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Enhancing kidney DDAH-1 expression by adenovirus delivery reduces ADMA and ameliorates diabetic nephropathy.

ABSTRACT: Endothelial dysfunction, characterized by reduced bioavailability of nitric oxide and increased oxidative stress, is a hallmark characteristic in diabetes and diabetic nephropathy (DN). High levels of asymmetric dimethylarginine (ADMA) are observed in several diseases including DN and are a strong prognostic marker for cardiovascular events in patients with diabetes and end-stage renal disease. ADMA, an endogenous endothelial nitric oxide synthase (NOS3) inhibitor, is selectively metabolized by dimethylarginine dimethylaminohydrolase (DDAH). Low DDAH levels have been associated with cardiac and renal dysfunction, but its effects on DN are unknown. We hypothesized that enhanced renal DDAH-1 expression would improve DN by reducing ADMA and restoring NOS3 levels. DBA/2J mice injected with multiple low doses of vehicle or streptozotocin were subsequently injected intrarenally with adenovirus expressing DDAH-1 (Ad-h-DDAH-1) or vector control [Ad-green fluorescent protein (GFP)], and mice were followed for 6 wk. Diabetes was associated with increased kidney ADMA and reduced kidney DDAH activity and DDAH-1 expression but had no effect on kidney DDAH-2 expression. Ad-GFP-treated diabetic mice showed significant increases in albuminuria, histological changes, glomerular macrophage recruitment, inflammatory cytokine and fibrotic markers, kidney ADMA levels, and urinary thiobarbituric acid reactive substances excretion as an indicator of oxidative stress, along with a significant reduction in kidney DDAH activity and kidney NOS3 mRNA compared with normal mice. In contrast, Ad-h-DDAH-1 treatment of diabetic mice reversed these effects. These data indicate, for the first time, that DDAH-1 mediates renal tissue protection in DN via the ADMA-NOS3-interaction. Enhanced renal DDAH-1 activity could be a novel therapeutic tool for treating patients with diabetes.

SUBMITTER: Wetzel MD

PROVIDER: S-EPMC7052661 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Enhancing kidney DDAH-1 expression by adenovirus delivery reduces ADMA and ameliorates diabetic nephropathy.

Wetzel Michael D MD Gao Ting T Stanley Kristen K Cooper Timothy K TK Morris Sidney M SM Awad Alaa S AS

American journal of physiology. Renal physiology 20200106 2

Endothelial dysfunction, characterized by reduced bioavailability of nitric oxide and increased oxidative stress, is a hallmark characteristic in diabetes and diabetic nephropathy (DN). High levels of asymmetric dimethylarginine (ADMA) are observed in several diseases including DN and are a strong prognostic marker for cardiovascular events in patients with diabetes and end-stage renal disease. ADMA, an endogenous endothelial nitric oxide synthase (NOS3) inhibitor, is selectively metabolized by ...[more]

PMID: 31904280

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Project description:Diabetic cardiomyopathy (DCM) is a form of idiopathic heart disease, with signs including hypertrophy of myocardial cells, hypertension‑independent fibrosis and coronary artery disease. Considering the involvement of dimethylarginine dimethylaminohydrolase 2 (DDAH2) in diabetes, it was hypothesized that DDAH2 may be beneficial to cardiac function and myocardial fibrosis during the progression of DCM with involvement of the DDAH/asymmetric NG, NGdimethyl‑L‑arginine (ADMA)/nitric oxide synthase (NOS)/nitric oxide (NO) signaling pathway. Following establishment of diabetic rat models, diabetes‑related blood biochemical indices and cardiac function were measured in diabetic rats treated with lentivirus expressing DDAH2, short hairpin RNA against DDAH2, or L‑NNA (inhibitor of NOS) to identify the roles of DDAH2 in DCM. The functional roles of DDAH2 in DCM were further determined through detection of the levels of collagen I, matrix metalloproteinase 2 (MMP2) and tissue inhibitor of metalloproteinase 2 (TIMP2). The H9C2 myocardial cell line was selected for in vitro experiments. The effects of DDAH2 on the migration of myocardial cells under high glucose conditions were also examined. To further investigate the underlying regulatory mechanism of DDAH2 in DCM, the contents of ADMA and NO, and the activities of DDAH and NOS were observed. The DCM model rats treated with DDAH2 exhibited reduced left ventricular end‑diastolic pressure, and decreased blood glucose, total cholesterol, triglyceride, fasting blood glucose, and fasting insulin levels, but exhibited increased left ventricular systolic pressure and maximum rate of left ventricular pressure rise/fall levels in myocardial tissues. Myocardial cells under high glucose conditions treated with DDAH2 showed reductions in collagen I, MMP2 and TIMP2, indicating that DDAH2 reduced cell migration. Decreased levels of ADMA and NO but increased levels of DDAH and NOS were observed following treatment with DDAH2, indicating that the DDAH/ADMA/NOS/NO pathway was activated. These results reveal that the overexpression of DDAH2 attenuates myocardial fibrosis and protects against DCM through activation of the DDAH/ADMA/NOS/NO pathway in DCM rats. These results indicate that DDAH2 is a potential therapeutic candidate for the treatment of DCM.

Project description:ObjectiveEndothelial dysfunction plays a pivotal role in the development of diabetic cardiovascular complications. Accumulation of endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) and inhibition of dimethylarginine dimethylaminohydrolase (DDAH) activity have been involved in diabetic endothelial dysfunction. This study was to investigate the effect of pyrrolidine dithiocarbamate (PDTC) on impairment of endothelium-dependent vasodilatation in diabetic rats and its potential mechanism.MethodsDiabetic rats were induced by a single intraperitoneal injection of streptozotocin (60mg/kg), and PDTC (10mg/kg) was given in drinking water for 8 weeks. Blood glucose and serum ADMA concentrations were measured in experimental rats. Recombinant adenovirus encoding human DDAH2 gene were constructed and ex vivo transferred to isolated rat aortas. The maximal relaxation (Emax) and half maximal effective concentration (EC50) of aortic rings response to accumulative concentrations of acetylcholine and vascular DDAH activity were examined before and after gene transfection.ResultsDiabetic rats displayed significant elevations of blood glucose and serum ADMA levels compared to control group (P<0.01). Vascular DDAH activity and endothelium-dependent relaxation of aortas were inhibited, as expressed by the decreased Emax and increased EC50 in diabetic rats compared to control rats (P<0.01). Treatment with PDTC not only decreased blood glucose and serum ADMA concentration (P<0.01) but also restored vascular DDAH activity and endothelium-dependent relaxation, evidenced by the higher Emax and lower EC50 in PDTC-treated diabetic rats compared to untreated diabetic rats (P<0.01). Similar restoration of Emax, EC50 and DDAH activity were observed in diabetic aortas after DDAH2-gene transfection.ConclusionsThese results indicate that PDTC could ameliorate impairment of endothelium-dependent relaxation in diabetic rats. The underlying mechanisms might be related to preservation of vascular DDAH activity and consequent reduction of endogenous ADMA in endothelium via its antioxidant action. This study highlights the therapeutic potential of PDTC in impaired vasodilation and provides a new strategy for treatment of diabetic cardiovascular complications.

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Enhancing kidney DDAH-1 expression by adenovirus delivery reduces ADMA and ameliorates diabetic nephropathy.

Publications

Enhancing kidney DDAH-1 expression by adenovirus delivery reduces ADMA and ameliorates diabetic nephropathy.

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