Mitsuo Tanimoto

Attenuating effect of angiotensin-(1–7) on angiotensin II-mediated NAD(P)H oxidase activation in type 2 diabetic nephropathy of KK-Ay/Ta mice

ANG-(1-7) is associated with vasodilation and nitric oxide synthase stimulation. However, the role of ANG-(1-7) in type 2 diabetes mellitus is unknown. In this study, we examined the hypothesis that ANG-(1-7) attenuates ANG II-induced reactive oxygen species stress (ROS)-mediated injury in type 2 diabetic nephropathy of KK-A(y)/Ta mice. KK-A(y)/Ta mice were divided into four groups: 1) a control group; 2) ANG II infusion group; 3) ANG II+ANG-(1-7) coinfusion group; and 4) ANG II+ANG-(1-7)+d-Ala(7)-ANG-(1-7) (A779) coinfusion group. In addition, primary mesangial cells were cultured and then stimulated with 25 mM glucose with or without ANG II, ANG-(1-7), and A779. The ANG II+ANG-(1-7) coinfusion group showed a lower urinary albumin/creatinine ratio increase than the ANG II group. ANG-(1-7) attenuated ANG II-mediated NAD(P)H oxidase activation and ROS production in diabetic glomeruli and mesangial cells. ANG II-induced NF-κB and MAPK signaling activation was also attenuated by ANG-(1-7) in the mesangial cells. These findings were related to improved mesangial expansion and to fibronectin and transforming growth factor-β1 production in response to ANG II and suggest that ANG-(1-7) may attenuate ANG II-stimulated ROS-mediated injury in type 2 diabetic nephropathy. The ACE2-ANG-(1-7)-Mas receptor axis should be investigated as a novel target for treatment of type 2 diabetic nephropathy.

Glomerular changes in the KK‐Ay/Ta mouse: A possible model for human type 2 diabetic nephropathy

Background:  In type 2 diabetic nephropathy, there is no animal model which has been completely matched with humans. Advanced glycation end products (AGE) and transforming growth factor‐beta (TGF‐β) are closely related to hyperglycaemia and their pathobiochemistry could explain diabetic nephropathy. The objective of the present study was to evaluate the KK‐Ay/Ta mouse as a suitable model for type 2 diabetic nephropathy including pathological changes and immunohistochemical analyses of AGE and TGF‐β, compared with the non‐diabetic BALB/cA mouse.

Increased serum endogenous secretory receptor for advanced glycation end-product (esRAGE) levels in type 2 diabetic patients with decreased renal function.

BACKGROUND The binding of advanced glycation end-products (AGEs) to their receptor for AGEs (RAGE) may play an important role in the development of diabetic vascular complications. Recently, soluble RAGE (sRAGE) has been identified as an alternative splicing form of RAGE. Furthermore, administration of sRAGE improved atherosclerosis in type 2 diabetic mice. OBJECTIVE The objective of the present study is to investigate the role of endogenous secretory RAGE (esRAGE) as a biological marker for type 2 diabetic nephropathy, and also to determine whether serum esRAGE levels are associated with serum AGEs [including Nepsilon-(carboxymethyl) lysine-protein adducts (CML) and pentosidine] levels. MATERIALS AND METHODS Serum esRAGE levels were examined in 107 type 2 diabetic patients including those on hemodialysis (HD). Diabetic patients were divided into three groups as follows: Group A [patients without nephropathy, i.e. normoalbuminuric stage (AER<30microg/mg creatinine)], Group B [patients with nephropathy (AER>30microg/mg creatinine) but excluding HD patients], and Group C (HD patients). RESULTS Serum esRAGE and AGEs (including CML and pentosidine) levels in Group C were significantly higher than in Group A or B. In single linear univariate correlation, serum esRAGE levels were correlated using body mass index (BMI), duration of diabetes, and serum creatinine, high-density lipoprotein (HDL)-cholesterol and AGEs (including CML and pentosidine) levels. Furthermore, in stepwise multivariate regression analysis, the levels of serum creatinine and duration of diabetes were independently associated with serum esRAGE levels. CONCLUSION Serum esRAGE levels are associated with the severity of renal dysfunction and duration of diabetes in type 2 diabetic patients.

Relationship between Abdominal Fat Accumulation and Insulin Resistance in Hemodialysis Patients

It is well known that obesity and insulin resistance are closely related to the development of type 2 diabetes. However, the exact pathogenic mechanism underlying the insulin resistance in renal disease has not been clarified. The purpose of the present study was to clarify the contribution of abdominal (visceral and subcutaneous) fat accumulation to insulin resistance and various clinical parameters, including C-reactive protein (CRP), in hemodialysis (HD) patients. Visceral and subcutaneous fat areas (VFA and SFA) were evaluated at the umbilical level by CT. Insulin resistance was estimated by the homeostasis model assessment−insulin resistance index (HOMA-IR) in 80 HD patients. Insulin resistance and CRP seemed to be closely correlated with fat-related parameters such as body mass index (BMI), VFA and SFA. HOMA-IR was positively correlated with BMI, VFA, SFA, triglycerides (TG), remnant-like particle (RLP)-cholesterol and CRP in simple regression analysis. In multiple stepwise regression analysis, SFA and RLP-cholesterol were predominant determinants of HOMA-IR in HD patients. Furthermore, CRP was positively correlated with BMI, VFA, SFA, TG, high-density lipoprotein (HDL)-cholesterol, atherosclerosis index (AI), immunoreactive insulin (IRI) and HOMA-IR in simple regression analysis. In multiple stepwise regression analysis, VFA and HDL-cholesterol were predominant determinants of CRP in HD patients. In conclusion, insulin resistance and CRP were related to fat-related parameters such as BMI, VFA and SFA in HD patients. Furthermore, the contribution of SFA to insulin resistance was much higher than that of VFA, while the opposite relation was recognized for CRP. (Hypertens Res 2008; 31: 83−88)

Effect of Combination Therapy with Angiotensin Receptor Blocker and 1,25-Dihydroxyvitamin D3 in Type 2 Diabetic Nephropathy in KK-Ay/Ta Mice

Background: Although angiotensin II type 1 receptor blockers (ARB) have beneficial effects in patients with diabetic nephropathy, they may induce a compensatory increase in renin. Renin exhibits profibrotic actions independent of angiotensin II, which is regulated by extracellular signal-regulated kinase 1 and 2 (ERK1/2). Calcitriol (1,25(OH)2D3) is a negative inhibitor of the renin-angiotensin system and the present study examined the effects of combination therapy with an ARB and 1,25(OH)2D3 on diabetic nephropathy in KK-Ay/Ta mice. Methods: KK-Ay/Ta mice were divided into four groups: ARB group, 1,25(OH)2D3 group, combination group, and control group. The urinary albumin/creatinine ratio (ACR) was measured and the renal expression of renin, p-ERK1/2 and TGF-β1 protein determined. Results: The levels of urinary ACR in the combination group were significantly lower than those in the ARB or control group. Renal expression of renin in the ARB group was significantly increased compared with the control group but was significantly decreased in both the 1,25(OH)2D3 and combination group. Renal expression of p-ERK1/2 in the combination group was significantly decreased compared with the control or ARB group. Expression of TGF-β1 protein in the ARB and combination groups, especially the combination group, was significantly decreased compared with those in the control group. Conclusions: These data suggest that the addition of 1,25(OH)2D3 to therapy with ARB further reduced proteinuria by suppressing the compensatory increase in renin expression in type 2 diabetic nephropathy. These effects might relate to suppression of renin, ERK1/2 and TGF-β1 expression which may or may not depend on angiotensin II.

Effect of Exercise on Kidney Function, Oxidative Stress, and Inflammation in Type 2 Diabetic KK-Ay Mice

Exercise is recommended for the management of type 2 diabetes, but its effects on diabetic nephropathy (DN) are still unknown. We hypothesized that appropriate exercise improves early DN via attenuation of inflammation and oxidative damage. Type 2 diabetic KK-A y mice, a spontaneous DN model, underwent two different kinds of exercise (i.e., moderate and low intensity). Sedentary mice or those undergoing an exercise regimen causing no significant body weight loss were used. We examined the urinary excretion of albumin, number of podocytes and macrophages, renal expressions of HIF-1α and MCP-1, and biomarkers of oxidative stress such as urinary 8-OHdG and serum SOD. Exercise reduced urinary levels of albumin and also maintained the number of podocytes in the exercised KK-A y mice independently of improvements of overweight and hyperglycemia, although moderate-intensity exercise increased expression of HIF-1α. Sedentary KK-A y mice showed increased expression of MCP-1 and infiltration of macrophage, increased urinary 8-OhdG, and decreased serum SOD levels compared with exercised KK-A y mice. On the whole, low-intensity exercise attenuates progression of early DN without affecting marked renal ischemia. Reduction rates of urinary albumin and maintained podocyte numbers, with parallel improvements in oxidative damage and inflammation, are related to beneficial effects of exercise in diabetic kidney disease.

Mindin: a novel marker for podocyte injury in diabetic nephropathy

BACKGROUND GeneChip Expression Analysis was employed to survey the glomerular gene expression profile in a type 2 diabetes (T2D) model of KK/Ta mice fed with a high-calorie diet (HC), and we focused on the role of mindin (also called spondin 2), whose expression is upregulated by HC. METHODS Isolated glomeruli from three 20-week-old KK/Ta mice fed with HC or a standard diet (SD) were dissected. Total RNA was extracted and labelled for hybridization using the Affymetrix GeneChip Mouse Genome 430 2.0 Array. The gene expression profile was compared between the HC and SD groups using GeneSpring 7.3.1 software. Mindin expression was examined using real-time PCR, western blot analysis and immunohistochemical staining in the glomeruli, cultured podocytes and urine samples of both mice and humans. RESULTS Podocyte foot process effacement was observed in mice fed with HC. The mindin protein expression levels in mice were localized in the podocytes, and their levels in the glomeruli were increased in the HC group compared with the SD group. The levels of urinary mindin in the HC group at 16 weeks of age were also significantly higher than those in the SD group although albumin/creatinine ratio (ACR) did not differ between the groups. Furthermore, the levels in patients with T2D were higher than those in healthy individuals and increased gradually with increases in ACR. CONCLUSIONS Mindin could be related to podocyte injury and appears to be an early biomarker of the progression of diabetic nephropathy.

Combination effects of enalapril and losartan on lipid peroxidation in the kidneys of KK-Ay/Ta mice.

Background: Adenosine monophosphate activated protein kinase (AMPK) has a protective effect on lipid peroxidation. Adiponectin and AMPK might have a role in the pathogenesis of diabetic nephropathy. Blockade of the renin-angiotensin system (RAS) increases adiponectin levels and reduces oxidative stress. The objective of the present study was to examine lipid peroxidation via adiponectin and AMPK activation in the kidneys of KK-Ay/Ta mice by RAS inhibitors, such as enalapril and/or losartan. Methods: KK-Ay/Ta mice were given enalapril (2.5 mg/kg/day) and/or losartan (25 mg/kg/day), or hydralazine (25 mg/kg/day) in the drinking water for 8 weeks starting at 8 weeks of age. They were divided into 5 groups as follows: enalapril 2.5 mg/kg/day treatment group (n = 5), losartan 25 mg/kg/day treatment group (n = 5), enalapril 2.5 mg/kg/day + losartan 25 mg/kg/day combination treatment group (n = 5), hydralazine 25 mg/kg/day treatment group (n = 5) and tap water group as the untreated group (n = 5). The urinary albumin/creatinine ratio (ACR), serum adiponectin and systemic blood pressure were measured as test parameters. Expressions of adiponectin, phospho-AMPKα (p-AMPKα) and phospho-acetyl CoA carboxylaseβ (p-ACCβ) in the kidneys were evaluated by Western blot analyses. Pathological changes of glomeruli were evaluated by light microscopy. Accumulations of Nε-(carboxymethyl) lysine (CML), malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE) in glomeruli were evaluated by immunohistochemical analyses. Results: Enalapril and/or losartan improved levels of urinary ACR with activation of adiponectin, p-AMPKα and p-ACCβ in the kidneys. CML, MDA and 4-HNE expressions in glomeruli were significantly suppressed by enalapril and/or losartan, especially in the combination treatment group. Conclusions: It appears that enalapril and/or losartan, especially in combination, inhibited accumulation of CML/MDA/4-HNE in diabetic renal tissues. These effects might be related to lipid peroxidation via tissue-specific activation of adiponectin and AMPK.

Pathogenesis and Novel Treatment from the Mouse Model of Type 2 Diabetic Nephropathy

Diabetic nephropathy (DN) is the leading cause of end-stage kidney disease worldwide. However, current treatments remain suboptimal. Many factors, such as genetic and nongenetic promoters, hypertension, hyperglycemia, the accumulation of advanced glycation end products (AGEs), dyslipidemia, and albuminuria/proteinuria itself, influence the progression of this disease. It is important to determine the molecular mechanisms and treatment of this disease. The development of diabetes results in the formation of AGEs, oxidative stress, and the activation of the renin-angiotensin-aldosterone system (RAAS) within the kidney, which promotes progressive inflammation and fibrosis, leading to DN and declining renal function. A number of novel therapies have also been tested in the experimental diabetic model, including exercise, inhibitors of the RAAS (angiotensin type 1 receptor blockers (ARB), angiotensin-converting enzyme (ACE) inhibitors), inhibitors of AGE (pyridoxamine), peroxisome proliferator-activated receptor (PPAR) γ agonists (pioglitazone), inhibitors of lipid accumulation (statins and eicosapentaenoic acid (EPA)), and the vitamin D analogues. This review summarizes the advances in knowledge gained from our studies and therapeutic interventions that may prevent this disease.

Contribution of subcutaneous fat accumulation to insulin resistance and atherosclerosis in haemodialysis patients

BACKGROUND Whereas visceral fat accumulation (VFA) is related to insulin resistance and atherosclerosis in both haemodialysis (HD) patients and the general population, little is known about the role of subcutaneous fat accumulation (SFA). The purpose of the present study was to examine and confirm the relationship between abdominal fat accumulation (AFA) and various clinical parameters in HD patients. METHODS Two hundred and thirty-three HD patients were recruited, including 120 with type 2 diabetes. Abdominal fat distribution was evaluated by computed tomography (CT) scans. Systemic atherosclerosis was assessed by intima-media thickness (IMT) using high-resolution B-mode ultrasonography. The insulin resistance was estimated by the homeostasis model assessment-insulin resistance (HOMA-IR). RESULTS Spearmans analysis revealed that both VFA and SFA showed a significant relationship with HOMA-IR and also that SFA was correlated significantly with IMT in all HD patients. SFA was an independent risk factor associated with HOMA-IR and IMT in multiple regression analysis. Neither body mass index (BMI) nor VFA was a predominant determinant of HOMA-IR and IMT. IMT in HD patients with high SFA/low BMI groups was significantly higher than in the low SFA/high BMI groups. CONCLUSION It appears that there is a close relationship between SFA and insulin resistance or atherosclerosis in HD patients. It was suggested that SFA plays important roles related to carbohydrate or lipid metabolism in HD patients.