Renal Hypertensive Angiopathy

AJH 1995; 8:167-176

Renal Hypertensive Angiopathy Comparison Between Chronic NO Suppression and DOCA-Salt Intoxication Yichun Xu, Jean-Franfois Arnal, Nicole Hinglais, Marie-Dominique Appay, Irene Laboulandine, Jean Bariety, and Jean-Baptiste Michel

NC-nitro-L-arginine methyl ester (L-NAME) and 11-desoxycorticosterone plus salt intake (DOCA- salt) hypertensive rat models were compared to study the possible involvement of model-specific factors in the development of renal angiopathy and left ventricular hypertrophy (LVH). Blood pressure was measured in L-NAME, DOCA-salt hypertensive, and control Wistar rats, and the lesions of nephroangiosclerosis and left ventricular hypertrophy were evaluated after 7 weeks. Arterial wall cyclic guanosine monophosphate, plasma renin activity (PRA), and renal renin storage were assessed in parallel. For the same level of hypertension in the two models, the renal arterial fibrinoid necrotic lesions were significantly more frequent in L-NAME than in DOCA-salt hypertensive rats. In DOCA-salt hypertensive rats, PRA was decreased and arterial cGMP increased compared to

controls. In the L-NAME model, arterial cGMP decreased and PRA showed a bimodal distribution in this intermediate stage of hypertensive disease. LVH was observed in DOCA-salt rats and only in the L-NAME rats with a high level of PRA. There was a close correlation between the lesions of nephroangiosclerosis, left ventricular index, and plasma renin activity in L-NAME rats. We therefore suggest that the activation of the renin-angiotensin system participates specifically in the development of the second stage of hypertension during chronic blockade of NO synthase involving nephroangiosclerosis and LVH. Am J Hypertens 1995;8:167-176

KEY WORDS: NG-nitro-r-arginine methyl ester (L-NAME), plasma renin activity, glomerulosclerosis, cyclic GMP, renin immunostaining.

I n vitro, NC-nitro-r-arginine methyl ester (L- NAME) is a potent inhibitor of both constitutive and inducible nitric oxide (NO) synthase. 1'2 In vivo L-NAME administration to rodents is ac-

companied by a rise in blood pressure and a gener- alized decrease in peripheral blood flow, indicating that a continuous release of NO of endothelial origin actively maintains vasodilator tone. 3"4 Constitutive

Received April 27, 1994. Accepted September 27, 1994. From INSERM U28 (XY, HN, AM-D, BJ), Paris; and INSERM

U367 (AJ-F, LI), Paris, France. This work was supported in part by the Institut National de la

Sant6 et de la Recherche M6dicale. Address correspondence and reprint requests to Dr. Jean-

Baptiste Michel, INSERM U367, 17 rue du Fer a Moulin, 75005 Paris, France.

NO-synthase has been also localized in macula densa cells, 5"6 involved in tubulo-glomerular feedback mod- ulation, 6"7 and in other tubular segments. 8 Moreover Tojo and coworkers 6 recently reported the presence of inducible NO-synthase in smooth muscle cells of the afferent arterioles in normal rats. Rats given L-NAME for 4 weeks showed a time- and dose- dependen t increase in blood pressure and thus L-NAME rats represent a novel experimental model of hypertension. 9'1° Six to eight weeks of L-NAME administration led to the development of nephroan- giosclerotic lesions. 1°-12

Desoxycorticosterone plus salt intake (DOCA-salt)- induced hypertension is a well established experimental model of hypertensive nephropathy. 13"14 In this model, circulating renin and subsequently an-

© 1995 by the American Journal of Hypertension, Ltd. 0895-7061/95/$9.50 0895-7061(94)00179-F

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168 XU E T A L AJH-FEBRUARY 1995-VOL. 8, NO. 2

giotensin II are both suppressed, whereas the endothelium appears to compensate for increased contrac- tile and reduced relaxant vascular muscle responses by increasing the release of endothelium-derived relaxing factor, NO. is

We have studied the L-NAME and DOCA-salt rat models of hypertension to determine whether blockade of the r-arginine-NO pathway has a specific con- tribution to the development of nephroangiosclerosis, beyond that of increased blood pressure. Blood pressure was therefore measu red in L-NAME, DOCA-salt, and control Wistar rats and nephroangiosclerosis and left ventricular hypertrophy (LVH) were evaluated after 7 weeks. The arterial wall cyclic guanosine monophosphate (cGMP) content (known to reflect NO-release9), plasma renin activity (PRA), and in situ renal renin histochemistry were assessed in parallel. Left ventricular mass and histologic structure were determined.

MATERIALS AND METHODS

Experimental Des ign and Animal Preparation Three groups of male Wistar rats (Iffa Credo, Lyon, France), weighing 120 to 130 g were used in this ex- periment. The L-NAME group consisted of 10 sham- operated rats given L-NAME (50 mg/100 mL, Sigma Chemical Company, St. Louis, MO) in the drinking water for 7 weeks, resulting in a daily intake of approximately 50 mg/kg of L-NAME.

Unilateral nephrectomy and subcutaneous DOCA pellet (200 mg/kg) implantation were performed in 10 anesthetized rats. The pellet contained 60% DOCA (Sigma) and 40% of a mixture of elastomere and cat- alyst M. These rats were given drinking water con- taining 1% NaC1 and a potassium supplement. The DOCA dose was chosen to increase blood pressure to the same extent as in rats given L-NAME (prelimi- nary studies). In the control group, 10 rats were sham-operated and received ordinary tap water for 7 weeks.

Systolic blood pressure was measured weekly by the tail-cuff method (W + W electronic recorder 8005, Apelab, Bagneux, France). At the end of the experimental protocol, all rats were placed in metabolic cages for a 24-h urine collection and then killed by decapitation. Blood samples were collected and the plasma stored at -20°C. The thoracic aorta was rap- idly excised, rinsed in cold phosphate buffered saline (PBS), frozen in liquid nitrogen, and stored at - 80°C for later cGMP detection. The kidneys were excised and a slice was fixed with alcoholic Bouin solution. The left ventricle was separated from the right ventricle and weighed. A transversal slice was then fixed in formaldehyde 4% for morphologic study.

Determination of Left Ventricular (LV) Hypertrophy Left ventricular hypertrophy was defined as a left ven-

tricular weight to body-weight ratio (left ventricular index) greater than 2 standard deviations above the mean ratio of the control group (ie, above 2.12 mg/g). Left ventricles were also studied histologically.

Determination of Aortic cGMP Concentrations The thoracic aorta was thawed and homogenized at 4°C in 10 vol 0.1 N HC1 with an all-glass homogenizer. Ho- mogenates were centrifuged at 15,000 g for 30 min and aliquots of the supernatants were stored at - 20°C until assayed. One of the aliquots was used to determine the protein concentration by the Bio-Rad Coomassie brilliant blue G-250 method (Biorad, Ivry/ seine, France) with bovine serum albumin (BSA) as the standard. 9 The cyclic GMP content was determined by radioimmunoassay as described by Cailla et a116 and modified by Honma et al. 17

Plasma and Urinary Parameters Plasma renin activity was assessed as previously described is by radioimmunoassay quantification of angiotensin I genera- tion. Plasma and urinary creatinine levels were measured by the method of Jaffe (Creatinine Combination Test, Boehringer, Mannheim, Germany). Blood urea nitrogen was measured using an enzymatic colori- metric method (Boehringer). Proteinuria was measured by the Coomassie brilliant blue method with a Biorad Kit (Biorad, Ivry/seine, France).

Morphologic Studies The fixed kidney and heart slices were embedded in paraffin and cut into 4-~m- thick sections. Kidney sections were stained with he- matoxylin-eosin-safran (HES), Masson's trichrome, and silver impregnation, while heart sections were stained with HES.

In order to evaluate the degree of renal damage, a method involving a semiquantitative score was de- veloped. At least 30 glomeruli and 30 arteries or arterioles were examined per section. The severity of each lesion was graded from 0.25 to 1 (0.25, 0.50, 0.75, 1) according to the percentage of the given structure affected by the pathologic process. Thus, 0.25 represented the involvement of 25% of the vascular wall section or glomerulus, whereas 1 indicated that the entire anatomic structure was involved. A global injury score was obtained by multiplying the number of lesions with a given degree of damage (0.25 to 1).

For analyzing the in situ renin storage in the juxtaglomerular apparatus, rabbit polyclonal antibodies to murine renin (mouse submandibular gland) 19 were used. Antibodies were visualized by the peroxidase

• 2 0 • antiperoxidase (PAP) complex techmque (Blolyon, Lyon, France). Monoclonal anti-o~-smooth muscle actin antibodies 21 were used to study the arterial and arteriolar lesions and visualized by the alkaline phosphatase anti-alkaline phosphatase (APAAP) complex method 22 (Dakopatts, Copenhagen, Denmark). Rab-

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AJH-FEBRUARY 1995-VOL. 8, NO. 2 NEPHROPATHY IN L-NAME HYPERTENSION 169

bit antirat albumin antibodies coupled to FITC (Nor- dic Diagnostics, Berchem) 23 were used to demonstrate absorption droplets in glomerular podocytes and tubular epithelial cells. Results were obtained by a direct immunofluorescent method.

Statistical Methods Results are expressed as mean + SEM. Differences in blood pressure were evaluated by repeated measures analysis of variance (ANOVA). An average of the systolic blood pressure at 5, 6, and 7 weeks (mean systolic blood pressure) was calcu- lated and used for correlations. One-way analysis of variance followed by Scheff~'s F-test was used to compare the effects of L-NAME and DOCA-salt on the different variables. Analysis of variance with mul- tiple regression involving several variables was also performed. Linear regression curves and correlation coefficients were obtained by the least-squares method.

RESULTS

Mortality Rate, Blood Pressure, and Body Weight Seven rats died before the end of the s tudy (3 L-NAME rats, 4 DOCA-salt rats); all had a systolic blood pressure ---220 mm Hg at least once before death. Both 7 weeks of oral L-NAME and DOCA-salt regimen induced a time-dependent increase in blood pressure (Figure 1). The mean systolic blood pressure (from the 5th, 6th, and 7th weeks) in DOCA-salt rats was not significantly different from that in L-NAME rats (Table 1). The DOCA-salt regimen reduced the rate of body weight increase compared to controls (Table 1).

PRA and Aortic cGMP Content Compared to control values mean PRA was significantly higher in the L-NAME group and lower in DOCA-salt rats (Table 1). PRA values were dispersed in the L-NAME rats, as demonstrated by the large standard deviation of this variable compared to control values. To further

"1- E E

03

O. O O JD .O O >,

co

200

150

1 2 0

~ ~ L-NAME D O C A - s a l t

~ ~ / ~ controls

2 3 4 5 6 7

Time (week)

FIGURE 1. Effects of L-NAME (-k) and DOCA-salt (A) administration on blood pressure. L-NAME and DOCA-salt induced a similar significant increase in blood pressure as compared to controls (0).

analyze the L-NAME group, we defined two subgroups according to the PRA value: if the PRA was more than 2 standard deviations above the mean value of controls, ie, >5.91 ng angiotensin I/mL/h, rats were classified in the group L-NAMEa; if not, they were classified in the group L-NAMED. Table 2 shows the higher PRA in the L-NAME a group (n = 5), and no significant difference between controls and L-NAME D rats (n = 5).

The cGMP content of the arterial wall was significantly altered in opposite directions in L-NAME and DOCA-salt rats (Table 1). Compared to controls, it was about 6.5-fold lower in L-NAME rats and 1.8-fold higher in DOCA-salt rats. Cyclic GMP content was not different between L-NAME a and b subgroups.

LV Weight and LV Index (Tables 1 and 2) The LV index (LV weight to body weight ratio) of DOCA-salt rats was significantly increased compared to that of controls (Table 1). There was a correlation between the LV index and the mean systolic blood pressure in this group (r = 0.7, P < .05).

LV weight and LV index were also significantly increased in L-NAME rats compared to controls (Table 1). Nevertheless, cardiac hypertrophy was lesser in L-NAME than in DOCA-salt rats (P < .001). In the L-NAME group, there was a close correlation between LV index and PRA (r = 0.9, P < .0005). LV index was only significantly increased in the L-NAME a group (Table 2). In contrast with DOCA-salt rats, no correlation was found between LV index and mean systolic blood pressure in the L-NAME group.

Creatinine Clearance, Plasma Urea Nitrogen, and Proteinuria (Table 3) Compared with controls, creatinine clearance was significantly lower only in DOCA-salt rats (Table 3). Plasma urea nitrogen levels in the L-NAME and DOCA-salt rats did not differ from those in the control group. Proteinuria was higher in both hypertensive rat groups. Once again renal function was more intensively impaired in the L-NAME a group compared to L-NAME b group (Table 4).

Renal Morphologic Alterations in L-NAME Rats Vascular Lesions (Table 5) Arterial and arteriolar medial fibrinoid necrosis were the most prominent lesions, the vessel walls being infiltrated by amorphous eosinophilic material. This fibrinoid necrosis was frequently accompanied by vascular wall cell proliferation and mild perivascular cellular infiltration (Figure 2A).

Arterial and arteriolar wall-thickening, constituted by arterial wall cell proliferation and extracellular matrix accumulation, was observed frequently. Immu- nohistochemistry using anti-m-smooth muscle actin antibodies showed that these proliferating cells were medial myocytes, strongly immunolabeled by anti-s-

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170 XU ET AL AJH-FEBRUARY 1995-VOL. 8, NO. 2

TABLE 1. COMPARISON OF PHYSIOLOGIC PARAMETERS IN L-NAME, DOCA-SALT RATS, AND IN CONTROLS

LVW/BW PRA Aortic cGMP BW (g) SBP (mm Hg) LVW (mg) (mg/g) (ngAI/mL/h) (fmolYmg protein)

Control 365.2 + 26.9 134 -+ 4 711 + 98 1.94 -+ 0.18 3.9 --- 2.0 1905 -+ 649 (n = 10)

L-NAME 353.6 + 39.5 204 + 12" 855 + 142" 2.45 + 0.47* 15.6 + 14.2"t 297 + 88"t (n = 10)

DOCA 304.1 + 54.61"t 203 -+ 11" 907 + 90* 3.04 + 0.37"t 0.5 + 0.4* 2811 + 904* (n = 10)

ANOVA P < .01 P = .0001 P < .005 P = .0001 P < .005 P < .0001

BW, body weight; SBP, an average of the systolic blood pressure at 5, 6, and 7 weeks; LVW, left ventricular weight; PRA, plasma renin activity; AI, angiotensin I; cGMP, cyclic guanosine monophosphate.

Data are mean +- SD, P value of one-way ANOVA.

*Significantly different from values in the control group, P < .05.

fSignificantly different from values in the DOCA-salt hypertensive rat group or different from values in the L-NAME hypertensive rat group, P < .05.

smoo th muscle-act in monoc lona l ant ibody, and ad- ventitial sp ind le - shaped fibroblasts which were not immuno labe l ed by this an t ibody (Figure 2B). No proliferative cells were seen in int ima. This medial and adventi t ial cell prol iferat ion and the concentric accu- mula t ion of extracellular matrix layers could result in "on ion skin"-l ike lesions that some t imes obli terated the vessel l u m e n (Figure 2C).

Glomerular Lesions (Table 5) Focal segmenta l g lomer- ulosclerosis was p resen t and its incidence was greater in rats wi th severe arterial or arteriolar lesions. Glo- meru la r tuft collapse was found in rats wi th severe arterial or arteriolar lesions. Ano the r podocyt ic lesion consisted of cytoplasmic hyal ine drople ts and detach- m e n t of the cy top lasm f rom the b a s e m e n t m e m b r a n e (Figure 3A). These podocyt ic hyal ine drople ts were detected by an t ia lbumin ant ibodies wi th a direct im- munof luo re scen t technique (Figure 3B). Hyal ine material and th rombi were obse rved in the capillary lu- mens of two rats and were capable of obli terating the g lomeru la r tuft (Figure 3C). This hyal ine material was also found in the ur inary space of B o w m a n ' s capsule. Along wi th arteriolar necrotic lesions, fibrinoid necrosis of the g lomeru la r tuft associated wi th crescentic

proliferat ion was occasionally obse rved in the Bow- m a n ' s ur inary space in one rat. A n d once again the vascular and the g lomerular lesions p r e d o m i n a t e d in the L-NAME a as c o m p a r e d to the L-NAME b sub- g roups (Table 6).

Tubuloin ters t i t ia l Lesions The tubulo in te rs t i t i a l lesions accompany ing vascular and g lomeru la r lesions were focal tubular a t rophy and dilatation, interstitial fibrosis, and mild mononuc l ea r cell infiltration. The severi ty of tubulointersti t ial lesions was propor t iona l to that of vascular and g lomeru la r lesions.

In Si tu Renin Storage Using rabbit polyclonal antibodies against mur ine renin, the extent of renin in the j u x t a g l o m e r u l a r a p p a r a t u s f r o m the m a j o r i t y of L-NAME rats was s h o w n to be similar to that in controls (Figure 4A). Howeve r , in the more severe cases (3 L-NAME a rats), there was an extension of renin- posit ive cell hyperp las ia in afferent arterioles (Figure 4B).

D O C A - S a l t Rats All p r e v i o u s l y desc r ibed al ter- ations were also obse rved in DOCA-sa l t rats, bu t the arterial and arteriolar lesions were less p r o n o u n c e d than in L-NAME rats, especial ly concern ing the ne-

TABLE 2. COMPARISON OF PHYSIOLOGIC PARAMETERS IN L-NAME RAT SUBGROUPS

SBP (mm Hg) LVW (mg) LVW/BW (mg/g) PRA (ng AI/mL/h)

Control (n = 10) 134 _+ 4 711.5 + 97.75 1.94 + 0.18 3.91 + 2.0 L-NAME a (n = 5) 207 + 16" 949.4 + 116.5"t 2.85 + 0.16"t 27.68 + 9.2"t L-NAME b (n = 5) 199 + 8* 761.4 + 98.79 2.05 + 0.28 3.43 + 1.87 ANOVA P < .0001 P < .0001 P < .0001 P < .0001

BW, body weight; SBP, an average of the systolic blood pressure at 5, 6, and 7 weeks; L VW, left ventricular weight; PRA, plasma renin activity; AI, angiotensin I.

Data are mean + SD, P value of one-way ANOVA.


fSignificantly different from values in the L-NAME b rats, P < .05.

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TABLE 3. RENAL FUNCTION PARAMETERS IN L-NAME, DOCA-SALT RATS, AND IN CONTROLS

Blood Urea Creatinine Nitrogen Proteinuria Clearance

(l~mol/mL) (mg/24 h) (mL/min)

Control 8.6 ± 1.69 9.39 -+ 4.66 1.04 ± 0.31 (n = 10)

L-NAME 13.1 -+ 4.3 80.75 ± 96.55* 0.80 -+ 0.15 (n = 10)

DOCA 12.44 ± 7.5 52.88 -+ 42.81 0.52 -- 0.23 (n = 10)

ANOVA NS P < .05

Data are mean +- SD, P value of one-way A N O V A .


crotic lesions (Table 5). In addition, in the DOCA-sal t rats, the glomerular diameter and the glomerular cap- i l lary tu f t v o l u m e w e r e i n c r e a s e d . G l o m e r u l a r podocyt ic lesions were more f requent ly observed than in L-NAME rats. On the other hand, immuno- h is tochemis t ry us ing antirenin antibodies s h o w e d that in situ renin storage by the juxtaglomerular apparatus was reduced in DOCA-sal t rats.

Results of the semiquantitative assessment of arterial and glomerular lesions in the two hypertensive rat models and the control group are shown in Table 5. The percentages of all vascular lesions were higher in the L-NAME and DOCA-sal t rats than in the control group. Necrotic arterial lesions were significantly more f r e q u e n t in L - N A M E - t r e a t e d rats t h a n in DOCA-sal t rats. In contrast, podocytic glomerular lesions were significantly more p ronounced in DOCA- salt than in L-NAME rats. In L-NAME rats, there were close correlations be tween PRA and necrotic arteriolar lesions (r = 0.98, P < .005) (Figure 5), arteri- oloproliferative lesions (r = 0.8, P < .01), and isch-

TABLE 4. RENAL FUNCTION PARAMETERS IN L-NAME RAT SUBGROUPS

Blood Urea Nitrogen Proteinuria

(lamol/mL) (mg/24 h)

Control (n = 10) 8.6 + 1.7 9.4 -+ 4.6 L-NAME a (n = 5) 16.0 + 4.1" 145 -+ 101"t L-NAME b (n = 5) 10.1 + 1.6 16.31 ± 19.8 ANOVA P < .0005 P < .0005

Data are mean +- SD, P value of one-way A N O V A .


tSignificantly different from values in the L-NAME b rats, P < .05.

emic glomerular lesions (r = 0.8, P < .01). These correlations did not exist in DOCA-sal t rats. There was no correlation be tween systolic blood pressure and necrotic arterial lesions in L-NAME rats. In these rats, vascular and glomerular lesions were also correlated. Both necrotic and proliferative arteriolar lesions were correlated with glomerular sclerosis (r = 0.7, P < .05 and r = 0.8, P < .001, respectively), ischemic glomerular lesions (r = 0.7, P < .005 and r = 0.8, P < .01), and podocyt ic glomerular cellular lesions (r = 0.6, P < .05 and r = 0.8, P < .01).

Once again, the structural alterations observed in L-NAME rats showed a bimodal distribution (Table 6) correlated with the bimodal distribution of functional alterations (Table 4) and of PRA and cardiac hyper- t rophy and fibrosis (Table 1).

Cardiac M o r p h o l o g i c S tudies L-NAME b rats had neither left ventricular hype r t rophy nor parenchymal myocardial necrosis (Figure 6A); L-NAMEa rats had left ventricular hype r t rophy and focal fibrosis (Figure 6B) accompanied by mononuclear cell infiltration. In this latter group, the same arterial alterations were observed in the heart as in the k idney (Figure 6C).

TABLE 5. RENAL ARTERIAL OR ARTERIOLAR AND GLOMERULAR LESIONS IN L-NAME, DOCA-SALT, AND CONTROL RATS

Arterial or Arteriolar Lesions (%) Glomerular Lesions (%)

Hyaline Droplet in Intracapillary

Necrosis Proliferation Fibrosis Sclerosis Podocyte Thrombi

Control 0 0.06 + 0.198 0.083 -+ 0.2 0.5 + 0.7 0 0 (n = 10)

L-NAME 10.6 -+ 10.6"t 17.5 + 9.66* 12.65 --- 10.8" 5.71 ± 5.72* 0.54 -+ 1.57 0.67 + 1.165 (n = 10)

DOCA 1.92 .+ 2.52 13.6 -+ 11.19" 11.33 + 11.5" 4.04 .+ 3.78 4.42 --- 4.15"t 2.5 --- 3.45* (n = 10)

ANOVA P < .005 P < .0005 P < .01 P < .05 P < .005 P < .05

Data are mean +_ SD, P value of one-way A N O V A .


tSignificantly different from values in the DOCA-salt hypertensive rats group or different from values in the L-NAME hypertensive rats group, P < .05.

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FIGURE 2. Renal arteriolar lesions in L-NAME rats. (A) Fibrinoid necrosis of the arteriolar medial layer; (B) hyperplasia of medial smooth muscle (oL actin staining); (C) "'onion skin aspect" ( x 400).

All hearts of DOCA-salt rats showed classical concentric left ventricular hypertrophy associated with coronary arterial wall hypertrophy (data not shown).

DISCUSSION

As arterial hypertension is an important determinant of renal and cardiac alterations, similar blood pressure levels had to be achieved in both the L-NAME and DOCA-salt groups to allow subsequent compar- isons. The tail-cuff method has been validated in this model in comparison with direct intraarterial assessment of blood pressure in conscious rats. 9'24 The lack of significant difference in mean systolic blood pressure between these two groups shows that this pre- requisite was obtained. However, renal arteriolar angiopathy (fibrinoid necrotic lesions) was significantly

more frequent in L-NAME rats than in DOCA-salt rats. Left ventricular hypertrophy was only observed in L-NAME rats with high levels of PRA and in DOCA-salt rats. Although these two hypertensive models could differ in many aspects, the fundamen- tal underlying systems involved (the renin-angiotensin system and the L-arginine-NO-cyclic GMP pathway) were clearly different in the present study.

NO release is dramatically decreased in L-NAME- induced hypertension, as previously demonstrated by the 10-fold reduction in aortic cyclic GMP content 9 and confirmed in the present study. In contrast, DOCA-salt rats had 1) increased arterial cyclic GMP content, probably reflecting, 2) increased basal NO release, which has been demonstrated elsewhere in isolated microvessels from this model using a phar- macologic approach. 15 This increased cyclic GMP

FIGURE 3. Renal glomerular lesions in L-NAME rats. (A) Epithelial droplets in the glomerular tuft; (B) immunostaining with antirat albumin antibodies; (C) glomerular hyalinosis (x 400).

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TABLE 6. RENAL ARTERIAL OR ARTERIOLAR A N D GLOMERULAR LESIONS IN L-NAME RAT SUBGROUPS

Arter ia l or Ar te r io la r Les ions (%)

Necrosis Proliferation Fibrosis Sclerosis

Glomerular Lesions (%)

Hyaline Droplet in Intracapillary

Ischemia Podocyte Thrombi

Control 0 0.06 -+ 0.198 0.083 -+ 0.2 0.5 -+ 0.7 0.29 -+ 0.68 0 0

(n - 10) L-NAME a 18.88 + 8.7"t" 23.8 -+ 8.5"t" 17.12 -+ 12.4" 8.08 + 7.3* 11 -+ 8.63"1" 1 +- 2.24 1 -+ 1.49

(n = 5) L-NAMEb 2.25 +- 2.19 11.21 -+ 6.22 8.17 -+ 7.72 3.33 -+ 2.52 1.17 + 1.12 0.08 +- 0.19 0.33 -+ 0.75

(n = 5) A N O V A P < .0001 P < .0001 P < .01 P < .05 P < .05 NS P < .05

Data are mean +- SD, P value of one-way ANOVA.


tSignificantly different from values in the L-NAME;; rats, P K .05.

content could also be due to activation of the partic- ulate guanylate cyclase, but we have recently shown that aortic cyclic GMP content was more responsive to endothelial NO production than to atrial natriuretic factor. 24 Cyclic GMP inhibits vascular contrac- tility and growth of cultured smooth muscle 25 and glomerular mesangial cells. 26 Thus, the blockade of NO release characterizing L-NAME-induced hypertension could be expected to favor renal arteriolar smooth muscle and mesangial cellular proliferation. Moreover, afferent renal arterioles probably process both the endothelial constitutive form and a smooth muscle cell-inductible form of NO synthase. 6 Con- versely, increased activity of the r-arginine-NO- cyclic GMP pathway in DOCA-salt rats could account for partial prevention of renal arteriolar smooth muscle cell proliferation in these rats. However, in the present study, we failed to demonstrate these effects

F I G U R E 4. Renin immunostaining. (A) Immunostaining of an afferent arteriole in an L-NAME rat (similar to controls); (B) hyperplasia of myoepithelioid renin positive cells in the L-NAME a subgroup (x250).

in our models, suggesting that hemodynamic and hormonal factors other than cyclic GMP predominate in the development of vascular proliferative lesions in DOCA-salt rats.

The renin-angiotensin-aldosterone system has been strongly implicated in the pathophysiology of different forms of hypertension. In the present study, chronic DOCA-salt administration decreased PRA as expected. In a previous study, hypertensive rats given L-NAME for 4 weeks had a slight decrease in PRA. 9 These data are in agreement with other studies, 27-29 whereas others observed an increase in PRA after blockade of NO synthase. 1°'12"3° The reason for this discrepancy may be due to the difference in time and L-arginine antagonist doses between these different studies and therefore to the difference in renal arteriolar tree damage. At this dose (approximately 50 mg/kg/day of L-NAME), 7 weeks represented an intermediate stage of the hypertensive disease between the initial well-compensated stage (1 month) and the later end-organ-diseased stage leading to death. It is an intermediate period during which each individual may or may not have evolved from the early to the

35

._o ~ 25

c

; 5 0 "v"

< 5 15 25 35

Plasma renin ac t i v i t y (ng AI/ml/h~

F I G U R E 5. Correlation (r = 0.98, P < .005) between arteriolar necrotic lesions and plasma renin activity in L-NAME rats. L-NAMEb group (0); L-NAME a group (-it).

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FIGURE 6. Left ventricular morphologic aspect. (A) Normal morphology in nonhypertrophic left ventricle of L-NAME b rats ( x 250); (B) subendocardial fibrosis area in a hypertrophied left ventricle of the L-NAME a group (x250); (C) medial hyperplasia of a coronary artery in the L-NAME~ group (x lO00).

decompensated stage of hypertension. Our early observed decrease in renin activity could be functionally attributed either to an increase in renal perfusion pressure or to the suppression of the potential direct stimulation of renin release by NO. However, the direct role of NO in inhibiting or stimulating renin synthesis remains very controversial. Gardes 31 and S c h o l z 32 demonstrated that NO could stimulate renin secretion, but Beierwaltes, 33 Vidal, 34 and Sigmon 3s have shown that NO suppresses renin secretion. In the present study, after 7 weeks of L-NAME, PRA remained normal in five rats (L-NAME b group) and was elevated in five others (L-NAME a group). There were close correlations between the bimodal distribution of PRA level and the bimodal distribution of arteriolar fibrinoid necrotic and proliferative lesions. Thus, the structural renal ischemia secondary to renal angiopathy could contribute to the increased renin secretion and angiotensin formation. This secondary rise in renin-angiotensin system activity could be attributed to the development of the nephroangioscle- rotic lesions (Tables 2, 4, and 6), forming the vicious circle depicted by Laragh and coworkers. 36 Con- versely, a significant increase in systemic and local (renal) production of a peptide such as angiotensin II could participate in the accelerated structural alterations observed in the L-NAME a subgroup. This would explain the close correlation between PRA and the frequency of angiopathy in the L-NAME rats. This correlation has also been found by Volpe and coworkers in stroke-prone spontaneously hypertensive rats (SHRSP). 37 Angiotensin II is known to increase vascular permeability, as demonstrated by several studies in hypertensive 38 and nonhypertensive models, 39 thus allowing the passage of fibrin and platelets through the vessel wall. 4° Indeed, in the present study, arterial fibrinoid necrotic lesions were significantly more frequent in the L-NAME rats, es- pecially in the L-NAME~ group characterized by an associated increased PRA.

Arterial blood pressure is considered to be an important determinant of LV hypertrophy in most rat

models of experimental hypertension and left ventricular hypertrophy is thought to be a universal mech- anism for adapting to cardiac overload. This was confirmed in the present DOCA-salt rats, whose LV index was 156% that of controls, and was correlated with systolic blood pressure. However, this correlation did not exist in L-NAME rats and LVH was not observed in L-NAME b rats, despite the same arterial pressure as in DOCA-salt rats, confirming previous observations after 8 weeks of L-NAME. 41 However, in the five L-NAME a rats, L-NAME induced left ventricular hypertrophy and focal fibrosis. PRA was significantly increased in this group and once again, Morton's study 12 also reported the concomitant ap- pearance of the renin angiotensin system activation, cardiac and vascular hypertrophy. The roles of angiotensin II in the development of left ventricular hypertrophy and cardiac fibrosis have been recently dis- c u s s e d . 42"43 Thus, the secondary increased renin- angiotensin activity in the L-NAMEa rats could participate in the development of left ventricular hypertrophy and fibrosis. In addition, coronary arterial lesions could also contribute to the development of myocardial fibrosis in these severely hypertensive L-NAME~ rats. These results suggest that factors other than arterial hypertension alone, such as the angiotensin peptidergic system, could also influence the development of left ventricular hypertrophy and cardiac fibrosis in this model of chronic NO synthase blockade.

Our earlier studies 9'4"44 and the present one suggest that the L-NAME model of hypertension could be described as a two-stage model depending on time and on the dose used. An early functional stage of hypertension without cardiac hypertrophy and without activation of the renin angiotensin system 9 is followed by a secondary stage associating structural damage of the renal and spinal arterioles, 12'44 activation of the renin angiotensin system, and the development of cardiac hypertrophy and fibrosis. 41 This suggests that renal angiopathy plays an important role in the secondary activation of renin release and

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the latter appea red to aggrava te the renal and cardiac consequences of N O blockade- induced hyper tens ion . It was therefore of interest to inhibit this sys tem using conver t ing e n z y m e inhibitors or angiotens in lI antag- onists to conf i rm the role of the RAS in the develop- men t of this N O blockade model . 12'27

ACKNOWLEDGMENTS

We thank Annie Depardieu for the artwork, and Georges Salmon and Liliane Louedec for their technical assistance.

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Renal Hypertensive Angiopathy

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