The nucleus pulposus (NP) of the human intervertebral disc (IVD) is a hyperosmotic tissue that is subjected to daily dynamic compressive loads. In order to survive within this environment the resident chondrocyte-like cells must be able to control their cell volume, whilst also controlling the anabolism and catabolism of their extra-cellular matrix. Recent studies have demonstrated expression of a range of bi-directional, transmembrane water and solute transporters, named aquaporins (AQPs), within chondrocytes of articular cartilage. The aim of this study was to use immunohistochemsitry to investigate the expression of aquaporins 1, 2 and 3 within the human IVD. Results demonstrated expression of both AQP-1 and -3 by cells within the NP and inner annulus fibrosus (AF), while outer AF cells lacked expression of AQP-1 and showed very low numbers of AQP-3 immunopositive cells. Cells from all regions were negative for AQP-2. Therefore this study demonstrates similarities in the phenotype of NP cells and articular chondrocytes, which may be due to similarities in tissue osmolarity and mechanobiology. The decrease in expression of AQPs from the NP to the outer AF may signify changes in cellular phenotype in response to differences in mechanbiology, osmolarity and hydration between the gelatinous NP and the fibrous AF.© 2008 Springer Science+Business Media B.V.
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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. Copyright© 2007 the American Physiological Society.
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Lithium treatment for 4 wk caused severe polyuria, dramatic downregulation in aquaporin-2 (AQP-2) expression, and marked decrease in AQP-2 immunoreactivity with the appearance of a large number of cells without AQP-2 labeling in the collecting ducts after lithium treatment. Surprisingly, this was not all due to an increase in AQP-2-negative principal cells, because double immunolabeling revealed that the majority of the AQP-2-negative cells displayed [H+]ATPase labeling, which identified them as intercalated cells. Moreover, multiple [H+]ATPase-labeled cells were adjacent, which was never seen in control rats. Quantitation confirmed a significant decrease in the fraction of collecting duct cells that exhibited detectable AQP-2 labeling compared with control rats: in cortical collecting ducts, 40 +/- 3.4 vs. 62 +/- 1.8% of controls (P<0.05; n = 4) and in inner medullary collecting ducts, 58 +/- 1.6 vs. 81 +/- 1.3% of controls (P<0.05; n = 4). In parallel, a significant increase in the fraction of intercalated ([H+]ATPase-positive) cells was shown. Urine output, whole kidney AQP- 2 expression, cellular organization, and the fractions of principal and intercalated cells in cortex and inner medulla returned to control levels after 4 wk on a lithium-free diet following 4 wk on a lithium-containing diet. In conclusion, lithium treatment not only decreased AQP-2 expression, but dramatically and reversibly reduced the fraction of principal cells and altered the cellular organization in collecting ducts. These effects are likely to be important in lithium-induced nephrogenic diabetes insipidus.
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Recent studies have shown that aquaporin water channels are expressed in human Meckel's cartilage. The aim of the present investigation was to determine if human articular chondrocytes and synoviocytes express aquaporin 1 (AQP1) water channels and to establish if there are any alterations in AQP1 expression in osteoarticular disorders such as osteoarthritis (OA) and rheumatoid arthritis (RA). Immunohistochemistry was employed semi-quantitatively to compare the expression of AQP1 in human chondrocytes derived from normal, OA and RA joints. PCR, cloning and sequencing confirmed the presence of AQP1 transcripts in chondrocytes. Normal human tissue microarrays including samples of kidney, choroid plexus and pancreas were used as positive controls for AQP1 expression. In most tissues AQP1 was expressed along endothelial barriers. In the kidney AQP1 was present in the glomerular capillary endothelium, proximal tubule and descending thin limbs. AQP1 was also localized to pancreatic ducts and acini and the apical membrane domain of the choroid plexus. Immunohistochemistry showed that AQP1 is expressed in synovial micro-vessels, synoviocytes and predominantly in chondrocytes located in the deep zone of articular cartilage. Image analysis of normal, OA and RA cartilage suggested that AQP1 may be upregulated in RA. This is the first report of AQP1 mRNA and protein expression in articular chondrocytes and synoviocytes. These findings suggest a potential role for AQP1 and possibly other members of the AQP gene family in the movement of extracellular matrix and metabolic water across the membranes of chondrocytes and synoviocytes for the purposes of chondrocyte volume regulation and synovial homeostasis.
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Aquaporin water channels are a family of membrane proteins that facilitate water movement across biological membranes. Aquaporin-1 (AQP-1) has been found to be important in osmotic water movement across cell membranes of epithelial and endothelial barriers. However, the distribution of AQP-1 in many normal human tissues is still unknown. The aim of this study was to use immunohistochemistry and semiquantitative histomorphometric analysis to determine the tissue distribution and relative expression of AQP-1 in normal human tissues using tissue microarray (TMA) technology. The normal human TMAs employed in this study included cardiovascular, respiratory, gastrointestinal, hepatic and pancreatobiliary, oral, salivary, nasal, mammary, fetal, endocrine, genital tract, central and peripheral nervous systems, urinary tract, skin, cartilage, and other soft connective tissues. Immunohistochemistry and semiquantitative histomorphometric analysis confirmed the presence of AQP-1 in endothelial barriers of almost all tissues and in many epithelial barriers. AQP-1 was highly expressed in the renal cortex, choroid plexus, and pancreatic ducts. AQP-1 expression levels were surprisingly high in the anus, gallbladder, and liver; moderate expression was also detected in the hippocampus and ependymal cells of the central nervous system. This is the first report of AQP-1 protein distribution in normal human TMAs. These findings confirm the presence of AQP-1 in human endothelia and selected water-transporting epithelia and several new locations, including mammary epithelium, articular chondrocytes, synoviocytes, and synovial microvessels where AQP-1 may be involved in milk production, chondrocyte volume regulation, synovial fluid secretion, and homeostasis, respectively.
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Chondrocytes exist in an unusual and highly variable ionic and osmotic environment in the extracellular matrix of articular cartilage. Alterations to the ionic and osmotic environment of chondrocytes influence the volume and ionic content of the cells, which, in turn, modifies the rate at which extracellular matrix macromolecules are synthesized and degraded. Thus, regulation of the water and solute content of chondrocytes will profoundly affect their anabolic and catabolic functions. The water content of cells is effectively influenced by the abundance of aquaporin (AQP) water channels. Recent studies have shown that several AQP water channel isoforms are expressed in chondrocytes from Meckel's cartilage, developing teeth and other orofacial tissues. The aim of the present investigation was to determine if chondrocytes from equine articular cartilage express AQP water channels. Polyclonal antibodies to AQP1, AQP2 and AQP3 were used in conjunction with immunohistochemistry, immunoblotting and quantitative flow cytometry to determine if AQP1, AQP2 and AQP3 are expressed in equine articular chondrocytes. Our studies show that AQP1 and AQP3 are expressed by chondrocytes in articular cartilage in situ and in isolated chondrocytes. We found no evidence for expression of AQP2. the vasopressin-regulated water channel in chondrocytes. AQP1 and AQP3 may be involved in the transport of water and small solutes and osmotically active metabolites across the chondrocyte plasma membrane during volume regulatory behaviour. AQP1 may be involved in transporting metabolic water. AQP3 may participate in the transport of glycerol and structurally related molecules.
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A new member of the aquaporin family (AQP10) has recently been identified in the human small intestine by molecular cloning and in situ hybridization. Ribonuclease protection assay and northern blotting have demonstrated that AQP10 is expressed in the human duodenum and jejunum. However, the subcellular distribution of the AQP10 protein and its plasma membrane polarization have not yet been established. The objective of this study was to determine the distribution of the AQP10 protein in the human ileum by immunohistochemistry and western blotting using a polyclonal antibody raised against a unique 17-amino acid peptide derived from the human AQP10 sequence. The distribution of the AQP1 and AQP3 proteins was also studied by immunohistochemical staining using affinity-purified polyclonal antibodies. Results revealed that the AQP10 protein is preferentially targeted to the apical membrane domain of absorptive intestinal epithelial cells, whereas AQP3 is located in the basolateral membrane of the cells and AQP1 expression is restricted to the mucosal microvascular endothelia. The presence of AQP10 in the apical membrane of intestinal villi suggests that this protein may represent an entry pathway for water and small solutes from the lumen across to the mucosal side.
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The antidiuretic hormone arginine vasopressin (AVP) increases the osmotic water permeability of the renal collecting ducts by inducing the shuttling of aquaporin-2 (AQP2) water channels from intracellular vesicles to the apical plasma membrane of the principal cells. This process has been demonstrated to be dependent upon the cytoskeleton and protein kinase A (PKA). Previous studies in the toad urinary bladder, a functional homologue of the renal collecting duct, have demonstrated that the sulphydryl reagent N-ethylmaleimide (NEM) is also able to activate the vasopressin-sensitive water permeability pathway in this tissue. The aim of the present study was to investigate the effects of NEM on AQP2 trafficking in a mammalian system. We show that NEM causes translocation of AQP2 from the cytosol to the plasma membrane in rat inner medullary collecting ducts; like the response to AVP, this action was also dependent upon an intact cytoskeleton and PKA. This effect is not mediated by cAMP, but results from direct activation of PKA by NEM.
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AVP increases the osmotic water permeability of renal collecting ducts by inducing the translocation of specific aquaporin-2 (AQP2) water channels from cytoplasmic vesicles to the apical plasma membrane of the principal cells. Here, we report a novel inner medullary tubule suspension for the study of this phenomenon that overcomes some of the drawbacks faced by present techniques; both primary cultures of inner medullary collecting duct cells and cell lines expressing AQP2 show aberrant trafficking and/or signaling pathways. The tubule suspensions were prepared by proteolytic digestion of inner medullas dissected from freshly isolated rat kidneys. After drug treatment, cellular distribution of AQP2 was determined by membrane fractionation and Western blotting or by immunocytochemistry. Treatment of suspensions with 1 nM AVP caused redistribution of AQP2 to the apical plasma membrane of the principal cells, a process inhibited by microtubule disruption or PKA inhibition. We conclude that this method provides a valuable new approach to the study of the cellular mechanisms involved in the response of the collecting duct to AVP.
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Aquaporin (AQP) water channels are important in the function of the kidney. Constitutively expressed AQP1 in the proximal tubule and descending limb is important in normal fluid absorption and in the counter-current multiplication system. The vasopressin-regulated shuttling of AQP2 is essential in antidiuresis and the regulation of water balance. Genetic damage to AQPs, or pathological changes in expression or function, impair renal water handling. The most striking examples of this involve disruption of AQP2 function, which can result in profound nephrogenic diabetes insipidus. Aquaporin 1 is present in capillaries and venules and appears to be important in peritoneal dialysis, where it appears to represent the "ultrasmall pores" of the three-pore model. Decreased expression or function of AQP1 may be responsible for some cases of ultrafiltration failure, but further evidence will be required to establish whether this is the case.
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