Korean J Urol.  2007 Oct;48(10):1050-1057. 10.4111/kju.2007.48.10.1050.

The Significance of Periurethral Fibrosis and the Change of Nitric Oxide Synthase Containing Nerves in the Urethra of Diabetic Rats

• Chung YG
• Yoo HS
• Kwon YH
• Park CS
• Lim WS
• Ryu JK
• Lee T
• Yoon SM

• KMID: 2139738
• DOI: http://doi.org/10.4111/kju.2007.48.10.1050

Abstract

PURPOSE: We have previously demonstrated that increased urethral resistance was more prominent in diabetic rats than in controls. This may result from a compressive obstruction such as damage of the urethral nerve containing nitric oxide. Another possible cause for urethral obstruction could be a constrictive obstruction such as a periurethral fibrosis. In the present study, we investigated the changes in the expression of nitric oxide synthase(NOS) isoforms(compressive obstruction) and collagen subtypes (constrictive obstruction) in the urethral tissues of non-insulin dependent diabetic rats.
MATERIALS AND METHODS
Thirty-six male Sprague-Dawley rats(18 diabetic rats and 18 control rats), bred from birth, were included in this study. Diabetes mellitus was induced by intraperitoneal administration of streptozotocin(90mg/kg) on the second day after birth. Urethral tissues were harvested at 12, 24 and 36 weeks after induction of diabetes and were stained for neuronal NOS(nNOS) and Masson trichrome. We also performed reverse transcriptase-polymerase chain reaction or Western blot analysis to evaluate mRNA or protein expression of NOS isoforms and collagen subtypes in the urethral tissues.
RESULTS
Immunohistochemical staining and Western blot analysis of nNOS revealed that the immunoreactivity and nNOS expression in the urethra was lower in the diabetic rats than in the controls. The Masson trichrome staining showed that there was urethral fibrosis in the diabetic rats. The mRNA or protein expression of collagen subtypes, especially type I collagen, were higher in the diabetic rat urethra than in the controls.
CONCLUSIONS
These data suggest that the increased urethral resistance in diabetic rats may be attributable to a decrease in the urethral nNOS expression and an increase in collagen content. Urethral dysfunction as well as a cystopathy may play an important role in the pathogenesis of diabetes- induced voiding dysfunction. (Korean J Urol 2007;48:1050-1057)

Keyword

Diabetic cystopathy; Urethra; Nitric oxide; Collagen

MeSH Terms

Animals
Blotting, Western
Collagen
Collagen Type I
Diabetes Mellitus
Fibrosis*
Humans
Male
Neurons
Nitric Oxide Synthase*
Nitric Oxide*
Parturition
Protein Isoforms
Rats*
Rats, Sprague-Dawley
RNA, Messenger
Urethra*
Urethral Obstruction
Collagen
Collagen Type I
Nitric Oxide
Nitric Oxide Synthase
Protein Isoforms
RNA, Messenger

Figure

• 

  Fig. 1 Immunohistochemical staining for the nNOS in the urethra of control (A series) and diabetic (B series) rats at 24 weeks of age. The nNOS immunoreactivities in each area were lower in the diabetic rats than the activities in the controls. Arrows denote nNOS-positive nerve fibers. Left figures, lamina propria (×200); Middle figures, smooth muscle layer (×400); Right figures, adventitia (×400). NOS: nitric oxide synthase.

• 

  Fig. 2 Western blot for nNOS and eNOS. (A) Representative gel pictures showing the protein expression of nNOS and eNOS in rat urethral tissues. (B) The nNOS protein expression was lower in the diabetic rats than in the controls at 24 and 36 weeks of age. (C) The eNOS protein expression was lower in diabetic rats than in the controls at 24 weeks of age. β-actin was used as a loading control. CON: control group, STZ: streptozotocin-induced diabetic group, NOS: nitric oxide synthase.

• 

  Fig. 3 Masson trichrome staining in 12-week-old control rats (A), 12-week-old diabetic rats (B), 24-week-old control rats (C), 24-week-old diabetic rats (D), 36-week-old control rats (E), and 36-week-old diabetic rats (F). Urethral fibrosis as evidenced by increase of collagen content compared to control rats was noted in diabetic rats.

• 

  Fig. 4 RT-PCR for type I and type III collagen subtypes. (A) Representative gel pictures showing the gene expression of type I and type III collagen in rat urethral tissues. (B) The gene expression of type I collagen was higher in the diabetic rats than in the controls at 12 and 24 weeks of age. (C) No significant difference was found in the gene expression of type III collagen between the groups. GAPDH was used as an internal control for the RT-PCR. CON: control group, STZ: streptozotocin-induced diabetic group, GAPDH: glyceraldehyde-3-phosphate dehydrogenase, RT-PCR: reverse transcript-polymerase chain reaction.

• 

  Fig. 5 Western blot analysis for type I collagen. (A) Representative gel pictures showing the protein expression of type I collagen in rat urethral tissues. (B) The expression of type I collagen was higher in the diabetic rats than in controls at 12 and 24 weeks of age. β-actin was used as a loading control. CON: control group, STZ: streptozotocin-induced diabetic group.

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