Renal Acid-base Regulation- New Insights From Animal Models

INVITED REVIEW

Renal acid-base regulation: new insights from animal models

Dominique Eladari & Yusuke Kumai

Received: 3 October 2014 /Revised: 2 December 2014 /Accepted: 3 December 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract Because majority of biological processes are de-pendent on pH, maintaining systemic acid-base balance is criti-cal. The kidney contributes to systemic acid-base regulation, byreabsorbing HCO3

(both filtered by glomeruli and generatedwithin a nephron) and acidifying urine. Abnormalities in thoseprocesses will eventually lead to a disruption in systemic acid-base balance and provoke metabolic acid-base disorders.Research over the past 30 years advanced our understandingon cellular and molecular mechanisms responsible for thoseprocesses. In particular, a variety of transgenic animal models,where target genes are deleted either globally or conditionally,provided significant insights into how specific transporters arecontributing to the renal acid-base regulation. Here, we broadlyoverview the mechanisms of renal ion transport participating toacid-base regulation, with emphasis on data obtained from trans-genic mice models.

Key words Kidney . Acid-base regulation . pRTA . dRTA .

Transgenic animal

Introduction

Maintaining acid-base homeostasis is critical for survival ofanimals, as changes in pH could have profound consequenceson all biological processes at levels of cell, tissue, and wholeanimal. The regulation of plasma pH is attained by respiratory(the lung) and urinary (the kidney) systems, which regulateplasma PCO2 and [HCO3

], respectively. The relationship isdescribed in the Henderson-Hasselbalch equation, as follows:

pH pK log HCO3

s PCO2

where s is solubility of CO2 at a given temperature, and pKthe dissociation constant of the CO2/HCO3

buffer system.1 Themajor advantage of CO2/HCO3

-buffer system is that arterialPCO2 and [HCO3

] could be regulated independent of eachother. Exhalation removes molecular CO2; and breathing ratecould have direct consequences on systemic acid-base status(e.g., respiratory acidosis induced by hypoventilation). The kid-ney plays amajor role in handlingHCO3

and this is achieved by(1) reabsorbing most of >4,000 mmol [HCO3

] filtered byglomeruli daily and (2) generating new [HCO3

], which isabsorbed and used to titrate acid yielded by protein breakdownduring metabolic processes.

The nephron (the structural unit of the kidney) consists ofseveral functionally distinct segments. The proximal tubule (PT)contributes to renal acid-base regulation by (1) absorbing most(85 %) of filtered bicarbonate, (2) generating new bicarbonatevia ammoniagenesis, and (3) secreting ammonia into lumen,which ultimately acts as the major base form of buffer (asNH3) used to titrate, trap, and excrete proton into urine (as

1 The actual acid form of this buffer system is not CO2 but rather iscarbonic acid H2CO3. However, H2CO3 is rapidly converted into CO2 bycarbonic anhydrase and, for the sake of simplicity, we will consider in thisreview that the acid form is CO2.

D. EladariDepartment of Physiology, Hopital Europen Georges Pompidou,AP-HP, 56 rue Leblanc, F-75015 Paris, France

D. EladariFacult de Mdecine Paris Descartes, Universit Paris Descartes,Sorbonne Paris Cit, 15 rue de lEcole de Mdecine,F-75006 Paris, France

D. Eladari (*)INSERM UMR_S 970, Equipe 12, Paris cardiovascular researchcenter (PARCC), 56 rue Leblanc, F-75015 Paris, Francee-mail: [email protected]

Y. KumaiDepartment of Physiology and Biophysics, School of MedicineCase Western Reserve University, Euclid Ave, Cleveland,OH 10900, USA

Pflugers Arch - Eur J PhysiolDOI 10.1007/s00424-014-1669-x

NH4+). The thick ascending limb (TAL) contributes to renal acid-

base regulation by (1) absorbing majority of remaining filteredbicarbonate (10 %) and (2) absorbing luminal ammonia toaccumulate sufficiently high [ammonia] in medullary interstitialfluid. Finally, the collecting duct (CD) contributes to renal acid-base balance by acidifying the urine via active secretion ofprotons. Because excreted H+ is generated via hydration reactionof cytosolic CO2, this process also generates HCO3

simulta-neously, which is absorbed through basolateral transporter (i.e.,the anion exchanger SLC4A1/AE1). The combination of ammo-nia accumulation into the interstitium by the TAL along with themarked difference of pH between interstitium and urinary fluiddue to urine acidification by the CD creates a steep interstitium-to-urine PNH3 gradient, which in turn drives its secretion intourine across collecting ducts epithelium.

In the present review, we will discuss major molecularmechanisms contributing to these processes within a nephron.Particular attention will be paid to physiological insightsgained through the use of transgenic animal models.

Acid-base regulation in proximal renal tubule

Figure 1a summarizes the molecular mechanisms of bicarbon-ate reabsorption at PT. PT cells secrete H+ into lumen, which

reacts with filtered bicarbonate to yield H2CO3. H2CO3 is thenimmediately dehydrated, a reaction mediated by extracellularcarbonic anhydrase type-IV (CA-IV), tethered to the apicalmembrane via glycosylphosphatidylinositol (GPI) linker. Theresulting CO2 enters the cell and is converted to H

+ andHCO3

via cytosolic carbonic anhydrase type-II (CA-II).HCO3

is then extruded from basolaterally expressed, elec-trogenic Na+-(HCO3

)3 co-transporter (NBCe1), while H+ is

again excreted to react with luminal HCO3. The net effect is

the reabsorption of HCO3 coupled by Na+. As expected from

this mechanism of bicarbonate reabsorption, disruption inapical acid extrusion and basolateral bicarbonate transportcould trigger proximal bicarbonate loss, thereby creatingproximal renal tubular acidosis (pRTA).

Apical H+ secretion in PT

Both Na+/H+ exchanger (NHE; members of SLC9 family) andvacuolar-type H+-ATPase (vH+-ATPase) contribute to the api-cal H+ secretion in PT. Currently, 13 isoforms have beenidentified in mammalian SLC9 family; they are widelyexpressed throughout the body and contribute to a numberof physiological functions [53]. Among those, NHE3(SLC9A3) plays a key role in apical H+ excretion in PT.Early studies (e.g., [176]) took advantage of different

Fig. 1 Molecular mechanisms ofbicarbonate reabsorption inproximal tubule (PT) and thickascending limb (TAL). PTexpresses (1) apical NHE3/8,vH+-ATPase and CA-IV, (2)cytosolic CA-II, and (3)basolateral NBC1. The net effectis the reabsorption of majority(85 %) of filtered HCO3

coupled with Na+ (Fig. 1a). TALexpresses (1) apical NHE3/8,vH+-ATPase, and CA-IV, (2)cytosolic CA-II, and (3)basolateral AE2. The net effect isthe reabsorption of remaining(10 %) bicarbonate (Fig. 1b).Abbreviations: AE: anionexchanger; CA: carbonicanhydrase, NBC1: Na+-HCO3

co-transporter; NHE: Na+-H+

exchanger

Pflugers Arch - Eur J Physiol

sensitivity of NHE isoforms to amiloride analogs and sug-gested Na+/H+ exchange activities in PT brush border vesiclesare primarily mediated by NHE3. Further, using specificantibodies raised against NHE3, two studies demonstratedthe presence of NHE3 in the brush border of PT cells [7,14]. In agreement with the proposed role of NHE3 in renalbicarbonate handling, its protein expression increased in ratcortical brush border vesicles following acid loading [6, 176].Finally, in 1998, Schultheis et al. [139] reported that globalknockout of NHE3 reduced bicarbonate reabsorption by60 % (measured by in situ perfusion of PT segment).While those animals exhibited acidosis even under the controlcondition, it was relatively mild. This is likely reflecting acompensatory increase in bicarbonate reabsorption in moredistal region of the nephron. Indeed, the tubuloglomerularfeedback remains intact [112], and contribution of NHE2 tobicarbonate reabsorption is dramatically enhanced in earlydistal tubule from NHE3 knockout mice [11]. Although theglobal NHE3-knockout mice provided useful insights on therole of NHE3 in bicarbonate reabsorption within PT, at thesame time NHE3 is also highly expressed in intestine andconsequently, mice suffer from fluid and bicarbonate absorp-tion defects along the gastrointestinal tract. As a result, loss ofNHE3 in the intestine could lead to hypovolemia and inducesystemic acidosis. Indeed, significant alkalinization of intesti-nal content was observed in NHE3 knockout mice [139]. Toclarify the role of NHE3 in PT electrolyte transport, a recentstudy created a new transgenic mouse line where NHE3 wasconditionally deleted in PT [109]. In agreement with theproposed role of NHE3, the knockout mice exhibited slightmetabolic acidosis, with plasma pH and [HCO3

] lowered by0.1 pH unit and 2 mMEq, respectively, and in isolatedperfused PT, the bicarbonate reabsorption rate was reducedby 36 %. The protein expression of NHE3 was significantlyelevated in the medulla, implying possible increase in theexpression in TAL. Taken together, it is safe to conclude thatH+-secretion mediated by NHE3 plays pivotal role in drivingbicarbonate reabsorption in PT.

PT also expresses several other isoforms of NHEs on itsapical membrane, and it has been long debated whether otherNHE isoforms could also contribute to bicarbonate reabsorp-tion at PT. Two studies using NHE3 knockout mice reporteddifferent results: Wang et al. [169] showed that treating PT ofNHE3 knockout mice with up to 100 M 5-(N-ethyl-N-isopropyl)amiloride (EIPA) did not reduce bicarbonate reab-sorption further, while Choi et al. [38] reported that there wassignificant EIPA-sensitive NHE activity in PT isolated fromNHE3, NHE2, and NHE2/3 double knockout mice. The dis-crepancy between these two studies might reflect difference inmethod (in situ vs. in vitro experiment and measurement ofluminal bicarbonate transport rate vs. magnitude of intracel-lular pH changes dependent on Na+ in [169] and [38], respec-tively), although this needs to be clarified [38]. Careful

examination of NHE2 expression along the renal tubule byimmunohistochemistry localized NHE2 only in the TAL andthe distal convoluted tubule (DCT) [34]. Moreover, althoughNHE2 knockout mice exhibited impaired ability to acidify thegut, no systemic acid-base disturbance was observed, suggest-ing that NHE2 is unlikely contributing to bicarbonate absorp-tion in PT significantly [138]. However, as stated above,NHE2 activity and NHE2-dependent bicarbonate absorptionwere markedly upregulated in the distal nephron of NHE3 KOmice, suggesting that NHE2 plays an important role in bicar-bonate absorption by the distal tubule and that this process isparticularly important when distal delivery of bicarbonate isincreased (e.g., in NHE3 knockout mice) [11].

In 2003, Goyal et al. [72] identified a new isoform of NHE(NHE8) in rabbit kidney, and a later detailed immunohisto-chemistry work showed that within a nephron, NHE8 isexpressed only in PT [71]. Although knockout of NHE8 alonedid not affect NHE activity in isolated PT (determined as theNa+-dependent pHi changes), NHE activity was significantlylower in NHE3/8 double knockout mice in comparison toNHE3 knockout mice [13]. Because NHE8 protein expressionwas significantly elevated in NHE3 knockout mice, it ispossible that NHE8 plays an additional role in H+-secretionin PT, especially when NHE3 function is impaired. Althoughthe role of NHE8 in PT function might be only supplementaryin adult, it might be playing more predominant role duringearly developmental stages of the animal. During the first24 days of development, protein expression of NHE3 andNHE8 within brush border membrane of PT increased anddecreased, respectively. Furthermore, brush border membraneprotein expression of NHE3 and NHE8 was both significantlyelevated in neonates following induction of acidosis. Thus, itis possible that the profile of NHE contributing to the bicar-bonate reabsorption in PT is altered during the development ofan animal [159]. More thorough profiling of NHE knockouttransgenic animals at various ages might provide an additionalinsight into the role played by multiple NHE isoforms in PT.

Early studies also observed that bicarbonate reabsorption inisolated PT could still occur even in the complete absence ofluminal Na+. In 1981, Chan and Giebisch [35] proposed thatelectrogenic mechanism of H+ excretion independent of Na+

could account for such bicarbonate reabsorption. The molec-ular mechanism of this Na+-independent acid secretion waslater corroborated by identification of a primary active protonpump (vH+-ATPase) in membrane vesicles isolated from ratkidney cortex [135]. Schwartz and Al-Awqati [142] latershowed that addition of CO2 increased the exocytotic fusionof vesicles containing H+-ATPase. The presence of a vH+-ATPase in the renal epithelium was subsequently confirmedby Ait-Mohamed et al. [1], where they reported N-ethylmalimide sensitive ATPase activity is widely distributedin rat kidney, including PT, and also by Brown et al. [25],where they localized a 56-kDa H+-ATPase subunit on apical


membrane invaginations (as well as inmicrovilli) in rat kidneyPT. The activity of the vH+-ATPase was markedly stimulatedin brush border membrane vesicles isolated from the kidney ofrats experiencing metabolic acidosis, further suggesting a rolefor vH+-ATPase in adaptive increase in bicarbonate reabsorp-tion capacity in response to metabolic acidosis [32].Subsequently, involvement of the vH+-ATPase in luminalacidification/bicarbonate reabsorption was demonstrated,where the authors treated NHE3 knockout mice withbafilomycin and observed inhibition in bicarbonate reabsorp-tion measured using in situ microperfusion [169].

vH+-ATPase is a large (900 kDa), multi-subunits trans-membrane protein complex that transports H+ across biolog-ical membranes at the expense of energy derived from hydro-lysis of cytosolic ATP. It consists of a peripheral, catalytic V1domain (consisting of subunits AH; donated in upper caseletters) and a membrane-bound V0 domain (consisting ofsubunits a, c, d, and e; donated in lower case letters) (forreviews, see [136, 168]). Each subunit of either V1 or V0domains has multiple isoforms exhibiting either broad or veryrestricted expression patterns. B1 and a4 play a key role inacid-base handling by the kidney and mutations in the genesencoding these proteins are known to induce serious distalrenal tubular acidosis (dRTA) in human patients [90, 151,153]. Although the role of specific subunits for H+-ATPasein acid-base regulation has been investigated largely in inter-calated cells within distal nephron (discussed below), werecently showed that the loss of an a4 subunit also affectsphysiological function in PT [76]. Specifically, in the a4knockout mice, we observed severe disruption of H+-ATPase assembly and expression in PT cells. The animalsalso exhibited proteinuria and elevated phosphate wasting inurine, possibly reflecting reduced endocytosis and lower api-cal expression of phosphate transporter, NaPi-lla, respectively.While transepithelial luminal transport of bicarbonate was notdirectly measured in [76], given that clinical cases of pRTA isoften associated with general disruption in PTs sodium-dependent apical transporter function (Fanconi syndrome)[75], more detailed examination of PT from a4 knockoutmice could provide useful insights into pathophysiology ofpRTA.

Role of carbonic anhydrase and apical influx of CO2

Carbonic anhydrase (CA) is a zinc-containing metalloproteinthat catalyzes the reversible hydration reaction of CO2: H2O+CO2 H

++HCO3. To date, 15 isoforms of CA have been

identified in mammals. Among those, cytosolic CA-II isexpressed ubiquitously in nephron, except in thin ascendingloop of Henle, and accounts for 95 % of CA activity innephron [27, 130]. PT (also in outer/inner medullarycollecting duct or OMCD/IMCD, and -intercalated cells incortical collecting duct, or CCD) also expresses CA-IV, an

isoform of CA bound to both apical and basolateral mem-branes via GPI linker [129]. As early as 1954, clinical studiesshowed the effect of CA inhibition on systemic acid-basebalance regulation [101]. Several subsequent studies (e.g.,[39, 41, 114]) also observed significant reduction in bicarbon-ate reabsorption rate in PT following CA inhibition.Furthermore, a mutation that disrupted CA-II function wasassociated with RTA (both pRTA and dRTA, presumablyreflecting the wide distribution of CA along the nephron)[150], thus confirming an important role played by CA inbicarbonate reabsorption in PT. In 1988, through a mutagen(N-thyl-N-nitrosourea) treatment, Lewis et al. [108] created amouse line carrying a mutation in CA-II. Interestingly, in suchmice, intercalated cells were greatly reduced in collecting duct[23], which is likely the reason for less acidified urine pro-duced by those mice as seen in the original study [108] (seethe section below for further discussion on intercalated cells inCD).

The molecular CO2 formed as a result of dehydration ofHCO3

needs to cross the apical membrane and enter thecytosol. While the traditional view has been small gaseousmolecules like CO2 can freely move across the cell membrane,there have been increasing interests in the role of severalmolecules acting as gas channels [19, 48]. By measuringchanges in surface pH in Xenopus laevis oocyte or by stop-flow analyses of recombinant cells, it has been shown thatsome Rhesus glycoproteins (Rhs), aquaporins (AQPs), andurea transporters could conduct CO2 and/or NH3 with varyingselectivity among isoforms [5860, 115, 117, 133, 178].Given those new data, it has been suggested that an apicallyexpressed AQP1 in PT is facilitating apical CO2 entry, thuscontributing to the overall bicarbonate reabsorption. A pre-liminary in vitro microperfusion experiment showed that bi-carbonate transport rate is significantly lower in PT isolatedfrom AQP1 null mice compared to the wild-type mice [19].On the other hand, based on stop-flow analysis, permeabilityto CO2 was not significantly different in apical membranevesicles isolated from wild-type and AQP1-null mice [51].One possible explanation for the negative result seen in [51] isthe considerably higher permeability to CO2 in isolated vesi-cles (due to their larger surface to volume ratio), in compari-son to the intact tubule. Regardless, given such contradictions,further experiments would be required to draw firm conclu-sions regarding the physiological significance of AQP1 as agas channel in PT.

Basolateral HCO3 extrusion in PT

Majority of basolateral HCO3 extrusion is mediated by Na+

HCO3 co-transporter (NBC or SLC4), which transports Na+

and HCO3 in both electrogenic- and electroneutral manner

depending on isoform [123]. The idea that there is a Na+-HCO3

co-transport activity on the basolateral membrane of


PT was experimentally demonstrated in 1983 [20], and thefirst NBC (NBCe1; but later named as NBCe1-A to accountfor splice variants) was cloned in 1997 from tiger salamander[134]. In 1999, Igarashi et al. [84] identified two nonfunction-al mutations in NBCe1 (R298S and R510H) as causes ofheritable pRTA. Those mutations in NBC induced very severeacidosis, where plasma [HCO3

] was as low as 5 mEq/L.Since then, additional 10 mutations in NBCe1 have beenidentified to cause pRTA, with eight missense mutations(R298S, S427L, T485S, G486R, R510H, L522P, A799V,and R881C), two nonsense mutations (Q29X and W516X),and two frame shift mutations (N721TfsX29 and S982NfsX4)[97, 145].

In agreement with these clinical cases of acidosis linkedwith NBCe1-A mutations, transgenic mice with alteredNBCe1 function also exhibit RTA. To date, two transgenicmice lines have been created: the first line lacks the expressionof NBCe1-A (the splice variant expressed in kidneys/eyes)and NBCe1-B (the splice variant expressed in pancreas/colon/duodenum) [55] and the second line harbors W516X-knockin, a point mutation that corresponds to one of thepRTA-causing mutations in human NBC [111]. Both trans-genic mice line suffered from early lethality, retarded growth,and severe pRTA, where blood pH and [HCO3

] were 6.8and 5 mEq/L, respectively. Given the shared phenotypebetween those two lines, it is safe to state that basolateralNBCe1-A expression is indispensable for proper bicarbonatereabsorption in PT and thus for systemic acid-base balanceregulation.

Bicarbonate handling by thick ascending limb

The TAL is responsible for reabsorbing about 10 % of filteredbicarbonate [62], as summarized in Fig. 1b. The region alsocontribute to the acid-base regulation by absorbing some ofthe luminal ammonia (see below for the role of ammoniahandling in renal acid-base regulation) [68]. Within TAL, aset of transporters similar to that in PT (apical NHE andcytosolic CA-II) are responsible for mediating the reabsorp-tion of bicarbonate, but it is likely that almost all apical H+

secretion is mediated by NHE, most notably NHE3 [7, 69,99]. NHE2 is also expressed on apical membrane of TAL [34],and given the compensatory response mediated by NHE2 inNHE3-knockout mice (as discussed above in the PT section)[11], it is also possible that NHE2 is also contributing to fine-tuning the reabsorption of bicarbonate in this region.

Two isoforms of NHEs, NHE1 and NHE4, are expressedon basolateral membrane of TAL. While the role of NHE4 inacid-base handling in TAL is likely to be mainly NH4

+ trans-port (see below) because of its lower sensitivity to pHi (pK fortransport activity was 6.21 and 6.75 for NHE4 and NHE1,

respectively) [33, 121], NHE1 is likely to be playing a role inbicarbonate reabsorption. The bicarbonate transport rate inisolated TAL was significantly lower when (1) amiloridewas added to the bath solution and (2) in NHE1 null mice[70].More importantly, treating TAL isolated fromNHE1 nullmice with amiloride (added to the bath) did not affect thebicarbonate reabsorption rate, suggesting that NHE1 is likelythe sole mediator of inhibitory effect from amiloride treatment[70]. A subsequent study by the same group also showed thatthe inhibitory effect from NHE1 inhibition was due to itsinteraction with apical NHE3 via actin cytoskeleton [172]. Itremains to be determined whether NHE1 could affect thefunction other apical transporters (e.g., Na+-K+-2Cl co-trans-porter, renal outer medullary K+ channel, epithelial Na+ chan-nels) in nephron and other parts of the body.

Molecular mechanisms for basolateral HCO3 extrusion in

TAL has been debated for a long time, and NBC (electrogenic,Na+-nHCO3

transport [92]), K+-HCO3 co-transport [105],

and Cl/HCO3 exchanger [155] have been proposed to be

involved with this process. Using enriched fraction of vesiclesisolated from basolateral membrane, we later confirmed thatbicarbonate transport activity is largely mediated by 4,4-diisothiocyano-2,2-stilbenedisulfonic acid (DIDS)-sensitive,Cl-dependent exchanger [44, 106]. Based on the localizationpattern, this exchange activity is likely to be mediated by AE2[5], and a later study demonstrated that acid loading signifi-cantly increased protein level in kidney cortex and basolateralexpression of AE2 in cortical TAL (but not in medullaryTAL). Although AE2-null mice have been generated, theglobal deletion of AE2 led to high mortality [56] and compre-hensive data regarding the effect of AE2-knockout on acid-base handling are not yet available. Generation of transgenicmice conditionally lacking AE2 in TAL would provide auseful model in addressing potential role played by AE2 inbicarbonate handling.

Renal ammonia handling

To maintain acid-base homeostasis, the kidney needs to notonly reabsorb filtered bicarbonate but also excrete the dailyacid load generated by protein catabolism (1 mmol/kg/day inhumans eating a typical western diet). Since minimal urine pHis 4.4 and urine volume rarely exceeds 2 L per day, theexcretion of acid as free protons is negligible and majorityof protons are rather excreted with the acid form of urinebuffers, particularly NH4

+. As a consequence, within thekidney, ammonia handling constitutes a major process foracid-base regulation (Figs. 2 and 3). Unlike bicarbonate, rel-atively minuscule amount of ammonia is filtered by the glo-merulus. Instead, ammonia is generated within renal epithelialcells (ammoniagenesis). Ammoniagenesis within PTalso gen-erates equimolar HCO3

as a by-product of enzymatic


reactions (Fig. 2a), which is transported via basolateral NBC(see above). The new bicarbonate ions added to the extracel-lular fluid (ECF) replenish the ECF bicarbonate buffer titratedby H+ generated through metabolic processes.

Ammoniagenesis in PT

The production of ammonia occurs throughout the nephron,but the rate of production is relatively higher in PT cells (inboth convoluted and straight segments; Fig. 2a). PT is also theonly region where activities of phosphate-dependent gluta-minase (PDG; an enzyme responsible for initiating the initialenzymatic reaction for conversion of glutamine to ammonia)and consequently, ammonia production, increase in responseto acid load [42, 67, 173, 175]. These observations suggest animportant role played by this segment in acid-base regulationthrough ammoniagenesis.

The excretion of ammonia into the lumen is likely to bepartially mediated by apical NHE, in the form of NH4

+. Usingrabbit brush border membrane vesicles, Kinsella and Aronsonobserved that Na+ uptake is competitively inhibited by external

NH4+ and Li+, hinting that the Na+/H+ exchanger could indeed

act as an exchanger between Na+ and NH4+ [95]. Subsequently

in isolated, microperfused mouse PT, it was observed that only asmall fraction of ammonia was secreted into lumen in the pres-ence of 100 M amiloride in perfusate [118]. Although resultsobtained from in vitro experiments often agree, in situmicroperfusion of PT in the presence of NHE inhibitors alonedid not affect ammonia secretion into the lumen [148, 149]. Theammonia secretionwas inhibited onlywhen bothNHE inhibitorsand barium (as a generic inhibitor for K+ conducting channels)were added to the lumen [149]. This observation suggests theremight bemultiple route ofNH4

+ transport on apicalmembrane ofPT (NHE and K+-conducting channel(s)); the molecular identifyof apical K+ channel contributing to NH4

+ secretion remains tobe identified. Additionally, a recent study showed that ammoniasecretion was not impaired in PT-specific NHE3 knockout mice,both under control and acid-loaded conditions [109]. While thisobservation possibly challenges the role of NHE3 as a mediatorof Na+/NH4

+ exchange in PT, it is important to realize that NHE8is also expressed on the apical membrane of PT. Assessing thepotential role played by other transporters able to mediate Na+/

Fig. 2 Molecular mechanisms of ammonia handling in proximal tubule(PT) and thick ascending limb (TAL). PT is the major site ofammoniagenesis with glutamine as an initial substrate. Newlysynthesized ammonia is secreted into lumen either as gaseous NH3 orionic NH4

+, carried by NHE3/8. H+ secreted by H+-ATPase is titrated byNH3 and trapped as NH4

+. The ammoniagenesis and its secretion isaccompanied by synthesis of equimolar HCO3

, which is absorbed viabasolatral NBC1(Fig. 2a). TAL absorbs luminal ammonia to establish

high concentration of ammonia in interstitium. This is accomplished byapical NKCC2, which can transport NH4

+ as a substitute for K+, andbasolateral (1) NHE4, which directly transports NH4

+ and (2) NBCn1,which transports HCO3

into epithelial cell and drives the conversion ofNH4

+ to NH3 (Fig. 2b). Abbreviations: CA: carbonic anhydrase, NBC1:Na+-HCO3

co-transporter; NKCC: Na+-K+-2Cl co-transporter; NHE:Na+-H+ exchanger


H+ exchange would therefore be critical to address the molecularmechanism of apical NH4

+ secretion in PT.It is also worth noting that at least in the in situ

microperfusion experiment, about 50 % of ammonia wassecreted into the lumen even after the addition of both NHEinhibitor and barium [149]. Given the role of AQP1 as a gaschannel (see discussion above on gas channels), it is alsopossible that AQP1 is responsible, at least partially, forconducting NH3 into the lumen. The potential role of AQP1in ammonia handling by PT remains to be testedexperimentally.

As a result of ammoniagenesis in PT followed by ammoniasecretion by PT cells into the tubular fluid, delivery rate ofammonia in urine leaving PT is almost equal to or slightlyhigher than the final excretion rate of ammonia in the urine[173].

Ammonia absorption in TAL

One of the major functions of TAL in acid-base regulation isto absorb ammonia (Fig. 2b). This absorption of ammonia

along with the counter-current multiplier mechanisms createsa sufficiently high ammonia concentration in medullary inter-stitial fluid, which, along with the protons secreted into thelumen, ultimately acts as the driving force for ammonia se-cretion into CD. Following the chronic acidosis, an ammoniaabsorption capacity in TAL is elevated [65], and consequently,contributes to generate steep ammonia content gradient acrossmedulla following acid treatment [122].

Because apical membrane of TAL is not very permeable toNH3 [91], it has been argued that primarily NH4

+ is transportedin TAL. It is now solidly established that an apically expressedNKCC2 constitutes a major entry route of NH4

+ in TAL cells[66, 68, 171]. The idea that NH4

+ could bind to a K+-binding sitein NKCC2 was demonstrated in Kinne et al. [94], where theymeasured the ion uptake kinetics using the apical membranefraction isolated from TAL. In 1987, Good et al. [63] demon-strated that ammonia transport in isolated TAL is inhibited byelevated potassium concentration in perfusate, thereby showingthat NH4

+ and K+ could indeed compete for the binding site of atransporter. The expression level of this co-transporter, as well asactivity, was found to be elevated following acid exposure, in

Fig. 3 Molecular mechanisms of urine acidification and ammoniasecretion in collecting duct (CD). CD is responsible for finalacidification of urine, which is largely accomplished by secretion of H+

into urine via apical vH+-ATPase expressed on -intercalated cells.Secreted acid is titrated by NH3, which is effectively secreted into urinethrough ammonia conducting Rhcg channels expressed on both apicaland basolateral membranes of -intercalated cells. Ammonia secretion is

also driven by high interstitium-to-urine ammonia gradient, establishedby TAL. Epithelia within CD also consists of -intercalated cells, whichexpress apical pendrin (SLC26A4) and principal cells, which expressapical channels for Na+ (epithelial Na+ channel), water (aquaporin 2), andK+ (renal outer medullary K+ channel). Abbreviations: AE: anion ex-changer; CA: carbonic anhydrase, IC: intercalated cell; PC: principal cell;Pds: pendrin


agreement with the increase in NH4+ absorption in acid-loaded

animal [9].For a long time, basolateral NHE was shown to contribute to

the ammonia absorption in TAL. Using enriched collection ofboth luminal and basolateral membrane vesicles, we showed,similar to PT, NH4

+ can substitute for H+ in Na+/H+ exchangeactivities in TAL cells [17]. Subsequently, we showed that NHE4is expressed on the basolateral side of TAL and observed that it ismuch less sensitive to internal H+ than the other basolateral Na+/H+ exchanger, NHE1 [33]. Based on this observation, we pro-posed that the low affinity of NHE4 for intracellular H+ makes itmore appropriately designed for NH4

+ transport across thebasolateral membrane of TAL. In the same study, we alsoshowed that NHE4 is less sensitive to EIPA than NHE1 (IC50for NHE1 andNHE4was 11 nMand 2.5M, respectively). Thisdifferential sensitivity to EIPA, as well as newly created NHE4-nullmice, was used to demonstrate the role ofNHE4 in ammoniaabsorption in TAL, especially under the acid-loaded mice [22].The NHE4-null mice developed and grew normally, althoughunder the control condition, they exhibited compensatedhyperchloremic metabolic acidosis. The urinary excretion rateof ammonia was significantly higher in NHE4-null mice underthe control condition, which might be reflecting more acidic (bypH 0.3 unit) urine. However, when challenged with an acidload (NH4Cl in drinking water), the ability to increase urinaryammonia output was significantly compromised in NHE4-nullmice, reflecting significantly lower ammonia accumulation inmedullae. The ammonia transport rate was significantly lowerin TAL isolated fromNHE4-null mice, andwas inhibited in TALisolated from wild-type mice only when treated with EIPA at adose high enough to inhibit NHE4 (10 M). Finally, metabolicacidosis increased mRNA expression and activities associatedwith NHE4 [22]. Those observations revealed critical role ofNHE4, especially when animals are challenged with acidosis, inammonia handling by TAL [22].

While those two transporters discussed above contribute toammonia absorption by directly transporting NH4

+,basolaterally expressed NBCn1, which acts as a base loader(i.e., mediates influx of HCO3

and Na+) also facilitatesammonia absorption in TAL [120]. The principle is thatHCO3

transported by NBCn1 reacts with intracellularNH4

+, and that gaseous NH3, rather than NH4+, exits through

the basolateral side of the membrane. Several indirect evi-dence supports this model: NBCn1 is highly expressed on thebasolateral membrane of TAL [167] and activity and proteinexpression of NBCn1 is elevated in TAL isolated from acid-loaded rats [98, 120]. While these observations strongly im-plies a role played by NBCn1 in physiological response ofTAL to acid load, for the moment, experimental evidence thatdirectly links the loss of NBCn1 function to abnormality inammonia handling within TAL is lacking. Thus, the involve-ment of NBCn1 in ammonia handling awaits furtherconfirmation.

Rhesus protein and ammonia secretion in collecting duct

CD is the last segment of a nephron and plays an importantrole in fine-tuning the final urine composition (Fig. 3).Ammonia diffuses into the CD lumen following the PNH3gradient established by TAL, and once in the lumen, it reactswith luminal proton and converted to NH4

+ (for mechanismsof urine acidification in CD, see the section below), thusmaintaining the PNH3 gradient. The ammonia handling inCD is controlled by several isoforms of ammonia conductingRhesus glycoproteins. Because Rhbg and Rhcg are two mainisoforms expressed in kidney, we will limit our discussion onthe function of those two isoforms.

Both human RhBG and mice Rhbg were first identified in2001 through sequence analysis and based on the result fromin situ hybridization, Rhbg was proposed to be distributed inconvoluted PTand loop of Henle [110]. However, it is unlike-ly that Rhbg protein is highly expressed in PT. A later study byQuentin et al. [131] detected low level of mRNA expression inPT segments isolated from rat kidney but no significant im-munohistochemical staining was observed in PT. Instead,staining was only seen in distal portion of the nephron, in-cluding connecting tubule (CNT), CCD, OMCD, and IMCD,where higher level of mRNA expression was also detected[131, 163]. Within those regions, Rhbg is highly expressed onbasolateral membrane of - and non- non--intercalatedcells, and also on basolateral side of principal cells to a lesserdegree (see the section below for classification of epithelialcells in CD). While it was not found to be expressed on -intercalated cells, it was expressed on the basolateral side ofCNT cells, which also express pendrin apically [131, 163].Because specific inhibitors are currently unavailable for Rhproteins, physiological significance of Rh proteins in ammo-nia handling has been studied using knockout animal model.In both Rhbg-null mice [30] and intercalated cell-specificRhbg knockout mice [15], no developmental detects or im-pairment in ammonia secretion into urine was observed undercontrol condition. Neither study observed compensatorychanges in Rhcg mRNA expression level or localization pat-tern (see below). Furthermore, no difference in permeability toNH3 or NH4

+ was observed in CCD isolated from Rhbg-nulland wild-type animals [30]. On the other hand, response toacid load differed between those two studies. WhileChambrey et al. [30] did not observe any difference in urineammonia concentration following acid load between Rhbg-null and wild-type animals, Bishop et al. [15] reported signif-icant (but transient) reduction in ammonia concentration inurine from Rhbg-null mice. It is worth noting that the methodof acid load was different between these two studies (NH4Clin water vs. HCl in chow), and possibly as a result of that,much higher urine ammonia secretion was seen in [15]. Takentogether, it is unlikely that Rhbg is playing a critical role inammonia handling under basal condition, or even when the


animals are challenged with mild acid load. Bishop et al. [15]observed that protein expression of glutamine synthetase (GS,a key enzyme for metabolizing ammonia) was reduced incortex and OMCD in Rhbg knockout mice. Although thisremains to be experimentally demonstrated, such response inGS could have elevated total ammonia content in medullaryinterstitial fluid and facilitated ammonia secretion into theurine, and this could be the reason for the lack of significanteffect in ammonia handling in Rhbg knockout mice.

Rhcg appears to have a distribution pattern similar to Rhbg.In segments of nephron isolated from rat kidney, Rhcg mRNAwas highly expressed in DCT, CNTand CD. In rat CNT, Rhcgwas expressed apically in all cells, but in CD, it was onlyexpressed on apical side of - and non-, non- intercalatedcells [47, 131]. A later study using mouse kidney showed thatRhcg distribution pattern was largely identical to that of rat(i.e., ubiquitous in CNT, - and non-, non- intercalatedcells in CD), but Rhcg also appeared to be expressed on apicalmembrane of principal cells [163]. While it was initiallyproposed that Rhcg is solely expressed on the apical mem-brane of those cells, and it creates, with basolaterallyexpressed Rhbg, an effective ammonia conducting pathwayfrom the interstitial fluid to the luminal fluid, it is now knownthat Rhcg is also expressed on basolateral side [21, 93, 146,147]. It is possible that this additional basolateral populationof Rhcg is alleviating the challenge when Rhbg was knockedout. Indeed, capacity to excrete ammonia was significantlymore impaired in CD-specific, Rhbg/Rhcg double knockoutmice in comparison to single Rhbg knockout mice [104].

Transgenic mice models have provided strong evidencesupporting the role of Rhcg in ammonia handling by kidneyeven under basal condition. To date, three lines, including global[16], collecting duct specific [102] and intercalated cell-specific[103] Rhcg knockout mice have been created. The Rhcg-nullmice were slightly smaller than wild-type mice, and exhibitedhypokalemia. Furthermore, their urine was significantly morealkaline and urine ammonia concentration was lower [16]. Theimpaired ability to secrete ammonia was exaggerated whenanimals were challenged with acid load. The urinary ammoniaexcretion remained about 60 % of wild-type animal (250 vs150 mol/day), and the animal suffered from continuous weightloss (up to 25 % in 7-days). Furthermore, permeability to NH3was significantly lower in CCD isolated from Rhcg-null mice,while there was no significant difference in permeability to NH4

+

[16]. Surprisingly, while Rhcg deletion impaired ammonia trans-port by CD and renal ammonia excretion, the urine pH of Rhcgknockout mice remained more alkaline than that of wild type (cf.Fig. 2d in [16]). This is not in line with a decrease in the amountof urine buffer since this should rather favor the development of amore acidic urine pH if proton secretion is normal. WhetherRhcg deletion could directly impair the capabilities of -intercalated cells to secrete protons into urine has not beenexamined. Because active proton secretion is required to

maintain PNH3 gradient (and drive ammonia secretion acrossCD epithelia), impaired proton secretion can directly reduceammonium secretion. Therefore, this point should be directlytested to clarify the molecular basis for impaired ammonia secre-tion in Rhcg-null mice. A similar reduction in urine ammoniaconcentration was observed in CD-specific Rhcg knockout micefollowing the acid-load treatment, although in their case, theurine pH was significantly lower in Rhcg knockout mice thanin wild type [102]. The protein expressions of Rhbg and phos-phoenolpyruvate carboxykinase (PEPCK), one of key enzymesfor ammoniagenesis, were also higher in acid-loaded Rhcgknockout animals than in wild type [102]. These could becompensatory response to facilitate ammonia excretion intourine, although clearly they could not fully compensate for thelost Rhcg [102]. When Rhcg was knocked out only in interca-lated cells, no impairment in ammonia excretion was observed inbasal condition [103], but when those animals were challengedwith acid load, the urine pH was significantly more acidic andammonia concentration was lower in knockout mice. The greaterurinary acidification is in contrast to what was reported with theglobal deletion of Rhcg [16], while similar to the CD-specificknockout mice [102]. More acidic urine would facilitate NH3excretion into lumen (as equilibrium between NH3 and NH4

+

would be skewed towardNH4+ and PNH3would bemaintained);

thus the lower urinary pH observed in [102, 103] might havepartially ameliorated the NH3 secretion due to Rhcg knockout.However, again, it remains to be tested why no such responsewas seen in global Rhcg knockout mice, especially because theRhcg-null mice exhibited systemic acidosis [16]. Under the basalcondition, no compensatory changes in Rhbg, PEPCK or PDGprotein expression was observed in intercalated cell-specificRhcg knockout mice either in cortex or medullae. These resultssuggest that Rhcg expressed in principal cell could compensatefor ammonia secretion under the control condition. It would be ofinterest to see if Rhcg distribution pattern was altered withinprincipal cells in knockout mice (i.e., elevated level of apicalexpression) using immuogold electron microscopy, given thatapical expression of Rhcg increased in chronically acid-loadedrat [147], presumably to facilitate ammonia secretion under suchcondition. Taken together, it is likely that Rhcg assumes a greaterrole in ammonia handling in CD than Rhbg; furthermore, theRhcg expressed on principal cells could compensate for the lossof Rhcg on intercalated cells.

Final urine acidification in collecting duct

The effective secretion of NH3 into the lumen via Rhcg iscritically dependent on urinary acidification, and this is thesecond major physiological function of CD in terms of acid-base regulation. Epithelia within CD are classified into twomorphologically distinctive cells types: principal cells and


intercalated cells, and intercalated cells are playing an impor-tant role in acid-base regulation [143]. While recent studiesshowed that intercalated cells could also contribute to system-ic fluid volume and salt balance [31, 73, 85, 107]; for reviews,see [4, 45, 46], we will focus here on the classical role ofintercalated cells in acid-base regulation.

Functional subtypes of intercalated cells

Depending on expressed transporters, intercalated cellscould be further subdivided into three subtypes: -intercalated cells, intercalated cells and non- non-intercalated cells [26, 143]. The -intercalated cells aremore predominant in medullary region of CD, columnarin shape and have an extensive apical microprojections andendocytotic activity. They express apical H+-ATPase andbasolateral kidney isoform of anion exchanger (kAE1), andis chiefly responsible for secretion of acid into lumen. The-intercalated cells are more predominant in cortical regionof CD, squamous in shape, and have smooth apical mem-brane without endocytosis. These cells could also bestained with peanut agglutinin. They express apical anionexchanger pendrin and basolateral H+-ATPase. The non-non- intercalated cells are more predominant in the lateportion of DCT, CNT and the proximal region of CD, andexpress both H+-ATPase and pendrin apically, and no clearfunction has been identified to this type of intercalated cells[4, 140]. While the discussion below will focus on - and-intercalated cells, it is worth noting that staining patternsseen in intercalated cells are not clear-cut; in fact, Bastaniet al. [12] listed as many as six different staining patternsof H+-ATPase within intercalated cells. Rather, the pheno-type of - and -intercalated cells should be considered astwo extreme ends within a spectrum of phenotype forintercalated cells. Indeed, as we discuss below, the interca-lated cells could be surprisingly plastic.

Disruption in -intercalated cell function and distal renaltubular acidosis

As stated above, H+-ATPase is a large, membrane-boundATPase consisting of multi-subunits. Within CD, mutationsin B1 and a4 subunits have been identified as causes ofheritable, recessive dRTA in humans [89, 90, 151, 153]. In2005, Finberg et al. [52] reported the consequence ofatp6v1b1 (the gene encoding for the B1 subunit) geneticdisruption in acid-base handling by mice. The urine producedby these knockout mice was significantly more alkaline evenunder the control condition, and unlike wild type, isolatedCCD from atp6v1b1 knockout mice exhibited virtually noconcanamycin-sensitive H+ excretion [52]. On the other hand,the atp6v1b1 knockout mice developed normally and exhib-ited metabolic acidosis only when the animals were

challenged with acid loading treatment [52]. They proposedthis relatively minor consequence at the systemic level (incomparison to symptoms seen in human clinical cases) mightbe reflecting the relatively alkali-rich content in the controldiet for mouse, in comparison to the typical Western diet forhuman consumption [52]. In addition, the authors also ob-served an apical enrichment of the B2 isoform in medullaryCD (but no changes in mRNA or protein expression levels insamples prepared from the whole kidney) [52, 126]. Theconsequences of B1 knockout on the H+-ATPase isoformprofile were reanalyzed more recently [162]. In this study,B1-subunit deficient mice were crossed with another line thatexpress enhanced green fluorescent protein (EGFP) under thecontrol of B1-subunit promoter. Because EGFP expression islimited to intercalated cells in suchmice, the authors were ableto collect EGFP-positive intercalated cells and assess theeffect of B1 deficiency on the expression of various subunitsspecifically in intercalated cells. Based on this approach, theyobserved significant reduction in the protein expression of A,E and H-subunits, but nearly 2-fold increase in B2 subunit[162]. The magnitude of reduction in the protein expression ofthose three subunits largely agreed with the H+-ATPase activ-ity in medullary CD [126, 162]. Although it remains to beinvestigated whether compensatory responses also occur inother segments of nephron, the increased B2 expression (bothat the total and apically expressed) within intercalated cellsmight be contributing to the little systemic acid-base distur-bance seen in Atp6v1b1 knockout mice under the controlcondition [162].

Previous studies showed that translocation of H+-ATPaseto apical membrane is stimulated by soluble adenylyl cyclase(sAC or AC 10) [124, 125]. As the name suggests, sAC isdistributed in cytosol, and is activated by rise in [HCO3

] [37].Because of its activation by rise in HCO3

, its role as a HCO3

(or more in general sense, acid-base sensor) has been ex-tensively investigated using both mammalian and non-mammalian models [28, 157, 158]. The role of sAC in adap-tation to metabolic acidosis is unclear. Indeed, under metabol-ic acidosis, blood bicarbonate, and hence urinary fluid bicar-bonate delivery, are both reduced and sAC should beinhibited. On the other hand, sAC might be playing an impor-tant role in stimulating acid secretion/bicarbonate reabsorptionin CD in response to elevated HCO3

delivery to CD. Thedelivery rate of HCO3

could increase when bicarbonatereabsorption in PT is compromised (e.g., in NHE3 knockoutmice; [119]) and treatment with acetazolamide [10]. WhilesAC knockout mice apparently did not develop abnormality inblood electrolyte balance under the control condition [50],their ability to respond acid/base challenge might be impaired.Thus, a more comprehensive investigation on the conse-quence of sAC-loss on acid-base handling in the kidney mightprovide additional insight into the molecular mechanisms ofproper acidification of urine in CD.


A kidney-specific anion exchanger (kAE1) is expressed onthe basolateral membrane of -intercalated cells. This is akidney-specific gene product of SLC4A1 gene which lacksthe N-terminal amino acids present on the erythroid AE1 (79AA in case of mice, 65 AA in case of humans) [24, 96]. In1997, a dominant dRTA associated with a mutation inkAE1 was identified [29]. The phenotype of kAE1 knockoutmice largely mirrored that of dRTA patients harboring amutation in their kAE [152]. Specifically, the animal sufferedfrom severe metabolic acidosis even under the control condi-tion. Further acidosis was induced when animals were chal-lenged with 1 day oral acid load (NH4Cl added to the drinkingwater), while wild type and kAE1 +/- mice were not signifi-cantly affected by the same treatment. Despite the severesystemic acidosis, urine produced by kAE-null mice remainedsignificantly more alkaline. The proportion of -intercalatedcells did not differ between wild-type and knockout mice, butDIDS-sensitive anion exchange activity (measured as Cl-dependent pH recovery from alkalosis) was significantly re-duced in -intercalated cells of kAE1 knockout mice.Although these phenotypes generally agree with the idea thatkAE is critical for proper acidification of urine, results in [152]should be interpreted with caution because of the severelydetrimental effects seen in these mice (the survival rate wasrather low in knockout mice, with about 16 % of postweaningmice surviving to 14-16 weeks). Furthermore, a previousstudy [128] also observed the loss of AE1 induced significantanemia, with hemoglobin and hematocrit about 80 (9.9 vs2.0 g/dl) and 90 % (33 vs 3 %) lower in AE1-null mice,respectively. Stehberger et al. [152] also observed that innermedullary mass was significantly lower in kAE1-null mice, asymptom that resembles chronic hydronephrosis. Therefore, itis possible that some of the observed phenotype was second-ary to general kidney failure. Development of CD-specifickAE knockout mice would provide a useful tool to assessthe role of kAE in urine acidification.

Acid-base disturbances and intercalated cell plasticity

In addition to the regulation of transporters within each cell,CD could remodel its cell population to better handle acid- orbase- challenge. That is, when animals are given acid load, -intercalated cells proliferate, while keeping the total numberof intercalated cells constant [26, 137, 143]. One interpretationof such observation was that one type of intercalated cellscould be converted to the other, when subjected to acidosis oralkalosis [2, 3]. Using immortalized -intercalated cells de-rived from rabbit, Al-Awqatis group investigated molecularmechanisms responsible for - to -intercalated cell conver-sion [43]. When such immortalized cells were plated at highdensity, the resulting confluent layer of cells exhibited -intercalated cell like characteristics (e.g., high apical endocy-tosis and basolateral immunostaining with an antibody against

band-3 anion exchanger). In addition, they detected a 230-kDaprotein in extracellular matrix (ECM) from cell culture platedat high density [161]. This protein was later purified andtermed hensin (or DMBT1 in Mouse Genome Project)[156]. Further studies using the same immortalized -intercalated cells demonstrated that soluble, monomeric formof hensin could not induce phenotype conversion in interca-lated cells and rather, polymerization of hensin is indispens-able for conversion of intercalated cells [77]. The polymeri-zation of hensin requires interaction with at last two factors: a29-kDa galectin-3 and cyclophilin A. Initially, Hikita et al.[78] suggested that galectin-3 needs to be secreted to bundlehensin fibers by binding to SPCR domain within hensinprotein. In kidney, galectin-3 is specifically expressed in CD,colocalizes with hensin and its expression in ECM is inducedfollowing acid treatment [141]. These observations agree withthe proposed role of galectin-3 as a mediator of hensin poly-merization [141]. Similarly, inhibition of cis trans prolyl isom-erization activity mediated by cyclophilin A eliminated thereduction in bicarbonate secretion by isolated rabbit CCDfollowing acid exposure [170], an effect most likely due toreduction in hensin secretion and polymerization [127].Although no comprehensive dataset have been publishedregarding the exact intracellular signaling induced by poly-merized hensin in ECM to initiate the conversion of interca-lated cell phenotype, strong candidates are integrin 6 and 1[166].

The functional significance of hensin as a regulator of acid-base balance in CCD has also been confirmed in isolatedtubules and transgenic animals. For example, inhibition ofhensin function by treating isolated rabbit CCD with hensinantibody prevented the reduction in bicarbonate reabsorptionfollowing 3-h acid treatment [144]. Although global knockoutof hensin proved lethal, intercalated cell-specific hensinknockout transgenic mice were viable [54]. These transgenicmice exhibited clear metabolic acidosis; urine pH in knockoutmice was higher by 0.5 pH unit, and blood pH and plasma[HCO3

] were lower by 0.1 pH unit and 5 mEq/L, respec-tively. Despite the symptoms of metabolic acidosis, net renalacid excretion was identical to that of wild-type mice, dem-onstrating that the renal response to metabolic acidosis oftransgenic mice was impaired by hensin deletion. More strik-ingly, in knockout mice no-intercalated cells were observed,while the density of -intercalated cells in cortex increased[54]. In agreement with the proposed role of 1-integrin,intercalated cell-specific integrin 1 knockout mice exhibitedthe identical phenotype as hensin knockout mice [54]. Theseobservations clearly show that CCD regulates the acid-basebalance not simply by altering the mRNA/protein expressionand activity levels of transporters expressed within each cell,but also by actively altering the profile of intercalated cells.Although many insights in the physiological role of hensinhave been gained through the use of immortalized rabbit -


intercalated cells, several questions remains to be exploredincluding the identify of molecular sensor that initially detectsan acid/base disturbance and initiates the hensin polymeriza-tion, and intracellular signaling induced by integrin 6 thatultimately results in the alteration of cellular phenotype (i.e.,morphology of the cell and trafficking of transporters toappropriate membranes).

Acidosis is one of the frequent side effects from clinicalapplication of calcineurine inhibitor and the series of study onhensin and intercalated cell differentiation could also shedinsights into the acidosis caused by calcineurine inhibitortreatment [116]. The treatment with FK506, an inhibitor forcalcineurine, did not disrupt blood or urine acid-base balancesignificantly, although the urine pH was slightly more acidicunder the control condition [116]. When the animals weresubjected to acid-load treatment, their ability to acidify urinewas significantly impaired, and consequently, blood pH wassignificantly lower (although their urinary and blood acid-base balance was restored in 7 days) [116]. Under the basalcondition, the frequency of pendrin-positive intercalated cells(i.e., non- intercalated cell) was significantly elevated inCCD in FK506-treated rat, although in acid-loaded animals,FK506 treatment did not affect frequency of intercalated cellsubtypes, either in CCD or OMCD [116]. The initial disrup-tion in the proportion of intercalated cell subtypesmight implyimpaired ability to appropriately control intercalated cell phe-notype, although it is hard to explain the significantly moreacidified urine in control condition in the presence of morependrin-positive intercalated cells. The molecular mecha-nisms of acidosis induced by calcineurine inhibition, in asso-ciation with regulation of intercalated cell phenotype, warrantsfurther investigations.

Differentiation of intercalated cells

Given the phenotype of hensin knockout mice as describedabove, it is expected that abnormalities in proper intercalatedcell differentiation could also lead to disruption in appropriaterenal acid-base regulation. While the complete molecularmechanisms for intercalated cell differentiation in mammalianCD are still investigated, several proteins, including adisintegrin and metalloproteinase domain 10 (Adam 10) [74]and mindbomb-1 (a mediator for Notch signaling) [87] havebeen proposed to be involved with the differentiation ofprincipal cells and intercalated cells within CD, as the relativedensity of principal vs. intercalated cells is significantly al-tered in CD when these proteins are knocked out. While it isexpected that those transgenic mice suffer from some disrup-tion in acid-base homeostasis, no physiological data regardingthat point have been published. On the other hand, the role offorkhead transcription factor 1 (Foxi1) in acid-base regulationis perhaps the most characterized. In normal mice, this tran-scription factor is expressed in both - and -intercalated

cells, but not in principal cells [18]. In CD, cells from Foxi1knockout mice were positively stained for both aquaporin 2 (amarker for principal cells) and carbonic anhydrase II (a markerfor intercalated cells), thus giving rise to the intermediatephenotype between fully differentiated principal cells andintercalated cells [18]. Further analysis of specific transportersrevealed Foxi1 knockout abolished expression of kAE1,pendrin and B1 subunit of H+-ATPase. As predicted fromthese abnormalities, ability to acidify their urine was impairedin Foxi1 knockout mice under control condition and also inresponse to acute acid load [18].

It is also of interest to note that Foxi1 appears to beinvolved with the differentiation and regulation of ion-transporting epithelial cells expressed in other organs. Forexample, Foxi1 knockout mice also lack pendrin expressionin endolymphatic duct/sac epithelium and are deaf [82]. InFoxi1 knockout mice, H+-ATPase staining was also absent inclear cells, which are responsible for luminal acidification inepididymis [165]. In addition, a similar role of delta-Notchsignaling and forkhead transcription factors in differentiatingion-transporting epithelial cells has also been reported fromfreshwater fish [49, 86, 81] and Xenopus [132]. Thus, mech-anisms for differentiation in ion-transporting epithelia in othertissues of non-mammalian models could potentially providenew insights into cellular differentiation in CD, which couldalso improve our understanding on the regulation of systemicacid-base balance.

Acidosis associated with hyperkalemia

While abnormalities discussed above are associated with type-I RTA (distal real tubular acidosis), and associated with hypo-kalemia, there is another class of RTA associated withhyperkalemia (type IV). The basis for this type of acidosis isthe improper action of aldosterone, which could occur eithera s r educ t i on in a ldos t e rone sec r e t i on ( t r u ehypoaldosteronism) or reduced sensitivity to aldosterone(pseudohypoaldosteronism; PHA) [88].

Type I PHA is caused by mutations in , or subunit ofepithelial Na+ channels [36, 154] or mutations in mineralo-corticoid receptor [57]. The role of -ENaC in developmentof PHAType-I was also confirmed in mice model [83]. Whileglobal knockout of -ENaC resulted in 100 % mortalitywithin 2 days after birth, the survival of knockout mice waspartially rescued by additionally inducing the expression ofENaC [83], although those animals still exhibited severePHA Type-I during first week after birth [83]. The loss offunction of ENaC results in excess delivery of Na+ out of thedistal nephron, thereby reversing the normal, lumen-negativepotential (a driving force for K+ and H+ secretion) and induc-ing hypotensive, hyperkalemic metabolic acidosis [88].

PHAType-II is caused by mutations in two members of thewith-no-lysine (K) kinase (WNK) family, WNK1 or WNK4


[174] or in their regulators KELCH3 and Cul3 [113].Physiological role of WNKs, especially in blood pressureregulation, has received considerable research interest since2001 and several in-depth reviews on this topic have beenpublished recently [8, 61, 79, 80, 160]. The precise mecha-nism by which PHA Type-II causing mutations leads to thedifferent phenotypic abnormalities characteristic of this syn-drome is not fully elucidated. However, mutations in WNK1,WNK4, KELCH3 and CUL3 genes all ultimately lead toabnormal phosphorylation of the Na+-Cl co-transporter(NCC) expressed on DCT, and an increased expression andactivity of NCC. Then on the one hand, excess reabsorption ofsalt in the DCT increases vascular volume resulting inthiazide-sensitive hypertenions. On the other hand reducedNa+ delivery to the lumen of downstream nephron segments(i.e., CNT and CD) reduces ENaC activity, and thereby, pre-vents the development of a lumen negative transepithelialvoltage. Simultaneously, it is proposed that the mutations alsoreduce the activity of renal outer medullary K+ channel(ROMK), expressed on apical membrane of principal cellsand that both effects act synergistically to block potassiumsecretion and provoke hyperkalemia. While the exact molec-ular basis that induces metabolic acidosis in this pathophysi-ological condition is not clearly understood, this could be thesecondary effect from hyperkalemia (e.g., due to the reducedammonia production and secretion following hyperkalemia[88], or from inhibition of ammonium absorption byNKCC2 in the TAL [63, 64]) or from the absence of ENaC-dependent transepithelial potential difference. Several differ-ent mouse models of PHAType-II have been now generated[40, 100, 164, 177] to test these possibilities directly.

Summary and perspectives

Biochemical transport kinetic analyses, in vitro and in situmicroperfusion of specific nephron segments, meticulous im-munohistochemical staining and use of transgenic mice overthe past three decades greatly expanded our understanding onthe mechanisms of acid-base regulation in a nephron. Despitethe significant progress, much remains to be learned, includ-ing exact molecular mechanism of transporters, such as K+/NH4

+ channel in TAL, relative contributions when multipleisoforms of transporters are co-expressed on the same region(e.g., NHE isoforms expressed on the apical membrane of PTfor bicarbonate reabsorption and ammonia excretion). In ad-dition, we have only partial understanding on molecularmechanisms of acid-base sensing and subsequent intracellularsignaling mechanism that is induced to initiate the physiolog-ical responses discussed above [28]. Results from Atp6v0a4knockout model [76] also serves as a reminder to consider thefunction of a nephron as a whole, not only within specific

functional segments. Indeed, interaction among segments,including tubuloglomerular feedback (TGF), is a key featureof renal function. Investigation on interaction among differentsegments, including various paracrine signaling occurringwithin a nephron, might show further mechanisms and regu-lation in acid-base handling in kidney.

Acknowledgments D Eladari and coworkers are funded by the InstitutNational de la Sant et de la Recherche Mdicale (INSERM). This workwas also funded by grants from lAgence Nationale de la Recherche(Appel projet BLANC 2010-HYPERCLO and Appel ProjetGnrique 2014-HYPERSCREEN to D.E.).

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