Expression of urinary exosomal miRNA-615-3p and miRNA-3147 in diabetic kidney disease and their association with inflammation and fibrosis

Figures & data

Figure 1. Characteristics of extracellular vesicle (EV) in subjects. 15 mL of morning urine from subjects was collected. Extracellular vesicles were extracted from urine by high-speed centrifugation (12000 × g, 30 min), ultrafiltration enrichment (100KD, 15 mL) and ExoQuick™ Exosome Precipitation Kit (EXOTC50A-1, SBI System) Resuspend in 1x PBS and store in −80 °C freezers until use. (a) WB results for exosome marker evaluation; PBS served as a negative control. (b) Electron Microscopy of Exosomes. (c) Particle size analysis of exosomes by nanoparticle tracking analyzer.

Figure 2. Expression levels of miRNA-615-3p and miRNA-3147 in urinary exosomes. DKD, T2DM and N represent the DKD group, T2DM group and control group, respectively. (a) Expression levels of urinary exosomal miRNA-615-3p in three groups of patients. (b) Expression levels of urinary exosomal miRNA-3147 in three groups of patients.

Table 1. Clinical characteristics of the included subjects.

Clinical parameters	N	T2DM	DKD	P-value
	(n = 20)	(n = 21)	(n = 42)
Gender (male, %)	14 (70.0%)	10 (47.6%)	28 (66.7%)	0.249
Age (years)	44.65 ± 13.60	55.33 ± 13.33	61.57 ± 11.90	<0.001
BMI	24.03 ± 2.66	24.37 ± 3.47	25.75 ± 0.66	0.177
Mean arterial pressure (mmHg)	100.00 ± 1.83	100.81 ± 2.48	105.93 ± 1.84	0.078
HbA1c (%)	4.90 (4.25–5.58)	8.50 (7.30–10.80)	6.80 (6.08–7.90)	<0.001
Blood glucose (mmol/L)	4.84 ± 0.27	6.89 ± 0.56	6.53 ± 0.43	0.017
HDL-C (mmol/L)	1.08 ± 0.07	1.11 ± 0.07	0.96 ± 0.05	0.179
LDL-C (mmol/L)	2.80 ± 0.18	2.92 ± 0.22	2.44 ± 0.17	0.160
Creatinine (umol/L)	76.53 (66.71–85.24)	57.30 (49.40–67.60)	185.55 (127.57–360.85)	<0.001
BUN (mmol/L)	5.54 (4.57–6.82)	5.90 (5.10–6.85)	11.90 (9.50–19.63)	<0.001
TC (mmol/L)	4.70 ± 0.16	4.79 ± 0.23	4.67 ± 0.23	0.935
TG (mmol/L)	1.40 ± 0.33	1.68 ± 0.19	2.04 ± 0.15	0.089
eGFR (CKD-EPIml·min-1·(1.73m2)-1)	101.00 (93.00–113.00)	104.00 (99.50–115.50)	35.50 (12.75–53.00)	<0.001
PCR	0.08 (0.06–0.11)	0.05 (0.04–0.09)	3.24 (0.71–5.82)	<0.001
ALP (U/L)	98.97 ± 6.39	67.44 ± 3.83	67.44 ± 7.77	0.001
Albumin (g/L)	42.77 (41.97–44.72)	38.80 (37.25–43.10)	33.55 (29.75–39.30)	<0.001
γ-GT(U/L)	22.82 (13.39–80.07)	20.00 (13.85–27.20)	21.35 (12.25–38.33)	0.878
Globulin(g/L)	27.23 ± 0.96	22.99 ± 0.73	26.43 ± 0.75	0.004
NLR	2.51 (1.83–3.09)	2.38 (1.97–3.04)	2.54 (2.09–3.37)	0.527
PLR	123.32 ± 8.49	122.30 ± 10.68	143.64 ± 8.80	0.184
LMR	3.62 (2.55–5.38)	5.32 (3.58–6.67)	3.05 (2.41–4.21)	0.001
ACR	2.19 (0.68–4.55)	2.96 (1.58–4.05)	1210.97 (158.64–3109.32)	<0.001
24-h urine protein(g/d)	–	–	2.78 (0.71–6.74)	–
MHR	0.50 ± 0.05	0.31 ± 0.05	0.60 ± 0.06	0.003

Measurement data were expressed as mean ± standard deviation (SD) or median and interquartile range according to the normal distribution statue, categorial data were expressed as numbers and percentages.

BMI body mass index, HbA1c glycated hemoglobin, HDL-C high density lipoprotein-cholesterol, LDL-C low density lipoprotein-cholesterol, BUN blood urea nitrogen, CKD-EPI The Chronic Kidney Disease Epidemiology Collaboration, TC total cholesterol, TG triglycerides, PCR Urine protein to creatinine ratio, ALP Alkaline phosphatase, γ-GT γ-Glutamyl transpeptidase, NLR Neutrophil to lymphocyte ratio, PLR Platelet to lymphocyte ratio, LMR Lymphocyte to monocyte ratio, ACR Albumin to creatinine ratio, MHR monocyte to HDL ratio.

Table 2. Expression levels of miRNA-615-3p and miRNA-3147 in urinary exosomes.

miRNA	N	T2DM	DKD	P-value
	(n = 20)	(n = 21)	(n = 42)
miRNA-615-3p	0.16	0.62	1.34	<0.001
	(0.04–0.62)	(0.17–1.44)	(0.68–5.15)
miRNA-3147	1.24	1.60	1.98	0.419
	(0.33–2.89)	(0.37–3.19)	(0.82–4.14)

Figure 3. Correlation analysis of urinary exosomal miRNA-615-3p with clinical indicators in DKD group. (a) Scr. (b) BUN. (c) eGFR. (d) PCR. (e) 24 h urine protein. (f) Albumin.

Table 3. Correlation analysis of urinary exosomal miRNA-615-3p and miRNA-3147 with clinical indicators in DKD group.

Clinical parameters	miRNA-615-3p		miRNA-3147
	r-value	P-value	r-value	P-value
Age (years)	0.006	0.957	0.040	0.799
Gender (male, %)	0.141	0.274	0.250	0.111
BMI	0.015	0.888	−0.053	0.739
Mean arterial pressure (mmHg)	0.022	0.837	0.143	0.367
HbA1c (%)	−0.139	0.200	−0.093	0.558
Blood glucose (mmol/L)	0.067	0.530	−0.079	0.619
HDL-C (mmol/L)	0.107	0.319	−0.079	0.621
LDL-C (mmol/L)	0.005	0.965	−0.170	0.282
Creatinine (umol/L)	0.403*	<0.001*	0.018	0.910
BUN (mmol/L)	0.367*	0.001*	−0.015	0.925
TC (mmol/L)	0.037	0.729	−0.070	0.660
TG (mmol/L)	−0.024	0.820	0.338*	0.029*
eGFR (CKD-EPIml·min-1·(1.73m2)-1)	−0.442*	<0.001*	−0.086	0.588
PCR	0.280*	0.009*	−0.061	0.701
ALP (U/L)	−0.125	0.246	−0.001	0.994
Albumin (g/L)	−0.276*	0.010*	0.075	0.637
γ-GT(U/L)	−0.088	0.410	−0.068	0.670
Globulin(g/L)	−0.091	0.398	0.063	0.694
NLR	0.136	0.205	−0.019	0.904
PLR	0.168	0.116	0.022	0.891
LMR	−0.062	0.566	0.071	0.657
ACR	0.193	0.221	−0.127	0.422
24-h urine protein(g/d)	0.395*	0.010*	−0.059	0.713
MHR	−0.078	0.468	−0.064	0.688

Table 4. Urinary exosomal miRNA-615-3p in patients with different stages of DKD.

Clinical parameters	Number of subjects (%)	Expression of miRNA-615-3p	P-value
Gender			0.284
Male	28 (66.67%)	1.13 (0.60–4.75)
Female	14 (33.33%)	2.00 (0.94–8.36)
Stage (ml·min-1·(1.73m2)-1)			0.009
G1 + G2 (>60)	7 (16.67%)	0.22 (0.12–1.34)
G3 (30–59)	16 (38.09%)	1.03 (0.72–1.90)
G4 (15–29)	8 (19.05%)	1.82 (0.91–6.44)
G5 (<15)	11 (26.19%)	13.42 (2.28–21.60)

Figure 4. Correlation of urinary exosomal miRNA-615-3p with serum Cystatin C and TGF-β1 in DKD group. (a) Cystatin C-miRNA-615-3p. (b) TGF-β1- miRNA-615-3p.

Table 5. Correlation of serum Cystatin C and TGF-β1 expression levels with miRNA-615-3p in DKD patients.

Clinical parameters	Cystatin C		TGF-β1
	r-value	P-value	r-value	P-value
miRNA-615-3p	0.610	<0.001	0.366	0.017

Figure 5. ROC curve analysis results of patients in the DKD group.

Data availability statement

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