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. Author manuscript; available in PMC: 2015 Jul 7.
Published in final edited form as: J Bone Miner Res. 2011 Aug;26(8):1904–1912. doi: 10.1002/jbmr.374

Chlorthalidone Improves Vertebral Bone Quality in Genetic Hypercalciuric Stone-Forming Rats

David A Bushinsky 1, Thomas Willett 2,3, John R Asplin 4, Christopher Culbertson 1, Sara PY Che 2, Marc Grynpas 2
PMCID: PMC4493760  NIHMSID: NIHMS342651  PMID: 21351146

Abstract

We have bred a strain of rats to maximize urine (U) calcium (Ca) excretion and model hypercalciuric nephrolithiasis. These genetic hypercalciuric stone-forming (GHS) rats excrete more UCa than control Sprague-Dawley rats, uniformly form kidney stones and, similar to patients, demonstrate lower bone mineral density. Clinically thiazide diuretics reduce UCa and prevent stone formation; however, whether they benefit bone is not clear. We used GHS rats to test the hypothesis that the thiazide diuretic chlorthalidone (CTD) would have a favorable effect on bone density and quality. Twenty GHS rats received a fixed amount of a 1.2% Ca diet and half were also fed CTD (4–5 mg/kg/day). Rats fed CTD had a marked reduction in UCa. The axial and appendicular skeletons were studied. An increase in trabecular mineralization was observed with CTD compared to controls. CTD also improved the architecture of trabecular bone. Using µCT, trabecular bone volume (BV/TV), trabecular thickness and trabecular number were increased with CTD. A significant increase in trabecular thickness with CTD was confirmed by static histomorphometry. CTD also improved the connectivity of trabecular bone. Significant improvements in vertebral strength and stiffness were measured by vertebral compression. Conversely, a slight loss of bending strength was detected in the femoral diaphysis with CTD. Thus, results obtained in hypercalciuric rats suggest that CTD can favorably influence vertebral fracture risk. CTD did not alter formation parameters suggesting that the improved vertebral bone strength was due to decreased bone resorption and retention of bone structure.

Keywords: rodent, nephrolithiasis, biomechanics, bone, hypercalciuria

INTRODUCTION

Hypercalciuria is the most common metabolic abnormality found in patients with nephrolithiasis (1). Hypercalciuria raises urine supersaturation with respect to the solid phases of calcium hydrogen phosphate (CaHPO4, brushite) and calcium oxalate (CaOx) enhancing the probability of nucleation and growth of crystals into clinically significant stones (1). Patients with hypercalciuria of unknown etiology, so called idiopathic hypercalciuria, often excrete more calcium than they absorb indicating a net loss of total body calcium (1;2). The source of this additional urine calcium is almost certainly the skeleton, by far the largest repository of calcium in the body (3). Indeed idiopathic hypercalciuria has been associated with markers of increased bone turnover (4). Urinary hydroxyproline is increased in unselected patients with idiopathic hypercalciuria (5) and serum osteocalcin levels are elevated in stone formers who have a defect in renal tubule calcium reabsorption but not in those with excessive intestinal calcium absorption (6). Bone turnover studies with 47Ca confirm increased formation and resorption (7). Cytokines known to increase bone resorption have also been shown to be elevated in patients with idiopathic hypercalciuria (8).

Bone mineral density is correlated inversely with urine calcium excretion in both men (9) and women (10). This relationship was confirmed in stone formers but not in non-stone formers (11). Patients with absorptive hypercalciuria have reduced bone formation and relatively increased bone resorption (12). A number of studies confirm that patients with nephrolithiasis have a reduction, though generally mild, in bone mineral density (BMD) compared to matched controls (4). After adjusting for a large number of variables an analysis of the 3rd National Health and Nutrition Examination Survey (NHANES III) demonstrated that men with a history of kidney stones have a lower femoral neck BMD than those without a history of stones (13). Analysis of almost 6000 older men demonstrated an association of kidney stones with decreased BMD (14). Stone formers have an increased risk of fractures (13;15). In NHANES III there was an increased risk of wrist and spine fractures in stone formers (13) and in a retrospective analysis stone formers had an increased incidence of vertebral fractures, but not fractures at other sites (15).

Thiazide diuretic agents such as chlorthalidone (CTD) reduce urine calcium excretion in normal people (16), patients with idiopathic hypercalciuria (17) and with hypoparathyroidism (18), and rats (19;20). These drugs act, at least in part, by stimulating calcium reabsorption in the distal convoluted tubule (16) and by producing extracellular fluid volume depletion (21). Thiazide diuretics are commonly used to treat calcium oxalate stone disease (1;22). Eight studies in humans have examined the effect of thiazide diuretics on preventing recurrent stone disease (23). A meta-analysis revealed that in all six studies in which treatment lasted for more than two years there was a significant reduction in stone recurrence rate (23). Thiazide diuretics are also used to treat hypertension (24). A number of studies have shown that in this clinical setting use of thiazide diuretics is associated with a reduction in osteoporotic fractures (25;26) and generally an increase in bone mineral density (27;28).

To establish an animal model of hypercalciuria we have successively inbred 82 generations of the most hypercalciuric progeny of hypercalciuric Sprague-Dawley rats, each of which now excretes 8 to 10 times as much urinary calcium as similarly fed controls (29–37). The hypercalciuria is due to increased intestinal calcium absorption (35), coupled to a defect in renal tubular calcium reabsorption (30;33) and enhanced bone mineral resorption (31), suggesting a systemic dysregulation of calcium homeostasis (34). Virtually all of these hypercalciuric rats form kidney stones while there was no evidence of stone formation in controls (29;32;36). We have termed the rats genetic hypercalciuric stone-forming (GHS) rats (29–37). We have recently demonstrated that GHS rats have a reduction in bone mineral density and bone strength even when fed a diet with ample calcium (37). Bones from the GHS rats had reduced density (measured with DXA), reduced trabecular volume and thickness, and were more brittle and fracture prone. Unlike in man, the diet of a rat can be precisely controlled while urine is quantitatively collected during studies lasting weeks to months and bone can be collected for detailed analysis. We used the GHS rats to ask whether, in addition to reducing urine calcium excretion, the thiazide diuretic chlorthalidone would improve bone density and quality in GHS rats.

Materials and Methods

Establishment of Hypercalciuric Rats

The genetic hypercalciuric stone forming rats were derived from Sprague-Dawley rats (Charles River Laboratories, Kingston, NY) by successively selecting and breeding the most hypercalciuric progeny of each generation as previously described (29–37).

Study Protocol

Twenty male GHS rats of the 82nd generation, initially weighing on average 215 g were housed in metabolic cages for 18 weeks. Rats were randomly divided into two groups:

All GHS rats were fed a High Calcium Diet (HCD, 1.2% calcium, 0.65% phosphorus, 0.24% magnesium, 0.4% sodium, 0.42% chloride, and 0.42% potassium) plus 5% hydroxyproline. All diets contained 2.2 IU vitamin D/g diet. With added hydroxyproline the GHS rats form calcium oxalate stones (29;36) and not calcium phosphate stones (32;38–40). By random allocation half of the rats were fed chlorthalidone (CTD, Sigma, St. Louis, MO) at a dose of 1mg CTD/15g of food. The rats not fed CTD served as controls (CTL). This amount of CTD provided approximately 4–5mg CTD/kg/24h, an amount which we have previously shown to decrease urine calcium excretion in rats (19;20).

Each rat was initially provided with 13 g/day of food, an amount that we have previously shown is completely consumed by a rat of this size (41). At 12 weeks, the amount of food was increased to 15 g/day to account for the increased dietary needs of the now larger rats. All rats were given deionized distilled water ad libitum. Any rat that ate less than 12 g of food per day until week 12 or ate less than 14 g of food per day from week 12 until the conclusion of the study or drank less than 15 mL of water on any day would have been excluded from the remainder of the study; however, all rats met these prospective criteria throughout the study.

At the end of weeks 6, 12, and 18, four consecutive 24-hour urine collections were obtained. Urine was collected with HCl on days 1 and 3 and Thymol on days 2 and 4. The samples collected in thymol were used for measurement of pH and chloride and the sample collected in HCl were used for all other measurements. All biochemical measurements were completed within two weeks.

At 18 weeks, the rats were killed and the lumbar spines (L1-L6) and femurs were removed, cleaned of connective tissue, and stored at −20° C. Bone quality was assessed as described below. The kidneys, ureters, and bladder were dissected en block, and radiographic analysis was performed. The presence of stones was determined in a blinded manner.

Chemical Determinations

Calcium, magnesium, phosphorus, ammonia, and creatinine, were measured spectrophotometrically using the Beckman CX5 Pro autoanalyzer (Beckman Instruments, Brea CA). Potassium, chloride, and sodium were measured by ion specific electrodes on the Beckman CX5. Urine pH was measured using a glass electrode. Citrate, oxalate, and sulfate were measured by ion chromatography using a Dionex ICS 2000 system (Dionex Corporation, Sunnyvale CA). Samples were loaded into a 25 µl loop using an autosampler and injected onto an AG-11 guard column and AS-11 analytical column in series, with KOH as the mobile phase. Ion peaks were detected using a conductivity meter with the eluent background conductivity suppressed using an anion self-regenerating suppressor.

Urine Supersaturation

The calcium oxalate ion activity product was calculated using the computer program EQUIL developed by B. Finlayson and associates (42). The computer program calculates free ion concentrations using the concentrations of measured ligands and known stability constants. Ion activity coefficients are calculated from ionic strength using the Davies modification of the Debye-Huckel solution to the Poisson-Boltzman equation. The program simultaneously solves for all known binding interactions among the measured substances. Oxalate, phosphorus and calcium ion activities were used to calculate the free-ion activity products. The free ions in solution are considered to be in equilibrium with the dissolved calcium oxalate governed by a stability constant (K) of 2.746 × 103M−1 and with the dissolved calcium hydrogen phosphate (brushite) governed by a K of 0.685 × 103M−1. The value of calcium oxalate in a solution at equilibrium with a solid phase of calcium oxalate, the solubility of calcium oxalate, is 6.16 × 10−6 M per liter. The value of brushite in a solution at equilibrium with a solid phase of brushite, the solubility of brushite, is 3.981 × 10−7 M per liter. The relative supersaturation for calcium oxalate is calculated as the ratio of the free-ion activity product of calcium and oxalate in the individual urine to the solubility of calcium oxalate. The relative supersaturation for brushite is calculated as the ratio of the free-ion activity product of calcium and phosphate in the individual urine to the solubility of calcium phosphate. Ratios of 1 connote a sample at equilibrium, above 1 supersaturation, and below 1 undersaturation.

Bone Quality Assessment

The effect of CTD treatment on bone quality was assessed in both the axial (vertebrae) and appendicular (femurs) skeleton from rats in each group as follows.

Micro-Computed Tomography (µCT)

To measure specimen geometry, architecture and volumetric bone mineral density (vBMD), the fifth lumber vertebra (L5) and right femur were scanned using a micro-computed tomography device (SkyScan 1174, SkyScan, Belgium). Three-dimensional model reconstruction and analyses were performed using software supplied by the µCT manufacturer (NRecon and CTan, SkyScan, Belgium). The vertebrae were scanned at 14.5µm and the femurs were scanned at 32.5µm camera spatial resolution. vBMD (g/cm3) was calibrated using two phantoms composed of known densities of calcium hydroxyapatite in epoxy.

The average height and cross-sectional area of the whole vertebra were determined using two-dimensional measurement tools in CTan. vBMD of each whole L5 was measured using a region of interest surrounding the whole bone. Using a region of interest defining only the trabecular bone in each L5, vBMD (g/cm3), trabecular bone volume (BV/TV; %), trabecular thickness (Tb.Th; µm), trabecular separation (Tb.Sp.; µm), and trabecular number (Tb.N.; 1/mm) were computed using three-dimensional analysis tools in CTan. These are all calculated using standard techniques (43). Femoral geometry and vBMD were measured at the mid-diaphysis over a 1mm range. The mean cross-sectional area (mm2), anterior-posterior diameter (mm), medial-lateral diameter (mm), cortical thickness (mm), minimum principle second moment of area (about the medial-lateral axis, Imin; mm4) and polar moment of area (J; mm4) were measured using CTan. Imin and J are measures of the structural resistance to bending or twisting respectively and are independent of the type of material.

Mechanical Testing

The mechanical properties of the vertebrae (L5) and right femur were measured using an Instron 4465 materials testing machine (Instron, Norwood, MA, USA) with a 1-kN load cell and a data acquisition card interfaced with a Pentium II PC sampling at 10Hz.

The body of each L5 was tested in compression using a modification of a technique previously published (44). Briefly, each vertebra was trimmed of soft tissue and processes leaving a quasi-cylindrical body with non-co-planar ends. The most orthogonal end was carefully sanded with fine grade sand paper until perpendicular with the long axis of the body. This end was then stood on a loading platen and held in place with a very small dot of cyanoacrylate glue. The opposing end of the body was then covered with a thin layer of PMMA, the top loading platen was then bought into contact to a 1-N preload allowing the PMMA to form a tight interface with the end. These steps ensured uniform loading of the specimens. After 10-minutes of PMMA curing time, the pre-load was increased to 5-N and the test run with a deformation rate of 1mm/min until failure, indicated by a >10% drop in load. Three-point-bending, load-deformation and stress-strain data were determined according to procedures described previously (45).

The diaphysis of each right femur was tested in three-point bending. Briefly, each femur was positioned posterior side down with a gauge length of 15.6mm. Each specimen was pre-loaded to a load of 1N and deformed at 1mm/min until complete failure. Stress-strain curves were generated from load-deformation curves by accounting for specimen geometry measured with µCT. From the load-deformation curve, ultimate load, failure displacement, energy-to-failure and stiffness were determined. From the stress-strain curve, ultimate stress, failure strain, toughness and modulus were determined.

Subsequently, the femoral neck of each right femur was tested to evaluate its mechanical properties. The diaphysis was embedded in PMMA with the long axis oriented concentric to the axis of a holder that mounted into the cross-head of the materials testing machine. The superior surface of the femoral head was then brought into contact with a loading edge of a hole in a thick metal plate. Each specimen was pre-loaded to 1N and then deformed at 1mm/min until fracture. Only load-deformation data could be determined for these tests because accounting for specimen geometry is prohibitive.

Histomorphometry

Static histomorphometry was performed on the L1 vertebrae to evaluate structural and formation parameters following the American Society for Bone and Mineral Research nomenclature and guidelines (46). The undecalcified vertebrae were first fixed in 70% ethanol, dehydrated in increasing concentrations of acetone (up to 100%) and then infiltrated with increasing concentrations of unpolymerized Spurr resin in acetone (up to 100% Spurr resin). Subsequently, the specimens were embedded in polymerized Spurr blocks. 5-µm sections were cut from the blocks using a rotary microtome (Leica RM 2265) with a tungsten carbide blade. The sections were stained with Goldner’s Trichrome stain. The trabecular bone was analyzed using a 25x objective and semi-automated bone histomorphometry system (BioQuant Nova Prime). The following data were measured: tissue volume (TV), bone volume (BV), bone surface (BS), osteoid surface (OS), osteoid volume (OV), and osteoid width (O.Wi). The following structural data were then calculated: bone volume (BV/TV;%), trabecular thickness (Tb.Th = BV/BS; µm), trabecular number (Tb.N; 1/mm), trabecular separation (Tb.Sp; µm). The following formation data were also calculated: osteoid volume (%OV; %), osteoid surface (OS/BS; %), osteoid thickness (O.Th.; µm).

Back Scattering Electron (BSE) Microscopy and Strut Analysis

After sections were cut for bone histomorphometry as discussed above, the plastic blocks were polished to a 1-um finish using diamond paste on a polisher (Phoenix BETA grinder/polisher). The blocks were imaged with a Philips XL30 SEM (Philips, USA) using a BSE detector (FEI, Hillsboro, Oregon, USA) with a voltage of 20kV, and a working distance of 15mm. The spot size was set to 6.2 to maintain a constant current of approximately 1nA in the beam as measured with a pico-ammeter connected to the SEM and a Faraday cup. A SiO2 standard and a MgF standard were used to calibrate the brightness and contrast. Six to eight fields were taken for each sample at 100x magnification, and were stitched together using software provided with the SEM. From each specimen, the grayscale histograms of the cortical and the trabecular bone were used for analysis. Mineralized bone exhibits a range of grayscale values, and the area at each gray scale was determined by image analysis using Quantimet software (Leica). Regions with higher grey levels (lighter) are more mineralized than those with lower grey levels (darker). The peak gray level represents the degree of mineralization and the full width at half maximum height (FWHMH) represents the heterogeneity of mineralization. Additionally, a logit function, as defined by Logit = ln (area under curve below cutoff / area under curve above cutoff) was used to compare the shift in mineralization profiles (47). The cutoff value was set to the average maximum grey level of the Vehicle group. A negative logit function represents a hypermineralized (i.e. more mineralized) distribution, while a positive logit function represents a hypomineralized (i.e. less mineralized) distribution.

Strut analysis was performed on the BSE images of L1 vertebrae to analyze the connectivity of the trabecular bone (48). Using Quantimet software, the images were thresholded and transformed into binary images. Subsequently, the trabecular bones were skeletonized to struts (49). The computer also automatically recognizes nodes, points at which three or more struts are connected, and free ends. Various connectivity parameters were measured, including total strut length, number of nodes, length of free-node strut, length of node-node strut, number of free ends and length of free-free strut.

Statistical Analysis

All values are expressed as mean + SE. Tests of significance were calculated by T tests, as appropriate, using conventional computer programs (SPSS, Chicago, IL, USA). p < 0.05 was considered significant. Tests of normality and equality of variance (Levene’s test) were performed to determine whether parametric or non-parametric tests should be utilized. For non-parametric tests, the Mann-Whitney U test was employed.

RESULTS

Urine Ion and Volume Excretion

Every six weeks, four successive 24-hr urine collections were obtained. The individual urine collections for the 20 rats divided into 2 groups were analyzed separately.

When compared to CTL, during all three-time periods, CTD induced a significant decrease in UCa excretion (Figure 1, top). When compared to CTL, during all three time periods CTD also induced a significant decrease in U phosphorus excretion (Figure 1, middle). When compared to CTL, during the first two time periods CTD induced a significant increase in oxalate excretion; however, during the third time period there was no difference in U oxalate excretion between the two groups (Figure 1, bottom).

Figure 1.

Figure 1

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Twenty Four hour Urine Calcium, Phosphorus and Oxalate. All GHS rats were fed a 1.2% Ca diet plus 5% hydroxyproline. Some rats were fed chlorthalidone (CTD) at a dose of 1mg CTD/15g of food while others were not fed CTD and served as controls (CTL). At the end of weeks 6, 12, and 18, four consecutive 24-hour urine collections were obtained and measured. All values are expressed as mean + SE. *, p < 0.05 compared with CTL.

Over the 18 wk experiment UNa was not different in CTD compared to CTL (1.63 +/− 0.06 mmole/24h vs. 1.49 +/− 0.08 respectively, p = NS) while UV was less in CTD compared to CTL (33.2 +/− 1.1 ml/24h vs. 41.6 +/− 0.9 respectively, p<0.01).

Supersaturation

When compared to CTL, CTD did not alter urine supersaturation with respect to the CaOx solid phase during any time period (Figure 2, top). When compared to Ctl, during all three-time periods CTD induced a significant decrease in urine supersaturation with respect to the CaHPO4 solid phase (Figure 2, bottom).

Figure 2.

Figure 2

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Urine Calcium Oxalate and Calcium Phosphate Supersaturation. All GHS rats were fed a 1.2% Ca diet plus 5% hydroxyproline. Some rats were fed chlorthalidone (CTD) at a dose of 1mg CTD/15g of food while others were not fed CTD and served as controls (CTL). At the end of weeks 6, 12, and 18, four consecutive 24-hour urine collections were obtained, measured and supersaturation (SS) for calcium oxalate (CaOx) and calcium phosphate calculated (CaP). All values are expressed as mean ± SE. *, p < 0.05 compared with CTL.

Stone Formation

Eight of the 10 CTL rats formed radiographically detectable stones while 4/10 CTD rats formed stones (p < 0.025).

Vertebral Bone Quality

Bone Mineral Density, Geometry, Architecture and Formation

The bone mineral density and geometry of the vertebrae and the structure of the trabecular bone of the vertebrae were evaluated using µCT and static histomorphometry. CTD did not alter vBMD at the whole bone level nor in the trabecular compartment (Table 1). CTD also did not alter the height and cross-sectional area. Trabecular bone volume, thickness and number, measured by µCT, were increased by CTD indicating denser trabecular architecture. Similarly, an increased trabecular thickness was measured by static histomorphometry. Formation parameters (%OV, OS/BS, O.Th) were not different suggesting that bone formation was not altered by CTD treatment.

Table 1.

Vertebrae: Bone Mineral Density, Geometry and Structure

µCT BMD Vehicle CTD
Whole L5 vBMD (g/cm3) 0.794 ± 0.042 0.793 ± 0.024
L5 Trab vBMD (g/cm3) 0.360 ± 0.047 0.385 ± 0.064
Geometry Vehicle CTD
Height H. (mm) 6.98 ± 0.16 6.98 ± 0.28
Cross-sectional Area C.A (mm2) 6.44 ± 0.41 6.74 ± 0.80
Trabecular Structure by µCT Vehicle CTD
Bone volume BV/TV (%) 34.0 ± 1.8 38.6 ± 4.1 *
Thickness Tb.Th (µm) 97.9 ± 1.8 102.0 ± 2.6 *
Number Tb.N (1/mm) 3.47 ± 0.19 3.78 ± 0.34 *
Separation Tb.S (µm) 235 ± 15 220 ± 26
Trabecular Structure by Static Histomorphometry Vehicle CTD
Bone volume BV/TV (%) 26.7 ± 4.5 29.2 ± 6.4
Thickness Tb.Th (µm) 56.8 ± 7.3 65.3 ± 10.5*
Number Tb.N (1/mm) 4.74 ± 0.91 4.45 ± 0.47
Separation Tb.S (µm) 162 ± 44 162 ± 29
Formation Parameters Vehicle CTD
Osteoid Volume %OV (%) 0.03 ± 0.04 0.04 ± 0.03
Osteoid Surface OS/BS (%) 0.48 ± 0.46 0.64 ± 0.56
Osteoid Thickness O.Th (µm) 2.23 ± 0.95 2.42 ± 1.10
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*

p-value<0.05 versus Vehicle

Vertebral Compression Testing

CTD induced a significant increase in ultimate load, yield load, and stiffness (Table 2). When specimen structure was accounted for, CTD significantly increased ultimate stress and yield stress i.e. the apparent strength of the material itself was increased.

Table 2.

Vertebrae: Compression Tests

Vehicle CTD
Structural Ultimate Load (N) 243 ± 29 298 ± 57*
Yield Load (N) 201 ± 29 252 ± 41*
Energy to Failure (mJ) 65 ± 31 68 ± 26
Stiffness (N/mm) 1272 ± 336 1705 ± 513*
Material Ultimate Stress (MPa) 37.8 ± 4.3 44.1 ± 6.0*
Yield Stress (MPa) 31.4 ± 4.8 37.8 ± 7.0*
Toughness (mJ/mm3) 1.42 ± 0.62 1.43 ± 0.45
Modulus (MPa) 1380 ± 380 1760 ± 480
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*

p-value<0.05 versus Vehicle

Mineralization and Strut Analysis

BSE imaging was conducted on the embedded L1 vertebrae to evaluate mineralization profiles and trabecular architecture. CTD did not alter mineralization heterogeneity (FWHM) in either the cortical or trabecular bone. Mineralization was significantly increased in both the cortical shell and the trabeculae (Table 3). In both the cortical and trabecular bone compartments, CTD increased the grey value of the histogram peak by 7% indicating a more highly mineralized mineral phase (Figure 3). This may have contributed to the higher apparent ultimate stress and yield stress measured in the vertebral compression tests.

Table 3.

Back Scattering Electron Microscopy and Strut Analysis

BSE Vehicle CTD
Cortical Logit (−) 0.24 ± 0.99 −0.93 ± 0.86*
Max Grey Level (pixel) 154.3 ± 8.0 165.8 ± 8.9*
FWHMH (pixel) 27.4 ± 3.2 24.50 ± 4.04
Trabecular Logit (−) −0.10 ± 0.80 −0.85 ± 0.61*
Max Grey Level (pixel) 151.2 ± 9.0 162.1 ± 8.6*
FWHMH (pixel) 26.3 ± 3.8 26.00 ± 1.8
Strut Analysis Vehicle CTD
Trabecular Total Strut Length (mm/mm2) 5.57 ± 0.59 6.26 ± 0.75*
Number of Nodes (mm−2) 13.72 ± 3.4 19.24 ± 5.2*
Length of Free-Node Strut (mm/mm2) 1.30 ± 0.26 1.09 ± 0.30
Length of Node-Node Strut (mm/mm2) 3.30 ± 0.76 4.23 ± 1.12*
Number of Free Ends (mm−2) 6.33 ± 0.87 6.0 ± 2.0
Length of Free-Free Strut (mm/mm2) 0.12 ± 0.09 0.07 ± 0.05
Total Strut Length (mm/mm2) 5.57 ± 0.59 6.26 ± 0.75*
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*

p-value<0.05 versus Vehicle

Figure 3.

Figure 3

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Averaged Histograms of Cortical and Trabecular Bone for Vehicle and CTD Group

Femoral Bone Quality

Bone Mineral Density, Geometry and Structure

Diaphysial volumetric bone mineral density, geometry and structure of the femora were measured using µCT. CTD did not alter vBMD (Table 4). However, CTD reduced the second moment of area (Imin) and polar moment of area (J) indicating an altered diaphysial structure.

Table 4.

Femur: Cortical Bone Mineral Density, Geometry and Structure Femur: Cortical Bone Mineral Density, Geometry and Structure

Vehicle CTD
µCT vBMD (g/cm3) 0.992 ± 0.030 1.002 ± 0.019
C.Th (mm) 0.655 ± 0.012 0.661 ± 0.011
C.A (mm2) 7.28 ± 0.26 7.03 ± 0.26
D.M/L (mm) 4.22 ± 0.18 4.10 ± 0.10
D.A/P (mm) 3.50 ± 0.09 3.41 ± 0.10
IMIN(mm4) 8.43 ± 0.75 7.45 ± 0.75*
J (mm4) 20.71 ± 1.92 18.45 ± 2.06*
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*

p-value<0.05 versus Vehicle

vBMD = volumetric bone mineral density

C.Th = cortical thickness

C.A = cortical area

D.M/L = medial-lateral diameter

D.A/P = anterior-posterior diameter

Imin = minimum principle second moment of area

J = polar moment of area

Mechanical Properties in Three-Point Bending

Three-point bending was used to evaluate the mechanical properties of the femoral diaphysial cortex of the right femurs. CTD decreased the ultimate load borne by the bone by 8% (Table 5). When structure was accounted for, this loss in strength was not reflected in the ultimate stress, indicating no difference in material properties but indicating a structural difference. The lower ultimate load was solely a result of the lower Imin and smaller anterior-posterior diameter.

Table 5.

Femur: Mechanical Tests

Vehicle CTD
Structural Ultimate load (N) 166.4 ± 9.2 153.2 ± 7.4*
Failure Displacement (mm) 0.56 ± 0.15 0.49 ± 0.09
Energy to failure (mJ) 61 ± 23 44.1 ± 9.6
Stiffness (N/mm) 569.8 ± 63 538.8 ± 34
Material Ultimate Stress (MPa) 135.2 ± 8.4 138.5 ± 8.6
Failure Strain (%) 4.8 ± 1.4 4.05 ± 0.74
Toughness (mJ/mm3) 4.2 ± 1.5 3.30 ± 0.60
Modulus (GPa) 5.4 ± 4.9 5.84 ± 5.83
Femoral Neck Fracture
Ultimate Load (N) 107 ± 22 95 ± 12
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*

p-value<0.05 versus Vehicle

Femoral Neck Fracture

The strength of the femoral necks of the right femora was evaluated using femoral neck fracture. CTD treatment did not alter the femoral neck strength (Table 4).

DISCUSSION

In this study we demonstrated that administration of the thiazide diuretic chlorthalidone to GHS rats not only reduces urine calcium excretion and supersaturation with respect to the calcium hydrogen phosphate (CaHPO4, brushite) solid phase, but has beneficial effects on vertebral bone quality, a common site of osteoporotic fracture in humans (50).

Bone quality assessment techniques were used to study the effect of chlorthalidone treatment on the axial and appendicular skeleton of GHS rats. Chlorthalidone had a significant positive effect on the trabecular bone volume of the lumbar vertebrae and on trabecular mineralization. Trabecular vBMD was not altered by chlorthalidone. The increased bone volume may be a direct result of decreased bone resorption due to CTD treatment as suggested by an early clinical study which demonstrated decreased serum tartrate resistant acid phosphatase and increased bone mass (51). As bone resorption is reduced, bone volume can be improved due to a shift in remodeling in favor of formation. Additionally the bone has more time to undergo secondary mineralization. As a result, the mineralization and the BV/TV are greater with CTD than with vehicle.

Chlorthalidone treatment improved the architecture of trabecular bones. As indicated by the structural parameters obtained from µCT, in addition to trabecular bone volume (BV/TV), trabecular thickness and trabecular number increased significantly. CTD also improved the connectivity of trabecular bone as found with strut analysis.

The cumulative effect of these positive changes in trabecular structure and secondary mineralization induced by chlorthalidone resulted in a significant improvement in vertebral strength and stiffness measured in compression. A greater number of positive changes were observed in the trabecular bone compared to cortical bone, likely due to the higher cellular activity in trabecular bone compared to cortical bone (52). The formation parameters measured with static histomorphometry were not altered by chlorthalidone indicating that the effect of chlorthalidone may be antiresorptive. Increased BV/TV without increased formation rate suggests less resorption: the only other known way to increase bone mass. Chlorthalidone increased the mineralization of the cortical bone and appeared to change diaphysial geometry, leading to a slightly weaker femur. Nevertheless, the material-level properties of the cortical bone remained unchanged. However, with femoral neck fracture, a more clinically representative test, no change in strength was detected. This loss of structural strength in the diaphysis does not correspond to clinical results (51;53) and may be explained by the differences in physiology between rats and humans. Rat cortical bone grows throughout life and lacks secondary osteal remodeling.

In a clinical study of post menopausal women, CTD use for an average of 2.6 years was associated with a significant reduction in annual bone loss rates compared with placebo use (53). In a large study of 3928 patients from the Hypertension Detection and Follow-up program, alkaline phosphatase declined progressively over 3 years leading to a more positive calcium balance and a reduction in bone turnover. The authors suggest that a beneficial effect of CTD could be decreased osteoporosis (54).

Two studies have examined the effect of thiazides on bone in hypercalciuric patients and their findings are both consistent with our observations in GHS rats (55;56). Adams et al treated 5 males with hypercalciuria and osteoporosis with thiazide and found an increase in BMD after an average of 9 months of follow up. Steiniche performed bone biopsies before and after a 6 month treatment with thiazide diuretics and found a reduction in bone turnover. Neither of these studies examined the effect of thiazides on bone quality or mechanics as was done in the current study.

The decrease in urine calcium excretion in the GHS rats fed chlorthalidone is almost certainly due to the increase in renal tubular calcium reabsorption induced by this medication (16;57). We have previously shown in rats (19) and man (17) that the decrease in urine calcium excretion is allowed to persist due to the chlorthalidone-induced reduction in intestinal calcium absorption. The increase in urine oxalate may be due to a reduction in calcium excretion. The chlorthalidone-induced reduction in urine calcium excretion, may have allowed less substrate for CaOx stone formation, less oxalate incorporation into stones and an increase in urine oxalate excretion. Previously we have shown that the GHS rats fed 5% hydroxyproline, an oxalate precursor, formed only calcium oxalate stones yet their urine oxalate excretion did not increase (29;36). In a prior study we have also shown that the increase in oxalate could be approximately accounted for by reduction in stone formation (20). While the increase in urinary oxalate may be ascribed to a reduction of oxalate consumption by the reduced number of calcium oxalate stones, further studies will be necessary to precisely define the physiologic basis for an increase in oxalate with CTD.

The decrease in urine phosphorus excretion after feeding the GHS rats chlorthalidone may be explained by our prior study in normal rats (19). As above when rats are fed chlorthalidone there is a decrease in intestinal calcium absorption, which allows the continued reduction in urine calcium excretion (19). The reduction in calcium absorption would lead to an increase in calcium in the intestinal lumen, which would be available to bind intestinal phosphate and reduce the available phosphate for absorption and subsequent excretion. In patients with chronic kidney disease, calcium has been shown to complex with intestinal phosphorus leading to a decrease in intestinal phosphorus absorption (58). We have also shown that the administration of chlorthalidone to humans also leads to a reduction in intestinal calcium absorption (59).

We have previously shown that the GHS rats fed a standard diet form calcium phosphate stones (32;38–40) which appears to be the favored initial ion complex in human calcium oxalate stone formation (60;61). The addition of the common amino acid and oxalate precursor, hydroxyproline, leads to formation of calcium oxalate kidney stones (29;36). In this study chlorthalidone induced a reduction only of supersaturation with respect to CaHPO4; there was no change in the supersaturation with respect to CaOx. In GHS rats not given additional hydroxyproline, we have previously shown that reduction of supersaturation with respect to the CaHPO4 solid phase leads to a reduction in stone formation (39). This study confirms our previous observation that CTD reduced stone formation in GHS rats (20).

Compared to the Sprague-Dawley strain from which they were derived GHS rats are polyuric (62) and this polyuria may be secondary to their hypercalciuria. Tubular calcium activates the calcium sensing receptor which induces movement of aquaporin 2 from the apical membrane into endocytic compartments. The reduction in apical membrane aquaporin 2 decreases trans tubular water reabsorption resulting in polyuria (63–65). In this study the decrease in urine volume in the GHS rats fed CTD may be related to the reduction in hypercalciuria induced by this diuretic.

Our study demonstrates that, in a model of hypercalciuric nephrolithiasis, CTD not only reduced urinary Ca excretion, calcium phosphate supersaturation and stone formation but also enhanced bone quality in trabecular bone. These data are in agreement with previous studies in patients showing a decrease in bone loss and a reduction in bone turnover. CTD may be able to reduce bone resorption (51) and increase bone mineralization which will subsequently lead to a decrease in bone formation (54), an effect found in most bone antiresorptive drugs.

Thus chlorthalidone affected the mineralization, geometry, architecture and strength of the vertebrae leading to positive effects on vertebral bone quality; most notably, increased compressive strength. Vertebral bone is the most common site of osteoporotic fracture and, if studies in the GHS rats can be replicated in humans, chlorthalidone may be useful in preventing vertebral fractures especially in hypercalciuric stone-formers. However, given the short duration of these studies and the known differences between rodent and human bone, long term, carefully controlled studies in hypercalciuric humans should be undertaken before these encouraging studies on the effects of chlorthalidone on the bone of hypercalciuric stone-forming rats are applied to hypercalciuric humans.

Acknowledgments

This work was supported by Grants RO1 DK 75462 and RO1 AR 46289 (both to D.A.B.) from the National Institutes of Health.

Footnotes

Conflict of Interest

David A. Bushinsky: none

Thomas Willett: none

John R. Asplin: employee of Litholink which measures urine supersaturation in stone formers

Christopher Culbertson: none

Sara P.Y. Che: none

Marc Grynpas: none

Contributor Information

David A. Bushinsky, Email: david_bushinsky@urmc.rochester.edu.

Thomas Willett, Email: willett@lunenfeld.ca.

John R. Asplin, Email: jasplin@litholink.com.

Christopher Culbertson, Email: christopher_culbertson@urmc.rochester.edu.

Sara P.Y. Che, Email: sara.py.che@gmail.com.

Marc Grynpas, Email: GRYNPAS@lunenfeld.ca.

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