AQP1 expression analysis in human diseases: implications for proteomic characterization

Aquaporin (AQP)1 belongs to a ubiquitous family of water channel proteins characterized by sequence similarity and the presence of two NPA (Asp-Pro-Ala) motifs existing in almost all organs and tissues. Currently, 13 human AQPs are known and they are divided into two subgroups according to their ability to transport only water molecules, such as AQP1, or also glycerol and other small solutes. The genomic, structural and functional aspects of AQP1 are briefly described. An in-depth discussion is devoted to proteomic approaches that are useful for identifying and characterizing AQP1, mainly through electrophoretic techniques combined with different extraction procedures followed by mass spectrometry analysis. Moreover, the relevance of AQP1 in human diseases is also explained. Its role in human tumors and, in particular, those of the kidney (e.g., clear cell renal carcinoma) is discussed.     

Expression and function of aquaporins in human skin: Is aquaporin-3 just a glycerol transporter?

The aquaporins (AQPs) are a family of transmembrane proteins forming water channels. In mammals, water transport through AQPs is important in kidney and other tissues involved in water transport. Some AQPs (aquaglyceroporins) also exhibit glycerol and urea permeability. Skin is the limiting tissue of the body and within skin, the stratum corneum (SC) of the epidermis is the limiting barrier to water loss by evaporation. The aquaglyceroporin AQP3 is abundantly expressed in keratinocytes of mammalian skin epidermis. Mice lacking AQP3 have dry skin and reduced SC hydration. Interestingly, however, results suggested that impaired glycerol, rather than water transport was responsible for this phenotype. In the present work, we examined the overall expression of AQPs in cells from human skin and we reviewed data on the functional role of AQPs in skin, particularly in the epidermis. By RT-PCR on primary cell cultures, we found that up to 6 different AQPs (AQP1, 3, 5, 7, 9 and 10) may be selectively expressed in various cells from human skin. AQP1, 5 are strictly water channels. But in keratinocytes, the major cell type of the epidermis, only the aquaglyceroporins AQP3, 10 were found. To understand the role of aquaglyceroporins in skin, we examined the relevance to human skin of the conclusion, from studies on mice, that skin AQP3 is only important for glycerol transport. In particular, we find a correlation between the absence of AQP3 and intercellular edema in the epidermis in two different experimental models: eczema and hyperplastic epidermis. In conclusion, we suggest that in addition to glycerol, AQP3 may be important for water transport and hydration in human skin epidermis.     

Expression and function of aquaporins in human skin: Is aquaporin-3 just a glycerol transporter?

The aquaporins (AQPs) are a family of transmembrane proteins forming water channels. In mammals, water transport through AQPs is important in kidney and other tissues involved in water transport. Some AQPs (aquaglyceroporins) also exhibit glycerol and urea permeability. Skin is the limiting tissue of the body and within skin, the stratum corneum (SC) of the epidermis is the limiting barrier to water loss by evaporation. The aquaglyceroporin AQP3 is abundantly expressed in keratinocytes of mammalian skin epidermis. Mice lacking AQP3 have dry skin and reduced SC hydration. Interestingly, however, results suggested that impaired glycerol, rather than water transport was responsible for this phenotype. In the present work, we examined the overall expression of AQPs in cells from human skin and we reviewed data on the functional role of AQPs in skin, particularly in the epidermis. By RT-PCR on primary cell cultures, we found that up to 6 different AQPs (AQP1, 3, 5, 7, 9 and 10) may be selectively expressed in various cells from human skin. AQP1, 5 are strictly water channels. But in keratinocytes, the major cell type of the epidermis, only the aquaglyceroporins AQP3, 10 were found. To understand the role of aquaglyceroporins in skin, we examined the relevance to human skin of the conclusion, from studies on mice, that skin AQP3 is only important for glycerol transport. In particular, we find a correlation between the absence of AQP3 and intercellular edema in the epidermis in two different experimental models: eczema and hyperplastic epidermis. In conclusion, we suggest that in addition to glycerol, AQP3 may be important for water transport and hydration in human skin epidermis.     

Junction-forming aquaporins

Aquaporins (AQPs) are a family of ubiquitous membrane channels that conduct water and solutes across membranes. This review focuses on AQP0 and AQP4, which in addition to forming water channels also appear to play a role in cell adhesion. We discuss the recently determined structures of the membrane junctions mediated by these two AQPs, the mechanisms that regulate junction formation, and evidence that supports a role for AQP0 and AQP4 in cell adhesion.     

Ontogeny of aquaporins in human fetal membranes

It has been proposed that four members of the aquaporin family (AQPs 1, 3, 8, and 9) are involved in the control of amniotic fluid (AF) homeostasis, as illustrated by their differential expression patterns in normal and pathological human term fetal membranes. However, there are no data available to date on their ontogeny throughout pregnancy. Our objective was to determine spatiotemporal expression profiles of the mRNA and proteins of all 13 members of this transmembrane channel family. For this purpose, we used healthy fetal membranes from the first, second, and third trimesters of pregnancy. Total mRNA and proteins were extracted from total membranes and from separated amnion and chorion. Quantitative PCR, Western blot, and immunohistochemistry experiments were carried out to determine the presence of AQPs and to quantify their spatiotemporal expression patterns throughout pregnancy. The WISH cell line was tested to propose a cellular model for the role of AQPs in the amnion compartment. AQP11 expression was established in amniotic membranes at term. Aquaporins 1, 3, 8, 9, and 11 mRNA and proteins were present in amnion and chorion throughout human gestation. Each AQP has a time-specific expression pattern, with AQP1 presenting the highest variation in terms of mRNA and protein levels. The WISH cell line also expressed the same five AQPs. Taken together, these results indicate that AQPs are expressed and potentially involved in the regulation of AF homeostasis throughout pregnancy. This also clearly supports the hypothesis that abnormal expression could occur at any time during pregnancy, ultimately leading to obstetrical pathologies such as polyhydramnios or oligohydramnios.     

水通道蛋白的生理功能 ——水通道基因敲除小鼠表型研究进展

水通道蛋白 (aquaporin, AQP) 是一族细胞膜上选择性高效转运水分子的特异孔道. 自从 Agre 等于 1992 年从红细胞膜发现第一个水通道蛋白 AQP1以来,有关水通道蛋白结构与功能的研究取得了迅速的、系列性的进展 . 已报道的哺乳动物 AQP 家族已有 11 个在蛋白质序列上有同源性成员 (AQP0~AQP10). AQP 在体内各系统组织中广泛表达,除了在与体液分泌和吸收密切相关的多种上皮和内皮细胞高表达外,在一些与体液转运无明显关系的组织细胞如红细胞、白细胞、脂肪细胞和骨骼肌细胞等处也有表达,提示 AQP 可能在多种器官生理和病理中发挥重要作用. 基因打靶技术是研究特定基因在体内生理功能的有力手段. 目前 AQP1、3、4、5 基因敲除和 AQP2 基因点突变的基因敲入小鼠模型 ( 模拟人类常染色体隐性遗传尿崩症 ) 已成功建立并广泛用于表型研究,在 AQP 水通道蛋白生理功能方面获得许多重要进展.     

New insight into the evolution of aquaporins from flowering plants and vertebrates: orthologous identification and functional transfer is possible

Aquaporins (AQPs) represent a family of channel proteins that transport water and/or small solutes across cell membranes in the three domains of life. In all previous phylogenetic analysis of aquaporin, trees constructed using proteins with very low amino acid identity (<15%) were incongruent with rRNA data. In this work, restricting the evolutionary study of aquaporins to proteins with high amino acid identity (>25%), we showed congruence between AQPs and organismal trees. On the basis of this analysis, we defined 19 orthologous gene clusters in flowering plant species (3 PIP-like, 7 TIP-like, 6 NIP-like and 3 SIP-like). We described specific conserved motifs for each subfamily and each cluster, which were used to develop a method for automatic classification. Analysis of amino acid identity between orthologous monocotyledon and dicotyledon AQPs from each cluster, suggested that PIPs are under high evolutionary constraint. The phylogenetic analysis allowed us the assignment of orthologous aquaporins for very distant animal lineages (tetrapods-fishes). We also demonstrated that the location of all vertebrate AQPs in the ortholog clusters could be predicted by comparing their amino acid identity with human AQPs. We defined four AQP subfamilies in animals: AQP1-like, AQP8-like, AQP3-like and AQP11-like. Phylogenetic analysis showed that the four animal AQPs subfamilies are related with PIP-like, TIP-like, NIP-like and SIP-like subfamilies, respectively. Thus, this analysis would allow the prediction of individual AQPs function on the basis of orthologous genes from Arabidopsis thaliana and Homo sapiens.     

Renal expression and functions of aquaporin 1 and aquaporin 4 in cattle

Abstract

Aquaporin (AQP) 1 and AQP 4 are members of the aquaporin water channel family that play an important role in reabsorption of water from the renal tubular fluid to concentrate urine. Studies of renal AQPs have been performed in human, rodents, sheep, dogs and horses. We studied nephron segment-specific expression of AQP 1 and AQP 4 using immunohistochemical staining on paraffin sections of bovine kidneys. AQP 1 was moderately expressed in endothelium of the cortical capillary network, vasa recta, and glomerular capillaries. AQP 4 was moderately expressed only in cytoplasm of epithelial cells in proximal tubules. We concluded that AQP 1 and AQP 4 in the bovine kidney showed some differences from other species in renal trans-epithelial water transport.     

Roles of aquaporins in the brain

It is now over 10 years ago that aquaporin 1 (AQP1) was discovered and cloned from the red blood cells, and in 2003 the Nobel price in Chemistry was awarded to Pr. Peter Agre for his work on AQPs, highlighting the importance of these proteins in life sciences. AQPs are water channels. To date this protein family is composed of 11 sub-types in mammalians. Three main AQPs described in the mammalian brain are AQP1, AQP4 and AQP9. Several recent studies have shown that these channels are implicated in numerous physiological functions. AQP1 has a role in cerebrospinal fluid formation, whereas AQP4 is involved in water homeostasis and extracellular osmotic pressure in brain parenchyma. AQP4 seems also to have an important function in oedema formation after brain trauma or brain ischemia. AQP9 is implicated in brain energy metabolism. The level of expression of each AQP is highly regulated. After a trauma or an ischemia perturbation of the central nervous system, the level of expression of each AQP is differentially modified, resulting in facilitating oedema formation. At present, the exact role of each AQP is not yet determined. A better understanding of the mechanisms of AQP regulation should permit the development of new pharmacological strategies to prevent oedema formation. AQP9 has been recently specifically detected in the catecholaminergic neurons of the brain. This new result strengthens the hypothesis that the AQPs are not only water channels, but that some AQPs may play a role in energy metabolism as metabolite channels.     

Effect of hyperosmotic stimulation on aquaporins gene expression in chick kidney

Birds can produce hyperosmotic urine, but their renal morphology differs from that of mammals. Recent studies in mammals, suggested that various aquaporins (AQPs) are present in the kidney and play crucial roles in urine production. To elucidate the role of AQPs in the avian kidney, we first examined for the presence of AQP1, 2, 3, 4, 7 and 9 mRNAs in the chick (Gallus gallus) kidney by RT-PCR analysis. Next, we quantified variations of AQPs mRNAs levels in chick kidney after hyperosmotic stimulation (water-deprivation or salt-loading) by real-time RT-PCR analysis. Our study showed that in addition to AQP1, 2, 3, 4 and 7, chick kidney also expressed AQP9 and that hyperosmotic stimulation induced changes in AQPs expression. In particular, water-deprivation increased AQP2 and AQP3 mRNAs levels, whereas salt-loading induced a significant increase in AQP1, AQP2 and AQP9 mRNAs levels. AQP4 and AQP7 mRNA levels were not affected by any hyperosmotic stimulation. Taken together, these results indicated that the presence of AQPs in chick kidney is similar to that in mammals, that the chick kidney has an additional AQP9 and that AQP1, 2, 3 and 9 may play a crucial but different role in water permeability in this organ.     

1,3‐propanediol binds deep inside the channel to inhibit water permeation through aquaporins

Aquaporins and aquaglyceroporins (AQPs) are membrane channel proteins responsible for transport of water and for transport of glycerol in addition to water across the cell membrane, respectively. They are expressed throughout the human body and also in other forms of life. Inhibitors of human AQPs have been sought for therapeutic treatment for various medical conditions including hypertension, refractory edema, neurotoxic brain edema, and so forth. Conducting all‐atom molecular dynamics simulations, we computed the binding affinity of acetazolamide to human AQP4 that agrees closely with in vitro experiments. Using this validated computational method, we found that 1,3‐propanediol (PDO) binds deep inside the AQP4 channel to inhibit that particular aquaporin efficaciously. Furthermore, we used the same method to compute the affinities of PDO binding to four other AQPs and one aquaglyceroporin whose atomic coordinates are available from the protein data bank (PDB). For bovine AQP1, human AQP2, AQP4, AQP5, and Plasmodium falciparum PfAQP whose structures were resolved with high resolution, we obtained definitive predictions on the PDO dissociation constant. For human AQP1 whose PDB coordinates are less accurate, we estimated the dissociation constant with a rather large error bar. Taking into account the fact that PDO is generally recognized as safe by the US FDA, we predict that PDO can be an effective diuretic which directly modulates water flow through the protein channels. It should be free from the serious side effects associated with other diuretics that change the hydro‐homeostasis indirectly by altering the osmotic gradients.     

Noncanonical binding of calmodulin to aquaporin-0: implications for channel regulation

Aquaporins (AQPs) are a family of ubiquitous membrane channels that conduct water across cell membranes. AQPs form homotetramers containing four functional and independent water pores. Aquaporin-0 (AQP0) is expressed in the eye lens, where its water permeability is regulated by calmodulin (CaM). Here we use a combination of biochemical methods and NMR spectroscopy to probe the interaction between AQP0 and CaM. We show that CaM binds the AQP0 C-terminal domain in a calcium-dependent manner. We demonstrate that only two CaM molecules bind a single AQP0 tetramer in a noncanonical fashion, suggesting a form of cooperativity between AQP0 monomers. Based on these results, we derive a structural model of the AQP0/CaM complex, which suggests CaM may be inhibitory to channel permeability by capping the vestibules of two monomers within the AQP0 tetramer. Finally, phosphorylation within AQP0's CaM binding domain inhibits the AQP0/CaM interaction, suggesting a temporal regulatory mechanism for complex formation.     

Expression and nephron segment-specific distribution of major renal aquaporins (AQP1-4) in Equus caballus, the domestic horse

Aquaporins (AQPs) play fundamental roles in water and osmolyte homeostasis by facilitating water and small solute movement across plasma membranes of epithelial, endothelial, and other tissues. AQP proteins are abundantly expressed in the mammalian kidney, where they have been shown to play essential roles in fluid balance and urine concentration. Thus far, the majority of studies on renal AQPs have been carried out in laboratory rodents and sheep; no data have been published on the expression of AQPs in kidneys of equines or other large mammals. The aim of this comparative study was to determine the expression and nephron segment localization of AQP1-4 in Equus caballus by immunoblotting and immunohistochemistry with custom-designed rabbit polyclonal antisera. AQP1 was found in apical and basolateral membranes of the proximal convoluted tubules and thin descending limbs of the loop of Henle. AQP2 expression was specifically detected in apical membranes of cortical, medullary, and papillary collecting ducts. AQP3 was expressed in basolateral membranes of cortical, medullary, and papillary collecting ducts. Immunohistochemistry also confirmed AQP4 expression in basolateral membranes of cells lining the distal convoluted and connecting tubules. Western blots revealed high expression of AQP1-4 in the equine kidney. These observations confirm that AQPs are expressed in the equine kidney and are found in similar nephron locations to mouse, rat, and human kidney. Equine renal AQP proteins are likely to be involved in acute and chronic regulation of body fluid composition and may be implicated in water balance disorders brought about by colic and endotoxemia.     

Structural Determinants of Oligomerization of the Aquaporin-4 Channel

The aquaporin (AQP) family of integral membrane protein channels mediate cellular water and solute flow. Although qualitative and quantitative differences in channel permeability, selectivity, subcellular localization, and trafficking responses have been observed for different members of the AQP family, the signature homotetrameric quaternary structure is conserved. Using a variety of biophysical techniques, we show that mutations to an intracellular loop (loop D) of human AQP4 reduce oligomerization. Non-tetrameric AQP4 mutants are unable to relocalize to the plasma membrane in response to changes in extracellular tonicity, despite equivalent constitutive surface expression levels and water permeability to wild-type AQP4. A network of AQP4 loop D hydrogen bonding interactions, identified using molecular dynamics simulations and based on a comparative mutagenic analysis of AQPs 1, 3, and 4, suggest that loop D interactions may provide a general structural framework for tetrameric assembly within the AQP family.     

Prediction of Aquaporin Function by Integrating Evolutionary and Functional Analyses

Aquaporins (AQPs) are a family of channel proteins, which transport water and/or small solutes across cell membranes. AQPs are present in Bacteria, Eukarya, and Archaea. The classical AQP evolution paradigm explains the inconsistent phylogenetic trees by multiple transfer events and emphasizes that the assignment of orthologous AQPs is not possible, making it difficult to integrate functional information. Recently, a novel phylogenetic framework of eukaryotic AQP evolution showed congruence between eukaryotic AQPs and organismal trees identifying 32 orthologous clusters in plants and animals (Soto et al. Gene 503:165–176, 2012). In this article, we discuss in depth the methodological strength, the ability to predict functionality and the AQP community perception about the different paradigms of AQP evolution. Moreover, we show an updated review of AQPs transport functions in association with phylogenetic analyses. Finally, we discuss the possible effect of AQP data integration in the understanding of water and solute transport in eukaryotic cells.