phosphodiesterase inhibitorscorrect resistance to...

582 Volume 7 ‘ Number 4 ‘ 1996

Phosphodiesterase Inhibitors Correct Resistance toNatriuretic Peptides in Rats with Heymann 1�2

Jean-Pierre Valentin, Wei-Zhong Ving, Leonardo A. Sechi, Kok-Tong Ling, Changbin Qiu,

William G. Couser, and Michael H. Humphreys3

J.-P. Valentin, W.-Z. Ying. K-I. Ling. C. Qiu, M.H. Hum-phreys. Division of Nephrology. San Francisco GeneralHospital. University of California at San Francisco, SanFrancisco. CA

L.A. Sechi, Hypertension Unit. Department of InternalMedicine, University of Udine, Udine, Italy

W.G. Couser. Division of Nephrology, University ofWashington. Seattle, WA

(J. Am. Soc. Nephrol. 1996; 7:582-593)

ABSTRACTExperimental nephrotic syndrome is characterized by

abnormal sodium metabolism, reflected in a bluntednatriuretic response both to volume expansion and to

infused atrial natriuretic peptide (ANP). The studies

presented here examined the relationships amongplasma ANP concentration and urinary sodium(UNaV) and cyclic GMP excretion (UCGMPV) in vivo,and the responsiveness of isolated glomeruli and

inner medullary collecting duct (IMCD) cells to ANP

and urodilatin (renal natriuretic peptide; RNP) in vitroin rats with Heymann nephrltis, an immunologicallymediated model of nephrotic syndrome. Nine to 14

days after ip injection of anti-Fx1A antiserum, rats wereproteinuric and had a blunted natriuretic response to

intravenous infusion of isotonic saline (2% bodyweight, given over 5 mm). Thirty mm after the onset of

the infusion, plasma ANP concentration was in-creased to the same extent in both normal andnephritic rats, compared with their respective hydra-

penic controls. Despite this increase, UCGMPV wassignificantly less in nephritic rats after the saline infu-slon. Accumulation of cGMP by isolated glomeruliand IMCD cells from nephritic rats after incubationwith ANP and RNP was also significantly reduced,

compared with normal rats. This difference was notrelated to differences in either density or affinity ofrenal ANP receptors, but was abolished when accu-mulation of cGMP was measured in the presence of

1 Received November 15, 1995. Accepted December 12, 1995.

2 Portions of this work were previously published in abstract form in din Res

1992;40:181A. and J Am Soc Nephroi 1991 ;3:448A.

3 Correspondence to Dr. M.H. Humphreys, Division of Nephrology. Box 1341,SFGH, University of Caiifornia at San Francisco, San Francisco, CA 94143.

1046.6673/0704.0582$03.OO/OJournal of the American Society of Nephroiogycopyright © 1996 by the American Society of Nephrology

io� M isobutylmethylxanthine orZaprinast, two differ-

ent inhibitors of cyclic nucleotide phosphodiesferases

(PDE). Infusion of Zaprinast into one renal artery innephritic rats normalized both the natriuretic response

to volume expansion and the increase in UCGMPV fromthe infused, but not the contralateral, kidney. Further-more, cGMP-PDE activity was increased in IMCD cell

homogenates from nephritic compared with normalrats (388 ± 32 versus 198 ± 93 pmol/min per mgprotein, P < 0.03). These results indicate that blunted

volume expansion natriuresis accompanied by cellu-lar resistance to ANP in vitro occurs in an immuno-logic model of renal injury. The resistance is notrelated to an alteration in ANP release or binding to itsrenal receptors, but is suppressed by PDE inhibitorsand is associated with increased renal cGMP PDEactivity, thus suggesting that enhanced cGMP-PDEactivity may account for resistance to the natriuretic

actions of ANP observed in vivo. This defect mayrepresent the intrinsic sodium transport abnormalitylinked to sodium retention in nephrotic syndrome.

Key Words: Atrial natriuretic peptide. cyclic GMP, inner med-

ullary collecting duct, phosphodiesterase. renal natriuretic

peptide

T he abnormal sodium metabolism that typifies the

nephrotic state is characterized in part by

blunted volume expansion natriuresis and a resis-

tance to the natriuretic actions of atrial natriuretic

peptide (ANP) (1,2). Although the mechanisms under-

lying these manifestations of abnormal sodium me-

tabohism are not fully understood, they have been

related to heightened renal sympathetic nerve activity

(3,4,5), activation of the renin-angiotensin system

(6,7), elevations in aldosterone activity (8), and re-

duced GFR (9). On the other hand, convincing evi-

dence argues that nephrotic sodium retention is as-sociated with an intrinsic abnormality in renal sodium

handling that leads to heightened sodium reabsorp-

tion (2, 10- 13). We have recently shown that in rats

with adriamycin nephrosis, inhibition of renal cyclic

guanosine 3’,5’-monophosphate (cGMP) phosphodi-

esterase (PDE) corrects both the resistance to ANP

observed in vitro and the blunted volume expansion

natriuresis in vivo (1 4). This led to the hypothesis thatthe blunted volume expansion natriuresis and ANP

resistance could each result from heightened activity

of renal cGMP-PDE, such that the cGMP-dependent

effects of ANP that mediate natriuresis are impaired,

when compared with normal rats (14).

The possibility that this could reflect a general

Valentin et al

Journal of the American Society of Nephrology 583

mechanism underlying abnormal sodium metabolism

in nephrotic syndrome is attractive. Adriamycin pro-

duces nephrosis in rats through a toxicity chiefly to

renal epithelial cells, which ultimately results in fibro-

sis and glomeruloscherosis ( 1 5). We consequently

sought to determine the basis for abnormal sodium

metabolism in an immunologically mediated form of

glomerular injury. We caused Heymann nephritis in

rats by injecting antiserum against Fx1A, a renal

brush border antigen, that results in 9 to 14 days in

stable proteinuria and a glomerular abnormality

closely resembling human membranous nephropathy

(1 6). Our results indicate that in this model, just as in

adriamycin nephrosis, blunted volume expansion na-

triuresis and resistance to ANP are corrected by inhib-itors of renal PDE. In addition, renal resistance to

urodilatin (renal natriuretic peptide: RNP) is also

present and corrected by PDE inhibitors. This renal

resistance to natriuretic peptides is associated with

heightened activity of renal cGMP PDE, thus support-

ing the possibility of a common underlying mecha-

nism contributing to sodium retention in various

forms of nephrotic syndrome.

MATERIALS AND METHODS

Animal Model

We carried out studies in male Sprague-Dawley rats (Ban-tin-Kingman, Fremont, CA) housed in climate-controlledconditions (20#{176}Cand 55% relative humidity with a 12-hlight/dark cycle) and provided standard rat chow and waterad Ubitum. Heymann nephritls was induced by the ip injec-tion of anti-FxlA antiserum (5 mL/kg of body weight) to ratsweighing 180 to 200 g, as described elsewhere (17,18).Control rats, which were matched for age and weight at thetime of the antiserum administration, received an equalvolume of normal saline. To determine 24-h water intake,urine volume, and urinary excretion of sodium, potassium,and protein, rats were placed in metabolic cages for the daybefore study. Rats were studied for 9 to 14 days after theinjection of antiserum or saline.

In Vivo Studies

On the day of the acute experiment, animals were anes-thetized with an ip injection of 100 mg/kg of Inactin#{174} (An-drew Lockwood and Associates, Sturtevant, WI) and placedon a heated table to maintain rectal temperature at 37 ±

0.5#{176}C.AnImals underwent tracheostomy and breathed spon-taneously: they were prepared for acute experimentation aspreviously described (14). In brief, catheters were insertedinto a femoral vein and artery, the right jugular vein, andcarotid artery for infusing solutions, sampling blood, andcontinuously measuring right atrial and arterial pressureswith Statham P23id pressure transducers (Gould Instru-ments, Oxnard, CA) connected to a polygraph (model 7D,Grass Instrument Co. , Quincy, MA). The jugular venouscatheter was positioned at the level of the right atrium. APE-60 catheter was inserted into the dome of the urinarybladder via a midline suprapubic incision for urine collec-tions. During the surgical preparation. a solution of 5% BSA(Sigma Chemicals, St. Louis, MO) in normal saline wasinfused via a syringe pump (Harvard Apparatus, SouthNatick, MA), until a total volume of 0.5% body weight was

administered to replace estimated fluid losses. After comple-tion of the surgery, albumin infusion was discontinued andreplaced by a solution of 0.9% sodium chloride containingmeglumine iothahamate to determine the renal clearance ofthis substance; the solution was infused for the rest of theexperiment at a rate of 40 �L/min. After a 45- to 60-ministabilization period, three urine collections of 20 mm eachwere obtained. At the end of this control period, a volume ofnormal saline equal to 2% of the rat’s weight was adminis-tered over a 5-mm interval. Urine collections continued forthree successive 10-mm periods, beginning with the onset ofthe infusion. The experiment was terminated by collecting a5-mL arterial blood sample into a chilled Vacutainer#{174} (Bec-ton-Dickinson Co., Rutherford, NJ) tube containing EDTAand 500 KIU aprotinin. Blood was immediately centrifuged(4,000 rpm) at 4#{176}Cfor 1 5 mm, and plasma was kept frozen at- 70#{176}CuntIl subsequently analyzed for immunoreactive ANPand cGMP.

This protocol was followed in three groups of rats. Group Iserved as time control subjects and consisted of eight normal(Ia) and seven nephritic (Ib) rats. They were studied over anidentical time period but did not undergo the 2% body weightsaline infusion. Group II consisted ofnine normal (Ila) and 13nephritic (lib) rats, undergoing the volume expansion proto-col described above. An additional group (Group III) under-went the same volume expansion protocol during intrarenalinfusion of Zaprinast (M&B 22,948; see Acknowledgmentsfor provider information). Through a heft paraspinal incision,the left renal artery was exposed and a curved 30-gaugeneedle inserted near its junction with the aorta. Promptreturn ofarterial blood through the PE- 10 tubing attached tothe needle confirmed successful placement. Zaprinast (1�g/�L) diluted in normal saline containing 1 mg/mL ofbacitracin and BSA was infused throughout the duration ofthe experiment at a rate of 10 �L/min. The left ureter wascannuhated with PE- 10 tubing for urine collection; bladderurine reflected excretion from the right contralateral kidney.Group lila consisted of nine normal, and Group 11Th, of tennephritic rats, undergoing the volume expansion protocoldescribed above.

Receptor Binding Studies

Rats were killed by decapitation, and their kidneys werequickly removed, decapsulated, snap-frozen in liquid nitro-gen. and stored at -70#{176}C.The density ofANP receptors wasassessed by a modification of an in situ receptor-bindingassay (19) as previously described (20). In brief, tissue sec-tions (10 jim) were cut on a cryostat at - 15#{176}C,thaw-mounted on poly-L-lyslne-coated slides, dried in vacua for 18h at -4 to +4#{176}Cover silica gel, and stored in sealed bakehiteboxes at -80#{176}C. Immediately before the assay, the slideswere brought to room temperature. Sections were preincu-bated for 10 mm at room temperature in 100 pL of a buffer(bufferA) containing 30 mM sodium phosphate (pH 7.2), 120mM sodium chloride, 0.3% bacitracin, and 0.5% receptor-grade BSA. After preincubation, the buffer was replaced with100 pL of fresh buffer A containing increasing concentra-tions (from 25 to 1500 pmol) of 1’25I1-rat-ANP1_28 (2200�tCi/mmole: DuPont-NEN, Boston, MA), and the sectionswere placed in a humidified chamber and incubated at roomtemperature for 1 5 mini. After incubation, the slides wererinsed in an ice-cold buffer containing 30 mM sodium phos-phate and 120 mM sodium chloride for 10 s, washed in thesame buffer for 5 mm, rinsed in deionized water for 20 s, anddried for 2 h in a stream of cool air. Nonspecific binding was

Resistance to Natriurefic Peptides in Heymann Nephritis

584 Volume 7 . Number 4 ‘ 1996

determined on adjacent sections under identical incubationconditions except for the addition of 1 MM unlabeled ANP1�28(Peninsula Laboratories, Belmont, CA). Thereafter, theamount of radioligand bound to the tissue sections wasdetermined by placing the slides in a gamma counter. Bind-ing characteristics were determined using the LIGAND pro-gram (21).

Determination of cGMP Generation by IsolatedGlomeruli and Inner Medullary CollectingDuct Cells

Rats were anesthetized with pentobarbital (50 mg/kg ip;Abbott Laboratories, Chicago, IL). Glomerull were isolated bya sieving procedure using a modffication (22) ofthe techniqueof Chaumet-Riffaud et a!. (23), as reported for our laboratory(14,20). Glomeruhi were suspended in ice-cold 20 mM Ths-(hydroxymethyl) aminomethane hydrochloride (‘Iris HC1)buffer, pH 7.4, contaIning 125 mM NaC1, 10 mM KC1, 10 mMsodium acetate, and 5 mM glucose (buffer B) and centrifugedat 120 g for 2 mini. The supernatant was discarded, and thepellet was resuspended in the same buffer and recentrifuged.Inner medullary collecting duct (IMCD) cells were isolated bycollagenase digestion of excised inner medullae according tothe method of Zeidel et aL (24) as carried out in our labora-tory (14). The suspension of inner medullary cells was lay-ered on a 16% Ficoll (Sigma) solution in nonbicarbonateRinger’s buffer (buffer C) and centrifuged at 2300 g for 40mm. Cells were subsequently washed through buffer C andthen a solution of buffer C containing 7.5% albumIn, toremove any traces of contaminating collagenase.

Freshly isolated glomeruli were resuspended in buffer Bcontaining 1 mM CaCl2 and 1 mg/mL bacitracin, and IMCDcells in buffer C containing bacitracin, 7.5 mM glucose, andno pyruvate or acetate, and preincubated for 10 mini at 37#{176}Cin a shaking water bath. In some preparations, 3-Isobutyl-1-methylxanthine (IBMX; Sigma) or Zaprinast, each at i0�M, was included in the incubation medium. Incubatlonswere carried out for 10 mIni at 37#{176}Cin the presence ofincreasing concentrations ( 1 0 ‘#{176} to 1 0� M) of syntheticrat-ANP1..28 (Peninsula Laboratories, Belmont, CA), urodila-tin (RNP; Peninsula Laboratories), or sodium nitroprusside(Sigma) dissolved in the incubation medium in volumes of 10pL. Incubatlons were terminated by adding 750 �L of ice-coldtrichloroacetic acid (final concentration, 6.6%) and cooling to4#{176}C.The precipitated protein was sedimented by centrifuga-tion at 4500 rpm for 15 mm at 4#{176}C,and the pellets weredissolved in 1 N NaOH and assayed for protein content by themethod of Lowiy et aL (25) using BSA as standard. Thesupernatants were stored at - 70#{176}Cuntil extracted andassayed for cGMP.

Determination of cGMP PhosphodiesteraseActIvity In IMCD Cells

Activity ofcGMP-PDE was determined in 12 normal and sixnephritic rats. Immediately after preparation, IMCD cellswere homogenized in a glass-Teflon� homogenizer. The ho-mogenization medium had the following composition: 0.25 Msucrose, 5 mM Ths, 3 mM magnesium chloride; 1 mM EDTA(pH, 7.4) (1 :3 wet wt/vol). Homogenization and all otherpreoperative procedures were conducted at 0 to 4#{176}C.Thehomogenate was centrifuged at 200 x g for 5 mm. Thesupernatant was incubated for 60 mm in a shaking waterbath, to facifitate the release of membrane-bound PDE. Thehomogenate was centrifuged at 100,000 x g for 60 mm; the

100,000 x g supernate (cytosol) was collected and immedi-ately frozen. The pellet was resuspended in a small volume(1: 10 vol) of the homogenization medium, centrifuged again(100,000 x g for 60 mm) and the supernatant collected andadded to the previous supernate: the pellet was discarded.

The cGMP-PDE activity was assayed according to theprocedure ofTorres et aL (26). The homogenate ofIMCD cells(10 to 15 jig of protein per tube) was incubated with 1 j.�M of[3Hj-cGMP (Dupont-NEN) in 200 j�L ofa buffer containing: 10mM magnesium sulfate, 2 mM EGTA, 0. 1% BSA and 50 mMThs hydrochloric acid (pH, 7.5). After a 20-mini incubationperiod at 37#{176}C,the reaction was stopped by heating at 95#{176}Cfor 3 mm, and the [3Hj-5’ -nucleotide products were con-verted to (3H1-nucleosldes by incubation with an excess of5’-nucleotidase (crotalus atox venom; Sigma). Nucleosideswere separated from nucleotides with use of QAE-Sephadexcolumns according to the procedure ofWells et aL (27), andthe column effluent counted for [3H1 activity in a liquidscintillation counter (Packard Instruments, Chicago, IL). Theenzyme activity was expressed in pmol of cGMP hydrolyzedper mini per mg of protein. The assays were run in triplicate.

Analytical Techniques

The hematocrit value was determined in triplicate on eachblood sample by spinning blood at 12,000 rpm in a ml-crofuge. Estimated changes in plasma volume were calcu-hated according to dV = flOO/(100 - Hi)) x 1100 X (Hi -

HO/HI], where dV is the percent ofchange in plasma volume,and Hi and Hf are the initial and final hematocrit values,respectively (28). Urine flow rate was determined gravimetri-cally, and urine sodium and potassium concentrations weremeasured by flame photometry (Instrument LaboratoriesModel 943, Lexington, MA). Plasma and urine iothalamateconcentrations were measured by fluorescence excitation(29), and the clearance of this substance was used as anindex of GFR. Clearance of sodium (CNa) and potassium (C�Jwere calculated as usual, and fractional excretions of Na andK were calculated as (CNa/GFR) X 100 and CK/(GFR) X 100,respectively.

Plasma concentration of immunoreactive ANP was deter-mined after extraction on a Sep-Pak C 1 8 column (WatersChromatography Division, Mililpore Corp., Miluord, MA) pre-equilibrated with 0. 1% tnifluoracetic acid. After ehution with75% methanol in 0. 1% tnifluoroacetic acid and evaporation todryness under a stream of nitrogen, the residue was recon-stituted in assay buffer and measured for ANP immunoreac-tivity with a commercially available kit (Peninsula Laborato-ries).

Cyclic GMP concentration in plasma and the supernatantof glomerular and IMCD cell incubations was determinedafter extraction with water-saturated ethyl ether to removethe TCA. After evaporation to dryness under a stream of air,the residue was reconstituted in 50 mM sodium acetatebuffer, pH 6.2, and measured for cGMP immunoreactivitywith a commercially available kit (Du Pont Company, Wilm-ington, DE). Urine concentration of cGMP was determineddirectly after acetylation.

Data are expressed as means ± SE. One-way analysis ofvariance with repeated measures and two-way analysis ofvariance with Dunnett’s and t test as post hoc tests were usedto assess significance among and between groups, respec-tively (StatvIew�: Brain Power, Calabasas, CA). P = 0.05 wasconsidered the minimum level ofsignificance. Area under thecurve was calculated according to the trapezoidal rule (30).

Valentin et al


RESULTS

Group Characteristics

Results from the metabolic cage study revealed that

nephritic rats had significantly higher urine volume

and urinary excretion of protein (Table 1). Water in-

take and urinary excretion of sodium (UNaV) did not

differ between groups, whereas urinary excretion of

potassium (UKV) was significantly lower in nephritic

rats.

Analysis of variance indicated that nephritic ani-

mahs had similar baseline values for most of the

measured variables (Table 2). Hematocrit values were

significantly lower in nephritic compared with normal

rats (39 ± 0.8 versus 44.8 ± 0.7%, P < 0.0001).

Urinary excretion of cGMP (U(.GMPV) was slightly, but

not signifIcantly, lower in nephritic compared with

normal rats (9.8 ± 2.3 versus 15.7 ± 2.7 pmol/min

respectively; P = not significant ENS]).

Effect of Volume Expansion in Normal andNephritic Rats

Renal function, urinary cation and cGMP excretion,

hematocrit value and heart rate remained stable in

time control experiments (Groups Ia and Ib; Table 2

and Figure 1). In normal rats, the volume expansion

protocol led to a brisk increase in urine flow and UNaV

(Table 2 and Figure 1). In nephritic rats, volume

expansion also increased both urine flow and UNaV,

but these increases were significantly reduced com-

pared with the normals. Within 30 mm, normal rats

eliminated 92 ± 15% of the infused load, whereas

nephritic animals excreted only 47 ± 6% (P = 0.004

between groups). GFR increased transiently in both

groups (by 43.2 ± 18.4 and 20.4 ± 7.4% respectively;

both P < 0.05 versus baseline and P = NS between

groups), then returned toward baseline values; conse-

quently, a large increase in fractional excretion of

sodium was detected in both groups. The blunted

TABLE 1 . Characteristics of normal and nephriticrats#{176}

Group Normal. . . b

SignificanceHeymann

..nephritis

Number 50 48BW (g) 302 ± 4 NS 290 ± 5WI (mL/24 h) 33.2 ± 1.3 NS 37.5 ± 1.7uv (mL/24 h) 17.7 ± 0.9 C 24.4 ± 1.7

UNaV (mmol/24 h) 2.04 ± 0.07 NS 2.05 ± 0.18UKV (mmol/24 h) 5.03 ± 0.17 C 443 � 0.18UprotV (mg/24 h) 13.7 ± 2.3 d 218.6 ± 36.5

a Values are means ± SE of variables obtained from the metabolic

cage during the 24 h preceding the acute experiment. BW, bodyweight; WI, water intake; UV, urine flow; UNaV, UKV, � urinaryexcretion of sodium, potassium. and proteins; NS, not significant.b Significance of difference between Normal and Heymann Nephritisgroups.cP< 0.05.d p < 0.005 between groups.

volume expansion natriuresis in nephritic rats was

associated with a marginally greater increase in

plasma volume, measured 10 mm after the onset of

the volume expansion compared with controls ( 1 3. 2 ±

3.7 versus 7.0 ± 2.3%, respectively; P = NS) as

calculated from changes in hematocrit values. Potas-

sium excretion increased transiently in both normal

and nephritic rats. As also presented in Table 2, mean

arterial pressure (MAP) increased slightly in both

groups as a consequence of the volume expansion

protocol.

Plasma ANP concentration, measured at the conclu-

sion of the experiment, was similar in time-control

normal and nephritic rats (Groups Ia and Ib) and was

increased to the same extent in both normal and

nephritic rats after the volume expansion procedure

(Figure 2). Plasma cGMP concentration in time-con-

trol normal (Ia) and nephritic (Ib) rats did not differ,

nor was it affected by the volume expansion procedure

(Group II; Figure 2). In addition, right atrial pressure

increased to the same extent during volume expan-

sion in both normal and nephritic rats (from 1 .2 ± 0.5

to 3.4 ± 0.6 and from 0.9 ± 0.3 to 3.4 ± 0.5 mm Hg,

respectively; both, P < 0.0001). Nephritic animals had

reduced natriuresis after volume expansion despite a

similar increase in the concentration of ANP in

plasma, indicating blunted ANP activity. To approach

this question directly, we measured the urinary excre-

tion of cGMP as an index of the renal action of ANP

(3 1). UCGMPV in nephritic animals was significantly

blunted compared with normal rats, paralleling tem-

porally the blunted natriuretic response (Figure 1).

These data are consistent with the hypothesis that

responsiveness to ANP is impaired in Heymann ne-

phritis.

Effect of ANP and RNP on cGMP Generation byIsolated Glomeruli and IMCD Cells

To test this hypothesis, we measured the accumu-

lation of cGMP by isolated glomeruli and IMCD cells in

response to increasing concentrations of synthetic

ANP and RNP. Basal cGMP accumulation was similar

in tissues from normal and nephritic rats, and in-

creased in a dose-dependent manner in response to

increasing concentrations of both ANP and RNP (Fig-

ure 3). However, both glomeruhi and IMCD cells iso-

hated from nephritic animals had a significantly

blunted response to both ANP and RNP when com-

pared with normal animals. Thus, impaired urinary

excretion of cGMP by nephritic rat kidneys in re-sponse to volume expansion in vivo was matched by

blunted responsiveness of target cells to ANP and RNP

in vitro.

Characteristics of ANP Receptors in the Kidney

We next asked whether the refractoriness to ANP

could be caused by an alteration in ANP receptor

characteristics in normal versus nephritic kidneys.

Incubation of rat kidney sections with increasing con-

Resistance to Natriuretic Peptides in Heymann Nephritis


TABLE 2. Effect of isotonic saline volume expansion in normal and nephritic rats on kidney function, systemichemodynamics, and hematocrit#{176}

uv UN V UKVPeriod . .

(pi/min) (�Eq/mln) (�Eq/min)GFR

(mL/mln)FENa (%) FEK (%)

MAP(mm Hg)

HR(beats/

mm)Hematocrit

�

Group Ia: Normal rats, time control (N = 8)Baseline 24.1 ±4.1 2.49±0.36 3.94±0.43 2.13±0.25 1.04±0.22 47.1 ±6.6 116±6 336±21 44.0± 1.1Oto 15 mm 19.8 ± 3.4 2.82 ± 0.42 3.50 ± 0.42 2.23 ± 0.23 1.00 ± 0.17 36.7 ± 4.6 119 ± 6 341 ± 21 44.1 ± 1.2l5to30min 19.7±3.0 2.93±0.52 3.46±0.27 2.12±0.17 1.12±0.27 38.5±4.6 119±6 338±20 43.9± 1.2

Group ib: Nephritic rats, time control (N = 7)Baseline 26.9 ± 7.7 2.42 ± 0.81 2.69 ± 0.52 1.96 ± 0.36 0.86 ± 0.19 33.0 ± 2.6 121 ± 10 306 ± 20 38.3 ± 1.30 to 15 mm 28.3 ± 6.5 2.58 ± 0.84 2.59 ± 0.40 2.06 ± 0.36 1.08 ± 0.32 31.3 ± 5.0 125 ± 1 1 310 ± 22 39.2 ± 1.315 to 30 mm 34.7 ± 8.1 2.92 ± 0.98 2.74 ± 0.37 1.66 ± 0.29 1.55 ± 0.51 39.7 ± 5.3 125 ± 10 320 ± 23 39.6 ± 1.5

Group la: Normal rats, volume expansion (N = 9)Baseline 25.9 ± 6.5 2.56 ± 0.85 2.97 ± 0.45 2.44 ± 0.29 0.95 ± 0.39 26.8 ± 3.0 131 ± 5 386 ± 12 45.5 ± 0.70 to 10 mm 270.6 ± 25.2C 31.13 ± 3.65C 95� ± 1.68C 3.20 ± 0.29b 7.98 ± 1.71C 79.2 ± 19.lb 139 ± 5b 393 � 9l0to20min 252.7±61.7C 30.67±5.24C 3.42±0.46 2.89±0.33 7.56±0.71C 28.1 ±5.1 138±5 404± 11 43.9± lOb2Oto3Omin 127.6±28.1C l9.63±3.56c 3.00±0.36 2.53±0.31 5.75± 1.1OC 29.9±3.3 139±5 403± 9 44.9±0.9

Group lIb: Nephritic rats, volume expansion (N 13)Baseline 23.2 ± 3.6 2.43 ± 0.56 3.43 ± 0.38 2.39 ± 0.31 0.83 ± 0.22 34.6 ± 3.4 122 ± 5 391 ± 11 39.4 ± 1.0

0 to 10 mm 130.0 ± 2l.9c 15.52 ± 3i�9C 5.98 ± O.67C 2.78 ± O.35b 4.09 ± O.88C 543 ± 6.4b 131 ± 6b 380 ± 1410 to 20 mm 137.5 ± 18.lC 19.58 ± 2.70c 355 ± 0.34 2.43 ± 0.30 6.47 ± O.89C 377 ± 5.6 128 ± 5b 386 ± 13 37.3 ± lob20 to 30 mm 84.3 ± 12.4C 11.67 ± l.72C 3.00 ± 0.31 1.94 ± O.23�’ 5.12 ± O.9OC 37.3 ± 3.8 127 ± 4b 388 ± 13 39.3 ± 1.4

a Values are means ± SE of multiple observations during the control period (baseline) and after either volume expansion or continuous hydropenia

(time control). UV. urine flow; UNaV. UKV. urinary excretion of sodium and potassium; GFP, glomerular filtration rate; FENa, FEK. fractional excretionof sodium and potassium; MAP. mean arterial pressure; HP. heart rate.b p < 0.05.

C p < 0.005 versus baseline.

centrations of [‘25I1-rat ANP1.28 resulted in an in-

crease in both total and nonspecific binding. Receptor

saturation, reflected by specific binding, was ap-

proached at 1 .2 nM in both normal and nephritic ratkidneys. Scatchard analysis of these data suggested

the presence of a single receptor site in each of the two

experimental groups. Neither the affinity, expressedas the apparent dissociation constant (Kd), nor the

maximum binding capacity (Bm�j for ANP differedbetween normal and nephritic rat kidney tissues (Ta-ble 3). These results suggest that the difference in ANP

responsiveness likely occurs at a post-receptor locus.

Influence of Cyclic Nucleotide PDE Inhibitionon the Response to ANP In Vitro and In Vivo

To examine whether differences in activity of cyclic

nucleotide PDE could account for the resistance to

ANP and RNP, we measured ANP-dependent accumu-

lation of cGMP by isolated glomeruhi and IMCD cells in

the presence of IBMX or Zaprinast. We predicted that

pharmacological inhibition of PDE would suppress oreliminate effects that operate through modulation of

PDE activity. Incubation in the presence of IBMXincreased basal cGMP production, but without appre-

ciable differences between tissues from normal or

nephritic kidneys (Figure 4). The stimulation of cGMP

production by ANP was amplified in the presence of

IBMX in both glomeruhi and IMCD cells from normal

and nephritic kidneys. However in both tissues, the

difference between normal and nephritic preparations

was no longer observed, as cGMP production by both

glomeruli and IMCD cells was statistically indistin-guishable from normals. These data were paralleled

by the results with Zaprinast. The effect of Zaprinaston basal cGMP production was modest, but stimula-

tion with 10_6 M ANP was again amplified, and onceagain no difference between normal and nephritic

glomeruli or IMCD cells occurred. Similar results were

obtained with respect to RNP (Figure 5). These find-

ings suggest that differences in cGMP catabolism existbetween normal and nephritic animals, which mayaccount, at least in part, for the resistance to ANP and

RNP seen in the latter. To assess further this possibil-

ity, we measured cGMP accumulation in normal and

nephritic tissues in response to sodium nitroprusside,

a compound that activates soluble guanylate cyclaserather than the particulate guanylate cyclase associ-

ated with the NPR-A (32). Incubation with sodium

nitroprusside caused a dose-dependent Increase in

cGMP accumulation by normal glomeruli and IMCD

cells (Figure 6), whereas accumulation by nephritic

cells was blunted at the highest dose used. The differ-

ence between normal and nephritic cells was abol-ished by addition of either IBMX or Zaprinast, thereby

duplicating the results observed with ANP- and RNP-

stimulated cGMP accumulation.

To determine if these in vitro observations reflected

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Valentin et al


Time (minutes)

Figure 1 . Urinary excretion of sodium (UNaV; top panel) andcGMP (UCGMPV; bottom panel) in normal (filled symbols)and nephritic rats (open symbols) after iv Infusion of normalsaline (squares) or continued euvolemia (circles). The salineload equal to 2% body weight was administered over 5 mmas indicated by the arrows. Both UNaV and UCGMPV increasedsignificantly less in rats with Heymann nephritis, comparedwith normal rats. � j� < 0.05 versus baseline. § P < 0.05between groups receiving the isotonic saline volume expan-sion.

the phenomenon of ANP resistance in vivo, we carriedout the volume expansion protocol in rats infused with

Zaprinast into the left renal artery (Group III). Theresults are presented in Figure 7 and Table 4. Intra-

renal infusion of Zaprinast increased baseline UV,UNaV, UKV, and UCGMPV similarly in both normal and

nephritic rats as indicated by the increase in the ratio

of infused to contralateral (R11�) kidney. The excretory

response to volume expansion by the contralateral

normal or nephritic kidney was similar to that ob-

served in the initial experiments (Figure 1). In normal

rats, R11� decreased significantly for UV and UNaV and

did not change significantly for UGMPV after volume

expansion, whereas R11� either did not change or

increased significantly in nephritic rats. R11� did not

change significantly for UKV in either normal or ne-phritic animals after volume expansion. Furthermore,

the values for both UNaV and UCGMPV from the ne-phritic kidneys infused with Zaprinast were statisti-

cally indistinguishable from either infused or con-

E0E

0.

ECs

0Ia Lb lIa LIb lila ilIb

GroupsFigure 2. Plasma concentrations of immunoreactive ANP(lR-ANP; top panel) and cGMP (lR-cGMP; bottom panel) innormal (Groups Ia, Ila, and lila) and nephritic rats (GroupsIb, llb, and lllb), measured 30 mm after either Initiation of thevolume expansion (Groups II and Ill) or continued hydrope-nia (Group I). Plasma ANP concentrations were increased tothe same extent in both normal and nephritic rats after thevolume expansion protocol, compared with their respectivehydropenic controls. Plasma cGMP concentration did notchange significantly after volume expansion in Group II, butincreased in both normal and nephritic rats infused withZaprinast into the left renal artery (Group Ill). NS, not signifi-cant. § P < 0.05 versus Group I; �) P < 0.005 versus Groups Iaand Ila; P1P < .01 versus Groups lb and lIb.

tralateral normal kidneys. Thus, intrarenal Zaprinastinfusion was able to restore the blunted excretory

responses to volume expansion in nephritic animals

in a manner similar to its restoration of ANP respon-

siveness in vitro. Plasma ANP concentration measured

at the conclusion of the experiment was not different

between normal and nephrltic rats, and was similar tothe value observed in Group II (Figure 2). PlasmacGMP was significantly increased in both normal andnephritlc animals, compared with both their hydro-

penic (Group I) and volume expanded (Group II) con-

trols (Figure 2). The volume expansion-associated in-

crease in MAP (group II) was blunted in both normaland nephritic rats infused with Zaprinast intrarenally

(Group III; Figure 8).

(;iomeruui IMCD cells Glomeruli IMCD cells

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log IRNPI, M

a Values are means ± SE. N, number of rats; NS, not significant.b Significance of difference between Normal and Heymann Nephritis

groups.



E450

Ot300 E

.3

,g150 �

Figure 3. Accumulation of cGMP in the medium of glomeruli(left side) and Inner medullary collecting duct (IMCD) cells(right side) Isolated from normal (filled squares) and ne-phrltlc rats (open squares) in response to increasing concen-trations of atrial natriuretic peptide (ANP; top panels) or renalnatriuretic peptide (RNP; bottom panels). Baseline cGMPaccumulation did not differ between normal and nephritictissues, and increased in a concentration-dependent man-ner with both ANP and RNP. However, for both ANP and RNP,the accumulation of cGMP by glomeruli and IMCD cellsisolated from nephritic kidneys was significantly less thanthat obtained from normal tissues. Data are means ± SE ofvalues obtained from four to eight rats in each group. � �0.05. � � P < 0.005 versus baseline. § P < 0.05 between groups.

TABLE 3. Binding characteristics of total ANPreceptors in the kidneys of normal and nephriticrats#{176}

Group Normal Significanceb HeymannNephritis

N 3 3

Kd (nmol) 7.3 ± 2.4 NS 5. 1 ± 1.2Bmax (pmol/mm3) 7.0 ± 2.2 NS 6.7 ± 3.7

Cyclic GMP-PDE Activity in IMCD CellHomogenates

Because Zaprinast is a selective inhibitor of theType V cGMP-specific PDE (33), the results of theseinhibitor studies suggested that ANP target sites inkidneys of rats with Heymann nephritis were charac-

terized by increased cGMP-PDE activity. To test this

conclusion, we measured cGMP-PDE activity in IMCD

cell homogenates isolated from normal and nephritic

rat kidneys. In 12 IMCD preparations from normal

L

I Ir2.

�4 �i�

II,.

III

I’. I.�

Log [ANPI, M

Figure 4. ANP-dependent accumulation of cGMP in glomer-uli (left side) and inner medullary collecting duct (IMCD)cells (right side) isolated from normal (black bars) andnephritic rats (hatched bars) incubated with vehicle alone(top panels), 3-isobutyl-l-methylxanthine (IBMX: middle pan-els), or Zaprinast (bottom panels). Both phosphodiesteraseinhibitors were used at iO� M. The blunted ANP-dependentcGMP accumulation seen in nephritlc compared with nor-mal tissues incubated with the vehicle alone was abolishedin the presence of each of the Inhibitors. Data are means ±

SE of values obtained from five to ten rats in each group. § P< 0.05. §� P < 0.005 versus baseline. � � < 0.05 betweennormal and nephritic tissues.

rats, the mean value ofcGMP-PDE activity was 198 ±

32 pmoh/min per mg protein, whereas in six prepara-

tions from rats with Heymann nephritis, the value was

approximately double at 388 ± 93 pmoh/min per mgprotein (P < 0.03). Thus, increased cGMP-PDE activityin target cells of ANP action in the kidney can be

demonstrated directly in nephritic rats with ANP re-

sistance in vivo and in vitro.

DISCUSSION

The results of this study demonstrate that bluntedvolume expansion natriuresis in rats with Heymannnephrltis can be related to renal resistance to natri-uretic peptides, in particular ANP, believed to be the

important mediator of the natriuretic response tovolume expansion (32,34). An earlier study first iden-

tified this blunted natriuretic response in Heymann

nephritis rats, and linked it to a defect in distal

nephron sodium reabsorption on the basis of mi-

cropuncture data (35). The IMCD is a major site ofaction ofANP within the kidney (32); as it is located in

the distal-most portion of the nephron, it is an impor-

tant regulator of the final composition of the urine.

Abnormal ANP action in this segment could contribute

Glomeruli IMCD cells Glomeruli IMCD cells

11�I:

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1�I

�

1

II

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1

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Valentin et al


yEs .7 .6 VUI .7 .6

Log [RNP], M

Figure 5. RNP-dependent accumulation of cGMP in glomer-uli (left side) and Inner medullary collecting duct (IMCD)cells (right side) isolated from normal (black bars) andnephritic rats (hatched bars) incubated with vehicle alone(top panels), 3-isobutyl-1-methylzanthine (IBMX: middle pan-els), or Zaprinast (bottom panels). Both phosphodiesteraseinhibitors were used at 10� M. The blunted RNP-dependentcGMP accumulation seen in nephritic compared with nor-mal tissues incubated with the vehicle alone was abolishedin the presence of each of the inhibitors. Data are means ±

SE or values obtained from five to ten rats In each group. § P< 0.05 versus baseline. * � < 0.05 between normal andnephrltic tissues.

to the blunted natriuretic response to volume expan-

sion which we observed. We therefore analyzed the

components of the ANP signaling system in rats with

Heymann nephritis.

The blunted response was not the result of impaired

secretion of ANP from the cardiac atria, as inferred

from the similar plasma concentrations ofANP seen innormal and nephritic rats. Right atrial pressure in-creased identically in the two groups as a result of thevolume expansion protocol. AtrIal stretch or disten-tion from intraluminal atrial pressure is the majordeterminant of ANP secretion (32); although we didnot measure peptide secretion directly, the equivalentincreases in plasma ANP concentration and right

atrial pressure offer strong evidence that the bluntednatriuretic response after volume expansion was not

the result of impaired secretion of ANP in nephriticrats. A qualitatively similar increase in plasma ANP

concentration after volume expansion was also ob-served in rats with experimental nephrotic syndrome

resulting from administration of adriamycin (14).

The increase in GFR that we measured after volume

expansion was somewhat smaller in neph.ritic rats,compared with normal controls, and this could have

YEN .� .7 .5 y55 .5 .� .5

Log [Sodium Nitroprusside], M

Figure 6. Accumulation of cGMP in glomeruli (left side) andinner medullary collecting duct (IMCD) cells (right side)isolated from normal (black bars) and nephritic rats(hatched bars) in response to sodium nitroprusside. Sampleswere incubated with vehicle alone (top panels), 3-Isobutyl-1-methylxanthine (IBMX; middle panels), or Zaprinast (bot-tom panels). The accumulation of cGMP by glomeruli andIMCD cells increased with sodium nitroprusside concentra-tion; however, the accumulation of cGMP was significantlyblunted in tissues obtained from nephritic rats. The bluntedsodium nitroprusside-dependent cGMP accumulation seenin nephritic compared with normal tissues incubated withthe vehicle alone was abolished In the presence of each ofthe inhibitors. Data are means ± SE of values obtained fromfive to ten rats in each group. § P < 0.05. §� P < 0.005 versusbaseline. * � < 0.05 between normal and nephritlc tissues.

contributed to the blunted natriuretic response. How-

ever, abundant older studies have shown that volume-expansion natriuresis still occurs when no change or

even a decrease in GFR takes place. Thus, a purelyhemodynamic basis for the blunted natriuresis doesnot seem likely. On the other hand, defective renalANP signaling was suggested by the observation thatUCGMPV was impaired in parallel with blunted UNaV in

nephritic rats after volume expansion (Figure 1). Cy-

clic GMP is the major currently recognized intracellu-lar second messenger for ANP (32), and UCGMPV is

believed to reflect ofANP action within the kidney (31).Although other mediators, notably nitric oxide, alsosignal through intracellular cGMP, the contribution ofthese other pathways to UCGMPV has not been estab-hished, but Is likely to be small relative to that of ANP.

This defective signaling was observed in isolated gb-

meruli and dispersed IMCD cells in vitro as well,

paralleling the in vivo results and indicating that the

latter were not simply the result of a failure of nor-maily secreted ANP to reach its target sites within the

10.0 -

7.5 -

5.0 -

2.5

0.0.

100-

75 -

50-

25.

0-

S

E0’

Csz

S

E0E0.

-50 .30 -10 _:�. �s 25

a Values are means ± SE of the ratio of the infused (left) to the

contralateral (right) kidney for urine flow rate (UV) and urinary excre-

tion of sodium (UNaV). potassium (UKV), and cGMP (UCGMPV). Leftkidneys received a continuous intraarterial Infusion of Zaprinast at therate of 10 �g/min. Ten normal and nine nephritlc rats underwent thevolume expansion protocol as described in the Methods section. NS,not significant; ND, not determined.b Significance of difference between Normal and Heymann Nephritisgroups.C p < 0.05 versus baseline.d p < 0.05 between normal and nephritic rats.



Time (minutes)

Figure 7. UrInary excretion of sodium (UNOV; top panel) andcGMP (UCGMPV; bottom panel) after volume expansion fromleft (circles) and right (squares) kidneys of normal (closedsymbols) and nephrltic (open symbols) rats. Left kidneysreceived a continuous infusion of Zaprinast at the dose of 10�g/min. The arrows indicate the period of Infusion of normal

saline (2% body weight iv over 5 mm). � � < 0.05 versusbaseline. § P < 0.05 between left and right nephrltlc kidney.

kidney, but rather reflected a cellular abnormality in

the action of ANP to increase cGMP accumulation.Basal cGMP accumulation in the absence of addedANP was not different in nephritic and control tissues,

again paralleling control values for UCGMPV in vivo,and suggesting that the nephritic defect was confinedto ANP-stimulated cGMP accumulation.

We attempted to establish the basis for this cellulardefect In nephritic renal tissue. Biologically activeNPR-A receptors contain guanylate cyclase as part of

the intracellular domain of the receptor protein (36),and occupancy of the receptor activates the enzyme tostimulate cGMP production. One possibility for ourresults therefore was an alteration in the number of

receptors or their binding characteristics. We tested

this possibility by carrying out a receptor-bindingassay on kidney slices by using I’25I1-ANP. Scatchard

analysis of the binding data could detect no differenceIn binding between nephritic and normal kidneys,suggesting that the cellular abnormality in cGMP

accumulation we observed in nephritic gbomeruli and

IMCD cells occurred at a postreceptor locus, a conclu-

sion also drawn in studies of rats with adriamycin

TABLE 4. Influence of cyclic GMP-PDE inhibition onthe renal response to volume expansion in vivo innormal and nephritic rats#{176}

Parameter and.

PeriodNormal

. , .

SignificanceHeymann

..Nephritis

Uv

Baseline 1.61 ± 0.20 NS 1.42 ± 0.110 to 10 mm 1.23 ± 0.1 lC 1.50 ± 0.11lOto2Omln l.12±O.06C d 1.49± 0.08

2Oto3Omin 1.48±0.23 NS 1.29± 0.11

UNaVBaseline 2.96 ± 0.51 NS 1.91 ± 0.320 to 10 mm i .�s ± 0. 12C d 2.52 ± O.36C10 to 20 mm 1.53 ± O.1OC d 3.06 ± 0.4520 to 30 mm i .89 ± 0.23C 2.37 ± 0.25

UKV

Baseline 1.21 ± 0.09 1.42 ± 0.230 to 10 mm i.i1 ± 0.09 1.31 ± 0.11

10 to 20 mm 1.04 ± 0.09 1.23 ± 0.0720 to 30 mm 1.32 ± 0.21 1.35 ± 0.05

UcGMPVBaseline 1.61 ± 0.16 1.38 ± 0.16OtolOmin ND ND10 to 20 mm 1.85 ± 0.27 2.27 ± 0.25C20 to 30 mm i .85 ± 0.29 2.43 ± 0.28C

nephrosis (13, 14), using different techniques of mea-

suring ANP binding.We then considered the possibility that the blunted

nephritic response was the result of rapid catabolismof cellular cGMP normally formed on the interaction ofANP with its receptor. To do this, we tested ANP-dependent cGMP accumulation in isolated glomeruliand dispersed IMCD cells that were incubated with

two different phosphodiesterase inhibitors, IBMX andZaprinast. In each case, the difference in cGMP accu-

mulation observed between normal and nephriticpreparations not incubated with PDE inhibitors was

abolished. IBMX is a nonspecific PDE inhibitor; basalcGMP accumulation in its presence was increasedthreefold, raising the possibility that the differencebetween normal and nephritic cells was obscured bythis much higher basal activity. However, this doesnot appear to be the case: incubation with Zaprinastled to only a modest increase in basal cGMP accumu-lation, yet also normalized ANP-dependent cGMP ac-cumulation by nephritic glomeruli and IMCD cells.

Because Zaprinast is a more specific inhibitor of theType V cGMP-specific PDE (33), this result suggested

5

0

Ila

[lb

5 10 15 20 25 30

Time (minutes)

I �

10

55

EE

< -S

.10

[Ia

(12 IIb0.Cs01.

� Lila

Ilib

-200 -100 0 100 200 300

Area Under the Curve (mmllg/min)

Figure 8. Time course of absolute changes in mean arterialpressure (�MAP; top panel) and corresponding area underthe curve (bottom panel) after isotonic volume expansion(as indicated by the arrows). �f P < 0.05 between Groups IIand Ill. The volume expansion-induced increase in MAP(Group II) was blunted In both normal and nephritic ratsinfused with Zaprinast into the left renal artery (Group Ill). NS,not significant. § P < .05 between Groups II and lllb. � � < ,�5between indicated groups.

that the impaired cGMP metabolism characteristic of

nephritic tissues was the result of heightened activityof this PDE enzyme.

This conclusion was further supported by the fInd-

ing that cGMP accumulation by isolated glomeruhi and

IMCD cells from nephritic animals in response tosodium nitroprusside was also blunted, compared

with normals, and this difference was again abolished

by incubation with either IBMX or Zaprinast. Sodium

nitroprusside increases cellular cGMP levels through

stimulation of soluble guanylate cyclase as opposed to

the membrane-bound enzyme that is an integral part

of the NPR-A. The fact that nitroprusside-dependent

cGMP accumulation was blunted in nephritic glomer-uli and IMCD cells, and was corrected by incubation

with Zaprinast, is consistent with increased cGMP-

PDE in nephritic cells, which catabolizes cGMP

formed normally from stimulation of the two different

guanylate cyclase enzymes. The demonstration of in-

creased cGMP hydrolytic activity in IMCD cell cytosol

provides direct evidence for this interpretation.We tested the physiological relevance of these in

Valentin et al


vitro observations by carrying out the volume expan-

sion protocol in nephritic rats, in which one kidney

received a constant intrarenal infusion of Zaprinast.The infused kidney responded to the volume expan-sion with a natriuresis and increase in UCGMPV thatwere statistically indistinguishable from either in-

lila fused or noninfused normal kidneys, whereas the

Hib contralateral noninfused kidney in these nephriticrats exhibited the same excretory defect identified in

the initial experiments. Thus, the nephritic abnormal-ity was Zaprinast-sensitive; this agent corrected therenal resistance to ANP which nephritic rats displayed

in response to volume expansion. Zaprinast has beenshown to enhance the natriuretic and UCGMPV re-

sponses to infusion of ANP in both normal rats and

rats with aortocaval fistula, a model of heart failure(37). We had shown earlier that it normalized theresponse to volume expansion in rats with experimen-

tal nephrotic syndrome resulting from injection ofadriamycin (14). These observations all suggest thatZaprinast or related compounds may offer a new

option in the pharmacological approach to pathologi-cal sodium retention, of which these conditions are

examples.The identification ofRNP (urodilatin) as a natriuretic

peptide synthesized within the kidney had led to thesuggestion that it is the intrarenal mediator of natri-

uretic actions previously ascribed to ANP (38). RNP

binds to and activates the same receptors in the

kidney as does ANP (16), and is more resistant to

degradation by neutral endopeptidase than is ANP

(39,40). Our observation that RNP-dependent cGMPaccumulation by nephritic glomeruli and IMCD cells

is also blunted, in parallel with the blunted response

to ANP, is consistent with the interaction of thesepeptides with the same receptor(s). Greenwald andassociates were able to demonstrate the presence ofincreased ANP-like immunoreactivity in the kidneys ofrats with experimental nephrotic syndrome (41). Pre-

sumably, this increased immunoreactivity was the

result of increased synthesis of RNP. Although thepathophysiologic significance of this observation isnot clear, it could be that resistance to natriureticpeptides, induced by the nephrotic (or nephritic) stateand mediated by heightened activity of cGMP-PDE,leads to a reactive increase in RNP synthesis in thekidney in an attempt to normalize sodium metabolismand maintain sodium balance. The relative roles ofRNP and ANP in governing renal sodium excretion ineither normal or pathologic conditions must awaitfurther study. However, acute volume expansion,such as utilized in the studies presented here, is the

one condition in which circulating ANP appears toplay a critical role in stimulating UNaV (34).

From our studies, it is unclear how an immunobog-ically (present studies) or toxicity-related (14) glomer-

ular injury is transduced into a cellular abnormality

in the IMCD. Proteinuria is common to both condi-

tions, and a toxic effect of filtered proteins in the

tubular lumen is certainly a possibility. Indeed, such


592 Volume 7 . Number 4 ‘ 1996

toxicity of proteinuria has been suggested to be anunderlying mechanism in progressive renal disease

(42,43). On the other hand, renal resistance to ANP,

and blunted volume expansion natriuresis, have beenobserved in rats with experimental liver disease re-

suiting from common bile duct ligation, a condition inwhich protein excretion is not increased (3,44,45). Inthis setting, renal denervation is reported to amelio-rate ANP resistance (3,44,45), so renal sympathetic

nerve activity could be mediating the increase incGMP hydrolysis in the inner medulla. Althoughheightened sympathetic nerve activity has also beenimplicated in nephrotic sodium retention (3,4,5), our

earlier study could not confirm any benefit of renaldenervation on volume expansion natriuresis in ratswith adriamycin nephrosis (14). Thus, the precise

signal by which gbomerubar injury leads to increasedcGMP hydrolysis in the IMCD is not known, but is animportant question for future research.

Volume expansion led to similar increases in MAP innormal and nephritic rats that did not occur duringintrarenal Zaprinast infusion. The infusion was asso-dated with a 2- to 3-fold increase in the plasmaconcentration of cGMP. Zaprinast possesses vasore-laxant properties in vitro (46) and hypotensive activityin vivo (47), presumably because of the accumulationof intracellular cGMP. It is thus conceivable that some

Zaprinast escaped from the renal circulation to inhibitcGMP-PDE in the systemic vasculature, resulting inthe increase in plasma cGMP and blunted systemichypertension. Alternatively, the increased circulatingcGMP concentration during intrarenal Zaprinast Infu-sion could be of renal origin. Preliminary studies

indicate that the hemoconcentrating but not the hy-potensive effect of exogenous ANP is blunted in ratswith Heymann nephritis, as is the increase in plasma

cGMP (48), suggestIng that extrarenal as well as renalresistance to ANP may exist in this model.

In summary, we have identified an abnormality incGMP metabolism in Isolated gbomeruli and dispersed

IMCD cells of rats with Heymann nephritis, which is

related to heightened activity of cGMP-PDE. This ab-normality may account, at least in part, for theblunted volume expansion natriuresis and renal re-sistance to ANP found in these nephritic rats in vivo,and lends further support to the contention that acomponent of nephrotic edema results from an intra-renal defect, leading to salt retention (1 ,2). Renal

resistance to ANP is characteristic of other states ofabnormal sodium metabolism, such as congestiveheart failure, diabetes meilitus, and liver disease, in

addition to nephrotic syndrome. In experimental heartfailure in the rat, decreased binding ofANP to its innermedullary receptors has been demonstrated (49), andwe have recently shown a decrease in renal ANP

receptors in rats with streptozotocin-induced diabetesthat could be corrected with insulin administration(50). These observations indicate that ANP resistancein these conditions may be related to a receptor ab-normality, in contrast to the post-receptor defect ob-

served in proteinuric renal disease (14, presentstudy). Thus, it seems likely that renal ANP resistancemay arise via different mechanisms in various condi-tions of altered sodium homeostasis. Further insight

into these mechanisms offers the hope of novel ap-proaches to the management of edema in these dis-eases.

ACKNOWLEDGMENTSThe authors acknowledge the secretarial support of Maria Narvaez.

This work was supported by Grants DK 31623, DK 34198, and DK

47659 from the National Institutes of Health, and by Grants-in-Aid

89-1 124 and 94-1229 from the American Heart Association. At the

time of these studies, Dr. Valentin was the recipient of fellowshipawards from ICI-Pharma and the American Heart Association, Call-

fornia Affiliate, and Dr. Sechi from the Itallan Society of Hypertensionand the Juvenile Diabetes Foundation International. Zaprinast (M&B22,948) was courteously provided by Dr. Taylor from the DagenhamResearch Center of Rhone-Poulenc Rorer, Ltd.

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