Effect of Denosumab on Bone Health, Vascular Calcification, and Health-Related Quality of Life in Hemodialysis Patients with Osteoporosis: A Prospective Observational Study

Background: Osteoporosis is common in hemodialysis (HD) patients, contributing to cardiovascular risks. Limited research exists on denosumab’s efficacy in this group. Our study explores denosumab’s effects on bone turnover markers (BTMs) and vascular calcification in chronic kidney disease–mineral bone disorder (CKD-MBD) patients. Methods: In a prospective single-center study, we investigated the effects of denosumab over 2 years on 30 HD patients from a cohort of 185. Annual assessments of bone mineral density (BMD), vascular calcification, and health-related quality of life (HRQL) were conducted and compared with an untreated group. Mineral and bone parameters were analyzed at specific intervals in the treatment group. Results: Denosumab notably raised femoral BMD in the initial year. Most bone turnover markers (BTMs) decreased, except for osteocalcin. Changes in T50 correlated with BTMs. Pre-denosumab supplementation of calcium and vitamin D helped manage mineral imbalances. Post denosumab, parathyroid hormone (PTH) levels increased initially, stabilizing after 3 months. No significant changes occurred in vascular calcification or HRQL. Conclusions: Denosumab exhibited varying effects on BMD improvement, with a stronger impact in the first year that diminished in the second year. Early PTH monitoring was crucial, and extending the administrative period may enhance BMD outcomes compared to the general population.


Introduction
Chronic kidney disease-mineral and bone disorder (CKD-MBD) is common in hemodialysis (HD) patients.However, compared to mineral marker management, there is limited research on bone health based on clinical guidelines [1].Limited research on the efficacy and safety of bisphosphonates in severe renal impairment restricts osteoporosis treatment options for HD patients [2].This limitation has delayed focus on the diagnosis and treatment of osteoporosis in this population.
Denosumab is a receptor activator of nuclear factor kappa-B ligand (RANKL) monoclonal antibody that has been increasingly used in patients with advanced CKD and HD patients due to its independence from renal function [3].Some studies have focused on short-term improvements in BMD, often noting hypocalcemia [4].A gap exists in understanding the holistic impact of denosumab on mineral parameters, bone health, and vascular calcification during treatment for CKD-MBD.Denosumab is used for impaired kidney function [5], leading HD patients to start osteoporosis treatment without considering the impact of CKD-MBD, and often outside of dialysis clinics [6].Therefore, nephrologists require comprehensive information and strategies regarding these treatment approaches and their ramifications.
Our study comprehensively assessed the impact of denosumab on BMD, vascular calcification, and HRQL over a two-year period, alongside specific evaluations of mineral parameters and BTMs at intervals.Limited research exists on the effects of denosumab in osteoporotic patients, necessitating a thorough investigation into its influence on mineral parameters, BTMs, and vascular calcification.

Study Design and Setting
In a prospective observational study at a single center in Korea, we investigated the relationships among denosumab treatment, BMD, biochemical parameters, BTMs, HRQL, and vascular calcification.This study received approval from the Institutional Review Board of Gachon University Gil Medical Center (GBIRB2020-342) and adhered to the Declaration of Helsinki, with written informed consent from all participants.

Study Population
Between March 2020 and February 2022, 185 HD patients at Gachon University Gil Medical Center underwent BMD analysis using dual-energy X-ray absorptiometry (DEXA).Osteoporosis was determined based on Korean Society for Bone and Mineral Research criteria [7] and World Health Organization T-scores for Asian countries, with a T-score ≤ -2.5 in the lumbar spine (LS), femoral neck (FN), or total hip (TH).Patients eligible for denosumab had been undergoing HD for ≥6 months and were free of conditions that would affect their biochemical parameters (e.g., catheters, malignancies, liver disease, infection, parathyroidectomy, or pretreatment with other anti-osteoporotic agents in the preceding 6 months).Patients with recent cardiovascular events, dental procedures, or upcoming dental interventions within 6 months were excluded.In the end, 30 patients were administered a subcutaneous dose of 60 mg/mL denosumab (Prolia ® Pre-filled Syringe, Amgen, Inc., Thousand Oaks, CA, USA) every 6 months.Following previous reports [8,9], patients presenting with hypocalcemia (corrected calcium < 8.4 mg/dL) before the denosumab injection received a combination of calcium and cholecalciferol supplements beginning 2 weeks before the administration of denosumab until the tracked corrected calcium level was >10 mg/dL.The corrected calcium level was calculated using Payne's formula [10].The other mineral parameters followed the CKD-MBD guidelines [1].In the end, of the 30 participants, 1 received only two doses over 1 year, while the remaining 29 received the scheduled four doses of denosumab over 2 years.Comparative analyses were conducted with the non-denosumab-administered group as the control, which refers to patients who have maintained stable HD for more than 6 months and whose BMD results do not correspond to osteoporosis or meet the exclusion criteria for denosumab administration.

Fracture Risk Assessment
The 10-year risk of major osteoporotic fracture (MOF) and hip fracture (HF) was computed using the Korean version of the FRAX ® calculator, accessible online at http://www.shef.ac.uk/FRAX/tool.aspx?country=25 (accessed on 26 October 2020), based on the completed questionnaires and medical chart review.

Determination of Serum Calcification Propensity (T50)
The T50 value was assessed utilizing a nephelometer (Nephelostar, BMG Labtech, Offenburg, Germany), measuring the time for the transformation from primary to secondary calciprotein particle (CPP) [11,12].In brief, serum (80 µL) was mixed with NaCl solution (20 µL) and then exposed to supersaturated concentrations of calcium (50 µL) and phosphate (50 µL) solutions in triplicate in a 96-well plate.The Nephelostar, operated via MARS software V2.41, followed the manufacturer's guidelines.Non-linear regression curves were computed to ascertain the T50 values.

Vascular Calcification Score
Plain X-ray images of the lateral lumbar spine were used to semiquantitatively calculate abdominal aortic calcification (AAC) scores for all subjects [13].Additionally, denosumab-treated patients underwent electron beam computed tomography (EBCT) to calculate the coronary artery calcium (CAC) score using the Agatston method [14].

Health-Related Quality of Life
The Kidney Disease Quality of Life Instrument-Short Form assesses HRQL in kidney disease patients using the validated Korean version [15].It includes a disease-specific section for dialysis patients, the Kidney Disease Component Summary (KDCS), which comprises health-related aspects divided into the Physical Component Summary (PCS) and the Mental Component Summary (MCS).

Definition of Outcomes
Primary outcomes were changes in BMD and BTMs following denosumab administration; secondary ones were changes in mineral parameters after denosumab administration in HD patients and the effect on established CKD-MBD treatment strategies that adhere to existing clinical guidelines.

Statistical Analyses
Continuous variables were assessed for normality using the Shapiro-Wilk test.Nonnormally distributed variables underwent either log transformation or non-parametric analysis.Normally distributed values are presented as mean ± standard deviation, while non-normally distributed values are reported as median and interquartile range.Group comparisons employed the χ2 test, Student's t-test, or analysis of variance (ANOVA) as appropriate.Within-group changes were evaluated using the paired t-test or Wilcoxon's signed-rank test.All statistical analyses were performed using R software, version 4.3.2., with packages available on the Comprehensive R Archive Network (http://cran.r-project.org, accessed on 31 October 2023).A p-value < 0.05 was considered statistically significant.
We compared the changes in bone resorption markers (CTx and TRACP5b) and bone formation markers (osteocalcin, tP1NP, and BAP) in treated patients relative to baseline (Figure 2).Both decreased by <50% 2 weeks after denosumab was initiated, and this was maintained for about 3 months; it then increased near the next dosing at 6 months.Among the bone formation markers, tP1NP and BAP gradually decreased during treatment compared to the more rapid changes in the bone resorption markers.Osteocalcin initially increased and then gradually decreased with the other markers.

Relationship between T50, Mineral Parameters, and Bone Turnover Markers after Denosumab Administration
The decreases in bone metabolism markers and calcium and phosphate levels were associated with an increase in T50.Subsequently, T50 tended to decrease as the next dosing cycle approached, and the bone metabolism markers and mineral parameters normalized toward baseline levels (Figure 2F,G).

Effect of Denosumab on Vascular Calcification
Vascular calcification was measured in the treated group via EBCT and plain X-ray, while only plain X-ray was conducted in the untreated group (Figure 5).No significant changes in vascular calcification were observed in either group during the observation period.

Effect of Denosumab on Quality of Life
HRQL scores were not significantly different between the groups at baseline (Figure 6).When assessed annually over the 2 years, neither group exhibited a significant difference in the KDCS, PCD, or MCS categories.

Discussion
The prevalence of osteoporosis in HD patients was 38.4% (71 of 185), with 16.2% receiving denosumab (7.2% of males and 26.1% of females).Denosumab boosted FN BMD after 12 months, which faded after 2 years.BTMs decreased after denosumab was administered; resorption markers initially dropped and then increased, and formation markers steadily decreased.T50 increased as BTMs decreased.Hypocalcemia (<8.4 g/dL) post-denosumab was prevented by 2 weeks of supplementation.PTH initially surged and improved despite vitamin D and calcimimetic administration.Vascular calcification scores and HRQL did not significantly change in either group.
ESKD patients often face bone disorders, leading to complications like fractures [16], attributable to irregular bone turnover and diminished BMD.While bone biopsy is the gold standard for turnover diagnosis, its invasiveness hampers practicality [1].Specific hormones like PTH and fibroblast growth factor-23 add complexity to bone marker regulation in ESKD [17], posing challenges in diagnosing and treating various CKD-MBD factors associated with BMD reduction.
Our study determined denosumab treatment targets based solely on BMD.However, additional assessment tools are necessary to evaluate fracture risk.A 10-year intervention threshold of 20% for MOF and 3% for HF is employed when selecting patients for osteoporosis treatment using the FRAX ® tool.We made this selection because FRAX ® exhibits superior performance in identifying patients unlikely to experience major osteoporotic or hip fractures within the next 10 years [18].However, this discriminatory method has only been applied to a subset of HD patients in studies with short observation periods [19].Therefore, further research is needed to assess its value as a tool for discerning indications for osteoporosis treatment and preventing fracture.
Osteoporosis has received less attention in ESKD patients due to the limitations of bisphosphonate use [20].The development of denosumab has led to increased efforts in diagnosing osteoporosis in ESKD patients [21,22], with previous reports indicating an associated increase in BMD [3,4,9,23].Our study demonstrated improved BMD in the FN area 1 year after denosumab was administered.However, the second-year results indicated a trend of non-sustainability.Long-term follow-up studies post denosumab administration have revealed that the initial improvements in hip region BMD gradually diminish, and discontinuing the drug over the following year leads to a decline in BMD [23].These findings suggest the need for prolonged administration to maintain improved BMD status.The absence of strategies to prevent the decrease in BMD after discontinuing denosumab underscores the need for further research in this area.
Previous research has consistently indicated a decrease in BTMs following denosumab administration [3,9,24].This decline persists until approximately 3 months postadministration, after which the BTMs stabilize as the next dosing at 6 months approaches [25].Given that denosumab inhibits bone resorption by targeting RANKL, our study reflected this, showing a swift reduction in bone resorption markers compared to bone formation markers in the early stages of denosumab treatment.This led to a secondary reduction in bone formation markers, which was repeated after each administration.T50, reflecting the transition time from primary to secondary CPPs in vitro [26], is a proposed serum marker indicating the propensity for calcification.A shorter T50 suggests a higher tendency for calcification, linked to increased cardiovascular disease (CVD) risk and all-cause mortality in CKD patients [11].A prior cross-sectional study indicated a weak correlation between T50 and BMD [12].The increase in T50 observed with denosumab treatment suggests prolonged maturation of CPP due to reduced bone resorption and lower calcium and phosphate levels.Early changes in bone metabolism post denosumab administration may affect the T50 level.Given the limited data on these effects on vascular health, further research is crucial for a comprehensive understanding of this area.
Denosumab often leads to hypocalcemia because of inhibited RANKL [2].Similar to previous research [3,8,9,25], we noted an early sharp drop in calcium levels after denosumab was initiated.Our treatment approach of providing calcium and cholecalciferol until the corrected calcium of 10 mg/dL is reached effectively regulated hypocalcemia.Despite efforts to mitigate hypocalcemia [3,17], the significant increase in PTH post-denosumab administration in our study suggests that mechanisms beyond hypocalcemia triggered this increase.As a mechanism to explain this, the possibility of an osteoanabolic effect of PTH due to a decrease in cortical porosity in the early stages of denosumab administration has been suggested [27,28], but the exact mechanism is not yet known.
CKD-MBD, coupled with osteoporosis and a high RANKL level, heightens bone loss, vascular inflammation, and calcification risk.Administering denosumab to CKD-MBD patients with osteoporosis lowered calcium and phosphate levels and inhibited vascular inflammation and calcification.In a small-scale study involving 21 HD patients, no improvement in CAC score was detected 6 months after denosumab administration when measured using EBCT [29].
The authors noted an inverse correlation between serum alkaline phosphatase level and coronary artery calcium (CAC) score, suggesting that the selective cessation of osteoclasts, along with intact osteoblast function and a high bone turnover rate, might create a reverse calcium paradox.This could lead to regressed ectopic calcification, stabilized vascular calcification, and increased bone mass [30,31].However, our 2-year study found no significant changes in CAC.In the non-denosumab group, there was a negative correlation between AAC and BMD_FN as well as BMD_TH.Surprisingly, in the denosumab group, no significant correlation was observed.The absence of a negative correlation in the denosumab treatment group may be attributed to their relatively lower bone mineral density.Therefore, this complexity implies mechanisms beyond straightforward changes in calcium content, underscoring the need for further investigation.
Enhancing HRQL and preventing fractures in HD patients with multiple comorbidities is vital when treating CKD-MBD, including osteoporosis.In a recent 24-month study with 332 non-dialysis osteoporotic patients, denosumab was associated with improved BMD and HRQL [32].While the BMD-HRQL link is promising, the lack of a control group and different patient populations necessitate further research to understand the factors involved in improving HRQL post-denosumab administration, particularly in patients undergoing HD.In our study, denosumab slightly improved FN BMD but did not significantly affect HRQL.The effects of comorbidities and other factors remain to be explored.
This first-of-its-kind Korean study examined denosumab's impact on BMD, mineral parameters, vascular calcification, and HRQL in HD patients, shedding light on osteoporo-sis treatment.However, this study has limitations, including a relatively small denosumab group, emphasis on measurable changes like BMD, and a potentially short 24-month study period to assess long-term denosumab safety.Additionally, there is a possibility of bias due to the selection of a control group that may not be well-matched to the denosumab treatment group.Furthermore, the lack of analysis of critical events such as new fractures or mortality after denosumab is a notable gap, underscoring the necessity for comprehensive exploration in future studies.

Conclusions
The initial effect of denosumab on BMD was less pronounced in HD patients.Denosumab affected BTMs by managing calcium and phosphate levels effectively, consistent with the CKD-MBD strategy.Notably, PTH levels initially increased.HD patients without denosumab maintained stable control over calcium, phosphate, and PTH levels for 24 months, but there were no significant improvements in vascular calcification or HRQL.Larger studies are necessary to appraise the changes in BMD and reduction in fracture risk associated with denosumab in osteoporotic HD patients.

Table 1 .
Baseline characteristics of the study group.

Table 2 .
Correlations between BMD and other variables.

Table 3 .
Correlations between BTMs and other variables in patients with denosumab administration.