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

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.


siRNA transfection
To deplete expression of ILK, AKT or WIP proteins by specific siRNAs, 1 THP-1 cells were cultured with fetal bovine serum-free medium at a concentration of 2.5 × 10 5 cells ml -1 . Cells were transfected in fetal bovine serum-free medium with 10 nM ILK-specific siRNA (which depleted ILK protein levels between 50-60%), 20 nM AKT-specific siRNA (which depleted AKT protein levels between 50-70%), 10 nM WIP-specific siRNA (which depleted WIP mRNA levels between 60-80%), or silencer-negative control (Scrambled RNA) using metafectene transfection reagent. After overnight incubation at 37°C in a 5% CO2 atmosphere with the RNA complex, cell suspension was centrifugated at 1,500 rpm for 5 min and cultured in medium containing fetal bovine serum for 24 h. Then, cells were treated as indicated. To verify the depletion of WIP mRNA levels, TaqMan gene expression assays were used to quantify WIPF1 (Hs00277097_m1) and β-actin (Hs01060665_g1) by RT-qPCR.

Western blot analysis
After treatments, cells were lysed in buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate) containing protease and phosphatase inhibitor cocktails. The resulting solution was spun at 11,000 rpm for 20 min at 4°C. Protein concentration for each sample was determined and equivalent amounts of protein (20-40 μg) were run onto 7.5-9% SDS-polyacrylamide gels under reducing conditions and then transferred onto polyvinylidene difluoride membranes. After membranes were blocked with 3% bovine serum albumin in TBS-T (50 mM Trizma, pH 7.6, 150 mM NaCl, 0.1% Tween 20) for 1 h at room temperature, the membranes were incubated overnight at 4°C with specific antibodies as previously described. 1 This incubation was followed by a second incubation for 1 h at room temperature with their corresponding HRP-conjugated secondary antibody). Membranes were reblotted with mouse anti-GAPDH antibody to normalize protein levels. 2 Antibodies dilutions ranged from 1:500 to 1:5000. Antibody-bound proteins were detected by chemiluminescence (Amersham Biosciences, Amersham, Little Chalfont, UK). Densitometry analyses were performed using ImageJ software (National Institutes of Health, USA).

Conditional ILK Knockdown Mice and study design
Conditional inactivation of the ILK gene was accomplished by crossing C57Bl/6 mice homozygous for the floxed ILK allele (LOX mice) with homozygous mice carrying a tamoxifeninducible CreER(T) recombinase gene, which express Cre under the control of the cytomegalovirus promoter (CRE mice). Tamoxifen was dissolved in a corn oil/ethanol (9:1) mixture. Male CRE-LOX mice (8-week-old), heterozygous for both transgenes, were injected intraperitoneally with 1.5 mg of tamoxifen once per day for 5 consecutive days to induce ILK deletion. Control animals were injected with vehicle, a corn oil/ethanol (9:1) mixture. Three weeks after the injections, tail DNA was genotyped by PCR with primers allow to distinguish excised ILK gene (230 bp) or to non-excised ILK (2100 bp): CCAGGTGGCAGAGGTAAGTA and CAAGGAATAAGGTGAGCTTCAGAA. 2 PCR DNA products were then analyzed by 1.5% agarose gel electrophoresis. Tamoxifen-treated CRE-LOX mice displaying successful depletion of ILK were termed cKD-ILK mice, and their control vehicle-treated CRE-LOX mice were termed wild-type (WT).

Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR)
Total RNA from THP-1 cells was extracted using TRIzol, transcribed to cDNA with a High-Capacity cDNA Reverse Transcription Kit, and 10 ng cDNA were amplified in a 7500 qPCR thermocycler.

Cell viability assay
The effect of pc, pCS and IS on cell viability was determined by Trypan Blue exclusion. 1 Briefly, 5 × 10 5 THP-1 cells were seeded on six-well culture plate and incubated for 6 or 24 h with the different concentrations of pc, pCS and IS. After incubation, cells were centrifuged, resuspended in 1 ml RPMI and diluted ½ in Trypan Blue. The percentage of cells excluding Trypan Blue was determined using a Countess™ Automated Cell Counter.

Reactive oxygen species (ROS) detection
The effect of pc and IS on ROS production, in particular hydrogen peroxide (H2O2), was determined by using the H2DCFDA probe. Catalase was applied as an antioxidant element to decreased intracellular H2O2. Catalase-treated THP-1 cells were pre-incubated overnight at a concentration of 250/500 units. Cells were incubated with H2DCFDA (5 μM) for 20 min in the dark and washed to eliminate remains of the probe. 5 × 10 4 cells were re-suspended in 100 μl RPMI with the different concentrations of pc and IS and seeded on 96-well culture plate. Fluorescence emission measurements were performed in a Victor X4 Multilabel plate reader (Perkin Elmer) using 490/535 nm excitation and emission filters. Measurements was taken at indicated times. Readings from 3 wells per treatment condition were used to mean fluorescence levels. ROS production was estimated using the mean fluorescence intensity of the cell population. Supplementary Fig. 8 ILK depletion prevents the ex vivo mice leukocyte increased podosome formation and the molecular mechanism downstream ILK activation induced by p-cresyl sulphate (pCS) plus indoxyl sulphate (IS) treatment. CRE-LOX mice were injected with tamoxifen (ILK conditional-knockdown mice, cKD-ILK) or vehicle (wild-type, WT) to induce ILK deletion. Leukocytes were obtained, seeded on fibronectin-coated coverslips, and incubated with a combination of high concentrations of pCS plus IS for 24 hours. (a and c) Podosome formation of leukocytes stained with phalloidin (red) and WASP (green) (a) or vinculin (red) and WIP (green) (c) and Hoeschst 33342 (blue), was determined by fluorescence confocal microscopy. A representative experiment is shown. Magnifications of the boxed area are shown at the bottom. Scale bars: 25 or 5 μm. (b) Histograms indicate the mean of the percentage of cells with podosomes per field of view treated as described above. (d and e) Median fluorescence intensity (MFI) of GSK-3β pS9 (d) and AKT pS473 (e) in the leukocyte cell population, analyzed by flow cytometry. Results are expressed as a percentage of WT control (untreated). Values are represented as mean ± SEM of 3 independent experiments. *P < 0.05 vs. WT control; #P < 0.05 vs. (pCS+IS) WT.