Multiple Antioxidants and L-Arginine Modulate Inflammation and Dyslipidemia in Chronic Renal Failure Rats

Abstract

The kidney is an important source of L-arginine, the endogenous precursor of nitric oxide (NO). Surgical problems requiring extensive renal mass reduction (RMR) decrease renal NO production, leading to multiple hemodynamic and homeostatic disorders manifested by hypertension, oxidative stress, and increased inflammatory cytokines. Using the RMR model of chronic renal failure (CRF), we assessed the effects of twelve weeks' administration of L-arginine and/or a mixture of antioxidants (L-carnitine, catechin, vitamins E and C) on plasma cytokines, soluble intercellular adhesion molecule-1 (sICAM-1), nitrate and nitrites (NO₂/NO₃), lipid profile, blood pressure, and renal function. CRF rats showed increased plasma IL-1α, IL1-β, IL-6, TNF-α, and sICAM-1 levels and decreased anti-inflammatory cytokines IL-4 and 10 levels, hypertension, and dyslipidemia. L-arginine treatment improved kidney functions, decreased systolic blood pressure, and decreased inflammatory cytokines levels. Antioxidants administration decreased inflammatory cytokines and sICAM-1 levels and increased IL-4 levels. Combined use of both L-arginine and the antioxidant mixture were very effective in their tendency to recover normal values of kidney functions, plasma cytokines, sICAM-1, blood pressure, NO₂/NO₃, cholesterol, and triglycerides concentrations. Indeed, the effects of L-arginine and the antioxidants on the reduction of proinflammatory cytokines may open new perspectives in the treatment of uremia.

Keywords:

• chronic renal failure
• L-arginine
• antioxidants
• inflammatory cytokines
• dyslipidemia

INTRODUCTION

NO is an inorganic gas that plays a crucial role in the regulation of vascular functions and homeostasis.^[1] Synthesis of NO is a function of a family of nitric oxide synthases (NOSs) catalyzing a semi-essential cationic amino acid L-arginine^[1,2] produced mainly by the proximal tubules of the kidney in mammals, and defects in its synthetic pathway have been implicated in several diseases.^[3] Loss of renal mass leads to hemodynamic alterations and hyperfiltration in the residual nephrons, which in turn leads to glomerular damage.^[4] In parallel with the progressive structural deterioration of the remaining kidney, a picture of chronic renal failure (CRF) develops with accumulation of urea, creatinine, and other guanidine compounds in blood.^[5] Limited availability of the substrate L-arginine due to decreased production after renal mass reduction (RMR) may reduce NO synthesis, and there is some evidence that superoxide production may be increased.^[6] Abnormalities in NO synthetic pathway like decreased synthesis, increased degradation due to oxidative stress, and reduced sensitivity to NO are among the common responsible mechanisms mediating the complex hemodynamic and homeostatic disorders associated with the progression of renal disease.^[7,8] Endothelial dysfunction due to decreased NO bioavailability occurs early after RMR and predispose to the major cardiovascular diseases.^[9] In rats with RMR, a major defect in inducible NO synthase (iNOS) content was suggested to underlie the progressive decreases in renal NO generation that correlate with signs of renal injury.^[10] Although renal NO production is deficient in RMR, increased iNOS activity can be induced in several cell types, including inflammatory and non-inflammatory cells, by a variety of cytokines (e.g., interleukin-1α [IL-1α], tumor necrosis factor-α [TNF-α], and microbial products like endotoxin) that can potentially induce iNOS via nuclear factor Kappa-B mediated pathway.^[11,12] Increased iNOS activity was reported to be associated with decrease L-arginine level in plasma and with enhanced inflammatory response that potentate tissue damage.^[13,14] On the other hand, L-arginine supplementation was reported to attenuate the progression of CRF^[15] and modulate the immune status in several pathological processes.^[16–18]

Other than the abnormalities in NO synthetic pathway, the pro-oxidant and antioxidant capacity is disturbed in CRF, resulting in increased oxidative stress.^[19,20] This potentially mediates cardiovascular, neurological, and several other complications of CRF and its byproducts, as advanced glycation end products (AGE), oxidized LDL, and lipoperoxide augment further oxidative damage and inflammation via activation of macrophages.^[21] Components of the anti-oxidant defense mechanisms, including vitamins C and E, selenium, and glutathione (GSH) scavenging system, are deficient in patients with CRF, and a protein oxidation patterns consistent with leukocyte myeloperoxidase-mediated events has been demonstrated in these patients.^[21,22] L-carnitine is another important antioxidant that gained specific interest in CRF and was found to be deficient in these patients.^[23] Uremic patients, as well as patients with CRF, appear to have abnormal renal handling of carnitine, leading to dyslipidemia, lethargy, muscular weakness, hypotension, cardiac dysfunction and arrhythmias, and recurrent cramps.^[24,25] Most of these symptoms were found to be improved by L-carnitine administration.^[25–27] Catechins are a group of flavonoids—present in vegetables and plant-derived beverages and food, like tea and chocolate—that have attracted particular attention due to their relative high antioxidant capacity and free radical scavenging activities in biological systems.^[28–30] Owing to the multiple complications caused by oxidative stress and inflammatory response associated with disordered L-arginine–NO pathway and deficient anti-oxidant defense mechanisms in CRF. This study aimed to evaluate the effects of L-arginine either alone or in combination with L-carnitine, catechin, and vitamins E and C on the pro-inflammatory cytokines production (IL-1α, IL-1β, IL-6, TNF-α) and anti-inflammatory cytokines (IL-4 and IL-10), soluble intercellular adhesion molecule-1 (sICAM-1), NO level, blood pressure, lipid profile, and renal function in CRF induced in rats by RMR.

MATERIALS AND METHODS

Animal Grouping and Treatment Regimen

Fifty male Wistar rats (2–3 months old, weighing 200–230 g), raised in the animal house of the Faculty of Medicine of King Saud University (KSU), were used in the study. Animals received tap water and food ad libitum and were housed four to a cage in a 12-h light-dark cycle. The study design was approved by the Research Ethics Committee of the Faculty of Medicine, KSU. The experimental protocol and housing facilities were conducted according to the standard established guidelines of laboratory animals of Collage of Medicine Research Center (CMRC), King Saud University. Rats were divided into five groups (n = 10 rats in each):

• Group A: healthy control rats receiving no treatment;

• Group A1: RMR rats receiving no treatment;

• Group A2: RMR rats receiving L-arginine treatment;

• Group A3: RMR rats receiving antioxidant treatment (L-carnitine + catechin + vitamins E and C); and

• Group A4: RMR rats receiving both L-arginine and antioxidant treatment.

Under pentobarbital anesthesia (40 mg/kg, i.p), RMR was induced in rats by removing approximately 5/6 of the kidney tissue, involving right nephrectomy and removal of the upper and lower poles of the left kidney.^[31] The RMR procedure was accomplished via dorsal incision and carried out under strict hemostasis and aseptic technique. Control rats were sham operated by flank incision under anesthesia. After the operation, rats were allowed to recover and have free access to a standard laboratory diet and tap water. L-arginine was given in a dose of 1.25 g/L in the drinking water (approximately 0.2g/kg/day),^[32] and vitamins E and C and catechin were mixed with food (400 mg/kg food, 500 mg/kg food, 100 mg/kg/day, respectively,^[33–36] and L-carnitine 500 mg/kg intraperitoneally).^[37] Treatment started one week after RMR and continued for 12 weeks. All drugs were purchased from Sigma Chemical Co. (Sigma, Ltd., USA).

Sampling

Blood samples were collected one week after the RMR procedure before drug supplementation (0 time) and at 12 weeks of treatment from the retro-orbital plexus in iced plain and EDTA-containing tubes. Serum and plasma were separated and stored at −70°C until assay. Urine was collected in sterile containers containing isopropranolol (0.1 mL) by putting the animals individually in sterile metabolic cages for 24 hours. Urine volume was measured in (mL/24), and the urine was then centrifuged. Part of the supernatant was used to measure creatinine and protein content, and the rest was stored at −70°C until assay of NO₂/NO₃.

Biochemical Analysis

Cytokine Immunoassays

Plasma IL-1α, IL-1β, IL-4, IL-6, and IL-10 (R&D Systems, UK) were assessed by a specific sandwich enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions. The optical density was read at 450 nm for IL-4, IL-6, and IL-1α and at 490 nm for IL-10 in an automated microplate reader (model ELX800, serial number 191720, Bio-Tek Instruments Inc., Vermont, USA)

Soluble Intercellular Adhesion Molecule-1 (sICAM-1)

Plasma sICAM-1 was measured using a specific ELISA kit produced by R&D Systems (UK). The optical density was read at 490 nm.

Serum and Urinary Nitric Oxides

Total body nitric oxide production was evaluated by measuring the stable end products of nitric oxide metabolism NO₂/NO₃ in serum and 24 hours urine by special kit (Cayman Chemical, USA) utilizing nitrate reductase and the Griess reagents as well as an ELIZA reader, as previously reported.^[38]

Kidney Function Tests

Serum urea and creatinine (Cr.) levels, plus urine Cr. and protein excretion, were measured by colorimetric methods using specific kits from Spinreact (Spain).^[39–41]

Serum Lipid Analysis

Total serum cholesterol and triglycerides levels were determined using a commercial kit (Spinreact, Spain) according to manufacture instructions.^[42,43]

Measurement of Blood Pressure in Conscious Rats

Systolic blood pressure was measured by a non-invasive blood pressure system (NIBP) using the tail-cuff plethysmography (Letica LE 5100, Panlab, Barcelona, Spain) one week after the surgery and every four weeks. The average of three measurements was used as a single value for each rat at each timepoint.^[44]

Statistical Analysis

Results were analyzed using the SPSS package for statistical analysis (version 12.0). A comparison between all the groups at each timepoint was done using one-way ANOVA. A post hoc LSD test was used when ANOVA gave significant differences. Comparison between the 0 and 12 dataset of the same group was one by paired sample t-test. All data are presented as mean ± SEM. Results were considered significant when p < 0.05.

RESULTS

Kidney Functions of RMR Rats after Various Treatments

One week after RMR (0 time), groups A1–A4 rats showed a picture of CRF characterized by significant reduction in creatinine clearance (cr. cl.) and significant marked increase in serum creatinine (cr.), urea, and urinary protein loss in comparison to sham-operated controls (p < 0.05). After 12 weeks, group A1 showed significant increase in serum urea, creatinine, urinary protein loss, and decreased creatinine clearance in comparison to week 0 (p < 0.05 for all variables). L-arginine supplementation caused serum urea and cr. levels and the proteinuria to remain significantly lower in group A2 rats compared to group A1 rats (p < 0.05), and also kept their cr. cl. significantly higher (p < 0.05). L-carnitine, catechin, and vitamins E and C treatment to group A3 also caused a significant reduction (p < 0.05) in serum urea and cr. levels and urinary protein and increased cr.cl. (p < 0.05) in comparison to the untreated rats. However, the effect of the antioxidant mixture on serum urea, cr., and cr.cl. was significantly less (p < 0.05) than that caused by L-arginine treatment. Combined treatment by L-arginine and the multiple antioxidants caused further improvement in kidney function in Group A4 rats, showing no significant difference from the sham operated control (p > 0.05) regarding serum cr., cr.cl., and urinary protein loss (see Table 1).

Table 1  Changes in kidney function, nitrate/nitrite (NO₂/NO₃) production, and lipid profile of chronic renal failure rats after 12 weeks of treatment with L-arginine and antioxidants mixture

Groups	Weeks	Control	RMR	RMR+L-arg.	RMR+antiox	RMR+L-arg.+antiox
S. urea (mg/dL)	0	34.96 ± 0.37	81.18 ± 0.37	82.12 ± 1.3	81.58 ± 1.43	82.42 ± 1.25
	12	34.7 ± 0.7	128.27 ± 0.6*^║	72.18 ± 1.8^†^║	102.76 ± 1.4^†^‡^║	42.1 ± 1.4^†^‡^§^║
S. cr (mg/dL)	0	0.43 ± 0.005	1.62 ± 0.07	1.57 ± 0.08	1.59 ± .08	1.58 ± .07
	12	0.43 ± 0.005	2.34 ± 0.1^║	1.25 ± 0.1^†	1.70 ± 0.06^†^‡	0.54 ± 0.02^†^‡^§^║
Cr. Cl (ml/min)	0	2.22 ± .09	1.12 ± .05	1.05 ± 0.04	1.01 ± 0.04	1.08 ± 0.04
	12	2.15 ± 0.06	0.77 ± 0.03^║	1.84 ± 0.11^†^║	1.83 ± 0.14^†^║	2.15 ± 0.07^†^‡^§^║
S. NO2/NO3 (μmol/L)	0	47.2 ± 0.85	39.87 ± 0.68	39.73 ± 0.55	39.84 ± 0.46	39.60 ± 0.50
	12	47.36 ± 0.80	20.84 ± 0.72^║	42.8 ± 1.78^†	29.6 ± 1.2^†^‡^║	46.17 ± 1.51^†^‡^§^║
Ur. vol (ml/24h)	0	13.53 ± 0.43	14.8 ± 0.39	14.63 ± 0.45	14.59 ± 0.45	14.54.4 ± 0.41
	12	15.09 ± 0.46	30.98 ± 0.4^║	27.7 ± 2.3^║	28.27 ± 0.72^║	13.38 ± 0.18^†^‡^§^║
Ur. protein (mg/24h)	0	8.19 ± .21	36.17 ± 1.4	36.12 ± 1.3	35.8 ± 1.32	37.78 ± 1.67
	12	8.56 ± .24	104.79 ± 3.24^║	49.22 ± 2.15^†^║	61.15 ± 3.2^†^║	16.63 ± 3.93^†^‡^§^║
Ur. NO2/NO3 (μmol/24h)	0	24.08 ± 0.59	18.12 ± 0.58	17.87 ± 0.58	17.65 ± 0.75	18.10 ± 0.46
	12	23.70 ± 0.52	8.98 ± 0.36^║	18.17 ± 0.38^†^║	15.06 ± 0.27^†^‡^║	20.46 ± 0.85^†^§^║
S. cholesterol (mg/dL)	0	101.67 ± 3.22	103.94 ± 3.16	95.73 ± 9.83	94. 37 ± 10.8	105.76 ± 2.9
	12	101.67 ± 3.22	155.87 ± 2.0^║	130.17 ± 3.04^†^║	133.31 ± 5.05^†^║	130.19 ± 5.31^†^║
S. triglyceride (mg/dL)	0	64.93 ± 2.00	78.10 ± 2.40	77.26 ± 1.59	77.43 ± 2.06	77.26 ± 1.70
	12	65.71 ± 1.62	112.98 ± 3.6^║	89.71 ± 0.88^†^║	74.31 ± 1.2^†^‡	68.65 ± 1.62^†^‡^§^║

Data are presented as mean ± SEM.

*p < 0.05 significant versus control animals.

^†p < 0.05 significant versus untreated renal mass reduction animals.

^‡p < 0.05 significant versus L-arginine-treated group.

^§p < 0.05 significant versus the antioxidants-treated group.

^║p < 0.05 significant versus the 0 time of this parameter in the same group.

Abbreviations: S. = serum, Ur. = urine, Cr = creatinine, Cl. = clearance, Vol = volume, Control = sham operated healthy rats receiving no treatment, RMR = renal mass reduction rats without treatment, RMR+L-arg = renal mass reduction rats receiving L-arginine treatment, RMR+antiox = renal mass reduction rats receiving antioxidants treatment, RMR+antiox+L-arg = renal mass reduction rats receiving L-arginine and antioxidant treatment.

NO₂/NO₃ Levels

As Table 1 shows, at week 0, serum and urinary NO₂/NO₃ levels decreased significantly in all rats subjected to RMR compared with the sham operated controls (p < 0.05). Group A1 rats showed a further decrease in their serum and urinary NO₂/NO₃ at week 12 (p < 0.05). Treatment with L-arginine significantly increased serum and urinary NO₂/NO₃ in group A2 rats, an effect that was greater than the one caused by L-carnitine, catechin, and vitamins E and C treatment in group A3 rats (p < 0.05). NO₂/NO₃ production in rats receiving combined L-arginine and antioxidants showed further significant increase (p < 0.05) in comparison to uremic rats receiving L-arginine alone.

Serum Cholesterol and Triglycerides Levels

At week 0, there was no significant difference in serum cholesterol levels between all the studied groups (p > 0.05), but serum triglycerides showed a significant increase (p < 0.05) in RMR rats in comparison to the sham operated group (p < 0.05). At week 12, serum cholesterol triglycerides levels increased significantly (p < 0.05) in untreated group A1 rats in comparison to sham operated controls. Antioxidant treatment caused greater reduction in serum triglyceride levels in comparison to L-arginine treatment (p < 0.05). The combined use of both L-arginine and the antioxidant mixture nearly normalized serum triglyceride levels to the control value (p = 0.4; see Table 1). On the other hand, all the used treatment regimens caused a significant reduction in serum cholesterol levels in comparison to untreated group A1 (p < 0.05). However, no significant difference between the effects of the three treatment regimens was found (see Table 1).

sICAM-1

Group A1 CRF rats showed marked increase in the plasma levels of sICAM-1in comparison to sham operated controls (p = 0.000). Use of the antioxidant treatment caused a significant reduction in the level of sICAM-1in group A2 in comparison to group A1 rats (p = 0.035). sICAM-1 level in the antioxidant-treated group A3 rats showed no significant difference from the control value (p = 0.51). L-arginine treatment either alone or in combination with the antioxidant mixture decreased sICAM-1 levels in treated rats in comparison to untreated group, but this reduction was not significant (see Table 2).

Table 2  Effect of L-arginine and multiple antioxidant treatments of plasma cytokines and soluble intercellular adhesion molecule-1 (sICAM-1) in chronic renal failure rats

Groups	Control	RMR	RMR+L-arg.	RMR+antiox	RMR+L-arg.+antiox
IL-1 alpha (pg/mL)	7.56 ± 0.25	69.40 ± 3.47	47.91 ± 1.23^†	59.10 ± 2.7^†^‡	53.61 ± 1.90^†
IL-1beta (pg/mL)	16.95 ± 0.49	86.68 ± 1.26	51.52 ± 1.61^†	39.75 ± 1.55^†^‡	31.17 ± 0.79^†^‡^§
Il-4 (pg/mL)	28.34 ± 0.44	27.14 ± 0.34	26.73 ± 0.2	27.43 ± 0.35	27.12 ± 0.34
IL-6 (pg/mL)	32.64 ± 0.98	89.30 ± 1.89	45.81 ± 1.31^†	44.39 ± 1.01^†	34.42 ± 1.03^†^‡^§
Il-10 (pg/mL)	116.00 ± 1.74	96.49 ± 0.89	98.66 ± 1.39	97.23 ± 3.8	97.88 ± 1.8
TNF-alpha (pg/mL)	6.06 ± 0.34	26.44 ± 1.01	17.97 ± 0.65^†	12.85 ± 0.78^†	19.12 ± 0.57^†^‡^§
sICAM-1 (pg/mL)	672.38 ± 63.49	1081.11 ± 65.56	956.11 ± 71.0	871.38 ± 76.22^†	1064.44 ± 70.39

Data are presented as mean ± SEM.

*p < 0.05 significant versus control animals.

^†p < 0.05 significant versus untreated renal mass reduction animals.

^‡p < 0.05 significant versus L-arginine-treated group.

^§p < 0.05 significant versus the antioxidants-treated group.

Abbreviations: control = sham operated healthy rats receiving no treatment, RMR = renal mass reduction rats without treatment, RMR+L-arg = renal mass reduction rats receiving L-arginine treatment, RMR+antiox = renal mass reduction rats receiving antioxidants treatment, RMR+antiox+L-arg = renal mass reduction rats receiving L-arginine and antioxidant treatment.

Cytokines Levels

Il-1α and IL-1β

Plasma IL-1α and 1β levels increased significantly in untreated group A1 rats in comparison to the sham operated group (p = 0.000; see Table 2). Treatment by L-arginine either alone or in combination with the antioxidants caused a significant decrease in IL-1α and IL-1β in treated rats in comparison to the untreated group (p = 0.000). The use of the antioxidant mixture also significantly decreases IL-1α and 1β levels in CRF rats, but their effect on IL-1α was less than that caused by L-arginine in contrast to their effect on IL-1β.

IL-6

As shown in Table 2, IL-6 increased significantly in CRF rats in comparison to the sham operated control (p = 0.000). Treatment by L-arginine and the antioxidants mixture either individually or in combination significantly decreased IL-6 levels in comparison to the untreated group. However, the reduction of IL-6 levels achieved by the combined use of L-arginine and the antioxidants was greater than that caused by either of them alone (see Table 2).

TNF-α

TNF-α level increased significantly in untreated CRF rats in comparison to the sham operated controls. Treatment by either L-arginine or antioxidants significantly decreased TNF-α levels in groups A2 and A3 rats, an effect that was comparable. But the combined use of L-arginine and the antioxidant treatment in group A4 rats caused a greater reduction in plasma TNF-α level in comparison to the use of either of the two treatments (see Table 2).

IL-4 and IL-10 Levels

As Table 2 shows, the anti-inflammatory cytokines IL-4 and IL-10 levels decreased significantly in CRF rats in comparison to the sham operated group (p = 0.019, p = 0.000, respectively). Treatment with the antioxidant mixture caused a significant increase in IL-4 in CRF rats to a level that that showed no significant difference from the control group (p = 0.07). However, L-arginine treatment either alone or in combination with the antioxidants failed to cause significant change in IL-4 levels, which remained significantly lower the control value (p = 0.002). None of the used treatment regimens caused significant changes in IL-10 levels.

Blood Pressure Changes at 0, 4, 8, and 12 Weeks of Various Treatments in CRF Rats

Marked elevation in arterial blood pressure was observed in CRF rats during the observation period (see Table 3). The use of either L-arginine or the antioxidant mixture significantly (p <0.05), but partially, attenuated the CRF-induced hypertension. The effect of both treatments was comparable (p > 0.05). The combined use of L-arginine and antioxidant therapy resulted in greater correction of CRF-induced hypertension in comparison to treatment by either of them (p < 0.05). Blood pressure remained higher than that of the sham operated group at 4 and 8 weeks of combined treatment (p < 0.05) but reached the control value after 12 weeks of the combined treatment (p > 0.05).

Table 3  Effect of L-arginine and multiple antioxidants on systolic blood pressure (BP) of chronic renal failure rats

	Systolic BP (mmHg) wk 0	Systolic BP (mmHg) wk 4	Systolic BP (mmHg) wk 8	Systolic BP (mmHg) wk 12
Control	112.3 ± 0.44	112.5 ± 0.60	113.0 ± 1.00	113.5 ± 0.47
RMR	149.2 ± 0.84	174.5 ± 1.84	197.0 ± 1.61	207.0 ± 1.85
RMR+L-arg	150.5 ± 0.61	147.9 ± 1.19^†	161.6 ± 1.55^†	169.1 ± 1.61^†
RMR+antiox	150.0 ± 0.66	150.1 ± 1.15^†	154.5 ± 2.1^†^‡	160.9 ± 1.9^†^‡
RMR+antiox+L-arg	150.4 ± 0.58	124.9 ± 0.64^†^‡^§	125.3 ± 0.81^†^‡^§	121.6 ± 1.09^†^‡^§

Data are presented as mean ± SEM.

*p < 0.05 significant versus control animals.

^†p < 0.05 significant versus untreated renal mass reduction animals.

^‡p < 0.05 significant versus L-arginine-treated group.

^§p < 0.05 significant versus the antioxidants-treated group.

Abbreviations: Control = sham operated healthy rats receiving no treatment, RMR = renal mass reduction rats without treatment, RMR+L-arg = renal mass reduction rats receiving L-arginine treatment, RMR+antiox = renal mass reduction rats receiving antioxidants treatment, RMR+antiox+L-arg = renal mass reduction rats receiving L-arginine and antioxidant treatment.

DISCUSSION

Chronic inflammation is a highly prevalent pathological process in CRF that predicts poor clinical outcome,^[45] enhancing cardiovascular complications and mortality in end stage renal disease.^[46,47] We reported previously^[48] that CRF induced in rats by subtotal nephrectomy depleted the antioxidant enzyme pool, manifested by declined catalase and superoxide dismutase enzymes activities, and reduced glutathione levels with the accumulation of the nitric oxide synthase inhibitor asymmetric dimethyl arginine (ADMA), which inhibits the activity of endothelial nitric oxide synthase (e-NOS). Accordingly, the combination of NO inactivation by reactive oxygen species (ROS) and depressed NO biosynthesis reduces the availability of NO and thus contributes to endothelial dysfunction and arteriosclerosis, which may lead to increased renovascular resistance and hypertension.^[7,48,49]

In the present study, RMR was followed by the development of CRF presented by systolic hypertension, increased serum urea and cr. levels, proteinuria, decreased cr.cl., and decreased urinary and serum NO₂/NO₃ content, suggesting that systemic NO production is impaired. The inappropriate NO level in CRF could be attributed in part to the accumulation of ADMA and methylguanidinen.^[48,50] In addition, the effects of NO may be limited by its rapid inactivation by oxygen free radicals, which are also increased in the uremia.^[21,48] These findings are in accordance of previous results documenting that NO release is impaired in CRF, and may be also involved in the pathogenesis of hypertension in this disease.^[7,8] It is well recognized that basal release of the endothelium-derived relaxing factor NO is crucial in the control of blood pressure. Indeed, mice with deficient e-NOS have elevated blood pressure.^[51] L-arginine administration (1.25 g/L in drinking water) for 12 weeks to group A2 rats in the current study was found to be associated with blunting the increase in systolic blood pressure, increased NO production measured by its stable metabolites NO₂/NO₃, decreased proteinuria, and increased cr.cl. The increased production of NO could explain the lowered blood pressure after L-arginine treatment and is consistent with reports of significant blood pressure reduction by L-arginine, whether it is provided through natural foods or as pharmacological preparations in humans.^[52] The reported increase in renal blood flow (RBF) and decreases glomerular capillary pressure and efferent arteriolar resistance in response to L-arginine treatment^[32] could provide further explanation of decreased arterial blood pressure and increased cr.cl. in CRF rats treated with L-arginine in the current study. Other than its ability to increase NO production, L-arginine is suggested to decrease the levels of the NOS antagonist ADMA, homocysteine, and malondialdehyde (MDA) and myeloperoxidase (MPO) activity in hemodialysis patients.^[53] Clinical and experimental evidence suggests that the enzymatic activity of NOS is regulated by the ratio between the concentration of L-arginine (the natural substrate) and that of ADMA (the endogenous inhibitor).^[54] Because ADMA is a competitive inhibitor of NOS, its inhibitory action can be overcome by increasing the concentration of the enzyme's substrate, L-arginine. Thus, nutritional supplementation with L-arginine may help to restore the physiological status by normalizing the L-arginine:ADMA ratio.^[54] The renoprotective and antihypertensive effect of L-arginine may be related in part to reduction in vascular and renal endothelin-1(ET-1) production through increasing NO release both in the circulation and in the renal tissues.^[32,55,56] Improvement of renal NO release may also prevent the development of glomerulosclerosis and the resulting hyperfiltration and proteinuria, which may be in part due to the reduction of glomerular ET-1 production.^[57] In fact, NO inhibits the mitogenic response and the expression of extracellular matrix proteins induced by various agonists in mesangial cells, including endothelin-1.^[58] This is supported by reduced sICAM-1 levels in association with increased NO levels in treated rats in the current study. Furthermore, L-arginine may decrease the oxidative stress^[53]; this effect is supported by previous reports of decreased urinary excretion of 8-iso-prostaglandin F_2α (PGF_2α), a non–invasive marker of oxidative stress in vivo after L–arginine treatment.^[59] Meanwhile, the blood pressure-lowering effect of the antioxidant mixture used in the current study could be due to decreased NO breakdown, potentially related to lower release of ROS.^[48] However, other effects like increased expression or activity of e-NOS are not rejected and are supported by increased serum concentration of NO metabolic end products (NO₂/NO₃) level in all of the CRF-treated rats. Oxidative stress related protein heme oxygenase-1 (HO-1)-mediated production of the vasodilator carbon monoxide (CO) may contribute to the regulation of the vascular tone and thereby blood pressure and endothelial function.^[60] In this regard, carnitine was reported to induce HO-1 gene and protein expression, together with parallel effect on eNOS, suggesting a possible mechanism for its antioxidant and protective effects from endothelial dysfunction.^[61] However, we observed that antioxidant therapy resulted in a partial correction of hypertension, as opposed to complete correction with the addition of L-arginine. This points to the role of numerous factors in the pathogenesis of CRF-induced hypertension.

Enhanced inflammatory condition in CRF rats in the current study was demonstrated by increased plasma pro-inflammatory cytokines IL-1α, IL-1β, IL-6, and TNF-α and decreased anti-inflammatory cytokines IL-4 and IL-10 levels. These findings are in accordance with previous reports in CRF patients and rats.^[46,62,63] The observed inflammatory response in CRF rats in the current study was found to be associated with an enhanced hyper-adhesive state demonstrated by increased levels of plasma sICAM-1. This increase may be due to inadequate clearance and enhanced synthesis/release,^[63,64] and it reflects the presence of CRF complications as malnutrition, inflammation, and cardiovascular diseases.^[63,64] A significant correlation between sICAM-1 and other inflammatory cytokines as TNF-α and C-reactive protein was reported,^[65,66] suggesting that the circulating level of this molecule may be regarded as a marker of inflammatory process. When malnutrition of uremia ensured, decreased plasma amino acids, including L-arginine,^[67] could enhance this inflammatory state. This assumption is supported by the reported increase of circulating cytokines such as TNF-α and IL-1β in malnourished uremic patients.^[68] This further exacerbates the oxidative and inflammatory milieu in uremia with increased aggregability of platelets taken from these patients.

We observed that L-arginine significantly decreases the inflammatory cytokines IL-1α, IL-1β, IL-6, and TNF-α production, but it did not influence the anti-inflammatory cytokines IL-4 and IL-10. This may indicate that the beneficial effects of L-arginine on CRF do not involve promoting anti-inflammatory cytokines production. However, in addition to a direct anti-inflammatory action of L-arginine that decreases the production of inflammatory cytokines in CRF, an indirect action could be attributed to its enhancing effect on NO production that was reported to decrease the leukocyte adhesion in post-ischemic tissue.^[69] L-carnitine, catechin, and vitamin E and C treatment in the present study decreased the inflammatory cytokines IL-1α, IL-1β, IL-6, and TNF-α levels and increased the anti-inflammatory cytokine IL-4 levels. This was associated with lowered plasma sICAM-1 levels. This inhibition of sICAM-1 up-regulation after antioxidant treatment could be attributed to increased NO levels according to the reported ability of NO to prevent the activation of nuclear transcription factor NF-KB^[70] and to its ability to act in an anti-adhesive manner through the down-regulation of neutrophil CD18/CD11 integrin,^[71] p-selectin,^[72] and E-selectin.^[73] These beneficial effects of the antioxidant therapy in CRF may result from the interruption of several pathophysiological mechanisms. Vitamin E protects glomerular basement membrane integrity, prevents neutrophil chemotaxis, and inhibits platelet aggregation.^[74] It also inhibits 5-lipoxygenase in peripheral blood monocytes of hemodialysis patients, resulting in decrease lipid peroxidation and leukotriene B₄ content.^[75] However, a vitamin E-enriched diet was reported to increase GSH level and inhibit oxidative endothelial damage.^[76] Regarding vitamin C, it functions as a cofactor for biosynthesis of carnitine,^[77,78] and it exerts a protective role against the peroxidative damage through its lipid-lowering effect.^[79] Carnitine itself has antioxidant action and a strong inhibitory effect on free radical production,^[80,81] and it exerts thiol and methionine-sparing activity.^[82] Furthermore, recent data showed that L-carnitine treatment is able to modify some particular aspects of the inflammatory status characterizing CRF patients.^[83] This is supported by the findings of the present study of decreased inflammatory cytokines in the plasma of CRF rats receiving L-carnitine in combination with the other antioxidant.

The catechin part of the antioxidant mixture—being a flavanoid—could help through the direct scavenging of some radical species, act as a chain-breaking antioxidant, or recycle other chain antioxidants such as α-tocopherol by donating a hydrogen atom to the tocopheryl radical.^[84] Therefore, the beneficial effects of antioxidant treatment in CRF rats in the current study may imply additive effects of vitamins E and C, catechin, and L-carnitine that modulate oxidant stress. Increased levels of these antioxidants could explain the improvement in kidney function and prevention of the oxidative stress induction of inflammatory cytokines in the treated uremic rats in the current study.

Hyperlipidemia in renal failure rats in the current study confirms previous reports of disordered lipid metabolism in this disease.^[85,86] Elevated serum cholesterol level is known to be associated with impaired endothelial function both in vivo and in vitro and may induce superoxide formation.^[87,88] When kidney fails, hepatic apo-A-I synthesis decreases, and high density lipoproteins (HDL) levels fall.^[89] HDL is an important antioxidant and defends the endothelium from the effects of cytokines.^[90] Inflammation causes further structural and functional abnormalities in HDL. The accumulation of intermediate-density lipoprotein (IDL) comprising very low-density lipoproteins (VLDL) and chylomicron remnants resulted in increased serum triglyceride levels.^[91] This disturbance of lipid metabolism is considered a characteristic feature of CRF. It impedes vascular relaxation and is associated with cardiovascular disease,^[92] and it may also be associated with the development of glomerular sclerosis and consequently with the progression of CRF.^[92] In mammalian cells, polyamines are synthesized from L-arginine by means of arginase in extra hepatic tissues.^[93] Dietary L-arginine supplementation appears to affect the metabolism of lipoproteins through the formation of polyamines by arginase enzyme in extrahepatic tissues.^[93] This may explain the reduction of cholesterol and triglycerides in CRF rats receiving L-arginine treatment either alone or in combination with antioxidants in the present study. Similar findings are recently reported in hemodialysis patients receiving oral L-arginine supplementation.^[81] Multiple recent studies reported the ability of each of the antioxidants used in the current study to decrease blood lipids in different diseases.^[94–98] Thus, the combined hypolipidemic effect of catechin, L-carnitine, and vitamins E and C may underlie the reduction of cholesterol and triglyceride levels in the antioxidant-treated group. This correction of dyslipidemia by antioxidant treatment in the current study was augmented by L-arginine addition, and these findings were in accordance with other studies that showed improvement in lipid, lipoprotein, and apoproteins profile during L-arginine treatment in diabetic rats and in healthy human volunteers.^[99,100]

CONCLUSIONS

Increased levels of pro-inflammatory cytokines and sICAM-1 in CRF are a marker of inflammation, malnutrition, and cardiovascular diseases and predict mortality. Both L-arginine and antioxidant mixture (L-carnitine, catechin, vitamins E and C) treatments showed beneficial effects in their tendency to lower proinflammatory cytokines and sICAM1 levels and recover normal values of kidney function, blood pressure, NO₂/NO₃, cholesterol, and triglycerides concentrations. Indeed, the effects of L-arginine and the antioxidants in CRF may open new perspectives in the treatment of uremia and raise the strong recommendation of immonutrition in critically ill patients. Diets containing high concentration of L-arginine are now commercially available,^[101] and findings of the present study support the beneficial effect of combined L-arginine and multiple antioxidants in CRF-induced by RMR. However, further studies in human patients are required.

ACKNOWLEDGMENT

The author is grateful to Mr. Cassemiro Victoria for his excellent help in the biochemical measurements and for Mr. Gamal Abdul Aal for his assistance and support during the experimental part of the work.

This study was supported by funds provided by King Abdulaziz City for Science and Technology, Saudi Arabia, Riyadh (LGP-10-3), and resources supplied by the College of Medicine Research Center (CMRC) of the Faculty of Medicine, King Saud University (07-580).

The author reports no conflict of interest. The author alone is responsible for the content and writing of the paper.