Varnika Jakka
Research Mentor: Jie Ren Gerald Har
Mentor Department: Biologic and Material Sciences and Prosthodontics, Dentistry
Author(s): Varnika Jakka, Lauren Surface, Gerald Har
Session: Session 3 (11:00 AM – 11:50 AM)
Presentation Type: Poster 48
Abstract
Chronic Kidney Disease increases bone loss, however, the mechanisms by which CKD changes osteocyte function and bone regulation are not fully understood. This study investigates how CKD-related conditions affect osteocyte metabolism and osteocyte gene expression. CKD was induced in mice using a 0.2% adenine diet. After disease development, kidney, liver, femur, and tibia samples were collected and protein was extracted. Bicinchoninic Acid (BCA) assays showed very high protein concentrations in kidney and liver tissues, confirming sufficient amounts of protein for future Western blot analysis. Protein was also successfully extracted from femur and tibia samples. To analyze osteocyte regulation under CKD-related conditions, differentiated OCY454 osteocyte cells were treated with parathyroid hormone (PTH), indoxyl sulfate (IS), or a combination of both. PTH and IS are two factors that are elevated in CKD and contribute to bone and mineral disorders, and were applied to examine how CKD-associated hormonal and metabolic changes affect osteocyte protein production. In osteocyte cultures, PTH treatment reduced the visible collagen layer compared to control wells, while overall protein levels remained similar between control and IS-treated groups. The combined PTH + IS group showed greater variability in protein levels, suggesting a possible interaction effect on osteocyte protein regulation. Based on an earlier metabolic assay, OCY454 cells consume a high amount of glutamine compared to asparagine. To further examine the role of glutamine and asparagine in osteocyte behavior, differentiated cells were cultured without asparagine, glutamine, or both amino acids. Removal of glutamine led to a significant reduction in total protein production, while removal of asparagine showed a smaller effect. These results suggest that osteocytes may require glutamine for biological processes such as protein synthesis and cell metabolism. Gene expression analysis via qPCR is currently ongoing to examine how removal of asparagine and glutamine may disrupt osteocyte gene expression and metabolic activity. Expression of eight genes is being analyzed, including RPLP0, Sost, Dkk1, RankL, OPG, Phex, Slc13a5, and Dmp1. These genes include markers involved in osteocyte signaling, bone remodeling, mineralization, and metabolic transport. Overall, these findings provide insight into how CKD-related conditions and changes in amino acids may influence osteocyte behavior. The results from these experiments will help to guide further analysis of osteocyte gene expression. Future work will focus on confirming gene expression trends and using Western blot analysis to better understand how CKD-related osteocyte changes affect function and bone regulation.
