JASN Express. Published on September 20, 2006 as doi: 10.1681/ASN.2005080878

HIV-1 and nef Synergistically Damage Podocytes, Leading to Glomerulosclerosis

Yiqin Zuo,* Taiji Matsusaka,*†§ Jianyong Zhong,** Ji Ma,* ** Li-jun Ma,‡ Zaher Hanna,†† ʈ Paul Jolicoeur,†† Agnes B. Fogo,*†‡ and Iekuni Ichikawa*† *Departments of Pediatrics, †Medicine, and ‡Pathology, Vanderbilt University Medical Center, Nashville, Tennessee; ʈ 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

This study aimed to identify the causative for HIV-1 associated nephropathy, a paradigmatic podocytopathy. A previous study demonstrated that transgenic expression of nonstructural HIV-1 genes selectively in podocytes in mice with FVB/N genetic background resulted in podocyte injury and glomerulosclerosis. In this study, transgenic mice that expressed individual HIV-1 genes in podocytes were generated. Five of six transgenic mice that expressed vpr developed podocyte damage and glomerulosclerosis. Analysis of an established vpr transgenic line revealed that transgenic mice on FVB/N but not on C57BL/6 genetic background developed podocyte injury by 8 wk of age, with later glomerulosclerosis. Four of 11 transgenic mice that expressed nef also developed podocyte injury. One transgenic line was established from the nef founder mouse with the mildest phenotype. Transgenic mice in this line developed mesangial expansion at 3 wk of age and mild focal podocyte damage at 10 wk of age. Mating with FVB/N mice did not augment nephropathy. None of the transgenic mice that expressed vif, , ,orvpu in podocytes, even with the FVB/N genetic background, developed podocyte injury. For testing effects of simultaneous expression of vpr and nef, these two lines were mated. All nef:vpr double-transgenic mice showed severe podocyte injury and glomerulosclerosis by 4 wk of age. In contrast, all vpr or nef single-transgenic mice in the same litter uniformly showed no or much milder podocyte injury. These findings indicate that vpr and nef each can induce podocyte injury with a prominent synergistic interaction. J Am Soc Nephrol 17: 2832–2843, 2006. doi: 10.1681/ASN.2005080878

ccumulating evidence indicates that podocyte dam- Tg26 and wild-type mice revealed that development of ne- age triggers progression of glomerular deterioration phropathy is due to intrarenal expression of HIV-1 mRNA that A that leads to glomerular sclerosis (1–10). However, is independent of circulating viral or dysregulated the nature of podocyte damage that causes irreversible progres- systemic cytokines (19). Transgenic rats that carry the same sion is poorly understood. HIV-1–associated nephropathy construct as Tg26 mice also develop nephropathy, confirming (HIVAN) is unique in that the etiology of podocyte injury is that gag and are not involved in HIVAN pathogenesis genetically well defined, namely the HIV-1 itself. Patients with (20,21). Moreover, the HIV-1 transgenic mouse line that lacks HIVAN present with heavy proteinuria and rapid progression gag, pol, and nef develops FSGS, and genetic complementation to end-stage renal failure with collapsing FSGS and microcystic with nef results in more severe glomerulosclerosis but less tubular dilation (11–17). HIV-1 encodes three structural genes severe tubulointerstitial injury (22). Conversely, the transgenic (gag, pol, and ), two essential regulatory genes (tat and rev), lines CD4C/HIV and CD4C/simian immunodeficiency virus and four accessory genes (vif, vpr, vpu, and nef). Studies on that express HIV nef or simian immunodeficiency virus nef, HIV-1 transgenic animal models have provided important in- respectively, develop tubulointerstitial injury and segmental sights into the pathogenesis of HIVAN. A transgenic line, Tg26, glomerulosclerosis (23–26). These findings suggest that al- that carries HIV-1 DNA that lacks gag and pol genes has been though nef may not be pathogenic in all settings, it may mod- studied extensively and is characterized by nephropathy that is ulate nephropathy. Furthermore, transgenic mice that carry similar to human HIVAN (18). The transgene of Tg26 is driven HIV-1 tat and vpr, but not mice that carry by the endogenous HIV-1 promoter, which is expressed in a HIV-1 genome defective in vpr develop glomerulosclerosis (27). wide range of cells. Reciprocal renal transplantation between Transgenic mice that carry macrophage-specific c-fms promoter and vpr also develop nephropathy (27). Collectively, these ob- Received August 24, 2005. Accepted August 1, 2006. servations indicate that vpr and nef are involved in the patho- genesis of HIVAN, although the specific cell type that is af- Published online ahead of print. Publication date available at www.jasn.org. fected directly by these gene products remains uncertain. Address correspondence to: Dr. Taiji Matsusaka, Departments of Pediatrics, Vanderbilt University Medical Center, MCN C4204, Nashville, TN 37232-3584. HIV-1 has been identified within renal cells, Phone: 615-343-0110; Fax: 615-322-7929; E-mail: [email protected] including podocytes, in humans and transgenic mice (19,28–

Copyright © 2006 by the American Society of Nephrology ISSN: 1046-6673/1710-2832 J Am Soc Nephrol 17: 2832–2843, 2006 Nephropathic HIV-1 Genes Cause Podocyte Damage 2833

31). In both settings, the podocytes appear dysregulated with loss of differentiation markers, proliferate, and undergo apo- ptosis (28,32–34). To evaluate the specific role of podocyte injury in HIVAN, we recently generated transgenic mice that carry a podocyte-specific nephrin gene (Nphs1) promoter com- bined with HIV-1 DNA that lacks gag, pol, and env (35). The mice on an FVB/N genetic background, termed HIV13FVB, developed proteinuria, podocyte injury, collapsing FSGS, and microcystic tubular dilation, indicating that selective expres- sion of the HIV-1 genes in podocytes alone 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 specific gene causes podocyte damage that leads to HIVAN. Using the nephrin promoter, we generated six series of transgenic mice, each expressing one of the above six genes in podocytes. The results demonstrate that selective podocyte expression of either vpr or nef alone can induce podocyte injury that leads to glo- merulosclerosis and that strain background also modulates the phenotype. In addition, our study reveals a prominent syner- Figure 1. Structure of the transgenes. All HIV-1 DNA fragments gistic effect of vpr and nef genes on podocyte injury. 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. Materials and Methods Nphs1-HIV1 is the transgene that was used in our previous Transgene Constructs study to generate the HIV13 line, which carries Nphs1 pro- moter; vif, vpr, vpu, nef, rev, and tat; and a poly A signal from All DNA fragments that encoded HIV-1 genes were generated from SV40 DNA. env in Nphs1-HIV1 is mutated (ϫ). The six trans- the transgene constructs that were used in our previous studies and gene constructs used in this study are shown. Each is composed were derived from pNL4–3 genomic DNA (Genbank accession no. of Nphs1 promoter, an individual HIV-1 gene (vif, vpr, vpu, nef, AF324493; hereafter, nucleotide [nt] number is shown on the basis of rev,ortat), internal ribosome entry site (IRES), enhanced green these data) (23,35). DNA fragments that encoded nef (nt 8787 to 9407), fluorescence (EGFP) gene, and a poly A signal. Nphs1- vif (nt 5041 to 5619), or vpr (nt 5559 to 5849) were generated by PCR. For rev and -tat include the respective introns, with disruptions of rev, SalI-XhoI fragment (nt 5785 to 8887) of the mutant B of CD4C/HIV1 env and vpu (ϫ). transgene construct was subcloned in pBR322. Mutant B construct has an insertion of a stop codon at the NdeI site (nt 6399), which terminates the translation of ENV protein after the 60th amino acid, whereas the Generation of Transgenic Mice length of wild-type ENV is 854 amino acids. For disruption of tat and Nphs1-vpu transgene DNA was injected into fertilized eggs of the vpu, the SalI-Acc65I (nt 5785 to 6343) portion was replaced with the FVB/N strain. DNA for Nphs1-rev, Nphs1-tat, Nphs1-vif, and Nphs1-vpr fragment that spanned from the initiation codon of rev (nt 5969) to the was injected into eggs of F2 hybrid between C57BL/6 and DBA/2. For Acc65I site (nt 6343), which was generated by PCR from the mutant C Nphs1-nef, both strains were used. The transgene integration was ex- of CD4C/HIV1 construct (23). This construct carries a deletion of a amined by PCR, using primers EGFP1 and EGFP2 (Table 1). For the fragment of nt 6063 to 6100, thus lacking the starting codon of vpu. The nef:vpr double-transgenic mouse study, transgenes nef and vpr were resultant SalI-HpaI (nt 8648) fragment contained the exons and the detected by PCR using the primer pairs nef1 and nef2, and vpr1 and intron of rev (nt 5969 to 8643) with disrupted tat, vpu, and env, thus vpr2, respectively (Table 1). encoding only rev.Atat fragment was generated from the above SalI-XhoI fragment of mutant B. For disruption of vpu, the SacI-Acc65I Detection of Transgene Expression portion (nt 5999 to 6343) was replaced with the corresponding fragment Total RNA was extracted from whole kidney and treated with DNase of the mutant C of CD4C/HIV1 construct. T at nt 5970 was mutated to I. RNA was reverse-transcribed using random primers, and HIV-1 C to disrupt the initiation codon of rev. The resultant SalI-BamHI genes Egfp and Gapdh (glyceraldehyde-3-phosphate dehydrogenase) fragment (nt 5785 to 8465) contains the exons and the intron of tat (nt were amplified by PCR using the primers listed in Table 1. In addition, 5830 to 8414) with mutations in rev, vpu, and env, thus encoding only a relative amount of nef mRNA to Gapdh mRNA was quantified by tat. For vpu, the HindIII-Acc65I fragment (nt 6026 to 6343) was used. real-time reverse transcription–PCR (RT-PCR), using the iCycler and Three nucleotides that preceded the initiation codon of rev, vif, nef, and SYBR-Green I assay (Bio-Rad Laboratories, Hercules, CA). The primers vpr were converted to ACC to match Kozak’s consensus sequence. Each for this analysis are shown in Table 1. of the six DNA fragments was combined with a 5.4-kb promoter EGFP fluorescence signal was not detectable in any of the transgenic fragment of the mouse nephrin gene (Nphs1), internal ribosome entry mice. We performed immunostaining for EGFP using mouse anti-GFP site, and enhanced green fluorescence protein (EGFP; Figure 1). The mAb (1:100; Chemicon, Temecula, CA) on paraffin tissues that were Nphs1 promoter contains the first and part of a second exon (up to XhoI heated by microwave. site) and the first intron. Two ATG sequences in the Nphs1 coding region were disrupted by site-directed . This promoter In Situ Hybridization segment directed podocyte-specific expression in our previous studies PCR product with nef1 and nef2 was subcloned into pGEM-T Easy (10,35). vector (Promega, Madison, WI). Plasmid DNA was linearized with 2834 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 2832–2843, 2006

Table 1. PCR primers useda

Target Gene Primer Name Primer Sequence (5Ј-3Ј)

Egfp EGFP1 GAAGCAGCACGACTTCTTCAAGTC EGFP2 TGGCGGATCTTAAGTTCACCTTG nef nef1 CAGTATCTCGAGACCTAGAA nef2 TAGCTTGTAGCACCATCCAA vpr vpr1 TGAAACTTACGGGGATACTTGG vpr2 GTCGAGTAACGCCTATTCTGC rev rev1 CGACGAAGAGCTCATCAGAA rev2 CGTCCCAGAAGTTCCACAAT tat tat1 TGGAGCCAGTAGATCCTAGACT tat2 CTATTCCTTCGGGCCTGTC vif vif1 GATGATTGTGTGGCAAGTAG vif2 GCTTGATATTCACACCTAGG vpu vpu1 AAAGAGCAGAAGACAGTGGCAAT vpu2 CATTTCCACCCCCATCTCCACA Gapdh GAPDH3 GAAGCAGCACGACTTCTTCAAGTC GAPDH4 TGGCGGATCTTGAAGTTCACCTTG nef nefFb GGTGGGTTTTCCAGTCACAC nefRb GGGAGTGAATTAGCCCTTCC Gapdh GAPDH1b TGGACCACCAACTGCTTAG GAPDH2b GGATGCAGGGATGATGTTC

aEgfp, the gene for enhanced green fluorescent protein (EGFP); Gapdh, the gene for glyceraldehyde-3-phosphate dehydrogenase, was used as an internal reference gene. bFor real-time reverse transcription–PCR (RT-PCR).

SpeI, and 35S-labeled sense and antisense RNA probes were synthe- washed, dehydrated, dried, dipped in photographic emulsion, and sized using T7 RNA polymerase. In situ hybridization was performed exposed at 4°C for 10 d. The sections were developed with D-19 as described previously (36). Briefly, hydrated paraffin sections were developer (Kodak, Rochester, NY) and counterstained with toluidine treated with proteinase K (10 ␮g/ml) for 8 min at 37°C, fixed in 4% blue O. buffered paraformaldehyde for 5 min, incubated in 0.1 M triethanol- amine/0.25% acetic anhydride, washed in PBS, dehydrated in serial Analysis of Transgenic Mice graded ethanol, and air-dried. The sections then were hybridized over- The Institutional Animal Care and Use Committee at Vanderbilt night at 50°C in buffer (40% formamide, 10% dextran sulfate, 8 mM University Medical School and the Animal Experimentation Committee dithiothreitol, 0.2 mg/ml tRNA, 300 mM NaCl, 10 mM Tris-HCl, 5 mM at Tokai University Medical School approved the protocol, in accor- EDTA, 0.02% polyvinylpyrrolidone, 0.02% Ficoll, and 0.02% BSA) that dance with the principles and procedures outlined in the National contained 35S-labeled probe (2 ϫ 104 cpm/␮l). Sections then were Institutes of Health Guide for the Care and Use of Laboratory Animals.

Table 2. Summary of transgenic founder mice that carried individual HIV-1 genes

No. of Founder Mice No. of Founder No. of with Transgene Mice with EGFP No. of Founder No. of Lines No. and Generation HIV-1 Founder Mice mRNA Detected by Detected by Mice with Backcrossed of FVB/N-Backcrossed Gene Analyzed RT-PCR in the Immunostaining Podocyte Injury with FVB/N Offspring Analyzed Kidney in Podocytes

vpr 6 5 1 5 2 55 (36 in N1, 19 in N2) nef 30a 11 0 4 1c 15 (3 in N1, 4 in N2, 8 in N3) rev 33b 1 0 3 15(7inN1,2inN2,6inN3) tat 33b 1 0 3 16 (10 in N1, 3 in N2, 3 in N3) vif 12 5 2 0 2 13 (10 in N1, 3 in N2) vpu 15 7 2 0 0d

aOne transgenic mouse, Nphs1-nef144, developed proteinuria and died at 7 wk of age. Postmortem histologic analysis showed FSGS. This mouse is not included in the total numbers. bOnly spliced mRNA was detected. cTwenty founder mice were generated on inbred FVB/N strain. dAll founder mice were generated on inbred FVB/N strain. J Am Soc Nephrol 17: 2832–2843, 2006 Nephropathic HIV-1 Genes Cause Podocyte Damage 2835

All transgenic mice that were analyzed in the study were heterozy- gous for each transgene. Transgenic mice that expressed tat, rev,orvif were backcrossed with FVB/N two or three times. The number, age, and generation of mice, respectively, were seven (at 4 mo of age in N1 generation), two (2, N2), and six (5, N3) for rev; 10 (4, N1), three (6, N2), and three (6, N3) for tat; and 10 (4, N1) and three (5, in N2) for vif. Seven founder mice that expressed vpu, which were generated in an inbred FVB/N strain, were analyzed at 5 mo of age. The Nphs1-vpr19 line was backcrossed with the C57BL/6 strain for seven generations. In N7 generation, two, six, and three transgenic 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 (at 1 wk of age), two (at 2 wk), Figure 2. Proteinuria in Nphs1-vpr and Nphs1-nef transgenic one (at 4 wk), six (at 6 wk), seven (at 8 wk), three (at 10 wk), four (at 12 mice. SDS-PAGE analysis of urinary protein. Urine samples (2 ␮ wk), and three (at 14 wk). Some were mated further with FVB/N mice, l) from Nphs1-vpr and Nphs1-nef transgenic mice and wild- generating 17 transgenic mice. Four, seven, five, and one mice were type mice were analyzed at 8 to 10 wk of age. Samples loaded analyzed at 2, 3, 4, and 8 wk of age, respectively. are molecular weight markers (lane 1), Nphs1-nef10 (lane 2), The Nphs1-nef10 founder mouse was mated with FVB/N, generating Nphs1-nef18 (lane 3), Nphs1-nef27 (lane 4), Nphs1-nef149 (lane 5), one transgenic offspring (Nphs1-nef10–40). Starting with this mouse, wild type (lane 6), Nphs1-vpr10 (lane 7), Nphs1-vpr16 (lane 8), the line was backcrossed with C57BL/6 mice four times. Six transgenic Nphs1-vpr19 (lane 9), and Nphs1-vpr21 (lane 10). Proteinuria mice of N4 generation were analyzed at 2 mo of age. One mouse of N1 was detected in Nphs1-vpr and Nphs1-nef transgenic mice but generation was mated with FVB/N three times. Eight transgenic mice not in Nphs1-nef10 or wild-type mice. of the third generation were analyzed at 2 mo of age. Twenty-four-hour urine was collected using metabolic cages, and 2 expressed vif, vpr, vpu, tat, rev,ornef in podocytes under the ␮l of urine samples was analyzed by SDS-PAGE usinga4to20% gradient gel (Bio-Rad). Increases in albumin and larger proteins were regulation of the Nphs1 promoter. We also aimed to establish considered as proteinuria, a marker of nephropathy. Plasma was col- transgenic lines that show nephropathy. Previous studies by lected when the mice were killed, and the blood urea nitrogen and others and us showed a strong predisposition of nephropathy creatinine were measured in a commercial laboratory. Histologic anal- ysis, immunostaining, electron microscopic analysis, and quantification of glomerular damage 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 generation of C57BL/6 backcrossing. A total of 126 mice were analyzed at 1, 7, 14, 21, and 28 d of age. Glomerular epithelial and mesangial injury indices were determined as previously reported (10). For mesangial changes, score 0 represents no lesion, whereas 1, 2, 3, and 4 represent mesangial matrix expansion, hyalinosis, or sclerosis involving Յ25, 25 to Յ50, 50 to Յ75, and Ͼ75% of the glomerular tuft area, respectively. The epi- thelial cell injury was graded as follows: 0, no lesion; 1, 2, 3, and 4, vacuolization, bleb, or proliferation of epithelial cells involving Յ25, 25 to Յ50, 50 to Յ75, and Ͼ75% of the glomerulus, respectively. Six and 13 wild-type mice, four and nine vpr mice, eight and five nef mice, and four and four nef:vpr double-transgenic mice were analyzed at 3 and 4 wk of age, respectively. For all types of mice that were subjected to the study and the parental mice, D3Mit203 genotype was determined by PCR on the basis of the information of the Mouse Genome Informatics database (MGI ID: 706352).

Statistical Analyses Figure 3. Transgene expression. (A and B) Transgene expression Results were expressed as mean Ϯ SE. Data were analyzed by one- in the transgenic line Nphs1-vpr19. The reporter gene product, way ANOVA followed by least significant difference test or Tamhane’s EGFP, was detected by immunostaining within the glomerulus correction test as appropriate. Differences were considered to be statis- (A). Staining for synaptopodin on the adjacent section (B) indi- tically significant at P Ͻ 0.05. cates that the transgene is expressed on podocytes. (C and D) Transgene expression in the transgenic line Nphs1-nef10. HIV-1 Results nef mRNA (C) was detected within the glomerulus. On the Variable Effect of Individual HIV-1 Genes on Development periphery of the tuft, the cell type that expressed nef mRNA of Nephropathy (arrows) was identified as podocytes from the anatomic loca- To identify any HIV-1 gene(s) that causes podocyte damage, tion. Sense control probe showed only background signal (D). we generated six series of transgenic mice that individually Magnification, ϫ400. 2836 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 2832–2843, 2006

Table 3. Summary of transgenic founder mice that expressed vpra

Mouse Sex Renal Histology Age at Analysis Genetic Background EGFP in Podocytes

Nphs1-vpr8 F FSGS 6 mo C57BL/6ϩDBA/2 (Ϫ) Nphs1-vpr10 F FSGS 3 mo C57BL/6ϩDBA/2 (Ϫ) Nphs1-vpr16 F FSGS 3 mo C57BL/6ϩDBA/2 ND Nphs1-vpr19 F FSGS 4 mo C57BL/6ϩDBA/2 (ϩ) Nphs1-vpr21 F FSGS 4 mo C57BL/6ϩDBA/2 (Ϫ)

aND, not done. of the FVB/N genetic background (35,37); therefore, establish- transgenic mice, transgene mRNA was detectable by RT-PCR in ment of a nephropathic transgenic line on the FVB/N back- the kidneys of 11 mice (Table 4). Three of them, Nphs1-nef27, ground was thought to be difficult, because mice would die of 149, and 162, had proteinuria (Figure 2) and advanced glomer- severe disease before producing offspring. We therefore gener- ulosclerosis with severe podocyte damage (see below). Nphs1- ated transgenic mice on a mixed genetic background of DBA/2 nef10 developed mild focal mesangial sclerosis with slight and C57BL/6, both of which are less susceptible to nephropa- podocyte damage at 5 mo of age. The remaining seven mice thy than FVB/N. We then backcrossed them with an FVB/N that expressed nef mRNA did not develop podocyte damage strain. We generated vpu transgenic mice in inbred FVB/N, (Table 4). The amount of nef mRNA was quantified by real-time because vpu mRNA was not detected in HIV13 transgenic mice RT-PCR. Nphs1-nef10 had higher expression of nef mRNA than and was unlikely to cause nephropathy. Because our previous other non-nephropathic Nphs1-nef mice (Table 4). Of note, studies found that penetrance of transgene expression by this Nphs1-nef149, 162, and 27, which showed severe FSGS, ex- promoter is not 100% (10,35), we examined the renal transgene pressed a lower level of nef mRNA than Nphs1-nef10. expression by RT-PCR for each HIV-1 gene and/or Egfp and In situ hybridization performed on the kidneys of the Nphs1- immunostaining for EGFP. The results are summarized in Ta- nef10 line detected nef mRNA on podocytes (Figure 3C). No ble 2. specific signal was detected with a sense probe (Figure 3D). Five of six founder Nphs1-vpr mice developed nonselective None of the transgenic mice that carried rev, tat, vif,orvpu proteinuria by 8 wk of age (Figure 2). Histologic analysis re- developed proteinuria. Nphs1-rev, -tat, and -vif mice were gen- vealed podocyte damage and FSGS (Table 3, see below). These erated on a mixed genetic background of C57BL/6 and DBA/2. mice expressed vpr mRNA in the kidney. In one mouse, Nphs1- Because development of nephropathy in HIV13 transgenic mice vpr19, EGFP was detectable by immunostaining that localized depends on the FVB/N genetic background (35), the lack of to podocytes (Figure 3, A and B). One Nphs1-vpr mouse did not nephropathy in these mice may reflect a lack of impact of the develop nephropathy, and the transgene was not detectable in FVB/N genetic background. To rule out this possibility, we the kidney of this mouse. chose founder transgenic mice that expressed the transgene in We obtained 10 Nphs1-nef founder transgenic mice on a kidneys and backcrossed them with wild-type FVB/N mice for mixed genetic background of C57BL/6 and DBA/2. After es- two (for Nphs1-vif) or three (for Nphs1-rev and Nphs1-tat) gen- tablishing a transgenic line (Nphs1-nef10 line), we additionally erations (Table 2). None of the transgenic offspring in N1 to N3 generated 20 Nphs1-nef founder transgenic mice on an inbred generations showed nephropathy (Figure 4, I through K). FVB/N genetic background. Of the total 30 Nphs1-nef founder Nphs1-vpu mice that were generated on the FVB/N genetic

Table 4. Summary of transgenic founder mice that expressed nef

Age at Genetic Amount of nef mRNA Mouse Sex Renal Histology Analysis Background (arbitrary unit)

Nphs1-nef149 M FSGS 6 mo FVB/N 64 Nphs1-nef162 M FSGS 5 wk FVB/N 416 Nphs1-nef10 M Mesangial sclerosis 6 mo C57BL/6 ϩ DBA/2 1352 Nphs1-nef15 M Normal 6 mo C57BL/6 ϩ DBA/2 588 Nphs1-nef27 F FSGS 2.5 mo C57BL/6 ϩ DBA/2 28 Nphs1-nef120 F normal 6 mo FVB/N 26 Nphs1-nef122 F normal 6 mo FVB/N 2 Nphs1-nef124 M normal 6 mo FVB/N 28 Nphs1-nef134 F normal 6 mo FVB/N Not measured Nphs1-nef151 M normal 6 mo FVB/N 10 Nphs1-nef164 M normal 6 mo FVB/N 21 J Am Soc Nephrol 17: 2832–2843, 2006 Nephropathic HIV-1 Genes Cause Podocyte Damage 2837

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 degen- eration 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: ϫ200 in A, C, E, G, and I through L; ϫ400 in B, D, F, and H (period acid-Schiff staining). background yielded seven founder mice that expressed the and nonselective proteinuria, severe podocyte injury, and seg- transgene. However, none of these mice developed nephropa- mental or global glomerulosclerosis. thy (Figure 4L). Taken together, these results indicate that vpr We previously observed that HIV13FVB mice showed imma- and nef cause podocyte injury. By contrast, vif, tat, rev, and vpu ture glomeruli at 2 wk of age (35). The pattern of glomerular are not nephropathic genes. maturation was normal in the Nphs1-vpr19 mice that were bred on FVB/N once or twice. Podocyte-Specific vpr-Transgene Nephropathy The Nphs1-vpr19 line was backcrossed with a C57BL/6 strain. Founder mice, Nphs1-vpr8, 10, 16, 19, and 21, showed mod- Eleven transgenic mice in N7 generation were analyzed at 2 to erate to severe segmental or global, noncollapsing sclerosis 7 mo of age. Most mice showed essentially normal renal mor- with tubulointerstitial injury (Figure 4). In these mice, nephrin phology, with the exception of one mouse, which showed mild and synaptopodin immunostaining was markedly suppressed. focal segmental sclerosis in 20% of glomeruli at 7 mo of age. By contrast, desmin was upregulated, indicating that the podo- The Nphs1-vpr8 founder mouse also produced a small num- cytes were damaged (Figure 5). In Nphs1-vpr10 and 19, podo- ber of transgenic offspring when mated with FVB/N. They cytes and parietal epithelial cells showed prominent vacuolar showed proteinuria and FSGS. degeneration (Figure 4D). In these mice, tubulointerstitial dam- age was severe, and the tubules were microcystically dilated Podocyte-Specific nef-Transgene Nephropathy (Figure 4C). Nphs1-nef27, 149, and 162 mice showed advanced noncollaps- Nphs1-vpr19 founder mouse produced transgenic offspring ing glomerulosclerosis with tubulointerstitial injury (Figure 4, E via mating with C57BL/6 mice. These were mated further with and F). Synaptopodin and nephrin were markedly reduced, FVB/N mice. We longitudinally analyzed 32 transgenic mice whereas desmin was upregulated within the podocytes (Figure that were obtained through this mating. Before 6 wk of age, 5, G through I). In addition, Nphs1-nef149 and 162 showed most transgenic mice had no proteinuria, and the renal histol- remarkable vacuolar degeneration in podocytes (Figure 4F). ogy was normal. At 8 wk of age, three of 10 transgenic mice These three founder mice did not produce offspring. developed mild proteinuria and had podocyte damage. After 8 Nphs1-nef10 showed focal proteinaceous casts and mild focal wk of age, all transgenic mice (n ϭ 9) had developed massive mesangial sclerosis at 5 mo of age (Figure 4, G and H). At 6 mo 2838 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 2832–2843, 2006

sis, three showed mild mesangial expansion, and the other one was normal. Of the eight transgenic mice with the FVB/N genetic background studied, only one showed mild mesangial expansion, and the other seven were normal. Therefore, mating with the FVB/N genetic background did not augment ne- phropathy in the Nphs1-nef10 line.

Synergy between vpr and nef in Causing Podocyte Damage and Nephropathy Our previous HIV-1 transgenic mice (HIV13FVB) had simul- taneous expression of both vpr and nef. To test whether vpr and nef have synergistic effects on podocyte injury, we mated Nphs1-vpr19 and Nphs1-nef10 lines and generated double-trans- genic 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 developed proteinuria by 1 wk of age, which progressed to massive and nonselective proteinuria by 3 wk of age. At 4 wk of age, nef:vpr mice had renal failure with markedly elevated blood urea nitrogen (260.7 Ϯ 33.5 mg/dl; n ϭ 3) compared with wild-type mice (24.5 Ϯ 9.3 mg/dl; n ϭ 4), vpr single-transgenic mice (26.0 Ϯ 7.2 mg/dl; n ϭ 6), and nef single-transgenic mice (24.3 Ϯ 0.58 mg/dl; n ϭ 3). Some nef:vpr mice had ascites. Histologically, none of the vpr or nef transgenic mice had abnormalities by 2 wk of age. Moreover, electron micro- scopic analysis at 2 wk of age detected no abnormalities in the single-transgenic mice. Eleven of 13 vpr mice and six of 13 nef mice did not have any abnormality by 4 wk of age. The Figure 5. Damage of podocytes in Nphs1-vpr and Nphs1-nef rest of the single-transgenic mice had only mild to moderate transgenic mice. Wild-type (A through C), Nphs1-vpr19 (D mesangial matrix expansion without remarkable podocyte through F), Nphs1-nef149 (G through I), and Nphs1-nef10 (J vacuolization. In contrast, by 1 wk of age, two of four nef:vpr through L) mice at 4 to 6 mo of age. There was prominent mice had remarkable mesangiolysis, dilated capillaries, mi- degeneration of podocytes with markedly reduced podocyte croaneurysms, and occasionally mesangial expansion and markers, synaptopodin, and nephrin in Nphs1-vpr19 (D and E) hypercellularity. In addition, one nef:vpr mouse had promi- and Nphs1-nef149 (G and H). In wild-type mice, desmin was nent hyperplastic lesions in the afferent arterioles and inter- faintly detectable only in mesangial cells (C), whereas in Nphs1- lobular artery. At 2 wk of age, all nef:vpr mice showed vpr19 and Nphs1-nef149, desmin was expressed in podocytes (F mesangial matrix expansion with an increase in type IV and I). In Nphs1-nef10, synaptopodin staining was preserved, collagen (data not shown). Synaptopodin and nephrin were but nephrin was slightly decreased. Desmin was stained in markedly downregulated, indicating that podocytes were expanded mesangial areas. Some podocytes also expressed desmin (arrows). A to C, D to F, H to I, and J to L are adjacent severely damaged. By electron microscopy, there was prom- sections, respectively. Magnification, ϫ400 (synaptopodin in A, inent podocyte degeneration with completely effaced foot D, G, and J; nephrin in B, E, H, and K; and desmin staining in processes and microvillous transformation (Figure 6). After 3 C, F, I, and L). wk of age, all nef:vpr mice developed severe glomeruloscle- rosis and tubulointerstitial injury with almost complete loss of synaptopodin staining (Figure 7). Quantitative analysis of age, podocytes showed focal injury with occasional protein for glomerular epithelial and mesangial injury at 3 and 4 wk reabsorption droplets and detachment from the glomerular of age is shown in Figure 8. basement membrane. Synaptopodin and nephrin were normal Because the nef mouse that was used for generating the or slightly downregulated. Desmin was slightly increased in double-transgenic mice had the FVB/N genetic background, podocytes of glomeruli with mesangial sclerosis (Figure 5, J the possibility existed that the severe nephropathy that was through L). observed in nef:vpr double-transgenic mice was attributed to Nphs1-nef10 founder mouse generated transgenic offspring. FVB/N genetic traits. We therefore determined the type of Transgenic mice that were obtained by backcrossing with D3Mit203 microsatellite, which is closely associated with the C57BL/6 strain for four generations and those with FVB/N major HIV-1 nephropathy susceptibility (37). Both Nphs1- strain for three generations were compared at 2 mo of age. Of nef10 and Nphs1-vpr19 founder mice were homozygous the six transgenic mice with C57BL/6 genetic background stud- C57BL/6 type (B6/B6), and the nef mouse that was used for the ied, two mice showed mild focal segmental glomerular sclero- mating had the B6/FVB type. The vpr mice that were used for J Am Soc Nephrol 17: 2832–2843, 2006 Nephropathic HIV-1 Genes Cause Podocyte Damage 2839

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 ϭ 2 ␮m.

the mating had B6/B6 type. Of the 21 nef:vpr double-transgenic vpu developed podocyte injury. FVB/N genetic background is mice analyzed, eight had the B6/B6 type and 13 had the B6/ a prerequisite for the development of nephropathy in the FVB type. We found no difference in the degree of renal injury HIV13 transgenic line (35) as well as in the Tg26 line (37). between B6/B6 and B6/FVB types at any age. This indicates Nphs1-vif, -tat, and -rev were generated in a mixed genetic that the synergistic effect of nef and vpr had a greater impact on background of DBA/2 and C57BL/6. We therefore backcrossed podocyte injury than the major genetic susceptibility factor. with the FVB/N strain for two to three generations. None of the Nphs1-vif, -tat, and -rev mice with the FVB/N genetic back- Rare Proliferation of Podocytes ground showed nephropathy. We could not detect VIF, TAT, It was reported that podocytes proliferate in human HIVAN REV, or with available antibodies. Therefore, we and Tg26 mice. However, none of the transgenic mice that were explored the possibility that these proteins were not generated. generated in this study, including Nphs1-vpr, Nphs1-nef, and We think that this is unlikely, because (1) mRNA and protein of nef:vpr mice, showed an appreciable degree of proliferation of the reporter gene EGFP were detected, (2) mRNA for each visceral epithelial cells. To detect proliferation of injured podo- HIV-1 gene was detected, (3) these antibodies failed to detect cytes, we performed double immunostaining for Ki-67 and transgene products also in the HIV13 mice, (4) the same Nphs1 desmin, a marker of injured podocytes. To distinguish injured promoter segment successfully generated functional proteins in podocytes from mesangial cells, which also were stained for podocytes in our previous studies (10,35,38), and (5) sequenc- desmin, the localization of doubly positive cells was deter- ing of the transgene cDNA found no accidental mutation or mined in periodic acid-Schiff–stained adjacent sections. Exten- unexpected splicing. These findings collectively indicate that sive study in more than 30 transgenic mice with advanced (1) vpr is a major pathogenic gene causing podocyte damage; (2) nephropathy found only one doubly positive cell in an ana- nef alone also can cause podocyte damage; and (3) vif, tat, rev,or tomic location of podocytes in one transgenic mouse of Nphs1- vpu alone does not cause podocyte damage, although possibil- vpr19 line with the FVB/N genetic background at 3 mo of age ities remain that these gene products may cause or modify (Figure 9). These indicated that podocyte proliferation is a very nephropathy in other genetic backgrounds. rare phenomenon in these transgenic mice. Notably, although nef mRNA was detected by RT-PCR in the Discussion kidney of 11 Nphs1-nef mice, only four developed nephropathy. Identification of Specific HIV-1 Genes That Cause Podocyte Nphs1-nef mice without nephropathy expressed lower amounts Damage of nef mRNA than did Nphs1-nef10, suggesting that a large In this study, we generated six series of transgenic mice, each amount of NEF is necessary to cause podocyte damage. However, individually expressing one of the candidate genes of HIV-1, Nphs1-nef mice with severe FSGS also expressed lower amounts of tat, rev, vif, vpu, vpr,ornef, selectively in podocytes. All founder nef mRNA. In these mice, podocytes were severely injured, so the transgenic mice that expressed vpr and four of 11 founder expression of nef RNA driven by Nphs1 promoter may be down- transgenic mice that expressed nef developed podocyte dam- regulated. Nevertheless, the possibility still remains that other age. Conversely, none of the mice that expressed vif, tat, rev,or genetic factors are required for the nephropathy to develop. 2840 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 2832–2843, 2006

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,ornef single-transgenic mice (P Ͻ 0.05).

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 synaptopo- din staining at this age (F). nef:vpr double-transgenic mouse showed global sclerosis with vacuolization in visceral and pa- rietal epithelial cells (G), with dramatically reduced synaptopo- Figure 9. Rare podocyte proliferation. One cell (arrow) was din staining (H). A to B, C to D, E to F, and G to H are adjacent doubly stained for synaptopodin (purple in A, brown in B) and sections, respectively. Magnification, ϫ400 (periodic acid-Schiff Ki-67 (brown in A) in one transgenic mouse of the Nphs1-vpr19 [PAS] in A, C, E, and G; and synaptopodin staining in B, D, F, line with FVB/N genetic background at 3 mo of age. PAS and H). staining in the adjacent section (B) confirms that the cell local- ization also is consistent with a podocyte. Extensive search in Ͼ30 transgenic mice found only one doubly positive cell. It is interesting that nef and vpr had remarkable synergistic effects on podocyte injury. Thus, nef:vpr double-transgenic mice developed substantially more accelerated and severe podocyte podocytes through activation of Src-dependent Stat and mito- damage than any transgenic mouse from the Nphs1-nef10 and gen-activated protein kinase pathways (39–41). Recently, Hu- Nphs1-vpr19 lines. sain et al. (42) reported that transgenic mice that expressed nef in podocytes showed expression of Ki-67 in podocytes and Characterization of Nephropathy downregulation of synaptopodin. These observations suggest It has been reported that podocytes undergo proliferation that dysregulation of cell cycles or may be involved and apoptosis in human HIVAN as well as in Tg26 mice in HIVAN pathogenesis. However, we found essentially no (28,32–34). In vitro experiments showed that NEF induces an- Ki-67–positive podocytes in Nphs1-vpr, Nphs1-nef, and nef:vpr chorage-independent proliferation and de-differentiation of mice. Similarly, we did not observe apoptotic podocytes by J Am Soc Nephrol 17: 2832–2843, 2006 Nephropathic HIV-1 Genes Cause Podocyte Damage 2841 terminal deoxynucleotidyl transferase–mediated dUTP nick- studies find no definitive proliferation or apoptosis of podo- end labeling staining in our transgenic mice (data not shown). cytes. However, even before the onset of proteinuria, podocytes Taken together, these findings do not support a prominent role in HIV13FVB mice were devoid of extension of primary pro- for proliferation or apoptosis in the injury of podocytes that cesses and foot processes. It is therefore possible that distur- was demonstrated in our transgenic mice. bance of the in podocytes is involved in the de- Segmental capillary collapse is a morphologic hallmark of velopment of nephropathy. In this regard, both VPR and NEF HIVAN and has been observed in HIV13FVB mice. In our have been reported to be associated with cytoskeletal alter- study, Nphs1-vpr, Nphs1-nef, and nef:vpr transgenic mice did not ations and changes in cell morphology (46–50). Elucidation of have collapsing features, suggesting that other HIV-1 genes, molecular mechanisms may also lead to understanding of the including vpu, tat, rev, and vif alone or in combination, may pathogenesis of more common glomerular diseases that result have an impact on producing tuft collapse. Injury of podocytes in glomerulosclerosis. by immunotoxin did not cause collapsing glomerulopathy (10), indicating that podocyte damage alone cannot cause this phe- Conclusion notype. The mechanism underlying the collapse is an intrigu- Our study demonstrates that both vpr and nef play an impor- ing unsolved question. tant role in pathogenesis of podocyte damage and subsequent One nef:vpr double-transgenic mouse showed hyperplastic FSGS. In addition, vpr and nef have a significant synergistic lesions in the afferent arteriole. It is interesting that the arterial effect on podocyte injury and subsequent development of glo- lesions were similar to those observed in angiotensinogen or merulosclerosis. AT1 receptor knockout mice (43), suggesting that these two different mouse models share a common secondary change in the artery. However, a precise mechanism for development of Acknowledgments this lesion is not clear. This study was supported by National Institutes of Health grants DK37868 and DK447577. Influence of Genetic Background Part of this study was presented in abstract form at the 5th Interna- tional Podocyte Conference, June 18 through 20, 2004; Seattle, WA; and Genetic background has a strong influence on the develop- the annual meeting of the American Society of Nephrology; October 27 ment of HIVAN. Black individual are especially susceptible to through November 1, 2004; St. Louis, MO. HIVAN (11–17). Genetic background also influences experi- We thank Dr. Valentina Kon for editorial assistance and discussions mental nephropathy such that the FVB/N genetic background and Dr. Kato and Ms. Tsuchida for microsatellite analysis. determines renal damage in Tg26 and HIV13 mice (35,37). In Tg26, a major nephropathy susceptibility locus is present on mouse 3A1 to 3 (37). In our study, mating with References FVB/N mice augmented nephropathy in the Nphs1-vpr19 line 1. Rennke HG: How does glomerular epithelial cell injury but not in the Nphs1-nef10 line. 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