Developing an In Vitro Model of CKD-MBD Induced αKlotho Suppression

Chronic Kidney Disease (CKD) affects approximately 1 in 10 Americans. Diabetic nephropathy is also associated with the development of chronic kidney disease-mineral bone disorder (CKD-MBD). CKD-MBD disrupts the normal bone-kidney endocrine axis responsible for regulating mineral metabolism, and hyperphosphatemia develops in late stage disease. Important clinical hallmarks of the CKD-MBD progression include elevated bioactive Fibroblast growth factor-23 (FGF23) and suppression of FGF23’s co-receptor, αKlotho (αKL). In healthy individuals the hormone FGF23, primarily produced by bone, and aKL aid in maintaining normal phosphate and vitamin D homeostasis. It is currently unknown what drives the suppression of αKL expression, however increasing αKL expression in CKD-MBD models is being investigated as a novel therapeutic. Our study sought to develop a novel in vitro model of one of the clinical hallmarks of the progression of CKD-MBD, αKL suppression, to investigate both possible stimuli of its repression and downstream signaling events. The Human Embryonic Kidney (HEK) cell line was used to determine if changes in fluid shear stress, similar to those that occur in diabetic nephropathy, could lead to reduced αKL expression. HEK cells were plated and exposed to oscillatory fluid shear stress (OFSS) for intervals between 0-60 min to examine protein expression or 0-2 hours to assess gene expression. HEK cells were sensitive to mechanical stimulation as pathways including increased ERK phosphorylation occurred in response to OFSS. In response to longer bouts of OFSS αKL expression was significantly (p\u3c0.05) reduced. Dramatic changes in fluid shear stress may serve as a stimulus for reduced αKL expression in CKD-MBD. Further studies are underway to investigate downstream signaling events related to αKL suppression. Understanding both the stimuli of αKL suppression and related downstream signaling events could provide novel therapeutic targets for the treatment of CKD-MBD

Osteocyte-Specific Deletion of the α2δ1 Auxiliary Voltage Sensitive Calcium Channel Subunit

Context: Skeletal unloading due to disuse, disease, or aging increases bone loss and the risk of skeletal fracture. Conversely, mechanical loading is anabolic to the skeleton, promoting skeletal integrity through increased bone formation. As calcium influx is the first measurable response of bone cells to mechanical stimuli, voltage sensitive calcium channels (VSCCs) play a critical role in bone formation. Given VSCC activity is influenced by its auxiliary α2δ1 subunit, regulating the gating kinetics of the channel’s pore-forming (α1) subunit and forward trafficking of VSCCs to cell membranes, the α2δ1 subunit may govern anabolic bone responses. Objective & Design: We hypothesized that osteocyte-specific α2δ1 deletion in a mouse model would impair skeletal development, decrease bone formation and mechanosensitivity. Methods: Generation of an osteocyte-specific α2δ1 knockout was accomplished by crossing mice (C57BL/6) harboring LoxP sequences flanking Cacna2d1, the gene encoding α2δ1, with mice expressing Cre recombinase under the control of the Dmp1 (10Kb) promoter (Cacna2d1fl/fl, Dmp1-Cre+). To assess skeletal phenotype and mechanosensitivity, longitudinal whole body and site-specific DXA, in vivo μCT (10wk old), and two weeks of tibial loading (16wks) will be conducted before femurs are collected at 20 wks for mechanical testing, ex vivo μCT, and quantitative histomorphometry. Results & Conclusion: Preliminary analyses show no differences in whole body or site-specific BMD and BMC values between mice over time, suggesting osteocyte-specific α2δ1 deletion may not influence skeletal development. However, key differences in mechanosensitivity following tibial loading are expected given the potential role of α2δ1 in mechanically-induced bone formation

Identification of Commercially Available Antibodies that Block Ligand Binding by BMPR2

Osteoporosis, a disease of low bone mineral density, affects 10 million Americans and triggers significant health problems and considerable socioeconomic burdens. Current treatments for osteoporosis have significant limitations, necessitating identifying new treatment strategies via building a better understanding of the endogenous mechanisms regulating bone mass. A recent study demonstrated that removal of the BMP type 2 receptor (BMPR2) in skeletal progenitor cells of Bmpr2-cKO mice during embryonic development leads to reduced age-related bone loss by sustained elevation in bone formation rate. This present study sought to advance the translational potential of the genetic model by identifying antibodies that neutralize the ligand-binding function of the BMPR2 extracellular domain (BMPR2-ECD). This study first established a modified, cell-free immunoprecipitation assay wherein the ligand BMP2 was pulled-down by BMPR2-ECD conjugated to Protein G beads; the unbound BMP2 (found in the supernatant) was subsequently quantified by ELISA. This yielded a standard assay wherein approximately 2 ug BMPR2-ECD leads to a 70% reduction in BMP2 signal. Next, the neutralizing ability of 3F6, a mouse monoclonal antibody raised against the ligand-binding region of BMPR2, was examined and was found to cause a dose-dependent inhibition of BMPR2-ECD ligand-binding. Given the ascites preparation of 3F6, specificity of this assay was confirmed by demonstrating that ligand-binding activity of BMPR2-ECD is unchanged in the presence of non-specific, negative-control ascites. Using these results as a guide, 1F12, another mouse monoclonal antibody raised against the ligand-binding region of BMPR2, was evaluated and was also found to neutralize the ligand-binding function of BMPR2-ECD. In contrast, no effect on ligand-binding function of BMPR2-ECD was observed with 9A10 even though this mouse monoclonal antibody is also raised against the ligand-binding region of BMPR2. These results provide proof-of-concept data for future studies evaluating inhibition of BMPR2 function in vivo as a means to reduce age-related bone loss

Identification of a bone morphogenetic protein type 2 receptor neutralizing antibody

The bone morphogenetic protein (BMP) signaling pathway comprises the largest subdivision of the transforming growth factor (TGFβ) superfamily. BMP signaling plays essential roles in both embryonic development and postnatal tissue homeostasis. Dysregulated BMP signaling underlies human pathologies ranging from pulmonary arterial hypertension to heterotopic ossification. Thus, understanding the basic mechanisms and regulation of BMP signaling may yield translational opportunities. Unfortunately, limited tools are available to evaluate this pathway, and genetic approaches are frequently confounded by developmental requirements or ability of pathway components to compensate for one another. Specific inhibitors for type 2 receptors are poorly represented. Thus, we sought to identify and validate an antibody that neutralizes the ligand-binding function of BMP receptor type 2 (BMPR2) extracellular domain (ECD)

Bringing Attention to Lesser-known Bone Remodeling Pathways

Osteoporosis, a disease of low bone mass, places individuals at enhanced risk for fracture, disability, and death. In the USA, hospitalizations for osteoporotic fractures exceed those for heart attack, stroke, and breast cancer and, by 2025, the number of fractures due to osteoporosis is expected to rise to nearly three million in the USA alone. Pharmacological treatments for osteoporosis are aimed at stabilizing or increasing bone mass. However, there are significant drawbacks to current pharmacological options, particularly for long-term management of this chronic condition. Moreover, the drug development pipeline is relatively bereft of new strategies. Consequently, there is an urgent and unmet need for developing new strategies and targets for treating osteoporosis. Casual observation led us to hypothesize that much of the bone remodeling research literature focused on relatively few molecular pathways. This led us to perform bibliometric analyses to determine the relative popularity of bone remodeling pathways in publications and US National Institutes of Health funding of the last 10 years. In this review article, we discuss these findings and highlight several less-examined signaling pathways that may hold promise for future therapies

Intramuscular versus Subcutaneous Injections of Testosterone in Transgender Men

Hormone replacement therapy (HRT) is a common treatment for gender dysphoria, a condition where a person’s gender assigned at birth does not match their gender identify. For transgender men seeking to achieve virilization, HRT is included in the standard of care as set forward by the World Professional Association for Transgender Health (WPATH). There are several routes of HRT administration including injection, pellet, gel, and patch. Many transgender men opt for the injectable testosterone for a variety of reasons including cost, insurance coverage, and impact on lifestyle. While the WPATH standards of care do include injectable testosterone as one of the HRT options, only intramuscular injections are mentioned in the most recent edition of standards published in 2012. Research has been put forth within the last several years exploring the effectiveness of not only intramuscular injections, but also subcutaneous injections. Though intramuscular injections continue to be the standard route by which testosterone is administered, there is substantial research that subcutaneous injections are equally, if not more, effective with a much higher patient preference. This primary literature review focuses primarily on the injectable methods of HRT and directly com-paring the effectiveness of intramuscular and subcutaneous testosterone for transgender men

Gestational Diabetes Leading to Cardiovascular Disease

Gestational diabetes mellitus (GDM) is an increasingly prevalent complication of preg-nancy leading to adverse maternal outcomes for years postpartum. This primary litera-ture review was conducted to explore what complications of GDM have significant cor-relation with an increased risk for eventual onset of cardiovascular disease (CVD). Overlapping risk factors for GDM and CVD such as obesity, sedentary lifestyle, and poor diet are largely the cause for this correlation. Obesity is a commonly recognized cause of the progression from GDM to CVD. However, there remains the need to iden-tify other factor(s) that elevate CVD risk in women who have had GDM but who are not obese and who lead an active life. The literature review explores the issues that may be secondary to obesity, along with outcomes of GDM that lie outside of obesity entire-ly and remain known exclusively as a by-product of GDM. This allowed for some pos-sible explanations for the increased cardiovascular risk seen in women who had GDM but would typically not be considered high risk for CVD or GDM. GDM results in vari-ous adverse outcomes including hypertension, dyslipidemia, inflammation, endothelial dysfunction, and type 2 diabetes, all of which promote progression to CVD. These may serve as intermediate factors in the onset of CVD up to 25 years postpartum. Seden-tary lifestyle and advanced maternal age are critical factors favoring the progression of GDM to CVD. Further identification of risk factors leading to CVD would allow for pre-vention of onset of CVD following a pregnancy affected by GDM

Perceptions of interprofessional education in the medical and nursing curriculums

Interprofessional education or IPE involves bringing individuals from two or more professions together to learn and communicate to improve future teamwork and collaboration. IPE research has shown that exposing future healthcare professionals to interprofessional training results in positive patient outcomes and reduced institutional costs. Our study will be a case based interprofessional simulation between Marian University nursing and medical students. The goals of our study include: 1) evaluating the perceptions of interprofessional education among nursing and medical students and 2) examining whether there are beneficial areas to overlap the nursing and medical training curriculums to implement effective IPE opportunities. The study will be conducted on January 22, 2020 in the Simulation Center at MU-COM, under the direction of David Patterson. Participants will be recruited via flyers and email communication. Incentivization could include using Amazon gift cards, funded via research funds available to BMS graduate students. Prior to the simulation, participants will be asked to complete an online module via Canvas. The module will pertain to the case, include anonymous demographics questions, as well as, a pre-survey. The Interdisciplinary Education Perception Scale (IEPS) survey will be used for pre and post simulation. Each participant will randomly be assigned a simulation team, balancing nursing students with medical students. On simulation day, participants will undergo a brief introduction/orientation to the required equipment. Once they are oriented to their surroundings they will work through the case as a team. The chosen case, which involves women’s reproductive health, will require students to not only use their clinical skills to treat the patient, but will also require them to handle a religious social aspect that will affect the available treatment options. When the teams have come to a conclusion with the case, they will enter a small conference room to undergo a debriefing session where facilitators, including both nursing and medical school faculty, will help them analyze their use of communication during the simulation. After this short discussion, participants will again take the IEPS survey, as well as provide feedback on the simulation.In the post-simulation analysis, we will compare pre-survey scores with post-survey scores and compare how perceptions of interprofessional education have changed. We believe that perceptions of IPE will improve from pre-simulation to post-simulation and that the participants will feel better prepared for future collaboration

The Role of Notum in Bone’s Response to Mechanical Loading and Aging

Osteoporosis is a silent, dangerous, pathology frequently undiagnosed until patients experience a major fracture. Fractures from osteoporosis lead to a 20% increase of mortality, revealing a need for novel anabolic therapies. Recently, mice lacking Notum protein expression were reported to have increased endocortical bone formation and strength. Notum is an inhibitory protein of the Wnt pathway, a major growth pathway in mammalians. The hypothesis for this work is Notum expression is affected by mechanical loading and aging. The goal of these studies is to investigate factors that could affect Notum expression, with the future direction of identifying factors that when pharmacologically modulated could promote anabolic bone growth. After establishing No-tum expression in the osteocyte cell line, MLOY4s, Notum expression was monitored in response to mechanical loading, mimicked via orbital shear stress for 15 min, 1hr, and 2hr. In addition, Notum expression was analyzed in aged bone. Our preliminary results suggest mechanical loading decreases Notum protein expression, while aging increases the expression of the Notum gene (p\u3c0.05). . With these results our hypothesis is supported, and future studies repressing Notum expression in bone as novel approach to promoting anabolic bone growth will be conducted