Therapeutic Effect of Artemether in an Experimental Model of Nephrosis

Abstract

This study was carried out to elucidate the therapeutic efficacy of a new antimalarial drug, artemether, in adriamycin-induced nephropathy. To induce experimental nephrosis, adriamycin was given once by a single intravenous injection through the tail vein. Six days after adriamycin injection, therapeutic protocol was developed by intramuscular (i.m.) administration of 5 mg/kg artemether (ART). The total of i.m. injections were 14, in which five injections were made every day and nine injections were carried out at regular 48-h intervals. The therapeutic protocol was terminated on day 28, and animals were sacrificed on day 49. Our results showed that treatment with ART caused a significant reduction in the level of proteinuria (118 ± 15 vs. 48 ± 21 mg/24 h), urine urea (723 ± 226 vs. 414 ± 185 mg/dL), and urine sodium (38 ± 8 vs. 28 ± 6 mEq/L) compared with nontreated controls. In addition, a decrease in serum cholesterol (250 ± 58 vs. 163 ± 58 mg/dL) and creatinine (1.4 ± 0.2 vs. 0.9 ± 0.2 mg/dL) and an increase in the level of serum albumin (3.3 ± 0.5 vs. 5.6 ± 0.5 g/dL) were significant in the group treated with ART compared with the patient group. Moreover, treatment with ART significantly reduced glomerular PMN and mononuclear cells infiltration, hypercellularity, karyorrhexis, wire loops and hydropic change in the capillary network within the renal cortex, as well as decreased hyalin casts. On the other hand, healthy controls receiving ART showed a significant decrease in amounts of serum triglyceride (174 ± 55 vs. 88 ± 27mg/dL), urine urea (720 ± 113 vs. 511 ± 211 mg/dL), urine sodium (45 ± 5 vs. 26 ± 6 mEq/L), and potassium (71 ± 14 vs. 53 ± 6 mEq/L) compared with the normal group, where significant reduction of urinary excretion of sodium and urea must be considered as a side effect of ART therapy. These data suggest that artemether therapy can ameliorate proteinuria and suppress the progression of glomerular lesions in an experimental model of nephrosis, and that it may be recommended as a lipid-lowering drug.

Keywords:

• Antimalarial
• artemether
• nephrosis treatment
• nephrotic syndrome
• proteinuria

Introduction

Artemisinin, a sesquiterpene lactone from Artemisia annua L. (“qinghao,” sweet wormwood), has raised considerable attention in past years (Efferth, 2005). The genus Artemisia L. belongs to the family Composite. More than 350 Artemisia species are known, many of which have been used in traditional folk medicines for various applications (cough, blood circulation, diuresis, hypertension, allergy, parasites, etc.). Sesquiterpene lactones, flavonoids, coumarins, acetylenes, and sterols have been isolated from Artemisia species, some of which reveal antimalarial, antiviral, antitumor, antipyretic, anticoagulant, antispasmodic, and other effects (Tan et al., 1998; Efferth et al., 2002a,b; Haynes et al., 2005; Mahmoud & Botros, 2005; Van der Meersch, 2005). The plant has been used in China for more than two millenia. Its first description dates back to the third century bc. Ge Hong (ad 281–340) recommended tea-brewed leaves to treat fever and chills in his “Handbook of Prescriptions for Emergency Treatment.” The “Compendium of Materia Medica” published by Li Shizen in 1596 cited Ge Hong's prescription. The fact that qinghao tea has withstood the centuries may be taken as a clue for the usefulness and activity of this prescription of traditional Chinese medicine (TCM) (Efferth, 2005).

A program for the discovery of new antimalarial drugs from TCM launched by the Chinese government led in 1972 to the identification of artemisinin (“qinghaosu”), the active principle of A. annua L. (Klayman, 1985; Butler & Wu, 1992). Because of the low solubility of artemisinin in oil and water, several semisynthetic derivatives have been developed, including artemether, arteether, artesunate, and others. The attractiveness of the artemisinin class of antimalarials is due to their activity against multidrug-resistant Plasmodium falciparum and Plasmodium vivax strains (Price et al., 1998; Guthmann et al., 2006; Haynes, 2006). Another salient feature is the lack of severe side effects in malaria patients (Ribeiro & Olliaro, 1998). Several million patients have been treated with these compounds during the past three decades (De Vries & Dien, 1996; Aceng et al., 2005; Falade et al., 2005; Shen & Thiero, 2005; Sutherland et al., 2005; Makanga et al., 2006; Meremikwu et al., 2006; Omari et al., 2006).

The antimalarial artemisinin derivatives also reveal antiangiogenic effect, as well as remarkable antineoplastic activity (Efferth et al., 2002b; Wartenberg et al., 2003; He & Liu, 2004). They are able to inhibit tumor necrosis factor (TNF)-α mRNA expression (Kwiatkowski & Bate, 1995). Artemisinin and its derivatives also inhibit growth of many transformed cell lines. Their growth-inhibitory activity correlated with the induction of apoptosis and prevention of tumor angiogenesis, whereas apoptosis was not observed in normal endothelial cells (Wartenberg et al., 2003; Wu et al., 2004). These drugs inhibit angiogenesis in a dose-dependent manner. They markedly reduce vascular endothelial growth factor (VEGF) binding to its receptors and diminish the expression levels of two major VEGF receptors, Flt-1 and KDR/flk-1, on the surface of human vein endothelial cells (Chen et al., 2003). In addition, it has been shown that they can suppress IL-2 production (Sun, 1991), as well as inhibit inducible nitric oxide synthase and NF-kB activation (Aldieri et al., 2003), mechanisms by which rheumatoid arthritis (RA) could be ameliorated.

The aim of the current research is to assess the therapeutic potency of artemether in an experimental model of nephrosis and whether this drug could be recommended for treatment of glomerular lesions.

Materials and Methods

Animals

A total of 40 female Sprague-Dawley rats weighing 180 ± 20 g were obtained from Razi Institute and housed in our animal facility (temperature, 20–22°C; humidity, 55–56%; 12-h dark cycle; unlimited access to food and water) for at least 1 week before the experiment. The rats were divided at random into four groups: N, normal group (n = 8); P, patient group (n = 11); At, patient group treated with artemether (n = 11); E, experimental healthy control receiving artemether (n = 8). Experimental procedures were performed in accordance with the recommendations and policies of the Iran Pasteur Institute for the protection of animals used for experimental and other scientific purposes.

Experimental protocol and treatment

To induce nephrotic syndrome, adriamycin (Adriablastina; Farmitalia, Milan, Italy) was given once by a single intravenous injection (7.5 mg/kg) through the tail vein. Six days after adriamycin injection, a therapeutic protocol was developed by intramuscular (i.m.) administration of artemether (5 mg/kg) to group At. The total of i.m. injections were 14, in which five injections were made every day and nine injections were carried out at regular 48-h intervals. The therapeutic protocol was terminated on day 28, and animals were sacrificed on day 49. Normal group (N) received nothing, and group (E) was experimental healthy control receiving artemether.

Assessment of kidney function

Measurement of proteinuria was done during the eight stages. With start of treatment, urine was collected before the i.m. injections of artemether. The urine protein was measured using precipitation by trichloroacetic acid. The creatinine clearance was calculated by measuring creatinine concentration in urine and plasma by the alkaline picrate method (Bousnes & Taussky, 1945). Sodium and potassium concentrations were determined by flame photometry. Urine and blood urea nitrogen (BUN) were assessed by the Veniamin method (Veniamin & Vakirtzi-Lemonias, 1970). Serum albumin was measured using the procedure of Bromcresol Green (Doumas et al., 1971).

Evaluation of serum lipid levels

Serum triglyceride and cholesterol were determined by routine laboratory tests on the day of sacrifice.

Renal histopathologic studies

Kidney specimens were processed using light microscopy. Renal tissues were fixed by immersion in 10% buffered formalin, embedded in paraffin, and 4-μ m sections were stained with hematoxylin-eosin and periodic acid–Schiff. The severity and extent of glomerular lesions were blindly evaluated for seven parameters: hypercellularity, glomerular infiltration of PMN and mononuclear cells, karyorrhexis, wire loops, hyalin casts, and cellular swelling (hydropic change) in capillary network within the renal cortex. They were evaluated by a semiquantitative method of renal histology using a grading scale of 0 to 3 (0, negative; 1, mild; 2, moderate; 3, severe).

Toxicologic studies

To evaluate the side effects of artemether in normal rats, the experimental group (E) was studied. These healthy controls received only intramuscular injections of artemether at one dose (5 mg/kg). In this group, the number and interval of injections was 12 and 48 h, respectively. Two days after the last injection, toxicologic studies were done as follows:

1. Assessment of kidney function was performed based on the measurements of urinary protein excretion, serum albumin, urine and plasma creatinine, urine and BUN, and sodium and potassium concentrations in urine and plasma.

2. Evaluation of serum lipid levels was carried out by determining serum triglyceride and cholesterol concentrations.

3. Renal tissues were assessed using light microscopy. Glomerular lesions were graded on a scale of 0 to 3 (0, negative; 1, mild; 2, moderate; 3, marked) according to seven parameters: glomerular infiltration of PMN and mononuclear cells, hypercellularity, karyorrhexis, wire loops, cellular swelling (hydropic change), and the presence of hyalin casts.

Statistical analysis

Data were expressed as means ± SD and ± SEM. Statistical analysis was performed using the Student's t-test for parametric data and Mann-Whitney test for nonparametric data. p values < 0.05 were considered significant.

Results

Effect of artemether on renal function

The changes of the mean levels of urinary protein excretion between N, P, and At are shown in Figure 1. This experiment showed that i.m. administration of artemether (5 mg/kg) could exert therapeutic effects on adriamycin-induced nephropathy. For exact evaluation of artemether (ART) effects, the experiment was terminated 20 days after the last ART injection. The urinary protein excretion was significantly less in ART-treated animals compared with that of nontreated controls (P < 0.05).

Figure 1  Time course of the mean values of proteinuria in different groups of N, P, At in experimental nephrosis. N, normal rats (n = 8); P, patient (nontreated) rats that show increase of proteinuria levels after day 6 (n = 11); At, treated group by intramuscular (i.m.) injection of artemether, 5 mg/kg (n = 11). Note: A single intravascular injection of adriamycin (7.5 mg/kg body weight) induced a severe nephrotic syndrome. Onset of i.m. administration of artemether was day 6 (after development of disease). There were significant differences between nontreated (P) and artemether-treated rats (At). p < .05 was considered to be significant.

Figure 2 shows the effect of ART on urine parameters in groups N, P, At, and E. In Figure 2A, comparison of antiproteinuric effect of ART is made between various groups at the end of the experiment (day 49). Here, reduction of proteinuria in At (48 ± 21) versus P (118 ± 15 mg/24 h) was significant. Figure 2B demonstrates the concentration of urine sodium in different groups, in which At (28 ± 6) vs. P (38 ± 8 mEq/L) was significant. In Figure 2C, the amounts of urine urea nitrogen have been compared between the groups (N, P, At, and E). This figure shows that the difference between treated rats (At) and nontreated animals (P) is significant (414 ± 185 vs. 723 ± 226 mg/dL) “P < 0.05”. In Figure 3, the comparison of serum creatinine concentration among various groups shows that there is a significant difference between At vs. P (0.9 ± 0.2 vs. 1.4 ± 0.2 mg/dL) (p < 0.05).

Figure 2  Effect of artemether on urine parameters in groups: N, normal (8); P, patient (11); At, treated rats with artemether (11); and E, experimental healthy control receiving artemether. Each bar represents mean ± SD. p < .05 vs. nontreated. (A) Comparison of antiproteinuric effect of artemether between groups N, P, At, and E at the end of experiment (day 49); At vs. P was significant. (B) Concentration of urine sodium in groups N, P, At, and E; At vs. P was significant. (C) Amounts of urine urea nitrogen in groups N, P, At, and E; At vs. P was significant.

Figure 3  Effect of artemether on serum creatinine in different groups: N, normal (8); P, patient (11); At, treated with artemther (11); E, experimental healthy control receiving artemther (8). Each bar represents the mean ± SD. Treatment with artemether showed a significant reduction in creatinine level compared with the patient group (p < .05).

Effect of artemether on serum lipid levels

Serum cholesterol and triglyceride levels were significantly elevated (p at least < 0.05) in nephrotic rats (P) when compared with the healthy controls (N) at the end of experiment (60 ± 8 vs. 250 ± 58 mg/dL and 174 ± 55 vs. 427 ± 62 mg/dL, respectively). Intramuscular injections of ART solution to patient rats significantly reduces serum cholesterol in nephrotic animals (250 ± 58 vs. 163 ± 58 mg/dL) (Figure 4).

Figure 4  Effect of artemether on serum cholesterol level in groups: N, normal (8); P, patient (11); At, Treated with artemther (11); E, experimental healthy control receiving artemther (8). Each bar represents the mean ± SD. Decrease in cholesterol concentration in group At vs. P was significant (p < .05).

Histopathologic findings

Light microscopic examination of renal tissue revealed the severity of hypercellularity, glomerular infiltration of PMN, karyorrhexis, wire loops, hydropic change in capillary network within the renal cortex, and existence of tubular casts in the various groups. The animals treated with ART showed a significant reduction in glomerular changes relative to nontreated controls (Figure 5 and Table 1).

Figure 5  Representative histopathologic slides of kidney specimens of a healthy rat (N), a control rat with adriamycin-induced nephropathy (P), a nephrotic animal treated with artemether (At), and healthy controls receiving ART (E). The rats that had been treated with artemether show significantly fewer signs of glomerular destruction (hematoxyline-eosin; original magnification × 40).

Table 1  Light microscopic findings of kidney histologic lesions in various groups.

Groups	No. of rats	Hypercellularity	PMN infiltration	Karyorrhexis	Wire loops	Hydropic change	Hyalin cast	Mononuclear cell infiltration
N	8	0.3 ± 0.3	0	0.2 ± 0.2	0.3 ± 0.2	0.3 ± 0.2	0	0
P	11	2.6 ± 0.3	1.3 ± 0.2	1.3 ± 0.3	2.0 ± 0.2	1.1 ± 0.1	2.6 ± 0.2	1.8 ± 0.3
At	11	0.5 ± 0.2	0.2 ± 0.1	0.5 ± 0.1	0.8 ± 0.1	0.3 ± 0.1	1.0 ± 0.1	1.0 ± 0.1
E	8	0.3 ± 0.2	0	0.1 ± 0.1	0.3 ± 0.1	0.1 ± 0.3	0	0

Values are expressed as mean ± SEM.Semiquantitative scoring of histologic lesions shows, a significant difference between group P vs. N and also P vs. At in hypercellularity, PMN infiltration, karyorrhexis, wire loops, hydropic change, hyalin cast, and mononuclear cell infiltration, whereas there was no significant difference between N vs. E. Treatment with artemether could significantly reduce histologic lesions in kidney.

Toxicologic findings

Table 2 and Table 3 show a comparison between four groups (N, P, At, and E) in serum and urine parameters. Table 2 summarizes the data concerning the effect of ART administration on serum parameters. In this examination, with the exception of triglyceride, there were no significant differences in the levels of serum determinants between the normal group (N) and healthy controls receiving ART (E), whereas, a decrease in triglyceride was significant (174 ± 55 vs. 88 ± 27 mg/dL) (p < 0.05). Table 3 represents the data concerning the effect of ART administration on urine parameters. Data analysis showed a significant difference in amounts of urine sodium (45 ± 5 vs. 26 ± 6 mEq/L), potassium (71 ± 14 vs. 53 ± 6 mEq/l), and urine urea (720 ± 113 vs. 511 ± 211 mg/dL), between the normal group (N) versus E (p < 0.05). Histologic examinations of kidney specimens obtained from N and E groups showed no glomerular changes in group E compared with group N (Table 1).

Table 2  Effect of artemether on serum determinants.

Groups	No. of	BUN rats(mg/dL)	Cholesterol (mg/dL)	Triglyceride (mg/dL)	Creatinine (mg/dL)	Potassium (mEq/L)	Sodium (mEq/L)	Albumin (g/dL)
N	8	26 ± 4	60 ± 8	174 ± 55	0.7 ± 0.2	4.7 ± 0.3	137 ± 6	6.5 ± 0.7
P	11	29 ± 3	250 ± 58	427 ± 62	1.4 ± 0.2	6.0 ± 0.7	159 ± 17	3.3 ± 0.5
At	11	25 ± 7	163 ± 58	360 ± 149	0.9 ± 0.2	5.1 ± 1.0	162 ± 5	5.6 ± 0.5
E	8	21 ± 5	75 ± 12	88 ± 27	0.7 ± 0.1	4.7 ± 1.2	143 ± 9	5.8 ± 0.3

Values are expressed as mean ± SD. N, normal; P, patient; At, artemether-treated rat; E, experimental healthy group under challenge with 12 intramuscular injections of 5 mg/kg/48 h artemether. Decrease in cholesterol and creatinine and increase in level of albumin in treated group with artemether compared with patient group was significant In addition, reduction of triglyceride in E compared with N group was significant (p < .05).

Table 3  Effect of artemether on urine determinants.

Groups	No. of rats	Protein (mg/24h)	Urea (mg/dL)	Creatinine (mg/dL)	Potassium (mEq/L)	Sodium (mEq/L)
N	8	6.0 ± 0.4	720 ± 113	151 ± 16	71 ± 14	45 ± 5
P	11	118 ± 15	723 ± 226	185 ± 45	64 ± 9	38 ± 8
At	11	48 ± 21	414 ± 185	150 ± 18	74 ± 10	28 ± 6
E	8	3.4 ± 0.5	511 ± 211	200 ± 10	53 ± 6	26 ± 6

Data represent mean ± SD. N, normal; P, patient; At, artemether-treated rat; E, experimental healthy group under challenge with 12 intramuscular injections of 5 mg/kg/48 h artemether. Treatment with artemether caused a significant reduction in level of urine protein, urea, and sodium compared with group P. Moreover, difference between N and E groups in amounts of urine sodium, potassium, and urea were significant. p < .05 was considered to be significant.

Discussion

This study was developed in an attempt to test the therapeutic effect of artemether as a new antimalarial drug in experimental nephrotic syndrome. This syndrome is characterized by massive proteinuria, edema, hyperlipidemia, and hypoalbuminemia (Cunard & Kelly, 2003). Adriamycin-induced nephropathy serves as an experimental model for minimal change nephropathy, and focal and segmental glomerulosclerosis (Remuzzi et al., 1985). Oxidative stress and superoxide, hydrogen peroxide, and, consequently, hydroxyl radical production are suggested to play a role in its pathogenesis (Okasora et al., 1992; Zima et al., 1998), and cytokines released by circulating blood mononuclear cells and/or resident renal cells could alter the permeability of the glomerular capillary wall (Bustos et al., 1995). Accumulated evidence indicates increased expression of IL-2 mRNA, a significant increase in TNF-α and nitric oxide (NO) production levels, as well as a significant increase in plasma and urinary VEGF levels in human and experimental model of nephrotic syndrome, suggesting that IL-2, TNF-α, NO, and VEGF, at least in part, might be involved in the pathophysiology of this disease (Ozen et al., 2001; Zhu et al., 2001; Lama et al., 2002; Zachwieja et al., 2002, 2003; Klahr & Morrissey, 2004; Shimoyama et al., 2004; Usta et al., 2004; Deepa & Varalakshmi, 2006; Wasilewska et al., 2006; Wasilewska & Zoch-Zwierz, 2006), whereas the antimalarial artemisinin derivatives markedly reduce VEGF binding to its receptors and diminish the expression levels of two major VEGF receptors, Flt-1 and KDR/flk-1, on the surface of human vein endothelial cells (Chen et al., 2003). In addition, it has been shown that they can suppress IL-2 production (Sun, 1991) and inhibit inducible nitric oxide synthase, and they are able to suppress (TNF-α mRNA expression (Kwiatkowski & Bate, 1995). Our findings show that artemether therapy not only is able to reduce proteinuria, but also it could significantly diminish urine urea and sodium compared with nontreated controls. In addition, decrease in serum creatinine and increase in the level of serum albumin were significant in the group treated with ART compared with the patient group. Moreover, our data in the current work revealed that treatment with ART significantly reduces serum cholesterol levels in the treated patient rats in experimental nephrosis. Nephrotic syndrome results in downregulation of LDL receptor expression, which, in turn, contributes to the pathogenesis of hypercholesterolemia and dysregulation of cholesterol synthesis and catabolism (Pesek-Diamond et al., 1992; Vaziri & Liang, 1996). Our results show that plasma cholesterol concentration in the nephrotic animals and normal group are in agreement with the studies of Vaziri and Liang (1996). Also, ART therapy not only did not exacerbate renal damage, but interestingly, administration of this drug could significantly diminish renal recruitment of leukocytes and reduce glomerular lesions in experimental model of nephrotic syndrome. On the other hand, healthy controls receiving ART showed a significant decrease in amounts of serum triglyceride, urine urea, and urine sodium and potassium compared with the normal group. Here, the significant reduction in amount of urine urea and urine sodium after ART therapy in the treated group and healthy controls receiving ART compared with the normal group may be an important factor for a further precision in administration of this drug.

In conclusion, a significant reduction in proteinuria, serum cholesterol, and serum creatinine, as well as suppression of glomerular lesions after artemether therapy suggests its therapeutic benefit in the treatment process of experimental nephrosis. But in long-term administration of this drug, reduction of urinary excretion of sodium and urine urea must be considered as a side effect of artesunate therapy.