Table Of Content

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 626 KidneyInternational(2013)83,626–634 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, KidneyInternational(2013)83,626–634 627 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 628 KidneyInternational(2013)83,626–634 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, KidneyInternational(2013)83,626–634 629 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 630 KidneyInternational(2013)83,626–634 SKMallipattuetal.:HIVtransgeneaggravatesdiabetickidneyinjury basic research 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. KidneyInternational(2013)83,626–634 631 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.32 632 KidneyInternational(2013)83,626–634 SKMallipattuetal.:HIVtransgeneaggravatesdiabetickidneyinjury basic research 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 Nephron2000;85:81–85. 28. NiewczasMA,GohdaT,SkupienJetal.CirculatingTNFreceptors1and2 REFERENCES predictESRDintype2diabetes.JAmSocNephrol2012;23:507–515. 1. RaoTK,FilipponeEJ,NicastriADetal.Associatedfocalandsegmental 29. BruggemanLA,DikmanS,MengCetal.Nephropathyinhuman glomerulosclerosisintheacquiredimmunodeficiencysyndrome.NEnglJ immunodeficiencyvirus-1transgenicmiceisduetorenaltransgene Med1984;310:669–673. expression.JClinInvest1997;100:84–92. 2. GardenswartzMH,LernerCW,SeligsonGRetal.Renaldiseaseinpatients 30. FengX,LuTC,ChuangPYetal.ReductionofStat3activityattenuates withAIDS:aclinicopathologicstudy.ClinNephrol1984;21:197–204. HIV-inducedkidneyinjury.JAmSocNephrol2009;20:2138–2146. 3. PardoV,AldanaM,ColtonRMetal.Glomerularlesionsintheacquired 31. WuKK,HuanY.Streptozotocin-induceddiabeticmodelsinmiceandrats. immunodeficiencysyndrome.AnnInternMed1984;101:429–434. CurrProtocPharmacol2008;Chapter5:Unit547;5.47.1–5.47.14. 4. CarboneL,D’AgatiV,ChengJTetal.Courseandprognosisofhuman 32. SandenSK,WigginsJE,GoyalMetal.Evaluationofathickandthin immunodeficiencyvirus-associatednephropathy.AmJMed1989;87: sectionmethodforestimationofpodocytenumber,glomerularvolume, 389–395. andglomerularvolumeperpodocyteinratkidneywithWilms’tumor-1 5. CantorKP,WeissSH,GoedertJJetal.HTLV-I/IIseroprevalenceandHIV/ proteinusedasapodocytenuclearmarker.JAmSocNephrol2003;14: HTLVcoinfectionamongU.S.intravenousdrugusers.JAcquirImmune 2484–2493. DeficSyndr1991;4:460–467. 33. AwazuM,OmoriS,IshikuraKetal.Thelackofcyclinkinaseinhibitor 6. GuptaSK,EustaceJA,WinstonJAetal.Guidelinesforthemanagementof p27(Kip1)amelioratesprogressionofdiabeticnephropathy.JAmSoc chronickidneydiseaseinHIV-infectedpatients:recommendationsofthe Nephrol2003;14:699–708. HIVMedicineAssociationoftheInfectiousDiseasesSocietyofAmerica. 34. SchifferM,MundelP,ShawASetal.Anovelrolefortheadaptormolecule ClinicalInfectiousDiseases2005;40:1559–1585. CD2-associatedproteinintransforminggrowthfactor-beta-induced 7. MallipattuSK,WyattCM,HeJC.ThenewepidemiologyofHIV-related apoptosis.JBiolChem2004;279:37004–37012. kidneydisease.JAIDSClinRes2012;S4:001–006. 35. KoopK,EikmansM,BaeldeHJetal.Expressionofpodocyte-associated 8. BerlinerAR,FineDM,LucasGMetal.ObservationsonacohortofHIV- moleculesinacquiredhumankidneydiseases.JAmSocNephrol2003;14: infectedpatientsundergoingnativerenalbiopsy.AmJNephrol2008;28: 2063–2071. 478–486. 36. ReinigerN,LauK,McCallaDetal.Deletionofthereceptorfor 9. RossMJ,KlotmanPE,WinstonJA.HIV-associatednephropathy:casestudy advancedglycationendproductsreducesglomerulosclerosisand andreviewoftheliterature.AIDSPatientCareSTDS2000;14:637–645. preservesrenalfunctioninthediabeticOVE26mouse.Diabetes2010;59: 10. FineDM,PerazellaMA,LucasGMetal.KidneybiopsyinHIV:beyondHIV- 2043–2054. associatednephropathy.AmJKidneyDis2008;51:504–514. 37. TakemotoM,AskerN,GerhardtHetal.Anewmethodforlargescale 11. ChoiAI,RodriguezRA,BacchettiPetal.Racialdifferencesinend-stage isolationofkidneyglomerulifrommice.AmJPathol2002;161:799–805. renaldiseaseratesinHIVinfectionversusdiabetes.JAmSocNephrol 38. ZuoY,MatsusakaT,ZhongJetal.HIV-1genesvprandnefsynergistically 2007;18:2968–2974. damagepodocytes,leadingtoglomerulosclerosis.JAmSocNephrol 12. MedapalliR,ParikhCR,GordonKetal.Comorbiddiabetesandtheriskof 2006;17:2832–2843. progressivechronickidneydiseaseinHIV-infectedadults:Datafromthe 39. PfafflMW.Anewmathematicalmodelforrelativequantificationinreal- VeteransAgingCohortStudy.JAcquirImmuneDeficSyndr2012;60: timeRT-PCR.NucleicAcidsRes2001;29:e45. 393–399. 40. PotapovaTA,SivakumarS,FlynnJNetal.Mitoticprogressionbecomes 13. DickieP,FelserJ,EckhausMetal.HIV-associatednephropathyin irreversibleinprometaphaseandcollapseswhenWee1andCdc25are transgenicmiceexpressingHIV-1genes.Virology1991;185:109–119. inhibited.MolBiolCell2011;22:1191–1206. 14. FornoniA,IjazA,TejadaTetal.Roleofinflammationindiabetic 41. GassmannM,GrenacherB,RohdeBetal.QuantifyingWesternblots: nephropathy.CurrDiabetesRev2008;4:10–17. pitfallsofdensitometry.Electrophoresis2009;30:1845–1855. 15. MartinkaS,BruggemanLA.PersistentNF-kappaBactivationinrenal epithelialcellsinamousemodelofHIV-associatednephropathy.AmJ This work is licensed under the Creative PhysiolRenalPhysiol2006;290:F657–F665. 16. BrosiusFC3rd,AlpersCE,BottingerEPetal.Mousemodelsofdiabetic Commons Attribution-NonCommercial-No nephropathy.JAmSocNephrol2009;20:2503–2512. Derivative Works 3.0 Unported License. To view a copy of 17. ZhongJ,ZuoY,MaJetal.ExpressionofHIV-1genesinpodocytesalone thislicense,visithttp://creativecommons.org/licenses/by-nc- canleadtothefullspectrumofHIV-1-associatednephropathy.KidneyInt 2005;68:1048–1060. nd/3.0/ 634 KidneyInternational(2013)83,626–634

ebook img

Expression of HIV transgene aggravates kidney injury in diabetic mice. PDF

1.9 MB·English
by  Mallipattu, Sandeep K
#journals #pubmed
Save to my drive
Quick download
Download
Upgrade Premium
Most books are stored in the elastic cloud where traffic is expensive. For this reason, we have a limit on daily download.

Preview Expression of HIV transgene aggravates kidney injury in diabetic mice.

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 626 KidneyInternational(2013)83,626–634 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, KidneyInternational(2013)83,626–634 627 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 628 KidneyInternational(2013)83,626–634 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, KidneyInternational(2013)83,626–634 629 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 630 KidneyInternational(2013)83,626–634 SKMallipattuetal.:HIVtransgeneaggravatesdiabetickidneyinjury basic research 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. KidneyInternational(2013)83,626–634 631 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.32 632 KidneyInternational(2013)83,626–634 SKMallipattuetal.:HIVtransgeneaggravatesdiabetickidneyinjury basic research 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 Nephron2000;85:81–85. 28. NiewczasMA,GohdaT,SkupienJetal.CirculatingTNFreceptors1and2 REFERENCES predictESRDintype2diabetes.JAmSocNephrol2012;23:507–515. 1. RaoTK,FilipponeEJ,NicastriADetal.Associatedfocalandsegmental 29. BruggemanLA,DikmanS,MengCetal.Nephropathyinhuman glomerulosclerosisintheacquiredimmunodeficiencysyndrome.NEnglJ immunodeficiencyvirus-1transgenicmiceisduetorenaltransgene Med1984;310:669–673. expression.JClinInvest1997;100:84–92. 2. GardenswartzMH,LernerCW,SeligsonGRetal.Renaldiseaseinpatients 30. FengX,LuTC,ChuangPYetal.ReductionofStat3activityattenuates withAIDS:aclinicopathologicstudy.ClinNephrol1984;21:197–204. HIV-inducedkidneyinjury.JAmSocNephrol2009;20:2138–2146. 3. PardoV,AldanaM,ColtonRMetal.Glomerularlesionsintheacquired 31. WuKK,HuanY.Streptozotocin-induceddiabeticmodelsinmiceandrats. immunodeficiencysyndrome.AnnInternMed1984;101:429–434. CurrProtocPharmacol2008;Chapter5:Unit547;5.47.1–5.47.14. 4. CarboneL,D’AgatiV,ChengJTetal.Courseandprognosisofhuman 32. SandenSK,WigginsJE,GoyalMetal.Evaluationofathickandthin immunodeficiencyvirus-associatednephropathy.AmJMed1989;87: sectionmethodforestimationofpodocytenumber,glomerularvolume, 389–395. andglomerularvolumeperpodocyteinratkidneywithWilms’tumor-1 5. CantorKP,WeissSH,GoedertJJetal.HTLV-I/IIseroprevalenceandHIV/ proteinusedasapodocytenuclearmarker.JAmSocNephrol2003;14: HTLVcoinfectionamongU.S.intravenousdrugusers.JAcquirImmune 2484–2493. DeficSyndr1991;4:460–467. 33. AwazuM,OmoriS,IshikuraKetal.Thelackofcyclinkinaseinhibitor 6. GuptaSK,EustaceJA,WinstonJAetal.Guidelinesforthemanagementof p27(Kip1)amelioratesprogressionofdiabeticnephropathy.JAmSoc chronickidneydiseaseinHIV-infectedpatients:recommendationsofthe Nephrol2003;14:699–708. HIVMedicineAssociationoftheInfectiousDiseasesSocietyofAmerica. 34. SchifferM,MundelP,ShawASetal.Anovelrolefortheadaptormolecule ClinicalInfectiousDiseases2005;40:1559–1585. CD2-associatedproteinintransforminggrowthfactor-beta-induced 7. MallipattuSK,WyattCM,HeJC.ThenewepidemiologyofHIV-related apoptosis.JBiolChem2004;279:37004–37012. kidneydisease.JAIDSClinRes2012;S4:001–006. 35. KoopK,EikmansM,BaeldeHJetal.Expressionofpodocyte-associated 8. BerlinerAR,FineDM,LucasGMetal.ObservationsonacohortofHIV- moleculesinacquiredhumankidneydiseases.JAmSocNephrol2003;14: infectedpatientsundergoingnativerenalbiopsy.AmJNephrol2008;28: 2063–2071. 478–486. 36. ReinigerN,LauK,McCallaDetal.Deletionofthereceptorfor 9. RossMJ,KlotmanPE,WinstonJA.HIV-associatednephropathy:casestudy advancedglycationendproductsreducesglomerulosclerosisand andreviewoftheliterature.AIDSPatientCareSTDS2000;14:637–645. preservesrenalfunctioninthediabeticOVE26mouse.Diabetes2010;59: 10. FineDM,PerazellaMA,LucasGMetal.KidneybiopsyinHIV:beyondHIV- 2043–2054. associatednephropathy.AmJKidneyDis2008;51:504–514. 37. TakemotoM,AskerN,GerhardtHetal.Anewmethodforlargescale 11. ChoiAI,RodriguezRA,BacchettiPetal.Racialdifferencesinend-stage isolationofkidneyglomerulifrommice.AmJPathol2002;161:799–805. renaldiseaseratesinHIVinfectionversusdiabetes.JAmSocNephrol 38. ZuoY,MatsusakaT,ZhongJetal.HIV-1genesvprandnefsynergistically 2007;18:2968–2974. damagepodocytes,leadingtoglomerulosclerosis.JAmSocNephrol 12. MedapalliR,ParikhCR,GordonKetal.Comorbiddiabetesandtheriskof 2006;17:2832–2843. progressivechronickidneydiseaseinHIV-infectedadults:Datafromthe 39. PfafflMW.Anewmathematicalmodelforrelativequantificationinreal- VeteransAgingCohortStudy.JAcquirImmuneDeficSyndr2012;60: timeRT-PCR.NucleicAcidsRes2001;29:e45. 393–399. 40. PotapovaTA,SivakumarS,FlynnJNetal.Mitoticprogressionbecomes 13. DickieP,FelserJ,EckhausMetal.HIV-associatednephropathyin irreversibleinprometaphaseandcollapseswhenWee1andCdc25are transgenicmiceexpressingHIV-1genes.Virology1991;185:109–119. inhibited.MolBiolCell2011;22:1191–1206. 14. FornoniA,IjazA,TejadaTetal.Roleofinflammationindiabetic 41. GassmannM,GrenacherB,RohdeBetal.QuantifyingWesternblots: nephropathy.CurrDiabetesRev2008;4:10–17. pitfallsofdensitometry.Electrophoresis2009;30:1845–1855. 15. MartinkaS,BruggemanLA.PersistentNF-kappaBactivationinrenal epithelialcellsinamousemodelofHIV-associatednephropathy.AmJ This work is licensed under the Creative PhysiolRenalPhysiol2006;290:F657–F665. 16. BrosiusFC3rd,AlpersCE,BottingerEPetal.Mousemodelsofdiabetic Commons Attribution-NonCommercial-No nephropathy.JAmSocNephrol2009;20:2503–2512. Derivative Works 3.0 Unported License. To view a copy of 17. ZhongJ,ZuoY,MaJetal.ExpressionofHIV-1genesinpodocytesalone thislicense,visithttp://creativecommons.org/licenses/by-nc- canleadtothefullspectrumofHIV-1-associatednephropathy.KidneyInt 2005;68:1048–1060. nd/3.0/ 634 KidneyInternational(2013)83,626–634

See more

The list of books you might like

Upgrade Premium
Most books are stored in the elastic cloud where traffic is expensive. For this reason, we have a limit on daily download.
DMCA & Copyright: Dear all, most of the website is community built, users are uploading hundred of books everyday, which makes really hard for us to identify copyrighted material, please contact us if you want any material removed.
Copyright @ PDFdrive.to, 2022 | We love our users