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MP82-04 TRANSCRIPTIONAL REGULATION OF CORTICOTROPIN RELEASING FACTOR
THE JOURNAL OF UROLOGYâ
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INTRODUCTION AND OBJECTIVES: The role of brain-derived neurotrophic factor (BDNF) in lower urinary tract dysfunction induced by spinal cord injury (SCI) is still unclear. Previous data showed that the neutralization of BDNF in SCI mice increased voided volume and improved the voiding efficiency (2016 AUA). BDNF might involve in the voiding phase via Ad-fiber afferents, on which the receptor of BDNF (tropomyosin receptor kinase B) is mainly distributed. Acid-sensing ion channels (ASIC), which can function as mechanosensors, are identified as a target of BDNF signaling. We therefore investigated the changes of urethral function and ASIC expression in dorsal root ganglia (DRG) after the neutralization of BDNF in SCI mice. METHODS: Female C57 BL/6N mice underwent Th8-9 spinal cord transection. Three weeks later, an osmotic pump was placed subcutaneously to administer 10mg/kg/hr of anti-BDNF antibody for 1 week. Four weeks after spinal cord transection, SCI mice were evaluated using single-filling cystometry and external urethral sphincter (EUS)-electromyogram (EMG) under an awake condition. CMG-EMG recordings were used to detect intermittent voiding coincided with reductions in intravesical pressure in CMG traces, which occurred during periods of reduced EUS-EMG activity. We measured voiding contraction time (VT), reduced EMG activity duration (RED) and the ratio of RED to VT. Bladder BDNF was measured and the transcripts of TRPV1 and ASIC1, 2, and 3 of L6/S1 DRG were also evaluated. RESULTS: Compared to vehicle-treated SCI mice, voided volume was significantly increased and voiding efficiency was significantly better in anti-BDNF antibody-treated SCI mice. In CMG-EMG recordings, the RED was significantly prolonged, and the ratio of RED to VT was significantly greater in anti-BDNF antibody-treated SCI mice than those in vehicle-treated SCI mice. Bladder BDNF levels of SCI mice were significantly increased compared with spinal intact mice, but decreased after anti-BDNF antibody treatment. The transcripts of TRPV1, ASIC2 and 3 were increased in SCI mice compared to spinal intact mice, and anti-BDNF treatment significantly decreased expressions of ASIC2 and 3, but not TRPV1. CONCLUSIONS: BDNF upregulation in the bladder is likely to be involved in dyssynergic activity of the EUS during voiding in association with ASIC overexpression in L6/S1 DRG in SCI mice. Thus, BDNF targeting treatments could be effective for voiding problems such as DSD and inefficient voiding after SCI. Source of Funding: NIH P01 DK093424
MP82-04 TRANSCRIPTIONAL REGULATION OF CORTICOTROPIN RELEASING FACTOR Lizath Aguiniga*, Anthony Schaeffer, David Klumpp, Chicago, IL INTRODUCTION AND OBJECTIVES: Interstitial cystitis (IC) patients suffer from chronic pelvic pain and bladder dysfunction. IC patients have altered cortisol levels suggesting dysregulation of the hypothalamic-pituitary adrenal (HPA) axis and suffer from exacerbated symptoms in response to high stress. Corticotropin-releasing factor (CRF) is the initiator of the HPA axis and mediates stress responses and voiding control, where increased CRF levels in Barrington’s nucleus induce bladder dysfunction. Arachidonic acid (AA) metabolites have been shown to induce CRF expression, however the transcriptional mediators of this modulation are unknown. Here we identify transcription factors that mediate AA-induced CRF gene expression. METHODS: We used MIRAGE software to identify candidate transcription factor binding sites in a 1kb region of the human CRF gene promoter. We identified a peroxisome proliferator activated hormone response element (PPRE) and two Xenobiotic Responsive Element (XRE) sites as candidate mediators of AA-dependent CRF induction. Site-directed mutations of the PPRE and XRE sites were generated in a CRF-luciferase reporter plasmid to evaluate responses to AA and the impact of AhR and PPAR gamma expressed in HEK 293T cells. The hypothalamic neuronal cell line N42 was used to evaluate the role of AhR and PPAR gamma in native promoter regulation by RT PCR. We
Vol. 197, No. 4S, Supplement, Monday, May 15, 2017
also generated mice that have PPAR gamma and AhR knocked-out in CRF-expressing cells and characterized voiding activity. RESULTS: AA induction in the PPRE mutant resulted in increased CRF promoter activity compared to WT, whereas XRE1 had decreased activity. The mutation of both XRE1 and XRE2 resulted in decreased responsiveness to AA. Over-expression of PPAR gamma alone showed no change, while over-expression of AhR alone showed a significant increase in AA-induced CRF expression; this was inhibited by over-expression of both AhR and PPAR gamma in HEK 293T cells. Over-expression of AhR in N42 cells resulted in increased CRF mRNA in response to AA induction. AhR conditional knockout mice showed increased voiding frequency. CONCLUSIONS: These results suggest AhR binding to the XRE sites modulates AA-dependent CRF gene expression. PPAR gamma inhibits AA-dependent CRF gene expression. Continued studies will use mouse models to examine the in vivo role of AhR and PPAR gamma inhibitors to modulate voiding activity. Source of Funding: Research reported in this publication was supported by the National Institute Of Allergy And Infectious Diseases of the National Institutes of Health under Award Number F31AI106357.
MP82-05 PSYCHOSOCIAL FACTORS, SLEEP, AND PHYSICAL FUNCTION IN WOMEN WITH LOWER URINARY TRACT SYMPTOMS Nazema Y. Siddiqui, MD, MHS*, Durham, NC; Anne P. Cameron, MD, Ann Arbor, MI; David Cella, PhD, Chicago, IL; Catherine S. Bradley, MD, MSCE, Iowa City, IA; H. Henry Lai, MD, Saint Louis, MO; Margaret E. Helmuth, MA, Jonathan Wiseman, MS, Ann Arbor, MI; James W. Griffith, PhD, Chicago, IL; Cindy L. Amundsen, MD, Durham, NC; Kimberly Kenton, MD, MS, Chicago, IL; J. Quentin Clemens, MD, FACS, MSCI, Ann Arbor, MI; Karl J. Kreder, MD, MBA, Iowa City, IA; Robert M. Merion, MD, FACS, Ann Arbor, MI; Ziya Kirkali, MD, John W. Kusek PhD, for the LURN, Bethesda, MD INTRODUCTION AND OBJECTIVES: We examined psychosocial health measures in women with lower urinary tract symptoms (LUTS), and compared them in groups of women based on presence and type of urinary incontinence (UI). METHODS: The Symptoms of Lower Urinary Tract Dysfunction Research Network (LURN) is conducting a prospective observational study in six clinical sites. This is a cross-sectional analysis of baseline information from 510 women seeking treatment for LUTS; those with urologic pain were excluded. The LUTS Tool was used to identify women with and without UI, and to categorize symptoms into: 1) stress (SUI); 2) urgency (UUI); and 3) mixed (MUI) subtypes. Patient-Reported Outcomes Measurement Information System (PROMIS) short forms assessed sleep disturbance, depression, anxiety and physical function. The International Physical Activity Questionnaire (IPAQ-SF), Perceived Stress Scale (PSS), and Childhood Traumatic Events Scale were also administered. RESULTS: Mean age was 5614 years; 82% were Caucasian, 46% were obese, and 15% reported diabetes. Women with UI (n¼425) reported more sleep disturbance (54 vs. 52, p<0.01), more depression (50 vs. 47, p¼0.03), more anxiety (51 vs. 48, p¼0.02), more perceived stress (13 vs. 11, p¼0.01), and poorer physical function (47 vs. 51, p<0.01) compared to those without UI (n¼85). There were no significant differences in childhood traumatic events (78% vs 70%, p¼0.12) or physical activity (1272 vs 1372 MET-minutes, p¼0.58) in women with UI compared to those without UI. Among UI subtypes, women with MUI reported the highest depression, anxiety and perceived stress, and the poorest physical function (table). CONCLUSIONS: Among women with LUTS, those with UI had more emotional distress and sleep disturbance, and worse physical function. Of women with UI, those with MUI reported more severe emotional distress. Frequency of at least one reported childhood traumatic event did not significantly differ across groups.
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INTRODUCTION AND OBJECTIVES: The role of brain-derived neurotrophic factor (BDNF) in lower urinary tract dysfunction induced by spinal cord injury (SCI) is still unclear. Previous data showed that the neutralization of BDNF in SCI mice increased voided volume and improved the voiding efficiency (2016 AUA). BDNF might involve in the voiding phase via Ad-fiber afferents, on which the receptor of BDNF (tropomyosin receptor kinase B) is mainly distributed. Acid-sensing ion channels (ASIC), which can function as mechanosensors, are identified as a target of BDNF signaling. We therefore investigated the changes of urethral function and ASIC expression in dorsal root ganglia (DRG) after the neutralization of BDNF in SCI mice. METHODS: Female C57 BL/6N mice underwent Th8-9 spinal cord transection. Three weeks later, an osmotic pump was placed subcutaneously to administer 10mg/kg/hr of anti-BDNF antibody for 1 week. Four weeks after spinal cord transection, SCI mice were evaluated using single-filling cystometry and external urethral sphincter (EUS)-electromyogram (EMG) under an awake condition. CMG-EMG recordings were used to detect intermittent voiding coincided with reductions in intravesical pressure in CMG traces, which occurred during periods of reduced EUS-EMG activity. We measured voiding contraction time (VT), reduced EMG activity duration (RED) and the ratio of RED to VT. Bladder BDNF was measured and the transcripts of TRPV1 and ASIC1, 2, and 3 of L6/S1 DRG were also evaluated. RESULTS: Compared to vehicle-treated SCI mice, voided volume was significantly increased and voiding efficiency was significantly better in anti-BDNF antibody-treated SCI mice. In CMG-EMG recordings, the RED was significantly prolonged, and the ratio of RED to VT was significantly greater in anti-BDNF antibody-treated SCI mice than those in vehicle-treated SCI mice. Bladder BDNF levels of SCI mice were significantly increased compared with spinal intact mice, but decreased after anti-BDNF antibody treatment. The transcripts of TRPV1, ASIC2 and 3 were increased in SCI mice compared to spinal intact mice, and anti-BDNF treatment significantly decreased expressions of ASIC2 and 3, but not TRPV1. CONCLUSIONS: BDNF upregulation in the bladder is likely to be involved in dyssynergic activity of the EUS during voiding in association with ASIC overexpression in L6/S1 DRG in SCI mice. Thus, BDNF targeting treatments could be effective for voiding problems such as DSD and inefficient voiding after SCI. Source of Funding: NIH P01 DK093424
MP82-04 TRANSCRIPTIONAL REGULATION OF CORTICOTROPIN RELEASING FACTOR Lizath Aguiniga*, Anthony Schaeffer, David Klumpp, Chicago, IL INTRODUCTION AND OBJECTIVES: Interstitial cystitis (IC) patients suffer from chronic pelvic pain and bladder dysfunction. IC patients have altered cortisol levels suggesting dysregulation of the hypothalamic-pituitary adrenal (HPA) axis and suffer from exacerbated symptoms in response to high stress. Corticotropin-releasing factor (CRF) is the initiator of the HPA axis and mediates stress responses and voiding control, where increased CRF levels in Barrington’s nucleus induce bladder dysfunction. Arachidonic acid (AA) metabolites have been shown to induce CRF expression, however the transcriptional mediators of this modulation are unknown. Here we identify transcription factors that mediate AA-induced CRF gene expression. METHODS: We used MIRAGE software to identify candidate transcription factor binding sites in a 1kb region of the human CRF gene promoter. We identified a peroxisome proliferator activated hormone response element (PPRE) and two Xenobiotic Responsive Element (XRE) sites as candidate mediators of AA-dependent CRF induction. Site-directed mutations of the PPRE and XRE sites were generated in a CRF-luciferase reporter plasmid to evaluate responses to AA and the impact of AhR and PPAR gamma expressed in HEK 293T cells. The hypothalamic neuronal cell line N42 was used to evaluate the role of AhR and PPAR gamma in native promoter regulation by RT PCR. We
Vol. 197, No. 4S, Supplement, Monday, May 15, 2017
also generated mice that have PPAR gamma and AhR knocked-out in CRF-expressing cells and characterized voiding activity. RESULTS: AA induction in the PPRE mutant resulted in increased CRF promoter activity compared to WT, whereas XRE1 had decreased activity. The mutation of both XRE1 and XRE2 resulted in decreased responsiveness to AA. Over-expression of PPAR gamma alone showed no change, while over-expression of AhR alone showed a significant increase in AA-induced CRF expression; this was inhibited by over-expression of both AhR and PPAR gamma in HEK 293T cells. Over-expression of AhR in N42 cells resulted in increased CRF mRNA in response to AA induction. AhR conditional knockout mice showed increased voiding frequency. CONCLUSIONS: These results suggest AhR binding to the XRE sites modulates AA-dependent CRF gene expression. PPAR gamma inhibits AA-dependent CRF gene expression. Continued studies will use mouse models to examine the in vivo role of AhR and PPAR gamma inhibitors to modulate voiding activity. Source of Funding: Research reported in this publication was supported by the National Institute Of Allergy And Infectious Diseases of the National Institutes of Health under Award Number F31AI106357.
MP82-05 PSYCHOSOCIAL FACTORS, SLEEP, AND PHYSICAL FUNCTION IN WOMEN WITH LOWER URINARY TRACT SYMPTOMS Nazema Y. Siddiqui, MD, MHS*, Durham, NC; Anne P. Cameron, MD, Ann Arbor, MI; David Cella, PhD, Chicago, IL; Catherine S. Bradley, MD, MSCE, Iowa City, IA; H. Henry Lai, MD, Saint Louis, MO; Margaret E. Helmuth, MA, Jonathan Wiseman, MS, Ann Arbor, MI; James W. Griffith, PhD, Chicago, IL; Cindy L. Amundsen, MD, Durham, NC; Kimberly Kenton, MD, MS, Chicago, IL; J. Quentin Clemens, MD, FACS, MSCI, Ann Arbor, MI; Karl J. Kreder, MD, MBA, Iowa City, IA; Robert M. Merion, MD, FACS, Ann Arbor, MI; Ziya Kirkali, MD, John W. Kusek PhD, for the LURN, Bethesda, MD INTRODUCTION AND OBJECTIVES: We examined psychosocial health measures in women with lower urinary tract symptoms (LUTS), and compared them in groups of women based on presence and type of urinary incontinence (UI). METHODS: The Symptoms of Lower Urinary Tract Dysfunction Research Network (LURN) is conducting a prospective observational study in six clinical sites. This is a cross-sectional analysis of baseline information from 510 women seeking treatment for LUTS; those with urologic pain were excluded. The LUTS Tool was used to identify women with and without UI, and to categorize symptoms into: 1) stress (SUI); 2) urgency (UUI); and 3) mixed (MUI) subtypes. Patient-Reported Outcomes Measurement Information System (PROMIS) short forms assessed sleep disturbance, depression, anxiety and physical function. The International Physical Activity Questionnaire (IPAQ-SF), Perceived Stress Scale (PSS), and Childhood Traumatic Events Scale were also administered. RESULTS: Mean age was 5614 years; 82% were Caucasian, 46% were obese, and 15% reported diabetes. Women with UI (n¼425) reported more sleep disturbance (54 vs. 52, p<0.01), more depression (50 vs. 47, p¼0.03), more anxiety (51 vs. 48, p¼0.02), more perceived stress (13 vs. 11, p¼0.01), and poorer physical function (47 vs. 51, p<0.01) compared to those without UI (n¼85). There were no significant differences in childhood traumatic events (78% vs 70%, p¼0.12) or physical activity (1272 vs 1372 MET-minutes, p¼0.58) in women with UI compared to those without UI. Among UI subtypes, women with MUI reported the highest depression, anxiety and perceived stress, and the poorest physical function (table). CONCLUSIONS: Among women with LUTS, those with UI had more emotional distress and sleep disturbance, and worse physical function. Of women with UI, those with MUI reported more severe emotional distress. Frequency of at least one reported childhood traumatic event did not significantly differ across groups.