Chronic exposure of human mesangial cells to high glucose environments activates the p38 MAPK pathway  William A. Wilmer, Cynthia L. Dixon, Courtney Hebert  Kidney International  Volume 60, Issue 3, Pages 858-871 (September 2001) DOI: 10.1046/j.1523-1755.2001.060003858.x Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 1 Seven-day high glucose (HG) treatment activates human mesangial cell (HMC) p38 mitogen-activated protein kinase (MAPK). HMCs were grown in normal glucose (NG) or HG media for seven days and were harvested as described in the Methods section. (A) Cell lysates immunoprecipitated with anti-p38 MAPK antibody (Ab) were separated by SDS-PAGE and immunoblotted with a monoclonal anti-phosphotyrosine antibody. The tyrosine phosphorylation of p38 MAPK protein [molecular radius (Mr) 39 kD] was increased in HG lysates compared to NG (N = 3). (B) Lysates harvested with SDS lysis buffer were separated by SDS-PAGE and immunoblotted with anti-phospho-threonine/tyrosine-p38 MAPK Ab (p-p38 MAPK) or a polyclonal anti-pan-p38 MAPK (p38 MAPK). Dual phosphorylation of a protein with Mr 39 kD (arrow) was greater in HG lysates compared with NG. The amount of total p38 MAPK did not differ between samples (N = 4). (C) p38 MAPK assays demonstrated increased p38 MAPK activity of HG versus NG cells. The means ± SE 32P-cpm of the phosphorylated peptide are represented. *P = 0.01 HG vs. NG (N = 8). Kidney International 2001 60, 858-871DOI: (10.1046/j.1523-1755.2001.060003858.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 2 Large osmotic gradients are necessary for p38 MAPK activation in HMCs. (A and B) Phospho-p38 MAPK Western blots. HMCs placed overnight in serum-free media were treated with increasing concentrations of D-mannitol for 30 minutes (A) or with 250 mOsm/L D-mannitol for 5 to 60 minutes (B). In both blots, treatment of HMCs with IL-1β (1.1 ng/mL × 30 min) served as a positive control. Relative to control (C) levels, D-mannitol (250 mOsm/L) increases phospho-p38 MAPK (p-p38 MAPK) within 15 minutes without changing total p38 MAPK content (p38 MAPK; N = 4). (C) p38 MAPK assays demonstrated increased p38 MAPK activity in cells treated with 250 mOsm/L D-mannitol relative to control values (C; N = 5). *P = 0.01. IL-1β treatment stimulated p38 MAPK activity threefold above control values. **P = 0.001. Kidney International 2001 60, 858-871DOI: (10.1046/j.1523-1755.2001.060003858.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 3 Acute HG treatment and chronic mannitol treatment fail to activate HMC p38 MAPK. (A) Phospho-p38 MAPK Western blot demonstrating the acute effects of HG treatment (hours) on HMC phospho-p38 MAPK levels (p-p38 MAPK) relative to total p38 MAPK levels (p38 MAPK). Experiments were performed in cells placed overnight in serum-free media to lower basal p38 MAPK phosphorylation and in cells kept in serum-containing media, similar to seven-day treatments of HG. IL-1β treatment (1.1 ng/mL × 30 min) served as positive controls (N = 4). (B) Phospho-p38 MAPK Western using lysates of HMC treated for seven days in NG media or in media supplemented with D-mannitol at concentrations iso-osmolar to HG media (M; N = 4). (C) p38 MAPK assays demonstrated similar levels of p38 MAPK activity in seven-day NG and seven-day D-mannitol cells (M; N = 5). Kidney International 2001 60, 858-871DOI: (10.1046/j.1523-1755.2001.060003858.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 4 Phorbol esters activate HMC p38 MAPK. (A) Phospho-p38 MAPK Western blot. HMCs were placed overnight in serum-free media (Control) and treated for 30 minutes with increasing concentrations of PMA. The phospho-p38 MAPK levels (p-p38 MAPK) versus total p38 MAPK content (p38 MAPK) are demonstrated (N = 3). (B) p38 MAPK assay using cell lysates of control and PMA-treated cells (N = 4, *P = 0.01). Kidney International 2001 60, 858-871DOI: (10.1046/j.1523-1755.2001.060003858.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 5 GF 109203 X (GFX) inhibits phorbol ester-mediated but not HG-mediated p38 MAPK activation. The PKC inhibitor GFX (2 μmol/L × 60 min) inhibits p38 MAPK phosphorylation (A) and p38 MAPK activity (B), as assessed by p38 MAPK assays, induced by PMA treatment (25 nmol/L × 30 min). *P = 0.02 PMA vs. serum-free control values; **P = 0.01 GFX + PMA vs. PMA values (N = 3). (C) Phospho-p38 MAPK Western blot demonstrating that the addition of GFX (2 μmol/L) to NG and HG cells for the last 12 hours of treatment did not alter the increased p38 MAPK phosphorylation of the HG cells (N = 4). (D) p38 MAPK assays demonstrated activity of NG (□) and HG (▪) lysates. No difference in HG p38 MAPK activity was present when GFX (2 μmol/L) was added to the HG cells for the last 1to 12 hours of treatment (N = 5; NS, not significant). Kidney International 2001 60, 858-871DOI: (10.1046/j.1523-1755.2001.060003858.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 6 Ro 32-0432 and rottlerin fail to inhibit HG-mediated p38 MAPK activation. The effects of the PKC inhibitors Ro 32-0432 (Ro) and rottlerin on p38 MAPK phosphorylation (p-p38 MAPK) were investigated using phospho-p38 MAPK westerns. (A) Relative to serum-free control levels, PMA (25 nmol/L × 30 min) increased p38 MAPK phosphorylation. Pretreatment (60 min) of cells with Ro (10 μmol/L) but not the vehicle for Ro (DMSO) inhibited the PMA-mediated p38 MAPK phosphorylation (N = 3). (B) The addition of Ro (10 μmol/L) for the last 12 hours of NG and HG treatment did not alter p38 MAPK phosphorylation (N = 3). NG and HG cells were treated with the vehicle DMSO (0.01%) during Ro treatment. (C) The addition of rottlerin (5 μmol/L), a PKC δ inhibitor, also failed to alter NG- and HG-mediated p38 MAPK phosphorylation. DMSO (0.05%) was present in the NG and HG cells during rottlerin treatment (N = 3). Kidney International 2001 60, 858-871DOI: (10.1046/j.1523-1755.2001.060003858.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 7 Role of reactive oxygen species (ROS) in HG-mediated p38 MAPK activation. (A) The phosphorylation of p38 MAPK (p-p38 MAPK) relative to total p38 MAPK levels (p38 MAPK) was increased in serum-free control cells treated for 30 minutes with diamide (2 mmol/L), which increases intracellular ROS, or H2O2 (N = 3). (B) The phosphorylation of p38 MAPK was not altered in NG cells treated for the last four hours of the seven-day treatment with L-NAC (NAC, 25 μmol/L) or DPI (10 μmol/L; N = 3). (C) The addition of these anti-oxidants for the last four hours of the seven-day HG treatment significantly lowered p38 MAPK phosphorylation relative to total p38 MAPK levels (N = 3). Kidney International 2001 60, 858-871DOI: (10.1046/j.1523-1755.2001.060003858.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 8 Activator protein (AP-1) activation by HG is attenuated by p38 MAPK inhibition. Prior to the AP-1 electrophoretic mobility shift assay (EMSA), NG and HG cells were treated with the vehicle DMSO (0.015%) or the p38 MAPK inhibitor SB 203580 (15 μmol/L) for four hours prior to nuclear protein harvest. AP-1 EMSAs were performed as described in the Methods section. The addition of 50× unlabeled AP-1 oligo to the nuclear proteins served as a competitor. AP-1–specific bands are demonstrated at the arrow. NS depicts the nonspecific bands (N = 3). Kidney International 2001 60, 858-871DOI: (10.1046/j.1523-1755.2001.060003858.x) Copyright © 2001 International Society of Nephrology Terms and Conditions