Jian-Ming Ouyang

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, β-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.

Reinjury risk of nano-calcium oxalate monohydrate and calcium oxalate dihydrate crystals on injured renal epithelial cells: aggravation of crystal adhesion and aggregation

Background Renal epithelial cell injury facilitates crystal adhesion to cell surface and serves as a key step in renal stone formation. However, the effects of cell injury on the adhesion of nano-calcium oxalate crystals and the nano-crystal-induced reinjury risk of injured cells remain unclear. Methods African green monkey renal epithelial (Vero) cells were injured with H2O2 to establish a cell injury model. Cell viability, superoxide dismutase (SOD) activity, malonaldehyde (MDA) content, propidium iodide staining, hematoxylin–eosin staining, reactive oxygen species production, and mitochondrial membrane potential (Δψm) were determined to examine cell injury during adhesion. Changes in the surface structure of H2O2-injured cells were assessed through atomic force microscopy. The altered expression of hyaluronan during adhesion was examined through laser scanning confocal microscopy. The adhesion of nano-calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) crystals to Vero cells was observed through scanning electron microscopy. Nano-COM and COD binding was quantitatively determined through inductively coupled plasma emission spectrometry. Results The expression of hyaluronan on the cell surface was increased during wound healing because of Vero cell injury. The structure and function of the cell membrane were also altered by cell injury; thus, nano-crystal adhesion occurred. The ability of nano-COM to adhere to the injured Vero cells was higher than that of nano-COD crystals. The cell viability, SOD activity, and Δψm decreased when nano-crystals attached to the cell surface. By contrast, the MDA content, reactive oxygen species production, and cell death rate increased. Conclusion Cell injury contributes to crystal adhesion to Vero cell surface. The attached nano-COM and COD crystals can aggravate Vero cell injury. As a consequence, crystal adhesion and aggregation are enhanced. These findings provide further insights into kidney stone formation.

Properties of mixed monolayer and LB films of chiral amino acid porphyrin

Abstract Mixed monolayers of a new chiral amino acid porphyrin (Py) and arachidic acid (AA) at the air/water interface were studied The diagrams obtained by plotting the mean area per molecule as a function of molar fraction of Py showed positive deviations from ideal conditions at low surface pressure (π) such as π=5 and 15 mN m−1 and showed ideal miscible behavior at high surface pressures, such as π=25 mN m−1. Vertical uniformity of the mixed LB films was demonstrated by UV–visible and fluorescence spectra. Aggregates were present when the mixed film was transferred at various surface pressures and at different molar ratios of Py and AA. When the molar ratio of Py was increased from 0.05 to 1.00, the absorption peak of the Soret band bathochromically shifted ca. 5.3 nm. When the deposited pressure is increased from 5 to 30 mN m−1, the absorption peak of Soret band of 1:4 Py–AA mixed LB film bathochromically shifted ca. 3.7 nm, and the absorbance increased linearly. Low angle X-ray diffraction results showed that the mixed LB films had a periodic layer structure with a layer spacing of ca. 5.5 nm.

Size-dependent cellular uptake mechanism and cytotoxicity toward calcium oxalate on Vero cells.

Urinary crystals with various sizes are present in healthy individuals and patients with kidney stone; however, the cellular uptake mechanism of calcium oxalate of various sizes has not been elucidated. This study aims to compare the internalization of nano-/micron-sized (50u2009nm, 100u2009nm, and 1u2009μm) calcium oxalate monohydrate (COM) and dihydrate (COD) crystals in African green monkey renal epithelial (Vero) cells. The internalization and adhesion of COM and COD crystals to Vero cells were enhanced with decreasing crystal size. Cell death rate was positively related to the amount of adhered and internalized crystals and exhibited higher correlation with internalization than that with adhesion. Vero cells mainly internalized nano-sized COM and COD crystals through clathrin-mediated pathways as well as micron-sized crystals through macropinocytosis. The internalized COM and COD crystals were distributed in the lysosomes and destroyed lysosomal integrity to some extent. The results of this study indicated that the size of crystal affected cellular uptake mechanism, and may provide an enlightenment for finding potential inhibitors of crystal uptake, thereby decreasing cell injury and the occurrence of kidney stones.

Preparation, characterization, and in vitro cytotoxicity of COM and COD crystals with various sizes

Calcium oxalate crystals in urine often differ in size and crystal phase between healthy humans and patients with kidney stones. In this work, calcium oxalate monohydrate (COM) and dihydrate (COD) with sizes of about 50 nm, 100 nm, 1 μm, 3 μm, and 10 μm were prepared by varying reactant concentration, reaction temperature, solvent, mixing manner, and stirring speed. These crystals mainly had a smooth surface and no obvious pore structure, except COM-1 μm. In cell culture medium, the zeta potential of crystals became increasingly negative with increasing size, and the absolute value of zeta potential of COD was greater than the same-sized COM. Results of cell viability and PI staining assays showed that the order of injury degree in African green monkey renal epithelial (Vero) cells caused by different sizes of COD was COD-50 nm>COD-100 nm>COD-1 μm>COD-3 μm>COD-10 μm, and that of different sizes of COM was COM-1 μm>COM-50~COM-100 nm>COM-3 μm>COM-10 μm. COM-1 μm presented the highest cytotoxicity in Vero cells, which was associated with its rougher surface, larger specific surface area (SBET), and larger pore volume. Overall, these findings indicated that the physical properties of crystals play an important role in their cytotoxicity.

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.

Adhesion and internalization differences of COM nanocrystals on Vero cells before and after cell damage.

The adhesion and internalization between African green monkey kidney epithelial (Vero) cells (before and after oxidative damage by hydrogen peroxide) and calcium oxalate monohydrate (COM) nanocrystals (97±35nm) were investigated so as to discuss the molecular and cellular mechanism of kidney stone formation. Scanning electron microscope (SEM) was used to observe the Vero-COM nanocrystal adhesion; the nanocrystal-cell adhesion was evaluated by measuring the content of malonaldehyde (MDA), the activity of superoxide dismutase (SOD), the expression level of cell surface osteopontin (OPN) and the change of Zeta potential. Confocal microscopy and flow cytometry were used for the observation and quantitative analysis of crystal internalization. In the process of adhesion, the cell viability and the SOD activity declined, the MDA content, Zeta potential, and the OPN expression level increased. The adhesive capacity of injured Vero was obviously stronger than normal cells; in addition the injured cells promoted the aggregation of COM nanocrystals. The capacity of normal cells to internalize crystals was obviously stronger than that of injured cells. Cell injury increased adhesive sites on cell surface, thereby facilitating the aggregation of COM nanocrystals and their attachment, which results in enhanced risk of calcium oxalate stone formation.

Property changes of urinary nanocrystallites and urine of uric acid stone formers after taking potassium citrate.

The property changes of urinary nanocrystallites in 20 cases of uric acid (UA) stone formers after 1 week of potassium citrate (K3cit) intake were comparatively studied by X-ray diffraction analysis, Fourier transform infrared spectroscopy, nanoparticle size analysis, and transmission electron microscopy. Before K3cit intake, the urinary crystallites mainly contained UA and calcium oxalate. After K3cit intake, the components changed to urate and UA; the qualities, species, and amounts of aggregated crystallites decreased; urine pH, citrate, and glycosaminoglycan excretions increased; and UA excretion, Zeta potential, and crystallite size decreased. The stability of crystallites followed the order: controls>patients after taking K3cit>patients before taking K3cit. Therefore, the components of urinary stones were closely related to the components of urinary crystallites.

Concave urinary crystallines: direct evidence of calcium oxalate crystals dissolution by citrate in vivo.

The changes in urinary crystal properties in patients with calcium oxalate (CaOx) calculi after oral administration of potassium citrate (K3cit) were investigated via atomic force microscopy (AFM), scanning electron microscopy (SEM), X-ray powder diffractometry (XRD), and zeta potential analyzer. The AFM and SEM results showed that the surface of urinary crystals became concave, the edges and corners of crystals became blunt, the average size of urinary crystallines decreased significantly, and aggregation of urinary crystals was reduced. These changes were attributed to the significant increase in concentration of excreted citrate to 492 ± 118u2009mg/L after K3cit intake from 289 ± 83u2009mg/L before K3cit intake. After the amount of urinary citrate was increased, it complexed with Ca2+ ions on urinary crystals, which dissolved these crystals. Thus, the appearance of concave urinary crystals was a direct evidence of CaOx dissolution by citrate in vivo. The XRD results showed that the quantities and species of urinary crystals decreased after K3cit intake. The mechanism of inhibition of formation of CaOx stones by K3cit was possibly due to the complexation of Ca2+ with citrate, increase in urine pH, concentration of urinary inhibitor glycosaminoglycans (GAGs), and the absolute value of zeta potential after K3cit intake.

Langmuir and Langmuir-Blodgett Films of Bilirubin

Abstract The surface pressure-area isotherms of bilirubin (H2BR) monolayers at an air-water interface on subphases with different pH values and on subphases containing metal ions such as Ca2+, Mg2+, Cu2+, Ni2+, Zn2+ and Pb2+ ion were investigated. H2BR can form expanded and stable monolayer on neutral and acidic subphases, while it can hardly form monolayer on basic subphases. The acid-base equilibrium of H2BR was discussed at the air-water interface. The association-dissociation of H2BR with H+ ions in the interfacial region was very different from that in the bulk solution. Some information regarding the packing density and degree of ionization of the head group under different experimental conditions were obtained. The formation of H2BR-metal complexes leads to changes in the shape of isotherms of H2BR and changes in UV-visible absorption spectra of the monolayer assemblies. In XPS spectra new XPS peaks assigned to the metal ions containing subphases appeared. Low-angle X-ray diffraction indicates that a Y-type LB films were formed with bilayer spacing of ca. 2.50 nm.