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.

Mechanism of hypercalciuria in genetic hypercalciuric rats. Inherited defect in intestinal calcium transport.

Excessive urine calcium excretion in patients with idiopathic hypercalciuria may involve a primary increase in intestinal calcium absorption, overproduction of 1,25-dihydroxyvitamin D3 or a defect in renal tubular calcium reabsorption. To determine the mechanism of hypercalciuria in an animal model, hypercalciuria was selected for in rats and the most hypercalciuric animals inbred. Animals from the fourth generation were utilized to study mineral balance and intestinal transport in relation to levels of serum 1,25(OH)2D3. Both urine calcium excretion and net intestinal calcium absorption were greater in hypercalciuric males (HM) than in normocalciuric males (NM) and in hypercalciuric females (HF) than in normocalciuric females (NF). However, serum 1,25(OH)2D3 was lower in HM than in NM and not different in HF than in NF. Net calcium balance was more positive in HM than in NM and in HF than in NF. In vitro duodenal calcium net flux was correlated with serum 1,25(OH)2D3 in HM and HF and in NM and NF. However, with increasing serum 1,25(OH)2D3 there was greater calcium net flux in hypercalciuric rats than in normocalciuric controls. Hypercalciuria in this colony of hypercalciuric rats is due to a primary intestinal overabsorption of dietary calcium and not an overproduction of 1,25(OH)2D3 or a defect in the renal tubular reabsorption of calcium.

Evidence that blood ionized calcium can regulate serum 1,25(OH)2D3 independently of parathyroid hormone and phosphorus in the rat.

This study asks whether arterial blood ionized calcium concentration (Ca++) can regulate the serum level of 1,25-dihydroxy-vitamin D3 [1,25(OH)2D3] independently of serum phosphorus and parathyroid hormone (PTH). We infused either PTH (bovine 1-34, 10 U/kg body wt/h) or saline into awake and unrestrained rats for 24 h, through a chronic indwelling catheter. PTH raised total serum calcium and arterial blood ionized calcium, yet serum 1,25(OH)2D3 fell from 35 +/- 6 (mean +/- SEM, n = 10) with saline to 12 +/- 3 pg/ml (n = 11, P less than 0.005 vs. saline). To determine if the decrease in serum 1,25(OH)2D3 was due to the elevated Ca++, we infused PTH into other rats for 24 h, along with varying amounts of EGTA. Infusion of PTH + 0.67 micron/min EGTA reduced Ca++, and 1,25(OH)2D3 rose to 90 +/- 33 (P less than 0.02 vs. PTH alone). PTH + 1.00 micron/min EGTA lowered Ca++ more, and 1,25(OH)2D3 increased to 148 +/- 29 (P less than 0.01 vs. saline or PTH alone). PTH + 1.33 micron/min EGTA lowered Ca++ below values seen with saline or PTH alone, and 1,25(OH)2D3 rose to 267 +/- 46 (P less than 0.003 vs. all other groups). Thus, during PTH infusion lowering Ca++ with EGTA raised 1,25(OH)2D3 progressively. There were no differences in serum phosphorus concentration or in arterial blood pH in any group infused with PTH. The log of serum 1,25(OH)2D3 was correlated inversely with Ca++ in all four groups infused with PTH (r = -0.737, n = 31, P less than 0.001), and also when the saline group was included (r = -0.677, n = 41, P less than 0.001). The results of this study indicate that serum 1,25(OH)2D3 may be regulated by Ca++ independent of PTH and serum phosphorus levels in the rat. Since 1,25(OH)2D3 regulates gastrointestinal calcium absorption, there may be direct feedback control of 1,25(OH)2D3, by its regulated ion, Ca++.

Evidence that serum calcium oxalate supersaturation is a consequence of oxalate retention in patients with chronic renal failure.

Serum oxalate rises in uremia because of decreased renal clearance, and crystals of calcium oxalate occur in the tissues of uremic patients. Crystal formation suggests that either uremic serum is supersaturated with calcium oxalate, or local oxalate production or accumulation causes regional supersaturation. To test the first alternative, we ultrafiltered uremic serum and measured supersaturation with two different methods previously used to study supersaturation in urine. First, the relative saturation ratio (RSR), the ratio of the dissolved calcium oxalate complex to the thermodynamic calcium oxalate solubility product, was estimated for 11 uremic (before and after dialysis) and 4 normal serum samples using a computer program. Mean ultrafiltrate oxalate predialysis was 89 +/- 8 microM/liter (+/- SEM), 31 +/- 4 postdialysis, and 10 +/- 3 in normals. Mean RSR was 1.7 +/- 0.1 (predialysis), 0.7 +/- 0.1 (postdialysis), and 0.2 +/- 0.1 (normal), where values greater than 1 denote supersaturation, less than 1, undersaturation. Second, the concentration product ratio (CPR), the ratio of the measured calcium oxalate concentration product before to that after incubation of the sample with calcium oxalate monohydrate crystal, was measured in seven uremic and seven normal serum ultrafiltrates. Mean oxalate was 91 +/- 11 (uremic) and 8 +/- 3 (normal). Mean CPR was 1.4 +/- 0.2 (uremic) and 0.2 +/- 0.1 (normal). Predialysis, 17 of 18 uremic ultrafiltrates were supersaturated with respect to calcium oxalate. The degree of supersaturation was correlated with ultrafiltrate oxalate (RSR, r = 0.99, r = 29, P less than 0.001; CPR, r = 0.75, n = 11, P less than 0.001). A value of ultrafiltrate oxalate of 50 microM/liter separated undersaturated from supersaturated samples and occurred at a creatinine of approximately 9.0 mg/dl.

Hyperresponsiveness of vitamin D receptor gene expression to 1,25-dihydroxyvitamin D3. A new characteristic of genetic hypercalciuric stone-forming rats.

Hypercalciuria in genetic hypercalciuric stone-forming (GHS) rats is accompanied by intestinal Ca hyperabsorption with normal serum 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] levels, elevation of intestinal, kidney, and bone vitamin D receptor (VDR) content, and greater 1,25(OH)2D3-induced bone resorption in vitro. To test the hypothesis that hyperresponsiveness of VDR gene expression to 1,25(OH)2D3 may mediate these observations, male GHS and wild-type Sprague- Dawley normocalciuric control rats were fed a normal Ca diet (0.6% Ca) and received a single intraperitoneal injection of either 1,25(OH)2D3 (10-200 ng/100 g body wt) or vehicle. Total RNAs were isolated from both duodenum and kidney cortex, and the VDR and calbindin mRNA levels were determined by Northern blot hybridization using specific cDNA probes. Under basal conditions, VDR mRNA levels in GHS rats were lower in duodenum and higher in kidney compared with wild-type controls. Administration of 1,25(OH)2D3 increased VDR gene expression significantly in GHS but not normocalciuric animals, in a time- and dose-dependent manner. In vivo half-life of VDR mRNA was similar in GHS and control rats in both duodenum and kidney, and was prolonged significantly (from 4-5 to > 8 h) by 1,25(OH)2D3 administration. Neither inhibition of gene transcription by actinomycin D nor inhibition of de novo protein synthesis with cycloheximide blocked the upregulation of VDR gene expression stimulated by 1,25(OH)2D3 administration. No alteration or mutation was detected in the sequence of duodenal VDR mRNA from GHS rats compared with wild-type animals. Furthermore, 1,25(OH)2D3 administration also led to an increase in duodenal and renal calbindin mRNA levels in GHS rats, whereas they were either suppressed or unchanged in wild-type animals. The results suggest that GHS rats hyperrespond to minimal doses of 1,25(OH)2D3 by an upregulation of VDR gene expression. This hyperresponsiveness of GHS rats to 1,25(OH)2D3 (a) occurs through an increase in VDR mRNA stability without involving alteration in gene transcription, de novo protein synthesis, or mRNA sequence; and (b) is likely of functional significance, and affects VDR-responsive genes in 1, 25(OH)2D3 target tissues. This unique characteristic suggests that GHS rats may be susceptible to minimal fluctuations in serum 1, 25(OH)2D3, resulting in increased VDR and VDR-responsive events, which in turn may pathologically amplify the actions of 1,25(OH)2D3 on Ca metabolism that thus contribute to the hypercalciuria and stone formation.

Increased intestinal vitamin D receptor in genetic hypercalciuric rats. A cause of intestinal calcium hyperabsorption.

In humans, familial or idiopathic hypercalciuria (IH) is a common cause of hypercalciuria and predisposes to calcium oxalate nephrolithiasis. Intestinal calcium hyperabsorption is a constant feature of IH and may be due to either a vitamin D-independent process in the intestine, a primary overproduction of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], or a defect in renal tubular calcium reabsorption. Selective breeding of spontaneously hypercalciuric male and female Sprague-Dawley rats resulted in offspring with hypercalciuria, increased intestinal calcium absorption, and normal serum 1,25(OH)2D3 levels. The role of the vitamin D receptor (VDR) in the regulation of intestinal calcium absorption was explored in 10th generation male genetic IH rats and normocalciuric controls. Urine calcium excretion was greater in IH rats than controls (2.9 +/- 0.3 vs. 0.7 +/- 0.2 mg/24 h, P < 0.001). IH rat intestine contained twice the abundance of VDR compared with normocalciuric controls (536 +/- 73 vs. 243 +/- 42 nmol/mg protein, P < 0.001), with no difference in the affinity of the receptor for its ligand. Comparable migration of IH and normal intestinal VDR on Western blots and of intestinal VDR mRNA by Northern analysis suggests that the VDR in IH rat intestine is not due to large deletion or addition mutations of the wild-type VDR. IH rat intestine contained greater concentrations of vitamin D-dependent calbindin 9-kD protein. The present studies strongly suggest that increased intestinal VDR number and normal levels of circulating 1,25(OH)2D3 result in increased functional VDR-1,25(OH)2D3 complexes, which exert biological actions in enterocytes to increase intestinal calcium transport. Intestinal calcium hyperabsorption in the IH rat may be the first example of a genetic disorder resulting from a pathologic increase in VDR.

Mechanism of amphotericin B stimulation of net calcium efflux from neonatal mouse calvariae.

Amphotericin B is a polyene antifungal agent that binds to membrane sterols, creating aqueous pores that permit ion fluxes sufficient to cause cell lysis. It has also been shown to alter ion transport in mammalian cells, including proton secretion from renal tubular cells. The latter effect can lead to distal renal tubular acidosis in patients treated for systemic fungal infections. Based on the understanding that osteoclast-mediated bone resorption is dependent on proton secretion, we examined the effect of amphotericin B on calcium efflux from neonatal mouse calvariae in organ culture. Amphotericin B (5 micrograms/ml) stimulated net calcium efflux from calvariae within 24 h to a level almost as great as that produced by a maximally effective concentration of parathyroid hormone. The stimulated calcium efflux was completely inhibited by both 10 ng/ml salmon calcitonin, a physiologic inhibitor of osteoclast activity, and 4 x 10(-4) M acetazolamide, a specific inhibitor of carbonic anhydrase, the enzyme necessary for substantial proton generation by osteoclasts. These results indicate a direct effect of amphotericin B on bone in vitro to stimulate osteoclast-mediated calcium efflux.

Conditioned medium from ras oncogene-transformed NIH 3T3 cells induces bone resorption in vitro.

Tumor-associated hypercalcemia is due, in part, to enhanced osteoclastic bone resorption induced by soluble factors elaborated from malignant cells. ras transformation of NIH 3T3 cells results in a 50-fold induction of cathepsin L mRNA and secretion of the corresponding protein. Since cathepsin L is an acid proteinase we asked whether conditioned medium from these cells would directly increase calcium release from bone in vitro. We tested conditioned medium obtained after 72 h culture of NIH 3T3 ras-transformed cells (DT) or nontransformed NIH 3T3 cells (3T3) and identical medium not exposed to cells (Ctl). Incubation of either live or dead neonatal mouse calvaria for 48 h in DT-conditioned medium increased calcium release compared to bones incubated with 3T3 medium. In both states the increased calcium release with DT medium was blocked by 0.25 mM E-64, a general cysteine proteinase inhibitor, and 1 microM Z-Phe-Ala-CH2F, a specific inhibitor of cathepsin L activity. Thus, conditioned medium from ras-transformed cells enhances calcium release in both live and dead bone. Since cathepsin L is the major protein secreted by these cells and the effect of DT-conditioned medium is blocked by a specific inhibitor of cathepsin L, these studies suggest that this acid proteinase acts directly on bone mineral to enhance net calcium release.

Regulation of 1,25-dihydroxyvitamin D3 by calcium in the parathyroidectomized, parathyroid hormone-replete rat.

Parathyroid hormone (PTH) is a major stimulus for the renal production of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Elevated arterial blood ionized calcium ([Ca2+]) depresses serum 1,25-(OH)2D3 in nonparathyroidectomized rats even when serum PTH is maintained at high levels by infusion. However, suppression by [Ca2+] of endogenous PTH, causing the fall in 1,25-(OH)2D, cannot be excluded. To determine whether [Ca2+] regulates 1,25-(OH)2D3 in the absence of a variation in PTH, we parathyroidectomized (PTX) rats (post-PTX calcium levels less than 7.0 mg/dl), inserted arterial and venous catheters, and then replaced PTH using an osmotic pump. We varied [Ca2+] by infusing either 75 mM sodium chloride (control), 0.61 mumol/min of EGTA (EGTA), or calcium chloride at 0.61 mumol/min (low calcium) or 1.22 mumol/min (high calcium) for 24 h 5 days after surgery. Blood was then drawn from the rat through the arterial catheter. Compared with the control, [Ca2+] fell with EGTA, remained constant with the low-calcium infusion, and rose with the high-calcium infusion. 1,25-(OH)2D3 was correlated inversely with [Ca2+] in all four groups together (r = -0.635, n = 34, p less than 0.001), within the control group alone (r = -0.769, n = 11, p less than 0.002), and within the EGTA group alone (r = -0.774, n = 10, p less than 0.003). Serum phosphorus, PTH, and arterial blood pH were not different in any group, and none correlated with serum 1,25-(OH)2D3. We conclude that 1,25-(OH)2D3 levels are regulated by [Ca2+] independently of serum PTH, phosphorus, and acid-base status, all of which support the hypothesis that [Ca2+] is a principal regulator of serum 1,25-(OH)2D3 in the rat.

Proton-induced physicochemical calcium release from ceramic apatite disks.

When bone is cultured in acid medium there is net calcium efflux (JCa) and proton influx (JH) relative to the mineral. The acid medium appears to induce physicochemical mineral dissolution as well as cell-mediated bone resorption. To determine the independent effect of acid medium on physicochemical dissolution, we utilized cell-free synthetic ceramic apatite (CAP) disks, which contain carbonate (5.5%) in an apatite structure chemically similar to mammalian bone. CAP disks were cultured in control (Ctl, pH approximately equal to 7.44) or acid (Met, pH approximately equal to 7.11) medium for 48 h and compared to similarly treated neonatal (4-6 days old) mouse calvariae. Medium was changed and analyzed at 3, 24, and 48 h. At 3, 24, and 48 h there was significantly greater JCa from the CAP disks and calvariae incubated in Met compared to Ctl; over the entire 48 h time period there was a greater progressive increase in JCa from the CAP disks than the calvariae incubated in Met. There was no significant JCa at 3, 24, or 48 h from CAP disks or calvariae incubated in Ctl. At 3 h there was significantly greater JH into the CAP disks and calvariae incubated in Met compared to Ctl; JH was greater into the CAP disks than the calvariae. Utilizing a synthetic model of bone mineral we demonstrated that acid medium induces physicochemical calcium efflux and proton influx relative to the mineral.

Hormonal regulation of Na(+)-Ca2+ exchange in osteoblast-like cells.

We proposed a role for Na-Ca exchange in hormonally mediated bone resorption and recently characterized Na-dependent Ca transport in an osteoblast-like rat osteosarcoma cell line (UMR-106). To test whether calcemic agents alter Na(+)-Ca2+ exchange in osteoblasts, UMR cells were treated acutely or cultured in the absence or presence of calcemic agent for 24 h. Cells were then loaded with the Ca-sensitive dye fura-2 in the presence of 140 mM NaCl, no Ca, and the absence or presence of 0.3 mM ouabain. Cells were resuspended at 22 degrees C, and the fluorescence ratio at excitation wavelength of 340 and 380 nm was measured. An outward Na gradient was generated by removing extracellular Na and maintaining isotonicity with choline chloride. Na(+)-Ca2+ exchange was demonstrated by enhanced Ca uptake in ouabain-treated (Na-loaded) cells after the addition of 1.5 mM Ca. Acute addition of 10(-7) M PTH or 10(-6) M PGE2 had no effect on Na-dependent Ca uptake. However, 24 h treatment of cells with PTH, PGE2, or 1,25(OH)2D3 caused a dose-dependent inhibition of Na(+)-Ca2+ exchange. Using the Na-sensitive dye, SBFI, we also demonstrated that the effect was bidirectional; PTH inhibited Ca-dependent Na uptake comparably to its inhibition of Na-dependent Ca uptake. The effects of the calcemic agents were mimicked by 24 h treatment of the cells with 1 microM forskolin or 2 microM PMA.(ABSTRACT TRUNCATED AT 250 WORDS)

Acidosis inhibits 1,25-(OH)2D3 but not cAMP production in response to parathyroid hormone in the rat.

Parathyroid hormone (PTH) is a major activator of renal proximal tubule 25-hydroxyvitamin D3-1-hydroxylase (1-OHase). Chronic metabolic acidosis (CMA) inhibits 1-OHase and reduces circulating 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] levels in rats fed a low-Ca diet (LCD, 0.002% Ca). To examine the cellular mechanism whereby CMA inhibits 1-OHase, PTH-dependent renal 1-OHase activity and cAMP were measured in proximal tubules isolated from rats fed LCD for 14 days and made acidotic by the addition of 1.5% ammonium chloride to the drinking water. Serum 1,25-(OH)2D3 and proximal tubule 1-OHase activity and cAMP content were lower in acidotic rats. hPTH-(1-34) (10(-7) M) in vitro increased cAMP content to equivalent concentrations in tubules from rats with CMA and from nonacidotic controls; however, PTH increased 1-OHase activity only in tubules from nonacidotic animals. Although forskolin increased tubule cAMP content to equivalent levels in tubules from acidotic and nonacidotic rats, 1-OHase activity declined in tubules from nonacidotic rats and remained suppressed in acidotic tubules. The results suggest that chronic metabolic acidosis inhibits the PTH activation of 1-OHase through alteration of one or more steps in a cAMP-independent messenger system. PTH and forskolin can increase cAMP production by acidotic and nonacidotic proximal tubules; however, 1-OHase activity is not restored to normal in acidotic tubules and nonacidotic tubule 1-OHase may be inhibited.

Effects of aluminum on bone surface ion composition.

Aluminum induces net calcium efflux from cultured bone. To determine whether aluminum alters the bone surface ion composition in a manner consistent with predominantly cell-mediated resorption, a combination of cell-mediated resorption and physicochemical dissolution or physicochemical dissolution alone, we utilized an analytic high-resolution scanning ion microprobe with secondary ion mass spectroscopy to determine the effects of aluminum on bone surface ion composition. We cultured neonatal mouse calvariae with or without aluminum (10(-7) M) for 24 h and determined the relative ion concentrations of 23Na, 27Al, 39K, and 40Ca on the bone surface and eroded subsurface. Control calvariae have a surface (depth approximately 6 nm) that is rich in Na and K compared with Ca(Na/Ca) = 24.4 + 1.4, mean + 95% confidence limit of counts per second of detected secondary ions, K+Ca = 13.2 + 0.9). Aluminum is incorporated into the bone and causes a depletion of surface Na and K relative to Ca (Na/Ca = 9.6 + 0.7, K/Ca = 4.9 + 0.4; each p < 0.001 versus control). After erosion (depth approximately 50 nm), control calvariae have more Na and K than Ca (Na/Ca = 16.0 + 0.1, K/Ca = 7.5 + 0.1); aluminum again depleted Na and K relative to Ca (Na/Ca = 4.1 + 0.1 K/Ca = 1.9 + 0.1; each p < 0.001 versus control). Aluminum produced a greater net efflux of Ca (362 +/- 53, mean +/- SE, nmol/bone/24 h) than control (60 +/- 30, p < 0.001). With aluminum, the fall in the ratios of both Na/Ca and K/Ca coupled with net Ca release from bone indicates that aluminium induces a greater efflux of Na and K than Ca from the bone surface and is consistent with an aluminum-induced removal of the bone surface. This alteration in surface ion concentration and calcium efflux is consistent with that observed when calcium is lost from bone through a combination of cell-mediated resorption and physicochemical dissolution.

Physicochemical effects of acidosis on bone calcium flux and surface ion composition.

Net calcium flux (JCa) from bone in vitro is pH dependent. When pH falls below 7.40, through a reduction in [HCO3-], there is both physicochemical and cell-mediated JCa. To characterize the physicochemical effect of acidosis on bone we inhibited the bone-resorbing cells (osteoclasts) with the specific inhibitor calcitonin and studied the effect of acidosis on JCa and bone ion composition using an analytic high-resolution scanning ion microprobe. Neonatal mouse calvariae were cultured for 48 h in physiologically neutral pH medium (Ntl, pH = 7.41, [HCO3-] = 25 nM) or in medium that modeled metabolic acidosis (Met, pH = 7.10, [HCO3-] = 12), each with or without calcitonin (CT, 3 x 10(-9) M). There was net calcium efflux in Ntl (JCa = 631 +/- 36 nmol per bone per 48 h), which increased in Met (1019 +/- 53, p < 0.01); CT inhibited JCa in Ntl (-54 +/- 11, p < 0.01 versus Ntl), which increased in Met (197 +/- 15, p < 0.01 versus Ntl + CT). In the presence of CT the increase in JCa in Met versus Ntl represents physiochemical bone dissolution. The Ntl bone surface (approximately 2 nm in depth) was rich in Na compared to Ca (Na/Ca = 11.9, count/s of detected secondary ions), which fell in Met (Na/Ca = 6.0, p < 0.05); CT caused a further reduction of Na/Ca (3.1, p < 0.01 versus Ntl and versus Met), which was not altered in Met (2.6, p < 0.05 versus Ntl + CT).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of metabolic acidosis on the potassium content of bone.

Metabolic acidosis induces resorption of cultured bone, resulting in a net efflux of calcium (Ca) from the bone and an apparent loss of mineral potassium (K). However, in these organ cultures, there is diffusion of K between the medium and the crystal lattice, causing difficulty in interpretation of the acid-induced changes in mineral ion composition. To determine the effects of acidosis on bone mineral K, we injected 4-day-old neonatal mice with pure stable isotope 41K, equal to approximately 5% of their total body K. Calvariae were dissected 24 h later and then cultured for 24 h in medium without added 41K, either at pH approximately 7.4 (Ctl) or at pH approximately 7.1 (Ac), with or without the osteoclastic inhibitor calcitonin (3 x 10(-9) M, CT). The bone isotopic ion content was determined with a high-resolution scanning ion microprobe utilizing secondary ion mass spectrometry. 41K is present in nature at 6.7% of total K. The injected 41K raised the ratio of bone 41K/(39K+41K) to 9.8+/-0.5% on the surface (ratios of counts per second of detected secondary ions, mean+/-95% confidence interval) but did not alter the ratio in the interior (6.9+/-0.4%), indicating biological incorporation of the 41K into the mineral surface. The ratios of 41K/40Ca on the surface of Ctl calvariae was 14.4+/-1.2, indicating that bone mineral surface is rich in K compared with Ca. Compared with Ctl, Ac caused a marked increase in the net Ca efflux from bone that was blocked by CT. Ac also induced a marked fall in the ratio of 41K/40Ca on the surface of the calvariae (43+/-0.5, p < 0.01 vs. Ctl), which was partially blocked by CT (8.2+/-0.9, p < 0.01 vs. Ctl and vs. Ac), indicating that Ac causes a greater release of bone mineral K than Ca which is partially blocked by CT. Thus, bone mineral surface is rich in K relative to Ca, acidosis induces a greater release of surface mineral K than Ca, and osteoclastic function is necessary to support the enriched levels of surface mineral K in the presence of acidosis.

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.

Physiological mechanisms for calcium-induced changes in systemic arterial pressure in stable dialysis patients.

The mechanisms by which variations in blood ionized calcium (Ca2+) influence systemic arterial pressures independent of changes in extracellular fluid volume, pH, and electrolytes are unknown. To study this issue, we dialyzed eight stable hemodialysis patients on three separate occasions during 1 week with dialysates differing only in calcium concentration. Ultrafiltration was adjusted to achieve the patient's estimated dry weight. Postdialysis Ca2+ was measured, as were arterial blood gases, electrolytes, magnesium, blood urea nitrogen, creatinine, and hematocrit. Blood pressures and two-dimensional, targeted M-mode echocardiograms were recorded with the patient in the supine position after 15 minutes of rest. Postdialysis, three different levels of Ca2+ were achieved. Other measured biochemical variables and body weight did not differ among the three study periods. Changes in Ca2+ correlated directly with changes in systolic, diastolic, and mean blood pressures, left ventricular stroke volume, and cardiac output. In contrast, heart rate, left ventricular end-diastolic dimension, and total systemic vascular resistance were not altered significantly by changes in Ca2+. Thus, alterations in Ca2+ within the physiological range affect systemic blood pressure primarily through changes in left ventricular output rather than in peripheral vascular tone in stable dialysis patients.

Hospital-acquired renal insufficiency: a prospective study.

Twenty-two hundred sixty-two consecutive medical and surgical admissions were evaluated prospectively to determine the contribution of iatrogenic factors to the development of renal insufficiency in hospital. Of 2,216 patients at risk, some degree of renal insufficiency developed in 4.9 percent. Decreased renal perfusion, postoperative renal insufficiency, radiographic contrast media, and aminoglycosides accounted for 79 percent of the episodes. Iatrogenic factors, broadly defined, accounted for 55 percent of all episodes. Poor prognostic indicators included oliguria, urine sediment abnormalities and, most importantly, severity of renal insufficiency; with an increase in serum creatinine of 3 mg/dl or greater, the mortality rate was 64 percent. Age, admission serum creatinine levels, and the number of episodes of renal insufficiency did not significantly affect outcome. We conclude that there is a substantial risk of the development of renal failure in hospital and that the mortality rate due to hospital-acquired renal insufficiency remains high.

Genetic hypercalciuric stone forming rats.

In humans, idiopathic hypercalciuria is associated with stone formation. To study the mechanisms that are responsible for the excess urine calcium excretion in ways that are difficult to impossible in humans, we have developed a rat model of idiopathic hypercalciuria. Hypercalciuric rats were successively inbred for more than 40 generations to produce a strain in which urine calcium excretion is far 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 absorption but an independent defect in renal tubular resorption and an increased tendency for bone demineralization. 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 appear to make the rats more sensitive to the effect of 1,25(OH)2D3. Whether the increased number of vitamin D receptors can be directly translated into hypercalciuria and whether the same abnormality is present in humans with idiopathic hypercalciuria 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.