A Mechanism Study Of The Protection By A Novel Curcumin Analog C66against Diabetic Nephropathy In Mice

The incidence of diabetic nephropathy (DN) shows an increasing tendency yearafter year along with the increase in diabetes. DN is a leading cause of end-stagerenal disease, resulting in high mortality and morbidity. However, it lacks effectiveapproaches for the prevention or treatment of this disease. Therefore, it is of greatimportance to develop new effective approaches to protect against DN. Oxidativestress is the main cause of DN. Under high blood glucose condition, excessivereactive oxygen species (ROS) accumulate in the kidney, which exceeds thescavenging activity of the antioxidant system, leading to the pathogenesis anddevelopment of DN. Nuclear factor (erythroid-derived2)-like2(Nrf2) plays acentral role in antioxidant-redox signaling. As a transcription factor, Nrf2translocates into the nucleus to bind the antioxidant response elements (AREs)within promotors of its downstream antioxidant genes, activating their transcriptions,therefore, attenuating diabetes-induced oxidative stress and slowing down theprogression of DN. Curcumin is a natural product that is beneficial for theprevention of a variety of diseases. However, the poor bioavailability extensivelylimits its clinical application. The effective dosage of curcumin in DN animal modelsvaried from50to200mg/kg daily. The novel curcumin analog C66has much lowereffective dose of only5mg/kg administered every other day. The much higherbioavailability of C66than native curcumin confers it a promising potential forclinical application. However, little is known about the protective mechanism of C66in DN.Previously we reported that C66up-regulated Nrf2expression in diabetic aorta,ameliorating diabetes-induced pathological changes. In order to difine whether C66up-regulates Nrf2to ameliorate DN, wild type (WT) C57BL6/J and Nrf2knockout(Nrf2-null) mice were induced to diabetes by multiple low-dose streptozotocininjection. Once diabetes was confirmed, both diabetic and non-diabetic mice were treated with C66(5mg/kg, every other day) or vehicle for6months in total. In WTmice, C66markedly reduced diabetes-induced proteinuria and renal fibrosis,whereas Nrf2deletion partially attenuated C66protection against DN. These resultsindicate that Nrf2plays a significant, though partial, role in C66protection andsuggest that C66has other protective mechanism in addition to Nrf2up-regulation.Our second aim is to determine how C66induces Nrf2expression and function.Nrf2is sequestered by Kelch-like ECH-associated protein1(Keap1) in thecytoplasm, hampering Nrf2nuclear translocation and the transcription of itsdownstream antioxidant genes, as well as promoting proteasomal degradation ofNrf2. Thus, reducing Keap1expression is a promising strategy to induce Nrf2activity. It is reported that microRNA-200a (miR-200a) is able to bind the3’untranslated region (3’UTR) of Keap1mRNA, leading to its degradation, followedby the decrease of Keap1protein. Interestingly, curcumin has been proven toup-regulate miR-200a. Therefore, increasing miR-200a may be the mechanism bywhich C66induces Nrf2function. To test this hypothesis, WT diabetic mice weretreated with locked nucleic acid-modified-anti-miR-200a (LNA-200a), a specificinhibitor of miR-200a, accompanying the administration of C66. In diabetic andnon-diabetic kidney, C66potently up-regulated miR-200a, decreased Keap1expression, up-regulated both total and nuclear Nrf2, as well as the mRNA levels ofNrf2downstream antioxidant genes Heme Oxygenase1and NAD(P)Hdehydrogenase quinone1, effects of which were completely abolished by LNA-200a.These results demonstrate that C66increases miR-200a to induce Nrf2.Our third aim is to explore other protective mechanism by C66other than theup-regulation of Nrf2. Among the microRNAs that are related to DN, curcumin isknown to decrease miR-21in addition to increasing miR-200a. It is noted thatmiR-21plays a key role in the pathogenesis of DN since it can bind the3’UTR ofSmad7mRNA, resulting in its degradation, followed by the decrease in Smad7protein. Since Smad7inhibits the activation of fibrotic transcription factor Smad3,miR-21indirectly activates Smad3to enhance the progression of renal fibrosis. Wethus hypothesized that C66down-regulates miR-21to ameliorate DN. We firstobserved the inhibitory effect of C66on miR-21and its primary transcriptionproduct primary-miR-21(pri-miR-21) in the kidney of WT mice. C66decreasedboth renal miR-21and pri-miR-21levels under both non-diabetic and diabetic conditions. These results confirmed the inhibitory effect of C66on miR-21andfurther indicates that C66regulates miR-21expression at transcription level, ratherthan miR-21post-trancriptional processing procedures. With the aim of evaluatingthe effect C66on Smad7/Smad3signaling pathway, Smad7mRNA and proteinlevels, along with p-Smad3protein level were culled for test. The results showed thatC66increased renal Smad7expression and decreased p-Smad3protein under bothnon-diabetic and diabetic conditions. In order to exclude the effect of Nrf2,Nrf2-null diabetic mice were treated with either C66or the specific miR-21inhibitorLNA-21, as positive control, for6months. C66and LNA-21similarly attenuatedmiR-21level and alleviated diabetes-induced proteinuria and renal injury in theabsence of Nrf2, demonstrating inhibition of miR-21as the additional protectivemechanism by C66in addition to Nrf2induction.Taken together, the present study demonstrates for the first time that the novelcurcumin analog C66has dual effects: inducing Nrf2and inhibiting miR-21.miR-200a was further identified to mediate the up-regulation of Nrf2by C66.