Publications by Author: Huan Chen

The bladder undergoes large shape changes as it fills and empties and experiences complex mechanical forces. These forces become abnormal in diseases of the lower urinary tract such as overactive bladder, neurogenic bladder, and urinary retention. As the primary mechanosensors linking the actin cytoskeleton to the extracellular matrix (ECM), integrins are likely to play vital roles in maintaining bladder smooth muscle (BSM) homeostasis. In a tamoxifen-inducible smooth muscle conditional knockout of β1-integrin, there was concomitant loss of α1- and α3-integrins from BSM and upregulation of αV- and β3-integrins. Masson's staining showed a reduction in smooth muscle with an increase in collagenous ECM. Functionally, mice exhibited a changing pattern of urination by voiding spot assay up to 8 wk after tamoxifen. By 8 wk, there was increased frequency with reductions in voided volume, consistent with overactivity. Cystometrograms confirmed that there was a significant reduction in intercontractile interval with reduced maximal bladder pressure. Muscle strip myography revealed a loss of contraction force in response to electrical field stimulation, that was entirely due to the loss of muscarinic contractility. Quantitative western blotting showed a loss of M3 receptor and no change in P2X1. qPCR on ECM and interstitial genes revealed loss of Ntpd2, a marker of an interstitial cell subpopulation; and an upregulation of S100A4, which is often associated with fibroblasts. Collectively, the data show that the loss of appropriate mechanosensation through integrins results in cellular and extracellular remodeling, and concomitant bladder dysfunction that resembles lower urinary tract symptoms seen in older people.

Acute urinary retention (AUR) is a common urological emergency and affects a significant patient population. The inability to eliminate urine may lead to permanent damage to the bladder's structure and functioning. However, we know little about the underlying molecular sequelae to the urine retention. To closely mirror the potential high pressures that patients with AUR could experience, we catheterized anesthetized female mice via the urethra and filled the bladder by pumping saline (25 µL/min) into the bladder lumen to 50 cm or 80 cm water pressure. A water column with designated height (50 or 80 cm) was then adjusted to maintain constant pressure in the bladder lumen for 30 minutes. Functional and morphological evaluations were performed from 0 to 24 hours after AUR treatment. Mice exhibited incontinence and overactivity with diminished voiding pressure. Significant injury was confirmed which revealed bladders with disrupted urothelial barrier, edematous lamina propria, and distorted muscle bundles. Bladder smooth muscle (BSM) from pressure-treated mice have significantly diminished contraction force, suggesting that bladder voiding dysfunction can be attributed to impaired BSM contractility. Indeed, dysregulation of acetylcholine and purinergic signaling pathways were demonstrated, indicating that reduced efficacy of these pathways contributes to impaired BSM contractility. Finally, altered expression of β1-integrin and extracellular matrix mediated mechanotransduction pathways were detected, suggesting a profound remodeling process. These data demonstrated an easy to perform, quantifiable, and reproducible AUR mouse model, which mimics well the characteristics of human AUR patients, and our data generate new insights into the molecular mechanisms that occur following AUR.

The voiding spot assay (VSA) is increasingly being adopted as a standard method for assessing mouse urinary function. However, VSA outcomes are highly sensitive to housing environment and procedural parameters. Many variables exist among laboratories, including analytical software, type of daily housing cage, transportation, and the time of the day. Some of these variables, such as the time of VSA and analytical software, have been shown to result in inconsistency and incomparability of data. In this study, we evaluated whether the results of VSA can be compared across laboratories by minimizing these variables. We found that analytical tools between Fiji and MATLAB are in good agreement in the quantification of VSA parameters, especially primary voiding spot (PVS) parameters. Unexpectedly, we found that mice housed in different daily home cages did not alter voiding patterns in a standard VSA cage. Nonetheless, we still recommend acclimation when performing VSA in unfamiliar cages. Notably, mice are highly sensitive to transportation and the time in the morning versus afternoon, which can induce significant changes in voiding patterns. Therefore, a standardized period among laboratories and allowing 2-3 days of rest for mice acclimation after transportation are necessary for VSA. Finally, we performed VSA using identical procedural parameters in two laboratories from two geographical locations to compare the results of VSA and concluded that it is possible to generate limited comparable VSA data, such as PVS volume.

Abnormalities in purine availability or purinergic receptor density are commonly seen in patients with lower urinary tract symptoms (LUTS), but the underlying mechanisms relating altered receptor function to LUTS are unknown. Here we provide extensive evidence for the reciprocal interplay of multiple receptors responding to ATP, ADP (adenosine diphosphate), and adenosine, agonists that regulate bladder function significantly. ADP stimulated P2Y12 receptors, causing bladder smooth muscle (BSM) contraction, whereas adenosine signaling through potentially newly defined A2b receptors, actively inhibited BSM purinergic contractility. The modulation of adenylyl cyclase-cAMP signaling via A2b and P2Y12 interaction actively regulated bladder contractility by modulating intracellular calcium levels. KO mice lacking the receptors display diametrically opposed bladder phenotypes, with P2Y12-KO mice exhibiting an underactive bladder (UAB) phenotype with increased bladder capacity and reduced voiding frequency, whereas A2b-KO mice have an overactive bladder (OAB), with decreased capacity and increased voiding frequency. The opposing phenotypes in P2Y12-KO and A2b-KO mice not only resulted from dysregulated BSM contractility, but also from abnormal BSM cell growth. Finally, we demonstrate that i.p. administration of drugs targeting P2Y12 or A2b receptor rescues these abnormal phenotypes in both KO mice. These findings strongly indicate that P2Y12 and A2b receptors are attractive therapeutic targets for human patients with LUTS.

Diabetic bladder dysfunction (DBD) is the most common complication in diabetes. Myogenic abnormalities are common in DBD; however, the underlying mechanisms leading to these remain unclear. To understand the importance of smooth muscle insulin receptor (IR)-mediated signaling in the pathogenesis of DBD, we conditionally deleted it to achieve either heterozygous (SMIR+/-) or homozygous (SMIR-/-) deletion in smooth muscle cells. Despite impaired glucose and insulin tolerance seen with SMIR-/- mice, both SMIR+/- and SMIR-/- mice exhibited normal blood glucose and plasma insulin levels. Interestingly, these mice had abnormal voiding phenotypes, that included urinary frequency and small voids, and bladder smooth muscle (BSM) had significantly diminished contraction force. Morphology revealed a dilated bladder with thinner BSM layer, and BSM bundles were disorganized with penetrating interstitial tissue. Deletion of IR elevated FoxO and decreased mTOR protein expression, which further decreased the expression of Chrm3, P2x1, Sm22, and Cav1.2, crucial functional proteins for BSM contraction. Furthermore, we determined the expression of adiponectin in BSM, and deletion of IR in BSM inhibited adiponectin-mediated signaling. In summary, disruption of IR-mediated signaling in BSM caused abnormalities in proliferation and differentiation, leading to diminished BSM contractility and a voiding dysfunction phenotype that recapitulates human DBD.

The general anesthetic ketamine has been repurposed by physicians as an anti-depressant and by the public as a recreational drug. However, ketamine use can cause extensive pathological changes, including ketamine cystitis. The mechanisms of ketamine's anti-depressant and adverse effects remain poorly understood. Here we present evidence that ketamine is an effective L-type Ca2+ channel (Cav1.2) antagonist that directly inhibits calcium influx and smooth muscle contractility, leading to voiding dysfunction. Ketamine prevents Cav1.2-mediated induction of immediate early genes and transcription factors, and inactivation of Cav1.2 in smooth muscle mimics the ketamine cystitis phenotype. Our results demonstrate that ketamine inhibition of Cav1.2 signaling is an important pathway mediating ketamine cystitis. In contrast, Cav1.2 agonist Bay k8644 abrogates ketamine-induced smooth muscle dysfunction. Indeed, Cav1.2 activation by Bay k8644 decreases voiding frequency while increasing void volume, indicating Cav1.2 agonists might be effective drugs for treatment of bladder dysfunction.