Review for 10.1186/1471-230X-1-10 (2001)

Reviewer's□report 
□Ileal□mucosal□bile□acid□absorption□is□increased□in□Cftr□knockout□mice Title: 
Authors: 
Matthias□Stelzner□( stelzner@u.washington.edu )
Sivagurunathan□Somasundaram□( siva_somasundaram@med.unc.edu )
Sum□P□Lee□( splee@u.washington.edu )
Rahul□Kuver□( kuver@u.washington.edu )
□2□ □17□Jul□2001 Version: Date: 
□Dr□Jacqueline□Hardcastle Reviewer: 
□not□specified Level□of□interest: 
□Unable□to□decide□on□acceptance□or□rejection□until□the□authors□have□ Advice□on□publication: 
responded□to□the□compulsory□revisions 
The□aim□of□this□investigation□is□to□determine,□using□a□CFTR□knockout□mouse□model,□whether□active□ 
bile□acid□uptake□by□the□murine□ileum□is□altered□in□CF.□Results□are□presented□which□appear□to□suggest 
that□both□active□uptake□of□taurocholate□and□expression□of□the□IBAT□(ileal□bile□acid□transporter)□are□ 
increased□in□CF□ileum.□However,□no□statisitcal□analysis□of□the□data□has□been□presented□and□it□is□ 
essential□that□this□is□provided□to□support□the□conclusions□drawn.□Examination□of□the□data□suggests□ 
that□some□of□the□observed□increases□in□taurocholate□uptake□are□not□actually□significant□and□this□ 
requires□clarification.□ 
SPECIFIC□POINTS□ 
Abbreviations□ 
CFTR:□cystic□fibrosis□transmembrane□conductance□regulator.□ 
This□is□the□protein□product□of□the□CF□gene□and□is□considered□to□act□as□a□Cl□channel.□ 
INTRODUCTION□ 
para1□ 
The□Na□channel□is□not□an□anion□channel.□The□sentence□should□read□"...regulation□of□other□ION□ channels,..."□ 
para2□ 
Bile□acid□malabsorption□is□not□a□consistent□finding□in□CF□as□the□authors□themselves□state□in□para2□of□ the□Discussion.□de□Rooij□et□al□(ref□11)□studied□BBMVs□from□2□CF□patients□and□concluded□that□bile□ 
acid□uptake□was□low,□but□not□abnormal.□ 
MATERIALS□AND□METHODS□ 
Animals□ 
Did□any□of□the□CFTR(-/-)□animals□suffer□from□intestinal□obstruction□as□they□were□fed□on□a□standard□ 
solid□diet?□ 
Preparation□of□intestinal□segments□ 
This□states□that□mice□were□anaesthetized□and,□if□this□was□so,□the□anaesthetic□used,□its□dose□and□ 
route□of□administration□should□be□given,□However,□the□previous□section□states□that□animals□were□ 
sacrificed□using□cervical□dislocation.□ 
Uptake□measurements□ 
The□time□between□exision□of□the□tissues□and□measurement□of□uptake□(1-2□hr)□seems□excessive,□ 
particularly□in□view□of□the□susceptibility□of□the□enterocytes□to□hypoxia.□ 
The□Na-free□medium□contained□an□additional□20mM□K,□an□ion□known□to□be□inhibitory□to□cell□function.□ It□would□be□useful□to□know□the□specific□activities□of□the□labelled□taurocholate□and□PEG□used.□ For□details□of□the□calculation□of□uptake□readers□are□referred□to□an□earlier□paper□(ref□16□-□this□has□now□ 
been□published□and□the□full□citation□should□be□added),□but□this□does□not□provide□the□information,□ 
simply□a□reference□to□yet□another□paper.□This□is□not□satisfactory.□ 
Immunohistochemistry□studies□ 
TBS□should□be□defined.□ 
How□selective□is□rabbit□antibody□for□mouse□IBAT?□ 
Western□blots□ 
Evidence□that□the□preparation□of□mouse□BBMVs□using□the□method□described□by□de□Rooij□et□al□(ref□ 
11)□for□human□tissue□pruduces□a□membrane□fraction□rich□in□brush□borders□(e.g.□enrichment□of□brush□ border□enzymes□such□as□sucrase,□alkaline□phosphatase)□should□be□presented.□ 
Expression□of□results□ 
There□is□no□indication□of□the□way□in□which□the□results□are□expressed,□e.g.□mean+/-s.e.m.,□nor□is□there 
any□evidence□of□statistical□analysis□of□the□data.□ 
RESULTS□ 
There□is□no□statistical□analysis□to□confirm□that□the□differences□in□taurochlolate□uptake□between□the□3□ 
groups□are□significant.□Examination□of□Fig□1□suggests□that□there□are□no□significant□differences□ 
between□wild-type□and□CFTR(-/-)□values□for□4,□5,□and□6□cm□tissues.□ 
Comparisons□between□the□groups□assumes□that□mucosal□area□is□not□altered□in□the□3□groups.□Is□this□ 
the□case?□ It□would□be□useful□to□include□the□rates□of□passive□uptake.□ 
There□is□no□statistical□analysis□of□the□quantification□of□the□Western□blot□data□(Fig□3).□ 
IBAT□immunostaining□was□found□at□the□base□of□the□crypt□in□CFTR(-/-)□ilea.□This□is□an□unusual□ 
location□for□a□nutrient□transporter□as□these□are□not□usually□expressed□until□the□enterocyte□reaches□the 
villus.□Could□this□indicate□a□lack□of□specificity□of□the□antibody?□ 
DISCUSSION□ 
Enhanced□bile□acid□absorption□in□CF□intestine□does□fit□in□with□observations□of□increased□ Na-dependent□sugar□and□amino□acid□absorption□in□jejunal□biopsies□from□CF□patients□(refs□25,□26).□ 
The□failure□to□observe□this□in□earlier□studies□(refs□28-30)□probably□reflects□the□smaller□number□of□ 
tissues□studied.□ 
Para6□states□that□CFTR(-/-)□mice□experience□bile□acid□malabsorption□although□Fig□1□shows□this□is□not 
the□case.□This□is□confusing.□ 
The□epithelial□Na□channel□is□ENaC,□not□EnaC. 
Competing□interests: 
None□declared.