Research ArticleMETABOLIC DISORDERS

Role of Toll-like receptors in diabetic renal lesions in a miniature pig model

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Science Advances  19 Jun 2015:
Vol. 1, no. 5, e1400183
DOI: 10.1126/sciadv.1400183

Figures

  • Fig. 1 Kidney PAS staining and glomerular area score results of diabetic miniature pigs.

    (A) PAS staining results of the kidneys of diabetic miniature pigs. (B) Score results of glomerular area. CON, normal control group; DM, diabetes group. Magnification, ×400. Glomerular areas are presented as means ± SD. *P < 0.05 versus CON.

  • Fig. 2 Expression of TLR endogenous ligand was analyzed by Western blot in the kidney tissues from diabetic miniature pigs.

    (A) Detection of TLR endogenous ligand expression by Western blot. (B) Semiquantitative analysis of expression levels of the TLR endogenous ligand. Protein expression data are presented as means ± SD. *P < 0.05 versus CON.

  • Fig. 3 Western blot analysis of the expression levels of MyD88-dependent TLRs in diabetic kidneys.

    (A) Detection of the MyD88-dependent TLR expression levels by Western blot. (B) Semiquantitative analysis of the MyD88-dependent TLR expression levels. Protein expression data are presented as means ± SD. *P < 0.05 versus CON.

  • Fig. 4 Immunohistochemical detection of the TLR2 and TLR4 expression levels in diabetic kidneys.
  • Fig. 5 Western blot analysis of the expression and activation of MyD88-dependent downstream signaling molecules in diabetic kidneys.

    (A) Detection of the expression and activation of MyD88-dependent downstream signaling molecules by Western blot. (B) Semiquantitative analysis of the expression and activation levels of MyD88-dependent downstream signaling molecules. Protein expression data are presented as means ± SD. *P < 0.05 versus CON.

  • Fig. 6 Western blot analysis of the expression of MyD88-independent TLRs and activation of downstream signaling pathway molecules in diabetic kidneys.

    (A) Detection of the expression of MyD88-independent TLRs and the activation of downstream signaling pathway molecules by Western blot. (B) Semiquantitative analysis of the expression of MyD88-independent TLRs and the activation of downstream signaling pathway molecules. Protein expression data are presented as means ± SD. *P < 0.05 versus CON.

  • Fig. 7 Western blot analysis of the expression and activation of NF-κB signaling pathway molecules in diabetic kidneys.

    (A) Detection of the expression and activation of NF-κB signaling pathway molecules by Western blot. (B) Semiquantitative analysis of the expression and activation of NF-κB signaling pathway molecules. Protein expression data are presented as means ± SD. *P < 0.05 versus CON.

  • Fig. 8 qRT-PCR detection of the mRNA expression levels of proinflammatory factors in the kidneys from diabetic miniature pigs.

    *P < 0.05 versus CON.

  • Fig. 9 Immunofluorescent (magnification, ×200) and immunohistochemical (magnification, ×400) detection of CD68 expression in diabetic kidneys.

    IHC, immunohistochemistry; IF, immunofluorescence.

Tables

  • Table 1 Blood biochemical results in the miniature swine diabetes model.

    Values are means ± SD. CON, normal control group; DM, diabetes group.

    ParamatersCONDM
    GLU (mM)5.29 ± 0.919.95 ± 0.96*
    INS (μIU/ml)4.97 ± 1.1823.63 ± 13.2*
    TG (mM)0.29 ± 0.040.62 ± 0.08*
    CHOL (mM)1.33 ± 0.142.22 ± 0.6*
    HDL-C (mM)0.39 ± 0.060.49 ± 0.11
    LDL-C (mM)0.64 ± 0.071.12 ± 0.37*
    Creatinine (μM)204.5 ± 15.93119.42 ± 18.82
    BUN (mM)8.21 ± 1.613.72 ± 1.78

    *P < 0.005 versus CON.

    • Table 2 Detailed information on the primary antibodies used.

      NA, unavailable amino acid sequence for pig proteins; Ab, antibody.

      AntibodyCompanyCatalog no.Pig homology with human (amino acid sequence)
      HMGB1Novus BiologicalsNB100-232299%; this Ab has been verified to react with pig HMGB1
      HSP70Santa Cruz Biotechnologysc-66048NA; this Ab has been verified to react with pig HSP70
      β-ActinCell Signaling Technology#4967100%; this Ab has been verified to react with pig β-actin
      TLR1AbnovaPAB021589%
      TLR2Abcamab10899887%
      TLR3Abcamab5342491%; this Ab has been predicted to react with pig TLR3
      TLR4Abcamab2204884%
      TLR5Abcamab6246086%
      TLR6Santa Cruz Biotechnologysc-3000188%; this Ab has been predicted to react with pig TLR6
      TLR7Abcamab11352492%; this Ab has been predicted to react with pig TLR7
      TLR8Santa Cruz Biotechnologysc-2546783%
      TLR9Abcamab1212186%
      TLR10Santa Cruz Biotechnologysc-3019889%
      TLR11AbnovaPAB11333NA
      MyD88Abcamab3607689%; this Ab has been predicted to react with pig MyD88
      Phospho–IRAK-1Santa Cruz Biotechnologysc-13019783%
      Phospho–IRF-3Cell Signaling Technology#494783%; this Ab has been verified to react with pig IRF-3
      Phospho-IKKβAbcamab59195NA
      Phospho-IκBαCell Signaling Technology#924695%; this Ab has been verified to react with pig IκBα
      NF-κBp65Abcamab31481NA; this Ab has been verified to react with pig NF-κBp65
      Phospho–NF-κBp65Cell Signaling Technology#3033NA; this Ab has been verified to react with pig NF-κBp65
      CD68Santa Cruz Biotechnologysc-9139NA