Abstract
Volume hyposensitivity resulting from impaired sympathetic detrusor relaxation during bladder filling contributes to detrusor underactivity (DU) associated with aging. Detrusor tension regulation provides an adaptive sensory input of bladder volume to the brainstem and is challenged by physiological stressors superimposed upon biological aging. We recently showed that HCN channels have a stabilizing role in detrusor sympathetic relaxation. While mature mice maintain homeostasis in the face of stressors, old mice are not always capable. In old mice, there is a dichotomous phenotype, in which resilient mice adapt and maintain homeostasis, while non-resilient mice fail to maintain physiologic homeostasis. In this DU model, we used cystometry as a stressor to categorize mice as old-responders (old-R, develop a filling/voiding cycle) or old-non-responders (old-NR, fail to develop a filling/voiding cycle; fluctuating high pressures and continuous leaking), while also assessing functional and molecular differences. Lamotrigine (HCN activator)-induced bladder relaxation is diminished in old-NR mice following HCN-blockade. Relaxation responses to NS 1619 were reduced in old-NR mice, with the effect lost following HCN-blockade. However, RNA-sequencing revealed no differences in HCN gene expression and electrophysiology studies showed similar percentage of detrusor myocytes expressing HCN (Ih) current between old-R and old-NR mice. Our murine model of DU further defines a role for HCN, with failure of adaptive recalibration of HCN participation and intensity of HCN-mediated stabilization, while genomic studies show upregulated myofibroblast and fibrosis pathways and downregulated neurotransmitter-degradation pathways in old-NR mice. Thus, the DU phenotype is multifactorial and represents the accumulation of age-associated loss in homeostatic mechanisms.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- β-AR:
-
β-Adrenergic receptor
- CNS:
-
Central nervous system
- CRH:
-
Corticotropin-releasing hormone
- CXCL14:
-
C-X-C Motif Chemokine Ligand 14
- DSM:
-
Detrusor smooth muscle cells
- DU:
-
Detrusor underactivity
- EFS:
-
Electric field stimulation
- FGF:
-
Fibroblast growth factor
- Gabrp :
-
Gamma-aminobutyric acid type A receptor subunit pi
- H-89:
-
N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide
- HBSS:
-
Hank’s balanced salt solution
- HCN:
-
Hyperpolarization-activated cyclic nucleotide-gated
- IP:
-
Intraperitoneal
- IPA:
-
Ingenuity Pathway Analysis
- Mb :
-
Myoglobin
- MMP:
-
Matrix metalloproteinase
- NS 1619:
-
1,3-Dihydro1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2Hbenzimidazol-2-one
- NO:
-
Nitric oxide
- Old-NR:
-
Old non-responder
- Old-R:
-
Old responder
- Pitx1 :
-
Paired like homeodomain 1
- PKA:
-
Protein kinase A
- TPM:
-
Transcripts per million
- VEGF:
-
Vascular endothelial growth factor
- ZD:
-
ZD7288
- PMC:
-
Pontine micturition center
References
Abrams P, Artibani W, Cardozo L, Dmochowski R, van Kerrebroeck P, Sand P (2009) Reviewing the ICS 2002 terminology report: the ongoing debate. Neurourol Urodyn 28(4):287. https://doi.org/10.1002/nau.20737
Abrams P, Cardozo L, Fall M, Griffiths D, Rosier P, Ulmsten U, van Kerrebroeck P, Victor A, Wein A (2002) The standardisation of terminology of lower urinary tract function: report from the standardisation sub-committee of the international continence society. Neurourol Urodyn 21(2):167–178. https://doi.org/10.1002/nau.10052
Al-Naggar IM, Hardy CC, Taweh OG, Grabauskas T, Mulkey DK, Kuchel GA, Smith PP (2019) HCN as a mediator of urinary homeostasis: age-associated changes in expression and function in adrenergic detrusor relaxation. J Gerontol–Ser A Biol Sci Med Sci 74(3):325–329. https://doi.org/10.1093/gerona/gly137
Augsten M, Hägglöf C, Olsson E, Stolz C, Tsagozis P, Levchenko T, Frederick MJ, Borg Å, Micke P, Egevad L, Östman A (2009) CXCL14 is an autocrine growth factor for fibroblasts and acts as a multi-modal stimulator of prostate tumor growth. Proc Natl Acad Sci USA 106(9):3414–3419. https://doi.org/10.1073/PNAS.0813144106
Baker, D., Al-Naggar, I. M., Sivajothi, S., Flynn, W. F., Amiri, A., Luo, D., Hardy, C. C., Kuchel, G. A., Smith, P. P., & Robson, P. (2021). A cellular reference resource for the mouse urinary bladder. BioRxiv, 2021.09.20.461121. https://doi.org/10.1101/2021.09.20.461121
Bayrak Ö, Dmochowski RR (2019) Underactive bladder: a review of the current treatment concepts. Turkish J Urol 45(6):401. https://doi.org/10.5152/TUD.2019.37659
Cao J, Papadopoulou N, Kempuraj D, Boucher WS, Sugimoto K, Cetrulo CL, Theoharides TC (2005) Human mast cells express corticotropin-releasing hormone (CRH) receptors and CRH leads to selective secretion of vascular endothelial growth factor. J Immunol 174(12):7665–7675. https://doi.org/10.4049/JIMMUNOL.174.12.7665
Chapple CR, Osman NI, Birder L, van Koeveringe GA, Oelke M, Nitti VW, Drake MJ, Yamaguchi O, Abrams P, Smith PP (2015) The underactive bladder: a new clinical concept? Eur Urol 68(3):351–353. https://doi.org/10.1016/J.EURURO.2015.02.030
Coolsaet B (1985) Bladder compliance and detrusor activity during the collection phase. Neurourol Urodyn 4(4):263–273. https://doi.org/10.1002/nau.1930040403
Coolsaet BLRA, van Mastrigt R, van Duyl WA, van Rees Vellinga F (1978) Some influences of the contractile element on the visco-elastic properties of bladder wall strips. Eur Urol 4(6):450–456. https://doi.org/10.1159/000474020
Daly DM, Nocchi L, Liaskos M, Mckay NG, Chapple C, Grundy D (2014) Age-related changes in afferent pathways and urothelial function in the male mouse bladder. J Physiol 592(3):537–549. https://doi.org/10.1113/JPHYSIOL.2013.262634
Dowling P, Ranson RN, Santer RM (2006) Age-associated changes in distribution of the P2X2 receptor in the major pelvic ganglion of the male rat. Neurosci Lett 404(3):320–323. https://doi.org/10.1016/j.neulet.2006.06.001
Drake MJ, Mills IW, Noble JG (2004) Melatonin pharmacotherapy for nocturia in men with benign prostatic enlargement. J Urol 171(3):1199–1202. https://doi.org/10.1097/01.JU.0000110442.47593.EA
Elewa RM, Abdallah M, Youssef N, Zouboulis CC (2012) Aging-related changes in cutaneous corticotropin-releasing hormone system reflect a defective neuroendocrine-stress response in aging. Rejuvenation Res 15(4):366–373. https://doi.org/10.1089/REJ.2011.1294
Gomez-Pinilla PJ, Gomez MF, Hedlund P, Swärd K, Hellstrand P, Camello PJ, Pozo MJ, Andersson KE (2007) Effect of melatonin on age associated changes in Guinea pig bladder function. J Urol 177(4):1558–1561. https://doi.org/10.1016/J.JURO.2006.11.071
Grammatopoulos DK (2007) The role of CRH receptors and their agonists in myometrial contractility and quiescence during pregnancy and labour. Front Biosci 12(2):561–571. https://doi.org/10.2741/2082
Hadley EC, Kuchel GA, Newman AB (2017) Report: NIA workshop on measures of physiologic resiliencies in human aging. J Gerontol Ser A, Biol Sci Med Sci 72(7):980–990. https://doi.org/10.1093/GERONA/GLX015
Hardy CC, Keilich SR, Harrison AG, Knight BE, Baker DS, Smith PP (2019) The aging bladder phenotype is not the direct consequence of bladder aging. Neurourol Urodyn 38(8):2121–2129. https://doi.org/10.1002/NAU.24149
Hardy CC, Al-Naggar IM, Kuo CL, Kuchel GA, Smith PP (2021) Aging changes in bladder hyperpolarization-activated cyclic nucleotide-gated channels are associated with increasing heterogeneity of adrenergic/mucosal influence on detrusor control in the mouse. J Gerontol Ser A, Biol Sci Med Sci 76(7):1153–1160. https://doi.org/10.1093/GERONA/GLAB070
Hendgen-Cotta UB, Kelm M, Rassaf T (2014) Myoglobin’s novel role in nitrite-induced hypoxic vasodilation. Trends Cardiovasc Med 24(2):69–74. https://doi.org/10.1016/J.TCM.2013.06.006
Huh Y, Cho J (2013) Urethane anesthesia depresses activities of thalamocortical neurons and alters its response to nociception in terms of dual firing modes. Front Behav Neurosci. https://doi.org/10.3389/FNBEH.2013.00141
Jeong SJ, Kim HJ, Lee YJ, Lee JK, Lee BK, Choo YM, Oh JJ, Lee SC, Jeong CW, Yoon CY, Hong SK, Byun SS, Lee SE (2012) Prevalence and clinical features of detrusor underactivity among elderly with lower urinary tract symptoms: a comparison between men and women. Korean J Urol 53(5):342. https://doi.org/10.4111/KJU.2012.53.5.342
Jeong SJ, Kim JS, Song SH, Kim H, Kim JH, Ye C, Lee SW, Kim JH, Kim H (2022) 10-year follow-up observation of idiopathic detrusor underactivity in the elderly living in community: treatment and urodynamic changes. Neurourol Urodyn 2:100272. https://doi.org/10.1016/J.CONT.2022.100272
Kashyap M, Yoshimura N, Smith PP, Chancellor M, Tyagi P (2015) Characterization of the role of HCN channels in β3-adrenoceptor mediated rat bladder relaxation. Bladder (San Francisco, Calif.) 2(2):15. https://doi.org/10.14440/BLADDER.2015.44
Kashyap M, Singh N, Yoshimura N, Chermansky C, Tyagi P (2020) Constitutively active HCN channels constrain detrusor excitability and modulate evoked contractions of human bladder. Am J Clin Exp Urol 8(5):163–176
Kirkland JL, Stout MB, Sierra F (2016) Resilience in aging mice. J Gerontol Ser A, Biol Sci Med Sci 71(11):1407–1414. https://doi.org/10.1093/GERONA/GLW086
Kolfschoten IGM, van Leeuwen B, Berns K, Mullenders J, Beijersbergen RL, Bernards R, Voorhoeve PM, Agami R (2005) A genetic screen identifies PITX1 as a suppressor of RAS activity and tumorigenicity. Cell 121(6):849–858. https://doi.org/10.1016/J.CELL.2005.04.017/ATTACHMENT/84C1B935-F02E-4FDC-B948-8EA216E1CCBE/MMC1.PDF
Kullmann FA, Daugherty SL, de Groat WC, Birder LA (2014) Bladder smooth muscle strip contractility as a method to evaluate lower urinary tract pharmacology. JoVE 90:e51807. https://doi.org/10.3791/51807
Larauche M, Kiank C, Tache Y (2009) Corticotropin Releasing Factor Signaling In Colon And Ileum: Regulation By Stress And Pathophysiological Implications. J Physiol Pharmacol 60(7):33
le Feber J, van Asselt E, van Mastrigt R (2004) Afferent bladder nerve activity in the rat: a mechanism for starting and stopping voiding contractions. Urol Res 32(6):395–405. https://doi.org/10.1007/s00240-004-0416-8
Lee CL, Jhang JF, Ho HC, Jiang YH, Hsu YH, Kuo HC (2022) Therapeutic outcome of active management in male patients with detrusor underactivity based on clinical diagnosis and videourodynamic classification. Sci Rep 12(1):1–9. https://doi.org/10.1038/s41598-021-04237-0
Lee CL, Kuo HC (2017) Pathophysiology of benign prostate enlargement and lower urinary tract symptoms: current concepts. Tzu-Chi Med J 29(2):79. https://doi.org/10.4103/TCMJ.TCMJ_20_17
Lemtiri-Chlieh F, Baker DS, Al-Naggar IM, Ramasamy R, Kuchel GA, Levine ES, Robson P, Smith PP (2022) The hyperpolarization-activated, cyclic nucleotide-gated channel resides on myocytes in mouse bladders and contributes to adrenergic-induced detrusor relaxation. Am J Physiol Regul, Integr Comp Physiol. https://doi.org/10.1152/AJPREGU.00277.2021
Liu DX, Lobie PE (2007) Transcriptional activation of p53 by Pitx1. Cell Death Differ 14(11):1893–1907. https://doi.org/10.1038/sj.cdd.4402209
Longhurst PA, Uvelius B (2001) Pharmacological techniques for the in vitro study of the urinary bladder. J Pharmacol Toxicol Methods 45(2):91–108. https://doi.org/10.1016/S1056-8719(01)00133-2
López-Otín C, Blasco MA, Partridge L, Serrano M, Kroemer G (2013) The hallmarks of aging. Cell 153(6):1194. https://doi.org/10.1016/J.CELL.2013.05.039
Lu J, Zhang Q, Tan D, Luo W, Zhao H, Ma J, Liang H, Tan Y (2016) GABA A receptor π subunit promotes apoptosis of HTR-8/SVneo trophoblastic cells: Implications in preeclampsia. Int J Mol Med 38(1):105. https://doi.org/10.3892/IJMM.2016.2608
Mader F, Müller S, Krause L, Springer A, Kernig K, Protzel C, Porath K, Rackow S, Wittstock T, Frank M, Hakenberg OW, Köhling R, Kirschstein T (2018) Hyperpolarization-activated cyclic nucleotide-gated non-selective (HCN) ion channels regulate human and murine urinary bladder contractility. Front Physiol 9(9):753. https://doi.org/10.3389/fphys.2018.00753
Michel MC (2011) β-Adrenergic receptor subtypes in the urinary tract. Handb Exp Pharmacol 202(202):307–318. https://doi.org/10.1007/978-3-642-16499-6_15
Morita T, Wheeler MA, Weiss RM (1986) Relaxant effect of forskolin in rabbit detrusor smooth muscle: role of cyclic AMP. J Urol 135(6):1293–1295. https://doi.org/10.1016/S0022-5347(17)46077-0
Nishii H (2021) A review of aging and the lower urinary tract: the future of urology. Int Neurourol J 25(4):273. https://doi.org/10.5213/INJ.2142042.021
Parks RJ, Howlett SE (2012) H-89 decreases the gain of excitation-contraction coupling and attenuates calcium sparks in the absence of beta-adrenergic stimulation. Eur J Pharmacol 691(1–3):163–172. https://doi.org/10.1016/J.EJPHAR.2012.07.012
Pfisterer MHD, Griffiths DJ, Schaefer W, Resnick NM (2006) The effect of age on lower urinary tract function: a study in women. J Am Geriatr Soc 54(3):405–412. https://doi.org/10.1111/J.1532-5415.2005.00613.X
Pfisterer MHD, Griffiths DJ, Rosenberg L, Schaefer W, Resnick NM (2007) Parameters of bladder function in pre-, peri-, and postmenopausal continent women without detrusor overactivity. Neurourol Urodyn 26(3):356–361. https://doi.org/10.1002/NAU.20381
Poolos NP, Migliore M, Johnston D (2002) Pharmacological upregulation of h-channels reduces the excitability of pyramidal neuron dendrites. Nat Neurosci 5(8):767–774. https://doi.org/10.1038/nn891
Postea O, Biel M (2011) Exploring HCN channels as novel drug targets. Nat Rev Drug Discovery 10(12):903–914. https://doi.org/10.1038/NRD3576
Propping S, Lorenz K, Michel MC, Wirth MP, Ravens U (2016) β-adrenoceptor-mediated relaxation of urinary bladder muscle in β2-adrenoceptor knockout mice. Front Pharmacol 7(MAY):118. https://doi.org/10.3389/FPHAR.2016.00118/BIBTEX
Ranson RN, Dodds AL, Smith MJ, Santer RM, Watson AHD (2003) Age-associated changes in the monoaminergic innervation of rat lumbosacral spinal cord. Brain Res 972(1–2):149–158. https://doi.org/10.1016/s0006-8993(03)02521-6
Rassouli O, Liapakis G, Lazaridis I, Sakellaris G, Gkountelias K, Gravanis A, Margioris AN, Karalis KP, Venihaki M (2011) A novel role of peripheral corticotropin-releasing hormone (CRH) on dermal fibroblasts. PLoS ONE. https://doi.org/10.1371/JOURNAL.PONE.0021654
Resnick NM, Yalla SV, Laurino E (1989) The pathophysiology of urinary incontinence among institutionalized elderly persons. N Engl J Med 320(1):1–7. https://doi.org/10.1056/NEJM198901053200101
Santer RM, Dering MA, Ranson RN, Waboso HN, Watson AHD (2002) Differential susceptibility to ageing of rat preganglionic neurones projecting to the major pelvic ganglion and of their afferent inputs. Autonomic Neurosci: Basic Clin 96(1):73–81. https://doi.org/10.1016/s1566-0702(01)00366-6
Schach C, Xu M, Platoshyn O, Keller SH, Yuan JXJ (2007) Thiol oxidation causes pulmonary vasodilation by activating K+ channels and inhibiting store-operated Ca2+ channels. Am J Physiol—Lung Cell Mol Physiol. https://doi.org/10.1152/ajplung.00276.2006
Semerciöz A, Onur R, Ayar A, Orhan I (2004) The inhibitory role of melatonin on isolated guinea-pig urinary bladder: an endogenous hormone effect. BJU Int 94(9):1373–1376. https://doi.org/10.1111/J.1464-410X.2004.05175.X
Shi B, Wu Y, Chen H, Ding J, Qi J (2022) Understanding of mouse and human bladder at single-cell resolution: integrated analysis of trajectory and cell-cell interactive networks based on multiple scRNA-seq datasets. Cell Prolif. https://doi.org/10.1111/CPR.13170
Smith PP, Kuchel GA (2010) Continuous uroflow cystometry in the urethane-anesthetized mouse. Neurourol Urodyn 29(7):1344–1349. https://doi.org/10.1002/nau.20850
Smith PP, Deangelis A, Kuchel GA (2012) Detrusor expulsive strength is preserved, but responsiveness to bladder filling and urinary sensitivity is diminished in the aging mouse. Am J Physiol—Regulat Integr Comp Physiol. https://doi.org/10.1152/ajpregu.00508.2011
Smith PP, DeAngelis A, Simon R (2015) Evidence of increased centrally enhanced bladder compliance with ageing in a mouse model. BJU Int 115(2):322–329. https://doi.org/10.1111/bju.12669
Smith PP, Birder LA, Abrams P, Wein AJ, Chapple CR (2016) Detrusor underactivity and the underactive bladder: symptoms, function, cause—what do we mean? ICI-RS think tank 2014. Neurourol Urodyn 35(2):312–317. https://doi.org/10.1002/NAU.22807
Takahashi K, Tanaka T, Yoshizawa Y, Fujisaki-Sueda-Sakai M, Son BK, Iijima K (2022) Lower urinary tract symptoms and functional ability in older adults: a community-based cross-sectional study. BMJ Open 12(4):e054530. https://doi.org/10.1136/BMJOPEN-2021-054530
Totzeck M, Hendgen-Cotta UB, Luedike P, Berenbrink M, Klare JP, Steinhoff HJ, Semmler D, Shiva S, Williams D, Kipar A, Gladwin MT, Schrader J, Kelm M, Cossins AR, Rassaf T (2012) Nitrite regulates hypoxic vasodilation via myoglobin-dependent nitric oxide generation. Circulation 126(3):325–334. https://doi.org/10.1161/CIRCULATIONAHA.111.087155
Tyson EK, Smith R, Read M (2009) Evidence that corticotropin-releasing hormone modulates myometrial contractility during human pregnancy. Endocrinology 150(12):5617–5625. https://doi.org/10.1210/EN.2009-0348
Ukraintseva S, Yashin AI, Arbeev KG (2016) Resilience versus robustness in aging. J Gerontol. Ser A, Biol Sci Med Sci 71(11):1533–1534. https://doi.org/10.1093/GERONA/GLW083
Ukraintseva S, Arbeev K, Duan M, Akushevich I, Kulminski A, Stallard E, Yashin A (2021) Decline in biological resilience as key manifestation of aging: potential mechanisms and role in health and longevity. Mech Ageing Dev. https://doi.org/10.1016/J.MAD.2020.111418
Wang Q, Zhao S, Gan L, Zhuang Z (2020) Bioinformatics analysis of prognostic value of PITX1 gene in breast cancer. Biosci Rep. https://doi.org/10.1042/BSR20202537
Warburton AL, Santer RM (1994) Sympathetic and sensory innervation of the urinary tract in young adult and aged rats: a semi-quantitative histochemical and immunohistochemical study. Histochem J 26(2):127–133. https://doi.org/10.1007/BF00157961
Whitson HE, Duan-Porter W, Schmader KE, Morey MC, Cohen HJ, Colón-Emeric CS (2016) Physical resilience in older adults: systematic review and development of an emerging construct. J Gerontol. Ser A, Biol Sci Med Sci 71(4):489–495. https://doi.org/10.1093/GERONA/GLV202
Yang Y, Ren L, Li S, Zheng X, Liu J, Li W, Fu W, Wang J, Du G (2022) GABRP is a potential prognostic biomarker and correlated with immune infiltration and tumor microenvironment in pancreatic cancer. Transl Cancer Res 11(4):649–668. https://doi.org/10.2037/TCR-21-2021/COIF
Zheng H, Lim JY, Seong JY, Hwang SW (2020) The role of corticotropin-releasing hormone at peripheral nociceptors: implications for pain modulation. Biomedicines 8(12):1–12. https://doi.org/10.3390/BIOMEDICINES8120623
Funding
National Institutes of Health R01AG058814; K02AG068375; K76AG054777. JB and GAK were supported by the UConn Claude D. Pepper Older Americans Independence Center (P30AG067988).
Author information
Authors and Affiliations
Contributions
Conceptualization—PPS, RR, DAR, IMA-N, CCH, GAK, JMB; Experiments: Cystometry and Pharmacomyography (RR, DAR, EW, IMA-N, CCH), RNAseq (DB), patch-clamp electrophysiology (FL-C), qPCR (RR, DSB); Analysis—RR (cystometry, pharmacomyography, qPCR), DSB (RNA seq), DAR (cystometry, pharmacomyography-preliminary); Interpretation—RR, PPS, JMB, FL-C and DSB; Writing—RR (95%), DB (RNAseq methods), FL-C (electrophysiology methods and results); Funding/Resources—PPS, GAK, ESL; Review and editing—all authors.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
10522_2022_10005_MOESM1_ESM.png
Supplementary file1 (PNG 317 kb) Expression of adrenergic, cholinergic, and muscarinic genes in old-R and old-NR bladders from bulk RNA sequencing. (A) Adrenergic receptors: Adra1a (p=0.286), Adra1d (p=0.905), Adra2b (p=0.730), Adra2c (p=0.556), Adrb2 (p=0.413) and Adrb3 (p=0.190); (B) Cholinergic receptors: Chrm1 (p=0.111), Chrm2 (p=0.556), Chrm3 (p=0.905); (C) Purinergic receptors: P2rx1 (p=0.111), P2rx2 (p=0.286), P2rx3 (p=0.556), P2rx4 (p=0.111), P2rx5 (p>0.999), P2rx6 (p=0.556) and P2rx7 (p=0.556). No significant difference was seen between the old responders (n=4) and old non-responders (n=5). Statistical analysis was done using multiple Mann-Whitney tests with significance set at P<0.05. TPM: transcripts per million
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Ramasamy, R., Baker, D.S., Lemtiri-Chlieh, F. et al. Loss of resilience contributes to detrusor underactivity in advanced age. Biogerontology 24, 163–181 (2023). https://doi.org/10.1007/s10522-022-10005-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10522-022-10005-y