The Effect Of AMPK On Protective Effects Of Sulforaphane In Diabetic Nephropathy

Background: Epidemiological studies have demonstrated that type 2 diabetes(T2D)accounts for more than 90% of diabetic patients and is an essential independent risk factor for chronic kidney disease(CKD).Diabetic nephropathy(DN)is the most common microvascular complication in patients with T2 D.It can severely damage renal function and then progress to the end-stage renal disease(ESRD),significantly threatening life.Particularly,DN is traditionally characterized by metabolic and hemodynamic changes caused by hyperglycemia.However,more and more emerging studies have revealed that the accumulation of lipids in the kidney plays a crucial role in the occurrence and development of DN,and is highly correlated with the decline of glomerular filtration rate and renal failure.The state of excess energy in the body caused by increased lipid intake,lipid metabolism disorder,and reduced energy consumption is the key to the occurrence of T2 D.When energy intake exceeds the fat storage capacity of adipose tissue,ectopic lipid deposition(ELD)will occur in non-fatty organs,triggering a series of pathological changes,which is called lipotoxicity.The long-term deposition of lipids in the kidney leads to renal lipotoxicity,which further induces oxidative stress damage,inflammation,and fibrosis,destroying the renal structure and function.Besides,more and more pieces of evidence confirmed that renal lipotoxicity is closely related to the activity of AMP-activated protein kinase(AMPK)and its downstream signals.AMPK is an essential nutrient and energy sensor and can be activated by energy deficiency.It promotes the production of ATP by increasing the activity or expression level of proteins related to catabolism and simultaneously reduce the consumption of ATP by inhibiting the biosynthetic pathway,contributing to maintaining energy balance in the body.The catalytic subunit α of AMPK is the center of the catalytic reaction.There is a threonine residue(Thr)in α subunit ring that can be phosphorylated,which mediates the activation of AMPK.Among the two subtypes of α subunits α1 and α2,only the α2 subtype plays an important role in regulating systemic energy metabolism and insulin sensitivity and is the core of the study of diabetes and other metabolic-related diseases.Moreover,α2 is more abundantly expressed in the kidney than α1 and plays a higher protective role in kidney injury.The activation of AMPK stimulates the biogenesis of mitochondria,increases the uptake of fatty acids(FAs)and subsequent fatty acid oxidation(FAO),and inhibits lipid biosynthesis at the same time.It is a potential target for the treatment of various metabolic syndromes related to lipid metabolism disorders.Sulforaphane(SFN),a natural isothiocyanate isolated from cruciferous vegetables such as cauliflower and cabbage,can activate nuclear factor(erythroid-derived 2)-like 2(Nrf2),an important transcription factor that regulates cellular oxidative stress.Our previous studies confirmed that SFN has a significant protective effect in T2D-induced nephropathy via the Nrf2-mediated antioxidant response.In addition,emerging studies suggest that SFN may also alleviate obesity and diabetic cardiomyopathy by inhibiting adipogenesis and improving dyslipidemia,which accompanied by AMPK activation,suggesting that SFN may play an important role in improving lipid metabolism through the activation of AMPK and its downstream signaling pathways.However,in DN,it is still uncertain whether the protective effect of SFN is achieved through AMPK-mediated lipid metabolism and whether AMPK,in particular α2 subtype,responsible for energy regulation,is indispensable for SFN function.Second,AMPK may also be a potential target for anti-oxidative stress therapy.It has been found that the activation of AMPK is closely related to up-regulation of the activation and function of Nrf2.However,the exact relationship between AMPK and Nrf2 is still unclear.In addition,previous studies mainly focused on the short-term effect of SFN on DN,but there was a lack of relevant studies on its long-term lasting effect.Objectives: The effects of SFN on T2D-induced nephropathy were investigated in wild-type(WT)and AMPKα2-knockout(Ampkα2-/-)T2D mice to:(1)determine whether the role and mechanism of AMPK(especially the α2 subtype)on protective effects of SFN in DN,and(2)investigate the lasting,post-treatment renoprotective effects of SFN after cessation of SFN therapy.Methods: Both WT and Ampkα2-/-mice were fed a high-fat diet(HFD)to induce insulin resistance,followed by streptozotocin(STZ)injection to induce hyperglycemia.After 7 days of STZ injection,mice with 3-hour fasting glucose≥250 mg/dl were deemed T2 D.Both diabetic and age-matched control mice(receiving normal diet,ND)were injected subcutaneously with either 0.5 mg/kg SFN for five days every week over the course of 3 months with continuous HFD or ND feeding.After 3 months of treatment(3 month time-point,3M),five mice from each group were sacrificed.SFN treatment of the remaining mice were discontinued and the mice were maintained on their respective HFD or ND for another 3 months before being sacrificed(6 month time-point,6M).We collected 24-hour urine by using metabolic cages upon sacrifice to detect urinary albumin.Histopathological and immunohistochemical staining were used to observe the morphological and structural changes in the kidney tissues collected.Oil red O staining was used to observe the lipid deposition in the kidneys.Western blot and real-time polymerase chain reaction(Real-time PCR)were used to detect kidney protein and m RNA changes in indicators related to inflammation,fibrosis,oxidative stress,and lipid metabolism.Results: In WT mice,SFN significantly alleviated lipid metabolism disorders and lipid deposition in diabetic kidneys at both 3M and 6M.Further,SFN also ameliorated renal inflammation,fibrosis,and oxidative stress injury caused by lipotoxicity.At 6M,SFN can effectively reduce the level of 24-hour urine albumin and kidney hypertrophy.Regarding the mechanism,SFN prevents T2D-induced nephropathy via AMPK-mediated activation of lipid metabolism pathways and Nrf2.However,these protective effects of SFN were not achieved in Ampkα2-/-mice.This suggests that both the regulation of lipid metabolism and the activation of the Nrf2 antioxidant system by SFN are dependent on the AMPKα2 subunit.Additionally,we also proved that the activation of Nrf2 is achieved through the protein kinase B(AKT)/glycogen synthase kinase-3β(GSK-3β)/src family tyrosine kinase(Fyn)signaling pathway mediated by activated AMPK.Conclusions:(1)SFN protects T2D-induced nephropathy by improving renal lipid metabolism and activating the Nrf2-mediated antioxidant pathway,which are both dependent on AMPKα2;(2)the activation of Nrf2 by SFN is achieved through AKT/GSK-3β/Fyn pathway mediated by AMPKα2;(3)the renal protection mediated by SFN continued to persist even after SFN was stopped for 3 months.