Biomedicines | Free Full-Text | Association between Serum GDF-15 and Cognitive Dysfunction in Hemodialysis Patients
Although there have been several studies on the relationship between GDF15 and mortality in hemodialysis patients, very few studies have evaluated serum GDF-15 as a marker for screening cognitive dysfunction in hemodialysis patients. This study investigated the relationship between cognitive dysfunction and serum GDF-15 levels in hemodialysis patients. In this study, our objective was to evaluate whether exposure to uremic toxins, observed in hemodialysis patients with an inclination towards elevated GDF-15 levels and cognitive dysfunction, might result in comparable outcomes in terms of GDF-15 expression in brain cells and tissues, as well as the survival of brain cells. The relationship between the expression of GDF-15 in mice and HT22 brain cells under conditions of increased uremia was investigated to determine whether uremic toxins were associated with an increase in brain GDF-15 levels.
In this study, we aimed to evaluate the potential of serum GDF-15 as a screening marker for cognitive decline in patients undergoing hemodialysis.
2. Materials and Methods
2.1. Study Population
This retrospective study analyzed data from hemodialysis patients treated at Chungnam National University Hospital, Daejeon, Republic of Korea, from January 2017 to June 2020. Ninety-two patients with end-stage renal disease on maintenance hemodialysis who underwent the Korean Mini-Mental Status Examination (K-MMSE) within 1 month of blood sample collection were included in the study population. The patients included in this study maintained hemodialysis three times a week, with each session lasting four hours. Patients diagnosed with dementia and taking medication were excluded. Patients diagnosed with depression or those taking medication for depression were excluded. This study conformed to the Declaration of Helsinki and was approved by the Ethics Committee of Chungnam National University Hospital (19 February 2021; Institutional Review Board approval no. CNUSH 2021-02-007).
2.2. Cognitive Function Assessment
2.3. Clinical Parameters
Patients’ clinical data were obtained from electronic medical records. Data collected included age, sex, body mass index, medical history, date of hemodialysis initiation, the period from hemodialysis initiation to the date of the K-MMSE, and education level. Medical history included hypertension, diabetes mellitus (DM), cerebral infarction, ischemic heart disease, and the causative disease of end-stage renal disease (ESRD). Blood sampling was performed immediately before hemodialysis. Laboratory data for serum GDF-15, serum creatinine, albumin, blood urea nitrogen (BUN), total protein, total cholesterol, total calcium, serum phosphorus, sodium, potassium, total CO2, and CRP were collected.
2.4. Measurement of Serum GDF-15 in Humans
The biospecimens and data used for further analysis on GDF-15 were provided by the Biobank of Chungnam National University Hospital, a member of the Korea Biobank Network. Blood samples for the measurement of serum GDF-15 were collected before dialysis on the day of hemodialysis. The samples were centrifuged immediately after collection and stored at −80 °C prior to use. Serum GDF-15 concentrations were determined in duplicate using a quantitative enzyme-linked immunosorbent assay kit (Human GDF-15 Quantikine ELISA Kit, R&D Systems, Minneapolis, MN, USA). All samples were analyzed in duplicate and measured according to the manufacturer’s instructions. In this study, the serum GDF-15 level was used as the average of the two replicates.
2.5. Mice and Drugs
To confirm that similar results were obtained in the in vitro and in vivo experiments regarding the analysis of GDF-15 measurement and cognitive decline in hemodialysis patients, experiments were also conducted in mice.
2.6. Blood and Tissue Preparation
The brain tissue from the sham and IRI mice was dissected and homogenized in lysis buffer. Then, the brain tissue lysates (15 μg) were separated by 10% or 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The primary antibodies (1:1000) used to probe the blots were anti-GDF-15 (Abcam, Cambridge, CB2 OAX, UK) and anti-α-tubulin (Cell Signaling Technology, Inc., Beverly, MA, USA). The blots were then incubated for 2 h with anti-rabbit IgG-HRP-linked antibody (1:1000) and anti-mouse IgG-HRP-linked antibody (1:1000) (Cell Signaling Technology, Inc.) as secondary antibodies. The protein bands were visualized using a chemiluminescence detection kit (Thermo Scientific, South Logan, UT, USA). The optical density of the proteins was determined using Gel-Pro Analyzer v3.1 software (Media Cybernetics, Silver Spring, MD, USA) to quantify the protein amount.
2.8. Cell Viability Assay
Brain cell injury was confirmed on the treatment of uremic toxin. Mouse hippocampal neuronal cell line HT22 was incubated with Dulbecco’s modified Eagle’s medium (DMEM; WELGENE, Gyeongsan-si, Republic of Korea) containing 10% fetal bovine serum (FBS; GenDEPOT, Katy, TX, USA) and 100 U/mL of penicillin–streptomycin (Gibco, Waltham, MA, USA) at 37 °C under 5% CO2. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma-Aldrich, St. Louis, MO, USA) assay was used to measure cell viability via mitochondrial reductase. Briefly, HT22 cells were seeded into a 96-well plate at a density of 5 × 103 cells/well in DMEM containing 10% FBS and incubated for 24 h. Then, the culture medium was treated with indoxyl sulfate (a representative uremic toxin) at concentrations of 1, 5, and 10 mM, respectively, for 24 h. Next, MTT solution was added to each well to a final concentration of 0.5 mg/mL and incubated for 2 h at 37 °C. After removing the culture medium containing MTT, dimethyl sulfoxide was added, and the plate was incubated to dissolve the reduced formazan crystals at 37 °C for 1 h. Finally, the absorbance was measured at 540 nm.
2.9. Statistical Analysis
This study confirmed the association between high levels of serum GDF-15 and cognitive dysfunction in ESRD on maintenance hemodialysis. Additionally, GDF-15 expression was revealed as increased in the brain tissue of uremic mice compared with normal mice.
In this study, using univariate and multivariate analyses, a statistically significant association was confirmed between high serum GDF-15 levels and a decline in cognitive function in hemodialysis patients. Furthermore, logistic regression analysis showed that the risk of cognitive dysfunction significantly increased by 2.912 times when the GDF-15 level was ≥5408.33 pg/mL in hemodialysis patients.
In this study, we were unable to identify antecedent factors that can be considered in the relationship between aging and the elevation of GDF-15. Furthermore, the specific mechanism through which GDF-15 may induce cognitive dysfunction can not be definitively confirmed. Consequently, a clear conclusion regarding whether GDF-15 triggers cognitive dysfunction or increases in association with cognitive dysfunction can not be drawn. While GDF-15 may rise in situations such as aging and conditions similar to chronic kidney disease (CKD), the increase in GDF-15 concentrations when brain cells and tissues are exposed to uremic toxins (such as indoxyl sulfate) suggests that elevated levels of GDF-15 may indicate brain damage in the context of cognitive decline. Especially in patients undergoing hemodialysis, considering GDF-15 elevation beyond a certain threshold as a potential marker for screening or suspicion of cognitive dysfunction appears plausible.
There are several limitations in this study. First, this study was a retrospective study and had a small sample size, which limited the analysis of GDF-15 trends by age. When the cutoff value of serum GDF-15 was set at 5408.33 pg/mL in the ROC curve, it showed 63.6% sensitivity and 64.4% specificity in predicting cognitive dysfunction. The applicability of these findings to a broader population is limited because the sample size was too small to set an accurate cutoff value that can be used as a marker of cognitive function. It is expected that the prediction rate can be further improved if a relatively larger amount of data is analyzed in future studies. Second, the results were analyzed based on patients from a single country, and consideration for other races was not taken into account. It seems necessary to consider multiple races in future studies. Third, as this study was retrospective, various tools can not be used to evaluate cognitive function, and only the K-MMSE was utilized. In future research, it is believed that richer results will be obtained by using multiple cognitive function assessment tools in retrospective studies. Fourth, among the patients included in this study, there were no individuals diagnosed with depression related to cognitive impairment. However, as undiagnosed depression was not assessed, it is considered crucial for future studies to evaluate not only cognitive function but also depression.
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