Chronic Kidney Disease Induces a Reduction In Hematopoietic Stem –and Progenitor Cell Frequencies.

Abstract 3857

The hematopoietic microenvironment (niche) plays a key role in the maintenance of hematopoietic stem cells (HSC). The bone lining-osteoblast has been identified as a crucial component of the stem cell niche and regulates the number of HSC in the niche via a variety of membrane-bound and secreted molecules. Chronic Kidney Disease (CKD) is marked by a specific Mineral Bone Disease (CKD-MBD), due to a sustained parathyroid hormone (PTH) release. This chronical hyperparathyroidy results in increased bone turnover due to increased osteoblast activity. We have established a mouse model of CKD to study the relationship between disturbed bone metabolism and hematopoiesis.

C57Bl/6 mice underwent surgically-induced CKD (thermocauterisation-nephrectomy). Twelve weeks after CKD-induction, bone structure was analyzed by micro-CT scan to confirm CKD-MBD. Indeed, all mice showed the features of CKD including significantly increased urea levels, increased trabecular bone volume, and a decrease in span incurvation and cortical thickness. In addition, CKD mice developed anemia. Subsequently, bone marrow cells (BMC) were harvested from CKD mice and sham-operated controls. Long-term HSC and short-term hematopoietic progenitor cells (HPC) were analyzed phenotypically by flowcytometry and functionally by cobblestone area forming cell (CAFC) assays and colony assays (CFU-C).

The frequencies of long-term repopulating Lin^negSca1^posc-Kit^HI (LSK) CD135^negCD34^neg HSC were significantly decreased in CKD mice compared to sham-operated controls (0.0028% ± 0.001 vs 0.0049% ± 0.001, p<0.05; n=5 per group), whereas the frequencies of HPC (LSK CD135^negCD34^pos) and multipotent progenitors (MPP; LSK CD135^posCD34^pos) remained stable. Also, total LSK cell number correlated positively with trabecular bone volume. However, CAFC analysis showed a decrease in both HSC and HPC frequencies (0.5 ± 0.1 vs 1.4 ± 0.7 CAFC week 5 per 10⁵ BMC, p<0.05; 0.6 ± 0.002 vs 1.5 ± 0.2 CAFC week 3 per 10⁵ BMC, p<0.05 for CKD vs controls respectively; n=4 per group), indicating a functional defect in their repopulating capacity. The colony-forming capacity of BMC obtained from CKD mice and controls was similar (4.4 × 10⁴ ± 1.5 × 10⁴ vs 4.6 × 10⁴ ± 1.7 × 10⁴ CFU-C per femur for CKD vs controls respectively; n=5). In contrast, a 3.6-fold increase in the frequency of peripheral blood CFU-C was found in CKD mice compared to sham-operated controls (42.7 ± 2.6 vs 11.8 ± 1.6 CFU-C per ml for CKD vs controls respectively; n=5).

CKD mice showed a 2.8-fold reduction in G-CSF-induced HSC/HPC mobilization compared to controls (355 ± 281 vs 1005 ± 476 CFU-C per ml peripheral blood for CKD vs controls respectively; n=5 per group). Analysis of BM extracellular fluid showed a 2.3-fold reduction in elastase activity and an increase of the protease inhibitor a1-antitrypsin in CKD mice compared to controls. No differences in MMP-9 activity were observed between the groups.

Together, our data show that in CKD 1) LSK CD135^negCD34^neg HSC frequencies are significantly decreased 2) LSK CD135^negCD34^pos HPC and LSK CD135^posCD34^pos MPP remain at similar levels 3) CAFC-week 3 HPC and CAFC week 5 HSC are significantly decreased 4) BMC CFU-C remain at similar levels, while peripheral blood CFU-C are increased and 5) G-CSF-induced stem cell mobilization is impaired in CKD. We hypothesize that the observed changes in the hematopoietic compartment are due to increase osteoblast activity.