Transport characteristics and morphology of the colon and coprodeum in two wild birds of different habitats, the rock ptarmigan (Lagopus mutus) and the common murre (Uria aalge).

Dietary salt intake in domestic fowl affects epithelial transport and morphology of the lower intestine (colon and coprodeum). This study investigated lower intestinal morphology and transport activity in two wild bird species with natural diets containing either low or high salt. Tissues from rock ptarmigan (Lagopus mutus) and common murres (Uria aalge) were sampled for histology and electrophysiological analyses. The ptarmigan exists on a low salt diet, while the murre lives on a high protein and high salt diet. The ptarmigan colon and coprodeum had villi/folds and crypts and the epithelium contained absorptive epithelial cells, mitochondria-rich cells and goblet cells. The colon had significant amiloride-inhibitable Isc, 5-15 µA/cm(2), with no glucose-stimulated Isc, and no significant phloridzin inhibition. The coprodeum also had high amiloride-inhibitable Isc. This transport pattern corresponded to that of chickens on low-salt diets. However, the ptarmigan colon also had a significant lysine/leucine-stimulated Isc of 3±1.0 µA/cm(2). The short U. aalge colon was similar to that of ptarmigans, but with no villi. It demonstrated a significant lysine/leucine-stimulated Isc (11±3.5 µA/cm(2)) with no amiloride-inhibitable Isc, similar to the high-salt chicken colon, but with no Na(+)-glucose cotransport. The murre coprodeum was inert to all substances and showed high resistance (1000 Ω·cm(2)), with a multilayered squamous epithelium. Despite some variations possibly associated with dietary protein intake, we conclude that natural high and low salt diets in different avian species are associated with different lower intestinal transport patterns, providing for post-renal adjustments in ion and water excretion.

Adaptation of teleosts to very high salinity.

A number of species of euryhaline teleosts have the remarkable ability to adapt and survive in environments of extreme salinity, up to two or even three times the osmolality of seawater. This review looks at some of the literature describing the adaptive changes that occur, primarily with intestinal water absorption and with the properties of the gill epithelium. While there is much that is still not completely understood, recent work has begun to look at these adaptations at the cellular and molecular level. As with seawater osmoregulation, fish adapting to hypersaline conditions generally increase drinking rates and water absorption across the intestine by solute-linked transport. This process requires increased activity, expression levels and possibly changes in subunit isoforms of Na(+)/K(+)-ATPase pumps, along with increases in other components of the NaCl absorptive pathway. Additionally, recent studies have demonstrated the importance of luminal anion exchange activity and of luminal alkalinization, which can support absorption against increasing osmotic gradients by promoting divalent ion precipitation (CaCO(3)) and by conversion of HCO(3)(-) ions to CO(2). The steepness of the lumen to blood osmotic gradient vis-a-vis the NaCl absorptive capacity, along with the accumulation of non-absorbed Mg(++) and SO(4)(--), likely become the limiting factors for survival in hypersaline conditions. Of interest is the observation of predicted hyperosmotic fluid absorption by the intestine in several species. Adaptive changes in the gill epithelium are also critical in this process, allowing for secretion of absorbed NaCl from the extracellular fluids. Most notably there are important changes in the numbers and size of mitochondrion-rich (MR) cells, the sites of active secretion of Cl(-), which ultimately drives the overall process of NaCl secretion. Balance studies of intake and output clearly indicate that a decrease in the osmotic permeability of the gill epithelium must also occur. The molecular correlates of this effect are not known, although decreased expression of one or more aquaporins seems to be a likely possibility. Finally, the regulatory changes seen with hypersaline adaptation may provide important new insights into epithelial function, including the role of organized transport assemblies ("metabolons") and changes in the expression of tight junction proteins such as claudins or occludins, which may modulate electrolyte permeabilities.

CFTR mediated chloride secretion in the avian renal proximal tubule.

In primary cell cultures of the avian (Gallus gallus) renal proximal tubule parathyroid hormone and cAMP activation generate a Cl(-)-dependent short circuit current (I(SC)) response, consistent with net transepithelial Cl(-) secretion. In this study we investigated the expression and physiological function of the Na-K-2Cl (NKCC) transporter and CFTR chloride channel, both associated with Cl(-) secretion in a variety of tissues, in these proximal tubule cells. Using both RT-PCR and immunoblotting approaches, we showed that NKCC and CFTR are expressed, both in proximal tubule primary cultures and in a proximal tubule fraction of non-cultured (native tissue) fragments. We also used electrophysiological methods to assess the functional contribution of NKCC and CFTR to forskolin-activated I(SC) responses in filter grown cultured monolayers. Bumetanide (10 µM), a specific blocker of NKCC, inhibited forskolin activated I(SC) by about 40%, suggesting that basolateral uptake of Cl(-) is partially mediated by NKCC transport. In monolayers permeabilized on the basolateral side with nystatin, forskolin activated an apical Cl(-) conductance, manifested as bidirectional diffusion currents in the presence of oppositely directed Cl(-) gradients. Under these conditions the apical conductance appeared to show some bias towards apical-to-basolateral Cl(-) current. Two selective CFTR blockers, CFTR Inhibitor 172 and GlyH-101 (both at 20 µM) inhibited the forskolin activated diffusion currents by 38-68%, with GlyH-101 having a greater effect. These data support the conclusion that avian renal proximal tubules utilize an apical CFTR Cl(-) channel to mediate cAMP-activated Cl(-) secretion.

NH4+ secretion in the avian colon. An actively regulated barrier to ammonium permeation of the colon mucosa.

Experiments were designed to characterize an active, electrogenic transport of NH(4)(+) ions across the colonic epithelium of the domestic fowl (Gallus gallus). Colonic segments were isolated and stripped of underlying muscle. The mucosal epithelia were mounted in Ussing chambers and voltage-clamped to measure the short-circuit currents (I(SC)) associated with transport. Bilateral addition of NH(4)(+) caused a dose-dependent outward current (negative I(SC)), with a Km of 34+/-8 mM and a maximal current response of 311+/-47 microA cm(-2) (12+/-2 microEq cm(-2) h(-1)). A similar effect was seen with unilateral addition of NH(4)(+) to the serosal (s) side, but not with mucosal (m) addition. Pre-treatment with 10(-4) M amiloride exposed a net outward (negative) I(SC), and serosal NH(4)(+) addition further increased this outward current with a Km of 53+/-24 mM. Decreasing the bath pH from 7.3 to 6.0 did not affect the I(SC) response to NH(4)(+). Unidirectional NH(4)(+) flux measurements revealed a net secretory flux (8.8+/-3.1 micromol cm(-2) h(-1) s-m, versus 2.6+/-1.4 micromol cm(-2) h(-1) m-s). Furthermore, the secretory flux closely matched the resulting change in I(SC) with serosal NH(4)(+), showing that the transepithelial flux of NH(4)(+) could account for the outward current response. Addition of 50 nM bafilomycin A to the mucosal solution completely eliminated serosal to mucosal NH(4)(+) transport, implicating an apical V-type H(+)-ATPase in this transport process. The I(SC) response to NH(4)(+) was partially inhibited by ouabain, a blocker of the Na(+)/K(+)-ATPase, but only minimally affected by bumetanide, an inhibitor of the serosal Na(+)-K(+)-2Cl(-) cotransporter. Active NH(4)(+) extrusion across the mucosal membrane, combined with low permeability to NH(3) in this tissue, allow for maintenance of steep ammonia gradients across the colonic epithelium and protection from ammonia toxicity. Furthermore, these studies indicate that the hen colon may be a useful new model system for the study of NH(4)(+) transport.

Adaptive strategies for post-renal handling of urine in birds.

Birds are a diverse vertebrate class in terms of diet and habitat, but they share several common physiological features, including the use of uric acid as the major nitrogenous waste product and the lack of a urinary bladder. Instead, ureteral urine refluxes from the urodeum into the more proximal coprodeum and portions of the hindgut (colon or rectum and ceca). This presents a potential problem in that hyperosmotic ureteral urine in contact with the permeable epithelia of these tissues would counteract renal osmotic work. This review describes and provides examples of different strategies used by avian species to balance renal and post-renal changes in urine composition. The strategies described include: 1. a "reptilian" mode, with moderate renal concentrating ability, but high rates of post-renal salt and water resorption; 2. the "mammalian" strategy, in which the coprodeum effectively functions like a mammalian urinary bladder, preserving the osmotic concentrating work of the kidney; 3. an interaction strategy, in which post-renal transport processes are hormonally regulated in order to optimize renal function under varying conditions of salt or water stress; 4. the salt gland strategy seen in marine or estuarine birds with functional salt glands, in which post-renal transport mechanisms are used to conserve urinary water and to recycle excess NaCl to the nasal salt glands. Finally, we also describe some features of an as-yet unstudied group of birds, the birds of prey. At least some species in this group are relatively good renal concentrators, and would be predicted to have post-renal mechanisms to preserve this work. This new synthesis illustrates the marked diversity of adaptive mechanisms used by avian species to maintain osmotic homeostasis.

Expression of SPAM1 (PH-20) in the murine kidney is not accompanied by hyaluronidase activity: evidence for potential roles in fluid and water reabsorption.

BACKGROUND: A role for Sperm Adhesion Molecule 1 (SPAM1) hyaluronidase in murine kidney, where Spam1 transcript levels have been reported to be higher in males, has not been clarified. METHODS: Spam1 RNA and protein were studied using RT-PCR, in situhybridization, Western blotting, immunohistochemistry and hyaluronic acid substrate gel electrophoresis. Urine volume and osmolality were studied in wild-type and Spam1 null mice. RESULTS: While RT-PCR supported a tendency of higher RNA expression in males, no sex difference for the protein was detectable in the cortex, medulla, and urine. Transcripts were predominantly localized in the proximal tubules and glomeruli, with lower levels in the medulla. Similarly, Western blotting and immunohistochemistry revealed that SPAM1 is more abundant in the cortex. Hyaluronidase activity was absent at neutral and acidic pH: suggesting non-enzymatic role(s) for SPAM1. Wild-type and Spam1 null mice given free access to water showed significantly reduced urine volumes (p < 0.01; n = 12) in the latter. Baseline urine osmolality was similar in both, leading to a significantly (p < 0.05) lower osmolar output in the nulls. After water deprivation (24 h), a significant (p < 0.01) increase in urine osmolality was seen only for wild-type mice. CONCLUSION: SPAM1 is implicated in fluid reabsorption and urine concentration.

Vitamin C transport and SVCT1 transporter expression in chick renal proximal tubule cells in culture.

The characteristics of vitamin C (ascorbic acid, ASC) transport were studied in polarized cultured monolayers of the chick (Gallus gallus) renal proximal tubule in Ussing chambers. Under voltage clamp conditions, monolayers responded to apical addition of ASC in a dose-dependent manner, with positive short circuit currents (I(SC)), ranging from 3 microA/cm(2) at 5 microM ASC to a maximal response of 27 microA/cm(2) at 200 microM, and a half-maximal response at 40 microM. There was no effect of basolateral addition of ASC, indicating a polarized transport process. The oxidized form of ASC, dehydroascorbic acid had negligible effects. The I(SC) response to ASC was completely eliminated with Na(+) ion replacement, and was also eliminated by bilateral reduction of bath Cl(-), from 137 to 2.6 mM. There was significant inhibition of the I(SC) responses to 30 microM ASC by the flavanoid quercetin (50 microM) and by 100 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 5-ethylisopropylamiloride (EIPA), blockers of anion exchangers and sodium-proton exchangers, respectively. There was no inhibition, however, by the chloride channel blocker 5-nitro-2(3-phenylpropylamino)benzoic acid (NPPB). Phorbol 12-myristate 13 acetate (PMA), the phorbol ester activator of protein kinase C, caused a 37% decrease in the I(SC) response to ASC. Chicken-specific primers to an EST homolog of the human vitamin C transporter SVCT1 (SLC23A1) were designed and used to probe transporter expression in these cells. RT-PCR analysis demonstrated the presence of chicken SVCT1 in both cultured cells and in freshly isolated proximal tubule fragments. These data indicate the presence of an electrogenic, sodium-dependent vitamin C transporter (SVCT1) in the chick renal proximal tubule. Vitamin C transport and conservation by the kidney is likely to be especially critical in birds, due to high plasma glucose levels and resulting high levels of reactive oxygen species.

Endocrine regulation of ion transport in the avian lower intestine.

The lower intestine (colon and coprodeum) of the domestic fowl maintains a very active, transporting epithelium, with a microvillus brush border, columnar epithelial cells, and a variety of transport systems. The colon of normal or high salt-acclimated hens expresses sodium-linked glucose and amino acid cotransporters, while the coprodeum is relatively inactive. Following acclimation to low salt diets, however, both colon and coprodeum shift to a pattern of high expression of electrogenic sodium channels, and the colonic cotransporter activity is simultaneously downregulated. These changes in the transport patterns seem to be regulated, at least in part, by aldosterone. Our recent work with this tissue has focused on whether aldosterone alone can account for the low salt pattern of transport. Other work has looked at the changes in morphology and in proportions of cell types that occur during chronic acclimation to high or low salt diets, and on a cAMP-activated chloride secretion pathway. Recent findings suggesting effects of other hormones on lower intestinal transport are also presented.

PTH stimulates a Cl(-)-dependent and EIPA-sensitive current in chick proximal tubule cells in culture.

The electrophysiological effects of parathyroid hormone (PTH) were studied in a primary cell culture model of the chick (Gallus domesticus) proximal tubule. In this model, confluent monolayers are grown on permeable filters and exhibit vectorial transport, including glucose-stimulated current. Under short-circuit conditions, PTH, at 10(-9) M, induced a positive current [short-circuit current (I(sc))] response, with an average 2-min peak response of 14.30 +/- 1.58 microA/cm(2) over the baseline I(sc,) followed by a slow decay. The PTH response was dose dependent, with a half-maximal response at 5 x 10(-9) M and maximal response at 5 x 10(-8) M. Forskolin and dibutyryl-cAMP also stimulated I(sc), as did the phosphodiesterase inhibitor IBMX. In contrast, the phorbol ester PMA inhibited baseline I(sc). The PTH response was nearly abolished by apical addition of 100 microM EIPA, an inhibitor of Na(+)/H(+) exchangers, and partially blocked by the Cl(-) channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 100 microM) and glibenclamide (300 microM). Higher doses of EIPA or NPPB alone (500 microM) were almost fully effective, with no or slight additional effects of NPPB or EIPA, respectively. The anion exchange inhibitor DIDS (100 microM) and the Na(+) channel blocker amiloride (10 microM) had no effect. Bilateral reduction of Cl(-) in the buffer, from 137 to 2.6 mM, abolished the PTH response; increasing Cl(-) concentration restored the I(sc) response, with a half-maximal effect at 50 mM. These data suggest that, in the chick proximal tubule, PTH activates both an Na(+)/H(+) exchanger and a Cl(-) channel that may be functionally linked.

Regulation of transepithelial phosphate transport by PTH in chicken proximal tubule epithelium.

The effect of parathyroid hormone (PTH) and activation of protein kinase C (PKC) and protein kinase A (PKA) on transepithelial P(i) transport was examined in monolayers of chick proximal tubule cells in primary culture (PTCs). Acute exposure of the PTCs to PTH (10(-9) M, basolateral side) significantly decreased the net reabsorption of P(i) by approximately 66%. There was no effect after the addition of PTH to the luminal side. Activation of PKC by phorbol 12-myristate 13-acetate (PMA; 0.1 microM) dramatically decreased net P(i) reabsorption by approximately 60%. Bisindolylmaleimide I (BIM; 1 microM), a highly selective PKC inhibitor, prevented PMA-induced inhibition. Activation of adenylate cyclase/PKA by forskolin (10 microM) mimicked the effect of PTH by significantly reducing net P(i) reabsorption by one-half. Addition of H-89 (10 microM), a potent inhibitor of PKA, abolished forskolin-induced inhibition. PTH inhibition was blocked by either BIM or H-89. Tissue electrophysiology remained stable after all treatments. There was a decreased immunoreactivity of the luminal Na+-P(i) cotransporter NaPi-IIa after PTH treatment. These data indicate that PTH inhibition of P(i) reabsorption in this in vitro system is mediated by PKC and PKA.

Carbonic anhydrase activity in kidney and lower intestine of the European starling.

A histochemical investigation of kidney and lower intestine of the European starling (Sturnus vulgaris) shows no carbonic anhydrase activity in proximal convoluted tubules, although activity is seen in similarly prepared sections of rat proximal tubules. Early distal tubule cells in the starling are stained throughout the cytoplasm and at the apical and highly infolded basolateral membranes. Late distal tubules lose apical activity and have reduced basolateral infolding, resulting in less intense staining. Darkly stained intercalated cells appear in the connecting tubules and cortical collecting ducts. Both of these segments also show intense basolateral staining. Medullary cones of the starling are highly organized, with central zones containing unstained thin descending limbs of loops of Henle, surrounded by both medullary collecting ducts with only scattered cells staining for enzyme, and by thick ascending limb segments. The latter contain many uniformly stained cells intermingled with occasional unstained cells. Scattered cells of the starling colonic villi demonstrate intense apical brush border membrane staining as well as cytoplasmic staining. Cells lining the cloaca stain less intensely. A biochemical assay for carbonic anhydrase was used to quantify enzyme activity in these tissues. Starling kidney contained 1.96 ± 0.33 (mean ± SEM) enzyme units/mg protein, less than half the activity seen in rat kidney. Stripped colonic epithelium contained 0.66 ± 0.15 enzyme units/mg protein. These quantitative results correlate well with the interpretations derived from the histochemical observations. The lack of proximal tubule carbonic anhydrase activity suggests that the avian kidney relies more on distal nephron segments to achieve net acidification of the urine.