Potential Role For Nrf2Activation In The Preventive Or Therapeutic Effects On Diabetic Nephropathy In Mice

Oxidative stress is a major cause for diabetic nephropathy. Up-regulation of the keyantioxidative transcription factor, nuclear factor-erythroid2-related factor2(Nrf2), was foundto prevent the development of diabetic nephropathy. The present study was to explore thepreventive and therapeutic effects of Nrf2activation induced by sulforaphane (SFN) andproteasomal inhibitor MG132at low dose (10μg/kg/day) on diabetic nephropathy.To explore the preventive effect of SFN on DN, type1diabetic mouse model wasinduced with multiple low-dose streptozotocin. Once the onset of hyperglycemia, diabetic andage-matched control mice were subcutaneously given SFN at0.5mg/kg daily for3months.At the end of3months treatment of SFN one set of mice were sacrificed to perform theexperimental measurements (3month time-point). The second set of both diabetic and controlmice were aged for additional3months without further SFN treatment (6month time-point).To explore the therapeutic effect of MG132on DN, transgenic type1diabetic (OVE26) micewere used. These mice display renal dysfunction, with albuminuria by3months of age, atwhich time MG132treatment was started. After3-months treatment with MG132, blood,urine and kidney samples were harvested. Rrenal function, morphology and biochemicalchanges were examined with Real-time PCR, Western blotting, and immunohistochemicalexamination. In order to explore the mechanism, we treated the human renal tubularepithelium cells (HK11) with Nrf2specific siRNA to knock down Nrf2gene at first, and thenadded HG/Pal, MG132and SFN.For SFN prevention experiment, SFN significantly prevented the development of diabetic nephropathy with elevated Nrf2expression and function in the kidney at3-monthtime-point, but not at6-month time-point. Blockade of pro-fibrotic and pro-inflammatoryproteins was documented concurrent with enhanced Nrf2expression in SFN-treated diabeticmice also at3-month time-point. For MG132treatment experiment, compared to age-matched,non-treated diabetic mice, MG132-treated diabetic mice showed significant improvements interms of renal structural and functional alterations. These therapeutic effects were associatedwith increased Nrf2expression and transcriptional up-regulation of Nrf2regulatedantioxidants. Furthermore, diabetes was found to significantly increase proteasomal activity inthe kidney, an effect that was significantly attenuated by3-months of treatment with MG132.Mechanistic study using HK11cells confirmed the role of Nrf2activation since silencing theNrf2gene with its specific siRNA abolished both SFN and MG132prevention of highglucose/palmitate-induced pro-fibrotic response.These results suggest that SFN’s preventive effects on diabetes-induced renal pathogenicdamage and Nrf2activation were not sustained, suggesting the requirement of continual useof SFN for its sustained effect. Additionally, MG132up-regulates Nrf2function via inhibitionof diabetes-increased proteasomal activity, which can provides the basis for the therapeuticeffect of MG132on the kidney against diabetes-induced oxidative damage, inflammation,fibrosis and eventual dysfunction.