ABSTRACT
Urinary calculi constitute one of the oldest afflictions of humans as well as animals, which are occurring globally. The calculi vary in shape, size and composition, which influence their clinical course. They are usually of the mixed-type with varying percentages of the ingredients. In medical management of urinary calculi, either the nature of calculi is to be known or the exact composition of calculi is required. In the present study, two selected calculi were recovered after surgery from two different patients for detailed examination and investigated by using Fourier-Transform infrared spectroscopy (FT-IR), thermo-gravimetric analysis (TGA), powder X-ray diffraction (XRD), scanning electron microscopy and energy dispersive analysis of X-rays (EDAX) techniques. The study demonstrated that the nature of urinary calculi and presence of major phase in mixed calculi could be identified by FT-IR, TGA and powder XRD, however, the exact content of various elements could be found by EDAX only.
Subject(s)
Aged , Calcium Oxalate/chemistry , Female , Humans , Male , Microscopy, Electron, Scanning/methods , Middle Aged , Powders , Spectrometry, X-Ray Emission/methods , Spectroscopy, Fourier Transform Infrared/methods , Thermogravimetry/methods , Urinary Calculi/chemistry , X-Ray Diffraction/methodsABSTRACT
Objective To investigate the protective effect of taurine on HK-2 cells exposed to oxalate (Ox) and calcium oxalate monohydrate crystal (COM) in vivo.Methods HK-2 cells,a proximal tubular epithelial cell line,were cultured.Five groups were divided in this study:control group (only HK-2 cells) ; Ox and COM group (HK-2 cells + Ox + COM) ; Taurine group (HK-2 cells + Ox + COM + Taurine) ; Apocynin group (HK-2 cells + Ox + COM + Apocynin) ; Catalase group (HK-2 cells + Ox + COM +Catalase).After 6 hrs,the cultures medias from each group were tested for LDH,H2O2,8-isoprostane,and MCP-1 protein.Cellular expression of MCP-1 mRNA and P47phox mRNA were determined by reverse transcriptase-polymerase chain reaction.After 24 hrs,cells livability was investigated by MTT.Results Compared with the control,cells livability was reduced when exposed to Ox and COM (P ＜ 0.05),Treatment with Taurine,Apocynin and Catalase significantly increased the cells livability (P ＜ 0.05).Compared with the control,the expression of LDH,H2O2,8-isoprostane,and cellular expression of MCP-1 mRNA and P47phox mRNA were increased following exposure to Ox and COM (P＜0.01,P＜0.01,P＜0.01,P＜0.01,P ＜0.05).Treatment with Taurine,Apocynin and Catalase significantly reduced the expression of LDH,H2O2,8-isoprostane,as well as the cellular expression of MCP-1 mRNA.Expression of P47phox mRNA in Taurine group was not reduced significantly (P ＞ 0.05).Conclusions This study showed that Taurine protected the HK-2 cells from oxidative injury exposed to Ox and COM by the pathway that may not be in relation to the inhibition of P47phox mRNA expression.
ABSTRACT
Objective To analyze and identify the differentially expressed proteins in human renal tubular epithelial cells (HK-2) after injury caused by oxalic acid and calcium oxalate monohydrate (COM) crystal, and to explore the potential role of renal tubular cell injury in kidney stone formation.Methods Normal HK-2 cells were cultured in vitro and the culture medium was changed with serum-free medium after cell growth to confluence. Oxalic acid and COM crystals (final concentration at 2 mmol/L and 200 mg/L, respectively) were added in the experimental group. Cells in both groups were then incubated at 37 ℃ for 12 h. The extracted proteins from both groups were separated by two dimensional electrophoresis followed by analysis, and the differentially expressed proteins were identified by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Two identified proteins were then verified by western blot. Results Reproducible two dimensional gel images of the proteins from both groups were successfully obtained. By using LC-ESI-MS/MS, 12 proteins: FK506-binding protein 4, isoform alpha-enolase of alpha-enolase, isoform M1 of pyruvate kinase isozymes M1/M2, ATP synthase subunit alpha, isoform 1 of 3′(2′), 5′-bisphosphate nucleotidase 1, isoform 2 of nucleophosmin, L-lactate dehydrogenase B chain, Budding uninhibited by benzimidazoles 3, Cofilin-1, Fascin, pyIsoform 1 of cytosol aminopeptidase, were identified. The deferentially expressed proteins were related to cellular processes including energy metabolism, cell multiplication, apoptosis, Ca2+ channel activity regulation, cell movement and signal transduction. Western blot verified that higher ENO1 but lower Cofilin-1 expressed in HK-2 cells after the injury. Conclusions High level oxalic acid and COM crystals can cause protein expression profile changes in normal human HK-2 cells. The changes of protein expression may not only protect HK-2 cells from being injured, but also be related to kidney stone formation.
ABSTRACT
Objective To evaluate the toxic effects of oxalic acid and calcium oxalate monohydrate crystals on human renal tubular epithelial cells (HK-2) as well as the influence on cell protein expression. Methods Normal HK-2 cells were cultured in vitro and the culture medium was changed to serum-free medium after cell growth to confluence. Oxalic acid with different concentration wasthen added and the formation of crystals and their adherence to cells were observed microscopically. A Fourier infrared spectrometer (FT-IR) was used to analyze the crystal composition. The toxic effects of 1, 2, 5 and 10 mmol/L oxalic acid on HK-2 cells after incubation for 4, 12 and 24 h were detected with a CCK-8 kit. Changes of protein express of HK-2 cells were determined using the Bradford method. Results Crystal formation and adherence to cell surface could be microscopically observed in a few minutes after oxalic acid was added to the DMED medium containing Caz+. The composition of the crystals was revealed to be calcium oxalate monohydrate by FT-IR. Oxalic acid and calcium oxalate monohydrate presented a concentration-dependent toxic effect on HK-2 cells which was, however, not merely increased with time lasting. The quantity of protein expressed by HK-2 cells incubated with 1,2, 5 mmol/L oxalic acid for 12 h and that of control was 358±51, 365±43, 328±52 and 329±60 mg/L, respectively (all P>0. 05), while the quantity of protein was significantly smaller than that of control after the incubation of 10 mmol/L oxalic acid for 12 h (264±76 vs 329±60 mg/L,P<0. 05).Conclusion Oxalic acid and calcium oxalate monohydrate crystals have toxic effects on normal human HK-2 cells and cause changes in protein expression,which may play an important role in the formation of renal calculi.