Part 2 Chronic Kidney Disease And Aging Differentially Diminish Bone Material And Microarchitecture in C57Bl/6 Mice
Mar 15, 2022
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3. Results
3.1 Confirmation of kidney disease
At the study endpoint three months from surgery, mice with 5/6 Nx had significantly elevated serum urea and diminished urine urea, consistent with impaired kidney function (Table 1). Serum PTH and phosphate were significantly increased as the main effects of CKD. Serum calcium was not significantly affected by CKD. For all serum and urine measures, age was not a significant main effect.
3.2 Aging and CKD reduce cortical and trabecular microarchitecture
Aging and CKD both negatively affected cortical geometry and trabecular microarchitecture (Tables 2 – 3, Figure 1). Aging from 6 mo. to 24 mo. significantly increased cortical bone cross-sectional dimensions (increased polar moment of inertia, PMI), porosity (Ct. Po) and tissue mineral density (TMD), and decreased cortical bone thickness (Ct. Th), cortical bone volume (decreased bone volume fraction (BV/TV), bone area fraction (BA/TA). Aging also reduced trabecular number (Tb. N) and thickness (Tb. Th), trabecular bone volume fraction (BV/TV), and volumetric bone mineral density (vBMD). As with aging, CKD also had a significant negative main effect on bone volume via decreased BV/TV, Tb. N, BA/TA, Ct. Th, and increased Ct.Po. These effects were additive, such that the worst bone volume was observed for old mice with CKD. Unlike aging, CKD significantly decreased BMI and TMD. Aging and CKD did not have a significant interaction for any cortical or trabecular microarchitecture measure.
3.3 Aging and CKD differently diminish whole-bone mechanical and material properties
At the whole-bone level, material and mechanical properties were both diminished by aging and CKD (Table 4, Supplementary Table 1). Aging from 6 mo. to 24 mo. significantly reduced most whole-bone material properties (e.g., modulus, yield stress, maximum stress) but did not affect toughness. CKD significantly reduced work to fracture, but most other material and mechanical parameters were not affected by CKD as the main effect. Toughness, the geometry-independent analog of work to fracture, had a significant interaction between aging and CKD (p = 0.011). From post-hoc testing, it was seen at 18 mo that CKD may lower toughness (−47.9%, p = 0.023) (Figure 2d).
3.4 Aging and CKD reduce FEA structural stiffness at the proximal tibia
We utilized FEA to evaluate how aging and CKD affect mechanical properties via changes to the trabecular and cortical structure. FEA structural stiffness at the proximal tibia significantly decreased with aging and CKD (Table 4). There was not a significant interaction between aging and CKD. FEA stiffness positively correlated with femoral stiffness from three-point bending (r2 = 0.67).
3.5 Aging lowers microscale bone stiffness while CKD reduces microscale bone tissue spatial variation
To assess how aging and CKD affect tissue-scale material properties of cortical bone, we employed nanoindentation and Raman spectroscopy at matched sites to evaluate microscale bone tissue modulus and composition (Table 5). Bone tissue heterogeneity was evaluated through the assessment of spatial variance of these properties. Mean reduced modulus (Er) and the standard deviation ofErwere significantly decreased and increased, respectively, with age. CKD significantly reduced the standard deviation, but not the mean, ofEr. Microscale bone tissue composition assessed by Raman spectroscopy (i.e., mean mineral: matrix, carbonate: phosphate, crystallinity) did not significantly change with either age or CKD. However, the standard deviation of crystallinity was significantly reduced with CKD.
3.6 Aging and CKD differently affect collagen fibril nanomechanics
We evaluated whether CKD and aging affect collagen nanomechanics via small-angle x-ray scattering (SAXS) concurrent with tensile loading. In agreement with results from bending, tensile measurements demonstrated that aging significantly reduced tissue yield stress and max stress. Collagen nanomechanics was affected differently by age and CKD (Table 6, Figure 2). Aging reduced the collagen strain measured at maximum tissue strain but did not affect the percent collagen strain (collagen strain / total tissue strain, %ColMax). By contrast, CKD significantly increased the %ColMax.
3.7 Collagen crosslinks did not differ with aging and CKD
We sought to understand whether the altered collagen nanomechanics observed in CKD could be attributed to changes in skeletal crosslinks. We, therefore, characterized enzymatic (hydroxyl lysyl pyridinoline, HP, and lysyl pyridinoline, LP) and non-enzymatic cross-linking (pentosidine) with high-performance liquid chromatography (HPLC). The crosslinks HP, LP, and pentosidine had higher mean concentrations with increasing age, but these changes were not significant (Table 7). Similarly, there was a trend of lower LP with CKD, but this effect was not significant. We further quantified total fluorescent advanced glycation end-products (AGEs) and found that total fluorescent AGEs did not differ with aging or CKD.
3.8 Age-related osteocyte lacunar geometry changes are not observed in aging mice with CKD
Osteocyte perilacunar/canalicular remodeling (PLR) responds to changes in mineral homeostasis and is important in determining bone fracture resistance. We evaluated osteocyte lacunar geometry to determine whether aging with CKD alters PLR (Table 8). The effects of aging alone (without CKD) for these Sham mice were reported previously (21). Briefly, for Sham mice, lacunae became smaller, more spherical, and less numerous with increased age. When aging and CKD were analyzed together for the present study, aging significantly decreased lacunar number density, volume, and surface area, and increased sphericity, oblateness, and span theta. There was not a significant effect of CKD for any measure. However, there was a significant interaction between age and CKD for sphericity. While sphericity increased with age for Sham mice, this measure was greatest for middle-aged mice with CKD and then decreased for old mice to a value similar to that observed in young mice. In other words, lacunae became smaller and rounder only in aging Sham mice, and not in aging mice with CKD. This pattern was also observed with CKD for lacunar number density, volume, and surface area, although the interaction between aging and CKD was not significant.
Osteocyte lacuna size did not increase with age for mice with CKD but instead increased with phosphate dysregulation. Specifically, for mice with CKD, osteocyte lacunar volume (Spearman ρ = + 0.584, p = 0.017), surface area (ρ = + 0.603 p = 0.013), and sphericity (ρ = - 0.500, p = 0.048) were correlated with serum phosphate assessed at the study endpoint. By contrast, measures related to lacunae prevalence and orientation (i.e., osteocyte number density, the closest center of mass, span theta) did not significantly correlate with circulating phosphate. No osteocyte lacunar measurements were significantly correlated with serum calcium, serum urea, or urine urea. For Sham mice, osteocyte lacunar number density was positively correlated with serum calcium (ρ = +0.681, p = 0.004). All other osteocyte lacunar measurements only weakly correlated with serum and urine chemistry.
The relationship between osteocyte lacuna size and serum phosphate for mice with CKD was not directly caused by increased PTH. When considering Sham and CKD mice together, phosphate significantly correlated with increased PTH (Spearman ρ = +0.562, p = 0.001). However, although phosphate and PTH were both increased as a main effect of CKD, these measures were not significantly correlated with each other for CKD mice (p > 0.05). No measures of osteocyte lacunar geometry significantly correlated with PTH for Sham or CKD mice.
3.9 Regression analysis reveal that altered collagen nanomechanics influence toughness
We sought to identify how changes to whole-bone toughness from bending were related to changes in nano-to microscale measures of bone quality for Sham and CKD mice. We performed a regression of whole-bone toughness as bone quality measures at smaller length- scales for Sham and CKD mice using age as a covariate. For Sham mice, no bone quality measures were significant explanatory variables for toughness. For CKD, the only significant predictor of toughness (T) was %ColMax (ln(T) = 2.437 – 0.00371*%ColMax; r2 = 49.4%, p < 0.05). The strength of the regression was not improved by adjusting for age.
3.10 Bone quality changes are not explained by variation in PTH
We assessed Spearman correlations between bone quality measures and PTH to understand if mice with higher PTH had worse bone quality. Though PTH was significantly higher with 5/6 Nx, there were no significant correlations between PTH and microscale bone quality.
4. Discussion
The purpose of this study was to investigate how 5/6 Nx – induced CKD and aging each diminishes bone quality, including bone microarchitecture as well as bone tissue material properties. CKD reduced bone quality, including microarchitecture and material, from the micrometer-length scale to the whole-bone scale. In our study, bone quality declines were observed across several modalities and length scales. The effect of CKD was to diminish cortical and trabecular microarchitecture, alter spatial variation in microscale material properties, and also alter collagen nanomechanics. Additionally, CKD and aging had an
interactive effect on a toughness that was greatest for middle-aged mice. Our results highlight that several key components of bone quality are impaired in CKD. These observations support a growing body of evidence from rodent and clinical studies that bone quality is reduced in CKD and likely contributes to loss of fracture resistance.
The loss of bone microarchitecture with CKD occurred in addition to the deleterious effects of aging and contributed to the loss of whole-bone strength. In this study, we found that aging and CKD both significantly diminished cortical and trabecular microarchitecture. Aging and CKD each resulted in thinner, more porous cortical bone, and fewer and thinner trabeculae. The only exception was bone mineral assessed by microCT (i.e., TMD), which increased with age and was diminished in CKD. For all cortical and trabecular metrics, the worst bone microarchitecture was apparent in old mice with CKD. The loss of bone microarchitecture influenced whole-bone structural integrity. From FEA, it was found that aging reduced bone microarchitecture at the proximal tibia while CKD reduced structural stiffness.
For measures relating to bone material quality, age and CKD had differing effects (Figure 2). With increased age, whole-bone modulus and maximum stress were both reduced, as was micrometer-scale tissue modulus. Yield and maximum stress from tensile testing of the ulna- radius also decreased with age. Further, consistent with human samples studied with SAXS, aging was associated with increased collagen strain at maximum strain, implying a stiffening of collagen fibers (26). CKD did not affect whole-bone modulus or strength, but instead significantly reduced work to fracture, and altered tissue-scale material spatial variation. Further, CKD may reduce toughness in middle-age mice, as with this disease the burden of tensile strain was shifted to collagen fibers. Increased percent collagen at maximum load was the only significant explanatory variable for reduced toughness in CKD, suggesting that the loss of whole-bone toughness may be at least partially explained by altered collagen mechanics.
The origin of altered collagen nanoscale mechanical behavior is uncertain. There was a trend of lower skeletal crosslinks with CKD. However, HP and LP measured from the humerus did not correlate with collagen mechanical properties from SAXS measured at the ulna/radius complex, perhaps due to different sites of analysis. This shift in strain may also result from altered mineral content or distribution, or changes to binding between collagen and mineral. Increased %ColMax may not be directly responsible for the loss of toughness in CKD but could be a symptom related to altered turnover and deficiencies in the mineral organization. For instance, altered mineralization could render the collagen more mobile, or may dissipate less energy through microcracking. These changes could produce the observed loss of toughness with CKD as well as the increased strain burden on collagen fibers. In future studies, it will be important to identify why CKD shifts strain to collagen fibers, but here we note that this change in collagen behavior appears to play an important role in how CKD diminishes whole-bone material properties.
As changes to bone material properties occur with CKD and the osteocyte is known to modulate tissue-scale bone quality through perilacunar/canalicular remodeling (PLR), we also aimed to understand whether 3D osteocyte lacunar morphologies were modified in CKD (15, 16). While osteocyte lacunae became smaller, sparser, and less aligned with increased age for Sham mice, lacunae remained prevalent and large for old mice with CKD.
Instead, for mice with CKD, serum phosphate was significantly and positively correlated with osteocyte lacuna size. Although both phosphate and PTH were significantly elevated
with CKD, mice with higher PTH did not have higher phosphate. These results are not necessarily discordant. Serum phosphate is the net result of phosphate added from intestinal absorption and bone resorption less phosphate removed by renal excretion and bone formation. Thus, although our CKD mice had generally elevated PTH, those with higher phosphate may have exhausted other systemic compensatory mechanisms.
Osteocytes express greater FGF23 in the context of impaired mineralization (e.g., DMP1 knockout, x-linked hypophosphatemia) (20,48,49) as well as in chronic kidney disease (14,48,50). Recent evidence suggests that in addition to this endocrine role, the osteocyte can directly participate in phosphate mineral homeostasis viaPLR (20). Osteocyte lacunae volumes were found to increase with x-linked hypophosphatemia. Treatment with either FGF23 or 1,25 dihydroxyvitamin D blocking antibodies restored osteocyte lacunar volumes to values closer to wild-type comparisons (20). In CKD, mineral dysregulation is initially spurred by the decreased phosphate filtration of the diseased kidney. Increased bone turnover is a maladaptive response that indicates that systemic compensatory mechanisms for maintaining phosphate control are overwhelmed (6,7). In this case, the relationship between heightened phosphate and increased osteocyte lacunar volume suggests that PLR could be an additional mineral homeostasis regulation strategy. Additional study is required to investigate how osteocyte lacunar geometries and PLR are related to altered bone turnover, bone tissue material quality, and fracture resistance in CKD.
Our findings suggest that processes affecting bone material are different in aging versus CKD. In aging humans as well as mice, bone turnover (i.e., osteoclastic resorption and osteoblastic formation) slows, and tissue maturity increases. In this study, 5/6 Nx produced distinct bone quality defects from aging, such as lower bone density from microCT, altered variability of tissue-scale material properties, and greater participation of collagen in tensile loading. Though PTH was significantly higher with CKD, mice with higher PTH did not have lower bone quality. Several reasons may explain this finding. First, mice in our study had much lower PTH than in studies with severe CKD and concomitant secondary hyperparathyroidism (9, 12). It is possible that changes in bone turnover were relatively modest in our study. However, we did not directly assess bone turnover, so this possibility is speculative. Second, CKD, regardless of turnover status, has been experimentally associated with altered bone quality. Iwasaki and coworkers reported that high-turnover CKD from 5/6 Nx as well as low-turnover CKD from 5/6 Nx and thyroparathyroidectomy both resulted in similarly altered bone mechanics, and attributed these changes to uremia (11). It is possible that bone quality loss is not predicated solely on secondary hyperphosphatemia but is also influenced by more modest changes in bone turnover and accumulation of uremic toxins.
We expected that several bone quality metrics, including whole bone toughness, pentosidine, mineral: matrix, and carbonate: phosphate would change from 6 to 24 months (25,26,30,51,52). While each of these metrics changed in the expected direction with increased age, the effect of age was not statistically significant. Post-hoc power analysis using G*Power (53) suggests that toughness, pentosidine, and mineral: matrix were underpowered to detect the effect of aging, while carbonate: phosphate was likely not affected by age in this study. In addition, the collagen crosslink LP was underpowered for the detection of an effect of CKD (Supplemental Table 2). There are several possibilities why some measures of bone quality were ultimately underpowered in this study. First, we utilized a custom Raman-nanoindentation instrument with the unique advantage of co-localized measurements. Signal intensity is lower on this custom instrument than standalone Raman instruments, and thus it is possible that our signal: noise ratio was not sufficient to discern age-related changes. Toughness (calculated from work to fracture) is a measure prone to high variability (54), and variability in mechanical properties is increased further for geriatric mice (25). Greater variability in pentosidine has also been observed with increased age in human and rodent studies (52,55,56). Thus it is likely that high variance in these measures obscured differences between evaluated groups.
Several limitations influenced the outcomes of this study. Dynamic quantitative histomorphometry was not performed, but future studies would enable the evaluation of osteoblast, osteoclast, and osteocyte activities as well as bone turnover. While we identified several bone quality impairments that emerge with CKD and deleteriously affect whole-bone toughness, further work is needed to confirm the origins of these impairments, as well as the role of the osteocyte in their occurrence. Analysis of gene and protein expression would be useful in identifying the origin of impaired collagen nanomechanics, as this impairment was not influenced by changes in collagen crosslinks. Additionally, our interpretation of how the osteocyte may participate in regulating mineral homeostasis and maintaining bone quality in CKD requires additional global (i.e., gene expression) and local measurements (i.e., histological assessment of bone resorption and formation).
In summary, we found that CKD lowers bone quality in addition to the effects of aging alone. CKD and aging similarly reduce cortical and trabecular bone microarchitecture such that the most profound loss of microarchitecture was observed in old mice with kidney disease. CKD also diminished bone material properties, resulting in bone with lowered apparent tissue maturity with altered collagen nanomechanics. These reductions in bone quality, including microarchitecture and bone material properties, provide insight into how fracture resistance is lost in CKD. Further, the combined effects of aging and CKD on bone quality help explain why older individuals with CKD have the highest risk of fracture.
Supplementary Material
Refer to the Web version on PubMed Central for supplementary material.
Acknowledgments:
We would like to thank William Schroeder for their excellent care of study animals, and William Schroeder and Ryan Clark for serum chemistry analyses. Support was provided for CMH through NIHT32 AG000279. Funding for this work was also provided to KBK and VLF by NIH/NCATS Colorado CTSA Grant Number UL1 TR001082, Co-Pilot Team Science Award. Funding to TA was provided by R01 DE019284. Combined analysis using nanoindentation and Raman spectroscopy was made possible through the use of a custom instrument funded by NSF Major Research Instrumentation award #1338154. X-ray scattering at Beamline 7.3.3 of the Advanced Light Source was supported by the U.S. Department of Energy Office basic Energy Sciences under Contract No. DE-AC02– 05CH11231. We also thank the A.Z. Weber group at LBL for providing their tensile stage, which was supported by DOE EERE Fuel-Cell Performance and Durability Consortium (FC-PAD).
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