Altered Bone Marrow Dynamics and Macrophage Polarization: Implications for Skeletal Health in Chronic Kidney Disease

Background: Chronic kidney disease (CKD) is the third fastest growing cause of death worldwide. CKD-mineral and bone disorder (CKD-MBD) parallels aging bone with marrow adipose tissue accumulation and bone loss. Bone dyshomeostasis in CKD is significantly linked with morbidity and mortality, exemplified by the increased fracture risk in patients with CKD. Moreover, patients with CKD that sustain fractures subsequently exhibit higher mortality rates. This phenotype suggests skewed differentiation of mesenchymal stromal cells (MSCs) towards adipogenesis with decreased osteoblast formation. Anemia of CKD and its accompanying iron deficiency are common co-morbidities independently increasing morbidity and mortality. 1 in 7 patients with CKD in the US are estimated to also be anemic. Crucially, as the severity of the disease progresses, as does the prevalence with 50% of patients in Stage V also suffering from anemia.

The adenine CKD-inducing rodent model displays co-perturbations of bone homeostasis and iron handling in CKD-MBD, a setting which mirrors patient phenotypes. It shows bone marrow (BM) iron accumulation demonstrating functional iron-deficiency, likely affecting the BM microenvironment. Over one third of the patients with CKD are also iron-deficient. Of note, functional iron-deficiency, due to elevation of hepatocyte-derived hepcidin promotes iron sequestration, particularly in macrophages (Mφs). Subsequently this can induce polarization and pro-inflammatory cytokine secretion. However, how and which polarized Mφs influence MSC differentiation remains controversial. We hypothesize the clinically observed pathophysiological decreased osteogenesis and increased marrow adipogenesis during CKD are due to macrophage-driven mechanisms in the BM.

Methods: To test this, we placed male C57Bl/6 mice on casein-control (CAS) and CKD-inducing 0.2% adenine (AD) diet for 6 weeks to assess BM and immune crosstalk at an early stage, capturing potentially causative mechanisms. We conduct CBC and iron panels on serum collected at necropsy to determine iron deficiency and anemia status. Likewise, the serum is also used to determine Hepcidin levels via an ELISA assay. To evaluate Mφ abundance and polarization in the BM we perform flow cytometry analysis. To better understand alterations in the BM niche at a single cell level we conducted single-cell RNA sequencing (scRNA-seq) of BM cells. Finally, RT-qPCR is used to measure relative mRNA levels of fibrotic and inflammatory markers in the kidney as well as osteogenic and adipogenic markers in cortical femur and whole tibia specimens.

Results: As expected, 6-week AD feeding in males show significantly elevated serum hepcidin and reciprocally decreased total serum iron versus CAS-fed mice. Kidney mRNA analysis showed elevated expression of the fibrosis marker type I collagen (Col1a1; 20-fold increase) and inflammatory markers, tumor necrosis factor α (Tnfα; 30-fold increase) and interleukin 6 (Il6; 50-fold increase) in AD-fed vs CAS mice. After noting an increase of total Mφs (CD45⁺CD11b⁺F4/80⁺) in the BM of mice on AD diet using flow cytometry, we assessed the percentage of Mφs that were CD80⁺ versus CD206⁺. While both subtypes were more abundant in AD mice vs. CAS mice, CD80⁺ Mφs exhibited a 3-fold increase vs 1.5-fold for CD206⁺ Mφs compared to their control counterparts. scRNA-seq of BM cells exposed large differences to the hematopoietic niche in AD-fed mice. Interestingly, KEGG pathway analysis of neutrophils shows upregulation of apoptosis, NF-κB, and cystolic DNA-sensing signaling, suggesting a heightened state of activation and inflammatory response. Conversely, there was a general lack of hematopoietic stem cells in CKD mice and a detrimental effect on B-cell maturation in this disease state. Additionally, CKD mice exhibited reduced mRNA expression in whole tibia osteogenic mRNA markers: osteocalcin (Bglap; 2-fold decrease) and alkaline phosphatase (Alpl) and increases in adipogenic markers: lipoprotein lipase (Lpl; 2.5-fold increase) and adiponectin (AdipoQ; 2.5-fold increase).

Conclusion: The characterization of BM cells in the setting of CKD is novel. By understanding alterations to the BM microenvironment, significant changes in cell populations and how Mφ polarization affects MSCs during CKD, we hope to reveal novel therapeutic targets to prevent bone loss.

No relevant conflicts of interest to declare.