Endolymphatic hydrops induced by different mechanisms responds differentially to spironolactone: a rationale for understanding the diversity of treatment responses in hydropic inner ear disease
Eva Degerman, René In ’t Zandt, Annki Pålbrink & Måns Magnusson
Received 10 Feb 2019, Accepted 03 May 2019, Published online: 30 May 2019
Download citation https://doi.org/10.1080/00016489.2019.1616819
Abstract
Background: The exact pathophysiological mechanism(s) underlying endolymphatic hydrops (EH) remain elusive. We have previously shown that chronic administration of vasopressin and inhibitors of the cAMP/cGMP degrading enzymes (PDE3, PDE4, PDE5) results in the development of EH to mice.
Aims/objectives: Evaluate the ability of spironolactone, an aldosterone antagonist, to prevent EH, when induced by different pathways.
Material and methods: Mice were treated for 4 weeks with vasopressin, the PDE3 inhibitor cilostamide and the PDE4 inhibitor rolipram in the presence or absence of spironolactone. EH was assessed using high resolution 9.4T MRI. The expression of proteins in human saccule sensory epithelium was studied with immunohistochemistry.
Results: Spironolactone prevents EH induced by vasopressin and rolipram, but not hydrops induced by cilostamide. The aldosterone target ENaC and the mineralocorticoid receptor were expressed in the human saccule sensory epithelium.
Conclusions: The effect of spironolactone on EH appears to be pathway-dependent and may provide explanations why certain drugs may be effective in some patients with hydropic ear disease while not in others.
Significance: Extrapolating this finding to the clinic supports that a personalized medicine approach is probably necessary in the treatment of diseases involving EH, as different pathways may be needed to be targeted for treatment.
Chinese abstract
背景：内淋巴积水（EH）的确切病理生理机制尚不清楚。我们之前已经证明, 长期服用加压素和cAMP/cGMP降解酶抑制剂（PDE3、PDE4、PDE5）会导致小鼠的内淋巴积水。
目的：评价醛固酮拮抗剂螺内酯在不同途径诱导下预防EH的能力。
材料和方法：小鼠在有或无螺内酯的情况下, 用加压素、PDE3抑制剂西洛他明和PDE4抑制剂罗利帕姆治疗4周。采用高分辨率9.4T MRI评估EH。用免疫组织化学方法研究蛋白质在人球囊感觉上皮中的表达。
结果：螺内酯对加压素和罗力普仑诱导的EH有抑制作用, 而对西洛他明诱导的水肿无抑制作用。醛固酮靶向ENAC和盐皮质激素受体在人球囊感觉上皮中被表达。
结论：螺内酯对EH的影响似乎是途径依赖性的, 这可能解释某些药物对某些水样耳病患者有效而对其他患者无效的原因。
意义：将这一发现外推到临床, 我们认为个人化医治法可能是治疗EH有关疾病所必需的, 因为治疗可能需要不同的途径。
Keywords: Magnetic resonance imaging, cochlea, endolymphatic hydrops, vasopressin, cilostamide, rolipram, PDE3, PDE4, spironolactone, Menière’s disease
Introduction
A characteristic finding of several inner ear disorders is the dilatation of the endolymphatic compartment of the inner ear, endolymphatic hydrops (EH), resulting from deteriorated ion homeostasis and fluid volume regulation [1 Oberman BS, Patel VA, Cureoglu S, et al. The aetiopathologies of Ménière's disease: a contemporary review. Acta Otorhinolaryngol Ital. 2017;37:250–263.
[PubMed], [Web of Science ®], , [Google Scholar]
]. EH may result from a number of processes such as autoimmune disorders, viral infections, electrolyte and fluid imbalance and trauma [1 Oberman BS, Patel VA, Cureoglu S, et al. The aetiopathologies of Ménière's disease: a contemporary review. Acta Otorhinolaryngol Ital. 2017;37:250–263.
[PubMed], [Web of Science ®], , [Google Scholar]
]. EH associated changes in the inner ear are believed to be of significance in relation to the symptoms associated with for example Menière’s disease such as fluctuating hearing loss, tinnitus, vertigo and aural fullness [1 Oberman BS, Patel VA, Cureoglu S, et al. The aetiopathologies of Ménière's disease: a contemporary review. Acta Otorhinolaryngol Ital. 2017;37:250–263.
[PubMed], [Web of Science ®], , [Google Scholar]
,2 Lopez-Escamez JA, Carey J, Chung WH, et al. Classification Committee of the Barany Society; Japan Society for Equilibrium Research; European Academy of Otology and Neurotology (EAONO); Equilibrium Committee of the American Academy of Otolaryngology-Head and Neck Surgery (AAO-HNS); Korean Balance Society. Diagnostic criteria for Menière's disease. J Vestib Res. 2015;25:1–7.
[PubMed], [Web of Science ®], , [Google Scholar]
].
Treatment regimes for diseases believed to involve EH such as Menière’s disease, involve a range of strategies. It includes low-salt diet restrictions, diuretics such as hydrochlorothiazide, furosemide and spironolactone, systemic and intratympanic steroids and ablative therapies and different types of surgery [3 Magnan J, Özgirgin ON, Trabalzini F, et al. European position statement on diagnosis, and treatment of Meniere's disease. Int Adv Otol. 2018;14:317–321.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
]. Despite the widespread use of diuretics, scientific evidence to support their efficacy is lacking, however, they are considered fairly safe options [3 Magnan J, Özgirgin ON, Trabalzini F, et al. European position statement on diagnosis, and treatment of Meniere's disease. Int Adv Otol. 2018;14:317–321.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
].
Although, the exact pathophysiological mechanism(s) underlying EH remain elusive, altered activities in the cAMP and cGMP signaling systems leading to dysregulation of fluid and ion homeostasis are believed to play important roles. For example, it has been suggested that EH in patients with Menière’s disease results from increased levels of, or increased sensitivity to vasopressin leading to activation of the cAMP/protein kinase A/aquaporin 2 (aqp2) system in the inner ear [4 Takeda T, Takeda S, Kakigi A, et al. Hormonal aspects of Ménière’s disease on the basis of clinical and experimental studies. ORL J Otorhinolaryngol Relat Spec. 2010;71:1–9.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
,5 Katagiri Y, Takumida M, Hirakawa K, et al. Long-term administration of vasopressin can cause Ménière's disease in mice. Acta Otolaryngol. 2014;134:990–1004.
[Taylor & Francis Online], [Web of Science ®], , [Google Scholar]
]. β-adrenergic receptors, known to mediate their effects via the cAMP signaling system, have also been suggested to be involved in the regulation of inner ear fluid ion homeostasis [5–7 Katagiri Y, Takumida M, Hirakawa K, et al. Long-term administration of vasopressin can cause Ménière's disease in mice. Acta Otolaryngol. 2014;134:990–1004.
Wangemann P. K(+) cycling and its regulation in the cochlea and the vestibular labyrinth. Audiol Neurootol. 2002;7:199–205.
Kim BG, Kim JY, Jung J, et al. β1- and β2-adrenergic stimulation-induced electrogenic transport by human endolymphatic sac epithelium and its clinical implications. Sci Rep. 2017;7:42217.
]. For example, activation of β1- and β2-adrenergic receptors have been shown to induce apical electrogenic transport in the human endolymphatic sac epithelium, effects that were impaired in patients with Menière’s disease [7 Kim BG, Kim JY, Jung J, et al. β1- and β2-adrenergic stimulation-induced electrogenic transport by human endolymphatic sac epithelium and its clinical implications. Sci Rep. 2017;7:42217.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
].
The nitric oxide/cGMP pathway is an established major regulatory system in cochlear pathophysiology and increased cGMP has been implicated in hearing loss [8 Fessenden JD, Schacht J. The nitric oxide/cyclic GMP pathway: a potential major regulator of cochlear physiology. Hear Res. 1998;118:168–176.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
]. A number of reports indicate that sildenafil and other inhibitors of PDE5, a cGMP degrading enzyme, is associated with sensorineural hearing loss [9 Mittal R, Bencie N, Shaikh N, et al. Role of cyclic nucleotide phosphodiesterases in inner ear and hearing. Front Physiol. 2017;8:1–3.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
].
We have previously shown that chronic administration of vasopressin and inhibitors of the cAMP/cGMP degrading enzymes PDE3, PDE4 and PDE5 to mice results in the development of EH [10 Degerman E, In 't Zandt R, Pålbrink AK, et al. Vasopressin induces endolymphatic hydrops in mouse inner ear, as evaluated with repeated 9.4 T MRI. Hear Res. 2015;330:119–124. Dec
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
,11 Degerman E, In 't Zandt R, Pålbrink A, et al. Inhibition of phosphodiesterase 3, 4, and 5 induces endolymphatic hydrops in mouse inner ear, as evaluated with repeated 9.4T MRI. Acta Otolaryngol. 2017;137:8–15.
[Taylor & Francis Online], [Web of Science ®], , [Google Scholar]
]. In the present study, we show that spironolactone, an aldosterone and mineralocorticoid receptor antagonist, prevents hydrops induced by vasopressin and the PDE4 inhibitor rolipram, but not by the PDE3 inhibitor cilostamide. The choice of spironolactone in the experimental study was inspired by the observation of improved hearing function associated with spironolactone treatment in some patients (unpublished) and by a number of studies on aldosterone, spironolactone and the inner ear with somehow contradictory results, for example Refs. [12–15 Salt AN, Plontke SK. Endolymphatic hydrops: pathophysiology and experimental models. Otolaryngol Clin North Am. 2010;43:971–983.
Qin L, Zhang B, Wang Q, et al. Effect of aldosterone on cochlear Af9 expression and hearing in guinea pig. Acta Otolaryngol. 2017;137:903–909.
Gross ND, Kempton JB, Trune DR. Spironolactone blocks glucocorticoid-mediated hearing preservation in autoimmune mice. Laryngoscope. 2002;112:298–303.
Mori N, Miyashita T, Inamoto R, et al. Ion transport its regulation in the endolymphatic sac: suggestions for clinical aspects of Meniere's disease. Eur Arch Otorhinolaryngol. 2017;274:1813–1820.
].
Material and methods
Induced hydrops in mice
Experimental design. Twenty female CBA/J mice of body weight 22–24 g (8 weeks of age) were used per study. The experiments were approved by the Regional Ethical Committee on Animal Experiments in Malmö/Lund.
Mice were subjected to continuous administration of either vasopressin (VP) ([Arg]8-VP Sigma Chemical, St Louis, MO) (0.5 mg/kg/day), rolipram (0.4mg/kg/day) (Otsuka pharmaceutical, Tokyo, Japan) or cilostamide (Abcam) (0.5 mg/kg/day) alone (10 mice for each condition) or in combination with spironolactone (1.2 mg/kg/day) (10 mice for each combination) via mini-osmotic pumps (model 2002; Alzet Corp., Palo Alto, CA) for 4 weeks. The pumps were implanted surgically in the subcutaneous tissues on the back between the scapulae. Gadolinium contrast agent (Dotarem, Guerbet, Villepinte, France) (gadoteric acid; 279.3 mg/ml, 0.5 mmol/ml) was administered intraperitoneally (100 μl/20g) in the left abdominal quadrant [10 Degerman E, In 't Zandt R, Pålbrink AK, et al. Vasopressin induces endolymphatic hydrops in mouse inner ear, as evaluated with repeated 9.4 T MRI. Hear Res. 2015;330:119–124. Dec
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
,11 Degerman E, In 't Zandt R, Pålbrink A, et al. Inhibition of phosphodiesterase 3, 4, and 5 induces endolymphatic hydrops in mouse inner ear, as evaluated with repeated 9.4T MRI. Acta Otolaryngol. 2017;137:8–15.
[Taylor & Francis Online], [Web of Science ®], , [Google Scholar]
]. 55–65 min after Gadolinium administration, contrast enhanced MR images were obtained at day zero and 4 weeks after initiation of hormone/drug administration. The inhibitors and spironolactone were dissolved in DMSO/EtOH 1:1.
Magnetic Resonance Imaging. The animals were anesthetized with 3.5% isoflurane in mixture of 200 ml/min oxygen and 200 ml/min nitrous oxide and maintained at 1.5–2% isoflurane inside the magnet. The induction chamber was kept warm at 37 °C. Inside the magnet, the respiratory rate of the animal was monitored (SA Instruments Inc, New York, NY) and the body temperature was maintained using circulating warm water bath (Lauda, Lauda-Köningshofen, Germany).
MR imaging was performed as previously described [10 Degerman E, In 't Zandt R, Pålbrink AK, et al. Vasopressin induces endolymphatic hydrops in mouse inner ear, as evaluated with repeated 9.4 T MRI. Hear Res. 2015;330:119–124. Dec
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
,11 Degerman E, In 't Zandt R, Pålbrink A, et al. Inhibition of phosphodiesterase 3, 4, and 5 induces endolymphatic hydrops in mouse inner ear, as evaluated with repeated 9.4T MRI. Acta Otolaryngol. 2017;137:8–15.
[Taylor & Francis Online], [Web of Science ®], , [Google Scholar]
] with a 9.4 T magnet (Agilent Inc., Palo Alto, CA) using Avance III electronics (Bruker, Ettlingen, Germany). The system is equipped with a 12 cm inner diameter gradient system having a maximum gradient strength of 670 mT/m. The animals were imaged using a quadrature transmit/receive cryoprobe (Bruker, Ettlingen, Germany). T1-weighted 3D images were acquired with a gradient echo 3D sequence; repetition time TR: 11 ms, echo time TE: 3.695 ms, number of averages: 2, data matrix size 220 × 220 × 147 pixels, field of view 15 × 15 × 10 mm3. Images were reconstructed by zero filling to increase the apparent resolution of the image to a matrix size of 440 × 440 × 294 pixels and an apparent pixel resolution of 0.034 mm.
Quantitative assessment of gadolinium in endolymphatic relative perilymphatic space. Synedra view personal 16 (Synedra, Innsbruck, Austria) as well as Adobe Photoshop CS5 (Adobe, San Jose, CA) were used for postproduction processing of images for the quantification of signal intensity in regions of interest and for labeling and demonstration of perilymph in the scala tympani and scala vestibuli and of endolymph in the scala media.
Images parallel to the modiolus of the cochleas of mice treated as previously described were used for measurements (exemplified in Figure 1, [10 Degerman E, In 't Zandt R, Pålbrink AK, et al. Vasopressin induces endolymphatic hydrops in mouse inner ear, as evaluated with repeated 9.4 T MRI. Hear Res. 2015;330:119–124. Dec
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
,11 Degerman E, In 't Zandt R, Pålbrink A, et al. Inhibition of phosphodiesterase 3, 4, and 5 induces endolymphatic hydrops in mouse inner ear, as evaluated with repeated 9.4T MRI. Acta Otolaryngol. 2017;137:8–15.
[Taylor & Francis Online], [Web of Science ®], , [Google Scholar]
]). To evaluate the effect of various treatments on the size of the endolymphatic fluid compartment, the relative area of scala media in the basal turn of the cochlea was estimated by calculating the ratio between scala media (endolymph, non-contrast enhanced) and scala media plus scala vestibuli (perilymph, contrast enhanced). The ratio of areas is subsequently converted to percentage as shown on the figures. The observer assessing and outlining the perilymphatic and endolymphatic space, was blinded to the treatments given. One ear in each group in the vasopressin substudy was lost to analysis. In the rolipram substudy a mouse in each group was lost before the first MRI.
Figure 1. Magnetic resonance imaging and quantitative assessment of gadolinium in endolymphatic relative perilymphatic space. Sagittal section of the mouse brain showing the inner ear and the contrast enhancement in the cochlea (left). To evaluate the effect of spironolactone on EH in different contexts, the size of the endolymphatic fluid compartment, the relative area of scala media in the basal turn of the cochlea, was estimated by calculating the ratio between scala media (noncontrast enhanced, lower dotted area) and scala media + scala vestibule (scala vestibule, upper dotted area, is contrast enhanced) (right). The ratio of areas is subsequently converted to percentage as shown in Figures 2–4. SV: scala vestibule; ST: scala tympani; SM: scala media; 1st: first turn; 2nd: second turn.
Display full size
Immunohistochemistry of human saccule
Tissue sampling and preparations. Using an operating microscope, two human saccules were dissected from the vestibules during the removal of vestibular schwannomas via the translabyrinthine approach. The sample was immediately fixed in HistoChoice (Amresco) in phosphate buffer saline (PBS) for 12 h, soaked in 30% sucrose in PBS at 4 °C over night and then embedded in Tissue Tec OCT Compound (Gene Research Lab). Eight μM thick sections were cut with a cryostat and collected on slides (super-frost-plus, Fischer Scientific). Eight consecutive tissue sections were placed on each glass slide. Tissue sections were stored at –80 °C until use. The procedures were evaluated by the responsible committee at Lund University on human experimentation and were in accordance with the Helsinki Declaration of 1975, as revised in 1983.
Immunohistochemistry. Before being immunohistochemically stained, sections were thawed and allowed to air-dry for 10 min. Next, a circle surrounding each tissue section was drawn with a hydrophobic pen (Dako, Denmark). Tissue sections were covered with 5% normal goat serum diluted in PBS for 1 h (approximately 30 μl per section). Subsequently, the specimens were exposed overnight at 4 °C in a humid chamber to antibodies specific for the mineralocorticoid receptor (Thermo Fisher) and alpha ENaC (Abcam) diluted 1:75 in 5% normal goat serum (Invitrogen). At the end of the incubations, the tissue sections were rinsed in PBS (3 × 15 min). Secondary goat anti rabbit antibodies (labeled with Alexa 555) or goat anti mouse antibodies (labeled with Alexa 488) (Invitrogen, diluted 1:200), were applied to the tissue sections and incubated for 2 h at room temperature in the dark. At the end of the incubation, sections were washed with PBS and 4,6-diamidino-2-phenylindole (DAPI, Invitrogen) was added for 10 min to visualize all cell nuclei. At the end of the incubation, the sections were washed in PBS and mounted with Vectashield (Vector Laboratories). Sections were observed using a camera-equipped fluorescence microscope. Images were obtained using a Zeiss Axiophot 2 microscope and a Hamamatsu C4742-95 camera with Openlab 5 software (Improvision) for image processing.
Statistical analysis
For statistical analysis, endolymph/perilymph ratios, performed before and after treatment in the same mouse, were compared using Student’s paired t-test with p < .05 accepted as an indication of statistical significance.
Results
In a recent study, using 9.4 T field strength MRI, we demonstrated the development of EH in mice, induced by chronic administration of vasopressin and PDE3,4,5 inhibitors [10 Degerman E, In 't Zandt R, Pålbrink AK, et al. Vasopressin induces endolymphatic hydrops in mouse inner ear, as evaluated with repeated 9.4 T MRI. Hear Res. 2015;330:119–124. Dec
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
,11 Degerman E, In 't Zandt R, Pålbrink A, et al. Inhibition of phosphodiesterase 3, 4, and 5 induces endolymphatic hydrops in mouse inner ear, as evaluated with repeated 9.4T MRI. Acta Otolaryngol. 2017;137:8–15.
[Taylor & Francis Online], [Web of Science ®], , [Google Scholar]
]. Here, we tested the ability of spironolactone to prevent the development of EH, when induced by three different mechanisms, two of which affect the cAMP-mediated pathways (vasopressin and the PDE4 inhibitor rolipram) and one that also affects the cGMP-pathway (the PDE3 inhibitor cilostamide) [16 Ahmad F, Murata T, Simizu K, et al. Cyclic nucleotide phosphodiesterases: important signaling modulators and therapeutic targets. Oral Dis. 2015;21(1):e25–e50.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
]. PDE3 and PDE4 are believed to control unique pools of cAMP and cGMP [16 Ahmad F, Murata T, Simizu K, et al. Cyclic nucleotide phosphodiesterases: important signaling modulators and therapeutic targets. Oral Dis. 2015;21(1):e25–e50.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
], possibly vasopressin acts in connection with PDE4 [17 Stefan E, Wiesner B, Baillie GS, et al. Compartmentalization of cAMP-dependent signaling by phosphodiesterase-4D is involved in the regulation of vasopressin-mediated water reabsorption in renal principal cells. J Am Soc Nephrol. 2007;18:199–212.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
].
Spironolactone prevents the development of vasopressin-induced endolymphatic hydrops in mice
Vasopressin or vasopressin + spironolactone were administrated to 10 plus 10 CBA/J mice for 28 d. As shown in Figure 2 (left), vasopressin treatment resulted in increased volume of the endolymphatic space as compared to pre-vasopressin (n = 19, p = .000068). However, in the presence of spironolactone, the vasopressin effect was prevented (n = 19, p = 0.20) (Figure 2, left). In Figure 2 (middle), prevalues and postvalues are indicated for each ear and in Figure 2 (right), the pretreatment value was set to 1 for each ear.
Figure 2. Spironolactone prevents vasopressin-induced endolymphatic hydrops in CBA/J mice. CBA/J mice were treated chronically with vasopressin for 28 d with or without SL and thereafter analyzed using MRI. Quantification of endolymphatic and perilymphatic cochlear compartments before and after vasopressin (VP) and before and after vasopressin plus spironolactone (VP + SL) administration was performed as described in Material and methods and in Figure 1. Data are presented as means (left), as individual values for each ear before and after treatment (middle) and as individual values for each ear with prevalues set to 1 (right).
Display full size
Spironolactone prevents the development of rolipram-induced endolymphatic hydrops in mice
Rolipram or rolipram + spironolactone were administrated to 10 plus 10 CBA/J mice for 28 d. As shown in Figure 3 (left), rolipram treatment resulted in increased volume of the endolymphatic space as compared to pre-rolipram (n = 18, p = .0085). However, in the presence of spironolactone, the rolipram effect was prevented (n = 18, p = 0.64) (Figure 3, left). In Figure 3 (middle), prevalues and postvalues are indicated for each ear and in Figure 3 (right), the pretreatment value was set to 1 for each ear.
Figure 3. Spironolactone prevents rolipram-induced endolymphatic hydrops in CBA/J mice. CBA/J mice were treated chronically with rolipram with or without SL for 28 d and thereafter analyzed using MRI. for 28 d. Quantification of endolymphatic and perilymphatic cochlear compartments before and after rolipram (rol) and before and after rolipram plus spironolactone (rol + SL) administration was performed as described in Material and methods and in Figure 1. Data are presented as means (left), as individual values for each ear before and after treatment (middle) and as individual values for each ear with prevalues set to 1 (right).
Display full size
Spironolactone does not prevent the development of cilostamide-induced endolymphatic hydrops in mice
Cilostamide or cilostamide + spironolactone were administrated to 10 plus 10 CBA/J mice for 28 d. As shown in Figure 4 (left), cilostamide treatment resulted in increased endolymphatic space as compared to pre-cilostamide (n = 20, p = .00043). In the presence of spironolactone, the cilostamide effect was maintained (Figure 4, left) (n = 20, p = .0000021). In Figure 4 (middle), prevalues and postvalues are indicated for each ear and in Figure 4 (right), the pretreatment value was set to 1 for each ear.
Figure 4. Spironolactone does not prevent cilostamide-induced endolymphatic hydrops in CBA/J mice. CBA/J mice were treated chronically with cilostamide with or without SL for 28 d and thereafter analyzed using MRI. Quantification of endolymphatic and perilymphatic cochlear compartments before and after cilostamide (Cil) and before and after cilostamide plus spironolactone (Cil + SL) administration was performed as described in Material and methods and in Figure 1. Data are presented as means (left), as individual values for each ear before and after treatment (middle) and as individual values for each ear with prevalues set to 1 (right).
Display full size
The aldosterone target ENaC and the mineralocorticoid receptor are expressed in the human saccule sensory epithelium
As shown in Figure 5, immunostaining of ENaC, a target for aldosterone action, was detected in the apical part of the sensory epithelium of the human saccule, presumably in both hair cells and supporting cells. The mineralocorticoid receptor was detected mainly in supporting cells as indicated by co-staining with the supporting cell marker glial acidic fibrillary protein (GFAP).
Figure 5. Pathway-dependent effect of spironolactone on the prevention of endolymphatic hydrops-working hypothesis. SL prevents EH induced by vasopressin believed to act via cAMP/protein kinas A mediated phosphorylation and translocation of the fluid transporter aqp2 to regulate inner ear functions. It is possible that PDE4 regulates the pool of cAMP utilized by vasopressin since (a) PDE4 interacts with aqp2 in kidney cells (b) PDE4 is expressed in the inner ear and (c) SL prevents hydrops induced by the PDE4 inhibitor rolipram. On the other hand, SL does not prevent hydrops induced by the PDE3 inhibitor cilostamide indicating that different pools of cyclic nucleotides are involved in hydrops induction in this model, cAMPPDE4, cAMPPDE3 and cGMPPDE3. Inhibition of aldosterone action could interfere with the vasopressin/PDE4 signaling systems at different levels. Since SL prevents rolipram-induced hydrops, one level would be downstream of cAMPPDE4 production. Mechanisms mediating cilostamide induced EH remain to be elucidated, however, most likely involve NO/cGMP as well as cAMP down-stream targets. Target proteins for the signaling systems described, of relevance for EH, presumably involve fluid and ion transporters such as aqp2, ENaC, NCC and NaKATPase. A key target for aldosterone action, ENaC and the mineralocorticoid receptor were expressed in the human saccule. Tissue sections were immunohistochemically stained with indicated antibodies. Merged photos of MR, DAPI (nuclear label) and GAFP (upper, left), merged photo of MR and DAPI (middle, left), merged photo of MR and GAFP (lower, left), merged photo with ENaC, DAPI and GAFP (right). NKA: NaKATPase; MR: mineralocorticid receptor; GAFP: glial acidic fibrillary protein; dapi stains nuclei; sensory epithelium, bar with two arrow heads.
Display full size
Discussion
Here, we present beneficial effects of the aldosterone antagonist spironolactone on EH hydrops induced by vasopressin and the PDE4 inhibitor rolipram. Increased action or sensitivity of vasopressin in the inner ear is believed to contribute to the development of EH via enhanced effects on aqp2, an important target for cAMP/PKA signaling [4 Takeda T, Takeda S, Kakigi A, et al. Hormonal aspects of Ménière’s disease on the basis of clinical and experimental studies. ORL J Otorhinolaryngol Relat Spec. 2010;71:1–9.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
]. In the kidney, aqp2 has been shown to connect to PDE4, a cAMP specific phosphodiesterase [17 Stefan E, Wiesner B, Baillie GS, et al. Compartmentalization of cAMP-dependent signaling by phosphodiesterase-4D is involved in the regulation of vasopressin-mediated water reabsorption in renal principal cells. J Am Soc Nephrol. 2007;18:199–212.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
]. It is, thus, possible that the ability of spironolactone to antagonize vasopressin induced EH is by interfering with a cAMP pool controlled by PDE4. In agreement with a connection between vasopressin and PDE4, hydrops induced by the PDE4 inhibitor rolipram, was also antagonized by spironolactone. The finding that EH induced by cilostamide, a PDE3 inhibitor hydrolyzing both cAMP and cGMP, was not prevented by spironolactone indicate that there should be different mechanisms for the development of EH. This is in agreement with the shown compartmentalization of cyclic nucleotides [16 Ahmad F, Murata T, Simizu K, et al. Cyclic nucleotide phosphodiesterases: important signaling modulators and therapeutic targets. Oral Dis. 2015;21(1):e25–e50.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
].
Indeed, cAMP and cGMP are believed to be tightly compartmentalized by the action of PDEs, belonging to presently defined 12 families, PDE1-12 [16 Ahmad F, Murata T, Simizu K, et al. Cyclic nucleotide phosphodiesterases: important signaling modulators and therapeutic targets. Oral Dis. 2015;21(1):e25–e50.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
]. We have previously shown the expression of PDE3 and PDE4 in the human inner ear and PDEs have been implicated in hearing dysfunction [9 Mittal R, Bencie N, Shaikh N, et al. Role of cyclic nucleotide phosphodiesterases in inner ear and hearing. Front Physiol. 2017;8:1–3.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
,11 Degerman E, In 't Zandt R, Pålbrink A, et al. Inhibition of phosphodiesterase 3, 4, and 5 induces endolymphatic hydrops in mouse inner ear, as evaluated with repeated 9.4T MRI. Acta Otolaryngol. 2017;137:8–15.
[Taylor & Francis Online], [Web of Science ®], , [Google Scholar]
]. Due to their distinct intrinsic characteristics and their intracellular targeting to different subcellular locations, different PDEs integrate multiple cellular inputs and modulate the amplitude, duration, termination and specificity of cAMP and cGMP signaling and actions. Thus, the cAMP/cGMP pools controlled by PDE3, appear not to cross-talk with spironolactone action which is the case for the cAMP-pool utilized by vasopressin and PDE4. Exactly how spironolactone interferes with cAMP signaling is not known. An interplay between cAMP and the mineralocorticoid receptor signaling pathways has been described, please see review and references herein [18 Hermidorff MM, de Assis LV, Isoldi MC. Genomic and rapid effects of aldosterone: what we know and do not know thus far. Heart Fail Rev. 2017;22:65–89.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
].
Although, our results indicate beneficial effects of spironolactone on the inner ear, contradictory results have been published with regards to effects of aldosterone and spironolactone on EH [12–15 Salt AN, Plontke SK. Endolymphatic hydrops: pathophysiology and experimental models. Otolaryngol Clin North Am. 2010;43:971–983.
Qin L, Zhang B, Wang Q, et al. Effect of aldosterone on cochlear Af9 expression and hearing in guinea pig. Acta Otolaryngol. 2017;137:903–909.
Gross ND, Kempton JB, Trune DR. Spironolactone blocks glucocorticoid-mediated hearing preservation in autoimmune mice. Laryngoscope. 2002;112:298–303.
Mori N, Miyashita T, Inamoto R, et al. Ion transport its regulation in the endolymphatic sac: suggestions for clinical aspects of Meniere's disease. Eur Arch Otorhinolaryngol. 2017;274:1813–1820.
]. In agreement with our results aldosterone has been reported to induce or worsen EH in different animal models with or without other EH inducing factors such as lipopolysaccharide and sac ablation, for review see [12 Salt AN, Plontke SK. Endolymphatic hydrops: pathophysiology and experimental models. Otolaryngol Clin North Am. 2010;43:971–983.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
]. On the other hand, aldosterone was reported to have beneficial effects on hearing preservation via the mineral corticoid receptor in the MRL/MpJ-Faslpr autoimmune disease mouse, an established model of spontaneous autoimmune sensorineural hearing loss [14 Gross ND, Kempton JB, Trune DR. Spironolactone blocks glucocorticoid-mediated hearing preservation in autoimmune mice. Laryngoscope. 2002;112:298–303.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
]. In some clinical studies, a low salt diet was advocated to be an effective treatment of Menière’s disease [15 Mori N, Miyashita T, Inamoto R, et al. Ion transport its regulation in the endolymphatic sac: suggestions for clinical aspects of Meniere's disease. Eur Arch Otorhinolaryngol. 2017;274:1813–1820.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
] which was actually correlated with increased aldosterone levels. On the other hand, diuretics are implicated in the treatment of Menière’s disease, although, evidence to support their efficacy is lacking [3 Magnan J, Özgirgin ON, Trabalzini F, et al. European position statement on diagnosis, and treatment of Meniere's disease. Int Adv Otol. 2018;14:317–321.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
].
Thus, different models, read-outs, methods, doses of aldosterone and study designs have been used in the context of spironolactone, aldosterone and the inner ear which may explain some of the contradictory results. One should have in mind that aldosterone is believed to act via the mineralocorticoid receptor as well as via cell surface receptors [18 Hermidorff MM, de Assis LV, Isoldi MC. Genomic and rapid effects of aldosterone: what we know and do not know thus far. Heart Fail Rev. 2017;22:65–89.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
] whereas spironolactone is believed to antagonize the effects induced by the mineralocorticoid receptor only. The exact arrangement and levels of key targets for aldosterone such as the amiloride sensitive Na + transporter ENaC, NCC and the NaK-ATPase in the inner ear presumably contribute to the impact of altered aldosterone level in different contexts in one or the other direction [19 Eckhard AH, Zhu M, O'Malley JT, et al. Inner ear pathologies impair sodium-regulated ion transport in Meniere's disease. Acta Neuropathol. 2019;137:343–357.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
]. Nevertheless, impaired Na + transport in the endolymphatic sac has been associated with Menière’s disease [15 Mori N, Miyashita T, Inamoto R, et al. Ion transport its regulation in the endolymphatic sac: suggestions for clinical aspects of Meniere's disease. Eur Arch Otorhinolaryngol. 2017;274:1813–1820.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
,20 Mittal R, Aranke M, Debs LH, et al. Indispensable role of ion channels and transporters in the auditory system. J Cell Physiol. 2017;232:743–758.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
].
From a clinical point of view the above results raise interesting perspectives. If the hydrops is a fundamental part of the development of pathology in Menière’s disease and hydrops may be developed due to more than one mechanism, the effect of treatment with diuretics or for that matter any other suggested treatments, could be dependent on if patients had hydrops developed from the mechanism that is targeted by the treatment. As this is not known, a cohort of patients may consist of any proportion of different etiologies to hydrops and treatment effects may be very difficult to ascertain. This points out the need for personalized medicine in Menière’s disease, as well as a difficulty in designing intervention studies.
Conclusions
We show that the aldosterone antagonist spironolactone prevents hydrops induced by vasopressin and the PDE4 inhibitor rolipram but not hydrops induced by the PDE3 inhibitor cilostamide in agreement with compartmentalization of cyclic nucleotides [16 Ahmad F, Murata T, Simizu K, et al. Cyclic nucleotide phosphodiesterases: important signaling modulators and therapeutic targets. Oral Dis. 2015;21(1):e25–e50.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
]. Thus, our study actually demonstrates that spironolactone is beneficial in one context but not in another which is in agreement with the contradictory results obtained in the context of aldosterone, spironolactone and the inner ear [12–15 Salt AN, Plontke SK. Endolymphatic hydrops: pathophysiology and experimental models. Otolaryngol Clin North Am. 2010;43:971–983.
Qin L, Zhang B, Wang Q, et al. Effect of aldosterone on cochlear Af9 expression and hearing in guinea pig. Acta Otolaryngol. 2017;137:903–909.
Gross ND, Kempton JB, Trune DR. Spironolactone blocks glucocorticoid-mediated hearing preservation in autoimmune mice. Laryngoscope. 2002;112:298–303.
Mori N, Miyashita T, Inamoto R, et al. Ion transport its regulation in the endolymphatic sac: suggestions for clinical aspects of Meniere's disease. Eur Arch Otorhinolaryngol. 2017;274:1813–1820.
]. Extrapolating this finding to the clinic, suggests that a personalized medicine approach is probably necessary in the treatment of diseases involving EH, such as Menière’s disease.
Acknowledgements
Lund University Bioimaging Center (LBIC) is gratefully acknowledged for providing experimental resources. ED and AP are affiliated with Lund University Diabetes Center (LUDC).
Disclosure statement
No potential conflict of interest was reported by the authors.
References
Oberman BS, Patel VA, Cureoglu S, et al. The aetiopathologies of Ménière's disease: a contemporary review. Acta Otorhinolaryngol Ital. 2017;37:250–263.[PubMed], [Web of Science ®], , [Google Scholar]
Lopez-Escamez JA, Carey J, Chung WH, et al. Classification Committee of the Barany Society; Japan Society for Equilibrium Research; European Academy of Otology and Neurotology (EAONO); Equilibrium Committee of the American Academy of Otolaryngology-Head and Neck Surgery (AAO-HNS); Korean Balance Society. Diagnostic criteria for Menière's disease. J Vestib Res. 2015;25:1–7.[PubMed], [Web of Science ®], , [Google Scholar]
Magnan J, Özgirgin ON, Trabalzini F, et al. European position statement on diagnosis, and treatment of Meniere's disease. Int Adv Otol. 2018;14:317–321.[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
Takeda T, Takeda S, Kakigi A, et al. Hormonal aspects of Ménière’s disease on the basis of clinical and experimental studies. ORL J Otorhinolaryngol Relat Spec. 2010;71:1–9.[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
Katagiri Y, Takumida M, Hirakawa K, et al. Long-term administration of vasopressin can cause Ménière's disease in mice. Acta Otolaryngol. 2014;134:990–1004.[Taylor & Francis Online], [Web of Science ®], , [Google Scholar]
Wangemann P. K(+) cycling and its regulation in the cochlea and the vestibular labyrinth. Audiol Neurootol. 2002;7:199–205.[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
Kim BG, Kim JY, Jung J, et al. β1- and β2-adrenergic stimulation-induced electrogenic transport by human endolymphatic sac epithelium and its clinical implications. Sci Rep. 2017;7:42217.[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
Fessenden JD, Schacht J. The nitric oxide/cyclic GMP pathway: a potential major regulator of cochlear physiology. Hear Res. 1998;118:168–176.[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
Mittal R, Bencie N, Shaikh N, et al. Role of cyclic nucleotide phosphodiesterases in inner ear and hearing. Front Physiol. 2017;8:1–3.[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
Degerman E, In 't Zandt R, Pålbrink AK, et al. Vasopressin induces endolymphatic hydrops in mouse inner ear, as evaluated with repeated 9.4 T MRI. Hear Res. 2015;330:119–124. Dec[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
Degerman E, In 't Zandt R, Pålbrink A, et al. Inhibition of phosphodiesterase 3, 4, and 5 induces endolymphatic hydrops in mouse inner ear, as evaluated with repeated 9.4T MRI. Acta Otolaryngol. 2017;137:8–15.[Taylor & Francis Online], [Web of Science ®], , [Google Scholar]
Salt AN, Plontke SK. Endolymphatic hydrops: pathophysiology and experimental models. Otolaryngol Clin North Am. 2010;43:971–983.[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
Qin L, Zhang B, Wang Q, et al. Effect of aldosterone on cochlear Af9 expression and hearing in guinea pig. Acta Otolaryngol. 2017;137:903–909.[Taylor & Francis Online], [Web of Science ®], , [Google Scholar]
Gross ND, Kempton JB, Trune DR. Spironolactone blocks glucocorticoid-mediated hearing preservation in autoimmune mice. Laryngoscope. 2002;112:298–303.[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
Mori N, Miyashita T, Inamoto R, et al. Ion transport its regulation in the endolymphatic sac: suggestions for clinical aspects of Meniere's disease. Eur Arch Otorhinolaryngol. 2017;274:1813–1820.[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
Ahmad F, Murata T, Simizu K, et al. Cyclic nucleotide phosphodiesterases: important signaling modulators and therapeutic targets. Oral Dis. 2015;21(1):e25–e50.[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
Stefan E, Wiesner B, Baillie GS, et al. Compartmentalization of cAMP-dependent signaling by phosphodiesterase-4D is involved in the regulation of vasopressin-mediated water reabsorption in renal principal cells. J Am Soc Nephrol. 2007;18:199–212.[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
Hermidorff MM, de Assis LV, Isoldi MC. Genomic and rapid effects of aldosterone: what we know and do not know thus far. Heart Fail Rev. 2017;22:65–89.[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
Eckhard AH, Zhu M, O'Malley JT, et al. Inner ear pathologies impair sodium-regulated ion transport in Meniere's disease. Acta Neuropathol. 2019;137:343–357.[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
Mittal R, Aranke M, Debs LH, et al. Indispensable role of ion channels and transporters in the auditory system. J Cell Physiol. 2017;232:743–758.[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
Additional information
Funding
The work was supported by grants from the Swedish Diabetes Foundation, the Swedish Foundation for Strategic Research Dnr IRC 15-0067, the Albert Påhlsson Foundation, Sweden, the Crafoord Foundation, Sweden, Diabetes Wellness Sweden, Skane County Council’s Research and Development Foundation, Sweden and the Medical Faculty, Lund University, Sweden.
Eva Degerman, René In ’t Zandt, Annki Pålbrink & Måns Magnusson
Received 10 Feb 2019, Accepted 03 May 2019, Published online: 30 May 2019
Download citation https://doi.org/10.1080/00016489.2019.1616819
Abstract
Background: The exact pathophysiological mechanism(s) underlying endolymphatic hydrops (EH) remain elusive. We have previously shown that chronic administration of vasopressin and inhibitors of the cAMP/cGMP degrading enzymes (PDE3, PDE4, PDE5) results in the development of EH to mice.
Aims/objectives: Evaluate the ability of spironolactone, an aldosterone antagonist, to prevent EH, when induced by different pathways.
Material and methods: Mice were treated for 4 weeks with vasopressin, the PDE3 inhibitor cilostamide and the PDE4 inhibitor rolipram in the presence or absence of spironolactone. EH was assessed using high resolution 9.4T MRI. The expression of proteins in human saccule sensory epithelium was studied with immunohistochemistry.
Results: Spironolactone prevents EH induced by vasopressin and rolipram, but not hydrops induced by cilostamide. The aldosterone target ENaC and the mineralocorticoid receptor were expressed in the human saccule sensory epithelium.
Conclusions: The effect of spironolactone on EH appears to be pathway-dependent and may provide explanations why certain drugs may be effective in some patients with hydropic ear disease while not in others.
Significance: Extrapolating this finding to the clinic supports that a personalized medicine approach is probably necessary in the treatment of diseases involving EH, as different pathways may be needed to be targeted for treatment.
Chinese abstract
背景：内淋巴积水（EH）的确切病理生理机制尚不清楚。我们之前已经证明, 长期服用加压素和cAMP/cGMP降解酶抑制剂（PDE3、PDE4、PDE5）会导致小鼠的内淋巴积水。
目的：评价醛固酮拮抗剂螺内酯在不同途径诱导下预防EH的能力。
材料和方法：小鼠在有或无螺内酯的情况下, 用加压素、PDE3抑制剂西洛他明和PDE4抑制剂罗利帕姆治疗4周。采用高分辨率9.4T MRI评估EH。用免疫组织化学方法研究蛋白质在人球囊感觉上皮中的表达。
结果：螺内酯对加压素和罗力普仑诱导的EH有抑制作用, 而对西洛他明诱导的水肿无抑制作用。醛固酮靶向ENAC和盐皮质激素受体在人球囊感觉上皮中被表达。
结论：螺内酯对EH的影响似乎是途径依赖性的, 这可能解释某些药物对某些水样耳病患者有效而对其他患者无效的原因。
意义：将这一发现外推到临床, 我们认为个人化医治法可能是治疗EH有关疾病所必需的, 因为治疗可能需要不同的途径。
Keywords: Magnetic resonance imaging, cochlea, endolymphatic hydrops, vasopressin, cilostamide, rolipram, PDE3, PDE4, spironolactone, Menière’s disease
Introduction
A characteristic finding of several inner ear disorders is the dilatation of the endolymphatic compartment of the inner ear, endolymphatic hydrops (EH), resulting from deteriorated ion homeostasis and fluid volume regulation [1 Oberman BS, Patel VA, Cureoglu S, et al. The aetiopathologies of Ménière's disease: a contemporary review. Acta Otorhinolaryngol Ital. 2017;37:250–263.
[PubMed], [Web of Science ®], , [Google Scholar]
]. EH may result from a number of processes such as autoimmune disorders, viral infections, electrolyte and fluid imbalance and trauma [1 Oberman BS, Patel VA, Cureoglu S, et al. The aetiopathologies of Ménière's disease: a contemporary review. Acta Otorhinolaryngol Ital. 2017;37:250–263.
[PubMed], [Web of Science ®], , [Google Scholar]
]. EH associated changes in the inner ear are believed to be of significance in relation to the symptoms associated with for example Menière’s disease such as fluctuating hearing loss, tinnitus, vertigo and aural fullness [1 Oberman BS, Patel VA, Cureoglu S, et al. The aetiopathologies of Ménière's disease: a contemporary review. Acta Otorhinolaryngol Ital. 2017;37:250–263.
[PubMed], [Web of Science ®], , [Google Scholar]
,2 Lopez-Escamez JA, Carey J, Chung WH, et al. Classification Committee of the Barany Society; Japan Society for Equilibrium Research; European Academy of Otology and Neurotology (EAONO); Equilibrium Committee of the American Academy of Otolaryngology-Head and Neck Surgery (AAO-HNS); Korean Balance Society. Diagnostic criteria for Menière's disease. J Vestib Res. 2015;25:1–7.
[PubMed], [Web of Science ®], , [Google Scholar]
].
Treatment regimes for diseases believed to involve EH such as Menière’s disease, involve a range of strategies. It includes low-salt diet restrictions, diuretics such as hydrochlorothiazide, furosemide and spironolactone, systemic and intratympanic steroids and ablative therapies and different types of surgery [3 Magnan J, Özgirgin ON, Trabalzini F, et al. European position statement on diagnosis, and treatment of Meniere's disease. Int Adv Otol. 2018;14:317–321.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
]. Despite the widespread use of diuretics, scientific evidence to support their efficacy is lacking, however, they are considered fairly safe options [3 Magnan J, Özgirgin ON, Trabalzini F, et al. European position statement on diagnosis, and treatment of Meniere's disease. Int Adv Otol. 2018;14:317–321.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
].
Although, the exact pathophysiological mechanism(s) underlying EH remain elusive, altered activities in the cAMP and cGMP signaling systems leading to dysregulation of fluid and ion homeostasis are believed to play important roles. For example, it has been suggested that EH in patients with Menière’s disease results from increased levels of, or increased sensitivity to vasopressin leading to activation of the cAMP/protein kinase A/aquaporin 2 (aqp2) system in the inner ear [4 Takeda T, Takeda S, Kakigi A, et al. Hormonal aspects of Ménière’s disease on the basis of clinical and experimental studies. ORL J Otorhinolaryngol Relat Spec. 2010;71:1–9.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
,5 Katagiri Y, Takumida M, Hirakawa K, et al. Long-term administration of vasopressin can cause Ménière's disease in mice. Acta Otolaryngol. 2014;134:990–1004.
[Taylor & Francis Online], [Web of Science ®], , [Google Scholar]
]. β-adrenergic receptors, known to mediate their effects via the cAMP signaling system, have also been suggested to be involved in the regulation of inner ear fluid ion homeostasis [5–7 Katagiri Y, Takumida M, Hirakawa K, et al. Long-term administration of vasopressin can cause Ménière's disease in mice. Acta Otolaryngol. 2014;134:990–1004.
Wangemann P. K(+) cycling and its regulation in the cochlea and the vestibular labyrinth. Audiol Neurootol. 2002;7:199–205.
Kim BG, Kim JY, Jung J, et al. β1- and β2-adrenergic stimulation-induced electrogenic transport by human endolymphatic sac epithelium and its clinical implications. Sci Rep. 2017;7:42217.
]. For example, activation of β1- and β2-adrenergic receptors have been shown to induce apical electrogenic transport in the human endolymphatic sac epithelium, effects that were impaired in patients with Menière’s disease [7 Kim BG, Kim JY, Jung J, et al. β1- and β2-adrenergic stimulation-induced electrogenic transport by human endolymphatic sac epithelium and its clinical implications. Sci Rep. 2017;7:42217.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
].
The nitric oxide/cGMP pathway is an established major regulatory system in cochlear pathophysiology and increased cGMP has been implicated in hearing loss [8 Fessenden JD, Schacht J. The nitric oxide/cyclic GMP pathway: a potential major regulator of cochlear physiology. Hear Res. 1998;118:168–176.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
]. A number of reports indicate that sildenafil and other inhibitors of PDE5, a cGMP degrading enzyme, is associated with sensorineural hearing loss [9 Mittal R, Bencie N, Shaikh N, et al. Role of cyclic nucleotide phosphodiesterases in inner ear and hearing. Front Physiol. 2017;8:1–3.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
].
We have previously shown that chronic administration of vasopressin and inhibitors of the cAMP/cGMP degrading enzymes PDE3, PDE4 and PDE5 to mice results in the development of EH [10 Degerman E, In 't Zandt R, Pålbrink AK, et al. Vasopressin induces endolymphatic hydrops in mouse inner ear, as evaluated with repeated 9.4 T MRI. Hear Res. 2015;330:119–124. Dec
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
,11 Degerman E, In 't Zandt R, Pålbrink A, et al. Inhibition of phosphodiesterase 3, 4, and 5 induces endolymphatic hydrops in mouse inner ear, as evaluated with repeated 9.4T MRI. Acta Otolaryngol. 2017;137:8–15.
[Taylor & Francis Online], [Web of Science ®], , [Google Scholar]
]. In the present study, we show that spironolactone, an aldosterone and mineralocorticoid receptor antagonist, prevents hydrops induced by vasopressin and the PDE4 inhibitor rolipram, but not by the PDE3 inhibitor cilostamide. The choice of spironolactone in the experimental study was inspired by the observation of improved hearing function associated with spironolactone treatment in some patients (unpublished) and by a number of studies on aldosterone, spironolactone and the inner ear with somehow contradictory results, for example Refs. [12–15 Salt AN, Plontke SK. Endolymphatic hydrops: pathophysiology and experimental models. Otolaryngol Clin North Am. 2010;43:971–983.
Qin L, Zhang B, Wang Q, et al. Effect of aldosterone on cochlear Af9 expression and hearing in guinea pig. Acta Otolaryngol. 2017;137:903–909.
Gross ND, Kempton JB, Trune DR. Spironolactone blocks glucocorticoid-mediated hearing preservation in autoimmune mice. Laryngoscope. 2002;112:298–303.
Mori N, Miyashita T, Inamoto R, et al. Ion transport its regulation in the endolymphatic sac: suggestions for clinical aspects of Meniere's disease. Eur Arch Otorhinolaryngol. 2017;274:1813–1820.
].
Material and methods
Induced hydrops in mice
Experimental design. Twenty female CBA/J mice of body weight 22–24 g (8 weeks of age) were used per study. The experiments were approved by the Regional Ethical Committee on Animal Experiments in Malmö/Lund.
Mice were subjected to continuous administration of either vasopressin (VP) ([Arg]8-VP Sigma Chemical, St Louis, MO) (0.5 mg/kg/day), rolipram (0.4mg/kg/day) (Otsuka pharmaceutical, Tokyo, Japan) or cilostamide (Abcam) (0.5 mg/kg/day) alone (10 mice for each condition) or in combination with spironolactone (1.2 mg/kg/day) (10 mice for each combination) via mini-osmotic pumps (model 2002; Alzet Corp., Palo Alto, CA) for 4 weeks. The pumps were implanted surgically in the subcutaneous tissues on the back between the scapulae. Gadolinium contrast agent (Dotarem, Guerbet, Villepinte, France) (gadoteric acid; 279.3 mg/ml, 0.5 mmol/ml) was administered intraperitoneally (100 μl/20g) in the left abdominal quadrant [10 Degerman E, In 't Zandt R, Pålbrink AK, et al. Vasopressin induces endolymphatic hydrops in mouse inner ear, as evaluated with repeated 9.4 T MRI. Hear Res. 2015;330:119–124. Dec
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
,11 Degerman E, In 't Zandt R, Pålbrink A, et al. Inhibition of phosphodiesterase 3, 4, and 5 induces endolymphatic hydrops in mouse inner ear, as evaluated with repeated 9.4T MRI. Acta Otolaryngol. 2017;137:8–15.
[Taylor & Francis Online], [Web of Science ®], , [Google Scholar]
]. 55–65 min after Gadolinium administration, contrast enhanced MR images were obtained at day zero and 4 weeks after initiation of hormone/drug administration. The inhibitors and spironolactone were dissolved in DMSO/EtOH 1:1.
Magnetic Resonance Imaging. The animals were anesthetized with 3.5% isoflurane in mixture of 200 ml/min oxygen and 200 ml/min nitrous oxide and maintained at 1.5–2% isoflurane inside the magnet. The induction chamber was kept warm at 37 °C. Inside the magnet, the respiratory rate of the animal was monitored (SA Instruments Inc, New York, NY) and the body temperature was maintained using circulating warm water bath (Lauda, Lauda-Köningshofen, Germany).
MR imaging was performed as previously described [10 Degerman E, In 't Zandt R, Pålbrink AK, et al. Vasopressin induces endolymphatic hydrops in mouse inner ear, as evaluated with repeated 9.4 T MRI. Hear Res. 2015;330:119–124. Dec
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
,11 Degerman E, In 't Zandt R, Pålbrink A, et al. Inhibition of phosphodiesterase 3, 4, and 5 induces endolymphatic hydrops in mouse inner ear, as evaluated with repeated 9.4T MRI. Acta Otolaryngol. 2017;137:8–15.
[Taylor & Francis Online], [Web of Science ®], , [Google Scholar]
] with a 9.4 T magnet (Agilent Inc., Palo Alto, CA) using Avance III electronics (Bruker, Ettlingen, Germany). The system is equipped with a 12 cm inner diameter gradient system having a maximum gradient strength of 670 mT/m. The animals were imaged using a quadrature transmit/receive cryoprobe (Bruker, Ettlingen, Germany). T1-weighted 3D images were acquired with a gradient echo 3D sequence; repetition time TR: 11 ms, echo time TE: 3.695 ms, number of averages: 2, data matrix size 220 × 220 × 147 pixels, field of view 15 × 15 × 10 mm3. Images were reconstructed by zero filling to increase the apparent resolution of the image to a matrix size of 440 × 440 × 294 pixels and an apparent pixel resolution of 0.034 mm.
Quantitative assessment of gadolinium in endolymphatic relative perilymphatic space. Synedra view personal 16 (Synedra, Innsbruck, Austria) as well as Adobe Photoshop CS5 (Adobe, San Jose, CA) were used for postproduction processing of images for the quantification of signal intensity in regions of interest and for labeling and demonstration of perilymph in the scala tympani and scala vestibuli and of endolymph in the scala media.
Images parallel to the modiolus of the cochleas of mice treated as previously described were used for measurements (exemplified in Figure 1, [10 Degerman E, In 't Zandt R, Pålbrink AK, et al. Vasopressin induces endolymphatic hydrops in mouse inner ear, as evaluated with repeated 9.4 T MRI. Hear Res. 2015;330:119–124. Dec
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
,11 Degerman E, In 't Zandt R, Pålbrink A, et al. Inhibition of phosphodiesterase 3, 4, and 5 induces endolymphatic hydrops in mouse inner ear, as evaluated with repeated 9.4T MRI. Acta Otolaryngol. 2017;137:8–15.
[Taylor & Francis Online], [Web of Science ®], , [Google Scholar]
]). To evaluate the effect of various treatments on the size of the endolymphatic fluid compartment, the relative area of scala media in the basal turn of the cochlea was estimated by calculating the ratio between scala media (endolymph, non-contrast enhanced) and scala media plus scala vestibuli (perilymph, contrast enhanced). The ratio of areas is subsequently converted to percentage as shown on the figures. The observer assessing and outlining the perilymphatic and endolymphatic space, was blinded to the treatments given. One ear in each group in the vasopressin substudy was lost to analysis. In the rolipram substudy a mouse in each group was lost before the first MRI.
Figure 1. Magnetic resonance imaging and quantitative assessment of gadolinium in endolymphatic relative perilymphatic space. Sagittal section of the mouse brain showing the inner ear and the contrast enhancement in the cochlea (left). To evaluate the effect of spironolactone on EH in different contexts, the size of the endolymphatic fluid compartment, the relative area of scala media in the basal turn of the cochlea, was estimated by calculating the ratio between scala media (noncontrast enhanced, lower dotted area) and scala media + scala vestibule (scala vestibule, upper dotted area, is contrast enhanced) (right). The ratio of areas is subsequently converted to percentage as shown in Figures 2–4. SV: scala vestibule; ST: scala tympani; SM: scala media; 1st: first turn; 2nd: second turn.
Display full size
Immunohistochemistry of human saccule
Tissue sampling and preparations. Using an operating microscope, two human saccules were dissected from the vestibules during the removal of vestibular schwannomas via the translabyrinthine approach. The sample was immediately fixed in HistoChoice (Amresco) in phosphate buffer saline (PBS) for 12 h, soaked in 30% sucrose in PBS at 4 °C over night and then embedded in Tissue Tec OCT Compound (Gene Research Lab). Eight μM thick sections were cut with a cryostat and collected on slides (super-frost-plus, Fischer Scientific). Eight consecutive tissue sections were placed on each glass slide. Tissue sections were stored at –80 °C until use. The procedures were evaluated by the responsible committee at Lund University on human experimentation and were in accordance with the Helsinki Declaration of 1975, as revised in 1983.
Immunohistochemistry. Before being immunohistochemically stained, sections were thawed and allowed to air-dry for 10 min. Next, a circle surrounding each tissue section was drawn with a hydrophobic pen (Dako, Denmark). Tissue sections were covered with 5% normal goat serum diluted in PBS for 1 h (approximately 30 μl per section). Subsequently, the specimens were exposed overnight at 4 °C in a humid chamber to antibodies specific for the mineralocorticoid receptor (Thermo Fisher) and alpha ENaC (Abcam) diluted 1:75 in 5% normal goat serum (Invitrogen). At the end of the incubations, the tissue sections were rinsed in PBS (3 × 15 min). Secondary goat anti rabbit antibodies (labeled with Alexa 555) or goat anti mouse antibodies (labeled with Alexa 488) (Invitrogen, diluted 1:200), were applied to the tissue sections and incubated for 2 h at room temperature in the dark. At the end of the incubation, sections were washed with PBS and 4,6-diamidino-2-phenylindole (DAPI, Invitrogen) was added for 10 min to visualize all cell nuclei. At the end of the incubation, the sections were washed in PBS and mounted with Vectashield (Vector Laboratories). Sections were observed using a camera-equipped fluorescence microscope. Images were obtained using a Zeiss Axiophot 2 microscope and a Hamamatsu C4742-95 camera with Openlab 5 software (Improvision) for image processing.
Statistical analysis
For statistical analysis, endolymph/perilymph ratios, performed before and after treatment in the same mouse, were compared using Student’s paired t-test with p < .05 accepted as an indication of statistical significance.
Results
In a recent study, using 9.4 T field strength MRI, we demonstrated the development of EH in mice, induced by chronic administration of vasopressin and PDE3,4,5 inhibitors [10 Degerman E, In 't Zandt R, Pålbrink AK, et al. Vasopressin induces endolymphatic hydrops in mouse inner ear, as evaluated with repeated 9.4 T MRI. Hear Res. 2015;330:119–124. Dec
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
,11 Degerman E, In 't Zandt R, Pålbrink A, et al. Inhibition of phosphodiesterase 3, 4, and 5 induces endolymphatic hydrops in mouse inner ear, as evaluated with repeated 9.4T MRI. Acta Otolaryngol. 2017;137:8–15.
[Taylor & Francis Online], [Web of Science ®], , [Google Scholar]
]. Here, we tested the ability of spironolactone to prevent the development of EH, when induced by three different mechanisms, two of which affect the cAMP-mediated pathways (vasopressin and the PDE4 inhibitor rolipram) and one that also affects the cGMP-pathway (the PDE3 inhibitor cilostamide) [16 Ahmad F, Murata T, Simizu K, et al. Cyclic nucleotide phosphodiesterases: important signaling modulators and therapeutic targets. Oral Dis. 2015;21(1):e25–e50.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
]. PDE3 and PDE4 are believed to control unique pools of cAMP and cGMP [16 Ahmad F, Murata T, Simizu K, et al. Cyclic nucleotide phosphodiesterases: important signaling modulators and therapeutic targets. Oral Dis. 2015;21(1):e25–e50.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
], possibly vasopressin acts in connection with PDE4 [17 Stefan E, Wiesner B, Baillie GS, et al. Compartmentalization of cAMP-dependent signaling by phosphodiesterase-4D is involved in the regulation of vasopressin-mediated water reabsorption in renal principal cells. J Am Soc Nephrol. 2007;18:199–212.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
].
Spironolactone prevents the development of vasopressin-induced endolymphatic hydrops in mice
Vasopressin or vasopressin + spironolactone were administrated to 10 plus 10 CBA/J mice for 28 d. As shown in Figure 2 (left), vasopressin treatment resulted in increased volume of the endolymphatic space as compared to pre-vasopressin (n = 19, p = .000068). However, in the presence of spironolactone, the vasopressin effect was prevented (n = 19, p = 0.20) (Figure 2, left). In Figure 2 (middle), prevalues and postvalues are indicated for each ear and in Figure 2 (right), the pretreatment value was set to 1 for each ear.
Figure 2. Spironolactone prevents vasopressin-induced endolymphatic hydrops in CBA/J mice. CBA/J mice were treated chronically with vasopressin for 28 d with or without SL and thereafter analyzed using MRI. Quantification of endolymphatic and perilymphatic cochlear compartments before and after vasopressin (VP) and before and after vasopressin plus spironolactone (VP + SL) administration was performed as described in Material and methods and in Figure 1. Data are presented as means (left), as individual values for each ear before and after treatment (middle) and as individual values for each ear with prevalues set to 1 (right).
Display full size
Spironolactone prevents the development of rolipram-induced endolymphatic hydrops in mice
Rolipram or rolipram + spironolactone were administrated to 10 plus 10 CBA/J mice for 28 d. As shown in Figure 3 (left), rolipram treatment resulted in increased volume of the endolymphatic space as compared to pre-rolipram (n = 18, p = .0085). However, in the presence of spironolactone, the rolipram effect was prevented (n = 18, p = 0.64) (Figure 3, left). In Figure 3 (middle), prevalues and postvalues are indicated for each ear and in Figure 3 (right), the pretreatment value was set to 1 for each ear.
Figure 3. Spironolactone prevents rolipram-induced endolymphatic hydrops in CBA/J mice. CBA/J mice were treated chronically with rolipram with or without SL for 28 d and thereafter analyzed using MRI. for 28 d. Quantification of endolymphatic and perilymphatic cochlear compartments before and after rolipram (rol) and before and after rolipram plus spironolactone (rol + SL) administration was performed as described in Material and methods and in Figure 1. Data are presented as means (left), as individual values for each ear before and after treatment (middle) and as individual values for each ear with prevalues set to 1 (right).
Display full size
Spironolactone does not prevent the development of cilostamide-induced endolymphatic hydrops in mice
Cilostamide or cilostamide + spironolactone were administrated to 10 plus 10 CBA/J mice for 28 d. As shown in Figure 4 (left), cilostamide treatment resulted in increased endolymphatic space as compared to pre-cilostamide (n = 20, p = .00043). In the presence of spironolactone, the cilostamide effect was maintained (Figure 4, left) (n = 20, p = .0000021). In Figure 4 (middle), prevalues and postvalues are indicated for each ear and in Figure 4 (right), the pretreatment value was set to 1 for each ear.
Figure 4. Spironolactone does not prevent cilostamide-induced endolymphatic hydrops in CBA/J mice. CBA/J mice were treated chronically with cilostamide with or without SL for 28 d and thereafter analyzed using MRI. Quantification of endolymphatic and perilymphatic cochlear compartments before and after cilostamide (Cil) and before and after cilostamide plus spironolactone (Cil + SL) administration was performed as described in Material and methods and in Figure 1. Data are presented as means (left), as individual values for each ear before and after treatment (middle) and as individual values for each ear with prevalues set to 1 (right).
Display full size
The aldosterone target ENaC and the mineralocorticoid receptor are expressed in the human saccule sensory epithelium
As shown in Figure 5, immunostaining of ENaC, a target for aldosterone action, was detected in the apical part of the sensory epithelium of the human saccule, presumably in both hair cells and supporting cells. The mineralocorticoid receptor was detected mainly in supporting cells as indicated by co-staining with the supporting cell marker glial acidic fibrillary protein (GFAP).
Figure 5. Pathway-dependent effect of spironolactone on the prevention of endolymphatic hydrops-working hypothesis. SL prevents EH induced by vasopressin believed to act via cAMP/protein kinas A mediated phosphorylation and translocation of the fluid transporter aqp2 to regulate inner ear functions. It is possible that PDE4 regulates the pool of cAMP utilized by vasopressin since (a) PDE4 interacts with aqp2 in kidney cells (b) PDE4 is expressed in the inner ear and (c) SL prevents hydrops induced by the PDE4 inhibitor rolipram. On the other hand, SL does not prevent hydrops induced by the PDE3 inhibitor cilostamide indicating that different pools of cyclic nucleotides are involved in hydrops induction in this model, cAMPPDE4, cAMPPDE3 and cGMPPDE3. Inhibition of aldosterone action could interfere with the vasopressin/PDE4 signaling systems at different levels. Since SL prevents rolipram-induced hydrops, one level would be downstream of cAMPPDE4 production. Mechanisms mediating cilostamide induced EH remain to be elucidated, however, most likely involve NO/cGMP as well as cAMP down-stream targets. Target proteins for the signaling systems described, of relevance for EH, presumably involve fluid and ion transporters such as aqp2, ENaC, NCC and NaKATPase. A key target for aldosterone action, ENaC and the mineralocorticoid receptor were expressed in the human saccule. Tissue sections were immunohistochemically stained with indicated antibodies. Merged photos of MR, DAPI (nuclear label) and GAFP (upper, left), merged photo of MR and DAPI (middle, left), merged photo of MR and GAFP (lower, left), merged photo with ENaC, DAPI and GAFP (right). NKA: NaKATPase; MR: mineralocorticid receptor; GAFP: glial acidic fibrillary protein; dapi stains nuclei; sensory epithelium, bar with two arrow heads.
Display full size
Discussion
Here, we present beneficial effects of the aldosterone antagonist spironolactone on EH hydrops induced by vasopressin and the PDE4 inhibitor rolipram. Increased action or sensitivity of vasopressin in the inner ear is believed to contribute to the development of EH via enhanced effects on aqp2, an important target for cAMP/PKA signaling [4 Takeda T, Takeda S, Kakigi A, et al. Hormonal aspects of Ménière’s disease on the basis of clinical and experimental studies. ORL J Otorhinolaryngol Relat Spec. 2010;71:1–9.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
]. In the kidney, aqp2 has been shown to connect to PDE4, a cAMP specific phosphodiesterase [17 Stefan E, Wiesner B, Baillie GS, et al. Compartmentalization of cAMP-dependent signaling by phosphodiesterase-4D is involved in the regulation of vasopressin-mediated water reabsorption in renal principal cells. J Am Soc Nephrol. 2007;18:199–212.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
]. It is, thus, possible that the ability of spironolactone to antagonize vasopressin induced EH is by interfering with a cAMP pool controlled by PDE4. In agreement with a connection between vasopressin and PDE4, hydrops induced by the PDE4 inhibitor rolipram, was also antagonized by spironolactone. The finding that EH induced by cilostamide, a PDE3 inhibitor hydrolyzing both cAMP and cGMP, was not prevented by spironolactone indicate that there should be different mechanisms for the development of EH. This is in agreement with the shown compartmentalization of cyclic nucleotides [16 Ahmad F, Murata T, Simizu K, et al. Cyclic nucleotide phosphodiesterases: important signaling modulators and therapeutic targets. Oral Dis. 2015;21(1):e25–e50.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
].
Indeed, cAMP and cGMP are believed to be tightly compartmentalized by the action of PDEs, belonging to presently defined 12 families, PDE1-12 [16 Ahmad F, Murata T, Simizu K, et al. Cyclic nucleotide phosphodiesterases: important signaling modulators and therapeutic targets. Oral Dis. 2015;21(1):e25–e50.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
]. We have previously shown the expression of PDE3 and PDE4 in the human inner ear and PDEs have been implicated in hearing dysfunction [9 Mittal R, Bencie N, Shaikh N, et al. Role of cyclic nucleotide phosphodiesterases in inner ear and hearing. Front Physiol. 2017;8:1–3.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
,11 Degerman E, In 't Zandt R, Pålbrink A, et al. Inhibition of phosphodiesterase 3, 4, and 5 induces endolymphatic hydrops in mouse inner ear, as evaluated with repeated 9.4T MRI. Acta Otolaryngol. 2017;137:8–15.
[Taylor & Francis Online], [Web of Science ®], , [Google Scholar]
]. Due to their distinct intrinsic characteristics and their intracellular targeting to different subcellular locations, different PDEs integrate multiple cellular inputs and modulate the amplitude, duration, termination and specificity of cAMP and cGMP signaling and actions. Thus, the cAMP/cGMP pools controlled by PDE3, appear not to cross-talk with spironolactone action which is the case for the cAMP-pool utilized by vasopressin and PDE4. Exactly how spironolactone interferes with cAMP signaling is not known. An interplay between cAMP and the mineralocorticoid receptor signaling pathways has been described, please see review and references herein [18 Hermidorff MM, de Assis LV, Isoldi MC. Genomic and rapid effects of aldosterone: what we know and do not know thus far. Heart Fail Rev. 2017;22:65–89.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
].
Although, our results indicate beneficial effects of spironolactone on the inner ear, contradictory results have been published with regards to effects of aldosterone and spironolactone on EH [12–15 Salt AN, Plontke SK. Endolymphatic hydrops: pathophysiology and experimental models. Otolaryngol Clin North Am. 2010;43:971–983.
Qin L, Zhang B, Wang Q, et al. Effect of aldosterone on cochlear Af9 expression and hearing in guinea pig. Acta Otolaryngol. 2017;137:903–909.
Gross ND, Kempton JB, Trune DR. Spironolactone blocks glucocorticoid-mediated hearing preservation in autoimmune mice. Laryngoscope. 2002;112:298–303.
Mori N, Miyashita T, Inamoto R, et al. Ion transport its regulation in the endolymphatic sac: suggestions for clinical aspects of Meniere's disease. Eur Arch Otorhinolaryngol. 2017;274:1813–1820.
]. In agreement with our results aldosterone has been reported to induce or worsen EH in different animal models with or without other EH inducing factors such as lipopolysaccharide and sac ablation, for review see [12 Salt AN, Plontke SK. Endolymphatic hydrops: pathophysiology and experimental models. Otolaryngol Clin North Am. 2010;43:971–983.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
]. On the other hand, aldosterone was reported to have beneficial effects on hearing preservation via the mineral corticoid receptor in the MRL/MpJ-Faslpr autoimmune disease mouse, an established model of spontaneous autoimmune sensorineural hearing loss [14 Gross ND, Kempton JB, Trune DR. Spironolactone blocks glucocorticoid-mediated hearing preservation in autoimmune mice. Laryngoscope. 2002;112:298–303.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
]. In some clinical studies, a low salt diet was advocated to be an effective treatment of Menière’s disease [15 Mori N, Miyashita T, Inamoto R, et al. Ion transport its regulation in the endolymphatic sac: suggestions for clinical aspects of Meniere's disease. Eur Arch Otorhinolaryngol. 2017;274:1813–1820.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
] which was actually correlated with increased aldosterone levels. On the other hand, diuretics are implicated in the treatment of Menière’s disease, although, evidence to support their efficacy is lacking [3 Magnan J, Özgirgin ON, Trabalzini F, et al. European position statement on diagnosis, and treatment of Meniere's disease. Int Adv Otol. 2018;14:317–321.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
].
Thus, different models, read-outs, methods, doses of aldosterone and study designs have been used in the context of spironolactone, aldosterone and the inner ear which may explain some of the contradictory results. One should have in mind that aldosterone is believed to act via the mineralocorticoid receptor as well as via cell surface receptors [18 Hermidorff MM, de Assis LV, Isoldi MC. Genomic and rapid effects of aldosterone: what we know and do not know thus far. Heart Fail Rev. 2017;22:65–89.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
] whereas spironolactone is believed to antagonize the effects induced by the mineralocorticoid receptor only. The exact arrangement and levels of key targets for aldosterone such as the amiloride sensitive Na + transporter ENaC, NCC and the NaK-ATPase in the inner ear presumably contribute to the impact of altered aldosterone level in different contexts in one or the other direction [19 Eckhard AH, Zhu M, O'Malley JT, et al. Inner ear pathologies impair sodium-regulated ion transport in Meniere's disease. Acta Neuropathol. 2019;137:343–357.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
]. Nevertheless, impaired Na + transport in the endolymphatic sac has been associated with Menière’s disease [15 Mori N, Miyashita T, Inamoto R, et al. Ion transport its regulation in the endolymphatic sac: suggestions for clinical aspects of Meniere's disease. Eur Arch Otorhinolaryngol. 2017;274:1813–1820.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
,20 Mittal R, Aranke M, Debs LH, et al. Indispensable role of ion channels and transporters in the auditory system. J Cell Physiol. 2017;232:743–758.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
].
From a clinical point of view the above results raise interesting perspectives. If the hydrops is a fundamental part of the development of pathology in Menière’s disease and hydrops may be developed due to more than one mechanism, the effect of treatment with diuretics or for that matter any other suggested treatments, could be dependent on if patients had hydrops developed from the mechanism that is targeted by the treatment. As this is not known, a cohort of patients may consist of any proportion of different etiologies to hydrops and treatment effects may be very difficult to ascertain. This points out the need for personalized medicine in Menière’s disease, as well as a difficulty in designing intervention studies.
Conclusions
We show that the aldosterone antagonist spironolactone prevents hydrops induced by vasopressin and the PDE4 inhibitor rolipram but not hydrops induced by the PDE3 inhibitor cilostamide in agreement with compartmentalization of cyclic nucleotides [16 Ahmad F, Murata T, Simizu K, et al. Cyclic nucleotide phosphodiesterases: important signaling modulators and therapeutic targets. Oral Dis. 2015;21(1):e25–e50.
[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
]. Thus, our study actually demonstrates that spironolactone is beneficial in one context but not in another which is in agreement with the contradictory results obtained in the context of aldosterone, spironolactone and the inner ear [12–15 Salt AN, Plontke SK. Endolymphatic hydrops: pathophysiology and experimental models. Otolaryngol Clin North Am. 2010;43:971–983.
Qin L, Zhang B, Wang Q, et al. Effect of aldosterone on cochlear Af9 expression and hearing in guinea pig. Acta Otolaryngol. 2017;137:903–909.
Gross ND, Kempton JB, Trune DR. Spironolactone blocks glucocorticoid-mediated hearing preservation in autoimmune mice. Laryngoscope. 2002;112:298–303.
Mori N, Miyashita T, Inamoto R, et al. Ion transport its regulation in the endolymphatic sac: suggestions for clinical aspects of Meniere's disease. Eur Arch Otorhinolaryngol. 2017;274:1813–1820.
]. Extrapolating this finding to the clinic, suggests that a personalized medicine approach is probably necessary in the treatment of diseases involving EH, such as Menière’s disease.
Acknowledgements
Lund University Bioimaging Center (LBIC) is gratefully acknowledged for providing experimental resources. ED and AP are affiliated with Lund University Diabetes Center (LUDC).
Disclosure statement
No potential conflict of interest was reported by the authors.
References
Oberman BS, Patel VA, Cureoglu S, et al. The aetiopathologies of Ménière's disease: a contemporary review. Acta Otorhinolaryngol Ital. 2017;37:250–263.[PubMed], [Web of Science ®], , [Google Scholar]
Lopez-Escamez JA, Carey J, Chung WH, et al. Classification Committee of the Barany Society; Japan Society for Equilibrium Research; European Academy of Otology and Neurotology (EAONO); Equilibrium Committee of the American Academy of Otolaryngology-Head and Neck Surgery (AAO-HNS); Korean Balance Society. Diagnostic criteria for Menière's disease. J Vestib Res. 2015;25:1–7.[PubMed], [Web of Science ®], , [Google Scholar]
Magnan J, Özgirgin ON, Trabalzini F, et al. European position statement on diagnosis, and treatment of Meniere's disease. Int Adv Otol. 2018;14:317–321.[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
Takeda T, Takeda S, Kakigi A, et al. Hormonal aspects of Ménière’s disease on the basis of clinical and experimental studies. ORL J Otorhinolaryngol Relat Spec. 2010;71:1–9.[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
Katagiri Y, Takumida M, Hirakawa K, et al. Long-term administration of vasopressin can cause Ménière's disease in mice. Acta Otolaryngol. 2014;134:990–1004.[Taylor & Francis Online], [Web of Science ®], , [Google Scholar]
Wangemann P. K(+) cycling and its regulation in the cochlea and the vestibular labyrinth. Audiol Neurootol. 2002;7:199–205.[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
Kim BG, Kim JY, Jung J, et al. β1- and β2-adrenergic stimulation-induced electrogenic transport by human endolymphatic sac epithelium and its clinical implications. Sci Rep. 2017;7:42217.[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
Fessenden JD, Schacht J. The nitric oxide/cyclic GMP pathway: a potential major regulator of cochlear physiology. Hear Res. 1998;118:168–176.[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
Mittal R, Bencie N, Shaikh N, et al. Role of cyclic nucleotide phosphodiesterases in inner ear and hearing. Front Physiol. 2017;8:1–3.[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
Degerman E, In 't Zandt R, Pålbrink AK, et al. Vasopressin induces endolymphatic hydrops in mouse inner ear, as evaluated with repeated 9.4 T MRI. Hear Res. 2015;330:119–124. Dec[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
Degerman E, In 't Zandt R, Pålbrink A, et al. Inhibition of phosphodiesterase 3, 4, and 5 induces endolymphatic hydrops in mouse inner ear, as evaluated with repeated 9.4T MRI. Acta Otolaryngol. 2017;137:8–15.[Taylor & Francis Online], [Web of Science ®], , [Google Scholar]
Salt AN, Plontke SK. Endolymphatic hydrops: pathophysiology and experimental models. Otolaryngol Clin North Am. 2010;43:971–983.[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
Qin L, Zhang B, Wang Q, et al. Effect of aldosterone on cochlear Af9 expression and hearing in guinea pig. Acta Otolaryngol. 2017;137:903–909.[Taylor & Francis Online], [Web of Science ®], , [Google Scholar]
Gross ND, Kempton JB, Trune DR. Spironolactone blocks glucocorticoid-mediated hearing preservation in autoimmune mice. Laryngoscope. 2002;112:298–303.[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
Mori N, Miyashita T, Inamoto R, et al. Ion transport its regulation in the endolymphatic sac: suggestions for clinical aspects of Meniere's disease. Eur Arch Otorhinolaryngol. 2017;274:1813–1820.[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
Ahmad F, Murata T, Simizu K, et al. Cyclic nucleotide phosphodiesterases: important signaling modulators and therapeutic targets. Oral Dis. 2015;21(1):e25–e50.[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
Stefan E, Wiesner B, Baillie GS, et al. Compartmentalization of cAMP-dependent signaling by phosphodiesterase-4D is involved in the regulation of vasopressin-mediated water reabsorption in renal principal cells. J Am Soc Nephrol. 2007;18:199–212.[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
Hermidorff MM, de Assis LV, Isoldi MC. Genomic and rapid effects of aldosterone: what we know and do not know thus far. Heart Fail Rev. 2017;22:65–89.[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
Eckhard AH, Zhu M, O'Malley JT, et al. Inner ear pathologies impair sodium-regulated ion transport in Meniere's disease. Acta Neuropathol. 2019;137:343–357.[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
Mittal R, Aranke M, Debs LH, et al. Indispensable role of ion channels and transporters in the auditory system. J Cell Physiol. 2017;232:743–758.[Crossref], [PubMed], [Web of Science ®], , [Google Scholar]
Additional information
Funding
The work was supported by grants from the Swedish Diabetes Foundation, the Swedish Foundation for Strategic Research Dnr IRC 15-0067, the Albert Påhlsson Foundation, Sweden, the Crafoord Foundation, Sweden, Diabetes Wellness Sweden, Skane County Council’s Research and Development Foundation, Sweden and the Medical Faculty, Lund University, Sweden.
Δεν υπάρχουν σχόλια:
Δημοσίευση σχολίου