Modulatory Effect of ZamZam Water on the Renal and Hepatic Function of Mice Challenged with Gentamicin

Gentamicin is very powerful aminoglycosides that is used for treatment of infections those caused by gram negative bacteria. It causes a dangerous nephrotoxic effect (Yousuf et al., 2018 and Mathew 1992). This adverse effect limits gentamicin clinical use. The exact and clear mechanism of gentamicin-induced nephrotoxicity is not fully understood yet, but a remarkable increase in of reactive oxygen species (ROS), inflammatory cytokines, lipid peroxidation increase which have an important role in this mechanism. Decrease of antioxidant renal enzymes as superoxide dismutase (SOD), glutathione (GSH) and glutathione peroxidase (GPx) catalase (Sanchez-Gonzalez et al., 2011 and Priyamvada et al., 2008). 30% of the aminoglycosides patients showed a nephrotoxic effect within 7 days of administration (Mathew 1992). Many studies revealed that anti-inflammatory agents and also antioxidants have a great role against gentamicin adverse effects (Feyissa et al., 2013; Lee et al., 2012; Anandan and Subramanian 2012 and Stojiljkovic et al., 2012).

Holy water was used long time ago for spiritual practices according to many different religions and beliefs. Sikhism, Hinduism and others use water for this purpose (Foley 2011; Clarke 2010 and Heinrich 2009).

Muslims believe that Zamzam water is blessed and they use it to satisfy thirst and hunger at the same time. Also Muslims believe that it cure any type of illness so millions pilgrims drink from Zamzam water as much as they can during pilgrimage (Careem 2005) from the time of the prophet Ibrahim.

Zamzam water is alkaline in nature that come from Zamzam well located in Makkah, Saudi Arabia but  now Zamzam water is available for all people throughout the Masjid Al Haram in  both Makkah and Medina. Every year millions of Muslims all over the world can drink from Zamzam water but the commercial export of water is prohibited by Saudi government (Basem Shomar 2012). Zamzam water is stated to have high concentrations of minerals like fluoride, calcium and magnesium some salts also recorded to be in high level like nitrate (ZSRC 2011). Arsenic level was reported to be very high may reach three times folds (LCC 2005). Many studies investigated Zamzam water and reported that the original water has high pH (average 8) and no bacteria. Zamzam water prevents the formation of kidney stones, cure from diabetes type 2 and also reduce the oxidative stress that is associated with many diseases (Arjinajarna et al., 2017; Tavafi et al., 2012; Abdel-Raheem et al., 2009 and Al-Majed et al., 2002).

In this work authors aim to clarify the ameliorative effect of Zamzam water on nephrotoxicity induced mice throughout biochemical studies for kidney and liver functions.  

Zamzam water was obtained from Zamzam well, Makkah, Kingdom of Saudi Arabia (KSA). Gentamicin was purchases from Sigma. Chemicals for biochemical investigation of different parameters (AST, ALT, CAT, Creatinine, urea, TNF, IL-6, IL-4, IL-10 and MDA) was purchased from Human Diagnostic Worldwide (Max-Planck-Ring 21, Wiessbaden, Germany).
 
Animals
 
Male Swiss Webster mice were purchased from King Saud University. Animals were kept under normal standard laboratory conditions of relative humidity and temperature. The dark/light cycle was as normal and animals allowed for free access to food and water. All experiments had been carried out according to the normal regulation.
 
Experimental protocol
 
In this study authors used twenty five male mice of 25-30 gm weight. Mice were randomly divided into five groups (5 mice for each). Mice allowed to be adapted to the experiment place condition for two weeks. The experimental continued for one month. Mice were grouped into the following groups.
 
Group I: Control group received Normal water for drinking and injected with phosphate buffer saline (PBS) intraperitoneal (IP).
Group II: Offered Zamzam water for drinking and injected with Gentamicin in concentration of 100 mg/kg of mice for 15 days.
Group III: Injected with Gentamicin in concentration of 100 mg/kg of mice for 15 days then offered a normal water.
Group IV: Injected with gentamicin for 15 days followed by Zamzam water drinking for another 15 days.

Group V: Offered Zamzam water for 15 days then injected with gentamicin for another 15 days during which, mice offered Zamzam water for drinking. After the end of the experiment, mice were sacrificed and blood samples were collected from each group and centrifuged at 4000 rpm for 10 min. Serum samples were stored in -20 for estimating different blood parameters. Tissues (kidney and spleen and liver) were collected and stored in -800- for PCR experiment.
 
Biochemical analysis
 
Kidney injury markers (creatinine and urea), proinflammatory cytokines, interleukin-6 (IL-6) and tumor necrosis factor (TNF), anti-inflammatory ones, interleukin-4 (IL-4), interleukin-10 (IL-10). Liver function was investigated throughout liver enzymes, aspartate transaminase (AST), alanine transaminase (ALT), catalase enzyme (CAT) and oxidative stress throughout lipid peroxidation and reduced glutathione were carried out using kits for each parameter from Human Diagnostic, Germany according to the manufacturer manual.
 
PCR
 
Total RNA was isolated from liver, spleen and kidney. mRNA was purified from total one and then cDNA was synthesized using oligo-dt (promega). Primers were designed for proinflammatory cytokines, IL-6 (5-cttcttgggactgatgtt-3) and (5-gtaagttgttcttcacaa-3) TNF (5-atccgagatgtggaactg-3) and (5-cacgtagtcggggcagcc-3), anti-inflammatory ones, IL-4 (5-ccccaccttgctgtcacc-3) and (5-tgagttcagaccgctgac-3) IL-10 (5-tgttgcctgccttactg-3) and (5-gcagttgatgaagatgtc-3) as forward and reverse primer respectively for each gene. The optimal annealing temperature was, 51, 49, 48, 51, for IL-6, TNF-α, IL-4 and IL-10 respectively. PCR amplification was done and the expression analysis was done in liver spleen and kidney.
 
Statistical analysis
 
Collected data were statistically analyzed using one way ANOVA test for comparison of the tested different groups. Significant level was considered at (P value £ 0.05). Statics were done for numerical results throughout mean, standard deviation and minimum and maximum of the range.

Gentamicin sulphate is used as antibacterial drugs especially for gram negative bacteria (Corona et al., 2014). Gentamicin antibiotic has a nephrotoxic and hepatotoxic side effect, for this reason it use is very limited. Its administration causes cell apoptosis, necrosis and many other physiological effect like macrophage infiltration and oxidative stress (Suzgec et al., 2005). The aim of this work is to show the effect of Zamzam water on the alteration of the renal and hepatic function of mice. Injection of 100 mg/kg of gentamicin through intraperitoneal rout results in nephrotoxicity and hepatotoxicity after 15 days by the elevation of serum creatinine, urea as kidney biomarkers, AST and ALT as liver function enzymes. An elevation of serum creatinine, urea as kidney biomarkers (preliminary unpublished data), AST and ALT as liver function enzymes was also recorded (Table 1).  



Gentamicin also increase the oxidative stress and inflammation which finally leads to apoptosis. These experimental results come in accordance with several previous studies (Arjinajarna et al., 2017). In this study a significant increase in MDA (peroxidation product) in mice injected by gentamicin when compared with the control ones. The oxidative stress conditions leads to attacking of polyunsaturated fatty acids (PUFAs) of the phospholipids found in the cellular membrane alerting the permeability characters of many intracellular components. These consequences finally leads to cell death. Degeneration of cells produces MDA which interact with nucleic acids resulting in impairing of the uniformity of protein and nucleic acid (Bharrhan et al., 2010). Many previous studies results support these results (Madrigal-Santillan et al., 2014 and Galaly et al., 2014). Also a significant decrease in the reduced glutathione was reported as in Table 1, Fig 1.

Pretreatment with ZamZam water for two weeks before gentamicin-challenged mice showed anti-per oxidative effect where results of the study stated a decrease in MDA and increase in catalase enzyme (CAT) in liver significantly. Plasma AST and ALT recorded an increase in gentamicin challenged more than those pretreated with ZamZam water Table 1, Fig 1. ZamZam water have a different ions and minerals concentration than normal water which may give Zamzam water its effect. Different minerals in Zamzam water like Lithium, lead, cadmium-zinc combination have an inhibitory effect on the proliferation of cancer cells (Corbit et al., 2006). ZamZam water has a high concentration of calcium. Ionized calcium is needed for many enzymatic reaction shared in many functions in the body like hepatic glycogen metabolism, hormone secretion, muscle contraction blood coagulation and other many functions (Schenck et al., 2012). Also Na+K ATPase enzyme that control and  regulate ions including calcium among them so the different proportion of calcium may affect the cellular activities including keeping hepatic enzymes ALT, AST and MDA.  

The level of both alanine transaminase and asparagine transaminase (ALT and AST) showed a highly significant (P<0.001) elevation in mice serum challenged with gentamicin. The elevation of these enzymes was modulated by using Zamzam water in other different group (Table 1). Results of this work showed also a significant increase in MDA (peroxidation product) in mice injected by gentamicin when compared with the control ones (Table 2).  

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A significant decrease in the reduced glutathione was reported as in Table 2. Pretreatment with ZamZam water for two weeks before gentamicin-challenged mice showed anti-per oxidative effect where results of the study stated a decrease in MDA and increase in reduced glutathione (Glu) in liver homogenate significantly (Table 2).

Results of this study revealed that the level of AST and ALT recorded a significant decrease in mice pretreated with Zamzam water. ZamZam water may reduce the oxidative stress throughout neutralization of free radicals those produced from oxidative stress. The different concentration of ZamZam water minerals than those of normal one may protect the integrity of the cell membrane of both liver and kidney these results were in agreement with that of (Rawi et al., 2011).

Both proinflammatory and anti-inflammatory cytokines such as IL-6, tumor necrosis factor alpha (TNF-α), IL-4 and IL-10 showed a marked change in both biochemical analysis and by semi quantitative PCR. Using ZamZam water in this experiment recorded a decrease in expression of TNF- and IL-6 (Fig 1). Increase in both IL-4 and IL-10 was also reported in this study Table 3 and 4 (Fig 1). Proinflammatory and anti-inflammatory cytokines increased after liver infection or liver damage. Differentially expression of proinflammatory cytokines in liver neutrophils and Kupffer cells was reported in previous experiments (Chiao et al., 2005 and Kim et al., 2006). The central mediator for the proinflammatory cytokines (TNF-α) and IL-4 has an anti-inflammatory effect. The expression of anti-inflammatory cytokines was reported to require induction of hem-oxygenase enzyme-1 (HO-1) throughout protein kinase pathway. In agreement with our results previous studies reported that ZamZam water significantly reduced the viability of cancer cells (Omar et al., 2017).

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Results of this study showed that pretreatment of ZamZam water leads to enhance antioxidative function. Proinflammatory and anti-inflammatory cytokines were changed using ZamZam water. Creatinine and urea also changed by using ZamZam water so we can say that ZamZam water has a protective effect for both kidney and liver cells.