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Keywords:

  • erectile dysfunction;
  • lower urinary tract symptoms;
  • oxidative stress;
  • partial bladder outlet obstruction;
  • transforming growth factor-β1

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

There is a growing body of evidence to support the direct link between obstructive bladder dysfunction and erectile dysfunction (ED). However, there have been few pathophysiological studies to determine the relationship between lower urinary tract syndrome (LUTS) and ED. As the transforming growth factor-β1 (TGF-β1) that induces the synthesis of collagen in the penile tissues is critical for the development of ED, the first aim of this study was to investigate the expression of TGF-β1 in the penis from male rabbits with chronic partial bladder outlet obstruction (PBOO). Besides, it has been suggested that oxidative stress plays a significant role in the pathophysiological mechanism of ED. Thus, the second aim of this study was to further investigate whether the urinary or serum oxidative stress markers are involved in chronic PBOO-induced penile dysfunction. A total of 16 male New Zealand White rabbits were separated equally into four groups: a control group and PBOO groups obstructed for 2, 4 and 8 weeks respectively. Using the RT-PCR and Western blot analysis, a progressive increase of TGF-β1 in penis was found at 2, 4 and 8 weeks after obstruction. Moreover, the biomarkers for oxidative stress or oxidative damage were significantly detected in the penis of rabbits after PBOO, which include the enhancement of 8-hydroxy-2′-deoxyguanosine (8-OHdG) in urine and plasma, plasma malondialdehyde (MDA) and total antioxidant capacity (TAC), as well as reduction of glutathione (GSH). On the basis of our results, the increase of TGF-β1 and elevated systemic oxidative stress may play key roles to contribute to penile dysfunction after chronic PBOO.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Epidemiological studies have provided clear evidence that lower urinary tract syndrome (LUTS) and erectile dysfunction (ED) are strongly linked (Rosen et al., 2003; Vallancien et al., 2003; Li et al., 2005) with obstructive LUTS being a better predictor of ED than irritative LUTS (Elliott et al., 2004; McVary, 2006). However, it is still unclear in our current understanding of the pathophysiological mechanisms, which show the relationship between LUTS and ED (McVary, 2006). Recently the mechanistic link between LUTS and ED proposed by Chang et al. (2002) was examined using the rabbit model with partial bladder outlet obstruction (PBOO). The results showed that the corpus cavernosum smooth muscle (CCSM) obtained from PBOO-treated rabbits with documented bladder dysfunction did not relax as well when subject to electrical field stimulation (EFS) when compared with sham-operated animals (Chang et al., 2002). Besides, Gur et al. (2008) also reported that rats with PBOO showed lower in vivo erectile responses than controls. Likewise, our previous study revealed that CCSM from rabbits with 8-week obstruction showed further decreased relaxation responses to EFS when compared with the 2-week group (Lin et al., 2008a). It is noteworthy that there was a visually apparent extensive increase in the density and distribution of collagen compared with the smooth muscle component in the penis after chronic PBOO. However, the molecular mechanism between the increased distribution of collagen and penile dysfunction is still poorly unknown. TGF-β1 is involved in the response to oxidative stress and is well known to play a fundamental role in the development of tissue fibrosis (Lijnen et al., 2000; Bujak & Frangogiannis, 2007). Thus, we hypothesize that TGF-β1 may be involved in the pathology of PBOO-induced penile dysfunction in the rabbit model.

Oxidative stress, which means an imbalance in the levels of reactive oxygen species (ROS) and the antioxidants, might play a significant role in the pathophysiological mechanism of ED (Agarwal et al., 2006). Improving the balance of oxidative status to decrease oxidative stress could be a beneficial therapeutic intervention in treating ED (Yasuda et al., 2008; Hirata et al., 2009). It has been proven that either serum or urinary oxidative stress markers were elevated in patients with ED (Agarwal et al., 2006). To our knowledge, so far there is no report regarding the oxidative stress markers in patients with ED and LUTS. Penile dysfunction has been shown in rabbits with chronic PBOO. In addition, the role of TGF-β1 on the pathology of ED and LUTS is still equivocal. Therefore, in this study, we first investigated whether the expression of TGF-β1 in the penis obtained from male rabbits with chronic PBOO is significantly changed from control levels. The second aim of this study was to investigate the serum oxidative stress markers in rabbits with chronic PBOO.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Experimental design

All animals were housed and treated according to the guidelines outlined in the Handbook of Laboratory Animal Breeding and Research Center, National Science Council of Taiwan. The Animal Ethics Committee of Chang Gung Medical Foundation approved all the protocols employed in these experiments. Sixteen young New Zealand White male rabbits (3–5 kg; 15–20 weeks old) were obtained from the Laboratory Animal Center of Chang Gung Medical Foundation (Chia-Yi, Taiwan). Sixteen rabbits were divided into four groups labelled as controls, 2, 4, and 8 weeks of obstruction respectively.

Surgical procedure for creating PBOO

Rabbits in the obstructed groups were anaesthetized by an intramuscular injection of a 1 mL/kg ketamine hydrochloride (Ketalar; Parke-Davis, Taipei, Taiwan) and Rompum (Bayer, Leverkusen, Germany) mixture. Each bladder was catheterized with an 8-French Foley catheter and the bladder was exposed through a midline incision. Mild bladder outlet obstruction was created by tying a 2-0 silk ligature loosely around the catheterized urethra and the catheter was then removed. The surgical induction procedure of the PBOO and all materials were the same in all of the groups. The wound was closed in layers and gentamicin (4 mg/kg, IM) was administered daily for the first 2 days postoperatively (Lin et al., 2007). Control rabbits received a sham operation. At 2, 4, and 8 weeks after the operation, blood and urine samples were immediately collected from the rabbits for further analysis.

Isolation of corpus cavernosum and Western blot

Each penis was surgically removed from the rabbits. The dissected penis was then washed with Tyrode’s buffer (124.9 mm NaCl, 2.5 mm KCl, 23.8 mm NaHCO3, 0.5 mm MgCl2, 0.4 mm NaH2PO4, 1.8 mm CaCl2, and 5.5 mm glucose) and incubated at 37 °C gassed with 95% O2 and 5% CO2. The corpus cavernosum was then dissected from the surrounding tunica and immediately frozen with liquid nitrogen and then stored at −80 °C. Equal amounts (40 μg/mL) of total proteins extracted from the corpus cavernosum of rabbits in all groups were separated on an 8% SDS-PAGE and transferred to a piece of PVDF membrane (Immobilon-P; Millipore, Billerica, MA, USA) with Towbin’s buffer (25 mm Tris, 192 mm glycine, and 20% methanol). The membranes were then blocked with 5% non-fat milk in Tween/Tris-buffered saline (TTBS, 10 mm Tris, pH 7.5, 100 mm NaCl, and 0.05% Tween-20). This was followed by hybridization with a rabbit polyclonal primary antibody to TGF-β1 (Abcam, Cambridge, UK) for 60 min at 37 °C in a rocking incubator followed by incubation with goat polyclonal secondary antibody to rabbit IgG (Abcam) for another 60 min at 37 °C. The membrane was washed with TTBS 5 times for 5 min. The protein bands on the membrane were visualized with enhanced chemiluminescence (ECL) substrate (Amersham Pharmacia Biotech, Buckinghamshire, UK) on a Kodak Image Station 440CF and analysed with Kodak ID Image Analysis Software (Scientific Image System, Rochester, NY, USA). To confirm equivalent protein loading and transfer, the membranes were stripped with 0.2 N NaOH for 20 min at room temperature, washed with TTBS, stained in Bradford reagent (0.01% Coomassie blue G-250, 9.5% ethanol, 8.5% H3PO4) for 30 min, and then destained (50% methanol, 1.0% acetic acid) until the background was reduced and the total proteins were clearly visible.

RNA extraction and RT-PCR

Penile tissue was homogenized with liquid nitrogen and total RNA was extracted from the resulting powder using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. First-strand cDNA was synthesized from 2 μg of total RNA by an oligo(dT) 12–18 primer and the High-Capacity cDNA Reverse Transcription Kit (M-MLV; Invitrogen) according to the manufacturer’s instructions (Invitrogen) in a total reaction volume of 20 μL. The cDNA was amplified by PCR in 25 μL of PCR Master Reaction Mix kit (Gene Mark, Taichung, Taiwan) containing 1.25 units of DNA polymerase, 200 μm dNTP, and 0.4 mm of each pair of the primers. Primers used for the amplification of the control, GAPDH (forward-ACTCTGGCAAAGTGGATG, reverse-TCCTGGAAGATGGTGATG) and target gene TGF-β1 (forward-AAGGGCTACCACGCCAACTT, reverse-CCGGGTTGTGCTGGTTGTAC) were designed on the basis of the nucleotide sequences in the rabbit species and their product sizes were 166 and 103 base pairs respectively. In previous experiments, we demonstrated that the amount of the DNA product amplified by PCR showed a linear relationship with the number of cycles using specific primers for each gene (not shown). The cycle number corresponding to the exponential phase of the reaction was determined to be 28 cycles at 56 °C annealing temperature for GAPDH and 28 cycles at 56 °C annealing temperature for TGF-β1. The PCR reaction was run at 95 °C for 5 min, followed by 28–30 cycles of 45 sec at 95 °C, 45 sec at 56 °C and 30 sec at 72 °C. The PCR products were electrophoresed on a 2% agarose gel stained with ethidium bromide and the intensities of the band signals were measured by an imaging system (Bio-Rad Laboratories, USA). To avoid unspecific annealing between different probes and to minimize the background, amplification was performed separately for each gene.

Analysis of lipid peroxides in blood plasma

The plasma from each rabbit was collected in the beginning of the experiment and before sacrifice at indicated time points. The lipid peroxidation product MDA was measured using an assay kit according to the manufacturer’s instruction (MDA-586; Oxis Research, Inc., Portland, OR, USA) (Erdelmeier et al., 1998). The method is specific for the determination of MDA.

Measurement of total antioxidant capacity (TAC) of blood plasma

The urine from each rabbit was collected in the beginning of the experiment and before sacrifice at indicated time points. The TAC in plasma was determined using the PAO kit from the Japan Institute for the Control of Aging (Nikken SEIL Co., Fukuroi, Japan) according to the manufacturer’s instructions (De Martino et al., 2001). Briefly, an aliquot of 100 μL plasma were mixed with 50 μL of Cu2+ solution which can be reduced to form Cu+ according to the antioxidant capacity of the sample after incubation at room temperature for 3 min. An aliquot of 50 μL of a chromogenic solution was then added to react with the Cu+ ion to form the stable product and its optimum absorbance at 490 nm was measured immediately. The TAC level of each plasma sample was quantified by using corresponding concentrations of uric acid to reduce the Cu2+ solution and therefore, the antioxidant power of the sample was estimated by multiplying the corresponding uric acid concentration by 2189 (1 mm uric acid = 2189 μm copper reducing power).

Determination of the 8-OHdG level in the plasma and urine samples

The amounts of 8-OHdG in plasma and urine were measured using an 8-OHdG enzyme-linked immunosorbent assay (ELISA) kit (Japan Institute for the Control of Aging, Fukuroi, Japan) according to the manufacturer’s instructions. The kit is a competitive in vitro ELISA system for the quantitative measurement of oxidative DNA adduct 8-OHdG and the specificity of the assay has been established (Tsai et al., 2007). The detection range of 8-OHdG was 0.5–200 ng/mL.

Determination of the GSH level in the plasma sample

The amount of reduced glutathione (GSH) was measured by the Bioxytech GSH/GSSG-412TM Kit (Oxis Research). Briefly, an aliquot of 50 μL plasma was mixed with 350 μL of 5% metaphosphoric acid (MPA) and then centrifuged at 3000 g at 4 °C for 10 min. The GSH level was determined from 50 μL of MPA extract which was incubated in the presence of 5-5′-dithiobis(2-nitrobenzoic acid) (DTNB), NADPH and glutathione reductase (GR) according to the supplied manufacturer’s protocol. The change in absorbance at 412 nm over 3 min was measured using a spectrophotometer for both samples and standards (0 to 3.0 μm of GSH) (Sordillo et al., 2007).

Statistical analysis

The results are expressed as mean ± SD. Statistical analysis was performed using analysis of variance followed by the Student’s t-test for individual differences. A value of p < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Expression of TGF-β1 in the penis from PBOO-treated rabbits

The expression level of TGF-β1 by RT-PCR from the penis was slightly increased after 2 weeks of obstruction (Fig. 1) but it is not statistically significant. However, the expression level of TGF-β1 determined by Western blot showed a significant increase after obstruction for 2, 4 and 8 weeks respectively (Fig. 2).

image

Figure 1.  Determination of the mRNA level of TGF-β1 in the rabbit penis by RT-PCR. Average mRNA expression of TGF-β1 in penis is shown as mean ± SD of the results obtained from four groups of rabbits.

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image

Figure 2.  Representative Western blots for determination of the protein expression level of TGF-β1 in rabbit penis. Average protein expression of TGF-β1 in penis is shown as mean ± SD of the results obtained from four groups of rabbits. *Significantly different from control (p < 0.05).

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Body weight and bladder weight after PBOO

There was no significant difference in the body weight of the rabbits among the four groups (Table 1). However, there were similar significant increases in bladder weight at 2 and 4 weeks of obstruction and a further increase (compared with 2 weeks) at 8 weeks obstruction (Table 1).

Table 1. Comparison of the 8-hydroxy-2′-deoxyguanosine (8-OHdG; urine), malondialdehyde (MDA; plasma), total antioxidant capacity (TAC; plasma), body weight and bladder weight of the rabbits in different groups
Mean ± SDGroupsControl2 weeks4 weeks8 weeks
  1. Each experiment was from four individual New Zealand White rabbits and expressed as mean ± SD (n = 4).

  2. * and **Significant difference from pre-OP (p < 0.05 and p < 0.01, respectively).

  3. ***Significant difference from control (p < 0.01).

  4. ****Significant difference from 2 and 4 weeks after post-OP (p < 0.01).

  5. 8-OHdG, 8-hydroxy-2′-deoxyguanosine; MDA, malondialdehyde; TAC, total antioxidant capacity; Pre-OP, pre-operation; Post-OP, post-operation.

Body weight (kg)Pre-OP3.56 ± 0.343.35 ± 0.293.34 ± 0.162.86 ± 0.42
Post-OP3.3 ± 0.063.31 ± 0.233.31 ± 0.123.46 ± 0.59
Bladder weight (g)Post-OP2.21 ± 0.076.76 ± 0.92***8.88 ± 0.92***12.47 ± 2.04***,****
Urinary 8-OHdG (ng/creatinine)Pre-OP149.47 ± 31.96160.38 ± 21.1163.55 ± 6.41142.21 ± 18.17
Post-OP135.71 ± 53.94210.93 ± 37.44327.59 ± 59.56**357.46 ± 51.69**
Plasma MDA (μm)Pre-OP28.37 ± 0.829.88 ± 3.131.18 ± 3.2523.89 ± 3.82
Post-OP23.61 ± 8.0732.72 ± 2.3343.85 ± 4.79*63.81 ± 8.14**
Plasma TAC (μm)Pre-OP3078.15 ± 295.732597.19 ± 267.83245.44 ± 768.143370.38 ± 463.59
Post-OP2450.81 ± 946.342262.61 ± 344.031760.74 ± 249.77*1380.68 ± 412.37**

Oxidative stress markers in the urine and plasma from PBOO-treated rabbits

There were significant increases in urinary 8-OHdG after 4 and 8 weeks of obstruction (Table 1). In addition, there were progressive significant increases in the MDA level in plasma of the rabbits with 4 and 8 week of obstruction groups, respectively (Table 1). We also found that there were significant increases in the 8-OHdG level in the plasma of the rabbits with 4 and 8 weeks obstruction groups (Fig 3). On the other hand, we found a significant decrease of TAC in the plasma of rabbits with 4 and 8 weeks of PBOO-induced obstruction (Table 1). There was also a significant reduction of GSH level in the plasma of rabbits with 4 and 8 weeks of obstruction respectively (Fig. 4).

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Figure 3.  Comparison of the 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels in the plasma of the rabbits with different durations of partial bladder outlet obstruction. Each bar is presented as the mean ± SD of the results obtained from four groups rabbits. * and **significantly different from pre-obstruction (p < 0.05 and 0.01). PR, pre-obstruction; PO, post-obstruction.

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image

Figure 4.  Comparison of the GSH level in the plasma of rabbits with different durations of partial bladder outlet obstruction. Each bar is presented as the mean ± SD of the results obtained from four groups of rabbits. **Significantly different from pre-obstruction (p < 0.01). PR, pre-obstruction; PO, post-obstruction.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

It has been reported that rats with chronic PBOO showed lower in vivo erectile responses than controls (Gur et al., 2008). In addition, penises obtained from rabbits with PBOO showed poor relaxation in response to different agents of stimulation (Chang et al., 2002). Furthermore, a time course study of chronic PBOO on the smooth muscle–collagen ratio of penis showed that with increased duration of obstruction, there was a decrease in smooth muscle–collagen ratio (Lin et al., 2008a). Also consistent with these previous findings, this current study illustrated that an increased expression of TGF-β1 in penis was prominent after chronic PBOO. On the other hand, we also noted that oxidative stress markers in both the plasma and urine were progressively increased after chronic PBOO. In general, any condition that results in systemic oxidative stress can damage specific physiological systems. We believe that because of the close proximity of the bladder and urethra to the corpus cavernosum, lower urinary tract oxidative stress can result in damage to the corpus cavernosum, which is consistent with ED.

The hypothesis of pathophysiology for the penile fibrosis of rabbits with chronic PBOO includes urethral ligation resulting in constant compression of the penile supply nerve travelling along the bladder neck and vascular compression from the over distended bladder (Chang et al., 2002; Lin et al., 2008a). The results from the sham operated group ruled out the denervation or ischaemia caused by surgery per se. Besides, this study demonstrated that chronic PBOO markedly induced and activated expression of TGF-β1 in the penis, which could also account for evolutionary penile fibrosis after 8 weeks of PBOO. Similarly, in either animal models or humans, increased penile expression of TGF-β1 was well illustrated in different aetiologies of ED, including spinal cord injury, cavernous injury and diabetes mellitus (DM) (Zhang et al., 2008; Jin et al., 2010; Shin et al., 2010). On the other hand, during chronic disease states including hepatic disease, pulmonary disease and peripheral vascular disease, there is an increase in oxidative stress and increased TGF-β1 (Moreland & Nehra, 2002). Taken together, in the above-mentioned literature there are considerable data that connect oxidative stress with increased TGF-β1 levels. Further research is warranted to unravel the mechanism that links PBOO and penile TGF-β1 expression.

There is a growing interest among researchers regarding the role of oxidative stress in the pathophysiological mechanism of ED. Oxidative stress occurs when there is an imbalance between production of ROS and the capability of the antioxidants and antioxidant enzymes to scavenge excess amounts of ROS (Azadzoi et al., 2005). Previous studies have shown that free radical scavengers can restore penile erectile function, indirectly suggesting that free radicals and increased oxidative stress are involved in the pathogenesis of ED (Hirata et al., 2009). Similarly, in this PBOO animal model with profound penile dysfunction, our studies showed strikingly increased oxidative stress markers in rabbits after 8 weeks of obstruction. This result also indicates that PBOO would increase systemic oxidative stress whose effect is also well known in DM, vascular disease or hypercholesterolaemia, and further influence penile function. By contrast, the oxidative stress markers were found to resume after 8 weeks reversal of PBOO (data not shown), and the penile function was also restored simultaneously (Lin et al., 2008b). Hence, the causal relationship between ED and LUTS can be explored from the changes seen in systemic oxidative stress. Additionally, we speculate that oxidative stress markers in plasma could serve as an alternative indicator for objective assessment of ED in patient with LUTS. The validity of clinical application of the above oxidative stress markers requires additional study.

Interestingly, studies conducted by Khan et al. (1999, 2003) showed a significant change in endothelin-1 (ET-1) receptor binding sites in the corpus cavernosum from the rabbits with PBOO. ET-1 and nitric oxide (NO) regulate each other’s synthesis and both are involved in the progression of urinary tract diseases, which include penis dysfunction (Khan et al., 2003). The disruption of the balance between ET-1 and NO is associated with various vascular pathogenesis (Hocher et al., 1997; Albertini & Clement, 1998; Khan et al., 1999, 2003). Therefore, we believe that after severe PBOO treatment, generation of free radicals as well as an imbalanced expression between ET-1 and NO could possibly contribute to the penile dysfunction (Lin et al., 2008a). Further studies to verify the molecular mechanism underlying the relationship between oxidative stress and penile dysfunction are warranted. In summary, we found that the expression of penile TGF-β1 progressively increased after chronic PBOO. Moreover, both urinary and plasma oxidative stress markers were prominent in the rabbits with chronic PBOO and profound penile dysfunction. Both increase in TGF-β1 and oxidative stress could account for, at least in part, the mechanism between LUTS and ED. Furthermore, oxidative stress markers could also be used for the evaluation of penile dysfunction associated with PBOO.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This study was supported by research grants NSC97-2320-B-010-038-MY3 and NSC99-2314-B-182A-039-MY3 from the National Science Council of Taiwan and by grant CMRP690421 from Chang Gung Medical Foundation of Taiwan. We would like to acknowledge the technical support and service of the Core Facilities at National Yang-Ming University.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References