Abstract
Background
Idiopathic intracranial hypertension is characterized by increased intracranial pressure. Its pathogenesis is largely unknown. Aquaporins may play a role in the homeostasis of cerebrospinal fluid.
Methods
We aimed to elucidate the role of aquaporins in idiopathic intracranial hypertension by measuring the level of aquaporin-1 and aquaporin-4 in the cerebrospinal fluid and plasma of 28 patients and 29 controls by enzyme-linked immunosorbent assay. The adipokines leptin and retinol-binding protein 4 were also measured.
Results
We found a reduction in aquaporin-4 in the cerebrospinal fluid of patients. Leptin levels were increased in the cerebrospinal fluid and plasma of patients and were correlated with weight, body mass index and body fat. There was no difference between patients and controls in the levels of aquaporin-4 and retinol-binding protein 4.
Conclusion
Our data suggest that an imbalance of aquaporin-4 in the cerebrospinal fluid of patients with idiopathic intracranial hypertension may contribute to the pathogenesis of this disorder.
Keywords
Introduction
Idiopathic intracranial hypertension (IIH) is a disorder with increased intracranial pressure; it mostly affects obese young women. Patients experience headaches and visual disturbances that may even result in visual loss. The pathogenesis is still unclear. Vitamin A (retinol) intoxication can cause secondary intracranial hypertension and defects of vitamin A metabolism have been hypothesized to be involved in the pathogenesis of IIH (1). A reduction in retinol-binding protein (RBP), an adipokine that is a transport protein of vitamin A, has been detected in the cerebrospinal fluid (CSF) of patients with IIH (2). Leptin, another adipokine, was reported to be increased in the CSF of patients with IIH (3). Aquaporins might provide a link between adipokines and increased intracranial pressure as they crucially contribute to water transport in brain tissue and the expression of aquaporin-1 is induced by retinol and leptin (4,5). However, the role of aquaporins in the pathogenesis of IIH is controversial (6). Aquaporins are involved in the development of cerebral oedema and there is no convincing evidence of cerebral oedema in patients with IIH (6). Venous hypertension is thought to play a role in the pathogenesis of IIH rather than cerebral oedema (6). Several studies (7–9) have focused on the role of aquaporins in the pathogenesis of IIH and have looked for autoantibodies against aquaporin-4 in IIH and for single nucleotide polymorphisms in the aquaporin-4 gene, without any positive results. We aimed to investigate the role of aquaporins in association with leptin and RBP-4 in the CSF of patients with IIH by measuring CSF and plasma levels of aquaporin-1, aquaporin-4, leptin and RBP-4.
Methods
Patients
Demographic data and mean levels of aquaporin-1, aquaporin-4, leptin and retinol-binding protein-4 in cerebrospinal fluid and plasma.
Data are presented as mean ± SD or mean (range) values.
BMI: body mass index; CSF: cerebrospinal fluid; IIH: idiopathic intracranial hypertension; RBP-4: retinol-binding protein 4.
Enzyme-linked immunosorbent assays
Plasma and CSF leptin and RBP-4 levels were measured by human Quantikine RBP-4 and leptin enzyme-linked immunosorbent assay (ELISA) kits (sensitivity: RBP-4, 0.628 ng/ml; leptin, 7.8 pg/ml; R&D, Minneapolis, MN, USA). ELISA kits for aquaporin-1 and aquaporin-4 were purchased from USCN (sensitivity: aquaporin-1, 0.11 ng/ml; aquaporin-4, 27.9 pg/ml; USCN, Wuhan, China). All ELISAs were conducted according to the manufacturers’ instructions. All samples were measured in duplicate. Plasma samples were diluted 1:1000 in the RBP-4 ELISA, 1:100 in the aquaporin-4 and leptin ELISA and 1:10 in the aquaporin-1 ELISA. CSF samples were diluted 1:10 in the aquaporin-4 ELISA and were used undiluted in the other ELISAs. Protein levels were measured as part of the routine diagnostic work-up. The CSF protein levels in the patients ranged from 0.176 to 0.793 g/l (mean 0.27 g/l, SD 0.131 mg/dl) and the plasma levels from 59 to 78 g/l (mean 72 g/l, SD 5.0 g/dl). The CSF protein levels of the controls ranged from 0.179 to 0.539 g/l (mean 0.332 g/l, SD 0.102 g/l) and the plasma levels from 63 to 78 g/l (mean 69 g/l, SD 4 g/l).
Statistical analysis
Statistical analysis was performed using SPSS 22 software (IBM, Armonk, NY, USA). The Mann–Whitney U-test was used for the comparison of continuous data. Categorical data were compared using a two-sided Fisher’s exact test. For correlation analysis, the bivariate Spearman correlation was used. A significance level of 5% was applied in all tests.
Results
Twenty-eight patients with a diagnosis of IIH and 29 controls were included in the study. Table 1 summarizes the demographic data. Patients with IIH were overweight with a mean BMI of 32.6 compared with 26.4 in the controls (p < 0.001) and a waist to hip ratio of 0.88 compared with 0.87 in the controls (p = 0.58). On the day of inclusion in the study, the patients reported the following signs and symptoms: headache (17), visual loss (seven), blurred vision (six), oculomotor symptoms (five), papilloedema (four), nausea/vomiting (three), vertigo (one) and tinnitus (one). Nine patients were asymptomatic at the day of inclusion. Eighteen patients were being treated with acetazolamide and three with topiramate.
In patients with IIH, the aquaporin-4 CSF to plasma ratio was reduced (p < 0.001) compared with the controls and the aquaporin-4 CSF levels were decreased (p = 0.032), whereas the aquaporin-4 plasma levels were slightly increased in the patients (p = 0.045) (Figure 1a and 1b; Table 1). We did not detect any difference in the aquaporin-1 CSF or plasma levels between the patients and controls (CSF, p = 0.11; plasma, p = 0.66) (Figure 1c and 1d; Table 1). The RBP-4 CSF and plasma levels did not differ between patients and controls (CSF, p = 0.45; plasma, p = 0.29) (Figure 1e and 1f; Table 1). CSF and plasma leptin levels were elevated in patients compared with controls (p < 0.001) (Figure 1g and 1h; Table 1), but the CSF to plasma ratio did not differ (p = 0.10) (Figure 1; Table 1). CSF and plasma leptin levels were correlated with weight (CSF, ρ = 0.38, p = 0.007; plasma, ρ = 0.612, p < 0.0001; Table 1), BMI (CSF, ρ = 0.486, p < 0.0001; plasma, ρ = 0.680, p < 0.0001; Table 1, Figure 2) and body fat (CSF, ρ = 0.378, p = 0.007; plasma, ρ = 0.612, p < 0.0001; Table 1). The scatter blot indicates that the elevated leptin levels were an effect of body weight/fat/BMI rather than of disease (Figure 2). There was no correlation between aquaporin-1, aquaporin-4, RBP-4 and weight, BMI or body fat. Aquaporin, RBP-4 and leptin levels did not differ between patients treated with acetazolamide and untreated patients.
Box plots of cerebrospinal fluid (CSF) and plasma levels of the measured parameters. CSF (a, c, e, g) and plasma levels (b, d, f, h) of aquaporin-4 (a, b), aquaporin-1 (c, d), leptin (e, f) and RBP-4 (g, h) of patients and controls. Aquaporin-4 was reduced in the CSF of patients compared with controls (a) and leptin CSF and plasma levels were increased in patients (e, f). *p < 0.05, **p < 0.001. Correlation between leptin cerebrospinal fluid (CSF) and plasma levels and body fat, body mass index (BMI) and weight in patients (open circles) and controls (closed circles). Dot blot showing the leptin CSF (a, c, e) and plasma (b, d, f) levels correlated with body fat, BMI and weight (x-axis) of patients and controls. CSF and plasma levels of leptin were positively correlated with body fat, BMI and weight. Open circles representing patients and closed circles representing controls are equally scattered along the regression line.
Discussion
Recent studies that have focused on a potential role of aquaporin-4 in IIH have given negative results with respect to aquaporin-4 autoantibodies and single nucleotide polymorphisms in the aquaporin-4 gene (7–9). We have provided here the first evidence that aquaporin-4 may play a role in the pathogenesis of IIH by our measurements of decreased levels of aquaporin-4 in the CSF of patients with IIH and a reduced CSF to plasma ratio of aquaporin-4. Aquaporin-4 is thought to be involved in water homeostasis in the brain. Mice deficient in aquaporin-4 are more resistant to cytotoxic oedema than their wild-type littermates, but are more susceptible to vasogenic oedema; damage to the blood–brain barrier led to increased intracranial pressure in these mice (11). Aquaporin expression is mediated by insulin, linking an imbalance of aquaporins with metabolic disorders (5). We did not find a correlation between levels of aquaporins and leptin or RBP-4 in our patients with IIH, so that our data do not support this link in IIH. As reported previously, we found increased CSF and plasma leptin levels in patients with IIH compared with controls (3). However, body fat, weight and BMI were higher in our patients than in the controls and leptin levels were correlated with these three parameters, suggesting that the elevated leptin levels in our patients are mostly explained by the higher weight, BMI and body fat in this group. Studies with weight-matched controls would have increased the quality of our study as they could have definitively resolved this aspect. However, it is difficult to obtain CSF from healthy controls and we took more than ten years to obtain an adequate number of age-matched controls. The leptin CSF to plasma ratio did not differ between the patients and controls, arguing against a CNS-specific role of the elevated leptin in this population.
A further limitation was the lack of reliable normal values for aquaporin levels in CSF. Our values differ from the normal values reported previously (12). Therefore more and larger studies are needed to establish reference data for the measurement of aquaporins in CSF.
Our study provides evidence of imbalances in the expression of aquaporin-4 in patients with IIH as a potential pathogenic factor. Larger studies allowing subgroup analysis are needed to further elucidate this aspect and to investigate aquaporin-4 as a therapeutic target.
Key findings
Aquaporin-4 levels are reduced in the cerebrospinal fluid of patients with idiopathic intracranial hypertension compared with controls.
Clinical implications
Aquaporin-4 levels in the cerebrospinal fluid of patients with idiopathic intracranial hypertension are reduced. Aquaporin-1 levels in cerebrospinal fluid do not differ between patients with idiopathic intracranial hypertension and controls. Leptin levels are increased in the cerebrospinal fluid and plasma of patients with idiopathic intracranial hypertension and are correlated with weight, body mass index and body fat.
Footnotes
Acknowledgements
We thank Sonja Mildner and Katharina Meder from the University Hospital Würzburg, Department of Neurology, for excellent technical assistance in the conduction of the ELISAs and Hiltrud Klüpfel and Kornelia Markert, University Hospital Würzburg, Department of Neurology, for organization of the preparation and storing of CSF and plasma samples. SM, KM, HK and KM did not receive any extra compensation for their work. KD and CS had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.
Declaration of conflicting interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article. The study was in part funded by an unrestricted grant from Novartis Pharma GmbH to MS. The sponsor had no influence on study conception, data collection or analysis, or interpretation.