heart failure (HF) is a multifaceted clinical syndrome characterized by reduced cardiac output and/or diastolic dysfunction associated with venous and capillary congestion. These initial alterations in cardiac function are accompanied by a progressive series of systemic abnormalities at both functional and structural levels. A number of physiological mechanisms are triggered in the face of impaired cardiovascular hemodynamics and sustained loss of myocardial contractility caused by heart disease. Many of these mechanisms are adaptive on a short-term basis but are often maladaptive over the long term. Underfilling of the arterial system, a major hemodynamic abnormality in HF evokes a number of neurohumoral responses to maintain sufficient blood flow delivery to peripheral organs and thereby compensate for the progressively declining cardiac performance (7, 8). Stimulation of these neurohumoral mechanisms, which include activation of the sympathetic and renin-angiotensin-aldosterone systems (RAAS) and enhanced vasopressin release, may, in fact, further aggravate HF through increased cardiac load and excessive sodium and water retention by the kidneys (16, 17, 21, 38, 39).

The apical entry of sodium constitutes the rate-limiting step in transepithelial sodium and fluid transport. Hence, any changes in either the number and/or the intrinsic activity of the proteins mediating this entry can affect the rate of renal tubular reabsorption (30). Recent studies provide compelling evidence that increased protein abundance of the predominant apical sodium transporters may play an important role in the pathogenesis of sodium and water retention in HF. It has been demonstrated by several investigators that the protein abundance of the Na⁺-K⁺-2Cl⁻ cotransporter NKCC2 is increased in the thick ascending limb of rats with HF induced by ligation of the left anterior descending coronary artery (LAD) (28, 31, 43, 48). Most recently, Lütken et al. (28) have shown that LAD-induced HF is associated with increased protein expression of Na⁺/H⁺ exchanger isoform 3 (NHE3) in renal cortex and outer medulla and altered expression of the epithelial sodium channel (ENaC) subunits. Nonetheless, to the best of our knowledge, there are no reports to date that have directly demonstrated that the activity of any of the major apical sodium transport proteins within the nephron is increased in HF.

The proximal tubule reabsorbs approximately two-thirds of the filtered sodium. NHE3-mediated Na⁺/H⁺ exchange is the principal apical membrane mechanism for sodium reabsorption in this nephron segment (4, 40, 51, 52). Considering the quantitative importance of NHE3 in mediating sodium reabsorption in the renal proximal tubule, changes in the activity of this exchanger for extended periods can have a great impact on extracellular volume homeostasis. We therefore hypothesized that NHE3 activity is increased in the renal proximal tubule of HF rats, potentially contributing to sodium retention, extracellular volume expansion, and ultimately to edema. In the present study, we made use of both in vivo and ex vivo approaches to measure NHE3 activity in an experimental model of HF, recently developed by Antonio et al. (2) in which rats are subjected to myocardial injury by means of radiofrequency ablation of the left ventricle. The molecular mechanisms underlying altered regulation of NHE3 in the renal proximal tubule of rats with HF were also explored.

MATERIALS AND METHODS

Animal protocols and surgical procedures.

All animal work was conducted in accordance with the ethical principles in animal research of the Brazilian College of Animal Experimentation and was approved by the institutional animal care and use committee. Experimental HF was induced in male Wistar rats (200–250 g) by left ventricular (LV) myocardial injury after radiofrequency catheter ablation (2). In brief, rats were anesthetized with halothane and a left thoracotomy was performed at the fourth intercostal space. The left pericardium was opened, and the tip of a custom-made catheter was placed on the LV anterolateral wall, perpendicular to the tissue, under constant manual pressure. LV lesions were created by delivering high-frequency currents (1,000 KHz, 12 watts, during 10 s) generated by a conventional radiofrequency generator (model TEB RF10; Tecnologia Eletrônica Brasileira, São Paulo, Brazil). The damaged area was immediately visualized by the presence of a clear, white disk-shaped region of coagulation necrosis. Thereafter, the heart was quickly returned to the thorax, and a suture was used to close the chest. Sham-operated rats underwent left thoracotomy and were mockingly ablated. All the following experimental procedures were performed 6 wk after myocardial injury for HF induction or sham operation.

Renal function studies.

Rats were anesthetized with ketamine-xylazine-acepromazine (64.9, 3.20, and 0.78 mg/kg sc, respectively) and placed on a heated surgical table to maintain body temperature. After tracheostomy, polyethylene (PE) catheters were inserted into the jugular vein and the urinary bladder for inulin infusion and urine collection, respectively. To control mean arterial pressure (MAP) and allow blood sampling, a PE-60 catheter was inserted into the right carotid artery. Glomerular filtration rate was determined by measuring the clearance of inulin as follows. First, a loading dose of inulin (100 mg/kg in 0.9% saline) was administered and, subsequently, continuous infusions of inulin (10 mg/kg in 0.9% saline) were made at 0.04 ml/min. Three consecutive 30-min periods of urine collection were performed. Blood samples were obtained at the beginning and at the end of the experiment. Plasma and urine sodium concentrations were measured by flame photometry (model B262; Micronal, São Paulo, Brazil), and inulin was determined by using the anthrone method (12).

Hemodynamic studies.

After renal function assessment, invasive hemodynamic studies were performed to evaluate cardiac performance. Rats were anesthetized using an intraperitoneal injection of ketamine (50 mg/kg) and xylazine (10 mg/kg) and placed on a heated rodent operating table (37°C) under oxygen-enriched mechanical ventilation. A MicroTip pressure-volume catheter (model 1.4 French SPR 839; Millar Instruments, Houston, TX) was then inserted into the right carotid artery and positioned immediately above the aortic valves to monitor aortic blood pressure. After 5 min of arterial blood pressure recording, the catheter was advanced into the LV cavity for simultaneous and continuous pressure and volume measurements. Volume calibration was accomplished by using a linear volume-conductance regression of the absolute blood volume (4 cylindrical chambers containing a specified volume of fresh heparinized rat blood) versus the correspondent signal acquired by the conductance catheter. Data were acquired for computer analysis (PVAN Software, Millar Instruments) using LabChart 7 Software System (PowerLab, ADInstruments, Bella Vista, NSW, Australia). The following data were obtained: heart rate (HR), MAP, LV end-systolic and end-diastolic pressures (LVEDP), maximal rate of LV pressure rise (+dP/dt_max) and decline (−dP/dt_max), time constant of LV pressure decay (Tau), stroke volume, cardiac output, and stroke work. Tau was calculated as the time constant of monoexponential pressure decay during the isovolumetric relaxation: P(t) = A + B exp(−t/Tau). The total peripheral resistance index (TPRI) was calculated (TPRI = MAP/cardiac output). Only rats with LVEDP > 15 mmHg were included in the HF group.

Stationary in vivo microperfusion.

Experiments were carried out essentially as described previously (10), including anesthesia and surgical preparation for in vivo micropuncture. Proximal tubules were punctured by means of a double-barreled micropipette, one barrel being used to inject FDC green-colored Ringer perfusion solution and the other to inject Sudan-black colored castor oil. To measure luminal pH, proximal tubules were impaled by a double-barreled asymmetric microelectrode, the larger barrel containing the H⁺-ion sensitive ion-exchange resin and the smaller barrel containing the reference solution (1 M KCl) colored by FDC green. By continuously measuring the luminal pH toward the steady-state level, the rate of tubular acidification, representing bicarbonate reabsorption, was evaluated in a solution, isolated, and blocked by oil. Thereby, luminal HCO₃⁻ activity, starting at 25 mM (perfusion solution) was progressively reduced to a stationary level by H⁺ secretion. The voltage between the microelectrode barrels, representing luminal H⁺ activity, was continuously recorded by means of a microcomputer equipped with an AD converter (Lynx, São Paulo, Brazil). Luminal bicarbonate was calculated from luminal pH and arterial blood Pco₂, and the rate of tubular acidification was expressed as the half time of the exponential reduction of the injected HCO₃⁻ concentration to its stationary level.

Postmortem analysis.

At the end of the experimental procedures, rats were killed by decapitation, and their hearts, lungs, and kidneys were quickly removed. Heart injury in HF rats was evaluated as follows. Right and left ventricle, including the interventricular septum were separately weighed, fixed in 10% formalin, and embedded in paraffin. Sections (5 μm) of paraffin-embedded tissue were mounted onto slides and stained with Picrossirius red for measurement of collagen scar and LV cavity perimeter. The slides were next scanned and digitally analyzed (Image Tool 3.0; University of Texas Health Science Center San Antonio, San Antonio, TX). Myocardial lesion was quantified by the percentage of LV perimeter containing scar tissue. Lung weights (indexed by body weight and tibia length) were taken as a marker of pulmonary congestion. Kidney cortices were cut off and used for the preparation of either renal cortical membrane vesicles or for RNA isolation.

Isolation of renal cortical membranes.

Renal cortical membranes were prepared by differential centrifugation of renal cortical homogenates from individual animals as described previously (11). Protein concentration was determined by the method of Lowry (27).

Microvillus membrane preparation.

Microvillus membrane vesicles (MMV) were isolated from renal cortices of individual animals as described previously (3). Membranes were isolated and stored in 200 mM mannitol, 80 mM HEPES, 41 mM KOH, 1 μM pepstatin, 1 μM leupeptin, 230 μM PMSF, pH 7.5 at −80°C. MMV preparations used in this study were 11- to 14-fold enriched in specific activity of the brush-border membrane marker enzyme γ-glutamyltranspeptidase relative to kidney homogenates. Protein concentration was determined by the method of Lowry (27).

Radioactive sodium uptake.

Renal cortical membranes were washed and equilibrated for 2 h at room temperature in 254 mM mannitol, 35 mM KOH, 68 mM HEPES, 50 mM MES, pH 6.0. The membranes were then centrifuged and resuspended in the same solution at a final protein concentration of 10 μg/μl. The uptake of ²²Na was then assayed after mixing 10 μl of the renal cortical membrane suspension with 90 μl of a hot solution containing 0.1 μCi of ²²Na, 300 mM mannitol, 42 mM KOH, 80 mM HEPES, pH 7.5. The uptake reaction was stopped after 10 s by the addition of 3 ml ice-cold solution (stop solution) containing 100 mM KCl, 42 mM KOH, 80 mM HEPES, pH 7.5. Renal cortical membrane vesicles were collected on 0.65-μm Millipore filters. After the filters were washed with an additional 9.0 ml of stop solution, ²²Na radioactivity was measured using a liquid scintillation counter. Some experiments were performed in the presence of 10 μM (S3226) to inhibit NHE3 activity.

SDS-PAGE and immunoblotting.

Protein samples were subjected to SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes (Immobilon-P; Millipore, Bedford, MA) and prepared for immunoblotting as previously described (10). The PVDF membranes were incubated with Blotto (5% nonfat dry milk and 0.1% Tween 20 in PBS, pH 7.4) for 1 h at room temperature to block nonspecific binding and were subsequently incubated overnight at 4°C with the primary antibody in Blotto at the following concentrations: 1:1,000 anti-PS552 (24), 1:1,000 anti-NHE3 (5), or 1:50,000 anti-actin. The membranes were then washed five times with Blotto and incubated for 1 h with horseradish peroxidase-conjugated immunoglobulin secondary antibody (1:2,000). Membranes were again washed five times with Blotto and then rinsed in PBS. Bound antibody was detected using an enhanced chemiluminescence system (GE Healthcare) according to the manufacturer's protocols. The visualized bands were digitized using the ImageScanner III (GE HealthCare) and quantified using Image Software (Scion, Federick, MD).

RNA isolation and real-time RT-PCR reaction.

Total RNA was isolated by the acid guanidinium thiocyanate phenol-chloroform method and cDNA synthesis was performed with random hexamers (High Capacity cDNA Archive kit-PE; Applied Biosystems) following the manufacturer's guidelines. Fifteen nanograms of cDNA were used for real-time RT-PCR reaction (SYBR Green PCR Master Mix-PE; Applied Biosystems) in an ABI Prism 7700 Sequence Detection System (Applied Biosystems). All samples were assayed in triplicate. The comparative threshold cycle method was used for data analyses. The following oligonucleotide primers were used: NHE3: 5′-ACTGCTTAATGACGCGGTGACTGT-3′ (forward) and 5′-AAAGACGAAGCCAGGCTCGATGAT-3′ (reverse), and 28S: 5′-TCATCAGACCCCAGAAAAGG-3′ (forward) and 5′-GATTCGGCAGGTGAGTTG-3′ (reverse).

Statistical analyses.

All results are reported as means ± SE with n indicating the number of observations. Comparisons between two groups were performed using unpaired t-tests. If more than two groups were compared, statistical significance was determined by ANOVA followed by Tukey's post hoc test. Results were considered significant when P < 0.05.

RESULTS

Experimental model, biometric, and cardiac parameters.

Herein, we used an experimental model of HF in which rats were subjected to myocardial injury by means of LV radiofrequency ablation (2). The major advantages of this model are the very consistent size of myocardial injury and low immediate (24 h) mortality rate. Accordingly, only 1 out of 22 rats died 24 h after being subjected to the procedure. Additionally, 6 wk after LV radioablation, we verified by standard histological analysis with Picrossirius red staining that LV radiofrequency ablation gave rise to a myocardial injury that was very reproducible in terms of both site (Fig. 1) and size (35.4 ± 2.6% of LV).

Table 1 summarizes the biometric parameters found in HF and sham-operated rats. Compared with sham animals, rats with HF did not display significant changes in body or absolute heart weight. However, when normalized by tibial length or body wt, relative heart weight was significantly increased in HF animals. In addition, the relative weight of the wet right lung was also significantly increased in HF animals compared with the sham group, suggestive of pulmonary congestion secondary to cardiac decompensation.

Figure 2 shows representative steady-state pressure-volume loops (P-V loops; Fig. 2A) and the pressure signal and dP/dt (Fig. 2B) obtained from individual HF and sham rats 6 wk after LV radioablation or sham operation, illustrating the typical hemodynamic changes that occur in HF. The rightward shift of the P-V loops and the decrease of the area within the P-V loop curves and of +dP/dt in HF rats indicates a decrease in cardiac performance. As shown in Table 2, HF was associated with a significant decrease of the maximal slope of the systolic pressure increment (+dP/dt_max), cardiac output, and stroke work when compared with sham animals, with no significant changes in HR, LV systolic pressure, and MAP, overall suggestive of relative LV systolic impairment. Furthermore, maximal rate of LV pressure decline (−dP/dt_max) was depressed (Fig. 2B and Table 2) and LVEDP (Fig. 2B and Table 2) and the time constant of isovolumetric relaxation (Tau) (Table 2) were increased in HF animals, indicating diastolic dysfunction. The TPRI was also significantly increased in HF rats. In line with previous findings (2), our data indicate that this experimental model of post-myocardial injury HF displays many abnormalities of cardiac function that resembles the human syndrome.

Renal function evaluation.

Renal function studies were carried out 6 wk after myocardial injury or sham operation. As shown in Table 3, urinary flow, glomerular filtration rate, urinary excretion of sodium, and fractional excretion of sodium were not statistically different between HF and sham, although a tendency to a decrease in rats with HF was observed. Daily sodium and water intake were also measured in sham and HF rats for three consecutive days prior to the experiments of renal function evaluation. Throughout this period, the daily average consumption of water (67.8 ± 4.4 vs. 69.4 ± 1.9 ml/kg, P < 0.05 vs. sham) and sodium (5.93 ± 0.41 vs. 5.97 ± 0.37 meq/kg, P < 0.05 vs. sham) were similar between these two groups of animals.

NHE3-mediated Na⁺/H⁺ exchange in renal proximal tubule.

A major fraction of proximal tubular bicarbonate reabsorption is mediated by proton secretion through NHE3 (40, 52). We therefore evaluated whether the in vivo NHE3 transport activity was altered in HF rats compared with sham by measuring the rate of bicarbonate reabsorption (J_HCO3⁻) in the native proximal tubules of these animals. To this end, stationary in vivo microperfusion experiments were conducted in individual proximal convoluted tubules from HF (n = 5) and sham-operated rats (n = 4). As indicated in Fig. 3A, J_HCO3⁻ was significantly increased (43 ± 6%) in HF rats compared with sham (2.71 ± 0.33 vs. 1.90 ± 0.11 nmol·cm²·s⁻¹; P < 0.05).

We next evaluated NHE3 activity by measuring the proton dependent uptake of ²²Na in renal cortical membrane vesicles isolated from HF (n = 4) and sham animals (n = 4) (Fig. 3B). In agreement with the micropuncture results, NHE3-mediated-²²Na uptake (= S3226-inhibitable ²²Na uptake) was higher in HF rats than in sham (58 ± 3%). No significant differences were observed between sham and HF rats in the component of ²²Na uptake that was insensitive to the specific inhibitor of NHE3 activity, S3226 (41). Taken together, these results suggest that altered renal handling of sodium in HF is at least partially attributed to enhanced NHE3-mediated sodium reabsorption in the renal proximal tubule.

NHE3 protein and mRNA expression in total renal cortex.

To investigate whether upregulation of NHE3 activity in HF was associated with increases of NHE3-protein abundance and/or mRNA levels, we carried out immunoblot and real-time RT-PCR analyses, respectively (Fig. 4). In accordance with results of a previous study in LAD-induced HF rats (28), we found that NHE3 protein abundance was 68 ± 8% higher in renal cortex of myocardial-injured HF rats compared with sham (Fig. 4A). Next, we determined whether the increase in NHE3 protein amount was accompanied by similar increases in the abundance of NHE3-mRNA in renal cortex. The data presented in Fig. 4B reveal that NHE3-mRNA expression relative to that of 28S ribosomal RNA was significantly higher in HF rats than in sham (145 ± 18%). Collectively, these findings suggest that the upregulation of proximal tubule NHE3 activity in HF rats is, at least in part, dependent on changes in NHE3 protein abundance and NHE3-mRNA expression levels.

Levels of NHE3 phosphorylated at the PKA consensus site serine 552.

One of the best-studied regulatory mechanisms affecting NHE3 activity is the inhibition resulting from agonists that activate cAMP-dependent protein kinase (PKA) (15, 24, 25). PKA directly phosphorylates NHE3 at serines 552 and 605, and it has been established that the residue 552 of NHE3 is phosphorylated to a much greater extent than the residue 605 at baseline in vivo (24). To determine whether altered phosphorylation of NHE3 may contribute to the increased activity of the exchanger in the renal proximal tubule of HF rats, we examined the levels of phosphorylated NHE3 in renal cortical membranes by means of immunoblotting. Experiments were carried out using a phosphospecific monoclonal antibody that recognizes NHE3 only when it is phosphorylated at serine 552 (24). As seen in Fig. 5, the relative ratio of NHE3-PS552/NHE3 total was significantly lower in renal cortical membranes isolated from HF compared with sham rats (−64 ± 4%).

The NHE3 phosphorylation status was also evaluated within the proximal tubule brush-border microvilli in HF and sham rats. As seen in Fig. 6, total NHE3 expression in MMV isolated from HF rats was higher (77 ± 10%, P < 0.001), and the PS552-NHE3-to-NHE3 ratio was lower than in sham (−60 ± 21%, P < 0.05). These results suggest that enhanced sodium reabsorption in the renal proximal tubule of HF rats is partially mediated through reduced baseline PKA-dependent phosphorylation of NHE3 at serine 552.

DISCUSSION

More than 50 years ago, Stead and Warren (44) noted that the most important causes of edema in HF are salt and water retention by the kidneys rather than the high venous pressure caused by impaired pumping of blood out of venous system. The potential causes of altered renal handling of salt and water may involve a decrease in glomerular filtration rate, an increase in tubular reabsorption of sodium, or both. In the present work, we demonstrate that the activity of the major proximal tubule sodium transporter NHE3 is increased in an experimental model of myocardial injury-induced HF. Higher NHE3 activity in the proximal tubule of HF rats is accompanied by enhanced renal cortical NHE3 expression at both mRNA and protein levels. In addition, the baseline PKA-dependent NHE3 phosphorylation at serine 552 is reduced in cortical membranes of rats with HF.

Numerous observations underscore the crucial role of NHE3 on extracellular volume control. NHE3 is acutely and chronically regulated by the renin-angiotensin system (14, 20, 37, 49, 54), sympathetic nervous system (55), as well as by an intrarenal dopamine natriuretic system (22, 29, 33). Moreover, NHE3 knockout mice are hypovolemic and exhibit reduced blood pressure (40). Furthermore, it has been previously demonstrated by other laboratories and ours that during the prehypertensive stage, NHE3 activity is increased in the renal proximal tubule of spontaneously hypertensive rats, possibly contributing to sodium retention and volume expansion in these animals (10, 26, 46). Here we show by both in vivo and ex vivo approaches that proximal tubule NHE3-mediated sodium reabsorption is increased in HF. Upregulation of NHE3 activity is accompanied by increases of NHE3 protein and mRNA expression. Of note, increased NHE3 protein expression have been previously observed in other experimental models of edematous disorders, including HF induced by ligation of the left anterior descending coronary artery (28) and cirrhosis (18). Despite upregulation of NHE3 function and expression, we did not find a statistically significant change of renal sodium handling after 6 wk of experimentally induced HF. Most likely, the animals might have experienced a positive sodium balance during the initial phase of heart failure and then reached a new steady state in which renal sodium output matches sodium intake.

NHE3 is phosphorylated on serine 552 under basal conditions by the adenylyl cyclase/cAMP-activated-protein kinase A (PKA) (24, 25) and the endogenous levels of phosphorylation is often affected as part of acute NHE3 regulation (15, 23, 24). Interestingly, baseline levels of NHE3 phosphorylation at this residue may also be associated with chronic regulation of NHE3 activity. In this regard, we have recently demonstrated that NHE3 activity is differentially regulated before and after development of hypertension in spontaneously hypertensive rats and that these changes of NHE3 activity occur concurrently with changes of NHE3 phosphorylation status in the spontaneously hypertensive rat model. In this work, we show that the levels of NHE3 phosphorylation at the PKA consensus site serine 552 in the proximal tubule apical membrane of HF rats are lower than in sham animals. Thus, the molecular mechanisms mediating enhanced sodium reabsorption in the renal proximal tubule of HF rats also appear to involve posttranslational covalent modification of NHE3. Moreover, decreased NHE3 phosphorylation at serine 552 is associated with an increase of NHE3 abundance in the brush-border microvilli, supporting the notion that phosphorylation of NHE3 at this specific PKA consensus site may play a role in subcellular trafficking of the transporter in vivo (23, 24). In congestive HF, volume expansion occurs in the venous side of the circulation, while the arterial underfilling due to reduced cardiac output is perceived as a plasma volume deficit by the arterial baroreceptors (7). Consequently, a series of neurohumoral adjustments is evoked that prevents the kidney to excrete accumulated salt and water. Among these neurohumoral responses are the activation of the renin-angiotensin-aldosterone (RAA) and the sympathetic nervous systems.

Both ANG II and norepinephrine have been shown to chronically increase NHE3 protein expression (42, 50, 54) and could be involved in upregulation of transporter expression in HF. Sonalker et al. (42) have demonstrated that chronic norepinephrine infusion resulted in a significant increase of NHE3 protein abundance in the rat renal cortex. Studies by several groups demonstrate that administration of ANG II increases NHE3 expression at both protein and mRNA levels in rat renal proximal tubule (14, 50) and cell culture models (34, 54). Additionally, LAD-induced HF rats treated with ANG II receptor antagonists display normalized levels of NHE3 expression (28) and renal sodium excretion (43), suggesting that ANG II influences renal sodium handling in cardiovascular disease at least, in part, via NHE3.

There is evidence that coupling of ANG II to the AT1 receptor inhibits cAMP accumulation and stimulates transcellular sodium transport in renal proximal tubule cells (45). β-Adrenoceptors also stimulate proximal tubule sodium reabsorption; however, these receptors are coupled to Gs proteins, which activate adenylyl cyclase to form cAMP. This paradox was explained by Hall et al (19) who demonstrated that β₂-receptors physically interact with the sodium proton exchanger regulatory factor (NHERF1), a protein that is required for PKA-mediated inhibition of NHE3. Stimulation of β₂-adrenoceptors by its ligands sequestrates NHERF and relieves the baseline inhibition of NHE3 by PKA. Thus, the decreased levels of endogenous NHE3 phosphorylation observed in the proximal tubule of HF rats might as well be mediated by activation of the RAAS and/or the sympathetic nervous system.

Both atrial and brain natriuretic peptides (ANP and BNP, respectively), originating in the myocardial cells and secreting into the plasma, play an important role in regulating extracellular fluid homeostasis and blood pressure by counterbalancing the RAAS and the sympathetic nervous system (13, 35, 53). Plasma levels of ANP and BNP have been shown to be increased in patients with HF and positively correlate with LV dysfunction (6, 35, 36, 47, 56). In fact, plasma concentrations of BNP have been widely used as a reliable prognostic indicator for HF patients in all stages of the disease (32). Notably, despite increases in the circulating levels of natriuretic peptides, renal sodium retention, volume expansion, and edema are continuously observed in patients and in experimental animal models of HF. Indeed, resistance to the natriuretic actions of ANP and BNP has been documented in HF subjects (1, 9). Numerous explanations for natriuretic peptide resistance in HF have been anticipated (7). Our results suggest that enhanced NHE3-mediated proximal tubular sodium reabsorption diminishes sodium delivery to the distal nephron site of natriuretic peptide action. Such decreased sodium load to the distal nephron may attenuate the natriuretic response to these peptides in HF patients.

Perspectives and Significance

The present study demonstrates that NHE3-mediated Na⁺/H⁺ exchange is significantly increased in the renal proximal tubule of an experimental model of post-myocardial injury HF. The molecular mechanisms underlying upregulation of NHE3 activity in HF seem to occur at transcriptional, translational, and posttranslational levels. These novel findings suggest that enhanced NHE3-mediated sodium reabsorption in the proximal tubule may contribute to extracellular volume expansion and ultimately to edema, one of the most important pathophysiological features of HF. Moreover, our study emphasizes the importance of undertaking a cardiorenal approach to contain progression of cardiac disease. Improving the understanding of the kidney's adaptive and maladaptive responses to cardiac dysfunction may lead to the development of new therapeutic strategies.

GRANTS

This research was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo, Conselho Nacional de Desenvolvimento Cientifico e Tecnológico, and by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

B.H.I., L.d.S., T.D.P., E.L.A., B.P.M.P., F.A.S., L.R.C.-L., P.J.F.T., G.M., and A.C.C.G. conception and design of research; B.H.I., L.d.S., T.D.P., E.L.A., B.P.M.P., F.A.S., L.R.C.-L., and G.M. performed experiments; B.H.I., L.d.S., T.D.P., E.L.A., B.P.M.P., F.A.S., L.R.C.-L., G.M., and A.C.C.G. analyzed data; B.H.I., L.d.S., T.D.P., E.L.A., G.M., and A.C.C.G. interpreted results of experiments; B.H.I., L.d.S., and A.C.C.G. prepared figures; B.H.I., L.d.S., T.D.P., E.L.A., B.P.M.P., F.A.S., L.R.C.-L., P.J.F.T., G.M., and A.C.C.G. edited and revised manuscript; B.H.I., L.d.S., T.D.P., E.L.A., B.P.M.P., F.A.S., L.R.C.-L., P.J.F.T., G.M., and A.C.C.G. approved final version of manuscript; A.C.C.G. drafted manuscript.