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Identification of the Nephropathy-Susceptibility Locus HIVAN4

Sindhuri Prakash,* Natalia Papeta,* Roel Sterken,* Zongyu Zheng,* Robert L. Thomas,‡ Zhenzhen Wu,‡ John R. Sedor,‡ Vivette D. DЈAgati,† Leslie A. Bruggeman,‡ and Ali G. Gharavi*

Department of *Medicine and †Pathology, Columbia University College of Physicians and Surgeons, New York, New York 10032, ‡Department of Medicine and the Rammelkamp Center for Education and Research, MetroHealth Medical Center, Case Western Reserve University School of Medicine, Cleveland, Ohio, 44109

ABSTRACT HIVAN1, HIVAN2, and HIVAN3 are nephropathy-susceptibility loci previously identified in the HIV-1 transgenic mouse, a model of collapsing glomerulopathy. The HIVAN1 and HIVAN2 loci modulate expression of Nphs2, which encodes podocin and several other podocyte-expressed . To identify additional loci predisposing to nephropathy, we performed a genome-wide scan in 165 backcross mice generated between the nephropathy-sensitive HIV-1-transgenic FVB/NJ (TgFVB) strain and the resistant Balb/cJ (BALB) strain. We identified a major susceptibility locus (HIVAN4) on 6 G3-F3, with BALB alleles conferring a twofold reduction in severity (peak LOD score ϭ 4.0). Similar to HIVAN1 and HIVAN2, HIVAN4 modulated expression of Nphs2, indicating a common pathway underlying these loci. We independently confirmed the HIVAN4 locus in a sister TgFVB colony that experienced a dramatic loss of nephropathy subsequent to a breeding bottleneck. In this low-penetrance line, 3% of the genome was admixed with BALB alleles, suggesting a remote contamination event. The admixture localized to discrete segments on chromosome 2 and at the HIVAN4 locus. HIVAN4 candidate genes include killer lectin-like receptor genes as well as A2m and Ptpro, whose products are enriched in the glomerulus and interact with HIV-1 . In summary, these data identify HIVAN4 as a major quantitative trait locus for nephropathy and a transregulator of Nphs2. Furthermore, similar selective breeding strategies may help identify further susceptibility loci.

J Am Soc Nephrol 22: 1497–1504, 2011. doi: 10.1681/ASN.2011020209

HIV-1 associated nephropathy (HIVAN) is an icans have identified a major susceptibility locus for AIDS-defining complication of HIV-1 infection. HIVAN, focal segmental glomerulosclerosis, and HIVAN is diagnosed based on kidney biopsy find- nondiabetic kidney failure on chromosome 22, within ings of collapsing focal segmental glomerulosclero- the APOL1 gene.3,4 It is hypothesized that the ne- sis with microcystic tubular dilation.1 In HIVAN, phropathy risk alleles propagated in West Africa be- the normally terminally differentiated glomerular podocytes display dedifferentiation, proliferation, and apoptosis; without antiretroviral therapy, this Received February 28, 2011. Accepted April 13, 2011. disorder frequently results in end-stage kidney fail- Published online ahead of print. Publication date available at ure.1 Patients with HIVAN are almost always of Af- www.jasn.org. rican ancestry and frequently have a strong family Correspondence: Ali G. Gharavi, Department of Medicine, Co- lumbia University College of Physicians and Surgeons, 1150 history of kidney failure, indicating an important Street Nicholas Avenue, Russ Berrie Pavilion Room 412, New role for genetic susceptibility in the pathogenesis of York, New York 10032. Phone: 212-851-5556; Fax: 212-305-5520; disease.2 Consistent with this epidemiology, recent Email: [email protected] genome-wide association studies in African Amer- Copyright © 2011 by the American Society of Nephrology

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cause the heterozygous genotype confers resistance to trypanoso- ceptibility loci, we generated an HIV1-transgenic backcross by miasis;4 it is not yet clear how homozygosity for these same breeding TgFVB with FVBxBALB F1 mice (52% male). Nearly APOL1 alleles produces susceptibility to histologically diverse ne- one-quarter of the 165 transgenic backcross mice were free of phropathies.5 nephropathy (total renal injury scores Յ3), while the remain- Heterozygous HIV-1 transgenic mice on the FVB/NJ ge- der had varying severity of kidney disease, indicating segrega- netic background (TgFVB) display virtually all of the clinical tion of nephropathy susceptibility alleles in this cross (Supple- and molecular features of HIVAN, enabling dissection of mo- mentary Figure 1a). Consistent with prior descriptions of the lecular pathways leading to disease.6–9 Studies in the murine TgFVB model and previous mapping studies, there were no model have indicated that HIVAN is caused directly by HIV-1 differences in severity of kidney disease based on gender.6,17,18 products, resulting in immune activation in the kidney and a A genome-wide scan and analysis of linkage with 548 infor- hyperplastic response in epithelial cells.6–9 These studies have mative markers identified a major locus on chromosome 6 identified a large number of dysregulated pathways F3-G3, with a peak lod score of 4.0 for the total renal injury (e.g., NF-kB,10,11 MAPK1,2,12,13 Stat312,13) and pathogenic score (peak at rs6329892, 143 Mb, Supplementary Figure 1b). mediators (bFGF,14 VEGF,15 TNF16) that link epithelial prolif- This locus, which we named HIVAN4, achieved genome-wide eration to glomerulosclerosis and are similarly activated in hu- significance based on accepted threshold (Ն3.3) and by 10,000 man disease (reviewed recently in references 7 through 9). The permutations of phenotype on genotype (genome-wide em- nature of the pathogenic process that encompasses these dys- piric p-value ϭ 0.004, Figure 1a). All of the subcomponents of regulated pathways, ultimately leading to proliferation and the histology score yielded peak lod scores of similar magni- scarring of the kidney, is not yet known. However, in conjunc- tude (lod range ϭ 3.7 to 4.9, Figure 1a). This interval was also tion with observations in humans, where very few HIV- significant for BUN (lod ϭ 3.0, empiric P ϭ 0.05) and sugges- infected individuals develop nephropathy, understanding the tive for serum albumin level (lod ϭ 2.4). The lod scores for unique host response to the virus likely holds the key. In this BUN and albumin diminished significantly (lod Ͻ0.5) after regard and similar to humans, genetic susceptibility has a pro- accounting for the histologic injury, indicating that these found effect on the development of nephropathy in the HIV-1 linkages do not represent a direct effect of HIVAN4 on BUN transgenic mouse model: the FVB/NJ strain is very susceptible, and albumin but are mediated by renal injury. Homozygos- but F1 hybrids with BALB/cJ, C57BL/6J and CAST/EiJ are pro- ity for the FVB allele at HIVAN4 conferred a twofold in- tected.17,18 Taking advantage of this strain dependence, we pre- crease in the histologic injury score compared with the het- viously generated mapping cohorts between TgFVB and two erozygote FVB/BALB genotype (Figure 1b). Finally, two counterstrains (C57BL/6J and CAST/EiJ strains), leading to intervals on chromosome 14 (peak lod 1.8 between identification of nephropathy quantitative trait loci (QTLs) on rs13459144 and rs6256423) and chromosome 15 (peak 3, 13, and 4 (HIVAN1, HIVAN2, and HIVAN3 lod ϭ 1.9 at rs13482585) demonstrated suggestive linkage loci, respectively).17,18 Further examination of these loci with for the histologic injury score (Supplementary Figure 1b). expression QTL analysis (expression profiling combined with linkage analysis) showed that the HIVAN1 and HIVAN2 loci HIVAN4 Transregulates Nphs2 Transcript Abundance regulate expression of Nphs2 (encoding Podocin) and several Previous studies have shown that HIVAN1 and HIVAN2 loci other podocyte-expressed genes.18 These data indicated the regulate transcript levels of Nphs2. To determine if HIVAN4 HIVAN1 and HIVAN2 susceptibility genes belong to a com- also transregulates Nphs2 expression, we measured Nphs2 mon regulatory pathway, and they likely introduce genetic le- transcripts abundance in 137 transgenic backcross mice with sions in an expression network involving multiple human ne- available kidney tissue. Nphs2 expression was inversely corre- phropathy genes. lated with renal histology score (r ϭϪ0.62, P ϭ 0.0001), and To identify additional loci predisposing to HIVAN and deter- genome-wide analysis of Nphs2 transcripts abundance re- mine whether Nphs2 transregulation is a common molecular fea- vealed suggestive linkage to the HIVAN4 locus (LOD score 2.1, ture of HIVAN loci, we conducted a new mapping study with the pointwise empiric p-value for linkage to HIVAN4 ϭ 0.006). third resistant counterstrain, Balb/cJ (BALB), leading to the iden- This result was obtained after adjustment for the severity of tification of a new HIVAN QTL. Furthermore, we obtained con- renal injury, indicating that this linkage is not a secondary firmation and higher resolution of this locus in an independent effect of kidney injury but truly represents transregulation. TgFVB colony selectively admixed with BALB alleles. In contrast, there was no evidence of an eQTL for Ptpro ( tryrosine phosphatase, receptor type, O, also known as GLEPP1), which is located within the HIVAN4 locus and en- RESULTS codes a podocyte expressed protein. While Ptpro transcript lev- els was highly correlated with histology injury score (r ϭ Ϫ Mapping a New HIVAN Locus to Chromosome 6 F3-G3 Ϫ0.46, p-value ϭ 3.6 ϫ 10 05) and Nphs2 transcript levels (r ϭ We had previously shown that TgFVBxBALB F1 hybrids do 0.67, P ϭ 2 ϫ 10Ϫ10), there were no differences in transcript not develop nephropathy, suggesting that the BALB genome levels between mice with FVB/FVB and FVB/BALB genotypes, confers resistance to this trait.8 To identify nephropathy sus- after correction for glomerulosclerosis (Lod Ͻ0.5). These find-

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Figure 1. (A) Lod plots for the nephropathy traits in the backcross. Map distance is presented on X-axis in Mb. The positions of informative markers are shown above the plot. The dashed horizontal lines indicate the thresholds for significant and suggestive linkage for a backcross based on 10,000 permutations (2.9 and 1.9, respectively). The bar above the lod plot indicates the 95th percentile confidence interval for the HIVAN4 locus. (B) Effect size for glomerulosclerosis and tubular injury score at the peak marker at the HIVAN4 locus. Homozygosity for FVB alleles is associated with a two-fold increase in injury scores.

ings suggest that HIVAN4 alleles do not directly influence homozygous and concordant with FVB alleles at 1292/1305 Ptpro transcript level and that the inverse correlation of Ptpro (99%) of markers across the genome. The 13 SNPs harboring transcript level with glomerulosclerosis occurs secondary to non-FVB alleles were clustered within two distinct intervals on kidney injury. chromosome 2 (85 to 152 Mb between rs3689658 and There were no other loci influencing Nphs2 or Ptpro tran- rs6247960) and chromosome 6 (121 to 139 Mb, between script abundance across the genome; particularly, there was no rs13479006 and rs13479071), and both of these intervals were evidence of an Nphs2 cis-eQTL in this cross, which is consistent fixed to homozygosity in all mice tested (Figure 2C and Sup- with FVB and BALB sharing the same haplotype at the Nphs2 plemental Figure 2). Consistent with the expectation of admix- locus. ture with an albino strain, comparison with reference strains revealed that all 13 non-FVB alleles were derived from the Independent Localization of HIVAN4 in an Admixed BALB genome. Most strikingly, the admixed interval on chro- Colony of TgFVB mosome 6 coincided perfectly with the HIVAN4 locus (Figure Concurrently, in a sister colony of TgFVB located at the Metro- 2C), strongly suggesting that introgression of BALB alleles in Health Medical Center (MHMC), investigators noted a dra- this region accounted for the loss of the nephropathy pheno- matic reduction in the penetrance of renal disease after the type. The lifespan of the low-penetrance TgFVB with the pro- colony was transferred to a new mouse facility in 2006; how- tective BALB alleles was Ͼ9 mo, whereas inbred TgFVB mice ever, two generation of backcrossing to FVB/NJ completely have considerably shorter life spans and require breeding at 6 restored the original nephropathy phenotype excluding trans- to 8 wk of age to sustain the colony (67% die or are moribund gene silencing as a mechanism (Figure 2A). Since the colony at a mean age of 71 d). Since there were no noticeable maintained the albino coat color of the FVB/N background changes in the severity of nephropathy before transfer to the strain, this suggested the occurrence of a spontaneous muta- new facility, this suggested that the contamination existed at tion(s) that conferred resistance to HIVAN or, alternatively, a low level, but BALB alleles rapidly propagated in the new inadvertent introgression of resistance alleles from one or colony because a survival constraint was placed on selection more albino strains. To study this further, the low penetrance of breeders, increasing the use of TgFVB with the protective mice were maintained by brother-sister mating as a separate BALB alleles. Consistent with this prediction, review of colony and 26 mice were genotyped across 1305 SNPs (Illu- breeding records showed that delays during transfer to a mina medium density panel). The low-penetrance mice were new facility in 2006 left older mice with minimal nephrop-

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Figure 2. (A) Breeding scheme in the TgFVB colony at the MetroHealth Medical Center (TgFVB MHMC). Mice ceased to manifest nephropathy after transfer of animals to a new facility in 2006. Histologic examination of mice age Ͼ7 months of age showed normal kidneys (center panel). Brother sister mating (shown on left side) led to the development of a colony showing no kidney disease and significant longevity. However, two generations of backcrossing to FVB/NJ (shown on right side) led to reemergence of early-onset nephropathy (Ͻ2 months of age), as demonstrated by prominent glomerulosclerosis, glomerular collapse, significant tubular dilation and microcysts, cast formation, and interstitial infiltrates. (B) Glomerulosclerosis and tubular injury scores (mean values Ϯ standard errors) in the TgFVB MHMC colony upon initiation of the colony in 2001–2006; a low-penetrance colony was identified after breeding delays during transfer of animals to a new facility in 2006 but the nephropathy phenotype reemerged after backcrossing to FVB/NJ for two generations in 2007. The injury scores in TgFVBxBALB F1 hybrids are shown for comparison. * PϽ0.05. **PϽ0.0001 versus Period 1 or Period 3 (C) Genotypes of the low penetrance colony demonstrate homozygosity for BALB alleles on chromosomes 2 and 6. Each line represents an individual mouse; each square represents genotypes for markers that are informative between FVB (top line, red) and BALB (second line, blue) on the Illumina medium density panel. The area of admixture on chromosome 6 coincides perfectly with the lod-2 interval for the HIVAN4 locus. athy as founders of the new colony, effectively creating a MHMC-TgFVB colony was approximately 35% smaller, genetic bottleneck. spanning a 19-Mb region containing 364 genes (Supple- mentary Table 1). Because a previous mapping study be- Annotation of Positional Candidates Within the tween TgFVB and C57BL/6J (B6) strains did not identify HIVAN4 Locus linkage to HIVAN4,18 this indicated that the FVB and B6 The refined HIVAN4 95th percentile confidence interval strains share the same susceptibility allele at this locus. Con- spans approximately 29 Mb between rs3695724 and sequently, we performed a three-way comparison of haplo- rs3711088 and contains 473 positional candidates. Impor- types, searching for regions within the HIVAN4 interval that tantly, the interval delineated by the low-penetrance are polymorphic between the FVB and BALB strains but are

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monomorphic between FVB and B6 strains. In total, 176/ interval-specific congenic and subcongenic lines for identi- 364 genes within the reduced HIVAN4 interval fulfilled fication of causal variants.20 These conditions were inadver- these selection criteria. We further annotated these genes tently recapitulated in the low-penetrance MHMC-TgFVB based on the presence of potentially deleterious amino-acid colony. The mapping resolution was comparable to studies substitutions (16 genes), evidence for interaction of human of outbred populations (approximately 19 Mb at HIVAN4) orthologs with HIV-1 (7 genes), and transcript enrichment and, simultaneously, this colony offered an incipient con- in the glomerulus (8 genes, Supplementary Table 1). Thus, genic strain for rapid generation of HIVAN4 congenic lines. in addition to Ptpro, which was studied further by expres- Our data indicate that selective breeding approaches may sion analysis (as described above), this annotation priori- prove very powerful for localizing and refining murine glo- tizes multiple candidates that can be pursued by sequencing, merulosclerosis susceptibility loci. expression analysis, and functional studies. For example, To date, three crosses with the TgFVB model have iden- the A2m gene, encoding Alpha-2 macroglobulin, harbors a tified four susceptibility loci for HIVAN on chromosomes potentially pathogenic variant, is enriched in the glomeru- 3A1-A3, 13A3-C2, 4A1-A5, and now 6F3-G3 (HIVAN1to4, lus and is predicted to interact with the HIV-1 protease. respectively17,18). We had previously shown that the HI- VAN1 and HIVAN2 loci regulate the expression of multiple podocyte-expressed genes in the absence of HIV-1.18 We DISCUSSION hypothesized that these changes were reac- tive to genetic lesions introduced by the HIVAN risk alleles, In this study, we describe a novel susceptibility locus for suggesting that the murine HIVAN susceptibility genes and nephropathy on mouse chromosome 6 F3-G3 using the podocyte genes belong to the same regulated network. Here HIV-1 transgenic mouse model. This finding was supported we show that HIVAN4 also transregulates Nphs2 expression, by a genome-wide significant detection of this interval in a thereby placing HIVAN1, 2, and 4 loci in the same pathway. backcross cohort derived from inbred strains, and examina- Although these intervals are large, the availability of multi- tion of a low-penetrance colony of TgFVB that demon- ple crosses now enables significant refinement of positional strated selective preservation of the BALB genome at the candidates by comparison of haplotypes among inbred HIVAN4 locus. Since the segments of admixture repre- strains.21 Thus, there are several noteworthy genes among sented Ͻ3% of the mouse genome, it is highly unlikely that the refined list of candidates at the HIVAN4 locus: a cluster the overlap with the HIVAN4 locus is coincidental. The Tg- of natural killer cell lectin-like receptor genes, which encode FVB mice usually have a limited lifespan because they de- proteins important for defense against pathogens,22–26 and velop nephropathy within 3 to 6 wk of age.6,19 This strongly several genes (Bid, A2m, Cd69, Grin2b, Pik3c2g, Plcz1, Itpr2) suggests that the protective BALB alleles at the HIVAN4 with potential interaction with HIV-1. Of note, variation in locus were retained because they conferred a survival and Klra8 gene, encoding a receptor important for natural killer reproductive advantage in the MHMC colony, owing to the cell cytoxicity, is associated with differential susceptibility absence of nephropathy. Moreover, this admixture re- to CMV infection in the mouse but is unlikely to be causal mained undetected due to the albino coat color in the BALB for HIVAN because FVB and BALB have a susceptibility and FVB strains. These findings illustrate the strong effect of haplotype to CMV infection23 Another positional candi- selection on beneficial alleles even within artificial colonies, date, Ptpro, encodes a podocyte expressed protein and has providing further evidence that HIVAN4 exerts a large effect dysregualed expression in HIVAN27,28 but did not demon- on the nephropathy phenotype. A second admixed segment strate a cis-eQTL on additional analysis. Finally, A2m, en- on chromosome 2 was also detected in the low penetrance coding Alpha-2 Macroglobulin, enriched in glomeruli,29 is a MHMC-TgFVB colony and thus represents a candidate lo- target for HIV-1 protease30 and harbors a missense variant cus for nephropathy. The BALB alleles at this locus would be that is predicted to affect protein function. While, to our predicted to have a recessive protective effect because this knowledge, none of the murine HIVAN susceptibility loci interval was not detected in the backcross, which can only overlap with other QTLs for nephropathy or proteinuria in identify additive effects. Further cross breeding can separate the mouse,31–34 the HIVAN4 locus coincides with a QTL for this chromosome-2 interval from the HIVAN4 locus and GFR identified in American Indians.35 Analysis of congenic formally determine whether this region also imparts an ef- lines will enable dissection of specific genes and variants fect on nephropathy. within the HIVAN4 interval, identify relationships to other Our findings indicate that murine nephropathy suscep- QTLs, and delineate common dysregulated pathways down- tibility alleles are under significant selection pressure and, stream of susceptibility alleles. given the opportunity, will be rapidly eliminated from the These studies also offer an interesting parallel to recent gene pool. Many intricate breeding strategies, such as selec- findings demonstrating the effect of natural selection on tive breeding or advanced intercross lines, aim to reproduce nephropathy susceptibility genes in humans. Studies have conditions of selection pressure to localize large-effect shown that genetic variation at the APOL1 locus on chro- QTLs.20 Alternatively, investigators sequentially generate mosome 22 is a major risk factor for nephropathy among

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African Americans with focal segmental glomerulosclerosis purification using the RNeasy kit (QIAGEN). cDNA was generated and nondiabetic end-stage kidney disease3,4 The APOL1 ne- with the Omni-Script kit (Qiagen). Gene expression was measured phropathy risk alleles have rapidly propagated because they in duplicates by quantitative PCR (qPCR) using SYBR-Green mix provide resistance to trypanosomiasis in heterozygotes, but and an iQ5 thermal Cycler (Bio-Rad). Expression values were stan- the increased susceptibility to kidney failure in homozy- dardized to an internal control reference sample (a male FVB/NJ) gotes exerts a balancing effect, maintaining the alternative included in each run, and ␤-actin was used as housekeeping con- alleles in the population.3,4 The mechanism of kidney injury trol (Pfaffl algorithm). Primers for Nphs2 and ␤-actin were previ- for the APOL1 risk-variants is not yet understood. APOL1 is ously described,18 and primers for Ptpro were Ptpro F, 5Ј ÐTGT- only present in hominoids and some Old World monkeys. CATGACGGCCATCCTTA-3Ј, and Ptpro-R, 5Ј-ACTGCGAAGA- About 10 to 15% of healthy African Americans are homozy- GTGACTTCGGA-3Ј. gous for the chromosome 22 risk alleles, and, conversely, 10 to 24% of African Americans with FSGS or hypertensive Genotyping and Analysis of Linkage end-stage kidney failure do not carry any APOL1 risk al- The backcross cohort was genotyped using Illumina medium-den- leles.3,4 These data imply additional genetic or environmen- sity SNP panel, which contains 1449 SNPs distributed across the tal requirements for the development of nephropathy. The genome (Center for Inherited Disease Research genotyping ser- recapitulation of virtually all phenotypic and molecular fea- vice, Baltimore, MD). Of these, 1305 were successfully genotyped ture of human disease in the TgFVB model further suggests and 548 SNPs were informative between FVB and BALB. Linkage that alternative genetic lesions, in the absence of APOL1, scans for histology score, BUN, and serum albumin were per- can predispose to HIVAN. Thus, identification of the mu- formed using R/QTL program with the nonparametric method. rine genetic variants may clarify additional risk factors and Lod scores of Ն3.3 were considered significant.36 In addition, we biologic pathways required for the development of ne- conducted 10,000 permutations of phenotypes on genotype to de- phropathy. termine the empirical significance of the linkage findings. Nphs2 transcript level was square-root transformed to achieve a normal distribution and analyzed using a parametric method (Haley- CONCISE METHODS Knott algorithm) with adjustment for the degree of glomeruloscle- rosis. Pointwise p-value for linkage of the Nphs2 transcript level to the HIVAN4 locus was calculated based on permutations analysis Animal Breeding and Phenotyping All inbred strains were purchased from the Jackson Laborator- limited to the HIVAN4 lod-2 interval. ies (Bar Harbor, ME). The HIV-1 transgenic mouse line TgN(pNL43d14)26Lom (TgFVB) was produced on the inbred Gene Annotation in the HIVAN4 Locus FVB/N genetic background and has been extensively character- SNP identification and annotation across the HIVAN4 interval were ized.6,17 The TgFVB colonies at Columbia University and Metro- obtained from the Mouse Phenome Database (http://phenome.jax. Health Medical Center were established from founders received org/). Segments that were not identical by descent were identified by from the Mount Sinai School of Medicine (courtesy of Dr. Paul filtering for SNPs that are polymorphic between FVB/NJ and BALB/ Klotman). The TgFVB x (FVB x BALB) F1 backcross was generated cByJ, but monomorphic between FVB/NJ and C57BL/6J strains. at Columbia University, and 165 heterozygous HIV-1 transgenic Gene annotation was performed using the PANTHER database progeny were phenotyped at 8 wk of age. Animals that were mor- (http://www.pantherdb.org/). Missense SNPs were evaluated for ibund were euthanized before this end point. Primary phenotypes potential to affect protein function using SIFT (http://blocks.fh- included renal histology, serum albumin, and blood urea nitrogen crc.org/sift/SIFT.html). HIV-1 interactions were obtained from (BUN). Renal histology was scored independently by an investiga- the HIV-1, Human Protein Interaction Database (http://www. tor (VDD) blinded to genetic background and other traits. Three ncbi.nlm.nih.gov/RefSeq/HIVInteractions/). Glomerular enriched genes traits related to tubulointerstitial disease (epithelial regeneration/ were annotated based on a curated list compiled by Lindenmeyer 29 degeneration, tubular casts and dilation, and interstitial infil- et al. trates) and one related to glomerular injury (glomerular sclerosis) were scored using a semiquantitative scale: 0 ϭ no disease; 1 ϭ 1to 25% of tissue showing abnormalities; 2 ϭ 26 to 50%; 3 ϭ 51 to ACKNOWLEDGMENTS 75%; and 4 ϭϾ75% of tissue affected. A global histologic score was calculated as an average of four renal injury scores (range 0 to This study was supported by National Institutes of Health grants 16). The protocol was approved by the IACUC committees at the DK087626 (to AGG) and DK061395 (to LB), and by the MetroHealth Columbia University Medical Center and Case Western Reserve Medical Center institutional funds and the histology core facility of University. the Columbia Diabetes and Endocrinology Research Center (NIH P30-DK63608). RLT was supported by training grant DK007470. Measurement of Podocyte Gene Expression LAB is a member of the Case Center for AIDS Research supported by Total RNA was extracted from mouse whole kidneys using TRIzol grant AI36219. Genotyping services were provided by the Center for reagent (Invitrogen), followed by DNaseI treatment and further Inherited Disease Research (CIDR). CIDR is funded through a federal

1502 Journal of the American Society of Nephrology J Am Soc Nephrol 22: 1497–1504, 2011 www.jasn.org BASIC RESEARCH contract from the National Institutes of Health to The Johns Hopkins TNFR2 interposes the proliferative and NF-kappaB-mediated in- University, contract number HHSN268200782096C. flammatory response by podocytes to TNF-alpha. Lab Invest 91: 413–425, 2011 17. Gharavi AG, Ahmad T, Wong RD, Hooshyar R, Vaughn J, Oller S, Frankel RZ, Bruggeman LA, D’Agati VD, Klotman PE, Lifton RP: DISCLOSURES Mapping a locus for susceptibility to HIV-1-associated nephropathy to mouse chromosome 3. Proc Natl Acad Sci U S A 101: 2488– None. 2493, 2004 18. Papeta N, Chan KT, Prakash S, Martino J, Kiryluk K, Ballard D, Bruggeman LA, Frankel R, Zheng Z, Klotman PE, Zhao H, D’Agati VD, Lifton RP, Gharavi AG: Susceptibility loci for murine HIV-associated nephropathy encode trans- REFERENCES regulators of podocyte gene expression. J Clin Invest 119: 1178–1188, 2009 19. 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J Am Soc Nephrol 22: 1497–1504, 2011 A Nephropathy-Susceptibility Locus on Mouse Chr. 6F3-G3 1503 BASIC RESEARCH www.jasn.org

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