| Objective1.To observe the histopathological characters of renal papilla and renal calcareous deposits in patients with various renal calculi,so as to explore the possible role of local histopathological changes and calcareous deposits in the formation of renal calculi.To explore the formation mechanism of renal calcareous deposits by determining the expression of osteopontin,bone morphogenetic protein-2(BMP-2) and typeⅡcollagen in the kidneys of patients with renal calculi.2.To evaluate the toxic effects of oxalic acid and/or calcium oxalate monohydrate (COM) crystals at different levels on human renal tubular epithelial cells(HK-2) as well as the influence on cell protein expression;to explore the potential role of epithelial cell injury in the formation of renal calculi.3.To identify the differently expressed proteins in HK-2 cells injured by oxalic acid and COM crystals,and to postulate and discuss the potential roles of these proteins in renal tubular epithelial cell injury as well as kidney stone formation,so as to provide new ways for further kidney stone formation.Methods1.Patients with renal calculi undergoing percutaneous nephrolithotomy(PCNL) were included,whose stones were analysed with a transform-infrared(FT-IR) spectrometer. Renal papilla biopsy specimens were obtained under a nephroscope during the operation, followed by staining with hematoxylin-eosin or alizarin bordeaux for light microscopy and electron microscopy to observe the histopathological characters.The expression of osteopontin,bone morphogenetic protein-2(BMP-2) and typeⅡcollagen in the kidneys of patients with renal calculi or without urolithiasis were determined by immunohistochemistry.2.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/or COM crystals with different concentrations were then added and the cells were incubated for 4h,12h or 24h,respectively.Crystal adherence to cells was observed microscopically.The toxic effects of oxalic acid and/or COM crystals with different concentrations on HK-2 cells after incubation for 4h,12h or 24h were detected with a CCK-8 kit.Changes of protein expression in HK-2 cells were determined using the Bradford method.3.The proteins of normal HK-2 cells and the HK-2 cells incubated with 2mM oxalic acid plus 200mg/L COM crystals for 12h were respectively separated using two-dimensional electrophoresis technology and visualized by silver staining.The digitized images were then analyzed with an Image Master software to obtain the differential protein profiling between normal HK-2 cells and the HK-2 cells incubated with oxalic acid plus COM crystals.4.The differential protein spots were identified using liquid chromatography-electrospray ionization-mass spectrometry/mass spectrometry(LC-ESI-MS/MS) conducted by a Finnigan LTQ mass spectrometer.Two identified proteins,ENO1 and Cofilin-1,were selected and their expression in normal HK-2 cells or injured HK-2 cells was determined by Western blotting.All identified proteins were classified by their subcellular location and biological function.The potential roles of these proteins in renal tubular epithelial cell injury as well as kidney stone formation were postulated and discussed.Results1.Renal papilla calcific plaques were found in a part of the patients with renal calculi during PCNL.Local calcareous deposits could be found in the biopsy specimens. Microscopically,in tissue sections with abundant calcareous deposits,calcareous deposits appear as tuberose or flakiness,localized in the tubular basement membranes and interstitial tissues;while in other sections,calcareous deposits could only be observed in the tubular basement membranes or spreading from the tubular basement membranes to the interstitium.Once the calcareous deposits pierced into the collection system,tiny stones growing on them could be observed.Immunohistochemistristry examination showed that osteopontin was positively expressed in the renal tubular epithelial cells of the patients with kidney stones and the normal controls,but BMP-2 and typeⅡcollagen were negatively expressed in the kidneys of both groups.2.Oxalic acid and/or 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 10mM oxalic acid or 10Mm plus 200mg/L crystals was significantly smaller than that of control(263.75±75.77ug/L vs 328.75±60.34ug/L and 227.5±54.97ug/ml vs 328.75±60.34ug/ml,respectively,P＜0.05).After incubated with 2mM oxalic acid plus 200mg/L COM crystals,HK-2 cells expressed more proteins than normal,but the different was not significant(332.5±51.48ug/ml vs 328.75±60.34ug/ml,P＞0.05). 3.The two-dimensional gel electrophoresis were successfully performed for isolating the proteins of normal HK-2 cells(Control Group) or HK-2 cells incubated with 2mM oxalic acid plus 200mg/L COM crystals(Experiment Group).Visualized spots were 2204±84 in the experiment group and 2347±57 in the control group.Software analysis revealed 16 protein spots showing differential expression between the two groups.In the experiment group,among these 16 protein spots,the up-regulated proteins were 9,while the down-regulated proteins were 7.4.Differential protein spots were identified using LC-ESI-MS/MS and analyzed by database searching.A total of 12 proteins were eventually identified,the function of which includes cellular energy metabolism,proliferation,apoptosis,Ca2+ channel activity regulation,cell movement and signal transduction.Western blotting confirmed that the injured HK-2 cells had higher expression of ENO1 but lower expression of Cofilin-1.Conclusions1.Renal papillary calcareous deposits in a part of patients with renal calculi may be initiated in the renal tubular basement membranes and spread into the interstitium.The role of renal calcareous deposits in kidney stone formation may include the following aspects:1) Calcareous deposits act as platforms for crystal adherence,aggregation and growth when piercing into the collection system;2) Detachment of calcareous deposits in the collection system may become the nucleus of stones;3) The interaction between calcareous deposits and local cells may be related to the formation of renal calculi.2.BMP-2 and typeⅡcollagen are negatively expressed in the kidney of patients with renal calculi,which suggests that the formation of renal calcareous deposits may not be an osteoblastic reaction resembling arteriosteogenesis.3.The toxic effect of oxalic acid and/or COM crystals on HK-2 cells was concentration dependent but is not merely increased with time lasting.4.High levels of oxalic acid and COM crystals can cause protein expression profile changes in normal human HK-2 cells.The function of concerned proteins includes energy metabolism,cell proliferation and apoptosis,cell movement,calcium channel activity regulation,signal transduction,protein synthesis and cellular stress reaction.The changes of protein expression may not only self-protect HK-2 cells from being injured,but also be related to kidney stone formation.Further studies on these proteins are required so as to find out new mechanism and pathways leading to stone formation.5.This study provides an important knowledge of oxalic acid and COM crystal-treated HK-2 cells' protein expression in vitro and should be helpful for the further elucidatation of the molecular mechanisms involved in the interaction between urinary stone ingredient crystals and renal epithelial cells and kidney stone formation,and provide new methods and for seeking effective precautionary measures for the recurrence of renal calculi. |
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