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. Author manuscript; available in PMC: 2022 Jul 1.
Published in final edited form as: Bone. 2021 Apr 17;148:115963. doi: 10.1016/j.bone.2021.115963

Strain-specific alterations in the skeletal response to adenine-induced chronic kidney disease are associated with differences in parathyroid hormone levels.

Corinne E Metzger 1, Elizabeth A Swallow 1, Alexander J Stacy 1, Matthew R Allen 1,2,3,4
PMCID: PMC8102422  NIHMSID: NIHMS1695097  PMID: 33878503

Abstract

Chronic kidney disease (CKD) leads to loss of cortical bone through cortical thinning and the development of cortical porosity. The goal of this current study was to assess cortical bone alterations to adenine-induced chronic kidney disease (CKD) in two strains of mice with known genetic differences in cortical thickness. We hypothesized that C3H mice with thicker cortices and baseline levels of intracortical remodeling would have greater cortical porosity in response to adenine-induced CKD compared to B6 animals.

Methods:

Female C57BL/6J (B6) and C3H/Hej (C3H) at 16-weeks of age were given a diet with 0.2% adenine to induce CKD for 6 weeks followed by a control diet for 4 weeks. Age- and strain-matched controls were fed the control diet without adenine for the 10-week period (n=8 per group per strain).

Results:

Both strains of adenine-fed mice had elevated blood urea nitrogen, demonstrating compromised kidney function, compared to strain-matched controls, but only B6 adenine mice had statistically higher parathyroid hormone (PTH), greater cortical porosity, high bone turnover rate, a greater percentage of osteocytes positive for RANKL and IL-17, and lower osteocyte apoptosis compared to B6 controls. C3H mice had intracortical remodeling present in both control and adenine mice, while B6 mice had intracortical remodeling present only in adenine mice. Adenine mice of both strains had lower cortical thickness and a higher percentage of osteocytes positive for TNF-α compared to controls.

Conclusion:

While both strains of mice had biochemical markers of kidney disease, only B6 mice developed a phenotype with significantly elevated PTH, high bone turnover, and cortical porosity development. This work, in a model of progressive CKD, further confirms the role of chronically elevated PTH in the development of cortical porosity and demonstrates adenine-induced increases in PTH contribute to intracortical remodeling in B6 mice. Adenine-induced changes that occurred in both strains of mice, notably lower cortical thickness and a higher percentage of osteocytes expressing TNF-α, indicate potential PTH-independent responses to CKD.

Keywords: cortical porosity, chronic kidney disease, parathyroid hormone

1. Introduction

Chronic kidney disease (CKD) is a complex disorder that leads to bone loss and increased fracture incidence [1] [2] [3] [4]. With approximately 15% of the adult population within the United States estimated to have CKD [5], understanding the impact of CKD on the skeleton is critical to developing approaches to reduce the disease burden. CKD results in abnormal mineral metabolism which can lead to secondary hyperparathyroidism. In both clinical and animal studies, high circulating parathyroid hormone (PTH), high bone turnover, and cortical porosity have been noted [6] [7] [8] [9] [10]. Although hyperparathyroidism is clearly a key player in the development of cortical porosity in CKD, other factors likely contribute to the preferential formation of cortical pores rather than loss of trabecular bone and endocortical resorption.

One concern of using rodent models is a lack of intracortical remodeling making cortical bone changes potentially not as comparable to humans. However, despite these differences, the formation and presence of cortical pores are seen in both humans and rodents in various conditions including CKD [6] [9] [10] [11] and aging [12] [13] [14] [15]. Whether basal levels of intracortical remodeling impacts cortical porosity formation remains largely unknown. Pre-clinical data have pointed to cortical thickness as a possible factor influencing intracortical remodeling, and, therefore, potentially cortical porosity. Studies using genetic mouse models have shown that C3H/Hej mice, with genetically thicker cortices, have baseline intracortical resorption spaces and undergo intracortical remodeling following OVX, while mice with thinner cortices (such as B6 and A/J) do not [16]. In our previous work with rodent models of CKD, we established that cortical pores are not static once formed and have the capacity to infill when PTH is suppressed (pore infilling paper); however, how this is related to the initiation of remodeling within the cortex is unknown. To further understand the dynamics of adenine-induced intracortical changes, we aimed to assess C3H mice with known differences in cortical thickness and remodeling compared to B6 mice.

PTH increases osteoclastic drive by increasing the expression of receptor activator of nuclear factor κB ligand (RANKL). Previously, we documented that RANKL was high in cortical osteocytes in mice with adenine-induced CKD [9]. We hypothesized these osteocytes releasing RANKL could drive osteoclasts to the cortex, stimulate intracortical resorption, and initiate the development of cortical porosity. Yet multiple other factors besides PTH, including pro-inflammatory cytokines, stimulate increased RANKL. In addition to disrupted mineral metabolism, CKD is considered a chronic pro-inflammatory state. C-reactive protein, a non-specific marker of inflammation, is elevated in patients with CKD [17] [18] [19] as are specific pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α) [20] [21] and interleukin-17 (IL-17) [22]. If/how inflammatory factors contribute to the development of skeletal phenotypes in CKD, specifically cortical porosity and cortical thinning, remains unknown.

In this current project, we aimed to compare cortical bone parameters in adenine-induced CKD in two strains – C57Bl/B6J mice and C3H/Hej mice with contrasting cortical thicknesses. We hypothesized that C3H/Hej animals, which have thicker cortical bone, would develop more cortical porosity with greater intracortical remodeling in response to adenine-induced CKD than C57Bl/B6 mice.

2. Methods

2.1. Animals:

Female C57Bl/6J mice (B6; JAX #000664; n=16) and female C3H/HeJ (C3H; JAX #000659; n=16) were ordered from Jackson Laboratories (Bar Harbor, ME, USA) at 15 weeks of age and group housed four per cage at an institutionally approved animal facility. Female mice were chosen as our previous work demonstrated increased PTH and cortical porosity over time in young (8 week old) female C57Bl/6J mice [9] and high PTH and cortical porosity after 10 weeks of adenine-induced CKD in skeletally mature C57Bl/6J female mice [10]. At 16 weeks of age, all mice were switched to a purified casein-based diet with adjusted calcium and phosphorous (0.9% phosphorous, 0.6% calcium) and half of the mice in each strain (n=8) were given the same casein-based diet with the addition of 0.2% adenine (Envigo Teklad Diets, Madison, WI, USA). After a 6-week induction on the adenine diet, all adenine mice were switched back to the control casein-based diet for four weeks as previously described [9] [10]. Our work in female B6 mice [9] [10] and other studies in rats with adenine-induced CKD have demonstrated an induction period of adenine followed by resumption of control diet is sufficient to maintain kidney disease with progressive increases in PTH [23] [24] [25] [26]. Furthermore, our previous work indicates mice regain some body weight and are observationally more active after removal of the adenine diet while maintaining high PTH and development of cortical bone phenotypes (y-adenine, Ca-H2O). Mice were monitored daily and body weights and food intake (averaged per cage) were measured weekly throughout the protocol. All mice were injected with fluorochrome calcein labels 7 and 2 days prior to euthanasia. After 10 weeks on diets, animals were anesthetized via vaporized inhaled isoflurane and euthanized via exsanguination. Kidney, right femurs, and right humeri were fixed in 10% neutral buffered formalin for 48 hours and then stored in 70% ethanol. All animal procedures were approved by the Indiana University School of Medicine Animal Use and Care Committee prior to the initiation of experimental protocols and methods were carried out in accordance with relevant guidelines and regulations.

2.2. Serum biochemistries:

Blood collected at time of euthanasia was used to measure serum blood urea nitrogen (BUN) via colorimetric assay to assess the presence of kidney disease (BioAssay Systems, Hayward, CA, USA). Serum calcium and phosphorous were assessed via colorimetric assays (Pointe Scientific, Canton, MI, USA). Serum 1–84 parathyroid hormone was measured via ELISA (Immnotopics Quidel, San Diego, CA, USA) which has an intra-assay coefficient of variation of 2.4–5.6%.

2.3. Ex vivo Micro-Computed Tomography of the Femur:

Right femurs were scanned on a SkyScan 1172 system (Bruker, Billerica, MA, USA) with a 0.5 aluminum filter and a 6 µm voxel size. Trabecular bone was analyzed in a 1 mm region starting proximal to the growth plate in the distal femur. Cortical bone properties (cortical bone area, cortical thickness) were analyzed on 5 contiguous slices located ~2.5 mm proximal to growth plate in the distal femur. Cortical porosity was determined by assessing void area between the periosteal and endosteal surfaces, presented as a % of overall cortical volume.

2.4. Ex vivo Micro-Computed Tomography of the Kidney:

Fixed adenine kidneys were scanned on a SkyScan 1176 system (Bruker, Billerica, MA, USA) with no filter and a 9 µm voxel size. A 0.5 mm volume of interest was selected in the middle of the kidney (transaxial plane obtained from µCT) for assessment of crystal volume/total kidney volume as previously described [9]. Control kidneys do not have anything detectable via µCT and, therefore, were not scanned.

2.5. Histomorphometry:

Undemineralized right distal femurs were fixed in neutral buffered formalin then subjected to serial dehydration and embedded in methyl methacrylate (Sigma Aldrich, St. Louis, MO). Serial frontal sections were cut 4 μm-thick and left unstained for analysis of fluorochrome calcein labels. Histomorphometric analyses were performed using BIOQUANT (BIOQUANT Image Analysis, Nashville, TN). All samples were analyzed by the same individual. All nomenclature for histomorphometry follows standard usage [27].

Intracortical parameters were measured within standardized regions that utilized both cortices at approximately the midshaft (approximately 600 mm2 of tissue analyzed). For dynamic data, the number of labeled pores, total labeled surface within pores, and interlabel distance were obtained. Calculations included pore number/bone area, mineral apposition rate (interlabel distance/5) and bone formation rate [100*MAR*(label length/2)/bone area]. For static intracortical assessment, TRAP-covered pores surfaces were measured and normalized to total pore surfaces within the region of interest (Ocs/Pore surface).

For trabecular measures, a standard region of interest of trabecular bone approximately 0.02 mm proximal to the primary spongiosa and excluding endocortical surfaces was utilized (approximately 1400 mm2). Total bone surface (BS), single-labeled surface (sLS), double-labeled surface (dLS), and interlabel distances were measured at 20x magnification. Mineralized surface to bone surface (MS/BS; [dLS+(sLS/2)]/BS*100), mineral apposition rate (MAR; average interlabel distance/5 days), and bone formation rate (BFR/BS; [MS/BS*MAR]*3.65) were calculated. A second 4 μm-thick section was stained with tartrate resistant alkaline phosphatase (TRAP) for assessment of osteoclasts. TRAP sections were analyzed as osteoclast-covered trabecular surfaces normalized to total trabecular bone surface (Oc.S/BS, %).

2.6. Immunohistochemistry:

Fixed right humeri were decalcified in 14% EDTA (~14 days) and embedded in paraffin. Serial 5 μm sections were taken. Sections were stained utilizing a standard avidin-biotin method as previously described [9]. Samples were stained for receptor activator of nuclear factor κB ligand (RANKL; Abcam, Cambridge, MA, USA), annexin V (Abcam), an early marker of cellular apoptosis, and two proinflammatory cytokines, tumor necrosis factor-α (TNF-α; Abcam) and interleukin-17 (IL-17; Abcam). Peroxidase development was performed with an enzyme substrate kit (DAB, Vector Laboratories, Burlingame, CA, USA). Counterstaining was conducted with methyl green (Vector Laboratories) which stain osteocyte nuclei. Negative controls for all antibodies were completed by omitting the primary antibody. Sections of the entire humeral midshaft cortical bone in cross-section were analyzed and results are reported as the percentage of osteocytes stained positively for the protein (DAB-positive) relative to all osteocytes (DAB-positive and methyl green-positive) in the cross-section. All analyses were completed by the same individual.

2.7. Statistical Analyses

All data were tested for normality and then analyzed using a 2×2 factorial ANOVA (strain-by-adenine). If the model 2×2 ANOVA p<0.05, main effects for strain, adenine, and strain-by-adenine interaction were recorded. Additionally, a post hoc Duncan analysis was conducted to determine which groups were different when the model p<0.05. Statistical analyses were completed on SPSS Statistics 26 (IBM; Armonk, NY, USA). All data are represented as mean ± standard deviation.

3. Results

3.1. Food intake and body mass

Both B6 and C3H adenine mice lost approximately equivalent amounts of weight while on the adenine diet, 22–25% lower than strain-matched controls. Both strains also had reduced food intake on the adenine diet with B6 mice eating approximately 1.5 g/day and C3H mice approximately 1.2 g/day while both sets of control mice ate approximately 2.3 g/day. Once mice returned to the control diet, C3H adenine mice more quickly regained body weight with no difference between C3H adenine and C3H control mice by week 8 (two weeks after resumption of control diet) and less than a 2% difference in body weight at study end point. B6 adenine mice remained statistically lower in body weight through week 9 and weighed ~8% less than B6 controls at 10 weeks. At study endpoint, there was a main effect of strain on body weight (p=0.004), but no effect of adenine (p=0.169) or strain-by-adenine interaction (p=0.476) with both groups of C3H mice weighing more than the adenine B6 mice with control B6 mice not different from any group.

3.2. Indices of kidney disease and serum measures

For serum BUN, there was a main effect of strain and adenine (p<0.0001 for both) and a strain-by-adenine interaction (p=0.028). The adenine group in both strains had higher BUN than the strain-matched control. Additionally, B6 mice had higher overall BUN than C3H mice (Figure 1A). For serum phosphorus, there was an effect of strain (p<0.0001), but no effect of adenine (p=0.115) or an interaction effect (p=0.183). Adenine C3H mice had lower serum phosphorus compared to control C3H mice with no difference between control and adenine in B6 mice (Figure 1B). Serum calcium also only had an effect of strain (p<0.0001) with no impact of adenine or interaction (p=0.315, p=0.895, respectively) with C3H mice having higher values than B6 (Figure 1C). There was no strain effect for serum PTH (p=0.543) nor an interaction effect (p=0.176), but there was an effect of adenine (p<0.0001). PTH was higher in B6 adenine mice vs. B6 control mice, but there was not a statistical difference between control and adenine in C3H mice (Figure 1D). Adenine mice in both strains had kidney crystals detectable by µCT – B6 had 0.27% while C3H had 1.54% (Figure 1E, Figure 1F).

Figure 1: Serum measures and indices of kidney disease.

Figure 1:

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A) Serum BUN was higher in both strains of adenine mice vs. strain-matched controls. B6 mice had higher BUN than C3H. B) Serum phosphorus was lower in C3H adenine mice vs. C3H controls, but not different between B6 control and adenine groups. C) Serum calcium was higher in C3H mice, but not different between control and adenine in either strain. D) Serum PTH was higher in B6 adenine mice vs. B6 controls. C3H mice did not have statistically higher PTH in adenine vs. control. E) Kidney crystals measured via micro-CT in B6 adenine and C3H adenine mice. F) Representative images of kidney crystals in B6 and C3H. Sample selected was closest to the mean in both groups. All data is mean ± standard deviation. Bars not sharing the same letter are statistically different. *p is the p-value for a significant strain-by-disease interaction effect.

3.3. Cortical bone structure

For cortical bone area at the distal 1/3 femur, there was an effect of strain (p<0.0001) and an interaction effect (p=0.028), but no adenine effect (p=0.729). C3H mice had higher cortical bone area than B6 with no statistical differences due to adenine in either strain (Figure 2A). For cortical thickness, there were effects of strain (p<0.0001), adenine (p<0.0001), and a strain-by-adenine interaction effect (p=0.029). Cortical thickness was higher in C3H mice vs. B6 with adenine groups in both strains having lower cortical thickness than strain-matched controls (Figure 2B). There was an adenine effect (p<0.0001) and a strain-by-adenine interaction effect (p<0.0001) for cortical porosity with no main effect of strain (p=0.527). B6 adenine mice had higher porosity than B6 controls while C3H mice had no differences in cortical porosity between control and adenine groups (Figure 2C).

Figure 2: Cortical bone parameters of the femur.

Figure 2:

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A) Cortical bone area was higher in C3H mice compared to B6 with no differences due to adenine. B) Cortical thickness was greater in C3H mice vs. B6 with lower cortical thickness in adenine mice of both strains compared to strain-matched controls. C) Cortical porosity was higher in B6 adenine mice with no differences between C3H mice. D) Representative images of cortical bone regions of interest. Images represent closest to the mean in porosity values of the group. All data is mean ± standard deviation. Bars not sharing the same letter are statistically different. *p is the p-value for a significant strain-by-disease interaction effect.

3.4. Intracortical bone turnover

For intracortical bone formation rate, there was a main effect of strain (p=0.033), a main effect of adenine (p<0.0001), and a strain-by-adenine interaction effect (p<0.0001). Both C3H mice groups had higher intracortical BFR compared to control B6 with no difference due to adenine in the C3H mice. B6 adenine mice had significantly higher intracortical BFR compared to B6 controls and both C3H groups (Figure 3A). The number of labeled pores normalized to bone area were significant for all factors (strain p=0.017, adenine p=0.018, strain-by-adenine interaction p<0.0001). Control C3H mice had higher values than B6 control mice. Adenine groups in both strains of mice had higher values than strain-matched controls (Figure 3B). For MAR, there were also main effects of strain (p=0.016), adenine (p<0.0001), and a strain-by-adenine interaction effect (p<0.0001). Both C3H groups were higher than B6 control mice with no difference between control and adenine in the C3H groups. B6 adenine mice had significantly higher intracortical MAR vs. control B6 mice (Figure 3C). Assessment of osteoclast-covered pore surfaces showed a main effect of strain (p=0.013), a main effect of adenine (p<0.0001), and a strain-by-adenine interaction effect (p<0.0001). Control B6 mice had no intracortical osteoclast-covered surfaces. C3H mice had intracortical osteoclast-covered surfaces, but no difference between control and adenine, while B6 adenine mice had the highest amount of TRAP-covered pore surfaces (Figure 3D).

Figure 3: Histomorphometric measures of intracortical bone turnover in the femur.

Figure 3:

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A) Labeled pores/bone area was higher in C3H control mice vs. B6 control mice. Adenine in B6 mice significantly elevated the number of labeled pores normalized to bone area. B) Mineral apposition rate (MAR) was higher in C3H control mice vs. B6 control mice. B6 adenine mice had the higher intracortical MAR with no difference due to adenine in C3H mice. C) Intracortical bone formation rate (BFR) was higher in C3H mice vs. B6 control mice. There was no impact of adenine in C3H mice, but higher BFR in B6 adenine mice compared to all other groups. D) TRAP-covered pores surfaces were not present in B6 control mice and not different between control and adenine in C3H mice. B6 adenine mice had the highest osteoclast-covered pores surfaces. E) Representative images of fluorescent calcein labels in cortical bone of control and adenine mice of both strains. Image taken at 20x magnification. All data is mean ± standard deviation. Bars not sharing the same letter are statistically different. *p is the p-value for a significant strain-by-disease interaction effect.

3.5. Cortical bone osteocyte RANKL and apoptosis measures

For %RANKL-positive osteocytes in the cortical bone of the humerus, there were main effects for strain and adenine and a strain-by-adenine interaction effect (p<0.0001 for all). B6 adenine had higher %RANKL-positive osteocytes vs. B6 control while C3H mice had no differences due to adenine (Figure 4A). For %Annexin V-positive osteocytes, there was a strain-by-adenine interaction effect (p=0.02), but no main effect of strain (p=0.478) or adenine (p=0.097). %Annexin V-positive osteocytes were lower in B6 adenine mice vs. B6 control mice with no differences between control and adenine in C3H mice (Figure 4B).

Figure 4: Immunohistochemical osteocyte markers in cortical bone of the humerus.

Figure 4:

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A) RANKL-positive osteocytes were higher in B6 adenine mice, but not different between adenine and control in C3H mice. B) Annexin V, an early marker of cellular apoptosis, was lower in B6 adenine mice vs. B6 controls with no differences in C3H mice. C) TNF-α-positive osteocytes were higher in both strains of adenine mice vs. strain-matched controls. D) IL-17-positive osteocytes were higher in B6 adenine mice, but not different between control and adenine in C3H mice. All data is mean ± standard deviation. Bars not sharing the same letter are statistically different. *p is the p-value for a significant strain-by-disease interaction effect.

3.6. Cortical bone osteocyte inflammatory factors

For %TNF-α-positive osteocytes in the cortical bone of the humerus, there was a main effect for strain (p=0.026) and adenine (p<0.0001), but no interaction effect (p=0.683). For both strains of mice, osteocytes positive for TNF-α were higher in adenine vs. control (Figure 4C). For %IL-17-positive osteocytes, there were main effects of strain (p=0.001) and adenine (p=0.004), but no strain-by-adenine interaction effect (p=0.115). IL-17-positive osteocytes were higher in B6 adenine mice vs. B6 controls with no statistical differences between control and adenine in C3H mice (Figure 4D).

3.7. Trabecular bone structure

For trabecular bone volume at the distal femur, there was an effect of strain (p<0.0001), adenine (p=0.017), and a strain-by-adenine interaction effect (p<0.0001). BV/TV was higher in C3H vs. B6. Additionally, B6 adenine mice had higher BV/TV than B6 controls while C3H adenine mice had lower BV/TV than strain-matched controls (Figure 5A). Trabecular thickness primarily showed effects of strain (p<0.0001) with an interaction effect (p=0.015), but no effect of adenine (p=0.762). Trabecular thickness was higher in C3H mice regardless of treatment, but C3H adenine mice had lower trabecular thickness while B6 mice had no difference due to adenine (Figure 5B). For trabecular separation, there was no effect of strain (p=0.674) and no interaction (p=0.085), but an effect of adenine (p=0.018). C3H adenine mice had higher trabecular separation than C3H controls with no difference between B6 mice (Figure 5C). For trabecular number, there was a strain effect and a strain-by-adenine interaction effect (p<0.0001 for both), but no effect of adenine (p=0.612). C3H mice had higher trabecular number than B6 mice. With adenine, B6 mice had higher trabecular number vs. B6 controls while C3H adenine mice had lower trabecular number vs. C3H controls (Figure 5D).

Figure 5: Trabecular bone parameters of the distal femur.

Figure 5:

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A) Trabecular bone volume was higher in C3H mice vs. B6 mice. B6 adenine mice had higher BV/TV than B6 controls while C3H adenine mice had lower BV/TV than controls. B) C3H mice had higher trabecular thickness than B6 with C3H adenine mice having lower trabecular thickness than C3H controls. C) C3H adenine mice had higher trabecular separation than C3H controls with no statistical differences between treatments in B6 mice. D) B6 adenine mice had higher trabecular number than strain-matched controls while C3H adenine mice had lower trabecular number vs. strain-matched controls. All data is mean ± standard deviation. Bars not sharing the same letter are statistically different. *p is the p-value for a significant strain-by-disease interaction effect.

3.8. Trabecular bone turnover

For trabecular bone formation rate at the distal femur, there was a strain-by-adenine interaction effect (p=0.003) with no independent effect of strain (p=0.086) or disease (p=0.178). B6 adenine mice had higher BFR than B6 control mice with no effect of adenine on BFR in C3H mice (Figure 6A). Mineralized surface had an effect of strain (p=0.001), but no effect of adenine (p=0.975) or interaction effect (p=0.135). MS/BS was higher in B6 mice vs. C3H, but not statistically different between control and adenine in either strain (Figure 6B). Differences in BFR were driven by MAR, with significant effects of strain (p<0.0001), adenine (p=0.026), and a strain-by-adenine interaction effect (p<0.0001). C3H mice had higher MAR than B6 mice. Adenine B6 mice had higher MAR than B6 controls while there was not a statistical difference between control and adenine in C3H mice (Figure 6C). For osteoclast-covered trabecular surfaces, there was an effect of strain, adenine, and a strain-by-adenine interaction (p<0.0001 for all). In both B6 and C3H mice, the adenine groups had higher osteoclast-covered surfaces (Figure 6D).

Figure 6: Histomorphometric measures of trabecular bone turnover in the distal femur.

Figure 6:

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A) Mineralized surface was not statistically different between control and adenine in either strain, but higher in B6 mice vs. C3H mice. B) Mineral apposition rate was higher in C3H mice vs. B6. B6 adenine mice had higher MAR than B6 controls with no differences between C3H mice. C) Bone formation rate was higher in B6 adenine mice vs. strain-matched controls, but not different due to adenine in C3H mice. D) Osteoclast-covered surfaces were statistically higher in both adenine groups regardless of strain. All data is mean ± standard deviation. Bars not sharing the same letter are statistically different. *p is the p-value for a significant strain-by-disease interaction effect.

4. Discussion

The primary finding of this study is that female C3H/HeJ do not develop increased PTH, cortical porosity or enhanced intracortical remodeling in response to adenine-induced CKD despite increased BUN. In comparison, adenine-induced CKD B6 mice have elevated PTH and develop intracortical remodeling, high bone turnover, and cortical porosity compared to strain-matched controls. These data support previous work regarding the importance of elevated PTH in the development of high bone turnover and cortical porosity associated with CKD [28] [29]. C3H mice did have reductions in cortical thickness and increased osteocyte TNF-α indicating disease processes in adenine-induced CKD that are likely independent of significantly elevated PTH.

Our primary goal for comparing C3H vs. B6 mice was to assess the impact of cortical thickness on porosity development in response to adenine-induced CKD. A previous study demonstrated that both sham and OVX C3H mice had intracortical resorption spaces, but these spaces were significantly higher in OVX mice [16]. In comparison, B6 mice with thinner cortices did not have intracortical resorption spaces with either sham or OVX treatment. Therefore, we hypothesized that C3H mice with thicker cortices would have more pronounced porosity in response to adenine-induced CKD. Contrary to our hypothesis, C3H mice did not have differences in cortical porosity between control and adenine-induced CKD groups. We hypothesize the lack of cortical porosity was due to PTH only being mildly higher (+50%) in C3H adenine mice compared to the 175% higher PTH in B6 adenine mice vs. strain-matched controls. Previously, we found approximately 40% of the variability in cortical porosity in female B6 mice was predicted by serum PTH [9] indicating the important role of PTH in porosity; therefore, we hypothesize the lack of significantly higher PTH in C3H mice is responsible for no difference in porosity. While we cannot answer questions regarding our initial hypothesis about cortical thickness influencing porosity, comparing C3H adenine mice to B6 adenine mice provides insight into strain-related responses in the skeletal phenotype of CKD. C3H adenine mice did, however, have lower cortical thickness compared to non-CKD C3H controls like the response in B6 adenine mice. We hypothesize that thinning of cortical bone in CKD may be independent of high PTH, or that the threshold for elevated PTH to trigger endocortical resorption is lower than it is to stimulate cortical pore formation.

Multiple studies have previously addressed differences between B6 and C3H mice in response to other stressors to the skeleton. For example, C3H mice are comparatively resistant to disuse-induced bone loss and to loading-induced bone formation in comparison to B6 mice [30] [31] [32] [33] [34]. With regards to OVX-induced bone loss, C3H mice have a greater magnitude of bone loss than B6 [35] and, as stated above, increased intracortical resorption spaces [16]. Studies have also shown C3H mice to have greater cortical bone area and higher trabecular mineral apposition rate compared to B6 mice [36], both also seen in this study. One study of interest to this work demonstrated B6 mice responded to a low dietary calcium challenge with high PTH, while C3H mice did not. This led the authors to hypothesize that C3H mice are less reliant on calcium mobilization from bone to maintain extracellular calcium which may be due to genetic differences in calcium handling, PTH, and vitamin D [37]. In our study, we found statistically higher PTH only in B6 adenine mice with no difference in serum calcium due to adenine in either strain. It is possible B6 mice, due to different calcium metabolism than C3H mice, maintained the same serum calcium levels in response adenine-induced CKD by increasing PTH.

Adenine-treated mice of both strains had higher BUN levels, approximately 50% higher than strain-matched controls. This indicates that both strains of mice had reduced kidney function due to adenine intake. Both strains of mice also had kidney crystals present from micro-CT analysis, although the amount and location of crystals varied, with C3H having more crystals that were more widely spread than B6. While beyond the scope of this study, this could indicate a strain-related difference in the kidney injury induced by adenine.

The high bone turnover phenotype seen in CKD is associated with cortical porosity and high PTH in both human clinical trials [7] and in animal models [8] [9] [10] [29]. In this study, B6 mice had high bone turnover characterized by increased bone formation rate and elevated osteoclast-covered surfaces. This high turnover was present in both trabecular bone and intracortical bone compartments. This points to the importance of PTH in the high bone turnover phenotype of CKD. Interestingly, B6 mice that do not typically have intracortical remodeling at this age were stimulated to induce it in the presence of adenine-induced CKD. In aged B6 mice, intracortical remodeling has also been noted along with cortical porosity, both these are not present in younger animals [14] [15]. What triggers the initiation of intracortical remodeling and cortical porosity formation in these conditions remains largely unknown. Also of interest is the fact that C3H mice had similar intracortical activity in both control and adenine groups. Therefore, the C3H mice with intracortical remodeling were not stimulated to increase it with adenine while the B6 mice without intracortical remodeling in controls had robust remodeling present when stimulated with adenine. This indicates that the baseline intracortical remodeling present in C3H mice did not appear to affect the degree of induced activity or pore formation in response to adenine.

Bone turnover in CKD can also be relatively low or normal compared to non-CKD patients, however, much less is understood about the mechanisms and outcomes of low or normal turnover phenotypes partially since many animal models of progressive CKD model high bone turnover. Interestingly, C3H adenine mice without statistically elevated PTH, had no statistical differences in bone formation rate between control and adenine in either the intracortical or trabecular compartment at the time point measured; however, C3H adenine mice did have 32% higher osteoclast surfaces than controls (significantly less of a difference than the 3.5-fold higher osteoclast surfaces in B6 adenine mice). Therefore, C3H adenine mice had neither higher turnover nor lower turnover in response to adenine-induced CKD. These differences between B6 and C3H mice with the same treatment allude to the possibility of genetic factors that could influence the response to CKD.

Previously, we hypothesized that signals from long-lived osteocytes embedded within the cortical bone drive osteoclastic bone resorption in the cortex leading to the formation of cortical porosity [9]. Over time in young female adenine mice, we found increased osteocyte RANKL and decreased osteocyte annexin V, an early marker of apoptosis [9]. Since PTH has known stimulatory effects on osteocyte RANKL and appears to inhibit osteocyte apoptosis when administered intermittently, we previously hypothesized these changes were due in part to chronically elevated PTH. The current study in skeletally mature female B6 mice supports our previous work in young female B6 mice [9] with higher osteocyte RANKL and lower osteocyte annexin V in response to adenine-induced CKD; however, this was only seen in the B6 adenine mice that had elevated PTH and no differences in these measures were detected in C3H mice. Additionally, only the B6 adenine mice had significantly elevated cortical porosity and ~30% of pore surfaces covered with osteoclasts. These data along with our previous data [9], demonstrate that elevated osteocyte RANKL and declines in osteocyte apoptosis accompany osteoclast activity and porosity formation within the cortex and, therefore, we hypothesize that osteocytes may play a key role in driving porosity development in CKD. Furthermore, it demonstrates the direction of changes are similar between the young mice previously reported and the skeletally mature female mice in this study although the magnitude of difference between control and adenine in osteocyte RANKL is slightly greater in the young mice – nearly 4-fold higher RANKL in adenine in young vs. 2.7-fold higher RANKL in adenine in skeletally mature. Additionally, it demonstrates that PTH is likely a major contributor to high osteocyte RANKL and cortical porosity development since C3H adenine mice without statistically elevated PTH did not have adenine-induced differences in osteocyte RANKL, cortical porosity, or osteoclast-covered pore surfaces.

In addition to PTH, key inflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-17 (IL-17), increase osteoclastogenesis directly [38] [39] as well as indirectly via increasing RANKL [40] [41]. Furthermore, continuously high PTH has been shown to upregulate IL-17A, contributing to high osteocytic RANKL and bone loss [42]. Therefore, we hypothesized that pro-inflammatory cytokines may contribute to the high RANKL and cortical osteoclastic drive in CKD. In this study, only B6 adenine mice had high IL-17-positive osteocytes in the cortical bone which could be related to the high PTH. Perhaps more interesting is that, like B6 adenine mice, C3H adenine mice also had high osteocyte TNF-α, 3-fold higher than controls. Osteocyte TNF-α is elevated in animal models of systemic inflammation including high fat-induced obesity [43], inflammatory bowel disease [44] [45], and spinal cord injury [46]. Since pro-inflammatory markers, like TNF-α, are elevated in CKD patients [20] and are associated with both the risk of developing CKD [47] and risk of mortality from CKD [48] [19], these results could potentially indicate CKD-related changes that are not associated with PTH. While we cannot discount other mechanisms that are intrinsic to the genetic differences between C3H and B6 animals, C3H mice have a response more like that of other conditions with inflammatory bone loss, namely trabecular bone loss and cortical thinning and no elevation in cortical porosity although the bone response to systemic inflammation in C3H mice is not fully known. Therefore, it is plausible that CKD-related inflammation impacts bone although the profound impact of high PTH is a stronger stimulus on bone turnover and cortical bone osteocytes than inflammation alone. There also could be a synergistic effect between inflammatory factors and chronically high PTH resulting in the unique phenotype seen in CKD.

Limitations of this study include only examining female mice. Females only were chosen based on our previous work with female B6 adenine mice [9] [10]. Future work could address sex differences particularly in male C3H mice as to our knowledge no previous studies have addressed adenine-induced CKD in C3H mice. Furthermore, multiple animal models of CKD demonstrate that males have a more severe phenotype than do females [49] [50] so the response could be more severe or different in males. Additionally, we chose to study C3H mice because of the thickness of their cortical bone, not due to potential differences in mineral metabolism compared to B6 mice; therefore, this study was not focused on assessment of differences in mineral metabolism nor the mechanisms underlying C3H mice responding to adenine-induced CKD differently than B6 mice. Future studies could address differences in calcium handling and vitamin D. Additionally, we have previously established a time-course assessment of PTH in young female B6 mice, but we do not know changes in PTH over time in female C3H mice. Therefore, our data can only assess differences at the single time point measured in this study. Nevertheless, we feel our data show that C3H mice can provide a unique comparison to B6 mice with respect to differences in PTH in relation to bone turnover and cortical porosity.

In conclusion, our study demonstrates strain-related differences between C57Bl/6J and C3H/Hej mice in response to adenine-induced CKD with only B6 mice developing high PTH, high bone turnover, and cortical porosity in CKD. Additionally, CKD with high PTH is associated with high osteocyte RANKL, pro-inflammatory factors, and lower osteocyte apoptosis which could contribute to the osteoclastic drive developing cortical porosity. Finally, both strains of mice had high osteocyte TNF-α indicating the potential for a PTH-independent pro-inflammatory status in bone due to CKD.

Highlights.

  • B6 adenine-CKD mice develop high PTH, cortical porosity, and high bone turnover

  • C3H adenine-CKD mice do not have significant elevations in PTH or porosity

  • Lower cortical thickness is present in all adenine mice regardless of strain

Acknowledgments

Funding: This work was supported by VA Merit Award (BX003025) from the U.S. Department of Veterans Affairs (Biomedical Laboratory Research and Development Service) to MRA and by the NIH (F32DK122731) to CEM.

Footnotes

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Competing Interests: The authors declare no competing interests.

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