A novel nanoparticle glutathione and Lepidium sativum treatment for gentamicin-induced acute renal failure in rats

Abstract

Acute renal failure (ARF) is a sudden, significant, and often reversible decline in kidney function, with 25% of all hospital-administered pharmaceuticals potentially causing nephrotoxicity. The study investigates the effectiveness of a novel nanoparticle (NP) formulation of glutathione (GSH) and Lepidium sativum (LS) in improving therapeutic outcomes in a rat model of ARF. Sixty adult male albino rats were allocated into ten groups, comprising six rats each, for the study. ARF was created by daily gentamicin (GN) administration for seven consecutive days and various treatment protocols, including chitosan (CS) NPs, spanlastics NPs, as well as conventional, NP formulations of GSH, LS, and their respective combinations. The effect was evaluated through various tests, and properties of nanoparticles were confirmed through characterization processes. The NP compositions markedly enhanced renal function, as seen by reduced urine concentrations of albumin and glucose. Furthermore, the serum concentrations of creatinine (SCr), blood urea nitrogen (BUN), and cystatin C were decreased. Tissue concentrations of nitrite, superoxide dismutase (SOD), and malondialdehyde (MDA), as markers of oxidative stress, were enhanced by both conventional and NP formulations. Additionally, they decreased inflammatory markers such as kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), tumor necrosis factor alpha (TNF-α), and interleukin-6 (IL-6). Histological analysis and immunohistochemical testing revealed that the combination therapy, particularly with the nanoforms, significantly decreased caspase 3 cellular immunoexpression, a sign of kidney cellular damage. The findings show that the ARF renal damage is considerably reduced when NPs containing GSH and LS are administered together. The study suggests a promising pharmacological approach for enhancing kidney regeneration and preserving renal function, potentially aiding in new therapeutic interventions for ARF treatment.

Keywords: ARF; Caspase 3; Gentamicin; IL-6; LS; Nanoparticles.

Fig. 1

TEM image of Lepidium sativum loaded nanospsnlastics (F1).

Fig. 2

Characterization of developed nanosystems (a) size distribution curve, (b) zeta potential of Lepidium sativum nanospanlastics (F1) and (c) zeta potential of glutathione chitosan nanoparticles.

Fig. 3

FTIR spectra of (A) Free Lepidium sativum, (B) plain nanospanlstics and (C) Lepidium sativum nanospanlastics (F1).

Fig. 4

X ray diffraction of (A) Free phytochemical (Lepidium sativum), (B) Physical mixture of spanlastics ingredients and (C) selected Lipidium sativum nanospanlastics (F1).

Fig. 5

Cumulative in-vitro release profiles of Lepidium sativum from nanospanlastics dispersion F1 and free drug solution in phosphate buffer pH 6.8 at 37 °C. Data are presented as mean ± SD (n = 3). The experiment was conducted in triplicate, and the data are shown as a mean ± standard deviation (SD).

Fig. 6

Urine albumin and glucose levels in rats with gentamicin (GN)-induced acute renal failure (ARF) as a function of conventional, nanoparticle (NP), and combination glutathione (GSH) and Lepidium sativum (LS) formulations. This data consists of the means with standard error of the mean (n = 6). ^ap < 0.0001 in comparison to the control group. ****p < 0.0001 in comparison to the ARF-induced group. ^#p < 0.05 when compared with the related nanoparticle group. ^$p < 0.05 when compared LS to GSH group. Non significance (p > 0.05). CS chitosan, O LS-GSH combination, GSH-NP glutathione nanoparticles, LS-NP Lepidium sativum nanoparticles, O-NP LS-GSH nanoparticle combinations.

Fig. 7

Impact of traditional, nanoparticle, and mixed glutathione (GSH) and Lepidium sativum (LS) formulations on creatinine, urea, and cystatin C serum levels in rats afflicted with gentamicin (GN)-induced acute renal failure (ARF). The data shown are the means with standard error of the mean (n = 6). ^ap < 0.0001 in comparison to the control group. ***p < 0.001 and ****p < 0.0001 in comparison to the ARF-induced group. ^#p < 0.05 and ^##p < 0.01 when compared with the related nanoparticle group. Non significance (p > 0.05). CS chitosan, O LS-GSH combination, GSH-NP glutathione nanoparticles, LS-NP Lepidium sativum nanoparticles, O-NP LS-GSH nanoparticle combinations.

Fig. 8

Acute renal failure (ARF) caused by gentamicin (GN) in rats: effects of conventional, nanoparticle (NP), and combination forms of glutathione (GSH) and Lepidium sativum (LS) on tissue levels of malondialdehyde, nitrite, and superoxide dismutase (SOD). The data consists of the means with standard error of the mean (n = 6). ^ap < 0.0001 in comparison to the control group. ****p < 0.0001 in comparison to the ARF-induced group. ^#p < 0.05, ^###p < 0.001 and ^####p < 0.0001 when compared with the related nanoparticle group. ^$p < 0.05 when compared LS to GSH group. Non significance (p > 0.05). ^$p < 0.05 when comparing the LS and LS-NP groups to the GSH and GSH-NP groups, respectively. Non significance (p > 0.05). CS chitosan, O LS-GSH combination, GSH-NP glutathione nanoparticles, LS-NP Lepidium sativum nanoparticles, O-NP LS-GSH nanoparticle combinations.

Fig. 9

Acute renal failure (ARF) induced by gentamicin (GN) in rats: effects of conventional, nanoparticle (NP), and combination forms of glutathione (GSH) and Lepidium sativum (LS) on tissue levels of neutrophil gelatinase-associated lipocalin (NGAL) and Kidney injury molecule-1 (KIM-1). The data shown are the means with standard error of the mean (n = 6). ^ap < 0.0001 in comparison to the control group. ****p < 0.0001 in comparison to the ARF-induced group. ^#p < 0.05 and ^####p < 0.0001 when compared with the related nanoparticle group. ^$p < 0.05 when comparing the LS and LS-NP groups to the GSH and GSH-NP groups, respectively. Non significance (p > 0.05). CS chitosan, O LS-GSH combination, GSH-NP glutathione nanoparticles, LS-NP Lepidium sativum nanoparticles, O-NP LS-GSH nanoparticle combinations.

Fig. 10

Assessment of the effects of traditional, nanoparticle (NP), and mixed forms of glutathione (GSH) and Lepidium sativum (LS) on tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) serum levels in rats induced acute renal failure (ARF) by gentamicin (GN). This data consists of the means with standard error of the mean (n = 6). ^ap < 0.0001 in comparison to the control group. ****p < 0.0001 in comparison to the ARF-induced group. ^#p < 0.05, ^##p < 0.01 and ^####p < 0.0001 when compared with the related nanoparticle group. ^$$p < 0.01 when comparing the LS to the GSH group. Non significance (p > 0.05). CS chitosan, O LS-GSH combination, GSH-NP glutathione nanoparticles, LS-NP Lepidium sativum nanoparticles, O-NP LS-GSH nanoparticle combinations.

Fig. 11

Histochemical staining of rat kidneys in different treatment groups. The bar representing the scale is 50 μm. (A) The renal histo-architecture is normal in the control group; the glomerulus (G) and tubules (RT) are also in good shape. On the other hand, the gentamicin (GN) (B) group shows massive coagulative tubular necrosis, loss of renal tubule architecture (asterick), and homogeneous eosinophilic protein fluid within the lumen of the kidney tubule (arrowheads). Necrosis of many tubules (arrow) and hydropic degeneration (asterick) in the CS and nanospanlastic groups, with protein fluid within the lumen of the kidney tubule (arrowheads) in sets (C) and (D). In (E) and (F), the GSH and LS groups demonstrate reduced tubular necrosis, while astericks indicate hydropic degeneration. Arrowheads indicate the presence of protein fluid within the lumen of the kidney tubule. The tubular architecture shows some improvement in the GSH-NPs and LS-NPs groups, but there are still some spots of tubular necrosis (asterisks) and protein fluid within the lumen of the renal tubule (arrowheads) in the G and H group. The tubular architecture of the kidneys significantly improved in the I and J groups treated with GSH-LS and GSH-LS-NPs, while there were some tubular epithelial degenerations (arrows) and fluid proteins within the lumen of the kidney tubule of certain tubules (arrowheads). GN gentamicin, GSH glutathione, LS Lepidium sativum, NPs nanoparticles.

Fig. 12

Tubular renal injury score. Pathological analysis using a semiquantitative scoring system with a range of 0 to 4. GN gentamicin, CS chitosan, GSH glutathione, GSH-NP glutathione nanoparticles, LS Lepidium sativum, LS-NP Lepidium sativum nanoparticles, O LS-GSH combination, O-NP LS-GSH nanoparticle combinations.

Fig. 13

Analysis of caspase 3 in rat kidney using immunohistochemistry. There was no caspase 3 expression in the control group (negative) (A). (B) The group of rats with gentamicin-induced acute renal failure (ARF) whose renal tubular epithelial cells showed an elevated level of caspase-3 immunoreactivity in their cytoplasm. Caspases-3 are positive when the color is brown. There was no statistically significant increase in caspase-3 immunostaining in the groups treated with nanospanlastic (D) and CS (C). There was a smaller reduction in caspase-3 immunostaining in the groups treated with GSH and LS (E) and (F). The immunostaining for caspase-3 was not significantly reduced in the GSH-NPs and LS-NPs groups. Groups I and J, which are combination of GSH-LS and GSH-LS-NPs, exhibited a significant reduction in caspase-3 immunostaining.

Fig. 14

(A) Caspases 3 positive cells’ IHC pixel intensity (B) The proportion of cells that are immunopositive for caspase 3. GN gentamicin, CS chitosan, GSH glutathione, LS (Lepidium sativum), O LS-GSH combination, GSH-NP glutathione nanoparticles, LS-NP Lepidium sativum nanoparticles, O-NP LS-GSH nanoparticle combinations.