Allosteric inhibition of carnosinase (CN1) by inducing a conformational shift

In humans, low serum carnosinase (CN1) activity protects patients with type 2 diabetes from diabetic nephropathy. We now characterized the interaction of thiol-containing compounds with CN1 cysteine residue at position 102, which is important for CN1 activity. Reduced glutathione (GSH), N-acetylcysteine and cysteine (3.2?±?0.4, 2.0?±?0.3, 1.6?±?0.2?µmol/mg/h/mM; p?C102S) and 229 (Mut2^C229S) revealed that only cysteine-102 is influenced by cysteinylation. Molecular dynamic simulation confirmed a conformational rearrangement of negatively charged residues surrounding the zinc ions causing a partial shift of the carnosine ammonium head and resulting in a less effective pose of the substrate within the catalytic cavity and decreased activity. Cysteine-compounds influence the dynamic behaviour of CN1 and therefore present a promising option for the treatment of diabetes.     

Reduced muscle carnosine content in type 2, but not in type 1 diabetic patients

Carnosine is present in high concentrations in skeletal muscle where it contributes to acid buffering and functions also as a natural protector against oxidative and carbonyl stress. Animal studies have shown an anti-diabetic effect of carnosine supplementation. High carnosinase activity, the carnosine degrading enzyme in serum, is a risk factor for diabetic complications in humans. The aim of the present study was to compare the muscle carnosine concentration in diabetic subjects to the level in non-diabetics. Type 1 and 2 diabetic patients and matched healthy controls (total n=58) were included in the study. Muscle carnosine content was evaluated by proton magnetic resonance spectroscopy (3 Tesla) in soleus and gastrocnemius. Significantly lower carnosine content (-45%) in gastrocnemius muscle, but not in soleus, was shown in type 2 diabetic patients compared with controls. No differences were observed in type 1 diabetic patients. Type II diabetic patients display a reduced muscular carnosine content. A reduction in muscle carnosine concentration may be partially associated with defective mechanisms against oxidative, glycative and carbonyl stress in muscle.     

Low plasma carnosinase activity promotes carnosinemia after carnosine ingestion in humans

A polymorphism in the carnosine dipeptidase-1 gene (CNDP1), resulting in decreased plasma carnosinase activity, is associated with a reduced risk for diabetic nephropathy. Because carnosine, a natural scavenger/suppressor of ROS, advanced glycation end products, and reactive aldehydes, is readily degraded in blood by the highly active carnosinase enzyme, it has been postulated that low serum carnosinase activity might be advantageous to reduce diabetic complications. The aim of this study was to examine whether low carnosinase activity promotes circulating carnosine levels after carnosine supplementation in humans. Blood and urine were sampled in 25 healthy subjects after acute supplementation with 60 mg/kg body wt carnosine. Precooled EDTA-containing tubes were used for blood withdrawal, and plasma samples were immediately deproteinized and analyzed for carnosine and β-alanine by HPLC. CNDP1 genotype, baseline plasma carnosinase activity, and protein content were assessed. Upon carnosine ingestion, 8 of the 25 subjects (responders) displayed a measurable increase in plasma carnosine up to 1 h after supplementation. Subjects with no measurable increment in plasma carnosine (nonresponders) had ～2-fold higher plasma carnosinase protein content and ～1.5-fold higher activity compared with responders. Urinary carnosine recovery was 2.6-fold higher in responders versus nonresponders and was negatively dependent on both the activity and protein content of the plasma carnosinase enzyme. In conclusion, low plasma carnosinase activity promotes the presence of circulating carnosine upon an oral challenge. These data may further clarify the link among CNDP1 genotype, carnosinase, and diabetic nephropathy.     

Carnosine enhances diabetic wound healing in the db/db mouse model of type 2 diabetes

Diabetes mellitus (DM) is a progressive disorder with severe late complications. Normal wound healing involves a series of complex and well-orchestrated molecular events dictated by multiple factors. In diabetes, wound healing is grossly impaired due to defective, and dysregulated cellular and molecular events at all phases of wound healing resulting in chronic wounds that fail to heal. Carnosine, a dipeptide of alanine and histidine and an endogenous antioxidant is documented to accelerate healing of wounds and ulcers. However, not much is known about its role in wound healing in diabetes. Therefore, we studied the effect of carnosine in wound healing in db/db mice, a mice model of Type 2 DM. Six millimeter circular wounds were made in db/db mice and analyzed for wound healing every other day. Carnosine (100?mg/kg) was injected (I.P.) every day and also applied locally. Treatment with carnosine enhanced wound healing significantly, and wound tissue analysis showed increased expression of growth factors and cytokines genes involved in wound healing. In vitro studies with human dermal fibroblasts and microvascular-endothelial cells showed that carnosine increases cell viability in presence of high glucose. These effects, in addition to its known role as an antioxidant and a precursor for histamine synthesis, provide evidence for a possible therapeutic use of carnosine in diabetic wound healing.     

Relevance of allosteric conformations and homocarnosine concentration on carnosinase activity

Activity of carnosinase (CN1), the only dipeptidase with substrate specificity for carnosine or homocarnosine, varies greatly between individuals but increases clearly and significantly with age. Surprisingly, the lower CN1 activity in children is not reflected by differences in CN1 protein concentrations. CN1 is present in different allosteric conformations in children and adults since all sera obtained from children but not from adults were positive in ELISA and addition of DTT to the latter sera increased OD450 values. There was no quantitative difference in the amount of monomeric CN1 between children and adults. Further, CN1 activity was dose dependently inhibited by homocarnosine. Addition of 80 μM homocarnosine lowered V _max for carnosine from 440 to 356 pmol/min/μg and increased K _m from 175 to 210 μM. The estimated K _i for homocarnosine was higher (240 μM). Homocarnosine inhibits carnosine degradation and high homocarnosine concentrations in cerebrospinal fluid (CSF) may explain the lower carnosine degradation in CSF compared to serum. Because CN1 is implicated in the susceptibility for diabetic nephropathy (DN), our findings may have clinical implications for the treatment of diabetic patients with a high risk to develop DN. Homocarnosine treatment can be expected to reduce CN1 activity toward carnosine, resulting in higher carnosine levels.     

Synthesis of functional dipeptide carnosine from nonprotected amino acids using carnosinase-displaying yeast cells

Carnosine (β-alanyl-l-histidine) is one of the bioactive dipeptides and has antioxidant, antiglycation, and cytoplasmic buffering properties. In this study, to synthesize carnosine from nonprotected amino acids as substrates, we cloned the carnosinase (CN1) gene and constructed a whole-cell biocatalyst displaying CN1 on the yeast cell surface with α-agglutinin as the anchor protein. The display of CN1 was confirmed by immunofluorescent labeling, and CN1-displaying yeast cells showed hydrolytic activity for carnosine. When carnosine was synthesized by the reverse reaction of CN1, organic solvents were added to the reaction mixture to reduce the water content. The CN1-displaying yeast cells were lyophilized and examined for organic solvent tolerance. Results showed that the CN1-displaying yeast cells retained their original hydrolytic activity in hydrophobic organic solvents. In the hydrophobic organic solvents and hydrophobic ionic liquids, the CN1-displaying yeast cells catalyzed carnosine synthesis, and carnosine was synthesized from nonprotected amino acids in only one step. The results of this research suggest that the whole-cell biocatalyst displaying CN1 on the yeast cell surface can be used to synthesize carnosine with ease and convenience.     

Mechanistic insights on anserine hydrolyzing activities of human carnosinases

Anserine and carnosine represent histidine-containing dipeptides that exert a pluripotent protective effect on human physiology. Anserine is known to protect against oxidative stress in diabetes and cardiovascular diseases. Human carnosinases (CN1 and CN2) are dipeptidases involved in the homeostasis of carnosine. In poikilothermic vertebrates, the anserinase enzyme is responsible for hydrolyzing anserine. However, there is no specific anserine hydrolyzing enzyme present in humans. In this study, we have systematically investigated the anserine hydrolyzing activity of human CN1 and CN2. A targeted multiple reaction monitoring (MRM) based approach was employed for studying the enzyme kinetics of CN1 and CN2 using carnosine and anserine as substrates. Surprisingly, both CN1 and CN2 can hydrolyze anserine effectively. The observed catalytic turnover rate (V_max/[E]_t) was 21.6 s^?1 and 2.8 s^?1 for CN1 and CN2, respectively. CN1 is almost eight-fold more efficient in hydrolyzing anserine compared to CN2, which is comparable to the efficiency of the carnosine hydrolyzing activity of CN2. The Michaelis constant (K_m) value for CN1 (1.96 mM) is almost three-fold lower compared to CN2 (6.33 mM), representing higher substrate affinity for anserine-CN1 interactions. Molecular docking studies showed that anserine binds at the catalytic site of the carnosinases with an affinity similar to carnosine. Overall, the present study elucidated the inherent promiscuity of human carnosinases in hydrolyzing anserine using a sensitive LC-MS/MS approach.     

Water extract of the root of Lindera strychnifolia slows down the progression of diabetic nephropathy in db/db mice

We examined a possible preventive effect of Linderae radix (LR), the root of Lindera strychnifolia, on the progression of diabetic nephropathy. Water extract of Linderae radix (LR extract) was orally administered to the C57BL/KsJ-db/db (db/db) mice, a model of genetic diabetes, at a dose of 730 mg/kg/day for 12 week. The LR extract treatment did not affect glucose metabolism and systolic pressure. However, it resulted in a better renal function as evaluated by creatinine clearance (Ccr) and serum creatinine than the control; Ccr and serum creatinine were progressively worsened in controls (0.13+/-0.01 (l/day) and 0.69+/-0.04 (mg/dl), respectively) whereas unchanged in the treated group (0.24+/-0.03 (l/day), p<0.05 and 0.53+/-0.04 (mg/dl), p<0.05, respectively). Kidneys of the LR extract-treated group showed glomeruli with greater area and cell population, smaller glomerular sclerotic index, and less fibrosis in glomeruli, where apoptotic rate of glomerular cells were decreased compared with the control kidneys. Furthermore, renal TGF-beta(1) expression was decreased in the LR extract-treated group. These findings suggest that the LR therapy can be a novel therapeutic approach against diabetic nephropathy.     

Vegetarianism,female gender and increasing age,but not <Emphasis Type="Italic">CNDP1</Emphasis> genotype,are associated with reduced muscle carnosine levels in humans

Carnosine is found in high concentrations in skeletal muscles, where it is involved in several physiological functions. The muscle carnosine content measured within a population can vary by a factor 4. The aim of this study was to further characterize suggested determinants of the muscle carnosine content (diet, gender and age) and to identify new determinants (plasma carnosinase activity and testosterone). We investigated a group of 149 healthy subjects, which consisted of 94 men (12 vegetarians) and 55 women. Muscle carnosine was quantified in M. soleus, gastrocnemius and tibialis anterior using magnetic resonance proton spectroscopy and blood samples were collected to determine CNDP1 genotype, plasma carnosinase activity and testosterone concentrations. Compared to women, men have 36, 28 and 82% higher carnosine concentrations in M. soleus, gastrocnemius and tibialis anterior muscle, respectively, whereas circulating testosterone concentrations were unrelated to muscle carnosine levels in healthy men. The carnosine content of the M. soleus is negatively related to the subjects’ age. Vegetarians have a lower carnosine content of 26% in gastrocnemius compared to omnivores. In contrast, there is no difference in muscle carnosine content between omnivores with a high or low ingestion of β-alanine. Muscle carnosine levels are not related to the polymorphism of the CNDP1 gene or to the enzymatic activity of the plasma carnosinase. In conclusion, neither CNDP1 genotype nor the normal variation in circulating testosterone levels affects the muscular carnosine content, whereas vegetarianism, female gender and increasing age are the factors associated with reduced muscle carnosine stores.     

Homocarnosinosis: Hypercarnosinuria

Abstract: Homocarnosine (γ-aminobutyrylhistidine) is a brain-specific dipeptide. Homocarnosinosis is a familial metabolic disorder in which spastic paraplegia, progressive mental deficiency, and retinal pigmentation coexist with increased CSF homocarnosine levels, i.e. approximately 20 times higher than the mean control level. In the present study, the urinary excretion of carnosine ( β -alanylhistidine) and anserine ( β -alanyl-1-methylhistidine) was determined in patients with homocarnosinosis and in their close relatives. Both the patients and their relatives were also loaded with chicken meat, which is rich in anserine and carnosine. The results were compared with those obtained in childhood hypercarnosinuria with serum carnosinase deficiency. Somewhat surprisingly, patients with homocarnosinosis were also shown to have hypercarnosinuria. Chicken meat loading in healthy individuals results in increased excretion of carnosine and anserine and, in addition, 1-methylhistidine in the urine. Homocarnosinosis patients and patients with serum carnosinase deficiency also showed an increased excretion of carnosine and anserine, but 1-methylhistidine was not detected in serum carnosinase deficiency, and it was present only in very small amounts in homocarnosinosis. This defect seems to be due to lack of the hydrolyzing enzyme activity. The biochemical and clinical similarities between adult homocarnosinosis and infantile serum carnosinase deficiency are intriguing. A complete insight into the relationship between them must await further investigation.     

Interactions among carnosine,anserine, ophidine and copper in biochemical adaptation

Recent findings indicate that carnosine, anserine and ophidine should chelate copper in the tissues where these dipeptides are present in high concentration. The observations that carnosine, anserine and ophidine are located in skeletal muscles exhibiting active oxidative metabolism and/or glycolysis and that their accumulation appears to occur ontogenetically at the same time as these tissues begin to function suggests that these dipeptides may be involved in the intracellular transport of copper for activation of cytochrome oxidase at the end of the electron transport chain and in the regulation of anaerobic glycolysis. This hypothesis provides explanations for the presence of ophidine in the skeletal muscle of whale, the presence of anserine in the flight muscles of birds, the regulatory mechanism that permits orderly replacement of the primary olfactory neuron within the nasal olfactory epithelium, and the high activity of carnosinase in the uterus.     

The antineoplastic effect of carnosine is accompanied by induction of PDK4 and can be mimicked by l-histidine

Carnosine (β-alanyl-l-histidine) is a naturally occurring dipeptide that shows antineoplastic effects in cell culture as well as in animal experiments. Since its mode of action and the targets at the molecular level have not yet been elucidated, we performed qRT-PCR experiments with RNA isolated from glioblastoma cell lines treated with carnosine, β-alanine, l-alanine, l-histidine and the dipeptide l-alanine-l-histidine. The experiments identified a strong induction of expression of the gene encoding pyruvate dehydrogenase 4 (PDK4) under the influence of carnosine and l-histidine, but not by the other substances employed. In addition, inhibition of cell viability was only detected in cells treated with carnosine and l-histidine, with the latter showing a significantly stronger effect than carnosine. Since the tumor cells expressed the tissue form of carnosinase (CN2) but almost no serum carnosinase (CN1), we conclude that cleavage by CN2 is a prerequisite for the antineoplastic effect of carnosine. In addition, enhanced expression of PDK4 under the influence of carnosine/l-histidine opens a new perspective for the interpretation of the ergogenic potential of dietary β-alanine supplementation and adds a new contribution to a growing body of evidence that single amino acids can regulate key metabolic pathways important in health and disease.     

Glomerular filtration rate determinations in conscious type II diabetic mice

Diabetic nephropathy is a major cause of end-stage renal disease worldwide. The current studies were performed to determine the later stages of the progression of renal disease in type II diabetic mice (BKS; db/db). Methodology was developed for determining glomerular filtration rate (GFR) in conscious, chronically instrumented mice using continuous intravenous infusion of FITC-labeled inulin to achieve a steady-state plasma inulin concentration. Obese diabetic mice exhibited increased GFR compared with control mice. GFR averaged 0.313 ± 0.018 and 0.278 ± 0.007 ml/min in 18-wk-old obese diabetic (n = 11) and control (n = 13) mice, respectively (P < 0.05). In 28-wk-old obese diabetic (n = 10) and control (n = 15) mice, GFR averaged 0.348 ± 0.030 and 0.279 ± 0.009 ml/min, respectively (P < 0.05). GFR expressed per gram BW was significantly reduced in 18- and 28-wk-old obese diabetic compared with control mice (5.9 ± 0.3 vs. 9.0 ± 0.3; 6.6 ± 0.6 vs. 7.8 ± 0.3 μl·min(-1)·g body wt(-1)), respectively (P < 0.05). However, older nonobese type II diabetic mice had significantly reduced GFR (0.179 ± 0.023 ml/min; n = 6) and elevated urinary albumin excretion (811 ± 127 μg/day) compared with obese diabetic and control mice (514 ± 54, 171 ± 18 μg/day), which are consistent with the advanced stages of renal disease. These studies suggest that hyperfiltration contributes to the progression of renal disease in type II diabetic mice.     

Involvement of heparan sulfate in the renoprotective effects of imidapril,an angiotensin-converting enzyme inhibitor,in diabetic db/db mice

Abstract

We investigated the renoprotective effects of imidapril hydrochloride ((-)-(4?S)-3-[(2?S)-2-[[(1?S)-1-ethoxycarbonyl-3-phenylpropyl] amino] propionyl]-1-methyl-2-oxoimidazolidine-4-carboxylic acid hydrochloride, imidapril), an angiotensin-converting enzyme inhibitor, in a diabetic animal model. We used BKS.Cg-+Lepr^db/+Lepr^db (db/db) mice, a genetic animal model of obese type 2 diabetes. Diabetic db/db mice suffered from glomerular hyperfiltration, albuminuria and hypoalbuminemia. Oral administration of 5?mg/kg/day of imidapril for 3 weeks suppressed renal hyperfiltration, reduced albuminuria and normalized hypoalbuminemia. Imidapril did not influence body weights, blood pressure or blood glucose concentrations in db/db mice. Urinary excretion of heparan sulfate (HS) in non-treated 11-week-old db/db mice was significantly lower than that in age-matched non-diabetic db/+m mice. HS is a component of HS proteoglycans, which are present in glomerular basement membranes and glycocalyx of cell surfaces. Reduced urinary HS excretion indicated glomerular HS loss in db/db mice. Imidapril increased urinary excretion of HS to concentrations observed in db/+m mice, indicating that imidapril prevented the loss of renal HS. These results suggest that imidapril ameliorates renal hyperfiltration and loss of renal contents of HS. Improvement of filtration function and maintenance of HS, which is an important structural component of glomeruli, may contribute to renoprotective effects of imidapril.     

Protection by carnosine-related dipeptides against hydrogen peroxide-mediated ceruloplasmin modification

Carnosine, homocarnosine, and anserine are present in high concentrations in the muscle and brain of many animals and humans. Previous studies showed that these compounds have an antioxidant function. We investigated the protective effects of carnosine and related compounds on the modification of human ceruloplasmin that is induced by H2O2. Carnosine, homocarnosine, and anserine significantly inhibited the fragmentation and inactivation of ceruloplasmin that is induced by H2O2. All three compounds also inhibited the release of copper ion from protein, and the formation of hydroxyl radicals in the ceruloplasmin/H2O2 system. These compounds inhibited the fragmentation of human serum albumin that is induced by the copper-catalyzed oxidation system, as well as by the iron-catalyzed oxidation system. These results suggest that carnosine, homocarnosine, and anserine might protect ceruloplasmin against H2O2-mediated oxidative damage through a combination of copper chelation and free radical scavenging.     

Biochemical and Physiological Evidence that Carnosine Is an Endogenous Neuroprotector Against Free Radicals

1. Carnosine, anserine, and homocarnosine are endogenous dipeptides concentrated in brain and muscle whose biological functions remain in doubt.2. We have tested the hypothesis that these compounds function as endogenous protective substances against molecular and cellular damage from free radicals, using two isolated enzyme systems and two models of ischemic brain injury. Carnosine and homocarnosine are both effective in activating brain Na, K-ATPase measured under optimal conditions and in reducing the loss of its activity caused by incubation with hydrogen peroxide.3. In contrast, all three endogenous dipeptides cause a reduction in the activity of brain tyrosine hydroxylase, an enzyme activated by free radicals. In hippocampal brain slices subjected to ischemia, carnosine increased the time to loss of excitability.4. In in vivo experiments on rats under experimental hypobaric hypoxia, carnosine increased the time to loss of ability to stand and breath and decreased the time to recovery.5. These actions are explicable by effects of carnosine and related compounds which neutralize free radicals, particularly hydroxyl radicals. In all experiments the effective concentration of carnosine was comparable to or lower than those found in brain. These observations provide further support for the conclusion that protection against free radical damage is a major role of carnosine, anserine, and homocarnosine.     

Specificity and distribution of mammalian carnosinase.

Hog kidney carnosinase (EC 3.4.13.3) was found to have a narrow specificity; it hydrolyzed carnosine, anserine and glycyl-L-histidine, but did not split L-alanyl-L-histidine or homocarnosine. The isoelectric point of this enzyme was 5.8 and its molecular weight was about 84 000. Carnosinase was found to be widely distributed in various tissues of the rat. Uterus, kidney, liver and lung contained high levels of carnosinase, whereas moderate concentrations were found in spleen, heart and brain, with low levels in small intestine, skeletal muscle and stomach, and none in blood.     

Hydrogen peroxide-mediated Cu,Zn-superoxide dismutase fragmentation: protection by carnosine, homocarnosine and anserine

The fragmentation of human Cu,Zn-superoxide dismutase (SOD) was observed during incubation with H(2)O(2). Hydroxyl radical scavengers such as sodium azide, formate and mannitol protected the fragmentation of Cu,Zn-SOD. These results suggested that *OH was implicated in the hydrogen peroxide-mediated Cu,Zn-SOD fragmentation. Carnosine, homocarnosine and anserine have been proposed to act as anti-oxidants in vivo. We investigated whether three compounds could protect the fragmentation of Cu,Zn-SOD induced by H(2)O(2). The results showed that carnosine, homocarnosine and anserine significantly protected the fragmentation of Cu,Zn-SOD. All three compounds also protected the loss of enzyme activity induced by H(2)O(2). Carnosine, homocarnosine and anserine effectively inhibited the formation of *OH by the Cu,Zn-SOD/H(2)O(2) system. These results suggest that carnosine and related compounds can protect the hydrogen peroxide-mediated Cu,Zn-SOD fragmentation through the scavenging of *OH.     

Sequence identification and characterization of human carnosinase and a closely related non-specific dipeptidase

Carnosine (beta-alanyl-L-histidine) and homocarnosine (gamma-aminobutyric acid-L-histidine) are two naturally occurring dipeptides with potential neuroprotective and neurotransmitter functions in the brain. Peptidase activities degrading both carnosine and homocarnosine have been described previously, but the genes linked to these activities were unknown. Here we present the identification of two novel cDNAs named CN1 and CN2 coding for two proteins of 56.8 and 52.7 kDa and their classification as members of the M20 metalloprotease family. Whereas human CN1 mRNA and protein are brain-specific, CN2 codes for a ubiquitous protein. In contrast, expression of the mouse and rat CN1 orthologues was detectable only in kidney. The recombinant CN1 and CN2 proteins were expressed in Chinese hamster ovary cells and purified to homogeneity. CN1 was identified as a homodimeric dipeptidase with a narrow substrate specificity for Xaa-His dipeptides including those with Xaa = beta Ala (carnosine, K(m) 1.2 mM), N-methyl beta Ala, Ala, Gly, and gamma-aminobutyric acid (homocarnosine, K(m) 200 microM), an isoelectric point of pH 4.5, and maximal activity at pH 8.5. CN2 protein is a dipeptidase not limited to Xaa-His dipeptides, requires Mn(2+) for full activity, and is sensitive to inhibition by bestatin (IC(50) 7 nM). This enzyme does not degrade homocarnosine and hydrolyzes carnosine only at alkaline pH with an optimum at pH 9.5. Based on their substrate specificity and biophysical and biochemical properties CN1 was identified as human carnosinase (EC ), whereas CN2 corresponds to the cytosolic nonspecific dipeptidase (EC ).     

Prevention of diabetic nephropathy by treatment with astaxanthin in diabetic db/db mice

Oxidative stress is implicated as an important mechanism by which diabetes causes nephropathy. Astaxanthin, which is found as a common pigment in algae, fish, and birds, is a carotenoid with significant potential for antioxidative activity. In this study, we examined whether chronic administration of astaxanthin could prevent the progression of diabetic nephropathy induced by oxidative stress in mice. We used female db/db mice, a rodent model of type 2 diabetes, and their non-diabetic db/m littermates. The mice were divided into three groups as follows: non-diabetic db/m, diabetic db/db, and diabetic db/db treated with astaxanthin. Blood glucose level, body weight, urinary albumin, and urinary 8-hydroxydeoxyguanosine (8-OHdG) were measured during the experiments. Histological and 8-OHdG immunohistochemical studies were performed for 12 weeks from the beginning of treatment. After 12 weeks of treatment, the astaxanthin-treated group showed a lower level of blood glucose compared with the non-treated db/db group; however, both groups had a significantly high level compared with the db/m mice. The relative mesangial area calculated by the mesangial area/total glomerular area ratio was significantly ameliorated in the astaxanthin-treated group compared with the non-treated db/db group. The increases in urinary albumin and 8-OHdG at 12 weeks of treatment were significantly inhibited by chronic treatment with astaxanthin. The 8-OHdG immunoreactive cells in glomeruli of non-treated db/db mice were more numerous than in the astaxanthin-treated db/db mice. In this study, treatment with astaxanthin ameliorated the progression and acceleration of diabetic nephropathy in the rodent model of type 2 diabetes. The results suggested that the antioxidative activity of astaxanthin reduced the oxidative stress on the kidneys and prevented renal cell damage. In conclusion, administration of astaxanthin might be a novel approach for the prevention of diabetes nephropathy.