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Soluble Form of the (Pro)Renin Receptor Is Augmented in the Collecting Duct and Urine of Chronic Angiotensin II–Dependent Hypertensive Rats

Originally publishedhttps://doi.org/10.1161/HYPERTENSIONAHA.110.167957Hypertension. 2011;57:859–864

Abstract

Renin synthesis and secretion by principal cells of the collecting duct are enhanced in angiotensin (Ang) II–dependent hypertension. The presence of renin/(pro)renin and its receptor, the (pro)renin receptor ([P]RR), in the collecting duct may provide a pathway for Ang I generation with further conversion to Ang II. To assess whether (P)RR activation occurs during Ang II–dependent hypertension, we examined renal (P)RR levels and soluble (P)RR excretion in the urine of chronic Ang II–infused rats (80 ng/min; for 2 weeks; n=10) and sham-operated rats (n=10). Systolic blood pressure and Ang II levels in the plasma and kidney were increased whereas plasma renin activity was suppressed in Ang II–infused rats. Renal (P)RR transcripts were upregulated in the cortex and medulla of Ang II–infused rats. (P)RR immunoreactivity in collecting duct cells and the protein levels of the full-length form (37-kDa band) were significantly decreased in the medulla of Ang II–infused rats. The soluble (P)RR (28-kDa band) was detected in the renal medulla and urine samples of Ang II–infused rats, which also showed increases in urinary renin content. To determine whether the soluble (P)RR could stimulate Ang I formation, urine samples were incubated with recombinant human (pro)renin. Urine samples of Ang II–infused rats exhibited increased Ang I formation compared with sham-operated rats. Thus, in chronic Ang II–infused rats, the catalytic activity of the augmented renin produced in the collecting duct may be enhanced by the intraluminal soluble (P)RR and cell-surface located (P)RR, thus contributing to enhanced intratubular Ang II formation.

Intrarenal formation of angiotensin (Ang) I occurs via actions of renal renin on angiotensinogen (AGT) delivered to the kidney and produced by the proximal tubule cells.1 In addition to its localization in juxtaglomerular cells, renin mRNA and protein are expressed in connecting tubules and cortical and medullary collecting ducts (CDs).25 In response to chronic Ang II infusions, the renin gene expression increases in principal cells of connecting tubules and CDs.4 Stimulation of CD renin in Ang II–dependent hypertension is mediated by an Ang II type 1 receptor mechanism, because treatment with Ang II type 1 receptor blockers prevents this response.6 The stimulation of CD renin in both kidneys of 2-kidney 1-clip Goldblatt hypertensive rats indicates that it occurs independent of changes in blood pressure.7

Nguyen et al8 cloned the (pro)renin receptor ([P]RR), a 350-amino acid protein with a single transmembrane domain, that binds renin or (pro)renin, thereby increasing their catalytic activity. In the human and rat kidney, (P)RR has been localized in glomerular mesangial cells, the subendothelium of renal arteries, podocytes, and distal nephron cells.810 Recently, Advani et al11 demonstrated by immunohistochemistry and in situ hybridization that (P)RR is predominantly expressed on the apical membranes of acid-secreting cells in the CD.11 Recent findings by Cousin et al12 have also demonstrated that the full-length form of (P)RR can be processed intracellularly by cleavage leading to a soluble form (s(P)RR), which can be secreted into the plasma and consequently bind renin. The discovery of the (P)RR has enhanced our understanding of the physiology of the tissue RAS and its contribution in cardiovascular and renal diseases1315; however, little is known regarding the changes in (P)RR in hypertension.

The demonstrations that in Ang II–dependent hypertension there is augmentation of CD renin gene expression4 and that most of the renin in medullary tissues of Ang II hypertensive rats is active7 suggest that (P)RR may be involved in the activation of (pro)renin and/or renin in the distal nephron segments, thus contributing to increased intrarenal and/or intratubular Ang I formation. In the present study, we tested the hypothesis that the (P)RR in the renal medulla and CD tubular fluid as reflected in the urine is augmented in chronic Ang II–infused rats.

Materials and Methods

Experimental Animals and Sample Collections

All of the experimental protocols were approved by the Tulane Institutional Animal Care and Use Committee. Male Sprague-Dawley rats (150 to 175 g; Charles River Laboratories) were cage housed and maintained in a temperature-controlled room with a 12-hour light/dark cycle, with free access to tap water and standard rat chow (Ralston Purina). Ten rats had osmotic minipumps subcutaneously implanted to infuse Ang II (80 ng/min, 14 days). Ten rats were sham operated. After a training period, the systolic blood pressures were monitored by tail-cuff plethysmography (IITC Instruments). On subsequent days, the rats were placed in metabolic cages for collection of urine both with and without a protease inhibitor mixture.16 At the end of the study, 5 rats were anesthetized with pentobarbital sodium (Ovation, Inc) for left kidney excision after unilateral ligature, and the right kidneys were sequentially perfused with saline solution (0.9% NaCl) and 4% paraformaldehyde. A similar set of rats was killed by conscious decapitation, and kidneys were dissected into the renal medulla and cortex for measurements of Ang II content, RNA, and protein extractions, as described.4,16 Trunk blood samples were collected for determination of plasma renin activity (PRA) and plasma Ang II concentration, as described previously.16

Urine Ang II and Renin Content

Urinary Ang II concentrations were determined by radioimmunoassay, as described previously,16 by incubating the samples with rabbit anti-Ang II antiserum (Peninsula Laboratories) and 125I-radiolabeled Ang II (Perkin Elmer Life and Analytic Sciences). Results are reported in femtomole per 24 hours of urine. To evaluate the contribution of the s(P)RR to enhance renin activity, we measured the Ang I formation after the addition of human recombinant (pro)renin (hPR; Lee BioSolutions, Inc) to urine samples of sham-operated and Ang II–infused rats collected with or without protease inhibitors. The procedure used was a modification of the PRA (Diasorin, Inc) assay. Briefly, 250 μL of rat urine with maleate generation buffer and PMSF from the PRA kit were spiked with 250 μL of renin substrate tetradecapeptide (4 μmol/L; Sigma-Aldrich, Inc). In addition, either 20 μL of WFML, a specific renin inhibitor (1 mmol/L; AnaSpec, Inc), or 10 μL of hPR (2.2 μmol/L) were added to identical aliquots of the urine-renin substrate tetradecapeptide mixture. Samples were incubated in a 37°C water bath, whereas replicate urine samples without the spike, being used to measure the background Ang I levels, were incubated at 4°C for 15 minutes. After the generation step, all of the samples were subjected to the remaining steps of the PRA assay. The measured values in nanograms of Ang I formed per milliliter per hour were converted into enzyme units excreted in 24-hour urine samples. The background Ang I levels were subtracted from all of the samples. For the samples spiked with hPR, the background renin and nonspecific enzyme activity were subtracted.

(P)RR Expression Studies

Quantitative Real-Time RT-PCR

Total RNA was isolated and quantified as described previously.4 The (P)RR mRNA levels were quantified by real-time RT-PCR (primers: 5′-ATCCTTGAGACGAAACAAGA-3′ [sense]; 5′-AGCCAGTCATAATCCACAGT-3′ [antisense], and 5′-6-carboxyfluorescein-ACACCCAAAGTCCCTACAACCTTG-black hole quencher1–3′ [fluorogenic probe]) and normalized against the expression level of rat GAPDH mRNA (primers: 5′-CAGAACATCATCCCTGCATC-3′ [sense]; 5′-CTGCTTCACCACCTTCTTGA-3′ [antisense]; and 5′-6-phosphoramidite-CCTGGAGAAACCTGCCAAGTATGATGA-black hole quencher2–3′ [fluorogenic probe]).

Immunohistochemistry

Rat kidney sections were stained by peroxidase techniques as described previously.17 The (P)RR immunostaining was performed using a rabbit anti-(P)RR antibody (No. 1623; Dr Genevieve Nguyen, College of France) at a 1:4000 dilution. For immunocolocalization, we used the rabbit anti (P)RR antibody as well as a goat anti-(P)RR (Abcam) at 1:400 dilution that were detected with diaminobenzidine reaction and antianion exchanger 1 (AE1) (Alpha Diagnostic International) for type A intercalated cells18 detected with Bajoran Purple Chromogen (Biocare Medical, LLC).

Immunoblotting Analysis

The renal (P)RR protein levels were examined using a polyclonal rabbit anti-(P)RR that recognizes the intracellular segment and the ectodomain (ATP6AP2, 1:400 dilution; Sigma-Aldrich) as described previously.19 In addition, (P)RR protein levels were examined in ×10 concentrated urine samples, collected previously into a protease inhibitor mixture, using similar protocol conditions. Because it has been reported recently that the soluble form of the (P)RR can be generated intracellularly by furin cleavage,12 we also measured the protein levels of furin in renal medullary tissues of these rats using a mouse antifurin, as described previously,20 at a dilution 1:100, overnight, followed by incubation with an antimouse secondary antibody at a 1:5000 dilution. All of the analyses by Western blotting were performed using the Odyssey detection system (LI-COR Biosciences).

Immunoprecipitation of the (P)RR

To assess whether renin or (pro)renin could bind the s(P)RR, urine samples were tested. Immunoprecipitation (IP) experiments were developed using Dynabeads M-280 (Invitrogen) covalently bound to purified sheep antirabbit IgG coated with the rabbit anti-(P)RR (ATP6AP2, 1:400 dilution; Sigma-Aldrich) and incubated with urine samples containing 100 μg of protein. Both supernatant and IP fractions were resolved by immunoblotting against 1:100 rabbit polyclonal antirenin H-105 antibody (Santa Cruz Biotechnologies). Human recombinant renin and (pro)renin (hPR) proteins (1 μL; Lee BioSolutions, Inc) were used as positive controls.

Statistical Analysis

The statistical significance defined at a value of P<0.05 was determined by using paired and unpaired Student t tests or by 1-way ANOVA with Tukey posttest.

Results

Body Weight, Systolic Blood Pressure, PRA, Plasma Ang II Levels, and Urine Ang II Levels

Body weights were similar at the study outset (sham: 221±5; Ang II–infused: 220±5 g; P value not significant) and significantly increased in both sham-operated and Ang II–infused rats, with a greater gain in sham-operated rats (Table). Systolic blood pressure averages were similar in both groups when the study began (sham: 109±4 mm Hg; Ang II–infused: 118±13 mm Hg; P value not significant); however, systolic blood pressure significantly increased in the Ang II–infused rats compared with the sham-operated rats (Table). At day 14, PRA was suppressed, whereas plasma and renal cortical and medullary Ang II levels, as well as urinary Ang II and renin content, were increased in the Ang II–infused rats compared with the sham-operated rats (Table).

Table. Physiological Parameters After 14 Days of Ang II Infusions

VariableShamAng II
Body weight, g275±8238±4*
Systolic blood pressure, mm Hg126±8228±8
PRA, ng Ang I · mL−1 · h−15.5±2.00.3±0.2*
Plasma Ang II, fmol/mL22±9179±30*
Urine Ang II excretion, fmol/24 h2079±3613813±431*
Kidney cortex Ang II, fmol/g229±50723±7*
Kidney medulla Ang II, fmol/g593±101898±34*
Urinary renin, enzymatic units ×10−6 in 24 h2.06±0.3410.01±2.18*

Values are mean±SEM.

*P<0.05 vs sham-operated rats.

P<0.001 vs sham-operated rats.

(P)RR Immunohistochemistry Studies

The (P)RR immunoreactivity was observed primarily in cells of the connecting tubules and CDs throughout the cortex, as well as in the outer and inner medulla (Figure 1). Negative control sections showed no (P)RR immunoreactivity (data not shown). Figure 1A shows bulging cells with intense apical (P)RR immunoreactivity (diaminobenzidine; brown and arrows), which were positive for basolateral AE1 (Bajoran Purple and asterisks), confirming the expression of (P)RR in type A intercalated cells in accordance with previous findings reported by Advani et al.11 The cells with specific apical positive staining for (P)RR were negative for aquaporin 2 (data not shown).

Figure 1.

Figure 1. (P)RR immunoreactivity in kidneys from sham-operated and Ang II–infused rats. A, Paraffin-embedded kidney sections (3 μm) showing (P)RR-specific staining (brown; arrowheads) on the apical membrane of the CD cells colocalizing with basolateral AE1 immunoreactivity (Bajoran Purple; *) in type-A intercalated cells (original magnification, ×1000). B, Densitometric analysis of the (P)RR intensity of sham-operated (B and D) and Ang II–infused rats (C and E) showing reduced number of positive cells in renal medullary tissues of Ang II–infused vs sham-operated rats (F) but not in the renal cortex (G). Scale bar: 100 μm. Values are mean±SE. *P<0.05 vs sham-operated rats.

Quantification of (P)RR Expression in Chronic Ang II–Infused Rats

Positive (P)RR immunoreactivity in CD cells was similar in the cortex of both the Ang II–infused and sham-operated rats (Ang II: 1.3±0.2; sham: 1.0±0.2 fold change compared with controls). This similarity was also reflected in the number of positive (P)RR-stained cells (Ang II: 21±1 versus sham: 22±1 positive cells per millimeter squared; Figure 1B, 1C, and 1F). In contrast, in the renal medulla, the (P)RR immunoreactivity was significantly decreased (Ang II: 0.7±0.1 versus sham: 1.0±0.1 fold change; P<0.001), and the number of positive cells with specific immunoreactivity was significantly less (Ang II: 14±2 versus sham: 34±2 positive cells per millimeter squared; P<0.001; Figure 1D, 1E, and 1G).

The (P)RR transcript levels were significantly greater in the renal medulla compared with the cortex in both groups. For both cortex and medulla, the (P)RR mRNA levels ([P]RR/GAPDH ratio) were significantly increased in the Ang II–infused rats compared with sham-operated rats (Ang II [cortex: 1.0±0.1; medulla: 1.6±0.1]; sham [cortex: 0.7±0.1; medulla: 1.2±0.1]; P<0.001; Figure 2A).

Figure 2.

Figure 2. (P)RR real-time quantitative RT-PCR and Western blot analyses in renal cortex and medulla. A, The mRNA levels for (P)RR were upregulated in the renal cortex and medulla in Ang II–infused rats. *P<0.001 (n=5). B, A representative blot of the (P)RR protein expression levels in the cortex and medulla as a fold change with respect to control rats showed a decrease in the full-length (P)RR and the presence of s(P)RR in the renal medulla of Ang II–infused rats. *P<0.05 (n=5).

Kidney (P)RR protein immunoblots from Ang II–infused and sham-operated rats showed the specific (P)RR band of 37 kDa. This band, although unchanged in the cortex, was significantly decreased in the medulla of Ang II–infused rats (cortex: 0.8±0.2 versus 1.0±0.2 fold change compared with control; P value not significant; medulla: 0.4±0.2 versus 1.0±0.2 fold change; P<0.05; Figure 2B). Furthermore, the presence of the s(P)RR form (28-kDa band; Figure 2B) became apparent while it was not detectable in the renal medulla of sham rats or in the cortex of either group.

Evidence of the Presence of Furin in the Renal Medullary Tissues of Ang II Hypertensive Rats

In an attempt to rule out whether furin, a protease recently involved in the intracellular cleavage of (P)RR,12 changes in the kidney of Ang II–hypertensive rats, we measured its protein levels in inner medullary tissues of the Ang II–infused and sham-operated rats. Furin:β-actin ratio was significantly augmented in Ang II–infused rats compared with sham-operated rats (Ang II–infused: 1.4±0.2 versus sham: 1.0±0.1 fold change; P<0.05).

Evidence of Functional s(P)RR in the Urine of Ang II Hypertensive Rats

Based on the results of the (P)RR expression data, we assessed the presence of s(P)RR in the urine of Ang II–infused hypertensive rats. Although urinary creatinine was not different between both groups (9246±1109 versus 10 201±388 μg/d; P value not significant), the (P)RR immunoblots using urine samples of Ang II–infused rats showed a transition from the 37-kDa band to the 28-kDa band, whereas in the sham-operated rats the s(P)RR form was not detectable (Figure 3A). These results suggest that secretion and urinary excretion of the s(P)RR form are induced in Ang II–infused rats. To determine whether the soluble form of (P)RR was bound to renin and/or (pro)renin in the urine of these rats, we immunoprecipitated the (P)RR. As shown in Figure 3B, using human recombinant (pro)renin and renin as positive controls, rat renin was detected in the IP, and its absence in the supernatant (S) fractions indicates that most of the renin was bound to (P)RR.

Figure 3.

Figure 3. Evidence of functional s(P)RR in the urine of Ang II hypertensive rats. A, Detection of the soluble form of the (P)RR (28 kDa) in urine samples of sham-operated and Ang II–infused rats. Eighty μg of protein from ×10 concentrated urine samples were loaded and incubated with a rabbit anti-(P)RR. B, IP of (P)RR in the urine samples of Ang II–infused and sham-operated rats demonstrated the absence of renin in the supernatant (S) fractions using the rabbit polyclonal antirenin H-105 antibody (Santa Cruz). C, A dose-response curve of hPR added in the experiment. D, Urine samples, initially collected with or without protease inhibitor mixture, measured in the presence and absence of 10 μL of hPR (2.2 pmol/μL), resulted in increased amounts of Ang I—forming enzymatic units excreted per day ×10−6 in Ang II–infused rats compared with sham-operated rats. In samples spiked with hPR, the background renin and nonspecific enzyme activity were subtracted. MWM indicates molecular weight marker; WB, Western blot; hR, recombinant human renin.

To examine whether the presence of the s(P)RR had functional implications in the urine of Ang II–infused rats, we further determined the renin activity in the presence or absence of hPR. Ten μL of 2.2 pmol/μL of hPR added in the experiment were determined based on a dose-response curve in urine samples of Ang II–infused rats (Figure 3C). In urine samples collected without protease inhibitors, the amount of Ang I–forming enzymatic units ×10−6 in 24-hour urine samples from Ang II–infused rats increased 5-fold compared with sham-operated rats (Table and Figure 3D) and increased even further in the presence of hPR in both groups (3118.33±886.25 versus 597.69±151.48; P<0.05; Figure 3D). To assess nonspecific enzymatic activation of the added hPR, the assay was repeated with urine samples collected into the same inhibitor mixture used for plasma Ang II determination.16 Under these conditions, urine samples from Ang II–infused rats still showed greater enzymatic activity than sham rat urine (18.21±6.41 versus 3.21±0.65 enzymatic units ×10−6 in 24-hour urine samples; P<0.05; Figure 3D), whereas no activity was detected in urine samples without hPR, indicating that the s(P)RR is functional and can enhance activity of (pro)renin leading to Ang I generation (Figure 3D).

Discussion

The present findings demonstrate that chronic infusion of Ang II for 14 days in Sprague-Dawley rats increased renal (P)RR transcript levels and augmented the soluble form of the (P)RR (28-kDa band) in renal inner medullary tissues and tubular fluid in distal nephron segments as reflected in the urine. Novel findings from this study suggest that the s(P)RR in the renal medulla is stimulated during Ang II–dependent hypertension and that this form of the (P)RR is secreted into the tubular fluid, which may contribute to increased renin activity in the CDs of Ang II hypertensive rats.

Using immunoblot analysis, we found that the protein levels of the full-length (P)RR (37-kDa band) were decreased in the renal medulla of chronic Ang II–infused rats. This finding was associated with increased protein levels of the s(P)RR in these tissues, suggesting that posttranscriptional changes in the intracellular processing of the (P)RR can be induced by a mechanism dependent on Ang II. Recently, it has been revealed that s(P)RR can be generated subcellularly by a furin-mediated cleavage.12 Moreover, it has been shown that Chinese hamster ovary cells exhibit the 28-kDa form, the amino-terminal fragment of the (P)RR on the endoplasmic reticulum and in the vesicle-like structures, but not on the plasma membrane, and that this form can be secreted into the extracellular space.21 In the present study, we found that furin protein levels were upregulated in inner medullary tissues of Ang II–infused rats compared with sham-operated rats. However, it cannot be excluded that other intracellular proteases may also be involved in the processing of (P)RR12 in this model of experimental hypertension.

Schefe et al22 reported that the promyelocytic leukemia zinc-finger transcription factor interacts directly with the C-terminal domain of the (P)RR, which is then translocated into the nucleus and represses transcription of the (P)RR.22 Accordingly, we found decreased protein levels of the full-length (P)RR (37-kDa band) and (P)RR immunoreactivity in the renal medulla of Ang II–infused rats. These findings contrasted with the augmentation of (P)RR mRNA levels in these tissues, further suggesting that posttranscriptional modifications of the full-length (P)RR occur to promote the formation of the s(P)RR in the renal medulla with its augmented secretion into the tubular fluid. It has been proposed that some soluble receptors behave like agonists, in which the complexes formed by the ligand and the soluble receptor target a second receptor on specific cells.23 The present study supports the notion that the (P)RR in chronic Ang II–dependent hypertensive rats contributes to increased intratubular Ang II formation by binding renin or (pro)renin produced and secreted by the neighboring principal cell,35 thus anchoring renin and/or (pro)renin at the cell surface, which may help to prevent or minimize renin washout into the urine.1 Furthermore, the secretion by the intercalated cells of the s(P)RR form into the tubular fluid may also increase binding to renin or (pro)renin, thereby enhancing even further the intraluminal conversion of AGT to Ang I and ultimately to Ang II, because Ang-converting enzyme is also present in the CD.24,25 Indeed, we found that the chronic Ang II–infused rats exhibited higher urinary renin activity than sham-operated rats. Shao et al16 demonstrated recently that the increases in intrarenal Ang II levels during chronic Ang II infusions involve substantial stimulation of endogenous Ang II formation, which contributes to overall augmentation of intrarenal and intratubular Ang II content.16 Increased urinary excretion of endogenous Ang II in Val5-Ang II–infused rats has been primarily attributed to an Ang II type 1 receptor–dependent mechanism and/or de novo formation of Ang II within the tubular lumen, because cotreatment with candesartan significantly prevented this response.26 Gonzalez-Villalobos et al,27,28 reported that Ang-converting enzyme inhibition with lisinopril (100 mg/L) in mice chronically infused with Ang II at a dose of 400 ng/kg per minute ameliorated mean arterial pressure and plasma and intrarenal Ang II concentrations. Importantly, these mice exhibited downregulation of renin expression in the CD cells.28 Taken together, the demonstration of the presence of s(P)RR and increased renin activity in the urine of Ang II–infused rats supports a functional role for the soluble (P)RR in the tubular fluid facilitating the generation of Ang I from AGT delivered to distal nephron segments from the proximal tubule during Ang II–dependent hypertension.

It has been shown that, in contrast to the species specificity for AGT cleavage, the binding of renin and (pro)renin to (P)RR shows an unpredicted low species specificity.29 Feldt et al29 showed that mouse (P)RR is able to bind human renin and (pro)renin. In the present study, we added 2.2 pmol/μL of hPR to urine samples from Ang II–infused and sham-operated rats to examine whether the soluble form of (P)RR was able to increase renin activity in the presence of renin substrate tetradecapeptide. Indeed, the renin activity in urine samples from Ang II–infused rats, in the absence of protease inhibitors, markedly increased. It has been reported that binding of renin to the (P)RR induced a 4-fold increase of the catalytic efficiency of AGT conversion to Ang I.30 The concentration of hPR used in these assays was markedly higher than the value of equilibrium dissociation constant reported for rat (P)RR and human (pro)renin, which is 3.7 nmol/L.31 These observations are of great relevance in light of recent demonstrations of renin upregulation in distal nephron segments of Ang II–dependent hypertensive rats4,7 and renin and/or (pro)renin secretion by CD cells,3,5 altogether providing a mechanistic basis for enhanced tubular Ang II formation in hypertension. The combined action of these RAS components on AGT delivered to the distal nephron segments might be key factors to explain the increases in distal tubular formation of Ang II during Ang II–dependent hypertension.16,32

Perspectives

The results of the current study provide evidence of a functional role for the soluble form of the (P)RR to enhance intrarenal and intratubular Ang II generation. Because the s(P)RR in the urine is primarily derived from the CD segments, the findings support an increased intraluminal renin activity, which may contribute to increased Ang II formation. The existence of an inducible and functional form of the s(P)RR in the renal medullary tissues and in the urine of chronic Ang II–infused rats reflects increased s(P)RR in CD tubular fluid and further supports the potential role of this soluble form to enhance intratubular renin activity in hypertension. The recent in vivo demonstration that increased urinary Ang II concentrations in mice infused chronically with Ang II enhance distal sodium reabsorption32 emphasizes further the importance that renin and (P)RR may have in the distal nephron segments contributing to the increases in intratubular Ang II formation, thus allowing for a greater distal tubular sodium reabsorption leading to progression of hypertension.

Acknowledgments

We extend thanks to Dr Genevieve Nguyen (College of France, Paris, France) for generously providing the (P)RR antibody. Also, we thank Victoria L. Martin and Kimberly Kavanagh for their excellent technical assistance.

Sources of Funding

This work was supported by the Institutional Developmental Award Program of the National Center for Research Resources (P20RR-017659), American Heart Association (09BGIA2280440), and the Eunice Kennedy Shriver National Institute of Child Health and Human Development (K12HD043451). A.A.G. is a recipient of a Comisión Nacional de Investigación Científica y Tecnológica postdoctoral fellowship from Chile. L.S.L. is a recipient of a Coordinación de Apoyo de Personas de Educación Superior Postdoctoral Fellowship from Brazil.

Disclosures

None.

Footnotes

Correspondence to Minolfa C. Prieto,
Department of Physiology, Tulane University, School of Medicine, 1430 Tulane Ave, SL39, New Orleans, LA 70112
. E-mail

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