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. 2020 Sep 23;11(1):410.
doi: 10.1186/s13287-020-01917-y.

Human amniotic epithelial cells ameliorate kidney damage in ischemia-reperfusion mouse model of acute kidney injury

Yifei Ren  1   2   3 , Ying Chen  1   2   3 , Xizi Zheng  1   2   3   4 , Hui Wang  1   2   3   5 , Xin Kang  1   2   3 , Jiawei Tang  1   2   3 , Lei Qu  1   2   3   4 , Xiaoyan Shao  6 , Suxia Wang  1   2   3   5 , Shuangling Li  7 , Gang Liu  1   2   3   4 , Li Yang  8   9   10   11
Affiliations

Human amniotic epithelial cells ameliorate kidney damage in ischemia-reperfusion mouse model of acute kidney injury

Yifei Ren et al. Stem Cell Res Ther. .

Abstract

Background: Acute kidney injury (AKI) is a common clinical disease with complex pathophysiology and limited therapeutic choices. This prompts the need for novel therapy targeting multiple aspects of this disease. Human amnion epithelial cell (hAEC) is an ideal stem cell source. Increasing evidence suggests that exosomes may act as critical cell-cell communicators. Accordingly, we assessed the therapeutic potential of hAECs and their derived exosomes (hAECs-EXO) in ischemia reperfusion mouse model of AKI and explored the underlying mechanisms.

Methods: The hAECs were primary cultured, and hAECs-EXO were isolated and characterized. An ischemic-reperfusion injury-induced AKI (IRI-AKI) mouse model was established to mimic clinical ischemic kidney injury with different disease severity. Mouse blood creatinine level was used to assess renal function, and kidney specimens were processed to detect cell proliferation, apoptosis, and capillary density. Macrophage infiltration was analyzed by flow cytometry. hAEC-derived exosomes (hAECs-EXO) were used to treat hypoxia-reoxygenation (H/R) injured HK-2 cells and mouse bone marrow-derived macrophages to evaluate their protective effect in vitro. Furthermore, hAECs-EXO were subjected to liquid chromatography-tandem mass spectrometry for proteomic profiling.

Results: We found that systematically administered hAECs could improve mortality and renal function in IRI-AKI mice, decrease the number of apoptotic cells, prevent peritubular capillary loss, and modulate kidney local immune response. However, hAECs showed very low kidney tissue integration. Exosomes isolated from hAECs recapitulated the renal protective effects of their source cells. In vitro, hAECs-EXO protected HK-2 cells from H/R injury-induced apoptosis and promoted bone marrow-derived macrophage polarization toward M2 phenotype. Proteomic analysis on hAECs-EXO revealed proteins involved in extracellular matrix organization, growth factor signaling pathways, cytokine production, and immunomodulation. These findings demonstrated that paracrine of exosomes might be the key mechanism of hAECs in alleviating renal ischemia reperfusion injury.

Conclusions: We reported hAECs could improve survival and ameliorate renal injury in mice with IRI-AKI. The anti-apoptotic, pro-angiogenetic, and immunomodulatory capabilities of hAECs are at least partially, through paracrine pathways. hAECs-EXO might be a promising clinical therapeutic tool, overcoming the weaknesses and risks associated with the use of native stem cells, for patients with AKI.

Keywords: Acute kidney injury; Cell therapy; Exosome; Human amnion epithelial cells; Ischemia.

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Conflict of interest statement

Li Yang has a patent pending for the use of hAECs in repair of the injured kidney after AKI. Shanghai iCELL Biotechnology company declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. The other authors have no financial conflicts of interest.

Figures

Fig. 1
Fig. 1
Establishment of IRI-AKI mouse model. a 7-day mortality rate in IRI mice with 30 min (30min-IRI, n = 7) or 33 min (33min-IRI, n = 21) ischemia. *P < 0.05. b Serum creatinine concentrations at day 1 and day 2 post-surgery in 30min-IRI group and 33min-IRI group. c Representative micrographs of PAS staining of kidneys from sham, 30min-IRI, and 33min-IRI group at day 1 after surgery. Scale bar, 25 μm. Arrows indicate damaged renal tubules
Fig. 2
Fig. 2
hAECs ameliorated acute renal IRI. a Morphology of hAECs was observed under bright field microscopy. Magnification, × 100 and × 400. b Flow cytometry analysis of cell surface markers SSEA4, CD324, HLA-DR, and CD146 on hAECs. The isotypes (ISO) were used as negative controls. hAECs are positive for SSEA4 and CD324 and negative for HLA-DR and CD146. c 7-day mortality rate in IRI mice with 33-min ischemia followed by vehicle (n = 19) or hAECs (n = 20) injection. *P < 0.05. d Serum creatinine concentrations in mice with 30 min ischemia followed by vehicle (n = 11) or hAECs injection (n = 13). *P < 0.05 vs IRI+Veh group, **P < 0.01 vs IRI+Veh group. e PAS staining of post-ischemic kidneys on day 1 after 30 min ischemia. Scale bar, 25 μm. Arrows indicate damaged renal tubules. f Renal pathological scores representing the degree of tubulointerstitial damage. *P < 0.05 vs IRI+Veh group. IRI+Veh: 30-min ischemia mice injected with vehicle alone (n = 3); IRI+hAECs: 30-min ischemia mice injected with 1 × 106 hAECs (n = 3). Data are shown as mean (SD)
Fig. 3
Fig. 3
Effects of intravenous injection of hAECs-EXO in mice with IRI-AKI. a Morphology of hAECs exosomes under transmission electron microscopy. Scale bar, 100 nm. b Mean diameter and concentration of hAEC exosomes analyzed by nanoparticle tracking system (NTA). Approximately 1.6 × 1010 particles were measured by NTA in hAECs exosomes, which came from a total of 48.2 × 106 hAECs. c Western blot detection of the exosome markers CD63, Flotillin, TSG101, and Alix. d 7-day mortality rate in mice with 33-min ischemia followed by vehicle (IRI+Veh, n = 11) or hAECs-EXO (IRI+hAECs-EXO, n = 12) injection. *P < 0.05. e Serum creatinine concentrations in mice with 30-min ischemia followed by vehicle (n = 5) or hAECs injection (n = 6). *P < 0.05 vs IRI+Veh group. f PAS staining of post-ischemic kidneys at day 1 after 30-min ischemia in different groups as indicated. Scale bar, 25 μm. Arrows indicate damaged renal tubules. g Renal pathological scores representing the degree of tubulointerstitial damage. *P < 0.05 vs IRI+Veh group. IRI+Veh: 30-min ischemia mice injected with vehicle alone; IRI+hAECs-EXO: 30-min ischemia mice injected with about 3 × 108 exosomes. Data are shown as mean (SD)
Fig. 4
Fig. 4
Anti-apoptotic effect of hAECs or hAECs-EXO in mice with 30-min ischemia and hAECs-EXO in H/R injured HK2 cells. a Representative micrographs of TUNEL staining in different groups as indicated at day 1, day 2, day 3, and day 7 post-ischemia. Scale Bar, 25 μm. b Quantification of TUNEL-positive cells/HPF. 3 different mice were used in each group with at least 6 images were taken on each mouse kidney. *P < 0.05 vs IRI+Veh group; **P < 0.01 vs IRI+Veh group; #P < 0.05 vs IRI+Veh group; ##P < 0.01 vs IRI+Veh group. c Representative Western blots showed protein expression of cleaved-caspase 3 and caspase 3 in different groups as indicated. d Graphic presentation showed the relative abundances of cleaved-caspase 3 in different groups (n = 3). *P < 0.05 vs normal control; #P < 0.05 vs H/R group. e Representative micrographs of Ki67 staining in different groups as indicated at day 1, day 2, day 3, and day 7 post-ischemia. Scale bar, 25 μm. f Quantification of Ki67-positive cells/HPF. 3 different mice were used in each group with at least 6 images were taken on each mouse kidney. *P < 0.05 vs IRI+Veh group; ***P < 0.001 vs IRI+Veh group; #P < 0.05 vs IRI+Veh group. g Representative Western blots showed protein expression of PCNA in different groups as indicated (n = 3). h Graphic presentation showed the relative abundances of PCNA in different groups. *P < 0.05 vs normal control; #P < 0.05 vs H/R group
Fig. 5
Fig. 5
hAECs or hAECs-EXO prevent peritubular capillary loss in mice with 30-min ischemia. a Representative micrographs of CD31 staining in different groups as indicated at day 7 post-ischemia. Arrows indicate CD31 positive peritubular capillaries (PTCs). Scale bar, 25 μm. b Quantification of CD31-positive signal/HPF. 3 different mice were used in each group with at least 6 images were taken on each mouse kidney. *P < 0.05 vs sham group; &P < 0.05 vs IRI+Veh group; #P < 0.05 vs IRI+Veh group. c Representative Western blot analyses showed protein expression of CD31 in different groups as indicated. d Graphic presentation showed the relative abundances of CD31 in different groups. *P < 0.05 vs sham group; &P < 0.05 vs IRI+Veh group; #P < 0.05 vs IRI+Veh group (n = 3). e Growth factors expression in post-ischemic kidneys on day 1, day 2, day 3, and day 7 after hAECs or hAECs-EXO administration in mice with IRI-induced AKI. mRNA transcripts of Egf, Fgf, Hgf, Igf-1, Pdgf, and Vegf were determined by qRT-PCR. *P < 0.05 vs IRI+Veh group; #P < 0.05 vs IRI+Veh group (n = 4)
Fig. 6
Fig. 6
hAECs altered chemokine expression and macrophage infiltration in kidneys with 30-min ischemia. a Macrophage populations were measured via flow cytometry. Representative gating strategy was shown. The percentages of macrophages from the total kidney cell population were calculated. b Percentage of F4/80+ macrophages in kidneys treated with hAECs or hAECs-EXO at day 1, day 2, day 3, and day 7 post-ischemia. &P < 0.05 vs sham group; &&P < 0.01 vs sham group; **P < 0.01 vs IRI+Veh group; #P < 0.05 vs IRI+Veh group; ##P < 0.01 vs IRI+Veh group. c Kidney chemokine concentrations from mice in different groups as indicated at day 1, day2, day 3, and day 7after IRI. *P < 0.05 vs IRI+Veh group; #P < 0.05 vs IRI+Veh group; ##P < 0.01 vs IRI+Veh group (n = 3)
Fig. 7
Fig. 7
hAECs-EXO induced M2 macrophage polarization. a CD206+/F4/80+ M2 macrophage population was measured via flow cytometry. Representative gating strategy was shown. The percentages of M2 macrophages from the total kidney cell population were calculated. b Percentage of M2 Macrophages in kidneys treated with hAECs or hAECs-EXO at day 1, day 2, day 3, and day 7 post-ischemia (n = 3). &P < 0.05 vs sham group; &&P < 0.01 vs sham group; &&&P < 0.001 vs sham group; **P < 0.01 vs IRI+Veh group. #P < 0.05 vs IRI+Veh group; ##P < 0.01 vs IRI+Veh group. c Kidney cytokine concentrations from mice in different groups as indicated at day 1, day2, day 3, and day 7 after IRI (n = 3). *P < 0.05 vs IRI+Veh group; #P < 0.05 vs IRI+Veh group; ##P < 0.01 vs IRI+Veh group. d Bone marrow monocytes were attached for 48 h and collected as control. Bone marrow-derived macrophages were cultured in hAECs-EXO conditioned medium for 7 days and collected. mRNA transcripts of macrophage marker (F4/80) and M1 (Ifnγ, iNos, Tnfα, Cd86) and M2 (Cd163, Cd206, Il4rα, Arg1) markers were determined by qRT-PCR (n = 3). *P < 0.05 vs control group; **P < 0.01 vs control group. Data are shown as mean (SD)
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References

    1. Bellomo R, Kellum JA, Ronco C. Acute kidney injury. Lancet. 2012;380(9843):756–766. - PubMed
    1. Sharfuddin AA, Molitoris BA. Pathophysiology of ischemic acute kidney injury. Nat Rev Nephrol. 2011;7(4):189–200. - PubMed
    1. Bonventre JV, Yang L. Cellular pathophysiology of ischemic acute kidney injury. J Clin Invest. 2011;121(11):4210–4221. - PMC - PubMed
    1. Zuk A, Bonventre JV. Acute kidney injury. Annu Rev Med. 2016;67:293–307. - PMC - PubMed
    1. Molitoris BA. Therapeutic translation in acute kidney injury: the epithelial/endothelial axis. J Clin Invest. 2014;124(6):2355–2363. - PMC - PubMed

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