Diabetic nephropathy is one of the major causes of renal failure, which is accompanied by the production of reactive oxygen species (ROS). Nrf2 is the primary transcription factor that controls the antioxidant response essential for maintaining cellular redox homeostasis. Here, we report our findings demonstrating a protective role of Nrf2 against diabetic nephropathy.
RESEARCH DESIGN AND METHODS
We explore the protective role of Nrf2 against diabetic nephropathy using human kidney biopsy tissues from diabetic nephropathy patients, a streptozotocin-induced diabetic nephropathy model in Nrf2−/− mice, and cultured human mesangial cells.
The glomeruli of human diabetic nephropathy patients were under oxidative stress and had elevated Nrf2 levels. In the animal study, Nrf2 was demonstrated to be crucial in ameliorating streptozotocin-induced renal damage. This is evident by Nrf2−/− mice having higher ROS production and suffering from greater oxidative DNA damage and renal injury compared with Nrf2+/+ mice. Mechanistic studies in both in vivo and in vitro systems showed that the Nrf2-mediated protection against diabetic nephropathy is, at least, partially through inhibition of transforming growth factor-β1 (TGF-β1) and reduction of extracellular matrix production. In human renal mesangial cells, high glucose induced ROS production and activated expression of Nrf2 and its downstream genes. Furthermore, activation or overexpression of Nrf2 inhibited the promoter activity of TGF-β1 in a dose-dependent manner, whereas knockdown of Nrf2 by siRNA enhanced TGF-β1 transcription and fibronectin production.
This work clearly indicates a protective role of Nrf2 in diabetic nephropathy, suggesting that dietary or therapeutic activation of Nrf2 could be used as a strategy to prevent or slow down the progression of diabetic nephropathy.
Diabetic nephropathy (DN) is one of the most common causes of end-stage renal failure. The pathogenesis of progressive renal damage is multifactorial and the mechanism by which hyperglycemia causes microangiopathy in diabetic glomeruli is still poorly understood. Because the renin angiotensin system has been reported to be an important contributory factor in the pathophysiology of DN, exogenous administration of angiotensin II receptor antagonist may be beneficial in counteracting some biochemical or functional changes of DN.
The present study was therefore undertaken to evaluate the preventive role of irbesartan in streptozotocin (STZ)-induced DN in rats.
Methods and material:
STZ-induced DN in rats was assessed biochemically by measuring urine volume, protein and electrolytes as well as blood urea and creatinine clearance.
Marked hyperglycemia, polyuria, proteinuria and uremia along with a reduction in urine electrolytes and creatinine clearance were observed in STZ diabetic rats. Pre-treatment with irbesartan (20 mg/kg, p.o. 5 days prior to STZ and continued for 16 weeks) also significantly altered these parameters towards normal, except blood glucose.
Pre-treatment with insulin reversed the parameters of DN. The data suggest that irbesartan prevents the development of STZ-induced DN in rats.
Diabetic nephropathy; irbesartan; streptozotocin
Xiao-Chai-Hu-Tang (XCHT), a traditional Chinese medicine formula consisting of seven medicinal plants, is used in the treatment of various diseases. We show here that XCHT could protect type-1 diabetic mice against diabetic nephropathy, using streptozotocin (STZ)-induced diabetic mice and high-glucose (HG)-exposed rat mesangial cell (RMC) as models. Following 4 weeks of oral administration with XCHT, renal functions and renal hypertrophy significantly improved in the STZ-diabetic mice, while serum glucose was only moderately reduced compared to vehicle treatment. Treatment with XCHT in the STZ-diabetic mice and HG-exposed RMC resulted in a decrease in expression levels of TGF-β1, fibronectin, and collagen IV, with concomitant increase in BMP-7 expression. Data from DPPH assay, DHE stain, and CM-H2DCFDA analysis indicated that XCHT could scavenge free radicals and inhibit high-glucose-induced ROS in RMCs. Taken together, these results suggest that treatment with XCHT can improve renal functions in STZ-diabetic mice, an effect that is potentially mediated through decreasing oxidative stress and production of TGF-β1, fibronectin, and collagen IV in the kidney during development of diabetic nephropathy. XCHT, therefore merits further investigation for application to improve renal functions in diabetic disorders.
Diabetic cardiomyopathy and nephropathy are two major causes of death of patients with diabetes. Extra generation of reactive oxygen species (ROS), induced by hyperglycemia, is considered as the main reason for the development of these diabetic complications. Transcription factor, NFE2-related factor 2 (Nrf2), is a master regulator of cellular detoxification response and redox status, and also provides a protective action from various oxidative stresses and damages. Recently we have demonstrated its important role in determining the susceptibility of cells or tissues to diabetes-induced oxidative stress and/or damage. Therefore, this review will specifically summarize the information available regarding the effect of Nrf2 on the diabetic complications with a focus on diabetic cardiomyopathy and nephropathy. Given the feature that Nrf2 is easily induced by several compounds, we also discussed the role of different Nrf2 activators in the prevention or therapy of various diabetic complications. These findings suggest that Nrf2 has a potential application in the clinic setting for diabetic patients in the short future.
Key features of diabetic nephropathy include the accumulation of extracellular matrix proteins. In recent studies, increased expression of type VIII collagen in the glomeruli and tubulointerstitium of diabetic kidneys has been noted. The objectives of this study were to assess whether type VIII collagen affects the development of diabetic nephropathy and to determine type VIII collagen–dependent pathways in diabetic nephropathy in the mouse model of streptozotocin (STZ)-induced diabetes.
RESEARCH DESIGN AND METHODS
Diabetes was induced by STZ injections in collagen VIII–deficient or wild-type mice. Functional and histological analyses were performed 40 days after induction of diabetes. Type VIII collagen expression was assessed by Northern blots, immunohistochemistry, and real-time PCR. Proliferation of primary mesangial cells was measured by thymidine incorporation and direct cell counting. Expression of phosphorylated extracellular signal–regulated kinase (ERK1/2) and p27Kip1 was assessed by Western blots. Finally, Col8a1 was stably overexpressed in mesangial cells.
Diabetic wild-type mice showed a strong renal induction of type VIII collagen. Diabetic Col8a1−/Col8a2− animals revealed reduced mesangial expansion and cellularity and extracellular matrix expansion compared with the wild type. These were associated with less albuminuria. High-glucose medium as well as various cytokines induced Col8a1 in cultured mesangial cells. Col8a1−/Col8a2− mesangial cells revealed decreased proliferation, less phosphorylation of Erk1/2, and increased p27Kip1 expression. Overexpression of Col8a1 in mesangial cells induced proliferation.
Lack of type VIII collagen confers renoprotection in diabetic nephropathy. One possible mechanism is that type VIII collagen permits and/or fosters mesangial cell proliferation in early diabetic nephropathy.
The present study was to investigate whether sulforaphane (SFN) can prevent diabetic nephropathy in type 1 diabetic mouse model induced by multiple low-dose streptozotocin. Diabetic and age-matched control mice were given SFN at 0.5 mg/kg body weight daily for 3 months. At the end of 3-month SFN treatment, the diabetic nephropathy, shown by renal inflammation, oxidative damage, fibrosis, and dysfunction, was significantly prevented along with an elevation of renal Nrf2 expression and transcription in diabetes/SFN group compared with diabetic group. However, this renal prevention by SFN was not seen when the 3-month SFN-treated diabetic mice were aged for additional 3 months without further SFN treatment. Nrf2-mediated renal protective effects in diabetes were evaluated in human renal tubular HK11 cells transfected with control and Nrf2 siRNA and treated with 27.5 mM mannitol or high glucose plus palmitate (300 μM). Blockade of Nrf2 expression completely abolished SFN prevention of the profibrotic effect induced by high glucose plus palmitate. These results support that renal Nrf2 expression and its transcription play important roles in SFN prevention of diabetes-induced renal damage. However, the SFN preventive effect on diabetes-induced renal pathogeneses is not sustained, suggesting the requirement of continual use of SFN for its sustained effect.
To determine 1) whether renal arginase activity or expression is increased in diabetes and 2) whether arginase plays a role in development of diabetic nephropathy (DN).
RESEARCH DESIGN AND METHODS
The impact of arginase activity and expression on renal damage was evaluated in spontaneously diabetic Ins2Akita mice and in streptozotocin (STZ)-induced diabetic Dilute Brown Agouti (DBA) and arginase-2–deficient mice (Arg2−/−).
Pharmacological blockade or genetic deficiency of arginase-2 conferred kidney protection in Ins2Akita mice or STZ-induced diabetic renal injury. Blocking arginases using S-(2-boronoethyl)-l-cysteine for 9 weeks in Ins2Akita mice or 6 weeks in STZ-induced diabetic DBA mice significantly attenuated albuminuria, the increase in blood urea nitrogen, histopathological changes, and kidney macrophage recruitment compared with vehicle-treated Ins2Akita mice. Furthermore, kidney arginase-2 expression increased in Ins2Akita mice compared with control. In contrast, arginase-1 expression was undetectable in kidneys under normal or diabetes conditions. Arg2−/− mice mimicked arginase blockade by reducing albuminuria after 6 and 18 weeks of STZ-induced diabetes. In wild-type mice, kidney arginase activity increased significantly after 6 and 18 weeks of STZ-induced diabetes but remained very low in STZ-diabetic Arg2−/− mice. The increase in kidney arginase activity was associated with a reduction in renal medullary blood flow in wild-type mice after 6 weeks of STZ-induced diabetes, an effect significantly attenuated in diabetic Arg2−/− mice.
These findings indicate that arginase-2 plays a major role in induction of diabetic renal injury and that blocking arginase-2 activity or expression could be a novel therapeutic approach for treatment of DN.
Vascular endothelial growth factor A (VEGFA) expression is increased in glomeruli in the context of diabetes. Here, we tested the hypothesis that this upregulation of VEGFA protects the glomerular microvasculature in diabetes and that therefore inhibition of VEGFA will accelerate nephropathy. To determine the role of glomerular Vegfa in the development and progression of diabetic nephropathy, we used an inducible Cre-loxP gene-targeting system that enabled genetic deletion of Vegfa selectively from glomerular podocytes of wild-type or diabetic mice. Type 1 diabetes was induced in mice using streptozotocin (STZ). We then assessed the extent of glomerular dysfunction by measuring proteinuria, glomerular pathology, and glomerular cell apoptosis. Vegfa expression increased in podocytes in the STZ model of diabetes. After 7 weeks of diabetes, diabetic mice lacking Vegfa in podocytes exhibited significantly greater proteinuria with profound glomerular scarring and increased apoptosis compared with control mice with diabetes or Vegfa deletion without diabetes. Reduced local production of glomerular Vegfa in a mouse model of type 1 diabetes promotes endothelial injury accelerating the progression of glomerular injury. These results suggest that upregulation of VEGFA in diabetic kidneys protects the microvasculature from injury and that reduction of VEGFA in diabetes may be harmful.
Human and animal studies support the idea that there are sex differences in the development of diabetic renal disease. Our lab and others have determined that in addition to Ang II (through the AT1R), growth hormone (GH) contributes to renal damage in models of renal failure; however, the impact of sex and GH on the mechanisms initiating diabetic renal disease is not known. This study examined the effect of sex and GH on parameters of renal damage in early, uncontrolled streptozotocin (STZ)-induced diabetes.
Adult male and female Sprague–Dawley rats were injected with vehicle (control), STZ, or STZ + GH and euthanized after 8 weeks.
Mild but significant glomerulosclerosis (GS) and tubulointerstitial fibrosis (TIF) was observed in both kidneys from male and female diabetic rats, with GH significantly increasing GS and TIF by 30% and 25% in male rats, but not in female rats. STZ increased TGF-β expression in both kidneys from male and female rats; however, while GH had no further effect on TGF-β protein in diabetic females, GH increased TGF-β protein in the male rat’s kidneys by an additional 30%. This sex-specific increase in renal injury following GH treatment was marked by increased MCP-1 and CD-68+ cell density. STZ also reduced renal MMP-2 and MMP-9 protein expression in both kidneys from male and female rats, but additional decreases were only observed in GH-treated diabetic male rats. The sex differences were independent of AT1R activity.
These studies indicate that GH affects renal injury in diabetes in a sex-specific manner and is associated with an increase in pro-inflammatory mediators.
Sex Differences; Gender; Glomerulosclerosis; Renal Injury; TGF-β; Inflammation
Effect of irbesartan, an angiotensin II receptor antagonist, was studied in streptozotocin (STZ) induced diabetic nephropathy. Polyuria, proteinuria, blood urea, creatinine clearance, and urinary electrolytes were determined to assess kidney damage. There was a significant increase in urine volume, urinary protein and blood urea in STZ induced diabetic rats. On the other hand, irbesartan treatment resulted in a significant reduction in urinary protein and blood urea in these rats. Irbesartan treatment also improved creatinine clearance and exhibited a natriuretic effect in these animals. Results suggest that irbesartan treatment ameliorate STZ induced diabetic nephropathic changes, in rats.
Irbesartan; Diabetic nephropathy; Streptozotocin
Diabetes in humans induces chronic complications such as cardiovascular damage, cataracts and retinopathy, nephropathy and polyneuropathy. The most common animal model of human diabetes is streptozotocin (STZ)-induced diabetes in the rat. The present study investigated the effects of Nigella sativa hydroalcholic extract on glucose concentrations in streptozotocin (STZ) diabetic rats.
In this study Twenty-five Wister-Albino rats (aged 8-9 weeks and weighing 200-250 g) were tested. Rats were divided into five experimental groups (control, untreated STZ-diabetic (60 mg/kg B.W., IP), treated STZ-diabetic with hydroalcholic extract of Nigella Sativa (NS) (5 mg/kg B.W, IP), treated STZ-diabetic with hydroalcholic extract of NS (10 mg/kg B.W., IP) and treated STZ-diabetic with hydroalcholic extract of NS (20 mg/kg B.W., IP and 32 days were evaluated to assess its effect on fasting blood glucose (FBG), and in different groups fasting blood glucose (FBG) and body weight (BW) were measured in the particular days (1, 16 and 32). At the end of the study, the animals were fasted overnight, anaesthetized with an intraperitoneal injection of sodium pentobarbital (60 mg/kg), and sacrificed for obtaining tissues samples (liver, pancreases). The number of islets and cells were counted and the islet diameters were determined by calibrated micrometer. The glycogen content in the liver was examined by Periodic Acid-Schiff (PAS) staining.
Treatment with NS (5 mg/kg b.w.) markedly increased BW gain and the FBG level was significantly (p<0.001) reduced when compared to the control. Histopathological examination showed that the NS (5 mg/kg b.w.) partially recovered hepatic glycogen content and protected the great deal of the pancreatic islet cells. The number of islets, cells and islets diameter were found statistically significant when compared to the control (p<0.01, p<0.05).
Higher doses of NS did not exhibit any therapeutic effect. These results showed that hydroalcholic extract of NS at low doses has hypoglycemic effect and ameliorative effect on regeneration of pancreatic islets and may be used as a therapeutic agent in the management of diabetes mellitus. The hypoglycemic effect observed could be due to amelioration of β-cell, thus leading to increased insulin levels. Consequently, N. sativa may prove clinically useful in the treatment of diabetics and in the protection of β-cells against streptozotocin.
The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1845133011104231
Nigella Sativa; Streptozotocin; Hypoglycemic; Rat
Diabetic nephropathy (DN) is one of the most common causes of end-stage renal failure. This study was performed to determine the effect of Dantonic Pill (DP) treatment on β-catenin expression in a rat model of streptozotocin- (STZ-) induced early-stage DN, with irbesartan treatment as a positive control. Including an analysis of the general metabolic index and renal function, immunohistochemical staining and reverse transcription real-time PCR for β-catenin were performed in the renal cortex of the rat models every 4 weeks. After the treatments of DP and irbesartan, the albuminuria level, kidney weight/body weight, and thickness of the glomerular basement membrane were decreased, but the expression of β-catenin was not downregulated in the renal cortex. The effective drug target of DP to ameliorate albuminuria and renal hypertrophy should not inhibit the upregulated expression of β-catenin in rats with STZ-induced early-stage diabetic damage.
Diabetic nephropathy is a complex and poorly understood disease process, and our current treatment options are limited. It remains critical, then, to identify novel therapeutic targets. Recently, a developmental protein and one of the bone morphogenetic protein antagonists, Gremlin, has emerged as a novel modulator of diabetic nephropathy. The high expression and strong co-localization with transforming growth factor- β1 in diabetic kidneys suggests a role for Gremlin in the pathogenesis of diabetic nephropathy. We have constructed a gremlin siRNA plasmid and have examined the effect of Gremlin inhibition on the progression of diabetic nephropathy in a mouse model. CD-1 mice underwent uninephrectomy and STZ treatment prior to receiving weekly injections of the plasmid. Inhibition of Gremlin alleviated proteinuria and renal collagen IV accumulation 12 weeks after the STZ injection and inhibited renal cell proliferation and apoptosis. In vitro experiments, using mouse mesangial cells, revealed that the transfect ion of gremlin siRNA plasmid reversed high glucose induced abnormalities, such as increased cell proliferation and apoptosis and increased collagen IV production. The decreased matrix metalloprotease level was partially normalized by transfection with gremlin siRNA plasmid. Additionally, we observed recovery of bone morphogenetic protein-7 signaling activity, evidenced by increases in phosphorylated Smad 5 protein levels. We conclude that inhibition of Gremlin exerts beneficial effects on the diabetic kidney mainly through maintenance of BMP-7 activity and that Gremlin may serve as a novel therapeutic target in the management of diabetic nephropathy.
Hyperglycemia induced reactive oxygen species (ROS) generation is believed as major factors leading to diabetic nephropathy (DN). DangGui (Angelica sinensis) is mentioned to show renal protective effect in combination with other herbs. Bone morphogenetic proteins-7 (BMP-7) is produced merit in protection of DN. The role of BMP-7 in DangGui-induced renal improvement is not clear. The present study investigated the effects of DangGui on renal functions, BMP-7 expression and the levels of ROS in streptozotocin (STZ)-induced diabetic rats and high glucose-exposed rat mesangial cells (RMCs). After 1- or 4-week treatment, DangGui improved renal functions and increased renal BMP-7 expression in diabetic rats. The BMP-7 expression in RMCs was reduced by high glucose treatment and this could be reversed by DangGui. Moreover, RMCs exposed to high glucose were expired by BMP-7 RNAi transfection but those cells remained alive by scramble transfection. Thus, we employed regular RMCs to knock down BMP-7 with RNAi and we found that DangGui increased BMP-7 expression in these RMCs. Direct activation of BMP-7 expression by DangGui could be considered. The results of DPPH assay, DHE stain and lucigenin assay indicated that DangGui could inhibit high glucose-induced ROS in RMCs. These results suggest that DangGui has an ability to improve renal functions in STZ-diabetic rats through increasing endogenous BMP-7 expression and decreasing oxidative stress in kidney. The present study suggest that DangGui could be applied to improve renal functions in diabetic disorders.
Background: Diabetic nephropathy (DN) is one of the most common chronic complications of diabetes and the leading cause of end-stage renal disease. Recent research has found that oxidative stress participates in the development of diabetic nephropathy. α-lipoic acid (α-LA), a powerful antioxidant, plays an important role in renal protection against DN, but the underlying mechanism remains unknown. This study modeled the renal protective effects of α-lipoic acid in streptozotocin (STZ) induced diabetic rats and explore the underlying mechanism, which provides new theoretical bases for clinical treatment of diabetic nephropathy. Methods: The diabetic model was induced by intraperitoneal injection of STZ on Male SD and then the diabetic rats were randomly divided into two groups: untreated-diabetic group (DM group), α-LA treated-diabetic group (α-LA group), and the normal rats served as control group (NC group). After 8 weeks of STZ induction, Blood glucose (BG), Blood Urea Nitrogen (BUN), Serum Creatinine (SCr) and urinary albumin excretion rate (UAER) were examined, and morphological changes were assessed by histology. The levels of malondialdehyde (MDA) and the activities of superoxide dismutase (SOD) were also evaluated in serum and renal cortex. Additionally, kidney mitochondrial membrane potential and mitochondrial swelling were measured for different groups. The expression of voltage-dependent anion channel (VDAC) on mitochondria were evaluated by both Western blotting and Immunohistochemistry. Results: After 8 weeks induction of STZ, significant reductions in BUN, SCr, UAER (P<0.01 or P<0.05) and histological improvement were observed in the α-LA group compared to the DM group. In the serum and renal cortex of α-LA group, the content of MDA and the activities of SOC were both significantly decreased (P<0.05). Compared to the DM group, the mitochondrial membrane potential in the α-LA group was significantly increased (P<0.05) and mitochondrial swelling was reduced. Meanwhile, the expression of VDAC on mitochondrial was significantly increased (P<0.05) in the α-LA group. Conclusion: Our findings indicate that antioxidant α-LA exerts a protective role against the development of DN, and the underlying mechanism may involve effective suppression of the generation of oxidants, protection of mitochondrial function, and up-regulating of VDAC expression.
Diabetic nephropathy; reactive oxygen species; mitochondria; voltage-dependent anion channel; α-lipoic acid
Diabetes mellitus affects the brain. Both type 1 and type 2 diabetic patients are associated with white matter (WM) damage observable to diffusion tensor imaging (DTI). The underlying histopathological mechanisms, however, are poorly understood. The objectives of this study are 1) to determine whether streptozotocin (STZ)-induced type 1 diabetes is associated with WM damage observable to DTI; and 2) to understand the pathophysiological aspects underlying STZ-induced brain injuries. Male Sprague–Dawley rats received a single intraperitoneal injection of STZ (62 mg/kg). DTI was used to assess brain abnormalities at 4 weeks after induction, combined with histological assessments and ultrastructural analysis. Compared to controls, the STZ-induced rats showed significantly reduced fractional anisotropy (FA) in the motor/somatosensory cortex and striatum. Histologically, the cortex and striatum of the diabetic animals are characterized by demyelination and axonal degradation. In conclusion, STZ-induced diabetes is associated with striatal/cortical injuries observable to DTI. The DTI abnormalities are likely manifestations of demyelination and axonal degradation in the affected brain regions, and can potentially be used as surrogates for evaluating diabetic brain injuries.
Chronic inflammation plays an important role in the progression of diabetic nephropathy (DN) and that the infiltration of macrophages in glomerulus has been implicated in the development of glomerular injury. We hypothesized that the plant polyphenolic compound curcumin, which is known to exert potent anti-inflammatory effect, would ameliorate macrophage infiltration in streptozotocin (STZ)-induced diabetic rats.
Diabetes was induced with STZ (55 mg/kg) by intraperitoneal injection in rats. Three weeks after STZ injection, rats were divided into three groups, namely, control, diabetic, and diabetic treated with curcumin at 100 mg/kg/day, p.o., for 8 weeks. The rats were sacrificed 11 weeks after induction of diabetes. The excised kidney was used to assess macrophage infiltration and expression of various inflammatory markers.
At 11 weeks after STZ injection, diabetic rats exhibited renal dysfunction, as evidenced by reduced creatinine clearance, increased blood glucose, blood urea nitrogen and proteinuria, along with marked reduction in the body weight. All of these abnormalities were significantly reversed by curcumin. Hyperglycemia induced the degradation of IκBα and NF-κB activation and as a result increased infiltration of macrophages (52%) as well as increased proinflammatory cytokines: TNF-α and IL-1β. Curcumin treatment significantly reduced macrophage infiltration in the kidneys of diabetic rats, suppressed the expression of above proinflammatory cytokines and degradation of IκBα. In addition, curcumin treatment also markedly decreased ICAM-1, MCP-1 and TGF-β1 protein expression. Moreover, at nuclear level curcumin inhibited the NF-κB activity.
Our results suggested that curcumin treatment protect against the development of DN in rats by reducing macrophage infiltration through the inhibition of NF-κB activation in STZ-induced diabetic rats.
Oxidative stress plays an important role in diabetes-induced vascular inflammation and pathogenesis. Nuclear factor E2-related factor-2 (Nrf2) is a transcription factor orchestrating antioxidant and cyto-protective responses to oxidative stress. In the present study, we tested whether sulforaphane (SFN) can protect the aorta from diabetes and, if so, whether the aortic protection is associated with up-regulation of Nrf2 and its down-stream antioxidants.
Type 1 diabetes was induced in FVB mice by multiple low-dose streptozotocin. Diabetic and age-matched control mice were treated with or without SFN at 0.5 mg/kg daily in five days of each week for three months. At the end of 3 months treatment of SFN one set of mice were sacrificed to perform the experimental measurements. The second set of both diabetic and control mice were aged for additional 3 months without further SFN treatment and then sacrificed to perform the experimental measurements. Aortas from these mice were assessed for fibrosis, inflammation, oxidative damage, and Nrf2 expression and transcription by immunohistochemical staining and real-time PCR method, respectively.
Diabetes induced significant increases in oxidative stress and inflammation in the aorta at both 3 and 6 months, and fibrotic response at 6 months. SFN completely prevented these diabetic pathogenic changes and also significantly up-regulated the expression of Nrf2 and its down-stream antioxidants.
These results suggest that diabetes-induced aortic fibrosis, inflammation, and oxidative damage can be prevented by SFN. The aortic protection from diabetes by SFN was associated with the up-regulation of Nrf2 and its downstream antioxidants.
Sulforaphane; Nrf2; aorta; Oxidative damage; Vascular inflammation
Sulforaphane-induced activation of the transcription factor NF-E2 related factor 2 (Nrf2 or the gene Nfe2l2) and subsequent induction of the phase II antioxidant system has previously been shown to exert neuroprotective action in a transient model of focal cerebral ischemia. However, its ability to attenuate functional and cellular deficits after permanent focal cerebral ischemia is not clear. We assessed the neuroprotective effects of sulforaphane in the photothrombotic model of permanent focal cerebral ischemia. Sulforaphane was administered (5 or 50 mg/kg, i.p.) after ischemic onset either as a single dose or as daily doses for 3 days. Sulforaphane increased transcription of Nrf2, Hmox1, GCLC and GSTA4 mRNA in the brain confirming activation of the Nrf2 system. Single or repeated administration of sulforaphane had no effect on the infarct volume, nor did it reduce the number of activated glial cells or proliferating cells when analyzed 24 and 72 h after stroke. Motor-function as assessed by beam-walking, cylinder-test, and adhesive test, did not improve after sulforaphane treatment. The results show that sulforaphane treatment initiated after photothrombosis-induced permanent cerebral ischemia does not interfere with key cellular mechanisms underlying tissue damage.
Cellular oxidative and electrophilic stress triggers a protective response in mammals regulated by NRF2 (nuclear factor (erythroid-derived) 2-like; NFE2L2) binding to deoxyribonucleic acid-regulatory sequences near stress-responsive genes. Studies using Nrf2-deficient mice suggest that hundreds of genes may be regulated by NRF2. To identify human NRF2-regulated genes, we conducted chromatin immunoprecipitation (ChIP)-sequencing experiments in lymphoid cells treated with the dietary isothiocyanate, sulforaphane (SFN) and carried out follow-up biological experiments on candidates. We found 242 high confidence, NRF2-bound genomic regions and 96% of these regions contained NRF2-regulatory sequence motifs. The majority of binding sites were near potential novel members of the NRF2 pathway. Validation of selected candidate genes using parallel ChIP techniques and in NRF2-silenced cell lines indicated that the expression of about two-thirds of the candidates are likely to be directly NRF2-dependent including retinoid X receptor alpha (RXRA). NRF2 regulation of RXRA has implications for response to retinoid treatments and adipogenesis. In mouse, 3T3-L1 cells’ SFN treatment affected Rxra expression early in adipogenesis, and knockdown of Nrf2-delayed Rxra expression, both leading to impaired adipogenesis.
Diabetes in humans and in experimental animals
produces changes in the function and structure of the
small intestine. The authors determined the activity of
intestinal disaccharidases (maltase and sucrase) and of
6-phosphofructo-1-kinase (PFK-1) in enterocytes isolated
from the small intestine of male Wistar rats (2.5 to 3 months
old) with experimental nonobese type 2 diabetes, induced by
streptozotocin (STZ) injection on the day of birth (n0-STZ)
or on the 5th day of life (n5-STZ), with different degrees
of hyperglycemia and insulinemia (n0-STZ and n5-STZ
models). The glycemia (mmol/L) of the diabetic rats
(n0-STZ: 8.77 ± 0.47; n5-STZ: 20.83 ± 0.63) was higher
(P < .01) than that of the nondiabetic (ND) rats
(5.99 ± 0.63); on the contrary, the insulinemia (ng/mL) was
significantly lower in both n0-STZ (1.74 ± 0.53; P < .05)
and n5-STZ (1.12 ± 0.44; P < .01) diabetic rats than in normal
rats (3.77 ± 0.22). The sucrase and maltase activities
(U/g protein) in diabetic rats (n0-STZ: 89 ± 9 and 266 ± 12;
n5-STZ: 142 ± 23 and 451 ± 57) were significantly higher
than those in the ND group (66 ± 5 and 228 ± 22). The
PFK-1 activities (mU/mg protein) in the diabetic models
(n0-STZ: 14.89 ± 1.51; n5-STZ: 13.35 ± 3.12) were significantly
lower (P < .05) than in ND rats (20.54 ± 2.83). The
data demonstrated enzymatic alterations in enterocytes isolated
fromthe small intestine of n0-STZ rats that are greater
(P < .05) than in the more hyperglycemic and hypoinsulinemic
n5-STZ animals. The results also show that nonobese type 2–like diabetes in the rat produces modifications that
favor an increase in glucose absorption rates.
Repetitive exposure of diabetic mice to low-dose radiation (LDR) at 25 mGy could significantly attenuate diabetes-induced renal inflammation, oxidative damage, remodeling, and dysfunction, for which, however, the underlying mechanism remained unknown. The present study explored the effects of LDR on the expression and function of Akt and Nrf2 in the kidney of diabetic mice. C57BL/6J mice were used to induce type 1 diabetes with multiple low-dose streptozotocin. Diabetic and age-matched control mice were irradiated with whole body X-rays at either single 25 mGy and 75 mGy or accumulated 75 mGy (25 mGy daily for 3 days) and then sacrificed at 1–12 h for examining renal Akt phosphorylation and Nrf2 expression and function. We found that 75 mGy of X-rays can stimulate Akt signaling pathway and upregulate Nrf2 expression and function in diabetic kidneys; single exposure of 25 mGy did not, but three exposures to 25 mGy of X-rays could offer a similar effect as single exposure to 75 mGy on the stimulation of Akt phosphorylation and the upregulation of Nrf2 expression and transcription function. These results suggest that single 75 mGy or multiple 25 mGy of X-rays can stimulate Akt phosphorylation and upregulate Nrf2 expression and function, which may explain the prevention of LDR against the diabetic nephropathy mentioned above.
The pathogenesis of diabetic nephropathy is complex and involves activation of multiple pathways leading to kidney damage. An important role for altered lipid metabolism via sterol regulatory element binding proteins (SREBPs) has been recently recognized in diabetic kidney disease. Our previous studies have shown that the farnesoid X receptor (FXR), a bile acid-activated nuclear hormone receptor, modulates renal SREBP-1 expression. The purpose of the present study was then to determine if FXR deficiency accelerates type 1 diabetic nephropathy in part by further stimulation of SREBPs and related pathways, and conversely, if a selective FXR agonist can prevent the development of type 1 diabetic nephropathy.
RESEARCH DESIGN AND METHODS
Insulin deficiency and hyperglycemia were induced with streptozotocin (STZ) in C57BL/6 FXR KO mice. Progress of renal injury was compared with nephropathy-resistant wild-type C57BL/6 mice given STZ. DBA/2J mice with STZ-induced hyperglycemia were treated with the selective FXR agonist INT-747 for 12 weeks. To accelerate disease progression, all mice were placed on the Western diet after hyperglycemia development.
The present study demonstrates accelerated renal injury in diabetic FXR KO mice. In contrast, treatment with the FXR agonist INT-747 improves renal injury by decreasing proteinuria, glomerulosclerosis, and tubulointerstitial fibrosis, and modulating renal lipid metabolism, macrophage infiltration, and renal expression of SREBPs, profibrotic growth factors, and oxidative stress enzymes in the diabetic DBA/2J strain.
Our findings indicate a critical role for FXR in the development of diabetic nephropathy and show that FXR activation prevents nephropathy in type 1 diabetes.
Decrease in endogenous hydrogen sulfide (H2S) was reported to participate in the pathogenesis of diabetic nephropathy (DN). This study is aimed at exploring the relationship between the abnormalities in H2S metabolism, hyperglycemia-induced oxidative stress and the activation of intrarenal renin-angiotensin system (RAS). Cultured renal mesangial cells (MCs) and streptozotocin (STZ) induced diabetic rats were used for the studies. The expressions of angiotensinogen (AGT), angiotensin converting enzyme (ACE), angiotensin II (Ang II) type I receptor (AT1), transforming growth factor-β1 (TGF-β1) and collagen IV were measured by real time PCR and Western blot. Reactive oxygen species (ROS) production was assessed by fluorescent probe assays. Cell proliferation was analyzed by 5'-bromo-2'-deoxyuridine incorporation assay. Ang II concentration was measured by an enzyme immunoassay. AGT, ACE and AT1 receptor mRNA levels and Ang II concentration were increased in high glucose (HG) -treated MCs, the cell proliferation rate and the production of TGF-β1 and of collagen IV productions were also increased. The NADPH oxidase inhibitor diphenylenechloride iodonium (DPI) was able to reverse the HG-induced RAS activation and the changes in cell proliferation and collagen synthesis. Supplementation of H2S attenuated HG-induced elevations in ROS and RAS activation. Blockade on H2S biosynthesis from cystathione-γ-lyase (CSE) by DL-propargylglycine (PPG) resulted in effects similar to that of HG treatment. In STZ-induced diabetic rats, the changes in RAS were also reversed by H2S supplementation without affecting blood glucose concentration. These data suggested that the decrease in H2S under hyperglycemic condition leads to an imbalance between oxidative and reductive species. The increased oxidative species results in intrarenal RAS activation, which, in turn, contributes to the pathogenesis of renal dysfunction.
Transcription factor Nrf2 is considered a master regulator of antioxidant defense in mammals. However, it is unclear whether this concept is applicable to nonmammalian vertebrates, because no animal model other than Nrf2 knockout mice has been generated to examine the effects of Nrf2 deficiency. Here, we characterized a recessive loss-of-function mutant of Nrf2 (nrf2fh318) in a lower vertebrate, the zebrafish (Danio rerio). In keeping with the findings in the mouse model, nrf2fh318 mutants exhibited reduced induction of the Nrf2 target genes in response to oxidative stress and electrophiles but were viable and fertile, and their embryos developed normally. The nrf2fh318 larvae displayed enhanced sensitivity to oxidative stress and electrophiles, especially peroxides, and pretreatment with an Nrf2-activating compound, sulforaphane, decreased peroxide-induced lethality in the wild type but not nrf2fh318 mutants, indicating that resistance to oxidative stress is highly dependent on Nrf2 functions. These results reveal an evolutionarily conserved role of vertebrate Nrf2 in protection against oxidative stress. Interestingly, there were no significant differences between wild-type and nrf2fh318 larvae with regard to their sensitivity to superoxide and singlet oxygen generators, suggesting that the importance of Nrf2 in oxidative stress protection varies based on the type of reactive oxygen species (ROS).