Atherosclerosis, Y 1 (1991) 241-246 P 1991 Elsevier Scientific Publishers
ATHERO
241 Ireland,
Ltd. All rights reserved
0021-Yl50/91/$03.50
04739
Effect of nifedipine on renal microvascular cholesterol accumulation and prostacyclin biosynthesis in cholesterol-fed rabbits Michael A. Kirschenbaum,
Daeyoung
D. Roh and Vaijinath
S. Kamanna
Nephrology Section, Department of Veterans Af7uir.y Medical Center, Long Beach, CA (U.S.A.), arld Dirkion CA (U.S.A.) Department ofMedicine, Unilvrsity of California, lurk,
of Nephrology,
(Received 18 July, 1991) (Revised, received 3 September, 1YY1) (Accepted 16 September, 1991)
Summary Studies, performed in rabbits, examined the effect of feeding a high cholesterol diet and/or a calcium antagonist, nifedipine, on renal microvascular prostacyclin biosynthesis and cholesterol accumulation. After 30 days, cholesterol-fed rabbits had elevated serum and tissue cholesterol levels associated with decreased microvascular prostacyclin biosynthesis and histologic evidence of microvascular and glomerular lipid accumulation. Nifedipine reduced tissue cholesterol levels, enhanced prostacyclin biosynthesis, and reduced the histologic evidence for lipid accumulation in renal microvessels and glomeruli. These studies suggest that calcium antagonists may have a beneficial effect in preventing the tissue cholesterol accumulation associated with a high-cholesterol diet and further suggest that these agents may have beneficial effects in the treatment of renal diseases associated with microvascular or glomerular lipid accumulation.
Key words:
Kidney; Microvasculature; Prostaglandins; Prostacyclin
Cholesterol;
Introduction It has been proposed metabolites are important
that arachidonic acid biochemical modula-
Correspondence to: Michael A. Kirschenbaum, M.D., Chief, Nephrology Section (11 IN), Department of Veterans Affairs Medical Center, 5901 East Seventh Street. Lone Beach. CA 90822, U.S.A.
Atherosclerosis;
Glomerulosclerosis;
Nifedipine;
tors of atherosclerosis [1,2] and previous studies have suggested that the capability of the vasculature to synthesize prostacyclin (PGI,) may ultimately protect it from developing atherosclerosis 13-91. In this regard, blood vessels obtained from animals with experimental atherosclerosis or human atherosclerotic subjects appear to demonstrate an ineffective capacity for PGI, biosynthesis [2,6,9,10]. Since PGI, appears to enhance cholesterol egress from ceils, in part, by stimulat-
242
ing cholesterol esterase activity [4,6,7], diminished microvascular PGI, activity associated with the atherosclerotic process may be a pivotal step which results in cholesterol and cholesteryl ester accumulation within microvascular cells. Calcium antagonists, including those having a dihydropyridine structure, have been proposed to prevent or at least alter process of atherosclerosis [ll-141. In addition to lowering blood pressure in hypertensive patients, and decreasing calcium-related cell damage, calcium antagonists may prevent atherosclerosis by enhancing the biosynthesis of PGI, and, by doing so, promote the hydrolysis of cholesteryl esters to cholesterol within the vascular cells and, eventually, the egress of cholesterol from vascular cells. The purpose of this study was to examine the effect of cholesterol feeding on renal microvascular cholesterol accumulation and to assess whether a dihydropyridine calcium antagonist, nifedipine, could alter both the biosynthesis of PGI, and the accumulation of cholesterol in this tissue. The results of this study indicate that similar to other vascular beds, the rabbit renal microvasculature exhibits the intracellular accumulation of both cholesterol and cholesteryl esters and the decreased biosynthesis of PGI, in response to 30 days of 2% cholesterol feeding. The results further suggest that these biochemical alterations could be prevented by the administration of a calcium antagonist, nifedipine.
Materials
and methods
Two groups of 6 New Zealand White rabbits (16-20 weeks, 3.0-3.5 kg) were allowed to eat either a standard chow (group I), or an isocaloric pellet diet containing 2% cholesterol (group II) for 30 days. Two additional groups of 6 rabbits were maintained on a similar diet except that the control rabbits, group III, and the 2% cholesterol-fed rabbits, group IV, were given a non-hypotensive dose of a calcium antagonist (nifedipine, 20 mg/day, added to the diet), for the 30-day period. Rabbits were weighed weekly and blood was drawn from an ear vein both at the start of the study and at the conclusion for measurement of serum cholesterol. Animals were
killed at 30 days and their kidneys were removed for the examination of renal histology and determination of microvascular cholesterol accumulation and PGI, biosynthesis. Renal cortical samples from the 4 treatment groups were sent for histological examination. All samples were coded and analyzed by a trained pathologist using standard light and electron microscopy procedures. Sections of renal cortex were stained with Oil red 0 to assess for the presence of lipids. Isolation of preglomerular microvessels was performed by injecting the kidneys with a suspension of magnetized iron oxide, homogenizing the cortex, and separating the iron-laden microvessels from non-vascular tissue with a strong magnet as previously described by this laboratory [ 151. The resulting preparation of vascular tissue was composed entirely of interlobular arteries and afferent arterioles and was free of glomeruli or non-vascular contaminants. The method for measuring 6-keto-prostaglandin F,, (6-keto-PGF,,), a stable nonenzymatic metabolite of PGI,, in freshly isolated renal microvasculature tissue by radioimmunoassay has been previously described [16] and the results were expressed as ng of the prostanoid produced/mg protein/5 min. Tissue pellets were digested overnight with 1 N NaOH at 40-45°C and neutralized with 1 N HCl before protein quantitation by the method of Lowry et al. [17] using bovine serum albumin as a standard. Determination of tissue cholesterol was performed using methods described by Heider and Boyett [18]. Tissue samples were homogenized in 0.25 M sucrose at 4°C using a Polytron tissue homogenizer. An aliquot of the microvessel homogenate was extracted with a chloroform methanol mixture [193. The organic phase was completely dried under N, and the dried lipid extract was reconstituted in 200 ~1 isopropanol. Total and free cholesterol was measured in the presence or absence of cholesteryl esterase (Boehringer Mannheim Biochemicals, Indianapolis, IN) respectively using the fluorometric method at excitation wavelength 325 nm, emission 415 nm [ 181. A standard curve was generated using 0.5- 10 pg cholesterol. The difference between total cholesterol and free cholesterol yielded the amount of cholesteryl ester.
243
The data in this report have been expressed as the mean iSEM. Statistical significance was determined by using Student’s f-test for unpaired data or by analysis of variance whenever appropriate. A P value < 0.05 was considered significant. Results
At the time of sacrifice, there were no differences in body weight when any of-the 4 groups of animals were compared (Table 1). However, the animals fed a 2% cholesterol diet (groups II and IV) had higher serum cholesterol levels when compared to either the control animals (group I) or those receiving only nifedipine (group III). Nifedipine appeared to have no effect on decreasing serum cholesterol levels in 2% cholesterol-fed rabbits (group II compared to group IV). Renal microvascular PGI, biosynthesis decreased significantly in the 2% cholesterol-fed rabbits (group II) when compared to control animals (group I). PGI, biosynthesis in the nifedipine-treated cholesterol-fed rabbits (group IV) was indistinguishable from that noted in control animals (group I). Nifedipine treatment to cholesterol-fed rabbits (group IV) showed a significant increase in PGI, biosynthesis when compared to a cholesterol-fed group not given nifedipine (group II). Nifedipine had no effect on PGI, biosynthesis in control animals fed a normal diet (group III>. There was a marked increase in free cholesterol and cholesteryl esters in the renal microves-
TABLE
Group
Group
II
Group
IV
sels of the 2% cholesterol-fed rabbits when compared to those fed a normal diet (Fig. 1). Treatment of the 2% cholesterol-fed rabbits with nifedipine resulted in a significant reduction in cholesterol accumulation (group IV vs. group II). Treatment of control-fed animals with nifedipine did not alter cholesterol accumulation (data not shown). There were no abnormalities in renal morphology seen in the control animals by light or electron microscopy and no lipid deposits were noted after staining with Oil red 0. After 30 days of cholesterol feeding, however, interlobular arteries and afferent arterioles in the group II rabbits showed mild intimal thickening and proliferation.
I
EFFECT OF 2% CHOLESTEROL FEEDING AND NIFEDIPINE RENAL MICROVASCULAR 6-KETO-PGF,, BIOSYNTHESIS (mean
I
Fig. 1. Effect of 2% cholesterol diet and nifedipine on rabbit renal microvascular cholesterol accumulation. Group I control diet, no nifedipine; group II 2% cholesterol diet, no nifedipine; and group IV 2% cholesterol diet plus nifedipine. Results are the mean + SEM. n = 6 for each group. *P < 0.05 when compared with group I; * * P < 0.05when compared to either group I or group II.
ON BODY
WEIGHT,
SERUM
CHOLESTEROL
k SEM)
Group
Diet
Drug
n
I II III IV
normal 2% cholesterol normal 2% cholesterol
none none nifedipine nifedipine
6 6 6 6
* P < 0.05 when group
II compared
to either
group
I or IV.
Body weight
Serum cholesterol
(kg)
(mg/dl)
Microvascular h-keto-PGF,,, (ng/mg protein)
46k 3 910+ 120 * 48+ 5 898k 168 *
18.2+3.1 11.5+1.8 * 17.4 f 4.0 18.7 k 4.6
3.2 3.4 3.3 3.4
+ * f f
0.2 0.3 0.3 0.3
AND
244 Oil red 0 staining revealed strongly birefringent lipid droplets in and around the microvascular intima, perivascular areas, glomerular capillaries and mesangium. Electron microscopy showed occasional histiocytic foam cells in the microvessels with varying degrees of lipid droplets. The calcium antagonist-treated animals showed noticeably less evidence of lipid deposition in the areas noted above as well as fewer foam cells seen in the vessel walls. Discussion Preglomerular renal microvessels obtained from rabbits fed a 2% cholesterol diet accumulate cholesterol and cholesteryl esters similar to other vascular beds. The cholesterol is deposited in the vascular endothelium, perivascular areas, glomerular capillaries, and mesangium. The cholesterol accumulation is associated with decreased PGI, biosynthesis. Finally, the data indicate that the administration of nifedipine to cholesterol-fed rabbits returned microvascular PGI, biosynthesis to baseline levels, decreased tissue cholesterol and cholesteryl ester accumulation, and decreased the histological evidence of lipid accumulation. These changes occurred in the absence of significant alterations in levels of serum cholesterol. Rabbits fed a high cholesterol diet have been extensively studied as model of human atherosclerosis [11,20] and these animals manifest biochemical abnormalities in lipids, lipoproteins and apolipoproteins similar to those seen in human hyperlipoproteinemic patients [ 11,211. Endothelium-derived vasoactive substances including arachidonic acid metabolites have been linked to atherosclerosis [1,5,101 and studies in non-renal blood vessels have suggested that the capacity of the vasculature to synthesize PGI, may protect it from atherosclerosis. Since PGI, appears to enhance cholesterol egress from cells by stimulating cholesterol esterase, diminished microvascular PGI, biosynthesis may be a pivotal step which results in cholesterol and cholesteryl ester accumulation within microvascular cells [4,6,7]. Calcium antagonists have been proposed to prevent the development of atherosclerosis [ll141. The precise role of calcium antagonists in the
management of hypertensive or normotensive patients with atherosclerotic cardiovascular disease is not fully understood. A number of studies over the past decade have suggested that calcium antagonists may exert beneficial antiatherosclerotic effects on blood vessels from animals with experimental atherosclerosis [11,22,23]. Other studies have suggested similar beneficial effects in human subjects by retarding the progression of atherosclerosis in patients with coronary artery disease or recent coronary artery bypass surgery [24-261. In addition to antihypertensive properties in hypertensive patients and their abilities to decrease calcium-related cell damage, calcium antagonists may protect the glomerulus and other renal microvessels from developing vascular injury (i.e., glomerulosclerosis) by mechanisms unrelated to their effects on tissue lipid abnormalities or glomerular hemodynamics [27]. Despite these other mechanisms, calcium antagonists have also been proposed to prevent atherosclerosis by enhancing the biosynthesis of PGI, and, by doing so, augment cholesterol esterase activity [4,6,7] and promoting cholesteryl ester hydrolysis. Finally, as has been shown by Henry and Bentley [ 111 and confirmed in this present study, the effect of calcium antagonists is not dependent on the ability of these agents to alter the serum cholesterol or a number of other cardiovascular parameters, but rather they are more closely related to the ability of these drugs to alter tissue levels of cholesterol and cholesteryl esters. The ability of calcium antagonists to alter the accumulation of lipid material in the renal microvasculature, glomeruli, and mesangium may have considerable implications on the use of these agents in patients with progressive renal disease in which the development of lipid deposition may be associated with the inexorable progression of their renal disease. In conclusion, similar to other vascular tissues which have been investigated, the rabbit renal microvasculature decreases PGI, biosynthesis and accumulates cholesterol and cholestetyl esters in response to cholesterol feeding. The lipid accumulation seen in these animals is primarily distributed in and around the endothelial cells, the perivascular areas, the glomerular capillary loops and the mesangium. The administration of a cal-
245 cium antagonist appears to prevent the accumulation of cholesterol in these tissue sites in the absence of any specific decrease in the levels of serum cholesterol. Because of their ability to reduce tissue cholesterol levels, calcium antagonists may have a potential use in the treatment of kidney patients with renal microvascular lipid accumulation in whom their renal disease may contribute to the progression of systemic atherosclerosis. Further studies are needed to understand whether calcium antagonists could modify lipidassociated progressive glomerulosclerosis.
IO
II
I2
13 14
Acknowledgements 15
We wish to thank Ms. Sunita Gupta for her technical assistance in performing these studies. We also wish to thank Dr. Raymond B. Wuerker for his expertise in the examination of the histologic specimens. This work was support by the Department of Veterans Affairs and a grant from the American Heart Association, California Affiliate and the National Kidney Foundation of Southern California.
I6
17
18
19
References 20 I Ross. R.. The pathogenesis of atherosclerosis, N. Engl. .I. Med., 314 (1986) 48X. 2 Chaudhari, A. and Kirschenbaum, M.A.. Effect of a 2% cholesterol diet on rabbit renal preglomerular microvascular PGI, and PGE, biosynthesis, Prostaglandin Leukotriene Essential Fatty Acids, 36 (1989) 81. 3 Schror. K., Prostaglandins, other eicosanoids and endothelial cells. Basic Res. Cardiol., 80 (1985) 502. 3 Hajjar. D.P.. Prostaglandins and cyclic nucleotides: modulators of arterial cholesterol metabolism, Biochem. Pharmacol., 34 (1985) 295. 5 Cannon, P.J.. Eicosanoids and the blood vessel wall, Circulation 70 (1984) 523. 6 Ilajjar. D.P.. Weksler. B.B., Falcone, D.J. et al., Prostacyclin modulates cholesteryl ester hydrolytic activity by its effect on cyclic adenosine monophosphate in rabbit aortic smooth muscle cells, J. Clin. Invest., 70 (1982) 479. 7 Hajjar. D.P.. Prostaglandins modulate arterial cholesteryl ester metabolism. Enzyme, 32 (1984) 218. X Mustard. J.F., Kinlough-Rathbone, R.L. and Packham, M.A.. Platelets, endothelium, and vessel injury. Adv. Prostaglandin Thromboxane Leukotriene Res., I3 (1985) 235. 4 Dembinska-Kiec. A.. Gtyglewska, T., Zmuda. A. and Gryglewsski, R.J.. The generation of prostacyclin by arteries
21
22
23
24
25
and by the coronary vascular bed is reduced in experimental atherosclerosis in rabbits, Prostaglandin, 14 (1977) 1025. Smith, W.L., Prostaglandin biosynthesis and its compartmentation in vascular smooth muscle and endothelial cells. Annu. Rev. Physiol., 48 (1986) 251. Henry, P.D. and Bentley. K.I., Suppression of atherogencsis in cholesterol-fed rabbit treated with nifedipine. J. Clin. Invest.. 68 (1981) 1366. Etingen, O.R. and Hajjar. D.P., Calcium channel blockers enhance cholesteryl ester hydrolysis and decrease total cholesterol accumulation in human aortic tissue, Circ. Res.. 66 (1990) 16.5. Henry, P.D.. Atherogenesis. calcium and calcium antagonists, Am. J. Cardiol.. 66 (lY90) 31. Parmley. W.W.. Vascular protection from atherosclerosis: potential of calcium antagonists, Am. J. Cardiol.. 66 (1990) 161. Chaudhari, A. and Kirschenbaum. M.A.. A rapid method for isolating rabbit renal microvessels. Am. J. Physiol.. 254 (10Xx) F29l. Chaudhari. A. and Kirschenbaum. M.A.. Altered glomerular eicosanoid biosynthesis in uranyl nitrate-induced acute renal failure, Biochim. Biophys. Acta. 792 (19X4) 135. Lowry. O.H.. Rosebrough, N.J., Farr. A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol.. Chem. 193 (1951) 265. Heider. J.G. and Boyett, R.L.. The picomole determination of free and total cholesterol in cells in culture. J. Lipid Res.. 19 (lY78) 514. Bligh. E.J. and Dyer, W.J.. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol., 37 (195’)) 911. Adams, C.W.M.. Bayliss, O.B. and Ibrahim, M.Z.M.. The distribution of lipids and enzymes in the aortic wall in dietary rabbit atheroma and human atherosclerosis. J. Pathol. Bacterial., 86 (1963) 421. Shore. V.G.. Shore. B. and Heart, R.C.. Changes in apolipoproteins and properties of rabbit very low density lipoproteins on induction of cholesterolemia. Biochemistry 13 (1974) 1579. Grodzinska, L., Basista. M.. Basista. E. et al.. Nitrendipine-stimulated release of prostacyclin-like substance in normal and atherosclerotic animals. Arzneimittelforschung, 37 (1987) 412. Etingen, O.R.. Hajjar, D.P.. Nifedipine increases cholesteryl ester hydrolytic activity in lipid-laden rabbit arterial smooth muscle cells. J. Clin. Invest.. 75 (1985) 1554. Loaldi. A., Polese, A.. Montorsi. P., Comparison of nifedipine, propranolol and isosorbide dinitrate on angiographic progression and regression of coronary arterial narrowing in angina pectoris. Am. J. Cardiol., 64 (1989) 433. Gottlieb, SO., Brinker, J.A. and Mellits, D., Effect of nifedipine on the development of coronary bypass graft stenoses in high-risk patients: a randomized, double-blind, placebo-controlled trial, Circulation. X0 (I 989) 11-223 (abstract).
246 26 Lichtlen, P.R., Hugenholtz, PG., Rafflenbeul, W., et al., Retardation of angiographic progression of coronary artery disease by nifedipine. Results of international nifedipine trial on anti-atherosclerotic therapy (INTACT), Lancet, 335 (1990) 1109.
27 Dworkin, L.D. and Benstein, J.A., Impact of antihypertensive therapy on progressive kidney damage, Am. J. Hypertens., 2 (1989) 1625.
ATHERO
241 Ireland,
Ltd. All rights reserved
0021-Yl50/91/$03.50
04739
Effect of nifedipine on renal microvascular cholesterol accumulation and prostacyclin biosynthesis in cholesterol-fed rabbits Michael A. Kirschenbaum,
Daeyoung
D. Roh and Vaijinath
S. Kamanna
Nephrology Section, Department of Veterans Af7uir.y Medical Center, Long Beach, CA (U.S.A.), arld Dirkion CA (U.S.A.) Department ofMedicine, Unilvrsity of California, lurk,
of Nephrology,
(Received 18 July, 1991) (Revised, received 3 September, 1YY1) (Accepted 16 September, 1991)
Summary Studies, performed in rabbits, examined the effect of feeding a high cholesterol diet and/or a calcium antagonist, nifedipine, on renal microvascular prostacyclin biosynthesis and cholesterol accumulation. After 30 days, cholesterol-fed rabbits had elevated serum and tissue cholesterol levels associated with decreased microvascular prostacyclin biosynthesis and histologic evidence of microvascular and glomerular lipid accumulation. Nifedipine reduced tissue cholesterol levels, enhanced prostacyclin biosynthesis, and reduced the histologic evidence for lipid accumulation in renal microvessels and glomeruli. These studies suggest that calcium antagonists may have a beneficial effect in preventing the tissue cholesterol accumulation associated with a high-cholesterol diet and further suggest that these agents may have beneficial effects in the treatment of renal diseases associated with microvascular or glomerular lipid accumulation.
Key words:
Kidney; Microvasculature; Prostaglandins; Prostacyclin
Cholesterol;
Introduction It has been proposed metabolites are important
that arachidonic acid biochemical modula-
Correspondence to: Michael A. Kirschenbaum, M.D., Chief, Nephrology Section (11 IN), Department of Veterans Affairs Medical Center, 5901 East Seventh Street. Lone Beach. CA 90822, U.S.A.
Atherosclerosis;
Glomerulosclerosis;
Nifedipine;
tors of atherosclerosis [1,2] and previous studies have suggested that the capability of the vasculature to synthesize prostacyclin (PGI,) may ultimately protect it from developing atherosclerosis 13-91. In this regard, blood vessels obtained from animals with experimental atherosclerosis or human atherosclerotic subjects appear to demonstrate an ineffective capacity for PGI, biosynthesis [2,6,9,10]. Since PGI, appears to enhance cholesterol egress from ceils, in part, by stimulat-
242
ing cholesterol esterase activity [4,6,7], diminished microvascular PGI, activity associated with the atherosclerotic process may be a pivotal step which results in cholesterol and cholesteryl ester accumulation within microvascular cells. Calcium antagonists, including those having a dihydropyridine structure, have been proposed to prevent or at least alter process of atherosclerosis [ll-141. In addition to lowering blood pressure in hypertensive patients, and decreasing calcium-related cell damage, calcium antagonists may prevent atherosclerosis by enhancing the biosynthesis of PGI, and, by doing so, promote the hydrolysis of cholesteryl esters to cholesterol within the vascular cells and, eventually, the egress of cholesterol from vascular cells. The purpose of this study was to examine the effect of cholesterol feeding on renal microvascular cholesterol accumulation and to assess whether a dihydropyridine calcium antagonist, nifedipine, could alter both the biosynthesis of PGI, and the accumulation of cholesterol in this tissue. The results of this study indicate that similar to other vascular beds, the rabbit renal microvasculature exhibits the intracellular accumulation of both cholesterol and cholesteryl esters and the decreased biosynthesis of PGI, in response to 30 days of 2% cholesterol feeding. The results further suggest that these biochemical alterations could be prevented by the administration of a calcium antagonist, nifedipine.
Materials
and methods
Two groups of 6 New Zealand White rabbits (16-20 weeks, 3.0-3.5 kg) were allowed to eat either a standard chow (group I), or an isocaloric pellet diet containing 2% cholesterol (group II) for 30 days. Two additional groups of 6 rabbits were maintained on a similar diet except that the control rabbits, group III, and the 2% cholesterol-fed rabbits, group IV, were given a non-hypotensive dose of a calcium antagonist (nifedipine, 20 mg/day, added to the diet), for the 30-day period. Rabbits were weighed weekly and blood was drawn from an ear vein both at the start of the study and at the conclusion for measurement of serum cholesterol. Animals were
killed at 30 days and their kidneys were removed for the examination of renal histology and determination of microvascular cholesterol accumulation and PGI, biosynthesis. Renal cortical samples from the 4 treatment groups were sent for histological examination. All samples were coded and analyzed by a trained pathologist using standard light and electron microscopy procedures. Sections of renal cortex were stained with Oil red 0 to assess for the presence of lipids. Isolation of preglomerular microvessels was performed by injecting the kidneys with a suspension of magnetized iron oxide, homogenizing the cortex, and separating the iron-laden microvessels from non-vascular tissue with a strong magnet as previously described by this laboratory [ 151. The resulting preparation of vascular tissue was composed entirely of interlobular arteries and afferent arterioles and was free of glomeruli or non-vascular contaminants. The method for measuring 6-keto-prostaglandin F,, (6-keto-PGF,,), a stable nonenzymatic metabolite of PGI,, in freshly isolated renal microvasculature tissue by radioimmunoassay has been previously described [16] and the results were expressed as ng of the prostanoid produced/mg protein/5 min. Tissue pellets were digested overnight with 1 N NaOH at 40-45°C and neutralized with 1 N HCl before protein quantitation by the method of Lowry et al. [17] using bovine serum albumin as a standard. Determination of tissue cholesterol was performed using methods described by Heider and Boyett [18]. Tissue samples were homogenized in 0.25 M sucrose at 4°C using a Polytron tissue homogenizer. An aliquot of the microvessel homogenate was extracted with a chloroform methanol mixture [193. The organic phase was completely dried under N, and the dried lipid extract was reconstituted in 200 ~1 isopropanol. Total and free cholesterol was measured in the presence or absence of cholesteryl esterase (Boehringer Mannheim Biochemicals, Indianapolis, IN) respectively using the fluorometric method at excitation wavelength 325 nm, emission 415 nm [ 181. A standard curve was generated using 0.5- 10 pg cholesterol. The difference between total cholesterol and free cholesterol yielded the amount of cholesteryl ester.
243
The data in this report have been expressed as the mean iSEM. Statistical significance was determined by using Student’s f-test for unpaired data or by analysis of variance whenever appropriate. A P value < 0.05 was considered significant. Results
At the time of sacrifice, there were no differences in body weight when any of-the 4 groups of animals were compared (Table 1). However, the animals fed a 2% cholesterol diet (groups II and IV) had higher serum cholesterol levels when compared to either the control animals (group I) or those receiving only nifedipine (group III). Nifedipine appeared to have no effect on decreasing serum cholesterol levels in 2% cholesterol-fed rabbits (group II compared to group IV). Renal microvascular PGI, biosynthesis decreased significantly in the 2% cholesterol-fed rabbits (group II) when compared to control animals (group I). PGI, biosynthesis in the nifedipine-treated cholesterol-fed rabbits (group IV) was indistinguishable from that noted in control animals (group I). Nifedipine treatment to cholesterol-fed rabbits (group IV) showed a significant increase in PGI, biosynthesis when compared to a cholesterol-fed group not given nifedipine (group II). Nifedipine had no effect on PGI, biosynthesis in control animals fed a normal diet (group III>. There was a marked increase in free cholesterol and cholesteryl esters in the renal microves-
TABLE
Group
Group
II
Group
IV
sels of the 2% cholesterol-fed rabbits when compared to those fed a normal diet (Fig. 1). Treatment of the 2% cholesterol-fed rabbits with nifedipine resulted in a significant reduction in cholesterol accumulation (group IV vs. group II). Treatment of control-fed animals with nifedipine did not alter cholesterol accumulation (data not shown). There were no abnormalities in renal morphology seen in the control animals by light or electron microscopy and no lipid deposits were noted after staining with Oil red 0. After 30 days of cholesterol feeding, however, interlobular arteries and afferent arterioles in the group II rabbits showed mild intimal thickening and proliferation.
I
EFFECT OF 2% CHOLESTEROL FEEDING AND NIFEDIPINE RENAL MICROVASCULAR 6-KETO-PGF,, BIOSYNTHESIS (mean
I
Fig. 1. Effect of 2% cholesterol diet and nifedipine on rabbit renal microvascular cholesterol accumulation. Group I control diet, no nifedipine; group II 2% cholesterol diet, no nifedipine; and group IV 2% cholesterol diet plus nifedipine. Results are the mean + SEM. n = 6 for each group. *P < 0.05 when compared with group I; * * P < 0.05when compared to either group I or group II.
ON BODY
WEIGHT,
SERUM
CHOLESTEROL
k SEM)
Group
Diet
Drug
n
I II III IV
normal 2% cholesterol normal 2% cholesterol
none none nifedipine nifedipine
6 6 6 6
* P < 0.05 when group
II compared
to either
group
I or IV.
Body weight
Serum cholesterol
(kg)
(mg/dl)
Microvascular h-keto-PGF,,, (ng/mg protein)
46k 3 910+ 120 * 48+ 5 898k 168 *
18.2+3.1 11.5+1.8 * 17.4 f 4.0 18.7 k 4.6
3.2 3.4 3.3 3.4
+ * f f
0.2 0.3 0.3 0.3
AND
244 Oil red 0 staining revealed strongly birefringent lipid droplets in and around the microvascular intima, perivascular areas, glomerular capillaries and mesangium. Electron microscopy showed occasional histiocytic foam cells in the microvessels with varying degrees of lipid droplets. The calcium antagonist-treated animals showed noticeably less evidence of lipid deposition in the areas noted above as well as fewer foam cells seen in the vessel walls. Discussion Preglomerular renal microvessels obtained from rabbits fed a 2% cholesterol diet accumulate cholesterol and cholesteryl esters similar to other vascular beds. The cholesterol is deposited in the vascular endothelium, perivascular areas, glomerular capillaries, and mesangium. The cholesterol accumulation is associated with decreased PGI, biosynthesis. Finally, the data indicate that the administration of nifedipine to cholesterol-fed rabbits returned microvascular PGI, biosynthesis to baseline levels, decreased tissue cholesterol and cholesteryl ester accumulation, and decreased the histological evidence of lipid accumulation. These changes occurred in the absence of significant alterations in levels of serum cholesterol. Rabbits fed a high cholesterol diet have been extensively studied as model of human atherosclerosis [11,20] and these animals manifest biochemical abnormalities in lipids, lipoproteins and apolipoproteins similar to those seen in human hyperlipoproteinemic patients [ 11,211. Endothelium-derived vasoactive substances including arachidonic acid metabolites have been linked to atherosclerosis [1,5,101 and studies in non-renal blood vessels have suggested that the capacity of the vasculature to synthesize PGI, may protect it from atherosclerosis. Since PGI, appears to enhance cholesterol egress from cells by stimulating cholesterol esterase, diminished microvascular PGI, biosynthesis may be a pivotal step which results in cholesterol and cholesteryl ester accumulation within microvascular cells [4,6,7]. Calcium antagonists have been proposed to prevent the development of atherosclerosis [ll141. The precise role of calcium antagonists in the
management of hypertensive or normotensive patients with atherosclerotic cardiovascular disease is not fully understood. A number of studies over the past decade have suggested that calcium antagonists may exert beneficial antiatherosclerotic effects on blood vessels from animals with experimental atherosclerosis [11,22,23]. Other studies have suggested similar beneficial effects in human subjects by retarding the progression of atherosclerosis in patients with coronary artery disease or recent coronary artery bypass surgery [24-261. In addition to antihypertensive properties in hypertensive patients and their abilities to decrease calcium-related cell damage, calcium antagonists may protect the glomerulus and other renal microvessels from developing vascular injury (i.e., glomerulosclerosis) by mechanisms unrelated to their effects on tissue lipid abnormalities or glomerular hemodynamics [27]. Despite these other mechanisms, calcium antagonists have also been proposed to prevent atherosclerosis by enhancing the biosynthesis of PGI, and, by doing so, augment cholesterol esterase activity [4,6,7] and promoting cholesteryl ester hydrolysis. Finally, as has been shown by Henry and Bentley [ 111 and confirmed in this present study, the effect of calcium antagonists is not dependent on the ability of these agents to alter the serum cholesterol or a number of other cardiovascular parameters, but rather they are more closely related to the ability of these drugs to alter tissue levels of cholesterol and cholesteryl esters. The ability of calcium antagonists to alter the accumulation of lipid material in the renal microvasculature, glomeruli, and mesangium may have considerable implications on the use of these agents in patients with progressive renal disease in which the development of lipid deposition may be associated with the inexorable progression of their renal disease. In conclusion, similar to other vascular tissues which have been investigated, the rabbit renal microvasculature decreases PGI, biosynthesis and accumulates cholesterol and cholestetyl esters in response to cholesterol feeding. The lipid accumulation seen in these animals is primarily distributed in and around the endothelial cells, the perivascular areas, the glomerular capillary loops and the mesangium. The administration of a cal-
245 cium antagonist appears to prevent the accumulation of cholesterol in these tissue sites in the absence of any specific decrease in the levels of serum cholesterol. Because of their ability to reduce tissue cholesterol levels, calcium antagonists may have a potential use in the treatment of kidney patients with renal microvascular lipid accumulation in whom their renal disease may contribute to the progression of systemic atherosclerosis. Further studies are needed to understand whether calcium antagonists could modify lipidassociated progressive glomerulosclerosis.
IO
II
I2
13 14
Acknowledgements 15
We wish to thank Ms. Sunita Gupta for her technical assistance in performing these studies. We also wish to thank Dr. Raymond B. Wuerker for his expertise in the examination of the histologic specimens. This work was support by the Department of Veterans Affairs and a grant from the American Heart Association, California Affiliate and the National Kidney Foundation of Southern California.
I6
17
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19
References 20 I Ross. R.. The pathogenesis of atherosclerosis, N. Engl. .I. Med., 314 (1986) 48X. 2 Chaudhari, A. and Kirschenbaum, M.A.. Effect of a 2% cholesterol diet on rabbit renal preglomerular microvascular PGI, and PGE, biosynthesis, Prostaglandin Leukotriene Essential Fatty Acids, 36 (1989) 81. 3 Schror. K., Prostaglandins, other eicosanoids and endothelial cells. Basic Res. Cardiol., 80 (1985) 502. 3 Hajjar. D.P.. Prostaglandins and cyclic nucleotides: modulators of arterial cholesterol metabolism, Biochem. Pharmacol., 34 (1985) 295. 5 Cannon, P.J.. Eicosanoids and the blood vessel wall, Circulation 70 (1984) 523. 6 Ilajjar. D.P.. Weksler. B.B., Falcone, D.J. et al., Prostacyclin modulates cholesteryl ester hydrolytic activity by its effect on cyclic adenosine monophosphate in rabbit aortic smooth muscle cells, J. Clin. Invest., 70 (1982) 479. 7 Hajjar. D.P.. Prostaglandins modulate arterial cholesteryl ester metabolism. Enzyme, 32 (1984) 218. X Mustard. J.F., Kinlough-Rathbone, R.L. and Packham, M.A.. Platelets, endothelium, and vessel injury. Adv. Prostaglandin Thromboxane Leukotriene Res., I3 (1985) 235. 4 Dembinska-Kiec. A.. Gtyglewska, T., Zmuda. A. and Gryglewsski, R.J.. The generation of prostacyclin by arteries
21
22
23
24
25
and by the coronary vascular bed is reduced in experimental atherosclerosis in rabbits, Prostaglandin, 14 (1977) 1025. Smith, W.L., Prostaglandin biosynthesis and its compartmentation in vascular smooth muscle and endothelial cells. Annu. Rev. Physiol., 48 (1986) 251. Henry, P.D. and Bentley. K.I., Suppression of atherogencsis in cholesterol-fed rabbit treated with nifedipine. J. Clin. Invest.. 68 (1981) 1366. Etingen, O.R. and Hajjar. D.P., Calcium channel blockers enhance cholesteryl ester hydrolysis and decrease total cholesterol accumulation in human aortic tissue, Circ. Res.. 66 (1990) 16.5. Henry, P.D.. Atherogenesis. calcium and calcium antagonists, Am. J. Cardiol.. 66 (lY90) 31. Parmley. W.W.. Vascular protection from atherosclerosis: potential of calcium antagonists, Am. J. Cardiol.. 66 (1990) 161. Chaudhari, A. and Kirschenbaum. M.A.. A rapid method for isolating rabbit renal microvessels. Am. J. Physiol.. 254 (10Xx) F29l. Chaudhari. A. and Kirschenbaum. M.A.. Altered glomerular eicosanoid biosynthesis in uranyl nitrate-induced acute renal failure, Biochim. Biophys. Acta. 792 (19X4) 135. Lowry. O.H.. Rosebrough, N.J., Farr. A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol.. Chem. 193 (1951) 265. Heider. J.G. and Boyett, R.L.. The picomole determination of free and total cholesterol in cells in culture. J. Lipid Res.. 19 (lY78) 514. Bligh. E.J. and Dyer, W.J.. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol., 37 (195’)) 911. Adams, C.W.M.. Bayliss, O.B. and Ibrahim, M.Z.M.. The distribution of lipids and enzymes in the aortic wall in dietary rabbit atheroma and human atherosclerosis. J. Pathol. Bacterial., 86 (1963) 421. Shore. V.G.. Shore. B. and Heart, R.C.. Changes in apolipoproteins and properties of rabbit very low density lipoproteins on induction of cholesterolemia. Biochemistry 13 (1974) 1579. Grodzinska, L., Basista. M.. Basista. E. et al.. Nitrendipine-stimulated release of prostacyclin-like substance in normal and atherosclerotic animals. Arzneimittelforschung, 37 (1987) 412. Etingen, O.R.. Hajjar, D.P.. Nifedipine increases cholesteryl ester hydrolytic activity in lipid-laden rabbit arterial smooth muscle cells. J. Clin. Invest.. 75 (1985) 1554. Loaldi. A., Polese, A.. Montorsi. P., Comparison of nifedipine, propranolol and isosorbide dinitrate on angiographic progression and regression of coronary arterial narrowing in angina pectoris. Am. J. Cardiol., 64 (1989) 433. Gottlieb, SO., Brinker, J.A. and Mellits, D., Effect of nifedipine on the development of coronary bypass graft stenoses in high-risk patients: a randomized, double-blind, placebo-controlled trial, Circulation. X0 (I 989) 11-223 (abstract).
246 26 Lichtlen, P.R., Hugenholtz, PG., Rafflenbeul, W., et al., Retardation of angiographic progression of coronary artery disease by nifedipine. Results of international nifedipine trial on anti-atherosclerotic therapy (INTACT), Lancet, 335 (1990) 1109.
27 Dworkin, L.D. and Benstein, J.A., Impact of antihypertensive therapy on progressive kidney damage, Am. J. Hypertens., 2 (1989) 1625.
