The protective role of estrogen and its receptors in gentamicin-induced acute kidney injury in rats

doi:10.1016/j.lfs.2019.117082

Life Sciences

Volume 239, 15 December 2019, 117082

https://doi.org/10.1016/j.lfs.2019.117082 Get rights and content

Highlights

•
Gentamicin induced acute kidney injury.
•
Megalin is implicated in gentamicin-endocytosis.
•
Genistien and estradiol ameliorated gentamicin-induced acute kidney injury.
•
Fulvestrant exaggerated gentamicin-induced acute kidney injury.

Abstract

Aim

Investigating the impact of 17β-Estradiol/estrogen receptors in gentamicin-induced nephrotoxicity.

Main methods

Three weeks post-ovariectomy or sham surgery for the Wistar albino female rats, thirty sham rats were randomly grouped (n = 6), received either vehicle or gentamicin; the estrogen receptors down regulator (fulvestrant); gentamicin plus fulvestrant; gentamicin plus the phytoestrogen (genistein). Forty–eight ovariectomized rats were randomly grouped (n = 6), treated with either vehicle or gentamicin; fulvestrant; gentamicin plus fulvestrant; genistein; gentamicin plus genistein; estradiol benzoate; gentamicin plus estradiol benzoate. Just post-treatment termination, the traditional kidney injury biomarkers (serum creatinine and blood urea nitrogen) and novel biomarkers (serum Kidney injury molecule −1, cystatin C, lactate dehydrogenase and, gamma-glutamyl transferase) were determined. Bovine serum albumin labeled with fluorescence isothiocyanate assessed megalin expression/endocytic functionality in the proximal tubules epithelial cells (PTECs). The immunohistochemical investigation for the same-sectioned slides of PTECs assessed the correlation between estrogen receptors α and megalin receptors expression. Histopathological examination of PTECs and subjective scoring system graded the damage markers.

Key findings

Estrogen receptor α expression was markedly dimensioned post-ovariectomy, co-localized and inversely correlated to megalin expression. Serum levels of the novel biomarkers were directly proportional to megalin expression in the PTECs and inversely correlated with estrogen receptor α expression. The injury was exaggerated in ovariectomized and intact rats received fulvestrant. Supplementation with estrogen or genistein ameliorated this injury.

Significance

Estrogen/estrogen receptors have a protective impact on gentamicin-induced acute kidney injury. Estrogen receptors antagonist exacerbate the injury, and oppositely, estrogens or phytoestrogens improve it.

Introduction

Gentamicin, one of the aminoglycoside antibiotics is highly effective in the treatment of severe gram-negative infections which are resistant to other antibiotics [1]. Single daily dose of 160 mg of gentamicin is effective in the treatment of urinary tract infections [2]. Unfortunately, the clinical use of this antibiotic is limited due to its nephrotoxicity mostly on the epithelial cells of the proximal tubules [3].

Megalin, a multi-ligand endocytic receptor, belonging to the low-density lipoprotein receptor family [4], is widely expressed in epithelial cells of the proximal tubules [5]. The uptake of aminoglycosides into the kidney in mice lacking megalin is eliminated and directly correlates with renal megalin expression/functionality; providing unequivocal evidence, that megalin is the only major pathway responsible for the renal accumulation of aminoglycosides and representing a unique target to prevent aminoglycoside-induced nephrotoxicity [6]. Given the indispensable endocytic and renal uptake role of megalin to aminoglycosides, it is rational to anticipate that modulation of this receptor expression/endocytic functionality in the epithelial cells of the proximal tubules could modulate gentamicin-induced AKI.

The kidney is a non-reproductive estrogenic organ with specific renal estrogen-induced gene activation. The estrogen receptor α expression in the epithelial cells of the proximal tubules is more pronounced than estrogen receptor – β subtype [7], however, both subtypes are co-expressed with megalin receptor [8,]. The co-localization of estrogen and megalin receptors, suggests that estrogen receptors, which are activated by ligand with 17-β estradiol are involved in regulating megalin expression in the epithelial cells of the proximal tubules. Besides, substantial evidence demonstrated that aminoglycosides exhibit sex-dependent nephrotoxicity, however, the exact molecular mechanisms whereby 17-β estradiol/estrogen receptors exert their effects are still unknown [[11], [12], [10]].

Serum creatinine is considered as delayed and unreliable indicator of acute kidney injury because it is influenced by multiple non-renal factors, such as age, gender, muscle mass, muscle metabolism, diet, and hydration status; it takes several days to reach a new steady-state and not rise until more than half of the kidney function is lost [13]. The blood urea nitrogen is relatively insensitive and unreliable to early kidney deterioration; its level is affected by high catabolic states and high protein diets. Besides, damage to 75% of nephrons is required before increase in its level [14].

Given the limitations of the traditional biomarkers (serum creatinine and blood urea nitrogen) as imprecise markers for acute kidney injury, they are substituted by the more sensitive and reliable biomarkers (kidney injury molecule-1, cystatin C, gamma-glutamyl transferase and lactate dehydrogenase).

Regarding the extremely efficacious effect of gentamicin in critical infections, it is imperative to achieve optimal renoprotective therapy against its nephrotoxicity. Our study aimed to estimate the role of 17-β estradiol/estrogen receptors, estrogen receptor down-regulator (fulvestrant), the phytoestrogen (genistein) and external supplementation of estrogen (estradiol benzoate) in megalin expression/endocytic functionality and consequently their roles in exaggerating or mitigating the gentamicin-induced acute injury.

Section snippets

Animal care and ethical approval

Adult female Wistar albino rats (2 months old, weighing approximately 180–200 g) were purchased from the experimental animal facility at the Nile for Pharmaceuticals & Chemical Industries (Cairo, Egypt). Rats were kept two per cage for two weeks for acclimatization in controlled housing conditions (room temperature 25 ± 2 °C, humidity (50–70%) and 12/12 h dark-light cycles) and kept free on a standard diet. Rats were given ad libitum access to food and water. The study was performed under the

Serum creatinine and BUN assessment

No statistically significant difference was noticed in serum levels of creatinine and BUN in Sham and OVX groups either received or did not receive the scheduled treatment regimen of gentamicin. At the proposed treatment dose level and duration of fulvestrant, genistein and estradiol benzoate, no significant difference in serum levels of creatinine and BUN was estimated between Sham and OVX groups either received or did not receive gentamicin compared to those received fulvestrant, genistein

Discussion

It is believed that gender disparity effects on nephrotoxic drugs could be attributed to the differences in the expression of drug transporters; thus, this sex-related difference in the expression of drug transporters could alter the renal uptake, accumulation, and nephrotoxicant-induced renal injury [31].

Megalin role in gentamicin endocytosis has received much attention. The estimated directly proportional correlation between megalin expression and the serum levels of the novel

Conclusion

The estimated directly proportional relationship between megalin expression and the serum levels of the novel AKI biomarkers, sharply confirmed that megalin is implicated in gentamicin-induced AKI. The protective effect of E2/ERs against gentamicin-induced AKI was clarified by our finding that E2 depletion post-ovariectomy and ERs blocking with fulvestrant elevates renal tissues megalin expression, linked directly with the apparent exaggerated gentamicin-induced AKI. The gentamicin-induced AKI

Declaration of competing interest

The authors declare that there was no conflict of interest.

Acknowledgments

The authors are greatly thankful to Dr. Wagdy Khalil, Professor of Molecular Genetics, Cell Biology Department, National Research Centre (NRC), Dokki, Giza, Egypt for his kind help in ELIZA assay and Dr. Sayed Abdel Raheem, Assistant professor of pathology, Al-Azhar University, Faculty of Medicine, Cairo for his kind help in histopathological investigation.

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Interference with megalin expression/endocytic function by montelukast mitigates gentamicin nephrotoxicity: Downregulation of ClC-5 expression
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Paraffin blocks (5 μm) were transversely sectioned using sledge microtome. Each section was stained with Hematoxylin and Eosin (H&E) as illustrated previously (Bancroft and Gamble 2008), followed by scoring of the lesions as previously demonstrated (Abd El-Lateef et al., 2019). The agar disc diffusion method was utilized to test the effect of MLK on the antibacterial activity of GM.
Megalin receptor-mediated endocytosis participates a crucial role in gentamicin (GM) uptake, accumulation, and toxicity. In this study, we investigated the potential effects of montelukast (MLK) on megalin expression/endocytic function against GM nephrotoxicity. Male Wistar rats were administered GM (120 mg/kg; i.p.) daily in divided doses along 4 hr; 30 mg/kg/hr; for 7 days. MLK (30 mg/kg/day) was orally administered 7 days before and then concurrently with GM. The protein expressions of megalin and chloride channel-5 (ClC-5); one of the essential regulators of megalin endocytic function; were determined by Western blotting. Besides, the endocytic function of megalin was evaluated by the uptake of bovine serum albumin labeled with fluorescein isothiocyanate (FITC-BSA) into proximal tubular epithelial cells. Moreover, kidney function biomarkers (Cr, BUN, GFR, KIM-1, cystatin-C) and apoptosis markers (p-AKT1, cleaved caspase-3) were estimated. Co-treatment with MLK downregulated ClC-5 expression leading to reduced recycling of megalin to the plasma membrane, reduced expression, and so impaired endocytic function that was evidenced by reduced uptake of FITC-BSA in proximal tubular epithelial cells. The protein expression of the apoptotic executioner cleaved caspase-3 was significantly reduced, while that of the antiapoptotic p-AKT1 was elevated. These results were confirmed by the improvement of kidney functions and histological findings. Our data suggest that MLK could interfere with megalin expression/endocytic function that could be attributed to downregulation of ClC-5 protein expression. That eventually reduces renal cell apoptosis and improves kidney functions after GM administration without affecting the antibacterial activity of GM. Therefore, reduced expression of ClC-5 and interference with megalin expression/endocytic function by MLK could be an effective strategy against GM nephrotoxicity.
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A p-value of <0.05 was considered to show a significant difference between groups. Our recent work indicates that megalin promotor region harbors response element for nuclear receptors [19]. Thus; we started our study by the computational analysis of the promotor region to detect whether it harbors the AR response element (ARE).
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The protective role of estrogen and its receptors in gentamicin-induced acute kidney injury in rats

Highlights

Abstract

Aim

Main methods

Key findings

Significance

Introduction

Section snippets

Animal care and ethical approval

Serum creatinine and BUN assessment

Discussion

Conclusion

Declaration of competing interest

Acknowledgments

J. Biol. Chem.

Gend. Med.

Br. J. Anaesth.

Kidney Int.

Prog. Histochem. Cytochem.

Toxicol. Lett.

Am. J. Kidney Dis.

Reprod. Biol.

J. Steroid Biochem. Mol. Biol.

J. Biol. Chem.

Vitam. Horm.

J. Biol. Chem.

Cell

J. Biol. Chem.

J. Steroid Biochem. Mol. Biol.

Outcomes in patients with gram-negative sepsis treated with gentamicin

Ther. Adv. Drug Saf.

Single-dose daily gentamicin therapy in urinary tract infection

Antimicrob. Agents Chemother.

Gentamicin nephrotoxicity in animals: current knowledge and future perspectives

EXCLI J.

An integrative overview on the mechanisms underlying the renal tubular cytotoxicity of gentamicin

Toxicol. Sci.

The LDL receptor related protein (LRP) family: an old family of proteins with new physiological functions

Ann. Med.

Estrogen and the inner ear: megalin knockout mice suffer progressive hearing loss

FASEB J.

Sex differences in acute kidney injury requiring dialysis

BMC Nephrol.

Sex hormones and their influence on chronic kidney disease

Curr. Opin. Nephrol. Hypertens.

Estradiol regulates AQP2 expression in the collecting duct: a novel inhibitory role for estrogen receptor α

Am. J. Physiol. Renal Physiol.

Experimental osteoporosis-different methods of ovariectomy in female white rats

Rocz. Akad. Med. Bialymst.

Metabolic effects of physical training in ovariectomized and hyperestrogenic rats

J. Appl. Physiol.

Estimation of some oxidative stress parameters in the serum and cerebellum of ovariectomized rats

J. Kerbala Univ.

Unique sex- and age-dependent effects in protective pathways in acute kidney injury

Am. J. Physiol. Renal Physiol.