Differentiation potential of urothelium from patients with benign bladder dysfunction

Abstract

Objective: To develop a novel in vitro approach to test the hypothesis that failure of urothelial differentiation underlies the aetiopathology of interstitial cystitis (IC), where there is evidence of compromised urinary barrier function, as benign dysfunctional bladder disease encompass several poorly understood clinically defined conditions, including IC, idiopathic detrusor overactivity (IDO) and stress urinary incontinence (SUI).

Materials and methods: Biopsy-derived urothelial cells from dysfunctional bladder biopsies were propagated as finite cell lines and examined for their capacity to differentiate in vitro, as assessed by the acquisition of a transitional cell morphology, a switch from a cytokeratin (CK)13(lo)/CK14(hi) to a CK13(hi)/CK14(lo) phenotype, expression of claudin 3, 4 and 5 proteins, and induction of uroplakin gene transcription.

Results: Two of 12 SUI cell lines showed early senescent changes in culture and were not characterized further; one of seven IC, one of five IDO and a further three SUI cell lines had some evidence of senescence at passage 3. Of the seven IC-derived cell lines, four showed a near normal range of differentiation-associated responses, but the remainder showed little or no response. Most IDO cell lines (four of five) showed a normal differentiation response, but at least three of the 10 SUI cell lines showed some compromise of differentiation potential.

Conclusion: This study supports the existence of a subset of patients with IC in whom a failure of urothelial cytodifferentiation might contribute to the disease, and provides a novel platform for investigating the cell biology of urothelium from SUI and other benign dysfunctional conditions.

Figure 1. Immunohistochemistry

Immunoperoxidase labelling of paraffin wax-embedded sections of IC, IDO and SUI, compared to normal ureter, as control. A panel of markers was used to assess the proliferation and differentiation status of urothelium in situ. Note the patchy presence of superficial cells in biopsy specimens. Two different IC biopsies for claudin 5 are shown, illustrating the variability of the labelling. In addition to urothelial reactivity, claudin 5 also labelled the vascular endothelium, which served as an internal positive control. Note that the stromal reactivity apparent with the rabbit anti-claudin 7 antibody is artefactual. Scale bar: 50μm.

Figure 2. Phase contrast micrographs

Phase contrast microscopy showing typical examples of cultures at passage 3 from the bladder-derived NHU cell line control, compared to biopsy-derived IDO, IC and SUI cultures in control growth medium (left panel) and after treatment for 3 days with TZ (1 μM) and PD153035 (1 μM) (right panel). Arrows indicate the differentiation-induced formation of rosettes. The third panel shows a typical senescent culture containing larger quiescent cells. Scale bar: 30 μm.

Figure 3. Localisation of PPARγ and RXRα in NHU and IC urothelial cells

Bladder-derived Y607 NHU cells and cystectomy-derived IC1 urothelial cells were seeded at 3×10⁵ cells/ml onto glass slides, grown to near-confluence and fixed in methanol:acetone. Indirect immunofluorescence was performed for PPARγ and RXRα and showed a predominantly nuclear localisation pattern. Scale bar: 10 μm.

Figure 4. Influence of differentiation on CK13 and CK14 expression in cultured IC, IDO and SUI urothelial cells

1. Urothelial cell cultures from IC (n = 6), IDO (n = 4) and SUI (n=10) were incubated in the presence or absence of TZ (1 μM) for 24 hours followed by with or without PD153035 (1 μM) for 6 days. A normal bladder NHU cell line was included as positive control. Media were changed every 3 days. Protein was extracted and 20 μg was resolved on 12.5% SDS polyacrylamide gels and transferred to nitrocellulose membranes. Bound primary antibody was detected with fluorescent-conjugated secondary antibodies and detected using the Li-CoR system. A representative Western blot is presented in the figure and illustrates both normal and atypical responses in terms of a switch from a CK13^lo/CK14^hi to a CK13^hi/CK14^lo phenotype.

2. Plots of individual cell lines analysed by Western blot analysis for expression of CK13 and CK14 proteins from undifferentiated (U) cells, and cells after differentiation-inducing (D) treatment with TZ and PD153035 for IC, IDO and SUI cultured urothelial cells. The cystectomy-derived IC1 cell line is indicated by a dashed line. Means are indicated by solid lines and medians by broken lines. The lysate from bladder NHU cell line (Y607) was included in each western blot and was used as an internal normalisation control. TZ and PD153035-treated Y607 lysate was taken as 1.0. There was no statistical significance between IC and IDO or SUI groups.

Figure 5. Effect of TZ and PD153035 on uroplakin expression in cultured IC, IDO and SUI urothelial cells

Urothelial cell cultures from IC (n = 6), IDO (n = 4) and SUI (n = 9) were treated with or without TZ (1μM) and fresh medium containing PD153035 (1μM) was added after 24 hours. After a further 3 days, RNA was extracted, cDNA generated and quantitative PCR was performed as outlined in the Materials and Methods, using GAPDH as the internal control. The cystectomy-derived IC1 cell line is indicated by a dashed line. The results are displayed as individual line plots and are expressed relative to the treated bladder-derived Y607 NHU cell line, which was included as control. There was a significant induction of uroplakin expression in differentiated versus non-differentiated conditions for all three patient groups, although note individual non-responder cell lines. The induction of UPK1b expression was significantly greater in the GSI than the IC group.

Figure 6. Influence of TZ and PD153035 on claudin expression in cultured NHU, IC and IDO urothelial cells

1. Claudin transcript expression. Cultures of IC (n = 3) and IDO (n = 2) cells were treated for 24 hours in the absence or presence of TZ (1 μM) and then in medium with or without PD153035 (1 μM). RNA was exacted at 3 days, cDNA was generated and RT-PCR was performed as outlined in the Materials and Methods, using claudin primers and GAPDH as the internal control. The PCR products were electrophoresed on a 2% agarose gel and visualised using ethidium bromide.

2. Western blot analysis. Urothelial IC, IDO and SUI cell cultures were treated with or without TZ (1 μM) for 24 hours and then in medium with or without PD153035 (1 μM) for 6 days. Medium was changed every 3 days with PD153035, as appropriate. Protein (20 μg) was resolved on 12.5% SDS polyacrylamide gels and transferred onto nitrocellulose membranes. Bound primary antibody was detected with fluorescent-conjugated secondary antibodies and quantified using the Li-CoR system. The bladder-derived Y607 NHU cell line was included in all blots as a control. A representative western blot is shown, which for comparison purposes, has the same samples shown in Figure 4.

3. Individual line plots of western blot data for claudin protein expression indicating relative change in claudin expression in response to treatment with TZ and PD153035 for IC, IDO and SUI cultured urothelial cells. The result for the Y607 bladder NHU cell line was taken as 1.0. The cystectomy-derived IC1 cell line is indicated by a dashed line. Means are indicated by solid lines and medians by broken lines. Induction of claudin 4 and 5 expression with differentiation was significant for claudin 4 and 5 in all three patient groups and for claudin 3 in the SUI group, however, note the presence of individual poor responder cell lines. There was no statistical significance between IC and IDO or SUI groups.

Figure 6. Influence of TZ and PD153035 on claudin expression in cultured NHU, IC and IDO urothelial cells

1. Claudin transcript expression. Cultures of IC (n = 3) and IDO (n = 2) cells were treated for 24 hours in the absence or presence of TZ (1 μM) and then in medium with or without PD153035 (1 μM). RNA was exacted at 3 days, cDNA was generated and RT-PCR was performed as outlined in the Materials and Methods, using claudin primers and GAPDH as the internal control. The PCR products were electrophoresed on a 2% agarose gel and visualised using ethidium bromide.

2. Western blot analysis. Urothelial IC, IDO and SUI cell cultures were treated with or without TZ (1 μM) for 24 hours and then in medium with or without PD153035 (1 μM) for 6 days. Medium was changed every 3 days with PD153035, as appropriate. Protein (20 μg) was resolved on 12.5% SDS polyacrylamide gels and transferred onto nitrocellulose membranes. Bound primary antibody was detected with fluorescent-conjugated secondary antibodies and quantified using the Li-CoR system. The bladder-derived Y607 NHU cell line was included in all blots as a control. A representative western blot is shown, which for comparison purposes, has the same samples shown in Figure 4.

3. Individual line plots of western blot data for claudin protein expression indicating relative change in claudin expression in response to treatment with TZ and PD153035 for IC, IDO and SUI cultured urothelial cells. The result for the Y607 bladder NHU cell line was taken as 1.0. The cystectomy-derived IC1 cell line is indicated by a dashed line. Means are indicated by solid lines and medians by broken lines. Induction of claudin 4 and 5 expression with differentiation was significant for claudin 4 and 5 in all three patient groups and for claudin 3 in the SUI group, however, note the presence of individual poor responder cell lines. There was no statistical significance between IC and IDO or SUI groups.

Figure 7. Immunofluorescence showing the effect of differentiation on the expression and localisation of CK13 and claudin proteins in cultured urothelial cells

Bladder-derived Y607 NHU and cystectomy-derived IC1 urothelial cells were seeded at 2×10⁵ cells/ml onto glass slides, allowed to adhere and then treated with TZ (1 μM) for 24 hours then PD153035 (1 μM) versus a no treatment control; slides were fixed after six days. Media were replaced every 3 days. Immunofluorescence was performed using the antibodies indicated and nuclei were counterstained with Hoechst 33258 (shown for the claudin 7 micrograph). Note the nuclear localisation of claudin 1 in untreated cultures, which may be a consequence of low E-cadherin expression by proliferating cell cultures (25). Scale bar, 20 μm.