Effect of angiotensin converting enzyme gene I/DpolymorphisMin South Indian children with nephrotic syndrome

Aravind Selvin Kumar Ramanathan,Balakrishnan Karuppiah,Murali Vijayan,Kamaraj Raju,Dhivakar Mani,Rathika Chinniah,Manikandan Thirunavukkarasu,Padma Malini Ravi,JeyaraMIlliayaraja Krishnan,Prabha Senguttuvan

1 Department of Pediatric Nephrology,Instituteof Child Health and Hospital for Children,Chennai,Tamil Nadu 600008,India;

2 Department of Medical Genetics,The Tamil Nadu Dr.M.G.R.Medical University,Chennai,Tamil Nadu 600032,India;

3 Department ofimmunology,School of Biological Sciences,Madurai Kamaraj University,Madurai,Tamil Nadu 625021,India;

4 Department of Biotechnology and Genetic Engineering,Bharathidasan University,Tiruchirappalli,Tamil Nadu 620024,India;

5 Department of Clinical Research,Narayana Health City,Bangalore,Karnataka 560099,India;

6 Department of Pediatric Nephrology,Dr.Mehta's Children's Hospital,Chennai,Tamil Nadu 600031,India.

AbstractNephrotic syndrome isoneof themostcommon childhood kidney diseases.Itismostly found in theagegroup of 2 to 8 years.Around 10%-15%of nephrotic syndrome cases are non-responders of steroid treatment(SRNS).Angiotensin converting enzyme(ACE)(I/D)geneassociation studiesare important for detecting kidney disease and herein weassessed theassociation of ACE(I/D)polymorphisMwithnephrotic syndromein South Indian children.We recruited 260 nephrotic syndrome(162 boysand 98 girls)and 218(140 boysand 78 girls)control subjects.ACE I/D polymorphisMwasanalyzed by PCRusing genotypeallele speci fi c primers.In ACE(I/D),wedid not fi nd signi fi cant association for the ungrouped data of nephrotic syndrome children and the control subjects.Kidney biopsies were done in 86 nephrotic syndrome cases(Minimal change disease,n=51;focal segmental glomerulosclerosis,n=27;diffuse mesangial proliferation,n=8).We segregated theMinto the minimal change disease/focal segmental glomerulosclerosisgroupsand observed thatthe ACE‘D’allelewasidenti fi ed with borderlinesigni fi cance in casesof focal segmental glomerulosclerosis and the‘I’allele was assessed as having very weak association in cases of minimal change disease.‘II’genotypewasweakly associated with Minimal changedisease.Gender speci fi c analysis revealed weak association of‘ID’genotypewith femalenephrotic syndrome in females.DoMinant expression of DD genotype was observed in males with nephrotic syndrome.Our fi nding indicated that ACE(I/D)has moderate association with focal segmental glomerulosclerosis.However,due to the limited number of biopsy proven focal segmental glomerulosclerosis subjects enrolled,further studies are required to con fi rMthese results.

Keywords:angiotensin converting enzyme,focal segmental glomerulosclerosis,Minimal change disease,nephrotic syndrome

Introduction

Nephrotic syndrome(NS)is a condition where damages are prominent in fi ltering units of kidney.NS ismorepredominantin children and commonly existsin the ages of 2 and 8 years.It seems to affect boys more often than girls[1-2].Theannual incidenceof NSin most countries isestimated to range froM2 to 7 new casesper 100,000 children.The annual incidence of NS in US,European,Africo-Caribbean and Asian children were2,2.6,3.4 and 16.9 per 100,000 children respectively and the cumulative prevalence was about 16 per 100,000[1-4].A geographically based epidemiological study of NS suggested that Asian children have higher incidence;mainly,it was higher in lower socioeconomic groups[5-6].Ethnic origin may play a major role of the histological modi fi cation and response to immunosuppressive treatment.Based on immunosuppressive treatment,NS is mainly divided into steroid sensitive,steroid dependent and steroid resistant.The histological evaluation of renal tissues in nephrotic caseshas threemain categories:minimal changedisease(MCD),focal segmental glomerulosclerosis(FSGS)and diffuse mesangial proliferation(DMP).MCD is idiopathic,with no change in the number of podocytes and it mostly responds to steroid treatment(remission).FSGS is a severe forMwhich decreases the number of podocytes and does not respond to steroid treatment.Finally,DMPisalso asevere forMof nephrotic which is rarely seen.

Genetic factors have been suggested to be important in determining the progression of NS.Recently,well documented evidences indicate that the renin-angiotensin system(RAS)is involved/implicated in pathogenesis of kidney disease[7-8].Angiotensin converting enzyme(ACE)is an important enzyme of RAS.The stability of plasma ACE levels,combined with marked inter individual differences and faMilial clustering of plasma ACE levels suggested its regulation to be under major gene control.Genes that are involved in the RAS systeMare functional candidates/close link with these phenotypes,it would be the prognostic factors of renal damage such as diabetic nephropathy,Immunoglobulin A(IgA)nephropathy[7],systeMic lupus erythematosus(SLE)[9]and/or lupus nephritis and fi nally lead to development of end stage renal disease.Some of the existing reports froMACE polymorphisMidenti fi ed the presence of‘D’allele is indicated for the risk of cardiovascular[10]and kidney damage[11].ACE DD genotype is responsible for hypertension[8],diabetes mellitus[12],IgA nephropathy[7],renal artery stenosis[7],cardiomyopathies[13]and carotid atherosclerosis[14].However,some previous studies evaluating/identify and proven relationship between steroid sensitive/FSGS and ACE gene polymorphisMof NS in different ethnic populations[15-16].ACE-IIgenotype was more frequent in steroid sensitivenephrotic syndrome(SSNS)patients as compared to controls in North India[17],Malaysia[18].DD genotype with NS has been reported froMTaiwan Province(China)[19],Egypt[20].However,the DD genotype was associated only with FSGS-SRNS in the Kuwaiti Arab children[21].In contrast to the previous studies,no such association of ACE genepolymorphisMwas found in Sw isschildren[22].Hence,weanalysed the distribution of ACE(I/D)gene polymorphisMof NS[SSNS/steroid resistant nephrotic syndrome(SRNS)]in South Indian children between theageof 2 and 12 years old.

Subjects and methods

Study subjects

In the present study,we recruited 260(mean age of 7.17±3.58)NS cases froMa single center;218 control subjects(mean age of 10.5±1.07)were recruited.Our institutional ethical committee approved the study and w ritten informed consent was obtained froMsubject’s parents.A ll enrolled children were born in South India,were of ethnically South Indian ancestry and belonged to the lower and Middle classes.Nephrotic cases were sporadic and itwasnoted that 85 enrolled(nephrotic 58;control 27)subjects were born froMsecond degree consanguineous parents.The enrolled subjects were between the ages of 2 and 12 years old.The inclusion criterion was the clinical presentation of NS.The diagnosis of NS was based on the presence of edema,urinary protein excretion≥40 mg/(m2·hour),spot albuMin to creatinine ratio＞2 mg,and hypoalbuMinemia＜2.5 g/dL.A ll the nephrotic cases received the standard steroid therapy and were classi fi ed into two categories on the basis of their clinical responses towards steroids:SSNS group and SRNS group.SSNSisde fi ned as(remission)strati fi ed into proteinuria negative to trace for three consecutive days or urine protein excretion＜4 mg/(m2·hour).SRNS isde fi ned as failure to achieve reMission after 4 week of daily oral prednisoloneat a dose of 2 mg/(kg·day).Children with NSand ahistory of positive HIV,HbsAg wereexcluded froMthe study.The control group consists of unrelated healthy individuals with no history of kidney disease/hypertension.

DNA isolation and ACE(I/D)genotyping

GenoMic DNA was isolated froM2 ML of blood using thestandard salting-outmethod.The16thintronof thepolymorphic ACE(I/D)genewasampli fi ed by PCR.The primers(forward/reverse)5’-TGGAGACCACTCCCATCCTTTCT-3’and 5’-GATGTGGCCATCACATTCGTCACGAT-3’were used to amplify the region ofintron 16 which produced a 287-bp insertion/deletion polymorphism.PCR reaction wasperformed in a fi nal volume of 12μL containing 7.64μL of milli Q water,3μL of genomic DNA(200 ng/ML),0.2μL of 5U Taq polymerase(Genet Bio,Korea),1.2μL of 10×PCR buffer,0.24μL of 10 mmol/L dNTP(Cinna Gen,Iran),0.36μL each of forward and reverse primer(10 mmol/L).PCR ampli fi cation was carried with an initial denaturation at 94°C for 5 minutes,followed by 30 cycles of denaturation at 94°C for 2 minutes,annealing at 58°C for 1 minute and extension at 72°C for 1 minute and a fi nal extension at 72°C for 5(Agilent,USA;model no:G8800A).Ampli fi ed productswere detected on a2%agarosegel containing 0.5 μg/ML of EthidiuMBromide.The amplicon containing the insertion allele(I)was visible as a band at approximately 490 bp;while deletion(D)at 190 bp;and 190-bp and 490-bp PCR products for ID heterozygotes.Due to preferential ampli fi cation of the D allele,it was possible that ID heterozygotes might be mistyped as DD homozygotes.Therefore,in order to increase the speci fi city of DD genotyping,all samples identi fi ed as DD after initial ampli fi cation were recon fi rmed with the second PCR containing an insertion speci fi c primers.

Statistical analysis

The statistical analysis was performed by STATA 11.1(College Station,TX,USA).The continuous variables of age,seruMcreatinine,protein and albumin and cholesterol level were expressed as mean and standard deviation.ACE(I/D)genotype and allele frequencies were compared between the cases and controls.Odds ratio with 95% con fi dence interval expressed as genotype and allele frequencies.Genotype and allele frequencies distribution were expressed as frequency and percentage.Hardy-Weinberg equilibrium(HWE)for ACE genotype was tested by Chisquare test in each group.Distribution of ACE(I/D)polymorphisMdoMinance and recessive model were expressed.Statistically signi fi cant accepted value was P＜0.05.

Results

The clinical and demographic data of the nephrotic cases are presented in Table 1.There were no statistically signi fi cant differences in the distribution of ACE(I/D)genepolymorphisMbetween the NScases and controls(Table2).Thedatawere strati fi ed based on steroid treatment response in NS cases.The differences in the frequencies for genotypesor alleles for SSNSand SRNS were found to be statistically insigni fi cant.We segregated NS biopsy cases into the MCD and FSGS groups.We found IIgenotype frequency was observed in 29.41%(15/51)of the MCD cases and in 11.11%(3/27)FSGS cases.This association wasestimated as[OR(95%CI)=3.33(0.80-19.61);P=0.06](Table 3).The frequency of DD genotype was 25.9%(7/27)in FSGS,but 11.7%(6/51)in MCD.The‘I’allele and‘D’allele were found to be weakly associated(both had borderline signi fi cance)with MCD and FSGS,respectively.Additionally,in 8 DMP cases,II and ID genotype frequencieswere50%(4/8)and 50%(4/8),respectively,no cases had DD genotype.However,when we segregated the groups into male and female populations of nephrotic cases and controls,we could not found a signi fi cant association for II,ID and DD genotype in males.In the female groups,ID genotype was slightly more prevalent(59.1%)in the nephrotic group than the control(51.2%)which showed a weak association[OR(95%CI)=1.74(0.93-3.24);P=0.06](Table 4).Logistic regression analysis of the genetic model was carried out in dominant[(II+ID)vs.DD],recessive[II vs.(DD+ID)],additive(II vs.DD)and co-dominant[ID vs.(DD+II)]for male,female and pooled NSand control subjects.The result of the doMinant model,which revealed a signi fi cant association,was estimated[OR(95%CI)=0.14(0.07-0.25);P=0.001]in male nephrotic cases(Table 5).Further,we calculated the degreeof doMinance(h)test to fi nd out the deviation of heterozygousstate froMthe risk of disease.Theanalysis revealed that the degree of doMinance was＜1 froMthe ACE(I/D)variant and NS in South Indian children.

Table 1 Clinical and demographic details of patients

Table 2 Distribution of ACE(I/D)genotype and allele frequencies in SSNS/SRNS patients and control subjects

Table 3 Distribution of ACE(I/D)genotype frequencies in MCD and FSGS subjects

Table 4 Distribution of ACE(I/D)genotype and allele frequencies in nephrotic syndrome patients and control strati fi ed by gender wise

Discussion

ACE is a glycoprotein and its stability in plasma,combined with marked inter-individual differences and the familial clustering of plasma ACE levels,suggests its regulation is under the control of a major gene.ACE I/D polymorphisMwasoriginally thoughtto account for varying degreesof phenotypic expression in circulation.The generation of Ang II depends on ACE.A ll nephrotic patients ultimately received the suggested treatment,with the synergic combination of ACE inhibitor and Ang IIreceptor blockers,as they decrease theproteinuria.Thus,itreduces theglomerular capillary pressure in addition to altering the glomerular permeability.Recently,Type 2 diabetes patients with nephropathy(proteinuria＞150 mg/24 hours)treated with standard ACE I therapy found progressive reduction of the proteinuria during 3-6 months study period[21].Effects of Ang II increased systemic and glomerular blood pressure,leading to tubule-interstitial fi brosisand glomerulosclerosis;fi nally progressed to loss of kidney function.DD homozygousor D allele isassociated with elevated circulating and tissue ACE activity compared to Iallele.Several studieshave found that D allele isan independent risk factor for hypertension,diabetic,IgA nephropathy,congenital renal malformations and NS[16,20,24].

In the present study,we observed that ACE(I/D)allelic distribution wasnot elevated in the control group when compared to the different subsets of nephrotic(SSNS/SRNS)cases,which is in agreement with previous studies from different ethnic populations such as Swedish and Egyptian[22,25-27].On the contrary,the ACE-IIgenotype was found more frequent in SSNS in North Indian and Pakistani children[17,28].However,DD genotype was higher in north Indian population[15]which also coincided with the Egyptein[25],Turkish[29]and Taiwanese[19]nephrotic children.Therefore,the ACE(I/D)polymorphisMand its linkage with NS in different ethnic populations are contentious.

Table 5 Risk factor analysis of ACE genotypic model

Podocyte loss is the main reason behind the development of FSGS.According to the histopathological condition,NS is distinguishable notably as MCD and FSGS.Thepathogenesisof MCD hasnotbeen clear so far;but there are evidences ofimmune dysfunction[30].Most MCD cases were in reMission,but a very few of theMwasadvanced to CKD.In our center,some of the SRNS cases had a slow progression,while most have fast progressions to end-stage renal disease.It seems that genetic markers/polymorphisMplays a susceptible/protective role in thediseasemechanism[31].Moreover,existing studies have identi fi ed the genetic markers and their linkage to the progression of kidney disease.Firstly,a study on PcKO mice,the Atg5 gene(functional block of autophagy)was associated with slow progression of podocyte degeneration and then to glomerulosclerosis[32].Secondly,the ACE II-A9570G,ACE-ID/DD and/or AGT-M235T polymorphisMwere associated with an increased risk factor for arterial hypertension in FSGSwith fast progression to chronic kidney disease(CKD)[16,31-34].In the present study,FSGS cases have‘D’allele association,which showed substantial persistence of the‘I’allele.However,the incidenceofiIgenotype cases(3/27)was lessdoMinant in FSGS.A ll three‘II’genotype FSGS cases had late onset of nephrotic below 10 years of age had slow progression to end-stage renal disease.In MCD cases,the‘I’allele showed strong association and the II genotype a weak association with very few of theMhaving DD genotype(11.7%).In thismanner MCD-DD genotype may play a role in the slow progression of MCD to FSGS.However,asecond biopsy is required to con fi rMthe conversion of MCD to FSGS.In one study froMKuwaiti,in Arab-Jew ish nephrotic children the DD genotype was associated with FSGS cases[21].In contrast,onemeta-analysisstudy showed DD genotype/‘D’allele is associated with MCD and II genotype played a protective role[35].In our study,the frequency ofiD genotypewasslightly increased in SSNS 32.71%(35/107)compared to SRNS 26.79%(41/153)butitwas not statistically signi fi cant.Moreover,a largenumber of biopsy proven MCD/FSGScasesmay be recommended along with routine follow-up for better interpretation of the role of the ACE ID/DD genotype.

In childhood,a preponderance of nephrotic in males is well-established.In an experimental rat model,an inactivating mutation in ACE resulted in lower ACE protein,compared with w ild type rats.In female rats,a low level of ACE protein did not affect the blood pressure,but male rats were protected froMhypertension[36].In gender based study,ACE DD genotype was associated with hypertension in males[37].Further,a large study(the Suita Study,Japan)which enrolled 14,200 individuals suggested a unique sex-speci fi c effect of ACE on hypertension.In addition,a metaanalysisstudy involving Asian maleswith hypertension revealed signi fi cant association between ACE(I/D)polymorphisMand CKD risk[37].On theother hand SLE in females(analysis of 644 families)was proven with ACE association[9].In various studies,established ACE(I/D)polymorphisMand gender based disease association have been documented in different ethnic populations.Lin etal.stated that ACE(I/D)polymorphisMand gender dependent effect have been observed very commonly among different populations.So,ACE locus is a sex-speci fi c candidate gene/association for differentdiseases.In thepresentstudy,thesubjectswere segregated,gender-w ise.The gender-w ise comparison results revealed that femalenephrotic children had weak association of‘ID’[OR(95%CI)=1.74(0.93-3.24);P=0.06].ACE ID genotype present in females with NS was 59.1%(58/98)and the control was 51.2%(40/78).In females,we observed ID genotype(subset of nephrotic)MCD,FSGS,DMP and pooled were 66.6%(12/18),63.6%(7/11),60%(3/5)and 64.7%(22/34)respectively.We conclude the ID genotype frequency was signi fi cantly increased in the NS and subset,compared to control females.

Finally,we found themoderatesigni fi cantassociation between the ACE‘D’alleleand FSGS cases.The study results are positive and suggest that future studies should recruit a larger number of biopsy proven FSGS cases froMdifferent geographical regions for a better understanding of NS.

Acknow ledgments

This work was supported by Networking Resource Centre in Biological Sciences(NRCBS;funded by UGC,Govt.ofindia).The author(RASK)thank the Co-ordinator,UGC-NRCBS,Madurai Kamaraj University,for providing the MKU-NRCBS visiting fellowship.