To determine the roles of endogenous cyclooxygenase 2 and prostaglandin E(2) in crystal-cell binding, which is considered to be an important step in the development of intratubular nephrocalcinosis.

An expression plasmid for human cyclooxygenase 2 was introduced into Madin-Darby canine kidney cells using the lipofection method. Cyclooxygenase activity was measured using thin-layer chromatography, and the prostaglandin E(2) concentration was determined with an enzyme immunoassay. In addition, crystal attachment was evaluated with a liquid scintillation counter using [(14)C] calcium oxalate monohydrate crystals, and immunohistochemistry and an enzyme immunoassay were used to analyze and quantify the expression of hyaluronan, a crystal-binding molecule.

Cyclooxygenase 2-overexpressing Madin-Darby canine kidney cells produced about 10-fold more prostaglandin E(2) than wild-type Madin-Darby canine kidney cells, and their hyaluronan production was also upregulated. The attachment of calcium oxalate monohydrate crystals to cyclooxygenase 2-overexpressing Madin-Darby canine kidney cells was significantly reduced compared with their attachment to wild-type and mock-transfected Madin-Darby canine kidney cells. Pre-incubation of the cyclooxygenase 2-overexpressing cells, as well as the mock-transfected and wild-type cells with the cyclooxygenase 2 selective inhibitor etodolac, increased the cellular attachment of calcium oxalate monohydrate crystals in a dose-dependent manner.

These findings suggest that cyclooxygenase 2 expression and the resultant increase in endogenous prostaglandin E(2), leading to increased hyaluronan production, help to prevent nephrocalcinosis by inhibiting the attachment of calcium oxalate monohydrate crystals to the surface of renal epithelial cells.

Findings are inconsistent in a few studies of the effect of n-3 fatty acid supplementation on urinary calcium and oxalate excretion in stone formers. We evaluated the physiological effects of supplementation with eicosapentaenoic acid and docosahexaenoic acid on urinary risk factors for calcium oxalate stone formation under standardized conditions.

We studied 15 healthy subjects initially while consuming a standardized diet for 5 days (control phase). During consecutive intervention phases 1-5-day standardized diet, 2-20-day free diet and 3-5-day standardized diet participants received 900 mg eicosapentaenoic acid and 600 mg docosahexaenoic acid daily. While ingesting the standardized diets, daily 24-hour urine samples were collected.

After short-term supplementation with eicosapentaenoic acid and docosahexaenoic acid in phase 1 we noted no changes in urinary parameters compared to the control phase. After 30-day supplementation with eicosapentaenoic acid and docosahexaenoic acid in phase 3 relative supersaturation with calcium oxalate decreased significantly by 23% from a mean ± SD of 2.01 ± 1.26 to 1.55 ± 0.84 due to significantly decreased urinary oxalate excretion (p = 0.023). Other urinary variables were not affected by supplementation.

Results show that 30-day n-3 fatty acid supplementation effectively decreases urinary oxalate excretion and the risk of calcium oxalate crystallization. The mechanism of the physiological effect may be decreased cellular oxalic acid exchange attributable to an altered fatty acid pattern of membrane phospholipids with concomitant changes in oxalate transporter activity. Calcium oxalate stone formers may benefit from long-term n-3 fatty acid supplementation.

Clinical and experimental investigations seem to underline the important role of fatty acids in the pathogenesis of hypercalciuria, a well-known risk factor for lithogenesis. To evaluate the relationships between the previously reported increase in plasma phospholipid arachidonic acid level and the factors responsible for calcium metabolism in idiopathic calcium nephrolithiasis, a best-fit model was constructed. This new statistical application shows a causal relationship between plasma phospholipid arachidonic acid content, intestinal calcium absorption, biochemical markers of bone turnover, urinary calcium excretion and bone mineral density at the lumbar spine. This model suggests that a defect in the phospholipid fatty acid composition could represent the primary event responsible for the mosaic of metabolic and clinical alterations that are distinctive features of renal stone formers, such as kidney, intestine, and bone calcium metabolism, and several forms of idiopathic hypercalciuria.

There is a growing body of evidence from the National Aeronautics and Space Administration and the Russian space program showing that humans exposed to the microgravity environment of space have a greater risk for developing renal stones. Increased bone resorption and the attendant hypercalciuria and hyperphosphaturia contribute significantly to raising the urinary state of saturation with respect to the calcium salts, namely calcium oxalate and calcium phosphate. In addition, other environmental and dietary factors may adversely affect urine composition and increase stone formation risk during space flight. For example, reductions in urinary volume, pH, and citrate contribute to raising stone formation risk. In addition to raising the risk for calcium stone formation, this metabolic profile is conducive to the formation of uric acid stones. Although observations to date have suggested that there may actually be a reduced food intake during the early phase of flight, crew members on longer-duration flights may increase food intake and be at increased risk for stone formation. Taken together, these findings support the use of nutritional recommendations for crew members that would serve to reduce the stone-forming propensity of the urinary environment. Pharmacologic intervention should be directed at raising urinary volumes, diminishing bone losses, and preventing reductions in urinary pH and citrate. Success in reducing the risk for stone formation in astronauts would also be of potential major benefit to the estimated 20 million Americans with nephrolithiasis.

Reports of an increase in plasma and erythrocyte phospholipid arachidonic acid content and in urinary prostaglandin E2 (PGE2) excretion in patients with idiopathic calcium nephrolithiasis suggested their crucial role in the pathogenesis of hypercalciuria, a well-known risk factor for lithogenesis.

To confirm this hypothesis, 15 healthy subjects and 20 nephrolithiasis patients were evaluated for plasma phospholipid polyunsaturated fatty acid content and PGE2 concentration, serum parathyroid hormone, 25 hydroxyvitamin D3, 1, 25-dihydroxyvitamin D3, and bone-specific alkaline phosphatase levels, as well as urinary excretion of calcium, biochemical markers of bone resorption (hydroxyproline and crossLaps), and intestinal calcium absorption. Furthermore, the effect of a 30-day fish-oil diet supplementation on the previously mentioned parameters was investigated in the patients.

At baseline, patients compared with controls showed higher levels of plasma phospholipid arachidonic acid content (P = 0.002), PGE2 (P = 0.0004), serum 25-vitamin D3 (P = 0.001), and 1,25-vitamin D3 (P = 0.001), urinary excretion of calcium (P = 0.001), hydroxyproline (P = 0.007), and crossLaps (P = 0.019), as well as intestinal calcium absorption (P = 0.03 at 60 min). Fish oil supplementation induced a reduction in the plasma phospholipid arachidonic acid level (P < 0.0001), and except for serum concentrations of 25-vitamin D3, normalized baseline blood and urinary parameters, including intestinal calcium absorption. A close correlation between plasma PGE2 and serum 1,25-vitamin D3 (P = 0.004) and between phospholipid arachidonic acid and intestinal calcium absorption (P = 0.0002) and calciuria (P = 0.007) was observed, as well as between urine excretion of crossLaps and hydroxyproline (P < 0.0001), crossLaps and calcium (P < 0.0001), and hydroxyproline and calcium (P < 0.0001).

These findings indicate that the phospholipid arachidonic acid content anomaly could represent the primary event responsible for the mosaic of metabolic and clinical alterations that are distinctive features of renal stone formers, and suggest that a common pathogenetic mechanism might account for the several forms of hypercalciuria detected in idiopathic calcium nephrolithiasis.

Essential fatty acid (EFA)-deficient animals develop severe osteoporosis coupled with increased renal and arterial calcification. This picture is similar to that seen in osteoporosis in the elderly, where the loss of bone calcium is associated with ectopic calcification of other tissues, particularly the arteries and the kidneys. Recent mortality studies indicate that the ectopic calcification may be considerably more dangerous than the osteoporosis itself, since the great majority of excess deaths in women with osteoporosis are vascular and unrelated to fractures or other bone abnormalities. EFAs have now been shown to increase calcium absorption from the gut, in part by enhancing the effects of vitamin D, to reduce urinary excretion of calcium, to increase calcium deposition in bone and improve bone strength and to enhance the synthesis of bone collagen. These desirable actions are associated with reduced ectopic calcification. The interaction between EFA and calcium metabolism deserves further investigation since it may offer novel approaches to osteoporosis and also to the ectopic calcification associated with osteoporosis which seems to be responsible for so many deaths.

As urinary prostaglandin excretion might be involved in idiopathic hypercalciuria, we studied the excretion of prostaglandin E2 and calcium together with serum prostaglandin E2, parathormone and 1,25 dihydroxyvitamin D in 20 patients and 11 controls Idiopathic hypercalciuric patients showed increased levels of urinary prostaglandin E2-like activity, which is correlated with calcium excretion. Although no change was observed in serum parathormone level in control and hypercalciuric patients, there was a positive correlation between serum 1,25 dihydroxyvitamin D and prostaglandin E2-like activity. These findings suggest that prostaglandin E2 could play a role in the hypercalciuria syndrome, possibly by increasing 1,25 dihydroxyvitamin D synthesis.

Decreased bone mass has been reported in patients with idiopathic hypercalciuria. Previous studies, using bioassays, have suggested a role of interleukin-1 (IL-1), in the decreased bone mineral density (BMD) of fasting hypercalciuria. The present study was designed to determine which IL-1 fraction (alpha or beta) correlates with bone resorption and whether other known bone resorting cytokines like interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) may play a role in this process. Cytokines production was determined by quantitative and specific analysis, enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase polymerase chain reaction (RT-PCR). Dual-energy X-ray absorptiometry and cytokine production by unstimulated and lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) were determined in a group of 29 patients with recurrent nephrolithiasis (17 hypercalciurics and 12 normocalciurics), and 12 healthy controls. The hypercalciuric subjects showed lower vertebral BMD than the normocalciuric or normal controls. There was no difference in spinal or femoral BMD between absorptive or fasting hypercalciurics. A significant negative correlation existed between urinary calcium excretion and vertebral BMD (r = -0.55, P < 0.01). Basal IL-1 alpha production correlated with vertebral BMD (r = -0.45, P < 0.02). This correlation was not seen with IL-1 beta, IL-6 or TNF-alpha production. LPS-induced IL-6 and TNF-alpha production were enhanced in the hypercalciuric patients, when compared to normocalciurics or controls. Control and normocalciuric subjects showed minimal amounts of IL-1 alpha mRNA. In contrast, hypercalciuric patients showed a significant increase of spontaneous IL-1 alpha mRNA transcription. These results suggest that different cytokines could be involved in the bone resorption process observed in hypercalciuria.

The possibility that dietary fish oil supplementation may benefit patients with hypercalciuric urolithiasis by decreasing calcium excretion and enhancing protective mechanisms has been studied in rats and humans. In experiments on rats in metabolic cages, fish oil inhibited experimental nephrocalcinosis induced by intraperitoneal calcium gluconate. There were no significant changes in urinary biochemistry. In a clinical study on 18 hypercalciuric recurrent stone patients fish oil significantly decreased urinary calcium excretion. This effect was accompanied by decreases in the excretion of magnesium and citrate. Oxalate excretion and urinary fibrinolytic activity were unchanged. Overall, fish oil had a limited impact on the risk profile for recurrent urolithiasis.

Children with hyperprostaglandin E syndrome, a neonatal variant of Bartter syndrome with enhanced renal and systemic formation of prostaglandin E2, have hypercalciuria, nephrocalcinosis, and osteopenia. Because prostaglandin E2 affects tubular calcium handling, stimulates the formation of calcitriol in vitro, and has osteolytic activity, we studied calcium homeostasis and the influence of prostaglandin E2 formation on hypercalciuria in nine patients with hyperprostaglandin E syndrome during long-term indomethacin treatment and after its withdrawal. Suppression of prostaglandin E2 formation by indomethacin resulted in improvement of biochemical and clinical features of hyperprostaglandin E syndrome. However, hypercalciuria, osteopenia, and nephrocalcinosis did not completely resolve. Despite a low calcium diet, daily urinary calcium excretion was enhanced during and after withdrawal of indomethacin treatment (median 6.3, range 5.3 to 14, and median 9.4, range 4.4 to 38 mg/kg per day, respectively). Daily urinary calcium excretion was greater after withdrawal than during indomethacin treatment. Urinary calcium excretion was not correlated with urinary prostaglandin E2 excretion. Plasma levels of intact parathyroid hormone (median 11, range 6.8 to 12 pmol/L) and calcitriol (median 157, range 108 to 236 pg/ml) were elevated during indomethacin treatment and decreased after withdrawal of indomethacin. These data suggest that hypercalciuria in hyperprostaglandin E syndrome is mainly due to a renal leak of calcium, which is caused by enhanced renal formation of prostaglandin E2 and a tubular defect not related to prostaglandin E2 formation. There is no evidence for prostaglandin-stimulated calcitriol formation. Decreasing plasma levels of parathyroid hormone in the presence of renal calcium losses after withdrawal of indomethacin treatment may be due to a bone resorption process caused by systemic prostaglandin formation; the process may contribute to hypercalciuria in the patient not receiving indomethacin.

Recently a technique to measure intact parathyroid hormone (PTH), i.e. the biologically active hormone, has been available. The aim of the present study was to apply this method to evaluate the parathyroid function in a material of recurrent renal stone formers (n = 324). Intact PTH was found to be inversely related to both urinary calcium (r = -0.15; p less than 0.01) and serum calcium (p less than 0.02) indicating that in the majority of the patients with hypercalciuria this was accounted for by intestinal hyperabsorption and not by high serum PTH. Hyperabsorption was also the likely explanation for the finding of a positive relationship between the urinary calcium and oxalate excretions (r = 0.22; p less than 0.001) in medication-free patients without intestinal disorders, i.e. without enteric hyperoxaluria. Altogether 25 patients (7%) had elevated serum PTH concentrations. They were followed up with fasting serum and urinary electrolytes and an oral calcium loading test (1 g of calcium) in order to evaluate the importance of renal and intestinal factors responsible for the elevated serum PTH concentrations. The investigation was carried out on a free diet and on low and high calcium intakes, respectively. The incidence of intestinal malfunction, which was sometimes present without clinical symptoms, was found to be approximately the same as that of impaired renal conservation of calcium. The findings in the patients with intestinal malfunction were a reduced intestinal absorption of calcium and an enhanced tubular reabsorption of calcium (TRCa), with greater reabsorption of calcium for higher PTH values. In patients with impaired renal conservation of calcium despite the raised PTH there was no correlation between PTH and TRCa. When PTH was suppressed during the oral calcium load the TRCa was found to be inappropriately low and the renal defect obvious. The intestinal calcium absorption was secondarily increased to compensate for the renal losses.

Prostanoids belong to the growing family of eicosanoids, which are all derived from arachidonic acid. Prostanoids act as modulators and mediators in a large spectrum of physiological and pathophysiological processes within the kidney. On the one hand, the potent vasoconstrictor and platelet-aggregating thromboxane (TX) A2 is involved in the pathophysiology of a variety of glomerular diseases, such as haemolytic-uraemic syndrome and immune-mediated glomerulopathies. Prostaglandin (PG) E2, on the other hand, interferes with tubular electrolyte and water handling. Clinical data support the hypothesis that this member of the prostanoid family contributes to the pathophysiology of Bartter's syndrome, hyperprostaglandin E syndrome, idiopathic hypercalciuria and renal diabetes insipidus. Both prostanoids, TXA2 and PGE2, are involved in the pathophysiology of obstructive uropathies. The physiological and protective role of renal vasodilator prostanoids (PGI2 and PGE2) has been studied during treatment with non-steroidal anti-inflammatory drugs. Part of the pharmacological effects of frusemide and converting enzyme inhibitors is mediated by PGI2 and PGE2. The role of renal prostanoids in cyclosporine toxicity is still equivocal. Future investigations on the physiological and pathophysiological role of renal prostanoids will have to consider the multiple interactions between prostanoids on the one hand, and classical hormones and other mediators (e.g. cytokines) on the other hand.

The low incidence of atherosclerosis and other degenerative diseases including stone disease in the Greenland Eskimo has been attributed to their high consumption of oily fish with its high concentration of eicosapentaenoic acid (EPA). Man cannot synthesis EPA from the precursor essential fatty acid, linolenic acid, and can only assimilate preformed EPA present in fish and fish oil, to bring about a change in the pathway of eicosanoid metabolism from the n-6 to the n-3 series. With a westernised diet the oxygenated products of renal prostaglandin synthesis are metabolites of the n-6 series and these are known to play an important role in several pathophysiological states including stone disease. Our previous studies have shown a relationship between prostaglandin activity and urinary calcium excretion and it would seem that the initiating factor/s for stone formation trigger the mechanisms for prostaglandin synthesis resulting in the biochemical abnormalities associated with stone disease. The Eskimo may be protected from these events by possession of an eicosanoid metabolism that follows an n-3 pathway. To test this hypothesis experiments were performed using an animal model of nephrocalcinosis. The animals were divided into three groups; one group was given an intra-peritoneal injection of 10% calcium gluconate daily for 10 days to induce nephrocalcinosis; a second group was fed MaxEPA fish oil before and during the calcium gluconate injections and a third group only received an intra-peritoneal injection of N saline. A group of 12 recurrent, hypercalciuric/hyperoxaluric stone-formers were treated with fish oil for eight weeks to study the effects on solute excretion. Nephrocalcinosis, which was readily produced in the control animals, was prevented in the experimental animals by pre-treatment with fish oil and urine calcium excretion was significantly reduced. The urinary calcium and oxalate excretion in the recurrent, hypercalciuric stone-formers was significantly reduced with fish oil treatment over an eight week period. There were no untoward side-effects. These studies indicate that the incorporation of EPA in the diet as a substitute metabolic pathway could be a unique way of correcting the biochemical abnormalities of idiopathic urolithiasis.

Nifedipine has been shown to lower urinary calcium in "essential" hypercalciuria. However, the mechanism(s) by which this action takes place is completely unknown. This study describes the effect of nifedipine on some calcium-controlling hormones in essential hypercalciuria. Nifedipine (20 mg/day) was administered to ten essential hypercalciuric patients, and urinary PgE2, plasma bicyclic PgE2, 1,25 vitamin D3, and PTH were assayed before and after drug administration. Nifedipine promoted a significant fall in urinary calcium (352.1 +/- 87.67 SD vs. 231.2 +/- 74.62 mg/hr; t = 7.35, p less than .0001) and PgE2 (343.92 +/- 42.71 vs. 245.03 +/- 35.41 SD ng/24 hr; t = 6.18, p less than .0002), as well as in plasma bicyclic PgE2 (310.00 +/- 30.91 vs. 200.00 +/- 31.62 SD pg/ml; t = 9.86, p less than .0001) and 1,25 (OH)2 vitamin D3 (32.77 +/- 3.23 vs. 26.94 +/- 2.94 SD pg/ml; t = 6.53, p less than .0001), while PTH remained unaltered (18.50 +/- 3.63 vs. 19.50 +/- 4.09 SD ng/ml; t = 0.85, p, ns). Urinary calcium and PgE2 correlated positively before (r = 0.81, p less than .005) but not after treatment. The fall in urinary PgE2 brought about by nifedipine seems to be due to an inhibition of PgE2 synthesis, since the absolute decrements in both urinary PgE2 and plasma PgE2 metabolites were positively correlated (r = 0.79, p less than .007). No correlation was found between the absolute decrements of plasma bicyclic PgE2 and 1,25 (OH)2 vitamin D3. These data seem to suggest that the fall in urinary calcium brought about by nifedipine is in some way related to PgE2 synthesis inhibition and to uncoupling of 1,25 (OH)2 vitamin D3 and PTH action.