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Functional Role of Metabolism, Osmotic Stress, and Sodium-Glucose Isoform- on Na+/H+ Exchanger Isoform 3 Activity in the Renal Proximal Tubule

† ‡ Thaissa Dantas Pessoa,* Luciene Cristina Gastalho Campos, Luciene Carraro-Lacroix, † Adriana C.C. Girardi, and Gerhard Malnic*

*Department of Physiology and Biophysics, Institute of Biomedical Sciences, and †Heart Institute (InCor) Medical School, University of São Paulo, São Paulo, Brazil; and ‡Department of Biological Sciences, Federal University of Paraná, Curitiba, Paraná, Brazil

ABSTRACT Na+-glucose cotransporter 1 (SGLT1)-mediated glucose uptake leads to activation of Na+-H+ exchanger 3 (NHE3) in the intestine by a process that is not dependent on glucose metabolism. This coactivation may be important for postprandial nutrient uptake. However, it remains to be determined whether SGLT- mediated glucose uptake regulates NHE3-mediated NaHCO3 reabsorption in the renal proximal tubule. Considering that this nephron segment also expresses SGLT2 and that the kidneys and intestine show significant variations in daily glucose availability, the goal of this study was to determine the effect of SGLT- mediated glucose uptake on NHE3 activity in the renal proximal tubule. Stationary in vivo microperfusion experiments showed that luminal perfusion with 5 mM glucose stimulates NHE3-mediated bicarbonate reabsorption. This stimulatory effect was mediated by glycolytic metabolism but not through ATP pro- duction. Conversely, luminal perfusion with 40 mM glucose inhibited NHE3 because of cell swelling. Notably, pharmacologic inhibition of SGLT activity by produced a marked inhibition of NHE3, even in the absence of glucose. Furthermore, immunofluorescence experiments showed that NHE3 colocalizes with SGLT2 but not SGLT1 in the rat renal proximal tubule. Collectively, these findings show that glucose exerts a bimodal effect on NHE3. The physiologic metabolism of glucose stimulates NHE3 transport activity, whereas, supraphysiologic glucose concentrations inhibit this exchanger. Additionally, Phlorizin-sensitive SGLT transporters and NHE3 interact functionally in the proximal tubule.

J Am Soc Nephrol 25: 2028–2039, 2014. doi: 10.1681/ASN.2013060588

The kidney proximal tubule (PT) is the site where glucagon like peptide-1 were shown to inhibit the reabsorption of approximately 70% of filtered NHE3 and promote natriuresis.3–8 Additionally, sodium bicarbonate occurs. It is mainly performed various conditions and substances related to glu- by the Na+/H+ exchanger isoform 3 (NHE3).1 The cose metabolism, including diabetes, , ATP, physiologic importance of NHE3 became evident and glucose, modulate NHE3 in different tissues, after the development of NHE3 knockout mice, which presented mild metabolic acidosis and vol- Received June 6, 2013. Accepted January 9, 2014. ume depletion with reduced BP, underscoring the Published online ahead of print. Publication date available at 2 role of NHE3 in volume . www.jasn.org. It has been shown that NHE3 physically and Correspondence: Prof. Gerhard Malnic, Department of Physiol- functionally interacts with dipeptidyl-peptidase IV, ogy and Biophysics, Institute of Biomedical Sciences, University an enzyme that degrades and inactivates the incretin of São Paulo, Avenida Professor Lineu Prestes, 1524, 2° andar, hormone glucagon like peptide-1.3 The inhibition Laboratório 231, São Paulo, Brazil. Email: [email protected] of dipeptidyl-peptidase IV and the action of Copyright © 2014 by the American Society of Nephrology

2028 ISSN : 1046-6673/2509-2028 JAmSocNephrol25: 2028–2039, 2014 www.jasn.org BASIC RESEARCH showing a close relationship between carbohydrate homeosta- glucose or a control solution (CTRL; solution without any sis and NHE3 activity.9–12 transported sugar) (Concise Methods). 2 Plasma glucose concentration is maintained at a constant As shown in Figure 1A, the rate of bicarbonate flux (JHCO3 ) level by a complex system, in which the kidneys perform a in the PTs perfused with 5 mM glucose (GLU) was significantly pivotal role by reabsorbing all the filtered glucose in the PT.13 In higher than in CTRL-perfused tubules (1.90360.083 versus addition, the kidneys and are the only organs that express 2.77960.093 nmol/cm2 per second). A progressive inhibition 2 the glucose-6-phosphatase enzyme, thus enabling them to of JHCO3 was found after the perfusion of higher glucose perform .14,15 This enzyme is only expressed concentrations (1.12560.13 and 1.2560.16 nmol/cm2 per sec- in the PT,16 highlighting the importance of this kidney seg- ond for GLU40 and GLU60, respectively). Because the stimu- ment in carbohydrate metabolism. latory effect was observed only on perfusion of GLU5 and the It has been shown that the kidneys metabolize 20% of the maximum inhibitory effect occurred on GLU40 perfusion, glucose consumed in a meal.14 The PT has a low expression of these concentrations were used in all the following experi- hexokinase but the highest concentration and activity of glucose- ments. Representative curves of pH changes are given in Sup- 6-phosphate dehydrogenase, indicating that this segment is able plemental Figure 1. to metabolize glucose.16,17 However, it is currently believed that the PT uses noncarbohydrate compounds as energy sources.17 With relation to glucose uptake, the majority of filtered glucose is reabsorbed by the low-affinity, high-capacity sodium- glucose cotransporter isoform 2 (SGLT2). Some glucose is also reabsorbed by the high-affinity, low-capacity sodium-glucose cotransporter isoform 1 (SGLT1).13 Recently, SGLT2 inhibitors have been approved for the treatment of hyperglycemia in di- abetic patients. The use of these inhibitors has been shown to decrease blood glucose, glycated hemoglobin, postprandial glucose, insulinemia, and body weight.18–20 The role of glucose uptake in the modulation of NHE3 activity in the small intestine has been extensively studied. Experiments have shown that glucose uptake through SGLT1 promotes intracellular NHE3-dependent alkalinization.21–26 However, functional differences between intestinal and renal NaHCO3 NHE3-mediated reabsorption have not been estab- lished. These two systems differ physiologically, because the gastrointestinal system is exposed to fluctuations in glucose concentration between the periods of fasting and after meals.13 The presence of large amounts of solutes within the intestinal cells after meals modulates transporters, such as 2 (GLUT2) and NHE3,21,27 an important process for nutrient absorption. Although the synergistic activation between SGLT1 and NHE3 has been observed in the intestine,21 it is not known if this process also occurs in the kidneys. Considering that the kidneys also express SGLT2 and the particularities of glucose availability in this organ, the goal of the present work was to 2 determine the effect of glucose and SGLTactivity on NHE3 in Figure 1. Proximal tubule NHE3-mediated HCO3 reabsorption 2 2 the renal PT. (JHCO3 ) is modulated by glucose. JHCO3 was evaluated by means of in vivo stationary microperfusion, and the continuous measurement of luminal pH was performed according to the RESULTS protocol described in Concise Methods. (A) Rat kidney proximal tubules were perfused with CTRL, GLU5, GLU20, GLU40, or 2 GLU60. The data are the means6SEMs. *P,0.001 versus CTRL; Glucose Modulates NHE3-Dependent JHCO3 in the **P,0.001 versus CTRL; #P,0.001 versus GLU5; &P,0.001 ver- Renal PT sus GLU20. (B) The tubules were perfused with CTRL, GLU5, or As an initial approach to study the effect of glucose on NHE3- GLU40 in the presence (+S3226) or absence (alone) of the spe- mediated bicarbonate reabsorption, Wistar rats were subjected cific NHE3 inhibitor S3226 (10 mM). The data are means6SEMs. to stationary microperfusion in vivo, and their PTs were per- The numbers of perfused tubules are indicated in the bars. fused with solutions containing different concentrations of *P,0.01 versus CTRL.

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2 To confirm that the glucose-dependent JHCO3 modula- Because is metabolized by galactokinase, an tion was NHE3-dependent, we performed experiments in the enzyme with expression and activity that decrease after presence of the specific NHE3 inhibitor S3226.28 The addition weaning,29 we postulated that differences in the metabolism 2 of 10 mM S3226 markedly inhibited JHCO3 in all groups of glucose and galactose could be responsible for the differ- (from 1.90360.083 to 0.70160.041 nmol/cm2 per second in ences in NHE3 modulation in the PT. To test this hypothesis, CTRL; from 2.77960.094 to 0.52160.070 nmol/cm2 per sec- we performed experiments in which the nonmetabolizable 2 ond in GLU5; from 1.12560.130 to 0.53860.069 nmol/cm glucose analog, a-methyl-D-glucopiranoside (a-MG), was per second in GLU40) (Figure 1B). The S3226-insensitive bi- perfused in the tubules. carbonate reabsorption component was equivalent among all As depicted in Figure 2B, a-MG5 was not able to stimulate different groups, indicating that NHE3 is the only H+ trans- NHE3. This result suggests that glucose metabolism is re- porter in the PT that is modulated by glucose. quired for 5 mM glucose-dependent NHE3 stimulation. How- ever, a-MG40 produced an inhibitory effect on NHE3 similar The Glucose-Dependent Stimulatory Effect on NHE3 to the effect produced by GLU40, showing that the glucose Occurs through Glucose Metabolism inhibitory effect occurs by a metabolically independent mech- To evaluate whether the glucose-dependent, NHE3-mediated anism (1.11360.10 nmol/cm2 per second for a-MG40 versus 2 2 JHCO3 modulation was caused by the intrinsic properties of 1.90360.083 nmol/cm per second for CTRL). glucose, we performed a set of experiments, in which glucose To evaluate if the mechanism by which GLU5 stimulates was replaced by galactose. The perfusion of different galactose NHE3 was by ATP generation, we analyzed if another substrate concentrations failed to modulate NHE3 activity (Figure 2A). capableofgeneratingATPwasabletomimicthestimulatory

Figure 2. Glucose-dependent NHE3 stimulation is dependent on glucose metabolism. The rate of bicarbonate reabsorption was 2 2 evaluated by stationary microperfusion and expressed as JHCO3 . The dotted horizontal lines indicate JHCO3 in GLU5- and GLU40- perfused tubules. (A) The experiments were performed in the presence of CTRL or galactose (GALAC; 5 and 40 mM), a C-4 epimer of glucose. (B) The tubules were perfused with CTRL or a-MG (5 and 40 mM), a nonmetabolizable glucose analog. (C) The experiments were conducted in the presence of CTRL or GLU5 alone or with 2-DG (10 mM), a hexokinase inhibitor. (D) PTs were perfused with glucose-free solution or GLU5 plus 2-DG in the presence or absence of 10 mM L-alanine alone or with 500 mM 7M DL-cycloserine, an alanine aminotransferase inhibitor. The numbers of perfused tubules are indicated above the bars. The data are the means6SEMs. *P,0.001 versus CTRL; #P,0.001 versus GLU5; ##P,0.01 versus GLU5.

2030 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 2028–2039, 2014 www.jasn.org BASIC RESEARCH effect of glucose on NHE3. We then performed experiments in which the PTs were perfused with amino acids that can be metabolically converted into ATP by specific aminotransfera- ses highly expressed in the kidney.30 Perfusion of 10 mM iso- leucine, alanine, or aspartate failed to stimulate NHE3 (data not shown), indicating that ATP generation is not the final step responsible for the stimulatory effect observed on GLU5 per- fusion. To determine the role of glucose metabolism on NHE3 activity, we performed experiments in which the PTs were perfused with CTRL and GLU5 alone or together with 10 mM 2-deoxy-D-glucose (2-DG), a hexokinase inhibitor. Addition of 10 mM 2-DG to GLU5 not only abolished the stimulatory effect of GLU5 on NHE3 but also, produced an additional inhibitory effect that was also observed on the perfusion of 2 CTRL+2-DG (JHCO3 from 1.90360.083 to 1.28960.054 nmol/cm2 persecondforCTRLandfrom2.77960.094 to 1.14360.073 nmol/cm2 per second for GLU5) (Figure 2C). Because 2-DG can result in ATP depletion,31 we performed experiments, in which alanine alone or plus DL-cycloserine (an alanine aminotransferase inhibitor) was added to the 2- DG– or GLU5+2-DG–containing solution as an ATP source. Addition of 10 mM alanine abolished the 2-DG–dependent Figure 3. High glucose-dependent NHE3 inhibition in the NHE3 inhibition in both the presence and absence of glucose 2 proximal tubule is caused by osmotic stress. (A) JHCO was (Figure 2D). The same effect was not observed in the presence 3 measured under hypotonic conditions (250 mOsM/kg H2O). For of the alanine aminotransferase inhibitor DL-cycloserine, 2 comparison, the dotted horizontal line indicates JHCO3 in showing that alanine conversion into pyruvate is necessary GLU40-perfused tubules. *P,0.001 versus CTRL. (B) The tubules to abolish the 2-DG–dependent NHE3 inhibition. Therefore, were perfused with CTRL, GLU40, or a-MG40 alone or together because the addition of alanine to the 2-DG+GLU5 solution with mannitol (40 mM). The dotted horizontal line indicates 2 6 did not produce a stimulatory effect, we confirmed that glu- JHCO3 in GLU5-perfused tubules. The data are means SEMs. cose metabolism is absolutely necessary for the NHE3 regula- The numbers of perfused tubules are indicated in the bars. tion, even after the addition of another source of energy. *P,0.05 versus CTRL; **P,0.01 versus CTRL; ***P,0.001 versus CTRL. High Glucose–Dependent NHE3 Inhibition Is Associated with Osmotic Stress highglucose-dependentinhibitoryeffectonNHE3and It is well established that SGLT1-mediated Na+-glucose uptake produced a stimulatory effect similar to the effect observed on 32 2 promotes cell swelling. As such, although all the buffers used GLU5 perfusion (Figure 3B, horizontal dotted line) (JHCO3 of in this study were osmotically balanced, an increase in cell 2.80260.216 nmol/cm2 per second for GLU40+40 mM manni- volume associated with SGLT-mediated glucose uptake was tol). As shown earlier, a-MG5 failed to mimic the stimulatory expected. To determine if cell swelling was associated with effect. Therefore, we conducted experiments where a-MG40 NHE3 inhibition in the PT, we performed experiments under was perfused alone or together with 40 mM mannitol. hypotonic conditions (250 mOsM/kg H2O). The perfusion of The addition of 40 mM mannitol to a-MG40 abolished the 2 the hypotonic solution inhibited JHCO3 to the same extent inhibitory effect of a-MG40 on NHE3 (1.11360.106 versus as GLU40 (Figure 3A, dotted horizontal line). This result 1.72660.119 nmol/cm2 per second) but was not able to shows that the cell swelling promoted by hypotonic shock produce a stimulatory effect. This finding further confirms inhibits NHE3 in the PT. that the stimulatory effect of glucose on NHE3 transport ac- To confirm that the GLU40-inhibitory effect was caused by tivity is dependent on glucose metabolism. cell swelling, we conducted experiments, in which 40 mM mannitol, a nonpermeable and osmotically active substance SGLTs Inhibition by Phlorizin Impairs NHE3-Dependent 2 that remains in the lumen after perfusion (thus producing an JHCO3 opposite osmotic effect) (Supplemental Figure 2), was added To verify the effect of SGLTs on NHE3, we used the SGLT to the GLU40 solution. competitive inhibitor, Phlorizin (Plz). Because of the high As depicted in Figure 3B, the addition of 40 mM mannitol to variability of Plz concentrations used in different re- 2 21,25,33,34 CTRL produced a slight inhibition of JHCO3 . Conversely, the ports and because of the fact that SGLTs are able to addition of 40 mM mannitol to GLU40 completely abolished the perform the electrogenic Na+ uptake in the absence of a

J Am Soc Nephrol 25: 2028–2039, 2014 Glucose, SGLT, and NHE3 2031 BASIC RESEARCH www.jasn.org sugar,35 we initially performed a Plz dose-response curve As depicted in Figure 6, the expression of NHE3 (Figure starting with the CTRL solution (absence of glucose). 6A) and SGLT2 (Figure 6B) was restricted to the PTapical BB, As illustrated in Figure 4A, 10 mM Plz produced a signifi- and the merged image (Figure 6C) showed a considerable co- 2 2 cant decrease of JHCO3 (to 1.38860.1283 nmol/cm per localization of these proteins. In contrast, the immunofluo- second versus CTRL) (Figure 4A, vertical dotted line). The rescence staining of SGLT1 (Figure 6F) was more diffuse and perfusion of higher Plz concentrations caused progressive de- could be detected at both BBs and in cytoplasm vesicles. Ac- 2 creases of JHCO3 with the most pronounced effect observed cordingly, the merged image of SGLT1 and NHE3 staining with 500 mMPlz. (Figure 6G) did not show a significant colocalization of these To establish whether Plz was capable of inhibiting NHE3 in two proteins. These results suggest that the inhibition of the presenceof a sugar, we performed a Plz dose-response curve NHE3 by Plz is most likely mediated by SGLT2. with the GLU5 and GLU40 solutions. As depicted in Figure 4B, the addition of 1 mM Plz to the GLU5 solution caused an inhibition similar to the inhibition observed by the addition DISCUSSION of 50 mM Plz to the CTRL solution. Because Plz is a compet- itive inhibitor, higher concentrations of the inhibitor were re- The present work shows that physiologic concentrations of quired to produce the same effect on NHE3 in the presence of glucose (5 mM) stimulate NHE3 transport activity by a process glucose. However, the addition of Plz was not able to produce that is dependent on glycolytic metabolism. Higher glucose any additional inhibitory effect when used together with the concentrations inhibit the transporter because of cell swelling. GLU40 solution (Figure 4C). In addition, we showed that the inhibition of the SGLT- 2 + Next, we examined the ability of Plz to inhibit JHCO3 in dependent Na uptake inhibits NHE3. Moreover, immuno- the presence of other sugars. For this purpose, we performed fluorescence experiments showed that NHE3 colocalizes with experiments with Plz and 5 mM galactose or a-MG5. The SGLT2 but not SGLT1. Figure 7 summarizes the major find- addition of 500 mM Plz to 5 mM galactose (Figure 4D, vertical ings of the present work. 2 dotted line) significantly inhibited NHE3-dependent JHCO3 . With regard to the effects of glucose metabolism on NHE3, However, only the addition of a higher concentration of Plz the results presented in this work are significantly different (1 mM) to a-MG5 (Figure 4E, vertical dotted line) was able to from the results reported in a previous study using the 2 21 significantly inhibit JHCO3 . Altogether, these results indicate intestinal cell line CaCo-2. In this intestinal cell model, that inhibition of SGLTactivity by Plz also inhibits NHE3. SGLT1-mediated Na+-glucose uptake led to NHE3-dependent cytoplasmic alkalinization that was not dependent on glucose Systemic Administration of Plz Produces metabolism. In contrast, we showed that glycolytic metabo- Bicarbonaturia lism is necessary for the stimulatory effect of GLU5 on NHE3, 2 To test whether Plz increases HCO3 excretion when adminis- because the perfusion of nonmetabolizable sugars did not pro- tered systemically, rats were treated with 0.4 mg/kg Plz by sub- duce the same stimulation and the addition of 2-DG to GLU5 cutaneous injection every 12 hours36,37 and placed in metabolic abolished the stimulatory effect. The generation of interme- cages. The renal function and other parameters are depicted in diary compounds of glycolytic metabolism may be involved in Supplemental Table 1. As expected, Plz increased the absolute this effect, because the generation of these compounds has and fractional excretions of glucose (Figure 5, A and B). As been reported to modulate intracellular proteins.38 depicted in Figure 5, C and D, Plz markedly increased urinary In agreement with our findings, some studies have shown + 2 2 Na and HCO3 excretion (HCO3 excretion of 0.298560.023 that glucose metabolism modulates other members of the NHE and Na+ excretion of 0.183660.0304 in Plz treatment versus family.9,31,39–44 These transporters have been shown to be in- 0.169060.038 and 0.091160.0108 mmol/d per 100 g body wt, hibited by glycolytic inhibitors, such as 2-DG,9,45–47 and have respectively, in vehicle) (Figure 5, C and D, *P,0.05) compared also been observed to be more sensitive to glycolytic inhibitors with controls. These findings are in accordance with our micro- than oxidative phosphorylation inhibitors.48–50 Additionally, perfusion data, showing that both systemic and local adminis- our study suggests, for the first time, that the apical uptake of trations of Plz inhibit NHE3 activity in the PT. glucose is important for maintaining NHE3 activity, because glucose was perfused only apically in the tubules, and that SGLT2 Colocalizes with NHE3 glucose metabolism occurs in the PT and is important for Plz was able to inhibit NHE3, even in the absence of a sugar, maintaining NHE3 activity. indicating that SGLT-dependent Na+ uptake can modulate NHE3 can also be modulated by ATP, and it is believed that NHE3. Given the functional interaction between SGLTs and the ancillary proteins bound to the C-terminal tail of NHE3 are NHE3, we then hypothesized that these transporters might be responsible for its modulation by ATP.41 Because alanine, a located in the same microdomain within the PT brush border noncarbohydrate source of energy, was able to abolish the in- (BB) membrane. Thus, we performed immunofluorescence hibitory effect produced by the perfusion of 2-DG only in the experiments to examine whether NHE3 colocalizes with absence of DL-cycloserine (an alanine aminotransferase in- SGLT1 and/or SGLT2. hibitor), we can assume that (1) 2-DG perfusion produced

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ATP depletion, which has been observed previously,41 and (2) alanine is converted into pyruvate and can be used by the PT cells as an energy source. In addition, these data suggest that decreased glycolytic metabolism as well as ATP depletion might inhibit NHE3 in the PT. Experiments conducted in absorptive epithelial cells and other heterologous systems have shown that SGLTs are capable of transporting water together with GLU.51–53 Thus, although all solutions used in this work were isotonic, cell swelling as- sociated with SGLT-mediated glucose uptake was expected. In the present study, we found that hypotonic shock inhibits NHE3, supporting the hypothesis that the GLU40- or a-MG40–dependent inhibitory effect on NHE3 is caused by an increase in cell volume. The abolishment of this inhibitory effect by the addition of mannitol confirmed that the osmotic flow of water was responsible for the GLU40-dependent in- hibitory effect on NHE3. The NHE family members, especially the housekeeping isoforms, are involved in the control of cell volume. Therefore, cell swelling might induce NHE3 inhibition as a cell survival response, although NHE3 itself is not considered a house- keeping protein. Alternatively, an increase in cell volume could signal excessive sodium uptake, leading to the inhibition of this transporter to maintain volume homeostasis. In the literature, there is no consensus regarding the effect of increased cell volume on NHE3 activity. Several studies have found that an increase in volume promotes NHE3 stimula- tion.54–57 However, other studies have found that the addition of an extracellular hypotonic solution inhibits or has no effect on NHE3.21,40,58 The differences in NHE3 activity caused by hypo-osmotic shock might be because of the involvement of different intermediaries responsible for NHE3 modula- tion.1,57,59,60 We showed that the pharmacological inhibition of SGLT by Plz is accompanied by marked NHE3 inhibition, indicating that SGLT activity plays a role in NHE3-dependent sodium bicarbonate reabsorption. Because the CTRL solution does not

solution in the presence of Plz (5, 10, 50, 100, 500 mM, or 2 mM). The 2 dotted horizontal lines indicate JHCO3 in GLU5- and GLU40- perfused tubules. (B) The experiments were performed in the presence of GLU5 instead of CTRL solution. The following con- centrations of Plz were used: 50 mM, 500 mM, 1 mM, 2 mM, or 3 2 mM. The dotted horizontal lines indicate JHCO3 in CTRL- and GLU40-perfused tubules. (C) Plz was added to GLU40 solution in the following concentrations: 500 mM,1mM,or2mM.Thedotted 2 horizontal lines indicate JHCO3 in CTRL- and GLU5-perfused tubules. (D and E) The tubules were perfused with Plz (50 mM, 500 mM, or 1 mM) together with GALAC5 and a-MG5, respectively. 2 Figure 4. The SGLTs inhibitor, Plz, inhibits NHE3-dependent The dotted horizontal lines indicate JHCO3 in CTRL-, GLU5-, and 2 HCO3 reabsorption in the renal proximal tubule. The graphs GLU40-perfused tubules. The dotted vertical lines indicate the fi 2 a , show the dose–response curves obtained for Plz treatment under concentration of Plz that signi cantly inhibited JHCO3 . P 0.05 b c different conditions. (A) The tubules were perfused with control versus no Plz; P,0.001 versus no Plz; P,0.001 versus no Plz.

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Therefore, although the increase of urinary excretion of sodium bicarbonate induced by Plz may have a glomerular component, the differences of the magnitude of changes (35% increase of GFR and 87% and 100% increase of bicarbonate and sodium urinary excretion, respectively) allied with the microperfusion finding that intraluminal perfusion of Plz inhibits NHE3 activity strongly suggest that there is an important proximal tubular component responsible for the Plz-mediated sodium bicarbon- ate urinary loss. Having defined a functional interaction between SGLTand NHE3, we next examined whether SGLT1 and/or SGLT2 are expressed in the same PT subcellular sites as NHE3. By immunostaining, we determined that, in adult rats, NHE3 and SGLT2 are localized in the apical membrane of PT and showed a clear colocalization. However, SGLT1 staining was not concordant with NHE3 staining. SGLT1 staining was observed in regions where NHE3 staining was not present, and its intracellular staining was observed in regions of strong apical NHE3 staining. Consistent with these findings, it has been shown that NHE3 is virtually absent in the PT S3 segment,64 the region with the highest expression of SGLT1. Additionally, although SGLT1 is expressed throughout the S1 Figure 5. Acute systemic administration of Plz increases Na+ and 2 and S2 segments of the PT, its location is markedly intracel- HCO urinary excretion. Wistar rats were placed in metabolic 3 64,65 cages for 24-hour urine collection during Plz (0.4 g/kg body wt lular, whereas the localization of NHE3 is mostly apical. every 12 hours) or vehicle treatment. After the 24-hour period, Having shown that SGLT2 and NHE3 colocalize in the rat rats were decapitated, and a blood sample was collected. (A) renal PT, we hypothesized that these two transporters may be Urinary glucose excretion (milligrams per day), (B) fractional ex- physically associated. Unfortunately, we were unable to vali- 2 cretion of glucose (%), (C) HCO3 excretion (millimoles per day/ date this hypothesis, because all commercially available anti- 100 g body wt), and (D) Na+ excretion (millimoles per day/100 g bodies recognizing SGLT2 that we tested were not suitable for body wt). Values are means6SEMs; n=7 rats/group. *P,0.05 immunoprecipitation procedures (data not shown). Although versus vehicle. BW, body wt; N.D., not detectable. the exact structural and functional features of the interaction of NHE3 and SGLT2 remain to be further characterized, our present data suggest that, because we could not find the apical contain any transportable sugar, the NHE3 inhibition by Plz colocalization of SGLT1 and NHE3, only SGLT2 inhibition is cannot be attributed to abolition of glucose uptake. In the involved in NHE3 inhibition. absence of glucose, SGLT1 and SGLT2 are still able to produce In summary, the findings of this study show a bimodal effect currents, which are attributed to Na+ leak.35 Then, NHE3 in- of glucose on NHE3 activity in the renal PT. The stimulatory hibition by Plz might occur through dissipation of this trans- effect of physiologic concentrations of glucose on NHE3 epithelial Na+ current. However, a direct action of Plz in the involves glucose metabolism by the glycolytic cascade, whereas exchanger cannot be discarded. the inhibitory effect of supraphysiological glucose concentra- Noteworthy, a recent report has shown that dapagliflozin, tionsinvolves the water influxcaused byglucose uptake on high an SGLT2 inhibitor, promotes diuresis and reduces BP.18 glucose perfusion. We also showed that SGLT2 colocalizes with Moreover, Plz prevents hypertension development in an ex- NHE3 in the PT and that SGLT inhibition is accompanied by perimental model of diabetes.61 Our results suggest that one NHE3 inhibition. Furthermore, these results provide evidence possible mechanism by which dapagliflozin and Plz could in- that the inhibition of NHE3 is the mechanism by which SGLT2 duce diuresis and attenuate hypertension development is inhibitors cause diuresis. + 2 through NHE3 inhibition of Na /HCO3 reabsorption. Asexpected,acutesystemicadministrationofPlzincreasesthe fractional excretion of glucose. We have found that Plz-induced CONCISE METHODS glicosuria is accompanied by natriuresis and bicarbonaturia. Interestingly, Plz also promoted an increase in GFR estimated by Reagents and Antibodies creatinineclearance.Althoughcreatinineclearancemaynotbean All chemicals were obtained from Sigma-Aldrich unless otherwise optimal filtration marker in rodents, the effect of Plz on GFR has noted.S3226wasdonatedbyJurgenPunter(Sanofi-AventisDeutschland been previously shown in earlier studies, in which Plz was GmbH, Frankfurt, Germany). Anti-SGLT2 polyclonal antibody and chronically administered to different animal models.62,63 anti-NHE3 monoclonal antibody were obtained from Santa Cruz

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Biotechnology. Anti-SGLT1 polyclonal antibody was obtained from Abcam, Inc. Alexafluor-588 and AlexaFluor-488 secondary antibodies were obtained from Life Technologies.

Animal Protocols Animal procedures and protocols followed the ethical principles in animal research of the Brazilian College of Animal Experimentation and were approved by the Institutional Animal Care and Use Committee. The experiments were performed using male Wistar rats (200–350 g) housed under standardized conditions of con- stant temperature of 22°C, 12:12-hour dark to light cycles, and relative humidity of 60% at the Institute of Biomedicine of the University of São Paulo animal facility. On the days of the ex- periment, the rats were anesthetized with ketamine-acepromazine (200 and 1.5 mg/kg in- tramuscularly, respectively) and Propofol (25 mg/kg intravenously as needed) and prepared for the stationary microperfusion experiments as described below.

Stationary Microperfusion Experiments The animals were prepared for in vivo micro- puncture as described previously.66 Supple- mental Figure 3 represents a model for the technique. In this technique, a double-barreled micropipette was used to puncture a PT. One pipette barrel was filled with the experimental

solutions (90 mM NaCl, 25 mM NaHCO3,5

mM KCl, 1 mM CaCl2,and1.2mMMgSO4) stained with FDC green. The glucose concentra- tions of these solutions varied between 0 (CTRL) and 60 mM or were substituted with 5 or 40 mM galactose or a-MG. Another group

confocal microscope. (A–C) SGLT2 (red) and NHE3 (green) staining were only present in the apical membrane of the proximal tubule. (C) The merged image shows the colocalization of SGLT2 and NHE3. (D) The merged staining images including the contrast phase channel of selected tubules and the nuclei staining (blue). (E–G) SGLT1 (red) staining was distrib- uted diffusely in the proximal tubules, whereas NHE3 (green) was only detected in the apical membrane of the proximal tubule. (G) The Figure 6. SGLT2, but not SGLT1, colocalizes with NHE3 in the renal proximal tubule. merged image shows that SGLT1 does not Kidney sections were obtained from adult rats. The kidney tissue was fixed, and the colocalize with NHE3. (H) The contrast phase sections obtained were subjected to immunofluorescence staining with antibodies channel of selected tubules with the nuclei against NHE3, SGLT2, and SGLT1. The images were obtained using a Carl Zeiss staining (blue).

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contained 1 mM KCl colored by FDC green. The voltage, representing tubular H+ activity, was measured by the electrode by a WPI electrom- eter. The voltage was measured continuously and recorded by an an- alog to digital converter (Lynx). Luminal bicarbonate was calculated

from luminal pH and arterial blood PCO2. The rate of tubular acid- ification was expressed as the half-time of the exponential reduction of 2 the injected HCO3 concentration to its stationary level (t1/2). The net 2 2 2 HCO3 reabsorption (JHCO3 ) per centimeter of tubule epithelium was calculated using the following equation: h i r JHCO 2 ¼ ln2 HCO 2 2 HCO 2 ∗ ; 3 t 3 0 3 s 1=2 2

where t1/2 is the half-time of bicarbonate absorption, r is the tubule 2 2 radius, and (HCO3 )0 and (HCO3 )s are the concentrations of the 2 2 injected HCO3 and HCO3 at the stationary level, respectively. The value of N is the number of perfused tubules. Each experimental group was composed of at least three animals, and each animal had one to four proximal tubules perfused.

Acute Phlorizin Treatment Male Wistar rats were placed in individual metabolic cages for the collection of urine samples, quantification of urine output, and food and water consumption. Animals were placed individually in a metabolic cage for a 48-hour period of adaptation. In the next 24-hour period, animals received Plz dissolved in a solution containing ethanol (2.5:1) in PBS injected subcutaneously at a dose of 0.4 g/kg two times Figure 7. Model of how physiological and supraphysiological (in 12-hour intervals) or vehicle. At the end of the period, animals concentrations of glucose modulate NHE3 activity in the renal PT. were immediately decapitated, and blood samples were collected for Perfused GLU enters the PT cell through SGLT1 and/or SGLT2. analysis. Intracellularly, it is metabolized by glucose metabolism through the action of hexokinase. Glucose metabolism stimulates the exchanger. The perfusion of supraphysiological concentrations of Renal Function Analysis ↑ Urine and blood samples, collected in metabolic cages, were used to glucose ( GLU) increases glucose uptake through SGLT2, the 2 low-affinity, high-capacity glucose transporter, because the Km of measure urine output, HCO3 excretion, plasma glucose, glucose this transporter is higher than the Km of physiological glucose excretion, fractional glucose excretion, sodium excretion, potassium concentration. Glucose uptake is known to be accompanied by excretion, and GFR. Creatinine clearance was used to estimate GFR. water flow, and as such, glucose uptake promotes cell swelling Serum and urinary creatinine and glucose concentrations were mea- and NHE3 inhibition. AQ, aquaporin. sured by a kinetic method (Labtest) using a ThermoPlate Analyzer Plus (ThermoPlate). Sodium and potassium were measured on an 2 electrolyte analyzer (Roche Diagnostic). Urine HCO3 was measured was perfused with the same CTRL solution and the 5 mM glucose in a gasometer Radiometer ABL5 (Radiometer Medical). solution with 5 or 10 mM isoleucine or with 10 mM 2-DG alone or plus 10 mM isoleucine. The 40 mM glucose and 40 mM a-MG so- Kidney Preparation for Immunohistochemistry lution were also perfused together with 40 mM mannitol. The osmo- The animals were anesthetized as described above, and the kidneys of lality of solutions ranged from 250 (hypotonic) to 300 (adjusted with all animals were perfused with PBS at 37°C through the abdominal raffinose), up to 340 (hypertonic) mosM/kg H2O. aorta for 10 minutes followed by periodate-lysine-paraformaldehyde The other pipette barrel was filled with castor oil stained with perfusion fixation for 20 minutes. The kidneys were removed and Sudan black. The tubules were perfused with the abovementioned immediately frozen in Tissue-Tek (Tissue-Tek OCT Compound) us- solutions depending on the experimental strategy, and the column of ing liquid nitrogen. injected fluid was isolated from tubular and glomerular fluid and held stationary until the next perfusion by droplets of castor oil. Immunohistochemistry To measure the tubular pH, a double-barreled, asymmetric The kidneys were frozen in Tissue-Tek and sectioned (6 mm) using a microelectrode was used. The larger barrel was silanized with Cryostat (Microm HM 505E). After fixation in 4% paraformalde- hexamethyldisilazane (Sigma Fluka) and contained an H+ ion- hyde, the sections were rehydrated in PBS for 5 minutes and incu- sensitive ion exchange resin (Sigma Fluka). The smaller barrel bated in 0.5% Triton X-100 solution for 10 minutes at room

2036 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 2028–2039, 2014 www.jasn.org BASIC RESEARCH temperature and then, PBS containing 2% casein (Sigma-Aldrich) for 5. Girardi AC, Knauf F, Demuth HU, Aronson PS: Role of dipeptidyl pep- 30 minutes to block nonspecific staining. The sections were incu- tidase IV in regulating activity of Na+/H+ exchanger isoform NHE3 in – bated with an antiserum raised against NHE3 mouse monoclonal proximal tubule cells. Am J Physiol Cell Physiol 287: C1238 C1245, 2004 antibody and SGLT1 rabbit polyclonal antibody (diluted 1:50 and 6. Pacheco BP, Crajoinas RO, Couto GK, Davel AP, Lessa LM, Rossoni LV, 1:25 in PBS containing 0.5% casein, respectively) or NHE3 and Girardi AC: Dipeptidyl peptidase IV inhibition attenuates blood pres- SGLT2 goat polyclonal antibody (1:25) overnight at 4°C in a humid sure rising in young spontaneously hypertensive rats. J Hypertens 29: chamber. This incubation was followed by four washes of 5 minutes 520–528, 2011 each in PBS. The sections were then incubated for 90 minutes at room 7. Crajoinas RO, Oricchio FT, Pessoa TD, Pacheco BP, Lessa LM, Malnic G, Girardi AC: Mechanisms mediating the diuretic and natriuretic actions temperature with AlexaFluor-488–coupled anti-mouse antibody, – – of the incretin hormone glucagon-like peptide-1. Am J Physiol Renal AlexaFluor-555 coupled anti-rabbit antibody, and AlexaFluor-555 Physiol 301: F355–F363, 2011 coupled anti-rabbit antibody at a final concentration of 1:500. The 8. Rieg T, Gerasimova M, Murray F, Masuda T, Tang T, Rose M, Drucker nuclei were stained with 20 mg/ml 49,6-diamidino-2-phenylindole DJ, Vallon V: Natriuretic effect by exendin-4, but not the DPP-4 in- (Life Technologies). Negative control slides were incubated with sec- hibitor alogliptin, is mediated via the GLP-1 receptor and preserved in – ondary antibody alone (data not shown). The sections were washed in obesetype2diabeticmice.Am J Physiol Renal Physiol 303: F963 F971, 2012 PBS and mounted in 50% glycerol solution diluted in PBS, and the 9. Cassel D, Katz M, Rotman M: Depletion of cellular ATP inhibits Na+/H+ images were captured using a Carl Zeiss 510 LMS confocal system antiport in cultured human cells. Modulation of the regulatory effect of connected to an Axiovert microscope. intracellular protons on the activity. J Biol Chem 261: 5460– 5466, 1986 10. Fuster DG, Bobulescu IA, Zhang J, Wade J, Moe OW: Characterization Statistical Analyses of the regulation of renal Na+/H+ exchanger NHE3 by insulin. Am J t Statistical comparisons were made by paired test by taking the prob- Physiol Renal Physiol 292: F577–F585, 2007 ability of 0.05 (5%) as the limit of significance. When more than two 11. Klisic J, Nief V, Reyes L, Ambuhl PM: Acute and chronic regulation of groups were compared, a one-way ANOVA followed by Tukey’s post the renal Na/H+ exchanger NHE3 in rats with STZ-induced diabetes – hoc test was used. For each group, a minimum of six tubules was mellitus. Nephron Physiol 102: 27 35, 2006 12. Dhar-Chowdhury P, Malester B, Rajacic P, Coetzee WA: The regulation analyzed (N=number of perfused tubules). of ion channels and transporters by glycolytically derived ATP. Cell Mol Life Sci 64: 3069–3083, 2007 13. Vallon V: Molecular determinants of renal glucose reabsorption. Focus on “Glucose transport by human renal Na+/D-glucose co- ACKNOWLEDGMENTS transporters SGLT1 and SGLT2.” Am J Physiol Cell Physiol 300: C6– C8, 2011 The authors thank Nancy Amaral Rebouças for her helpful support. 14. Stumvoll M, Meyer C, Mitrakou A, Gerich JE: Important role of the This work was supported by Fundação de Amparo à Pesquisa do kidney in human carbohydrate metabolism. 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