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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.

Renin or a renin-like enzyme evolved in ancestral vertebrates and is conserved along the vertebrate phylogeny. The ontogenic development of renin, however, is not well understood in nonmammalian vertebrates. We aimed to determine the expression patterns and relative abundance of renin mRNA in pre- and postnatal chickens (Gallus gallus, White Leghorn breed). Embryonic day 13 (E13) embryos show renal tubules, undifferentiated mesenchymal structures, and a small number of developing glomeruli. Maturing glomeruli are seen in post-hatch day 4 (D4) and day 30 (D30) kidneys, indicating that nephrogenic activity still exists in kidneys of 4-week-old chickens. In E13 embryos, renin mRNA measured by quantitative polymerase chain reaction in the adrenal glands is equivalent to the expression in the kidneys, whereas in post-hatch D4 and D30 maturing chicks, renal renin expressions increased 2-fold and 11-fold, respectively. In contrast, relative renin expression in the adrenals became lower than in the kidneys. Furthermore, renin expression is clearly visible by in situ hybridization in the juxtaglomerular (JG) area in D4 and D30 chicks, but not in E13 embryos. The results suggest that in chickens, renin evolved in both renal and extrarenal organs at an early stage of ontogeny and, with maturation, became localized to the JG area. Clear JG structures are not morphologically detectable in E13 embryos, but are visible in 30-day-old chicks, supporting this concept.

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.

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.

Nephrin, a major intercellular junction (ICJ) molecule of mammalian podocytes in the renal glomerulus, is absent in the avian genome. We hypothesized that birds use ICJ molecules other than nephrin in their podocytes. Therefore, in the present study, we examined the possible involvement of adherens junction (AJ) proteins in the ICJs of avian podocytes. We found the AJ proteins N-cadherin and α- and β-catenins in podocytes of quail and chickens but not in those of rats, pigs or humans. The AJ proteins were prominent in avian glomerulus-rich fractions in immunoblot analyses, and in immunofluorescence microscopy analyses, they were localized along glomerular capillary walls appearing in at least two staining patterns: weakly diffuse and distinctly granular. Immunoelectron microscopy demonstrated that the significant accumulation of immunogold particles for the AJ proteins were especially evident in avian slit diaphragms and AJs. Furthermore, N-cadherin was found to be expressed in all nephron cells in the early developmental stage but became confined to podocytes during maturation. These results indicate that avian slit diaphragms clearly express AJ proteins as compared with that in the mammal—where AJ proteins are suppressed to an extremely low level—and that avian podocytes are interconnected by AJs per se in addition to slit diaphragms.

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.

Chickens (males more than females) have higher blood pressure (BP) than most mammals and spontaneously develop vascular neointimal plaques (NP) and diffuse subendothelial thickening in the lower segment of the abdominal aorta (AbA, referred to as 'NP-prone area') that partly resemble atherosclerotic lesions in mammals. NP areas, which are larger in males, have a causal relationship with incremental increases in BP during maturation. We hypothesize that decreased wall distensibility and altered hemodynamic forces at the NP-prone area may contribute to the NP formation. We measured pressure pulse wave (PW) and systolic and diastolic BP along the descending aorta in anesthetized chickens at different ages using an intravascular microtip transducer and calculated pulse pressure (PP) as an indicator for artery distensibility. At all ages examined and in both sexes, the PW showed a sharper peak at the more peripheral locations and the amplitude of the PW increased as it descended the aorta. PP, expressed as relative increases from the PP in the aortic arch (%), was 40.4+/-12.6 and 71.4+/-18.6 at the AbA and ischiadic artery, respectively, in young males (24-27 weeks); 23.5+/-8.6 and 43.8+/-16.2 in adults (72-75 weeks); and 5.4+/-3.4 and 9.1+/-4.9 in chicks (5-7 weeks). Location-dependent increases in PP were significantly higher in young males (P0.05). The PP increases in females were not different among the three age groups. The contour of the PW in the proximal aorta changes in older birds, exhibiting steeper increases in the ascending and descending limbs, suggesting that faster wave reflection from the periphery augments peak systolic pressure. NP was most frequently seen in the lower segment of the abdominal aorta in older males. These results suggest that: (1) site-dependent increases in PP amplitude are marked in young males, possibly reflecting a reduction in arterial wall elasticity enhanced by incremental rises in BP, and (2) NP formation may contribute to the stiffness of aortic walls in the NP-prone area.

We previously reported that birds may concentrate urine by a countercurrent multiplier mechanism using single-solute (NaCl) recirculation, in which the thick ascending limb of Henle (TAL) of mammalian-type nephrons provides an energy source. The Japanese quail TAL has a higher lumen-to-bath Cl flux (JCl,lb) than that of mammals; the mechanism for maintaining the osmotic gradient along the medullary cone is unknown. We investigated whether salt delivery alters the NaCl reabsorption rate in the TAL dissected from both normal and salt-loaded Japanese quail, Coturnix coturnix, 3-7 wk old. When salt loading to the TAL was increased by increasing the perfusion flow rate (PFR), the lumen-to-bath Na and Cl flux coefficients (KNa,lb or KCl,lb, 10(-7) cm2/s) increased, respectively (P < 0.05), from 5.1 +/- 0.9 to 7.6 +/- 0.7 and from 6.8 +/- 0.9 to 8.9 +/- 1.4. Ouabain addition significantly reduced the KNa,lb. A significant correlation existed between PFR and JCl,lb (r = 0.69, P < 0.01) and PFR and JNa,lb. When the TAL was perfused at a low PFR, increasing Cl concentration in the perfusate and bathing medium from 125 to 175 (P < 0.05) or 225 mM (P < 0.01) increased JCl,lb but not KCl,lb, while decreasing Cl concentration decreased JCl,lb but not KCl,lb. Salt loading of intact birds (0.2 M NaCl drinking water) for 7 days increased the plasma Na level and the cloacal fluid-to-plasma osmolality ratio from 0.28 +/- 0.07 to 1.06 +/- 0.05 (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

In the domestic fowl, angiotensin II (ANG II) decreases blood pressure in vivo and causes endothelium-dependent relaxation of aortic smooth muscles in vitro. To characterize ANG II-induced vasorelaxation, we compared endothelium-dependent vasodilatory effects of [Asp1,Val5]-ANG II (fowl ANG II) and acetylcholine (ACh) with the endothelium-independent vasorelaxing effect of sodium nitroprusside (SNP) on isometric tension of fowl aortic rings. Hemoglobin (Hb), gossypol, and N omega-nitro-L-arginine methyl ester (L-NAME), inhibitors for endothelium-derived relaxing factor (EDRF) in mammalian blood vessels, partially inhibited vasorelaxation induced by ANG II and ACh in fowl. Hb also markedly attenuated SNP-induced vasorelaxation, but not 8-bromoguanosine 3',5'-cyclic monophosphate-induced relaxation. 3,4,5-Trimethoxybenzoic acid 8-(diethylamino)octyl ester hydrochloride (TMB-8) or the removal of Ca2+ from the bathing medium attenuated the ACh-induced relaxation but did not significantly reduce vasorelaxation induced by ANG II or SNP. In the zero Ca2+ medium, aortic rings showed tachyphylaxis to ACh, while ANG II caused tachyphylaxis regardless of the presence or absence of external Ca2+. Furthermore, pretreatment of the ring with a high dose of ACh abolished the vasorelaxation response to ANG II, suggesting that ACh and ANG II may share a common Ca2+ pool. Calmidazolium, a calmodulin antagonist, abolished the vasorelaxation induced by ANG II and ACh but not that by SNP. Comparison of the vasodilatory effects of several ANG II analogues on fowl aortic rings showed an approximate potency order of [Asp1,Val5]-ANG II = [Asp1,Ile5]-ANG II > [Asn1,Ile5]-ANG II = [Sar1,Ile5]-ANG II > [Val5]-ANG III.(ABSTRACT TRUNCATED AT 250 WORDS)

In domestic fowl, angiotensin II (ANG II) produces a unique vasodepressor response in vivo and endothelium-dependent relaxation of aortic rings in vitro that appear to be a direct effect on vascular smooth muscle mediated through vascular angiotensin receptors. To explore the possible role of the endothelium in ANG II-induced vasodilation, ANG II binding to aortic membrane fractions and intact endothelium and prostaglandin (PG) production were examined in fowl aortas. 125I-[Ile5]ANG II binding by endothelium-intact aortic membrane fractions was consistently higher than binding by identically prepared endothelium-deleted membrane fractions at virtually all concentrations of ligand (10 pM-0.20 microM). Incubation of intact aortic rings with 125I-[Ile5]ANG II (0.50 nM) resulted in specific endothelial binding that increased linearly with time from 5.5 +/- 1.7 (SE) fmol/mg protein at 5 min to 13.7 +/- 1.8 at 30 min. Endothelial ANG II binding increased linearly with the dose of ligand, from 2.7 +/- 0.3 fmol/mg protein at 0.1 nM to 21.0 +/- 2.2 at 1.0 nM. Specific ANG II binding to aortic endothelium was competitively displaced 73 +/- 11% by unlabeled ANG II (0.1 microM) but not by bradykinin (0.1 microM). Incubation of intact aortic rings with [14C]arachidonic acid resulted in the formation of radioactive metabolites that comigrated in thin-layer chromatography with authentic PGE2 but not with 6-keto-PGF1 alpha. PGE2 production by aortic rings (44.4 +/- 4.5 ng.mg dry tissue-1.h-1) was not stimulated by addition of ANG II. These results suggest that specific receptors for ANG II exist in fowl aortic endothelium and that PGs are not involved in ANG II-induced vasodilation of the fowl aorta.

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.

Kidneys play an essential role in fluid-ion balance, but the mechanisms of renal handling of water vary depending on structural organization of kidneys and the environment. Fishes and amphibians in a hypoosmotic environment excrete excess water by forming dilute urine, whereas terrestrial tetrapods require water conservation by the kidney for survival. Diluting segments operated by a luminal Na(+)-K(+)-2Cl(-) cotransporter coupled with a basolateral Na(+)-K(+) pump are essential in forming dilute urine. In birds and mammals, the diluting segment that has the same transport characteristics now serves, with the development of additional architectural organization, for countercurrent urine concentration and water conservation. Recently, a number of aquaporin (AQP) water channels have been identified in various transporting epithelia. AQPs conserve the NPA (asparagine-proline-alanine) motif, forming pores selective to water. Although all vertebrate kidneys presumably possess AQP water channels, AQP homologues have been cloned only from amphibian, avian and mammalian renal systems. Studies on expression sites, function and regulation of AQPs will provide important insight into cellular and molecular mechanisms of epithelial water transport and its control by humoral, neural and hemodynamic mechanisms.

Fowl show spontaneous elevation of blood pressure (BP) and neointimal plaque formation in the abdominal aorta at young ages. Maturation/age-dependent modulation of vascular lesions and a causal relationship between elevated BP and neointima formation, however, have not been clarified. We therefore intended to characterize, first, maturation/age-dependent neointimal plaque formation and vascular lesions and, second, their relationship to BP elevation. The BP measured in conscious domestic fowl, Gallus gallus, White Leghorn breed, DeKalb strain, via an indwelling catheter inserted into the ischiadic artery, increased with maturation in males; and at plateau level, BP (mmHg) was significantly (P0.01) higher in males (194.0+/-4.6, n=11) than in females (169.3+/-3.1, n=10). Neointimal plaques consisting of neointimal cells and abundant extracellular matrix appeared initially in the distal segment of the abdominal aorta (lesion-prone area) of chicks as early as 6 weeks old. The area (size) of neointimal plaques right above the ischiadic bifurcation increased with maturation, whereas the plaque area became smaller with some degenerative changes in adult birds. In some birds, diffuse subendothelial hyperplasia and more extensive plaque formation at the branching points of the aorta were observed. The plaque area appears to be larger in birds, particularly in males that have higher BP (r=0.68). The width of aortic smooth muscle (SM) layers, measured in regions with no plaque, increased with age, whereas the number of cells per unit of area decreased, suggesting that hypertrophy of vascular SM occurs in response to exposure of the vascular wall to high BP. The number of cells was significantly (P0.01) higher in the plaque than in underlying aortic SM layers or in layers with no plaque formation. Both neointimal plaques and underlying SM layers are immunohistochemically positive for alpha SM actin, suggesting that neointimal cells are modulated SM cells, whereas the staining with SM myosin heavy chain antibody is low in neointimal plaques. Furthermore, plasma arginine levels dropped in accordance with the time of neointimal plaque formation, whereas plasma cholesterol levels showed an age-dependent increase. The results suggest that spontaneous development of neointimal plaques may be a consequence of exposure to high BP and associated local hemodynamic changes.

Many bird species show the spontaneous development of high arterial pressure and vascular lesions in the aorta and other large arteries. In chickens, arterial pressure tends to increase with age/maturation (particularly in males), and subendothelial hyperplasia (neointima) in the abdominal aorta is often seen prior to sexual maturation. The mechanisms involved, however, are not known. Our aim, therefore, was to determine (1) whether cytosolic Ca2+ signaling (CCS) responses to vasoactive substances in fowl aortic smooth muscle differ among chickens at different maturation stages and (2) whether CCS responses to Ca2+ channel agonists in neointimal plaques differ from those in normal aortic smooth muscle. K+ increased CCS in a dose-dependent manner in isolated and superfused abdominal aortic smooth muscle tissue from chicks (5–9 weeks old), pullets (11–18 weeks old) and adult hens (20 weeks and older); CCS responses increased as chickens matured. The addition of Bay K 8644 (10−6 mol l−1) to Ringer’s solution containing 50 mmol l−1 K+ further increased CCS, and this response was reduced to half by nifedipine (10−6 mol l−1). Norepinephrine did not alter CCS in chicks, whereas marked dose-dependent increases in CCS were noted in pullets. In contrast to the CCS responses to K+, the norepinephrine-induced CCS responses became smaller in adult hens. Isolated neointimal plaques showed only slight increases in CCS in response to 50 mmol l−1 K+ plus Bay K 8644, whereas clear responses were noted in aortic smooth muscle tissue underlying the plaques. These results suggest (1) that CCS responses to Ca2+ channel agonists increased with sexual maturation in fowl, but (2) that CCS responses to norepinephrine were low in mature hens and to K+ plus Bay K 8644 were low in spontaneously developed neointima, suggesting that phenotypic modulation of Ca2+ channel/norepinephrine receptors may have occurred during maturation/aging and in neointima.

Previously, we reported that the countercurrent urine concentration mechanism in birds appears to operate by recycling of a single solute (NaCl), in which the thick ascending limb of looped nephrons provides an energy source. To determine the importance of the medullary collecting duct (MCD) in the countercurrent multiplier system, we examined in isolated and perfused MCDs from Japanese quail, Coturnix coturnix, the osmotic and/or diffusional water permeability and whether arginine vasotocin (AVT) regulates water permeability. We noted that dark cells that possess electron-dense cytoplasm and numerous mitochondria and light mucus-secreting cells exist in the cortical collecting duct (CD), whereas only mucus-secreting cells are present in the MCDs. The volume flux (Jv) in the MCDs from intact birds and that from the water-deprived birds were nearly zero; after exposure to a hyperosmotic bath and AVT (2 x 10(-5) M), the Jv was significantly higher in water-deprived birds. The diffusional water permeability (Pdw) was moderately high in MCDs bathed in an isosmotic bath in which the Pdw was increased slightly by AVT (10(-5) M, bath) and more markedly (10(-5) M) by forskolin (Fsk), whereas 1,9-dideoxy Fsk (an inactive analogue) showed no effect. Furthermore, the basal adenosine 3',5'-cyclic monophosphate (cAMP) levels were higher in the medulla than in the cortex and were stimulated only slightly by AVT (10(-5) M) and markedly by Fsk (10(-4) M) in both the cortex and medulla. These results in the C. coturnix CD indicate the following. 1) Two types of cells are present; whereas dark cells resemble mammalian intercalated cells morphologically, it is not certain whether mucus-secreting cells are equivalent to principal cells. 2) AVT increases Pdw via a cAMP mechanism; the relatively high basal Pdw and minor effect of AVT on Jv and Pdw suggest, however, that diffusional water movement across the MCD may occur without significant direct control by AVT.

Systemic hypovolemia and hypotension increase plasma renin activity (PRA) in fowl, but it is not clear whether this response is mediated directly by reduced renal arterial perfusion pressure (RAPP) or indirectly via renal nerves activated when systemic baroreceptors detect hypotension. To evaluate the influence of RAPP on renin release, arterial and renal venous blood samples were collected as RAPP was reduced step-wise from 108 mm Hg (control) to 71 and 47 mm Hg. PRA in systemic arterial (aPRA) and renal venous (vPRA) plasma was measured as the rate of fowl angiotensin I (ANG I) generation. Basal vPRA (2.47 +/- 0.6 ng x ml-1 x min-1) tended to be higher than aPRA (1.24 +/- 0.3 ng x ml-1 x min-1). When RAPP was reduced to 47 mm Hg both vPRA (4.35 +/- 0.6 ng x ml-1 x min-1) and aPRA (1.91 +/- 0.3 ng x ml-1 x min-1) increased significantly. Significant negative slopes (P = 0.01) were obtained when changes in aPRA or vPRA were regressed on RAPP. Mean systemic arterial pressure did not change during reductions in RAPP, nor did angiotensinogen concentrations differ when systemic arterial (472 +/- 30 ng/ml) and renal venous (460 +/- 27 ng/ml) plasma values were compared. Renal plasma flow was fully autoregulated as RAPP was reduced from 108 to 47 mm Hg; consequently, RAPP-induced increases in vPRA cannot be attributed to hemoconcentration of secreted renin. These results demonstrate that reductions in RAPP directly stimulate renin release from domestic fowl kidneys.

A superfusion cascade system was used to determine whether endothelium-dependent vasorelaxation induced by acetylcholine (ACh) is mediated by a humoral factor(s) in the domestic fowl. An abdominal aorta (5 cm in length) with intact endothelium, excised from the donor bird, was mounted in an organ bath and perfused with avian Ringer solution. The vasorelaxing activity of the effluent was determined by an endothelium-denuded aortic ring (bioassay ring) equilibrated and precontracted with phenylephrine (PHE) (10(-6) M). The effluent (containing PHE) from the donor aorta with intact endothelium did not significantly change the PHE-induced tension of the bioassay rings. ACh (10(-6) M), when added to the perfusion medium that runs through the control polyethylene tubing, produced further contraction of the PHE-precontracted endothelium-denuded bioassay rings, whereas ACh caused relaxation of the rings when added to the perfusate that passes through the endothelium-intact donor aorta. The ACh-induced relaxation of the bioassay ring was slightly potentiated by superoxide dismutase (100 U/ml). Hemoglobin (10(-5) M) increased the basal tension of the bioassay rings and completely inhibited the ACh-induced relaxation of the rings. These results suggest that ACh-induced relaxation of fowl aorta is, at least partially, mediated by a humoral factor(s) that resembles endothelium-derived relaxing factor demonstrated in mammals.

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.

The renin-angiotensin system (RAS) in mammals may participate in the controls of blood pressure and aldosterone secretion, and possibly in the regulation of renal function. It has been shown that renin release is controlled by:1) two intrarenal receptors, the renal arteriolar receptor and the macula densa; 2) the sympathetic nervous system; and 3) several humoral agents. Recent studies indicate interrelations between the RAS and renal prostaglandins and the kallikrein-kinin system. Comparative studies have revealed that renal renin and the juxtaglomerular (JG) cells emerged during the early evolution of bony fishes, wherease the macula densa evolved later in the vertebrate phylogney. Exogenously administered angiotensins and renin produce vasopressor actions in representative species of all vertebrate classes from elasmobranchs to mammals, and increase secreations of mineralocorticoids from the adrenal cortex (interrenal) in amphibians, repitles, and possibly in teleosts. Angiotensin causes glomerular diuresis in teleosts and lung-fishes, which may be ascribed to increased dorsal aortic pressure, while angiotensin may have both glomerular and tubular actions in some amphibians. Intracranial injection of angiotensin stimulates drinking in teleosts, repites, and birds, but not in amphibians. Hemorrage and acute hypotension are potent stimuli for causing renin release in an aglomerular teleost and a bird. When we consider this fact together with the anatomical evidence that the evolution of the JG cells precedes that of the macula densa, it appears that the RAS HAS EVOLVED WITH A CLOSE RELATIONSHIP TO BLOOD PRESSURE HOEMOSTASIS. On the other hand, there is no clear evidence that the RAS is activated in depleted teleosts and amphibians. Although the RAS appears to exert several functions in man and other mammals, some of them may be more important in primitive animals, while a similar function remains in mammals as a relic of the primitive system. Comparative approaches provide a perspective of biological history and unique experimental model that will eventually aid in understanding of the underlying mechanisms operating in mammals.

Renin-like activity has been found in the plasma and kidneys of both marine and freshwater teleosts. A reliable and sensitive method is needed for the determination of plasma renin activity to study the physiological function of the renin-angiotensin system in fishes. Partially purified angiotensin (presumably angiotensin I) from the toadfish, Opsanus tau, was bound by a human angiotensin I antibody, whereas angiotensin from the eel was not. The activity of toadfish angiotensin, measured by the vasopressor bioassay in the rat with angiotensin II-amide as a standard and by the radioimmunoassay method for human angiotensin I, was nearly equal in the range of 5–70 ng (angiotensin eq)/ml. Angiotensin was generated from toadfish plasma or plasma and kidney extract by incubating in vitro with Dowex resin and EDTA (bioassay system) or EDTA and phenylmethylsulfonylfluoride (radioimmunoassay system). Formation of angiotensin increased with time initially in both methods and then reached a plateau. The optimum pH for incubating plasma in the radioimmunoassay system was 6.2–6.5. Plasma renin activity (rate of angiotensin generation) and plasma angiotensinogen levels determined by the radioimmunoassay system were well-correlated with those determined by the bioassay system in the same plasma, although the levels were higher in the radioimmunoassay. The amounts of plasma necessary to measure plasma renin activity and angiotensinogen with the radioimmunoassay method are 50 and 5 μl, respectively, and the measurements showed good reproducibility. Thus, plasma renin activity may be determined in the same fish by drawing small amounts of blood repeatedly, without producing significant hemodynamic changes. The radioimmunoassay method also allows the measurement of a large number of samples simultaneously, since angiotensin generated in the incubated mixture can be determined directly without extraction.

Angiotensin I (ANG I) from various nonmammalian vertebrates does not bind the antibody to human ANG I, and thus radioimmunoassay (RIA) is not applicable for measurement of renin activity. It is largely due to this difficulty that biochemistry and physiology of the renin-angiotensin system of nonmammalian vertebrates have not been studied. The present work was therefore undertaken to develop a system to determine plasma renin activity by measuring the ANG I generation rate with RIA in fowl plasma. Antibody to [Asp 1 ,Val 5 ,Ser 9 ]-angiotensin I (fowl ANG I) was produced by immunizing goats and rabbits with a conjugate of human immunoglobulin and fowl ANG I. An antiserum, with a maximum binding of 86.3%, was obtained from the goat after six intradermal injections of the conjugate (each contained 0.25 mg fowl ANG I). This antiserum (at a final dilution of 1:200,000), and 125 I-labeled fowl ANG I, purified by DEAE-cellulose and Sephadex G-25 column chromatography, were used for the RIA of natural fowl ANG I. Fowl ANG I generated by incubating plasma and kidney extract, was measured by both RIA and rat vasopressor assay. The values obtained from these two methods correlated well. The formation of angiotensin increased when the duration of incubation was prolonged, and was optimum at pH 5.0–5.5. Plasma renin activity (rate of fowl ANG I generation) in 50 μl plasma from anesthetized female white leghorn chickens (24 weeks, n = 6) was 15.04 ± 2.26 ng/ml·hr, and that in plasma from chronically cannulated conscious chickens (30 weeks, n = 5) was 4.58 ± 0.61 ng/ml·hr. The plasma angiotensinogen level was 500–600 ng/ml in both groups. Thus this sensitive RIA method may provide a useful tool to study the functions and regulation of the renin-angiotensin system in fowl.

The toadfish,Opsanus tau, lacks renal glomeruli and macula densa, but has high renal renin activity and abundant granulated cells in renal arteries and arterioles. Reduction of blood pressure (BP) or blood volume by hemorrhage or vasodilatory drugs causes renin release, indicating that an intrarenal or extrarenal pressure- or volume-sensitive mechanism exists for controlling renin release in the toadfish. Thus, we examined whether 1) β-adrenergic receptor-mediated activation of renin release, and 2) calcium influx which may underlie the baroreceptor mechanism are involved in the cellular control of renin release. Acute injection of isoproterenol (1 μg/kg, n = 6) decreased BP and increased plasma renin activity (PRA) 4-5 fold in unanesthetized toadfish. Propranolol abolished both effects, but did not decrease basal PRA levels.In vitro superfusion of renal slices with bicarbonate Ringer's solution showed a steady secretion of renin, and addition of 50 mM K(+) (K(+) methylsulfate replacing NaCl, n = 10) to the superfusate markedly suppressed renin secretion. Nifedipine (10(-5) M, n = 8) completely restored the high K(+)-induced inhibition of renin secretion from renal slices, whereas isoproterenol (10(-4) M, n = 6) neither increased basal renin secretion nor restored K(+)-induced renin suppression. These results suggest that calcium influx may mediate inhibitory messages for renin secretion, while the β-adrenoceptor-mediated activation of granulated cells appears absent in toadfish.

The plasma and kidney angiotensinases were compared in the rat. Effects of acid and alkali treatments and of enzyme inhibitors on the angiotensinase activities and the renin-angiotensionogen reaction were studied. Angiotensinase activity of the plasma was greatest near neutral pH at 37°C and was blocked almost completely by dialysis against disodium ethylenediaminetetraacetic acid (EDTA). The kidney angiotensinase at neutral pH was also inactivated by acidification and dialysis against EDTA. The activity having a optimum pH of 4.5 to 5.0 was not affected by the dialysis but was inhibited by alkali or diisopropyl fluorophosphate. The renin-angiotensinogen reaction was least affected by incubation of the acid-treated and dialysed kidney extract with the dialysed plasma at pH 7.4 and 37°C.

Renin and juxtaglomerular (JG) cells are present in tetrapod vertebrates and teleosts the macula densa (MD) and extraglomerular mesangium (EGM) are seen only in mammals. Detailed studies of the evolution of juxtaglomerular apparatus (JGA) are presented in this paper. No granules were stained with Bowie's method in the kidneys of Chondrostei: Polypterus senegalus, Calamoichthys calabaricus, Acipenser brevirostris, and Holostein: Amia calva, Lepisosteus osseus. In the kidney of the lungfish, Protopterus aethiopicus, JG cells were found in the arterial wall distant from the glomeruli. Three types of distribution of JG cells were observed in aglomerular teleosts. In the kidney of Opsanus tau, JG cells were distributed in the walls of small arteries. In Lophius litulon, JG cells were distributed in several regions of the kidney. In Phrynelox nox, P. tridens and Histrio histrio, JG cells were concentrated in a limited area of the kidney. The MD site was examined with the light microscope in Gallus gallus v. domesticus and Coturnix coturnix japonica, and with the electron microscope in G. gallus. The avian cells possess some of the characteristics of mammalian MD cells, and can be considered as structurally transitional between typical MD cells in mammals and the ordinary cells of the distal tubule.

A series of the DCA-treated rats were unilaterally nephrectomized, given 1% NaCl solution for drinking fluid, and killed periodically to observe the evolution and progress of vascular lesions in relation to the elevation of blood pressure.(1) Acute necrotizing vascular damage appeared in bursts in the early period of DCA treatment. The evolution of lesions diminished thereafter while interstitial cells and fibers proliferated in the areas involved and continuously accumulated in parallel with time. In the kidney, lesions further extended with destruction of the glomeruli and tubules secondary to vascular damage.(2) The necrotizing vascular damage evoluted in the period when blood pressure was elevating rapidly. The histological features and localization of lesions highly suggest that the rise in blood pressure, which results in altered vascular permeability, and the status of the musculature of the peripheral vascular bed related to some maldistribution of sodium ion are of fundamental importance in the pathogenesis of the damage.(3) On the other hand, the fact that the evolution of the damage diminished in the period when hypertension became established points out that high blood pressure is not an only dertermning factor.

A simple, accurate technique has been devised to sex externally channel catfish, Ictalurus punctatus, regardless of body size or state of gonadal maturation. Male catfish have a single urogenital opening; females have two openings partitioned by a septum—an anterior genital pore, and a posterior urinary pore. In male fish, a single probe can be inserted into the urinary bladder through the urogenital pore. Female fish are differentiated from males by the insertion of two probes, one into the urinary bladder through the urinary pore and the second into the oviduct through the genital pore.

Renal tubules from the Japanese quail, Coturnix coturnix, were perfused in vitro to characterize the transepithelial voltage (Vt). The thick limb (TL) of the mammalian-type (MT) nephron showed Vt positive in the lumen (+9.1 +/- 0.7 mV, n = 35). The Vt decreased with increases in hydrostatic perfusion pressure. Furosemide (lumen), Na cyanide (bath), and ouabain (bath) reversibly reduced Vt. Removal of Cl or Na from the perfusate and the bath decreased Vt of the TL from +10.3 +/- 3.0 to -0.3 +/- 0.3 (n = 5) and from +7.4 +/- 1.5 to +1.2 +/- 0.2 mV (n = 8), respectively. The distal tubule of the reptilian-type (RT) nephron showed two types of Vt: a lumen-negative Vt in the late segment and a lumen-positive Vt in the early segment that is in close contact with the parent glomerulus. Both voltages were reversibly reduced by ouabain and Na cyanide. These results suggest that in quail the TL of the MT nephron resembles the thick ascending limb of the mammalian kidney with both Na and Cl required for generation of luminal positivity and the distal tubule of the RT nephron appears functionally heterogeneous.

Renin, a protein enzyme synthesized by the juxtaglomerular cells in the kidney, is released into the circulation and possibly into the renal lymph (Morgan et al., 1982). In mammals, renin acts on angiotensinogen (renin substrate), an a -globulin in plasma, to form angiotensin I, a decapeptide, which is subsequently converted by converting enzymes to active octapeptide angiotensin II (ANG II), which plays an important role in maintaining cardiovascular and hydromineral homeostasis (Fig. 1) (for review, see Peach, 1977; Skeggs et al., 1974; Vane, 1974). A similar renin-angiotensin system has been shown in teleost and nonmammalian tetrapods (for review, see Sokabe et al., 1969; Nishimura et al., 1973), but evolution and function of this enzyme cascade among more primitive fishes is uncertain (Nishimura et al., 1970).

Isolated segments of the renal tubules from the freshwater trout, Salmo gairdneri, were perfused in vitro to characterize ion and water transport. The distal tubule showed a transepithelial voltage (Vt) positive in the lumen (+17.8 +/- 1.4 mV). Furosemide added to the lumen and Na cyanide and ouabain added to the bath reduced the lumen-positive Vt of the distal tubule. Removal of either Cl- or Na+ from both perfusate and bathing medium abolished the lumen-positive Vt. When the distal tubule was perfused and bathed with isosmotic solution, net water flux (Jv) was nearly zero. Jv and hydraulic conductivity remained low when the osmolality of the bathing fluid was increased with raffinose. Neurohypophysial hormones added to the bath showed no effect. Chloride efflux (lumen to bath, 171.1 +/- 17.1 peq x mm-1. min-1) was significantly higher than chloride influx (bath to lumen, 105.6 +/- 12.3 peq x mm-1 x min-1), suggesting that net chloride reabsorption exists. These results suggest that in the freshwater trout, which lack the loop of Henle, the distal tubule acts as a diluting segment. The presence of sodium, in addition to chloride, is required to generate the lumen-positive Vt in the distal tubule.

Avian kidneys show features of both mammalian and non-mammalian kidneys, possessing cortical reptilian-type (RT) and medullary mammalian-type (MT) nephrons, blood supplies from renal arterial and renal portal systems, and a primitive macula densa. To determine how these morphological characteristics contribute to unique renal functions, we examined first a possible functional link between the renal tubules and the preglomerular vasculature and, second, the function of the loop of Henle of the MT nephrons. Infusion of 5% NaCl (2.0 ml/kg/h) into the renal portal system of the pullet Gallus domesticus caused diuresis and natriuresis in the infused side, whereas infusion of the same dose into the systemic circulation showed no effect. Infusion of 10% NaCl at a higher flow rate into either the renal portal or systemic routes caused hypernaturemia and a prolonged antidiuresis, presumably due to a release of arginine vasotocin. Plasma renin activity (PRA) decreased during renal portal infusion of hypertonic saline, suggesting that increases in intratubular or peritubular NaCl levels may suppress PRA. The thick limb of the loop of Henle (TLH) isolated from the MT nephron of the quail Coturnix coturnix revealed a lumen-positive, furosemide-sensitive transepithelial voltage (Vt) that requires the presence of both Na and Cl. Net water flux (Jv) was nearly zero when the TLH was perfused and bathed with isosmotic solution. Net water flux increased only slightly and osmotic water permeability (Lp) was low when the osmotic gradient was imposed. Vasotocin altered neither Vt, Jv, nor Lp. Chloride efflux was higher than Cl influx, and net Cl absorption was comparable to that of the TLH of the mammalian kidney.(ABSTRACT TRUNCATED AT 250 WORDS)

Birds and mammals can produce hyperosmotic urine, but their renal morphology and urine-concentrating mechanisms differ. To elucidate the countercurrent urine concentration mechanism in birds, we examined the structure and transport properties of the descending limb (DL) of Henle of mammalian-type nephrons in Japanese quail, Coturnix coturnix. In the avian renal medulla, a prominent ring of collecting ducts and scattered thick limbs surrounds a core of capillaries and DLs. Epithelial cells in the upper DL (DLu) have abundant microvilli and shallow, tight junctions; cells in the lower DL are flat and have little interdigitation. Transepithelial voltage was zero when the DLu was perfused and bathed in isosmotic avian Ringer solution. The efflux coefficients (10(-7) cm2/s) for Na (31.7 +/- 2.3) and Cl (24.9 +/- 3.6) were not significantly different and were unaltered by ouabain (10(-4) M) (32.5 +/- 2.2). Diffusional water permeability measured by [3H]H2O was low (73.0 +/- 7.8, 10(-7) cm2/s). Volume flux was nearly zero and increased only slightly when an osmotic gradient was imposed. These results suggest the DLu is highly permeable to Na and Cl and virtually impermeable to water; thus NaCl extruded actively from the thick ascending limb may enter the DL unaccompanied by water. This countercurrent multiplication system by use of single-solute recycling and a transport cascade of graded hairpin turns may help establish an osmotic gradient along the medullary cone. Thus avian and mammalian renal countercurrent multiplication systems may differ.

Temporary tinnitus and/or hearing loss elicited by contraction of facial mimetic muscles are often observed on the affected side in patients recovering from facial nerve paralysis and in those with facial spasm. In order to clarify the mechanism of this phenomenon, changes in auditory threshold and middle ear compliance during voluntary contraction of mimetic muscles were investigated. The results suggest that this type of tinnitus and/or hearing loss is due to nonacoustic contraction of the stapedial muscle synchronous with contraction of mimetic muscles.

The renin contents of the corpuscles of Stannius (CS) and kidney were determined in Cyprinus carpio, Carassius auratus , and Lophius litulon . The tissue extracts were incubated with homologous plasmas as the angiotensinogen source, and the resulting vasopressor activity was bioassayed in anesthetized rats. Renin activities per g of the corpuscular tissue were comparable those of the kidney, but total amounts were 0.13. 0.081, and 0.72% of those in the kidney of Cyprinus, Carassius , and Lophius , respectively. The activity in the corpuscles is specific to them, since no pressor activity was found in the heart, spleen, hepatopancreas, testis, ovary, and head kidney treated by the same procedures. The pressor activity formed in the incubation media showed some characteristics of angiotensin. Granules were demonstrated by Bowie's method in the corpuscles of Carassius, Lophius , and four other teleosts. The role of the renin in the corpuscles is not known.

A transcutaneous electrical stimulation method was used to treat hemifacial spasms. This treatment was applied concomitantly with drug therapy to 21 patients, and was found effective in 17 patients (81%). Electrotherapy is considered to be useful in patients with mild spasms and in those with severe spasms for whom surgical treatment is not possible.