Prevalence of resistant hypertension and associated factors in Japanese subjects with type 2 diabetes.

OBJECTIVE: The prevalence of treatment resistant hypertension (RH) depends on methods used for blood pressure (BP) measurements, goals of BP, and therapeutic efforts in terms of medication and adherence. We focused on diabetic subjects and explored the prevalence of RH in primary care practice. METHODS: In 1737 subjects with type 2 diabetes who continued regular visits, office BP was evaluated by multiple measurements over one year. RH was defined as using more than four antihypertensive drugs or failure to achieve the goal with three antihypertensive drugs from different classes. The RH prevalence was investigated with BP goals <130/80 and 140/90 mmHg. RESULTS: The percentage of subjects who achieved BP goals <130/80 and 140/90 were 70.5% and 93.8% with adherence to medication ≥95%, and the corresponding prevalence rates of RH in treated subjects were 28.4% and 21.8%, respectively. Factors independently associated with RH were age (odds ratio 1.02 [95% CI 1.01-1.04]), body mass index (1.10 [1.06-1.13]), variability in systolic BP (1.06 [1.02-1.09]), triglycerides (2.86 [1.34-6.11]), macroalbuminuria (3.33 [2.03-5.48]), estimated glomerular filtration rate (0.98 [0.97-0.99]), retinopathy (1.91 [1.39-2.61]), and family history of hypertension (1.85 [1.23-2.21]). Worsening albuminuria and glomerular filtration rate enhanced the prevalence of RH in a graded manner. CONCLUSION: Careful estimation of office BP values over one year with a high achievement of BP goals and adequate adherence revealed that the prevalence of RH in type 2 diabetes is high. RH was characterized by accumulation of cardiovascular genetic and environmental risks.

Chronic kidney disease categories and renal-cardiovascular outcomes in type 2 diabetes without prevalent cardiovascular disease: a prospective cohort study (JDDM25).

AIMS/HYPOTHESIS: In type 2 diabetic patients at low risk for cardiovascular disease (CVD), the relationship between the clinical course of nephropathy by stage of chronic kidney disease (CKD) and onset of CVD remains unclear. Clarification of this relationship is important for clinical decision-making for both low- and high-risk diabetic patients. METHODS: This 4 year prospective study enrolled 2,954 type 2 diabetic patients with no prevalent CVD, and serum creatinine <176.8 µmol/l. The risk for CVD onset (non-fatal and fatal CVD and stroke, and peripheral arterial disease) was assessed according to CKD stage categorised by urinary albumin-to-creatinine ratio (ACR; mg/mmol) and estimated GFR (eGFR; ml min(-1) 1.73 m(-2)). Association of progression from 'no CKD' stage (ACR <3.5 mg/mmol and eGFR ≥ 90 ml min(-1) 1.73 m(-2)) with risk for CVD onset was also evaluated. RESULTS: During follow-up (median 3.8 years), 89 CVD events occurred. Compared with patients with 'no CKD' as reference, those with ACR ≥ 35.0 mg/mmol with co-existing eGFR 60-89 ml min(-1) 1.73 m(-2) or <60 ml min(-1) 1.73 m(-2) showed increased risk for CVD onset, whereas those with eGFR ≥ 90 ml min(-1) 1.73 m(-2) did not. Those with ACR <3.5 mg/mmol and eGFR <60 ml min(-1) 1.73 m(-2) did not show any increased risk. Among patients with 'no CKD' stage at baseline, those who progressed to ACR ≥ 3.5 mg/mmol during follow-up showed an increased risk compared with those who did not, whereas those who progressed to eGFR <90 ml min(-1) 1.73 m(-2) did not have increased risk. CONCLUSIONS/INTERPRETATION: The risk for CVD was associated with progression of albuminuria stage rather than eGFR stage in type 2 diabetic patients at relatively low risk for CVD.

Reduction of both beta cell death and alpha cell proliferation by dipeptidyl peptidase-4 inhibition in a streptozotocin-induced model of diabetes in mice.

AIMS/HYPOTHESIS: Incretins stimulate insulin secretion in a glucose-dependent manner but also promote pancreatic beta cell protection. Dipeptidyl peptidase-4 (DPP-4) inhibitors are a new glucose-lowering treatment that blocks incretin degradation by DPP-4. We assessed whether DPP-4 inhibition suppresses the progression to hyperglycaemia in a low-dose streptozotocin (STZ)-induced diabetic mouse model, and then investigated how DPP-4 inhibition affects islet function and morphology. METHODS: The DPP-4 inhibitor, des-fluoro-sitagliptin (SITA), was administered to mice during and after STZ injections, and in some mice also before STZ. RESULTS: In control mice, STZ resulted in hyperglycaemia associated with impaired insulin secretion and excess glucagon secretion. In SITA-treated STZ mice, these metabolic abnormalities were improved, particularly when SITA administration was initiated before STZ injections. We observed beta cell loss and dramatic alpha cell expansion associated with decreased insulin content and increased glucagon content after STZ administration. In SITA-treated mice, islet architecture and insulin content were preserved, and no significant increase in glucagon content was observed. After STZ exposure, beta cell apoptosis increased before hyperglycaemia, and SITA treatment reduced the number of apoptotic beta cells. Interestingly, alpha cell proliferation was observed in non-treated mice after STZ injection, but the proliferation was not observed in SITA-treated mice. CONCLUSIONS/INTERPRETATION: Our results suggest that the ability of DPP-4 inhibition to suppress the progression to STZ-induced hyperglycaemia involves both alleviation of beta cell death and alpha cell proliferation.

Effects of olmesartan on renal and cardiovascular outcomes in type 2 diabetes with overt nephropathy: a multicentre, randomised, placebo-controlled study.

AIMS/HYPOTHESIS: The renal and cardiovascular protective effects of angiotensin receptor blocker (ARB) remain controversial in type 2 diabetic patients treated with a contemporary regimen including an angiotensin converting enzyme inhibitor (ACEI). METHODS: We examined the effects of olmesartan, an ARB, on primary composite outcome of doubling of serum creatinine, endstage renal disease and death in type 2 diabetic patients with overt nephropathy. Secondary outcome included composite cardiovascular outcomes, changes in renal function and proteinuria. Randomisation and allocation to trial group were carried out by a central computer system. Participants, caregivers, the people carrying out examinations and people assessing the outcomes were blinded to group assignment. RESULTS: Five hundred and seventy-seven (377 Japanese, 200 Chinese) patients treated with antihypertensive therapy (73.5% [n = 424] received concomitant ACEI), were given either once-daily olmesartan (10-40 mg) (n = 288) or placebo (n = 289) over 3.2 ± 0.6 years (mean±SD). In the olmesartan group, 116 developed the primary outcome (41.1%) compared with 129 (45.4%) in the placebo group (HR 0.97, 95% CI 0.75, 1.24; p = 0.791). Olmesartan significantly decreased blood pressure, proteinuria and rate of change of reciprocal serum creatinine. Cardiovascular death was higher in the olmesartan group than the placebo group (ten vs three cases), whereas major adverse cardiovascular events (cardiovascular death plus non-fatal stroke and myocardial infarction) and all-cause death were similar between the two groups (major adverse cardiovascular events 18 vs 21 cases, all-cause deaths; 19 vs 20 cases). Hyperkalaemia was more frequent in the olmesartan group than the placebo group (9.2% vs 5.3%). CONCLUSIONS/INTERPRETATION: Olmesartan was well tolerated but did not improve renal outcome on top of ACEI. TRIAL REGISTRATION: ClinicalTrials.gov NCT00141453.

Long-term effect of modification of dietary protein intake on the progression of diabetic nephropathy: a randomised controlled trial.

AIMS/HYPOTHESIS: There is currently insufficient evidence to recommend a low-protein diet for type 2 diabetic patients with diabetic nephropathy. We assessed whether a low-protein diet could prevent the progression of diabetic nephropathy. METHODS: This was a multi-site parallel randomised controlled trial for prevention of diabetic nephropathy progression among 112 Japanese type 2 diabetic patients with overt nephropathy. It was conducted in Japan from 1 December 1997 to 30 April 2006. The participants were randomly assigned using a central computer-generated schedule to either low-protein diet (0.8 g kg(-1) day(-1)) and normal-protein diet (1.2 g kg(-1) day(-1)), and were followed for 5 years. The participants and investigators were not blinded to the assignment. The primary outcomes were the annual change in estimated GFR and creatinine clearance, the incidence of doubling of serum creatinine and the time to doubling of baseline serum creatinine. RESULTS: The study was completed by 47 (84%) of 56 participants in the low-protein diet group and 41 (73%) of 56 participants in the normal-diet group. During the study period, the difference in mean annual change in estimated GFR between the low-protein diet and the normal-protein diet groups was -0.3 ml min(-1) 1.73 m(-2) (95% CI -3.9, 4.4; p = 0.93). The difference in mean annual change in creatinine clearance between the low-protein diet and the normal-protein diet groups was -0.006 ml s(-1) 1.73 m(-2) (95% CI -0.089, 0.112; p = 0.80). A doubling of serum creatinine was reached in 16 patients of the low-protein group (34.0%), compared with 15 in the normal-protein group (36.6%), the difference between groups being -2.6% (95% CI -22.6, 17.5; p = 0.80). The time to doubling of serum creatinine was similar in both groups (p = 0.66). CONCLUSIONS/INTERPRETATION: It is extremely difficult to get patients to follow a long-term low-protein diet. Although in the low-protein group overall protein intake was slightly (but not significantly) lower, it did not confer renoprotection. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov NCT00448526. FUNDING: Research grant from the Ministry of Health, Labour and Welfare of Japan.

Pretransplant pharmacodynamic analysis of immunosuppressive agents using CFSE-based T-cell proliferation assay.

The impact of pretransplant T-cell sensitivity testing using carboxylfluorescein diacetate succinimidyl ester (CFSE)-based flow cytometry was studied in 32 patients with chronic renal failure. There was considerable interindividual variation in the inhibitory effects of cyclosporine (CSA), tacrolimus (TAC), and prednisolone (PRD) but only a small amount of interindividual variation for mycophenolic acid (MPA). Patients with high sensitivity to CSA tended to experience viral reactivation. In addition to post-transplant blood-level monitoring, pretransplant pharmacodynamics could provide useful information on optimal and safe immunosuppressive therapy.

Prooxidant action of furanone compounds: implication of reactive oxygen species in the metal-dependent strand breaks and the formation of 8-hydroxy-2'-deoxyguanosine in DNA.

Prooxidant properties of furanone compounds including 2,5-furanone (furaneol, 4-hydroxy-2,5-dimethyl-furan-3-one), 4,5-furanone (4,5-dimethyl-3-hydroxy-2(5H)-furanone) (sotolone) and cyclotene (2-hydroxy-3-methyl-2-cyclopenten-1-one) were analyzed in relation to the metal-reducing activity. Only 2.5-furanone known as a "strawberry or pineapple furanone" inactivated aconitase the most sensitive enzyme to active oxygen in the presence of ferrous sulfate, suggesting the furaneol/iron-mediated generation of reactive oxygen species. 2,5-Furanone caused strand scission of pBR322 DNA in the presence of copper. Treatment of calf thymus DNA with 2,5-furanone plus copper produced 8-hydroxy-2'-deoxyguanosine in DNA. 2,5-Furanone showed a potent copper-reducing activity, and thus, DNA strand breaks and the formation of 8-hydroxy-2'-deoxyguanosine by 2,5-furanone can be initiated by the production of superoxide radical through the reduction of cupric ion to cuprous ion, resulting in the conversion to hydrogen peroxide and hydroxyl radical. However, an isomer and analog of 2,5-furanone, 4,5-furanone and cyclotene, respectively, did not show an inactivation of aconitase, DNA injuries including strand breakage and the formation of 8-hydroxy-2'-deoxyguanosine, and copper-reducing activity. Cytotoxic effect of 2,5-furanone with hydroxyketone structure can be explained by its prooxidant properties: furaneol/transition metal complex generates reactive oxygen species causing the inactivation of aconitase and the formation of DNA base damage by hydroxyl radical.

Predictive impact of elevated serum level of IL-18 for early renal dysfunction in type 2 diabetes: an observational follow-up study.

AIMS/HYPOTHESIS: The early identification of type 2 diabetic patients at risk of developing microalbuminuria-an independent risk factor for renal and cardiovascular diseases-is important to improve the patients' outcomes. We investigated whether serum levels of IL-18, a proinflammatory cytokine, were a predictor of early renal dysfunction. MATERIALS AND METHODS: A total of 249 Japanese type 2 diabetic patients without overt proteinuria were enrolled in an observational follow-up study (median follow-up 7 years), and their stage of diabetic nephropathy was classified and their estimated glomerular filtration rate (eGFR) was calculated annually. RESULTS: At baseline, serum levels of IL-18 were higher in subjects with microalbuminuria (n = 76) than in those with normoalbuminuria (n = 173). Elevated serum levels of IL-18 were associated with the progression of nephropathy to a higher stage in normoalbuminuric subjects (118 [interquartile range 91-159] ng/l vs 155 [interquartile range 121-205] ng/l, p = 0.003), but not in microalbuminuric subjects (154 [interquartile range 113-200] ng/l vs 160 [interquartile range 101-190] ng/l, p = 0.50). The adjusted risk for developing microalbuminuria was 3.6 (95% CI 1.2-10.4) in normoalbuminuric subjects with serum IL-18 levels above the median (>/=134.6 ng/l), and was significantly enhanced in those urinary AERs at the upper end of the normal range (7.5 mug/min

Glucocorticoid-induced diabetes mellitus: prevalence and risk factors in primary renal diseases.

BACKGROUND/AIMS: In patients with primary renal diseases the current knowledge of hyperglycemia associated with corticosteroid therapy is limited. We therefore examined the prevalence and risk factors of glucocorticoid-induced diabetes mellitus (DM) in primary renal diseases. METHODS: Patients were recruited with primary renal diseases who were started on corticosteroids between April 2002 and June 2005. In patients with DM, an impaired fasting glucose level and/or positive urinary glucose analyses before corticosteroids therapy were excluded. RESULTS: During corticosteroid therapy (initial dose: prednisolone 0.75 +/- 0.10 mg/kg/day), DM was newly diagnosed in 17 (40.5%) of 42 patients. All of the 17 patients were diagnosed as having DM by postprandial hyperglycemia at 2 h after lunch, although they had normal fasting blood glucose levels. Age (OR 1.40, 95% CI 1.06-1.84) and body mass index (OR 1.87, 95% CI 1.03-3.38) were determined as independent risk factors for glucocorticoid-induced DM. CONCLUSION: Over 40% of patients with primary renal disease developed DM during treatment with corticosteroids. A high age and high body mass index are the independent risk factors for glucocorticoid-induced DM. 24-hour urinary glucose analyses and postprandial plasma glucose are useful for detecting glucocorticoid-induced DM.

Protective effect of NADP-isocitrate dehydrogenase on the paraquat-mediated oxidative inactivation of aconitase in heart mitochondria.

Protective role of NADP-isocitrate dehydrogenase in the oxidative inactivation of mitochondrial enzymes was analyzed. Administration of paraquat to the rat inactivated liver mitochondrial enzymes: the aconitase activity decreased to one quarter, and citrate synthase and fumarase to half, whereas cytosolic enzymes were not affected. Activities of heart mitochondrial and cytosolic enzymes were not at all changed in the rat treated with paraquat, but paraquat directly inactivated aconitase in the heart mitochondria isolated from the non-treated rats. The paraquat-dependent inactivation of aconitase was prevented by activating NADP-isocitrate dehydrogenase in the presence of oxidized glutathione. NADP-isocitrate dehydrogenase could regenerate glutathione in isolated heart mitochondria, indicating that paraquat-mediated inactivation depends on the glutathione-regenerating activity by enhanced NADPH supply. Lower NADP-isocitrate dehydrogenase activity in liver mitochondria cannot regenerate reduced glutathione for scavenging reactive oxygen species, resulting in the paraquat-induced oxidative inactivation of mitochondrial enzymes. However, higher activity of NADP-isocitrate dehydrogenase participates in the regeneration of reduced glutathione causing stabilization of enzymes in heart mitochondria.

The telmisartan renoprotective study from incipient nephropathy to overt nephropathy--rationale, study design, treatment plan and baseline characteristics of the incipient to overt: angiotensin II receptor blocker, telmisartan, Investigation on Type 2 Diabetic Nephropathy (INNOVATION) Study.

We planned the INNOVATION study to determine whether telmisartan, an angiotensin-2-receptor blocker, delays the progression of renal disease from incipient nephropathy to overt nephropathy in hypertensive or normotensive Japanese patients with type 2 diabetes mellitus. The INNOVATION study is a randomized, double-blind, placebo-controlled trial. Eligible patients must have incipient nephropathy (defined as a urinary albumin to creatinine ratio of 100-300 mg/g creatinine) and a serum creatinine concentration of < 1.5 mg/dl for men and < 1.3 mg/dl for women. Patients who need treatment with angiotensin II receptor blockers or angiotensin-converting enzyme inhibitors are excluded. Eligible patients are randomly assigned to three groups: telmisartan titrated to 40 mg; telmisartan titrated to 80 mg; or placebo. The primary endpoint is the time from baseline visit to first detection of overt nephropathy (defined by a urinary albumin to creatinine ratio that is > 300 mg/g creatinine and 30% higher than the baseline on at least two consecutive visits). A total of 1855 patients have been enrolled from 160 study centres. In 527 randomized patients (28.4% of the enrolled patients), mean (SD) urinary albumin to creatinine ratio and serum creatinine concentration at baseline were 173.3 (47.2) mg/g creatinine and 0.78 (0.19) mg/dl. Sixty-eight per cent of the patients had hypertension at baseline. Mean (SD) systolic and diastolic blood pressures at baseline were 137.1 (14.6) and 77.5 (10.3) mmHg. The INNOVATION study will determine whether telmisartan, an angiotensin II receptor blocker, provides clinical benefits in hypertensive or normotensive patients with diabetes mellitus and diabetic nephropathy.

Intestinal thrombotic microangiopathy after allogeneic bone marrow transplantation: a clinical imitator of acute enteric graft-versus-host disease.

Thrombotic microangiopathy after bone marrow transplantation (post-BMT TMA) is a serious transplant-related complication. We identified 16 patients with TMA after allogeneic BMT who showed histopathological evidence of intestinal TMA in their gut specimens (six autopsies, 10 biopsies). In all, 14 patients had grade II-IV acute graft-versus-host disease (GVHD). The first seven patients were retrospectively diagnosed with TMA. Since six of them were diagnosed with progressive GVHD at that time because there was no awareness of the existence of intestinal TMA, they received more intensive treatment for GVHD, but all died between days +49 and +253. In contrast, the remaining nine patients were recently diagnosed with intestinal TMA on the basis of colonoscopic biopsies. For eight of these patients, the immunosuppressants were reduced, and the patients' intestinal symptoms improved gradually. Six of the nine patients were still alive 12 months after the diagnosis of TMA. Our findings suggest that the gut may be a site involved in post-BMT TMA, presenting as ischemic enterocolitis. Differentiating intestinal TMA from acute GVHD is important in patients suffering from severe and refractory diarrhea after BMT.

Prooxidant action of gallic acid compounds: copper-dependent strand breaks and the formation of 8-hydroxy-2'-deoxyguanosine in DNA.

Gallic acid and its alkylesters, polyphenolic compounds with antioxidative activity, acted as a prooxidant causing a copper-dependent DNA damage. Treatment of DNA from plasmid pBR322 and calf thymus with gallic acid plus copper ion caused strand scission and the formation of 8-hydroxy-2'-deoxyguanosine in DNA. Addition of catalase protected DNA from the gallic acid/copper-dependent strand breaks and the formation of 8-hydroxy-2'-deoxyguanosine, indicating that hydroxyl radical may participate in the DNA damage. Ethyl-, propyl- and butylgallates showed only a little DNA damage. Octyl- and laurylgallates caused negligible damage of DNA. DNA strand breaks and formation of 8-hydroxy-2'-deoxyguanosine were closely related to the reduction of copper by gallate compounds. These results imply that cuprous ion reduced by gallate derivatives may play a key role in the oxidative cleavage of DNA and the formation of base adduct. The cytotoxic effect of gallate compounds can be explained by their prooxidant action dependent on the reducing activity.

Increased arterial wall stiffness limits flow volume in the lower extremities in type 2 diabetic patients.

OBJECTIVE: To document an association between arterial wall stiffness and reduced flow volume in the lower-extremity arteries of diabetic patients. RESEARCH DESIGN AND METHODS: We recruited 60 consecutive type 2 diabetic patients who had no history or symptoms of peripheral arterial disease (PAD) in the lower extremities and normal ankle/brachial systolic blood pressure index at the time of the study (non-PAD group) and 20 age-matched nondiabetic subjects (control group). We used an automatic device to measure pulse wave velocity (PWV) in the lower extremities as an index of arterial wall stiffness. At the popliteal artery, we evaluated flow volume and the resistive index as an index of arterial resistance to blood flow using gated two-dimensional cine-mode phase-contrast magnetic resonance imaging. RESULTS: Consistent with previous reports, we confirmed that the non-PAD group had an abnormally higher PWV compared with that of the control group (P < 0.001). To further demonstrate decreased flow volume and abnormal flow pattern at the popliteal artery in patients with a higher degree of arterial wall stiffness, we assigned the 60 non-PAD patients to tertiles based on their levels of PWV. In the highest group, magnetic resonance angiograms of the calf and foot arteries showed decreased intravascular signal intensity, indicating the decreased arterial inflow in those arteries. The highest group was also characterized by the lowest late diastolic and total flow volumes as well as the highest resistive index among the groups. From stepwise multiple regression analysis, PWV and autonomic function were identified as independent determinants for late diastolic flow volume (r(2) = 0.300; P < 0.001). CONCLUSIONS: Arterial wall stiffness was associated with reduced arterial flow volume in the lower extremities of diabetic patients.

Identification of a novel GH isoform: a possible link between GH and melanocortin systems in the developing chicken eye.

GH is considered to play a role in the pathogenesis of diabetic retinopathy, causing neovascularization in the retina. The present study was conducted to assess the possibility that GH may play a role in ocular development by determining whether GH is expressed in the eye of the chicken during development. In the 17-d-old embryo, immunocytochemistry detected immunoreactive GH in retinal pigment epithelial (RPE) cells. Characterization of GH mRNA expressed in the eye by RT-PCR and rapid amplification of cDNA 5'-ends revealed it to be a novel GH mRNA transcribed from the middle of the intron 3 of the chicken GH (cGH) gene. The deduced protein, designated small GH isoform (s-cGH), was a cytosolic protein of 16.5 kDa with 140 amino acid (aa) residues, lacking the signal peptide and the N-terminal 71 aa residues of 22-kDa cGH, replacing them with 20 aberrant aa residues, and identical to 22-kDa cGH for the C-terminal 120-aa residue portion. Western blotting determined the molecular size of immunoreactive GH in RPE cells to be 80-84 kDa, similar to the computed molecular mass of s-cGH/GH receptor complex. Furthermore, RT-PCR demonstrated that GH receptor mRNA, but not s-cGH mRNA, was expressed in RPE cells. These results suggest that RPE cell is one of the target cells of s-cGH in the eye. During embryonic development, the immunoreactivity for s-cGH in RPE cells was initially observed on embryonic d 10, and the staining intensity increased and peaked on embryonic d 17. By hatching, s-cGH immunoreactivity in RPE cells was gradually decreased, and it was not detectable after hatching. This ontogenetic staining pattern correlates well with the pattern of the production of alpha MSH in RPE cells. The cell type expressing s-cGH remains to be identified; however, our findings imply a possible involvement of GH in the regulation of ocular development by acting on the intraocular melanocortin system in the chicken.

Cellular mechanisms in the development and progression of diabetic nephropathy: activation of the DAG-PKC-ERK pathway.

Diabetic nephropathy is characterized functionally by glomerular hyperfiltration and albuminuria and histologically by the expansion of glomerular mesangium. We and others have found that protein kinase C (PKC) is activated through an increase in de novo synthesis of diacylglycerol (DAG) from glucose in glomerular mesangial cells cultured under high glucose conditions and in glomeruli of diabetic rats. The activation of PKC could activate further various intracellular signal transduction systems, such as extracellular regulated kinase (ERK). The activation of the DAG-PKC-ERK pathway is considered to be one of the important molecular mechanisms of the development and progression of diabetic nephropathy. To prove this hypothesis, we examined whether the inhibition of the DAG-PKC-ERK pathway could prevent the development of glomerular dysfunction in diabetic animals. First, we found that thiazolidinedione compounds could inhibit PKC activation by activating DAG kinase. Thiazolidinedione compounds were able to prevent glomerular hyperfiltration, albuminuria, and excessive production of extracellular matrix proteins in glomeruli in streptozotocin-induced diabetic rats, a model for type 1 diabetes. Second, we tried to inhibit PKC directly by oral administration of PKC beta inhibitor. PKC beta inhibitor could prevent albuminuria and mesangial expansion in db/db mice, a model for type 2 diabetes. These results confirmed the importance of the activation of the DAG-PKC-ERK pathway in the development of glomerular dysfunction in diabetes.

Dinucleotide repeat polymorphism of matrix metalloproteinase-9 gene is associated with diabetic nephropathy.

BACKGROUND: Although genetic susceptibility has been proposed as an important factor for the development and progression of diabetic nephropathy, the definitive gene has not been identified. To identify the genetic marker for diabetic nephropathy, we examined the association between the (A-C)n dinucleotide repeat polymorphism upstream of the matrix metalloproteinase-9 (MMP-9) gene and diabetic nephropathy in a group of Japanese patients with type 2 diabetes. METHODS: Patients were divided into three groups based on their urinary albumin excretion rate (AER) and the stage of diabetic retinopathy as follows: uncomplicated group (U), normal albuminuria (AER <20 microg/min) without proliferative retinopathy and with the duration of diabetes more than 20 years (N = 32); microalbuminuria group (M), 20 < or = AER < 200 microg/min (N = 155); overt nephropathy group (O), AER > or = 200 microg/min (N = 63). The region containing the dinucleotide repeat upstream of MMP-9 gene was amplified by polymerase chain reaction (PCR). The amplified products were analyzed with 7% formamide/urea acrylamide gel electrophoresis. The promoter constructs of the MMP-9 gene were transfected with the CMV-beta-galactosidase construct into 293 cells using the liposome method. Twenty-four hours after transfection, cells were harvested, and luciferase and beta-galactosidase activities were measured. RESULTS: Nine alleles of the dinucleotide repeat polymorphism (17 to 25 repeats) were identified, and the frequency of each allele in diabetic subjects was not different from that in nondiabetic controls. The frequency of the allele containing 21 repeats (A21) was most abundant (42.4% in control and 45.6% in diabetic subjects), followed by the allele with 23 repeats (A23; 35.4% in control and 27.6% in diabetic subjects). The A21 allele was less frequent in M and O than U (O, 38.9%; M, 45.5%; U, 59.3%, chi2 = 7.18; P < 0.05, O vs. U), while the frequency of the alleles other than A21 was not different among each group. The calculated odds ratio for nephropathy in the noncarrier, heterozygote, or homozygote of A21 allele was 3.38, 1.97, and 0.2, respectively. Furthermore, the promoter assay for the MMP-9 gene revealed that the A21 allele had a higher promoter activity compared with other alleles. No significant correlation was observed between serum MMP-9 concentrations and the MMP-9 gene polymorphism. CONCLUSION: These results indicate that the patients with A21 allele of the MMP-9 gene may be protected from the development and progression of diabetic nephropathy. Thus, the microsatellite polymorphism upstream of the MMP-9 gene could be a useful genetic marker for diabetic nephropathy.

Aluminum-induced apoptosis in PC12D cells.

The addition of aluminum-maltol complex to PC12D cells induced a time-dependent and concentration-dependent growth inhibition as well as cell death, whereas aluminum chloride or maltol alone did not affect the viability of PC12D cells. Apoptosis of differentiated PC12D cells was assessed by using terminal deoxynucleotidyltransferase-mediated 2'-deoxyuridine-5'-triphosphate nick end labeling (TUNEL) technique to detect DNA strand breaks in situ. The number of TUNEL-positive cells treated with aluminum-maltol increased with time in the treatment cultures. The ability of aluminum ion to elevate intracellular reactive oxygen species was determined by fluorescence in PC12D cells loaded with the oxidant-sensitive dye 2',7'-dichlorofluorescin diacetate. Aluminum ion incorporated to PC 12D cells causes apoptotic cell death by enhancing the generation of reactive oxygen species.