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. 2020 Aug 6:8:739.
doi: 10.3389/fcell.2020.00739. eCollection 2020.

Inflammation, Senescence and MicroRNAs in Chronic Kidney Disease

Andres Carmona  1 , Fatima Guerrero  1   2 , Maria Jose Jimenez  1 , Francisco Ariza  1 , Marisa L Agüera  1   3 , Teresa Obrero  1 , Victoria Noci  4 , Juan Rafael Muñoz-Castañeda  1   3 , Mariano Rodríguez  1   2   3   5 , Sagrario Soriano  1   3   5 , Juan Antonio Moreno  1   6 , Alejandro Martin-Malo  1   2   3   5 , Pedro Aljama  1   2
Affiliations

Inflammation, Senescence and MicroRNAs in Chronic Kidney Disease

Andres Carmona et al. Front Cell Dev Biol. .

Abstract

Background: Patients with chronic kidney disease (CKD) show a chronic microinflammatory state that promotes premature aging of the vascular system. Currently, there is a growth interest in the search of novel biomarkers related to vascular aging to identify CKD patients at risk to develop cardiovascular complications.

Methods: Forty-five CKD patients were divided into three groups according to CKD-stages [predialysis (CKD4-5), hemodialysis (HD) and kidney transplantation (KT)]. In all these patients, we evaluated the quantitative changes in microRNAs (miRNAs), CD14+C16++ monocytes number, and microvesicles (MV) concentration [both total MV, and monocytes derived MV (CD14+Annexin V+CD16+)]. To understand the molecular mechanism involved in senescence and osteogenic transdifferentation of vascular smooth muscle cells (VSMC), these cells were stimulated with MV isolated from THP-1 monocytes treated with uremic toxins (txMV).

Results: A miRNA array was used to investigate serum miRNAs profile in CKD patients. Reduced expression levels of miRNAs-126-3p, -191-5p and -223-3p were observed in CKD4-5 and HD patients as compared to KT. This down-regulation disappeared after KT, even when lower glomerular filtration rates (eGFR) persisted. Moreover, HD patients had higher percentage of proinflammatory monocytes (CD14+CD16++) and MV derived of proinflammatory monocytes (CD14+Annexin V+CD16+) than the other groups. In vitro studies showed increased expression of osteogenic markers (BMP2 and miRNA-223-3p), expression of cyclin D1, β-galactosidase activity and VSMC size in those cells treated with txMV.

Conclusion: CKD patients present a specific circulating miRNAs expression profile associated with the microinflammatory state. Furthermore, microvesicles generated by monocytes treated with uremic toxins induce early senescence and osteogenic markers (BMP2 and miRNA-223-3p) in VSMC.

Keywords: chronic kidney disease; microRNAs; microvesicles; monocytes CD14+CD16++; vascular smooth muscle cells.

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Figures

FIGURE 1
FIGURE 1
Heat map of differentially expressed miRNAs from serum samples of CKD patients. Circulating miRNAs expression was downregulated in CKD4-5 and HD patients whereas was upregulated in KT patients. Red indicates downregulated miRNAs expression and green indicates upregulated miRNAs expression. n = 4 each group.
FIGURE 2
FIGURE 2
Expression relative miRNAs levels in CKD patients and elderly subjects. The results are represented as the means ± SEM. Relative expression levels of circulating: (A) miRNA-126-3p; (B) miRNA-191-3p; (C) miRNA-223-3p; (D) miRNA-363-3p and (E) miRNA-495-3p are shown. The data were analyzed using an ANOVA test and Post hoc Bonferroni to evaluate statistical significance between groups. If the normality or equal variance test was violated, a comparison was made using the non-parametric Mann–Whitney U-test. p-value < 0.05 was considered on the borderline of statistical significance. *p < 0.03 vs. kidney transplantation.
FIGURE 3
FIGURE 3
Comparison of miRNAs expression between the CKD4-5 and KT patients with a similar eGFR [“KT (CKD4-5 equivalent)]”, eGFR between 6 and 29 [ml/min/1.73m2]). (A) miRNA-126-3p, (B) miRNA-191-5p and (C) miRNA-223-3p. The data were analyzed using student’s t-test. Data are depicted as means ± SEM. p-value < 0.05 was considered on the borderline of statistical significance. *p ≤ 0.02.
FIGURE 4
FIGURE 4
Selected 5 microRNA ratios were analyzed in the whole cohort, including 45 CKD patients and 10 elderly subjects. (A) miRNA-126-3p/miRNA-363-3p, (B) miRNA-223-3p/miRNA-363-3p, (C) miRNA-223-3p/miRNA-495-5p, (D) miRNA-191-5p/miRNA-363-3p and (E) miRNA-191-5p/miRNA-495-5p. The data were analyzed using an ANOVA test and Post hoc Bonferroni to evaluate statistical significance between groups. p-value < 0.05 was considered on the borderline of statistical significance. *p < 0.05 vs. CKD4-5; †p < 0.05 vs. kidney transplantation.
FIGURE 5
FIGURE 5
Representative flow cytometry monocytes subsets. The results are represented as the means ± SEM. Percentage of (A) CD14++ CD16-/dim and (B) CD14+CD16++ monocytes in elderly patients, CKD4-5, hemodialysis and kidney transplantation patients. The data were analyzed using an ANOVA test and Post hoc Bonferroni to evaluate statistical significance between groups. p-value < 0.05 was considered on the borderline of statistical significance. *p < 0.05 vs. all the groups.
FIGURE 6
FIGURE 6
Representative histogram and quantification of microvesicles (MV/μl) in plasma by flow cytometry. The results are represented as the means ± SEM. (A) Number of microvesicles per microliter (Log Annexin V + MV) in elderly subjects, CKD4-5, hemodialysis and kidney transplantation patients. (B) Number of microvesicles per microliter (Log CD14 + Annexin V + MV) in elderly subjects, CKD4-5, hemodialysis and kidney transplantation patients. (C) Number of microvesicles per microliter (Log CD14 + Annexin V + CD16 + MV) in elderly patients, CKD4-5, hemodialysis and kidney transplantation patients. The data were analyzed using an ANOVA test and Post hoc Bonferroni to evaluate statistical significance between groups. p-value < 0.05 was considered on the borderline of statistical significance. *p ≤ 0.03 vs. elderly subjects; †p < 0.001vs. all groups.
FIGURE 7
FIGURE 7
Correlation between proinflammatory monocytes (% CD14+CD16++) and microvesicles (Log CD14 + Annexin V + CD16 + MV).
FIGURE 8
FIGURE 8
Microvesicles derived from uremic toxins-treated THP-1 induces senescence of vascular smooth muscle cells. Senescent-associated β-Gal activity staining of different of MV. (A–C) Representative images of inverted optical microscopy of the senescence studies of (A) control VSMC, (B) VSMC treated with cnMV and (C) txMV. (D) Quantification Total Area β-Gal/count and (E) Average size (μm) of β-Gal positive cells. (F) Expression level of cyclin D1was measured in VSMC. The data were analyzed using an ANOVA test and Post hoc Bonferroni to evaluate statistical significance between groups. If the normality or equal variance test was violated, a comparison was made using the non-parametric Mann–Whitney U-test. Data are the means ± SEM of six independent experiments. *p < 0.001 vs. VSMC; †p < 0.001 vs. VSMC + cnMV; ‡p = 0.009 vs. VSMC; #p = 0.015; &p = 0.01 vs. VSMC + cnMV.
FIGURE 9
FIGURE 9
Expression levels of BMP2 and miRNA-223-3p in vascular smooth muscle cells (VSMC). Histogram of (A) BMP2 mRNA and (B) miRNA-223-3p levels were measured in VSMC. txMV induces differential expression of BMP2 and miRNA-223-3p in VSMC. The data were analyzed using an ANOVA test and Post hoc Bonferroni to evaluate statistical significance between groups. Data are the means ± SEM of six independent experiments. *p < 0.02 vs. VSMC.
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