The Effects Of Dexamethasone On Podocytes Injury Induced By Puromycin Aminonucleoside And Its Possible Mechanism

Podocytes and slit diaphragm between foot processes are important components of the glomerular filtration barrier. The related molecules in them, such as nephrin, podocin, CD2AP andα-actinin-4, etc., play an important role during the development of proteinuria. The establishment of human podocyte cell line makes it possible to study the mechanism of proteinuria on the molecular level for our research. Currently, there were few reports about the using of cultured podocytes in vitro, in particular the research of mechanism of glucocorticoid with human podocyte cell. This study was to establish an in vitro podocyte injury model induced by puromycin aminonucleoside (PAN), which can provide the conditions for further study of the molecular mechanism of proteinuria. Glucocorticoids are widely used in the treatment of most primary and some secondary glomerular diseases characterized by proteinuria. However, the specific molecular mechanism of dexamethasone on podocyte is not fully clear. Therefore, we further use of cultured human podocytes in vitro to discuss the molecule mechanism of anti-proteinuria of dexamethasone.【Aim】1. To establish the an in vitro podocyte injury model induced by PAN, providing the conditions for further study on the role of related molecules of podocytes in the molecular mechanism during the development of the proteinuria.2. To observe the effects of dexamethasone on podocyte viability and apoptotic percentage induced by PAN. 3. To discuss the relationship of dexamethasone mechanism of anti-proteinuria and the key molecular slit diaphragm nephrin.【Methods】1. Podocyte culture:Cells were grown on dishes at 33℃in RPMI 1640 medium with 1U/ml ITS in supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 U/ml strieptomycin. To induce differentiation, podocytes were maintained at 37℃under 5% CO2 and 95% air to allow differentiation for at least 10 days. Differentiated podocytes were cultured for 12h in RPMI 1640 medium before exposed to various experimental conditions.2. The viability of podocytes were detected with MTT assay : Podocytes were divided into 4 groups :①normal control group;②puromycin (PAN 0.5mg/ml) group;③P AN(4.5μg/ml) group;④P AN (1.5μg/ml)+DEX(1×10-7～1×10-5 M)group ; The viability of cells was determined by MTT colorimetry at 12h, 24h, 48h, and 72h respectively.3. Detection of apoptosis by DAPI staining: Groups are the same with MTT. Since the effects of injury of PAN and the protection of DEX in each group with MTT method are not significant after 12h, the group at 12h was removed in this experiment.4.In early experiments,by testing the podocyte viability and apoptosis after puromycin intervention, found podocyte injury is not significant in the PAN (0.5μg/ml) group.However, in the PAN (4.5μg/ml),a great of podocyte death after 12h.Therefor, cultured human podocytes were divided into 3 groups as follows:①n ormal control group;②PAN(1.5mg/ml)group: PAN treated podocytes for 24h, 48h, and 72h respectively;③PAN (1.5μg/ml) + DEX (1×10-7～1×10-5 M) group:Podocytes were treated with PAN plus different concentrations of DEX for 48h. The expression of podocytes nephrin in three groups was detected by Western Bolt respectively.【Results】1. Viability of podocytes treated with PAN.The viability of podocytes decreased significantly after treated with the 4.5μg/ml PAN for 12h. After 48h, compared with the normal control group (0.96±0.08), the viability of podocytes of PAN 1.5μg/ml group (0.33±0.07) decreased significantly(p<0.01)。PAN 0.5μg/ml group(0.75±0.03) didn't decrease comparing with the normal control group (1.05±0.11)until 72h later (p<0.01).2. The change of the apoptotic percentage of podocytes treated with PAN.The apoptotic percentage of podocytes increased significantly after treated with PAN 4.5μg/ml for 12h. Compared with the normal control group (3.41%±1.28%), the apoptotic percentage of podocytes of PAN 1.5μg/ml group (21.61%±3.98%) increased significantly(p<0.01) after 48h。After 72h, it is hard to find normal podocytes in the PAN (4.5μg/ml) group. Although the apoptotic percentage of podocytes of PAN (0.5μg/ml) group increased, there is no significance compared with the normal control group.3. The change of the expression of the slit diaphragm protein nephrin treated with PAN. (Western Blot)Compared with the normal control group, the expression of nephrin began to decrease in PAN-treated podocytes after 24h. The expression of nephrin decreased significantly after 48h and 72h respectively.4. Viability of podocytes treated with dexamethasone pretreatmentPodocytes were pretreated with DEX for 30 minutes before treated with PAN. After 48h, the viability of podocytes treated with dexamethasone pretreatment and PAN were divided as follows: DEX 1×10-5M(0.96±0.06), DEX 1×10-6 M(0.86±0.05), DEX 1×10-7 M(0.78±0.03). Compared with the normal control group and PAN 1.5μg/ml(0.34±0.05)group, there is significance even among the three groups. Also, between the DEX 1×10-6M group and the DEX 1×10-7M group (p<0.05), other (p<0.01), the difference had significance.5. The protection of the apoptotic percentage of podocytes treated with dexamethasone pretreatmentAfter 48h, compared with the PAN1.5μg/ml group (20.60%±5.49%), the apoptotic percentage of podocytes of DEX 1×10-5M group decreased significantly(p<0.05). After 72h, compared between the other two groups treated with DEX and the single group treated with PAN, the apoptotic percentage of podocytes decreased too and the difference is significant.6. The change of the expression of nephrin treated with dexamethasone pretreatment (Western Blot)The expression of nephrin increased significantly with dexamethasone pretreatment and the effect of DEX 1×10-6 M group is the strongest. The effect of DEX decreased along with the decreasing of DEX concentration.【Conclusions】1. The podocyte injury by PAN treatment depends on the time and the concentration. To establish the podocyte injury model induced by PAN, the appropriate concentration of PAN was 1.5μg/ml, the reaction time was 48h.2. DEX has direct effects on podocytes to increase in the expression of nephrin and to decrease apotosis which may relate with the protection of podocytes and the improvement of proteinuria.