Maki Murakoshi

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.

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.

Prediction of diagnosis of immunoglobulin A nephropathy prior to renal biopsy and correlation with urinary sediment findings and prognostic grading.

Several clinical markers correlate well with the diagnosis and prognosis of IgA nephropathy (IgAN). In the present study, we re‐evaluated the usefulness of these four clinical markers for prediction of the diagnosis of patients with IgAN through a comparison between many more patients with IgAN and those with other types of renal diseases. 364 patients with IgAN and 289 with other types of renal disease were examined. An analysis was performed prior to renal biopsy, using clinical markers including, serum IgA, serum IgA/C3 ratio, number of red blood cells in urinary sediments, and urinary protein. Patients with IgAN were divided into four groups according to histopathological findings. Presence of microscopic hematuria, persistent proteinuria, high serum IgA levels, and the serum IgA/C3 ratios are useful for prediction of diagnosis of IgAN and distinguishing it from other renal diseases. Blood pressure, urinary protein, serum uric acid, renal function, and urinary sediment findings may be useful for prediction of prognostic grading in patients with IgAN. J. Clin. Lab. Anal. 22:114–118, 2008.

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.

Role of the TNF pathway in the progression of diabetic nephropathy in KK-Ay mice

Chronic inflammation promotes the progression of diabetic nephropathy (DN). However, the role of TNF-α remains unclear. The objectives of the present study were to examine whether TNF-α inhibition with a soluble TNF receptor (TNFR)2 fusion protein, i.e., etanercept (ETN), improves the early stage of DN in the type 2 diabetic model of the KK-A(y) mouse and to also investigate which TNF pathway, TNFR1 or TNFR2, is predominantly involved in the progression of this disease. ETN was injected intraperitoneally into mice for 8 wk. Renal damage was evaluated by immunohistochemistry, Western blot analysis, and/or real-time PCR. In vitro, mouse tubular proximal cells were stimulated by TNF-α and/or high glucose (HG) and treated with ETN. ETN dramatically improved not only albuminuria but also glycemic control. Renal mRNA and/or protein levels of TNFR2, but not TNF-α and TNFR1, in ETN-treated KK-A(y) mice were significantly decreased compared with untreated KK-A(y) mice. mRNA levels of ICAM-1, VCAM-1, and monocyte chemoattractant protein-1 and the number of F4/80-positive cells were all decreased after treatment. Numbers of cleaved caspase-3- and TUNEL-positive cells in untreated mice were very few and were not different from ETN-treated mice. In vitro, stimulation with TNF-α or HG markedly increased both mRNA levels of TNFRs, unlike in the in vivo case. Furthermore, ETN partly recovered TNF-α-induced but not HG-induced TNFR mRNA levels. In conclusion, it appears that ETN may improve the progression of the early stage of DN predominantly through inhibition of the anti-inflammatory action of the TNF-α-TNFR2 pathway.

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.

Pleiotropic effect of pyridoxamine on diabetic complications via CD36 expression in KK-Ay/Ta mice

AIM Pyridoxamine inhibits the development of diabetic complications. CD36 is a receptor/transporter which binds fatty acids of lipoproteins. The objective of the present study was to examine the pleiotropic effects of pyridoxamine, especially CD36 expression in KK-A(y)/Ta mice with type 2 diabetic nephropathy. METHODS KK-A(y)/Ta mice were divided into 2 groups as follows: pyridoxamine treatment group and a tap water group as controls. The urinary ACR, fasting serum insulin, TG and lipoprotein subclasses were measured as biochemical parameters. The renal expressions of malondialdehyde (MDA) were evaluated by immunohistochemistry. CD36 mRNA expressions in kidney and adipose tissue were also evaluated using real-time PCR. RESULTS Pyridoxamine decreased levels of urinary ACR, serum TG, especially VLDL and fasting serum insulin. MDA accumulation in the pyridoxamine treated group was significantly lower than those in the non-treatment group. The CD36 accumulation and mRNA expressions in kidney and adipose tissue in the treatment group were significantly higher than those in the non-treatment group. CONCLUSIONS It appears that pyridoxamine ameliorated lipid peroxidation and insulin resistance in KK-A(y)/Ta mice. This pleiotropic effect of pyridoxamine was related to CD36 expression in the kidney and adipose tissue.

Role of mindin in diabetic nephropathy.

A number of studies have shown that proinflammatory cytokines have important roles in determining the development of microvascular diabetic complications, including nephropathy. Inflammatory biomarkers should be useful for diagnosis or monitoring of diabetic nephropathy. Mindin (spondin 2) is a member of the mindin-/F-spondin family of secreted extracellular matrix (ECM) proteins. Recent studies showed that mindin is essential for initiation of innate immune response and represents a unique pattern-recognition molecule in the ECM. Previously, we demonstrated that the levels of urinary mindin in patients with type 2 diabetes were higher than those in healthy individuals. We propose that urinary mindin is a potent biomarker for the development of diabetic nephropathy.