Interactions Between Aging and Chronic Kidney Disease on the Skeleton

Indiana University-Purdue University Indianapolis (IUPUI)In the US, 15% of adults have chronic kidney disease (CKD). While CKD occurs across all ages, the prevalence is highest in the aged, with ~40% of individuals over age 65 having some form of CKD. CKD and aging are each independently associated with higher fracture risk, and thus overlaying CKD in the aging population presents an additive fracture risk. Cortical porosity is a central tenet underlying skeletal fragility and occurs in CKD and aging. Previous research on cortical porosity has focused on preventing pore formation, while research on pore reversal (infilling) is lacking. Pore infilling is dependent on proper osteoblast function, and previous research has shown that infilling is possible in young mice. However, it is unclear whether infilling is possible in aging mice due to aging-associated osteoblast dysfunction. Therefore, we proposed that aging animals with CKD may require both suppression of CKD-induced elevations in parathyroid hormone (PTH) and anabolic therapy to infill cortical pores. Romosozumab, a humanized monoclonal sclerostin antibody, uses PTH-independent mechanisms to increase osteoblast activity, making it an attractive therapeutic for CKD. CKD was induced by feeding aging (78-week) male mice 0.2% adenine for six weeks followed by two weeks of maintenance on control diet for a total study duration of eight weeks of CKD; mice were then treated with calcium water, romosozumab, or the combination and their effectiveness in improving skeletal quantity and quality was evaluated. Romosozumab treatment was associated with higher trabecular bone volume, lower cortical porosity, and higher mechanical properties compared to control animals. Combination treatment also resulted in benefits to trabecular bone volume and mechanical properties. These results demonstrate that both romosozumab alone and when combined with PTH suppression can be effective at improving bone microarchitecture and mechanical properties in aged individuals with CKD who are at high risk of fracture

Erythropoietin and a hypoxia-inducible factor prolyl hydroxylase inhibitor (HIF-PHDi) lowers FGF23 in a model of chronic kidney disease (CKD)

Iron‐deficiency anemia is a potent stimulator of the phosphaturic hormone Fibroblast growth factor‐23 (FGF23). Anemia, elevated FGF23, and elevated serum phosphate are significant mortality risk factors for patients with chronic kidney disease (CKD). However, the contribution of anemia to overall circulating FGF23 levels in CKD is not understood. Our goal was to investigate the normalization of iron handling in a CKD model using the erythropoiesis stimulating agents (ESAs) Erythropoietin (EPO) and the hypoxia‐inducible factor prolyl hydroxylase inhibitor (HIF‐PHDi) FG‐4592, on the production of, and outcomes associated with, changes in bioactive, intact FGF23 (“iFGF23”). Our hypothesis was that rescuing the prevailing anemia in a model of CKD would reduce circulating FGF23. Wild‐type mice were fed an adenine‐containing diet to induce CKD, then injected with EPO or FG‐4592. The mice with CKD were anemic, and EPO improved red blood cell indices, whereas FG‐4592 increased serum EPO and bone marrow erythroferrone (Erfe), and decreased liver ferritin, bone morphogenic protein‐6 (Bmp‐6), and hepcidin mRNAs. In the mice with CKD, iFGF23 was markedly elevated in control mice but was attenuated by >70% after delivery of either ESA, with no changes in serum phosphate. ESA treatment also reduced renal fibrosis markers, as well as increased Cyp27b1 and reduced Cyp24a1 mRNA expression. Thus, improvement of iron utilization in a CKD model using EPO and a HIF‐PHDi significantly reduced iFGF23, demonstrating that anemia is a primary driver of FGF23, and that management of iron utilization in patients with CKD may translate to modifiable outcomes in mineral metabolism

Reversing cortical porosity: Cortical pore infilling in preclinical models of chronic kidney disease

Purpose Chronic kidney disease (CKD) patients have a high incidence of fracture due in part to cortical porosity. The goal of this study was to study cortical pore infilling utilizing two rodent models of progressive CKD. Methods Exp 1: Female C57Bl/6J mice (16-week-old) were given dietary adenine (0.2%) to induce CKD for 10 weeks after which calcium water supplementation (Ca-H2O; 1.5% and 3%) was given to suppress PTH for another 4 weeks. Exp 2: Male Cy/+ rats were aged to ~30 weeks with baseline porosity assessed using in vivo μCT. A second in vivo scan followed 5-weeks of Ca-H2O (3%) supplementation. Results Exp 1: Untreated adenine mice had elevated blood urea nitrogen (BUN), parathyroid hormone (PTH), and cortical porosity (~2.6% porosity) while Ca-H2O lowered PTH and cortical porosity (0.5–0.8% porosity). Exp 2: Male Cy/+ rats at baseline had variable porosity (0.5%–10%), but after PTH suppression via Ca-H2O, cortical porosity in all rats was lower than 0.5%. Individual pore dynamics measured via a custom MATLAB code demonstrated that 85% of pores infilled while 12% contracted in size. Conclusion Ca-H2O supplementation causes net cortical pore infilling over time and imparted mechanical benefits. While calcium supplementation is not a viable clinical treatment for CKD, these data demonstrate pore infilling is possible and further research is required to examine clinically relevant therapeutics that may cause net pore infilling in CKD

SUN-351 Sex Is a Strong Variable in the Mineral Metabolism Defects and Endocrine Dysfunction Associated with the Murine Adenine Diet Model of Chronic Kidney Disease (CKD)

The adenine diet is widely used in animal models to produce a tubulointerstitial fibrosis and inflammation that mimics human CKD in many aspects. These include the biochemical manifestations hyperphosphatemia and anemia, as well as endocrine dysfunction with elevated FGF23 and hyperparathyroidism. Male rodents are known to be less tolerant of adenine diet regimen than females, however the underlying mechanisms driving the sex differences remain unclear. Additionally, much of the data for adenine studies arises from rats, whereas mice are more commonly used in laboratory settings and are far easier to manipulate genetically. To this end, as part of a larger study to test the effects of iron-handling in CKD, we assessed the biochemical, molecular, and physical differences between male and female mice receiving an adenine diet to induce CKD. Flox-Fgf23 mice (8 weeks of age, n=4-6/group; mice were Cre negative, thus phenotypically wild type) were placed on a 0.2% adenine-containing diet (CKD); a matching casein-based diet served as control. After 6 weeks, mice were euthanized, and blood and tissues were collected for analysis. As expected, body weight at baseline was initially higher in males than in females, however males lost significantly more weight. Serum BUN was also elevated in both sexes receiving adenine, although males were higher (1.2 fold; p<0.01). Males also had elevated creatinine and lower total serum iron from baseline whereas females had no significant changes. FGF23 was elevated in all mice, with no significant differences between sexes. Kidney fibrosis and inflammation markers were elevated in the CKD mice, with males having higher expression of Col1a1 and -3a1 versus females (3.5/1.5 fold; p<0.001) and TNFα mRNA (2 fold; p<0.001). Renal expression of the anabolic vitamin D metabolizing enzyme Cyp27b1 (1α-hydroyxlase) and early growth response 1 (Egr1) were increased in CKD mice, with males having higher expression over females. Conversely, CKD males had lower kidney Klotho mRNA expression, and both sexes fed adenine expressed significantly lower NPT2a (sodium- phosphate co-transporter2a) mRNA. Liver expression of ferritin (Fth1) was elevated in male CKD mice compared to diet controls, whereas female mice had no differences. Elevated FGF23 has been linked to ventricular hypertrophy, and CKD males had significantly higher heart weight to femur ratio at completion of the study. Our results support that male mice succumb more rapidly than females to adenine diet mediated CKD phenotypes, potentially enhanced by fibrosis and inflammation. It remains to be determined whether the more rapid onset of defects in iron handling parameters accelerate the severe male CKD phenotype

Integrative genomic analysis of adult mixed phenotype acute leukemia delineates lineage associated molecular subtypes

Mixed phenotype acute leukemia (MPAL) is a rare subtype of acute leukemia characterized by leukemic blasts presenting myeloid and lymphoid markers. Here we report data from integrated genomic analysis on 31 MPAL samples and compare molecular profiling with that from acute myeloid leukemia (AML), B cell acute lymphoblastic leukemia (B-ALL), and T cell acute lymphoblastic leukemia (T-ALL). Consistent with the mixed immunophenotype, both AML-type and ALL-type mutations are detected in MPAL. Myeloid-B and myeloid-T MPAL show distinct mutation and methylation signatures that are associated with differences in lineage-commitment gene expressions. Genome-wide methylation comparison among MPAL, AML, B-ALL, and T-ALL sub-classifies MPAL into AML-type and ALL-type MPAL, which is associated with better clinical response when lineage-matched therapy is given. These results elucidate the genetic and epigenetic heterogeneity of MPAL and its genetic distinction from AML, B-ALL, and T-ALL and further provide proof of concept for a molecularly guided precision therapy approach in MPAL