Fig 1.
High glucose levels inhibit scratch-wound healing in cultured kidney tubular cells.
RPTC, NRK-52E and HEK 293 cells were cultured for 2 days in low glucose (5.5 mM) or high glucose (30 mM) medium, followed by a scratch-wound healing experiment. (A) RPTC were scratch-wounded with a sterile pipette tip and then incubated in low glucose or high glucose medium. Representative wounds immediately after scratching and after 6 and 18 h of healing were recorded with a phase-contrast microscope. (B) The wound width was measured at 6 and 18 h after scratching to determine the distance over which healing occurred in RPTC. (C) High glucose inhibited scratch-wound healing in RPTC in a concentration-dependent manner at 18 h after scratching. (D) NRK-52E were scratch-wounded with a sterile pipette tip and then incubated in low glucose or high glucose medium, the wound width was measured at 18 h after scratching. (E and F) HEK 293 were scratch-wounded with a sterile pipette tip and then incubated in low glucose or high glucose medium, the wound width was measured at 18 h after scratching. Data are expressed as the mean ± S.D. (n = 4). *, p<0.05 versus the low glucose group.
Fig 2.
High glucose inhibits transwell migration in cultured kidney tubular cells.
RPTC, NRK-52E and HEK 293 cells were cultured for 2 days in low glucose (5.5 mM) or high glucose (30 mM) medium, and then used for a transwell migration experiment. (A) Representative PI staining of migratory cells was recorded with a fluorescence microscope. A total of 3x105 RPTC were seeded in a transwell insert, which was put in a 24-well plate containing 600 μL low glucose or high glucose medium for 6 h. The cells that migrated to the undersurface were stained with PI and counted. (B) Migratory cells attached to the undersurface were counted after PI staining in RPTC. (C) Migratory cells attached to the undersurface were counted after PI staining in HEK 293 cells. (D) Migratory cells attached to the undersurface were counted after PI staining in NRK-52E. Data are expressed as the mean ± S.D. (n = 4). *, p<0.05 versus the low glucose group.
Fig 3.
TLR4 upregulation during high glucose treatment in RPTC.
TLR4 mRNA and protein levels were examined via real-time PCR and Western blotting after low gucose or high glucose treatment. (A and B) TLR4 mRNA upregulation in the high glucose medium. (C) TLR4 protein levels increased after the high glucose treatment. (D) Experiments were analyzed via densitometry and normalized to the control (0 h). (E) MCP-1 mRNA upregulation in high glucose medium for 24 h. (F) Immunofluorescence analysis of TLR4 expression. PRTC cells were cultured for 2 days in low glucose (5.5 mM) or high glucose (30 mM) medium.The cells were fixed for immunofluorescent staining of TLR4 (red). Data are expressed as the mean ± S.D. (n≥3). *, p<0.05 versus the low glucose group; **, p<0.05 versus the control (0 h).
Fig 4.
Effect of PKC on high glucose induced TLR4 expression.
RPTC were treated for 24 hours with low glucose (5.5 mM), high glucose (30 mM), or the PKC inhibitor staurosporine (10 nM, added 1 hour before high glucose treatment), and the cell lysates were collected, TLR4 mRNA, phospho-PKC (pan), p-p38, p38 levels were examined via real-time PCR and Western blotting. (A) PKC phosphorylation and p-p38 upregulation after high glucose treatment. The PKC inhibitor staurosporine decreased phospho-PKC and p-p38 levels but not total PKC levels. (B) Staurosporine reduced TLR4 mRNA levels under the high glucose condition. (C) RPTC were pretreated with staurosporine (10 nM), then treated with high glucose (30mM) for 6, 12, 24 h, and cell lysates were collected at different time points. phospho-PKC (pan), p-p38 levels were examined via Western blotting. (D) Effect of staurosporine on scratch wound healing in low glucose and high glucose. Data are expressed as the mean ± S.D. (n = 4). *, p<0.05 versus the low glucose group; **, p<0.05 versus the high glucose group without staurosporine treatment.
Fig 5.
TLR4 inhibitor promotes scratch-wound healing and migration in high-glucose-treated RPTC.
RPTC were cultured for 2 days in low glucose (5.5 mM) or high glucose (30 mM) medium, and then used for following experiments. (A) Titration of TAK-242 concentration. RPTC cells were cultured for 12 h in low glucose or high glucose medium with or without TAK-242. (B) MyD88 upregulation was also inhibited by TAK-242 under high glucose conditions by Western blotting. (C) TLR4 mRNA upregulation was also inhibited by TAK-242 under high glucose conditions by real-time PCR analysis. (D) Enhanced wound healing in the high glucose condition with the TLR4 inhibitor. RPTC cells were scratch-wounded and incubated in low glucose or high glucose medium with or without 100 nM TAK-242 for 18 h and then the healing distance was measured. (E) Enhanced cell migration under the high glucose condition with the TLR4 inhibitor. A total of 3x105 RPTC were seeded in a transwell insert, which was put in a 24-well plate containing 600 μL of medium with or without 100 nM TAK-242 in low glucose or high glucose medium for 6 h. The cells that migrated to the undersurface of the insert were stained with PI and counted. In C, D and E, data are expressed as the mean ± S.D. (n = 4). *, p<0.05 versus the low glucose group; **, p<0.05 versus the high glucose group without TAK-242 treatment.
Fig 6.
TLR4 shRNA abrogates impaired wound healing in high-glucose-treated RPTC.
RPTC were infected with lentiviruses containing a scrambled control sequence (Scr) or the TLR4 shRNA sequence (shRNA) and then cultured for 2 days in low glucose or high glucose medium for the following experiments. (A) TLR4 knockdown caused by the TLR4 shRNA lentivirus. After infection and a high glucose treatment, whole-cell lysates were collected for an immunoblot analysis of TLR4 and MyD88. (B) Scratch-wound healing. After lentiviral infection, RPTC were scratch-wounded and incubated in low glucose or high glucose medium for 18 h to measure the distance over which healing occurred. (C) Transwell cell migration. After lentiviral infection, a total of 3x105 RPTC were seeded in a transwell insert, which was put in a 24-well plate containing 600 μL of low glucose or high glucose medium for 6 h. The cells that migrated to the undersurface were stained with PI and counted. In B and C, data are expressed as the mean ± S.D. (n = 4). *, p<0.05 versus the low glucose group; **, p<0.05 versus high glucose group infected with the scrambled sequence.
Fig 7.
Overexpression of TLR4 in low-glucose-treated RPTC suppresses wound healing.
RPTC were infected with lentiviruses containing TLR4 or control lentivirus and then subjected to low glucose and high glucose treatment for 2 days, followed by a scratch-wound healing assay. (A) Increased TLR4 levels were detected in the low-glucose- and high-glucose-treated RPTC. (B) MCP-1 mRNA level in low-glucose-treated and high-glucose-treated RPTC cells after TLR4 overexpression. (C) Overexpression of TLR4 in low-glucose-treated cells also suppressed wound healing, mimicking the effect of high glucose levels. (D) RPTC cells were infected with lentivirus containing TLR4 or control lentivirus, then cultured for 18 h in low glucose or high glucose medium with or without TAK-242, followed by a scratch-wound healing assay. Data are expressed as the mean ± S.D. (n = 4). *, p<0.05 versus the low glucose group; #, p<0.05 versus the group without TAK-242.