Derivation of the immortalized cell line-UM51-PrePodo-hTERT and its responsiveness to Angiotensin II and activation of RAAS

Abstract

Recent demographic studies predict there will be a considerable increase of elderly people within the next decades. Aging has been recognized as one of the main risk factors of the world's most prevalent diseases, including neurodegenerative disorders, cancer, cardiovascular disease, and metabolic disease. 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 the telomere length with progressive cell division is critically shortened, the cell becomes proliferative arrested and DNA damage response and cellular senescence are triggered. At this time point the so called "Hayflick limit" is attained. Podocytes are a cell type that is found in the kidney glomerulus where they have major implications in blood filtration. Mature podocytes are terminal differentiated cells that are unable to undergo cell division in vivo. For this reason, the establishment of 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 cell cells were isolated directly from the urine of a 51-year-old male. The immortalized cell line has been cultured over the course of one year (~100 passages) with high proliferation capacity, while still endowed with contact inhibition and P53 expression and activation. Furthermore, by immunofluorescent-based expression and quantitative Real-Time PCR for the podocyte markers 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 (RAS). Elevated levels of ANGII have been identified as a main risk factor for the initiation and progression of chronic kidney disease (CKD). CKD is characterized by an impairment of podocyte function and subsequently podocyte apoptosis. As a major risk factor for patients to develop CKD, diabetes, hypertension, heart disease and stroke have been recognized - all of which show increased incidence in elderly people. In conclusion, we have shown that it is possible to by-pass cellular replicative senescence (Hayflicks limit) by TERT-driven immortalization of human urine-derived pre-podocyte cells from a 51-year-old African male.

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

“…Furthermore, another study carried out by Pierzchalski et al, showed that p53 is able to induce apoptosis via the activation of the Renin-Angiotensin System 29 . In two recent studies carried out in our laboratory, we demonstrated the devastating effect of the activated Renin-Angiotensin-System on podocyte morphology and functionality 51,52 . Therefore, we propose that the observed upregulated secretion of EGFR and VEGF mainly trigger angiogenesis and the activation of p53 and the Renin-Angiotensin system.…”