Single-cell transcriptomic atlas of Kidney-Yang and Kidney-Yin deficiency syndromes reveals distinct osteoimmune-stromal programs underlying osteopenia and osteoporosis

Abstract

Osteopenia and osteoporosis are escalating health burdens lacking precise therapeutic strategies owing to pronounced disease heterogeneity. Kidney-Yang deficiency (KYangD, “cold syndrome”) and Kidney-Yin deficiency (KYinD, “heat syndrome”) represent two major traditional Chinese medicine (TCM)-defined syndromes underlying these conditions, but their molecular basis remains obscure. Here, we performed single-cell RNA sequencing on bone tissue from osteopenia/osteoporosis patients stratified by KYangD, KYinD, or non-deficiency group, generating the first transcriptomic atlas of syndrome-specific osteoimmune and stromal programs. KYangD exhibited an energy- and perfusion-limited, low-grade inflammation in type H-like capillary endothelial cells (CD31^highEMCN^high). This inflammation was characterized by incomplete angiogenic activation, reduced phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT)/hypoxia-inducible factor 1 (HIF-1)/nitric oxide (NO) signaling despite compensatory Notch signaling, and weakened extracellular matrix/adhesion that restricts osteoblast maturation. KYinD was dominated by inflammation and endoplasmic reticulum (ER) stress with nuclear factor kappa B (NF-κB)/tumor necrosis factor (TNF)/interleukin (IL)-17 activation, protease-rich matrix remodeling, and secreted phosphoprotein 1 (SPP1)-mediated adhesion, alongside broad attenuation of Notch signaling, vascular endothelial growth factor (VEGF), angiopoietin 1 (ANGPT1), and C-X-C motif chemokine ligand 12 (CXCL12) scaffolds and loss of bone morphogenetic protein (BMP), transforming growth factor beta (TGF-β), and Wnt osteogenic pathways. These alterations spanned endothelial, stromal, osteolineage, and immune compartments with network-level rewiring of intercellular communication. Collectively, our findings establish the first single-cell molecular bridge between TCM-defined syndromes and osteoporotic pathology, providing a framework for syndrome-specific precision interventions, namely, restoring energy metabolism and vascular-osteogenic coupling in KYangD, and suppressing hyper-inflammation while rebuilding endothelial scaffolds in KYinD.

Keywords

osteopenia and osteoporosis / Kidney-Yang deficiency / Kidney-Yin deficiency / single-cell RNA sequencing / bone microenvironment

References

[1] Cui J , Shibata Y , Zhu T , Zhou J , Zhang J . Osteocytes in bone aging: advances, challenges, and future perspectives. Ageing Res Rev 2022; 77: 101608

[2] Cosman F , Crittenden DB , Adachi JD , Binkley N , Czerwinski E , Ferrari S , Hofbauer LC , Lau E , Lewiecki EM , Miyauchi A , Zerbini CAF , Milmont CE , Chen L , Maddox J , Meisner PD , Libanati C , Grauer A . Romosozumab treatment in postmenopausal women with osteoporosis. N Engl J Med 2016; 375(16): 1532–1543

[3] Xiao PL , Cui AY , Hsu CJ , Peng R , Jiang N , Xu XH , Ma YG , Liu D , Lu HD . Global, regional prevalence, and risk factors of osteoporosis according to the World Health Organization diagnostic criteria: a systematic review and meta-analysis. Osteoporos Int 2022; 33(10): 2137–2153

[4] Si L , Winzenberg TM , Jiang Q , Chen M , Palmer AJ . Projection of osteoporosis-related fractures and costs in China: 2010–2050. Osteoporos Int 2015; 26(7): 1929–1937

[5] Zhao JG , Zeng XT , Wang J , Liu L . Association between calcium or vitamin D supplementation and fracture incidence in community-dwelling older adults: a systematic review and meta-analysis. JAMA 2017; 318(24): 2466–2482

[6] Odén A , McCloskey EV , Johansson H , Kanis JA . Assessing the impact of osteoporosis on the burden of hip fractures. Calcif Tissue Int 2013; 92(1): 42–49

[7] Ge JR , Xie LH , Chen J , Li SQ , Xu HJ , Lai YL , Qiu LL , Ni CB . Liuwei Dihuang Pill (六味地黄丸) treats postmenopausal osteoporosis with Shen (Kidney) Yin deficiency via Janus kinase/signal transducer and activator of transcription signal pathway by up-regulating cardiotrophin-like cytokine factor 1 expression. Chin J Integr Med 2018; 24(6): 415–422

[8] Zhang JH , Xin J , Fan LX , Yin H . Intervention effects of Zuoguiwan containing serum on osteoblast through ERK1/2 and Wnt/β-catenin signaling pathway in models with Kidney-Yang-deficiency, Kidney-Yin-deficiency osteoporosis syndromes. China J Chin Mater Med (Zhongguo Zhong Yao Za Zhi) 2017; 42(20): 3983–3989

[9] Qi XN. Diagnostic criteria and congenital and acquired risk factors for Kidney-Yang deficiency syndrome in primary osteoporosis. Liaoning University of Traditional Chinese Medicine, 2022 (in Chinese)

[10] Zhao T , Yang X , Yu L , Wang H , Wang H , Yu C , Meng J . Analysis on reliability and validity of epidemiologic questionnaire on Kidney-Yin deficiency syndrome. Tianjin J Tradit Chin Med (Tianjin Zhong Yi Yao) 2012; 29(05): 496–498

[11] Yuan C , Yu XT , Wang J , Shu B , Wang XY , Huang C , Lv X , Peng QQ , Qi WH , Zhang J , Zheng Y , Wang SJ , Liang QQ , Shi Q , Li T , Huang H , Mei ZD , Zhang HT , Xu HB , Cui J , Wang H , Zhang H , Shi BH , Sun P , Zhang H , Ma ZL , Feng Y , Chen L , Zeng T , Tang DZ , Wang YJ . Multi-modal molecular determinants of clinically relevant osteoporosis subtypes. Cell Discov 2024; 10(1): 28

[12] Li Y , Liang W , Li X , Gao B , Gan H , Yin L , Shen J , Kang J , Ding S , Lin X , Liao L , Li C . Effect of serum from postmenopausal women with osteoporosis exhibiting the Kidney-Yang deficiency pattern on bone formation in an hFOB 1.19 human osteoblastic cell line. Exp Ther Med 2015; 10(3): 1089–1095

[13] Shang G , Zhou X , Yin J , Niu X , Zhao Y , Li X , Tong Q , Bao B , Cao Y , Cheng F , Li Z , Yao W . Multi-omics analysis of kidney, bone and bone marrow explored potential mechanisms of Erzhi Wan against osteoporosis with Kidney-Yin deficiency. J Pharm Biomed Anal 2024; 246: 116211

[14] Zhao DF , Shu B , Tang DZ , Zhang Y , Zhao YJ , Li CG , Wang Q , Liang QQ , Cui XJ , Yang YP , Xu H , Shi Q , Wang YJ . Drug discovery investigation of nourishing Kidney-Yin and warming Kidney-Yang granules in prevention and treatment of primary osteoporosis based on theory of “treating both disease and traditional Chinese medicine syndrome. ” Mod Tradit Chin Med Mater Med-World Sci Technol (Shi Jie Ke Xue Ji Shu—Zhong Yi Yao Xian Dai Hua) 2017; 11: 1768–1772

[15] Yang W , Wang M , Hu J , Mo K , Xie X . The complex association between the immune system and the skeletal system in osteoporosis: a study of single-cell RNA sequencing. Int Immunopharmacol 2024; 138: 112611

[16] Rodrigues J , Wang YF , Singh A , Hendriks M , Dharmalingam G , Cohen-Solal M , Kusumbe AP , Ramasamy SK . Oestrogen enforces the integrity of blood vessels in the bone during pregnancy and menopause. Nat Cardiovasc Res 2022; 1(10): 918–932

[17] Wang L , Zhou F , Zhang P , Wang H , Qu Z , Jia P , Yao Z , Shen G , Li G , Zhao G , Li J , Mao Y , Xie Z , Xu W , Xu Y , Xu Y . Human type H vessels are a sensitive biomarker of bone mass. Cell Death Dis 2017; 8(5): e2760

[18] Acuña-Pilarte K , Koh MY . The HIF axes in cancer: angiogenesis, metabolism, and immune-modulation. Trends Biochem Sci 2025; 50(8): 677–694

[19] Liu ZL , Chen HH , Zheng LL , Sun LP , Shi L . Angiogenic signaling pathways and anti-angiogenic therapy for cancer. Signal Transduct Target Ther 2023; 8(1): 198

[20] Martins BR , Pinto TS , da Costa Fernandes CJ , Bezerra F , Zambuzzi WF . PI3K/AKT signaling drives titanium-induced angiogenic stimulus. J Mater Sci Mater Med 2021; 32(1): 18

[21] Higashi Y . Roles of oxidative stress and inflammation in vascular endothelial dysfunction-related disease. Antioxidants 2022; 11(10): 1958

[22] Maamoun H , Abdelsalam SS , Zeidan A , Korashy HM , Agouni A . Endoplasmic reticulum stress: a critical molecular driver of endothelial dysfunction and cardiovascular disturbances associated with diabetes. Int J Mol Sci 2019; 20(7): 1658

[23] Qu Z , Zhao S , Zhang Y , Wang X , Yan L . Natural compounds for bone remodeling: targeting osteoblasts and relevant signaling pathways. Biomed Pharmacother 2024; 180: 117490

[24] Luo J , Li L , Shi W , Xu K , Shen Y , Dai B . Oxidative stress and inflammation: roles in osteoporosis. Front Immunol 2025; 16: 1611932

[25] Wang L , Ruan M , Bu Q , Zhao C . Signaling pathways driving MSC osteogenesis: mechanisms, regulation, and translational applications. Int J Mol Sci 2025; 26(3): 1311

[26] Shang J , Yu Z , Xiong C , Zhang J , Gong J , Yu C , Huang Y , Zhou X . Resistin targets TAZ to promote osteogenic differentiation through PI3K/AKT/mTOR pathway. iScience 2023; 26(7): 107025

[27] Chen R , Wang J , Zhan R , Zhang L , Wang X . Fecal metabonomics combined with 16S rRNA gene sequencing to analyze the changes of gut microbiota in rats with Kidney-Yang deficiency syndrome and the intervention effect of You-gui pill. J Ethnopharmacol 2019; 244: 112139

[28] Li XL , Jin Y , Gao R , Zhou QX , Huang F , Liu L. . Wenjing decoction: mechanism in the treatment of dysmenorrhea with blood stasis syndrome through network pharmacology and experimental verification.. J Ethnopharmacol 2025; 337(Pt 1): 118818

[29] Jing Y , Cai D , Chen Q , Xiong Q , Hu T , Yao Y , Lin C , Sun X , Lu Y , Kong X , Wu X , Li Y , Bian H . Liuwei Dihuang soft capsules attenuates endothelial cell apoptosis to prevent atherosclerosis through GPR30-mediated regulation in ovariectomized ApoE-deficient mice. J Ethnopharmacol 2017; 208: 185–198

[30] Ding Y , Dong Y , Wei W . Protective role of Erzhi Pills in H₂O₂-induced oxidative stress, inflammation, and angiogenesis in ARPE-19 cells. Biochem Genet 2026; 64: 4538–4555

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