The monocyte chemoattractant protein-1/CCR2 loop, inducible by TGF-beta, increases podocyte motility and albumin permeability

Abstract

The role of monocyte chemoattractant protein-1 (MCP-1) in diabetic nephropathy is typically viewed through the lens of inflammation, but MCP-1 might exert noninflammatory effects on the kidney cells directly. Glomerular podocytes in culture, verified to express the marker nephrin, were exposed to diabetic mediators such as high glucose or angiotensin II and assayed for MCP-1. Only transforming growth factor-beta (TGF-beta) significantly increased MCP-1 production, which was prevented by SB431542 and LY294002, indicating that signaling proceeded through the TGF-beta type I receptor kinase and the phosphatidylinositol 3-kinase pathway. The TGF-beta-induced MCP-1 was found to activate the podocyte's cysteine-cysteine chemokine receptor 2 (CCR2) and, as a result, enhance the cellular motility, cause rearrangement of the actin cytoskeleton, and increase podocyte permeability to albumin in a Transwell assay. The preceding effects of TGF-beta were replicated by treatment with recombinant MCP-1 and blocked by a neutralizing anti-MCP-1 antibody or a specific CCR2 inhibitor, RS102895. In conclusion, this is the first description that TGF-beta signaling through PI3K induces the podocyte expression of MCP-1 that can then operate via CCR2 to increase cellular migration and alter albumin permeability characteristics. The pleiotropic effects of MCP-1 on the resident kidney cells such as the podocyte may exacerbate the disease process of diabetic albuminuria.

Fig. 1.

Podocyte cell line expresses nephrin. A: nephrin protein in the podocyte lysate can be detected by Western blotting at the expected 175- to 185-kDa double immunoband. The nephrin band is competitively diminished by the addition of a nephrin-blocking peptide and unaffected by an irrelevant blocking peptide (BSA); 1:50 refers to the ratio of anti-nephrin antibody to blocking peptide (either nephrin-blocking or irrelevant BSA). β-Actin is shown as a loading control. B: nephrin gene expression was verified in podocytes by RT-PCR, using primers that span exons 19 and 22. Mouse kidney total RNA was used as a positive control. Sequencing of the entire 372-bp RT-PCR band was found to match perfectly with the published sequence (accession no. NM_019459). C: nephrin by immunofluorescence can be seen to localize preferentially at the cell-cell interface between 3 podocytes in culture (left). Nuclei are stained by DAPI. In contrast, nephrin shows less tendency to organize at the margins when the podocyte is solitary (right). Magnification: ×400.

Fig. 2.

TGF-β stimulates monocyte chemoattractant protein-1 (MCP-1) expression. A: cultured, differentiated mouse podocytes were treated with 2 ng/ml of recombinant transforming growth factor (TGF)-β1 for 48 h. Compared with control, TGF-β1 markedly increased MCP-1 production as measured by ELISA of cell lysate. The TGF-β-stimulated MCP-1 production was significantly blunted by concurrent treatment with 1 μM SB431542 (n = 3). *P < 0.05 vs. control. †P < 0.05 vs. TGF-β. B: TGF-β1 at 2 ng/ml progressively increased podocyte MCP-1 content over time, most evident at 48 h (n = 5) when the step-up in MCP-1 production was greatest between TGF-β and vehicle treatment (control for each time point set at 100%). ‡P < 0.01 vs. either 8 (n = 3) or 24 h (n = 5). C: high ambient glucose (25 mM) for 2 wk (n = 4), angiotensin II (10⁻⁸ M) for 6 h (n = 3), or VEGF (8 ng/ml) for 48 h (n = 3) had no significant effects on the production of MCP-1.

Fig. 3.

Phosphatidylinositol 3-kinase (PI3K) mediates TGF-β1-stimulated MCP-1. A: a specific inhibitor of PI3K, LY294002 (25 μM), completely inhibited TGF-β-stimulated MCP-1 production by cultured podocytes (n = 3). The modest decrease in MCP-1 due to LY294002 alone was not significantly different from control. *P < 0.05 vs. control. †P < 0.05 vs. TGF-β. B: TGF-β1 treatment of podocytes activated the PI3K pathway, evident in the increased amount of phospho-Akt compared with the constant level of total Akt.

Fig. 4.

Cysteine-cysteine chemokine receptor 2 (CCR2) protein and mRNA in podocytes. A: CCR2 staining is evident in podocytes as a red signal. The intense nuclear signal abates when the cells are not permeabilized before staining (inset). B: no fluorescence is detected when the primary antibody is omitted. C: staining is competitively obliterated by a blocking peptide, indicating the specificity of the primary antibody for CCR2. D: staining is not affected, however, by an irrelevant blocking peptide (in this case, VEGFR-1 antigen). Magnification: ×400. E: RT-PCR confirms the expression of CCR2 mRNA in podocytes (200-bp band). RT-PCR performed on monocyte RNA, a positive control, shows a CCR2 band of identical size. The negative control, water, showed no RT-PCR band.

Fig. 5.

MCP-1 stimulates podocyte motility as evaluated by a scratch-wound assay. A: in the counting of the number of cells that had repopulated a consistently defined area of the scratch, MCP-1 was seen to significantly stimulate podocyte migration at all time points, resulting in quicker wound closure. A similar effect was seen with TGF-β1 treatment. Both MCP-1- and TGF-β1-induced motility increases were prevented by CCR2 inhibition with RS102895 (n = 4). *P < 0.05 vs. control. †P < 0.05 vs. MCP-1. ‡P < 0.05 vs. TGF-β. B: in a follow-up scratch assay at 42 h, the TGF-β-induced motility gain (gray vs. white bar) was nullified by neutralizing MCP-1 with a specific antibody (α-MCP-1 Ab; red vs. gray bar). A species- and isotype-matched irrelevant antibody (Irr Ab) was used as the control (n = 3). *P < 0.05 vs. control+Irr Ab. ‡P < 0.05 vs. TGF-β+Irr Ab.

Fig. 6.

MCP-1-induced actin cytoskeleton reorganization in podocytes. A: filamentous (F)-actin strands were visible as cytoplasmic stress fibers in control cells. B: exposure to MCP-1 decreased the density of stress fibers and increased the localization of actin to bundles at the cell periphery (arrows). C: TGF-β-treated cells also showed a loss of stress fibers and increased peripheral actin. D–F: changes in F-actin appearance described above were not affected by an irrelevant hamster IgG, an isotype control. G–I: neutralizing anti-MCP-1 antibody (hamster, 30 μg/ml) greatly reduced the arrangement of F-actin near the cell margins that was secondary to MCP-1 or TGF-β. J–L: RS102895 (6 μM), a specific CCR2 inhibitor, also effectively blocked the MCP-1- or TGF-β-induced actin cytoskeletal reorganization. Magnification: ×400.

Fig. 7.

MCP-1 increases podocyte permeability to albumin. A: Evans blue-labeled albumin (EBA) can be quantified by the absorbance characteristics of the Evans blue dye, which absorbs light most strongly at 620 nm. In our tests, the A₆₂₀ measurement is oblivious to DMEM but is quite sensitive to the mixture of DMEM+EBA. B: in a Transwell setup to assay the cellular permeability to EBA, performed after the transepithelial electrical resistance had plateaued at 114.5 ± 24.6 Ω·cm², significantly more EBA had diffused from the lower into the upper chamber across the podocyte monolayer at 24 h as a result of MCP-1 treatment (50 ng/ml) vs. control. TGF-β (2 ng/ml) had a similar effect on EBA transit, although not statistically significant. The permeability to albumin induced by MCP-1 or TGF-β was returned to control levels by concurrent treatment with RS102895 (6 μM), an inhibitor of CCR2 signaling (n = 4). *P < 0.05 vs. control. †P < 0.05 vs. MCP-1. ‡P < 0.05 vs. TGF-β.

Fig. 8.

Proposed TGF-β-induced MCP-1/CCR2 loop in podocytes. 1: The diabetic milieu stimulates mesangial cells to produce TGF-β. 2: TGF-β binds to its type II receptor, which is upregulated in the podocytes in diabetes. 3: Podocytes are stimulated to produce MCP-1 via TGF-β type I receptor signaling and the PI3K pathway. 4: Via the CCR2 receptor, MCP-1 causes increased podocyte migration, actin cytoskeletal changes, and albumin hyperpermeability.