The Impairments Of High Glucose On Kidney Stem Cells And Its Injury Mechanism

Objective: To isolate and steadily culture kidney stem cells (KSC) from rat renalpapilla, and to identify the biological characteristics of KSC.The effects of highglucose on the basic features and paracrine action of KSC, and the impacts of highglucose on differentiation of KSC into renal tubular epithelial cell (RTEC) wereinvestigated.Methods:①The KSC were isolated from the tips of the renal papilla in4weeksSprague-Dawley rats. The morphology of KSC was observed through inversionmicroscope, and the phenotye characteristics of KSC were identified through flowcytometry and immunofluorescence. The ability of adipogenic and osteogenicdifferentiation was evaluated. The differences of gene expression between KSCand rat RTEC were compared using quantitative real time polymerase chainreaction (qRT-PCR).②The KSC were cultured in the media with high glucose(30mmol/L) and with normal glucose (5.6mmol/L) respectively. The changes ofthe cell morphology, proliferation, phenotypic markers, chemokine expression andanti-hypoxia ability were investigated.③The KSC were cultured in media withhigh glucose or with normal glucose respectively. The supernatant of thepre-treated KSC will be collected as the conditional media. Thehypoxia/reoxygenation (H/R) model of rat RTEC was established using theNRK-52E cell line. The effects of KSC conditional media on the H/R RTEC wereinvestigated.④The differentiation of the KSC was induced using the inducedmedium (containing Activin A, BMP-7, etinoic acid) and renal epithelial cellgrowth medium (REGM) alternately. KSC co-culture with hypoxia injured RTECin induced medium and REGM alternately were also observed. The KSC culturedusing the MSC conditional medium as a control. The KSC differentiation rates ofall groups were calcaulated in this study. Meanwhile, the effects of high glucoseculture medium and normal glucose on the induced KSC gene expression ofmature epithelial cell marker E-Cadherin、CK-18、AQP-1were also investigated. Results:①The KSC showed a spindle-shaped and arborization-like growth；immunofluorescence indicated that KSC staining with α-SMA, Vimentin,N-Cadherin, Nestin, CD133marker, and without E-Cadherin, CK-18, ZO-1. Theresults of flow cytometry showed the positive staining of CD29, CD90, CD73andCD45were99%,95.8%,99.9%and3.4%respectively. The positive stainings ofstem cell marker CD133and Nestin were33.2%and70.2%respectively，theirdouble staining rate was31.4%. After adipogenic differentiation, the KSC showedpositive staining by oil red O. Also, the KSC showed orange calcium depositionby alizarin red staining and showed black as staining by Von kossa afterosteogenic differentiation. qRT-PCR showed that the expression of embryonicstem cell marker Nanog, Oct4/pou5f1, Sox2/sry-box-2and mesenchymal markerα-SMA、Vimentin in KSC were higher than that in RTEC (P <0.01). Meanwhile,compared with RTEC, the mature epithelium marker E-Cadherin、CK18werelower expression in the KSC (P <0.01).②The KSC grew slowly in the highglucose media. However, the synthesis of Fibronectin、Collagean I and CollageanIV of the KSC cultured in high glucose media were increased. qRT-PCR indicatedthat the expression of Fibronectin、Collagean I and Collagean IV mRNAincreased.The gene expression of mesenchymal marker-SMA、Vimentin and fibrosisfactors marker TGF-β、CTGF、PAI-1were higher than that cultured in the normalmedia. The anti-hypoxia ability of KSC was decreased after treated with highglucose media. when KSC were cultured in hypoxia environment for24hours, thecell proliferation index and the gene expression of SDF-1and CXCR4were lowerin high glucose pre-treated KSC than that in the cultured KSC with normal media(<0.01).③The idea H/R model of RTEC were established using hypoxia for4hours and reoxygenation2hours in this study. After hypoxia, the apoptosis rate ofthe H/R RTEC, the LDH and MDA level of the H/R RTEC supernatant wereincreased， the SOD level of the H/R RTEC supernatant were decreased.Comparied with control group, the cell apoptosis rate, the LDH and MDA levelwere lower and the SOD level were much higher in the H/R RTEC afterco-cultured with KSC conditional media (P <0.01). When compare between thenormal and high glucose group, the cell apoptosis rate, the LDH and MDA levelof H/R RTEC in high glucose group were much higher than that in normal glucose group (P <0.01), the SOD level of H/R RTEC supernatant in high glucose groupwere much lower than that in normal glucose group (P <0.05).④When KSC wasco-cultured with hypoxia injured RTEC in induced medium and REGMalternately, the positive rate of CK-18was44.2%. Immunofluorescence resultsindicated that the mature epithelial cell marker CK-18、E-Cadherin、ZO-1wererpartly positive in the co-culture induced group. KSC were pre-treated with normalglucose and high glusoe culture medium respectively, then induced by theco-culture induce proposal, the positive rate of CK-18in normal glucose KSCgroup was (52.37±1.23)%， in high glucose KSC group was (39.86±7.44)%respectively. The positive rate of CK-18in high glucose KSC group wassignificantely lower than that in normal glucose KSC group（P=0.045）. Theresults of qRT-PCR showed that the gene expression of mature epithelial cellmarker E-Cadherin and AQP-1were significantely lower in the high glucose KSCgroup than that in the normal glucose KSC group（P <0.05）.Conclusion:①The kidney stem cells were isolated successfully and steadilycultured from the rat renal papilla. Their biological characteristics were identifiedin this study. In the high glucose medium, the KSC proliferation ability weredecrease, the gene expression of fibrosis promote factors were increase, theanti-hypoxia ability was impaired and the gene expression of chemokin weredecrease. The KSC conditional media could repair the hypoxia/reoxygenationinjury of RTEC. These effects were mainly by inhibiting cell apoptosis, reducingthe oxidative stress. The cell apoptosis inhibiting rate and the anti-oxidative stresscapacity were supressed when KSC were pre-treated by media with high glucose.The differentiation of KSC into RTEC was successfully induced in vitro. The bestcell differentiation proposal was that KSC co-culture with hypoxia injured RTECin the induced medium and REGM alternately. High glucose in the medium mayinjured the differentiation of KSC.