Dysregulation of dimethylargininedimethylaminohydrolase/asymmetric dimethylarginine pathway in rat type II diabetic nephropathy

Abstract

An impaired generation of nitric oxide has been associated with diabetic renal disease. In order to elucidate the underlying molecular mechanisms into how nitric oxide synthesis is impaired in diabetic renal disease, we examined changes in activities and expressions of some renal enzymes involved in nitric oxide production during the development of diabetic nephropathy in type II diabetic Otsuka Long-Evans Tokushima Fatty rats. Ten-week old Otsuka Long-Evans Tokushima Fatty (n = 40) and control Long-Evans Tokushima Otsuka rats (n = 20) were given drinking water containing 20% sucrose to accelerate the development of diabetic nephropathy. Otsuka Long-Evans Tokushima Fatty rats developed diabetic nephropathy in an age-dependent manner. Renal nitric oxide synthase activities in Otsuka Long-Evans Tokushima Fatty rats gradually declined with the progression of diabetic mellitus and were significantly lower than those of age-matched Long-Evans Tokushima Otsuka rats after 22 weeks of age. The lower activities of renal nitric oxide synthase in Otsuka Long-Evans Tokushima Fatty rats were correlated with relatively higher levels of renal free asymmetric dimethylarginine, an endogenous nitric oxide synthase inhibitor, and were also correlated with decreased activities of dimethylargininedimethylaminohydrolase which metabolizes asymmetric dimethylarginine to citrulline. These results imply that dimethylargininedimethylaminohydrolase dysregulation may play an important role in the development of diabetic nephropathy by increasing asymmetric dimethylarginine levels, which leads to inhibition of renal nitric oxide synthesis.

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

Fig. 1

Alteration of pathophysiological parameters in LETO and OLETF rats. A, Body weight changes of LETO rats (○, n = 4 for each time point) and OLETF rats (I, n = 8 for each point) during the course of the experiment. B, Plasma glucose AUC of LETO rats (open bars, n = 4 in each time points) and OLETF rats (closed bars, n = 8 in each points) in OGTT. C, Alteration of the pancreatic islet area of LETO (open bars, n = 3 in each time point) and OLETF (closed bars, n = 3 in each time point) in OGTT. Areas of islet (3–46 in each rats) on pancreatic section after HE staining were measured by NIH image software. D–G, Histological features of the glomeruli in LETO rats and OLETF rats. PAS staining demonstrates expansion of mesangial area (arrows) in LETO rat at 10 (D), 27 (E) weeks and in OLETF rats at 19 (F), 27 (G) weeks. Values are mean ± SD. Statistically significant: *p<0.05, **p<0.01 when compared with age-matched LETO rats.

Fig. 2

Renal NOS activities and protein expression levels in LETO and OLETF rats. A, NOS activities measured in whole kidney homogenates of LETO rats (open bars, n = 4) and OLETF rats (closed bars, n = 7–8). B, Expression of eNOS, iNOS and β-actin proteins in the kidney of LETO rats (n = 2) and OLETF rats (n = 2) determined by western blotting analyses. C and D, summarize eNOS/β-actin iNOS/β-actin expressions. Values are mean ± SD. Statistically significant: *p<0.05, **p<0.01 when compared to age-matched LETO rats, and ^#p<0.05, ^##p<0.01 when compared with 10 week old rats.

Fig. 3

Renal DDAH activities and protein expression levels in LETO and OLETF rats. A, DDAH activities in whole kidney homogenates of LETO rats (open bars, n = 4) and OLETF rats (closed bars, n = 7–8). B, Expression of DDAH1, DDAH2, and β-actin proteins in the kidney in LETO rats (n = 2) and OLETF rats (n = 2). C and D, summarize DDAH1/β-actin and DDAH2/β-actin expressions. Values are mean ± SD. Statistically significant: *p<0.05, **p<0.01 when compared to age-matched LETO rats, and ^#p<0.01 when compared with 10 week old rats.

Fig. 4

Levels of renal free ADMA, SDMA, and arginine in LETO and OLETF rats. Renal free ADMA, SDMA, and arginine (Arg) in LETO rats (open bars, n = 3–4) and OLETF rats (closed bars, n = 5–6) were analyzed by LC-ESI-MS/MS as described in Materials and Methods section. Levels of ADMA (A), ratio of Arg/ADMA (B) and ADMA/SDMA (C) were summarized. Values are mean ± SD. Statistically significant: *p<0.05, **p<0.01 when compared to age-matched LETO rats, and ^#p<0.05 when compared with 10 week old rats.