| Previous studies suggested that hyperuricemia (HUA) was only considered as amarker of metabolic abnormalities, but in clinical patients with hyperuricemia whichare associated with kidney diseases,such as hypertension、cardiovascular disease anddiabetes.Although there is a debate about which hyperuricemia is a cause of thesediseases or merely a "sign". More and more evidences proved that hyperuricemia hasa direct injury in kidney and cardiovascular system,even as an independent risk factorof kidney disease and cardiovascular disease.Kidney injury was caused not only by uric acid or urate but also by theobstruction from urate crystals. Kang and other researchers shared a view that themechanism of kidney disease caused by uric acid including microvascular disease ofthe glomerular afferent artery、kidney inflammation、activating renin-angiotensinsystem (RAS)and cyclooxygenase-2(COX-2).Uric acid crystals can also activate the complement system, start an inflammatory response, stimulate renal tubular epithelial cells in the synthesis ofprostaglandin E2(PGE2) and monocyte chemoattractant protein-1(MCP-1),amplifythe local inflammatory reaction, eventually lead to renal interstitialfibrosis and renalfailure.As we all known,the cell-cell junction is achieved by some junctions such astight junction(TJ)、adherens junction(AJ）、gap junction、desmosome junction.TJ isa cell junction which can form a circumferential belt around an epithelial orendothelial cell, separating the plasma membrane into an apical and a basolateraldomain. What＇s more,it plays a critical roles in maintaining permeability and barrierfunction of epidermis and endothelial cells.Tight junctions is a dynamic adjustment structure which is composite withmembrane protein (ZO-1, ZO-2, ZO-3) and transmembrane protein (occludin,claudins). The tight junction (TJ) has other major functions:first, as a “gate,” it servesas a regulatory barrier separating and maintaining biological fluid compartments ofdifferent composition.Second,as a“fence,”it generates and maintains the apicobasalpolarity of cells、regulates cell growth、proliferation、differentiation and participatein epithelial cell EMT process.Tight junction can be destrcted by a variety of factors such as oxidative stress、pathogens、proinflammatory cytokines,which can damage the tight junction proteinstructure and function, and the destruction of the tight junction protein related to avariety of diseases, including gastrointestinal tract, lung, and kidney disease.Paleerathput forward that calcium oxalate crystals can damage barrier function and fencefunction of TJ and play an important role in rennal interstitial fibrosis caused bykidney stones.Hower, according to current study, the relationship between uric acidstones and tight junction are not clear.【Objective】1.Explore to the change of ZO-1and Occludin’s expression,distribution and function on NRK-52E cells under Monosodium urate monohydrate (MSU) crystals.2. Explore the impact of renal tubular epithelial cell transdifferentiation induced byMonosodium urate monohydarte crystals.【Methods】NRK-52E cells were exposed to different concentration of MSU (100μg/ml,300μg/ml,500μg/ml,700μg/ml) for24h、48h、72h，and then:1. Detected cell viability by MTT,selecting one of the best concentration for nextExperiment;2. Observing NRK52Ecell morphological changes by exposing to MSU undermicroscope;3. Detected the expression of ZO-1,E-cadherin and α-SMA protein expressionby immunofluroescence;4. Detected the expression of ZO-1、Occludin、E-cad、α-SMA mRNA by RT-PCR.【Results】1.Cell viability by MTT;1.1.Compared with the control group,different concentrations of MSU(100μg/ml、300μg/ml、500μg/ml、700μg/ml) stimulate the cells24h, cellviability decreased to89.13±7.7%;85.60±12%;78.54±13%;47.82±14%;(P<0.05);1.2.Compared with the control group,MSU (500μg/ml) stimulate cells24h、48h、72h, cell viability were reduced to69.79±8.34%;66.43±10.15%;65.73±10.05%;(p <0.05);2.Cell Morphological ChangesObserved NRK-52E cell morphology change by MSU induced at different times2.1.Normal control group NRK-52E cells were round or oval, with a typicalepithelial cell cobblestone morphology.at each time point,shape was no significantchange. 2.2.Compared with blank group, MSU(500μg/ml)concentrations induced cellsafter24h, its shape did not change significantly; After48h, the cells becamehypertropy and gradually lost the typical cobblestone morphology,group of500μg/ml and700μg/ml were obvious；after72h, the cells become spindle-shaped,lost their cobblestone appearance, with fibroblast characteristics.500μg/ml groupwas obvious and700μg/ml group can be visible part of the cell death from cellculture bottles.3. Indirect immunofluorescence on ZO-1, E-cadherin, α-SMA protein’s expressionand distributionTight junction proteins (ZO-1, Occludin) and E-cadherin protein (E-cad) expresson the cell membrane; a-smooth muscle actin (α-SMA), express a few oncytoplasmic in normal cell, even no expression.MSU (500μg/ml) were stimulated cells24h,48h,72h fluorescence intensity ofZO-1, E-cadherin gradually decreased; for72h, and even can be observed of ZO-1,E-cadherin membrane proteins fracture and appeared cytoplasmic expressionphenomenon.MSU (500μg/ml) stimulated cells24h、48h、72h, immunofluorescence showedα-SMA fluorescence intensity gradually increased, at72hours, the cytoplasmshows a great number of α-SMA expression.4．ZO-1mRNA、Occludin mRNA,E-cad mRNA and α-SMA mRNA expression byRT-PCRNormal NRK52E cells can express a great number of ZO-1、 Occludin andE-cadherin mRNA, but α-SMA have little or no expression.Compared with the control group,MSU(500μg/ml) induced in NRK-52E12h、24h、48h、72h: ZO-1mRNA expression levels were reduced to0.80±0.20fold,、0.62±0.31fold、0.47±0.27fold、0.34±0.16fold.Compared with the control group,Occludin mRNA expression were reduced to0.80±0.17fold、0.69±0.16fold、0.62±0.21-fold、0.50±0.19-fold. Compared with the control group,E-cad mRNA expression were reduced to0.78±0.13fold、0.64±0.20-fold、0.56±0.23-fold、0.41±0.18-fold.Compared with the control group,-SMA mRNA expression were reduced to1.17±0.13fold、1.82±0.12fold、2.49±0.10-fold、4.16±0.17-fold.【Conclusion】1. Monosodium sodium urate crystals (MSU) in the renal tubular epithelial cells candecrease expression of tight junction proteins, distribution of abnormalities;2. Cell-cell connectivity were destructed and accompanied by a decline in E-cadexpression and a increase of α-SMA expression, which suggest that the destructionof tight junction structure may be an important role in the uric acid crystals whichstart renal tubular epithelial cell damage and transdifferentiation. |
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