Mechanisms and physiological relevance of acid-base exchange in functional units of the kidney

Proximal convoluted tubule

The proximal convoluted tubule (PCT) is engaged in reabsorption and recycling of various ions and solutes. About two-thirds of filtered water, NaCl, Ca²⁺, as well as all glucose, phosphates and amino acids, are reabsorbed by extensive apparatus of specialized transporters acting at the brush-bordered apical and the basolateral surfaces of the epithelial cells. Different PCT segments are coordinated by the electrochemical gradient created by basolateral Na⁺-K⁺-ATPase (Van Der Wijst et al., 2019). These transport processes require high amounts of energy in the form of ATP generated by abundant elongated mitochondria of the epithelial cells (Kriz & Kaissling, 2007). In addition, PCTs reabsorb substantial amounts of albumins and low-molecular-weight plasma proteins, e.g., hormones, enzymes, lipoproteins, vitamin carriers, passing through the glomerular basement membrane into the filtrate. Normally, human urine is almost totally cleared of plasma proteins by the clathrin-dependent endocytosis-mediated reabsorption in PCT (Eshbach & Weisz, 2017).

The glomerular filtrate that contacts the apical surfaces of PCT cells is initially similar in composition to the interstitial fluid that contacts the basolateral surfaces. With the total filtrate volume of about 180 liters per day, almost the entire quantity of bicarbonate ion must be reabsorbed from the filtrate. The HCO₃^– reabsorption is tightly coupled to Na⁺ and water reabsorption. The transport of HCO₃^–, about 80% of which occurs in PCT, is provided by two pathways. The major indirect CO₂ pathway has been conventionally considered as the unique route of HCO₃^– reabsorption, with H⁺ is secreted into the lumen via the apical Na⁺/H⁺ exchanger NHE3 and the vacuolar-type H⁺-ATPase. Secreted H⁺ is combined with HCO₃^– and then converted to CO₂ and H₂O by carbonic anhydrase IV; CO₂ then enters PCT cells by diffusion to recreate HCO₃^– and H⁺; the reaction is catalyzed by carbonic anhydrase II. The other pathway is direct HCO₃^– transport into PCT cells by the apically expressed Na⁺/HCO₃^– cotransporter NBCn2 (Guo et al., 2017); HCO₃^– is further transferred to the blood via the basolateral Na⁺/HCO₃^– cotransporter NBCe1-A (Rajkumar & Pluznick, 2018). The NHE3 sodium/proton exchanger and Н⁺-ATPase located at the apical membranes of PCT cells acidify the lumen and alkalize the cells thereby ensuring about 80% of bicarbonate reabsorption. Transport of bicarbonate ions via the basolateral membrane to the interstitium by NBC, a 1Na⁺/3HCO₃⁻ transporter, maintains the pH gradient (Boron, 2006; Romero et al., 2013). The PCT cells also produce new bicarbonate to neutralize the metabolically released mineral acids. The remaining HCO₃⁻ ions are reabsorbed in the ascending loop of Henle (10%) and distal tubules (10%). Apart from carbonic anhydrase and NHE3 sodium/proton exchanger, the process involves the proton-pumping ATPase V. From the interstitial fluid HCO₃⁻ is pumped to the blood mostly by NBCe1-A Na⁺/HCO₃⁻ electrogenic co-transporter, renal splice variant. The protons released by PCT cells into the tubular lumen react with HCO₃⁻ in the filtrate to form CO₂ and H₂O; the reaction is catalyzed by carbonic anhydrase IV expressed at the apical membrane. Water molecules cross the apical membrane exclusively via aquaporin (AQP1) channel, which also apparently transports the major portion of CO₂. In combination, these processes ensure NaHCO₃ reabsorption. A small proportion of H⁺ released by PCT cells into the lumen is saturated by various luminal buffers (NH₃, inorganic phosphate, creatinine) where both NH₄⁺ excretion and acid formation can be measured (Boron, 2006). The transport processes in PCT are summarized in Fig. 2.

The regulation of AB balance in PCT involves several pH-sensitive molecules: calcium-sensing receptor CaSR, G-protein coupled receptor Gprc5c, receptor tyrosine kinases ErbB1/2, proline-rich tyrosine kinase Pyk2, pH-sensitive ion channel TASK2 and bicarbonate-stimulated soluble adenylyl cyclase (Brown & Wagner, 2012; Rajkumar & Pluznick, 2018). Pyk2 is a key mediator of increased NHE3 activity following acid stimulation. Pyk2 shows maximal kinase activity at low pH within the physiological range, decreasing from 7.4 to 7.0; experiments indicate that Pyk2 directly responds to the change in pH. Pyk2 knockdown with either dominant-negative pyk2 or siRNA prevented the acid-induced NHE3 activation. The Pyk2 signaling cascade presumably involves endothelin receptor B (ETRB) known as NHE3 activity modulator (Brown & Wagner, 2012; Rajkumar & Pluznick, 2018). ETRB stimulation leads to RhoA-dependent cytoskeletal remodeling and NHE3 membrane accumulation followed by increased proton efflux from PCT cells (Wesson, 2011). Another pH regulation pathway includes ErbB1/2- and angiotensin II receptor type 1-mediated signaling cascade. ErbB1/2 is a putative CO₂/bicarbonate sensing molecule at the basolateral membrane of PCT cells (Zhou et al., 2006a, 2006b; Skelton & Boron, 2015), consistently with the observed activation of ErbB2 by mildly alkaline extracellular media (pH 8–9) (Serova et al., 2019). Analysis of ErbB1/2 tyrosine phosphorylation in rabbit PCT also indicates a response to acute AB imbalance (Skelton & Boron, 2015).

The proximal bicarbonate reabsorption is regulated by several hormones acting for either minutes-to-hours or days. The process can be stimulated by adrenergic agonists and angiotensin II, inhibited by parathyroid hormone via cAMP signaling and stimulated by hypercalcemia. In chronic acidosis, the binding of renal endothelin 1 to its receptor ETRB has been identified as the trigger of increased Na⁺/H⁺ exchange in PCT (Wesson, 2011). Glucocorticoids participate in the regulation as well, notably during the chronic response to acidosis (Bobulescu et al., 2005).

Of course, other ions, notably oxalate and phosphate, contribute to the maintenance of AB balance by the kidney as carriers. Expression of SLC26A6 at the apical membranes of PCT epithelium is crucial for NaCl reabsorption and facilitates oxalate secretion. SLC26A6 also plays a role in transtubular NaCl reabsorption and oxalate transport potentially involving SLC26A7 and AE1 at the basolateral membrane. In addition, SLC26A6 interacts with NaDC-1 to regulate citrate absorption from the urinary lumen thereby preventing Ca²⁺ oxalate crystallization and kidney stone formation (Wang et al., 2021). Systemic levels of inorganic phosphate (P_i) are primarily adjusted by reabsorption in PCT involving SLC34A1 (NaPi-IIa, main), while SLC34A3 (NaPi-IIc, developmental) and SLC20A2 (PiT-2, accessory) P_i transporters (Burns & Werner, 2020).

TASK2, another pH-sensitive molecule regulating bicarbonate reabsorption in PCT, is a two-pore domain K⁺ channel activated by extracellular alkalization. TASK2-null mice show decreased water and sodium reabsorption in the kidneys under alkali load, leading to metabolic acidosis and significantly reduced arterial blood pressure compared to normal mice (Warth et al., 2004). TASK2 is coexpressed with Na⁺/HCO₃^– cotransporter NBCe1-A. The release of Na⁺ and HCO₃^– ions via NBCe1-A causes basolateral membrane depolarization. With the increase in extracellular HCO₃^– concentration pH rises, activating TASK2. The release of accumulated K⁺ via TASK2 leads to repolarization of the membrane providing a driving force for Na⁺ and HCO₃^– reabsorption (Warth et al., 2004). Double inactivation of NBCe1-A and TASK2 results in metabolic acidosis (Rajkumar & Pluznick, 2018).

Soluble adenylyl cyclase (sAC) is expressed in kidney tubules and activated by HCO₃^– ions; the response can be enhanced by calcium. The protein may function as a CO₂ sensor involved in cAMP-dependent V-ATPase membrane accumulation and proton secretion (Levin et al., 2021).

Calcium-sensing receptor CaSR is a G protein-coupled receptor (GPCR) expressed at the apical membrane. CaSR is sensitive to extracellular pH, with alkali-enhanced response to calcium and magnesium ions. CaSR stimulation increases NHE3 activity in PCT (Rajkumar & Pluznick, 2018). Gprc5c, also a GPCR at the apical membrane in PCT, is functionally similar to CaSR. Gprc5c knockout mice have abnormal pH homeostasis with lower blood pH, higher urine pH and reduced renal NHE3 activity compared to wild type, suggesting a role in regulation of systemic AB balance effectuated through NHE3 (Rajkumar & Pluznick, 2018).

Bicarbonate reabsorption in the kidney is also coupled to glucose reabsorption. A Na⁺/glucose cotransporter SGLT2 (Slc5a2) is the main glucose transporter in the kidney. About 90% of glucose is reabsorbed in PCT via SGLT2 expressed at the apical membrane. SGLT2 appears to functionally interact with NHE3 at the apical membrane, possibly via PDZK1 and 17-kDa membrane-associated protein (MAP17). NHE3 mediates bicarbonate reabsorption and ammonia secretion and also contributes to Na⁺ and water reabsorption. The SGLT2/NHE3 multiprotein complex may represent a functional unit that affords concomitant regulation of AB balance, extracellular fluid volume and glucose homeostasis. A nonspecific competitive SGLT inhibitor phlorizin markedly increased the urinary Na⁺ and HCO₃^– excretion (Silva et al., 2020). Selective SGLT2 inhibitors reduce both glucose reabsorption rates and blood pressure (Burnier, Oe & Vallon, 2022).

It should be mentioned that transport and metabolism of amino acids (AA) in PCT also contribute to AB balance regulation. The total amount of amino acids in the renal filtrate is reabsorbed in PCT and partially metabolized (mainly glutamine) producing ammonia and bicarbonate. Glutamine transport and metabolism are coupled to proton excretion (Harris et al., 2023). The glutamine transfer by apical Na⁺-dependent neutral amino acid transporter 1 and basolateral Na⁺-coupled neutral amino acid transporter 3 is complemented by glutaminase activity. In acidosis, glutaminase activity is significantly enhanced to afford the clearance of H⁺ as NH₄⁺ (Li, Zheng & Wu, 2020).

The proximal reabsorption of bicarbonate is extensively regulated; however, residual amounts of this ion are efficiently reabsorbed in the distal PCT through different regulatory mechanisms.

Loop of Henle

The proximal convoluted tubule continues into the loop of Henle, a U-shaped epithelial tubule running into the medulla and returning to the cortex where it continues into the distal convoluted tubule of the nephron. The loop of Henle consists of two limbs—a descending and an ascending, connected by a thin-walled middle segment much narrower than other renal tubules (Pallone, Zhang & Rhinehart, 2003). The descending limb does not participate in ion transport: it is only permeable to water, which flows through AQP1 and AQP3 channels in the apical and basolateral membranes of the descending thin tubule; the driving force is provided by small constant osmotic gradients of solutes maintained locally (Agre et al., 2002).

The thick ascending limb of the loop of Henle (TAL) is largely responsible for the water content of the urine: from highly concentrated small volumes of urine in anti-diuresis to large volumes of very low osmolarity in water diuresis. Specifically, TAL is responsible for diluting the content of the lumen while mounting the osmotic pressure in the interstitium. TAL is impermeable to water; the dilution occurs through active reabsorption of NaCl which feeds the hypertonic gradient. The resulting interstitial hypertonicity of the medulla provides a driving force for water reabsorption from the urine at the final stages of its formation in collecting tubules of the medulla. In water diuresis, collecting tubules become impermeable to water and the urine stays diluted (Greger, 1985).

The loop of Henle (particularly TAL) reabsorbs about 40% of Na⁺ present in the glomerular filtrate. Sodium ions enter cytosol mostly by Na⁺/K⁺/2Cl⁻ electroneutral cotransporter NKCC2 (encoded by SLC12A1) at the apical cell membrane. Unlike its closely related isoform NKCC1 (SLC12A2) expressed in multiple organs and tissues, NKCC2 is a TAL-specific protein (Zacchia et al., 2018). Sodium ions subsequently leave the cells by sodium pumps at the basolateral surface; the parallel basolateral efflux of chloride ions is mediated by ClC-Ka and ClC-Kb channels with Barttin subunits (Hennings et al., 2017). By contrast, potassium ions flow back into the lumen via the renal outer medullary K⁺ (ROMK) channels thereby recovering the gradient required for the sodium-chloride symport and simultaneously the positive potential on the apical membrane which coordinates the paracellular reabsorption of cations (Zacchia et al., 2016).

The combined activity of essential transporters and ion channels involved in the salt reabsorption (NKCC2, ROMK, ClC-Kb) ensures the electrolyte balance. A defect in any of these components may cause a salt-wasting phenotype. However, as the reabsorption in TAL is regulated by hormones and paracrine factors through multiple intracellular signaling pathways, renal salt wasting often reflects abnormal regulation of the non-affected transport machinery. Moreover, activating mutations in transporter-encoding genes can ultimately lead to salt-wasting nephropathy, which illustrates the complex nature of the water-salt homeostasis. Defects in uromodulin, Ste20-related proline alanine rich kinase (SPAK) and oxidative stress responsive kinase (OSR1) molecules may affect water-salt homeostasis by influencing NKCC2 activity (Watanabe et al., 2002).

The loop of Henle contributes to AB homeostasis by reabsorbing bicarbonate (up to 15% of the initial content in the glomerular filtrate). In rats, the descending limb is fairly permeable to bicarbonate; nevertheless, its concentration inside the loop decreases toward the U-turn, reflecting the high-rate water reabsorption (Capasso et al., 1991). In TAL, bicarbonate is reabsorbed transcellularly; the mechanisms (NHE-mediated exchange) are similar to those in PCT. Of NHE2 and NHE3 isoforms found in the membranes, NHE3 is essential to AB homeostasis as indicated by targeted inactivation of corresponding genes in mice (Watts & Good, 1994; Ledoussal et al., 2001). In vivo and ex vivo perfusion experiments show that bicarbonate reabsorption requires carbonic anhydrase and can be stimulated by bumetanide. The bicarbonate efflux from cells is mediated by Cl⁻/HCO₃⁻ exchanger 2 (AE2). A similar exchanger, AE1, is expressed at the apical (luminal) surface and coupled to Na⁺/H⁺ activity thereby particiapting in Na⁺ reabsorption (Eladari et al., 1998; Houillier & Bourgeois, 2012). The major contribution of NHE3 is complemented by several accessory transporters. Functional and immunohistochemical tests reveal H⁺ ATPase expression along TAL. Its role in bicarbonate reabsorption is considered secondary, under physiological conditions often superfluous, but still significant for AB homeostasis. Shifts in AB balance modulate the rates of bicarbonate reabsorption along TAL: the process is stimulated in metabolic acidosis and suppressed in alkalosis, independently of the acute or chronic nature of the condition. According to functional studies, both NHE and H⁺-ATPase activities adapt to changes in AB status, apparently under hormonal stimulation (glucocorticoids, aldosterone) (Capasso et al., 1994). Another phosphate transporter, sodium-dependent SLC34A2 co-localized with uromodulin in TAL, is also active in distal renal tubules (Burns & Werner, 2020).

NH₃ trafficking is another important regulatory factor of ion exchange in the loop of Henle. The bulk of excreted ammonium is absent in the glomerular filtrate, but produced from glutamine in the kidney and secreted to the urine (Weiner & Verlander, 2013). TAL plays a decisive role in NH₃ reabsorption; the process involves the NKCC2-mediated potassium binding. The K⁺/NH₄⁺ exchange and K⁺ transfer in TAL have been described as well, albeit provide a minor contribution compared to the NKCC2-mediated paracellular effects. The basolateral efflux of cations in TAL depends on NHE4 (Na⁺/NH₄⁺), NBCn1 (Na⁺/HCO₃⁻) and chloride transporters (Blanchard et al., 1998). The routes of ion exchange in the loop of Henle are summarized in Fig. 3.

Distal convoluted tubule

The loop of Henle continues into the distal convoluted tubule—a relatively short portion of the nephron critically important for sodium, potassium and divalent cation homeostasis. DCT also participates in NHE2-dependent bicarbonate reabsorption. DCT cells are rich in mitochondria supporting the electrolyte transporters, notably the basolateral Na⁺/K⁺-ATPase. The basolateral efflux of bicarbonate proceeds by AE2-mediated exchange with chloride, but may also involve basolateral chloride channels partially permeable to HCO₃⁻. The cells express cytosolic carbonic anhydrase CA II, but lack the membrane-bound CA IV at the apical surface (Subramanya & Ellison, 2014). The electrolyte trafficking in DCT involves sodium, potassium and chloride ions. Sodium transport in DCT (up to 10% of the overall Na⁺ reabsorption) is mediated by thiazide-sensitive NaCl cotransporter (NCC, SLC12A3); sodium-dependent Cl⁻/HCO₃⁻ exchanger (NDCBE, SLC4A8) and amiloride-sensitive epithelial sodium channel (ENaC) (Leviel et al., 2010); NCC is regulated by a complex cascade of WNK-dependent serine-threonine kinases SPAK and OCR-1. Chloride transport uses ClC-Kb channels and KCl cotransporter 4 (KCC4, SLC12A7). Potassium secretion in DCT involves the ‘big K⁺’ and ROMK channels (Meneton, Loffing & Warnock, 2004). The routes of ion transport in DCT are summarized in Fig. 4.

Collecting tubules and collecting ducts

The epithelial collecting system of the kidney connects nephrons to the urinary tract. This system substantively contributes to the water-electrolyte balance by means of aldosterone/ADH-dependent reabsorption and secretion (Olde Engberink et al., 2023). The walls of collecting tubules comprise two types of epithelial cells, principal cells (PC) and intercalated cells (IC) distinguished morphologically and expressing specific sets of ion channels and transporters (Fig. 5). PC express the epithelial Na⁺ channel ENaC and aquaporin AQP2 at the apical membrane, and AQP3/AQP4 at the basolateral membrane. AQP2 marks the entire length of the epithelial collecting system, from connecting tubules through cortical and outer medullary collecting tubules to inner medullary collecting tubules and ducts. IC, which express H⁺-ATPase V and carbonic anhydrase CAII, are engaged in the acid/bicarbonate transport: V-ATPases acidify certain cytoplasmic compartments and pump protons across plasma membranes, while carbonic anhydrases catalyze the production of H⁺ and HCO₃⁻ from carbon dioxide and water. Intercalated cells of the collecting system can be further subdivided into IC-α and IC-β based on molecular markers and functional differences specified in Table 1.

IC-α express H⁺-ATPase V at the apical membrane and Cl⁻/HCO₃⁻ anion exchanger at the basolateral membrane, responsible for proton secretion and bicarbonate reabsorption, respectively. ATPase V is regulated via soluble adenylate cyclase cascade. Electrogenic chloride transporter A11 (SLC26A11) is also expressed at the apical membranes of IC-α (Roy, Al-Bataineh & Pastor-Soler, 2015a). The acidified urine is less susceptible to microbial colonization. Moreover, though antimicrobial defense cannot be regarded as a direct function of IC-α, these cells participate in the innate immunity reactions by producing lipocalin, a neutrophil gelatinase-associated protein that binds and inhibits siderophores—small chelating compounds secreted by bacteria and fungi to promote iron transfer across cell membranes (Paragas et al., 2014).

IC-β are marked by apical expression of pendrin SLC26A4, which mediates bicarbonate secretion, and basolateral expression of V-ATPase engaged in H⁺ reabsorption and sodium-dependent Cl⁻/HCO₃⁻ exchanger NDCBE (Eladari et al., 1998; Petrenko et al., 2013). Apart from pendrin, the base excretion involves cystic fibrosis transmembrane conductance regulator (CFTR); both proteins are found at the apical membrane of IC-β. In human patients and experimental mice with cystic fibrosis this functionality is impared. CFTR-dependent transport is crucial for HCO₃^– secretion to the urine (Berg et al., 2021). Anoctamins ANO1/6 are required for proper expression and function of CFTR. Thus, in renal collecting system, bicarbonate secretion occurs through a synergistic action of CFTR and pendrin supported by ANO (Kunzelmann et al., 2023). Anion exchanger 4 (AE4), also implicated in bicarbonate transport, is specifically expressed at the basolateral membrane of IC-β. Although sodium reabsorption via AE4 is too weak for plasma volume regulation, the response of IC-β to AB-related metabolic challenges depends on AE4 (Vitzthum, Meyer-Schwesinger & Ehmke, 2024).

Conventionally, major cell types of the collecting system were assigned distinct functionalities, with IC-α and -β ensuring, respectively, acid and base secretion and principal cells reabsorbing sodium and water and secreting potassium. However, recent studies reveal broader functional profiles. In particular, IC-β are capable of NaCl absorption and participate in regulation of extracellular fluid volume and blood pressure. In β-ICs, the apical influx of Cl^– via pendrin is coupled to the apical Na⁺ influx via NDCBE (Almomani et al., 2014; Roy, Al-Bataineh & Pastor-Soler, 2015b). An increasing amount of evidence indicates that IC control the maintenance of Na⁺ balance by adjacent principal cells. In particular, Na⁺ absorption via ENaC in principal cells is coordinated to Cl⁻ absorption via pendrin in IC to operate the NaCl absorption (Wall, Verlander & Romero, 2020), while luminal bicarbonate levels have been positively associated with ENaC expression and activity. Therefore, pendrin can modulate ENaC; the link is mediated by luminal HCO₃⁻ concentrations or pH. Pendrin gene ablation leads to decreased abundance of H⁺-ATPase in IC-β and a concomitant increase in the intracellular ATP levels. The luminal ATP levels subsequently rise due to enhanced ATP secretion via connexin 30 which contains a CO₂ binding site. When pCO₂ rises, the hemichannel opens and releases ATP (Serova et al., 2020). In the lumen, ATP binds purinergic receptors at the apical surface to promote calcium-dependent production of prostaglandin E2 which downregulates ENaC in principal cells. This loop links bicarbonate transport to regulation of sodium balance, extracellular fluid volume and blood pressure (Gueutin et al., 2013). Another signaling molecule expressed by IC-β, the insulin-receptor related receptor (IRR), participates in bicarbonate excretion regulation and provides a pH-sensor of extracellular fluid alkalinization (Gantsova et al., 2022; Korotkova et al., 2022). In mice, IC-α of cortical collecting ducts can secrete Na⁺ and Cl^– via NKCC1, probably HKA2 and a Cl^– channel (Morla et al., 2016). Recent studies indicate that principal cells are involved in the control of AB homeostasis. For instance, high systemic acid loads stimulate ADH production. Subsequent activation of Avpr2 receptor for ADH in principal cells triggers production of Gdf15 which activates the ErbB2 signaling pathway in IC-α to induce their proliferation (Cheval et al., 2021).

A minor subpopulation of IC is neither α, nor β (Table 1); these cells express both pendrin and H⁺-ATPase in the apical membrane and apparently secrete both bicarbonate and protons. IC have more restricted representation in the collecting system than principal cells. IC-α are found in connecting tubules, cortical collecting tubules and initial portions of medullary collecting tubules. IC-β are found in connecting tubules and cortical collecting tubules, but typically absent in the medullary collecting tubules. PC and IC also express alternative Cl⁻/HCO3⁻ exchangers (SLC26A7, SLC26A11), AE4 (SLC4A9) and Na⁺/K⁺-ATPase (Chen, Higgins & Zhang, 2017).

Another G-protein-coupled receptor implicated in AB homeostasis, Gpr116, is expressed in cortical collecting ducts by IC-α. Tubule-specific Gpr116 inactivation in mice leads to production of acidic urine and mild systemic alkalosis; the analysis reveals impaired distribution of V-ATPase proton pumps at the surface of IC-α. Gpr4, another acid-sensing molecule expressed in collecting tubules, is significant as well: genetic inactivation of Gpr4 reduces net acid secretion leading to acidosis (Zaidman & Pluznick, 2022).

The epithelial collecting system also implements NH₃/NH₄⁺ transport regulated by a complex process that involves at least two Rhesus glycoproteins: RhB (SLC42A2) and RhC (SLC42A3) (Harris et al., 2023; Takvam et al., 2023). Rhbg expressed in IC-α and non-α, non-β intercalated cells, principal cells in the basolateral plasma membrane. Rhbd is not found in IC-β. Rhcg is found in the apical and basolateral membrane in type IC-α and principal cells, but also it is expressed in the apical plasma membrane in non-α, non-β intercalated cells. Recent reports show that RhC is specifically involved in transport of the molecular form (NH₃), whereas RhB participates in transport of both NH₃ and NH₄⁺ (Weiner & Verlander, 2014). Collecting duct-specific double knockout of these proteins confirms the role of RhB/C in kidney response to metabolic acidosis. Compared with wild type mice, mice with collecting duct Rhbg and Rhcg deletion had significantly more severe metabolic acidosis more severe HCl-induced metabolic acidosis (Lee et al., 2014).