Role of Detrusor PDGFR α + Cells in Cyclophosphamide-Induced Detrusor Overactivity

Cyclophosphamide (CYP)-induced cystitis is a rodent model that shares many features common to the cystitis occurring in patients, including detrusor overactivity (DO). Platelet-derived growth factor receptor alpha positive ( PDGFRα + ) cells regulate muscle excitability in murine bladders during filling. PDGFRα + cells express small conductance Ca 2+ -activated K + channels (predominantly SK3) that provide stabilization of membrane potential during filling. We hypothesized that down-regulation of the regulatory functions of PDGFR α + cells and/or loss of PDGFRα + cells generates the DO in CYP-treated mice. After CYP treatment, transcripts of Pdgfr α and Kcnn3 were reduced, and PDGFR α and SK3 protein was also reduced in detrusor muscle extracts. The distribution of PDGFRα + cells was also reduced. Inflammatory markers were increased in CYP-treated detrusor muscles. An SK channel agonist, CyPPA, increased outward current and hyperpolarization in PDGFRα + cells. This response was significantly depressed in PDGFRα + cells from CYP-treated bladders. E x vivo cystometry showed increased transient contractions in CYP-treated bladders, and the sensitivity of these bladders to apamin was reduced, reflecting the reduction in the SK conductance expressed by PDGFRα + cells. In summary, PDGFRα + cells were reduced and the SK3 conductance was downregulated in CYP-treated bladders. These changes are consistent with the development of DO after CYP treatment.


Introduction
Interstitial cystitis (IC) is characterized by suprapubic and/or bladder pain that is accompanied with an increase in urinary urgency, frequency and nocturia 1,2 . The pathology of IC does not follow that of other diseases/syndromes in the bladder, such as carcinoma, urinary tract infections or cystitis introduced by radiation or medication 3,4 .
Although the pathogenesis of IC is still not fully understood, several factors have been suggested which include neuronal, urothelium, and myogenic causes with likely associated inflammation 4 .
CYP can also induce detrusor overactivity (DO) 23 , but the functional changes in the detrusor smooth muscle responsible for DO have not been determined.
Recently, a unique population of interstitial cells were identified in the bladder.
These cells are immunopositive for platelet-derived growth factor receptor alpha (PDGFRα), and therefore are referred to by this chemical coding, i.e. PDGFRα + cells.
PDGFRα + cells have been identified in murine, pig and human detrusor muscles 24,25 .
These cells are located on the edges of smooth muscle bundles and have multiple branches that couple to and form an electrical syncytium with smooth muscle cells (SMCs) 24,26,27 . The function of PDGFRα + cells in detrusor muscles has been investigated with molecular and electrophysiological techniques. PDGFRα + cells displayed higher current density of small conductance Ca 2+ -activated K + (SK) channels 25,28 , as compared to detrusor SMCs. Activation of SK channels in PDGFRα + cells provides a stabilizing influence on smooth muscle excitatiiby. We hypothesized that loss-of-function in PDGFRα + cells or SK channels could lead to DO in cystitis. This study describes investigation into the molecular and functional changes that occur in detrusor PDGFRα + cells in CYP-injected bladder.

Downregulation of PDGFRα and SK channel in murine bladders from CYPinduced cystitis.
We compared the transcriptional expression of Pdgfra and Kcnn1-4 in detrusor muscles from CYP-treated and saline-treated mice. Pdgfra and Kcnn3 (SK3) were significantly downregulated, but neither SK1-2 (Kcnn1 and Kcnn2) nor IK (Kcnn4) were changed significantly after CYP-treatment (n=4, Fig. 1A and B). Detrusor muscles from CYP-treated mice displayed a significant increase in Il6 and Tnf (inflammatory markers) (n= 4, Fig. 1C) suggesting the onset of bladder inflammation after CYP treatment.
Levels of transcripts do not necessarily translate linearly into protein expression. Therefore, we also employed immunohistochemistry and Wes analysis to confirm parallel changes in protein expression. Immunohistochemistry revealed PDGFRα immune-positive cells were down-regulated in CYP-treated detrusor compared with age-matched saline-injected controls (Fig. 2A& B). Wes analysis showed in a more quantitative way that PDGFRα and SK3 were significantly downregulated in CYPtreated detrusor muscles, as compared to controls (n=4, Fig. 3)). These data are consistent with transcriptional data, and suggested the possibility that down-regulation of Pdgfrα and/or Kcnn3 transcripts could be involved in generation of DO.

The effect of apamin on CYP-treated bladders using ex vivo preparation.
We also examined the pressure-volume relationships of excised bladders in ex vivo preparations to investigate changes in functional expression of SK channel in control and CYP-treated bladders. Ex vivo pressure-volume measurements exclude extrinsic neural regulation during filling, so this technique highlights regulation via myogenic mechanisms. In in vivo cystometry, voiding contractions start around 20 cmH2O in murine bladder 29 . Thus, we analyzed the pressure amplitude and frequency up to 15 cmH2O (see Fig. 5A  CYP-treated bladders displayed increased amplitude of TCs with high frequency before apamin treatment ( Fig. 6A-C). SKA-31 and apamin treatment did not significantly affect the amplitude or frequency of TCs in CYP-treated bladders (n=6, Fig. 6B & C).

Discussion
This study investigated the mechanisms of DO in CYP-induced cystitis. We found the downregulation of gene transcripts and protein levels of PDGFRα and SK3 in CYPtreated detrusor muscles. The current density from SK channels evoked by a SK channel activator, CyPPA, was depressed in PDGFRα + cells isolated from CYP-treated detrusor muscles suggesting that gene and protein expression levels have an important functional effect on bladder function. In ex vivo experiments, an SK channel antagonist, apamin, increased the amplitude and frequency of TCs during filling in control bladders.
However, the effects of apamin were reduced significantly in the CYP-treated bladders.
Similarly the effects of an SK agonist were also minimal in CYP-treated bladders. These data suggest that loss of PDGFRα + cells and the major functional conductance provided by SK channels in these cells in CYP-treated bladders can lead to increased TCs and induction of DO.
Chemical cystitis is one of the adverse effects observed after administration of CYP chemotherapy in humans 30 . CYP also induces cystitis in mice and rats 13,31 .
Therefore, CYP treatments of rodents has been used widely as an experimental model of IC/BPS. Single CYP intraperitoneal injection leads to urinary bladder inflammation 32 , visceral pain 8 and DO 23 . However, the mechanisms of DO induced by CYP have not been clarified by previous studies. In the current study we tested the hypothesis that functional causes of DO after CYP treatment could be related to loss-of-function of bladder regulation provided by PDGFRα + cells.
Previous studies have shown that as the bladder fills, volume increases and the walls are stretched, but intravesical pressure remains low during much of the period of filling 33 . This accommodation occurs even though there is a natural tendency for detrusor SMCs to contract in response to stretch 34,35 . During bladder filling, NVCs are detected in cystometric records from various species [36][37][38][39] . NVCs appear to correspond to localized contractions that are also observed in ex vivo bladder preparations and have been termed 'spontaneous phasic contractions', 'micromotions' or 'TCs' [40][41][42][43][44] . TCs increase as bladder filling proceeds 40 . Activation of non-selective cation channels expressed in detrusor SMCs 34,35 generates TCs which initiate sensory inputs via activation of afferent nerves 40 that propagate to the central nervous system during bladder filling [45][46][47][48] . However, under physiological condition, development of TCs can be restrained due to activation of SK channels in detrusor muscles 25 . For instance, SK channel activators reduce and SK channel blocker increase detrusor muscle contractions [49][50][51][52][53] . Furthermore, Kcnn3 knockout mice show an increase in NVCs in in vivo cystometry and TCs in ex vivo bladders 40,54 . Previous reports demonstrate that Kcnn3 are highly expressed in detrusor PDGFRα + cells 25,28,55 . Since the current density of SK channels is very low in SMCs compared to detrusor PDGFRα + cells 25 , it seems clear that PDGFRα + cells serve an important stabilizing role in regulating contractile activity of SMCs during bladder filling. Therefore, disruption of PDGFRα + cells and/or downregulation of SK channels and intracellular signaling pathways regulating SK channel activity could lead to DO.
In the present study we found that Kcnn3 and Pdgfra transcripts were downregulated in CYP-treated detrusor muscles. Western analysis confirmed that both PDGFRα and SK3 proteins were reduced in CYP-treated detrusor muscles.
Immunohistochemistry also showed a reduction in the density of PDGFRα + cells in CYP-injected detrusor muscle. At present we do not know what factors, activated by CYP, might be responsible for loss of PDGFRα + cells and downregulation of SK channels in detrusor muscles, however the upregulation of inflammatory factors, such as Tnfα and Il6, suggest that an inflammatory mechanism may be involved.
Functional studies to evaluate the state of the SK conductance in detrusor PDGFRα + cells before and after CYP treatment were performed using the patch clamp technique. Our recordings confirmed the presence of an SK conductance in control (untreated) PDGFRα + cells, and the availability of this conductance was decreased in PDGFRα + cells isolated from CYP-treated bladders. As above, CYP treatment led to reduced PDGFRα + cell density, and the patch clamp experiments showed that there was concomitant reduction in the SK conductance normally prominent in these cells.
Since SK channels provide stabilization of membrane potential and excitability of SMCs during bladder filling, reducing the availability of the SK conductance would lead to increased generation of TCs. This hypothesis was confirmed using ex vivo bladder preparations of mice treated with CYP. We found an increase in TCs as the bladders were filled and less sensitivity of apamin in comparison to the control mice.
In conclusion, we found that CYP treatments induced DO was caused by reduced detrusor PDGFRα + cells and reduction in the prominent SK conductance expressed by these cells that is utilized to regulate SMC excitability during bladder filling. These experiments provide a novel understanding of detrusor PDGFRα + cells and how defects in these cells can contribute to the development of abnormal bladder activity.

Induction of CYP-induced cystitis
Murine CYP-induced cystitis was established according to previously described protocols 23,32,56 . To induce acute cystitis, mice were injected with 1.2 mg of CYP per 100 μl of saline solution and control mice were injected only with saline solution. Both mice were sacrificed on day 7 after CYP treatment.

RNA isolation, reverse-transcription PCR and quantitative PCR
Total RNA was isolated from the detrusor smooth muscle tissue using Direct-zol RNA miniPrep Kit (Zymo Research, Irvine, CA, USA), and first-strand cDNA was synthesized using qScriptTM cDNA SuperMix (Quanta, Gaithersburg, MD, USA) according to the manufacturer's instructions. Endpoint PCR was performed with specific primers (Table 1)

Wes Simple Western automated capillary electrophoresis and immunodetection
Tissue samples were prepared by homogenizing at 4 o C in 0.3ml radioimmune precipitation assay (RIPA) buffer (1X/ type) with added protease inhibitor tablet (Thermo-Fisher mini tablets EDTA free) with a Bullet Blender (5 mins, speed 5, 1 stainless steel bead per detrusor muscle). The homogenate was centrifuged at 4ºC, 3000 x g for 10 minutes, to remove cell debris. The supernatant was aliquoted and stored at -80°C 57 . Other tissue samples were subjected to differential centrifugation to obtain a plasma membrane-enriched fraction for PDGFRα and SK3 detection. In this case, the tissues were homogenized in ice cold lysis buffer (mM; 50 Tris HCl pH 8.0, 60 beta-glycerophosphate, 100 NaF, 2 EGTA, 25 Na-pyrophosphate, 1 DTT, and protease inhibitor tablet). Each tissue was homogenized in 0.3 ml lysis buffer, centrifuged at 16,000 x g at 4 o C for 10 min, and the supernatants centrifuged at 100,000 x g for 1 hour The plate was then loaded onto the automatic size-based Simple Western system for protein separation, antibody incubation and imaging using the Wes default parameters.
Image reconstruction of the detected proteins was generated by Compass software (ProteinSimple). The protein signals were quantified from the eletropherogram of the area under the chemiluminescent intensity peak obtained by Compass software.

Electrophysiological recordings
Whole cell currents and membrane potentials were recorded using whole cell voltage-and current-clamp techniques. Cells were placed in a 0.5 ml chamber mounted on an inverted microscope (Nikon, Japan). PDGFRα + cells, isolated from Pdgfra tm11(EGFP)Sor /J mice, were identified by the fluorescence of eGFP in nuclei. Pipette tip resistances were: 4-6 MΩ for PDGFRα + cells. An Axopatch 200B amplifier with a CV-4 headstage (Molecular Devices, Sunnyvale, CA, USA) was used. All data were analysed using pCLAMP software (Axon Instruments, USA) and Graphpad Prism (v. 3.0, Graphpad Software Inc., SanDiego, CA, USA). All recordings were made at room temperature of ∼21°C.

Ex Vivo Preparation
This technique is used to establish the relationship between storage volume and pressure. The bladders were extracted and the urethras ligated, close to the vesicoureteric junctions. A catheter was placed in the urethral opening to record pressure and filling. Intravesical pressure was recorded in reference to atmospheric pressure. An amplifier was connected to a transducer that has been filled with water. A syringe filled with KRB solution was connected to a pump. The infusion rate of the KRB solution was 15-25 µL/min by automatic infusion and this was kept at 37°C. The filling of the bladder with KRB solution was stopped when the pressure reaches 45-50 cmH2O. This is to avoid distending the bladder which can cause permanent tissue damage. Recordings obtained were analyzed by Clampfit (Molecular device) which had the baseline adjusted to examine the amplitude and frequency of pressures during filling (see Fig. 5).

Solutions and Chemicals
Whole-cell configuration was achieved in Ca 2+

Statistical Analyses
All Patch clamp data were expressed as means ± SEM. All statistical analyses were performed using Graphpad Prism. Student's paired or non-paired t test were used to compare groups of data and differences were considered to be significant at P < 0.05. Data analysis for the westerns was performed using Compass software (ProteinSimple, San Jose, CA, USA), and expressed as intensity area/ug of protein.
Lane view images of the western blots were saved as JPEGs, opened with Adobe Photoshop, converted to TIFFs; and saved after adjusting the image resolution with the Auto Res function.