Collecting Duct Intercalated Cell Function and Regulation

RenalCJASN Physiology ePress. Published on January 28, 2015 as doi: 10.2215/CJN.08880914

Ankita Roy,* Mohammad M. Al-bataineh,* and Nu´ria M. Pastor-Soler*†

Abstract Intercalated cells are kidney tubule epithelial cells with important roles in the regulation of acid-base homeostasis. However, in recent years the understanding of the function of the intercalated cell has become greatly enhanced and has shaped a new model for how the distal segments of the kidney tubule integrate salt and water *Renal-Electrolyte Division, Department reabsorption, potassium homeostasis, and acid-base status. These cells appear in the late distal convoluted tubule of Medicine; and or in the connecting segment, depending on the species. They are most abundant in the collecting duct, where †Department of Cell they can be detected all the way from the cortex to the initial part of the inner medulla. Intercalated cells are Biology, University of interspersed among the more numerous segment-specific principal cells. There are three types of intercalated Pittsburgh School of Medicine, Pittsburgh, cells, each having distinct structures and expressing different ensembles of transport proteins that translate into Pennsylvania very different functions in the processing of the urine. This review includes recent findings on how intercalated A.R. and M.M.A. cells regulate their intracellular milieu and contribute to acid-base regulation and sodium, chloride, and contributed equally to potassium homeostasis, thus highlighting their potential role as targets for the treatment of hypertension. Their this work. novel regulation by paracrine signals in the collecting duct is also discussed. Finally, this article addresses their role as part of the innate immune system of the kidney tubule. Correspondence: Dr. Clin J Am Soc Nephrol ccc–ccc Nu´ria M. Pastor-Soler, 10: , 2015. doi: 10.2215/CJN.08880914 Renal-Electrolyte Division, A915 Scaife Hall, 3550 Terrace Street, Pittsburgh, PA Introduction ammonia/ammonium, a topic reviewed in a separate 15261. Email: Intercalated cells are epithelial cells traditionally asso- article in this series (9). [email protected] ciated with the regulation of acid-base homeostasis in The relevance of intercalated cell dysfunction in distal segments of the kidney tubule (Figure 1) (1). clinical scenarios is often not as evident as the relevance These cells also participate in potassium and ammonia of principal cell dysfunction, such as in patients who transport and have a role in the innate immune system. present with diabetes insipidus or the syndrome of in- Many early studies emphasized that these tubule seg- appropriate antidiuretic hormone secretion. In clini- ments were not part of the classic nephron because they cal practice, intercalated cell dysfunction is most often arise from the mesonephric kidney or Wolffian duct, associated with metabolic acidosis, although histologic which also gives origin to the male excurrent duct (2). or laboratory confirmation of this dysfunction is seldom Most collecting duct cells express the epithelial sodium performed in the general acute care setting. Moreover, channel (ENaC) and are grouped together as principal the contribution of intercalated cells in preventing cells (3,4). Until recently, our understanding of the col- acidemia is often eclipsed by the coordinated compen- lecting duct and the roles of intercalated cells has lagged satory roles of the lung, bone, and more proximal kid- behind that of other segments. The collecting duct was ney tubule segments. Nonetheless, animals subjected to initially described as not having a specialized function dietary acid loading have significant increases in the or as having a role only in water reabsorption (5,6). luminal (facing the urine) surface area of intercalated Intercalated cells are essential in the response to acid- cells, changes that begin within a few hours from the base status of the organism, and they help dispose of change in diet (reviewed in references 7,10). acid that is generated by dietary intake and cannot be Until very recently, intercalated cells were not thought eliminated via the lungs, the so-called fixed or nonvol- to contribute to extracellular fluid volume regulation, yet atile acid (Figure 2). The kidney contributes to acid- now they are firmly established as important contrib- base homeostasis by recovering filtered bicarbonate in utors to collecting duct NaCl transepithelial transport the proximal tubule. Distally, intercalated cells generate and the protection of intravascular volume in concert new bicarbonate, which is consumed by the titration of with principal cells (Figure 2) (reviewed by Eladari et al. nonvolatile acid (7). Dysfunction of the proximal tu- [4]). An impressive new study has now established that, bule, where approximately 90% of the bicarbonate is in vivo, intercalated cells regulate their intracellular reabsorbed, leads to proximal renal tubular acidosis (8). volume by a mechanism involving the vacuolar The connecting segment and collecting duct rely H1-ATPase (H1-ATPase; also referred to as V-ATPase mostly on their intercalated cells to reabsorb the nor- in the literature). This study is important because it es- mally smaller amount of residual bicarbonate. In addi- tablishes that although most animal cells rely on the tion, intercalated cells participate in the excretion of Na1/K 1-ATPase (Na,K-ATPase) to energize other www.cjasn.org Vol 10 February, 2015 Copyright © 2015 by the American Society of Nephrology 1 2 Clinical Journal of the American Society of Nephrology

intercalated cells have been performed in cell lines of inner medullary origin, such as the mIMCD3 cell line (18,19). In the collecting duct, principal and intercalated cells form a “salt and pepper” epithelium (Figures 1 and 2) (20). This more primitive appearance of the collecting duct is similar to that of frog skin, reptilian bladder, and fish gills (20–22). In the kidney, the transition from the early part of the distal convoluted tubule to an epithelium containing both principal and intercalated cells coincides with the transition to segments that are derived from the ureteric bud outpouch- ing of the Wolffian duct (2). The ratio of principal to intercalated cells varies slightly between tubular segments, with a ratio of 2:1 in outer medullary collecting duct segments and 3:1 in the cortical collecting duct. The ratio also varies between species (10,23). Intercalated cells have had obscure names such as “dark cell” and “special cell,” a terminology that reflected how little was known about their function from the time of their discovery in 1876 until the late 1960s (24). Their ultrastructural description, however, has remained quite consistent Figure 1. | Intercalated cells in rat collecting duct. The cartoon and over the years. Intercalated cells are also called “mitochondria- confocal micrograph illustrate the intercalated cell distribution along rich cells,” reflecting the high levels of round mitochondria the kidney tubule and within the epithelium. Intercalated cells were at their apical pole, or in their cytoplasm, a distribution that detected in the cortical and outer medullary collecting duct (green oval) was quite different from that of other kidney tubule cells, by immunoflorescence labeling using an antibody against one of the which accumulated mitochondria around the basolateral H1-ATPase subunits (green). Intercalated cells are also located in membrane (Figure 3) (24–27). These cells also have “numer- the connecting segment (red circle). The apical or luminal labeling of ” the H1-ATPase indicates that these cells are mostly type A intercalated ous irregular apical microvilli compared with the surround- fi cells (A-IC). Principal cells were labeled with an antibody against the ing segment-speci c cells (24). Moreover, it became clear that water channel aquaporin-2 (red). Modified from reference 9, with intercalated cells lacked a central cilium, at least in the cortex, permission. which also differentiated them from the adjacent principal cells (Figure 3) (10,28–30). Their peculiar morphology was reminiscent of acid-secreting cells in the turtle bladder transport processes, intercalated cells instead rely on the (31,32) and frog skin (33), and, like these cells, intercalated H1-ATPase (11). Therefore, now it also must be consid- cells participate in urinary acid secretion, bicarbonate reab- ered that cell types with high H1-ATPase levels may use sorption, and bicarbonate secretion (5). this pump for more than just acid-base regulation. The question remains of how these two disparate cell types coordinate their function in these epithelia (20,34). The plasma membrane of each epithelial cell organizes into api- Intercalated Cell Distribution, Nomenclature, cal and basolateral domains that develop distinct membrane Morphology, and Developmental Considerations lipid and transport protein compositions. Thus, within a The cells lining the proximal tubule, thin limb, and thick homogeneous epithelium of one cell type, the apical mem- ascending limb have a homogeneous ultrastructural ap- branes (facing the tubular lumen) of all cells could function pearance. Tubule-specific cells function as a unit within each as a unit, and similarly for the basolateral domains of the segment. Distal to the macula densa, however, the epithelia same cells facing the interstitium. The maintenance of the of the distal convoluted tubule, connecting segment, and apical and basolateral domains of the kidney tubular epithe- collecting duct are lined by different types of cells. Most cells lium depends on the presence of functional tight junctions, in these segments are considered segment-specificprincipal the differential permeabilities of the two membranes, and cells, while the other cells are grouped together and called the generation of gradients between the lumen and the in- intercalated cells (5,12). An impediment to the study of these terstitium. Recent findings have uncovered the paracrine cells has been their pleomorphism and their isolation within signals that integrate the function of intercalated and prin- the epithelium; they sit surrounded by principal cells. Inter- cipal cells in the collecting duct (35). calated cells vary in morphology, localization, and abun- Three types of intercalated cells are traditionally recog- dance, and these differences are evident not only among nized, based largely on cell morphology and localization species but also among individuals from the same species. (reviewed in reference 36). These cells fall into the catego- In addition, few cell lines replicate their phenotypes in cul- ries of type A (also known as “a”), type B (or “b”), and ture. Some of the available immortalized intercalated cell non-A, non-B intercalated cells (Figure 2) (reviewed else- fi models include the rabbit Clone-C cells, the mouse OMCDis, where [10,36]). The initial morphologic classi cation was and the canine MDCKC11 (13–15). The Clone-C cell line is upheld after both functional and immunolabeling studies particularly useful because it can switch phenotypes from once inhibitors and antibodies for individual transport type B to type A intercalated cells (Figure 2) depending on proteins became available. Currently, intercalated cells culture conditions (16,17). Of note, many very relevant studies are classified with respect to the presence of the chloride- that have uncovered important regulatory pathways in bicarbonate exchanger AE1 (Slc4a1) and to the subcellular Clin J Am Soc Nephrol 10: ccc–ccc, February, 2015 Intercalated Cell Physiology, Roy et al. 3

Figure 2. | Transepithelial transport processes and regulatory mechanisms in type A-IC and type B intercalated cells (B-IC). This cartoon illustrates the major transport proteins expressed in the three main epithelial cell types present in the collecting duct: the principal cell, which expresses the epithelial sodium channel; the acid-secreting type A-IC; and type B-IC, which secretes bicarbonate while reabsorbing NaCl. In 1 1 1 the cortical and outer medullary collecting duct, type A-ICs express H -ATPase and the H /K -ATPase at the apical/luminal membrane, while 2 2 they express the Cl /HCO3 exchanger AE1 at their basolateral membrane. The bicarbonate sensor soluble adenylyl cyclase (sAC) and protein 1 2 kinase A (PKA) play important roles in the regulation of the H -ATPase (see Figure 5A). Slc26a11 (A11), an electrogenic Cl transporter, as well 2 2 as a Cl /HCO3 anion exchanger, are also expressed at the apical membrane of the type A-IC. On the other hand, the type B-ICs display an 2 2 electroneutral NaCl transport/reabsorption pathway at their apical membrane that involves pendrin, a Cl /HCO3 exchanger, and the 1 2 2 1 Na -driven Cl /HCO3 exchanger (NDCBE). The proposed basolateral Na extrusion pathway would involve the cotransporter Slc4a9 (AE4). The 2 1 mechanism of Cl exit remains to be elucidated. In type B-ICs, reabsorption of NaCl from the lumen is energized by the basolateral H -ATPase rather 1 1 than by Na /K -ATPase. 4 Clinical Journal of the American Society of Nephrology

the expression of AE1 at their basolateral membrane. On the other hand, the intercalated cells involved in bicarbonate secretion express the chloride/bicarbonate exchanger pendrin at their apical membrane. These cells are further classified as type B and non-A, non-B intercalated cells. While type B intercalated cells express the H1-ATPase at their basolateral pole, the non-A, non-B intercalated cells express both the H1-ATPase and pendrin at their apical membrane. Moreover, non-A, non-B intercalated cells are located in the connecting segment or connecting tubule (10,38). All three types of intercalated cells express carbonic anhydrase in their cytoplasm (36,39). This metalloenzyme reversibly catalyzes the hydration of CO2 (40) and thus leads to the for- mation of bicarbonate. At least 13 isoforms of carbonic anhydrase have been identified in mammals. Carbonic an- 1 hydrase II, acting in tandem with H -ATPase, facilitates the coordinated proton and bicarbonate secretion from intercalated cells. Table 1 summarizes the current findings on transport or other proteins that can help in the characterization and classification of intercalated cell types.

Intercalated Cell Development and Plasticity Interest in intercalated cell development arose, in part, because of the intriguing presence of the non-A, non-B intercalated cell, which some viewed as an intermediate type. Moreover, other intercalated cells did not fit into any of the three available classifications because of the expression of marker proteins in unexpected subcellular localiza- tions. These other types of intercalated cells were thought to represent a series of intermediate forms that could arise if, for example, a type B intercalated cell were transforming into a type A intercalated cell under the pressure of metabolic acidosis. One such “intermediate” cell might express the H1-ATPase diffusely in the cytoplasm or at both the apical and basolateral domains (41,42). There is now evidence that Figure 3. | Morphology of rat cortical collecting duct intercalated type A and type B intercalated cell types represent different cells. (A) The scanning electron micrograph shows the luminal sur- states of differentiation, as proposed by Al-Awqati and con- face of the rat collecting duct. In this image principal cells can be firmed by others (16). In some of these studies carried out in easily identified by their small microprojections into the lumen and the rabbit model, adaptation to acidosis was not accompa- by the presence of a single cilium. Two configurations of intercalated nied by changes in the number of intercalated cells but cells are present in this tubule. The type A-ICs (arrows) have a large rather by a change from type B to type A intercalated cells luminal surface covered mostly with microplicae, while a type B-IC (7,43). The transition process from type B to type A interca- (arrowhead) has a more angular cellular outline and smaller apical microvilli (original magnification, 35500). Reproduced with per- lated cell depends on the basolateral deposition of the extra- mission from reference 10. (B) This transmission electron micrograph cellular matrix proteins hensin (DMBT1), galectin 3, and of rat cortical collecting duct illustrates further the two configurations other proteins (7,44). Overall, induction of chronic metabolic of intercalated cells. The type A-IC (right) shows a well developed acidosis increases the proportion of type A intercalated cells apical tubulovesicular membrane compartment, with prominent while metabolic alkalosis causes an increase of type B inter- microprojections that are part of the microplicae on the luminal calated cells (7,45,46). Moreover, mice with a hensin defect membrane. In this image, the type B-IC (left) presents a denser cytoplasm in intercalated cells indeed developed metabolic acidosis. In with more abundant mitochondria and many vesicles throughout the this mouse model, intercalated cells in the cortex had a type cytoplasm. The luminal membrane of the type B cell has a quite smooth B phenotype (44). Interestingly, the hensin-deficient mice luminal membrane (original magnification, 35000). Reproduced with had modified the phenotype of medullary epithelial cells. permission from reference 10. These modified medullary cells had the ultrastructure of the type B intercalated cells usually found in cortex, while localization of the multisubunit H1-ATPase (36,37). This they differed from type B cells in that they did not express classification has been further modified to reflect whether pendrin (47). cells express the transport protein pendrin, a subtype of In addition to the above factors, the Notch signaling path- chloride-bicarbonate exchanger (37). Therefore, another def- way also controls the development of primitive epithelia, inition for type A intercalated cells combines their function such as the ones in the connecting segment and the collecting in urinary acidification, their lack of pendrin expression, duct (20,48). The Notch signaling cascade is essential for a the presence of H1-ATPase at their apical membrane, and developmental process called lateral inhibition, where Clin J Am Soc Nephrol 10: ccc–ccc, February, 2015 Intercalated Cell Physiology, Roy et al. 5

Table 1. Relevant proteins expressed in kidney intercalated cells

Intercalated Cell Type Protein Expressed Type A Type B Non-A, Non-B

Carbonic anhydrase II: catalyzes the reversible hydration of CO2 Apical Cytoplasmic Cytoplasmic to bicarbonate and water 1 1 H -ATPase: pumps H across the (plasma) membrane, and into Apicala Basolaterala Apical and diffuse the extracellular space vesiculara AE1 (Slc4a1): anion exchanger 1, exchanges a Cl- for Basolaterala abicarbonate Pendrin (Slc26a4): exchanges a Cl- for a bicarbonate Apicala Apicala H,K-ATPase: exchanges an H1 for a K1 at the expense of ATP Apical Apical? AE4 (Slc4a4): anion exchanger 4, exchanges a Cl- for (n) Basolateral bicarbonate RhBG: Rh B glycoprotein, ammonia transporter Basolateral Basolateral RhCG: Rh C Glycoprotein, ammonia transporter Apical Apical NDCBE (Slc4a8): Na1-driven Cl2/bicarbonate exchanger Apical 2 2 - A11 (Slc26a11): electrogenic Cl transporter and Cl /HCO3 Apical anion exchanger NKCC1: Na-K-2Cl cotransporter Basolateral BK channel or Maxi-K: K1 secretion Apical

Adapted from reference 36. BK, big potassium. aThe differential localization patterns pertaining to the transport proteins identify a combination of key markers that aid in the clas- siﬁcation of the different intercalated cell subtypes.

neighboring cells undergo one final step that gives them very cell characteristics. For example, this undifferentiated cell different phenotypes. Notch signaling disruption indeed al- type expresses the water channel aquaporin-2, which is ters collecting duct cellular composition, resulting in more expressed in principal cells, and the cytosolic enzyme car- intercalated cells and fewer principal cells; in addition, prob- bonic anhydrase II, which is used as a marker of interca- ably because of the decreased numbers of principal cells, the lated cells. Foxi1-null mice did not express H1-ATPase or mice also display a nephrogenic diabetes insipidus pheno- pendrin in their collecting ducts. type (48). The developmental events would involve ureteric bud cells differentiating into principal cells, with active Notch Type A Intercalated Cells, Their Major Transport signaling, while intercalated cells would appear because of Proteins, and Their Hormonal Regulation inactive Notch signaling (49,50). Intercalated cell development Type A intercalated cells are present in the late distal goes one step further to generate cells of type B lineage in the convoluted tubule, the connecting segment, cortical and cortex and outer medulla. The expression of grainyhead genes outer medullary collecting duct, and, in some reports, the results in the type B intercalated cell phenotype (21,51,52). early part of the inner medullary collecting duct. They are In rodent kidney, the appearance of intercalated cells dur- the more abundant type of intercalated cell in the outer ing development has traditionally been tracked by following stripe of the outer medulla in most mammalian species (10). the expression of several of the intercalated cell–specific Morphologically, they lack a cilium, they have numerous H1-ATPase subunits and other markers. This multisubunit apical microplicae, and mitochondria are abundant near proton pump is ubiquitous, but several of its subunits are the apical pole (10,28). Their most characterized role is as specific to distal nephron intercalated cells (47,53). These in- cells that secrete protons into urine, and they can easily be tercalated cell–specificH1-ATPase subunits (such as a4 and identified for their lack of pendrin expression. 1 B1) appear early in the medulla at embryonic day 15.5 (E15.5), Type A intercalated cells can secrete H equivalents 1 1 1 after the detection of the expression of Foxi1, a forkhead fam- into urine via the H -ATPase or the H /K -ATPase ily transcription factor that is detected in adult intercalated (H,K-ATPase) at their apical membrane. The latter pump ex- cells (54). Pendrin-positive intercalated cells have been detect- changes one potassium ion for each extruded proton. In ad- ed in the mouse kidney at E14. There was also a debate on dition, these cells express Slc4a1, a splice variant of erythroid whether intercalated cells developed from principal cells or band 3, at the basolateral membrane (Figure 1) (42). The vice versa, or whether one cell population could contribute to secretion of a proton into the tubular lumen, whether it is the repopulation of the collecting duct after injury. Some of in exchange for potassium reabsorption or not, results in the this controversy has been clarified by the characterization of a generation of intracellular bicarbonate via carbonic mouse strain lacking a transcription factor of the forkhead anhydrase II, which is reabsorbed into the interstitium in family, Foxi1 (55). In adult mice, Foxi1 is detected only in exchange for chloride by AE1. The H1-ATPase is very intercalated cells, suggesting that this transcriptional reg- abundant at the apical membrane of type A intercalated ulator is required for intercalated cell development. In- cells and in subapical vesicles or tubulovesicular structures, deed, Foxi1 knockout mice lack intercalated cells, and and they appear as 10-nm spherical structures or “studs” their collecting duct epithelium instead expresses a more coating these membranes, also described as rod-shaped primitive cell type with some principal and intercalated particles (56,57). 6 Clinical Journal of the American Society of Nephrology

1 a a a The H -ATPase facilitates the movement of protons of their two subunit isoforms: HK 1 (gastric) or HK 2 (co- across the apical membrane of type A intercalated cells. lonic) (reviewed in reference 61). Type A intercalated cells 2 a – fi Other ion movements, such as Cl and/or bicarbonate ex- from HK 1,2 null mice had signi cantly slower acid extrusion 1 a trusion, compensate H transport in proton-secreting cells compared with A-type intercalated cells from HK 1-null or 1 a a (32,58). In the case of H -ATPase, two other main factors HK 2-null mice (62), although the lack of either subunit affect its function at the plasma membrane: the pH differ- affected the rate of acid extrusion from both type A and ence across the apical membrane and the transepithelial po- type B cells (63). tential difference (59). For example, this pump mediates H1 Therefore, type A intercalated cells in the collecting duct 1 1 transport at a rate that is 0 when the luminal pH is ,4.5, participate in active potassium reabsorption via the H /K - while the transport rate of the pump is saturated at a pH of ATPases (Figure 2), and they undergo hypertrophy when 7.0–8.0. In addition, when the lumen potential is 120 mV animals are fed a low-potassium diet. However, the contri- relative to the interstitium, the H1 transport rate is negligible. bution of each a isoform in kidney potassium and acid han- On the other hand, at an applied potential of 230 mV the H1 dling remains to be determined, as the null mice for either a a fi pumping rate saturates. A hypothesis was then presented that the 1 or 2 subunits did not have signi cant changes in Na1 reabsorption through ENaC would create a more lumen their acid-base or potassium excretion (62). In the setting negative transtubular potential difference along the distal of acidosis, the activity of H1/K1-ATPase increases in the nephron. This potential difference would favor H1-ATPase collecting duct (64,65). These findings indicate that this function in type A intercalated cells. Wagner and colleagues pump represents a key mechanism of proton secretion by elucidated in vivo that ENaC function in the connecting seg- the kidney in metabolic acidosis. ment was sufficient to induce this upregulation (60). In addition to the H1/K1-ATPase, another important Both AE1 and H1-ATPase are upregulated in the kidney regulator of K1 homeostasis, the high-conductance big po- during metabolic acidosis (45), and the morphology of tassium (BK) or maxi-K channel is also highly expressed at type A intercalated cells changes (Figure 4) (10). Although the apical membrane of type A intercalated cells (66), es- the entire collecting duct contributes to urinary acidifica- pecially in animals fed a high-potassium diet. These chan- tion, the outer medullary collecting duct is very important nels, which are less abundant in principal cells, are in this process, and most intercalated cells in this segment activated by depolarization, stretch/luminal flow, intracel- are of type A expressing apical H1-ATPase (5,42). In ad- lular calcium increases, or hypoosmotic stress (reviewed in dition, in collecting duct cells, including intercalated cells, reference 67). Moreover, the BK channel is inhibited by the H1/K1-ATPases can be detected by following the expression with-no-lysine kinase 4 (WNK4) in a phosphorylation- dependent manner (68,69). In a later study performed in a cell line of intercalated cell characteristics, WNK4 inhibited BK channel activity in part by increasing channel ubiquiti- nation and degradation (70). The researchers investigating flow-induced K secretion from the BK channel in type A intercalated cells found evidence that a basolateral Na-K-Cl 1 cotransporter (NKCC1) could support entry of K into the cell, especially in light of the almost negligible levels of 1 1 Na /K -ATPase in this type of intercalated cells (Figure 2) (71).

Acid-Base Sensing in Intercalated Cells The activity and subcellular localization of acid-base transport proteins can be quickly altered by changes in extracellular or intracellular pH or bicarbonate changes. These ﬁndings have led to an intense search for the pH- sensing mechanism within the intercalated cells. For exam- 1 ple, the H -ATPase in type A intercalated cells is acutely activated (within minutes to hours) by kinases such as pro- Figure 4. | Change in intercalated cell morphology in response to tein kinase A (PKA), while it is acutely inhibited by the chronic acid-base status changes. This transmission electron micro- metabolic sensor AMP activated protein kinase (AMPK) graph shows the apical membrane of type A cells from the collecting (Figure 5) (72–74). This downregulation of the proton duct from a normal rat (A) and from a rat with acute respiratory acid- pump by AMPK is preliminary evidence that intercalated osis (B). In the control animal prominent studs (arrowheads) are cell function can be altered when the tubule is under meta- observed on tubulovesicular structures in the control rat (A), while bolic stress, such as under conditions of ischemia. These two they are more abundant on the apical membrane in the experimental kinases also regulate the H1-ATPase in proximal tubule and animal (B). In another study, Brown and colleagues showed that these epididymis (75,76). We have characterized two major phos- studs are H1-ATPase, both at the membrane and in the tubulove- phorylation sites in the H1-ATPase A subunit; Ser-175 is sicular subapical structures (57). The increase in the number of H1-ATPases (studs) at the apical membrane in the animal with acidosis required for PKA-mediated pump activation while Ser-384 (B) is coupled to an increase in membrane microprojections and a de- is required for downregulation of the pump by AMPK crease in the number of tubulovesicular structures in the acidotic rat (B). (72,73). In particular, Ser-175 is in part responsible for the 1 (Original magniﬁcation: A, 348,000; B, 342,400.) Reproduced with activation of H -ATPase in endosomes downstream of G permission from reference 10. protein–coupled signaling via cAMP/PKA (77). Clin J Am Soc Nephrol 10: ccc–ccc, February, 2015 Intercalated Cell Physiology, Roy et al. 7

Figure 5. | Model of H1-ATPase coregulation at the apical membrane of A-ICs by two kinases, downstream of acid-base status and of cellular metabolic stress. (A) Acute increases in the level of intracellular bicarbonate activates the bicarbonate-sensor sAC, which generates cAMPand then activates PKA. Studies using pharmacological activators have shown that exchange protein directly activated by cAMP (Epac) is not likely to play a role in H1-ATPase regulation (183). Carbonic anhydrase II (CAII) is involved in the generation of intracellular bicarbonate. Downstream of PKA, the A subunit of H1-ATPase is then phosphorylated at Ser-175 (S175) (72). This phosphorylation event is involved in 1 1 activating H -ATPase at the apical membrane. (B) This acute stimulatory effect of the sAC/cAMP/PKA signaling cascade on apical H -ATPase activity is counterbalanced by the inhibitory effect of the metabolic sensor AMP activated protein kinase (AMPK), downstream of ischemia or metabolic stress, for example. Acute metabolic stress leads to an elevation of the levels of AMP compared with ATP, and the increase in cellular 1 [AMP]/[ATP] directly activates AMPK. AMPK mediates the downregulation of H -ATPase activity by phosphorylating Ser-384 (S384) in the proton pump’s A subunit (73).

1 In addition, PKA activation of the H -ATPase has also been Another important sensor of extracellular pH in the kidney linked to the acute activation of the bicarbonate-sensor soluble is the G protein–coupled receptor 4 (GPR4). Its function was adenylyl cyclase (sAC) in type A intercalated cells (Figure 5A) identiﬁed and characterized in cell lines in studies that used (74). This cascade links physiologic changes in plasma and/or the mOMCD1 intercalated cell model (18). GPR4-null mice intracellular bicarbonate to acute intercalated cell phenotypic had decreased kidney net acid secretion and a nongap met- changes. For example, the PKA activator cAMP directly stim- abolic acidosis, suggesting that GPR4 is a pH sensor with a ulates proton secretion in primary cultures of mouse interca- likely important role in promoting acid secretion in kidney lated cells (78) while signiﬁcantly increasing the size of apical collecting duct intercalated cells in vivo. Moreover, the non- microplicae in these cells (78). The role of bicarbonate- receptor tyrosine kinase Pyk2 was studied as a potential activated sAC in intercalated cell function is also supported sensor of pH changes in the distal tubule. Pyk2 is expressed in cases of increased chronic luminal bicarbonate delivery in kidney, and it becomes autophosphorylated by several (over days) to the collecting duct (79). A recent study found stimuli, including decreased pH (81). Studies also performed that Slc26a11, recognized as an electrogenic Cl2 transporter as in mOMCD1 cells revealed that Pyk2 and downstream ef- 2 2 1 1 1 1 well as a Cl /HCO3 anion exchanger, facilitates H -ATPase fectors increased H -ATPase but not H /K -ATPase in this function in type A intercalated cells (80). intercalated cell model. 8 Clinical Journal of the American Society of Nephrology

Type B Intercalated Cells and Their Transport have confirmed that the paracellular pathway plays an im- Processes portant role in Cl2 reabsorption in the collecting duct (93). The classic type B intercalated cells express a chloride- On the other hand, the role of type B intercalated cells in Cl2 bicarbonate exchanger, pendrin, at their apical membrane reabsorption became clear when pendrin was identified in 1 and express H -ATPase at their basolateral membrane these cells (82). Because both sodium and chloride intake can (Figure 6) (41,82). These cells are thought to be responsible modulate kidney function, pendrin was considered immedi- for the secretion of OH2 equivalents. Pendrin is encoded ately as a potential mediator in the pathogenesis of hyper- by the SLC26A4 PDS gene. This transport protein is a mem- tension (90,94). Several studies have confirmed that pendrin ber of the multifunctional sulfate transporter solute carrier protein expression decreases with maneuvers that increase 26 family (SLC26). Although it does not transport sulfate urinary Cl2 excretion, while protocols that caused Cl2 (83), it can transport anions such as iodide, Cl2, and bicar- depletion, such as furosemide treatment, significantly in- bonate (84). Pendrin is also expressed in epithelial cells in the creased pendrin levels. Later studies showed that metabolic inner ear (85) and the thyroid, as well as in other tissues acidosis induced by acetazolamide or ammonium sulfate ad- (82,86). This differential tissue expression pattern highlights ministration reduced the levels of pendrin irrespective of low different transport roles for pendrin. For example, in kidney Cl2 levels in urine (95). Aldosterone and angiotensin II also intercalated cells and in the inner ear, pendrin acts as a increase the levels of pendrin (96,97), thus linking this trans- chloride-bicarbonate exchanger (85), while in the thyroid port protein to the maintenance of adequate extracellular gland it mediates iodide transport (86). Therefore, mutations volume and potentially to the development of hypertension. in SLC26A4 involve various organs to differing degrees. Recently, it has been proposed that pendrin may be activated Vaughan Pendred described this disorder as the combina- via akidney-specific mechanism that does not include circu- tion of deafness and goiter (87,88). Remarkably, both pa- lating aldosterone (98). Furthermore, angiotensin II mediated tients with Pendred syndrome, which is inherited in an pendrin upregulation and subcellular localization changes autosomal recessive pattern, and pendrin-null mice have in kidney occur via the angiotensin 1a receptor (99). Pendrin- normal kidney function under baseline conditions (82,89). mediated Cl2 uptake leads to vascular volume expansion However, upon bicarbonate administration, which in- and pendrin-induced modification of ENaC abundance and duces pendrin upregulation in wild-type mice, pendrin- function (98). Changes in luminal bicarbonate concentration null mice developed a severe metabolic alkalosis. These and/or pH could also contribute to this signaling (100). alkali-loaded animals deficient in pendrin could not secrete Thus, pendrin expressed in the kidney might represent a bicarbonate, suggesting a critical role for pendrin in the potential target for blood pressure control. This role is illus- modulation of acid-base status that protects against meta- trated in pendrin-null mice that were resistant to mineralo- bolic alkalosis (90,91). corticoid-induced hypertension and in turn became Initially, the reabsorption of Cl2 in the collecting duct was hypotensive when fed a low-sodium diet. attributed to the paracellular pathway, with some studies Both type B and non-A, non-B intercalated cells (Table 1) indicating that there was transcellular Cl2 absorption (92) express pendrin at their apical membrane and intracellu- (reviewed in references 4,37). Indeed, very recent studies lar vesicles (Figure 6) (82,101). Pendrin is more abundant

Figure 6. | Model of major transport processes and regulatory mechanisms in B-ICs. This cartoon is modiﬁed from the model proposed by 1 Chambrey and colleagues (35). These cells participate in electroneutral NaCl absorption, which is energized by H -ATPase. Two cycles of 1 2 transport via pendrin, when coordinated with one cycle of NDCBE results in net uptake of one Na and one Cl and the extrusion of two bicarbonate 2 2 2 (HCO3 )ions.TheCl ions recycle across the apical membrane. A basolateral channel (not shown) mediates Cl exit. The basolateral anion 1 1 exchanger 4 (AE4 or Slc4a9), together with the H -ATPase also facilitates Na and bicarbonate exit. Clin J Am Soc Nephrol 10: ccc–ccc, February, 2015 Intercalated Cell Physiology, Roy et al. 9

1 at the apical plasma membrane than intracellularly in medullary small-conductance K channel (ROMK) expressed non-A, non-B cells under baseline conditions. This find- in principal cells and the flow-mediated, large-conductance, ing suggests that pendrin-mediated anion exchange oc- calcium-activated potassium channel or BK channel, which curs to a greater extent in non-A, non-B cells than in type is expressed in intercalated cells (Figure 7) (71,115). Thus, B cells (102). This difference in pendrin subcellular dis- potassium secretion is promoted without retaining sodium. tribution also suggests that its function is likely regu- It is paradoxical that activation of the same MR in the in- lated by trafficking between the plasma membrane and tercalated cell would result in increases of both NaCl reab- cytoplasmic vesicles. Nitric oxide also contributes to inter- sorption and regulation of acid-base transport proteins calated cell transport protein regulation as it reduces (110). The RAAS is activated during metabolic acidosis pendrin protein abundance in the kidney without modu- (116), while RAAS blockade inhibits kidney acid excretion lating pendrin subcellular localization in a cAMP-dependent in the setting of acidosis (112,117). The regulatory effects of manner (103). The regulation of pendrin also involves steroid hormones on individual transport proteins in inter- glycosylation-dependent processes (104,105). In addition to calated cells have been extensively studied. For example, the regulation of acid-base status, it appears that pendrin decreased dietary potassium increases type A intercalated expressed in kidney intercalated cells plays a role in con- cell function apical projections and increases the activity of trolling iodide homeostasis as well, especially during high H1/K1-ATPase and the BK channel (66). However, under water intake (106). potassium-replete conditions, which are known to increase Type B intercalated cells have recently come to promi- aldosterone levels, changes in dietary sodium and serum nence with the discovery that the Na1-driven chloride/ aldosterone did not affect BK expression in intercalated bicarbonate exchanger (NDCBE) (107) and pendrin colo- cells (118). calize at the apical membrane of these cells (Figure 6). There are several important findings to consider in the Together these two transport proteins mediate the until regulation of H1-ATPase and other transport proteins recently unexplained thiazide-sensitive electroneutral in intercalated cells by steroid hormones. For example, NaCl reabsorption in the collecting duct (4,11,107). More- Wagner and colleagues showed that angiotensin II in- over, pendrin deletion in type B intercalated cells disturbs creases the expression of the H1-ATPase (119). More- the function of ENaC in adjacent principal cells (98,100). over, mineralocorticoids, such as aldosterone, which is As in intercalated cells, NaCl absorption is energized by released in metabolic acidosis, also increase the expres- the H1-ATPase and not by the Na1/K1-ATPase (11); sion of pendrin and the H1-ATPase (58,97). Recently a 1 Eladari and colleagues hypothesized that Na transport in novel modulatory pathway that affects the MR in inter- type B intercalated cells was mediated by a bicarbonate- calated cells has been uncovered. This pathway, which dependent transport protein at the basolateral membrane. involves MR phosphorylation, is likely the key to the 1 This transport protein therefore would be energized by H - differential response of these cells when only aldosterone ATPase.Membersofthesameresearchgrouphadpre- is present (as in hyperkalemia), in contrast to when both viously shown that the anion exchanger 4 (AE4/Slc4a9) aldosterone and angiotensin II are present (as in volume was expressed basolaterally in type B intercalated cells (Fig- depletion) (see Figure 8) (69,120). The MR can be acti- ure 1) (108). vated by both aldosterone and cortisol (121–123). While Acidosis induces important changes in the transport pro- principal cells express the enzyme 11b hydroxysteroid teins expressed in type B intercalated cells. For example, dehydrogenase (11bHSD2), which rapidly converts cor- acidosis downregulates pendrin expression at their apical tisol to a less active hormone, cortisone, intercalated cells membrane and in apical recycling endosomes, and more- lack this enzyme (69,110,121). Thus, in principal cells over, AE4 expression is diminished at their basolateral the MR is mostly activated by aldosterone and not cor- pole (109). tisol, and this activation leads to the coordinated action of ENaC and the Na,K-ATPase. A recent study elegantly showed that a single phosphorylation site, Ser-843, in the Regulation of Intercalated Cell Transport Proteins by MR inactivates it, and this inactive receptor is the major Hormones and Other Mediators: Effects of (Pro)renin, species in intercalated cells (69). When there is volume Renin, Angiotensin, and Aldosterone depletion (Figure 8B), both angiotensin II and WNK4 Steroid hormone receptors, such as the mineralocorticoid promote dephosphorylation/activation of the MR. Once receptor (MR), function downstream of the renin-angiotensin- active, this receptor binds aldosterone and mediates the aldosterone system (RAAS). Together with the glucocorticoid increase in H1-ATPase and chloride-bicarbonate exchanger receptor these hormones elicit signaling events in the kidney activity. This coordinated action increases plasma volume that regulate salt and potassium homeostasis, intravascular while inhibiting potassium secretion. In the absence of angio- volume (110), and cell metabolism (111–113). Aldosterone, tensin II, the receptor remains phosphorylated and thus aldo- via the MR, in turn activates ENaC in principal cells, result- sterone cannot promote H1-ATPase activity. In this scenario, ing in an increase in paracellular Cl2 transport, H1 secre- aldosterone promotes K1 secretion from principal cells. An- tion from intercalated cells, and activation of basolateral giotensin II also increases net transcellular chloride absorption Na1/K1-ATPase (reviewed in references 60,110,114). How- across type B cells via a mechanism that involves both pendrin ever, these transcellular transport cascades are mostly rel- and activation of the H1-ATPase (96). Together these findings evant to principal cells (11). show the physiologic relevance of selective mineralocorticoid During hyperkalemia, aldosterone is also secreted. Its activation in intercalated cells. stimulation of ENaC via the MR leads to lumen electro- The role of renin on intercalated cell function has come to 1 negativity, which promotes K secretion via the renal outer the forefront because of the identification of the (Pro)renin 10 Clinical Journal of the American Society of Nephrology

1 Figure 7. | Intercalated cells are involved in K secretion in the collecting duct. This cartoon illustrates the location of the voltage- 1 dendent renal outer medullary small-conductance K (ROMK) and the flow-dependent big potassium (BK) channels in the intercalated 1 cells (type A-IC and type B-IC) and principal cells. (A) Under normal dietary conditions, ROMK predominantly secretes K . However, 1 1 1 under the influence of a K -rich diet, because of the increased activity of the basolateral Na /K -ATPase, the transport via the epithelial 1 sodium channel (ENaC)– transport increases, generating a driving force for ROMK to secrete more K from the principal cells. (B) When 1 high K secretion is accompanied by increased flow, it stimulates BK channel synthesis and function in the intercalated cells (type A-IC and type B-IC). receptor [(P)RR] as an accessory protein of H1-ATPase. prorenin-induced activation of Erk1/2 in a cultured cell However, and just like H1-ATPase, (P)RR is ubiquitously model of intercalated cells (133). (P)RR has also been expressed. This receptor protein has a large extracellular shown to play a role in vasopressin-mediated H1-ATPase domain that binds the enzymes renin and pro-renin (124– activity and cAMP accumulation, in a prorenin-independent 126). (P)RR was referred to as the ATP6AP2 (ATPase, H1 manner in the intercalated cells. The (P)RR has been im- transporting, lysosomal accessory protein 2) (127). (P)RR plicated in several pathologic conditions, such as diabetic undergoes proteolytic cleavage to generate soluble (P)RR nephropathy, hypertension, albuminuria, and pre- (128,129). (P)RR is easily detected in type A intercalated eclampsia. However, (P)RR blockade would likely have 1 cells and not in principal cells (130,131). Binding of renin adverse effects through its activation of H -ATPase and prorenin to (P)RR increases the catalytic efficiency of (130,134–136). these enzymes in the conversion of angiotensinogen to angiotensin by 4-fold. Prorenin secreted from the principal cells binds to the (P)RR located in the apical mem- Intercalated Cell Dysfunction and the Pathogenesis of brane of intercalated cells or to the soluble (P)RR, and Distal Renal Tubular Acidosis thus enhances local angiotensin I and angiotensin II for- Mutations in certain intercalated cell transport proteins, mation in the collecting duct (Figure 9) (114,132). A (P) or associated enzymes, can lead to distal renal tubular ac- RR interaction with the H1 -ATPase is required for idosis (dRTA) type 1, a syndrome characterized by a decreased Clin J Am Soc Nephrol 10: ccc–ccc, February, 2015 Intercalated Cell Physiology, Roy et al. 11

Figure 8. | Model of regulatory pathways for the mineralocorticoid receptor (MR) in intercalated cells: hyperkalemia and volume depletion. This figure depicts pathways that involve the differential phosphorylation state of the MR in principal versus intercalated cells (type A-IC; type B-IC), that are likely the key to the distinct responses of these cells in two different scenarios. (A) The first scenario involves conditions when only aldosterone is present (as in hyperkalemia). In this case, hyperkalemia leads to aldosterone secretion while no angiotensin II is present. Here, MR is phosphorylated in intercalated but not in principal cells. These conditions lead to aldosterone-mediated Na1 reabsorption via the epithelial sodium channel in principal cells, which drives K1 secretionalsoinprincipal cells. (B) In contrast, when both angiotensin II and aldosterone are present (as in intravascular volume depletion), the MR is dephos- phorylated downstream of angiotensin II, and the activity of this receptor is thus restored in intercalated cells. In addition, as a result of aldosterone signaling both pendrin and H1-ATPase are upregulated, and in turn there is a decrease drive for K1 secretion. Modified from reference 69. capability of urinary acidification and variable degrees of H1-ATPase a4 and B1 subunits result in early-onset dRTA hyperchloremic, hypokalemic metabolic acidosis (137). When (140–142). These findings suggest that the differential distri- this condition is severe, the patient cannot excrete the daily bution of isoforms of H1-ATPase in intercalated versus acid load, which varies from 50 to 100 mEq/d depending on other epithelial cells may be acquired during early infancy the patient’s diet. This continued proton accumulation leads in humans. Another important transport protein in inter- to a normal anion gap metabolic acidosis, with values of calated cells is the chloride-bicarbonate exchanger AE1. A plasma bicarbonate that can fall below 10 mEq/L. The treat- large screen of kindreds with dRTA revealed that some mu- ment involves potassium and alkali repletion. When the dis- tations in AE1 cause autosomal dominant dRTA, while other ease is not so severe, it is called incomplete, type 1 dRTA. defects identified in AE1 appear to result in autosomal reces- These patients still have an inability to acidify the urine sive dRTA (143–145). In a mouse animal model, that deletion (pH.5.5) without a significant drop in plasma bicarbonate. of carbonic anhydrase II reduces the number of intercalated Some forms of dRTA are due to a back-leak of protons re- cells in both type A and type B intercalated cells (146). sulting from the exposure of the patient to pharmacologic In humans, dRTA is often accompanied by some degree agents that damage the tubular plasma membrane, such as of salt, water, and potassium losses, and the causes for these amphotericin (138). However, these patients will often dis- findings were not well understood (147). Recently, a mouse play signs and symptoms associated with principal as well as model of dRTA, which was deficient in the intercalated cell– 1 intercalated cell dysfunction. specific B1 subunit of H -ATPase (ATP6V1B1), also present- Inherited forms of dRTA most often result from the dis- ed with urinary losses, leading to hypovolemia, hypokalemia, ruption of normal type A intercalated cell physiology. Pa- and polyuria (Figure 10) (35). Although these mice did not tients with dRTA tend to have acidemia because of the lack present with acidosis unless challenged with an acid load of acid secretion from type A intercalated cells. This lack of (148), the molecular defect in H1-ATPase led to dysfunction acid secretion, and alkalinization of the urine, can present of both ENaC and the combined pendrin/NDCBE (35). Re- with nephrocalcinosis and/or nephrolithiasis, as well as searchers studying this mouse model discovered that the hypokalemia (139). The inherited forms of dRTA have NaCl urinary loss coincided with increased prostaglandin been described as having autosomal dominant or recessive E2 (PGE2) and ATP levels in urine. PGE2 is a known inhib- modes of transmission. Some disease-causing mutations of itor of Na1 transport in the collecting duct (149–151). 12 Clinical Journal of the American Society of Nephrology

Figure 9. | Intercalated cells are targets of paracrine angiotensin II signaling. Angiotensin II stimulates secretion of prorenin and renin from principal cells. Prorenin binds to the prorenin receptor at the apical membrane of type A-IC, where in turn it stimulates the synthesis of angiotensin II from angiotensin I via the action of angiotensinogen, which originates in proximal segments of the nephron. Angiotensin II then binds to the angiotensin receptor 1a (AT1aR) in the type A-IC. Moreover, prorenin binds to its receptor in type A-IC to stimulate signaling via p58 or cAMP.

in vivo Indeed, when PGE2 synthesis was blocked ,the dysfunction. The estimates of patients with Sjögren syn- animals displayed restored ENaC levels in the cortex drome and dRTA vary from 30% to 70%; this condition (35). This elegant study proposed a mechanism wherein often presents in the context of tubulointerstitial nephritis type B intercalated cells would sense increases in NaCl (154–156). Autoimmune antibodies isolated from patients delivery to the distal nephron, leading to release of ATP interact with the intercalated cells, but the precise antigens from intracellular stores. This ATP release would be fol- have not been determined (157). Kidney biopsies of pa- lowedbyanincreaseinPGE2fromthetypeBintercalated tients with Sjögren syndrome and dRTA reveal a reduced cells, and then to a decrease in Na1 absorption by the expression of H1-ATPase and AE1 (158,159). There is also adjacent principal cells. decrease in the total number of intercalated cells in these pa- In addition, rare genetic defects, such as carbonic anhy- tients. Taken together, Sjögren syndrome is associated with drase II deficiency, can lead to intercalated cell as well as loss of intercalated cell transport activity associated with de- proximal tubular dysfunction. This renal tubular acidosis is fects in acid-base balance, although the precise events in the also referred to as type 3 or mixed renal tubular acidosis developmentofthesefindings are yet to be explored. (137,152). The patients with this genetic defect also present Lithium is a proven treatment for bipolar disorder, and with osteopetrosis and cerebral calcification. approximately 40% of patients receiving this treatment develop urinary concentrating defects and eventually nephrogenic diabetes insipidus, which can contribute to ESRD Acquired Intercalated Cell Dysfunction (reviewed in references 160,161). One of the many renal man- Amphotericin can cause a generalized dysfunction of ifestations of long-term lithium treatment is that patients the kidney tubule, including intercalated cells, because it develop an incomplete form of renal tubular acidosis that damages the plasma membrane and causes an H1 back-leak is usually not clinically relevant (162). Despite this clinical of protons due to the exposure (138,153). On the other syndrome, the findings in rats treated with lithium reveal hand, some conditions, such as Sjögren syndrome and increased numbers of type A intercalated cells that are pres- some cases of systemic lupus erythematosus, that can present even in the inner medulla (163). It has been proposed ent with dRTA due to more predominant intercalated cell that lithium decreases the gradient for proton secretion Clin J Am Soc Nephrol 10: ccc–ccc, February, 2015 Intercalated Cell Physiology, Roy et al. 13

Figure 10. | Intercalated cells are necessary to maintain body fluid and electrolyte balance. This model summarizes the recent findings by 1 Gueutin and colleagues (35), which showed how H -ATPase dysfunction in type B-IC leads to the urinary water and sodium losses in patients 1 with distal renal tubule acidosis. This group studied mice with significantly decreased H -ATPase activity in the collecting duct intercalated 1 cells due to knockout of the B1 of the H -ATPase subunit (ATP6V1B1–/–mice). The specific knockout of the B1 subunit does not disturb 1 H -ATPase expression in the proximal tubule. These animals presented with a significant urinary loss of NaCl, revealing impaired function of epithelial sodium channel (ENaC) in principal cells, as well as decreased pendrin and NDCBE function in type B-IC in the cortical collecting duct. These animals had an upregulation of ENaC in the medulla. High levels of prostaglandin E2 (PGE2) and ATP were detected in the urine of these animals. When PGE2 was normalized using pharmacologic agents these animals also normalized their ENaC levels in the cortex, and they had improved 1 polyuria and hypokalemia. When the H -ATPase was inactivated in type B-IC, it resulted in ATP release with the subsequent increases in PGE2 release from these cell as well.

(a voltage-dependent defect) along with a concomitant de- Findings similar to those observed with long-term crease in H1-ATPase activity. Therefore, the observed increase lithium treatment were described after dietary K1 deple- in the number of intercalated cells may be a potential defense tion, with increases in intercalated cells that were reversed mechanism against the initial mechanism of lithium-induced upon dietary K1 repletion (167). These researchers concluded acidosis. Moreover, lithium inhibits glycogen synthase kinase that principal cells and intercalated cells may interconvert 3b (reviewed in reference 164). Inhibition of this kinase dur- in response to changes in dietary K1 and that autophagy ing kidney development induces nephron differentiation via was likely involved in the pathways driving this intercon- the Wnt signaling pathway (165). Wnt signaling, a very rel- version. evant cascade needed for normal embryonic development, The antirejection medications cyclosporine and tacrolimus has been reviewed elsewhere (166). Therefore, another expla- affect intercalated cell function and result in the development nation for the increased number of intercalated cells seen of dRTA, numerous changes in proximal and distal nephron with lithium treatment could be the disruption of Wnt sig- epithelial transport proteins, and a decrease in the non–type naling downstream of lithium-mediated glycogen synthase A intercalated cells (168,169). Cyclosporine also inhibits cy- kinase 3b inhibition. clophilin and thus affects hensin polymerization after 14 Clinical Journal of the American Society of Nephrology

Figure 11. | The intercalated cells as key effectors of the innate immune system. This model presents various signaling cascades triggered in intercalated cells by pathologic conditions that can result in the release of defensins. Collecting duct epithelial cells respond to gram-negative bacteria (uropathogenic Escherichia coli [UPEC]) by activating cascades drownstream from the toll-like receptor 4 (TLR4) and some TLR4- independent pathways (lower). Ischemic injury also induces release of the defensin neutrophil gelatinase-associated lipocalin from intercalated cells (NGAL) (upper), and type A-IC also secrete the defensin RNAase7, which is a key molecule to prevent urinary tract infection. 1 Funtional apical H -ATPase is also relevant for the type A intercalated cell anti-microbial function. extracellular deposition, a step that when defective reduces lipopolysaccharides in the outer membrane of gram-negative terminal differentiation of intercalated cells (170). bacteria, is expressed in mouse collecting duct intercalated cells (172). TLRs induce a dynamic immune response via sev- Intercalated Cells and the Innate Immune Response eral signaling pathways, including the production of antimi- The epithelial cells lining the urinary tract play a major crobial peptides (AMPs) (Figure 11) (172,173). role in maintaining the sterility of urine, which is achieved AMPs are polypeptides of ,100 amino acids that at phys- by mucus production, urinary flow, and bladder emptying, iologic concentrations are active against bacteria, enveloped all of which provide immediate defense against microbial viruses, fungi, and protozoa. AMPs are expressed by neutrophils invasion. This innate defense cascade is less specificthan or epithelial cells constitutively or after induction by exposure of the adaptive immune response. Among the better-known the cells to pathogens (174). Urinary tract and kidney epithelial components of the innate immune response are the Toll-like cells synthesize several AMPs, including defensins, cathelicidin, receptors (TLRs). TLRs play a key role in containing urinary hepcidin, and ribonuclease 7 (RNAse7). Among these AMPs, tract infections and pyelonephritis. More than 13 TLRs have defensins and RNAse7 are expressed in the kidney collecting been identified in mammals, and kidney tubular epithelial duct (175). Defensins, which have broad-spectrum antimi- cells express most of them. Among these TLRs, TLR4 has been crobial activity, are classified into two main subfamilies: implicated in the innate immune defense against uropatho- a-andb-defensins. Of the b-defensins, human b-defensin- genic Escherichia coli, the most frequent pathogen responsible 1andb-defensin-2 are expressed in the loop of Henle, distal for urinary tract infection (171). TLR4, which recognizes tubule, and collecting duct. In contrast to human b-defensin-1, Clin J Am Soc Nephrol 10: ccc–ccc, February, 2015 Intercalated Cell Physiology, Roy et al. 15

human b-defensin-2 is not constitutively expressed in the This work was supported in part by the National Institutes of kidney, but instead its upregulation has been implicated Health grant R01-DK084184 and P30-DK079307, as well as by a in the immune response during chronic kidney infection Junior Scholar Award of the Department of Medicine at the Uni- (176). On the other hand, RNAse7 is a potent AMP con- versity of Pittsburgh School of Medicine (N.M.P.-S.) and F32- stitutively expressed by the uroepithelium of the bladder, ure- DK097889 (M.M.A.). ter, and intercalated cells. Urinary concentrations of RNAse7 are much greater than those of other AMPs, exhibiting a Disclosures bactericidal activity against both gram-negative and Recent funding was received from Sanofi (for a postdoctoral gram-positive bacteria. fellowship grant to M.M.A.). Another process that implicates type A intercalated cells in the immune response during urinary tract infection is that these cells secrete the bacteriostatic protein lipocalin 2, References also known as neutrophil gelatinase–associated lipocalin 1. Crayen ML, Thoenes W: Architecture and cell structures in the (NGAL). An important difference between NGAL and distal nephron of the rat kidney. Cytobiologie 17: 197–211, 1978 other AMPs is that NGAL expression is intensively upre- 2. Davies JA, Davey MG: Collecting duct morphogenesis. Pediatr gulated by both septic and aseptic causes of AKI (177,178). Nephrol 13: 535–541, 1999 Barasch and colleagues showed that type A intercalated cells 3. Meneton P, Loffing J, Warnock DG: Sodium and potassium are key elements in the production of this bacteriostatic pro- handling by the aldosterone-sensitive distal nephron: The piv- tein. They used a mouse model expressing an NGAL re- otal role of the distal and connecting tubule. Am J Physiol Renal Physiol 287: F593–F601, 2004 porter gene, which showed NGAL expression after kidney 4. Eladari D, Chambrey R, Peti-Peterdi J: A new look at electrolyte ischemia (179,180). In contrast to most of the AMPs, NGAL transport in the distal tubule. Annu Rev Physiol 74: 325–349, 2012 is specific in its binding to enterochelin (Ent), a product of 5. Madsen KM, Tisher CC: Structural-functional relationship along gram-negative bacteria that removes Fe31 from transferrin the distal nephron. Am J Physiol 250: F1–F15, 1986 31 6. Smith H: The Kidney: Structure and function in Health and (181,182). When NGAL is secreted, it binds the Ent:Fe Disease, Oxford, Oxford University Press, 1951 complex and thus it exerts its bacteriostatic effects (181,182). 7. Al-Awqati Q: Terminal differentiation in epithelia: The role of In addition to its effects on Ent, NGAL secretion is required integrins in hensin polymerization. Annu Rev Physiol 73: 401– for TLR4 to undergo activation (179). Moreover, mice that 412, 2011 lack type A intercalated cells have reduced urinary acidifica- 8. Rodrı´guez Soriano J: Renal tubular acidosis: The clinical entity. J Am Soc Nephrol 13: 2160–2170, 2002 tion and lipocalin 2/NGAL secretion into urine, as well as 9. Weiner ID, Mitch WE, Sands JM: Urea and ammonia metabo- decreased clearance of bacteria from urine (179). The impor- lism and the control of renal nitrogen excretion [published tance of intercalated cells in the innate immune response is online ahead of print July 30, 2014). Clin J Am Soc Nephrol doi: highlighted by the recent characterization of yet another 10.2215/CJN.10311013 10. Madsen KM, Verlander JW, Tisher CC: Relationship between scavenger receptor called secretory glycoprotein S5D-SRCRB structure and function in distal tubule and collecting duct. derived from these cells (183). J Electron Microsc Tech 9: 187–208, 1988 11. Chambrey R, Kurth I, Peti-Peterdi J, Houillier P, Purkerson JM, Summary and Conclusions Leviel F, Hentschke M, Zdebik AA, Schwartz GJ, Hu¨bner CA, fi fi Eladari D: Renal intercalated cells are rather energized by a When intercalated cells were rst identi ed, their mor- proton than a sodium pump. Proc Natl Acad Sci U S A 110: phology was distinctive and reminiscent of acid-secreting 7928–7933, 2013 cells in more primitive epithelia. Since the 1960s, the es- 12. Kaissling B, Kriz W: Structural analysis of the rabbit kidney. Adv sential role of the intercalated cells in acid-base homeostasis Anat Embryol Cell Biol 56: 1–123, 1979 was solidified, first with the characterization of the trans- 13. Edwards JC, van Adelsberg J, Rater M, Herzlinger D, Lebowitz J, al-Awqati Q: Conditional immortalization of bicarbonate- port processes in the distal nephron and later with the iden- secreting intercalated cells from rabbit. Am J Physiol 263: tification of individual membrane transport proteins, such C521–C529, 1992 1 as H -ATPase, AE1, and pendrin, contributing to those pro- 14. Guntupalli J, Onuigbo M, Wall S, Alpern RJ, DuBose TD Jr: 1 1 1 cesses. The role of intercalated cells in the pathogenesis of Adaptation to low-K media increases H( )-K( )-ATPase but not H(1)-ATPase-mediated pHi recovery in OMCD1 cells. Am J distal (type 1) and mixed renal tubule acidosis has been Physiol 273: C558–C571, 1997 clearly established. In the last few years, the acid-base cel- 15. Gekle M, Wu¨nsch S, Oberleithner H, Silbernagl S: Character- lular sensors such as sAC and GPR4, and downstream- ization of two MDCK-cell subtypes as a model system to study activated kinases such as PKA or the metabolic sensor principal cell and intercalated cell properties. Pflugers Arch AMPK have been recognized as key regulators of interca- 428: 157–162, 1994 16. Al-Awqati Q: Plasticity in epithelial polarity of renal in- lated cell function. Moreover, the role of intercalated cells tercalated cells: Targeting of the H(1)-ATPase and band 3. Am J as regulators of salt reabsorption and intravascular vol- Physiol 270: C1571–C1580, 1996 ume preservation was established with the identification of 17. van Adelsberg J, Edwards JC, Takito J, Kiss B, al-Awqati Q: An NDCBE and pendrin in type B intercalated cells. Lately, it induced extracellular matrix protein reverses the polarity of band 3 in intercalated epithelial cells. Cell 76: 1053–1061, 1994 has come to the forefront that both intercalated cell volume 18. Sun X, Yang LV, Tiegs BC, Arend LJ, McGraw DW, Penn RB, regulation and the functional integrity of the distal nephron 1 Petrovic S: Deletion of the pH sensor GPR4 decreases renal acid depend on H -ATPase function in these fascinating cells. excretion. J Am Soc Nephrol 21: 1745–1755, 2010 19. Schwartz JH, Li G, Yang Q, Suri V, Ross JJ, Alexander EA: Role of 1 Acknowledgments SNAREs and H -ATPase in the targeting of proton pump-coated vesicles to collecting duct cell apical membrane. Kidney Int 72: We thank Dr. Kenneth R. Hallows for his critical reading of the 1310–1315, 2007 manuscript and Dr. Arohan Subramanya for helpful suggestions. 20. Sampogna RV, Al-Awqati Q: Salt and pepper distribution of cell We thank Mr. Andrew P. Zerby for bibliography research support. types in the collecting duct. J Am Soc Nephrol 24: 163–165, 2013 16 Clinical Journal of the American Society of Nephrology

21. Al-Awqati Q, Gao XB: Differentiation of intercalated cells in the beta- to alpha-intercalated cells and induces distal renal tubular kidney. Physiology (Bethesda) 26: 266–272, 2011 acidosis. Proc Natl Acad Sci U S A 107: 21872–21877, 2010 22. Wieczorek H, Brown D, Grinstein S, Ehrenfeld J, Harvey WR: 45. Bastani B, Purcell H, Hemken P, Trigg D, Gluck S: Expression Animal plasma membrane energization by proton-motive and distribution of renal vacuolar proton-translocating adeno- V-ATPases. BioEssays 21: 637–648, 1999 sine triphosphatase in response to chronic acid and alkali loads 23. Liu W, Xu S, Woda C, Kim P, Weinbaum S, Satlin LM: Effect of in the rat. J Clin Invest 88: 126–136, 1991 flow and stretch on the [Ca21]i response of principal and in- 46. Sabolic I, Brown D, Gluck SL, Alper SL: Regulation of AE1 anion tercalated cells in cortical collecting duct. Am J Physiol Renal exchanger and H(1)-ATPase in rat cortex by acute metabolic Physiol 285: F998–F1012, 2003 acidosis and alkalosis. Kidney Int 51: 125–137, 1997 24. Griffith LD, Bulger RE, Trump BF: Fine structure and staining of 47. Forgac M: Vacuolar ATPases: Rotary proton pumps in physiol- mucosubstances on “intercalated cells” from the rat distal con- ogy and pathophysiology. Nat Rev Mol Cell Biol 8: 917–929, voluted tubule and collecting duct. Anat Rec 160: 643–662, 1968 2007 25. Hancox NM, Komender J: Quantitative and qualitative changes 48. Jeong HW, Jeon US, Koo BK, Kim WY, Im SK, Shin J, Cho Y, Kim in the “dark” cells of the renal collecting tubules in rats deprived J, Kong YY: Inactivation of Notch signaling in the renal col- of water. Q J Exp Physiol Cogn Med Sci 48: 346–354, 1963 lecting duct causes nephrogenic diabetes insipidus in mice. 26. Clark SL Jr: Cellular differentiation in the kidneys of newborn J Clin Invest 119: 3290–3300, 2009 mice studies with the electron microscope. J Biophys Biochem 49. Chen L, Al-Awqati Q: Segmental expression of Notch and Hairy Cytol 3: 349–362, 1957 genes in nephrogenesis. Am J Physiol Renal Physiol 288: F939– 27. Flume JB, Ashworth CT, James JA: An electron microscopic F952, 2005 study of tubular lesions in human kidney biopsy specimens. Am 50. Liu Y, Pathak N, Kramer-Zucker A, Drummond IA: Notch sig- J Pathol 43: 1067–1087, 1963 naling controls the differentiation of transporting epithelia and 28. Latta H, Maunsbach AB, Madden SC: Cilia in different segments multiciliated cells in the zebrafish pronephros. Development of the rat nephron. J Biophys Biochem Cytol 11: 248–252, 1961 134: 1111–1122, 2007 29. Pfaller W, Klima J: A critical reevaluation of the structure of the 51. Yamaguchi Y, Yonemura S, Takada S: Grainyhead-related tran- rat uriniferous tubule as revealed by scanning electron mi- scription factor is required for duct maturation in the salivary croscopy. Cell Tissue Res 166: 91–100, 1976 gland and the kidney of the mouse. Development 133: 4737– 30. Schwartz EA, Leonard ML, Bizios R, Bowser SS: Analysis and 4748, 2006 modeling of the primary cilium bending response to fluid shear. 52. Quigley IK, Stubbs JL, Kintner C: Specification of ion transport Am J Physiol 272: F132–F138, 1997 cells in the Xenopus larval skin. Development 138: 705–714, 31. Stetson DL, Steinmetz PR: Alpha and beta types of carbonic 2011 anhydrase-rich cells in turtle bladder. Am J Physiol 249: F553– 53. Breton S, Brown D: Regulation of luminal acidification by the F565, 1985 V-ATPase. Physiology (Bethesda) 28: 318–329, 2013 32. Schwartz JH, Steinmetz PR: CO2 requirements for H1 secretion 54. Vidarsson H, Westergren R, Heglind M, Blomqvist SR, Breton S, by the isolated turtle bladder. Am J Physiol 220: 2051–2057, Enerba¨ck S: The forkhead transcription factor Foxi1 is a master regulator of vacuolar H-ATPase proton pump subunits in the 1971 inner ear, kidney and epididymis. PLoS ONE 4: e4471, 2009 33. Harvey BJ: Energization of sodium absorption by the 55. Blomqvist SR, Vidarsson H, Fitzgerald S, Johansson BR, H(1)-ATPase pump in mitochondria-rich cells of frog skin. JExp Ollerstam A, Brown R, Persson AE, Bergstro¨m GG, Enerba¨ck S: Biol 172: 289–309, 1992 Distal renal tubular acidosis in mice that lack the forkhead 34. Kleyman TR, Satlin LM, Hallows KR: Opening lines of com- transcription factor Foxi1. J Clin Invest 113: 1560–1570, 2004 munication in the distal nephron. J Clin Invest 123: 4139–4141, 56. Brown D, Weyer P, Orci L: Nonclathrin-coated vesicles are in- 2013 volved in endocytosis in kidney collecting duct intercalated 35. Gueutin V, Vallet M, Jayat M, Peti-Peterdi J, Cornie`re N, Leviel F, b cells. Anat Rec 218: 237–242, 1987 Sohet F, Wagner CA, Eladari D, Chambrey R: Renal - 57. Brown D, Gluck S, Hartwig J: Structure of the novel membrane- intercalated cells maintain body fluid and electrolyte balance. coating material in proton-secreting epithelial cells and iden- J Clin Invest 123: 4219–4231, 2013 tification as an H1ATPase. JCellBiol105: 1637–1648, 1987 36. Kriz W, Kaissling B: Structural Organization of the Mammalian 58. Wagner CA, Finberg KE, Breton S, Marshansky V, Brown D, Kidney. In: Seldin and Giebisch’s The Kidney: Physiology and Geibel JP: Renal vacuolar H1-ATPase. Physiol Rev 84: 1263– Pathophysiology, edited by Alpern RJ, Caplan MJ, Moe OW, 1314, 2004 London, Academic Press, 2013, pp 595–691 59. Andersen OS, Silveira JE, Steinmetz PR: Intrinsic characteristics 37. Wall SM, Weinstein AM: Cortical distal nephron Cl(-) transport of the proton pump in the luminal membrane of a tight urinary in volume homeostasis and blood pressure regulation. Am J epithelium. The relation between transport rate and delta mu H. Physiol Renal Physiol 305: F427–F438, 2013 J Gen Physiol 86: 215–234, 1985 38. Kim J, Kim YH, Cha JH, Tisher CC, Madsen KM: Intercalated cell 60. Kovacikova J, Winter C, Loffing-Cueni D, Loffing J, Finberg KE, subtypes in connecting tubule and cortical collecting duct of Lifton RP, Hummler E, Rossier B, Wagner CA: The connecting rat and mouse. J Am Soc Nephrol 10: 1–12, 1999 tubule is the main site of the furosemide-induced urinary 39. Spicer SS, Stoward PJ, Tashian RE: The immunohistolocalization acidification by the vacuolar H1-ATPase. Kidney Int 70: 1706– of carbonic anhydrase in rodent tissues. J Histochem Cytochem 1716, 2006 27: 820–831, 1979 61. Gumz ML, Lynch IJ, Greenlee MM, Cain BD, Wingo CS: The 40. Schwartz GJ: Physiology and molecular biology of renal car- renal H1-K1-ATPases: physiology, regulation, and structure. bonic anhydrase. J Nephrol 15[Suppl 5]: S61–S74, 2002 Am J Physiol Renal Physiol 298: F12–F21, 2010 1 41. Brown D, Hirsch S, Gluck S: An H -ATPase in opposite plasma 62. Lynch IJ, Rudin A, Xia SL, Stow LR, Shull GE, Weiner ID, Cain membrane domains in kidney epithelial cell subpopulations. BD, Wingo CS: Impaired acid secretion in cortical collecting Nature 331: 622–624, 1988 duct intercalated cells from H-K-ATPase-deficient mice: role of 42. Alper SL, Natale J, Gluck S, Lodish HF, Brown D: Subtypes of HKalpha isoforms. Am J Physiol Renal Physiol 294: F621–F627, intercalated cells in rat kidney collecting duct defined by anti- 2008 bodies against erythroid band 3 and renal vacuolar H1-ATPase. 63. Greenlee MM, Lynch IJ, Gumz ML, Cain BD, Wingo CS: The Proc Natl Acad Sci U S A 86: 5429–5433, 1989 renal H,K-ATPases. Curr Opin Nephrol Hypertens 19: 478–482, 43. Purkerson JM, Tsuruoka S, Suter DZ, Nakamori A, Schwartz GJ: 2010 Adaptation to metabolic acidosis and its recovery are associated 64. Silver RB, Frindt G, Mennitt P, Satlin LM: Characterization and with changes in anion exchanger distribution and expression in regulation of H-K-ATPase in intercalated cells of rabbit cortical the cortical collecting duct. Kidney Int 78: 993–1005, 2010 collecting duct. J Exp Zool 279: 443–455, 1997 44. Gao X, Eladari D, Leviel F, Tew BY, Miro´-Julia` C, Cheema FH, 65. Silver RB, Mennitt PA, Satlin LM: Stimulation of apical H-K- Miller L, Nelson R, Paunescu TG, McKee M, Brown D, Al- ATPase in intercalated cells of cortical collecting duct with Awqati Q: Deletion of hensin/DMBT1 blocks conversion of chronic metabolic acidosis. Am J Physiol 270: F539–F547, 1996 Clin J Am Soc Nephrol 10: ccc–ccc, February, 2015 Intercalated Cell Physiology, Roy et al. 17

66. Najjar F, Zhou H, Morimoto T, Bruns JB, Li HS, Liu W, Kleyman mediates bicarbonate secretion. Proc Natl Acad Sci U S A 98: TR, Satlin LM: Dietary K1 regulates apical membrane expres- 4221–4226, 2001 sion of maxi-K channels in rabbit cortical collecting duct. Am J 83. Everett LA, Glaser B, Beck JC, Idol JR, Buchs A, Heyman M, Physiol Renal Physiol 289: F922–F932, 2005 Adawi F, Hazani E, Nassir E, Baxevanis AD, Sheffield VC, Green 67. Woda CB, Miyawaki N, Ramalakshmi S, Ramkumar M, Rojas R, ED: Pendred syndrome is caused by mutations in a putative Zavilowitz B, Kleyman TR, Satlin LM: Ontogeny of flow- sulphate transporter gene (PDS). Nat Genet 17: 411–422, stimulated potassium secretion in rabbit cortical collecting 1997 duct: functional and molecular aspects. Am J Physiol Renal 84. Scott DA, Wang R, Kreman TM, Sheffield VC, Karniski LP: The Physiol 285: F629–F639, 2003 Pendred syndrome gene encodes a chloride-iodide transport 68. Zhuang J, Zhang X, Wang D, Li J, Zhou B, Shi Z, Gu D, Denson protein. Nat Genet 21: 440–443, 1999 DD, Eaton DC, Cai H: WNK4 kinase inhibits Maxi K channel 85. Everett LA, Morsli H, Wu DK, Green ED: Expression pattern of activity by a kinase-dependent mechanism. Am J Physiol Renal the mouse ortholog of the Pendred’s syndrome gene (Pds) Physiol 301: F410–F419, 2011 suggests a key role for pendrin in the inner ear. Proc Natl Acad 69. Shibata S, Rinehart J, Zhang J, Moeckel G, Castaneda-Bueno~ M, Sci U S A 96: 9727–9732, 1999 Stiegler AL, Boggon TJ, Gamba G, Lifton RP: Mineralocorticoid 86. Royaux IE, Suzuki K, Mori A, Katoh R, Everett LA, Kohn LD, receptor phosphorylation regulates ligand binding and renal Green ED: Pendrin, the protein encoded by the Pendred syn- response to volume depletion and hyperkalemia. Cell Metab drome gene (PDS), is an apical porter of iodide in the thyroid 18: 660–671, 2013 and is regulated by thyroglobulin in FRTL-5 cells. Endocrinol- 70. Wang Z, Subramanya AR, Satlin LM, Pastor-Soler NM, Carattino ogy 141: 839–845, 2000 MD, Kleyman TR: Regulation of large-conductance 87. Pendred V: Deaf-mutism and goitre. Lancet 148: 532, 1896 Ca21-activated K1 channels by WNK4 kinase. Am J Physiol 88. Morgans ME, Trotter WR: Association of congenital deafness Cell Physiol 305: C846–C853, 2013 with goitre; the nature of the thyroid defect. Lancet 1: 607–609, 71. Palmer LG, Frindt G: High-conductance K channels in in- 1958 tercalated cells of the rat distal nephron. Am J Physiol Renal 89. Kim YH, Verlander JW, Matthews SW, Kurtz I, Shin W, Weiner Physiol 292: F966–F973, 2007 ID, Everett LA, Green ED, Nielsen S, Wall SM: Intercalated cell 72. Alzamora R, Thali RF, Gong F, Smolak C, Li H, Baty CJ, Bertrand H1/OH- transporter expression is reduced in Slc26a4 null CA, Auchli Y, Brunisholz RA, Neumann D, Hallows KR, Pastor- mice. Am J Physiol Renal Physiol 289: F1262–F1272, 2005 Soler NM: PKA regulates vacuolar H1-ATPase localization and 90. Verlander JW, Kim YH, Shin W, Pham TD, Hassell KA, activity via direct phosphorylation of the a subunit in kidney Beierwaltes WH, Green ED, Everett L, Matthews SW, Wall SM: cells. J Biol Chem 285: 24676–24685, 2010 Dietary Cl(-) restriction upregulates pendrin expression within 73. Alzamora R, Al-Bataineh MM, Liu W, Gong F, Li H, Thali RF, the apical plasma membrane of type B intercalated cells. Am J Joho-Auchli Y, Brunisholz RA, Satlin LM, Neumann D, Hallows Physiol Renal Physiol 291: F833–F839, 2006 KR, Pastor-Soler NM: AMP-activated protein kinase regulates 91. Wall SM, Kim YH, Stanley L, Glapion DM, Everett LA, Green the vacuolar H1-ATPase via direct phosphorylation of the A ED, Verlander JW: NaCl restriction upregulates renal Slc26a4 subunit (ATP6V1A) in the kidney. Am J Physiol Renal Physiol through subcellular redistribution: Role in Cl- conservation. 305: F943–F956, 2013 Hypertension 44: 982–987, 2004 74. Gong F, Alzamora R, Smolak C, Li H, Naveed S, Neumann D, 92. Sansom SC, Weinman EJ, O’Neil RG: Microelectrode assess- Hallows KR, Pastor-Soler NM: Vacuolar H1-ATPase apical ac- ment of chloride-conductive properties of cortical collecting cumulation in kidney intercalated cells is regulated by PKA and duct. Am J Physiol 247: F291–F302, 1984 AMP-activated protein kinase. Am J Physiol Renal Physiol 298: 93. Gong Y, Yu M, Yang J, Gonzales E, Perez R, Hou M, Tripathi P, F1162–F1169, 2010 Hering-Smith KS, Hamm LL, Hou J: The Cap1-claudin-4 regu- 75. Al-bataineh MM, Gong F, Marciszyn AL, Myerburg MM, Pastor- latory pathway is important for renal chloride reabsorption and Soler NM: Regulation of proximal tubule vacuolar H(1)-ATPase blood pressure regulation. Proc Natl Acad Sci U S A 111: by PKA and AMP-activated protein kinase. Am J Physiol Renal E3766–E3774, 2014 Physiol 306: F981–F995, 2014 94. Kurtz TW, Al-Bander HA, Morris RC Jr: “Salt-sensitive” essential 76. Hallows KR, Alzamora R, Li H, Gong F, Smolak C, Neumann D, hypertension in men. Is the sodium ion alone important? NEngl Pastor-Soler NM: AMP-activated protein kinase inhibits alkaline JMed317: 1043–1048, 1987 pH- and PKA-induced apical vacuolar H1-ATPase accumula- 95. Hafner P, Grimaldi R, Capuano P, Capasso G, Wagner CA: tion in epididymal clear cells. Am J Physiol Cell Physiol 296: Pendrin in the mouse kidney is primarily regulated by Cl- C672–C681, 2009 excretion but also by systemic metabolic acidosis. Am J 77. Gidon A, Al-Bataineh MM, Jean-Alphonse FG, Stevenson HP, Physiol Cell Physiol 295: C1658–C1667, 2008 Watanabe T, Louet C, Khatri A, Calero G, Pastor-Soler NM, 96. Pech V, Zheng W, Pham TD, Verlander JW, Wall SM: Angio- Gardella TJ, Vilardaga JP: Endosomal GPCR signaling turned off tensin II activates H1-ATPase in type A intercalated cells. JAm by negative feedback actions of PKA and v-ATPase. Nat Chem Soc Nephrol 19: 84–91, 2008 Biol 10: 707–709, 2014 97. Mohebbi N, Perna A, van der Wijst J, Becker HM, Capasso G, 78. Păunescu TG, Ljubojevic M, Russo LM, Winter C, McLaughlin Wagner CA: Regulation of two renal chloride transporters, AE1 MM, Wagner CA, Breton S, Brown D: cAMP stimulates apical and pendrin, by electrolytes and aldosterone. PLoS ONE 8: V-ATPase accumulation, microvillar elongation, and proton e55286, 2013 extrusion in kidney collecting duct A-intercalated cells. Am J 98. Kim YH, Pech V, Spencer KB, Beierwaltes WH, Everett LA, Physiol Renal Physiol 298: F643–F654, 2010 Green ED, Shin W, Verlander JW, Sutliff RL, Wall SM: Reduced 79. Paunescu TG, Da Silva N, Russo LM, McKee M, Lu HA, Breton ENaC protein abundance contributes to the lower blood pres- S, Brown D: Association of soluble adenylyl cyclase with the sure observed in pendrin-null mice. Am J Physiol Renal Physiol V-ATPase in renal epithelial cells. Am J Physiol Renal Physiol 293: F1314–F1324, 2007 294: F130–F138, 2008 99. Verlander JW, Hong S, Pech V,Bailey JL, Agazatian D, Matthews 80. Xu J, Barone S, Li H, Holiday S, Zahedi K, Soleimani M: SW, Coffman TM, Le T, Inagami T, Whitehill FM, Weiner ID, Slc26a11, a chloride transporter, localizes with the vacuolar Farley DB, Kim YH, Wall SM: Angiotensin II acts through the H(1)-ATPase of A-intercalated cells of the kidney. Kidney Int angiotensin 1a receptor to upregulate pendrin. Am J Physiol 80: 926–937, 2011 Renal Physiol 301: F1314–F1325, 2011 81. Fisher KD, Codina J, Petrovic S, DuBose TD Jr: Pyk2 regulates 100. Pech V, Pham TD, Hong S, Weinstein AM, Spencer KB, Duke BJ, H1-ATPase-mediated proton secretion in the outer medullary Walp E, Kim YH, Sutliff RL, Bao HF, Eaton DC, Wall SM: Pendrin collecting duct via an ERK1/2 signaling pathway. Am J Physiol modulates ENaC function by changing luminal HCO3-. JAm Renal Physiol 303: F1353–F1362, 2012 Soc Nephrol 21: 1928–1941, 2010 82. Royaux IE, Wall SM, Karniski LP, Everett LA, Suzuki K, Knepper 101. Wall SM, Hassell KA, Royaux IE, Green ED, Chang JY, Shipley MA, Green ED: Pendrin, encoded by the Pendred syndrome GL, Verlander JW: Localization of pendrin in mouse kidney. Am gene, resides in the apical region of renal intercalated cells and J Physiol Renal Physiol 284: F229–F241, 2003 18 Clinical Journal of the American Society of Nephrology

102. Kim YH, Kwon TH, Frische S, Kim J, Tisher CC, Madsen KM, along the distal tubule. Am J Physiol Renal Physiol 299: F1473– Nielsen S: Immunocytochemical localization of pendrin in in- F1485, 2010 tercalated cell subtypes in rat and mouse kidney. Am J Physiol 121. Bostanjoglo M, Reeves WB, Reilly RF, Vela´zquez H, Robertson Renal Physiol 283: F744–F754, 2002 N, Litwack G, Morsing P, Dørup J, Bachmann S, Ellison DH: 103. Scott DA, Karniski LP: Human pendrin expressed in Xenopus 11Beta-hydroxysteroid dehydrogenase, mineralocorticoid re- laevis oocytes mediates chloride/formate exchange. Am J ceptor, and thiazide-sensitive Na-Cl cotransporter expression Physiol Cell Physiol 278: C207–C211, 2000 by distal tubules. J Am Soc Nephrol 9: 1347–1358, 1998 104. Azroyan A, Laghmani K, Crambert G, Mordasini D, Doucet A, 122. Farman N, Rafestin-Oblin ME: Multiple aspects of mineralo- Edwards A: Regulation of pendrin by pH: dependence on gly- corticoid selectivity. Am J Physiol Renal Physiol 280: F181– cosylation. Biochem J 434: 61–72, 2011 F192, 2001 105. Alesutan I, Daryadel A, Mohebbi N, Pelzl L, Leibrock C, Voelkl 123. Stanton BA: Regulation by adrenal corticosteroids of sodium J, Bourgeois S, Dossena S, Nofziger C, Paulmichl M, Wagner and potassium transport in loop of Henle and distal tubule of rat CA, Lang F: Impact of bicarbonate, ammonium chloride, and kidney. J Clin Invest 78: 1612–1620, 1986 acetazolamide on hepatic and renal SLC26A4 expression. Cell 124. Nguyen G, Delarue F, Burckle´ C, Bouzhir L, Giller T, Sraer JD: Physiol Biochem 28: 553–558, 2011 Pivotal role of the renin/prorenin receptor in angiotensin II 106. Kim YH, Pham TD, Zheng W, Hong S, Baylis C, Pech V, production and cellular responses to renin. J Clin Invest 109: Beierwaltes WH, Farley DB, Braverman LE, Verlander JW, Wall 1417–1427, 2002 SM: Role of pendrin in iodide balance: going with the flow. Am J 125. Batenburg WW, Krop M, Garrelds IM, de Vries R, de Bruin RJ, Physiol Renal Physiol 297: F1069–F1079, 2009 Burckle´ CA, Mu¨ller DN, Bader M, Nguyen G, Danser AH: 107. Leviel F, Hu¨bner CA, Houillier P, Morla L, El Moghrabi S, Prorenin is the endogenous agonist of the (pro)renin receptor. Brideau G, Hassan H, Parker MD, Kurth I, Kougioumtzes A, Binding kinetics of renin and prorenin in rat vascular smooth Sinning A, Pech V, Riemondy KA, Miller RL, Hummler E, Shull muscle cells overexpressing the human (pro)renin receptor. GE, Aronson PS, Doucet A, Wall SM, Chambrey R, Eladari D: J Hypertens 25: 2441–2453, 2007 The Na1-dependent chloride-bicarbonate exchanger SLC4A8 126. Jan Danser AH, Batenburg WW, van Esch JH: Prorenin and the mediates an electroneutral Na1 reabsorption process in the (pro)renin receptor—an update. Nephrol Dial Transplant 22: renal cortical collecting ducts of mice. J Clin Invest 120: 1627– 1288–1292, 2007 1635, 2010 127. Ludwig J, Kerscher S, Brandt U, Pfeiffer K, Getlawi F, Apps DK, 108. Hentschke M, Hentschke S, Borgmeyer U, Hu¨bner CA, Kurth I: Scha¨gger H: Identification and characterization of a novel The murine AE4 promoter predominantly drives type B in- 9.2-kDa membrane sector-associated protein of vacuolar tercalated cell specific transcription. Histochem Cell Biol 132: proton-ATPase from chromaffin granules. J Biol Chem 273: 405–412, 2009 10939–10947, 1998 109. Purkerson JM, Heintz EV, Nakamori A, Schwartz GJ: Insights 128. Yoshikawa A, Aizaki Y, Kusano K, Kishi F, Susumu T, Iida S, into acidosis-induced regulation of SLC26A4 (pendrin) and Ishiura S, Nishimura S, Shichiri M, Senbonmatsu T: The (pro) SLC4A9 (AE4) transporters using three-dimensional morpho- renin receptor is cleaved by ADAM19 in the Golgi leading to its metric analysis of b-intercalated cells. Am J Physiol Renal secretion into extracellular space. Hypertens Res 34: 599–605, Physiol 307: F601–F611, 2014 2011 110. Verrey F, Hummler E, Schild L, Rossier B: Control of Na1 129. Cousin C, Bracquart D, Contrepas A, Corvol P,Muller L, Nguyen transport by aldosterone. In: The Kidney: Physiology and G: Soluble form of the (pro)renin receptor generated by in- Pathophysiology, edited by Seldin DW, Giebisch G, Phila- tracellular cleavage by furin is secreted in plasma. Hypertension delphia, Williams & Wilkins, 2000, pp 1441–1471 53: 1077–1082, 2009 111. Karim Z, Szutkowska M, Vernimmen C, Bichara M: Renal 130. Ichihara A, Kaneshiro Y, Suzuki F: Prorenin receptor block- handling of NH3/NH41: Recent concepts. Nephron, Physiol ers: effects on cardiovascular complications of diabetes and 101: 77–81, 2005 hypertension. Expert Opin Investig Drugs 15: 1137–1139, 112. Sebastian A, Sutton JM, Hulter HN, Schambelan M, Poler SM: 2006 Effect of mineralocorticoid replacement therapy on renal acid- 131. Gonzalez AA, Lara LS, Luffman C, Seth DM, Prieto MC: Soluble base homeostasis in adrenalectomized patients. Kidney Int 18: form of the (pro)renin receptor is augmented in the collecting 762–773, 1980 duct and urine of chronic angiotensin II-dependent hyperten- 113. Pearce D, Soundararajan R, Trimpert C, Kashlan OB, Deen PM, sive rats. Hypertension 57: 859–864, 2011 Kohan DE: Collecting duct principal cell transport processes 132. Rohrwasser A, Morgan T, Dillon HF, Zhao L, Callaway CW, and their regulation [published online ahead of print May 29, Hillas E, Zhang S, Cheng T, Inagami T, Ward K, Terreros DA, 2014]. Clin J Am Soc Nephrol doi: 10.2215/CJN.05760513 Lalouel JM: Elements of a paracrine tubular renin-angiotensin 114. Prieto-Carrasquero MC, Botros FT, Kobori H, Navar LG: Col- system along the entire nephron. Hypertension 34: 1265–1274, lecting duct renin: a major player in angiotensin II-dependent 1999 hypertension. J Am Soc Hypertens 3: 96–104, 2009 133. Advani A, Kelly DJ, Cox AJ, White KE, Advani SL, Thai K, 115. Frindt G, Palmer LG: Low-conductance K channels in apical Connelly KA, Yuen D, Trogadis J, Herzenberg AM, Kuliszewski membrane of rat cortical collecting tubule. Am J Physiol 256: MA, Leong-Poi H, Gilbert RE: The (Pro)renin receptor: site- F143–F151, 1989 specific and functional linkage to the vacuolar H1-ATPase in 116. Schambelan M, Sebastian A, Katuna BA, Arteaga E: Adreno- the kidney. Hypertension 54: 261–269, 2009 cortical hormone secretory response to chronic NH4Cl-induced 134. Ichihara A, Sakoda M, Kurauchi-Mito A, Kaneshiro Y, Itoh H: metabolic acidosis. Am J Physiol 252: E454–E460, 1987 Renin, prorenin and the kidney: a new chapter in an old saga. 117. Wagner CA, Devuyst O, Bourgeois S, Mohebbi N: Regulated J Nephrol 22: 306–311, 2009 acid-base transport in the collecting duct. Pflugers Arch 458: 135. Kaneshiro Y, Ichihara A, Takemitsu T, Sakoda M, Suzuki F, 137–156, 2009 Nakagawa T, Hayashi M, Inagami T: Increased expression of 118. Estilo G, Liu W, Pastor-Soler N, Mitchell P, Carattino MD, cyclooxygenase-2 in the renal cortex of human prorenin re- Kleyman TR, Satlin LM: Effect of aldosterone on BK channel ceptor gene-transgenic rats. Kidney Int 70: 641–646, 2006 expression in mammalian cortical collecting duct. Am J Physiol 136. Deinum J, Tarnow L, van Gool JM, de Bruin RA, Derkx FH, Renal Physiol 295: F780–F788, 2008 Schalekamp MA, Parving HH: Plasma renin and prorenin and 119. Rothenberger F, Velic A, Stehberger PA, Kovacikova J, Wagner renin gene variation in patients with insulin-dependent diabetes CA: Angiotensin II stimulates vacuolar H1 -ATPase activity in mellitus and nephropathy. Nephrol Dial Transplant 14: 1904– renal acid-secretory intercalated cells from the outer medullary 1911, 1999 collecting duct. J Am Soc Nephrol 18: 2085–2093, 2007 137. DuBose TD Jr, Goode AK: Isolated renal tubular disorder: 120. Ackermann D, Gresko N, Carrel M, Loffing-Cueni D, Mechanisms and clinical expression. In: Schrier’s Diseases of Habermehl D, Gomez-Sanchez C, Rossier BC, Loffing J: In vivo the Kidney, edited by Coffman TM, Falk RJ, Molitoris B, Neilson nuclear translocation of mineralocorticoid and glucocorticoid EG, Schrier RW, Philadelphia, Wolters Kluwer Lippincott, receptors in rat kidney: Differential effect of corticosteroids Williams & Wilkins, 2007, pp 587-614. Clin J Am Soc Nephrol 10: ccc–ccc, February, 2015 Intercalated Cell Physiology, Roy et al. 19

138. Douglas JB, Healy JK: Nephrotoxic effects of amphotericin B, 154. Pertovaara M, Korpela M, Kouri T, Pasternack A: The occurrence including renal tubular acidosis. Am J Med 46: 154–162, 1969 of renal involvement in primary Sjo¨gren’s syndrome: A study of 139. Sebastian A, McSherry E, Morris RC Jr: Renal potassium wasting 78 patients. Rheumatology (Oxford) 38: 1113–1120, 1999 in renal tubular acidosis (RTA): Its occurrence in types 1 and 2 155. Ren H, Wang WM, Chen XN, Zhang W, Pan XX, Wang XL, Lin Y, RTA despite sustained correction of systemic acidosis. J Clin Zhang S, Chen N: Renal involvement and followup of 130 pa- Invest 50: 667–678, 1971 tients with primary Sjo¨gren’s syndrome. J Rheumatol 35: 278– 140. Karet FE, Finberg KE, Nayir A, Bakkaloglu A, Ozen S, Hulton SA, 284, 2008 Sanjad SA, Al-Sabban EA, Medina JF, Lifton RP: Localization of a 156. Cohen EP, Bastani B, Cohen MR, Kolner S, Hemken P, Gluck SL: gene for autosomal recessive distal renal tubular acidosis with Absence of H(1)-ATPase in cortical collecting tubules of a normal hearing (rdRTA2) to 7q33-34. Am J Hum Genet 65: patient with Sjogren’s syndrome and distal renal tubular 1656–1665, 1999 acidosis. J Am Soc Nephrol 3: 264–271, 1992 141. Karet FE, Finberg KE, Nelson RD, Nayir A, Mocan H, Sanjad SA, 157. Devuyst O, Lemaire M, Mohebbi N, Wagner CA: Autoanti- Rodriguez-Soriano J, Santos F, Cremers CW, Di Pietro A, bodies against intercalated cells in Sjo¨gren’s syndrome. Kidney Hoffbrand BI, Winiarski J, Bakkaloglu A, Ozen S, Dusunsel R, Int 76: 229, 2009 Goodyer P, Hulton SA, Wu DK, Skvorak AB, Morton CC, 158. DeFranco PE, Haragsim L, Schmitz PG, Bastani B: Absence of 1 Cunningham MJ, Jha V, Lifton RP: Mutations in the gene en- vacuolar H( )-ATPase pump in the collecting duct of a patient coding B1 subunit of H1-ATPase cause renal tubular acidosis with hypokalemic distal renal tubular acidosis and Sjo¨gren’s with sensorineural deafness. Nat Genet 21: 84–90, 1999 syndrome. J Am Soc Nephrol 6: 295–301, 1995 142. Stover EH, Borthwick KJ, Bavalia C, Eady N, Fritz DM, Rungroj 159. Walsh S, Turner CM, Toye A, Wagner C, Jaeger P, Laing C, N, Giersch AB, Morton CC, Axon PR, Akil I, Al-Sabban EA, Unwin R: Immunohistochemical comparison of a case of in- Baguley DM, Bianca S, Bakkaloglu A, Bircan Z, Chauveau D, herited distal renal tubular acidosis (with a unique AE1 muta- Clermont MJ, Guala A, Hulton SA, Kroes H, Li Volti G, Mir S, tion) with an acquired case secondary to autoimmune disease. Mocan H, Nayir A, Ozen S, Rodriguez Soriano J, Sanjad SA, Nephrol Dial Transplant 22: 807–812, 2007 Tasic V, Taylor CM, Topaloglu R, Smith AN, Karet FE: Novel 160. Trepiccione F, Christensen BM: Lithium-induced nephrogenic ATP6V1B1 and ATP6V0A4 mutations in autosomal recessive diabetes insipidus: new clinical and experimental findings. distal renal tubular acidosis with new evidence for hearing loss. J Nephrol 23[Suppl 16]: S43–S48, 2010 J Med Genet 39: 796–803, 2002 161. Gru¨nfeld JP, Rossier BC: Lithium nephrotoxicity revisited. Nat 143. Karet FE, Gainza FJ, Gyo¨ry AZ, Unwin RJ, Wrong O, Tanner MJ, Rev Nephrol 5: 270–276, 2009 Nayir A, Alpay H, Santos F, Hulton SA, Bakkaloglu A, Ozen S, 162. Batlle D, Gaviria M, Grupp M, Arruda JA, Wynn J, Kurtzman Cunningham MJ, di Pietro A, Walker WG, Lifton RP: Mutations NA: Distal nephron function in patients receiving chronic lithium therapy. Kidney Int 21: 477–485, 1982 in the chloride-bicarbonate exchanger gene AE1 cause 163. Christensen BM, Marples D, Kim YH, Wang W, Frøkiaer J, autosomal dominant but not autosomal recessive distal renal Nielsen S: Changes in cellular composition of kidney collecting tubular acidosis. Proc Natl Acad Sci U S A 95: 6337–6342, duct cells in rats with lithium-induced NDI. Am J Physiol Cell 1998 Physiol 286: C952–C964, 2004 144. Bruce LJ, Cope DL, Jones GK, Schofield AE, Burley M, Povey S, 164. Kishore BK, Ecelbarger CM: Lithium: A versatile tool for un- UnwinRJ,WrongO,TannerMJ:Familialdistalrenaltubular derstanding renal physiology. Am J Physiol Renal Physiol 304: acidosis is associated with mutations in the red cell anion F1139–F1149, 2013 exchanger (Band 3, AE1) gene. J Clin Invest 100: 1693–1707, 165. Kuure S, Popsueva A, Jakobson M, Sainio K, Sariola H: Glyco- 1997 gen synthase kinase-3 inactivation and stabilization of beta- 145. Ribeiro ML, Alloisio N, Almeida H, Gomes C, Texier P,Lemos C, catenin induce nephron differentiation in isolated mouse and Mimoso G, Morle´ L, Bey-Cabet F, Rudigoz RC, Delaunay J, rat kidney mesenchymes. J Am Soc Nephrol 18: 1130–1139, Tamagnini G: Severe hereditary spherocytosis and distal renal 2007 tubular acidosis associated with the total absence of band 3. 166. Pulkkinen K, Murugan S, Vainio S: Wnt signaling in kidney Blood 96: 1602–1604, 2000 development and disease. Organogenesis 4: 55–59, 2008 146. Breton S, Alper SL, Gluck SL, Sly WS, Barker JE, Brown D: 167. Park EY, Kim WY, Kim YM, Lee JH, Han KH, Weiner ID, Kim J: Depletion of intercalated cells from collecting ducts of carbonic Proposed mechanism in the change of cellular composition in anhydrase II-deficient (CAR2 null) mice. Am J Physiol 269: the outer medullary collecting duct during potassium homeo- F761–F774, 1995 stasis. Histol Histopathol 27: 1559–1577, 2012 147. Sebastian A, McSherry E, Morris RC Jr: Impaired renal conser- 168. Heering P, Ivens K, Aker S, Grabensee B: Distal tubular vation of sodium and chloride during sustained correction of acidosis induced by FK506. Clin Transplant 12: 465–471, systemic acidosis in patients with type 1, classic renal tubular 1998 acidosis. J Clin Invest 58: 454–469, 1976 169. Mohebbi N, Mihailova M, Wagner CA: The calcineurin in- 148. Finberg KE, Wagner CA, Bailey MA, Paunescu TG, Breton S, hibitor FK506 (tacrolimus) is associated with transient meta- Brown D, Giebisch G, Geibel JP, Lifton RP: The B1-subunit of bolic acidosis and altered expression of renal acid-base 1 the H( ) ATPase is required for maximal urinary acidification. transport proteins. Am J Physiol Renal Physiol 297: F499–F509, Proc Natl Acad Sci U S A 102: 13616–13621, 2005 2009 149. Guan Y, Zhang Y, Breyer RM, Fowler B, Davis L, He´bert RL, 170. Peng H, Vijayakumar S, Schiene-Fischer C, Li H, Purkerson Breyer MD: Prostaglandin E2 inhibits renal collecting duct Na1 JM, Malesevic M, Liebscher J, Al-Awqati Q, Schwartz GJ: absorption by activating the EP1 receptor. J Clin Invest 102: Secreted cyclophilin A, a peptidylprolyl cis-trans isomerase, 194–201, 1998 mediates matrix assembly of hensin, a protein implicated 150. He´bert RL, Jacobson HR, Fredin D, Breyer MD: Evidence that in epithelial differentiation. JBiolChem284: 6465–6475, separate PGE2 receptors modulate water and sodium transport 2009 in rabbit cortical collecting duct. Am J Physiol 265: F643–F650, 171. Korhonen TK, Virkola R, Holtho¨fer H: Localization of binding 1993 sites for purified Escherichia coli P fimbriae in the human kid- 151. Ando Y, Asano Y: Luminal prostaglandin E2 modulates sodium ney. Infect Immun 54: 328–332, 1986 and water transport in rabbit cortical collecting ducts. Am J 172. Chassin C, Goujon JM, Darche S, du Merle L, Bens M, Cluzeaud Physiol 268: F1093–F1101, 1995 F, Werts C, Ogier-Denis E, Le Bougueńec C, Buzoni-Gatel D, 152. Krupin T, Sly WS, Whyte MP, Dodgson SJ: Failure of acetazol- Vandewalle A: Renal collecting duct epithelial cells react to amide to decrease intraocular pressure in patients with car- pyelonephritis-associated Escherichia coli by activating distinct bonic anhydrase II deficiency. Am J Ophthalmol 99: 396–399, TLR4-dependent and -independent inflammatory pathways. 1985 J Immunol 177: 4773–4784, 2006 153. Zietse R, Zoutendijk R, Hoorn EJ: Fluid, electrolyte and acid- 173. Gluba A, Banach M, Hannam S, Mikhailidis DP, Sakowicz A, base disorders associated with antibiotic therapy. Nat Rev Rysz J: The role of Toll-like receptors in renal diseases. Nat Rev Nephrol 5: 193–202, 2009 Nephrol 6: 224–235, 2010 20 Clinical Journal of the American Society of Nephrology

174. Ali AS, Townes CL, Hall J, Pickard RS: Maintaining a sterile Ratner AJ, Barasch J: a-Intercalated cells defend the urinary urinary tract: The role of antimicrobial peptides. J Urol 182: system from bacterial infection. J Clin Invest 124: 2963–2976, 21–28, 2009 2014 175. Spencer JD, Schwaderer AL, Dirosario JD, McHugh KM, 180. Paragas N, Qiu A, Zhang Q, Samstein B, Deng SX, Schmidt-Ott McGillivary G, Justice SS, Carpenter AR, Baker PB, Harder J, KM, Viltard M, Yu W, Forster CS, Gong G, Liu Y, Kulkarni R, Hains DS: Ribonuclease 7 is a potent antimicrobial Mori K, Kalandadze A, Ratner AJ, Devarajan P, Landry DW, peptide within the human urinary tract. Kidney Int 80: 174– D’Agati V, Lin CS, Barasch J: The Ngal reporter mouse detects 80, 2011. the response of the kidney to injury in real time. Nat Med 17: 176. Lehmann J, Retz M, Harder J, Krams M, Kellner U, Hartmann J, 216–222, 2011 Hohgra¨we K, Raffenberg U, Gerber M, Loch T, Weichert- 181. Goetz DH, Holmes MA, Borregaard N, Bluhm ME, Raymond Jacobsen K, Sto¨ckle M: Expression of human beta-defensins 1 KN, Strong RK: The neutrophil lipocalin NGAL is a bacterio- and 2 in kidneys with chronic bacterial infection. BMC Infect static agent that interferes with siderophore-mediated iron ac- Dis 2: 20, 2002 quisition. Mol Cell 10: 1033–1043, 2002 177. Mishra J, Ma Q, Prada A, Mitsnefes M, Zahedi K, Yang J, Barasch 182. Barasch J, Mori K: Cell biology: Iron thievery. Nature 432: 811– J, Devarajan P: Identiﬁcation of neutrophil gelatinase-associated 813, 2004 lipocalin as a novel early urinary biomarker for ischemic renal 183. Miro´-Julia` C, Escoda-Ferran C, Carrasco E, Moeller JB, Vadekaer injury. J Am Soc Nephrol 14: 2534–2543, 2003 DF, Gao X, Paragas N, Oliver J, Holmskov U, Al-Awqati Q, 178. Mori K, Lee HT, Rapoport D, Drexler IR, Foster K, Yang J, Lozano F: Expression of the innate defense receptor S5D-SRCRB Schmidt-Ott KM, Chen X, Li JY, Weiss S, Mishra J, Cheema FH, in the urogenital tract. Tissue Antigens 83: 273–285, 2014 Markowitz G, Suganami T, Sawai K, Mukoyama M, Kunis C, 184. Pastor-Soler NM, Hallows KR, Smolak C, Gong F, Brown D, D’Agati V, Devarajan P, Barasch J: Endocytic delivery of Breton S: Alkaline pH- and cAMP-induced V-ATPase lipocalin-siderophore-iron complex rescues the kidney from membrane accumulation is mediated by protein kinase A in ischemia-reperfusion injury. J Clin Invest 115: 610–621, 2005 epididymal clear cells. Am J Physiol Cell Physiol 294: 179. Paragas N, Kulkarni R, Werth M, Schmidt-Ott KM, Forster C, C488–C494, 2008 Deng R, Zhang Q, Singer E, Klose AD, Shen TH, Francis KP, Ray S, Vijayakumar S, Seward S, Bovino ME, Xu K, Takabe Y, Amaral FE, Mohan S, Wax R, Corbin K, Sanna-Cherchi S, Mori K, Published online ahead of print. Publication date available at www. Johnson L, Nickolas T, D’Agati V, Lin CS, Qiu A, Al-Awqati Q, cjasn.org.