ACE Inhibition Prevents and Reverses L-NAMEExacerbated Nephrosclerosis in Spontaneously Hypertensive Rats
Abstract Chronic nitric oxide inhibition exacerbates hypertension and nephrosclerosis in spontaneously hypertensive rats (SHRs). In this study, we determined whether angiotensin-converting enzyme (ACE) inhibition could prevent or reverse the systemic, renal, and glomerular hemodynamic alterations and the pathological changes of nephrosclerosis. Four groups of 20-week-old SHRs were studied: group 1, untreated controls; group 2, treated with Nω-nitro-l-arginine methyl ester (L-NAME, 50 mg/L for 3 weeks); group 3, L-NAME cotreated with quinapril (3 mg·kg−1·d−1 for 3 weeks); and group 4, L-NAME for 3 weeks followed by quinapril for 3 weeks (same doses). The results of this study demonstrated that both cotreatment (group 3) and posttreatment (group 4) with quinapril reduced mean arterial pressure (186±9 and 192±9 mm Hg, respectively, compared with group 2 SHRs, 221±5 mm Hg) and total peripheral resistance index associated with significant reductions in afferent and efferent arteriolar resistances; nephrosclerosis pathological scores; and urinary protein excretion (all at least P<.01). ACE inhibition also significantly increased stroke index, single-nephron glomerular filtration rate, and ultrafiltration coefficient compared with the L-NAME SHRs. Most notable were the findings that cotreatment with quinapril completely prevented the renal glomerular hemodynamic alterations with reduced glomerular capillary hydrostatic pressure and efferent arteriolar resistance compared with both the untreated and the L-NAME–treated SHRs (all at least P<.01). Posttreatment with quinapril also reversed the glomerular injury (subcapsular, −83%; juxtamedullary, −56%) and arteriolar (−87%) injury scores obtained from renal biopsy specimens (P<.005 and P<.0001, respectively). These changes were associated with decreased periarteriolar fibronectin and increased afferent arteriolar α-smooth muscle actin deposition (immunohistochemistry). These data, therefore, demonstrate that ACE inhibition not only prevents but also reverses L-NAME–exacerbated severe nephrosclerosis in SHRs, as indicated by improved systemic, renal, and glomerular hemodynamic changes, proteinuria, and histological alterations.
- renal micropuncture
- rats, inbred, SHR
- angiotensin-converting enzyme inhibitors
Antihypertensive drug therapy has reduced morbidity and mortality from stroke, congestive heart failure, and coronary heart disease in patients with hypertension, but it has been ineffective in reducing end-stage renal disease.1 In this respect, it is unclear whether goal treatment pressures have been realistic; whether only certain antihypertensive agents can reduce or may even exacerbate the renal microcirculatory and structural involvement of hypertension; or whether the nephrosclerosis of hypertension is, in fact, irreversible. Studies that used several animal models of renal disease with hypertension have shown that renal ischemia, glomerular hypertension, and glomerular filtration were associated with leakage of protein molecules, thereby promoting glomerular sclerosis and progressive functional renal deterioration.2 3 These reports have also demonstrated renal protection using ACE inhibitors, but they all involved reduced renal mass or experimental and clinical diabetes mellitus. Often the responses were better than one would expect from the hypotensive effect of the drug alone.4 5 6 7
Recently, using aged (73-week-old) SHRs as a model of human essential hypertension, we demonstrated a natural progression of the functional and structural evidence of severe hypertensive nephrosclerosis and its reversibility after only 3 weeks of ACE inhibition therapy.8 Since it is neither possible to purchase nor practical to maintain such aged SHRs because it is extremely costly and time-consuming, we developed a similar model of naturally developing nephrosclerosis in younger (20-week-old) SHRs with 3 weeks of intervention with the NO synthase inhibitor L-NAME.9 The control and L-NAME–treated SHR groups in that study are included in the present report for the purpose of reference. The present report not only demonstrates similar reversibility of all aspects of the severe nephrosclerosis in that L-NAME SHR model within 3 weeks with the same ACE inhibitor, but we have also been able to prevent renal involvement with the simultaneous administration of ACE inhibitor with L-NAME.
Male 17-week-old SHRs (Charles River Laboratories, Wilmington, Mass) were housed in plastic cages and maintained in a temperature- and light-controlled room. Throughout the study, the rats had free access to standard rat chow (Ralston-Purina). All experiments had been approved by our institutional animal care committee.
All surgical procedures were performed under sterile conditions with pentobarbital (10 mg/kg IP) anesthesia for renal biopsy or with inactin (100 mg/kg IP; Byk-Gulden) for the hemodynamic and micropuncture studies. The rats (290 to 330 g body weight) were divided into four experimental groups: group 1 (control, n=10) rats were given the standard vehicle, tap water, alone; and group 2 (L-NAME, n=12) rats were administered L-NAME (Sigma Chemical Co) in drinking water (50 mg/L) for 3 weeks. These two SHR groups were the subject of our earlier study9 and are included in this report so that they may serve as a frame of reference for the evaluation of the subsequent two groups, described as follows: group 3 (L-NAME plus the ACE inhibitor quinapril, 3 mg·kg−1·d−1, by gastric gavage for 3 weeks; n=8) and group 4 (L-NAME, then the ACE inhibitor; n=9) rats that were administered the same dose of L-NAME in drinking water for 3 weeks followed by a 3-week course of treatment with quinapril (the same dose as in group 3) after the L-NAME was discontinued. The dose of L-NAME (Sigma) was arrived at in an earlier study9 that demonstrated its effectiveness in mimicking the naturally occurring and severe nephrosclerosis in the 73-week-old SHRs, manifested by its associated clinical, physiological, and pathological changes.8
The drinking water with L-NAME was changed daily to quantify the precisely measured dose of L-NAME, volume intake, and urinary volume. Thus, this volume intake permitted a daily L-NAME dose of 7.6±0.7 mg/kg body wt. Quinapril (3 mg/kg) was initiated only after repeated measurements of daily UProtV were obtained, after which it was administered daily for 3 weeks by gastric gavage when measurements of daily UProtV were repeated. Thus, the ACE inhibition therapy of groups 3 and 4 was for a 3-week period, although the latter SHR group was 3 weeks older at the conclusion of this study because of the prior 3-week intervention with L-NAME. The selection of a 3-week period of treatment with the ACE inhibitor was chosen because, in our previous studies, this 3-week period was shown to be of sufficient duration to reduce arterial pressure and to reverse LV hypertrophy and the renal histopathological lesions as well as the associated systemic, renal, and glomerular hemodynamic alterations.8 10 11 12 To determine the histological effects of quinapril treatment in group 4, open renal biopsy was performed under pentobarbital anesthesia through a midline abdominal incision under sterile conditions after the 3-week intervention with L-NAME. In that procedure, a small portion of the lower pole of the right kidney was removed surgically, and bleeding was controlled by gelfoam application.7 Renal tissue from the same rat was also examined from the autopsy specimen obtained 3 weeks later, after quinapril treatment, which permitted comparison of both renal histological findings in each rat. The 24-hour UProtV was measured before the renal micropuncture study by the method of Lowry et al,13 and the 24-hour UNaV was also determined with a Beckman Astra 8 flame photometer.
In addition, in a preliminary study for this report (and as a control group for group 4), we studied four untreated SHRs that were given L-NAME for 3 weeks and thereafter only tap water instead of quinapril for 3 weeks. Four other SHRs died during this post–L-NAME period without further study. This preliminary work was done to determine whether the L-NAME–induced changes reversed themselves spontaneously during a 3-week (non–L-NAME) period in SHRs. Pollock et al14 had reported earlier, in normotensive rats, that blood pressure returned to normal when L-NAME was discontinued. However, in our preliminary study, the findings in the SHRs were not similar to those in the normal rats. Thus, LV hypertrophy (LV index, 3.36 mg/g); cardiac (reduced CI, 245 mL·min−1·kg−1; SI, 0.69 mL per beat per square meter) and renal (reduced GFR of 0.74 mL/min and elevated renal vascular resistance of 147 mm Hg·mL−1·min−1) dysfunction; and the severe nephrosclerosis were not different from those findings obtained at the conclusion of 3 weeks of L-NAME, although their MAP (208 mm Hg) was slightly lower. Histologically, in this group, the pathological changes of nephrosclerosis were similar to those findings obtained in untreated 73-week-old SHRs8 or the younger SHRs that were treated with L-NAME for 3 weeks (group 2).9 Thus, they were all characterized by severe glomerular sclerosis and lesser glomerular thrombosis, marked glomerular ischemic changes, and tubulointerstitial changes. Therefore, L-NAME hypertensive nephrosclerosis in the SHRs did not reverse naturally during the 3-week period after L-NAME withdrawal.
All rats were deprived of food overnight before the renal hemodynamic and micropuncture studies, although they were allowed free access to water. They were anesthetized with inactin and then placed on a heating pad to maintain rectal temperature at 37°C throughout the study. After a tracheostomy, a polyethylene catheter (PE-50) was inserted into the abdominal aorta through the right femoral artery to permit blood sampling and measurement of MAP and heart rate. The right carotid artery and right jugular vein were also cannulated with PE-50 catheters for determination of cardiac output with a thermocouple microprobe connected to a thermodilution device (Cardiotherm 500, Columbus Instrument) that was calibrated twice, at the beginning and end of each study.9 Cardiac output was calculated by use of a conversion factor that depended on the injected volume and intravascular catheter length; it was then normalized for body weight and expressed as CI in mL·min−1·kg−1. Pressures were measured with Gould-Statham transducers (model P23 Db, Statham Instruments) connected to a multichannel polygraph (Sensor Medics R612, Beckman Instruments Inc).
The left kidney was exposed through a ventral midline abdominal incision. Its ureter was catheterized, and the kidney was immobilized and prepared for micropuncture as described previously.8 9 10 The renal surface was illuminated and bathed in 0.9% NaCl at 34°C to 36°C. The left femoral vein was used for [3H]methoxyinulin (850 μCi/mL) infusion at a rate of 0.1 mL·100 g body wt−1·h−1, and the right femoral vein was also cannulated with PE-50 polyethylene tubing for infusion of a saline solution containing 5.6% p-aminohippurate (Merck Sharp & Dohme) at a rate of 0.2 mL·100 g body wt−1·h−1 for the initial 45 to 60 minutes of the procedure and then at a rate of 0.1 mL·100 g body wt−1·h−1 for maintenance infusion.
After physiological equilibration, urine was collected over two 30-minute periods, and blood samples were drawn at the midpoint of each period. Simultaneously, the following micropuncture measurements were made: (1) efferent glomerular arteriolar blood was withdrawn by direct puncture of two or three superficially located “star vessels”; (2) precisely timed (90-second) samples of fluid were collected from four to six selected superficial proximal tubules for determination of SNGFR; and (3) PE, PT, and SFP were measured directly by a servo-null system (Instrumentation for Physiology & Medicine). The PT and PE measurements were obtained from proximal convoluted tubules and the star vessels, respectively. Because the SHR glomerular capillaries are not superficial in the renal location, PG was calculated from the sum of the SFP and the systemic ΠA. The PE, PT, and SFP measurements were made three times, and their averages were determined.
The tubular fluid, urine, and plasma samples were counted for [3H]inulin radioactivity by placement in 10-mL scintillation vials (Bio-Safe II) for counting in a β-scintillation counter, which allowed calculation of SNGFR, GFR, and ERPF. These measurements permit calculation of ΠA and ΠE, RA and RE, and the glomerular capillary Kf. At the termination of each study, blood was drawn for measurement of serum creatinine and uric acid concentrations by a 747-100 Analyzer (Boehringer Mannheim/Hitachi).
Light microscopy was performed after completion of each micropuncture study. Kidneys, heart, and thoracic aorta were removed, weighed, and fixed in 10% neutral buffered formalin. Two midcoronal slices of the right kidney, 2 to 3 mm thick, were embedded in paraffin after conventional processing. Sections (3 μm thick) were stained with hematoxylin-eosin and by the periodic acid–Schiff reaction. Sections obtained from the left kidney were stained with hematoxylin-eosin, periodic acid–Schiff, periodic acid–methenamine–silver, and phosphotungstic acid–hematoxylin for specific staining of extracellular matrix, fibrin, and collagen. Grading of GIS and of AIS was performed as previously described.8 9 GIS was graded from 0 to 3+, in which 0 was no injury, 1+ was injury of up to one third (≤1/3) of the glomerulus, 2+ was one third to two thirds glomerular injury, and 3+ was injury of more than two thirds (≥2/3) of glomerular involvement. The AIS was also graded from 0 to 3+, in which 0 was no injury at all, 1+ demonstrated hyalinosis of the arteriolar wall up to 50% of the mural circumference, 2+ demonstrated hyalinosis between 50% and 100% of the wall circumference but without luminal narrowing, and 3+ was complete mural hyalinosis with luminal encroachment. The overall nephrosclerosis score was calculated by adding the GIS and AIS for each rat. These scores were obtained by independent study by two investigators, and scoring of all tissue was conducted in a blinded manner.
For immunohistochemistry, immunostaining of the sections of each group was carried out with the streptavidin/biotin immunoperoxidase method (LSAB kit, DAKO) after deparaffinization. Antibodies used for immunohistochemical analysis were as follows: anti–α-SMA was detected with murine monoclonal antibody 1A4 (DAKO) diluted 1:50, and fibronectin with mouse monoclonal antibody NCL-FIB (Novocastra, Vector) diluted 1:100.
One-way ANOVA, followed by Duncan’s multiple range test, was performed for between-group significance, and linear regression analysis was used to examine for correlation between CI and micropuncture data and between CI and morphological data.15 16 All data are expressed as mean±SEM. A probability level of <5% was considered to be of statistical significance.
Body and Organ Weights
Body weight was greater in group 4 rats, no doubt because of their additional 3 weeks in age, but this was associated with decreased LV and aortic indexes. Otherwise, the body and organ weight data were not significantly different among the other three groups (Table 1⇓).
Systemic, Cardiac, and Whole-Kidney Hemodynamics
L-NAME treatment of group 2 significantly increased MAP, TPRI, and renal vascular resistance index and reduced CI, SI, ERPF, and GFR (Table 2⇓).9 Cotreatment with L-NAME and quinapril (group 3) significantly prevented the L-NAME–induced increases in MAP, TPRI, renal vascular resistance index, and the reductions of CI, SI, ERPF, and GFR (Table 2⇓). Furthermore, when quinapril was administered after the 3-week L-NAME period (group 4), the increases in MAP, TPRI, and SI and the decreases in GFR were reversed significantly; heart rate decreased, although ERPF and CI remained unchanged (Table 2⇓). Finally, there was a direct correlation between the changes in CI and ERPF (r=.558; P<.005).
At the superficial nephron glomerular dynamic level, the L-NAME group (group 2) demonstrated decreased SNPF, SNGFR, PT, and Kf and increased RA, RE, and SFP, but ΔP and PG remained unchanged (Table 3⇓).9 This unchanged PG was associated with a reduced SNPF and an intense RA increase with a lesser RE increase (Table 3⇓; Fig 1⇓). Cotreatment with L-NAME and quinapril (group 3) significantly prevented the foregoing L-NAME–induced changes in whole kidney, single nephron, and glomerular dynamic functions. Furthermore, the ΔP and PG were significantly reduced and associated with a decreased SFP and RE (compared with the control untreated SHRs; Table 3⇓ and Fig 1⇓). However, when quinapril treatment followed L-NAME administration (group 4), SNPF was not significantly changed during the 3-week quinapril period compared with the L-NAME treatment group (group 2) (Table 3⇓). Other glomerular dynamic alterations (including SNGFR, PT, SFP, ΔP, PG, RA, RE, and Kf) were reversibly changed after that 3-week period of quinapril treatment.
UProtV and other indexes were increased significantly by L-NAME (Table 4⇓), but both treatment groups receiving quinapril (groups 3 and 4) demonstrated significantly reduced protein excretion compared with the untreated SHRs (group 1) or the SHR group treated only with L-NAME (group 2) (Table 4⇓; P<.01). Furthermore, there was a direct correlation between UProtV and nephrosclerosis score (see below) (r=.550, P<.001). UNaV, increased by L-NAME (Table 4⇓; P<.05), still remained increased despite the reduced MAP and UProtV after the 3 weeks of quinapril (group 4). There was no difference in UNaV between the untreated SHRs and those given L-NAME simultaneously with quinapril (group 3). It was also of interest that serum creatinine and uric acid concentrations were increased significantly by L-NAME (group 2), and the uric acid concentrations were reduced by quinapril (in both groups 3 and 4), with the creatinine level remaining unchanged (Table 3⇑).
Renal Morphological Findings
The morphological appearance of glomeruli and interstitium remained entirely normal or displayed only a few minor alterations of the afferent arterioles in the 20-week-old control SHRs (group 1; Fig 2A⇓). However, the glomeruli of the L-NAME–treated SHRs (group 2) exhibited a diffuse increase in mesangial matrix with minimal evidence of cellular proliferation (Fig 2B⇓).9 L-NAME cotreatment with quinapril (group 3; Fig 2C⇓) displayed only minimal histological alterations. The frequency of segmental glomerular lesions was reduced by 58%, a value significantly less severe than that seen in the L-NAME–treated SHRs (group 2). However, two rats of this group did develop severe glomerulosclerosis with mesangiolysis or an increased mesangial matrix that was similar to that in the L-NAME–treated SHRs (group 2). Afferent arteriolar alterations in all rats were reduced by 77% (average), significantly less than that in the untreated group 1 SHRs (P<.05; Table 4⇑). The tubule and interstitial alterations were virtually absent in the group 3 rats.
Despite the unchanged ERPF and PG, all rats in the group receiving quinapril after L-NAME administration (group 4) demonstrated reversible changes within 3 weeks in the segmental glomerular and arteriolar lesions (Fig 2D⇑) compared with the renal biopsy lesions from the same rats after only 3 weeks of L-NAME (Fig 3⇓). The glomeruli (obtained by renal biopsy after week 3) showed severe nephrosclerotic lesions that were similar in severity to the lesions observed in the L-NAME–treated SHRs (group 2). However, on autopsy, the glomeruli after week 6 (after quinapril treatment; group 4) showed only minimal structural alterations and only irregularity of the capillary loops with adhesions to Bowman’s capsule (Fig 2D⇑). In addition, there were only occasional glomeruli that demonstrated sclerosis within the interstitium and also shunt glomeruli associated with interstitial fibrosis, tubular atrophy, and inflammatory cell infiltration.
Immunoreactive Renal α-SMA and Fibronectin
Immunoreactive fibronectin deposition was increased in the surrounding area of injured afferent arterioles and interlobular arteries in association with fibrinoid necrosis and the onionskin appearance of severe nephrosclerosis in the L-NAME–treated SHRs (group 2). Furthermore, staining for fibronectin was increased in the interstitium (Fig 4⇓, upper right). However, in control untreated group 1 (Fig 4⇓, upper left) and cotreated L-NAME and quinapril SHRs (group 3), fibronectin was deposited minimally in the periarteriolar adventitia of the afferent arterioles, and there was no staining in the interstitium. In the group receiving quinapril after L-NAME (group 4), fibronectin deposition was decreased in the adventitia of afferent arterioles and interlobular arteries, which was associated with a decreased AIS compared with group 2.
In control SHRs (group 1; Fig 4⇑, lower left), the α-SMA staining was localized to the afferent arterioles and to the interlobular arterial smooth muscular cells; similar staining was demonstrated in group 3. By contrast, in group 2 (Fig 4⇑, lower right), this α-SMA staining was reduced in the injured afferent arterioles, with fibrinoid degeneration. The decreased afferent arteriolar α-SMA staining in the L-NAME SHRs was reversed significantly by quinapril (group 4). Hence, immunoreactive fibronectin and α-SMA depositions were evident in afferent arterioles and associated with an increased AIS.
Glomerular and Arteriolar Injury Scores
Quantitative histological severity grading revealed more severe GIS and AIS (P<.01, Table 4⇑) in the L-NAME SHRs (group 2) than the untreated SHRs (group 1). Quinapril cotreatment (group 3) significantly reduced the severity scoring of the cortical glomerular lesions. The GIS of L-NAME SHRs that were later treated with quinapril (group 4) also demonstrated a significant reduction in the subcapsular glomeruli; however, the juxtamedullary glomerular GIS remained unchanged. The AIS was also reduced significantly in both quinapril-treated groups (groups 3 and 4) than in the L-NAME SHRs (group 2). Thus, the overall nephrosclerosis score (GIS+AIS) was significantly greater in the L-NAME SHRs than the untreated SHRs (P<.01), and this score was reduced significantly by both quinapril treatment protocols (P<.01).
Finally, there was a strongly positive correlation between the glomerular and arteriolar injury indexes (r=.744, P<.0005). Moreover, direct correlations were also demonstrated between nephrosclerosis score and RA (r=.680, P<.0005), RE (r=.649, P<.0005), SFP (r=.552, P<.001), ΔP (r=.630, P<.0005), and PG (r=.548, P<.001), and there were indirect correlations between nephrosclerosis score and SNPF (r=−.544, P<.001) and SNGFR (r=−.581, P<.0005). These important pathophysiological correlations demonstrated a strong and close relation between the degree of renal ischemia and the pathological evidence of nephrosclerosis.
The effects of the ACE inhibitor quinapril on intrarenal hemodynamics in rats with cardiac failure and in young and aged SHRs have been reported earlier from our laboratory,8 10 11 but the effects on intrarenal hemodynamics, proteinuria, and histopathology are still unclear in the young SHRs with chronic NO synthase inhibition.17 We therefore designed the present study with two purposes in mind: to determine whether a similar length of ACE inhibition treatment prevents or reverses the whole-kidney and glomerular dynamic alterations of nephrosclerosis as well as the pathological changes associated with prolonged blockade of NO synthase in young SHRs. The data presented demonstrate convincingly that ACE inhibition almost totally prevented the damage induced by L-NAME and that it also significantly reversed those effects within only a 3-week treatment period. These prevention and reversal changes applied to clinical and functional (ie, proteinuria and serum uric acid concentration) indexes, although serum creatinine concentration remained unchanged during the 3-week treatment period with the ACE inhibitor. One possible explanation for the unchanged serum creatinine level despite an improved GFR is that the treatment period of 3 weeks was not sufficiently long. However, the reversible changes of serum uric acid concentration by the ACE inhibitor were most likely related to functional changes associated with the increase in renal blood flow.18 19
In an earlier study involving 73-week-old SHRs with naturally developing nephrosclerosis, we demonstrated that quinapril also decreased PG, RA, and RE, increased SNPF, and decreased glomerular and arteriolar injury scores within a 3-week treatment period.8 The responses of the 20-week-old SHRs used in the present report that were cotreated with L-NAME and quinapril (group 3) are consistent with that earlier study.8 Thus, RA and PG were significantly reduced by quinapril, suggesting that the clinical, hemodynamic, glomerular dynamic, and histological damage of L-NAME–induced nephrosclerosis in the younger SHRs can be prevented by ACE inhibition. These changes were confirmed by the striking pathophysiological changes that clearly indicated renal prevention. Moreover, when the quinapril was administered after L-NAME (group 4), these same clinical, hemodynamic, glomerular dynamic, and histological alterations revealed at least partial reversal of these alterations within the same 3-week period. To be sure, there were some differences between groups 3 and 4 with respect to the functional glomerular dynamics and pathological changes in response to the ACE inhibitor: ACE inhibition (group 3) completely prevented the effects of L-NAME, whereas ACE inhibitor after L-NAME (group 4) did not fully reverse these changes. As indicated above, these differences may reflect the brief treatment period (only 3 weeks) in group 4, which was already damaged by L-NAME. Nevertheless, even during this brief period, some significant functional and structural improvements were demonstrated.
In our earlier study of 20-week-old SHRs, MAP and RA were elevated, but the PG (53±1 mm Hg) and RE were normal.10 These younger rats demonstrated only minimal pathological changes that were manifested by hyaline deposition and increased media wall thickness of the afferent arterioles, and there was no evidence of glomerular sclerosis (similar to the changes in group 1). In that earlier study, ACE inhibition with quinapril subsequently reduced PG and the glomerular arteriolar resistances.11 In other reports, cilazapril, captopril, perindopril, and other ACE inhibitors also diminished intrarenal and mesenteric arteriolar hypertrophy and glomerular damage in young SHRs20 given DOCA salt,21 in one-kidney, one clip Goldblatt hypertensive rats,22 and in subtotally nephrectomized rats.7 However, until recently, little information was known concerning the renal effects of ACE inhibitors in chronic NO inhibited models. Arnal et al23 reported that chronic adaptations to NO synthase inhibition were not uniform in normotensive rats. Specifically, ≈25% of their rats treated with L-NAME developed cardiac hypertrophy and increased PRA, whereas their remaining rats did not. In another report, L-NAME–treated SHRs demonstrated increased PRA, severely elevated arterial pressure, and a high mortality that they attributed to developed nephrosclerosis.24 In yet another model of chronic renal failure, using subtotal nephrectomy with L-NAME (with dosing schedule and duration of treatment similar to our present study), Fujihara et al25 produced severe glomerulosclerosis and arterial pressure elevation but without a changed PRA. Hence, the physiological responses to chronic NO synthase blockade do not appear to be uniform in all studies and may be related to the experimental models used and the alterations induced pathophysiologically.
The most intriguing observation of our present study is that treatment with quinapril prevented the rise in arterial pressure and the pathophysiological renal alterations provoked by the inhibition of NO synthesis induced by L-NAME. It is important to recognize that even though MAP was not fully normalized, quinapril prevented not only the systemic and local hemodynamic changes but also the renal histological alterations and the proteinuria induced by inhibited NO synthesis. Quinapril inhibits the degradation of bradykinin as well as angiotensin II generation, and it produces endothelium-dependent relaxation involving the stimulation of NO release mediated through the bradykinin B2 receptor or by certain prostaglandins.26 27 28 29 30 31 32 The vascular protective capacity of NO is attributed not only to its vasodilator action but also to its antimitotic, antiproliferative, and antiplatelet properties,27 which probably contribute to the overall effects of ACE inhibition on vascular smooth muscle.28 Thus, our study does not provide the precise mode of action subserved by ACE inhibition in these L-NAME SHRs.
Glomerular thrombosis frequently occurred in the afferent arteriolar branching capillaries (Fig 5⇓) of L-NAME SHR nephrosclerosis (group 2) that are associated with increased mesangial matrix and endothelial cells despite an unchanged PG. Other studies with chronic NO synthase inhibition in normotensive rats have also reported the relation between glomerular sclerosis and increased PG without associated glomerular thrombosis.25 33 34 35 36 These PG and pathological changes with NO inhibition might be considered to be inconsistent with the concept of Baylis et al.33 The SHRs in our study, however, did not demonstrate an increased PG.9 The normal PG that we found in the L-NAME SHRs could be explained by the reduction in whole-kidney renal blood flow that was associated with a reduction in SNPF and an exacerbation of severely elevated RA that was more intense than the RE increase. However, we recently reported that when a thiazide diuretic was added to L-NAME, RE increased even more, despite a further reduction in SNPF, which then resulted in an increased PG.37 However, Raij and coworkers38 also reported that NO synthase inhibition with L-NAME in normotensive rats resulted in diffuse glomerular thrombosis after lipopolysaccharide administration.
It is not known why glomerular thrombosis occurs in SHRs treated chronically with L-NAME without associated glomerular hypertension. However, it has been suggested that intraglomerular tumor necrosis factor, interleukin-1, and platelet-activating factor released locally (by either activated mesangial cells or blood-borne macrophages) can induce endothelial synthesis of tissue factor and plasminogen inactivator.39 Therefore, it would appear that, under these circumstances, the local release of NO associated with ACE inhibition may be important in preventing glomerular thrombosis. Moreover, the precise mechanisms whereby ACE inhibition prevents or reverses the severe hypertensive nephropathy in L-NAME SHRs remain to be elucidated. In support of ascribing these to renin-angiotensin mechanisms, local or systemic, is the prevention of angiotensin II synthesis as well as the more recent finding that thiazide cotreated with L-NAME exacerbates the glomerulopathy.37 Further support may be offered by recent observations that demonstrated in vitro that angiotensin promoted intravascular thrombosis.40 Thus, it was of great interest to see that cotreatment or posttreatment with ACE inhibitors either prevented or significantly reversed the intravascular thrombosis associated with L-NAME. However, until studies are reported with an angiotensin II receptor antagonist or with a bradykinin or prostaglandin antagonist, the role of the kinins or prostaglandins cannot be separated from the effects of angiotensin II.
In summary, chronic ACE inhibition with quinapril prevented as well as reversed the systemic, whole-kidney and glomerular dynamic and pathological alterations that resulted from 3 weeks of L-NAME intervention. These changes occurred even though arterial pressure RA and RE were not always normalized. Quinapril therefore protected and reversed the nephrosclerosis and afferent arteriolar injuries in SHRs that were associated with increased fibronectin and decreased α-SMA deposition induced by chronic L-NAME treatment.
Selected Abbreviations and Acronyms
|α-SMA||=||α-smooth muscle actin|
|ACE||=||angiotensin converting enzyme|
|AIS||=||arteriolar injury score|
|ERPF||=||effective renal plasma flow|
|GFR||=||glomerular filtration rate|
|GIS||=||glomerular injury score|
|L-NAME||=||Nω-nitro-l-arginine methyl ester|
|MAP||=||mean arterial pressure|
|PE||=||efferent arteriolar pressure|
|PG||=||glomerular capillary hydrostatic pressure|
|PRA||=||plasma renin activity|
|PT||=||proximal tubular pressure|
|ΔP||=||pressure gradient across glomerular capillary wall|
|ΠA||=||afferent colloid osmotic pressures|
|ΠE||=||efferent colloid osmotic pressure|
|RA||=||afferent glomerular arteriolar resistances|
|RE||=||efferent glomerular arteriolar resistances|
|SHR||=||spontaneously hypertensive rat|
|SNGFR||=||single-nephron glomerular filtration rate|
|SNPF||=||single-nephron plasma flow|
|TPRI||=||total peripheral vascular resistance index|
|UNaV||=||urinary sodium excretion|
|UProtV||=||urinary protein excretion|
This study was supported by funds from the Hypertension Research Trust Fund, Alton Ochsner Medical Foundation. We also acknowledge the Parke-Davis Research Laboratories for their generous gift of quinapril. We deeply appreciate the valuable technical assistance and support of Drs Gordon B. McFarland and Gladden W. Willis of our Departments of Orthopedic Surgery and Pathology.
Reprint requests to Edward D. Frohlich, MD, Vice President for Academic Affairs, Alton Ochsner Medical Foundation, 1516 Jefferson Hwy, New Orleans, LA 70121.
This manuscript from Alton Ochsner Medical Foundation was sent to Theodore Kotchen, MD, Consulting Editor, for review by expert referees, for editorial decision, and for final disposition.
- Received September 7, 1995.
- Revision received October 19, 1995.
- Accepted November 29, 1995.
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