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2009 Homer W. Smith Award: Minerals in Motion:From New Ion Transporters to New Concepts

Rene J.M. Bindels

Department of Physiology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands

Calcium (Ca2�) and magnesium (Mg2�)minerals are essential for many physio-logic processes.1 Ca2� plays a pathologicrole in osteoporosis, nephrolithiasis, vas-cular calcification, nephrocalcinosis, andchronic kidney disease, and disturbancesin Mg2� contribute to muscle cramps,paraesthesia, convulsions, arrhythmias,and cardiac arrest. Their overall mineralbalance is regulated by the concertedactions of kidneys, intestine, and bone.2

The kidneys determine the final excre-tion of these cations and fulfills, andtherefore, an important step in homeo-static control. This role was recognizedby Homer Smith, who wrote “…Little isknown concerning Ca2� excretion ex-cept that the total excretion can be in-creased or decreased by a variety of cir-cumstances. Much that has been saidabout Ca2� applies to Mg2�. The mech-anism by which renal excretion of Mg2�

is controlled is unknown…”3

In the last decade, considerable

progress has been made in elucidatingthe molecular mechanisms underlyingthe reabsorption of these minerals by thekidney. Instrumental in this respect arestudies of rare monogenic diseases re-lated to defective renal Mg2� handlingand genetically modified mice with de-leted Ca2� transport proteins.4,5 Thesestudies identified new transport proteinsand have led to the development of newconcepts for the renal handling of min-erals.

IMPORTANCE OF THE DISTALPART OF THE NEPHRON

Active reabsorption of Mg2� and Ca2�

takes place in the distal part of thenephron only. More precisely, this partof the nephron is comprised of the distalconvoluted tubule (DCT) and the con-necting tubule (CNT) leading to the col-lecting duct.6 The former can be further

subdivided into early (DCT1) and late(DCT2) segments. Based on micropunc-ture experiments and the conspicuous lo-calization of transport proteins, activeMg2� transport is confined to the DCT1and DCT2 segments, whereas active Ca2�

reabsorption mainly occurs along theDCT2 and CNT segments (Figure 1).1

Thus, DCT2 functions as a transition areabetween Mg2� and Ca2� reabsorption.

Mg2� REABSORPTION IN DCT1AND DCT2 SEGMENTS

The DCT is famous for the presence ofthe thiazide-sensitive NaCl co-trans-porter (NCC) along the luminal mem-brane, which is energized by a Na� gra-dient generated by the basolateralNa�-K�-ATPase.7,8 Active transcellu-lar Mg2� transport along the DCT isenvisaged by the following sequentialsteps (Figure 2).9 Driven by a favorablemembrane potential, Mg2� enters theDCT cell through an apical epithelialMg2� channel. The chemical drivingforce for Mg2� is limited because theextra- and intracellular Mg2� concen-trations are in the same millimolarrange. Importantly, Mg2� entry intothe cells seems to be the rate-limiting

Published online ahead of print. Publication dateavailable at www.jasn.org.

Correspondence: Dr. Rene J.M. Bindels, 286 Phys-iology, Radboud University Nijmegen Medical Cen-tre, POB 9101, 6500 HB, Nijmegen, The Nether-lands. Phone: 31-24-3614211; Fax: 31-24-3616413; E-mail: r.bindels@fysiol.umcn.nl

Copyright © 2010 by the American Society ofNephrology

ABSTRACTThe kidneys play a critical role in maintaining the systemic balance of Mg2� andCa2� cations. The reabsorptive capacity of these divalent cations adapt to changesin their plasma concentrations. Active reabsorption of Mg2� and Ca2� takes placein the distal convoluted and connecting tubules, respectively, and is initiated bycellular transport through selective transient receptor potential (TRP) channelslocated along the luminal membrane and modulated by hormonal stimuli. Recentcharacterization of underlying molecular defects in renal Mg2� handling illuminatecomplex transport processes in the kidney and their contribution to the overallmineral balance. Likewise, studies of Ca2� transport proteins in null mice disclosemolecular mechanisms maintaining normal plasma Ca2� levels and the hypercalci-uria-related adaptations important in the prevention of kidney stones. Currentknowledge of Mg2� and Ca2� transport is summarized here as comprehensivecellular models of the distal nephron.

J Am Soc Nephrol 21: 1263–1269, 2010. doi: 10.1681/ASN.2010010001

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step and thus the site of regulation.Subsequently, Mg2� diffuses throughthe cytosol to be extruded actively

against an electrochemical gradientacross the basolateral membrane. Forthe Mg2� extrusion, unidentified can-

didates could be a Na�-dependent ex-change mechanism or an ATP-depen-dent Mg2� pump. Some of the salientfeatures are described below.

Transient Receptor PotentialMelastatin, Subtype 6The apical epithelial Mg2� channel isknown as the transient receptor potentialmelastatin, subtype 6 (TRPM6). TRPM6is a cation channel composed of sixtransmembrane-spanning domains anda conserved pore-forming region that as-sembles in a tetrameric configuration.Studies of families with autosomal reces-sive hypomagnesemia with secondaryhypocalcemia identified mutations inTRPM6.10,11 TRPM6 is one of eightmembers of the identified TRPM cationchannel subfamily and is composed of2022 amino acids encoded by a largegene containing 39 exons.10 –12 TRPM6displays a restricted expression patternand is predominantly present in reab-sorbing epithelia.10,11,13 In the kidney,TRPM6 localizes along the apical mem-brane of the DCT.13 This channel is aunique bifunctional protein consisting ofan Mg2� permeable cation channel withprotein kinase activity and is occasionallyreferred to as chanzymes.14 Electrophysi-ologic characterization of TRPM6 showsthat TRPM6-transfected human embry-onic kidney 293 cells exhibit outwardly rec-tifying currents. Mg2� itself has a profoundeffect on the activity of TRPM6. For in-stance, intracellular Mg2� levels tightlyregulate TRPM6 activity with an apparentKi of 0.5 mM that is comparable to physi-ologic intracellular Mg2� concentra-tions.13 Furthermore, extracellular Mg2�

also affects TRPM6, because Mg2� restric-tion significantly up-regulates levels ofmRNA encoding renal TRPM6.15

Kv1.1The voltage-gated K� channel, Kv1.1, isa new protein thought to regulate Mg2�

influx through TRPM6. Recently, therehas been evidence that a mutation inKCNA1 encoding Kv1.1 causes autoso-mal dominant hypomagnesemia.16 Thephenotype detectable from infancy con-sists of recurrent muscle cramps, tetany,tremor, muscle weakness, cerebellar at-

DCT1 DCT2 CNT

Transepithelial Mg2+

transportTransepithelial Ca2+

transport

Figure 1. Overview of Mg2� and Ca2� handling in the distal nephron. The activereabsorption of the minerals Mg2� and Ca2� takes place in the distal part of thenephron only. More precisely, this part of the nephron is comprised of the DCT and theCNT to the collecting duct. The former can be further subdivided into an early (DCT1)and late (DCT2) portion. Active Mg2� transport is confined to the DCT1 and DCT2,whereas active Ca2� reabsorption mainly occurs in the DCT2 and CNT segments. Thus,DCT2 functions as a transition area between Mg2� and Ca2� reabsorption.

NCC

DCT1/DCT2

TRPM6

Apical BasolateralEGF

EGFR

?

HNF1B

1.4riK1.1vK

Mg2+

Mg2+

K+K+

Na+

Transcription

γ-subunitof Na+,K+-ATPase

DCT1/DCT

Figure 2. Mechanism of active Mg2� reabsorption in DCT1 and DCT2 segments.Apical membrane TRPM6 channels are located in the apical membrane, which facili-tates transport of Mg2� from the tubular fluid into the cell. Mg2� reabsorption isprimarily driven by the luminal membrane potential established by the voltage-gatedK� channel, Kv1.1. The Na�-K�-ATPase, situated in the basolateral membrane, pro-vides a sodium (Na�) gradient that is used by the thiazide-sensitive NCC to facilitatetransport of Na� from the tubular fluid into the cytoplasm and a K� gradient togenerate local membrane potential. K� is supplied to the Na�-K�-ATPase throughrecycling through Kir4.1. The �-subunit of the Na�-K�-ATPase regulates the functionof Na� pump. Transcription factor HNF1B (hepatocyte nuclear factor 1 homeobox B)regulates the expression of the �-subunit of the Na�-K�-ATPase. EGF is the firstmagnesiotropic hormone to regulate active Mg2� reabsorption through the TRPM6channel.

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1264 Journal of the American Society of Nephrology J Am Soc Nephrol 21: 1263–1269, 2010

rophy, and myokymia. The K� channelco-localizes with TRPM6 along the lumi-nal membrane of the DCT. The identi-fied mutation results in a nonfunctionalchannel with a dominant-negative effecton wild-type channel function.17 Thus,Kv1.1 is a new luminal K� channel in theDCT that establishes favorable luminalmembrane potential controlling TRPM6-mediated Mg2� reabsorption.

�-Subunit of the Na�-K�-ATPaseWe also identified FXYD2 as being in-volved in hypomagnesemia.18 FXYD2encodes the �-subunit of the basolateralNa�-K�-ATPase and is mutated in pa-tients with autosomal dominant renalhypomagnesemia associated with hy-pocalciuria. Currently, the exact molec-ular mechanism by which the �-subunitcontrols Mg2� handling in the DCT re-mains elusive. It is postulated this trans-membrane protein facilitates the basolat-eral extrusion of Mg2� in renal epithelialcells.19 Others suggest the �-subunit regu-lates additional transport mechanisms thatlocalize to the basolateral membrane suchas the Na�-K�-ATPase, Kir4.1/5.1, or theunidentified basolateral Mg2� extrusionmechanism (Figure 2).1,5

Hepatocyte Nuclear Factor 1BFurther support for an active role of the�-subunit in Mg2� reabsorption is sug-gested by the observation that a tran-scription factor, hepatocyte nuclear fac-tor 1B (HNF1B), modulates the FXYD2gene.20 Hypomagnesemia, hypermag-nesuria, and hypocalciuria are observedin one half of the HNF1B mutation car-riers. Analyses of the FXYD2 promoterregion identify two highly conservedHNF1B recognition sites. Future studiesshould confirm the role of HNF1B in theregulation of FXYD2 and possibly othercomponents of the molecular machineryinvolved in renal Mg2� handling.

Kir4.1Two independent studies recently de-scribed a mutation within the KCNJ10gene as the underlying cause of a hypomag-nesemia syndrome.21,22 The first study de-scribed two nonrelated consanguineousfamilies with a disorder characterized by

epilepsy, ataxia, sensorineurol deafness,and tubulopathy (also referred to asSeSAME), whereas the other study de-scribed four kindreds with similar clinicalfindings. The KCNJ10 gene encodes a K�

channel called Kir4.1, expressed in brain,ear, and kidney, in keeping with the pheno-type observed in these patients. The renalphenotype of EAST syndrome (a syn-drome characterized by epilepsy, ataxia,sensorineural deafness, and tubulopathy)is similar to the Gitelman’s syndrome phe-notype and consists of polyuria, hypokale-mic metabolic alkalosis, hypomagnesemia,and hypocalciuria.22 In kidney, Kir4.1 isexpressed along the basolateral membraneof DCT cells with the Na�-K�-ATPase.Kir4.1 is thought to recycle K� into the in-terstitium to allow a sufficient supply of K�

for optimal Na�-K-ATPase activity.

EGF

We identified EGF as the first magne-siotropic hormone directly stimulatingTRPM6 activity.23 Genetic analysesshowed that a point mutation in thepro-EGF gene causes a rare inheritedautosomal recessive form of renal hy-pomagnesemia. EGF acts as an auto-crine/paracrine magnesiotropic hor-mone, specifically increasing TRPM6activity by engagement of its receptoralong the basolateral membrane ofDCT cells. This activation relies onboth the Src family of tyrosine kinasesand the downstream effector, Rac1.Activation of Rac1 increases the mobil-ity of TRPM6, assessed by fluorescencerecovery after photobleaching, and aconstitutively active mutant of Rac1mimics the stimulatory effect of EGFon TRPM6 mobility and activity. Ulti-mately, TRPM6 activation results fromincreased cell surface abundance.24

These findings provide the first insightinto the molecular regulation ofTRPM6 by extracellular EGF. More-over, it shows the molecular basis forthe hypomagnesemia after treatment withcetuximab, an EGF receptor blocking anti-body used in the treatment of colorectalcancer, and indicates TRPM6 is a potentialpharmacologic target during cetuximabtherapy.23,25

COMPREHENSIVE MODEL OFTRANSCELLULAR Mg2�

REABSORPTION ALONG THE DCT

The recent knowledge concerning themolecular nature of Mg2� transportingproteins offers for the first time a com-prehensive cellular model for transepi-thelial Mg2� reabsorption (Figure 2).The epithelial Mg2� channel TRPM6 fa-cilitates Mg2� entry from tubular fluidthrough an energized local electrochem-ical gradient. Importantly, the DCT celllacks a substantial chemical gradient forMg2�. The luminal membrane potentialin the DCT favoring luminal Mg2� in-flux is approximately �70 mV and likelyestablished by the luminal Kv1.1. chan-nel. The basolateral extrusion mecha-nism for Mg2� remains elusive and is asubject for further study. The Na�-K�-ATPase in the basolateral membranegenerates opposing K� and Na� gradi-ents. Importantly, Kir4.1 enables the ba-solateral recirculation of K�, therebysupplying sufficient K� during hightransport rates of the Na�-K�-ATPase;the basolateral �-subunit in all likelihoodsupports the Na� pump. Apparently,these special features are necessary to en-able the substantial transport of NaCl byNCC and Mg2� by TRPM6 in the DCTcell. Finally, EGF stimulates transcellulartransport of Mg2�. Activation of the ba-solateral EGF receptor promotes the in-sertion of TRPM6 channels into the lu-minal membrane to stimulate this Mg2�

reabsorption. Thus, in the last two de-cades, many Mg2� transport proteinshave been identified and characterizedby several research groups; the next stepwill be to develop specific therapeutics totreat the corresponding forms of hypo-magnesemia.

Ca2� REABSORPTION IN DCT2AND CNT SEGMENTS

In DCT2 and CNT segments, Ca2� reab-sorption takes place against its chemicalgradient, indicating that the transport isactive.26 In addition to the ubiquitouslyexpressed Na�-K�-ATPase, the Na�/Ca2� exchanger (NCX1) and the plasma

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membrane ATPase type 1b (PMCA1b)are also found along the basolateral siteof the DCT2 and CNT segments.2 DCT2shares similarities with the CNT seg-ment, because both segments express thetransient receptor potential vanilloidsubtype 5 (TRPV5) channel and theCa2�-binding protein, calbindin-D28K.Transepithelial transport of Ca2� is athree-step procedure and is outlined inmore detail below (Figure 3).

Ca2� influx across the apical mem-brane is mediated by TRPV5. Subse-quently, the specialized intracellular car-rier protein, calbindin-D28K, sequestersCa2� entering the cell, and this complexdiffuses toward the basolateral mem-brane. Finally, transporter proteins, suchas NCX1 and PMCA1b, extrude Ca2�

from the epithelial cell back into the cir-culation.2

Apical Entry of Ca2� by TRPV5To identify the apical Ca2� influx chan-nel involved in transcellular Ca2� reab-sorption, we performed functional ex-pression cloning using a cDNA libraryfrom rabbit primary CNT and the corti-cal collecting duct.27 Injection of totalmRNA from this isolation into Xenopuslaevis oocytes induces a 45Ca2� uptaketwo to three times above background.Subsequently, the entire cDNA librarywas screened for 45Ca2� uptake, and asingle transcript was isolated that en-codes for a novel epithelial Ca2� channelcalled eCaC1 and later renamed TRPV5,as a member of the TRP channel super-family.27,28 This channel comprises in-tracellular amino and carboxyl-terminaltails flanking six transmembrane do-mains and an additional hydrophobicstretch between domains 5 and 6, pre-dicted to be the pore-forming region.Furthermore, the first extracellular loopbetween transmembrane domains 1 and2 contains an evolutionary conserved as-paragine (N358) crucial for its complex-glycosylation, and in turn, for regulatingchannel activity.29 –31 The carboxyl andamino-terminal tails contain several reg-ulatory sites including protein kinase Cand A sites, which suggests an importantrole for phosphorylation in the regula-tion of channel activity. Moreover, in

cultured mammalian cells, as well as inoocytes, TRPV5 assembles into ho-motetramers to acquire an active confor-mational state.29,32

The TRPV5-null (TRPV�/�) mouseprovides compelling evidence for the phys-iologic function of this channel. ActiveCa2� reabsorption in DCT2 and CNT seg-ments is severely impaired in these null an-imals, because TRPV5�/� mice excrete�10-fold more Ca2� than their wild-typelittermates, in line with a postulated gate-keeper function for TRPV5 in active Ca2�

reabsorption.33 Electrophysiologic studiesshowed constitutive activity of TRPV5 atlow intracellular Ca2� concentrations andphysiologic membrane potentials.34 Thecurrent–voltage relationship of TRPV5shows strong inward rectification.2,34 An-other important functional feature is thatTRPV5 is the most Ca2�-selective memberof the TRP superfamily.34 The single chan-nel conductance, Po, and the number ofchannels at the plasma membrane deter-mines cellular TRPV5 activity. This activityis under the control of various factors likehormones, intracellular Ca2�, and otherintracellular messengers.

Vitamin DAmple evidence of a direct role for vita-min D in the positive regulation of

TRPV5 comes from several animal stud-ies, particularly those involving 25-hy-droxyvitamin D3-1�-hydroxylase– andvitamin D receptor–null mice.35,36 A di-rect relationship between 1,25(OH)2D3-induced expression of Ca2� transportproteins and transcellular Ca2� trans-port is known from studies of culturedcells from DCT and CNT cells.37,38 To-gether these studies suggest a consistent1,25(OH)2D3 sensitivity of TRPV5 and thecalbindins, and, to a lesser extent, the baso-lateral extrusion systems involving NCX1,a Na�/Ca2�exchanger, and PMCA1b, aplasma membrane Ca2�-ATPase.

Thiazide DiureticsThiazide diuretics, in contrast to loop di-uretics, have the unique characteristic ofdecreasing Na� reabsorption while in-creasing Ca2� reabsorption. In addition,mutations in the NCC gene encoding theNaCl co-transporter cause Gitelman’s syn-drome. Patients with Gitelman’s syn-drome exhibit hypovolemia, hypokalemicalkalosis, hypomagnesemia, and hypocal-ciuria.39 Intriguingly, the molecular mech-anisms responsible for the hypocalciuriaand hypomagnesemia with thiazide ad-ministration or in Gitelman’s syndromeremain elusive. Two hypotheses exist withrespect to the Ca2�-sparing effect of thia-

DCT2/CNT

Apical Basolateral

DCT2/CNT

PTH

PTHR

PMCA1b

NCX1

TRPV5Ca2+

Ca2+

Ca2+

CaBP

ATP

ADP

1, 25(OH)2D3

Figure 3. Mechanism of active Ca2� reabsorption in DCT2 and CNT. A three-stepprocess facilitates active and transcellular Ca2� transport. The first step is entry ofluminal Ca2� at the apical side of the cell through the TRPV5 channel. Subsequently,calbindin (CaBP) buffers Ca2�, and the Ca2� diffuses to the basolateral membrane. Atthe basolateral membrane, Ca2� is extruded by PMCA1b and NCX1. This process iscontrolled by calciotropic hormones including parathyroid hormone and 1,25(OH)2D3.

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zides.40,41 First, renal salt and water losscaused by thiazide treatment results incontraction of the extracellular volume(ECV), which triggers a compensatory in-crease of proximal Na� reabsorption. Thisin turn enhances the electrochemical gra-dient driving passive Ca2� transport inproximal tubular segments.6,39,42 Second,thiazide treatment stimulates Ca2� reab-sorption in DCT, possibly through theTRPV5 channel, that could explain theCa2�-sparing effect.40 We showed in ratsthat hydrochlorothiazide-induced hy-pocalciuria is accompanied by a significantdecrease in body weight compared withcontrols, confirming ECV contraction.43,44

Because sodium depletion results in a sim-ilar hypocalciuria, it is likely that the ECVcontraction by itself is responsible for thethiazide-induced hypocalciuria. Furtherevidence supporting this notion is the find-ing that sodium repletion during thiazidetreatment, thereby preventing the ECVcontraction, normalizes the calciuresis. Adirect role for TRPV5 in the thiazide-in-duced hypocalciuria seems unlikely, be-cause thiazides also have a hypocalciuric ef-fect in TRPV5�/� mice, and the overlap inthe expression of NCC and TRPV5 in thedistal part of the nephron is restricted toDCT2.44 Taken together, enhanced proxi-mal tubular Na� transport as a conse-quence of ECV contraction stimulatesparacellular Ca2� transport and best ex-plains the tubular mechanism for thiazide-induced hypocalciuria.

Activation of the Ca2�-SensingReceptor Prevents NephrolithiasisTRPV5�/� mice display hypercalciuriafrom impaired active Ca2� reabsorptionbut also hyperphosphaturia, polyuria,and increased urinary acidification.33

The latter two adaptations seem highlybeneficial because there are no renal cal-cium precipitates. Polyuria also dimin-ishes the risk of renal stone formation byreducing urinary Ca2� concentration. Inmice, calciuresis linearly correlates withurinary volume because an increase inCa2� excretion leads to an enhanced uri-nary volume. The consistent polyuria inhypercalciuric TRPV5�/� mice, noted bya substantial decrease in urinary osmola-lity, is caused by downregulation of renal

AQP2 water channels, possibly a result ofactivating the Ca2�-sensing receptoralong the luminal membrane of the col-lecting duct.45 Furthermore, gene abla-tion of the collecting duct-specific B1subunit of H�-ATPase in TRPV5�/�

mice abolishes enhanced urinary acidifi-cation, which resulted in severe tubularprecipitations of Ca2�-phosphate in therenal medulla.45 Thus, in TRPV5�/�

mice, activation of the renal Ca2�-sens-ing receptor promotes H�-ATPase–me-diated H� excretion and downregulationof AQP2, leading to urinary acidificationand polyuria, respectively (Figure 4).

FUTURE PERSPECTIVES ONRENAL Ca2� HANDLING

Ca2� reabsorption in the kidney, andparticularly in the distal DCT2 andCNT segments, is crucial for the main-tenance of the Ca2� balance. The iden-tification and characterization of the

proteins mediating this active Ca2�

transport provides novel insight andmeans to study molecular relation-ships. In these segments, TRPV5 facil-itates the gatekeeper function of Ca2�

entry, and therefore, a tight control ofits activity enables the organism to ad-just Ca2� reabsorption according tothe demands of Ca2� load. The molec-ular mechanism of Ca2� shuttling be-tween calbindin-D28K on one site andNCX1 and PMCA1b on the other site isnot clear. Another interesting and un-addressed question is the regulation ofNCX1 and PMCA1b in DCT2 and CNTcells. Whether there is a crosstalk be-tween apical Ca2� entry and basolat-eral Ca2� extrusion regulatory systemsis not known. The next step is to inves-tigate how these Ca2� transport pro-teins communicate with each other tofacilitate optimal and regulated Ca2�

reabsorption under conditions of dis-turbed Ca2� homeostasis. Finally, therole of TRPV5 in Ca2�-related disor-ders needs further study.

Polyuria Acidification

LowCa2+

HighCa2+

CaSR

H+

ATPase

AQP2

+ +

Figure 4. Molecular mechanism of Ca2�-induced polyuria and urinary acidification.The Ca2�-sensing receptor (CaSR) is localized at the apical site of principal andintercalated cells of the collecting duct. AQP2 proteins are responsible for waterreabsorption, whereas H�-ATPases pump H� into the tubular fluid. During hypercal-ciuria, increased urinary Ca2� levels activate the CaSR. CaSR activation leads to AQP2downregulation and polyuria. Furthermore, the CaSR triggers urinary acidification byincreasing the H�-ATPase activity. Both polyuria and increased urinary acidificationprevent the precipitation of renal Ca2�-phosphate.

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ACKNOWLEDGMENTS

There are many people I would like to ac-

knowledge and thank for their invaluable

contribution to my work. It has been an

honor and privilege to be working with

each one of them over the last 25 years. In

particular, I would like to thank my col-

league, Joost Hoenderop, for critical read-

ing of this manuscript before submission

and for his continuous support during our

long-term collaboration. Furthermore, I

would like to acknowledge the contribution

of many colleagues who spent some time in

my laboratory including technicians, PhD

students, postdocs, and staff members. Fi-

nally, I would like to thank all my collabo-

rators for their seminal help and making

science so much fun. It was and is a great

pleasure to work with you all.

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