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Biomineralization of Calcium Oxalate Modulated by Damaged Renal Epithelial Cell and Defective Langmuir-Blodgett Film

Author: DengSuiPing
Tutor: OuYangJianMing
School: Jinan University
Course:
Keywords: monolayer calcium oxalate Langmuir-Blodgett film defect renal tubular epithelial cell injury biomineralization
CLC: R692.4
Type: PhD thesis
Year: 2009
Downloads: 119
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Abstract

Urolithiasis is a complex process controlled by chemical factors and cells in a complicated system in vivo. In the present study, the biomineralization of calcium oxalate (CaOxa) crystals, the most abundant constituent of kidney stones, was investigated systematically in vitro models of urines from healthy volunteers and stone-former patients, Langmuir monolayers, defective Langmuir-Blodgett (LB) films, and injured renal epithelial cells, respectively. Using such models, it is helpful to understand the relationships between urolithiasis and the injury of renal epithelial membrane at the molecular level. The findings in this study may provide important insights into the pathogenesis of nephrolithiasis.1. Crystallization of CaOxa was comparatively studied in vitro in diluted lithogenic urine and in diluted healthy urine by means of scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared (FT-IR) spectroscopy. In healthy urine, the crystallization was a growth-controlled process in the early stage of crystallization and a nucleation-controlled process in the middle and late stage. However, the crystallization of CaOxa was always a growth-controlled process in the lithogenic urine. That is, the size of CaOxa particles grow gradually and at last large size of CaOxa stones formed. Comparing the CaOxa crystals grown in lithogenic urine and in healthy urine, three differentiations were observed. First, the average particle size of CaOxa crystals precipitated in lithogenic urine is larger than that in healthy urine. Second, the morphology of calcium oxalate monohydrate (COM) crystals changes from sharp hexagonal in lithogenic urine to round and blunt in healthy urine. Third, calcium oxalate dihydrate (COD) crystals were induced in healthy urine. The results in this study may provide important clues to cure urinary stones.2. The crystallization of COM crystals grown beneath stearic acid (SA) monolayers at different surface pressures in the presence of potassium citrate (K3cit) and at varied pH value was investigated. The size of the COM crystals grown at low surface pressures, such as 0.1 and 1 mN/m, was larger than those grown at high surface pressures. This result was due to a perfect match between the monolayer lattice and the crystal plane, and the monolayers at low surface pressure had a greater dynamic freedom and compressibility than those at high pressure to reorganize themselves in order to optimize the geometrical and stereochemical fit and then to accommodate the nucleating or growing crystals. K3cit inhibit COM growth at SA monolayers because K3cit disturbed the ordered array of the SA molecules at the monolayer surface, leading to a mismatch between the SA monlayer and COM nuclei. Furthermore, the variety of pH changed the fraction of citrate species, consequently affecting the growth of COM faces. Calcium oxalate dihydrate (COD) was dominant at pH 12. It was attributed to the induction of K3cit to COD and a high molar ratio of calcium to oxalate at the SA monolayer-water interface at high pH.The crystallization of COM crystals beneath dipalmitoylphosphatidylcholine (DPPC) monolayers in the presence of urinary macromolecule chondroitin sulfate C (C6S) was first examined. C6S can inhibit the growth and aggregation of COM crystals and induce the preferential growth of COM ((?)) faces. It was contributed to the co-action of negative-charged monolayer and C6S to the calcium-rich ((?)) face of COM. 3. Although the LB technique has been used for decades, applications of the method have been frustrated by defects. However, the defective LB film of DPPC was first used as a model for injured renal epithelial membrane to induce CaOxa growth. Atomic force microscopy (AFM) and fluorescence microscopy were used to observe the microstructures of liquid condensed (LC) domains and liquid expanded (LE) phases in defective monolayers of DPPC transferred to a mica surface after treatment by potassium oxalate (K2C2O4). The orderly molecular arrays in the monolayers were destroyed by K2C2O4, especially at the LC/LE boundaries. As a result, the circular defective domains were formed. SEM results showed that the defective domains could induce the formation of circular patterns of COM crystals. As the concentration of K2C2O4 [c(K2C2O4)] increased from 0.3 mmol/L to 5.0 mmol/L, the effect of K2C2O4 on the monolayers was gradually strengthened. The monolayers treated by 0.3 mmol/L K2C2O4 induced solid circular patterns of COM crystals, that is, the COM crystallites compactly arrayed in the domains. When c(K2C2O4) was increased to 5.0 mmol/L, the patterns turned to a ring shape, that is, there were crystals only at the LE/LC boundaries and few crystals were found in center of the domains, and the number of small patterns with a diameter of less than 20μm increased remarkably. The effect of a series of parameters, such as the kinds of substrates, temperature, deposition parameters etc., on the defects in LB films and the effect of these parameters on the formation of COM crystals were also investigated.4. In an oxidative model with human kidney tubular epithelial cells (HKC) in vitro, the changes of HKC cells after oxidative injury, as well as the different controllability between normal cells and injured cells to the nucleation and growth of CaOxa crystals from surpersaturated solutions, were investigated by means of cell counting kit-8 (CCK-8) assay, SEM, confocal laser microscopy, inverted microscopy and Zeta potential analyzer, respectively. Results indicate that H2O2 could significantly injure HKC cells, and decrease the cell viability in a dose-and time-dependent manner in the concentrations range from 0.3 mmol/L to 0.5 mmol/L and in the exposure time from 0.5 h to 1.5 h. The increased damage level of HKC cells could significantly promote the quantity of CaOxa crystals, but could hardly promote the size of crystals (P> 0.05). It suggested that the underlying mechanism by which the injured HKC cell promoted stone formation was by providing more nucleating sites for crystals, and not by promoting crystal growth. Moreover, COD crystals were endocytosed by the normal HKC cells without measurable injury. It indicated that the normal cells could eliminate a certain amount of COD crystals. However, the endocytosis capacity of the cells was weaken when they were injured, and osteopontin (OPN) were observed on the injured cell surface by the confocal microscopy, leading to the increase of the quantity of crystals on the cell surface. It shows that the injured cells could promote crystals retention on the cell surface and facilitate the formation of renal calculi.

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CLC: > Medicine, health > Surgery > Urology ( urinary and reproductive system diseases) > Kidney disease > Kidney stones
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