Research Article Upregulation of Oxidative Stress Related...

Research ArticleUpregulation of Oxidative Stress Related Genes ina Chronic Kidney Disease Attributed to Specific GeographicalLocations of Sri Lanka

Saravanabavan Sayanthooran1 Dhammika N Magana-Arachchi1

Lishanthe Gunerathne2 Tilak D J Abeysekera3 and Suneth S Sooriyapathirana4

1Cell Biology National Institute of Fundamental Studies Kandy Sri Lanka2Renal Research Centre District Hospital Girandurukotte Sri Lanka3Department of Pharmacology Faculty of Medicine University of Peradeniya Peradeniya Sri Lanka4Department of Molecular Biology and Biotechnology Faculty of Science University of Peradeniya Peradeniya Sri Lanka

Correspondence should be addressed to Dhammika N Magana-Arachchi cellbioifsaclk

Received 10 May 2016 Revised 29 September 2016 Accepted 16 October 2016

Academic Editor Leon Spicer

Copyright copy 2016 Saravanabavan Sayanthooran et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Objective To infer the influence of internal and external oxidative stress in chronic kidney disease patients of unknown etiology(CKDu) in Sri Lanka by analyzing expression of genes related directly or indirectly to oxidative stress glutamate-cysteine ligasecatalytic subunit (GCLC) glutathione S-transferasemu 1 (GSTM1) glucose-6-phosphate dehydrogenase (G6PD) fibroblast growthfactor-23 (FGF23) and NLR family pyrin domain containing 3 (NLRP3)Methods Reverse transcription quantitative polymerasechain reaction (RT-qPCR) was carried out for the selected populations CKDu patients (119899 = 43) chronic kidney disease patients(CKD 119899 = 14) healthy individuals from a CKDu endemic area (GHI 119899 = 9) and nonendemic area (KHI 119899 = 16) Fold changeswere quantified relative to KHI Results GCLC had greater than threefold upregulation in all three study groups with a maximumof 727-fold upregulation in GHI (119901 = 0000) GSTM1 was not expressed in 256 of CKDu and 429 of CKD patients but CKDupatients expressing GSTM1 showed upregulation of 260-fold (119901 lt 005) Upregulation of FGF23 and NLRP3 genes in CKD andCKDu was observed (119901 lt 001) with greater fold changes in CKD Conclusion Results suggest higher influence of external sourcesof oxidative stress in CKDu possibly owing to environmental conditions

1 Introduction

Chronic kidney disease (CKD) is on the increase globallywith 10 percent of the worldwide population being affectedby CKD and millions dying each year due to lack of accessto treatment and to end-stage renal disease [1] This globalincrease of CKD is majorly secondary to an increase indiabetes infections and cardiovascular and autoimmunedisorders [2]

In developed countries generally the risk factors includetype 2 diabetes hypertension obesity and ageing Glomeru-lar nephritis and interstitial nephritis are the major causes ofCKD in developing countries and point towards microbialinfections and environmental toxins as risk factors [3] Dia-betes and hypertension are also risk factors in the developing

world with diabetes causing 91 to 299 percent of the cases ofESRD and hypertension causing 13 to 21 percent of the cases[3]

In Sri Lanka between 1990 and 2007 hospital admissionsdue to all diseases of the genitourinary system doubled withhospital deaths due to such diseases rising from 26 to 91per 100000 people [4] CKD has gained a lot of importancein Sri Lanka due its rise in alarming rates in many regionsof the country in which its cause is yet unknownuncertainThe Sri Lankan government and World Health Organization(WHO) hence have given it the name chronic kidney diseaseof unknown etiology (CKDu) It is estimated that around400000 people in Sri Lankarsquos north central region may beaffected by the disease [5]

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 7546265 9 pageshttpdxdoiorg10115520167546265

2 BioMed Research International

This peculiar form of CKD is not specific to Sri Lankaand similar cases have been recorded across three continentsaround the world [6] mostly in agricultural communities ofLatin America [7 8] Egypt [9] and India [10 11]

Reactive oxygen species (ROS) and nitrogen species(RNS) are essential for maintaining the healthy physiologicalstate of an individual however when it exceeds the necessaryamounts it leads to oxidative stress which is the pathophys-iological mechanism for many chronic diseases includingCKD [12]The expressions of the oxidative stress related genesalter depending on both the internal and external oxidativestress We therefore hypothesize that studying the differentialexpression of genes related to oxidative stress response inCKD CKDu and healthy individuals of a CKDu-affectedendemic region and comparing them to healthy individualsof an unaffected region would reveal the contribution ofoxidative stress from the environment As majority of thehypotheses for causes of CKDu are environmental relatedstudying the expressions of oxidative stress related genes inthese populations of different geographical areas would resultin better understanding the influence of the environment inthis disease

The oxidative stress related genes selected for the studyinclude the glutamine cysteine ligase C subunit (GCLC)glutathione S transferase mu 1 (GSTM1) and the glucose 6phosphate dehydrogenase (G6PD) genes Two of these genesGCLC and GSTM1 are related to the glutathione (GSH)protein The G6PD gene was selected as it plays a role inprotection against reactive oxygen species by the productionof NADPH which is required for the generation of reducedGSH as well as due to its deficiency being linked to CKDu inanother study [13] Polymorphisms of these genes althoughnot in combination have been seen in cases of CKDu andend-stage renal disease (ESRD) in Mexican [14] Indian [15]and Sri Lankan populations [13]

ROS is however generated in CKD by several internalprocesses including hyperactivation of NADPH oxidaseincreased production of oxidative stress markers and therelease of uremic toxins [16] We therefore analyzed theexpression pattern of genes reacting to internal sources ofoxidative stress production to better understand the environ-mental versus physiological sources of oxidative stress in thestudy groups

The increased oxidative stress in chronic kidney diseaseis in part attributed to the dysfunctional mitochondria whichcause an increased electron leakage from the respiratorychain during oxidative phosphorylation with a consequentgeneration of ROS [16] NLR family pyrin domain containing3 (NLRP3) is known to be activated by mitochondrial ROS[16] and therefore it was selected to study the influence ofmitochondrial oxidative stress Fibroblast growth factor-23(FGF23) a bone-derived hormone although not directlylinked to oxidative stress is believed to influence systemicphosphate homeostasis vitamin D metabolism and alphaklotho expression [18] all factors which play roles in theoxidative stress balance These genes were thus selected toprovide an exhaustive spectrum of basic conditions

Dry zone of Sri LankaNorth Central ProvinceUva Province

GirandurukotteKandy

N

Figure 1 Map of Sri Lanka highlighting dry zones of the countryand study locations

2 Materials and Methods

21 Study Population and Selection Criteria Ethical clearancewas obtained from the Ethics Review Committee of thePostgraduate Institute of Science University of PeradeniyaInformed consent for the study was obtained from eachsubject prior to blood collection The samples were collectedin different batches from June 2013 to January 2015

The study population was recruited in three study groupsCKDu CKD and healthy groups from Girandurukotte(GHI) which is a high prevalent area for CKDu located inthe dry zone of Sri Lanka The CKD and CKDu patientswere recruited from the District Hospital GirandurukotteA control group of healthy individuals was selected fromthe Kandy District (KHI) which is not an endemic area ofCKDu (Figure 1)The two healthy groups GHI and KHI wereapparently healthy at the time of recruitment and did notshow symptoms of any disease did not have any past medicalhistory and were not previously diagnosed with any chronicdiseases or infection All study subjects belonged to the samerace and ethnicity and only difference was on their place ofresidence

The diagnosis of patients was carried out by the consult-ing nephrologist at the hospital and CKDu was diagnosedbased on criteria set by the Ministry of Health Sri Lanka nopast history of diabetes mellitus chronic or severe hyperten-sion snake bite glomerulonephritis or urological diseasesnormal HBA1C (lt65) blood pressure lt 160100mmHguntreated or lt14090mmHg on up to two antihypertensivemedications

22 Inclusion and Exclusion Criteria Inclusion criteria forthe study were patients diagnosed by the physician as CKDu

BioMed Research International 3

Table 1 Primer and probe sequences used for the study

Name Accession number Amplicon length(base pairs)

Oligonucleotide sequences 51015840ndash31015840(F forward R reverse P probe) References

GCLC NM 0014983 149F ACAAGGACGTTCTCAAGTG

Self-designedR AGGATGGTTTGGGTTTGTCP CCTGTCTGGGGAGAAAGTTCTTGAAACTCT

GSTM1 NM 0005613 121F GCATGATCTGCTACAATCCA

Self-designedR TTGTTTCCTGCAAACCATP CCTGAAAAGCTAAAGCTCTACTCAGAG

G6PD NM 0004024 76F TGCCCCCGACCGTCTAC

RTPrimer DBR ATGCGGTTCCAGCCTATCTGP ACTCGTGAATGTTCTTGGTGACGGCC

NLRP3 NM 0048954 225 F CTCCTTTACGCCAGGGTGAG Self-designedR AGATAGCGGGAATGATGATATGAG

FGF23 NM 0206382 120 F AATCTCAGCACCAGCCACTC Self-designedR GGAGGCATTGGGATAGGCTC

B2M NM 0040482 98F TGCCGTGTGAACCATGTGA

Koop et al 2003 [17]R CCAAATGCGGCATCTTCAAP TGATGCTGCTTACATGTCTCGATCCCACT

GAPDH NM 0012897451 151F TGACCTCAACTACATGGTTTA

Self-designedR GCCCCACTTGATTTTGGAP CCATGGCACCGTCAAGGCTGA

(irrelevant of the stage) and CKD patients with etiologiesof diabetes or severe hypertension Other etiologies of CKDwere excluded from the study to limit variation The qualityand quantity of the extracted RNA were also factors deter-mining inclusion to the study The integrity of RNA as seenon agarose gel electrophoresis had to have 28s rRNA bandapproximately twice as intense as the 18s rRNA band andabsorbance A260A280 ratio of above 18 The quantity hadto be sufficient for the preparation of cDNA Samples that didnot fulfill the above criteria were excluded

23 Specimen Collection and RNA Extraction RNA wasextracted from whole blood (1mL) using TRIzol LS reagent(Invitrogen USA) according to manufacturer instructionsThe RNA was further purified using PAXgene spin columns(PreAnalytix USA)

24 Reverse Transcription Quantitative PCR (RT-qPCR)cDNA was prepared by reverse transcription of 1120583g of totalRNA using QuantiTect Reverse Transcription kit (Qiagen) asdescribed in themanufacturer protocol and quantitative PCRwas carried out using QuantiTect Probe PCR kit (Qiagen)on a Rotor-Gene Q PCR cycler (Qiagen) Hydrolysis probeslabeled with 51015840FAM fluorescent dye and 31015840BHQ quencher aswell as SYBR greenmediated fluorescent detection were usedfor qPCR Details of primerprobe pairs used in the study arelisted (Table 1)

25 Data Analysis Fold changes were calculated using thecomparative Ct method [19] Normalization was carriedout against two reference genes B2M and GAPDH and

individual fold changes were calculated using geometricmean of KHI males as calibrators The log

2normalized fold

changes were used to calculate outliers The median valueswere calculated and extreme values extending to either sideof 150 of the interquartile range (IQR) were consideredoutliers and not used in further calculations Geometricmeans of expression fold changes for each gene in betweenthe groups were calculated The significance of the foldchanges was calculated using one-way ANOVA followedby post hoc Tukey analysis of significant results Significantresults were considered as those with 119901 values lt 005

3 Results

The population included for the study from the Giran-durukotte area consisted of CKDu patients (119899 = 43) CKDpatients (119899 = 14) having etiologies of either hypertensiondiabetes or both and healthy individuals (GHI 119899 = 9)Healthy population were volunteers from the Kandy district(KHI 119899 = 16)

It is notable that the majority of the CKD and CKDupatients were farmers whereas the controls were of otheroccupations The study population consisted of more malesin accordance with an association that has been foundwith gender and proteinuric-CKD patients in a Sri Lankanpopulation [20] Patient demographics have been presentedin Table 2

31 Statistical Analysis Fold changes and statistical signif-icance as per one-way ANOVA and post hoc Tukey HSDanalysis are summarized in Table 2 Of the studied genes


Table 2 Demographics of study population

CKDu (119899 = 43) CKD (119899 = 14) GHI (119899 = 9) KHI (119899 = 16)

Gender Males = 37 Males = 13 Males = 8 Males = 15Females = 6 Females = 1 Females = 1 Females = 1

Age (years) 520 plusmn 92 582 plusmn 107 400 plusmn 113 34 plusmn 81

Occupation Farmers = 38 Farmers = 12 Farmers = 0 Farmers = 0Nonfarmers = 5 Nonfarmers = 2 Nonfarmers = 9 Nonfarmers = 16

Area of residence Girandurukotte Girandurukotte Girandurukotte Kandy

Table 3 Fold change in expression of genes in the study groupscompared to KHI and their statistical significance as per one-wayANOVA followed by post hoc Tukey analysis

GeneFold changecompared toKHI group

Statisticalsignificance 119901

G6PDlowast CKDu 073 0703119873 = 75 CKD 062 0562119874 = 1 GH 042 0137GSTM1 CKDu 260 0036119873 = 53 CKD 158 0729119873119864 = 25 GH 237 0177119874 = 4

GCLC CKDu 316 0001119873 = 74 CKD 410 0002119873119864 = 1 GH 727 0000119874 = 7

NLRP3lowast CKDu 655 0006119873 = 54 CKD 1130 0010119873119864 = 3 GH 238 0553FGF23lowast CKDu 2194 0003119873 = 49 CKD 13497 0001119873119864 = 6 GH 252 0904119873 number included for one-way ANOVA119874 outliers119873119864 number of samples not expressing genelowasttotal sample less than 82 due to insufficient sample amount

one-way ANOVA showed significant difference in betweenthe groups for the GCLC (F370 = 9659 119901 = 0000) GSTM1(F349 = 2917 119901 = 0043) NLRP3 (F351 = 5111 119901 = 0004)and FGF23 (F345 = 7460 119901 = 0000) genes The G6PD genedid not show significant difference within the groups (F348= 1839 119901 = 0153) Post hoc Tukey analysis showed that theupregulation of theGCLCgene in theCKDu (316-fold) CKD(410-fold) andGHI (727-fold) groupswhen compared to theKHI group was significant at 119901 lt 001 Notable was the highupregulation of this gene (727 fold) in the GHI compared toKHI (119901 lt 001) (Table 3)

The GSTM1 gene did not show Ct values in 11 outof 43 CKDu patients (256) six out of 14 CKD patients(429) and eight of sixteen (50) KHI The population ofthe Girandurukotte area who expressed the gene howevershowed increased expression of the gene with CKDu patients

showing 260-fold upregulation compared to KHI (119901 =0036) and GHI showing 240-fold upregulation compared toKHI (119901 = 0177 Table 2)

Post hoc analysis of the NLRP3 gene showed 655-fold(119901 = 0006) and 1130-fold (119901 = 0010) upregulation in theCKDu andCKDgroups respectively whereas FGF23 showeda similar pattern with 2191-fold (119901 = 0003) and 13497-fold(119901 = 0001) upregulation in the CKDu and CKD groups

Box and whisker plots depicting median values and out-liers (150 IQR and 200 IQR) for each gene in each studygroup are presented (Figure 2) Average log

2normalized fold

changes calculated following outlier removal are depictedgraphically in Figure 3

4 Discussion

Gene expression studies based on renal biopsies have madean important contribution to understanding the role oftranscriptional regulation in renal disease [21] Studying theexpression patterns of genes in CKDu-endemic populationscould give a better understanding of the influential factorsof chronic kidney disease in this population For this studygenes known to be functioning in the oxidative stress balancewere selected for their expressions to be studied in bloodof populations of both a CKDu-endemic region as well as anonendemic region Genes not directly related to oxidativestress but indicative of the sources of oxidative stress werealso selected to be studied As CKDu is believed to bea result of occupational and environmental conditions itwas hypothesized that the study of oxidative stress relatedgenes would help better understand the influence of theenvironment in this disease

The GCLC and GSTM1 genes were selected for the studybased on their relation to the glutathione (GSH) proteinGSH functions in protecting the cells from toxic compoundsoxidative damage and radiation and forms complexes withseveral metal ions thus protecting cells from metal toxicityas well [22] It is used as a cosubstrate in the detoxificationof electrophilic xenobiotics and peroxides [23] GSH is alsoconsidered the first line of defense in metal toxicity beforeeven metallothionein [22] The expression of the GCLC genewas seen to be upregulated more than 316-fold in all threestudy groups from the Girandurukotte region the CKDuCKD and GHI when compared to the KHI (119901 lt 001)GCLC catalyzes the rate-limiting step in the production ofGSH and synthesis of GSH is not possible in the absenceof GCLC Its activity is highest in the liver followed by thelungs and kidneys and the steady state levels of the enzyme


27

CKDu GHI KHICKDDiagnosis

minus400

minus200

000

200

400

600G

6PD

(a)

10

8

5113

CKD GHI KHICKDuDiagnosis

minus1000

minus500

000

500

GST

M1

⋆

⋆

(b)

56

635139

23

177

minus1000

minus500

000

500

1000

GCL

C


⋆

(c)


minus500

minus250

000

250

500

750

NLR

P3

(d)

FGF2

3

1500

1000

500

000

minus500CKD GHI KHICKDu

Diagnosis

(e)

Figure 2 Box plots showing median values and range of log2normalized fold changes of (a) G6PD (b) GSTM1 (c) GCLC (d) NLRP3 and

(e) FGF23 genes in the four groups CKDu CKD GHI and KHI Outliers in each group are depicted by (∘) for values exceeding 150 IQRand (⋆) for values exceeding 200 IQR Numbers alongside symbols indicate sample ID

vary in different tissue types and stages of development[23] The GSH effect depends on the balance of depletionregeneration and synthesis of themolecule [24] andwhen theGSH is depleted the GSH synthesizing systems start makingmore GSH [25] The increased production of this enzyme in

blood of theGirandurukotte study groups indicates increasedproduction of GSH to counter oxidative stress The GHIgroup had the highest expression of this gene at 727-foldupregulation and this is possibly a reason for their beinghealthy and not showing symptoms of the disease


G6PD GSTM1 GCLCStudy gene

NLRP3 FGF23

CKDuCKDGHI

minus2

minus1

0

1

2

3

4

5

6

7

8

log 2

norm

aliz

ed fo

ld ch

ange

Figure 3 log2normalized fold change of the study genes in the four

study groups

The GSTM1 gene belongs to a group of enzymes that isresponsible for the conjugation of GSH to electrophilic xeno-biotics thereby playing a key role in the biotransformationand detoxification of xenobiotics and endogenous substances[14] It was seen in our study population that GSTM1 genedid not show Ct values in 11 out of 43 CKDu patients (256)six out of 14 CKD patients (429) and eight out of sixteen(50) of the KHI All nine individuals of the GHI group hadexpression of the geneTheCKDu patients who expressed thegene however showed increased expression with 260-foldupregulation (119901 lt 001) compared to KHI It can be inferredthat absence of the gene in a risk environmentmay be furtheraggravating disease progression

The lack of the GSTM1 gene expression in 256 of theCKDupatients and 429 of the CKDpatients and 50of theKHI population indicates the possibility of a null mutationin this ethnicity The GSTM1 null mutation is a commonpolymorphism and has also been associated with the end-stage renal disease of unknown etiology in Mexican patients[14] The GSTM1 null polymorphism individuals were alsofound to be more vulnerable to organochlorine pesticidesleading to the development of CKDu in a study carried outin an Indian population [15] The lack of expression of thegene observed in 256 of our CKDu patients and 429of the CKD patients indicates that they are vulnerable andsusceptible to oxidative stressors While 50 of the KHI alsodid not express the gene their good health indicates that theyare not being exposed to the same level of oxidative stressThegroups who had expression of the gene including the GHIgroup although not statistically significant had upregulationof the gene This again indicates regulation taking placeto counter oxidative stress The individuals of the KHIgroup who expressed the GSTM1 gene had lower expressioncompared to the study groups from Girandurukotte

The transcription of genes encoding the detoxifying andantioxidant enzymes such as the GSTs and GLCs are stimu-lated in response to cytoplasmic oxidative stress [26] Among

other oxidative stressors the expression of GCLC gene hasbeen associated with countering metal toxicity due to itsmetal binding sites and has especially been correlated withcadmium toxicity [24] which has been a major contender asthe causative agent of CKDu

The alteration of expressions of the GSH related genes inthe populations of the Girandurukotte region both healthyand diseased could be further interpreted based on thefunctions of GSH and the current hypotheses of CKDu Theupregulation of the GSH related genes in these groups indi-cates that there isare certain environmental factors causingdepletion of GSH thereby inducing expression of genes tocounteract it GSH acts as a metal chelating agent and oxygenradical scavenger [24] Redox-inactive metals including leadmercury cadmium and arsenic indirectly cause oxidativestress by depletion of the cellsrsquo major sulfhydryl reserves [27]TheGSH tripeptidemoleculemakes upmore than 90 of thetotal nonprotein sulfur and the redox-inactivemetals interactwith the GSH metabolism as an essential part of its toxicresponse [25]

Another gene that we studied that is related to oxidativestress and plays a role in protection against reactive oxygenspecies is the gene responsible for the production of theglucose-6-phosphate dehydrogenase (G6PD) enzyme G6PDdeficiency is a very common enzymopathy in the world aswell as in CKDu prevalent areas of Sri Lanka [13] The studyby Jayasekara et al found 20 of the CKDu cases to havesome severity of G6PD deficiency whereas only 2 of thehealthy controls had the deficiency [13] This enzyme is thefirst rate-limiting enzyme in the pentose phosphate pathwayand is responsible for the production of NADPH which isneeded in the glutathione reduction process [28] Howeverthe differential expressions seen in our study groups were notstatistically significant on the one-way ANOVA

Our finding based on the GSH related gene expressionindicates comparatively less oxidative stress in the KHI andthe possibility that the oxidative stress factors isare ofan environmental source Many studies have based theirhypotheses on the fact that the drinking water sourcesdiffer in the affected communities The Kandy populationconsumes water supplied via pipelines after water treat-ment whereas the Girandurukotte population had mainlygroundwater sources Studies have analyzed the amountof trace and ultra trace elements in drinking water andhave found moderate to high levels of fluoride in affectedregions [29]Wasana et al [30] found strong relation betweensynergistic effects of fluorine cadmium and hardness ofdrinking water and prevalence of CKDu Dharma-wardanaet al [31] hypothesize that CKDu in the NCP is linkedwith fertilizer runoff into the river systems from agriculturalactivity in the central hills which increases the ionicity ofthe drinking water which has potential to further damagethe protein structures in the kidney Exposure to pesticideshas also been implicated in the induction of oxidative stressand they can disturb the oxidative balance through eitherdirect or indirect pathways including thiol oxidation [32]Urine cadmium and arsenic concentrations in individualshave been in some studies found to be at levels known tocause kidney damage [33] The role of GSH in counteracting


arsenic toxicity has also been documented Exogenous GSHadministered to rats has also helped to reduce arsenic speciesin liver and to stop the decrease of blood and hepaticglutathione and total antioxidant capacity [34] Algal toxinsare another hypothesized cause for CKDu as cyanotoxinproducing algae have been found in drinking water of certainCKDu-affected regions [35] GSTM3 another member of theGST family was one of the phase II enzymes found to beupregulated in hepatoma HepG2 cells in the detoxificationresponse to a cyanotoxin cylindrospermopsin [36]

The sources of the rice which is the main staple of theSri Lankan people and the drinking water were the main dif-ferences between the two groups and therefore the oxidativestress could also be related to this factorThe Girandurukottepopulation had rice from their own production whereasrice purchased in Kandy comes from different parts of thecountry The climate between the two areas is also differentGirandurukotte is of the dry zone and has temperaturesaveraging 281∘Cand 22mmrainfall in thewarmest and driestperiods of May and June respectively [37] whereas Kandybeing of the wet zone and having temperatures averaging259∘C and 100mm rainfall in the warmest and driest periodsof April and February respectively [38]The people of Giran-durukotte especially those involved in farming are exposedto the high temperatures and have a threat of dehydration[39] Consumption of contaminated water in a dehydratedstate further concentrates the effect of the possible toxins

ROS are however not only from external sources but alsogenerated within cells by aerobic respiration as well as byanabolic and catabolic reactions taking place within the cells[40] The influence of metabolic dysregulation might also bea major contributor to oxidative stress in these patients TheNLRP3 gene was selected due to its activity being dependentonmitochondrial ROS productionThemitochondria are thelargest producer of the ROS within cells due to the electronleak from the electron transport chain [40] The increasedoxidative stress in chronic kidney disease is in part attributedto the dysfunctional mitochondria which cause an increasedelectron leakage from the respiratory chain during oxidativephosphorylation with a consequent generation of ROS [16]We observed an upregulation of the NLRP3 gene in both theCKDu and CKD patients with fold changes of 655 and 1130respectively (119901 lt 001) ROS generation by mitochondriaplays an important role in the NLRP3 inflammasome whereblocking the ROS generation by mitochondria was seen toeven stop the NLRP3 inflammasome activation [41] Furtherhyperuricemia which occurs with deteriorating renal func-tion [42] induces oxidative stress as a primary deleteriouseffect [43] Chronic hyperuricemia promotes mitochondrialdysfunction and decreased ATP content which results inrenal oxidative stress The mitochondrial dysfunction causedby hyperuricemia could further increase electron leakage andincrease ROS productionThe fold changes observed suggestmore involvement of these metabolic pathways and cellularinfluence of oxidative stress in the CKD patients

FGF23 a bone-derived hormone is believed to influencesystemic phosphate homeostasis vitamin D metabolismand alpha klotho expression [18 44 45] and is thereforeindirectly linked to ageing and oxidative stress generated

by dysregulation of these metabolic processes It is believedto have a protective response to prevent excess phosphateretention which leads to a reduction in vitamin D [18 46]which could further worsen oxidative stress conditions [18]Serum FGF23 levels are seen to rise exponentially as renalfunction declines [44] A significant level of upregulation ofthe FGF23 gene was seen in our CKD and CKDu populationsof 13497-fold and 2194-fold respectively (119901 lt 001) FGF23levels have even been seen to reach 10000-fold higher in end-stage renal disease when compared to healthy individuals[47] however the comparatively lower fold changes in ourstudy could be due to the fact that the gene expression levelswere analyzed in whole blood which is not the primarytissue of production of the FGF23 The increase seen ishowever sufficient to observe the regulation of this gene inrelation to CKD andCKDu Similar to the NLRP3 gene CKDpopulation showed higher expression of the FGF23 gene thanthe CKDu population which again points towards differingpathophysiological conditions and sources of oxidative stressin the two disease groups

The stage of disease and administered medication willfurther have an impact on the expression of the said genesThe patients in our study both CKD and CKDu are treatedconservatively to manage symptoms and prescribed supple-mental iron folic acid and calcium while patients identifiedwith diabetes and cholesterol are further given respectivemedications while monitoring blood sugar and cholesterollevels Calcium supplementation is an influencing factor inFGF23 regulation [48] and this could therefore be influencingresults observed of the gene but the effect should becomenullified when comparing the CKD and CKDu groups whoare both given the supplementation

The cellular source of oxidative stress is playing a biggerrole in CKDpatients whereas there is a possible environmen-tal source of oxidative stress which is not present in theKandyregion that is causing the upregulation of the GSH relatedgenes in the GHI group The GHI population althoughshowing increase of GCLC expression showed relativelyslight increase of NLRP3 and FGF23 genes which was notof statistical significance This shows the lack of cellularmediated oxidative stress in this group but the presenceof other factors that could influence GCLC upregulationSeveral extracellular stimuli cause ROSproduction includinggrowth factors inflammatory cytokines ionizing radiationUV chemical oxidants chemotherapeutics hyperoxia tox-ins and transition metals [40] The oxidative stressors couldbe any of the risk factors hypothesized in the literatureincluding but not limited to metal toxicity fluoride toxicityagrochemical toxicity and algal toxicity which have beencorrelated with disease prevalent areas and linked to watersources

5 Limitations of Study

The physiological and pathological conditions of an individ-ual is a summed effect of many different genes and influencesof external and internal factors and therefore cannot simplybe linked to the few genes analyzed in this study HoweverGSH plays a significant role in the antioxidant protective


system of the body and as GCLC and GSTM1 genes playsignificant roles in the production and function of GSH theirpatterns can be indicative of the physiological condition ofan individual The FGF23 gene expression was studied inblood tissue which is not its primary tissue of production butthe expression seen is sufficient to infer possible influencesof the gene in the study groups Other limitations includedsmall study population and also age difference in betweenthe healthy groups and the disease groups The two healthygroups were however of the same age range and thereforedoes not affect the conclusion of this study The groupscould be further divided stage-wise in a larger sample size toobserve changes of the genes with disease progression

6 Conclusion

The most significant finding of the study was the higherupregulation of the GCLC gene in the GHI group comparedto the other study groups The GSTM1 null mutation in theGirandurukotte population resulted in disease but this wasnot so in the Kandy population Patients showed effects ofmitochondrial source of oxidative stress as well as metabolicdysregulation as seen with NLRP3 and FGF23 upregulationmore in CKD than in CKDu pointing at different triggersof oxidative stress in the two disease subtypes GHI did notshow significant upregulation of the NLRP3 or FGF23 genestherefore indicating that theGCLCupregulation is externallystimulated From this pilot study we can therefore concludethat there is an environmental source of oxidative stress inthe Girandurukotte region which is not present in the Kandyregion It can be inferred that GSH plays a protective rolein keeping individuals of the Girandurukotte area healthyand the lack thereof promotes disease Studies with a widerarray of genes and stage-wise classification are currently beingcarried out to further understand molecular mechanisms ofthe disease and to identify causative etiology

Competing Interests

The authors declare that there are no competing interestsregarding the publication of this paper

Acknowledgments

Theauthors thank theNational ResearchCouncil of Sri Lanka(Grant no 11-059) for financial assistance and the staff ofthe District Hospital Girandurukotte for their support andtechnical assistance in sample collection

References

[1] G Garcia-Garcia and V Jha ldquoChronic kidney disease indisadvantaged populationsrdquo Indian Journal of Nephrology vol25 no 2 pp 65ndash69 2015

[2] R AHamer andAM ElNahas ldquoThe burden of chronic kidneydiseaserdquo British Medical Journal vol 332 no 7541 pp 563ndash5642006

[3] R S Barsoum ldquoChronic kidney disease in the developingworldrdquo The New England Journal of Medicine vol 354 no 10pp 997ndash999 2006

[4] A Bandarage ldquoPolitical economy of epidemic kidney disease inSri Lankardquo SAGE Open vol 3 no 4 2013

[5] H Ranasinghe and M Ranasinghe ldquoStatus gaps and wayforward in addressing theChronic KidneyDiseaseUnidentified(CKDu) in Sri Lankardquo Journal of Environmental Professionals SriLanka vol 4 no 2 pp 58ndash68 2015

[6] VMWeaver J J Fadrowski and B G Jaar ldquoGlobal dimensionsof chronic kidney disease of unknown etiology (CKDu) amodern era environmental andor occupational nephropathyrdquoBMC Nephrology vol 16 no 1 article 145 2015

[7] D E Weiner M D McClean J S Kaufman and D R BrooksldquoThe Central American epidemic of CKDrdquo Clinical Journal ofthe American Society of Nephrology vol 8 no 3 pp 504ndash5112013

[8] J Wijkstrom R Leiva C-G Elinder et al ldquoClinical andpathological characterization of mesoamerican nephropathy anew kidney disease in central americardquo American Journal ofKidney Diseases vol 62 no 5 pp 908ndash918 2013

[9] O El-Minshawy ldquoEnd stage renal disease in El-Minia Gover-norate Egypt data of the year 2007rdquo Nephro-Urology Monthlyvol 3 no 2 pp 118ndash121 2011

[10] M M Rajapurkar G T John A L Kirpalani et al ldquoWhat dowe know about chronic kidney disease in India first report ofthe Indian CKD registryrdquo BMCNephrology vol 13 no 1 article10 2012

[11] D V Reddy and A Gunasekar ldquoChronic kidney disease intwo coastal districts of Andhra Pradesh India role of drinkingwaterrdquo Environmental Geochemistry and Health vol 35 no 4pp 439ndash454 2013

[12] D Drozdz P Kwinta K Sztefko et al ldquoOxidative stressbiomarkers and left ventricular hypertrophy in children withchronic kidney diseaserdquo Oxidative Medicine and CellularLongevity vol 2016 Article ID 7520231 8 pages 2016

[13] J M K B Jayasekara D M Dissanayake M D N GunaratneR Sivakanesan and D M T S Dissanayake ldquoPrevalenceof G6PD deficiency in patients with chronic kidney diseaseof unknown origin in North Central region of Sri Lankacase control studyrdquo International Journal of Recent ScientificResearch vol 4 no 4 pp 455ndash458 2013

[14] B E Gutierrez-Amavizca R Orozco-Castellanos R Ortız-Orozco et al ldquoContribution of GSTM1 GSTT1 and MTHFRpolymorphisms to end-stage renal disease of unknown etiologyin Mexicansrdquo Indian Journal of Nephrology vol 23 no 6 pp438ndash443 2013

[15] M Siddarth S K Datta M D Mustafa et al ldquoIncreased levelof organochlorine pesticides in chronic kidney disease patientsof unknown etiology role of GSTM1GSTT1 polymorphismrdquoChemosphere vol 96 pp 174ndash179 2014

[16] S Granata A Dalla Gassa P Tomei A Lupo and G ZazaldquoMitochondria a new therapeutic target in chronic kidneydiseaserdquo Nutrition amp Metabolism vol 12 no 1 p 49 2015

[17] K Koop M Eikmans H J Baelde et al ldquoExpression ofpodocyte-associated molecules in acquired human kidney dis-easesrdquo Journal of the American Society of Nephrology vol 14 no8 pp 2063ndash2071 2003

[18] L D Quarles ldquoRole of FGF23 in vitamin D and phosphatemetabolism implications in chronic kidney diseaserdquo Experi-mental Cell Research vol 318 no 9 pp 1040ndash1048 2012

[19] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the2(minusDelta Delta C(T)) MethodrdquoMethods vol 25 no 4 pp 402ndash408 2001


[20] N T C Athuraliya T D J Abeysekera P H Amerasinghe etal ldquoUncertain etiologies of proteinuric-chronic kidney diseasein rural Sri LankardquoKidney International vol 80 no 11 pp 1212ndash1221 2011

[21] B J Keller S Martini J R Sedor and M Kretzler ldquoAsystems view of genetics in chronic kidney diseaserdquo KidneyInternational vol 81 no 1 pp 14ndash21 2012

[22] R K Singhal M E Anderson and A Meister ldquoGlutathione afirst line of defense against cadmium toxicityrdquo FASEB Journalvol 1 no 3 pp 220ndash223 1987

[23] E L Dahl and R T Mulcahy ldquoCell-type specific differencesin glutamate cysteine ligase transcriptional regulation demon-strate independent subunit controlrdquo Toxicological Sciences vol61 no 2 pp 265ndash272 2001

[24] N Franchi D Ferro L Ballarin and G Santovito ldquoTran-scription of genes involved in glutathione biosynthesis in thesolitary tunicate Ciona intestinalis exposed to metalsrdquo AquaticToxicology vol 114-115 pp 14ndash22 2012

[25] N Ercal H Gurer-Orhan and N Aykin-Burns ldquoToxic metalsand oxidative stress part I mechanisms involved in metal-induced oxidative damagerdquo Current Topics in Medicinal Chem-istry vol 1 no 6 pp 529ndash539 2001

[26] H Saito ldquoToxico-pharmacological perspective of the Nrf2-Keap1 defense system against oxidative stress in kidney dis-easesrdquo Biochemical Pharmacology vol 85 no 7 pp 865ndash8722013

[27] S J Stohs and D Bagchi ldquoOxidative mechanisms in the toxicityof metal ionsrdquo Free Radical Biology and Medicine vol 18 no 2pp 321ndash336 1995

[28] H Budak H Ceylan E F Kocpinar N Gonul andO ErdoganldquoExpression of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in oxidative stress inducedby long-term iron toxicity in rat liverrdquo Journal of Biochemicaland Molecular Toxicology vol 28 no 5 pp 217ndash223 2014

[29] R Chandrajith S Nanayakkara K Itai et al ldquoChronic kidneydiseases of uncertain etiology (CKDue) in Sri Lanka geo-graphic distribution and environmental implicationsrdquo Environ-mentalGeochemistry andHealth vol 33 no 3 pp 267ndash278 2011

[30] H M S Wasana D Aluthpatabendi W M T D KularatneP Wijekoon R Weerasooriya and J Bandara ldquoDrinkingwater quality and chronic kidney disease of unknown etiology(CKDu) synergic effects of fluoride cadmium and hardness ofwaterrdquo Environmental Geochemistry and Health vol 38 no 1pp 157ndash168 2016

[31] M W C Dharma-wardana S L Amarasiri N Dharmawar-dene and C R Panabokke ldquoChronic kidney disease ofunknown aetiology and ground-water ionicity study based onSri Lankardquo Environmental Geochemistry and Health vol 37 no2 pp 221ndash231 2015

[32] D Braconi G Bernardini M Fiorani et al ldquoOxidative damageinduced by herbicides is mediated by thiol oxidation andhydroperoxides productionrdquo Free Radical Research vol 44 no8 pp 891ndash906 2010

[33] N Jayatilake S Mendis P Maheepala and F R MehtaldquoChronic kidney disease of uncertain aetiology prevalence andcausative factors in a developing countryrdquoBMCNephrology vol14 no 1 article 180 2013

[34] D Wang L Lin X Li and G-F Sun ldquoEffects of glutathione onthe in vivo metabolism and oxidative stress of arsenic in micerdquoThe Journal of Toxicological Sciences vol 40 no 5 pp 577ndash5832015

[35] D N Magana Arachchi and H M Liyanage ldquoDetermining thepresence of cyanotoxins in water reservoirs of anuradhapurausing molecular and bioassay methodsrdquo Journal of the NationalScience Foundation of Sri Lanka vol 40 no 2 pp 157ndash167 2012

[36] A Straser M Filipic and B Zegura ldquoCylindrospermopsininduced transcriptional responses in human hepatoma HepG2cellsrdquo Toxicology in Vitro vol 27 no 6 pp 1809ndash1819 2013

[37] Climate Girandurukotte 2016 httpenclimate-dataorgloca-tion765861

[38] ldquoClimate Kandyrdquo 2016 httpenclimate-dataorglocation5671[39] E A R I E Siriwardhana P A J Perera R Sivakanesan T

Abeysekara D Nugegoda and J A A S Jayaweera ldquoDehydra-tion andmalaria augment the risk of developing chronic kidneydisease in Sri Lankardquo Indian Journal of Nephrology vol 25 no3 pp 146ndash151 2015

[40] HCui Y Kong andH Zhang ldquoOxidative stressmitochondrialdysfunction and agingrdquo Journal of Signal Transduction vol2012 Article ID 646354 13 pages 2012

[41] R Zhou A S Yazdi P Menu and J Tschopp ldquoA role formitochondria inNLRP3 inflammasome activationrdquoNature vol469 no 7329 pp 221ndash225 2011

[42] I Ohno ldquoRelationship between hyperuricemia and chronickidney diseaserdquo Nucleosides Nucleotides and Nucleic Acids vol30 no 12 pp 1039ndash1044 2011

[43] M Cristobal-Garcıa F E Garcıa-Arroyo E Tapia et al ldquoRenaloxidative stress induced by long-term hyperuricemia altersmitochondrial function and maintains systemic hypertensionrdquoOxidative Medicine and Cellular Longevity vol 2015 Article ID535686 8 pages 2015

[44] K Nitta N Nagano and K Tsuchiya ldquoFibroblast growth factor23klotho axis in chronic kidney diseaserdquo Nephron ClinicalPractice vol 128 no 1-2 pp 1ndash10 2014

[45] M Wolf ldquoForging forward with 10 burning questions onFGF23 in kidney diseaserdquo Journal of the American Society ofNephrology vol 21 no 9 pp 1427ndash1435 2010

[46] O Gutierrez T Isakova E Rhee et al ldquoFibroblast growthfactor-23 mitigates hyperphosphatemia but accentuates cal-citriol deficiency in chronic kidney diseaserdquo Journal of theAmerican Society of Nephrology vol 16 no 7 pp 2205ndash22152005

[47] T E Larsson ldquoFGF23 beyond mineral metabolism a bridge tocardiovascular diseaserdquo Clinical Journal of the American Societyof Nephrology vol 6 no 12 pp 2735ndash2737 2011

[48] M E Rodriguez-Ortiz I Lopez J R Munoz-Castaneda etal ldquoCalcium deficiency reduces circulating levels of FGF23rdquoJournal of the American Society of Nephrology vol 23 no 7 pp1190ndash1197 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


This peculiar form of CKD is not specific to Sri Lankaand similar cases have been recorded across three continentsaround the world [6] mostly in agricultural communities ofLatin America [7 8] Egypt [9] and India [10 11]

Reactive oxygen species (ROS) and nitrogen species(RNS) are essential for maintaining the healthy physiologicalstate of an individual however when it exceeds the necessaryamounts it leads to oxidative stress which is the pathophys-iological mechanism for many chronic diseases includingCKD [12]The expressions of the oxidative stress related genesalter depending on both the internal and external oxidativestress We therefore hypothesize that studying the differentialexpression of genes related to oxidative stress response inCKD CKDu and healthy individuals of a CKDu-affectedendemic region and comparing them to healthy individualsof an unaffected region would reveal the contribution ofoxidative stress from the environment As majority of thehypotheses for causes of CKDu are environmental relatedstudying the expressions of oxidative stress related genes inthese populations of different geographical areas would resultin better understanding the influence of the environment inthis disease

The oxidative stress related genes selected for the studyinclude the glutamine cysteine ligase C subunit (GCLC)glutathione S transferase mu 1 (GSTM1) and the glucose 6phosphate dehydrogenase (G6PD) genes Two of these genesGCLC and GSTM1 are related to the glutathione (GSH)protein The G6PD gene was selected as it plays a role inprotection against reactive oxygen species by the productionof NADPH which is required for the generation of reducedGSH as well as due to its deficiency being linked to CKDu inanother study [13] Polymorphisms of these genes althoughnot in combination have been seen in cases of CKDu andend-stage renal disease (ESRD) in Mexican [14] Indian [15]and Sri Lankan populations [13]

ROS is however generated in CKD by several internalprocesses including hyperactivation of NADPH oxidaseincreased production of oxidative stress markers and therelease of uremic toxins [16] We therefore analyzed theexpression pattern of genes reacting to internal sources ofoxidative stress production to better understand the environ-mental versus physiological sources of oxidative stress in thestudy groups

The increased oxidative stress in chronic kidney diseaseis in part attributed to the dysfunctional mitochondria whichcause an increased electron leakage from the respiratorychain during oxidative phosphorylation with a consequentgeneration of ROS [16] NLR family pyrin domain containing3 (NLRP3) is known to be activated by mitochondrial ROS[16] and therefore it was selected to study the influence ofmitochondrial oxidative stress Fibroblast growth factor-23(FGF23) a bone-derived hormone although not directlylinked to oxidative stress is believed to influence systemicphosphate homeostasis vitamin D metabolism and alphaklotho expression [18] all factors which play roles in theoxidative stress balance These genes were thus selected toprovide an exhaustive spectrum of basic conditions

Dry zone of Sri LankaNorth Central ProvinceUva Province

GirandurukotteKandy

N

Figure 1 Map of Sri Lanka highlighting dry zones of the countryand study locations

2 Materials and Methods

21 Study Population and Selection Criteria Ethical clearancewas obtained from the Ethics Review Committee of thePostgraduate Institute of Science University of PeradeniyaInformed consent for the study was obtained from eachsubject prior to blood collection The samples were collectedin different batches from June 2013 to January 2015

The study population was recruited in three study groupsCKDu CKD and healthy groups from Girandurukotte(GHI) which is a high prevalent area for CKDu located inthe dry zone of Sri Lanka The CKD and CKDu patientswere recruited from the District Hospital GirandurukotteA control group of healthy individuals was selected fromthe Kandy District (KHI) which is not an endemic area ofCKDu (Figure 1)The two healthy groups GHI and KHI wereapparently healthy at the time of recruitment and did notshow symptoms of any disease did not have any past medicalhistory and were not previously diagnosed with any chronicdiseases or infection All study subjects belonged to the samerace and ethnicity and only difference was on their place ofresidence

The diagnosis of patients was carried out by the consult-ing nephrologist at the hospital and CKDu was diagnosedbased on criteria set by the Ministry of Health Sri Lanka nopast history of diabetes mellitus chronic or severe hyperten-sion snake bite glomerulonephritis or urological diseasesnormal HBA1C (lt65) blood pressure lt 160100mmHguntreated or lt14090mmHg on up to two antihypertensivemedications

22 Inclusion and Exclusion Criteria Inclusion criteria forthe study were patients diagnosed by the physician as CKDu






















2normalized fold


3 Results






CKDu (119899 = 43) CKD (119899 = 14) GHI (119899 = 9) KHI (119899 = 16)









GCLC CKDu 316 0001119873 = 74 CKD 410 0002119873119864 = 1 GH 727 0000119874 = 7







2normalized fold


4 Discussion




27


minus400

minus200

000

200

400

600G

6PD

(a)

10

8

5113


minus1000

minus500

000

500

GST

M1

⋆

⋆

(b)

56

635139

23

177

minus1000

minus500

000

500

1000

GCL

C


⋆

(c)


minus500

minus250

000

250

500

750

NLR

P3

(d)

FGF2

3

1500

1000

500

000


Diagnosis

(e)







NLRP3 FGF23

CKDuCKDGHI

minus2

minus1

0

1

2

3

4

5

6

7

8

log 2

norm

aliz

ed fo

ld ch

ange


study groups




















6 Conclusion


Competing Interests


Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





































2normalized fold


3 Results






CKDu (119899 = 43) CKD (119899 = 14) GHI (119899 = 9) KHI (119899 = 16)









GCLC CKDu 316 0001119873 = 74 CKD 410 0002119873119864 = 1 GH 727 0000119874 = 7







2normalized fold


4 Discussion




27


minus400

minus200

000

200

400

600G

6PD

(a)

10

8

5113


minus1000

minus500

000

500

GST

M1

⋆

⋆

(b)

56

635139

23

177

minus1000

minus500

000

500

1000

GCL

C


⋆

(c)


minus500

minus250

000

250

500

750

NLR

P3

(d)

FGF2

3

1500

1000

500

000


Diagnosis

(e)







NLRP3 FGF23

CKDuCKDGHI

minus2

minus1

0

1

2

3

4

5

6

7

8

log 2

norm

aliz

ed fo

ld ch

ange


study groups




















6 Conclusion


Competing Interests


Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















CKDu (119899 = 43) CKD (119899 = 14) GHI (119899 = 9) KHI (119899 = 16)









GCLC CKDu 316 0001119873 = 74 CKD 410 0002119873119864 = 1 GH 727 0000119874 = 7







2normalized fold


4 Discussion




27


minus400

minus200

000

200

400

600G

6PD

(a)

10

8

5113


minus1000

minus500

000

500

GST

M1

⋆

⋆

(b)

56

635139

23

177

minus1000

minus500

000

500

1000

GCL

C


⋆

(c)


minus500

minus250

000

250

500

750

NLR

P3

(d)

FGF2

3

1500

1000

500

000


Diagnosis

(e)







NLRP3 FGF23

CKDuCKDGHI

minus2

minus1

0

1

2

3

4

5

6

7

8

log 2

norm

aliz

ed fo

ld ch

ange


study groups




















6 Conclusion


Competing Interests


Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















27


minus400

minus200

000

200

400

600G

6PD

(a)

10

8

5113


minus1000

minus500

000

500

GST

M1

⋆

⋆

(b)

56

635139

23

177

minus1000

minus500

000

500

1000

GCL

C


⋆

(c)


minus500

minus250

000

250

500

750

NLR

P3

(d)

FGF2

3

1500

1000

500

000


Diagnosis

(e)







NLRP3 FGF23

CKDuCKDGHI

minus2

minus1

0

1

2

3

4

5

6

7

8

log 2

norm

aliz

ed fo

ld ch

ange


study groups




















6 Conclusion


Competing Interests


Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















NLRP3 FGF23

CKDuCKDGHI

minus2

minus1

0

1

2

3

4

5

6

7

8

log 2

norm

aliz

ed fo

ld ch

ange


study groups




















6 Conclusion


Competing Interests


Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























6 Conclusion


Competing Interests


Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Upregulation of Oxidative Stress Related...

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