The primary finding of this study is that two weeks of continuous opioid exposure in mice with adenine-induced CKD leads to an exacerbated inflammatory state in osteocytes and increased osteoclasts. Without altering indices of kidney function or circulating PTH levels, two weeks of continuous oxycodone treatment resulted in a greater percentage of osteocytes positive for both the mu opioid receptor and TLR4. Both adenine-induced CKD and oxycodone treatment led to greater levels of osteocytes positive for RANKL resulting in opioid-treated CKD mice having the highest values of all. Overall, two weeks of short-term exposure to oxycodone led to elevated osteoclasts which, when combined with the pro-osteoclast setting of CKD could eventually lead to detrimental effects on bone.

As opioids have been widely used and misused over many years, epidemiological studies demonstrate connections to reduced bone mass[28,29,30,31,32,33] and elevated fracture risk with long-term use [34]. For example, individuals with an opioid use disorder are 4.13 × more likely to fracture than those without [35]. Meta-analyses have reported ~ 1.88 relative risk of any fracture in patients with chronic opioid use for non-cancer pain [15] and an approximate 1.54 relative risk of specifically hip fractures with opioid use [14]. Additionally, studies have shown increased fracture risk in older adults with opioid treatment [18, 36, 37]. Importantly, the association between opioid use and fractures is stronger than opioid use and falls/fall injuries [18] indicating that the increased fracture rate in older adults is not solely due to an increase in falls. In CKD where fracture rates are already higher than an age-matched population [2, 3, 38, 39], the compounding influence of opioids on bone mass and bone fragility require exploration. Two studies have shown increased fracture rates in CKD patients with both short- and long-term use of opioids [5] as well as an opioid dose-dependent increase in fractures [6]. Therefore, epidemiological studies demonstrate increased bone loss and elevated fracture risk with long-duration opioid use.

In the clinical setting, oxycodone is considered a moderately safe opioid to use for chronic analgesia in CKD when dosed properly [24]; therefore, in our current study, we aimed to assess oxycodone treatment in mice with established adenine-induced CKD. All CKD mice in this study had elevated serum BUN indicating the presence of kidney disease as well as high PTH which matches our previous work with this model of CKD [25]. Two weeks of oxycodone treatment had no effect on BUN nor PTH in our current study. Additionally, the oxycodone treatment also had no impact on body weight, food intake, or overall animal appearance/behavior.

CKD with secondary hyperparathyroidism is associated with high bone turnover in both human [40] and animal models [25] and in the current study, both P1NP and TRAcP 5b, serum markers of bone turnover, were elevated in all adenine mice. In vitro studies have demonstrated an inhibitory effect of endogenous opioids (met-enkephalin) on osteoblasts and this inhibitory effect is abolished with the addition of the naltrexone, a competitive antagonist of opioid receptors [21]. Similarly, mice lacking dynorphin, an endogenous opioid that binds to the kappa opioid receptor, had higher bone mass and bone turnover than wild-type counterparts [41]. Therefore, opioids may have a direct effect on bone cells, particularly an inhibitory effect on osteoblasts. We were unable to directly measure bone formation rate with dynamic histomorphometry in the current study due in part to the shorter time of treatment. Our systemic marker of formation was not altered due to oxycodone treatment, but our 2-week period of exposure could have been too short to influence systemic markers of formation. For example, sustained morphine delivery for 25 days in healthy male mice reduced bone formation rate [19], while our study only had 14 days of treatment. In the current study, we measured trabecular osteoid and found that both osteoid-covered trabecular surfaces and osteoid width were elevated in our CKD mice. While there was not a statistical difference in osteoid-covered trabecular surfaces, likely due to the high variability in the adenine group, the oxycodone-treated CKD group had ~ 40% lower osteoid-covered trabecular surfaces than the saline-treated adenine group which may indicate a mild suppression of bone forming in these mice. Future work should include studies with fluorochrome labels to be able to directly assess bone formation rate via dynamic histomorphometry.

After the 2-week period of treatment, the oxycodone-treated mice in our study showed different mu opioid receptors presence in the osteocytes. Previous work has established that cultured osteoblast-like cells express opioid receptors [20]. To our knowledge, no other studies have shown mu opioid receptor expression in mouse osteoblasts or osteocytes; however, the antibody utilized is well-reported with specificity to the receptor. In our current study, we found osteocytes, terminally differentiated cells from the osteoblast lineage, positively stained for the mu opioid receptor and the percentage of osteocytes that stained positively was ~ 30% higher in the oxycodone-treated mice. Therefore, from our immunohistochemical analysis, it appears that continuous opioid treatment increases mu receptor protein expression. Interestingly, there were also main effects of adenine on the mu receptor in both trabecular and cortical bone indicating the disease process itself may alter endogenous opioid activity or responses. Further work needs to be done to validate the presence and role of the mu opioid receptor in bone cells in various species.

Beyond direct effects of opioids acting through opioid receptors, evidence points to indirect effects of opioids leading to increased inflammation. CKD alone is characterized by a chronic pro-inflammatory state and inflammatory factors in CKD are associated with overall mortality [42, 43] and TLR4 signaling is induced by the uremic environment [44]. Apart from CKD, opioid receptor agonists also activate the TLR4 pathway by binding to an accessory protein on TLR4 [22]. This, in turn, could activate pro-inflammatory signaling, upregulate NF-κB expression, and increase overall pro-inflammatory cytokines [23]. In the condition of CKD, this increase in inflammatory drive could exacerbate CKD-induced inflammation. In our study, osteocytes positive for TLR4 were not different due to only CKD; however, TLR4-positive osteocytes were nearly twofold higher in oxycodone-treated mice regardless of CKD status. Importantly, circulating PTH was not associated with TLR4-positive osteocytes (R2 = 0.004) indicating the alterations were likely primarily driven by opioid exposure. The elevated TLR4 may have a compounding effect on TNF-α-positive osteocytes in CKD mice as levels were 44% higher in oxycodone-treated adenine mice vs. untreated adenine mice, while oxycodone treatment only caused an 11% difference between the non-adenine groups. Additionally, protein expression of RANKL, a downstream target of the NF-κB signaling pathway activated by TLR4, was higher due to both CKD and oxycodone treatment resulting in nearly ~ 2.7-fold higher osteocyte RANKL in adenine–oxycodone-treated mice vs. control saline-treated mice. Regression analyses showed that both TLR4-positive osteocytes and TNF-positive osteocytes were significant predictors of the variability in RANKL-positive osteocytes; however, TLR4 alone was not a predictor of osteoclast-covered surfaces. Therefore, in our model of continuous oxycodone treatment, oxycodone alone increased TLR4, but the combination with adenine-induced CKD led to higher TNF-α and RANKL indicating a greater pro-inflammatory state than CKD alone.

A key hallmark of the bone phenotype of CKD with high PTH is rampant osteoclastic resorption [25, 26] which leads to bone loss and cortical bone resorption in the form of cortical porosity. After only two weeks of oxycodone treatment in mice with established CKD, there were not notable differences in bone structural parameters beyond expected changes due to CKD except for trabecular bone volume a 14% lower in AD + OXY than untreated adenine, but only statistically lower than both CON groups. Cortical bone area was 8% lower in oxycodone-treated adenine mice compared to untreated adenine mice, but this did not approach statistical difference. These changes allude to potential resorption-driven bone loss with oxycodone treatment which could become more apparent with longer duration of treatment. Oxycodone treatment did result in 40–70% higher osteoclast-covered trabecular surfaces compared to matched controls. Due to both CKD-induced increases in osteoclasts and oxycodone-induced increases in osteoclasts, adenine mice treated with oxycodone had 4.5-fold  higher osteoclast-covered surfaces than the saline control group. Exacerbating the already high osteoclastic drive of CKD with chronic opioids could lead to even greater bone loss over time than CKD alone.

A limitation of the current study is that we only assessed male mice. We previously have shown similar phenotypes in male and female C57Bl/6 mice with adenine-induced CKD [25], but the impact of opioids in the context of CKD may vary between sexes. In humans, the impact of long-term opioid exposure on bone appears to be stronger in males vs. females [30, 33, 45]. Additionally, 25 days of sustained morphine administration in mice impacted the bone of male mice, but not female mice [19]. Therefore, future studies should assess opioids in the context of the CKD-induced bone phenotype in both males and females. As previously mentioned, we were unable to directly measure bone formation rate via dynamic histomorphometry and, therefore, cannot directly assess tissue-specific changes in bone formation rate. These measures would add valuable information about bone-specific changes in future studies. Furthermore, our study assessed only a 2-week chronic exposure to oxycodone and longer durations will need to be assessed in future studies which would also allow for better assessment of tissue-specific changes in bone formation rate.

Overall, this study demonstrates that two weeks of continuous oxycodone treatment in mice with biochemical and skeletal manifestations consistent with CKD resulted in an elevated pro-inflammatory state within bone and increased osteoclasts. With prolonged treatment, these changes could lead to significantly greater bone loss and skeletal fragility than CKD alone. These data allude to the importance of understanding the mechanisms of opioid-induced bone changes as well as how those changes interact with different disease states like CKD. Additionally, these data highlight the need for the development of safer, alternative analgesic options for CKD patients with high pain burden.