A novel role of peroxiredoxin 2 in diabetic kidney disease progression by activating the classically activated macrophages

Figures

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  Fig. 1. A mice DKD model was successfully constructed. (a) Model construction flowchart. A DM mice model was constructed through an intraperitoneal injection of STZ (55 mg/kg), once per day for 5 consecutive days. Mice in the control group were given the same volume of STZ solvent. After detection for 2 consecutive days, mice with a random blood glucose level ≥ 16.7 mmol/L and positive urine glucose were considered as DM. Then, mice with DM were fed normally and executed under anaesthesia at 20, 28, and 36 w, respectively. Serum and kidney tissues were collected for biochemical and pathological analyses. (b) Serological analyses. Including random blood glucose (BG), blood urea nitrogen (BUN), and blood creatinine (CRE) in mice; n = 4–6, #, a, b, c, DM vs. NC, p < 0.05; ns, not significant. (c) Pathological analysis. Bars: 50 μm; black arrows, damaged renal tubular brush border microvillus; red arrows, thickening of the glomerular basement membrane. A quantitative analysis of positive areas of Masson staining was performed using image J software. n = 3, * p < 0.05, **** p < 0.0001.

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  Fig. 2. PRDX2 showed a positive correlation with M1 polarization during DKD progression. (a) Immunohistochemical analysis. At 20, 28 and 36 w after DM model construction, the infiltration of macrophages (F4/80) and the distribution of inflammatory-related factors (IL-1β, IL-6 and TNF-α) in the kidney tissues were detected. Positive cell counts and positive area analyses were performed using image J software. Data were represented by x ± SD. n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (b) The expression level of PRDX2, M1 polarization, inflammatory and fibrosis-related factors were detected using WB at 20, 28, and 36 w after DM model construction. Macrophage markers, F4/80; molecules related to M1 polarization signaling pathway, TLR4, NF-κB p65, NF-κB p-p65, STAT1; marker of M1 polarization, iNOS. Marker of M2 polarization, Arg-1; inflammatory factors, IL-6, TNF-α, IL-1β. Grayscale value was quantified using image J software. β-actin was used as an internal reference. Data was represented as x ± SD, n = 3. Pearson correlation analysis was performed for the correlation analysis of each line. n = 3; red values, p < 0.05.

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  Fig. 3. HG and TNF-α can stimulate renal tubular epithelial cells to upregulate PRDX2 under the DM condition. (a) IFA analysis of DKD renal tissues. Purplish red, α- SMA; red, AQP1; green, PRDX2; blue, DAPI. Observation and collection of images were performed using a laser confocal microscope with the same laser power. Bars: 50 μm; white arrows, co-localization of PRDX2 and α-SMA. (b) Detection of the protein expression level of PRDX2 in NRK52E cells. Cells were treated using DMEM with HG (30 mM) or TNF-α (50 ng/mL), and DMEM with NG (5.5 mM) was used as a negative control (NC). After treatment for 6, 12, 24, and 36 h, cells were collected and the lysate was subjected to WB analysis. Use image J software for grayscale value detection. β-actin was used as an internal reference for relative expression level analysis, and the data was represented by x ± SD. n = 3, ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.0001. (c) Detection of PRDX2 localization in NRK52E cells. Cells (on coverslips) were treated using DMEM with HG (30 mM) or TNF-α (50 ng/mL), and DMEM with NG (5.5 mM) was used as a negative control. After 24 h of treatment, coverslips were detected using IFA. Observation and…

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  Fig. 4. M1 polarization stimulated by rPRDX2. (a) Detection of M1-polarization-related protein expression level using WB. J774A.1 cells were treated using rPRDX2 (5 µg/mL), with an equal volume of PBS as a negative control. After 12 and 24 h of treatment, cells were collected and the lysate was subjected to WB analysis. Molecules related to the M1 polarization signaling pathway, TLR4, NF-κB p65, NF- κB p-p65, STAT1, iNOS. Marker of M2 polarization, Arg-1. Inflammatory factors, IL-6, TNF-α, IL-1β. Grayscale value was quantified using image J software. β-actin was used as an internal reference. Data was represented by x ± SD, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (b) Localization of PRDX2 and TLR4 in J774A.1 cells was detected using IFA. Cells (on coverslips) were pre-treated w/ or w/o the TLR4 specific inhibitor TAK242 (1 µM) for 1 h, which were then were treated w/ or w/o rPRDX2 (5 µg/mL), with an equal volume of PBS as a negative control (NC). After 24 h, coverslips were detected using IFA and immediately observed using a laser confocal microscope with same power. Red, TLR4; green, PRDX2; blue, DAPI. Bars: 5 μm. White arrows, co-localization.

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  Fig. 5. M1 polarization of macrophages stimulated by rPRDX2 through TLR4. (a) M1 polarization in J774A. 1 cells stimulated by rPRDX2 through TLR4. Cells were pre-treated w/ or w/o the TLR4-specific inhibitor TAK242 (1 µM) for 1 h. Afterwards, cells were treated using rPRDX2 (5 µg/mL), with an equal volume of PBS as a negative control. After 24 h, cells were collected and the lysate was subjected to WB analysis. Molecules related to M1 polarization signaling pathway, NF-κB p65, NF- κB p-p65, STAT1, iNOS. Marker of M2 polarization, Arg-1. Inflammatory factors, IL-6, TNF-α, IL-1β. Grayscale value was quantified using image J software. β-actin was used as an internal reference. Data was represented by x ± SD, n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (b) The localization of M1-polarization-related key molecules was detected using IFA. J774A. 1 cells (on coverslips) were pre-treated w/ or w/o TAK242 (1 µM) for 1 h, which were then treated w/ or w/o rPRDX2 (5 µg/mL), with an equal volume of PBS as a negative control (NC). After 24 h, coverslips were detected using IFA and immediately observed using a laser confocal microscope with the same laser power. For macrophage…

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  Fig. 6. Macrophage migration and phagocytosis could be enhanced by rPRDX2 through TLR4. (a) A scratch experiment was used to detect the cell migration ability. Cells were pre-treated w/ or w/o the TLR4-specific inhibitor TAK242 (1 µM) for 1 h. Afterwards, cells were treated using rPRDX2 (5 µg/mL), with an equal volume of PBS as negative control. Cells were observed and recorded at the same position of each well at 0 and 24 h. The distance between the red dashed lines represented the width of the scratch. Bars, 20 μm; Data was represented by x ± SD, n = 3, ns, not significant, **** p < 0.0001. (b) The fluorescence microsphere experiment was used to detect macrophage phagocytosis. Cells (on coverslips) were pre-treated w/ or w/o the TLR4-specific inhibitor TAK242 (1 µM) for 1 h. Afterwards, the cells were treated using rPRDX2 (5 µg/mL), with an equal volume of PBS as a negative control. Meanwhile, green fluorescent latex microspheres (1:500) were added into wells. After 24 h of treatment, coverslips were fixed with 4% PFA and stained with DAPI, then immediately observed using an upright fluorescence microscope. Green, green latex microspheres; blue, DAPI; bars: 20 μm.