Toxic Effect And Mechanism Of Triptolide On Podocytes In Vitro And In Vivo

Objective: To observe the direct effect of triptolide(TP)on normal immortalized podocytes in vitro and normal podocytes in vivo,and to explore its mechanism and clinical significance.Methods: We treated the human immortalized podocytes with 10,30 and 50 ng/ml of TP to take the first look of the cell death under a light microscope.We used Annexin V staining and flow cytometry to detect apoptosis.We used Western blot to examine the expression and activation of MAPK p38,p53,bax and caspase3.At the same time,podocytes were treated with different doses of glucocorticoids(1 ?mol,10 ?mol,100 ?mol).qPCR was used to compare the effects of TP and dexamethasone on the transcriptional levels of some podocytes,including CD2 AP,SYNPO,NPHS2,VEGFA,WT1 and other genes that have been verified by our previous work,such as ENPEP,PAK1,FGFR1,GAD45 A,CERS6,ZNF277,TSC22D1 and SEPT10.TP and glucocorticoids were used together to treat cells followed by examination of the parameters mentioned above to determine whether glucocorticoids could reverse the injurious effect of TP on podocytes.In vivo,mice were treated by intraperitonial injection of TP(100-600 ?g/kg/day)for 28 days,and urine samples were collected once a week to monitor proteinuria development.At the end of the treatment for 28 days,urine,blood and renal tissue samples were collected for biochemical and histological analyses.At the same time,we isolated glomeruli of the mice to analyze the expression of podocyte specific and essential genes.We also isolated glomeruli from normal rats and treated them with 30 ng/ml TP for 24 h followed by qPCR analysis of the expression of podocyte-specific essential genes.Results: We found that treatment of TP at 10 ng/ml,a concentration that is routinely used for podocyte protection,was sufficient to activate pro-apoptotic signaling of MAPK p38,p53 and BAX and induced apoptosis in cultured podocytes;and higher concentrations of TP exacerbated the p38,p53 and BAX activations and cell apoptosis.Moreover,TP severely downregulated the genes that are essential for podocyte structure and function.Interestingly,TP-induced apoptosis of podocytes,activation of apoptotic signal and down-regulation of important genes in podocytes could not be reversed by glucocorticoids.In vivo,high-dose TP treatment for prolonged time did not cause podocyte injury,essential genes downregulation,and proteinuria in mice.And we found that TP did not down-regulate the important genes of podocytes in isolated glomeruli ex vivo,that is,it had no toxic effect on podocytes in isolated glomeruli ex vivo.TP was neither toxic to the podocytes with isolated glomeruli ex vivo.Conclusion: TP is toxic to immortalized podocytes in culture but not to the podocytes in animals or isolated glomeruli ex vivo.Our study suggests that immortalized podocytes might have genetically evolved to become sensitive to TP toxicity and thus caution should be taken in interpreting data from immortalized podocytes.On contrary,in vivo TP could be as safe as glucocorticoids for podocytes when it is used for disease treatment.Finally,TP may be used as a unique in vitro model for studying steroid-resistant podocytopathies.