Circulating and renal vein levels of microRNAs in patients with renal artery stenosis

Abstract

Background

MicroRNAs (miRs) are small non-coding RNAs that are important regulators of gene expression and have been implicated in atherosclerosis. Kidney injury distal to atherosclerotic renal artery stenosis (ARAS) is aggravated by atherosclerosis. Therefore, this study tested the hypothesis that renal miR expression would be altered in patients with ARAS.

Methods

Patients with essential hypertension (EH; n = 13) or ARAS (n = 13) underwent a 3-day protocol study under controlled conditions. For miR levels, blood samples were collected from EH and ARAS renal vein (RV) and inferior vena cava or peripheral vein of matched normotensive healthy volunteers (HV; n = 13) and patients with coronary atherosclerosis (CA; n = 11). Single-renal blood flow was measured in EH and ARAS using computer tomography to calculate renal gradients and release of miRs.

Results

Glomerular filtration rate (GFR) was lower in ARAS compared with the other groups. Systemic levels of most miRs were elevated in CA. RV miR levels were lower than systemic levels in both ARAS and EH. GFR-adjusted RV levels of miR-21, 155 and 210 were reduced only in ARAS patients compared with systemic levels in HV, although cross-kidney gradients were not different from EH. RV levels of miR-21, 126, 155 and 210 correlated with GFR.

Conclusions

Levels of atherosclerosis-related miR-21, 126, 155 and 210 are decreased in the stenotic-kidney vein of ARAS compared with EH patients, likely due to decreased GFR. Yet, these miRs might be implicated in modulating renal injury in ARAS, and their RV level may be a marker reflecting their renal expression.

INTRODUCTION

Atherosclerotic renal artery stenosis (ARAS) accounts for ∼90% of cases of renal occlusive vascular disease, and its prevalence increases with age, particularly in patients with diabetes, hyperlipidemia, diffuse atherosclerosis or hypertension [1]. ARAS results in a progressive loss of renal mass and function, and up to 27% of patients will develop chronic renal failure within 6 years [2]. Atherosclerosis elicits microvascular and macrovascular dysfunction as well as tissue structural remodeling, which likely activate similar mechanisms, interact and often exacerbate renal injury [3]. Their pathophysiological mechanisms include oxidative stress and inflammation, with several downstream sequences of feed-forward interactions, activating transcription factors that lead to vascular, tubulointerstitial and glomerular injury. Moreover, atherosclerosis may blunt intrinsic defense mechanisms designed to preserve renal structural integrity, and thereby facilitate renal scarring [3].

MicroRNAs (miRs) are small noncoding RNAs that post-transcriptionally regulate gene expression by binding to the target mRNA and play a critical function during development through effects on cell proliferation, differentiation, apoptosis, carcinogenesis, angiogenesis and hematopoiesis [4, 5]. Recent studies have demonstrated dysregulated expression of miRs in the vascular wall in patients and animal models of atherosclerosis, implicating several miRs in pivotal mechanisms regulating the progression of disease [6–9]. Accumulating evidence also suggests that miRs have an important role in controlling blood pressure, kidney injury and progression of chronic kidney disease (CKD) [10–12].

In recent years, miRs previously identified in specific tissues were also detected in extracellular fluids including plasma and serum and have been proposed to be potential biomarkers for a range of diseases, particularly given their stability in plasma and the availability of quantitative methods such as real-time polymerase chain reaction (PCR) [13, 14]. Several miRs have been implicated in atherosclerosis [6, 9, 15]. In the atherosclerotic lesion, miR-21 may protect endothelial function and increase nitric oxide (NO) synthesis, although it has also been suggested to promote atherosclerosis by inducing oxidative stress and inhibiting apoptosis in vascular smooth muscle cells [15] and has been implicated in the development of fibrosis [16]. miR-126 and miR-155 act as negative modulators of vascular inflammation and progression of atherosclerosis, and their systemic levels are down-regulated in patients with coronary artery disease [8, 15]. miR-210 is up-regulated in hypoxic atherosclerotic plaques, promotes angiogenesis and plaque progression and its systemic levels are increased in patients with atherosclerosis [9, 17]. Originally considered to be brain-specific, miR-124 possesses anti-inflammatory properties and is up-regulated in aortic atherosclerotic tissue but down-regulated in the plasma of the same patients [18–20].

Circulating levels of miRs may also be reduced in patients with impaired kidney function, although the underlying mechanism is unclear [21]. We have previously shown increased levels of inflammatory cytokines and decreased numbers of endogenous endothelial cells in the vein draining the stenotic human kidney [22]. Given the prominent injury and decreased function in the post-stenotic kidney, it is not unlikely that the expression of protective miR is reduced in its venous effluent.

However, neither the systemic nor renal levels of miRs in patients with ARAS and EH have been previously shown. Therefore, this study was designed to test the hypothesis that circulating levels of miRs would be lower in patients with ARAS compared with patients with essential hypertension (EH) or to normotensive controls.

MATERIALS AND METHODS

Patient populations

Patients identified with EH (n = 13) or ARAS (n = 13) were prospectively enrolled from August 2008 to October 2010. Informed written consent was obtained after receiving approval from the Institutional Review Board of the Mayo Clinic (07-005156). ARAS was defined using entry criteria analogous to enrollment in Cardiovascular Outcomes for Renal atherosclerotic Lesions (CORAL) trial [7]. Imaging criteria included renal artery Doppler ultrasound velocity acceleration (peak systolic velocity>200 cm/s), or MR/CT angiography with evident stenosis >60% and/or post-stenotic dilation. Exclusion criteria included estimated glomerular filtration rate (eGFR, modification of diet in renal disease) <30 mL/min/1.73 m² (because of subsequent use of contrast media), uncontrolled hypertension [systolic blood pressure (SBP) >180 mmHg, despite antihypertensive therapy], diabetes requiring medications, recent cardiovascular event (myocardial infarction, stroke, congestive heart failure within 6 months), pregnancy and kidney transplant. Patients were admitted to the Clinical Research Unit (CRU) for 3 days. Dietary intake of sodium (150 mEq) was controlled throughout the duration of the study and samples collected under controlled conditions. Hypertensive patients underwent imaging studies during their CRU stay. Antihypertensive medications including angiotensin-converting enzyme inhibitor or angiotensin receptor blocker were continued. Normotensive healthy control subjects [SBP <130 and diastolic blood pressure (DBP) <80 mmHg] were prospectively recruited through the Mayo Clinic Biobank. In addition, patients identified during coronary angiography with early and non-obstructive (<40%) coronary atherosclerosis (CA) [23] but without ARAS were enrolled and matched to EH and ARAS according to age, weight and body mass index (BMI).

Clinical data collection and laboratory measurement

Clinical data collected by physical examination or via the electronic medical records included age, sex, height, weight, BMI and use of concomitant medications. Systolic, diastolic and mean arterial pressures were arranged from three consecutive measurements taken at the CRU. Serum creatinine, eGFR, low-density lipoprotein, high-density lipoprotein, total cholesterol and triglyceride levels were determined by standard procedures.

Blood sampling and measurement of renal blood flow

Blood samples were obtained in hypertensive patients prior to measurement of renal blood flow (RBF) by multi-detector computer tomography (MDCT). In brief, a guide catheter was placed via the femoral or internal jugular vein (using a 6F sheath) and blood samples were drawn from the right and left renal vein (RV) and inferior vena cava (IVC) with a 5F pigtail Cobra catheter (Cook, Inc., Bloomington, IN, USA). Levels of miR, neutrophil gelatinase-associated lipocalin (NGAL) and inflammatory cytokines were obtained from the RV and IVC of all ARAS and EH patients, and from a peripheral (antecubital) vein in healthy volunteers (HV) and CA patients. The catheter was then moved to the superior vena cava for contrast media injections during imaging. Single-kidney RBF was measured in ARAS and EH patients using MDCT (Somatom Sensation-64, Siemens Medical Solutions, Germany) [22, 24]. Images (45 consecutive scans) were obtained after central venous injection of iopamidol-370 (0.5 mL/kg, up to 40 mL and 15 mL/s) using a power injector. Single-kidney RBF was calculated by multiplying kidney volume by renal perfusion (milliliters per minute per milliliter of tissue), as we have previously detailed [25]. Perfusion was calculated from time attenuation curves obtained in the kidney after contrast injection, and cortical and medullary volumes calculated with stereology. Single-kidney volume was obtained by the summing cortical and medullary volumes.

Measurement of miRs with quantitative real-time PCR

Total RNA was isolated from 400 μL human plasma samples by the mirVana PARIS total RNA isolation kit (Life Technologies, Cat. # AM1556) according to the kit protocol. In the absence of established endogenous small RNA control for plasma samples, we spiked 25 fmol of cel-mir-39 (Life Technology, Cat. # 4464066) into each sample just after adding a 2× denaturing solution. A fixed volume of 1.5 μL of RNA elute was reverse transcribed by using the TaqMan MicroRNA reverse transcription kit (Life Technologies, Cat. # 4366596). For PCR, 1.33 μL of RT product was combined with 10 μL of TaqMan universal master mix (Cat. # 4440038), 7.67 μL of H₂O and 1 μL of primers, including miR-21, miR-124a, miR-126, miR-155 and miR-210 (Life Technologies, Cat. # 4427975, assay ID: 000397, 001182, 000451, 002623 and 000512, respectively), to make up a 20 μL reaction. Real-time PCR was carried out on an Applied Biosystems ViiA7 Real-Time PCR system at 50°C for 2 min, 95°C for 10 min and 40 cycles of 95°C for 15 s and 60°C for 1 min. Fold changes of miR levels in ARAS and EH patients relative to Biobank patients were calculated using the 2^−ΔΔCt method.

In some experiments, miR mimics were run together with the samples and used to generate standard curves, and calculate absolute numbers of copies of target genes in the samples. These mimics included (all purchased from Life Technology): miR-21 (MC10206), miR-124 (MC10691), miR-126 (MC10401), miR-155 (MC12601) and miR-210 (MC10516). We then normalized these data using a median normalization procedure [14]. Briefly, the median values obtained from cel-mir-39 Ct values of all the samples were used to calculate the normalization factor = 2^{(median cel-mir-39 Ct value − average Ct value of the given sample)}. The number of copies of a given miR in each sample was then divided by the normalization factor to generate the normalized copies of genes in each sample.

Based on the assumption that the difference between infra-renal IVC and RV levels reflects the net release of miR within the affected kidney, we estimated a renal miR gradient (RV-IVC) and the net release (gradient × RBF) for each measured product [22].

Measurement of plasma cytokines

For tumor necrosis factor (TNF)-α, 25 μL of plasma samples were used and measured by human cytokine/chemokine Luminex kit from Millipore (Cat. # MPXHCYTO-60K). Samples for NGAL were diluted 1 : 500 before being added to wells, and NGAL levels (ng/mL) tested by enzyme-linked immunosorbent assay (BioPorto Diagnostics, Cat. # KIT 036), as we have shown before [26].

Statistical analysis

Results were expressed as mean ± SD and presented with a bar chart for normally distributed data, or median (interquartile range) and box plot for non-normally distributed data. Comparisons between independent groups were performed using two-sample t-test with unequal variance (or the Wilcoxon rank-sum test for skewed data) and a χ²-test or Fisher's exact test for categorical variables as appropriate. Comparisons among four groups were performed using analysis of variance (ANOVA). For adjusting level of miRs by GFR, we used analysis of covariance (ANCOVA). For performing ANCOVA, skewed data were transformed to logarithmic values. We used non-parametric paired (Wilcoxon signed-rank) tests for comparing bilateral RV levels of miRs in patients with ARAS. Spearman rank correlation analysis was used to test for associations between miRs and other variables with adjustment for GFR. All tests were two-tailed, and P-values ≤0.05 were considered statistically significant.

RESULTS

Patient characteristics

Table 1 summarizes the characteristics of patients included in this study. Nine of thirteen patients with ARAS had bilateral stenoses, and the more severe side was considered as ARAS, while the unaffected or not severely affected side was considered as contra-lateral kidney (CLK). SBP was similarly elevated in ARAS and CA compared with HV, but not different from EH, which also tended to be higher than in HV (P = 0.09). There were no differences in antihypertensive regimens between patient groups, except for the lower rate of angiotensin blockade in CA. ARAS patients tended to have more coronary (stenosis ≥50%) or peripheral (other than renal) arterial disease than EH patients. Serum creatinine levels were elevated and eGFR reduced in ARAS patients compared with EH, CA and HV. Single-kidney RBF was also lower in the stenotic compared with EH kidney. Otherwise, urinary protein excretion did not differ among the groups.

Table 1.

Clinical, laboratory and demographic data in HV and patients with ARAS or EH (n = 13 each group), or early CA (n = 11)

Parameters	HV	EH	CA	ARAS	P-valuea	P-valueb	P-valuec
Age, years	70.1 ± 6.9	68.9 ± 6.4	67.5 ± 5.4	70.6 ± 6.4	0.9	0.9	0.2
Men, n (%)	5 (38.5)	7 (53.9)	2 (18.2)	9 (69.2)***	0.7	0.2	0.02
Body weight, kg	75.0 ± 18.1	80.7 ± 15.8	81.1 ± 23.3	83.9 ± 16.7	0.9	0.6	0.7
BMI, kg/m2	25.5 ± 4.6	27.9 ± 3.7	29.5 ± 6.8	28.6 ± 3.8	0.9	0.6	0.6
SBP, mmHg	120.9 ± 10.1	131.2 ± 18.6	146 ± 14.4**	142.1 ± 19.1**	0.3	0.007	0.5
DBP, mmHg	70.5 ± 8.0	70.5 ± 13.9	74.2 ± 10.1	68.4 ± 9.6	0.9	0.9	0.7
Mean blood pressure, mmHg	87.3 ± 7.8	90.7 ± 13.3	100.6 ± 11.5**	92.9 ± 11.7	0.9	0.6	0.8
Use of concomitant medication, n (%)
ACEI/ARB		13 (100)	3 (27.3)*	13 (100)***	0.2		<0.001
CCB		4 (30.8)	4 (36.4)	6 (46.2)	0.7		0.8
β-blocker		8 (61.5)	5 (45.5)	10 (76.9)	0.7		0.2
Statins		8 (61.5)	8 (72.7)	10 (76.9)	0.7		0.9
Laboratory measures
Total cholesterol, mg/dL	186.5 ± 30.8	180.9 ± 33.1	202.1 ± 56.1	181.6 ± 32.2	0.9	0.9	0.2
Triglycerides, mg/dL	103.5 ± 3.25	128.1 ± 42.2	145.1 ± 81.3	140.6 ± 77.8**	0.4	0.04	0.6
High-density lipoprotein, mg/dL	64.2 ± 13.4	49.9 ± 13.8	53.9 ± 13.6	50.1 ± 23.6	0.9	0.2	0.6
Low-density lipoprotein, mg/dL	101.5 ± 29.1	105.4 ± 23.0	119.3 ± 44.3	99.1 ± 21.0	0.9	0.9	0.1
Renal function
Serum creatinine, mg/dL	0.9 ± 0.2	1.0 ± 0.3	0.8 ± 0.1	1.5 ± 0.3*,**,***	0.001	<0.001	<0.001
eGFR, mL/min per 1.73 m2	73.2 ± 13.2	73.4 ± 24.6	78.9 ± 15.8	48.1 ± 10.3*,**,***	0.003	0.003	<0.001
24 h urine protein, mg/day	70 (52–112)	51 (47–62)		84 (59–110)	0.2	0.9
Single RBF, mL/min		369.1 ± 171.1		198.5 ± 102.6*	0.007

Values are expressed as mean ± SD, or median (25–75%).

BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; ACEI, angiotensin-converting enzyme inhibitor; ARB, angiotensin receptor blocker; CCB, calcium channel blocker; eGFR, estimated glomerular filtration rate; RBF, renal blood flow.

^aP-value ARAS versus EH, ^bP-value ARAS versus HV, ^cP-value ARAS versus CA.

*P ≤ 0.05 versus EH, **P ≤ 0.05 versus HV, ***P ≤ 0.05 versus CA.

We also analyzed complications of long-lasting uncontrolled hypertension, including left ventricular hypertrophy (LVH) in ARAS and EH. In ARAS, only one patient had LVH, and two patients in EH, suggesting comparable rates. Eye examination was documented in only four patients (none of whom had any evidence of hypertensive changes).

Table 2 summarizes inflammatory and tubular injury makers. In ARAS patients, systemic levels of TNF-α (P = 0.001 and P = 0.003, respectively) and NGAL (P < 0.02 and P = 0.004, respectively) were higher than in both EH and HV, and their RV and CLK levels were similarly elevated compared with EH RV levels. Systemic levels of TNF-α and NGAL in CA were also higher than in EH.

Table 2.

Cytokine levels measured in HV and patients with ARAS, EH or CA

Parameters	HV (n = 13)	EH (n = 13)		CA (n = 11)	ARAS (n = 13)
	Systemic	Systemic	RV	Systemic	Systemic	RV	CLK
TNF-α (pg/mL)	4.4 (3.2–5.5)	3.1 (1.9–6.3)	3.4 (2.4–6.1)	8.6 (5.2–10.4)*,**	9.4 (6.8–11.1)*,**	9.3 (6.9–11.5)*	11.5 (7.2–14.7)*
NGAL (ng/mL)	65.9 ± 27.1	78.2 ± 66.3	71.0 ± 39.3	124.2 ± 8.2*,**	141.7 ± 132.5*,**	147.9 ± 43.6*	146.9 ± 11.8*

Values are expressed as mean ± SD, median (25–75%).

ARAS, atherosclerotic renal artery stenosis; EH, essential hypertension; HV, healthy volunteers; CA, coronary atherosclerosis; TNF, tumor necrosis factor; NGAL, neutrophil gelatinase-associated lipocalin; RV, renal vein; IVC, inferior vena cava.

*P ≤ 0.05 versus EH, **P ≤ 0.05 versus HV.

Circulating miRs

Systemic levels of miR-21, miR-126, miR-155 and miR-210 were elevated or tended to increase in CA compared with the other groups, whereas miR-124a level was reduced in CA compared with EH and ARAS (Figure 1), and miR-126 in EH was higher than in HV (Figure 1, P = 0.03). RV levels of miR-21, miR-126, miR-155 and miR-210 were reduced in the stenotic (but not CLK) ARAS kidney compared with EH (Figure 1, all P ≤ 0.03), and RV miR-21 and miR-124a levels were reduced in the stenotic ARAS kidney compared with the CLK. RV levels of miR-124a in EH were lower than in the systemic circulation in HV (P = 0.02).

FIGURE 1:

Levels of miRs (unadjusted for GFR) in the systemic circulation of HV, EH, CA and ARAS patients, and in the RV of EH, stenotic kidney (ARAS) and CLK. RV level of miR-21 was decreased in the ARAS kidney compared with EH and CLK and to systemic levels in HV. RV level of miR-124a was also reduced in the ARAS kidney compared with the CLK, and in EH kidneys compared with systemic levels in HV. RV levels of miR 126, 155 and 210 were significantly decreased in ARAS compared with EH or HV. Systemic levels all miRs in CA were higher than in EH and ARAS, whereas in EH only systemic level of miR-126 was significantly increased compared with HV. *P ≤ 0.05, #P ≤ 0.05 versus systemic levels of HV.

Figure 2 shows the levels of miRs after GFR adjustment. RV levels of miR-21, miR-124a, miR-126, miR-155 and miR-210 were no longer different between ARAS and EH (P = 0.3, P = 0.2, P = 0.5, P = 0.4 and P = 0.1, respectively), but RV levels of miR-21, miR-155 and miR-210 remained reduced in ARAS (but not in EH) compared with their systemic level in HV (P = 0.03, P = 0.04 and P = 0.005, respectively). RV level of miR-124a in EH was significantly lower than in HV (P = 0.008), while systemic level of miR-126 in ARAS, CA and EH were increased compared with HV (P = 0.03 and P = 0.002, respectively). RV level of miR-21 and miR-124a remained decreased in ARAS kidney compared with CLK (P = 0.04 and P < 0.001, respectively). Systemic miR-155 and miR-21 level in CA remained elevated compared with other groups, and their level of miR-124a tended to be reduced compared with EH and ARAS (P = 0.08 and P = 0.09, respectively).

FIGURE 2:

Levels of miRs adjusted for GFR in all groups. RV level of miR-21, 155 and 210 were significantly decreased in the stenotic ARAS kidney compared with HV, and miR-21 and miR-124a compared with the CLK. RV level of miR-124a was significantly decreased in EH compared with HV. Systemic level of miR-126 was increased in all patient groups compared with HV. *P ≤ 0.05, #P ≤ 0.05 versus systemic levels of HV.

RV levels of miR-21, miR-124a and miR-210 were significantly reduced in the stenotic ARAS kidney compared with their levels in the CLK (P = 0.006, P = 0.004 and P = 0.05, respectively), and level of miR-155 tended to decrease (P = 0.08).

Renal release of miRs

In both ARAS and EH, levels of all miRs were consistently lower in the stenotic RV than in the IVC of the same patient, with the exception of RV levels of miR-210 (Figure 3) in EH. Unadjusted or adjusted gradient and net release of miR-124a were elevated in CLK compared with EH and stenotic kidney, and unadjusted gradient miR-21 in CLK was also higher than stenotic kidney. However, none of these miRs were different between ARAS and EH, whether unadjusted (Figure 3) or adjusted (Figure 4) for GFR.

FIGURE 3:

GFR-unadjusted gradients and net release of miRs were not significantly different between patients with EH and ARAS. Gradient of miR-21was increased in CLK compared with stetenotic kidney, and both gradient and net release of miR-124a in CLK were higher than EH and stenotic kidney.

FIGURE 4:

GFR-adjusted gradients and net release of miRs were not significantly different between EH and ARAS patients. However, gradient and net release of miR-124a remained elevated in CLK compared with other groups, after adjustment.

Factors influencing circulating level of miRs

Table 3 summarizes the correlation between unadjusted miRs levels and other variables in all groups. Age or gender was not associated with these miRs. SBP was inversely correlated with RV levels of miR-21, 126, 155 and 210 and eGFR significantly correlated with them directly. RV level of miR-124a was not correlated with blood pressure or renal function. RV levels of miR-21, 126 and 155 inversely correlated with NGAL and TNF-α.

Table 3.

Bivariate correlation analysis between miR (copy number) and variables

Variables	miR-21 (RV)		miR-21 (systemic)		miR-124a (RV)		miR-124a (systemic)		miR-126 (RV)		miR-126 (systemic)		miR-155 (RV)		miR-155 (systemic)		miR-210 (RV)		miR-210 (systemic)
	r	P-value	r	P-value	r	P-value	r	P-value	r	P-value	r	P-value	r	P-value	r	P-value	r	P-value	r	P-value
Age		0.3		0.8		0.9		0.8		0.4		0.7		0.5		0.9		0.9		0.9
Gender		0.2		0.06		0.8		0.1		0.1		0.2		0.2		0.09		0.1		0.3
BMI		0.5		0.4	−0.37	0.02		0.3		0.9		0.06		0.4		0.1		0.1		0.9
SBP	−0.49	0.001		0.9		0.3		0.08	−0.43	0.006		0.5	−0.46	0.003		0.6	−0.42	0.008		0.6
DBP		0.9		0.8		0.9		0.3		0.8		0.7		0.6		0.9		0.9		0.6
Total cholesterol	0.33	0.04		0.8		0.3		0.5		0.06		0.5		0.7		0.2		0.2		0.2
TG		0.4		0.9		0.1		0.5		0.9		0.2		0.9		0.1		0.1		0.6
HDL		0.07		0.9	0.34	0.04		0.9		0.2		0.3		0.1		0.6	−0.37	0.02		0.9
LDL	0.36	0.03		0.8		0.5		0.4		0.06		0.1		0.07		0.3		0.3		0.2
eGFR (MDRD)	0.45	<0.001	0.37	0.008		0.1		0.8	0.49	0.002	0.35	0.01	0.58	<0.001	0.42	0.003	0.36	0.02	0.34	0.02
RBF		0.2		0.9		0.8		0.8		0.2		0.5	0.41	0.05		0.4		0.9		0.7
NGAL (RV)	−0.59	0.002		0.6		0.6		0.7	−0.54	0.005		0.2	−0.63	0.001		0.3		0.06		0.5
NGAL (IVC)	−0.44	0.03	0.42	0.02		0.6		0.8		0.3	0.35	0.04		0.06		0.1		0.3	0.46	0.006
TNF-α (RV)	−0.46	0.02		0.3		0.4		0.7	−0.61	0.01	−0.48	0.02	−0.64	0.001	−0.49	0.01		0.4		0.3
TNF-α (IVC)		0.2		0.2		0.3		0.4		0.1		0.2	−0.39	0.02		0.2		0.3	0.34	0.02

BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; TG, triglyceride; HDL, high-density lipoprotein; LDL, low-density lipoprotein; eGFR, estimated glomerular filtration rate; MDRD, modification of diet in renal disease; NGAL, neutrophil gelatinase-associated lipocalin; TNF-α, tumor necrosis factor-α.

After adjustment for GFR using analysis of partial correlation, none of these miRs correlated with other clinical or laboratory variables (data not shown).

DISCUSSION

This study shows that when measured under well-controlled conditions, levels of several miRs, postulated to play protective roles in atherosclerotic disease, are decreased in patients with ARAS compared with the CLK, to patients with EH, and to healthy controls. These observations might implicate miRs in the pathogenesis of renal disease in ARAS. However, these decreases largely disappeared after adjustment for GFR, suggesting that they were driven mainly by reduced renal function.

Because circulating miRs might be correlated with the local miR expression signature in a specific pathology, miRs can be useful clinical biomarkers [27]. In atherosclerosis, circulating miRs released from the lesion may reflect their expression at the tissue [9], yet their uptake into an atherosclerotic lesion might reduce their circulating level, despite up-regulated tissue expression [20]. These previous reports emphasize the importance of distinguishing these expression sites and imply that plasma level could depend on the sampling site or circumstances. In this study, we could not evaluate expression of miRs in the tissue, but measured their plasma level in the RV. Indeed, discordant RV and systemic levels of miRs may suggest that differential RV level might be a more sensitive marker for local expression or uptake of miRs in the kidney.

In this study, GFR-unadjusted levels of some miRs in patients with ARAS differed from their levels in patients with EH or with non-renal atherosclerosis. Apart from miR-124a, RV levels of all miRs were lower in the stenotic ARAS kidney compared with the EH kidney and systemic levels in HV. Of these, miR-126 and miR-155 have been shown to be protective against progression of atherosclerosis [8, 15]. Furthermore, levels of both miR-124a and miR-21, which is considered to have a bi-directional effect on progression of atherosclerosis, were reduced in stenotic compared with the less affected kidney. In fact, miR-124a levels were elevated in the CLK compared with EH. Interestingly, miR-124a plays a role in regulation of inflammation [19] and may participate in regulation of blood pressure by attenuating the renin–angiotensin–aldosterone system, but RV level did not correlate with GFR, suggesting that factors other than GFR could affect its expression. Notably, prominent differences in RV rather than systemic levels between groups suggest that miRs may act locally, either on the atherosclerotic lesion or within the kidney, and that their renal expression might manifest better in the RV rather than in the systemic circulation levels.

Interestingly, the differences in these miR levels between the two groups were abolished after adjustment for GFR, indicating that renal function may affect their expression. Moreover, with the exception of miR-124a, the RV level of all measured miRs correlated directly with eGFR, supporting the link between renal function and miR levels. Indeed, although previously reported to be up-regulated in atherosclerosis [9], in this study RV miR-210 levels were decreased in ARAS, possibly because of their decreased renal function. While in CKD with severely reduced renal function, accumulated enzymes such as RNases may increase degradation of miRs [21, 28], the mechanism for the decreased levels of miRs in these patients remains unclear, especially given the modest impairment in renal function in our patients. However, the difference between their levels in the stenotic kidney and CLK may implicate local mechanisms in regulation of some miRs in ARAS, and that reduced expression or increased uptake of some miRs is more prominent in the stenotic kidney.

miR-126 regulates the function of endothelial cells and maintains the vascular integrity and angiogenesis [29, 30], inhibits leukocyte adherence and blunts progression of atherosclerosis [31]. In contrast, circulating levels of miR-126 increased in patients with acute coronary syndromes, because of systemic endothelial activation [32]. We observed similar systemic levels of miR-126 in EH, CA and ARAS, which were all significantly elevated compared with levels in healthy controls, possibly due to systemic hypertension. Previous studies reported conflicting effects of miR-21 and miR-155 on atherosclerosis. miR-21 may play a protective role in tissue injury [33], although it may conversely induce neointimal formation [15]. miR-155 may affect vascular remodeling, down-regulates the angiotensin II type-1 receptor and suppress inflammatory and immune reaction in the kidney [34–36]. Inflammation has been known as an important pathway that mediates deleterious processes in the stenotic kidney, and TNF-α tissue expression and release are up-regulated in ARAS [37, 38]. The reduced levels of miR-21 and miR-155 that we found in the RV of our ARAS patients and their inverse correlation with NGAL and TNF-α may indicate that a reduction in their levels is permissive for renal injury in ARAS. miR-210 may promote angiogenesis and cell survival [39], but also atherosclerotic plaque progression [40]. As also observed in this study, its levels are elevated in patients with arteriosclerosis [9] and acute kidney injury [12]. Interestingly, we found a decreased RV level of miR-210 in the stenotic ARAS kidney compared with EH and HV, suggesting attenuation of this defense mechanism, which might promote injury of the affected kidney. Despite its attenuation by GFR adjustment, decreased levels in the stenotic kidney RV might imply some roles in ARAS. Possibly, diminished expression of these miRs leads to suppression of protective effects, and thereby promotes vascular and renal injury. In addition, decreased expression of miRs in the injured kidney might be partly responsible for differences in circulating levels of miRs in patients with CKD. Notably, the correlation with different cytokines was likely also mediated by the impact of GFR.

Importantly, systemic levels of most miRs were significantly elevated in patients with CA compared with HV, whereas only miR-126 was also elevated in EH and ARAS. These observations suggest that the decreased levels observed in ARAS patients are unlikely to result from the atherosclerotic process alone.

This study had several limitations. First, the cost and comprehensive nature of our studies (3-day CRU protocol, dietary intervention, CT scanning, exposure to radiation and contrast media) precluded a larger-scale study. For a similar reason, we could not define cause–effect or evaluate the effects of miRs on clinical outcomes, which were also not our goal. Second, kidney tissue was unavailable, and renal expression of miRs might differ from their plasma level. Third, GFR levels were significantly decreased in ARAS compared with EH, CA or healthy controls, and we cannot exclude the effect of GFR on the level of miRs in non-ARAS CKD. Fourth, urinary concentrations and excretions of miRs were not evaluated, yet the excreted miRs would not contribute to further decreasing their RV level in ARAS with already reduced kidney function compared with EH. Finally, the lack of data regarding the duration of hypertension and its systemic effects makes it difficult to evaluate the possible effects of hypertension on nephroangiosclerosis in ARAS and EH patients. Yet, the clinical profile of the groups makes it unlikely for the ARAS group to have less cardiovascular risk. Nevertheless, application of our results to other cohorts should be done with caution, and further studies are needed. Notably, in a pilot study (unpublished data) we have found that levels of cytokines are comparable in the IVC and peripheral vein of the same individuals, supporting this comparison.

In conclusion, miRs implicated in atherosclerosis may also be involved in developing and modulating renal injury and impairment of renal function in ARAS. Our results demonstrated for the first time the differential expression of miRs in ARAS compared with EH, and in the RV compared with the systemic circulation. Relation of RV miRs level with GFR and the different level of miRs between the RV and IVC may implicate miR in kidney injury. Better understanding of the role of miR in the post-stenotic kidney may provide us with novel biomarkers for outcomes or therapeutic strategies for renal repair in ARAS.

CONFLICT OF INTEREST STATEMENT

None declared.