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Bladder and bowel dysfunction (BBD) is a common yet underdiagnosed paediatric entity that describes lower urinary tract symptoms (LUTS) accompanied by abnormal bowel patterns manifested as constipation and/or encopresis. LUTS usually manifest as urgency, urinary frequency, incontinence, and urinary tract infections (UTI). Despite increasing recognition of BBD as a risk factor for long-term urinary tract problems including recurrent UTI, vesicoureteral reflux, and renal scarring, the mechanisms underlying BBD have been unclear, and treatment remains empirical. We investigated how constipation affects the lower urinary tract function using a juvenile murine model of functional constipation. Following four days of functional constipation, animals developed LUTS including urinary frequency and detrusor overactivity evaluated by awake cystometry. Physiological examination of detrusor function in vitro using isolated bladder strips, demonstrated a significant increase in spontaneous contractions without affecting contractile force in response to electrical field stimulation, carbachol, and KCl. A significant upregulation of serotonin receptors, Htr2a and Htr2c, was observed in the bladders from mice with constipation, paralleled with augmented spontaneous contractions after pre-incubation of the bladder strips with 0.5 µM of serotonin. These results suggest that constipation induced detrusor overactivity and increased excitatory serotonin receptor activation in the urinary bladder, which contributes to the development of BBD.

Chronic lung diseases (CLDs), such as chronic obstructive pulmonary disease, interstitial lung disease, and lung cancer, are among the leading causes of morbidity globally and impose major health and financial burdens on patients and society. Effective treatments are scarce, and relevant human model systems to effectively study CLD pathomechanisms and thus discover and validate potential new targets and therapies are needed. Precision-cut lung slices (PCLS) from healthy and diseased human tissue represent one promising tool that can closely recapitulate the complexity of the lung's native environment, and recently, improved methodologies and accessibility to human tissue have led to an increased use of PCLS in CLD research. Here, we discuss approaches that use human PCLS to advance our understanding of CLD development, as well as drug discovery and validation for CLDs. PCLS enable investigators to study complex interactions among different cell types and the extracellular matrix in the native three-dimensional architecture of the lung. PCLS further allow for high-resolution (live) imaging of cellular functions in several dimensions. Importantly, PCLS can be derived from diseased lung tissue upon lung surgery or transplantation, thus allowing the study of CLDs in living human tissue. Moreover, CLDs can be modeled in PCLS derived from normal lung tissue to mimic the onset and progression of CLDs, complementing studies in end-stage diseased tissue. Altogether, PCLS are emerging as a remarkable tool to further bridge the gap between target identification and translation into clinical studies, and thus open novel avenues for future precision medicine approaches.

Introduction: Idiopathic Pulmonary Fibrosis (IPF) is an age-related disease, which displays several hallmarks of aging, such as cellular senescence. The lung epithelium represents a major site of tissue injury in IPF resulting in functional changes including aberrant activity of the developmental WNT/β-catenin pathway. The potential impact of WNT/β-catenin signaling on epithelial senescence in aging and IPF remains elusive. Methods: Single Cell RNA Sequencing (scRNAseq) from EpCAM-enriched cell populations was performed from IPF and donor lungs. RNAseq, qPCR and flow cytometry was applied to characterize ATII cells from young (3 months) and old (18 months) wild-type or WNT-reporter mice, respectively. Progenitor cell potential was investigated by organoid assay. ATII cells were treated with WNT3A for up to 7 days. Cellular senescence was monitored by qPCR and senescence-associated s-Galactosidase activity. Results: ScRNAseq revealed a concomitant enrichment of both WNT/β-catenin signaling and cellular senescence in distinct cell populations in IPF. Similarly, aged ATII cells showed increased cellular senescence and increased WNT/β-catenin activity. Moreover, aged ATII cells exhibit an impaired organoid forming capacity. Bulk RNA sequencing revealed 416 differentially expressed genes in aged ATII cells, many of which are further known to be dysregulated in fibrosis. Chronic WNT/β-catenin activation induced cellular senescence in primary ATII cells. Simultaneously, chronic but not acute WNT activation induced a profibrotic state with increased expression of the impaired ATII cell marker Keratin8. Conclusions: These results suggest that WNT/β-catenin signaling contributes to progenitor cell dysfunction and impaired lung repair in the aged lung, which is further enhanced in IPF.

Introduction: Chronic lung diseases (CLD) are linked to aging and are characterized by different hallmarks of aging, such as cellular senescence. Within CLD, several cell types have been shown to become senescent, however, it is unknown whether these are disease-specific, and moreover, potential disease- and/or cell-specific senescence inducers, remain unexplored. WNT/β-catenin is an oncogenic pathway increased in pulmonary fibrosis and has been reported to induce senescence. Here, we investigate which cells are affected by WNT/β-catenin induced senescence in the lung. Methods: Precision-cut lung slices (PCLS) were treated with WNT stimulators for up to 7 days. PCLS were characterized by qPCR, Immunofluorescence and Flow Cytometry directly after slicing or after 24h, 5 days or 7 days of culture. Results: WNT-stimulation in PCLS led to significant induction of WNT target genes, such as Axin2. After 7 days of treatment, cellular senescence was assessed by p16 and p21 gene expression and Immunofluorescence. In particular, we found strong p21 staining in lung epithelial cells in PCLS. In order to characterize single cells from PCLS, we established a protocol to isolate single cells from PCLS for subsequent flow cytometry and sorting. Single cell suspensions generated from PCLS showed similar proportions of epithelial (EpCam+), inflammatory (CD45+), endothelial (CD31+) and mesenchymal (CD90+) cells as compared to the native lung. Conclusions and Outlook: Cellular senescence can be induced in PCLS ex vivo and PCLS further allow mechanistic analysis of single cell-specific senescence. Ongoing analysis of single cell sequencing data from human aging and CLDs will be utilized to validate cell- / disease-specific senescence.

Cytotoxic chemotherapy is the foundation for the treatment of the wide variety of childhood malignancies; however, these therapies are known to have a variety of deleterious side effects. One common chemotherapy used in children, doxorubicin (DOX), is well known to cause cardiotoxicity and cardiomyopathy. Recent studies have revealed that DOX impairs skeletal and smooth muscle function and contributes to fatigue and abnormal intestinal motility in patients. In this study, we tested the hypothesis that systemic DOX administration also affects detrusor smooth muscle (DSM) function in the urinary bladder, especially when administered at a young age. The effects on the DSM and bladder function were assessed in BALB/cJ mice that received six weekly intravenous injections of DOX (3 mg·kg−1·wk−1) or saline for the control group. Systemic DOX administration resulted in DSM hypertrophy, increased voiding frequency, and a significant attenuation of DSM contractility, followed by a slower relaxation compared with the control group. Gene expression analyses revealed that unlike DOX-induced cardiotoxicity, the bladders from DOX-administered animals showed no changes in oxidative stress markers; instead, downregulation of large-conductance Ca2+-activated K+channels and altered expression of myosin light-chain kinase coincided with reduced myosin light-chain phosphorylation. These results indicate that in vivo DOX exposure caused DSM dysfunction by dysregulation of molecules involved in the detrusor contractile-relaxation mechanisms. Collectively, our findings suggest that survivors of childhood cancer treated with DOX may be at increased risk of bladder dysfunction and benefit from followup surveillance of bladder function.

Aims Mechanosensitivity of the urinary bladder is regulated by many factors including mechano-gated two-pore domain (K2 P, KCNK) potassium channels. TWIK-related K+ channel, TREK-1, is a predominantly expressed member of K2 P channel family in the human detrusor, and its expression and function are diminished in patients with overactive lower urinary tract symptoms (LUTS). The changes in channel activity may result from spontaneously occurring gene mutations. The aim of this study was to compare single nucleotide polymorphisms (SNPs) in TREK-1 channel between patients with LUTS and healthy donors. Methods Six SNPs (rs370266806, rs373919966, rs758937019, rs769301539, rs772497750, and rs775158737) in two pore domains of human TREK-1 gene were analyzed using TaqMan SNP genotyping assay with manufacturer-designed primers and allele-specific probes. The screening was done in control bladders and detrusor specimens from patients with overactive LUTS. Statistical analyses were performed using R, Fisher's exact test and Hardy-Weinberg Equilibrium. Results Six SNPs in two pore domains of the human TREK-1 gene were analyzed in human bladder specimens. The frequencies of rs758937019-CT genotype (P = 0.0016) and rs758937019-T allele (P = 0.0022) were significantly higher in the group with overactive LUTS. There was no significant association of rs775158737-GA genotype and rs775158737-A allele with the overactive LUTS, though they were present only in the overactive LUTS group. Conclusions Our results provide evidence that altered expression and function of TREK-1 channel in patients with overactive LUTS could be due to genetic polymorphisms in the pore domains of TREK-1 channel (rs758937019).

Background Previously published results from our laboratory identified a mechano-gated two-pore domain potassium channel, TREK-1, as a main mechanosensor in the smooth muscle of the human urinary bladder. One of the limitations of in vitro experiments on isolated human detrusor included inability to evaluate in vivo effects of TREK-1 on voiding function, as the channel is also expressed in the nervous system, and may modulate micturition via neural pathways. Therefore, in the present study, we aimed to assess the role of TREK-1 channel in bladder function and voiding patterns in vivo by using TREK-1 knockout (KO) mice. Methods Adult C57BL/6 J wild-type (WT, N = 32) and TREK-1 KO (N = 33) mice were used in this study. The overall phenotype and bladder function were evaluated by gene and protein expression of TREK-1 channel, in vitro contractile experiments using detrusor strips in response to stretch and pharmacological stimuli, and cystometry in unanesthetized animals. Results TREK-1 KO animals had an elevated basal muscle tone and enhanced spontaneous activity in the detrusor without detectable changes in bladder morphology/histology. Stretch applied to isolated detrusor strips increased the amplitude of spontaneous contractions by 109% in the TREK-1 KO group in contrast to a 61% increase in WT mice (p ≤ 0.05 to respective baseline for each group). The detrusor strips from TREK-1 KO mice also generated more contractile force in response to electric field stimulation and high potassium concentration in comparison to WT group (p ≤ 0.05 for both tests). However, cystometric recordings from TREK-1 KO mice revealed a significant increase in the duration of the intermicturition interval, enhanced bladder capacity and increased number of non-voiding contractions in comparison to WT mice. Conclusions Our results provide evidence that global down-regulation of TREK-1 channels has dual effects on detrusor contractility and micturition patterns in vivo. The observed differences are likely due to expression of TREK-1 channel not only in detrusor myocytes but also in afferent and efferent neural pathways involved in regulation of micturition which may underly the “mixed” voiding phenotype in TREK-1 KO mice.

Detrusor overactivity (DO) is the abnormal response of the urinary bladder to physiological stretch during the filling phase of the micturition cycle. The mechanisms of bladder smooth muscle compliance upon the wall stretch are poorly understood. We previously reported that the function of normal detrusor is regulated by TREK-1, a member of the mechanogated subfamily of two-pore-domain potassium (K2P) channels. In the present study, we aimed to identify the changes in expression and function of TREK-1 channels under pathological conditions associated with DO, evaluate the potential relationship between TREK-1 channels and cytoskeletal proteins in the human bladder, and test the possibility of modulation of TREK-1 channel expression by small RNAs. Expression of TREK-1 channels in DO specimens was 2.7-fold decreased compared with control bladders and was associated with a significant reduction of the recorded TREK-1 currents. Isolated DO muscle strips failed to relax when exposed to a TREK-1 channel opener. Immunocytochemical labeling revealed close association of TREK-1 channels with cell cytoskeletal proteins and caveolins, with caveolae microdomains being severely disrupted in DO specimens. Small activating RNA (saRNA) tested in vitro provided evidence that expression of TREK-1 protein could be partially upregulated. Our data confirmed a significant downregulation of TREK-1 expression in human DO specimens and provided evidence of close association between the channel, cell cytoskeleton, and caveolins. Upregulation of TREK-1 expression by saRNA could be a future step for the development of in vivo pharmacological and genetic approaches to treat DO in humans.