Kidney Mesenchymal Stem Cell‐derived Extracellular Vesicles Engineered to Express Erythropoietin Improve Renal Anemia in Mice with Chronic Kidney Disease

Abstract

Extracellular vesicles (EVs) shed from kidney mesenchymal stem cells (KMSCs) show protective effects against acute kidney injury and progressive kidney fibrosis via mRNA transfer. Previous studies report improvement of renal anemia following administration of genetically modified MSCs or peritoneal mesothelial cells that secrete erythropoietin (EPO). Here, we determined whether EPO-secreting KMSC-derived EVs (EPO(+)-EVs) can improve renal anemia in mouse models of chronic kidney disease (CKD). The mouse CKD and renal anemia model was induced by electrocoagulation of the right renal cortex and sequential left nephrectomy. At six weeks post-nephrectomy, we observed significantly lower hemoglobin (10.4 ± 0.2 vs. 13.2 ± 0.2 g/dL) and significantly higher blood urea nitrogen and serum creatinine levels in CKD mice relative to controls (60.5 ± 0.5 and 0.37 ± 0.09 mg/dL vs. 19.9 ± 0.5 and 0.12 ± 0.02 mg/dL, respectively). Genetically engineered EPO(+)–KMSCs secreted 71 IU/mL EPO/106 cells/24 h in vitro, and EPO(+)–EVs isolated by differential ultracentrifugation expressed EPO mRNA and horizontally transferred EPO mRNA into target cells in vitro and in vivo. Furthermore, at two weeks post-injection of EPO(+)–KMSCs or EPO(+)-EVs into CKD mice with renal anemia, we observed significant increases in hemoglobin levels (11.7 ± 0.2 and 11.5 ± 0.2 vs. 10.1 ± 0.2 g/dL, respectively) and significantly lower serum creatinine levels at eight weeks in comparison to mice receiving vehicle control (0.30 ± 0.00 and 0.23 ± 0.03 vs. 0.43 ± 0.06 mg/dL, respectively). These results demonstrate that intraperitoneal administration of EPO(+)-EVs significantly increased hemoglobin levels and renal function in CKD mice, suggesting the efficacy of these genetically engineered EVs as a promising novel strategy for the treatment of renal anemia.

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Funding

This study was supported by a faculty research grant from Yonsei University College of Medicine (6-2014-0042), a research grant from CHA Bundang Medical Center (2015 − 0301), and the Basic Science Research Program through the National Research Foundation of Korea (2012R1A1A2009345) funded by the Ministry of Education of the Republic of Korea.

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Conceptualization: Hoon Young Choi and Hyeong Cheon Park; Methodology: Hoon Young Choi, Hyeong Cheon Park, Tae Yeon Kim, Mirae Lee, Soo Hyun Kim, and Jong Hyun Jhee; Formal analysis and investigation: Hoon Young Choi, Tae Yeon Kim, Mirae Lee, Soo Hyun Kim, and Hyung Jong Kim; Writing - original draft preparation: Hoon Young Choi and Hyeong Cheon Park; Writing - review and editing: Hoon Young Choi, Yong Kyu Lee, Hyung Jong Kim, and Hyeong Cheon Park; Funding acquisition: Yong Kyu Lee, Hyung Jong Kim, and Hyeong Cheon Park; Supervision: Hyung Jong Kim, Jong Hyun Jhee ,and Hyeong Cheon Park.

All authors read and approved the final manuscript.

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Correspondence to Hyeong Cheon Park.

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The animal study protocol was performed in accordance with guidelines for laboratory animals and approved by the Institutional Animal Care and Use Committee, Department of Laboratory Animal Medicine, Medical Research Center, Yonsei University College of Medicine (2010-0226-1).

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This article belongs to the Topical Collection: Special Issue on Exosomes and Microvesicles: from Stem Cell Biology to Translation in Human Diseases

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Supplementary Information

Supplemental Figure S1
figure9

Immunofluorescence staining of cytokeratin in mouse peritoneal mesothelial cells (PMCs). Immunofluorescence microscopy showing cytokeratin-labeled (red) mouse PMCs. DAPI, 4’,6-diamidino-2-phenylindole. (PNG 315 kb)

Supplemental Figure S2
figure10

Anemia and renal function in normal mice and mouse models of chronic kidney disease (CKD). (A) Hemoglobin levels and (B) blood urea nitrogen levels (BUN) and serum creatinine (Cr) levels in normal and CKD mice. *P < 0.05 vs. vehicle control, Kruskal–Wallis test. (PNG 60 kb)

Supplemental Figure S3
figure11

Masson’s trichrome staining of tissue from mouse models of chronic kidney disease (CKD). (A) Extensive fibrotic lesions were present in the cortex and corticomedullary junction of kidney sections from CKD mice after 6 and 9 weeks. (B) Semiquantitative injury scoring of the cortex and corticomedullary junction of kidney sections from CKD mice after 6 and 9 weeks. (PNG 2399 kb)

Supplement Figure S4
figure12

Erythropoietin (EPO)(+)–kidney-derived mesenchymal stem cell (KMSC)-derived extracellular vesicles (EPO(+)-EVs) mediate horizontal EPO mRNA transfer into human tubular cells (HK-2) and human podocytes in vitro. (A) Immunofluorescence microscopy showing red CellTracker™ -labeled EVs in human tubular cells (HK-2) and human podocytes. (B) EPO mRNA levels in HK-2 and human podocytes treated in vitro with EPO(+)-EVs. EPO(−)-EVs were used as negative controls.(PNG 413 kb)

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Choi, H.Y., Kim, T.Y., Lee, M. et al. Kidney Mesenchymal Stem Cell‐derived Extracellular Vesicles Engineered to Express Erythropoietin Improve Renal Anemia in Mice with Chronic Kidney Disease. Stem Cell Rev and Rep (2021). https://doi.org/10.1007/s12015-021-10141-x

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Keywords

  • Extracellular vesicles
  • Kidney mesenchymal stem cells
  • Erythropoietin
  • mRNA transfer
  • Renal anemia
  • Chronic kidney disease