Pharmacodynamic interaction of ethyl acetate fraction from alcoholic extract of Phyllanthus niruri leaves with gentamicin-induced nephrotoxicity in Wister albino rats

INTRODUCTION

Drug-induced nephrotoxicity represents a significant clinical challenge, impacting individuals across diverse demographics and necessitating extensive medical intervention, often leading to prolonged hospital stays and substantial economic strain.^[1] The nephrotoxic effects of certain pharmacological agents underscore the urgent need for effective strategies to alleviate this condition.^[2] In the modern era, there has been a growing interest in the potential of both synthetic and natural compounds to counteract the detrimental effects of nephrotoxic drugs.^[3]

The kidneys play a critical role in the elimination of waste products and excess substances.^[4] Certain drugs and antibiotics, including penicillins, cephalosporins, tetracyclines, sulphonamides and aminoglycosides, have the potential to cause nephrotoxicity.^[5]

phyllanthus niruri plant originated in India and used widely in traditional medicine.^[6] Extracts of phyllanthus species have been studied for the treatment of urolithiasis, diabetes, hypertension, jaundice and hypercalciuria.^[7] The species possess antimutagenic and anticarcinogenic properties,^[8] anticancer properties,^[9] antioxidant properties,^[10] hepatoprotective properties,^[11,12] antihyperuricaemic properties^[13] and antihyperlipidaemic activity.^[14] phyllanthus species from India exhibit high antioxidant activity in their methanol extracts.^[15]

The current work was plotted to fulfil the need for more efficient and affordable nephroprotection as there has been little research conducted in this area.^[16] The findings from this study pave the way for innovative treatment approaches that leverage natural extracts to enhance kidney protection during aminoglycoside therapy. The results may provide valuable insights into the protective mechanisms of P. niruri.^[17]

MATERIALS AND METHODS

Study design

After getting ethical approval (Reg. no. SGTU/IAEC/2023/09), a total of twenty-four albino rats were randomly divided into four groups, each consisting of six. The group serving as the control was administered normal saline, while negative control group was given gentamicin. The third group was pre-treated with an extract ethyl acetate fraction of P. niruri (EAFPN) at 200 mg/kg, followed by gentamicin, and the fourth group received 400 mg/kg of EAFPN, followed by gentamicin [Table 1]. After 9 days of treatment, the rats were sacrificed for blood sample collection and the kidneys were excised.

Table 1: Groups & Treatment Schedule

RESULTS

The control group maintained stable levels across all parameters. Conversely, the gentamicin-treated group showed significant increases in serum creatinine, urea and blood urea nitrogen (BUN), alongside a reduction in body weight, indicating renal impairment. EAFPN at 200 and 400 mg/kg exhibited significant results in nephrotoxicity caused by gentamicin [Table 2].

Table 2: Comparison of serum-urea, BUN, body weight at baseline and 10^th day within the groups

While EAFPN treatments, both 200 mg/kg (p = 0.014) and 400 mg/kg (p = 0.009) doses, exhibit significant reductions in serum creatinine levels. However, EAFPN groups (200 and 400 mg/kg) exhibited non-substantial differences in urea levels. Negative control group with the EAFPN 200 and 400 mg both comparisons exhibit significant decreases in urea levels (p = 0.006 and p = 0.003, individually), implying a protective effect of EAFPN against gentamicin-induced nephrotoxicity. Pairwise comparisons of BUN levels among various treatment groups have been done as it is a key biomarker for assessing kidney function [Figure 1]. The gentamicin group revealed a highly significant increase in BUN levels compared to the control group. While comparing the gentamicin group with the EAFPN 200 mg/kg and 400 mg/kg groups, there was a significant reduction in BUN levels, while comparing EAFPN 200 mg/kg and 400 mg/kg groups exhibited no significant difference in BUN levels (p = 0.688), implying similar efficacy at these doses.

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Figure 1: Bar graph with scatter overlay showing level of significance: Annotated as *p<0.05, **p<0.01, (a) serum creatinine (mg/dL), (b) BUN: blood urea nitrogen (mg/dL) and (c) Urea (mg/dL)). EAFPN: Ethyl acetate fraction of P. niruri

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In Table 2b presents the pairwise distribution of body weight data. Significant differences were observed between the control group and both the gentamicin and EAFPN (200 mg/kg and 400 mg/kg) groups. However, comparisons between gentamicin and EAFPN doses, as well as between the two EAFPN doses, showed no significant differences. On the 10^th day, the gentamicin group demonstrated a highly significant difference in baseline body weight compared to the control (p = 0.001), with similar results for both EAFPN doses (p = 0.001), indicating significant variations from the control.

Table 2a: Post hoc analysis by using the least significant difference method for serum creatinine, Urea, BUN levels

Table 2b: Post hoc analysis by using the least significant difference method for body weights

The study’s findings from Table 3 indicate that treatments did not significantly affect potassium (p = 0.683) or sodium levels (p = 0.089). However, there was a significant change in erythrocyte sedimentation rate (ESR) among the groups (p = 0.002), with the gentamicin group showing higher ESR (4.67 ± 0.81) compared to the control (3.00 ± 0.89) and EAFPN groups (2.67 ± 0.81 for 200 mg/kg and 2.50 ± 1.04 for 400 mg/kg). This indicates that gentamicin may cause inflammation or an immune response, while EAFPN appears to mitigate these effects.

Table 3: Comparison of potassium, sodium, and ESR within the groups

Table 3a: Post hoc analysis by using the least significant difference method

Gentamicin treatment significantly increased ESR (p = 0.004), indicating inflammation. In contrast, EAFPN at 200 mg/kg and 400 mg/kg did not differ significantly from the control (p = 0.528 and p = 0.347), suggesting no inflammatory effect. Compared to gentamicin, both EAFPN doses significantly reduced ESR (p = 0.001), demonstrating anti-inflammatory or protective potential. No difference was observed between the two EAFPN doses (p = 0.751), implying comparable efficacy.

Table 4 presents the effects of various treatments on urine output and kidney weights. At baseline, urine output did not differ significantly among groups (p = 0.739), indicating comparable renal function before intervention. By the 9^th day, a marked reduction in urine output was observed in the gentamicin-treated group (3.67 ± 1.63 mL), demonstrating significant nephrotoxicity (p = 0.001). In contrast, rats receiving EAFPN at 200 mg/kg and 400 mg/kg exhibited notably higher urine outputs (7.33 ± 1.21 mL and 8.17 ± 2.31 mL, respectively), suggesting a dose-dependent nephroprotective effect. Kidney weights did not differ significantly among groups for either the right (p = 0.379) or left kidney (p = 0.350), indicating that treatment did not markedly influence renal mass [Figure 2].

Table 4: Comparison of right and left kidney weight and urine at baseline and 10^th day within the groups.

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Figure 2: Bar graph with scatter overlay showing level of significance: Annotated as **p<0.01, ****p<0.001 (a) body weight (g) at baseline and (b) Final body weight (g) 10^th day, EAFPN: Ethyl acetate fraction of P. niruri

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Urine output on day 9 showed marked intergroup differences. Gentamicin caused a severe decline in urine output versus control (p = 0.001), confirming nephrotoxicity. Both EAFPN 200 mg/kg and 400 mg/kg groups had lower outputs than control (p = 0.007, p = 0.044) but significantly higher than gentamicin (p = 0.002, p = 0.001), demonstrating a dose-related reno-protective effect. Comparable efficacy was indicated by the non-significant difference between the two EAFPN dosages (p = 0.418).

Histopathological examination

For histopathological evaluation, kidney samples were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned at 3–4 μm before being stained with hematoxylin and eosin (H&E). The microsections were examined under both low power (10x magnification) and high power (40x magnification) and representative photos were taken using camera.

The control groups kidney histology reveled normal tubules and glomeruli [Figure 3].The group treated with gentamicin (80 mg/kg) exhibited harmaturia, tubular epithelial necrosis, desquamation, proteinaceous casts, blood in the tubules and inflammatory infiltrate [Figure 4]. The treatment group receiving EAFPN (200 mg/kg) + gentamicin (80 mg/kg) showed hematuria, desquamation, and mild inflammatory infiltrate [Figure 5]. The second treatment group receiving EAFPN (400 mg/kg) + gentamicin (80 mg/kg) showed glomeruli and minimal inflammatory infiltrate [Figure 6]. Finally, EAFPN (400 mg/kg) demonstrated better nephroprotective activity compared to EAFPN (200mg) against gentamicin-induced nephrotoxicity.

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Figure 3: Histopathology of kidney observed (a-b) Hematoxylin and eosin, at (a)10× (b) 40×: Control group (normal Saline) showing normal glomeruli & tubules.

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Figure 4: Histopathology of kidney observed (a-b) Hematoxylin and eosin, at (a) 10× (b) 40×: gentamicin group showing haematuria, tubular epithelial necrosis, desquamation, proteinaceous case and blood in the tubules.

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Figure 5: Histopathology of kidney observed (a-b) Hematoxylin and eosin, at (a) 10× (b) 40×: EAFPN (200mg/kg) + gentamicin (80mg/kg) group showing haematuria, desquamation and mild inflammatory infiltrate. EAFPN: Ethyl acetate fraction of P. niruriI.

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Figure 6: Histopathology of kidney observed (a-b) Hematoxylin and eosin, at (a) 10× (b) 40×: EAFPN (400mg/kg) + gentamicin (80mg/kg) group showing glomeruli and minimal inflammatory infiltrate, EAFPN: Ethyl acetate fraction of P. niruri

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DISCUSSION

An Indian investigation reveals that pharmacological agents are implicated in 20% of all instances of acute kidney injury.^[19]Renal toxicity attributed to pharmaceutical substances affects 14–16% of the populace, as evidenced by prospective cohort studies.^[20,21] The prevalence of nephrotoxicity associated with aminoglycoside antibiotics escalated from 3% in 1969 to a range of 10–20% by the year 2010.^[22,23] Approximately 27 million people worldwide suffer from chronic renal disease, with a 30% rise in drug-induced nephrotoxicity reported over the last decade. Nephrotoxic agents include penicillin, cephalosporin, tetracycline, sulphonamide and aminoglycosides.^[24] Nephrotoxic agents such as penicillin, cephalosporin, tetracycline, sulphonamide and aminoglyco-sides can cause kidney damage.^[5] Clinical doses of gentamicin (10–20 mg/kg) can lead to tubular alterations and potential kidney failure, while higher doses (40 mg/kg or more) are necessary for rapid cortical necrosis.^[25] According to current literature, approximately 30% of individuals administered gentamicin therapy for a duration exceeding 7 days demonstrate indications of nephrotoxicity.^[26] The renal impairment induced by gentamicin is correlated with elevated serum concentrations of creatinine and urea, alongside increased serum cystatin-C and a reduction in glomerular filtration rate.^[27] Renal tubular epithelial cell apoptosis and necrosis, glomerular damage, cast formation and inflammatory cell infiltration are significant histological changes associated with gentamicin-mediated renal injury.^[28] Acute tubular necrosis, interstitial nephritis, glomerulonephritis, renal vascular damage and intrarenal obstruction are other classifications of the renal compartments that are principally impacted by drug-induced renal failure.^[29]

The study’s findings suggest that P. niruri methanol extract may have potential as a nephroprotective agent. P. niruri has a protective effect against gentamicin-induced nephrotoxicity, according to histopathological studies. It is reasonable to believe that the strong antioxidant action of P. niruri extract plays an important role in nephron protection against gentamicin-induced nephrotoxicity. Several experimental animal models have shown strong evidence of a relationship between oxidative stress and nephrotoxicity. In these investigations, gentamicin-induced lipid peroxidation was inhibited by both drugs. Diffuse glomerular congestion, tubular casts, peritubular congestion, epithelial desquamation and blood vessel congestion were all evident in the gentamicin-treated group. The treatment group has a limited degree of dilated and congested blood vessels inside the interstitium.^[30]

Another study indicates that the aqueous leaf and seed extract of phyllanthus amarus offers protection against the deleterious renal side effects associated with paracetamol and gentamicin.^[31]

In control group of rats, body weight increased significantly [Table 2]. Urine output also increased [Table 4]. ESR was 3.00 ± 0.89 mm/h at the end of 2 h [Table 3]. Serum potassium and sodium levels were showing normal values [Table 3]. Blood urea and serum creatinine levels were normal, respectively [Table 2]. The right and left kidney weights were 1.21 ± 0.25 and 1.21 ± 0.18 g [Table 4], respectively. The histopathology of the kidney of the control group showed normal glomeruli and normal tubules.

Animals treated with gentamicin (80 mg/kg i.p.) for 9 days showed a significant decrease in urine output [Table 4]. Renal function insufficiency is known to decrease the excretion of electrolytes. There was a non-significant increase in serum potassium and a non-significant decrease in sodium as compared to control rats [Table 3]. However, the body weights of these rats were decreased [Table 2]. These rats also exhibited a significant rise in ESR at the end of 2 h [Table 3]. Blood urea and serum creatinine are biochemical parameters for the evaluation of renal function. Gentamicin-treated rats also had a significant increase in blood urea and serum creatinine, respectively [Table 2] as compared to the control group. This biochemical evidence is also suggestive of renal function insufficiency. The histopathology of the kidney showed haematuria, tubular epithelial necrosis, desquamation, proteinaceous cast and blood in the tubules and inflammatory infiltrate.

Animals treated with EAFPN (200 mg/kg orally) + gentamicin (80 mg/kg i.p.) for 10 days showed a decrease in urine output [Table 4]. This has been a non-significant decrease in serum potassium and non-significant increase in sodium as compared to gentamicin [Table 3]. However, the body weights of these rats were increased, respectively [Table 2]. These rats also exhibited a significant decrease in ESR at the end of 2 h [Table 3] as compared to the gentamicin group. As compared to the gentamicin group, the treatment group also showed a significant decrease in blood urea and serum creatinine [Table 2]. The histopathology of the kidney showed haematuria, desquamation and mild inflammatory infiltrate.

Animals treated for 9 days exhibited decline in potassium levels and elevation in sodium levels in negative control group [Table 3]. However, decrease in urine output was observed in EAFPN (400mg/kg orally) and negative control group [Table 4]. However, the body weights of these rats were increased, respectively [Table 2]. These rats exhibited a significant decrease in ESR at the end of 2 h [Table 3]. As compared to the gentamicin group, the treatment group also showed a decrease in blood urea and serum creatinine [Table 2]. The histopathology of the kidney showed glomeruli and minimal inflammatory infiltrate.

CONCLUSION

The findings of the present study reveal that the ethyl acetate fraction of the alcoholic extract of P. niruri leaves reduces kidney damage against gentamicin-induced nephrotoxicity. The biochemical and histopathological results of EAFPN (400 mg/kg) have shown better nephroprotective activity compared to EAFPN (200 mg/kg) against gentamicin-induced nephrotoxicity. However, further studies should be designed to explore the molecular mechanisms associated with the nephroprotective activity along with dose modifications of EAFPN.