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Original research article

H2S donors attenuate diabetic nephropathy in rats:Modulation of oxidant status and polyol pathway

Marwa M. Safar *, Rania M. Abdelsalam

Department of Pharmacology and Toxicology, Faculty of Pharmacy, Cairo University, Cairo, Egypt

Introduction

Nephropathy is one of the serious long-term complications ofdiabetes which affects up to 40% of patients [1]. Albeit severalfactors are involved in the genesis of diabetic nephropathy,oxidative stress is regarded as a major culprit in the onset andprogression of kidney damage [2]. Chronic hyperglycemiaenhances the polyol pathway which comprises the conversion ofglucose to sorbitol by aldose reductase (AR) and the subsequentmetabolism of sorbitol to fructose by sorbitol dehydrogenase (SD).Activation of polyol pathway was formerly recognized as animportant factor in the development of diabetic nephropathy [3].

Despite its long-standing reputation as a foul smelling gas,hydrogen sulfide (H2S) is now considered the third gaseous

signaling molecule after nitric oxide and carbon monoxide [4]. In

this regard, it has emerged as a chief modulator of cardiovascular

homoeostasis, neurotransmission, as well as insulin secretion

[5,6]. Anti-apoptotic effect of H2S has also been claimed [7]. H2S

deficiency was observed in various animal models of arterial and

pulmonary hypertension, Alzheimer’s disease, gastric mucosal

injury, as well as liver cirrhosis. On the other hand, its excessive

production may contribute to the pathogenesis of inflammatory

diseases, septic shock, and cerebral stroke [8]. Hence H2S regulation

might provide a therapeutic prospect in various conditions. In the

past few years, the decline of blood H2S level in diabetic patients and

experimental animals has been demonstrated and was speculated to

contribute to vascular inflammation [9,10].Sulfurous mineral water has been long employed in thermal

medicine mainly for the treatment of dermatological and skeletal

muscle disorders in the form of mud and bath therapies [11,12]. In

addition, drinking of S containing water (hydroponic treatment) is

utilized especially in cases of gastroenteric disturbances [13]. A

promising role of S-based therapies in defending oxidative damage

Pharmacological Reports 67 (2015) 17–23

A R T I C L E I N F O

Article history:

Received 20 December 2013

Received in revised form 26 July 2014

Accepted 1 August 2014

Available online 20 August 2014

Keywords:

Diabetic nephropathy

Sulfurous mineral water

H2S

Insulin like growth factor-I (IGF-I)

Polyol pathway

A B S T R A C T

Background: Sulfurous mineral water and its main active ingredient sodium hydrosulfide (NaHS) are

major sources of H2S. The present study aimed to explore their protective effect on one of the serious

long-term complications of diabetes; diabetic nephropathy.

Methods: Sulfurous mineral water (as drinking water), NaHS (14 mmol/kg/day; ip), and gliclazide

(10 mg/kg; po) were administered daily for 6 weeks to streptozotocin (STZ)-diabetic rats.

Results: STZ-induced diabetes was associated with body weight reduction, hyperglycemia, overproduc-

tion of glycated hemoglobin, as well as decline in serum insulin, C-peptide, and insulin like growth

factor-I. Besides, diabetes impaired kidney functions and imposed oxidative and nitrosative stress as

manifested by elevated contents of renal thiobarbituric acid reactive substances and nitric oxide, parallel

to reduced glutathione content. These deleterious effects were antagonized by sulfurous water and to a

better extent by NaHS. Activities of myeloperoxidase and sorbitol dehydrogenase were not altered by

STZ or any of the treatments. However, STZ-induced diabetes was accompanied by an increment of

aldose reductase which was only mitigated by gliclazide and NaHS. Histopathological examination of

kidney sections corroborated the biochemical findings.

Conclusion: This study suggests a novel therapeutic approach for diabetic nephropathy using H2S

donors.

� 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp.

z o.o. All rights reserved.

Abbreviations: AR, aldose reductase; HbA1C %, glycated hemoglobin; IGF-I, insulin

like growth factor-I; MPO, myeloperoxidase; GSH, reduced glutathione; NaHS,

sodium hydrosulfide; SD, sorbitol dehydrogenase; STZ, streptozotocin; TBARs,

thiobarbituric acid reactive substance content; NOx, total nitrate/nitrite content.

* Corresponding author.

E-mail addresses: marwa.safar@pharma.cu.edu.eg, marwasafar@gmail.com

(M.M. Safar).

Contents lists available at ScienceDirect

Pharmacological Reports

jou r nal h o mep ag e: w ww .e lsev ier . co m / loc ate /p h arep

http://dx.doi.org/10.1016/j.pharep.2014.08.001

1734-1140/� 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

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associated with aging and age-related degenerative diseases hasbeen reported [14]. Nevertheless, until now, the response of theantioxidant defense system to drinking therapies involving H2S-rich water has been poorly scrutinized. Thus, the present studyaimed to investigate the effects of sulfurous mineral water and itsmain active ingredient NaHS on diabetic nephropathy in rats ascompared to the widely used and well tolerated sulfonyl urea;gliclazide. Modulation of renal oxidant status and polyol pathwayhas been targeted.

Materials and methods

Animals

Sixty-six male Wistar rats weighing 200–220 g were used inthis study. The study was conducted in accordance with the ethicalprocedures and policies approved by Animal Care and UseCommittee of Faculty of Pharmacy Cairo University, Cairo, Egypt.Animals had free access to standard rat chow and waterthroughout the study.

Drugs and chemicals

Streptozotocin was purchased from Sigma Chemicals Company,St. Louis, MO, USA. Sulfurous mineral water was obtained from theThermal Center of Helwan (Helwan Kabritage, Helwan Province;south of Cairo, Egypt), which has a sulfuric degree of 8.4 mg/L, asreported in Table 1. Gliclazide (Servier, France) was also used inthis study. All other chemicals used were of the highest purity andanalytical grade.

Experimental design

Diabetes was induced by a single intraperitoneal injection ofSTZ (50 mg/kg), freshly prepared in 0.1 M citrate buffer, pH 4.5[15]. A normal control group (n = 10) was injected with theappropriate volume of the citrate buffer. The STZ-treated rats wereallowed to drink 5% glucose solution during the first 24 h ofdiabetes induction to overcome the drug-induced hypoglycemia[16]. Four days later, blood samples were collected from rats’ tailsand hyperglycemia was confirmed by a blood glucose levelreaching 300 mg/dl. Glucose was measured using an analyzer(Roche Diagnostic Accu-Check test strips, Germany).

Diabetic rats were randomly divided into four groups (n = 10).The first one served as a control untreated diabetic group. Thesecond group was supplied daily with sulfurous mineral waterinstead of their drinking water [17]. Rats of the third group wereintraperitoneally injected with NaHS at a dose of 14 mmol/kg/day[18], while those of the fourth group were treated with gliclazide(10 mg/kg/day, po) [19]. Since NaHS is considered a toxicsubstance, we injected NaHS (14 mmol/kg/day) alone to controlnon-diabetic rats (n = 8) as the treatment control. [20] reported anLD50 value of 15 mg/kg (approximately 192 mmol/kg) for NaHS in

rats, which is far beyond the used dosage. Another control group(n = 8) was supplied with sulfurous mineral water to exclude anytoxic effect of H2S rich water consumption.

After 6 weeks and immediately before scarification, the bodyweight of each rat was determined. Blood samples werewithdrawn from the retro-orbital plexus and divided on twospecimens; one was processed for serum preparation used forassaying the levels of glucose, insulin, IGF-I, C-peptide, urea, andcreatinine. The other was collected in heparinized tubes and usedfor the estimation of glycated hemoglobin (HbA1C %). Immediatelyafter blood sampling, animals were euthanized by decapitation,and their kidneys were excised, washed with ice-cold physiologicalsaline, blotted dry, and weighed. The left kidneys were homoge-nized in ice-cold saline (10%, w/v) and used for assessment ofthiobarbituric acid reactive substance content (TBARs), reducedglutathione (GSH), total nitrate/nitrite content (NOx), as well asmyeloperoxidase (MPO), SDH, and AR activities.

Biochemical investigations in blood and serum

Serum level of glucose and the percentage of HbA1C wereassayed using commercially available kits provided by Stanbio, SanAntonio, TX, USA. Serum insulin level was measured by immunor-adiometric assay using kits provided by Immunotech, France.Serum C-peptide was measured using solid phase, two sitechemiluminescent immunometric assay using kits supplied byDPC, USA on Immulite analyzer. IGF-I was assessed using an ELISAkit provided by DRG Instruments GmbH, Germany. Serum levels ofurea and creatinine were estimated using commercial test reagentkits provided by Biodiagnostic, Giza, Egypt.

Biochemical investigations in kidney homogenate

Renal content of TBARS was determined according to themethod described by [21] using malondialdehyde (MDA) as astandard. GSH content was measured using Ellman’s reagent asdescribed by [22]. Total tissue nitrite and nitrate content wasestimated according to the method described by [23] based on theGriess reaction and employing vanadium trichloride as reducingagent. The kinetic method described by [24] was employed todetermine MPO activity. Renal activities of AR and SDH weredetermined according to the methods of [25,26], respectively. Theprotein content was measured by the method of [27].

Histopathological studies

Immediately following sacrifice, sections of the left kidneyswere fixed in 10% formalin/saline, embedded in paraffin wax andcut at 5 mm thickness. Sections were processed, stained withhematoxylin and eosin dye (H&E), and examined under lightmicroscope.

Statistical analysis

Data were expressed as means � SEM. Comparisons betweenmeans were carried out using one-way ANOVA followed by Tukey–Kramer multiple comparisons test using Instat software, version 3(GraphPad Software, Inc., San Diego, USA); probability level of lessthan 0.05 was accepted as statistically significant.

Results

No difference was detected between the normal rats and thosewhich received sulfurous mineral water or NaHS (at the useddosage) alone in any of the tested parameters, indicating that theyare safe and had no toxic effects.

Table 1Physico-chemical characteristics of the sulfurous mineral water of Helwan

Kabritage.

Parameter Unit Parameter Unit

pH 6.69 K+ 2 mg/L

Conductivity 6590 mS/Cm HCO3� 180 mg/L

Fixed residue (at 180 8C) 6480 mg/L F� 1.5 mg/L

Sulfuric degree 8.4 mg/L Cl� 1560 mg/L

CO2 80 mg/L NO2� <0.01 mg/L

Ca2+ 403 mg/L SO42� 884 mg/L

Mg2+ 49 mg/L NO3� 0.04 mg/L

Na+ 1300 mg/L Iron 0.006 mg/L

M.M. Safar, R.M. Abdelsalam / Pharmacological Reports 67 (2015) 17–2318

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General features (Table 2)

Diabetic rats were severely hyperglycemic and exhibitedoverproduction of glycated hemoglobin accompanied by reducedserum levels of insulin (80.5%), C-peptide (70%), and IGF-I (54%) ascompared to normal animals. These were associated with a markeddecline in body weight (33%) and increment of relative kidneyweight (77%). Sulfurous mineral water, NaHS, as well as thereference antidiabetic drug, gliclazide, antagonized hyperglycemiaby 47%, 59%, and 57%, respectively. In addition, the treated groupsrevealed reduced levels of HbA1C by 50%, 54%, and 33%,respectively, as compared to the diabetic group. Similarly, relativekidney weight was lowered by sulfurous mineral water (28%),NaHS (35%), and gliclazide (36%) without normalizing the bodyweight. Albeit none of the treatments restored the C-peptide level,NaHS leveled up serum insulin and IGF-I by nearly 4 folds and2 folds, respectively, as compared to the diabetic rats. Bothsulfurous mineral water and gliclazide tended to increase IGF-Ilevel but did not reach statistical significance.

Alterations of kidney function biomarkers (Fig. 1)

STZ-induced diabetes provoked a pronounced spike in serumurea (2.3 folds) along with a significant elevation of serumcreatinine (49%). Sulfurous mineral water, NaHS, and gliclazidecounteracted these changes. Reduction of urea reached 39.5%, 59%,and 61%; while that of creatinine was 27%, 31%, and 34.5% bysulfurous mineral water, NaHS, and gliclazide, respectively ascompared to diabetic rats.

Effects on renal content of TBARS, GSH, NOx, and MPO (Table 3)

A prominent increment of renal TBARS content (43.5%), ahallmark of lipid peroxidation, was observed in the untreateddiabetic group which was hampered by sulfurous mineral water(52%), NaHS (38%), and gliclazide (41%). Likewise, STZ-treated ratsshowed an elevation in renal NOx content which mounted to 27%,but only NaHS and gliclazide mitigated this effect by 30% and 36%respectively as compared to diabetic animals. On the other hand,

Table 2Final body weight, relative kidney weight, serum glucose, insulin, C-peptide, IGF-I, and glycated hemoglobin of normal, diabetic, and diabetic treated rats.

Parameters/groups Final body

weight (g)

Relative kidney

weight (g %)

Glucose (mg/dl) HbA1C (%) Insulin (mIU) C-peptide

(ng/ml)

IGF-I (ng/ml)

Normal 262 � 14.8 0.52 � 0.02 130.12 � 3.45 5.97 � 0.18 15.81 � 1.39 3.81 � 0.75 187.29 � 21.43

Sulfurous water 223 � 23 0.63 � 0.04 105.48 � 7.96 6.56 � 0.54 22.8 � 3.11 3.54 � 0.7 180.39 � 19.76

NaHS 232 � 7.7 0.56 � 0.02 127.64 � 16 5.83 � 0.6 12.63 � 0.15 3.52 � 0.55 181.25 � 19.51

Diabetic 176 � 13.3a 0.92 � 0.04a,a1,a2 412.80 � 24.12a,a1,a2 16.08 � 1.43a,a1,a2 3.09 � 0.2a,a1,a2 1.14 � 0.09a,a1,a2 85.93 � 2.37a,a1,a2

Diabetic + gliclazide 212 � 10.5 0.59 � 0.06b 178.58 � 31.15b 10.73 � 0.54a,a1,a2,b 9.43 � 3.34a1 1.96 � 1 144.41 � 9.2

Diabetic + sulfurous

water

249 � 12.5 0.66 � 0.05b 218.49 � 22.78a,a1,a2,b 8.01 � 0.51b 4.69 � 0.27a,a1 1.56 � 0.06 143.62 � 22.55

Diabetic + NaHS 202 � 14.2 0.6 � 0.04b 170.4 � 14.44b 7.37 � 0.58b 12.68 � 0.44a1,b 2 � 0.11 185.57 � 25.23b

Values are expressed as mean � SEM of 7–10 animals in each group. avs normal, a1vs sulfurous water, a2vs NaHS, bvs diabetic (one-way ANOVA followed by Tukey–Kramer multiple

comparisons test; p < 0.05).

Table 3Renal contents of TBARS, GSH, NOx, and MPO of normal, diabetic, and diabetic treated rats.

Parameters/groups TBARS (nmol/g tissue) GSH (mg/g tissue) NOx (mmol/g tissue) MPO (U/g tissue)

Normal 100.33 � 4.43 967.74 � 97.32 163.49 � 9.95 0.41 � 0.03

Sulfurous water 116.92 � 4.37 901.94 � 92.13 123.63 � 2.62 0.36 � 0.05

NaHS 71.18 � 7.58 1387.1 � 68.28 126.65 � 2.57 0.37 � 0.02

Diabetic 144.03 � 17.87a,a2 327.1 � 68.55a,a1,a2 207.29 � 10.05a,a1,a2 0.39 � 0.03

Diabetic + gliclazide 85.36 � 15.47b 1354.84 � 234.53b 132.31 � 4.79b 0.44 � 0.03

Diabetic + sulfurous water 68.76 � 7.35b 929.03 � 80.43b 203.32 � 1.792a1,a2 0.35 � 0.03

Diabetic + NaHS 89.46 � 8.31b 1329.95 � 159.53b 144.57 � 7.81b 0.4 � 0.03

Values are expressed as mean � SEM of 7–10 animals in each group. avs normal, a1vs sulfurous water, a2vs NaHS, bvs diabetic (one-way ANOVA followed by Tukey–Kramer multiple

comparisons test; p < 0.05).

Fig. 1. Kidney function tests of normal, diabetic, and diabetic treated rats. Each bar with vertical line represents the mean � SEM of 7–10 animals in each group. avs normal,a1vs sulfurous water, a2vs NaHS, bvs diabetic (one-way ANOVA followed by Tukey–Kramer multiple comparisons test; p < 0.05).

M.M. Safar, R.M. Abdelsalam / Pharmacological Reports 67 (2015) 17–23 19

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renal GSH was depleted in diabetic rats by 66%. All tested drugsreinstated normal GSH content. No significant change wasdetected in MPO content.

Effects on polyol pathway (Fig. 2)

STZ-induced diabetes instigated a significant increase in renalAR activity (66%) without affecting the SDH activity. NaHS andgliclazide reduced the elevated AR activity by 46% and 50%,respectively. No improvement was depicted in the sulfurousmineral water treated group.

Histopathological examination of kidney

Fig. 3 illustrates photomicrographs of kidneys from normal,diabetic, and treated rats. A normal architecture of the glomeruliand tubules in the cortex and the tubules at the corticomedullaryjunction was manifested in the normal group (a and b), whilediabetic rats demonstrated focal inflammatory cells infiltration inbetween the glomeruli, tubules, and blood vessels at the cortex (c).There were swelling and vacuolization in the lining endothelium ofthe glomerular tufts as well as swelling and degeneration in thelining epithelium of the tubules (d). The tubular lumen of themedullary portion had homogenous eosinophilic casts as well asred blood cells (e). Congestion and dilatation were detected in theblood vessels of both the cortex and the medulla (f and g). No majorhistopathological alteration was observed in treated rats whichportrayed normal histological structures of the glomeruli andtubules at the cortex (h–j).

Discussion

The current investigation depicts antidiabetic effects ofsulfurous mineral water and to a better extent its main activeconstituent NaHS which are quite comparable to gliclazide. Thisgoes in line with the findings of [28]. A recent study demonstratedthat NaHS possesses an insulin sensitizing effect both in vitro andin vivo [29]. Decline in plasma H2S level and its vascular productionhave been previously reported in diabetic animals and humans[10]. Consequently, the exogenous administration H2S donors inthis study might have provided compensation. Sulfur is alsonecessary for the formation of insulin and IGF-I [30]. Several linesof evidence accentuate the antidiabetic effects of sulfur containingcompounds via increasing insulin secretion and insulin sensitivity[31,32].

STZ-induced diabetes was associated with marked kidneyfunction impairment along with renal lipid peroxidation, excessivenitric oxide production, and depletion of GSH; effects which clearly

indicate enhanced oxidative stress. Similar findings have beenpreviously documented [33,34]. Glucose auto-oxidation, non-enzymatic glycation, and the overproduction of reactive oxygenspecies (ROS) by mitochondria are potential sources of hypergly-cemia-induced oxidative stress [3,35]. Increased HbA1C levelobserved herein represents a key point in diabetes associatednephropathy. Oxidant injury of proteins and lipids along with theaccumulation of AGEs instigate dysfunction of apical transportersin renal proximal tubule cells [36], proliferation of various renalcell types [37], microvascular complications [38], and apoptosis[39]. C-peptide, a peptide hormone that possibly acts through a Gprotein-coupled membrane receptor, was shown to protect againstapoptosis in kidney proximal tubular cells [40]. In this context, thereduced serum level of C-peptide in diabetic rats has contributed tothe kidney malfunction and histopathological changes. None of thetested drugs antagonized this effect.

In the present study, sulfurous mineral water and NaHScounteracted the elevation of renal TBARS and replenished GSHcontents; the latter was also able to normalize the NOx content.Improvement in renal redox balance was reflected on kidneyfunction tests, its hypertrophy and histopathological features.Several lines of evidence emphasize these findings where sulfurousmineral water drinking therapy improved body redox status inhealthy volunteers [17] and in cardiac tissue of diabetic rats[28]. Similarly, [41] reported the decrease in NOx production inosteoarthritic mice subjected to mud therapy. The protectiveeffects of sulfur water against the free-radical damage of lipids andproteins may be possibly related to the increase in total –SH levels,which include both protein (principally albumin) and non-protein(cysteine and GSH) thiol groups [17].

H2S may react with reactive oxygen and/or nitrogen specieslimiting their toxic effects and might be involved in the reductionof thiols, thus being directly implicated in redox reactions as anantioxidant [8]. In vitro, the H2S donor NaHS directly scavengedhydrogen peroxide and superoxide anions [18] while in vivo, itsadministration alleviated ischemia/reperfusion injury-inducedrenal dysfunction [42] and mitigated ureteral obstruction-induced kidney fibrosis [43]. The endogenously produced H2Sapparently ameliorates oxidative injury by increasing theintracellular concentrations of GSH via increasing the activityof g-glutamylcysteine synthetase and upregulating cystinetransport [5]. Besides its observed effect as an antioxidant dueto its H2S content’s, sulfurous mineral water may combat the stateof hypomagnesaemia often encountered in diabetes [44] due to itsenrichment with Mg2+. The sulfur content might also play a role;sulfur compounds have been found to be beneficial in counter-acting oxidative stress and ameliorating diabetic nephropathy[45,46].

Fig. 2. Sorbitol dehydrogenase and aldose reductase activities of normal, diabetic, and treated rats. Each bar with vertical line represents the mean � SEM of 7–10 animals in

each group. avs normal, a1vs sulfurous water, a2vs NaHS, bvs diabetic (one-way ANOVA followed by Tukey–Kramer multiple comparisons test; p < 0.05).

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Elevation of renal AR along with a deranged oxidant status iscoherent with previous studies [33]. Activation of NADPH-dependent aldose reductase (AR) diminishes the quantity ofNADPH available for the reduction of oxidized glutathione (GSSG)by glutathione reductase [47]. This suggests that the reduced renalGSH content observed herein is due to its reduced synthesis ratherthan its increased degradation. Taken in consideration that GSHrepresents the key non-enzymatic tissue antioxidant, its depletionindicates a reduced capacity of kidney antioxidant defensesystem making it more vulnerable to the hyperglycemic ROS- and

RNS-induced damage. This is confirmed by the finding thatabnormal polyol pathway in diabetic rats resulted in glomerulo-pathy and renal hypofunction [3]. Notably, aldose reductaseinhibitors exhibited protective effect on diabetic nephropathypossibly via counteracting oxidative and nitrosative stress indiabetic kidney [48] and/or reducing renal vascular endothelialgrowth factor VEGF overexpression [49]. Normalization of ARactivity by NaHS is a key finding in this study.

Renal SD was not altered in this study demonstrating that ARactivity, the first step in the polyol pathway, makes a greater

Fig. 3. Photomicrographs of sections from the kidneys of normal, diabetic, diabetic treated rats Kidneys from (a) normal rat treated with 1% Tween 80 showing normal

histological structure of the glomeruli (g) and tubules (t) at the cortex; (b) normal rat treated with 1% Tween 80 showing normal histological structure of the tubules (t) at the

corticomedullary portion; (c) diabetic rat showing focal inflammatory cells infiltration (m) in between the glomeruli (g) and blood vessels (v); (d) diabetic rat showing

vacuolization and swelling in the lining endothelium of the glomerular tuft (g) with swelling and degeneration in the lining epithelium of the tubules (d); (e) diabetic rat

showing homogenous eosinophilic casts (c) and red blood cells (r) in the lumen of the tubules at the medullary portion; (f) diabetic rat showing severe congestion in cortical

blood vessels (v); (g) diabetic rat showing severe congestion in medullary blood vessels (v); (h–j) rats treated with gliclazide, sulfurous water, NaHS, respectively showing

normal histological structure of the glomeruli (g) and tubules (t) at the cortex (H&E �40 except f and g �80).

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contribution to the etiology of diabetic nephropathy than does thesecond step involving SD. Interestingly, it was claimed that geneexpression of the two enzymes are not regulated via a commonmechanism and are tissue-specific; where STZ-diabetes increasedSD gene expression in testis but not in kidney or brain [50].

In conclusion, this study suggests that H2S donors possess anti-diabetic effects and ameliorate associated nephropathy possiblyvia modulation of renal redox status and polyol pathway. Hence,may promise a novel and applicable approach in preventing and/ortreating diabetic complications.

Conflicts of interest statement

The authors declare that there are no conflicts of interest thatcould prejudice the impartiality of this scientific work.

Funding

This study received no funds from any sources. It was carried onthe author’s own expenses from their salaries they receive from thefaculty.

Acknowledgments

The authors gratefully acknowledge Dr. Tarek Samir, WaterPollution Control Department, National Research Center, Cairo,Egypt, for the analysis of sulfurous mineral water. The authors alsothank Prof. Dr. Adel Bakeir, Histology Department, Faculty ofVeterinary Medicine, Cairo University, for performing the histo-pathological examinations of this study.

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