Size-Dependent Cytotoxicity, Adhesion, and Endocytosis of Micro-/Nano-hydroxyapatite Crystals in HK-2 Cells.

Nano-hydroxyapatite (nano-HAP) is often used as a crystal nest to induce calcium oxalate (CaOx) kidney stone formation, but the mechanism of interaction between HAP crystals of different properties and renal tubular epithelial cells remains unclear. In this study, the adhesion and endocytosis of HAP crystals with sizes of 40 nm, 70 nm, 1 µm, and 2 µm (HAP-40 nm, HAP-70 nm, HAP-1 µm, and HAP-2 µm, respectively) to human renal proximal tubular epithelial cells (HK-2) were comparatively studied. The results showed that HAP crystals of all sizes promoted the expression of osteopontin and hyaluronic acid on the cell surface, destroyed the integrity of the lysosomes, and induced the apoptosis and necrosis of cells. Nano-HAP crystals had a higher specific surface area, a smaller contact angle, a higher surface energy, and a lower Zeta potential than those of micro-HAP. Therefore, the abilities of HK-2 cells to adhere to and endocytose nano-HAP crystals were greater than their abilities to do the same for micro-HAP crystals. The order of the endocytosed crystals was as follows: HAP-40 nm > HAP-70 nm > HAP-1 µm > HAP-2 µm. The endocytosed HAP crystals entered the lysosomes. The more crystal endocytosis and adhesion there is, the more toxic it is to HK-2 cells. The results of this study showed that nanosized HAP crystals greatly promoted the formation of kidney stones than micrometer-sized HAP crystals.

Shape-dependent adhesion and endocytosis of hydroxyapatite nanoparticles on A7R5 aortic smooth muscle cells.

The interaction between nanohydroxyapatite (HAP) and smooth muscle cells is an important step in vascular calcification. However, the effect of the shape of HAP on adhesion and endocytosis to aortic smooth muscle cells has been rarely reported. Four different morphological HAP crystals (H-Rod, H-Needle, H-Sphere, and H-Plate) were selected to interact with rat aortic smooth muscle cells (A7R5). Fluorescence-labeled HAP was used to detect crystal adhesion and endocytosis and then pretreated with different endocytic inhibitors to explore the pathway of endocytotic crystals. The distribution of crystals inside and outside the cells and the crystal localization in lysosomes was observed through laser confocal microscopy. The effect of crystal on the cell cycle and the changes in the expression of phosphatidylserine, osteopontin, α-actin, core binding factor alpha 1, and osterix on the surface of A7R5 cells were detected. The adhesion and endocytosis of HAP on A7R5 cells were closely related to crystal shapes and ranked as follows: H-Plate > H-Sphere > H-Needle > H-Rod. H-Sphere and H-Needle were internalized into the cells mainly via the clathrin-mediated pathway, whereas H-Plate and H-Rod were internalized into the cells mainly via macropinocytosis. The endocytosed nano-HAP was mainly distributed in the cell lysosome. The adhesion and endocytosis of HAP to A7R5 cells were positively correlated with the specific surface area, and contact area of HAP and negatively correlated with the absolute value of Zeta and contact angle of HAP. This study provided insights into the effect of crystal morphology on vascular calcification and its mechanism.

Repair Effects of Astragalus Polysaccharides with Different Molecular Weights on Oxidatively Damaged HK-2 Cells.

This study investigated the repair effects of three Astragalus polysaccharides (APSs) with different molecular weights (Mws) on injured human renal proximal tubular epithelial (HK-2) cells to reveal the effect of Mw of polysaccharide on cell repair. A damage model was established by injuring HK-2 cells with 2.6 mM oxalate, and APS0, APS1, and APS2 with Mw of 11.03, 4.72, and 2.61 KDa were used to repair the damaged cells. After repair by APSs, the morphology of damaged HK-2 cells gradually returned to normal, the destruction of intercellular junctions recovered, intracellular reactive oxygen species production amount decreased, and their mitochondrial membrane potential increased. In addition, the cell cycle progression gradually normalized, lysosome integrity increased, and cell apoptotic rates obviously declined in the repaired cells. All three APSs could promote the expression of Keap1, Nrf2, SOD1, and CAT. In addition, the expression levels of inflammation markers containing MCP-1 and IL-6 decreased after APS repair. We deduced that APSs exert their repair function by activating the Nrf2-Keap1 signaling pathway and inhibiting inflammation. Among the APSs, APS1 with a moderate Mw provided the strongest repair effect. APSs may have a preventive effect on kidney stones.

Comparison of the adhesion and endocytosis of calcium oxalate dihydrate to HK-2 cells before and after repair by Astragalus polysaccharide.

This work investigated the effects of repairing injured renal proximal tubular epithelial (HK-2) cells by using three Astragalus polysaccharides (APS) with different molecular weights and the adhesion and endocytosis of HK-2 cells to the calcium oxalate dihydrate (COD) nanocrystals before and after repair to develop new products that can protect against kidney stones. HK-2 cells cultured in vitro were injured with 2.6 mmol/L oxalic acid to establish a damaged cell model. Three kinds of APS (APS0, APS1, and APS2 with molecular weights of 11.03, 4.72, and 2.60 kDa, respectively) were used to repair the damaged cells. The changes in the adhesion and endocytosis of 100 nm COD crystals to cells before and after the repair were detected. After the repair of HK-2 cells by the APS, the speed of wound healing of the damaged HK-2 cells increased, and the amount of phosphatidylserine (PS) ectropion decreased. In addition, the proportion of cells with adhered COD crystals decreased, whereas the proportion of cells with internalized crystals increased. As a result of the repair activity, APS can inhibit the adhesion and promote the endocytosis of COD nanocrystals to damaged cells. APS1, which had a moderate molecular weight, displayed the strongest abilities to repair the cells, inhibit adhesion, and promote endocytosis. Thus, APS, particularly APS1, may serve as potential green drugs for preventing kidney stones.

Diethyl citrate and sodium citrate reduce the cytotoxic effects of nanosized hydroxyapatite crystals on mouse vascular smooth muscle cells.

OBJECTIVE: This study aimed to investigate the damage mechanism of nanosized hydroxyapatite (nano-HAp) on mouse aortic smooth muscle cells (MOVASs) and the injury-inhibiting effects of diethyl citrate (Et2Cit) and sodium citrate (Na3Cit) to develop new drugs that can simultaneously induce anticoagulation and inhibit vascular calcification. METHODS: The change in cell viability was evaluated using a cell proliferation assay kit, and the amount of lactate dehydrogenase (LDH) released was measured using an LDH kit. Intracellular reactive oxygen species (ROS) and mitochondrial damage were detected by DCFH-DA staining and JC-1 staining. Cell apoptosis and necrosis were detected by Annexin V staining. Intracellular calcium concentration and lysosomal integrity were measured using Fluo-4/AM and acridine orange, respectively. RESULTS: Nano-HAp decreased cell viability and damaged the cell membrane, resulting in the release of a large amount of LDH. Nano-HAp entered the cells and damaged the mitochondria, and then induced cell apoptosis by producing a large amount of ROS. In addition, nano-HAp increased the intracellular Ca2+ concentration, leading to lysosomal rupture and cell necrosis. On addition of the anticoagulant Et2Cit or Na3Cit, cell viability and mitochondrial membrane potential increased, whereas the amount of LDH released, ROS, and apoptosis rate decreased. Et2 Cit and Na3Cit could also chelate with Ca+ to inhibit the intracellular Ca2+ elevations induced by nano-HAp, prevent lysosomal rupture, and reduce cell necrosis. High concentrations of Et2Cit and Na3Cit exhibited strong inhibitory effects. The inhibitory capacity of Na3Cit was stronger than that of Et2Cit at similar concentrations. CONCLUSION: Both Et2Cit and Na3Cit significantly reduced the cytotoxicity of nano-HAp on MOVASs and inhibited the apoptosis and necrosis induced by nano-HAp crystals. The chelating function of citrate resulted in both anticoagulation and binding to HAp. Et2Cit and Na3Cit may play a role as anticoagulants in reducing injury to the vascular wall caused by nano-HAp.

Comparison of the Inhibitory Mechanisms of Diethyl Citrate, Sodium Citrate, and Phosphonoformic Acid on Calcification Induced by High Inorganic Phosphate Contents in Mouse Aortic Smooth Muscle Cells.

OBJECTIVE: This study aimed to investigate the differences and inhibitory effects of diethyl citrate (Et2Cit), sodium citrate (Na3Cit), and phosphonoformic acid (PFA) on calcification induced by high inorganic phosphate (Pi) contents in mouse aortic smooth muscle cells (MOVAS) and to develop drugs that can induce anticoagulation and inhibit vascular calcification (VC). METHODS: Alive and fixed MOVAS were assessed for 14 days in the presence of high Pi with increasing Et2Cit, Na3Cit, and PFA concentrations. Calcification on MOVAS was measured through Alizarin red staining and the deposited calcium amount; apoptosis was detected by annexin V staining; and cell transdifferentiation was examined by measuring smooth muscle lineage gene (α-SMA) expression and alkaline phosphatase activity. RESULTS: Coincubation of MOVAS with Et2Cit, Na3Cit, and PFA significantly decreased Pi-induced VC in live MOVAS, and the apoptotic rate was reduced by low inhibitor concentrations. The 3 inhibitors could prevent the alkaline phosphatase activity induced by high Pi contents and increased the expression of α-smooth muscle actin genes. Thus, the transdifferentiation of MOVAS into osteoblast-like cells was blocked. Their inhibitory effects exhibited concentration dependence. The inhibitory effect of each inhibitor at the same concentration showed the following trend: PFA > Na3Cit > Et2Cit. CONCLUSIONS: Et2Cit, Na3Cit, and PFA prevented the calcification of MOVAS and inhibited the osteochondrocytic conversion of vascular smooth muscle cells. Thus, Et2Cit and Na3Cit as anticoagulants may alleviate VC in clinical applications.

Effect of Content of Sulfate Groups in Seaweed Polysaccharides on Antioxidant Activity and Repair Effect of Subcellular Organelles in Injured HK-2 Cells.

This study aims to investigate the repair effect of subcellular structure injuries of the HK-2 cells of four degraded seaweed polysaccharides (DSPs), namely, the degraded Porphyra yezoensis, Gracilaria lemaneiformis, Sargassum fusiform, and Undaria pinnatifida polysaccharides. The four DSPs have similar molecular weight, but with different content of sulfate groups (i.e., 17.9%, 13.3%, 8.2%, and 5.5%, resp.). The damaged model was established using 2.8 mmol/L oxalate to injure HK-2 cells, and 60 µg/mL of various DSPs was used to repair the damaged cells. With the increase of sulfate group content in DSPs, the scavenging activity of radicals and their reducing power were all improved. Four kinds of DSPs have repair effect on the subcellular organelles of damaged HK-2 cells. After being repaired by DSPs, the release amount of lactate dehydrogenase was decreased, the integrity of cell membrane and lysosome increased, the Δψm increased, the cell of G1 phase arrest was inhibited, the proportion of S phase increased, and cell apoptotic and necrosis rates were significantly reduced. The greater the content of sulfate group is, the stronger is the repair ability of the polysaccharide. These DSPs, particularly the polysaccharide with higher sulfate group content, may be a potential drug for the prevention and cure of kidney stones.

Inhibition of urinary macromolecule heparin on aggregation of nano-COM and nano-COD crystals.

PURPOSE: This research aims to study the influences of heparin (HP) on the aggregation of nano calcium oxalate monohydrate (COM) and nano calcium oxalate dihydrate (COD) with mean diameter of about 50 nm. METHOD: The influences of different concentrations of HP on the mean diameter and Zeta potential of nano COM and nano COD were investigated using a nanoparticle size Zeta potential analyzer. RESULTS: HP could be adsorbed on the surface of nano COM and nano COD crystals, leading to an increase in the absolute value of Zeta potential on the crystals and an increase in the electrostatic repulsion force between crystals. Consequently, the aggregation of the crystals is reduced and the stability of the system is improved. The strong adsorption ability of HP was closely related to the -OSO3- and -COO- groups contained in the HP molecules. X-ray photoelectron spectroscopy confirmed the coordination of HP with Ca2+ ions of COM and COD crystals. CONCLUSION: HP could inhibit the aggregation of nano COM and nano COD crystals and increase their stability in aqueous solution, which is conducive in inhibiting the formation of calcium oxalate stones.

Activation of endogenous anti-inflammatory mediator cyclic AMP attenuates acute pyelonephritis in mice induced by uropathogenic Escherichia coli.

The pathogenesis of pyelonephritis caused by uropathogenic Escherichia coli (UPEC) is not well understood. Here, we show that besides UPEC virulence, the severity of the host innate immune response and invasion of renal epithelial cells are important pathogenic factors. Activation of endogenous anti-inflammatory mediator cAMP significantly attenuated acute pyelonephritis in mice induced by UPEC. Administration of forskolin (a potent elevator of intracellular cAMP) reduced kidney infection (ie, bacterial load, tissue destruction); this was associated with attenuated local inflammation, as evidenced by the reduction of renal production of proinflammatory mediators, renal infiltration of inflammatory cells, and renal myeloperoxidase activity. In primary cell culture systems, forskolin not only down-regulated UPEC-stimulated production of proinflammatory mediators by renal tubular epithelial cells and inflammatory cells (eg, monocyte/macrophages) but also reduced bacterial internalization by renal tubular epithelial cells. Our findings clearly indicate that activation of endogenous anti-inflammatory mediator cAMP is beneficial for controlling UPEC-mediated acute pyelonephritis in mice. The beneficial effect can be explained at least in part by limiting excessive inflammatory responses through acting on both renal tubular epithelial cells and inflammatory cells and by inhibiting bacteria invasion of renal tubular epithelial cells.

Nanouric acid or nanocalcium phosphate as central nidus to induce calcium oxalate stone formation: a high-resolution transmission electron microscopy study on urinary nanocrystallites.

PURPOSE: This study aimed to accurately analyze the relationship between calcium oxalate (CaOx) stone formation and the components of urinary nanocrystallites. METHOD: High-resolution transmission electron microscopy (HRTEM), selected area electron diffraction, fast Fourier transformation of HRTEM, and energy dispersive X-ray spectroscopy were performed to analyze the components of these nanocrystallites. RESULTS: The main components of CaOx stones are calcium oxalate monohydrate and a small amount of dehydrate, while those of urinary nanocrystallites are calcium oxalate monohydrate, uric acid, and calcium phosphate. The mechanism of formation of CaOx stones was discussed based on the components of urinary nanocrystallites. CONCLUSION: The formation of CaOx stones is closely related both to the properties of urinary nanocrystallites and to the urinary components. The combination of HRTEM, fast Fourier transformation, selected area electron diffraction, and energy dispersive X-ray spectroscopy could be accurately performed to analyze the components of single urinary nanocrystallites. This result provides evidence for nanouric acid and/or nanocalcium phosphate crystallites as the central nidus to induce CaOx stone formation.

Inhibition of the K+ channel K(Ca)3.1 reduces TGF-ß1-induced premature senescence, myofibroblast phenotype transition and proliferation of mesangial cells.

OBJECTIVE: K(Ca)3.1 channel participates in many important cellular functions. This study planned to investigate the potential involvement of K(Ca)3.1 channel in premature senescence, myofibroblast phenotype transition and proliferation of mesangial cells. METHODS & MATERIALS: Rat mesangial cells were cultured together with TGF-ß1 (2 ng/ml) and TGF-ß1 (2 ng/ml) + TRAM-34 (16 nM) separately for specified times from 0 min to 60 min. The cells without treatment served as controls. The location of K(Ca)3.1 channels in mesangial cells was determined with Confocal laser microscope, the cell cycle of mesangial cells was assessed with flow cytometry, the protein and mRNA expression of K(Ca)3.1, α-smooth muscle actin (α-SMA) and fibroblast-specific protein-1 (FSP-1) were detected with Western blot and RT-PCR. One-way analysis of variance (ANOVA) and Student-Newman-Keuls-q test (SNK-q) were used to do statistical analysis. Statistical significance was considered at P<0.05. RESULTS: Kca3.1 channels were located in the cell membranes and/or in the cytoplasm of mesangial cells. The percentage of cells in G0-G1 phase and the expression of K(ca)3.1, α-SMA and FSP-1 were elevated under the induction of TGF-ß1 when compared to the control and decreased under the induction of TGF-ß1+TRAM-34 when compared to the TGF-ß1 induced (P<0.05 or P<0.01). CONCLUSION: Targeted disruption of K(Ca)3.1 inhibits TGF-ß1-induced premature aging, myofibroblast-like phenotype transdifferentiation and proliferation of mesangial cells.

Changes in urinary nanocrystallites in calcium oxalate stone formers before and after potassium citrate intake.

The property changes of urinary nanocrystallites in 13 patients with calcium oxalate (CaOx) stones were studied before and after ingestion of potassium citrate (K3cit), a therapeutic drug for stones. The analytical techniques included nanoparticle size analysis, transmission electron microscopy, X-ray diffraction, and Fourier-transform infrared spectroscopy. The studied properties included the components, morphologies, zeta potentials, particle size distributions, light intensity autocorrelation curves, and polydispersity indices (PDIs) of the nanocrystallites. The main components of the urinary nanocrystallites before K3cit intake included uric acid, ß-calcium phosphate, and calcium oxalate monohydrate. After K3cit intake, the quantities, species, and percentages of aggregated crystals decreased, whereas the percentages of monosodium urate and calcium oxalate dehydrate increased, and some crystallites became blunt. Moreover, the urinary pH increased from 5.96 ± 0.43 to 6.46 ± 0.50, the crystallite size decreased from 524 ± 320 nm to 354 ± 173 nm, and the zeta potential decreased from -4.85 ± 2.87 mV to -8.77 ± 3.03 mV. The autocorrelation curves became smooth, the decay time decreased from 11.4 ± 3.2 ms to 4.3 ± 1.7 ms, and the PDI decreased from 0.67 ± 0.14 to 0.53 ± 0.19. These changes helped inhibit CaOx calculus formation.

Uremic anorexia and gastrointestinal motility dysfunction correlate with the changes of ghrelin system in hypothalamus.

AIM: Ghrelin can act as a signal for meal initiation and play a role in the regulation of gastrointestinal (GI) motility via hypothalamic circuit. This study investigated the correlation between changes of hypothalamic ghrelin system and GI motility dysfunction and anorexia in rats with chronic renal failure (CRF). METHODS: Sprague-Dawley (SD) rats (male/female 1:1, 180 ± 20 g) were randomly classified into a CRF group and control group (n = 8 per group). 5/6 nephrectomy was used to construct the CRF model. When plasma creatinine concentration (PCr) and blood urea nitrogen (BUN) in the CRF group were twice higher than the normal, food intake (g/24 h) and gastrointestinal interdigestive myoelectric complex (IMC) were detected. Then all rats were killed for assessment of the mRNA expression of ghrelin and growth hormone secretagogue receptor (GHS-R) in hypothalamus using reverse transcription-polymerase chain reaction. Analysis of variance, Student-Newman-Keuls-q-test and Correlation Analysis were used to do statistical analysis. P < 0.05 was considered as statistically significant. RESULTS: Compared to the control group, the CRF group was obviously decreased in the food intake (g/24 h), the phase III duration and amplitude and the ghrelin and GHS-R expression in the hypothalamus (P < 0.05). There was a positive correlation between them (P < 0.05). CONCLUSION: Changes of ghrelin and GHS-R in the hypothalamus correlate with gastrointestinal motility dysfunction and anorexia in rats with CRF.

Coordination dynamics and coordination mechanism of a new type of anticoagulant diethyl citrate with ca(2+) ions.

Diethyl citrate (Et2Cit) is a new potential anticoagulant. The coordination dynamics and coordination mechanism of Et2Cit with Ca(2+) ions and the effect of pH on the complex were examined. The result was compared with that for the conventional anticoagulant sodium citrate (Na3Cit). The reaction order (n) of Et2Cit and Na3Cit with Ca(2+) was 2.46 and 2.44, respectively. The reaction rate constant (k) was 120 and 289 L·mol(-1) ·s(-1). The reverse reaction rate constant (k re) was 0.52 and 0.15 L·mol(-1) ·s(-1), respectively. It is indicated that the coordination ability of Et2Cit with Ca(2+) was weaker than that of Na3Cit. However, the dissociation rate of the calcium complex of Et2Cit was faster than that of Na3Cit. Increased pH accelerated the dissociation rate of the complex and improved its anticoagulant effect. The Et2Cit complex with calcium was synthesized and characterized by elemental analysis, XRD, FT-IR, (1)H NMR, and ICP. These characteristics indicated that O in -COOH and C-O-C of Et2Cit was coordinated with Ca(2+) in a bidentate manner with 1 : 1 coordination proportion; that is, complex CaEt2Cit was formed. Given that CaEt2Cit released Ca(2+) more easily than Na3Cit, a calcium solution was not needed in intravenous infusions using Et2Cit as anticoagulant unlike using Na3Cit. Consequently, hypocalcemia and hypercalcemia were avoided.

Anticoagulation of diethyl citrate and its comparison with sodium citrate in an animal model.

AIMS: To improve the side effects caused by sodium citrate (Na(3)Cit), the anticoagulant effects of diethyl citrate (Et(2)Cit) were investigated. METHODS: The in vitro anticoagulant effects and dissociation capacity of the chelate of Et(2)Cit with calcium ions were compared with those of Na(3)Cit in rabbits. RESULTS: The activated coagulation time test showed that blood clotting time exceeded 1,200 s when the concentrations of Et(2)Cit and Na(3)Cit were greater than 87.2 and 8.72 mmol/l, respectively. The concentrations of free calcium ions in blood c(Ca(2+)) were reduced when Et(2)Cit was injected into the rabbits. CONCLUSIONS: Et(2)Cit reduces the concentration of ionized Ca(2+) in blood and has anticoagulant effects. The dissociation of the chelate of Et(2)Cit with Ca(2+) was faster than that of Na(3)Cit with Ca(2+) within 10 min after injection. The recovery speed of blood calcium concentration with Et(2)Cit was more rapid than that with Na(3)Cit. The findings show that Et(2)Cit prevents hypocalcemia.

[Effect of ghrelin on duodenal migrating myoelectric complex in rats with chronic renal failure].

OBJECTIVE: To investigate the effect of ghrelin on duodenal migrating myoelectric complex (MMC) in rats with chronic renal failure (CRF). METHODS: Thirty healthy male SD rats were randomly assigned into sham-operated group (n=6) and CRF group (n=24), and the latter group was divided into 4 subgroups according to ghrelin doses administered with or without pretreatment with the receptor antagonist D-Lys(3)-GHRP-6. After a 18-h fasting, the rats with or without pretreatment with D-Lys(3)-GHRP-6 were given subcutaneous injections of ghrelin at different doses to observe the changes in duodenal MMC recorded using a multi lead physiological recording system. RESULTS: Ghrelin significantly increased the MMC cycle duration and dose-dependently enhanced the frequency, amplitude and percentage of phase III MMC cycle. This effect was inhibited by the pretreatment with ghrelin receptor antagonist D-Lys(3)-GHRP-6. CONCLUSION: Ghrelin can promote gastrointestinal motilities of rats with CRF, and the receptor of ghrelin can regulate the activity of MMC.

Measurement of urine crystallites and its influencing factors by nanoparticle size analyzer.

The difference of urine crystallites under 1000 nm in 10 patients with urolithiasis and 10 healthy subjects with no history of urolithiasis was comparatively studied with the nanoparticle size analyzer. By comparing the differences of intensity-autocorrelation curve, polydispersity index (PDI), Zeta potential, and relative error of average diameter of the two kinds of urine crystallites, it was concluded that the urine crystallites of healthy subjects were more stable than those of patients. The urine crystallites of healthy subjects had a narrower size distribution from 100 nm to 350 nm and a better dispersion (PDI < 0.3). However, the urine crystallites of patients with urolithiasis had a wider distribution from dozens of nanometers to 1000 nm and a worse dispersion (PDI > 0.5). The best processing method for urine crystallites detection was found: after antisepticising and protein-coagulating with formaldehyde, the urine was diluted with distilled water of the same volume, then filtrated through a micropore film of 3 microm, and the filtrate was centrifugalized at 4000 rpm for 15 minutes. This method can remove the cell fragments and macromolecular substances in the urine without affecting the detection of the urine crystallites under 1000 nm. The results were consistent with those obtained by transmission electron microscope (TEM).

[Effects of linoleic acid on intracellular calcium concentration in primarily cultured rat pancreatic ß-cells and underlying mechanism].

In this study, we investigated the mechanism of linoleic acid-stimulated increase in intracellular calcium concentration ([Ca(2+)](i)) in pancreatic islet ß-cells. Pancreatic islet cells were primarily isolated from rats and cultured for the experiments. The cells were loaded with Fluo-3/AM, the indicator of [Ca(2+)](i), and the intensity of Fluo-3 was measured using confocal microscope. The islet ß-cells were identified by immunocytochemical staining with insulin antibody after recording. The drugs were given by perfusion system. The results showed that linoleic acid (20 µmol/L) stimulated [Ca(2+)](i) increase with the first peak increase and the following plateau increase. Linoleic acid-stimulated [Ca(2+)](i) increase was partly inhibited by removal of extracellular calcium and by transient receptor potential (TRP) channel blocker, La(3+), and it was totally blocked by exhaustion of intracellular calcium stores and inhibition of phospholipase C. It is concluded that linoleic acid stimulates [Ca(2+)](i) increase in islet ß-cells through both extracellular calcium influx via TRP channels and calcium release from intracellular calcium stores.

Composition and morphology of nanocrystals in urines of lithogenic patients and healthy persons.

The composition and morphology of nanocrystals in urines of healthy persons and lithogenic patients were comparatively investigated by means of X-ray diffraction (XRD) and transmission electron microscopy (TEM). It was shown that the main composition of urinary nanocrystals in healthy persons were calcium oxalate dihydrate (COD), uric acid, and ammonium magnesium phosphate (struvite). However, the main compositions of urinary nanocrystals in lithogenic patients were struvite, beta-tricalcium phosphate, uric acid, COD, and calcium oxalate monohydrate (COM). According to the XRD data, the size of nanocrystals was calculated to be 23 approximately 72 nm in healthy urine and 12 approximately 118 nm in lithogenic urine by Scherer formula. TEM results showed that the nanocrystals in healthy urine were dispersive and uniform with a mean size of about 38 nm. In contrast, the nanocrystals in lithogenic urine were much aggregated with a mean size of about 55 nm. The results in this work indicated that the urinary stone formation may be prevented by diminishing the aggregation and the size differentiation of urinary nanocrystals by physical or chemical methods.