Natriuretic peptide receptor A mediates renal sodium excretory responses to bloo - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:46 |只看该作者 |倒序浏览 |打印

作者：Shang-Jin Shi, Elangovan Vellaichamy, So Yeon Chin, Oliver Smithies, L. Gabriel Navar,  Kailash N. Pandey,作者单位：Department of Physiology and Hypertension and Renal Center of Excellence, TulaneUniversity Health Sciences Center School of Medicine, New Orleans, Louisiana70112; and Department of Pathology, University ofNorth Carolina, Chapel Hill, North Carolina 27599

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      【摘要】- Z( t8 F5 s. K9 \
   The deficiency of Npr1 [genetic determinant of natriuretic peptide receptorA (NPRA)] increases arterial pressures and causes hypertensive heart diseasein mice similar to those seen in untreated human hypertensive patients.However, the quantitative role of NPRA in mediating the renal responses toblood volume expansion remains uncertain. To determine the specificcontribution of NPRA in mediating the signaling mechanisms responsible for natriuretic and diuretic responses to nondilutional intravascular expansion,we administered whole blood to anesthetized Npr1 homozygous null mutant(0-copy), wild-type (2-copy), and gene-duplicated (4-copy) mice. In wild-type(2-copy) animals, urinary flow (µl ·min - 1 · g kidneywt - 1 ) increased from 4.9 ± 1.0 to 14.4± 1.8 and sodium excretion (µeq ·min - 1 · g kidney wt - 1 ) from 1.15 ± 0.22 to 3.11 ±0.60, associated with a rise in glomerular filtration rate (GFR; ml ·min - 1 · g kidneywt - 1 ) from 0.63 ± 0.03 to 0.82 ±0.09 and renal plasma flow (RPF; ml · min - 1 · g kidney wt - 1 ) from 2.96 ± 0.17 to4.36 ± 0.41, whereas arterial pressure did not significantly increase.After volume expansion, 0-copy mice showed significantly lesser increases inurinary flow ( P P even though the increases in arterial pressures were greater ( P augmented responses in urinary flow ( P excretion ( P P 0.01) and RPF ( P These results establish that NPRA activation is the predominant mechanismmediating the natriuretic, diuretic, and renal hemodynamic responses to acuteblood volume expansion. 5 V. c. f( I* }  N0 K9 ^$ J" ^' o
      【关键词】 sodium excretion cGMP gene disruption gene duplication
   IN RESPONSE TO AN increase in atrial distension, the peptide hormone atrial natriuretic peptide (ANP) is released into the circulation andelicits natriuresis, diuresis, and vasodilation ( 8, 25 ). Acting on natriureticpeptide receptors, ANP inhibits salt and water reabsorption in proximal tubuleand inner medullary collecting duct cells and inhibits renin and vasopressinrelease as well as aldosterone synthesis and secretion( 1, 4, 7, 47 ).% {( T' j9 y( W# H% f; z# P
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Natriuretic peptides belong to a family of three homologous peptidehormones. ANP and brain natriuretic peptide (BNP) are released by the heart;and C-type natriuretic peptide (CNP) is produced in endothelial cells( 10, 20, 46 ). All three natriureticpeptides are thought to exert important roles in the maintenance of bloodpressure and cardiovascular homeostasis. Distinct natriuretic peptidereceptors have been identified and characterized by molecular cloning( 39, 41 ). These include natriureticpeptide receptor A, B, and C, also designated as NPRA, NPRB, and NPRC,respectively ( 11, 19, 33 ). Both ANP and BNPspecifically bind to NPRA, whereas CNP binds to NPRB; nevertheless, all threenatriuretic peptides show affinity to NPRC. The hormone binding to NPRA andNPRB results in the production of intracellular second messenger cGMP byguanylyl cyclase activity that resides in the intracellular domains of thesereceptors ( 22, 36, 43 ). NPRA is thought to be the primary ANP/BNP signaling molecule and has been suggested as the principalmediator of natriuretic peptide activities.

Previous experimental data established that ANP plays an important role inregulation of renal function by its vasodilatory and natriuretic responses andits ability to counteract the renin-angiotensin-aldosterone system in atissue-specific manner ( 25 ).Attempts have been made to define physiological responses of ANP using several experimental approaches. It has been possible to correlate the effects ofchanges in blood hormone levels commensurate with those found inpathophysiological states by infusing the exogenous hormones( 40 ). Cardiac appendectomy hasbeen used to prevent ANP release; however, the problem in this setting is thatthe missing normal cardiac function results in a lack of physiological reflexes that are normally elicited by atria( 42 ). Other studies usedmonoclonal antibodies against circulating ANP and agents that specificallyinhibit the signaling pathway of NPRA by blocking cGMP production. Althoughtwo compounds, A-71915 and HS-142-1, have been shown to diminish theeffect of ANP by antagonizing NPRA, these compounds do not completely inhibit NPRA and may have nonspecific effects( 9, 30 ). Gene-targeting strategiesin mice provide novel approaches in the study of the physiological responsescorresponding to gene dosage in vivo( 15, 45 ).; r) R/ i1 U$ F: |
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Genetic mouse models with disruption of the ANP/NPRA system have providedstrong support for a physiological role of this hormone-receptor system in theregulation of arterial pressure and other pathophysiological functions( 13, 14, 21, 24, 31, 37, 44 ). Therefore, the geneticdefects that reduce the activity of ANP and its receptor system can beconsidered as candidate contributors to essential hypertension and congestive heart failure ( 12, 14, 17, 18, 31, 44, 48 ). To examine the regulatoryrole of NPRA in kidney function and blood pressure homeostasis at themolecular level, we performed studies evaluating the changes in renal functionusing Npr1 (coding for NPRA) gene-disrupted and gene-duplicated mutant mousemodels. We hypothesized that the quantitative genetic alterations in NPRA expression levels in vivo mediate the primary ANP signaling mechanismresponsible for the natriuretic and hemodynamic responses to intravascularexpansion. To test this hypothesis, we administered whole blood to nullhomozygous mutant (0-copy), wild-type (2-copy), and gene-duplicated (4-copy)mice to produce intravascular volume expansion not accompanied byhemodilution.
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MATERIALS AND METHODS; G$ V. Y) s  L1 B& W2 y

Generation of mice and genotyping. Npr1 gene-disrupted and gene-duplicated mice were generated by homologous recombination in embryonicstem cells as previously described( 31, 32 ). Animals were bred andmaintained at the Animal Care Facility of Tulane University Health SciencesCenter and handled under protocols approved by the Institutional Animal Careand Use Committee. Npr1 genotypes used in the present studies were littermate progenies of a mixed 129/C57BL6 genetic background and have been designated asfollows: homozygous mutant allele (-/-; 0-copy), wild-type allele( / ; 2-copy), and gene-duplicated allele (  /  ; 4-copy). The breeding of1-copy ( /-) heterozygous animals generated progenies consisting of 0-, 1-, and 2-copy mice. These animals were genotyped by multiple PCR analysis ofDNA isolated from tail biopsies using primer A (5'-GCT CTC TTGTCG CCG AAT CT-3'), corresponding to a sequence 5' to the mouseNpr1 gene common to both alleles (2-copy); primer B (5'-TGT CACCAT GGT CTG ATC GC-3'), corresponding to an exon 1 sequence only presentin the intact mouse allele (1-copy); and primer C (5'-GCT TCCTCG TGC TTT ACG GT-3'), a sequence in the neomycin resistance cassetteonly present in the null allele (0-copy). The PCR reaction from tail DNA included 50 mM Tris · HCl (pH 8.5), 20 mM ammonium sulfate, 1.5 mMMgCl 2, 10% DMSO, 100 µM each of dNTPs, 2 U of Taq DNApolymerase, and 40 nM primers. The PCR for 0-, 1-, and 2-copy mice wasperformed by the use of a 60-s denaturation step at 94°C, a 60-s annealingstep at 60°C, and 60-s extension step at 72°C, respectively, for 35cycles using DNA Thermal Cycler 480 as previously described( 44 ) with modifications. PCRproducts were resolved on 2% agarose gels with the endogenous band of 500 bpand targeted band of 200 bp ( Fig.1 A ).

Fig. 1. A : analysis of genomic tail DNA obtained from the 0-, 1-, and2-copy mice demonstrating the deletion event of Npr1 gene ( lanes 2, 3, and 4 ). B : analysis of tail DNA obtained fromthe 2-, 3-, and 4-copy mice showing the duplication event of Npr1 gene( lanes 2, 3, and 4 ). Lane 1 represents DNAmarkers in both A and B.* h- l7 N/ z2 @8 a! J  `2 c
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The breeding of gene-duplicated 3-copy (  / ) heterozygous mice generatedprogenies consisting of 2-, 3-, and 4-copy animals. The Npr1 gene-duplicatedmice were genotyped using upstream (5'-CCT CTA GAT GCA TAC ATG TCGC-3') and downstream (5'-GGT CAA GTT AAG TGT ATT TTT TTCCC-3') primers. The PCR reaction from tail DNA included 10 mM Tris· HCl, pH 8.3, 50 mM KCl, 4 mM MgCl 2, 0.4 mM each of dNTPs,2.5 U of Taq DNA polymerase, and 40 nM primers. The PCR was performedby the use of a 30-s denaturation step at 94°C, a 30-s annealing step at60°C, and a 30-s extension step at 72°C, respectively, for 35 cyclesas previously described ( 32 )with modifications. The amplified genomic fragments were separated onnondenaturing acrylamide gels and corresponded to 108- and 124-bp bands from 2- and 4-copy genotypes, respectively, whereas the heterozygous (3-copy)animals contained both 108- and 124-bp amplified genomic fragments( Fig. 1 B )., T/ J2 a- r7 n- m# T1 @
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Animal preparation. The mice were housed under a 12: 12-hlight-dark cycle at 25°C and fed regular chow (Purina Laboratory) and tapwater ad libitum. In the present experiments, 16- to 24-wk-old male miceweighing 30-35 g were used. Animals were anesthetized with Inactin (100mg/kg ip thiobutabarbital sodium salt). A supplemental dosage of anesthetic (5mg/kg im ketamine) was administered as required. The animals were placed on aservo-controlled surgical table that maintained body temperature at 37°C, and a tracheotomy was performed. The animals were allowed to breathehumidified 95% O 2 -5% CO 2 by placing the exterior end ofthe tracheal cannula inside a small plastic chamber. The right jugular veinwas catheterized with PE-10 tubing for fluid infusion. After catheterization,0.9% NaCl containing 10% Inutest (Laevasom-Gesellschoft, Ling, Austria), 3%PAH (Sigma), and 1% BSA was infused at a rate of 2.5 µl/min. The left carotid artery was cannulated (PE-10 tubing connected to PE-50 tubing) formeasurements of arterial pressure. Blood pressures were recorded on a pressuretransducer connected to a Grass polygraph (Grass Instrument, Quincy, MA).Blood pressures were determined continuously throughout the duration of theexperiment. The bladder was catheterized with PE-50 tubing via a supra pubic incision for urinary collections as previously described( 6 ). After equilibration for 45min, eight consecutive 20-min urinary collections were obtained. At the end ofthe experiment, blood was collected from the carotid artery into hematocrittubes and chilled tubes containing 5 µl of 0.2 M sodium-EDTA and wasimmediately centrifuged at 4°C. Plasma was removed and stored at-80°C until used to assay ANP, total protein content, Inutest, andPAH concentrations.
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Volume expansion. Npr1 homozygous mutant (0-copy; n = 9),wild-type (2-copy; n = 9), and gene-duplicated (4-copy; n =9) mice received whole blood obtained from age-matched 1-copy ( n =18) or 3-copy ( n = 9) male donor mice. The donor animals wereanesthetized with Inactin (100 mg/kg ip), and the whole blood was drawn by cardiac puncture using a heparinized syringe. Hemodynamic and kidney functionswere determined before (0-60 min), during (60-80 min), and after(80-160 min) blood volume expansion. Before the volume expansion period(0-60 min), three initial consecutive 20-min urine collections and blood pressure measurements were performed, after which the blood volume expansionwas carried out beginning at 60 min. Fresh whole blood was infused into 0-,2-, and 4-copy mice over a 20-min volume expansion period (60-80 min) toexpand the circulating blood volume by an estimated 15% (450 µl, 1.5%of body wt). Five consecutive 20-min recovery (60-160 min) bloodpressure measurements and urinary collections were performed during and afterthe blood infusion. One hundred microliters of blood were collected at 60 and100 min, and total blood was collected at 160 min after the completion of theexperiment.

Determination of plasma and urinary inulin (Inutest) and PAH. Tenmicroliters of plasma were mixed with 110 µl of 3.2% TCA, centrifuged at3,000 rpm for 30 min, and the supernatant was collected. Each 5 µl ofcollected urine were diluted with 9.995 ml of water. For Inutest measurements,25 µl of TCA-precipitated plasma supernatant, diluted urine samples, orInutest standards were added to a 96-well plate. Two hundred fifty microlitersof 0.1% anthrone were added to each well, and samples were incubated at60°C for 10 min. The plates were counted at 620-nm wavelength with amultiscan plate reader (Lab System, Franklin, MA). Similarly, for PAHmeasurement, 50-µl samples and PAH standards were added to a 96-well plate.Simultaneously, water (150 µl), 0.2 N HCl (40 µl), 0.1% sodium nitrite(20 µl), 0.5% ammonium sulfamate (20 µl), and 0.1% N -(naphthyl)ethylenediamine (20 µl) were added to each well. After each addition, theplate was shaken and then counted at 540 nm. Glomerular filtration rate (GFR)was calculated as urine-to-plasma Inutest concentration ratio times urinaryflow and was factored per gram kidney weight. Renal plasma flow (RPF) wascalculated as urine-to-plasma PAH concentration ratio times urinary flow andwas factored per gram kidney weight. The plasma protein concentrations ofdonor and recipient mice were determined in a 96-well plate using a Bio-Radprotein assay kit (Hercules, CA).

Analytic procedures. The hematocrit of each blood sample from donor or recipient mice was measured after centrifugation with Adams Autocrit.Urinary volumes were determined by gravimetry. The sodium and potassiumconcentrations of all samples were determined by flame photometry (model 443,Instrumentation Laboratory). Fractional sodium excretion was calculated by dividing the urine/plasma sodium concentration ratio by the urine/plasmaInutest ratio. Similarly, fractional potassium excretion was calculated bydividing the urine/plasma potassium concentration ratio by the urine/plamaInutest ratio.
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Urinary cGMP assay. Ten microliters of acetylated urine samples were mixed with 90 µl of 0.05 M sodium acetate buffer, pH 6.2, and cGMP inthe samples was determined with a radioimmunoassay kit (Peninsula, Belmont,CA) as previously described( 35 ).
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Plasma ANP assay. A plasma sample (400 µl) was applied toSep-Pak C-18 column that was prewashed with methanol and 0.1% trifluoroaceticacid (TFA). The column was washed with 5 ml of 0.1% TFA and eluted with a 3-mlmixture of methanol:water:TFA (80:19.9:0.1) as previously described( 34, 44 ). The eluates were broughtto dryness in a speed vac centrifuge. The residues were dissolved into 100 mMTris buffer, pH 7.5, containing 2.5 mM EDTA, 1 mM PMSF, 0.02% sodium azide,and 0.1% BSA. ANP was quantitated using ANP antibodies (Peninsula;10,000 x dilution) and 125 I-ANP as previously described( 38 ). One hundred-microliter plasma samples were mixed with 100 µl antibody at 4°C for 48 h, afterwhich 100 µl 125 I-ANP (15,000 cpm) were added and samples wereincubated further for 24 h at 4°C. On day 4, 100 µl (500-folddilution) goat anti-rabbit immunoglobulin serum and 10 µl (500-folddilution) normal rabbit serum were added and mixed. After centrifugation at 1,500 g for 30 min at 4°C, the supernatant was aspirated andradioactivity in the pellet was counted with a gamma counter.

Statistical analyses. The results are presented as means ± SE. Statistical analyses were performed by factorial one-way ANOVA followed bythe Student-Newman-Keuls post hoc test for multiple comparisons (GraphPadInstat Software, GraphPad Software, San Diego, CA). For all tests, statisticalsignificance was set at P+ \7 M/ X% B5 M5 a* H& q5 L3 B, P

RESULTS, [! m/ v9 w2 z! e( R/ n' S

Urinary cGMP and plasma ANP concentrations. As shown in Fig. 2, before volumeexpansion, urinary cGMP excretion rates (pmol ·min - 1 · gkidney - 1 ) were significantly lower in 0-copy mice(3.5 ± 0.9; P higher in 4-copymice (14.7 ± 1.3; P with 2-copy wild-typemice (7.6 ± 1.0). Interestingly, during volume expansion, urinary cGMPexcretion rates were dramatically increased in both 2- and 4-copy mice;however, the cGMP values were significantly higher in 4-copy mice (27.8 ± 2.1; P (14.2 ± 1.9). On the other hand, the urinary cGMP excretion rate wassignificantly reduced in 0-copy mice (1.0 ± 0.05; P excretion rates were 0.7± 0.1, 3.6 ± 1.6, and 10.5 ± 2.0 in 0-, 2-, and 4-copymice, respectively.* s) ~" g- `  M2 K/ c, B0 Y( X

Fig. 2. Effect of volume expansion on urinary cGMP excretion in Npr1 homozygousnull mutant (0-copy; n = 9), wild-type (2-copy; n = 9), andgene-duplicated (4-copy; n = 9) mice. Blood volume expansion wasadministered for a 20-min period (between 60 and 80 min). Urine was collectedat each 20-min interval throughout the entire duration of the experiment(0-160 min). The contents of cGMP in urinary samples were determined byradioimmunoassay. KW, kidney weight. * P P; R/ k( K. V# \1 [% `+ n# t

Figure 3 shows that aftervolume expansion, the plasma ANP concentrations (pmol/ml) were significantlyhigher in 0-copy mice (2.5 ± 0.2; P ± 0.2) and 4-copy (0.7 ± 0.1) mice,respectively.% k- ~: F8 X4 G' C% e2 d- M
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Fig. 3. Effect of volume expansion on plasma atrial natriuretic peptide (ANP)levels in Npr1 homozygous mutant (0-copy), wild-type (2-copy), andgene-duplicated (4-copy) mice. Blood was collected from the cannulated carotidartery at the end of the volume expansion experiment. Plasma was separated andANP levels were determined by radioimmunoassay. The number of animals used ineach experiment is indicated within the vertical bars ( n = 9).* P P P  P. X" Q- e; f, j/ ?1 A, z& Z; K, ]

Plasma protein concentrations and hematocrits. The plasma protein concentrations were not significantly different among either donor (1- and3-copy) or recipient (0-, 2-, and 4-copy) mice ( Fig. 4 A ). Similarly,there were no significant differences in hematocrits in the donor mice.However, after volume expansion, the hematocrits in recipient 2- and 4-copymice were significantly higher compared with recipient 0-copy mice( Fig. 4 B ).% r! f0 d$ R9 J! A/ z  u0 x! b
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Fig. 4. Level of plasma protein ( A ) and hematocrit ( B ) in 1- and3-copy donor mice and 0-, 2-, and 4-copy recipient mice. The number of animalsused in each experiment is indicated within the vertical bars ( n =9). * P P- A; g1 v+ b+ C, Z
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Mean arterial blood pressures before, during, and after whole bloodvolume expansion. The mean arterial pressures during all conditions were30-40 mmHg higher in Npr1 homozygous null mutant (0-copy) mice than inwild-type (2-copy) mice ( Fig.5 ). In contrast, the mean arterial pressures were 15-20 mmHglower in Npr1 gene-duplicated (4-copy) mice than in wild-type (2-copy) controlanimals. Before volume expansion (0-60 min), the mean arterial pressures(mmHg) were significantly higher in 0-copy mice (129 ± 4; P lower in 4-copy mice (77 ± 2; P with 2-copy wild-type mice (92 ± 3). During volumeexpansion (60-80 min), the mean arterial pressures increased and remained at significantly higher levels in 0-copy mice (140 ± 4; P levels in 4-copymice (90 ± 3; P with 2-copy wild-type mice(108 ± 3). Even after volume expansion (80-160 min), the meanarterial pressures remained at significantly higher levels in 0-copy mice (136± 4; P mice (81 ± 3; P animals (95 ± 4).7 d0 t: D! x* [7 H' R2 x4 p8 R4 y
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Fig. 5. Effect of volume expansion on mean blood pressures in Npr1 homozygousmutant (0-copy; n = 9), wild-type (2-copy; n = 9), andgene-duplicated (4-copy; n = 9) mice. Mean blood pressures measuredby cannulated carotid arterial methods were continuously recorded for 160 minthroughout the duration of the experiment. ** P P, K2 E( f. d2 t" A3 B

GFR and RPF. As shown in Fig.6 A, before volume expansion, the baseline (0-60min) GFR (ml · min - 1 · g kidneywt - 1 ) was significantly lower in 0-copy mice (0.49± 0.03; P in 4-copy mice(0.81 ± 0.04; P 2-copy wild-typecounterparts (0.63 ± 0.03). During the volume expansion period, GFRincreased in all groups, which was maintained at a significantly lower levelin 0-copy mice (0.58 ± 0.04; P level in 4-copy mice (1.19 ± 0.12; P with 2-copy wild-type animals (0.82 ± 0.09).Nevertheless, after volume expansion, GFR still remained at a significantly lower level in 0-copy mice (0.49 ± 0.05; P and at asignificantly higher level in 4-copy mice (0.87 ± 0.08; P (0.68 ± 0.06).
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Fig. 6. Effect of volume expansion (VE) on glomerular filtration rate (GFR; A ) and renal plasma flow (RPF; B ) in Npr1 null mutant(0-copy; n = 9), wild-type (2-copy; n = 9), andgene-duplicated (4-copy; n = 9) mice. Both GFR and RPF were measuredin 3 consecutive periods at before (0-60 min), during (60-80 min),and after (80-160 min) pure blood VE. * P P+ \4 J+ B4 c/ i' B8 }2 X, s  w; C

Similarly, as shown in Fig.6 B, the baseline RPF (ml · min - 1 · g kidneywt - 1 ) was significantly lower in 0-copy mice (2.24± 0.41; P significantly higher in 4-copy mice(4.00 ± 0.49; P 0.17). During volume expansion, the RPF remained at asignificantly lower level in 0-copy mice (2.72 ± 0.37; P and increased significantly in 4-copy mice (5.78 ± 0.50; P ± 0.41). Again, after volume expansion, the RPF was lower in 0-copymice (2.29 ± 0.30; P significantlyelevated in 4-copy mice (4.48 ± 0.47; P 0.34).

Urinary flow and urinary sodium and potassium excretion. During volume expansion, urinary flow (µl ·min - 1 · g kidneywt - 1 ) in 2-copy animals increased from 4.9± 1.0 to 14.4 ± 1.8 and sodium excretion rate (µeq ·min - 1 · g kidneywt - 1 ) from 1.15 ± 0.22 to 3.11 ±0.60 ( Fig. 7, A and B ). In contrast, 0-copy animals exhibited only a smallchange in urinary flow (3.4 ± 0.2 to 5.0 ± 1.0; P 0.001) and sodium excretion (0.69 ± 0.21 to 1.10 ± 0.18; P pressures.Interestingly, 4-copy mice showed significantly higher urinary flow (6.5± 0.6 to 24.0 ± 2.5; P ± 0.60; P ( Fig. 7, A and B ). Even after volume expansion, both 2- and 4-copymice had significantly higher urinary flows and sodium excretion ratescompared with 0-copy mice. In contrast, urinary potassium excretion rates werenot significantly different in any of the Npr1 genotypes before, during, andafter volume expansion and were not increased by blood volume expansion ( Fig. 7 C ).8 e" c2 l- ^# w# D- e) k
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Fig. 7. Effect of volume expansion on urinary flow ( A ), urinary sodiumexcretion ( B ), and urinary potassium excretion ( C ) in Npr1homozygous mutant (0-copy; n = 9), wild-type (2-copy; n =9), and gene-duplicated (4-copy; n = 9) mice. Pure blood volumeexpansion was administered in a 20-min period (60-80 min). Urinaryvolume was collected at a 20-min period throughout the entire duration of the160-min experiment at before, during, and after volume expansion. * P P P; e: O2 g% j) G6 x9 h

As shown in Fig. 8, beforevolume expansion, the percent baseline fractional sodium excretions remainedat a significantly lower level in 0-copy mice (0.76 ± 0.14%) and werenot significantly different in 4-copy (1.51 ± 0.14%) mice compared with 2-copy (1.32 ± 0.13%) wild-type mice. However, during volume expansion,fractional sodium excretions were significantly elevated in 4-copy mice (3.65± 0.22%; P and remained significantly lower in0-copy mice (1.23 ± 0.16%; P (2.45 ± 0.16%). After volume expansion, fractionalsodium excretions were still significantly higher in 2-copy (1.64 ± 0.16%) and 4-copy (1.90 ± 0.18%) mice compared with 0-copy mice (1.14± 0.15%). No significant differences in fractional potassium excretionswere observed among the three groups of Npr1 mice (data not shown).( J7 t$ e4 ~+ P- i' P' p

Fig. 8. Effect of VE on fractional sodium excretion in Npr1 homozygous mutant(0-copy; n = 9), wild-type (2-copy; n = 9), andgene-duplicated (4-copy; n = 9) mice. * P P

DISCUSSION

The present study examined the role of NPRA in the renal and arterialpressure responses to blood volume expansion in Npr1 homozygous null mutant(0-copy), wild-type (2-copy), and gene-duplicated (4-copy) mice. The Npr1gene-disrupted (0-copy) mice showed the highest blood pressures, whereas theNpr1 gene-duplicated (4-copy) mice had the lowest blood pressures comparedwith wild-type (2-copy) mice. During the period of volume expansion with wholeblood infusion, the mean arterial blood pressures increased in all threegenotypes; however, the mean arterial pressures were always significantlylower in 4-copy mice and significantly higher in 0-copy mice compared with2-copy animals. Because of the slow infusion rate, the changes in arterial pressure in response to blood volume expansion were modest, although theyappeared to be slightly greater in 2- and 4-copy mice. The renal hemodynamicfunction was different in the three groups. GFR was 25 to 35% lower in 0-copymice and 30 to 45% higher in gene-duplicated (4-copy) mice compared with2-copy wild-type control mice. Similarly, RPF was 25 to 30% lower in 0-copymice and 45 to 70% higher in 4-copy mice compared with 2-copy control animals.It is noteworthy that 4-copy mice had higher GFR and RPF values during thevolume expansion period, whereas 0-copy mice showed significantly lower GFR orRPF responses to the volume expansion. Furthermore, during the volumeexpansion, 2-copy wild-type animals exhibited significantly higher urinary flow and sodium excretory responses compared with 0-copy null mutant micedespite the greater mean arterial pressures in the 0-copy mice. Interestingly,4-copy mice showed even greater urinary flow and sodium excretory responsesthan 2-copy mice. Urinary potassium excretion rates were not significantlydifferent among three Npr1 genotypes before, during, or after volume expansion.
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A number of factors influence the kidney's ability to excrete sodium andwater in response to blood volume expansion( 2, 3, 23, 27, 28 ). Activation of natriureticsystems such as natriuretic peptides (ANP, BNP) and nitric oxide enhances the pressure-natriuresis relationship and reduces arterial pressures. It has alsobeen suggested that chloride-mediated feedback control of NPRA occurs in thekidney and probably plays a role in the regulation of ANP-mediated natriuresis( 26 ). Initial studies haveshown that ANP suppresses renin and decreases blood pressures( 5, 24, 29 ). Our earlier findings withNpr1 gene-disrupted mice demonstrated that, at birth, the absence of NPRAallows greater renin and ANG II levels and increased renin mRNA expressioncompared with 2-copy control mice( 44 ). However, at 3-16wk of age, both circulating and kidney renin and ANG II levels were decreaseddramatically in 0-copy mice compared with 2-copy control animals. Thisdecrease in the renin activity in adult 0-copy mice could be due toprogressive elevation in arterial pressures leading to inhibition of renin synthesis and release from the kidney juxtaglomerular cells. However,aldosterone levels in 0-copy mice were dissociated from the circulating ANG IIlevels and were elevated. It is also possible that the renin in 0-copy micemight be suppressed as a consequence of aldosterone-mediated sodium retentionwith associated fluid retention( 44 ). These and other varioussystems could be responsible for the differences in basal values among thethree groups.
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Previous studies suggested that increased levels of ANP released into theplasma in response to blood volume expansion in rats were mainly responsiblefor the natriuretic and diuretic responses ( 3, 4, 40 ). Furthermore, Paul et al.( 40 ) showed that both ANP andacute blood volume expansion act on the kidney through a similar saturablemechanism. However, direct evidence demonstrating the quantitativecontribution of ANP working through NPRA to the natriuresis and diuresisresulting from an isohemic, isooncotic blood volume expansion has not beenobtained. In a study using Npr1 homozygous null mutant and wild-type mice, itwas demonstrated that infusion of ANP, while causing substantial natriuresis and diuresis in wild-type mice, did not cause significant increases in sodiumexcretion or urinary flow in NPRA-deficient mice ( 16 ). Furthermore, urinaryflow and sodium excretion rapidly increased in response to volume expansionwith albumin containing Ringer solution in wild-type (2-copy) mice comparedwith homozygous null mutant (0-copy) mice. In the present study, we examined the quantitative contribution and possible mechanisms mediating the responsesof NPRA by determining the RPF, GFR, urinary flow, and sodium and potassiumexcretion patterns following blood volume expansion in 0-, 2-, and 4-copy micein a Npr1 gene dose-dependent manner. By using whole blood, hemodilution didnot occur and plasma protein levels were not reduced. Thus other natriureticmechanisms related to plasma dilution, such as decreases in colloid osmoticpressure or decreases in hematocrit, were not activated with this protocol.Although the blood volume expansion stimulated the release of ANP in all threeNpr1 genotypes of mice, significant functional responses occurred only in 2- and 4-copy mice but not in 0-copy animals. These results demonstrate that theANP/NPRA axis is primarily responsible for mediating the renal hemodynamic andsodium excretion responses to intravascular blood volume expansion.Furthermore, the sodium excretion responses appear to be due to the combinedcontribution of increases in filtered load as well as reductions in tubularfractional reabsorption. The associated changes in urinary cGMP excretion rates are consistent with the activation of tubular NPRA leading to increasedformation of cGMP, which might pass into tubular fluid and be excreted in theurine. In addition, it should be noted that the increase in urinary flow couldbe due, in part, to an inhibition of antidiuretic hormone levels via the low-pressure volume receptors stimulated by intravascular volume expansion.4 y) l* }9 R9 N! _

The finding that the absence of NPRA almost completely prevented the sodiumexcretory responses to blood volume expansion is somewhat surprising in thatthere are multiple systems that respond to volume expansion. An important partof the experimental design was to minimize nonspecific responses that could be associated with hemodilution, reductions in plasma colloid osmotic pressure,and other compositional changes in the blood that could directly affect sodiumreabsorption and/or GFR. In addition, the blood volume infusion period wasextended over a 20-min period to minimize reflexogenic alterations insympathetic tone and volume expansion-mediated inhibition of vasopressin release. In this setting, it was possible to demonstrate the critical rolethat NPRA exerts in mediating the sodium excretory responses to a selectivestimulus associated with blood volume expansion, which is presumably due toincreases in right atrial pressure caused by the blood infusion.4 y' W# P4 x1 t4 D! E8 r

Earlier studies suggested that the ANP/NPRA system plays an important rolein blood pressure homeostasis by direct natriuretic, diuretic, andvasodilatory actions on the kidneys( 4, 25 ). ANP-deficient geneticstrains of mice demonstrated that a defect in ANP synthesis can causehypertension in homozygous null mutant mice with no circulating or cardiac ANP( 14 ). Therefore, geneticdefects that reduce the activity of the natriuretic peptide system can beconsidered as candidate contributors to essential hypertension. Mice lackingthe ANP gene function and kept on a high-salt diet (8% NaCl) showedhypertension with increased arterial pressures of 22 mmHg, whichsuggested that genetically reduced production of ANP can lead tosalt-sensitive hypertension( 14 ). Npr1 gene-deficient miceused in the present study exhibited a higher mean arterial pressure onhigh-salt diet compared with animals kept on medium- or low-salt diets ( 32 ). The absence of NPRAexpression in 0-copy mice provoked salt-sensitive increases in bloodpressures, whereas an increased expression of NPRA in 4-copy mice was able tolower the blood pressures and protected against high dietary salt intake.These previous studies provide evidence that ANP can be considered a majorsystem contributing an important role in the regulation of blood volume andaltered blood pressure. However, it should be acknowledged that Lopez et al.( 21 ) failed to showsalt-sensitive hypertension in their Npr1 gene-deficient mice. Thus it is likely that ANP/NPRA axis induces hemodynamic and excretory parametersincluding sodium excretion, which reduces the intravascular fluid volume andis thus responsible for the decreased blood pressures observed ingene-duplicated (4-copy) mice. Our data demonstrate that ANP/NPRA axis servesan important mediator in acute natriuresis and diuresis after blood volumeexpansion.
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In conclusion, our present results demonstrate that both GFR and RPF weresignificantly lower in 0-copy and higher in 4-copy mice compared with 2-copywild-type animals before, during, and after volume expansion. The data showthat ANP responses to volume expansion led to the significantly lesserexcretion of sodium and water in 0-copy mice and significantly greater excretory responses along with reduced tubular reabsorption in 4-copy micecompared with 2-copy (wild type) mice. Similarly, during the volume expansion,urinary cGMP concentration was significantly lower in 0-copy mice and greaterin 4-copy mice compared with 2-copy control animals. Thus the higher cGMP concentrations in urinary samples were increased corresponding to Npr1 genecopy numbers. Our findings establish that NPRA is critical in mediating thenatriuresis, diuresis, and renal hemodynamic responses to acute blood volumeexpansion.
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DISCLOSURES
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This research was supported by National Institutes of Health GrantsHL-62147 and HL-26371 and the Louisiana Board of Regents Health ExcellenceFund.8 b# F; {) h1 N- z
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ACKNOWLEDGMENTS

The authors thank H. T. Nguyen for technical assistance and B. M. Harborfor secretarial assistance. We also thank L. Dupepe for help in animal surgeryand tail biopsies.2 R$ @  i7 m  m/ G
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