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Published in final edited form as: Neurosci Lett. 2023 Nov 28;818:137563. doi: 10.1016/j.neulet.2023.137563

Lumbosacral spinal proteomic changes during PAR4-induced persistent bladder pain

Shaojing Ye a, Nilesh M Agalave b, Fei Ma a, Dlovan F D Mahmood a, Asma Al-Grety b, Payam Emani Khoonsari b, Camila I Svensson c, Kim Kultima b, Pedro L Vera a,d,*
PMCID: PMC10929774  NIHMSID: NIHMS1949196  PMID: 38036085

Abstract

Repeated intravesical activation of protease-activated receptor-4 (PAR4) in mice results in persistent bladder hyperalgesia (BHA). We investigated spinal proteomic changes associated with persistent BHA. Persistent BHA was induced in female mice by repeated (3x; days 0,2,4; n = 9) intravesical instillation of PAR4 activating peptide (PAR4-AP) while scrambled peptide served as the control (no pain; n = 9) group. The threshold to lower abdominal von Frey stimulation was recorded prior to and during treatment. On day 7, L6-S1 spinal segments were excised and examined for proteomic changes using LC-MS/MS. In-depth, unbiased proteomic tandem-mass tag (TMT) analysis identified and relatively quantified 6739 proteins. We identified significant changes with 29 decreasing and 51 increasing proteins in the persistent BHA group and they were associated with neuroprotection, redox modulation, mitochondrial factors, and neuronal-related proteins. In an additional experiment, decreases in protein levels were confirmed by immunohistochemistry for metallothionein 1/2. Our results show that persistent bladder pain is associated with central (spinal) protein changes. Previous work showed that PAR4-induced bladder pain is mediated, at least in part by spinal MIF. Further functional studies of these top changing proteins may lead to the discovery of novel potential therapeutic targets at the spinal level to modulate persistent bladder pain. Future studies will examine the effect of spinal MIF antagonism on PAR4-induced spinal proteomics associated with persistent bladder pain.

Keywords: Persistent bladder pain, Spinal proteins, Metallothioneins

1. Introduction

We previously described an animal model of persistent bladder pain where repeated (3x; every other day) intravesical instillations of a protease-activated receptor-4 activating peptide (PAR4-AP) resulted in pain that extended to day 9 [1]. In this model, bladder pain persisted a full five days after the last stimulus yet there were minimal or no histological changes in the bladder that could be associated with bladder inflammation [1].

Bladder pain was reversed by treatment with a systemic inhibitor of macrophage migration inhibitory factor (MIF) or by systemic administration of an inhibitor of high-mobility group box-1 (HMGB1) [1]. Moreover, we also showed that bladder pain could be reversed temporarily by intrathecal injections into the lumbosacral spine of an anti-MIF monoclonal antibody to neutralize MIF or a HMGB1-inhibitor [2] with maximal analgesic effect at 2 h post-injection. This evidence strongly suggested to us that lumbosacral spinal cord changes are associated with this persistent bladder pain model and furthermore, showed that spinal MIF and HMGB1 mediated bladder pain.

As a first step in discovering the spinal mechanisms of persistent bladder pain, the present study aims to determine spinal proteomics changes associated with PAR4-induced persistent bladder pain.

2. Material and methods

2.1. Experiment 1. Lumbosacral spinal proteomics changes in persistent bladder pain

All animal experiments were approved by the Lexington VA Health Care System Institutional Animal Care and Use Committee (VER-19-005-AF) and performed according to the guidelines of the National Institutes of Health.

We used repeated intravesical PAR4-AP instillations to induce acute bladder hyperalgesia (BHA; n = 9) as described earlier [1]. Briefly, C57BL/6 female mice were anesthetized with isoflurane (3% induction, 1.5% maintenance) and transurethrally catheterized (PE10, 11 mm length). Urine was drained by gently applying pressure to the lower abdomen. Bladders were slowly instilled with 100 μl of PAR4-AP (AYPGKF-NH2; Peptides International, Louisville, KY; 100 μM in sterile PBS) (pH 7.4, 100 μl) and remained in the bladder for 1 h. Intravesical scrambled peptide (YAPGKP-NH2; Peptides International, Louisville, KY; 100 μM in sterile PBS) was instilled in the same manner and served as the control (no pain) group (n = 9). Mice were allowed to recover and returned to their cages. Intravesical treatments were repeated another 2 times at 48 h intervals.

2.1.1. Abdominal mechanical sensitivity

Mice were acclimated to the testing conditions as follows:

  • (Days 1,2) Acclimation to testing room:

    –Mice placed in testing room and left undisturbed for 3 h.

  • (Days 3,4) Acclimation to testing chamber:

    –Mice placed in testing chamber and left undisturbed for 2 h.

  • (Day 5) Acclimation to von Frey monofilaments:

    –Mice placed in testing chamber and von Frey monofilaments applied to lower abdominal area.

  • Day 7) Baseline von Frey testing

Testing of lower abdominal mechanical hypersensitivity (an index of bladder pain) was performed as previously described [1–4]. Briefly, 50% mechanical threshold [5] was calculated by measuring the response to von Frey fibers (0.008, 0.02, 0.07, 0.16, 0.4, 1.0, 2.0 and 6.0 g) applied to the lower abdominal region. A positive response was defined as any one of three behaviors: (1) licking the abdomen, (2) flinching/jumping, or (3) abdomen withdrawal. Whenever a positive response to a stimulus occurred, the next smaller von Frey filament was applied. Otherwise, the next higher filament was applied. 50% thresholds were measured at baseline (day 0; prior to any treatment) and on days 2,3,4 and 7.

2.1.2. Proteomics sample preparation

Sham intrathecal injections were carried out to serve as control for future experiments that will test the effect of intrathecal treatments. On day 7, two hours after sham intrathecal injection (shown previously as the maximum analgesic treatment effect time [2]) L6/S1 spinal segments from both treatment groups were excised, snap frozen and stored at −80° C.

Spinal cord tissue was collected in a 1.5 ml Eppendorf tube and stored at −80°C. On experiment day, spinal cord tissue was trimmed and immersed in a lysis buffer (1% SDS in 1X PBS with protease inhibitor). Three freeze–thaw cycles were performed using ethanol and dry ice baths (in a Styrofoam box) and a temperature-controlled thermomixer. A tip sonicator was used to homogenize samples on wet ice for 10–15 s (0.3 s ON/0.7 s OFF pulse). Samples were clarified by centrifugation at 13,000 rpm for 30 min at 4°C. Supernatant containing total proteins were collected in a new labeled Eppendorf tube. Protein concentration was measured using a standard BCA assay kit from Pierce (ThermoFisher, 23225). An equal amount of protein was taken from each sample with a similar volume for the analysis. Samples were reduced by adding 10 mM dithiothreitol (DTT, Sigma, D9779), followed by incubation at 56°C for 30 min at a thermomixer with 500 rpm. Samples were alkylated by adding 25 mM iodoacetamide (IAA, Sigma, I6125) for 30 min at 37° C in the dark. Reduced/alkylated proteins were precipitated by adding one volume of pre-chilled acetone and another 5 volumes stepwise with vigorous vortexing and incubating at −20°C overnight. Protein pellet was obtained at bottom of Eppendorf tube by centrifuging at 20,000 g for 30 min at 4° C. Supernatant was discarded and the protein pellet was washed with one volume of 5:1, acetone: water. Pellets were resuspended in a digestion buffer (50 mM Triethylammonium bicarbonate, Sigma, T7408, pH 8.0). Enzymatic digestion was performed by adding Trypsin/Lyc protease mix (ThermoFisher, A41007) to each sample at 1:50 ratio and incubated for 4 h at 37°C with 500 RPM shaking followed by overnight digestion adding one more part of the enzyme. Digested protein samples were frozen at −80°C until the TMT labeling step.

2.1.3. TMT labelling and fractionation

Samples were randomly assigned to the TMTsix plex label (ThermoFisher, 90061) including samples from scramble control and PAR4-AP group on every TMT plex. The reagents from the TMT kit were equilibrated at room temperature. Six individual TMT labels were hydrated by adding 41 μl of anhydrous acetonitrile and incubated for 5 min at room temperature with occasional vortexing. Each sample was labeled with the respective assigned TMT label and incubated for 2 h at room temperature. TMT labeling reaction was stopped by adding 5% Hydroxylamine for 15 min. Quenched individual samples were pooled together from respective sets. All sets were further cleaned with Strata-X-C columns (8B-S029-TAK-TN) using wash solution (30%MeOH + 0.1% formic acid) and elution solution (30% MeOH + 5% ammonium hydroxide). Resultant eluted pools were fractionated using a high-performance LC system Ultimate-3000™ (Dionex Sunnyvale CA, USA), by trapping them first on a pre-column (3 μm C18 particles) and by eluting the peptides through a 50 cm EasySpray PepMap RSLC C-18 column (Thermo Scientific, Bremen, Germany). The eluted peptides were acquired using a high-resolution quadrupole orbitrap Q-Exactive mass spectrometer (Thermo Scientific, Bremen, Germany) in a data-dependent mode where the top 12 intense peptide ions were selected in full MS mode and further fragmented in MS/MS mode. The full MS was acquired with the orbitrap set at 70,000 resolution power (scan range 300–1600 m/z) and the MS/MS was performed by collision-induced dissociation and acquired at 35,000 resolution (scan range of 100–2000 m/z). In total 22 fractions were run from each pool. The experimental pipeline is briefly illustrated in Fig. 1.

Fig. 1.

Fig. 1.

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Graphical illustration shows the experimental pipeline for proteomics, with processing the spinal cord tissue, TMT labeling, fractionation under the uHPLC system, MS run, and data analysis.

2.1.4. Protein identification and quantification

Protein identification and quantification were conducted using Proteome Discoverer (Version 2.2.0.388, Thermo Fisher Scientific, Waltham, MA). The spectral data were searched against the Mus musculus protein data set and for known human contaminants. The analysis workflow incorporated Spectrum Files (data input), Spectrum Selector (spectrum and feature retrieval), Percolator (peptide spectral match or PSM Validation and FDR analysis), and Reporter Ions Quantifier (quantification). TMT 6-plex options were selected for respective analyses in all nodes. Only proteins with high or medium confidence, corresponding to a p-value ⩽0.05 were included in subsequent analyses. Results from Proteome Discoverer were exported and unique peptides and their quantification values were appended to their corresponding protein accession number.

2.1.5. Pathway analysis using STRING database

In order to visualize the predicted relationship between top increasing and decreasing proteins, we performed web-based enrichment analysis using the STRING database (www.string-db.org). As a statistical background, we used all detected proteins to perform pathway analysis on top increasing and decreasing regulated proteins.

2.2. Experiment 2: Preliminary validation of targets using immunohistochemistry

We replicated part of the experimental paradigm as described including two groups (n = 4/group): group 1: Intravesical scrambled peptide + sham i.t. (no pain); group 2: Intravesical PAR4-AP + sham i.t. (persistent BHA; unalleviated pain group). Two hours after sham intrathecal injections [2] the mice were transcardially perfused with saline followed by 4% paraformaldehyde. L6/S1 spinal cord segments were excised, placed in 4% paraformaldehyde overnight and then processed for paraffin embedding.

Paraffin sections (5 μm) of paraformaldehyde fixed L6/S1 spinal cord were dewaxed and processed for antigen retrieval using citrate buffer, pH 6.0 at 94°C for 20 min. Sections were treated with 3% H2O2 for 10 min to quench endogenous peroxidase and then with normal donkey serum for 1 h followed by incubation with primary antibody (rabbit anti-metallothionein; ab192385; Abcam, at 1:400 overnight at 4°C). Bound primary antibody was detected using a VECTASTAIN® ABC Kits (PK-4001, Vector Labs).

Two spinal cord sections were processed for immunohistochemistry from each animal. One section was selected randomly for photographs (20x) of dorsal horn and ventral horn, respectively. Digital images were captured with a Leica Microscope. Densitometry was performed using Image J.

2.3. Statistical analysis

Statistical analysis was performed using R [6]. For experiment 1, in each TMT set, the ratios between the samples and pools were utilized, and the data was transformed to the log2 scale. Proteins with less than 50% missing values were included for analysis. For each protein, a linear model was fitted where the TMT set was included as a covariate to adjust for differences between sets, and then t-statistics was used to determine any significant differences between persistent BHA and control.

Changes in histological scores (Experiment 2) were analyzed using Student’s t-test.

3. Results

3.1. Experiment 1. Lumbosacral spinal proteomics changes in persistent bladder pain

3.1.1. Persistent bladder hyperalgesia in the PAR4-AP treated group

We tested changes in von Frey lower abdominal mechanosensitivity in the 2 groups in this experiment up to day 7 prior to intrathecal treatment and lumbosacral spinal segment excision. Mice treated (3x) with intravesical scrambled peptide showed no change in von Frey threshold over the course of 7 days (Fig. 2). However, mice treated with intravesical PAR4-AP showed persistent BHA (Fig. 2).

Fig. 2.

Fig. 2.

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Persistent BHA induced by repeated intravesical PAR4 activation prior to lumbosacral spinal segment excision. Control mice treated with intravesical scrambled peptide (Scramb.) did not developed persistent BHA.

3.1.2. Global spinal proteome at lumbosacral region of spinal cord in persistent bladder hyperalgesia

We used LC-MS/MS method to investigate the regulation of proteome at the lumbosacral region of the spinal cord after induction of PAR4-induced BHA (PAR4 group). Lumbosacral region was collected on day 7 after induction of BHA model, control mice were treated with scrambled peptide. Tissues were subsequently processed for LC-MS/MS analysis. We found 6739 proteins were identified and relatively quantified. Out of 6739, 29 proteins were statistically significantly decreasing (Fig. 3A) whereas 51 proteins were statistically significantly increasing (Fig. 3B) in the PAR4-AP group compared to scrambled peptide control. The proteins that showed the highest increase were related to neuro-degeneration, synaptic function, mitochondrial activity, oxidative stress, pain induction, inflammation, and muscle and actin regulation, as shown in Fig. 3B. On the other hand, the proteins with the highest decrease were associated with neuroprotection, redox modulation, neuronal function, mitochondrial function, and spinal atrophy, as depicted in Fig. 3A. The full list of increasing and decreasing proteins is provided as Supplementary Table 1 (increasing) and Supplementary Table 2 (decreasing).

Fig. 3.

Fig. 3.

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Heatmap shows top decreasing (A) and increasing (B) proteins from mice treated with PAR4-AP (PAR4) compared with scrambled peptide control.

3.1.3. Enrichment pathway analysis on top increasing and decreasing proteins using STRING database

As a result of pathway analysis, five major clusters were identified including neuroprotective proteins, ribosomal proteins, mitochondrial-related proteins, and transcriptional regulators/caspase-related proteins (Fig. 4). Interestingly, many of the proteins on the provided list show a higher level of interaction than expected. We found protein–protein interaction (PPI) enrichment had a p-value = 0.0846 and average local clustering coefficient of 0.215. Moreover, we did not observe the functional enrichment in our network of top increasing and decreasing proteins. Eventually, GO-analysis will not include biological processes, molecular functions, cellular components, KEGG pathways and Reactome pathways.

Fig. 4.

Fig. 4.

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STRING pathway analysis shows the five clusters and pathway interactions within top increasing and Top decreasing proteins those are related to ribosomal protein cluster, Neuroprotective proteins cluster, Mitochondrial related proteins cluster, neuronal proteins cluster and transcription regulators and caspase related cluster.

3.2. Experiment 2. Preliminary validation of specific proteomics targets

In order to validate the results of our proteomics experiment, we replicated the experimental paradigm as described: group 1: Intravesical scrambled peptide + sham i.t. (no pain; n = 4); group 2: Intravesical PAR4-AP + sham i.t. (persistent BHA; unalleviated pain group; n = 4) and we examined pan-metallothionein (MT) levels in the L6/S1 cord using immunoperoxidase.

MT staining was prominent through the L6/S1 spinal segment with motorneurons and dorsal horns showing substantial immunostaining in mice treated with intravesical scrambled peptide (no pain group; Fig. 5A). Mice treated with PAR4-AP showed a significant decrease in MT immunostaining all throughout the cord (Fig. 5B). Densitometry analysis confirmed a statistically significant decrease in the dorsal horn area of PAR4-AP treated animals compared to scrambled peptide treatment (Fig. 5C).

Fig. 5.

Fig. 5.

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Decreased metallothionein (MT) staining in L6/S1 spinal cord in persistent BHA. A) MT immunostaining in ventral and dorsal horns of mice treated with scrambled peptide (no pain). B) Mice with persistent BHA had significant reductions in MT immunostaining consistent with decreases observed using unbiased proteomics analysis. C) Box plot showing median and IQR from densitometry analysis (* = p < 0.05). Scramb = Scramble peptide treated; PAR4 = PAR4-AP treated.

4. Discussion

Our results show that repeated intravesical treatment with PAR4-AP results in persistent bladder pain and proteomic changes at the level of the lumbosacral spinal cord. It should be noted that as we reported previously[1,2], this model of persistent bladder pain results in little or no inflammatory changes in the bladder and no changes in micturition.

We observed that mice with PAR4-induced persistent BHA had a different lumbosacral spinal proteomics profile than those in the control group that exhibited no bladder pain (intravesical scrambled peptide treated). We identified 29 proteins that were decreased and 51 proteins that were increased in the persistent BHA group when compared to control (scrambled peptide; no pain).

The lumbosacral proteins observed to decrease after persistent BHA included prothymosin-α (PTMA), metallothionein(MT)-1, MT-2, V-set and transmembrane domain-containing protein 2-like protein (VSTM2L), V-type proton ATPase subunit S1 (ATP6A1) and mitochondrial import inner membrane translocase subunit tim8 A and others listed in Table 2.

Metallothioneins (MT) are small, cysteine-rich proteins involved in metal homeostasis, oxidative stress and inflammation [7–11]. They are involved in mediating pain with conflicting reports in the literature. A decrease in MT-2 was reported in spinal cord neurons [12] and peripheral nerves [12] in neuropathic pain while MT-1/2 was reported increased in spinal cord blood vessels in another study [13]. We verified using immunohistochemistry, that two of those proteins MT-1/2, were in fact decreased in the dorsal horn of the lumbosacral spinal cord. Since we used a pan-metallothionein antibody we were unable to identify individual metallothionein proteins. Nevertheless, this finding provides initial validation of our proteomics findings and indicates that further work should concentrate on validating and determining the functional significance of the proteins identified for their role in persistent bladder pain.

PTMA is a ubiquitous protein with numerous roles that include neuroprotection [14–16], modulation of immune responses (possible through binding TLR4 receptors [17]) and may function as an alarmin [18]. More recently, PTMA was reported to mediate post-stroke pain [19]. VSTM2L is expressed in multiple tissues (including brain) although its role is still unclear. It has been implicated in modulating neuroprotection (as an antagonist of the neuroprotective peptide Humanin [20]), neurodegenerative and metabolic diseases [21–23]. ATP6A1 is a highly conserved and ubiquitous protein with multiple functions including acidification of intracellular compartments, iron metabolism, bone erosion, inflammation and cancer [24–26]. In the brain, ATP6AP1 is involved in neurotransmitter loading into vesicle and neurotransmitter release although the exact mechanisms are not clear [27]. To our knowledge our current findings are the first to associate VSTM2L and ATP6A1 with pain.

Among the top proteins identified as increased during persistent BHA (Table 1) we noted claudin-19, caveolin-2 and superoxide dismutase. Claudin-19 is one of the tight-junction proteins that maintain the blood–brain barrier and spinal cord claudin-19 was shown to decline mRNA expression in a rodent model of neuropathy [28] while a different report showed no change in expression in the dorsal root ganglia using the same model [29]. Our findings that claudin-19 protein levels actually increased after persistent BHA is intriguing and warrants further investigation.

Caveolin-2 was also reported increased in the brains of old mice and was associated with neuroinflammation [30]. Superoxide dismutase is modulator of reactive oxygen species [31] and mediates trigeminal pain centrally [32]. We recently reported that oxidative stress mediates PAR4-induced bladder pain [4] and our current findings support a spinal locus of action.

Proteomics analysis in Interstitial Cystitis/Bladder Pain Syndrome has focused on urinary, bladder or serum proteins in search for biomarkers for this disease [33–35]. Meanwhile, analysis of spinal proteomics changes is a powerful tool to discover novel proteins involved in chronic pain conditions [36–38] including work from members of our study team [39,40]. Our approach of focusing on lumbosacral spinal proteomics in a persistent bladder pain model that results in little or no bladder inflammation and may mimics non-Hunner IC/BPS represents, to our knowledge, a novel approach in using unbiased proteomics analysis to understand persistent bladder pain.

Thus, our results show that spinal proteomics changes accompany persistent bladder pain and our previous work [2] showed that persistent bladder pain was modulated by spinal MIF. The specific role of each of the identified potential targets in the development of persistent bladder pain remain to be elucidated. Further study of these possible targets may help us understand the mechanisms of persistent bladder pain.

5. Conclusions

We identified spinal proteomics changes associated with persistent bladder pain after repeated intravesical infusion with PAR4-AP and we provided preliminary validation for two of the targets identified. Together our results show that spinal changes due to peripheral manipulation to induce persistent bladder pain may result in discovery of novel targets that can be used to modulate persistent bladder pain. Future studies will validate the proteomics changes outlined here and determine their functional role in mediating persistent bladder pain as well as determine specific spinal proteomic changes mediated by intraspinal MIF antagonism.

Acknowledgments

This work for supported by funding from NIH (DK121695; PLV), the Knut and Alice Wallenberg Foundation (CIS) and the Magnus Bergvall Foundation (KK). The material is the result of work supported with the resources and facilities at the Lexington (Kentucky) VA Health Care System.

Footnotes

CRediT authorship contribution statement

Shaojing Ye: Conceptualization, Methodology, Validation, Investigation, Data curation, Formal analysis, Writing - original draft, Writing - review & editing. Nilesh M. Agalave: Conceptualization, Methodology, Validation, Investigation, Data curation, Formal analysis, Software, Writing - original draft, Writing - review & editing. Fei Ma: Conceptualization, Methodology, Validation, Investigation, Writing - original draft, Writing - review & editing. Dlovan F. D Mahmood: Investigation, Data curation, Writing - original draft, Writing - review & editing. Asma Al-Grety: Investigation, Data curation, Writing - original draft, Writing - review & editing. Payam Emani Khoonsari: Investigation, Data curation, Writing - original draft, Writing - review & editing. Camila I. Svensson: Conceptualization, Methodology, Validation, Writing - original draft, Writing - review & editing, Funding acquisition. Kim Kultima: Conceptualization, Methodology, Validation, Resources, Software, Formal analysis, Writing - original draft, Writing - review & editing, Funding acquisition. Pedro L. Vera: Conceptualization, Methodology, Validation, Resources, Software, Formal analysis, Writing - original draft, Writing - review & editing, Funding acquisition.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.

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