Changes in bone characteristics precede serum mineral deterioration in a mouse model of early stage chronic kidney disease
Lieve Verlinden, Ingrid Stockmans, Karen Moermans, Geert Carmeliet
TL;DR
In a mouse model of early chronic kidney disease, bone changes occur before serum mineral levels are affected, showing early signs of bone deterioration.
Contribution
This study reveals that bone formation declines and bone marrow adiposity increases before serum calcium levels change in early CKD.
Findings
Bone mass decreases in trabecular and later cortical bone in Col4a3−/− mice.
Osteoblast number and activity are reduced, while osteoclast numbers remain unchanged.
Increased bone marrow adiposity is associated with reduced bone formation in early CKD.
Abstract
Calcium metabolism is tightly regulated and involves hormonal communication between the intestine, kidney, and bone. Renal failure leads to impaired calcium homeostasis, but how each of the calcium-handling tissues adapts during the initial phases of the disease, remains to be explored. To this end, we used Col4a3−/− mice in which CKD develops progressively, as shown by the gradually decreased glomerular filtration rate and increased uremia. Mineral homeostasis was disturbed with increased serum levels of phosphaturic hormones, FGF23, and PTH and decreased 1,25(OH)2D3 levels. These hormonal adaptations in Col4a3−/− mice preserved normal serum calcium levels and maintained intestinal calcium absorption and renal fractional clearance of calcium. However, bone mass was already affected, starting with a decrease in trabecular bone mass and later evolving to additional cortical bone loss.…
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| 14.9 ± 2.7 | 26.8 ± 7.7 | 22.8 ± 3.8 | 68.7 ± 28.8 |
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| 312.9 ± 68.6 | 822.8 ± 545.7 | ||||||
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| 170.3 ± 74.4 | 898.3 ± 442.1 | ||||||
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| 77.9 ± 17.8 | 59.1 ± 17.4 | 54.6 ± 11.9 | 25.5 ± 20.1 |
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| 9.8 ± 1.1 | 9.4 ± 1.3 | 9.8 ± 0.7 | 9.5 ± 0.3 |
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| 10.6 ± 1.7 | 11.4 ± 2.6 | 9.8 ± 0.7 | 10.5 ± 1.8 |
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- —Fund of Scientific Research-Flanders
- —KU Leuven10.13039/501100004040
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Taxonomy
TopicsParathyroid Disorders and Treatments · Bone health and osteoporosis research · Vitamin D Research Studies
Introduction
The progressive impairment of kidney function in CKD is accompanied by dysregulation of mineral and bone metabolism (also known as CKD-MBD).^1,2^ Preservation of a normal calcium balance is challenged in CKD-MBD patients, and the resulting hypocalcemia may impact cellular functioning in several organs. Serum calcium levels are, therefore, maintained as long as possible by different hormones regulating calcium handling in the intestine, kidneys, and bone. When serum calcium levels drop, PTH is released by the parathyroid glands and stimulates renal calcium reabsorption, increases bone resorption, and, hence, skeletal calcium release.^3,4^ Moreover, PTH induces the expression of renal Cyp27b1, the enzyme that converts 25OHD to its active form 1,25(OH)2_D_3. 1,25(OH)2_D_3, in turn, enhances intestinal calcium absorption and increases renal calcium reabsorption,^5^ in order to restore serum calcium levels. The calcium balance is highly intertwined with phosphate homeostasis, which is mainly controlled by PTH and FGF23, two phosphaturic hormones that enhance renal phosphate excretion.^6,7^ PTH and FGF23 regulate Cyp27b1 expression in opposite ways, and 1,25(OH)2_D_3 in turn stimulates intestinal phosphate absorption.
Perturbations in mineral and bone metabolism start at an early stage of CKD and are considered to be initiated by altered phosphate clearance resulting in elevated levels of FGF23 and PTH, which are later accompanied by decreased 1,25(OH)2_D_3 levels.^8,9^ These hormonal alterations serve to maintain serum calcium and phosphate concentrations within the normal range as long as possible. Changes in calcium homeostasis should be avoided, as both a positive and a negative calcium balance can have important health implications in CKD patients. A negative calcium balance, with calcium excretion exceeding calcium intake, might advance the development of CKD-MBD and increased fracture risk.^10^ Different types of bone defects might occur with changes in turnover, mineralization, and volume. On the other hand, a positive calcium balance enhances the risk of extraskeletal calcifications and cardiovascular complications.^11–13^ The reduced 1,25(OH)2_D_3 levels are considered to decrease intestinal calcium absorption in CKD patients, but, conflicting data have been reported, ranging from no changes to calcium malabsorption already present at the early stages of CKD, even before an apparent increase in serum PTH levels.^14^ As calcium malabsorption is suggested to induce bone mineral loss in CKD, it is important to carefully investigate the dysregulation of calcium balance during CKD development and its possible impact on bone homeostasis.
To study how calcium metabolism in the intestine, kidney, and bone is changed during the early stages of progressive renal fibrosis, we used Col4a3^−/−^ mice, containing a deletion of the α3 chain of type IV collagen.^15^ The Col4a3^−/−^ knockout model was initially developed as a murine model of Alport syndrome, a human condition exhibiting hereditary nephritis associated with various ocular and auditory defects. Interestingly, Col4a3^−/−^ mice develop chronic kidney damage with predictable onset and progression, which leads to reduced trabecular and cortical BMD.^16^ A detailed examination of calcium and bone homeostasis in this study shows that serum calcium and phosphate levels are preserved at an early stage of CKD, likely because of adaptations in PTH and FGF23. However, bone loss was already present and was linked to decreased bone formation and increased BM adiposity. These findings highlight that bone properties should be evaluated rigorously from the early stage of CKD onward.
Materials and methods
Genetic mouse model
Mice with a targeted deletion of the C-terminal non-collagenous cross-linking NC1 domain of type IV collagen α-3 chain^15^ were obtained from the Jackson Laboratory (Col4a3^tm1Dec^, JAX stock #002908, Bar Harbor, USA). The WT Col4a3 allele was identified as a 900 bp PCR-fragment using the forward (FW) primer 5′-CCA GGC TTA AAG GGA AAT CC-3′ and the reverse (RV) primer 5′-CCT GCT AAT ATA GGG TTC GAG A-3′, whereas the mutant Col4a3 allele resulted in a 230 bp PCR-fragment using the same FW primer combined with the RV primer 5′-CTA TCA GGA CAT AGC GTT GG-3′. Phenotypic analysis was performed on female Col4a3^−/−^ mice and their WT littermates using different cohorts for the analysis at 8 and 10 wk of age. These ages represent the early stage of CKD in this model, which also shows limited survival after 14 wk of age.^15,17^ All mice were maintained on a diet containing 0.6% Ca, 0.54% Pi, and 3000 IU vitamin D per kg (Ssniff, Soest, Germany), as described in Stubbs et al.^17^ To analyze dynamic bone parameters, calcein (16 mg/kg body weight; Sigma-Aldrich) was administrated via intraperitoneal injection 4 d and 1 d before euthanasia.^18–26^ Mice were housed in our animal facility (Proefdierencentrum Leuven, Belgium) with 12 h dark/light cycles, a constant temperature (22-24 °C), and humidity (55%-60%). Food and water were supplied ad libitum. Animal welfare was continuously monitored throughout the experiment. Mice exhibiting clinical signs of distress, such as excessive urine production, ruffled fur, and a hunched posture, or experiencing rapid weight loss, were promptly removed from the study to minimize suffering. All animal experiments were approved by the Institutional Animal Care and Research Advisory Committee of the KU Leuven (P234/2013).
Serum and urine biochemistry
Urine of 8-wk-old female mice was collected during a 24 h stay in individual metabolic cages (Tecniplast, Buguggiate, Italy), and food and water intake were monitored during this timeframe. Following euthanasia, blood was obtained from 8- and 10-wk-old female mice via cardiac puncture. Serum and urinary calcium (OSR60117), phosphate (OSR6122), and creatinine (OSR6178) concentrations and serum urea (BUN, OSR6134) were analyzed on an AU640 chemistry analyzer (Analis, Vilvoorde, Belgium). Serum PTH (Quidel, San Diego, USA) and intact FGF23 (iFG23, Kainos, Tokyo, Japan) levels were measured with ELISA according to the manufacturer’s recommendations. Serum 1,25(OH)2_D_3 concentrations were measured by liquid chromatography-tandem mass spectrometry at the University Hospital of Leuven.
Renal function
Inulin clearance was measured as described by Rieg et al.^27^ in 8-wk-old female Col4a3^−/−^ mice and their WT littermates. Briefly, FITC-inulin (Merck, Overijse, Belgium) in 0.9% NaCl was injected via the tail vein (22 mg/mL, 3.75 μL/g body weight). Blood was sampled from the tail vein at 3, 7, 10, 15, 35, 55, 75, 100, 120, 150, and 180 min after injection, and the fluorescence of HEPES-buffered serum samples was measured (excitation wavelength of 485 nm and emission wavelength of 535 nm, Victor Wallac plate reader, Perkin Elmer, Zaventem, Belgium). A 2-phase exponential decay curve was fitted for each mouse, and the glomerular filtration rate was calculated based on this curve as described by Rieg et al.^27^
Functional calcium absorption assay
Intestinal Ca^2+^ absorption was assessed in 10-wk-old female mice as described previously.^5^ Briefly, 15 μL/g body weight of a radioactive solution containing 0.1 mM CaCl_2_, 125 mM NaCl, 17 mM Tris, 1.8 g/L fructose, and 20 μCi ^45^CaCl_2_ (20 Ci/g; Perkin Elmer) was given by oral gavage, and radioactivity in serum was analyzed after 2, 4, 6, 8, and 10 min by liquid scintillation counting. The change in serum ^45^Ca^2+^ concentration (Δμmol) was calculated from the ^45^Ca^2+^ content of the serum and the specific activity of the administered ^45^Ca^2+^.
Intestinal permeability assay
Transepithelial electrical resistance (TEER), a measure of duodenal permeability, was assessed in 8-wk-old female mice, as described previously.^28^ Briefly, duodenal tissue was dissected and mounted in triplicate in Ussing chambers with 0.096 cm^2^ of tissue exposed. The mucosal side was exposed to 10 mM mannitol and the serosal side to 10 mM glucose in a Krebs-Ringer bicarbonate buffer. To preserve tissue viability, samples were maintained at 37 °C and oxygenated with O_2_/CO_2_ (95/5%) at all times. After a 20-min stabilization period, TEER was monitored after the induction of bipolar constant-current pulses of 50 μA every minute during a 2 h period.
Gene expression analysis
Total RNA was isolated from the kidney, duodenum, and bone (osteocyte-enriched fraction from the femur) of 10-wk-old female mice by snap-freezing freshly dissected tissues in liquid nitrogen, followed by homogenization and extraction using TRIzol (Invitrogen) and phenol/chloroform purification.^19,21–24,26,29–32^ RNA from cultured cells was isolated using an RNeasy Mini Kit (Qiagen, Germantown, USA). cDNA was synthesized using reverse transcriptase SuperScript II RT (Thermo Fisher Scientific) and qRT-PCR was performed as described^24^ on the 7500 Fast Real-Time PCR System (Applied Biosystems) using specific forward and reverse oligonucleotide primers (sequences available upon request). The 2^−ΔΔCT^ method was used to calculate relative gene expression, using the expression of hypoxanthine-guanine phosphoribosyltransferase (Hprt) as an internal control.
Bone structure and histomorphometric analysis
Upon dissection, right tibiae were fixed in freshly prepared paraformaldehyde (2% in PBS solution), whereas left tibiae were fixed in Burkhardt’s solution. Paraformaldehyde-fixed tibiae were used for ex vivo micro-CT (μCT) analysis and subsequently decalcified for histological examination.
Ex vivo μCT analysis was performed on paraformaldehyde-fixed tibiae using the high-resolution SkyScan 1172 system (50 kV, 200 μA, 0.5 mm Al filter; Bruker, Kontich, Belgium). Scans were taken with a pixel size of 5 μm with an angular increment of 0.4° and a frame averaging of 2. Serial tomographs were reconstructed from raw projection data with the cone-beam reconstruction software (NRecon, Bruker) using the following settings: smoothing = 1; ring artifact reduction = 7, and beam-hardening correction = 30%. Reconstructed datasets were reoriented in DataViewer (Bruker) to position each bone in a straight, upright orientation. Trabecular and cortical morphometric parameters were quantified using CTAn (Bruker). Bone was segmented from soft tissue using a global threshold of 80-255 (grayscale units), and ROI corresponding to trabecular and cortical compartments were manually delineated. Trabecular analyses were performed in the proximal metaphysis (1-2.5 mm below the distal end of the growth plate), and cortical analyses were performed in the mid-diaphysis (2.5-3 mm below the distal end of the growth plate).^19–24,29,30,32–34^ Cortical porosity was quantified using the 2D analysis module in CTAn (Bruker). Fully enclosed pores were detected in the binarized objects, and porosity was calculated as the area of these enclosed pores expressed as a percentage of the total area of the binarized object. Analysis was performed according to the guidelines of the American Society for Bone and Mineral Research.^35^ Three-dimensional models were constructed with the CTvox software (Bruker).
After scanning, paraformaldehyde-fixed tibiae were decalcified for 2 wk in 0.5 M EDTA in PBS, pH 7.4, prior to dehydration in graded alcohol concentrations, embedding in paraffin, and sectioning at 4 μm. Osteoblast parameters were quantified on H&E-stained sections and osteoclasts on sections stained for TRAP activity and counterstained with Light Green SF Yellowish (Merck) as described previously.^19–22,24,26,32,36^
Burkhardt-fixed tibiae were embedded in MMA and sectioned at 4 μm. MMA sections were stained according to the von Kossa and Goldner method or left unstained for dynamic histomorphometry.^19–22,24,26,32,36^ All histomorphometric measurements were performed in 3 consecutive fields along the vertical axis of the central proximal metaphysis, starting 0.2 mm from the distal end of the growth plate, and this analysis was done on 3 longitudinal bone sections, each at least 40 μm apart. Adipocyte numbers were quantified on 3 longitudinal Goldner-stained bone sections per mouse, with sections being at least 40 μm apart. Per section, the number of adipocytes was quantified in 3 consecutive fields along the vertical axis of the central metaphysis, starting 0.2 mm from the distal end of the growth plate of the proximal tibia, summed per section, and expressed relative to tissue volume. Per mouse, the average number of the 3 sections is depicted. Adipocyte size was determined in a rectangle (600 × 400 μm situated 1 mm below the distal end of the growth plate) using the Adiposoft plugin of the ImageJ software.^37,38^
All quantifications were expressed according to the American Society for Bone and Mineral Research standardized histomorphometry nomenclature.^39^ Images were acquired on an Axioplan 2 imaging system with an Axiocam MRc5 camera and Axiovision (release 4.8) software (Zeiss, Zaventem, Belgium).
Calcium content in bone
Femurs were dried overnight at 100°C, ashed for 8 h at 500° C, dissolved in 1 mL of 1 M HCl, and diluted 1/50 in milliQ water prior to calcium measurement on the AU640 chemistry analyzer.
In vitro osteogenic differentiation
Primary trabecular osteoblasts were isolated from the long bones of the hind limbs of 8-wk-old Col4a3^−/−^ mice and their WT littermates.^22^ First, periosteal cells were removed by incubating the bones with 2 mg/mL collagenase type II (Thermo Fisher Scientific) for 30 min at 37° C. Thereafter, the epiphyses were removed, and the bones were flushed with PBS. The remaining bone fragments were minced and digested with collagenase type II for 40 min at 37° C to release skeletal stem and progenitor cells (SSPCs). These SSPCs were cultured in αMEM supplemented with 10% FBS, 100 IU/mL of penicillin, and 100 μg/mL of streptomycin (Thermo Fisher Scientific). At confluency, osteogenic differentiation was induced by adding 50 μg/mL of ascorbic acid and 10 mM ß-glycerophosphate (Merck, osteogenic medium) for 10 d, after which cultures were stained with Alizarin Red. To assess differences, 3-4 biological repeats, each consisting of 3 technical replicates, were included. For each biological repeat, the technical replicates were averaged and used as one value in the subsequent statistical analysis.
In vitro adipogenic differentiation
BM SSPCs were isolated from the long bones of the hind limbs of 8-wk-old Col4a3^−/−^ mice and their WT littermates, and cultured until confluency in αMEM supplemented with 10% FBS, 100 IU/mL of penicillin, and 100 μg/mL of streptomycin. Confluent cultures were trypsinized and seeded at 26 000 cells/cm^2^, and adipogenic differentiation was induced by adding 10 nM of dexamethasone, 10 μg/mL of human insulin, 50 μm of indomethacin, and 500 nM of 3-isobutyl-1-methylxanthine (all from Sigma Aldrich). After 14 d, cultures were stained with Oil Red O or used for RNA extraction. To assess differences in adipogenic differentiation, 4-7 biological repeats were included, each consisting of 2-4 technical replicates depending on the yield of primary cells. For each biological repeat, the technical replicates were averaged and used as one value in the subsequent statistical analysis.
Statistical analysis
All statistical analyses were performed with Prism 10.4.2 (Graphpad Software, La Jolla, USA). Power calculations were based on using 2-sided independent t-tests for data analysis (β = 0.80, α = 0.05) and the means and SD reported in previous studies. The number of mice calculated to be sufficient for each specific analysis is as follows: for GFR evaluation, 7 mice/genotype to detect a 40% difference between genotypes^27^; for analysis of the bone phenotype, a minimum of 9 mice/group to detect a 30% difference in trabecular bone mass^24^; for radioactive calcium absorption experiments, 6 mice per genotype to detect a 40% difference^24^; for TEER evaluation, 5 mice per genotype to detect a 30% difference.^40^ All in vivo data were expressed as the mean ± SD. All in vitro data were expressed as the mean ± SEM. Differences between 2 experimental groups were analyzed by the Student’s t-test (normal data distribution) or the Mann-Whitney’s-test. The difference between the median adipocyte sizes was determined by the Kolmogorov-Smirnov test. Differences between genotypes across ages were assessed using 2-way ANOVA with Sidak’s post hoc test. When a significant interaction between age and genotype was detected, data were further analyzed using 1-way ANOVA followed by Sidak’s multiple comparisons test, and the results of these post hoc comparisons were plotted. Spearman correlation analysis was used to assess relationships between adipocyte number, osteoblast number, and bone volume fraction (BV/TV). Differences were considered significant at p < .05.
Results
Col4a3−/− mice develop mineral-related hormonal adaptations of CKD
Before we investigated how the mineral and bone parameters were altered in Col4a3^−/−^ mice, we measured body weight and verified the progressive deterioration of renal function, typifying CKD. Col4a3^−/−^ mice gained significantly less weight than Col4a3^+/+^ mice, despite comparable food intake (Figure S1A and B). We, furthermore, confirmed that serum BUN levels gradually increased in Col4a3^−/−^ mice compared to Col4a3^+/+^ mice, with higher values at 10 than at 8 wk (Table 1). To evaluate renal function per se, we intravenously injected FITC-inulin and noted that its clearance was slower and less efficient in 8-wk-old Col4a3^−/−^ mice (Figure S1C). Accordingly, the GFR, calculated from a 2-phase exponential curve fit of the plasma FITC-inulin concentrations,^27^ was significantly lower in Col4a3^−/−^ mice than in Col4a3^+/+^ mice (Figure S1D).
Next, we assessed parameters of phosphate homeostasis, as phosphate retention, due to reduced kidney function, is considered an early trigger for the adaptive responses in mineral and bone homeostasis in CKD. Fractional phosphate excretion (Figure 1A) and serum phosphate levels (Table 1) were still within the normal range, whereas the levels of the phosphaturic hormones FGF23 and PTH were manifestly increased in 10-wk-old Col4a3^−/−^ mice (Table 1), indicating that these hormonal changes can still preserve normal phosphate homeostasis. Accordingly, the renal expression of the sodium-phosphate cotransporters Npt2a and Npt2c, mediating phosphate reabsorption in the proximal renal tubules, was significantly reduced in Col4a3^−/−^ mice (Figure 1B). Renal expression of the FGF23 receptor Fgfr1 was not changed, whereas transcript levels of its coreceptor Klotho were significantly reduced in Col4a3^−/−^ mice (Figure 1B).
Normal renal phosphate and calcium handling. (A) Fractional phosphate excretion in 8- and 10-wk-old mice (n = 5-9). (B) Renal expression of Fgfr1, Klotho, Npt2a, and Npt2c in 10-wk-old Col4a3−/− mice relative to Col4a3+/+ levels set as 100% in (n = 7-12). (C) Renal mRNA levels of Vdr, Cyp27b1, and Cyp24a1 in 10-wk-old Col4a3−/− mice relative to Col4a3+/+ levels set as 100% (n = 7-12). (D) Fractional calcium excretion in 8- and 10-wk-old mice (n = 5-9). (E) Renal transcript levels of the calcium transporters Trpv5, Calb1, Pmca1, and Ncx-1 in 10-wk-old Col4a3−/− mice relative to Col4a3+/+ levels set as 100% (n = 7-12). All data points represent data from individual mice and mean and SD are indicated.
As FGF23 and PTH regulate the renal expression of the 1,25(OH)2_D_3 synthesizing enzyme Cyp27b1, but in opposite ways, we measured circulating concentrations of 1,25(OH)2_D_3 as well as the transcript levels of Cyp27b1 and of the vitamin D inactivating enzyme Cyp24a1 in the kidney. Serum 1,25(OH)2_D_3 levels were significantly reduced in 10-wk-old Col4a3^−/−^ mice (Table 1), despite increased Cyp27b1 levels and decreased Cyp24a1 and Vdr expression in Col4a3^−/−^ kidneys (Figure 1C).
FGF23, PTH, and 1,25(OH)2_D_3 also stimulate renal calcium reabsorption, but fractional calcium excretion was not altered in 10-wk-old Col4a3^−/−^ mice (Figure 1D), likely because the expression of the various classes of renal calcium transporters was altered differently (Figure 1E). We noticed an increase in the apical calcium influx channel Trpv5, but a decrease in the intracellular calcium transporter Calb1, whereas the basolateral calcium export protein Pmca1 was unchanged and Ncx1 was decreased. In addition, serum calcium levels were normal in Col4a3^−/−^ mice (Table 1).
These data indicate that Col4a3^−/−^ mice develop renal dysfunction that mimics the early stage of CKD, when hormonal changes regulate the expression of renal phosphate and calcium transporters to maintain normal serum levels of phosphate and calcium.
Intestinal calcium absorption is normal in Col4a3−/− mice
Next, we investigated whether intestinal calcium absorption was altered in response to reduced 1,25(OH)2_D_3 levels. We detected significantly reduced transcript levels of the apical calcium entry channel Trpv6, the intracellular calcium transporter S100g, and of the basolateral ATP-dependent calcium extruder Pmca1 in 10-wk-old Col4a3^−/−^ mice (Figure 2A). However, despite this reduced expression of calcium transporters, we could not detect compromised intestinal calcium absorption in Col4a3^−/−^ mice, as orally administered radioactive calcium was equally well absorbed in Col4a3^−/−^ and Col4a3^+/+^ mice (Figure 2B). To examine whether this normal intestinal calcium absorption in Col4a3^−/−^ mice could be attributed to elevated paracellular transport, we examined epithelial integrity by measuring transepithelial electric resistance (TEER) of duodenal samples. However, TEER measurements were not different between genotypes (Figure 2C). In addition, the gene expression of the tight junction proteins Cldn 2 and Cldn12, which contribute to paracellular intestinal calcium absorption, was similar in Col4a3^−/−^ and Col4a3^+/+^ mice (Figure 2D). Together, these findings indicate that intestinal calcium absorption is normal in Col4a3^−/−^ mice, despite reduced transcript levels of known calcium transporters.
Normal intestinal calcium absorption despite decreased transcript levels of calcium transport genes. (A) Duodenal mRNA levels of Trpv6, S100g, and Pmca1 in 10-wk-old Col4a3−/− mice relative to Col4a3+/+ levels set as 100% (n = 9-12). Data points represent data from individual mice and mean and SD are indicated. (B) In vivo intestinal calcium absorption assessed by the appearance of 45Ca2+ (Δμmol) in serum within 10 min after oral gavage in 10-wk old Col4a3−/− mice and WT littermates (n = 6-8). Data are expressed as mean and SD. (C) Transepithelial electrical resistance, as a measure of membrane permeability, was identical in 8-wk-old Col4a3−/− mice and WT littermates (n = 2-3). Data are expressed as mean and SD. (D) Duodenal transcript levels of Cldn2 and Cldn12 in 10-wk-old Col4a3−/− mice relative to Col4a3+/+ levels set as 100% (n = 9-12). Data points represent data from individual mice and mean and SD are indicated.
Col4a3−/− mice display a low bone mass due to decreased osteoblast number and activity
Besides the kidney and intestine, bone also contributes to calcium homeostasis, but often at the expense of its structure, which depends on a sufficient calcium supply. Mineralized bone mass was investigated by μCT analysis (Figure 3A) and on von Kossa-stained bone sections (Figure 3B). We observed that trabecular bone mass was significantly reduced in 10-wk-old Col4a3^−/−^ mice (Figure 3C). A reduction in trabecular number (Figure 3C) accounted for the decline in trabecular bone mass, whereas no significant decrease was observed for trabecular thickness (Figure 3C). In addition, cortical thickness was significantly lower in 10-wk-old Col4a3^−/−^ mice, whereas cortical porosity decreased with age in Col4a3^+/+^ but not in Col4a3^−/−^ mice (Figure 3D). This reduced bone mass was confirmed by a significant decrease in total femur calcium in 8-wk-old, and more markedly in 10-wk-old Col4a3^−/−^ mice (Figure 3E).
Reduced bone mass in Col4a3−/− mice. (A, B) Representative cross-sectional 3D μCT images of the tiba (A) and representative Von Kossa-stained sections (B) of 10-wk-old mice. (C, D) Quantification of trabecular bone volume (BV/TV), trabecular number, and trabecular thickness (C), and cortical thickness and porosity (D) of 8- and 10-wk old mice (n = 9-13). (E) Femoral calcium content of 8- and 10-wk-old mice (n = 8-12). All data points represent data from individual mice and mean and SD are indicated.
To investigate the cellular processes responsible for the lower bone mass in Col4a3^−/−^ mice, tibia sections were analyzed histomorphometrically. We detected a significant decrease in osteoblast number, quantified on H&E-stained sections, in Col4a3^−/−^ mice (Figure 4A and B) and this decrease was confirmed by reduced gene expression of the osteoblast marker Bglap2, but not Alpl, in osteocyte-enriched bone fractions, suggesting a decrease in mature mineralizing osteoblasts (Figure 4C). Osteoblast-produced extracellular matrix was analyzed on Goldner-stained bone sections (Figure 5A). We noticed a significant thinning of osteoid seems in Col4a3^−/−^ mice, whereas the osteoid surface was not changed (Figure 5A). Analysis of calcein labels revealed no differences in the mineral apposition rate (MAR) and the extent of mineralizing surface between genotypes (Figure 5B). In addition, we observed a decrease in the femoral calcium content normalized to its dry weight in 10-wk-old Col4a3^−/−^ mice, suggesting a decrease in bone matrix mineralization (Figure 5C). However, skeletal transcript levels of Enpp1 and Spp1, 2 inhibitors of bone mineralization, were not significantly changed in Col4a3^−/−^ mice (Figure 5D).
Reduced osteoblast number in Col4a3−/− mice. (A, B) Representative H&E-stained section of the tibia (A) and histomorphometric analysis of osteoblast numbers (B) in 10-wk-old mice (n = 9-13) (scale bar is 100 μm for upper pictures and 30 μm for detailed pictures). (C) mRNA levels of the osteoblast markers Alpl and Bglap2 in osteocyte-enriched fractions isolated from femurs of 10-wk-old Col4a3−/− mice relative to Col4a3+/+ levels set as 100% (n = 5). All data points represent data from individual mice and mean and SD are indicated.
Decreased osteoblast activity and mineralization in Col4a3−/− mice. (A) Representative Goldner-stained section of the tibia of 10-wk-old mice. Black arrowheads point to thicker osteoid seems; white arrowhead points to adipocytes. Histomorphometric analysis of osteoid surface (OsteoidS/BS) and osteoid width (n = 9-13). (B) Representative pictures of calcein-labeled bone sections of tibia of 10-wk-old mice, and histomorphometric quantification of dynamic bone parameters after calcein injections; mineralizing surface and mineral apposition rate. (C) Femur calcium content normalized to dry weight as a measure for bone mineralization of 8- and 10-wk-old mice (n = 8-12). (D) mRNA levels of the mineralization inhibitors Enpp1 and Spp1 in osteocyte-enriched fractions isolated from femurs of 10-wk-old Col4a3−/− mice relative to Col4a3+/+ levels set as 100% (n = 5). (E) Histomorphometric quantification of osteoclast surface (OcS/BS) on TRAP-stained sections, and mRNA levels of Rankl, Opg, and Ctsk in osteocyte-enriched fractions isolated from femurs of 10-wk-old Col4a3−/− mice relative to Col4a3+/+ levels set as 100% (n = 5). (F) Rankl/Opg ratio in femurs of 10-wk-old mice. (G) Histomorphometric quantification of adipocyte number and size on Goldner-stained sections, and mRNA levels of Pparg and Fabp4 in femurs of 10-wk-old Col4a3−/− mice relative to Col4a3+/+ levels set as 100% (n = 5). (H) Visualization of the correlation between adipocyte number (AdN) and trabecular bone volume (BV/TV), between AdN and osteoblast number (ObN), and between ObN and BV/TV, including Pearson correlation coefficients and corresponding p-values. All data points represent data from individual mice and mean and SD are indicated.
On the other hand, analysis of TRAP-stained bone sections did not reveal changes in the osteoclast surface between genotypes, but only a decrease with age (Figure 5E). Gene expression of Rankl was increased without a manifest change in Opg in the osteocyte-enriched bone fraction of Col4a3^−/−^ mice (Figure 5E), but the ratio of Rankl/Opg, a major determinant of osteoclastic bone resorption, was similar between genotypes (Figure 5F). In addition, the expression of the osteoclast marker Ctsk was not changed in Col4a3^−/−^ mice (Figure 5E).
Histological examination of bone sections of Col4a3^−/−^ mice revealed a major increase in the number of BM adipocytes and in their median size in 10-wk-old Col4a3^−/−^ mice (Figure 5A and G and Figure S2), whereas food intake was similar between both genotypes (Figure S1B). This elevated BM adiposity was, however, not reflected in the transcript levels of Pparg and of the adipocyte marker Fabp4 (Figure 5G).
In conclusion, Col4a3^−/−^ mice progressively develop a low-turnover bone mass phenotype, with a reduction in bone formation and likely matrix mineralization, rather than an increase in bone resorption, underlying the decreased bone mass.
In vitro osteoblastic and adipogenic differentiation potential is normal
As described above, the number of osteoblasts was strongly reduced in Col4a3^−/−^ mice, whereas BM adipocytes were significantly elevated. Moreover, the adipocyte number correlated inversely with both trabecular bone volume and the osteoblast number, whereas the osteoblast number showed a positive correlation with trabecular bone volume (Figure 5H). A potential explanation is that the osteogenic differentiation potential of skeletal SSPCs derived from Col4a3^−/−^ mice was compromised in favor of their adipogenic differentiation. In vitro osteogenic differentiation of trabecular SSPCs derived from Col4a3^−/−^ was normal, as evidenced by Alizarin Red staining and the expression of the osteoblast markers Spp1, Alpl, and Bglap2 (Figure 6A). Furthermore, the adipogenic differentiation of BM SSPCs was similar between genotypes, as shown by Oil Red O staining and the expression of Pparg, Fabp4, Fasn, Plin1, and Adipoq (Figure 6B). These data indicate that the reduction in osteoblasts and the increase in adipocytes in Col4a3^−/−^ mice are likely caused by an altered bone microenvironment, which promotes adipogenic over osteogenic differentiation of SSPCs during the development of renal osteodystrophy.
Normal in vitro osteogenic and adipogenic differentiation potential of Col4a3−/− SSPCs. (A) Representative alizarin red staining and mRNA expression of Spp1, Alpl, and Bglap2 after osteogenic differentiation of SSPCs derived from 8-wk-old mice (n = 2-3). Each data point represents the average of 3 technical replicates. The mean and SEM of the biological replicates is depicted. (B) Representative Oil Red O staining and transcript levels of Pparg, Fabp4, Fasn, Plin1, and Adipoq after adipogenic differentiation of BM stromal cells derived from 8-wk-old mice (n = 2-3). Each data point represents the average of 2-4 technical replicates. The mean and SEM of the biological replicates are depicted.
Discussion
During the early stage of CKD, serum calcium and phosphate levels remain normal, due to hormonal adaptations. Further renal deterioration during the late stages of CKD disturbs these mineral balances, which together with the hormonal alterations result in CKD-MBD, comprising different types of bone pathology.^41–43^ To investigate whether the early stages of renal dysfunction already affect bone properties, or rather, intestinal mineral homeostasis, we used the Col4a3^−/−^ mouse model. We here report that when serum calcium and phosphate are still normal at the early stage of CKD, intestinal calcium absorption is not affected, but bone mass already decreases gradually due to a reduced osteoblast number, and that, concurrently, BM adiposity increases.
Bone mass decreases progressively in Col4a3^−/−^ mice, paralleling the gradual decline in renal function, which is in agreement with previous findings of Dussold et al.^16^ Trabecular bone mass is reduced and cortical bone is also affected, characterized by cortical thinning. Mechanistically, we noticed a decreased osteoblast number, which together with the normal osteoclast parameters, points to a low bone turnover phenotype, where reduced bone formation cannot match the unaltered bone resorption and leads to decreased bone mass in CKD mice. The impaired osteoblast function, with reduced matrix formation and mineralization, results in lower calcium incorporation in the bone matrix and a decreased calcium content of bone. This reduced calcium deposition in bone might have contributed to maintaining normal serum calcium levels at this early stage of CKD. Indeed, the preservation of serum calcium at the expense of calcium content in the bone has been observed in mouse models with a negative calcium balance, due to the conditional deletion of the vitamin D receptor (VDR) in intestinal epithelial cells.^18^ In the latter model, the increased 1,25(OH)2_D_3 levels induce the expression of mineralization inhibitors like Enpp1 and Spp1, and thus hinder calcium deposition in the bone. These mineralization inhibitors show a trend to be increased in the Col4a3^−/−^ mice, but it remains to be investigated which factor induces their expression, as 1,25(OH)2_D_3 levels are decreased in Col4a3^−/−^ mice. On the other hand, FGF23 can also reduce matrix mineralization as it decreases, in osteoblasts, the expression of ALPL (TNAP) that converts the mineralization inhibitor PPi to inorganic phosphate and thus increases the ratio of PPi versus Pi.^44^ However, the expression of Alpl was not significantly decreased in Col4a3^−/−^ mice. The exact trigger that reduces bone formation and mineralization at this early stage of CKD remains, thus, to be elucidated.
Interestingly, the decrease in bone formation is associated with an increase in BMAT in Col4a3^−/−^ mice. An expansion of BMAT is classically linked to aging, osteoporosis, anorexia nervosa, and certain medications, but recent preclinical^45^ and clinical studies reported that CKD is also associated with greater marrow adiposity.^46–48^ Importantly, an excess of BMAT is linked to lower BMD and an elevated fracture risk.^49^ BM adipocytes and osteoblasts are derived from a common bipotential SSPC^50,51^ and the observed inverse relation between the numbers of osteoblasts and BM adipocytes in Col4a3^−/−^ mice suggests that the adipogenic lineage commitment of SSPCs is stimulated in CKD.
However, our in vitro studies show that the intrinsic capacity of SSPCs to differentiate into osteoblasts or adipocytes is not altered in Col4a3^−/−^ mice, as has also been reported in cultures derived from mice with adenine-induced CKD^45^ and in pediatric CKD patients.^52^ In contrast, several studies using osteoblasts derived from rats or patients with advanced CKD have reported reduced mineralization capacity. Therefore, we postulate that the epigenetic alterations observed in late-stage CKD may not yet be present in the Col4a3^−/−^ early-stage disease model.^51,53,54^ Likely, CKD-induced changes in hormone and mineral metabolism and/or the altered local bone microenvironment contribute to the increased number of BM adipocytes in vivo.
Indeed, recent evidence shows that acute renal damage increases glycerol-3-P production in the kidney, which is converted to lysophosphatidic acid (LPA) in bone cells, and LPA on its turn induces FGF23 production after binding to the LPA receptor 1.^55^ Given that glycerol-3-P is also a precursor for the synthesis of triglycerides, stored in adipocytes, it might be interesting to further investigate the link between a renal damage-induced altered metabolic profile and the increase in BM adiposity in CKD.
The decrease in bone mass at this early stage of CKD is not associated with impaired calcium handling in the intestine or kidney as intestinal calcium absorption and fractional calcium excretion are normal. Nevertheless, the gene expression of several calcium transporters is decreased in the intestine and kidney, likely because of reduced 1,25(OH)2_D_3 levels.^56,57^ This discrepancy between normal functional calcium (re)absorption and altered expression of calcium transporters suggests that other, still unidentified, calcium handling pathways exist and contribute to preserving normal serum calcium levels in the early stages of CKD. A similar observation was made in mice with the inactivation of Trpv6 and S100g, which did not reduce intestinal calcium absorption,^58^ indicating that other proteins contribute to intestinal calcium absorption.
Taken together, serum calcium and phosphate levels remain normal during the early stage of CKD because the altered levels of FGF23, PTH, and 1,25(OH)2_D_3 preserve serum calcium but at the expense of bone mass. The decrease in bone formation is associated with increased BM adiposity, but further investigation is necessary to delineate the underlying mechanisms. These findings suggest that bone properties, including BM adiposity, should be evaluated already at an early stage of CKD in patients.
Supplementary Material
ziag017_Supplemental_Files
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