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Exosomes: Advances, development and potential therapeutic strategies in diabetic nephropathy

Open AccessPublished:July 01, 2021DOI:https://doi.org/10.1016/j.metabol.2021.154834

      Highlights

      • Exosomes are involved in the pathophysiological processes associated with diabetic nephropathy.
      • Exosomes play an important role on the crosstalk between the kidney cells.
      • Exosomes potentially provide novel non-invasive biomarkers for diabetic nephropathy, especially urinary exosomes.
      • Currently, exosomes derived from stem cells have shown great therapeutic potential in diabetic nephropathy.

      Abstract

      Exosomes, a major type of extracellular vesicles (EVs), are nanoscale vesicles excreted by almost all cell types via invagination of the endosomal membrane pathway. Exosomes play a crucial role in the mediation of intercellular communication both in health and disease, which can be ascribed to their capacity to be transported to neighboring or distant cells, thus regulating the biological function of recipient cells through cargos such as DNA, mRNA, proteins and microRNA. Diabetic nephropathy (DN) is a serious microvascular complication associated with diabetes mellitus as well as a significant cause of end-stage renal disease worldwide, which has resulted in a substantial economic burden on individuals and society. However, despite extensive efforts, therapeutic approaches that prevent the progression of DN do not exist, which implies new approaches are required. An increasing number of studies suggest that exosomes are involved in the pathophysiological processes associated with DN, which may potentially provide novel biomarkers and therapeutic targets for DN. Hence, this review summarizes recent advances involving exosome mechanisms in DN and their potential as biomarkers and therapeutic targets.

      Abbreviations:

      EVs (extracellular vesicles), DN (diabetic nephropathy), SGLT2 (sodium-glucose co-transporter 2), GLP-1 (glucagon-like peptide-1), MVBs (multivesicular bodies), ESRD (end-stage renal disease), GBM (glomerular basement membrane), GECs (glomerular endothelial cells), GMCs (glomerular mesangial cells), EMT (epithelial-mesenchymal transition), PTECs (proximal tubular epithelial cells), NF-κB (nuclear factor κB), TGF-β (transforming growth factor-β), VEGF (vascular endothelial growth factor), ECM (extracellular matrix), mTOR (mammalian target of rapamycin), AMPK (adenosine monophosphate- activated protein kinase), MSCs (mesenchymal stem cells), ADSCs (adipose-derived stem cells), IR (insulin resistance), hUSCs (human urine-derived stem cells), PPARγ (peroxisome proliferator-activated receptor γ), ATM (adipose tissue macrophages), GLUT4 (glucose transporter 4)

      Keywords

      1. Introduction

      DN, a serious microvascular complication of diabetes, is a chronic, progressive disorder and a common cause of end-stage renal disease (ESRD), which has high morbidity and mortality [
      • Webster A.C.
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      Chronic kidney disease.
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      • Umanath K.
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      Update on diabetic nephropathy: core curriculum 2018.
      ]. Approximately 30% of people with type 1 diabetes and 40% of people with type 2 diabetes will develop DN [
      • Alicic R.Z.
      • Rooney M.T.
      • Tuttle K.R.
      Diabetic kidney disease: challenges, progress, and possibilities.
      ]. The clinical diagnosis of DN chiefly depends on the presence of proteinuria, reduction of estimated glomerular filtration rate (eGFR), and along with a long history of diabetes [

      American Diabetes Association. 11. Microvascular complications and foot care: standards of medical care in diabetes-2020. Diabetes Care. 2020;43:S135-s51. https://doi.org/10.2337/dc20-S011.

      ,
      • Anders H.J.
      • Huber T.B.
      • Isermann B.
      • Schiffer M.
      CKD in diabetes: diabetic kidney disease versus nondiabetic kidney disease.
      ], which will be enhanced by the coexistence of diabetic retinopathy and the exclusion of other chronic kidney diseases [
      • Muskiet M.H.A.
      • Wheeler D.C.
      • Heerspink H.J.L.
      New pharmacological strategies for protecting kidney function in type 2 diabetes.
      ]. In particular, microalbuminuria has been recognized as a conventional predictor for renal diseases including DN; however, in reality, renal function is damaged or even deteriorated before the microalbuminuria is detected [
      • Pavkov M.E.
      • Knowler W.C.
      • Lemley K.V.
      • Mason C.C.
      • Myers B.D.
      • Nelson R.G.
      Early renal function decline in type 2 diabetes.
      ]. Renal biopsy, the gold standard for the diagnosis of kidney disease, is also accompanied with the disadvantages of an invasive procedure and the inability to track disease progression. Therefore, more specific and sensitive biomarkers for the diagnosis of DN are required. To date, DN therapeutic strategies rely on comprehensive control, including amelioration of albuminuria, hyperglycemia, and hypertension, administration of renin–angiotensin–aldosterone system (RAAS) inhibitors, and, new renoprotective drugs [
      • Muskiet M.H.A.
      • Wheeler D.C.
      • Heerspink H.J.L.
      New pharmacological strategies for protecting kidney function in type 2 diabetes.
      ,
      • DeFronzo R.A.
      • Reeves W.B.
      • Awad A.S.
      Pathophysiology of diabetic kidney disease: impact of SGLT2 inhibitors.
      ], such as sodium-glucose co-transporter 2 (SGLT2) inhibitors and glucagon-like peptide-1 (GLP-1). However, there remains a high incidence of ESRD even with these treatments. All in all, it is urgent to explore new approaches to diagnose and treat DN. An increasing number of studies suggest that exosomes are involved in DN [
      • Wen J.
      • Ma Z.
      • Livingston M.J.
      • Zhang W.
      • Yuan Y.
      • Guo C.
      • et al.
      Decreased secretion and profibrotic activity of tubular exosomes in diabetic kidney disease.
      ,
      • Su H.
      • Qiao J.
      • Hu J.
      • Li Y.
      • Lin J.
      • Yu Q.
      • et al.
      Podocyte-derived extracellular vesicles mediate renal proximal tubule cells dedifferentiation via microRNA-221 in diabetic nephropathy.
      ]. In our review, we summarize the potential roles of exosomes on the pathogenesis, biomarkers and therapies in DN.

      2. The biogenesis of exosomes

      Exosomes were first discovered early in 1981 by Trams et al. using a transmission electron microscope; however, only minor structural features (e.g., vesicle-like) were reported at this stage [
      • Trams E.G.
      • Lauter C.J.
      • Salem N.J.
      • Heine U.
      Exfoliation of membrane ecto-enzymes in the form of micro-vesicles.
      ]. Exosomes were first defined more clearly six years later by Johnstone et al. during a study of the processes associated with reticulocyte maturation in sheep [
      • Johnstone R.M.
      • Adam M.
      • Hammond J.R.
      • Orr L.
      • Turbide C.
      Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes).
      ]. However, they were considered little more than “junk” excreted by cells and attracted minimal attention from researchers at the time. Currently, exosomes are widely recognized as extracellular vesicles (EVs) surrounded by lipid bilayers with diameters ranging between ~40 and 160 nm (average, ~100 nm), which are released by a variety of cell types [
      • Kalluri R.
      • LeBleu V.S.
      The biology, function, and biomedical applications of exosomes.
      ]. Exosomes usually originate from the endosomal system via mechanisms including internal budding and invagination of the plasma membrane, and are often accompanied with the formation of multivesicular bodies (MVBs) [
      • Kalluri R.
      • LeBleu V.S.
      The biology, function, and biomedical applications of exosomes.
      ] (Fig. 1). Initially, endosomes are generated by internal budding of the plasma membrane, which leads to the formation of early-sorting endosomes (ESEs) and late-sorting endosomes (LSEs). Next, after sequential or double inward invagination of the plasma membrane, MVBs are formed; this process enables MVBs containing exosome precursors known as intraluminal vesicles (ILVs). As a result, exosomes are excreted into the extracellular space following fusion of the MVBs outer membrane with the cellular plasma membrane and exocytosis [
      • Février B.
      • Raposo G.
      Exosomes: endosomal-derived vesicles shipping extracellular messages.
      ,
      • Kourembanas S.
      Exosomes: vehicles of intercellular signaling, biomarkers, and vectors of cell therapy.
      ]. Regulation of exosome formation follows a mechanism known as endosomal sorting complexes required for transport [
      • Shao H.
      • Im H.
      • Castro C.M.
      • Breakefield X.
      • Weissleder R.
      • Lee H.
      New technologies for analysis of extracellular vesicles.
      ]. Ral GTPases [
      • Hyenne V.
      • Apaydin A.
      • Rodriguez D.
      • Spiegelhalter C.
      • Hoff-Yoessle S.
      • Diem M.
      • et al.
      RAL-1 controls multivesicular body biogenesis and exosome secretion.
      ], Rab GTPases [

      Ostrowski M, Carmo NB, Krumeich S, Fanget I, Raposo G, Savina A, et al. Rab27a and Rab27b control different steps of the exosome secretion pathway. Nat Cell Biol. 2010;12:19–30; sup pp 1–13. https://doi.org/10.1038/ncb2000.

      ] and exosome markers (e.g., CD9, CD81, CD63, TSG101 and flotillin) also participate in exosome biogenesis [
      • Kalluri R.
      • LeBleu V.S.
      The biology, function, and biomedical applications of exosomes.
      ].
      Fig. 1
      Fig. 1The formation of exosomes. Exosomes originate from the endosomal system, including invagination of the plasma membrane, early-sorting endosomes (ESEs), late-sorting endosomes (LSEs), and ultimately multivesicular bodies (MVBs). Subsequently, exosomes can be released to the extracellular space by fusion of MVBs with the cellular plasma membrane.
      Initially, exosomes were only considered to be responsible for the removal of unwanted cellular material such as proteins and lipids. However, the development of research in this field over recent decades has shown that exosomes are involved in the regulation of intercellular communication [
      • Huang-Doran I.
      • Zhang C.Y.
      • Vidal-Puig A.
      Extracellular vesicles: novel mediators of cell communication in metabolic disease.
      ,
      • Valadi H.
      • Ekström K.
      • Bossios A.
      • Sjöstrand M.
      • Lee J.J.
      • Lötvall J.O.
      Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.
      ,
      • Tkach M.
      • Théry C.
      Communication by extracellular vesicles: where we are and where we need to go.
      ]. Depending on the cell from which they originate, exosomes carry complex molecular cargos such as proteins, lipids, RNA, non-coding RNA, microRNA and small RNA, which are carefully controlled by the parent cell allowing information relating to specific cellular functions to pass from parent cell to acceptor cell [
      • Akers J.C.
      • Gonda D.
      • Kim R.
      • Carter B.S.
      • Chen C.C.
      Biogenesis of extracellular vesicles (EV): exosomes, microvesicles, retrovirus-like vesicles, and apoptotic bodies.
      ]. The surface molecules of exosomes vary considerably, which results from the diverse range of cells they originate from, and endow them with high selectivity towards specific receptor cells. The general exosome uptake mechanism by acceptor cells follows three main pathways: membrane fusion with acceptor cells, internalization triggered by targeting to acceptor cells with specific molecules on the surface of exosomes, and endocytosis [
      • Mathieu M.
      • Martin-Jaular L.
      • Lavieu G.
      • Théry C.
      Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication.
      ].
      The varying sizes, content, function, and cellular origin of exosomes contributes to their heterogeneity [
      • Kalluri R.
      • LeBleu V.S.
      The biology, function, and biomedical applications of exosomes.
      ]. Although our understanding of exosome biogenesis, release, uptake, and function are not completely clear [
      • Mathieu M.
      • Martin-Jaular L.
      • Lavieu G.
      • Théry C.
      Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication.
      ,
      • Cocucci E.
      • Meldolesi J.
      Ectosomes and exosomes: shedding the confusion between extracellular vesicles.
      ,
      • Colombo M.
      • Raposo G.
      • Théry C.
      Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles.
      ,
      • van Niel G.
      • D’Angelo G.
      • Raposo G.
      Shedding light on the cell biology of extracellular vesicles.
      ], exosome-focused research remains topical and extensive, which can be ascribed to the potential capability of exosomes in the diagnosis and treatment of various diseases, including neurodegeneration [
      • Yuan L.
      • Li J.Y.
      Exosomes in Parkinson’s disease: current perspectives and future challenges.
      ], metabolic disorders [
      • Castaño C.
      • Kalko S.
      • Novials A.
      • Párrizas M.
      Obesity-associated exosomal miRNAs modulate glucose and lipid metabolism in mice.
      ], cardiovascular dysfunction [
      • Wu R.
      • Gao W.
      • Yao K.
      • Ge J.
      Roles of exosomes derived from immune cells in cardiovascular diseases.
      ], and cancer [
      • Wu H.
      • Fu M.
      • Liu J.
      • Chong W.
      • Fang Z.
      • Du F.
      • et al.
      The role and application of small extracellular vesicles in gastric cancer.
      ]. Furthermore, exosomes have been widely used in targeted drug delivery through surface engineering techniques, which provides effective strategies for diagnosis and treatment, especially in cancer [
      • Liang Y.
      • Duan L.
      • Lu J.
      • Xia J.
      Engineering exosomes for targeted drug delivery.
      ].
      Exosomes are released by most cell types into the extracellular space and can be detected in various body fluids and tissues such as urine, blood, saliva and adipose tissue [
      • Liang Y.
      • Duan L.
      • Lu J.
      • Xia J.
      Engineering exosomes for targeted drug delivery.
      ]. The foundation for research into exosome function is based on the isolation and purification of exosomes. Several advanced techniques have been reported for the isolation and purification of exosomes, including ultracentrifugation, density gradient ultracentrifugation, immunoaffinity capture, size exclusion chromatography, and polymer precipitation [
      • Yang D.
      • Zhang W.
      • Zhang H.
      • Zhang F.
      • Chen L.
      • Ma L.
      • et al.
      Progress, opportunity, and perspective on exosome isolation - efforts for efficient exosome-based theranostics.
      ]. Each technique has unique advantages and disadvantages. For example, ultracentrifugation is the most widely used and is considered the “gold standard” isolation method for exosome extraction, with the capacity to simultaneously extract large sample volumes. However, this method has disadvantages such as lengthy operational times and extensive instrument requirements. Furthermore, the exclusion of microvesicles is common using this method, which leads to impure isolated exosomes [
      • Ferguson S.W.
      • Nguyen J.
      Exosomes as therapeutics: the implications of molecular composition and exosomal heterogeneity.
      ]. Immunoaffinity capture techniques rely on specific binding between markers and antibodies and are suitable for the isolation of exosomes from specific sources; shortcomings include expensive antibodies and low productivity [
      • Yang D.
      • Zhang W.
      • Zhang H.
      • Zhang F.
      • Chen L.
      • Ma L.
      • et al.
      Progress, opportunity, and perspective on exosome isolation - efforts for efficient exosome-based theranostics.
      ]. As such, due to the heterogeneity of exosomes, thus far no exosome isolation method has been consistently recommended by researchers [
      • Ludwig N.
      • Whiteside T.L.
      • Reichert T.E.
      Challenges in exosome isolation and analysis in health and disease.
      ]. The choice of which technique to use is driven by a multitude of factors including starting matrix (cell culture media versus biofluid), starting volume and downstream analyses and research question. Additionally, after exosome isolation, exosome characterization is required using a combination of transmission electron microscopy and protein markers, such as CD63, CD9 and CD81 [

      Lötvall J, Hill AF, Hochberg F, Buzás EI, Di Vizio D, Gardiner C, et al. Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles. J Extracell Vesicles. 2014;3:26913. https://doi.org/10.3402/jev.v3.26913.

      ]. In summary, the extraction of exosomes remains a challenge, which requires further improvement by researchers.

      3. Exosomes in DN pathogenesis

      The main pathological hallmarks of renal injury in diabetes include functional (such as proteinuria and glomerular filtration rate decline) and structural (extracellular matrix accumulation, glomerular basement membrane thickening, podocyte injury and depletion, tubulointerstitial inflammation, and fibrosis) changes [
      • Alicic R.Z.
      • Rooney M.T.
      • Tuttle K.R.
      Diabetic kidney disease: challenges, progress, and possibilities.
      ]. Unfortunately, knowledge surrounding the multiple and complicated pathological mechanisms remains limited. Long-term hyperglycemia is the main contributory factor in the development of DN; however, strict glycemic management may not always slow or prevent the progression of DN [
      • DeFronzo R.A.
      • Reeves W.B.
      • Awad A.S.
      Pathophysiology of diabetic kidney disease: impact of SGLT2 inhibitors.
      ]. Other factors underlying the mechanisms of DN, including inflammation, fibrosis, mitochondrial dysfunction, impaired autophagy and oxidative stress, also play a pivotal role in the progression of DN [
      • DeFronzo R.A.
      • Reeves W.B.
      • Awad A.S.
      Pathophysiology of diabetic kidney disease: impact of SGLT2 inhibitors.
      ]. In addition, emerging evidence implicates abnormal crosstalk between renal cells in the pathogenesis of DN [
      • Chen S.J.
      • Lv L.L.
      • Liu B.C.
      • Tang R.N.
      Crosstalk between tubular epithelial cells and glomerular endothelial cells in diabetic kidney disease.
      ].
      Nevertheless, despite the currently available therapies the residual risk of DN progression remains high [
      • Muskiet M.H.A.
      • Wheeler D.C.
      • Heerspink H.J.L.
      New pharmacological strategies for protecting kidney function in type 2 diabetes.
      ], indicating additional potential molecular mechanisms contributing to DN progression that require further investigation. The stimulation of renal cells with high concentrations of glucose alters the composition and communication, which further modifies and injures intact cells, indicating that exosomes may participate in the pathophysiological mechanisms of diabetic kidney disease [
      • da Silva Novaes A.
      • Borges F.T.
      • Maquigussa E.
      • Varela V.A.
      • Dias M.V.S.
      • Boim M.A.
      Influence of high glucose on mesangial cell-derived exosome composition, secretion and cell communication.
      ].

      3.1 Exosomes and intercellular communication

      Exosomes are cell-derived, membrane-bound delivery vesicles, which play a crucial role in the regulation of cellular biofunction by mediating cell-to-cell communication via the transfer of cargos including microRNA, mRNA and proteins [
      • Yang B.
      • Chen Y.
      • Shi J.
      Exosome biochemistry and advanced nanotechnology for next-generation theranostic platforms.
      ]. Evidence exists that these cargos are carried into neighboring or distant cells to mediate the biofunction of recipient cells [
      • Karpman D.
      • Ståhl A.L.
      • Arvidsson I.
      Extracellular vesicles in renal disease.
      ]. Recent studies also indicate that exosomes transfer their cargos between the kidney cells [
      • Gildea J.J.
      • Seaton J.E.
      • Victor K.G.
      • Reyes C.M.
      • Bigler Wang D.
      • Pettigrew A.C.
      • et al.
      Exosomal transfer from human renal proximal tubule cells to distal tubule and collecting duct cells.
      ] (Fig. 2).
      Fig. 2
      Fig. 2The mechanisms of exosomes in DN through cell-cell crosstalk. Previous studies implied that exosomes involve in cell to cell crosstalk in kidney in the context of high glucose, such as glomerular endothelial cells (GECs) to glomerular mesangial cells (GMCs),GECs to podocytes, GMCs to podocytes, tubular epithelial cells (TECs) to fibroblasts, TECs to macrophages, TECs to GECs and podocytes to TECs, which contribute to kidney injury, including epithelial-mesenchymal transition (EMT), extracellular matrix (ECM) overproduction, cell apoptosis, inflammation, and eventually renal fibrosis.
      An important component of the glomerular filtration barrier, glomerular endothelial cells (GECs) have been reported to participate in the pathogenesis of DN through crosstalk with other glomerular cells, such as glomerular mesangial cells (GMCs) and podocytes [
      • Fu J.
      • Lee K.
      • Chuang P.Y.
      • Liu Z.
      • He J.C.
      Glomerular endothelial cell injury and cross talk in diabetic kidney disease.
      ]. However, the delicate molecular mechanisms underlying these interactions are poorly understood. Recently, researchers have found exosomes secreted from high glucose-induced GECs delivered circRNA or mRNA to GMCs, which leads to the activation, proliferation and extracellular matrix (ECM) protein overproduction of GMCs and promotes renal fibrosis [
      • Ling L.
      • Tan Z.
      • Zhang C.
      • Gui S.
      • Cui Y.
      • Hu Y.
      • et al.
      CircRNAs in exosomes from high glucose-treated glomerular endothelial cells activate mesangial cells.
      ,
      • Wu X.M.
      • Gao Y.B.
      • Cui F.Q.
      • Zhang N.
      Exosomes from high glucose-treated glomerular endothelial cells activate mesangial cells to promote renal fibrosis.
      ]. Similarly, under high glucose conditions, GECs-derived exosomes, enriched in transforming growth factor-β (TGF-β) mRNA, lead to the epithelial-mesenchymal transition (EMT) and dysfunction of podocyte via Wnt/β-catenin signaling pathway [
      • Wu X.
      • Gao Y.
      • Xu L.
      • Dang W.
      • Yan H.
      • Zou D.
      • et al.
      Exosomes from high glucose-treated glomerular endothelial cells trigger the epithelial-mesenchymal transition and dysfunction of podocytes.
      ]. Furthermore, due to a lack of proliferative capacity when confronted with injury, podocyte apoptosis and depletion is a hallmark of DN [

      Anil Kumar P, Welsh GI, Saleem MA, Menon RK. Molecular and cellular events mediating glomerular podocyte dysfunction and depletion in diabetes mellitus. Front Endocrinol (Lausanne). 2014;5:151. https://doi.org/10.3389/fendo.2014.00151.

      ]. A previous study demonstrated that HG-activated GMCs interact with podocytes through secreting exosomes, thus resulting in podocyte apoptosis, and that TGF-β/PI3K-AKT signaling maybe implicated in this process [
      • Wang Y.Y.
      • Tang L.Q.
      • Wei W.
      Berberine attenuates podocytes injury caused by exosomes derived from high glucose-induced mesangial cells through TGFβ1-PI3K/AKT pathway.
      ].
      In addition, tubular epithelial cells (TECs) and their interaction with fibroblasts contribute to interstitial fibrosis in kidney diseases, such as DN [
      • Humphreys B.D.
      Mechanisms of renal fibrosis.
      ,
      • Venkatachalam M.A.
      • Weinberg J.M.
      • Kriz W.
      • Bidani A.K.
      Failed tubule recovery, AKI-CKD transition, and kidney disease progression.
      ,
      • Liu B.C.
      • Tang T.T.
      • Lv L.L.
      • Lan H.Y.
      Renal tubule injury: a driving force toward chronic kidney disease.
      ]. Wen et al. [
      • Wen J.
      • Ma Z.
      • Livingston M.J.
      • Zhang W.
      • Yuan Y.
      • Guo C.
      • et al.
      Decreased secretion and profibrotic activity of tubular exosomes in diabetic kidney disease.
      ] found that exosomes extracted from HG-treated renal tubular cells have the capacity to stimulate the transformation of fibroblasts into myofibroblasts in renal, suggesting that exosomes may involve in the communication between the two cell types. Likewise, dedifferentiation of renal proximal tubular epithelial cells (PTECs) also plays a crucial role in interstitial fibrosis. After treatment with high glucose, podocyte-derived EVs induced dedifferentiation of PTECs through transporting miR-221-3p via the Wnt/β-catenin signaling pathway [
      • Su H.
      • Qiao J.
      • Hu J.
      • Li Y.
      • Lin J.
      • Yu Q.
      • et al.
      Podocyte-derived extracellular vesicles mediate renal proximal tubule cells dedifferentiation via microRNA-221 in diabetic nephropathy.
      ]. Moreover, evidence exists that implies TECs exosomal miR-19b-3p mediates the crosstalk between TECs and macrophages as well as contributes to the activation of M1 macrophages by targeting nuclear factor κB (NF-κB)/SOCS-1, which accounts for tubulointerstitial inflammation [
      • Lv L.L.
      • Feng Y.
      • Wu M.
      • Wang B.
      • Li Z.L.
      • Zhong X.
      • et al.
      Exosomal miRNA-19b-3p of tubular epithelial cells promotes M1 macrophage activation in kidney injury.
      ]. TECs also secrete EVs, which stimulates an inflammatory response through NF-κB and induces GECs injury [
      • Chen S.J.
      • Lv L.L.
      • Liu B.C.
      • Tang R.N.
      Crosstalk between tubular epithelial cells and glomerular endothelial cells in diabetic kidney disease.
      ].

      3.2 Exosomes and inflammation

      Inflammatory responses are commonly characterized by the release of proinflammatory cytokines, chemokines, adhesion molecules and inflammatory signal transduction [
      • Alicic R.Z.
      • Johnson E.J.
      • Tuttle K.R.
      Inflammatory mechanisms as new biomarkers and therapeutic targets for diabetic kidney disease.
      ]. Initially, DN was considered a non-inflammatory disease; however, mounting evidence indicates that chronic inflammatory stimulation contributes to the initiation and ongoing injury caused by DN [
      • Alicic R.Z.
      • Johnson E.J.
      • Tuttle K.R.
      Inflammatory mechanisms as new biomarkers and therapeutic targets for diabetic kidney disease.
      ,
      • Van J.A.
      • Scholey J.W.
      • Konvalinka A.
      Insights into diabetic kidney disease using urinary proteomics and bioinformatics.
      ,
      • Navarro-González J.F.
      Mora-Fernández C, Muros de Fuentes M, García-Pérez J. inflammatory molecules and pathways in the pathogenesis of diabetic nephropathy.
      ]. Specifically, macrophages, can be classified as: M1 macrophages (mainly secrete pro-inflammatory mediators) and M2 macrophages (that possess anti-inflammatory functions), and are types of immune inflammatory cells closely associated with the regulation of inflammatory processes [
      • Viola A.
      • Munari F.
      • Sánchez-Rodríguez R.
      • Scolaro T.
      • Castegna A.
      The metabolic signature of macrophage responses.
      ]. Macrophages migrate and infiltrate the kidney tissue during incipient DN [
      • Nguyen D.
      • Ping F.
      • Mu W.
      • Hill P.
      • Atkins R.C.
      • Chadban S.J.
      Macrophage accumulation in human progressive diabetic nephropathy.
      ], which promotes the release of TGF-β, reactive oxygen species (ROS), vascular endothelial growth factor (VEGF) and cytokines such as IL-1 and TNF [
      • Pérez-Morales R.E.
      • Del Pino M.D.
      • Valdivielso J.M.
      • Ortiz A.
      • Mora-Fernández C.
      • Navarro-González J.F.
      Inflammation in diabetic kidney disease.
      ], subsequently accelerating the progression of DN; however, the mechanisms require clarification [
      • Hickey F.B.
      • Martin F.
      Role of the immune system in diabetic kidney disease.
      ]. In contrast, activation of TGF-β1 and IL-1β drives macrophages activation and accumulation, thus a negative feedback loop occurs. Recent studies have also reported that EVs may also mediate these mechanisms. After treatment with high glucose, mouse macrophage-derived EVs miR-21-5p, elevated the levels of inflammasome NLRP3 and IL-1β, thereby causing podocyte pyroptosis through targeting A20, both in DN model mice and in vitro [
      • Ding X.
      • Jing N.
      • Shen A.
      • Guo F.
      • Song Y.
      • Pan M.
      • et al.
      MiR-21-5p in macrophage-derived extracellular vesicles affects podocyte pyroptosis in diabetic nephropathy by regulating A20.
      ]. NF-κB, a ubiquitous transcription factor, is one major component of inflammatory reactions [
      • Navarro-González J.F.
      Mora-Fernández C, Muros de Fuentes M, García-Pérez J. inflammatory molecules and pathways in the pathogenesis of diabetic nephropathy.
      ]. Research has indicated that in the context of high glucose concentrations, macrophage-related exosomes induce macrophages activation, proliferation, and the secretion of inflammatory factors and pro-fibrotic cytokines, including IL-1β, TNF-α and TGF-β1 via regulating the NF-κB p65 pathway, resulting in the kidney injury and inflammation [
      • Zhu M.
      • Sun X.
      • Qi X.
      • Xia L.
      • Wu Y.
      Exosomes from high glucose-treated macrophages activate macrophages and induce inflammatory responses via NF-κB signaling pathway in vitro and in vivo.
      ]. Another study conducted by the same team also reported that these exosomes can be ingested and internalized by mesangial cell and trigger mesangial cell activation, extracellular matrix (ECM) excessive deposition, and inflammatory factors release, such as IL-1β and TNF-α through TGF-β1/Smad3 signaling, indicating such alterations contribute to mesangial expansion and renal fibrosis [
      • Zhu Q.J.
      • Zhu M.
      • Xu X.X.
      • Meng X.M.
      • Wu Y.G.
      Exosomes from high glucose-treated macrophages activate glomerular mesangial cells via TGF-β1/Smad3 pathway in vivo and in vitro.
      ]. As described above [
      • Lv L.L.
      • Feng Y.
      • Wu M.
      • Wang B.
      • Li Z.L.
      • Zhong X.
      • et al.
      Exosomal miRNA-19b-3p of tubular epithelial cells promotes M1 macrophage activation in kidney injury.
      ], in mice models of acute and chronic kidney injury, exosomes derived from mouse TECs also drive activating macrophage by targeting NF-κB, thus resulting in tubulointerstitial inflammation.

      3.3 Exosomes and autophagy

      Autophagy is characterized as a cellular self-protection mechanism involving the degradation of intracellular materials such as organelles and proteins, which depends on the presence of proteolytic enzymes in lysosomes, and recycled as energy sources [
      • DeFronzo R.A.
      • Reeves W.B.
      • Awad A.S.
      Pathophysiology of diabetic kidney disease: impact of SGLT2 inhibitors.
      ,
      • Kimura T.
      • Isaka Y.
      • Yoshimori T.
      Autophagy and kidney inflammation.
      ]. As such, autophagy plays a significant role in normal cell function, survival and homeostasis [
      • Choi A.M.
      • Ryter S.W.
      • Levine B.
      Autophagy in human health and disease.
      ]. Autophagic activity is precisely regulated by signaling pathways that mainly include one negative regulator- mammalian target of rapamycin (mTOR), two positive regulators- adenosine monophosphate-activated protein kinase (AMPK) and sirtuins [
      • Tang C.
      • Livingston M.J.
      • Liu Z.
      • Dong Z.
      Autophagy in kidney homeostasis and disease.
      ]. Especially, under energy depletion (e.g., starvation), increased autophagy is crucial to meet cell energy demands [
      • DeFronzo R.A.
      • Reeves W.B.
      • Awad A.S.
      Pathophysiology of diabetic kidney disease: impact of SGLT2 inhibitors.
      ]. In addition, the process of autophagy is initiated from the formation of the phagophore, then requires the formation of autophagosome, which has a double membrane framework, and autophagosome-lysosome fusion, thus resulting in degradation of the materials enclosed in the autophagosome [
      • Yu L.
      • Chen Y.
      • Tooze S.A.
      Autophagy pathway: cellular and molecular mechanisms.
      ].
      The essential roles of autophagy in the pathogenesis of DN have been previously reported [
      • Tagawa A.
      • Yasuda M.
      • Kume S.
      • Yamahara K.
      • Nakazawa J.
      • Chin-Kanasaki M.
      • et al.
      Impaired podocyte autophagy exacerbates proteinuria in diabetic nephropathy.
      ]. For instance, diabetic model mice developed podocyte-specific, autophagy-deficient aggravated podocyte damage and proteinuria after receiving a high-fat diet [
      • Tagawa A.
      • Yasuda M.
      • Kume S.
      • Yamahara K.
      • Nakazawa J.
      • Chin-Kanasaki M.
      • et al.
      Impaired podocyte autophagy exacerbates proteinuria in diabetic nephropathy.
      ]. In streptozotocin (STZ)-induced mice, increased autophagy was observed in PTECs which could offset mitochondrial dysfunction and fibrosis [
      • Sakai S.
      • Yamamoto T.
      • Takabatake Y.
      • Takahashi A.
      • Namba-Hamano T.
      • Minami S.
      • et al.
      Proximal tubule autophagy differs in type 1 and 2 diabetes.
      ]. Of note, autophagic activity showed different outcomes in type 1 and type 2 diabetes [
      • Sakai S.
      • Yamamoto T.
      • Takabatake Y.
      • Takahashi A.
      • Namba-Hamano T.
      • Minami S.
      • et al.
      Proximal tubule autophagy differs in type 1 and 2 diabetes.
      ,
      • He Q.
      • Wang L.
      • Zhao R.
      • Yan F.
      • Sha S.
      • Cui C.
      • et al.
      Mesenchymal stem cell-derived exosomes exert ameliorative effects in type 2 diabetes by improving hepatic glucose and lipid metabolism via enhancing autophagy.
      ]; that is, autophagic kidney activity was enhanced in type 1 diabetes (include STZ-induced mice and Akita mice), which exerted a protective effect, whereas, autophagic induction was suppressed in db/db mice and high-fat diet-induced rats' models (both are type 2 diabetes), which insults the kidney. Similarly, short-term exposure of podocytes in a high glucose environment may trigger autophagy and ameliorate the kidney lesions; conversely, prolonged hyperglycemia may inhibit autophagy in podocytes, thus accounting for the progression of DN [
      • Tang C.
      • Livingston M.J.
      • Liu Z.
      • Dong Z.
      Autophagy in kidney homeostasis and disease.
      ].
      As the key regulator of autophagy, mTORC1 activated by high glucose contributes to the kidney damage through suppressing autophagy [
      • Huang C.
      • Zhang Y.
      • Kelly D.J.
      • Tan C.Y.
      • Gill A.
      • Cheng D.
      • et al.
      Thioredoxin interacting protein (TXNIP) regulates tubular autophagy and mitophagy in diabetic nephropathy through the mTOR signaling pathway.
      ]. However, exosomes secreted from mesenchymal stem cells (MSCs) induced autophagy by suppressing the mTOR pathway, thus attenuating renal injury in rats' models of diabetes [
      • Ebrahim N.
      • Ahmed I.A.
      • Hussien N.I.
      • Dessouky A.A.
      • Farid A.S.
      • Elshazly A.M.
      • et al.
      Mesenchymal stem cell-derived exosomes ameliorated diabetic nephropathy by autophagy induction through the mTOR signaling pathway.
      ]. A similar effect was observed for exosomes extracted from adipose-derived stem cells (ADSCs) [
      • Jin J.
      • Shi Y.
      • Gong J.
      • Zhao L.
      • Li Y.
      • He Q.
      • et al.
      Exosome secreted from adipose-derived stem cells attenuates diabetic nephropathy by promoting autophagy flux and inhibiting apoptosis in podocyte.
      ]. Previous studies have also indicated that autophagy deficiency in adipose tissue may induce increased exosome production, which promotes the development of obesity and insulin resistance (IR) [
      • Li F.
      • Li H.
      • Jin X.
      • Zhang Y.
      • Kang X.
      • Zhang Z.
      • et al.
      Adipose-specific knockdown of Sirt1 results in obesity and insulin resistance by promoting exosomes release.
      ].

      3.4 Exosomes and insulin resistance

      A typical feature and an independent risk factor of type 2 diabetes, IR is characterized by a decreased glucose uptake and utilization efficiency accompanied with an increase in insulin concentration. Generally, IR exists long before type 2 diabetes is diagnosed. Increased insulin resistance can also be observed in type 1 diabetes, which is classically defined as an absolute insulin deficiency, when microalbuminuria is present. Therefore, it follows that the severity of IR strongly correlates with DN processes [
      • Karalliedde J.
      • Gnudi L.
      Diabetes mellitus, a complex and heterogeneous disease, and the role of insulin resistance as a determinant of diabetic kidney disease.
      ]. As well as the well-known insulin-targeted organs (e.g., liver, white adipose tissue, and skeletal muscle), almost all kidney cells including GMCs, podocytes, and tubular cells, respond to insulin stimulation. In the context of IR triggered by high glucose or obesity, these renal cells are affected, which results in reduced podocyte viability and tubular function [
      • Artunc F.
      • Schleicher E.
      • Weigert C.
      • Fritsche A.
      • Stefan N.
      • Häring H.U.
      The impact of insulin resistance on the kidney and vasculature.
      ].
      In addition, exosomes are believed to participate in the mediation of insulin sensitivity associated with obesity or diabetes [

      Ying W, Riopel M, Bandyopadhyay G, Dong Y, Birmingham A, Seo JB, et al. Adipose tissue macrophage-derived exosomal miRNAs can modulate in vivo and in vitro insulin sensitivity. Cell. 2017;171:372–84.e12. https://doi.org/10.1016/j.cell.2017.08.035.

      ,
      • Ge Q.
      • Xie X.X.
      • Xiao X.
      • Li X.
      Exosome-like vesicles as new mediators and therapeutic targets for treating insulin resistance and β-cell mass failure in type 2 diabetes mellitus.
      ]. Protein kinase B (also known as Akt) and peroxisome proliferator-activated receptor γ (PPARγ) are potent insulin signaling regulators. For instance, in 2017, a study by Ying W et al. focusing on lean and obese mice showed that exosomes obtained from adipose tissue macrophages (ATM) can regulate insulin sensitivity and glucose tolerance through insulin pathway. The lean mice exhibited IR and glucose intolerance after administration of obese ATM-exosomes via alterations to the insulin signaling pathway, including decreased phosphorylation of Akt, suppressed expression of PPARγ and its target gene, glucose transporter 4 (GLUT4). Conversely, the obese mice treated with ATM-exosomes from lean mice manifested an improvement in IR and an alleviation of glucose tolerance [

      Ying W, Riopel M, Bandyopadhyay G, Dong Y, Birmingham A, Seo JB, et al. Adipose tissue macrophage-derived exosomal miRNAs can modulate in vivo and in vitro insulin sensitivity. Cell. 2017;171:372–84.e12. https://doi.org/10.1016/j.cell.2017.08.035.

      ]. There is also evidence that suggests elevation of podocyte-derived EVs containing nephrin and podocalyxin in the urine may be considered early markers of kidney disease associated with IR [
      • Zhang L.H.
      • Zhu X.Y.
      • Eirin A.
      • Nargesi A.A.
      • Woollard J.R.
      • Santelli A.
      • et al.
      Early podocyte injury and elevated levels of urinary podocyte-derived extracellular vesicles in swine with metabolic syndrome: role of podocyte mitochondria.
      ]. Overall, there remains barely publications with regard to the mechanism of interaction between exosomes with IR in DN.

      4. Exosomes as potential biomarkers of DN

      The employment of exosomes as liquid biopsies is especially promising due to their ubiquitous presence in biological fluids and potential for multicomponent analyses [
      • Kalluri R.
      The biology and function of exosomes in cancer.
      ]. Lv et al. [
      • Lv L.L.
      • Cao Y.
      • Liu D.
      • Xu M.
      • Liu H.
      • Tang R.N.
      • et al.
      Isolation and quantification of microRNAs from urinary exosomes/microvesicles for biomarker discovery.
      ] found exosomes was stable at 4 °C 24 h for shipping before stored at −80 °C and was stable in urine when stored at −80 °C for 12 months. Exosomal miRNA was detectable despite 5 repeat freeze-thaw cycles. An increasing body of literature is emerging that addresses urinary exosomes as potential non-invasive biomarkers for the diagnosis of DN.
      microRNAs (miRNAs) packaged in exosomes are considered a form of cell-to-cell communication and have received increasing attention over the past decade. miRNAs are small, stable, non-coding, single-stranded RNAs which negatively regulate gene and protein expression at a post-transcriptional level [
      • Lorenzen J.M.
      • Thum T.
      Circulating and urinary microRNAs in kidney disease.
      ]. Cheng et al. [
      • Cheng L.
      • Sun X.
      • Scicluna B.J.
      • Coleman B.M.
      • Hill A.F.
      Characterization and deep sequencing analysis of exosomal and non-exosomal miRNA in human urine.
      ] reported that miRNAs were found to be significantly enriched and intact in urine-derived exosomes compared with cell-free urine. To date, an increasing number of differentially-expressed miRNAs have been found in exosomes extracted from urine obtained from animals' models as well as patients with DN (Table 1). Previous studies have demonstrated that in both type 1 DN(defined as persistent microalbuminuria) and type 2 diabetic patients with DN (eGFR<60 ml/min/1.73m2), the urinary exosomal miRNA content was altered and that urinary exosomal miR-145-5p [
      • Barutta F.
      • Tricarico M.
      • Corbelli A.
      • Annaratone L.
      • Pinach S.
      • Grimaldi S.
      • et al.
      Urinary exosomal microRNAs in incipient diabetic nephropathy.
      ] or miR-320c [
      • Delić D.
      • Eisele C.
      • Schmid R.
      • Baum P.
      • Wiech F.
      • Gerl M.
      • et al.
      Urinary exosomal miRNA signature in type II diabetic nephropathy patients.
      ] levels were higher, thus indicating their potential as novel biomarkers in the diagnosis of DN. Eissa et al. [
      • Eissa S.
      • Matboli M.
      • Bekhet M.M.
      Clinical verification of a novel urinary microRNA panal: 133b, -342 and -30 as biomarkers for diabetic nephropathy identified by bioinformatics analysis.
      ] observed significantly increased levels of urinary exosomal miR-133b-3p, miR-342-3p, and miR-30a-5p in patients of type 2 diabetes with micro- or macro- albuminuria. Meanwhile, these miRNAs were also altered in diabetic patients with normoalbuminuria, indicating their potential as early biomarkers in diagnosis of DN. Another study found three miRNAs isolated from urinary exosomes (miR-362-3p, miR-877-3p, and miR-150-5p) were increased and one (miR-15a-5p) was decreased in diabetic patients with macroalbuminuria compared to that of patients with type 2 diabetes. Furthermore, these miRNAs might mediate DN processes via the p53, mTOR, and AMPK pathways, indicating their potential capability in the early diagnosis of DN [
      • Xie Y.
      • Jia Y.
      • Cuihua X.
      • Hu F.
      • Xue M.
      • Xue Y.
      Urinary exosomal microRNA profiling in incipient type 2 diabetic kidney disease.
      ]. In a recent study, when compared to patients with type 2 diabetes, a notable upregulation in miR-21-5p, let-7e-5p and miR-23b-3p was observed in urinary samples from patients with type 2 DN (eGFR <60 ml/min/1.73m2); in contrast, miR-30b-5p and miR-125b-5p expression was significantly decreased in type 2 DN [
      • Zang J.
      • Maxwell A.P.
      • Simpson D.A.
      • McKay G.J.
      Differential expression of urinary exosomal microRNAs miR-21-5p and miR-30b-5p in individuals with diabetic kidney disease.
      ]. Similarly, two of the most prevalent upregulation miRNAs, namely miR-188-5p and miR-150-3p, were found in the urinary exosomes of DN patients who underwent a renal biopsy, in contrast, the miR-153-3p levels were downregulated [
      • Lee W.C.
      • Li L.C.
      • Ng H.Y.
      • Lin P.T.
      • Chiou T.T.
      • Kuo W.H.
      • et al.
      Urinary exosomal microRNA signatures in nephrotic, biopsy-proven diabetic nephropathy.
      ]. Last year, Tsai et al. [
      • Tsai Y.C.
      • Kuo M.C.
      • Hung W.W.
      • Wu L.Y.
      • Wu P.H.
      • Chang W.A.
      • et al.
      High glucose induces mesangial cell apoptosis through miR-15b-5p and promotes diabetic nephropathy by extracellular vesicle delivery.
      ] also investigated that miR-15b-5p harvested from EVs in urine was higher in type 2 diabetic patients; moreover, the levels were correlated with the urinary albumin-to-creatinine ratio, enabling it to be used as a novel indicator to forecast the severity of kidney damage in DN. Significantly, another study reported that levels of miR-451-5p in urinary exosomes were increased in diabetic rats while they were decreased in the kidneys of diabetic rats, implying this change is involved in kidney damage; moreover, the detection of increased miR-451-5p in urinary exosomes may offer a sensitive biomarker for incipient diabetic kidney disease instead of monitoring albumin excretion levels [
      • Mohan A.
      • Singh R.S.
      • Kumari M.
      • Garg D.
      • Upadhyay A.
      • Ecelbarger C.M.
      • et al.
      Urinary exosomal microRNA-451-5p is a potential early biomarker of diabetic nephropathy in rats.
      ]. In addition to the analysis of urine, reports relating to serum exosome analysis also exist. In 2019, Kim and his team reported that in comparison to healthy controls, circulating miRNAs (e.g. miR-1246, miR-642a-3p, let-7c-5p, miR-1255b-5p, miR-5010-5p, miR-150-3p, let-7i-3p) were significantly up-regulated in DN patients with micro- or macro-albuminuria compared to patients without DN, suggesting the potential of these miRNAs in the diagnosis and treatment of DN [
      • Kim H.
      • Bae Y.U.
      • Jeon J.S.
      • Noh H.
      • Park H.K.
      • Byun D.W.
      • et al.
      The circulating exosomal microRNAs related to albuminuria in patients with diabetic nephropathy.
      ].
      Table 1miRNAs in urinary exosomes as potential biomarkers of DN.
      miRNAsLevelType of DNSpeciesReference
      miR-145-5pType 1 DNHuman, mice, cell[
      • Barutta F.
      • Tricarico M.
      • Corbelli A.
      • Annaratone L.
      • Pinach S.
      • Grimaldi S.
      • et al.
      Urinary exosomal microRNAs in incipient diabetic nephropathy.
      ]
      miR-320cType 2 DNHuman[
      • Delić D.
      • Eisele C.
      • Schmid R.
      • Baum P.
      • Wiech F.
      • Gerl M.
      • et al.
      Urinary exosomal miRNA signature in type II diabetic nephropathy patients.
      ]
      miR-133b-3p, miR-342-3p, miR-30a-5pType 2 DNHuman[
      • Eissa S.
      • Matboli M.
      • Bekhet M.M.
      Clinical verification of a novel urinary microRNA panal: 133b, -342 and -30 as biomarkers for diabetic nephropathy identified by bioinformatics analysis.
      ]
      miR-362-3p, miR-877-3p, miR-150-5pType 2 DNHuman[
      • Xie Y.
      • Jia Y.
      • Cuihua X.
      • Hu F.
      • Xue M.
      • Xue Y.
      Urinary exosomal microRNA profiling in incipient type 2 diabetic kidney disease.
      ]
      miR-15a-5pType 2 DNHuman[
      • Xie Y.
      • Jia Y.
      • Cuihua X.
      • Hu F.
      • Xue M.
      • Xue Y.
      Urinary exosomal microRNA profiling in incipient type 2 diabetic kidney disease.
      ]
      miR-21-5p, let-7e-5p, miR-23b-3pType 2 DNHuman[
      • Zang J.
      • Maxwell A.P.
      • Simpson D.A.
      • McKay G.J.
      Differential expression of urinary exosomal microRNAs miR-21-5p and miR-30b-5p in individuals with diabetic kidney disease.
      ]
      miR-30b-5p, miR-125b-5pType 2 DNHuman[
      • Zang J.
      • Maxwell A.P.
      • Simpson D.A.
      • McKay G.J.
      Differential expression of urinary exosomal microRNAs miR-21-5p and miR-30b-5p in individuals with diabetic kidney disease.
      ]
      miR-188-5p, miR-150-3pHuman[
      • Lee W.C.
      • Li L.C.
      • Ng H.Y.
      • Lin P.T.
      • Chiou T.T.
      • Kuo W.H.
      • et al.
      Urinary exosomal microRNA signatures in nephrotic, biopsy-proven diabetic nephropathy.
      ]
      miR-153-3pHuman[
      • Lee W.C.
      • Li L.C.
      • Ng H.Y.
      • Lin P.T.
      • Chiou T.T.
      • Kuo W.H.
      • et al.
      Urinary exosomal microRNA signatures in nephrotic, biopsy-proven diabetic nephropathy.
      ]
      miR-15b-5pType 2 DNHuman, cell, rat[
      • Tsai Y.C.
      • Kuo M.C.
      • Hung W.W.
      • Wu L.Y.
      • Wu P.H.
      • Chang W.A.
      • et al.
      High glucose induces mesangial cell apoptosis through miR-15b-5p and promotes diabetic nephropathy by extracellular vesicle delivery.
      ]
      miR-451-5pType 1 DNRat[
      • Mohan A.
      • Singh R.S.
      • Kumari M.
      • Garg D.
      • Upadhyay A.
      • Ecelbarger C.M.
      • et al.
      Urinary exosomal microRNA-451-5p is a potential early biomarker of diabetic nephropathy in rats.
      ]
      Abbreviation: DN: Diabetic nephropathy; ↑: Increased; ↓: Decreased.
      Proteomic studies of urinary exosomes have suggested that proteins carried within exosomes may also be candidate biomarkers for DN. For instance, the levels of Xaa-Pro dipeptidase (or Prolidase, PEPD) were found to be substantially increased in the urinary exosomes of diabetic patients, which appeared to correlate with the severity of DN [
      • Raimondo F.
      • Corbetta S.
      • Morosi L.
      • Chinello C.
      • Gianazza E.
      • Castoldi G.
      • et al.
      Urinary exosomes and diabetic nephropathy: a proteomic approach.
      ]. similarly, reports concerning Wilm's tumor 1 (WT1) protein indicated that WT1 participated in the homeostasis of mature podocytes, and could be detected in the urinary exosomes of diabetic patients to reflect renal function damage [
      • Abe H.
      • Sakurai A.
      • Ono H.
      • Hayashi S.
      • Yoshimoto S.
      • Ochi A.
      • et al.
      Urinary exosomal mRNA of WT1 as diagnostic and prognostic biomarker for diabetic nephropathy.
      ,
      • Kalani A.
      • Mohan A.
      • Godbole M.M.
      • Bhatia E.
      • Gupta A.
      • Sharma R.K.
      • et al.
      Wilm’s tumor-1 protein levels in urinary exosomes from diabetic patients with or without proteinuria.
      ]. Zubiri et al. [

      Zubiri I, Posada-Ayala M, Benito-Martin A, Maroto AS, Martin-Lorenzo M, Cannata-Ortiz P, et al. Kidney tissue proteomics reveals regucalcin downregulation in response to diabetic nephropathy with reflection in urinary exosomes. Transl Res. 2015;166:474–84.e4. https://doi.org/10.1016/j.trsl.2015.05.007.

      ] revealed that urinary exosomal regucalcin was downregulated in a rat model of early DN versus healthy individuals, indicating that regucalcin may represent a novel tool to diagnose and monitor the progression of diabetic kidney diseases. Epithelium-specific transcription factor-Elf3 from urinary exosomes was only found in patients with T2DN, reflecting the podocyte injury associated with DN [
      • Sakurai A.
      • Ono H.
      • Ochi A.
      • Matsuura M.
      • Yoshimoto S.
      • Kishi S.
      • et al.
      Involvement of Elf3 on Smad3 activation-dependent injuries in podocytes and excretion of urinary exosome in diabetic nephropathy.
      ]. Against a background of high urinary albumin, another three proteins were found to be expressed to differing levels in urinary exosomes of patients with DN, these were: α-microglobulin/bikunin precursor (increased); histone-lysine N-methyltransferase (observed only in patients with DN); and, voltage-dependent anion-selective channel protein 1(decreased) [
      • Zubiri I.
      • Posada-Ayala M.
      • Sanz-Maroto A.
      • Calvo E.
      • Martin-Lorenzo M.
      • Gonzalez-Calero L.
      • et al.
      Diabetic nephropathy induces changes in the proteome of human urinary exosomes as revealed by label-free comparative analysis.
      ].
      Taken together, there is accumulating evidence to support the theory that cargos carried in urinary exosomes, such as miRNA and proteins, can be used as early markers of DN and have the potential to predict disease progression. However, the origin and mechanism concerning these cargos are not fully understood and further in-depth investigations are required to explore and verify the clinical value of these new biomarkers.

      5. Therapeutic perspectives of exosomes in DN

      Although current remedies have a significant effect on the progression of DN, the incidence of ESRD associated with diabetes remains high and effective tools to halt (or even reverse) the progression of DN are lacking [
      • Ahmad J.
      Management of diabetic nephropathy: recent progress and future perspective.
      ,
      • Fernandez-Fernandez B.
      • Ortiz A.
      • Gomez-Guerrero C.
      • Egido J.
      Therapeutic approaches to diabetic nephropathy—beyond the RAS.
      ]. Therefore, there is a pressing need to explore and recognize emerging, potent renoprotective therapeutic strategies to slow the progression of DN. Recently, several studies have confirmed that exosomes offer potential as therapeutic delivery vehicles for the treatment of DN (Table 2).
      Table 2Potential therapeutic roles of exosomes in DN.
      Origin of exosomesCargoMechanismOrganismReference
      ADSCsmiR-486-5pFacilitating autophagy flux through Smad1/mTOR signaling pathwayMice[
      • Ebrahim N.
      • Ahmed I.A.
      • Hussien N.I.
      • Dessouky A.A.
      • Farid A.S.
      • Elshazly A.M.
      • et al.
      Mesenchymal stem cell-derived exosomes ameliorated diabetic nephropathy by autophagy induction through the mTOR signaling pathway.
      ]
      ADSCsmiR-215-5pRelieving EMT of podocyte through downregulating ZEB2Cells[
      • Fernandez-Fernandez B.
      • Ortiz A.
      • Gomez-Guerrero C.
      • Egido J.
      Therapeutic approaches to diabetic nephropathy—beyond the RAS.
      ]
      hUSCsInhibiting podocyte apoptosis by suppressing caspase-3 overexpression and promoting vascular regeneration and cell survivalRats, cells[
      • Phinney D.G.
      • Pittenger M.F.
      Concise review: MSC-derived exosomes for cell-free therapy.
      ]
      hUSCsmiR-16-5pAlleviating podocyte injury via suppressing VEGFA expressionRats, cells[
      • Yin K.
      • Wang S.
      • Zhao R.C.
      Exosomes from mesenchymal stem/stromal cells: a new therapeutic paradigm.
      ]
      MSCsSuppressing proinflammatory cytokine, epithelial damage in proximal tubules, tubular EMT, apoptosis and degeneration of TECsMice[
      • Duan Y.R.
      • Chen B.P.
      • Chen F.
      • Yang S.X.
      • Zhu C.Y.
      • Ma Y.L.
      • et al.
      Exosomal microRNA-16-5p from human urine-derived stem cells ameliorates diabetic nephropathy through protection of podocyte.
      ]
      MSCsActivating autophagy through suppressing mTOR signaling pathwayRats[
      • Huang C.
      • Zhang Y.
      • Kelly D.J.
      • Tan C.Y.
      • Gill A.
      • Cheng D.
      • et al.
      Thioredoxin interacting protein (TXNIP) regulates tubular autophagy and mitophagy in diabetic nephropathy through the mTOR signaling pathway.
      ]
      BMSCsmiR-let-7a-3pInhibiting oxidative stress and represses renal cell apoptosis via downregulating USP22Rats[
      • Paulini J.
      • Higuti E.
      • Bastos R.M.
      • Gomes S.A.
      • Rangel É.B.
      Mesenchymal stem cells as therapeutic candidates for halting the progression of diabetic nephropathy.
      ]
      M2 macrophagemiR-25-3pProtecting podocytes injury through activating autophagy of the cells by suppressing DUSP1 expressionCells[
      • Bochon B.
      • Kozubska M.
      • Surygała G.
      • Witkowska A.
      • Kuźniewicz R.
      • Grzeszczak W.
      • et al.
      Mesenchymal stem cells-potential applications in kidney diseases.
      ]
      MusclemiR-23a-3p/27a-3pAttenuating renal fibrosis through muscle-kidney crosstalkMice[
      • Nagaishi K.
      • Mizue Y.
      • Chikenji T.
      • Otani M.
      • Nakano M.
      • Konari N.
      • et al.
      Mesenchymal stem cell therapy ameliorates diabetic nephropathy via the paracrine effect of renal trophic factors including exosomes.
      ]
      Abbreviation: ADSCs: adipose-derived stem cells; EMT: epithelial-mesenchymal transition; ZEB2: zinc finger E-box-binding homeobox-2; hUSCs: human urine-derived stem cells; VEGFA: vascular endothelial growth factor A; MSCs: mesenchymal stem cells; TECs: tubular epithelial cells; BMSCs: bone marrow mesenchymal stem cells; USP22: ubiquitin-specific protease 22; DUSP1: dual specificity protein phosphatase1.
      Stem cells, which can self-renew and develop into a wide variety of functional cells, have been considered as potential therapeutic agents for DN due to their beneficial effects being mainly exerted through paracrine mechanisms. There is a growing mount of strong evidence that demonstrates exosomes derived from stem cells, which can transport exogenous miRNAs to recipient cells [
      • Phinney D.G.
      • Pittenger M.F.
      Concise review: MSC-derived exosomes for cell-free therapy.
      ], are efficacious and safe for the treatment of renal diseases in rat and mouse models [
      • Yin K.
      • Wang S.
      • Zhao R.C.
      Exosomes from mesenchymal stem/stromal cells: a new therapeutic paradigm.
      ]. One report indicated that exosomal miR-486-5p produced from ADSCs mitigates podocyte damage in db/db mice by facilitating autophagy flux through the Smad1/mTOR signaling pathway [
      • Jin J.
      • Shi Y.
      • Gong J.
      • Zhao L.
      • Li Y.
      • He Q.
      • et al.
      Exosome secreted from adipose-derived stem cells attenuates diabetic nephropathy by promoting autophagy flux and inhibiting apoptosis in podocyte.
      ]. In a follow-up study, the same team also found that miR-215-5p in ADSC exosomes may relieve podocyte EMT when treated with high concentrations of glucose via inhibiting zinc finger E-box-binding homeobox-2, which is the target gene of miR-215-5p [
      • Jin J.
      • Wang Y.
      • Zhao L.
      • Zou W.
      • Tan M.
      • He Q.
      Exosomal miRNA-215-5p derived from adipose-derived stem cells attenuates epithelial-mesenchymal transition of podocytes by inhibiting ZEB2.
      ]. Moreover, human urine-derived stem cells (hUSCs)-extracted exosomes may have the potential to inhibit podocyte apoptosis by suppression of caspase-3 overexpression and promotion of vascular regeneration and cell survival, which resulted in protection from kidney damage in type 1 diabetic rats [
      • Jiang Z.Z.
      • Liu Y.M.
      • Niu X.
      • Yin J.Y.
      • Hu B.
      • Guo S.C.
      • et al.
      Exosomes secreted by human urine-derived stem cells could prevent kidney complications from type I diabetes in rats.
      ]. A similar study also showed that exosomal miR-16-5p secreted by hUSCs effectively alleviated high glucose-induced podocyte injury via suppression of vascular endothelial growth factor A (VEGFA) expression [
      • Duan Y.R.
      • Chen B.P.
      • Chen F.
      • Yang S.X.
      • Zhu C.Y.
      • Ma Y.L.
      • et al.
      Exosomal microRNA-16-5p from human urine-derived stem cells ameliorates diabetic nephropathy through protection of podocyte.
      ]. MSCs have recently attracted increasing interest as a novel regenerative therapy against kidney injury [
      • Paulini J.
      • Higuti E.
      • Bastos R.M.
      • Gomes S.A.
      • Rangel É.B.
      Mesenchymal stem cells as therapeutic candidates for halting the progression of diabetic nephropathy.
      ,
      • Bochon B.
      • Kozubska M.
      • Surygała G.
      • Witkowska A.
      • Kuźniewicz R.
      • Grzeszczak W.
      • et al.
      Mesenchymal stem cells-potential applications in kidney diseases.
      ]. Nagaishi and his colleagues found that MSCs suppressed proinflammatory cytokines (such as TNF-α) expression, epithelial damage in the proximal tubules, tubular EMT, apoptosis and degeneration of TECs in the kidneys of diabetic mice, which may contribute to the level of exosomes found in the MSCs [
      • Nagaishi K.
      • Mizue Y.
      • Chikenji T.
      • Otani M.
      • Nakano M.
      • Konari N.
      • et al.
      Mesenchymal stem cell therapy ameliorates diabetic nephropathy via the paracrine effect of renal trophic factors including exosomes.
      ]. Another study concluded that exosomes derived from MSCs may activate autophagy through suppression of the mTOR signaling pathway, thus exerting their nephroprotective and antifibrotic effect in rats' models of diabetic nephropathy [
      • Ebrahim N.
      • Ahmed I.A.
      • Hussien N.I.
      • Dessouky A.A.
      • Farid A.S.
      • Elshazly A.M.
      • et al.
      Mesenchymal stem cell-derived exosomes ameliorated diabetic nephropathy by autophagy induction through the mTOR signaling pathway.
      ]. A more recent report suggested that bone marrow mesenchymal stem cells (BMSCs)-derived exosomal miR-let-7a-3p inhibited oxidative stress and repressed renal cell apoptosis in the renal tissues of diabetic rats via downregulation of ubiquitin-specific protease 22 [
      • Mao R.
      • Shen J.
      • Hu X.
      BMSCs-derived exosomal microRNA-let-7a plays a protective role in diabetic nephropathy via inhibition of USP22 expression.
      ].
      In addition to exosomes from various stem cells, there are also several reports concerning exosomes secreted from other cell types as potential therapies for DN. Huang et al. suggested that exosomal miR-25-3p derived from M2 macrophages may protect podocytes against high glucose-induced injury via activation of cell autophagy through suppression of dual specificity protein phosphatase 1 expression [
      • Huang H.
      • Liu H.
      • Tang J.
      • Xu W.
      • Gan H.
      • Fan Q.
      • et al.
      M2 macrophage-derived exosomal miR-25-3p improves high glucose-induced podocytes injury through activation autophagy via inhibiting DUSP1 expression.
      ]. Another report demonstrated that exosomal miRNA-23a-3p/27a-3p shed from muscle is involved in crosstalk between muscle and kidney cells, thus attenuating renal fibrosis in diabetic mice [
      • Zhang A.
      • Li M.
      • Wang B.
      • Klein J.D.
      • Price S.R.
      • Wang X.H.
      miRNA-23a/27a attenuates muscle atrophy and renal fibrosis through muscle-kidney crosstalk.
      ]. Taken together, these findings suggest that exosomes secreted from stem cells, M2 macrophages and tissues offer the capacity to suppress the vitro and in vivo progression of DN through various mechanisms, which provides the basis for future applications of exosomes as a novel biological therapeutic approach for the treatment of DN. However, more detailed studies on the protective mechanism of exosomes for DN are required and further basic and clinical trials are required to validate the promising results described above.

      6. Summary

      In conclusion, DN is a global public health problem due to its high incidence and substantial economic burden. Despite decades of effort, effective, targeted therapeutic strategies for DN remain elusive. Nephrologists and endocrinologists are continually searching for new tools to improve their ability to rapidly and accurately diagnose renal disease caused by diabetes via noninvasive methodologies as well as for novel therapeutic approaches via exploration of the molecular mechanisms of DN. As explained in this review, exosomes possess various properties including modulation of cell-to-cell crosstalk, cargo transfer to specific target cells, and alteration of the biofunction of acceptor cells, which has exhibited an important influence on the pathogenesis, diagnosis and treatment of DN. Therefore, exosomes provide considerable promise for the future development of DN treatments, although some potential exosome biomarkers and therapeutic strategies for renal diseases associated with diabetes remain limited and require further validation in comprehensive basic and clinical trials. In the near future, we believe that the intrinsic properties of exosomes will be fully revealed and utilized, allowing more patients suffering from diabetic kidney disease to benefit.

      Funding

      This study was supported by National Science and Technology Major Project of China (Grant No. 2020ZX09201-009 ).

      CRediT authorship contribution statement

      LZS and LDW conceived the idea. CJF and ZQ prepared the figures and tables. LDW and CJF drafted the manuscript. LZS and LDW edited and revised the manuscript. All authors read and approved the final manuscript.

      Declaration of competing interest

      The authors have no conflict of interest to declare.

      Acknowledgements

      We thank the Research Institute of Nephrology personnel for their guidance and help.

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