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High sodium reduced the expression of PTH1R and Klotho by inhibiting 1,25 (OH)2D3 synthesis in cultured proximal tubule epithelial cells

ABSTRACT: The proximal tubule is the sensing site of sodium and phosphorus and the main place for the synthesis and metabolism of 1,25 (OH)2D3. Sodium may share the sensing mechanism with phosphate in proximal tubule epithelial cells; whether sodium cooperates with phosphate or plays an independent role in the regulation of phosphorus homeostasis remains unknown. In this in vitro study, we investigated the effects of high sodium on the synthesis and function of active vitamin D and local phosphorus regulation in proximal tubular epithelial cells. Human proximal tubule epithelial (HK-2) cells were treated with different concentrations of sodium/phosphorus. The expression of 1α-OHase (Cyp27b1) and 24-OHase (Cyp24a1) was determined by RT-PCR and Western Blot. Liquid chromatography/mass spectrometry (LC/MS) and enzyme-linked immunosorbent assay (ELISA) were used to detect the levels of 1,25 (OH)2D3. Intracellular Ca2+ ([Ca2+]i) was detected with the Ca2+ indicator dye Fura-4. Chromatin samples were immunoprecipitated with antibodies against parathyroid receptor 1 (PTH1R) and Klotho. High sodium decreased the expression of 1,25 (OH)2D3 by reducing 1α-OHase and 24-OHase in HK-2 cells, reduced the expression of PTH1R and Klotho, and increased the intracellular calcium concentration. These effects were reversed by sodium phosphorus transporter inhibitor (PFA), sodium hydrogen transporter inhibitor (Caliporide), and a chelator of the extracellular calcium, whereas enhanced by ouabain. Vitamin D receptor agonists significantly increased the recruitment of VDR to the Vitamin D response element (VDRE) of PTH1R and Klotho promoter, thus increasing the expression of PTH1R and Klotho. High extracellular sodium can decrease the synthesis of active vitamin D in the proximal tubules, affect the gene regulation of 1,25 (OH)2D3/VDR, and significantly reduce the expression of PTH1R and Klotho. The data reveal the influence of a high-sodium diet on mineral metabolism and the core role of vitamin D in kidney mineral metabolism.

ORGANISM(S): Homo sapiens

PROVIDER: GSE186286 | GEO | 2023/10/20

REPOSITORIES: GEO

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Project description:Heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2 plays a pivotal role in vitamin D receptor (VDR) signaling by acting as a vitamin D response element (VDRE)-binding protein (VDRE-BP). Transcriptional regulation by active 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) involves occupancy of VDRE by VDRE-BP or 1,25(OH)2D3 bound-VDR. This relationship is disrupted by over-expression of VDRE-BP and can cause a form of human hereditary vitamin D-resistant rickets (HVDRR). DNA array analyses using B-cells from an HVDRR patient and matched control defined a sub-cluster of genes where 1,25(OH)2D3-regulated transcription was abrogated by over-expression of VDRE-BP. Amongst these, the DNA-damage-inducible transcript 4 (DDIT4), an inhibitor of mammalian target of rapamycin (mTOR) signaling, was also induced by 1,25(OH)2D3 in human osteoblasts. Chromatin immunoprecipitation using 1,25(OH)2D3-treated osteoblasts confirmed that liganded VDR and VDRE-BP compete for binding to the proximal promoter of the DDIT4 gene in a similar fashion to other known 1,25(OH)2D3-target genes. Treatment of osteoblasts with 1,25(OH)2D3 induced DDIT4 expression and suppressed phosphorylated S6K1T389 protein (a downstream target of mTOR). The functional importance of this for 1,25(OH)2D3 responses in osteoblasts was underlined by the fact that siRNA knockdown of DDIT4 expression suppressed antiproliferative and cell growth responses to 1,25(OH)2D3. These data confirm that VDRE-BP is required for normal 1,25(OH)2D3-mediated transcription and cell function in osteoblasts. Conversely over-expression of VDRE-BP exerts a dominant-negative effect on transcription of 1,25(OH)2D3-target genes. Characterization of VDRE-BP action in 1,25(OH)2D3-treated osteoblasts highlights an entirely novel role for vitamin D as a regulator of mTOR – a known ‘master regulator’ of cell function. We performed gene expression microarray analysis in HVDRR EBV-transformed B-cells and control cells in the presence or absence of vitamin D.

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High sodium reduced the expression of PTH1R and Klotho by inhibiting 1,25 (OH)2D3 synthesis in cultured proximal tubule epithelial cells

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