| Background:Aristolochic acid (AA), belonged to Aristolochia, can cause kidney tubular necrosis,renal interstitial fibrosis and then result in chronic renal failure in patients who have AAor AA-containing drugs for a long-term. This type of kidney disease is generally definedas aristolochic acid nephropathy (Aristolochic Acid Nephropathy, AAN) by scholarsthroughout the world. The most of AAN patients showed renal tubular epithelial celldegeneration, necrosis, the lack of obvious epithelial cell regeneration, bare basementmembrane, diffuse interstitial widening and progressive fibrosis, which are different fromthe other kidney diseases or toxic nephropathy. Currently, the mechanism of these specialpathological injury is not very clear and there are not effective clinical treatments forAAN patients. So, AAN patients often delayed healing and eventually progress to end-stage renal failure. Therefore, it’s necessary to explore the tubular injury and repairmechanisms of AAN deeply. Scholars conducted a lot of researches about the tubules, themajor and important target of AA, and established theories including the renal tubularepithelial cell apoptosis, transition, intracellular AA-DNA adduct formation and urinariatumor formation. These theories can explain part of the pathological features of AAN, butthe reasons for inhibition of tubules repair still need to be further clarified. Recent years,the role of both peritubular capillary(PTC) injure, loss and endothelial cells damage inacute and chronic kidney disease(especially the progression of chronic kidney disease)attracted the attentions of scholars. Accordingly, it is necessary to observe the PTC injuryand loss in the process of AAN and analyze the relationship between the PTC injury, lossand tubular damage, proliferation and inhibition of tubules. It’s also essential to reserchthe cell biology and toxicity mechanisms of AA for PTC injury deeply and propose a newmechanism for the treatment and a new protective therapeutic strategies of aristolochicacid nephropathy. Objective:The study plan to observe the PTC injury, loss and analyze the relationship betweenthe PTC injury, loss and tubular damage, proliferation and inhibition of tubules throughestablishing an AAN animal models, then examine the ultrastructural changes of PTCendothelial cells by transmission electron microscopy. The later study proposes toestablish AA injure (human umbilical vein endothelial cells, HUVEC) model induced byaristolochic acid, observe endothelial cell proliferation, apoptosis and migration, tubeformation under the influence of AA toxicity; to reserch the toxicity mechanisms of AAfor PTC injury deeply and propose a new mechanism for clinic treatment and therapeuticstrategies of aristolochic acid nephropathy.Method:The study including vivo and vitro two parts.Part I: To obsever the PTC injure induced by AA and analyze the relationshipbetween PTC injure and tubular damage, repair in vivo.1. To establish AAN mouse model.C57/BL/6mouse were divided into4groups,including the control2weeks group, the AA2weeks group, the control4weeks groupand the AA4weeks group. The AA group were intraperitoneal injected AA dissolved inphosphate-buffered saline at a dose of5mg·kg-1omni biduo for up to2weeks or4weeks,while the control group were received saline only;2. The blood, urine and kidney of mouse were collected at2and4weeks. The bloodand urine were detected by an automatic biochemical analyzer and kidney sections wereinvestigated pathologically;3. The correlation between PTC injure and tubular damage, repair were analyzed.Part II: To observe the mechanism of the vascular endothelial cells injure inducedby AA in vitro.1. According to the reserches, the study grouped with different concentrations of AA(0，5g/ml，10g/ml，20g/ml) and at different times (24h,48h,72h) to observe therole of AA on human umbilical vein endothelial cells (HUVEC), MTT assay theinfluence of AA on cell proliferation ability, then screening drugs optimum time;2. LDH release: different concentrations of AA(0，5g/ml，10g/ml，20g/ml)treat HUVEC for48h (optimal time), then detect LDH release in cytoplasm; 3. Tube formation: cells were collected after different concentrations of AA(0，5g/ml，10g/ml，20g/ml)treated for48h (optimal time), then inoculated intomatrigel-coated96-well plate(103to104cells per well) and incubated for6h, the tubeformation was observed through microscope;4. Endothelial cell migration: cells were collected after different concentrations ofAA(0，5g/ml，10g/ml，20g/ml)treated for48h, then inoculated into Transwellchamber(105cells per well) and the lower chamber containing low concentration ofserum (2.5%FBS) in DMEM high glucose medium stimulated cell migration, thenincubated for18~24h. After fixation and staining counted cells in the lower chamberlower under an inverted microscope;5. Endothelial cell apoptosis: cells(≥105cells per group) were collected afterdifferent concentrations of AA(0，5g/ml，10g/ml，20g/ml)treated for48h, thendetect apoptotic cells in flow cytometry after the Annexin V-FITC and PI staining;6. Mitochondrial membrane potential: cells were collected after differentconcentrations of AA(0，5g/ml，10g/ml，20g/ml)treated for48h, then detectedchanges in mitochondrial membrane potential by laser scanning confocal after the JC-1staining;7. Mitochondria-mediated apoptosis pathway: cells were collected after differentconcentrations of AA(0，5g/ml，10g/ml，20g/ml)treated for48h, then detected theexpression of Caspase-3、Caspase-9、Bax、Bcl-2、Apaf-1、Cytochrome C via westernbloting.Result:Part I:1. Compared with control group, serum Scr, BUN and urine NAG of AA group wereextraordinary increased(especially the AA4W group).2. Compared with control group, tubular damage, interstitial fibrosis, PTC injureand loss of AA group were extraordinary increased(especially the AA4W group); theproliferation and repair of injure tubular epithelial cells in AA2W group are better thanwhich in AA4W group. The PTC loss were significantly positively correlated withtubular injury scored(P＜0.05), and they were negatively correlated with the positiveexpression of PCNA(P<0.05). 3. The swelling of mitochondria and part of mitochondrial spinal disorders,disappeared were visible in PTC endothelial cells of AA group.Part II:1. AA can reduce the viability of HUVEC and increase LDH release in a dose-dependent manner.2. AA can decrease the ability of HUVEC tube formation and the ability ofendothelial migration in vitro, both of them showed a dose-dependent manner.3. AA can increase the expression of apoptotic marker proteins (BAX、caspase-3),induce mitochondrial membrane potential in endothelial cells decrease, leadmitochondrial apoptosis pathway-related proteins (Cytochrome C, Apaf-1, Caspase-9)upregulated and anti-apoptotic protein Bcl-2downregulated, which suggesting that AAcan induce HUVEC apoptosis through activate the mitochondrial apoptotic pathway.Conclusion:1. AA can induce PTC injury and loss of mouse kidney in a dose-dependent manner,and PTC injury and loss have a markely positive correlation with tubular injury,proliferation and inhibition of tubule recovery;2. AA can inhibit HUVEC proliferation, tube formation and migration, restrain theregeneration of vascular when endothelial cells damaged, which toxicity may affect PTCrepair and functional recovery;3. AA may induce HUVEC apoptosis through activate the mitochondrial apoptoticpathway. |
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