Triptolide Protects Podocytes From Interleukin-13 Induced Injury In Vitro

Podocyte injury is the characteristic feature of minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS). Interleukin-13 (IL-13) has been strongly implicated in the pathogenesis of MCD. Triptolide have been used to treat glomerulonephritis for more than 30 years in China with dramatic antiproteinuric effects. We study the effect of triptolide in the podocyte injury induced by IL-13.Part OneInterleukin-13 indcued podocyte injury via a signal transducer and activator of transcription-6-dependent mechanismObjects:Podocyte injury is the characteristic feature of minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS). Interleukin-13 (IL-13) , one of the Th2 cytokines, has been strongly implicated in the pathogenesis of MCD and FSGS. To examine a direct role for IL-13 in podocyte injury, the expression of IL-13 receptor in podocyte and direct effects of IL-13 on podocyte were studied. Further- more, we also want to study the mechanism of action preliminarily.Methods:The expression of IL-13 receptor was studied by RT-PCR and immunohisto -chemistry. After being treated with 10ng/m, 50ng/ml or 100ng/ml IL-13 for various time (8h, 12h ,24h ), changes in both F-actin and ZO-1 patterns in podocytes were observed by confocal microscopy. In order to understand the underlying mechanism of IL-13 effect on podocytes, the activity of JAK-STAT6 signal pathway were analyzed by measuring the extent of phosphorylation of STAT6 using western blotting.Results:The expression of IL-13 receptorα1 mRNA and protein were detected in podocytes. IL-13 induced reorganization of F-actin fibers and redistribution of ZO-1 in a dose and time -dependent manner. 50ng/ml IL-13 treated for 24 hours can damage podocyte distinctly. The pattern of cytoskeleton changed into reorganization of F-actin to the cell periphery at the expense of transcytoplasmic microfilaments. ZO-1 staining appeared markedly fragmented and its density decreased. Western blotting demonstrated phosphorylation of STAT6 in podocytes upon incubation with IL-13. STAT6 signaling pathway inhibitor leflunomide can block the effect of IL-13 on F-actin and ZO-1 in podocytes.Conclusion:Podocyte expressed IL-13 receptor. IL-13 damaged the cytoskeleton structure and tight junction protein. The activation of the STAT6 signal pathway might play an important role in the podocyte injury induced by IL-13.Part Two Triptolide protects podocytes from IL-13 induced injury in vitroObjects:Extract of Tripterygium wilfordii Hook F(TWHF) has been used in treatment of glomerulonephritis for more than 30 years in China, showing dramatic antiproteinuric effect. Triptolide, a diterpene triepoxide, purified from TWHF, has been identified as one of the major active components and has a direct protective effect to podocytes. To study the protective effect of triptolide to podocytes from IL-13 induced injury, cultured mouse podocytes were used to explore underlying mechanism of triptolide action in vitro.Methods:Conditionally immortalized mouse podocytes were treated with IL-13. The podocytes were pretreated for 30 minutes with triptolide (1ng/m, 3ng/ml or 10ng/ml) or dexamethasone (1μM) before IL-13 (50ng/ml) exposure for 24hrs. Both F-actin and ZO-1 patterns in podocytes were observed by confocal microscopy. In order to understand the underlying mechanism of IL-13 effect on podocytes, the activity of JAK-STAT6 signal pathway were analyzed by measuring the extent of phosphory- lation of STAT6 using western blotting.Results:In cultured mouse podocytes, we found that IL-13 almost completely distrupted the podocyte actin cytoskeleton, and made the disappearance of ZO-1 expression , while triptolide stabilized the actin filments , and improved the ZO-1 expression. Furthermore , triptolide can inhibit the activation of STAT6 phosphorylation induced by IL-13.Conclusion:Triptolide prevented the IL-13-induced podocyte injury. The above action of triptolide might contribute to JAK-STAT6 signaling pathway.