Fig 1.
The viability of HK-2 cells with exogenous PTX3.
Treatments with low (A) and high (B) doses of exogenous PTX3 (P) were compared. Cont, control; T50, 50 ng/mL TGF-β (positive control). Data are represented as means ± s.e.m from each HK-2 group (n = 3). *p < 0.05. 'CON' is abbreviated 'control' which is treated nothing.
Fig 2.
Exogenous PTX3 improves the viability of HK-2 cells after ischemic and hypoxic injuries.
Cell viabilities were measured with and without PTX3 at 1, 5, or 10 nm (P1, P5, and P10, respectively) (A) 24 and 48 h after treatment with 0.3 μg/mL A23187 (A0.3) and (B) after exposure to a hypoxic environment (H). We show that PTX3 provides significant cell viability increasing against HK-2 cell injury after 48hr. Values are expressed as means ± s.e.m of at least four independent experiments, *p < 0.05 (n = 4). 'CON' is abbreviated 'control' which is treated nothing.
Fig 3.
Exogenous PTX3 attenuates calcium responses to ischemic injury.
(A) Intracellular calcium indicated by fluo-4 fluorescence was enhanced in HK-2 cells treated with 0.3 μg/mL A23187 and reduced by the addition of 1 and 5 nM PTX3 (P1 and P5, respectively). (B) Calpain activity as measured by fluorimetry increased in cells treated with 0.3 μg/mL and 0.6 μg/mL A23187 (A0.3 and A0.6, respectively), which was attenuated by treatment with PTX3. Results represent the mean ± s.e.m of four independent experiments (n = 4). The statistical signification was marked as *p < 0.05 respectively, compared with control. 'CON' is abbreviated 'control' which is treated nothing.
Fig 4.
(A) DCF staining for ROS increased with A23187 treatment and was attenuated with exposure to 1 and 5 nM PTX3 (P1 and P5, respectively). (B) ROS activity as measured by fluorimetry increased(F.I. of DCF) in cells treated with 0 nM/mL and 5 nM/mL PTX3 (A0.3, A0.3+P1 and A0.3+P5, respectively), which was attenuated by treatment with PTX3. *p < 0.05 versus A23187 (0.3μg/mL) subjected to administrate PTX3. The statistical signification was marked as * for p < 0.05 compared with the control. 'CON' is abbreviated 'control' which is treated nothing.
Fig 5.
(A) Mitochondrial membrane potentials were visualized by the intensity of fluorescing JC-1 aggregates (red) and monomers (green), which indicate high and low potentials, respectively, after treatment with 0.3 or 1 μg/mL A23187 (A0.3 and A1, respectively) and exposure to PTX3. (B) Numerical data were expressed as % Red/Green fluorescence cells which were increased with increasing doses of PTX3. Data is representative of five independent experiments and expressed as means ± s.e.m, *p < 0.05 as compared with their respective A23187 (0.3μg/mL) only. **p < 0.001 as compared control group and A23187administrated group (0.3μg/mL and 1μg/mL). 'CON' is abbreviated 'control' which is treated nothing.
Fig 6.
Treatment of HK-2 cells with 0.3 μg/mL A23187 (A0.3) induces apoptosis as determined by TUNEL assays.
Representative micrographs (A) and quantification (B) of TUNEL staining revealed that apoptotic processes were attenuated in ischemic cells exposed to 1 and 5 nM PTX3 (P1 and P5, respectively). Numerical data were expressed as mean intensity apoptotic cells respective to their control. Data expressed means ± s.e.m, *p < 0.05 as compared with their respective A23187 (0.3μg/mL) only. 'CON' is abbreviated 'control' which is treated nothing.
Fig 7.
Active caspase-3 and PARP cleavage were detected by Western blotting of supernatants from untreated cells and cells treated with 0.3 μg/mL A23187.
The corresponding increases in PARP cleavage, and active caspase-3 were attenuated in cells treated with 1 or 5 nM PTX3 (P1 and P5, respectively). GAPDH was used as a loading control. 'CON' is abbreviated 'control' which is treated nothing.
Fig 8.
Proposed model of the renoprotective effects of PTX3 on apoptosis-mediated signaling in AKI.