Mitochondrial permeability transition pore opening induces the initial process of renal calcium crystallization

Renal tubular cell injury induced by oxidative stress via mitochondrial collapse is thought to be the initial process of renal calcium crystallization. Mitochondrial collapse is generally caused by mitochondrial permeability transition pore (mPTP) opening, which can be blocked by cyclosporine A (CsA). Definitive evidence for the involvement of mPTP opening in the initial process of renal calcium crystallization, however, is lacking. In this study, we examined the physiological role of mPTP opening in renal calcium crystallization in vitro and in vivo. In the in vitro study, cultured renal tubular cells were exposed to calcium oxalate monohydrate (COM) crystals and treated with CsA (2 μM). COM crystals induced depolarization of the mitochondrial membrane potential and generated oxidative stress as evaluated by Cu-Zn SOD and 4-HNE. Furthermore, the expression of cytochrome c and cleaved caspase 3 was increased and these effects were prevented by CsA. In the in vivo study, Sprague-Dawley rats were administered 1% ethylene glycol (EG) to generate a rat kidney stone model and then treated with CsA (2.5, 5.0, and 10.0 mg/kg/day) for 14 days. EG administration induced renal calcium crystallization, which was prevented by CsA. Mitochondrial collapse was demonstrated by transmission electron microscopy, and oxidative stress was evaluated by measuring Cu-Zn SOD, MDA, and 8-OHdG generated by EG administration, all of which were prevented by CsA. Collectively, our results provide compelling evidence for a role of mPTP opening and its associated mitochondrial collapse, oxidative stress, and activation of the apoptotic pathway in the initial process of renal calcium crystallization.     

Urinary glycosaminoglycans and glycoproteins in a calcium oxalate crystallization system

This study measures the effects of total urinary glycosaminoglycans (GAGs), glycoproteins (GPs) and individual GAGs on the nucleation rates (Bo), growth rates (G) and suspension densities (Mт) of calcium oxalate (CaOx) crystallization by the mixed suspension mixed product removal (MSMPR) system. Total urinary GAGs, glycoproteins and individual GAGs including heparan sulfate (HS), chondroitin sulfate (CS) and Hyaluronic acid (HA) were added into the artificial urine (AU) and then introduced into the MSMPR test chamber and the crystal sizes and numbers were analyzed by a particle counter. The effects of added GAGs and GPs on CaOx crystallization were reflected by the changes on the crystallization indexes including the Bo, G and Mт of CaOx that were calculated based on the crystal size and numbers. Total urinary GAGs showed no statistical significance on CaOx crystallization. However, individual GAGs such as HA, CS and HS enhanced Bo and suppressed the G when measured individually. CS and HS enhanced the Mт while HA shown no significant change in the Mт of CaOx. Total urinary GPs showed an increase in the G and Mт of crystals. Although total urinary GAGs showed no statistically significant effect on CaOx crystallization, individual GAGs (CS, HS) promoted the CaOx crystallization by increasing the suspension density of smaller crystals, indicative of reduced risk of stones while HA showed no significance in the M(T) of CaOx formed. Urinary GPs indicated increased sizes and M(T) suggesting larger crystals and/or aggregates.     

Sialylation of urinary prothrombin fragment 1 is implicated as a contributory factor in the risk of calcium oxalate kidney stone formation

Urinary glycoproteins are important inhibitors of calcium oxalate crystallization and adhesion of crystals to renal cells, both of which are key mechanisms in kidney stone formation. This has been attributed to glycosylation of the proteins. In South Africa, the black population rarely form stones (incidence < 1%) compared with the white population (incidence 12-15%). A previous study involving urinary prothrombin fragment 1 from both populations demonstrated superior inhibitory activity associated with the protein from the black group. In the present study, we compared N-linked and O-linked oligosaccharides released from urinary prothrombin fragment 1 isolated from the urine of healthy and stone-forming subjects in both populations to elucidate the relationship between glycosylation and calcium oxalate stone pathogenesis. The O-glycans of both control groups and the N-glycans of the black control samples were significantly more sialylated than those of the white stone-formers. This demonstrates a possible association between low-percentage sialylation and kidney stone disease and provides a potential diagnostic method for a predisposition to kidney stones that could lead to the implementation of a preventative regimen. These results indicate that sialylated glycoforms of urinary prothrombin fragment 1 afford protection against calcium oxalate stone formation, possibly by coating the surface of calcium oxalate crystals. This provides a rationale for the established roles of urinary prothrombin fragment 1, namely reducing the potential for crystal aggregation and inhibiting crystal-cell adhesion by masking the interaction of the calcium ions on the crystal surface with the renal cell surface along the nephron.     

Attenuation of oxalate-induced nephrotoxicity by eicosapentaenoate-lipoate (EPA-LA) derivative in experimental rat model

Hyperoxaluria is one of the major risk factors for the formation of urinary calcium oxalate stones. Calcium oxalate crystals and their deposition have been implicated in inducing renal tubular damage. Lipoic acid (LA) and eicosapentaenoic acid (EPA) have been shown to ameliorate the changes associated with hyperoxaluria. This prompted us to investigate the nephroprotectant role of EPA-LA, a new derivative, in vivo in hyperoxaluric rats. Elevation in the levels of calcium, oxalate and phosphorus, the stone-forming constituents, were observed in calculogenic rats as a manifestation of crystal deposition.Tubular damage to the renal tissue was assessed byassaying the excretion of marker enzymes in the urine. Damage to the tubules was indicated by increased excretion of alkaline phosphatase (ALP), lactate dehydrogenase (LDH), gamma-glutamyl transferase (gamma-GT), beta-Glucuronidase (beta-GLU) and N-Acetyl beta-D glucosaminidase (NAG). Fibrinolytic activity was found to be reduced. Administration of EPA, LA and EPA-LA reduced the tubular damage and decreased the markers of crystal deposition markedly, which was substantiated by the reduction in weight of bladder stone formed. Our results highlight that EPA-LA is the most effective drug in inhibiting stone formation and mitigating renal damage caused by oxalate toxicity, thus confirming it as a nephroprotectant. Further work in this direction is warranted to establish the therapeutic effectiveness of this new derivative.     

Effect of hydrochlorothiazide on urine saturation with brushite,in vitro collagen calcification by urine,and urinary inhibitors of collagen calcification

To clarify further the beneficial effect of thiazide diuretics on recurrent calcium nephrolithiasis, the effect of short-term hydrochlorothiazide therapy on urine saturation with brushite (CaHPO₄·2H₂O), in vitro collagen calcification by urine, and urinary inhibitors of calcification was studied.In 22 patients with idiopathic calcium oxalate/phosphate stones the urine calcium excretion decreased, the urine magnesium excretion increased and the urine magnesium/calcium ratio increased significantly (P < 0.001) during hydrochlorothiazide therapy. Supersaturation of the urine with brushite, which was present in 19 of the 22 patients, was reduced significantly (P < 0.001) in all during thiazide therapy, and to the undersaturated range in 16. The ability of urine to calcify collagen in vitro also decreased significantly (P < 0.001) during thiazide therapy, a change that correlated significantly (r = 0.4513, P < 0.05) with the decrease in brushite saturation. The concentration of urinary inhibitors of calcification, as determined with an in vitro collagen calcification system, was decreased significantly (P < 0.01) by thiazide therapy.It was concluded that, in addition to decreasing urine calcium excretion and increasing urine magnesium excretion, thiazide diuretics decrease the urinary brushite saturation and thus may prevent spontaneous nucleation or crystal growth, or both, of calcium phosphate. The ability of thiazides to decrease collagen calcification in vitro suggests that they may also prevent crystal growth on a nidus of organic matrix. Thiazides do not appear to act by increasing the excretion of urinary inhibitors of calcification.     

A bout of resistance exercise increases urinary calcium independently of osteoclastic activation in men

Ashizawa, Noriko, Rei Fujimura, Kumpei Tokuyama, andMasashige Suzuki. A bout of resistance exercise increases urinary calcium independently of osteoclastic activation in men.J. Appl. Physiol. 83(4):1159-1163, 1997.Metabolic acidosis increases urinary calciumexcretion in humans as a result of administration of ammonium chloride,an increase in dietary protein intake, and fasting-inducedketoacidosis. An intense bout of exercise, exceeding aerobic capacity, also causes significant decrease in blood pH as aresult of increase in blood lactate concentration. In this study weinvestigated changes in renal calcium handling, plasma parathyroidhormone concentration, and osteoclastic bone resorption after a singlebout of resistance exercise. Ten male subjects completed about of resistance exercise with an intensity of 60% of one repetitionmaximum for the first set and 80% of one repetition maximum for thesecond and third sets. After exercise, blood and urine pH shiftedtoward acidity and urinary calcium excretion increased.Hypercalciuria was observed in the presence of an increased fractionalcalcium excretion and an unchanged filtered load of calcium. Therefore,the observed increase in urinary calcium excretion was due primarily todecrease in renal tubular reabsorption of calcium. Likely causes of theincrease in renal excretion of calcium are metabolic acidosis itselfand decreased parathyroid hormone. When urinary calcium excretionincreased, urinary deoxypyridinoline, a marker of osteoclastic boneresorption, decreased. These results suggest that1) strenuous resistance exerciseincreased urinary calcium excretion by decreasing renal tubular calciumreabsorption, 2) urinary calciumexcretion increased independently of osteoclast activation, and3) the mechanism resulting inpostexercise hypercalciuria might involve non-cell-mediatedphysicochemical bone dissolution.

     

Silent Renal Stones in Primary Hyperparathyroidism: Prevalence and Clinical Features

Objective(1) To evaluate the prevalence of silent nephrolithiasis in patients with primary hyperparathyroidism (PHPT) compared with controls, and (2) To characterize clinically PHPT patients with silent renal stones.MethodsWe reviewed clinical data for 141 patients with PHPT and without symptoms or history of nephrolithiasis in whom renal ultrasonography was performed at diagnosis. A total of 141 sex- and age- matched subjects with abdomen ultrasonography obtained for reasons different from urinary symptoms served as controls.ResultsSilent nephrolithiasis was more prevalent in PHPT patients than in controls (11.35% vs. 2.13%, P = .003). Among patients with PHPT, those with silent renal stones showed higher serum calcium and parathyroid hormone levels and met surgical criteria, regardless of nephrolithiasis, more frequently than those without renal stones.ConclusionThe prevalence of silent nephrolithiasis is increased in patients with PHPT as compared with controls. Moreover, it seems likely that silent renal stone disease could identify a subset of PHPT patients with more severe disease. Accordingly, we suggest ultrasonographic screening of nephrolithiasis in all PHPT patients. Further studies are needed to better characterize this clinical entity. (Endocr Pract. 2014;20:1137-1142)     

Kidney Stones

The prevalence of kidney stones has steadily risen during this century; passage of a calculus and a positive family history increase the probability of recurrence. Findings from recent studies on the cause of renal calculi have stressed crystallization and crystal aggregation of stone minerals from supersaturated urine, rather than excessive organic matrix. Absence of normal urine inhibitors of calcium salts is also stressed. Formation of calcium oxalate stones is the major problem. Therapy with decreased calcium and oxalate intake, thiazides, phosphate salts and allopurinol in various combinations has substantially decreased the prevalence of recurrent stones. The rationale for the use of allopurinol is that uric acid salts enhance the tendency for calcium oxalate to crystallize from supersaturated urine. The hypercalciuria seen in 30 percent to 40 percent of patients with oxalate stones is usually caused by intestinal hyperabsorption of calcium. Although patients with uric acid calculi constitute only a small fraction of those in whom stones form, they represent a group in whom good medical therapy, based on sound physiologic principles, has proved extremely successful. Renal tubular syndromes lead to nephrocalcinosis and lithiasis through hypercalciuria, alkaline urine and hypocitraturia, the latter an inhibitor of calcium salt precipitation. Recent advances in surgical techniques are discussed, including the rationale for removing staghorn calculi. The ileal ureter and coagulum pyelolithotomy deserve special emphasis.     

The effect of seed crystals of hydroxyapatite and brushite on the crystallization of calcium oxalate in undiluted human urine in vitro: implications for urinary stone pathogenesis

BACKGROUND: The aim of this study was to determine whether crystals of hydroxyapatite (HA) or brushite (BR) formed in urine promote the epitaxial deposition of calcium oxalate (CaOx) from undiluted human urine in vitro and thereby explain the occurrence of phosphate in the core of urinary stones consisting predominantly of CaOx. MATERIALS AND METHODS: Crystals of HA, BR, and CaOx were generated from human urine and their identity confirmed by X-ray analysis. Standard quantities of each crystal were then added to separate aliquots of pooled undiluted human urine and CaOx crystallization was induced by the addition of identical loads of sodium oxalate. Crystallization was monitored by Coulter Counter and (14) C-oxalate analysis and the precipitated crystals were examined by scanning electron microscopy. RESULTS: In comparison with the control to which no seeds were added, addition of CaOx crystals increased the deposition of (14) C-oxalate by 23%. On the other hand, seeds of HA and BR had no effect. These findings were supported by Coulter Counter analysis, which showed that the average modal sizes of crystal particles precipitated in the presence of HA and BR seeds were indistinguishable from those in the control, whereas those deposited in the presence of CaOx were significantly larger. Scanning electron microscopy confirmed these results, demonstrating that large aggregates of CaOx dihydrates were formed in the presence of CaOx seeds, whereas BR and to a lesser extent HA seeds were scattered free on the filtration membrane and attached like barnacles on the surface of the freshly precipitated CaOx crystals. CONCLUSION: Seed crystals of HA or BR do not promote CaOx deposition in urine in vitro and are therefore unlikely to influence CaOx crystal formation under physiologic conditions. However, binding of HA and BR crystals to, and their subsequent enclosure within, actively growing CaOx crystals might occur in vivo, thereby explaining the occurrence of mixed oxalate/phosphate stones.     

Vitexin exerts protective effects against calcium oxalate crystal-induced kidney pyroptosis in vivo and in vitro

BackgroundNephrolithiasis is a common urinary disease with a high recurrence rate of secondary stone formation. Several mechanisms are involved in the onset and recurrence of nephrolithiasis, e.g., oxidative stress, inflammation, apoptosis, and epithelial-mesenchymal transition (EMT). Vitexin, a flavonoid monomer derived from medicinal plants that exert many biological effects including anti-inflammatory and anticancer effects, has not been investigated in nephrolithiasis studies. Moreover, pyroptosis, a form of programmed cell death resulting from inflammasome-associated caspase activation, has not been studied in mice with nephrolithiasis.PurposeWe aimed to investigate the protective effect and underlying mechanisms of vitexin in nephrolithiasis, and the related role of pyroptosis in vivo and in vitro.MethodsMouse models of nephrolithiasis were established via intraperitoneal injection of glyoxylate, and cell models of tubular epithelial cells and macrophages were established using calcium oxalate monohydrate (COM). Crystal deposition and kidney tissue injury were evaluated by hematoxylin and eosin, and von Kossa staining. Renal oxidative stress indexes including malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), and catalase (CAT), were analyzed. The renal expression of interleukin-1 beta (IL-1β), gasdermin D (GSDMD), osteopontin (OPN), CD44, and monocyte chemotactic protein 1 (MCP-1), and EMT-related proteins in renal tubular epithelial cells was assessed. Cell viability and the apoptosis ratio were evaluated.ResultsIn vivo, vitexin alleviated crystal deposition and kidney tissue injury, and decreased the level of MDA, and increased the levels of SOD, GSH, and CAT. Vitexin also reduced the levels of the pyroptosis-related proteins GSDMD, NLRP3, cleaved caspase-1, and mature IL-1β, which were elevated in mice with nephrolithiasis, and repressed apoptosis and the expression of OPN and CD44. Moreover, vitexin mitigated F4/80-positive macrophage infiltration and MCP-1 expression in the kidneys. Furthermore, an in vitro study showed that vitexin increased the viability of HK-2 cells and THP-1-derived macrophages, which was impaired by treatment with COM crystals, decreased the medium lactate dehydrogenase (LDH) level, and inhibited the expression of pyroptosis-related proteins in HK-2 cells and macrophages. Vitexin repressed EMT of HK-2 cells, with increased expression of pan-cytokeratin (Pan-ck) and decreased expression of Vimentin and alpha-smooth muscle actin (α-SMA), and downregulated the Wnt/β-catenin pathway. Moreover, vitexin suppressed tumor necrosis factor-α (TNF-α) and IL-1β mRNA expression, which was upregulated by COM in macrophages.ConclusionVitexin exerts protective effects against nephrolithiasis by inhibiting pyroptosis activation, apoptosis, EMT, and macrophage infiltration. In addition, GSDMD-related pyroptosis mediates nephrolithiasis.     

Structure of Proteus mirabilis biofilms grown in artificial urine and standard laboratory media

Proteus mirabilis is a urinary pathogen that can differentiate from a swimmer cell into a swarmer cell morphotype and can form biofilms on the surfaces of urinary catheters. These biofilms block these catheters due to crystals trapped within these structures. The effect of encrustation on biofilm formation and structure has not been studied using confocal scanning laser microscopy (CSLM). Therefore, a comparison of biofilm structure in artificial urine (AU) and laboratory media was undertaken. We compared the structure of P. mirabilis biofilms in AU and Luria-Bertani broth using CSLM and 3D imaging. Biofilms grown in Luria-Bertani broth formed mushroom structures at 24 h and contained nutrient channels. AU biofilms were observed to form a different structure at 24 h. AU biofilm structure was observed to be a flat layer, almost devoid of nutrient channels. Swarmer cells were observed protruding out of the biofilm into the bulk fluid. This could be due to nutrient depravation within the biofilm or a means of further colonizing the surface. This study has demonstrated that two markedly different biofilm structures are formed, depending on the growth media utilized.     

Systematic comparisons of artificial urine formulas for in vitro cellular study

Artificial urine (AU) is widely used for in vitro cellular study to simulate normal physiological environment of the kidney and urinary tract. However, some compositions of many of previously established AU formulas are out of their physiological ranges in normal human urine. Therefore, we established a new AU formula, named “AU-Siriraj,” and then performed systematic comparisons of AU-Siriraj with other six previously established AU formulas, ultrafiltrated (UF) urine, and blank-control to determine their compatibility with MDCK cells. The data indicate that AU-Siriraj is the best, and AU-3 is the second best, AU formula for in vitro cellular study.     

Association of vitamin D receptor gene polymorphism with the urine calcium level in nephrolithiasis patients

Abstract

Association of vitamin D receptor (VDR) gene polymorphism with the urine calcium level in nephrolithiasis patients from the published reports are still conflicting. This study was conducted to evaluate the relationship between VDR BsmI (rs1544410), Fok1 (rs2228570), TaqI (rs731236) and ApaI (rs7975232) gene polymorphism and urine calcium level in nephrolithiasis patients using meta-analysis method. The association studies were identified from PubMed, and Cochrane Library on 1 April 2014, and eligible investigations were included and synthesized using meta-analysis method. Four reports were recruited into this meta-analysis for the association of VDR BsmI, Fok1, TaqI and ApaI gene polymorphism with urine calcium level in nephrolithiasis patients. In this meta-analysis, VDR BsmI B allele and BB genotype, Fok1 f allele and ff genotype, TaqI, and ApaI gene polymorphism were not associated with urine calcium level in nephrolithiasis patients. However, the BsmI bb genotype and Fok1 FF genotype were associated with the urine calcium level in nephrolithiasis patients. In conclusion, VDR BsmI bb genotype and Fok1 FF genotype were associated with the urine calcium level in nephrolithiasis patients. However, more studies should be conducted to confirm it.     

Inhibition of prostaglandin synthesis in man

7α-Hydroxy-5,11-diketotetranor-prostane-1,16-dioic acid, the major urinary metabolite of prostaglandins E₁ and E₂ in man, was determined in human urine by a method based on the use of the bis (O-²H₃-methyloxime) derivative of dimethyl 7α-hydroxy-5,11-diketotetranor-prostane-1,16-dioate as internal standard and determination of the ratio between unlabeled and deuterium-labeled molecules by multiple-ion analysis. Male subjects excreted larger amounts of the metabolite (6.5–46.7 μg/24 hours, n=10) than did female subjects (2.5–5.3 μg/24 hours, n=10). The excretion rate was strongly suppressed following oral administration of therapeutic doses of indomethacin, aspirin and sodium salicylate.     

Nephrolithiasis and pyelonephritis in two West Indian manatees (Trichechus manatus spp.)

Two West Indian manatees (Trichechus manatus spp.) were reported with severe emaciation. One animal was a Florida manatee from the Everglades; the other was an Antillean manatee from Cuba. On necropsy, both animals had nephrolithiasis, pyelonephritis, and moderate to severe renomegaly. Histopathology revealed multifocal to diffuse pyelonephritis, interstitial nephritis, and nephrocalcinosis. The stones were analyzed and consisted primarily of calcium carbonate. Serum chemistry values for the Florida animal revealed no renal abnormalities. The mechanism of calculus formation remains unclear in manatees. In horses, another hindgut fermenter, the most common urolith is also calcium carbonate. Urinalyses performed on manatees are very similar to those of horses (i.e., alkaline urine, low specific gravity, and calcium carbonate crystals). Formation of uroliths in manatees may have a pathogenesis similar to equine urolithiasis.     

Macropinocytosis is the Major Mechanism for Endocytosis of Calcium Oxalate Crystals into Renal Tubular Cells

During an initial phase of kidney stone formation, the internalization of calcium oxalate (CaOx) crystals by renal tubular cells has been thought to occur via endocytosis. However, the precise mechanism of CaOx crystal endocytosis remained unclear. In the present study, MDCK renal tubular cells were pretreated with inhibitors specific to individual endocytic pathways, including nystatin (lipid raft/caveolae-mediated), cytochalasin D (actin-dependent or macropinocytosis), and chlorpromazine (CPZ; clathrin-mediated) before exposure to plain (non-labeled), or fluorescence-labeled CaOx monohydrate (COM) crystals. Quantitative analysis by flow cytometry revealed that pretreatment with nystatin and CPZ slightly decreased the crystal internalization, whereas the cytochalasin D pretreatment caused a marked decrease in crystal uptake. Immunofluorescence study and laser-scanning confocal microscopic examination confirmed that the cytochalasin D-pretreated cells had dramatic decrease of the internalized crystals, whereas the total number of crystals interacted with the cells was unchanged (crystals could adhere but were not internalized). These data have demonstrated for the first time that renal tubular cells endocytose COM crystals mainly via macropinocytosis. These novel findings will be useful for further tracking the endocytosed crystals inside the cells during the course of kidney stone formation.     

Gastrin-releasing peptide receptor gene silencing inhibits the development of the epithelial–mesenchymal transition and formation of a calcium oxalate crystal in renal tubular epithelial cells in mice with kidney stones via the PI3K/Akt signaling pathway

Between 1% and 15% of people are globally affected by kidney stones, and this disease has become more common since the 1970s. Therefore, this study aims to investigate the effects of gastrin-releasing peptide receptor (GRPR) gene silencing via the PI3K/Akt signaling pathway on the development of the epithelial–mesenchymal transition (EMT) and formation of a calcium oxalate crystal in renal tubular epithelial cells (TECs) of kidney stones. A total of 70 clean and healthy C57BL/6J mice were assigned into the normal ( n = 10) and kidney stones groups ( n = 60). The underlying regulatory mechanisms of GRPR were analyzed in concert with the treatment of shGRPR-1, LY294002, and shGRPR-1 + LY294002 in TECs isolated from mice with kidney stones. A series of experiments were conducted for the measurement of urinary oxalate and urinary calcium, the renal calcium salt deposition, the positive rate of GRPR, the expressions of renal TECs related genes and calcium oxalate regulation related genes, and the growth of calcium crystals induced by cells. After treatment of shGRPR-1 and shGRPR-1 + LY294002, levels of urinary oxalate and urinary calcium in the serum, as well as positive rate of GRPR, became relatively low, levels of E-cadherin enhanced, whereas levels of Akt, PI3K, GRPR, extents of PI3K and Akt phosphorylation, α-SMA, Vimentin and FSP-1, OPN, MCP-1, and CD44 decreased and a number of crystals reduced. Taken together, we conclude that GRPR gene silencing suppresses the development of the EMT and formation of the calcium oxalate crystal in renal TECs of kidney stones through the inactivation of the PI3K/Akt signaling pathway.     

Idiopathic calcium nephrolithiasis. 1. Differences in urine crystalloids,urine saturation with brushite and urine inhibitors of calcification between persons with and persons without recurrent kidney stone formation.

The propensity of urine to promote calcium stone formation was compared in 64 patients with recurrent idiopathic calcium nephrolithiasis and 30 healthy individuals without such a history. The rates of excretion of urine crystalloids, the urine saturation with brushite (CaHPO4-2H2O), the ability of the urine to calcify collagen in vitro, and the concentration of urine inhibitors of collagen calcification were measured. The patients had a reduced urine citrate excretion rate in addition to an increased urine calcium excretion rate, while the rates for urine magnesium, phosphate, uric acid and oxalate were not significantly different in the two groups of subjects. The urine concentration of magnesium, phosphate and uric acid was decreased in the patients because of the higher urine volume. The urine creatinine excretion rate correlated with the rates of excretion of urine calcium, magnesium, phosphate, uric acid and oxalate in both groups, which suggested that increased lean body mass, possibly associated with greater food intake, may be an important determinant of crystalloid excretion. The urine of the patients was significantly more saturated with brushite than the urine of the control subjects and resulted in greater collagen calcification when incubated in vitro. The urine concentration of inhibitors of collagen calcification, however, was not significantly different in the two groups. Thus, the urine of patients with recurrent idiopathic calcium nephrolithiasis is more highly saturated with brushite, largely as a result of an increased urine calcium excretion rate, and contains a lower concentration of magnesium and citrate, substances that tend to prevent the precipitation and growth of crystals in urine.