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Protective effect of l -arginine on hypercholesterolemia-enhanced renal ischemic injury
Atherosclerosis 143 (1999) 327 – 334
Protective effect of L-arginine on hypercholesterolemia-enhanced renal ischemic injury Silvia Bernardete Campos *, Maria Ori, Egı´dio Lima Do´rea, Antonio Carlos Seguro Laborato´rio de Pesquisa Ba´sica da Disciplina de Nefrologia da Faculdade de Medicina da Uni6ersidade de Sa˜o Paulo-LIM 12, A6 Ange´lica 2121 apto 12, CEP 01227 -200 -Higieno´polis, Sa˜o Paulo, Brazil Received 6 April 1998; received in revised form 12 November 1998; accepted 1 December 1998
Abstract The effects of hypercholesterolemia on ischemic renal failure were evaluated in rats subjected to 60 min of left renal artery clamping and contralateral nephrectomy. One group of rats (HC) was kept on a cholesterol-supplemented diet for 3 weeks before renal injury and compared to a group fed a regular diet (ND). Two days after renal ischemia, inulin clearance (Cin, ml/min per 100 g BW) was lower in HC-rats (0.03390.011) than in ND-rats (0.2279 0.037; PB 0.01). indicating that hypercholesterolemia potentiated renal ischemic injury. Twenty-one days after renal ischemia the Cin of HC-rats did not differ from ND-rats, suggesting that hypercholesterolemia did not limit late recovery. Since nitric oxide production is impaired in HC, L-arginine (50 mg/kg BW i.v.) was administered immediately after ischemia. Two days after ischemia, L-arg did not protect ND-rats from ischemia, while the Cin and renal blood flow were higher in L-arg-treated HC rats than in untreated HC rats (Cin = 0.1259 0.013 rats vs. 0.033 90.011; P B 0.001) (RBF = 3.9690.64 vs. 2.40 90.20 ml/min per 100 g BW; PB 0.05), indicating that L-arg protects HC rats from renal ischemia. The administration of D-arginine to ND rats induced a significant decrease of the Cin and a significant increase of FE H2O, FE Na and FE K compared to the L-arginine and not treated groups. Cultures of inner medullary collecting duct cells from ND rats were resistant to 24-h hypoxia. In contrast, IMCD cell cultures from HC rats showed higher LDH release after 24-h hypoxia than normoxic cells (69.2 93.4 vs. 30.9 93.6%, PB 0.001); 1 mM L-arg added to the medium attenuated LDH release (44.3 9 2.4%, P B0.01). These data demonstrate that HC predisposes renal tubular cells to hypoxic injury and L-arg protects cells of HC. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Hypercholesterolemia; Nitric oxide; L-arginine; Acute renal failure
1. Introduction Several studies have demonstrated that short-term experimental hypercholesterolemia caused by cholesterol feeding leads to spontaneous arterial constriction and/or increased reactivity to various vasoconstrictors, including renal vasoconstriction associated with a decrease in glomerular filtration rate in normal rats [1–3]. A potential clinical implication of these findings is that hypercholesterolemia can aggravate ischemic renal failure. Recent studies have shown that the synthesis, release or metabolism of nitric oxide (NO) is altered by * Corresponding author. Fax: +55-11-2802267.
hypercholesterolemia (HC), which could explain the impaired vasodilatation induced by acetylcholine under similar experimental conditions [4,5]. Not only these hemodynamic effects, but also alterations in membrane lipid composition which influence membrane fluidity, cation transport and Na + –K + -ATPase activity in all cells of the organism, can predispose renal tubular cells to the injury of hypoxia and may be an additional mechanism explaining a possible deleterious role of hypercholesterolemia in ischemic renal failure [6,7]. The purpose of the present investigation was to determine the possible effect of hypercholesterolemia on ischemic renal failure determined by clearance studies and by renal blood flow measurements in rats and in
0021-9150/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 2 1 - 9 1 5 0 ( 9 8 ) 0 0 3 1 9 - 0
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Table 1 Serum levels of total cholesterol and fractions and triglycerides in ND and HC ratsa Group
Cholesterol (mg/dl)
ND (n= 50.9 93.8 7) HC (n= 7) 163.4 925.5b
Cholesterol, HDL (mg/dl)
Cholesterol, LDL (mg/dl)
Triglyceride, (mg/dl)
Albumin(g/dl)
27.79 4.2
11.5 95.1
100 910
2.9 90.1
20.492.9
126.0 9 27.0b
63 9 9c
3.2 9 0.1
Results are expressed as means 9S.E.M. PB0.05 versus ND. c PB0.01 versus ND. a
b
Table 2 Renal function studies after 2 days of right nephrectomy in ND and HC ratsa Group
V (ml/min)
BW (g)
Cin (ml/min per 100 g BW)
FE H2O (%)
FE Na (%)
FE K (%)
ND (n = 5) HC (n= 5)
0.00790.001 0.011 90.003
282 910 264 99
0.3790.03 0.38 9 0.05
1.04 9 0.09 1.33 90.16
1.10 9 0.19 1.58 9 0.33
20.00 9 5.40 26.10 95.60
a Results are expressed as means 9 S.E.M. V, urine volume; BW, body weight; Cin, inulin clearance rate; FE H2O, fractional excretion of water; FE Na, fractional excretion of sodium; FE K, fractional excretion of potassium.
primary cultures of inner medullary collecting duct (IMCD) cells of rats subjected to hypoxia in vitro. The role of nitric oxide was evaluated by the intravenous administration of L-arginine, the precursor of nitric oxide synthesis, to the intact animal and by its addition to the culture medium.
2. Methods Male Wistar rats (180 – 250 g) were maintained in individual metabolic cages with free access to tap water and fed a regular rat diet or a pellet diet supplemented with 4% cholesterol and 1% cholic acid for 3 weeks. The composition of the two diets was exactly the same except for cholesterol and cholic acid. At the end of the third week, the animals were anesthetized and submitted to a suprapubic incision. Ischemic renal failure was induced by clamping the left renal artery followed by the removal of the right kidney. After 60 min, the clamp was removed. In another five animals of the normal diet (ND) group and cholesterol-supplemented diet (HC) group right kidney was removed, but the left renal artery was not clamped. The animals were allowed to recover, were returned to their metabolic cages and kept on their original diets. Clearance studies were performed in both ND and HC rats on the 2nd and 21st days following renal ischemia. Rats were anesthetized with sodium thionembutal (50 mg/kg BW, i.p.). The trachea was cannulated with a PE-240 catheter and the animals were maintained under conditions of spontaneous breathing. The right femoral artery was catheterized with a PE-60 catheter to control mean arterial pressure and to allow blood sampling. The urinary bladder was cannulated
with a PE-240 catheter by a suprapubic incision in order to collect urine samples. After completion of the surgical procedure, a loading dose of inulin (100 mg/kg BW diluted in 1 ml of 0.9% saline) was administered through the jugular vein, followed by a constant infusion of inulin (10 mg/kg BW in 0.9% saline) at 0.04 ml/min throughout the experiment. Three urine samples were collected at 30-min intervals and blood samples were obtained at the start, beginning, in the middle and at the end of the collection periods. In order to study the potential protective effect of nitric oxide in ischemic renal failure, a group of ND and HC rats was injected with L-arginine (50 mg/kg BW) and another group was injected with D-arginine (50 mg/kg BW), an inert isomer that is not a substrate for NO synthesis, through the femoral vein immediately after the induction of ischemic renal failure. Inulin concentrations were determined by the anthrone method (inulin clearance), and sodium and potassium were measured with a flame spectrophotometer (Instrumentation Lab). Total cholesterol was determined by the enzymatic method of Abbell-Kendal, modified. HDL cholesterol by the phosphotungstate precipitation method, triglycerides by the colorimetric enzymatic method, and albumin by the bromocresol green method.
2.1. Measurements of renal blood flow Measurements of RBF were also made. Briefly, rats weighing 150–250 g were fed a regular diet or a cholesterol-supplemented diet as described previously. At the end of the third week, the animals were submitted to ischemic renal failure induced by clamping the left renal artery for 60 min followed by the removal of the right kidney. With the aim to study the role of nitric oxide,
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Table 3 Renal function studies after 2 and 21 days of ischemia in ND and HC ratsa Group
BW (g)
V (ml/min)
Cin (ml/min per 100 g BW)
FE H2O (%)
FE Na (%)
FE K (%)
ND, 2 days (n =11) ND, 21 days (n = 5) HC, 2 days (n = 8) HC, 21 days (n = 5)
242 9 10 282910 214 9 12 264 9 9
0.0089 0.001 0.0079 0.001 0.006 90.001 0.0119 0.003
0.2279 0.037 0.340 9 0.080 0.033b 90.011 0.360e 90.040
2.08 90.24 1.06d 90.21 19.26b 96.23 1.17d 90.08
1.84 90.26 0.92d 90.20 17.16c 94.24 1.92d 91.10
28.4 95.8 46.5d 917.0 172.8c 936.8 36.6d 917.5
Results are expressed as means 9 S.E.M. BW, body weight; V, urine volume; Cin, inulin clearance rate; FE H2O, fractional excretion of water; FE Na, fractional excretion of sodium; FE K, fractional excretion of potassium. b PB0.001, ND vs. HC. c PB0.01, ND vs. HC. d PB0.05, 2 days vs. 21 days. e PB0,001, 2 days vs. 21 days. a
Table 4 Renal function studies after 2 days of ischemia in ND and HC rats, treated with L-arginine and
D-argininea
Group
BW (g)
V (ml/min)
Cin (ml/min per 100 g BW)
FE H2O (%)
FE Na (%)
FE K (%)
ND (n = 11) ND+L-arg (n = 9) ND+D-arg (n = 5) HC (n=8) HC+L-arg (n =7) HC+D-arg (n = 6)
242 9 10 235 9 13 230 915 214 9 12 215 99 23199
0.0089 0.001 0.0279 0.009 0.02190.008 0.0069 0.001 0.0189 0.004e 0.01390.003
0.2279 0.037 0.1239 0.027b 0.0969 0.048b 0.0339 0.011 0.1259 0.013f 0.03190.011g
2.08 90.24 11.48 93.70 3.74 914.02b,c 19.26 96.23 6.65 9 1.03e 26.91 95.34h
1.84 90.26 6.50 91.49 13.30 94.94b,c 17.16 94.24 5.23 9 0.88e 17.62 94.11
28.4 95.8 65.7 922.5 239.5 968.1b,d 172.8 936.8 57.9 9 14.6e 124.5 923.9
a Results are expressed as means 9 S.E.M. BW, body weight; V, urine volume; Cin, inulin clearance rate; FE H2O, fractional excretion of water; FE Na, fractional excretion of sodium; FE K, fractional excretion of potassium. b PB0.01 vs. ND. c PB0.05 L-arg vs. D-arg. d PB0.01 L-arg vs. D-arg. e PB0.05 vs. HC. f PB0.001 vs. HC. g PB0.001 L-arg vs. D-arg. h PB0.05 L-arg vs. D-arg.
a group of ND and HC rats received L-arginine (50 mg/kg BW) by the femoral vein immediately after the induction of ischemia. Another group of ND and HC rats received D-arginine (50 mg/kg BW). After 48 h, the rats were anesthetized with sodium thionembutal (50 mg/kg BW, i.p.) and the trachea was cannulated with a PE-240 catheter and a catheter (PE 60) was placed in the left femoral artery. By way of a median incision, the left renal pedicle was carefully dissected and the renal artery was isolated with care to avoid disturbing the renal nerves. An electromagnetic flow probe (Transonic Systems, Bethesda, MD) was placed around the exposed renal artery, and RBF was measured with an electromagnetic flowmeter (Transonic Systems, T 106 XM).
0.2% collagenase for 20 min at 37°C. Fetal calf serum (Laborclin) was added to neutralize collagenase and the cell suspension was centrifuged at 1500 rpm for 5 min. The pellet was placed in bottles containing RPMI 1640 supplemented with T3 (6× 10 − l2 M), T4 (6 ×10 − 8 M), 2 mg/ml transferrin, 1.9× 10 − 11 M prolactin, 0.66 mU/ ml insulin, 10 − 8 M dexamethasone, 100 mU/ml penicillin, 100 mg/ml streptomycin and 2.5 mg/ml amphotericin. Culture medium was changed every day and cells were studied 6 days after the initial plating. Cells were exposed to hypoxia by bubbling the medium with 100% N2 for 60 min and maintained at low P O2 for the subsequent 23 h. In a group of cells, L-arginine or D-arginine was added to the culture medium on the day of hypoxia. Cell injury was evaluated by lactate dehydrogenase (LDH) release.
2.2. IMCD culture 2.3. LDH release measurement Immediately after weaning, male Wistar rats were provided with a regular or HC diet for 3 weeks. IMCD segments were isolated from the renal papilla by microdissection with a sharpened Dumond forceps no. 5 in PBS solution and the fragments were digested with
After 24 h of incubation, the supernatant was separated for LDH determination in medium containing 100 ml sodium pyruvate, 100 ml Tris/EDTA, 100 ml NADH, 400 ml deionized water and 300 ml supernatant.
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Cells were washed three times with PBS and 1 ml of ATV (0.02% versene and 0.2% trypsin) was added. After 15 min, the preparation was centrifuged at 1000 rpm for 5 min. The pellet was lysed with 1.5% triton X-100 and LDH activity was determined in medium containing 100 ml sodium pyruvate, 100 ml Tris/EDTA, 100 ml NADH, 650 ml deionized water and 50 ml pellet. LDH activity was also determined in the ATV supernatant in medium containing 100 ml sodium pyruvate, 100 ml Tris/EDTA, 100 ml NADH, 600 ml deionized water and 100 ml of ATV supernatant. LDH release was converted to percent release by dividing supernatant activity by total activity according to Bergmeyer [8,9].
2.4. Statistical analysis All values are reported as means 9S.E.M. Data were analyzed by ANOVA test and the Student Newman Keuls multiple comparison post-test, with the level of significance set at P B0.05.
3. Results Rats fed a regular or cholesterol-supplemented diet for 3 weeks did not show a significant difference in weight gain. Measurements of total serum, HDL and LDL cholesterol and triglycerides are shown in Table 1. The values did not differ from those obtained before the study. After 21 days, rats fed the HC showed a significant increase in total serum cholesterol levels compared to ND rats (163.4 9 25.5 vs. 50.9 9 3.8 mg/ dl, PB0.05). HDL levels did not differ between groups, whereas the LDL level was significantly higher in the HC group than in ND rats (126.0927.0 vs. 11.59 5.1 mg/dl, PB 0.05). The HC rats showed significant, lower serum triglyceride levels compared with ND rats (639 9 vs. 100910 mg/dl, P B0.05). Serum albumin levels did not differ between groups. Renal function studies after 2 days of right nephrectomy showed that inulin clearance did not differ between ND and HC rats (0.37 90.03 and 0.38 90.05
ml/min per 100 g BW). Also, fractional excretion of water (ND, 1.04 9 0.09%; HC, 1.33 9 0.16%, NS), sodium (ND, 1.109 0.19%; HC, 1.589 0.33%, NS) and potassium (ND, 20.009 5.40%; HC, 26.1095.60%, NS) did not differ between groups (Table 2).
3.1. Effect of hypercholesterolemia on ischemic renal failure Hypercholesterolemic rats showed a significant decrease in inulin clearance compared to control rats after 2 days of 60-min left renal ischemia and contralateral nephrectomy (0.0339 0.011 vs. 0.2279 0.037 ml/min per 100 g BW, P B0.001). Hypercholesterolemia also induced an impairment in tubular function compared with ND rats, as shown by the high fractional excretion of water (19.26 9 6.23 vs. 2.08 9 0.24%, PB0.001), sodium (17.169 4.24 vs. 1.8490.26%, PB 0.01) and potassium (172.89 36.8 vs. 28.4 9 5.8%, PB0.01) (Table 3). Recovery from renal ischemia was studied after 21 days. The inulin clearance of the HC group did not differ from that of ND rats (0.360 90.040 vs. 0.340 9 0.080 ml/min per 100 g BW). The tubular parameters studied also did not show significant differences between groups in terms of the fractional excretion of water (HC, 1.179 0.08; ND, 1.0690.21%), sodium (HC, 1.929 1.10; ND, 0.929 0.20%) and potassium (HC, 36.69 17.5; ND, 46.59 17.0%) (Table 3).
3.2. Effect of L -arginine and ischemic renal failure
D -arginine
infusion on
The response of post-ischemic kidneys to L-arginine and D-arginine is depicted in Table 4. In HC rats, L-arginine produced a lower decrease in inulin clearance 2 days after ischemia (0.125 9 0.013 ml/min per 100 g BW) as compared with HC rats which did not receive L-arginine (0.0339 0.011 ml/min per 100 g BW, PB0.001) and D-arginine group (0.031 90.011 ml/min per 100 g BW, PB0.001). A significant protection of tubular function was observed in hypercholesterolemic
Fig. 1. Renal blood flow (RBF) after 2 days of 60-min ischemia in rats fed a normal (ND) and cholesterol supplemented diet (HC).
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Fig. 2. Percentage of lactate dehydrogenase (LDH) released from inner medullary collecting duct (IMCD) cell cultures of rats kept on a normal diet (ND) after 24 h of normoxia, hypoxia or hypoxia plus L-arginine (1 mM).
rats treated with L-arginine since fractional excretion of water, sodium and potassium was lower than in the untreated group (FE H2O: 6.6591.03 vs. 19.269 6.23%, P B 0.001; FE Na: 5.23 9 0.88 vs. 17.16 9 4.24%, P B 0.001; FE K: 57.9914.6 vs. 172.8 936.8%, PB 0.001). This protection was not observed with the D-arginine-treated rats. In the ND group, the effect of L-arginine administration was opposite to that observed in HC rats. Inulin clearance was lower, even though not significantly in L-arginine-treated rats than in the group that did not receive L-arginine (0.1239 0.027 vs. 0.2279 0.037 ml/min per 100 g BW, NS). The fractional excretion of water and sodium was significantly higher in these rats compared with the untreated group (FE H2O: 11.4893.70 vs. 2.0890.24%, P B 0.001; FE Na: 6.509 1.49 vs. 1.8490.26%, P B 0.001). The fractional excretion of potassium did not differ between groups. The administration of D-arginine to ND rats also caused a significant decrease of the Cin (0.0969 0.048 vs. 0.22790.037 ml/min per 100 g BW, PB 0.01). The tubular injury, assessed by a significant increase of FE H2O, FE Na and FE K, was greater in this group than in the L-arginine and not treated rats.
3.3. Measurement of renal blood flow (RBF) The RBF of ND and HC rats did not differ after 2 days of ischemia (2.30 90.39 vs. 2.40 9 0.20 ml/min per 100 g, NS). Treatment with L-arginine caused vasodilation only in the HC group after 2 days of ischemia (3.96 90.64 vs.2.409 0.20 ml/min per 100 g, P B0.05) (Fig. 1). A vasodilatory effect was not observed with the D-arginine administration.
3.4. Inner medullary collecting duct cell culture IMCD cell cultures were resistant to 24-h hypoxia in vitro, as demonstrated by LDH release which did not
differ from that observed in normoxic cells (hypoxic: 22.29 3.6% and normoxic: 21.49 0.8%). L-Arginine (1 mM) added to the culture medium had no effect on LDH release by hypoxic cells (19.69 3.7%). In contrast, IMCD cell cultures from HC rats submitted to 24-h hypoxia presented higher LDH release than normoxic cells (69.293.4 vs. 30.99 3.6%; PB 0.001) while the addition of 1 mM L-arginine to the medium of hypoxic cells attenuated LDH release (44.392.4 vs. 69.293.4%, PB 0.01) (Figs. 2 and 3). The addition of D-arginine to the culture medium did not have any protective effect (Fig. 4).
4. Discussion Recent evidence has shown that short-term induced hypercholesterolemia and hyperlipemia can promote vasoconstriction and/or increased vascular reactivity [1–3]. In vitro studies demonstrated that LDL cholesterol inhibits endothelium-dependent relaxation, suggesting that LDL might be responsible for the vasoconstriction that occurs with hypercholesterolemia. These studies suggest that these vascular effects are mainly functional in nature, since the duration of the experimental hypercholesterolemia was too short for gross pathologic lesions to develop in the blood vessels. Factors responsible for it could be decreased production of prostacyclin, increased production of thromboxane and impaired synthesis, release, metabolism and action of nitric oxide [4,10,11]. Nitric oxide is extremely important in the pathogenesis of this vasoconstriction and several studies on animals and humans have demonstrated that infusion of L-arginine could prevent the vasoconstriction induced by hypercholesterolemia [5,12]. The role of hypercholesterolemia in renal function has been studied by Kaplan et al. [10] who demon-
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Fig. 3. Percentage of lactate dehydrogenase (LDH) released from inner medullary collecting duct (IMCD) cell cultures of rats kept on a cholesterol supplemented diet (HC) after 24 h of normoxia, hypoxia and hypoxia plus L-arginine (1 mM).
strated that short-term HC in rats resulted in marked vasoconstriction of renal blood vessels with a consequent fall in GFR attributed to the reduction in QA and probably in part to a fall in Kf, as evidenced by micropuncture measurements. These results have important clinical implications since patients with increased cholesterol levels could develop more serious acute renal failure when submitted to renal ischemia or nephrotoxic drugs. In the present study, we used a well-known model of post-ischemic acute renal failure induced by 60 min of left renal artery clamping and contralateral nephrectomy, which allows a fixed time of ischemia. We clearly demonstrated that HC rats had a more important reduction in renal function compared with the ND group, studied 2 days after ischemia, as assessed by the lower inulin clearance as well as by the higher fractional sodium, potassium and water excretion, demonstrating that hypercholesterolemia potentiated renal ischemic damage. To examine the direct effect of hypercholesterolemia on hypoxic tubular cells, we studied cultures of IMCD cells submitted to 24-h hypoxia and demonstrated that cultures of IMCD cells are resistant to hypoxia as assessed by LDH release. However, when IMCD cells from HC rats were submitted to the same level of hypoxia there was an increase in LDH release, indicating that hypercholesterolemia sensitized these cells to hypoxia. It is well established that lipid membranes are in dynamic equilibrium with plasma lipids. Hypercholesterolemia can induce alterations in membrane composition, chemical properties and ion transport and permeability. An increase in intracellular calcium content, a recognized mediator of ischemic lesion, had been described [13,14], perhaps due to its increased transport by calcium receptor operated channels (ROC) [15], as well as inhibition of Na + – K + -ATPase [6,7] and Ca2 + ATPase activities.
Another possible factor involved in the great susceptibility to hypoxia of IMCD cells from HC rats is LDL cholesterol, which when oxidized could lead to production of deleterious oxygen radicals [16,17]. It is interesting to observe that after 3 weeks of recovery, the renal function of the HC group was similar to that of the ND group, as assessed by measurements of glomerular filtration and fractional sodium, potassium and water excretion. The importance of lipids in compensatory renal growth is recognized. Some studies have demonstrated that the phospholipid content of the hypertrophic kidney is increased [18]. It is known that amino acid supplementation enhances renal phospholipid biosynthesis after acute tubular necrosis increasing regeneration of renal tubular cells [19]. The present results demonstrate that hypercholesterolemia did not limit late recovery from renal ischemia. Several studies have shown the role of nitric oxide in ischemic acute renal failure. The results are controversial depending on the model studied. Schramm et al. [20] demonstrated that rats treated with L-arginine (300 mg/kg BW) immediately after 40-min bilateral renal ischemia showed improvement of GFR for 180 min after ischemia. This effect is stereospecific, since it is not observed with the D-arginine isomer. The protection afforded by NO might be explained by its vasodilatory effect counteracting vasoconstrictors such as angiotensin, catecholamins and endothelin. Another effect is the action on mesangial cells, causing an increase of Kf. Nevertheless, a study using proximal tubule suspensions exposed to 15-min hypoxia and 35-min reoxygenation demonstrated that L-arginine as well as sodium nitroprusside (NO donor) enhanced the hypoxia/reperfusion injury. Previous treatment with LNAME prevented the tubular injury, as did hemoglobin, indicating that NO is a possible mediator
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Fig. 4. Percentage of lactate dehydrogenase (LDH) released from inner medullary collecting duct (IMCD) cell cultures of rats kept on a cholesterol supplemented diet (HC) after 24 h of normoxia, hypoxia and hypoxia plus D-arginine (1 mM).
of hypoxia/reperfusion injury. This effect is attributed to the increased production of peroxynitrite radical in this situation [21].Yu [22] suggested that rats supplemented with L-arginine for 1 week showed a greater decrease in renal function, perhaps due to an excess production of NO and its potent reactive oxygen species, peroxynitrite radical. In the present study, the acute infusion of L-arginine to the ND group did not protect against the ischemic insult. This is in agreement with the results reported by Lopez-Neblina et al. [23] in a model of 75-min ischemia and contralateral nephrectomy in rats. It is well-known that remnant kidneys become hypertrophic and increase the synthesis of Larginine per nephron. Alternatively, other sources of production of L-arginine, such as liver and brain, may enhance their synthesis of this amino acid in this remnant kidney model, maintaining an adequate level of this amino acid. Thus, it is possible that in this model, the administration of L-arginine increased the production of NO and peroxynitrite radical. The increased fractional excretion of sodium and water observed in ND group treated with L-arginine could be due to proximal tubular damage [21,22]. The administration of L-arginine to HC protected the animals against the ischemic injury as assessed by inulin clearance, which was higher than the value observed in untreated HC rats. The mechanism of protection observed in the present study possibly was vasorelaxation since supplementation with L-arginine increased the RBF, possibly by the production of NO, since it was not observed with the inert isomer D-arginine which is impaired in a hypercholesterolemic environment. In addition, a direct protective effect was also demonstrated since the injury caused by hypoxia in IMCD cells from hypercholesterolemic rats was prevented by the addition of L-arginine to the culture medium, but
not by the addition of D-arginine. This protection may have been related to a reduction in the transport functions of epithelial renal tubular cells. It is recognized that NO inhibits Na + –K + -ATPase and sodium transport reducing oxygen consumption by these cells and diminishing their susceptibility to hypoxic injury [24,25]. Another possibility is that there may be some endogenous inhibitor of NO synthase that may be displaced by exogenous L-arginine. According to this hypothesis, a recent study showed that cholesterol feeding increases plasma concentrations of asymmetrical dimethylarginine (ADMA), an endogenous NO synthesis inhibitor in the rabbit [26]. ADMA may competitively inhibit NO synthase and/or L-arginine uptake into cells. These findings at least partly explain the protective effect of the higher extracellular concentration of L-arginine in hypoxic injury in IMCD cells from hypercholesterolemic rats. Rats treated with D-arginine showed a significant increase in FE H2O, FE Na and FE K. Andrade et al. [27] had similar results in a model of radiocontrast nephrotoxicity. These results suggest an aggravation of tubular injury, perhaps by an amino acid-induced effect. This was not observed with Larginine, since it was metabolized into nitric oxide. In conclusion, our results suggest that hypercholesterolemia is an aggravating factor of postischemic acute renal failure, partially mediated by a direct effect on epithelial tubular cells, which become more susceptible to ischemic injury. The recovery was similar in both groups despite the more severe insufficiency of the hypercholesterolemic group, which implies a beneficial role of cholesterol during the phase of recovery from acute renal failure. The acute infusion of L-arginine protected only the HC group and part of this protection was due to the vasodilatory renal effect and part to a direct effect on tubular cells.
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Acknowledgements These studies were supported by the Instituto dos Laborato´rios de Investigac¸a˜o Me´dica HC/FMUSP/ LIM-12, Fundac¸a˜o Faculdade de Medicina, Conselho Nacional de Pesquisa (CNPq), Fundac¸a˜o Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES), Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo and by Universiade Federal de Sa˜o Paulo (UNIFESP).
References [1] Casino PR, Kilcoyne CM, Quyyumi AA, Hoeg JM, Panza JA. The role of nitric oxide in endothelium-dependent vasodilation of hypercholesterolemic patients. Circulation 1993;88:2541 – 7. [2] Chowienczyk PJ, Watts GF, Cockcroft JR, Ritter JM. Impaired endothelium-dependent vasodilatation of forearm resistance vessels in hypercholesterolemia. Lancet 1992;340:1430–2. [3] Rossitch E Jr, Alexander E III, Black P M, Cooke JP. LArginine normalizes endothelial function in cerebral vessels from hypercholesterolemic rabbits. J Clin Invest 1991;87:1295– 9. [4] O’Hara Y, Peterson TE, Harrison DG. Hypercholesterolemia increases endothelial superoxide anion production. J Clin Invest 1993;91:2546 – 51. [5] Creager MA, Cooke JP, Mendelsohn ME, Gallagher SJ, Coleman SM, Loscalzo J, Dzau VJ. Impaired vasodilation of forearm resistance vessels in hypercholesterolemic humans. J Clin Invest 1990;86:228 – 34. [6] Lijnen P, Celis H, Fagard R, Staessen J, Amery A. Influence of cholesterol lowering on plasma membrane lipids and cationic transport systems. J Hypertens 1994;12:59–64. [7] Broderick R, Bialecki R, Tulenko TN. Cholesterol-induced changes in rabbit arterial smooth muscle sensitivity to adrenergic stimulation. Am J Physiol 1989;257(Heart Circ Physiol 26):170 – 8. [8] Bergmeyer HV. Methods in Enzymatic Analysis, 2nd edn. New York: Academic Press, 1974:574–89. [9] Beutler E. Lactate dehydrogenase (LD 12). In: Beutler E, editor. Red Cell Metabolism; A Manual of Biochemical Methods, 3nd edn. New York: Grune & Stratton, 1984:65–6. [10] Kaplan R, Aynedjian HS, Schiondorff D, Bank N. Renal vasoconstriction caused by short-term cholesterol feeding is corrected by thromboxane antagonist or probucol. J Clin Invest 1990;86:1707 – 14.
.
[11] Bank N, Aynedjian H. Role of thromboxane in impaired renal vasodilatation response to acetylcholine in hypercholesterolemic rats. J Clin Invest 1992;89:1636 – 42. [12] Cooke JP, Tsao P. Cytoprotective effects of nitric oxide. Circulation 1993;88(5, part 1):2451 – 4. [13] Bonventre JV. Mechanism of ischemic acute renal failure. Kidney Int 1993;43:1160 – 78. [14] Molitoris BA. Cellular basis of ischemic acute renal failure. In: Lazarus JM, Brenner BM, editors. Acute Renal Failure. Philadelphia, PA: Saunders, 1993:1 – 32. [15] Bialecki RA, Tulenko TN, Colucci W. Cholesterol enrichment increases basal and agonist-stimulated calcium influx in rat vascular smooth muscle cells. J Clin Invest 1991;88:1894 –900. [16] Peer G, Blum M, Iaina A. Nitric oxide and acute renal failure. Nephron 1996;73:375 – 81. [17] Keaney JF Jr, Gaziano JM, Xu A, Frei B, Curran-Celentano J, Shwaery GT, Loscalzo J, Vita JA. Dietary antioxidants preserve endothelium-dependent vessel relaxation in cholesterol-fed rabbits. Proc Natl Acad Sci USA 1993;90:11880– 4. [18] Bucher LR, Malt RA. Regeneration of liver and kidney. Foreword by Ingelfinger FJ, editor. New Engl J Med 1971:1–278. [19] Toback FG. Amino acid enhancement of renal regeneration after acute tubular necrosis. Kidney Int 1977;12:193 – 8. [20] Schramm L, Eidbreder E, Schmitt A, Kartenbender K, Zimmermann J, Ling H, Hdeidlan A. Role of L-arginine-derived NO in ischemic acute renal failure in the rat. Renal Fail 1994;16(5):555– 69. [21] Yu L, Gengaro PE, Niederberger M, Burke TJ, Shultz P, Schrier RW. Nitric oxide: a mediator in rat tubular hypoxia/reoxygenation injury. Proc Natl Acad Sci USA 1994;91:1691 – 5. [22] Yu L. Nitric oxide in acute renal failure: Foe or friend? Kidney Int 1997;52(Suppl 61):S39 – 40. [23] Lo´pez-Neblina F, Paez AJ, Toledo AH, Toledo-Pereyra LH. Role of nitric oxide in ischemia/reperfusion of the rat kidney. Circ Shock 1995;44:91 – 5. [24] Reyes AA, Karl IE, Klahr S. Role of arginine in health and in renal disease. Am J Physiol 1994;267:F331– 46. [25] Stoos BA, Carretero AO, Fahry RD, Scicli G, Garvin JL. Endothelium-derived relaxing factor inhibits transport and increases cGMP content in cultured mouse cortical collecting duct cells. J Clin Invest 1992;89:761 – 5. [26] Boger RH, Bode-Boger SM, Brandes RP, Phivthong-ngam L, Bohme M, Nafe R, Mugge A, Frolich JC. Dietary L-arginine reduces the progression of atherosclerosis in cholesterol-fed rabbits: comparison with lovastatin. Circulation 1997;96:1282–90. [27] Andrade LC, Campos SB, Seguro AC. Hypercholesterolemia aggravates radiocontrast nephrotoxicity: protective role of Larginine. Kidney Int 1998;53:1736 – 42.
Protective effect of L-arginine on hypercholesterolemia-enhanced renal ischemic injury Silvia Bernardete Campos *, Maria Ori, Egı´dio Lima Do´rea, Antonio Carlos Seguro Laborato´rio de Pesquisa Ba´sica da Disciplina de Nefrologia da Faculdade de Medicina da Uni6ersidade de Sa˜o Paulo-LIM 12, A6 Ange´lica 2121 apto 12, CEP 01227 -200 -Higieno´polis, Sa˜o Paulo, Brazil Received 6 April 1998; received in revised form 12 November 1998; accepted 1 December 1998
Abstract The effects of hypercholesterolemia on ischemic renal failure were evaluated in rats subjected to 60 min of left renal artery clamping and contralateral nephrectomy. One group of rats (HC) was kept on a cholesterol-supplemented diet for 3 weeks before renal injury and compared to a group fed a regular diet (ND). Two days after renal ischemia, inulin clearance (Cin, ml/min per 100 g BW) was lower in HC-rats (0.03390.011) than in ND-rats (0.2279 0.037; PB 0.01). indicating that hypercholesterolemia potentiated renal ischemic injury. Twenty-one days after renal ischemia the Cin of HC-rats did not differ from ND-rats, suggesting that hypercholesterolemia did not limit late recovery. Since nitric oxide production is impaired in HC, L-arginine (50 mg/kg BW i.v.) was administered immediately after ischemia. Two days after ischemia, L-arg did not protect ND-rats from ischemia, while the Cin and renal blood flow were higher in L-arg-treated HC rats than in untreated HC rats (Cin = 0.1259 0.013 rats vs. 0.033 90.011; P B 0.001) (RBF = 3.9690.64 vs. 2.40 90.20 ml/min per 100 g BW; PB 0.05), indicating that L-arg protects HC rats from renal ischemia. The administration of D-arginine to ND rats induced a significant decrease of the Cin and a significant increase of FE H2O, FE Na and FE K compared to the L-arginine and not treated groups. Cultures of inner medullary collecting duct cells from ND rats were resistant to 24-h hypoxia. In contrast, IMCD cell cultures from HC rats showed higher LDH release after 24-h hypoxia than normoxic cells (69.2 93.4 vs. 30.9 93.6%, PB 0.001); 1 mM L-arg added to the medium attenuated LDH release (44.3 9 2.4%, P B0.01). These data demonstrate that HC predisposes renal tubular cells to hypoxic injury and L-arg protects cells of HC. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Hypercholesterolemia; Nitric oxide; L-arginine; Acute renal failure
1. Introduction Several studies have demonstrated that short-term experimental hypercholesterolemia caused by cholesterol feeding leads to spontaneous arterial constriction and/or increased reactivity to various vasoconstrictors, including renal vasoconstriction associated with a decrease in glomerular filtration rate in normal rats [1–3]. A potential clinical implication of these findings is that hypercholesterolemia can aggravate ischemic renal failure. Recent studies have shown that the synthesis, release or metabolism of nitric oxide (NO) is altered by * Corresponding author. Fax: +55-11-2802267.
hypercholesterolemia (HC), which could explain the impaired vasodilatation induced by acetylcholine under similar experimental conditions [4,5]. Not only these hemodynamic effects, but also alterations in membrane lipid composition which influence membrane fluidity, cation transport and Na + –K + -ATPase activity in all cells of the organism, can predispose renal tubular cells to the injury of hypoxia and may be an additional mechanism explaining a possible deleterious role of hypercholesterolemia in ischemic renal failure [6,7]. The purpose of the present investigation was to determine the possible effect of hypercholesterolemia on ischemic renal failure determined by clearance studies and by renal blood flow measurements in rats and in
0021-9150/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 2 1 - 9 1 5 0 ( 9 8 ) 0 0 3 1 9 - 0
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Table 1 Serum levels of total cholesterol and fractions and triglycerides in ND and HC ratsa Group
Cholesterol (mg/dl)
ND (n= 50.9 93.8 7) HC (n= 7) 163.4 925.5b
Cholesterol, HDL (mg/dl)
Cholesterol, LDL (mg/dl)
Triglyceride, (mg/dl)
Albumin(g/dl)
27.79 4.2
11.5 95.1
100 910
2.9 90.1
20.492.9
126.0 9 27.0b
63 9 9c
3.2 9 0.1
Results are expressed as means 9S.E.M. PB0.05 versus ND. c PB0.01 versus ND. a
b
Table 2 Renal function studies after 2 days of right nephrectomy in ND and HC ratsa Group
V (ml/min)
BW (g)
Cin (ml/min per 100 g BW)
FE H2O (%)
FE Na (%)
FE K (%)
ND (n = 5) HC (n= 5)
0.00790.001 0.011 90.003
282 910 264 99
0.3790.03 0.38 9 0.05
1.04 9 0.09 1.33 90.16
1.10 9 0.19 1.58 9 0.33
20.00 9 5.40 26.10 95.60
a Results are expressed as means 9 S.E.M. V, urine volume; BW, body weight; Cin, inulin clearance rate; FE H2O, fractional excretion of water; FE Na, fractional excretion of sodium; FE K, fractional excretion of potassium.
primary cultures of inner medullary collecting duct (IMCD) cells of rats subjected to hypoxia in vitro. The role of nitric oxide was evaluated by the intravenous administration of L-arginine, the precursor of nitric oxide synthesis, to the intact animal and by its addition to the culture medium.
2. Methods Male Wistar rats (180 – 250 g) were maintained in individual metabolic cages with free access to tap water and fed a regular rat diet or a pellet diet supplemented with 4% cholesterol and 1% cholic acid for 3 weeks. The composition of the two diets was exactly the same except for cholesterol and cholic acid. At the end of the third week, the animals were anesthetized and submitted to a suprapubic incision. Ischemic renal failure was induced by clamping the left renal artery followed by the removal of the right kidney. After 60 min, the clamp was removed. In another five animals of the normal diet (ND) group and cholesterol-supplemented diet (HC) group right kidney was removed, but the left renal artery was not clamped. The animals were allowed to recover, were returned to their metabolic cages and kept on their original diets. Clearance studies were performed in both ND and HC rats on the 2nd and 21st days following renal ischemia. Rats were anesthetized with sodium thionembutal (50 mg/kg BW, i.p.). The trachea was cannulated with a PE-240 catheter and the animals were maintained under conditions of spontaneous breathing. The right femoral artery was catheterized with a PE-60 catheter to control mean arterial pressure and to allow blood sampling. The urinary bladder was cannulated
with a PE-240 catheter by a suprapubic incision in order to collect urine samples. After completion of the surgical procedure, a loading dose of inulin (100 mg/kg BW diluted in 1 ml of 0.9% saline) was administered through the jugular vein, followed by a constant infusion of inulin (10 mg/kg BW in 0.9% saline) at 0.04 ml/min throughout the experiment. Three urine samples were collected at 30-min intervals and blood samples were obtained at the start, beginning, in the middle and at the end of the collection periods. In order to study the potential protective effect of nitric oxide in ischemic renal failure, a group of ND and HC rats was injected with L-arginine (50 mg/kg BW) and another group was injected with D-arginine (50 mg/kg BW), an inert isomer that is not a substrate for NO synthesis, through the femoral vein immediately after the induction of ischemic renal failure. Inulin concentrations were determined by the anthrone method (inulin clearance), and sodium and potassium were measured with a flame spectrophotometer (Instrumentation Lab). Total cholesterol was determined by the enzymatic method of Abbell-Kendal, modified. HDL cholesterol by the phosphotungstate precipitation method, triglycerides by the colorimetric enzymatic method, and albumin by the bromocresol green method.
2.1. Measurements of renal blood flow Measurements of RBF were also made. Briefly, rats weighing 150–250 g were fed a regular diet or a cholesterol-supplemented diet as described previously. At the end of the third week, the animals were submitted to ischemic renal failure induced by clamping the left renal artery for 60 min followed by the removal of the right kidney. With the aim to study the role of nitric oxide,
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Table 3 Renal function studies after 2 and 21 days of ischemia in ND and HC ratsa Group
BW (g)
V (ml/min)
Cin (ml/min per 100 g BW)
FE H2O (%)
FE Na (%)
FE K (%)
ND, 2 days (n =11) ND, 21 days (n = 5) HC, 2 days (n = 8) HC, 21 days (n = 5)
242 9 10 282910 214 9 12 264 9 9
0.0089 0.001 0.0079 0.001 0.006 90.001 0.0119 0.003
0.2279 0.037 0.340 9 0.080 0.033b 90.011 0.360e 90.040
2.08 90.24 1.06d 90.21 19.26b 96.23 1.17d 90.08
1.84 90.26 0.92d 90.20 17.16c 94.24 1.92d 91.10
28.4 95.8 46.5d 917.0 172.8c 936.8 36.6d 917.5
Results are expressed as means 9 S.E.M. BW, body weight; V, urine volume; Cin, inulin clearance rate; FE H2O, fractional excretion of water; FE Na, fractional excretion of sodium; FE K, fractional excretion of potassium. b PB0.001, ND vs. HC. c PB0.01, ND vs. HC. d PB0.05, 2 days vs. 21 days. e PB0,001, 2 days vs. 21 days. a
Table 4 Renal function studies after 2 days of ischemia in ND and HC rats, treated with L-arginine and
D-argininea
Group
BW (g)
V (ml/min)
Cin (ml/min per 100 g BW)
FE H2O (%)
FE Na (%)
FE K (%)
ND (n = 11) ND+L-arg (n = 9) ND+D-arg (n = 5) HC (n=8) HC+L-arg (n =7) HC+D-arg (n = 6)
242 9 10 235 9 13 230 915 214 9 12 215 99 23199
0.0089 0.001 0.0279 0.009 0.02190.008 0.0069 0.001 0.0189 0.004e 0.01390.003
0.2279 0.037 0.1239 0.027b 0.0969 0.048b 0.0339 0.011 0.1259 0.013f 0.03190.011g
2.08 90.24 11.48 93.70 3.74 914.02b,c 19.26 96.23 6.65 9 1.03e 26.91 95.34h
1.84 90.26 6.50 91.49 13.30 94.94b,c 17.16 94.24 5.23 9 0.88e 17.62 94.11
28.4 95.8 65.7 922.5 239.5 968.1b,d 172.8 936.8 57.9 9 14.6e 124.5 923.9
a Results are expressed as means 9 S.E.M. BW, body weight; V, urine volume; Cin, inulin clearance rate; FE H2O, fractional excretion of water; FE Na, fractional excretion of sodium; FE K, fractional excretion of potassium. b PB0.01 vs. ND. c PB0.05 L-arg vs. D-arg. d PB0.01 L-arg vs. D-arg. e PB0.05 vs. HC. f PB0.001 vs. HC. g PB0.001 L-arg vs. D-arg. h PB0.05 L-arg vs. D-arg.
a group of ND and HC rats received L-arginine (50 mg/kg BW) by the femoral vein immediately after the induction of ischemia. Another group of ND and HC rats received D-arginine (50 mg/kg BW). After 48 h, the rats were anesthetized with sodium thionembutal (50 mg/kg BW, i.p.) and the trachea was cannulated with a PE-240 catheter and a catheter (PE 60) was placed in the left femoral artery. By way of a median incision, the left renal pedicle was carefully dissected and the renal artery was isolated with care to avoid disturbing the renal nerves. An electromagnetic flow probe (Transonic Systems, Bethesda, MD) was placed around the exposed renal artery, and RBF was measured with an electromagnetic flowmeter (Transonic Systems, T 106 XM).
0.2% collagenase for 20 min at 37°C. Fetal calf serum (Laborclin) was added to neutralize collagenase and the cell suspension was centrifuged at 1500 rpm for 5 min. The pellet was placed in bottles containing RPMI 1640 supplemented with T3 (6× 10 − l2 M), T4 (6 ×10 − 8 M), 2 mg/ml transferrin, 1.9× 10 − 11 M prolactin, 0.66 mU/ ml insulin, 10 − 8 M dexamethasone, 100 mU/ml penicillin, 100 mg/ml streptomycin and 2.5 mg/ml amphotericin. Culture medium was changed every day and cells were studied 6 days after the initial plating. Cells were exposed to hypoxia by bubbling the medium with 100% N2 for 60 min and maintained at low P O2 for the subsequent 23 h. In a group of cells, L-arginine or D-arginine was added to the culture medium on the day of hypoxia. Cell injury was evaluated by lactate dehydrogenase (LDH) release.
2.2. IMCD culture 2.3. LDH release measurement Immediately after weaning, male Wistar rats were provided with a regular or HC diet for 3 weeks. IMCD segments were isolated from the renal papilla by microdissection with a sharpened Dumond forceps no. 5 in PBS solution and the fragments were digested with
After 24 h of incubation, the supernatant was separated for LDH determination in medium containing 100 ml sodium pyruvate, 100 ml Tris/EDTA, 100 ml NADH, 400 ml deionized water and 300 ml supernatant.
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Cells were washed three times with PBS and 1 ml of ATV (0.02% versene and 0.2% trypsin) was added. After 15 min, the preparation was centrifuged at 1000 rpm for 5 min. The pellet was lysed with 1.5% triton X-100 and LDH activity was determined in medium containing 100 ml sodium pyruvate, 100 ml Tris/EDTA, 100 ml NADH, 650 ml deionized water and 50 ml pellet. LDH activity was also determined in the ATV supernatant in medium containing 100 ml sodium pyruvate, 100 ml Tris/EDTA, 100 ml NADH, 600 ml deionized water and 100 ml of ATV supernatant. LDH release was converted to percent release by dividing supernatant activity by total activity according to Bergmeyer [8,9].
2.4. Statistical analysis All values are reported as means 9S.E.M. Data were analyzed by ANOVA test and the Student Newman Keuls multiple comparison post-test, with the level of significance set at P B0.05.
3. Results Rats fed a regular or cholesterol-supplemented diet for 3 weeks did not show a significant difference in weight gain. Measurements of total serum, HDL and LDL cholesterol and triglycerides are shown in Table 1. The values did not differ from those obtained before the study. After 21 days, rats fed the HC showed a significant increase in total serum cholesterol levels compared to ND rats (163.4 9 25.5 vs. 50.9 9 3.8 mg/ dl, PB0.05). HDL levels did not differ between groups, whereas the LDL level was significantly higher in the HC group than in ND rats (126.0927.0 vs. 11.59 5.1 mg/dl, PB 0.05). The HC rats showed significant, lower serum triglyceride levels compared with ND rats (639 9 vs. 100910 mg/dl, P B0.05). Serum albumin levels did not differ between groups. Renal function studies after 2 days of right nephrectomy showed that inulin clearance did not differ between ND and HC rats (0.37 90.03 and 0.38 90.05
ml/min per 100 g BW). Also, fractional excretion of water (ND, 1.04 9 0.09%; HC, 1.33 9 0.16%, NS), sodium (ND, 1.109 0.19%; HC, 1.589 0.33%, NS) and potassium (ND, 20.009 5.40%; HC, 26.1095.60%, NS) did not differ between groups (Table 2).
3.1. Effect of hypercholesterolemia on ischemic renal failure Hypercholesterolemic rats showed a significant decrease in inulin clearance compared to control rats after 2 days of 60-min left renal ischemia and contralateral nephrectomy (0.0339 0.011 vs. 0.2279 0.037 ml/min per 100 g BW, P B0.001). Hypercholesterolemia also induced an impairment in tubular function compared with ND rats, as shown by the high fractional excretion of water (19.26 9 6.23 vs. 2.08 9 0.24%, PB0.001), sodium (17.169 4.24 vs. 1.8490.26%, PB 0.01) and potassium (172.89 36.8 vs. 28.4 9 5.8%, PB0.01) (Table 3). Recovery from renal ischemia was studied after 21 days. The inulin clearance of the HC group did not differ from that of ND rats (0.360 90.040 vs. 0.340 9 0.080 ml/min per 100 g BW). The tubular parameters studied also did not show significant differences between groups in terms of the fractional excretion of water (HC, 1.179 0.08; ND, 1.0690.21%), sodium (HC, 1.929 1.10; ND, 0.929 0.20%) and potassium (HC, 36.69 17.5; ND, 46.59 17.0%) (Table 3).
3.2. Effect of L -arginine and ischemic renal failure
D -arginine
infusion on
The response of post-ischemic kidneys to L-arginine and D-arginine is depicted in Table 4. In HC rats, L-arginine produced a lower decrease in inulin clearance 2 days after ischemia (0.125 9 0.013 ml/min per 100 g BW) as compared with HC rats which did not receive L-arginine (0.0339 0.011 ml/min per 100 g BW, PB0.001) and D-arginine group (0.031 90.011 ml/min per 100 g BW, PB0.001). A significant protection of tubular function was observed in hypercholesterolemic
Fig. 1. Renal blood flow (RBF) after 2 days of 60-min ischemia in rats fed a normal (ND) and cholesterol supplemented diet (HC).
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Fig. 2. Percentage of lactate dehydrogenase (LDH) released from inner medullary collecting duct (IMCD) cell cultures of rats kept on a normal diet (ND) after 24 h of normoxia, hypoxia or hypoxia plus L-arginine (1 mM).
rats treated with L-arginine since fractional excretion of water, sodium and potassium was lower than in the untreated group (FE H2O: 6.6591.03 vs. 19.269 6.23%, P B 0.001; FE Na: 5.23 9 0.88 vs. 17.16 9 4.24%, P B 0.001; FE K: 57.9914.6 vs. 172.8 936.8%, PB 0.001). This protection was not observed with the D-arginine-treated rats. In the ND group, the effect of L-arginine administration was opposite to that observed in HC rats. Inulin clearance was lower, even though not significantly in L-arginine-treated rats than in the group that did not receive L-arginine (0.1239 0.027 vs. 0.2279 0.037 ml/min per 100 g BW, NS). The fractional excretion of water and sodium was significantly higher in these rats compared with the untreated group (FE H2O: 11.4893.70 vs. 2.0890.24%, P B 0.001; FE Na: 6.509 1.49 vs. 1.8490.26%, P B 0.001). The fractional excretion of potassium did not differ between groups. The administration of D-arginine to ND rats also caused a significant decrease of the Cin (0.0969 0.048 vs. 0.22790.037 ml/min per 100 g BW, PB 0.01). The tubular injury, assessed by a significant increase of FE H2O, FE Na and FE K, was greater in this group than in the L-arginine and not treated rats.
3.3. Measurement of renal blood flow (RBF) The RBF of ND and HC rats did not differ after 2 days of ischemia (2.30 90.39 vs. 2.40 9 0.20 ml/min per 100 g, NS). Treatment with L-arginine caused vasodilation only in the HC group after 2 days of ischemia (3.96 90.64 vs.2.409 0.20 ml/min per 100 g, P B0.05) (Fig. 1). A vasodilatory effect was not observed with the D-arginine administration.
3.4. Inner medullary collecting duct cell culture IMCD cell cultures were resistant to 24-h hypoxia in vitro, as demonstrated by LDH release which did not
differ from that observed in normoxic cells (hypoxic: 22.29 3.6% and normoxic: 21.49 0.8%). L-Arginine (1 mM) added to the culture medium had no effect on LDH release by hypoxic cells (19.69 3.7%). In contrast, IMCD cell cultures from HC rats submitted to 24-h hypoxia presented higher LDH release than normoxic cells (69.293.4 vs. 30.99 3.6%; PB 0.001) while the addition of 1 mM L-arginine to the medium of hypoxic cells attenuated LDH release (44.392.4 vs. 69.293.4%, PB 0.01) (Figs. 2 and 3). The addition of D-arginine to the culture medium did not have any protective effect (Fig. 4).
4. Discussion Recent evidence has shown that short-term induced hypercholesterolemia and hyperlipemia can promote vasoconstriction and/or increased vascular reactivity [1–3]. In vitro studies demonstrated that LDL cholesterol inhibits endothelium-dependent relaxation, suggesting that LDL might be responsible for the vasoconstriction that occurs with hypercholesterolemia. These studies suggest that these vascular effects are mainly functional in nature, since the duration of the experimental hypercholesterolemia was too short for gross pathologic lesions to develop in the blood vessels. Factors responsible for it could be decreased production of prostacyclin, increased production of thromboxane and impaired synthesis, release, metabolism and action of nitric oxide [4,10,11]. Nitric oxide is extremely important in the pathogenesis of this vasoconstriction and several studies on animals and humans have demonstrated that infusion of L-arginine could prevent the vasoconstriction induced by hypercholesterolemia [5,12]. The role of hypercholesterolemia in renal function has been studied by Kaplan et al. [10] who demon-
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Fig. 3. Percentage of lactate dehydrogenase (LDH) released from inner medullary collecting duct (IMCD) cell cultures of rats kept on a cholesterol supplemented diet (HC) after 24 h of normoxia, hypoxia and hypoxia plus L-arginine (1 mM).
strated that short-term HC in rats resulted in marked vasoconstriction of renal blood vessels with a consequent fall in GFR attributed to the reduction in QA and probably in part to a fall in Kf, as evidenced by micropuncture measurements. These results have important clinical implications since patients with increased cholesterol levels could develop more serious acute renal failure when submitted to renal ischemia or nephrotoxic drugs. In the present study, we used a well-known model of post-ischemic acute renal failure induced by 60 min of left renal artery clamping and contralateral nephrectomy, which allows a fixed time of ischemia. We clearly demonstrated that HC rats had a more important reduction in renal function compared with the ND group, studied 2 days after ischemia, as assessed by the lower inulin clearance as well as by the higher fractional sodium, potassium and water excretion, demonstrating that hypercholesterolemia potentiated renal ischemic damage. To examine the direct effect of hypercholesterolemia on hypoxic tubular cells, we studied cultures of IMCD cells submitted to 24-h hypoxia and demonstrated that cultures of IMCD cells are resistant to hypoxia as assessed by LDH release. However, when IMCD cells from HC rats were submitted to the same level of hypoxia there was an increase in LDH release, indicating that hypercholesterolemia sensitized these cells to hypoxia. It is well established that lipid membranes are in dynamic equilibrium with plasma lipids. Hypercholesterolemia can induce alterations in membrane composition, chemical properties and ion transport and permeability. An increase in intracellular calcium content, a recognized mediator of ischemic lesion, had been described [13,14], perhaps due to its increased transport by calcium receptor operated channels (ROC) [15], as well as inhibition of Na + – K + -ATPase [6,7] and Ca2 + ATPase activities.
Another possible factor involved in the great susceptibility to hypoxia of IMCD cells from HC rats is LDL cholesterol, which when oxidized could lead to production of deleterious oxygen radicals [16,17]. It is interesting to observe that after 3 weeks of recovery, the renal function of the HC group was similar to that of the ND group, as assessed by measurements of glomerular filtration and fractional sodium, potassium and water excretion. The importance of lipids in compensatory renal growth is recognized. Some studies have demonstrated that the phospholipid content of the hypertrophic kidney is increased [18]. It is known that amino acid supplementation enhances renal phospholipid biosynthesis after acute tubular necrosis increasing regeneration of renal tubular cells [19]. The present results demonstrate that hypercholesterolemia did not limit late recovery from renal ischemia. Several studies have shown the role of nitric oxide in ischemic acute renal failure. The results are controversial depending on the model studied. Schramm et al. [20] demonstrated that rats treated with L-arginine (300 mg/kg BW) immediately after 40-min bilateral renal ischemia showed improvement of GFR for 180 min after ischemia. This effect is stereospecific, since it is not observed with the D-arginine isomer. The protection afforded by NO might be explained by its vasodilatory effect counteracting vasoconstrictors such as angiotensin, catecholamins and endothelin. Another effect is the action on mesangial cells, causing an increase of Kf. Nevertheless, a study using proximal tubule suspensions exposed to 15-min hypoxia and 35-min reoxygenation demonstrated that L-arginine as well as sodium nitroprusside (NO donor) enhanced the hypoxia/reperfusion injury. Previous treatment with LNAME prevented the tubular injury, as did hemoglobin, indicating that NO is a possible mediator
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Fig. 4. Percentage of lactate dehydrogenase (LDH) released from inner medullary collecting duct (IMCD) cell cultures of rats kept on a cholesterol supplemented diet (HC) after 24 h of normoxia, hypoxia and hypoxia plus D-arginine (1 mM).
of hypoxia/reperfusion injury. This effect is attributed to the increased production of peroxynitrite radical in this situation [21].Yu [22] suggested that rats supplemented with L-arginine for 1 week showed a greater decrease in renal function, perhaps due to an excess production of NO and its potent reactive oxygen species, peroxynitrite radical. In the present study, the acute infusion of L-arginine to the ND group did not protect against the ischemic insult. This is in agreement with the results reported by Lopez-Neblina et al. [23] in a model of 75-min ischemia and contralateral nephrectomy in rats. It is well-known that remnant kidneys become hypertrophic and increase the synthesis of Larginine per nephron. Alternatively, other sources of production of L-arginine, such as liver and brain, may enhance their synthesis of this amino acid in this remnant kidney model, maintaining an adequate level of this amino acid. Thus, it is possible that in this model, the administration of L-arginine increased the production of NO and peroxynitrite radical. The increased fractional excretion of sodium and water observed in ND group treated with L-arginine could be due to proximal tubular damage [21,22]. The administration of L-arginine to HC protected the animals against the ischemic injury as assessed by inulin clearance, which was higher than the value observed in untreated HC rats. The mechanism of protection observed in the present study possibly was vasorelaxation since supplementation with L-arginine increased the RBF, possibly by the production of NO, since it was not observed with the inert isomer D-arginine which is impaired in a hypercholesterolemic environment. In addition, a direct protective effect was also demonstrated since the injury caused by hypoxia in IMCD cells from hypercholesterolemic rats was prevented by the addition of L-arginine to the culture medium, but
not by the addition of D-arginine. This protection may have been related to a reduction in the transport functions of epithelial renal tubular cells. It is recognized that NO inhibits Na + –K + -ATPase and sodium transport reducing oxygen consumption by these cells and diminishing their susceptibility to hypoxic injury [24,25]. Another possibility is that there may be some endogenous inhibitor of NO synthase that may be displaced by exogenous L-arginine. According to this hypothesis, a recent study showed that cholesterol feeding increases plasma concentrations of asymmetrical dimethylarginine (ADMA), an endogenous NO synthesis inhibitor in the rabbit [26]. ADMA may competitively inhibit NO synthase and/or L-arginine uptake into cells. These findings at least partly explain the protective effect of the higher extracellular concentration of L-arginine in hypoxic injury in IMCD cells from hypercholesterolemic rats. Rats treated with D-arginine showed a significant increase in FE H2O, FE Na and FE K. Andrade et al. [27] had similar results in a model of radiocontrast nephrotoxicity. These results suggest an aggravation of tubular injury, perhaps by an amino acid-induced effect. This was not observed with Larginine, since it was metabolized into nitric oxide. In conclusion, our results suggest that hypercholesterolemia is an aggravating factor of postischemic acute renal failure, partially mediated by a direct effect on epithelial tubular cells, which become more susceptible to ischemic injury. The recovery was similar in both groups despite the more severe insufficiency of the hypercholesterolemic group, which implies a beneficial role of cholesterol during the phase of recovery from acute renal failure. The acute infusion of L-arginine protected only the HC group and part of this protection was due to the vasodilatory renal effect and part to a direct effect on tubular cells.
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Acknowledgements These studies were supported by the Instituto dos Laborato´rios de Investigac¸a˜o Me´dica HC/FMUSP/ LIM-12, Fundac¸a˜o Faculdade de Medicina, Conselho Nacional de Pesquisa (CNPq), Fundac¸a˜o Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES), Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo and by Universiade Federal de Sa˜o Paulo (UNIFESP).
References [1] Casino PR, Kilcoyne CM, Quyyumi AA, Hoeg JM, Panza JA. The role of nitric oxide in endothelium-dependent vasodilation of hypercholesterolemic patients. Circulation 1993;88:2541 – 7. [2] Chowienczyk PJ, Watts GF, Cockcroft JR, Ritter JM. Impaired endothelium-dependent vasodilatation of forearm resistance vessels in hypercholesterolemia. Lancet 1992;340:1430–2. [3] Rossitch E Jr, Alexander E III, Black P M, Cooke JP. LArginine normalizes endothelial function in cerebral vessels from hypercholesterolemic rabbits. J Clin Invest 1991;87:1295– 9. [4] O’Hara Y, Peterson TE, Harrison DG. Hypercholesterolemia increases endothelial superoxide anion production. J Clin Invest 1993;91:2546 – 51. [5] Creager MA, Cooke JP, Mendelsohn ME, Gallagher SJ, Coleman SM, Loscalzo J, Dzau VJ. Impaired vasodilation of forearm resistance vessels in hypercholesterolemic humans. J Clin Invest 1990;86:228 – 34. [6] Lijnen P, Celis H, Fagard R, Staessen J, Amery A. Influence of cholesterol lowering on plasma membrane lipids and cationic transport systems. J Hypertens 1994;12:59–64. [7] Broderick R, Bialecki R, Tulenko TN. Cholesterol-induced changes in rabbit arterial smooth muscle sensitivity to adrenergic stimulation. Am J Physiol 1989;257(Heart Circ Physiol 26):170 – 8. [8] Bergmeyer HV. Methods in Enzymatic Analysis, 2nd edn. New York: Academic Press, 1974:574–89. [9] Beutler E. Lactate dehydrogenase (LD 12). In: Beutler E, editor. Red Cell Metabolism; A Manual of Biochemical Methods, 3nd edn. New York: Grune & Stratton, 1984:65–6. [10] Kaplan R, Aynedjian HS, Schiondorff D, Bank N. Renal vasoconstriction caused by short-term cholesterol feeding is corrected by thromboxane antagonist or probucol. J Clin Invest 1990;86:1707 – 14.
.
[11] Bank N, Aynedjian H. Role of thromboxane in impaired renal vasodilatation response to acetylcholine in hypercholesterolemic rats. J Clin Invest 1992;89:1636 – 42. [12] Cooke JP, Tsao P. Cytoprotective effects of nitric oxide. Circulation 1993;88(5, part 1):2451 – 4. [13] Bonventre JV. Mechanism of ischemic acute renal failure. Kidney Int 1993;43:1160 – 78. [14] Molitoris BA. Cellular basis of ischemic acute renal failure. In: Lazarus JM, Brenner BM, editors. Acute Renal Failure. Philadelphia, PA: Saunders, 1993:1 – 32. [15] Bialecki RA, Tulenko TN, Colucci W. Cholesterol enrichment increases basal and agonist-stimulated calcium influx in rat vascular smooth muscle cells. J Clin Invest 1991;88:1894 –900. [16] Peer G, Blum M, Iaina A. Nitric oxide and acute renal failure. Nephron 1996;73:375 – 81. [17] Keaney JF Jr, Gaziano JM, Xu A, Frei B, Curran-Celentano J, Shwaery GT, Loscalzo J, Vita JA. Dietary antioxidants preserve endothelium-dependent vessel relaxation in cholesterol-fed rabbits. Proc Natl Acad Sci USA 1993;90:11880– 4. [18] Bucher LR, Malt RA. Regeneration of liver and kidney. Foreword by Ingelfinger FJ, editor. New Engl J Med 1971:1–278. [19] Toback FG. Amino acid enhancement of renal regeneration after acute tubular necrosis. Kidney Int 1977;12:193 – 8. [20] Schramm L, Eidbreder E, Schmitt A, Kartenbender K, Zimmermann J, Ling H, Hdeidlan A. Role of L-arginine-derived NO in ischemic acute renal failure in the rat. Renal Fail 1994;16(5):555– 69. [21] Yu L, Gengaro PE, Niederberger M, Burke TJ, Shultz P, Schrier RW. Nitric oxide: a mediator in rat tubular hypoxia/reoxygenation injury. Proc Natl Acad Sci USA 1994;91:1691 – 5. [22] Yu L. Nitric oxide in acute renal failure: Foe or friend? Kidney Int 1997;52(Suppl 61):S39 – 40. [23] Lo´pez-Neblina F, Paez AJ, Toledo AH, Toledo-Pereyra LH. Role of nitric oxide in ischemia/reperfusion of the rat kidney. Circ Shock 1995;44:91 – 5. [24] Reyes AA, Karl IE, Klahr S. Role of arginine in health and in renal disease. Am J Physiol 1994;267:F331– 46. [25] Stoos BA, Carretero AO, Fahry RD, Scicli G, Garvin JL. Endothelium-derived relaxing factor inhibits transport and increases cGMP content in cultured mouse cortical collecting duct cells. J Clin Invest 1992;89:761 – 5. [26] Boger RH, Bode-Boger SM, Brandes RP, Phivthong-ngam L, Bohme M, Nafe R, Mugge A, Frolich JC. Dietary L-arginine reduces the progression of atherosclerosis in cholesterol-fed rabbits: comparison with lovastatin. Circulation 1997;96:1282–90. [27] Andrade LC, Campos SB, Seguro AC. Hypercholesterolemia aggravates radiocontrast nephrotoxicity: protective role of Larginine. Kidney Int 1998;53:1736 – 42.