<div>Expression of AQP8 in serum of patients with Meniere’s disease and its
value in evaluating the degree of hydrolabyrinth and predicting
prognosis</div><div>Abstract</div><div>Objective To explore the value of serum aquaporin 8 (AQP8) expression in
evaluating the degree of hydrolabyrinth and predicting prognosis in
patients with Meniere’s disease.</div><div>Methods 105 patients with Meniere’s disease in our hospital were
included in the Meniere’s disease group.Another 102 healthy subjects in
our hospital were included as the control group. The multivariate
Logistic regression was used to analyze the influencing factors of poor
prognosis in patients with Meniere’s disease.</div><div>Results The expression of serum AQP8 mRNA in the Meniere’s disease group
was greatly higher than that in the control group
(<i>P</i> &lt;0.05). Serum AQP8 mRNA expression in severe hydrops
group was greatly higher than that in mild hydrops group and no
endolymphatic hydrops group, and serum AQP8 mRNA expression in mild
hydrops group was greatly higher than that in no endolymphatic hydrops
group (<i>P</i> &lt;0.05). The course of disease, the proportion
of severe hydrops and the expression of serum AQP8 mRNA in the poor
prognosis group were greatly higher than those in the good prognosis
group (<i>P</i> &lt;0.05). The area under the curve (AUC) of serum
AQP8 mRNA for predicting poor prognosis in Meniere’s disease patients
was 0.812 (95%<i>CI:</i> 0.702-0.922), with a sensitivity of 81.0% and
a specificity of 70.2%. AQP8 mRNA was an independent risk factor for
poor prognosis in patients with Meniere’s disease
(<i>P</i> &lt;0.05).</div><div>Conclusion AQP8 mRNA is associated with the degree of hydrolabyrinth in
patients with Meniere’s disease, and has a high performance in
predicting prognosis.</div><div>Key words Meniere’s disease; Aquaporin 8; Degree of hydrolabyrinth;
Prognosis; Serum</div><div>key points</div><ul><li>Serum AQP8 is highly expressed in Meniere’s disease patients</li><li>The high expression of serum AQP8 in patients with Meniere’s disease
is closely related to the aggravation of endolymphatic hydrops</li><li>AQP8 mRNA was an independent risk factor in poor prognosis of
Meniere’s patients</li><li>AQP8 expression has important predictive value in poor prognosis of
Meniere’s disease, which features high predictive sensitivity and
specificity</li><li>AQP8 mRNA is associated with the degree of hydrolabyrinth in patients
with Meniere’s disease</li></ul><div>1 Introduction</div><div>Meniere’s disease is a clinical syndrome characterized by recurrent
episodes of spontaneous vertigo, unilateral fluctuating sensorineural
hearing loss, tinnitus, and sufficient hearing <sup>1,2</sup>.
Endolymphatic hydrops is considered as the pathophysiological basis of
the disease, which has been confirmed in anatomical pathology studies
and recent magnetic resonance imaging (MRI) <sup>3,4</sup>.
However, MRI is not appropriate for repeated detections, and there are
certain risks in detection. Also, the image quality will be affected by
the dose of contrast agent. In clinical practice, it is necessary to
find relevant auxiliary detection indexes and then improve the accuracy
in Meniere’s disease assessment. Aquaporin (AQP) is a transmembrane
protein originally identified as a water channel with the ability to
regulate the transport of water, glycerin, urea and other small
molecules <sup>5</sup>. Recent studies have shown that, AQP plays
an important role in the treatment of liver cirrhosis, heart failure,
Meniere’s disease, cancer, bullous pemphigoid, eczema, and Sjogren
syndrome <sup>5</sup>. Considering the lack of laboratory indexes
for Meniere’s disease, this study detects the expression of serum AQP8
mRNA in Meniere’s disease patients, preliminarily discusses the value of
serum AQP8 mRNA in evaluating the degree of endolymphatic hydrops in
Meniere’s disease and predicting its prognosis.</div><div>2 Research objects and methods</div><div>2.1 Research objects</div><div>From February 2016 to April 2022, 105 Meniere’s disease patients treated
in our hospital were included in the Meniere’s disease group. The
patients were 40~73 years old, with an average age of
(60.32±8.34) years old, including 40 males and 65 females. The gender,
age, body mass index, smoking history, drinking history, hypertension,
hyperlipidemia, diabetes, disease site and disease course were collected
for Meniere’s disease patients. A total of 102 healthy subjects were
included in the control group, ranging in age from 40 to 75 years old,
with an average age of (60.45±7.96) years old and including 36 males and
66 females.</div><div>Inclusion criteria: ① The Meniere’s disease patients were diagnosed
based on the relevant diagnostic criteria formulated by the European
Society of Otoneurology and the American Society of Otolaryngology Head
and Neck Surgery<sup>6</sup>; ② The study complies with ethical
standards, the patient follow-up data is complete, the patients and
their families can cooperate with the treatment and follow-up; ③The
control group has no abnormal vestibular function in various hearing
tests, no Meniere’s disease and other vertigo diseases. Exclusion
criteria: ① Patients with vestibular neuritis and other vertigo
diseases; ② Patients previously with chronic diseases such as otitis
media; ③ Patients with cerebrovascular disease, acoustic neuroma, and
sudden deafness. The study follows transparent reporting of a
multivariable prediction model for individual prognosis or diagnosis
(TRIPOD): The TRIPOD statement</div><div>2.2 Methods</div><div>2.2.1 Determination of AQP8 mRNA expression level in serum</div><div>10 mL peripheral venous blood samples were collected from Meniere’s
disease patients before treatment, placed in a dry centrifuge tube, left
standing at room temperature for 30 min, and centrifuged at 3000 r/min
for 15 min at room temperature to collect the upper serum, and stored at
-80 °C. After freeze-thaw on ice, RNA extraction kit (Beijing Yita
Biotech Co., Ltd., article number: YT9197) was used to isolate and
extract total serum RNA. After testing its concentration, purity and
integrity, the first-strand cDNA synthesis kit (Norgen Biotek, Canada,
article number: NGB-54420) was used for reverse transcription into cDNA,
and the operation was carried out in strict accordance with the
instructions. Serum AQP8 mRNA expression was determined by V115896
quantitative real-time PCR (qRT-PCR) instrument produced by Applied
Biosystems, the United States, and the internal reference was GAPDH.
Loading system: cDNA (50 ng/μL) 1 μL, 2×SYBR Green qPCR Master Mix
(Beijing Bairuiji Biotechnology Co., Ltd., article number:
BN12014-20μL×100T) 10 μL, upstream and downstream primers 0.5 μL each,
ddH<sub>2</sub>O 8 μL. Reaction procedure: pre-denaturation at 95
°C for 1 min; denaturation at 95 °C for 30 s, annealing at 60 °C for 30
s, extension at 72 °C for 30 s, a total of 45 cycles. For the reaction
results, the relative expression of AQP8 was statistically calculated by
the 2<sup>-∆∆CT</sup> method. The primer sequences are shown in
Table 1.</div><div>2.2.2 The degree of endolymphatic hydrops in Meniere’s disease,
evaluation and grouping of poor prognosis</div><div>All Meniere’s disease patients underwent inner ear scans using MRI
technology (the instrument was a 3.0T Magnetom Verio scanner produced by
Siemens, Germany) to evaluate the degree of endolymphatic hydrops. The
degree of endolymphatic hydrops was divided into grade 0, without
vestibular membrane displacement and endolymphatic hydrops
(non-endolymphatic hydrops group, 29 cases); grade 1, with mild
endolymphatic hydrops and vestibular membrane displacement, but spatium
endolymphaticum area is smaller than the vestibular canal area (mild
hydrops group, 48 cases); grade 2, with severe endolymphatic hydrops,
vestibular membrane displacement and spatium endolymphaticum area is
greater than the vestibular canal area (severe hydrops group, 28 cases).
The Meniere’s disease patients were evaluated after 3 months of
treatment. If the symptoms tinnitus and dizziness were not significantly
improved, the speech recognition rate increased by &lt;5%, and
the pure tone audiometry improved by &lt;15 dB, the prognosis was
poor (poor prognosis group, 21 cases). Otherwise, the prognosis was good
(good prognosis group, 84 cases).</div><div>2.3 Statistical processing</div><div>All data were processed using SPSS 25.0. The enumeration data was
expressed by n (%), and tested by; the measurement data obeyed the
normal distribution, and was expressed by (± ). The two groups were
subjected to independent sample t test, the three groups were subjected
to one-way analysis of variance, and SNK-q test was used for further
comparison between the two groups. Receiver operator characteristic
(ROC) curve analysis was used to analyze the predictive value of serum
AQP8 mRNA expression for poor prognosis in Meniere’s disease patients;
Multivariate Logistic regression was used to analyze the factors
influencing poor prognosis in Meniere’s disease patients. <i>P</i>&lt; 0.05 indicates that the difference is statistically
significant.</div><div>3 Results</div><div>3.1 Comparison of serum AQP8 mRNA expression in control group and
Meniere’s disease group The serum AQP8 mRNA expression was significantly
higher in the Meniere’s disease group than in the control group
(<i>P</i> &lt;0.05). See Table 2.</div><div>3.2 Comparison of serum AQP8 mRNA expression in Meniere’s disease
patients with different degrees of endolymphatic hydrops Serum AQP8 mRNA
expression was significantly higher in severe hydrops group than in mild
hydrops group and non-endolymphatic hydrops group. See Table 3.</div><div>3.3 Comparison of general data and serum AQP8 mRNA expression between
the good prognosis group and the poor prognosis group Differences in
gender, age, body mass index, smoking history, drinking history,
hypertension, hyperlipidemia, diabetes, and disease site between the two
groups were not statistically significant (<i>P</i> &gt;0.05).
The disease course, the proportion of severe hydrops and the expression
of serum AQP8 mRNA were significantly higher in the poor prognosis group
than in the good prognosis group (<i>P</i> &lt;0.05). See Table 4.</div><div>3.4 Predictive value of serum AQP8 mRNA expression for poor prognosis in
Meniere’s disease patients</div><div>Using serum AQP8 mRNA as the detection variable, and using poor
prognosis as the state variable, the ROC curve was plotted to analyze
the predictive value of serum AQP8 mRNA expression in the poor prognosis
of Meniere’s disease patients. The results
showed that, in prediction of poor prognosis of Meniere’s disease
patients based on serum AQP8 mRNA, the area under the curve (AUC) was
0.812 (95%CI: 0.702-0.922), the critical value was 2.52, the
sensitivity was 81.0%, and the specificity was 70.2%. See Figure 1.</div><div>3.5 Multivariate Logistic regression analysis of poor prognosis in
Meniere’s disease patients Using poor prognosis of Meniere’s disease
patients after 3 months of treatment as the dependent variable, and
using AQP8 mRNA, disease course, and endolymphatic hydrops degree as
independent variables, a multivariate Logistic regression analysis was
performed. The results showed that, AQP8 mRNA was an independent risk
factor in poor prognosis of Meniere’s patients (P &lt; 0.05). See
Table 5.</div><div>4 Discussion</div><div>In 2015, European Society of Otoneurology developed guidelines for the
diagnosis of Meniere’s disease. Meniere’s disease patients should have
the following symptoms: two or more spontaneous vertigo episodes
(lasting 20 min to 12 h); mid-low frequency sensorineural hearing loss
in one ear in audiometry records; fluctuating auditory symptoms
(tinnitus or fullness) in the ear. The standardized definition marks an
important research milestone for clinicians and researchers<sup>6,7</sup>. However, the above criteria are subjective or
based on subjective hearing tests. Due to the lack of objective criteria
for Meniere’s disease, its diagnosis and disease assessment is sometimes
controversial or unclear.</div><div>Traditionally, endolymphatic hydrops has been viewed as an objective
histopathological evidence of Meniere’s disease, but histopathology can
only be performed after death and cannot be used to evaluate patients
with episodes of vertigo. The development of active Meniere’s disease
and its time gap from autopsy assessments limit our understanding of
disease progression<sup>8,9</sup>. Many otologic biomarkers are
currently used to differentiate Meniere’s disease from vestibular
migraine and assess the progression of Meniere’s
disease<sup>10,11</sup>. AQP is transmembrane water channel that
affects electrolyte balance and thus plays a central role in the
regulation of transcellular water flux <sup>12,13</sup>. Previous
studies have shown that, the underlying pathological mechanisms leading
to Meniere’s disease may include genetics, blood vessel, immunity,
abnormal AQP expression, or a combination of multiple factors<sup>10</sup>. Mom et al.<sup>14</sup>showed that
dexamethasone can affect inner ear water flux in Meniere’s disease
patients by targeting AQP2. Asmar et al. <sup>15</sup>showed that
the expression of AQP2 was up-regulated in the endolymphatic sac of
Meniere’s disease patients and involved in the regulation of endolymph
volume. It is speculated that AQP8 is also closely related to Meniere’s
disease in this study, which may be involved in the occurrence and
development of Meniere’s disease.</div><div>The results of this study showed that, serum AQP8 was highly expressed
in Meniere’s disease patients, and its expression increased with the
aggravation of endolymphatic hydrops. It suggests that high expression
of AQP8 may promote the occurrence and development of Meniere’s disease.
According to the AQP regulation of transcellular water flux, it is
speculated that the up-regulation of AQP8 expression may disrupt the
electrolyte balance, affect the permeability of endolymphatic sac
epithelium against water, which harms absorption of endolymph, results
in retention of endolymph, aggravates the degree of endolymphatic
hydrops. The ROC curve analysis results showed that, AQP8 has a high
predictive value in the prognosis of Meniere’s disease, indicating that
AQP8 may be used as a potential auxiliary biomarker for poor prognosis
of Meniere’s disease. In clinical practice, prognosis of Meniere’s
disease can be preliminarily predicted via serum AQP8 detection level,
thus making it possible to better identify high-risk groups with poor
prognosis. Close monitoring and special care should be given to patients
with AQP8 expression above 2.52, so that the clinical treatment plan can
be adjusted in a timely manner. The multi-factor results showed that,
compared with factors such as disease course, degree of endolymphatic
hydrops, etc. the elevated AQP8 expression was more closely related to
the poor prognosis of Meniere’s disease, suggesting that AQP8 could be
used as a biological therapy target for Meniere’s disease for further
in-depth research in clinical practice.</div><div>To conclude, serum AQP8 is highly expressed in Meniere’s disease
patients, and the increased expression is closely related to the
aggravation of endolymphatic hydrops. AQP8 expression has important
predictive value in poor prognosis of Meniere’s disease, which features
high predictive sensitivity and specificity. This study provides a
theoretical basis for the targeted therapy of Meniere’s disease. There
will be more in-depth and flexible application of AQP8 in research of
Meniere’s disease and related diseases in the future. However, this
study has the following limitations: endolymphatic hydrops has not been
confirmed by pathological results, and no histopathological evidence has
been found; the sample size is small, and the optimal critical value of
serum AQP8 still needs to be further optimized for prognosis prediction.</div><div>References</div><div>1. Desiato VM, Patel JJ, Nguyen SA, Meyer TA, Lambert PR. Cochlear
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prognosis in patients with Meniere’s disease</div><div>Table 1 Primer sequence</div>