Wang Yujia, Di Yan, Li Yongqi, Lu Jing, Ji Bofan, Zhang Yuxia, Chen Zhiqing, Chen Sijie, Liu Bicheng, Tang Rining
Institute of Nephrology, Zhong Da Hospital, Southeast University School of Medicine, Nanjing, Jiangsu 210003, China.
Institute of Nephrology, NanJing LiShui People's Hospital, Zhongda Hospital Lishui Branch, Southeast University School of Medicine, Nanjing, Jiangsu 211200, China.
Chin Med J (Engl). 2025 Jan 5;138(1):68-78. doi: 10.1097/CM9.0000000000003261. Epub 2024 Aug 16.
Renal osteodystrophy (ROD) is a skeletal pathology associated with chronic kidney disease-mineral and bone disorder (CKD-MBD) that is characterized by aberrant bone mineralization and remodeling. ROD increases the risk of fracture and mortality in CKD patients. The underlying mechanisms of ROD remain elusive, partially due to the absence of an appropriate animal model. To address this gap, we established a stable mouse model of ROD using an optimized adenine-enriched diet and conducted exploratory analyses through ribonucleic acid sequencing (RNA-seq).
Eight-week-old male C57BL/6J mice were randomly allocated into three groups: control group ( n = 5), adenine and high-phosphate (HP) diet group ( n = 20), and the optimized adenine-containing diet group ( n = 20) for 12 weeks. We assessed the skeletal characteristics of model mice through blood biochemistry, microcomputed tomography (micro-CT), and bone histomorphometry. RNA-seq was utilized to profile gene expression changes of ROD. We elucidated the functions of differentially expressed genes (DEGs) using gene ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and gene set enrichment analysis (GSEA). DEGs were validated via quantitative real-time polymerase chain reaction (qRT-PCR).
By the fifth week, adenine followed by an HP diet induced rapid weight loss and high mortality rates in the mouse group, precluding further model development. Mice with optimized adenine diet-induced ROD displayed significant abnormalities in serum creatinine and blood urea nitrogen levels, accompanied by pronounced hyperparathyroidism and hyperphosphatemia. The femur bone mineral density (BMD) of the model mice was lower than that of control mice, with substantial bone loss and cortical porosity. ROD mice exhibited substantial bone turnover with an increase in osteoblast and osteoclast markers. Transcriptomic profiling revealed 1907 genes with upregulated expression and 723 genes with downregulated expression in the femurs of ROD mice relative to those of control mice. Pathway analyses indicated significant enrichment of upregulated genes in the sphingolipid metabolism pathway. The significant upregulation of alkaline ceramidase 1 ( Acer1 ), alkaline ceramidase 2 ( Acer2 ), prosaposin-like 1 ( Psapl1 ), adenosine A1 receptor ( Adora1 ), and sphingosine-1-phosphate receptor 5 ( S1pr5 ) were successfully validated in mouse femurs by qRT-PCR.
Optimized adenine diet mouse model may be a valuable proxy for studying ROD. RNA-seq analysis revealed that the sphingolipid metabolism pathway is likely a key player in ROD pathogenesis, thereby providing new avenues for therapeutic intervention.
肾性骨营养不良（ROD）是一种与慢性肾脏病 - 矿物质和骨异常（CKD - MBD）相关的骨骼病变，其特征为骨矿化和重塑异常。ROD增加了CKD患者骨折和死亡的风险。ROD的潜在机制仍然不清楚，部分原因是缺乏合适的动物模型。为了填补这一空白，我们使用优化的高嘌呤饮食建立了稳定的ROD小鼠模型，并通过核糖核酸测序（RNA - seq）进行了探索性分析。
将8周龄雄性C57BL / 6J小鼠随机分为三组：对照组（n = 5）、腺嘌呤和高磷（HP）饮食组（n = 20）以及优化的含腺嘌呤饮食组（n = 20），持续12周。我们通过血液生化、显微计算机断层扫描（micro - CT）和骨组织形态计量学评估模型小鼠的骨骼特征。利用RNA - seq分析ROD的基因表达变化。我们使用基因本体（GO）分析、京都基因与基因组百科全书（KEGG）通路分析和基因集富集分析（GSEA）阐明差异表达基因（DEG）的功能。通过定量实时聚合酶链反应（qRT - PCR）验证DEG。
到第五周时，腺嘌呤加HP饮食导致小鼠组体重快速下降和高死亡率，无法进一步进行模型开发。优化的腺嘌呤饮食诱导的ROD小鼠血清肌酐和血尿素氮水平出现明显异常，伴有明显的甲状旁腺功能亢进和高磷血症。模型小鼠的股骨骨矿物质密度（BMD）低于对照小鼠，伴有大量骨质流失和皮质骨孔隙率增加。ROD小鼠表现出大量的骨转换，成骨细胞和破骨细胞标志物增加。转录组分析显示，与对照小鼠相比，ROD小鼠股骨中有1907个基因表达上调，723个基因表达下调。通路分析表明上调基因在鞘脂代谢通路中显著富集。通过qRT - PCR在小鼠股骨中成功验证了碱性神经酰胺酶1（Acer1）、碱性神经酰胺酶2（Acer2）、类prosaposin 1（Psapl1）、腺苷A1受体（Adora1）和鞘氨醇 - 1 - 磷酸受体5（S1pr5）的显著上调。
优化的腺嘌呤饮食小鼠模型可能是研究ROD的有价值的替代模型。RNA - seq分析表明鞘脂代谢通路可能是ROD发病机制中的关键因素，从而为治疗干预提供了新途径。
