EMT Of Renal Tubular Epithelial Cells In The Crystals Of Calcium Oxalate Kidney Injury Mice Model And The Protective Mechanism Of Fasudil

Obstructive nephropathy is one of the most common causes of urinary calculi,and with the improvement of living standards, the incidence of stones are rising, theincidence of left and right kidney stones are equal, and the incidence of bilateralkidney stones is about7%-10%, of which the proportion of kidney stones of calciumoxalate stones account for approximately80%. The hazards to human body by Kidneystones is not only limited to obstruction,the renal insufficiency will be more seriously.As the mechanism of stones mediated renal damage remains unclear,meanwhile, inorder to find new treatment measures, this study try to observe the occurrence ofEMT in renal tubular epithelial cell by crystallization of calcium oxalate kidney injurymodel.To provide a new therapeutic approach for clinical medicine,we also try tolooking for drugs and to explore its possible mechanism.Part I: EMT of renal tubular epithelial cells and the expression ofROCK1in the crystals of calcium oxalate kidney injury mice model[Objective] To study the EMT occurrence and ROCK1expression in the glyoxylateinduced crystallization of calcium oxalate kidney injury mice model[Methods] To select C57BL/6J mice to build the glyoxylate induced crystallization ofcalcium oxalate kidney injury model and get the specimens from kidney tissue at theend of the experiment. HE staining and VON KOSSA staining were used to observerenal structural changes and calcium deposition separately, and the double staining ofimmunofluorescence was used to monitor the occurrence of the EMT, while theexpression of ROCK1was detected by Western blot and RT-PCR.[Results] With the continuous intraperitoneal injection of glyoxylate time prolonged,HE staining displayed that in the lumen of proximal tubular there were refractiveirregular crystals adhesion gradually, and the tubular epithelial cells become swellingand deformation,and also the basement membrane exposed gradually; VON KOSSAstaining showed that the black calcium deposition more and more in the proximallumen; the double staining of immunofluorescence, RT-PCR, Western blot showedthat the renal tubular epithelial markers E-cadherin Pan-ck gradually lost, and theexpression of interstitial sign α-SMA and Vimentin gradually increase. In addition,the Western blot refected that with the glyoxylate intervention enhanced, theexpression of ROCK1increased and on the third day of intervention that reached a peak and maintain.[Conclusion] Glyoxylate induced crystallization of calciumoxalate kidney injury model exists EMT occurrence in the early stage, which starts theprocess of fibrosis, and Rho/ROCK signaling transduction pathway is involved in thisprocess.PartⅡ: Intervention effect of Rho kinase specific inhibitor, fasudil,on the injury kidney associated with calcium oxalate crystals[Objective] To observe the intervention effect of Rho kinase specific inhibitor, fasudil,on the injury kidney associated with calcium oxalate crystals[Methods] C57BL/6J mice were selected and randomly divided into normal controlgroup, the glyoxylate group, the glyoxylate+fasudil group. At the end of theexperiment, specimens of kidney tissue were obtained, and HE staining, VONKOSSA staining, picric acid-Sirius red, immunohistochemistry, RT-PCR,Western-blot detection were used to detect the intervention effect of fasudil.[Results] The three groups of mice experimental results showed that: compared withthe normal control group, the glyoxylate+fasudil group showed slightly damaged inrenal tissue, and calcium salt deposited with little fibrosis. The expression ofepithelial markers E-cadherin and Pan-ck declined, and interstitial flag α-SMA andVimentin expressed slightly as well. In addition, the expression of PAI-1, OPN, CD44and ROCK1increased as well; compared with the simple glyoxylate group, theglyoxylate+fasudil group showed significantly reduced structural damage of thekidney tissue, and decreased deposited calcium salt. The expression the α-SMA,Vimentin, PAI-1, OPN, CD44and ROCK1reduced significantly, while theexpression of E-cadherin and Pan-ck increased with alleviated renal fibrosis.[Conclusion] Fasudil, as the Rho kinase specific inhibitor, has a protective effect onthe nephropathy associated with calcium oxalate crystalsPart III: Mechanism study of fasudil on the crystalline kidney injuryinduced by glyoxylate[Objective] To investigate the protective mechanism of fasudil on the crystallinekidney injury induced by glyoxylate.[Methods] C57BL/6J mice were selected and randomly divided into normal controlgroup, the glyoxylate group alone, the glyoxylate+fasudil group. At the end of the experiment, specimens of kidney tissue were obtained, and Western-blot was appliedto detect the expression of smad2, smad3, p-smad2, p-smad3and PAI-1, whileimmunohistochemical methods were used to detect cell proliferation and apoptosis.[Results] Western blot analysis showed no significant differences in smad2andsmad3expression of the three groups. While the expression of PAI-1, p-smad2andp-smad3increased compared with the normal control group, but was significantlyreduced as compared with simple glyoxylate group. The PCNA and Tunel detectionshowed that the glyoxylate+fasudil group manifested increased apoptosis and moreactive cell proliferation compared with the normal control group, while significantlyreduced apoptosis and cell proliferation compared to the glyoxylate alone group[Conclusion] Fasudil can protect the crystalline kidney injury that induced byglyoxylate, by means of affecting cell proliferation and apoptosis, and Rho/ROCK-smad2/smad3-PAI-1signal transduction pathway.