Essential regulatory elements for NHE3 gene transcription in renal proximal tubule cells

The objectives of the present study were to identify the cis-elements of the promoter absolutely required for the efficient rat NHE3 gene transcription and to locate positive and negative regulatory elements in the 5’-flanking sequence (5’FS), which might modulate the gene expression in proximal tubules, and to compare this result to those reported for intestinal cell lines. We analyzed the promoter activity of different 5’FS segments of the rat NHE3 gene, in the OKP renal proximal tubule cell line by measuring the activity of the reporter gene luciferase. Because the segment spanning the first 157 bp of 5’FS was the most active it was studied in more detail by sequential deletions, point mutations, and gel shift assays. The essential elements for gene transcription are in the region -85 to -33, where we can identify consensual binding sites for Sp1 and EGR-1, which are relevant to NHE3 gene basal transcription. Although a low level of transcription is still possible when the first 25 bp of the 5’FS are used as promoter, efficient transcription only occurs with 44 bp of 5’FS. There are negative regulatory elements in the segments spanning -1196 to -889 and -467 to -152, and positive enhancers between -889 and -479 bp of 5’FS. Transcription factors in the OKP cell nuclear extract efficiently bound to DNA elements of rat NHE3 promoter as demonstrated by gel shift assays, suggesting a high level of similarity between transcription factors of both species, including Sp1 and EGR-1.

Transport of methotrexate by the in vitro isolated rabbit proximal tubule.

The tubular transport of [3H]methotrexate was studied in isolated nonperfused and perfused superficial proximal tubular segments of rabbit kidneys. Reabsorption represented only 5% of perfused methotrexate, and appeared to be mostly of passive nature inasmuch as it was not modified by reducing the temperature or by ouabain. Cellular accumulation in nonperfused segments and secretion in perfused tubules were highest in the S2 segment and lower in the S3 and S1 segments. Secretion against a bath-to-lumen concentration gradient was observed only in S2 segments (with a maximum methotrexate secretory rate of 478 +/- 48 fmol/mm.min and an apparent Km of transport of 363 +/- 32 microM), and was inhibited by probenecid and folate. The low capacity for methotrexate secretion may be explained by a low capacity of transport across the basolateral membrane of the proximal cell as methotrexate was accumulated only to a low extent in nonperfused tubules (tissue water to medium concentration ratio of 8.2 +/- 1 in S2 segments). During secretion a small amount of methotrexate was metabolized; the nature of the metabolite(s) remains to be defined.

CO-EXPRESSION OF GCDH AND OAT1 IN NEURONS AND PROXIMAL TUBULE CELLS

Tissue-specific expression studies of Glutaryl-CoA dehydrogenase (Gcdh) in adult rats revealed expression in the whole rat brain, almost exclusively in neurons, and surprisingly high expression in the juxtamedullar cortex of the kidney. The organic anion transporter 1 (OAT1) mediates basolateral uptake of glutarate derivatives from proximal tubule cells and contributes to their renal clearance. In brain, OAT1 is expressed at the choroid plexus, in neurons of cortex and hippocampus. We hypothesized that Gcdh and Oat1 are co-expressed in the same cells in kidney and brain and analyzed their mRNA expression by in situ hybridization on cryosections of adult rat brain, kidney and liver. In brain, Gcdh and Oat1 were found co-expressed in most neurons. Only the Purkinje neurons of the cerebellum were found to be Oat1 negative. In the kidney Gcdh and Oat1 are widely co-expressed with a specific high expression in proximal tubule cells. In conclusion there seems to be a functional coupling of Gcdh and Oat1 on a renal and neuronal level. Further studies are ongoing to confirm these findings in human tissues.

Transport of the organic cation N1-methylnicotinamide by the rabbit proximal tubule. I. Accumulation in the isolated nonperfused tubule.

Isolated nonperfused rabbit renal proximal tubules were used to investigate the basolateral step of transport of the organic cation N1-methylnicotinamide (NMN). NMN accumulation was highest and saturable in S2 and S3 segments, but lowest and nonsaturable in S1 segments. In S1 segments, accumulation of [3H]-NMN (0.5-8 microM in the bath) resulted in an average tubular water/medium concentration ratio (T/M) of 8.2, whereas in S2 and S3 segments T/M averaged 19.5 and 18.6, respectively. At these concentrations, about 30% of the label was attached in all segments to a metabolite comigrating with nicotinamide. KCN (10(-2) M) or ouabain (10(-4) M) reduced T/M to about 8 for all segments. NMN accumulation was inhibited (to a T/M of about 3 with mepiperphenidol) by other organic cations (10(-5)-10(-3) M) with the potency sequence mepiperphenidol greater than tetraethylammonium = quinine greater than morphine, these organic cations having no effect on p-aminohippurate accumulation, except for the highest concentration of quinine (10(-3) M). After correction for metabolism, NMN accumulation could be accounted for by simple electrochemical equilibrium across the basolateral membrane. The basolateral step of NMN transport appears therefore to be a carrier-mediated diffusion, in opposition to the active basolateral accumulation described for tetraethylammonium.

Regulation of the renal proximal tubule second sodium pump by angiotensins

For several years it was believed that angiotensin II (Ang II) alone mediated the effects of the renin-angiotensin system. However, it has been observed that other peptides of this system, such as angiotensin-(1-7) (Ang-(1-7)), present biological activity. The effect of Ang II and Ang-(1-7) on renal sodium excretion has been associated, at least in part, with modulation of proximal tubule sodium reabsorption. In the present review, we discuss the evidence for the involvement of Na+-ATPase, called the second sodium pump, as a target for the actions of these compounds in the regulation of proximal tubule sodium reabsorption.

Essential regulatory elements for NHE3 gene transcription in renal proximal tubule cells

The objectives of the present study were to identify the cis-elements of the promoter absolutely required for the efficient rat NHE3 gene transcription and to locate positive and negative regulatory elements in the 5’-flanking sequence (5’FS), which might modulate the gene expression in proximal tubules, and to compare this result to those reported for intestinal cell lines. We analyzed the promoter activity of different 5’FS segments of the rat NHE3 gene, in the OKP renal proximal tubule cell line by measuring the activity of the reporter gene luciferase. Because the segment spanning the first 157 bp of 5’FS was the most active it was studied in more detail by sequential deletions, point mutations, and gel shift assays. The essential elements for gene transcription are in the region -85 to -33, where we can identify consensual binding sites for Sp1 and EGR-1, which are relevant to NHE3 gene basal transcription. Although a low level of transcription is still possible when the first 25 bp of the 5’FS are used as promoter, efficient transcription only occurs with 44 bp of 5’FS. There are negative regulatory elements in the segments spanning -1196 to -889 and -467 to -152, and positive enhancers between -889 and -479 bp of 5’FS. Transcription factors in the OKP cell nuclear extract efficiently bound to DNA elements of rat NHE3 promoter as demonstrated by gel shift assays, suggesting a high level of similarity between transcription factors of both species, including Sp1 and EGR-1.

Long-term regulation of vacuolar H(+)-ATPase by angiotensin II in proximal tubule cells

Long-term effects of angiotensin II (Ang II) on vacuolar H(+)-ATPase were studied in a SV40-transformed cell line derived from rat proximal tubules (IRPTC). Using pH(i) measurements with the fluorescent dye BCECF, the hormone increased Na(+)-independent pH recovery rate from an NH(4)Cl pulse from 0.066 +/- 0.014 pH U/min (n = 7) to 0.14 +/- 0.021 pH U/min (n = 13; p < 0.05) in 10 h Ang II (10(-9) M)-treated cells. The increased activity of H(+)-ATPase did not involve changes in mRNA or protein abundance of the B2 subunit but increased cell surface expression of the V-ATPase. Inhibition of tyrosine kinase by genistein blocked Ang II-dependent stimulation of H(+)-ATPase. Inhibition of phosphatidylinositol-3-kinase (PI3K) by wortmannin and of p38 mitogen-activated protein kinase (MAPK) by SB 203580 also blocked this effect. Thus, long-term exposure of IRPTC cells to Ang II causes upregulation of H(+)-ATPase activity due, at least in part, to increased B2 cell surface expression. This regulatory pathway is dependent on mechanisms involving tyrosine kinase, p38 MAPK, and PI3K activation.

Direct action of aldosterone on bicarbonate reabsorption in in vivo cortical proximal tubule

Pergher PS, Leite-Dellova D, de Mello-Aires M. Direct action of aldosterone on bicarbonate reabsorption in in vivo cortical proximal tubule. Am J Physiol Renal Physiol 296: F1185-F1193, 2009. First published February 18, 2009; doi:10.1152/ajprenal.90217.2008.-The direct action of aldosterone (10(-12) M) on net bicarbonate reabsorption (J(HCO3)(-)) was evaluated by stationary microperfusion of an in vivo middle proximal tubule (S2) of rat kidney, using H ion-sensitive microelectrodes. Aldosterone in luminally perfused tubules caused a significant increase in J(HCO3)(-) from a mean control value of 2.84 +/- 0.08 [49/19 (n degrees of measurements/n degrees of tubules)] to 4.20 +/- 0.15 nmol.cm(-2).s(-1) (58/10). Aldosterone perfused into peritubular capillaries also increased J(HCO3)(-), compared with basal levels during intact capillary perfusion with blood. In addition, in isolated perfused tubules aldosterone causes a transient increase of cytosolic free calcium ([Ca(2+)](i)), monitored fluorometrically. In the presence of ethanol ( in similar concentration used to prepare the hormonal solution), spironolactone (10(-6) M, a mineralocorticoid receptor antagonist), actinomycin D (10(-6) M, an inhibitor of gene transcription), or cycloheximide (40 mM, an inhibitor of protein synthesis), the J(HCO3)(-) and the [Ca(2+)](i) were not different from the control value; these drugs also did not prevent the stimulatory effect of aldosterone on J(HCO3)(-) and on [Ca(2+)](i). However, in the presence of RU 486 alone [10(-6) M, a classic glucocorticoid receptor (GR) antagonist], a significant decrease on J(HCO3)(-) and on [Ca(2+)](i) was observed; this antagonist also inhibited the stimulatory effect of aldosterone on J(HCO3)(-) and on [Ca(2+)](i). These studies indicate that luminal or peritubular aldosterone (10(-12) M) has a direct nongenomic stimulatory effect on J(HCO3)(-) and on [Ca(2+)](i) in proximal tubule and that probably GR participates in this process. The data also indicate that endogenous aldosterone stimulates J(HCO3)(-) in middle proximal tubule.

Dipeptidyl peptidase IV inhibition downregulates Na(+)-H(+) exchanger NHE3 in rat renal proximal tubule

In the microvillar microdomain of the kidney brush border, sodium hydrogen exchanger type 3 (NHE3) exists in physical complexes with the serine protease dipeptidyl peptidase IV (DPPIV). The purpose of this study was to explore the functional relationship between NHE3 and DPPIV in the intact proximal tubule in vivo. To this end, male Wistar rats were treated with an injection of the reversible DPPIV inhibitor Lys [Z(NO(2))]-pyrrolidide (I40; 60 mg center dot kg(-1)center dot day(-1) ip) for 7 days. Rats injected with equal amounts of the noninhibitory compound Lys[ Z(NO(2))]-OH served as controls. Na(+) -H(+) exchange activity in isolated microvillar membrane vesicles was 45 +/- 5% decreased in rats treated with I40. Membrane fractionation studies using isopycnic centrifugation revealed that I40 provoked redistribution of NHE3 along with a small fraction of DPPIV from the apical enriched microvillar membranes to the intermicrovillar microdomain of the brush border. I40 significantly increased urine output ( 67 +/- 9%; P < 0.01), fractional sodium excretion ( 63 +/- 7%; P < 0.01), as well as lithium clearance ( 81 +/- 9%; P < 0.01), an index of end-proximal tubule delivery. Although not significant, a tendency toward decreased blood pressure and plasma pH/HCO(3)(-) was noted in I40-treated rats. These findings indicate that inhibition of DPPIV catalytic activity is associated with inhibition of NHE3-mediated NaHCO(3) reabsorption in rat renal proximal tubule. Inhibition of apical Na(+) -H(+) exchange is due to reduced abundance of NHE3 protein in the microvillar microdomain of the kidney brush border. Moreover, this study demonstrates a physiologically significant interaction between NHE3 and DPPIV in the intact proximal tubule in vivo.

Role of CFTR and ClC-5 in Modulating Vacuolar H(+)-ATPase Activity in Kidney Proximal Tubule

Background/Aims: It has been widely accepted that chloride ions moving along chloride channels act to dissipate the electrical gradient established by the electrogenic transport of H(+) ions performed by H(+)-ATPase into subcellular vesicles. Largely known in intracellular compartments, this mechanism is also important at the plasma membrane of cells from various tissues, including kidney. The present work was performed to study the modulation of plasma membrane H(+)-ATPase by chloride channels, in particular, CFTR and ClC-5 in kidney proximal tubule. Methods and Results: Using in vivo stationary microperfusion, it was observed that ATPase-mediated HCO(3)(-) reabsorption was significantly reduced in the presence of the Cl(-) channels inhibitor NPPB. This effect was confirmed in vitro by measuring the cell pH recovery rates after a NH(4)Cl pulse in immortalized rat renal proximal tubule cells, IRPTC. In these cells, even after abolishing the membrane potential with valinomycin, ATPase activity was seen to be still dependent on Cl(-). siRNA-mediated CFTR channels and ClC-5 chloride-proton exchanger knockdown significantly reduced H(+)-ATPase activity and V-ATPase B2 subunit expression. Conclusion: These results indicate a role of chloride in modulating plasma membrane H(+)-ATPase activity in proximal tubule and suggest that both CFTR and ClC-5 modulate ATPase activity. Copyright (C) 2010 S. Karger AG, Basel

Regulation of Na(+)/H(+) exchanger NHE3 by glucagon-like peptide 1 receptor agonist exendin-4 in renal proximal tubule cells

Carraro-Lacroix LR, Malnic G, Girardi AC. Regulation of Na(+)/H(+) exchanger NHE3 by glucagon-like peptide 1 receptor agonist exendin-4 in renal proximal tubule cells. Am J Physiol Renal Physiol 297: F1647-F1655, 2009. First published September 23, 2009; doi:10.1152/ajprenal.00082.2009.-The gut incretin hormone glucagon-like peptide 1 (GLP-1) is released in response to ingested nutrients and enhances insulin secretion. In addition to its insulinotropic properties, GLP-1 has been shown to have natriuretic actions paralleled by a diminished proton secretion. We therefore studied the role of the GLP-1 receptor agonist exendin-4 in modulating the activity of Na(+)/H(+) exchanger NHE3 in LLC-PK(1) cells. We found that NHE3-mediated Na(+)-dependent intracellular pH (pH(i)) recovery decreased similar to 50% after 30-min treatment with 1 nM exendin-4. Pharmacological inhibitors and cAMP analogs that selectively activate protein kinase A (PKA) or the exchange protein directly activated by cAMP (EPAC) demonstrated that regulation of NHE3 activity by exendin-4 requires activation of both cAMP downstream effectors. This conclusion was based on the following observations: 1) the PKA antagonist H-89 completely prevented the effect of the PKA activator but only partially blocked the exendin-4-induced NHE3 inhibition; 2) the MEK1/2 inhibitor U-0126 abolished the effect of the EPAC activator but only diminished the exendin-4-induced NHE3 inhibition; 3) combination of H-89 and U-0126 fully prevented the effect of exendin-4 on NHE3; 4) no additive effect in the inhibition of NHE3 activity was observed when exendin-4, PKA, and EPAC activators were used together. Mechanistically, the inhibitory effect of exendin-4 on pHi recovery was associated with an increase of NHE3 phosphorylation. Conversely, this inhibition took place without changes in the surface expression of the transporter. We conclude that GLP-1 receptor agonists modulate sodium homeostasis in the kidney, most likely by affecting NHE3 activity.

Distinct mechanisms underlie adaptation of proximal tubule Na+/H+ exchanger isoform 3 in response to chronic metabolic and respiratory acidosis

The Na+/H+ exchanger isoform 3 (NHE3) is essential for HCO3- reabsorption in renal proximal tubules. The expression and function of NHE3 must adapt to acid-base conditions. The goal of this study was to elucidate the mechanisms responsible for higher proton secretion in proximal tubules during acidosis and to evaluate whether there are differences between metabolic and respiratory acidosis with regard to NHE3 modulation and, if so, to identify the relevant parameters that may trigger these distinct adaptive responses. We achieved metabolic acidosis by lowering HCO3- concentration in the cell culture medium and respiratory acidosis by increasing CO2 tension in the incubator chamber. We found that cell-surface NHE3 expression was increased in response to both forms of acidosis. Mild (pH 7.21 +/- 0.02) and severe (6.95 +/- 0.07) metabolic acidosis increased mRNA levels, at least in part due to up-regulation of transcription, whilst mild (7.11 +/- 0.03) and severe (6.86 +/- 0.01) respiratory acidosis did not up-regulate NHE3 expression. Analyses of the Nhe3 promoter region suggested that the regulatory elements sensitive to metabolic acidosis are located between -466 and -153 bp, where two consensus binding sites for SP1, a transcription factor up-regulated in metabolic acidosis, were localised. We conclude that metabolic acidosis induces Nhe3 promoter activation, which results in higher mRNA and total protein level. At the plasma membrane surface, NHE3 expression was increased in metabolic and respiratory acidosis alike, suggesting that low pH is responsible for NHE3 displacement to the cell surface.

Increased NHE3 abundance and transport activity in renal proximal tubule of rats with heart failure

Inoue BH, dos Santos L, Pessoa TD, Antonio EL, Pacheco BPM, Savignano FA, Carraro-Lacroix LR, Tucci PJF, Malnic G, Girardi ACC. Increased NHE3 abundance and transport activity in renal proximal tubule of rats with heart failure. Am J Physiol Regul Integr Comp Physiol 302: R166-R174, 2012. First published October 26, 2011; doi:10.1152/ajpregu.00127.2011.-Heart failure (HF) is associated with a reduced effective circulating volume that drives sodium and water retention and extracellular volume expansion. We therefore hypothesized that Na(+)/H(+) exchanger isoform 3 (NHE3), the major apical transcellular pathway for sodium reabsorption in the proximal tubule, is upregulated in an experimental model of HF. HF was induced in male rats by left ventricle radiofrequency ablation. Sham-operated rats (sham) were used as controls. At 6 wk after surgery, HF rats exhibited cardiac dysfunction with a dramatic increase in left ventricular end-diastolic pressure. By means of stationary in vivo microperfusion and pH-dependent sodium uptake, we demonstrated that NHE3 transport activity was significantly higher in the proximal tubule of HF compared with sham rats. Increased NHE3 activity was paralleled by increased renal cortical NHE3 expression at both protein and mRNA levels. In addition, the baseline PKA-dependent NHE3 phosphorylation at serine 552 was reduced in renal cortical membranes of rats with HF. Collectively, these results suggest that NHE3 is upregulated in the proximal tubule of HF rats by transcriptional, translational, and posttranslational mechanisms. Enhanced NHE3-mediated sodium reabsorption in the proximal tubule may contribute to extracellular volume expansion and edema, the hallmark feature of HF. Moreover, our study emphasizes the importance of undertaking a cardiorenal approach to contain progression of cardiac disease.