-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 IIϩANG-(1-7) coinfusion group; and 4) ANG IIϩANG-(1-7)ϩD-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 IIϩANG-(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-B 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-␤1 production in response to ANG II and suggest that ANG-(1-7) may attenuate ANG II-stimulated ROSmediated 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.THE RECENT DISCOVERY OF THE renal renin-angiotensin system (RAS) (41), angiotensin-converting enzyme-related carboxypeptidase (ACE2), and ANG-(1-7) has changed the way the RAS is viewed. ANG-(1-7) is present in kidneys at concentrations comparable to ANG II and is associated with vasodilation, modulation of sodium and water transport, and stimulation of nitric oxide synthase (NOS) (7,9,24,34). The effect of ANG-(1-7) on diabetes is still not clear. In a recent study using the streptozotocin-induced diabetic rat model, ANG-(1-7) infusion prevented diabetes-induced abnormal vascular responses to norepinephrine, endothelin-1, and ANG II in the perfused mesenteric bed and renal arteries. This study also reported decreased urinary albumin excretion in response to ANG-(1-7) (6, 7, 13). Another study in the same streptozotocin-induced diabetic rat model showed that chronic injection of ANG-(1-7) accelerated diabetic renal injury (36). In db/db mice, glomerular expression of ACE2 was increased, and ACE2 inhibitor use showed increased glomerular staining for fibronectin and an extracellular matrix protein (42). However, there have been no studies on the direct effect of ANG-(1-7) on ANG II-induced diabetic glomerular changes.

To determine correlations among the levels of urinary MMP-9 and type-IV collagen, hyperglycemia, urinary protein excretion, and renal injuries in patients with type 2 diabetic nephropathy, we measured levels of urinary MMP-9 and protein, blood urea nitrogen (BUN), serum creatinine (s-Cr), fasting plasma glucose (FPG), and glycohemoglobin A1c (HbA1c) in 47 diabetic patients and 14 healthy adults. Urinary type-IV collagen was also measured in 28 diabetic patients and seven healthy adults. Patients with diabetic nephropathy were divided into two groups: 1). patients with normoalbuminuria or microalbuminuria (0-299 mg/g.Cr; n=27), and 2). patients with macroalbuminuria (>300 mg/g.Cr; n=20). The mean level of urinary MMP-9 in group 2 was significantly higher than those in healthy adults (P<0.05), and the levels of urinary MMP-9 in patients with diabetic nephropathy increased in accordance with the clinical stage of the disease. The levels of urinary MMP-9 tended to be correlated with HbA1c in these patients, but the correlation was not statistically significant. The mean level of urinary type-IV collagen in group 2 of patients with diabetic nephropathy was significantly higher than that in group 1 and healthy adults. Levels of urinary type-IV collagen in patients with diabetic nephropathy also increased in accordance with the clinical stage of the disease. The results suggest that measurements of urinary MMP-9, as well as urinary type-IV collagen, may be useful for evaluating the degree of renal injuries in patients with type 2 diabetic nephropathy, especially in the early stage.

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-A^y/Ta mice. Methods: KK-A^y/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.

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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-A^y/Ta mice by RAS inhibitors, such as enalapril and/or losartan. Methods: KK-A^y/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 carboxylase_β (p-ACC_β) in the kidneys were evaluated by Western blot analyses. Pathological changes of glomeruli were evaluated by light microscopy. Accumulations of N^ε-(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-ACC_β 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.