4. Aquaporins and Their Implication in Health

Aquaporins are a family of membrane water channel proteins that osmotically modulate water fluid homeostasis in several tissues; some of them also transport small solutes such as glycerol. The human body expresses 13 AQPs with specific organ, tissue, and cellular localization. The regulation of AQPs takes place either through gating or through controlled transport and integration to specific membrane compartments. AQPs are involved in inflammation, water balance disorders, brain oedema, cancer, and many other health conditions.

Overview

Regarding their gene and protein structure, AQPs are a family of highly conserved transmembrane proteins. AQPs consist of six alpha-helices which are spanning the cell membrane, and which are connected via five loops A to E (Figure 4.1 A). So-called asparagine-proline-alanine sequences (NPA motifs) in loops B and E are evolutionary highly conserved features. There is evidence that the NPA motifs are functionally important for water permeation. The six helices form a pore structure (Figure 4.1 B), and for most of the AQPs, the known quaternary structure is a homo-tetramer (Fig. 4.1 C), with the exception of AQP11 forming a homodimer. For AQP4 it has been shown that the tetramers can form oligomeric arrays in membranes. The AQP structure can mediate transport of water and other molecules across cell membranes (Figure 4.2).

Depictions of the Secondary (A), Tertiary (B), and Quaternary (C) structure of an AQP channel. Ex = extracellular space, In = intracellular space, N = N-Terminus of protein, C = C-terminus (adapted from Wikipedia).⁵⁴

The human body expresses 13 AQPs with specific functions and organ, tissue, and cellular localization as summarized in table 4.1. The regulation of AQPs takes place either through gating or through controlled transport and integration to specific membrane compartments.

Several modulators for AQPs are known. Among these are heavy metal ions (Hg, Pt), some of them antineoplastic drugs (e.g., cisplatin), B-nonylglucoside, tetraethylammonium, thiazolidinediones, sennosides, and auto-antibodies (AQP4) (table 4.1).

Loss of Function - Modulation of Function

In this paragraph, we aim to assess how many AQP-based drugs are currently under clinical development for AQP as drug targets. Soon after their discovery, AQPs were considered as potential drug targets.^{56 57 58 59 60}

It is notable that AQPs are of fundamental importance for cellular and body function, and that loss of function would lead to pathological conditions. This can be caused by alterations of a gene, e.g., an AQP gene, which in most cases leads to disruption of the molecular function of the AQP protein. For example, alterations in the Major Intrinsic Protein of Lens Fiber (MIP; Aquaporin 0) gene lead to cataract formation.⁶¹ Another way that loss of function can happen is through the formation of autoantibodies. Auto-antibodies are produced by the immune system and are directed against one or more of an individual’s own proteins.

A well-known immune pathological condition arises when autoantibodies are directed against aquaporin 4 (AQP4). This can lead to demyelination and diseases such as optic neuritis or neuromyelitis optica.⁶²

In order to get a more comprehensive idea of which diseases are related to AQP gene or protein loss of function, we assessed a database which serves as an amalgamated human disease compendium with diverse clinical and genetic annotation as well as a structured search information on human diseases from various sources.⁶³ This integrated compendium of annotated diseases was compiled from 68 data sources and holds more than ∼20, 000 disease entries. The results of this research regarding AQPs are listed in table 4.2.

AQPs: Potential Implications in Inflammation and Other Health Conditions

As a family of membrane water channels, AQPs are involved in a wide range of physiological functions. AQP-mediated osmotic water transport across cell plasma membranes facilitates transepithelial fluid transport, cell migration, and neuroexcitation; AQP-mediated glycerol transport regulates cell proliferation, adipocyte metabolism, and epidermal water retention.⁶⁵ In general, AQPs play a role in maintaining body water within a suitable range (homeostasis) and in many physiological processes. Through these physiological processes, AQPs affect many health conditions. Inflammation, water balance disorders, brain oedema, and cancer are among the areas that have been extensively studied. There have been very promising developments in connection with small-molecule AQP modulators for clinical applications, including sepsis and cytokine storm, refractory oedema, cancer, glaucoma, certain brain conditions, obesity, etc.⁶⁶

Inflammation is a multifaceted phenomenon in which the immune system plays a pivotal role. The immune system can be divided into two interconnected subsystems:

• The innate or non-specific immune system includes cells and processes that protect the host from infections by pathogen organisms.

• The adaptive immune response, triggered by the signals from the innate system, releases numerous substances as the real messengers of the inflammation process. The adaptive response, which relies mainly on tissue-resident macrophages and is intermediate between the basal homeostatic state and a classic inflammatory response, is seen as the cause of the chronic inflammatory conditions.^{67 68}

Inflammation is initiated by the alteration of cellular and tissue homeostasis. Cells can undergo modifications of the osmotic microenvironment, causing an increase in the cell’s hydraulic permeability and size.⁶⁹ Clinically, the swelling of cells and tissues with a surplus of extracellular fluids (oedema) is a clear sign of homeostasis disturbance and inflammation. In some cases, chronic inflammation can be caused by a more intense response and damage.

There is a great deal of data showing that AQPs participate in maintaining constant water homeostasis in many physiological processes related to secretive and absorptive activities.⁷⁰ AQPs also control cellular processes, including cell adhesion, signaling, volume regulation, and protein expression.⁷¹

AQPs and phagocytic functions of immune cells

Cell volume and shape modifications of macrophages promptly occur during phagosome development.⁷² Aqua-glyceroporins (AQGPs) have been identified as novel key elements in macrophage immune function. Researchers found that AQP3 facilitates water and glycerol transportation and participates in phagocytic and migration activity.⁷³ AQP9 was also confirmed to play a role in macrophages during infection and as a mediator between bacteria and macrophages.⁷⁴

AQPs and migration of immune cells

Plenty of data has demonstrated that AQPs facilitate migration of immune cells. AQP9 is found to be involved in the migration of neutrophils.⁷⁵ AQP3 and AQP1 are involved in macrophage migration.^{76 77}

Potential role of AQPs in inflammation of different organs

Researchers have demonstrated that AQPs contribute to regulating fluid trafficking and the inflammation process in lung infection⁷⁸ and asthma.⁷⁹ The potential contribution of AQPs in neuroinflammation has also been widely investigated, examining several diseases caused by a failure of innate immunity, such as neuromyelitis optica (NMO) and multiple sclerosis.⁸⁰ The expression of AQPs and their relevance in physiological and pathological processes have been evidenced in the gastrointestinal tract of human and mammalian species.⁸¹ In 2007, Guttman et al. proved the direct correlation between AQPs and diarrhea for the first time, finding that AQP contributes to diarrhea by attaching and effacing bacteria pathogenesis.⁸² AQPs are also shown to be involved in several inflammatory diseases affecting bone and cartilage. In 2017, Cai et al. identified AQP4 as being potentially responsible for RA pathogenesis in an adjuvant-induced arthritis (AIA) rat model.⁸³

Inflammation is a universal response mechanism that serves as an inter-communicator of biological systems. Uncontrolled or dysregulated inflammation addresses chronic low-grade effects, eventually resulting in multimorbidity. AQPs are clearly potential targets for drugs, paving the way for AQP-based therapies.⁸⁴

Thirteen AQPs have been identified in mammalian tissues. They are involved in a wide range of physiological functions. With our increasing knowledge of the roles of AQPs in health and disease, AQP-based diagnostics and therapeutics are beginning to be explored. The possibility of regulating AQP expression and function offers potential therapeutic approaches for many pathologies.

In kidney and water balance disorders

Kidneys play a key role in body water and electrolyte homeostasis. Among the most closely studied functions of AQPs are their physiological functions in the kidneys. One of the main AQPs expressed in the kidney is AQP1 to 4.^{85 86} Mutations that cause a lack of AQP1 and AQP2 can result in a urinary concentrating function.^{87 88} The expression of AQP2 is regulated by vasopressin. Mutations of either the vasopressin or AQP2 gene can lead to congenital nephrogenic diabetes insipidus (NDI). Patients with this condition are unable to concentrate their urine due to lack of AQP2 function, which leads to a high risk of dehydration.⁸⁹ AQP inhibitors are thus predicted to have “aquaretic” activity, producing a water > salt diuresis. An AQP inhibitor could be a drug (target) for “aquaretic” therapy of refractory oedema.⁹⁰

In hepatobiliary tract, salivary glands, and pancreas

Recent research has found that AQPs play a role in digestive fluid secretion in the hepatobiliary tract⁹¹, salivary glands, and pancreas.⁹² AQPs are also involved in hepatic gluconeogenesis and fat metabolism in the liver.⁹³ This data indicates that AQPs contribute to the pathogenesis of disorders including cholestasis, gallstone formation, insulin resistance, fatty liver disease, hepatic cirrhosis, and hepatocarcinoma, as well as diabetes.⁹⁴

In the central nervous system and brain oedema

The association between AQPs, especially AQP4, and brain oedema has been one of the most promising areas for AQPs-based therapy. AQP4 is expressed in astrocytes throughout the Central Nervous System, particularly at the interfaces between brain parenchyma and cerebrospinal fluid in the ventricular and subarachnoid compartments.⁹⁵

As a bidirectional water channel, AQP4 plays a double role in water accumulation and clearance. In cytotoxic oedema, such as water intoxication, ischemic stroke, and bacterial meningitis, water moves into the brain through an intact blood-brain barrier. AQP4 plays a facilitating role in water accumulation. Underexpression of AQP4 can improve this kind of brain swelling.^{96 97} Conversely, in vasogenic oedema, such as brain tumors and brain abscesses, water moves into the brain through a leaky blood-brain barrier and exits the brain through AQP4-rich glia limitans. AQP4 plays an important role in water clearance. Underexpression of AQP4 in this model worsens clinical outcome.⁹⁸ (Papadopoulos et al. 2004). This model also applies to obstructive hydrocephalus.⁹⁹ Similarly, in spinal cord injury, AQP4 underexpression is associated with reduced swelling in compression injury (cytotoxic oedema), but with increased swelling in contusion injury (vasogenic oedema).¹⁰⁰

In cancer

The involvement of AQPs in tumor angiogenesis, local invasion, and metastasis is through AQP-facilitated cell migration.¹⁰¹ Many tumors express AQPs, with the expression correlating with tumor grade.¹⁰² Researchers have found impaired tumor growth and angiogenesis in mice lacking AQP1.¹⁰³ AQP-facilitated cell migration appears to be relevant not only to angiogenesis but also to tumor spread, glial scarring, wound healing, and immune-cell chemotaxis.¹⁰⁴

Other than AQPs, AQGP was also found to be involved in cancer. AQGP3 was also found to facilitate cell proliferation in various AQP3-expressing cell types, including skin, colon, and cornea. AQP3-null mice showed complete resistance to formation of skin tumors in response to a tumor initiator-promoter protocol.¹⁰⁵ It is possible that AQP3 inhibition can reduce both tumor-cell migration and proliferation. In recent years, various chemicals have been tested as AQP inhibitors for their therapeutic effects on cancer, especially those targeting AQP5 and AQP3.¹⁰⁶ Reports also suggest that AQPs may play a role as prognostic markers.¹⁰⁷ In the near future, we may see these studies enter a clinical study phase.

Involvement in other conditions

AQPs are involved in many other physiological conditions. Although the mechanisms have not been clearly established, many AQPs provide novel therapeutic approaches for different health conditions.

In neural tissues, AQP4 is expressed in supportive cells adjacent to excitable cells in the brain, retina, inner ear, and olfactory epithelium. Studies have shown impairment in vision, hearing, and olfaction in AQP4-deficient mice.108 (Verkman 2012). Other studies found that AQP4 deficiency reduced the threshold for seizure initiation and increased seizure duration and intensity.109 (Binder et al. 2006).

Studies on AQGP also found that AQP-regulated membrane glycerol permeability is involved in certain physiological functions. AQP3-facilitated glycerol transport in the skin is an important determinant of epidermal and stratum corneum hydration.¹¹⁰ AQP7 is expressed in the plasma membrane of adipocytes, and its deficiency can cause an increase in fat mass and adipocyte hypertrophy.¹¹¹ AQP9 has also been shown to play an important role in hepatic glycerol uptake.¹¹²

Angiogenesis: The formation of new blood vessels from existing vessels is called angiogenesis. It is a normal part of growth and healing. It also plays a role in several diseases, including cancer.¹¹³

Gluconeogenesis: A process by which our body forms glucose from non-carbohydrate precursors. This process can help to maintain the glucose level when we are not consuming enough carbohydrates.

Macrophage: Macrophage is a type of the blood cells involved in the detection, phagocytosis, and destruction of bacteria and other harmful organisms. They can also activate other immune cells and present antigens to T cells and initiate inflammation by releasing molecules (known as cytokines).¹¹⁴

Neutrophils: Neutrophils constitute another type of blood cell in the immune system. They are one of the first cells that travel to the site of acute inflammation during a bacterial infection. They are part of the innate immune system and the most abundant type of white blood cells in humans. They help to heal damaged tissues and resolve infections, and they form an essential part of the innate immune system. Neutrophils are the first white blood cells recruited to sites of acute inflammation, in response to chemotactic cues produced by stressed tissue cells and tissue-resident immune cells such as macrophages.¹¹⁵

Subarachnoid space: The subarachnoid space is the interval between the arachnoid membrane and the pia mater. CSF pass through the ventricular system and eventually enters the subarachnoid space.¹¹⁶ It is occupied by delicate connective tissue trabeculae and intercommunicating channels containing CSF as well as branches of the arteries and veins of the brain.

Ventriculus of the brain: The ventricles of the brain are a communicating network of cavities filled with cerebrospinal fluid (CSF). They are interconnected and located within the brain parenchyma.¹¹⁷

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