Browsing by Subject "Renal disease"

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Cellular stress regulates fibroblast growth factor 23 (FGF23) und αklotho
(2023) Münz, Sina; Föller, Michael
Cellular stress is defined as the impairment of regular cell function by internal or external stimuli including critical temperatures, energy deficiency, infections, mechanic injury, or chemical noxae. The present thesis aims to investigate the influence of cellular stress on the expression of FGF23 and αklotho. FGF23 is predominantly produced in bone and regulates the phosphate excretion in the kidney. Thereby, αklotho functions as a co-receptor for FGF23. By binding to the FGF receptor-αklotho complex, FGF23 reduces the reabsorption of phosphate from the tubular lumen by decreasing the abundance of sodium-phosphate co-transporters. Furthermore, FGF23 decreases the synthesis of 1,25(OH)2D3, active vitamin D, and increases its degradation. 1,25(OH)2D3 is a regulator of intestinal phosphate absorption and therefore, FGF23 additionally reduces dietary phosphate uptake. Chronically elevated FGF23 is associated with numerous disorders such as kidney disease or CVD. Beside its function as a co-receptor of FGFR, αklotho has many beneficial FGF23-independent functions. It has originally been identified as an anti-aging hormone, as a loss-of-function mutation in the αklotho gene causes numerous aging-like symptoms such as vascular and tissue calcification, osteoporosis, sterility, and an early death. The present papers investigated the influence of cytostatic drugs cisplatin, paclitaxel, and doxorubicin as well as apoptosis inducers PAC-1 and serum depletion on the regulation of FGF23 and αklotho. In UMR106 rat osteoblast-like osteosarcoma cells, a 24 or 48 h-treatment with cisplatin, doxorubicin, PAC-1, or serum reduction and depletion significantly up-regulated Fgf23 expression. Under serum depletion, also FGF23 protein secretion was increased. In addition to FGF23, cisplatin and doxorubicin also increased gene expression of pro-inflammatory cytokine Il6 hinting at the presence of necrotic cell death. By inhibiting Il-6 membrane receptor gp130 it has been shown, that FGF23 stimulation partially depended on IL-6 signaling. The stimulation of FGF23 by inflammatory mediators including IL-6, TNFα, TGF-β, or IL-1β has already been reported by others. Furthermore, inflammatory diseases such as rheumatoid arthritis, CKD, or inflammatory bowel disease are associated with excess FGF23 serum concentrations. In this regard, we investigated gene expression and activation of the transcription factor NFκB, which regulates numerous inflammatory functions. Cisplatin and doxorubicin increased the expression of NFκB subunit Rela and cisplatin also stimulated the phosphorylation of NFκB. Independently, NFκB inhibitors wogonin and withaferin A attenuated cisplatin-mediated stimulation of FGF23 indicating, that FGF23 excess was in part promoted by NFκB signaling. These investigations confirmed a strong impact of cisplatin or doxorubicin-induced inflammation on FGF23 synthesis, whereas PAC-1 and serum depletion have reported to directly induce apoptosis, which is commonly not associated with inflammation. Known factors, induced by all cytotoxic substances used here, are the formation of ROS and activation of HIF1α. Both are positive regulators of FGF23, leading to the conclusion, that cellular stress might regulate FGF23 via HIF1α or oxidative stress. FGF23 excess results in increased bone resorption and suppressed bone formation. Likewise, also chemotherapeutic drugs and serum deficiency reduce bone density. Therefore, the stimulation of FGF23 may cause or further stimulate bone resorption. In paper 2, the influence of the cytostatic drugs cisplatin, paclitaxel, and doxorubicin as well as apoptosis inductors PAC-1 or serum depletion on αklotho expression in renal MDCK, NRK-52E, and HK-2 cells has been investigated. In fact, all cytotoxic compounds stimulated gene expression of αklotho while decreasing cell proliferation and viability. By using a combined apoptosis and necrosis assay, we confirmed the induction of apoptosis but also necrosis to a variable extent. Additionally, the transcriptional regulation of apoptotic proteins of the BCL-2 family was assessed and confirmed apoptosis stimulation. Transcription factor PPARγ is a known positive regulator of αklotho. In MDCK cells, we detected a significant influence of cisplatin-mediated stimulation of PPARγ mRNA on the αklotho increase. Furthermore, cisplatin, doxorubicin, PAC-1, and serum deprivation also up-regulated FGFR production in MDCK cells. In cancer cells, overexpression of FGFR is associated with enhanced resistance against chemotherapeutic drugs. Consequently, αklotho and FGFR1 stimulation may be a protective mechanism to prevent hyperphosphatemia during diseases. However, human HK-2 cells treated with cisplatin, paclitaxel, doxorubicin, or serum depletion significantly down-regulated αklotho expression and protein secretion. PAC-1 did not change the expression or production of αklotho in HK-2 cells, which might be explained by the minor effect of PAC-1 on non-carcinogenic cells lacking an overexpression of procaspase-3. The differential regulation of αklotho in MDCK and NRK-52E versus HK-2 cells by cytotoxic stress might have numerous causes. For instance, there is evidence of an increased sensitivity of HK-2 cells to stress stimuli but a better comparability to the animal model. However, immortalized cell lines can not completely reflect the conditions of native tissue especially with regard to cell death. Furthermore, the species, sex or age of the donor organism as well as passage number of the cells and drug transporter expression might impact αklotho regulation. Additionally, the mode of cell death determined by intracellular ATP homeostasis and its regulation of AMPK might play an important role in αklotho regulation. However, all these theories need to be further addressed. In summary, inflammation, ROS formation, or the activation of HIF1α are all reported to correlate in a negative manner with αklotho production or serum levels. αklotho down-regulation may be a tool to increase cell proliferation or prevent hypophosphatemia. In contrast, AMPK activation by intracellular ATP restriction may positively regulate αklotho to promote cell protection and avoid hyperphosphatemia.