- Academic Editors
Background: Diabetic bladder dysfunction (DBD) is driven in part by
inflammation which dysregulates prostaglandin release in the bladder. Precise
inflammatory mechanisms responsible for such dysregulation have been elusive.
Since prostaglandins impact bladder contractility, elucidating these mechanisms
may yield potential therapeutic targets for DBD. In female Type 1 diabetic Akita
mice, inflammation mediated by the nucleotide-binding domain,
leucine-rich–containing family, pyrin domain–containing-3 (NLRP3) inflammasome
is responsible for DBD. Here, we utilized female Akita mice crossbred with
NLRP3 knock-out mice to determine how NLRP3-driven inflammation impacts
prostaglandin release within the bladder and prostaglandin-mediated bladder
contractions. Methods: Akita mice were crossbred with
NLRP3
Diabetic bladder dysfunction (DBD) develops in half of all patients with diabetes [1, 2, 3]. Their symptoms are variable with underactive bladder (UAB) being one of the more bothersome phenotypes. Patients may experience a decreased sensation of bladder fullness, slow urine stream, and overflow incontinence indicative of hypocontractile bladders and often rely on self-catheterization to void, since no targeted pharmacological therapies are available. In order to develop much-needed therapies, further elucidation of the mechanisms responsible for DBD are warranted.
A hallmark of diabetes is low-grade inflammation mediated by a host of pro-inflammatory immune responses [4, 5, 6]. Of particular interest is inflammation mediated by the nucleotide-binding domain, leucine-rich–containing family, pyrin domain–containing-3 (NLRP3) inflammasome. This component of the innate immune system is highly expressed in bladder urothelial cells. Diabetic metabolites, such as uric acid and high-mobility group box 1 protein, activate NLRP3 to release pro-inflammatory cytokines [7]. Notably, we have shown NLRP3 to be a critical inflammatory pathway responsible for DBD in female type 1 diabetic Akita mice [7, 8]. Despite its critical importance, it is unclear what downstream signaling pathways are dysregulated by NLRP3 activation in the bladder. However, in the kidney, NLRP3 activation within murine proximal tubule cells increases prostaglandin production [9, 10], suggesting similar pathways may be active in the bladder.
Prostaglandins are eicosanoids derived from fatty acid synthesis via cyclooxygenases and they are produced throughout the body, including urothelia and bladder smooth muscle [11, 12]. Inflammatory conditions such as diabetes have been shown to upregulate prostaglandin production and release [4, 13, 14, 15]. Once released, prostaglandins activate receptors in an autocrine and paracrine manner within close proximity from where they are released and are quickly degraded into metabolites which accumulate in urine. Such metabolites are upregulated in patients with either type 1 or type 2 diabetes [4, 13, 14, 15]. Moreover, controlling blood glucose also reduces the levels of prostaglandin metabolites in type 1 diabetic urine [14]. Despite clinical evidence of increased prostaglandin production in diabetic bladders, the physiological effects of prostaglandins on diabetic bladder function are not well understood.
Several prostaglandins have been the focus of scientific inquiry in the diabetic
bladder - particularly prostaglandin E2 (PGE2) and prostaglandin F2
Animal procedures were conducted in accordance to guidelines set forth by the
National Institutes of Health Guide for the Care and Use of Laboratory Animals
and approved by the Duke University Medical Center Institutional Animal Care and
Use Committee (approval number: A088-22-05). Type 1 diabetic Akita mice
(C57BL/6J-Ins2Akita/J; stock #003548; Jackson Laboratory, Bar Harbor, ME, USA) have a heterozygous mutation of the
insulin 2 (Ins2) gene which causes them to spontaneously develop
hyperglycemia by 4–5 weeks of age. To determine the specific role of NLRP3 in the
progression of diabetes, Akita mice and NLRP3
(1) Non-diabetic NLRP3
(2) Diabetic NLRP3
(3) Non-diabetic NLRP3
(4) Diabetic NLRP3
Blood obtained from the lateral tail vein was collected to measure blood glucose using the AimStrip Plus glucometer and testing strips (Lot number 396051.Germaine Laboratories, San Antonio, TX, USA).
Bladders were excised and immediately put in ice cold Krebs solution
made up of 118.5 mM NaCl, 58.44 mM KCl, 1.2 mM MgCl
Bladders were excised and placed in ice cold Krebs solution consisting
of 118.5 mM NaCl, 58.44 mM KCl, 1.2 mM MgCl
Stock solutions of PGE2 (Cayman Chemical Co.; item #14010), PGF2
At 30 weeks, mice were euthanized and bladders were removed. The detrusor was
carefully separated from the mucosa and stored at –80 °C. Detrusors
were homogenized in ice cold RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5%
sodium deoxycholate, 0.1% SDS, and 50 mM Tris; pH 7.4) supplemented with a
protease and phosphatase inhibitor cocktail at 1
Proteins (100 µg) from each detrusor were reduced in lithium dodecyl sulfate
sample buffer and heated at 70 °C for 5 minutes. Reduced proteins were
then loaded into two separate 4–12% Bis-Tris SDS polyacrylamide gels
(Invitrogen, Waltham, MA, USA) with each gel containing 50 µg of protein per detrusor. Both
gels containing the molecular weight marker, Precision Plus Protein WesternC
Blotting Standard (Bio-Rad; Hercules, CA, USA), underwent simultaneous
electrophoresis in a duel chamber tank containing 1
Both membranes were probed simultaneously overnight at 4 °C in PBS containing 5% bovine serum albumin, 0.1% Tween 20, and either a primary antibody or primary antibody plus a blocking peptide. The first membrane was probed with a primary antibody against the FP receptor (1:200; Cayman Chemical Co.; item #101802) while the second membrane was probed with a premixed solution containing the FP receptor primary antibody (1:200) and a FP receptor blocking peptide (1:100; Cayman item #301802). Following the overnight primary antibody +/- blocking peptide incubation, secondary antibody incubation was performed at room temperature for 1 hour using a goat anti-rabbit alexa fluor 488 antibody (1:5,000; Jackson Immunoresearch item #111-545-144) in PBS containing 5% bovine serum albumin and 0.1% Tween 20. Labeled proteins were detected using the ChemiDoc MP system (Bio-Rad). Membranes were re-probed with a primary antibody against the housekeeping protein, GAPDH (1:5000; GeneTex item #GTX100118, Irvine, CA, USA). Protein expression analysis was conducted using Image Lab 6.1 software (Bio-Rad) and target protein expression was normalized to GAPDH expression. Representative western blot images were converted from color images to gray scale using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Uncropped western blots used for data analysis are provided as Supplementary Figs. 1,2,3 section.
Prism 9 software (GraphPad; San Diego, CA, USA) was used to create all graphs
and perform statistical analysis. All data is reported as mean
Consistent with prior studies [8], female diabetic Akita mice exhibit marked hyperglycemia by 30 weeks of age (Fig. 1A). Although NLRP3 gene deletion has previously been demonstrated to prevent diabetic females from developing bladder inflammation [7, 8], NLRP3 gene deletion has no impact on blood glucose (Fig. 1B). This suggests hyperglycemia-induced inflammation is a key factor responsible for the development and progression of DBD in this model.
NLRP3 gene deletion does not impact blood glucose of
female Akita mice. Blood glucose was measured in all groups at 30 weeks of age.
(A) Female diabetics demonstrate marked hyperglycemia compared to non-diabetic
controls. (B) In the absence of the NLRP3 gene, and therefore
NLRP3-mediated inflammation, blood glucose remains significantly
elevated. Data are presented as mean
The increased levels of prostaglandin metabolites found in the urine of patients
with diabetes suggests diabetes increases prostaglandin production [13, 14, 15];
however, it is unclear where prostaglandin upregulation occurs – either
systemically and/or locally within the bladder. In this set of experiments, we
sought to determine if this increase may result from a change in its release from
urothelia and/or detrusors. Diabetes increases the amount of PGE2 released from
urothelia compared to non-diabetics (Fig. 2A). Surprisingly, diabetes
dichotomously decreases the amount of PGE2 released from bladder smooth muscle
(Fig. 2B). In order to determine if these changes were due to NLRP3-mediated
inflammation, we performed the same experiments in diabetic females lacking the
NLRP3 gene. We have previously shown that diabetic mice lacking the
NLRP3 gene do not develop inflammation in the bladder, even at 30 weeks,
and the mice are protected from developing UAB [7, 8]. Here, deletion of the
NLRP3 gene also prevents diabetes from dysregulating PGE2 release in
both urothelia and detrusors (Fig. 2C,D) as there are no significant differences
in PGE2 release compared to non-diabetics without the NLRP3 gene. The
same pattern does not apply to the release of PGF2
Diabetes increases prostaglandin E2 (PGE2) release from
urothelia but conversely decreases PGE2 release from detrusors due to
NLRP3-dependent inflammation. Strips of urothelia-lined mucosa and detrusors
were stretched ex vivo to release PGE2, which was then quantified using
an enzyme linked immunosorbent assay (ELISA). (A) Diabetes nearly doubles the
amount of PGE2 released from urothelia. (B) However, in the detrusor, the amount
of PGE2 released from detrusors is significantly reduced. (C) In diabetic mice
lacking the NLRP3 gene, no significant changes in PGE2 release are
detected in either the urothelia or (D) detrusors. Data are presented as mean
Prostaglandin F2
Prostaglandins exert physiological effects on tissues within limited proximity
to the site from which they are released and, in the bladder, prostaglandins
regulate detrusor contractility. To determine how prostaglandin-mediated
contractile force is impacted by diabetes, ex vivo concentration
response curves to PGE2 and PGF2
Bladder contractions mediated by PGE2 are not impacted by
NLRP3-mediated inflammation attributed to diabetes. Strips of bladder tissue
with intact mucosa and urothelia were used to assess smooth muscle function
ex vivo in a myograph apparatus. Contractile force was measured in
response to cumulative increases in concentrations of PGE2. (A) Contractile force
generated in response to PGE2 and the (B) potency (EC
Diabetes increases PGF2
Although PGF2
FP receptor activation facilitates the increase in
PGF2
FP receptors are highly expressed in human bladder smooth muscle [23]. Multiple
FP receptor isoforms have been identified in mouse liver, spleen, kidneys,
testes, and vas deferens [24]. The most abundant of which are the FP
Diabetes increases FP
DBD is a multifactorial pathology for which the precise mechanisms remain elusive. One well-regarded contributor to DBD is inflammation, which has been linked to aberrant prostaglandin synthesis and release [4, 13, 14, 15]. However, a link between DBD, the contribution of specific inflammatory signaling pathways, and aberrant prostaglandin regulation in the bladder has not been established in current literature. Here, we demonstrate that diabetes-associated inflammation mediated by the NLRP3 inflammasome may be responsible for dysregulated prostaglandin release in the bladder, enhanced prostaglandin-mediated detrusor contractions, and increased prostaglandin receptor expression in the female Type 1 diabetic Akita model that develops UAB [8]. Our data suggests NLRP3 inhibition may prevent the development of DBD in part by preventing such dysregulation of prostaglandin signaling pathways. In the pursuit of elucidating these NLRP3-dependent mechanisms, we demonstrate an upregulation of FP receptors in diabetic bladders. Pharmacological agonists targeting these FP receptor populations may increase the contractile force of otherwise underactive bladders and possibly prove to be an effective therapeutic option to treat existing DBD.
Systemic low-grade inflammation is a well-appreciated characteristic of diabetes. Patients with diabetes and refractory overactive bladder (OAB) have been shown to have significant urothelial inflammation as indicated by an elevated number of activated mast cells [26]. Our laboratory has extensively investigated the critical role of the NLRP3 inflammasome in mediating urothelial inflammation [7, 8, 27, 28, 29]. NLRP3 is ubiquitously expressed in urothelia and can be activated by metabolites commonly found in urine of patients with diabetes [7]. Upon activation, NLRP3 causes urothelial cell death and releases pro-inflammatory cytokines which subsequently recruit mast cells to further perpetuate an inflammatory cascade. In female Akita mice, NLRP3 is a critical factor responsible for the development of both OAB and UAB. Genetic deletion of NLRP3 in female Akita mice prevents bladder inflammation assessed using an Evans blue assay and the development of either DBD phenotype [7, 8]. This poses the novel possibility that NLRP3 inhibitors may prevent the development of DBD in human patients. Unfortunately, there are currently no clinical biomarkers to predict the development of DBD and patients generally do not seek urological consult until after symptoms of DBD have developed. Thus, in order to identify novel therapeutic targets to treat existing DBD, our efforts focused on elucidating the poorly understood NLRP3-dependent downstream signaling pathways which regulate bladder function.
NLRP3-dependent inflammation has been shown to facilitate murine proximal tubule
cell injury by increasing cyclooxygenase-2-mediated PGE2 synthesis [10]. High
levels of prostaglandin metabolites found in the urine of patients with diabetes
suggest this signaling pathway may be consistent with pathology contributing to
clinical DBD. Interestingly, inflammatory conditions are often associated with
increased prostaglandin synthesis, and high levels of PGE2 and PGF2
Since prostaglandins exert physiological effects on tissues near the site of
release before rapidly degrading, we postulated that changes in prostaglandin
release would associate with changes in the prostaglandin-mediated contractile
force of the detrusor. Multiple studies demonstrated prostaglandins directly
contract detrusor ex vivo [16, 19, 20, 21]. Given the significant increase in
stretch-mediated PGE2 release, the lack of significant changes in PGE2-mediated
contractile force in diabetic bladders was unanticipated. This could be due to
changes in the expression of multiple forms of EP receptors in the detrusor.
Changes in the expression of EP1 and EP3 receptors, which are pro-contractile,
may be masked by changes in the expression of EP2 and EP2 receptors which
facilitate relaxation [30]. However, since the potency of PGE2, indicated by the
EC
Diabetes-associated pathological changes in the mechanisms by which
prostaglandins generate contractile force in the bladder are poorly understood,
particularly regarding PGF2
Inflammation mediated by NLRP3 is attributed to a host of negative pathologies contributing the development of DBD in female Akita mice [7, 8]. However, the NLRP3-dependent increase in contractile force generated by FP receptor activation as well as the increased production of urothelial PGE2 appear more likely to be compensatory mechanisms rather than causes of UAB in diabetics. In an upcoming study, we will investigate the nature of prostaglandin synthesis and receptor expression at an earlier time point when the Akita develops overactive bladder. We have previously demonstrated that female Akita mice demonstrate a progression from OAB at 15 weeks of age to UAB by 30 weeks of age [7, 8]. If we see results similar to what is observed at 30 weeks, that would suggest that this is a compensatory mechanism that is overwhelmed after a further 15 weeks of disease progression.
Regardless of the reasons why FP receptors are upregulated, our data suggests FP
receptor agonists may be effective therapies to treat existing underactive DBD.
This is interesting given the availability of FP receptor agonists currently
being used to treat non-urological conditions such as glaucoma. One commonly
prescribed FP receptor agonist is latanoprost. This medication is available as an
ophthalmic solution which reaches a peak systemic half-life in 5 minutes and has
an elimination half-life of only 17 minutes [33]. Despite this short half-life
and low potential for systemic effects, OAB symptoms can develop as a consequence
of latanoprost therapy. In one case report, a 62-year-old female patient began
voiding small amounts of urine at an increased frequency and exhibited urge
incontinence upon taking latanoprost [34]. Conventional challenge-rechallenge
testing confirmed latanoprost was the cause of her OAB symptoms, which subsided
upon terminating latanoprost therapy. Furthermore, similar to the ex
vivo PGF2
NLRP3-dependent inflammation associated with diabetes dysregulates the release of prostaglandins and increases the capacity of underactive bladders to generate contractile force in response to prostaglandins that activate FP receptors within the detrusors due, in part, to a four-fold upregulation of FP receptor protein expression. These novel findings suggest FP receptor agonists – of which there are commercially available examples [34] – may be utilized as a novel therapeutic to treat existing underactive DBD.
Uncropped western blot images used for analysis are provided as Supplementary Figs. 1, 2, and 3. All raw numerical data used for analysis and reported within figure graphs are provided as Supplementary Tables 1, 2, 3, 4.1, 4.2, 5.1, 5.2, 6, and 7.
MRO: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Data curation, Writing – original draft, Writing – review & editing, Visualization, Supervision, Project administration, Funding acquisition. FMH Jr.: Methodology, Investigation, Supervision, Project administration, Writing – review & editing. NP: Methodology, Investigation, Writing – review & editing. HJ: Methodology, Investigation. JTP: Conceptualization, Validation, Formal analysis, Resources, Writing – review & editing, Supervision, Project administration, Funding acquisition. All authors have participated sufficiently in the work to take public responsibility for appropriate portions of the content and agreed to be accountable for all aspects of the work in ensuring that questions related to its accuracy or integrity. All authors read and approved the final manuscript. All authors contributed to editorial changes in the manuscript.
Animal procedures were conducted in accordance to guidelines set forth by the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Duke University Medical Center Institutional Animal Care and Use Committee (approval number: A088-22-05).
We would like to thank the Duke University Breeding Core Facility for their invaluable assistance in breeding the mice generated for this manuscript.
This work was supported by National Institute of Diabetes and Digestive and Kidney Disease grants RO1 DK117890 and K12 DK100024.
The authors declare no conflict of interest.
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