Liver X receptor-activating ligands modulate renal and intestinal sodium–phosphate transporters
2011; Elsevier BV; Volume: 80; Issue: 5 Linguagem: Inglês
10.1038/ki.2011.159
ISSN1523-1755
AutoresYupanqui Caldas, Héctor Giral, Michael A. Cortázar, Eileen Sutherland, Kayo Okamura, Judith Blaine, Vı́ctor Sorribas, Hermann Koepsell, Moshe Levi,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoCholesterol is pumped out of the cells in different tissues, including the vasculature, intestine, liver, and kidney, by the ATP-binding cassette transporters. Ligands that activate the liver X receptor (LXR) modulate this efflux. Here we determined the effects of LXR agonists on the regulation of phosphate transporters. Phosphate homeostasis is regulated by the coordinated action of the intestinal and renal sodium–phosphate (NaPi) transporters, and the loss of this regulation causes hyperphosphatemia. Mice treated with DMHCA or TO901317, two LXR agonists that prevent atherosclerosis in ApoE or LDLR knockout mice, significantly decreased the activity of intestinal and kidney proximal tubular brush border membrane sodium gradient-dependent phosphate uptake, decreased serum phosphate, and increased urine phosphate excretion. The effects of DMHCA were due to a significant decrease in the abundance of the intestinal and renal NaPi transport proteins. The same effect was also found in opossum kidney cells in culture after treatment with either agonist. There was increased nuclear expression of the endogenous LXR receptor, a reduction in NaPi4 protein abundance (the main type II NaPi transporter in the opossum cells), and a reduction in NaPi co-transport activity. Thus, LXR agonists modulate intestinal and renal NaPi transporters and, in turn, serum phosphate levels. Cholesterol is pumped out of the cells in different tissues, including the vasculature, intestine, liver, and kidney, by the ATP-binding cassette transporters. Ligands that activate the liver X receptor (LXR) modulate this efflux. Here we determined the effects of LXR agonists on the regulation of phosphate transporters. Phosphate homeostasis is regulated by the coordinated action of the intestinal and renal sodium–phosphate (NaPi) transporters, and the loss of this regulation causes hyperphosphatemia. Mice treated with DMHCA or TO901317, two LXR agonists that prevent atherosclerosis in ApoE or LDLR knockout mice, significantly decreased the activity of intestinal and kidney proximal tubular brush border membrane sodium gradient-dependent phosphate uptake, decreased serum phosphate, and increased urine phosphate excretion. The effects of DMHCA were due to a significant decrease in the abundance of the intestinal and renal NaPi transport proteins. The same effect was also found in opossum kidney cells in culture after treatment with either agonist. There was increased nuclear expression of the endogenous LXR receptor, a reduction in NaPi4 protein abundance (the main type II NaPi transporter in the opossum cells), and a reduction in NaPi co-transport activity. Thus, LXR agonists modulate intestinal and renal NaPi transporters and, in turn, serum phosphate levels. 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In this study, we document a novel role for the LXR-activating ligands, DMHCA and TO901317, in the inhibition of the major renal and intestinal Na-Pi transporters, resulting in a decrease of serum phosphate levels. This study along with our previous findings of reduction of atherosclerosis, suggests that LXR-activating ligands, such as DMHCA, capable of inducing reverse cholesterol transport without the lipogenic effects might be a promising therapeutic agent in the prevention of hyperphosphatemia and its cardiovascular consequences. In our initial studies, we determined the effects of both T0901317 and DMHCA on potential LXR targets in the kidney and the intestine.22.Kratzer A. Buchebner M. Pfeifer T. et al.Synthetic LXR agonist attenuates plaque formation in apoE-/- mice without inducing liver steatosis and hypertriglyceridemia.J Lipid Res. 2009; 50: 312-326Crossref PubMed Scopus (116) Google Scholar We found that both T0901317 and DMHCA increased ABCA1 and ABCG1 mRNA abundance in the kidney and the ileum (Figure 1). As previously shown in hepatocytes and macrophages,32.Quinet E.M. Savio D.A. Halpern A.R. et al.Gene-selective modulation by a synthetic oxysterol ligand of the liver X receptor.J Lipid Res. 2004; 45: 1929-1942Crossref PubMed Scopus (115) Google Scholar T0901317 also increased sterol-regulatory-element-binding protein 1c, FAS, and SCD-1 mRNA abundance in the kidney and the ileum; however, the effects of DMHCA on sterol-regulatory-element-binding protein 1c, FAS, and SCD-1 were minimal (Figure 1). These results were also in agreement with the previous data, showing the activation of LXR target genes in the kidney when the mice were treated with TO901317.37.Zhang Y. Zhang X. Chen L. et al.Liver X receptor agonist TO-901317 upregulates SCD1 expression in renal proximal straight tubule.Am J Physiol Renal Physiol. 2006; 290: F1065-F1073Crossref PubMed Scopus (43) Google Scholar Upregulation of ABCA1 and stearoyl-CoA desaturase 1 protein in kidney and ileum was also confirmed by western blotting and immunofluorescence microscopy (Figure 1c–e). No significant effects were observed in the expression of carbohydrate-responsive-element-binding protein and liver pyruvate kinase. In kidney BBM, sodium-dependent Pi uptake was reduced by 20% and 15% in the DMHCA- and TO901317-treated mice, respectively. Treatment with DMHCA had even more marked effects in the ileum where sodium-dependent Pi uptake was reduced by 56% and by 51% when mice were treated with TO901317 (Figure 2). In all cases, sodium-gradient-independent Pi transport was measured by using choline chloride rather than sodium chloride. An average of 5% of total uptake in the kidney BBM and an average of 14% of the total uptake in the ileum BBM was due to Na-independent Pi uptake, and this Na-independent component of total uptake was modified by neither DMHCA nor TO901317. Therefore, both drugs are inhibiting the transport of Pi in both epithelia. The DMHCA- or TO901317-induced decrease in renal and intestinal NaPi transport activity was paralleled by an increase in urinary Pi excretion and a small but significant decrease in serum Pi concentration (Figure 3). Changes in the urinary glucose or protein excretion were not detectable after treatment; however, a small significant decrease in the urine pH was detected in the treated mice (Supplementary Figure A online). Download .jpg (.05 MB) Help with files Supplementary Figure To determine the serum levels of Pi, Ca, FGF23, and parathyroid hormone, blood was collected when animals were killed. Treatment with TO901317 caused a 20% decrease in serum phosphate concentration, with a smaller reduction of 14% after treatment with DMHCA (Figure 3a). This is associated with an increase of ∼30% in the urine phosphate excretion with either compound (Figure 3b). No significant changes in the serum calcium concentration were observed (Figure 3c). Additionally treatment with TO901317 caused a 64% increase in serum FGF23 concentration, with a smaller increase of 46% after treatment with DMHCA (Figure 4a). No significant changes in the serum parathyroid hormone levels were observed after treatment with either compound (Figure 4b). To determine the mechanism of the LXR-agonist-mediated decrease in renal BBM NaPi cotransport activity, we determined the abundance of BBM NaPi cotransporters by western blotting. We found that treatment with DMHCA or TO901317 caused significant decreases in the protein abundance of all of the three renal transporters, namely, NaPi-2a, NaPi-2c, and Pit-2 (Figure 5a). The effect of these compounds on the renal NaPi transporter abundance was independent of alterations in the protein abundance of the PDZ-domain-interacting proteins, namely, NHERF-1 and PDZK-1 (Figure 5c). These proteins are well known for the regulation of the renal phosphate transporters. In addition, we found that DMHCA did not alter renal Na-glucose SGLT-2 protein levels, whereas TO901317 caused a decrease of SGLT-2 protein abundance (Supplementary Figure C online). Measurements of the mRNA abundance of these transporters in parallel samples of the kidney cortex by real-time quantitative PCR indicate that these ligands decrease the mRNA abundance of NaPi-2a and NaPi-2c but not Pit-2 (Figure 5b). Treatment with DMHCA or TO901317 also caused a significant decrease in ileum BBM NaPi-2b protein abundance, with no significant effects on Pit-1 protein abundance (Figure 6a). The effect of these ligands on the intestinal NaPi-2b transporter abundance was independent of the alterations in the protein abundance of the PDZ-domain-interacting proteins, namely, NHERF-1 and PDZK-1 (Figure 6c). In addition, we found no effects of these LXR agonists on the intestinal Na-glucose SGLT1 transporter protein levels (Supplementary Figure B online). The effect of DMHCA or TO901317 on NaPi-2b protein abundance was associated with a parallel decrease in NaPi-2b mRNA; 75% after treatment with TO901317 and 50% with DMHCA (Figure 6b). To determine whether DMHCA or TO901317 has direct modulatory effects on NaPi cotransport activity, independent of systemic metabolic and hormonal factors, we studied their effects in OK cells. OK cells are a well-established model of the renal proximal tubule, which expresses the endogenous type IIa NaPi cotransporter, also known as NaPi-4. Treatment of OK cells with DMHCA induced translocation of LXR to the nucleus (Figure 7a). This effect was also observed with TO901317 compound (data not shown). Both LXR agonists caused a dose-dependent decrease in OK cell NaPi cotransport activity (Figure 7b), measured by whole-confluent cells 32P uptake. Western blot of apical membranes isolated from OK cells and immunofluorescence studies indicate that these agonists caused parallel decreases in the apical membrane NaPi-4 protein abundance (Figure 7c, d and Supplementary Figure D online). Hyperphosphatemia is a major risk factor for cardiovascular disease. Any interventions that decrease serum Pi concentration and possibly prevent the cardiovascular consequences of hyperphosphatemia are welcomed. In this study, we show that TO901317 and DMHCA, two LXR-activating ligands, which have been previously described and shown to prevent atherosclerosis in ApoE-knockout mice,22.Kratzer A. Buchebner M. Pfeifer T. et al.Synthetic LXR agonist attenuates plaque formation in apoE-/- mice without inducing liver steatosis and hypertriglyceridemia.J Lipid Res. 2009; 50: 312-326Crossref PubMed Scopus (116) Google Scholar also causes a significant decrease in serum Pi concentration by inhibiting the activity of the renal and intestinal NaPi transporters. The effects of TO901317 or DMHCA in decreasing renal proximal tubular BBM NaPi cotransport activity are reflected by an increase in the urinary Pi excretion and significant decreases in the abundance of NaPi-2a, NaPi-2c, and Pit-2. Although the decreases in NaPi-2a and NaPi-2c protein abundance may be mediated by the transcriptional mechanisms, the decrease in Pit-2 protein abundance seems to be independent of Pit-2 transcriptional regulation. These results are associated with a significant increase of the FGF23 serum levels after treatment with LXR agonist. FGF23 is a well-known phosphaturic hormone38.Komaba H. Fukagawa M. FGF23-parathyroid interaction: implications in chronic kidney disease.Kidney Int. 2010; 77: 292-298Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar capable of downregulating the expression of the renal NaPi transporters. In addition, it is also important to mention that the protein levels of the Na-glucose transporters SGLT2 in kidney BBM are also reduced after treatment with TO901317, with no significant effects with DMHCA. It is now quite well established that NaPi–PDZ-type (PSD-95, discs-large, and ZO-1) protein interactions are important for the regulation of NaPi-2a and NaPi-2c protein expression in the proximal tubular apical BBM.39.Hernando N. Deliot N. Gisler S.M. et al.PDZ-domain interactions and apical expression of type IIa Na/P(i) cotransporters.Proc Natl Acad Sci USA. 2002; 99: 11957-11962Crossref PubMed Scopus (163) Google Scholar, 40.Lanaspa M.A. Giral H. Breusegem S.Y. et al.Interaction of MAP17 with NHERF3/4 induces translocation of the renal Na/Pi IIa transporter to the trans-Golgi.Am J Physiol Renal Physiol. 2007; 292: F230-F242Crossref PubMed Scopus (48) Google Scholar, 41.Villa-Bellosta R. Barac-Nieto M. Breusegem S.Y. et al.Interactions of the growth-related, type IIc renal sodium/phosphate cotransporter with PDZ proteins.Kidney Int. 2008; 73: 456-464Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar Our studies indicate that the effects of these LXR agonists on NaPi-2a, NaPi-2c, and NaPi-2b protein abundance are independent of alterations on BBM expression of the PDZ proteins, NHERF-1 or PDZK-1. However, LXR-induced modifications in NHERF-1 or PDZK-1–NaPi interactions cannot be ruled out. DMHCA and TO901317 also have marked effects in inhibiting intestinal BBM NaPi cotransport activity. This inhibition occurs via a major decrease in the BBM protein abundance of the major intestinal NaPi transporter NaPi-2b. There is also a parallel decrease in NaPi-2b mRNA abundance, which indicates that DMHCA and TO901317 may downregulate NaPi-2b via transcriptional mechanisms. To determine whether LXR activation has also direct effects in modulating NaPi cotransport activity, we have performed parallel studies in OK cells, a well-established model of the renal proximal tubule.39.Hernando N. Deliot N. Gisler S.M. et al.PDZ-domain interactions and apical expression of type IIa Na/P(i) cotransporters.Proc Natl Acad Sci USA. 2002; 99: 11957-11962Crossref PubMed Scopus (163) Google Scholar, 42.Sorribas V. Markovich D. Hayes G. et al.Cloning of a Na/Pi cotransporter from opossum kidney cells.J Biol Chem. 1994; 269: 6615-6621Abstract Full Text PDF PubMed Google Scholar We found that both of the LXR agonists cause a dose-dependent decrease in OK cells NaPi cotransport activity by decreasing the apical BBM abundance of the OK cell type II NaPi cotransporter NaPi-4 protein. Additional studies indicate that DMHCA as well as TO901317 induce increased nuclear expression of LXR protein, further supporting that these drugs are LXR-activating ligands; this is correlated with the upregulation of LXR target genes, including ABCA1 (data not shown), after treatment of the OK cells with this agonist. Our study demonstrates that LXR has a novel role in inhibiting renal and intestinal NaPi transporters and decreasing serum Pi concentration through multiple mechanisms, including phosphatonin modulation and direct control of Pi transporter abundance in epithelia. This effect of LXR, along with its well-established effects in mediating reverse cholesterol transport, inhibiting inflammatory cytokines, and preventing atherosclerosis, establishes it as a major target for the prevention of hyperphosphatemia and the associated cardiovascular complications. Male C57Bl/6 mice were purchased from Jackson Laboratory (Bar Harbor, ME) and maintained in a clean environment on a regular 12-h light–12-h dark cycle. Before the initiation of the corresponding diets, mice were kept on a standard laboratory chow diet (Harland Teklad 2019 chow diet) with 0.9% of Ca and 0.7% of Pi. Male C57Bl/6 mice were fed chow diet containing (a) no ligands, or (b) DMHCA (80 mg/kg body weight/day), or (c) T0901317 (35 mg/kg body weight/day) for 15 days. Diets were supplemented with the respective LXR ligand at a level sufficient to provide the appropriate mg/kg food dose on consumption of a 5 g diet by a 25 g mouse/day. Body weight and food intake were monitored regularly. We studied n=24 mice in each treatment group: 12 mice for renal and intestinal BBM isolation and 12 mice for renal and intestinal RNA isolation. On the 14th day of the treatment, the mice were placed in metabolic balance cages for urine collection. Animal experiments were approved by the Institutional Animal Care and Research Advisory Committee of the University of Colorado at Denver. OK proximal tubule cells were grown in DMEM-F-12 (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, 50 U/ml penicillin, and 50 μg/ml streptomycin. For experimental work, cells were seeded on porous membrane inserts (Corning, Lowell, MA). After confluency, cells were placed in DMEM-F-12 supplemented with 0.2% fetal bovine serum and penicillin/streptomycin to get them quiescent for 24–48 h before treatment. Cells were treated with different concentration of LXR agonist, either DMHCA or TO901317 (stocks were resuspended in dimethyl sulfoxide). Working solutions of these agonists were prepared in DMEM-F-12 supplemented with 0.2% fetal bovine serum and penicillin/streptomycin. Cells were treated with 1:1000 dilution of dimethyl sulfoxide (control) or DMHCA or TO901317 for 24 h. All chemicals were obtained from Sigma (Saint Louis, MI), except when noted. A polyclonal rabbit anti-NaPi-IIa antibody was generated by Affinity Bio Reagents (Golden, CO) and used at 1:5,000 for western blotting.13.Breusegem S.Y. Takahashi H. Giral-Arnal H. et al.Differential regulation of the renal sodium-phosphate cotransporters NaPi-IIa, NaPi-IIc, and PiT-2 in dietary potassium deficiency.Am J Physiol Renal Physiol. 2009; 297: F350-F361Crossref PubMed Scopus (64) Google Scholar A rabbit anti-NaPi-IIc antibody was custom-made by Davids Biotechnologie (Regensburg, Germany), as previously described,13.Breusegem S.Y. Takahashi H. Giral-Arnal H. et al.Differential regulation of the renal sodium-phosphate cotransporters NaPi-IIa, NaPi-IIc, and PiT-2 in dietary potassium deficiency.Am J Physiol Renal Physiol. 2009; 297: F350-F361Crossref PubMed Scopus (64) Google Scholar and was used at 1:1000 for western blotting. The polyclonal rabbit anti-NaPi-2b, anti-PiT1, and anti-PiT2 antibodies were also custom generated by Davids Biotechnologie (Regensburg, Germany) as described before,14.Giral H. Caldas Y. Sutherland E. et al.Regulation of the Rat Intestinal Na-dependent Phosphate Transporters by Dietary Phosphate.Am J Physiol Renal Physiol. 2009; 297: F1466-F1475Crossref PubMed Scopus (125) Google Scholar and was used at 1:1,000 dilution for western blotting. The goat anti-LXRα/β was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The rabbit anti-ABCA1 was purchased from Novus Biologicals (Littleton, CO), and the rabbit anti-stearoyl-CoA desaturase 1 was purchased from Cell Signaling Technology (Danvers, MA). The rabbit anti-Na/H exchange regulatory factor-1 antibody was purchased from Sigma. The rabbit anti-PDZK1 was a kindly gift from Dr David Silver (Columbia University). Mice were anesthetized via an intraperitoneal injection of 50 mg/kg pentobarbital sodium (Pentothal, Abbott Laboratories, Abbott Park, IL). After clamping of the renal vessels, blood was drawn for biochemical analysis, the kidneys and the ileum were removed, and the ileum mucosa was scraped for BBM isolation. Kidney slices from two mice were combined in 7.5 ml isolation buffer consisting of 15 mmol/l Tris·HCl (pH 7.4), 300 mmol/l mannitol, 5 mmol/l ethylene glycol tetraacetic acid, and 1 Roche Complete inhibitor tablet per 250 ml buffer. The kidney slices were homogenized using a Potter-Elvejham homogenizer with 8–10 rapid strokes and transferred to a chilled capable tube. Kidney residues remaining on the homogenizer were rinsed off with 10 ml water that was then added to the kidney homogenate. BBM was prepared by a double Mg2+ precipitation. For the first Mg2+ precipitation, MgCl2 was added to the homogenate (final concentration of 15 mmol/l), and the homogenate was shaken every 5 min on ice for 20 min before centrifugation at 2500 g for 15 min. The supernatant was subjected to a second Mg2+ precipitation; and from the resulting supernatant, the BBM was recovered by centrifugation at 38,000 g for 40 min. The BBM was resuspended, and its protein content quantified. Ileum BBM was similarly isolated by double Mg2+ precipitation as describe above. BBM of the OK cells was isolated by Mg2+ precipitation. Briefly, OKP cells were grown to confluence in 100 mm dishes. At 24 h before the experiment, the cells were placed in DMEM medium containing 0.2% fetal bovine serum to synchronize them. Cells were incubated with control media (1:1000 dimethyl sulfoxide) or DMHCA or TO901317 for 24 h. After treatment, the cells were washed in ice cold PBS and scraped into isolation buffer (15 mmol/l Tris (pH7.4), 300 mmol/l mannitol, 5 mmol/l ethylene glycol tetraacetic acid, and one Mini-Complete tablet (Roche)) on ice. The cells were then homogenized by aspirating 30 times through a 23-gauge needle. MgCl2 was added to a final concentration of 15 mmol/l, and the homogenate was shaken on ice for 20 min. The homogenate was centrifuged at 2500 × g at 4 C for 15 min. The supernatant was removed and spun at 60,000 × g for 40 min. The final pellet was resuspended in isolation buffer. Phosphate transport from kidney or ileum was measured by rapid filtration of radioactive 32Pi uptake in freshly isolated BBM vesicles.43.Sorribas V. Lotscher M. Loffing J. et al.Cellular mechanisms of the age-related decrease in renal phosphate reabsorption.Kidney Int. 1996; 50: 855-863Abstract Full Text PDF PubMed Scopus (38) Google Scholar The BBM and the uptake solution were incubated for 10 s at 25°C for kidney and 30 s at 37°C for ileum. Phosphate transport in OK cells was measured by radioactive. 32Pi uptake in treated confluent cells for 6 min at 25°C, as described.14.Giral H. Caldas Y. Sutherland E. et al.Regulation of the Rat Intestinal Na-dependent Phosphate Transporters by Dietary Phosphate.Am J Physiol Renal Physiol. 2009; 297: F1466-F1475Crossref PubMed Scopus (125) Google Scholar, 40.Lanaspa M.A. Giral H. Breusegem S.Y. et al.Interaction of MAP17 with NHERF3/4 induces translocation of the renal Na/Pi IIa transporter to the trans-Golgi.Am J Physiol Renal Physiol. 2007; 292: F230-F242Crossref PubMed Scopus (48) Google Scholar, 44.Breusegem S.Y. Halaihel N. Inoue M. et al.Acute and chronic changes in cholesterol modulate Na-Pi cotransport activity in OK cells.Am J Physiol Renal Physiol. 2005; 289: F154-F165Crossref PubMed Scopus (27) Google Scholar Blood samples were collected in heparin-containing tubes during sacrifice. The 24 h and spot urine was collected in animals treated for 2 weeks. Plasma obtained after centrifugation and urine samples were analyzed for phosphate (Pi) concentrations by using the commercial kit Stanbio Liqui-UV (Stanbio; Boerne, TX). Creatinine concentration in urine was determined using QuantiChrom Creatinine Assay (BioAssay Systems; Hayward, CA). FGF-23 (C-Term) and intact parathyroid hormone were determined with specific ELISA kits from Immunotopics (San Clemente, CA). n=10–12 animals per group was used in these assays. BBM proteins (20 or 30 μg) were separated by 10% SDS-polyacrylamide gel electrophoresis, and transferred onto nitrocellulose membranes. Membranes were blocked with 5% milk in PBS Tween 20 before incubation with primary antibodies diluted in PBTS overnight at 4 °C. After washes with phosphate buffered saline and Tween 20, membranes were incubated with Licor-conjugated (LI-COR, Lincoln, NE) donkey secondary antibodies diluted 1:5,000 for 1 h. Membranes were scanned using Licor system. Densitometry data are presented as average ± s.d. Total RNA was isolated from kidney cortex and ileum using the Qiagen RNeasy Mini Kit, and complementary DNA was synthesized using reverse transcription reagents from Bio-Rad (Hercules, CA). The mRNA level was quantified using a Bio-Rad iCyCler real-time PCR machine. Cyclophilin A was used as an internal control, and the amount of RNA was calculated by the comparative threshold cycle method as recommended by the manufacturer. All of the data were calculated from duplicate reactions of three different experiments. Cells were washed with PBS before blocking for 30 min with 5% goat serum and permeabilized with 0.1% saponin in PBS. Cells were then incubated overnight at 4°C with primary antibodies. After washing with saponin solution, sections were incubated with secondary fluorescent goat antibodies for 1 h. After washing three times, the cells were mounted in Vectashield (Vector Labs, Burlingame, CA). Confocal images were acquired on a Zeiss 510 NLO-META LSM laser scanning confocal microscope (Carl Zeiss, Thornwood, NY), and the Olympus Fluoview 1000 confocal microscope (Olympus, Center Valley, PA). Data are expressed as means±s.d., *P<0.05, **P<0.005, and ***P<0.001. Data were analyzed for statistical significance by unpaired Student's t-test or one-way analysis of variance. We thank Makoto Miyazaki for help and advice. This work was supported by grants from the National Institutes of Health (NIH) 3R01 AG026529 supplemental grant to Yupanqui Caldas and NIH 2R01 DK066029-6 to Moshe Levi. Figure A. Treatment with DMHCA or TO901317 compound induced a small significant decrease in the urine pH compared to control samples. Figure B. Protein abundance by western blotting of the renal Na-glucose transporter SGLT2 in BBM showed a significant reduction after treatment with TO901317 compound. No significant change was observed with DMHCA. Figure C. Protein abundance by western blotting of the Na-glucose transporter SGLT1 in intestinal BBM shows no significant changes after treatment with neither compound. Figure D. Western blotting of NaPi4 in OK cells BBM showing down-regulation after treatment with either LXR agonist. Supplementary material is linked to the online version of the paper at http://www.nature.com/ki
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