Tissue and Renal Response to Chronic Respiratory Acidosis

TISSUE AND RENAL RESPONSE TO CHRONIC RESPIRATORY ACIDOSIS

Norman W. Carter, … , Donald W. Seldin, H. C. Teng

J Clin Invest. 1959;38(6):949-960. https://doi.org/10.1172/JCI103878.

Research Article

Find the latest version: https://jci.me/103878/pdf TISSUE AND RENAL RESPONSE TO CHRONIC RESPIRATORY ACIDOSIS * By NORMAN W. CARTER,t DONALD W. SELDIN AND H. C. TENG (From the Department of Internal Medicine, The University of Texas Southwestern Medical School, Dallas, Texas) (Submitted for publication October 27, 1958; accepted February 26, 1959) The elevation of pCO2 which occurs during to enter during respiratory acidosis is the red cell respiratory acidosis is accompanied by a rise in (1, 3, 9). The role of renal losses in the produc- the concentration of bicarbonate and a fall in the tion of hypochloremia is less certain. In short concentration of chloride in serum (1). The term human experiments, chloruresis has not increased bicarbonate concentration results from been observed during respiratory acidosis (5, both cellular and renal responses. The cellular 10). However, in the rat, Epstein, Branscome response is characterized by the exchange of ex- and Levitin observed increased chloride excre- tracellular hydrogen ions for intracellular cations, tion during the first 24 hours of an imposed re- thereby contributing to the elevation of the ex- spiratory acidosis (11). tracellular concentration of bicarbonate (2, 3). The present study in the rat was undertaken in The renal factors responsible for the elevated an attempt to delineate the pattern of renal and bicarbonate concentration may be divided into extrarenal responses to respiratory acidosis. The two phases: 1) acid excretion, and 2) bicarbonate excretion of sodium, potassium and chloride was reabsorption. In acute human experiments, aug- studied during the early and late phases. The mented acid excretion, as evidenced by increased contribution of acid loss, as ammonia and titrata- excretion of ammonia and titratable acid, has been ble acid, to the elevation of plasma bicarbonate reported (4). Barker, Singer, Elkinton and concentration was examined. The activities of Clark observed these changes also, but noted that the renal enzymes concerned with acid excretion, if the urine was acid prior to breathing carbon glutaminase and carbonic anhydrase, were deter- dioxide, further increase in acid excretion may not mined in order to ascertain whether adaptation of occur (5). Perhaps this explains, in part at these enzymes occurs as a result of chronic re- least, the failure of Longson and Mills to find in- spiratory acidosis. Finally, possible extrarenal creased acid excretion during short term human contribution to the buffering of carbon dioxide experiments (6). was examined in both muscle and bone. In acute respiratory acidosis tubular reabsorption of bicarbonate is increased (7). Moreover, PROCEDURE in chronic respiratory acidosis in dogs, there is Male Sprague-Dawley rats weighing between 300 and a further augmentation of reabsorption of bicarbo- 400 Gm. were used in all experiments. In early experi- nate that varies directly with the duration of the ments, the rats were tube-fed a liquid diet twice daily imposed acidosis (8). that provided a total of 2.0 Gm. of protein, 1.5 Gm. of fat and 2.0 Gm. of glucose per day together with essential The fall in the concentration of serum chloride minerals and vitamins. Later experiments used the same during respiratory acidosis may also result from diet; however, the diet was offered in a standard drink- both cellular and renal responses. The only cell ing fountain, a measured amount of diet being offered into which chloride has been clearly demonstrated twice daily. Particular care was taken to ascertain that each rat took the entire amount of diet offered. The * This work was supported in part by a research grant intake of equal amounts of diet by both control and ex- from the National Institutes of Health, Public Health perimental animals is essential, for, as has been previ- Service, and in part by a grant from the Dallas Heart ously pointed out, variations in dietary intake result in Association. altered excretion of acid by the kidneys (12). With t This work was done during a Traineeship granted by respect to sodium, potassium and chloride, two different the Institute of Arthritis and Metabolic Diseases, Na- diets were used. The standard diet (Std. Diet) provided tional Institutes of Health, United States Public Health 2.0 mEq. of sodium, 1.0 mEq. of potassium and 3.0 mEq. Service. of chloride per day. The electrolyte deficient diet (E.D. 949 950 NORMAN W. CARTER, DONALD W. SELDIN AND H. C. TENG Diet) contained only negligible amounts of these ions. breathing 10 per cent carbon dioxide. Groups 7 and 8 All rats were given distilled water ad libitum. were a control group and a group subjected to 10 per A large box was constructed for use as a metabolic cent carbon dioxide, respectively. In an attempt to mag- cage for those animals subjected to respiratory acidosis. nify changes in plasma and muscle electrolytes, as well The design was such that daily urines could be collected as in urinary excretion of electrolytes, these two groups from individual rats. The proper content of carbon received the E.D. Diet. Urines were collected and dioxide within the box was maintained by automatic elec- analyzed as in Groups 4 and 5. trical controls which intermittently released carbon dioxide into the box. By this method, the carbon dioxide METHODS content of the box could consistently be maintained within ± 1.0 per cent of the desired value. Oxygen content of Rats subjected to carbon dioxide were killed while the atmosphere in the box was frequently checked with breathing an atmosphere having the same carbon diox an Orsat gas analysis apparatus, and was found to ide content as was used for the experiment. This was range from 19 to 21 per cent. It was not necessary to accomplished by supplying an appropriate oxygen-carbon remove rats from the box during an experiment; feed- dioxide mixture to a small box into which the anesthe- ing and cage cleaning were carried out through air- tized rat's head was placed by way of a snugly fitting tight arm ports. By means of air-conditioning, both con- neck port. Both control and experimental animals were trol and experimental animals were kept in an atmosphere anesthetized by an intraperitoneal injection of pentobarbi- maintained nearly constant at 730 F. tal. Blood was received anaerobically into a previously aorta. were collected from individual heparinized syringe from the abdominal Muscle Twenty-four hour urines removed from the hind legs. In some ani- rats under oil and preserved with phenylmercuric nitrate samples were this mals, the bones of the hind legs were taken for electrolyte and toluene. Approximately midway through study, analysis. The kidneys were perfused with chilled iso- changes in the urine collection system were made that for tonic saline in order to wash out all red blood cells, then resulted in different values urinary constituents. frozen for enzyme assays. of a silicone to removed and immediately The change was the application coating Methods for the determination of plasma electrolytes, the surface of all glassware used in the collection system. from rat urine ammonia, pH and titratable acidity, muscle elec- This gave rise to an immediate flow of urine the trolytes and kidney glutaminase activity were those into the collection vessels and made it unnecessary to previously described from this laboratory (12, 13). rinse the collection system at the end of each collection of urine Urine sodium and potassium concentrations were deter- period, since there was no significant evaporation mined by flame photometry (14). Urine chloride con- the new urine collec- on the siliconized surfaces. Using centration was determined by a modification of the Vol- tion system, it was found that apparent ammonia excre- method (15). Whole blood pH was anaero- times than that found hard-Harvey tion was approximately 1.66 greater determined at 37° C. by means of a Cambridge An con- bically using the old urine collection system. additional water-jacketed glass electrode and a Cambridge pH meter. trol study, using the new urine collection system, was Plasma carbon dioxide tension was estimated by means made in order to facilitate comparison with experimental of the Henderson-Hasselbalch equation using the blood groups. The study included eight groups of rats. Groups pH, plasma carbon dioxide content and a pK of 6.11. 1 through 3 were those in which the original urine col- Buffer anion was estimated by means of a Singer- lection method was used (Table II). The new urine col- Hastings nomogram assuming a red cell hematocrit of lection method was used in the remaining five groups 50 per cent in each case (16). (Table II). Electrolyte composition of marrow-free bone was de- All groups were observed for a period of 11 days. termined in Groups 4 and 5. The method of analysis Group 1 served as a control for Groups 2 and 3. In these was a modification of the ion-exchange column method three groups, all rats received the Std. Diet, and daily of Forbes and D'Ambruso (17). urines were collected for determination of pH, ammonia Renal carbonic anhydrase activity was determined in and titratable acidity. Group 2 was subjected to an at- water homogenates of kidney tissue. The method de- mosphere of 10 per cent carbon dioxide. Group 3 was scribed by Ashby and Chan (18) was modified in the fol- subjected to 15 per cent carbon dioxide. lowing way. A veronal-sodium veronal buffer (0.03 M) Group 4 served as a control for Groups 5 and 6. These as suggested by Roughton and Booth (19) was substituted three groups received the Std. Diet, and as in previous for the carbonate-bicarbonate buffer of the original groups daily urines were collected for the determination method because the former provides a better pH range of pH and acid excretion. In addition, sodium, potassium for the enzymatic reaction and does not adversely affect and chloride were determined in the urines from Groups the enzyme. Since the reaction was carried out from 4 and 5. Group 5 was subjected to 10 per cent carbon pH 7.9 to pH 6.3, water soluble brom-thymol-blue was used dioxide. In an attempt to produce a marked additive as the indicator in a final concentration of 1.57 mg. per effect on acid excretion, Group 6 was subjected to 10 100 ml. This concentration was below that demonstrated per cent carbon dioxide for two days, then 15 per cent for by Wilbur and Anderson to cause inhibition of the en two days, these cyclic changes being maintained through- zyme (20). The reaction was carried out at 30 C. out the experiment. These rats were sacrificed while (+ 0.20 C.). The exact time for the reaction CO2 + H2O ELECTROLYTE EXCHANGES IN RESPIRATORY ACIDOSIS 95-1

TABLE I Plasma electrolyte concentrations

C02 Whole Buffer Group Procedure Na K C1 contents blood pHI pC02I anion mEq./L. mEq./L. mEq./L. mEq.1L. mm. Hg mEq./L. 1 and 4* Std. Diet t144.0 (10)t 5.0 (10) 108.8 (10) 24.7 (10) 7.43 (5) 35.4 (5) 48 (5) only 42.2 ±.44 4:2.8 :1 1.8 41.02 4:2.6 -41.4 2 and 5* Std. Diet 151.4 (10) 4.73 (10) 91.8 (10) 45.2 (10) 7.28 (9) 102.3 (9) 58 (9) + 10% CO2 13.9 :+:.46 ±3.0 i2.3 4±.04 :+:6.6 :+:4.0 3 Std. Diet 149.3 (4) 4.31 (5) 85.3 (4) 52.4 (4) 7.15 (5) 147.7 (5) 60 (5) + 15% CO2 13.1 :1:.25 =14.5 ±3.2 ±.07 :1:29.3 i1.3 6 Std. Diet 150.9 (4) 3.60 (4) 93.5 (4) 42.3 (4) 7.19 (4) 108.3 (4) 55 (4) + 10 and ±4.0 4.34 -3.9 ±2.9 ±.01 ±9.2 ±2.2 15% C02 7 E.D. Diet 145.6 (5) 4.81 (5) 104.6 (5) 22.2 (5) 7.39 (5) 36.7 (5) 46 (5) only ±1.8 ±.42 +1.4 ±1.9 ±.01 ±1.8 ±1.4 8 E.D. Diet 156.4 (9) 2.85 (9) 89.1 (9) 46.2 (9) 7.24 (9) 102.5 (9) 58 (9) + 10% CO2 ±3.1 ±.47 ±2.4 ±1.0 ±.04 ±2.7 ±2.4 * Groups 1 and 4 both received the same Std. Diet. Methods for urine collection differed in the two groups. The electrolyte composition in blood was identical in both groups, and these data were pooled. Since the same circum- stances existed for Groups 2 and 5, their data were also pooled. t Mean and standard deviations. t Number in parentheses, number of animals used. § The mean and standard deviation for pCO2 was not estimated from the mean values for CO2 content and pH. Instead, the pCO2 was estimated for each rat from the CO2 content and pH; then the mean and standard deviations were determined. As a consequence, there is an apparent disparity between the mean values for CO2 content and pH on the one hand and pCO2 on the other as these values appear in this table. = HCOS to proceed sufficiently far to the right to lower dosis as evidenced by increased plasma CO2 con- the pH of the buffer to 6.3 without added enzyme was tent, decreased blood pH and increased plasma the control time. The amount of enzyme (kidney ho- mogenate) necessary exactly to halve this time was de- pCO2 (Table I). Fifteen per cent carbon dioxide fined as a unit of enzyme activity. gave rise to a more profound acidosis than did Throughout, results are reported as mean values for 10 per cent carbon dioxide in that the mean blood respective groups together with the standard deviation of pH for Group 3 was significantly lower (p < the mean. P values were obtained from appropriate 0.01) than those values found in Groups 2 and 5. tables after the t value was calculated. The somewhat higher CO2 content found for Group 3 was, however, of only doubtful signifi- RESULTS cance (p > 0.02) when compared with the CO2 Rats used in these experiments were able to content of Groups 2 and 5. Although the mean withstand an atmosphere of 10 per cent carbon blood pH of Group 6 (10 and 15 per cent carbon dioxide for 11 days without obvious untoward dioxide) was significantly lower (p < 0.001) than effect. Experiments lasting 14 days gave rise to that value for Groups 2 and 5, the CO2 content a mortality of approximately 5 per cent. An at- and pCO2 were not significantly different. These mosphere of 15 per cent carbon dioxide resulted data suggest that in both Group 3 and Group 6 in a mortality of approximately 40 per cent within the acidosis was more severe and the compensa- 11 days. A mortality of 100 per cent occurred tion less complete than in Groups 2 and 5. within 30 hours when rats were subjected to 20 There was no significant difference found in the per cent carbon dioxide. degree of acidosis produced with 10 per cent carbon dioxide between groups that received the Degree of acidosis Std. Diet (Groups 2 and 5) and the E.D. Diet Rats subjected to 10 or 15 per cent carbon diox- (Group 8) (Table I). ide atmosphere developed severe respiratory aci- As was expected, respiratory acidosis produced 952 NORMAN W. CARTER, DONALD W. SELDIN AND H. C. TENG an elevation of whole blood buffer anion (16). groups with respiratory acidosis, significantly Since the value found for 10 per cent carbon lower than control values (Table I). The only dioxide was not significantly different from that abnormally low potassium concentrations occurred found for 15 per cent carbon dioxide (Table I), in those rats receiving the potassium deficient it would appear that under these experimental con- E.D. Diet (Group 8). There was no definite re- ditions, buffer anion concentration displayed a lationship between plasma pCO2 and potassium maximal concentration not entirely proportionately concentration (Table I). Potassium concentration related to the degree of hypercapnea. and blood pH likewise had no definite relationship (Table I). Plasma sodium, potassium and chloride In all our experimental groups, the expected In all groups subjected to respiratory acidosis, hypochloremia of respiratory acidosis was ob- the plasma sodium concentration was significantly served (Table I). The decrement in chloride increased above control values (Table I). Al- concentration was, for the most part, only slightly though acute respiratory acidosis has been demon- less than the increment in carbon dioxide content strated to accelerate the excretion of water (10), of the plasma. no increased urine volumes were observed in rats during chronic respiratory acidosis. The hyper- Acid excretion natremia may have been the consequence of the Table II summarizes the cumulative acid excre- inability of rats who are breathing rapidly to in- tion for experimental and control groups during crease the intake of water sufficiently to replenish the 11 days of observation. The actual differences deficits resulting from enhanced respiratory water between rats subjected to respiratory acidosis and loss. control animals were small, although significant Plasma potassium concentrations were, in all for the most part. Ammonia excretion was sig-

TABLE II Cumulative acid excretion during respiratory acidosis for 11 days

Group (No. of rats) Procedure NH,* TA* NHs + TA* mEq. mEq. mEq. Group It Std. Diet 14.0 =1 0.8 3.87 0.52 17.71 :at 0.9 (5) only Group 2t Std. Diet 18.0 4t 2.4 4.05 :1: 0.38 22. 1 2.9 (10) +10%C02 p = <0.001 p = >0.5 p = 0.01 Group 3t Std. Diet 20.4 i 2.2 4.09 :1: 0.69 24.1 4 2.8 (5) +15%C02 p = <0.001 p = >0.5 p = <0.01 Group 4t Std. Diet 24.6 i: 0.9 3.26 41 1.09 27.7 41 1.6 (5) only Group 5t Std. Diet 26.2 :1: 0.5 5.98 4 1.04 31.5 4 0.8 (4) + 10%C02 p = <0.02 p = 0.01 p = <0.01 > 0.01 Group 6$ Std. Diet 28.9 :1: 3.4 5.77 d: 0.84 32.9 4: 6.8 (5) + l0and p = <0.05 p = <0.01 p = <0.20 15% CO2 > 0.02 > 0.10 Group 7$ E.D. Diet 23.5 :1: 1.0 4.59 :i= 0.83 28.1 ± 1.7 (6) only Group 8t E.D. Diet 25.6 2.5 4.40 ± 0.84 29.7 :1: 3.0 (10) +10%CO2 p= <0.001 p= <0.20

* Cumulative excretion is expressed as mean and sstandard deviations. Statistical expressions compare experimental groups with their respective controls. t Groups 1, 2 and 3, original urine collection method. $ Groups 4 through 8, later urine collection method. ELECTROLYTE EXCHANGES IN RESPIRATORY ACIDOSIS 953 nificantly increased in most of the groups subjected to either 10 or 15 per cent CO2 (Groups 2, 3, 5 and 8), although in one instance the significance of the increase was borderline (Group 6). The cumulative excretion of titratable acid (TA) was increased in two groups (Groups 5 and 6). In all experimental groups excepting Group 8, the i | | z SJTD. Diet conl Irv a-- cumulative excretion of acid (NH3 plus TA) rose significantly. The daily excretion of total acid 2.10--

0.0-

t5.565 75_

6D t _- 5-

I z 3 4 5 6 7 8 9 to it LD A Y .5 FIG. 2. MEAN DAILY URINARY Acm EXCRETION DURING RESPIRATORY ACIDOSIS

ammonia and titratable acid excretion. Ammonia excretion tended to remain slightly above control values throughout the study. Early in respiratory acidosis, urine pH tended to be lower than in the control groups but rose to control values or above, more or less paralleling the fall in ammonia excretion, as the study was continued.

D A Y .S FIG. 1. DAILY TOTAL ACID EXCRETION (NH3 PLUS TA) Diet - E 1(STD. Only Gx'oup DURING RESPIRATORY ACIDOSIS (fMeo.r.L4 S.D.j Ffl2l STD. Diet with 10% CO., rhsoup 5 (Me i-. S.-D.-) for three representative groups (2, 3, 8) of rats subjected to respiratory acidosis is plotted in Fig- ure 1. There was a tendency for the highest acid excretion to occur during the first two days of respiratory acidosis, after which acid excretion tended to fall to a level only slightly greater than that observed in control animals. To examine the daily pattern of urine pH, NH3 and titratable acid during respiratory acidosis, data from rats subjected to 10 per cent CO2 are com- 4 6 pared with their controls in Figure 2. All other D CLy5 groups behaved in similar fashion. The early rise FIG. 3. MEAN DAILY URINARY EXCRETION OF NA, K in total acid excretion was the result of a rise in AND CL DURING RESPIRATORY ACIDosIs 954 NORMAN W. CARTER, DONALD W. SELDIN AND H. C. TENG

during the first day. Thereafter, chloride excre-

.25'- E.0. Diat 0nly G zou-p tion fell to control values while potassium excre- 4 M0a( rL S.D.) 0 tion remained slightly elevated throughout the 7 A %ED. Diat with 10%/ CO2 ~~~~Gxro"p 8 1.6 jM aQ S.D.) study. Similar findings have been obtained by

10 others (11). In Table III, the total cumulative 1.0 _ mean excretions of sodium, potassium and chloride 5 ad are given for the rats during respiratory acidosis on Std. and E.D. Diet. The excess potassium excreted in the groups with respiratory acidosis was strikingly similar for both dietary regimens (AK Std. Diet = 3.76 mEq. and AK E.D. Diet = 3.50 mEq.). To a lesser extent, this was true for chloride excretion (ACl Std. Diet = 2.65 mEq. and ACl E.D. Diet = 2.18 mEq.). This suggests that urinary potassium and chloride loss incident to a given degree of respiratory acidosis is essen- tially independent of the availability of these ions in the diet.

Da~ys Muscle and bone electrolytes FIG. 4. MEAN DAILY URINARY EXCRETION OF NA, K AND CL DURING RESPIRATORY ACIDOSIS Intracellular sodium of muscle was significantly decreased in all groups with respiratory acidosis Sodium, potassium and chloride excretion as compared with control values (Table IV). The mean daily urinary excretion of sodium, This is in accord with previous findings in rats acidosis potassium and chloride was compared in rats sub- with chronic respiratory (23). was a of jected to 10 per cent carbon dioxide while on There slight lowering muscle potassium in all groups to aci- Std. Diet (Figure 3) and while on E.D. Diet subjected respiratory (Figure 4) with their respective controls. So- dosis, and this was significant in those groups dium excretion was not significantly changed given the Std. Diet and 10 or 15 per cent carbon by respiratory acidosis. Both potassium and dioxide (Table IV, Groups 2 and 5, and Group chloride excretion were significantly increased 3). Previously, high normal muscle potassium

TABLE III Total cumulative mean excretion of Na, K and Cl

Procedure Sodium Potassium Chloride mEq. mEq. mEq. Group 4 Std. Diet 21.61t 10.63 34.28 only (5)* :1:1.21 4-0.92 :1:1.59 Group 5 Std. Diet + 21.73 14.39 36.93 10% CO2 (4) -10.92 :1:0.92 4:0.22 p= >0.01 p= >0.01 <0.001 <0.001 Group 7 E.D. Diet 2.35 3.27 2.28 only (6) 410.51 :1.38 4-0.27 Group 8 E.D. Diet + 2.07 6.77 4.46 10% CO2 (5) 4:0.42 411.30 :1:0.60 P= >0.50 p= >0.01 p= >0.001 <0.30 <0.001 * Number in parentheses, number of animals used. t Mean and standard deviation. ELECTROLYTE EXCHANGES IN RESPIRATORY ACIDOSIS 955

TABLE IV Muscle and bone electrolytes

Group...... 1 and 4 (5)* 2 and 5 (5) 3 (5) 6 (5) 7 (5) 8 (5)

Procedure ...... Std. Diet Std. Diet Std. Diet Std. Diet E.D. Diet E.D. Diet only + 10% C02 + 15% C02 + 1O and only + 10% C02 15% C02 Musclet Nait 4.45§ 3.31 3.16 2.82 4.26 2.86 =0.43 ±0.53 40.78 ±0.45 -0.42 40.54 K 45.1 42.0 40.3 43.8 46.8 43.2 ±i1.3 41.6 ±1.7 ±2.3 ±1.2 ±-2.9 Bonell Nai¶ 254 258 ±13 ±8 K 16.6 20.0 ±1.6 ±2.1 Mg 578 480 ±13 ±21 Ca 13,070 13,320 ±100 ±100 * Number in parentheses, number of animals used. t mEq. per 100 Gm. fat-free dry muscle. $ Nai in muscle was calculated from the total muscle sodium by correcting for the sodium in the extracellular fluid of the muscle sample after the method of Yannet and Darrow (21). § Group mean value and standard deviation. mEq. per Kg. fat-free dry bone. ¶ Nai in bone was calculated from the total bone sodium by correcting for the sodium in the extracellular fluid of the bone sample. Extracellular fluid volume was derived by assuming all bone chloride to be extracellular (22). values have been reported for rats with chronic + 10.2 mm. Hg; plasma chloride, 93.6 ± 0.8 respiratory acidosis (23). mEq. per L.; blood pH, 7.25 + 0.08. Ammonia In Table IV, electrolyte composition of bone excretion was elevated by about 3 mEq. per day during respiratory acidosis is compared with con- above those levels found in animals given 10 per trols. The most striking finding was a marked fall in bone magnesium. TABLE V Renal glutaminase and carbonic anhydrase activities Renal glutaminase and carbonic anhydrase Renal Renal carbonic Renal carbonic anhydrase activity failed to show Group Procedure glutaminase* anhydraset any significant change after 11 days of respiratory 1 and 4 Std. Diet t608 (10) § 370 (5) acidosis (Table V). The increased bicarbonate only ±60 ±6 reabsorption which occurred in the animals sub- 2 and 5 Std. Diet 657 (13) 365 (5) jected to respiratory acidosis was not, therefore, + 10% C02 ±i119 ±40 mediated by adaptive increase in renal carbonic 3 Std. Diet 535 (5) 378 (4) anhydrase activity as measured by the in vitro + 15% C02 ±79 ±4 method employed. 6 Std. Diet 781 (4) Despite prolonged and persistent respiratory +l0and ±113 acidosis, the activity of renal glutaminase did 15% C02 not rise (Table V). 7 E.D. Diet 537 (4) 313 (5) To explore the significance of chloride loads only ±t77 44 during respiratory acidosis, rats subjected to 10 8 E.D. Diet 811 (9) 314 (5) + 10%C02 ±176 ±3 per cent CO2 were given in addition 3 mMole NH4C1 daily and were killed after 11 days. The *,uMole NH3 per 100 mg. dry kidney tissue per hour. following results were obtained: plasma CO2 con- t Units per Gm. wet kidney tissue. I Mean and standard deviation. tent, 39.2 + 2.4 mEq. per L.; plasma pCO2, 90.9 § Number in parentheses, number of animals used. 956 NORMAN W. CARTER, DONALD W. SELDIN AND H. C. TENG cent CO2 alone. Urine pH was lower (mean, pCO2 increases, carbonic acid can be excreted via 5.8) than that found in respiratory acidosis alone the lungs and therefore would not require renal (mean, 6.2 to 6.8). Renal glutaminase activity compensation beyond the initial period, since the was 2,228 + 371 mMole per 100 mg. dry tissue blood pH was markedly acid throughout the pe- per hour as contrasted to a mean value of 657 for riod of study. respiratory acidosis alone. Unlike metabolic acidosis, the serum bicarbonate rises during CO2-rebreathing. This rise begins DISCUSSION within the first hour and reaches a maximum The pattern of urinary acid excretion during value in about one day, after which the elevated respiratory acidosis differs markedly from that serum concentration is maintained throughout the resulting from other forms of acid loading. In duration of exposure to CO2 (25). It is probable the present study, there was initially a small but that the absence of greater augmentation of acid significant rise in the excretion of acid (both as excretion is linked with this elevation of serum ammonia and titratable acid) during the first day bicarbonate. During the early period, before the or two of acidosis; thereafter, despite continued, concentration of serum bicarbonate has reached severe, systemic acidosis, urinary acid excretion maximum values, mean urine pH falls below con- returned virtually to control values. By control levels; the excretion of ammonia and titratable trast, the administration of an acid buffer, such as acid is therefore accelerated. After the first day, sodium acid phosphate, promptly increases the ex- the urine becomes more alkaline. Although the cretion of titratable acid as long as the acid load elevated pCO2 of blood accelerates bicarbonate re- is given, even though the amounts of acid cause absorption, it is probable that a portion of the only a slight lowering of the concentration of se- greatly increased load of filtered bicarbonate es- rum bicarbonate (24). capes into the urine, especially when the serum bi- The character of acid excretion during am- carbonate concentration is maximal, rendering it monium chloride acidosis differs even more strik- more alkaline. In turn, the more alkaline urine ingly from that encountered during respiratory may serve to suppress the excretion of ammonia acidosis. The administration to rats of small and titratable acid. amounts of a strong acid, e.g., 2 mMole NH4Cl The early rise in the concentration of serum daily, results in the following response (13): 1) bicarbonate is roughly paralleled by a commensu- Acid excretion (almost entirely as ammonia) rate fall in serum chloride. This fall in filtered rises over a five day period to a maximum level. chloride and rise in filtered bicarbonate could re- 2) This level is maintained throughout the dura- sult in a sharp reduction in the proportion of tion of the acid loading. 3) Despite the absence of chloride as compared with bicarbonate reaching changes in the pH or concentrations of bicarbonate the exchange site in the distal tubule. It is es- in blood (or at most, minor depressions), far more pecially noteworthy, in this connection, that the acid is excreted than in control animals. In- administration of 3 mMole NH4C1 to rats during 4) respiratory deed, acid excretion is directly proportional to the acidosis markedly accelerated NH3 magnitude of the strong acid load (whereas in excretion and lowered urine pH, at the same time respiratory acidosis, 10 and 15 per cent CO2 elicit that renal glutaminase was activated to levels the same minor rise in urinary acid). The sharp (2,228 + 371 mMole NH3 per 100 mg. dry tis- dichotomy between the profound degree of sys- sue per hour) not significantly different from that temic acidosis (blood pH 7.15 + 0.07 in 15 per found in rats subjected to 3 mMole NH4C1 daily cent CO2 group) on the one hand and the com- alone (Figure 5). It is likely that the NH4C1 paratively minor rise in acid excretion on the other load, by reducing the concentration of bicarbonate constitutes one of the most striking features of the and raising the concentration of chloride in se- pattern of acid excretion during respiratory aci- rum, diminished the amount of bicarbonate reach- dosis. The failure of respiratory acidosis to pro- ing the distal tubule, at the same time that the de- duce a sustained increase in renal acid excretion livery of NaCl to the exchange site was increased. cannot be attributed to the fact that as plasma As a result, ammonia excretion was accelerated ELECTROLYTE EXCHANGES IN RESPIRATORY ACIDOSIS 957

win pH ..4377.27 l7.28 7. 15 ±.02 ±-.04 ±.04 ±.07 CO2 (Conten) 24.7 24.0 19.6 18.0 17.8 20.5 25.2 45.2 52.4 < mEq ±1.8 ±1.2 ±1.4 ±1.6 ±3.3 ±1 .6 ± 1.1 ±2.3 ±3.2

0e i2PCO2 35.4 40.0 102.3 147.7 mmHg ±2.6 ±2.3 6.6 ±29.3

L 3500- ;N. 1,,3000.

<-2500- L&JL 2000.

0o 1500-

zzx 21Q000 500F::

STD. 2mM 3mM 5mM 6mM 3.75mM 6mM I 0% CO2 15% CO2 TREATMENT DIET NaH2PO4 NaHCO3 RESPIRATORY ONLY NH4CI LOADS ACIDOSIS

FIG. 5. RESPONSE OF RAT RENAL GLUTAMINASE ACTIVITY TO VARIOUS EXPERIMENTAL CONDITIONS and this, in turn, could have activated renal glu- then result in an activation of the ammonia-pro- taminase. ducing enzyme systems of the rat. In keeping with the minor and transient rise Although acid excretion is elevated to only a in ammonia excretion during respiratory acidosis minor extent in respiratory acidosis, the reab- is the finding that renal glutaminase is not acti- sorption of large amounts of filtered bicarbonate vated. Figure 5 summarizes the response of this indicates that hydrogen ion secretion is increased enzyme system to various acid-base loads, as (26, 27). This augmentation in bicarbonate reab- found in our laboratory. Glutaminase adapta- sorption and hydrogen ion secretion is best cor- tion is observed only when ammonia excretion is related with the rise in tension of CO2 in plasma persistently increased, a circumstance that prevails (5, 7, 28, 29). In chronic respiratory acidosis in during chronic administration of a strong acid the dog, there is a further increase in bicarbonate (NH4CI) load. Since CO2-rebreathing results in reabsorption above that which might be predicted a far more severe systemic acidosis (and intra- for any particular plasma pCO2 value (8). That cellular acidosis as well), it seems likely that acti- this apparent adaptation to prolonged respiratory vation of the glutaminase enzyme system cannot acidosis might be mediated by means of the specific be ascribed either to extracellular or intracellular adaptation of renal carbonic anhydrase would seem acidosis alone. It is possible that the activation a distinct possibility. Killion and Schaefer have of glutaminase is attributable to product removal reported increased renal carbonic anhydrase ac- (i.e., ammonia excretion); any circumstance tend- tivity occurring within one hour in the kidneys of ing to accelerate the excretion of ammonia should rats and guinea pigs exposed to an atmosphere 958 NORMAN W. CARTER, DONALD W. SELDIN AND H. C. TENG containing 30 per cent carbon dioxide (30). How- tion to blood pH observed in human subjects with ever, in our more chronic experiments, in at- disturbances in acid-base balance (34) was not mospheres with less carbon dioxide content, we apparent in this study. It seems likely that re- were unable to show increased activity of this en- spiratory acidosis does in fact cause a transfer zyme. The explanation of the progressively more of potassium into the extracellular space, but in efficient bicarbonate reabsorption at any given our chronic studies renal losses probably prevent pCO2 during chronic respiratory acidosis is there- the usually observed hyperkalemia. fore not forthcoming from these studies. Our finding of a slightly decreased muscle po- In vitro studies with whole blood have shown tassium is in agreement with the observation of that when exposed to carbon dioxide, chloride in Levitin, Jockers and Epstein (35). The previ- the plasma enters the erythrocyte (31, 32). In ously noted potassium shift into the extracelluar acute respiratory acidosis in man (2, 3) and the space, together with the observed loss of potas- nephrectomized dog (3), this mechanism has been sium in the urine, is in keeping with the finding shown to be important in producing hypochloremia. of lowered muscle potassium. The lowering of However, that an added mechanism for hypo- both muscle potassium and sodium in respiratory chloremia is the urinary loss of chloride had not acidosis could be a consequence of exchange of been clearly demonstrated until the studies of extracellular hydrogen ion for intracellular anions. Epstein, Branscome and Levitin (11). In our Decreased muscle sodium in metabolic acidosis experiments, an early, marked, negative balance has been previously reported, whereas with an of chloride was likewise observed. Although the adequate intake of potassium, muscle potassium is shift of chloride from the extracellular space into usually not lowered by metabolic acidosis (13, the erythrocyte does promote hypochloremia, it is 36, 37). Thus, in both metabolic and respiratory apparent that the early loss of chloride in urine is acidoses, the sodium of muscle is decreased, also an important factor. whereas only respiratory acidosis significantly The excretion of potassium is slightly increased lowers muscle potassium. The apparent restric- throughout the period of study. In acute respira- tion of pH change principally to the extracellular tory acidosis in man of one hour duration, potas- fluid in metabolic acidosis (38, 39) may in some sium excretion is depressed (5), presumably be- way explain the difference in response of muscle cause increased H+, generated from CO2 in the potassium in metabolic and respiratory acidoses. tubular cell, rather than K+, exchanges for reab- As suggested by Fenn, Rogers and Ohr, the lower- sorbed Na+. The persistently elevated potas- ing of muscle sodium in both metabolic and re- sium excretion, despite lowered serum concentra- spiratory acidoses may not represent a simple tions, may be the consequence of accelerated de- exchange of intracellular sodium for extracellular livery of NaHCO3 to the distal tubule as serum hydrogen ion, but rather an increased ability of bicarbonate rises, a circumstance which could in- the sodium pump to deliver sodium into a more crease the Nat-K+ exchange and thus account acid medium (40). for the potassium loss. If increased amounts of Bone, as an extracellular repository for sodium NaHCO3 are delivered to the distal tubule, the and potassium, has been recognized as a potential exchange of Na+ for H+, as well as for K+, is in all source from which the urinary excretions of these likelihood augmented, but the enhanced secretion anions during acidosis can be derived (22). That of He into bicarbonate-containing tubular fluid in metabolic acidosis bone sodium is released, ap- would not augment urinary acid (NH3 plus ti- parently in exchange for hydrogen ion, has been tratable acid) excretion. demonstrated (22, 37, 41). Bergstrom and Wal- The finding of a lowered plasma concentration lace (22) observed a similar role for bone potas- of potassium in the experimental groups of this sium, but Levitt, Turner, Sweet and Pandiri (37) study contrasts with the elevated concentra- failed to find changes in bone potassium in rats tions found in acute respiratory acidosis (2, 3, with metabolic acidosis. In respiratory acidosis, 33), where potassium has been shown to enter no change was found by us in bone sodium, con- the extracellular space, presumably from cells. firming a previous observation (35). It is pos- Similarly, the relationship of potassium concentra- sible that the chronicity of our experiments, as ELECTROLYTE EXCHANGES IN RESPIRATORY ACIDOSIS 959

compared with the more acute studies of metabolic 3. Giebisch, G., Berger, L., and Pitts, R. F. The extra- acidosis, would explain the differences observed renal response to acute acid-base disturbances of in bone sodium. Bone potassium was slightly respiratory origin. J. clin. Invest. 1955, 34, 231. 4. Davies, H. W., Haldane, J. B. S., and Kennaway, elevated in respiratory acidosis. Thus, in chronic E. L. Experiments on the regulation of the blood's respiratory acidosis, participation of bone sodium alkalinity. J. Physiol. (Lond.) 1920, 54, 32. and potassium in the buffering of carbonic acid is 5. Barker, E. S., Singer, R. B., Elkinton, J. R., and not apparent. Clark, J. K. The renal response in man to acute experimental respiratory alkalosis and acidosis. SUM MARY J. clin. Invest. 1957, 36, 515. 6. Longson, D., and Mills, J. N. The failure of the kid- The pattern of renal and cellular responses of ney to respond to respiratory acidosis. J. Physiol. electrolytes to chronic respiratory acidosis was (Lond.) 1953, 122, 81. 7. Dorman, P. J., Sullivan, W. J., and Pitts, R. F. studied in rats. The renal response to acute respiratory acidosis. Urinary acid excretion (NH. plus titratable J. clin. Invest. 1954, 33, 82. acid) increased transiently for the first day or 8. Sullivan, W. J., and Dorman, P. J. The renal re- two and then returned to control values despite sponse to chronic respiratory acidosis. J. clin. continued severe respiratory acidosis. A slight rise Invest. 1955, 34, 268. in urine pH, presumably due to increased bicar- 9. Platts, M. M., and Greaves, M. S. The composition of the blood in respiratory acidosis. Clin. Sci. 1957, bonate excretion- as serum bicarbonate reached 16, 695. maximal values, appeared to be chiefly responsible 10. Barbour, A., Bull, G. M., Evans, B. M., Jones, N. for the rapid return of urinary ammonia to control C. H., and Logothetopoulos, J. The effect of values. Renal glutaminase was not activated dur- breathing 5 to 7% carbon dioxide on urine flow and ing respiratory acidosis; this is strong evidence mineral excretion. Clin. Sci. 1953, 12, 1. against the role of intracellular or extracellular 11. Epstein, F. H., Branscome, W., and Levitin, H. The mechanism of hypochloremia in chronic respiratory pH as regulatory factors in the adaptation of renal acidosis. Clin. Res. Proc. 1957, 5, 17. ammonia-producing enzymes. In spite of in- 12. Rector, F. C., Jr., Seldin, D. W., Roberts, A. D., Jr., creased tubular bicarbonate reabsorption, as evi- and Copenhaver, J. H. Relation of ammonia ex- denced by the stabilization of serum bicarbonate cretion to urine pH. Amer. J. Physiol. 1954, 179, at high levels, adaptation of renal carbonic anhy- 353. drase did not occur. 13. Rector, F. C., Jr., Seldin, D. W., and Copenhaver, J. H. The mechanism of ammonia excretion dur- Chloride excretion was greatly elevated the ing ammonium chloride acidosis. J. clin. 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