Redox-Active Bacteria Alleviated Ethylene Glycol-Induced Kidney Calculi in Rats

Introduction

Kidney stones, also known as nephrolithiasis, are one of the most common and chronic diseases known to man for a long time. It is estimated that approximately 1–15% of people will have some form of nephrolithiasis during their lifetime (1). In recent years, the prevalence of the disease has increased, with different rat in women (7.1%) and men (10.6%) (2, 3). In addition, the prevalence of this disease increases with age. For example, in the United States, the prevalence of the disease is 1.3% in people aged 20–29 years, but 19.1% in men and 9.4% in women aged 60–69 years ⁽⁴⁾. In the Islamic Republic of Iran, the prevalence of this disease was 5.7% (145 per hundred thousand people) in 2013, and the recurrence rate of the disease in patients was about 36%, while the overall recurrence rate of the disease is estimated to be about 50% (5, 6).

The cellular and molecular mechanisms of kidney stone formation have not yet been elucidated, but histologically, a multistep process involving saturation, crystal nuclei formation, growth and accumulation of particle has been observed (7). Several studies have mentioned the role of reactive oxygen species (ROS) in the pathogenesis of nephrolithiasis. Although the consumption of oxygen in the human body is vital and necessary, it is associated with the production of reactive oxygen species (ROS). An imbalance between ROS production and the body's antioxidant capacity can damage biomolecules and eventually body cells. Recent studies have shown that the extent of oxidative stress and urinary tract damage is higher in patients with kidney stones than in individuals who do not form stones (8). Several approaches have been investigated to inhibit the formation of kidney stones using antioxidant compounds and drugs (9-11).

In this study, we investigated the effect of oral administration of a bacterium with extracellular electron transfer (EET) ability on kidney stone formation. The hypothesis of the study was based on the interaction between the oxidation-reduction, or redox activity of an EET bacterium and kidney stone formation. Recently, the relationship between the gut microbiome and kidney stones has been studied (12). The lake of mitochondria in bacteria has led them to use the inner membrane to perform the electron transfer cycle for their ATP production. Some bacteria have the ability to transfer respiratory chain electrons to extracellular solids, which is called extracellular electron transfer (EET) (13). The mechanism of electron transfer in Shewanella oneidensis MR-1 is well known. In this bacterium, the outer membrane appendages in contact with the external environment have two proteins, including mtrC and OmcA, which transfer electrons to external mineral acceptors containing Mn⁴⁺, Fe³⁺, or Mn⁵⁺ with the participation of secreted flavins  (13, 14).

The association between EET bacteria in the gut microbiome and such pathological conditions has been noted in other studies. Faecalibacterium prausnitzii is one of the most abundant bacteria in the gut microbiome of humans and is known as an electrogenic bacterium. The low abundance of this bacterium in the gut has been associated with the occurrence of Crohn's disease. This bacterium uses flavin and thiol for its EET process, so the important effects of a diet rich in flavin- or antioxidant suggested for the treatment of patients with Crohn's disease (15). Since the EET process alters environmental oxidation and reduction reactions and oxidants are involved in the pathogenesis of kidney stones, we investigated the effect of oral administration of an EET bacterium on kidney stone formation in this study.

Methods

Bacterial culture and colony count

Shewanella oneidensis strain number IBRC-M 10991 was purchased from Iranian Biological Resource Center (IBRC) and cultured in Luria-Bertani (LB) broth at 37 °C under aerobic conditions. Riboflavin (Sigma Aldrich), erythromycin (Sigma Aldrich), co-trimoxazole (Sobhan Darou), and ampicillin were dissolved in deionized water and stored at -20 °C.

For serial dilution (1/100, 1/1000, 1/10000, etc.) and colony counting, bacteria were resuspended in 0.5ml LB broth media and then cultured in dishes containing LB agar media at 37 °C in an aerobic tent. After one night, colony-forming units (CFU) in the dishes were visually counted. CFU was calculated by multiplying the bacterial colonies by the dilution used on the plate. The counted bacteria were dissolved in LB broth media containing 15% glycerol and stored at -20 °C until use for administration by oral gavage.

Animal model and experimental design.

The experiment was done based on the Guide for the Care and Use of Laboratory Animals (CULA), and the animal procedures used were approved by the Ethics Committee of Kashan University of Medical Sciences (IR.KAUMS.AEC.1401.011). Healthy male Wistar rats weighing 139-239 g were used and maintained at 25 ± 2°C. Animals were randomly divided into three groups (n=9), including group 1, which had free access to standard rat chow and water throughout the study period; group 2, which was treated with 1% ethylene glycol (EG) in drinking water; group 3, which was treated with 1% EG and oral administration of 0.5ml of a bacterial suspension.

Urinary oxalate was measured using commercially available kits according to the manufacturer’s instructions (Darman Faraz Kav, Iran).

Histological studies

Thirty-two days after the treatment began, the animals were sacrificed using CO₂ inhalation in an anesthesia chamber. The kidneys were then removed and weigh for further analysis. To conduct histological examination, the kidneys were fixed in 10% formalin, dehydrated in a gradient of ethanol, embedded in paraffin, and cut into 5μ serial sections. For each group, four to five slides containing 3-5 sections were deparaffinized, stained with hematoxylin and eosin, and examined under a light microscope. The number of calcium oxalate (CaOx) deposits in the renal tubules was counted in microscopic fields with a 400x magnification.

Statistical analysis

All statistical analyses were conducted using Graph Pad Prism version 8. One-way ANOVA was performed for statistical analysis. Differences were considered statistically significant at P-value<0.05.

Results

Histopathology and oxalate load

Analysis of kidney tissue sections under polarized light microscope displayed no kidney crystals in any microscopic field in the control group. In addition, microscopic analysis at 100X magnification showed normal structures of glomeruli and renal tubules in the control group (Figure 1). In ethylene glycol-treated rats, crystal deposition (indicated by arrows) and tubule destruction were observed mainly in the renal cortex.

Figure 1. Histopathological examination of calcium oxalate crystals in kidney sections of study groups. a. Neither abnormal-glomerulus and -renal tubules nor crystal deposition were observed in the 100 and 40x magnifications of the control group. b. Abnormal renal tubules and jagged deposition of crystals were widely observed in the cortex of the ethylene glycol group. c. The group that was treated with ethylene glycol and Sh.O bacteria

A total of 215 microscopic fields (at 100X magnification) were examined for crystal deposits, including 40 fields in the control group, 86 in the ethylene glycol group, and 89 in the ethylene glycol+Sh. O bacteria treated group. Quantitative analysis of crystal deposits showed a significant increase in the ethylene glycol treated group compared to the ethylene glycol+Sh. O treated group (P-value<0.05) (Figure 2).

Figure 2. Statistics analysis of crystal deposition centers in groups of study

After 23 days of ethylene glycol treatment, urine samples were collected from rats and the urinary oxalate levels were measured using commercially available kits. Although oxalate levels were lower in the ethylene glycol + bacteria treated groups than in the ethylene glycol alone treated group, this difference was not statistically significant (Figure 3).

Figure 3. Urinary oxalate was determined in three groups of study, including ethylene glycol (EG) rats, ethylene glycol + bacteria (Sh. O), and control. On the day of sampling, 23 days after the beginning of treatment, levels of urinary oxalate were increased significantly in rats receiving ethylene glycol in comparison to the control rats.

Whole body and kidney weight

Kidneys were weighed at the end of the paper, and total animal weight was recorded on days 0, 3, 9, 15, 21, and 29. The graph of total animal weight showed no significant difference in weight gain or loss among the studied groups at the measured time points (Figure 4).

Figure 4. Animal weight graphs at different times after treatment. Weight loss/gain was measured for groups of study, including ethylene glycol-treated, ethylene glycol-Sh. O treated and a control group that did not receive ethylene glycol and Sh. O.

Evaluation of kidney weight of all rats after slaughter showed significant weight gain in the group that received both ethylene glycol and bacteria compared to the groups that received only ethylene glycol and the control group (Figure 5).

Figure 5. Graph of the kidney weight at the end of the study. Animals that co-received ethylene glycol and Sh. O bacteria showed significant weight gain in comparison to the control and ethylene glycol groups.

Discussion

Nephrolithiasis is a urinary tract disease that has become a significant concern for human health in recent years. Calcium oxalate stones are the most common, accounting for approximately 75% of all kidney stones (16). ROS-induced oxidative stress can contributes to the formation of calcium oxalate stones. A study conducted in rats found that diets low in antioxidant substances ameliorate the formation of calcium oxalate stones by increasing ROS levels, causing oxidative stress, and enhancing the binding of calcium oxalate within the kidney (17).  Prevention of kidney stone formation through the use of antioxidant compounds has been investigated in different investigations. In one research conducted by Gulshan et al., it was found that administering 200mg/kg of an extract of an antioxidant plant known as Cynodon dactylon resulted in a reduction in the production of oxalate stones in an animal model (11). Similarly, Khajowi Rad and colleagues utilized pure fractions of this herbal drug to effectively decrease the formation of kidney stones in an animal model. According to their results, Cynodon dactylon fractions were identified as potential therapeutic substances for preventing the formation of kidney stones (10). In addition, other antioxidants such as banana stem extract, citrate and their combinations, vitamin E, quercetin and taurine have also been examined; however, it has been reported that in clinical trials, increasing the level of antioxidants simply cannot eliminate oxidative stress (18-21).

NADPH oxidase is one of the main enzymes included in the production of ROS in the body, especially in the kidneys. Metformin, which inhibits ROS and NADPH oxidase (22) was successfully used in an animal model of kidney stones, and the results indicated that metformin alleviates the formation of oxalate stones (9).

investigations have shown that the intestinal microbiome of individuals with kidney stones differs from that of non-stone-forming individuals. In stone-forming individuals, the abundance of bacteria such as Bacterioides was 3.4 times higher than in non-stone-forming individuals, while the abundance of Provetella bacteria in non-stone-forming individuals was 2.8 times that of stone-forming individuals (23). Studies have shown a relationship between the microbiome and kidney stone formation (12).

There are some bacteria in the human gut microbiome that have the ability to transfer electrons outside the cell (EET) and modulate the redox potential of the surrounding environment (15, 24). Microorganisms with the ability to transfer electrons outside the cell (electrogenic) have been studied for bioremediation of environmental pollution, producing nanoparticles with new characteristics, and producing biological energy (25). However, in vivo studies on these bacteria are limited. For instance, in a study by Khan et al., the effect of an electrogenic bacterium, Faecalibacterium prausnitzii, on the process of IBD was investigated (15) and it was found that electrogenic bacteria use thiol and flavin compounds to transfer electrons to oxygen.

 As oxidant substances are included in the pathogenesis of kidney stones, this research was designed to screen the effect of administering electrogenic bacteria on oxalate stone formation in the kidney. The hypothesis of this experiment was based on the fact that the oxidative capacity induced by EET bacteria changes the rate of stone formation in the kidneys.

We demonstrated that oral gavage of Sh. O bacteria decrease the rate of ethylene glycol-induced oxalate stones in the kidneys. The investigation’s results indicated that administration of Sh. O can alleviate renal tubule injury, urinary oxalate, and renal crystal deposition, which is consistent with the results of previous studies on antioxidant administration.

Since oxidative damage occurs in the kidney tissue of rats with nephrolithiasis during the development of stones (26), the potential protective mechanism of Sh. O involved in riboflavin-mediated antioxidant activity. The function of electron transfer from the cell to the extracellular compounds in the gut border has been previously demonstrated for flavin compounds (15). In a previous study, we demonstrated that the Sh. O bacteria require riboflavin to effectively transfer cellular electrons to external acceptors (unpublished data). The results showed that kidney weight significantly increased in rats receiving Sh .O, and ethylene glycol in comparison to the control and ethylene glycol alone groups. According to these results, the administration of the compound of Sh. O and riboflavin increases kidney health.

Conclusion

Our results showed that oral gavage of Sh. O in combination with riboflavin can alleviate renal crystal deposition and tubular damage in rats with ethylene glycol-induced nephrolithiasis.

Authors’ Contribution

Conceptualization, investigation and Writing–review: M. Kh; Methodology: M. ZB and T. M

Acknowledgement

Special thanks to Kashan University of Medical Sciences, Kashan, Iran.

Conflicts of Interest 

Authors declare that they have no conflict of interest.

Funding

No funding.

Ethical statement

This study was financially supported by Kashan University of Medical Sciences (grant number 401080). Animal procedures were approved by the Ethics Committee of Kashan University of Medical Sciences (IR.KAUMS.AEC.1401.011).

Data Availability Statement

The data supporting these study findings are available from the corresponding author upon reasonable request.

Abbreviations

CFU      Colony-Forming Units

CULA   Care and Use of Laboratory Animals

EET      Extracellular Electron Transfer

IBRC     Iranian Biological Resource Center

ROS      Reactive Oxygen Species

Sh. O     Shewanella Oneidensis