Effect of prothrombin and its activation fragments on calcium oxalate crystal growth and aggregation in undiluted human urine in vitro: relationship between protein structure and inhibitory activity. (41/363)

In recent years there has been great interest in the putative role of prothrombin and its activation peptides, especially the urinary form of prothrombin fragment 1, in the pathogenesis of calcium oxalate (CaOx) urolithiasis. Previously, we showed that prothrombin and its activation peptides inhibit CaOx crystallization in inorganic conditions in vitro. The aim of the present study was to determine if this inhibitory activity is retained in undiluted human urine and, therefore, whether it is likely to have any influence under physiological conditions. A secondary objective was to assess the relationship between the structures of the proteins and their inhibitory activities. Prothrombin was purified from Prothrombinex-HT, cleaved with thrombin and the resulting fragment 1 (F1) and fragment 2 (F2) were purified. The purity of each protein was confirmed by SDS/PAGE, and their effects on CaOx crystallization in undiluted ultrafiltered human urine were determined at a final concentration 80.65 nmol/l using Coulter Counter and [(14)C]oxalate analysis. The precipitated crystals were visualized using scanning electron microscopy. The Coulter Counter data revealed that, whereas prothrombin and its activation peptides did not affect the urinary metastable limit and the size of the precipitated particles, F1 did significantly reduce the latter. These findings were corroborated with scanning electron microscopy which also revealed that the reduction in particle size caused by F1 resulted from a decrease in the degree of crystal aggregation, rather than in the size of the individual crystals. The [(14)C]oxalate data showed that none of the proteins added significantly inhibited the mineral deposition. It was concluded that with the exception of F1, which does inhibit CaOx crystal aggregation, prothrombin and its activation peptides do not alter the deposition and aggregation of CaOx crystals in ultrafiltered human urine in vitro. Also, the gamma-carboxyglutamic acid domain of prothrombin and F1, which is absent from thrombin and F2, is the region of the molecules that determines their potent inhibitory effects. The superior potency of F1, compared with prothrombin, probably results from the molecule's greater charge-to-mass ratio.  (+info)

Reduced crystallization inhibition by urine from women with nephrolithiasis. (42/363)

BACKGROUND: Human urine is known to inhibit growth, aggregation, nucleation, and cell adhesion of calcium oxalate monohydrate (COM) crystals, the main solid phase of human kidney stones. This study tested the hypothesis that low levels of inhibition are present in women with calcium oxalate stones and, therefore, could promote stone production. METHODS: In 17 stone-forming women and 17 normal women matched in age within five years, inhibition by dialyzed urine proteins of COM growth and aggregation was examined, as well as whole urine upper limits of metastability (ULM) for COM and calcium phosphate (CaP) in relation to the corresponding supersaturation (SS). RESULTS: Compared to normals, patient urine showed a reduced ULM in relation to SS. In contrast to men, there was no difference in growth inhibition. CONCLUSIONS: Reduced CaP and CaOx ULM values in relation to SS are a characteristic of female stone formers. This defect could promote stones by facilitating crystal nucleation. Abnormal inhibition may well be a very important cause of human nephrolithiasis.  (+info)

Predicting the crystallization potential of urine from cats and dogs with respect to calcium oxalate and magnesium ammonium phosphate (struvite). (43/363)

The objective of this study was to validate two programs (SUPERSAT and EQUIL 2) for calculation of calcium oxalate (CaOx) and magnesium ammonium phosphate (struvite; MAP) relative supersaturation (RSS) in dog and cat urine. Healthy adult cats (n = 10) and dogs (n = 9) were fed standard diets for a 3-wk period. Urine was collected (24 h, dogs; 48 h, cats) and filtered, and the pH was measured. A 20-mL aliquot was titrated to pH 2 and frozen for analysis. Additional aliquots were incubated with 1 g seed crystals at 38 degrees C; CaOx for 24 h (cat) and 2, 6 and 9 d (dog); MAP for 48 h (dog) and 6 d (cat). Samples were analyzed for 10 substances. RSS was calculated using EQUIL 2 and SUPERSAT. CaOx RSS (SUPERSAT): dog urine was initially supersaturated, whereas cat urine was undersaturated with the diets used. Cat urine reached the solubility product (K(sp)), (RSS = 1) after 24-h incubation, whereas dog urine was still approaching K(sp) at 9 d. MAP RSS (SUPERSAT): urine from both species was undersaturated and increased toward K(sp) during incubation. Final RSS values were compared for both programs. SUPERSAT resulted in values close to 1 for both CaOx and MAP; EQUIL 2 gave similar values for CaOx RSS, although MAP RSS values were considerably higher than 1. In conclusion, EQUIL 2 and SUPERSAT both calculated reasonably accurate RSS values for CaOx, whereas only SUPERSAT provided an accurate measure of MAP RSS.  (+info)

Analysis of Ca(2+) uptake into the smooth endoplasmic reticulum of permeabilised sternal epithelial cells during the moulting cycle of the terrestrial isopod Porcellio scaber. (44/363)

In terrestrial isopods, large amounts of Ca(2+) are transported across anterior sternal epithelial cells during moult-related deposition and resorption of CaCO(3) deposits. Because of its toxicity and function as a second messenger, resting cytosolic Ca(2+) levels must be maintained below critical concentrations during epithelial Ca(2+) transport, raising the possibility that organelles play a role during Ca(2+) transit. We therefore studied the uptake of Ca(2+) into Ca(2+)-sequestering organelles by monitoring the formation of birefringent calcium oxalate crystals in permeabilised anterior and posterior sternal epithelium cells of Porcellio scaber during Ca(2+)-transporting and non-transporting stages of the moulting cycle using polarised-light microscopy. The results indicate ATP-dependent uptake of Ca(2+) into organelles. Half-maximal crystal growth at a Ca(2+) activity, a(Ca), of 0.4 micromol l(-1) and blockade by cyclopiazonic acid suggest Ca(2+) uptake into the smooth endoplasmic reticulum by the smooth endoplasmic reticulum Ca(2+)-ATPase. Analytical electron microscopical techniques support this interpretation by revealing the accumulation of Ca(2+)-containing crystals in smooth membranous intracellular compartments. A comparison of different moulting stages demonstrated a virtual lack of crystal formation in the early premoult stage and a significant fivefold increase between mid premoult and the Ca(2+)-transporting stages of late premoult and intramoult. These results suggest a contribution of the smooth endoplasmic reticulum as a transient Ca(2+) store during intracellular Ca(2+) transit.  (+info)

Glandular hairs of Sigesbeckia jorullensis Kunth (Asteraceae): morphology, histochemistry and composition of essential oil. (45/363)

Long-stalked glandular hairs of outer and inner involucral bracts of Sigesbeckia jorullensis, which are important for epizoic fruit propagation, were investigated using light and scanning electron microscopy. The essential oil secreted by the hairs was analysed by chromatographic methods including gas chromatography/mass spectrometry and with a laser microprobe mass analyser. The glandular hairs consisted of a large multicellular stalk and a multicellular secreting head. The apical layer of glandular head cells was characterized by leucoplasts and calcium oxalate crystals. Below the apical cells there were up to six layers of cells containing many chloroplasts around the nucleus and surrounded by vacuoles filled with flavonoids and tannins. The essential oil originating in the head cells was secreted into the subcuticular space and may be liberated by rupture of the cuticle. It was mainly composed of sesqui- and diterpenes, with the sesquiterpene hydrocarbon germacrene-D as the main component. Monoterpenes, n-alkanes and their derivatives as well as flavonoid aglycones were also detected. The stickiness of the essential oil is probably associated with the high content of oxygenated sesqui- and diterpenes. In addition to long-stalked trichomes, small biseriate trichomes occurred, secreting small quantities of essential oil into a subcuticular space.  (+info)

COM crystals activate the p38 mitogen-activated protein kinase signal transduction pathway in renal epithelial cells. (46/363)

Interaction of calcium oxalate monohydrate (COM) crystals with renal cells has been shown to result in altered gene expression, DNA synthesis, and cell death. In the current study the role of a stress-specific p38 MAP kinase-signaling pathway in mediating these effects of COM crystals was investigated. Exposure of cells to COM crystals (20 microg/cm(2)) rapidly stimulated strong phosphorylation and activation of p38 mitogen-activated protein kinase (p38 MAP kinase) and re-initiation of DNA synthesis. Inhibition of COM crystal binding to the cells by heparin blocked the effects of COM crystals on p38 MAPK activation. We also show that specific inhibition of p38 MAPK by 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl) imidazole (SB203580) or by overexpression of a dominant negative mutant of p38 MAP kinase abolishes COM crystal-induced re-initiation of DNA synthesis. The inhibition is dose-dependent and correlates with in situ activity of native p38 MAP kinase, determined as mitogen-activated protein kinase-activated protein kinase-2 (MAPKAP kinase-2) activity in cell extracts. In summary, inhibiting activation of p38 MAPK pathway abrogated the DNA synthesis in response to COM crystals. These data are the first demonstrations of activation of the p38 MAPK signaling pathway by COM crystals and suggest that, in response to COM crystals, this pathway transduces critical signals governing the re-initiation of DNA synthesis in renal epithelial cells.  (+info)

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. (47/363)

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.  (+info)

Pathophysiologic basis for normouricosuric uric acid nephrolithiasis. (48/363)

BACKGROUND: Low urinary pH is the commonest and by far the most important factor in uric acid nephrolithiasis but the reason(s) for this defect is (are) unknown. Patients with uric acid nephrolithaisis have normal acid-base parameters according conventional clinical tests. METHODS: We studied steady-state plasma and urinary parameters of acid-base balance in subjects with normouricosuric pure uric acid stones. We also tested the ability of these subjects to excrete ammonium in response to an acute acid load. We compared these parameters in patients with pure uric acid stones to patients with mixed uric acid/calcium oxalate stones, pure calcium stones, and normal volunteers. RESULTS: Pure uric acid stone formers have a much higher incidence of either diabetes or glucose intolerance. After equilibration to a control diet, patients with uric acid stones have lower urinary pH and they excrete less of their acid as ammonium. This is compensated by higher titratable acidity and hypocitraturia. Despite their low baseline urinary pH, uric acid stone formers further acidify their urine after an acid load because of a severely impaired ammonia excretory response. Their characteristics are significantly different from normal volunteers and pure calcium stone formers. Patients with mixed uric acid/calcium stones exhibit intermediate characteristics. CONCLUSION: We propose that certain patients with normouricosuric uric acid nephrolithiasis have a renal acidification disease. The primary defect lies in renal ammonium excretion, which may be linked to the insulin-resistant state. Although net acid excretion is maintained at the expense of increased titratable acidity and to some degree hypocitraturia, the compromise is acid urine pH and may result in uric acid nephrolithiasis.  (+info)