Hypoxia modulates the undifferentiated phenotype of human renal inner medullary CD133(+) progenitors through Oct4/miR-145 balance
Low-oxygen tension is an important component of the stem cell microenvironment. In rodents, renal resident stem cells have been described in the papilla, a relatively hypoxic region of the kidney. In the present study, we found that CD133(+) cells, previously described as renal progenitors in the human cortex, were enriched in the renal inner medulla and localized within the Henle's loop and thin limb segments. Once isolated, the CD133(+) cell population expressed renal embryonic and stem-related transcription factors and was able to differentiate into mature renal epithelial cells. When injected subcutaneously in immunodeficient mice within Matrigel, CD133(+) cells generated canalized structures positive for renal specific markers of different nephron segments. Oct4A levels and differentiation potential of papillary CD133(+) cells were higher than those of CD133(+) cells from cortical tubuli. Hypoxia was able to promote the undifferentiated phenotype of CD133(+) progenitors from papilla. Hypoxia stimulated clonogenicity, proliferation, vascular endothelial growth factor synthesis, and expression of CD133 that were in turn reduced by epithelial differentiation with parallel HIF-1α downregulation. In addition, hypoxia downregulated microRNA-145 and promoted the synthesis of Oct4A. Epithelial differentiation increased microRNA-145 and reduced Oct4 level, suggesting a balance between Oct4 and microRNA-145. MicroRNA-145 overexpression in CD133(+) cells induced downrelation of Oct4A at the protein level, inhibited cell proliferation, and stimulated terminal differentiation. This study underlines the role of the hypoxic microenvironment in controlling the proliferation and maintaining a progenitor phenotype and stem/progenitor properties of CD133(+) cells of the nephron. This mechanism may be at the basis of the maintenance of a CD133(+) population in the papillary region and may be involved in renal regeneration after injury.
[Show abstract] [Hide abstract] ABSTRACT: Experimental models are inevitably a compromise between accurately reproducing a pathological situation and schematically simplifying it, which is intended to provide both relevance and conclusiveness. In-vivo models are very relevant, but multiple cell-types undergoing various changes may hinder the observation of individual molecular events. Here, we describe a method for analyzing and isolating specific cell types from the kidney and studying the phenotype they have acquired in vivo. Using flow cytometry, immunofluorescence, and RT-PCR, we show that our method is suitable for studying and isolating proximal tubular cells with an anti Prominin-1 antibody. Kidneys are subjected to mechanical dissociation followed by flow-cytometry analysis. Hundreds of thousands of proximal tubular cells are then isolated by magnetic separation followed by direct analysis or primary cell culture. Using our method, we detect phenotypic changes in the proximal tubular cells after renal ischemia reperfusion, and we isolate the proximal tubular cells, with a purity over 80%. This method is efficient, quick, simple, and cheap, and should be useful for studying cell-type specific parameters after in vivo experimental studies. It is also a simple method to obtain a specific primary cell culture from any animal strain.0Comments 2Citations
- "Ex-vivo analysis of proximal tubular cells in the whole kidney suspension Proximal tubular epithelial cells are highly differentiated renal cells. Prominin-1 is a surface glycoprotein expressed in the brush border101112. Anti-prominin-1 antibodies are theoretically appropriate for use in that a) they are commercially available (anti-Prominin-1 rat monoclonal IgG1 antibody, clone MB9-3G8, Miltenyi Biotec, Bergisch Gladbach, Germany), b) they have been conjugated with a number of tags, and c) they target the extracellular regionFigure 1 Flowchart of the method detailing the three applications of dissociated cells: direct analysis, isolation of a subpopulation of cells for subsequent analysis, or for primary cell culture.Table 1 Protocol for direct dissociation of a whole kidney into a cell suspension of prominin-1, which makes it possible to work with live, unpermeabilized cells. There are several isoforms of Prominin-1, and some anti- Prominin-1 antibodies show clone-dependent specificities for renal epithelial cellular subtypes . "
[Show abstract] [Hide abstract] ABSTRACT: Extracellular vesicles (EVs) present in the urine are mainly released from cells of the nephron and can therefore provide information on kidney function. We here evaluated the presence of vesicles expressing the progenitor marker CD133 in the urine of normal subjects and of patients undergoing renal transplant. We found that EV expressing CD133 were present in the urine of normal subjects, but not of patients with end stage renal disease. The first day after transplant, urinary CD133+ EVs were present at low levels, to increase thereafter (at day 7). Urinary CD133+ EVs significantly increased in patients with slow graft function in respect to those with early graft function. In patients with a severe pre-transplant vascular damage of the graft, CD133+ EVs did not increase at day 7. At variance, the levels of EVs expressing the renal exosomal marker CD24 did not vary in the urine of patients with end stage renal disease or in transplanted patients in respect to controls. Sorted CD133+ EVs were found to express glomerular and proximal tubular markers. These data indicate that urinary CD133+ EVs are continuously released during the homeostatic turnover of the nephron and may provide information on its function or regenerative potential.0Comments 5Citations
- "Cytofluorimetric analysis of the EVs using latex beads showed presence of typical exosome markers such as CD9, CD81 and Rab5, and of the renal vesicle marker CD24  (Fig. 1B).Figure 1C shows Western Blot analysis of EVs expressing the tetraspanins CD9, CD63 and CD81. We focused our attention on CD133, a stem cell marker expressed in the kidney by progenitors cells101112 and previously described in the urinary EVs . We found that CD133 + EVs were detectable at high levels in the urine of normal subjects using cytofluorimetric assay (Fig. 2A). "
[Show abstract] [Hide abstract] ABSTRACT: Recent approaches of regenerative medicine can offer a therapeutic option for patients undergoing acute kidney injury. In particular, mesenchymal stem cells were shown to ameliorate renal function and recovery after acute damage. We here evaluated the protective effect and localization of CD133+ renal progenitors from the human inner medulla in a model of glycerol-induced acute tubular damage and we compared the results with those obtained with bone marrow-derived mesenchymal stem cells. We found that CD133+ progenitor cells promoted the recovery of renal function, preventing tubular cell necrosis and stimulating resident cell proliferation and survival, similar to mesenchymal stem cells. In addition, by optical imaging analysis, CD133+ progenitor cells accumulated within the renal tissue, and a reduced entrapment in lung, spleen, and liver was observed. Mesenchymal stem cells were detectable at similar levels in the renal tissue, but a higher signal was present in extrarenal organs. Both cell types produced several cytokines/growth factors, suggesting that a combination of different mediators is involved in their biological action. These results indicate that human CD133+ progenitor cells are renotropic and able to improve renal regeneration in acute kidney injury.0Comments 3Citations
- "This is also suggested by the lack of acquirement of epithelial or endothelial markers. Although further studies are required to analyze the possible integration of CD133 + cells from inner medulla in the murine kidney and their differentiation at later time, these results may suggest that these cells display differential properties with respect to those from cortex, as previously described (Bussolati et al. 2012). Indeed, CD133 + cells from the inner medulla were shown in vitro to be responsible for erythropoietin production (Bussolati et al. 2013 ). "