The kidneys play two major roles in the maintenance of normal acid-base balance: reabsorption of HCO3− and excretion of H+. The first role of the kidneys is to reabsorb the filtered HCO3− so that this important extracellular buffer is not excreted in urine. The second role of the kidneys is to excrete fixed H+ that is produced from protein and phospholipid catabolism. There are two mechanisms for excretion of this fixed H+: (1) excretion of H+ as titratable acid (i.e., buffered by urinary phosphate) and (2) excretion of H+ as NH4+. Excretion of H+ by either mechanism is accompanied by synthesis and reabsorption of new HCO3−. The purpose of synthesis and reabsorption of new HCO3− is to replenish the HCO3− stores that were used in buffering fixed H+.
Reabsorption of Filtered HCO3−
Almost 99.9% of the filtered HCO3− is reabsorbed, ensuring that the major extracellular buffer is conserved, rather than excreted. The reabsorption rate can be calculated (as explained in Chapter 6) by comparing the filtered load of HCO3− with the excretion rate of HCO3−. If the glomerular filtration rate (GFR) is 180 L/day and the plasma HCO3− concentration is 24 mEq/L, then the filtered load is 4320 mEq/day (180 L/day × 24 mEq/L). The measured excretion rate of HCO3− is merely 2 mEq/day. Therefore, the reabsorption rate of HCO3− is 4318 mEq/day, which is 99.9% of the filtered load. Most filtered HCO3− reabsorption occurs in the proximal tubule, and only small quantities are reabsorbed in the loop of Henle, distal tubule, and collecting duct.
Mechanism of HCO3− Reabsorption in the Proximal Tubule
Figure 7-5 is a diagram of a cell of the early proximal tubule, where filtered HCO3− is reabsorbed. Reabsorption of filtered HCO3− involves the following steps and includes conversion of HCO3− to CO2 in the lumen, diffusion of CO2 into the cell, conversion back to HCO3− in the cell, and reabsorption of HCO3− into the blood:
Figure 7–5 Mechanism for reabsorption of filtered HCO3− in a cell of the proximal tubule. ATP, Adenosine triphosphate.
1. The luminal membrane contains an Na+-H+ exchanger, which is one of several Na+-dependent secondary active transport mechanisms in the early proximal tubule. As Na+ moves from the lumen into the cell down its electrochemical gradient, H+ moves from the cell into the lumen against its electrochemical gradient.
2. The H+ secreted into the lumen combines with filtered HCO3− to form H2CO3. The H2CO3 then decomposes into CO2 and H2O, catalyzed by a brush border carbonic anhydrase. (Carbonic anhydrase inhibitors such as acetazolamide inhibit the reabsorption of filtered HCO3− by interfering with this step.) The CO2 and H2O that are formed in this reaction readily cross the luminal membrane and enter the cell.
3. Inside the cell, the reactions occur in reverse. CO2 and H2O recombine to form H2CO3, catalyzed by intracellular carbonic anhydrase. H2CO3 is converted back to H+ and HCO3−. The fates of the H+and HCO3− are different. H+ is secreted by the Na+-H+ exchanger to aid in the reabsorption of another filtered HCO3−. The HCO3− is transported across the basolateral membrane into the blood (i.e., the HCO3− is reabsorbed) by two mechanisms: Na+-HCO3− cotransport and Cl−-HCO3− exchange. Special features of the mechanism for reabsorption of filtered HCO3− include the following:
The process results in net reabsorption of Na+ and HCO3−. Thus, a portion of the Na+ reabsorption in the proximal tubule is linked directly to the reabsorption of filtered HCO3−. (The rest of the Na+reabsorption is linked to reabsorption of glucose, amino acids, Cl−, and phosphate.)
There is no net secretion of H+ via this mechanism. Each H+ secreted by the Na+-H+ exchanger in the luminal membrane combines with a filtered HCO3− to form CO2 and H2O, which enter the cell and are converted back to H+ and HCO3−. The H+ is recycled across the luminal membrane on the Na+-H+ exchanger to reabsorb more filtered HCO3−.
Because there is no net secretion of H+ by this mechanism, it produces little change in tubular fluid pH.
Effect of Filtered Load of HCO3−
The filtered load of HCO3− is the product of GFR and the plasma HCO3− concentration. Over a wide range of filtered loads, virtually all of the HCO3− is reabsorbed. However, when the plasma HCO3−concentration is greater than 40 mEq/L, the filtered load becomes so high that the reabsorption mechanism is saturated; any filtered HCO3− that cannot be reabsorbed is excreted. For example, in metabolic alkalosis where the blood HCO3− concentration is elevated, restoration of normal acid-base balance requires excretion of the excess HCO3− in the urine. This is accomplished because, as the concentration of HCO3− in the blood increases, the filtered load increases and exceeds the reabsorptive capacity. The nonreabsorbed HCO3− is excreted, lowering the blood HCO3− concentration to normal.
Effect of Extracellular Fluid Volume
Most of the filtered HCO3− is reabsorbed in the proximal tubule, where changes in ECF volume alter isosmotic reabsorption via changes in the Starling forces in the peritubular capillaries (see Chapter 6). Because HCO3− is part of this isosmotic reabsorption, changes in ECF volume alter HCO3− reabsorption in a predictable way. For example, ECF volume expansion inhibits isosmotic reabsorption in the proximal tubule and, therefore, inhibits HCO3−reabsorption. Conversely, ECF volume contraction stimulates isosmotic reabsorption in the proximal tubule and stimulates HCO3− reabsorption.
A second mechanism, involving angiotensin II, participates in the response of HCO3− reabsorption to ECF volume contraction. Recall that decreases in ECF volume activate the renin–angiotensin II–aldosterone system. Angiotensin II stimulates Na+-H+ exchange in the proximal tubule, thus stimulating HCO3− reabsorption and increasing the blood HCO3− concentration. This mechanism explains the phenomenon of contraction alkalosis, which literally means metabolic alkalosis that occurs secondary to ECF volume contraction. Contraction alkalosis occurs during treatment with loop diuretics orthiazide diuretics, and it is a complicating factor in the metabolic alkalosis caused by vomiting. Contraction alkalosis is treated by infusing isotonic NaCl to restore ECF volume.
Effect of PCO2
Chronic changes in PCO2 alter the reabsorption of filtered HCO3− and explain the phenomenon of renal compensation for chronic respiratory acid-base disorders. Increases in PCO2 increase the reabsorption of HCO3−, and decreases in PCO2 decrease the reabsorption of HCO3−.
The mechanism underlying the effect of CO2 is not completely understood. One explanation, however, involves the supply of CO2 to the renal cells. In respiratory acidosis, the PCO2 is increased. Because more CO2 is available in the renal cells to generate H+ for secretion by the Na+-H+ exchanger, more HCO3− can be reabsorbed. Thus, the plasma HCO3− concentration increases, which increases the arterial pH (a compensation). In respiratory alkalosis, the PCO2 is decreased. As less CO2 is available in the renal cells to generate H+ for secretion, less HCO3− is reabsorbed. In this case, the plasma HCO3−concentration decreases, which decreases the arterial pH (a compensation).
Excretion of H+ as Titratable Acid
By definition, titratable acid is H+ excreted with urinary buffers. Inorganic phosphate is the most important of these buffers because of its relatively high concentration in urine and its ideal pK. Recall that there is a significant amount of phosphate in urine because only 85% of the filtered phosphate is reabsorbed; 15% of the filtered phosphate is left to be excreted as titratable acid.
Mechanism of Excretion of Titratable Acid
Titratable acid is excreted throughout the nephron, but primarily in the α-intercalated cells of the late distal tubule and collecting ducts. The cellular mechanism for this process is illustrated in Figure 7-6 and is described as follows:
Figure 7–6 Mechanism for excretion of H+ as titratable acid. ATP, Adenosine triphosphate.
1. The luminal membrane of α-intercalated cells of the late distal tubule and collecting ducts has two primary active transport mechanisms for secreting H+ into tubular fluid. The first mechanism for H+secretion is H+ ATPase,which is stimulated by aldosterone. Aldosterone not only acts on the principal cells in stimulation of Na+ reabsorption and K+ secretion but also stimulates H+ secretion in the α-intercalated cells. The other mechanism for H+secretion is H+-K+ ATPase, the transporter responsible for K+ reabsorption in α-intercalated cells (see Chapter 6). In the lumen, the secreted H+ combines with the A− form of the phosphate buffer, HPO4−2, to produce the HA form of the buffer, H2PO4−. H2PO4− is titratable acid, which is excreted.
For this mechanism to be useful, it is essential that most of the filtered phosphate be in the form that can accept an H+ (i.e., in the HPO4−2 form). Is this so? By calculating the relative concentrations of HPO4−2 and H2PO4− at pH 7.4, it can be confirmed that the concentration of HPO4−2 is almost fourfold the concentration of H2PO4− in the glomerular filtrate (pH = pK + log HPO4−2/H2PO4−, where pK = 6.8; at pH 7.4, HPO4−2/H2PO4− = 3.98).
2. The H+ secreted by the H+ ATPase is produced in the renal cells from CO2 and H2O, which combine to form H2CO3 in the presence of intracellular carbonic anhydrase. H2CO3 dissociates into H+, which is secreted, and HCO3−, which is reabsorbed into the blood via Cl−-HCO3− exchange.
3. For each H+ excreted as titratable acid, one new HCO3−is synthesized and reabsorbed. This new HCO3− replenishes extracellular HCO3− stores, which previously had been depleted from buffering fixed H+. Because the generation, or synthesis, of new HCO3− is an ongoing process, HCO3− is continuously replaced as it is used for buffering the fixed acids produced from protein and phospholipid catabolism.
Amount of Urinary Buffer
The amount of H+ excreted as titratable acid depends on the amount of urinary buffer available. Although it may not be immediately obvious why this is so, the underlying principle is that the minimum urine pH is 4.4. Because blood pH is 7.4, a urine pH of 4.4 represents a 1000-fold difference in H+ concentration across the renal tubular cells. This 1000-fold difference is the largest concentration gradient against which H+ can be secreted by the H+ATPase. When the urine pH is reduced to 4.4, net secretion of H+ ceases.
To understand this principle, it is important to distinguish between the amount of H+excreted and the value for urine pH. To illustrate this distinction, consider the following two examples: First, imagine that there are no urinary buffers. In that case, the first few H+ secreted, finding no urinary buffers, would be free in solution and cause the pH to decrease to the minimum value of 4.4, and thereafter, no additional H+ could be secreted. Next, imagine that urinary buffers are plentiful. In that case, large quantities of H+ could be secreted and buffered in urine before the pH would be reduced to 4.4.
This point is further illustrated in Figure 7-7. Figure 7-7A shows the range of tubular fluid pH (shaded area) superimposed on the phosphate titration curve. Begin with the glomerular filtrate, which has a pH of 7.4: Both HPO4−2and H2PO4− are present, with the concentration of HPO4−2 considerably higher than that of H2PO4−. As H+ is secreted into tubular fluid, it combines with the HPO4−2 form of the phosphate buffer and converts it to H2PO4−. In the linear portion of the titration curve (pH 7.8 to 5.8), the addition of H+ to tubular fluid causes the pH to decrease only modestly. However, once most of the HPO4−2 has been converted to H2PO4−, further secretion of H+ causes the tubular fluid pH to decrease precipitously to 4.4. At that point, no additional H+ can be secreted. The only way to secrete more H+ would be to provide more HPO4−2. Thus, the amount of H+ excreted as titratable acid depends on the amount of available urinary buffer.
Figure 7–7 Comparison of effectiveness of phosphate (A) and creatinine (B) as urinary buffers. The pK of the phosphate buffer is 6.8; the pK of the creatinine buffer is 5.0. The shaded areas show the total amount of H+ that is secreted into tubular fluid between the glomerular filtrate (pH 7.4) and the final urine (pH 4.4).
pK of Urinary Buffers
The pK of the urinary buffers also affects the amount of H+ that is excreted. Robert Pitts demonstrated the importance of pK by comparing the effectiveness of creatinine (with a pK of 5.0) as a urinary buffer with the effectiveness of phosphate (with a pK of 6.8). He found that for a given quantity of urinary buffer, more H+ was excreted when the buffer was phosphate than when the buffer was creatinine (see Fig. 7-7).
The difference in the amount of H+ excreted is attributed to the different pKs of the two buffers. Remember that phosphate is an almost ideal urinary buffer. The linear range of its titration curve overlaps almost perfectly with the range of tubular fluid pH. In Figure 7-7A, the shaded area under the phosphate titration curve represents the total amount of H+ secreted as the tubular fluid pH decreases from pH 7.4 in glomerular filtrate to pH 4.4 in the final urine.
Figure 7-7B shows the titration curve for creatinine. Again, the pH of tubular fluid ranges from 7.4 (in glomerular filtrate) to 4.4 in the final urine. The pK of creatinine, at 5.0, is close to the minimum urine pH; therefore, the total amount of H+ that can be secreted (shaded area) before the pH falls to 4.4 is much less than the amount secreted when phosphate is the buffer.
Excretion of H+ as NH4+
If titratable acid were the only mechanism for excreting H+, then excretion of fixed H+ would be limited by the amount of phosphate in urine. Recall that fixed H+ production from protein and phospholipid catabolism is approximately 50 mEq/day. On average, however, only 20 mEq/day of this fixed H+ is excreted as titratable H+. The remaining 30 mEq/day is excreted by a second mechanism, as NH4+.
Mechanism of Excretion of H+ as NH4+
Three segments of the nephron participate in the excretion of H+ as NH4+: the proximal tubule, the thick ascending limb of Henle’s loop, and α-intercalated cells of the collecting ducts. In the proximal tubule,NH4+ is secreted by the Na+-H+ exchanger. In the thick ascending limb, NH4+ that was previously secreted by the proximal tubules is reabsorbed and added to the corticopapillary osmotic gradient. In the α-intercalated cells of the collecting duct, NH3and H+ are secreted into the lumen, combine to form NH4+, and are excreted.
Proximal tubule. In the cells of the proximal tubule, the enzyme glutaminase metabolizes glutamine to glutamate and NH4+ (Fig. 7-8). The glutamate is metabolized to α-ketoglutarate, which is ultimately metabolized to CO2and H2O and then to HCO3−. The HCO3− is reabsorbed across the basolateral membrane into the blood via Na+- HCO3− cotransport. Similar to the titratable acid mechanism, this HCO3− is newly synthesized and helps to replenish HCO3− stores in the ECF. For each NH4+ generated (and ultimately excreted), one new HCO3− is reabsorbed.
Figure 7–8 Mechanism of excretion of H+ as NH4+. In the proximal tubule, NH3 is produced from glutamine in the renal cells. H+ is secreted by Na+-H+ exchanger and NH3 diffuses into the lumen. NH4+ is reabsorbed by Na+-K+-2Cl− cotransporter in the TALH and deposited in the medullary interstitial fluid (not shown). In the collecting ducts, NH3 diffuses from the medullary interstitium into the lumen, combines with secreted H+ in the lumen, and is excreted as NH4+. ATP, Adenosine triphosphate. TALH, thick ascending limb of loop of Henle.
The fate of the NH4+ requires several additional steps. In the proximal tubule cell, NH4+ is in equilibrium with NH3 and H+. The NH3 form, being lipid soluble, diffuses down its concentration gradient from cell to lumen, and the H+ is secreted into the lumen on the Na+-H+ exchanger. Once in the lumen, NH3 and H+ recombine into NH4+. The fate of the NH4+, once in the lumen of the proximal tubule, is as follows. A portion of the NH4+ is excreted directlyin the urine. The remainder follows a circuitous route and is excreted indirectly: It is first reabsorbed by the thick ascending limb, then deposited in the medullary interstitial fluid, and then secreted from the medullary interstitial fluid into the collecting ducts for final excretion.
Thick ascending limb. As previously noted but not shown in Figure 7-8, a portion of the NH4+ that is secreted in the proximal tubule and delivered to the loop of Henle is reabsorbed by the thick ascending limb. At the cellular level, NH4+ is reabsorbed by substituting for K+ on the Na+-K+-2Cl− cotransporter. As a result of this substitution, NH4+ participates in countercurrent multiplication (much like NaCl) and is concentrated in the interstitial fluid of the inner medulla and papilla of the kidney.
Collecting duct. As described for the titratable acid mechanism, the luminal membrane of α-intercalated cells of the collecting duct contains two transporters that secrete H+ into tubular fluid (see Fig. 7-8): H+ ATPase and H+-K+ATPase. The H+ ATPase is stimulated by aldosterone.
As H+ is secreted into tubular fluid, NH3 diffuses from its high concentration in the medullary interstitial fluid into the lumen of the collecting duct, where it combines with the secreted H+ to form NH4+. The question that arises is Why does only the NH3 form of the NH3/NH4+buffer diffuse from the medullary interstitium? The answer is that although both NH4+ and NH3 are present in the medullary interstitial fluid, only the NH3 form is lipid soluble and can diffuse across the collecting duct cells into tubular fluid. Once in the tubular fluid, NH3 combines with the secreted H+ to form NH4+. NH4+ is not lipid soluble and, thus, is trapped in the tubular fluid and excreted. The overall process is termed diffusion trapping because the lipid-soluble form of the buffer (NH3) diffuses and the water-soluble form of the buffer (NH4+) is trapped and excreted.
Note that the source of the H+ secreted by the α-intercalated cells is CO2 and H2O. For each H+ produced in the cells and secreted, one new HCO3− is synthesized and reabsorbed. As with the titratable acid mechanism, this new HCO3− helps to replenish depleted HCO3− stores.
Effect of Urinary pH on Excretion of NH4+
As urinary pH decreases, the excretion of H+ as NH4+ increases. The effect of urine pH on the excretion of NH4+ is advantageous: In acidosis, where urine pH tends to be low, there are large quantities of H+ to be excreted. The mechanism underlying the effect of urine pH is based on diffusion trapping of NH3/NH4+. As the pH of urine decreases, more of the urinary buffer is present in the NH4+ form and less is present in the NH3 form. The lower the luminal concentration of NH3, the larger the gradient for diffusion of NH3 from medullary interstitial fluid into tubular fluid. Thus, the lower the pH of tubular fluid, the greater the amount of NH3 diffusion and the greater the amount of H+ excreted as NH4+.
Effect of Acidosis on NH3 Synthesis
The rate of NH3 synthesis changes, depending on the quantity of H+ that must be excreted. In chronic acidosis, there is an adaptive increase in NH3 synthesis in the cells of the proximal tubule. The mechanism involves a decrease in intracellular pH, which induces the synthesis of enzymes involved in glutamine metabolism. When NH3 synthesis is augmented in this way, more H+ is excreted as NH4+ and more new HCO3− is reabsorbed. For example, in diabetic ketoacidosis, fixed acid production is increased. The ability of the kidneys to excrete this additional fixed acid load is attributable, in large part, to an adaptive increase in NH3 synthesis.
Effect of Plasma K+ Concentration on NH3 Synthesis
Plasma K+ concentration also alters NH3 synthesis. Hyperkalemia inhibits NH3 synthesis and reduces the ability to excrete H+ as NH4+, causing type 4 renal tubular acidosis (RTA). Hypokalemiastimulates NH3 synthesis and increases the ability to excrete H+ as NH4+. These effects are most likely mediated by the exchange of H+ and K+ across renal cell membranes, which in turn alters intracellular pH. In hyperkalemia, K+ enters the renal cells and H+leaves. The resulting increase in intracellular pH inhibits NH3 synthesis from glutamine. In hypokalemia, K+ leaves renal cells and H+ enters. The resulting decrease in intracellular pH stimulates NH3 synthesis from glutamine.
Comparison of Titratable Acid and NH4+ Excretion
On a daily basis, H+ is excreted as both titratable acid and NH4+ so that normally all of the fixed H+ produced from protein and phospholipid catabolism is eliminated from the body (and all of the HCO3− used to buffer that fixed H+is replaced). Table 7-1 summarizes and compares the rates of excretion of H+ as titratable acid and NH4+ in normal persons and in those with different types of metabolic acidosis (i.e., diabetic ketoacidosis and chronic renal failure).
Table 7–1 Comparison of H+ Excretion as Titratable Acid and NH4+
In normal persons eating a relatively high protein diet, approximately 50 mEq of fixed H+ is produced daily. The kidneys excrete all (100%) of the fixed acid that is produced: 40% is excreted as titratable acid (20 mEq/day) and 60% as NH4+ (30 mEq/day).
In persons with diabetic ketoacidosis, fixed acid production may be increased as much as 10-fold, to 500 mEq/day. To excrete this additional acid load, excretion of both titratable acid and NH4+ is increased. NH4+ excretion is increased because acidosis induces the enzymes involved in glutamine metabolism, thereby increasing NH3 synthesis. As more NH3 is produced by the renal cells, more H+ is excreted as NH4+.
It is less apparent why titratable acid excretion is increased. In diabetic ketoacidosis, β-OH butyric acid and acetoacetic acid are overproduced, which causes metabolic acidosis. The salts of these ketoacids (i.e., butyrate and acetoacetate) are themselves filtered and serve as urinary buffers, similar to phosphate, increasing the total amount of H+ excreted as titratable acid.
Chronic renal failure is another cause of metabolic acidosis. A person in chronic renal failure who continues to eat a relatively high protein diet will produce 50 mEq of fixed acid daily. In this disease, there is a progressive loss of nephrons, and the renal mechanisms for excreting fixed acid are severely impaired for two reasons: (1) Titratable acid excretion is reduced because glomerular filtration is reduced, which reduces the filtered load of phosphate and, thus, the amount of phosphate that can serve as a urinary buffer; (2) NH4+ excretion is reduced because synthesis of NH3 is impaired in the diseased nephrons.
Notice that the total fixed acid excretion in chronic renal failure is only 15 mEq/day (10 mEq as titratable acid plus 5 mEq as NH4+), which is much less than the amount of fixed acid produced from protein catabolism (50 mEq/day). In chronic renal failure, the cause of the metabolic acidosis is, in fact, the inability of the kidneys to excrete all of the fixed acid produced daily. Logically, persons with chronic renal failure are placed on a low-protein diet to reduce daily fixed acid production and thereby reduce the demand on the kidneys for fixed acid excretion and new HCO3− reabsorption.