Augmented efficacy of exogenous extracellular vesicles targeted to injured kidneys

All data relevant to this work are included in this paper and Supplementary Information. Choi, H. & Lee, D. S. Illuminating the physiology of extracellular vesicles. Stem Cell Res. Ther. 7, 55 (2016). Article Google Scholar Zou, X. et al. Targeting murine mesenchymal stem cells to kidney injury molecule-1 improves their therapeutic efficacy in chronic ischemic kidney injury. Stem Cells Transl. Med. 7, 394–403 (2018). CAS Article Google Scholar Eirin, A. et al. Mesenchymal stem cell-derived extracellular vesicles attenuate kidney inflammation. Kidney Int. 92, 114–124 (2017). CAS Article Google Scholar Dimke, H. et al. Tubulovascular cross-talk by vascular endothelial growth factor a maintains peritubular microvasculature in kidney. J. Am. Soc. Nephrol. 26, 1027–1038 (2015). CAS Article Google Scholar He, F. F. et al. Angiopoietin-Tie signaling in kidney diseases: an updated review. FEBS Lett. 593, 2706–2715 (2019). CAS Article Google Scholar Download references This work was partly supported by NIH Grants Numbers DK122734, DK102325, and DK120292. Division of Nephrology and Hypertension, Mayo Clinic, Rochester, MN, USA Xiao-Jun Chen, Kai Jiang, Christopher M. Ferguson, Hui Tang, Xiangyang Zhu & Lilach O. Lerman Department of Nephrology, The Second Xiangya Hospital of Central South University, Changsha, Hunan, China Xiao-Jun Chen Department of Cardiovascular Diseases, Mayo Clinic, Rochester, MN, USA Amir Lerman & Lilach O. Lerman You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar X.-J.C., X.Z., and L.O.L. conceived and designed the study. X.-J.C. and H.T. analyzed and interpreted experiments. K.J. and C.M.F. performed and interpreted MRI experiments. X.Z., A.L., and L.O.L. supervised, interpreted, and provided intellectual input. X.-J.C. and L.O.L. wrote the manuscript with input from all coauthors. L.O.L. oversaw experimental design, data analyses, and manuscript preparation. Correspondence to Lilach O. Lerman. L.O.L. receives funding from Novo Nordisk and advises Weijian Technologies and AstraZeneca. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Reprints and Permissions Chen, X., Jiang, K., Ferguson, C.M. et al. Augmented efficacy of exogenous extracellular vesicles targeted to injured kidneys. Sig Transduct Target Ther 5, 199 (2020). https://doi.org/10.1038/s41392-020-00304-6 Download citation Received: 28 January 2020 Revised: 03 August 2020 Accepted: 20 August 2020 Published: 14 September 2020 DOI: https://doi.org/10.1038/s41392-020-00304-6


Dear Editor,
Extracellular vesicles (EVs) derived from mesenchymal stem/ stromal cells (MSCs) contain genetic and protein material that stimulate tissue repair and ameliorate injury in recipient cells. Advantages of particulate MSC-EVs over MSCs in treating kidney disease include better penetration of injured glomerular filtration barrier to access podocytes or tubular cells. However, systemic EV delivery yields low kidney retention efficiency, limiting their regenerative benefits. 1 Previously, we coated adipose tissue-derived (AD)-MSC with antibodies against kidney injury molecule (KIM)-1 (ab-KIM1), a protein upregulated in damaged kidneys. 2 Conjugating ab-KIM1 did not impair MSC function but increased their retention and reparative potency in murine renal artery stenosis (RAS). 2 We hypothesized that ab-KIM1 conjugation would similarly enhance retention of exogenously delivered EVs in ischemic kidneys and confer superior therapeutic benefits.
Congruently, KIM-EV-treated mice manifested greater attenuation of STK tubular injury, capillary loss, oxidative stress, and fibrosis ( Fig. 1h-l). Tubular cells are susceptible to injury, and inadequately repaired cells may prompt fibrosis and inflammation. While AD-MSC-EV blunt STK tubular injury and fibrosis, 3 ab-KIM-1 conjugation enhanced their STK retention. Double staining indicated that intact KIM-EV engrafted in tubules and might have possibly conferred resistance to injury and improved STK-GFR.
Remodeling and loss of microcirculation also mediate renal ischemic disease progression, yet MSC-derived EVs are endowed with pro-angiogenic properties. In this study, EV delivery improved peritubular capillary density. The robust proangiogenic effects of KIM-EV might stem from repair of tubular cells that regulate microvascular development 4 and reduced oxidative stress and fibrosis. 2 Furthermore, while both decreased the upregulated STK gene expression of VEGF and Flk-1 (likely compensatory to ischemia), only KIM-EV normalized Angpt-1 expression (Supplementary Fig. 1e). Angiopoietin-1 is expressed in renal cortex epithelia and upregulated by ischemia and angiotensin-II. 5 Angiopoietin-1 can blunt angiogenesis when pro-angiogenic factors are upregulated and promote fibrosis and inflammation. 5 Therefore, augmented KIM-EV tubular engraftment might have downregulated Angpt-1 and Mcp-1 and, in turn, STK oxidative stress, inflammation, capillary loss, and fibrosis.
Interestingly, EVs and KIM-EV both attenuated proinflammatory gene expression, including intercellular celladhesion molecule-1, interleukin-6, and tumor necrosis-factor-α ( Supplementary Fig. 2e). However, KIM-EVs were more effective in downregulating monocyte chemotactic-protein-1, a mediator of ischemic injury in renal tubules, suggesting a greater antiinflammatory potency of KIM-EV and efficacy to attenuate kidney damage.
Therefore, we introduce a novel approach to target MSCderived EVs to injured kidneys. EV coating with ab-KIM1, a specific marker of kidney injury, increased their retention in the ischemic kidney and enhanced their therapeutic effects. Our study extends strategies for EV-based treatment in ischemic kidney injury, as well as their broad applications using comparable strategies.

DATA AVAILABILITY
All data relevant to this work are included in this paper and Supplementary Information.

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Signal Transduction and Targeted Therapy (2020) 5:199 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. © The Author(s) 2020 Fig. 1 a Flow gating strategy: a side-scatter [SSC](low) population (P1), events <1 µm, were gated from total events; b P1 population was gated to exclude doublets (P2); c, d SSC(low) DiO(+) events were considered EVs. Allophycocyanin [APC](−)DiO(−) and APC(+)DiO(+) populations were sorted from P2, with 99% unstained extracellular vesicles (EVs) APC(−)DiO(−) and 90% DiO(+) EV coated with ab-KIM1; e Images of ab-KIM1-coated EV (scale bar = 7 μm) double positive for allophycocyanin (red) and Dio (green). BF bright field, ab-KIM1 antibodies against kidney injury molecule-1. b Percentages of cells engrafting EVs detected in the heart, lungs, liver, spleen, and kidneys by imaging flow cytometry. *p < 0.05 vs. native-EV. c Immunofluorescent co-staining with Phaseolus vulgaris erythroagglutinin (PHA-E) and peanut agglutinin (PA) (magenta) identified EV fragments (green, DiO) within stenotic kidney (STK) proximal and distal tubular cells. d STK/ contralateral kidney (CLK) volume ratio decreased similarly in all the RAS groups. e STK-RBF decreased in RAS, increased similarly by native-EVs and KIM-EV, but remained lower than Sham. f STK-GFR decreased in RAS and improved only by KIM-EV. g Higher plasma creatinine in RAS vs. Sham was unaffected by EVs but normalized by KIM-EVs. h STK cortex and medulla staining of trichrome (×20), PAS (×20), CD31 (red, ×40), and dihydroethidium (DHE) [×40, pink, nuclei blue] after treatment with Vehicle (V), EV, or KIM-EV. i, j Renal fibrosis and tubular injury increased in RAS + Vehicle. KIM-EV attenuated them further than RAS + EV. k STK CD31 staining showed a decrease in microvascular density, which improved with EV, and significantly more by KIM-EV. l Elevated STK-DHE indicated increased superoxide production, which decreased by EV and tended to decrease further by KIM-EV. *p < 0.05 vs. Sham, † p < 0.05 vs. RAS + vehicle, ‡ p < 0.05 vs. RAS + EV, # p = 0.1 vs. RAS + EV Letter