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3930 Bone Marrow Remodeling and Pro-Inflammatory Megakaryocytes in a Murine Model of Chronic Kidney Disease

Program: Oral and Poster Abstracts
Session: 301. Platelets and Megakaryocytes: Basic and Translational: Poster III
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Bleeding and Clotting, Diseases
Monday, December 9, 2024, 6:00 PM-8:00 PM

Estelle Carminita, PhD1,2*, Virginia Camacho, PhD1,3, Isabelle C Becker, PhD, MSc1,3, Maria Barrachina, PhD4,5, Karen Guo4*, Kellie R. Machlus, PhD6 and Joseph E. Italiano, PhD1,3

1Vascular Biology Program, Boston Children's Hospital, Boston, MA
2Surgery Departement, Harvard Medical School, Boston, MA
3Harvard Medical School, Boston, MA
4Vascular Biology Program, Boston Children's Hospital, Boston
5Surgery Department, Harvard Medical School, Boston
6Vascular Biology Program, Harvard Medical School and Boston Children's Hospital, Boston, MA

Background: Chronic Kidney Disease (CKD) is a significant medical problem characterized by loss of kidney function. It is a silent, progressive, and irreversible disease with no current cure. Hemostatic disorders and cardiovascular disease are the main complications leading to increased mortality in patients suffering from CKD. Moreover, severe calcium and vitamin D deficiencies weaken the bones of these patients, resulting in mineral bone disease. This bone atrophy is the second most common complication during CKD. While the role of platelets in cardiovascular events associated with CKD remains important but controversial, whether defective platelet production from bone marrow-resident megakaryocytes (MKs) affects CKD progression has not been investigated to date.

Aim: Determine the contribution of megakaryocytes in the production of dysfunctional platelets responsible for hemostatic disorders in CKD.

Method: An aristolochic acid nephropathy (AAN)-CKD model was utilized to mimic CKD development in mice. Whole blood and bones were collected from AAN-CKD mice and PBS-injected control mice. Flow cytometry was used to evaluate the total blood cell count, platelet reactivity, and composition of the hematopoietic stem and progenitor cell (HSPC) compartment. MK numbers and shape were assessed in femoral cryosections by immunofluorescence imaging.

Results: Our data reveals a progressive increase in platelet counts following CKD development, as reported in CKD patients. Platelets from CKD mice display hyper-reactivity to platelet agonists (ADP, U46619, thrombin, and CRP). CKD mice also displayed a decrease in newly generated Thiazole Orange (TOhigh) platelets and increased platelet neutrophil aggregates (PNAs). Platelet releasate analysis revealed a decreased level of P-selectin suggesting that either CKD-Platelets are deficient in alpha-granules or the platelet secretory system is altered. Flow cytometric analysis of the bone marrow hematopoietic compartment revealed significant reductions in cellularity in CKD animals compared to control. Critically, we observed a reduction in the total numbers of megakaryocyte progenitors (MkPs) and mature MKs (CD41+/CD42+) in AAN-CKD bone marrow indicating that MK maturation is impaired during CKD progression. Immunofluorescence imaging of femoral cryosections confirmed the reduced number of megakaryocytes (CD41+) in AAN-CKD, while no difference in size was observed. Despite an unaltered MK size, we observed a significant increase in medium ploidy 16N MKs. Bone marrow fluid analysis revealed significant changes to cytokine composition in CKD mice compared to the controls and culture of MKs with CKD-bone marrow fluid drove an increase in proplatelet formation. MKs incubated with CKD-bone marrow fluid are more elaborated with an increased number of branches and proplatelet tips.

Conclusion: Altogether, our results reveal disrupted bone marrow homeostasis during CKD development, which might explain the dysfunctional phenotype of platelets leading to hemostatic disorders in CKD patients.

Disclosures: Italiano: StellularBio: Membership on an entity's Board of Directors or advisory committees; SpryBio: Membership on an entity's Board of Directors or advisory committees.

*signifies non-member of ASH