Enalapril and Renal Injury in Spontaneously
Hypertensive Rats
Leonard G. Feld, Steven Cachero, Judith B. Van Liew, Marianna Zamlauski-Tucker, and Bernice Noble
Rats of the spontaneously hypertensive strain develop kidney damage that resembles the nephropathy seen in some cases of human essential hypertension. Previous studies with a triple drug antihypertensive regimen indicated that proteinuria and glomerular histopathology in spontaneously hypertensive rats might develop despite long-term effective control of systemic blood pressure. To investigate further the relation between hypertension and kidney disease, a group of spontaneously hypertensive rats were treated with enalapril at 15 weeks of age. Blood pressure, protein excretion, and kidney function were measured in those rats at regular intervals during the next year and a half and were compared with untreated spontaneously hypertensive rats and the normotensive Wlstar-Kyoto parent strain. Kidney tissue samples from all three groups, collected at autopsy, were stained by immunohistochemical and conventional methods to assess the relative severity and nature of kidney damage. Although enalapril therapy was completely effective in controlling the blood pressure of spontaneously hypertensive rats, it only postponed the onset of kidney disease. Enalapril-treated spontane-ously hypertensive rats eventually exhibited albuminuria as severe as that found in hyperten-sive rats. Kidney vessel pathology was completely prevented with enalapril, but the abnormal accumulation of mononuclear cells in tubulointerstitlal and periglomerular sites was the same as in untreated spontaneously hypertensive rats. We have concluded that elevated protein excretion in rats of the spontaneously hypertensive rat strain is not a secondary consequence of systemic hypertension. Structural abnormalities of renal vessels also do not appear to contribute significantly to the pathogenesis of albuminuria in spontaneously hypertensive rats. Other explanations must be sought to account for the close link between spontaneous hypertension and kidney damage in this animal model. The clear dissociation of kidney disease from systemic hypertension exhibited by spontaneously hypertensive rats may also be relevant for human disease (Hypertension 1990;16:544-554)
I
n male spontaneously hypertensive rats (SHR), systolic blood pressure reaches hypertensive levels (above 150 mm Hg) at 2 months of age, continues to increase until 3 months, and remainsFrom the Departments of Pediatrics, Physiology, Microbiology and Pathology, State University of New York at Buffalo School of Medicine and Biomedical Sciences, Veteran's Administration Medical Center, Children's Hospital of Buffalo, Buffalo, N.Y.
Presented in part at the Society of Pediatric Research Meeting in Anaheim, California, 1988, and the International Society of Nephrology Meeting in London, England, 1987.
Supported by the Veteran's Administration, the New York State Affiliate of the American Heart Association, the New York Health Research Council, Biomedical Research Support Grants from SUNY at Buffalo and Children's Hospital of Buffalo, the James H. Cummings Foundation, and by a Senior International Fogarty Fellowship to B.N.
Address for reprints: Leonard G. Feld, MD, PhD, Children's Kidney Center, Children's Hospital of Buffalo, 219 Bryant Street, Buffalo, NY 14222.
Received November 20, 1989; accepted in revised form June 4, 1990.
abnormally elevated (220 mm Hg) throughout the life of the rat (16-18 months).1 Proteinuria, predom-inantly albuminuria, which invariably develops, is first detectable in 5-month-old SHR and becomes progressively worse with age.1 Renal function, as assessed by creatinine and inulin clearances, deteri-orates significantly after 1 year of age. Pathological changes in the kidneys of SHR, most severe in glomeruli and vessels of the inner (juxtamedullary) cortex, also become progressively more severe with age.1 Those changes include glomerular sclerosis, tubule atrophy and cast formation, and mononuclear cell infiltration of the kidney interstitium. Renal arteries and arterioles become thickened with hyper-plasia of medial muscle layers, destruction of internal elastic lamellae, and perivascular inflammation. Kid-ney histopathology in the SHR resembles that found in human cases of kidney insufficiency associated with essential hypertension. It is generally believed that the vascular lesions, including glomerular
age, result from increased intravascular pressure, and that proteinuria is the consequence of hypertensive injury to the glomerular capillaries.2"6
To determine whether control of blood pressure could prevent nephropathy in SHR, we instituted an antihypertensive drug regimen (a combination of hydralazine, reserpine, and hydrochlorothiazide) in young animals before the onset of hypertension.7 With that therapy, the onset of proteinuria was delayed by many months. However, despite success-ful control of systemic blood pressure to normal levels, SHR on this triple drug antihypertensive ther-apy eventually developed kidney histopathology and pathophysiology as severe as that found in untreated animals. Those observations led us to speculate that both the vascular pathology and the glomerular dys-function exhibited by SHR were not secondary to elevated systemic blood pressure but had another underlying cause.
To investigate further the relation between sys-temic hypertension and kidney damage in SHR, we tested the effect of another antihypertensive drug on the development of nephropathy. We chose enala-pril, an angiotensin I converting enzyme inhibitor, for several reasons.8-11 First, it reduces systemic blood pressure by mechanisms completely different from the triple drug therapy. Second, it has been shown to be effective in reducing glomerular capillary pressure as well as systemic blood pressure, and third, it has been found to reduce glomerular damage and pro-teinuria in several other experimental models of renal insufficiency. Because the pathogenesis of pro-teinuria has also been linked to glomerular infiltra-tion with mononuclear cells,12 we used immunohis-tochemical methods to compare the subset composition and microanatomic distribution of mononuclear cell infiltration in the kidneys of enal-april-treated SHR (SHRE), untreated SHR, and Wistar-Kyoto (WKY) controls.
Methods Rats
Males of the SHR strain (n=19) and the WKY normotensive parent strain (n=9) were obtained from Harlan Sprague Dawley, Indianapolis, Ind. at 10-14 weeks of age. All animals were housed in separate cages with free access to food and water. All rats were fed a 24% protein diet (Teklad, Madison, Wis.). Ex-perimental protocols were examined and approved by the Laboratory Animal Care Committee, State Univer-sity of New York at Buffalo, Buffalo, N.Y.
Blood Pressure Measurements
Indirect systolic blood pressure was determined by the tail-cuff method (Narco Biosystems, Houston, Tex.) without anesthesia, and the animals soon be-came accustomed to the procedure. The average of at least three readings, taken in a quiescent state, was recorded every 10-20 weeks.
Inulin Clearance
Inulin clearance was measured in conscious SHRE (n=8) rats by modification of the single-injection method described by Hall et al.13 We injected 50 ^ G of methoxy-[3H]inulin (ICN Radiochemicals, Irvine, Calif., sp. act. 42 mCi/g) in 0.5 ml sterile 0.9% saline into the tail vein and then collected blood samples from the cut tail tip over a 2-hour period. Blood samples were taken at 2, 5, 8, 11, 15, 20, 40, 60, 90, and 120 minutes. Inulin clearance was then deter-mined by Q/A ratio, where Q is amount of isotope given in microcuries and A is the area under the disappearance curve from zero to 120 minutes, ex-pressed as microcuries per milliliterx minute. The amount of isotope given was confirmed by counting a 10 ^.1 aliquot of the injectate. Measurement of [3H]inulin in samples was done on a Packard (Tri-Carb) liquid scintillation counter (Downers Grove, 111.). The scintillation fluid consisted of 10 ml Ecol-ume (ICN Radiochemicals, Irvine, Calif.) to which was added 10 /A plasma. Tritium quench standards were used to determine counting efficiency.
Analytical Methods
A microcontinuous gradient gel electrophoresis pro-cedure was used for separation and quantitation of proteins in plasma (diluted 1:51) and urine.1 The acrylamide continuous gradient gels were made in 5 fil microcaps. The nonpolymerized acrylamide in the mi-crocap was drawn off and replaced by an equivalent amount of sample or standard. The remaining empty portion of the microcap was filled with buffer, and electrophoresis was run for 40 minutes. The gels were extruded from the capillary tubes, stained overnight, and scanned directly in an ultramicrodensitometer (Joyce-Loebl, Gates Head-on-Tyne, England). The electrophoresis scans were quantitated using a specially designed program for the Northstar Horizon computer. Plasma and urine creatinine were analyzed after ab-sorption and elution from Lloyd's reagent.14
Histology and Immunopathology
One half of each kidney was fixed in buffered formalin and embedded in paraffin, the other was flash frozen and stored at -70° C. Sagittal sections of fixed tissues were stained with periodic acid-Schiff, methenamine silver, and hematoxylin-eosin reagents, and evaluated independently by two of us (B.N., L.G.F.). Measurements of vessel wall thickness were made with a filar ocular micrometer (American Op-tical, Buffalo, N.Y.), using an oil immersion (xlOO) objective lens. Slides stained with periodic acid-Schiff reagent were used for these measurements. Two or three sections were examined from each rat kidney, with a minimum of five rats per group. Only vessels cut in full circular cross-section were selected for measurement. A minimum of five arterioles and three cortical radial arteries were measured on each section. For the cortical radial arteries, only the thickness of the media was measured. With these
observations the mean value of vessel wall thickness for each rat was calculated, from which the mean value per group was determined.
Monoclonal antibody staining of frozen tissue sec-tions was used to evaluate mononuclear cell infiltra-tion, replacing the more subjective scoring system used in the past.1715 The following monoclonal anti-bodies (Bioproducts for Science, Indianapolis, Ind.) were used: OX6, which recognizes major histocom-patibility Class II (la) antigens16; 0X19, which stains T lymphocytes17; 0X33, an antibody to B lympho-cytes18; EDI, a marker for monocytes and most macrophages; ED2, which stains mature connective tissue macrophages; and ED3, which stains a subset of macrophages normally limited to lymphoid tis-sues.19 A peroxidase-labeled rabbit antimouse immu-noglobulin reagent was used, with diaminobenzidine as substrate, to detect binding of the monoclonal antibodies. For each rat, two or more tissue sections were stained with each of the monoclonal antibodies. Positive cells in glomeruli and Bowman's capsule were counted individually in at least 10 glomeruli per section. Clusters of infiltrating cells (containing 10 or more mononuclear inflammatory cells) in periglomer-ular and interstitial sites were also counted. Superfi-cial and deep areas of the cortex were representa-tively sampled.
Design of Experiment
Three groups of animals were studied: WKY rats (n=9), untreated SHR (n=10), and SHRE (n=9). Enalapril (75 mg/1; Merck Sharp and Dohme, West Point, Pa.) was added to the drinking water at 15 weeks of age. The dose was adjusted as needed to maintain normotension. Because long-term adminis-tration of enalapril does not in itself cause an in-crease in protein excretion,20-21 we chose not to include a group of WKY rats treated with enalapril among the controls. Urinary protein excretion and creatinine clearance were measured from single over-night (18 hour) urine collections at selected intervals from 10 to 95 weeks of age. During those collections, food was removed and urine was separated from feces by a baffle system. At the end of the collection period, blood was taken from the cut tail tip. At 95 weeks of age, SHRE rats were killed by inhalation anesthesia (Metofane, Pittman Moore, Washington Crossing, N.J.) to obtain tissue for histological stud-ies. As life span is shorter in untreated SHR (70-75 weeks of age), final measurements of function and studies of histology in those rats were carried out when they were killed at 60 and 70-75 weeks of age. WKY rats were killed at 75 and 95 weeks of age. Statistical Analysis
Statistical comparisons were done using the unpaired Student's t test and multivariate repeated-measures analysis of variance, as appropriate. Values are given as mean±SEM. In the text, when statistical significance is ascribed to a particular value, it is at/?<0.05. All data
500 400 300 40 60 Ago, weefca 100
FIGURE 1. Line graph showing body weight versus age in Wistar-Kyoto rats (•), spontaneously hypertensive rats (SHR) ( • ) , and enalapril-treated SHR ( • ) .
were analyzed using the SPSSX program (SPSS Inc., Chicago, 111.) on an IBM PC computer.
Results
There was no significant difference in body weight between the groups at any time (Figure 1). Enalapril was always effective in maintaining blood pressure in SHRE at the level of normotensive WKY rats (Fig-ure 2), whereas systolic blood press(Fig-ure in untreated SHR was always significantly higher, as previously described.1
Urinary albumin excretion and creatinine clear-ances in WKY rats and in untreated SHR were similar to our previous reports.1715 Abnormal albu-min excretion was detected first at 20 weeks of age in untreated SHR and rose continuously thereafter (Figure 3A). Albumin excretion in enalapril-treated rats was not different from WKY controls until 75 weeks of age (SHRE, 16.7±1.5; WKY rats, 7.0±1.0 mg/24 hrxlOO g body wt (Figure 3A). However, albumin excretion in SHRE increased rapidly during the subsequent 20 weeks to equal the values seen in 75-week-old untreated SHR. High molecular weight protein (high molecular weight is molecular weights greater than albumin) excretion also increased over time in both SHR and SHRE groups (SHR, 40.1 ±3.0
230
SHR
20 40 60 80 100
FIGURE 2. Line graph showing blood pressure versus age in Wistar-Kyoto (WKY) rats, spontaneously hypertensive rats (SHR), and enalapril-treated SHR (SHRE). Systolic blood pressure is determined by indirect tail-cuff method
k i 30 -8 UJ K E 0 40 60 Age, weekj B 50 r 40 C 3 20 10 WKY O SHRE • SHR • 20 40 60 80 Age. weeks 100
FIGURE 3. Panel A: Line graph showing albumin excretion versus age. Panel B: Line graph showing high molecular weight (HMW) protein excretion versus age. WKY, Wistar-Kyoto rats; SHRE, enalapril-treated spontaneously hypertensive rats; SHR, spontaneously hypertensive rats; BW, body weight.
versus SHRE, 22.3 ±3.0 mg/24 hrxlOO g body wt, /?<0.005) (Figure 3B).
As we have previously described,15 estimates of glomenilar filtration rate using creatinine clearance and inulin clearance were similar in the untreated SHR and WKY rat strains. As shown in Table 1, creatinine clearances in untreated SHR at 75 weeks of age were significantly lower than age-matched WKY rats and SHRE. At 95 weeks of age, creatinine clearance (0.51 ±0.03 ml/minxlOO g body wt) and inulin clearance (0.50±0.06 ml/minx 100 g body wt) were similar in eight SHRE for which both analyses were performed. Creatinine clearances and plasma creatinine concentrations in those SHRE were not significantly different from WKY rats at 95 weeks (Table 1). Therefore glomenilar filtration rate in SHRE was maintained throughout the study.
Untreated SHR had glomerular pathology that, as has been described before,1-715 was most severe in the juxtamedullary zone and consisted of fibrinoid necrosis, sclerosis (Table 2), and pericapsular fi-brosis. Renal arteries exhibited intimal proliferation and hyperplasia of medial muscle layers (Table 2, Figure 4A). At 95 weeks of age, glomenilar sclerosis in SHRE was somewhat less severe than in 75-week-old untreated SHR (Table 2). The thickness of the media layer of arterioles and cortical radial arteries in the SHRE group was not significantly different from WKY rats (Figure 4B, Table 2), and all larger TABLE 1. Effect of Enalapril on Creatinine Clearance and Plasma Creatinine Concentration in Spontaneously Hypertensive Rats
Group WKY SHR SHRE p WKY vs. SHR pWKY vs. SHRE pSHR vs. SHRE Creatinine 60 wk 0.76±0.03 0.59±0.02 0.67±0.03 NS NS NS
clearance (ml/minx 100 g body wt) 75 wk 0.68±0.06 0.31 ±0.05 0.46±0.03 <0.01 <0.01 <0.05 95 wk 0.55±0.04 0.44±0.02 NS 60 wk 0.31 ±0.02 0.41 ±0.01 0.35 ±0.01 •cO.01 NS <0.01 Plasma creatinine (mg/dl) 75 wk 0.29±0.004 0.69+0.11 0.46+0.02 <0.01 <0.01 <0.01 95 wk 0.41 ±0.02 0.44+0.02 NS
A mininum of five animals were used for each analysis. WKY, Wistar-Kyoto normotensive controls; SHR, u n t r e a t e d spontaneously hypertensive rats; S H R E , enalapril-treated spontaneously hypertensive rats.
TABLE 2. Effect of Enalapril Treatment on Glomerular and Vascular Histopathology in
Spontaneously Hypertensive Rats
Glomerulosclerosis (%) Vessel wall thickness
Group (age in weeks) WKY (75) SHR (60) SHR (75) SHRE (95) Global 0 3±2* 7±2t 2±1* Segmental 0 15±3* 32±3t 17+3* Arteriole 6.8±0.2 9.6±1.3f 12.7±1.2t 6.2±0.5 Cortical radial artery 14.2±0.5 21.3±1.2f 24.1±2.0| 16.4±1.2 A minimum of five rats were used for each measurement. WKY, Wistar-Kyoto normotensive controls; SHR, untreated spontaneously hypertensive rats; SHRE, enalapril-treated spontaneously hypertensive rats.
'Significantly different from WKY rats atp<0.01.
tSignificantly different from WKY rats and SHRE atp<0.01.
B
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FIGURE 4. Panel A: Photomicrograph of formalin-fixed kidney tissue section, stained with silver methenamine, from the cortex of a 75-week-old, untreated sponta-neously hypertensive rat (SHR). Charac-teristic thickening of an arterial wall is seen
(arrow). There is an abnormal accumula-tion of mononuclear cells within the inter-stitium. Magnification x50. Panel B: Photomicrograph of formalin-fixed kidney tissue section, stained with silver methe-namine, from the cortex of a 95-week-old SHR treated with enalapril. Artery appears normal (small arrow), but there are abnor-mal numbers of mononuclear cells in the interstitium. One of the glomeruli is se-verely sclerotic (large arrow). Magnifica-tion x50.
arteries had a completely normal appearance. At 75 and 95 weeks of age, WKY rats had no evidence of glomerular sclerosis, interstitial infiltration, or vascu-lar pathology.
At all times, the intraglomerular population of mononuclear cells was identical in SHR, SHRE, and WKY rats, consisting of two to three la-positive and EDl-positive cells, few if any T cells, and no pheno-typically abnormal (ED2-positive or ED3-positive) macrophages (Table 3). The frequency of mononu-clear cells in glomeruli was also identical to that in normal Lewis rats.12 In and around Bowman's cap-sule, however, the number of both lymphocytes and macrophages was significantly elevated in 75-week-old SHR, with increased numbers of macrophages detectable at 60 weeks (Table 3). The number of periglomerular mononuclear cells was the same in 95-week-old, enalapril-treated SHR as in
75-week-old, untreated SHR (Table 3, Figure 5). Because small numbers of macrophages have been found in close association with the juxtaglomerular apparatus in normal kidneys,22 we estimated the severity of abnormal mononuclear cell infiltration at the vascu-lar pole by counting only clusters containing 10 or more positive cells. A few la-positive mononuclear cells, but none stained by 0X19 or EDI, were detected at the glomerular vascular pole in WKY rats. (Those la-positive mononuclear cells were mac-rophages as they also stained with W3/25, a mono-clonal antibody to T helper cells and some macro-phages.23 Data not shown.) The frequency of mononuclear cell clusters associated with the vascu-lar pole was significantly elevated in 60-week-old untreated SHR and increased by 75 weeks (Table 3). An identical frequency was noted at 95 weeks of age in SHR treated with enalapril (Figure 6A). In
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t^ q * d oi d o +| +| +| rn \c> Tt CN d ^ t ci r^ oj *b o d -H -H +1 +t +1 +1 (N •* (N O\ O +1 +1 +1 p >o •* -H O Tt o +l +l +l O H N d r>i +1 +1 >O 00 4 +1 +1 Tf O o n o VI +1 ( N rn # rN d •H m +1 12 . # d •H +1 00 # rN +1 a. ^H (S p VO d d >-J d +1 +1 +1 +1 CO ^ 00 O r-1 Tt O f^ « e i^ iq o d d d +1 +1 +1 +1 p c<-) o q v o «S v o •**' o d rn cs ^o o\ d d d c> +1 +1 +H -H U-) O) - H CT; «-H CS «S Tf rn m rt n d d d d VI VI VI VI o o o o +1 +1 +1 +1 v } vq en w-j (N (N fn d p tn fn CT; .-i d d d +1 +H -H -H oq « » H o d o d VI VI VI VI d c*S ^H in +1 +1 +1 +1 O\ 00 t*** Q\week-old SHR and 95-week-old SHRE, the mac-rophage population at the vascular pole included cells expressing antigens recognized by ED2 (Figure 6B) and ED3 not normally found in the kidney cortex. Because those phenotypically abnormal mac-rophages, which were only a small subset of the total, were never present in large numbers, they were not counted as "clusters."
Interstitial foci of mononuclear cells, not present in WKY rats, were diffusely distributed in SHR by 60 weeks and were present in high frequency by 75 weeks (Table 3). The extent of interstitial infiltration in SHRE at 95 weeks was significantly greater than in 60-week-old SHR but somewhat less severe than in 75-week-old SHR. Interstitial aggregates of mononu-clear cells always included some macrophages (5-10%) stained with ED2 or ED3. Foci of mononuclear cell accumulation were prominently associated with the arcuate arteries in untreated SHR, with apparent increases in size and frequency of those aggregates from 60 to 75 weeks. In enalapril-treated SHR, perivascular infiltration was either very infrequent or entirely absent. No attempt was made to quantitate the periarterial component of interstitial inflamma-tion because of intrinsic sampling problems. Besides T cells and macrophages, the large periarterial foci in SHR also contained many B lymphocytes (Figure 7).
Discussion
Treatment with enalapril had a number of benefits for spontaneously hypertensive rats. Blood pressure was easily maintained within normal limits, life span was prolonged, and deterioration of kidney function (glomerular filtration rate) was prevented. In those respects, the effects of enalapril were similar to the triple-drug antihypertensive therapy we have re-ported previously.7 In addition, SHR treated with enalapril were completely protected from the renal vessel histopathology that develops, despite success-ful control of systemic blood pressure, with the triple-drug regimen. However, although enalapril was effective in postponing the onset of albuminuria and high molecular weight proteinuria from the fifth to the 12th month of life, it was no more effective than the triple drug therapy in preventing the devel-opment of albuminuria, which eventually became as severe in normotensive as in untreated animals (Fig-ure 3A). This study confirms and extends our earlier observations that albuminuria in SHR can be com-pletely dissociated from systemic hypertension and supports our contention that kidney damage in SHR must result from another mechanism. Furthermore, albumin excretion was just as severe in enalapril-treated rats with vessels of normal diameter and moderate glomerulosclerosis as in hypertensive rats with highly thickened vessels and severe sclerosis (Table 2).
In a number of animal models of kidney disease, glomerular hypertension is thought to contribute significantly to the progression of chronic renal insuf-ficiency, proteinuria, and glomerulosclerosis.4-6-9'10
*r<
FIGURE 5. Panel A: Photomicrograph of peroxidase staining of EDl-positive macrophages in a frozen section of kidney cortex from a 95-week-old spontaneously hypertensive rat (SHR) treated with enal-april. Abnormal numbers of macrophages are present within and around Bowman's capsule. Two macrophages (arrows) are also visible within the glomerular tuft (G), a frequency not different from normal. Magnification x]25. Panel B: Photomi-crograph of peroxidase staining of 0X19-positive T lymphocytes (arrows) in a frozen section of kidney cortex from a 95-week-old SHR treated with enalapril. T lympho-cytes are visible within and around Bow-man's capsule and the urinary space but not inside the glomerular capillary tuft. Magnification x725.
What is the evidence that it may be important in the pathogenesis of proteinuria (albuminuria) in SHR? Based on the measurements of glomerular capillary pressure in superficial nephrons in young SHR (4 months of age),24-25 superficial proximal tubular albu-min concentrations,17 and glomerular filtration rates,17 there are no hemodynamic abnormalities in superficial nephrons that can be implicated in the etiology of functional or structural renal injury in the two-kidney SHR during the first 6 months of life.
Even though juxtameduUary glomeruli are not accessible for direct measurements of glomerular capillary pressure, there is indirect evidence derived from our work17 and that of others4*26-27 to support the premise that an increase in glomerular capillary pressure in juxtameduUary nephrons may be one of the factors responsible for renal pathology in the SHR. We have previously shown that a distinctive pattern of juxtameduUary glomerular injury
devel-oped in both untreated and antihypertensive drug-treated SHR. Because the magnitude of the albu-minuria correlated with the severity of deep nephron histopathology, damage to the glomerular capillary wall of the juxtameduUary glomeruli resulting in an increase in macromolecular permeability was most likely the cause of proteinuria.
Recently, Dworkin et al26 compared the effects of angiotensin converting enzyme inhibitors (captopril, enalapril) and triple drug therapy. In contrast to the two-kidney SHR, glomerular hypertension developed in superficial nephrons of untreated, uninephrecto-mized rats. Therefore, unilateral nephrectomy im-poses a severe physiological stress4 that results in an accelerated form of renal injury in the SHR. The two treatments were equivalent in their ability to prevent glomerular hypertension in the uninephrectomized SHR at 11 weeks of age. However, information on glomerular capillary pressure is lacking beyond 11
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• * • ~ . B *-FIGURE 6. Panel A: Photomicrograph of peroxidase staining of OX6-positive (la antigen-bearing) cells in a frozen section of kidney cortex from a 95-week-old sponta-neously hypertensive rat (SHR) treated with enalapril. Clusters containing abnor-mal numbers of OX6-positive mononu-clear cells are prominent at the vascular pole of glomeruli (arrows) and between some tubules. Weak staining of the cyto-plasm of some proximal tubule celb is not different from normal. Isolated OX6-pos-itive cells within glomeruli (G) and be-tween tubules are also normal. Magnifica-tion x50. Panel B: Photomicrograph of peroxidase staining of ED2-positive mac-rophages within and around Bowman's capsule. Several ED2-positive celb are at the vascular pole (arrows) of the glomeru-lus (G). Distribution of ED3-positive mac-rophages was similar, but the frequency of ED3-positive cells was somewhat less. Magnification x]25.
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weeks. The distribution of glomerular structural changes at 36 weeks of age was most prominent in deep glomeruli and agrees with our findings at 60-70 weeks in the intact SHR.1 7 This injury can be ame-liorated equally by the three therapies as glomerular sclerosis was reduced by 50% at 36 weeks in treated rats. Protein excretion in untreated SHR was signif-icantly greater at 32 and 36 weeks of age when compared with treated rats. At these times there was no significant difference in urinary protein excretion between the three drug-treated groups. Protein ex-cretion rates in treated groups increased from 32 to 36 weeks of age. It is interesting to speculate that if the study had been carried out longer there could have been a further increase in urinary protein excretion similar to that observed at 60-70 weeks of age in the SHR with two kidneys given triple therapy or enalapril.
Anderson et al21 also showed that both triple drug therapy (hydralazine, chlorothiazide, and reserpine) and angiotensin converting enzyme inhibitor (capto-pril) reduced glomerular capillary pressure to control values in superficial nephrons in streptozotocin-induced diabetic rats at 6-10 weeks of diabetes. How-ever, albuminuria and glomerular histopathological changes occurred at 28 weeks of diabetes in the triple drug diabetic group but not in the captopril-treated diabetic rats followed up to 70 weeks of diabetes. Persistent control of glomerular capillary pressure in the captopril-treated group was also evident at this time. In contrast, the older diabetic animals on triple drug therapy escaped from glomerular capillary hyper-tensive control. Therefore SHR and streptozotocin models differ in long-term response to therapies.
We reported that triple drug therapy was only able to delay but not prevent renal injury in the
FIGURE 7. Photomicrograph showing peroxi-dase staining of OX33-positive B lymphocytes in an aggregate of mononuclear cells alongside an artery (A) in the kidney cortex of a sponta-neously hypertensive rat. Magnification xl25.
kidney SHR. The present study describes a similar delay in renal injury with enalapril therapy. If triple drug and enalapril therapies are equally effective in reducing glomerular capillary pressure and juxta-medullary glomerular hypertension is a factor in the renal injury in the SHR, then the delay produced by these antihypertensive drug treatments may only last until there is an escape from control of glomerular capillary pressure in deep nephrons.
The use of dietary protein restriction in untreated, uninephrectomized SHR slowed progression of pro-teinuria and glomerulosclerosis by a reduction in glomerular capillary pressure.4 However, no benefit of dietary protein restriction could be demonstrated in the two-kidney, untreated SHR.15 A low protein diet was only effective in reducing albuminuria and glomerular histopathology when combined with tri-ple drug therapy. Juxtamedullary glomerular blood flow, derived from deep cortical afferent arterioles, is not autoregulated but increases with increments in renal artery pressure.28^29 It is possible that the severe systemic hypertension in untreated SHR would in-crease glomerular capillary pressure in these deep nephrons sufficiently to be impervious to the effects of a low protein diet. However, the combination of a low protein diet with triple drug therapy may further delay the escape from glomerular capillary hyperten-sive control that occurs with antihypertenhyperten-sive therapy alone because systemic hypertension is not a factor. Until the appropriate direct measurements of glo-merular capillary pressure have been made in older normotensive SHR with two kidneys, the relation of increased albumin excretion to glomerular hyperten-sion in this model will remain unresolved.
There are also other possible factors in the devel-opment of albuminuria and histopathology in the SHR. The abnormal intraglomerular accumulation of mononuclear cells, which has been linked to protein-uria in other forms of kidney disease,30 was found in
this study to be entirely absent in both treated and untreated SHR. In contrast, extraglomerular mono-nuclear cell infiltration of the kidney, which has also been correlated with the severity of kidney dysfunc-tion,31 was prominent in both groups. Because many of the macrophages infiltrating Bowman's capsule and the vascular pole of glomeruli expressed differ-entiation antigens (la, ED2, ED3) suggestive of an activated state, it is tempting to speculate that vaso-active mediators, released by macrophages into the glomerular milieu, could account for increased per-meability of the glomerular capillary wall. This in-triguing possibility remains to be evaluated in SHR. In a rat model of acute ureteral obstruction, it has also been proposed that alterations in kidney func-tion might result from secretory activities of macro-phages infiltrating the cortical interstitium.31 Using a diet deficient in essential fatty acids to deplete inter-stitial macrophages in SHR, we have collected pre-liminary evidence that albuminuria is delayed by reducing periglomerular inflammation in the pres-ence of high systemic blood pressure.32
It is possible that enalapril may delay albuminuria and reduce kidney vessel histopathology in SHR by a nonhemodynamic mechanism. The amelioration by captopril (another angiotensin converting enzyme inhibitor) of proteinuria in lupuslike nephritis of MRL/lpr mice has been attributed to an immuno-modulatory effect of the drug, rather than normaliza-tion of blood pressure.33 A poorly understood, cen-tral dysregulation of the immune system, with some similarities to the immunoregulatory disorders of lupus-prone mice, has been implicated in the patho-genesis of hypertension in the SHR,34-35 and might be involved in kidney damage as well. Better under-standing of the benefits of enalapril and triple drug therapy in SHR will depend on more information about 1) the possible effect of antihypertensive drug
therapies on the immune system and 2) the immuno-logical status of SHR.
In summary, long-term control of hypertension in SHR by enalapril delayed but could not prevent the eventual development of albuminuria and increased excretion of high molecular weight proteins. Enala-pril therapy did protect the SHR completely against vessel wall hypertrophy. We conclude that the cause of kidney damage in SHR is probably multifactorial involving both hemodynamic and nonhemodynamic factors. Our findings in the SHR model of essential hypertension may be relevant for the subset of pa-tients with essential hypertension in which renal function deteriorates progressively despite effective control of systemic blood pressure.36
Acknowledgments
We appreciate the technical assistance of Nancy Manz, Susan Bemben, Susan Alder, and Sheila Rosenberry. We acknowledge the generosity of Merck Sharp & Dohme Research Laboratories, West Point, Pa., for supplying Vasotec (enalapril). We greatly appreciate the statistical expertise of Mary Jane Feldman, PhD, and the secretarial assistance of Teresa M. Schuster.
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