Mechanisms used to dispose of progressively increasing alkali load in rats

Surinder Cheema-Dhadli1, Shih-Hua Lin2, and Mitchell L. Halperin1

1 Renal Division, St. Michael's Hospital, University of Toronto, Toronto, Ontario, Canada M5B 1A6; and 2 Renal Division, Tri-Service General Hospital, National Defense Medical Center, Taipei 100, Taiwan


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Our objective was to describe the process of alkali disposal in rats. Balance studies were performed while incremental loads of alkali were given to rats fed a low-alkali diet or their usual alkaline ash diet. Control groups received equimolar NaCl or KCl. Virtually all of the alkali was eliminated within 24 h when the dose exceeded 750 µmol. The most sensitive response to alkali input was a decline in the excretion of NH<UP><SUB>4</SUB><SUP>+</SUP></UP>. The next level of response was to increase the excretion of unmeasured anions; this rise was quantitatively the most important process in eliminating alkali. The maximum excretion of citrate was ~70% of its filtered load. An even higher alkali load augmented the excretion of 2-oxoglutarate to >400% of its filtered load. Only with the largest alkali load did bicarbonaturia become quantitatively important. We conclude that renal mechanisms eliminate alkali while minimizing bicarbonaturia. This provides a way of limiting changes in urine pH without sacrificing acid-base balance, a process that might lessen the risk of kidney stone formation.

acid-base balance; ammonium; bicarbonate; citrate; net acid excretion; organic anions; 2-oxoglutarate


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

TO ACHIEVE ACID-BASE balance, the rate of appearance and elimination of H+ must be equal (11, 26). One can readily identify metabolic pathways that produce H+, because the overall valence of all end products other than H+ is more anionic than all of its substrates (11, 13). In this analysis, cofactors such as adenine nucleotides and redox couples are ignored because they are present in catalytic amounts and are both formed and removed in a metabolic process (13). Said another way, for the net accumulation of H+, there would have to be an equivalent rise in the concentration of the more anionic form of these catalysts. Nevertheless, this cannot produce a large H+ load, because these catalysts are below the millimolar range in the body.

In quantitative terms, the net appearance of H+ is equal to the number of milliequivalents of SO<UP><SUB>4</SUB><SUP>2−</SUP></UP>, inorganic divalent HPO<UP><SUB>4</SUB><SUP>2−</SUP></UP>, and organic anions that are excreted in urine in the steady state (26). There are two major renal processes that eliminate H+. First, H+ can bind to urinary anions if their pK is in the range of the urine pH; in effect, this means HPO<UP><SUB>4</SUB><SUP>2−</SUP></UP> for the most part, because most organic anions have a pK that is appreciably lower than urine pH. Excretion of HPO<UP><SUB>4</SUB><SUP>2−</SUP></UP> is the most important determinant of titratable acid (TA) excretion (14). Second, disposal of H+ also occurs when NH<UP><SUB>4</SUB><SUP>+</SUP></UP> is excreted, because new HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> is added to the body in a 1:1 stoichiometry with NH<UP><SUB>4</SUB><SUP>+</SUP></UP> excretion (10).

Acid production and net acid excretion do not explain mass balance of H+, because these terms do not include production and elimination of dietary alkali (8, 22). One's diet contains organic anions ingested largely as their K+ salts (8, 24). The daily alkali load is the result of metabolism of many of these anions to neutral end products (13). This alkali input can be removed by titrating some of the daily H+ production, or it can be eliminated directly (bicarbonaturia) or indirectly (involving a metabolic and renal process; Fig. 1). The indirect removal of alkali begins with H+ and organic anion production from neutral precursors such as glucose. Next, these organic anions are converted into end products of metabolism by being excreted into urine as their Na+ or K+ salts (i.e., not with H+ or NH<UP><SUB>4</SUB><SUP>+</SUP></UP>; Fig. 1) (13). The excretion of these organic anions represents the elimination of "potential HCO3-" in the urine (Ref. 17; Eq. 1). In fact, organic anion excretion in rats consuming their usual diet is an order of magnitude larger than net acid excretion (22, 25)
 NAE = NH<SUP>+</SUP><SUB>4</SUB> + TA − (HCO<SUP>−</SUP><SUB>3</SUB> + potential HCO<SUP>−</SUP><SUB>3</SUB>) (1)
where NAE is net acid excretion.


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Fig. 1.   Base balance: elimination of exogenous alkali. Diet provides a net load of K+ and unmeasured anions. After these organic anions are metabolized to neutral end products, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> is formed (left). Once HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reacts with endogenously produced H+ (made from neutral precursors such as glucose), it is eliminated as CO2+H2O. The renal excretion of citrate anions produced in this metabolic process is promoted by the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> load and/or K+ (bottom).

The present study was designed to evaluate mechanisms that eliminate a progressively increasing alkali load. Two groups of rats were studied; one group consumed a regular alkaline ash diet, and the second group consumed a low-alkali diet. Pair feeding was an essential component of the protocol. The incremental exogenous alkali load was given by the intraperitoneal (IP) route to bypass the uncertainties of absorption in the gastrointestinal tract (see METHODS). With respect to total unmeasured anion excretion, two were singled out for more detailed evaluation. Our rationale was that each one represented a significant proportion of the total unmeasured anions when alkali was provided and, also, being trivalent (citrate) or divalent [2-oxoglutarate (2-OG)], they could chelate ionized Ca2+ in the urine (6).

Our results indicate that the mechanisms to remove lower doses of alkali decreased the rate of excretion of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and increased the rate of excretion of total unmeasured anions. Somewhat larger doses of alkali primarily augmented the excretion of citrate. Two processes seemed to play a role in citrate excretion. First, there was an increased net synthesis of citrate, as reflected by a higher concentration of citrate in plasma. Second, the fractional excretion of citrate rose from <10 to ~70%. A further increase in alkali intake caused the excretion of 2-OG to become quantitatively important. Again, two processes were involved. First, there was enhanced production of 2-OG, because its concentration in plasma rose. Second, renal secretion of 2-OG was required to achieve these high rates of excretion (its fractional excretion was ~400%). Bicarbonaturia became a quantitatively important form of alkali elimination only when alkali intake was very large.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Rats

Rats were cared for in accordance with the guidelines of the Canadian Council on Animal Care. St. Michael's Hospital Animal Care Committee approved the study protocol.

Procedure

Adult male Wistar rats (300-400 g) were housed in individual metabolic cages and given 20 ml of 5% sucrose to drink at 0900 and 1600 on the experimental days. The amounts of food given and remaining were weighed daily. The completeness of urine collection was assessed by examining the creatinine excretion rate (5); thymol was added as a urine preservative. Blood was drawn under light anesthesia before and after the balance period.

Experimental Protocols

Because the regular laboratory rodent diet has more listed cations than anions (Table 1), it contains an appreciable unknown anion load. Many of these unknown anions are absorbed and converted by metabolism to HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, so they constitute a daily alkali load (22). For these reasons, we also studied a group of rats that consumed a low-alkali diet. By necessity, this diet was also low in K+. When alkali was given by adding it to the drinking water, only 76 ± 12% (means ± SE) of this alkali was ingested even when the drinking water was supplemented with 5% sucrose. Similarly, rats consuming their regular chow ingested only 83 ± 13% of their daily load of NaHCO3. This ingestion was even lower if the alkaline salts were added to the drinking water in hypertonic form. Because 100% of the alkali had to be absorbed relatively early in the 24-h period to achieve 24-h balances, we elected to use the IP route of alkali administration.

                              
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Table 1.   Diet composition

Low-alkali diet. The experimental period (2 days) was preceded by a 2-day accommodation to the diet. One control group (n = 6) continued to consume it without supplements on each of the 2 experimental days. Two other groups of 6 rats each received 3,000 µmol KCl or NaCl in 2 equally divided doses by the IP route to control for Na+ or K+ inputs.

The alkali load was given as a hypertonic solution (300 mM) by the IP route on each of the 2 experimental days. The doses were (in µmol) 750 (n = 12), 1,125 (n = 8), 1,500 (n = 12), 2,250 (n = 6), or 3,000 (n = 10). The 1,125-, 1,500-, and 2,250-µmol doses were divided into two equal portions and given at 0900 and 1600 to minimize pulse increase in the plasma HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration. The 3,000-µmol dose was divided into three doses given at 0900, 1300, and 1700. The results are reported for day 2 of KHCO3 supplementation.

Regular laboratory rat diet. The design of these experiments was similar; pair-fed rats consumed 18 ± 2 g of regular chow (n = 9). Rats in the experimental groups were given (in µmol) 750 (n = 6), 1,500 (n = 6), or 3,000 (n = 6) of NaHCO3 by the IP route as described for the previous protocol. An additional group (n = 6) received 3,000 µmol of NaCl to control for the Na+ load.

Analytic techniques. Na+ and K+ in plasma and urine were determined by flame photometry, whereas Cl- was determined by electromimetic titration. Blood-gas analysis was performed at 37°C with a digital pH/blood-gas analyzer (model 178 blood/pH analyzer; Corning). The concentration of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> was calculated from the pH and PCO2 by using a solubility factor of 0.0301 (plasma) or 0.0309 (urine) and a pK' adjusted for ionic strength (22). NH<UP><SUB>4</SUB><SUP>+</SUP></UP>, citrate, creatinine, Ca2+, Mg2+, HPO<UP><SUB>4</SUB><SUP>2−</SUP></UP>, and SO<UP><SUB>4</SUB><SUP>2−</SUP></UP> were measured as previously described (4, 17). The concentrations of citrate and 2-OG were measured in a 5% perchloric acid extract of plasma; each assay was performed on the experimental day (2).

Calculations

Total unmeasured anions. This total includes citrate and 2-OG and was calculated as the sum of the excretion of the measured cations (Na+ + K+ + Ca2+ + Mg2+ + NH<UP><SUB>4</SUB><SUP>+</SUP></UP>) minus measured anions (Cl- + HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>+ HPO<UP><SUB>4</SUB><SUP>2−</SUP></UP>+ SO<UP><SUB>4</SUB><SUP>2−</SUP></UP>) in microequivalent terms.

Urinary unmeasured anion excretion rate that reflects elimination of alkali load. This excretion rate was the total unmeasured anion excretion rate per day with an alkali supplement minus the daily excretion of total unmeasured anions with no alkali supplement while the low-alkali diet was consumed.

Total alkali excretion. Net acid and total unmeasured anion excretions were measured in rats that did and did not receive alkali supplements. The difference in these excretion rates compared with alkali-supplemented rats was used to calculate how much alkali was eliminated by the process of a decrease in net acid excretion and by an increase in the excretion of unmeasured anions.

Fractional excretion of citrate and 2-OG. The fractional excretion of citrate and 2-OG was measured in urine collected over 4 h and midpoint blood collected from an identically treated subgroup to avoid studying anesthetized rats. The blood acid-base parameters were comparable at 2 and 24 h. The fractional excretion was calculated as
100 × (urine/plasma)<SUB>citrate</SUB>/(urine/plasma)<SUB>creatinine</SUB>

Statistical Analysis

Results are reported as means ± SE. Statistical analysis was performed by using an unpaired Student's t-test. A P value <0.05 was considered to be statistically significant.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Rats fed the low-alkali diet had a plasma pH, PCO2, and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>concentration in the normal range (Table 2). Although these values tended to be lower than corresponding values in rats fed their usual chow, the differences were not statistically significant. Over the wide range of alkali input, there were only small changes in the net appearance of SO<UP><SUB>4</SUB><SUP>2−</SUP></UP> and H2PO<UP><SUB>4</SUB><SUP>−</SUP></UP>+HPO<UP><SUB>4</SUB><SUP>2−</SUP></UP> (Tables 3 and 4).

                              
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Table 2.   Values in blood of fed rats given intraperitoneal alkali


                              
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Table 3.   Effect of a graded alkali load on its disposal in rats fed a low-alkali diet


                              
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Table 4.   Effect of a graded alkali load on its disposal in rats fed their usual laboratory diet

Rats consuming the low-alkali diet had the highest rate of excretion of net acid and the lowest rate of excretion of total unmeasured anions, citrate, and 2-OG. With the lower doses of alkali, the decline in net acid plus the rise in the daily total unmeasured anion excretion were somewhat lower than the rise in alkali intake (Fig. 2). At these lower doses, alkali was eliminated primarily by lowering the excretion of NH<UP><SUB>4</SUB><SUP>+</SUP></UP>; there were only small increments in the excretion of total unmeasured anions (Table 3). TA excretion declined by a much smaller amount because of the rise in urine pH from 6.3 to 6.9. Notably, there was only a small rise in the excretion of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> with the 750-, 1,500-, and 2,250-µmol KHCO3 loads. In contrast, there was an appreciable rise in the rate of excretion of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> at the largest dose of KHCO3 (3,000 µmol), perhaps related to a rapid absorption rate from the peritoneal cavity.


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Fig. 2.   Comparison of the alkali load and renal excretion of alkali (for details, see Tables 3 and 4). Solid line, line of identity for the 2 parameters. Rats virtually eliminated the total alkali dose in their 24-h urine output. Alkali elimination was equal to the fall in net acid excretion+rise in unmeasured anion excretion.

The remaining studies were carried out in rats that consumed their usual diet. These rats had a higher excretion of citrate and total unmeasured anions than did alkali-loaded rats fed the low-alkali diet. The rise in citrate excretion was in part due to an increased filtered load and a rise in its fractional excretion (peak ~70% of its filtered load; Table 5). With the 750 µeq of NaHCO3 load, the major pathway for alkali elimination was a further rise in the excretion of total unmeasured anions. With 1,500 µeq of NaHCO3, there was a large increment in 2-OG excretion (total 800 ± 64 µeq/day) to values approaching those of citrate (976 ± 124 µeq/day; Table 4; Fig. 3). Interestingly, the fractional excretion of 2-OG approached 400%, and there was also an increase in the plasma level of 2-OG (Table 5). The acid-base status in plasma reported in Table 1 was similar in rats used to obtain plasma levels of citrate and 2-OG. Again, bicarbonaturia became prominent with the largest alkali dose.

                              
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Table 5.   Excretion of citrate and 2-oxoglutarate



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Fig. 3.   Effect of an alkali load on the excretion of acids and bases. Alkali load (in µeq/day) is shown on the x-axis, and the urinary excretions (in µeq/day) are shown on the y-axis. open circle , Total unmeasured anions; black-triangle, NH<UP><SUB>4</SUB><SUP>+</SUP></UP>; , citrate; *, 2-oxoglutarate.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

There have been many studies in which acid or alkali loads were given to rats to measure the rates of excretion of net acid and total unmeasured anions in general as well as citrate in particular (e.g., reviewed in Refs. 3, 8, 19, 25). What was not addressed in previous studies was the sensitivity of each of these response elements to an alkali load while a low-alkali diet was consumed. Furthermore, it was not clear what renal mechanisms (secretion and/or reabsorption) participated in the excretion of individual organic anions, because these processes were obscured in 24-h urine collections. Another emphasis of the present study was to evaluate how the defense of acid-base balance would affect the composition of urine with respect to the risk of formation of kidney stones.

Alkali is present in the diet, primarily as K+ salts of organic anions; its source is usually fruits and vegetables. Therefore, to remove alkali from the diet, one must reduce its K+ content. To control for the separate effects of K+ depletion and alkali removal, two control groups were employed. The first received KCl in equimolar amounts to KHCO3. The second control group received NaCl instead of KCl to control for the Cl- load. In all cases, the acid-base composition of the urine of rats receiving KCl or NaCl was similar to that in the non-alkali-treated rats fed the low-alkali diet. Hence, the effects of KHCO3 administration were likely due to its alkali rather than its K+ load.

The traditional view of acid-base balance focuses on the production of acids (sulfuric and phosphoric acids) and H+ removal by the renal excretion of net acid (14). This view ignores the large net alkali load of the diet despite the fact that all the urinary indicators of net H+ production (SO<UP><SUB>4</SUB><SUP>2−</SUP></UP> + inorganic HPO<UP><SUB>4</SUB><SUP>2−</SUP></UP> + organic anions) exceed net acid excretion by ~10-fold in the steady state in rats (Table 4; Refs. 8, 22, 25). Part of the reason for excluding total urine unmeasured anions in the net acid excretion formula was the uncertainty as to whether all of them were involved in acid-base balance. For example, some of these anions may be ingested and excreted unchanged as their Na+ or K+ salts; they do not participate in acid-base balance. By giving rats NH4Cl and measuring H+ balance, Lin et al. (22) demonstrated that ~90% of these total unmeasured urinary anions represented the overall response to eliminating the daily alkali load. They also suggested that the large nonrenal source of alkali needed to achieve acid-base balance was not from bone (low daily excretion of Ca2+; Tables 3 and 4). They concluded that there were two independently regulated renal processes to achieve acid-base balance. The quantitatively smaller process was acid balance as reflected by the excretion of net acid. Base balance, the quantitatively larger process, was primarily the response to dietary alkali. It resulted in the excretion of a family of unmeasured anions with pK < pH 5; these are represented by the term potential HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in the urine (Eq. 1).

All alkali supplements were eliminated in the 24-h period of observation, except in rats fed the low-alkali diet and given <1,500 µmol of KHCO3 (Fig. 2). This small positive alkali balance might be due to a titration of H+ that was retained when the diet was devoid of its alkali supplements. In rats fed the low-alkali diet, the most sensitive major response to the alkali load was a decline in the excretion of NH<UP><SUB>4</SUB><SUP>+</SUP></UP>; with the higher alkali doses, there was a rise in excretion of total unmeasured anions (Table 3; Fig. 3). With the use of 3,000 µeq of alkali, there was now evidence of an appreciable increment in the rate of excretion of citrate and a smaller rise in the rate of excretion of 2-OG (Table 3; Fig. 3).

When rats were fed their regular chow, the intake of alkali was large enough to have a near-maximal rate of excretion of citrate (948 µeq/day). Two mechanisms participated in this response. First, there was an increased net production of citric acid, as evidenced by the near doubling of its concentration in plasma. Second, the reabsorption of filtered citrate was inhibited because its fractional excretion rose to ~70% (Table 5). Even larger alkali loads were required to augment the excretion of 2-OG (800 µeq/min; Table 4). Again, two mechanisms were involved in this process. First, more 2-OG was produced because there was a twofold rise in its concentration in plasma. Second, there was a net renal secretion of 2-OG because its fractional excretion was ~400% (Table 5). Bicarbonaturia rose by an appreciable amount in rats that received the largest doses of alkali (Tables 3 and 4). This was not simply due to an expansion of extracellular fluid volume (7), because rats given equimolar NaCl did not have an appreciable rise in their rate of excretion of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (results not shown).

There is a much smaller daily rate of excretion of total unmeasured anions in humans than in rats; in general, this excretion rate is quantitatively similar to that of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (20, 21, 24). This lower total unmeasured anion excretion rate could reflect the much lower alkali load per kilogram of body weight in humans compared with rats. Nevertheless, there are qualitative similarities between the rate in humans and that in rats. For example, citrate excretion also declines markedly with an acid load (9) and rises appreciably with an alkali load (29). In the setting of prolonged fasting in obese human subjects, there is a very high rate of excretion of ketoacid anions (18). The rate of ketoacid anion excretion is also markedly decreased with an acid load (15, 17) and increased with an alkali load (27). These changes in excretion rates involved changes in both renal reabsorption and plasma levels (17). Another example of modulation of the rate of excretion of unmeasured anions (potential HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>) by acid-base factors was suggested by Kamel et al. (16). These authors proposed that acid-base balance was achieved in patients with type IV renal tubular acidosis by having parallel decreases in the rate of excretion of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and unmeasured (organic) anions (potential HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>). Hence, the physiology described in this paper does not appear to be unique to rats.

Physiological Perspectives

We identified two advantages of minimizing bicarbonaturia with an alkali load. First, having a urine pH ~6 could decrease the likelihood of nephrolithiasis by diminishing the risk of precipitation of Ca3(PO4)2 and uric acid in the urinary tract (1, 6). To form Ca3(PO4)2, ionized Ca2+ must react with divalent inorganic HPO<UP><SUB>4</SUB><SUP>2−</SUP></UP> (the second pK of inorganic HPO<UP><SUB>4</SUB><SUP>2−</SUP></UP> is in the 6.7-6.8 range in urine, Ref. 28). Patients who have a defect in distal H+ secretion (distal renal tubular acidosis) and a higher urine pH have an increased risk of nephrolithiasis. One risk factor is acidemia because it leads to a diminished rate of excretion of citrate (trivalent organic anions chelate ionized Ca2+) (6).

The second potential role for minimizing bicarbonaturia and/or having a higher urine pH concerns the regulation of the excretion of K+ (12). Kaliuresis is augmented when the urine pH is high and aldosterone acts (4). The main reason for a higher rate of excretion of K+ in this setting is a higher concentration of K+ in the lumen of the cortical collecting duct (4, 30). Lin et al. (23) demonstrated that the rate of excretion of K+ and the transtubular K+ concentration ratio rose appreciably when bicarbonaturia and/or a more alkaline luminal pH was present during prolonged fasting. This high transtubular K+ concentration ratio could not be attributed to enhanced excretion of Na+ or anions that were not reabsorbed in the cortical collecting duct (ketoacid anions). Lin et al. speculated that distal delivery of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and/or a higher luminal fluid pH was the means by which the NaCl-retaining (little HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> delivery) or kaliuretic actions (more distal delivery of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> of aldosterone) could be selected (12). Hence, having unmeasured anions rather than HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> appear in the urine could be important for the fine-tuning of the kaliuresis in subjects in which aldosterone actions are needed for the reabsorption of NaCl without sacrificing K+ balance.

Concluding Remarks

The traditional analysis of acid-base balance focuses primarily on acids and largely ignores the disposition of dietary alkali; the excretion of total unmeasured anions (potential HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>) in general and citrate, in particular, are not included in the net acid excretion formula (14). In fact, alkali loads are dealt with by both decreasing NH<UP><SUB>4</SUB><SUP>+</SUP></UP> excretion and increasing total unmeasured anion excretion. In quantitative terms in rats, modulation of total organic unmeasured anions excretion is 10-fold larger than changes in net acid excretion.

With either an acid or an alkali load, the overall purpose seems to be avoiding large swings in urine pH (6). Minimizing bicarbonaturia despite large changes in alkali intake could be important in lessening the risk of kidney stone formation and permitting HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and/or luminal pH to regulate K+ excretion when aldosterone acts. Hence, a better understanding is possible when acid-base balance is viewed from the perspective of integrative physiology.


    ACKNOWLEDGEMENTS

We are grateful to Dr. Kamel S. Kamel for helpful discussions and suggestions during the preparation of this manuscript. We are also indebted to Stella Tang and Chee Keong Chong for technical assistance and Jolly Mangat for secretarial assistance.


    FOOTNOTES

This research was supported by Canadian Institutes for Health Research Grant MT-15485.

Address for reprint requests and other correspondence: M. L. Halperin, Lab No. 1, Research Wing, St. Michael's Hospital, 38 Shuter St., Toronto, Ontario, Canada M5B 1A6 (E-mail: mitchell.halperin{at}utoronto.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published January 8, 2002;10.1152/ajprenal.00006.2001

Received 9 January 2001; accepted in final form 18 December 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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