David L. Goldstein

Comparative Functional Analysis of Aquaporins/Glyceroporins in Mammals and Anurans

Maintenance of fluid homeostasis is critical to establishing and maintaining normal physiology. The landmark discovery of membrane water channels (aquaporins; AQPs) ushered in a new area in osmoregulatory biology that has drawn from and contributed to diverse branches of biology, from molecular biology and genomics to systems biology and evolution, and from microbial and plant biology to animal and translational physiology. As a result, the study of AQPs provides a unique and integrated backdrop for exploring the relationships between genes and genome systems, the regulation of gene expression, and the physiologic consequences of genetic variation. The wide species distribution of AQP family members and the evolutionary conservation of the family indicate that the control of membrane water flux is a critical biological process. AQP function and regulation is proving to be central to many of the pathways involved in individual physiologic systems in both mammals and anurans. In mammals, AQPs are essential to normal secretory and absorptive functions of the eye, lung, salivary gland, sweat glands, gastrointestinal tract, and kidney. In urinary, respiratory, and gastrointestinal systems, AQPs are required for proper urine concentration, fluid reabsorption, and glandular secretions. In anurans, AQPs are important in mediating physiologic responses to changes in the external environment, including those that occur during metamorphosis and adaptation from an aquatic to terrestrial environment and thermal acclimation in anticipation of freezing. Therefore, an understanding of AQP function and regulation is an important aspect of an integrated approach to basic biological research.

Effect of wind on avian metabolic rate with particular reference to Gambel's quail

The metabolic rate of Gambels quail (Callipepla gambelii) increases linearly with increasing wind speed at both 10 and 20 C. These results are compared with data from the literature for seven other avian species. The square root of wind speed, though often used as the independent variable, does not provide the best description of metabolic rate in wind for most species and temperatures; however, presentation of all data in a common form does reveal patterns among and within species. The effect of convective heat loss on metabolic rate-that is, the slope of metabolism (in watts, W) on the square root of wind speed (m/s)-increases as mass increases. This slope also increases within a given species as ambient temperature (Ta) decreases. These relationships are the result of relative changes in surface area, thermal conductance, and the temperature difference driving heat flux. The slope of the regression of metabolism on the square root of wind speed [b, in W/(m/s)1/2] may be described as b = .0092M.66ΔT.32, where M is mass in grams and ΔT is the difference between the lower critical temperature and Ta (in °C). This equation predicts the metabolic rate of a bird at any wind speed in temperatures below thermoneutrality.

Osmoregulation in the Field by Salt-Marsh Savannah Sparrows Passerculus sandwichensis beldingi

We studied the osmoregulatory strategy of salt-marsh savannah sparrows (Passerculus sandwichensis beldingi) in the field in Baja California, Mexico, and we compared these birds with a subspecies of savannah sparrow found concurrently in the uplands surrounding the salt marsh. Passerculus sandwichensis beldingi had higher plasma osmolalities (349 vs. 339 mmol/kg), higher ureteral urine osmolalities (577 vs. 477 mmol/kg), higher urine sodium concentrations (126 vs. 74 meq/L), higher urine flow rates (7–10 vs. 1.7 μL/min), and larger kidneys (0.36vs. 0.21 g/pair of kidneys) than the upland sparrows. Other unusual findings in the salt-marsh birds were the presence of substantial volumes of hyperosmotic fluids (up to 811 mmol/kg) in the lower intestine and uric acid extending into the ileum. The rectal epithelium of P. s. beldingi was relatively smooth, with few low apical membrane extensions, whereas that of the upland sparrows had a dense apical microvillous brush border. The maximum urine concentration that we measured in freshly captured P. s. beldingi was 2.7 times plasma osmolality. Our findings suggest an osmoregulatory strategy that includes a high intake of solute, a copious urine flow to excrete this solute load, and, at least at times, substantial refluxing into the intestines of hyperosmotic urinary fluids. This unusual strategy presumably reflects the unusual circumstance of a bird without salt glands living in a salt marsh.

Renal function in red wattlebirds in response to varying fluid intake

Abstract Red wattlebirds (Anthochaera carunculata) are among the more nectarivorous of the Australian honeyeaters (family Meliphagidae). As such, they potentially ingest large and dilute fluid loads as food, and they produce copious dilute urine in the field. We examined in the laboratory the renal mechanisms by which such fluid loads are processed. Wattlebirds received one of three liquid diets [a mix of honey, water, and Complan (Boots) complete dietary supplement] of varying concentration (250, 1000, and 1750 mmol/kg, Na+/K+ concentrations of 4/4, 12/15, and 23/30 mmol/l, respectively). We measured renal function via infusion of a filtration marker (14C-polyethylene glycol) from osmotic minipumps implanted intraperitoneally. Wattlebirds consumed volumes of the three diets sufficient to provide nearly equal caloric intakes (approximately 200 kJ/day), and as a consequence had water turnover rates varying from 30 to 200 ml/day (approximately 50–335% of total body water per day). Renal function in the morning, before feeding, did not differ among diet groups (glomerular filtration rate =18 ml/h, urine flow rate =0.4 ml/h). In the afternoon, after feeding, urine flow did vary, from 3 ml/h in birds on the most concentrated diet to 6 ml/h on the most dilute. This was accomplished by varying the rate of tubular reabsorption of water (from a high of >90% on the most concentrated diet to a low of just over 70% on the most dilute), with little variation in the rate of glomerular filtration (mean ∼23 ml/h). Comparisons between dietary intakes and urinary outputs of water and electrolytes suggest that not all dietary water was absorbed from the gut, but that there was significant post-renal reabsorption of Na+. The reduced fractional water reabsorption on the dilute diet was accompanied by a decrease in the circulating concentration of arginine vasotocin (from >4 pg/ml in birds on the two more concentrated diets to <1 pg/ml on the most dilute diet). In contrast, concentrations of aldosterone (10–20 pg/ml) did not differ among diets. Perhaps in consequence, renal fractional absorption of Na+ also did not differ, and so birds on the dilute diet, with their higher urine flows, had higher rates of Na+ excretion and suffered a decreased concentration of Na+ in the plasma.

Taking Physiology to the Field: Using Physiological Approaches to Answer Questions about Animals in Their Environments*

Both technological and conceptual advances continue to enhance our ability to evaluate physiological mechanisms in free‐living animals. Although complex and uncontrolled natural environments may challenge our ability to define causal mechanistic relationships, they provide opportunities not available in more conventional laboratory settings. Among these opportunities are the ability to observe the interplay between physiology and behavior, the potential inspiration to physiological studies from novel observations in the field, and the ability to evaluate the extent to which particular physiological systems are challenged under natural conditions. As we accumulate information about physiological function in the field, we are often forced to reconsider established paradigms: hibernating bears may contract their muscles to maintain strength and tone, testosterone levels in male stonechats maintaining territories in winter are exceptionally low, wintering emperor penguins may risk overheating, and large desert mammals may eschew brain‐cooling mechanisms. Measuring and quantifying the organismal response to a changing environment provides a link between mechanistic physiology and behavior, ecology, and evolution and gives us new tools to understand population, community, and ecosystem‐level processes.

Regulation of Water and Sodium Balance in the Field by Australian Honeyeaters (Aves: Meliphagidae)

We evaluated the use of water and sodium by free‐living individuals of several species of Australian honeyeaters (Acanthorhynchos superciliosus, Phylidonyris novaehol‐landiae, Phylidonyris nigra, Manorina flavigula, and Antho‐chaera carunculata). Water and Na fluxes were highly variable between species, largely reflecting differences in diet. Water fluxes ranged from approximately 300% of total body water per day in 10‐g, nectarivorous A. superciliosus to approximately 45% of total body water per day, typical of a desert species, in M. flavigula, a 50‐g, insectivorous, arid‐zone bird. Similarly, Na fluxes ranged from nearly 60% of Na pool per day in A. superciliosus to about 25% per day in M. flavigula. Despite these different fluxes, values of regulated osmoregulatory variables, including plasma osmolality, hematocrit, plasma concentrations of Na+ and K+, and exchangeable Na pool, were relatively invariant both between species and within species at different seasons. In contrast, values of variables reflecting the operation of regulatory systems did differ between species and seasons. Urine concentrations were highest in M. flavigula and, in A. carunculata, varied seasonally (higher in summer than winter). Plasma concentrations of aldosterone were lowest in A. carunculata (5–25 pg/mL), highest in P. novaehollandiae (70–200 pg/ mL), and in the latter species were higher in winter than summer. Concentrations of arginine vasotocin ranged from 5 pg/mL in A. carunculata to greater than 30 pg/mL in M. flavigula. Our data demonstrate that within the family Meli‐phagidae, there exists substantial variation in the fluxes of water and Na and that these relate in part to body size variation but more importantly to diet. The different fluxes between species are reflected in the values of numerous os‐moregulatory variables.

Daily rhythms in rates of glomerular filtration and cloacal excretion in captive and wild song sparrows (Melospiza melodia)

We infused polyethylene glycol (PEG) into laboratory-housed song sparrows (Melospiza melodia) via intraperitoneally implanted osmotic minipumps. The plasma concentration of PEG was significantly higher at the end of the dark phase of the photoperiod than at the end of light, indicating a nighttime reduction in the rate of glomerular filtration (GFR, the route of PEG elimination). The rate of cloacal voiding of PEG also varied through the day. The rate was similar to the rate of PEG infusion from the minipump during most of the daylight hours. However, PEG excretion was signifcantly reduced during the dark phase and elevated during the first hours of light, a pattern that resulted from sequestering urine in the lower intestine during the night. To examine patterns of osmoregulation in the field, we also implanted minipumps into free-living sparrows. One to fiue days later we recaptured the birds and measured concentrations of PEG in plasma and urine. As in the laboratory, the GFR (and urine flow rate) tended to be lowest in the early morning and higher later in the day. This nocturnal reduction in GFR and cloacal voiding should contribute to minimizing water losses during the night.

Expression and immunolocalization of aquaporins HC-1, -2, and -3 in Cope's gray treefrog, Hyla chrysoscelis

We have previously identified two aquaporins (HC-1, HC-2) and a glyceroporin (HC-3), homologs, respectively, of mammalian AQP1, AQP2, and AQP3, from the freeze-tolerant treefrog Hyla chrysoscelis. The objective of the present study was to investigate by Western blotting and immunohistofluorescence the expression and localization of these proteins in warm-acclimated, hydrated treefrogs. We hypothesized that patterns of protein expression would reflect unique osmoregulatory roles for the three aquaporins. Western blots revealed a spectrum of protein bands from 28 kDa to 65+ kDa; treatment with N-glycosidase suggested that this reflected variable glycosidation of the aquaporins. HC-1 was expressed in all organs, including dermis of skin, sinusoids and septa of liver, Bowmans capsule of kidney, intestinal lacteal vessels, and perimysium and vasculature of muscle. HC-3 expression was also widespread, but with different localization, including epidermis and dermis of skin, renal collecting ducts, and colonic villous epithelium. HC-2 expression was limited to osmoregulatory organs (renal collecting ducts and epidermis). In many ways, the expression of these proteins paralleled their mammalian homologs. For example, HC-2 and HC-3 expression in collecting ducts appeared similar to the mammalian pattern (the former more apical, the latter more basal). However, some aspects of localization (e.g. HC-1 in Bowmans capsule) were unique, and the ubiquity of HC-3 expression may relate to its facilitation of glycerol transport in this animal that possesses glycerol-dependent freeze tolerance.

Renal Response to Dietary Protein in the House Sparrow Passer domesticus

Many birds switch seasonally or during ontogeny between diets of varying protein content. In mammals, high‐protein diets induce hypertrophy of the kidney in general and of the thick ascending limbs (TAL) in particular, along with increases in glomerular filtration rate (GFR) and urine flow. A hypothesis to explain these phenomena is that the TAL become increasingly sensitive to peptide hormones (glucagon and antidiuretic hormone [ADH]) released in response to protein feeding; the consequent enhancement of ion reabsorption dilutes urine reaching the macula densa, thereby suppressing tubulo‐glomerular feedback (TGF) and causing a rise in GFR. Avian kidneys possess most of the elements involved in this mechanism, including loops of Henle with TAL, sensitivity of TAL to ADH (arginine vasotocin [AVT] in birds), and the elements of TGF. We therefore hypothesized that switching from a low‐protein to a high‐protein diet would induce responses in birds similar to those found in mammals. We tested this hypothesis by feeding house sparrows, Passer domesticus, isocaloric diets containing either 8% or 30% protein. Birds on high‐protein food had larger renal medullae, both in mass and in TAL diameter, but no increase in whole‐kidney mass. Urine flow was approximately doubled on high‐protein food, but there was no change in GFR. We were not able to detect an increased sensitivity of AVT‐induced adenylyl cyclase activity in TAL from high‐protein animals, and responsiveness to glucagon was higher in TAL from birds eating low‐protein food. We are unable to conclude that a suppression of TGF is responsible for the rise in urine flow in birds eating high‐protein foods, and the mechanisms behind the medullary hypertrophy and the diuresis remain to be fully explored.

Renal Glomerular and Tubular Effects of Antidiuretic Hormone and Two Antidiuretic Hormone Analogues in House Sparrows (Passer domesticus)

We infused arginine vasotocin, the natural avian antidiuretic hormone, and two antidiuretic hormone analogues into house sparrows (Passer domesticus) to evaluate the vascular and tubular components of antidiuresis in a small (25-g) bird. During control infusion of 25 mmol L⁻¹ NaCl (0.6 mL h⁻¹), urine flow rate was 0.73 mL h⁻¹, glomerular filtration rate was 10.0 mL h⁻¹, the ratio of polyethylene glycol (PEG) in the urine relative to that in the plasma was 16.4, and urine osmolality was 279 mOsmol kg⁻¹. Infusion of arginine vasotocin (0.4 ng kg⁻¹ min⁻¹) decreased urine flow rate by 50% and glomerular filtration rate by 27%, while urine osmolality and the ratio of urine PEG to plasma PEG rose to 150% and 140% of control values, respectively. A higher dose of arginine vasotocin (1.6 ng kg⁻¹ min⁻¹) accentuated these changes. Infusion of the antidiuretic hormone analogue dPTyr(Me)AVT, designed as an antagonist to the V₁ (mammalian vascular) receptors for arginine vasopressin, by itself (4.0 ng kg⁻¹ min⁻¹) had no effect on any measured variable (P ≥ 0.1). Infusion of the analogue along with arginine vasotocin (0.4 ng kg⁻¹ min⁻¹) abolished the effect of arginine vasotocin on glomerular filtration rate, which suggests that this analogue blocked vascular receptors for arginine vasotocin in house sparrows. Under these circumstances, changes in urine flow rate, the ratio of urine PEG to plasma PEG, and urine osmolality were reduced to nonsignificance. The analogue d(CH₂)₅[D-Ile²,Ile⁴,Ala-MH₂]AVP, designed as an antagonist to the effects of arginine vasopressin at V₂ (mammalian renal tubular) receptors, also was without effect by itself. However, in the presence of this analogue, the effects of arginine vasotocin on urine flow rate and the ratio of urine PEG to plasma PEG were significantly enhanced, and this occurred without any enhanced diminution of glomerular filtration rate. Thus, this analogue appeared to activate a tubular mechanism of antidiuresis. Overall, the data suggest that action of arginine vasotocin at renal vascular receptors plays an important role in effecting antidiuresis in house sparrows. Blockade of renal vascular actions of arginine vasotocin by a V₁ antagonist suggests that these receptors may be similar to the mammalian vascular (V₁) receptor. The data also suggest a separate action of arginine vasotocin at the renal tubules, but the receptors there apparently differ from the mammalian tubular (V₂) receptor.