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• W2987023410 abstract "Acute kidney injury, defined by a rapid deterioration of renal function, is a common complication in hospitalized patients. Among the recent therapeutic options, the use of extracellular vesicles (EVs) is considered a promising strategy. Here we propose a possible therapeutic use of renal-derived EVs isolated from normal urine (urine-derived EVs [uEVs]) in a murine model of acute injury generated by glycerol injection. uEVs accelerated renal recovery, stimulating tubular cell proliferation, reducing the expression of inflammatory and injury markers, and restoring endogenous Klotho loss. When intravenously injected, labeled uEVs localized within injured kidneys and transferred their microRNA cargo. Moreover, uEVs contained the reno-protective Klotho molecule. Murine uEVs derived from Klotho null mice lost the reno-protective effect observed using murine EVs from wild-type mice. This was regained when Klotho-negative murine uEVs were reconstituted with recombinant Klotho. Similarly, ineffective fibroblast EVs acquired reno-protection when engineered with human recombinant Klotho. Our results reveal a novel potential use of uEVs as a new therapeutic strategy for acute kidney injury, highlighting the presence and role of the reno-protective factor Klotho. Acute kidney injury, defined by a rapid deterioration of renal function, is a common complication in hospitalized patients. Among the recent therapeutic options, the use of extracellular vesicles (EVs) is considered a promising strategy. Here we propose a possible therapeutic use of renal-derived EVs isolated from normal urine (urine-derived EVs [uEVs]) in a murine model of acute injury generated by glycerol injection. uEVs accelerated renal recovery, stimulating tubular cell proliferation, reducing the expression of inflammatory and injury markers, and restoring endogenous Klotho loss. When intravenously injected, labeled uEVs localized within injured kidneys and transferred their microRNA cargo. Moreover, uEVs contained the reno-protective Klotho molecule. Murine uEVs derived from Klotho null mice lost the reno-protective effect observed using murine EVs from wild-type mice. This was regained when Klotho-negative murine uEVs were reconstituted with recombinant Klotho. Similarly, ineffective fibroblast EVs acquired reno-protection when engineered with human recombinant Klotho. Our results reveal a novel potential use of uEVs as a new therapeutic strategy for acute kidney injury, highlighting the presence and role of the reno-protective factor Klotho. Acute kidney injury (AKI), a common complication in hospitalized patients, is characterized by a rapid deterioration of renal function.1Morigi M. Rota C. Remuzzi G. Mesenchymal Stem Cells in Kidney Repair.Methods Mol. Biol. 2016; 1416: 89-107Crossref PubMed Scopus (33) Google Scholar Around 8%–16% of the patients suffering from AKI are estimated to progress to chronic renal disease, with high mortality risk and related costs.2Rewa O. Bagshaw S.M. Acute kidney injury—epidemiology, outcomes and economics.Nat. Rev. Nephrol. 2014; 10: 193-207Crossref PubMed Scopus (362) Google Scholar, 3Grange C. Iampietro C. Bussolati B. Stem cell extracellular vesicles and kidney injury.Stem Cell Investig. 2017; 4: 90Crossref PubMed Scopus (27) Google Scholar, 4Pozzoli S. Simonini M. Manunta P. Predicting acute kidney injury: current status and future challenges.J. Nephrol. 2018; 31: 209-223Crossref PubMed Scopus (38) Google Scholar Although the renal tissue shows an intrinsic ability to regenerate after damage, full recovery in most of the cases is not achieved. Therefore, multiple clinical approaches for renal regeneration are nowadays being considered. Among the new therapeutic options for AKI, the use of stem cell-derived extracellular vesicles (EVs) is gaining increasing interest.5Zhao L. Hu C. Zhang P. Jiang H. Chen J. Genetic communication by extracellular vesicles is an important mechanism underlying stem cell-based therapy-mediated protection against acute kidney injury.Stem Cell Res. Ther. 2019; 10: 119Crossref PubMed Scopus (12) Google Scholar For example, EVs from mesenchymal stromal cells (MSC EVs) have been found to exert a promising therapeutic effect.6Bruno S. Grange C. Collino F. Deregibus M.C. Cantaluppi V. Biancone L. Tetta C. Camussi G. Microvesicles derived from mesenchymal stem cells enhance survival in a lethal model of acute kidney injury.PLoS ONE. 2012; 7: e33115Crossref PubMed Scopus (429) Google Scholar, 7Gatti S. Bruno S. Deregibus M.C. Sordi A. Cantaluppi V. Tetta C. Camussi G. Microvesicles derived from human adult mesenchymal stem cells protect against ischaemia-reperfusion-induced acute and chronic kidney injury.Nephrol. Dial. Transplant. 2011; 26: 1474-1483Crossref PubMed Scopus (558) Google Scholar, 8Bruno S. Grange C. Deregibus M.C. Calogero R.A. Saviozzi S. Collino F. Morando L. Busca A. Falda M. Bussolati B. et al.Mesenchymal stem cell-derived microvesicles protect against acute tubular injury.J. Am. Soc. Nephrol. 2009; 20: 1053-1067Crossref PubMed Scopus (889) Google Scholar, 9Bruno S. Tapparo M. Collino F. Chiabotto G. Deregibus M.C. Soares Lindoso R. Neri F. Kholia S. Giunti S. Wen S. et al.Renal Regenerative Potential of Different Extracellular Vesicle Populations Derived from Bone Marrow Mesenchymal Stromal Cells.Tissue Eng. Part A. 2017; 23: 1262-1273Crossref PubMed Scopus (94) Google Scholar, 10Shen B. Liu J. Zhang F. Wang Y. Qin Y. Zhou Z. Qiu J. Fan Y. CCR2 Positive Exosome Released by Mesenchymal Stem Cells Suppresses Macrophage Functions and Alleviates Ischemia/Reperfusion-Induced Renal Injury.Stem Cells Int. 2016; 2016: 1240301Crossref PubMed Scopus (90) Google Scholar, 11Nassar W. El-Ansary M. Sabry D. Mostafa M.A. Fayad T. Kotb E. Temraz M. Saad A.N. Essa W. Adel H. Umbilical cord mesenchymal stem cells derived extracellular vesicles can safely ameliorate the progression of chronic kidney diseases.Biomater. Res. 2016; 20: 21Crossref PubMed Scopus (161) Google Scholar, 12Tapparo M. Bruno S. Collino F. Togliatto G. Deregibus M.C. Provero P. Wen S. Quesenberry P.J. Camussi G. Renal Regenerative Potential of Extracellular Vesicles Derived from miRNA-Engineered Mesenchymal Stromal Cells.Int. J. Mol. Sci. 2019; 20: E2381Crossref PubMed Scopus (20) Google Scholar MSC EVs may act through multiple regenerative processes, including induction of tubular cell survival, proliferation, suppression of inflammation, and inhibition of fibrosis.7Gatti S. Bruno S. Deregibus M.C. Sordi A. Cantaluppi V. Tetta C. Camussi G. Microvesicles derived from human adult mesenchymal stem cells protect against ischaemia-reperfusion-induced acute and chronic kidney injury.Nephrol. Dial. Transplant. 2011; 26: 1474-1483Crossref PubMed Scopus (558) Google Scholar, 8Bruno S. Grange C. Deregibus M.C. Calogero R.A. Saviozzi S. Collino F. Morando L. Busca A. Falda M. Bussolati B. et al.Mesenchymal stem cell-derived microvesicles protect against acute tubular injury.J. Am. Soc. Nephrol. 2009; 20: 1053-1067Crossref PubMed Scopus (889) Google Scholar, 9Bruno S. Tapparo M. Collino F. Chiabotto G. Deregibus M.C. Soares Lindoso R. Neri F. Kholia S. Giunti S. Wen S. et al.Renal Regenerative Potential of Different Extracellular Vesicle Populations Derived from Bone Marrow Mesenchymal Stromal Cells.Tissue Eng. Part A. 2017; 23: 1262-1273Crossref PubMed Scopus (94) Google Scholar,13Tomasoni S. Longaretti L. Rota C. Morigi M. Conti S. Gotti E. Capelli C. Introna M. Remuzzi G. Benigni A. Transfer of growth factor receptor mRNA via exosomes unravels the regenerative effect of mesenchymal stem cells.Stem Cells Dev. 2013; 22: 772-780Crossref PubMed Scopus (222) Google Scholar The transfer of EV biological cargo (proteins, RNAs, and DNAs) into target cells appears fundamental in cell fate modulation.14Collino F. Bruno S. Incarnato D. Dettori D. Neri F. Provero P. Pomatto M. Oliviero S. Tetta C. Quesenberry P.J. Camussi G. AKI recovery induced by mesenchymal stromal cell-derived extracellular vesicles carrying microRNAs.J. Am. Soc. Nephrol. 2015; 26: 2349-2360Crossref PubMed Scopus (147) Google Scholar, 15Lai R.C. Yeo R.W. Lim S.K. Mesenchymal stem cell exosomes.Semin. Cell Dev. Biol. 2015; 40: 82-88Crossref PubMed Scopus (289) Google Scholar, 16Ratajczak J. Miekus K. Kucia M. Zhang J. Reca R. Dvorak P. Ratajczak M.Z. Embryonic stem cell-derived microvesicles reprogram hematopoietic progenitors: evidence for horizontal transfer of mRNA and protein delivery.Leukemia. 2006; 20: 847-856Crossref PubMed Scopus (1051) Google Scholar Interestingly, microRNAs (miRNAs), transferred by MSC EVs, have been considered the main effectors in the enhancement of proliferation and survival of renal tubular cells.9Bruno S. Tapparo M. Collino F. Chiabotto G. Deregibus M.C. Soares Lindoso R. Neri F. Kholia S. Giunti S. Wen S. et al.Renal Regenerative Potential of Different Extracellular Vesicle Populations Derived from Bone Marrow Mesenchymal Stromal Cells.Tissue Eng. Part A. 2017; 23: 1262-1273Crossref PubMed Scopus (94) Google Scholar,14Collino F. Bruno S. Incarnato D. Dettori D. Neri F. Provero P. Pomatto M. Oliviero S. Tetta C. Quesenberry P.J. Camussi G. AKI recovery induced by mesenchymal stromal cell-derived extracellular vesicles carrying microRNAs.J. Am. Soc. Nephrol. 2015; 26: 2349-2360Crossref PubMed Scopus (147) Google Scholar Renal cells lining the nephron also release bioactive EVs able to target downstream cells, suggesting the presence of an intra-nephron communication between cells along the urinary lumen.17Sallustio F. Costantino V. Cox S.N. Loverre A. Divella C. Rizzi M. Schena F.P. Retraction: Human renal stem/progenitor cells repair tubular epithelial cell injury through TLR2-driven inhibin-A and microvesicle-shuttled decorin.Kidney Int. 2015; 87: 482Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar,18Borges F.T. Melo S.A. Özdemir B.C. Kato N. Revuelta I. Miller C.A. Gattone 2nd, V.H. LeBleu V.S. Kalluri R. TGF-β1-containing exosomes from injured epithelial cells activate fibroblasts to initiate tissue regenerative responses and fibrosis.J. Am. Soc. Nephrol. 2013; 24: 385-392Crossref PubMed Scopus (242) Google Scholar EVs released by proximal tubular cells were shown to be internalized by distal tubule and collecting duct cells in vitro.19Gildea J.J. Seaton J.E. Victor K.G. Reyes C.M. Bigler Wang D. Pettigrew A.C. Courtner C.E. Shah N. Tran H.T. Van Sciver R.E. et al.Exosomal transfer from human renal proximal tubule cells to distal tubule and collecting duct cells.Clin. Biochem. 2014; 47: 89-94Crossref PubMed Scopus (54) Google Scholar Moreover, in collecting duct cells, the functional transfer of AQP2 via EVs has been reported.20Street J.M. Birkhoff W. Menzies R.I. Webb D.J. Bailey M.A. Dear J.W. Exosomal transmission of functional aquaporin 2 in kidney cortical collecting duct cells.J. Physiol. 2011; 589: 6119-6127Crossref PubMed Scopus (94) Google Scholar Urine-derived EVs (uEVs) have been extensively characterized for their cargo21Pisitkun T. Shen R.F. Knepper M.A. Identification and proteomic profiling of exosomes in human urine.Proc. Natl. Acad. Sci. USA. 2004; 101: 13368-13373Crossref PubMed Scopus (1463) Google Scholar, 22Gonzales P.A. Pisitkun T. Hoffert J.D. Tchapyjnikov D. Star R.A. Kleta R. Wang N.S. Knepper M.A. Large-scale proteomics and phosphoproteomics of urinary exosomes.J. Am. Soc. Nephrol. 2009; 20: 363-379Crossref PubMed Scopus (499) Google Scholar, 23Miranda K.C. Bond D.T. McKee M. Skog J. Păunescu T.G. Da Silva N. Brown D. Russo L.M. Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease.Kidney Int. 2010; 78: 191-199Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 24Merchant M.L. Rood I.M. Deegens J.K.J. Klein J.B. Isolation and characterization of urinary extracellular vesicles: implications for biomarker discovery.Nat. Rev. Nephrol. 2017; 13: 731-749Crossref PubMed Scopus (174) Google Scholar and found to contain kidney cell-specific markers from different nephron segments, such as megalin, cubilin, aminopeptidase, aquaporin-1 (AQP1), aquaporin-2 (AQP2), podocin, and podocalyxin (PDX).25Hoorn E.J. Pisitkun T. Zietse R. Gross P. Frokiaer J. Wang N.S. Gonzales P.A. Star R.A. Knepper M.A. Prospects for urinary proteomics: exosomes as a source of urinary biomarkers.Nephrology (Carlton). 2005; 10: 283-290Crossref PubMed Scopus (141) Google Scholar Indeed, EVs, present in the urine, are considered to mainly derive from renal cells rather than serum, considering the small size (6-nm diameter) of renal filtration pore.26Huang J. Brenna C. Khan A.U.M. Daniele C. Rudolf R. Heuveline V. Gretz N. A cationic near infrared fluorescent agent and ethyl-cinnamate tissue clearing protocol for vascular staining and imaging.Sci. Rep. 2019; 9: 521Crossref PubMed Scopus (14) Google Scholar However, the potential role of uEVs in renal recovery after damage is currently unknown. Interestingly, a proteomic analysis revealed that uEVs contain Klotho,21Pisitkun T. Shen R.F. Knepper M.A. Identification and proteomic profiling of exosomes in human urine.Proc. Natl. Acad. Sci. USA. 2004; 101: 13368-13373Crossref PubMed Scopus (1463) Google Scholar,22Gonzales P.A. Pisitkun T. Hoffert J.D. Tchapyjnikov D. Star R.A. Kleta R. Wang N.S. Knepper M.A. Large-scale proteomics and phosphoproteomics of urinary exosomes.J. Am. Soc. Nephrol. 2009; 20: 363-379Crossref PubMed Scopus (499) Google Scholar a single-pass transmembrane protein crucial in renal tissue regeneration.27Hu M.C. Kuro-o M. Moe O.W. Renal and extrarenal actions of Klotho.Semin. Nephrol. 2013; 33: 118-129Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar Klotho was initially identified as an anti-aging gene,28Kuro-o M. Matsumura Y. Aizawa H. Kawaguchi H. Suga T. Utsugi T. Ohyama Y. Kurabayashi M. Kaname T. Kume E. et al.Mutation of the mouse klotho gene leads to a syndrome resembling ageing.Nature. 1997; 390: 45-51Crossref PubMed Scopus (2528) Google Scholar highly expressed in the kidney.29Li S.A. Watanabe M. Yamada H. Nagai A. Kinuta M. Takei K. Immunohistochemical localization of Klotho protein in brain, kidney, and reproductive organs of mice.Cell Struct. Funct. 2004; 29: 91-99Crossref PubMed Scopus (269) Google Scholar The protein’s extracellular domain, when cleaved, is released into the circulation and acts as a soluble factor, exerting a reno-protective role in tissue regeneration.30Hu M.C. Kuro-o M. Moe O.W. Secreted klotho and chronic kidney disease.Adv. Exp. Med. Biol. 2012; 728: 126-157Crossref PubMed Scopus (92) Google Scholar, 31Panesso M.C. Shi M. Cho H.J. Paek J. Ye J. Moe O.W. Hu M.C. Klotho has dual protective effects on cisplatin-induced acute kidney injury.Kidney Int. 2014; 85: 855-870Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 32Lu X. Hu M.C. Klotho/FGF23 Axis in Chronic Kidney Disease and Cardiovascular Disease.Kidney Dis. (Basel). 2017; 3: 15-23Crossref PubMed Google Scholar, 33Sakan H. Nakatani K. Asai O. Imura A. Tanaka T. Yoshimoto S. Iwamoto N. Kurumatani N. Iwano M. Nabeshima Y. et al.Reduced renal α-Klotho expression in CKD patients and its effect on renal phosphate handling and vitamin D metabolism.PLoS ONE. 2014; 9: e86301Crossref PubMed Scopus (84) Google Scholar, 34Sugiura H. Yoshida T. Mitobe M. Yoshida S. Shiohira S. Nitta K. Tsuchiya K. Klotho reduces apoptosis in experimental ischaemic acute kidney injury via HSP-70.Nephrol. Dial. Transplant. 2010; 25: 60-68Crossref PubMed Scopus (79) Google Scholar Klotho is modulated within renal tissue after injury, and its levels decline in blood, urine, and kidney in acute31Panesso M.C. Shi M. Cho H.J. Paek J. Ye J. Moe O.W. Hu M.C. Klotho has dual protective effects on cisplatin-induced acute kidney injury.Kidney Int. 2014; 85: 855-870Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar and chronic damage.32Lu X. Hu M.C. Klotho/FGF23 Axis in Chronic Kidney Disease and Cardiovascular Disease.Kidney Dis. (Basel). 2017; 3: 15-23Crossref PubMed Google Scholar,33Sakan H. Nakatani K. Asai O. Imura A. Tanaka T. Yoshimoto S. Iwamoto N. Kurumatani N. Iwano M. Nabeshima Y. et al.Reduced renal α-Klotho expression in CKD patients and its effect on renal phosphate handling and vitamin D metabolism.PLoS ONE. 2014; 9: e86301Crossref PubMed Scopus (84) Google Scholar On the other side, forced expression of exogenous Klotho has been shown to exert a reno-protective role, indicating that Klotho administration can be considered as a promising approach for kidney repair.34Sugiura H. Yoshida T. Mitobe M. Yoshida S. Shiohira S. Nitta K. Tsuchiya K. Klotho reduces apoptosis in experimental ischaemic acute kidney injury via HSP-70.Nephrol. Dial. Transplant. 2010; 25: 60-68Crossref PubMed Scopus (79) Google Scholar,35Satoh M. Nagasu H. Morita Y. Yamaguchi T.P. Kanwar Y.S. Kashihara N. Klotho protects against mouse renal fibrosis by inhibiting Wnt signaling.Am. J. Physiol. Renal Physiol. 2012; 303: F1641-F1651Crossref PubMed Scopus (106) Google Scholar The aim of the present study is to investigate the possible therapeutic use of human uEVs in the recovery of renal damage in an experimental model of AKI, generated by glycerol injection. Moreover, the specific role of the reno-therapeutic factor Klotho, present in EV cargo, is dissected. To determine whether uEVs have therapeutic action, the effect of uEVs was evaluated in a murine model of rhabdomyolysis-induced AKI.8Bruno S. Grange C. Deregibus M.C. Calogero R.A. Saviozzi S. Collino F. Morando L. Busca A. Falda M. Bussolati B. et al.Mesenchymal stem cell-derived microvesicles protect against acute tubular injury.J. Am. Soc. Nephrol. 2009; 20: 1053-1067Crossref PubMed Scopus (889) Google Scholar This model is characterized by muscle injury and the subsequent release of nephrotoxic substances and iron deposition on renal tissue (Figure S1). uEVs were obtained by ultracentrifugation of urine from healthy volunteers (n = 25 different pools). In selected experiments, uEVs were further purified by floating on density gradient (uEV float), to exclude the effect of non-vesicular contaminants.36Kowal J. Arras G. Colombo M. Jouve M. Morath J.P. Primdal-Bengtson B. Dingli F. Loew D. Tkach M. Théry C. Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes.Proc. Natl. Acad. Sci. USA. 2016; 113: E968-E977Crossref PubMed Scopus (1522) Google Scholar The mean diameter of uEVs and uEVs float was 175.1 ± 69 nm (mode: 149 ± 30 nm) and 160.1 ± 34 nm (mode: 150 ± 28 nm), respectively, as evaluated by NanoSight analysis (Figure 1A). The identity of the EVs was confirmed by the expression of CD63 and lack of calreticulin, an endoplasmic reticulum marker (Figure 1B). Moreover, transmission electron microscopy of uEVs demonstrated their spheroid morphology and confirmed their size in the nano-range (Figure 1C). EVs from urine expressed the characteristic renal markers AQP1, AQP2, and Klotho, absent in MSC EVs used as a control (Figure 1B). Moreover, cytofluorimetric analyses of uEVs confirmed the presence of renal tubular and podocyte surface markers (AQP1, AQP2, and PDX), as well as of renal exosomal markers (CD24,37Keller S. Rupp C. Stoeck A. Runz S. Fogel M. Lugert S. Hager H.D. Abdel-Bakky M.S. Gutwein P. Altevogt P. CD24 is a marker of exosomes secreted into urine and amniotic fluid.Kidney Int. 2007; 72: 1095-1102Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar CD81, and CD63). No detection of markers of platelets, endothelial cells, and monocytes/leukocytes, reported in serum EVs,38Cavallari C. Ranghino A. Tapparo M. Cedrino M. Figliolini F. Grange C. Giannachi V. Garneri P. Deregibus M.C. Collino F. et al.Serum-derived extracellular vesicles (EVs) impact on vascular remodeling and prevent muscle damage in acute hind limb ischemia.Sci. Rep. 2017; 7: 8180Crossref PubMed Scopus (27) Google Scholar was observed (Figure 1D). One day after AKI induction, concomitant with the peak of the damage, mice received the following treatments: (1) uEVs, (2) uEVs float, (3) EVs from MSCs, and (4) EVs from fibroblasts. MSC and fibroblast EVs were used as positive and negative controls, respectively. All mice received 2.0 × 108 uEVs/100 μL (a reported effective dose for MSC EVs14Collino F. Bruno S. Incarnato D. Dettori D. Neri F. Provero P. Pomatto M. Oliviero S. Tetta C. Quesenberry P.J. Camussi G. AKI recovery induced by mesenchymal stromal cell-derived extracellular vesicles carrying microRNAs.J. Am. Soc. Nephrol. 2015; 26: 2349-2360Crossref PubMed Scopus (147) Google Scholar) into the tail vein. The control group was treated with 100 μL of saline (vehicle). Mice injected with uEVs and floating uEVs showed a significant reduction of blood urea nitrogen (BUN) and creatinine at day 3 (48 h after treatment) compared with untreated mice (Figure 2A). The amelioration in physiological parameters induced by uEVs was similar to that obtained by the administration of pro-regenerative MSC EVs (Figure 2A). In contrast, fibroblast EVs did not have any effect on the recovery (Figure 2A). The histological evaluation of kidney sections in the vehicle group revealed the presence of intra-tubular protein casts and tubular epithelial cell necrosis (Figure 2B). Mice treated with uEVs or MSC EVs, but not with fibroblast EVs, showed preserved tissue architecture similar to that of healthy mice (Figure 2B). The number of casts and necrotic tubules was significantly reduced in uEV- and MSC EV-treated mice, confirming their beneficial effect (Figure 2C). No significant difference was observed between animals treated with uEVs and MSCs. On the same line, uEV and MSC EV treatments improved tubular cell proliferation (Figures 3A and 3B ) and reduced tubular cell apoptosis (Figure S2) compared with the vehicle group. The pro-regenerative effect of uEVs was further confirmed by using float uEVs (Figures 2 and 3). We further analyzed, at mRNA level, the effect of uEVs on markers of renal damage and inflammation. In particular, AKI mice showed an increased expression of renal injury markers such as NGAL, PAI, and caspase-3 (Figure 3C). Moreover, SOX9, a transcription factor induced in tubular cells as a consequence of tissue damage,39Kumar S. Liu J. Pang P. Krautzberger A.M. Reginensi A. Akiyama H. Schedl A. Humphreys B.D. McMahon A.P. Sox9 Activation Highlights a Cellular Pathway of Renal Repair in the Acutely Injured Mammalian Kidney.Cell Rep. 2015; 12: 1325-1338Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar was also upregulated (Figure 3C). In contrast, mice treated with uEVs, as well as MSC EVs, showed a significant downregulation of injury markers (Figure 3C). In addition, renal tissue from AKI mice showed a significant increase of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and IL-6 (Figure 3D). The administration of uEVs prevented the inflammatory cascade in a manner similar to MSC EVs, with a significant reduction of all inflammatory markers analyzed (Figure 3D). Moreover, the pro-inflammatory driver nuclear factor κB (NF-κB) was significantly upregulated during AKI and downregulated by uEV treatment (Figure 3D). To determine the localization of uEVs within injured renal tissue, uEVs labeled with a fluorescent dye were injected intravenously and tracked by optical imaging in both healthy and AKI mice. The biodistribution analysis, performed 4 h after the administration of uEVs, revealed the presence of fluorescent signal in whole-body images, as well as in ex vivo isolated kidneys (Figure 4A). The fluorescence, acquired in live animals, was detectable in spleen and kidney of healthy and AKI mice. However, the injured kidneys showed higher fluorescent intensity compared with the healthy ones (Figure 4A). The analysis of dissected kidneys revealed the preferential uEV localization in damaged organs (Figure 4B). To confirm the effective localization of uEVs into injured kidneys, we analyzed the transfer of their miRNA cargo to renal tissue. In particular, from the 204 miRNAs, identified by array within uEVs (Table S1), we confirmed the expression of the top 10 uEV miRNAs by quantitative real-time PCR, and we evaluated their transfer to renal tissue (Table S2; Figure 4C). We observed a significant increase of miR-30 family and miR-151 in renal tissue, 4 h after uEV injection, demonstrating the transfer of four uEV miRNAs to injured kidneys (Figure 4D). Predictive target analysis, using the FunRich tool, performed on the four highly expressed and transferred uEV miRNAs, showed 1,545 selectively predicted target genes. Subsequently, interactome network analysis was conducted to identify relevant pathways involved in uEV biological function (Figure S3). The analysis revealed a strong representation (p < 0.001) of molecules involved in pro-regenerative pathways, such as proteoglycan-syndecan signaling events, insulin growth factor 1 (IGF-1) and hepatocyte growth factor (HGF). The effect of EV cargo within the renal tissue was further confirmed by the downregulation of selected miRNA targets. In fact, SMAD1 and SMAD2, central factors in the interactome, cyclin D1 (CCND1) (miR-30a target), and c-Myc (miR-30 family and miR-151 targets) were downregulated in the renal tissue of uEV-treated mice (Figure 4E). We then focused our attention on a specific renal factor, Klotho, involved in tissue regeneration. uEVs were found to contain Klotho, mainly the short soluble isoform (Figure 5A), at levels of 0.5–10 pg/2.0 × 108 EV particles, as determined by ELISA. In addition, Klotho mRNA was also detected within uEVs (32.05 ± 0.75 cycle threshold [Ct] value). The expression of Klotho in renal tissue is known to decrease during acute and chronic damage.40Hu M.C. Kuro-o M. Moe O.W. Klotho and kidney disease.J. Nephrol. 2010; 23: S136-S144PubMed Google Scholar On the same line, in our data, renal tissue derived from AKI mice showed a significant reduction of Klotho at both mRNA and protein levels, compared with the tissue derived from healthy mice (Figures 5B–5E). Of interest, only uEV treatment, and not MSC EV treatment, significantly increased murine Klotho mRNA 4 h after injection (Figure 5B). However, no human Klotho mRNA was detected in injured renal tissue, excluding mRNA direct transfer. Moreover, the expression of Klotho protein, lost upon damage, was significantly restored to normal levels 4 and 48 h after uEV administration (Figures 5C and 5D). The immunohistochemical analysis confirmed the restoration of Klotho levels, 48 h after EV injection, and showed the predominant localization of the protein to distal and proximal tubules (Figure 5E). We further analyzed the expression of two factors linked with the pro-regenerative effects of Klotho: CTGF35Satoh M. Nagasu H. Morita Y. Yamaguchi T.P. Kanwar Y.S. Kashihara N. Klotho protects against mouse renal fibrosis by inhibiting Wnt signaling.Am. J. Physiol. Renal Physiol. 2012; 303: F1641-F1651Crossref PubMed Scopus (106) Google Scholar,41 and α-SMA.41Wu Y.L. Xie J. An S.W. Oliver N. Barrezueta N.X. Lin M.H. Birnbaumer L. Huang C.L. Inhibition of TRPC6 channels ameliorates renal fibrosis and contributes to renal protection by soluble klotho.Kidney Int. 2017; 91: 830-841Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar uEV treatment inhibited both CTGF and α-SMA upregulation observed in AKI mice (Figure 5F). To sustain the role of Klotho present in uEVs and to assess whether the administration of Klotho carried by EVs may have an advantage over the use of the recombinant Klotho, we compared their effect on renal regeneration. Mice were treated with uEVs (containing Klotho at 1 pg/2.0 × 108 EV particles) or human recombinant Klotho at the same quantity (1 pg/mouse). The administration of recombinant Klotho alone, at variance with uEVs, did not exert any protective effect in damaged renal tissue, as evaluated by BUN, creatinine, and tissue histology (Figure 6). We further confirmed the presence of Klotho in EVs released by human proximal tubular epithelial cells (HK2) (Figure S4). Tubular cells are considered the major source of EVs found in urine.19Gildea J.J. Seaton J.E. Victor K.G. Reyes C.M. Bigler Wang D. Pettigrew A.C. Courtner C.E. Shah N. Tran H.T. Van Sciver R.E. et al.Exosomal transfer from human renal proximal tubule cells to distal tubule and collecting duct cells.Clin. Biochem. 2014; 47: 89-94Crossref PubMed Scopus (54) Google Scholar,20Street J.M. Birkhoff W. Menzies R.I. Webb D.J. Bailey M.A. Dear J.W. Exosomal transmission of functional aquaporin 2 in kidney cortical collecting duct cells.J. Physiol. 2011; 589: 6119-6127Crossref PubMed Scopus (94) Google Scholar Of interest, when quantified by ELISA, the amount of Klotho present in HK2 EVs was at the same range as the one in uEVs (1–2 pg/2.0 × 108 EV particles). We then analyzed the effect of HK2 EVs in our AKI model. As expected, HK2-derived EVs displayed a beneficial effect comparable with uEVs (Figure 6). To dissect the role of Klotho in the reno-protective effect of uEVs, we compared the regenerative effect of uEVs isolated from the urine of wild-type and Klotho null mice. Whereas the effect of wild-type murine uEVs (muEVs) was superimposable to human uEVs, no effect was observed using Klotho null muEVs (Figure 7). In particular, Klotho null murine uEVs were completely ineffective in restoring tissue morphology and renal function (Figure 7). Klotho expression in muEVs isolated from the urine of Klotho null mice was restored by engineering EVs with human recombinant Klotho. In order to associate the role of Klotho, present in human uEVs, with the regenerative effect of uEVs, we then treated AKI mice with Klotho engineered EVs, deriving from the urine of Klotho null mice. The number of particles injected was determined based on the r" @default.
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• W2987023410 date "2020-02-01" @default.
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• W2987023410 title "Urinary Extracellular Vesicles Carrying Klotho Improve the Recovery of Renal Function in an Acute Tubular Injury Model" @default.
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• W2987023410 doi "https://doi.org/10.1016/j.ymthe.2019.11.013" @default.
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