Nrf2-ARE Signaling Pathway Protect Diabetic Kidney Via Regulator Antioxidant Gene Expression

Objective: Diabetic nephropathy (DN) is one of the most common complications and the leading cause of mortality and morbidity of diabetes mellitus. The pathologic manifestations of DN include glomerular hypertrophy, basement membrane thickening, mesangial dilation, and glomerularsclerosis and interstitial fibrosis in the terminal stage. The excessive accumulation of extracellular matrix (ECM) is the common pathophysiological foundation. Recent studies showde that the oxidative stress plays an important role in the pathogenesis of DN. The excessive production of reactive oxygen species (ROS) not only stimulate all signaling pathways known to be associated with diabetic microvascular complications including DN, such as PKC pathway, polyol pathway, hexosamine pathway and AGEs formation, but also activate NF-κB and upregulate the transcription of adhesive molecule and inflammatory factors. That is the common mechanism of diabetic microvascular complications including DN. Persistent existence of those abnormal changes will increase the synthesis of mesangial matrix and basement membrane, decrease degradation of them, thus leading to the development and progression of DNOrganism has developed a complex response system to the oxidative stress induced by ROS damage. When exposed to ROS, cells can produce a series of protective proteins to mitigate cellular injury. This coordinated response is controlled by antioxidant responsive element (ARE) in the upstream of protective genes. More recently, nuclear factor erythroid 2-related factor 2 (Nrf2) had been found to be the activator of ARE. The Nrf2-ARE signaling pathway has been proved to be the key regulator of antioxidant and cytoprotective proteins. Protective effect of Nrf2-ARE signal pathway to anti- environment stress, anti-apoptosis, neuro-protection, anti-carcinoma and antii-ageing in vivo and in vitro have been reported, but little is known about the protective role of Nrf2 in DN.Disequilibrium between oxidation and reduction systems is pathophysiological foundation of DN. Some studies indicated that activation of antioxidant protein is effective strategies for the management of DN. The present study try to clarify whether Nrf2-ARE is related to the oxidative stress in DN, and whether diabetic renal injury can be ameliorated by stimulating Nrf2-ARE, in order to provide experimental basis for prevention and trentment of DN.Methods:1 Induction of diabetic model and examination of relative parameters.Uninephrectomized male CD1 mice were randomly divided into two groups: Control group, DM group. The mice of DM group received a single intraperitoneal injection of STZ dissolved in 0.1mol/L citrate buffer (pH 4.5) at a dose of 130mg/kg. The mice of control group only received an injection of the same volume of 0.1mol/L sodium citrate. The model of diabetes was considered to be successful when the blood glucose was≥16.7mmol/L and the glucose in urine was+++～++++after 72 hours of the injection. Six mice of control or DM group were respectively sacrificed at the 4th and 12th weeks after STZ injection. At the end of the study, 24 h urines were collected in metabolic cage for the measurement of urine albumin (Ualb, at the 4th week ) and urine protein (Upro, at the 12th week); parameters of kidney function and lipid peroxidation marker malondialdehyde (MDA) were evaluated in serum samples .The renal cortex were removed and used for MDA content measurement, histopathology examination, immohistochemistry to detected the location and expression of Nrf2, HO-1,γ-GCS in renal cortex, and western blot to detected the expression of Nrf2(total and nucleus),HO-1,γ-GCS protein .2 Transient transfection of pcDNA3/mNrf2 plasmid into MMC cells cultured in high glucose medium and and examination of relative parameters.Mouse mesangial cells (MMC) were maintained at 37℃in a humidified atmosphere of 5% CO2 in DME-F12 medium containing penicillin/ streptomycin (100 U/ml and 100μg/ml, respectively) and 10% fetal bovine serum (FBS), with a normal D-glucose concentration of 5.5mM. Prior to use, cells at 70～80% confluence were incubated in serum-free medium for 12 hours. Transient transfection of MMC cells was carried out according to the Lipofectamine 2000 manufacturer's instruction. Six hours after transfection, the medium was replaced by normal DME-F12 medium with 10% FBS for 12 hours. MMC cells were randomly divided into 5 groups: Control group (media containing 5.5mM glucose), Mannitol control group (media containing 5.5mM glucose and 24.5mM mannitol), HG group (media containing 30mM glucose), HG+ pcDNA3 group (media containing 30mM glucose and transfected pcDNA3). HG+pcDNA3/mNrf2 group (media containing 30mM glucose and transfected pcDNA3/Nrf2).Cells were collected at 48 hours after high glucose stimulation. Immunocytochemistry were used to analyze expressed location of Nrf2 andγ-GCS. Western-blot were used to detected the expression of Nrf2(total and nucleus),HO-1,γ-GCS protein. Reverse transcription polymerse chain reaction (RT-PCR) were used to detected the expression of Nrf2,HO-1,γ-GCS mRNA. 2'7'-Dichloro-fluorescein diacetate (2'7'-DCFH-DA) and MDA were assessed as cellular ROS generation and lipid peroxidation. The contents of TGF-β1 in the supernatants were measured by Enzyme linked immunosorben assay (ELISA). Proliferation activity of mesangial cells were determined by flow cytometry and analyzed by DNA analysis software modeling program.3 Transient transfection of siRNA Nrf2(m) into MMC cells cultured in high glucose medium and examination of relative parameters.siRNA targeting mouse Nrf2 mRNA was used to silence Nrf2 gene expression. Transient transfection of MMC cells was carried out according to the Lipofectamine 2000 manufacturer's instruction. After optimizing transfection conditions, MMC cells were divided into 5 groups: Control group (media containing 5.5mM glucose), Mannitol control group (media containing 5.5mM glucose and 24.5mM mannitol), HG group (media containing 30mM glucose), HG+ siRNA control group (media containing 30mM glucose and transfected non-specific siRNA). HG+ siRNANrf2(m)group(media containing 30mM glucose and transfected siRNA Nrf2(m)). Cells were collected at 48 hours after high glucose stimulation. Exam methods of Nrf2,HO-1,γ-GCS expression and ROS generation ,lipid peroxidation , cell proliferation , TGF-β1 secretion in different groups were described as former.4 Treatment of diabetic mice with 1%tBHQ food and examination of relative parameters.Uninephrectomized male CD-1 mice were randomly assigned to three groups: Control group(C group); Diabetes mellitus group (DM group); tBHQ intervention group (DM+tBHQ group).The mice of DM group and DM+tBHQ group were induced by single-dose intraperitoneal injection of STZ.Mice of DM+tBHQ group were fed with food containing 1%tBHQ(w/w)three days after model establishment. Specimens were harvested at 4th and 12thweek after the model establishment. The glomeruli were separated by sieving method .Renal weight/body index, quantification of 24 hour urine albumin (of 4th week) and protein(of 12th week), MDA contents in the serum and glomerular homogenate of mice were evaluated as indication of ROS injury. The localization and expressions of Nrf2, HO-1 andγ-GCS proteins in the glomerulus were measured by immunohistochemical assay andwestern blot. Expressions of Nrf2, HO-1 andγ-GCS mRNA in the glomerulus were detected by RT-PCR. ECM deposition in the glomerulus were measured by HE and PAS staining, transmission electron microscopy examination, immunohistochemical assay of FN in the glomerulus.Results:1 The expression of Nrf2,HO-1,γ-GC in the renal tissue and relative to oxidative stress in the kidneys of STZ-induced diabetic mice①Compared with C group, renal weight/body index, 24h urine albumin quantification of 4th week and 24h urine protein quantification of 12th week in DM group were significantly higher.②The mice showed slightly enlarged glomeruli at 4th week , and the number of MMC cells was increased with glomeruli hypertrophy, thickened glomerular basement membrane and expanded mesangium matrix were observed in DM group at 12thweek vs C group by HE and PAS staining .③Immunohistochemistry staining indicated that Nrf2 protein was presented in both nuclear and cytoplasm of renal glomeruli cells; Nrf2 nucleus protein expressions were significantly higher than those in C group of the same time; HO-1 protein was mainly expressed in cytoplasm of renal glomeruli cells and the expression was significantly lower than that in C group at the 4th week but higher at the 12th week;γ-GCS protein presented in cytoplasm of renal glomeruli and tubular cells, and the protein expressions were significantly higher than those in C group of the same time.④MDA contents of the serum and renal cortex in DM group were significantly higher than those in C group at the corresponding time .⑤Western blot showed that: At the 4thweek, Nrf2 total protein expression in DM group was similar to that in C group; HO-1 expression was significantly lower than that in C group;γ-GCS and Nrf2 nucleus protein expressions were significantly higher than those in C group; At the 12th week, Nrf2 total protein expression of DM group was similar to that in C group; HO-1,γ-GCS and Nrf2 nucleus protein expressions were significantly higher than those in C group.2 The expression of Nrf2,HO-1,γ-GCS protein and effects of Nrf2-ARE on ROS generation,proliferation,TGF-β1 secretion of MMC cells cultured in high glucose medium.①MMC cells incubated in high-glucose medium resulted higher ROS production and MDA content in time-dependent manner than that incubated in normal-glucose medium.②Activation of Nrf2 by transfected pcDNA3/Nrf2(m) plasmid induced nuclear translocation of Nrf2 and increased ARE-linked gene HO-1,γ-GCS protein and mRNA expression after 48h-incubation in high-glucose media.This induction was markedly attenuated by pretreatment with siRNA Nrf2(m)③Immunochemistry showed increased Nrf2 andγ-GCS expression in cytoplasm meanwhile increased Nrf2 nuclear expression in transfected pcDNA3-Nrf2 plasmid group vs high-glucose stimulating group. This induction was markedly attenuated by pretreatment with siRNA Nrf2(m)④Hyperglycemia increased the formation of ROS, MDA, proliferation ,TGF-β1 secretion of MMC were markedly prevented by pcDNA3-Nrf2 plasmid transfection while this prevention were attenuated by knockdown of Nrf2 expression through siRNA Nrf2(m) transfection.3 tBHQ attenuates glomerular ROS injury of diabetic mice via activating Nrf2-ARE pathway①Renal weight/body index, 24h urine albumin quantification of the 4thweek and 24h urine protein quantification of the 12thweek in DM group were significantly higher than those in group C; and these parameters were markedly improved in DM+tBHQ group compared to those in DM group.②MDA contents of the serum and renal glomeruli homogenate in DM group were significantly higher than those in C group at the corresponding time, and these parameters decreased in the tBHQ intervention group.③Compared with group DM at corresponding time, expressions of glomerular total protein Nrf2, HO-1,γ-GCS, nuclear protein Nrf2 and Nrf2, HO-1,γ-GCS mRNA in the tBHQ intervention group increased significantly;④Transmission electron microscope at 4th week, HE and PAS staining at 12th week and semi-quantitative FN immunohistochemical assay showed that tBHQ intervention could reduce the ECM deposition in glomerulus of the diabetic mice.Conclusion: Our study showed that the renal tissues of STZ induced diabetic model presented ROS injury and elevation of Nrf2 levels. Nrf2-ARE signaling pathway and the downstream antioxidant protein HO-1 andγ-GCS play an important role in antioxidant mechanism of DN. tBHQ is able to ameliorate renal injury induced by ROS and reduce the ECM deposition in glomerulus of diabetic kidney, The mechanism may be associated with activating Nrf2-ARE signaling pathway and initiating the downstream genes HO-1 andγ-GCS transcription and expression. In the mesangial cells of mice, high glucose induced ROS production, and overexpression of Nrf2 by transient transfection of pcDNA3/Nrf2 plasmid activated expression of Nrf2 and its downstream antioxidant genes HO-1 andγ-GCS, inhibited ROS production, lipid peroxidation , cell proliferation and TGF-β1 secretion whereas knockdown of Nrf2 by siRNA displayed the adverse effects. The above results suggested that activation of Nrf2 could be an effective method to prevent and slow down the progression of DN in vivo and in vitro.