Renal Ammonium Ion Production and Excretion

The maintenance of systemic acid-base balance within relatively narrow limits is essential for life. However, the development of metabolic acidosis, which is characterized by a significant decrease in plasma pH and bicarbonate ions, is a common clinical condition. Metabolic acidosis is caused by the overproduction of acid due to a high protein diet, increased catabolism of endogenous proteins during prolonged fasting, sepsis or cachexia, and various alterations in metabolism such as diabetic ketoacidosis, lactic acidosis, or genetically determined acidurias. It also results from loss of base due to excessive diarrhea or renal defects in bicarbonate reabsorption. The onset of metabolic acidosis triggers an essential adaptive response in the kidney that is characterized by a pronounced increase in catabolism of plasma glutamine and an increased synthesis of bicarbonate and ammonium ions that occur predominantly within the proximal convoluted tubule. These adaptations are sustained, in part, by increased expression of the genes that encode a basolateral glutamine transporter, the mitochondrial glutaminase and glutamate dehydrogenase, and the cytoplasmic phospho enol pyruvatecarboxykinase and by activation of the mitochondrial glutamine transporter, the apical Na ⁺ /H ⁺ exchanger, and the basolateral Na ⁺ -3HCO ₃ ^– co-transporter. The resulting increases in the corresponding activities facilitate the basolateral uptake of glutamine, an increased reabsorption of bicarbonate ions, the increased synthesis of ammonium and bicarbonate ions, and their vectoral transport across the apical and basolateral membranes, respectively. Nearly 80% of the generated ammonium ions are subsequently reabsorbed within the medullary thick ascending limb to produce a high interstitial concentration of ammonium ions within the renal medulla. This gradient provides the driving force for the final transport of ammonium ions into the urine that occurs via specific ammonia channels located within the basolateral and apical membranes of the collecting ducts. All of these steps are finely regulated to ensure that the levels of renal ammonium ion production and excretion are appropriate to sustain normal acid–base balance.

Key Words

ammoniagenesis, acid–base balance, glutaminase, phosphoenolpyruvate carboxykinase, ammonia transporters

Introduction

The maintenance of systemic acid–base balance is essential for survival. Increased renal ammoniagenesis and gluconeogenesis from plasma glutamine constitute an essential physiological response to metabolic acidosis that partially restores acid–base balance. Onset of acidosis triggers a rapid and pronounced increase in extraction and catabolism of plasma glutamine within the renal proximal convoluted tubule. The mitochondrial phosphate-activated glutaminase (GA) catalyzes the initial reaction in the primary pathway for the renal catabolism of glutamine and is a key regulator of the increased renal ammoniagenesis and gluconeogenesis. Glutamine extracted by the kidney is both deamidated and deaminated to yield two ammonium ions. The increased renal ammoniagenesis provides an expendable cation that facilitates the excretion of titratable acid while conserving sodium and potassium ions. In rats and humans, the resulting α-ketoglutarate is primarily converted to glucose. This pathway generates HCO ₃ ^– ions that partially compensate the systemic acidosis. During chronic acidosis, this adaptive response is sustained, in part, by cell-specific increases in expression of various enzymes and transport proteins. The adaptive increases in GA and the cytosolic phospho enol pyruvate carboxykinase (PEPCK) are paradigms for characterization of the mechanisms that mediate the pH-responsive regulation of renal gene expression. The pronounced increase in GA results from stabilization of the GA mRNA, while the more rapid increase in PEPCK is initiated by activating transcription of the PCK1 gene and sustained, in part, by a gradual stabilization of the PEPCK mRNA. A decrease in intracellular pH activates multiple signal transduction pathways within proximal tubule cells that mediate the increased expression of PEPCK and the exocytosis and increased expression of NHE3, the apical Na ⁺ /H ⁺ exchanger. The latter protein contributes to the active translocation of H ⁺ and NH ₄ ⁺ ions into the urine. A significant portion of the ammonium ions, generated in the proximal tubule, is subsequently reabsorbed within the medullary thick ascending limb to produce a high interstitial concentration of ammonium ions within the renal medulla. This gradient provides the driving force for the final transport of ammonium ions into the urine that occurs via specific ammonia channels located within the basolateral and apical membranes of the collecting ducts. All of these steps are subject to fine regulation to ensure that the level of renal ammonium ion production and excretion are appropriate to sustain normal acid–base balance. As a result, excess production of acid or insufficient adaptations lead to various pathophysiologies.

Role of Renal Ammonium Ion Production and Excretion in the Maintenance of Acid–Base Balance

All higher eukaryotes maintain the pH of the extracellular fluid and of various intracellular compartments within narrow limits. For example, in humans, normal arterial plasma pH is set at 7.40±0.04. Blood pH values outside this narrow range define an acidosis (pH<7.36) or an alkalosis (pH>7.44). The relative concentrations of HCO ₃ ^– and CO ₂ are the primary determinants of plasma pH. The reaction of H ⁺ with HCO ₃ ^– to form H ₂ CO ₃ has a pK of 6.1 and thus would appear to be a relatively ineffective buffer at pH 7.4. However, the resulting carbonic acid is rapidly equilibrated with CO ₂ and H ₂ O by multiple carbonic anhydrases. The combined reactions provide an effective buffer system due to the ability of the lungs to maintain a constant CO ₂ concentration through changes in the rate of respiration. A constant blood pH of 7.40 is produced by maintaining a pCO ₂ of 40 mmHg and a blood HCO ₃ ^– concentration of 24 mM. The kidney contributes to the maintenance of a constant blood HCO ₃ ^– concentration by recovering nearly all of the HCO ₃ ^– that is filtered by the glomeruli. In humans, the daily filtered load of HCO ₃ ^– is approximately 4000 mmol.

About 70–80 percent of the filtered HCO ₃ ^– is reabsorbed in the proximal tubule. This process requires the pumping of H ⁺ ions into the tubular lumen via NHE3, the apical Na ⁺ /H ⁺ exchanger, and various H ⁺ -ATPases ( Figure 57.1 ). A luminal carbonic anhydrase uses the H ⁺ ions to protonate the filtered HCO ₃ ^– to form CO ₂ and H ₂ O. After diffusing into the epithelial cells of the proximal tubule, a cytosolic carbonic anhydrase hydrates the CO ₂ to reform HCO ₃ ^– and regenerate the excreted H ⁺ ions. The HCO ₃ ^– ions are then transported across the basolateral membrane by NBC1, a Na ⁺ /3HCO ₃ ^– cotransporter. The overall process of HCO ₃ ^– absorption is inhibited by alkalosis and hypocalcemia and is increased by acidosis, hypercalcemia and by increased levels of angiotensin II. Additional regulation occurs in the distal nephron segments that are responsible for the recovery of the HCO ₃ ^– that remains in the fluid leaving the proximal tubule. The type A intercalated cells within the cortical and outer medullary collecting ducts secrete H ⁺ ions via the apical H ⁺ -ATPase and an electroneutral H ⁺ /K ⁺ -ATPase. The reabsorbed HCO ₃ ^– is subsequently transported across the basolateral membrane by AE1, a HCO ₃ ^– /Cl ^– antiporter.

Catabolic pathways result in a net production of volatile and non-volatile acids. The major acid generated as a product of carbohydrate and fat catabolism is CO ₂ which is reversibly hydrated to form carbonic acid. A normal individual generates approximately 20 moles of CO ₂ per day. However, this acid is volatile and is effectively expelled by the lungs. In contrast, the catabolism of protein also produces non-volatile acids including H ₂ SO ₄ and H ₃ PO ₄ . These are strong acids that rapidly dissociate to generate H ⁺ ions and the corresponding anions. The H ⁺ ions combine with HCO ₃ ^– to form CO ₂ and H ₂ O. However, this process causes a decrease in blood HCO ₃ ^– concentration and a corresponding decrease in blood pH. The average person eating a western diet generates about 70 mmol per day of non-volatile acids from the catabolism of proteins and amino acids. To maintain normal acid–base balance, the kidneys of this individual must accomplish the net synthesis of an equivalent amount of HCO ₃ ^– ions.

The renal production of HCO ₃ ^– is primarily accomplished by the extraction and catabolism of plasma glutamine. This process occurs largely within the proximal convoluted tubule and is primarily initiated by the mitochondrial glutaminase (GA) and glutamate dehydrogenase (GDH). The combined reactions accomplish the deamidation and oxidative deamination of glutamine and generate two ammonium ions and α-ketoglutarate:

The subsequent conversion of α-ketoglutarate to phospho enol pyruvate is accomplished by reactions of the tricarboxylic acid cycle and the phospho enol pyruvate carboxykinase (PEPCK). This process also generates two HCO ₃ ^– and two H ⁺ ions:

The two H ⁺ ions that are produced in these reactions are consumed when the phospho enol pyruvate is either converted to glucose, a neutral molecule, or is oxidized to CO ₂ and H ₂ O. The net effect of the combined reactions is the catabolism of glutamine to yield two NH ₄ ⁺ , two HCO ₃ ^– ions, and one-half molecule of glucose. For this process to result in the net production of HCO ₃ ^– ions, the resulting NH ₄ ⁺ ions must be excreted in the urine. Ammonium ions that are added to the renal venous blood are utilized in the liver to form urea, a process that consumes HCO ₃ ^– ions. Therefore, the amount of ammonium ions excreted in the urine is equivalent to the net production of HCO ₃ ^– ions by the kidney.

Pathways of Renal Ammoniagenesis

During normal acid–base balance, the kidneys extract and metabolize very little of the plasma glutamine. The measured rat renal arterial-venous difference is less than three percent of the arterial concentration of glutamine, whereas only seven percent of the plasma glutamine is extracted by the human kidneys even after an overnight fast. Therefore, renal uptake is significantly less than the fraction of plasma glutamine that is filtered by the glomeruli and enters the lumen of the nephron ( Figure 57.1 ). Most of the filtered glutamine is reabsorbed within the proximal convoluted tubule. This uptake is probably mediated by B ^o AT1, a Na ⁺ -dependent neutral amino acid transporter that is localized to the apical membrane of the small intestine and the renal proximal tubule. Mutations in the SLC6A19 gene that encodes this transporter are associated with Hartnup's disorder. Most of the recovered glutamine is subsequently transported across the basolateral membrane via the LAT2 isoform (SLC7A8) of the Na ⁺ -independent system L family of amino acid transporters. Transporters of this family have a preference for leucine, but also recognize a broad range of neutral amino acids including glutamine. The LAT2 isoform is expressed predominantly in the basolateral membrane of intestinal epithelial cells and the renal proximal tubule. Thus, it functions in the cellular release of glutamine that was absorbed from the intestinal lumen or the glomerular filtrate. However, it also catalyzes an exchange of extracellular and intracellular amino acids that may contribute to basolateral glutamine uptake by intestinal epithelial cells in a post-absorptive state and by the proximal tubule during metabolic acidosis.

Utilization of the small fraction of extracted plasma glutamine requires its transport into the mitochondrial matrix where glutamine is deamidated by a phosphate-activated GA and then oxidatively deaminated by GDH ( Fig. 57.1 ). Glutamine uptake occurs via a mersalyl-sensitive electroneutral uniporter. The mitochondrial glutamine transporter has been purified from rat kidney and was shown by reconstitution in lipid vesicles to be specific for glutamine and asparagine and inhibited by various thiol reagents. Kinetic measurements using isolated rat renal mitochondria indicated that the rate of glutamine transport is not rate limiting for glutamine catabolism. However, the basal level of GA is much greater than what is required to accomplish the basal catabolism of glutamine. Therefore, either the activity of the mitochondrial glutamine transporter or the GA must be largely inhibited or inactivated in vivo during normal acid–base balance to account for the effective reabsorption of glutamine. Finally, during normal acid–base balance, the urine is only slightly acidified. Thus, only two-thirds of the ammonium ions produced from glutamine are trapped in the tubular lumen and are excreted. The remainder is added to the renal venous blood.

Acute Regulation of Renal Ammoniagenesis

Acute onset of a metabolic acidosis produces acute alterations in the interorgan metabolism of glutamine which support the rapid and pronounced changes in glutamine catabolism that occur in the kidney ( Figure 57.2 ). Typically, acute acidosis has been induced by stomach loading rats with 2.0 mmol NH ₄ Cl/100 g body weight. Within 1 to 3 h, the arterial plasma glutamine concentration is increased two-fold, primarily due to an increased release of glutamine from muscle tissue. Significant renal extraction of glutamine becomes evident as the arterial plasma concentration is increased. After 3 h, net extraction reaches 25% of the plasma glutamine, a level that slightly exceeds the percent filtered by the glomeruli. Thus, the direction of the basolateral glutamine transport must be reversed in order for the proximal convoluted tubule cells to extract glutamine from both the glomerular filtrate and the venous blood. Adrenalectomized rats exhibit an impaired ability to increase plasma glutamine and to extract glutamine during onset of acute acidosis, suggesting that this response may be mediated, at least in part, by an adrenal hormone. Within 4 h, plasma HCO ₃ ^– decreased from 28 mM to 17 mM, urine pH decreased from 7.2 to 5.8, and renal ammonium ion excretion increased 6-fold. The prompt acidification of the urine may result from the rapid translocation and acute activation of NHE3, the apical Na ⁺ /H ⁺ exchanger. This process facilitates the rapid removal of cellular ammonium ions and ensures that the bulk of the ammonium ions generated from the amide and amine nitrogens of glutamine are excreted in the urine.

The acute increase in renal ammoniagenesis from plasma glutamine is initiated more rapidly than the adaptive increases in the levels of GA and GDH. The rapid increase in ammoniagenesis may result from an acute activation of the mitochondrial glutamine transporter. In addition, the cellular concentrations of glutamate and α-ketoglutarate within the renal cortex are decreased significantly in acute acidosis. The latter compounds are products and potent inhibitors of the GA and GDH reactions, respectively. The decrease in concentrations of the two regulatory metabolites may result from a pH-induced activation of α-ketoglutarate dehydrogenase and the rapid induction of PEPCK. The increased level of the latter enzyme would facilitate the cataplerotic removal of intermediates of the tricarboxylic acid cycle. Thus, increased renal catabolism of glutamine initially results from a rapid activation of key transport processes, an increased availability of glutamine, and a decrease in product inhibition of the GA and GDH activities. The net effect is a rapid and pronounced increase in the excretion of ammonium ions and titratable acid in the urine.

Various indirect studies also support the hypothesis that changes in product inhibition may contribute to the acute regulation of renal catabolism of glutamine. For example, inhibition of glutamate uptake both in vivo and in renal cell culture reduces the cellular glutamate concentration and stimulates flux through GA leading to increased ammoniagenesis. In addition, a mutation in GDH, that increases in vivo activity by reducing GTP inhibition, is responsible for the hyperinsulinism/hyperammonium syndrome. Similarly, systemic activation of GDH can be produced by feeding rats a non-metabolizable analog of leucine that functions as an allosteric activator of GDH. The latter protocol produced a mild hyperammonemia that resulted from a renal specific increase in ammoniagenesis. All of these observations support the concept that acute activation of flux through GA and GDH may contribute to the acute regulation of renal ammoniagenesis.

Only a few studies have characterized the changes in renal glutamine extraction and ammoniagenesis that occur in humans during acute and chronic acidosis. Both studies enrolled hypertensive patients who were undergoing renal vein catheterization to assess plasma renin activity. In the acute study, half of the patients were fed 3 doses of NH ₄ Cl during the 24 h before catheterization.

This protocol produced a significant decrease in arterial pH (7.41 to 7.33), arterial HCO ₃ ^– (23 mM to 15 mM), and urine pH (5.6 to 4.7), but increased total renal ammonium ion production by only 60%. The acidotic patients exhibited no significant changes in arterial glutamine or renal extraction of glutamine (~6%), but did exhibit an increased extraction of plasma glycine, proline and ornithine. During chronic acidosis, glutamine extraction by the human kidney was increased even though blood glutamine levels were decreased. The combined data indicate that the mechanism to increase renal catabolism of plasma glutamine is not acutely activated in humans and that amino acids in addition to glutamine may contribute to total renal ammoniagenesis.

Chronic Adaptations to Metabolic Acidosis

Role of Increased Gene Expression

During chronic metabolic acidosis, the acute decreases in the renal concentrations of glutamate and α-ketoglutarate are partially compensated and the arterial plasma glutamine concentration is decreased to 70% of normal. However, the kidneys now extract nearly 40% of the total plasma glutamine in a single pass through this organ ( Figure 57.3 ). Renal catabolism of glutamine is now sustained by increased expression of the genes that encode various ion transporters and key enzymes of glutamine metabolism. Following onset of acidosis, a rapid induction of PEPCK gene expression occurs only within the S1 and S2 segments of the proximal tubule. The more gradual increase in the levels of the mitochondrial GA and GDH also occur solely within the proximal convoluted tubule. Previous micropuncture studies and assays using microdissected nephron segments established that the preponderance of renal ammoniagenesis in normal or acidotic rats occurs within the convoluted portion of the proximal tubule. The decreases in plasma pH and HCO ₃ ^– concentration during metabolic acidosis produce a comparable and sustained decrease in the intracellular pH (pH _i ) of the proximal convoluted tubule. Thus, the adaptive increases in gene expression may be initiated by a decrease in pH _i . This hypothesis is supported by the findings that similar changes in gene expression occur in response to feeding a high protein diet or chronic hypokalemia, conditions that decrease intracellular pH but produce either no change in extracellular pH or a pronounced alkalosis, respectively. ^6,60 The adaptations in GA and PEPCK levels result from increased rates of synthesis of the proteins ^61,62 that correlate with comparable increases in the levels of their respective mRNAs. However, the increase in GA results from the selective stabilization of the GA mRNA, whereas the more rapid increase in PEPCK activity is initiated by enhanced transcription of the PCK1 gene. The activities of the apical Na ⁺ /dicarboxylate co-transporter, NaDC-1, the mitochondrial glutamine transporter, the basolateral SNAT3 glutamine transporter (SLC38A3), the apical Na ⁺ /H ⁺ exchanger, NHE3, the basolateral Na ⁺ -3HCO ₃ ^– co-transporter, NBC1, the medullary Na ⁺ K ⁺ -2Cl ^– co-transporter, and the ammonia channel, RhCG are also increased during chronic acidosis.

The adaptation in the NaDC-1 transporter contributes to an increased reabsorption and metabolism of citrate within the proximal tubule. This reduces the excretion of a weak acid in the urine. Following cellular uptake, citrate is metabolized through one of two pathways, a cytoplasmic pathway involving citrate lyase or a mitochondrial pathway involving the citric acid cycle. During acidosis, the activities of cytoplasmic citrate lyase and mitochondrial aconitase are also increased. Since both pathways generate HCO ₃ ^– , the increased reabsorption of citrate is equivalent to a decrease in base excretion. Enhanced catabolism of citrate also produces substrates that support the increased gluconeogenesis. By contrast, the onset of acidosis produced a decreased transcription of the apical sodium-dependent phosphate transporter, NaPi-II, in rat kidney and in OK cells. This adaptation may contribute to an increased excretion of phosphate, a titratable acid. However, the onset of acidosis in mice decreased the expression of NaPi-IIa and NaPi-IIb mRNAs, but increased levels of both isoforms in isolated brush border membranes. Thus, the transient increase in phosphaturia may be caused by a direct effect of H ⁺ ion concentration on the activity of the phosphate transporter.

Chronic acidosis also leads to a pronounced increase in the levels of the SNAT3 glutamine transporter (SLC38A3) in rat kidney. Adaptive increases in this transporter also occur in response to chronic dietary potassium restriction and a high protein intake, conditions that also cause an increase in renal ammonia synthesis and excretion. A comprehensive survey of the adaptive response of known amino acid transporters in mouse kidney, demonstrated that only the basolateral SNAT3 transporter exhibits a rapid and pronounced increase in both mRNA and protein levels during onset of acidosis. Surprisingly, expression of the y ⁺ -LAT1 (SLC7A7), a basolateral cationic/neutral amino acid exchanger subunit, was decreased significantly during acidosis. The SNAT3 transporter has a high affinity for glutamine. It was initially cloned by its homology to the vesicular GABA transporter. It catalyzes a Na ⁺ -dependent uptake of glutamine that is coupled to the efflux of H ⁺ ions. However, under physiological conditions, this reaction is reversible. Thus, it can also catalyze H ⁺ ion uptake coupled to glutamine efflux. In brain, the SNAT3 transporter is expressed primarily in glial cells, while in liver, it is found solely in the perivenous hepatocytes. Under normal acid–base conditions, it is localized solely to the basolateral membrane of the proximal straight tubules within the outer stripe of the outer medulla in rat kidney. All of these cell types express high levels of glutamine synthetase. Thus, the SNAT3 transporter may function primarily to catalyze a pH-dependent release of glutamine. However, during chronic acidosis, increased expression of the SNAT3 transporter occurs primarily in the basolateral membranes of the S1 and S2 segments of proximal tubule, the site of increased glutamine catabolism. Given the sustained increase in H ⁺ ion concentration within these cells, the increased expression of the SNAT3 transporter is likely to contribute to the basolateral uptake of glutamine. Therefore, the adaptive increase in the SNAT3 transporter may contribute to both the rapid reversal of glutamine transport across the basolateral membrane that occurs during acute acidosis and the elevated and sustained extraction of plasma glutamine during chronic acidosis.

The increase in apical Na ⁺ /H ⁺ exchanger activity sustains the acidification of the fluid in the tubular lumen and contributes to the active transport of ammonium ions. Thus, the increased renal ammoniagenesis continues to provide an expendable cation that facilitates the excretion of titratable acids while conserving sodium and potassium ions. The increased Na ⁺ /H ⁺ exchanger activity also promotes the tubular reabsorption of HCO ₃ ^– ions. In humans, the α-ketoglutarate generated from glutamine is primarily converted to glucose and to a lesser extent oxidized to CO ₂ ⁵ . Either process requires the cataplerotic activity of PEPCK to convert intermediates of the tricarboxylic acid cycle to phospho enol pyruvate. This product is either utilized to initiate gluconeogenesis or converted to pyruvate that re-enters the mitochondria and is oxidized to CO ₂ . However, both pathways generate 2 HCO ₃ ^– ions per mole of α-ketoglutarate. Activation of NBC1, the basolateral Na ⁺ /3HCO ₃ ^– co-transporter, facilitates the translocation of reabsorbed and of de novo -synthesized HCO ₃ ^– ions into the renal venous blood. However, this process occurs without an increase in the level of the NBC1 protein. Thus, the combined adaptations also create a net renal release of HCO ₃ ^– ions that contribute to the ability of the kidney to partially restore acid–base balance. In addition to these adaptations, the renal proximal tubule undergoes an extensive hypertrophy during chronic acidosis.

Difference gel electrophoresis along with proteomic and bioinformatic techniques were used to identify proteins that are differentially expressed in the rat renal proximal convoluted tubule during metabolic acidosis. Tubules were prepared by incubation of minced rat renal cortex with collagenase and purified by Percoll density gradient centrifugation. The purity of the isolated proximal convoluted tubules was confirmed by western blot analyses using antibodies to proteins that are expressed only within a single cortical segment of the nephron. This analysis indicated that the purified tubules retained proteins that are expressed only in this segment, but essentially lacked proteins specific for the thick ascending limb, distal tubule, and cortical collecting duct. Nearly 2000 protein spots were resolved when the proximal tubule lysates were fractionated by two-dimensional gel electrophoresis. The protein spots that were differentially expressed were picked and analyzed by mass spectrometry. This analysis identified 21 proteins which are increased between 1.5- and 8.4-fold, including GA (8.4-fold), PEPCK (7.0-fold) and GDH (3.0-fold), and 16 proteins that are decreased between .67- and .03-fold. The observed changes indicate that amino acid catabolism, an ER-stress response, and Ca ⁺⁺ -signaling are activated, while conversion of glycine to creatine, oxidation of pyruvate, and fatty acid catabolism are decreased in the proximal convoluted tubule during chronic metabolic acidosis. Many of the observed changes, including activation of an ER-stress response, were confirmed by western blot analysis.

A recent microarray analysis characterized the mRNA isolated from whole kidneys of control mice and of mice that were made acidotic by providing 0.28 M NH ₄ Cl as the drinking water. After 2 d, this protocol produced a significant acidosis (arterial blood pH of 7.10 and 12 mM HCO ₃ ^– ) that was partially compensated by seven days. Microarray analysis detected 13,000 mRNAs or ~40% of the genes on the mouse genomic array. The levels of 333 mRNAs were up-regulated and another 342 were down-regulated during both acute and chronic acidosis. Cluster analysis indicated that a large proportion of the regulated genes encode solute transporters and proteins involved in cell growth, proliferation, apoptosis, ammoniagenesis, water homeostasis, and energy metabolism. As expected, the mRNAs that encode GA, GDH, PEPCK and the SNAT3 transporter were among those that exhibited the most pronounced increases. The changes in five mRNAs were confirmed by RT-PCR of mRNA isolated from dissected S1 and S2 segments. It remains uncertain which of the other changes occur within the proximal convoluted tubule, within other cell types, or within multiple segments of the nephron. However, this analysis clearly established that expression of a very large number of genes is altered in the kidney in response to acidosis.