Aquaporins in the digestive system

Fluid transfer such as secretion and absorption is one of the major functions of the digestive system. Aquaporins are water channel proteins providing water transfer across the cellular membrane. At least six aquaporin isoforms are expressed in the digestive system. Aquaporin-1 (AQP1) is widely distributed in endothelial cells of capillaries and small vessels as well as in the central lacteals in the small intestine. AQP1 is also present in the duct system in the pancreas, liver, and bile duct. AQP3 is mainly expressed in the epithelia of the upper digestive tract from the oral cavity to the stomach and of the lower digestive tract from the distal colon to the anus. AQP4 is present in the parietal cells of the stomach and in the intestinal epithelia. AQP5 is expressed in acinar cells of the salivary, pyloric, and duodenal glands. AQP8 is expressed in the intestinal epithelia, salivary glands, pancreas, and liver. AQP9 is present in the liver and intestinal goblet cells. Aquaporins have important roles in the digestive system, such as AQP5 in saliva secretion, as shown by the studies on AQP5-null mice. In addition, water transfer across the digestive epithelia seems to occur not only via aquaporins but also via other transporter or channel systems.     

Role of water channels in fluid transport studied by phenotype analysis of aquaporin knockout mice

Aquaporin-type water channels are expressed widely in mammalian tissues, particularly in the kidney, lung, eye and gastrointestinal tract. To define the role of aquaporins in organ physiology, we have generated and analysed transgenic mice lacking aquaporins (AQP) 1, 3, 4 and 5. Multiple phenotype abnormalities were found in the null mice. For example, in kidney, deletion of AQP1 or AQP3 produced marked polyuria whereas AQP4 deletion produced only a mild concentrating defect. Deletion of AQP5, the apical membrane water channel in the salivary gland, caused defective saliva production. Deletion of AQP1 or AQP5, water channels in lung endothelia and epithelia, resulted in a 90% decrease in airspace-capillary water permeability. In the brain, deletion of AQP4 conferred marked protection from brain swelling induced by acute water intoxication and ischaemic stroke. The general paradigm that has emerged from these phenotype studies is that aquaporins facilitate rapid near-isosmolar transepithelial fluid absorption/secretion, as well as rapid vectorial water movement driven by osmotic gradients. However, we have found many examples in which the tissue-specific expression of an aquaporin is not associated with any apparent phenotypic abnormality. The physiological data on aquaporin null mice suggest the utility of aquaporin blockers and aquaporin gene replacement in selected human diseases.     

Screening of aquaporin 7 and aquaporin 8 expression in 35 organs using semi-quantified RT-PCR methods

Screening of aquaporin 7 and aquaporin 8 expression in 35 organs usingsemi-quantified RT-PCR methods     

Natriuretic peptides up-regulate aquaporin 3 in a human colonic epithelial cell line

Several specialized channels termed aquaporins (AQPs) facilitate water transport in the gastrointestinal tract. AQP3 localizes to epithelial cells in the human small intestine and colon. However, the regulatory mechanisms responsible for AQP3 function in the gastrointestinal tract are not well understood. To characterize the regulation of AQP3 expression by atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), we studied mRNA expression by Northern blotting, protein expression by Western blotting and DNA binding activity by electrophoretic mobility shift assay (EMSA) in the human colonic epithelial cell line HT-29. We also used several inhibitors to investigate signal transduction. AQP3 mRNA was up-regulated in addition to ANP (>100 nM) and BNP (>10 nM). The expression of AQP3 protein was enhanced at 1 h after the addition of ANP and BNP. The combination of protein kinase-A (PK-A) and protein kinase-G (PK-G) inhibitors completely inhibited the expression of AQP3 mRNA enhanced by ANP or BNP to its basal level. The EMSA of the cyclic-AMP response element (CRE) in HT-29 cells revealed a single band. These results indicate that ANP and BNP up-regulated the expression of AQP3 mRNA and protein, and both PK-A and PK-G dependent pathways mediated this effect.     

Aquaporin water channels and endothelial cell function

The aquaporins (AQP) are a family of homologous water channels expressed in many epithelial and endothelial cell types involved in fluid transport. AQP1 protein is strongly expressed in most microvascular endothelia outside of the brain, as well as in endothelial cells in cornea, intestinal lacteals, and other tissues. AQP4 is expressed in astroglial foot processes adjacent to endothelial cells in the central nervous system. Transgenic mice lacking aquaporins have been useful in defining their role in mammalian physiology. Mice lacking AQP1 manifest defective urinary concentrating ability, in part because of decreased water permeability in renal vasa recta microvessels. These mice also show a defect in dietary fat processing that may involve chylomicron absorption by intestinal lacteals, as well as defective active fluid transport across the corneal endothelium. AQP1 might also play a role in tumour angiogenesis and in renal microvessel structural adaptation. However, AQP1 in most endothelial tissues does not appear to have a physiological function despite its role in osmotically driven water transport. For example, mice lacking AQP1 have low alveolar-capillary water permeability but unimpaired lung fluid absorption, as well as unimpaired saliva and tear secretion, aqueous fluid outflow, and pleural and peritoneal fluid transport. In the central nervous system mice lacking AQP4 are partially protected from brain oedema in water intoxication and ischaemic models of brain injury. Therefore, although the role of aquaporins in epithelial fluid transport is in most cases well-understood, there remain many questions about the role of aquaporins in endothelial cell function. It is unclear why many leaky microvessels strongly express AQP1 without apparent functional significance. Improved understanding of aquaporin-endothelial biology may lead to novel therapies for human disease, such as pharmacological modulation of corneal fluid transport, renal fluid clearance and intestinal absorption.     

Comparative distribution of carbonic anhydrase isozymes III and II in rodent tissues

Carbonic anhydrase (CA) III was demonstrated immunocytochemically in epithelium in some regions of salivary gland ducts, colon, bronchi, and male genital tract and in adipocytes, in addition to skeletal muscle and liver where the isozyme was previously localized. Basal cells beneath the submandibular gland's excretory ducts in guinea pig stained for CA III. Carbonic anhydrase III occurred alone in some and with CA II in other sites but was often absent from CA-II-containing types of cells. This was exemplified by CA III's abundance in CA-II-positive proximal colon and its sparsity in the CA-II-rich distal colon of the mouse. Striated ducts in guinea pig, but not mouse salivary glands, stained darker for CA and appeared accordingly to function more actively in ion transport compared with excretory ducts. Carbonic anhydrase content varied among genera in liver and pancreas and between mouse species and strains in salivary glands and kidney. Newly observed murine sites of CA II activity included Auerbach's plexus and a population of leukocytes infiltrating the lamina propria in small intestine, and several types of cells in the male genital tract. In immunoblot tests, antisera to CA III showed no cross reactivity with antisera to CA II, but those to CA II disclosed weak cross reactivity with CA III.     

Cellular localization of aquaporins along the secretory pathway of the lactating bovine mammary gland: An immunohistochemical study

In this study we examined the cellular localization of aquaporins (AQPs) along the secretory pathway of actively lactating bovine mammary glands using immunohistochemistry. Mammary tissues examined included secretory ducts and acini, gland cisterns, teats, stromal and adipose tissues. Aquaporin 1 (AQP1) was localized in capillary endothelia throughout the mammary gland in addition to myoepithelial cells underlying teat duct epithelia. AQP2 and AQP6 were not detected and AQP9 was found only in leukocytes. AQP3 and AQP4 were observed in selected epithelial cells in the teat, cistern and secretory tubuloalveoli. AQP5 immunopositivity was prominent in the cistern. AQP3 and AQP7 were found in smooth muscle bundles in the teat, secretory epithelial cells and duct epithelial cells. These immunohistochemical findings support a functional role for aquaporins in the transport of water and small solutes across endothelial and epithelial barriers in the mammary gland and in the production and secretion of milk.     

Genetic deletion of aquaporin-1 results in microcardia and low blood pressure in mouse with intact nitric oxide-dependent relaxation,but enhanced prostanoids-dependent relaxation

The water channels, aquaporins (AQPs) are key mediators of transcellular fluid transport. However, their expression and role in cardiac tissue is poorly characterized. Particularly, AQP1 was suggested to transport other molecules (nitric oxide (NO), hydrogen peroxide (H₂O₂)) with potential major bearing on cardiovascular physiology. We therefore examined the expression of all AQPs and the phenotype of AQP1 knockout mice (vs. wild-type littermates) under implanted telemetry in vivo, as well as endothelium-dependent relaxation in isolated aortas and resistance vessels ex vivo. Four aquaporins were expressed in wild-type heart tissue (AQP1, AQP7, AQP4, AQP8) and two aquaporins in aortic and mesenteric vessels (AQP1–AQP7). AQP1 was expressed in endothelial as well as cardiac and vascular muscle cells and co-segregated with caveolin-1. AQP1 knockout (KO) mice exhibited a prominent microcardia and decreased myocyte transverse dimensions despite no change in capillary density. Both male and female AQP1 KO mice had lower mean BP, which was not attributable to altered water balance or autonomic dysfunction (from baroreflex and frequency analysis of BP and HR variability). NO-dependent BP variability was unperturbed. Accordingly, endothelium-derived hyperpolarizing factor (EDH(F)) or NO-dependent relaxation were unchanged in aorta or resistance vessels ex vivo. However, AQP1 KO mesenteric vessels exhibited an increase in endothelial prostanoids-dependent relaxation, together with increased expression of COX-2. This enhanced relaxation was abrogated by COX inhibition. We conclude that AQP1 does not regulate the endothelial EDH or NO-dependent relaxation ex vivo or in vivo, but its deletion decreases baseline BP together with increased prostanoids-dependent relaxation in resistance vessels. Strikingly, this was associated with microcardia, unrelated to perturbed angiogenesis. This may raise interest for new inhibitors of AQP1 and their use to treat hypertrophic cardiac remodeling.     

Immunolocalization of the water channel, aquaporin-5 (AQP5), in the rat digestive system

Aquaporin-5 (AQP5), an isoform of membrane water channel aquaporins, is expressed in the salivary and lacrimal glands. We surveyed the expression and immunohistochemical localization of AQP5 in the rat digestive system. RT-PCR analysis revealed that AQP5 is expressed in the submandibular gland, tongue, gastric corpus, pyloric region, duodenum, and liver. Immunofluorescence microscopy using AQP5-specific antibodies showed that AQP5 protein is present in the minor salivary glands of the tongue, the pyloric glands, and duodenal glands. To distinguish apical and basolateral domains of the plasma membrane of epithelial cells, double-immunofluorescence staining for AQP5 and tight junction protein occludin was performed. In the minor salivary gland, AQP5 was present in both the serous and mixed secretory end portions. AQP5 was found in the apical membrane of the secretory cells including intercellular secretory canaliculi demarcated with occludin. At higher magnifications, omega-shaped indentations of AQP5 labeling were seen along the apical membrane, suggesting a dynamic process for the apical membrane in exocytosis. Only weak labeling for AQP5 was detected in the basolateral domain. In the stomach, AQP5 was detected in the apical membrane of the pyloric gland secretory cells. In the duodenum, AQP5 was restricted to duodenal glands, where it was localized to the apical membrane. AQP5 was not detected in the intestinal glands or cells in the villi. These observations show that AQP5 is localized mainly in the apical membrane, including intercellular secretory canaliculi of secretory cells in the minor salivary glands, pyloric glands, and duodenal glands. AQP5 appears to play an important role in water transfer in these glands.     

Use of aquaporins 1 and 5 levels as a diagnostic marker in mild-to-moderate adult-onset asthma

Characteristic features of asthma include airway inflammation and hyperactivity, mucus hypersecretion, mucosal edema, and airway remodeling. These features could be due to pathological water transport across pulmonary epithelia and aquaporins (AQPs) have recently been isolated as key proteins in fluid transportation in the human respiratory tract. We aimed to evaluate the role of aquaporins in the pathogenesis of asthma and their possible use a diagnostic marker of the disease. A total of 110 hospitalized and outpatients with mild to moderate adult-onset asthma were invited to participate in this study and 34 submitted an induced sputum sample adequate for analysis. The amount of AQP1, AQP5 and MUC5AC were measured with ELISA assay. The amount of IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ and IL-17 in both serum and sputum were measured with Cytometry Bead Array (CBA kit). Our results suggest that sputum AQP5, AQP1 and MUC5AC are all in a good correlation (r=0.498 between AQP5 and AQP1, r=0.529 and r=0.661 between MUC5AC and AQP5 or AQP1, respectively, all P<0.05). The AUC value for AQP1 and AQP5 to diagnose asthma were 0.729 and 0.745, respectively. In conclusion, water homeostasis plays an important role in maintaining adequate fluid transportation within the lung and is involved in the pathogenesis of asthma. Our results suggest that AQP may influence pulmonary physiology that their dysfunction can contribute to pulmonary pathogenesis, such as asthma. Moreover, their quantification could serve as biomarkers for the diagnosis of asthma.     

NH3 and NH 4 + permeability in aquaporin-expressing Xenopus oocytes

We have shown recently, in a yeast expression system, that some aquaporins are permeable to ammonia. In the present study, we expressed the mammalian aquaporins AQP8, AQP9, AQP3, AQP1 and a plant aquaporin TIP2;1 in Xenopus oocytes to study the transport of ammonia (NH₃) and ammonium (NH₄⁺) under open-circuit and voltage-clamped conditions. TIP2;1 was tested as the wild-type and in a mutated version (tip2;1) in which the water permeability is intact. When AQP8-, AQP9-, AQP3- and TIP2;1-expressing oocytes were placed in a well-stirred bathing medium of low buffer capacity, NH₃ permeability was evident from the acidification of the bathing medium; the effects observed with AQP1 and tip2;1 did not exceed that of native oocytes. AQP8, AQP9, AQP3, and TIP2;1 were permeable to larger amides, while AQP1 was not. Under voltage-clamp conditions, given sufficient NH₃, AQP8, AQP9, AQP3, and TIP2;1 supported inwards currents carried by NH₄⁺. This conductivity increased as a sigmoid function of external [NH₃]: for AQP8 at a bath pH (pH_e) of 6.5, the conductance was abolished, at pH_e 7.4 it was half maximal and at pH_e 7.8 it saturated. NH₄⁺ influx was associated with oocyte swelling. In comparison, native oocytes as well as AQP1 and tip2;1-expressing oocytes showed small currents that were associated with small and even negative volume changes. We conclude that AQP8, AQP9, AQP3, and TIP2;1, apart from being water channels, also support significant fluxes of NH₃. These aquaporins could support NH₄⁺ transport and have physiological implications for liver and kidney function.     

Aquaporins in the central nervous system

In this review, we have tried to summarize most available data dealing with the aquaporin (AQP) family of water channels in the CNS. Two aquaporins have been identified so far in the CNS, AQP1 and AQP4. AQP1 is restricted to the choroid plexus of the lateral ventricles, which raises a role for this aquaporin in cerebrospinal fluid formation. AQP4 is the predominant water channel in the brain and it is more widely distributed than originally believed, with a marked prevalence over periventricular areas. In the first part of this review, we examine the complete distribution pattern of AQP4 in the CNS including its rostro-caudal localization to end with its subcellular location. After discussing scarce data dealing with regulation of aquaporins in the CNS, we focus in potential roles for aquaporins. Novel recent data highlights very important roles for this aquaporin in the normal and pathological brain including, among others, role in potassium buffering, body fluid homeostasis, central osmoreception and development and restoration of brain edema.     

Role of aquaporins in lung liquid physiology

Aquaporins (AQPs) are small, integral membrane proteins that facilitate water transport across cell membranes in response to osmotic gradients. Water transport across epithelia and endothelia in the peripheral lung and airways occurs during airway hydration, alveolar fluid transport and submucosal gland secretion. Several AQPs are expressed in the lung and airways: AQP1 in microvascular endothelia, AQP3 and AQP4 in airway epithelia, and AQP5 in type I alveolar epithelial cells, submucosal gland acini, and a subset of airway epithelial cells. Phenotype analysis of transgenic knockout mice lacking AQPs has defined their roles in the lung and airways. AQP1 and AQP5 provide the principal route for osmotically driven water transport between airspace and capillary compartments; however, alveolar fluid clearance in the neonatal and adult lung is not affected by their deletion, nor is lung fluid accumulation in experimental models of lung injury. In the airways, though AQP3 and AQP4 facilitate osmotic water transport, their deletion does not impair airway hydration, regulation of airway surface liquid, or fluid absorption. In contrast to these negative findings, AQP5 deletion in submucosal glands reduced fluid secretion by >50%. The substantially slower fluid transport in the lung compared to renal and secretory epithelia probably accounts for the lack of functional significance of AQPs in the lung and airways. Recent data outside of the lung implicating the involvement of AQPs in cell migration and proliferation suggests possible new roles for lung AQPs to be explored.     

Expression and localization of epithelial aquaporins in the adult human lung

Aquaporins (AQPs) facilitate water transport across epithelia and play an important role in normal physiology and disease in the human airways. We used in situ hybridization and immunofluorescence to determine the expression and cellular localization of AQPs 5, 4, and 3 in human airway sections. In nose and bronchial epithelia, AQP5 is expressed at the apical membrane of columnar cells of the superficial epithelium and submucosal gland acinar cells. AQP4 was detected in basolateral membranes in ciliated ducts and by in situ in gland acinar cells. AQP3 is present on basal cells of both superficial epithelium and gland acinus. In these regions AQPs 5, 4, and 3 are appropriately situated to permit transepithelial water permeability. In the small airways (proximal and terminal bronchioles) AQP3 distribution shifts from basal cell to surface expression (i.e., localized to the apical membrane of proximal and terminal bronchioles) and is the only AQP identified in this region of the human lung. The alveolar epithelium has all three AQPs represented, with AQP5 and AQP4 localized to type I pneumocytes and AQP3 to type II cells. This study describes an intricate network of AQP expression that mediates water transport across the human airway epithelium.     

Evidence that aquaporin-8 is located in the basolateral membrane of rat submandibular gland acinar cells

It is possible that, during primary saliva formation, aquaporins (AQPs) facilitate transcellular water flow across acinar cells to the lumina of salivary glands. In the rat submandibular gland (rSMG) AQP5 is localized in the apical membranes of acinar cells. The presence of a basolateral AQP in the same cell type has not been reported. We have therefore used immunofluorescence confocal microscopy to determine the subcellular localization of a newly discovered aquaporin, AQP8, in rSMG epithelial cells. The antibodies we used were made against the amino- or carboxyl-terminus (anti-rAQP8NT and anti-rAQP8CT, respectively) of an AQP8 cloned from rat pancreas and liver (rAQP8). Two lines of evidence suggest that both antibodies are suitable for immunolocalization studies. First, results of immunofluorescence confocal microscopy studies show that both antibodies bind to the plasma membranes of 293 cells infected with an adenovirus encoding rAQP8. Second, results of immunoblots of membranes from infected cells suggest that both antibodies bind to glycosylated and non-glycosylated forms of rAQP8. When tested in frozen sections of rSMG, we could not detect the binding of anti-rAQP8NT to any membranes. In contrast, anti-rAQP8CT binds to the basolateral membranes of acinar (but not ductal) epithelia, suggesting that rAQP8 resides in the basolateral membranes of acinar cells. Lack of anti-rAQPNT binding to basolateral membranes suggests that this epitope is not available in the membranes. Our evidence for the basolateral localization of rAQP8 in acinar cells, coupled with previous findings that AQP5 is localized apically in the same cells, raises the possibility that water crosses the acinar epithelium through these channels during primary saliva formation.     

Expression of heterologous aquaporins for functional analysis in Saccharomyces cerevisiae

In this study the yeast Saccharomyces cerevisiae, which is a genetically tractable model for analysis of osmoregulation, has been used for analysis of heterologous aquaporins. Aquaporin water channels play important roles in the control of water homeostasis in individual cells and multicellular organisms. We have investigated the effects of functional expression of the mammalian aquaporins AQP1 and AQP5 and the aquaglyceroporins AQP3 and AQP9. Expression of aquaporins caused moderate growth inhibition under hyperosmotic stress, while expression of aquaglyceroporins mediated strong growth inhibition due to glycerol loss. Water transport was monitored in protoplasts, where the kinetics of bursting was influenced by presence of aquaporins but not aquaglyceroporins. We observed glycerol transport through aquaglyceroporins, but not aquaporins, in a yeast strain deficient in glycerol production, whose growth depends on glycerol inflow. In addition, a gene reporter assay allowed to indirectly monitor the effect of AQP9-mediated enhanced glycerol loss on osmoadaptation. Transport activity of certain aqua(glycero)porins was diminished by low pH or CuSO₄, suggesting that yeast can potentially be used for screening of putative aquaporin inhibitors. We conclude that yeast is a versatile system for functional studies of aquaporins, and it can be developed to screen for compounds of potential pharmacological use.     

Aquaporins in the kidney: from molecules to medicine.

The discovery of aquaporin-1 (AQP1) answered the long-standing biophysical question of how water specifically crosses biological membranes. In the kidney, at least seven aquaporins are expressed at distinct sites. AQP1 is extremely abundant in the proximal tubule and descending thin limb and is essential for urinary concentration. AQP2 is exclusively expressed in the principal cells of the connecting tubule and collecting duct and is the predominant vasopressin-regulated water channel. AQP3 and AQP4 are both present in the basolateral plasma membrane of collecting duct principal cells and represent exit pathways for water reabsorbed apically via AQP2. Studies in patients and transgenic mice have demonstrated that both AQP2 and AQP3 are essential for urinary concentration. Three additional aquaporins are present in the kidney. AQP6 is present in intracellular vesicles in collecting duct intercalated cells, and AQP8 is present intracellularly at low abundance in proximal tubules and collecting duct principal cells, but the physiological function of these two channels remains undefined. AQP7 is abundant in the brush border of proximal tubule cells and is likely to be involved in proximal tubule water reabsorption. Body water balance is tightly regulated by vasopressin, and multiple studies now have underscored the essential roles of AQP2 in this. Vasopressin regulates acutely the water permeability of the kidney collecting duct by trafficking of AQP2 from intracellular vesicles to the apical plasma membrane. The long-term adaptational changes in body water balance are controlled in part by regulated changes in AQP2 and AQP3 expression levels. Lack of functional AQP2 is seen in primary forms of diabetes insipidus, and reduced expression and targeting are seen in several diseases associated with urinary concentrating defects such as acquired nephrogenic diabetes insipidus, postobstructive polyuria, as well as acute and chronic renal failure. In contrast, in conditions with water retention such as severe congestive heart failure, pregnancy, and syndrome of inappropriate antidiuretic hormone secretion, both AQP2 expression levels and apical plasma membrane targetting are increased, suggesting a role for AQP2 in the development of water retention. Continued analysis of the aquaporins is providing detailed molecular insight into the fundamental physiology and pathophysiology of water balance and water balance disorders.     

Role of gastrin in gastrointestinal adaptation after small bowel resection

Gastrin is a trophic hormone for the mucosa of the oxyntic gland area of the stomach, the proximal small intestine, and the colon. It also has trophic effects on the pancreas. All these tissues undergo hyperplasia to some extent following distal small bowel resection. This study evaluates the role of gastrin in postresectional hyperplasia by examining the growth of these tissues in antrectomized rats following intestinal resection. Antrectomy itself caused atrophy of the oxyntic gland mucosa, colonic mucosa, and the pancreas but had no effect on ileal mucosa. Resection by itself stimulated growth of all tissues and significantly increased serum gastrin levels. After resection in antrectomized animals, all tissues underwent an adaptational response. The increase in total DNA content after resection in antrectomized rats was as great in all tissues, except the colon, as it was in animals with intact antrums and normal gastrin levels. These results indicate that gastrin plays no role in the postresectional hyperplasia observed in the various tissues of the gastrointestinal tract.     

Cell lineage specificity of newly raised monoclonal antibodies against gastric mucins in normal, metaplastic, and neoplastic human tissues and their application to pathology diagnosis

The specificity of monoclonal antibodies against gastric mucins (designated as HIK1083, PGM 36, and PGM 37) was studied immunohistochemically in normal, metaplastic, and neoplastic human tissues. These antibodies labeled class III mucin-producing cells identified by paradoxical concanavalin A staining in normal stomach, duodenum (Brunner gland), biliary tract, and main pancreatic duct; in mucinous metaplasia of pancreas and gallbladder; and in adenocarcinomas of stomach (90%), bile duct (80%), gallbladder (100%), pancreas (80%), lung (100% of goblet cell type adenocarcinomas), ovary (67% of mucinous carcinomas), and uterine cervix (100% of adenoma malignum tumors). Normal and neoplastic cells of esophagus, colon, salivary gland, kidney, endometrium, breast, prostate, and liver, as well as normal small intestine, lung, and uterine cervix, were all negative. The antibodies used should be valuable for the detection of class III mucin and class III mucin-producing cells in normal, metaplastic, and neoplastic tissues.