Employing extracellular vesicles for non-invasive renal monitoring: A captivating prospect

Table 1 Methods used for the isolation of urinary extracellular vesicles

Technique	Type	Advantage(s)	Disadvantage (s)	Purity	Ref.
Centrifugation	Differential	Easy to perform	Co-purification of protein aggregates (THP) Inability to separate exosomes from microvesicles Lengthy Impractical for large-scale studies	++	[37,44,50,52,69,92]
	Sucrose gradient	No mechanical stress and hence allows the collection of morphologically intact particles Removes THP	High sucrose concentration may affect the biological functions of exosomes Inability to separate exosomes from particles with similar density and size Difficult and lengthy Impractical for large-scale studies	+++++	[23,50,54,55]
	Sucrose cushion	No mechanical stress and hence allows the collection of morphologically intact particles	Inability to separate exosomes from particles with similar density and size Difficult and lengthy Impractical for large-scale studies	+++++	[33,56,57,78,80]
Filtration	Nanofiltration	Easy to perform	Low purity grade Co-purification of protein aggregates (THP) Low exosome yield due to their lost on the surface of the nano-membrane	+++	[94]
	Microfiltration	Easy to perform Rapid Maintain vesicle structure	Co-purification of protein aggregates (THP) Inability to separate exosomes from microvesicles	+++	[59]
Immunoaffinity separation	Immunobeads	Allow rapid semi quantitative characterization of the surface phenotype can be tissue-specific	Not suited for large sample volumes Captured extracellular vesicles may not retain biological functionality even if successfully eluted from bead surface Co-purification of protein aggregates (THP) Low yield	++	[57,61,62]
Commercial kits	Total Exosome Isolation Reagent-Invitrogen™ Exomir™ Exo-Spin™ Exotest™ Exosome Dynabeads® miRCURY™ ExoQuick-TC™ and DiagExo®	Rapid and requires low sample volume Practical	Impractical for large-scale studies Reproducibility, yield and sample quality should be checked case by case Co-purification of protein aggregates (THP)	++

THP: Tamm-Horsfall protein.

Table 2 Putative disease-associated protein and RNA biomarkers identified in urinary extracellular vesicles

Name of the putative marker	UniProt ID/RNA name	Molecular and biological function	Associated Kidney disease(s )	Disease/model	Ref.
Adenine phosphoribosyltransferase	APT_HUMAN	Enzyme involved in the purine nucleotide salvage pathway. Its deficiency can lead to urolithiasis and renal failure	APRT deficiency	Healthy	[62]
Low-density lipoprotein receptor- related protein 2 mRNA	LRP2_HUMAN (megalin), LRP2	Multi-ligand binding receptor; mediates endocytic uptake of complexes between the steroid 25(OH) vitamin D3 and vitamin D-binding protein in kidney proximal tubules	Donnai-Barrow syndrome; Renal aminoglycoside accumulation; Nephrotoxicity	Healthy	[38,78]
Polycystin-1, Polycystin-2	PKD1_HUMAN PKD2_HUMAN	Integral membrane glycoproteins, associated with structural and/or functional defects in the primary apical cilium of epithelia and polycystic kidney disease	ADPKD	Healthy	[45,46,54,62]
Solute carrier family 12 member 2 (Sodium potassium chloride cotransporter-2)	S12A2_HUMAN	Membrane transporter: aids in the active transport of sodium, potassium, and chloride into and out of cells; NKCC2 mutations lead to type I Bartter syndrome	Antenatal Bartter syndrome type 1	Healthy	[33,41,62]
Myosin-9	MYH9_HUMAN	MYH9 polymorphisms have been shown to associate with glomerulosclerosis and non-diabetic end stage renal disease	Fechtner syndrome and Epstein syndrome	Healthy	[62]
Aquaporin-2 mRNA	AQP2_HUMAN AQP2	Molecular water channel in the basolateral and apical plasma membranes of the collecting duct	Autosomal dominant and autosomal recessive nephrogenic diabetes insipidus	Healthy; Various kidney diseases	[24,33,38,39,44 ,54,58,62,95]
Neprilysin (CD10)	NEP_HUMAN	Surface metallo-endopeptidase highly expressed in the kidney brush-border membranes	Membranous glomerulonephritis	Healthy; Diabetic nephropathy, Renal cell carcinoma	[25,33,77,95]
Osteoprotegerin	TR11B_HUMAN	Decoy receptor of proximal tubular cells	chronic kidney disease-mineral and bone disorder	Healthy; ADPKD CKDD patients	[49]
Podocalyxin	PODXL_HUMAN	he major sialoprotein of kidney glomerulus; Involved in the regulation of cell adhesion, cell morphology and cancer progression	Autosomal recessive steroid–resistant nephrotic syndrome	Healthy; Glomerular disease; Renal cell carcinoma	[42,58,62,77,95]
Solute carrier family 12 member 3 (Thiazide- sensitive Na-Cl cotransporter; NCC)	S12A3_HUMAN	Membrane transporter highly expressed in the kidney: aids reabsorbing sodium and chloride ions from the tubular fluid into the cells of the distal convoluted tubule of the nephron	Gitelman syndrome; Aldosteronism	Healthy; High blood pressure; Aldosteronism	[41,62,79]
Protein AMBP (alpha-1-microglobulin; bikunin precursor)	AMBP_HUMAN	Membrane glycoprotein with serine protease inhibitor activity	Diabetic nephropathy	Healthy; DN patients in advanced disease stages	[16,45]
Leucine-rich alpha-2-glycoprotein-1	LRG1_HUMAN	Involved in protein-protein interaction, signal transduction, and cell adhesion and development; Expressed during granulocyte differentiation	NSCLC	Healthy; NSCLC	[71]
Matrix metalloproteinase 9	MMP9_HUMAN	Involved in extracellular matrix remodeling	Renal cell carcinoma	Healthy; Renal cell carcinoma	[77]
Basigin , Extracellular Matrix Metallo- proteinase Inducer	BASI_HUMAN	Involved in extracellular matrix remodeling	Renal cell carcinoma	Healthy; Renal cell carcinoma	[77]
Ceruloplasmin	CERU_HUMAN	A ferroxidase enzyme in serum	Renal cell carcinoma	Healthy; Renal cell carcinoma	[77]
Dickkopf related protein 4	DKK4_HUMAN	Involved in extracellular matrix remodeling	Renal cell carcinoma	Healthy; Renal cell carcinoma	[77]
Carbonic Anhydrase 9	CAH9_HUMAN	A metalloenzyme that catalyzes the reversible hydration of carbon dioxide	Renal cell carcinoma	Healthy; Renal cell carcinoma	[77]
Dipeptidase 1	DPEP1_HUMAN	A kidney membrane enzyme that hydrolyzes a variety of dipeptides and is implicated in renal metabolism of glutathione and its conjugates	Renal cell carcinoma	Healthy; Renal cell carcinoma	[77]
Syntenin-1	SDCB1_HUMAN	Scaffold protein Pbp1 Syndecan-binding protein	Renal cell carcinoma	Healthy; Renal cell carcinoma	[77]
Leucine-rich repeatkinase2	LRRK2	A member of the leucine-rich repeat kinase family; Associated to risks to inflammation-linked diseases that include Crohn’s disease and mycobacterium infection	Parkinson’s disease	Healthy; Parkinson’s disease	[50]
Activating transcription factor 3	ATF3_HUMAN miRNA494	Transcription factor with protective role in renal ischemia-reperfusion injury	AKI	AKI	[22]
Fetuin-A	FETUA_HUMAN	Plasma binding glycoprotein; inhibitor of calcification, regulator of cell-dependent process of osteogenesis	AKI	AKI	[46,96]
Wilm’s tumor protein	WT1_HUMAN	Zinc finger protein with essential role in the normal development of the urogenital system	Early podocyte injury in diabetic nephropathy; steroid responsiveness or renal pathological conditions in patients with idiopathic nephrotic syndrome	FSGS patients; Diabetic Patients with or without Proteinuria; idiopathic nephrotic syndrome	[22,30,92]
Isoform 1 of histone-lysine N-methyltransferase	MLL3_HUMAN	Histonemethyltransferase which methyl- ates “Lys-4” of histone H3, a specific tag for epigenetic transcriptional activation	Diabetic nephropathy	DN patients in advanced disease stages	[16]
Voltage-dependent anion-selective channel protein 1	VDAC1_HUMAN	Porin ion channel of the outer mitochondrial membrane	Diabetic nephropathy	DN patients in advanced disease stages	[16]
GPRC5B	GPC5B_HUMAN	G protein coupled receptor required for tubulogenesis	Acute Kidney Disease	AKD	[94]
Aquaporin-1 mRNA	AQP1_HUMAN AQP1	Molecular water channel in the basolateral and apical plasma membranes of the proximal tubules.	Renal ischaemia reperfusion injury	Renal transplantation; Renal cell carcinoma	[38,77,97]
Neutrophil gelatinase-associated lipocalin	NGAL_HUMAN	Iron-trafficking protein involved in multiple processes such as apoptosis, innate immunity and renal development. In the kidney it is produced by distal nephron	kidney transplantation	kidney allograft recipients	[91]
Immunoglobulin light chain	IGLL1_HUMAN IGKC_HUMAN	Immunoglobulin light chain is the small polypeptide subunit of an antibody	Light Chain Amyloidosis	Light chain amyloidosis; multiple myeloma; monoclonal gammopathy; non-paraproteinemia related kidney disease	[65]
Panel of 24 proteins _HUMAN	SERPINA1, A2M, APOA1, APOA2, CA1, CD5L, DPEP1, FGA, FGB, FGG, FN1, GSTT1, SLC2A1, HBA1, HBB, SERPIND1, HP, HPR, ITIH1, PON1, RAB27B, S100A8, S100A9, TACSTD2	-	Bladder cancer	Bladder cancer; Hernia	[77]
PSA	KLK3_HUMAN	Serine protease with diverse physiological functions; implicated in carcinogenesis	Prostate cancer	Prostate cancer	[56]
Glutamate carboxypeptidase 2 (Prostate-specific membrane antigen-PSMA)	FOLH1_HUMAN	Folate hydrolase and N-acetylated-alpha-linked-acidic dipeptidase activity; Involved in prostate tumor progression	Prostate cancer	Prostate cancer	[56]
microRNA	MicroRNA-29c	-	Renal fibrosis	Healthy; CKD	[40]
microRNA	miR-130A	-	Type 1 diabetes	type 1 diabetic patients with and without incipient diabetic nephropathy	[81]
microRNA	miR-145	-	Type 1 diabetes	type 1 diabetic patients with and without incipient diabetic nephropathy	[81]
microRNA	miR-155	-	Type 1 diabetes	type 1 diabetic patients with and without incipient diabetic nephropathy	[81]
microRNA	miR-424	-	Type 1 diabetes	type 1 diabetic patients with and without incipient diabetic nephropathy	[81]
mRNA (CD2-associated protein)	CD2AP	-	Podocyte injury	Healthy; diabetic nephropathy; focal segmental glomerulosclerosis; IgA nephropathy;membranous nephropathy	[39]
mRNA (Prostate cancer antigen 3)	PCA-3	-	Prostate cancer	Healthy; Prostate cancer	[32]
mRNA (TMPRSS2/ERGa fusion transcript)	TMPRSS2:ERG	-	Prostate cancer	Healthy; Prostate cancer	[32]

CKD: Chronic kidney disease; AKI: Acute kidney injury; PSA: Prostate specific antigen; NSCLC: Non-small cell lung cancer; APRT: Adenine phosphoribosyltransferase deficiency; CD2AP: CD2-associated protein; PCA-3: Prostate cancer antigen 3; ADPKD: Autosomal dominant polycystic kidney disease.