- Email: [email protected]
Evidence of Nonuniformity in Urothelium Barrier Function between the Upper Urinary Tract and Bladder
Evidence of Nonuniformity in Urothelium Barrier Function between the Upper Urinary Tract and Bladder Nicholas A. Williams, Luke Barnard, Chris J. Allender,* Jenna L. Bowen, Mark Gumbleton, Tim Harrah, Aditya Raja and Hrishi B. Joshi From the School of Pharmacy and Pharmaceutical Sciences, Cardiff University (NAW, LB, CJA, JLB, MG) and Department of Urology, University Hospital of Wales (AR, HBJ), Cardiff, Wales, United Kingdom, and Department of Research and Development, Boston Scientific, Urology and Women’s Health Division (TH), Marlborough, Massachusetts
Purpose: We compared the relative permeability of upper urinary tract and bladder urothelium to mitomycin C. Materials and Methods: Ex vivo porcine bladder, ureters and kidneys were dissected out and filled with 1 mg mle1 mitomycin C. At 60 minutes the organs were emptied and excised tissue samples were sectioned parallel to the urothelium. Sectioned tissue was homogenized and extracted mitomycin C was quantified. Transurothelial permeation across the different urothelia was calculated by normalizing the total amount of drug extracted to the surface area of the tissue sample. Average mitomycin C concentrations at different tissue depths (concentration-depth profiles) were calculated by dividing the total amount of drug recovered by the total weight of tissue. Results: Mitomycin C permeation across the ureteral urothelium was significantly greater than across the bladder and renal pelvis urothelium (9.07 vs 0.94 and 3.61 mg cme2, respectively). Concentrations of mitomycin C in the ureter and kidney were markedly higher than those achieved in the bladder at all tissue depths. Average urothelial mitomycin C concentrations were greater than 6.5-fold higher in the ureter and renal pelvis than in the bladder. Conclusions: To our knowledge we report for the first time that the upper urinary tract and bladder show differing permeability to a single drug. Ex vivo porcine ureter is significantly more permeable to mitomycin C than bladder urothelium and consequently higher mitomycin C tissue concentrations can be achieved after topical application. Data in this study correlate with the theory that mammalian upper tract urothelium represents a different cell lineage than that of the bladder and it is innately more permeable to mitomycin C. Key Words: urinary bladder, kidney, ureter, urothelium, mitomycin
Abbreviations and Acronyms HPLC ¼ high performance liquid chromatography MMC ¼ mitomycin C RNU ¼ radical nephroureterectomy UP ¼ uroplakin UTUC ¼ upper tract urothelial carcinoma Accepted for publication October 6, 2015. No direct or indirect commercial incentive associated with publishing this article. The corresponding author certifies that, when applicable, a statement(s) has been included in the manuscript documenting institutional review board, ethics committee or ethical review board study approval; principles of Helsinki Declaration were followed in lieu of formal ethics committee approval; institutional animal care and use committee approval; all human subjects provided written informed consent with guarantees of confidentiality; IRB approved protocol number; animal approved project number. * Correspondence: Room 0.45, School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Redwood Building, King Edward VII Ave., Cardiff, Wales, United Kingdom, CF10 3NB (telephone: þ44 (0)29 208 75824; FAX: þ44 (0)29 208 74149; e-mail: [email protected]).
See Editorial on page 544.
UROTHELIAL carcinoma, the fourth most common tumor type, can develop in the lower or the upper urinary tract.1 Bladder cancer accounts for 90% to 95% of all urothelial carcinomas while those originating in the upper tract represent 5% to 10%. Although rare, the incidence of UTUC
has increased in the last 3 decades and is now about 2 cases per 100,000 person-years.2 Due to restricted symptomology the disease is commonly advanced at diagnosis. Consequently prognosis is poor with an overall 5-year survival rate of less than 50%.3 Although the urinary tract is lined by
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1 continuous urothelium, UTUC shows different pathological findings than those of the bladder. Most importantly UTUC is significantly more aggressive and invasive.4 Regardless of tumor location EAU (European Association of Urology) guidelines state that the gold standard treatment for UTUC is RNU.1 In certain patients endoscopic management has emerged as a new treatment option and in 2009 it accounted for greater than 10% of all UTUC surgical interventions in England.3 This conservative approach allows for preservation of the kidney while sparing the patient the complications and morbidity associated with major surgery.1 Although to our knowledge no randomized, controlled trials comparing endoscopic management with RNU have been performed, a systematic review of oncologic outcomes suggested that in specific favorable low grade UTUC elective cases endoscopic treatment can yield effective oncologic control and renal preservation.5 This is supported by comparable 5-year disease specific survival for immediate RNU and endoscopic management.5,6 Unfortunately these benefits come at the expense of unfavorable tumor progression5 with 1 group reporting recurrence in 68% of the cohort.6 In an attempt to decrease recurrence after endoscopic management the postoperative administration of adjuvant topical chemotherapy with agents such as MMC6e11 and immunotherapy with bacillus Calmette-Gu erin12 has been reported. The rationale behind this stems from the established efficacy of these agents in treating bladder cancer.13,14 However, the efficacy of topical chemotherapy in UTUC is not proven. The poor quality of the studies, that is small, retrospective series with limited followup and no control arm, has prevented results from demonstrating unequivocal benefit.1,5 If topical drug delivery is to be beneficial in decreasing the recurrence of UTUC, efficacious concentrations of drug must be achieved in the target tissue.5 Currently the accepted dogma is that urothelial permeability is consistent throughout the urinary tract. This is largely based on the assumption that histologically the urothelium is unchanged in the upper and the lower urinary tract.15 To our knowledge no group to date has investigated the relative permeability of bladder, ureter and renal pelvis urothelium. However, evidence suggests that despite apparent histological homology protein expression on the surface of urothelial umbrella cells is not consistent.16,17 Therefore, given the important role of umbrella cells in maintaining barrier function, we hypothesized that this may give rise to varying transurothelial permeation at these distinct locations. In this study we compared the relative permeability of upper urinary tract urothelium and bladder urothelium to MMC.
MATERIALS AND METHODS Mitomycin C Topical Instillation in Isolated Porcine Bladder, Ureter and Kidney. En bloc porcine urinary tracts from pigs weighing 70 to 90 kg were obtained fresh from a local abattoir within 5 minutes of sacrifice and immediately immersed in cold oxygenated Krebs buffer. Working in a shallow bed of Krebs buffer the excess perivesical fat was trimmed, and the bladder, ureters and kidneys were dissected out. Ureters (about 10 cm) were dissected out with approximately 2 cm remaining attached to the bladder and kidney. Organs were rinsed with saline to remove residual urine and filled with MMC solution (1 mg mle1 in normal saline, that is MMC 40 mg powder for solution for injection, ProStrakan, Galashiels, United Kingdom) using a 5Fr 70 cm open-ended ureteral catheter (Cook Medical, Bloomington, Indiana). The bladder, kidney and ureter were filled through the urethra, ureteral orifice and directly into the ureter, respectively. Since the volume of the renal pelvis is variable, preexperimental test instillations with methylene blue (1 mg mle1 in normal saline) were done to ensure adequate contact with the renal pelvis urothelium. After instillation the entry orifices were sutured and the organs were submerged in oxygenated Krebs buffer maintained at 37C in a waterbath for 60 minutes. Four experiments were performed, each representing a different ex vivo porcine urinary tract. Time from tissue recovery to start of the experiment was approximately 30 minutes. Distribution in Bladder, Ureter and Kidney Wall. Following 60-minute instillation the organs were removed, emptied and opened by a single vertical incision. To remove surface adsorbed drug the urothelium was thoroughly rinsed with saline. Tissue samples from areas of drug contact (observed due to purple staining conferred by MMC) were excised and surface area was measured. Tissue samples were immediately snap frozen between 2 metal plates using liquid nitrogen and fixed to cork mounts with Tissue-TekÒ CRYO-OCT Compound. Tissue was sectioned using a CM3050 S cryostat (Leica Microsystems, Buckinghamshire, United Kingdom). The time between experiment end and freezing was less than 2 minutes. Samples were serially sectioned parallel to the urothelial surface at 50 mm and sections were collected in preweighed 1.5 ml Eppendorf tubes. Two 50 mm tissue sections between 0 and 100 mm were grouped for analysis as were the 2, 50 mm sections between 100 and 200 mm. Groups of 6, 50 mm tissue sections between 200 and 1,400 mm, and groups of 12, 50 mm tissue sections between 1,400 and 7,400 mm were also grouped. For all groups the tissues sections were weighed and homogenized in a PrecellysÒ24 homogenizer. Drug was extracted in 1 ml of mobile phase for 24 hours with 10-minute sonication per sample. Samples were then centrifuged at 7,000 rpm at 2,680 gravity and supernatant was isolated for analysis by HPLC. Average drug concentrations at different tissue depths were calculated by dividing the total amount of drug recovered by the total weight of tissue. Transurothelial permeation was calculated by normalizing the total
NONUNIFORMITY IN UROTHELIUM BARRIER FUNCTION
amount of drug extracted from all tissue sections to the surface area of the tissue sample. HPLC Analysis. MMC was analyzed by HPLC using a 5 mm, 250 4.6 mm KromasilÒ C18 I.D column. The mobile phase consisted of 80% 5 mM phosphate buffer (pH 7):20% acetonitrile with ultraviolet detection at 365 nm. Injection volume was 20 ml and the flow rate was 1 ml minutee1.
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Table 1. Individual organ dimensions and MMC volume instilled in each of 4 experiments Wt (gm)
Size (cm)
Instillation (ml)
Extraction Validation. Deionized water (0.5 ml) was added to the tissue homogenate of previously extracted sectioned urinary tract tissue. Samples were immediately vortexed and centrifuged, and supernatant was discarded. Ethyl acetate (0.25 ml) was added to the tissue homogenate and MMC was extracted for 12 hours with 10-minute sonication per sample. Samples were centrifuged, supernatant was isolated and any extracted MMC was quantified by HPLC.
Urinary tract 1: Bladder Ureter 1/ureter 2 Kidney 1/kidney 2 Urinary tract 2: Bladder Ureter 1/ureter 2 Kidney 1/kidney 2 Urinary tract 3: Bladder Ureter 1/ureter 2 Kidney 1/kidney 2 Urinary tract 4: Bladder Ureter 1/ureter 2 Kidney 1/kidney 2
Quantifying Ureter, Bladder and Kidney Tissue Layer Depth
Validating Extraction. To validate the MMC extrac-
Samples of ureter, bladder and kidney (about 1 cm2) were taken from porcine urinary tracts excised immediately after sacrifice on site at the abattoir. Samples were fixed, sectioned and stained with Masson trichrome before examination by light microscopy. Mean depth of the different tissue layers of the ureter, bladder and kidney were measured directly from photomicrographs using NIS-Elements Basic Research imaging software (NikonÒ).
Statistical Analysis All statistical analyses were performed using PrismÒ, version 6.0c. For all comparisons 1-way ANOVA with the Tukey post hoc test for multiple comparisons was used.
RESULTS Mitomycin C Topical Instillation to Isolated Porcine Bladder, Ureter and Kidney. Using a ureteral catheter 1 mg mle1
26.32 0.87/1.13 147.5/151.5
Whole 5/6 Whole
16.5 1/1.9 8.5/9.5
29.13 1.64/2.22 104.94/103.86
Whole 10/10 Whole
17 1.8/2.0 7.1/10.9
40.56 2.24/2.31 159.5/143.9
Whole 10/10 Whole
24.3 2.25/2.75 14/14.5
32.9 2.2/2.3 145.2/144.5
Whole 10/10 Whole
26 2/3.3 11/16.25
tion protocol a second extraction in ethyl acetate was done in samples from each tissue type, including 2, 50 mm sections between 100 and 200 mm tissue depth for each of the 4 urinary tracts. Before adding ethyl acetate a washing step was included to remove any adsorbed MMC from the surface of the tissue homogenate. MMC is highly soluble in ethyl acetate and the solvent has been used by other groups to extract MMC from bladder tissue.18 For all tissue sections the amount of MMC extracted in the secondary step was below the lower limit of detection, suggesting that complete extraction of MMC had been achieved by analytical extraction. Quantifying Ureter, Bladder and Kidney Tissue Layer Depth Although the relative thickness of tissue layers in the bladder wall is well established, the upper
MMC was instilled in isolated porcine bladder, ureter and kidney. Due to natural intraspecies variation the urinary organs varied in size and, thus, the volume of MMC instilled varied (table 1). Preexperimental test instillations with methylene blue demonstrated complete exposure of the renal pelvis urothelium after filling the kidney through the ureteral orifice (supplementary fig. 1, http:// jurology.com/). MMC stained the urothelium purple, making it easy to identify tissue areas exposed to drug solution (fig. 1). HPLC Analysis. HPLC analysis of MMC produced
sharp, almost symmetrical peaks that eluted at a stable retention time (supplementary fig. 2, http:// jurology.com/). The lower limits of detection and quantification were 0.003 and 0.01 mg mle1, respectively, with good sensitivity in homogenized tissue (supplementary fig. 2, http://jurology.com/).
Figure 1. Ex vivo porcine bladder (A), kidney (B) and ureter (C ) after 60-minute instillation of 1 mg mle1 MMC. Purple staining of tissue surface enabled easy identification of drug contact areas.
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urinary tract is less well characterized. Sections of tissue were stained with Masson trichrome (fig. 2). Tissue layer depth was measured directly from photomicrographs using NIS-Elements (fig. 2, D). Measurements calculated as the distance between the top and the base of the layer were made across the whole of the micrograph and the mean thickness of the tissue layers was calculated (table 2). MMC Distribution in Bladder, Ureter and Kidney Wall Concentration-depth profiles were constructed to examine the relative distribution of MMC in the different urinary tract tissues (fig. 3). Average concentrations of MMC in the ureter and kidney were markedly higher than those achieved in the bladder at all tissue depths investigated (fig. 3, A). This was the case in each of the 4 experiments performed (fig. 4). Variation in the relative proportion and composition of tissue layers of the upper and the lower urinary tract makes comparison of drug concentrations in the lamina propria and detrusor muscle difficult. However, the urothelium of the upper and the lower porcine urinary tract was of
Table 2. Tissue layer measurements of ex vivo porcine ureter, bladder and kidney Mean Tissue Layer Thickness (mm)* Ureter Urothelium Lamina propria Smooth muscle Cortex Adventitia Whole wall
Bladder
186.9 182.6 355.8 1,252.0 652.9 3,433.8 Not applicable 1,104.1 357.9 2,299.7 5,226.3
Kidney 176.5 3,470.1 Not applicable 5,635.4 Not applicable 9,282.1
* Total of 20 measurements per layer from 2 whole porcine urinary tracts.
similar size (table 2). Average urothelial MMC concentrations were greater than 6.5-fold higher in the ureter and renal pelvis than in the bladder (48.37, 45.00 and 6.86 mg gme1, respectively, fig. 3, B). Considering the large variation in thickness of the bladder, ureter and kidney wall (table 2), drug recovered from tissue was normalized to surface area, enabling permeation to be compared (table 2 and fig. 3, C ). MMC permeation across ureteral urothelium was significantly greater than across
Figure 2. Representative photomicrographs show bladder (A), kidney (B) and ureter (C and D) sections from single ex vivo porcine urinary tract. Note example of ureteral smooth muscle layer measurements calculated by NIS-Elements (D). Masson trichrome, scale bar indicates 3,000 (A and B) and 2,000 (C and D) mm.
NONUNIFORMITY IN UROTHELIUM BARRIER FUNCTION
A
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B
C
Figure 3. Mean SEM findings after 60-minute instillation of 1 mg mle1 MMC in 4 urinary tracts. Average concentration-depth profiles, that is mass of drug per gm tissue, of MMC in ex vivo porcine bladder, ureter and kidney wall (A) with average urothelial concentration calculated at 150 mm deep (B). Asterisks indicate 1-way ANOVA with Tukey post hoc test for multiple comparisons p <0.01 ureter and kidney vs bladder. Transurothelial permeation of MMC in ex vivo porcine bladder, ureter and kidney wall (C ). Triple asterisks indicate 1-way ANOVA with Tukey post hoc test for multiple comparisons p <0.001 ureter vs bladder. Double asterisks indicate 1-way ANOVA with Tukey post hoc test for multiple comparisons p <0.01 ureter vs kidney.
bladder urothelium (9.07 vs 0.94 mg cme2, p <0.001). Transurothelial permeation across renal pelvis urothelium was markedly greater (3.8-fold higher) than across bladder urothelium, although this was not statistically significant when assessed at an a level of 0.05 (p ¼ 0.08). This was likely due to the relatively small sample size in relation to the natural variability in permeation of the urinary tracts investigated. Permeation across ureteral urothelium was significantly greater than across renal pelvis urothelium (p <0.01). At tissue depths beyond 350 mm MMC concentrations in the ureter were greater than in the kidney (fig. 3, A).
DISCUSSION To our knowledge no studies have compared the relative permeability of the upper and the lower urinary tract to topically delivered drugs. MMC is one of the few drugs that is used topically to treat disease of the upper urinary tract10 and the bladder.19 As such it serves as a clinically relevant exemplar to investigate transurothelial permeability at these different sites. Transurothelial permeation of MMC across porcine ureteral and renal pelvis urothelium was
markedly greater than that of the bladder, although significance was only found for the ureter. Consequently drug tissue concentrations achieved in the upper urinary tract were significantly greater than in the bladder at all tissue depths investigated (fig. 3, A). Our reported bladder wall concentrations of MMC are similar to those reported by other groups (supplementary fig. 3, http://jurology.com/).18 However, because of differences in experimental design, it is not reasonable to make direct comparisons. Unfortunately to our knowledge no previous group has investigated the MMC concentrations achieved in the upper urinary tract following local delivery. Therefore, no ureteral or kidney data are available for comparison. Indeed to our knowledge this is the first report of a study comparing urothelial permeability between the upper urinary tract and bladder for any molecule. The greater permeability of the porcine ureter and renal pelvis urothelium might be explained by the relative UP content of the different regions of the urinary tract.16 Urothelium forms a continual lining of the renal pelvis, ureter, bladder and proximal urethra. It was generally believed that the upper and the lower urinary tracts were lined by 1 homogenous urothelium.16 Urothelia at these
NONUNIFORMITY IN UROTHELIUM BARRIER FUNCTION
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A
B
C
D
Figure 4. Mean SD of all raw data on concentration-depth profiles, that is mass of drug per gm tissue, of MMC in ex vivo porcine bladder, ureter and kidney wall after 60-minute instillation of 1 mg mle1 MMC. Tissue samples analyzed included 6 bladder, 5 ureter and 5 kidney replicates (A), 5 bladder, 8 ureter and 5 kidney replicates (B), 4 bladder, 8 ureter and 3 kidney replicates (C ), and 6 bladder, 4 ureter and 5 kidney replicates (D).
different regions are morphologically similar in thickness and were presumed to perform a similar barrier function.15 Histology of the ex vivo porcine urinary tract showed no discernible difference in the thickness of the urothelium of the upper urinary tract or the bladder (table 2 and fig. 4). However, recently it was found that the urothelium of the mammalian urinary tract can be divided into at least 3 cell lineages based on ultrastructure and UP content, including renal pelvis/ureter, bladder/trigone and proximal urethra.16 Immunofluorescence and transmission electron microscopy of bovine urothelium indicated that urothelial cells of the bladder contain more UPs than those of the ureter.16 Additionally, immunoblot analysis of bovine urothelium cultured in vitro revealed that the bladder contained approximately 10 times more UP than the ureter or the renal pelvis. When maintained under identical in vitro conditions, bovine urothelium from the bladder and the ureter showed different proliferative potential and formed morphologically distinct colonies. Conversely in vitro cultured urothelium from the renal pelvis showed indistinguishable growth potential from that of the ureter. Preliminary work by the same group suggested that the concept of urothelial heterogeneity also extended to monkeys and humans.
Subsequently Riedel et al found that with respect to UP composition urothelial heterogeneity was indeed more prominent in umbrella cells of the human ureter than in those of the bladder.17 The renal pelvis was not investigated. Immunohistochemical staining revealed that 15 of the 18 ureters investigated had a significant subpopulation of ureteral umbrella cells that lacked UPIII and UPIb. The group concluded that the UPIII/UPIb pair may in fact be completely absent from the ureters. In comparison only 2 of the 10 bladder samples investigated lacked UPIII and UPIb, and both of these samples were taken from the ureteral orifice or the immediately surrounding area, suggesting that the urothelium may have been of ureteral origin. UPIII is integral to the formation of an effective urothelial barrier.20 UPIII knockout mice show a more permeable urothelium as demonstrated by increased penetration of methylene blue in umbrella cells and higher transurothelial permeability to water and urea.20,21 Similar to findings in the human ureter UPIII knockout mice demonstrated decreased production of the UPIII partner protein UPIb. It is possible that lack of the UPIII/UPIb pair in the human ureter might render human ureteral urothelium more permeable than bladder urothelium. Interestingly umbrella cell associated CK20 (cytokeratin 20), an additional marker of
NONUNIFORMITY IN UROTHELIUM BARRIER FUNCTION
urothelial differentiation, showed more extended expression among umbrella cells of the bladder than among those of the ureter.22 Therefore, evidence suggests that mammalian ureteral urothelium is less differentiated than bladder urothelium. Although UP content of the human renal pelvis was not investigated, bovine data16 and evidence that the renal pelvis and ureter are from the same cell lineage suggest that it may show similar heterogeneity and increased permeability. It should be pointed out that to our knowledge UP expression studies have not been done in the pig. Urothelial heterogeneity is suggested as an explanation of the results in our study based on results in other mammalian species in the literature as discussed. Furthermore, pigs are an established and well characterized model of the human urinary tract23 with physiology,24 tissue structure and composition similar to those of humans.25 In addition to the presence of UPs, the umbrella cells of the urothelium have intercellular TJs (tight junctions) and a GAG (glycosaminoglycan) layer, which are both believed to increase the impermeability of the urothelium.26,27 Investigating relative TJ protein expression and GAG density in the lower and the upper urinary tract may yield further information regarding the observed difference in permeability. Such study is encouraged.
CONCLUSIONS To our knowledge we report the first evidence that the upper urinary tract and the bladder show differing permeability to a single drug. Because
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ex vivo porcine ureter is significantly more permeable to MMC than bladder urothelium, higher MMC tissue concentrations can be achieved after topical application. The renal pelvis was also found to be markedly more permeable, although significance was not achieved. The data presented in this study correlate with the theory that the mammalian renal pelvis and the ureter represent a different cell lineage than the bladder and they are innately more permeable. A less differentiated urothelium may have no major functional consequences for the ureter or renal pelvis since compared to the bladder, upper tract urothelium has fewer barrier requirements (lower intraluminal pressure, less distension and storage requirements) so that the presence of fully functional UPs may not be essential.17 However, there may be distinct advantages when considering topical administration of drug to the upper urinary tract. Increased urothelial permeability to chemotherapies such as MMC would potentially allow for higher drug concentrations to be achieved in the ureteral and kidney wall. Unfortunately unlike bladder urothelial carcinoma28e30 to our knowledge the MMC concentrations necessary to effectively treat UTUC have not been established. Nonetheless, if delivered to the upper urinary tract in an effective manner, the increased permeability of the ureter and renal pelvis urothelium could have important ramifications for conservative treatment of UTUC.
ACKNOWLEDGMENTS Derek Scarborough provided technical assistance.
REFERENCES 1. Roupr^et M, Babjuk M, Comperat E et al: European guidelines on upper tract urothelial carcinomas: 2013 update. Eur Urol 2013; 63: 1059.
6. Cutress ML, Stewart GD, Wells-Cole S et al: Long-term endoscopic management of upper tract urothelial carcinoma: 20-year single-centre experience. BJU Int 2012; 110: 1608.
11. Eastham JA and Huffman JL: Technique of mitomycin C instillation in the treatment of upper urinary tract urothelial tumors. J Urol 1993; 150: 324.
2. Raman JD, Messer J, Sielatycki JA et al: Incidence and survival of patients with carcinoma of the ureter and renal pelvis in the USA, 1973-2005. BJU Int 2011; 107: 1059.
7. Aboumarzouk OM, Somani B, Ahmad S et al: Mitomycin C instillation following ureterorenoscopic laser ablation of upper urinary tract carcinoma. Urol Ann 2013; 5: 184.
3. Eylert MF, Hounsome L, Verne J et al: Prognosis is deteriorating for upper tract urothelial cancer: data for England 1985-2010. BJU Int 2013; 112: E107.
8. Carmignani L, Bianchi R, Cozzi G et al: Intracavitary immunotherapy and chemotherapy for upper urinary tract cancer: current evidence. Rev Urol 2013; 15: 145.
12. Giannarini G, Kessler TM, Birkh€auser FD et al: Antegrade perfusion with bacillus CalmetteGuerin in patients with non-muscle-invasive urothelial carcinoma of the upper urinary tract: who may benefit? Eur Urol 2011; 60: 955.
4. Stewart GD, Bariol SV, Grigor KM et al: A comparison of the pathology of transitional cell carcinoma of the bladder and upper urinary tract. BJU Int 2005; 95: 791.
9. Patel A and Fuchs GJ: New techniques for the administration of topical adjuvant therapy after endoscopic ablation of upper urinary tract transitional cell carcinoma. J Urol 1998; 159: 71.
5. Cutress ML, Stewart GD, Zakikhani P et al: Ureteroscopic and percutaneous management of upper tract urothelial carcinoma (UTUC): systematic review. BJU Int 2012; 110: 614.
10. Keeley FX Jr and Bagley DH: Adjuvant mitomycin C following endoscopic treatment of upper tract transitional cell carcinoma. J Urol 1997; 158: 2074.
13. Williams SK, Hoenig DM, Ghavamian R et al: Intravesical therapy for bladder cancer. Expert Opin Pharmacother 2010; 11: 947. 14. Shelley MD, Mason MD and Kynaston H: Intravesical therapy for superficial bladder cancer: a systematic review of randomised trials and meta-analyses. Cancer Treat Rev 2010; 36: 195. 15. Hicks RM: The fine structure of the transitional epithelium of rat ureter. J Cell Biol 1965; 26: 25.
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16. Liang FX, Bosland MC, Huang H et al: Cellular basis of urothelial squamous metaplasia. J Cell Biol 2005; 171: 835.
uroplakin ablation elevates urothelial permeability. Am J Physiol Renal Physiol 2002; 283: F1200.
injury of surface epithelial cells. Am J Physiol Ren Physiol 2002; 283: F242.
17. Riedel I, Liang FX, Deng FM et al: Urothelial umbrella cells of human ureter are heterogeneous with respect to their uroplakin composition: different degrees of urothelial maturity in ureter and bladder? Eur J Cell Biol 2005; 84: 393.
22. Moll R, L€owe A, Laufer J et al: Cytokeratin 20 in human carcinomas. A new histodiagnostic marker detected by monoclonal antibodies. Am J Pathol 1992; 140: 427.
27. Parsons CL, Boychuk D, Jones S et al: Bladder surface glycosaminoglycans: an epithelial permeability barrier. J Urol 1990; 143: 139.
23. Tscholl R, Tettamanti F and Zingg E: Ileal substitute of ureter with reflux-plasty by terminal intussusception of bowel: animal experiments and clinical experience. Urology 1977; 9: 385.
28. Schmittgen TD, Wientjes MG, Badalament RA et al: Pharmacodynamics of mitomycin C in cultured human bladder tumors. Cancer Res 1991; 51: 3849.
18. Wientjes MG, Badalament RA, Wang RC et al: Penetration of mitomycin C in human bladder. Cancer Res 1993; 53: 3314. 19. Tolley DA, Parmar MK, Grigor KM et al: The effect of intravesical mitomycin C on recurrence of newly diagnosed superficial bladder cancer: a further report with 7 years of follow up. J Urol 1996; 155: 1233. 20. Hu P, Deng FM, Liang FX et al: Ablation of uroplakin III gene results in small urothelial plaques, urothelial leakage, and vesicoureteral reflux. J Cell Biol 2000; 151: 961. 21. Hu P, Meyers S, Liang FX et al: Role of membrane proteins in permeability barrier function:
24. Crowe R and Burnstock G: A histochemical and immunohistochemical study of the autonomic innervation of the lower urinary tract of the female pig. Is the pig a good model for the human bladder and urethra? J Urol 1989; 141: 414. 25. Dixon JS and Gosling JA: Histology and fine structure of the muscularis mucosae of the human urinary bladder. J Anat 1983; 136: 265. 26. Lavelle J, Meyers S, Ramage R et al: Bladder permeability barrier: recovery from selective
29. Schmittgen TD, Weaver JM, Badalament RA et al: Correlation of human bladder tumor histoculture proliferation and sensitivity to mitomycin C with tumor pathobiology. J Urol 1994; 152: 1632. 30. Yen WC, Schmittgen T and Au JL: Different pH dependency of mitomycin C activity in monolayer and three-dimensional cultures. Pharm Res 1996; 13: 1887.
Purpose: We compared the relative permeability of upper urinary tract and bladder urothelium to mitomycin C. Materials and Methods: Ex vivo porcine bladder, ureters and kidneys were dissected out and filled with 1 mg mle1 mitomycin C. At 60 minutes the organs were emptied and excised tissue samples were sectioned parallel to the urothelium. Sectioned tissue was homogenized and extracted mitomycin C was quantified. Transurothelial permeation across the different urothelia was calculated by normalizing the total amount of drug extracted to the surface area of the tissue sample. Average mitomycin C concentrations at different tissue depths (concentration-depth profiles) were calculated by dividing the total amount of drug recovered by the total weight of tissue. Results: Mitomycin C permeation across the ureteral urothelium was significantly greater than across the bladder and renal pelvis urothelium (9.07 vs 0.94 and 3.61 mg cme2, respectively). Concentrations of mitomycin C in the ureter and kidney were markedly higher than those achieved in the bladder at all tissue depths. Average urothelial mitomycin C concentrations were greater than 6.5-fold higher in the ureter and renal pelvis than in the bladder. Conclusions: To our knowledge we report for the first time that the upper urinary tract and bladder show differing permeability to a single drug. Ex vivo porcine ureter is significantly more permeable to mitomycin C than bladder urothelium and consequently higher mitomycin C tissue concentrations can be achieved after topical application. Data in this study correlate with the theory that mammalian upper tract urothelium represents a different cell lineage than that of the bladder and it is innately more permeable to mitomycin C. Key Words: urinary bladder, kidney, ureter, urothelium, mitomycin
Abbreviations and Acronyms HPLC ¼ high performance liquid chromatography MMC ¼ mitomycin C RNU ¼ radical nephroureterectomy UP ¼ uroplakin UTUC ¼ upper tract urothelial carcinoma Accepted for publication October 6, 2015. No direct or indirect commercial incentive associated with publishing this article. The corresponding author certifies that, when applicable, a statement(s) has been included in the manuscript documenting institutional review board, ethics committee or ethical review board study approval; principles of Helsinki Declaration were followed in lieu of formal ethics committee approval; institutional animal care and use committee approval; all human subjects provided written informed consent with guarantees of confidentiality; IRB approved protocol number; animal approved project number. * Correspondence: Room 0.45, School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Redwood Building, King Edward VII Ave., Cardiff, Wales, United Kingdom, CF10 3NB (telephone: þ44 (0)29 208 75824; FAX: þ44 (0)29 208 74149; e-mail: [email protected]).
See Editorial on page 544.
UROTHELIAL carcinoma, the fourth most common tumor type, can develop in the lower or the upper urinary tract.1 Bladder cancer accounts for 90% to 95% of all urothelial carcinomas while those originating in the upper tract represent 5% to 10%. Although rare, the incidence of UTUC
has increased in the last 3 decades and is now about 2 cases per 100,000 person-years.2 Due to restricted symptomology the disease is commonly advanced at diagnosis. Consequently prognosis is poor with an overall 5-year survival rate of less than 50%.3 Although the urinary tract is lined by
0022-5347/16/1953-0763/0 THE JOURNAL OF UROLOGY® Ó 2016 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION AND RESEARCH, INC.
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1 continuous urothelium, UTUC shows different pathological findings than those of the bladder. Most importantly UTUC is significantly more aggressive and invasive.4 Regardless of tumor location EAU (European Association of Urology) guidelines state that the gold standard treatment for UTUC is RNU.1 In certain patients endoscopic management has emerged as a new treatment option and in 2009 it accounted for greater than 10% of all UTUC surgical interventions in England.3 This conservative approach allows for preservation of the kidney while sparing the patient the complications and morbidity associated with major surgery.1 Although to our knowledge no randomized, controlled trials comparing endoscopic management with RNU have been performed, a systematic review of oncologic outcomes suggested that in specific favorable low grade UTUC elective cases endoscopic treatment can yield effective oncologic control and renal preservation.5 This is supported by comparable 5-year disease specific survival for immediate RNU and endoscopic management.5,6 Unfortunately these benefits come at the expense of unfavorable tumor progression5 with 1 group reporting recurrence in 68% of the cohort.6 In an attempt to decrease recurrence after endoscopic management the postoperative administration of adjuvant topical chemotherapy with agents such as MMC6e11 and immunotherapy with bacillus Calmette-Gu erin12 has been reported. The rationale behind this stems from the established efficacy of these agents in treating bladder cancer.13,14 However, the efficacy of topical chemotherapy in UTUC is not proven. The poor quality of the studies, that is small, retrospective series with limited followup and no control arm, has prevented results from demonstrating unequivocal benefit.1,5 If topical drug delivery is to be beneficial in decreasing the recurrence of UTUC, efficacious concentrations of drug must be achieved in the target tissue.5 Currently the accepted dogma is that urothelial permeability is consistent throughout the urinary tract. This is largely based on the assumption that histologically the urothelium is unchanged in the upper and the lower urinary tract.15 To our knowledge no group to date has investigated the relative permeability of bladder, ureter and renal pelvis urothelium. However, evidence suggests that despite apparent histological homology protein expression on the surface of urothelial umbrella cells is not consistent.16,17 Therefore, given the important role of umbrella cells in maintaining barrier function, we hypothesized that this may give rise to varying transurothelial permeation at these distinct locations. In this study we compared the relative permeability of upper urinary tract urothelium and bladder urothelium to MMC.
MATERIALS AND METHODS Mitomycin C Topical Instillation in Isolated Porcine Bladder, Ureter and Kidney. En bloc porcine urinary tracts from pigs weighing 70 to 90 kg were obtained fresh from a local abattoir within 5 minutes of sacrifice and immediately immersed in cold oxygenated Krebs buffer. Working in a shallow bed of Krebs buffer the excess perivesical fat was trimmed, and the bladder, ureters and kidneys were dissected out. Ureters (about 10 cm) were dissected out with approximately 2 cm remaining attached to the bladder and kidney. Organs were rinsed with saline to remove residual urine and filled with MMC solution (1 mg mle1 in normal saline, that is MMC 40 mg powder for solution for injection, ProStrakan, Galashiels, United Kingdom) using a 5Fr 70 cm open-ended ureteral catheter (Cook Medical, Bloomington, Indiana). The bladder, kidney and ureter were filled through the urethra, ureteral orifice and directly into the ureter, respectively. Since the volume of the renal pelvis is variable, preexperimental test instillations with methylene blue (1 mg mle1 in normal saline) were done to ensure adequate contact with the renal pelvis urothelium. After instillation the entry orifices were sutured and the organs were submerged in oxygenated Krebs buffer maintained at 37C in a waterbath for 60 minutes. Four experiments were performed, each representing a different ex vivo porcine urinary tract. Time from tissue recovery to start of the experiment was approximately 30 minutes. Distribution in Bladder, Ureter and Kidney Wall. Following 60-minute instillation the organs were removed, emptied and opened by a single vertical incision. To remove surface adsorbed drug the urothelium was thoroughly rinsed with saline. Tissue samples from areas of drug contact (observed due to purple staining conferred by MMC) were excised and surface area was measured. Tissue samples were immediately snap frozen between 2 metal plates using liquid nitrogen and fixed to cork mounts with Tissue-TekÒ CRYO-OCT Compound. Tissue was sectioned using a CM3050 S cryostat (Leica Microsystems, Buckinghamshire, United Kingdom). The time between experiment end and freezing was less than 2 minutes. Samples were serially sectioned parallel to the urothelial surface at 50 mm and sections were collected in preweighed 1.5 ml Eppendorf tubes. Two 50 mm tissue sections between 0 and 100 mm were grouped for analysis as were the 2, 50 mm sections between 100 and 200 mm. Groups of 6, 50 mm tissue sections between 200 and 1,400 mm, and groups of 12, 50 mm tissue sections between 1,400 and 7,400 mm were also grouped. For all groups the tissues sections were weighed and homogenized in a PrecellysÒ24 homogenizer. Drug was extracted in 1 ml of mobile phase for 24 hours with 10-minute sonication per sample. Samples were then centrifuged at 7,000 rpm at 2,680 gravity and supernatant was isolated for analysis by HPLC. Average drug concentrations at different tissue depths were calculated by dividing the total amount of drug recovered by the total weight of tissue. Transurothelial permeation was calculated by normalizing the total
NONUNIFORMITY IN UROTHELIUM BARRIER FUNCTION
amount of drug extracted from all tissue sections to the surface area of the tissue sample. HPLC Analysis. MMC was analyzed by HPLC using a 5 mm, 250 4.6 mm KromasilÒ C18 I.D column. The mobile phase consisted of 80% 5 mM phosphate buffer (pH 7):20% acetonitrile with ultraviolet detection at 365 nm. Injection volume was 20 ml and the flow rate was 1 ml minutee1.
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Table 1. Individual organ dimensions and MMC volume instilled in each of 4 experiments Wt (gm)
Size (cm)
Instillation (ml)
Extraction Validation. Deionized water (0.5 ml) was added to the tissue homogenate of previously extracted sectioned urinary tract tissue. Samples were immediately vortexed and centrifuged, and supernatant was discarded. Ethyl acetate (0.25 ml) was added to the tissue homogenate and MMC was extracted for 12 hours with 10-minute sonication per sample. Samples were centrifuged, supernatant was isolated and any extracted MMC was quantified by HPLC.
Urinary tract 1: Bladder Ureter 1/ureter 2 Kidney 1/kidney 2 Urinary tract 2: Bladder Ureter 1/ureter 2 Kidney 1/kidney 2 Urinary tract 3: Bladder Ureter 1/ureter 2 Kidney 1/kidney 2 Urinary tract 4: Bladder Ureter 1/ureter 2 Kidney 1/kidney 2
Quantifying Ureter, Bladder and Kidney Tissue Layer Depth
Validating Extraction. To validate the MMC extrac-
Samples of ureter, bladder and kidney (about 1 cm2) were taken from porcine urinary tracts excised immediately after sacrifice on site at the abattoir. Samples were fixed, sectioned and stained with Masson trichrome before examination by light microscopy. Mean depth of the different tissue layers of the ureter, bladder and kidney were measured directly from photomicrographs using NIS-Elements Basic Research imaging software (NikonÒ).
Statistical Analysis All statistical analyses were performed using PrismÒ, version 6.0c. For all comparisons 1-way ANOVA with the Tukey post hoc test for multiple comparisons was used.
RESULTS Mitomycin C Topical Instillation to Isolated Porcine Bladder, Ureter and Kidney. Using a ureteral catheter 1 mg mle1
26.32 0.87/1.13 147.5/151.5
Whole 5/6 Whole
16.5 1/1.9 8.5/9.5
29.13 1.64/2.22 104.94/103.86
Whole 10/10 Whole
17 1.8/2.0 7.1/10.9
40.56 2.24/2.31 159.5/143.9
Whole 10/10 Whole
24.3 2.25/2.75 14/14.5
32.9 2.2/2.3 145.2/144.5
Whole 10/10 Whole
26 2/3.3 11/16.25
tion protocol a second extraction in ethyl acetate was done in samples from each tissue type, including 2, 50 mm sections between 100 and 200 mm tissue depth for each of the 4 urinary tracts. Before adding ethyl acetate a washing step was included to remove any adsorbed MMC from the surface of the tissue homogenate. MMC is highly soluble in ethyl acetate and the solvent has been used by other groups to extract MMC from bladder tissue.18 For all tissue sections the amount of MMC extracted in the secondary step was below the lower limit of detection, suggesting that complete extraction of MMC had been achieved by analytical extraction. Quantifying Ureter, Bladder and Kidney Tissue Layer Depth Although the relative thickness of tissue layers in the bladder wall is well established, the upper
MMC was instilled in isolated porcine bladder, ureter and kidney. Due to natural intraspecies variation the urinary organs varied in size and, thus, the volume of MMC instilled varied (table 1). Preexperimental test instillations with methylene blue demonstrated complete exposure of the renal pelvis urothelium after filling the kidney through the ureteral orifice (supplementary fig. 1, http:// jurology.com/). MMC stained the urothelium purple, making it easy to identify tissue areas exposed to drug solution (fig. 1). HPLC Analysis. HPLC analysis of MMC produced
sharp, almost symmetrical peaks that eluted at a stable retention time (supplementary fig. 2, http:// jurology.com/). The lower limits of detection and quantification were 0.003 and 0.01 mg mle1, respectively, with good sensitivity in homogenized tissue (supplementary fig. 2, http://jurology.com/).
Figure 1. Ex vivo porcine bladder (A), kidney (B) and ureter (C ) after 60-minute instillation of 1 mg mle1 MMC. Purple staining of tissue surface enabled easy identification of drug contact areas.
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urinary tract is less well characterized. Sections of tissue were stained with Masson trichrome (fig. 2). Tissue layer depth was measured directly from photomicrographs using NIS-Elements (fig. 2, D). Measurements calculated as the distance between the top and the base of the layer were made across the whole of the micrograph and the mean thickness of the tissue layers was calculated (table 2). MMC Distribution in Bladder, Ureter and Kidney Wall Concentration-depth profiles were constructed to examine the relative distribution of MMC in the different urinary tract tissues (fig. 3). Average concentrations of MMC in the ureter and kidney were markedly higher than those achieved in the bladder at all tissue depths investigated (fig. 3, A). This was the case in each of the 4 experiments performed (fig. 4). Variation in the relative proportion and composition of tissue layers of the upper and the lower urinary tract makes comparison of drug concentrations in the lamina propria and detrusor muscle difficult. However, the urothelium of the upper and the lower porcine urinary tract was of
Table 2. Tissue layer measurements of ex vivo porcine ureter, bladder and kidney Mean Tissue Layer Thickness (mm)* Ureter Urothelium Lamina propria Smooth muscle Cortex Adventitia Whole wall
Bladder
186.9 182.6 355.8 1,252.0 652.9 3,433.8 Not applicable 1,104.1 357.9 2,299.7 5,226.3
Kidney 176.5 3,470.1 Not applicable 5,635.4 Not applicable 9,282.1
* Total of 20 measurements per layer from 2 whole porcine urinary tracts.
similar size (table 2). Average urothelial MMC concentrations were greater than 6.5-fold higher in the ureter and renal pelvis than in the bladder (48.37, 45.00 and 6.86 mg gme1, respectively, fig. 3, B). Considering the large variation in thickness of the bladder, ureter and kidney wall (table 2), drug recovered from tissue was normalized to surface area, enabling permeation to be compared (table 2 and fig. 3, C ). MMC permeation across ureteral urothelium was significantly greater than across
Figure 2. Representative photomicrographs show bladder (A), kidney (B) and ureter (C and D) sections from single ex vivo porcine urinary tract. Note example of ureteral smooth muscle layer measurements calculated by NIS-Elements (D). Masson trichrome, scale bar indicates 3,000 (A and B) and 2,000 (C and D) mm.
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A
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B
C
Figure 3. Mean SEM findings after 60-minute instillation of 1 mg mle1 MMC in 4 urinary tracts. Average concentration-depth profiles, that is mass of drug per gm tissue, of MMC in ex vivo porcine bladder, ureter and kidney wall (A) with average urothelial concentration calculated at 150 mm deep (B). Asterisks indicate 1-way ANOVA with Tukey post hoc test for multiple comparisons p <0.01 ureter and kidney vs bladder. Transurothelial permeation of MMC in ex vivo porcine bladder, ureter and kidney wall (C ). Triple asterisks indicate 1-way ANOVA with Tukey post hoc test for multiple comparisons p <0.001 ureter vs bladder. Double asterisks indicate 1-way ANOVA with Tukey post hoc test for multiple comparisons p <0.01 ureter vs kidney.
bladder urothelium (9.07 vs 0.94 mg cme2, p <0.001). Transurothelial permeation across renal pelvis urothelium was markedly greater (3.8-fold higher) than across bladder urothelium, although this was not statistically significant when assessed at an a level of 0.05 (p ¼ 0.08). This was likely due to the relatively small sample size in relation to the natural variability in permeation of the urinary tracts investigated. Permeation across ureteral urothelium was significantly greater than across renal pelvis urothelium (p <0.01). At tissue depths beyond 350 mm MMC concentrations in the ureter were greater than in the kidney (fig. 3, A).
DISCUSSION To our knowledge no studies have compared the relative permeability of the upper and the lower urinary tract to topically delivered drugs. MMC is one of the few drugs that is used topically to treat disease of the upper urinary tract10 and the bladder.19 As such it serves as a clinically relevant exemplar to investigate transurothelial permeability at these different sites. Transurothelial permeation of MMC across porcine ureteral and renal pelvis urothelium was
markedly greater than that of the bladder, although significance was only found for the ureter. Consequently drug tissue concentrations achieved in the upper urinary tract were significantly greater than in the bladder at all tissue depths investigated (fig. 3, A). Our reported bladder wall concentrations of MMC are similar to those reported by other groups (supplementary fig. 3, http://jurology.com/).18 However, because of differences in experimental design, it is not reasonable to make direct comparisons. Unfortunately to our knowledge no previous group has investigated the MMC concentrations achieved in the upper urinary tract following local delivery. Therefore, no ureteral or kidney data are available for comparison. Indeed to our knowledge this is the first report of a study comparing urothelial permeability between the upper urinary tract and bladder for any molecule. The greater permeability of the porcine ureter and renal pelvis urothelium might be explained by the relative UP content of the different regions of the urinary tract.16 Urothelium forms a continual lining of the renal pelvis, ureter, bladder and proximal urethra. It was generally believed that the upper and the lower urinary tracts were lined by 1 homogenous urothelium.16 Urothelia at these
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A
B
C
D
Figure 4. Mean SD of all raw data on concentration-depth profiles, that is mass of drug per gm tissue, of MMC in ex vivo porcine bladder, ureter and kidney wall after 60-minute instillation of 1 mg mle1 MMC. Tissue samples analyzed included 6 bladder, 5 ureter and 5 kidney replicates (A), 5 bladder, 8 ureter and 5 kidney replicates (B), 4 bladder, 8 ureter and 3 kidney replicates (C ), and 6 bladder, 4 ureter and 5 kidney replicates (D).
different regions are morphologically similar in thickness and were presumed to perform a similar barrier function.15 Histology of the ex vivo porcine urinary tract showed no discernible difference in the thickness of the urothelium of the upper urinary tract or the bladder (table 2 and fig. 4). However, recently it was found that the urothelium of the mammalian urinary tract can be divided into at least 3 cell lineages based on ultrastructure and UP content, including renal pelvis/ureter, bladder/trigone and proximal urethra.16 Immunofluorescence and transmission electron microscopy of bovine urothelium indicated that urothelial cells of the bladder contain more UPs than those of the ureter.16 Additionally, immunoblot analysis of bovine urothelium cultured in vitro revealed that the bladder contained approximately 10 times more UP than the ureter or the renal pelvis. When maintained under identical in vitro conditions, bovine urothelium from the bladder and the ureter showed different proliferative potential and formed morphologically distinct colonies. Conversely in vitro cultured urothelium from the renal pelvis showed indistinguishable growth potential from that of the ureter. Preliminary work by the same group suggested that the concept of urothelial heterogeneity also extended to monkeys and humans.
Subsequently Riedel et al found that with respect to UP composition urothelial heterogeneity was indeed more prominent in umbrella cells of the human ureter than in those of the bladder.17 The renal pelvis was not investigated. Immunohistochemical staining revealed that 15 of the 18 ureters investigated had a significant subpopulation of ureteral umbrella cells that lacked UPIII and UPIb. The group concluded that the UPIII/UPIb pair may in fact be completely absent from the ureters. In comparison only 2 of the 10 bladder samples investigated lacked UPIII and UPIb, and both of these samples were taken from the ureteral orifice or the immediately surrounding area, suggesting that the urothelium may have been of ureteral origin. UPIII is integral to the formation of an effective urothelial barrier.20 UPIII knockout mice show a more permeable urothelium as demonstrated by increased penetration of methylene blue in umbrella cells and higher transurothelial permeability to water and urea.20,21 Similar to findings in the human ureter UPIII knockout mice demonstrated decreased production of the UPIII partner protein UPIb. It is possible that lack of the UPIII/UPIb pair in the human ureter might render human ureteral urothelium more permeable than bladder urothelium. Interestingly umbrella cell associated CK20 (cytokeratin 20), an additional marker of
NONUNIFORMITY IN UROTHELIUM BARRIER FUNCTION
urothelial differentiation, showed more extended expression among umbrella cells of the bladder than among those of the ureter.22 Therefore, evidence suggests that mammalian ureteral urothelium is less differentiated than bladder urothelium. Although UP content of the human renal pelvis was not investigated, bovine data16 and evidence that the renal pelvis and ureter are from the same cell lineage suggest that it may show similar heterogeneity and increased permeability. It should be pointed out that to our knowledge UP expression studies have not been done in the pig. Urothelial heterogeneity is suggested as an explanation of the results in our study based on results in other mammalian species in the literature as discussed. Furthermore, pigs are an established and well characterized model of the human urinary tract23 with physiology,24 tissue structure and composition similar to those of humans.25 In addition to the presence of UPs, the umbrella cells of the urothelium have intercellular TJs (tight junctions) and a GAG (glycosaminoglycan) layer, which are both believed to increase the impermeability of the urothelium.26,27 Investigating relative TJ protein expression and GAG density in the lower and the upper urinary tract may yield further information regarding the observed difference in permeability. Such study is encouraged.
CONCLUSIONS To our knowledge we report the first evidence that the upper urinary tract and the bladder show differing permeability to a single drug. Because
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ex vivo porcine ureter is significantly more permeable to MMC than bladder urothelium, higher MMC tissue concentrations can be achieved after topical application. The renal pelvis was also found to be markedly more permeable, although significance was not achieved. The data presented in this study correlate with the theory that the mammalian renal pelvis and the ureter represent a different cell lineage than the bladder and they are innately more permeable. A less differentiated urothelium may have no major functional consequences for the ureter or renal pelvis since compared to the bladder, upper tract urothelium has fewer barrier requirements (lower intraluminal pressure, less distension and storage requirements) so that the presence of fully functional UPs may not be essential.17 However, there may be distinct advantages when considering topical administration of drug to the upper urinary tract. Increased urothelial permeability to chemotherapies such as MMC would potentially allow for higher drug concentrations to be achieved in the ureteral and kidney wall. Unfortunately unlike bladder urothelial carcinoma28e30 to our knowledge the MMC concentrations necessary to effectively treat UTUC have not been established. Nonetheless, if delivered to the upper urinary tract in an effective manner, the increased permeability of the ureter and renal pelvis urothelium could have important ramifications for conservative treatment of UTUC.
ACKNOWLEDGMENTS Derek Scarborough provided technical assistance.
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