Nephroprotective Effect of Withania somnifera: A Dose-Dependent Study

Abstract

In the present study, we investigated the protective effect of Withania somnifera, an indigenous medicinal herb used in ayurvedic traditional systems for more than 3000 years in India, on gentamicin (GEN)-induced nephrotoxicity. The root extract of three different doses of W. somnifera (viz., 250, 500, and 750 mg/kg) was administered orally to rats for 14 days before GEN treatment and thereafter concurrently with GEN (100 mg/kg) for 8 days. Nephrotoxicity was evident in GEN-treated rats by significant increase in kidney weight, urea, creatinine, urinary protein, and glucose, and significant reduction in body weights and potassium, which was histopathologically confirmed by tubular necrosis. In contrast W. somnifera (500 mg/kg) significantly reversed these changes as evidenced microscopically when compared to other two doses of W. somnifera (250 and 750 mg/kg), and there were no significant changes in the levels of sodium in the experimental animals compared to control. Thus, our results suggested the nephroprotective effect of Withania somnifera, which could be by enhancing antioxidant activity with natural antioxidants and scavenging the free radicals.

Keywords:

• nephrotoxicity
• W. somnifera
• GEN
• rats

INTRODUCTION

Gentamicin (GEN), an aminoglycoside antibiotic that occupies fourth place among antibiotics, was found to be effective in treating gram-negative bacterial infections,^[1] and is also widely used for its low cost and the low levels of resistance, especially among the Enterobacteriaceae family bacterium.^[2] Despite their beneficial effects, they are well known to cause high incidence of nephrotoxicity and ototoxicity. The administration of GEN for more than seven days shows symptoms of nephrotoxicity,^[3] which complicates and increases the cost of the treatment. This shows a need for research to find effective practices that can protect or reverse gentamicin-induced nephrotoxicity. GEN-induced nephrotoxicity is characterized by an increase in serum creatinine, urea concentration, and renal tubular necrosis, followed by deterioration and renal failure.^[4,5] Although many reports suggest that the pathophysiology of GEN-induced nephrotoxicity is unclear, researchers found that in GEN-treated rats, reactive oxygen species (ROS) including superoxide anion (O₂^−•),^[6] hydrogen peroxide (H₂O₂),^[7–9] and hydroxyl radicals (HO) were found to be enhanced in both in vivo (renal cortex) and in vitro (mitochondrial) models. It was also confirmed by others that ROS participate in all pathological conditions, including glomerular disease,^[4] and in renal ischemia and reperfusion injury^[10] and other toxic renal failure.^[11] In addition, oxidative stress releases iron from mitochondria of renal cortex and forms an iron-GEN complex known as ferrous iron, which is a potent catalyst of free radicals and enhance the generation of ROS.^[12–14] Thus, previous studies confirm that there is a relationship between oxidative stress and nephrotoxicity.

The World Health Organization found that 80% of the world's population depends on medicinal plants for their healthcare needs, and more than 30% of the pharmaceutical preparations are based on plants.^[15] Withania somnifera, commonly known as ashwagandha or winter cherry, is used for therapeutic purpose in Indian traditional medicine for more than 3000 years for its ability to strengthen the immune system. The ashwagandha, belonging to the family Solanceae, is an erect, evergreen tomentose shrub, grown wild and also cultivated for medicinal use in many parts of India and all over the world. Among the Indian medicinal plants, thirteen positive alkaloids and to date around 138 withanolides have been reported from W. somnifera (see Table 1),^[16] and they are reported to have many medicinal properties, such as immunomodulatory,^[17] anti-inflammatory,^[18] endocrine,^[19] anti-stress,^[20] anti-cancer,^[21 adaptogenic,^[22] anti-tumor,^[23] central nervous system,^[24,25] cardiovascular,^[26,27] and neuroprotective^[28] activities, respectively. Among the other parts of this plant, the root has been considered to be the most active part for therapeutic purposes.^[26,29]

Table 1  Active compounds of Withania somnifera^[35]

Name / S. No.
Withaferin A
Diacetylwithaferin A
2,3-Dihydrowithaferin A
Withanolide viscosa lactone B
27-o-β-D-Glucopyranosyl viscosa lactone B
Dihydrowithaferin A
27-o-Glucopyranosylwithaferin A
Withanoside I-VII
24,25-Dihydro-27-desoxywithaferin A
Physagulin D(1→6)-β-D-glucopyranosyl-(1→4)-β-D-glucopyranoside
Sitoindosides VII-X
27- o-β-D-Glucopyranosylphysagulin D
5-Dehydroxywithanolide R
Withasomniferin A
1-Oxo-5β,6β-epoxy-witha-2-ene-27-ethoxy-olide
4-(1-Hydroxy-2,2-dimethylcyclopropanone)-2,3-dihydrowithaferin A
D-Glucopyranosyl
D-Glucopyranoside
4,16-Dihydroxy-5β,6β-epoxyphysagulin D

Investigators also observed that the kidneys from GEN-treated rats are more vulnerable to ROS due to deficiency of the antioxidant enzymes.^5,[30–32] In general, it was found that flavonoids and polyphenolic compounds are potent antioxidants and are known to modulate the activities of various enzyme systems due to their interaction with various biomolecules and scavenge the free radicals.^[33] Previously, Prakash et al.^[34] stated that W. somnifera extract, for its antioxidant status and free radical scavenging property on 7,12-dimethylbenz(a)anthracene, induced skin cancer in Swiss albino mice. Furthermore, the antioxidant and free radical scavenging properties of W. somnifera were also studied in fore stomach and skin carcinogenesis in mice.^[35–37] Taking into account the medicinal properties of W. somnifera, we hypothesize that W. somnifera would work in a dynamic way to give maximum therapeutic efficacy in the prevention or reversal of drug-induced nephrotoxicity without causing any side effects.

MATERIALS AND METHODS

Plant Material

The commercially available powdered root of W. somnifera obtained from Indian Medical Practitioners Co-operative Society (IMCOPS), Adyar, Chennai, India, was used, and its aqueous suspension in 2% gum acacia was administered orally at 250 mg, 500 mg, and 750 mg/kg body weight daily.

Chemicals

GEN sulphate was purchased from Ranbaxy Laboratories Limited, New Delhi, India. All other chemicals used in the study were of analytical grade.

Animal Maintenance

Adult male albino Wistar rats, weighing 180–200g and bred in the Central Animal House, Rajah Muthiah Medical College, Annamalai University, were used. The animals were housed in polycarbonate cages in a room with a 12 h day-night cycle, temperature of 22 ± 2°C, and humidity of 45–64%. Animals were fed with a standard pellet diet (Hindustan Lever Ltd., Mumbai, India) and water ad libitum. All animal experiments were approved by the ethical committee (Vide. No. 538/2008) of Annamalai University and were in accordance with the guidelines of the National Institute of Nutrition (NIN), Indian Council of Medical Research (ICMR), Hyderabad, India.

Experimental Design

Male albino Wistar rats (body weight 180–200g) will be used in our study. After one week of acclimatization period, rats were stratified by body weight and assigned into eight groups of six animals each:

• group 1: control rats were injected with 0.5 ml isotonic saline;

• groups 2–4: rats administered with Withania somnifera (250 mg, 500 mg, and 750 mg/kg, respectively) via intragastric intubations;

• group 5: rats treated with GEN intraperitoneally (100 mg/kg) for last eight days;

• groups 6–8: rats treated with Withania somnifera (250 mg, 500 mg, and 750 mg/kg, respectively, for 14 days before GEN) + GEN (100 mg/kg from day 15 to 22 days) via intragastric intubations.

After dosing on the eighth day, rats were maintained in metabolic cages with a 12-hour light:dark cycle to collect 24-hour urine, which was stored at -20°C for the determination of protein and glucose using Spectrophotometer and Hitachi 912 auto analyzer (Borinhger, Germany). Animals were sacrificed on day 9; before sacrifice, changes in body weight were recorded and blood was collected to obtain plasma, which was stored at −80°C until creatinine, urea, sodium, and potassium levels were determined by using Spectrophotometer and Hitachi 912 auto analyzer (Borinhger, Germany). The kidneys were removed immediately after sacrifice, weighed, and processed for histopathological examination.

Biochemical Determinations

Levels of urea, sodium and potassium were measured in the plasma according to standard methods stated elsewhere.^[38] Creatinine levels were estimated in plasma following an alkaline picrate method.^[38] The absorbance of each of the previous parameters was read at 480 nm using UV spectrophotometer-Hitachi 912. Blank and a series of standards were processed similarly.

Histopathological Examination

The left kidney was sectioned longitudinally in two halves and fixed in 10% buffered neutral formalin, dehydrated in graded alcohol, embedded in paraffin wax, sectioned at 5μm-thickness, and stained with hematoxylin and eosin (H&E) for light microscopic examination. Renal tubular necrosis was assessed on a score previously described^[39] and as follows: +, mild usually single-cell necrosis in sparse tubules; ++, moderate, more than one cell involved in sparse tubules; +++, severe necrosis in almost every power field; ++++, massive total necrosis.

Statistical Analysis

All data were expressed as mean ± SD. The statistical significance was evaluated by one-way of analysis of variance (ANOVA) using SPSS version 11.5 (SPSS, Cary, North Carolina, USA), and the individual comparison was done by Duncan's multiple range test (DMRT).

RESULTS

Effects of GEN and W. somnifera on Kidney Weights and Body Weights

GEN-treated rats were found to be significantly higher in kidney weights and significantly lower in body weights in comparison with control rats. Treatment with W.somnifera (500 mg) reversed the effects caused by GEN, significantly when compared with low (W. somnifera, 250 mg) and high (W. somnifera, 750 mg) doses, as shown in Figure 1.

Figure 1.  A. Effect of W. somnifera on changes in body weight of normal and experimental rats. Values are given as mean ± SD from six rats in each group. Values not sharing a common superscript (a, b, c, d) differ significantly at p < 0.05 (DMRT). B. Effect of W. somnifera on changes in kidney weight of normal and experimental rats. Values are given as mean ± SD from six rats in each group. Values not sharing a common superscript (a, b, c, d) differ significantly at p < 0.05 (DMRT).

Effects of GEN and W. somnifera on Biochemical Variables

The levels of plasma urea and creatinine were significantly elevated, and plasma potassium levels were significantly declined in GEN-treated rats when compared with control. On the other hand, treatment with W. somnifera (500 mg) significantly reduced the urea and creatinine when compared with low (W. somnifera, 250 mg) and high (W. somnifera, 750 mg) doses. Sodium levels show no significant changes in all the groups compared to control (see Figure 2).

Figure 2.  C. Effect of W. somnifera on plasma urea of normal and experimental rats. Values are given as mean ± SD from six rats in each group. Values not sharing a common superscript (a, b, c, d) differ significantly at p < 0.05 (DMRT). D. Effect of W. somnifera on the levels of plasma creatinine of normal and experimental rats. Values are given as mean ± SD from six rats in each group. Values not sharing a common superscript (a, b, c, d) differ significantly at p < 0.05 (DMRT). E. Levels of urinary protein of normal and experimental rats. Values are given as mean ± SD from six rats in each group. Values not sharing a common superscript (a, b, c, d) differ significantly at p < 0.05 (DMRT). F. Levels of urinary glucose of normal and experimental rats. Values are given as mean ± SD from six rats in each group. Values not sharing a common superscript (a, b, c, d) differ significantly at p < 0.05 (DMRT). G. Effect of W. somnifera on changes in sodium and potassium levels of normal and experimental rats. Values are given as mean ± SD from six rats in each group. Values not sharing a common superscript (a, b, c, d) differ significantly at p < 0.05 (DMRT).

Urinary protein and glucose levels were significantly increased in GEN-treated rats when compared with control, and treatment with W. somnifera (500 mg) significantly decreased the urinary protein and glucose levels when compared with low (W. somnifera, 250 mg) and high (W. somnifera, 750 mg) doses (see Figure 2).

Effects of GEN and W. somnifera Treatment on Kidney Histology

Table 2 and Figures 3A–3E show the histological changes in kidneys. The renal histology of control rats appears normal (see Figure 3A), and GEN-treated rats show a moderate degree of tubular necrosis (+++) (see Figure 3B). After pretreatment of W. somnifera (250, 500, and 750 mg/kg b.wt), W. somnifera (500 mg/kg b.wt) significantly reduced the tubular necrosis (+) (see Figure 3C) compared to other two doses (250 mg and 750 mg/kg b.wt), where slightly decreased the GEN-induced tubular necrosis (++) (see Figures 3D and 3E).

Figure 3.  A. Control rats (renal tubules appears normal) (H & E 100×). B. GEN (→) severe renal tubular necrosis and interstitial inflammatory infilterate (H & E 100×). C. GEN + W. somnifera (250 mg/kg b.wt) moderate necrosis and interstitial inflammatory infiltrate (H & E 100×). D. GEN + W. somnifera (500 mg/kg b.wt) mild necrosis, no casts and inflammatory infiltrate (H & E 100×). E. GEN + W. somnifera (750 mg/kg b.wt) less necrosis, cast with tubules and mild inflammatory infiltrate (H & E 100×).

Table 2  Renal histological changes in rat kidney

Group	Tubular necrosis (arbitory score)
Control rats	―
GEN	+++
GEN + W. somnifera (250 mg/kg b.wt)	++
GEN + W. somnifera (500 mg/kg b.wt)	+
GEN + W. somnifera (750 mg/kg b.wt)	++

DISCUSSION

Gentamicin is an aminoglycoside antibiotic for which therapeutic use is limited, due to its severe nephrotoxicity.^[1] The present study confirmed that GEN at a dose of 100 mg/kg b.w for eight days produces nephrotoxicity,^[40,41] as evidenced by the impairment in glomerular function. Researchers also observed that creatinine (Cr) level is a more accurate measurement for glomerular impairment than urea levels/Cr clearance.^[42,43] Our study results coincide with earlier reports that a significant increase in plasma urea and creatinine or decrease in potassium levels^[40,41,44] might indicate a nephrotoxic condition in GEN-treated rats and may be due to kidney damage caused by the enhanced generation of ROS. These findings correlated well with the renal histological examination, which revealed renal tubular necrosis and interstitial nephritis. Previous studies found that GEN is capable of enhancing the formation of ROS, including O₂^−•[6] and H₂O₂^[7–9] radicals that stimulate^[45] the mesangial cells contraction, and decreasing the GFR without alternating the glomerular structure. It was also observed that increased O₂^−• could react with nitric oxide (NO, a vasodilator) to form peroxynitrite, a cytotoxic oxidant radical specie, which decreases the GFR.^[46] However, many investigators strongly reported that potent natural or synthetic agents that scavenge or interfere with ROS production can successfully ameliorate GEN-induced nephropathy.^{[5,6,32,47–49]}

In our study, after pretreatment of W. somnifera at three different dose levels of 250, 500, and 750 mg/kg b.wt, the administration of W. somnifera 500 mg/kg b.wt was more effective in decreasing the raised plasma urea and creatinine levels, bringing about marked recovery in kidneys, as evidenced microscopically when compared to other two doses of W. somnifera (250 and 750 mg). Marked reduction in these levels might be due to the presence of W. somnifera's potent antioxidant substances, such as its alkaloids, withanolides, and few flavonoids. In particular, glycowithnolide substances sitoindosides VII-X and withaferin A, which are rich in antioxidant properties,^[34–36] enhance the antioxidant status and in turn may quench the free radicals generated in GEN-induced nephrotoxicity. Former reports from our lab showed that W. somnifera normalized the levels of urea, creatinine, uric acid, and non-protein nitrogen by increasing the antioxidant status in hyperammonemic conditions.^[50] In addition, W. somnifera proved to have nephroprotective effect in cyclophosamide-induced urotoxicity,^[51] and in lead-induced oxidative damage proved its hepatoprotective effects.^[52]

Our results showed an increase in kidney weight and decrease in body weight in GEN-treated rats. This is in agreement with the previous reports, that GEN-treated rats showed a significant increase in kidney weight due to edema caused by tubular necrosis^[39] and a decrease in body weight due to loss of appetite.^[39,43] On the other hand, treatment with W. somnifera (250, 500, and 750 mg/kg b.wt) dose-dependently increased the body weight, where W. somnifera (500 mg/kg b.wt) showed a marked increase in the body weight compared to other two doses. Previously, Sharma et al.^[53] noticed in their work that the rats treated with W. somnifera have gained weight and were also found to be healthier than normal rats. More recently, W. somnifera, an Indian ginseng sold in the U.S. market without prescription as a dietary supplement,^[37,54] has structural similarities with the bioactive components present in panax ginseng called ginsenosides. Many toxicological studies in animal models reported that Withania somnifera is nontoxic and proved to be very safe to humans as expected. Our present findings corroborate these reports.

Furthermore, it has been observed that the phytochemicals present in W. somnifera are responsible for overcoming the excitotoxicity and oxidative damage.^[36,55] Therefore, our results are in accordance with these earlier reports, and the therapeutic activity of W. somnifera was also supported by its numerous bioactive components, including withaferin A, sitoindosides VII–X, 5-dehydroxy withanolide-R, withasomniferin-A, 1-oxo-5β, 6β-epoxy-witha-2-ene-27-ethoxy-olide, 2,3-dihydrowithaferin A, 24,25-dihydro-27-desoxy withaferin A, 27-Ο-β-D-glucopyranosyl physagulin D, physagulin D, withanoside I–VII,27-O-β-D-glucopyranosylviscosalactoneB,4,16-dihydroxy-5β,6β-epoxyphysagulinD, viscosa lactone B, and diacetyl withaferin A,^[56,57] which might reverse the damage caused in renal cells.

Our study results on selecting the effective dose of W. somnifera (i.e., 250, 500, or 750 mg/kg) on GEN-induced nephrotoxicity confirmed that 500 mg of W. somnifera was more prominent in protecting kidney against GEN-induced nephrotoxicity, though the exact mechanism underlying this is unclear. We believe that the concentration of low dose W. somnifera (250 mg) might not be sufficient to scavenge the radicals, yet the high dose of W. somnifera (750 mg) might interact with some other molecules instead of radicals. Thus, in this work, we conclude that the maximum therapeutic efficacy by which W. somnifera exhibits the nephroprotective effect could be attributed first by promoting enhanced antioxidant activity with natural antioxidants and then by scavenging the free radicals. Hence, the exact mechanism by which W. somnifera exerts a nephroprotective effect has to be elucidated.

ACKNOWLEDGMENTS

T. Jeyanthi gratefully acknowledges the financial assistance awarded by Annamalai University in the form of a research student fellowship.