1. Volume 83, Issue 5, Pages 887-900 (May 2013)
The TLR4 antagonist CRX-526 protects against advanced diabetic nephropathy 
Miao Lin, Wai Han Yiu, Rui Xi Li, Hao Jia Wu, Dickson W.L. Wong, Loretta Y.Y. Chan, Joseph C.K. Leung, Kar Neng Lai, Sydney C.W. Tang 
Kidney International 
Volume 83, Issue 5, Pages (May 2013)
DOI: /ki
Copyright © 2013 International Society of Nephrology Terms and Conditions

2. Figure 1
Albuminuria and blood glucose levels in wild-type (WT) and endothelial nitric oxide synthase knockout (eNOSKO) mice. Urinary albumin/creatinine ratios (ACRs) and fasting blood glucose were determined at 4 weekly intervals. CRX-526 treatment significantly prevented the progressive increase of urine albumin-to-creatinine ratio in both (a) diabetic (DM) WT mice and (b) diabetic eNOSKO mice. Fasting glucose levels were significantly increased 2 weeks after streptozotocin (STZ) injection and maintained at similar levels over the 12 weeks in both (c) diabetic WT mice and (d) diabetic eNOSKO mice. CRX-526 treatment did not affect the blood glucose levels. *P<0.05 versus the corresponding diabetic group treated with vehicle.
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3. Figure 2
Morphological changes in wild-type (WT) mice. Representative periodic acid-Schiff staining of the renal cortical sections in nondiabetic (Non-DM) mice treated with (a) vehicle or (b) CRX-526, and in diabetic (DM) mice treated with (c) vehicle or (d) CRX-526. Original magnification × 400. Tubulointerstitial lesions in nondiabetic mice treated with (e) vehicle or (f) CRX-526, and in diabetic mice treated with (g) vehicle or (h) CRX-526. Original magnification × 100. For quantification, 50 glomeruli were randomly selected for assessing the (i) glomerular tuft area and (j) glomerulosclerotic index. (k, l) A total of 400 renal cortical tubules were assessed and graded in an observer-blinded manner for tubular and interstitial injury. **P<0.01 versus the corresponding group of nondiabetic mice; †P<0.05 versus diabetic mice treated with vehicle.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

4. Figure 3
Morphological changes in endothelial nitric oxide synthase knockout (eNOSKO) mice. Representative periodic acid-Schiff staining of the renal cortical sections in nondiabetic (Non-DM) mice treated with (a) vehicle or (b) CRX-526, and in diabetic (DM) mice treated with (c) vehicle or (d) CRX-526. Original magnification × 400. Tubulointerstitial lesions in nondiabetic mice treated with (e) vehicle or (f) CRX-526, and in diabetic mice treated with (g) vehicle or (h) CRX-526. Original magnification × 100. For quantification, 50 glomeruli were randomly selected for assessing the (i) glomerular tuft area and (j) glomerulosclerotic index, and (k, l) 400 renal cortical tubules were assessed in an observer-blinded manner for tubular and interstitial injury. **P<0.01 versus the corresponding group of nondiabetic mice; †P<0.05 and ††P<0.01 vs. diabetic mice treated with vehicle.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

5. Figure 4
Renal cortical high-mobility group box 1 (HMGB1) and Toll-like receptor 4 (TLR4) expression in wild-type (WT) mice and endothelial nitric oxide synthase knockout (eNOSKO) mice. (a) Representative renal cortical immunostaining for HMGB1. Renal cortical HMGB1 expression in nondiabetic (Non-DM) WT mice (a, e) or diabetic (DM) WT mice (b, f), and in nondiabetic eNOS mice (c, g) or diabetic eNOS mice (d, h), determined by real-time PCR. (i) Negative control by omission of the corresponding primary antibodies demonstrated no nonspecific staining. (j) Quantitative analysis of cortical HMGB1 staining. (k) Renal cortical TLR4 mRNA expression determined by real-time PCR. Original magnification: × 400 (a–d); × 1000 (e–h) of the marked area in the corresponding upper panels. *P<0.05, **P<0.01 versus the corresponding nondiabetic animals. CCL-2, chemokine (C-C motif) ligand 2; IOD, integrated optical density; OPN, osteopontin.
Kidney International  , DOI: ( /ki )
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6. Figure 5
Renal cortical CCL-2 and OPN expression in wild-type (WT) mice. (a) Renal cortical CCL-2 mRNA expression, determined by real-time PCR. (b) Quantitative analysis of cortical CCL-2 staining. (c) Renal cortical OPN mRNA expression, determined by real-time PCR. (d) Quantitative analysis of cortical OPN staining. (e) Representative renal cortical immunostaining for CCL-2 and OPN. Original magnification × 400. *P<0.05, **P<0.01 versus the corresponding nondiabetic (Non-DM) animals; †P<0.05 and ††P<0.01 versus diabetic (DM) mice treated with vehicle. CCL-2, chemokine (C-C motif) ligand 2; IOD, integrated optical density; OPN, osteopontin.
Kidney International  , DOI: ( /ki )
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7. Figure 6
Renal cortical CCL-2 and OPN expression in endothelial nitric oxide synthase knockout (eNOSKO) mice. (a) Renal cortical CCL-2 mRNA expression, determined by real-time PCR. (b) Quantitative analysis of cortical CCL-2 staining. (c) Renal cortical OPN mRNA expression, determined by real-time PCR. (d) Quantitative analysis of cortical OPN staining. (e) Representative renal cortical immunostaining for CCL-2 and OPN. Original magnification × 400. *P<0.05, **P<0.01 versus the corresponding nondiabetic (Non-DM) animals; †P<0.05 ††P<0.01 versus diabetic (DM) mice treated with vehicle. CCL-2, chemokine (C-C motif) ligand 2; IOD, integrated optical density; OPN, osteopontin.
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8. Figure 7
Renal cortical CCL-5 expression in wild-type (WT) and endothelial nitric oxide synthase knockout (eNOSKO) mice. Renal cortical CCL-5 mRNA expression, determined by real-time PCR in (a) WT mice and (b) eNOSKO mice. *P<0.05, **P<0.01 versus the corresponding nondiabetic (Non-DM) animals; †P<0.05 versus diabetic (DM) mice treated with vehicle.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

9. Figure 8
Macrophage infiltration into the kidney. (a) Representative renal cortical sections of F4/80 immunostaining. Original magnification × 400. Number of glomerular F4/80+ cells in (b) wild-type (WT) group and (d) endothelial nitric oxide synthase knockout (eNOSKO) group. Number of interstitial F4/80+ cells in (c) WT group and (e) eNOSKO group. **P<0.01 versus the corresponding nondiabetic (Non-DM) animals; ††P<0.01 versus diabetic (DM) mice treated with vehicle.
Kidney International  , DOI: ( /ki )
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10. Figure 9
Renal cortical collagen deposition and transforming growth factor (TGF)-β expression in wild-type (WT) mice. (a) Representative Masson’s trichrome staining in renal cortical sections and immunostaining for collagen IV (COL IV) and TGF-β. Original magnification × 400. (b) Renal cortical COL IV mRNA expression, determined by real-time PCR (left), and renal cortical immunostaining for glomerular COL IV (middle) and tubulointerstitial COL IV (right). (c) Renal cortical TGF-β mRNA expression, determined by real-time PCR (left), and renal cortical immunostaining for TGF-β (right). *P<0.05, **P<0.01 versus the corresponding nondiabetic (Non-DM) animals; †P<0.05, ††P<0.01 versus diabetic (DM) mice treated with vehicle. IOD, integrated optical density.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

11. Figure 10
Renal cortical collagen deposition and transforming growth factor (TGF)-β expression in endothelial nitric oxide synthase knockout (eNOSKO) mice. (a) Representative Masson’s trichrome staining in renal cortical sections and immunostaining for collagen IV (COL IV) and TGF-β. Original magnification, × 400. (b) Renal cortical COL IV mRNA expression, determined by real-time PCR (left), and renal cortical immunostaining for glomerular COL IV (middle) and tubulointerstitial COL IV (right). Original magnification × 400. (c) Renal cortical TGFβ mRNA expression, determined by real-time PCR (left); and renal cortical immunostaining for TGFβ (right). **P<0.01 vs. the corresponding nondiabetic (Non-DM) animals; ††P<0.01 vs. diabetic (DM) mice treated with vehicle. IOD, integrated optical density.
Kidney International  , DOI: ( /ki )
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12. Figure 11
Renal cortical phosphorylated nuclear factor-κB (NF-κB)/p65 nuclear staining in diabetic and nondiabetic mice. (a) Representative renal cortical immunostaining for nuclear phosphorylated NF-κB/p65 in wild-type (WT) mice and endothelial nitric oxide synthase knockout (eNOSKO) mice. Original magnification × 400. Quantitative analysis of phosphorylated NF-κB/p65 nuclear staining in the glomeruli (b, d) and tubulointerstitium (c, e). **P<0.01 versus the corresponding nondiabetic (Non-DM) animals, ††P<0.01 versus diabetic (DM) mice treated with vehicle.
Kidney International  , DOI: ( /ki )
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13. Figure 12
Cortical nuclear factor-κB (NF-κB)/p65 signaling in diabetic and nondiabetic mice. Western blot analysis of NF-κB/p65 phosphorylation in (a) wild-type (WT) mice and (b) endothelial nitric oxide synthase knockout (eNOSKO) mice. Quantitative analysis of phosphorylated NF-κB/p65 in (c) WT mice and (d) eNOSKO mice. Levels of phosphorylation were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Results are expressed as means±s.d. **P<0.01 versus the corresponding nondiabetic (Non-DM) animals, ††P<0.01 versus diabetic (DM) mice treated with vehicle.
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14. Figure 13
Effect of CRX-526 on high glucose-induced OPN expression in cultured PTEC. PTEC were pretreated with CRX-526 (5, 10, or 20μg/ml) or Toll-like receptor 4 (TLR4)-neutralizing antibody (20μg/ml) before the addition of HG (30mmol/l). OPN mRNA expression was determined by (a) real-time PCR after 24h of incubation, and their protein levels in culture supernatants were determined by (b) enzyme-linked immunosorbent assay after 48h of incubation. Results are represented by means±s.d of three to five independent experiments. *,** Denote significant differences at P<0.05 and P<0.01, respectively. HG, high glucose; NG, normal glucose; OPN, osteopontin; PTEC, proximal tubular epithelial cells.
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15. Figure 14
Effect of Toll-like receptor 4 (TLR4) inhibition by CRX-526 on high glucose-mediated nuclear factor-κB (NF-κB) nuclear translocation in PTEC. PTEC were pretreated with CRX-526 (20μg/ml) for 1h, incubated with HG (30mmol/l) for 8h, and stained by immunofluorescence for the p65 subunit of NF-κB (green, upper panels) and for cell nuclei with 4′,6-diamidino-2-phenylindole (blue, lower panels). PTEC-treated normal glucose (NG, 5.5mmol/l) or HG plus anti-TLR4 antibody served as baseline and positive controls, respectively. Results shown are representative of three independent experiments. HG, high glucose; PTEC, proximal tubular epithelial cells.
Kidney International  , DOI: ( /ki )
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