STUDY ON MECHANISM OF ACID AND AMMONIA EXCRETION BY KIDNEY AFTER ACID LOAD. Hisato YOSHIMURA, Mamoru FUJIMOTO AND Junichi SUGIMOTO

Transcription

1 STUDY ON MECHANISM OF ACID AND AMMONIA EXCRETION BY KIDNEY AFTER ACID LOAD Hisato YOSHIMURA, Mamoru FUJIMOTO AND Junichi SUGIMOTO Department of Physiology, Kyoto Prefectural University of Medicine, Kyoto It is well known that animals can put up with a great deal of acid load not only by buffer actions of body fluid, but by pulmonary output of volatile acid and renal excretion of nonvolatile acid; and that the kidney plays the most important role in excretion of excessive fixed acid radicals when the animal is going to recover from acidosis. In the previous paper, the authors reported that the homestatic function of body fluid after acid loading is such a well arranged process that the infused acid is neutralized at first by extracellular buffer, secondly by intracellular buffer, and finally removed by the kidneys as ammonia and acid; being referred to as the three-step-regulation of acid-base balance1). The finding that the renal excretion of the exessive acid increases during the period of acid loading and even thereafter until the acid-base states of the animal restore normal conditions seems to be quite reasonable. Though a number of investigators have observed this phenomenon, the mechanism whereby the renal acidification and ammonia production would be enhanced in acidosis has not yet been fully discussed. It is well known that PITTS and his colleague2) had clearly explained the urinary acidification in terms of sodium-hydrogen ion exchange mechanism in the distal tubule. However, it is not yet certain why the activity of sodium-hydrogen ion exchange mechanism whereby hydrogen ions are secreted into tubular urine is augmented in acidosis. Now, since adrenocortical hormone is known of its strong sodium retaining and potassium eliminating action, it might be suggested that this hormone could act directly on some pump mechanism of sodium reabsorption, so that the intracellular hydrogen ions, as well as potassium ions, would be more actively secreted into the acid urine by the exchange mechanism under the influence of this hormone. According to PITTS3) and BERLINER et al.4) the urinary ammonia has been claimed to be secreted in the form of neutral molecule by diffusion from tubular cell into luminal fluid. However, there are some points about the ammonia excretion that can hardly be accounted for with this theory but explained more Received for publication October 3,

2 144 H. YOSHIMURA, M. FUJIMOTO AND J. SUGIMOTO reasonably by a sodium-ammonium ion exchange mechanism on the luminal surface of distal tubule, as is offered by RYBERG5). Though the fact that ammonia is secreted in increasing amount by the renal tubule in chronic acidosis in rats was accounted for by an adaptive increase of renal glutaminase activity (RECTOR et al.6)), discussion on the increased excretion of ammonia in prolonged acidosis of dog is not yet settled. On the other hand, there are many reports on the adrenalectomized animal unable to produce much amount of the urinary acid and ammonia and to regulate the acid-base balance of body fluid7,8). The discussion on this mechanism is rather complicated and remained unsolved. The present paper is undertaken to study the mechanism of regulatory systern of renal acid handling with special reference to effects of adrenalectomy. EXPERIMENTAL METHOD Forty five healthy adult mongrel dogs weighing 5 to 12 Kg were divided into the following three groups: (1) Normal thirty dogs were infused isotonic hydrochloric acid solution (0. 16 normal) intravenously at the rate of 1-8mMol/Kg of body weight. (2) The other ten dogs were adrenalectomized on both sides prior to the acid infusion, and on the fourth day after adrenalectomy the same amount of acid as in the normal dog was loaded intravenously to the adrenalectomized dogs and its effect was cornpared with that of normal dogs. (3) The remaining five dogs were adrenalectomized and desoxycorticosterone acetate was administered at the rate of 1mg/Kg of body weight. Two hours after DCA injection, acid was infused into them as described above. In the first group, various control experiments were made as follows: The animals were anesthetized with an intravenous injection of mg/Kg sodium methyl-hexabarbiturate, and were kept supine under loose restraint. The special care being paid on the corneal reflex and abdominal muscle tone, too deep anesthesia was avoided. To avoid a variation of acid-base balance in the animals due to diet, the dogs were fed on the neutral diet with proper amount of water (9) and were kept in metabolic cages for about a week or longer prior to the infusion of acid. The infusion of 0.16 normal hydrochloric acid was started after an adequate control period of several ten minutes, and sustained for 1 to 6 hours at a constant rate of 1-2 ml/min. (10-60ƒÊEq/min./Kg) with a calibrated constant infusion pump. Urine samples were collected under liquid paraffine from indwelling catheter of urinary bladder (with manual compression on abdomen) at intervals of 5-30 minutes throughout the experiment. Blood samples were withdrawn from the jugular vein or from an artificial side-tube made on the carotid artery during the experiment when the circulating blood electrode was applied there. Blood centrifuge was made under liquid paraffine to avoid an escape of CO2 from the sample. In order to study the possible role of mineralcorticoid on the acid-base regulation by the kidney, the acid was given to the other groups, i.e. the adrenalectomized dogs and DCA administered dogs, under the same circumstances and with the methods that had been established in the first group. In the second group, both sides of adrenal glands are removed one by one unilaterally at interval of about a couple of weeks. Adrenalectomy was made by flank incision under hexabarbiturate anesthesia. No supplementary fluid was added to the

3 ACID AND AMMONIA EXCRETION 145 animal. Acid infusion experiment was made on the adrenalectomized dog on the fourth day after adrenalectomy, when the direct effects of operative stress had almost disappeared. For the survey of blood ph, a tube-type glass electrode was especially designed for the measurement of circulating blood. Blood was prevented from its clotting by heparin. injection ( Unit/Kg) and was led directly to this glass electrode through polyethylene tube and changes in ph were recorded automatically and continuously in SHI- MADZU Electronic Balancing Recorder10) in combination with vacuum tube ph meter (SHIMADZU GM-1 type11). However, in some experiments blood ph was estimated from values in the plasma which was taken intermittently from the animal and ph was determined with YOSHIMURA'S syringe type glass electrode30) immediately after the experiment was over. The ph measurement in urine samples were made with this type electrode in all cases. The total CO2 contents of blood and urine were determined by VAN SLYKE'S manometric method modified by SAITO12). The partial pressure of CO2 (pco2) in plasma and urine was calculated from the Henderson-Hasselbalch's equation, utilizing the actually measured ph and total CO2 contents values and 6.10 for plasma pk' and the equation of pk'(= ãƒê.) for urine, where ƒê is ionic strength calculated from total ionic concentration and varied within the range of in the urine. Sodium and potassium level in plasma as well as in urine were measured with LANGE'S flame photometer13), while chloride was determined by modified SCHALES and SCHALES method14). Ammonia was determined by CONWAY'S method15), while titratable acidity was estimated electrometrically by the modified method of ASADA in our laboratory16). In order to study changes of renal functions during the experiment, either endogeneous creatinine clearance or inulin clearance method was employed and its values were regarded as glomerular filtration rate. The determination of creatinine was dependent on FOLIN and WU method17), while inulin was measured by SCHREINER'S method18). The renal glutaminase activity was determined with kidney homogenate from normal dogs before and after acid load and also from adrenalectomized ones. The measurement was performed by RECTOR'S method at optimal ph 8. 00). With the same samples for glutaminase, renal carbonic anhydrase was determined by the method used by IWASAKI in our laboratory with WARBURG'S manometer,9). For the calculation of enzyme activity, nitrogen contents in the kidney homogenate were analysed by means of Semi-micro Kjeldahl method and enzyme activities were expressed in terms of per 1 milligram of tissue nitrogen, or of per 1 gram of fresh tissue weight. EXPERIMENTAL RESULT Effect of acid infusion on urinary electrolytes of normal and adrenalectomized dogs. FIG. 1 shows a typical example of urinary changes in normal and adrenal - ectomized dogs during HCI infusion. The left half of FIG. 1 represents an example of a normal dog which was infused intravenously with isotonic HCl solution (6.7mM/Kg). The most of the results were essentially similar among other thirty experiments. On the other hand, the right half of FIG. 1 represents an observation on urine electrolytes in an adrenalectomized dog to which the acid was infused at the same rate as that on the left side. The experiment was

4 146 H. YOSHIMURA, M. FUJIMOTO AND J. SUGIMOTO

5 ACID AND AMMONIA EXCRETION 147 performed on the fourth day after adrenalectomy (acid infusion at the rate of 10-60ƒÊEq/min./Kg). In the normal dog in FIG. 1, the urinary ph has begun to decrease about an hour after the initiation of acid infusion and attained 5.7 or so towards the end of infusion. A sign of gradual restoration of urinary ph appeared shortly after the end of infusion. A gradual increment of titratable acidity and ammonia output was associated with the decrease of urinary ph. The CO2 contents in the urine reduced almost to disappearance. Generally speaking, sodium excretion (open Na space in the figure) tends to decrease gradually, while chloride excretion increases. Though the excretion of Na and Cl are influenced more or less by urine volume, these changes can not be explained solely by this effect. The excretion of chloride keeps an equilibrium with that of Na and K in the first half of experiment, but these partners of Cl have been replaced more and more by NH4 ions in the latter half as the acidosis proceeds, so that a part of chloride was excreted as NH4Cl in the urine at the final stage of infusion. The output of K tends to increase, resulting in the gradual decrease of the urinary Na/K ratio. Juding from this change of the urinary Na/K ratio, it is suggested that the adrenal activity would be stimulated by putting the animal into acidosis and action of some mineralcorticoid might be potentiated. In the adrenalectomized dog, on the other hand, the acidification of urine appeared for a short period but the decreased urinary ph began to rise and is maintained almost neutral in spite of acid infusion. This is perhaps due to a failure in renal reabsorption of bicarbonate by adrenalectomy, because, the bicarbonate contents in urine were hardly effected by acid infusion. Urinary ammonia and titratable acidity in this case does not so much increase as in normal acid infused dogs. Urinary sodium output was considerably increased while potassium excretion was rather depressed. Consequently, the urinary Na/K ratio was increased after acid infusion in the adrenalectomized dog, while it was depressed in the normal dog, as can be seen in FIG. 1. After all, it can be said that the adrenalectomized dog can not adapt to the acute acid load and it fails to potentiate an acid removal from the body. In order to compare quantitatively the effects of acid load among normal, adrenalectomized animal and DCA administered dog, the extent of blood ph decrease at the end of acid infusion was plotted against acid load in FIG. 2, where normal dogs are represented by solid circles, adrenalectomized dogs by cross marks and DCA administered dogs by triangles. The decrease in blood ph bears a close positive correlation with the acid load, and thus the strength of acidosis is roughly proportional to the excess amount of acid in blood. The rate of acid infusion seems to have no influence on the final effect of acid load to decrease blood ph. This suggests that the infused acid can diffuse rapidly out of the vascular system to be neutralized by extracellular and

6 148 H. YOSHIMURA, M. FUJIMOTO AND J. SUGIMOTO FIG. 2. Relationship between Decrease of Blood ph and Acid Load on Normal, Adrenalectomized and DCA Administered Dogs at the end of Acid Infusion. FIG. 3. Relationship between Acid Load and Change of Titratable Acidity in Urine Collected during the whole period of Acid Infusion from Normal, Adrenalectomized and DCA Administered Dog. possibly intracellular buffers, and consequently the changes of blood ph remains relatively indifferent to the rate of infusion. The blood ph decrease in adrenalectomized dogs in FIG. 2, however, was larger than in normal dogs, and their regression line related with acid load was located lower than that in normal dog group. In DCA administered dogs (DCA 1mg/Kg was given 2 hours before infusion), however, the blood ph fall is less than normal dog group. FIG. 3 represents the change of titratable acidity in acidosis from the

7 ACID AND AMMONIA EXCRETION 149 respective control value before acid infusion which was plotted against the amount of acid load (+ sign for its increase and - for its decrease). Solid circles in the figure represent the cases of control urine ph above 7, while open circles are cases of the control ph under 7. The increase of titratable acidity after acid load seems to have some bearing on the amount of acid load, but the correlation is not so good as to have a clearcut regression line. The reason is as follows. In cases of urine ph under 7, there had already been produced a certain amount of titratable acidity prior to acid infusion, so that the acid infusion could not potentiate the titratable acidity so much as in those cases where neutral or alkaline urine are excreted prior to acid infusion. Thus the effect of acid infusion may differ according to the ph of control urine. The fact can be confirmed in FIG. 3. It is reasonable that in adrenalectomized dogs most of titratable acidity ( ~ in FIG. 3) were at lower level than those of normal dogs because of their inability of increasing the urinary titratable acid after acid infusion. The same fact can be confirmed in FIG. 4 in which the titratable acidity in urine is plotted against the blood ph. The titratable acidity increases as the blood ph decreases in normal dogs as well as in adrenalectomized dogs. The titratable acidity is less in the adrenalectomized dogs than the normal acidotic dogs at the same blood ph. The administration of DCA to the adrenalectomized dog can restore the acid excretion to the same level as that in the normal dog. FIG. 4. Correlation between Blood ph and Titratable Acidity in Urine of the Normal and Adrenalectomized Dog collected during the whole period of Acid Infusion.

8 150 H. YOSHIMURA, M. FUJIMOTO AND J. SUGIMOTO FIG. 5. Correlation between ph and ammonia of urine in normal and adrenalectomized dog collected during the whole period of HCl infusion. The relationship between ammonia output and ph of urine collected during acid infusion with normal as well as adrenalectomized dogs is illustrated in FIG. 5. Ammonia output not only in total amount but in concentration bears a good inverse correlation with urinary ph in normal dogs as well as in adrenalectomized dogs. Comparison between plotts in left and right hand sides in the figure reveals that the ammonia output increases as the urine ph decreases and vice versa, and that the amount of ammonia excreted in urine of the adrenalectomized dog is always lower than in the normal dog urine at the same urine ph.

9 ACID AND AMMONIA EXCRETION 151 In order to investigate the cause of the difference of ammonia-ph correlation between normal and adrenalectomized dogs, renal glutaminase and carbonic anhydras activities were determined on both normal and adrenalectomized dog kidneys. In order to determine these enzymes, kidneys were removed from the body and were immediately perfused with saline solution to wash out the blood remaining in the organs and their homogenized samples were analysed. The two columns at the left hand side in TABLE 1 show a comparison of renal glutaminase and carbonic anhydrase activities in normal dog kidneys (without infusion of acid) with those in adrenalectomized ones. No significant difference in either renal glutaminase or carbonic anhydrase activity was found between normal and adrenalectomized dogs. The fact suggests that the ability of renal production of ammonia and hydrogen ion in adrenalectomized animal remains normal, and that the lowered output of ammonia in adrenalectomized dog should be due to the reduction in transport mechanism of ammonia from the tubular cell into the lumen but not to the reduction of glutaminase activity. From the current view that the adrenal corticoid can promote the reabsorption of Na from luminal fluid, it is reasonable to explain this reduction of ammonia output in adrenalectomized dog as due to the decrease in Na+-NH4+ ion exchange through tubular cell membrane. The restoration of ammonia output by administration of DCA to these dogs supports this view, as will be explained in the next section. The adaptive increase of renal glutaminase in prolonged acidosis was verified by RECTOR et al.6) with rats which were acidotic by daily administration of ammonium chloride. They failed, however, to prove it in dogs29). The authors investigated this problem with 8 acidotic dogs, into which about 3mEq/Kg body weight of hydrochloric acid was infused intravenously on the 3rd day before the analysis of kidney. The decrease of blood ph was restored to the normal level after 24 hours, but the acid radicals infused remained in the intracellular fluid about a week before it has been excreted by kidney (Refer to YOSHMURA et al.1)). The glutaminase and carbonic anhydrase contents of these dogs after 3 days of acidotic state are summarized to the right column in TABLE 1. Glutaminase activity in acidosis dog was increased significantly in all examples, while carbonic anhydrase remains unchanged. The difference between the results of RECTER et al.29) and the present authors' is suggested to be due to difference of experimental conditions, especially to different kind of acid administered. 2. Effect of DCA administration on adrenalectomized dog. As stated previously, it was demonstrated that adrenalectomized animals lost the normal function of acid radical handling of the kidney, and permitted its blood ph to be lower than normal dogs. These effects seem to be due to lack of mineralcorticoid in adrenalectomized dogs. In order to confirm the fact, the acid was infused into DCA administered adrenalectomized dogs and changes

10 152 H. YOSHIMURA, N.I. FUJIMOTO AND J. SUGIMOTO FIG. 6. Influence of DCA on Uninary and Blood Electrolytes of Adrenalectomized Dog. in the acid-base balance were observed. DCA was given to dogs 2 hours prior to acid infusion. A part of the experimental results are shown in FIG. 6. The acid infusion was made at the rate of 6.4mEq/Kg (64ƒÊEq/min./Kg) for 100 minutes. Urinary ph was already acid (about 5.6 even in the control period, nevertheless, urinary acidification was further pointiated during acid infusion, and simultaneously an increase of titratable acidity and ammonia excretion was also demonstrated. Sodium was effectively retained in the body, while potassium was excreted copiously; therefore, urinary Na/K ratio was considerably lowered, suggesting that the administered DCA brought the restoration of renal function of ammonia output and urine acidification.

11 ACID AND AMMONIA EXCRETION 153 DISCUSSION It was demonstrated in this study that the urinary output of acid and ammonia increases in acute acidosis. These responses to acidosis decrease in adrenalectomized dogs; and further it was confirmed that DCA, when it is administered in proper amount to the adrenalectomized animals, initiates an increase of the urinary titratable acidity and ammonia. As for the mechanism of acidification, the theoretical concept which has been prevailing at present is as follows: 1) Hydrogen ions are secreted into tubular urine in exchange for sodium ions in the distal tubule, where sodium ions undergo the facultative reabsorption. In this case the hydrogen ions which are secreted into urine are derived from metabolically produced carbonic acid under the presence of carbonic anhydrase within the distal tubular cells20). 2) Potassium ions are also secreted into distal tubular fluid in exchange for sodium ions and thus a competitive secretion occurs between potassium and hydrogen ions so long as normal acid-base balance of the body fluid is maintained (BERLINER21)). In order to criticize the above concept of urinary acidification, discussion will be made in the following two important points: (1) the ion exchange proc ss between luminal sodium and intracellular hydrogen or potassium, (2) the regulation of this exchange process in acidosis. And it will be extended with special reference to the experimental results with adrenalectomized dogs which are loaded with acid. To explain the reason why the ions exchange process between Na and H on the luminal surface of the renal tubule can be changed in acidosis, PITTS and others22) stated that the intracellular K deficiency which is associated with acidosis might initiate an augmentation of bicarbonate reabsorption as well as of acid production. When the acid is infused, hydrogen ions would probably enter the cell in exchange for intracellular potassium, so that the plasma potassium level would rise in proportion to the degree of potassium emigration from the cells. Therefore, the higher the plasma acid level goes up, the lower the intracellular potassium concentration falls down. Consequently, the cation which is excreted from the cell in exchange for reabsorbed Na+ is not K+ but accelerated. In order to examine the relationship between the intracellular K concentration and the urinary titratable acid output in the present experiments, the titratable acidities were plotted against the plasma K concentration as shown in FIG. 7. The titratable acidity is expressed in the amount of output per Kg of body weight per minute. It is demonstrated that the titratable acidity bears a close positive correlation with serum potassium concentration. Thus, PITTS'

12 154 H. YOSHIMURA, M. FUJIMOTO AND J. SUGIMOTO FIG. 7. Correlation between Plasma Potassium Concentration and Urinary Titratable Acidity of Normal and Adrenalectomized Dog. hypothesis is verified. On the other hand, it should be reminded that the intracellular H+ concentration may be increased after the acid infusion. Therefore, the increased urinary titratable acidity might be due to the increase of hydrogen ion concentration in the cells. It is very difficult to decide which causes of the two may predominates. In the present experiments, however, it was verified that the correlation between plasma K concentration and the urinary output of titratable acid varies according to the physiological condition of the experimental dog. It is demonstrated in FIG. 7 that adrenalectomized dogs can produce less amount of titratable acidity than normal dogs at the same plasma potassium level but administration of DCA can permit the animal to produce even greater amount of the acidity than the normal dog. Therefore, one of the factors governing the production of titratable acidity is certainly the mineralcorticoid. Since the mineralcorticoid is known as an activator of Na reabsortion in the renal tubule, it is extremely reasonable that Na+-H+ ion exchange process may be promoted by mineralcorticoid, and thus the acid production is accelerated. As is presented in FIG. 4, the gradient of regression line or regression coefficient of acid excretion against blood ph (the degree of acidosis) varied with

13 ACID AND AMMONIA EXCRETION 155 or without the presence of adrenal gland. Consequently, it is presumed that the blood acidosis itself might accelerate the secretory activity of this adrenal gland and thus the urinary acid production is promoted in acidosis. The mineralcorticoid can not be considered as the only factor which governs the acidification of urine, because the titratable acid output increases in proportion to the degree of acidosis even in absence of adrenal gland. Therefore, some direct effect of acidosis on the mechanism of acid production in urine might be concerned there. The direct effect may be explained either by the decrease of intracellular K+ concentration or the increase of intracellular H+ concentration as already discussed above. In short, the following two reasons are proposed to explain the increased urine acidification in acidosis: 1) the acidosis itself gives a direct effect on Na+- K+ and Na+-H+ ion exchange mechanism in the distal tubular cells, and 2) acidosis probably alters a secretory activity of adrenal gland and secondarily the ion exchange of Na+ and H+ is promoted in acidosis. The mechanism of the direct effect of acidosis upon the urinary acidification is a matter of future study. Concerning the secretory mechanism of urinary ammonia, there have been presented two hypotheses: (1) capture-by-diffusion (PITTS2)), (1) Na+-NH4+ ions exchange hypothesis (RYBERG5)). According to the former concept, the intracellular ammonia molecules (NH3) are secreted by diffusion along the concentration gradient established between renal tubule cell and tubular urine. In the latter theory, however, the intracellular ammonia ions which occupy almost all the share of ammonia at the cellular ph can be secreted directly in exchange for sodium ions when the filtered sodium ions are actively reabsorbed by the distal tubule. The capture-by-diffusion theory is based on the assumption that NH3 is freely permeable to the renal tubular cell wall but NH4+ is relatively impermeable to it. This assumption is dependent upon the classical experiment with red cells by JACOBS24) when the concept of active transport of electrolytes has not yet been established. Therefore, it is not certain how far this assumption may be available to renal tubular cell. Recently, YOSHIMURA et al.25) studied on the acid-base regulation of frog after acid load and verified that the ammonia is excreted in the form of neutral molecule by diffusion into tubular lumen where it is captured by the acid, chiefly carbonic acid. The ammonia thus captured is transformed. into ammonium ion and equilibrates with bicarbonate ion in the form of NH4HCO3. In the urine of mammals, however, the ammonia is excreted in the form of NH4Cl. PITTS explained the mechanism of production of ammonium chloride as due to the exchange of luminal Na+ with intracellular H+ to produce HCl which is neutralized with neutral ammonia to form NH4Cl. There is, however, no evidence of HCl production in the tubular lumen. On the contrary, the urinary ph of tubular fluid measured indicates that it is not less than 4.5 or

14 156 H. YOSHIMURA, M. FUJIMOTO AND J. SUGIMOTO so in mammals. It follows that free HCl can not exist in an appreciable amount at such a weak acid reaction. Especially it is unreasonable to consider the formation of HCl at the ph of 5-7 which is indicated in FIG. 5. In the present experiments, it was proved that the urinary acidification is effected by the exchange mechanism of luminal Na+ with intracellular H+ and the reduced acid formation in adrenalectomized animal was explained as due to the decrease of the exchange mechanism by the lack of mineralcorticoid. Similar reduction of ammonium chloride formation was demonstrated in the adrenalectomized animal in FIG. 5. Why is it unreasonable to explain this reduction as due to the decreased ion exchange mechanism of Na+ for intracellular NH4+? If the JACOB'S concept of impermeability of NH4+ through tubular cell membrane had lost its basis in the light of recent concept of active transport, it seems rather reasonable to account for the formation of ammonium chloride in urine as due to the exchange of tubular Na+ with intracellular NH4+. Concerning the existence of NH4+ in the tubular cells, RECTOR et al.26) claimed that approximately 99% of ammonia present in the cell at the physiologic ph is in the form of ammonium ion. An experimental evidence of Na+-NH4+ exchange is presented in FIG. 8, in which the ammonia output in urine is correlated with the reabsorption of Na+ as is the case for K+ output in urine. The reabsorption of Na+ was calculated with all the dogs experimented in this study of which glomerular filtration rate was estimated from the endogeneous creatinine clearance or the inulin clearance. Thus the increased output of ammonia in FIG. 8. Correlation of urinary ammonia and potassium output with sodium reabsorption by renal tubule in normal dogs.

15 ACID AND AMMONIA EXCRETION 157 acidosis is explained by the increased exchange of Na+ with NH4+ which is potentiated by mineralcorticoid in acidosis. Another possible explanation of the enhanced production of ammonia in acidosis is a possibility of increase of glutaminase in the acidotic animal. Though the level of renal glutaminase activity at the end of acid infusion has not been measured, however, the duration of acid infusion seems to be too short to permit the adaptative increase of glutaminase content. Thus the authors decline to neglect the possibility of increase of glutaminase during the period of acid infusion, and the increased ammonia production seems to be solely dependent on the increase of ammonia secretory mechanism. As an adaptive increase of renal glutaminase was demonstrated in the dog kidney on the 3rd day after acid infusion in TABLE 1, this change in enzyme activity should play the important role in the augmentation of ammonia excretion at the latter stage of acidosis. TABLE 1. Enzyme contents in kidney of normal and adrenalectomized dogs As stated previously, RECTOR et al.29) could not find any adaptive increase of glutaminase in daily NH4Cl ingested dog. Therefore, it is suggested that the administration of strong acid like HCl might exert a stimulation for an adaptive increase of renal enzyme. Several years ago, YOSHIMURA and his coworkers27) observed the following facts: (1) there was a stochastically significant inverse correlation between urinary ammonia and urine Na/K ratio, or between urinary Na and NH3, (2) when ammonium chloride was in jected intravenously, by which intracellular NH4+ level would be raised, urinary ammonia was increased, (3) ammonia output in urine was depressed when Diamox (acetazolamide) was applied to dogs, whereby intracellular hydrogen ion concentration and consequently intracellular ammonium ion concentration would be depressed. (4) On the assumption that free ammonia may passively diffuse out of tubular cell of which the intracellu-

16 158 H. YOSHIMURA, M. FUJIMOTO AND J. SUGIMOTO lar ph is approximate 7.0 and keep a diffusion equilibrium with free ammonia molecule in tubular urine, a calculation of intracellular concentration of total ammonia was made with the experimentally estimated urinary ammonia concentration by aid of the Henderson-Hasselbalch equation (pk'=8.90). The calculation showed that the ammonia concentration in cells is more than 10.5mEq/l even when the urinary free ammonia was only 0.5mEq/l, and this value exceeds far beyond the poisoning limit of ammonia for the cell, 3mEq/l28). All these facts strongly support the view of NH4+ exchange with Na+ in the formation of NH4Cl. However, the postulation that ammonia might diffuse out of renal tubular cell more or less to acid urine can not be denied. Therefore until the intracellular ph of renal tubules is determined, the secretion of ammonia by diffusion cannot be denied. In short the mechanism of ammonia secretion by the tubule should be explained predominantly by the ion exchange theory, though the diffusion process may partly play a role. It was confirmed in the present experiments with dogs that adaptive increase of renal glutaminase is responsible for the increased excretion of urinary ammonia in the later stage of acidosis. SUMMARY After isotonic hydrochloric acid was infused intravenously into 30 normal, 10 adrenalectomized and 5 DCA administered adrenalectomized dogs, changes of acid-base balance in the blood and urine were observed and the urinary acidification as well as of ammonia secretion was investigated. The results obtained are summarized as follows: 1. Urinary output of titratable acidity and ammonia increased after the acid. infusion and bore an inverse correlation with the blood ph. Acid urine formation was less remarkable in adrenalectomized dogs, while it was restored after the administration of DCA. 2. A positive correlation was found between plasma potassium concentration and urinary titratable acidity. The amount of titratable acidity is, however, lower in adrenalectomized dogs than normal dogs at the same plasma potassium concentration, while it is higher in DCA administered dogs. 3. It follows that the mineralcorticoid from the adrenal glands can play an importantant role in acid urine formation, probably by promoting an ion exchange mechanism of H+ for Na+ across the cell membrane of the renal tubule in acidosis. The fact that the acid urine can also be produced even in the adrenalectomized dog, though to a lesser extent, indicates that the urinary acidification can be initiated by the other factor than the mineralcorticoid. The direct effect of acidosis was suggested as the other factor, which may influence the H+ ion secretion mechanism of tubular cell in association with either K+ deficiency or ph decrease in the cells.

17 ACID AND AMMONIA EXCRETION Urinary ammonia bears a clear inverse correlation with urinary ph, and an appreciable depression in urinary output of ammonia appeared in adrenalectomized dog, and this fact can be explained as due to the decrease of Na+-NH4+ exchange in adrenalectomized dog. The predominance of the ion exchange theory over the capture-by-diffusion theory in mammals for the urinary ammonia excretion was discussed. 5. About the renal glutaminase and carbonic anhydrase activity, no difference could be found between normal and adrenalectomized dogs, suggesting no difference in the ability of production of acid and ammonia within the renal tubular cell in two kinds of animals. 6. It follows that a disturbance of Na+-H+ and Na+-NH4+ ion exchange mechanism in the renal tubule in adrenalectomized dogs would result in a failure of adaptive increase of urinary output of acid and ammonia in acute acidosis. 7. It is confirmed with dog experiments that the increase of urinary ammonia in prolonged acidosis is due to an adaptive increase of renal glutaminase. The authors wish to express their cordial thanks to Dr. YADA and Dr. KUWADA for their technical assistance of measuring glutaminase in the experiments. REFERENCES 1) YOSHIMURA, H. et al. Jap. J. Physiol. 11: 109, ) PITTS, R. F. AND ALEXANDER, R. S. Am. J. Physiol. 144: 239, ) PITTS, R. F. Am, J. Med. 9: 356, ) ORLOFF, J. AND BERLINER, R. W. J. Clin. Invest. 35: 223, ) RYBERG, C. Acta Physiol. Scand. 14: 114, ) RECTOR, F. C. et al. Am. J. Physiol. 179: 353, ) JIMINEZ-DIAZ, C. Lancet 2: 1135, ) SALTORIUS, O. W. et al. Endocrinol. 52: 256, ) IWANAMI, M. Biochemistry 30: 337, 1958 (Japanese). 10) YOSHIMURA, H. AND FUJIMOTO, M. _Jap. J. Med. Progr. 46: 33, 1959 (Japanese). 11) YOSHIMURA, H. AND FUJIMOTO, M. J. Soc. Inst. Tech. Jap. 9: 138, 1959 (Japanese). 12) SAITO, K. I. Physiol. Soc. Jap. 2: 213, 1937 (Japanese). 13) YOSHIMURA, H. AND INOUE, T. Jap. J. Med. Progr. 46: 1, 1959 (Japanese). 14) ASPER, S. P. et al. J. Biol. Chem. 168: 779, ) CONWAY, E. J. Microdiffusion Analysis and Volumetric Error, 1950 (London). 16) ASADA, T. Jap. J. Med. Progr. 43: 513, 1956 (Japanese). 17) FOLIN, O. AND WU, H. J. Biol. Chem. 38: 81, ) SCHREINER, G. E. Proc. Soc. Exper. Biol. Med. 74: 117, (H. SMITH: Principles of Renal Physiology, p.209, 1956). 19) IWASAKI, H. J. Physiol. Soc. Jap. 19: 143, 1957 (Japanese). 20) SMITH, H. W. Principles of Renal Physiology, 1956, (New York Oxford Univ. Press). 21) BERLINER, R. W. et al. Am. J. Physiol. 162: 348, ) GIEBISCH, G. et al. Am. J. Physiol. 183: 377, ) ROBERTS, K. E. et al. Metabolism 5: 404, ) JACOBS, M. H. Cold Spring Harbor Symposia On Quantitative Biology 8: 30, ) YOSHIMURA, H. et al. Am. J. Physiol. 201: 980, 1961.

18 160 H. YOSHIMURA, M. FUJIMOTO AND J. SUGIMOTO 26) RECTOR, F. C. et al. Am. J. Physiol. 179: 353, ) YOSHIMURA, H. et al. J. Physiol. Soc. Jap. 20: 988, 1958 (Japanese). 28) BALDWIN, E. Dynamic Aspects of Biochemistry, 1949 (Cambridge). 29) RCTOR, F. C., JR. AND ORLOFF J. J. Clin. Invest. 38: 366, ) YOSHIMURA, H. J. Biochem. 23: 335, 1935.