A Human Conditionally Immortalized Proximal Tubule Epithelial Cell Line as a Novel Model for Studying Senescence and Response to Senolytics

Abstract

Accumulating evidence suggests that senescence of kidney tubule epithelial cells leads to fibrosis. These cells secrete senescence-associated secretory phenotype (SASP) factors that are involved in diverse signaling pathways, influencing kidney fibrosis. Here, we investigated whether our previously established conditionally immortalized proximal tubule epithelial cell line overexpressing the organic anion transporter 1 (ciPTEC-OAT1) can be used as a valid in vitro model to study kidney senescence and senolytics response. CiPTEC-OAT1 proliferates rapidly at 33°C and exhibits a “senescence-like” arrest at 37°C, most likely due to suppression of SV40T expression and subsequent reactivation of the p53 and Rb pathways. To understand how permissive (33°C) and non-permissive (37°C) temperatures of the cell culture affect the senescence phenotype, we cultured ciPTEC-OAT1 for up to 12 days and evaluated the apoptosis and SASP markers. Day 0 in both groups is considered as the non-senescence group (control). Further, the potential of navitoclax, dasatinib, quercetin, and the combination of the latter two to clear senescent cells was evaluated. Maturation of ciPTEC-OAT1 at non-permissive temperature affected mRNA and protein levels of senescence markers. A remarkable upregulation in p21 gene expression was found in the non-permissive temperature group, whereas expression of Lamin B1 decreased significantly. SASP factors, including PAI-1A, IL-1β, CTGF, and IL-6 were upregulated, but no significant difference in Bcl-2 and Bcl-xl were found in the non-permissive temperature group. After culturing ciPTEC-OAT1 up to 12 days, cells in the non-permissive temperature group showed an upregulation in the apoptosis-associated proteins Bcl-2, BID, and Bax, and a downregulation in Mcl-1, Bad, Bak, and Bim at various time points. Further, Bcl-xl, Puma, Caspase 3, Caspase 7, and Caspase 9 showed initial upregulations followed by downregulations at later time points. The loss of Lamin B1, upregulation of SA-β-gal expression and increase in its activity, upregulation of p21 levels and downregulation of p53, along with the upregulation of SASP factors, confirmed that maturation at 37°C promotes senescence features. Finally, the senolytics response was evaluated by testing cell viability following exposure to senolytics, to which cells appeared dose-dependently sensitive. Navitoclax was most effective in eliminating senescent cells. In conclusion, culturing ciPTEC-OAT1 at 37°C induces a senescence phenotype characterized by increased expression of cell cycle arrest and anti-apoptosis markers, SASP factors, and responsiveness to senolytics treatment. Therefore, ciPTEC-OAT1 represents a valid model for studying kidney senescence by simply adjusting culture conditions.

The Uremic Toxin Indoxyl Sulfate Accelerates Senescence in Kidney Proximal Tubule Cells

“…Compared to the non-senescent group (Day 0), LaminB1 was downregulated at 37 • C at Day 9 for protein level (Figure 2A,B) and on Day 6 and Day 9 for mRNA level (Figure 2F), whereas p21 was upregulated at 37 • C for both protein level (Figure 2C,D) and mRNA level (Figure 2E) at different time points. These findings suggest that the cells obtained a senescence phenotype over time, consistent with our previous results [34]. This effect was sustained when cells were treated with IS, as shown by clear differences and upregulation and downregulation trends (Figure 2A-F).…”

“…During maturation for 9 days, many senescence related genes are changed (Figure 5A). The upregulation of SASP factors and p21 observed is consistent with our previous findings [34]. The expression levels of common senescence markers are shown in Figure 5B.…”

“…We recently demonstrated that a human conditionally immortalized proximal tubule epithelial cell line overexpressing the organic anion transporter 1 (ciPTEC-OAT1) acquires a conditional senescent phenotype when cells are cultured at a non-permissive temperature [34]. Considering the facts that the main focus of the present work is the effect of IS in kidney senescence and that (primary) PTEC rapidly lose the expression of OAT1 when cultured in vitro, the ciPTEC-OAT1 cell model, showing stable OAT1 expression and function, was used in this study [35].…”

Involvement of Bcl-2 Family Proteins in Tetraploidization-Related Senescence

“…Cellular models have recently allowed the study of alterations in the expression of Bcl-2 family proteins in relation to senescence in different cell lines and in response to a variety of stimuli. For instance, the conditionally immortalized proximal tubule epithelial cell line overexpressing organic anion transporter 1 (ciPTEC-OAT1) shows a remarkable upregulation of SASP factors and p21 Waf1/Cip1 , as well as upregulation of Bcl-2, Bid and Bax and downregulation of Mcl-1, Bad, Bak and Bim, when entering senescence at 37 °C [ 21 ]. Senescence initiated by the suppression of heat shock protein 70-2 in epithelial ovarian cancer cells is characterized by an increased expression of p21 Waf1/Cip1 and p16 INK4a ; upregulation of Bax, Bim, Bak, Bad, Bid, Puma and Noxa and downregulation of Bcl-2, Bcl-xL and Mcl-1 [ 22 ].…”

Connective Tissue Growth Factor in Idiopathic Pulmonary Fibrosis: Breaking the Bridge

“…AECII cells augmented the expression of CTGF in bleomycin-induced lung fibrosis [ 110 ]. The expression of CTGF was significantly elevated on the mRNA level, suggesting epithelial senescence in kidney fibrosis [ 111 ]. Furthermore, CTGF was shown to decrease mitochondrial metabolism, resulting in ER-stress-associated pro-fibrotic effects in cardiovascular fibrosis [ 112 ].…”