Long-term multiple risk factor interventions in Japanese elderly diabetic patients: the Japanese Elderly Diabetes Intervention Trial--study design, baseline characteristics and effects of intervention.

AIM: To evaluate long-term, multiple risk factor intervention on physical, psychological and mental prognosis, and development of complications and cardiovascular disease in elderly type 2 diabetes patients. METHODS: Our randomized, controlled, multicenter, prospective intervention trial included 1173 elderly type 2 diabetes patients who were enrolled from 39 Japanese institutions and randomized to an intensive or conservative treatment group. Glycemic control, dyslipidemia, hypertension, obesity, diabetic complications and atherosclerotic disease were measured annually. Instrumental activity of daily living, cognitive impairment, depressive symptoms and diabetes burden were assessed at baseline and 3 years. RESULTS: There was no significant difference in clinical or cognitive parameters at baseline between the two groups. The prevalence of low activities of daily living, depressive symptoms and cognitive impairment was 13%, 28% and 4%, respectively, and was similar in the two groups. A small, but significant difference in HbA1c between the two groups was observed at 1 year after the start of intervention (7.9% vs 8.1%, P < 0.05), although this significant difference was not observed after the second year. With the exception of coronary revascularization, there was no significant difference in fatal or non-fatal events between the two groups. Composite events were also similar in the two groups. CONCLUSIONS: This study showed no significant differences in fatal or non-fatal events between intensive and conventional treatment. The present study might clarify whether treatment of risk factors influences function and quality of life in elderly diabetic patients.

Non-high-density lipoprotein cholesterol: an important predictor of stroke and diabetes-related mortality in Japanese elderly diabetic patients.

AIMS: To evaluate the association of low-density lipoprotein, high-density lipoprotein and non-high-density lipoprotein cholesterol with the risk of stroke, diabetes-related vascular events and mortality in elderly diabetes patients. METHODS: This study was carried out as a post-hoc landmark analysis of a randomized, controlled, multicenter, prospective intervention trial. We included 1173 elderly type 2 diabetes patients (aged ≥ 65 years) from 39 Japanese institutions who were enrolled in the Japanese elderly diabetes intervention trial study and who could be followed up for 1 year. A landmark survival analysis was carried out in which follow up was set to start 1 year after the initial time of entry. RESULTS: During 6 years of follow up, there were 38 cardiovascular events, 50 strokes, 21 diabetes-related deaths and 113 diabetes-related events. High low-density lipoprotein cholesterol was associated with incident cardiovascular events, and high glycated hemoglobin was associated with strokes. After adjustment for possible covariables, non-high-density lipoprotein cholesterol showed a significant association with increased risk of stroke, diabetes-related mortality and total events. The adjusted hazard ratios (95% confidence intervals) of non-high-density lipoprotein cholesterol were 1.010 (1.001-1.018, P = 0.029) for stroke, 1.019 (1.007-1.031, P < 0.001) for diabetes-related death and 1.008 (1.002-1.014; P < 0.001) for total diabetes-related events. CONCLUSIONS: Higher non-high-density lipoprotein cholesterol was associated with an increased risk of stroke, diabetes-related mortality and total events in elderly diabetes patients.

The effects of blood pressure control levels on the renoprotection of type 2 diabetic patients without overt proteinuria.

BACKGROUND: There is little evidence regarding the target blood pressure level in patients with type 2 diabetes mellitus without overt proteinuria. METHODS AND RESULTS: We followed 608 Japanese patients with type 2 diabetes without apparent cardiovascular disease and overt proteinuria who underwent cerebral magnetic resonance imaging for a mean of 7.5 years. The patients were categorized according to their mean systolic blood pressure during the follow-up period (strict: <130 mm Hg, moderate: ≥130 and <140 mm Hg, poor: ≥ 140 mm Hg). The risks for the primary composite outcome of death or end-stage renal disease were not different among the three groups. The renal risk of the doubling of serum creatinine for the poor group was significantly higher than those in other groups. In addition, among the patients without silent cerebral infarction (SCI), the renal risk was significantly lower in the strict group than in the moderate group. Further, in both the SCI and non-SCI groups, strict blood pressure control slowed the progression of albuminuria. CONCLUSIONS: In nonproteinuric diabetic patients without SCI, strict blood pressure control was associated with improved renal outcomes. There may be different effects of intensive blood pressure control on the renoprotection of diabetic patients according to their complications.

Cerebral microvascular disease predicts renal failure in type 2 diabetes.

Abnormalities in small renal vessels may increase the risk of developing impaired renal function, but methods to assess these vessels are extremely limited. We hypothesized that the presence of small vessel disease in the brain, which manifests as silent cerebral infarction (SCI), may predict the progression of kidney disease in patients with type 2 diabetes. We recruited 608 patients with type 2 diabetes without apparent cerebrovascular or cardiovascular disease or overt nephropathy and followed them for a mean of 7.5 years. At baseline, 177 of 608 patients had SCI, diagnosed by cerebral magnetic resonance imaging. The risk for the primary outcome of ESRD or death was significantly higher for patients with SCI than for patients without SCI [hazard ratio, 2.44; 95% confidence interval (CI) 1.36 to 4.38]. The risk for the secondary renal end point of any dialysis or doubling of the serum creatinine concentration was also significantly higher for patients with SCI (hazard ratio, 4.79; 95% CI 2.72 to 8.46). The estimated GFR declined more in patients with SCI than in those without SCI; however, the presence of SCI did not increase the risk for progression of albuminuria. In conclusion, independent of microalbuminuria, cerebral microvascular disease predicted renal morbidity among patients with type 2 diabetes.

Causes of death in Japanese diabetics: A questionnaire survey of 18,385 diabetics over a 10-year period.

We collated and analysed data from hospital records regarding the cause of death of 18,385 patients with diabetes who died in 282 medical institutions throughout Japan over the 10-year period between 1991 and 2000. Autopsy was carried out in 1750 cases. The most frequent cause of death in all 18,385 cases was malignant neoplasia, accounting for 34.1% of cases, followed by vascular diseases (including diabetic nephropathy, ischemic heart diseases and cerebrovascular diseases) in 26.8%, infections in 14.3%, and then diabetic coma in 1.2%. The most common malignancy was liver cancer, accounting for 8.6% of all the deaths. Of the deaths from vascular diseases, diabetic nephropathy was the cause of death in 6.8% of cases, and the frequency as cause of death for ischemic heart diseases and cerebrovascular diseases were similar at 10.2% and 9.8%, respectively. Myocardial infarction accounted for almost all the deaths from ischemic heart diseases, whereas deaths from cerebral infarction were 2.2-fold as common as those from cerebral hemorrhage. In the analyses of the relationship between age and causes of death in diabetic patients who underwent autopsy, the overall mortality rate as a result of vascular diseases increased with age, although the mortality rates from diabetic nephropathy and cerebrovascular diseases increased little from the fifth decade of life. The mortality rate from ischemic heart diseases increased with age, however, and was higher than the other forms of vascular diseases from the sixth decade of life, accounting for approximately 50% of vascular deaths in the eighth decade. Malignant neoplasia was the most frequent cause of death from the fifth decade of life, and was extremely common in the seventh decade, accounting for 46.3% of all the deaths. The mortality rate from infections varied little between age groups from the fifth decade of life. In the analyses of glycemic control and the age at the time of death, lifespans were 2.5 years shorter in males, and 1.6 years shorter in female diabetics with poor glycemic control than in those with good or fair glycemic control. This difference was greater for deaths as a result of infections and vascular diseases, particularly diabetic nephropathy, than for malignant neoplasia. Analysis of the relationship between glycemic control and the duration of diabetes and deaths as a result of vascular diseases showed no correlation between the level of glycemic control and death from diabetic nephropathy, ischemic heart diseases or cerebrovascular diseases. In diabetics with disease durations of less than 10 years, the mortality rate from macroangiopathy was higher than that as a result of diabetic nephropathy, a form of microangiopathy. Treatment for diabetes comprised of diet alone in 21.5%, oral hypoglycemic agents in 29.5%, and insulin with or without oral hypoglycemic agents in 44.2%, which was the most common. In particular, 683/1170 (58.4%) diabetics who died from diabetic nephropathy were on insulin therapy, a higher proportion than the 661/1687 (39.2%) who died from ischemic heart diseases, or the 659/1622 (40.6%) who died from cerebrovascular diseases. The average age at the time of death in the survey population was, 68 years for males and 71.6 years for females. These were 9.6 and 13 years, respectively, short of the average life expectancy for the Japanese general population. In comparison with the previous survey (1981-1990), the average age at the time of death had increased 1.5 years for males, and 3.2 years for females. The average life expectancy for the Japanese general population had also increased 1.7 and 2.7 years, respectively, over that period, showing that advances in the management and treatment of diabetes have not led to any improvement in patients' life expectancies. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2010.00019.x, 2010).

Clinical impact of reducing microalbuminuria in patients with type 2 diabetes mellitus.

Diabetic nephropathy in type 2 diabetes is a leading cause of end-stage renal disease worldwide. Its early clinical sign is microalbuminuria, which is not only a predictor for progression of nephropathy but also an independent risk factor for cardiovascular disease. A few decades ago, diabetic nephropathy was believed to be progressive and irreversible. Thus, the main therapeutic objective for type 2 diabetic patients with microalbuminuria was to prevent progression to overt proteinuria. However, there is now growing evidence regarding remission/regression of diabetic nephropathy. In recent clinical trials using the renin-angiotensin system blockade drugs, a reduction in microalbuminuria by the use of these drugs has been noted. We also reported that a reduction in microalbuminuria was more frequent than progression to overt proteinuria and that multifactorial control approach was important to the reduction of microalbuminuria. These results for type 2 diabetes are similar to those previously reported for type 1 diabetes. Furthermore, our recent study showed that the 8-year cumulative incidence rate of renal and cardiovascular events was significantly lower in patients with remission than in those without it. The annual decline rate of estimated glomerular filtration rate in patients with remission was also significantly slower. These studies provide clinical evidence implying that the reduction of microalbuminuria in type 2 diabetic patients occurs frequently and brings about renal and cardiovascular risk reduction. Reducing microalbuminuria is therefore considered to be an important therapeutic objective and may be a biomeasure of therapeutic success in type 2 diabetic patients.

SNPs in KCNQ1 are associated with susceptibility to type 2 diabetes in East Asian and European populations.

We conducted a genome-wide association study using 207,097 SNP markers in Japanese individuals with type 2 diabetes and unrelated controls, and identified KCNQ1 (potassium voltage-gated channel, KQT-like subfamily, member 1) to be a strong candidate for conferring susceptibility to type 2 diabetes. We detected consistent association of a SNP in KCNQ1 (rs2283228) with the disease in several independent case-control studies (additive model P = 3.1 x 10(-12); OR = 1.26, 95% CI = 1.18-1.34). Several other SNPs in the same linkage disequilibrium (LD) block were strongly associated with type 2 diabetes (additive model: rs2237895, P = 7.3 x 10(-9); OR = 1.32, 95% CI = 1.20-1.45, rs2237897, P = 6.8 x 10(-13); OR = 1.41, 95% CI = 1.29-1.55). The association of these SNPs with type 2 diabetes was replicated in samples from Singaporean (additive model: rs2237895, P = 8.5 x 10(-3); OR = 1.14, rs2237897, P = 2.4 x 10(-4); OR = 1.22) and Danish populations (additive model: rs2237895, P = 3.7 x 10(-11); OR = 1.24, rs2237897, P = 1.2 x 10(-4); OR = 1.36).

MAPK/AP-1-dependent regulation of PAI-1 gene expression by TGF-beta in rat mesangial cells.

BACKGROUND: Receptor-regulated Smads and/or mitogen-activated protein kinases (MAPKs) are involved in transforming growth factor-beta (TGF-beta)-induced expression of various genes, including plasminogen activator inhibitor-1 (PAI-1). Because the sequence of the promoter region in rat PAI-1 gene differs from that in the human gene, we examined the mechanisms of TGF-beta-induced rat PAI-1 expression in rat mesangial cells. METHODS: TGF-beta1-induced PAI-1 and c-fos mRNA expressions were determined by Northern blot analysis. Activation of MAPKs and Smad proteins was evaluated by an immunoblot analysis. DNA binding activities of nuclear protein were examined by using an electrophoretic mobility shift assay (EMSA). The activities of PAI-1 promoter were measured by a luciferase reporter assay. RESULTS: Extracellular-regulated kinase (ERK) and c-Jun NH-terminal kinase (JNK) phosphorylation, c-fos mRNA expression, and activator protein-1 (AP-1) DNA binding activity stimulated by TGF-beta1 were completely suppressed by the ERK kinase (MEK) inhibitors. EMSA and reporter analysis revealed that an AP-1-like sequence located in the proximal region of the rat PAI-1 promoter was the target for TGF-beta1, and the disruption of this AP-1-like sequence suppressed basal and TGF-beta1-induced promoter activation. TGF-beta1 also stimulated nuclear translocation of Smads and binding to palindromic Smad binding element (SBE) located in the rat PAI-1 promoter, without being affected by MEK inhibitor. Point mutation and deletion of palindromic SBE did not affect TGF-beta1-induced rat PAI-1 promoter activity. Moreover, interferon-gamma (IFN-gamma) inhibited TGF-beta1-induced PAI-1 expression through selectively suppressing the ERK-AP-1 pathway. CONCLUSION: These results suggest that the essential requirement of MAPK/AP-1 activation for TGF-beta1-induced PAI-1 expression is unique to rat mesangial cells.

Genetic variations in the gene encoding TFAP2B are associated with type 2 diabetes mellitus.

To search a gene(s) conferring susceptibility to type 2 diabetes mellitus, we genotyped nearly 60,000 gene-based SNPs for Japanese patients and found evidence that the gene at chromosome 6p12 encoding transcription-factor-activating protein 2beta (TFAP2B) was a likely candidate in view of significant association of polymorphism in this gene with type 2 diabetes. Extensive analysis of this region identified that several variations within TFAP2B were significantly associated with type 2 diabetes [a variable number of tandem repeat locus: chi(2)=10.9, P=0.0009; odds ratio=1.57, 95% CI 1.20-2.06, intron 1+774 (G/T); chi(2)=11.6, P=0.0006; odds ratio=1.60, 95% CI 1.22-2.09, intron 1+2093 (A/C); chi(2)=12.2, P=0.0004; odds ratio=1.61, 95% CI 1.23-2.11]. The association of TFAP2B with type 2 diabetes was also observed in the UK population. These results suggest that TFAP2B might be a new candidate for conferring susceptibility to type 2 diabetes and contribute to the pathogenesis of type 2 diabetes.

Genetic variations in the gene encoding ELMO1 are associated with susceptibility to diabetic nephropathy.

To search for a gene(s) conferring susceptibility to diabetic nephropathy (DN), we genotyped over 80,000 gene-based single nucleotide polymorphisms (SNPs) in Japanese patients and identified that the engulfment and cell motility 1 gene (ELMO1) was a likely candidate for conferring susceptibility to DN, in view of the significant association of an SNP in this gene with the disease (intron 18+9170, GG vs. GA+AA, chi(2) = 19.9, P = 0.000008; odds ratio 2.67, 95% CI 1.71-4.16). In situ hybridization (ISH) using the kidney of normal and diabetic mice revealed that ELMO1 expression was weakly detectable mainly in tubular and glomerular epithelial cells in normal mouse kidney and was clearly elevated in the kidney of diabetic mice. Subsequent in vitro analysis revealed that ELMO1 expression was elevated in cells cultured under high glucose conditions (25 mmol/l) compared with cells cultured under normal glucose conditions (5.5 mmol/l). Furthermore, we identified that the expression of extracellular matrix protein genes, such as type 1 collagen and fibronectin, were increased in cells that overexpress ELMO1, whereas the expression of matrix metalloproteinases was decreased. These results indicate that ELMO1 is a novel candidate gene that both confers susceptibility to DN and plays an important role in the development and progression of this disease.

Successful recovery of infective endocarditis-induced rapidly progressive glomerulonephritis by steroid therapy combined with antibiotics: a case report.

BACKGROUND: The mortality rate among patients with infective endocarditis, especially associated with the presence of complications or coexisting conditions such as renal failure and the use of combined medical and surgical therapy remains still high. Prolonged parenteral administration of a bactericidal antimicrobial agent or combination of agents is usually recommended, however, the optimal therapy for infective endocarditis associated with renal injury is not adequately defined. CASE PRESENTATION: Patient was a 24-years old man who presented to our hospital with fever, fatigue, and rapidly progressive glomerulonephritis. He had a history of ventricular septum defect (VSD). A renal biopsy specimen revealed crescentic glomerulonephritis and echocardiogram revealed VSD with vegetation on the tricuspid valve. Specimens of blood demonstrated Propionibacterium Acnes. The intensive antibiotic therapy with penicillin G was started without clinical improvement of renal function or resolution of fever over the next 7 days. After the short-term treatment of low dose of corticosteroid combined with continuous antibiotics, high fever and renal insufficiency were dramatically improved. CONCLUSION: Although renal function in our case worsened despite therapy with antibiotics, a short-term and low dose of corticosteroid therapy with antibiotics was able to recover renal function and the patient finally underwent tricuspid valve-plasty and VSD closure. We suggest that the patients with rapidly progressive glomerulonephritis associated with infective endocarditis might be treated with a short-term and low dose of corticosteroid successfully.

Antiproteinuric effect of candesartan cilexetil in Japanese subjects with type 2 diabetes and nephropathy.

The effect of the angiotensin II receptor blocker, candesartan cilexetil, on proteinuria was examined in a prospective, multicenter, randomized, double-blind study in Japanese subjects with type 2 diabetes. This study enrolled diabetic subjects with confirmed proteinuria into four groups for 12 weeks of treatment with placebo or candesartan cilexetil 2, 4, or 8 mg. The contribution of the angiotensin converting enzyme (ACE) gene polymorphism to the effect of candesartan cilexetil was also examined. In 127 subjects, candesartan cilexetil showed a dose-related reduction in proteinuria after 12 weeks of treatment (F = 9.45, P = 0.0013), with a 18.1% reduction in the 4-mg group, and a 5.8% reduction in the 8-mg group, in contrast to a 32.2% increase in the placebo group, and a 0.8% increase in the 2-mg group. These results indicate that candesartan cilexetil is useful in reducing proteinuria in diabetic subjects when compared with placebo. In addition, candesartan cilexetil seems to be effective in subjects with both the II and DD genotypes of the ACE gene.

Association of the gene encoding wingless-type mammary tumor virus integration-site family member 5B (WNT5B) with type 2 diabetes.

Recent reports have suggested that WNT signaling is an important regulator for adipogenesis or insulin secretion and might be involved in the pathogenesis of type 2 diabetes. To investigate possible roles of the WNT genes in conferring susceptibility to type 2 diabetes, we examined the association of the genes that encode members of the WNT family with type 2 diabetes in the Japanese population. First, 40 single-nucleotide polymorphism (SNP) loci within 11 WNT genes were analyzed in 188 subjects with type 2 diabetes (case-1) and 564 controls (control-1). Among them, six SNP loci exhibited a significant difference (P<.05) in the allele and/or genotype distributions between case and control subjects. These SNP loci were further analyzed in another set of case (case-2; n=733) and control (control-2; n=375) subjects to confirm their statistical significance. As a result, one SNP locus in the WNT5B gene was strongly associated with type 2 diabetes ( chi 2=15.6; P=.00008; odds ratio=1.74; 95% confidence interval 1.32-2.29). Expression of the WNT5B gene was detectable in several tissues, including adipose, pancreas, and liver. Subsequent in vitro experiments identified the fact that expression of the Wnt5b gene was increased at an early phase of adipocyte differentiation in mouse 3T3-L1 cells. Furthermore, overexpression of the Wnt5b gene in preadipocytes resulted in the promotion of adipogenesis and the enhancement of adipocytokine-gene expression. These results indicate that the WNT5B gene may contribute to conferring susceptibility to type 2 diabetes and may be involved in the pathogenesis of this disease through the regulation of adipocyte function.

A simple and sensitive method for glutamine:fructose-6-phosphate amidotransferase assay.

For measuring glutamine:fructose-6-phosphate amidotransferase (GFAT) activity in cultured cells, an enzyme method -GDH method- was set up with high-efficiency, high-sensitivity and simple operation by determining the formed glutamate. During the process of making samples, reduced glutathione (GSH, 5 mM) and glucose-6-phosphate Na2 (5 mM) were added to the buffer for scraping the cells. The range of protein content in the samples was 80-150 microg. In the GFAT activity assay, the end product reduced acetylpyridine adenine dinucleotide (APADH) was determined at 370 nm directly. The suitable concentrations of the reactants fructose-6-phosphate (F-6-P), glutamine, acetylpyridine adenine dinucleotide (APAD) and glutamate dehydrogenase (GDH) were 0.8, 6 and 0.3 mM and 6 U, respectively. However, the excess of APAD may interfere with the APADH measurement. The reaction time course was 90 min. The GFAT activity in 3T3-L1, L6, HepG2 and HIRc cells were 1.84-8.51 nmol glutamate/mg protein.min.

Association of solute carrier family 12 (sodium/chloride) member 3 with diabetic nephropathy, identified by genome-wide analyses of single nucleotide polymorphisms.

To identify genetic elements that might confer susceptibility to diabetic nephropathy, we performed a genome-wide analysis of gene-based single nucleotide polymorphisms (SNPs) in a large cohort of Japanese patients with diabetes. In case-control association studies, patients with type 2 diabetes were divided into two groups, one having retinopathy as well as overt nephropathy and the other (the control group) having diabetic retinopathy but with no signs of renal involvement. Genotyping of these patients at >55,000 SNP loci indicated a gene encoding solute carrier family 12 member 3 (SLC12A3) to be a good candidate for the susceptibility to diabetic nephropathy, in view of a significant association of one landmark SNP located in the 24th intron (chi(2) = 15.4, P = 0.000087, odds ratio = 2.53 [95% CI 1.57-4.09]). Subsequent analysis of additional genetic variations in this gene identified several SNPs that were significantly associated with nephropathy, especially one in exon 23 (+78 G to A: Arg913Gln, chi(2) = 18.5, P = 0.00002, odds ratio = 2.53 [95% CI 1.64-3.90]). The results implicated that substitution of Arg913 to Gln in the SLC12A3 gene might reduce the risk to develop diabetic nephropathy and suggested that the gene product might be a potential target for the prevention or treatment of this disease.

APOE polymorphism and the progression of diabetic nephropathy in Japanese subjects with type 2 diabetes: results of a prospective observational follow-up study.

OBJECTIVE: The aim of this study is to clarify the conflicting results of the epsilon2/epsilon3/epsilon4 APOE polymorphism as a risk factor on diabetic nephropathy by a cohort study. RESEARCH DESIGN AND METHODS: A total of 429 Japanese subjects with type 2 diabetes and with normoalbuminuria (n = 299) or with microalbuminuria (n = 130) were enrolled in a prospective observational follow-up study during 1995-1998 and followed until 2001 (for at least 3 years). The endpoint was the occurrence of a renal event defined as the progression to a higher stage of diabetic nephropathy. RESULTS: During the study (the mean follow-up period: 4.4 +/- 1.0 years), 31 of 429 subjects progressed: 21 from normoalbuminuria to microalbuminuria and 10 from microalbuminuria to overt proteinuria. The allele frequency of the APOE polymorphism was significantly different between the progressors and the nonprogressors. Eight of 42 epsilon2 carriers (19%) progressed, whereas 23 of 387 noncarriers (6%) progressed with a relative risk of 3.2 (95% CI 1.5-6.7). When subjects were stratified by renal status at baseline, each relative risk for the progression in the epsilon2 carriers was 2.7 (0.99-7.4) in those with normoalbuminuria and 4.2 (1.3-13.3) in those with microalbuminuria. Furthermore, when analyzed only in subjects with normoalbuminuria and short duration of diabetes (<15 years) at baseline, the risk in the epsilon2 carriers became higher to 3.2 (1.2-8.8). CONCLUSIONS: Our follow-up study indicates that the epsilon2 allele of the APOE polymorphism is a prognostic risk factor for both the onset and the progression of diabetic nephropathy in Japanese type 2 diabetes.

Effects of antioxidants in diabetes-induced oxidative stress in the glomeruli of diabetic rats.

Numerous reports have demonstrated that oxidative stress induced by diabetes plays an important role in the development and progression of diabetic vascular complications including nephropathy. Indeed, there is emerging evidence that the formation of reactive oxygen species (ROS) is a direct consequence of hyperglycemia. Biomarkers for oxidative damage to DNA, lipids, and proteins are also supporting the concept of increased oxidative stress in diabetes and diabetic nephropathy. However, there is an unanswered question: When does oxidative stress as a pathogenetic event occur in the process of diabetic nephropathy? To answer this question, glomerular ROS was imaged with the use of 2', 7'-dichlorofluorescein diacetate (DCFH-DA). The image of DCF fluorescence was strong in glomeruli from diabetic rats as compared with that of glomeruli from nondiabetic control rats. mRNA expression of antioxidant enzymes such as catalase, glutathione peroxidase, Cu/Zn superoxide dismutase, and heme oxygenase-1 (HO-1) was also determined because oxidative stress definitely refers to the situation of an imbalance between the production of ROS and antioxidant defense. The mRNA expression of catalase, glutathione peroxidase, and Cu/Zn superoxide dismutase 2 wk after the induction of diabetes was not significantly different from that in control rats. Alternatively, mRNA and protein expression of HO-1 was strongly induced by 16-fold in diabetic glomeruli after the induction of diabetes. Antioxidant treatment with either vitamin E or probucol almost completely normalized HO-1 overexpression in diabetic glomeruli, supporting the existence of oxidative stress in the glomeruli of early diabetes. Furthermore, It has reported that antioxidant treatment with vitamin E, probucol, alpha-lipoic acid, or taurine normalized diabetes-induced not only renal dysfunction such as albuminuria and glomerular hypertension but also glomerular pathologies. In summary, oxidative stress by diabetes could play a crucial role in the development and progression of diabetic nephropathy, and antioxidant treatment could be a potential therapeutic procedure for diabetic nephropathy.

Activation of Src kinase in platelet-derived growth factor-B-dependent tubular regeneration after acute ischemic renal injury.

We previously reported that the platelet-derived growth factor B-chain (PDGF-B)/PDGF receptor (PDGFR) axis is involved in tubular regeneration after ischemia/reperfusion injury of the kidney. In the present study, we examined the activation of Src tyrosine kinase, a crucially important signaling molecule for PDGFR, and assessed the role of Src in PDGF-B-dependent renal tubular regeneration afterischemia/reperfusion injury. Immunoblot using clone 28, a monoclonal antibody specific for the active form of Src kinases, demonstrated increased active Src expression in the injured rat kidney 6 hours after reperfusion with peak activation at 12 hours. In vitro kinase assay confirmed increased Src activity that concurred with PDGFR-beta activation as detected by the increment of receptor-phosphorylated tyrosine. Immunohistochemistry using clone 28 demonstrated that active Src was preferentially expressed in the S3 segment of the proximal tubule in reperfused kidney, where it is not normally expressed. This enhanced expression of active Src was co-localized with the increased PDGFR expression in the tubular cells that were undergoing cell proliferation cycle. Trapidil administration suppressed Src and PDGFR-beta activation in the reperfused kidney and resulted in deteriorated renal function. These findings suggest that active Src participates in PDGF-B-dependent regeneration of tubular cells from acute ischemic injury.