Relationship of Urinary and Blood Carbon Dioxide Tensionduring Hypercapnia in the RatIts Significance in the Evaluation of Collecting Duct Hydrogen Ion Secretion
Daniel C. Batile, Mary Downer, Cory Gutterman, and Neil A. KurtzmanWest Side Veterans Administration Hospital and the University of Illinois College of Medicine,Department of Medicine, Section of Nephrology, Chicago, Illinois 60612
Abstract
This study was designed to establish the relationship betweenurinary pCO2and systemic blood pCO2 during acute hypercap-nia and to investigate the significance of this relationship tocollecting duct hydrogen ion (He) secretion when the urine isacid and when it is highly alkaline. In rats excreting a highlyalkaline urine, an acute increase in blood pCO2 (from 42±0.8to 87±0.8 mmHg) resulted in a significant fall in urine minusblood (U-B) pCO2 (from 31±2.0 to 16±4.2 mmHg, P< 0.005), a finding which could be interpreted to indicateinhibition of collecting duct H' secretion by hypercapnia. Theurinary pCO2 of rats with hypercapnia, unlike that of normo-capnic controls, was significantly lower than that of blood whenthe urine was acid (58±6.3 and 86±1.7 mmHg, P < 0.001)and when it was alkalinized in the face of accelerated carbonicacid dehydration by infusion of carbonic anhydrase (78±2.7and 87±1.8 mmHg, P < 0.02). The finding of a urinary pCO2lower than systemic blood pCO2 during hypercapnia suggestedthat the urine pCO2 prevailing before bicarbonate loadingshould be known and the blood pCO2 kept constant to evaluatecollecting duct H' secretion using the urinary pCO2 technique.
In experiments performed under these conditions, sodiumbicarbonate infusion resulted in an increment in urinary pCO2(i.e., a ApCO2) which was comparable in hypercapnic andnormocapnic rats (40±7.2 and 42±4.6 mmHg, respectively)that were alkalemic (blood pH 7.53±0.02 and 7.69±0.01,respectively). The U-B pCO2, however, was again lower inhypercapnic than in normocapnic rats (15±4.0 and 39±2.5mmHg, respectively, P < 0.001). In hypercapnic rats in whichblood pH during bicarbonate infusion was not allowed tobecome alkalemic (738±0.01), the ApCO2 was higher thanthat of normocapnic rats which were alkalemic (70±5.6 and42±4.6 mmHg, respectively, P < 0.005) while the U-B pCO2was about the same (39±3.7 and 39±2.5 mmHg). Wefurtherexamined urine pCO2 generation by measuring the differencebetween the urine pCO2of a highly alkaline urine not containingcarbonic anhydrase and that of an equally alkaline urinecontaining this enzyme. Carbonic anhydrase infusion to hyper-capnic rats that were not alkalemic resulted in a fall in urine
Portions of this work were presented at the National Meeting of theAmerican Federation of Clinical Research (Washington, DC, 1982).
Address correspondence to Dr. BatIle, University of Illinois Hospital.Received for publication 8 August 1983 and in revised form 4
January 1985.
PCO2(from 122±5.7 to 77±2.2 mmHg)which was greater (P< 0.02) than that seen in alkalemic normocapnic controls (from73±1.9 to 43±13 mmHg)with a comparable urine bicarbonateconcentration and urine nonbicarbonate buffer capacity. CO2generation, therefore, from collecting duct H+ secretion andtitration of bicarbonate, was higher in hypercapnic rats thanin normocapnic controls.
Weconclude that in rats with acute hypercapnia, the U-BPCO2 achieved during bicarbonate loading greatly underesti-mates collecting duct H+ secretion because it is artificiallyinfluenced by systemic blood pCO2. The ApCO2 is a betterqualitative index of collecting duct H+ secretion than the U-BpCO2, because it is not artificially influenced by systemic bloodPCO2 and it takes into account the urine pCO2 prevailingbefore bicarbonate loading.
Introduction
The carbon dioxide tension (pCO2) of a highly alkaline urinereaches a value substantially higher than that of systemic blood(1-8). Pitts and Lotspeich (1) proposed that the existence ofan appreciable gradient between urine and blood pCO2 wasthe consequence of distal H+ secretion into bicarbonate-richtubular urine in the face of delayed carbonic acid dehydration.They reasoned that, in the absence of carbonic anhydrase inthe lumen of the distal nephron, carbonic acid would notcompletely dehydrate to carbon dioxide during the time thattubular fluid is in transit through the distal nephron. Completionof carbonic acid dehydration in the renal pelvis would ultimatelyincrease urinary pCO2 because the surface to volume relation-ship in this region is not favorable for CO2 back diffusion (1).
It is now known from recent direct intrarenal pCO2determinations that during alkalinization of the urine a nearmaximal urinary pCO2 is achieved in the papillary collectingduct (9, 10). That the rise in urinary pCO2 is an intrarenalrather than a postpapillary event, however, does not invalidatethe classic notion that distal H+ secretion is largely responsiblefor the high urinary pCO2 achieved in a highly alkaline urine.Although other explanations have been offered to account forthe development of a urine to blood PCO2 gradient (3, 6, 7,1 1-17), it is generally accepted that distal H+ secretion largelyaccounts for this phenomenon (18-29). The recent finding ofa significant disequilibrium pH in the papillary collecting ductunder conditions of bicarbonate loading has been proposed asproof of the notion that H+ secretion by the collecting duct isthe most likely determinant of the urine minus blood (U-B)'PCO2gradient observed in an alkaline urine (9).
The U-B pCO2gradient achieved in response to bicarbon-
1. Abbreviations used in this paper: GFR, glomerular filtration rate;U-B, urine minus blood.
Urinary Carbon Dioxide Tension during Hypercapnia 1517
ate loading, however, may not be the ideal way to express therelationship between urinary pCO2 generation and distal H'secretion (30). The use of the U-B pCO2 as a marker of distalH' secretion assumes that blood pCO2 and urine PCO2 areabout the same before alkalinization of the urine. There aresituations, however, where the urinary pCO2 can be lowerthan blood pCO2 as in metabolic and respiratory acidosis (3,6, 31-33). It also assumes that during bicarbonate loadingmedullary blood pCO2 is similar to that of systemic blood.The latter assumption was supported by the findings of Uhlichet al. (34), which showed that during bicarbonate loading vasarecta blood pCO2 was not substantially different from thatprevailing in systemic blood (as determined by renal arterypCO2). Using a recently developed pCO2 microelectrode (35-39), DuBose (9), however, found that during bicarbonateloading the pCO2 of the papillary collecting duct is equal tothat of its adjacent vasa recta but is markedly higher thansystemic blood pCO2. He suggested a role for trapping of CO2in the medullary countercurrent system to explain the similarvalues for pCO2 in the papillary collecting duct and its adjacentvasa recta (9).
Although the contribution of CO2 trapping (if any) tourinary pCO2 generation during hypercapnia is unknown,there is no a priori reason to believe that systemic blood pCO2would reflect vasa recta pCO2 in hypercapnic subjects excretingan alkaline urine. Moreover, it is now known that in rats withacute respiratory acidosis, inner collecting duct pCO2 is lowerthan that of the blood (38, 39). Based on these considerations,we reasoned that the use of the U-B pCO2 achieved duringbicarbonate loading could be a poor marker of collecting ductH+ secretion under conditions where systemic blood pCO2 isaltered. Accordingly, we examined urinary pCO2 generationand U-B pCO2 gradients in hypercapnic rats studied underconditions where the urine was acid and when it was alkalinizedin the presence and in the absence of accelerated carbonic aciddehydration.
MethodsAdult Sprague-Dawley rats (250-380 g) were allowed free access tofood (Purina Lab Chow, Ralston Purina Co., St. Louis, MO) and wateruntil the morning of each experiment. Studies using conventionalclearance techniques were performed in anesthetized rats. Anesthesiawas accomplished by the intraperitoneal administration of Inactin (100mg/kg body weight). Tracheostomy was performed and one carotidartery and jugular vein were cannulated. Rats were connected to arodent respirator (model 680, Harvard Apparatus Co., Inc., S. Natick,MA) to sustain a blood pCO2 at the desired level. At the start of theexperiment, '251I-iothalamate diluted in saline (0.75 gCi/ml) was infusedby an infusion pump at a rate of 0.024 ml/min throughout the courseof the experiment as a marker of glomerular filtration rate (GFR). Anequilibration period of 60 min was allowed before any collection wasstarted. Urine samples were collected via a suprapubic catheter anddrained into preweighed glass vials containing mineral oil; urinevolume was determined gravimetrically. Blood samples were collectedfrom the carotid artery during the middle portion of each clearanceperiod.
Timed urine collections of -20-30 min duration were obtainedin the following groups of rats: in group Ia, eight normocapnic ratswere infused with sodium bicarbonate (0.9-M solution) at a rate of 6ml/h. At least two clearance collections were obtained from each ratafter a urine pH of 7.8 or higher was achieved. The first urine collectionwith a urine pH of 7.8 or higher was discarded to eliminate mixing ofurine of different pH's. After completion of this protocol, acutehypercapnia was induced in the eight rats by making them inhale amixture of 10% C02, balance air via tracheal intubation. After 2 h of
stable hypercapnia (blood pCO2 range, 80-100 mmHg), urine collectionswere obtained while sodium bicarbonate infusion was continuedthroughout the experiment to keep urinary pH above 7.8.
In group Ib, nine rats were studied during normocapnia in responseto a sodium bicarbonate infusion as described above. After obtainingat least two control clearance collections at a urinary pH of 7.8 orhigher, moderate hypercapnia was induced using a mixture of 5%CO2, balance air. Blood PCO2was kept constant between 68 and 78mmHg for 2 h. Thereafter, experimental urine collections werecompleted while sodium bicarbonate infusion was administered as ingroup Ia.
In group Ila, seven rats were studied before and after induction ofsevere hypercapnia (blood pCO2, 80-100 mmHg). Urine collectionswere obtained during the first and the second hour of sustainedrespiratory acidosis to determine the effect of this acid-base alterationon urinary pCO2 and renal acid excretion.
In group lIb, seven normocapnic rats excreting a urine of lowbicarbonate concentration (<3 meq/liter) were studied during 2 hand served as controls for hypercapnic rats excreting an acid urine(group Ila).
In group IIIa, seven rats were studied during severe hypercapnia asin group Ila except that a bolus dose of 5 mg of carbonic anhydrasefollowed by a continuous infusion (5 mg/h) was administered beforeinduction of severe hypercapnia. Thereafter, carbonic anhydrase wasinfused at a rate of 5 mg/h during 2 h of sustained hypercapnia toexamine urinary pCO2 generation in the face of accelerated carbonicacid dehydration.
In group IlIb, six normocapnic rats were studied during the infusionof carbonic anhydrase as in group IIIb and served as normocapniccontrols.
After completion of the above protocols, groups Iha, Ilb, IIa, andIlIb were infused with sodium bicarbonate. In groups Ila and IlIb,the infusion of carbonic anhydrase was continued throughout theexperiment. At least three urine collections were completed during theinfusion of sodium bicarbonate (0.9-M solution) at a rate of 6 ml/hwhile blood PCO2was held constant.
In group IV, eleven rats were studied during 2 h of acute respiratoryacidosis as in group Iha. Thereafter, they were infused with sodiumbicarbonate at a slower rate (3 ml/h) in an effort to prevent a rise inblood pH to the alkalemic range that occurred in all previous groupsstudied during the course of sodium bicarbonate infusion.
In group IVa, a subset of six rats of group IV were further studiedduring carbonic anhydrase infusion (5 mg/h for 2 h after an initialbolus dose of 5 mg). Three collections of -30 min duration wereobtained while the rate of sodium bicarbonate infusion was maintainedat - 3 ml/h.
In groups Va and Vb, ten normocapnic rats served as controls forgroup IVa hypercapnic rats. After completion of three urine collectionsduring the infusion of sodium bicarbonate, five of them were givencarbonic anhydrase (group Va) and five were not (group Vb, timecontrols). The rate of sodium bicarbonate infusion was maintained at-3 ml/h throughout the protocol.
Analytical methods. The urine and arterial blood samples for pHand PCO2 were measured on a Radiometer system (RadiometerAmerica, Inc., Westlake, OH) immediately after collection. Bicarbonateconcentration was calculated from the pH and PCO2 using the Hen-derson-Hasselbalch equation as previously described (40, 41). Sodiumand potassium were measured by flame photometry. Titratable aciditywas calculated from the amount of 0.1 NaOHused to titrate a 1-mlsample (0.1 ml urine plus 0.9 ml distilled H20) up to a pH of 7.4.Ammoniumwas measured by a formulin titrimetric method (42).
Nonbicarbonate buffer capacity was measured using an automatictitrator (Radiometer America, Inc., model TTT 60, ABU 12) aspreviously described by Stinebaugh et al. (20). Briefly, HCl was addedin sufficient amounts as to lower the pH of each urine specimen tobelow 2.5. The specimens were agitated for 30 min to ensure carbondioxide loss. Thereafter, a titration curve from 2.5 to 8.5 was obtainedby adding NaOH(0.1 M) to each of the urine samples. The nonbicar-bonate buffer capacity was calculated from the pH 7.0-8.5 portion of
1518 D. C. Batlle, M. Downer, C. Gutterman, and N. A. Kurtzman
the titration curve as well as from 3.0 to 8.5 pH U. Either calculationyielded similar results, owing to the linearity of the titration curve.Carbonic anhydrase B from bovine erythrocytes was obtained fromSigma Chemical Co. (St. Louis, MO). Carbonic anhydrase activity inthe urine was measured by the method of Maren et al. (43). Thistechnique measures carbonic anhydrase activity by the rate of carbondioxide hydration. Statistical analyses were performed using the t testfor paired and unpaired data when appropriate. Values are means±SE.
Results
Effect of an acute elevation of blood pCO2 on urine pCO2in rats excreting a highly alkaline urine (Table I, Fig. 1)Effect of severe hypercapnia (group Ia). The data of rats infusedwith sodium bicarbonate before induction of hypercapnia arepresented in Table I. In these rats, urine pCO2 after alkaliniza-tion of the urine was 75±1.6 mmHgand U-B pCO2 was31±2 mmHg. Induction of acute hypercapnia in the face ofcontinuous sodium bicarbonate infusion resulted in a fall inblood pH and a rise in plasma bicarbonate. The increase inblood PCO2 (from 42±0.8 to 87±0.8 mmHg) was associatedwith an increase in urine pCO2 (from 75±1.6 to 103±4.2mmHg,P < 0.001) and a decrease in U-B pCO2 (from 31±2to 16±4.2 mmHg, P < 0.005). The rise in urine pCO2 was ofa greater magnitude than the rise in urine bicarbonate concen-tration (from 163±5.2 to 196±12 meq/liter); consequently,urinary pH fell (from 7.93±0.009 to 7.87±0.02, P < 0.01).
Effect of moderate hypercapnia (group Ib). In these rats,blood PCO2 was increased to a lesser degree in order toinvestigate whether the low U-B pCO2 observed in rats withsevere hypercapnia had been, at least in part, the consequenceof their higher blood pCO2 per se, rather than failure togenerate a sufficiently large urinary pCO2.
The increase in blood pCO2 (from 40±0.6 to 73±0.06mmHg) resulted in a fall in blood pH despite an increase inplasma bicarbonate secondary to continuous sodium bicarbon-ate infusion. Urinary pCO2 increased from 79±2.8 to 103±5.2mmHg, P < 0.005; that is, to a value virtually identical tothat observed in rats with severe hypercapnia (group Ia). Thisincrease in urine pCO2 occurred in the face of a modest risein urine bicarbonate concentration (from 145±4.5 to 199±11meq/liter) which was also virtually identical to that observedin rats with severe hypercapnia (group Ia) (Fig. 1).
In contrast to rats with severe hypercapnia, however, theU-B pCO2 did not decrease significantly in response toinduction of hypercapnia of moderate severity (from 39±2.5to 30±4.9 mmHg). Hence, the U-B pCO2 of rats withmoderate hypercapnia was significantly higher than that ofrats with severe hypercapnia (30±4.9 and 16±4.2 mmHg,respectively; P < 0.05). This difference was likely the conse-quence of a blood pCO2 lower in rats with moderate hyper-capnia than in rats with severe hypercapnia (73±0.6 and87±0.8 mmHg,P < 0.001) because urinary pCO2was virtuallyidentical between both groups (Fig. 1). Blood pH, plasmasodium, plasma potassium, and urine flow were not significantlydifferent between these two groups of hypercapnic animals.After induction of hypercapnia GFR fell to a comparablelevel in both groups. A fall in GFR was also observed innormocapnic time control rats (group Vb) that are presentedin Table V (see below).
Acid excretion during acute respiratory acidosis (Table II)Induction of severe hypercapnia (group Iha) resulted in asignificant fall in blood pH, a fall in urinary pH, and an
increase in net acid excretion which were observed during thefirst hour of acute hypercapnia. The increase in titratable acidexcretion was associated with an increase in phosphate excre-tion. A significant increase in ammonium excretion and afurther increase in titratable acid and net acid excretion wereobserved during the second hour of sustained hypercapnia.The increase in ammonium excretion observed during thesecond hour of sustained hypercapnia was associated with aurinary pH identical to that observed during the first hour ofhypercapnia. As anticipated, plasma K and plasma phosphateincreased in response to hypercapnia. GFRdid not fall signif-icantly after induction of acute respiratory acidosis.
Urinary pCO2 during acute respiratory acidosisThe urinary pCO2 of group Ila rats was 57±9.1 mmHgin thefirst hour and 58±6.3 mmHgduring the second hour ofsustained hypercapnia. These values of urinary pCO2 weresignificantly lower than blood pCO2 (57±9.1 vs. 84±2.2 mmHg,P < 0.02, and 58±6.3 vs. 86±1.7 mmHg,P < 0.005). Therefore,in hypercapnic rats the U-B pCO2 difference was markedlynegative throughout the 2 h of observation (Fig. 2). Theurinary pCO2 of control rats (group Ilb) was not significantlydifferent than that of the blood (37±3.2 and 41±1.3 mmHg,respectively).
The urinary pCO2 observed in group Ila rats during thesecond hour of hypercapnia was compared to that of normo-capnic control rats (group Ilb) which were excreting an acidurine. The urinary pCO2 of hypercapnic rats was higher thanthat of normocapnic control rats (58±6.3 and 37±3.2 mmHg,respectively, P < 0.02) with a comparably low urine bicarbonateconcentration (1.1±0.3 and 1.6±0.3 meq/liter, respectively).Consequently, urinary pH was lower in hypercapnic rats thanin control rats (5.78±0.06 and 6.17±0.07, respectively, P< 0.005). Hypercapnic rats had a urinary phosphate concen-tration higher than that of normocapnic controls (72±17 and29±6.3 mmol/liter, respectively, P < 0.05).
Urinary pCO2 during acute respiratory acidosis andcarbonic anhydrase infusionIn rats with acute respiratory acidosis infused with carbonicanhydrase (group I1la), urinary pCO2 was 50±4.3 and 55±4.7mmHgduring the first and second hour of hypercapnia,respectively. These values of urinary pCO2 were significantlylower than blood PCO2(50±4.3 vs. 80±1.5 mmHg,P < 0.001,and 55±4.7 vs. 86±2.2 mmHg, P < 0.001, respectively). Theurinary pCO2 of these rats was also higher than that ofnormocapnic controls (group IlIb) infused with carbonic an-hydrase (55±4.7 and 30±3.6 mmHg, respectively, P < 0.005)(Table III). The urinary pCO2 of hypercapnic rats infused withcarbonic anhydrase (group IlIa) was not significantly differentfrom that of hypercapnic rats not receiving this enzyme (groupIha) (55±4.7 and 58±6.3 mmHg, respectively). Hence, theurinary pCO2 of hypercapnic rats excreting a urine of lowbicarbonate content was lower than that of the blood regardlessof whether carbonic anhydrase was given or not. This resultedin a markedly negative U-B pCO2 difference in hypercapnicbut not in normocapnic rats (Fig. 2).
Effect of alkalinization of the urine on urinary pCO2 inrats infused with carbonic anhydrase (Table III)In hypercapnic rats (group IIla), sodium bicarbonate infusionresulted in a significant rise in urine pCO2 (from 55±4.7 to78±2.7 mmHg, P < 0.005). In normocapnic control rats
Urinary Carbon Dioxide Tension during Hypercapnia 1519
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Figure 1. Blood pCO2, U-B pCO2, urine pCO2, and urine bicarbon-ate concentration in rats with severe hypercapnia (open bars) and inrats with moderate hypercapnia (dark bars) infused with sodiumbicarbonate. The lower U-B pCO2 of rats with severe hypercapniawas totally accounted by their higher blood pCO2 because urinePCO2was the same as that of rats with moderate hypercapnia.
(group IIb), sodium bicarbonate infusion also resulted in asignificant rise in urine pCO2 (from 30.5±3.6 to 42±1.7mmHg, P < 0.02). The increment in urinary pCO2 elicited bysodium bicarbonate loading was higher in hypercapnic thanin normocapnic rats (24±5.2 and 12±3.1 mmHg, respectively),but this difference did not achieve statistical significance(P > 0.05). During sodium bicarbonate loading, the urinePCO2 of hypercapnic rats was significantly lower than thatsystemic blood pCO2 (78±2.7 and 87±1.8 mmHg, respectively,P < 0.02), while that of normocapnic rats was not (42±1.7and 40±0.3 mmHg, respectively). This resulted in a negativeU-B pCO2 in hypercapnic rats but not in controls (Fig. 2).
Urine pH was higher in normocapnic rats than in hyper-capnic rats (8.08±0.05 and 7.90±0.02, respectively, P < 0.005)while urine bicarbonate concentration was not significantlydifferent. GFR did not change significantly in either groupwhile plasma sodium and plasma bicarbonate increased inboth groups.
Effect of alkalinization of the urine on urinary pCO2 inrats not infused with carbonic anhydrase (Table IV)In the set of experiments presented in Table I, hypercapniawas induced in normocapnic rats that were excreting an
alkaline urine and, therefore, had a high urine pCO2. Thus,the additional rise in urinary pCO2 that occurred in these ratscould have been the result of abruptly raising the blood pCO2rather than the consequence of alkalinizing the urine. In thefollowing set of experiments, sodium bicarbonate was infusedinto hypercapnic rats that were excreting a urine with lowbicarbonate content. In hypercapnic rats (group Ila) in whichsodium bicarbonate infusion resulted in a high blood pH,urinary pCO2 rose from 58±6.3 to 98±4.9 mmHg(P < 0.005)in the absence of any significant change in blood pCO2. Thisrise in urinary pCO2 was therefore virtually identical to therise in U-B pCO2 (from -28±4.7 to 15±4.0 mmHg). Theurinary pCO2 achieved under these conditions was comparableto that of rats with moderate (group Ia) and severe hypercapnia(group Ib) in which blood PCO2was increased after alkaliniza-tion of the urine (compare Table IV with Table I). Blood pHafter bicarbonate loading was also not significantly different(7.55±0.02, 7.58±0.01, and 7.53±0.02 in groups Ia, Ib, andIha, respectively). Plasma sodium was higher in the hypercapnicrats presented in Table I (groups Ia and Ib) than in thosepresented in Table IV (group Ila). GFR fell significantly ingroups Ia and lb but not in group Ila, probably because theduration of the experiment was longer in groups Ia and lb.
In normocapnic control rats (group TIb), urinary pCO2increased from 37±3.2 to 79±2.6 mmHg. Hence, the incrementin urinary pCO2 elicited by bicarbonate infusion while bloodPCO2was kept constant (thereafter to be referred to as ApCO2)was almost identical in hypercapnic rats (group Ila) andnormocapnic rats (40±7.2 and 42±4.6 mmHg, respectively).After bicarbonate loading, urinary pCO2 was higher in hyper-capnic rats than in controls (98±4.9 and 79±2.6 mmHg, P< 0.001), but the U-B pCO2 was lower in hypercapnic ratsthan in controls (15±4.0 and 39±2.5 mmHg, P < 0.001).
In hypercapnic rats (group IV) in which bicarbonate infusionresulted in a normal blood pH (7.38±0.01), urinary pCO2increased from 55±6.9 to 125±3.6 mmHg, P < 0.001. Thus,the ApCO2of these rats (70±5.6 mmHg)was greater than thatobserved in normocapnic rats (42±4.6 mmHg)(Fig. 3, left). Itwas also greater (P < 0.005) than that of hypercapnic rats(40±7.2 mmHg) with a higher blood pH (group Ila). The
Table II. Effect of Acute Respiratory Acidosis (Group Ila) on Acid Excretion
Blood
Plasma Plasma UrneGFR V pH pCO2 HCO3 K P04 UPO4X V pH TA NH4 Net acid
* Statistical analysis (P value) between hypercapnia (2nd h) and prehypercapnia.
Urinary Carbon Dioxide Tension during Hypercapnia 1521
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Figure 2. The U-B pCO2 of hypercapnic rats (groups Ila, Illa, andIVa) is compared with that of normocapnic control rats (groups Ilb,IIb, and Va). The U-B pCO2 of hypercapnic rats was lower thanthat of normocapnic control rats when the urine was acid (with andwithout carbonic anhydrase infusion) and when it was alkalinized inthe face of a continuous carbonic anhydrase (CA) infusion. 0, con-
trol; *, hypercapnia.
U-B pCO2 of group IV hypercapnic rats (39±3.7 mmHg)was
significantly higher than that of group Ila hypercapnic rats(15±4.0 mmHg) and not significantly different than that ofnormocapnic controls (group Ilb) (39±2.5 mmHg). Thesedifferences in urine pCO2 and U-B pCO2 were observed inthe face of a comparable increase in urine bicarbonate concen-
tration in the three groups studied (Fig. 4). Plasma sodiumalso increased to a comparable level in the three groups.Plasma K was higher in hypercapnic rats than in controls.
Effect of carbonic anhydrase infusion on urine pCO2 in ratsexcreting an alkaline urine (Table V)In normocapnic control rats (group Va), carbonic anhydraseinfusion resulted in a fall in urine pCO2 (from 73±1.9 to43±1.3 mmHg,P < 0.001) while urine bicarbonate concentra-tion did not change significantly. Consequently, urine pHincreased (from 7.87±0.03 to 8.04±0.04, P < 0.02). In hyper-capnic rats (group IVa), the infusion of carbonic anhydrasealso resulted in a fall in urine pCO2 (from 122±5.7 to 77±2.2mmHg,P < 0.001), while urine bicarbonate concentration didnot change and urine pH increased (from 7.65±0.03 to7.85±0.03, P < 0.005). The fall in urine pCO2 elicited bycarbonic anhydrase infusion was greater in hypercapnic ratsthan in normocapnic controls (-45±4.3 and -30±2.6 mmHg,respectively, P < 0.02) (Fig. 3, right).
Nonbicarbonate buffer capacity was not significantly dif-ferent between normocapnic and hypercapnic rats either before(2.4±0.3 and 2.3±0.6 meq/liter per pH unit, respectively) or
after carbonic anhydrase infusion (2.5±0.5 and 3.9±2.3 meq/liter per pH unit, respectively).
Carbonic anhydrase activity in the urine was not signifi-cantly different between hypercapnic and normocapnic rats
(275±96 vs. 362±103 gg/ml, respectively). Likewise, the ex-
cretion of this enzyme was not significantly different betweenthe two groups (31±8.3 vs. 23±4.0 ,ug/min, respectively).Carbonic anhydrase infusion was associated with a significantfall in GFRand urine flow in both hypercapnic and normo-capnic rats. Neither parameter, however, was significantlydifferent between hypercapnic and normocapnic rats.
In time control rats (group Vb) GFRalso fell significantly,but urine, pCO2, and U-B pCO2 did not. The fall in GFRwas likely the result of the prolonged duration of this experi-ment. Nonbicarbonate buffer capacity in the time controlgroup was not significantly different than that of hypercapnic(group IVa) and normocapnic rats (group Va) infused withcarbonic anhydrase.
After carbonic anhydrase infusion, the urine pCO2 ofhypercapnic rats (group IVa) fell to a level lower than that ofthe blood (77±2.2 and 86±0.9 mmHg, respectively, P< 0.025), while that of normocapnic rats (group Va) did not(43±1.3 and 40±1.0 mmHg, respectively). Thus, the relation-ship between urine pCO2 and blood pCO2 was virtuallyidentical to that observed in hypercapnic (group Illa) andnormocapnic rats (group IIb) in which the infusion of carbonicanhydrase antedated the infusion of bicarbonate (Fig. 2). Asanticipated, the urinary pCO2 achieved in both hypercapnicand normocapnic rats infused with sodium bicarbonate andcarbonic anhydrase was markedly lower than that observedwhen this enzyme was not administered (compare Tables IIIand V with Table IV).
DiscussionThis study demonstrates a clear dissociation between urinePCO2 and blood pCO2 during acute hypercapnia. In normo-capnic rats excreting a highly alkaline urine, induction ofsevere hypercapnia resulted in a fall in the U-B pCO2gradientdespite an increase in urine pCO2(group Ia, Table I). Accordingto current views (9, 16-29, 36), the finding that the U-BPCO2 achieved during bicarbonate loading is lower in hyper-capnic rats than in normocapnic rats (Tables I and IV) requiresthe conclusion that collecting duct H' secretion is decreasedby hypercapnia. In fact, such a conclusion was recently reachedby another group of investigators who noted that the U-BPCO2gradient of hypercapnic dogs infused with sodium bicar-bonate was lower than normal (23).
That the low U-B pCO2 observed during bicarbonateloading reflects decreased collecting duct H' secretion duringhypercapnia, however, is not likely correct. The urine pCO2of hypercapnic rats was higher than that of normocapnic rats,a finding that could be interpreted as indicative of enhancedcollecting duct H' secretion in hypercapnic rats (i.e., theopposite interpretation of that portrayed by the U-B pCO2gradient). Alternatively, it could be argued that the higherurine pCO2 observed in hypercapnic rats was purely the resultof their higher blood pCO2. The following findings, however,mitigate against the latter possibility.
First, rats with moderate hypercapnia (group Tb) exhibiteda urinary pCO2 virtually identical to that of rats with severe
hypercapnia (group Ia) in the face of a comparable urinebicarbonate concentration (Fig. 1). This finding indicates thatthe U-B pCO2 was influenced by changes in systemic bloodPCO2 rather than by changes in urinary pCO2 generation.Second, urinary pCO2 did not increase to the level prevailingin the blood in hypercapnic animals excreting a urine of low
1522 D. C. Batle, M. Downer, C. Gutterman, and N. A. Kurtzman
Urinary Carbon Dioxide Tension during Hypercapnia 1523
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Figure 3. The relationship of urine pCO2 to blood pCO2 duringnormocapnia and hypercapnia. The left panel depicts the increase inurine PCO2elicited by sodium bicarbonate loading (i.e., the ApCO2)in normocapnic rats (group Ilb) and hypercapnic rats (group IV).Open circles denote urine pCO2 when the urine was acid; closedcircles denote the urine pCO2 achieved during bicarbonate loading.The ApCO2was significantly higher in hypercapnic rats than innormocapnic controls (P < 0.005). The right panel depicts the fall in
bicarbonate content; therefore, their U-B pCO2 gradient was
markedly negative (Fig. 2). This finding is in agreement withrecent studies in dogs with acute hypercapnia (32) and in ratswith chronic hypercapnia (33). Third, in hypercapnic ratsexcreting a urine of low bicarbonate content, the U-B pCO2was also negative when carbonic anhydrase was given to ensure
complete carbonic acid dehydration and achievement of dif-fusion equilibrium between tubular and peritubular CO2(TableIII, Fig. 2). Thus, even in the face of accelerated carbonic aciddehydration, urinary pCO2 and blood PCO2did not equilibrateduring hypercapnia. This finding differs from the study ofAdrogue et al. (23) in dogs with severe hypercapnia. Theseauthors reported that during carbonic anhydrase infusion,urine pCO2 before bicarbonate loading was not significantlydifferent from arterial blood pCO2 within 30 min of inductionof hypercapnia (23). Accordingly, they proceeded to use theU-B pCO2 achieved after bicarbonate loading as a marker ofdistal acidification (23). The reasons for the discrepancy betweenour results and those of Adrogue et al. (23) are not readilyapparent except that their studies were performed in only fouranimals of a different species. Fourth, in hypercapnic ratsinfused with carbonic anhydrase and sodium bicarbonate,urinary pCO2 was still significantly lower than blood PCO2(78±2.7 vs. 87±1.8 mmHg, P < 0.02), which resulted in a
negative U-B pCO2 (Table III, Fig. 2). This finding furthersuggests that systemic blood pCO2 does not reflect intrarenalPCO2even in the face of accelerated carbonic acid dehydration
APC02 (- 30 + 2.6mmHg)
I I I I I40 50 60 70 80 90 100 110
Blood PCO2
urine pCO2 (-ApCO2) caused by carbonic anhydrase infusion tonormocapnic rats (group Va) and hypercapnic rats (group IVa).Closed circles denote urine pCO2 in an alkaline urine. Closed boxesdenote that urine pCO2 was measured during carbonic anhydraseinfusion. The fall in urine pCO2 elicited by carbonic anhydraseinfusion was significantly higher in hypercapnic rats than in normo-
capnic controls (P < 0.02).
and in the presence of large urinary bicarbonate concentrations.Thus, factors other than equilibration between systemic bloodPCO2 and renal PCO2 determine, at least in part, the urinePCO2 in hypercapnic rats (see below).
Having established that the urinary pCO2 of hypercapnicrats is lower than systemic blood pCO2 we sought to determinethe effect of alkalinization of the urine on urinary pCO2generation when blood pCO2 was kept constant. Wereasonedthat if blood pCO2 were not allowed to change the incrementin urinary pCO2 observed in response to sodium bicarbonateloading (i.e., the ApCO2), it would provide an indirect quali-tative measurement of collecting duct H+ secretion. The ApCO2of hypercapnic rats with a high blood pH (group Ila) was
comparable to that of normocapnic rats with a high blood pH(group IIb) (Table IV). The ApCO2 of hypercapnic rats with a
normal blood pH (group IV) was higher than that of normo-
capnic rats (Fig. 3, left), and than that of hypercapnic rats witha high blood pH (group Ila) (Table IV). The U-B pCO2achieved by hypercapnic rats with a high blood pH (groupIla), however, was again lower than that of normocapnic rats(group Ilb) (15±4.0 and 39±1.5 mmHg, respectively), whilethat of hypercapnic rats with a normal blood pH (group IV)was the same (39±3.7 and 39±2.5 mmHg, respectively).
Urinary bicarbonate concentration, a major determinantof urinary pCO2generation (13-30), increased to a comparablelevel in both groups of hypercapnic rats and in normocapniccontrols (Fig. 4). Since blood pCO2 was not allowed to change,
Urinary Carbon Dioxide Tension during Hypercapnia 1525
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the rise in urine pCO2 was the consequence, at least in part,of collecting duct H' secretion into a bicarbonate-rich urineand not the consequence of equilibration between intrarenaland systemic blood pCO2.
Urine pCO2 and its derivatives as indices of distal acidifi-cation. The possible pitfalls of the urine pCO2and its derivativesas markers of collecting duct hydrogen ion secretion can besummarized as follows:
(a) U-B pCO2 during bicarbonate loading. The use of theU-B pCO2 observed in a highly alkaline urine as an index ofcollecting duct H+ secretion requires that during bicarbonateloading, systemic blood pCO2 be similar to that of medullaryblood. DuBose (9), however, has recently demonstrated thatin a highly alkaline urine the pCO2 in the papillary collectingduct and adjacent vasa recta is markedly higher than that ofthe systemic blood (9). Thus, in normocapnic subjects infusedwith bicarbonate, intrarenal blood pCO2 and systemic bloodPCO2 dissociate. Since the rise in urine pCO2 in an alkalineurine occurs in the collecting duct and is, therefore, associatedwith a rise in vasa recta pCO2, the use of the U-B pCO2serves no purpose because systemic blood pCO2 is not a
reflection of intrarenal blood pCO2 (30). This fact, in our view,invalidates the use of the U-B pCO2 gradient in a highlyalkaline urine as an index of distal H+ secretion even duringnormocapnia. This is not to say that the conclusions derivedfrom numerous studies in normocapnic subjects using theU-B pCO2 gradient as a marker of distal acidification needto be altered. When urinary pCO2 and blood pCO2 are aboutthe same before bicarbonate loading (as is usually the case
of hypercapnic rats (group Ila) (open triangles) and normocapnic rats(group Ilb) (open circles) that were alkalemic. The U-B pCO2 ofgroup Ila rats was lower, while that of group IV was equal to that ofnormocapnic control rats (group Ilb) with a comparably high urinebicarbonate concentration.
during normocapnia), the rise in urinary pCO2 elicited bysodium bicarbonate infusion is equivalent to the U-B pCO2achieved under this condition (e.g., normocapnic rats, TableIV). Therefore, both parameters yield the same information inregards to the ability to generate CO2 as a result of collectingduct H+ secretion. When blood pCO2 and urine pCO2 are
widely different, however, the U-B pCO2 gradient achievedduring bicarbonate loading becomes an artifact that gives no
physiological information about collecting duct H+ secretion.The artifactual nature of the U-B pCO2 gradient is illustratedby our studies during hypercapnia, where it clearly underesti-mates distal H+ secretion (Table IV, Fig. 4). This situation isthe opposite of hypocapnia, where the U-B pCO2 gradientcan overestimate collecting duct H+ secretion (44).
(b) Urine pCO2 after bicarbonate loading. To take intoaccount the relationship between urine PCO2and blood pCO2that prevailed before administration of sodium bicarbonate,we used the ApCO2as an index of collecting duct H+ secretion(Table IV, Fig. 3, left). Blood pCO2 was no longer a variablebecause it was not allowed to change, and the base-line urinePCO2 (i.e., the pCO2 of an acid urine) was known. Thus, theAPCO2is appropriate as a qualitative index of collecting ductH+ secretion, in that it integrates the effects of both an acidand a highly alkaline urine on intrarenal CO2generation whileit eliminates the blood pCO2 as a variable. If the ApCO2were
the best way to express the relationship between urinary pCO2generation and collecting duct H+ secretion, one would haveto conclude that collecting duct H+ secretion was normal in
1526 D. C. Batlle, M. Downer, C. Gutterman, and N. A. Kurtzman
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The ApCO2 may be problematic, in that a number ofvariables (blood pH, plasma sodium, plasma potassium, volumestatus, medullary blood flow rate, etc.) change during bicar-bonate diuresis as compared with base-line conditions. In thisregard, the end point urine pCO2 observed under identicalconditions of bicarbonate diuresis, urine flow rate, and volumestatus is a more suitable marker of collecting duct H' secretionthan the ApCO2. If one were to use the end-point urine pCO2,one would have to conclude that collecting duct H+ secretionwas enhanced in both groups of hypercapnic rats because theend-point urine pCO2 was higher than that of normocapniccontrols (Fig. 4). It is readily apparent, however, that the urinePCO2 (i.e., that observed in a highly alkaline urine) fails totake into account the fact that the pCO2 prevailing in theurine of hypercapnic animals before bicarbonate loading ishigher than that of normocapnic rats. Thus, during hypercapnia,the end point urine pCO2 observed after bicarbonate loadingis uncertain as a marker of collecting duct H+ secretion becauseit could overestimate it as opposed to the U-B pCO2, whichunderestimates it.
Weattempted to equalize urine pCO2 and blood pCO2 inhypercapnic rats by alkalinizing the urine in the presence ofcarbonic anhydrase infusion. We reasoned that the rise inurinary pCO2 attributable to H+ secretion into a bicarbonate-rich urine would be dissipated by accelerating carbonic aciddehydration, and that urinary pCO2 and blood pCO2 wouldbe approximately the same. Urinary pCO2 was about that ofthe blood in normocapnic rats but not in hypercapnic rats(Tables III and V, Fig. 2). The finding of a urinary pCO2 lowerthan that of the blood in a highly alkaline urine rich incarbonic anhydrase deserves consideration. We think thataccelerated consumption of CO2 within collecting duct cellsaccounts for lowering of tubular CO2 (and final urine C02)below systemic blood levels during hypercapnia. CO2 is likelyconsumed and bicarbonate regenerated because of enhancedsecretion of H+, which under these conditions is titrated bybicarbonate and by nonbicarbonate buffer. The titration of H+by bicarbonate, in the presence of carbonic anhydrase, doesnot increase urine pCO2 because of accelerated carbonic aciddehydration; the titration of H+ by nonbicarbonate bufferconsumes CO2 in the process of bicarbonate regeneration andlowers urine CO2. These two processes likely occur in bothhypercapnic and normocapnic rats. That urine pCO2 is lowerthan that prevailing in the blood in hypercapnic rats but notin normocapnic rats requires either enhanced collecting ductH+ secretion or excess nonbicarbonate buffer in the hypercapnicanimals. The urinary nonbicarbonate buffer capacity was com-parable between hypercapnic and normocapnic rats (Table V),which suggests that enhanced collecting duct H+ secretion was,at least in part, the cause of tubular CO2 removal duringhypercapnia.
The basic principle in using the urinary pCO2 in a highlyalkaline urine as a marker of distal acidification is to estimate
the amount of pCO2generated from H+ secretion and titrationof bicarbonate buffer in the face of delayed carbonic aciddehydration (i.e., in the absence of carbonic anhydrase in thelumen of the distal nephron). In this regard, the difference inurine pCO2 obtained in a highly alkaline urine not containingcarbonic anhydrase and in an equally alkaline urine containingcarbonic anhydrase should provide the best possible estimateof PCO2 generated from H' secretion and titration of bicar-bonate buffer. To attack this issue, we gave carbonic anhydraseto hypercapnic and normocapnic rats that were excreting analkaline urine (Table V). This maneuver resulted in a greaterfall in urine pCO2 (i.e., -ApCO2) in hypercapnic rats than innormocapnic rats (Fig. 3, right). This clearly demonstrates thatthe amount of pCO2 generated in the face of delayed carbonicacid dehydration (i.e., before carbonic anhydrase infusion) wasgreater in these hypercapnic rats thap in controls, a findingwhich denotes enhanced collecting duct H+ secretion. BloodpH was normal in the hypercapnic rats and alkalemic innormocapnic rats. Since changes in serosal pH (21, 45, 46)are known to modulate H' secretion, it seems likely that thedifference in urinary pCO2 generation noted between hyper-capnic and normocapnic rats was determined by the prevailingblood pH rather than blood pCO2. This is further suggestedby the finding of a higher ApCO2 in hypercapnic rats with anormal blood pH (group IV) than ip those with a high bloodpH (group Ila), while blood pCO2 was identical in both groups(Table IV).
(c) Urinary pCO2 before bicarbonate loading. The findingthat urinary pCO2 is lower than systemic blood pCO2 inhypercapnic rats excreting an acid urine (Fig. 2, Tables III andIV) also deserves some comment. In an acid urine, secretedH+ must react with buffers other than bicarbonate because theamount of filtered bicarbonate reaching the collecting duct isvery low. Carbonic acid formation is necessarily low, therebylimiting the amount of CO2 generated from the dehydrationof this acid. In keeping with this notion, carbonic anhydraseinfusion to hypercapnic rats in which the urine contained littlebicarbonate did not result in a urinary pCO2 different fromthat of hypercapnic rats not infused with the enzyme. In otherwords, the enzyme had no effect on urinary pCO2 becausebicarbonate was virtually absent from the lumen of the col-lecting duct. Collecting duct H+ secretion when luminal bicar-bonate is negligible results in the titration of nonbicarbonatebuffer, a process that causes consumption of CO2 because CO2combines with hydroxyl anions in the cell to regeneratebicarbonate.
Note, however, that the mechanism(s) whereby urine pCO2is lower than that of the blood in animals excreting an acidicurine has never been definitively elucidated. Rector (47) con-sidered several possibilities other than collecting duct H+secretion. Enhanced bicarbonate reabsorption by a nonhydrogenion secretory mechanism could lower tubular CO2 (31, 47).This is not likely, because the amount of bicarbonate reachingthe collecting duct in our experiments was minimal, as judgedby the extremely low urinary bicarbonate concentration ob-tained during acute respiratory acidosis.
Another factor that could lower urine pCO2 is mixing ofurine containing excess of nonbicarbonate buffer with a poorlybuffered acid urine (3, 47). Assuming that the nonbicarbonatebuffer in excess during acute respiratory acidosis is phos-phate, carbonic acid present in acidic urine will be consumedin the following reaction: H2CO3 + Na2HPO4 - NaHCO3+ NaH2PO4. We cannot exclude a role for excess urinary
1528 D. C. Batlle, M. Downer, C. Gutterman, and N. A. Kurtzman
phosphate buffer in lowering urine pCO2 below systemic bloodpCO2, because urine phosphate concentration was higher inrats with acute respiratory acidosis than in controls. It mustbe emphasized that the hypothetical role of admixture ofheterogeneous urine as a mechanism of lowering tubular CO2tensions does not negate the role of enhanced H' secretion asthe main mechanism responsible for this phenomenon.
It is now known that during acute respiratory acidosis,renal cortical pCO2 exceeds systemic blood pCO2 (36) whileinner medullary collecting duct pCO2 is lower than arterialblood PCO2 (38, 39). Therefore, tubular CO2 is lost in transitfrom the superficial distal tubule to the inner medullarycollecting tubule. Bengele et al. (39) and Gougoux et al. (32)explained the findings of an inner medullary collecting ductPCO2 lower than that of the blood by postulating a loss ofluminal CO2by diffusional flux into the lining epithelium. Wethink that this loss of tubular CO2 (and thereby final urineC02) requires that H' secretion be enhanced and cell CO2consumed in the process of bicarbonate regeneration. As H'is secreted into a bicarbonate-poor luminal fluid, ammoniumand acid phosphate are preferentially formed, thereby resultingin cell CO2 consumption. This results in the creation of alumen-to-cell and peritubular blood-to-cell CO2 gradient andtubular CO2 removal.
The titration of secreted hydrogen ions by nonbicarbonatebuffer must result in the formation of either ammonium ortitratable acidity or both. That urinary pH fell while titratableacidity and ammonium excretion increased in response tohypercapnia (Table II) is also consistent with the notion thatthe low U-B pCO2 of these rats was achieved in associationwith enhanced collecting duct H+ secretion. The increase inammonium excretion observed in the second hour of sustainedhypercapnia was unassociated with a fall in urine pH (TableII), which suggests that ammonia production increased. Thisis in agreement with several studies showing that overall kidneyacidification is increased during hypercapnia (48-56) and withrecent work showing a prompt adaptive increase in ammonia-genesis in response to acute hypercapnia (57). While proximalnephron segments are the major site of ammonia production,it is now known that a large portion of ammonium and netacid excreted in the final urine are added to the medullarycollecting during respiratory acidosis (39) as well as metabolicacidosis (58, 59).
Based on all the above considerations, we believe that thereis no evidence for the notion that distal acidification isdecreased during acute hypercapnia (23). In view of recentdirect studies showing that hypercapnia does not increaseacidification above normal in either the superficial distal tubule(60) or in the inner medullary collecting duct (39), it seemslikely that during hypercapnia acidification is either normal orenhanced only in the outer portion of the medullary collectingduct and/or the cortical collecting duct.
In conclusion, this study shows that hypercapnic rats havea urinary pCO2 lower than systemic blood pCO2 when theurine is acid and when it is alkalinized in the face of acceleratedcarbonic acid dehydration. These findings suggest that duringacute hypercapnia, CO2 is removed from the collecting ductowing to consumption of cell CO2in the process of bicarbonateregeneration via collecting duct H+ secretion and titration ofnonbicarbonate buffer. During bicarbonate loading, rats withhypercapnia have a U-B pCO2 lower than or equal to thatof normocapnic control rats, but the rise in urine pCO2 elicitedby this maneuver when blood pCO2 is kept constant is equal
or higher in hypercapnic rats than in normocapnic rats,depending on the prevailing blood pH. In hypercapnic rats,the U-B pCO2 underestimates collecting duct H' secretionbecause this parameter is artificially influenced by systemicblood pCO2, a variable that does not reflect vasa recta pCO2during bicarbonate loading. The ApCO2 is a more appropriatequalitative index of collecting duct H' secretion than the U-BpCO2, because it is not influenced by systemic blood pCO2and takes into account the urine pCO2 prevailing beforebicarbonate loading. A more precise evaluation of collectingduct H' secretion requires the infusion of carbonic anhydraseto estimate the amount of PCO2generated from H' secretionand titration of bicarbonate buffer when carbonic acid dehy-dration is accelerated and when it is delayed.
AcknowledgmentsThis paper received support from a National Institutes of Health grant(AM20170) and a Veterans Administration Merit Review Board grant.
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