Korean J Urol.  2012 Sep;53(9):636-642. 10.4111/kju.2012.53.9.636.

Differential Expression of Nerve Injury-Induced Protein 1 (Ninjurin 1) in In Vivo and In Vitro Models for Diabetic Erectile Dysfunction

• Kim DK
• Yin GN
• Ryu JK
• Suh JK

• KMID: 2061703
• DOI: http://doi.org/10.4111/kju.2012.53.9.636

Abstract

PURPOSE
Endothelial dysfunction and peripheral neuropathy are important mechanisms responsible for diabetes-induced erectile dysfunction (ED). Nerve injury-induced protein 1 (Ninjurin 1) is known to be related to neuroinflammatory processes and is also reported to induce vascular regression during the developmental period. In the present study, we determined the differential expression of Ninjurin 1 in penile tissue of streptozotocin (STZ)-induced diabetic mice with ED.
MATERIALS AND METHODS
Diabetes was induced in 8-week-old C57BL/6J mice by intraperitoneal injections of STZ (50 mg/kg for 5 days). Eight weeks later, erectile function was measured by electrical stimulation of the cavernous nerve (n=6 per group). The penis was then harvested for immunohistochemical analysis and Western blot analysis for Ninjurin 1 (n=4 per group). We also determined Ninjurin 1 expression in primary cultured mouse cavernous endothelial cells (MCECs) incubated under the following conditions: normal glucose condition (5 mM), high-glucose condition (30 mM), and high-glucose condition (30 mM)+insulin (1 nM).
RESULTS
The expression of Ninjurin 1 protein was significantly higher in both cavernous endothelial cells and the dorsal nerve bundle of diabetic mice than in those of controls. In the in vitro study in MCECs, Ninjurin 1 expression was also significantly increased by the high-glucose condition and was returned to baseline levels by treatment with insulin.
CONCLUSIONS
Regarding the role of Ninjurin 1 in neuropathy and vascular regression, it would be interesting to examine the effects of inhibition of Ninjurin 1 on erectile function in animal models of ED with a vascular or neurogenic cause.

Keyword

Diabetes mellitus; Endothelium; Erectile dysfunction; Nerve; Ninj1

MeSH Terms

Animals
Blotting, Western
Caves
Diabetes Mellitus
Electric Stimulation
Endothelial Cells
Endothelium
Erectile Dysfunction
Glucose
Injections, Intraperitoneal
Male
Mice
Models, Animal
Penis
Peripheral Nervous System Diseases
Streptozocin
Glucose
Streptozocin

Figure

• 

  FIG. 1 Decrease in erectile function in diabetic mice. (A) Representative intracavernous pressure (ICP) responses to electrical stimulation of the cavernous nerve for the age-matched control and diabetic groups. The stimulus interval is indicated by a solid bar. (B) The ratios of mean maximal ICP and total ICP to mean systolic blood pressure (MSBP) stimulated at 5 V and 12 Hz in each group. Each bar depicts the mean values (±standard error) from n=6 animals per group. DM, diabetes mellitus. a:p<0.01 compared with the control group.

• 

  FIG. 2 Increase in Ninjurin 1 expression in diabetic mouse penis. (A) Representative Western blot analysis showing the protein expression of Ninjurin 1. (B) Data are presented as the relative density of Ninjurin 1 protein compared with that of β-actin. The relative ratio measured in the control group is arbitrarily presented as 1. Each bar depicts the mean values (±standard error) from n=4 animals per group. DM, diabetes mellitus. a:p<0.01 compared with the control group.

• 

  FIG. 3 Differential distribution of Ninjurin 1 in the corpus cavernosum tissue of diabetic mice. Immunofluorescent double-staining of penile tissue performed with antibody to PECAM-1 (an endothelial cell marker) and Ninjurin 1. Note increase in Ninjurin 1 expression and decrease in PECAM-1 expression in diabetic corpus cavernosum tissue (×200). DM, diabetes mellitus; PECAM-1, platelet/endothelial cell adhesion molecule-1.

• 

  FIG. 4 Differential distribution of Ninjurin 1 in the dorsal nerve bundle of diabetic mice. Immunofluorescent double-staining of penile tissue performed with antibody to neurofilament (an axonal marker) and Ninjurin 1. Note increase in Ninjurin 1 expression and decrease in nerve content in diabetic dorsal nerve bundle (×400). DM, diabetes mellitus; DAPI, 4,6-diamidino-2-phenylindole.

• 

  FIG. 5 Increase in Ninjurin 1 expression in mouse cavernous endothelial cells exposed to the high-glucose (HG) condition, which was reversed by treatment with insulin. (A) Characterization of primary cultured mouse cavernous endothelial cells. Fluorescent immunocytochemistry of primary mouse cavernous endothelial cells with antibody against platelet/endothelial cell adhesion molecule-1 (PECAM-1) (positive marker) and antibody against CD90 and myosin (negative markers) (×200). (B) Representative Western blot analysis showing the protein expression of Ninjurin 1. After serum starvation for 24 hours, cavernous endothelial cells were incubated under the following conditions for 48 hours: normal glucose condition (NG, 5 mM); high-glucose condition (30 mM); high-glucose condition (30 mM)+insulin (IN, 1 nM). (C) Data are presented as the relative density of Ninjurin 1 protein compared with that of β-actin. The relative ratio measured in the normal glucose condition is arbitrarily presented as 1. Each bar depicts the mean values (±standard error) from four independent experiments. DAPI, 4,6-diamidino-2-phenylindole. a:p<0.01 compared with the other groups, b:p<0.01 compared with HG group.

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