Expression of HIV transgene aggravates kidney injury in diabetic mice.

1.9 MB·English

by  Mallipattu, Sandeep K

basic research http://www.kidney-international.org
&2013InternationalSocietyofNephrology
OPEN
Expression of HIV transgene aggravates kidney injury
in diabetic mice
Sandeep K. Mallipattu1, Ruijie Liu1,2, Yifei Zhong3, Ed Y. Chen4, Vivette D’Agati5, Lewis Kaufman1,
Avi Ma’ayan4, Paul E. Klotman6, Peter Y. Chuang1 and John C. He1,2,4
1DivisionofNephrology,DepartmentofMedicine,MountSinaiSchoolofMedicine,NewYork,NewYork,USA;2RenalSection,JamesJ.
PetersVAMedicalCenter,NewYork,NewYork,USA;3DepartmentofNephrology,LonghuaHospital,ShanghaiUniversityofTraditional
Chinese Medicine, Shanghai, China; 4Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine,
NewYork,NewYork,USA;5DepartmentofPathology,ColumbiaUniversity,NewYork,NewYork,USAand6BaylorCollegeofMedicine,
Houston, Texas, USA
Withthe widespread use ofcombination antiretroviral It had been nearly three decades since the first published
agents,the incidence ofHIV-associatednephropathy has reports of HIV-associated nephropathy (HIVAN), an aggres-
decreased.Currently, HIV-infected patients livemuchlonger sive form of focal segmental glomerulosclerosis in patients
andoftensufferfromcomorbiditiessuchasdiabetesmellitus. with AIDS.1–3 Withinyears,HIVAN was recognized not only
Recent epidemiologicalstudies suggest that concurrent HIV as an AIDS-defining illness4 but also as a disease
infectionanddiabetesmellitusmayhaveasynergisticeffect predominant in patients of African descent.5 The incidence
onthe incidence of chronickidney disease. Toaddressthis, of HIVAN and the progression to end-stage renal disease
wedetermined whether HIV-1 transgeneexpression continued to rise until the advent and use of combination
accelerates diabetickidneyinjuryusing a diabetic HIV-1 antiretroviral therapy (cART) in the mid 1990s. In fact, with
transgenic (Tg26)murine model.Diabetes was initially thewidespreaduseofcART,theincidenceofend-stagerenal
inducedwithlow-dosestreptozotocininbothTg26andwild- disease attributed to HIVAN has reduced to approximately
typemiceona C57BL/6 background, which isresistant to 800–900 cases per year in the United States. However, the
classic HIV-associated nephropathy. Although diabetic prevalence of end-stage renal disease in this population has
nephropathyisminimally observedonthe C57BL/6 continued to rise, likely because of an increase in patient
background, diabetic Tg26miceexhibited a significant survival.6 In addition, as patients live longer with HIV, they
increase inglomerular injury comparedwith nondiabetic are at increased risk for comorbidities such as hypertension
Tg26miceand diabetic wild-type mice. Validationof and diabetes mellitus (DM).7
microarray gene expression analysisfromisolated glomeruli Several studies have shown that many patients who
showed a significant upregulationofproinflammatory undergo a clinically indicated kidney biopsy are diagnosed
pathways indiabeticTg26mice. Thus, our studyfoundthat with a non-HIVAN-related kidney disease in the post-ART
expressionofHIV-1genesaggravatesdiabetickidneydisease. era,rangingfromimmunecomplex–mediatedkidneydiseases
KidneyInternational(2013)83,626–634;doi:10.1038/ki.2012.445; to arterionephrosclerosis, and diabetic nephropathy.8–10
publishedonline16January2013 Studies such as these suggest that the spectrum of kidney
KEYWORDS:diabeticnephropathy;glomerulopathy;HIV disease has changed considerably in the last 15 years. With
thischange,theclinicalcourseofkidneydiseaseinthecART
era has been more indolent, a slow progressive decline in
kidney function with lower levels of proteinuria. Although
the Office of AIDS Research Advisory Council has clearly
stated in the 2012 guidelines that the presence of HIVAN is
anindicationforinitiationofcARTregardlessofCD4count,
the decision to start cART in non-HIVAN-related kidney
disease independent of other factors remains unclear
(Aidsinfo.nih.gov).
Studies have estimated that nearly 15% ofHIV-1-infected
patients have comorbid DM. In fact, several studies have
Correspondence:JohnC.He,DepartmentofMedicine/Nephrology,Mount
suggestedthatconcurrentHIVandDMmayhaveanadditive
Sinai School of Medicine, One Gustave L Levy Place, Box 1243, New York,
NewYork10029,USA.E-mail:[email protected] effect on the incidence of chronic kidneydisease (CKD).11,12
For example, Choi et al.11 showed that HIV and DM may
Received 16 July 2012; revised 14 September 2012; accepted 18
October2012;publishedonline16January2013 have a synergistic effect on the incidence of end-stage renal
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SKMallipattuetal.:HIVtransgeneaggravatesdiabetickidneyinjury basic research
disease in both Caucasians and African Americans. 250
*
Specifically, in a recent study using the Veterans Aging
Cohort Study database, we demonstrated that concurrent mg) 200
occurrenceofHIVandDMhasagreatereffectontheriskof μg/
CKD progression compared with the occurrence of either cr ( 150
condition alone.12 ein/
ot 100
SincethederivationoftheHIV-1transgenicmicebearing pr
adefectiveproviruslackinggag-pol(Tg26)intheearly1990s, e
studies have shown that classical features of HIVAN are Urin 50
exhibited primarily on the FVB background strain.13 These
0
Tg26miceatanearlyageexhibitnephroticrangeproteinuria,
STZ – + – +
podocytededifferentiation andproliferation, collapsingfocal
segmental glomerulosclerosis, and microcystic tubular WT Tg26
dilatation and injury, consistent with the features observed Figure1|DiabeticHIV-1transgenic(Tg26)miceexhibitan
in HIVAN.13 However, these changes are not observed in increaseinproteinuria.Wild-type(WT)andTg26miceweretreated
withstreptozotocin(STZ).UrinewascollectedatbaselinebeforeSTZ
Tg26 mice on the C57BL/6 background. Therefore, Tg26
orsodiumcitratebufferinjection,andtheurineprotein/creatinine
mice on the C57BL/6 background strain were used to study ratiowascheckedmonthly.Urineprotein/creatinineratioat6
non-HIVAN kidney disease. monthsofage(n¼6,*Po0.01versusmicetreatedwithsodium
Although epidemiological studies suggest that HIV citratebuffer).
infection is associated with the progression of CKD in
patients with DM, the cause–effect relationship and the (Figure 2). Histologically and by quantification, glomerular
mechanism behind this association have yet to be described. size, mesangial expansion, and glomerular basement mem-
Previous studies suggest that chronic inflammation con- brane (GBM) thickening were significantly increased in
tributestodiseasepathogenesisinbothdiabeticnephropathy diabetic Tg26 mice compared with all other groups (Figures
and HIVAN.14,15 Thus, we hypothesized that chronic 3a–c).Inaddition,diabeticandnondiabeticTg26micehada
infection with HIV-1 leads to a proinflammatory state, significant increase in foot process effacement as compared
thereby accelerating diabetic glomerular disease. Here, we withWTmicewithorwithoutdiabetes(Figure3d).Also,the
demonstrate that streptozotocin (STZ)-induced diabetic podocyte number per glomerulus was significantly reduced
mice exhibit more glomerular injury in the presence of in diabetic Tg26 mice as compared with all other groups
HIV-1 transgene expression. We also provide evidence (Figure3e).Combined,thesefindingssuggestthatexpression
suggesting that this process is associated with an of HIV-1 transgene in C57BL/6 diabetic mice induces
upregulation of proinflammatory pathways. glomerular injury, typically observed in early diabetic
nephropathy.
RESULTS
Diabetic Tg26mice exhibit anincrease in glomerularinjury Increase in the activation of pathwaysinvolved in
C57BL/6micearetypicallyresistanttoSTZ-induceddiabetic inflammationfromenrichmentanalysisofgenearraydatain
nephropathy16 and Tg26 mice on the C57BL/6 background diabetic Tg26mice
are resistant to HIVAN, typically observed on the FVB To identify unique or synergistic pathways from the
background.17,18 To study non-HIVAN-related kidney differentially expressed genes in diabetic Tg26 mice, we used
disease, Tg26 mice on the C57BL/6 background were used oligonucleotide microarrays to profile gene expression in
to test our hypothesis. To confirm that STZ treatment glomeruli isolated from WT mice, diabetic WT mice, Tg26
inducedDMinmice,physiologicalparametersofthemicein mice, and diabetic Tg26 mice. Similarly, microarrays from
eachofthefourgroupsweremeasured(SupplementaryTable patients with diabetic nephropathy (glomeruli) were re-
S1 online). Also, to verify that HIV-1 viral gene expression trieved from the Woroniecka Database in Nephromine
was unchanged after induction of diabetes with STZ, nef (http://www.nephromine.org). Also, to compare changes in
mRNA expression was measured using real-time PCR gene expression in known murine models of diabetic
(Supplementary Figure S1 online). A lack of change in nef nephropathy, we acquired microarrays deposited in Gene
expression in Tg26 mice with or without diabetes suggests Expression Omnibus (GSE710).19 For the differentially
that an increase in glomerular injury observed in diabetic expressed genes in each experimental group, we performed
Tg26 mice was not a result of an increase in local viral gene gene enrichment analysis using the Expression2Kinases
expression. nef mRNA expression was undetectable in wild- software (www.maayanlab.net/X2K/) program to identify
type (WT) mice (data not shown). At 6 months of age, pathwaysinvolvedininflammation and fibrosis.Acombina-
diabetic Tg26 mice exhibited a significant increase in tion of gene-list libraries such as Wikipathway, MGI
proteinuria as compared with all other groups (Figure 1). Mammalian Phenotype, KEGG Pathway, and GO molecular
Histologicalchangesbylightandelectronmicroscopyatlow- function revealed a significant increase in the activation of
andhigh-powermagnificationareshownat6months ofage pathways involving inflammation in diabetic Tg26 mice,
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basic research SKMallipattuetal.:HIVtransgeneaggravatesdiabetickidneyinjury
WT Tg26
STZ – + – +
×20
×40
×3k
×10k
Figure2|DiabeticHIV-1transgenic(Tg26)miceexhibitanincreaseinglomerularinjury.Paraffin-embeddedsectionswerestainedwith
periodicacid–Schiff(PAS)andimagesweretakenatlowpower((cid:2)20)andhighpower((cid:2)40).Ultrastructuralchangesareshownatlowpower
((cid:2)3000)andhighpower((cid:2)10,000)bytransmissionelectronmicroscopy.
a b c
m 2.0 500 0.6
μ
6merular volume 10 110...505 * GBM thickess (nm)432100000000 * * Mesangial area/glomerular area (%)00..42 *
o
Gl 0.0 0 0.0
STZ – + – + STZ – + – + STZ – + – +
WT Tg26 WT Tg26 WT Tg26
d e
2000 100
m) **
process width (n 11505000000 * ** docyte number/glomerulus 864000 *
ot Po 20
o
F
0 0
STZ – + – + STZ – + – +
WT Tg26 WT Tg26
Figure3|QuantificationofglomerularinjuryindiabeticHIV-1transgenic(Tg26)mice.Quantificationof(a)glomerularvolume,
(b)glomerularbasementmembrane(GBM)thickness,(c)andmesangialexpansionisshown(n¼3,*Po0.0001versusallothergroups).
(d)Quantificationofpodocyteeffacementisshown(n¼4,*Po0.0001versusallgroups,**Po0.0001versusdiabeticandnondiabeticwild-type
(WT)mice).(e)Quantificationofpodocytenumberisshown(n¼3,*Po0.0001versusallgroups).
consistent with those observed in human diabetic nephro- Upregulationofproinflammatorygenesisvalidatedbyreal-
pathy and a known murine model of diabetic nephropathy time PCR
(db/db mice) (Figure 4). These findings suggest that HIV-1 Tovalidatetheactivationofproinflammatorypathwaysfrom
anddiabetesmayhaveasynergiceffectontheinductionofa gene enrichment analysis, primers for selected genes
proinflammatory state leading to the pathogenesis observed implicated in inflammation were designed (Supplementary
in diabetic Tg26 mice. Table S2 online) and quantitative real-time PCR was
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SKMallipattuetal.:HIVtransgeneaggravatesdiabetickidneyinjury basic research
Abnormal cell death (MP0000313)
Abnormal cell cycle (MP0003077)
Abnormal immune system (MP0002722)
Abnormal innate immunity (MP0002419)
Abnormal antigen presenting (MP0002452)
Diabetic + Tg26 Abnormal survival (MP0010769)
Abnormal immune cell (MP0001819)
Abnormal adaptive immunity (MP0002420)
TNFR1 Pathway
Abnormal inflammatory response (MP0001845)
Abnormal cell death (MP0000313)
Abnormal cell cycle (MP0003077)
Abnormal immune system (MP0002722)
Abnormal innate immunity (MP0002419)
Tg26 only Abnormal antigen presenting (MP0002452)
Abnormal survival (MP0010769)
Abnormal immune cell (MP0001819)
Abnormal adaptive immunity (MP0002420)
TNFR1 Pathway
Abnormal inflammatory response (MP0001845)
Abnormal cell death (MP0000313)
Abnormal cell cycle (MP0003077)
Abnormal immune system (MP0002722)
Abnormal innate immunity (MP0002419)
Diabetic only Abnormal antigen presenting (MP0002452)
Abnormal survival (MP0010769)
Abnormal immune cell (MP0001819)
Abnormal adaptive immunity (MP0002420)
TNFR1 Pathway
Abnormal inflammatory response (MP0001845)
Abnormal cell death (MP0000313)
Abnormal cell cycle (MP0003077)
Abnormal immune system (MP0002722)
DN db/db Abnormal innate immunity (MP0002419)
Abnormal antigen presenting (MP0002452)
(GSE 710) Abnormal survival (MP0010769)
Abnormal immune cell (MP0001819)
Abnormal adaptive immunity (MP0002420)
TNFR1 Pathway
Abnormal inflammatory response (MP0001845)
Abnormal cell death (MP0000313)
Abnormal cell cycle (MP0003077)
Human DN Abnormal immune system (MP0002722)
Abnormal innate immunity (MP0002419)
(woroniecka Abnormal antigen presenting (MP0002452)
database) Abnormal survival (MP0010769)
Abnormal immune cell (MP0001819)
Abnormal adaptive immunity (MP0002420)
TNFR1 Pathway
Abnormal inflammatory response (MP0001845)
0 2 4 6 8 1015 20 25 30 35
–log (P-value)
Figure4|ActivationofpathwaysinvolvedininflammationfromgeneenrichmentanalysisindiabeticHIV-1transgenic(Tg26)mice,
db/dbmice(GSE710),andhumandiabeticnephropathy.Analysisoftermsthatareoverrepresentedamongdifferentiallyexpressedgenesin
eachofthegroupsascomparedwithuntreatedwild-type(WT)miceusingacombinationofthefollowinggene-listlibraries:Wikipathway,MGI
Mammalianphenotype,KEGGPathway,andGOmolecularfunction(Po0.05þBenjamini–Hochbergcorrection).
performed. These proinflammatory genes include VCAM1, Tg26 mice, elevation of circulating inflammatory cytokines
FAS, FASL, c-MYC, CCL2, and ICAM1. Real-time PCR may still contribute to the glomerular changes observed in
revealedasignificantincreaseintheseproinflammatorygenes this group.
inisolatedglomeruliofdiabeticTg26miceascomparedwith
allothergroups(Figure5).Combined,thesefindingsconfirm Upregulation of profibrotic genes in diabeticTg26mice by
that a local proinflammatory state is associated with the real-time PCR
glomerular disease observed in diabetic Tg26 mice. To assess for the activation of profibrotic pathways, primers
for genes involved in mesangial expansion were designed
Circulating levelsof inflammatory markers (Supplementary Table S2 online) and quantitative real-time
Toclarifytheroleofcirculatinginflammatorymarkersinthe PCR was performed. These genes include TGFb1, CTGF,
progressionofkidneydiseaseobservedindiabeticTg26mice, CollagenTypeI,anda-SMA.Real-timePCRfromglomerular
serumwascollectedfromallfour groups ofmiceat thetime extracts revealed a significant increase in the mRNA
that they were being killed. The sandwich enzyme immu- expression of these genesin diabetic Tg26 miceas compared
noassay technique was used to measure the circulating levels with all other groups (Figure 7).
ofTNFR1,TNFR2,andIL-6.AsignificantincreaseinTNFR1
and IL-6 serum levels was observed in diabetic-only, Tg26- Collagen TypeIV expression is increased indiabetic Tg26
only, and diabetic Tg26 mice as compared with WT mice mice
(Figures 6a, c). This finding was not observed in circulating CollagenTypeIVisconstitutivelyexpressed intheGBMand
levelsofTNFR2(Figure6b).Althoughtheactivationoflocal in the mesangial matrix.20 As we observed an increase in
inflammatory genes is predominantly observed in diabetic GBM thickness and mesangial matrix in diabetic Tg26 mice,
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basic research SKMallipattuetal.:HIVtransgeneaggravatesdiabetickidneyinjury
Fold change in VCAM1mRNA expression 543210 * Fold change in FASmRNA expression211002......050505 * Fold change in FASLmRNA expression432105 *
STZ – + – + STZ – + – + STZ – + – +
WT Tg26 WT Tg26 WT Tg26
Fold change in MYCmRNA expression11002.....50500 * Fold change in CCL2mRNA expression 3210 * Fold change in ICAM1mRNA expression0426 *
STZ – + – + STZ – + – + STZ – + – +
WT Tg26 WT Tg26 WT Tg26
Figure5|UpregulationofproinflammatorygenesindiabeticHIV-1transgenic(Tg26)miceisvalidatedbyreal-timePCR.Glomeruli
wereisolatedfromallgroupsofmiceandtotalRNAwasextracted.Real-timePCRwasperformedfor(a)VCAM1,(b)FAS,(c)FASL,
(d)c-MYC,(e)CCL2,and(f)ICAM1(n¼3,*Po0.01versusallothergroups).
R1 (ng/ml) 221...505 * * * R2 (ng/ml) 1150 6 (pg/ml)1105 * * *
m TNF 1.0 m TNF 5 um IL- 5
eru 0.5 eru Ser
S 0.0 S 0 0
STZ – + – + STZ – + – + STZ – + – +
WT Tg26 WT Tg26 WT Tg26
Figure6|Measurementofcirculatingseruminflammatorymarkers.Bloodwasdrawnandserumwasisolatedfromallgroupsofmice.
Thequantitativesandwichenzymeimmunoassaywasusedtomeasureserum(a)tumornecrosisfactorreceptor1(TNFR1),(b)tumor
necrosisfactorreceptor2(TNFR2),and(c)interleukin6(IL-6)levels(n¼6,*Po0.005versuswild-type(WT)mice).
a b c d
βFold change in TGF1mRNA expression 21100.....05050 * Fold change in CTGFmRNA expression 3210 * αFold change in -SMAmRNA expression 43210 * Fold change in collagenype 1 mRNA expression022110......050505 *
STZ – + – + STZ – + – + STZ – + – + t STZ – + – +
WT Tg26 WT Tg26 WT Tg26 WT Tg26
Figure7|UpregulationofprofibroticgenesindiabeticHIV-1transgenic(Tg26)micebyreal-timePCR.Glomeruliwereisolatedfromall
groupsofmiceandtotalRNAwasextracted.Real-timePCRwasperformedfor(a)transforminggrowthfactorbeta1(TGFb1),(b)connective
tissuegrowthfactor(CTGF),(c)alphasmoothmuscleactin(a-SMA),and(d)CollagenTypeI(n¼3,*Po0.01versusallothergroups).
Type IV collagen expression was measured to assess for Smad3 phosphorylation was significantly increased in
changesinexpressionanddistribution.Inisolatedglomeruli, diabetic Tg26 mice (Figure 9a). These findings were
Collagen Type IV mRNA expression was significantly quantified by densitometric analysis (Figure 9b). Combined,
increased in diabetic Tg26 mice (Figure 8a). These findings these findings suggest a significant activation of the
were validated by immunofluorescence qualitatively and profibrotic pathway in diabetic Tg26 mice.
quantitatively (Figures 8b and c).
DISCUSSION
Smad3phosphorylation isincreased in diabetic Tg26mice A large body of epidemiological studies indicates that the
Since there was an upregulation of local profibrotic genes spectrum of HIV-related kidney disease has changed
involved in matrix expansion and GBM thickening, we markedly in the last decade. Further, as patients with HIV
assessed for activation of the TGFb pathway by measuring live longer, they are more susceptible to non-HIVAN-related
Smad3 phosphorylation from the isolated renal cortex. kidney diseases such as diabetic nephropathy. Although a
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a b
n collagenexprtession 21..05 *
ge iNA 1.0
nR
Fold chatype IV m 00S..50TZ – + – +
WT Tg26
WT WT + STZ
c
Fold change in opticaldensity (arbitrary units) S3210TZ – +* – *+*
WT Tg26
Tg26 Tg26 + STZ
Figure8|CollagenTypeIVexpressionisincreasedindiabeticHIV-1transgenic(Tg26)mice.Glomeruliwereisolatedfromallgroupsof
miceandtotalRNAwasextracted.(a)Real-timePCRwasperformedforCollagenTypeIV(n¼3,*Po0.05versusallothergroups).Therenal
cortexwasisolatedfromallgroupsofmiceand(b)immunofluorescencestainingwasperformedforCollagenTypeIVwith(c)quantification
usingImageJ(n¼3,*Po0.05versuswild-type(WT),**Po0.05versusallothergroups).
WT Tg26 d3 6
STZ – + – + mas) *
Totapl--SSmmaadd33 5500 kkDDaa mad3/total Sarbitrary unit 24
S(
β-Actin 43 kDa p- 0
STZ – + – +
WT Tg26
Figure9|Smad3phosphorylationisincreasedindiabeticHIV-1transgenic(Tg26)mice.Thenuclearproteinwasinitiallyisolatedfromthe
renalcortexandp-Smad3(Smad3phosphorylation),total-Smad3,andbeta-actinproteinexpressionlevelsweremeasuredby(a)westernblot.
Therepresentativeblotsofthreeindependentexperimentsareshown.(b)Thedensitometricanalysesoftheseblotsareshown
(n¼3,*Po0.05versusallothergroups).
diagnosis of HIVAN is an indication for cART initiation resistant to diabetic nephropathy and HIVAN.16–18 Although
regardless of CD4 count, it remains unclear whether this these findings suggest that HIV infection contributes to the
remains applicable to patients with non-HIVAN-related accelerationofdiabeticnephropathy,weacknowledgethatthe
kidney disease. Further, mechanisms that contribute to this pathology is not overwhelming. In fact, these findings are
disease process have yet to be described. Here, we provide more representative of an early stage of glomerular disease
evidence that HIV-1 may contribute to or aggravate early and are likely attributable to the sclerosis-resistant mouse
stagesofglomerulardiseaseobservedindiabeticnephropathy background, C57BL/6.21 Diabetes-related glomerulosclerosis
probably through activation of proinflammation pathways. is typically observed in sclerosis-prone genetic
Although epidemiological studies suggest that HIV infec- backgrounds.16,22,23 Further studies using a similar approach
tion is associated with the progression of diabetic nephro- in sclerosis-prone genetic backgrounds will be required to
pathy, the cause–effect relationship remains unclear. We determine the progression to advanced glomerulosclerosis.
provide direct evidence showing that expression of HIV-1 Nevertheless, we believe that the changes observed in this
transgeneindiabeticmicecontributestoasignificantincrease study are unique and likely attributable to the presence of
in glomerular injury, mimicking early diabetic nephropathy. concomitant diabetes and HIV-1 transgene expression,
The observed changes in glomerular injury in the diabetic especially as these pathological changes are typically not
Tg26micewereontheC57BL/6background,astraintypically observed on this background.
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basic research SKMallipattuetal.:HIVtransgeneaggravatesdiabetickidneyinjury
Toidentifygenesorsynergisticpathwaysthathavearolein transgenic mouse line (Tg26 mice) that bears a defective HIV-1
the disease progression observed in diabetic Tg26 mice, proviruslackinggag-pol(Tg26)hasbeendescribed.13Tg26micein
microarrayexpressionstudieswereperformed.Usingthegene the FVB/N background were backcrossed six generations to a
enrichment analysis tool from the Expression2Kinases soft- C57BL/6background.WTmicegeneratedfromthesamelitterofTg26
ware,24 we confirmed that proinflammatory genes were micewereusedascontrolsinthestudies.Genotypingbytailprepand
PCRwasperformedat2weeksofageaspreviouslydescribed.30
significantly upregulated in diabetic Tg26 mice, suggesting
an underlying mechanism mediating the observed disease
Low-dose STZ-induced diabetes
process.Similarinflammatory pathwayswerealsoactivatedin
Induction of diabetes using STZ has been described previously.31
human diabetic nephropathy as well as in a known murine
Briefly,onday1,Tg26andWTmiceat6weeksofagewereinitially
model of diabetic nephropathy. This suggests that diabetic
deprivedoffood for 4hours. Thereafter, low-doseSTZ (50mg/kg)
nephropathymayinpartbeactivatedbycomplementingthese
in 50mmol/l sodium citrate buffer (pH 5.4) was administered
inflammatory pathways in C57BL/6 mice in the setting of
intraperitoneally. STZwas administered similarly at the same dose
concomitant diabetes and HIV-1 transgene expression. Also, ondays2–5.Onday14,allmiceweredeprivedoffoodfor6hand
theactivationofinflammatorypathwayswithHIVinfectionis fastingbloodglucoselevelfromthetailveinwascheckedusingthe
consistent with previous studies.25 Further, HIV-associated OneTouchBloodGlucoseMonitoringSystem.Repeatfastingblood
inflammationhasbeenimplicatedinprematureonsetofother glucosemeasurementsweretakenmonthlytoverifyhyperglycemia.
end-organ abnormalities.25,26 Finally, we observed an Diabetes mellitus was defined as sustained fasting blood glucose
upregulation of genes involved in GBM thickening and above 250mg/dl at two distinct time points 2 weeks post STZ
injection.31 Allmicewerekilled at6 monthsofage.
mesangial expansion, as well as the activation of the TGFb
pathway,indiabeticTg26mice.Therefore,wespeculatethata
Measurement of urinealbuminand creatinine
stateoflocalchronicinflammationinducedbyHIVaccelerates
Urine albumin was quantified by ELISA using a kit from Bethyl
diabetic kidney disease.
Laboratory(Houston,TX).Urinecreatininelevelsweremeasuredin
CirculatinglevelsofTNFR1havebeenassociatedwiththe
the same samples using the QuantiChrom Creatinine Assay Kit
progression of diabetic nephropathy.27,28 Minimal, yet signi-
(DICT-500) (BioAssay Systems, Hayward, CA) according to the
ficant,elevationinserumTNFR1andIL-6maycontributeto
manufacturer’s instructions. The urine albumin excretion rate was
theearlyglomerularchangesobservedindiabeticTg26mice. expressedastheratio ofalbumin tocreatinine.
However, it is likely not primarily responsible for the
histological changes observed in the diabetic Tg26 mice, as Bright-field lightmicroscopy and morphometric studies
a significant increase in serum TNFR1 and IL-6 levels was Micewereperfused with Hank’s buffered salt solution (HBSS)and
also observed in diabetic-only and Tg26-only mice. The role fixed with 4% paraformaldehyde overnight. Kidney tissue was
of HIV-induced systemic inflammatory response in kidney embedded in paraffin by American Histolabs (Gaithersburg, MD)
injury still remains to be defined until other circulating and 3mm-thick sections were stained with periodic acid–Schiff
inflammatory markers are measured. However, in HIVAN, (Sigma, StLouis,MO).
Estimation of glomerular volumeand mesangial areawas made
reciprocal renal transplantation between WTand Tg26 mice
as previously described.32,33 Briefly, digitized images were scanned
revealed that intrarenal expression of HIV-1 genes, not
and profile areas were traced using ImageJ. The mean glomerular
systemic inflammation, is responsible for progression to
tuft volume was determined from the mean glomerular cross-
HIVAN.29Therefore,thekidneyhasbeenconsideredareser-
sectional area by light microscopy. The glomerular cross-sectional
voir for HIV-1. In this study, a lack of change in nef expres-
areawascalculatedonthebasisoftheaverageareaof30glomeruli
sion in Tg26 mice with or without diabetes suggests that an ineachgroup,andglomerulartuftvolumewascalculatedusingthe
increaseinglomerularinjuryobservedindiabeticTg26mice following equation:
is not a result of an increase in local viral gene expression.
In summary, we report a murine model to study non- GV¼ b(cid:2)GA3/2
HIVAN-related kidney disease. Our studies suggest that the k
presenceoftheHIV-1transgeneacceleratestheprogressionof (b¼1.38,theshapecoefficientofspheres(theidealizedshapeof
diabetic nephropathy. Further, the activation of local proin- glomeruli), and k¼1.1, the size distribution coefficient). Also,
flammatorypathwaysinthesettingofconcurrentdiabetesand mesangial expansion was defined as a periodic acid–Schiff-positive
HIV-1 transgene expression maycontributetothe glomerular andnuclei-freeareainthemesangium.Quantificationofmesangial
injury observed in this murine model. This study provides a expansionwasbasedon20glomerulicutatthevascularpoleineach
group.
novel insight into the potential mechanisms involved in the
Quantification of podocyte number per glomerulus was
progression of non-HIVAN-related kidney disease.
determined using Wilm’s tumor-1-stained podocytes. First, kidney
sections from these mice were prepared in an identical manner.
MATERIALS AND METHODS Thereafter, 4mm-thick sections were stained with rabbit anti-WT-1
Genotyping of Tg26mice (Novus, Littleton, CO) as previously described.34 Counting of
The Mount Sinai School of Medicine Animal Institute Committee podocytes and quantification of glomerular area and volume were
approved all animal studies, and the NIH Guide for the Care and performedusingImageJandbythemethodstandardizedbyAnimal
Use of Laboratory Animals was followed strictly. Derivation of a Modelsof DiabeticComplications Consortium.32
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Transmissionelectronmicroscopyandmorphometricstudies (1.5mg)usingtheSuperScriptIIIFirst-StrandSynthesisKit(Invitrogen),
Mice were perfused with HBSS and immediately fixed in 2.5% and cDNA (1ml) was amplified in triplicate using SYBR GreenER
glutaraldehydeforelectronmicroscopy.Sectionsweremountedona qPCR Supermix on an ABI PRISM 7900HT (Applied Biosystems,
copper grid and photographed under a Hitachi H7650 microscope Foster City, CA). Primers were designed using PrimerBlast (http://
(Tokyo, Japan). www.ncbi.nlm.nih.gov/tools/primer-blast/) and purchased through
Quantification of podocyte effacement was performed as pre- Sigma(SupplementaryTable2online),withtheexceptionofpreviously
viously described.35 Briefly, negatives were digitized, and images publishedprimersfornefandGAPDH.7,38Lightcycleranalysissoftware
with a final magnitude of approximately X15,000 were obtained. (Carlsbad,CA)wasusedtodeterminecrossingpointsusingthesecond
ImageJ1.26tsoftware(NationalInstituteofHealth,rsb.info.nih.gov/ij) derivativemethod.Efficiencywascalculatedforallthedesignedprimers
wasusedtomeasurethelengthoftheperipheralGBM,andthenumber usingtherelativestandardcurvemethod.Datawerenormalizedtothe
of slit pores overlying this GBM length was counted. The arithmetic housekeepinggene(GAPDH)andpresentedasfoldincreasecompared
meanofthefootprocesswidth(WFP)wascalculatedasfollows: with RNAisolated fromWTanimals using thePfafflmethod.39
P
p GBMlength
WFP¼ 4(cid:2) Pslits MIneitaiaslulyr,e2m00enmtl ooffbinlofloadmwmaastcioolnle-rceteldatferdomsemruimcematatrhkeertisme that
theywerebeingkilled.Bloodsampleswereallowedtoclotfor2hat
(Sslitsindicatesthetotalnumberofslitscounted,SGBMlength
room temperature before centrifuging for 20min at 2000g. Serum
indicatesthetotalGBMlengthmeasuredinoneglomerulus,andp/4
wasextractedandquantitativesandwichenzymeimmunoassaywas
isthecorrectionfactorfortherandomorientationbywhichthefoot
performed as per manufacturers’ protocol to quantify serum
processesweresectioned).35
TNFR1, TNFR2, and IL-6 levels (no. MRT10, no. MRT20, no.
Sections stained with uranyl acetate and lead citrate were
M6000B; R&Dsystems, Minneapolis, MN).
mounted on a copper grid and photographed under a JEOL 1011
transmission electron microscope using Gatan imaging software
Immunofluorescence
(Gatan, Warrendale, PA). Quantification of GBM thickness was
performed as previously described.36 The thickness of multiple Kidney sections from these mice were prepared in an identical
manner.Immunostainingwasperformedusingrabbitanti-collagen
capillaries was measured in 6–8 glomeruli per mouse (n¼3 per
IV (Santa Cruz, Santa Cruz, CA). After washing, sections were
group). A mean of 114 measurements was taken per mouse (from
incubated with a fluorophore-linked secondary antibody (Alexa
podocytetoendothelialcellmembrane)atrandomsiteswhereGBM
Fluor 488 anti-rabbit IgG from Invitrogen). After staining, slides
wasdisplayed inthebest cross-section.
weremountedinAquaPoly/Mount(Polysciences,Warrington,PA)
and photographed under an AxioVision IIe microscope with a
Microarray geneexpression and geneontology analysis
digital camera (Munchen, Germany).
Gene expression profiling using Illumina (San Diego, CA) gene
expressionbeadchipswasperformedattheMountSinaiInstitution
Quantification of immunofluorescence
Microarray Core Facility. The Illumina MouseWG-6 v2.0 was used
Aftersectionswerestainedwithanti-CollagenTypeIVantibody,nega-
to profile gene expression in the glomeruli from all four experi-
tivesweredigitized,andimageswithafinalmagnitudeofBX40were
mental groups: WTmice, diabetic-only mice, Tg26-only mice, and
obtained. ImageJ 1.26t software (National Institute of Health,
diabetic Tg26 mice. In brief, the raw data were processed using a
rsb.info.nih.gov/ij)wasusedtomeasurethelevelofimmunostaining
Python program in which quantile normalization was performed.
in the glomeruli. First, the images were converted to an 8-bit
Theunpairedt-testwasusedtoassessstatisticalsignificancebetween
grayscale.Next,theglomerularregionwasselectedformeasurement
thegroups.Alistofdifferentiallyexpressedgeneswasgeneratedfor
of area and integrated density. Then, the background intensity was
eachgroupusingacutoffofPo0.05withtheBenjamini–Hochberg
measuredbyselectingthreedistinctareasinthebackgroundwithno
correction.EnrichmentanalysiswasperformedusingtheExpression2-
staining.Thecorrectedopticaldensitywasdeterminedasfollows:
Kinases software, and Fisher’s exact test was used to determine the
terms that were overrepresented among the differentially expressed
COD¼ID(cid:3)ðA(cid:2)MGVÞ
genesineachofthegroupscomparedwithuntreatedWTmice.24
(IDistheintegrateddensityoftheselectedglomerularregion;A
Isolation of glomeruli from mice for RNAextraction istheareaoftheselectedglomerularregion;andMGVisthemean
Mouseglomeruliwereisolatedasdescribed.37Inbrief,animalswere grayvalueofthebackgroundreadings).40
perfusedwithHBSScontaining2.5mg/mlironoxideand1%bovine
serum albumin. At the end of perfusion, kidneys were removed, Western blot
decapsulated,mincedinto1-mm3pieces,anddigestedinHBSScon- Glomeruliwerelysedwithabuffercontaining1%Triton,aprotease
taining 1mg/ml collagenase A and 100U/ml deoxyribonuclease I. inhibitor cocktail, and tyrosine and serine-threonine phosphoryla-
Digestedtissuewasthenpassedthrougha100-mmcellstrainerand tion inhibitors. Lysates were subjected to an immunoblot analysis
collectedbycentrifugation.Thepelletwasresuspendedin2mlofHBSS using rabbit anti-phospho Smad3 (Cell Signaling, Danvers, MA),
andglomeruliwerecollectedusingamagnet.Thepurityoftheglo- rabbit anti-total Smad3 (Cell Signaling), and mouse anti-b-actin
meruliwasverifiedbymicroscopy.TotalRNAwasisolatedfromkidney antibodies (Abcam, Cambridge, MA). Densitometric analysis for
glomeruliofmiceusingTRIzol(Invitrogen,GrandIsland,NY). quantificationwasperformed aspreviouslydescribed.41
Real-time PCR Statistical analysis
Total RNA was extracted using TRIzol (GIBCO Life Technology, Data were expressed as mean±s.e.m. (X±s.e.m.). The unpaired
GrandIsland,NY).First-strandcDNAwaspreparedfromtotalRNA t-testwasusedtoanalyzedatabetweentwogroups.Theanalysisof
KidneyInternational(2013)83,626–634 633
basic research SKMallipattuetal.:HIVtransgeneaggravatesdiabetickidneyinjury
variance followed by Bonferroni’s correction was used when more 18. GharaviAG,AhmadT,WongRDetal.Mappingalocusforsusceptibility
thantwogroupswerepresent.Allexperimentswererepeatedatleast toHIV-1-associatednephropathytomousechromosome3.ProcNatl
AcadSciUSA2004;101:2488–2493.
three times, and representative experiments are shown. Statistical
19. SusztakK,BottingerE,NovetskyAetal.Molecularprofilingofdiabetic
significancewasconsidered whenPo0.05. mousekidneyrevealsnovelgeneslinkedtoglomerulardisease.Diabetes
2004;53:784–794.
20. ButkowskiRJ,WieslanderJ,KleppelMetal.Basementmembrane
DISCLOSURE
collageninthekidney:regionallocalizationofnovelchainsrelatedto
Alltheauthorsdeclarednocompetinginterests.
collagenIV.KidneyInt1989;35:1195–1202.
21. HeC,EspositoC,PhillipsCetal.Dissociationofglomerularhypertrophy,
ACKNOWLEDGMENTS cellproliferation,andglomerulosclerosisinmousestrainsheterozygous
WethankDrAGharaviforuseofthebackcrossedTg26mice.This foramutation(Os)whichinducesa50%reductioninnephronnumber.
workwassupportedbyfundsfromtheNIH/NIDDK(1R01DK078897- JClinInvest1996;97:1242–1249.
22. YabukiA,TanakaS,MatsumotoMetal.Morphometricstudyofgender
01and973fund2012CB517601toJCH,P01DK056492toPEKand
differenceswithregardtoage-relatedchangesintheC57BL/6mouse
JCH,5K08DK082760toPYC,andT32DK07757-12and1F32
kidney.ExpAnim2006;55:399–404.
DK094635-01toSKM). 23. YangHC,ZuoY,FogoAB.Modelsofchronickidneydisease.DrugDiscov
TodayDisModels2010;7:13–19.
24. ChenEY,XuH,GordonovSetal.Expression2Kinases:mRNAprofiling
SUPPLEMENTARYMATERIAL
linkedtomultipleupstreamregulatorylayers.Bioinformatics2012;28:
TableS1.Physiologicalparametersofmicetreatedwithandwithout
105–111.
Streptozotocin(STZ).
25. DeeksSG.HIVinfection,inflammation,immunosenescence,andaging.
TableS2.Oligonucleotideprimersusedforquantitativereal-time AnnuRevMed2011;62:141–155.
PCRanalysis. 26. RobertsonKR,SmurzynskiM,ParsonsTDetal.Theprevalenceand
FigureS1.nefexpressionwasunchangedindiabeticand incidenceofneurocognitiveimpairmentintheHAARTera.AIDS2007;21:
nondiabeticTg26mice. 1915–1921.
27. ShikanoM,SobajimaH,YoshikawaHetal.Usefulnessofahighlysensitive
Supplementarymaterialislinkedtotheonlineversionofthepaperat
urinaryandserumIL-6assayinpatientswithdiabeticnephropathy.
http://www.nature.com/ki
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28. NiewczasMA,GohdaT,SkupienJetal.CirculatingTNFreceptors1and2
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634 KidneyInternational(2013)83,626–634