Derivation of the Immortalized Cell Line UM51-PrePodo-hTERT and Its Responsiveness to Angiotensin II and Activation of the RAAS Pathway

Abstract

Recent demographic studies predict there will be a considerable increase in the number of elderly people within the next few decades. Aging has been recognized as one of the main risk factors for the world’s most prevalent diseases such as neurodegenerative disorders, cancer, cardiovascular disease, and metabolic diseases. During the process of aging, a gradual loss of tissue volume and organ function is observed, which is partially caused by replicative senescence. The capacity of cellular proliferation and replicative senescence is tightly regulated by their telomere length. When telomere length is critically shortened with progressive cell division, cells become proliferatively arrested, and DNA damage response and cellular senescence are triggered, whereupon the “Hayflick limit” is attained at this stage. Podocytes are a cell type found in the kidney glomerulus where they have major roles in blood filtration. Mature podocytes are terminal differentiated cells that are unable to undergo cell division in vivo. For this reason, the establishment of primary podocyte cell cultures has been very challenging. In our present study, we present the successful immortalization of a human podocyte progenitor cell line, of which the primary cells were isolated directly from the urine of a 51-year-old male. The immortalized cell line was cultured over the course of one year (~100 passages) with high proliferation capacity, endowed with contact inhibition and P53 expression. Furthermore, by immunofluorescence-based expression and quantitative real-time PCR for the podocyte markers CD2AP, LMX1B, NPHS1, SYNPO and WT1, we confirmed the differentiation capacity of the immortalized cells. Finally, we evaluated and confirmed the responsiveness of the immortalized cells on the main mediator angiotensin II (ANGII) of the renin–angiotensin system (RAAS). In conclusion, we have shown that it is possible to bypass cellular replicative senescence (Hayflick limit) by TERT-driven immortalization of human urine-derived pre-podocyte cells from a 51-year-old African male.

Unveiling Angiotensin II and Losartan-Induced Gene Regulatory Networks Using Human Urine-Derived Podocytes

“…In a previous manuscript, we reported the existence of urine-derived renal progenitor cells (UdRPCs) inherently capable of differentiating into podocytes [ 19 , 24 , 28 ]. UdRPCs adopt the typical “fried egg” morphology typical of podocytes after seven days of continuous culturing in adv.…”

“…We differentiated primary SIX2-positive UdRPC UM51 and the transformed counterpart UM51-hTERT into podocytes [ 28 ]. The expression of NPHS1 was analyzed to confirm the differentiation of both cell types into mature podocytes, and phalloidin staining was employed to reveal cytoskeletal morphologies ( Figure 1 A,B).…”

Cytokines in the Urine of AKI patients regulate TP53 and SIRT1 and can be used as biomarkers for the early detection of AKI

“…A number of the chemokines, such as VEGF or EGFR, that were found to be upregulated are capable of either activating or being activated by either TP53 [27][28][29][30] or SIRT1 31,32 . To test the hypothesis that the chemokines found to be upregulated in the AKI stage2/3 urine have a direct influence on the expression levels of TP53 and SIRT1, we incubated our recently published immortalized podocyte cell line UM51 hTERT 26 with medium composed of 10% of the patient urine 72hrs post-surgery for 120 h. Relative protein expression normalized to GAPDH for TP53, SIRT1 and the phosphorylation levels of Histone 2A (pH2A.X), which is an established biomarker of DNA-damage 33 , were detected by Western blotting (fig. 3a) and full images of Western blots are shown in supplementary figure 3.…”

“…At 72 hrs post-surgery, common biomarkers for the detection of AKI in both male female patients include, VEGF, SERPIN3A, TNFSF12, ANPEP, CXCL1, REN, CLU and PLAU. Furthermore, we applied the urine to our recently published hTERT immortalized podocyte cell line 26 and evaluated the effects of the urine on TP53 and SIRT1 expression.…”

Renal progenitors derived from urine for personalized diagnosis of kidney diseases

“…These cells exhibit a limited expandability and lifespan due to their mature differentiation state, requiring immortalization to maintain them in vitro [5‒10]. Immortalized urinary podocytes and tubular cells of healthy volunteers and patients established by hTERT, temperature-sensitive SV40 T antigen transfection or the introduction of viral oncogenes possess strong proliferative potential and could acquire the phenotype of fully differentiated cells in response to changes in culture conditions (such as temperature, exposure to retinoic acid, retinoic acid + vitamin D3, hydrocortisone + EGF + tri-iodothyronine) [9, 11‒15]. Even if several studies highlight the usefulness of these immortalized-kidney cells for disease modeling [6, 12, 16, 17], drug screening [12, 14], and bioengineering application [13, 18], the invasive procedure of immortalization could affect the transcriptome and interfere with cellular pathways of cells, which might lose their sensitivity to external stimuli [5, 19].…”