Indoxyl sulfate- and P-cresol-induced monocyte adhesion and migration is mediated by integrin-linked kinase-dependent podosome formation

Abstract

Cardiovascular disease is an important cause of death in patients with chronic kidney disease (CKD). Protein-bound uremic toxins, such as p-cresyl and indoxyl sulfate (IS), are poorly removed during hemodialysis, leading to vascular endothelial dysfunction and leukocyte extravasation. These processes can be related to dynamic adhesion structures called podosomes. Several studies have indicated the role of integrin-linked kinase (ILK) in the accumulation of integrin-associated proteins in podosomes. Here, we investigated the involvement of ILK and podosome formation in the adhesion and extravasation of monocytes under p-cresol (pc) and IS exposure. Incubation of THP-1 human monocyte cells with these toxins upregulated ILK kinase activity. Together, both toxins increased cell adhesion, podosome formation, extracellular matrix degradation, and migration of THP-1 cells, whereas ILK depletion with specific small interfering RNAs suppressed these processes. Interestingly, F-actin colocalized with cortactin in podosome cores, while ILK was colocalized in podosome rings under toxin stimulation. Podosome Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP) and AKT protein depletion demonstrated that monocyte adhesion depends on podosome formation and that the ILK/AKT signaling pathway is involved in these processes. Ex vivo experiments showed that both toxins induced adhesion and podosome formation in leukocytes from wild-type mice, whereas these effects were not observed in leukocytes of conditional ILK-knockdown animals. In summary, under pc and IS stimulation, monocytes increase podosome formation and transmigratory capacity through an ILK/AKT signaling pathway-dependent mechanism, which could lead to vascular injury. Therefore, ILK could be a potential therapeutic target for the treatment of vascular damage associated with CKD.

Fig. 1. p-Cresol (pc) and indoxyl sulfate (IS) upregulate ILK activity in THP-1 cells.

THP-1 cells were incubated with a pc, b IS, or c low concentrations of pc plus IS or high concentrations of pc plus IS for different times. d THP-1 cells were transfected with scrambled RNA (Sc) or were depleted of ILK with specific siRNA and treated as in c for 24 h. Representative western blots of GSK-3β phosphorylated on the serine-9 residue (P-GSK-3β) or ILK are shown. GAPDH was used as the endogenous control. The bars represent the normalized densitometric values of the blots against the endogenous control values. All values are presented as the mean ± SEM from 4 independent experiments. *P < 0.05 vs. untreated control (CT); ^#P < 0.05 vs. pc (10 μg ml⁻¹).

Fig. 2. p-Cresol (pc) and indoxyl sulfate (IS) induce THP-1 cell adhesion to a fibronectin matrix.

THP-1 cells were transfected with scrambled RNA (Sc) as a control (CT) (upper microphotographs, b black bars) or were depleted of ILK with specific siRNA (lower microphotographs, b white bars). Afterward, the cells were seeded on fibronectin-coated coverslips and incubated with low concentrations of pc plus IS or high concentrations of pc plus IS for 24 h. a Adhesion of THP-1 cells stained with phalloidin (red) and Hoechst 33342 (blue) to the fibronectin matrix was determined by fluorescence confocal microscopy. The results of a representative experiment are shown. Scale bar, 50 µm. b Bar graphs indicating the average percentages of attached THP-1 cells treated as in a. The results are expressed as a percentage of the number of untreated CT cells. All values are presented as the mean ± SEM from 4 independent experiments. *P < 0.05 vs. CT; ^#P < 0.05 vs. Sc. TGF-β1 was used as a positive control.

Fig. 3. p-Cresol (pc) and indoxyl sulfate (IS) induce THP-1 cell podosome formation in a fibronectin matrix.

THP-1 cells were transfected with scrambled RNA (Sc) as a control (CT) (a, c upper microphotographs, b black bars) or were depleted of ILK with specific siRNA (a, c lower microphotographs, b white bars). Afterward, the cells were seeded on fibronectin-coated coverslips and incubated with low or high concentrations of pc plus IS (a, b) or high concentrations of pc plus IS (c) for 24 h. a, c Podosome formation of THP-1 cells stained with phalloidin (red) and Hoechst 33342 (blue) (a) or phalloidin (red), WASP (green) and Hoechst 33342 (blue) (c) was determined by fluorescence confocal microscopy. A representative experiment is shown. Scale bar, 25 or 5 µm, respectively. Magnifications of the boxed area are shown at the bottom left. b Bar graphs showing the mean of the percentage of cells with podosomes per field of view for cells treated as in a. All values are presented as the mean ± SEM from 4 independent experiments. *P < 0.05 vs. untreated CT; ^#P < 0.05 vs. Sc. TGF-β1 was used as a positive control.

Fig. 4. p-Cresol (pc) and indoxyl sulfate (IS) induce THP-1 cell matrix degradation and cell migration.

THP-1 cells were transfected with scrambled RNA (Sc) as a control (CT) (upper microphotographs, b–d black bars) or were depleted of ILK with specific siRNA (lower microphotographs, b–d white bars). a–c Afterward, the cells were seeded on TRITC-gelatin-coated coverslips and incubated with high concentrations of pc plus IS for 24 h. a Confocal micrographs showing the distribution of TRITC gelatin (red) and THP-1 cells stained with phalloidin (green) and Hoechst 33342 (blue). The results of a representative experiment are shown. Scale bar, 50 µm. b, c Bar graphs indicating the average percentage of THP-1 cells with an associated subjacent area of gelatin degradation (b) or the total degraded area divided by the total cell area (µm²) (c) per field of view for cells treated as in a. d Afterward, the cells were loaded in the upper chamber of the filter and incubated with high concentrations of pc plus IS for 24 h. Cell migration was determined by Transwell migration assay. The bar graphs indicate the average percentage of THP-1 cells that migrated across the filter toward MCP-1 cells treated as in a. b, c The results are expressed as a percentage of the number of untreated CT cells. All values are presented as the mean ± SEM from 4 or 6 independent experiments. *P < 0.05 vs. CT; ^#P < 0.05 vs. Sc. TGF-β1 was used as a positive control. MCP-1 was used as a chemoattractant.

Fig. 5. Podosome-specific WIP protein depletion impairs p-cresol (pc)- and indoxyl sulfate (IS)-induced cell adhesion to a fibronectin matrix.

THP-1 cells were transfected with scrambled RNA (Sc) as a control (CT) (upper microphotographs, b, c black bars) or were depleted of WIP with specific siRNA (lower microphotographs, b, c white bars). Afterward, the cells were incubated with high concentrations of pc plus IS for 24 h. a Adhesion of THP-1 cells stained with phalloidin (red) and Hoechst 33342 (blue) to the fibronectin matrix was determined by fluorescence confocal microscopy. The results of a representative experiment are shown. Scale bar, 50 µm. b Bar graphs indicating the average percentage of attached THP-1 cells treated as in a. c Bar graphs showing the mean percentage of cells with podosomes per field of view for cells treated as in a. The results are expressed as a percentage of the number of untreated CT cells. All values are presented as the mean ± SEM from 4 independent experiments. *P < 0.05 vs. CT; ^#P < 0.05 vs. Sc. TGF-β1 was used as a positive control.

Fig. 6. ILK is localized in the podosome rings of THP-1 cells induced by p-cresol (pc) and indoxyl sulfate (IS).

THP-1 cells were seeded on fibronectin-coated coverslips and incubated with high concentrations of pc plus IS for 24 h. Confocal micrograph showing the distribution of ILK (green) in the podosome ring and colocalization of phalloidin-stained F-actin (red) and cortactin (blue) in the podosome core in THP-1 cells. Scale bar, 5 µm. Magnifications of the boxed area are shown at the bottom left. The experiment was repeated five times.

Fig. 7. Molecular mechanism downstream of ILK activation.

a THP-1 cells were incubated with high concentrations of p-cresol (pc) plus indoxyl sulfate (IS) for different times. b THP-1 cells were transfected with scrambled RNA (Sc) as a control (CT) (black bars) or were depleted of ILK with specific siRNA (white bars). Afterward, the cells were incubated with high concentrations of pc plus IS for 3 h. a, b Representative Western blots of AKT phosphorylated on the serine-473 residue (P-AKT) are shown. GAPDH was used as the endogenous control. The bars represent the normalized densitometric values of the blots against the endogenous control values. c–f THP-1 cells were transfected with scrambled RNA (Sc) as a control (CT) (c upper microphotographs, d, e black bars) or were depleted of AKT with specific siRNA (c lower microphotographs, d, e white bars). Afterward, the cells were seeded on fibronectin-coated coverslips and incubated with high concentrations of pc plus IS. c Adhesion of THP-1 cells stained with phalloidin (red) and Hoechst 33342 (blue) to the fibronectin matrix was determined by fluorescence confocal microscopy. The results of a representative experiment are shown. Scale bar, 50 µm. d Bar graphs indicating the average percentage of attached THP-1 cells treated as in c. e Bar graphs showing the mean percentage of cells with podosomes per field of view for cells treated as in c. The results are expressed as a percentage of the number of untreated CT cells. All values are presented as the mean ± SEM from 3, 4, or 5 independent experiments. *P < 0.05 vs. CT; ^#P < 0.05 vs. Sc. TGF-β1 was used as a positive control. f Total AKT expression was measured by western blot analysis (AKT). GAPDH was used as the endogenous control.

Fig. 8. ILK depletion prevents ex vivo increases in podosome formation and adhesion to a fibronectin matrix in mouse leukocytes and the molecular mechanism downstream of ILK activation induced by p-cresol (pc) plus indoxyl sulfate (IS) treatment.

CRE-LOX mice were injected with tamoxifen (ILK conditional-knockdown [cKD-ILK] mice) or vehicle (wild-type [WT] mice) to induce ILK deletion. Leukocytes were obtained, seeded on fibronectin-coated coverslips, and incubated with high concentrations of pc plus IS for 24 h. a Bar graphs indicating the average percentage of leukocytes attached to the fibronectin matrix as determined by fluorescence confocal microscopy. b, c The podosome formation of leukocytes stained with phalloidin (red) and a WASP antibody (green) (b) or vinculin (red) and WIP (green) antibodies (c) as well as Hoechst 33342 (blue) was determined by fluorescence confocal microscopy. The results of a representative experiment are shown. Magnifications of the boxed area are shown at the bottom. Scale bars: 25 and 5 μm. d Bar graphs indicating the mean percentage of cells with podosomes per field of view for cells treated as described above. e, f Median fluorescence intensity (MFI) of GSK-3β pS9 (e) and AKT pS473 (f) in the leukocyte cell population as analyzed by flow cytometry. The results are expressed as a percentage of the WT control (untreated). g Uncleaved ILK mRNA expression in leukocytes was quantified by RT–qPCR. The relative fold changes in mRNA content vs. those in the WT group after normalization to total β-actin content (the endogenous control) are presented. The values are presented as the mean ± SEM from 3 or 5 independent experiments. *P < 0.05 vs. WT control; ^#P < 0.05 vs. (pc + IS) WT.