Role of the effect of inhibition of neutral endopeptidase on vascular and neural complications in streptozotocin-induced diabetic rats

Abstract

We have previously shown that treating streptozotocin-induced diabetic rats, an animal model of type 1 diabetes, with Ilepatril (an inhibitor of neutral endopeptidase and angiotensin converting enzyme (ACE)) improves vascular and neural function. In this study we sought to determine the individual effect of inhibition of neutral endopeptidase and ACE on diabetes-induced vascular and neural dysfunction. After 4 weeks of untreated diabetes, rats were treated for 12 weeks with Ilepatril, Enalapril (ACE inhibitor) or Candoxatril (neutral endopeptidase inhibitor) followed by analysis of neural and vascular function. Diabetes caused slowing of motor and sensory nerve conduction, thermal hypoalgesia, reduction in intraepidermal nerve fiber density in the hindpaw and impairment in vascular relaxation to acetylcholine and calcitonin gene-related peptide in epineural arterioles of the sciatic nerve and to atrial natriuretic peptide and calcitonin gene-related peptide in renal arteries. Inhibition of neutral endopeptidase or ACE improved neural function; however, dual inhibition of neutral endopeptidase and ACE with Ilepatril tended to have the greatest efficacy. Ilepatril and Candoxatril treatment of diabetic rats was more efficacious in improving vascular responsiveness in epineurial arterioles than treatment with Enalapril. Ilepatril, Enalapril or Candoxatril treatment of diabetic rats were all efficacious in renal arteries. These studies suggest that combination therapy may be the most effective approach for treatment of diabetic neural and vascular complications.

Fig. 1. Effect of treatment of streptozotocin-diabetic rats with Ilepatril, Enalapril or Candoxatril on motor and sensory nerve conduction velocity

At 12 weeks of age rats were made diabetic using streptozotocin and 4 weeks later were divided into 4 groups. One group was fed a normal diet and the other three groups were fed diets containing Ilepatril, Enalapril or Candoxatril, respectively for 12 weeks. A non-diabetic control group was also included. Afterwards, motor and sensory nerve conduction velocity was examined as described. Data are presented as the mean ± S.E.M in m/sec. The number of rats in each group was the same as shown in Table 1. * P < 0.05, compared to control rats fed the standard diet; + P < 0.05, compared to untreated diabetic rats.

Fig. 2. Effect of treatment of streptozotocin-diabetic rats with Ilepatril, Enalapril or Candoxatril on thermal nociception and intraepidermal nerve fiber density

Rats were treated as describe in Fig. 1 and the group identification is the same. Thermal nociception and intraepidermal nerve fiber density was examined as described. Data are presented as the mean ± S.E.M for thermal nociception in sec and intra epidermal nerve fiber profiles per mm. The number of rats in each group was the same as shown in Table 1. * P < 0.05, compared to control rats fed the standard diet ; + P < 0.05, compared to untreated diabetic rats.

Fig. 3. Effect of treatment of streptozotocin-diabetic rats with Ilepatril, Enalapril or Candoxatril on vascular relaxation by acetylcholine in epineurial arterioles

Rats were treated as describe in Fig. 1. Pressurized arterioles (40 mm Hg and ranging from 60–100 µm luminal diameter) were constricted with U46619 (30–50%) and incremental doses of acetylcholine were added to the bathing solution while recording steady state vessel diameter. Data are presented as the mean of % relaxation ± S.E.M. For these studies two vessels were collected from each rat, studied and the data combined. The number of rats in each group was the same as shown in Table 1. * P < 0.05, compared to control rats fed the standard diet; + P < 0.05, compared to untreated diabetic rats.

Fig. 4. Effect of treatment of streptozotocin-diabetic rats with Ilepatril, Enalapril or Candoxatril on vascular relaxation by calcitonin gene-related peptide in epineurial arterioles

Arterioles were derived from rats as described in Fig. 3. Incremental doses of calcitonin gene-related peptide (CGRP) were added to the bathing solution while recording steady state vessel diameter. Data are presented as the mean of % relaxation ± S.E.M. The number of rats in each group was the same as shown in Table 1. * P < 0.05, compared to control rats fed the standard diet; + P < 0.05, compared to untreated diabetic rats.

Fig. 5. Effect of treatment of streptozotocin-diabetic rats with Ilepatril, Enalapril or Candoxatril on vascular relaxation by atrial natriuretic peptide in renal arteries

Rats were treated as described in Fig. 1. Renal arteries were harvested as described and relaxation to incremental doses of atrial natriuretic peptide (ANP) was determined. Data are presented as the mean of % relaxation ± S.E.M. The number of rats in each group was the same as shown in Table 1. * P < 0.05, compared to control rats fed the standard diet; + P < 0.05, compared to untreated diabetic rats.

Fig. 6. Effect of treatment of streptozotocin-diabetic rats with Ilepatril, Enalapril or Candoxatril on vascular relaxation by calcitonin gene-related peptide in renal arteries

Rats were treated as described in Fig. 1. Renal arteries were harvested as described and relaxation to incremental doses of calcitonin gene-related peptide (CGRP) was determined. Data are presented as the mean of % relaxation ± S.E.M. The number of rats in each group was the same as shown in Table 1. * P < 0.05, compared to control rats fed the standard diet; + P < 0.05, compared to untreated diabetic rats.