Effect of bolus and divided feeding on urine ions and supersaturation in genetic hypercalciuric stone-forming rats.

Because urine ion excretion varies throughout the day, clinicians monitor 24 h urine samples to measure ion excretion and supersaturation in kidney stone patients. However, these results are averages and may not reflect maximal supersaturation which drives stone formation. We measured ion excretion and saturation in genetic hypercalciuric stone-forming rats on both a normal or low calcium diet over 0-3, 3-6 and 6-24 h using two feeding protocols, where the daily food allotment was fed either as a bolus or divided into three portions. With a normal calcium diet, urine calcium, oxalate, volume, and calcium oxalate supersaturation were significantly greater on the bolus compared to the divided feeds in the prandial and postprandial periods. Bolus eaters also excreted more calcium and oxalate and had increased volume over 24 h. Maximal calcium oxalate supersaturation was greater during the initial time periods than during the entire 24 h, regardless of the feeding schedule. With the low calcium diet, the effect of bolus feeding was reduced. Thus, urine ion excretion and supersaturation vary with the type of feeding. If these results are confirmed in man, it suggests that eating as a bolus may result in greater prandial and postprandial calcium oxalate supersaturation. This may increase growth on Randall's plaques and promote stone disease.

Effect of cinacalcet on urine calcium excretion and supersaturation in genetic hypercalciuric stone-forming rats.

Idiopathic hypercalciuria is the most common metabolic abnormality in patients with nephrolithiasis. Through successive inbreeding, we have developed a strain of rats whose urine calcium (UCa) excretion is approximately 8-10-fold greater than that of control rats and who spontaneously form kidney stones. We have termed these rats genetic hypercalciuric stone-forming (GHS) rats. The physiology of the hypercalciuria in the GHS rats closely parallels that of man. We have recently shown that the GHS rat kidneys have an increased number of receptors for calcium (CaR) compared to Sprague-Dawley rats, the strain of rats originally bred to develop the GHS rats. Calcimimetics, such as cinacalcet (Cin), increase the sensitivity of the CaR to Ca. The effects of Cin on UCa are complex and difficult to predict. We tested the hypothesis that Cin would alter urinary (U) Ca and supersaturation with respect to calcium hydrogen phosphate (CaHPO(4)) and calcium oxalate (CaOx). GHS or control rats were fed a normal Ca diet (0.6% Ca) for 28 days with Cin (30 mg/kg/24 h) added to the diet of half of each group for the last 14 days. The protocol was then repeated while the rats were fed a low Ca (0.02% Ca) diet. We found that Cin led to a marked reduction in circulating parathyroid hormone and a modest reduction in serum Ca. Cin did not alter UCa when the GHS rats were fed the normal Ca diet but lowered UCa when they were fed the low Ca diet. However, Cin did not alter U supersaturation with respect to either CaOx or CaHPO(4) on either diet. If these findings in GHS rats can be confirmed in man, it suggests that Cin would not be an effective agent in the treatment of human idiopathic hypercalciuria and resultant stone formation.

Metabolic, but not respiratory, acidosis increases bone PGE(2) levels and calcium release.

A decrease in blood pH may be due to either a reduction in bicarbonate concentration ([HCO(3)(-)]; metabolic acidosis) or to an increase in PCO(2) (respiratory acidosis). In mammals, metabolic, but not respiratory, acidosis increases urine calcium excretion without altering intestinal calcium absorption, indicating that the additional urinary calcium is derived from bone. In cultured bone, chronic metabolic, but not respiratory, acidosis increases net calcium efflux (J(Ca)), decreases osteoblastic collagen synthesis, and increases osteoclastic bone resorption. Metabolic acidosis increases bone PGE(2) production, which is correlated with J(Ca), and inhibition of PGE(2) production inhibits this acid-induced J(Ca). Given the marked differences in the osseous response to metabolic and respiratory acidosis, we hypothesized that incubation of neonatal mouse calvariae in medium simulating respiratory acidosis would not increase medium PGE(2) levels, as observed during metabolic acidosis. To test this hypothesis, we determined medium PGE(2) levels and J(Ca) from calvariae incubated at pH approximately 7.1 to model either metabolic (Met; [HCO(3)(-)] approximately 11 mM) or respiratory (Resp; PCO(2) approximately 83 Torr) acidosis, or at pH approximately 7.5 as a control (Ntl). We found that after 24-48 and 48-51 h in culture, periods when cell-mediated J(Ca) predominates, medium PGE(2) levels and J(Ca) were increased with Met, but not Resp, compared with Ntl, and there was a direct correlation between medium PGE(2) levels and J(Ca). Thus metabolic, but not respiratory, acidosis induces the release of bone PGE(2), which mediates J(Ca) from bone.

Effect of acidosis on urine supersaturation and stone formation in genetic hypercalciuric stone-forming rats.

BACKGROUND: We have successively inbred over 45 generations a strain of rats to maximize urine calcium excretion. The rats now consistently excrete 8 to 10 times as much calcium as controls and uniformly form poorly crystalline calcium phosphate kidney stones. In humans with calcium nephrolithiasis, consumption of a diet high in acid precursors is often cited as a risk factor for the development of calcium-based kidney stones; however, the effect of this diet on urinary supersaturation with respect to the common solid phases found in kidney stones has not been determined. METHODS: To determine the effect of the addition of an acid precursor on urine ion excretion, supersaturation, and stone formation, we fed these genetic hypercalciuric stone-forming (GHS) rats 13 g/day of a 1.2% calcium diet with 0.0, 0.5, 1.0, or 1.5% NH4Cl in the drinking water for 14 weeks (N = 8 for each). Urine was collected and analyzed every two weeks. RESULTS: As expected, the addition of dietary NH4Cl led to a progressive fall in urine pH and urine citrate, while urine ammonium increased. Urine calcium and phosphorus increased, while urine oxalate fell. Increasing dietary NH4Cl led to a fall in supersaturation with respect to CaHPO4 (brushite) and CaOx and a rise in supersaturation with respect to uric acid. In spite of differences in supersaturation, most rats in each group formed stones that contained calcium phosphate and not calcium oxalate. CONCLUSIONS: Thus, while the provision of additional dietary acids alters urinary ion excretion and lowers supersaturation with respect to CaHPO4 and CaOx, it does not change the character or rate of stone formation in the GHS rats.

Acid-base imbalance and the skeleton.

Humans generally consume a diet that generates metabolic acids leading to a reduction in the concentration of systemic bicarbonate and a fall in pH. In vitro experiments indicate that this metabolic acidosis causes a release of calcium from bone that initially is simply due to physicochemical dissolution of the mineral. On a more chronic basis metabolic acidosis alters bone cell function; there is an increase in osteoclastic bone resorption and a decrease in osteoblastic bone formation. Concomitant with the dissolution and resorption of the bone mineral there is buffering of the addition protons by bone leading to restoration of the systemic pH. Interestingly respiratory acidosis, caused by an increase in the partial pressure of carbon dioxide induces far less bone dissolution and resorption and the additional hydrogen ions are not buffered by bone. As we age we are less able to excrete these metabolic acids due to the normal decline in renal function. We hypothesize that a slight, but significant, metabolic acidosis leads to greater loss of bone mineral and increase potential to fracture.

Prostaglandins regulate acid-induced cell-mediated bone resorption.

Metabolic acidosis induces bone calcium efflux initially by physicochemical dissolution and subsequently by cell-mediated mechanisms involving inhibition of osteoblasts and stimulation of osteoclasts. In rat kidney, acidosis increases endogenous prostaglandin synthesis, and in bone, prostaglandins are important mediators of resorption. To test the hypothesis that acid-induced bone resorption is mediated by prostaglandins, we cultured neonatal mouse calvariae in neutral or physiologically acidic medium with or without 0.56 microM indomethacin to inhibit prostaglandin synthesis. We measured net calcium efflux and medium PGE(2) levels. Compared with neutral pH medium, acid medium led to an increase in net calcium flux and PGE(2) levels after both 48 h and 51 h, a time at which acid-induced net calcium flux is predominantly cell mediated. Indomethacin inhibited the acid-induced increase in both net calcium flux and PGE(2). Net calcium flux was correlated directly with medium PGE(2) (r = 0.879, n = 29, P < 0.001). Exogenous PGE(2), at a level similar to that found after acid incubation, induced net calcium flux in bones cultured in neutral medium. Acid medium also stimulated an increase in PGE(2) levels in isolated bone cells (principally osteoblasts), which was again inhibited by indomethacin. Thus acid-induced stimulation of cell-mediated bone resorption appears to be mediated by endogenous osteoblastic PGE(2) synthesis.

Contribution of organic material to the ion composition of bone.

Studies of bone mineral ranging from cadaveric analysis to the use of high-resolution ion microprobe with secondary ion mass spectroscopy (SIMS) have concluded that bone is rich in sodium and potassium relative to calcium. Exposure of bone to acid conditions either in vitro or in vivo leads to an exchange of hydrogen ions for sodium and potassium buffering the acidity of the medium or blood, respectively. Whether these monovalent ions reside within the mineral or organic phases of bone has never been determined. To determine the contribution of organic material to bone ion composition, we dissected calvariae from 4- to 6-day-old mice, removed organic material of some with hydrazine (Hydr), and prepared all bones for analysis using a high-resolution scanning ion microprobe coupled to a secondary ion mass spectrometer. We found that in non-Hydr-treated calvariae (Ctl) there was far more surface sodium and potassium than calcium (23Na/ 40Ca = 15.7 + 1.9, ratio of counts of detected secondary ions, mean + 95% CI, 39K/40Ca = 44.0 + 1.5). Removal of organic material with hydrazine (Hydr) led to a marked fall in the ratio of sodium to calcium and potassium to calcium (23Na/40Ca = 5.9 + 1.4, p < 0.025 vs. respective Ctl and 39K/40Ca = 1.1 + 1.5, p < 0.001 vs. respective Ctl). Similarly, when examining the cross-section of the calvariae there was more sodium and potassium than calcium (23Na/40Ca = 8.6 + 1.6, 39K/40Ca = 26.7 + 1.8). Treatment with Hydr again caused a marked fall in both ratios (23Na/40Ca = 0.3 + 1.6, p < 0.001 vs. respective Ctl and 39K/40Ca = 0.02 + 1.9, p < 0.001 vs. respective Ctl). Thus, within bone the organic material contains the majority of the sodium and potassium. This suggests that the organic material in bone and not the mineral itself is responsible for the acute buffering of the additional hydrogen ions during metabolic acidosis.

The effects of acid on bone.

Metabolic acidosis induces calcium efflux from bone and in the process buffers the additional hydrogen ions. Initially metabolic acidosis stimulates physicochemical mineral dissolution and then cell-mediated bone resorption. Acidosis increases activity of the bone resorbing cells, the osteoclasts, and decreases activity of the bone forming cells, the osteoblasts. Osteoblastic immediate early response genes are inhibited as are genes controlling matrix formation.

Isolated hypercalciuria with mutation in CLCN5: relevance to idiopathic hypercalciuria.

UNLABELLED: Isolated hypercalciuria with mutation in CLCN5: Relevance to idiopathic hypercalciuria. BACKGROUND: Idiopathic hypercalciuria (IH) is the most common risk factor for kidney stones and often has a genetic component. Dent's disease (X-linked nephrolithiasis) is associated with mutations in the CLCN5 chloride channel gene, and low molecular weight (LMW) proteinuria was universally observed in affected males. We sought to identify mutations in CLCN5 or abnormalities in LMW protein excretion in a large group of patients with IH and in a rat model of genetic hypercalciuria. METHODS: One hundred and seven patients with IH (82 adults and 25 children) and one asymptomatic hypercalciuric man with a known inactivating mutation in CLCN5 were studied. Secondary causes of hypercalciuria were excluded in all. The excretion of retinol-binding protein and beta2-microglobulin was measured by immunoassay in 101 patients with IH. Mutation analysis of the CLCN5 gene was performed in 32 patients with IH and in the genetic hypercalciuric stone-forming (GHS) rat strain. RESULTS: LMW protein excretion was normal in 92 patients with IH, and only slight abnormalities were found in the other nine, none of whom had a mutation in CLCN5. One 27-year-old man who had a CLCN5 mutation was found to have isolated hypercalciuria without LMW proteinuria, renal failure, or other evidence of renal disease. Mutation analysis was normal in 32 patients with IH. The CLCN5 sequence was normal in the GHS rat. CONCLUSIONS: Inactivation of CLCN5 can be found in the setting of hypercalciuria without other features of X-linked nephrolithiasis. However, mutations in CLCN5 do not represent a common cause of IH.

Calcium phosphate supersaturation regulates stone formation in genetic hypercalciuric stone-forming rats.

BACKGROUND: Hypercalciuria is the most common metabolic abnormality observed in patients with nephrolithiasis. Hypercalciuria raises urine supersaturation with respect to the solid phases of calcium oxalate and calcium phosphate, leading to an enhanced probability for nucleation and growth of crystals into clinically significant stones. However, there is little direct proof that supersaturation itself regulates stone formation. Through successive inbreeding of the most hypercalciuric progeny of hypercalciuric Sprague-Dawley rats, we have established a strain of rats, each of which excrete abnormally large amounts of urinary calcium and each of which forms calcium phosphate kidney stones. We used these hypercalciuric (GHS) rats to test the hypothesis that an isolated reduction in urine supersaturation, achieved by decreasing urine phosphorus excretion, would decrease stone formation in these rats. METHODS: Thirty 44th-generation female GHS rats were randomly divided into three groups. Ten rats received a high-phosphorus diet (0.565% phosphorus), 10 a medium-phosphorus diet (0.395% phosphorus), and 10 a low-phosphorus diet (0.225% phosphorus) for a total of 18 weeks. The lowered dietary phosphorus would be expected to result in a decrease in urine phosphorus excretion and a decrease in urinary supersaturation with respect to the calcium phosphate solid phase. Every two weeks, 24-hour urine collections were obtained. All relevant ions were measured, and supersaturation with respect to calcium oxalate and calcium hydrogen phosphate were determined. At the conclusion of the experiment, each rat was killed, and the kidneys, ureters, and bladder were dissected en block and x-rayed to determine whether any stones formed. A decrease in stone formation with a reduction in urinary supersaturation would support the hypothesis that supersaturation alone can regulate stone formation. RESULTS: Decreasing the dietary phosphorus intake led to a progressive decrease in urine phosphorus excretion and an increase in urine calcium excretion, the latter presumably caused by decreased intestinal calcium phosphate binding and increased calcium absorption. With decreasing dietary phosphorus intake, there was a progressive decrease in saturation with respect to the calcium phosphate solid phase. Fifteen of the 20 kidneys from the 10 rats fed the high-phosphorus diet had radiographic evidence of kidney stone formation, whereas no kidneys from the rats fed either the medium- or low-phosphorus diet developed kidney stones. CONCLUSIONS: A decrease in urine phosphorus excretion not only led to a decrease in urine supersaturation with respect to the calcium phosphate solid phase but to an elimination of renal stone formation. The results of this study support the hypothesis that variation in supersaturation alone can regulate renal stone formation. Whether a reduction of dietary phosphorus will alter stone formation in humans with calcium phosphate nephrolithiasis remains to be determined.

Diuretic effects on calcium metabolism.

Diuretics have numerous effects on calcium metabolism. The loop diuretics promote, and the thiazide diuretics inhibit, renal calcium excretion. In this review we detail the basic mechanisms of renal calcium excretion and then explain how diuretics influence this excretion. Finally we review how these agents can be used to alter calcium homeostasis in a clinically efficacious manner.

In vitro metabolic and respiratory acidosis selectively inhibit osteoblastic matrix gene expression.

Clinically, a decrease in blood pH may be due to either a reduction in bicarbonate concentration ([HCO(-)(3)], metabolic acidosis) or an increase in PCO(2) (respiratory acidosis). In mammals, metabolic acidosis induces a far greater increase in urine calcium excretion than respiratory acidosis. In cultured bone, metabolic acidosis induces a marked increase in calcium efflux and a decrease in osteoblastic collagen synthesis, whereas isohydric respiratory acidosis has little effect on either parameter. We have shown that metabolic acidosis prevents the normal developmental increase in the expression of RNA for matrix Gla protein and osteopontin in chronic cultures of primary murine calvarial bone cells (predominantly osteoblasts) but does not alter expression of osteonectin. To compare the effects of isohydric metabolic and respiratory acidosis on expression of these genes, bone cell cultures were incubated in medium at pH approximately 7.2 to model metabolic ([HCO(-)(3)], approximately 13 mM) or respiratory (PCO(2), approximately 80 mmHg) acidosis or at pH approximately 7.4 as a control. Cells were sampled at weeks 4, 5, and 6 to assess specific RNA content. At all time periods studied, both metabolic and respiratory acidosis inhibited the expression of RNA for matrix Gla protein and osteopontin to a similar extent, whereas there was no change in osteonectin expression. In contrast to the significant difference in the effects of metabolic and respiratory acidosis on bone calcium efflux and osteoblastic collagen synthesis, these two forms of acidosis have a similar effect on osteoblastic RNA expression of both matrix Gla protein and osteopontin. Thus, although several aspects of bone cell function are dependent on the type of acidosis, expression of these two matrix genes appears to be regulated by extracellular pH, independently of the type of acidosis.

Effects of in vivo metabolic acidosis on midcortical bone ion composition.

Chronic metabolic acidosis increases urine calcium excretion without altering intestinal calcium absorption, suggesting that bone mineral is the source of the additional urinary calcium. During metabolic acidosis there appears to be an influx of protons into bone mineral, lessening the magnitude of the decrement in pH. Although in vitro studies strongly support a marked effect of metabolic acidosis on the ion composition of bone, there are few in vivo observations. We utilized a high-resolution scanning ion microprobe with secondary ion mass spectroscopy to determine whether in vivo metabolic acidosis would alter bone mineral in a manner consistent with its purported role in buffering the increased proton concentration. Postweanling mice were provided distilled drinking water with or without 1.5% NH(4)Cl for 7 days; arterial blood gas was then determined. The addition of NH(4)Cl led to a fall in blood pH and HCO(-)(3) concentration. The animals were killed on day 7, and the femurs were dissected and split longitudinally. The bulk cortical ratios Na/Ca, K/Ca, total phosphate/carbon-nitrogen bonds [(PO(2) + PO(3))/CN], and HCO(-)(3)/CN each fell after 1 wk of metabolic acidosis. Because metabolic acidosis induces bone Ca loss, the fall in Na/Ca and K/Ca indicates a greater efflux of bone Na and K than Ca, suggesting H substitution for Na and K on the mineral. The fall in (PO(2) + PO(3))/CN indicates release of mineral phosphates, and the fall in HCO(-)(3)/CN indicates release of mineral HCO(-)(3). Each of these mechanisms would result in buffering of the excess protons and returning the systemic pH toward normal.

Genetic hypercalciuric stone-forming rats.

In humans, idiopathic hypercalciuria is associated with stone formation. In order to study the mechanisms that are responsible for excess urine calcium excretion, in ways that are difficult or impossible in humans, we have developed a rat model of hypercalciuria. Spontaneously hypercalciuric rats have been successively inbred for over 50 generations to produce a strain in which urine calcium excretion is over 10 times greater than that of controls, and all rats form kidney stones. Analysis of the model has revealed that the rats not only exhibit increased intestinal calcium reabsorption but an independent defect in renal tubular calcium resorption and an increased tendency for bone resorption. These findings closely parallel those in patients with idiopathic hypercalciuria. In the intestine, bone and kidney there is an increased number of vitamin D receptors which are hyperresponsive to 1,25-dihydroxyvitamin D3. Whether the increased number of vitamin D receptors is directly responsible for the hypercalciuria and whether the same abnormality is present in humans with idiopathic hypercalciuria is under investigation. Hypercalciuric rats appear to be an excellent model to provide insights into the mechanisms causing hypercalciuria, and to delineate treatments for stone disease.

Increased dietary oxalate does not increase urinary calcium oxalate saturation in hypercalciuric rats.

BACKGROUND: Human calcium oxalate (CaOx) nephrolithiasis may occur if urine is supersaturated with respect to the solid-phase CaOx. In these patients, dietary oxalate is often restricted to reduce its absorption and subsequent excretion in an effort to lower supersaturation and to decrease stone formation. However, dietary oxalate also binds intestinal calcium which lowers calcium absorption and excretion. The effect of increasing dietary oxalate on urinary CaOx supersaturation is difficult to predict. METHODS: To determine the effect of dietary oxalate intake on urinary supersaturation with respect to CaOx and brushite (CaHPO4), we fed 36th and 37th generation genetic hypercalciuric rats a normal Ca diet (1.2% Ca) alone or with sodium oxalate added at 0.5%, 1.0%, or 2.0% for a total of 18 weeks. We measured urinary ion excretion and calculated supersaturation with respect to the CaOx and CaHPO4 solid phases and determined the type of stones formed. RESULTS: Increasing dietary oxalate from 0% to 2.0% significantly increased urinary oxalate and decreased urinary calcium excretion, the latter presumably due to increased dietary oxalate-binding intestinal calcium. Increasing dietary oxalate from 0% to 2.0% decreased CaOx supersaturation due to the decrease in urinary calcium offsetting the increase in urinary oxalate and the decreased CaHPO4 supersaturation. Each rat in each group formed stones. Scanning electron microscopy revealed discrete stones and not nephrocalcinosis. X-ray and electron diffraction and x-ray microanalysis revealed that the stones were composed of calcium and phosphate; there were no CaOx stones. CONCLUSION: Thus, increasing dietary oxalate led to a decrease in CaOx and CaHPO4 supersaturation and did not alter the universal stone formation found in these rats, nor the type of stones formed. These results suggest the necessity for human studies aimed at determining the role, if any, of limiting oxalate intake to prevent recurrence of CaOx nephrolithiasis.

Alendronate decreases urine calcium and supersaturation in genetic hypercalciuric rats.

BACKGROUND: The mechanism of excess urine calcium excretion in human idiopathic hypercalciuria (IH) has not been determined but may be secondary to enhanced intestinal calcium absorption, decreased renal calcium reabsorption, and/or enhanced bone demineralization. We have developed a strain of genetic hypercalciuric stone-forming (GHS) rats as an animal model of human IH. When these GHS rats are placed on a low-calcium diet (LCD), urinary calcium (UCa) excretion exceeds dietary calcium intake, suggesting that bone may contribute to the excess UCa excretion. We used the GHS rats to test the hypothesis that bone contributes to the persistent IH when they are fed an LCD by determining if alendronate (Aln), which inhibits bone resorption, would decrease UCa excretion. METHODS: GHS rats (N = 16) and the parent strain (Ctl, N = 16) were fed 13 g/day of a normal (1.2%) calcium diet (NCD) for seven days and were then switched to a LCD (0. 02%) for seven days. Ctl and GHS rats in each group were then continued on LCD for an additional seven days, with or without injection of Aln (50 micrograms/kg/24 hrs). UCa excretion was measured daily during the last five days of each seven-day period. To determine the effects of Aln on urine supersaturation, the experiment was repeated. All relevant ions were measured, and supersaturation with respect to calcium oxalate and calcium hydrogen phosphate was determined at the end of each period. RESULTS: UCa was greater in GHS than in Ctl on NCD (7.4 +/- 0.5 mg/24 hrs vs. 1.2 +/- 0.1, GHS vs. Ctl, P < 0.01) and on LCD (3.9 +/- 0.2 mg/24 hrs vs. 0. 7 +/- 0.1, GHS vs. Ctl, P < 0.01). LCD provides 2.6 mg of calcium/24 hrs, indicating that GHS rats are excreting more calcium than they are consuming. On LCD, Aln caused a significant decrease in UCa in GHS rats and brought GHS UCa well below calcium intake. Aln caused a marked decrease in calcium oxalate and calcium hydrogen phosphate supersaturation. CONCLUSION: Thus, on a LCD, there is a significant contribution of bone calcium to the increased UCa in this model of IH. Aln is effective in decreasing both UCa and supersaturation. The Aln-induced decrease in urine supersaturation should be beneficial in preventing stone formation in humans, if these results, observed in a short-term study using the hypercalciuric stone-forming rat can be confirmed in longer term human studies.