- Email: [email protected]
Early molecular changes in bladder hypertrophy due to bladder outlet obstruction
BASIC SCIENCE
EARLY MOLECULAR CHANGES IN BLADDER HYPERTROPHY DUE TO BLADDER OUTLET OBSTRUCTION BRIAN J. FLYNN, HAMAYUN S. MIAN, PETER J. CERA, RONALD L. KABLER, JOSEPH J. MOWAD, ALICE H. CAVANAUGH, AND LAWRENCE I. ROTHBLUM
ABSTRACT Objectives. To determine the temporal relationship between the increase in bladder mass and the expression of growth-associated gene products during bladder hypertrophy due to partial bladder outlet obstruction. Methods. Adult female rats, subjected to partial bladder outlet obstruction, were killed at defined points, and their bladder weight and total protein were determined and compared with sham-operated and nonoperated controls. Hyperplasia was determined by the expression of proliferating cell nuclear antigen, transcription factors, and cyclins in obstructed rat bladders. Bladder protein was fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and expression of the indicated proteins was determined by Western analysis and immunohistochemistry. Results. The mean bladder weight in sham-operated rats remained at 127 ⫾ 17 mg, and the weight in the obstructed animals increased to 239 ⫾ 56 mg at 12 hours, increasing to 486 ⫾ 168 mg by 168 hours. The total bladder protein increased 1.8-fold after 12 hours and continued to increase for the duration of obstruction. The expression of proliferating cell nuclear antigen in the obstructed group did not begin until 24 hours of obstruction. The expression of the transcription factors, upstream binding factor, and c-Jun followed a similar pattern. Cyclin E and C expression increased most significantly after 48 hours. Conclusions. Bladder growth after 12 hours of partial outlet obstruction represents cellular hypertrophy based on the increases in bladder weight and total protein accumulation. Cellular hyperplasia occurs after 24 hours of obstruction as represented by increases in transcription factors and cell cycle-specific proteins. UROLOGY 59: 978–982, 2002. © 2002, Elsevier Science Inc.
T
he urinary bladder functions to store urine at low pressure and expel urine periodically by detrusor contractions.1 The bladder is composed of epithelium, smooth muscle, connective tissue, and extracellular matrix and is capable of responding to mechanical stresses by increasing in mass through numerous cellular and structural changes.2 This rapid and substantial increase in bladder mass is referred to as bladder hypertrophy (BH). Mechanical stress such as diuresis, diabetes, and
This study was supported in part by a Geisinger Health System Multidisciplinary grant to J. Mowad and R. Kabler, and a grant from the NIH (GM-46991) to L. I. Rothblum. From the Departments of Urology and Pathology, and Weis Center for Research, Geisinger Health System, Danville, Pennsylvania Reprint requests: Alice H. Cavanaugh, Ph.D., Weis Center for Research, Geisinger Health System, 100 North Academy Avenue, Danville, PA 17822-2618 Submitted: October 25, 2001, accepted (with revisions): February 6, 2002
978
© 2002, ELSEVIER SCIENCE INC. ALL RIGHTS RESERVED
neuronal injury, as well as bladder outlet obstruction, can all lead to BH.2 Bladder outlet obstruction, which affects 50% to 80% of men older than age 50, is most commonly due to benign prostatic hyperplasia.3 BH is initially compensatory; however, this process can eventually lead to bladder decompensation and subsequent urinary retention.2 Levin et al.4 in their pioneering studies have divided the bladder’s progressive response to partial outlet obstruction into three phases: (a) an initial response during which substantial alterations in bladder mass, pharmacology, and physiology occur; (b) a compensated phase, characterized by a stable bladder mass and stable (or augmented) contractility; and (c) a decompensated phase characterized by a progressive deterioration in contractile and functional status. Clinically, the decompensated bladder may loose its functional ability to empty, resulting in chronic urinary retention. A better understanding of the molecular biology of 0090-4295/02/$22.00 PII S0090-4295(02)01619-9
TABLE I. Bladder weight and total protein accumulation following partial bladder outlet obstruction Sham-Operated Animals Time (hr) 12 24 72 168
n 9 18 13 20
Weight (mg)* 132 122 128 124
⫾ ⫾ ⫾ ⫾
13 19 19 17
Obstructed Animals
Protein (mg)† 10.3 8.7 10.1 8.8
⫾ ⫾ ⫾ ⫾
2.4 3.1 2.1 2.7
n 10 20 17 12
Weight (mg)* 239 295 348 486
⫾ ⫾ ⫾ ⫾
56 88 104 168
Protein (mg)†
P Value‡
⫾ ⫾ ⫾ ⫾
0.005 0.001 0.001 0.001
18.3 23.4 28.6 40.3
6.4 9.8 9.1 14.3
Mean bladder weight and total protein ⫾ standard deviation of sham and obstructed rats from time 0 to the time of bladder harvest. The nonoperated control group (n ⫽ 25) had a mean bladder weight of 101 ⫾ 15 mg and total protein of 5.8 ⫾ 2.6 mg. * Bladder wet weight determined after removing surrounding fat and connective tissue. † Total protein determined by Bio-Rad DC protein assay. The ratio of total bladder protein to bladder wet weight remained the same among control, sham, and obstructed animals, suggesting that edema was not responsible for the increase in bladder weight. This ratio (mg/g) was 70 (control), 76 (12-hr obstructed), 72 (12-hr sham), 79 (24-hr obstructed), 79 (24-hr sham), 82 (72-hr obstructed), 79 (72-hr sham), 82 (168-hr obstructed), and 83 (168-hr sham). ‡ P value performed with paired Student’s t test to compare differences in total protein between sham-operated and obstructed rats at the respective points. Differences were regarded as statistically significant at P ⬍0.05. No statistical difference was found in total protein between sham-operated and control rats (P ⫽ 0.08).
BH may result in the ability to reverse the cascade of events that lead to irreversible decompensation. Previous reports have indicated that the mRNAs for several growth factors may increase or decrease in response to BH.4 –13 However, these studies did not determine whether the protein expression paralleled the changes in mRNA. Because an increase in the amount of mRNA does not necessarily demonstrate an increase in protein, our goal was to investigate the expression of cell cycle-specific and growth-related proteins during BH. We examined the accumulation of mitotic markers, transcription factors, and cyclins in the bladders of rats subjected to partial bladder outlet obstruction. Our results indicate that BH occurs by two distinct processes, hypertrophy followed by hyperplasia. MATERIAL AND METHODS ANIMALS Female Sprague-Dawley rats (⬃60 days old) were obtained from Charles River Laboratories (Charleston, SC). Rats were divided into three groups: controls, sham-operated, and obstructed. The obstructed group underwent a partial, infravesical, standardized degree of obstruction using a modification of the technique of Mattiasson and Uvelius.14 –16 The urethra was intubated with a 3.5F polyvinyl chloride feeding tube. A midline incision was made and the retropubic space developed. A double 4-0 silk ligature was placed loosely around the proximal urethra and secured. The feeding tube was removed, and the midline fascia and skin were reapproximated. Sham-operated animals underwent identical surgical procedures without ligation. The control animals had no surgery. The animals were housed in accordance with the Association and Accreditation of Laboratory Animal Care International (AALAC). Bladders were harvested from each group at 12, 24, 72, and 168 hours after surgery.
TISSUE HARVESTING Individual bladders were harvested after the rats were humanely killed. The bladder was placed in 4°C phosphate-buffered saline solution containing protease inhibitors (1 mM benzamidine, 10 M leupeptin, and 1 g/mL aprotinin). The surrounding fat and connective tissue were removed. The UROLOGY 59 (6), 2002
weight was determined, and the tissue was frozen in liquid nitrogen and stored at ⫺80°C.
MOLECULAR ANALYSIS Individual bladders from the indicated times were homogenized in 20 mM Tris-HCl (pH 7.5), 1% Triton X-100, 100 mM NaCl, and 0.5% NP-40 containing protease inhibitors. Protein concentrations of bladder homogenates were determined by the Bio-Rad DC protein assay (Bio-Rad Laboratories, Hercules, Calif). Protein from each sample was fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto Immobilon P (Millipore Corporation, Bedford, Mass) membranes. The membranes were blocked with 5% milk in phosphate-buffered saline containing 0.05% Tween20. The blots were probed with an antibody to the indicated protein: proliferating cell nuclear antigen (PCNA; Neomarkers, Fremont, Calif), upstream binding factor (UBF),17 c-Jun (BD Transduction Laboratories, Lexington, Ky) and cyclins E, C, and D3 (Santa Cruz Biotechnology, Santa Cruz, Calif). Proteins were visualized using the appropriate secondary antibodies and enhanced chemiluminescence (Amersham-Pharmacia, Piscataway, NJ).
IMMUNOHISTOCHEMISTRY Whole bladders from each group were fixed in formalin after harvest. Paraffin-embedded sections were immunostained using an antibody to PCNA.
STATISTICAL ANALYSIS Statistical analysis was performed using Student’s t test. Statistical significance was at P ⬍0.05.
RESULTS The data in Table I compared the bladder wet weight and total protein of the sham and obstructed animals at the indicated points. The bladder weight of the obstructed animals increased 80% by 12 hours after surgery, with a 78% increase in protein compared with the sham-operated animals. These values continued to increase 24, 72, and 168 hours after obstruction. The difference in bladder weight and protein between the obstructed animals and sham animals was significant at all points (P ⬍0.05). The ratio of total bladder protein 979
FIGURE 1. Western analysis of protein expression: 100 g of protein from homogenates of control, sham, and obstructed bladders were prepared as described in the Material and Methods section, fractionated on 10% sodium dodecyl sulfate-polyacrylamide gels and transferred to Immobilon P membranes. Blots were probed with antibodies to PCNA (1:1000), cyclin E (1:1000), cyclin C (1:1000), cyclin D3 (1:1000), UBF (1:5000), or c-Jun (1:1000). Lane 1, control bladders; lane 2, bladders from sham-operated animals; lanes 3 and 4, 12hour obstructed bladders; lane 5, 24-hour obstructed bladders; lane 6, 72-hour obstructed bladders; and lane 7, 168-hour obstructed bladders. Representative blots are shown. Each analysis was repeated at least three times with samples from different surgical or control groups.
to bladder wet weight remained constant in all groups, suggesting that edema was not responsible for the increase in bladder weight (Table I). The expression of PCNA, a marker for cell division, was determined by Western analysis. PCNA expression showed no difference in the obstructed animals compared with the nonoperated and sham-operated controls (Fig. 1, lanes 1 and 2). It is important to note that the expression of PCNA at 12 hours was not elevated (Fig. 1, lanes 3 and 4). Increased amounts of PCNA were observed after 24 hours of obstruction (lane 5). This increase peaked at 72 hours (lane 6) and remained elevated 168 hours after obstruction (lane 7). The expression of cyclins E, C, and D3 during partial urethra obstruction showed similar patterns (Fig. 1). Cyclins C and E demonstrated significant accumulation by 24 hours, peaked after 72 hours, and were still elevated after 168 hours (Fig. 1, lanes 5, 6, and 7). Cyclin D3 exhibited a slightly 980
different pattern of expression in that significant levels were not apparent until the 72-hour point (lane 7). It should be noted that the cyclin levels in the nonoperated and sham-operated controls were barely detectable (lanes 1, 2). We next determined whether changes in the components of ribosomal RNA (rRNA) synthesis occurred as a result of BH. The expression of the rRNA transcription factor termed UBF was determined (Fig. 1). UBF exists as two forms, UBF 1 and UBF 2, and therefore migrates as a doublet. We found that the amount of UBF increased by 24 hours in obstructed animals (lane 5) but not in the sham-operated rats or controls (lanes 1 and 2). This increase peaked at 72 hours (lane 6) and began to decline by 168 hours of obstruction (lane 7). Cellular division is often accompanied by increase in RNA polymerase II and its associated transcription factors.18 One such factor, c-Jun, was barely detectable in control and sham-operated animals (Fig. 1, lanes 1 and 2). However, protein levels increased in obstructed animals at 24 hours (lane 5) and continued to increase at 168 hours (lane 7). Immunohistochemistry was performed at all points. No difference was found in the PCNA staining between the sham-operated and obstructed animals at 12 hours. However, after 24 hours and for the duration of the obstruction, the immunoreactivity for PCNA was significantly greater in the obstructed animals than in the controls (compare Fig. 2A with 2B). Immunostaining revealed a predominant distribution of PCNA in the urothelium. COMMENT Partial urethral obstruction has been used to induce BH in various animal models.8,12,14 –16,19 –24 Hypertrophy and hyperplasia have both been reported to contribute to the increase in bladder mass that occurs after obstruction.2,4,5,10,12,14,19 –22 We attempted to determine whether these processes occurred simultaneously or in succession. The significant increase in bladder weight and protein accumulation demonstrated that significant bladder growth occurs within 12 hours. However, PCNA expression was unchanged. This mitotic marker did not increase until 24 hours of obstruction. This suggests that the initial increase in bladder mass and protein observed during the first 24 hours is predominantly hypertrophic. Hyperplasia does not occur until after at least 24 hours of obstruction. The immunohistochemical localization of PCNA suggests the increased expression of this mitotic cell marker may originate from the urothelium. The expression of PCNA and cyclins E, C, and D3 were used as the reporters for mitosis in our UROLOGY 59 (6), 2002
FIGURE 2. (A) Immunohistochemical localization of PCNA: photomicrograph from an unobstructed animal showing little immunoreactivity in the urothelium. Reduced from ⫻313. (B) Immunohistochemical localization of PCNA: photomicrograph from an obstructed rat bladder (72 hours) showing significant immunoreactivity concentrated in the urothelium. Reduced from ⫻313.
study. Saito et al.21 and Monson et al.22 both demonstrated an increase in 3H-thymidine incorporation by 24 hours, peaking at 72 hours, and decreasing to control levels by 7 to 14 days. The increased expression of PCNA at 24 hours of obstruction is in agreement with the results of these studies. However, we did not observe a return of protein expression of these markers to control levels at 7 days. It has been demonstrated by Levin et al.4 that virtually all bladder compartments respond to mechanical stress by increasing 3H-thymidine incorporation, thereby suggesting hyperplasia. However, the urothelium is the most reactive. Our immunohistochemical localization of PCNA is in agreement with this report. Cyclins are growth-related proteins that are expressed at specific phases of the cell cycle.25 Sequential formation, activation, and inactivation of cyclins and the regulation of cyclin-dependent kiUROLOGY 59 (6), 2002
nase activity are integral in regulating the mammalian cell cycle.25,26 To our knowledge, the role of cyclins in BH due to any stimulus has never been examined. In our study, the cyclins showed the greatest increase in expression after 72 hours of obstruction. These results again suggest that hyperplasia occurs only after 24 hours of obstruction. An increase in bladder mass, by hypertrophy or hyperplasia, requires an accumulation of protein. This is accomplished by increasing the rate of protein synthesis, with only minor changes in the rate of protein degradation.27 Furthermore, this is usually achieved by increased ribosome biogenesis, including rDNA transcription. Previous studies have demonstrated that changes in UBF gene expression parallel the changes in rDNA transcription during cardiac hypertrophy and also during differentiation of L-6 myoblasts.27,28 Therefore, we sought to determine whether the increase in protein synthesis that occurs during BH was associated with changes in the rDNA transcription apparatus. We did observe a significant increase in the RNA polymerase I transcription factor, UBF, in the bladders of obstructed rats. This change occurred at 24 hours and was sustained for the duration of the experiment. Our findings are consistent with reports of other organs demonstrating increases in UBF during cellular growth.29 However, this is the first report of changes in an RNA polymerase I transcription factor during BH. We continued to examine transcriptional regulation in BH by determining the expression of c-Jun. The Jun and fos family of proteins compose the transcription factor AP-1, which is a major target of mitogen-activated signal transduction. A recent study by Persson et al.30 of nuclear factor B and AP-1 complexes demonstrated increased DNA binding activity of these transcription factors in rat bladder smooth muscle after outlet obstruction and mechanical stretching. Levin et al.,4 in their studies on rabbit BH did not detect any change in the mRNA levels of c-Jun during the first 24 hours. In contrast, we saw a significant increase in the protein expression of c-Jun by 24 hours in obstructed rats. Many critical issues need to be addressed to enable the clinician to treat patients with end-stage bladders. Notably, what are the cellular/molecular events at which point the bladder loses its ability to empty after prolonged obstruction? If we can better understand the molecular biology of BH, it may become possible to reverse the cascade of events that lead to irreversible decompensation. CONCLUSIONS Collectively, our experiments begin to describe the temporal relationship between the increase in 981
bladder mass and the processes responsible for this growth. The increase in bladder mass in the first 12 hours of BH is due to cellular hypertrophy, as evidenced by the increase in bladder weight and total protein and lack of expression of mitotic markers. Future studies will determine whether the expression of these proteins returns to control levels after the obstruction is relieved and at what point this is still possible. Answers to these questions may facilitate the development of novel therapies designed to prevent or reverse the processes that lead to the end-stage bladder. REFERENCES 1. Steers WD: Physiology and pharmacology of the bladder and urethra, in Walsh PC, Retik AB, Vaughn ED, et al (Eds): Campbell’s Urology, 7th ed. Philadelphia, WB Saunders, 1998, vol 1, pp 870 –915. 2. Levin RM, Wein AJ, Buttyan R, et al: Update on bladder smooth-muscle physiology. World J Urol 12: 226 –232, 1994. 3. McConnell JD: Epidemiology, etiology, pathophysiology, and diagnosis of benign prostatic hyperplasia, in Walsh PC, Retik AB, Vaughn ED, et al (Eds): Campbell’s Urology, 7th ed. Philadelphia, WB Saunders, 1998, vol 2, pp 1429 –1452. 4. Levin RM, Monson FC, Haugaard N, et al: Genetic and cellular characteristics of bladder outlet obstruction. Urol Clin North Am 22: 263–283, 1995. 5. Santarosa R, Colombel MC, Kaplan S, et al: Hyperplasia and apoptosis: opposing cellular processes that regulate the response of the rabbit bladder to transient outlet obstruction. Lab Invest 70: 503–510, 1994. 6. Abdel-Gawad M, Elhilali MM, and Huynh H: Alterations of the insulin-like growth factor system of mitogens in hyperplastic bladders of paraplegic rats. J Urol 161: 699 –705, 1999. 7. Chen Y, Gustafsson B, and Arnqvist HJ: IGF-binding protein-2 is induced during development of the urinary bladder hypertrophy in the diabetic rat. Am J Physiol 272: E297– E303, 1997. 8. Chen Y, Bornfeldt KE, Arner A, et al: Increase in insulin-like growth factor I in hypertrophying smooth muscle. Am J Physiol 266: E224 –E229, 1994. 9. Sterle M, Kreft ME, and Batista U: The effect of epidermal growth factor and transforming growth 1 on proliferation and differentiation of urothelial cells urinary bladder explant culture. Biol Cell 89: 263–271, 1997. 10. Chen MW, Levin RM, and Buttyan R: Peptide growth factors in normal and hypertrophied bladder. World J Urol 13: 344 –348, 1995. 11. Koo HP, Santarosa RP, Buttyan R, et al: Early molecular changes associated with streptozocin-induced diabetic bladder hypertrophy in the rat. Urol Res 21: 375–381, 1993. 12. Baskin LS, Sutherland RS, Thomson AA, et al: Growth factors and receptors in bladder development and obstruction. Lab Invest 75: 157–166, 1996. 13. Buttyan R, Jacobs BZ, Blaivas JG, et al: Early molecular response to rabbit bladder outlet obstruction. Neurourol Urodyn 11: 225–238, 1992. 14. Uvelius B, Persson L, and Mattiasson A: Smooth muscle
982
cell hypertrophy and hyperplasia in the rat detrusor after short-time infravesical outflow obstruction. J Urol 131: 173– 176, 1984. 15. Mattiasson A, and Uvelius B: Changes in contractile properties in hypertrophic rat urinary bladder. J Urol 128: 1340 –1342, 1982. 16. Malmgren A, Sjo¨ gren C, Uvelius B, et al: Cystometrical evaluation of bladder instability in rats with infravesical outflow obstruction. J Urol 137: 1291–1294, 1987. 17. O’Mahony DJ, Xie WQ, Smith SD, et al: Differential phosphorylation and localization of the transcription factor UBF in vivo in response to serum deprivation. J Biol Chem 267: 35–38, 1992. 18. Paule MR: Regulation of ribosomal RNA expression, in Paule MR (Ed): Transcription of Ribosomal RNA Genes by Eukaryotic RNA Polymerase I. New York, Springer-Verlag, 1998, pp 195–200. 19. Kato K, Monson FC, Longhurst PA, et al: The functional effects of long-term outlet obstruction on the rabbit urinary bladder. J Urol 14: 600 – 606, 1990. 20. Malkowicz SB, Wein AJ, Elbadawi A, et al: Acute biochemical and functional alterations in the partially obstructed rabbit urinary bladder. J Urol 136: 1324 –1329, 1986. 21. Saito M, Longhurst PA, Tammela TL, et al: Effects of partial outlet obstruction of the rat urinary bladder on micturition characteristics, DNA synthesis and the contractile response to the field stimulation and pharmacological agents. J Urol 150: 1045–1051, 1993. 22. Monson FC, McKenna BA, Wein AJ, et al: Effect of outlet obstruction on 3H-thymidine uptake: a biochemical and radioautographic study. J Urol 148: 158 –162, 1992. 23. Steers WD, and de Groat WC: Effect of bladder outlet obstruction on micturition reflex pathways in the rat. J Urol 140: 864 – 871, 1988. 24. Mostwin JL, Karim OM, Van Koeveringe G, et al: Guinea pig as an animal model for the study of urinary bladder function in the normal and obstructed state. Neurourol Urodyn 13: 137–145, 1994. 25. Li JM, and Brooks G: Cell cycle regulatory molecules (cyclins, cyclin-dependent kinases and cyclin-dependent kinase inhibitors) and the cardiovascular system: potential targets for therapy? Eur Heart J 20: 406 – 420, 1999. 26. Hunter T, and Pines J: Cyclins and cancer II: cyclin D and CDK inhibitors come of age. Cell 79: 573–582, 1994. 27. Hannan RD, Luyken J, and Rothblum LI: Regulation of ribosomal DNA transcription during contraction-induced hypertrophy of neonatal cardiomyocytes. J Biol Chem 271: 3213–3220, 1996. 28. Larson DE, Xie W, Glibetic M, et al: Coordinated decreases in rRNA gene transcription factors and rRNA synthesis during muscle cell differentiation. Proc Natl Acad Sci USA 90: 7933–7936, 1993. 29. Glibetic M, Taylor L, Larson D, et al: The RNA polymerase I transcription factor UBF is the product of a primary response gene. J Biol Chem 270: 4209 – 4212, 1995. 30. Persson K, Dean-McKinney T, Steers WD, et al: Activation of the transcription factors nuclear factor-B and activator protein-1 in bladder smooth muscle exposed to outlet obstruction and mechanical stretching. J Urol 165: 633– 639, 2001.
UROLOGY 59 (6), 2002
EARLY MOLECULAR CHANGES IN BLADDER HYPERTROPHY DUE TO BLADDER OUTLET OBSTRUCTION BRIAN J. FLYNN, HAMAYUN S. MIAN, PETER J. CERA, RONALD L. KABLER, JOSEPH J. MOWAD, ALICE H. CAVANAUGH, AND LAWRENCE I. ROTHBLUM
ABSTRACT Objectives. To determine the temporal relationship between the increase in bladder mass and the expression of growth-associated gene products during bladder hypertrophy due to partial bladder outlet obstruction. Methods. Adult female rats, subjected to partial bladder outlet obstruction, were killed at defined points, and their bladder weight and total protein were determined and compared with sham-operated and nonoperated controls. Hyperplasia was determined by the expression of proliferating cell nuclear antigen, transcription factors, and cyclins in obstructed rat bladders. Bladder protein was fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and expression of the indicated proteins was determined by Western analysis and immunohistochemistry. Results. The mean bladder weight in sham-operated rats remained at 127 ⫾ 17 mg, and the weight in the obstructed animals increased to 239 ⫾ 56 mg at 12 hours, increasing to 486 ⫾ 168 mg by 168 hours. The total bladder protein increased 1.8-fold after 12 hours and continued to increase for the duration of obstruction. The expression of proliferating cell nuclear antigen in the obstructed group did not begin until 24 hours of obstruction. The expression of the transcription factors, upstream binding factor, and c-Jun followed a similar pattern. Cyclin E and C expression increased most significantly after 48 hours. Conclusions. Bladder growth after 12 hours of partial outlet obstruction represents cellular hypertrophy based on the increases in bladder weight and total protein accumulation. Cellular hyperplasia occurs after 24 hours of obstruction as represented by increases in transcription factors and cell cycle-specific proteins. UROLOGY 59: 978–982, 2002. © 2002, Elsevier Science Inc.
T
he urinary bladder functions to store urine at low pressure and expel urine periodically by detrusor contractions.1 The bladder is composed of epithelium, smooth muscle, connective tissue, and extracellular matrix and is capable of responding to mechanical stresses by increasing in mass through numerous cellular and structural changes.2 This rapid and substantial increase in bladder mass is referred to as bladder hypertrophy (BH). Mechanical stress such as diuresis, diabetes, and
This study was supported in part by a Geisinger Health System Multidisciplinary grant to J. Mowad and R. Kabler, and a grant from the NIH (GM-46991) to L. I. Rothblum. From the Departments of Urology and Pathology, and Weis Center for Research, Geisinger Health System, Danville, Pennsylvania Reprint requests: Alice H. Cavanaugh, Ph.D., Weis Center for Research, Geisinger Health System, 100 North Academy Avenue, Danville, PA 17822-2618 Submitted: October 25, 2001, accepted (with revisions): February 6, 2002
978
© 2002, ELSEVIER SCIENCE INC. ALL RIGHTS RESERVED
neuronal injury, as well as bladder outlet obstruction, can all lead to BH.2 Bladder outlet obstruction, which affects 50% to 80% of men older than age 50, is most commonly due to benign prostatic hyperplasia.3 BH is initially compensatory; however, this process can eventually lead to bladder decompensation and subsequent urinary retention.2 Levin et al.4 in their pioneering studies have divided the bladder’s progressive response to partial outlet obstruction into three phases: (a) an initial response during which substantial alterations in bladder mass, pharmacology, and physiology occur; (b) a compensated phase, characterized by a stable bladder mass and stable (or augmented) contractility; and (c) a decompensated phase characterized by a progressive deterioration in contractile and functional status. Clinically, the decompensated bladder may loose its functional ability to empty, resulting in chronic urinary retention. A better understanding of the molecular biology of 0090-4295/02/$22.00 PII S0090-4295(02)01619-9
TABLE I. Bladder weight and total protein accumulation following partial bladder outlet obstruction Sham-Operated Animals Time (hr) 12 24 72 168
n 9 18 13 20
Weight (mg)* 132 122 128 124
⫾ ⫾ ⫾ ⫾
13 19 19 17
Obstructed Animals
Protein (mg)† 10.3 8.7 10.1 8.8
⫾ ⫾ ⫾ ⫾
2.4 3.1 2.1 2.7
n 10 20 17 12
Weight (mg)* 239 295 348 486
⫾ ⫾ ⫾ ⫾
56 88 104 168
Protein (mg)†
P Value‡
⫾ ⫾ ⫾ ⫾
0.005 0.001 0.001 0.001
18.3 23.4 28.6 40.3
6.4 9.8 9.1 14.3
Mean bladder weight and total protein ⫾ standard deviation of sham and obstructed rats from time 0 to the time of bladder harvest. The nonoperated control group (n ⫽ 25) had a mean bladder weight of 101 ⫾ 15 mg and total protein of 5.8 ⫾ 2.6 mg. * Bladder wet weight determined after removing surrounding fat and connective tissue. † Total protein determined by Bio-Rad DC protein assay. The ratio of total bladder protein to bladder wet weight remained the same among control, sham, and obstructed animals, suggesting that edema was not responsible for the increase in bladder weight. This ratio (mg/g) was 70 (control), 76 (12-hr obstructed), 72 (12-hr sham), 79 (24-hr obstructed), 79 (24-hr sham), 82 (72-hr obstructed), 79 (72-hr sham), 82 (168-hr obstructed), and 83 (168-hr sham). ‡ P value performed with paired Student’s t test to compare differences in total protein between sham-operated and obstructed rats at the respective points. Differences were regarded as statistically significant at P ⬍0.05. No statistical difference was found in total protein between sham-operated and control rats (P ⫽ 0.08).
BH may result in the ability to reverse the cascade of events that lead to irreversible decompensation. Previous reports have indicated that the mRNAs for several growth factors may increase or decrease in response to BH.4 –13 However, these studies did not determine whether the protein expression paralleled the changes in mRNA. Because an increase in the amount of mRNA does not necessarily demonstrate an increase in protein, our goal was to investigate the expression of cell cycle-specific and growth-related proteins during BH. We examined the accumulation of mitotic markers, transcription factors, and cyclins in the bladders of rats subjected to partial bladder outlet obstruction. Our results indicate that BH occurs by two distinct processes, hypertrophy followed by hyperplasia. MATERIAL AND METHODS ANIMALS Female Sprague-Dawley rats (⬃60 days old) were obtained from Charles River Laboratories (Charleston, SC). Rats were divided into three groups: controls, sham-operated, and obstructed. The obstructed group underwent a partial, infravesical, standardized degree of obstruction using a modification of the technique of Mattiasson and Uvelius.14 –16 The urethra was intubated with a 3.5F polyvinyl chloride feeding tube. A midline incision was made and the retropubic space developed. A double 4-0 silk ligature was placed loosely around the proximal urethra and secured. The feeding tube was removed, and the midline fascia and skin were reapproximated. Sham-operated animals underwent identical surgical procedures without ligation. The control animals had no surgery. The animals were housed in accordance with the Association and Accreditation of Laboratory Animal Care International (AALAC). Bladders were harvested from each group at 12, 24, 72, and 168 hours after surgery.
TISSUE HARVESTING Individual bladders were harvested after the rats were humanely killed. The bladder was placed in 4°C phosphate-buffered saline solution containing protease inhibitors (1 mM benzamidine, 10 M leupeptin, and 1 g/mL aprotinin). The surrounding fat and connective tissue were removed. The UROLOGY 59 (6), 2002
weight was determined, and the tissue was frozen in liquid nitrogen and stored at ⫺80°C.
MOLECULAR ANALYSIS Individual bladders from the indicated times were homogenized in 20 mM Tris-HCl (pH 7.5), 1% Triton X-100, 100 mM NaCl, and 0.5% NP-40 containing protease inhibitors. Protein concentrations of bladder homogenates were determined by the Bio-Rad DC protein assay (Bio-Rad Laboratories, Hercules, Calif). Protein from each sample was fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto Immobilon P (Millipore Corporation, Bedford, Mass) membranes. The membranes were blocked with 5% milk in phosphate-buffered saline containing 0.05% Tween20. The blots were probed with an antibody to the indicated protein: proliferating cell nuclear antigen (PCNA; Neomarkers, Fremont, Calif), upstream binding factor (UBF),17 c-Jun (BD Transduction Laboratories, Lexington, Ky) and cyclins E, C, and D3 (Santa Cruz Biotechnology, Santa Cruz, Calif). Proteins were visualized using the appropriate secondary antibodies and enhanced chemiluminescence (Amersham-Pharmacia, Piscataway, NJ).
IMMUNOHISTOCHEMISTRY Whole bladders from each group were fixed in formalin after harvest. Paraffin-embedded sections were immunostained using an antibody to PCNA.
STATISTICAL ANALYSIS Statistical analysis was performed using Student’s t test. Statistical significance was at P ⬍0.05.
RESULTS The data in Table I compared the bladder wet weight and total protein of the sham and obstructed animals at the indicated points. The bladder weight of the obstructed animals increased 80% by 12 hours after surgery, with a 78% increase in protein compared with the sham-operated animals. These values continued to increase 24, 72, and 168 hours after obstruction. The difference in bladder weight and protein between the obstructed animals and sham animals was significant at all points (P ⬍0.05). The ratio of total bladder protein 979
FIGURE 1. Western analysis of protein expression: 100 g of protein from homogenates of control, sham, and obstructed bladders were prepared as described in the Material and Methods section, fractionated on 10% sodium dodecyl sulfate-polyacrylamide gels and transferred to Immobilon P membranes. Blots were probed with antibodies to PCNA (1:1000), cyclin E (1:1000), cyclin C (1:1000), cyclin D3 (1:1000), UBF (1:5000), or c-Jun (1:1000). Lane 1, control bladders; lane 2, bladders from sham-operated animals; lanes 3 and 4, 12hour obstructed bladders; lane 5, 24-hour obstructed bladders; lane 6, 72-hour obstructed bladders; and lane 7, 168-hour obstructed bladders. Representative blots are shown. Each analysis was repeated at least three times with samples from different surgical or control groups.
to bladder wet weight remained constant in all groups, suggesting that edema was not responsible for the increase in bladder weight (Table I). The expression of PCNA, a marker for cell division, was determined by Western analysis. PCNA expression showed no difference in the obstructed animals compared with the nonoperated and sham-operated controls (Fig. 1, lanes 1 and 2). It is important to note that the expression of PCNA at 12 hours was not elevated (Fig. 1, lanes 3 and 4). Increased amounts of PCNA were observed after 24 hours of obstruction (lane 5). This increase peaked at 72 hours (lane 6) and remained elevated 168 hours after obstruction (lane 7). The expression of cyclins E, C, and D3 during partial urethra obstruction showed similar patterns (Fig. 1). Cyclins C and E demonstrated significant accumulation by 24 hours, peaked after 72 hours, and were still elevated after 168 hours (Fig. 1, lanes 5, 6, and 7). Cyclin D3 exhibited a slightly 980
different pattern of expression in that significant levels were not apparent until the 72-hour point (lane 7). It should be noted that the cyclin levels in the nonoperated and sham-operated controls were barely detectable (lanes 1, 2). We next determined whether changes in the components of ribosomal RNA (rRNA) synthesis occurred as a result of BH. The expression of the rRNA transcription factor termed UBF was determined (Fig. 1). UBF exists as two forms, UBF 1 and UBF 2, and therefore migrates as a doublet. We found that the amount of UBF increased by 24 hours in obstructed animals (lane 5) but not in the sham-operated rats or controls (lanes 1 and 2). This increase peaked at 72 hours (lane 6) and began to decline by 168 hours of obstruction (lane 7). Cellular division is often accompanied by increase in RNA polymerase II and its associated transcription factors.18 One such factor, c-Jun, was barely detectable in control and sham-operated animals (Fig. 1, lanes 1 and 2). However, protein levels increased in obstructed animals at 24 hours (lane 5) and continued to increase at 168 hours (lane 7). Immunohistochemistry was performed at all points. No difference was found in the PCNA staining between the sham-operated and obstructed animals at 12 hours. However, after 24 hours and for the duration of the obstruction, the immunoreactivity for PCNA was significantly greater in the obstructed animals than in the controls (compare Fig. 2A with 2B). Immunostaining revealed a predominant distribution of PCNA in the urothelium. COMMENT Partial urethral obstruction has been used to induce BH in various animal models.8,12,14 –16,19 –24 Hypertrophy and hyperplasia have both been reported to contribute to the increase in bladder mass that occurs after obstruction.2,4,5,10,12,14,19 –22 We attempted to determine whether these processes occurred simultaneously or in succession. The significant increase in bladder weight and protein accumulation demonstrated that significant bladder growth occurs within 12 hours. However, PCNA expression was unchanged. This mitotic marker did not increase until 24 hours of obstruction. This suggests that the initial increase in bladder mass and protein observed during the first 24 hours is predominantly hypertrophic. Hyperplasia does not occur until after at least 24 hours of obstruction. The immunohistochemical localization of PCNA suggests the increased expression of this mitotic cell marker may originate from the urothelium. The expression of PCNA and cyclins E, C, and D3 were used as the reporters for mitosis in our UROLOGY 59 (6), 2002
FIGURE 2. (A) Immunohistochemical localization of PCNA: photomicrograph from an unobstructed animal showing little immunoreactivity in the urothelium. Reduced from ⫻313. (B) Immunohistochemical localization of PCNA: photomicrograph from an obstructed rat bladder (72 hours) showing significant immunoreactivity concentrated in the urothelium. Reduced from ⫻313.
study. Saito et al.21 and Monson et al.22 both demonstrated an increase in 3H-thymidine incorporation by 24 hours, peaking at 72 hours, and decreasing to control levels by 7 to 14 days. The increased expression of PCNA at 24 hours of obstruction is in agreement with the results of these studies. However, we did not observe a return of protein expression of these markers to control levels at 7 days. It has been demonstrated by Levin et al.4 that virtually all bladder compartments respond to mechanical stress by increasing 3H-thymidine incorporation, thereby suggesting hyperplasia. However, the urothelium is the most reactive. Our immunohistochemical localization of PCNA is in agreement with this report. Cyclins are growth-related proteins that are expressed at specific phases of the cell cycle.25 Sequential formation, activation, and inactivation of cyclins and the regulation of cyclin-dependent kiUROLOGY 59 (6), 2002
nase activity are integral in regulating the mammalian cell cycle.25,26 To our knowledge, the role of cyclins in BH due to any stimulus has never been examined. In our study, the cyclins showed the greatest increase in expression after 72 hours of obstruction. These results again suggest that hyperplasia occurs only after 24 hours of obstruction. An increase in bladder mass, by hypertrophy or hyperplasia, requires an accumulation of protein. This is accomplished by increasing the rate of protein synthesis, with only minor changes in the rate of protein degradation.27 Furthermore, this is usually achieved by increased ribosome biogenesis, including rDNA transcription. Previous studies have demonstrated that changes in UBF gene expression parallel the changes in rDNA transcription during cardiac hypertrophy and also during differentiation of L-6 myoblasts.27,28 Therefore, we sought to determine whether the increase in protein synthesis that occurs during BH was associated with changes in the rDNA transcription apparatus. We did observe a significant increase in the RNA polymerase I transcription factor, UBF, in the bladders of obstructed rats. This change occurred at 24 hours and was sustained for the duration of the experiment. Our findings are consistent with reports of other organs demonstrating increases in UBF during cellular growth.29 However, this is the first report of changes in an RNA polymerase I transcription factor during BH. We continued to examine transcriptional regulation in BH by determining the expression of c-Jun. The Jun and fos family of proteins compose the transcription factor AP-1, which is a major target of mitogen-activated signal transduction. A recent study by Persson et al.30 of nuclear factor B and AP-1 complexes demonstrated increased DNA binding activity of these transcription factors in rat bladder smooth muscle after outlet obstruction and mechanical stretching. Levin et al.,4 in their studies on rabbit BH did not detect any change in the mRNA levels of c-Jun during the first 24 hours. In contrast, we saw a significant increase in the protein expression of c-Jun by 24 hours in obstructed rats. Many critical issues need to be addressed to enable the clinician to treat patients with end-stage bladders. Notably, what are the cellular/molecular events at which point the bladder loses its ability to empty after prolonged obstruction? If we can better understand the molecular biology of BH, it may become possible to reverse the cascade of events that lead to irreversible decompensation. CONCLUSIONS Collectively, our experiments begin to describe the temporal relationship between the increase in 981
bladder mass and the processes responsible for this growth. The increase in bladder mass in the first 12 hours of BH is due to cellular hypertrophy, as evidenced by the increase in bladder weight and total protein and lack of expression of mitotic markers. Future studies will determine whether the expression of these proteins returns to control levels after the obstruction is relieved and at what point this is still possible. Answers to these questions may facilitate the development of novel therapies designed to prevent or reverse the processes that lead to the end-stage bladder. REFERENCES 1. Steers WD: Physiology and pharmacology of the bladder and urethra, in Walsh PC, Retik AB, Vaughn ED, et al (Eds): Campbell’s Urology, 7th ed. Philadelphia, WB Saunders, 1998, vol 1, pp 870 –915. 2. Levin RM, Wein AJ, Buttyan R, et al: Update on bladder smooth-muscle physiology. World J Urol 12: 226 –232, 1994. 3. McConnell JD: Epidemiology, etiology, pathophysiology, and diagnosis of benign prostatic hyperplasia, in Walsh PC, Retik AB, Vaughn ED, et al (Eds): Campbell’s Urology, 7th ed. Philadelphia, WB Saunders, 1998, vol 2, pp 1429 –1452. 4. Levin RM, Monson FC, Haugaard N, et al: Genetic and cellular characteristics of bladder outlet obstruction. Urol Clin North Am 22: 263–283, 1995. 5. Santarosa R, Colombel MC, Kaplan S, et al: Hyperplasia and apoptosis: opposing cellular processes that regulate the response of the rabbit bladder to transient outlet obstruction. Lab Invest 70: 503–510, 1994. 6. Abdel-Gawad M, Elhilali MM, and Huynh H: Alterations of the insulin-like growth factor system of mitogens in hyperplastic bladders of paraplegic rats. J Urol 161: 699 –705, 1999. 7. Chen Y, Gustafsson B, and Arnqvist HJ: IGF-binding protein-2 is induced during development of the urinary bladder hypertrophy in the diabetic rat. Am J Physiol 272: E297– E303, 1997. 8. Chen Y, Bornfeldt KE, Arner A, et al: Increase in insulin-like growth factor I in hypertrophying smooth muscle. Am J Physiol 266: E224 –E229, 1994. 9. Sterle M, Kreft ME, and Batista U: The effect of epidermal growth factor and transforming growth 1 on proliferation and differentiation of urothelial cells urinary bladder explant culture. Biol Cell 89: 263–271, 1997. 10. Chen MW, Levin RM, and Buttyan R: Peptide growth factors in normal and hypertrophied bladder. World J Urol 13: 344 –348, 1995. 11. Koo HP, Santarosa RP, Buttyan R, et al: Early molecular changes associated with streptozocin-induced diabetic bladder hypertrophy in the rat. Urol Res 21: 375–381, 1993. 12. Baskin LS, Sutherland RS, Thomson AA, et al: Growth factors and receptors in bladder development and obstruction. Lab Invest 75: 157–166, 1996. 13. Buttyan R, Jacobs BZ, Blaivas JG, et al: Early molecular response to rabbit bladder outlet obstruction. Neurourol Urodyn 11: 225–238, 1992. 14. Uvelius B, Persson L, and Mattiasson A: Smooth muscle
982
cell hypertrophy and hyperplasia in the rat detrusor after short-time infravesical outflow obstruction. J Urol 131: 173– 176, 1984. 15. Mattiasson A, and Uvelius B: Changes in contractile properties in hypertrophic rat urinary bladder. J Urol 128: 1340 –1342, 1982. 16. Malmgren A, Sjo¨ gren C, Uvelius B, et al: Cystometrical evaluation of bladder instability in rats with infravesical outflow obstruction. J Urol 137: 1291–1294, 1987. 17. O’Mahony DJ, Xie WQ, Smith SD, et al: Differential phosphorylation and localization of the transcription factor UBF in vivo in response to serum deprivation. J Biol Chem 267: 35–38, 1992. 18. Paule MR: Regulation of ribosomal RNA expression, in Paule MR (Ed): Transcription of Ribosomal RNA Genes by Eukaryotic RNA Polymerase I. New York, Springer-Verlag, 1998, pp 195–200. 19. Kato K, Monson FC, Longhurst PA, et al: The functional effects of long-term outlet obstruction on the rabbit urinary bladder. J Urol 14: 600 – 606, 1990. 20. Malkowicz SB, Wein AJ, Elbadawi A, et al: Acute biochemical and functional alterations in the partially obstructed rabbit urinary bladder. J Urol 136: 1324 –1329, 1986. 21. Saito M, Longhurst PA, Tammela TL, et al: Effects of partial outlet obstruction of the rat urinary bladder on micturition characteristics, DNA synthesis and the contractile response to the field stimulation and pharmacological agents. J Urol 150: 1045–1051, 1993. 22. Monson FC, McKenna BA, Wein AJ, et al: Effect of outlet obstruction on 3H-thymidine uptake: a biochemical and radioautographic study. J Urol 148: 158 –162, 1992. 23. Steers WD, and de Groat WC: Effect of bladder outlet obstruction on micturition reflex pathways in the rat. J Urol 140: 864 – 871, 1988. 24. Mostwin JL, Karim OM, Van Koeveringe G, et al: Guinea pig as an animal model for the study of urinary bladder function in the normal and obstructed state. Neurourol Urodyn 13: 137–145, 1994. 25. Li JM, and Brooks G: Cell cycle regulatory molecules (cyclins, cyclin-dependent kinases and cyclin-dependent kinase inhibitors) and the cardiovascular system: potential targets for therapy? Eur Heart J 20: 406 – 420, 1999. 26. Hunter T, and Pines J: Cyclins and cancer II: cyclin D and CDK inhibitors come of age. Cell 79: 573–582, 1994. 27. Hannan RD, Luyken J, and Rothblum LI: Regulation of ribosomal DNA transcription during contraction-induced hypertrophy of neonatal cardiomyocytes. J Biol Chem 271: 3213–3220, 1996. 28. Larson DE, Xie W, Glibetic M, et al: Coordinated decreases in rRNA gene transcription factors and rRNA synthesis during muscle cell differentiation. Proc Natl Acad Sci USA 90: 7933–7936, 1993. 29. Glibetic M, Taylor L, Larson D, et al: The RNA polymerase I transcription factor UBF is the product of a primary response gene. J Biol Chem 270: 4209 – 4212, 1995. 30. Persson K, Dean-McKinney T, Steers WD, et al: Activation of the transcription factors nuclear factor-B and activator protein-1 in bladder smooth muscle exposed to outlet obstruction and mechanical stretching. J Urol 165: 633– 639, 2001.
UROLOGY 59 (6), 2002