Drugs and chemicals

MF (Metformin Hexal; Germany) was supplied as a white powder, soluble in distilled water, and freshly prepared as an aqueous solution to be given as a single daily oral dose of 100 mg/kg/day. ⁸ GM treatment protocols used in the present study have been reported earlier. ²¹

Animals

In a preclinical study, 50 male Wistar rats with a weight range of 200-250 g, purchased from Jundishapur University of Medical Sciences, Ahvaz, Iran, were transferred to the animal house of Shahrekord University of Medical Sciences, Shahrekord, Iran. Animals were housed at an environment of controlled temperature (25 ± 3°C) and humidity (50-60%) with a 12-hour dark-light cycle (lights on at 7 AM) and free access to pelleted diet and tap water for 2 weeks to acclimatize to the new environment. The animal experimentation was conducted in accordance with the National Institute of Health guide for the careful use of laboratory animals.²² Their general health state and activity were monitored closely during the experiment.

Experimental design

The animals were divided into five groups (10 rats each) as follows:

[INLINE:1]

On the first day (before experiment) and the final day (day of sacrificing), serum samples were obtained to measure blood urea nitrogen (BUN) and serum creatinine (Cr) for all the rats. Rats were sacrificed (i.p.) under general anesthesia with ketamine. The kidneys were removed for histologic examinations.

Determination of serum BUN and Cr level

BUN and Cr levels were measured by a colorimetric method using commercial kits on an autoanalyzer.

Histopathologic evaluations

The kidneys of each animal were dissected out, then fixed in buffered formalin for 12 h and processed for histopathologic examination. Three-micrometer-thick paraffin sections were stained with hematoxylin and eosin (H and E) for light microscope examination using conventional protocol. Histopathologic studies were performed under a light microscope. Slides were coded and examined by a histopathologist who was blinded to the treatment groups. All specimens were examined for six morphologic parameters including epithelial cell vacuolization, degeneration, tubular cell flattening, hyaline cast, tubular dilatation, and debris materials in tubular lumen on a semi-quantitative score from 1 to 5, while the score of zero was assigned to the normal tissue without damage.⁹,²³,²⁴

Statistical analyses

Data were expressed as mean ± SEM. The t0-paired test was used to compare the serum BUN and Cr levels before and after the experiments. One-way analysis of variance (ANOVA) was applied to compare the serum BUN and Cr levels between the groups. To compare the pathology damage score between the groups, Kruskal-Wallis and Mann-Whitney U-test were applied. P values <0.05 were considered statistically significant.

The effects of MF on BUN and Cr levels

The data for serum levels of BUN and Cr are shown in Figure 1. No significant differences were observed before the experiment. The levels of BUN in groups II, IV, and V, and the serum levels of Cr in groups II and V were increased significantly after the experiment (P < 0.05).Figure 1

The serum levels of BUN and Cr before and after the experiment in five groups of animals. Group I, sham group; group II, positive control group treated with gentamicin; group III, treated with metformin; group IV, treated with gentamicin for 10 days and post-treatment with metformin for the next 10 days; and group V, co-administration of metformin and gentamicin for 10 days. The symbols (FNx01) and (†) stand for significant difference before the experiment (P < 0.05) and significant difference from positive control group (P < 0.05), respectively

In this study, we found that MF could ameliorate GM-induced kidney tissue toxicity. We also found that MF was still effective when the drug was administered after progression of tubular damage by GM. MF is used for the treatment of diabetes as a sugar-lowering agent.¹⁵,¹⁶ In addition, MF is recommended as the drug of first choice in type 2 diabetes.¹⁶,¹⁷,¹⁸,¹⁹ There are potential survival benefits associated with the use of MF, in addition to the benefits in respect to cardiovascular outcomes and metabolic parameters as suggested by recent studies. ²,³,⁴,⁵ MF exerts its metabolic activity through the induction of the adenosine monophosphate (AMP)-activated protein kinase (AMPK) pathway which acts as a sensor detecting variations of intracellular energy levels.¹⁹

It was suggested that hypoxia-induced hypoxia-inducible factor (HIF)-1α accumulation in diabetic nephropathy could be suppressed by GM through the repression of oxygen consumption.⁹,²³,²⁴,²⁵,²⁶,²⁷ HIF-1α plays an important role in chronic hypoxia and tubulointerstitial fibrosis, which are presently considered to be the common pathways for various progressive kidney diseases, including diabetic nephropathy.⁹,²³,²⁴,²⁵,²⁶,²⁷ Alterations in epithelial cell polarity and in the subcellular distributions of epithelial ion transport proteins are key molecular consequences of acute kidney injury and intracellular energy depletion.²⁸ AMPK, a cellular energy sensor, is rapidly activated in response to renal ischemia, and AMPK activity may influence the maintenance or recovery of epithelial cell organization in mammalian renal epithelial cells subjected to energy depletion.²⁸,²⁹ At a molecular level, energy deprivation causes key energy-dependent membrane proteins to become displaced and dysfunctional. ¹³,²⁹,³⁰ Specially, in the proximal tubule, the Na-K-ATPase is internalized from the basolateral membrane, disrupting the cell′s capacity to maintain normal transepithelial sodium transport. ¹³,²⁸,²⁹ Preservation of a polarized plasma membrane distribution of Na-K-ATPase in renal epithelia is essential for the maintenance of both solute reabsorption and volume homeostasis. It was shown that Na-K-ATPase becomes mislocalized after energy deprivation. ⁷,⁸,¹⁵ ATP depletion also perturbs the distribution of tight junction proteins, further disrupting epithelial cell polarity and organization ⁷ and leading to back leak of extracellular fluid into the urinary space. Such molecular insults result in accumulation of potentially harmful toxins. ⁸ MF activates AMPK in rat kidney lysates. ²⁵,²⁶ MF treatment increases detectable p-AMPK in a dose-dependent manner, and MF-induced AMPK activation occurs in proximal tubules as well as in distal segments. ²⁸,²⁹ Mitochondria represents one of the major cellular sources of ROS generation ⁷,¹⁵ and mitochondrial toxicity can also be mediated by ROS. ROS are normally produced at low levels by mitochondria themselves. But under pathological conditions, the intracellular and intramitochondrial ROS content may be amplified.⁷,¹⁵ GM as a mitochondrial toxin can exert various morphological damages to the kidney. Indeed, after GM treatment, intracellular ROS content can reach a toxic level, thus causing cell death and malfunctioning of the organ.¹¹,³⁰,³¹,³²,³³ Literature review shows that the renal protective effects of MF in diabetic nephropathy have not been fully evaluated. Meanwhile, in accordance with our results, prevention of histologic changes due to GM toxicity by MF was shown by Morales et al.⁸ They found that control and MF-treated rats showed no structural alterations in renal tissues, while massive and diffuse cell necrosis was observed in the proximal tubules of kidneys from rats injected with GM.⁸ They could show that tubular lumen was frequently filled with hyaline casts or heterogeneous cellular debris, while in rats treated with GM and MF, most of the proximal tubules were saved and manifestations of necrosis were observed in less than 10% of cells.⁸ Similar results were obtained in our study also.

Based on the above in vivo results, it was concluded that MF ameliorates GM-induced acute renal failure. Also, the present study shows that delayed treatment of MF for GM-induced acute renal failure was still effective as when MF was given in combination with the GM. Hence, MF is a nephroprotective drug to prevent or attenuate the tubular damage caused by GM or other nephrotoxic agents which act through the same mechanisms as this aminoglycoside. However, these studies are promising and warrant a more comprehensive trial.

This study was supported by a grant from Research Deputy and Medical Plants Research Center of Shahrekord University of Medical Sciences (Grant #994).