Characterization and determination of cadmium resistance of CD133+/CD24+ and CD133−/CD24+ cells isolated from the immortalized human proximal tubule cell line, RPTEC/TERT1

Abstract

Stem/progenitor cells are involved in the regeneration of the renal tubules after damage due to a toxic insult. However, the mechanism involved in the regeneration of the tubules by the stem cells is not well understood due to the lack of immortal cell lines that represent the stem/progenitor cells of the kidney. A previous study from our laboratory has shown that the immortalized cell line RPTEC/TERT1 contains two populations of cells, one co-expressing CD24 and CD133, the other expressing CD24 only. The goal of the present study was to determine if both these populations could be sorted into separate independent cultures and if so, determine their characteristic features and response to the nephrotoxicant cadmium. The results of our study show that both the populations of cells could grow as independent cultures and maintain their phenotype after extended sub-culture. The CD133 + /CD24 + co-expressing cells formed multicellular spheroids (nephrospheres), a characteristic feature of stem/progenitor cells, and formed branched tubule-like structures when grown on the surface of matrigel, whereas the CD133 − /CD24 + cells were unable to form these structures. The CD133 + /CD24 + cells were able to grow and undergo neurogenic, adipogenic, osteogenic, and tubulogenic differentiation, whereas the CD133 − /CD24 + cells expressed some of the differentiation markers but were unable to grow in some of the specialized growth media. The CD133 + / CD24 + co-expressing cells had a shorter doubling time compared to the cells that expressed only CD24, and were more resistant to the toxic effects of the heavy metal, cadmium. In conclusion, the isolation and characterization of these two cell populations form the RPTEC/TERT1 cell line will facilitate the development of studies that determine the mechanisms involved in tubular damage and regeneration particularly after a toxic insult.

Elevated glucose represses lysosomal and mTOR-related genes in renal epithelial cells composed of progenitor CD133+ cells

“…The present study shows that elevated glucose concentrations can reduce the expression of genes associated with the lysosome and the related mTOR pathway in human cultured renal epithelial cells. The hRTE cell culture model was chosen for the present studies since they have recently been shown to be composed of approximately 60–70% of cells that possess both CD133 and CD24, with the remaining cells expressing CD24 and no CD133 [ 6 , 7 ]. This is important, since as detailed in the introduction, in vivo studies have shown that progenitor cells capable of tubular regeneration always co-express CD133 and CD24.…”

“…As methodology reported before [ 6 ], cells were grown on coverslips and fixed with 3.7% formaldehyde for 15 min, followed by permeabilization using 0.1% Triton-X 100 for 10 min. The coverslips were washed 3 times using PBS for 5 min; blocked in 3% BSA for 30 min and incubated with primary antibody for 40 min at 37°C.…”

“…These human studies, as well as studies utilizing animal models, have been complimented by the employment of in vitro cell culture models of putative proximal tubular cells to study a large range of factors associated with DKD. For humans, the most frequently used model is mortal cultures of renal tubular epithelial cells (RTE) isolated from the renal cortex of the human kidney, an HPV16 E 6 /E 7 immortalized culture from the above cells identified as HK-2 cells, and a recently established immortalized culture of the above primary cultures using a construct containing hTERT called RPTEC/TERT1 [ 6 ]. All the three models express many of the features of proximal tubule differentiation, but they fail to express other important features such as a brush border on the apical surface, the SLC5A2 Na + -coupled glucose transporter, and an altered transepithelial resistance [ 7 ].…”

“…In contrast, the number of HK-2 cells co-expressing CD133 and CD24 was below 20%. In a subsequent study, the RPTEC/TERT1 cells were sorted into two cell lines, one that expresses CD133 and CD24 and another one that expresses only CD24 [ 6 ]. The cells co-expressing CD133 and CD24 were shown to produce nephrospheres, formed branched tubule-like structures when grown on the surface Matrigel™, and were able to undergo neurogenic, adipogenic, and tubulogenic differentiation.…”

Meta-analysis of gene expression profiling reveals novel basal gene signatures in MCF-10A cells transformed with cadmium

“…These nodules form 5-8 days post injection and start to regress after 8-12 days. The cellular morphology within these nodules is being used by this laboratory [31,20], and others [32] to judge the ability of cells to form differentiated structures. Thus, the ability to form nodules with histologically differentiated structures was determined for the MCF-10 parent and the MCF-10ACd cells.…”