Fig 1.
Distribution of allograft function in each study group.
(A) Distribution of subjects according to allograft function and post-transplant years in LTS group and CAD group. Closed triangle mean the average value of MDRD eGFR at each post-transplant year in 587 patients who took kidney transplantation between 1995 and 2010 and current laboratory data is available in our center. (B) Comparison of allograft function assessed by MDRD eGFR in each study and control group. Note that allograft function was significantly superior in LTS group compared to CAD group. * P<0.05 for each comparison. LTS, long term stable; CAD, chronic allograft dysfunction; ES, early stable; ESRD, end stage renal disease; HC, healthy control; MDRD eGFR; Modification of Diet in Renal Disease estimated glomerular filtration rate.
Table 1.
Baseline characteristics of the patient populations.
Fig 2.
Distribution of lymphocyte and CD4+ T cell in each patient group.
PBMC from each patient group were stimulated for 4h ex vivo with PMA and ionomycin in the presence of Golgi Stop. The percentage of target cells was measured by flowcytometry. (A) The representative figure of flowcytometric analysis for lymphocyte and CD4+ T cell. (B) The proportion (%) of Lymphocyte/Total cells and (C) CD4+ T/Lymphocyte cells in each group. * P<0.05 for each comparison. LTS, long term stable; CAD, chronic allograft dysfunction; ES, early stable; ESRD, end stage renal disease; HC, healthy control.
Fig 3.
Distribution of Th1, Th2, Th17 and Treg subpopulations out of CD4+ T lymphocytes.
(A) PBMCs were stained with anti-CD4 PE-cy7, anti-CD25 APC, anti-IFN-γ FITC, anti-IL-17 PE, anti-IL-4 APC and anti-Foxp3 FITC. CD4+ T cells were gated for further analysis. (B) The proportion (%) of IFN-γ+/CD4+ T cells (C) IL-4+/CD4+ T cells (D) IL-17+/CD4+ T cells (E) CD25+FOXP3+/CD4+T cells in each patient group. * P<0.05 for each comparison. LTS, long term stable; CAD, chronic allograft dysfunction; ES, early stable; ESRD, end stage renal disease; HC, healthy control.
Fig 4.
Distribution of Tnaïve, TCM, TEM subpopulations of CD4+T lymphocytes and IL-17+/TEM subpopulations of CD4+ T lymphocytes.
(A) PBMCs were stained with anti-CD4 PE-cy7, anti-CD45RA–FITC, anti-CCR7 APC and anti-IL-17 PE. CD4+ T cells were gated for further analysis. (B) The proportion (%) of Tnaïve/CD4+ T (CD45RA+CCR7+/CD4+ Tcells) (C) TCM/CD4+ T (CD45RA–CCR7+/CD4+Tcells) (D) TEM/CD4+ T (CD45RA–CCR7–/CD4+ Tcells) (E) After surface staining with CD45 and CCR7 mAbs, analysis of IL-17 in CD4+ T cell subsets by intracellular flow cytometry was done. (F) The proportion (%) of IL-17+/TEM. in each patient group. * P<0.05 for each comparison. LTS, long term stable; CAD, chronic allograft dysfunction; ES, early stable; ESRD, end stage renal disease; HC, healthy control.
Fig 5.
Distribution of chemokine receptor CCR4+CCR6–, CCR4–CCR6+ and CCR4+CCR6+ subpopulations of CD4+ T lymphocytes.
(A) PBMCs were stained with anti-CD4 PE-cy7, anti-CCR4 PE, anti-CCR6 APC and anti-IL-17 FITC. CD4+ T cells were gated for further analysis. (B) The proportion (%) of CCR4+CCR6–/CD4+ T cells (C) CCR4–CCR6+/CD4+ T cells (D) CCR4+CCR6+/CD4+ T cells in each patient group. (E) After surface staining with anti-CD4, CCR4 and CCR6 mAbs, analysis of IL-17 in CD4+ T cell subsets by intracellular flow cytometry was done. (F) The proportion (%) of IL-17+/CCR4+CCR6+CD4+ T cells in each patient group. * P<0.05 for each comparison. LTS, long term stable; CAD, chronic allograft dysfunction; ES, early stable; ESRD, end stage renal disease; HC, healthy control.
Fig 6.
Expression of Th17 pathway molecules and serum Th17 associated cytokine level in LTS and CAD group.
The expression of (A) IL-1beta (B) RAGE (C) HMGB1 mRNA was measured using real-time PCR. In addition, concentrations of (D) IL-17 (E) IL-33 and (F) RAGE were determined using ELISA in the serum of LTS and CAD patient group. Bars show the means. * P<0.05 vs. LTS. CAD, chronic allograft dysfunction; LTS, long term stable.
Fig 7.
Expression of acute and chronic injury markers in renal tubular cells treated with recombinant human IL-17.
Renal tubular epithelial cell were cultured with rhIL-17 (0, 10, 50, or 100 ng/ml) for 72 hours, and the production of (A) IL-6 and (B) IL-8 was measured using ELISA. Renal tubular epithelial cell were cultured with rhIL-17 (0, 10, 50, or 100 ng/ml) for 72 hours, and the expression of (C) ACTA-2 and (D) CTGF mRNA, relative to β-actin, was measured using real-time polymerase chain reaction. Bars show the means. *P<0.05 vs. Nil, †P<0.05 vs. IL-17 10.