Abstract: PD12-05
Sources of Funding: Naratil Pioneer Award from Women's Health Research at Yale

Introduction

Urothelial cells grown as a monolayer cannot recapitulate the differential function of urothelial cells (e.g. apical, intermediate, basal urothelial cells). Lipopolysaccharide (LPS) has been used as a surrogate for E. coli in study of host response to urinary tract infections. We isolated basal membrane of apical urothelial cells from urothelial tissue for patch clamp studies. We measured potassium channel activities in the apical cell and how the large conductance potassium channel activity changed when the urothelium was exposed to LPS.

Methods

Female C57BL/6 mice 8 to 12 weeks of age were used. Pure urothelium was dissected out using our published technique. Urothelium was placed luminal side down and umbrella cells were exposed after removing basal and intermediate cells with a glass micropipette (Fig. 1). Both inside-out and cell attached configurations were used. Urothelium was exposed to 40 µg/ml of LPS for 30 minutes. Blockers of BK (paxilline, iberiotoxin), LPS (polymyxin B), protein kinase A (H89) were also used. RT-PCR was performed to assess expression of BK and LPS-receptors (TLR4, CD14, and MD-2).

Results

Two K conductances were found, 28.3±1pS and 200.6±4pS. The 200pS channel was sensitive to intracellular calcium, voltage and blocked with iberiotoxin and paxilline. RT-PCR confirmed presence of BK in urothelium. This suggested that the 200pS channel was the BK channel. LPS significantly increased BK channel activity of NPo from 0.50±0.13 to 1.62±0.31 (p<0.001) (Fig. 2); this effect was abrogated by polymyxin B (Fig. 2) and H89. TLR4, CD14 and MD-2 mRNAs were expressed only in the urothelium and not lamina propria or detrusor smooth muscle.

Conclusions

To our knowledge, this is the first time basal membranes of umbrella cells have been patch clamped. LPS significantly increased BK channel activity. This change in apical membrane activity represents an early host urothelial cell response to UTI. While we did not study the downstream results of increased BK activity, it is likely that this would have shown increased urothelial cytokine release. This BK-dependent cytokine release mechanism has been described in macrophages.

Funding

Naratil Pioneer Award from Women's Health Research at Yale