Citation: Lu M, Chai TC. A method to study bladder urothelial cellular function in situ. Bladder 2014;1(1):e3. doi: 10.14440/bladder.2014.27
A method to study bladder urothelial cellular function in situ
Department of Urology, Yale School of Medicine, New Haven, CT 06519, USA
Competing Interests: The authors have declared that no competing interests exist.
Received July 19, 2014; Revision received November 2, 2014; Accepted November 3, 2014; Published November 7, 2014
Objective: The bladder urothelium is comprised of basal, intermediate, and apical urothelial cells. Ability to functionally study an individual urothelial cell while preserving its in situ location would represent an advance in bladder urothelial biology.
Materials and Methods: Mice were euthanized and cardiac perfused with phosphate-buffered saline. Bladders were then excised. Urothelial sheets were dissected off the lamina propria using forceps with a microscope (5 × magnification). Sheets were stained with H&E. In separate experiments, cell-attached electrophysiology was performed by placing urothelial sheets in Ringer’s bath solution, with either basal or apical surface down. Using 40 × magnification, individual urothelial cells from different layers were identified. Potassium currents on these cells were measured in situ using single channel patch-clamp technique.
Results: Histology revealed that urothelial sheets were free of lamina propria and smooth muscles. The proportion of apical cells, compared to proportion of intermediate and basal cells, with measurable potassium currents was considerably higher in apical cells (69% of apical versus 16% of intermediate and 18% of basal cells). Of the active patches detected in apical cells, 100% of these patches showed a 43 pS current conductance. For intermediate and basal cells, 75-83% demonstrated a 43 pS current and 17-25% demonstrated a 22 pS current. Single cells, from all 3 layers, could also be individually microdissected completely off the urothelium.
Conclusions: A novel approach was developed in which individual urothelial cells within the multi-layered urothelium were identified and functionally studied in situ. Electrophysiologic characterization revealed different phenotype between the cells from different layers. Single cells from an identified layer can be harvested off the urothelium allowing for other studies. This technique allows investigators to study various cellular functions while preserving cellular location within the urothelium.
Keywords: bladder, urothelium, urothelial cells, potassium current
The bladder urothelium is a multi-layered epithelium, with the apical cells lining the bladder lumen, the intermediate cells just below, and the basal cells abutting the basement membrane. The apical urothelial cells have specialized function that maintain bladder-blood impermeability  via expression of uroplakin plaques  and tight junction proteins . Because the urothelium is theorized to have additional properties, such as urothelial-afferent signaling  and immune surveillance , the ability to dissect out individual cells from the multi-layered urothelium would represent an advance in the field of urothelial biology. Physiologic studies on bladder urothelial function (such as patch clamp electrophysiology and calcium microfluorimetry) have often utilized primary dissociated cell cultures which loses cellular specificity. Cultured urothelial cells undergo changes that de-differentiate from their in situ phenotype .
A new technique was developed in which we were able to dissect a urothelial sheet of pure urothelial cells without lamina propria nor smooth muscle and to patch-clamp a single urothelial cell, in situ within the urothelium, all the while, knowing which specific layer this cell resided. Furthermore, this technique allows the complete removal of a single cell off the urothelium entirely; thus allowing possibility of other single cell studies. This technique will increase the granularity of our understanding of urothelial cellular biology, and allow the ability to measure potential differential function between the cells from different layers of the urothelium.
Dissection of Urothelial Sheet from Mouse Bladder
This protocol was approved by Yale’s Institutional Animal Care and Use Committee. Male C57BL/6 WT mice, 10 weeks old, were purchased from Jackson Labs and housed in animal facility on normal diet for one week. After the mouse was anesthetized with intraperitoneal pentobarbital sodium (0.07 mg/mg body weight), animal was cardiac (left ventricle puncture, right atriotomy) perfused with PBS solution at 0.2 mg/ml for 5 minutes at a perfusion speed of 10 ml/min. Then, the whole bladder was excised and placed into PBS. The bladder was initially cross-sectioned into three pieces. Each cross-sectional piece (which was a horizontal slice giving a ring configuration to the section) was opened up and then cut into four equal pieces. Each of these pieces was used for urothelial sheet dissection. The urothelium was peeled off under microscope (5x magnification) with microforceps, preserving the orientation of the apical surface (meaning that the apical side of the urothelium was identified and maintained at all times). The urothelial sheet was then bathed in a solution containing 0.9 mM CaCl2, 0.4 mM MgCl2, 2.68 mM KCl, 1.47 mM KH2PO4, 136.89 mM NaCl and 8.1 mM Na2HPO4.
To assess the quality for the above described dissection, urothelial sheets were fixed in 4% paraformaldehyde in 0.14 M Sorensen’s phosphate buffer for 10 min before placing in 30% sucrose in 0.01 M phosphate buffer saline (PBS) for overnight. Then the urothelial sheets were paraffin embedded and sectioned at 8 µm and mounted on Fischer Superfrost Plus glass slides. Standard H&E staining was used to assess the architecture and anatomy of the dissected urothelial sheet. A full thickness bladder wall was also sectioned and H&E stained for comparison to the dissected urothelial sheet.
Localization and microdissection of apical, intermediate, and basal cells within the urothelial sheet
A urothelial sheet was transferred to a Cell-Tak (Sigma) coated cover glass (5 × 5 mm) to immobilize it for cell identification during patch clamping which was performed under an inverted microscope with 40 × magnifications. Basal and apical layer cells were directly patched because we oriented either apical or basal side of the urothelial face up (closer to eyepiece). For localization of intermediate cells, another glass micropipette (Sutter Model P-97 micropuller to a tip diameter of 20 µm), controlled with a micromanipulator (Narshige), was used to physically remove either basal or apical cells (based on orientation of the sheet) to expose underlying intermediate cells. Removal of cells was facilitated by applying suction through the micropipette in addition to applying physical motion to the micropipette (see Movie S1: removal of apical cell, Movie S2: removal of intermediate cell, and Movie S3: removal of basal cell).
Electrophysiologic Protocols and Solutions
The experiments were conducted at room temperature (22-24oC) on an inverted microscope (IMT-2, Olympus). Electrodes were pulled from borosilicate glass capillaries on a Sutter Instrument Co (Model P-97, USA) and polished on a MF-830 microforge (Narishige, Tokyo). Patch electrodes had resistances of 6-7 MΩ when filled with pipette solution (in mM: 140 KCl, 1.8 MgCl2, 10 HEPES, pH 7.4 with KOH). Single channel currents were recorded by using Axopatch 200B and digitized by Digidata 1322A with pCLAMP10.2. All the chemicals were purchased from Sigma.
Single channel patch clamp recording were performed in cell-attached configuration to measure potassium currents. Urothelial sheet were bathed in solution contained of 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM glucose, 10 mM HEPES, pH 7.4.
Figure 1. Mouse bladder wall cross-section in H&E staining showing urothelium, lamina propria and muscularis. The adventitia was not shown in this section. 5 × and 40 × magnifications.
Signals were digitized at a sampling rate 4 kHz (DigiData 1322A, Axon Instruments) and saved in a PC for further analysis. Patch data were analyzed using Clampfit 10.2 at a digital filter frequency of 250 Hz. Channel activity was calculated by the following equation during sampling periods of 10-30 seconds. NPo= Σ( t1 + t2+ … tn), where N is the number of observed current levels in the patch, Po is the single channel open probability; tn is the fractional open time spent at each of the current levels. Results are given as means ± SE. Channel conductance was calculated by using GraphPad Prism 5 software from linear regression analysis of single channel current-voltage curves. Voltage (V) applied to the pipette was referenced to the bath potential and reported as –V.
Figure 2. H&E staining of mouse bladder urothelial sheet cross-section cut showing urothelial purity of the dissection without lamina propria and without detrusor smooth muscle. Right panel is magnification (40 ×) showing details of the mouse bladder urothelium.
Dissected urothelial sheets are devoid of any lamina propria
From a full thickness section, the urothelium, lamina propria, and muscularis can be seen on H&E (Fig. 1). The urothelium was comprised of three layers of cells: a single layer of apical cells, two to three layers of intermediate cells and a single layer of basal cells.
A dissected mouse urothelial sheet was devoid of any lamina propria tissue contamination (Fig. 2). Urothelial sheets were harvested repetitively with the same results of having a non-contaminated multi-layered urothelium. Figure 3 shows a urothelial sheet under a microscope, with 5x and 10x magnifications.
Visualization of apical, intermediate and basal cells
After harvesting urothelial sheets, and maintaining orientation of urothelium, the sheets were immobilized to the bottom of inverted microscope chamber bathed with perfusion solution. A glass micropipette was used to expose cells from different layers. Three cell layers are shown in Figure 4, right panel, at 40 × magnification. The basal cells faced closest to the eyepiece, and are indicated by “b”. Between the basal cell and apical cell (indicated by “a”), are the two to three cells layer comprising the intermediate layer indicated by “i”. Videos of the micropipette completely dissecting off an apical, intermediate and basal cells are included in the supplemental data uploaded on journal website (Movie S1：removal of apical cell, Movie S2：removal of intermediate cell, and Movie S3: removal of basal cell). A photomicrograph of a single apical cell, dissected off a urothelial sheet, is shown in Figure 5.
Figure 4. Mouse urothelial sheet under inverted microscope with basal urothelium facing up, towards eyepiece. Left panel is 4 x magnification and the patch clamp micropipette is seen. Right panel is 40 x magnification. Representative cells from the three layers of the urothelium can be seen after removing appropriate neighboring cells. a = apical cells, i = intermediate cells, b = basal cells.
Figure 5. Microphotograph image showing a single apical cell microdissected off the urothelial sheet. Videos showing removal of individual cells from the urothelium are at the journal website (Movie S1 = removal of apical cell, Movie S2 = removal of intermediate cell,Movie S3=removal of basal cell).
Electrophysiologic measurements of the different types of urothelial cells
From 14 mice, 168 total cells were patched; 64 cells (38%) showed active channel activities (Table 1). There were, proportionally, many more cells with active patches in the apical population (40/58, 69%), compared to intermediate (12/55, 22%) and basal (12/55, 22%) populations. The active patches were characterized by two different potassium conductances. Two representative tracings are shown in Figure 6A and 6B at holding voltages of -20 mV, -40 mV and -80 mV. The I-V (pA vs mV) curves are shown in Figure 6A and 6B. These tracings reflected channel conductances of 43 pS and 22 pS, respectively. The channel open probabilities of these types of currents were different with 0.05 (for 43 pS conductance current) and 0.44 (for 22 pS conductance current). When active patches were detected, apical cells only expressed the 43 pS potassium current (100%), whereas intermediate and basal cells had similar dichotomy with ~80% expressing the 43 pS and ~20% expressing the 22 pS potassium current (Table 1).
The ability to characterize any function of an individual apical, intermediate or basal urothelial cell would significantly increase the details of our understanding of bladder urothelial biology. The orientation of the cells within the bladder urothelium is presumed to be related to differentiation, as the cells go through the layers, they become terminally differentiated in the apical layer. It was shown, using embryonic mice manipulations, that existence of a single layer of urothelial cells in the bladder expressing the terminally differentiated uroplakins III (UPIII) was possible in a p63 knock-out transgenic animal . p63 is a protein that is a phenotypic marker for basal epithelial cells and is a master regulator for epithelial development. Because UPIII, a marker for apical (umbrella) cells was expressed in a single layer urothelium with no intermediate and no basal cells, the authors in reference 7 suggested that the apical cells arise from a certain stem cell whereas basal/intermediate urothelial cells arise from a different stem cell. However, others have shown that apical cells can arise from basal and intermediate cells [8, 9]. The biology of how the urothelium becomes stratified and whether specific functions are attributed to different layers remain complex and incompletely understood.
Most functional studies of bladder urothelial cells have used monolayer cell cultures derived from urothelial biopsies (numerous studies) or using chambers to measure permeability/transmembrane epithelial resistance of the entire thickness of the urothelium . Antibody localization or antibody-related purification techniques have found certain proteins associated with certain layers, thus attributing function based on the specific proteins being differentially expressed (e.g. findings of tight junction proteins in the apical cells only , finding of uroplakins on apical cells ). It was suggested that the bladder urothelium adsorb proteins, including antibodies, in a non-specific manner which can give rise to inconsistent results . None of these techniques permit layer-specific single cell functional studies. We described our technique of obtaining urothelial sheets devoid of lamina propria, by microforceps without any additional treatments or specialized techniques.
|n = 14 mice||Number of Cells Patched||Number of Cells with Active/Inactive Patches||Number of Cells with 43 pS K channels (% of only active patches)||Number of Cells with 22 pS K channels (% of only active patches)|
|Apical cells||58||40/18||40 (100%)||0 (0%)|
|Intermediate cells||55||12/43||9 (75%)||3 (25%)|
|Basal cells||55||12/43||10 (83%)||2 (17%)|
|Total||168||64/104||59 (92%)||5 (8%)|
Our data not only shows the feasibility of measuring electrophysiologic function of individual cells within the urothelial sheet (in situ), but the data suggested differential potassium ion conductance between the cells of the different urothelial layers. The specific type of potassium channels mediating these currents were not determined in this study because the primary intent of this work was to demonstrate the feasibility of this new technique. Certainly, use of specific pharmacologic blockers in the future can potentially identify specific channels mediating these potassium currents. However, the importance of this study was to demonstrate a technique which provided a novel approach to study urothelial cellular function in situ within the milieu of the urothelium.
From these data, it appeared that the phenotype of potassium currents were different based on cellular location within the stratified urothelium. Compared to intermediate/basal cells, apical cells had a higher percentage of active patches (40 out of 58, 69%) and no expression of the 22pS potassium conductance current (Table 1). The possible reasons for lack of detecting active patches could be due to the fact that no potassium channels were present on the patched area or the channels were closed (silent). The role of potassium channel in the urothelial cells could be related to observation of potassium absorption across the urothelium . As stated, the specific potassium ion channels mediating these currents were not addressed in this study. However, presence of active potassium channels, including Kir2.1 and BK, have been detected in cultured human bladder urothelial cells, with altered function of these channels in interstitial cystitis and overactive bladder [15, 16]. The conductance of Kir2.1 is small (~27 pS) , whereas conductance of BK is large (~180 pS) , so the 22 pS potassium current detected in this study might be Kir2.1. It should be noted that these channel activities in the prior urothelial studies were measured in cultured monolayer of cells from human subjects. Whereas in this study, patch clamp studies were performed on urothelial cells in situ and from a different animal species.
Figure 6. Two types of potassium channel activities were detected in the urothelial cells. Representative single channel recording traces were chosen at V = -20 mV, -40 mV and -80 mV in cell-attached configuration. “C” indicates channel closed state. “O” indicates channel open state. The I-V curves showed different channel conductance slopes
The other utility of this technique involve possibilities other functional studies on individual cells. Cells dissected completely free (see Movie S1, Movie S2 and Movie S3 on Bladder website) using this technique can also be functionally studied with calcium microfluorimetry. Primary cultured or dissociated urothelial cells have been shown to have increased intracellular calcium in response to capsaicin [17, 18], muscarinic agonists [19, 20, 21], ATP , stretch , and TRPV4 agonist . This technique could allow investigators to be able to perform calcium imaging on single cells from identified layers of the urothelium.
It is recognized that heterogeneity of neighboring cells within the same tissue may be important in explaining the biology of certain diseases and embryonic development. For example, heterogeneity of neoplastic cells within the same tumor might drive the biologic behavior of the tumor. In embryogenesis, heterogeneity of neighboring cells is a necessity for normal development. Single cell analysis allows the genome sequence using the relatively new technique of amplification method-multiple annealing and looping-based amplification cycles (MALBAC) . The ability to obtain viable single cells from the bladder urothelium would allow single cell genome sequencing which will increase our biologic understanding of the urothelium.
We described a microdissection technique enabling functional studies of individual urothelial cells in situ within the urothelium. We found that membrane potassium permeability properties were unique depending on the location of the cells. Basal and intermediate cells exhibited one type of potassium conductivity phenotype whereas apical cells exhibited another phenotype suggesting different membrane properties between cells in different layer of the urothelium. This technique can be further applied to study other single cell functional studies to increase our understanding of the complexity of the bladder urothelium.
- Kreft ME, Hudoklin S, Jezernik K, Romih R (2010) Formation and maintenance of blood-urine barrier in urothelium. Protoplasma 246: 3-14. doi: 10.1007/s00709-010-0112-1. [View Article] [PubMed] [Google Scholar]
- Sun TT, Zhao H, Provet J, Aebi U, Wu XR (1996) Formation of asymmetric unit membrane during urothelial differentiation. Mol Biol Rep 23: 3-11. [PubMed] [Google Scholar]
- Carattino MD, Prakasam HS, Ruiz WG, Clayton DR, McGuire M, et al. (2013) Bladder filling and voiding affect umbrella cell tight junction organization and function. Am J Physiol Renal Physiol 305: 305-1158. doi: 10.1152/ajprenal.00282.2013. [View Article] [PubMed] [Google Scholar]
- Birder L, Wyndaele J (2012) From urothelial signalling to experiencing a sensation related to the urinary bladder. Acta Physiol (Oxf) 207: 34-39. doi: 10.1111/apha.12011. [View Article] [PubMed] [Google Scholar]
- Bäckhed F, Söderhäll M, Ekman P, Normark S, Richter-Dahlfors A (2001) Induction of innate immune responses by Escherichia coli and purified lipopolysaccharide correlate with organ- and cell-specific expression of Toll-like receptors within the human urinary tract. Cell Microbiol 3: 153-158. [PubMed] [Google Scholar]
- Sun TT (2006) Altered phenotype of cultured urothelial and other stratified epithelial cells: implications for wound healing. Am J Physiol Renal Physiol 291: 291-1. doi: 10.1152/ajprenal.00035.2006. [View Article] [PubMed] [Google Scholar]
- Signoretti S, Pires MM, Lindauer M, Horner JW, Grisanzio C, et al. (2005) p63 regulates commitment to the prostate cell lineage. Proc Natl Acad Sci U S A 102: 11355-11360. doi: 10.1073/pnas.0500165102. [View Article] [PubMed] [Google Scholar]
- Lavelle J, Meyers S, Ramage R, Bastacky S, Doty D, et al. (2002) Bladder permeability barrier: recovery from selective injury of surface epithelial cells. Am J Physiol Renal Physiol 283: 283-242. doi: 10.1152/ajprenal.00307.2001. [View Article] [PubMed] [Google Scholar]
- Acharya P, Beckel J, Ruiz WG, Wang E, Rojas R, et al. (2004) Distribution of the tight junction proteins ZO-1, occludin, and claudin-4, -8, and -12 in bladder epithelium. Am J Physiol Renal Physiol 287: 287-305. doi: 10.1152/ajprenal.00341.2003. [View Article] [PubMed] [Google Scholar]
- Yu J, Manabe M, Wu XR, Xu C, Surya B, et al. (1990) Uroplakin I: a 27-kD protein associated with the asymmetric unit membrane of mammalian urothelium. J Cell Biol 111: 1207-1216. [PubMed] [Google Scholar]
- Yu W, Hill WG (2011) Defining protein expression in the urothelium: a problem of more than transitional interest. Am J Physiol Renal Physiol 301: 301-932. doi: 10.1152/ajprenal.00334.2011. [View Article] [PubMed] [Google Scholar]
- Spector DA, Deng J, Stewart KJ (2013) Hydration status affects sodium, potassium, and chloride transport across rat urothelia. Am J Physiol Renal Physiol 305: 305-1669. doi: 10.1152/ajprenal.00353.2013. [View Article] [PubMed] [Google Scholar]
- Sun Y, Chen M, Lowentritt BH, Van Zijl PS, Koch KR, et al. (2006) EGF and HB-EGF modulate inward potassium current in human bladder urothelial cells from normal and interstitial cystitis patients. Am J Physiol Cell Physiol 292: 292-106. doi: 10.1152/ajpcell.00209.2006. [View Article] [PubMed] [Google Scholar]
- Li M, Sun Y, Simard JM, Wang J, Chai TC (2009) Augmented bladder urothelial polyamine signaling and block of BK channel in the pathophysiology of overactive bladder syndrome. Am J Physiol Cell Physiol 297: 297-1445. doi: 10.1152/ajpcell.00259.2009. [View Article] [PubMed] [Google Scholar]
- Kullmann FA, Shah MA, Birder LA, de Groat WC (2009) Functional TRP and ASIC-like channels in cultured urothelial cells from the rat. Am J Physiol Renal Physiol 296: 296-892. doi: 10.1152/ajprenal.90718.2008. [View Article] [PubMed] [Google Scholar]
- Li M, Sun Y, Simard JM, Chai TC (2011) Increased transient receptor potential vanilloid type 1 (TRPV1) signaling in idiopathic overactive bladder urothelial cells. Neurourol Urodyn 30: 606-611. doi: 10.1002/nau.21045. [View Article] [PubMed] [Google Scholar]
- Li M, Sun Y, Tomiya N, Hsu Y, Chai TC (2013) Elevated polyamines in urothelial cells from OAB subjects mediate oxotremorine-evoked rapid intracellular calcium rise and delayed acetylcholine release. Am J Physiol Renal Physiol 305: 305-445. doi: 10.1152/ajprenal.00345.2012. [View Article] [PubMed] [Google Scholar]
- Gupta GN, Lu S, Gold MS, Chai TC (2009) Bladder urothelial cells from patients with interstitial cystitis have an increased sensitivity to carbachol. Neurourol Urodyn 28: 1022-1027. doi: 10.1002/nau.20726. [View Article] [PubMed] [Google Scholar]
- Kullmann FA, Artim D, Beckel J, Barrick S (2008) de Groat WC, Birder LA (2008) Heterogeneity of muscarinic receptor-mediated Ca2+ responses in cultured urothelial cells from rat. Am J Physiol Renal Physiol 294: 294-971. doi: 10.1152/ajprenal.00313.2007. [View Article] [PubMed] [Google Scholar]
- Wu C, Gui GP, Fry CH (2011) Intracellular Ca(2+) regulation and electrophysiolgical properties of bladder urothelium subjected to stretch and exogenous agonists. Cell Calcium 49: 395-399. doi: 10.1016/j.ceca.2011.03.008. [View Article] [PubMed] [Google Scholar]
- Mochizuki T, Sokabe T, Araki I, Fujishita K, Shibasaki K, et al. (2009) The TRPV4 cation channel mediates stretch-evoked Ca2+ influx and ATP release in primary urothelial cell cultures. J Biol Chem 284: 21257-21264. doi: 10.1074/jbc.M109.020206. [View Article] [PubMed] [Google Scholar]
- Zong C, Lu S, Chapman AR, Xie XS (2012) Genome-wide detection of single-nucleotide and copy-number variations of a single human cell. Science 338: 1622-1626. doi: 10.1126/science.1229164. [View Article] [PubMed] [Google Scholar]
Supplementary information of this article can be found online at http://www.bladderj.org.