Na /H Exchanger Regulatory Factor 3 Is Critical for Multidrug Resistance Protein 4–Mediated Drug Efflux in the Kidney

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Na+/H+ Exchanger Regulatory Factor 3 Is Critical for Multidrug Resistance Protein 4–Mediated Drug Efﬂux in the Kidney

† ‡ ‡ Joonhee Park,* Jin-Oh Kwak,* Brigitte Riederer, Ursula Seidler, Susan P.C. Cole,§ † Hwa Jeong Lee, and Min Goo Lee*

*Department of Pharmacology and Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea; †College of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul, Korea; ‡Department of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, Hannover, Germany; and §Department of Pathology and Molecular Medicine and Division of Cancer Biology and Genetics, Queen’s University, Kingston, Ontario, Canada

ABSTRACT Na+/H+ exchanger regulatory factor 3 (NHERF3) is a PSD-95/discs large/ZO-1 (PDZ)-based adaptor protein that regulates several membrane-transporting proteins in epithelia. However, the in vivo physiologic role of NHERF3 in transepithelial transport remains poorly understood. Multidrug resistance protein 4 (MRP4) is an ATP binding cassette transporter that mediates the efflux of organic molecules, such as nucleoside analogs, in the gastrointestinal and renal epithelia. Here, we report that Nherf3 knockout 2/2 (Nherf3 ) mice exhibit profound reductions in Mrp4 expression and Mrp4-mediated drug transport in the kidney. A search for the binding partners of the COOH-terminal PDZ binding motif of MRP4 among several epithelial PDZ proteins indicated that MRP4 associated most strongly with NHERF3. When expressed in HEK293 cells, NHERF3 increased membrane expression of MRP4 by reducing internalization of cell surface MRP4 and consequently, augmented MRP4-mediated efflux of adefovir, a nucleoside-based 2/2 antiviral agent and well known substrate of MRP4. Examination of wild-type and Nherf3 mice revealed that Nherf3 is most abundantly expressed in the kidney and has a prominent role in modulating Mrp4 levels. Deletion of Nherf3 in mice caused a profound reduction in Mrp4 expression at the apical membrane of renal proximal tubules and evoked a significant increase in the plasma and kidney concentrations of adefovir, with a corresponding decrease in the systemic clearance of this drug. These results suggest that NHERF3 is a key regulator of organic transport in the kidney, particularly MRP4-mediated clearance of drug molecules.

J Am Soc Nephrol 25: 726–736, 2014. doi: 10.1681/ASN.2013040438

Assembly of protein complexes by adaptor proteins contain either two or four PDZ domains and were with PSD-95/discslarge/ZO-1(PDZ)domains plays the ﬁrst family of PDZ-containing proteins shown an important role in the regulation of many membrane proteins, especially in epithelial and Received April 30, 2013. Accepted August 21, 2013. neuronal tissues.1,2 For example, PDZ-based adapter proteins in epithelia, such as Shank2, syn- Published online ahead of print. Publication date available at aptic scaffolding molecule (S-SCAM), and Na+/H+ www.jasn.org. exchanger regulatory factors (NHERFs), are known Correspondence: Dr. Hwa Jeong Lee, College of Pharmacy, to be involved in the regulation of cell surface ex- Graduate School of Pharmaceutical Sciences, Ewha Womans Uni- versity, 52, Ewhayeodae-gil, Seodaemun-gu, Seoul 120-750, Korea, pression and the activity of many membrane trans- or Prof. Min Goo Lee, Department of Pharmacology, Yonsei Uni- porters and receptors in the respiratory, digestive, versity College of Medicine, 134 Sinchon-Dong, Seoul 120-752, urinary, and reproductive organs.3–6 The four Korea. Email: [email protected] or [email protected] members of the NHERF proteins (NHERF1 to 4) Copyright © 2014 by the American Society of Nephrology

726 ISSN : 1046-6673/2504-726 J Am Soc Nephrol 25: 726–736, 2014 www.jasn.org BASIC RESEARCH to be involved in epithelial transport. NHERF3, also known as terminus of MRP4 comprising the PDZ binding motif PDZK1 or CAP70, has four PDZ domains and is normally (MRP4-DETAL), indicated that a PDZ-based interaction medi- localized to the apical microdomains of gastrointestinal and ates the association between MRP4 and NHERF3 in HEK293 kidney epithelia. Each PDZ domain of NHERF3 has been pro- cells. posed to bind independently to the PDZ binding motif of various To determine whether MRP4 directly associates with membrane proteins, such as the cystic fibrosis transmembrane NHERF3 and, if so, which PDZ domain of NHERF3 is conductance regulator (CFTR), the Na+/H+ exchanger 3, the ur- responsible for the interaction, pull-down assays were per- 7–10 ate transporter, and the organic anion transporter 4. However, formed with recombinant proteins. Thus, four His6-tagged 2 2 Nherf3 knockout (Nherf3 / ) mice do not have a discernible recombinant proteins encoding individual PDZ domains of phenotype except mild hypercholesterolemia,11 and, conse- NHERF3 (Figure 1, B and C) and a glutathione S-transferase quently, the in vivo role of NHERF3 in transepithelial transport (GST) fusion protein containing the last 40 amino acids of the remains poorly understood. COOH terminus of MRP4 (GST-MRP4-C40) (Figure 1D) Transporters belonging to either the ATP binding cassette were expressed in Escherichia coli and purified. Pull-down as- (ABC) or the solute-linked carrier superfamilies of membrane says with the recombinant proteins and glutathione agarose proteins play diverse roles in the pharmacokinetic and beads indicated that the COOH terminus of MRP4 directly pharmacodynamic pathways of drugs and their metabo- associates with PDZ1 of NHERF3 (Figure 1E). lites.12 Multidrug resistance protein 4 (MRP4/ABCC4) is a member of the C subfamily of ABC transporters and mediates NHERF3 Increases the Expression and Function of the efflux of a group of organic molecules using energy gen- MRP4 in HEK293 Cells erated from the binding and hydrolysis of ATP.13 Cumulative Next, we examined the effects of NHERF3 on the expression evidence suggests that MRP4 pumps out purine compounds levels and function of MRP4 in HEK293 cells. First, cell surface and plays an important role in the renal elimination of purine- and total MRP4 levels were measured in the presence and based antiviral and antineoplastic agents.14,15 However, the absence of transfected NHERF3. As shown in Figure 2A, regulatory mechanisms for MRP4 expression and function NHERF3 greatly augmented the total and cell surface expres- have not been extensively studied. sion of MRP4. Quantitative analyses of multiple experiments The COOH terminus of MRP4 contains a class 1 PDZ are summarized in Figure 2, B–D, and show that coexpression interaction motif (-S/T-X-Ф, where Ф is a hydrophobic amino of NHERF3 increased the amount of MRP4 in cell lysates and acid),16 indicating that MRP4 may interact with PDZ-based the cell surface by 4.0- and 5.9-fold, respectively (Figure 2, B adaptors in epithelia. In preliminary studies using a yeast two- and C). As a consequence, cell surface expression of MRP4 hybrid assay, MRP4 was found to strongly interact with relative to the total amount in cell lysates was also increased by NHERF3 among several PDZ adaptor proteins expressed in 38% (Figure 2D). epithelial tissues. The aim of the present study was to identify Second, the effect of NHERF3 coexpression on the func- the physiologic and pharmacologic roles of the interaction of tional activity of MRP4 in HEK293 cells was measured. The NHERF3 with MRP4 using a variety of in vitro and in vivo cells were loaded with the dipivoxil ester form of [3H]adefovir experimental approaches. The results obtained provide strong (Figure 2E), an antiviral agent and well known substrate of evidence that NHERF3 is a key regulator of organic transport MRP4,17 and the efflux activity of MRP4 was indirectly esti- in the kidney, particularly the MRP4-mediated clearance of mated by measuring the radioactivity remaining in the cells purine-based drug molecules. after 2 hours at 37°C. As shown in Figure 2F, expression of MRP4 strongly decreased the amount of radioactivity (and hence, adefovir) that remained in the cells. Notably, coexpres- RESULTS sion of NHERF3 further decreased the [3H]adefovir radioactivity from 4.0860.793105 to 2.0360.523105 counts per NHERF3 Interacts with MRP4 minute/mg protein (P,0.05). These results indicate that Interactions between the COOH termini of ABC transporters NHERF3 can increase the functional activity of MRP4 in that have a PDZ binding motif and the PDZ domains of adaptor HEK293 cells, which correlates well with the results obtained proteins expressed in epithelia were initially identified by a from the cell surface labeling experiments. yeast two-hybrid screening assay. The results indicated that the PDZ domains of S-SCAM, NHERF1, and NHERF3 might Molecular Mechanism of MRP4 Upregulation by interact with MRP4. NHERF3 showed the strongest interac- NHERF3 tion when examined by reporter gene activities (Supplemental Enhanced levels of MRP4 could be explained by transcriptional Table 1). The interaction between MRP4 and NHERF3 found upregulation or increased stability of MRP4 mRNA. However, in the yeast two-hybrid system was confirmed in mammalian results from quantitative RT-PCR revealed that the amount of human embryonic kidney (HEK) 293 cells by a coimmunopre- MRP4 mRNA in HEK293 cells was not affected by NHERF3 cipitation assay (Figure 1A). Experiments with an expression coexpression (Supplemental Figure 1). Several PDZ-based vector, which lacks the last four amino acids of the COOH adaptors have shown that they increase the cell surface stability

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Figure 1. MRP4 directly binds to NHERF3 through a PDZ-based interaction. (A) Immunoprecipitation assays using transfected HEK293 cells confirmed the interaction between MRP4 and NHERF3 in mammalian cells. The interaction was eliminated by the deletion of the PDZ binding motif at the COOH terminus of MRP4 (MRP4-DETAL). (B–E) Pull-down assay using recombinant proteins. (B) Schematic diagram of the four NHERF3 PDZ domain constructs linked to a 6X His tag. (C) Polypeptides corresponding to each of the four PDZ domains in NHERF3 were purified using a nickel-nitrilotriacetic acid protein purification system. (D) Peptides containing the GST- conjugated COOH-terminal 40 amino acids of MRP4 (arrow) and a GST control protein were purified using glutathione-Sepharose beads. (E) GST pull-down showing the direct association between the COOH terminus of MRP4 and the first PDZ domain (PDZ1) of NHERF3.

of associated membrane proteins.6,18 Therefore, the effects of To explore mechanisms responsible for the NHERF3- NHERF3 on the cell surface stability of MRP4 were exam- induced increase in MRP4 expression, internalization and ined. HEK293 cells transfected with Myc-tagged MRP4 were recycling of cell surface MRP4 were examined. As shown in treated with cycloheximide (0.1 mg/ml) for up to 36 hours to Figure 4, NHERF3 significantly reduced the internalization inhibit new protein synthesis; proteins on the cell surface of surface MRP4; however, surface recycling of MRP4 was were biotinylated, and cell surface MRP4 was detected with not affected by NHERF3. To further explore the mecha- an anti-Myc antibody. As shown in Figure 3, A and B, only nisms responsible, protein degradation assays were per- 8.662.1% of MRP4 remained at the cell surface 36 hours formed with inhibitors of lysosomal (bafilomycin A) and after the cycloheximide treatment. Interestingly, coexpres- proteasomal (MG132) degradation. As shown in Supple- sion of NHERF3 increased this value to 35.765.3%. Coex- mental Figure 2, treatment with bafilomycin A, but not pression of NHERF3 also enhanced the stability of MRP4 in with MG132, greatly inhibited the degradation of MRP4. total cell lysate, as well as the surface protein. Thus, the The effect of bafilomycin A was significantly reduced in cells amount of total MRP4 remaining 36 hours after treatment where NHERF3 was coexpressed (Supplemental Figure 2). with cycloheximide was increased from 37.464.7% to Collectively, the above results indicate that NHERF3 re- 67.966.1% by coexpression of NHERF3 (Figure 3, C and duces internalization of surface MRP4, an effect associated D), indicating that degradation of MRP4 was attenuated by with a decrease in the lysosome-mediated degradation of NHERF3 coexpression. MRP4.

728 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 726–736, 2014 www.jasn.org BASIC RESEARCH

Figure 2. NHERF3 upregulates MRP4 expression and function in HEK293 cells. (A–D) The effects of NHERF3 coexpression on the total and cell surface expression of MRP4 were examined in HEK293 cells. (A) A representative immunoblot of cell surface and lysate proteins in the presence and absence of NHERF3. (B) Densitometric analyses of total MRP4 protein levels in the lysate immunoblots (n=8). MRP4 levels were corrected relative to aldolase levels. (C) Densitometric analyses of biotinylated surface MRP4 protein levels (n=7). The amount of surface MRP4 was corrected for the amount of aldolase in the corresponding lysate blots. (D) Relative abundance of MRP4 at the cell surface (corrected for the total MRP4 in the lysate blots; n=7). (E and F) Functional activity of MRP4 was measured by loading HEK293 cells with [3H]adefovir and measuring remaining tritium in the cells after 2 hours. (E) Chemical structure of [3H]adefovir. (F) Efﬂux of [3H]adefovir, which represents MRP4 function, was augmented in the cells coexpressing NHERF3. *P,0.05; **P,0.01.

NHERF3 Upregulates MRP4 in the Mouse Kidney We next examined the effect of Nherf3 ablation. Both We next investigated whether the profound in vitro effect of immunohistochemistry and immunoblotting experiments NHERF3 on MRP4 expression could be reproduced in vivo showed that the absence of Nherf3 evoked a profound decrease in a mouse model. NHERF3 has been reported to regulate in Mrp4 expression in the mouse kidney. Immunohistochem- membrane proteins in the digestive organs and the kid- ical images showed that Mrp4 is densely localized at the apical ney,7,8,10,19 and therefore, we first examined expression pat- membrane of proximal tubules in wild-type mice in contrast to 2 2 terns of NHERF3 using tissues from wild-type (Nherf3+/+)and kidneys from Nherf3 / mice, where the staining intensity of 2 2 Nherf3 / mice. Immunoblotting experiments confirmed the Mrp4 is reduced (Figure 5B). Quantification of the immuno- 2 2 absence of Nherf3 protein in the Nherf3 / mice (Supplemen- blotting results further indicated that the total and apical sur- tal Figure 3). The amount of Nherf3 protein varied among the face (brush border membrane [BBM]) Mrp4 protein are 2 2 four tissues examined, with the highest levels of protein in the greatly reduced in Nherf3 / mice (Figure 5, C–F). NHERF3 kidney, comparatively lower levels in the ileum and liver, and ablation decreased total and BBM MRP4 by 62.167.5% and 2 2 very low levels in the colon (Supplemental Figure 3). Because 94.161.8%, respectively, in the Nherf3 / kidney. As a con- Nherf3 was most highly expressed in the kidney, subsequent sequence, BBM expression of MRP4 relative to the total morphologic and biochemical studies were focused mainly on amount in the kidney tubules was also decreased by that organ. Immunofluorescence imaging of wild-type mice 85.463.2% (Figure 5F). revealed that Nherf3 is highly expressed in the apical regions of most tubules of the kidney (Figure 5A). Similarly, Mrp4 was Effect of Nherf3 Ablation on Mrp4-Mediated Adefovir observed in the apical membrane of some tubules, consistent Transport in Mice with previous reports that MRP4 is principally expressed in MRP4 has been reported to transport a broad spectrum of en- the proximal tubules.20 Thus, Nherf3 and Mrp4 colocalize in dogenous and exogenous substrates, particularly nucleoside- the apical regions of kidney tubules (Figure 5A). based molecules.14,21 We chose adefovir for evaluating the in

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Figure 3. NHERF3 increases the stability of MRP4 protein in HEK293 cells. (A and B) The stability of MRP4 protein at the cell surface was examined using a biotinylation assay. After treatment with cycloheximide (0.1 mg/ml), protein samples were harvested at the indicated times. Immunoblotting for aldolase confirmed the absence of cytosolic proteins in the biotinylated samples. Results obtained in multiple experiments (n=4) are summarized in B. (C and D) The stability of MRP4 protein in total cell lysates was examined up to 36 hours. Results of multiple experiments (n=4) are summarized in D. Stability of MRP4 both on the cell surface and in the cytosol was increased by coexpression of NHERF3. For more accurate densitometric quantification of MRP4 levels, a 5-fold larger amount of protein sample was loaded in lanes without NHERF3 (aldolase immunoblot). *P,0.05; **P,0.01. CHX Tx, cycloheximide. vivo role of NHERF3-mediated MRP4 regulation for following plasma time concentration curve was increased from reasons. (1) Renal excretion is a major route for systemic clear- 64.668.4 to 84.7612.7 hzmg/ml (P,0.05) (Figure 6B), and ance of adefovir.22 (2)MRP4playsasignificant role in the systemic clearance of adefovir was decreased from 20.862.8 to renal elimination of adefovir.14 (3) Metabolic enzymes do 15.961.8 ml/h (Figure 6C). In addition, the tissue concentra- not play a major role in the systemic clearance of adefovir.23 tion of adefovir 3 hours after injection was 2.0-fold greater in 2 2 (4) As a hydrophilic molecule, adefovir is barely permeable the kidney of Nherf3 / mice (Figure 6D). Taken together, to cell membrane by itself,21 and (5) adefovir causes a dose- these results indicate that the Mrp4-mediated adefovir efflux 2 2 dependent nephrotoxicity.24 in renal tubules is significantly reduced in Nherf3 / mice. [3H]Adefovir was intravenously injected into wild-type 2 2 and Nherf3 / littermates, and pharmacokinetic parameters were analyzed using blood samples. As shown in Figure 6 DISCUSSION and Table 1, plasma concentrations of adefovir and some pharmacokinetic parameters were significantly altered in the Although several reports suggest that NHERF3 is involved in 2 2 Nherf3 / mice. For example, the plasma concentration of the regulation of many epithelial transporters, its precise 2 2 adefovir in Nherf3 / mice was 14.363.8 mg/ml compared physiologic roles are still poorly understood. In the normal 2 2 with 7.862.0 mg/ml in wild-type mice at 90 minutes after circumstances, the only discernible phenotype of Nherf3 / injection (Figure 6A). As a consequence, the area under the mice is mild hypercholesterolemia that is possibly because

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Figure 4. NHERF3 reduces internalization of surface MRP4. (A and B) The internalization of cell surface MRP4 protein was assayed in HEK293 cells with or without NHERF3 coexpression. Cell surface proteins were biotinylated and then internalized for indicated times at 37°C. Proteins remaining at the cell surface were stripped of biotin with the MESNA stripping buffer. Internalized, biotinylated proteins were immunoblotted. (A) A representative MRP4 immunoblot of internalization assays is shown. Lane 1, total cell surface MRP4; lane 2, MESNA stripping control; lanes 3–5, internalized MRP4 that remained in the cells after the indicated times. (B) Shown are the quantitative analyses of internalized MRP4 in cells with control or NHERF3 coexpression (n=4). MRP4 internalization was significantly reduced by NHERF3 coexpression. (C and D) The recycling of cell surface MRP4 protein was assayed in HEK293 cells with or without NHERF3 coexpression. Cell surface proteins were biotinylated and then internalized for 1 hour at 37°C. Proteins remaining at the cell surface were stripped of biotin with the MESNA stripping buffer. Nonrecycled, biotinylated proteins were collected at the indicated times after another round of MESNA treatment. (C) A representative MRP4 immunoblot of recycling assays in HEK293 cells is shown. Lane 1, total cell surface MRP4; lane 2, MESNA stripping control; lane 3, internalized MRP4 after 1 hour at 37°C; lanes 4–6, internalized MRP4 that remained in the cells after 1, 2, and 4 hours, respectively. The difference between lane 3 and lanes 4–6 represents recycled MRP4. (D) Shown are the quantitative analyses of recycled MRP4 in cells with control or NHERF3 coexpression (n=4). MRP4 recycling was not significantly altered by NHERF3 coexpression. *P,0.05; **P,0.01. of a dysregulation of the hepatic HDL receptor, scavenger re- was more clearly evident in kidney epithelia that natively ceptor class B type 1.11,25 In the present study, we provide express MRP4 on the BBM (Figure 5). Results from the MRP4 evidence that NHERF3 is crucial for the regulation of MRP4 internalization and recycling assays indicated that NHERF3 expression and activity both in cell culture model as well as in stabilizes cell surface MRP4 primarily by reducing its internal- vivo in the mouse kidney. Physical interactions between ization rather than augmenting its membrane insertion (Fig- NHERF3 and MRP4 were shown using three different exper- ure 4). Thus, the decrease in internalization-associated cellular imental approaches (Figure 1, Supplemental Table 1). Aug- degradation of MRP4 (Supplemental Figure 2) seems to be mented expression of NHERF3 was associated with elevated responsible for the increase in total MRP4. MRP4 levels in both human HEK293 cells and the mouse Interestingly, unlike NHERF3, several PDZ-containing kidney (Figures 2 and 5). Finally, the Mrp4-mediated systemic proteins have been reported to reduce the expression of 2 2 clearance of adefovir was significantly reduced in Nherf3 / MRP4 by facilitating internalization of cell surface MRP4. For mice (Figure 6, Table 1). example, NHERF1/EBP50, a member of the NHERF family The most notable finding of this study is that NHERF3 that also binds to MRP4 through its COOH-terminal PDZ enhanced the cell surface expression of MRP4. This finding motif, decreases the cellular and surface expression of MRP4.16

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2 2 Figure 5. Mrp4 levels are decreased in the kidney tubules of Nherf3 / mice. (A) Immunoﬂuorescence image of Mrp4 (green) and Nherf3 (red) in the kidney of Nherf3+/+ (wild-type) mice. Nherf3 colocalizes with Mrp4 at the apical membrane in the kidney tubules. 2 2 DAPI, 49,6-diamidino-2-phenylindole. (B) Immunohistochemical detection of Mrp4 in the kidney tissues of Nherf3+/+ and Nherf3 / mice. Mrp4 stained with 3,39-diaminobenzidine is localized at the apical membrane of proximal tubules of Nherf3+/+ mice (arrow). Note 2 2 that Mrp4 levels are reduced in the Nherf3 / mice (arrowheads). (C–F) The effects of Nherf3 deletion on the total and apical surface (BBM) expression of MRP4 in kidney tubules were examined. (C) A representative immunoblot of total (lysate) and apical surface (BBM) 2 2 MRP4 in the kidneys of Nherf3+/+ and Nherf3 / mice. (D) Densitometric analyses of total MRP4 protein levels in the lysate immunoblots (n=6). (E) Densitometric analyses of MRP4 protein levels in the BBM preparations (n=6). (F) Relative abundance of MRP4 at the BBM (corrected for the total MRP4 in the lysate blot; n=6). **P,0.01.

In addition, sorting nexin 27, a member of the sorting nexin Mrp4, ablation of Nherf3 did not affect NaPi-2a and NaPi-2c family of proteins known to be involved in the cellular traf- levelsinmiceonnormaldiets.19 One of the unique character- ficking and sorting of cargo proteins, also binds to cell surface istics of the NaPi protein is that its levels are regulated by the 26 MRP4 and increases its internalization. Therefore, it is pos- amount of Pi in the diet, where diets low in Pi resultinan 2 2 sible that NHERF3 expression may inhibit the associations upregulation of NaPi. When Nherf3 / mice were fed a low between these internalization-promoting PDZ proteins and Pi-containing diet, however, the upregulation of NaPi was at- MRP4. Especially in cells that express both NHERF1 and tenuated for NaPi-2c but not NaPi-2a.19 Interestingly, NaPi-2c NHERF3, such as kidney tubules, ablation of NHERF3 may interacts with the PDZ2 domain of NHERF3, whereas NaPi-2a play a particularly important role in disrupting the balance interacts with PDZ3. In this study, we have shown that MRP4 between the MRP4-NHERF1 and MRP4-NHERF3 associa- predominantly binds to the PDZ1 domain of NHERF3. As tions, increasing the internalization and degradation of MRP4. suggested in the case of NaPi,19 the association of different As mentioned earlier, NHERF3 has four PDZ domains, but transporters with different NHERF3 PDZ domains may de- no other distinct domain structures. The specific roles of each termine the differences in the regulation of transporters by this of the four PDZ domains in cell physiology are not currently adaptor protein. well understood. The two sodium-dependent phosphate As a nucleotide transporter, MRP4 has been shown to (Na/Pi) transporters (NaPi-2a and NaPi-2c) are known to function as an efflux pump for cAMP and at least in some bind to NHERF3 in kidney tubules.19 However, the patterns circumstances, be involved in the termination of cellular cAMP of NaPi-2a and NaPi-2c regulation by NHERF3 differ. Unlike signals.27–29 For example, the cAMP-induced activation of

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association in the gut and that the MRP4- NHERF3 interaction has the highest physiologic relevance in the kidney tubules. In the present study, ablation of Nherf3 caused a .80% increase in the plasma concentration of adefovir and a 24% reduction in its systemic clearance from mice. These results are highly consistent with results 2 2 obtained from Mrp4 / mice, in which the systemic clearance of adefovir was de- 2 2 creased by 24%.14 In Mrp4 / mice, the reduction in the systemic clearance of adefovir was mainly caused by a 62% decrease in the kidney tubular clearance.14 Although MRP4-mediated renal clearance is the major route for systemic elimination of adefovir, it should be remembered that there are other routes for adefovir clearance, such as glomerular filtration and non- MRP4–mediated tubular secretions. Therefore, a partial decrease in the systemic 2 2 Figure 6. Plasma clearance of adefovir is reduced in the Nherf3 / mice. (A) [3H]Adefovir clearance of adefovir may not be clinically 2 2 was administered intravenously to Nherf3+/+ and Nherf3 / littermates, and plasma meaningful in itself. However, the reduced concentrations of adefovir were measured at the indicated times. (B) Shown are the plasma and renal clearances of adefovir in 2 2 values of area under the plasma time concentration curve (AUC) from five littermate Mrp4 / mice were associated with accu- pairs. (C) Shown are the values of plasma clearance of adefovir from five littermate mulation of adefovir and its phosphory- pairs. (D) The concentration of adefovir in kidney tissue was measured 3 hours after lated toxic metabolites in the kidney,14 an , , administration of the drug using a different set of mice (n=5). *P 0.05; **P 0.01. observation that was reproduced in the 2 2 current study of Nherf3 / mice (Figure 6D). Nephrotoxicity is a major adverse Table 1. Pharmacokinetic parameters of adefovir in +/+ 2/2 Nherf3 and Nherf3 mice reaction of adefovir, which is often observed with the use of this drug.30 Thus, it is reasonable to conclude that NHERF3- Nherf3+/+ n Nherf32/2 n P Parameter ( =5) ( =5) Value mediated upregulation of MRP4 could play a critical role in 6 6 ke (1/h) 1.70 0.24 1.37 0.12 0.13 preventing toxicities associated with adefovir and possibly, 6 6 t1/2 (h) 0.44 0.05 0.52 0.04 0.09 other related MRP4 substrates. Together, the observations zm 6 6 a AUCinf (h g/ml) 64.62 8.37 84.66 12.72 0.03 that NHERF3 is most highly expressed in the kidney and Vd (ml) 12.4660.67 11.5860.93 0.19 2 2 that Nherf3 / mice exhibit a mild phenotype are consistent Cl (ml/h) 20.8162.75 15.9561.84 0.02a with the conclusion that a major physiologic/pharmacologic Data are mean6SEM. ke, elimination rate constant; t1/2,apparentterminal half-life; AUCinf, area under the concentration time curve with the last con- role of NHERF3 is the regulation of organic transporters in the centration extrapolated based on the elimination rate constant; Vd, volume kidney epithelia, especially those transporters involved in the of distribution; Cl, clearance. disposition and/or elimination of xenobiotic substrates, such aP,0.05. as MRP4.

CFTR is augmented by the MRP4 inhibitor MK571 in HT29- CONCISE METHODS 2 2 CL19A colonic epithelial cells and the intestines of Mrp4 / mice.29 Based on results from in vitro studies, Li et al.29 sug- Plasmids, Cell Culture, and Animals gested that NHERF3 may mediate the association between HEK293 cells were maintained in DMEM-HG (Invitrogen, Carlsbad, CFTR and MRP4 in the gut. As shown in Supplemental Figure CA) supplemented with 10% FBS. Plasmids were transiently trans- 3, however, levels of NHERF3 expression in the gut, especially fected into HEK293 cells using Lipofectamine Plus Reagent (Invi- in the colon, are very low compared with levels in the kidney. trogen). The pcDNA3.1/Hygro-MRP4 plasmid16 was subcloned into In addition, ablation of Nherf3 does not signiﬁcantly affect the pCMV-myc-N using the EcoRI/KpnI sites to generate pCMV-N-myc- cAMP-induced activation of Cftr in the mouse intestine and wt-MRP4. The MRP4-DETAL plasmid was generated by a PCR-based colon.10 These observations suggest that PDZ adaptor pro- site-directed mutagenesis. GST-MRP4-C40 containing the DNA se- teins other than NHERF3 may mediate the CFTR-MRP4 quence for the COOH-terminal 40 amino acids (amino acids

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1286–1325) of MRP4 was generated by inserting PCR fragments am- analyzed using an imaging software package (Multi Gauge Version 3.0; plified from pCMV-N-myc-wt-MRP4 into the bacterial expression FujiFilm, Valhalla, NY). vector pGEX-4T-1 (GE Healthcare) using the BamHI/EcoRI sites. Plasmids encoding for the four PDZ domains of NHERF3 fused Surface Protein Internalization and Recycling Assay with His6 were generated by inserting PCR fragments amplified The internalization assay of cell surface MRP4 was performed as from each PDZ domain (amino acids 1–105, 119–230, 231–348, described previously with a slight modification.32 Briefly, transfected and 365–474) into the bacterial expression vector pET-28c(+) cells were biotinylated using EZ-Link sulfo-NHS-SS-biotin (Pierce) (Novagen, Darmstadt, Germany) using the BamHI/KpnI sites. The for 30 minutes in the dark at 4°C. The cells were then warmed to 37°C open reading frame of human NHERF3 was PCR-amplified from for the indicated times to induce internalization, and the remain- pOTB7-hNHERF3 (Thermo Scientific, Waltham, MA) and subcloned ing disulphide bonds on sulfo-NHS-SS-biotinylated proteins were into pcDNA3.1(+)-N-hemagglutinin (HA) using the BamHI/XbaI stripped of biotin with four 15-minute washes in the sodium 2- 2 2 sites to generate pcDNA3.1(+)-N-HA-hNHERF3. Nherf3 / mice mercaptoethanesulfonate (MESNA) stripping buffer of 50 mM were handled and genotyped as previously described.10,11 Animal MESNA, 150 mM NaCl, 1 mM EDTA, 0.2% BSA, and 20 mM Tris use and welfare adhered to the National Institutes of Health Guide (pH 8.6). Subsequently, the cells were lysed with lysis buffer. The for the Care and Use of Laboratory Animals, and the protocol was lysates were centrifuged for 10 minutes (13,0003g), and the pellet reviewed and approved by the Committee on Animal Research at was discarded. Avidin solution (20 ml, Streptavidin beads; Pierce) Yonsei Medical Center, Seoul, Korea (no. 2010–0217). was added to the supernatant (200 mg protein per 200 ml lysis buffer), and the mixture was incubated for 3 hours with gentle agitation. Immunoblotting, Immunoprecipitation, and Surface Avidin-bound complexes were pelleted by centrifugation, washed Biotinylation three times with lysis buffer, and immunoblotted. For immunoblotting, cell and tissue lysates were suspended in an SDS Cell surfacerecycling of internalized MRP4 was assayed in HEK293 buffer and separated by SDS-PAGE. The separated proteins were cells as described previously.33 Because it is difficult to positively transferred and immobilized onto a nitrocellulose membrane and visualize the recycled MRP4, the amount of recycled MRP4 was es- blotted with anti-MRP4 (Alexis Biochemical, San Diego, CA), anti- timated by subtracting nonrecycled MRP4 from total internalized aldolase (Santa Cruz Biotechnology, Santa Cruz, CA), and anti-HA MRP4. Briefly, the plasma membrane proteins were biotinylated (Santa Cruz Biotechnology) monoclonal antibodies. Protein bands and then internalized for 1 hour at 37°C. Proteins remaining at the were detected by enhanced chemiluminescence (Amersham Bio- cell surface were stripped of biotin with the MESNA buffer as de- sciences). scribed earlier. To detect the recycling of internalized, biotinylated For immunoprecipitation, cell lysates were mixed with the anti- MRP4, cells were incubated at 37°C for indicated times and then HA polyclonal antibody (Santa Cruz Biotechnology) and incubated quickly cooled to 4°C. Biotinylated MRP4 that is recycled to the overnight at 4°C in a lysis buffer containing 50 mM TriszHCl (pH 7.4), plasma membrane was again stripped of biotin by the MESNA buffer 150 mM NaCl, 1% (vol/vol) Nonidet P-40, 0.25% (vol/vol) sodium washes. The remaining nonrecycled biotinylated MRP4 up to 4 hours deoxycholate, 1 mM EDTA, and a complete protease inhibitor mix- was measured, and the recycled MRP4 was calculated as described in ture (Roche Applied Science). Immune complexes were collected by Figure 4. Because the cellular degradation rate of MRP4 was very slow binding to mixed protein G/A beads and washed three times with lysis (Figure 3D), intracellular degradation of biotinylated MRP4 was not buffer before electrophoresis. considered. Surface biotinylation was performed as previously described.31 Briefly, transfected cells were washed with ice-cold PBS containing Immunohistochemistry +/+ 0.1 mM CaCl2 and 1 mM MgCl2, and the plasma membrane proteins For immunofluorescence detection, kidney tissues from Nherf3 2 2 were biotinylated by sulfo-NHS-SS-biotin (Pierce) in PBS for 30 mi- (wild type) and Nherf3 / mice were first embedded in tissue-freezing nutes at 4°C. After biotinylation, the cells were washed extensively medium (TissueTec OCT; Sakura), frozen in liquid nitrogen, and then with PBS to remove excess biotin. The cells were lysed, and an avidin- cut into 4-mm sections. The sections were fixed and permeabilized by containing solution (Streptavidin Agarose Resins; Pierce) was added incubation in ethanol-acetone solution (1:1; vol/vol) for 10 minutes at to the supernatant; the mixture was incubated at 4°C overnight. 220°C. Nonspecific binding sites were blocked by incubation for 1 Avidin-bound complexes were washed three times, eluted in SDS hour at room temperature with 0.1 ml PBS containing 5% goat serum, sample buffer, resolved by SDS-PAGE, electrotransferred, and im- 1% BSA, and 0.1% gelatin (blocking medium). After blocking, the munoblotted with appropriate antibodies. sections were stained by incubating them with anti-MRP4 (Alexis To determine the stability of MRP4 on the cell surface and in total Biochemical) and anti-NHERF3 (Novous Biologic) antibodies fol- cell lysates, cells were treated with cycloheximide (0.1 mg/ml; Sigma- lowed by fluorophore-tagged secondary antibodies. DNA was stained Aldrich) to prevent additional protein synthesis 72 hours after with 49,6-diamidino-2-phenylindole. Images were obtained with a transfection with pCMV-N-myc-wt-MRP4. Surface proteins were Zeiss LSM 710 confocal microscope. biotinylated, and cells were harvested at 0, 12, 24, and 36 hours after For detection of Mrp4 by 3,39-diaminobenzidine staining, kidney the cycloheximide treatment. MRP4 on the cell surface and in lysates tissue was fixed in formalin, and paraffin blocks were prepared. Sec- was detected by immunoblotting with anti-Myc antibody (Santa Cruz tions were incubated with the anti-MRP4 antibody (Alexis Biochem- Biotechnology). The density of signals on the immunoblots was ical) and then followed by anti-rat IgG (H+L) conjugated with

734 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 726–736, 2014 www.jasn.org BASIC RESEARCH horseradish peroxidase (Thermo Scientific); 3,39-diaminobenzidine Statistical Analyses signals were developed with a peroxidase substrate kit (Vector The results of multiple experiments are presented as the mean6SEM. Laboratories, Burlingame, CA) according to the manufacturer’s Statistical analyses of in vitro data were performed with t test or instructions. ANOVA followed by Tukey’s multiple comparison test as appropriate. Pharmacokinetic parameters were determined using WinNonlin fi Pull-Down Assays (Pharsight), and testing for statistical signi cance was performed All recombinant fusion proteins were produced in BL-21 (DE3) E. coli using the paired t test. P values,0.05 were considered statistically fi strain. The synthesis of GSTand His6 fusion proteins was induced by signi cant. isopropyl b-D-1-thiogalactopyranoside (1 mM) at 37°C. Recombi- nant proteins were purified with glutathione-Sepharose beads (Amersham Bioscience) or a nickel-nitrilotriacetic acid protein pu- ACKNOWLEDGMENTS rification system (Qiagen) according to the manufacturer’sinstruc- tions. Eluted His fusion proteins were mixed with the GST fusion 6 We thank the Yonsei-Carl Zeiss Advanced Imaging Center for tech- recombinant proteins bound to glutathione-Sepharose. After over- nical assistance. night incubation at 4°C, the bead-bound complexes were washed, This work was supported by National Research Foundation, eluted in SDS sample buffer, and immunoblotted. Ministry of Science, ICT and Future Planning, Korea Grants 2013R1A3A2042197 and 2007-0056092 (to M.G.L.); Deutsche For- Adefovir Cell Accumulation Assay schungsgemeinschaftGrant SFB621/C9 (to U.S.); Canadian Institutes of HEK293cellswereseededin triplicatein 12-well platesand transfected Health Research Grant MOP-106513 (to S.P.C.C.); and National with pCMV-N-myc-wt-MRP4. Cells were exposed to the dipivoxil Project for Personalized Genomic Medicine, Korea Health 21 Research ester form of [3H]adefovir ([3H]bis(POM)PMEA; specific activity 5.5 and Development Project, Ministryof Health and Welfare,KoreaGrant Ci/mmol; Moravek Biochemicals, Brea, CA) mixed with a 100-fold A111218-PG03 (to M.G.L.). greater amount of nonradiolabeled adforvir dipivoxil (total concentration of 1 mM; Sigma-Aldrich) for 2 hours at 37°C in HBSS, and the transport reaction was terminated by washing cells with ice-cold PBS and solubilizing in 1 M NaOH. Radioactivity was measured in a liquid DISCLOSURES scintillation counter (Beckman Coulter, Inc., Fullerton, CA) after the None. addition of scintillation cocktail (PerkinElmer Life and Analytical Sciences). Data were expressed relative to the amount of cell protein (counts per minute per milligram protein) in each sample, which was REFERENCES determined by the Bradford colorimetric method. 1. 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736 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 726–736, 2014

SUPPLEMENTAL INFORMATION

NHERF3/PDZK1 is critical for MRP4 expression and MRP4-mediated drug efflux in the kidney

Joonhee Park1,2, Jin-Oh Kwak1, Brigitte Riederer3, Ursula Seidler3, Susan P. C. Cole4, Hwa Jeong Lee2,*, and Min Goo Lee1,*.

1Department of Pharmacology and Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752, Korea 2Division of Life and Pharmaceutical Sciences and College of Pharmacy, Ewha Womans University, Seoul 120-750, Korea 3Department of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, Hannover, Germany 4Department of Pathology & Molecular Medicine, and Division of Cancer Biology &

Genetics, Queen’s University, Kingston, Canada

1 SUPPLEMENTAL METHODS

Yeast two-hybrid assay

The L40 yeast strain harboring the reporter genes HIS3 and LacZ, under control of the upstream LexA DNA-binding domain was used. To semi-quantify the interaction, HIS3 activity was determined by the percentage of yeast colonies growing on histidine-lacking medium. cDNA sequences containing the COOH-terminal 50 amino acids of MRP4 were amplified by PCR and subcloned into pBHA construct (a bait vector containing the LexA

DNA-binding domain). The pGAD10 (a prey vector, Clontech) constructs containing the

PDZ domains S-SCAM-PDZ1-2, NHERF1-PDZ1, NHERF1-PDZ2, full-length NHERF2 and full-length NHERF3 were generated.

Real-time quantitative PCR analysis

Total RNA was extracted from HEK 293 cells by using Purelink RNA minikit (Ambion,

Carlsbad), and cDNA was synthesized with an AccuScript High Fidelity 1st Strand cDNA

Synthesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. Real- time PCR was performed with an ABI PRISM 7300 system (Applied Biosystems, Foster City,

CA). The reaction was measured by detecting the binding of TaqMan probe to double-strand

DNA. Amplification was performed with the following cycling condition: 50 °C for 2 min;

95 °C for 10 min; 40 cycles of 95 °C for 15 s and 60 °C for 1 min. According to the comparative cycle threshold (Ct) method, gene expression was normalized to the expression of the housekeeping gene GAPDH, yielding a ΔCt value. Primers used for real-time RT-PCR were purchased from Applied Biosystems (catalog number: 4333764F for GAPDH,

Hs00988734_m1 for MRP4 ).

2 Protein degradation assay with lysosome and proteasome inhibitors

HEK 293 cells were treated with cycloheximide (Sigma, 0.1 mg/mL) to prevent further protein synthesis 72 h after transfection with pCMV-N-myc-wt-MRP4, and were harvested

36 h after the cycloheximide treatment. Some cells were treated with bafilomycin A (Sigma;

0.1 μg/mL) (an inhibitor of vacuolar H+-ATPase) or the proteasome inhibitor MG132 (Sigma;

2 μg/mL) in addition to cycloheximide. MRP4 in lysates were detected by immunoblotting with anti-myc antibody (Santa Cruz Biotechnology). The density of signals on the immunoblots was analyzed using an imaging software package (Multi Gauge Version 3.0,

Fujifilm, Valhalla, NY).

Supplemental Table 1. Results from a yeast two-hybrid assay.

PDZ adaptor ABCA1 ABCA7 ABCC2/MRP2 ABCC4/MRP4 proteins His ß-gal His ß-gal His ß-gal His ß-gal

S-SCAM PDZ1-2 + + + + ++ ++ ++ ++

PDZ1 ------++ ++ NHERF1 PDZ2 ------

NHERF2 PDZ1-2 ------+ -

NHERF3 PDZ1-4 - - - - ++ ++ +++ +++

Polypeptides corresponding to the COOH-terminal 50 amino acids of human ABCA1, ABCA7, ABCC2/MRP2, and ABCC4/MRP4 were tested for their ability to bind the PDZ-based adaptor proteins that were found in epithelial tissues using yeast two-hybrid assays. The intensity of the binding was determined by the induction of the yeast reporter genes HIS3 (HIS) and -galactosidase (-gal). His activity (measured as percentage of colonies growing on histidine-lacking medium) was rated as follows: +++ (>40%), ++ (10-40%), + (<10%), - (no significant growth); -gal activities (time needed for yeast colonies to turn blue in X-gal filter lift assays at room temperature) were as follows: +++ (<30 min), ++ (30-90 min), + (>90 min), - (no detection).

mRNA

-1

-2

-3 Ct (GAPDH-MRP4) Ct  -4

-5 MRP4 MRP4+NHERF3

Supplemental Figure 1. mRNA levels of MRP4 using a quantitative real-time PCR. HEK293 cells were transfected with plasmids encoding MRP4 and/or NHERF3. The relative MRP4 mRNA levels are presented using the cycle threshold values normalized to those of the housekeeping gene

GAPDH (Ct). Lines represent means ± SEM (n=6).

Myc-MRP4 ++++ ++++ ** NHERF3 ---- ++++ Cycloheximide -+++ -+++ 120 Bafilomycin A --+- --+- 100 MG132 ---+ ---+ kDa 80 -225 60 (%) -150 40

MRP4 (Anti-Myc) 20

relative expression level level expression relative 0 -76 Myc-MRP4 ++++++++ NHERF3 NHERF3 - - - -++++ Cycloheximide -+++-+++ -52 Bafilomycin A --+---+- -38 MG132 ---+---+ Aldolase

Supplemental Figure 2.

NHERF3 reduces the lysosomal degradation of MRP4. The stability of MRP4 in the total cell lysates was examined in the presence of inhibitors of lysosomal (bafilomycin A, 0.1 μg/mL) and proteasomal (MG132, 2 μg/mL) degradation. Protein samples were prepared 36 h after the inhibitor treatments. A representative immunoblot is shown in panel A, and the results of multiple experiments (n=4) are summarized in the bar graph shown in panel B. Bafilomycin inhibited the degradation of MRP4 by 39% and 23% in cells without and with NHERF3 coexpression. The effect of bafilomycin was significantly reduced by NHERF3 co-expression

(**p<0.01). For an accurate densitometric quantification of MRP4 levels, a 5-fold larger amount of protein sample was loaded in lanes without NHERF3 (see aldolase immunoblot).

+/+ -/- +/+ -/- +/+ -/- +/+ -/- Nherf3

-76 kDa NHERF3

Supplemental Figure 3.

Expression of Nherf3 in the ileum, colon, kidney and liver of Nherf3+/+ and Nherf3-/- mice. The same amount of protein sample (30 g) was loaded in each tissue lane. A protein sample from HEK 293 cells transfected with pcDNA3.1-HA-NHERF3 (3 g) was used as a control.

Nherf3 was predominantly expressed in the kidney tissue of Nherf3+/+ (wild-type) mice.