Hyaluronan Regulates The Adhesion Of Renal Epithelial Cells Through P38MAPK And JNK Signaling Pathways In Urinary Stone Formation

Objective To evaluate the toxic effect of oxalate and calcium oxalate monohydrate （COM） crystals on human renal tubular epithelial cells （HK-2）.Methods HK-2cells were cultured in co-culture system to confluence. Oxalic acid and COM crystals with different concentration （0,1,5and10mmol/L） were then added. The toxic effect of Oxalic acid and COM crystals on HK-2cells at24h after incubation were examined by measuring the activity of lactic dehydrogenase （LDH） and dyeing with4’,6-diamidino-2-phenylindole（DAPI）.Results Dyeing with DAPI showed that the number of cells was not significant decreased until the concentration of oxalic acid up to10mmol/L. The number of cells was reduced dramatically after treating with1mmol/L COM. Cells were almost dead after incubation24h with5and10mmol/L COM. LDH activity was increased significantly in5and10mmol/L oxalic acid group （p<0.01）. Comparing with oxalic acid group, LDH activity was increased significantly in1,5and10mmol/L COM group （p<0.01）.Conclusion Oxalic acid and COM crystals have toxic effect on HK-2cells in a concentration dependent manner. COM crystals are more toxic than oxalic acid. Objective To evaluate the MAPK signaling pathways involved in the process of calcium oxalate monohydrate （COM） crystals in human renal tubular epithelial cells （HK-2）. To research the effect of SB203580on regulation of HAS1-3, CD44and adhesive influence between COM crystal and HK-2cells.Methods HK-2cells were cultured in serum-starved DMEM for12h before exposure to calcium oxalate monohydrate （lmM） for different time course （0,15min,30min,60min and120min）. The activity of MAPK signaling pathways were evaluated by phosphorylation of p38, JNK and ERK. SB203580was used to block the transduction of p38. RT-PCR was used to measure the mRNA level of HAS1-3and CD44. Ion chromatograph was performed to measure the adhesion effect between COM crystal and HK-2cells.Results Exposure to calcium oxalate monohydrate （COM） crystal rapidly activated p38-MAPK. Calcium oxalate monohydrate （COM） crystal induced modest activation of JNK. In contrast, COM crystal had no effect on phosphorylation of ERK. The adhesive effect between COM crystal and HK-2cells was reduced by the use of SB203580（50μM）.The mRNA levels of HAS1-3and CD44were diminished by the use of SB203580. Conclusion Exposure to calcium oxalate monohydrate （COM） crystal selectively activates p38-MAPK and JNK signaling pathways. SB203580reduces the mRNA levels of HAS1-3, CD44and diminish the adhesive effect between COM crystal and HK-2cells. Objective To evaluate the expression of hyaluronan, CD44and hyaluronan synthase1-3in kidney stone patients. The effect of HAS1on the adhesion between renal epithelial cells （HK-2） and calcium oxalate monohydrate （COM） crystals was studied. Methods The expression of hyaluronan, CD44and hyaluronan synthase1-3were measured between kidney stone patients and normail kidney patients by immunohistochemistry. HK-2cells were exposed to calcium oxalate monohydrate （1mM） for different time course （Oh,24h,48h）. The mRNA levels of HAS1-3and CD44were measured by RT-PCR, and the protein level of HAS1was measured by western blot. The expression of hyaluronan was detected by Elisa kit. siRNA methods was used to influent the content of hyaluronan and the adhesion of renal epithelial cells. Ion chromatograph and confocal laser scanning microscopy were used to measure the adhesive effect between COM crystal and HK-2cells. Results The expression of hyaluronan, CD44and hyaluronan synthase1-3were higher in kidney stone group by immunohistochemistry. At the24h after exposure to calcium oxalate monohydrate（COM） crystal, the mRNA levels of HAS1-3and CD44were highest by RT-PCR, which diminished at the48h after exposure to COM crystals. The protein level of HAS1was highest at48h after exposure to COM crystals. It could be blocked by the HAS siRNA. The content of hyaluronan were measured at different time course （Oh,12h,24h,36h,48h） by ELISA kit. It began to increase at12h and reached its peak at24h. At36h after exposure it began to decrease. The adhesive effect between COM crystal and HK-2cells were reduced by the use of HAS1siRNA through confocal laser scanning microscopy and ion chromatagraph. Conclusion The expression of hyaluronan, CD44and hyaluronan synthase1-3were higher in kidney stone patients. Hyaluronan plays a crucial role in the formation of kidney stone. The adhesion of between COM crystal and HK-2cells can be decreased by the use of HAS1siRNA. Objective long noncoding RNAs （IncRNAs） are very important in biological functions. Microarray was used to reveal the changes of HK-2cells induced by COM crystals, so as to provide candidate IncRNAs involved in the molecular mechanisms concerning kidney stone formation.Methods HK-2cells were oxposed to COM crystals（lmmol/L） for24h. The microarray was used to detect the change of lncRNAs. RT-qPCR was used to validate the results.Results From the data we found there were2971lncRNAs that differentially expressed in HK-2cells exposed to lmmol/L COM crystals.1630lncRNAs were upregulated and1341lncRNAs were downregulated. Four lncRNAs （ENST00000430583、ENST00000428930、 NR₀29401and chr13） validated by qPCR were matched the result of microarray.Conclusion Our study is the first one to determine lncRNAs expression patterns in HK-2cells exposed to COM crystals. The results displayed that clusters of lncRNAs were aberrantly expressed may exert a partial role in stone formation. Taken together, this study may provide potential targets for future treatment of stone disease and novel insights into stone formation biology.