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The pulmonary epithelial glycocalyx, an anionic cell surface layer enriched in glycosaminoglycans such as heparan sulfate and chondroitin sulfate, contributes to the alveolar barrier. Direct injury to the pulmonary epithelium induces shedding of heparan sulfate into the air space; the impact of this shedding on recovery after lung injury is unknown. Using mass spectrometry, we found that heparan sulfate was shed into the air space for up to 3 wk after intratracheal bleomycin-induced lung injury and coincided with induction of matrix metalloproteinases (MMPs), including MMP2. Delayed inhibition of metalloproteinases, beginning 7 days after bleomycin using the nonspecific MMP inhibitor doxycycline, attenuated heparan sulfate shedding and improved lung function, suggesting that heparan sulfate shedding may impair lung recovery. While we also observed an increase in air space heparanase activity after bleomycin, pharmacological and transgenic inhibition of heparanase in vivo failed to attenuate heparan sulfate shedding or protect against bleomycin-induced lung injury. However, experimental augmentation of airway heparanase activity significantly worsened post-bleomycin outcomes, confirming the importance of epithelial glycocalyx integrity to lung recovery. We hypothesized that MMP-associated heparan sulfate shedding contributed to delayed lung recovery, in part, by the release of large, highly sulfated fragments that sequestered lung-reparative growth factors such as hepatocyte growth factor. In vitro, heparan sulfate bound hepatocyte growth factor and attenuated growth factor signaling, suggesting that heparan sulfate shed into the air space after injury may directly impair lung repair. Accordingly, administration of exogenous heparan sulfate to mice after bleomycin injury increased the likelihood of death due to severe lung dysfunction. Together, our findings demonstrate that alveolar epithelial heparan sulfate shedding impedes lung recovery after bleomycin.

Water conservation is one of the most challenging processes for terrestrial vertebrates and is necessary for their survival. Birds are the only vertebrate animals other than mammals that have the ability to concentrate their urine. Previously, we identified and characterized aquaporins (AQP)1–4 responsible for urine concentration in Japanese quail kidneys. Today, a total of 13 orthologs for these genes have been reported in birds. Bird AQPs can be classified into four subfamilies: 1) Classical AQPs (AQP0–5 and novel member, AQP4-like) that conserve the selectivity filter; 2) aquaglyceroporins (AQP3, 7, 9 and 10) that retain an aspartic acid residue in the second NPA box and expand the pore to accept larger molecules; 3) unorthodox AQPs (AQP11–12) which structurally resemble their mammalian counterparts; 4) AQP8-type, a subfamily that differs from mammalian AQP8. Interestingly, over the course of time, birds lost their mammalian counterpart AQP6 but obtained a novel AQP4-like aquaporin member. In quail and/or chicken kidneys, at least six AQPs are expressed. Quail AQP1 (qAQP1) is expressed in both cortical and medullary proximal tubules but is absent in the descending limb (DL) and the thick ascending limb (TAL), supporting our previous finding that the DL and TAL are water impermeable. AQP2, an arginine vasotocin (AVT)-sensitive water channel, is exclusively expressed in the principal cells of the collecting duct (CD). AQP4 is unlikely to participate in free water resorption from the collecting duct (CD), and only AQP3 may represent an exit pathway for water reabsorbed apically via AQP2. While AQP9 is not expressed in mammalian kidneys, AQP9 was recently found in chicken kidneys. This review summarizes the current knowledge of the structure, function and expression of bird AQPs.

The lung epithelial glycocalyx is a carbohydrate-enriched layer lining the pulmonary epithelial surface. Although epithelial glycocalyx visualization has been reported, its composition and function remain unknown. Using immunofluorescence and mass spectrometry, we identified heparan sulfate (HS) and chondroitin sulfate within the lung epithelial glycocalyx. In vivo selective enzymatic degradation of epithelial HS, but not chondroitin sulfate, increased lung permeability. Using mass spectrometry and gel electrophoresis approaches to determine the fate of epithelial HS during lung injury, we detected shedding of 20 saccharide-long or greater HS into BAL fluid in intratracheal LPS-treated mice. Furthermore, airspace HS in clinical samples from patients with acute respiratory distress syndrome correlated with indices of alveolar permeability, reflecting the clinical relevance of these findings. The length of HS shed during intratracheal LPS-induced injury (≥20 saccharides) suggests cleavage of the proteoglycan anchoring HS to the epithelial surface, rather than cleavage of HS itself. We used pharmacologic and transgenic animal approaches to determine that matrix metalloproteinases partially mediate HS shedding during intratracheal LPS-induced lung injury. Although there was a trend toward decreased alveolar permeability after treatment with the matrix metalloproteinase inhibitor, doxycycline, this did not reach statistical significance. These studies suggest that epithelial HS contributes to the lung epithelial barrier and its degradation is sufficient to increase lung permeability. The partial reduction of HS shedding achieved with doxycycline is not sufficient to rescue epithelial barrier function during intratracheal LPS-induced lung injury; however, whether complete attenuation of HS shedding is sufficient to rescue epithelial barrier function remains unknown.

Intratracheal (IT) administration of experimental agents is an essential technique in murine models of diffuse lung diseases, such as bleomycin-induced pulmonary fibrosis. However, distribution of intratracheally-administered agents to the distal mouse lung is often asymmetric, with lung parenchymal concentrations increased in the smaller (but equally accessible) left lung of the mouse. Described in this report is a novel intrabronchial (IB) approach to cannulate the left and/or right lungs of living mice non-operatively. It is also demonstrated how this approach can be used to selectively administer agents to one lung or adapted (via dose-adjusted IB delivery) to improve the left-right symmetry of lung delivery of experimental agents, thereby improving models of diffuse lung disease such as bleomycin-induced pulmonary fibrosis.

The mechanistic target of rapamycin (mTOR) is a central regulator of cellular responses to environmental stress. mTOR (and its primary complex mTORC1) is, therefore, ideally positioned to regulate lung inflammatory responses to an environmental insult, a function directly relevant to disease states such as the acute respiratory distress syndrome. Our previous work in cigarette smoke-induced emphysema identified a novel protective role of pulmonary mTORC1 signaling. However, studies of the impact of mTORC1 on the development of acute lung injury are conflicting. We hypothesized that Rtp801, an endogenous inhibitor of mTORC1, which is predominantly expressed in alveolar type II epithelial cells, is activated during endotoxin-induced lung injury and functions to suppress anti-inflammatory epithelial mTORC1 responses. We administered intratracheal lipopolysaccharide to wild-type mice and observed a significant increase in lung Rtp801 mRNA. In lipopolysaccharide-treated Rtp801(-/-) mice, epithelial mTORC1 activation significantly increased and was associated with an attenuation of lung inflammation. We reversed the anti-inflammatory phenotype of Rtp801(-/-) mice with the mTORC1 inhibitor, rapamycin, reassuring against mTORC1-independent effects of Rtp801. We confirmed the proinflammatory effects of Rtp801 by generating a transgenic Rtp801 overexpressing mouse, which displayed augmented inflammatory responses to intratracheal endotoxin. These data suggest that epithelial mTORC1 activity plays a protective role against lung injury, and its inhibition by Rtp801 exacerbates alveolar injury caused by endotoxin.

For terrestrial vertebrates, water economy is a prerequisite for survival, and the kidney is their major osmoregulatory organ. Birds are the only vertebrates other than mammals that can concentrate urine in adaptation to terrestrial environments. Aquaporin (AQP) and glyceroporin (GLP) are phylogenetically old molecules and have been found in plants, microbial organisms, invertebrates, and vertebrates. Currently, 13 AQPs/aquaGLPs and isoforms are known to be present in mammals. AQPs 1, 2, 3, 4, 6, 7, 8, and 11 are expressed in the kidney; of these, AQPs 1, 2, 3, 4, and 7 are shown to be involved in fluid homeostasis. In avian kidneys, AQPs 1, 2, 3, and 4 have been identified and characterized. Also, gene and/or amino acid sequences of AQP5, AQP7, AQP8, AQP9, AQP11, and AQP12 have been reported in birds. AQPs 2 and 3 are expressed along cortical and medullary collecting ducts (CDs) and are responsible, respectively, for the water inflow and outflow of CD epithelial cells. While AQP4 plays an important role in water exit in the CD of mammalian kidneys, it is unlikely to participate in water outflow in avian CDs. This review summarizes current knowledge on structure and function of avian AQPs and compares them to those in mammalian and nonmammalian vertebrates. Also, we aim to provide input into, and perspectives on, the role of renal AQPs in body water homeostasis during ontogenic and phylogenetic advancement.

Sepsis outcomes are heavily dependent on the development of septic organ injury, but no interventions exist to interrupt or reverse this process. microRNA-223 (miR-223) is known to be involved in both inflammatory gene regulation and host-pathogen interactions key to the pathogenesis of sepsis. The goal of this study was to determine the role of miR-223 as a mediator of septic kidney injury. Using miR-223 knockout mice and multiple models of experimental sepsis, we found that miR-223 differentially influences acute kidney injury (AKI) based on the model used. In the absence of miR-223, mice demonstrated exaggerated AKI in sterile models of sepsis (LPS injection) and attenuated AKI in a live-infection model of sepsis (cecal ligation and puncture). We demonstrated that miR-223 expression is induced in kidney homogenate after cecal ligation and puncture, but not after LPS or fecal slurry injection. We investigated additional potential mechanistic explanations including differences in peritoneal bacterial clearance and host stool virulence. Our findings highlight the complex role of miR-223 in the pathogenesis of septic kidney injury, as well as the importance of differences in experimental sepsis models and their consequent translational applicability.

Water deprivation or arginine vasotocin upregulates aquaporin-2 (AQP2) expression in apical and subapical regions of medullary collecting duct (CD) cells of Coturnix coturnix quail (q) kidneys. We therefore aimed to determine whether the CD has AQPs mediating water exit from the intracellular to the extracellular (interstitial) space. Using a homologue cloning technique, we isolated two distinct qAQP4 cDNAs from quail medullary cones; long (L, open reading frames) and short (S) cDNA encoded 335 (qAQP4-L) and 301 (qAQP4-S) amino acids with, respectively, 80% and 87% identity to human long- and short-form AQP4. qAQP4-S is identical to qAQP4-L from the second initiation site. Both isoforms have two NPA motifs, but lack cysteine at the known mercury-sensitive site. qAQP4-L and qAQP4-S are expressed in membranes of Xenopus laevis oocytes, but both failed to increase the water permeability ( P f ) of oocytes exposed to a hypotonic solution. Glutamate (Q242) replacement with histidine did not increase P f . With conventional RT-PCR and real-time PCR, qAQP4-L/S mRNA signals were detected in the brain, lung, heart, intestine, adrenal gland, skeletal muscle, liver, and kidney (higher in medulla than in cortical region). qAQP4-L mRNA was detected only in the brain and adrenal gland. Orthogonal arrays of intramembranous particles were not detected in quail CDs. The results suggest that although qAQP4-L and qAQP4-S have high homology to mammalian AQP4, their physiological function may be different.

Both mammals and birds can concentrate urine hyperosmotic to plasma via a countercurrent multiplier mechanism, although evolutionary lines leading to mammals and birds diverged at an early stage of tetrapod evolution. We reported earlier (Nishimura H, Koseki C, and Patel TB. Am J Physiol Regul Integr Comp Physiol 271: R1535–R1543, 1996) that arginine vasotocin (AVT; avian antidiuretic hormone) increases diffusional water permeability in the isolated, perfused medullary collecting duct (CD) of the quail kidney. In the present study, we have identified an aquaporin (AQP) 2 homolog water channel in the medullary cones of Japanese quail, Coturnix coturnix (qAQP2), by RT-PCR-based cloning techniques. A full-length cDNA contains an 822-bp open reading frame that encodes a 274-amino acid sequence with 75.5% identity to rat AQP2. The qAQP2 has six transmembrane domains, two asparagine-proline-alanine (NPA) sequences, and putative N-glycosylation (asparagine-124) and phosphorylation sites (serine-257) for cAMP-dependent protein kinase. qAQP2 is expressed in the membrane of Xenopus laevis oocytes and significantly increased its osmotic water permeability (Pf), inhibitable ( P < 0.01) by mercury chloride. qAQP2 mRNA (RT-PCR) was detected in the kidney; medullary mRNA levels were higher than cortical levels. qAQP2 protein that binds to rabbit anti-rat AQP2 antibody is present in the apical/subapical regions of both cortical and medullary CDs from normally hydrated quail, and the intensity of staining increased only in the medullary CDs after water deprivation or AVT treatment. The relative density of the ∼29-kDa protein band detected by immunoblot from the medullary cones was modestly higher in water-deprived/AVT-treated quail. The results suggest that 1) medullary CDs of quail kidneys express a mercury-sensitive functioning qAQP2 water channel, and 2) qAQP2 is at least partly regulated by an AVT-dependent mechanism. This is the first clear identification of AQP2 homolog in nonmammalian vertebrates.

Systemic inflammatory illnesses (such as sepsis) are marked by degradation of the endothelial glycocalyx, a layer of glycosaminoglycans (including heparan sulfate, chondroitin sulfate, and hyaluronic acid) lining the vascular lumen. We hypothesized that different pathophysiologic insults would produce characteristic patterns of released glycocalyx fragments. We collected plasma from healthy donors as well as from subjects with respiratory failure due to altered mental status (intoxication, ischemic brain injury), indirect lung injury (non-pulmonary sepsis, pancreatitis), or direct lung injury (aspiration, pneumonia). Mass spectrometry was employed to determine the quantity and sulfation patterns of circulating glycosaminoglycans. We found that circulating heparan sulfate fragments were significantly (23-fold) elevated in patients with indirect lung injury, while circulating hyaluronic acid concentrations were elevated (32-fold) in patients with direct lung injury. N-Sulfation and tri-sulfation of heparan disaccharides were significantly increased in patients with indirect lung injury. Chondroitin disaccharide sulfation was suppressed in all groups with respiratory failure. Plasma heparan sulfate concentrations directly correlated with intensive care unit length of stay. Serial plasma measurements performed in select patients revealed that circulating highly sulfated heparan fragments persisted for greater than 3 days after the onset of respiratory failure. Our findings demonstrate that circulating glycosaminoglycans are elevated in patterns characteristic of the etiology of respiratory failure and may serve as diagnostic and/or prognostic biomarkers of critical illness.

Previous studies have shown that the calcitonin gene-related peptide (CGRP) immunoreactivity in the central nervous system (CNS) of adult rats is sexually dimorphic and regulated by sex steroid. In the present study, we used immunocytochemistry to investigate the sex difference in CGRP-immunoreactive (IR) neurons in rat dorsal root ganglia (DRG). The numbers of CGRP-IR neurons at the cervical, lumbar and sacral levels in the female rats were significantly lower than those of the male rats. We also found that the number of CGRP-IR neurons at the lumbar level was increased in ovariectomized (OVX) rats, but was decreased in estradiol (E2)-treated rats (OVX+E2). A large number of estrogen receptor (ER)-IR neurons at the lumbar level were found in the female rats, and its number was greater than that in the male rats. We also investigated the change in the number of ER-IR neurons of OVX rats after estrogen treatment. The number of ER-IR neurons in the OVX+E2 rats was consistent with that of the intact female rats, but was significantly increased in the OVX rats. As shown by a double-labeling immunocytochemical method, over 80% of the CGRP-IR neurons at the lumbar level showed ER immunoreactivity in the female, OVX and OVX+E2 rats, compared to only about 46% in the male rats. These results indicate that there is a gender difference in CGRP expression in the rat DRG, and that this CGRP expression might be downregulated by estrogen (at least in part) through its receptor.

In this study, we observed the changes of glucocorticoid receptor (GR)-immunoreactivity (ir) and cell death in the rat hippocampal granule cell layer at various periods after adrenalectomy (ADX). Our results revealed that all of the rats shortly after ADX showed a rapid loss of GR-ir and subsequent appearance of degenerating cells in the granule cell layer. One month after ADX, however, about 80% of the rats displayed a restoration of GR-ir and the absence of degenerating cells in the granule cell layer, and this phenomenon was successively noted for 6 months. Hippocampal structural destruction 3 and 6 months after ADX was found in about 20% of the rats with loss of GR-ir in the granule cell layer; the ADX rats with even weak GR-ir in this area had a normal hippocampus. The treatment of rats with synthetic GR agonist, dexamethasone, immediately after ADX prevented the loss of GR-ir and significantly reduced the number of degenerating cells in the granule cell layer. Our results clarified that granule cell death after ADX was necessarily accompanied by the disappearance of GR-ir in the granule cell layer, suggesting that ADX-induced granule cell death is predicated on the loss of GR-ir and that the presence of GR-ir in this area may be important for granule cell survival.

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.

Sepsis, a systemic inflammatory response to infection, commonly progresses to acute lung injury (ALI), an inflammatory lung disease with high morbidity. We postulated that sepsis-associated ALI is initiated by degradation of the pulmonary endothelial glycocalyx, leading to neutrophil adherence and inflammation. Using intravital microscopy, we found that endotoxemia in mice rapidly induced pulmonary microvascular glycocalyx degradation via tumor necrosis factor-α (TNF-α)-dependent mechanisms. Glycocalyx degradation involved the specific loss of heparan sulfate and coincided with activation of endothelial heparanase, a TNF-α-responsive, heparan sulfate-specific glucuronidase. Glycocalyx degradation increased the availability of endothelial surface adhesion molecules to circulating microspheres and contributed to neutrophil adhesion. Heparanase inhibition prevented endotoxemia-associated glycocalyx loss and neutrophil adhesion and, accordingly, attenuated sepsis-induced ALI and mortality in mice. These findings are potentially relevant to human disease, as sepsis-associated respiratory failure in humans was associated with higher plasma heparan sulfate degradation activity; moreover, heparanase content was higher in human lung biopsies showing diffuse alveolar damage than in normal human lung tissue.

We immunohistochemically studied the distribution of nucleoporin p62 in the hippocampus and hypothalamus of rat brain. Previous reports have shown the presence of p62-immunoreactivity (ir) in the nuclear rim in the non-neuronal cells, but the present study showed that of p62-ir within the nucleus in addition to the nuclear rim in the neuronal cells of the hippocampus and hypothalamic nuclei; in these areas the glucocorticoid receptor (GR) undergoes nucleocytoplasmic translocation determined by ligand. We analyzed the expression of p62-ir after adrenalectomy (ADX). ADX changed the localization of GR-ir from the nucleus to the cytoplasm, but did not change the localization or immunoreactivity of p62, suggesting that nucleoporin p62 is stable regardless of intracellular signal transduction between the cytoplasm and the nucleus.

Avian kidneys have loopless and looped nephrons; a countercurrent multiplier mechanism operates in the latter by NaCl recycling. We identified an aquaporin-2 (AQP2) homolog in apical/subapical regions of cortical and medullary collecting duct (CD) cells in kidneys of Japanese quail (q), Coturnix japonica. We investigated whether undernutrition during the embryonic/maturation period retards kidney and AQP2 development in quail and programs impaired volume regulation in adults. Protocols included 1) time course and 2) effects of 5–10% egg white withdrawal (EwW) or 48-h post-hatch food deprivation (FD) on nephron growth and qAQP2 mRNA expression, and 3) effects of EwW and FD on qAQP2 mRNA responses to 72-h water deprivation in adults. In metanephric kidneys, qAQP2 mRNA is expressed in medullary CDs at embryonic day 10; distribution and intensity increase during maturation. The number and size of glomeruli continue to increase after birth, whereas nephrogenic zones decrease. In EwW embryos, qAQP2 mRNA expression is initially delayed, then restored; birth weight and hatching rate are lower than in controls. Adults from EwW embryos and FD chicks have fewer ( P < 0.01) glomeruli. Water deprivation reduces body weight more in EwW birds than in controls. The results suggest that qAQP2 evolved in metanephric kidneys and that undernutrition may retard nephrogenesis, leading to impaired adult water homeostasis.

Fowl (males more than females) show maturation-dependent rises in blood pressure (BP) and formation of neointimal plaques (NPs), resembling balloon catheter injury-induced neointima, in the abdominal aorta (AbA) just above the bifurcation. The plaque comprises neointimal cells containing abundant endoplasmic reticulum and extracellular matrix. Hence, we investigated whether rapid incremental BP increases in male chicks trigger NP formation, possibly via endothelial injury in hemodynamically selective areas. In 6-wk-old chicks ( n = 8) treated 4 wk with solvent (Sv; minipump) or arginine supplement (Arg; 0.3% in drinking water), BP increased from 140 ± 5 to 159 ± 4 (Sv) and from 138 ± 4 to 157 ± 3 (Arg) mmHg, whereas propranolol treatment (Prop, 8 mg·kg−1·day−1; minipump) prevented the rise. Arg and Prop groups had, respectively, 73% and 77% smaller ( P < 0.05) NP areas and 19% and 25% less ( P < 0.01) AbA medial thickness than Sv controls. In 16-wk-old cockerels, established BP remained high after Sv and Arg treatments. In the Prop group, BP decreased, but neither NP area nor medial thickness was lower than in the Sv group, whereas the Arg group showed greater NP area and medial thickness. Pulse pressure, determined by intravascular transducer, increased as the pulse wave descended the aorta. The results suggest that maturation-dependent rises in BP in chicks may trigger NP formation in the lower segment of the AbA, which was prevented by inhibition of BP increase, or via a possible increase in nitric oxide availability. BP reduction exerts no effect once BP reaches a plateau. Involvement of endothelial injury leading to NP formation and hemodynamic forces selective for the lesion-prone area remain to be determined.

To clarify the role of nitric oxide (NO) in the ontogeny of arginine vasopressin (AVP) and oxytocin (OXT) neurons in the hypothalamo-neurohypophysial system (HNS), we observed the coexpression pattern of NADPH-diaphorase (NADPH-d) activity and AVP- or OXT-immunoreactivity (IR) in the rat hypothalamus and posterior pituitary during the postnatal period. The enzymatic activity of NADPH-d was observed in the supraoptic nucleus (SON), paraventricular nucleus (PVN), median eminence (ME) and posterior pituitary throughout the postnatal development. AVP-containing neurons were clearly observed from postnatal day 1 in both the SON and PVN, while OXT-containing neurons were recognized from postnatal day 14. The coexistence of NADPH-d and AVP or OXT was detected in the SON from postnatal day 14. At postnatal day 21, the coexpression pattern was approximately the same as that of the SON and PVN in adult rats. Our findings indicated that the expression of NADPH-d and OXT was observed from almost the same postnatal period in both the SON and PVN. In addition, the pattern of increased numbers of NADPH-d positive fibers was similar to that of OXT-immunoreactive fibers in both the inner layer of the ME and the posterior pituitary. A good correlation was thus obtained between OXT expression and NADPH-d activity in the HNS during postnatal development. The present study suggests that NO is more closely involved in the expression and regulation of secretion of OXT than AVP.

Purpose To determine the effects of water deprivation and vasotocin (AVT, avian ADH) treatment on the regulation of quail aquaporin 2 (qAQP2) mRNA expressions in looped (advanced) and loopless (primitive) nephrons of adult quail kidneys. Introduction Birds and mammals are the only vertebrates that can concentrate urine. Avian kidneys contain both primitive loopless (mainly found in the cortical regions) and more advanced looped (found in the medullary regions) nephrons. Only the looped nephrons can produce hyperosmolar urine. We have previously identified an AQP2 homologue in quail kidney with ˜77% homology to rat AQP2. Immunoreactive qAQP2 is expressed on the apical side of the collecting ducts (CD) in both cortical and medullary regions and is enhanced by dehydration and AVT only in medullary CDs, suggesting that the regulatory role of AVT in water transport may have evolved in looped nephrons. Methods Sexually matured quails at 6 weeks old were examined in 4 groups (n = 10-12) as follows: (1) normal hydration (C); (2) vasotocin treated (V) (50 ng/kg/d, by subcutaneous mini-pump, 72 h); (3) water deprived (WD) for 72 h; (4) WD plus V as above. RNA in both cortical and medullary regions (normalized with 18S) was measured by real-time PCR after reverse transcription. Results In all groups, qAQP2 mRNA levels are higher (3.6 to 4.7 times) in the medulla than cortex. Differences in mean (6 SE) qAQP2 mRNA quantities in cortex among the groups are not significant; C (0.19 6 0.06), V (0.16 6 0.05), WD (0.37 6 0.05), and WD plus V (0.34 6 0.11). Mean qAQP2 mRNA levels are significantly higher (p Conclusions The results suggest that qAQP2 expressions are higher in medullary regions with the looped nephrons. Advanced tubular structures may be required for water control by ADH-axis via AQP2 expressions in avian kidneys.