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.

Keywords: asymmetric dimethylarginine; diabetic nephropathy; dimethylarginine dimethylaminohydrolase; nitric oxide.

Fig. 1.

Diabetes increases kidney asymmetric dimethylarginine (ADMA) and reduces kidney dimethylarginine dimethylaminohydrolase (DDAH) activity and DDAH-1 expression. A: kidney ADMA levels were determined by ELISA. B: DDAH activity was determined as described in materials and methods. C and D: levels of DDAH-1 and DDAH-2 protein in normal and diabetic mouse kidneys were determined by Western blot analysis and normalized to GAPDH. Western blots are representative of 5 mice/group. Data are presented as means ± SE. *P < 0.05 compared with normal.

Fig. 2.

Administration of adenovirus expressing dimethylarginine dimethylaminohydrolase-1 (Ad-h-DDAH-1) reduces albuminuria in diabetic mice. Urine was collected from mice and analyzed for the urine albumin-to-creatinine ratio. Data are presented as means ± SE; n = 10–13 mice/group. *P < 0.005 compared with normal; #P < 0.05 compared with adenovirus expressing green fluorescent protein (Ad-GFP)-treated diabetic mice.

Fig. 3.

Administration of adenovirus expressing dimethylarginine dimethylaminohydrolase-1 (Ad-h-DDAH-1) improves mouse kidney DDAH activity and reduces renal asymmetric dimethylarginine (ADMA) in diabetic mice. A: kidneys were analyzed for DDAH activity as described in materials and methods. B: ADMA levels were measured in mouse kidney samples by ELISA. Data are presented as means ± SE; n = 8–14 mice/group. *P < 0.05 compared with normal; #P < 0.05 compared with adenovirus expressing green fluorescent protein (Ad-GFP)-treated diabetic mice.

Fig. 4.

Administration of adenovirus expressing dimethylarginine dimethylaminohydrolase-1 (Ad-h-DDAH-1) increases kidney endothelial nitric oxide synthase (NOS3) mRNA and reduces urinary thiobarbituric acid-reactive substances (TBARS) in diabetic mice. A: total RNA was isolated from mouse kidneys, and RT-PCR was performed to determine NOS3 mRNA levels normalized to 18S rRNA. B: mouse 24-h urine was collected for the measurement of TBARS. Data are presented as means ± SE; n = 5–10 mice/group. *P < 0.05 and **P < 0.001 compared with normal; #P < 0.005 compared with adenovirus expressing green fluorescent protein (Ad-GFP)-treated diabetic mice.

Fig. 5.

Administration of adenovirus expressing dimethylarginine dimethylaminohydrolase-1 (Ad-h-DDAH-1) reduces kidney pathology in diabetic mice. Paraffin-embedded mouse kidney sections were subjected to periodic acid-Schiff staining. Scale bar = 50 µm (×40) and 20 µm (×100). Data are presented as means ± SE; n = 8–11 mice/group. *P < 0.005 compared with normal; #P < 0.005 compared with adenovirus expressing green fluorescent protein (Ad-GFP)-treated diabetic mice.

Fig. 6.

Administration of adenovirus expressing dimethylarginine dimethylaminohydrolase-1 (Ad-h-DDAH-1) reduces glomerular macrophage infiltration in diabetic mice. A: glomerular macrophage recruitment was visualized by immunohistochemical staining using Mac-2 antibody, and pictures were taken at ×100 magnification. Red arrows indicate macrophages. Scale bar = 20 µm. B: macrophage numbers per glomerulus were counted in a blinded manner. The number of glomerular macrophages was counted in 40 glomeruli/section (number of macrophages in glomeruli divided by the number of glomeruli) under ×40 magnification and averaged. Data are presented as means ± SE; n = 8–12 mice/group. *P < 0.001 compared with normal; #P < 0.005 compared with adenovirus expressing green fluorescent protein (Ad-GFP)-treated diabetic mice.

Fig. 7.

Administration of adenovirus expressing dimethylarginine dimethylaminohydrolase-1 (Ad-h-DDAH-1) reduces inflammatory cytokines and fibrotic markers in diabetic mice. RT-PCR was performed on RNA extracted from kidney and analyzed for the indicated genes using indicated Taqman primers. Threshold cycle (C_t) values were normalized to 18S rRNA values and then compared with normal mice. Data are presented as means ± SE; n = 6–7 mice/group. *P < 0.01 and **P < 0.001 compared with normal; #P < 0.05 compared with adenovirus expressing green fluorescent protein (Ad-GFP)-treated diabetic mice.