Urinary exosomes derived circRNAs as biomarkers for chronic renal fibrosis

Figures & data

Figure 1. Urinary exosomes identification. (A) TEM image of exosomes from healthy controls and CKD patients. (B) NTA analysis of urinary exosomes. (C) Western blot showed that exosomes from healthy controls and CKD patients expressed CD9 and TSG101.

Figure 2. Human circRNAs microarray. (A) Volcano map of circRNAs microarray. (B) Heat map of circRNAs microarray.

Table 1. Clinical profile of patients with CKD and healthy controls.

	CKD (n = 110)	Control (n = 54)	P value
Age (years)	43.8 ± 15.6	39.2 ± 15.3	.075
Gender (male/female)	62/48	38/16	.084
24h Proteinuria (g/day)	2.315(0.010–24.580)	/	/
Scr (mmol/l)	108.9 ± 83.6	62.1 ± 6.8	<.001
BUN (mmol/L)	7.1 ± 3.8	4.6 ± 1.2	<.001
Cystatin C (mg/L)	1.44 ± 0.88	0.67 ± 0.10	<.001
eGFR (ml/min per 1.73 m^2)	82.8 ± 35.2	121.7 ± 21.7	<.001
SBP (mmHg)	131.2 ± 15.7	120.3 ± 7.3	<.001
DBP (mmHg)	85.8 ± 12.8	73.7 ± 6.5	<.001
Score of TIF	30 (0–91)	/
Score of glomerular sclerosis	1.0 (0.0–4.0)	/	/
hsa_circ_0036649	0.642	0.951	/
	(0.187–3.848)	(0.889–0.977)	<.001

Scr: serum creatinine; eGFR: estimated glomerular filtration rate; BUN: blood urea nitrogen; SBP: systolic blood pressure; DBP: diastolic blood pressure.

Figure 3. Expression of hsa_circ_0036649 in urine exosomes from CKD patients and healthy control. (A) Expression of hsa_circ_0036649 was significantly decreased in CKD patients compared to healthy controls. (B) In CKD patients, urinary exosomes hsa_circ_0036649 expression was significantly decreased in mild-moderate and severe renal fibrosis patients compared to none fibrosis (#p < .001 vs control; *p = .012 vs none fibrosis; **p = .041 vs mild-moderate, p < .001 vs none fibrosis).

Table 2. Clinical and pathological parameters of CKD patients.

	None	Mild-Moderate	Severe	P value
	(n = 33)	(n = 42)	(n = 35)
Age, y	42.0 ± 18.9	41.7 ± 13.4	48.4 ± 15.0	.153
Sex (M/F)	16/17	23/19	23/12	.346
Scr, μmol/L	85.8 ± 67.6	111.2 ± 38.3	134.6 ± 73.5	.036
BUN, mmol/L	5.6 ± 2.8	7.4 ± 4.3	8.6 ± 3.6	.001
Cystatin C, mg/L	1.08 ± 0.60	1.39 ± 0.99	1.92 ± 0.86	<.001
eGFR (ml/min per 1.73 m^2)	99.9 ± 30.1	88.5 ± 38.3	57.2 ± 20.8	<.001
24h proteinuria (g/day)	1.800	1.595	2.850	.093
	(0.010–9.160)	(0.100–21.580)	(0.220–13.210)
SBP, mmHg	130.1 ± 15.7	126.6 ± 13.3	137.9 ± 16.7	.006
DBP, mmHg	84.2 ± 12.3	82.1 ± 10.6	91.9 ± 13.7	.002
Urine pH	6.2 ± 0.7	6.2 ± 0.6	6.0 ± 0.6	.324
Usage of diuretic	12	16	13	.988
Usage of ACEI/ARB	17	22	16	.826
hsa_circ_0036649	0.703	0.635	0.602	.001
	(0.429–3.848)	(0.281–1.250)	(0.187–1.111)
Score of TIF	0	30 (0–45)	70 (52–91)	<.001
Score of glomerular sclerosis	0.6 (0.0–0.9)	1.0 (0.6–2.4)	2.2 (0.9–4.0)	<.001

Scr: serum creatinine; eGFR: estimated glomerular filtration rate; BUN: blood urea nitrogen; SBP: systolic blood pressure; DBP: diastolic blood pressure.

Figure 4. Correlation between hsa_circ_0036649 expression and clinical parameters. (A) Spearman correlation between hsa_circ_0036649 and Scr (r_s = −0.366, p < .001). (B) Spearman correlation between hsa_circ_0036649 and BUN (r_s = −0.215, p = .006). (C) Spearman correlation between hsa_circ_0036649 and cystatin c (r_s = −0.424, p < .001). (D) Spearman correlation between hsa_circ_0036649 and eGFR (r_s = 0.374, p < .001).

Figure 5. Correlation between hsa_circ_0036649 expression and 24 h Proteinuria, renal fibrosis pathological parameters. (A) Spearman correlation between hsa_circ_0036649 and 24 h Proteinuria (r_s = −0.214, p = .024). (B) Spearman correlation between hsa_circ_0036649 and score of TIF (r_s = −0.360, p < .001). (C) Spearman correlation between hsa_circ_0036649 and score of glomerular sclerosis (r_s = −0.273, p = .004).

Table 3. Multivariate logistic regression analysis of selected variables for renal fibrosis.

	OR	95% CI	P Value
Hsa_circ_0036649	0.125	0.026–0.599	.009
Scr	0.995	0.983–1.007	.383
BUN	0.888	0.714–1.105	.287
Cystatin C	1.465	0.319–6.737	.623
eGFR	0.986	0.965–1.008	.217
24h proteinuria	1.080	0.904–1.289	.398

Scr: serum creatinine; eGFR: estimated glomerular filtration rate; BUN: blood urea nitrogen; OR: odds ratio; CI: confidence interval.

Figure 6. The receiver operating characteristic (ROC) curve showed the diagnosis value of the urine exosomes hsa_circ_0036649 for renal fibrosis. ROC curve showed the urine exosomes hsa_circ_0036649 distinguished renal fibrosis (AUC of 0.706, 95% CI, 0.606–0.807; p = .001).

Data availability statement

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.