Alternation in the Abundance and Phosphorylation Status of Aquaporin-2 in the Serum Exosome of Patients with Chronic Kidney Disease

Research Article

J Dis Markers. 2023; 8(2): 1057.

Alternation in the Abundance and Phosphorylation Status of Aquaporin-2 in the Serum Exosome of Patients with Chronic Kidney Disease

Yao-Te Chung1,5; Chiung-Hu Kuo2; Ru-Yi Tsai3; Jin-Wun Chen4; Chin Li5; Wen-Yi Li6*

1Department of First Common Laboratory, National Taiwan University Hospital, Yunlin Branch, Yun-Lin, Taiwan

2Department of Laboratory Medicine, National Taiwan University Hospital Chutung Branch, Hsinchu, Taiwan

3Department of Cardiovascular Center, National Taiwan University Hospital Yunlin Branch, Yun-Lin, Taiwan

4Department of First Common Laboratory, National Taiwan University Hospital Yunlin Branch, Yun-Lin, Taiwan

5Department of Biomedical Sciences, National Chung-Cheng University, Chia-Yi, Taiwan

6Department of Internal Medicine, National Taiwan University Hospital Yunlin Branch, Yun-Lin, Taiwan

*Corresponding author: Wen-Yi Li Department of Internal Medicine, National Taiwan University Yunlin Branch, 579, Sec 2, Yunlin Road, Douliu City, Yunlin 640, Taiwan Tel: 886-5-5323911; Fax: 886-5-5373257 Email: Y00905@ms1.ylh.gov.tw

Received: August 01, 2023 Accepted: August 28, 2023 Published: September 04, 2023

Abstract

Background: Exosomes, which are vesicles ranging from 40 to 160 nm in size, are released from cellular membranes and serve as a means of cell-to-cell communication by delivering their contents, such as microRNA and protein, to recipient cells. These extracellular vesicles are present in human blood and urine and their composition can be altered in specific pathogenic conditions. Aquaporin-2 (AQP2) is a critical aquaporin involved in water homeostasis and has been identified as an important biomarker in chronic kidney disease. However, it is unclear whether AQP2 is transported via exosomes.

Method: This study included 16 participants with chronic kidney disease and 11 healthy volunteers. Serum exosomes were isolated through polymer precipitation and imaged using transmission electron microscopy. The diameter of purified serum exosomes was measured via flow cytometry, and their identity was further confirmed by identifying exosome markers through immunoblotting. Additionally, the participants’ hepatic B and C virus infection status was determined using chemiluminescent microparticle immunoassay.

Results: The precipitation method was utilized to isolate serum exosomes, and successful purification was confirmed through transmission electron microscopy imaging. Size estimation was conducted via flow cytometry, and the presence of exosome markers CD63, TSG101, CD9, and CD81 was confirmed through immunoblotting. Furthermore, the presence of AQP2 was detected in serum exosomes, and its abundance was found to significantly decrease in patients with chronic kidney disease. However, despite the decreased abundance, the percentage of phosphorylated AQP2 at serine 256 was significantly increased.

Conclusion: Our findings suggest that changes in the abundance and phosphorylation status of AQP2 on serum exosomes are associated with the development of chronic kidney disease, and could potentially serve as a signal for exosome transport.

Keywords: CKD; AQP2; Exosme

Lay Summary

Exosomes, small vesicles involved in cell communication, carry molecules between cells. This study examined Aquaporin-2 (AQP2), a protein important in water balance, and its presence in exosomes, specifically in Chronic Kidney Disease (CKD). Blood samples were collected from CKD patients and healthy individuals, and exosomes were isolated and characterized. AQP2 was found in exosomes, and its levels were significantly lower in CKD patients compared to healthy individuals. Interestingly, phosphorylation of AQP2 at a specific site was increased in CKD patients. These findings suggest that changes in AQP2 abundance and phosphorylation in exosomes may be linked to CKD development. Understanding exosome-associated changes in AQP2 could offer insights into CKD progression and potentially lead to new diagnostic markers or treatment strategies. Further research is needed to explore the underlying mechanisms and clinical implications of these findings.

Introduction

Exosomes are a type of extracellular vesicle that range in size from 40 to 160 nm in diameter, depending on the cell type they originate from. Monocytes and macrophages, for example, can produce exosomes that are larger, with an average size of up to 200 nm [1]. Endothelial cells, on the other hand, tend to produce exosomes that are around 150 nm in diameter [2]. These vesicles are capable of carrying various types of biomolecules, including DNA, mRNA, microRNA, and proteins, and can act as vehicles to transport cargo to target cells in a paracrine or endocrine manner [3,4]. Additionally, exosomes have been found to play a role in viral infections [5,6], making them a versatile and multi-functional means of intercellular communication.

Exosomes often exhibit disease-specific changes, making them potential diagnostic or prognostic biomarkers. In the context of kidney-related pathologies, various biomarkers have been investigated. Studies have demonstrated significant changes in exosomal microRNAs during the progression of diabetic nephropathy [7], while exosomal AQP2 has been evaluated as a potential biomarker for renal dysfunction [8]. In addition, general circulation microparticles have been utilized to monitor vascular dysfunction in end-stage renal failure [9]. However, whether there are discernable alterations in circulating exosomes of individuals with chronic kidney disease remains unclear.

Ultracentrifugation and precipitation are the two primary methods for isolating exosomes from liquid biopsies. Precipitation, which involves the use of high-molecular-weight polymers such as PEG, can be performed without special equipment and is a popular alternative to ultracentrifugation [10]. While ultracentrifugation yields a lower quantity of exosomes, it produces a much purer sample. However, due to the requirement for an ultra-high-speed centrifuge, this method is less commonly used for routine exosome isolation. Confirmation of isolated exosomes can be achieved through various experimental techniques. For instance, flow cytometry analysis can be employed to determine particle diameter. In addition, immunoblotting can be used to detect known exosome transmembrane protein markers, such as CD63, CD9, and CD81, as well as the cargo protein TSG101, which serve as positive identification for exosomes in purified samples [11-13].

Aquaporins are integral membrane proteins that regulate water homeostasis by serving as water channels. AQP2, a member of the aquaporin family, is involved in endocytosis and exocytosis and is also incorporated into exosomes that are subsequently released into the blood or urine. The activity of AQP2 is modulated by phosphorylation and ubiquitination. Studies have demonstrated that serine-256 phosphorylation of AQP2 is associated with trafficking between intracellular spaces via vesicles [14], while threonine-269 and serine-261 phosphorylation is involved in secretion [15]. However, the relationship between AQP2 phosphorylation and exosome secretion is not well understood. In this study, we isolated and analyzed exosomes from serum samples collected from healthy volunteers and patients with chronic kidney disease. Our findings revealed that the concentration of exosomes was significantly lower in patients with chronic kidney disease than in healthy individuals. Despite the lower exosome concentration, the ratio of serine-256 phosphorylated AQP2 to unphosphorylated AQP2 was considerably higher in patients compared to healthy volunteers. Our study highlights the potential of using the concentration of serum exosomes and phosphorylated AQP2 as a signal for evaluating chronic kidney disease.

Materials and Methods

Study Design

The flow chart depicts the essential components of the study design (Figure 1). In this study, serum exosomes were isolated using polymer precipitation and visualized using transmission electron microscopy. The size of purified serum exosomes was determined using flow cytometry, while their identity was confirmed through the identification of exosome markers via immunoblotting.