Fig 1: PGRN blockade by anti-PGRN antibody showed therapeutic effect on invasive C.albicans infection.WT mice were intravenously injected with 1×106 CFU of C. albicans, and PGRN blockade was performed by intraperitoneally administration of 5 μg of anti-PGRN monoclonal antibody on day 0 (same day as C. albicans infection), followed by a booster dose of 2.5 μg 24 hour later. (A) Survival of these mice (n = 10 for each group) was monitored. (B) Mouse Clinical Assessment Score for Sepsis (M-CASS) scores of WT mice (n = 5) treated with or without anti-PGRN antibody after intravenous infection with 1 × 106 CFU of C.albicans. (C) Kidney fungal loading was performed in WT mice (n = 5) treated with or without anti-PGRN antibody at the indicated times after intravenous infection with 1 × 106 CFU of C.albicans. (D) Kidney IL-6, CXCL1 and CCL2 levels were quantified by ELISA in WT mice (n = 5) treated with or without anti-PGRN antibody at the indicated times after intravenous infection with 1 × 106 CFU of C.albicans. (E) The numbers of macrophages (CD11b+F4/80+) and neutrophils (CD11b+Ly-6G+) in the kidneys were quantified by flow cytometry analysis in WT mice (n = 5) treated with or without anti-PGRN antibody at the indicated times after intravenous infection with 1 × 106 CFU of C.albicans. (F) The gene expression levels of kidney injury marker-1 (KIM-1) were determined by quantitative PCR in WT mice (n = 5) treated with or without anti-PGRN antibody at the indicated times after intravenous infection with 1 × 106 CFU of C.albicans. (G) Serum urea levels were measured in WT mice (n = 5) treated with or without anti-PGRN antibody at the indicated times after intravenous infection with 1 × 106 CFU of C.albicans. (H) Serum creatinine levels were measured in WT mice (n = 5) treated with or without anti-PGRN antibody at the indicated times after intravenous infection with 1 × 106 CFU of C.albicans. All data were pooled from three independent experiments. Log-rank test was used to analyze the difference between survival curves. The Mann–Whitney U test was used to analyze the difference of other parameters between anti-PGRN-treated and control IgG-treated mice at the same time point after C.albicans infection, and p values were shown when compared between groups denoted by horizontal lines.
Fig 2: GSK3ß and ERK phosphorylation in cortical neurons due to high glucose and PGRN treatment. Cells were treated with high glucose and PGRN for 24 or 72 h before harvest and western blot analysis. (A,B) GSK3ß phosphorylation at serine nine was unaltered by high glucose or PGRN at 24 h (A) but showed significant change at 72 h (B). At 72 h, high glucose increased phosphorylation from 1.000 ± 0.078 AU to 2.044 ± 0.445 AU, while PGRN treatment lowered phosphorylation in both control (1.000 ± 0.078 AU to 0.550 ± 0.112 AU) and high-glucose conditions (2.044 ± 0.445 AU to 0.628 ± 0.178 AU). N = 5-6 samples. (C) ERK1/2 phosphorylation at threonine 202/tyrosine 204 increased with simultaneous PGRN and high-glucose conditions from 1.000 ± 0.145 AU to 2.403 ± 0.381 AU at 24 h of treatment. Upon closer analysis, ERK2, but not ERK1, phosphorylation was significantly affected, with ERK2 phosphorylation increasing from 1.000 ± 0.197 AU to 3.401 ± 0.544 AU. N = 3 samples. (D) ERK1/2 phosphorylation at threonine 202/tyrosine 204 was not significantly altered by high glucose or PGRN after 72 h. N = 6 samples. **p < 0.01; ***p < 0.001.
Fig 3: PGRN deletion reduced immune cell apoptosis and tissue injury in the kidneys after invasive C.albicans infection.PGRN KO (n = 5) and WT (n = 5) mice were infected intravenously with 4 × 105 colony forming units (CFU) of C. albicans, and then immune cell apoptosis in the kidney and tissue injury markers were assessed at the indicated times. (A) The kidneys from WT (n = 5) and PGRN KO (n = 5) mice at the indicated times after intravenous infection with 4 × 105 CFU of C. albicans were subjected to DNA fragmentation analysis (terminal deoxynucleotidyltransferase dUTP nick end labeling [TUNEL]). Representative examples were shown. (B) TUNEL-positive cells were counted (n = 5 per group) at the indicated times after intravenous infection with 4 × 105 CFU of C. albicans. (C) Kidney total RNA was analysed for gene expression of kidney injury marker-1 (KIM-1) at the indicated times after intravenous infection with 4 × 105 CFU of C. albicans. (D) Serum urea levels in WT (n = 5) and PGRN KO (n = 5) mice after intravenous infection with 4 × 105 CFU of C. albicans. (E) Serum creatinine levels in WT (n = 5) and PGRN KO (n = 5) mice after intravenous infection with 4 × 105 CFU of C. albicans. All data were pooled from three independent experiments. The Mann–Whitney U test was used to analyze the difference between WT and PGRN KO mice at the same time point after C.albicans infection, and p values were shown when compared between groups denoted by horizontal lines.
Fig 4: Local and systemic PGRN production in mice after invasive C.albicans infection.Wild type (WT, n = 5) mice were infected intravenously with 4 × 105 colony forming units (CFU) of C. albicans. (A) Organs were removed at the indicated time points, and blood was collected by cardiac puncture. Samples were assayed for PGRN content by specific sandwich enzyme-linked immunosorbent assay (ELISA). (B) PGRN concentrations in the kidney and blood from Toll-like receptor (TLR) 2 knockout (KO), TLR4 KO, TLR7 KO, type I IFN-α/β receptor (IFNAR) KO, and WT mice at the indicated times after intravenous infection with 4 x105 CFU of C. albicans. All data were pooled from three independent experiments. The statistical differences were determined by Kruskal-Wallis test followed by Dunn’s multiple comparisons post test, and p values were shown when compared between groups denoted by horizontal lines.
Fig 5: LAMP2A protein levels and punctate localization in cortical neurons and astrocytes due to high glucose and PGRN treatment. (A) Total protein LAMP2A levels are unchanged among treatment groups in primary cortical neurons treated for 24 h. N = 6 samples. (B) Perinuclear LAMP2A punctate formation increased in neurons under HG (from 1.000 ± 0.137 AU to 1.463 ± 0.108 AU), an effect that was attenuated with PGRN (from 1.031 ± 0.083 AU to 1.246 ± 0.096 AU). N = 12-27 cells. (C) Representative immunofluorescence images of primary neurons after 72 h of treatment, with blue as DAPI, green as MAP2, and red as LAMP2A. Scale bar, 10 µm. (D) Perinuclear LAMP2A punctate formation appeared to show a trend toward an increase due to PGRN but was not statistically significant (F = 1.825, p = 0.151). N = 16-22 cells. (E) Representative immunofluorescence images of primary astrocytes after 72 h of treatment, with blue as DAPI, green as GFAP, and red as LAMP2A. Scale bar, 10 µm. **p < 0.01.
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Available conjugates: Sizes Available: 50 ug