The Cystic Fibrosis of Exocrine...

The Cystic Fibrosis of Exocrine Pancreas

Michael Wilschanski1 and Ivana Novak2

1Pediatric Gastroenterology, Hadassah University Hospital, Jerusalem 91240, Israel2Molecular Integrative Physiology, Department of Biology, University of Copenhagen, DK 2100Copenhagen Ø, Denmark

Correspondence: [email protected]

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is highly expressedin the pancreatic duct epithelia and permits anions and water to enter the ductal lumen.This results in an increased volume of alkaline fluid allowing the highly concentratedproteins secreted by the acinar cells to remain in a soluble state. This work will expoundon the pathophysiology and pathology caused by the malfunctioning CFTR protein withspecial reference to ion transport and acid–base abnormalities both in humans and animalmodels. We will also discuss the relationship between cystic fibrosis (CF) and pancreatitis,and outline present and potential therapeutic approaches in CF treatment relevant to thepancreas.

The pancreas is one of the organs earliest andmost seriously affected by cystic fibrosis

(CF). Although cystic fibrosis transmembraneconductance regulator (CFTR) is expressedonly in a very small percentage of exocrine cells,its malfunction has catastrophic effects on thewhole organ, resulting in its eventual destruc-tion, leading to maldigestion and malnutrition.In recent years, new therapeutic approaches arebeing developed to improve anion/fluid bal-ance, especially in the airways. Whether thesewill have any value for the pancreas requiresa more detailed understanding of pancreaticfunction drawn from clinical and genetic stud-ies and cell/organ studies of ion channels andtransporters specific for pancreatic cells. In thepresent work, we try to raise some of the criticalissues of the physiology and pathophysiology ofthe pancreas in CF.

EXOCRINE PANCREATIC ABNORMALITIES

Exocrine Pancreatic Function

The CFTR protein is highly expressed in pancre-aticductal epithelia and permits anionsand fluidto enter the ductal lumen. There is evidence thatCFTR is associated with bicarbonate transportdirectly or indirectly (see below). Indeed accord-ing to the Quinton hypothesis, it is the defectin bicarbonate transport that is the primary de-fect in CF leading to mucoviscidosis (Quinton2008). The net result of ductal function is anincreased volume of alkaline fluid, allowing thehighly concentrated proteins secreted by the ac-inar cells to remain in a soluble state. Absentor reduced CFTR channel function impairschloride and bicarbonate transport of the ducts,which results in reduced volume and hypercon-centration of macromolecules (Kopelman et al.

Editors: John R. Riordan, Richard C. Boucher, and Paul M. Quinton

Additional Perspectives on Cystic Fibrosis available at www.perspectivesinmedicine.org

Copyright # 2013 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a009746

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1985, 1988). The consequences of mutations inthe CFTR gene have been shown by pancreaticfunction studies that indicate that CF patientshave a low flow of secretions with a high proteinconcentration, which presumably will precipi-tate in the duct lumina causing obstruction anddamage (Fig. 1).

These changes in the CF pancreas begin inutero and after delivery the process of small ductobstruction leading to large duct obstructioncontinues. At birth, and for several months af-terward, there is a release into the blood streamof proteins originating in the pancreas. An ex-ample of this is immune reactive trypsinogen(IRT) that forms the basis for the neonatalscreening test for CF. Interestingly, with thiswholesale destruction of the exocrine pancreasoccurring, the infant is asymptomatic. The rea-

son for this silent destruction is yet to be deter-mined. Eventually, this process results in severeinflammation, obstruction of ducts by mucusand calcium containing debris, the destructionof acini, and generalized fibrosis. Contrary topopular belief that the pancreas is entirely non-functioning at birth, the high IRT does showthat some exocrine pancreatic tissue is stillpresent and this may have a bearing on possiblesmall molecule therapies targeted at the remain-der of the pancreas that may rescue enough tis-sue to preserve viability of the remaining pan-creas.

One of the most remarkable observations isthat genetic factors exquisitely influence the de-gree of pancreatic disease and its rate of pro-gression. Large studies of CF patients resultedin their classification as pancreatic insufficient

Duct

Protein precipitationand obstruction

Concentrated acidicprotein secretion

Ductal cellCentroacinar cell

High proteinconcentration

H2O

Acinar cell

ZymogengranulesCI–

HCO3–

HCO3–

X

HCO3–

HCO3–Secretin Cl–

Cl–

CFTR

Na+

cAMPCa2+

CCKAChSecretin/VIP

Acinus

Figure 1. Pathogenesis of pancreatic disease in CF. Acinar cells secrete large quantities of protein, primarily in theform of digestive enzymes, into the acinar lumen. Under normal circumstances anions (Cl2 and HCO3

2) aresecreted into the ductal lumen (see detailed model in Fig. 3). This provides a driving force for the movement offluid into the lumen of the duct and maintains the solubility of secreted proteins in a dilute, alkaline solution. InCF, impaired anion transport into the proximal ducts results in decreased secretion of more acidic fluid, whichleads to precipitation of secreted proteins. Intraluminal obstruction of the ducts then causes progressive pan-creatic damage and atrophy. (From Wilschanski and Durie 2007; reprinted, with permission.)

M. Wilschanski and I. Novak

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(PI) or pancreatic sufficient (PS). PI patientscomprise 85% of all CF patients and havemaldigestion as defined by evidence of steat-orrhea following 72-hr fat balance studies.These PI patients require pancreatic enzyme re-placement therapy with meals. In contrast, PSpatients have evidence of pancreatic damage(these patients may be diagnosed by the highneonatal IRT test, which means that damage isoccurring), but retain sufficient endogenousexocrine pancreatic function to sustain normaldigestion.

Exocrine pancreatic status is directly linkedto genotype (Kerem et al. 1990; Kristidis et al.1992). Analysis of particular CFTR mutationsin patients with these pancreatic phenotypes(PI vs. PS) revealed two categories of alleles:“severe” and “mild.” Patients homozygous orcompound heterozygous for severe alleles be-longing to classes I, II, III, or VI confer pan-creatic insufficiency, whereas a mild class IV orV allele sustains pancreatic function in a domi-nant fashion, even if the second mutation issevere. This observation appears plausible be-cause all known mild alleles belong to class IVor V, all of which are (or predicted to be) asso-ciated with some residual chloride channel ac-tivity at the epithelial apical membranes. How-ever, this classification system is not entirelyconsistent as there are some class I mutationswith the stop codon at the end of the gene thatare in fact PS. A small proportion (2%–3%) ofpatients carrying severe mutations on both al-leles are PS at diagnosis, but most experiencegradual transition from PS to PI. A few missensemutations (e.g., G85E) confer a variable pancre-atic phenotype.

Although mild mutations confer sufficientCFTR function to prevent the pancreas frombeing completely destroyed, many PS patientshave reduced exocrine pancreatic capacity andare associated with an increased risk of pancre-atitis. Recurrent acute and chronic pancreatitisis a relatively infrequent complication of CF,first reported by Shwachman et al. (1975). Inthis retrospective study, only 0.5% of CF patientshad pancreatitis. More recently, Durno et al.(2002) reported in a cohort of more than 1000patients, followed over a period of 30 years, that

the incidence was 1.7%. All the patients withpancreatitis were PS. In fact, this subgroup ofPS patients appears to be highly susceptible topancreatitis because almost one in five was af-fected by this complication. There have beensuggestions in the literature that PI patientsmay also have pancreatitis, but most probablyin these patients pancreatic function was notfully investigated (De Boeck et al. 2005). In thelargest study to date of CF PS patients, Ooi et al.(2011), in a seminal paper, determined the as-sociation between severity of CFTR genotypeand the risk of pancreatitis. They examined alarge cohort of 277 PS patients from two CFcenters of which 62 had well-documented pan-creatitis. Using a novel pancreatic insufficiencyprevalence score, the mutations were dividedinto three main groups: severe, moderate-se-vere, and mild. They found that the proportionof patients who developed pancreatitis was sig-nificantly greater for genotypes in the mildgroup than the moderate-severe group. Thus,the more mild mutations are associated withincreased risk of pancreatitis.

Recurrent “Idiopathic” Pancreatitis

Several studies have shown that patients withidiopathic acute, recurrent, and chronic pancre-atitis carry a significantly higher frequency ofCFTR gene mutations than the general popu-lation (Cohn et al. 1998; Sharer et al. 1998).Bishop et al. (2005) prospectively examined56 patients with idiopathic recurrent acute orchronic pancreatitis by performing extensivegenotyping and transepithelial measures of ionchannel function and comparing the findingswith healthycontrols, obligate CF heterozygotes,and patients with a prior diagnosis of CF-dis-ease (PS and PI phenotypes). Genetic analysisrevealed that 24 (40%) patients carried at leastone CFTR mutation or variant, while six (10%)carried alterations on both alleles.

The sweat chloride and nasal potential dif-ference (NPD) results in the patients with pan-creatitis ranged from the values for healthycontrols and obligate heterozygotes to the val-ues for CF patients with PS and PI. Mediansweat chloride and NPD results in patients

CF of the Exocrine Pancreas

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with none or one mutation were clustered withvalues obtained in controls and obligate hetero-zygotes. In contrast, in patients with pancreati-tis carrying CFTR mutations on both alleles,median ion transport values were intermediatebetween those of the controls and obligate het-erozygotes and those of CF PS patients. Someindividual values overlapped with the CF pa-tients, and the diagnosis of CF could be con-firmed in 21% of patients byabnormal ion chan-nel measurements. Thus, CFTR-mediated ionchannel abnormalities are influenced by thenumber or severity of the CFTR mutations andshow a range of abnormalities similar to those inpatients with mild or severe classic CF at oneextreme, and controls and obligate CF heterozy-gotes on the other. This continuum of electro-physiological abnormalities is not surprising asPS patients have a 17% risk of developing pan-creatitis and many of these presentations are inadulthood.

Similar observations have been made in in-dividuals with other CF-like phenotypes, suchas men with infertility caused by congenitalbilateral absence of the vas deferens who areknown to carry a high frequency of CFTR-gene mutations (Wilschanski et al. 2006). Arelatively large population was examined, andsimilar to the patients with idiopathic pancrea-titis, a wide range of electrophysiological abnor-malities was observed. Abnormalities of CFTRfunction correlated closely with the number andseverity of CFTR mutations.

In a recent publication, Schneider et al.(2011) investigated a large group of patientswith “idiopathic” chronic pancreatitis and con-firmed other studies that the combination ofCFTR and serine protease inhibitor Kazal-type 1 (SPINK1) mutations markedly increasethe risk of pancreatitis. However, a novel find-ing was that the variant R75Q of CFTR in-creases the risk of pancreatitis markedly. Patch-clamp studies on cells expressing this mutationshowed that bicarbonate current is significantlyimpaired. This mutation is not associated withCF but this CFTR variant may be a new class ofmutation that is specific to the pancreas, partic-ularly correlating with the electrophysiologicalstudies.

DUODENAL ACIDITY—PANCREASAND OTHER ORGANS

One of the hallmarks of CF in the digestive sys-tem is hyperacidity in the duodenum. Hyper-acidity reflects multiorgan contributions. Thelow duodenal pH has most severe consequencesfor the activity of pancreatic enzymes and, in thelong run, for the energy balance of a patient.The malfunctioning pancreas may further con-tribute to this acid/base imbalance.

Problems with Pancreatic Enzymesbut Not with Ulcers

The acidic duodenal condition contributes toinactivation of pancreatic enzymes, if still pres-ent in PS patients, precipitation of bile acids, andthe development of meconium ileus (Freedmanet al. 2001). In PI patients, duodenal acidity lim-its the action of replacement enzymes, especiallythat of lipase. Decreased lipase activity causessteatorrhea and fat malabsorption that are diffi-cult to treat (Robinson et al. 1990). In addition,there are increased circulating and tissue levels of(n-6) fatty acids and inflammatory mediators(leukotrienes B4, IL, TNF-a), and there is oxi-dant stress and redox imbalance.

One would predict that the acidic duodenalenvironment would lead to ulcers. Paradoxical-ly, it seems that CF patients do not have a higherincidence of duodenal ulcers (and even pepticulcers may be diminished) (Kaunitz and Akiba2006). Possibly, cellular pH buffering is elevat-ed, as HCO3

2 is trapped because of dysfunc-tional CFTR and down-regulated Cl2/HCO3

2

exchangers. For example, in DF508 human ormouse models, pancreatic duct cells and entero-cytes have higher resting intracellular pH (El-gavish 1991; Hirokawa et al. 2004). In addition,although HCO3

2 secretion is reduced, the duo-denal acid/base barrier is only slightly impaired(Hirokawa et al. 2004).

How Does Hyperacidity Arise?

In CF patients, duodenal hyperacidity (belowpH 4) is prominent in the postprandial period;however, resting gastric and duodenal pH values



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are normal (Robinson et al. 1990; Barracloughand Taylor 1996).

At first, duodenal hyperacidity was thoughtto be caused by gastric hypersecretion (Coxet al. 1982). Therefore, to lower the acid loadto duodenum, gastric acid production is curbedby use of proton pump inhibitors and H2 recep-tor antagonists. Indeed, omeprazole treatmentseemed to improve fat digestion/absorptionand improved patient weight (Barraclough andTaylor, 1996; Proesmans and De Boeck, 2003).Also in a recent successful model of CF, the fer-ret CFTR knockout, it was shown that omepra-zole and ursodeoxycholic acid improved weightand survival of animals (Sun et al. 2010). Nev-ertheless, it seems that CFTR is also one of theCl2 channels or transporters that is necessaryfor gastric acid secretion (Heitzmann and War-th, 2007; Kopic and Geibel 2010). For example,the DF508 mutation in mice leads to decreasedacid secretion (Sidani et al. 2007). As discussedin a review (Heitzmann and Warth 2007), itseems that the effect of CFTR on acid secretionmay depend on the particular CFTR mutationand rescue by other Cl2 transporters/channels.

Normally, acid chyme is neutralized byHCO3

2 secreted by duodenal epithelia, pancre-atic duct and bile duct secretions (Ainsworthet al. 1991). Therefore, duodenal hyperacidityin CF has been ascribed to loss-of-function inthe CFTR transporter, especially in duodenaland pancreatic epithelia.

Pancreas—Lack of Bicarbonate Secretionand Other Effects

One may ask whether, in CF, lack of pancreaticHCO3

2 secretion contributes to duodenal acid-ification and whether acidity, in turn, has con-sequences for overall pancreatic function. Here,we will deal with pH-related effects on acini andwhole pancreas function. Hþ/HCO3

2transportin pancreatic ducts will be discussed in the nextsection.

Secretion originating from healthy acini(e.g., stimulated with cholecystokinin) is neu-tral or even alkaline in pH, and contains en-zymes (Sewell and Young 1975; You et al. 1983;Case and Argent 1993). In the pancreas with

impaired duct function, secretion is not onlylow in volume and high in enzyme concentra-tion, but it also has a relatively low pH. For ex-ample, CFTR2/2 (CFTRtm1UNC) mouse pan-creatic-biliary juice after secretin stimulationwas four-fold lower in volume and had pH 6.6compared with pH 8.1 in wild-type mice (Freed-man et al. 2001). In the CFTR2/2 pig, pancreaticjuice also had pH 5.7 compared with pH 8.4 inwild-type pigs (Uc et al. 2011). In addition, acid-ification of ductal and acinar lumens (CFTR2/2

mice) can lead to secondary impairment ofapical trafficking of zymogen granule mem-branes and solubilization of secretory (pro)en-zymes (Freedman et al. 2001). Nevertheless,recent data on isolated acini of normal miceshow that luminal/extracellular space is acidic,presumably owing to acidic secretory granulesthat contain the vacuolar type Hþ pump (Beh-rendorff et al. 2010). Taken together, it seemsthatoptimal enzyme secretion processes relies onneutralizing fluid secretion of adjacent ductsand/or normal fluid secretion of acini.

In addition to the role of the pancreas in theduodenal pH/enzyme environment, duodenalhyperacidity has also indirect effects on pancre-as. Increased release of gut hormones, such assecretin, results in increased signaling to the ex-ocrine pancreas that would upregulate pancre-atic HCO3

2 secretion. Indeed, in CF patients,increased plasma secretin levels are detected(Windstetter et al. 1997). In agreement, onestudy on CFTR2/2 mice shows that mRNAfor secretin (in duodenum) and vasoactive in-testinal peptide (VIP) (in pancreas) were signif-icantly increased, as well as pancreatic cAMPlevels (De Lisle et al. 2001). Another study showsthat such a situation may lead also to addedstress, as indicated by increased expression ofstress-/inflammation-related genes (Kaur et al.2004). When the duodenal pH was experimen-tally corrected, expression of stress genes wasalso corrected.

Pancreatic dysfunction in CF involves thedefective coupling of both ductal and acinarfunctions in the exocrine pancreas. BecauseCFTR is mainlyexpressed in ducts, the followingsection will consider pancreatic duct functionon the cellular and integrated level.



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CFTR AND OTHER TRANSPORTERSIN PANCREATIC DUCT SECRETION—A CELLULAR APPROACH

Pancreatic juice HCO32 concentrations vary

with secretory rates, and they are inversed tochanges in Cl2 concentrations. Interestingly, therelation between secretory rates and HCO3

2con-centrations in pancreatic juice collected fromthe pancreas of various species fall within thesame pattern (Fig. 2). This indicates that thebasic mechanism of HCO3

2secretion/salvagingmay be similar (Fig. 3), but perhaps the ductmass is different. Taking this as a starting point,ion transport models based on studies of cellsand tissues from various animals are suitablemodels for basic secretion mechanisms andfor CF models.

CFTR and Anion Exchanger

The fingerprinting of cellular mechanisms forpancreatic duct ion transport began when it wasdiscovered that secretin-/cAMP- activated Cl2

channels were functionally located on the lumi-nal membranes of isolated rat pancreatic ducts,and the first ion transport models were proposed

(Gray et al. 1988; Novak and Greger 1988b). Al-most at the same time, CFTR was discovered(Kerem et al. 1989; Riordan et al. 1989), andsoon thereafter CFTR was shown to have prop-erties of a Cl2 channel, including in the pan-creatic ducts (Gray et al. 1989, 1993; Tabcharaniet al. 1991). Subsequently, CFTR was immuno-localized in rodent and human pancreas tointercalated and small intralobular ducts thatalso express aquaporins and carbonic ahydra-ses (Kumpulainen and Jalovaara 1981; Marinoet al. 1991; Hyde et al. 1997; Burghardt et al.2003).

The question of how CFTR Cl2 channelscould underlie HCO3

2 secretion of pancreas,has been a challenging problem ever since.The first proposal was that by coupling of Cl2

channels to Cl2/HCO32 exchange operating in

parallel would result in efflux of HCO32into the

lumen. The anion exchangers belonging to thesolute carrier families SLC26A6 and SLC26A3were found expressed in pancreatic ducts, andthe proposed transport ratio of 2HCO3

2:1Cl2

for the first transporter would make it a can-didate for secreting ducts (Lohi et al. 2000;Greeley et al. 2001; Dorwart et al. 2008). Untilnow, studies of SLC26A6 null mice showed

00

20

40

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80

HC

O3– c

once

ntra

tion

(mm

ol/L

)

100

120

140

160

180

20 40 60

Secretory rate (μL/min-kg body weight)

80 100

Rat

Human

PigDogCatGuinea pigRabbitHamster

120 140

Figure 2. The relation between secretory rates and HCO32concentrations in pancreatic juice of various species.

Secretion was stimulated by secretin and secretory rate was collected for body weight. For details, see Novak et al.(2011).



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different effects on duct/pancreas secretion(Wang et al. 2006; Ishiguro et al. 2007; Stewartet al. 2009; Yang et al. 2009). Nevertheless, itseems that there is a functional coupling be-tween the R domain of CFTR and sulfate trans-porter anti-sigma (STAS) domains of this ex-changer (Dorwart et al. 2008).

There are several studies that indicate otherways to achieve HCO3

2 secretion. For exam-ple, the CFTR channel is HCO3

2 permeable orCFTR regulates anion selectivity of anotherchannel/transporter. Thus, guinea pig pancre-atic ducts secrete fluid (and presumably HCO3

2)in the absence of luminal Cl2, in which Cl2/HCO3

2exchange would not favor HCO32secre-

tion (Ishiguro et al. 1998, 2009). Another studyon HEK293 cells expression system showed thatCFTR mutants associated with PI (e.g., I1489T)did not support HCO3

2 transport, whereasthose associated with PS (e.g., R117H) showreduced transport (Choi et al. 2001). Indeed,many studies, also on pancreatic ducts, inves-tigated whether CFTR conducts HCO3

2 (Becq

et al. 1993; Gray et al. 1993; O’Reilly et al. 2000).Until very recently, the consensus was that thepermeability ratio PHCO3

2/PCl2 was .0.2–0.4 in conditions with intracellular Cl2 andpH values expected for most epithelia. A newerstudy also shows similar permeability ratio andit remains independent of ionic conditions(Tang et al. 2009).

Nevertheless, a recent study shows that hu-man and rodent tissues express volume/Cl 2

i

sensitive WNK1 kinase and two downstreamkinases, SPAK and OSR1. Using patch-clamprecording, it was reported that PHCO3

2/PCl2

increased from 0.24 to 1.09 when the Cl2 con-centration in the pipette was decreased from 150to 4 mM, and investigators ascribed this perme-ability to CFTR. The investigators postulatedthat WNK1-OSR1/SPAK pathway is a molecu-lar switch operating in distal ducts, which leadsto increased concentration of incoming HCO3

2

from 80 to 150 mM (Park et al. 2010). Althoughattractive, the model does not yet explain howincreased concentration of HCO3

2 would asso-ciate with fluid transport, and it cannot accountfor the fact that proximal small ducts are therichest sites of CFTR/AQP/carbonic anhydraseexpression. Also, other studies show that WNKsinhibit CFTR (Yang et al. 2007, 2011). No doubtthis exciting field of volume-sensitive kinasesmay not only be of relevance to kidneyand bloodpressure regulation, but also to salt and watertransport in other epithelia.

NHE and NBC

In any case, HCO32transport across the luminal

membrane relies on provision of cellular HCO32.

This source could be achieved by carbonic-an-hydrase catalyzed hydration of CO2, and extru-sion of resulting Hþ to the interstitium via abasolateral Naþ/Hþ exchanger, NHE1. Alterna-tively, or in addition, HCO3

2could enter acrossthe basolateral membrane by pancreatic Naþ-HCO3

2 cotransporter, pNBC, which transports1Naþ:2HCO3

2 (also named NBCe1) (Zhao etal. 1994; Ishiguro et al. 1996; Abuladze et al.1998). Recent studies show that IRBIT (inosi-tol-3-phosphate receptor-binding protein) ac-tivates pNBC and CFTR, and thus could be

K+K+

CA

cAMP

CFTR

Ca2+

+

H+

K+

K+

K+

Cl–

Cl–

Cl–

H+

H+

H+H+

~ ~

~

Na+

Na+

InterstitiumLumen

Na+

Na+

HCO3–

HCO3– H2O

CO2

HCO3–

?

HCO3–

+

Figure 3. Cellular model for HCO32secretion in pan-

creatic ducts. Primary active transporters (pumps)and several secondary active transporters and ionchannels, including Ca2þ-activated Kþ and Cl2

channels, are involved in creating the chemical andelectrical driving gradient necessary for productionof NaHCO3-rich pancreatic juice in healthy pancreas,as described in text. The CFTR has central role pan-creatic ducts; in CF, pancreatic juice volume and pHare reduced.



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a coordinating factor for transepithelial iontransport (Shirakabe et al. 2006; Yang et al.,2009, 2011). Thus, Ca2þ/IP3 activating agonistscould regulate HCO3

2 secretion, if the appro-priate ion transporters on the luminal mem-brane are present (see below).

Proton Pumps

In addition to the secondary active transport-ers, NHE1 and pNBC, several earlier studiessearched for primary active transporters (Fig.3). One obvious candidate is the vacuolar Hþ-pump, but evidence at the molecular level ismissing, and the function in stimulated ductsis unclear (Zhao et al. 1994; Villanger et al.1995; de Ondarza and Hootman 1997; Chenget al. 1998). Recently, another study focused onother types of pumps, and it was shown thatrodent pancreatic ducts express both gastricand nongastric Hþ/Kþ pumps, which signifi-cantly contribute to secretin-stimulated duct se-cretion (Novak et al. 2011).

Other Roles of CFTR

CFTR can regulate other transporters includingCl2/HCO3

2 exchange in pancreatic tissue (Leeet al. 1999). In other respiratory epithelia, CFTRand ENaC are inversely regulated (Donaldsonand Boucher 2007). Freshly isolated rodent ductand in vivo pancreas studies show no evidencefor functional ENaC (Novak and Hansen 2002),although on culture, interlobular murine ductsdevelop sensitivity to amiloride and in somesituations can even absorb (Zeiher et al. 1995;Pascua et al. 2009).

One important feature of epithelial secre-tion of CF patients is the high mucus content.This phenomenon may not be only a conse-quence of lack of hydration or increased num-ber of mucus secreting cells. It is proposed thatHCO3

2 is a chaotropic anion important in mu-cus expansion and, therefore, lack of CFTRfunction will impair mucus hydration (Quinton2008; De Lisle 2009; Garcia et al. 2009; Chonget al. 2013). This may be of relevance to distalpancreatic ducts that contain numerous mucussecreting cells.

A number of studies on various cells indi-cate that CFTR is involved in release of ATPfrom intracellular to extracellular spaces, andCFTR has been ascribed the role of an ATPtransporter or regulator. Currently, a numberof other ATP release mechanisms are more fa-vored (Novak 2011).

Ca2þ-Activated Cl2 Channels

In addition to CFTR, which is mainly regulatedby cAMP/PKA signaling, Ca2þ-activated Cl2

channels (CaCC) could potentially drive secre-tion (Fig. 3). In rodent and human pancreaticducts, many studies have shown that a numberof agonists increase cellular Ca2þ, increase Cl2

conductance(althoughtransiently),changepHi,and evoke fluid production in isolated ducts(Pahl and Novak 1993; Hug et al. 1994; Win-penny et al. 1998; Szalmay et al. 2001; Pascuaet al. 2009). The question is whether this secre-tion is also HCO3

2 rich, that is, if machinerysimilar to that operated by CFTR could be re-cruited, and/or if such CaCC are also HCO3

2

permeable. If IRBIT stimulates pNBC (seeabove), HCO3

2 permeability on the luminalmembrane would be required.

Interestingly, the identity of such CaCC hasbeen elusive and a number of candidates wereproposed earlier, including ClC-2 and bestro-phins (Duran et al. 2010). Recently, three inde-pendent reports have shown that the TMEM16/Anoctamine families are good candidates forCaCC (Caputo et al. 2008; Schroeder et al. 2008;Yang et al. 2008). TMEM16A is expressed inrodent acinar cells (Yang et al. 2008) and knock-out of TMEM16A in mice caused defects inCaCC in pancreatic acini (Ousingsawat et al.2009). TMEM16A is also expressed in CFPAC-1 cells (Caputo et al. 2008) and in human pan-creatic duct cell lines expressing normal CFTR,in which it determines transepithelial transport(Wang et al. 2013).

Kþ Channels

Kþ channels are not usually included in pancre-atic duct cell models. Nevertheless, there are twoindications that Kþ transport is also important



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in pancreatic ducts. First of all, pancreatic juicecontains Kþhigher than in plasma. The Kþ con-ductance is increased with several agonists, andit is clear that Kþ channels are not only impor-tant for setting the resting membrane poten-tial, but they also keep the driving force for an-ion secretion (Novak and Greger 1988a, 1991).We know the identity of some Kþ channels;this includes BK (maxi-K, SLO, KCNMA1), IK(KCNN4), TASK-2 (KCNK5), and others. Someare located on both the luminal and basolateralmembranes, which may be operated by differentregulatory systems (Gray et al. 1990; Fong et al.2003; Hede et al. 2005; Jung et al. 2006; Hayashiet al. 2012).

Regulation of Pancreatic Duct Secretion

The classical bicarbonate-evoking secretagogueis secretin, although a number of other hor-mones and transmitters can also evoke andcoregulate (HCO3

2) secretion. Even cholinergicstimulation and CCK can evoke HCO3

2 secre-tion in some species, and they can potentiate thesecretin effect on the volume of secretion (Youet al. 1983; Holst 1993; Park et al. 1998; Cheyand Chang 2001), although there are exceptions(Evans et al. 1996). Whether human pancreaticjuice is also rich in HCO3

2 under these condi-tions is not clear.

One of the novel additions to regulation ofpancreatic fluid secretion is the purinergic sig-naling, which coordinates acini-duct functions.This pathway relies on extracellular ATP andadenosine that, via purinergic and adenosinereceptors, regulates specific epithelial transport-ers (Novak 2008, 2011). Pancreatic acini releaseATP, some of which is stored in zymogen gran-ules (Sørensen and Novak 2001; Haanes andNovak 2010). Pancreatic ducts express severaltypes of P2 receptors, including P2Y2, P2Y4,P2Y11, P2X4, and P2X7 receptors, and adeno-sine A2A and A2B receptors. Luminal ATP canthen upregulate anion and fluid secretion, andthis activity involves regulation of CaCC and IK(Hug et al. 1994; Ishiguro et al. 1999; Hede et al.2005; Jung et al. 2006; Novak et al. 2010; Hay-ashi et al. 2012; Wang et al. 2013). ATP alsostimulates luminal anion exchange in duct epi-

thelia expressing functional CFTR (Namkunget al. 2003). In addition, ATP/uridine triphos-phate (UTP) also potentiate cAMP-evoked mu-cin secretion (Jung et al. 2010). From the baso-lateral membrane, ATP may be released by nervesand/or distended epithelium; some purinergicreceptors are inhibitory to secretion (e.g., P2Y2receptors inhibit BK channels) whereas otherreceptors may have positive effects on secretion(Hede et al. 1999, 2005; Ishiguro et al. 1999;Wang et al. 2013).

ANIMAL MODELS FOR CF OF PANCREAS—INTEGRATED APPROACH

Although physiological studies on isolated ductsand human duct cell lines have taught us muchabout ion transport and regulation thereof, it isnot enough to understand the impact of CFTRmutations on whole gland pathophysiology inCF. That is, we have to understand the link be-tween dysfunction in HCO3

2secretion, enzymesecretion, mucus plugging, and changes in pan-creas morphology at an integrated level. There-fore, CF animal models have been invaluable,although challenging our understanding attimes. The first mouse models of CF were de-veloped shortly after the discovery of CFTR, andrecently they were followed by very promisingCF models in pigs and ferrets.

Mouse CF Models

There are a number of murine CF models de-veloped, although a variety are gene-target-ing strategies. These mice showed processingof mutated proteins, presence of other rescuingtransporters, and last but not least, diversityin murine genetic background. These variableshave led to heterogeneity of disease manifesta-tions in mouse models (Ostedgaard et al. 2007).Generally, defects in airways and pancreas havebeen milder and more subtle to detect, where-as abnormalities in ion transport and morphol-ogy of intestinal tissues are marked and new-born mice suffer from intestinal obstruction.This is quite different from humans, in whichonly 10% of newborns have meconium ileus but



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already 80%–90% have PI. Thus, there is ap-parent discordance between intestinal and pan-creatic CF phenotype in newborns. However,the rodent pancreas is different from the humanpancreas in structure and development. The ro-dent pancreas is immature at birth and under-goes significant weight increase and matura-tion development postnatally, regarding enzymeand bicarbonate secretion (Githens 1994; Sca-glia et al. 1997).

In the following section, we will focus onCF mouse models with respect to pancreas.The first CF mouse model was generated atthe University of North Carolina and is denotedCFTRtm1UNC. Only 5% of animals survived, ex-hibiting severe intestinal problems, but the pan-creas morphology was not markedly affected atthe point of the initial examination (Snouwaertet al. 1992). In a similar knockout model gen-erated in Cambridge, CFTRtm1CAM, about 40%of animals survived and about half of the miceshowed some pancreatic pathology (i.e., block-age of pancreatic ducts), possibly because theseanimals lived longer (Ratcliff et al. 1993). In theBaylor College model (CFTRtm1BAY), newbornknockout animals had normal pancreas, butwith increasing age, some animals showed dila-tation and inflammation of main ducts as wellas some acinar atrophy (O’Neal et al. 1993). Inthe Edinburgh model (CFTRtm1HGU), there re-mained 10% of wild-type CFTR mRNA, 95%mice survived, and the phenotype was milder(Dorin et al. 1992, 1994).

As the reports above indicate, developmentof CF in murine pancreas may depend on time.If animals were put on a complete supplement-ed liquid diet, they were able to survive longer,develop manifestations of CF in many organs,including pancreas, such as duct lumen dilata-tion and obstruction and acinar atrophy (Durieet al. 2004). Knockout animals still had lowerbody weight, lower pancreas mass and pancre-atic enzyme content. Possibly, this lower weightwas not only caused by the CF defect, but waspartly attributable to malnutrition (Ip et al.1996).

First, we summarize what is known about theacinar-related pathology. Acini of CFTRtm1UNC

homozygous mice had fewer zymogen granules

and the major sulfated glycoprotein, gp300,normally present in ZG, was lining distendedacinar lumens. There was also impaired apicalmembrane trafficking compared to wild-typemice (De Lisle 1995, 2001). In isolated acinifrom knockout animals, there was enhanced se-cretory protein response with dibutyryl-cAMP(dbcAMP) and carbachol, which may contrib-ute to micro-precipitation and development ofacinar dysfunction (Tang et al. 1999). Increasedlevels of mRNA for secretin and VIP and pan-creatic cAMP levels indicated that pancreas waschronically stimulated (De Lisle et al. 2001), andthere was impaired stress-gene expression (Kauret al. 2004). Also knockout animals had mildPI, higher baseline proinflammatory states,and an antiapoptotic phenotype, which maysensitize them to develop more severe acutepancreatitis with a marked pancreatic inflam-matory response (DiMagno et al. 2005).

Second, let us examine whether knockoutmice also showed signs of pancreatic ductal dis-orders. CFTRtm1UNC knockout mice producedlower volume and acidic pancreatic-biliary juiceafter secretin stimulation, and there was alsoreduced response to CCK (Freedman et al.2001; DiMagno et al. 2005). In cultured ductmonolayer from CFTRtm1UNC knockout mice,forskolin stimulated small short-circuit cur-rents (Isc) compared to the wild type, but ion-omycin stimulated currents that were 3� higherin knockout than in wild-type preparation, in-dicating that CaCC was up-regulated (Clarkeet al. 1994). Pancreatic ducts isolated from an-other CFTR-null mouse (CFTRtm1CAM) secret-ed 60% less fluid when stimulated with forsko-lin, and apparently about 30% less with secretin,although the secretin response was much lowereven in wild-type mice. In the same preparation,carbachol also induced secretion, but to aboutthe same extent in both wild-type and CFTR-null ducts, indicating that CaCC was well ex-pressed and not up-regulated in knockouts(Pascua et al. 2009). Similar conclusions werereached using whole-cell patch-clamp on ductcells from the same mutant type. Ducts ofknockout mice did not have any cAMP-activat-ed currents (i.e., CFTR), but ionomycin-stimu-lated currents that were about 10� higher than



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CFTR-currents, and these are about the samein both wild-type and knockout cells (Win-penny et al. 1995). In CFTRtm1HGU model, thereis residual CFTR activity (10% CFTR mRNA isexpressed), but ionomycin-induced currents(i.e., CaCC) were larger and again similar inboth wild-type and knockout cells (Gray et al.1994). Thus, most studies show that CaCC wasnot up-regulated in CFTR2/2.

Nevertheless, the duct studies mentionedabove leave some issues. First, the ducts usedwere short- and long-term cultured, and someendogenous receptors were down-regulated.Therefore, experiments necessitate use of agentsthat stimulate “simple” signaling pathways thanone might expect for receptors. Second, allmouse studies are performed on larger ductsthat would not be primary secretors and caneven absorb (Zeiher et al. 1995; Pascua et al.2009).

In murine models of DF508 (CFTRtm2CAM,CFTRtm2KTH, CFTRtm1EUR), there can be a gra-dient for severity of CFTR-DF508 processingdefects that is more serious in humans than inmouse or pig (Ostedgaard et al. 2007). That is,DF508 CFTR is partially processed in mouse.Animals survive into adulthood and displayseveral abnormalities seen in human DF508patients. They also show little or no pancreatichistopathology (van Doorninck et al. 1995;Zeiher et al. 1995), although functional studiesindicate some abnormalities. Cultured duct epi-thelium from CFTRtm2KTH mice shows that acAMP agonist can induce secretion and Iscchanges in wild type but not in DF508. Thus,one could expect at least mild pancreas pheno-type (Zeiher et al. 1995). Also, forskolin stimu-lated anion exchanger activity in luminal mem-branes of perfused main ducts was lower inmutants compared to wild type (Lee et al.1999). In contrast, similar ducts express lessNHE3, and CFTR interaction with NHE3 pos-tulated in HCO3

2 salvage mechanisms (Ahnet al. 2001) could affect anionic compositionof juice. In humans, CFTR gene mutations as-sociate with recurrent acute pancreatitis in pa-tients with PS. Both CFTR2/2 and DF508 micedevelop caerulein-induced pancreatitis (Di-Magno et al. 2005, 2010).

G551D and G480C mutants would be ofinterest for pancreas, as these mutations are as-sociated with PI. However, mice with this mu-tation have milder symptoms compared to nullmice (e.g., reduced risk of fatal intestinal block-age), and they have residual (�4%) mutant ac-tivity, and no pancreas and lung pathology wasdetected or studied (Delaney et al. 1996; Dick-inson et al. 2002).

Pig and Ferret CF Models

Relatively mild changes in airway and pancreaticepithelia of mouse models spurred develop-ment of CF models in other animals. In contrastto mice, pigs have many physiological and ana-tomical features similar to humans. Recently, apig model of CF was made by targeted disrup-tion of both CFTR alleles. Newborn CFTR2/2

pigs exhibit many CF features, including exo-crine pancreatic pathology (dilated and ob-structed ducts with mucus incretion, residualacini, interstitial zymogen material, limited in-flammation, and fibrosis (Rogers et al. 2008;Meyerholz et al. 2010). There were also intesti-nal lesions (meconium ileus and microcolon),and if surgically treated, animals reached de-veloping lung abnormalities similar to CF pa-tients. Also, degeneration of the pancreas con-tinued, becoming more fatty, with markedlydecreased exocrine cells, and obstructed ducts.Pig “cystic fibrosis of the pancreas” closely re-sembled Dorothy Anderson’s original descrip-tions (Stoltz et al. 2010).

The ferret CFTR2/2 model also has manycharacteristics of human CF disease includingdefective airway Cl2 transport and submucosalgland secretion, pancreatic, liver, and vas defer-ens disease, and variable intestinal disorder (Sunet al. 2010). In the ferret, the pancreas of new-born animals showed dilated acini and ductswith inspissated eosinophilic zymogen secre-tions, but there was significantly less destructionas seen in newborn CF pigs.

Taken together, animal models are invalu-able tools for studying basic and CF-relatedmechanisms of integrated pancreatic function,and the effects of therapeutics. Although pigand ferret models may develop CF more in line



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with humans, mouse models still offer a lotof potential especially in our understanding ofregulation of cAMP versus Ca2þ-driven anionsecretion.

THERAPEUTIC POTENTIALSFOR CF OF PANCREAS

Therapeutic approaches for pancreatic treat-ments are very challenging, as most CF patientsare born with PI or get it soon after, and thepancreatic mass is destroyed. In worst cases,when the liver is also affected, pancreas–livertransplantations are performed in a few centers.Another approach would be to reconstitute thepancreas mass using stem cell approaches. Thiswould involve both acini and duct regeneration.At the moment, this task is not high on theresearch priority list in the CF field, perhapsbecause very good advances have been madewith symptomatic strategies. The focus on treat-ing PI patients is to provide sufficient nutrition,vitamins, and pancreatic enzyme replacementtherapy to PI patients to improve nutritionalstatus of the patient. Nevertheless, more prog-ress is still needed, especially for lipase prepara-tions, as enzymes do not work optimally inacidic duodenal environments. Therefore, thereare special efforts to design improved lipasepreparations by molecular design and recombi-nant technology (Colin et al. 2010). For exam-ple, recombinant acid-resistant lipase of plantorigin Merispase (Meristem Therapeutics) isbeing evaluated in phase II trials. Another prod-uct in clinical trials contains microbial lipase,and also protease and amylase preparation (Li-promatase, Lilly). These types of products maybe essential as an alternative to porcine-basedproducts.

In patients that still have some pancreasfunction (PS), the strategy must be to preventduct/acini degradation, inflammation, and fi-brosis, that is, development of pancreatitis. Theproblem is that PS patients have increased riskof pancreatitis. A number of anti-inflammatoryand antioxidant agents are currently in use, in-cluding gluthathione, sildenafil, simavastatin,etc. However, further research and novel strate-gies are needed to improve the clinical care of

CF patients (Innis and Davidson 2008; Jonesand Helm 2009).

Nevertheless, because the culprit is CFTRexpressed in ducts, the main therapeutic strate-gies should be to modulate the basic defect here,which could possibly improve duct function inPI and PS patients and allow the pancreas toregenerate and develop. For CFTR protein res-cue strategies, see Rowe and Verkman (2013).

Another approach is to bypass defectiveCFTR and activate CaCC (e.g., TMEM16A),although it is not clear whether the optimaltarget is the channel, intracellular Ca2þ levels,or a receptor regulating either or both (Cuthbert2011). Activation of CaCC by the lantibioticduramycin (Moli1901, Lancovutide, Lantibio)is being tested as a strategy and is in phase II trialin Europe. (The effect may be via raising intra-cellular calcium levels.) The outcome for pan-creas is unclear, as tests on cell lines Panc-1 andCFPAC-1 indicate that the effects are nonspecif-ic (Oliynyk et al. 2010). Also, studies on CFmouse models and human pancreatic cell linesindicate that CaCC does not take over fromCFTR (see above). Another channel that may berelevant for some epithelia is ClC-2 that is acti-vated by lubiprostone, although the effect is viathe prostanoid receptor EP4 (Cuthbert 2011),and ClC-2 is only expressed in pancreatic acini.

An extension of this strategy would be toenhance natural regulator of CaCC, such as P2receptors (e.g., P2Y2 receptors), as has been en-visaged for airways (Deterding et al. 2007; Laz-arowski and Boucher 2009). Although the basicresearch and strategy was well planned, Denu-fasol tetrasodium (INS37217, Inspire) was tak-en out of phase III trials recently. For pancreas,one would need to target the receptors of theluminal membrane because the basolateral re-ceptors can, in fact, inhibit secretion rather thanstimulate it (see above). For obvious reasons,delivery of intraluminal UTP derivative is un-realistic.

CONCLUDING REMARKS

Translational research is identifying a numberof compounds that offer promising pharma-cotherapy for CF. However, regarding the pan-



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creas, therapeutic approaches are more com-plicated than for airways as they require system-ic administration, and testing of pancreatic pa-rameters in PS patients would need to beimplemented. The possibility of increasing thefunction of a mutation that ordinarily confersPI to one that confers PS via a potentiator orcorrector is appearing. However, this may resultin increasing the risk of pancreatitis in a CFpatient who did not suffer from this before.Before we move in this direction, we clearlyneed more drug studies on pancreatic functionin good animal models.

ACKNOWLEDGMENTS

Work cited (I.N.) is supported by the DanishNatural Science Council and the LundbeckFoundation.

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2013; doi: 10.1101/cshperspect.a009746Cold Spring Harb Perspect Med Michael Wilschanski and Ivana Novak The Cystic Fibrosis of Exocrine Pancreas

Subject Collection Cystic Fibrosis

Cystic FibrosisAntibiotic and Anti-Inflammatory Therapies for

ElbornJames F. Chmiel, Michael W. Konstan and J. Stuart

The Cystic Fibrosis IntestineRobert C. De Lisle and Drucy Borowitz

System of the LungStructure and Function of the Mucus Clearance

Brenda M. Button and Brian ButtonCorrectors and PotentiatorsCystic Fibrosis Transmembrane Regulator

Steven M. Rowe and Alan S. Verkman

Other than CFTRNew Pulmonary Therapies Directed at Targets

Scott H. Donaldson and Luis Galietta

The Cystic Fibrosis of Exocrine PancreasMichael Wilschanski and Ivana Novak

The Cystic Fibrosis Airway MicrobiomeSusan V. Lynch and Kenneth D. Bruce

and StabilityFunctionTransmembrane Conductance Regulator

Dynamics Intrinsic to Cystic Fibrosis

Dokholyan, et al.P. Andrew Chong, Pradeep Kota, Nikolay V.

Regulator (ABCC7) StructureCystic Fibrosis Transmembrane Conductance

John F. Hunt, Chi Wang and Robert C. FordPerspectiveThe Cystic Fibrosis Gene: A Molecular Genetic

Lap-Chee Tsui and Ruslan Dorfman

Cystic FibrosisStatus of Fluid and Electrolyte Absorption in

M.M. Reddy and M. Jackson StuttsAnion PermeationThe CFTR Ion Channel: Gating, Regulation, and

Tzyh-Chang Hwang and Kevin L. Kirk

PhenotypesThe Influence of Genetics on Cystic Fibrosis

Michael R. Knowles and Mitchell DrummCFTRAssessing the Disease-Liability of Mutations in

Claude Ferec and Garry R. Cutting

Lessons from the Biochemical World−−Perspectives on Mucus Properties and Formation

Gustafsson, et al.Daniel Ambort, Malin E.V. Johansson, Jenny K.

Supramolecular Dynamics of MucusPedro Verdugo

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