Acid-base balance and urinary acidification in birds

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ACID-BASE BALANCE AND URINARY ACIDIFICATION IN BIRDS SCOTT LONG Laboratoire de Physiologie Cellulaire et Cornparke, Facultt des Sciences, Universit& de Nice. 06034 Nice Cedex, France and Department of Medical Physiology A, Panum Institute, University of Copenhagen, 3C Blegdamsvej, 2200 Copenhagen, Denmark (Recrioed 6 July 1981) Abstract-This essay will treat, first, the defended parameters of acid-base status in avian blood and their modification under conditions pertinent to the life of birds and, second, urinary acidification and its role in maintenance of acid-base balance. Of these two topics, urinary acidification is of particular interest to the author, has received less sustained attention experimentally and has been infrequently reviewed ISvkes. 1971). so it will receive more attention here. The reports cited in the essay concern primarily ‘a&It birds outside periods of egg-laying.

I. INTRODUCTION

Birds and mammals arose from cotylsoaurs, a now extinct reptilian group which flourished during the Carboniferous epoch roughly 300 million years ago (Romer, 1955). Common traits among extant reptiles, birds and mammals represent features retained and developed from common ancestral stock and/or convergent evolution, especially relevant in comparisons of birds and mammals. Reptiles, birds and mammals are primarily terrestrial classes although secondary return to aquatic habitats is found in all three classes. With terrestriality is associated increased reliance upon pulmonary respiration for oxygen uptake and excretion of metabolically produced carbon dioxide (Johansen, 1979); this reliance is nearly total in birds and mammals. These two classes can also be characterized by complete separation of systemic and pulmonary circulations, elevation of systemic blood pressure (Johansen, 1979), maintenance of body temperature at relatively stable values generally above ambient temperature and increased use of aerobic metabolism with consequently greater demands for food and oxygen than found in reptiles (Bennett & Ruben, 1979). Nitrogen metabolism leads to excretion of poorly soluble uric acid and urates in birds and terrestrial reptiles and to urea excretion in mammals (Dantzler & Braun, 1980). Uricotelism is commonlybut not always (Sykes, 197lt_thought to be advantageous to recuperation of filtered water in kidneys with limited (birds) or no (reptiles) ability for elaboration of concentrated urines. Solute-free water reabsorption in mammals is accomplished by the medullary countercurrent mechanism, present but less powerful in birds and absent in reptiles (Dantzler & Braun, 1980). Compared to reptiles, birds and especially mammals are characterized by a greater stability of the composition of body fluids, including their acid-base status.

However in poikilothermic vertebrates like reptiles extracellular pH generally rises with decreasing temperatures (Howell & Rahn, 1976), as observed also for mammalian blood and bicarbonate-protein solutions in closed (constant COZ), in vitro systems (Reeves & Rahn, 1979). Reeves and Rahn have proposed that such shifts in blood pH in poikilotherms favor constant ionization of amino acid radicals (of histidine in particular), important for maintenance of enzymatic activity at a variety of body temperatures. The relative constancy of extracellular pH observed in birds and mammals thus represents one limited aspect of this general temperature-dependent system, favored by homeothermy in these two classes and controlled in large part by respiratory and renal regulation of Pco, and bicarbonate. Several other physiological characteristics listed above for reptiles, birds and mammals are relevant to acid-base status of these classes. In general one notes an overall increase in hemoglobin and total CO2 contents in birds and mammals with predominantly pulmonary respiration (Lenfant et al., 1970) and hence a greater contribution of these buffer systems to maintenance of acid-base balance. In aquatic, diving reptiles, birds and mammals both hemoglobin and total CO2 contents are generally elevated relative to values in terrestrial representatives and serve to maintain acid-base status during periods of hypoxia (Wood & Johansen, 1974). The potential offset of acid-base status by increased production of CO2 at the higher metabolic rates of birds and mammals is reduced by passage of the entire cardiac output through the highly developed pulmonary circulation at increased perfusion and ventilation rates relative to reptilian

Based originally on observation in mammals, the term acid-base equilibrium has implied maintenance of a nearly constant pH value in extracellular fluid and of a somewhat lower value in intracellular fluid. 519

values (Johansen, 1979; Bennett & Ruben, 1979). Renal participation in acid-base balance, i.e. recuperation of filtered bicarbonate and excretion of acid, is favored by the effects of elevated body temperature

and by increased systemic arterial presssure producing filtration rates in birds and mammals both increased and generally less variable (especially in mammals) than those found in reptiles (Dantzler & Braun, 1980). Finally, uricotelism may contribute to acid

SCOTTLONG

520 Table

1. Arterial

blood

gas, pH and

body temperature birds PC02

Species Gullus domesticus

7.52 )

(chicken) N = 5 Cairina moschuta

7.49 * 0.01

(duck) N = 6 Kawashiro & Scheid,

PO,

0.01

Comments

(mmHg)

(mmHg)

PH

under control and experimental conditions in

33 * 1

82 + 2

41.0

At rest

I

82 + 1

41.0

At rest

38 i

1975

Anus olatvrhwchos

7.50

(duck) N = 10 Kiley et al., 1979 Pigeon N = 5-6 4-5 N=2 Lutz & Schmidt-Nielsen, Anus platxrhynchos (duck) N = 18

7.50

iJv=

( + 0.02) (+ 0.02)

25.1 (kO.5) 18.7(+1)

lOO(k2) llS(k2)

41.0 43.2

Running,

At rest 705, maximal

At rest, P,,, P,,, P,,,

effort

760 mmHg 282 mmHg 235 mmHg

7.60 + 0.04 7.66 + 0.02 7.85

28.7 t 1.1 13.5 * 1.0 10.5

81.2 + 0.5 29.5 f 2.2 23.2

4G41 40-41 4&41

7.50 + 0.003 7.52 * 0.006

30.4 + 0.4 27.5 +_ 0.8

102 + 0.8 105 i 0.8

41.4 41.7

At rest, Tam,, 2o’C Panting, Tamb 35°C

7.52 + 0.01 7.69 & 0.01

28.5 + 0.6 12.3kO.9

39.7 43.1

At rest, Tamb 22-26 C Panting, T.,mb 48-54’C

1977

iv=it Bouverot

rt a/.. 1974 livia (pigeon) N = 14 Calder & Schmidt-Nielsen, 1966 Columba

Mean

+ SEM. Values

in parentheses

are estimated

excretion by reptilian and avian kidneys since the pK of this buffer system is about 5.8. 2.

ACID-BASE PARAMETERS IN AVIAN BLOOD AND THEIR RESPIRATORY CONTROL

Table 1 presents control values of acid-base parameters and their change, if any, during exposure of birds to laboratory conditions similar to those encountered in natural settings, e.g. exercise, increased ambient temperature and varying altitudes. Inspection of the table shows that under control conditions blood has both a more alkaline pH and lower PcoI than values found in placental mammals. This trend was first noted in eight species by Calder and Schmidt-Nielsen (1968). who reported average blood pH of 7.52 and Pcoi of 28 mmHg; the individual values showed no correlation with body weight (0.3-7.5 kg) in their series. Reported measurements for blood [HCO,] are fewer but give an value of 24-25 mmol/l and a range of average 16-32 mmol/l, encompassing mammalian values (Tucker. 1968; Kiley et al., 1979; Beth & Johansen, 1980; Lenfant et ul., 1969; Krausz et u1., 1977; Marder & Arad, 1975; Scheipers et ul., 1975; Sauveur, 1969; Cohen et al., 1972: Siggaard-Andersen, 1976). Relative to mammals. birds -appear to be in a state of acute respiratory alkalosis. Explanation for the differences between birds and mammals undoubtedly involves many parameters, such as temperature effects, partition of CO, among the different body compartments, differences in non-bicarbonate buffering systems, etc. It has been suggested that a more effective ventilation in birds than in mammals creates the higher pH and lower P,,, observed in the former (Calder & SchmidtNielsen, 1968). Recent results demonstrate a higher Pco, in exhaled air than in arterialized blood leaving the lungs due to the cross-current relationship of blood and air flows reinforced by the Haldane effect (Scheid & Piiper, 1980). This phenomenon, in keeping with the suggestion of Calder & Schmidt-Nielsen, may play an important role in determining acid-base status in avian blood.

-

from graphs.

Non-bicarbonate buffering in avian blood is assured primarily by protein, although inositol pentaphosphate in erythrocytes may contribute (Lykkeboe & Johansen, 1975). The reported values of plasma proteins (3.5-6.0gm”i,) (Griminger, 1976) and of hemoglobin (6.5-19 grn:t”) (Sturkie, 1976a) encompass many mammalian values. but in general the average values in birds appear intermediate between reptilian and mammalian values (Lenfant et ul., 1970; Dessauer, 1974; Griminger, 1976). It is not surprising that the same generality appears to hold for buffer values in true plasma. The reported values in birds range from 20-33 mEq per liter per pH unit, with average values in non-diving birds (Lykkeboe & Johansen, 1975) somewhat lower than those for terrestrial mammals and higher than those in reptiles, including diving species (Wood & Johansen, 1974). Buffer values of intracellular fluid (muscles of breast. leg, heart) show considerable diversity in pigeons and chicken, not only between species but also among tissues within each animal. The authors of this study (Lykkeboe & Johansen, 1975) relate the range of values to metabolic activity of the different tissues. their myoglobin content and the buffering role of the dipeptides, carnosine and anserine, present in high concentrations in tissues of low respiratory activity (e.g. breast muscle of chicken). A similar diversity of intracellular buffer values exists in mammals (Siggaard-Andersen, 1976). As indicated in Table 1, control values may be changed by a variety of factors such as exercise, changes in ambient gas concentrations and elevated body and ambient temperatures. All the changes in that table are related to acute changes in respiration which in birds is sensitive to variatlon m arterial Pcoz and PO, (detected by carotid bodies), Pco2 in respiratory gas (intrapulmonary receptors) and directly or indirectly to body temperature (receptors in the hypothalamus and midbrain) (Fedde. 1976). The altered respiratory patterns induce varying degrees of offset in the control acid-base values through their effects on the ratio presumably secondary to the animals‘ (HCO,)/r*Pco,> adaptation to imposed conditions requiring sustained or increased delivery of Oz. dissipation of CO2 accumulated

Avian acid-base balance and urinary acidification during exercise and heat dissipation through panting. In general the results in the table indicate a rather effective protection of arterial pH and Po, with greater variation noted for Pco, and (HCO,). In ducks running on a treadmill at 1.5 km/hr (about 70% maximal effort), a Cfold increase in minute volume due to increased frequency and decreased tidal volume produced a si~ni~cant increase in Po, and decrease in Pco, in arterial blood and significant decreases in both in venous blood at the end of 20 min periods. Concomitant decreases in P,?, and [HC03] in both arterial and venous blood contrtbuted to stability of blood pH. although a slight increase in the arter~ovenous difference for blood pH occurred. The authors point out that the commonly accepted avian control mechanisms for respiratory regulation cannot explain the increased ventilation in these animals (Kiley rr al., 1979). A similar pattern of acid-base and blood gas changes has been described for the pigeon in flight {Butler et ni., 1977). Exposure to simulated altitude of 6100m for an hour demonstrated superior tolerance for such conditions in the house sparrow2 relative to its mouse companion (Tucker, 1968). Recent experiments have extended observations of the effects of altitude on acid-base balance (Lutz & Schmidt-Nielsen, 1977). Pigeons exposed acutely for an hour or more to hypobaric pressures of 349mm~g (6100 m) and 282 mmHg (cu. 7700 m) showed only slight alkalinization of arterial pH (0.06 pH units) accompanied by significant decreases in arterial Po, and Pco,. However, exposure of two birds to barometric pressures of 23.5mmHg (9150 m) demonstrated that the pigeons were able to tolerate even greater reduction in arterial Po, and an jmportant alkalosis (+0.25 pH units) although some stress, indicated by panting, was evident. In Tucker’s experiments on sparrows (1968), exposure to atmospheric pressure of 344mmHg at 5’C also produced alkalosis (+O.lO pH unit) and lowered P,,, in mixed arterial and venous blood, accompanied by increases in both Frequency and tidal volume. Many birds pant at elevated ambient temperatures (Calder and Schmidt-Nielsen, 1968). At moderate elevation of ambient temperature up to 30-3X body temperature in ducks remained nearly constant (Bouverot et af., 1974). The rise in pulmonary ventilation with rising ambient temperature is composed of increased respiratory frequency (20-25 times that at 20°C) and decreased tidal volume (&fold); arterial pH, Pox. Pco, oxygen consumption, and CC& excretion remained nearly constant relative to controls, while pulmonary water loss increased nearly &fold as ambient temperature rose from 20 to 35°C. Similar responses were evoked in the swan over the same range of ambient temperatures (Beth & Johansen, 1980), as well as in two desert birds (Marder & Arad. 197.5; Krausz et a/., 1977) with increases in average blood pH of no more than 0.04 units even at ambient temperatures of 45°C. Before equilibration in the swan, however, a pronounced reduction in arterial Pco, and alkaiosis was observed during the first 5 min of panting. In these birds the relatively stable blood pH during panting was associated with near constancy of body temperature. When body temperature was raised by 3°C in pigeons in response to ambient temperature of 5i”C, the decrease in tidal volume observed was apparently unable to protect blood pH which rose nearly 0.2 units (Calder & Schmidt-Nielsen, 1966). A rise in body temperature of more than 2°C was also measured in the running duck but without significant change in arterial pH despite a 4-fold increase in minute volume (Kiley et al., 1979). The large air sacs represent anatomical dead space in the avian respiratory system and probably play an important role in protecting acid-base balance during panting (Bouverot PI al., 1974). Dead space in the mammalian respiratory system accounts for a smaller per cent of total volume

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and even mild panting in dogs is accompanied by respiratory alkalosis (Adams. 1971). These experiments indicate that in many situations birds can accommodate to needs of changing oxygen consumption or of evaporative water loss by changing respiratory patterns without severe offset of arterial pH. In some cases increased frequency is accompanied by shallower breathing, i.c reduced tidal volume. which minimizes CO, loss. In other situations where the need for oxygen (e.g. at high altitudes) or for water loss (e.g. at elevated body temperature) overcomes these mechanjsms, frank respiratory alkalosis ensues. 3. URINARY ACBJlFlCATlON IN BIRDS In birds as in mammals pulmonary excretion of acid as COZ accounts for 997,;)or more of the total daily acid excretion. In birds. it seems likely that the strong acids, H2S0, derived from catabolism of sulfur-containing amino acids and H3P04 from phospholipids. represent the major threat to acid-base balance by the non-volatile acids derived from diet. The protons liberated from these acids are excreted by the gastrointestinal tract and the kidneys, primarily in association with buffers, e.g. ammonia, phosphate and probably urates and uric acid (U + UA). in chicken, quail, starling and turkey (Dantzler & Braun, 1980; Vogel et al., 1965) glomerular filtrates equivalent to plasma volume enter the nephron every 1@60min, rates several times greater than maximal rates observed in water loaded reptiles (Dantzler & Braun, 1980). Glomerular filtration rates in birds are somewhat reduced relative to mammalian values. but tubular secretion tends to maximize use of these volumes for excretion; for example, tubular cellular secretion of ammonia, phosphate, U i IJA and H’ all contribute to net renal acid excretion, In birds and reptiles the nephrons are perfused not only with postglomerufar arterial blood {sole origin of peritubular blood Row in mammaJs) but also with venous blood delivered from posterior regions of the body by the renal portal system; the content of this blood in Pco, and metabolites may influence tubular events in ways not observed in mammals. The avian kidney effects two major operations contributing to maintenance of stable acid-base status. recuperation of filtered bicarbonate and excretion of buffer-bound acid. Urinary acidification can be quantitated in terms of two interdependent variables, urinary pH and net buffer-bound acid excretion. The rate of net acid excretion is defined as the product of urine flow rate and urinary buffer concentrations: EH = V([NH4] j. [T.A.] - [HCO,]). Titratable acidity (T.A.) in birds is composed primarily of the nhosohate (HPO,/H,POd) and mate/uric acid (UIUA) buffer systems ‘(Wolbach: 19%). Bicarbonate is assigned a negative value in the definition above because it represents base excretion. The interdependence of acid excretion and urinary pH is evident; urinary pH determines the relative proportions of the salt and acid components of these two buffer systems. Furthermore, urinary pH plays a major role in NH, excretion and may participate in determining the excretion rates of phosphates and U + UA (Woibach, 1955; Long & Skadhauge, 198Oa). it will be assumed in this essay that the primary if not sole mechanism responsible for both bicarbonate reabsorption and net acid excretion is the tubular secretion of H’ ions. Urinary pH. pH of whole urine in birds (generally domestic fowl) reveals a wide range from 5 to 8 under experimental conditions (Sykes, 1971; Sturkic, 1976b). The maximal pH gradient between blood and urine is thus around 2.5 units, comparable to mammalian values (Pit&, 1974). The choice of a single urinary pH (or any other urinary

SCOTT LONG

521

variable) as “average” or “normal” has less meaning than the same variable in blood due to the varying renal role in maintenance of homeostasis in the extracellular fluid. The reported control values for urinary pH generally fall in the range 5.5-7.5, but some of the higher values may be due to delayed measurement of pH with consequent loss of CO,

from the samples. Urinary pH is inAuenced by acid-base status, sex of the animal and reproductive cycles, urine flow rates, water and electrolyte balance and diet (Sauveur, 1969; Simkiss, 1970; Skadhauge. 1977; McNabb et al., 1973; Okumura & Tasaki, 1968). Bicurbonure. Reabsorption of bicarbonate in mammals appears to be based ,primarily on secretion of H’ from intracellular into tubular fluid. The intracciluiar reactions.

.

r”

1

H,O+CO,=H,CO,%H+

+HCOJ.

are catalyzed by carbonic anhydrase (CA) found in the cells of the proximal and distat tubules and in the collecting duct. The secreted proton combines with filtered bicarbonate to form water and COz. Carbon dioxide diffuses into the cell, is rehydrated by carbonic anhydrase with the resultant bicarbonate moving back into extracellular fluid and the proton available for another cycle of secretion (Pitts, 1974). This mammalian model is the result of many experiments using micropuncture techniques and perfusion of isolated nephron segments. Such experiments are lacking in birds. However. some information from clearance and histological studies suggests that the mammalian model may be relevant to bicarbonate reabsorption in birds. Carbonic anhydrase has been found in the proximal tubule of the pigeon (Rosen 1972), but reports of its presence or absence in more distal portions of the nephron are lacking. Infusion of carbonic anhydrase inhibitors alkalinizes the urine (Wolbach, 1955; Orloff & Davidson. lY.59). Unilateral infusion of hydrochloric or acetic acid into the renal portal supply of the kidney was associated with a reduction of urinary pH in the experin~ental kidney relative to controi values in the contralateral kidney (Orloff & Davidson 1959); the authors suggested that acidification increases P co, in the blood perfusing the experimental kidney with consequent increase in intracellular PcoZ and H” secretion according to the schema outlined above. This mechanism may be of importance in less experimental settings. Pc,, m mixed venous blood is 48 mmHg higher than ;n arterial blood (Lutz & Schmidt-Nielsen, 1977: Butler et al.. 1977: Beth & Johansen. 1980: Kilev rr ai., 1979); portal perfusion of renal tubules may thus favor the secretion of Hi. The acid urinary pH commonly reported in birds suggests that bicarbonate reabsorption is very efficient. With the assumptions of equilibration between blood and urinary Pco, and a (U/P) for inulin of 50. 99.704 of filtered bicarbonate has been reabsorbed from urine at pH 6. Absolute rates of bicarbonate reabsorption. of net acid excretion and of H” secretion (calculated as bicarbonate reabsorption + acid excretion) are variable functions in the avian kidney. Reasonable assumptions for calcuiation of urinary (HCO3) allow one to conclude that the rates of H’ secretion and bicarbonate reabsorption decline while net acid excretion rates rise in metabolic acidosis (HCI infusion) (Wolbach, 1955). Data on plasma and urinary composition in laying hens support the same conclusion (Anderson, 1967; Simkiss, 1970). Amntonio. Ammonia accounts for 10_3Ou/;,of nitrogen excreted in ducks and chickens (Okumura & Tasaki, 1968; McNabb & McNabb, 1975; Stewart et a/., 1969). It exists in micromolar quantities in the plasma (McNabb et al., 1972) and is found at concentrations 80-300 times higher in urines of these animals. With (U/P) ratios for inulin of 3 in maximal diuresis and 100 in maximal antidiuresis. most

of the excreted ammonia appears to be the result of tubular secretion rather than filtration. Reported excretion rates of ammonia vary greatly from 10 to 500pmol/hr/kg body weight and reflect the commonly reported influences of diet, urinary flow rates and pH (Skadhauge. 1977; McNabb et al., 1972: Stewart et al., Long & Skadhauge, 1980a). With a pK greater than 9 ammonia binds Hi effectively in the range of urinary pH. As in mammals ammonia excretion is inversely related to urinary pH both at normal and diuretic urinary Bow rates (Long & Skadhauge. 1980a; Wolbach, 1955). The observations cited suggest that the mechanism of ditfusion trapping, used to explain the correlation in mammals, is also applicable to birds. According to this explanation. the liposoluble ammonia (NHJ) diffuses along its concentration gradient from its site of cellular synthesis into the tubular fluid (and peritubular blood) where its degree of protonation depends-upon intratubular DH. The charged form. NH:. is much less lioosoluble and it thus trapped in the excreted urine (Pit&, 1974). The mechanism of diffusion trapping does not appear to describe ammonia excretion in some aquatic reptiles which excrete very large quantities of ammonia in alkaline urines (Dantzler, 1976). Infusion of D,L-alanine, L-leucine and glycine increased ammonia excretion without marked changes in either urinary pH or flow rates (Wolbach. 1955). These results suggest increased cellular synthesis of ammonia as the basis for increased ammonia excretion observed in these experiments and on protein-rich diets (Okumura & Tasaki, 1968; McNabb et ut., 1972). The stimulation of ammonium excretion with ingestion of protein is of obvious advantage for excretion of the acid generated on such a diet. Under conditions of the experiments cited, urinary (NH,) is determined by urinary pH; but the converse does not appear to be true, i.e. ammonium does not play a role in determining urinary pH. This is turn suggests that ammonia is added to tubular fluid proximal to site of final tubular acidification and/or at rates below those of H secretion responsible for the final blood-to-urine pH gradient. Phosphate. The buffer system HP04/H,P04 with a pK 6.887.0 represents an effective means of acid excretion in avian urines and makes a major contribution to titratable acidity (Prashad & Edwards. 1973: Wolbach. 195% Rates of phosphate excretion are quite variable in birds, ranging _ from near zero to 1.50I,cmol/hr/kg in chickens (Martindale, 1973: Prashad 8c Edwards, 1973; Skadhauge, 1977) and several times that value in starlings (Wideman et ui., 1980). Calcification of the eggshell greatly increases phosphate excretion and serves to mitigate the concomitant acidosis (Prashad & Edwards, 1973). Net renal reabsorption characterizes phosphate handling in non-laying hens, net secretion during laying periods (Prashad & Edwards, 1973; Martindale, f973). although net secretion has been observed in non-laying hens (Levinsky & Davidson. 1957). Both net secretion and net reabsorption of phosphate have been reported in control (i.e. non-laying) starlings (Wideman rr ui.. 1980). Parathyroidectomy reduces net phosphate excretion while administration of parathyroid extract or hormone (PTE or PTH) and phosphate loading increase its excretion in association with net tubular secretion (Wideman et al., 1980; Martindale, 1973; Levinsky & Davidson, 1957). In starlings both secretory and reabsorptive mechanisms show tubular transport maxima (Wideman et a!., 1980). Recent micropuncture studies (the first in birds!) indicate that proximal tubules of superficial nephrons participate in both secretion and reabsorption of phosphate (Laverty & Dantzter, 1981f. Wideman and his associates conclude from their studies that hypocalcemia (rather than hyperphosphatemia)? induced by phosphate loading. is probably responsible for

Avian acid-base balance and urinary ac~di~cation liberation of PTH which in turn induces net phosphate secretion and phosphaturia. They further conclude that PTH acts by inhibition of the phosphate reabsorptive mechanism rather than by stimulation of secretion. Phosphate handling in reptiles shows many similarities to avian patterns, e.g. net secretion stimulated by PTE and by phosphate loading, net reabsorption in parathyroidectomized animals (Dantzler, 1976). in contrast net phosphate reabsorption characterizes renal handling in mammals; phosphaturia following PTH administration is associated with decreased phosphate reabsorption as in birds (Pit@ 1974; Wideman ct al., 1980). Thus some species of birds and reptiles appear to share in common a mechanism for phosphate reabsorption controlled by the parathyroid glands and another for phosphate secretion, perhaps retained from a common ancestor. In mammals the former only appears to play a major role in phosphate excretion although evidence for some transtubular secretion is accumulating. The model of Wideman et al., is relevant to acid excretion in birds since metabolic acidosis (HCI infusion) increases and metabolic alkalosis (HCOJ infusion) decreases free Ca in the plasma and should thus decrease and increase phosphate excretion respectively in the acid and alkaline urines formed in those conditions. Such responses, it should be pointed out, do not help to resolve the induced disturbances of acid-base balance. In contrast, in hens not subjected to metabolic disturbances of acid-base status, a significant negative correlation between urinary pH and phosphate excretion has been reported (Long & Skadhauge, 1980a), as well as between urinary pH and phosphate/inulin clearance ratios (unpublished), which approached unity at pH 5 and zero at pH 7. These data suggest that intratubular titration of phosphate may interact with hormonal control of tubular phosphate reabsorption and secretion in determining the excreted phosphate load, perhaps by setting substrate levels for preferential reabsorption of HPOI. Urates and uric acid. Birds, like terrestrial reptiles, excrete a significant fraction of their nitrogenous wastes as uric acid and urates, generally more than 507; in chickens, turkeys and ducks (McNabb & McNabb, 1975a; Vogel et al., 1965; Stewart et al., 1969). These animals excrete 50-350 ~moI/hr~g with variation due to protein content of diets, water availability, time of day. urinary Bow rates and DH (McNabb & McNabb. 1975b: McNabb et al., 1973; Vogel er al., 1965, Long & Skadhauge, 1980a). U + UA is secreted by the renal tubles with a transport maximum of roughly 150 pmol/hr/kg occurring at plasma concentrations of 1%2.0mmol/l (Sykes, 1971). Normal plasma levels are less than 1 mmol,l, and (U + UA)~inulin clearance ratios are found in the range of 5-20, indicative of tubular secretion of SO-95:/bof excreted loads (Berger er al., 1960; Stewart et al., 1969). In some birds de nova synthesis of U + UA in renal tubular cells contributes an important fraction, up to 20% in normally fed chickens, to the excreted load (Dantzler, 1978). The low solubility of uric acid and of urates and their consequent precipitation reduces the contribution of these compounds to urinary osmotic content relative to urinary osmolality expected were the same amount of nitrogen excreted as urea. This recuperation of solute-free water in avian and reptilian kidneys, especially under conditions of water deprivation has been often discussed (Sykes, 1971; McNabb & McNabb, 1975b; Dantzler, 1978). However, recent findings have raised questions concerning the role of precipitated U + UA in excretion of electrolytes and acid (McNabb & McNabb, 1975b; Dantzler & Braun, 1980; Long & Skadhauge, 1980b; Anderson & Braun, 1980). The pK of the U/UA buffer system is approx 5.8, so that the proportion of acid and salt forms may theoretically show considerable variation in the range of urinary pH in birds. Unfortunately, the complicated physical chemistry of

523

U + UA and of their association with electrolytes and urinary mucoids does not permit a simple prediction of the U/UA ratio based on the Henderson-Hasselbalch equation (McNabb & McNabb, f980). The reports of significant molar ratios for iNa)/ (U -t UA) and (K)/(U + UA) in urinary precipitates of chickens (McNabb et al.. 1973) and starlings (Braun, 1978) in some cases much greate; than unity, raise the possibility that urates may constitute an important fraction if not all the precipitated U + UA; divalent cations also show significant concentration in the precipitates (McNabb & McNabb, 1975). However, most if not all U + UA in avian urinary precipitates examined by X-ray crystailography exists as uric acid dihydrate (Lonsdale & Sutor, 1971). Recent studies in chickens (Long & Skadhauge, 1980b) and quail (Anderson & Braun, 1980) reveal these same molar ratios to be less than 0.15; in the former study these ratios, which concern the major urinary cations, showed no correlation with urinary pH. Taken together, these studies suggest that urinary precipitates may play a major role in acid excretion in birds, but the binding of divalent cations and possible species differences remain to be settled before general assignment of such a role. The predominance of precipitated U + UA in the acid form does not preclude an important excretion of electrolytes in precipitates since the cations may be trapped between UA layers and/or associated with urinary mucoids within the precipitates (Lonsdale & Sutor, 1971; McNabb & McNabb. 1975b. 1980). Limits of acid excretion as UA can be estimated according to two assumptions. Of the 150 PM U + UA excreted/hr/kg in a urine of pH 6.2, 85% was found in the precipitate fraction (Long & Skadhauge, unpublished data). If all precipitated U + UA exists as UA, then application of the Henderson-Hasselbalch equation to the remaining supernatant U + UA indicates that 897” of the total U + UA excreted is used for acid excretion. On the other hand, application of that equation to the total U + UA excreted would reduce UA to 28% of the total. In the first case UA accounts for about half of net renal acid excretion measured and to about 25% in the second case. With either assumption UA clearly plays an important role in net renal acid excretion at this pH in chickens, as suggested also by the non-phosphate titratable acidity measured by Wolbach (1955). Participation of U + UA in renal resolution of acid-base disturbances is not clear. Total excretion of U + UA does not show significant change in cockerels made alkalotic or acidotic bv addition of NaHCO, or HCI to a 20% casein diet for 3 hays (Okumura & Tasaki, 1968). In acute metabolic acidosis (HCI infusion), however, phosphate excretion dwindled and titratable acidity increased with falling urinary pH; Wolbach (1955) concluded that the additional H’ was bound as UA with obvious advantage in resolution of the acidosis. The significance of these results to less experimental conditions remains in question since the animals used were severely diuretic which would favor an increased fraction of total U + UA in the supernatant. In contrast to these findings in chickens, metabolic alkalosis in water snakes increased U + UA excretion while metabolic acidosis was without effect (Dantzler, 1976). In the antidiuretic chicken not subjected to metabolic disturbances of acid-base status, a positive correlation of total U + UA excretion and pH 5-7 in ureteral urines has been reported (Long & Skadhauge, 1980a). If indeed U + UA in precinitates is present as UA, the continued presence of precipitates observed in urines at pH 6-7 will maintain a significant level of net acid excretion despite decreases in NH,+ and H2P04 and rising HC03 excretion (Long 8~ Skadhauge, 1980a). The effects of this renal loss Of acid on systemic acid-base balance could be met by small changes in respiratory loss of C02, supplemented by intestinal and cloaca1 recuperation of acid-bearing buffers (see below).

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U + UA secretion has been less thoroughly studied at the tubular level in birds than in snakes. The complexities of U -t UA secretion and reabsorption are many and beyond the scope of this review; interested readers should consult Dantzler’s review (1978). Sufftce it to say that birds and reptiles secrete U + UA, and evidence for reabsorption is lacking. Secretion occurs in the proximal tubule of the snake, while ureteral stop-flow studies in the turkey suggest a more distal site, evidenced by coincidence of lower (Na) in samples with elevated (U + UA) (Vogel et ol., 1965). A more distal site might be advantageous in view of the low solubility of U t UA since the avian kidney reabsorbs more water than does the reptilian. In ureotelic mammals U + UA excretion is greatly reduced. and both tubular reabsorption and secretion have been observed in micropuncture studies which reveal great diversity in the patterns of tubular handling of these compounds (Dantzler, 1968). Net renal acid rxcretion. Few determinations of net acid excretion exist for any vertebrate class other than mammals. The value for the chicken, - IO PEqlhrjkg (net base excretion), estimated from the data of Wolbach (1955), is in the same range observed in most vertebrates where average values of up to +50/rEq/hr/kg are found (Long & Giebisch, 1980). However. in less diuretic chickens producing more acid urines, the data suggest a much greater net acid excretion. Values of 15@200pEq/hr/kg can be calculated in urines at pH 5.0-5.5 for chickens on two diets, without taking the excretion of U t- UA into account (Skadhauge, 1977). More recent determination of acid excretion in hens including U + UA gives values in excess of 200 pEq/hrjkg at pH 5.7 (Long & Skadhauge, 1980a), several times greater than that expected in man, for example, at the same pH. Acid excretion is influenced by diet and a flexible urinary pH, as observed in mammals (Long & Giebisch, 1980) and presumed for reptiles where urinary pH and (NH,) vary with diet (Minnich, 1972). In birds a high protein diet increases urinary excretion of NH, and U + UA, lowers urinary pH and increases acid excretion, all factors which promote stability of acid-base status in face of the increased acid production on such a diet (McNabb et ul., 1972; McNabb et al., 1973; Okumura & Tasaki, 1968; Long & Skadhauge. 1980a). 4.

MODIFICATION OF RENAL ACID EXCRETION IN GUT AND CLOACA

The final excretion of acid in feces is obviously more important to acid-base balance in birds than the delivery of ureteral urine into the cloaca. Urine passes anteriorly from cloaca into the gastrointestinal tract as high as the junction of colon, ileum and caeca (Sykes, 1971). pH along the gastrointestinal system and cloaca becomes increasingly alkaline with values from 6-7 commonly reported for the large intestine and cloaca (Sturkie, 19%~). Average pH of cloaca1 feces in the chicken was almost a pH unit more alkaline than the ureteral urines collected in the same groups of birds (Long & Skadhauge, unpublished results). This alkalinization may back-titrate some of the buffers excreted by the kidney, e.g. phosphate, to decrease final acid excretion. Furthermore, Barnes (1972) has reported significant numbers of anaerobic, uricolytic bacteria (109/gm caecal content) in the caeca of a variety of birds; the CO2 and NH3 liberated are both highly diffusible compounds and may return to the general circulation or may be incorporated into bacterial metabolism. These processes would reduce net acid excretion. In a recent study on the desert quail, Anderson & Braun (1980) have compared U + UA content of ureteral urine and feces to find that roughly 60y0 of renally excreted U f UA was degraded before evacuation in the feces. In summary. arterial pH in birds appears relatively stable in many conditions despite significant offset for

blood gas tensions from normal values, e.g. in exercise, in thermoregulation at moderate elevations of ambient temperature, in acute exposure to reduced atmospheric pressure. Under moderate stress, arterial (H+) changes from 0 to 15% are accompanied by much greater changes in Pco, (from 10 to almost 500/,). Under severe stress with heavy panting, respiratory protection breaks down with frank alkalosis resulting. Part of this protection is inherent in the HC%/Pco, buffer system in the blood and part from interaction of respiratory alkalosis with lactic acid production in exercise; but it seems likely that the large anatomical dead space of air sacs, peculiar to birds, plays an important role. The avian kidney also contributes to stability of acidbase status through excretion of buffer-bound HC derived from non-volatile acids produced by metabolism. Net acid excretion rates in birds may be quite high relative to mammalian values, but at present this generality rests on calculations from a single species, the chicken. Uricotelism in birds appears to make an important contribution to net acid excretion, and the high rates of U + UA excretion in many birds argues for the relevance of the values of net acid excretion in chickens for other avian species. Although relatively little work has been done so far, the intestine and cloaca appear to play an important role in controlling the loss of acid excreted by the kidney. In a comparative context, birds and mammals show many similarities in acid-base balance and its control. Parallel, or convergent, evolution from a common reptilian ancestor, 300 million years ago, has produced many different anatomical designs in these two classes; of particular relevance here are differences in renal and especially in pulmonary design, as well as differences in metabolic patterns (uricotelism versus ureotelism) and in some transport systems (renal handling of U + UA and phosphate). Even so, the physiology of acid-base balance and its dependence on and control by several systems shows more similarities than differences, in my opinion, and argues for the perspective that evolution selects for physiological integration of many systems rather than for the design and function of its individual components. AcknoMlledyements-The author thanks the Fulbright Commission of Denmark, the NOVO fund, and NATO grant 1795 for support during his work at the Panum Institute. REFERENCES ADAMST. (1971) Carnivores. In Co~~#~a~jue P~~~~o~ogyof ~~~~~~or~gu~a~iun(edited by WHITTOW G. C.), pp. 151-189. Academic Press, New York. ANDERSON G. L. & BRAUNE. J. (1980) Renal function and post-renal modification of urine in the desert quail. Fed. Proc. 39, 58%. ANDERSON R. S. (1967) Acid-base changes in the excreta of the laying hen. Vet. Rec. 80, 314. BARNESE. M. (1972) The avian intestinal flora with particular reference to the possible ecological significance of the cecal anaerobic bacteria. iZm. JI. &in. Nutr. 25, 1475-1479. BECHC. & JOHANSEN K. (1980) Ventilatory and circulatory responses to hyperthermia in the mute swan (Cygnus olor). J. exp. Biol. 88, 195-204. BENNETT A. F. & RUBENJ. A. (1979) Endothermy and activity in vertebrates. Science, N.Y. 206, 649-654. BERGERL., Yii T. S. & GUTMANA. B. (1960) Effect of drugs that alter uric acid excretion in man on uric acid clearance in the chicken. Am. J. Physiol. 198, 575-580. BOUVER~TP., H~LDWEING. & LE GOFF D. (1974) Evaporative water loss, respiratory pattern, gas exchange and acid-base balance during thermal panting in Pekin ducks exposed to moderate heat. Resp. Physiol. 21, 25.5-269.

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