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. 2013 May 22;8(5):e64558.
doi: 10.1371/journal.pone.0064558. Print 2013.

Decreased microRNA is involved in the vascular remodeling abnormalities in chronic kidney disease (CKD)

Neal X Chen  1 , Kraiwiporn Kiattisunthorn, Kalisha D O'Neill, Xianming Chen, Ranjani N Moorthi, Vincent H Gattone 2nd, Matthew R Allen, Sharon M Moe
Affiliations

Decreased microRNA is involved in the vascular remodeling abnormalities in chronic kidney disease (CKD)

Neal X Chen et al. PLoS One. .

Abstract

Patients with CKD have abnormal vascular remodeling that is a risk factor for cardiovascular disease. MicroRNAs (miRNAs) control mRNA expression intracellularly and are secreted into the circulation; three miRNAs (miR-125b, miR-145 and miR-155) are known to alter vascular smooth muscle cell (VSMC) proliferation and differentiation. We measured these vascular miRNAs in blood from 90 patients with CKD and found decreased circulating levels with progressive loss of eGFR by multivariate analyses. Expression of these vascular miRNAs miR-125b, miR-145, and miR-155 was decreased in the thoracic aorta in CKD rats compared to normal rats, with concordant changes in target genes of RUNX2, angiotensin II type I receptor (AT1R), and myocardin. Furthermore, the expression of miR-155 was negatively correlated with the quantity of calcification in the aorta, a process known to be preceded by vascular de-differentiation in these animals. We then examined the mechanisms of miRNA regulation in primary VSMC and found decreased expression of miR-125b, 145, and 155 in VSMC from rats with CKD compared to normal littermates but no alteration in DROSHA or DICER, indicating that the low levels of expression is not due to altered intracellular processing. Finally, overexpression of miR-155 in VSMC from CKD rats inhibited AT1R expression and decreased cellular proliferation supporting a direct effect of miR-155 on VSMC. In conclusion, we have found ex vivo and in vitro evidence for decreased expression of these vascular miRNA in CKD, suggesting that alterations in miRNAs may lead to the synthetic state of VSMC found in CKD. The decreased levels in the circulation may reflect decreased vascular release but more studies are needed to confirm this relationship.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Circulating miRNA levels in controls, CKD patients and hemodialysis patients in freshly isolated samples.
Sera were collected from stage 3–4 CKD patients (n = 10), hemodialysis patients (n = 10) and healthy volunteers (n = 8) and total RNA isolated and real time PCR performed to determine the expression of circulating levels of miR-125b (A), miR-145 (B) and miR-155 (C) normalized by U6. Total serum miRNA concentration in the three groups was measured using Agilent Bioanalyzer (D) to demonstrate that the proportion of miRNA to total RNA analyzed is not the etiology of decreased expression of these specific miRNA. Each sample was assayed in triplicate. Data were expressed as mean ± SEM. * p<0.01 compared to healthy volunteers; ** p<0.05 compared to healthy volunteers.
Figure 2
Figure 2. Decreased expression of miRNAs and downstream genes in thoracic aorta from CKD animals.
Thoracic aorta were collected at 35 weeks of age from CKD (n = 8) and normal rats (n = 8) and total RNA was isolated and miRNA expression determined by real time PCR and normalized by U6. The results demonstrated that there is significantly reduction in expression in miR-125b, miR-145, and in thoracic aorta from CKD compared to that from normal rats (Figure 2A). Aortic calcification was determined biochemically and results demonstrated that aorta calcification is increased in CKD rats and the greater the calcification, the lower the miR-155 expression level (r = 0.54, p = 0.04; Figure 2B). Real time PCR was also performed to determine the expression of several target genes and normalized by β-actin. The results demonstrated that the expression of AT1R (Figure 2C) and RUNX2 (Figure 2E) is increased whereas myocardin expression is decreased (Figure 2D) in aorta from CKD rats compared to normal rats, corresponding to known physiologic roles of these vascular miRNA. Data were expressed as mean ± SEM. * p<0.05, CKD vs. normal.
Figure 3
Figure 3. The Expression of vascular microRNAs and target genes in VSMCs from normal and CKD rats.
Rat VSMC isolated from normal or CKD rats were cultured in growth media for 4 days and total RNA isolated. Real time PCR was performed to determine the expression of miR-125b, miR-145, miR-155 and miR-210 and normalized by U6 (A). The expression of myocardin (B), RUNX2 (C) and AT1R (D) was also determined by real time PCR and normalized by β-actin. Each sample (n = 9 with cells isolated from 3 different normal or CKD animals) was assayed in triplicate. Data were expressed as mean ± SEM. * p<0.05, CKD vs. normal.
Figure 4
Figure 4. Overexpression of miR-155 and AT1R expression in VSMC from CKD rats.
Rat VSMC isolated from CKD rats were transfected with 30 nM of miR-155 mimic or miR negative control for 48 hrs. Non-transfected VSMC (NT) was also used as control. The overexpression of miR-155 in VSMC was confirmed by real time PCR (A). The miR-155 target gene AT1R expression in VSMC was also determined by real time PCR, demonstrating a significant inhibition of AT1R expression compared to that with negative control or non-transfected VSMC (B). Data were expressed as mean ± SEM (n = 3 separate experiments). *p<0.05, miR-155 mimic vs. negative control or NT.
Figure 5
Figure 5. Upregulation of miR-155 decreases cellular proliferation in VSMC from CKD rats.
Rat VSMC isolated from CKD rats were transfected with 30 nM of miR-155 mimic or miR negative control. Non-transfected VSMC (NT) was also used as control. The cellular proliferation was determined 48, 72 and 96 hrs after transfection using Cell Titer 96 Proliferation Assay Kit. The results demonstrated that the transfection of miR-155 mimic significantly inhibited cellular proliferation at 72 and 96 hrs in VSMC from CKD rats compared to that with negative control or non-transfected VSMC. Data were expressed as mean ± SEM (n = 3 separate experiments). *p<0.05, miR-155 mimic vs. negative control or NT.
Figure 6
Figure 6. Hypothesis of the impact of vascular miRNAs on the pathogenesis of cardiovascular disease of CKD.
This figure is our hypothesis of how the miRNAs may affect cardiovascular disease in CKD. During development, normal differentiation of VSMC is controlled in part by the ‘master’ regulator, myocardin. In adulthood, the majority of VSMC are in a quiescent, synthetic state. During acute insults, these synthetic VSMC change to a more proliferative state, returning to a quiescent state after the insult. However, in the setting of kidney disease, VSMC appear to stay in a more proliferative state with corresponding increased expression of AT1R and RUNX2, and decreased myocardin expression. The decreased expression forces the VSMC to remain in a continual proliferative or de-differentiated state. MiRNAs are known to be important in regulating such differentiation during development, but the low levels of miR-155, 145, and 125b observed in CKD arteries and VSMC and in the circulation of patients with CKD in the present study may lead to further propagation of de-differentiated VSMC, potentiating the development of hypertension, cardiovascular disease and vascular calcification.
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