Nox5, a novel target in diabetes research? !Chelsea Nadeau-Towaij, Kidney Research Centre !

Acknowledgements!Literature cited! Contact information!

1  Fulton(D.(Nox5(and(the(regula4on(of(cellular(func4on.(An#ox&&&Redox&Signal&11:2443;2452,(2009.(((

2  Montezano(A,(Burger(D,(Ceravolo(G,(et&al.(Novel(Nox(homologues(in(vasculature:(focusing(of(Nox4(and(Nox5.(Clin&Sci&120:131;141,(2011.((

3  Sedeek(M,(Hébert(R,(Kennedy(C,(et&al.(Molecular((((mechanisms(of(hypertension:(role(of(Nox(family(NADPH(oxidases.(Cur&Opin&in&Neph&and&Hyper&18:122;127,(2009.((

Thank(you(to(the(Touyz(lab(for(their(lucigenin(assay(protocol.(Thank(you( to( Dr.( Kennedy,( the( principle( inves4gator,( along( with( Dr.(Holterman,(my(supervisor,(and(the(other(members(of(the(Kennedy(lab.((Thank(you(to(Jessmyn(Niergarth(for(the(illustra4ons.((

For(more(informa4on:((The(Kidney(Research(Centre’s(web;site:(hYp://www.ohri.ca/centrres/KRC/default.asp((Email:(cnade046@uoYawa.ca(

Introduction! Results and discussion!

Conclusion!

The(Nox(family(of(NADPH(oxidases(is(a(group(of(enzymes(that(generate(reac4ve(oxygen(species((ROS).(Nox5(is(of(interest(in(diabe4c(kidney(disease(research(for(numerous(reasons:(

!  It(generates(superoxide,(which(can(cause(oxida4ve(stress(ensuing(in(renal(damage1,3.(

!  It(is(the(only(isoform(that(operates(independently(of(the(cytosolic(subunit(p22phox.3(

! Compared(to(the(other(Nox(isoforms,(it(has(a(more(localized(4ssue(distribu4on,(and(is(expressed(in(podocytes,(which(are(in4mately(involved(in(the(progression(of(renal(disease2.((

!  It(appears(to(be(upregulated(in(pa4ents(with(DKD(as(compared(to(non;diabe4c(controls(using(immunofluorescence(detec4on.(

We(hypothesize(that(Nox5(is(upregulated(in(response(to(diabe4c(s4muli.(The(goal(of(this(study(is(to(confirm(this(in&vitro(by(s4mula4ng(human(podocytes(with(Ang(II(or(TGFβ(and(quan4fying(changes(in(gene(expression(and(superoxide(produc4on(using(real;4me(PCR(and(lucigenin(assays(respec4vely.(Expression(of(the(other(Nox(isoforms(present(in(human(glomerular(podocytes(will(also(be(measured.(

Methods!

Future steps!

S4mula4on(of(hPODS(in&vitro(with(AngII(and(TGFβ(caused(increased(gene(expression(of(Nox5(and(increased(produc4on(of(ROS(which(agrees(with(our(an4cipated(results.(Increased(gene(expression(of(Nox4(was(also(observed.(Although(more(research(is(required,(preliminary(data(suggests(that(Nox5(might(be(an(appropriate(target(in(aYempts(to(slow(the(progression(of(diabe4c(kidney(disease((((

!  S4mula4on(of(hPODs(under(other(diabe4c(condi4ons,(including(high(glucose(and(stretch(

!  siRNA(knockdowns(of(NOX5(and(p22phox(in(hPODs(to(confirm(the(func4onal(contribu4ons(of(the(various(NOX(isoforms(to(ROS(produc4on(

!  (In&vivo&characteriza4on(of(NOX5(in(a(line(of(transgenic(mice(with(the(human(gene(inserted(under(control(of(the(nephrin(promoter(

Podocytes(in(culture(were(

s4mulated(with(AngII(or(TGFβ(

Cells(were(lysed(and(scraped(aher(predetermined(periods(of(4me(

For(lucigenin(assay,(samples(were(loaded(in(a(96(well(plate(and(luminescence(was(

detected(

For(gene(expression,(RNA(was(isolated(using(Qiagen’s(RNeasy(

Micro(kit((

First(strand(synthesis(was(

performed(using(RT;PCR(

Rela4ve(gene(expression(was(measured(using(

qPCR(

AngII(s4mula4on(of(hPODS(for(6h(provoked(a(3(fold(induc4on(of(Nox5,(and(a(30%(increase(in(ROS(produc4on(compared(to(an(uns4mulated(control.(For(gene(expression,(results(were(normalized(against(GAPDH,(a(housekeeping(gene.(In(the(ROS(assay,(results(were(normalized(to(the(protein(concentra4on.(AngII(did(not(induce(significant(induc4on(of(Nox1(or(Nox4.(((In(considering(the(lucigenin(assay,(it(is(important(to(note(that(it(provides(a(measure(of(total(superoxide(produc4on(in(the(podocyte(and(is(therefore(not(specific(to(Nox5(generated(ROS.(Nox1(and(Nox2,(also(present(in(podocytes,(produce(superoxide(that(may(be(detected(in(the(assay.(Note(that(Nox4(produces(primarily(hydrogen(peroxide(which(is(not(detected(by(this(assay.((((((((((A(24h(TGFβ(s4mula4on(provoked(an(4(fold(induc4on(of(Nox5(and(an(8(fold(increase(in(Nox4(expression.(For(gene(expression,(results(were(normalized(against(GAPDH,(a(housekeeping(gene.((

0(

1(

2(

3(

4(

Control( Vehicle( 500nM(AngII(

Rela%v

e'Fold'In

duc%on

'

Treatment'

Fig'1.'AngII's%mula%on'of'hPODs'–'gene'expression'(n=3)'

Nox1(Nox4(Nox5(

0(

2(

4(

6(

8(

10(

Untreated( Vehicle( 5ng/mL(TGFβ(

Rela%v

e'Fold'Cha

nge'

Treatment'

Fig'3.'TGFβ's%mula%on'of'hPODs'–'gene'expression'

Nox2(Nox4(Nox5(

0(

50(

100(

150(

200(

Control( Vehicle( 500nM(2h( 500nM(4h( 500nM(8h(

%'of'con

trol'

Treatment'

Fig'2.'AngII's%mula%on'of'hPODS'–'ROS'produc%on'(n=3)'

Nox5, a novel target in diabetes research? !Chelsea Nadeau-Towaij1, Chet E. Holterman1, Christopher R.J. Kennedy1 !1Dept of CMM, University of Ottawa, Kidney Research Centre!

Acknowledgements!Literature cited! Contact information!

1  Fulton(D.(Nox5(and(the(regula4on(of(cellular(func4on.(An#ox&&&Redox&Signal&11:2443;2452,(2009.(((

2  Montezano(A,(Burger(D,(Ceravolo(G,(et&al.(Novel(Nox(homologues(in(vasculature:(focusing(of(Nox4(and(Nox5.(Clin&Sci&120:131;141,(2011.((

3  Sedeek(M,(Hébert(R,(Kennedy(C,(et&al.(Molecular((((mechanisms(of(hypertension:(role(of(Nox(family(NADPH(oxidases.(Cur&Opin&in&Neph&and&Hyper&18:122;127,(2009.((

Thank(you(to(the(Touyz(lab(for(their(lucigenin(assay(protocol.(Thank(you( to( Dr.( Kennedy,( the( principle( inves4gator,( along( with( Dr.(Holterman,(my(supervisor,(and(the(other(members(of(the(Kennedy(lab.((((

For(more(informa4on:((The(Kidney(Research(Centre’s(web;site:(hXp://www.ohri.ca/centres/KRC/default.asp((Email:(cnade047@uoXawa.ca((

Introduction! Results and Discussion!

Conclusion!

The(Nox(family(of(NADPH(oxidases(is(a(group(of(enzymes(that(generate(reac4ve(oxygen(species((ROS).(Nox5(is(of(interest(in(diabe4c(kidney(disease(research(for(numerous(reasons:(!  It(generates(superoxide,(which(can(cause(oxida4ve(stress(

ensuing(in(renal(damage1,3.(!  It(is(the(only(isoform(that(operates(independently(of(the(

cytosolic(subunit(p22phox.3(!  Compared(to(the(other(Nox(isoforms,(it(has(a(more(localized(

4ssue(distribu4on,(and(is(expressed(in(podocytes,(which(are(in4mately(involved(in(the(progression(of(renal(disease2.((

!  It(appears(to(be(upregulated(in(pa4ents(with(DKD(as(compared(to(non;diabe4c(controls(using(immunofluorescence(detec4on.(

We(hypothesize(that(Nox5(is(upregulated(in(response(to(diabe4c(s4muli.(The(goal(of(this(study(is(to(confirm(this(in&vitro(by(s4mula4ng(human(podocytes(with(Ang(II(or(TGFβ(and(quan4fying(changes(in(gene(expression(and(superoxide(produc4on(using(real;4me(PCR(and(lucigenin(assays(respec4vely.(Expression(of(the(other(Nox(isoforms(present(in(human(glomerular(podocytes(will(also(be(measured.(

Methods!

Future Steps!

(n=3,(mean(±(StDev)(((

S4mula4on(of(hPODS(in&vitro(with(AngII(and(TGFβ(caused(increased(gene(expression(of(Nox5(and(increased(produc4on(of(ROS(which(agrees(with(our(an4cipated(results.(Increased(gene(expression(of(Nox4(was(also(observed.(Although(more(research(is(required,(preliminary(data(suggests(that(Nox5(might(be(an(appropriate(target(in(aXempts(to(slow(the(progression(of(diabe4c(kidney(disease((((

!  S4mula4on(of(hPODs(under(other(diabe4c(condi4ons,(including(high(glucose(and(stretch(

!  siRNA(knockdowns(of(NOX5(and(p22phox(in(hPODs(to(confirm(the(func4onal(contribu4ons(of(the(various(NOX(isoforms(to(ROS(produc4on(

!  (In&vivo&characteriza4on(of(NOX5(in(a(line(of(transgenic(mice(with(the(human(gene(inserted(under(control(of(the(nephrin(promoter(

0(

1(

2(

3(

4(

Control( Vehicle( 500nM(AngII(

Rela%v

e'Fold'In

duc%on

'

Treatment'

Fig'1.'AngII's%mula%on'of'hPODs'–'gene'expression'

Nox1(Nox4(Nox5(

0(

2(

4(

6(

8(

10(

Untreated( Vehicle( 5ng/mL(TGFβ(

Rela%v

e'Fold'Cha

nge'

Treatment'

Fig'3.'TGFβ's%mula%on'of'hPODs'–'gene'expression'

Nox2(Nox4(Nox5(

hPODs(in(culture(were(s4mulated(with(AngII(or(TGFβ(

Qiagen's(RNeasy(miniprep(kit(was(used(to(isolate(RNA(

Reverse(transcriptase(PCR(was(performed(to(make(cDNA(

Real;4me(PCR(was(used(to(measure(the(expression(levels(of(

desired(transcripts(

Expression(levels(were(normalized(to(GAPDH(

Lucigenin(assay(was(performed(to(measure(superoxide(produc4on(

Produc4on(levels(were(normalized(to(protein(

concentra4on(

Cells(were(lysed(for(RNA(or(protein(quan4fica4on(

0(

50(

100(

150(

200(

Control( Vehicle( 500nM(2h( 500nM(4h( 500nM(8h(

%'of'con

trol'

Treatment'

Fig'2.'AngII's%mula%on'of'hPODS'–'ROS'produc%on'

(n=3,(mean(±(StDev)(((

AngII(s4mula4on(of(hPODS(for(6h(provoked(a(3(fold(induc4on(of(Nox5,(and(a(30%(increase(in(ROS(produc4on(compared(to(an(uns4mulated(control.(For(gene(expression,(results(were(normalized(against(GAPDH,(a(housekeeping(gene.(In(the(ROS(assay,(results(were(normalized(to(the(protein(concentra4on.(((((A(24h(TGFβ(s4mula4on(provoked(an(4(fold(induc4on(of(Nox5(and(an(8(fold(increase(in(Nox4(expression.(For(gene(expression,(results(were(normalized(against(GAPDH,(a(housekeeping(gene.((((In(considering(the(lucigenin(assay,(it(is(important(to(note(that(it(provides(a(measure(of(total(superoxide(produc4on(in(the(podocyte(and(is(therefore(not(specific(to(Nox5(generated(ROS.(Noxes(1(and(2,(also(present(in(podocytes,(produce(superoxide(that(may(be(detected(in(the(assay.(Note(that(Nox4(produces(primarily(hydrogen(peroxide(which(is(not(detected(by(this(assay.(((