Expression of components of the urothelial cholinergic system in bladder and cultivated primary urothelial cells of the pig

Over the last years, the use of urinary pig bladders, and the pig itself as a large animal model, have become extremely popular for urological examinations. Especially pharmacological studies depend on the testing of drugs using porcine material [1‐3]. This is justified on the basis that the porcine bladder closely resembles the human organ both anatomically and physiologically [4, 5]. Nevertheless, not much research on the molecular basics has as yet been conducted which justifies unlimited use and comparison of the porcine material [5].

Some molecular examinations of detrusor and urothelial tissue have been performed on human material concerning transporters and receptors of anticholinergic drugs, such as trospium chloride, which are used as the clinical standard in the treatment of overactive bladder (OAB) [6‐9]. Although it is clear that anticholinergic drugs block muscarinic (M-) acetylcholine (ACh) receptors, it is not completely understood how these drugs interact with the cellular machinery of the urothelium and the detrusor. Goepel et al. [10], as well as Sellers et al. [11], showed that muscarinic receptors M2 and M3 are prominent in porcine as well as human detrusor tissue, and in the human urothelium all five known M-receptor subtypes are present [12‐14].

Besides the M-receptors, other possible drug targets exist in the human bladder. Lips et al. [15] found proof for different transporters and enzymes responsible for synthesis and storage of ACh in the murine and human urothelium, including the carnitine acetyl-transferase (CarAT), whereas the classical ACh synthesising enzyme of neuronal cells, choline acetyltransferase (ChAT), seems to be absent in the urothelium. Furthermore, they demonstrated expression of the organic cation transporters OCT1–3 in the human urothelium. Interestingly, all of them are active in transporting TrCL [16]. Moreover, the solute carrier organic anion transporting polypeptide 1A2 (OATP1A2), involved in the cellular uptake of TrCL, as well as P-glycoprotein (syn. ATP-binding cassette transporter ABCB1, encoded by the multi-drug resistance gene MDR1), involved in the efflux of TrCL, are expressed in the normal urothelium [17‐19]. The vesicular ACh transporter VAChT, which transports ACh in synaptic vesicles of neuronal cells, appears to be absent in the urothelium [13].

With regard to the pig as a model for pharmacological studies on anticholinergic drugs in the bladder, our aim was to determine whether these relevant transporters, enzymes and receptors are present in the porcine urothelium with the same expression pattern and to the same extent as in the human. Furthermore, their molecular stability during cell culture was to be examined.

Previously, systematic examinations on the urothelium as an active component of the bladder storage and micturition process have been overlooked. This might be due to the fact, that this special tissue was only assumed to be a barrier with no physiological impact on the bladder function [21, 22]. As it has become increasingly clear that the urothelium plays an essential role, it is even more important to re-examine the animal models already used for translational examinations applicable to the human bladder.

So far, we have only established the M2 and M3 immunohistostaining for the targets also analysed via qRT-PCR. As it is not easy to find antibodies that are applicable for pig tissue, it is often necessary to use antibodies for human or other species and hope for cross-reactions. Therefore, we also tried a GAPDH antibody which turned out to react very unspecific. As the use of immune staining is more for visual effects, we postponed the establishment of other antibodies.

Using qRT-PCR expression analysis of selected enzymes, transporters and receptors of the urothelial cholinergic system, a comparable expression pattern was detected in the pig in the present study, as has previously been described for humans [13, 15, 18, 19]. Although VAChT is an important vesicular transporter for ACh, it could not be detected in the porcine urothelium. This too, is in accordance with the findings in the human urothelium, where the presence of VAChT could not be proved [15]. However, as a limitation it has to be mentioned that the gene expression assay for VAChT was derived from a predicted cDNA sequence (see Table 1) and could not be confirmed in any porcine tissue so far. Additionally, in the present study, expression of the classical ACh synthesising enzyme ChAT could not be detected in the porcine urothelium as has previously been reported for the human [13]. However, the porcine urothelium showed high mRNA expression levels for CarAT, which is an alternative source of ACh synthesis in the urothelium [13]. In the pig, all three OCTs (OCT1–3) were detected in the urothelium in the order OCT3 > OCT2 > OCT1, and all of them were also present in the human urothelium [13]. The solute carrier OATP1A2, uptake carrier for endogenous substances and pharmaceuticals in the human urothelium, has been identified as a transporter for TrCL by Bexten et al. [19], and could also be detected in the pig urothelium by our group. ABCB1 plays an important role in the efflux of many drugs, also including the anticholinergic drug TrCL [23]. ABCB1 is highly expressed in normal human urothelium [18, 19] and the present study also confirmed its expression in the porcine urothelium. Finally, all 5 M-receptors have been reported in the human urothelium [13, 14], and could be detected in the present study with a similar expression pattern in the pig, with the highest expression levels for M2 and M3.

Two different pig races were analysed i.e. GL and GM, and both showed comparable expression patterns to each other and the human. Taking this into account, it could be expected that pharmacological studies in pigs, for example with anticholinergic drugs, would substantially reflect the situation in man.

Cultivation of urothelial cells has previously been established in order to avoid repeated in vivo experiments in pigs, and to enable in vitro studies with urothelial derived cells. For this reason, the expression of the relevant enzymes, transporters, and receptors was also analysed under cell culture conditions during repeated passaging. Unfortunately, most of the analysed targets revealed continuous downregulation over time, which in general limits the usability of these cells, for example, for transport or receptor binding experiments at higher passaging frequencies. Bexten et al. were able to show that, on the one hand, TrCL is a substrate of the aforementioned solute carriers OCT1 and OATP1A2 (uptake), but on the other hand also for the efflux carrier ABCB1 [19]. The in vitro downregulation of the uptake transporters in combination with the still relatively high amount of the ABCB1 efflux carrier would lead to a non-physiological shift compared to the actual in vivo situation. This cultivation-related downregulation could also be observed for the muscarinic receptors, especially M2 and M3, which play a crucial role in the storing and voiding mechanisms of the bladder. M3 is known to mediate the contractile response and thus is addressed by the main muscarinic receptor antagonist TrCL [12]. This has to be considered for uptake studies with anticholinergic drugs like TrCL. However, downregulation of M1-M5 has also been demonstrated for human urothelial cells in culture by Tyagi et al. [14].

Interestingly, ABCB1 showed slight upregulation during the first round of cultivation in comparison to the tissue samples. This upregulation of ABCB1 might be triggered by cell culture supplements such as antibiotics, as the main role of this transporter is the efflux of potential hazardous substances out of the cell [23, 24].

UCs in culture are a promising model for pharmacological in vitro studies with anticholinergic drugs. However, such studies would be disadvantaged by down-regulation of relevant transporters and receptors for ACh and / or anticholinergic drugs. Nevertheless, Mukerji et al. [25], as well as Gupta et al. [26], could show that urothelial cells of patients with interstitial cystitis (IC) retained their phenotype under cell culture conditions. Furthermore, the animals in this study were mature but not old [27, 28] and thus, do not represent the aged population which suffer from bladder dysfunctions. Therefore, a corresponding animal model with IC or OAB could provide more insight in the mechanisms of these diseases at a molecular level.

It remains that physiological and pharmacological studies on the urinary bladder are mainly conducted in rodent models [29‐32]. These animals are cheap, easy to handle, and a large number of individuals can be investigated. However, data generated in rodents may not be fully comparable to the situation in humans, as the anatomy, physiology and the day-night-rhythm of these animals is somewhat different. Therefore, pigs were analysed in the present study as these show a more comparable bladder physiology to humans [4].

Initially the study was intended only with GL pigs as these are cheaper to obtain and therefore used more commonly in animal studies. Additionally, it is common practice to perform whole bladder experiments and drug studies using pig bladders from an abattoir [1‐3, 33]. However, the GM has proven itself to be a comparable animal model for urological in vivo long-term studies, not only on the physiological [4] but also on the molecular level, as was also demonstrated in the present study. Based on the data presented here, both pig races are appropriate as a pharmacological animal model concerning the investigated targets.

This study was able to show that the porcine urothelium of the GL and GM pig is very similar to the human urothelium concerning the investigated ACh-dependent targets. We therefore conclude that both pig races are appropriate as a pharmacological animal model for in vivo and ex vivo investigations. Nevertheless, the use of UCs from healthy animals is limited due to the down-regulation of the afore-mentioned targets. Therefore, the development and use of an animal model with OAB or IC could provide more insight into the working mechanism of these diseases.