#3048 INDOXYL SULFATE INHIBITS OSTEOGENESIS OF BONE MARROW DERIVED MESENCHYMAL STEM CELLS THROUGH AHR/HES1/BMP2 PATHWAY
Authors: Hsieh Chin Wen , Ho Mei-Ling , Chen Chung-Hwan , Chang Je-Ken 
Published: June 2023

Abstract

Background and Aims
There is a high global burden of chronic kidney disease (CKD). The increased risk of bone fracture in patients with CKD suggests that CKD-associated factors, such as uremic toxins, might be the cause. Recent studies demonstrated that low turnover osteodystrophy and bone formation defect develop in early CKD, which indicates that uremic toxins, particularly in relatively low concentrations, can potentially be a cause. Thus far, studies about defected osteogenesis adopted high concentrations of uremic toxins (for example, indoxyl sulfate (ISx) &gt;100 μM). Furthermore, the pathology is not yet fully investigated. Uremic toxin induced aryl hydrocarbon receptor (AHR) has been linked to bone formation defect, however, the mechanisms are incompletely understood. AHR inhibit osteogenesis during early processes of differentiation of early cells (stem cells), but whether uremic toxins induced AHR to affect stem cell in osteogenesis lineage has yet to be studied. The present study aims to clarify whether ISx at concentrations approximate to average in CKD patients (20 μM in free form) affect osteogenesis of bone marrow-derived mesenchymal stem cells (BMSCs), and further survey the underline signal transduction of AHR-induced defected osteogenesis.

Method
Mineralization of D1 cells (mouse BMSCs) was evaluated by alizarin red S staining (ARS). The mRNA expressions and protein levels of osteogenic markers including Runx2, BMP2, ALP, and osteocalcin were determined by quantitative real-time polymerase chain reaction (RT-PCR) and western blotting. AHR translocation into the nucleus was evaluated by an immunofluorescence confocal microscope. Reporter gene assays were adopted to study the transcriptional regulation of Runx2 and BMP2.

Results
ISx at concentrations ≤50 μM significantly downregulated mineralization of BMSCs with the most influence in the early stage of osteogenesis. ISx decreased the mRNA expressions and protein levels of osteogenic markers Runx2, BMP2, ALP, and osteocalcin. ISx of 50 μM induced AHR translocation into the nucleus and upregulated AHR target genes CYP1a1 and CYP1b1. The ISx-induced mineralization defects of BMSCs were counteracted by AHR antagonist CH223191, while downregulated gene expressions of osteogenic markers were recovered by AHR antagonist and knockdown (KD) of AHR. In mechanistic studies, ISx upregulated mRNA levels of hairy and enhancer of split (HES1), which is a primary target of NOTCH signaling. Although there were no significant differences in expressions of Notch1∼4, the upregulated Hes1 was attenuated by the antagonist and KD of AHR, which indicated that Hes1 was directly regulated by AHR. We identified putative binding sites of transcription repressor Hes1 in the promoter area of BMP2 and Runx2. Following experimental treatment of ISx, expression levels of BMP2 and Runx2 monitored by reporter assays were downregulated, which indicated that Hes1 plays as a transcription repressor to downregulated BMP2 and Runx2.

Conclusion
ISx under clinically relevant concentrations attenuated mineralization of BMSCs, particularly in the early stage of osteogenesis. ISx-induced AHR upregulated Hes1, which inhibited expressions of not only Runx2, but also BMP2, which has not been published before. A study of the underline signal transduction pathways relevant to the AHR genomic control system may provide potential therapy for low bone formation in early CKD.