Long noncoding RNA PR11-387H17.6 as a potential novel diagnostic biomarker of atherosclerotic renal artery stenosis

Figures & data

Figure 1. Differential expression of lncRNAs in ARAS patients and control individuals. (A) The scatter plots showed LncRNA expression in test samples versus normal samples. X-axis depicted data values of control samples; Y-axis depicted data values of test samples. Dots were located above the upper green line and below the under green line represent fold change ≥1.5, ‘Test’ indicates ARAS samples; ‘Normal,’ control samples. (B) Heat map of lncRNA expression from microarray analysis of combined renal artery tissue samples of patients with ARAS and control subjects (T, renal atherosclerosis tissue; C, normal renal artery tissue). Each row represented one lncRNA and each column represents a sample. The color scale shown at the top illustrated the relative expression level of a lncRNA; red represents high expression and green represented low expression. (C) The volcano plots showed thousands of lncRNAs were significantly different by using lncRNA expression thresholds of more than 1.5-fold change with p<.05. The red point in the plot represented the deferentially expressed Coding genes with statistical significance.

Table 1. Characteristics of a training group population.

	Healthy volunteer (n = 18)	Non-ARAS (n = 18)	ARAS (n = 18)	p Value
Age, years	42.50 ± 8.50	58.00 ± 10.23*	64.56 ± 9.81*^,^+	<.001
Female, n (%)	7 (38.89)	5 (27.78)	6 (33.33)	.709
Physical data
Body mass index, kg/m^2	23.76 ± 2.49	26.89 ± 4.72*	25.49 ± 3.16	.046
Systolic blood pressure, mmHg	125.39 ± 13.81	135.56 ± 24.40	149.94 ± 13.10*^,^+	.002
Diastolic blood pressure, mmHg	74.39 ± 12.54	76.39 ± 13.68	77.44 ± 9.32	.741
Pulse pressure, mmHg	51.83 ± 5.77	59.44 ± 16.86	70.78 ± 12.31*^,^+	<.001
Smoking, n (%)	–	10 (55.56)	11 (61.11)	.735
Drinking, n (%)	–	2 (11.11)	4 (22.22)	.371
Laboratory data
CK-MB, U/L	–	14.61 ± 5.20	17.28 ± 14.33	.466
WBC (×10^9/L)	6.04 ± 1.33	7.64 ± 2.23*	7.98 ± 1.45*	.004
TC, mmol/L	4.44 ± 0.53	4.10 ± 0.88	4.91 ± 1.58^+	.091
TG, mmol/L	0.90 ± 0.34	1.85 ± 0.60*	2.91 ± 1.96*^,^+	<.001
LDL, mmol/L	2.77 ± 0.41	2.34 ± 0.58	2.99 ± 1.21^+	.058
HDL, mmol/L	2.33 ± 0.25	1.62 ± 0.30*	1.48 ± 0.33*	<.001
Creatinine, µmol/L	69.11 ± 13.40	76.05 ± 16.31	84.31 ± 19.16*	.456
eGFR, mL/min	127.70 ± 30.84	104.01 ± 26.15*	99.44 ± 5.34*	.001
NT-ProBNP, pg/mL	48.58	90.45	164.90*	.002
lncRP11-387H17.6, log change	0.02	0.02	0.15*	.001
Comorbid conditions, n (%)
Prior MI	–	1 (5.56)	3 (16.67)	.596
Hypertension	–	8 (44.44)	14 (77.78)	.040
Diabetes mellitus	–	3 (16.67)	5 (27.78)	.688
Dyslipidemia	–	10 (55.6)	10 (55.6)	1.000
Stroke	–	2 (11.11)	5 (27.78)	.238
Peripheral artery disease	–	0 (0.00)	3 (16.67)	.070
Coronary artery disease vessels	–
1 vessel	–	1 (5.56)	3 (16.67)	.596
2 vessel	–	5 (33.33)	7 (22.22)	.480
3 vessel	–	7 (38.89)	8 (44.44)	.735

ARAS: atherosclerotic renal artery stenosis; TC: total cholesterol; TG: triglyceride; LDL: low-density lipoprotein cholesterol; HDL: high-density lipoprotein cholesterol; eGFR: estimated glomerular filtration rate using the Chronic Kidney Disease Epidemiology Collaboration; NT-ProBNP: N-terminal pro-brain natriuretic peptide; MI: myocardial infarction.

Data are presented as means (±SD) or number (%).

* p<.05 vs. healthy controls.

+ p<.05 vs. non-ARAS.

Figure 2. Expression levels of lncRNAs RP11-387H17.6, BC080653, RP1-32B1.4, RP5-1068H6.3, GHRLOS, and XLOC_009769 were assessed by quantitative polymerase chain reaction (qPCR) using GAPDH as a reference gene for normalization among patients with ARAS (n = 18), non-ARAS (n = 18), and HV(n = 18). (A–F) Expression levels of lncRNAs: (A) RP11-387H17.6, (B) BC080653, (C) RP1-32B1.4, (D) RP5-1068H6.32, (E) GHRLOS, and (F) XLOC_009769. ARAS: atherosclerotic renal artery stenosis; HV: healthy volunteers.

Figure 3. Receiver operating characteristic (ROC) curve analyses of LncRNAs RP11-387H17.6, BC080653, RP1-32B1.4, RP5-1068H6.3, GHRLOS, and XLOC_009769 for diagnosis of ARAS among patients with ARAS (n = 18), non-ARAS (n = 18), and HV (n = 18). AUC: area under the curve, ARAS: atherosclerotic renal artery stenosis. (A–F) Expression levels of lncRNAs: (A) RP11-387H17.6, (B) BC080653, (C) RP1-32B1.4, (D) RP5-1068H6.32, (E) GHRLOS, and (F) XLOC_009769. ARAS: atherosclerotic renal artery stenosis; HV: healthy volunteers.

Figure 4. Expression levels of lncRP11-387H17.6 and ROC curve analyses of lncRP11-387H17.6 alone and lncRP11-387H17.6 combined with risk factors for the diagnosis of ARAS among patients with ARAS (n = 99), non-ARAS (n = 45), and HV (n = 50). (A) Expression levels of lncRNAs RP11-387H17.6 among patients with ARAS (n = 99), non-ARAS (n = 49), and HV (n = 50). GAPDH was used as the normalization control. (B) ROC curves showing the diagnostic performance of lncRP11-387H17.6 alone and lncRP11-387H17.6 combined with risk factors. ARAS: atherosclerotic renal artery stenosis; HV: healthy volunteers.

Table 2. Characteristics of a testing group population.

	Healthy volunteers (n = 50)	Non-ARAS (n = 49)	ARAS (n = 99)	p Value
Age, years	49.56 ± 11.97	60.67 ± 9.63*	64.85 ± 8.86*^,^+	<.001
Female, n (%)	18 (36.00)	11 (22.45)	40 (40.40)	.096
Physical data
Body mass index, kg/m^2	23.00 ± 3.39	26.91 ± 2.86*	25.46 ± 3.40*^,^+	<.001
Systolic blood pressure, mmHg	119.64 ± 12.03	138.00 ± 19.68*	146.23 ± 21.90*^,^+	.016
Diastolic blood pressure, mmHg	74.30 ± 9.23	77.43 ± 12.26	79.32 ± 12.58	.051
Pulse pressure, mmHg	45.34 ± 10.91	60.57 ± 16.53*	66.24 ± 20.82*	<.001
Smoking, n (%)	–	30 (61.22)	42 (42.42)	.031
Drinking, n (%)	–	17 (34.69)	19 (19.19)	.120
Laboratory data
CK-MB, U/L	–	13.69 ± 8.69	14.81 ± 13.30	.594
WBC (×10^9/L)	5.92 ± 1.61	7.08 ± 1.78*	7.31 ± 1.74*	<.001
TC, mmol/L	4.04 ± 0.58	3.95 ± 1.02	4.35 ± 1.09*^,^+	.038
TG, mmol/L	0.91 ± 0.36	2.09 ± 2.12*	1.94 ± 1.92*	.001
LDL, mmol/L	2.08 ± 0.58	2.29 ± 0.76	2.47 ± 1.00*	.032
HDL, mmol/L	3.08 ± 0.65	0.79 ± 0.16*	0.91 ± 0.39*	<.001
Creatinine, µmol/L	65.88 ± 13.35	77.45 ± 17.66*	77.85 ± 24.44*	.002
eGFR, mL/min	134.52 ± 43.46	98.95 ± 22.47*	94.02 ± 28.63*	<.001
NT-ProBNP, pg/mL	58.80	93.74*	188.60*^,^+	<.001
lncRP11-387H17.6, log change	0.02	0.02	0.09*^,^+	<.001
Comorbid conditions, n (%)
Prior MI	–	17 (34.69)	19 (19.19)	.039
Hypertension	–	21 (42.86)	31 (31.31)	.084
Diabetes mellitus	–	31 (63.27)	76 (76.77)	.084
Dyslipidemia	–	33 (67.35)	48 (48.48)	.030
Stroke	–	8 (16.33)	27 (27.27)	.140
Peripheral artery disease	–	2 (4.08)	8 (8.16)	.354
Coronary artery disease vessels	–
1 vessel	–	13 (26.53)	20 (20.20)	.439
2 vessel	–	17 (34.69)	35 (35.35)	.834
3 vessel	–	19 (38.78)	40 (40.40)	.737

Data are presented as means (±SD) or number (%).

ARAS: atherosclerotic renal artery stenosis; TC: total cholesterol; TG: triglyceride; LDL: low-density lipoprotein cholesterol; HDL: high-density lipoprotein cholesterol; eGFR: estimated glomerular filtration rate using the Chronic Kidney Disease Epidemiology Collaboration; NT-ProBNP: N-terminal pro-brain natriuretic peptide; MI: myocardial infarction.

* p<.05 vs. healthy controls.

+ p<.05 vs. non-ARAS.

Table 3. Univariate analysis for lncRP11-387H17.6 in patients with ARAS.

	Univariate analysis
Variable	OR (95% CI)	p Value
Sex	1.992 (1.015–3.908)	.045
Age, years	1.058 (1.022–1.095)	.001
SBP, mmHg	1.022 (1.006–1.037)	.005
Pulse pressure, mmHg	1.020 (1.003–1.038)	.021
Smoking	0.559 (0.304–1.028)	.061
Dyslipidemia	0.549 (0.296–1.017)	.057
TC, mmol/L	1.460 (1.088–1.960)	.012
LDL, mmol/L	1.714 (1.147–2.561)	.009
lncRP11-387H17.6, log change	3.249 (1.268–8.324)	.014
NT-ProBNP, pg/mL	1.000	.531
Prior MI	0.630 (0.309–1.285)	.204

ARAS: atherosclerotic renal artery stenosis; SBP: systolic blood pressure; TC: total cholesterol; LDL: low-density lipoprotein cholesterol; NT-ProBNP: N-terminal pro-brain natriuretic peptide; MI: myocardial infarction; OR: odds ratio; CI: confidence interval.

Dichotomous variables (yes = 1, no = 0): smoking, dyslipidemia, prior MI; categorical variables: sex (female = 1, male = 0).

Table 4. Multivariate analysis for lncRP11-387H17.6 in patients with ARAS.

	Multivariate analysis
Variable	OR (95% CI)	p Value
Sex	1.209 (0.500–2.922)	.673
Age, years	1.051 (1.010–1.095)	.016
SBP, mmHg	1.028 (1.010–1.047)	.003
Smoking	0.697 (0.319–1.520)	.364
TC, mmol/L	1.373 (0.597–3.162)	.456
LDL, mmol/L	1.296 (0.432–3.886)	.644
lncRP11-387H17.6, log change	3.039 (1.388–6.654)	.006

ARAS: atherosclerotic renal artery stenosis; SBP: systolic blood pressure; TC: total cholesterol; LDL: low-density lipoprotein cholesterol; NT-ProBNP: N-terminal pro-brain natriuretic peptide; OR: odds ratio; CI: confidence interval.

Dichotomous variables (yes = 1, no = 0): smoking; categorical variables: sex (female = 1, male = 0).