Mesenchymal Stem Cells-Derived Exosomes Ameliorate Ischemia/Reperfusion Induced Acute Kidney Injury in a Porcine Model

Abstract

Exosomes are membrane-enclosed vesicles secreted by cells, containing a variety of biologically active ingredients including proteins, nucleic acids and lipids. In this study, we investigated the therapeutic effects of the exosomes and underlying mechanisms in a miniature pig model of ischemia/reperfusion-induced acute kidney injury (I/R-AKI). The exosomes were extracted from cultured human umbilical cord derived mesenchymal stem cells (hUC-MSCs) and infused into a miniature pig model of I/R AKI. Our results showed that 120 min of unilateral ischemia followed by reperfusion and contralateral nephrectomy resulted in renal dysfunction, severe kidney damage, apoptosis and necroptosis. Intravenous infusion of one dose of exosomes collected from about 4 × 10⁸ hUC-MSCs significantly improved renal function and reduced apoptosis and necroptosis. Administration of hUC-MSC exosomes also reduced the expression of some pro-inflammatory cytokines/chemokines, decreased infiltration of macrophages to the injured kidneys and suppressed the phosphorylation of nuclear factor-κB and signal transducer and activator of transcription 3, two transcriptional factors related to inflammatory regulation. Moreover, hUC-MSC exosomes could promote proliferation of renal tubular cells, angiogenesis and upregulation of Klotho and Bone Morphogenetic Protein 7, two renoprotective molecules and vascular endothelial growth factor A and its receptor. Collectively, our results suggest that injection of hUC-MSC exosomes could ameliorate I/R-AKI and accelerate renal tubular cell repair and regeneration, and that hUC-MSC exosomes may be used as a potential biological therapy for Acute kidney injury patients.

Keywords: acute kidney injury; apoptosis; exosomes; human umbilical cord derived mesenchymal stem cells; ischemia/reperfusion; macrophages; necroptosis; transcriptional factors.

FIGURE 1

Kidney injury indicators elevated after surgical establishment of I/R Induced AKI in a porcine Model. (A,B) Renal function of pigs after different ischemia duration and reperfusion (each group n = 4). Blood was sampled before operation and 24, 48, 72 h after reperfusion. Values at different time points vs. baseline value (D0) from the same group for statistical analysis. SCr, serum creatinine; BUN, blood urea nitrogen. (C) Photomicrographs illustrating hematoxylin and eosin staining of kidney tissue from each group. Arrows point to injured tubules with intraluminal casts. (D) Tubule Injury was scored and graphed to analyze the severity of renal tubular injury. (E) Photomicrographs illustrating immunohistochemical staining of NGAL. Scale bar = 100 µm. (F) Quantification of the numbers of NGAL positive tubules from immunohistochemical staining. (G) The kidney tissue lysates from four groups were subject to immunoblot analysis with specific antibodies against NGAL. (H) Expression levels of NGAL were quantified by densitometry analysis and then normalized with GAPDH (3 independent trials). Data are means ± sem.*p < 0.05; **p < 0.01 versus sham controls.

FIGURE 2

Apoptosis and necroptosis levels in miniature pigs with acute kidney injury induced by ischemia-reperfusion. (A) Representative images of TUNEL staining of kidney tissue from each group. Scale bar = 100 µm. (B) Quantification and plot of the TUNEL stained images. (C) Photomicrographs illustrating immunofluorescence staining of phospho-MLKL. Scale bar = 200 µm. (D) Quantification and plot of the immunofluorescence integrated density of phospho-MLKL. (E) Porcine kidney tissue lysates from four groups were subject to immunoblot analysis with specific antibodies against cleaved-caspase 3, MLKL, phospho-MLKL, RIPK3 and phospho-RIPK3. Expression levels of cleaved-caspase 3 (F), MLKL (H) and RIPK3 (J) were quantified by densitometry analysis and then normalized with GAPDH. The phosphorylation levels of MLKL (G) and RIPK3 (I) were calculated quantitatively by densitometry. Data are means ± sem.*p < 0.05; **p < 0.01 versus sham controls.

FIGURE 3

Extraction and identification of exosomes from mesenchymal stem cells derived from human umbilical cord. (A) Pictures of the 6th passage of mesenchymal stem cells after 24 and 72 h of culture. Scale bar = 100 µm. (B) The expression of the marker molecules CD9 and CD81 in the exosome lysate was detected by immunoblotting. Two samples from different extraction batches was tested. (C) Morphology of exosomes observed under transmission electron microscope. (D) The particle size distribution of exosomes by Nanoparticle Tracking Analysis. (E) Biodistribution of exosomes labeled by DiI in different organs. Scale bar = 100 µm.

FIGURE 4

The renal ischemia-reperfusion injury in miniature pigs was significantly reduced after exosomes treatment. (A,B) Serum creatinine (SCr) and blood urea nitrogen (BUN) at 72 h after treatment. (C) Photomicrographs illustrating hematoxylin and eosin staining of kidney tissue from four groups. Arrows point to injured tubules with intraluminal casts. Scale bar = 100 µm. (D) Tubule Injury was scored and graphed to analyze the severity of renal tubular injury. (E) Photomicrographs illustrating immunohistochemical staining of NGAL. Scale bar = 100 µm. (F) Quantification and plot of the percentage of NGAL positive tubules from immunohistochemical staining. (G) Porcine kidney tissue lysates from four groups were subject to immunoblot analysis with specific antibodies against NGAL. (H) Expression levels of NGAL were quantified by densitometry analysis and then normalized with GAPDH. Data are means ± sem.*p < 0.05; **p < 0.01 versus sham controls.

FIGURE 5

Exosomes down-regulated apoptosis and necroptosis levels in pigs with acute kidney injury induced by ischemia-reperfusion. (A) Representative images of TUNEL staining of kidney tissue from four groups. Scale bar = 100 µm. (B) Quantification and plot of the TUNEL stained images. (C) Photomicrographs illustrating immunofluorescence staining of phospho-MLKL. Scale bar = 200 µm. (D) Quantification and plot of the Immunofluorescence integrated density of phospho-MLKL. (E) Porcine kidney tissue lysates from four groups were subject to immunoblot analysis with specific antibodies against cleaved-caspase 3, MLKL, phospho-MLKL. Expression levels of cleaved-caspase 3 (F) and MLKL (H) were quantified by densitometry analysis and then normalized with GAPDH. The phosphorylation level of MLKL (G) was calculated quantitatively by densitometry. Data are means ± sem.*p < 0.05; **p < 0.01 versus sham controls.

FIGURE 6

MSCs-derived exosomes promote renal regeneration and the expression of renal protection factors. (A) Photomicrographs illustrating immunohistochemical staining of regeneration biomarker PCNA. Scale bar = 100 µm. (B) Quantification and graph of the number of PCNA positive cells from immunohistochemical staining. (C) Porcine kidney tissue lysates from four groups were subject to immunoblot analysis with specific antibodies against PCNA, Klotho and BMP-7. Expression levels of PCNA (D), Klotho (E) and BMP-7 (F), were quantified by densitometry analysis and then normalized with α-Tubulin. Data are means ± sem.*p < 0.05; **p < 0.01 versus sham controls.

FIGURE 7

MSCs-derived exosomes inhibited production of pro-inflammatory factors. (A) Photomicrographs illustrating immunohistochemical staining of macrophage biomarker F4/80 Scale bar = 100 µm. Arrows point to F4/80-positive macrophages. (B) Quantification and graph of the number of F4/80 positive cells from immunohistochemical staining. RT-qPCR was applied to detect the expression level of the inflammatory factor MCP-1 (C), TNF-α (D), IL-1β (E) and IL10 (F). (H) Porcine kidney tissue lysates from four groups were subject to immunoblot analysis with specific antibodies against phospho-NF-κb, NF-κB, phospho-STAT3 and STAT3. Expression levels of NF-κB (I) and STAT3 (K) were quantified by densitometry analysis and then normalized with GAPDH. The phosphorylation levels of NF-κB (G) and STAT3 (J) were calculated quantitatively by densitometry. Data are means ± sem.*p < 0.05; **p < 0.01 versus sham controls.

FIGURE 8

MSCs-derived exosomes restore loss of renal angiogenesis. (A) Photomicrographs illustrating co-immunofluorescence staining of endothelial markers CD31 and VEGFA. Scale bar = 200 µm. Quantification and plot of immunofluorescence density of VEGFA (B) and CD31 (C). (D) Porcine kidney tissue lysates from each group were subject to immunoblot analysis with specific antibodies against CD31, VEGFA and VEGFR2. Expression levels of CD31 (E), VEGFA (F) and VEGFR2 (G), were quantified by densitometry analysis and then normalized with α-Tubulin. Data are means ± sem.*p < 0.05; **p < 0.01 versus sham controls.