Preglomerular Sudanophilia in L-NAME Hypertensive Rats
Involvement of Endothelin
Abstract To characterize alterations of renal vessels occurring during systemic hypertension elicited in rats by 5, 10, and 25 days of treatment by the nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester (L-NAME) (20 mg/kg daily), preglomerular vasculatures, consisting of arcuate arteries and their branches, interlobular arteries, and afferent arterioles, were isolated by HCl maceration. Blockade of nitric oxide synthase significantly increased tail-cuff systolic blood pressure by 21±2% and 42±3% after 5 and 25 days, respectively. Medias of hypertensive arcuate arterial branches and interlobular arteries but not of afferent arterioles had focal deposits of Sudan black–positive lipid droplets. At 25 days, vessel wall thickness increased by 72±6% along the sudanophilic areas. Immunostaining of sudanophilic lesions with a panel of antibodies unveiled medial cell proliferation, macrophage invasion, immunoreactive vascular cell adhesion molecule-1, and low-density lipoprotein. The frequency of sudanophilic lesions increased with time to affect 26±2% and 36±3% of arcuate arterial branches and interlobular arteries, respectively, at 25 days. Hypertensive L-NAME–treated rats developed glomerular injury probed by albuminuria and glomerular immunostaining for α-smooth muscle actin. Administration of the nonselective endothelin antagonist bosentan (30 mg/kg daily) blunted the development of sudanophilic lesions during L-NAME treatment without affecting arterial hypertension or degree of glomerular injury. Therefore, L-NAME hypertension leads to rapid development of focal, inflammatory, proliferative, and sudanophilic lesions along preglomerular vessels, suggesting atherosclerosis-like processes. Furthermore, endothelin is a likely mediator in the development of these lesions.
Endothelial dysfunction, as often manifested by alterations in the l-arginine/NO pathway, plays a key role in the genesis of structurofunctional alterations of the vessel wall associated with atherosclerosis or hypertension (for reviews, see References 1 through 4). This holds true for the renal vasculature,1 in which, in addition, the interplay between NO production, Ang II, and cytokines is a central determinant of glomerular and tubulointerstitial responses to injury.5 In rats, a sustained and reversible systemic hypertension can be induced by chronic inhibition of endothelial NO production6 7 8 9 10 by use of recently available l-arginine analogs like L-NAME. This new model of hypertension is characterized by marked renal vasoconstriction, reduction of glomerular filtration rate, glomerular hypertension, and albuminuria.6 7 8 9 10 Furthermore, although the precise pathogenesis of L-NAME hypertension remains unknown, its development requires an intact renin-angiotensin system,9 10 and endothelin may be involved.11
Various renal morphological alterations were described in rats receiving a daily dose of 5 to 70 mg/kg of L-NAME over a period of 1 to 2 months, including glomerulosclerosis, focal glomerular hyalinization, arteriolar wall thickening and occlusion, and focal interstitial expansion.6 7 12 Although vascular lesions observed during chronic L-NAME hypertension were similar to those encountered in other rat models such as two-kidney, one clip or Ang II–induced hypertension,13 14 no detailed study is presently available concerning their development and segmental distribution, and no immunohistochemical characterization has been undertaken thus far. Recent studies by our group conducted in L-NAME hypertensive rats10 led us to examine isolated renal vasculatures.15 One striking feature of preglomerular vessels was the presence of focal wall thickening loaded with SB+ lipid droplets. To the best of our knowledge, such SB+ lesions have never been described in morphological studies on hypertensive rat models.6 7 12 13 14
The aim of the present study was to document the existence and assess the segmental and time-dependent distribution of preglomerular lesions in rats receiving L-NAME for a period of up to 25 days. This was achieved by direct microscopic observation of large, intact segments of the preglomerular vasculature isolated by our maceration-dissection technique.15 Vascular lesions were further characterized with a panel of antibodies by use of isolated vasculatures and cryostat sections. Albuminuria and intraglomerular expression of α-SM actin were used as markers of glomerular injury. In addition, we assessed reversibility and prevention of preglomerular lesions and examined the potential pathogenic role of endothelin by using the nonselective endothelin antagonist bosentan.
The present animal experiments complied with French law and guiding principles for experimental procedures as set forth in the Declaration of Helsinki. All animals were purchased from Iffa Credo, Lyon, France, and were fed a standard rat chow. SBP was assessed in conscious rats by tail-cuff manometry (Narco BioSystems). Three groups of 10 adult male Wistar rats weighing 250 to 350 g received 20 mg · kg−1 · d−1 of L-NAME (Sigma Chemical Co) by gavage for 5, 10, and 25 days (L-NAME 5, L-NAME 10, and L-NAME 25 groups, respectively). Controls were 10 body weight–matched normotensive Wistar rats (Control group) whose SBP was measured only on the day they were killed. The stability of SBP was verified over a period of 25 days in a separate group of 5 Time-Control rats. The reversibility of L-NAME hypertension was assessed in 6 rats (Reversibility group) 10 days after interruption of a 10-day period of treatment by L-NAME (20 mg · kg−1 · d−1). A group of 5 rats simultaneously received losartan (30 mg · kg−1 · d−1) and L-NAME (20 mg · kg−1 · d−1) by gavage for 10 days (L-NAME+Losartan group). Losartan (DuP 753) was kindly provided by Dr Ronald D. Smith (DuPont Merck Pharmaceutical Co). Finally, a group of 8 rats simultaneously received the nonspecific endothelin antagonist bosentan (30 mg · kg−1 · d−1) and L-NAME (20 mg · kg−1 · d−1) by gavage for 25 days (L-NAME+B group). In pilot studies, this treatment inhibited peak pressor responses to endothelin 1 by ≈80% (Sigma, 300 pmol/kg body wt IV bolus). Bosentan was kindly supplied by Dr Jean-Paul Clozel (Hoffman-LaRoche).
Albuminuria was determined on 24-hour urine collections in the L-NAME 25, Time-Control, and L-NAME+B groups. Albumin was measured by laser nephelometry (086-105 Laser Nephelometer, PDQ, American Instruments Co) as described earlier16 after its immunoprecipitation by a rabbit polyclonal anti-albumin antibody (RARa/Alb, Tebu). Albuminuria was expressed as micrograms of albumin excreted per 24 hours.
Blood lipids were determined on plasma collected from anesthetized rats after an overnight fasting period and were expressed as millimoles per liter of plasma. Total plasma cholesterol levels were determined colorimetrically (cholesterol enzymatic PAP 250 kit, Biomerieux). Plasma LDL cholesterol and plasma HDL cholesterol levels were determined after precipitation (LDL/Cholesterol/Phospholipids kit; HDL/Cholesterol/Phospholipids kit; Biomerieux).
Renal tissue renin concentration was measured in cortical tissue homogenates in the presence of an excess of renin substrate at pH 6.5 with a commercially available radioimmunoassay (CIS Biointernational). Renin concentration was expressed as micrograms of Ang I generated per hour at 37°C per gram of renal cortex.
Isolation of Vessels
Detailed study and characterization of vascular lesions was undertaken in kidneys only. Nevertheless, the presence of SB+ lesions was also assessed in three additional regional vasculatures in L-NAME hypertensive rats. Heart, brain, mesentery, and kidneys were removed and processed as described.15 Brain, heart, and kidney tissues were macerated in 5 mol/L HCl for 1 hour at 37°C, rinsed, and immersed in distilled water at 4°C for 24 to 48 hours. Mesenteric vessels were macerated for 3 hours at room temperature in 3 mol/L HCl before water immersion. Under stereoscopic observation, arteries located at the base and at the surface of the brain were separated from the tissue. In hearts, only vessels situated between tissue layers were isolated. In mesentery, three or four branching orders were explored from the mesenteric artery. In kidneys, preglomerular vessels were separated from tubules. Most glomeruli and postglomerular vessels were removed by this procedure.15 Isolated preglomerular vasculatures were largely intact and kept their three-dimensional topology. They comprised the first divisions of the renal artery into arcuate arteries. In rats, arcuate arteries are not single, unbranched vessels but rather constitute highly dichotomous “trees” that spread at the corticomedullary junction parallel to the kidney surface. We therefore distinguished ArcAs from their smaller ArcBs. Isolated “arcuate trees” were still connected to ILAs and AAs. Vessels were permeabilized with 1% Triton X-100 (Sigma) and immersion-fixed in 10% buffered formalin (Accustain, Sigma) before microscopic examination, Sudan black staining, or immunohistochemistry.
Under pentobarbital anesthesia, kidneys from Control normotensive and L-NAME hypertensive rats were successively rinsed via an aortic catheter with albuminated (1% bovine serum albumin, fraction V, Sigma) PBS, pH 7.4, and PBS containing 0.5 mol/L sucrose. Kidneys were cut transversely and kept overnight at 4°C in PBS-sucrose for cryoprotection, embedded in O.C.T. compound (Miles), and frozen in liquid nitrogen–cooled isopentane. Immunohistochemistry and Sudan black staining were performed on 10-μm-thick cryostat sections.
Sudan Black Staining
Isolated vessels or cryostat sections were first exposed to 60% ethanol for 5 minutes and then to a filtered solution of Sudan black B (Sigma) in 60% ethanol for 15 minutes. Staining was differentiated in 60% ethanol. Vessels and sections were brought back to distilled water. In some instances, cryostat sections were first stained with Sudan black, then photographed without a coverslip under a Leitz Laborlux microscope and further processed for immunohistochemistry (see below).
Quantification of SB+ Lesions
After Sudan black staining, portions of dissected renal vasculatures were observed under a Leitz M7 stereoscope or a Leitz compound microscope. During microscopic examination of isolated vessels, to avoid dimensional distortion resulting from compression, we used fragments of coverslips or pieces of 50-μm microsutures (Ethicon) as spacers between slide and coverslip. SB+ lesions were quantified along the ArcB and ILAs. For a given type of preglomerular vessel, relative frequencies of damaged vessels were calculated as the number of vascular segments with at least one SB+ lesion divided by the total number of vascular segments observed. The percentage of total SB+ lesions specifically located at branching points along the ArcB and ILAs was assessed. During the course of these studies, we also found another type of lesion that will be referred to as “aneurysms,” and we assessed their relative frequency with respect to the total number of lesions (ie, SB+ lesions plus aneurysms). Furthermore, under microscopic observation with a ×25 long-working-distance Leitz objective and using a videomicroscopic measuring system,15 we assessed vessel wall thickness (precision of ±0.5 μm) along SB+ lesions and aneurysms and along adjacent “control” segments. Injured wall thickness was expressed as percent of nearby control value. Thus, values <100% indicated a relative distension, whereas values >100% denoted a relative hypertrophy of the vessel wall.
Immunohistochemical stainings were performed on isolated renal vasculatures and on renal cryostat sections by the avidin-biotin-immunoperoxidase method, as has been described for renin in isolated vessels.15 A series of six primary monoclonal and polyclonal antibodies were used: (1) ED1 (1:500; Serotec), a mouse monoclonal IgG antibody that recognizes cytoplasmic antigen in monocytes and macrophages17 ; (2) PC10 (1:1000; DAKO A/S), a mouse monoclonal IgG antibody directed against PCNA/cyclin. PCNA/cyclin is a 36-kD nuclear polypeptide expressed mostly by proliferating cells during the late G1-S phase and, to a lesser degree, during the G2-M phase of the cell cycle18 ; (3) a mouse monoclonal anti–α-SM–specific actin antibody (1:200; clone No. 1A4, lot No. A-2547, Sigma) characterized in rat kidneys,19 intraglomerular expression of α-SM actin being a good index of glomerular injury14 ; (4) a specific mouse monoclonal IgG antibody directed against human VCAM-1 (1:200; clone BBIG-V1, R&D Systems); (5) a specific polyclonal rabbit anti-rat renin antibody diluted 1:2500,15 kindly provided by Dr Tadashi Inagami (Vanderbilt University, Nashville, Tenn); and (6) a specific polyclonal rabbit anti–apolipoprotein B-100, the major protein component of LDL, displaying specific high-affinity binding to LDL (dilution 1:200; ref. no. 20-AR40, lot No. P4100322; Fitzgerald Industries International, Inc). Its distribution was taken as an index of vascular LDL infiltration. Antibodies 2 and 5 were used in isolated renal vessels, and antibodies 1 through 4 and 6 were used in cryostat sections. Tissues were exposed for 60 minutes to all primary antibodies except 3, 4, and 6, to which tissues were exposed for 120 minutes. Depending on the primary antibody used, samples were incubated with either anti-mouse or anti-rabbit IgG biotinylated antibody. They were incubated with avidin–horseradish peroxidase complex (Vectastain ABC kit, Vector Laboratories) and exposed to 0.01% diaminobenzidine tetrachloride and 0.02% H2O2 as a source of peroxidase substrate (DAB Substrate kit, Vector Laboratories). Sections and vessels were washed in tap water. Negative controls were indicated by absence of staining when the primary antibody was omitted. Sections were counterstained with hematoxylin and mounted in glycerol-Mowiol medium (Calbiochem-Novabiochem Corp). Vascular lipid deposits were visible on freshly prepared sections but were extracted by the mounting medium within a few days. Paired successive stainings with Sudan black and ED1 or Sudan black and PC10 were performed on the same cryostat section. Paired stainings with ED1 and PC10, ED1 and anti–VCAM-1, and ED1 and anti-LDL were performed on two consecutive sections.
Between-group comparisons were performed by one-way ANOVA; pairwise mean comparisons were performed with Fisher’s protected least significant difference test. Where appropriate, within-group analyses were carried out with paired t test or ANOVA for repeated measures, followed by Fisher’s protected least significant difference test for pairwise multiple comparisons. Linear regression analysis was performed by the least-squares method. A value of P<.05 was considered to be significant. Values are given as mean±SEM.
Systemic Blood Pressure
Mean SBPs obtained in the various experimental groups are given in Fig 1⇓. No significant time-related change in SBP was found in the Time-Control group. No between-group difference (one-way ANOVA, eight groups) was found in mean baseline SBP. Mean SBP was significantly increased by 5, 10, and 25 days of L-NAME treatment. In the Reversibility group, SBP returned to baseline after discontinuation of treatment. In the L-NAME+Losartan group, concomitant administration of the angiotensin type 1 antagonist prevented the hypertensive effect of L-NAME. Of importance, bosentan did not prevent L-NAME–induced hypertension, since similar mean SBPs were achieved in L-NAME 25 and L-NAME+B rats.
Time-Related Changes in Urine, Plasma, and Tissue Biochemistry
As depicted in Fig 2⇓, albumin excretion increased significantly at day 5 and remained elevated for 25 days of L-NAME treatment, which suggested early occurrence and persistent alteration of glomerular barrier function. Treatment with bosentan did not affect this pattern. In these rats, albumin excretion increased significantly from a mean baseline of 301±61 to a value of 697±207 μg/24 h (n=8) after 25 days of treatment. This terminal value was not significantly different from that achieved in L-NAME 25 rats (729±108 μg/24 h, n=5). No significant time-related change in albumin excretion was found in the Time-Control group (Fig 2⇓).
As shown in Fig 3⇓, L-NAME treatment had no significant effect on total plasma cholesterol, HDL cholesterol, and LDL cholesterol levels. In the Reversibility and L-NAME+Losartan groups, plasma cholesterol averaged 1.12±0.09 (n=6) and 1.18±0.07 (n=5) mmol/L, respectively. These values did not differ significantly from the 1.37±0.16 mmol/L obtained in the Control group.
Renal renin concentration was measured in 5 rats per group. Renin concentration averaged 1048±90 and 1169±175 μg Ang I · h−1 · g−1 in the Control and L-NAME 5 groups, respectively (P=NS). However, lower values of 628±45 and 472±75 μg Ang I · h−1 · g−1 were found after 10 and 25 days of treatment, respectively. In L-NAME+B rats, renal renin concentration averaged 168±32 μg Ang I · h−1 · g−1 (n=8), a value significantly lower than in L-NAME 25 rats.
Characteristics of Vascular Lesions
Fig 4A⇓ through 4D illustrates the light-microscopic appearance of preglomerular vascular lesions after 25 days of L-NAME treatment. Under low-angle epi-illumination, conspicuous, light-scattering, focal deposits were observed along the ArcB and ILAs, in addition to renin deposits localized along some terminal AAs (Fig 4A⇓). Higher magnification unveiled the granular nature of arterial deposits and vessel wall hypertrophy (Fig 4B⇓). Staining with Sudan black demonstrated the lipidic nature of the granules (Fig 4C⇓ and 4D⇓). These lipid droplets were always localized within the media of the vessel wall (Fig 4D⇓), followed the general orientation of smooth muscle cells, and were not limited to flow dividers but rather entirely encircled the vessels. Of importance, SB+ lesions were never detected along the main ArcA and AAs. Examination of renal vessels from the L-NAME 5 and L-NAME 10 groups indicated the regular occurrence of focal aneurysms (Fig 4E⇓ and 4F⇓). The aneurysms were devoid of lipid deposits, often adjacent to SB+ regions (Fig 4E⇓), and characterized by thinned vessel wall and enlarged lumen (Fig 4F⇓).
In L-NAME–treated rats, aneurysms and SB+ lesions were never encountered in brain or heart vessels; only a few SB+ lesions were detected at the level of third- or fourth-order branchings from the mesenteric artery.
No aneurysms and few residual SB+ lesions were found in the Reversibility group, which demonstrated a complete reversibility of vascular lesions. No vascular lesions were observed in the L-NAME+Losartan group. Hence, in the absence of high blood pressure, L-NAME per se cannot induce formation of vascular lesions. In L-NAME+B rats, some SB+ lesions and aneurysms were encountered along the ArcB and ILAs. No lesions were observed in Control, normotensive rats.
Quantification of Vascular Lesions
Relative frequencies of SB+ vessels were assessed in 5 rats per group. On average, 39±4 and 185±9 ArcB and ILAs, respectively, were observed per rat (n=20). As shown in Fig 5⇓, the frequency of SB+ vessels time-dependently increased during L-NAME treatment. Frequencies of SB+ ArcB and SB+ ILAs were highly correlated (r=.92, P=.0001, n=25; analysis including Control and L-NAME+B rats). The percentage of SB+ lesions located at branching points along the ArcB and ILAs showed no significant time-related trend and indicated that such localization was not a salient feature of SB+ lesions. At days 5, 10, and 25 of treatment, mean percentages were 29±13%, 23±7%, and 18±7%, respectively, in ArcB and 19±5%, 19±5%, and 18±1% in ILAs. As shown in Fig 5⇓, there was a striking reduction in frequencies of SB+ vessels in L-NAME+B rats.
Aneurysms were examined in 4 rats in each of the three L-NAME groups, and their relative frequency decreased significantly with time. When observations made in ArcB and ILAs were pooled, aneurysms accounted for 39±11%, 25±9%, and 8±8% of total vascular lesions in rats treated with L-NAME for 5, 10, and 25 days, respectively. In fact, aneurysms were found in only 1 of 4 L-NAME 25 rats. Hence, aneurysms were progressively replaced by SB+ lesions, suggesting that the latter developed from the former. This view was reinforced by our finding of complete circular rupture of the internal elastic lamina at the level of several SB+ lesions as a testimony of vessel wall stretching. Evolution of relative wall thickness of SB+ lesions and aneurysms is shown in Fig 6⇓. Only relative hypertrophy showed significant time-related change, and if one assumes that SB+ lesions developed from aneurysms, this represented an approximately threefold increase in wall thickness after 25 days of L-NAME treatment. Aneurysms were present in all L-NAME+B rats. They outnumbered SB+ lesions, and in 3 of 8 rats, extensive portions of the preglomerular vasculature exhibited more or less continuous ballooning.
Immunohistochemical Characterization of SB+ Lesions
As shown in Fig 7A⇓, none of the granules located within vascular lesions contained immunoreactive renin; renin cells present at the distal end of some AAs (Fig 4A⇑) were used as “internal controls.” Immunostaining of isolated renal vasculatures of L-NAME 5, L-NAME 10, and L-NAME 25 rats with PC10 revealed intense cell proliferation in areas of increased wall thickness, whereas scattered PC10-positive endothelial and medial cell nuclei were present elsewhere, including in glomeruli (Fig 7B⇓ through 7D). Only scattered PC10-positive cells were observed in Control rats and in rats from the Reversibility and L-NAME+Losartan groups.
To increase the probability of sectioning vascular lesions, immunostaining of cryostat sections was performed in L-NAME 25 rats only. Paired studies demonstrated colocalization of vessel wall thickening, Sudan black staining, and ED1- and PC10-positive immunostainings (Fig 8A⇓ through 8D). Staining of successive sections with ED1 and PC10 confirmed these observations and allowed better preservation of structures. Glomerular accumulation of SB+ lipids was not observed. As shown in Fig 8E⇓ and 8F⇓, distinct macrophages were present periarterially, whereas staining of vascular lesions was always diffuse, probably owing to tissue smearing during cutting. Individual macrophages were also identified within renal interstitium and glomeruli. However, typical glomerular foam cells, as recently demonstrated in rat glomerulonephritis,20 were not observed in L-NAME rats, consistent with our finding of a lack of glomerular lipid deposits. As shown in Fig 8G⇓ and 8H⇓, ED1-positive lesions were also positive for VCAM-1 and for apolipoprotein B-100. The immunostainings for VCAM-1 and apolipoprotein B-100 were observed throughout vessel media, as reported by others within typical atherosclerotic lesions of mouse aorta.21 Therefore, SB+ lesions were characterized by simultaneous macrophage accumulation, proliferation of medial cells, expression of VCAM-1, and LDL infiltration.
Expression of α-SM Actin
On average, 205±12 glomerular profiles were explored per rat (n=20), and the distribution of immunoreactive α-SM actin was assessed in 4 rats per group. Our present observations confirmed and extended previous studies performed in L-NAME 25 rats only.22 In Control rats, as described earlier,14 19 22 α-SM actin was expressed along preglomerular and postglomerular vessels, and 17±1% of glomerular profiles had a few scattered α-SM actin–positive cells. In L-NAME 5 rats, consistent with reported increases in glomerular capillary pressure,6 86±2% of glomeruli showed prominent mesangial staining. This ratio did not change significantly in L-NAME 10 and L-NAME 25 rats; it averaged 86±3% and 87±1%, respectively. Therefore, time-related changes in albuminuria (Fig 2⇑) and intraglomerular α-SM actin expression converged in showing early and persistent glomerular injury in our model of L-NAME hypertension. In L-NAME+B rats, 85±1% of glomeruli exhibited mesangial α-SM actin staining, a value not significantly different from that achieved in L-NAME 25 rats.
Chronic administration of the NO synthase inhibitor L-NAME has provided a new experimental rat model of systemic hypertension.6 7 8 9 10 Chronic L-NAME blockade was shown to decrease endothelial NO synthase abundance23 and impair acetylcholine-induced vasodilation along the renal microvasculature.22 Thus, endothelial dysfunction is central to this new hypertension model. Further, this hypertensive state is reversible after cessation of L-NAME administration7 9 and can be prevented7 9 10 or reversed7 9 by pharmacological interference with the renin-angiotensin system. The present report provides, for the first time, a characterization of renal preglomerular lesions associated with chronic L-NAME–induced hypertension in the rat; examines their prevention and reversibility; and provides clues as to the underlying pathogenic mechanism(s). Instrumental to our analysis was the use of a maceration-dissection technique15 that provided an unprecedented topographic overview of preglomerular vessels from the main ArcA to the AAs and allowed us to find early, infrequent lesions that might have escaped detection by conventional histology.
Our basic finding in L-NAME hypertensive rats was the systematic presence of focal deposition of SB+ lipids within the media of the ArcB and ILAs, associated with increased vessel wall thickness. Use of specific antibodies allowed us to demonstrate that lipid deposits were associated with monocyte/macrophage invasion, expression of VCAM-1, cell proliferation, and LDL infiltration and hence indicated the presence of key elements of the early atherosclerotic process.3 21 In addition, SB+ lesions were found in mesenteric but not in brain or heart vessels. Our results agree with earlier observations made in hypertensive and diabetic patients by Wilens and Elster.24 These authors documented the existence of conspicuous lipid deposits along renal arterioles undergoing hyaline sclerosis and suggested that similar processes underlie atherosclerosis and arteriolar sclerosis. Since then, similarities between glomerulosclerotic and atherosclerotic processes have been demonstrated.25 In rats, hypercholesterolemia promotes aortic fatty streaks26 and aggravates experimental glomerulopathy,27 suggesting a link between these two processes. By contrast, in a recent study on hypercholesterolemic rabbits, Kamanna et al28 concluded that preglomerular vessels were relatively immune to atherosclerosis. Besides potential species differences suggested by the latter study,28 our present work provides a unifying paradigm by which to interpret the large spectrum of preglomerular vascular lesions currently described in hypertensive rat models,13 14 including L-NAME hypertension.6 7 12
Development of SB+ lesions was highly focal and extremely rapid in onset; 8% to 9% of the ArcB and ILAs are affected after 5 days of L-NAME treatment, ie, at a time when SBP had increased significantly, by 21%. Medial cell proliferation was present at that early stage, as reflected by a 40% increase in wall thickness and a positive staining for PCNA/cyclin. Our present results do not allow us to define the exact moment of appearance of SB+ lesions. However, they demonstrate that SB+ lesions develop within a time frame considerably shorter than that required to induce “classic” atherosclerosis in rodents via atherogenic diets.21 26 Of importance, our present results show that vascular lesions induced by 10 days of L-NAME treatment are fully reversible. Further studies are necessary to establish whether a stage of irreversible vascular alteration is achieved later, during L-NAME hypertension.
After 25 days of L-NAME treatment, neither glomerular lipid deposits nor typical glomerular foam cells20 27 could be detected in the present studies. Such dissociation between vascular and glomerular lipid deposition was noted earlier,24 and a dissociation between vascular and glomerular proliferative responses was recently shown in a model of chronic Ang II–induced hypertension.14 Nevertheless, our observation of parallel increases in albuminuria and in intraglomerular staining of α-SM actin, starting at day 5 of L-NAME treatment, unequivocally demonstrates early alterations in glomerular function and structure. Furthermore, bosentan selectively suppressed SB+ lesions without affecting the degree of glomerular injury. Therefore, both preglomerular vessels and glomeruli developed lesions within the same time frame, but distinct pathophysiological processes were probably involved.
Examination of renal preglomerular vasculatures after 5 or 10 days of L-NAME treatment revealed the presence of aneurysms devoid of lipid droplets and often contiguous to SB+ lesions. Frequencies of these two types of lesions showed reciprocal time-related changes, aneurysms being progressively and almost totally replaced by SB+ lesions, suggesting that SB+ lesions developed rapidly from preexisting focal aneurysms. This concept was reinforced by the occasional finding of ruptured internal elastic laminae at the site of SB+ lesions. Furthermore, when blood pressure was clamped to normotensive levels by losartan, L-NAME per se did not induce SB+ lesions. Conversely, formation of aneurysms persisted and was even amplified in hypertensive bosentan-treated rats. Various rat studies have examined the effects of an acute Ang II–induced hypertension on mesenteric29 30 and renal vasculatures31 and demonstrated alternating zones of constriction and dilation along arteries and arterioles. Only dilated zones exhibited increased permeability to colloidal carbon or ferritin and smooth muscle cell rarefaction and necrosis and thus represented the earliest forms of hypertensive vascular lesions.29 30 31 Our present observations are consistent with these acute studies and provide, on a long-term basis, the subsequent vascular response that leads to an atherosclerotic process.
Besides systemic hypertension, several pathogenic mechanisms may contribute to the development of SB+ lesions during prolonged L-NAME treatment. First, chronic L-NAME blockade increases oxidative endothelial stress4 and interferes with the well-recognized antiatherogenic and antimitotic role of NO.2 32 Second, Ang II has a potent mitogenic effect on renal smooth muscle cells32 and promotes LDL transfer into vascular wall33 and LDL peroxidation.34 Our study confirms that Ang II is involved during the early phase of L-NAME hypertension, since renal renin remained stable for 5 days of treatment and losartan-treated rats did not become hypertensive. Interestingly, our results allow us to rule out the possibility that vascular accumulation of lipids was due to excesses in plasma cholesterol, HDL cholesterol, or LDL cholesterol. Third, use of the endothelin antagonist bosentan permitted identification of endothelin as a specific mediator of SB+ lesions. Our results are in agreement with recent studies performed in deoxycorticosterone acetate–salt hypertensive rats35 36 showing that endothelin controls vascular hypertrophy/remodeling processes and, to a lesser degree, blood pressure levels. Along the same lines, it was recently suggested that endothelin promotes the early inflammatory phase of atherosclerosis in hamsters fed cholesterol.37 Finally, an increase in urinary excretion of immunoreactive endothelin was demonstrated during L-NAME hypertension in rats.11
In summary, our results demonstrate that L-NAME hypertension in rats is associated with rapid, focal, and reversible development of SB+ lesions along renal preglomerular vessels. These lesions are characterized by medial cell proliferation, macrophage invasion, and infiltration of LDL, thus providing the hallmark of an early atherosclerotic process. Furthermore, SB+ lesions most likely develop from focal, pressure-induced aneurysms. Ang II contributes crucially to raising blood pressure during the early phase of hypertension. Whereas endogenous endothelin contributes little to raising blood pressure, it selectively mediates the formation of SB+ lesions.
Selected Abbreviations and Acronyms
|Ang I||=||angiotensin I|
|Ang II||=||angiotensin II|
|ArcA||=||main trunk of arcuate arteries|
|ArcB||=||lateral or terminal branches of arcuate arteries|
|L-NAME||=||NG-nitro-l-arginine methyl ester|
|PCNA||=||proliferating cell nuclear antigen|
|SB+||=||Sudan black positive; sudanophilic|
|SBP||=||systolic blood pressure|
|VCAM-1||=||vascular cell adhesion molecule-1|
These studies were supported by a grant from Institut National de la Santé et de la Recherche Médicale to Dr Casellas (INSERM CRE 930404). During the course of these studies, N. Bouriquet was successively supported by research fellowships from the Société d’Hypertension Artérielle and from the Société de Néphrologie. We acknowledge Annie Artuso and Agnès Robert for their expert technical assistance. Histological equipment was kindly made available to us by Dr Jean Léger (INSERM U300, Montpellier). Micrographs were printed by Patrick Schuman (INSERM, Service d’Iconographie, Montpellier).
Presented in part at the 27th and 28th Annual Meetings of the American Society of Nephrology, held in Orlando, Fla, October 26-29, 1994, and in San Diego, Calif, November 5-8, 1995, respectively, and published in abstract form (J Am Soc Nephrol. 1994;5:535; J Am Soc Nephrol. 1995;6:619).
- Received September 25, 1995.
- Revision received November 17, 1995.
- Accepted December 7, 1995.
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