| Objective: Renal interstitial fibrosis is a common result of all chronickidney disease, which is an important reason for irreversible renal injury. It ismeaningful to search for therapeutics to delay or even reverse the renalinterstitial fibrosis progression, thus to slow disease progression and extendthe patients’ linves. Kidney injury molecule-1(Kim-1)，a type I membraneglycoprotein that is expressed at negligible levels in normal rat kidneys, ismassively induced in many acute kidney injury(AKI) in rats, such as ischemiareperfusion injury, toxic or contrast-induced nephropathy, with resolutionduring the restoration of structural and functional integrity. Because of itshigh sensitivity and specificity, it is becoming a better biomarker for AKI.Recently it has become increasingly clear that KIM-1is also expressed inchronic renal disease(CKD). This has been established in experimental modelssuch as protein-overload nephropathy, adriamycin-induced nephropathy,angiotensin Ⅱ-induced renal damage in homozygous Ren2rats, and murinepolycystic kidney disease, but the molecular mechanisms and role of KIM-1inCKD remain obscure. Domestic scholars have observed the expression andrelationship of KIM-1and hypoxia-inducible factor1α (HIF-1α) in contrast-induced nephropathy and the hypoxia/reoxygenation phases of humanproximal tubular epithelial cells (HK2). The results suggested that there wassome correlation between the two, which illustrated that KIM-1waspotentially a downstream molecule of HIF-1α involved in processes of kidneyinjury and repair. In this study we sought to observe the morphologicalchanges of kidney tissue and investigate the expressions of KIM-1, HIF-1αand α-SMA with immunochemical staining and RT-PCR in unilateral ureteralobstruction (UUO) rats kidneys, and we investigated their alteration under theintervention of HIF-1α inhibitor (rapamycin), thus further to explore the relationship between KIM-1and HIF-1α, α-SMA. Otherwise, the renalprotection of rapamycin can be further established, which is meaningful forclinical advances in the prevention and treatment of chronic kidney disease.Methods:72healthy clean grade male Sprague-Dawley rats weighing (200±20)g were randomly divided into three groups. They were sham-operatedrats group (Sham group, n=24), unilateral ureteral obstruction model group(UUO group, n=24), and rapamycin treatment group (Rap group, n=24). Theleft ureter of each UUO group and Rap group rat was ligated and cut, but insham group rats the left ureter was only separated, no ligated or cut. From firstday before surgey until the killed day, rapamycin (2mg/kg/d) wasadministrated to Rap group rats, and the same volume of physiological salinewas administrated to UUO group and Sham group rats as control. Six ratswere randomly selected out of each group on days1，4，7and14after UUO orsham operation, and the left kidneys were harvested. Moiety tissue was fixedin4%paraformaldehyde and embedded in paraffin for pathomorphology byHE staining. The expressions of KIM-1, HIF-1α and α-SMA protein wereanalyzed by immunohistochemical method. The remnant renal tissue wasflashed-frozen by liquid nitrogen and stored in-80℃for RT-PCR to examinethe levels of KIM-1, HIF-1α and α-SMA mRNA. Image analysis system andGel imaging system were used for semi-quantitative analysis. Experimentaldata were shown as mean±standard deviation. SPSS13.0software was usedfor statistical analysis. P value<0.05was considered to have statisticalsignificance.Results:1Morphological changes of kidney tissuesIn Sham group there were no difference between the left and the rightkidney. In UUO group and Rap group the size of the obstructed kidney wasbigger than the other in the same rat, and the obstructed kidneys becomebigger as time went on since the first day after surgery. In the bigger kidneyrenal cortex became thinner, and renal pelvis expanded, and kidney hydropsappeared. Especially, the changes were most obvious at14day after operation.Under the light microsope, the structure of glomeruli and interstitial tubule showed no obvious abnormalities in Sham group. In UUO and Rap group, themorphological change was slight at1day. From the4day after surgery thelesion became obvious. There were renal tubule expansion, loss of brushborder in tubular epithelial cells, inflammatory infiltration, interstitial edema,and the lesion become aggravated as time went on. The relativetubulointerstitial area increased gradually at1，4，7and14day. Compared withUUO group, the morphological changes were alleviated in the correspondingRap group.2Immunohistochemistry resultsOur findings showed that the expressions of KIM-1and α-SMA were atnegligible levels, and the expression of HIF-1α was little in Sham group ratkidneys. In UUO group, KIM-1, HIF-1α and α-SMA began to rise at1dayafter operation and reached the peak at14day (P<0.05). After administrationof rapamycin, the levels of KIM-1，HIF-1α and α-SMA were lower than thosein UUO group at the same time points of1day,4day,7day and14day(P<0.05) yet were higher than those in Sham group rats(P<0.05).3RT-PCR resultsCompared with Sham group, the levels of KIM-1mRNA, HIF-1α mRNAand α-SMA mRNA increased significantly at every time points in UUO group(P<0.05). Moreover, compared with UUO group, the expressions of KIM-1mRNA, HIF-1α mRNA and α-SMA mRNA were all downregulated in Rapgroup (P<0.05) yet still higher than those in Sham group (P<0.05).4Correlations of KIM-1with HIF-1α and α-SMAWe performed Pearson correlation anlysis on KIM-1mRNA level, HIF-1αand α-SMA mRNA level. Results showed that KIM-1mRNA expression waspositively associated with the level of HIF-1α mRNA (r=0.874, P<0.05) andα-SMAmRNA(r=0.886, P<0.05).Conclusion: In UUO group rat kidneys, the expressions of HIF-1α, KIM-1and α-SMA were upregulated. Rapamycin could ameliorates kidney fibrosis，downregulated the expressions of HIF-1α, KIM-1and α-SMA. Besides, as amolecule downstream of HIF-1α, KIM-1potentially contributed to the development of renal interstitial fibrosis. |
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