HIV-1 Genes vpr and nef Synergistically Damage Podocytes, Leading to Glomerulosclerosis
Yiqin Zuo*,
Taiji Matsusaka*,,,
Jianyong Zhong**,
Ji Ma*,**,
Li-jun Ma,
Zaher Hanna,
Paul Jolicoeur,
Agnes B. Fogo*,, and
Iekuni Ichikawa*,,||
* Departments of Pediatrics, Medicine, and Pathology, Vanderbilt University Medical Center, Nashville, Tennessee; Departments of Metabolic and Kidney Diseases and || Pediatrics and ¶ Institute of Medical Science, Tokai University, Isehara, Kanagawa, Japan; ** Department of Nephrology, Huashan Hospital, Fudan University, Shanghai, China; and Laboratory of Molecular Biology, Clinical Research Institute of Montreal, Montreal, Quebec, Canada
Address correspondence to: Dr. Taiji Matsusaka, Departments of Pediatrics, Vanderbilt University Medical Center, MCN C4204, Nashville, TN 37232-3584. Phone: 615-343-0110; Fax: 615-322-7929; E-mail: matsusaka_taiji{at}yahoo.com
Received for publication August 24, 2005.
Accepted for publication August 1, 2006.
This study aimed to identify the causative gene for HIV-1 associatednephropathy, a paradigmatic podocytopathy. A previous studydemonstrated that transgenic expression of nonstructural HIV-1genes selectively in podocytes in mice with FVB/N genetic backgroundresulted in podocyte injury and glomerulosclerosis. In thisstudy, transgenic mice that expressed individual HIV-1 genesin podocytes were generated. Five of six transgenic mice thatexpressed vpr developed podocyte damage and glomerulosclerosis.Analysis of an established vpr transgenic line revealed thattransgenic mice on FVB/N but not on C57BL/6 genetic backgrounddeveloped podocyte injury by 8 wk of age, with later glomerulosclerosis.Four of 11 transgenic mice that expressed nef also developedpodocyte injury. One transgenic line was established from thenef founder mouse with the mildest phenotype. Transgenic micein this line developed mesangial expansion at 3 wk of age andmild focal podocyte damage at 10 wk of age. Mating with FVB/Nmice did not augment nephropathy. None of the transgenic micethat expressed vif, tat, rev, or vpu in podocytes, even withthe FVB/N genetic background, developed podocyte injury. Fortesting effects of simultaneous expression of vpr and nef, thesetwo lines were mated. All nef:vpr double-transgenic mice showedsevere podocyte injury and glomerulosclerosis by 4 wk of age.In contrast, all vpr or nef single-transgenic mice in the samelitter uniformly showed no or much milder podocyte injury. Thesefindings indicate that vpr and nef each can induce podocyteinjury with a prominent synergistic interaction.
Accumulating evidence indicates that podocyte damage triggersprogression of glomerular deterioration that leads to glomerularsclerosis (1–10). However, the nature of podocyte damagethat causes irreversible progression is poorly understood. HIV-1–associatednephropathy (HIVAN) is unique in that the etiology of podocyteinjury is genetically well defined, namely the HIV-1 itself.Patients with HIVAN present with heavy proteinuria and rapidprogression to end-stage renal failure with collapsing FSGSand microcystic tubular dilation (11–17). HIV-1 encodesthree structural genes (gag, pol, and env), two essential regulatorygenes (tat and rev), and four accessory genes (vif, vpr, vpu,and nef). Studies on HIV-1 transgenic animal models have providedimportant insights into the pathogenesis of HIVAN. A transgenicline, Tg26, that carries HIV-1 DNA that lacks gag and pol geneshas been studied extensively and is characterized by nephropathythat is similar to human HIVAN (18). The transgene of Tg26 isdriven by the endogenous HIV-1 promoter, which is expressedin a wide range of cells. Reciprocal renal transplantation betweenTg26 and wild-type mice revealed that development of nephropathyis due to intrarenal expression of HIV-1 mRNA that is independentof circulating viral proteins or dysregulated systemic cytokines(19). Transgenic rats that carry the same construct as Tg26mice also develop nephropathy, confirming that gag and pol arenot involved in HIVAN pathogenesis (20,21). Moreover, the HIV-1transgenic mouse line that lacks gag, pol, and nef developsFSGS, and genetic complementation with nef results in more severeglomerulosclerosis but less severe tubulointerstitial injury(22). Conversely, the transgenic lines CD4C/HIV and CD4C/simianimmunodeficiency virus that express HIV nef or simian immunodeficiencyvirus nef, respectively, develop tubulointerstitial injury andsegmental glomerulosclerosis (23–26). These findings suggestthat although nef may not be pathogenic in all settings, itmay modulate nephropathy. Furthermore, transgenic mice thatcarry HIV-1 long terminal repeat tat and vpr, but not mice thatcarry HIV-1 genome defective in vpr develop glomerulosclerosis(27). Transgenic mice that carry macrophage-specific c-fms promoterand vpr also develop nephropathy (27). Collectively, these observationsindicate that vpr and nef are involved in the pathogenesis ofHIVAN, although the specific cell type that is affected directlyby these gene products remains uncertain.
HIV-1 gene expression has been identified within renal cells,including podocytes, in humans and transgenic mice (19,28–31).In both settings, the podocytes appear dysregulated with lossof differentiation markers, proliferate, and undergo apoptosis(28,32–34). To evaluate the specific role of podocyteinjury in HIVAN, we recently generated transgenic mice thatcarry a podocyte-specific nephrin gene (Nphs1) promoter combinedwith HIV-1 DNA that lacks gag, pol, and env (35). The mice onan FVB/N genetic background, termed HIV13FVB, developed proteinuria,podocyte injury, collapsing FSGS, and microcystic tubular dilation,indicating that selective expression of the HIV-1 genes in podocytesalone can produce the full spectrum of HIVAN.
HIV13FVB mice carry six HIV-1 genes: vif, vpr, vpu, tat, rev,and nef. In this study, we aimed to determine which specificgene causes podocyte damage that leads to HIVAN. Using the nephrinpromoter, we generated six series of transgenic mice, each expressingone of the above six genes in podocytes. The results demonstratethat selective podocyte expression of either vpr or nef alonecan induce podocyte injury that leads to glomerulosclerosisand that strain background also modulates the phenotype. Inaddition, our study reveals a prominent synergistic effect ofvpr and nef genes on podocyte injury.
Transgene Constructs
All DNA fragments that encoded HIV-1 genes were generated fromthe transgene constructs that were used in our previous studiesand were derived from pNL4–3 genomic DNA (Genbank accessionno. AF324493; hereafter, nucleotide [nt] number is shown onthe basis of these data) (23,35). DNA fragments that encodednef (nt 8787 to 9407), vif (nt 5041 to 5619), or vpr (nt 5559to 5849) were generated by PCR. For rev, SalI-XhoI fragment(nt 5785 to 8887) of the mutant B of CD4C/HIV1 transgene constructwas subcloned in pBR322. Mutant B construct has an insertionof a stop codon at the NdeI site (nt 6399), which terminatesthe translation of ENV protein after the 60th amino acid, whereasthe length of wild-type ENV is 854 amino acids. For disruptionof tat and vpu, the SalI-Acc65I (nt 5785 to 6343) portion wasreplaced with the fragment that spanned from the initiationcodon of rev (nt 5969) to the Acc65I site (nt 6343), which wasgenerated by PCR from the mutant C of CD4C/HIV1 construct (23).This construct carries a deletion of a fragment of nt 6063 to6100, thus lacking the starting codon of vpu. The resultantSalI-HpaI (nt 8648) fragment contained the exons and the intronof rev (nt 5969 to 8643) with disrupted tat, vpu, and env, thusencoding only rev. A tat fragment was generated from the aboveSalI-XhoI fragment of mutant B. For disruption of vpu, the SacI-Acc65Iportion (nt 5999 to 6343) was replaced with the correspondingfragment of the mutant C of CD4C/HIV1 construct. T at nt 5970was mutated to C to disrupt the initiation codon of rev. Theresultant SalI-BamHI fragment (nt 5785 to 8465) contains theexons and the intron of tat (nt 5830 to 8414) with mutationsin rev, vpu, and env, thus encoding only tat. For vpu, the HindIII-Acc65Ifragment (nt 6026 to 6343) was used. Three nucleotides thatpreceded the initiation codon of rev, vif, nef, and vpr wereconverted to ACC to match Kozak’s consensus sequence.Each of the six DNA fragments was combined with a 5.4-kb promoterfragment of the mouse nephrin gene (Nphs1), internal ribosomeentry site, and enhanced green fluorescence protein (EGFP; Figure 1).The Nphs1 promoter contains the first and part of a second exon(up to XhoI site) and the first intron. Two ATG sequences inthe Nphs1 coding region were disrupted by site-directed mutagenesis.This promoter segment directed podocyte-specific expressionin our previous studies (10,35).
Figure 1. Structure of the transgenes. All HIV-1 DNA fragments that were used in our study are derived from an HIV-1 DNA clone, pNL4-3. The structure of pNL4-3 is shown at the top. Nphs1-HIV1 is the transgene that was used in our previous study to generate the HIV13 line, which carries Nphs1 promoter; vif, vpr, vpu, nef, rev, and tat; and a poly A signal from SV40 DNA. env in Nphs1-HIV1 is mutated (x). The six transgene constructs used in this study are shown. Each is composed of Nphs1 promoter, an individual HIV-1 gene (vif, vpr, vpu, nef, rev, or tat), internal ribosome entry site (IRES), enhanced green fluorescence protein (EGFP) gene, and a poly A signal. Nphs1-rev and -tat include the respective introns, with disruptions of env and vpu (x).
Generation of Transgenic Mice Nphs1-vpu transgene DNA was injected into fertilized eggs ofthe FVB/N strain. DNA for Nphs1-rev, Nphs1-tat, Nphs1-vif, andNphs1-vpr was injected into eggs of F2 hybrid between C57BL/6and DBA/2. For Nphs1-nef, both strains were used. The transgeneintegration was examined by PCR, using primers EGFP1 and EGFP2(Table 1). For the nef:vpr double-transgenic mouse study, transgenesnef and vpr were detected by PCR using the primer pairs nef1and nef2, and vpr1 and vpr2, respectively (Table 1).
Detection of Transgene Expression
Total RNA was extracted from whole kidney and treated with DNaseI. RNA was reverse-transcribed using random primers, and HIV-1genes Egfp and Gapdh (glyceraldehyde-3-phosphate dehydrogenase)were amplified by PCR using the primers listed in Table 1. Inaddition, a relative amount of nef mRNA to Gapdh mRNA was quantifiedby real-time reverse transcription–PCR (RT-PCR), usingthe iCycler and SYBR-Green I assay (Bio-Rad Laboratories, Hercules,CA). The primers for this analysis are shown in Table 1.
EGFP fluorescence signal was not detectable in any of the transgenicmice. We performed immunostaining for EGFP using mouse anti-GFPmAb (1:100; Chemicon, Temecula, CA) on paraffin tissues thatwere heated by microwave.
In Situ Hybridization
PCR product with nef1 and nef2 was subcloned into pGEM-T Easyvector (Promega, Madison, WI). Plasmid DNA was linearized withSpeI, and 35S-labeled sense and antisense RNA probes were synthesizedusing T7 RNA polymerase. In situ hybridization was performedas described previously (36). Briefly, hydrated paraffin sectionswere treated with proteinase K (10 µg/ml) for 8 min at37°C, fixed in 4% buffered paraformaldehyde for 5 min, incubatedin 0.1 M triethanolamine/0.25% acetic anhydride, washed in PBS,dehydrated in serial graded ethanol, and air-dried. The sectionsthen were hybridized overnight at 50°C in buffer (40% formamide,10% dextran sulfate, 8 mM dithiothreitol, 0.2 mg/ml tRNA, 300mM NaCl, 10 mM Tris-HCl, 5 mM EDTA, 0.02% polyvinylpyrrolidone,0.02% Ficoll, and 0.02% BSA) that contained 35S-labeled probe(2 x 104 cpm/µl). Sections then were washed, dehydrated,dried, dipped in photographic emulsion, and exposed at 4°Cfor 10 d. The sections were developed with D-19 developer (Kodak,Rochester, NY) and counterstained with toluidine blue O.
Analysis of Transgenic Mice
The Institutional Animal Care and Use Committee at VanderbiltUniversity Medical School and the Animal Experimentation Committeeat Tokai University Medical School approved the protocol, inaccordance with the principles and procedures outlined in theNational Institutes of Health Guide for the Care and Use ofLaboratory Animals.
All transgenic mice that were analyzed in the study were heterozygousfor each transgene. Transgenic mice that expressed tat, rev,or vif were backcrossed with FVB/N two or three times. The number,age, and generation of mice, respectively, were seven (at 4mo of age in N1 generation), two (2, N2), and six (5, N3) forrev; 10 (4, N1), three (6, N2), and three (6, N3) for tat; and10 (4, N1) and three (5, in N2) for vif. Seven founder micethat expressed vpu, which were generated in an inbred FVB/Nstrain, were analyzed at 5 mo of age.
The Nphs1-vpr19 line was backcrossed with the C57BL/6 strainfor seven generations. In N7 generation, two, six, and threetransgenic mice were analyzed at 2, 4, and 7 mo of age, respectively.Transgenic mice in N1 generation were mated with FVB/N mice,generating 32 transgenic offspring. These were analyzed longitudinally.The numbers of mice that were studied at each age were six (at1 wk of age), two (at 2 wk), one (at 4 wk), six (at 6 wk), seven(at 8 wk), three (at 10 wk), four (at 12 wk), and three (at14 wk). Some were mated further with FVB/N mice, generating17 transgenic mice. Four, seven, five, and one mice were analyzedat 2, 3, 4, and 8 wk of age, respectively.
The Nphs1-nef10 founder mouse was mated with FVB/N, generatingone transgenic offspring (Nphs1-nef10–40). Starting withthis mouse, the line was backcrossed with C57BL/6 mice fourtimes. Six transgenic mice of N4 generation were analyzed at2 mo of age. One mouse of N1 generation was mated with FVB/Nthree times. Eight transgenic mice of the third generation wereanalyzed at 2 mo of age.
Twenty-four-hour urine was collected using metabolic cages,and 2 µl of urine samples was analyzed by SDS-PAGE usinga 4 to 20% gradient gel (Bio-Rad). Increases in albumin andlarger proteins were considered as proteinuria, a marker ofnephropathy. Plasma was collected when the mice were killed,and the blood urea nitrogen and creatinine were measured ina commercial laboratory. Histologic analysis, immunostaining,electron microscopic analysis, and quantification of glomerulardamage were performed as described previously (10,35).
Analysis of Synergy between vpr and nef Nphs1-nef10–40 was mated with Nphs1-vpr19 mice of N2 generationof C57BL/6 backcrossing. A total of 126 mice were analyzed at1, 7, 14, 21, and 28 d of age. Glomerular epithelial and mesangialinjury indices were determined as previously reported (10).For mesangial changes, score 0 represents no lesion, whereas1, 2, 3, and 4 represent mesangial matrix expansion, hyalinosis,or sclerosis involving 25, 25 to 50, 50 to 75, and >75% ofthe glomerular tuft area, respectively. The epithelial cellinjury was graded as follows: 0, no lesion; 1, 2, 3, and 4,vacuolization, bleb, or proliferation of epithelial cells involving25, 25 to 50, 50 to 75, and >75% of the glomerulus, respectively.Six and 13 wild-type mice, four and nine vpr mice, eight andfive nef mice, and four and four nef:vpr double-transgenic micewere analyzed at 3 and 4 wk of age, respectively. For all typesof mice that were subjected to the study and the parental mice,D3Mit203 genotype was determined by PCR on the basis of theinformation of the Mouse Genome Informatics database (MGI ID:706352).
Statistical Analyses
Results were expressed as mean ± SE. Data were analyzedby one-way ANOVA followed by least significant difference testor Tamhane’s correction test as appropriate. Differenceswere considered to be statistically significant at P < 0.05.
Variable Effect of Individual HIV-1 Genes on Development of Nephropathy
To identify any HIV-1 gene(s) that causes podocyte damage, wegenerated six series of transgenic mice that individually expressedvif, vpr, vpu, tat, rev, or nef in podocytes under the regulationof the Nphs1 promoter. We also aimed to establish transgeniclines that show nephropathy. Previous studies by others andus showed a strong predisposition of nephropathy of the FVB/Ngenetic background (35,37); therefore, establishment of a nephropathictransgenic line on the FVB/N background was thought to be difficult,because mice would die of severe disease before producing offspring.We therefore generated transgenic mice on a mixed genetic backgroundof DBA/2 and C57BL/6, both of which are less susceptible tonephropathy than FVB/N. We then backcrossed them with an FVB/Nstrain. We generated vpu transgenic mice in inbred FVB/N, becausevpu mRNA was not detected in HIV13 transgenic mice and was unlikelyto cause nephropathy. Because our previous studies found thatpenetrance of transgene expression by this promoter is not 100%(10,35), we examined the renal transgene expression by RT-PCRfor each HIV-1 gene and/or Egfp and immunostaining for EGFP.The results are summarized in Table 2.
Table 2. Summary of transgenic founder mice that carried individual HIV-1 genes
Five of six founder Nphs1-vpr mice developed nonselective proteinuriaby 8 wk of age (Figure 2). Histologic analysis revealed podocytedamage and FSGS (Table 3, see below). These mice expressed vprmRNA in the kidney. In one mouse, Nphs1-vpr19, EGFP was detectableby immunostaining that localized to podocytes (Figure 3, A and B).One Nphs1-vpr mouse did not develop nephropathy, and the transgenewas not detectable in the kidney of this mouse.
Figure 2. Proteinuria in Nphs1-vpr and Nphs1-nef transgenic mice. SDS-PAGE analysis of urinary protein. Urine samples (2 µl) from Nphs1-vpr and Nphs1-nef transgenic mice and wild-type mice were analyzed at 8 to 10 wk of age. Samples loaded are molecular weight markers (lane 1), Nphs1-nef10 (lane 2), Nphs1-nef18 (lane 3), Nphs1-nef27 (lane 4), Nphs1-nef149 (lane 5), wild type (lane 6), Nphs1-vpr10 (lane 7), Nphs1-vpr16 (lane 8), Nphs1-vpr19 (lane 9), and Nphs1-vpr21 (lane 10). Proteinuria was detected in Nphs1-vpr and Nphs1-nef transgenic mice but not in Nphs1-nef10 or wild-type mice.
Figure 3. Transgene expression. (A and B) Transgene expression in the transgenic line Nphs1-vpr19. The reporter gene product, EGFP, was detected by immunostaining within the glomerulus (A). Staining for synaptopodin on the adjacent section (B) indicates that the transgene is expressed on podocytes. (C and D) Transgene expression in the transgenic line Nphs1-nef10. HIV-1 nef mRNA (C) was detected within the glomerulus. On the periphery of the tuft, the cell type that expressed nef mRNA (arrows) was identified as podocytes from the anatomic location. Sense control probe showed only background signal (D). Magnification, x400.
We obtained 10 Nphs1-nef founder transgenic mice on a mixedgenetic background of C57BL/6 and DBA/2. After establishinga transgenic line (Nphs1-nef10 line), we additionally generated20 Nphs1-nef founder transgenic mice on an inbred FVB/N geneticbackground. Of the total 30 Nphs1-nef founder transgenic mice,transgene mRNA was detectable by RT-PCR in the kidneys of 11mice (Table 4). Three of them, Nphs1-nef27, 149, and 162, hadproteinuria (Figure 2) and advanced glomerulosclerosis withsevere podocyte damage (see below). Nphs1-nef10 developed mildfocal mesangial sclerosis with slight podocyte damage at 5 moof age. The remaining seven mice that expressed nef mRNA didnot develop podocyte damage (Table 4). The amount of nef mRNAwas quantified by real-time RT-PCR. Nphs1-nef10 had higher expressionof nef mRNA than other non-nephropathic Nphs1-nef mice (Table 4).Of note, Nphs1-nef149, 162, and 27, which showed severe FSGS,expressed a lower level of nef mRNA than Nphs1-nef10.
Table 4. Summary of transgenic founder mice that expressed nef
In situ hybridization performed on the kidneys of the Nphs1-nef10line detected nef mRNA on podocytes (Figure 3C). No specificsignal was detected with a sense probe (Figure 3D).
None of the transgenic mice that carried rev, tat, vif, or vpudeveloped proteinuria. Nphs1-rev, -tat, and -vif mice were generatedon a mixed genetic background of C57BL/6 and DBA/2. Becausedevelopment of nephropathy in HIV13 transgenic mice dependson the FVB/N genetic background (35), the lack of nephropathyin these mice may reflect a lack of impact of the FVB/N geneticbackground. To rule out this possibility, we chose founder transgenicmice that expressed the transgene in kidneys and backcrossedthem with wild-type FVB/N mice for two (for Nphs1-vif) or three(for Nphs1-rev and Nphs1-tat) generations (Table 2). None ofthe transgenic offspring in N1 to N3 generations showed nephropathy(Figure 4, I through K). Nphs1-vpu mice that were generatedon the FVB/N genetic background yielded seven founder mice thatexpressed the transgene. However, none of these mice developednephropathy (Figure 4L). Taken together, these results indicatethat vpr and nef cause podocyte injury. By contrast, vif, tat,rev, and vpu are not nephropathic genes.
Figure 4. Renal lesions in Nphs1-vpr and Nphs1-nef transgenic mice. (A and B) Wild-type mouse at 4 mo of age showing normal histology. (C and D) Nphs1-vpr19 at 4 mo of age had noncollapsing glomerulosclerosis, proteinaceous casts, and tubulointerstitial injury (C) and vacuolar degeneration of glomerular epithelial cells (D). (E and F) Nphs1-nef149 at 6 mo of age showed noncollapsing focal segmental glomerulosclerosis, tubular microcystic dilation, and interstitial fibrosis (E) and vacuolar degeneration of glomerular epithelial cells (F). (G and H) Nphs1-nef10 at 5 mo of age showed focal casts and focal mesangial sclerosis. (I through L) Nphs1-vif, -tat, -rev, and -vpu mice showed normal renal histology. Magnifications: x200 in A, C, E, G, and I through L; x400 in B, D, F, and H (period acid-Schiff staining).
Podocyte-Specific vpr-Transgene Nephropathy
Founder mice, Nphs1-vpr8, 10, 16, 19, and 21, showed moderateto severe segmental or global, noncollapsing sclerosis withtubulointerstitial injury (Figure 4). In these mice, nephrinand synaptopodin immunostaining was markedly suppressed. Bycontrast, desmin was upregulated, indicating that the podocyteswere damaged (Figure 5). In Nphs1-vpr10 and 19, podocytes andparietal epithelial cells showed prominent vacuolar degeneration(Figure 4D). In these mice, tubulointerstitial damage was severe,and the tubules were microcystically dilated (Figure 4C).
Figure 5. Damage of podocytes in Nphs1-vpr and Nphs1-nef transgenic mice. Wild-type (A through C), Nphs1-vpr19 (D through F), Nphs1-nef149 (G through I), and Nphs1-nef10 (J through L) mice at 4 to 6 mo of age. There was prominent degeneration of podocytes with markedly reduced podocyte markers, synaptopodin, and nephrin in Nphs1-vpr19 (D and E) and Nphs1-nef149 (G and H). In wild-type mice, desmin was faintly detectable only in mesangial cells (C), whereas in Nphs1-vpr19 and Nphs1-nef149, desmin was expressed in podocytes (F and I). In Nphs1-nef10, synaptopodin staining was preserved, but nephrin was slightly decreased. Desmin was stained in expanded mesangial areas. Some podocytes also expressed desmin (arrows). A to C, D to F, H to I, and J to L are adjacent sections, respectively. Magnification, x400 (synaptopodin in A, D, G, and J; nephrin in B, E, H, and K; and desmin staining in C, F, I, and L).
Nphs1-vpr19 founder mouse produced transgenic offspring viamating with C57BL/6 mice. These were mated further with FVB/Nmice. We longitudinally analyzed 32 transgenic mice that wereobtained through this mating. Before 6 wk of age, most transgenicmice had no proteinuria, and the renal histology was normal.At 8 wk of age, three of 10 transgenic mice developed mild proteinuriaand had podocyte damage. After 8 wk of age, all transgenic mice(n = 9) had developed massive and nonselective proteinuria,severe podocyte injury, and segmental or global glomerulosclerosis.
We previously observed that HIV13FVB mice showed immature glomeruliat 2 wk of age (35). The pattern of glomerular maturation wasnormal in the Nphs1-vpr19 mice that were bred on FVB/N onceor twice.
The Nphs1-vpr19 line was backcrossed with a C57BL/6 strain.Eleven transgenic mice in N7 generation were analyzed at 2 to7 mo of age. Most mice showed essentially normal renal morphology,with the exception of one mouse, which showed mild focal segmentalsclerosis in 20% of glomeruli at 7 mo of age.
The Nphs1-vpr8 founder mouse also produced a small number oftransgenic offspring when mated with FVB/N. They showed proteinuriaand FSGS.
Podocyte-Specific nef-Transgene Nephropathy Nphs1-nef27, 149, and 162 mice showed advanced noncollapsingglomerulosclerosis with tubulointerstitial injury (Figure 4, E and F).Synaptopodin and nephrin were markedly reduced, whereas desminwas upregulated within the podocytes (Figure 5, G through I).In addition, Nphs1-nef149 and 162 showed remarkable vacuolardegeneration in podocytes (Figure 4F). These three founder micedid not produce offspring.
Nphs1-nef10 showed focal proteinaceous casts and mild focalmesangial sclerosis at 5 mo of age (Figure 4, G and H). At 6mo of age, podocytes showed focal injury with occasional proteinreabsorption droplets and detachment from the glomerular basementmembrane. Synaptopodin and nephrin were normal or slightly downregulated.Desmin was slightly increased in podocytes of glomeruli withmesangial sclerosis (Figure 5, J through L).
Nphs1-nef10 founder mouse generated transgenic offspring. Transgenicmice that were obtained by backcrossing with C57BL/6 strainfor four generations and those with FVB/N strain for three generationswere compared at 2 mo of age. Of the six transgenic mice withC57BL/6 genetic background studied, two mice showed mild focalsegmental glomerular sclerosis, three showed mild mesangialexpansion, and the other one was normal. Of the eight transgenicmice with the FVB/N genetic background studied, only one showedmild mesangial expansion, and the other seven were normal. Therefore,mating with the FVB/N genetic background did not augment nephropathyin the Nphs1-nef10 line.
Synergy between vpr and nef in Causing Podocyte Damage and Nephropathy
Our previous HIV-1 transgenic mice (HIV13FVB) had simultaneousexpression of both vpr and nef. To test whether vpr and nefhave synergistic effects on podocyte injury, we mated Nphs1-vpr19and Nphs1-nef10 lines and generated double-transgenic mice (nef:vpr).
Compared with transgenic mice that carried either vpr or nef,which had no proteinuria by 4 wk of age, the nef:vpr developedproteinuria by 1 wk of age, which progressed to massive andnonselective proteinuria by 3 wk of age. At 4 wk of age, nef:vprmice had renal failure with markedly elevated blood urea nitrogen(260.7 ± 33.5 mg/dl; n = 3) compared with wild-type mice(24.5 ± 9.3 mg/dl; n = 4), vpr single-transgenic mice(26.0 ± 7.2 mg/dl; n = 6), and nef single-transgenicmice (24.3 ± 0.58 mg/dl; n = 3). Some nef:vpr mice hadascites.
Histologically, none of the vpr or nef transgenic mice had abnormalitiesby 2 wk of age. Moreover, electron microscopic analysis at 2wk of age detected no abnormalities in the single-transgenicmice. Eleven of 13 vpr mice and six of 13 nef mice did not haveany abnormality by 4 wk of age. The rest of the single-transgenicmice had only mild to moderate mesangial matrix expansion withoutremarkable podocyte vacuolization. In contrast, by 1 wk of age,two of four nef:vpr mice had remarkable mesangiolysis, dilatedcapillaries, microaneurysms, and occasionally mesangial expansionand hypercellularity. In addition, one nef:vpr mouse had prominenthyperplastic lesions in the afferent arterioles and interlobularartery. At 2 wk of age, all nef:vpr mice showed mesangial matrixexpansion with an increase in type IV collagen (data not shown).Synaptopodin and nephrin were markedly downregulated, indicatingthat podocytes were severely damaged. By electron microscopy,there was prominent podocyte degeneration with completely effacedfoot processes and microvillous transformation (Figure 6). After3 wk of age, all nef:vpr mice developed severe glomerulosclerosisand tubulointerstitial injury with almost complete loss of synaptopodinstaining (Figure 7). Quantitative analysis for glomerular epithelialand mesangial injury at 3 and 4 wk of age is shown in Figure 8.
Figure 6. Glomerular ultrastructure of nef:vpr mouse. Electron microscopy of the kidney of a wild-type (A) and nef:vpr double-transgenic mouse (B) at 2 wk of age. In contrast to normal podocytes with fine foot processes in (A), podocytes in nef:vpr showed degeneration with complete effacement of foot processes and microvillous transformation (B). Bar = 2 µm.
Figure 7. Glomerular histology of vpr, nef, and nef:vpr mice at 4 wk of age. Compared with wild-type (A and B), nef single-transgenic mouse displayed mild mesangial expansion (C) with downregulation of synaptopodin (D). vpr single-transgenic mouse had normal renal histology (E) and normal synaptopodin staining at this age (F). nef:vpr double-transgenic mouse showed global sclerosis with vacuolization in visceral and parietal epithelial cells (G), with dramatically reduced synaptopodin staining (H). A to B, C to D, E to F, and G to H are adjacent sections, respectively. Magnification, x400 (periodic acid-Schiff [PAS] in A, C, E, and G; and synaptopodin staining in B, D, F, and H).
Figure 8. Quantitative analysis of glomerular injury. Indices for glomerular epithelial injury and mesangial injury at 3 and 4 wk of age are shown. Note prominent epithelial and mesangial injury in nef:vpr double-transgenic mice compared with wild-type (WT), vpr, or nef single-transgenic mice (P < 0.05).
Because the nef mouse that was used for generating the double-transgenicmice had the FVB/N genetic background, the possibility existedthat the severe nephropathy that was observed in nef:vpr double-transgenicmice was attributed to FVB/N genetic traits. We therefore determinedthe type of D3Mit203 microsatellite, which is closely associatedwith the major HIV-1 nephropathy susceptibility locus (37).Both Nphs1-nef10 and Nphs1-vpr19 founder mice were homozygousC57BL/6 type (B6/B6), and the nef mouse that was used for themating had the B6/FVB type. The vpr mice that were used forthe mating had B6/B6 type. Of the 21 nef:vpr double-transgenicmice analyzed, eight had the B6/B6 type and 13 had the B6/FVBtype. We found no difference in the degree of renal injury betweenB6/B6 and B6/FVB types at any age. This indicates that the synergisticeffect of nef and vpr had a greater impact on podocyte injurythan the major genetic susceptibility factor.
Rare Proliferation of Podocytes
It was reported that podocytes proliferate in human HIVAN andTg26 mice. However, none of the transgenic mice that were generatedin this study, including Nphs1-vpr, Nphs1-nef, and nef:vpr mice,showed an appreciable degree of proliferation of visceral epithelialcells. To detect proliferation of injured podocytes, we performeddouble immunostaining for Ki-67 and desmin, a marker of injuredpodocytes. To distinguish injured podocytes from mesangial cells,which also were stained for desmin, the localization of doublypositive cells was determined in periodic acid-Schiff–stainedadjacent sections. Extensive study in more than 30 transgenicmice with advanced nephropathy found only one doubly positivecell in an anatomic location of podocytes in one transgenicmouse of Nphs1-vpr19 line with the FVB/N genetic backgroundat 3 mo of age (Figure 9). These indicated that podocyte proliferationis a very rare phenomenon in these transgenic mice.
Figure 9. Rare podocyte proliferation. One cell (arrow) was doubly stained for synaptopodin (purple in A, brown in B) and Ki-67 (brown in A) in one transgenic mouse of the Nphs1-vpr19 line with FVB/N genetic background at 3 mo of age. PAS staining in the adjacent section (B) confirms that the cell localization also is consistent with a podocyte. Extensive search in >30 transgenic mice found only one doubly positive cell.
Identification of Specific HIV-1 Genes That Cause Podocyte Damage
In this study, we generated six series of transgenic mice, eachindividually expressing one of the candidate genes of HIV-1,tat, rev, vif, vpu, vpr, or nef, selectively in podocytes. Allfounder transgenic mice that expressed vpr and four of 11 foundertransgenic mice that expressed nef developed podocyte damage.Conversely, none of the mice that expressed vif, tat, rev, orvpu developed podocyte injury. FVB/N genetic background is aprerequisite for the development of nephropathy in the HIV13transgenic line (35) as well as in the Tg26 line (37). Nphs1-vif,-tat, and -rev were generated in a mixed genetic backgroundof DBA/2 and C57BL/6. We therefore backcrossed with the FVB/Nstrain for two to three generations. None of the Nphs1-vif,-tat, and -rev mice with the FVB/N genetic background showednephropathy. We could not detect VIF, TAT, REV, or VPU proteinwith available antibodies. Therefore, we explored the possibilitythat these proteins were not generated. We think that this isunlikely, because (1) mRNA and protein of the reporter geneEGFP were detected, (2) mRNA for each HIV-1 gene was detected,(3) these antibodies failed to detect transgene products alsoin the HIV13 mice, (4) the same Nphs1 promoter segment successfullygenerated functional proteins in podocytes in our previous studies(10,35,38), and (5) sequencing of the transgene cDNA found noaccidental mutation or unexpected splicing. These findings collectivelyindicate that (1) vpr is a major pathogenic gene causing podocytedamage; (2) nef alone also can cause podocyte damage; and (3)vif, tat, rev, or vpu alone does not cause podocyte damage,although possibilities remain that these gene products may causeor modify nephropathy in other genetic backgrounds.
Notably, although nef mRNA was detected by RT-PCR in the kidneyof 11 Nphs1-nef mice, only four developed nephropathy. Nphs1-nefmice without nephropathy expressed lower amounts of nef mRNAthan did Nphs1-nef10, suggesting that a large amount of NEFis necessary to cause podocyte damage. However, Nphs1-nef micewith severe FSGS also expressed lower amounts of nef mRNA. Inthese mice, podocytes were severely injured, so the expressionof nef RNA driven by Nphs1 promoter may be downregulated. Nevertheless,the possibility still remains that other genetic factors arerequired for the nephropathy to develop.
It is interesting that nef and vpr had remarkable synergisticeffects on podocyte injury. Thus, nef:vpr double-transgenicmice developed substantially more accelerated and severe podocytedamage than any transgenic mouse from the Nphs1-nef10 and Nphs1-vpr19lines.
Characterization of Nephropathy
It has been reported that podocytes undergo proliferation andapoptosis in human HIVAN as well as in Tg26 mice (28,32–34).In vitro experiments showed that NEF induces anchorage-independentproliferation and de-differentiation of podocytes through activationof Src-dependent Stat and mitogen-activated protein kinase pathways(39–41). Recently, Husain et al. (42) reported that transgenicmice that expressed nef in podocytes showed expression of Ki-67in podocytes and downregulation of synaptopodin. These observationssuggest that dysregulation of cell cycles or apoptosis may beinvolved in HIVAN pathogenesis. However, we found essentiallyno Ki-67–positive podocytes in Nphs1-vpr, Nphs1-nef, andnef:vpr mice. Similarly, we did not observe apoptotic podocytesby terminal deoxynucleotidyl transferase–mediated dUTPnick-end labeling staining in our transgenic mice (data notshown). Taken together, these findings do not support a prominentrole for proliferation or apoptosis in the injury of podocytesthat was demonstrated in our transgenic mice.
Segmental capillary collapse is a morphologic hallmark of HIVANand has been observed in HIV13FVB mice. In our study, Nphs1-vpr,Nphs1-nef, and nef:vpr transgenic mice did not have collapsingfeatures, suggesting that other HIV-1 genes, including vpu,tat, rev, and vif alone or in combination, may have an impacton producing tuft collapse. Injury of podocytes by immunotoxindid not cause collapsing glomerulopathy (10), indicating thatpodocyte damage alone cannot cause this phenotype. The mechanismunderlying the collapse is an intriguing unsolved question.
One nef:vpr double-transgenic mouse showed hyperplastic lesionsin the afferent arteriole. It is interesting that the arteriallesions were similar to those observed in angiotensinogen orAT1 receptor knockout mice (43), suggesting that these two differentmouse models share a common secondary change in the artery.However, a precise mechanism for development of this lesionis not clear.
Influence of Genetic Background
Genetic background has a strong influence on the developmentof HIVAN. Black individual are especially susceptible to HIVAN(11–17). Genetic background also influences experimentalnephropathy such that the FVB/N genetic background determinesrenal damage in Tg26 and HIV13 mice (35,37). In Tg26, a majornephropathy susceptibility locus is present on mouse chromosome3A1 to 3 (37). In our study, mating with FVB/N mice augmentednephropathy in the Nphs1-vpr19 line but not in the Nphs1-nef10line. Although our study was performed on a mixed genetic backgroundwith a limited number of mating, these findings indicate thatthe vpr gene has a genetic background dependence of nephropathy.We previously showed that HIV13 mice on the C57BL/6 geneticbackground showed only mild mesangial expansion, which was ina manner similar to Nphs1-nef10 mice. Mild mesangial expansionin HIV13 mice with the C57BL/6 genetic background may primarilyreflect the activity of the nef gene. Similar interplay of viralgenes and host response, influenced by genetic background, maycontribute to the variable manifestations of HIV-1 infectionin the kidney in various human populations.
Possible Molecular Mechanisms
Mechanisms by which HIV-1 genes cause nephropathy are not known.VPR is thought to play an important role in the intracellulartransport of the HIV-1 preintegration complex and has been reportedto affect several cellular functions, including G2 cell-cyclearrest, regulation of apoptosis, transcriptional activation,co-activation of glucocorticoid receptor, ion channel formation,and disruption of cytoskeleton (44). Similarly, NEF has multiplefunctions, including enhancement of viral infectivity, perturbationof endosome trafficking pathways, and modulation of cell signaling(45). How these cellular mechanisms are modulated in podocyteshas not been addressed. Our studies find no definitive proliferationor apoptosis of podocytes. However, even before the onset ofproteinuria, podocytes in HIV13FVB mice were devoid of extensionof primary processes and foot processes. It is therefore possiblethat disturbance of the cytoskeleton in podocytes is involvedin the development of nephropathy. In this regard, both VPRand NEF have been reported to be associated with cytoskeletalalterations and changes in cell morphology (46–50). Elucidationof molecular mechanisms may also lead to understanding of thepathogenesis of more common glomerular diseases that resultin glomerulosclerosis.
Our study demonstrates that both vpr and nef play an importantrole in pathogenesis of podocyte damage and subsequent FSGS.In addition, vpr and nef have a significant synergistic effecton podocyte injury and subsequent development of glomerulosclerosis.
Acknowledgments
This study was supported by National Institutes of Health grantsDK37868 and DK447577.
Part of this study was presented in abstract form at the 5thInternational Podocyte Conference, June 18 through 20, 2004;Seattle, WA; and the annual meeting of the American Societyof Nephrology; October 27 through November 1, 2004; St. Louis,MO.
We thank Dr. Valentina Kon for editorial assistance and discussionsand Dr. Kato and Ms. Tsuchida for microsatellite analysis.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
Rennke HG: How does glomerular epithelial cell injury contribute to progressive glomerular damage?
Kidney Int Suppl 45
: S58
–S63, 1994[Medline]
Kriz W, Gretz N, Lemley KV: Progression of glomerular diseases: Is the podocyte the culprit?
Kidney Int 54
: 687
–697, 1998[CrossRef][Medline]
Boute N, Gribouval O, Roselli S, Benessy F, Lee H, Fuchshuber A, Dahan K, Gubler MC, Niaudet P, Antignac C: NPHS2, encoding the glomerular protein podocin, is mutated in autosomal recessive steroid-resistant nephrotic syndrome.
Nat Genet 24
: 349
–354, 2000[CrossRef][Medline]
Kaplan JM, Kim SH, North KN, Rennke H, Correia LA, Tong HQ, Mathis BJ, Rodriguez-Perez JC, Allen PG, Beggs AH, Pollak MR: Mutations in ACTN4, encoding alpha-actinin-4, cause familial focal segmental glomerulosclerosis.
Nat Genet 24
: 251
–256, 2000[CrossRef][Medline]
Kim YH, Goyal M, Kurnit D, Wharram B, Wiggins J, Holzman L, Kershaw D, Wiggins R: Podocyte depletion and glomerulosclerosis have a direct relationship in the PAN-treated rat.
Kidney Int 60
: 957
–968, 2001[CrossRef][Medline]
Koziell A, Grech V, Hussain S, Lee G, Lenkkeri U, Tryggvason K, Scambler P: Genotype/phenotype correlations of NPHS1 and NPHS2 mutations in nephrotic syndrome advocate a functional inter-relationship in glomerular filtration.
Hum Mol Genet 11
: 379
–388, 2002[Abstract/Free Full Text]
Rantanen M, Palmen T, Patari A, Ahola H, Lehtonen S, Astrom E, Floss T, Vauti F, Wurst W, Ruiz P, Kerjaschki D, Holthofer H: Nephrin TRAP mice lack slit diaphragms and show fibrotic glomeruli and cystic tubular lesions.
J Am Soc Nephrol 13
: 1586
–1594, 2002[Abstract/Free Full Text]
Kim JM, Wu H, Green G, Winkler CA, Kopp JB, Miner JH, Unanue ER, Shaw AS: CD2-associated protein haploinsufficiency is linked to glomerular disease susceptibility.
Science 300
: 1298
–1300, 2003[Abstract/Free Full Text]
Ichikawa I, Ma J, Motojima M, Matsusaka T: Podocyte damage damages podocytes: Autonomous vicious cycle that drives local spread of glomerular sclerosis.
Curr Opin Nephrol Hypertens 14
: 205
–210, 2005[Medline]
Matsusaka T, Xin J, Niwa S, Kobayashi K, Akatsuka A, Hashizume H, Wang QC, Pastan I, Fogo AB, Ichikawa I: Genetic engineering of glomerular sclerosis in the mouse via control of onset and severity of podocyte-specific injury.
J Am Soc Nephrol 16
: 1013
–1023, 2005[Abstract/Free Full Text]
Bourgoignie JJ: Renal complications of human immunodeficiency virus type 1.
Kidney Int 37
: 1571
–1584, 1990[Medline]
Humphreys MH: Human immunodeficiency virus-associated glomerulosclerosis.
Kidney Int 48
: 311
–320, 1995[Medline]
Ross MJ, Klotman PE: Recent progress in HIV-associated nephropathy.
J Am Soc Nephrol 13
: 2997
–3004, 2002[Free Full Text]
Kimmel PL, Barisoni L, Kopp JB: Pathogenesis and treatment of HIV-associated renal diseases: Lessons from clinical and animal studies, molecular pathologic correlations, and genetic investigations.
Ann Intern Med 139
: 214
–226, 2003[Abstract/Free Full Text]
Weiner NJ, Goodman JW, Kimmel PL: The HIV-associated renal diseases: Current insight into pathogenesis and treatment.
Kidney Int 63
: 1618
–1631, 2003[CrossRef][Medline]
Ross MJ, Klotman PE: HIV-associated nephropathy.
AIDS 18
: 1089
–1099, 2004[CrossRef][Medline]
Dickie P, Felser J, Eckhaus M, Bryant J, Silver J, Marinos N, Notkins AL: HIV-associated nephropathy in transgenic mice expressing HIV-1 genes.
Virology 185
: 109
–119, 1991[CrossRef][Medline]
Bruggeman LA, Dikman S, Meng C, Quaggin SE, Coffman TM, Klotman PE: Nephropathy in human immunodeficiency virus-1 transgenic mice is due to renal transgene expression.
J Clin Invest 100
: 84
–92, 1997[Medline]
Reid W, Sadowska M, Denaro F, Rao S, Foulke J Jr, Hayes N, Jones O, Doodnauth D, Davis H, Sill A, O’Driscoll P, Huso D, Fouts T, Lewis G, Hill M, Kamin-Lewis R, Wei C, Ray P, Gallo RC, Reitz M, Bryant J: An HIV-1 transgenic rat that develops HIV-related pathology and immunologic dysfunction.
Proc Natl Acad Sci U S A 98
: 9271
–9276, 2001[Abstract/Free Full Text]
Ray PE, Liu XH, Robinson LR, Reid W, Xu L, Owens JW, Jones OD, Denaro F, Davis HG, Bryant JL: A novel HIV-1 transgenic rat model of childhood HIV-1-associated nephropathy.
Kidney Int 63
: 2242
–2253, 2003[CrossRef][Medline]
Kajiyama W, Kopp JB, Marinos NJ, Klotman PE, Dickie P: Glomerulosclerosis and viral gene expression in HIV-transgenic mice: Role of nef.
Kidney Int 58
: 1148
–1159, 2000[CrossRef][Medline]
Hanna Z, Kay DG, Rebai N, Guimond A, Jothy S, Jolicoeur P: Nef harbors a major determinant of pathogenicity for an AIDS-like disease induced by HIV-1 in transgenic mice.
Cell 95
: 163
–175, 1998[CrossRef][Medline]
Simard MC, Chrobak P, Kay DG, Hanna Z, Jothy S, Jolicoeur P: Expression of simian immunodeficiency virus nef in immune cells of transgenic mice leads to a severe AIDS-like disease.
J Virol 76
: 3981
–3995, 2002[Abstract/Free Full Text]
Hanna Z, Priceputu E, Kay DG, Poudrier J, Chrobak P, Jolicoeur P: In vivo mutational analysis of the N-terminal region of HIV-1 Nef reveals critical motifs for the development of an AIDS-like disease in CD4C/HIV transgenic mice.
Virology 327
: 273
–286, 2004[CrossRef][Medline]
Priceputu E, Rodrigue I, Chrobak P, Poudrier J, Mak TW, Hanna Z, Hu C, Kay DG, Jolicoeur P: The Nef-mediated AIDS-like disease of CD4C/human immunodeficiency virus transgenic mice is associated with increased Fas/FasL expression on T cells and T-cell death but is not prevented in Fas-, FasL-, tumor necrosis factor receptor 1-, or interleukin-1beta-converting enzyme-deficient or Bcl2-expressing transgenic mice.
J Virol 79
: 6377
–6391, 2005[Abstract/Free Full Text]
Dickie P, Roberts A, Uwiera R, Witmer J, Sharma K, Kopp JB: Focal glomerulosclerosis in proviral and c-fms transgenic mice links Vpr expression to HIV-associated nephropathy.
Virology 322
: 69
–81, 2004[CrossRef][Medline]
Barisoni L, Bruggeman LA, Mundel P, D’Agati VD, Klotman PE: HIV-1 induces renal epithelial dedifferentiation in a transgenic model of HIV-associated nephropathy.
Kidney Int 58
: 173
–181, 2000[CrossRef][Medline]
Bruggeman LA, Ross MD, Tanji N, Cara A, Dikman S, Gordon RE, Burns GC, D’Agati VD, Winston JA, Klotman ME, Klotman PE: Renal epithelium is a previously unrecognized site of HIV-1 infection.
J Am Soc Nephrol 11
: 2079
–2087, 2000[Abstract/Free Full Text]
Ross MJ, Bruggeman LA, Wilson PD, Klotman PE: Microcyst formation and HIV-1 gene expression occur in multiple nephron segments in HIV-associated nephropathy.
J Am Soc Nephrol 12
: 2645
–2651, 2001[Abstract/Free Full Text]
Winston JA, Bruggeman LA, Ross MD, Jacobson J, Ross L, D’Agati VD, Klotman PE, Klotman ME: Nephropathy and establishment of a renal reservoir of HIV type 1 during primary infection.
N Engl J Med 344
: 1979
–1984, 2001[Free Full Text]
Barisoni L, Kriz W, Mundel P, D’Agati V: The dysregulated podocyte phenotype: A novel concept in the pathogenesis of collapsing idiopathic focal segmental glomerulosclerosis and HIV-associated nephropathy.
J Am Soc Nephrol 10
: 51
–61, 1999[Abstract/Free Full Text]
Barisoni L, Mokrzycki M, Sablay L, Nagata M, Yamase H, Mundel P: Podocyte cell cycle regulation and proliferation in collapsing glomerulopathies.
Kidney Int 58
: 137
–143, 2000[CrossRef][Medline]
Shankland SJ, Eitner F, Hudkins KL, Goodpaster T, D’Agati V, Alpers CE: Differential expression of cyclin-dependent kinase inhibitors in human glomerular disease: Role in podocyte proliferation and maturation.
Kidney Int 58
: 674
–683, 2000[CrossRef][Medline]
Zhong J, Zuo Y, Ma J, Fogo AB, Jolicoeur P, Ichikawa I, Matsusaka T: Expression of HIV-1 genes in podocytes alone can lead to the full spectrum of HIV-1-associated nephropathy.
Kidney Int 68
: 1048
–1060, 2005[CrossRef][Medline]
Ma LJ, Nakamura S, Aldigier JC, Rossini M, Yang H, Liang X, Nakamura I, Marcantoni C, Fogo AB: Regression of glomerulosclerosis with high-dose angiotensin inhibition is linked to decreased plasminogen activator inhibitor-1.
J Am Soc Nephrol 16
: 966
–976, 2005[Abstract/Free Full Text]
Gharavi AG, Ahmad T, Wong RD, Hooshyar R, Vaughn J, Oller S, Frankel RZ, Bruggeman LA, D’Agati VD, Klotman PE, Lifton RP: Mapping a locus for susceptibility to HIV-1-associated nephropathy to mouse chromosome 3.
Proc Natl Acad Sci U S A 101
: 2488
–2493, 2004[Abstract/Free Full Text]
Asano T, Niimura F, Pastan I, Fogo AB, Ichikawa I, Matsusaka T: Permanent genetic tagging of podocytes: Fate of injured podocytes in a mouse model of glomerular sclerosis.
J Am Soc Nephrol 16
: 2257
–2262, 2005[Abstract/Free Full Text]
Husain M, Gusella GL, Klotman ME, Gelman IH, Ross MD, Schwartz EJ, Cara A, Klotman PE: HIV-1 Nef induces proliferation and anchorage-independent growth in podocytes.
J Am Soc Nephrol 13
: 1806
–1815, 2002[Abstract/Free Full Text]
Sunamoto M, Husain M, He JC, Schwartz EJ, Klotman PE: Critical role for Nef in HIV-1-induced podocyte dedifferentiation.
Kidney Int 64
: 1695
–1701, 2003[CrossRef][Medline]
He JC, Husain M, Sunamoto M, D’Agati VD, Klotman ME, Iyengar R, Klotman PE: Nef stimulates proliferation of glomerular podocytes through activation of Src-dependent Stat3 and MAPK1,2 pathways.
J Clin Invest 114
: 643
–651, 2004[CrossRef][Medline]
Husain M, D’Agati VD, He JC, Klotman ME, Klotman PE: HIV-1 Nef induces dedifferentiation of podocytes in vivo: A characteristic feature of HIVAN.
AIDS 19
: 1975
–1980, 2005[Medline]
Matsusaka T, Miyazaki Y, Ichikawa I: The renin angiotensin system and kidney development.
Annu Rev Physiol 64
: 551
–561, 2002[CrossRef][Medline]
Andersen JL, Planelles V: The role of Vpr in HIV-1 pathogenesis.
Curr HIV Res 3
: 43
–51, 2005[CrossRef][Medline]
Wei BL, Arora VK, Foster JL, Sodora DL, Garcia JV: In vivo analysis of Nef function.
Curr HIV Res 1
: 41
–50, 2003[CrossRef][Medline]
Macreadie IG, Castelli LA, Hewish DR, Kirkpatrick A, Ward AC, Azad AA: A domain of human immunodeficiency virus type 1 Vpr containing repeated H(S/F)RIG amino acid motifs causes cell growth arrest and structural defects.
Proc Natl Acad Sci U S A 92
: 2770
–2774, 1995[Abstract/Free Full Text]
Fackler OT, Kienzle N, Kremmer E, Boese A, Schramm B, Klimkait T, Kucherer C, Mueller-Lantzsch N: Association of human immunodeficiency virus Nef protein with actin is myristoylation dependent and influences its subcellular localization.
Eur J Biochem 247
: 843
–851, 1997[Medline]
Zhao Y, Yu M, Chen M, Elder RT, Yamamoto A, Cao J: Pleiotropic effects of HIV-1 protein R (Vpr) on morphogenesis and cell survival in fission yeast and antagonism by pentoxifylline.
Virology 246
: 266
–276, 1998[CrossRef][Medline]
Chang F, Re F, Sebastian S, Sazer S, Luban J: HIV-1 Vpr induces defects in mitosis, cytokinesis, nuclear structure, and centrosomes.
Mol Biol Cell 15
: 1793
–1801, 2004[Abstract/Free Full Text]
Olszewski A, Sato K, Aron ZD, Cohen F, Harris A, McDougall BR, Robinson WE Jr, Overman LE, Weiss GA: Guanidine alkaloid analogs as inhibitors of HIV-1 Nef interactions with p53, actin, and p56lck.
Proc Natl Acad Sci U S A 101
: 14079
–14084, 2004[Abstract/Free Full Text]
This article has been cited by other articles:
S. N. Korgaonkar, X. Feng, M. D. Ross, T.-c. Lu, V. D'Agati, R. Iyengar, P. E. Klotman, and J. C. He HIV-1 Upregulates VEGF in Podocytes
J. Am. Soc. Nephrol.,
May 1, 2008;
19(5):
877 - 883.
[Abstract][Full Text][PDF]
T.-c. Lu, J. C. He, Z.-h. Wang, X. Feng, T. Fukumi-Tominaga, N. Chen, J. Xu, R. Iyengar, and P. E. Klotman HIV-1 Nef Disrupts the Podocyte Actin Cytoskeleton by Interacting with Diaphanous Interacting Protein
J. Biol. Chem.,
March 28, 2008;
283(13):
8173 - 8182.
[Abstract][Full Text][PDF]
H. Ideura, K. Hiromura, N. Hiramatsu, T. Shigehara, S. Takeuchi, M. Tomioka, T. Sakairi, S. Yamashita, A. Maeshima, Y. Kaneko, et al. Angiotensin II provokes podocyte injury in murine model of HIV-associated nephropathy
Am J Physiol Renal Physiol,
October 1, 2007;
293(4):
F1214 - F1221.
[Abstract][Full Text][PDF]
L. A. Bruggeman Viral Subversion Mechanisms in Chronic Kidney Disease Pathogenesis
Clin. J. Am. Soc. Nephrol.,
July 1, 2007;
2(Supplement_1):
S13 - S19.
[Abstract][Full Text][PDF]
K. Kiryluk, J. Martino, and A. G. Gharavi Genetic Susceptibility, HIV Infection, and the Kidney
Clin. J. Am. Soc. Nephrol.,
July 1, 2007;
2(Supplement_1):
S25 - S35.
[Abstract][Full Text][PDF]
,
Top
Abstract
Introduction
25, 25 to 
