Downregulation of Ca2+ and Mg2+ Transport Proteins in the Kidney Explains Tacrolimus (FK506)-Induced Hypercalciuria and Hypomagnesemia
Tom Nijenhuis,
Joost G.J. Hoenderop and
René J.M. Bindels
Department of Physiology, Nijmegen Center for Molecular Life Sciences, University Medical Center Nijmegen, Nijmegen, the Netherlands
Correspondence to René J.M. Bindels, 160 Cell Physiology, University Medical Center Nijmegen, P.O. Box 9101, NL-6500 HB Nijmegen, the Netherlands. Phone: +31-24-3614211; Fax: +31-24-3616413; E-mail: r.bindels{at}ncmls.kun.nl
ABSTRACT. FK506 (tacrolimus) and dexamethasone are potent immunosuppressantsknown to induce significant side effects on mineral homeostasis,including hypercalciuria and hypomagnesemia. However, the underlyingmolecular mechanisms remain unknown. The present study investigatedthe effects of FK506 and dexamethasone on the expression ofproteins involved in active Ca2+ reabsorption: the epithelialCa2+ channel TRPV5 and the cytosolic Ca2+-binding protein calbindin-D28K.In addition, the renal expression of the putative Mg2+ channelTRPM6, suggested to be involved in transcellular Mg2+ reabsorption,was determined. Administration of FK506 to rats by daily oralgavage during 7 d significantly enhanced the urinary excretionof Ca2+ and Mg2+ and induced a significant hypomagnesemia. FK506significantly decreased the renal mRNA expression of TRPV5 (62± 7% relative to controls), calbindin-D28K (9 ±1%), and TRPM6 (52 ± 8%), as determined by real-timequantitative PCR analysis. Furthermore, semiquantitative immunohistochemistryshowed reduced renal protein abundance of TRPV5 (24 ±5%) and calbindin-D28K (29 ± 4%), altogether suggestingthat downregulation of these transport proteins is responsiblefor the FK506-induced Ca2+ and Mg2+ wasting. In contrast, dexamethasonesignificantly enhanced renal TRPV5 (150 ± 15%), calbindin-D28K(177 ± 23%), and TRPM6 (156 ± 20%) mRNA levelsalong with TRPV5 (211 ± 8%) and calbindin-D28K (176 ±5%) protein abundance in the presence of significantly increasedCa2+ and Mg2+ excretion. This indicated that these proteinsare directly or indirectly regulated by dexamethasone. In conclusion,FK506 and dexamethasone induce renal Ca2+ and Mg2+ wasting,albeit by different mechanisms. Downregulation of specific Ca2+and Mg2+ transport proteins provides a molecular mechanism forFK506-induced hypercalciuria and hypomagnesemia, whereas dexamethasonepositively regulates these proteins.
Immunosuppressants such as the calcineurin inhibitors cyclosporinA and FK506 (tacrolimus) along with glucocorticoids such asdexamethasone are widely prescribed in numerous disorders andto organ transplant recipients. Although their immunosuppressiveactions are achieved by distinct mechanisms, FK506 and dexamethasoneboth are known to induce significant side effects on mineralhomeostasis. These drugs are associated with an increased boneturnover, a negative Ca2+ balance and hypercalciuria, perturbationsthat can ultimately result in osteoporosis (1–3). Furthermore,hypomagnesemia is a widely known additional side effect of FK506treatment (4, 5). The kidney is crucial to both Ca2+ and Mg2+homeostasis by providing the main excretory route for thesedivalent ions. However, the exact mechanisms by which theseimmunosuppressants provoke renal cation wasting are unknown.
The hypercalciuria during treatment with these drugs has beenattributed to increased bone resorption as well as decreasedrenal Ca2+ reabsorption (2, 6, 7). In the kidney, the bulk ofCa2+ reabsorption is accomplished by Na+-driven passive paracellularCa2+ transport in the proximal tubule and thick ascending limbof Henle (TAL) (8). Fine-tuning of Ca2+ excretion is achievedby active transcellular reabsorption of Ca2+ in the distal convolutedtubule (DCT) and connecting tubule (CNT), which involves apicalCa2+ entry through the epithelial Ca2+ channel TRPV5 (previouslyECaC1), intracellular buffering and facilitated diffusion bythe Ca2+-binding protein calbindin-D28K, and basolateral extrusionby the Na+/Ca2+ exchanger (NCX1) and the plasma membrane Ca2+ATPase (PMCA1b) (9, 10). Theoretically, inhibition of activeCa2+ reabsorption could be involved in the immunosuppressant-inducedhypercalciuria.
FK506 treatment has been associated with an inappropriatelyhigh fractional excretion of Mg2+, suggesting that inhibitionof passive or active Mg2+ reabsorption could contribute to thehypomagnesemia (11). Active reabsorption of Mg2+ has been localizedto the DCT, but in contrast to Ca2+ reabsorption, little isknown about the specific proteins that mediate transcellularMg2+ transport (12, 13). Recently, it was shown that autosomalrecessive hypomagnesemia, characterized by disturbed intestinalMg2+ absorption and inappropriately high fractional Mg2+ excretionrates, is caused by mutations in the gene encoding TRPM6 (14, 15). Like other ion channels in the transient receptor potential(TRP) family, this protein contains six transmembrane domainsand is highly homologous to the Mg2+-permeable ion channel TRPM7,and TRPM6 mRNA expression was detected in kidney and intestine(15). Recently, we demonstrated that TRPM6 confines a Mg2+ permeablechannel that is exclusively expressed in the apical membraneof mouse DCT and small intestine, suggesting that TRPM6 constitutesthe apical Mg2+ entry channel in transcellular Mg2+ (re)absorption(16). The identification of TRPM6 provides an important toolto study renal active Mg2+ transport during FK506 treatmenton a molecular level.
Taken together, the cascade of cellular and molecular eventsthat lead to impaired renal Ca2+ and Mg2+ handling during FK506and glucocorticoid treatment is largely unknown. Hypothetically,downregulation of Ca2+ and Mg2+ transport proteins in the distalpart of the nephron may be involved in the pathogenesis of hypercalciuriaand hypermagnesuria during drug treatment. The aim of the presentstudy, therefore, was to determine the effects of FK506 anddexamethasone on the expression of the Ca2+ transport proteinsTRPV5 and calbindin-D28K and the putative Mg2+ channel TRPM6in the kidney. To this end, rats were treated for 7 d with theseimmunosuppressants and housed in metabolic cages to collectsamples for urine analysis. Subsequently, expression levelsof TRPV5, calbindin-D28K, and TRPM6 were determined by real-timequantitative PCR and immunohistochemical analysis.
Animal Studies
Young adult male Wistar rats with an initial weight of 225 to250 g were housed individually in metabolic cages to collect24-h urine samples. The animals were kept in a light- and temperature-controlledroom with ad libitum access to standard pelleted diet and water.The animals were randomly assigned to either the control group(n = 10) or the two treatment groups, receiving FK506 (n = 6)or dexamethasone (n = 6). These immunosuppressants were dissolvedin peanut oil and administered daily by oral gavage. Controlanimals received vehicle only, whereas the two treatment groupsreceived either FK506 (Fujisawa Pharmaceutical Co., Osaka, Japan)at a single dose of 1 mg/d or dexamethasone (Sigma-Aldrich,Zwijndrecht, the Netherlands) at 300 µg/d. Animals weretreated for 7 d, after which blood samples were taken and theanimals were killed. Kidney cortex and duodenum were sampledand immediately frozen in liquid nitrogen. In addition, kidneycortex was fixed for immunohistochemistry by immersion in 1%(wt/vol) periodate-lysine-paraformaldehyde for 2 h and 15% (wt/vol)sucrose in PBS overnight (17). Subsequently, samples were storedat -80°C until further processing. The animal ethics boardof the University Medical Center Nijmegen approved all experimentalprocedures.
Analytical Procedures
Serum and urine Ca2+ concentrations were determined using acolorimetric assay as described previously (18). Serum and urine[Mg2+], [Na+], [creatinine] [glucose], alkaline phosphatase,and -glutamyl transpeptidase were measured on a Hitachi autoanalyzer(Hitachi Corp., Tokyo, Japan).
Real-Time Quantitative PCR
Total RNA was extracted from kidney cortex and duodenum usingTrizol Total RNA Isolation Reagent (Life Technologies BRL, Breda,the Netherlands). The obtained RNA was subjected to DNAse treatmentto prevent genomic DNA contamination. Thereafter, 2 µgof RNA was reverse transcribed by Moloney-murine leukemia virus-reversetranscriptase (Life Technologies BRL) as described previously(19). The obtained cDNA was used to determine TRPV5, calbindin-D28K,and TRPM6 mRNA levels in kidney cortex as well as TRPV6, calbindin-D9K,and TRPM6 mRNA expression in duodenum. Expression levels werequantified by real-time quantitative PCR on an ABI Prism 7700Sequence Detection System (PE Biosystems, Rotkreuz, Switzerland).PCR primers and fluorescence probes were designed using thecomputer program Primer Express (Applied Biosystems, FosterCity, CA) and are listed in Table 1 (Biolegio, Malden, the Netherlands).
Table 1. Sequences of primers and Taqman probes for real-time quantitative PCRa
Immunohistochemistry
Immunohistochemical staining was performed as described previously(17). In short, either single or double staining of sectionsfor TRPV5, calbindin-D28K, kallikrein, and the Na+-Cl- co-transporter(NCC) was performed on 7-µm sections of fixed frozen kidneysamples. TRPV5 staining involved immersion of the kidney sectionsin boiled citrate target retrieval buffer (0.01 M sodium citrateand 0.01 M citric acid [pH 6.0]), which was then left to coolfor 30 min and subsequent incubation in 0.3% (vol/vol) H2O2in buffer (0.15 M NaCl, 0.1 M Tris-HCl [pH 7.5]) for 30 min.Sections were incubated for 16 h at 4°C with primary antibody,affinity-purified guinea pig TRPV5 antibody (1:1000) (17), mouseanti–calbindin-D28K (Swant, Bellinzona, Switzerland; 1:500),rabbit anti-NCC (1:300) (20), and rabbit anti-kallikrein (1:5000;Calbiochem, San Diego, CA). After incubation with biotin-coatedgoat anti–guinea pig secondary antibody, TRPV5 was visualizedusing a tyramide signal amplification kit (NEN Life ScienceProducts, Zaventem, Belgium). For detection of calbindin-D28K,NCC, and kallikrein, sections were incubated with an Alexa 488–conjugatedgoat anti-rabbit or an Alexa 594–conjugated goat anti-mousesecondary antibody. Images were made using a Zeiss fluorescencemicroscope equipped with a digital photo camera (Nikon DMX1200).For semiquantitative determination of protein levels, imageswere analyzed with the Image Pro Plus 4.1 image analysis software(Media Cybernetics, Silver Spring, MD), resulting in quantificationof the protein levels as the mean of integrated optical density.
Statistical Analyses
Data are expressed as means ± SEM. Statistical comparisonswere tested by one-way ANOVA and Fisher multiple comparison.P < 0.05 was considered statistically significant. Statisticalanalysis was performed using the Statview Statistical Packagesoftware (Power PC version 4.51; Berkeley, CA) on a Macintoshcomputer.
Body Weight, Urine, and Serum Analysis
Net urinary excretion of Ca2+ and Mg2+ is depicted in Figure 1.Additional biochemical analysis of the serum and 24-h urinesamples of the experimental groups is shown in Table 2. In theFK506 group, net and fractional excretion of Ca2+ and Mg2+ wassignificantly enhanced. Na+ excretion was reduced in this treatmentgroup, whereas urine volume did not significantly differ fromcontrols. Although serum Ca2+ and Na+ levels were unaffected,FK506 treatment induced a striking hypomagnesemia. Dexamethasoneadministration enhanced urinary Ca2+ as well as Mg2+ excretion.Likewise, Na+ excretion and urine volume were significantlyincreased. In the dexamethasone-treated animals, serum Ca2+levels did not significantly differ from controls, whereas serumNa+ levels were decreased. Serum Mg2+ levels were significantlyreduced relative to controls but remained within the normalrange. Both serum creatinine and GFR, as determined by creatinineclearance, did not significantly differ from controls in boththe FK506 and dexamethasone groups. Serum glucose levels weresignificantly increased in both the FK506- and dexamethasone-treatedgroups, resulting in increased glucose excretion in the dexamethasonegroup only. In addition, urinary excretion of markers of tubulardamage (alkaline phosphatase and -glutamyl transpeptidase) wasdetectable, albeit not significantly increased in the FK506group, whereas the dexamethasone-treated group showed significantenzymuria (21). Although baseline body weight did not differsignificantly between the groups (data not shown), at the endof the experiment, dexamethasone-treated animals had a significantlylower mean body weight compared with controls, whereas FK506treatment did not affect body weight.
Figure 1. Effect of FK506 and dexamethasone treatment on net urinary Ca2+ and Mg2+ excretion in the rat. Net excretion of Ca2+ and Mg2+ was determined by analysis of 24-h urine samples of rats individually housed in metabolic cages. Ctr, controls; FK506, FK506 1 mg/d by oral gavage; Dex, dexamethasone 300 µg/d by oral gavage. Data are presented as means ± SEM. **P < 0.05 versus controls.
Table 2. Urine and serum analyses and body weighta
Renal mRNA Expression Levels of TRPV5, Calbindin-D28K, and TRPM6
For evaluating the possible association between increased urinaryexcretion of Ca2+ and Mg2+ and the expression levels of therespective transport proteins, renal mRNA levels of the epithelialCa2+ channel TRPV5, the cytosolic Ca2+-binding protein calbindin-D28Kand the putative epithelial Mg2+ channel TRPM6 were determinedby real-time quantitative PCR analysis. FK506 significantlydecreased TRPV5, calbindin-D28K, and TRPM6 mRNA levels in thekidney (Figure 2). Conversely, dexamethasone treatment significantlyenhanced the renal mRNA expression of both Ca2+ transport proteinsand TRPM6.
Figure 2. Effect of FK506 and dexamethasone on mRNA expression levels of Ca2+ and Mg2+ transport proteins in the rat kidney. Renal mRNA expression levels of the epithelial Ca2+ channel TRPV5, the cytosolic Ca2+-binding protein calbindin-D28K (CaBP28), and the putative Mg2+ channel TRPM6 were determined by real-time quantitative PCR analysis. Ctr, controls; FK506, FK506 1 mg/d by oral gavage; Dex, dexamethasone 300 µg/d by oral gavage. Data are presented as means ± SEM. **P < 0.05 versus controls.
Renal Protein Abundance of TRPV5 and Calbindin-D28K
For assessing whether the changes in renal mRNA levels resultedin altered protein expression, TRPV5 and calbindin-D28K proteinabundance was semiquantified by immunohistochemistry (Figure 3A).These results illustrated that the above-mentioned mRNAresults were indeed accompanied by similar effects on the proteinlevel. Computerized analysis of the immunohistochemical stainingshowed significantly reduced TRPV5 and calbindin-D28K proteinexpression during FK506 treatment, whereas dexamethasone significantlyenhanced the abundance of these Ca2+ transport proteins in thekidney (Figure 3B). Of note, the effects on TRPV5 protein expressionwere more pronounced compared with the mRNA level, suggestingthat in addition to affecting gene transcription, additionaltranslational changes might have occurred.
Figure 3. Effect of FK506 and dexamethasone on protein abundance in the rat kidney. (A) Representative images showing kidney sections stained for the epithelial Ca2+ channel TRPV5, the cytosolic Ca2+-binding protein calbindin-D28K (CaBP28), kallikrein, and the Na+-Cl- cotransporter (NCC). (B) Protein abundance was determined by computerized analysis of immunohistochemical images and is presented as integrated optical density (IOD; arbitrary units). Ctr, controls; FK506, FK506 1 mg/d by oral gavage; Dex, dexamethasone 300 µg/d by oral gavage. Data are presented as means ± SEM. **P < 0.05 versus controls.
For confirming the specificity of the observed effects on theCa2+ and Mg2+ transport proteins, protein abundance of kallikreinand NCC were determined by immunohistochemistry (Figure 3A).The renal abundance of tissue kallikrein, a specific markerfor the DCT and CNT (22), did not significantly differ fromcontrols in both the FK506 and dexamethasone groups (Figure 3B).In addition, the protein expression of NCC, exclusivelyexpressed in the DCT, was not significantly affected by FK506treatment but showed a significant, albeit modest, increasein the dexamethasone-treated animals (Figure 3B). Furthermore,no signs of a general deleterious effect of FK506 (inclusionbodies, tubular vacuolization, and atrophy) were detected onlight microscopic analysis (4). GFR was unaffected and enzymuriawas not significantly increased, although markers of tubulardamage were detectable. Importantly, the unaltered expressionof kallikrein and NCC suggested that no overt FK506 nephrotoxicitywas present in DCT.
In addition, co-staining of kidney sections for the presenceof TRPV5 and kallikrein was performed, which showed a full co-localizationof these proteins in DCT and CNT in the control animals (Figure 4A).Importantly, Figure 4B clearly illustrates that TRPV5 proteinabundance in DCT and CNT is significantly reduced in the FK506-treatedanimals, whereas kallikrein expression levels are not affected.Dexamethasone treatment did not influence the co-localizationof TRPV5 and kallikrein, substantiating that the dexamethasone-inducedincrease of TRPV5 abundance is confined to these nephron segments(Figure 4C). Co-staining of kidney sections for kallikrein andcalbindin-D28K showed similar results, with the exception thatcalbindin-D28K expression extended further into the corticalcollecting duct (data not shown).
Figure 4. Co-immunohistochemical staining of rat kidney for the epithelial Ca2+ channel TRPV5 and kallikrein. Co-staining of kidney sections for TRPV5 and kallikrein, a marker for the distal convoluted tubule (DCT) and connecting tubule (CNT), in controls (A), FK506-treated animals (B), and dexamethasone-treated animals (C). Controls, rats receiving vehicle only; FK506, rats receiving FK506 1 mg/d by oral gavage; Dexamethasone, rats receiving dexamethasone 300 µg/d by oral gavage.
Intestinal mRNA Expression Levels of TRPV6, Calbindin-D9K, and TRPM6
Theoretically, upregulation of Ca2+ transport proteins in theintestine could contribute to the increased Ca2+ wasting, anddecreased intestinal TRPM6 expression could contribute to thehypomagnesemia. Therefore, the duodenal mRNA expression of theepithelial Ca2+ channel TRPV6, the cytosolic Ca2+-binding proteincalbindin-D9K, and TRPM6 were determined (Figure 5). mRNA expressionof all three transporters was not significantly altered by FK506treatment. Dexamethasone significantly increased TRPV6 and calbindin-D9KmRNA expression levels, whereas TRPM6 mRNA expression was notsignificantly affected.
Figure 5. Effect of FK506 and dexamethasone treatment on mRNA expression of Ca2+ and Mg2+ transport proteins in rat duodenum. Duodenal mRNA expression levels of the epithelial Ca2+ channel TRPV6, the cytosolic Ca2+-binding protein calbindin-D9K (CaBP9), and the putative Mg2+ channel TRPM6 were determined by real-time quantitative PCR analysis. Ctr, controls; FK506, FK506 1 mg/d by oral gavage; Dex, dexamethasone 300 µg/d by oral gavage. Data are presented as means ± SEM. * P < 0.05 versus controls.
The present study demonstrated that FK506 and dexamethasonesignificantly enhance renal Ca2+ and Mg2+ excretion, albeitby different molecular mechanisms. The decreased expressionof the Ca2+ transport proteins TRPV5 and calbindin-D28K andthe putative Mg2+ channel TRPM6 during FK506 treatment suggestedthat downregulation of these transport proteins contributesto the pathogenesis of FK506-induced hypercalciuria and hypomagnesemia.In contrast, dexamethasone treatment increased renal expressionlevels of TRPV5, calbindin-D28K, and TRPM6, indicating thatthese transport proteins are either directly or indirectly regulatedby dexamethasone.
FK506 treatment significantly increased urinary Ca2+ excretion,accompanied by a downregulation of the renal mRNA expressionof TRPV5 and calbindin-D28K and a specific reduction of theprotein abundance of these Ca2+ transport proteins in DCT andCNT, recognized as the main sites of transcellular Ca2+ reabsorption.Previous reports showed reduced calbindin-D28K levels duringFK506 treatment, suggesting that FK506 could affect active Ca2+transport (23). That serum Ca2+ concentrations and GFR did notdiffer from controls confirmed that impaired Ca2+ reabsorptionrather than an increased filtered load caused the hypercalciuria.Morphologic features of tubular toxicity were not detected.Furthermore, the excretion of urinary markers of tubular damagewas not significantly increased and the expression of specificmarkers for DCT and CNT was unaltered, excluding that a generaldeleterious effect of FK506 on DCT is responsible for the observeddownregulation. In addition, the expression of the major intestinalCa2+ transport proteins was not increased by FK506 treatment,excluding that hypercalciuria is secondary to upregulation ofactive Ca2+ absorption. The present data strongly supportedour hypothesis that FK506 induces a primary defect of renalactive Ca2+ reabsorption by specifically downregulating theproteins involved in active Ca2+ transport.
The molecular mechanism underlying the downregulation of theCa2+ transport proteins by FK506 remains elusive. In previousstudies, plasma calcitriol (1,25(OH)2D3) was either unalteredor moderately increased, whereas plasma parathyroid levels werenot affected by similar doses of FK506, which excludes thatthe reduced Ca2+ transport protein expression levels are secondaryto decreased circulating levels of these calcitropic hormones(11, 23, 24). The immunosuppressive action of FK506 dependson the inhibition of the Ca2+-dependent phosphatase calcineurinin T lymphocytes (25–27). Calcineurin is not known tobe involved in renal Ca2+ reabsorption, but another calcineurininhibitor, cyclosporine A, increased urinary Ca2+ excretionand decreased calbindin-D28K protein levels, suggesting thatcalcineurin inhibition may play a role in the impairment ofCa2+ reabsorption by these drugs (23, 28). In addition, FK506binds to intracellular immunophilins called FK506-binding proteins(FKBP), which have been implicated as ion channel regulators(29–31). In particular, FKBP4 was shown to bind and regulatethe Ca2+-permeable Drosophila TRPL channel, and this bindingwas disrupted by the addition of FK506 (32). Furthermore, severalintracellular Ca2+-release channels were shown to be modulatedby binding and dissociation of FKBP (33–35). Therefore,it is tempting to speculate that FKBP are potential associatedproteins regulating TRPV5 expression or activity.
Hypomagnesemia is a widely known adverse effect of FK506 treatment(4, 5). The increased fractional excretion of Mg2+ in the presenceof a profound hypomagnesemia indicated that renal Mg2+ reabsorptionis seriously impaired by FK506. This renal Mg2+ wasting wasaccompanied by reduced renal expression of the putative Mg2+channel TRPM6. Mutations in the gene encoding TRPM6 were recentlyshown to cause hereditary hypomagnesemia, which was also accompaniedby an inappropriately increased fractional excretion of Mg2+(14, 15). Importantly, RT-PCR analysis of microdissected ratnephrons showed strong TRPM6 expression in the DCT, the mainsite of active Mg2+ reabsorption (15). Together with the highstructural homology to TRPM7, which has been previously identifiedto constitute a Mg2+ permeable cation channel, these data indicatedthat this protein constitutes the apical Mg2+ entry channelin renal transcellular Mg2+ transport in DCT (36, 37). Of note,the DCT has been previously indicated as a possible site ofthe tubular Mg2+ leak during FK506 treatment (13). The duodenalTRPM6 mRNA levels were not significantly altered by FK506 treatment,which is at variance with primary role of duodenal TRPM6 inthe pathogenesis of hypomagnesemia. Altogether, our data showedthat in addition to mutations in TRPM6, drug-induced downregulationof this ion channel in kidney is associated with increased urinaryMg2+ excretion and hypomagnesemia, further substantiating theimportance of TRPM6 in renal Mg2+ reabsorption.
Dexamethasone-treated animals displayed a significant hypercalciuria,but, in contrast to FK506 treatment, dexamethasone increasedthe expression levels of TRPV5 and calbindin-D28K. However,in line with previous data that showed decreased tubular reabsorptionof Ca2+ during glucocorticoid treatment, the unaltered serumCa2+ levels and GFR suggested that a primary impairment of Ca2+reabsorption is responsible for the hypercalciuria observedin our study (2, 6). In the proximal tubule and TAL, passivereabsorption of Ca2+ takes place, which is altogether responsiblefor reabsorbing approximately 70 to 90% of the filtered Ca2+(8). In this paracellular pathway, Ca2+ is transported downan electrochemical gradient that is dependent on Na+ reabsorption,and impairment of the latter is known to result in increasedCa2+ excretion. In this study, net Na+ excretion was indeedconsiderably increased and serum Na+ levels were reduced, whereasNCC expression was enhanced. Previously, glucocorticoid excesswas demonstrated to enhance the expression of the distally locatedNCC and epithelial Na+ channel, whereas dexamethasone was shownto inhibit the function and expression of the proximal tubularNa+/phosphate co-transporter (38–41). Therefore, reducedreabsorption of Na+ in the proximal tubule or TAL could be responsiblefor the increased Na+ excretion and, by decreasing passive Ca2+reabsorption, the dexamethasone-induced hypercalciuria. In addition,enzymuria was significantly increased by dexamethasone, suggestingthat structural damage to the proximal tubule or TAL could contributeto the increased Na+ and Ca2+ excretion. The increased expressionof the proteins involved in active Ca2+ transport in DCT indicatedthat these proteins are directly or indirectly regulated bydexamethasone. The latter might reflect a compensatory effectsecondary to the increased distal Ca2+ load, similar to theincreased expression of distally located Na+ transporters, whichhas been shown after inhibition of Na+ reabsorption in the loopof Henle (42–44). The concomitant increase in expressionof the major intestinal Ca2+ transport proteins suggests a directstimulatory effect of dexamethasone on active Ca2+ (re)absorptionin kidney and intestine.
It is interesting that dexamethasone treatment resulted in anincreased urinary excretion of Mg2+ with a concomitant decreaseof serum Mg2+ levels. However, the latter effect was much smallerthan that observed in the FK506-treated animals and remainedwithin the normal range, possibly because of the milder renalMg2+ leak. Alternatively, increased intestinal Mg2+ absorptionmediated by upregulated intestinal TRPM6 expression levels mightcounterbalance renal Mg2+ losses, but this is not supportedby the present study. In analogy to Ca2+ reabsorption, a substantialpart of Mg2+ filtered by the glomerulus is reabsorbed via theparacellular Na+-driven pathway (12). Therefore, impairmentof the passive reabsorption of divalent ions in proximal tubuleand TAL could explain both the hypercalciuria and hypermagnesuria.Subsequently, the enhanced TRPM6 expression levels could resultfrom a direct stimulatory effect of dexamethasone or a compensatoryresponse, serving to limit the renal Mg2+ wasting.
Indeed, serum glucose levels were significantly increased inboth the FK506- and dexamethasone-treated animals. In the FK506-treatedgroup, urinary glucose excretion did not significantly differfrom controls, but the trend toward increased glycosuria couldvery well explain the concomitant trend toward increased urinevolume. Furthermore, dexamethasone significantly increased glycosuria,which in conjunction with the increased Na+ excretion mightcause the observed polyuria.
In conclusion, FK506-induced downregulation of Ca2+ and Mg2+transport proteins could be a critical factor in the pathogenesisof hypercalciuria and hypomagnesemia. Elucidation of the molecularmechanism responsible for these inhibitory effects will extendour knowledge of the in vivo regulation of these transportersand will identify novel targets for pharmacologic therapy.
Acknowledgments
This study was supported by the Dutch Kidney Foundation (C10.1881)and the Dutch Organization of Scientific Research (Zon-Mw 016.006.001).
We thank the Fujisawa Pharmaceutical Co. for generously supplyingtacrolimus (FK506).
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Received for publication June 13, 2003.
Accepted for publication November 27, 2003.
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Abstract
Introduction
-glutamyl transpeptidase were measured on a Hitachi autoanalyzer (Hitachi Corp., Tokyo, Japan). 
-hydroxylase knockout mice. FASEB J 16: 1398–1406, 2002
