Treatment of Experimental Nephrotic Syndrome with Artesunate

Abstract

The present study was designed to test the therapeutic effect of a new antimalarial drug, artesunate in experimental model of nephrotic syndrome. To induce this experimental model, Adriamycin was given once by a single intravenous injection (7.5 mg/kg) through the tail vein. Six days after injection of Adriamycin, therapeutic protocol was developed by intraperitoneally (IP) administration of 5 mg/kg artesunate (ARS). Total of IP injections were 14, of which 5 injections were made every day and 9 injections were carried out at regular 48-h intervals. Therapeutic protocol was terminated on day 28 and animals were killed on day 49. The results showed that treatment with ARS caused a significant reduction in the level of proteinuria, urine urea and urine sodium compared with nontreated controls. In addition, decrease in serum triglyceride and increase in the level of serum albumin was significant in treated group with ARS compared with nontreated controls. Moreover, treatment with ARS significantly reduced glomerular polymorphonuclear (PMN) and mononuclear cells infiltration, hypercellularity, karyorrhexis, wire loops, and hydropic change in capillary network within the renal cortex, as well as decreased hyalin casts. On the other hand, healthy controls receiving ARS showed a significant decrease in amounts of serum triglyceride, urine urea, and urine sodium and potassium compared with normal group. These data suggest that artesunate therapy can ameliorate proteinuria, and suppress the progression of glomerular lesions in experimental model of nephrotic syndrome; it may also be recommended as a lipid-lowering drug.

Artesunate (ARS) is a semisynthetic derivative of the sesquiterpene artemisinin used for the second line therapy of malaria infections (Dell’Eva et al. 2004). The story starts in China with Artemisia annua L. A crude extract of the wormwood plant Artemisia annua (qinghao) was first used as an antipyretic 2000 years ago (Davis et al. 2005; Van der Meersch 2005). Ge Hong (281–340 ad) 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 (Efferth 2005). The specific effect of this plant on the fever of malaria was reported in the 16th century and active constituent of the extract was identified and purified in the 1970s, and named qinghaosu, or artemisinin (Davis et al. 2005; Klayman 1985). Although artemisinin proved effective in clinical trials in the 1980s, a number of semisynthetic derivatives (artesunate, arteether, artemether, artelinate, artemisitene, dihydroartemisinylester stereoisomers) were developed to improve the drug’s pharmacological properties and antimalarial potency (Hien and White 1993). Several million patients have been treated with these compounds during the past three decades (De Vries and Dien 1996; Denis et al. 2006; Bhatt et al. 2006; Guthmann et al. 2006; Duffy and Mutabingwa 2006). The antimalarial artemisinin derivatives also reveal remarkable antineo-plastic activity (Efferth et al. 2002). Of the available derivatives, artesunate has the most favorable pharmacological profile for use in artemisinin-based combination therapy of uncomplicated malaria. The presence of a hemisuccinate group in this molecule confers water solubility and relatively high oral bioavailability. It is rapidly and quantitatively converted in vivo to the potent active metabolite dihydroartemisinin (Davis et al. 2001). It has been reported that ARS has protective effect on lipopolysaccharide (LPS)-induced vascular endothelial cells activation and injury, which might be related with its inhibition on tumor necrosis factor alpha (TNF-α) mRNA expression (Kwiatkowski and Bate 1995; He and Liu 2004). ARS also inhibits growth of many transformed cell lines. Its growth inhibitory activity correlated with the induction of apoptosis and prevention of tumor angiogenesis, whereas apoptosis was not observed in normal endothelial cells (Dell’Eva et al. 2004; He and Liu 2004; Wu et al. 2004). Artemisinin and its derivatives significantly 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, artemisinin and its derivative are able to suppress interleukin (IL)-2 production (Sun 1991), as well as inhibit inducible nitric oxide synthase and nuclear factor (NF)-κB activation (Aldieri et al. 2003). Nephrotic syndrome is characterized by glomerular epithelial cell injury and a decrease in the glomerular basement membrane proteoglycan content (Bustos et al. 1995; Vasudevan et al. 2004). The characteristic of this syndrome is massive proteinuria with fusion of the epithelial cells, edema, hyperlipidemia, and hypoalbuminemia (Kanwar 1991; Cunard and Kelly 2003; Franceschini et al. 2006). Focal segmental glomerulosclerosis (FSGS) is the leading cause of nephrotic syndrome in an adult worldwide. The prevalence of FSGS is estimated as being 20% to 30% in adults over the age of 15 years and slightly higher (30% to 35%) in the elderly “age > 60 years” (Hirayama and Koyama 2004; Kanjanabuch et al. 2006). The minimal change nephrotic syndrome is the most common diagnosis associated with nephrotic syndrome in children and accounts for 10% to 15% of nephrosis in adults (Nolasco et al. 1986; Bagga and Mantan 2005; Saha and Singh 2006). A long-term cortico-steroid therapy is a first therapeutic approach for patients with nephrotic syndrome. In patients who have contraindication to steroids or in those who do not respond to steroids, immunosuppressive agents such as cyclosporine, mizoribine, azathioprine, and cyclophosphamide are the next therapeutic approach for inducing the remission of the nephrotic syndrome (Takeda and Tomino 2004). There are several experimental models for evaluating the therapeutic efficacy of various drugs and study of pathophysiology of this syndrome. The experimental model of nephrotic syndrome can be induced by Adriamycin (Bertani et al. 1986a; Bertani et al. 1986b; Okuda et al. 1986; Tsuchida et al. 2004) and/or puromycin aminonucleoside (Nakayama et al. 2004). Rats that are given Adriamycin develop heavy proteinuria within a few weeks after Adriamycin administration. Reactive oxygen species produced during metabolism of Adriamycin are purported to play an important role in the pathogenesis of experimental Adriamycin nephropathy in rats (Zima et al. 1998; Javaid et al. 2001). This study was designed based on the pathogenesis of the nephrotic syndrome, the association of chronic renal failure with abnormal activation of NF-κB, nitric oxide synthase, and proinflammatory cytokines production (especially TNF-α and IL-2), and the inhibitory effect of artesunate on mentioned parameters (Furusu et al. 1998; Cao et al. 2001; Zachwieja et al. 2003; Pogan et al. 2005; Zhao et al. 2005; Koriakova et al. 2006). The aim of the present research was to determine whether the artesunate could suppress the development of nephrotic syndrome caused by injection of Adriamycin in experimental model.

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°C to 22°C; humidity 55% to 56%; 12-h light/12-h dark cycle; unlimited access to food and water) for at least one week before the experiment. The rats were divided at random into four groups: (1) N: normal group (vehicle; n = 8); (2) P: patient group (Adriamycin + vehicle; n = 11); (3) As: treated group (Adriamycin + artesunate; n = 11); (4) E: experimental group (artesunate + vehicle; 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 on day 0. Six days after injection of Adriamycin, therapeutic protocol was developed by intraperitoneally (IP) administration of artesunate (5 mg/kg) to treatment group (As). Total of IP injections were 14, of which 5 injections were made every day and 9 injections were carried out at regular 48-h intervals. Therapeutic protocol was terminated on day 28. In order to have a final evaluation of serum and urine determinants, blood samples were obtained at sacrifice by heart puncture after collecting urine on day 49. Animals were killed on day 49 and kidneys were removed.

Assessment of Kidney Function

Measurement of proteinuria was carried out during the eight stages (weeks 0 to 7). With start of treatment on day 6, 24-h urine was collected from rats placed in metabolic cages once a week. The urine protein was measured using trichloroacetic acid turbidimetric method for microproteinuria screening in rat (Cheung et al. 1987; Shishido and Sudo 1988; Khatami et al. 2005). The creatinine clearance was calculated by measuring creatinine concentration in urine and plasma by the alkaline picrate method (Bousnes and Taussky, 1945). Sodium and potassium concentrations were determined by flame photometry. Urine and blood urea nitrogen (BUN) were assessed by the Veniamin method (Veniamin and Vakirtzi-Lemonias, 1970). Serum albumin was measured using the Doumas et al. procedure (Doumas et al. 1971).

Evaluation of Serum Lipid Levels

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

Renal Histopathological Studies

Kidney specimens were processed using light microscopy. Renal tissues were fixed by immersion in 10% buffered formalin, embedded in paraffin, and 4-micron sections were stained with hematoxylineosin and periodic acid–Schiff. The severity and extent of glomerular lesions were blindly evaluated in seven parameters: hypercellularity, glomerular infiltration of polymorphonuclear (PMN) and mononuclear cells, karyorrhexis, wire loops, hyalin casts, and cellular swelling (hydropic change) in capillary network within the renal cortex. These parameters 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).

Toxicological Studies

To evaluate the side effects of artesunate in normal rats, experimental group (E) was studied. These healthy controls received only intraperitoneally injections of artesunate at 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, toxicological 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 blood urea nitrogen (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 < .05 were considered significant.

RESULTS

Effect of Artesunate on Renal Function

The changes of the mean levels of urinary protein excretion between N (vehicle), P (Adriamycin + vehicle), and As (Adriamycin + artesunate; treated group) are shown in Fig. 1. This experiment showed that IP administration of artesunate (5 mg/kg) could exert its therapeutic effects on Adriamycin-induced nephropathy based on significant reduction of proteinuria in treatment group (As). For exact evaluation of artesunate (ARS) effects, the experiment was terminated 20 days after the last ARS injection. The urinary protein excretion was significantly less in ARS-treated animals compared to nontreated controls (p < .05).

Fig. 2 shows the effect of ARS on urine parameters (protein excretion, Sodium and urea nitrogen) with significant differences in groups N, P, As, and E. In Fig. 2A , comparison of antiproteinuric effect of ARS is made between various groups at the end of experiment (day 49). Here As versus P was significant. Fig. 2B demonstrates the concentration of urine sodium in different groups, in which As verus P was significant. In Fig. 2C , the amounts of urine urea nitrogen have been compared between the groups (N, P, As, and E). This figure shows that the difference between treated rats (As) and nontreated animals (P) is significant (p < .05).

Effect of Artesunate on Serum Lipid Levels

Serum cholesterol and triglyceride levels were significantly elevated (p at least <. 05) in nephritic rats “P” (Adriamycin + vehicle) when compared with the healthy controls at the end of experiment. Intraperitoneally injections of ARS solution to patient rats significantly reduce serum triglyceride in nephrotic animals (Fig. 3).

Histopathological 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 ARS showed a significant reduction in glomerular changes than nontreated controls (Fig. 4 and see Tab. 3).

Toxicological Findings

Tab. 1 and 2 show a comparison between four groups (N, P, As, and E) in serum and urine parameters. Tab. 1 summarizes the data concerning the effect of ARS 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 ARS (E), whereas a decrease in triglyceride was significant (p < .05). Tab. 2 represents the data on the effect of ARS administration on urine parameters. Data analysis showed a significant difference in amounts of urine sodium and potassium and urine urea between the normal group (N) and group E (p < .05). A significant decrease in urinary excretion of sodium as well as urine urea following treatment with artesunate could be proposed as side effects of this drug. Histological examinations of kidney specimens obtained from N and E groups showed no glomerular changes in group E compared with group N following ARS administration (Tab. 3).

DISCUSSION

The literature data assembled in this article document the therapeutic effect of artesunate as a new antimalarial drug in experimental model of nephrotic syndrome. This syndrome is characterized by massive proteinuria, edema, hyperlipidemia, and hypoalbuminemia (Cunard and 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 super-oxide, hydrogen peroxide, and, consequently, hydroxyl radical production are suggested to play a role in glomerulosclerosis pathogenesis (Okasora et al. 1992; Zima et al. 1998); also 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 vascular endothelial growth factor (VEGF) levels in humam 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 (Zhu et al. 2001; Ozen et al. 2001; Zachwieja et al. 2002; Lama et al. 2002; Zachwieja et al. 2003; Shimoyama et al. 2004; Usta et al. 2004; Klahr and Morrissey 2004; Deepa and Varalakshmi 2006; Wasilewska and Zoch-Zwierz 2006; Wasilewska et al. 2006). On the other hand, 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 and Bate 1995). Our findings show that artesunate therapy not only is able to reduce proteinuria, but it could also significantly diminish urine urea and urine sodium and potassium compared with nontreated controls. In addition, increase in the level of serum albumin was significant in group treated with ARS compared with patient group. Moreover, our data in the current work revealed that treatment with ARS significantly reduced serum triglyceride levels in treated rats in the experimental model of nephrotic syndrome. This syndrome results in down-regulation of low-density lipoprotein (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 and Liang 1996). Our results showing that plasma triglyceride concentration in the nephrotic animals and normal group are in agreement with the studies of Vaziri and Liang (1996). Also, ARS 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 this experimental model. On the other side, healthy controls receiving ARS showed a significant decrease in amounts of serum triglyceride, urine urea, and urine sodium and potassium compared with normal group. This study (Tab. 1 and 2) showed that treatment with artesunate caused an elevation in serum sodium as well as a decrease in urinary excretion of sodium as further indication of dehydration. Here, the significant reduction in amounts of urine sodium and urine urea following ARS therapy in treated group and healthy controls receiving ARS compared with normal group may be an important factor for a further investigation of the precise administration of this drug.

To conclude, a significant reduction in proteinuria and serum triglyceride as well as suppression of glomerular lesions following artesunate therapy suggests its therapeutic benefit in the treatment process of experimental nephrotic syndrome. But in long-term administration of this drug, reduction of urinary excretion of sodium and urine urea must be considered as side effects of artesunate therapy.