Uremia Impacts VE-Cadherin and ZO-1 Expression in Human Endothelial Cell-to-Cell Junctions
Abstract
:1. Introduction
2. Results
2.1. Clinical and Laboratory Characteristics of the Study Population
2.2. Clinical and Biochemical Characteristics of Each Uremic Pool
2.3. Concentration of Systemic and Vascular Inflammatory Biomarkers
2.4. Multivariate Analysis of Independent Determinants of Chemokines, Adhesion Molecules
2.5. Correlations between Uremic Toxins Serum Concentration and eGFR
2.6. VE-Cadherin and ZO-1 Expression Increased in CKD Iliac and Renal Arteries
2.7. Cell Viability
2.8. Uremic Milieu Increases Endothelial Cell Permeability
2.9. Uremia Impacts the Intercellular Adhesion and the Endothelial Cell Phenotype
2.10. Uremic Environment Modifies the Endothelial Cell Cytoskeleton
2.11. Uremic Environment Impacts Endothelial Cell Adherent Junction and VE-Cadherin Expression
2.12. Uremic Environment Differently Modulates ZO-1 Gene and Protein Expression
3. Discussion
4. Conclusions
5. Materials and Methods
5.1. Patients
5.1.1. Patients’ Samples Collection and Processing for In Vivo and In Vitro Assays
5.1.2. Clinical and Biochemical Characteristics of the Patients
5.2. Materials
5.3. Uremic Toxins’ Preparation
5.4. PCS and IS Serum Measurement
5.5. Measurement of MCP-1, IL-8, sVCAM-1, and sICAM-1 Serum Concentrations
5.6. Endothelial Cell Culture and Treatment
5.7. MTT Cell Viability Assay
5.8. Cell Permeability Assay
5.9. Scanning Electron Microscopy (SEM)
5.10. F-actin Staining by Fluorescence Microscopy
5.11. Immunochemical Analysis of VE-Cadherin and ZO-1 On Arteries
5.12. VE-Cadherin and ZO-1 Gene Expression
5.13. VE-Cadherin and ZO-1 Western Blot Analysis
5.14. VE-Cadherin and ZO-1 Immunofluorescence Analysis
5.15. VE-Cadherin and ZO-1 Flow Cytometry Analysis
5.16. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Analyzed Parameters | Mean Or Percent |
---|---|
Patients (n) | 80 |
Traditional risk factors | |
Mean age ± SEM, years | 62.7 ± 1.3 |
Gender, % male | 61.0 |
Race, % caucasians | 86.0 |
Smoking, % | 33.0 |
Alcoholism, % | 14.0 |
Diabetes mellitus, % | 43.0 |
Hypertension, % | 79.0 |
Dyslipidemia, % | 56.3 |
Primary kidney disease | |
Nephrosclerosis, % | 30.0 |
Diabetic nephropathy, % | 21.3 |
Chronic glomerulonephritis, % | 10.0 |
Polycystic kidney disease, % | 12.5 |
Others and unknown, % | 26.2 |
Analyzed Parameters | Media ± SEM | Range |
---|---|---|
Biochemical characterization | ||
Uric acid, mg/dL | 7.0 ± 2.0 | 1.7–15.6 |
Albumin, g/dL | 4.2 ± 0.6 | 2.2–5.7 |
Calcium, mg/dL | 8.6 ± 3.0 | 1.0–12.7 |
Creatinine, mg/dL | 2.3 ± 1.5 | 0.5–7.6 |
Glucose, mg/dL | 116.0 ± 52.1 | 53.0–284.0 |
Potassium, mmol/L | 5.0 ± 0.6 | 3.5–7.0 |
Sodium, mmol/L | 141.0 ± 4.5 | 132.0–158.0 |
Urea, mg/dL | 77.0 ± 40.6 | 13.0–189.0 |
PCS, mg/L | 20.5 ± 19.0 | 0.01–92.35 |
IS, mg/L | 4.6 ± 6.5 | 0.25–40.26 |
Pi, mg/dL | 4.4 ± 1.1 | 2.4–9.9 |
Analyzed Parameters | GI (n = 7) Latent CKD | GII (n = 42) Mild CKD | GIII (n = 31) Severe CKD |
---|---|---|---|
Traditional risk factors | |||
Smoking, % | 52 | 36 | 45 |
Alcoholism, % | 22 | 12 | 26 |
Diabetes mellitus, % | 43 | 32 | 58 |
Hypertension, % | 78 | 100 | 94 |
Dyslipidemia, % | 57 | 56 | 58 |
CVD, % | 43 | 36 | 55 |
Biochemical characterization | |||
Uric acid (mg/dL) | 6.1 | 7.0 | 7.7 |
Albumin (g/dL) | 4.5 | 4.2 | 4.0 |
Calcium (mg/dL) | 9.7 | 7.7 | 8.6 |
Creatinine (mg/dL) | 1.0 | 1.6 | 3.7 |
Glucose (mg/dL) | 137.1 | 95.3 | 120.8 |
hs-CRP (mg/L) | 5.1 | 5.9 | 7.4 |
Potassium (mmol/L) | 5.0 | 5.0 | 5.0 |
Sodium (mmol/L) | 141.0 | 142.0 | 139.0 |
Urea (mg/dL) | 42.3 | 63.9 | 114.0 |
PCS (mg/L) | 11.1 | 15.4 | 33.9 |
IS (mg/L) | 1.5 | 2.1 | 9.6 |
Pi (mg/dL) | 4.1 | 4.1 | 5.0 |
Target Gene | Primers | Amplicon |
---|---|---|
VE-cadherin | 5’-CAGCCCAAAGTGTGTGAGAA-3’ (F) 5’-CGGTCAAACTGCCCATACTT-3’ (R) | 185 pb |
ZO-1 | 5’-GCGGTCAGAGCCTTCTGATC-3’ (F) 5’-CATGCTTTACAGGAGTTGAGACAG-3’ (R) | 122 pb |
p120 | 5’-GATGCTGTCAAGTCCAATGCAG-3’ (F) 5’-AGTACTGGGATGCCCTTGAGC-3’ (R) | 101 pb |
β-catenin | 5’-GTGCTATCTGTCTGCTCTAGTA-3’ (F) 5’-CTTCCTGTTTAGTTGCAGCATC-3’ (R) | 154 pb |
Vinculin | 5’-TCAGATGAGGTGACTCGGTTGG-3’ (F) 5’-GGGTGCTTATGGTTGGGATTCG-3’ (R) | 109 pb |
HPRT | 5’-GAACGTCTTGCTCGAGATGTGA-3’ (F) 5’-TCCAGCAGGTCAGCAAAGAAT-3’ (R) | 101 pb |
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Maciel, R.A.P.; Cunha, R.S.; Busato, V.; Franco, C.R.C.; Gregório, P.C.; Dolenga, C.J.R.; Nakao, L.S.; Massy, Z.A.; Boullier, A.; Pecoits-Filho, R.; et al. Uremia Impacts VE-Cadherin and ZO-1 Expression in Human Endothelial Cell-to-Cell Junctions. Toxins 2018, 10, 404. https://doi.org/10.3390/toxins10100404
Maciel RAP, Cunha RS, Busato V, Franco CRC, Gregório PC, Dolenga CJR, Nakao LS, Massy ZA, Boullier A, Pecoits-Filho R, et al. Uremia Impacts VE-Cadherin and ZO-1 Expression in Human Endothelial Cell-to-Cell Junctions. Toxins. 2018; 10(10):404. https://doi.org/10.3390/toxins10100404
Chicago/Turabian StyleMaciel, Rayana A. P., Regiane S. Cunha, Valentina Busato, Célia R. C. Franco, Paulo C. Gregório, Carla J. R. Dolenga, Lia S. Nakao, Ziad A. Massy, Agnès Boullier, Roberto Pecoits-Filho, and et al. 2018. "Uremia Impacts VE-Cadherin and ZO-1 Expression in Human Endothelial Cell-to-Cell Junctions" Toxins 10, no. 10: 404. https://doi.org/10.3390/toxins10100404