Stratification of neurogenic bladder risk in spina bifida using the urinary peptidome.

Neurogenic bladder poses a major morbidity in children with spina bifida (SB), and videourodynamic studies (VUDS) are used to stratify this risk. This small-scale pilot study utilized current mass-spectrometry-based proteomic approaches to identify peptides or proteins in urine that may differentiate children at high risk of developing renal complications from a neurogenic bladder. Twenty-two urine samples of which nine had high bladder pressure storage that put the upper urinary tract at risk, while 13 with a lower risk for renal compromise were analyzed. More than 1,900 peptides across all 22 samples were quantified, and 115 peptides differed significantly (P < 0.05) between the two groups. Using machine learning approaches five peptides that showed the greatest differences between these two clinical categories were used to build a classifier. We tested this classifier by blind analysis of an additional six urine samples and showed that it correctly assigned the unknown samples in their proper risk category. These promising results indicate that a urinary screening test based on peptides could be performed on a regular basis to stratify the neurogenic bladder into low or high-risk categories. Expanding this work to larger cohorts as well as across a broad spectrum of urodynamics outcomes may provide a useful diagnostic test for neurogenic bladder.NEW & NOTEWORTHY This approach could help risk stratify the neurogenic bladder in patients with spina bifida and could allow us to safely defer on up to 1/3 of urodynamic studies. These pilot data justify a larger trial before this approach becomes a clinical tool.

Corticotropin-Releasing Hormone from the Pontine Micturition Center Plays an Inhibitory Role in Micturition.

Lower urinary tract or voiding disorders are prevalent across all ages and affect >40% of adults over 40 years old, leading to decreased quality of life and high health care costs. The pontine micturition center (PMC; i.e., Barrington's nucleus) contains a large population of neurons that localize the stress-related neuropeptide, corticotropin-releasing hormone (CRH) and project to neurons in the spinal cord to regulate micturition. How the PMC and CRH-expressing neurons in the PMC control volitional micturition is of critical importance for human voiding disorders. To investigate the specific role of CRH in the PMC, neurons in the PMC-expressing CRH were optogenetically activated during in vivo cystometry in unanesthetized mice of either sex. Optogenetic activation of CRH-PMC neurons led to increased intermicturition interval and voided volume, similar to the altered voiding phenotype produced by social stress. Female mice showed a significantly more pronounced phenotype change compared with male mice. These effects were eliminated by CRH-receptor 1 antagonist pretreatment. Optogenetic inhibition of CRH-PMC neurons led to an altered voiding phenotype characterized by more frequent voids and smaller voided volumes. Last, in a cyclophosphamide cystitis model of bladder overactivity, optogenetic activation of CRH-PMC neurons returned the voiding pattern to normal. Collectively, our findings demonstrate that CRH from PMC spinal-projecting neurons has an inhibitory function on micturition and is a potential therapeutic target for human disease states, such as voiding postponement, urinary retention, and underactive or overactive bladder.SIGNIFICANCE STATEMENT The pontine micturition center (PMC), which is a major regulator of volitional micturition, is neurochemically heterogeneous, and excitatory neurotransmission derived from PMC neurons is thought to mediate the micturition reflex. In the present study, using optogenetic manipulation of CRH-containing neurons in double-transgenic mice, we demonstrate that CRH, which is prominent in PMC-spinal projections, has an inhibitory function on volitional micturition. Moreover, engaging this inhibitory function of CRH can ameliorate bladder hyperexcitability induced by cyclophosphamide in a model of cystitis. The data underscore CRH as a novel target for the treatment of voiding dysfunctions, which are highly prevalent disease processes in children and adults.

Murine social stress results in long lasting voiding dysfunction.

Repeated exposure to social stress shifts the voiding phenotype in male mice leading to bladder wall remodeling and is associated with increased expression of the stress neuropeptide, corticotropin-releasing factor (CRF) in Barrington's nucleus neurons. In these studies, we set out to determine if the voiding phenotype could recover upon removal from the stressor. Male mice were exposed for 1h daily to an aggressor and the voiding phenotype was assessed at one month followed by randomization to three groups. One group underwent immediate sacrifice. Two groups were allowed a one month recovery from the social stress exposure with or without the addition of fluoxetine (1.2mg/ml) in their drinking water and repeat voiding patterns were measured prior to sacrifice. Social stress significantly increased bladder mass, bladder mass corrected for body weight, voided volumes, and decreased urinary frequency. The abnormal voiding phenotype persisted after a 1month recovery with no effect from the addition of fluoxetine. CRF mRNA in Barrington's nucleus was increased by social stress and remained elevated following recovery with no effect from the addition of fluoxetine. The mRNA and protein expression for the alpha 1 chains of type 1 and type III collagen was unchanged across all groups suggesting that changes in the extracellular matrix of the bladder are not responsible for the voiding phenotype. This persisting voiding dysfunction correlates with the persistent elevation of CRF mRNA expression in Barrington's nucleus.

Putting the past behind us: Social stress-induced urinary retention can be overcome.

INTRODUCTION: To study the pathophysiology of dysfunctional voiding, we have previously developed a model of stress-induced voiding dysfunction. We have shown that cyclosporine A (CsA), an inhibitor of the Ca(2+)-calmodulin complex, can prevent social stress-induced urinary retention. However, treatment with cyclosporine has not had an effect on the increase in the stress peptide corticotrophin-releasing factor (CRF) in Barrington's nucleus, which is involved in the micturition pathway. OBJECTIVE: We now investigate whether cyclosporine administered after stress can reverse the abnormal voiding phenotype, and whether it has effects on the bladder wall itself, or on the stress response within Barrington's nucleus. MATERIALS AND METHODS: Six-week old Swiss-Webster mice were exposed to aggressor males for 1 h a day, followed by 23 h of barrier separation. In a long-term trial, 1 month of stress was followed by single-cage housing for 6 months. In a separate CsA reversal trial, mice either received CsA in drinking water or had plain drinking water during 1 month of single-cage housing during recovery. Bladder contractile function was examined on a Guth myograph. Nuclear translocation of myocyte enhancing factor (MEF)-2 and NFAT (nuclear factor of activated T cells) in the bladder was assessed using electrophoretic mobility shift assays (EMSAs). The expression of CRF was determined in Barrington's nucleus using in situ hybridization. RESULTS: Voiding dysfunction persisted for up to 6 months after stress exposure while mice recovered in single-cage housing. In the CsA reversal trial, voiding patterns improved when they received CsA in water during single-cage housing following stress, whereas those that underwent single-cage housing alone had persistent abnormal voiding (Fig. A). There was no difference between CRF levels in Barrington's nucleus between reversal groups (p = 0.42) (Fig. B), possibly indicating a direct effect on the bladder rather than a persistent stress effect. There were no differences in the contractility of bladder wall muscle. CsA decreased the nuclear translocation of MEF-2 and NFAT induced by stress (Fig. C,D). CONCLUSION: CsA reverses stress-induced urinary retention, but does not change the stress-induced CRF increase in Barrington's nucleus. Furthermore, bladder smooth muscle contractility is unchanged by CsA; however, there are changes in the levels of the downstream transcription factors MEF-2 and NFAT. We suspect that additional CsA responsive neural changes play a pivotal role in the abnormal voiding phenotype following social stress.

Evaluation of different alpha-Galactosyl glycoconjugates for use in xenotransplantation.

Porcine organs are rapidly rejected after transplantation into primate recipients due to the presence of preexisting immunoglobulins that bind to terminal galactose alpha1,3 galactose residues (alpha-galactosyl) present on porcine glycoproteins and glycolipids. Currently available immunosuppressive reagents have been largely ineffective at controlling the synthesis of these anti-Gal antibodies. Nonantigenic hapten polymers have been shown to be effective materials for blocking humoral immune responses in various model systems. We have developed a series of alpha-galactosyl glycoconjugate polymers and tested their ability to block anti-Gal antibody binding in vitro and in vivo. A galactose alpha1,3 galactose beta 1,4 GlcNAc trisaccharide free acid (TRFA) with a hexanoic acid spacer, containing five methylene groups and a carboxylic acid, was produced and coupled to a variety of polymeric backbones including dextran, branched poly(ethylene glycol) (PEG), and poly-L-lysine. The ability of monomeric TRFA and the alpha-galactosyl conjugates to block anti-Gal IgG and IgM binding was determined using a competition ELISA assay on defined HSA-Gal glycoconjugates and porcine microvascular endothelial cell substrates. We show that branched PEG carriers, with a TRFA sugar attached to each branch, exhibit enhanced antibody blocking ability compared to TRFA, but at higher target antigen densities these simple PEG conjugates are no more effective then an equivalent amount of TRFA in blocking anti-Gal IgM antibody interactions. In contrast, polymers of the branched PEG conjugates and linear conjugates made using dextran and poly-L-lysine were 2000 to 70000-fold more effective inhibitors of anti-Gal antibodies. In a study using nonhuman primates, a single dose infusion of polymeric PEG or dextran glycoconjugates dramatically reduced the level of circulating anti-Gal antibodies in cynomologus monkeys for at least 72 h. Glycoconjugates similar to these might be useful both to block anti-Gal interactions in vivo and to specifically control the induced anti-Gal immune response.