Specific renal parenchymal-derived urinary extracellular vesicles identify age-associated structural changes in living donor kidneys

Figures & data

Fig. 1.  Example of fluorescence dot (quadrant) plots showing fluorophore spectra separate of markers used to identify urinary EVs in this study. (a) Isotype controls (fluorescein (FITC)-conjugated immunoglobulin G (IgG) versus phycoerythrin (PE)-conjugated IgG); (b) annexin-V (phosphatidylserine) versus CD63 (exosome); (c) intercellular adhesion molecule-1 (ICAM-1) versus monocyte chemoattractant protein-1 (MCP-1); (d) tissue factor (TF) versus vascular cell adhesion molecule -1 (VCAM-1); (e) beta-adrenergic receptor-1 (β-1 AR) versus renin; (f) SM22 alpha versus annexin-V-PE; (g) nephrin versus podocin; (h) cytokeratin 8 (CK 8) versus caludin-1; (i) URAT-1 versus megalin; (j) aquaporin-1 versus SLC14A2; (k) uromodulin versus epidermal growth factor receptor (EGFR); (l) SLC12A3 versus prominin; (m) aquaporin-2 versus V-ATPase; (n) cytokeratin 20 versus cytokeratin 19.

Table I. Clinical and renal biopsies characteristics of living kidney donors

Clinical characteristics	Women (n=69)	Men (n=69)	p
Age (years)	46 (38, 56)	42 (32, 51)	0.142
Body mass index (kg/m^2)	25 (23, 30)	28 (25, 31)	0.016
Systolic blood pressure (mmHg)	113 (104, 127)	119 (110, 130)	0.088
Diastolic blood pressure (mmHg)	69 (63, 74)	72 (64, 78)	0.151
Haemoglobin (g/dL)	14 (13, 14)	15 (15, 16)	<0.001
Total cholesterol (mg/dL)	192 (171, 210)	195 (174, 221)	0.438
Low density lipoprotein cholesterol (mg/dL)	109 (90, 121)	117 (98, 136)	0.017
High density lipoprotein cholesterol (mg/dL)	65 (52, 74)	48 (39, 55)	<0.001
Triglycerides (mg/dL)	86 (70, 106)	123 (75, 157)	0.003
Fasting blood glucose (mg/dL)	93 (89, 99)	97 (93, 101)	0.013
Serum creatinine (mg/dL)	0.8 (0.7, 0.9)	1.0 (1.0, 1.1)	<0.001
Uric acid (mg/dL)	4 (4, 5)	6 (6, 7)	<0.001
Estimated glomerular filtration rate (mL/min/1.73 m^2)	86 (79, 100)	91 (82, 98)	0.500
Corrected iothalamate (mL/min/1.73 m^2)	100 (90, 114)	100 (92, 108)	0.685
Urine protein excretion (mg/24 h)	42 (26, 68)	43 (29, 66)	0.665
Urine albumin excretion (mg/24 h)	2 (0, 6)	3 (0.0, 9.0)	0.547
Urine creatinine (mg/dL)	77 (60, 120)	152 (111, 215)	<0.001
Renal biopsy characteristics
Non-sclerotic glomeruli volume (mm^3)	0.0020 (0.0017, 0.0025)	0.0029 (0.0023, 0.0037)	<0.001
Cortex per glomerulus (mm^3)	0.058 (0.045, 0.078)	0.071 (0.054, 0.094)	0.005
Mean profile tubular area (µm^2)	3,978 (3,294, 4,842)	4,619 (3,675, 6,086)	0.026
Detectable fibrosis	18 (26%)	11 (17%)	0.198
Any detectable glomerulosclerosis	20 (29%)	21 (31%)	0.807
Arteriosclerosis (Intimal thickening, %)	33 (14, 42)	36 (20, 46)	0.301

Data are presented as the median (25th percentile, 75th percentile).

p<0.05; significantly different between women and men.

Table II. Sex differences in urinary EVs from living kidney donors

Urinary EVs	Markers	Women (n=69)	Men (n=69)	p
Microvesicles/phosphatidylserine	Annexin-V	14.0 (13.3, 14.6)	13.1 (12.4, 13.9)	0.000
Exosomes	CD63	13.2 (12.8, 13.7)	12.7 (11.9, 13.3)	0.000
EVs positive for cellular adhesion/inflammatory molecules
Cell adhesion	ICAM-1	11.0 (10.3, 11.6)	10.7 (10.0, 11.4)	0.079
Cell adhesion	VCAM-1	9.1 (7.7, 10.6)	9.3 (8.2, 10.3)	0.535
Inflammation/pro-coagulant	TF	12.4 (11.4, 13.5)	12.4 (11.3, 13.6)	0.997
Inflammation	MCP-1	10.3 (8.9, 11.2)	10.1 (9.5, 10.6)	0.797
EVs from renal cells
Juxtaglomerular cells	β-1 AR	10.1 (9.4, 12.0)	10.4 (9.0, 12.4)	0.904
Mesangial cells	SM22 alpha	10.9 (10.2, 11.5)	10.1 (9.5, 10.9)	0.000
Podocytes	Nephrin	10.5 (8.8, 11.4)	9.9 (8.5, 10.9)	0.157
	Podocin	10.4 (8.4, 11.3)	9.9 (8.7, 10.6)	0.245
Bowman's capsule – parietal cells	Claudin-1	10.2 (8.0, 10.9)	9.5 (7.7, 10.5)	0.048
	Cytokeratin 8	10.4 (9.4, 11.1)	10.0 (8.3, 10.8)	0.078
Proximal tubule – simple cuboidal epithelium	Megalin	9.2 (7.9, 10.8)	9.0 (8.0, 10.2)	0.757
	URAT1	10.4 (9.7, 11.0)	10.3 (9.8, 11.0)	0.755
Descending limb of Henle's loop – simple squamous epithelium	SLC14A2	10.3 (9.5, 11.0)	10.2 (9.7, 10.6)	0.595
	Aquaporin-1	10.5 (9.8, 10.9)	10.2 (9.7, 10.6)	0.121
Ascending limb of Henle's loop – simple cuboidal epithelium	EGFR	10.4 (9.1, 11.9)	10.6 (9.6, 12.5)	0.386
	Uromodulin	12.5 (11.2, 13.8)	12.2 (11.5, 13.4)	0.649
Distal tubule – simple cuboidal epithelium	Prominin-2	9.9 (8.5, 10.6)	9.7 (8.9, 10.6)	0.960
	SLC12A3	10.6 (9.8, 11.4)	10.4 (9.8, 11.0)	0.138
Collecting duct – principal/intercalated cells	Aquaporin-2	10.4 (9.4, 11.4)	10.4 (8.6, 11.0)	0.451
	V-ATPase	9.7 (8.4, 10.9)	9.5 (8.3, 10.4)	0.360
Renal pelvis – transitional epithelium	Cytokeratin 19	10.6 (9.6, 11.3)	10.4 (9.6, 11.2)	0.705
	Cytokeratin 20	8.1 (7.2, 10.6)	8.3 (7.4, 9.7)	0.594

Data are presented as median (25th percentile, 95th percentile) of natural log of respective markers positive for urinary vesicles/mg creatinine.

p<0.05; significantly different between women and men.

Table III. Changes of urinary EVs with older age

EVs versus older age	Spearman's correlation (ρ)	p
Total EVs (microvesicle and exosome)
Annexin-V microvesicles/phosphatidylserine	−0.114	0.182
CD63 exosomes	−0.198	0.021
EVs from cells associated with renal capillaries
β-1 Adrenergic receptor (β-1 AR) – juxtaglomerular cells	−0.461	0.001
SM22 alpha – mesangial cells	0.091	0.302
Nephrin – podocytes	−0.238	0.006

p<0.05; significantly decreased with older age.

Fig. 2.  Example of biopsies morphometric observation indices of nephron hypertrophy and nephrosclerosis/glomerulosclerosis. (a) Two functional non-sclerotic glomeruli; (b) 2 globally sclerosed glomeruli; (c) 5 consecutive 0.2 mm² circles (green) in which full (red) or partial (cyan) tubules are labelled, and their area subsequently quantified after exclusion of all non-tubular structures (yellow); (d) area of cortical fibrosis and tubular atrophy outlined in blue dashed line; (e) the percent intimal thickening (arteriosclerosis) was determined by the area of intima (between yellow and blue boundaries) divided by the area of intima and lumen (within blue boundary). Examples of different degrees of intimal thickening are shown in E1–no thickening, E2–moderate thickening and E3–severe thickening.

Table IV. Changes of phosphatidylserine, exosome, cellular adhesion and inflammatory markers positive for urinary EVs with nephron hypertrophy

	Biopsy measurements
	Non-sclerotic glomeruli volume		Cortex per glomerulus		Mean profile tubular area
	rho	p	rho	p	rho	p
EVs positive for phosphatidylserine (annexin-V) and exosome (CD63) marker
Annexin-V	−0.254	0.003	−0.200	0.020	−0.081	0.349
CD63	−0.176	0.044	−0.178	0.041	−0.116	0.185
EVs positive for cellular adhesion/inflammatory molecules
ICAM-1	−0.018	0.834	−0.111	0.204	0.022	0.803
VCAM-1	−0.001	0.999	−0.108	0.216	0.073	0.403
Tissue factor	0.043	0.622	−0.027	0.756	−0.028	0.751
MCP-1	−0.028	0.755	−0.065	0.465	−0.180	0.042

−rho_s, p<0.05: values in italic.

Table V. Changes of urinary EVs–derived from different types of cells surrounded by renal capillaries, nephron and renal pelvis with nephron hypertrophy

	Biopsy measurements
	Non-sclerotic glomeruli volume		Cortex per glomerulus		Mean profile tubular area
	rho	p	rho	p	rho	p
EVs from cells associated with renal capillaries
β-1 AR	0.029	0.847	−0.204	0.174	−0.098	0.515
SM22 alpha	−0.188	0.033	−0.207	0.019	−0.062	0.486
Nephrin	−0.033	0.710	−0.151	0.085	−0.052	0.554
Podocin	−0.108	0.222	−0.104	0.241	−0.068	0.440
EVs from parietal cells of Bowman's capsule
Claudin-1	−0.038	0.669	−0.132	0.138	−0.059	0.511
Cytokeratin 8	−0.127	0.157	−0.279	0.002	−0.096	0.286
EVs from epithelial cells of proximal tubule
Megalin	−0.053	0.547	−0.126	0.149	0.059	0.501
URAT-1	0.035	0.695	−0.042	0.639	0.119	0.179
EVs from epithelial cells of descending and ascending limb of Henle's loop
Aquaporin-1	−0.064	0.492	−0.179	0.054	−0.198	0.033
EGFR	0.045	0.610	0.074	0.406	−0.022	0.808
Uromodulin	−0.004	0.965	−0.070	0.429	−0.110	0.217
SLC14A2	0.003	0.972	−0.082	0.349	0.083	0.341
EVs from epithelial cells of distal tubule
Prominin-2	0.034	0.711	−0.106	0.248	−0.003	0.974
SLC12A3	0.013	0.887	−0.094	0.289	0.040	0.653
EVs from principal and intercalated cells of collective tubule
Aquaporin-2	0.003	0.974	−0.122	0.199	−0.082	0.388
V-ATPase	−0.068	0.439	−0.216	0.013	−0.053	0.542
EVs from epithelial cells of renal pelvis
Cytokeratin19	−0.060	0.519	−0.123	0.190	−0.029	0.757
Cytokeratin 20	−0.084	0.342	−0.123	0.163	0.038	0.671

−rho_s, p<0.05: values in italic.

Table VI. Changes of phosphatidylserine, exosome, cellular adhesion and inflammatory markers positive for urinary EVs with nephrosclerosis

	Biopsy measurements
	Cortical fibrosis groups		Number of fibrosis foci		Globally sclerotic glomeruli (%)		Arteriosclerosis (% intimal thickening)
	rho	p	rho	p	rho	p	rho	p
EVs positive for phosphatidylserine (annexin-V) and exosome (CD63) marker
Annexin-V	0.193	0.027	0.189	0.030	−0.077	0.370	−0.201	0.030
CD63	0.161	0.069	0.166	0.059	−0.083	0.347	−0.180	0.055
EVs positive for cellular adhesion/inflammatory molecules
ICAM-1	0.113	0.203	0.103	0.244	−0.183	0.036	0.022	0.818
VCAM-1	−0.016	0.856	−0.012	0.894	−0.133	0.128	0.072	0.441
Tissue factor	0.146	0.097	0.147	0.094	−0.145	0.094	−0.246	0.008
MCP-1	0.188	0.036	0.187	0.037	−0.076	0.392	0.016	0.864

+rho_s, p<0.05: values in bold.

−rho_s, p<0.05: values in italic.

Table VII. Changes of urinary EVs–derived from different types of cells surrounded by renal capillaries, nephron and renal pelvis with nephrosclerosis

	Biopsy measurements
	Cortical fibrosis groups		Number of fibrosis foci		Globally sclerotic glomeruli (%)		Arteriosclerosis (% intimal thickening)
	rho	p	rho	p	rho	p	rho	p
EVs from cells associated with renal capillaries
β-1 AR	0.075	0.623	0.052	0.733	−0.467	0.001	−0.076	0.642
SM22 alpha	0.080	0.376	0.087	0.336	−0.054	0.544	−0.033	0.733
Nephrin	0.106	0.235	0.109	0.222	−0.265	0.002	−0.112	0.236
Podocin	0.008	0.931	0.005	0.955	−0.142	0.107	0.138	0.146
EVs from parietal cells of Bowman's capsule
Claudin-1	0.153	0.089	0.165	0.068	−0.247	0.005	−0.092	0.342
Cytokeratin 8	0.096	0.293	0.110	0.230	−0.093	0.304	0.045	0.643
EVs from epithelial cells of proximal tubule
Megalin	−0.035	0.697	−0.024	0.790	−0.178	0.041	0.015	0.870
URAT-1	−0.098	0.274	0.089	0.321	−0.140	0.114	−0.141	0.139
EVs from epithelial cells of descending and ascending limb of Henle's loop
Aquaporin-1	−0.065	0.492	−0.065	0.492	0.014	0.884	0.128	0.201
EGFR	0.091	0.311	0.102	0.256	−0.025	0.776	−0.074	0.438
Uromodulin	0.126	0.160	0.140	0.120	−0.060	0.503	−0.125	0.192
SLC14A2	0.004	0.964	0.014	0.879	−0.001	0.989	−0.062	0.511
EVs from epithelial cells of distal tubule
Prominin-2	0.006	0.947	−0.002	0.981	−0.205	0.025	−0.062	0.536
SLC12A3	−0.017	0.847	−0.021	0.814	−0.132	0.133	−0.118	0.215
EVs from principal and intercalated cells of collective tubule
Aquaporin-2	−0.044	0.651	−0.042	0.666	−0.187	0.048	−0.083	0.416
V-ATPase	0.048	0.589	0.056	0.526	−0.187	0.032	−0.033	0.725
EVs from epithelial cells of renal pelvis
Cytokeratin19	0.157	0.097	0.156	0.100	−0.006	0.950	0.039	0.698
Cytokeratin 20	0.020	0.827	0.026	0.773	−0.096	0.279	0.069	0.468

−rho_s, p<0.05: values in italic.

Fig. 3.  Shows the overall changes of specific populations of urinary EVs between sexes and with older age, nephron hypertrophy and nephrosclerosis. (−) indicates decrease and (+) indicates increase. Abbreviations/renal cell markers: annexin-V, microvesicles; aquaporin-1, simple squamous epithelium of descending limb of Henle's loop; aquaporin-2, principal cells of collecting tubule; β1-AR (beta-1 adrenergic receptor), juxtaglomerular cells; CD63, exosomes; CK8 (cytokeratin 8), parietal cells; claudin-1, parietal cells; GSG, globally sclerotic glomeruli; ICAM-1, intercellular adhesion molecule-1; megalin, simple cuboidal epithelium of proximal tubule; MCP-1, monocyte chemoattractant protein-1; nephrin, podocytes; NSG, non-sclerotic glomeruli; prominin-2, simple cuboidal epithelium of distal tubule; SM22 alpha, mesangial cells; TF, tissue factor; V-ATPase, intercalated cells of collecting tubule.