| Proteinuria is an important indicator of impaired glomerular filtration barrier. Podocyte is the essential part of the glomerular filtration barrier and charge barrier. Impaired podocyte adhesion to glomerular basement membrane (GBM) may contribute to podocyte detachment from GBM which finally causes proteinuria and then leads to glomerulosclerosis. Podocytes adhere to the GBM principally viaα3β1 integrin, which is abundantly expressed in the podocyte plasma membrane, and believed to be a major receptor for GBM extracellular matrix components.Statins are also called HMG-CoA reductase and clinical studies suggest that statins reduce proteinuria and slow the decline in kidney function in chronic kidney disease. Recently people have paid their attention to the direct effect of statins on podocytes but the mechanisms are little known. Injection of puromycin aminonucleoside (PAN) to rats produces severe proteinuria and mimics the lesions of FSGS. In particular, PAN specifically injures podocytes, leading to foot process effacement, actin cytoskeleton disorganization and abnormal distribution of slit diaphragm proteins.Aim1. To investigate the effect of PAN on expression ofβ1 integrin and synthesis of ROS in podocytes.2. To observe the effect of fluvastatin on the expression ofβ1 integrin and synthesis of ROS in PAN-treated podocytes.3. To explore the association between the expression ofβ1 integrin and the synthesis of ROS. Methods:1.Cell culture:Cells were grown on dishes at 33℃in the presence of 1U/ml ITS in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 U/ml streptomycin. 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. Cultured human podocytes were divided into 4 groups:①normal control group;②DMSO(1mg/ml)control group;③PAN (1.0μg/ml) group:PAN treated podocytes for 12h, 24h, 48h, and 72h respectively;④PAN(1.0μg/ml) +FLV(1×10-8～1×10-5 M) group : podocytes were treated with PAN plus different concentrations of FLV for 48h. The expression ofβ1 integrin and reactive oxygen species (ROS) in four groups was detected by Western Bolt and DCFHDA (2'7'-Dichlorofluoresecein 3'6'-diacetate) respectively.3. Podocytes were divided into 5 groups:①normal control group;②DMSO(1mg/ml)control group;③PAN(1.0μg/ml) group;④PAN (1.0μg/ml)+FLV(1×10-8～1×10-5 M)group ;⑤FLV(1×10-8～1×10-5 M)group. The viability of cells was determined by MTT colorimetry at 12h, 24h, 48h, and 72h respectively.Results:1. Effect of PAN on the expression ofβ1 integrin Compared with the normal control group, the expression ofβ1 integrin in PAN-treated podocytes began to decrease after 24h(P<0.05),and this effect continued in 48h and 72h group(P<0.05),the expression of podocyte in 12h group didn't change significantly(P>0.05).2. Effect of fluvastatin on expression ofβ1 integrin in PAN -treated podocytes Compared with the normal control group, the expression ofβ1 integrin were significantly down regulated in PAN-treated podocytes after 48h DMSO had no significant effect on podocytes. After co-treating podocytes with fluvastatin and PAN, the expression ofβ1 integrin was up regulated significantly when the concentration of fluvastatin was 1×10-7 mol/L(P<0.05).3. Effect of PAN on the synthesis of ROS Compared with the normal control group, exposure to PAN significantly increased the synthesis of ROS after 24h and 48h (p<0.05).Then at 72h the expression of ROS decreased but was still more significant than the normal control group(P<0.05).4. Effect of fluvastatin on synthesis of ROS in PAN-treated podocytes Compared with normal control group, the synthesis of ROS was significantly inhibited by co-treating podocytes with fluvastatin and this effect was statistically conspicuous when the concentration of fluvastatin was 1×10-7 mol/L(P<0.05).5. The expression ofβ1 integrin had an inverse correlation with the synthesis of ROS in PAN group and PAN+FLV group.6.Effect of fluvastatin on viability of podocytes The results of MTT revealed that DMSO and lower concentration of fluvastatin(1×10-8M and 1×10-7M) had no significant effect on viability of podocyte ( P>0.05),while lower podocyte viability was found in higher concentration of fluvastatin (1×10-6M and 1×10-5M) group, PAN group and PAN+FLV group at 24h(P<0.05). Compared with the PAN group ,the viability of podocyte improved significantly in PAN+FLV group when the concentration of fluvastatin was 1×10-7 M(P<0.05)at the time of 48h.Conclusions:1. PAN can down regulate the expression ofβ1 integrin and induce the synthesis of ROS in podocytes.3.Fluvastatin can up regulate the expression ofβ1 integrin and inhibit the synthesis of ROS in PAN-treated podocytes.5. The expression ofβ1 integrin had an inverse correlation with the synthesis of ROS in PAN-treated podocytes.6. Fluvastatin protected podocytes by up regulating the expression ofβ1 integrin and the mechanism maybe correlated with fluvastatin inhibiting the synthesis of ROS.
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