- to threefold by rmOPN, P<0.05. However, only ERK and not JNK expression was significantly inhibited after preincubation with the inhibitor (Figures 10a–d). As c-Jun is phosphorylated by JNK at serine 63,13.Johnson G.L. Nakamura K. The c-jun kinase/stress-activated pathway: regulation, function and role in human disease.Biochim Biophys Acta. 2007; 1773: 1341-1348Crossref PubMed Scopus (314) Google Scholar we looked at the expression of c-Jun to determine whether the ability of activated JNK to phosphorylate c-Jun is altered. We showed a significant downregulation of phosphorylated c-Jun, P<0.05 (Figures 10e, f) suggesting that OPN impacts the downstream activity of JNK. These results are in keeping with previous investigation in osteoblastic cells, in which activation of ERK/MAPK and c-Jun occurred during cellular adhesion to OPN-coated plates.14.Lee Y.J. Park S.J. Lee W.K. et al.MG63 osteoblastic cell adhesion to the hydrophobic surface precoated with recombinant osteopontin fragments.Biomaterials. 2003; 24: 1059-1066Crossref PubMed Scopus (43) Google Scholar In this investigation, two PPARγ ligands, rosiglitazone and pioglitazone, attenuated albuminuria in the db/db C57BLKS/Jlepr mouse which, to date, is one of the better known models of human type 2 DN.12.Breyer M.D. Bottinger E. Brosius III, F.C. et al.Mouse models of diabetic nephropathy.J Am Soc Nephrol. 2005; 16: 27-45Crossref PubMed Scopus (412) Google Scholar,15.Breyer M.D. Bottinger E. Brosius F.C. et al.Diabetic nephropathy: of mice and men.Adv Chronic Kidney Dis. 2005; 12: 128-145Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar Although insulin administration to db/db mice lowered plasma glucose to the levels comparable to TZDs, it did not attenuate albuminuria to the same extent as PPARγ activation. In previous studies, TZDs decreased albuminuria in humans and db/db mice better than other oral antihyperglycemic agents16.Bakris G.L. Ruilope L.M. McMorn S.O. et al.Rosiglitazone reduces microalbuminuria and blood pressure independently of glycemia in type 2 diabetes patients with microalbuminuria.J Hypertens. 2006; 24: 2047-2055Crossref PubMed Scopus (90) Google Scholar, 17.Hsueh W.A. Nicholas S.B. Peroxisome proliferator-activated receptor-gamma in the renal mesangium.Curr Opin Nephrol Hypertens. 2002; 11: 191-195Crossref PubMed Scopus (19) Google Scholar, 18.Sarafidis P.A. Lasaridis A.N. Nilsson P.M. et al.The effect of rosiglitazone on urine albumin excretion in patients with type 2 diabetes mellitus and hypertension.Am J Hypertens. 2005; 18: 227-234Crossref PubMed Scopus (46) Google Scholar and are effective in STZ-treated mice without changes in plasma glucose.3.Nicholas S.B. Kawano Y. Wakino S. et al.Expression and function of peroxisome proliferator-activated receptor-gamma in mesangial cells.Hypertension. 2001; 37: 722-727Crossref PubMed Google Scholar Our and other observations that PPARγ is expressed in mesangial cells help to explain these findings, although the mechanisms for the TZD effect has been obscure.3.Nicholas S.B. Kawano Y. Wakino S. et al.Expression and function of peroxisome proliferator-activated receptor-gamma in mesangial cells.Hypertension. 2001; 37: 722-727Crossref PubMed Google Scholar In vitro, PPARγ activation impaired mesangial cell growth; TGF-β, plasminogen-activator inhibitor-1 and vascular endothelial growth factor production; and TGF-β activity.3.Nicholas S.B. Kawano Y. Wakino S. et al.Expression and function of peroxisome proliferator-activated receptor-gamma in mesangial cells.Hypertension. 2001; 37: 722-727Crossref PubMed Google Scholar, 19.Onozaki A. Midorikawa S. Sanada H. et al.Rapid change of glucose concentration promotes mesangial cell proliferation via VEGF: inhibitory effects of thiazolidinedione.Biochem Biophys Res Commun. 2004; 317: 24-29Crossref PubMed Scopus (37) Google Scholar, 20.Weigert C. Brodbeck K. Bierhaus A. et al.c-Fos-driven transcriptional activation of transforming growth factor beta-1: inhibition of high glucose-induced promoter activity by thiazolidinediones.Biochem Biophys Res Commun. 2003; 304: 301-307Crossref PubMed Scopus (27) Google Scholar We found that TZD or insulin administration both decreased glomerular OPN expression in db/db mice across all treatment groups, but OPN was reduced to a greater extent by the TZDs, paralleling their effects on albuminuria. The decrease in glomerular OPN in vivo can be attributed, at least in part, to glucose lowering, as OPN expression is increased by hyperglycemia.21.Mori S. Takemoto M. Yokote K. et al.Hyperglycemia-induced alteration of vascular smooth muscle phenotype.J Diabetes Complications. 2002; 16: 65-68Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar,22.Sodhi C.P. Phadke S.A. Batlle D. et al.Hypoxia and high glucose cause exaggerated mesangial cell growth and collagen synthesis: role of osteopontin.Am J Physiol Renal Physiol. 2001; 280: F667-F674PubMed Google Scholar However, this study suggests that TZDs suppress OPN expression beyond glucose lowering. In cultured mesangial cells, TZDs suppressed AngII-induced OPN expression, whereas insulin had no effect. Thus, TZDs have direct actions to inhibit mesangial OPN production, whereas the insulin effect in vivo is likely mediated through glucose lowering. To define the role of OPN in albuminuria, we induced STZ diabetes in OPN-null mice, which were on a mixed background. As STZ has been suggested to cause renal damage beyond its diabetogenic effect, we studied OPN effects in the Ins2akita/+ type 1 diabetic mouse, which has a severe insulin deficiency due to a β-cell insulin processing defect with subsequent renal damage (http://www.amdcc.org). STZ-diabetic OPN−/− mice developed albuminuria that was only 20–50% of diabetic OPN+/+, which was associated with decreased glomerular collagen, fibronectin, and TGF-β, suggesting decreased renal profibrotic changes. Similarly, loss of OPN in the Ins2akita/+ resulted in ACRs that were 30–50% of diabetic controls and attenuated the diabetes-induced increase in glomerular TGF-β and extracellular matrix proteins. Deletion of OPN in the genetic Leprdb/db type 2 diabetic mouse also caused a 30% reduction in albuminuria and was similarly associated with decreased glomerular collagen, fibronectin, and TGF-β expression. Thus, in three strains of diabetic mice, loss of OPN resulted in substantial reduction of albuminuria and mesangial fibrosis, demonstrating a critical role of OPN in DN. It is important to note here that as mixed backgrounds were used in these studies, genetic differences in the mice cannot be fully excluded as explanations for some of the effects observed. However, the in vitro data appear to support the in vivo observations. Addition of rmOPN to mesangial cells resulted in a dose- and time-dependent stimulation of TGF-β, whereas OPN antibodies inhibited the hyperglycemia-induced rise in OPN. Taken together, these results suggest that OPN is a key profibrotic factor contributing to the development of glomerulosclerosis and albuminuria in diabetes. OPN was first isolated from bone23.Hunter G.K. Hauschka P.V. Poole A.R. et al.Nucleation and inhibition of hydroxyapatite formation by mineralized tissue proteins.Biochem J. 1996; 317: 59-64Crossref PubMed Scopus (507) Google Scholar, 24.Hunter G.K. Kyle C.L. Goldberg H.A. Modulation of crystal formation by bone phosphoproteins: structural specificity of the osteopontin-mediated inhibition of hydroxyapatite formation.Biochem J. 1994; 300: 723-728Crossref PubMed Scopus (356) Google Scholar, 25.Prince C.W. Oosawa T. Butler W.T. et al.Isolation, characterization, and biosynthesis of a phosphorylated glycoprotein from rat bone.J Biol Chem. 1987; 262: 2900-2907Abstract Full Text PDF PubMed Google Scholar and later identified in other tissues and cells, including the vasculature, heart, as well as renal tubules and glomerular epithelial cells, and several body fluids suggesting it is secreted by cells.26.Higashibata Y. Sakuma T. Kawahata H. et al.Identification of promoter regions involved in cell- and developmental stage-specific osteopontin expression in bone, kidney, placenta, and mammary gland: an analysis of transgenic mice.J Bone Miner Res. 2004; 19: 78-88Crossref PubMed Scopus (23) Google Scholar,27.Weber G.F. The metastasis gene osteopontin: a candidate target for cancer therapy.Biochim Biophys Acta. 2001; 1552: 61-85Crossref PubMed Scopus (116) Google Scholar Its diverse functions regulate tissue calcification and promote inflammation, tissue remodeling, fibrosis, and angiogenesis.28.Canfield A.E. Farrington C. Dziobon M.D. et al.The involvement of matrix glycoproteins in vascular calcification and fibrosis: an immunohistochemical study.J Pathol. 2002; 196: 228-234Crossref PubMed Scopus (101) Google Scholar Many of these effects are mediated by the binding of OPN to cell surface integrin receptors, such as ανβ1, ανβ3, ανβ5, which activate intracellular signaling pathways.29.Yokosaki Y. Tanaka K. Higashikawa F. et al.Distinct structural requirements for binding of the integrins alphavbeta6, alphavbeta3, alphavbeta5, alpha5beta1 and alpha9beta1 to osteopontin.Matrix Biol. 2005; 24: 418-427Crossref PubMed Scopus (111) Google Scholar In the kidney, OPN appears to be protective against accumulation of calcium oxalate stones.30.Beshensky A.M. Wesson J.A. Worcester E.M. et al.Effects of urinary macromolecules on hydroxyapatit
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