Human extracellular microvesicles from renal tubules reverse kidney ischemia-reperfusion injury in rats
Hypoxic acute kidney injury, a major unresolved problem, initiates, or aggravates, renal functional and structural decline. There is no treatment for hypoxic acute renal injury and its sequelae. We tested the hypothesis that human kidney tubular cells, or their extracellular vesicles (exosomes), prevent renal injury when infused intravenously 24 hours after 50 minutes of bilateral renal ischemia in Nude rats. Cells and their exosomes were from harvested human kidneys declined for
... for transplantation. Injections of either cells or exosomes, given after 24 and 48 hours of reperfusion, preserved renal function and structure in both treatment groups. However, exosomes were superior to cells; and maintained renal vascular and epithelial networks, prevented renal oxidant stress, and apoptosis; and restrained activation of pro-inflammatory and pro-fibrogenic pathways. Exosomes worked in 24 hours, consistent with functional rather than regenerative activity. Comprehensive proteomic analysis identified 6152 renal proteins from all cellular compartments; and 628 were altered by ischemia at all cell levels, while 377 were significantly improved by exosome infusions. We conclude that renal damage from severe ischemia was broad, and human renal exosomes prevented most protein alterations. Thus, exosomes seem to acutely correct a critical and consequential abnormality during reperfusion. In their absence, renal structure and cells transition to a chronic state of fibrosis and extensive renal cell loss. OPEN ACCESS Citation: Dominguez JM II, Dominguez JH, Xie D, Kelly KJ (2018) Human extracellular microvesicles from renal tubules reverse kidney ischemiareperfusion injury in rats. PLoS ONE 13(8): e0202550. https://doi.org/10.Institute. The funders had no role in study design, models of acute and chronic renal injury      . We also recognized an apparent paradox in our data, as a relatively small number of infused cells had broad beneficial renal effects  . Hence, we hypothesized that transplanted primary renal cells amplified their actions through released extracellular vesicles (EVs) in situ  . Exosomes, the best characterized type of EVs; are secreted nanovesicles (30-150nm in diameter) that contain proteins, lipids, mRNA and miRNAs  . Secreted exosomes interconnect cells, and convey the metabolic state of the originating cells, including protective responses to hypoxia    . For example, we have showed that renal exosomes released from hypoxia pre-conditioned renal tubular cells (i.e., HPC exosomes) prevented most manifestations of severe AKI  . We now compared the effects of HPC human kidney tubular cells with their exosomes on athymic Nude rats with severe hypoxic AKI. We found that after 24 hours of reperfusion, peripheral intravenous infusion of human kidney tubular cells, or their exosomes, protected severely post-ischemic kidneys at multiple levels, including structure, function and expressed proteins. Methods Complete description of methods can be found in the Supplemental section. Results Human renal tubular cells and HLA A1 expression in rat kidneys Cultured human renal tubular cells expressed key epithelial features prior to infusion, Fig 1. The donor cells were positive for Human Leukocyte Antigen A1 and for pan-cytokeratin, consistent with their human epithelial derivation. They were 77 ± 4% positive for the organic anion transporter 1, a renal proximal tubule marker. We presume the remaining epithelial cells were from other nephron segments or had precursor cell background , although they did not express the stem cell marker nanog. In addition, they did not express endothelial markers CD31 or E-selectin or glomerular podocyte markers nephrin or synaptopodin or the fibroblast markers alpha smooth muscle actin or fibroblast specific protein-1. Cells were also Fig 1. Human renal cells and HLA A1 expression in rat kidneys. Top, human kidney tubular cells were 99 ± 1% positive for HLA-A1 (red, left), 99 ± 1% positive for Pan-cytokeratin (green, second from left), 77 ± 4% positive for OAT1 (red, second from right), and 63 ± 3% positive for transfected GFP (right), n = 4. Bottom, HLA-A1 immunoreactivity (green) was not detected in sham rats (SHAM; left). In contrast, HLA-A1 (green) was expressed in renal tubules of ischemic rats injected with kidney cell exosomes (huEXO; middle, arrows), or their originating transplanted cells (huCELLS, right, arrow heads). Renal F actin was stained with rhodamine phalloidin (red) to outline cells, and Hoechst 33342 (blue) was added to label nuclei.