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Accelerated development of collapsing glomerulopathy in mice congenic for the HIVAN1 locus
original article
http://www.kidney-international.org & 2009 International Society of Nephrology
see commentary on page 353
Accelerated development of collapsing glomerulopathy in mice congenic for the HIVAN1 locus Ka T. Chan1, Natalia Papeta1, Jeremiah Martino1, Zongyu Zheng1, Rachelle Z. Frankel1, Paul E. Klotman2, Vivette D. D’Agati3, Richard P. Lifton4 and Ali G. Gharavi1 1
Department of Medicine, Columbia University College of Physicians and Surgeons, New York, New York, USA; 2Department of Medicine, Mount Sinai School of Medicine, New York, New York, USA; 3Department of Pathology, Columbia University College of Physicians and Surgeons, New York, New York, USA and 4Departments of Genetics and Medicine and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut, USA
HIV-1 transgenic mice on the FVB/NJ background (TgFVB) are a well validated model of HIV-associated nephropathy (HIVAN). A mapping study between TgFVB and CAST/EiJ (CAST) strains showed this trait to be influenced by a major susceptibility locus on chromosome 3A1–A3 (HIVAN1), with CAST alleles associated with increased risk of disease. We introgressed a 50 Mb interval, encompassing this HIVAN1 locus, from CAST into the TgFVB genome (TgFVB-HIVAN1CAST congenic mice). Compared to the TgFVB strain, these congenic mice developed an earlier onset of proteinuria, a rapid progression to kidney failure, and increased mortality. A prospective study of these congenic mice also showed that they had a significantly greater histologic and biochemical evidence of glomerulopathy with one-third of mice developing global glomerulosclerosis by 6 weeks of age. An F2 cross between TgFVB and the congenic mice identified a significant linkage (LOD ¼ 3.7) to a 10 cM interval within the HIVAN1 region between D3Mit167 and D3Mit67 resulting in a 60% reduction of the original interval. These data independently confirm that a gene on chromosome 3A1-A3 increases susceptibility to HIVAN, resulting in early onset and rapid progression of kidney disease. These mice represent a new model to study the development and progression of collapsing glomerulopathy. Kidney International (2009) 75, 366–372; doi:10.1038/ki.2008.625; published online 17 December 2008 KEYWORDS: genetic susceptibility; HIV-associated nephropathy; mouse model
Correspondence: Ali G. Gharavi, Department of Medicine, Columbia University College of Physicians and Surgeons, 1150 St Nicholas Ave, Russ Berrie Pavillion 302 New York, New York 10032, USA. E-mail: [email protected] Received 31 July 2008; revised 1 October 2008; accepted 21 October 2008; published online 17 December 2008 366
HIV-1 infection is associated with a number of pathologically distinct glomerulopathies that can lead to renal failure.1–6 Among these glomerulopathies, HIV-associated nephropathy (HIVAN) is the most common form and is characterized by the development of collapsing glomerulopathy and microcystic tubular dilatation.5,6 Most patients with HIVAN progress to end-stage renal failure without antiretroviral therapy, directly implicating HIV-1 in the pathogenesis of disease.4,7,8 Data suggest that HIVAN is produced by HIV-1induced dysregulation of glomerular podocytes, the highly differentiated visceral epithelial cells lining the glomerular filtration barrier.4 In HIVAN, podocytes exhibit enhanced proliferation and apoptosis and lose signature proteins of maturity such as podocin and synaptopodin.7,9,10 The mechanism underlying the dysregulated podocyte phenotype is not fully understood. HIV-1 has been shown to interfere with multiple pathways that are critical for maintaining cellular quiescence, with the HIV-1 nef protein as an important pathogenic mediator.10–18 Host genetic factors also have an important impact on the course of HIV-1 infection and the development of complications.3,19,20 For example, the progression of HIV-1 disease is influenced by HLA genotype21 or copy number variation at the CCL3L1 locus,20 and the development of dementia is influenced by APOE alleles.22 Several lines of evidence also suggest that host genetic factors are also important for the development of HIVAN. Contrary to other glomerular diseases associated with HIV-1 infection, HIVAN almost exclusively occurs in individuals of African ancestry.1,2,23,24 Moreover, family members of HIVAN patients also have a higher incidence of end-stage renal disease, suggesting segregation of nephropathy-predisposing alleles in these families.25 Most recently, studies have shown that sequence variants in the podocyte-expressed non-muscle myosin heavy chain gene (MYH9) are associated with multiple nephropathies including HIVAN, explaining these epidemiological observations among African Americans.26,27 Kidney International (2009) 75, 366–372
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KT Chan et al.: Accelerated nephropathy in HIVAN1 congenic mice
Multiple murine models of HIVAN have been developed and highlight the importance of permissive genetic background on the development of this trait. Introduction of the HIV-1 proviral genome, or specific HIV-1 genes (such as nef) into the mouse genome reproduces all the clinical and pathological features of HIVAN.15–18,28 However, the development of disease is highly strain dependent. Studies using independent transgene constructs have shown that the FVB/ NJ (FVB) genetic background is highly permissive, resulting in all the manifestations of collapsing glomerulopathy.17,18,28,29 To examine the impact of host genetic factors, we previously produced F1 hybrids between HIV-1 transgenic FVB lines (TgFVB) and six inbred strains and found wide variation in penetrance of disease.29 Using a cross between TgFVB and the CAST/EiJ (CAST) strain, we identified a major susceptibility locus for HIVAN on chromosome 3A1–A3, which we named HIVAN1.29 Interestingly, at the HIVAN1 locus, CAST alleles were associated with increased severity of disease. However, the HIVAN1 95th percentile confidence interval spanned over 50 Mb, necessitating additional experiment for identification of the underlying gene. Derivation of congenic lines trapping susceptibility loci is a powerful approach for replication of linkage findings and subsequent elucidation of complex traits.30 Congenic lines are produced by introgressing chromosomal segments encompassing the quantitative trait loci (QTL) of interest from a donor strain into a recipient strain, thereby enabling analysis of alternative alleles of the QTL within the same genetic background.31 This procedure provides independent verification of the initial linkage studies, enables better characterization of the phenotypic effect imparted by the QTL, and facilitates gene identification by fine mapping or derivation of subcongenic lines.30,31 Toward identification of the HIVAN1 gene, we produced mice congenic for the HIVAN1 interval and report genetic and phenotypic analysis of this new model of nephropathy.
RESULTS
We produced HIVAN1 mice congenic (named the TgFVBHIVAN1CAST strain) by introducing the chromosome segment encompassing this interval from CAST into the FVB genome by 10 generations of backcrossing. A genome scan with 82 informative loci in the 10th backcross generation confirmed that the TgFVB-HIVAN1CAST strain possessed a pure FVB genetic background outside the introgressed region. The proximal and distal loci homozygous for CAST alleles were delimited by rs6372626 (located at 4.2 Mb on the mouse reference sequence) and rs13477119 (54.6 Mb), respectively; the next proximal and distal markers homozygous for FVB alleles were rs6171250 (3 Mb) and rs30305237 (55.8 Mb), respectively. Thus the size of the congenic segment spans from a minimum of 50.4 Mb to a maximum of 52.8 Mb. Nontransgenic FVB-HIVAN1CAST mice were phenotypically normal with no evidence of nephropathy or proteinuria. However, HIV-1 transgenic counterparts developed rapid onset of kidney disease (Figure 1) with B40% mortality rate at 8 weeks of age (compared to o5% for TgFVB). Moreover, by 4 weeks of age, HIV-1 transgenic TgFVB-HIVAN1CAST mice displayed increased incidence of proliferative epidermal lesions characteristic of animals expressing this HIV-1transgene construct,32 necessitating euthanasia based on humane criteria. Therefore, to prospectively study the impact of HIVAN1 alleles on the development of kidney disease, a cohort of TgFVB-HIVAN1CAST mice were studied from birth to 6 weeks of age and compared to TgFVB counterpart. At 3 weeks of age, all HIV-1 transgenic mice displayed significant proteinuria compared to nontransgenic mice, but homozygosity for CAST alleles was associated with a twofold increase in albuminuria (Po0.04; Figure 2a). At 6 weeks of age, TgFVB-HIVAN1CAST mice continued to display significantly greater proteinuria compared to TgFVB mice (Po0.02; Figure 2a). On histologic assessment, TgFVB-
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Figure 1 | Histology of TgFVB-HIVAN1CAST mice (PAS stain). Congenic mice display advanced global glomerulosclerosis (a–c) or collapsing glomerulosclerosis with podocyte hyperplasia (d–f). (a and d) Low power view ( 200) demonstrates diffuse and global glomerulosclerosis, extensive microcystic tubular dilatation and severe tubulointerstitial injury. (b and c) High power views ( 400) of glomeruli showing global sclerosis and podocyte depletion. Higher power views ( 600) of glomeruli showing collapsing sclerosis (e) with podocyte hyperplasia and cytoplasmic vacuolization (f). Kidney International (2009) 75, 366–372
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Figure 2 | Phenotypic comparison of TgFVB and TgFVB-HIVAN1 mice. Comparison of proteinuria (a) and glomerulosclerosis scores (b) in HIV-1 transgenic mice carrying different alleles at the HIVAN1 locus on chromosome 3A1–A3. Values for a cohort of nontransgenic (nonTg) mice of all three genotypes are shown for comparisons. (c) Comparison of HIV-1 Env expression in mice carrying different alleles at the HIVAN1 locus. #Po0.005, *Po0.05 vs Tg FVB/FVB genotype.
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Figure 3 | Fine mapping of the HIVAN1 locus. (a) LOD plots for renal histology scores in the F2 intercross between TgFVB and TgFVBHIVAN1CAST strains. Map distance is presented in cM. The positions of informative markers are shown above each plot. The dashed horizontal lines indicate the empiric threshold for 95th and 99th percentile significance level based on 10,000 permutations (1.9 and 3.2, respectively). The 95th confidence interval for the location of the HIVAN1 gene is shown above the LOD plot. (b) Phenotypic effects of HIVAN1 genotype in F2 cross is consistent with an additive model (mean±s.e.m.).
HIVAN1CAST demonstrated hallmarks of collapsing glomerulopathy, including tuft collapse and overlying podocyte proliferation and hypertrophy (Figure 1). Quantitative comparison of renal histopathology demonstrated significantly increased glomerulosclerosis (P ¼ 0.003; Figure 2b) and tubular injury with microcystic dilatation (P ¼ 0.002; Figure 2b) in TgFVB-HIVAN1CAST compared to TgFVB mice. Interestingly, 9/25 TgFVB-HIVAN1CAST animals also demonstrated global glomerulosclerosis with extensive podocyte depletion at 6 weeks of age, a finding that was not observed in their TgFVB counterparts (P ¼ 0.002 on Fisher’s exact test). Heterozygous mice also displayed increased pathology, intermediate between FVB/FVB and CAST/CAST, consistent with an additive effect of CAST alleles (Figure 2a and b). Previous studies in TgFVB mice have shown that the development of nephropathy coincides with increased expres368
sion of the HIV-1 transgene, followed by gradual reduction in expression secondary to loss of renal epithelial cells accompanying progressive renal injury.27 To assess whether increased severity of kidney disease in TgFVB-HIVAN1CAST mice is attributable to enhanced transgene expression, we quantitated transgene expression in 10 CAST/CAST and 10 FVB/FVB congenic mice with mild to moderate renal injury. These mice were matched for severity of renal histology scores to avoid bias introduced by secondary decrease in transgene expression due to nephropathy. There were no differences in transgene expression between the two genotype groups (Figure 2c), suggesting that increased severity of kidney disease is mediated through variation in host response to HIV-1 rather than enhanced expression of the transgene. These data demonstrate successful trapping of the HIVAN1 susceptibility alleles in our congenic strain, providKidney International (2009) 75, 366–372
KT Chan et al.: Accelerated nephropathy in HIVAN1 congenic mice
ing a novel tool for identifying the underlying susceptibility allele. We next utilized this strain to achieve further refinement of the HIVAN1 interval by meiotic mapping. We produced an F2 intercross between TgFVB and FVBHIVAN1CAST strains and phenotyped 68 F2 progeny. Genotypic analysis with seven informative markers demonstrated significant linkage of renal histology score with the HIVAN1 locus (peak LOD 3.7, empiric P ¼ 0.001 and 3.3, empiric P ¼ 0.004, at D3Mit224 for glomerular and tubular injury scores, respectively; Figure 3a). As a single interval was tested, these LOD scores achieve pointwise significance for replication (Po0.01) and remarkably, also exceed published genome-wide significance.33 In this cross, the HIVAN1 alleles demonstrated an additive effect (Figure 3b), explaining 20 and 12% of the variance in the glomerulosclerosis and tubular injury scores. The effect sizes were also consistent with observations in the congenic lines (Figure 3b). As this cross was produced on a homogeneous background, the linkage peak was much narrower than the original study, with the 95% confidence limit localizing the HIVAN1 gene within a B10 cM interval delimited by D3Mit167 and D3Mit67 (corresponding to B32–52 Mb position in mouse reference sequence), a region containing 126 genes. This represents a 60% reduction in the original HIVAN1 interval. As laboratory mouse strains are derived from a limited set of ancestors, comparison of haplotype structure across inbred strains can identify segments that are identical by descent (IBD), enabling further higher resolution mapping of linkage regions.34,35 We therefore examined the haplotype structure of the refined region and identified genes that contain informative single nucleotide polymorphisms (SNPs) between FVB and CAST. We identified 46,004 SNPs within the critical regions that are polymorphic between CAST and FVB (1 SNP/394 bp). Among these, 3684 SNPs are located within 78 NCBI annotated genes, but the remaining genes could not be studied due to lack of SNP data in either FVB or CAST strains. Among these 78 genes with non-IBD haplotypes, 22 genes harbor nonsynonymous variants, providing an initial selection tool for prioritization of genes within this interval (total of 95 SNPs; Supplementary Data). The high polymorphism rate between the CAST and FVB strains is consistent with the wild-derived origin of CAST strain and its relatively low contribution to the laboratory mouse genome, suggesting that comparison of IBD structure between FVB and CAST would not significantly refine the interval further.34 DISCUSSION
In this study, we characterized a novel congenic strain that exhibits rapid onset and increased severity of HIVAN. Introgression of CAST alleles at the HIVAN1 locus into the FVB genome resulted in earlier onset of disease, as evidenced by greater proteinuria at 3 weeks of age, worse histologic features and proteinuria at 6 weeks of age and early demise. In addition, many animals developed global glomerulosclerosis and podocyte depletion, a feature rarely observed in Kidney International (2009) 75, 366–372
original article
younger TgFVB mice. These congenic lines enabled better resolution of the QTL by isolating it in a homogeneous genetic background. Analysis of TgFVB-HIVAN1CAST congenic lines also enabled better characterization of the QTL effect size, demonstrating that it accounts for 12–20% of the variance in renal injury and is associated with a twofold higher glomerulosclerosis score and proteinuria by 6 weeks of age. These effects are very significant considering that our original mapping study relied on phenotypic characterization at 12 weeks of age, allowing more time for nephropathy to develop.29 Analysis of heterozygotes supported an additive genetic model, which was further confirmed by an F2 mapping cohort. These data independently confirm our initial mapping study, demonstrating that a gene on chromosome 3A1–A3 mediates susceptibility to HIVAN. The TgFVB-HIVAN1CAST congenic strain now offers a novel tool for identifying the gene(s) mediating increased susceptibility to HIVAN and studying the pathogenesis of this disease. As both TgFVB and TgFVB-HIVAN1CAST strains develop nephropathy, it is unclear whether HIVAN1 alleles directly contribute to the development of HIVAN or primarily affect progression of disease. Therefore, it will be interesting to determine whether HIVAN1 alleles exacerbate other forms of nephropathy, or whether similar to older haploinsufficient Cd2ap or Actn4 mice,36,37 the underlying susceptibility state will manifest with mild proteinuria or subtle ultrastructural defects in aged nontransgenic FVBHIVAN1CAST mice. Additional refinement of the HIVAN1 interval can be achieved by systematic derivation of subcongenic lines that can further dissect the region into smaller segments and can determine whether this genetic interval harbors a single- or multiple-linked QTLs. Alternatively, meiotic mapping in congenic lines can further resolve the interval to a tractable segment for gene identification. Although analysis of haplotype structure had previously been helpful for refinement of a nephropathy locus among classic laboratory strains,35 this strategy was not useful in this study because the wild-derived CAST strain has a relatively minor contribution to the laboratory mouse genome. Hence, most segments of the HIVAN1 interval harbor polymorphisms between FVB and CAST, complicating identification of the functional allele(s). Meiotic mapping appears to be the most feasible option, as demonstrated by significant linkage to smaller, well defined region obtained with our modest sized F2 cohort. This F2 cross indicated that a single QTL segregates within the HIVAN1 locus, suggesting that generation of larger mapping cohorts would rapidly resolve the interval further. An examination of positional candidates in the current 95% confidence interval reveals that nearly half are minimally annotated. Interestingly, ontology searches of the annotated genes reveal significant enrichment for genes encoding calcium dependent proteins (Anxa5, Trpc3, Pcdh18, Pcdh10, Cetn4, Fat4) or JAK-STAT signaling (Spry1, Il21, Il2). Other potentially relevant candidates include genes with known interaction with HIV-1 such as Fgf2, Pik3ca, Il2, and 369
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Ccna2, which are involved in some of the principle pathways affected by HIV-1 infection such as cell cycle regulation or cytokine production. We anticipate that gene expression analysis in FVB-HIVAN1CAST strains will provide additional information for prioritization of genes by identifying differentially expressed renal transcripts. Any genes that are differentially expressed and located within the critical interval would represent strong positional candidates that should be pursued by functional studies or transgenesis experiments. Collapsing glomerulopathy can be caused by many different primary insults to glomerular podocytes. In HIVAN and other forms of collapsing glomerulopathy, the proliferating podocytes are gradually depleted, undermining the architecture of the glomerular filtration barrier. Ultrastructurally, collapsing glomerulopathy is characterized by actin cytoskeletal effacement, whereas other forms of focal segmental glomerulosclerosis are associated with reorganization of the actin cytoskeleton into mats.38 Known environmental triggers for this lesion include infectious agents (HIV-1, cytomegalovirus) or drugs (bisphosphonates, interpheron-a).4,39 Inherited disorders featuring collapsing glomerulopathy have also been reported.40 In humans, noncoding variants in the MYH9 gene were recently associated with HIVAN, primary focal segmental glomerulosclerosis or nondiabetic end-stage renal failure.26,27 Mutations in the coding sequence of MYH9 have dominant, pleiotropic effects in extrarenal tissues but are sometimes associated with nephropathy.41 Interestingly, Myh9 haploinsufficient mice are phenotypically normal, suggesting that additional genetic or environmental modifier may be required for manifestation of the complications observed in humans.42,43 Myh9 is located on chromosome 15 in the mouse and is therefore distinct from the HIVAN1 gene, but given the close similarity of the TgFVB model to the human disease, it is likely that HIVAN1 operates in the same pathway as Myh9. Cross-breeding studies with HIV-1 transgenic mice and the FVB-HIVANCAST strain can determine the relationship among HIV-1, Myh9, and HIVAN1. In addition to HIV-1 transgenic mice, several animal models have also been developed, highlighting novel pathways underlying this lesion. For example, collapsing glomerulopathy in kd/kd mice is due to a mutation in the gene encoding prenyl diphosphate synthase, subunit 2 (Pdss2), resulting defective mitochondrial CoQ biosynthesis.44 Tissue specific expression of mutant alleles resulted in biochemical defects of CoQ9 and CoQ10 deficiency, but expression in podocytes was necessary and sufficient to recapitulate the full renal phenotype.45 Similarly, Henderson et al. have reported that mice homozygous for Actn4 null alleles, or a human disease associated Actn4 mutation also develop collapsing glomerulopathy, whereas mice heterozygous for the human mutation only manifest mild ultrastructural changes.37 Actn4 and Pssd2 operate in widely disparate pathways, complicating inference of a common mechanism for the development of collapsing glomerulopathy. Moreover, as both Actn4 and Pssd2 are ubiquitously expressed, the reasons for the unique 370
KT Chan et al.: Accelerated nephropathy in HIVAN1 congenic mice
vulnerability of podocytes are not known. It is generally hypothesized that podocytes are particularly susceptible to injury because they are terminally differentiated and cannot repopulate subsequent to genetic or environmental insults.4 The remaining podocytes must therefore undergo hypertrophy to cover a larger surface area, leaving denuded segments of the basement membrane that promote the development of sclerotic lesions. Finally, beyond as certain threshold, podocyte injury may spread locally, further propagating the segmental sclerotic process.46 However, the molecular pathways linking podocyte injury, cytoskeletal remodeling, propagation of injury, and glomerulosclerosis remains unclear. The availability of different models now enables systematic cross-comparative studies, which may provide additional insight into shared mechanisms underlying collapsing glomerulopathy. MATERIALS AND METHODS Animal breeding The HIV-1 transgenic mouse line TgN(pNL43d14)26Lom 26 (TgFVB) was produced on the inbred FVB/NJ genetic background and has been extensively characterized. The TgFVB colony at Columbia University has been continuously backcrossed to FVB/NJ for at least 30 generations. Congenic mice for the HIVAN1 locus were generated by generating F1 hybrid between FVB/NJ and CAST/ EiJ followed by 10 generations of backcrossing to FVB/NJ with selection of CAST alleles across the HIVAN1 locus at each generation. As HIV-1 transgenic mice develop kidney disease, wild-type FVB/NJ mice were utilized for nine generations of backcrossing, yielding mice that were heterozygotes for CAST alleles the HIVAN1 locus at each generation. We then utilized TgFVB for the 10th backcross generation, yielding HIV-1 transgenic mice with FVB/CAST genotype at the HIVAN1 locus. Trangenic heterozygous mice were intercrossed with nontransgenic heterozygous mice to yield transgenic mice with CAST/CAST, CAST/FVB, or FVB/FVB genotypes at the HIVAN1 locus. Mice carrying the full-length haplotype across the interval were selected for study. The markers utilized for derivation of the congenic intervals were D3Mit61 (5.1 Mb), D3Mit203 (26.8 Mb), and D3Mit67 (52.8 Mb). Subsequently the boundaries of the congenic region were better delineated by SNP genotyping. Successful introgression of the HIVAN1 interval into the FVB/NJ genetic background was further confirmed by typing 82 informative loci across the genome in the 10th backcross generation (Kbioscience, Hoddesdon, UK), which confirmed that congenic mice carried only FVB alleles outside the HIVAN1 interval. For characterization of congenic mice, the study endpoint was limited to 6 weeks of age because of increased mortality and development of skin lesions. Histology was compared between mice of differing genotypes at the HIVAN1 locus (FVB/FVB, N ¼ 30; FVB/CAST, N ¼ 26; CAST/CAST, N ¼ 22). In a subset of congenic mice, we quantitated albumin/creatinine ratio (Exocell, Philadelphia, PA, USA) at 3 and 6 weeks of age (FVB/FVB, N ¼ 16; FVB/ CAST, N ¼ 17; CAST/CAST, N ¼ 19). Fifteen nontransgenic mice of all three genotype class were also tested as controls. We also produced an independent cohort of 68 F2 mice for meiotic mapping. For this cross, we produced F1 hybrids between FVB-HIVAN1CAST congenic mice and TgFVB and cross-bred a transgenic to a nontransgenic F1 hybrid to generate F2 intercross mice. Renal histology was scored independently by an investigator Kidney International (2009) 75, 366–372
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KT Chan et al.: Accelerated nephropathy in HIVAN1 congenic mice
(VDD) blinded to genetic background and other traits, scoring the severity of tubulointerstitial disease (tubular casts, tubular dilatation, epithelial regeneration/degeneration) and glomerular injury (segmental and global glomerulosclerosis and collapse, podocyte hyperplasia and collapse) using a continuous and a semiquantitative scale: 0 ¼ no disease, 1 ¼ 1–25% of glomeruli or tubular area showing abnormalities, 2 ¼ 26–50%, 3 ¼ 51–75%, and 4475% of tissue affected. The protocol was approved by the IACUC committee at the Columbia University Medical Center. Genotyping and analysis of linkage The F2 cohort was genotyped using seven informative microsatellite markers across the HIVAN1 locus (Figure 3). Multipoint analysis of linkage was performed using the R/QTL package.47 As the renal injury scores were not normally distributed, a nonparametric model was used. In addition, we conducted 10,000 permutations of phenotypes on genotype to determine the empirical significance of the linkage findings.
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Statistical and bioinformatics analysis Statistical analyses were performed using SPSS. The primary endpoints were the comparison of proteinuria and histologic injury between TgFVB and FVB-HIVAN1CAST strains using two-sided t-test. P-valuesp0.05 were considered significant. SNP ID and annotation across the HIVAN1 interval were obtained from the Mouse Phenome Database (http://aretha.jax.org/pub-cgi/phenome/ mpdcgi?rtn=docs/home). Non-IBD segments were identified by filtering for polymorphic SNP between FVB/NJ and CAST/eiJ strains. Gene annotation was performed using the PANTHER database (http://www.pantherdb.org/).
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DISCLOSURE
All the authors declared no competing interests. ACKNOWLEDGMENTS
This work is supported by NIH 1PO1 DK56492-01. AGG is supported by the Columbia Center for Glomerular diseases and the Emerald Foundation. SUPPLEMENTARY MATERIAL Supplementary Data Table S. Positional candidates within the 95th percent confidence interval in F2 cross. Table S1. Positional candidates with nonsynonymlous variants.
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Supplementary material is linked to the online version of the paper at http://www.nature.com/ki
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Accelerated development of collapsing glomerulopathy in mice congenic for the HIVAN1 locus Ka T. Chan1, Natalia Papeta1, Jeremiah Martino1, Zongyu Zheng1, Rachelle Z. Frankel1, Paul E. Klotman2, Vivette D. D’Agati3, Richard P. Lifton4 and Ali G. Gharavi1 1
Department of Medicine, Columbia University College of Physicians and Surgeons, New York, New York, USA; 2Department of Medicine, Mount Sinai School of Medicine, New York, New York, USA; 3Department of Pathology, Columbia University College of Physicians and Surgeons, New York, New York, USA and 4Departments of Genetics and Medicine and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut, USA
HIV-1 transgenic mice on the FVB/NJ background (TgFVB) are a well validated model of HIV-associated nephropathy (HIVAN). A mapping study between TgFVB and CAST/EiJ (CAST) strains showed this trait to be influenced by a major susceptibility locus on chromosome 3A1–A3 (HIVAN1), with CAST alleles associated with increased risk of disease. We introgressed a 50 Mb interval, encompassing this HIVAN1 locus, from CAST into the TgFVB genome (TgFVB-HIVAN1CAST congenic mice). Compared to the TgFVB strain, these congenic mice developed an earlier onset of proteinuria, a rapid progression to kidney failure, and increased mortality. A prospective study of these congenic mice also showed that they had a significantly greater histologic and biochemical evidence of glomerulopathy with one-third of mice developing global glomerulosclerosis by 6 weeks of age. An F2 cross between TgFVB and the congenic mice identified a significant linkage (LOD ¼ 3.7) to a 10 cM interval within the HIVAN1 region between D3Mit167 and D3Mit67 resulting in a 60% reduction of the original interval. These data independently confirm that a gene on chromosome 3A1-A3 increases susceptibility to HIVAN, resulting in early onset and rapid progression of kidney disease. These mice represent a new model to study the development and progression of collapsing glomerulopathy. Kidney International (2009) 75, 366–372; doi:10.1038/ki.2008.625; published online 17 December 2008 KEYWORDS: genetic susceptibility; HIV-associated nephropathy; mouse model
Correspondence: Ali G. Gharavi, Department of Medicine, Columbia University College of Physicians and Surgeons, 1150 St Nicholas Ave, Russ Berrie Pavillion 302 New York, New York 10032, USA. E-mail: [email protected] Received 31 July 2008; revised 1 October 2008; accepted 21 October 2008; published online 17 December 2008 366
HIV-1 infection is associated with a number of pathologically distinct glomerulopathies that can lead to renal failure.1–6 Among these glomerulopathies, HIV-associated nephropathy (HIVAN) is the most common form and is characterized by the development of collapsing glomerulopathy and microcystic tubular dilatation.5,6 Most patients with HIVAN progress to end-stage renal failure without antiretroviral therapy, directly implicating HIV-1 in the pathogenesis of disease.4,7,8 Data suggest that HIVAN is produced by HIV-1induced dysregulation of glomerular podocytes, the highly differentiated visceral epithelial cells lining the glomerular filtration barrier.4 In HIVAN, podocytes exhibit enhanced proliferation and apoptosis and lose signature proteins of maturity such as podocin and synaptopodin.7,9,10 The mechanism underlying the dysregulated podocyte phenotype is not fully understood. HIV-1 has been shown to interfere with multiple pathways that are critical for maintaining cellular quiescence, with the HIV-1 nef protein as an important pathogenic mediator.10–18 Host genetic factors also have an important impact on the course of HIV-1 infection and the development of complications.3,19,20 For example, the progression of HIV-1 disease is influenced by HLA genotype21 or copy number variation at the CCL3L1 locus,20 and the development of dementia is influenced by APOE alleles.22 Several lines of evidence also suggest that host genetic factors are also important for the development of HIVAN. Contrary to other glomerular diseases associated with HIV-1 infection, HIVAN almost exclusively occurs in individuals of African ancestry.1,2,23,24 Moreover, family members of HIVAN patients also have a higher incidence of end-stage renal disease, suggesting segregation of nephropathy-predisposing alleles in these families.25 Most recently, studies have shown that sequence variants in the podocyte-expressed non-muscle myosin heavy chain gene (MYH9) are associated with multiple nephropathies including HIVAN, explaining these epidemiological observations among African Americans.26,27 Kidney International (2009) 75, 366–372
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KT Chan et al.: Accelerated nephropathy in HIVAN1 congenic mice
Multiple murine models of HIVAN have been developed and highlight the importance of permissive genetic background on the development of this trait. Introduction of the HIV-1 proviral genome, or specific HIV-1 genes (such as nef) into the mouse genome reproduces all the clinical and pathological features of HIVAN.15–18,28 However, the development of disease is highly strain dependent. Studies using independent transgene constructs have shown that the FVB/ NJ (FVB) genetic background is highly permissive, resulting in all the manifestations of collapsing glomerulopathy.17,18,28,29 To examine the impact of host genetic factors, we previously produced F1 hybrids between HIV-1 transgenic FVB lines (TgFVB) and six inbred strains and found wide variation in penetrance of disease.29 Using a cross between TgFVB and the CAST/EiJ (CAST) strain, we identified a major susceptibility locus for HIVAN on chromosome 3A1–A3, which we named HIVAN1.29 Interestingly, at the HIVAN1 locus, CAST alleles were associated with increased severity of disease. However, the HIVAN1 95th percentile confidence interval spanned over 50 Mb, necessitating additional experiment for identification of the underlying gene. Derivation of congenic lines trapping susceptibility loci is a powerful approach for replication of linkage findings and subsequent elucidation of complex traits.30 Congenic lines are produced by introgressing chromosomal segments encompassing the quantitative trait loci (QTL) of interest from a donor strain into a recipient strain, thereby enabling analysis of alternative alleles of the QTL within the same genetic background.31 This procedure provides independent verification of the initial linkage studies, enables better characterization of the phenotypic effect imparted by the QTL, and facilitates gene identification by fine mapping or derivation of subcongenic lines.30,31 Toward identification of the HIVAN1 gene, we produced mice congenic for the HIVAN1 interval and report genetic and phenotypic analysis of this new model of nephropathy.
RESULTS
We produced HIVAN1 mice congenic (named the TgFVBHIVAN1CAST strain) by introducing the chromosome segment encompassing this interval from CAST into the FVB genome by 10 generations of backcrossing. A genome scan with 82 informative loci in the 10th backcross generation confirmed that the TgFVB-HIVAN1CAST strain possessed a pure FVB genetic background outside the introgressed region. The proximal and distal loci homozygous for CAST alleles were delimited by rs6372626 (located at 4.2 Mb on the mouse reference sequence) and rs13477119 (54.6 Mb), respectively; the next proximal and distal markers homozygous for FVB alleles were rs6171250 (3 Mb) and rs30305237 (55.8 Mb), respectively. Thus the size of the congenic segment spans from a minimum of 50.4 Mb to a maximum of 52.8 Mb. Nontransgenic FVB-HIVAN1CAST mice were phenotypically normal with no evidence of nephropathy or proteinuria. However, HIV-1 transgenic counterparts developed rapid onset of kidney disease (Figure 1) with B40% mortality rate at 8 weeks of age (compared to o5% for TgFVB). Moreover, by 4 weeks of age, HIV-1 transgenic TgFVB-HIVAN1CAST mice displayed increased incidence of proliferative epidermal lesions characteristic of animals expressing this HIV-1transgene construct,32 necessitating euthanasia based on humane criteria. Therefore, to prospectively study the impact of HIVAN1 alleles on the development of kidney disease, a cohort of TgFVB-HIVAN1CAST mice were studied from birth to 6 weeks of age and compared to TgFVB counterpart. At 3 weeks of age, all HIV-1 transgenic mice displayed significant proteinuria compared to nontransgenic mice, but homozygosity for CAST alleles was associated with a twofold increase in albuminuria (Po0.04; Figure 2a). At 6 weeks of age, TgFVB-HIVAN1CAST mice continued to display significantly greater proteinuria compared to TgFVB mice (Po0.02; Figure 2a). On histologic assessment, TgFVB-
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Figure 1 | Histology of TgFVB-HIVAN1CAST mice (PAS stain). Congenic mice display advanced global glomerulosclerosis (a–c) or collapsing glomerulosclerosis with podocyte hyperplasia (d–f). (a and d) Low power view ( 200) demonstrates diffuse and global glomerulosclerosis, extensive microcystic tubular dilatation and severe tubulointerstitial injury. (b and c) High power views ( 400) of glomeruli showing global sclerosis and podocyte depletion. Higher power views ( 600) of glomeruli showing collapsing sclerosis (e) with podocyte hyperplasia and cytoplasmic vacuolization (f). Kidney International (2009) 75, 366–372
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Figure 2 | Phenotypic comparison of TgFVB and TgFVB-HIVAN1 mice. Comparison of proteinuria (a) and glomerulosclerosis scores (b) in HIV-1 transgenic mice carrying different alleles at the HIVAN1 locus on chromosome 3A1–A3. Values for a cohort of nontransgenic (nonTg) mice of all three genotypes are shown for comparisons. (c) Comparison of HIV-1 Env expression in mice carrying different alleles at the HIVAN1 locus. #Po0.005, *Po0.05 vs Tg FVB/FVB genotype.
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Figure 3 | Fine mapping of the HIVAN1 locus. (a) LOD plots for renal histology scores in the F2 intercross between TgFVB and TgFVBHIVAN1CAST strains. Map distance is presented in cM. The positions of informative markers are shown above each plot. The dashed horizontal lines indicate the empiric threshold for 95th and 99th percentile significance level based on 10,000 permutations (1.9 and 3.2, respectively). The 95th confidence interval for the location of the HIVAN1 gene is shown above the LOD plot. (b) Phenotypic effects of HIVAN1 genotype in F2 cross is consistent with an additive model (mean±s.e.m.).
HIVAN1CAST demonstrated hallmarks of collapsing glomerulopathy, including tuft collapse and overlying podocyte proliferation and hypertrophy (Figure 1). Quantitative comparison of renal histopathology demonstrated significantly increased glomerulosclerosis (P ¼ 0.003; Figure 2b) and tubular injury with microcystic dilatation (P ¼ 0.002; Figure 2b) in TgFVB-HIVAN1CAST compared to TgFVB mice. Interestingly, 9/25 TgFVB-HIVAN1CAST animals also demonstrated global glomerulosclerosis with extensive podocyte depletion at 6 weeks of age, a finding that was not observed in their TgFVB counterparts (P ¼ 0.002 on Fisher’s exact test). Heterozygous mice also displayed increased pathology, intermediate between FVB/FVB and CAST/CAST, consistent with an additive effect of CAST alleles (Figure 2a and b). Previous studies in TgFVB mice have shown that the development of nephropathy coincides with increased expres368
sion of the HIV-1 transgene, followed by gradual reduction in expression secondary to loss of renal epithelial cells accompanying progressive renal injury.27 To assess whether increased severity of kidney disease in TgFVB-HIVAN1CAST mice is attributable to enhanced transgene expression, we quantitated transgene expression in 10 CAST/CAST and 10 FVB/FVB congenic mice with mild to moderate renal injury. These mice were matched for severity of renal histology scores to avoid bias introduced by secondary decrease in transgene expression due to nephropathy. There were no differences in transgene expression between the two genotype groups (Figure 2c), suggesting that increased severity of kidney disease is mediated through variation in host response to HIV-1 rather than enhanced expression of the transgene. These data demonstrate successful trapping of the HIVAN1 susceptibility alleles in our congenic strain, providKidney International (2009) 75, 366–372
KT Chan et al.: Accelerated nephropathy in HIVAN1 congenic mice
ing a novel tool for identifying the underlying susceptibility allele. We next utilized this strain to achieve further refinement of the HIVAN1 interval by meiotic mapping. We produced an F2 intercross between TgFVB and FVBHIVAN1CAST strains and phenotyped 68 F2 progeny. Genotypic analysis with seven informative markers demonstrated significant linkage of renal histology score with the HIVAN1 locus (peak LOD 3.7, empiric P ¼ 0.001 and 3.3, empiric P ¼ 0.004, at D3Mit224 for glomerular and tubular injury scores, respectively; Figure 3a). As a single interval was tested, these LOD scores achieve pointwise significance for replication (Po0.01) and remarkably, also exceed published genome-wide significance.33 In this cross, the HIVAN1 alleles demonstrated an additive effect (Figure 3b), explaining 20 and 12% of the variance in the glomerulosclerosis and tubular injury scores. The effect sizes were also consistent with observations in the congenic lines (Figure 3b). As this cross was produced on a homogeneous background, the linkage peak was much narrower than the original study, with the 95% confidence limit localizing the HIVAN1 gene within a B10 cM interval delimited by D3Mit167 and D3Mit67 (corresponding to B32–52 Mb position in mouse reference sequence), a region containing 126 genes. This represents a 60% reduction in the original HIVAN1 interval. As laboratory mouse strains are derived from a limited set of ancestors, comparison of haplotype structure across inbred strains can identify segments that are identical by descent (IBD), enabling further higher resolution mapping of linkage regions.34,35 We therefore examined the haplotype structure of the refined region and identified genes that contain informative single nucleotide polymorphisms (SNPs) between FVB and CAST. We identified 46,004 SNPs within the critical regions that are polymorphic between CAST and FVB (1 SNP/394 bp). Among these, 3684 SNPs are located within 78 NCBI annotated genes, but the remaining genes could not be studied due to lack of SNP data in either FVB or CAST strains. Among these 78 genes with non-IBD haplotypes, 22 genes harbor nonsynonymous variants, providing an initial selection tool for prioritization of genes within this interval (total of 95 SNPs; Supplementary Data). The high polymorphism rate between the CAST and FVB strains is consistent with the wild-derived origin of CAST strain and its relatively low contribution to the laboratory mouse genome, suggesting that comparison of IBD structure between FVB and CAST would not significantly refine the interval further.34 DISCUSSION
In this study, we characterized a novel congenic strain that exhibits rapid onset and increased severity of HIVAN. Introgression of CAST alleles at the HIVAN1 locus into the FVB genome resulted in earlier onset of disease, as evidenced by greater proteinuria at 3 weeks of age, worse histologic features and proteinuria at 6 weeks of age and early demise. In addition, many animals developed global glomerulosclerosis and podocyte depletion, a feature rarely observed in Kidney International (2009) 75, 366–372
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younger TgFVB mice. These congenic lines enabled better resolution of the QTL by isolating it in a homogeneous genetic background. Analysis of TgFVB-HIVAN1CAST congenic lines also enabled better characterization of the QTL effect size, demonstrating that it accounts for 12–20% of the variance in renal injury and is associated with a twofold higher glomerulosclerosis score and proteinuria by 6 weeks of age. These effects are very significant considering that our original mapping study relied on phenotypic characterization at 12 weeks of age, allowing more time for nephropathy to develop.29 Analysis of heterozygotes supported an additive genetic model, which was further confirmed by an F2 mapping cohort. These data independently confirm our initial mapping study, demonstrating that a gene on chromosome 3A1–A3 mediates susceptibility to HIVAN. The TgFVB-HIVAN1CAST congenic strain now offers a novel tool for identifying the gene(s) mediating increased susceptibility to HIVAN and studying the pathogenesis of this disease. As both TgFVB and TgFVB-HIVAN1CAST strains develop nephropathy, it is unclear whether HIVAN1 alleles directly contribute to the development of HIVAN or primarily affect progression of disease. Therefore, it will be interesting to determine whether HIVAN1 alleles exacerbate other forms of nephropathy, or whether similar to older haploinsufficient Cd2ap or Actn4 mice,36,37 the underlying susceptibility state will manifest with mild proteinuria or subtle ultrastructural defects in aged nontransgenic FVBHIVAN1CAST mice. Additional refinement of the HIVAN1 interval can be achieved by systematic derivation of subcongenic lines that can further dissect the region into smaller segments and can determine whether this genetic interval harbors a single- or multiple-linked QTLs. Alternatively, meiotic mapping in congenic lines can further resolve the interval to a tractable segment for gene identification. Although analysis of haplotype structure had previously been helpful for refinement of a nephropathy locus among classic laboratory strains,35 this strategy was not useful in this study because the wild-derived CAST strain has a relatively minor contribution to the laboratory mouse genome. Hence, most segments of the HIVAN1 interval harbor polymorphisms between FVB and CAST, complicating identification of the functional allele(s). Meiotic mapping appears to be the most feasible option, as demonstrated by significant linkage to smaller, well defined region obtained with our modest sized F2 cohort. This F2 cross indicated that a single QTL segregates within the HIVAN1 locus, suggesting that generation of larger mapping cohorts would rapidly resolve the interval further. An examination of positional candidates in the current 95% confidence interval reveals that nearly half are minimally annotated. Interestingly, ontology searches of the annotated genes reveal significant enrichment for genes encoding calcium dependent proteins (Anxa5, Trpc3, Pcdh18, Pcdh10, Cetn4, Fat4) or JAK-STAT signaling (Spry1, Il21, Il2). Other potentially relevant candidates include genes with known interaction with HIV-1 such as Fgf2, Pik3ca, Il2, and 369
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Ccna2, which are involved in some of the principle pathways affected by HIV-1 infection such as cell cycle regulation or cytokine production. We anticipate that gene expression analysis in FVB-HIVAN1CAST strains will provide additional information for prioritization of genes by identifying differentially expressed renal transcripts. Any genes that are differentially expressed and located within the critical interval would represent strong positional candidates that should be pursued by functional studies or transgenesis experiments. Collapsing glomerulopathy can be caused by many different primary insults to glomerular podocytes. In HIVAN and other forms of collapsing glomerulopathy, the proliferating podocytes are gradually depleted, undermining the architecture of the glomerular filtration barrier. Ultrastructurally, collapsing glomerulopathy is characterized by actin cytoskeletal effacement, whereas other forms of focal segmental glomerulosclerosis are associated with reorganization of the actin cytoskeleton into mats.38 Known environmental triggers for this lesion include infectious agents (HIV-1, cytomegalovirus) or drugs (bisphosphonates, interpheron-a).4,39 Inherited disorders featuring collapsing glomerulopathy have also been reported.40 In humans, noncoding variants in the MYH9 gene were recently associated with HIVAN, primary focal segmental glomerulosclerosis or nondiabetic end-stage renal failure.26,27 Mutations in the coding sequence of MYH9 have dominant, pleiotropic effects in extrarenal tissues but are sometimes associated with nephropathy.41 Interestingly, Myh9 haploinsufficient mice are phenotypically normal, suggesting that additional genetic or environmental modifier may be required for manifestation of the complications observed in humans.42,43 Myh9 is located on chromosome 15 in the mouse and is therefore distinct from the HIVAN1 gene, but given the close similarity of the TgFVB model to the human disease, it is likely that HIVAN1 operates in the same pathway as Myh9. Cross-breeding studies with HIV-1 transgenic mice and the FVB-HIVANCAST strain can determine the relationship among HIV-1, Myh9, and HIVAN1. In addition to HIV-1 transgenic mice, several animal models have also been developed, highlighting novel pathways underlying this lesion. For example, collapsing glomerulopathy in kd/kd mice is due to a mutation in the gene encoding prenyl diphosphate synthase, subunit 2 (Pdss2), resulting defective mitochondrial CoQ biosynthesis.44 Tissue specific expression of mutant alleles resulted in biochemical defects of CoQ9 and CoQ10 deficiency, but expression in podocytes was necessary and sufficient to recapitulate the full renal phenotype.45 Similarly, Henderson et al. have reported that mice homozygous for Actn4 null alleles, or a human disease associated Actn4 mutation also develop collapsing glomerulopathy, whereas mice heterozygous for the human mutation only manifest mild ultrastructural changes.37 Actn4 and Pssd2 operate in widely disparate pathways, complicating inference of a common mechanism for the development of collapsing glomerulopathy. Moreover, as both Actn4 and Pssd2 are ubiquitously expressed, the reasons for the unique 370
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vulnerability of podocytes are not known. It is generally hypothesized that podocytes are particularly susceptible to injury because they are terminally differentiated and cannot repopulate subsequent to genetic or environmental insults.4 The remaining podocytes must therefore undergo hypertrophy to cover a larger surface area, leaving denuded segments of the basement membrane that promote the development of sclerotic lesions. Finally, beyond as certain threshold, podocyte injury may spread locally, further propagating the segmental sclerotic process.46 However, the molecular pathways linking podocyte injury, cytoskeletal remodeling, propagation of injury, and glomerulosclerosis remains unclear. The availability of different models now enables systematic cross-comparative studies, which may provide additional insight into shared mechanisms underlying collapsing glomerulopathy. MATERIALS AND METHODS Animal breeding The HIV-1 transgenic mouse line TgN(pNL43d14)26Lom 26 (TgFVB) was produced on the inbred FVB/NJ genetic background and has been extensively characterized. The TgFVB colony at Columbia University has been continuously backcrossed to FVB/NJ for at least 30 generations. Congenic mice for the HIVAN1 locus were generated by generating F1 hybrid between FVB/NJ and CAST/ EiJ followed by 10 generations of backcrossing to FVB/NJ with selection of CAST alleles across the HIVAN1 locus at each generation. As HIV-1 transgenic mice develop kidney disease, wild-type FVB/NJ mice were utilized for nine generations of backcrossing, yielding mice that were heterozygotes for CAST alleles the HIVAN1 locus at each generation. We then utilized TgFVB for the 10th backcross generation, yielding HIV-1 transgenic mice with FVB/CAST genotype at the HIVAN1 locus. Trangenic heterozygous mice were intercrossed with nontransgenic heterozygous mice to yield transgenic mice with CAST/CAST, CAST/FVB, or FVB/FVB genotypes at the HIVAN1 locus. Mice carrying the full-length haplotype across the interval were selected for study. The markers utilized for derivation of the congenic intervals were D3Mit61 (5.1 Mb), D3Mit203 (26.8 Mb), and D3Mit67 (52.8 Mb). Subsequently the boundaries of the congenic region were better delineated by SNP genotyping. Successful introgression of the HIVAN1 interval into the FVB/NJ genetic background was further confirmed by typing 82 informative loci across the genome in the 10th backcross generation (Kbioscience, Hoddesdon, UK), which confirmed that congenic mice carried only FVB alleles outside the HIVAN1 interval. For characterization of congenic mice, the study endpoint was limited to 6 weeks of age because of increased mortality and development of skin lesions. Histology was compared between mice of differing genotypes at the HIVAN1 locus (FVB/FVB, N ¼ 30; FVB/CAST, N ¼ 26; CAST/CAST, N ¼ 22). In a subset of congenic mice, we quantitated albumin/creatinine ratio (Exocell, Philadelphia, PA, USA) at 3 and 6 weeks of age (FVB/FVB, N ¼ 16; FVB/ CAST, N ¼ 17; CAST/CAST, N ¼ 19). Fifteen nontransgenic mice of all three genotype class were also tested as controls. We also produced an independent cohort of 68 F2 mice for meiotic mapping. For this cross, we produced F1 hybrids between FVB-HIVAN1CAST congenic mice and TgFVB and cross-bred a transgenic to a nontransgenic F1 hybrid to generate F2 intercross mice. Renal histology was scored independently by an investigator Kidney International (2009) 75, 366–372
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(VDD) blinded to genetic background and other traits, scoring the severity of tubulointerstitial disease (tubular casts, tubular dilatation, epithelial regeneration/degeneration) and glomerular injury (segmental and global glomerulosclerosis and collapse, podocyte hyperplasia and collapse) using a continuous and a semiquantitative scale: 0 ¼ no disease, 1 ¼ 1–25% of glomeruli or tubular area showing abnormalities, 2 ¼ 26–50%, 3 ¼ 51–75%, and 4475% of tissue affected. The protocol was approved by the IACUC committee at the Columbia University Medical Center. Genotyping and analysis of linkage The F2 cohort was genotyped using seven informative microsatellite markers across the HIVAN1 locus (Figure 3). Multipoint analysis of linkage was performed using the R/QTL package.47 As the renal injury scores were not normally distributed, a nonparametric model was used. In addition, we conducted 10,000 permutations of phenotypes on genotype to determine the empirical significance of the linkage findings.
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Statistical and bioinformatics analysis Statistical analyses were performed using SPSS. The primary endpoints were the comparison of proteinuria and histologic injury between TgFVB and FVB-HIVAN1CAST strains using two-sided t-test. P-valuesp0.05 were considered significant. SNP ID and annotation across the HIVAN1 interval were obtained from the Mouse Phenome Database (http://aretha.jax.org/pub-cgi/phenome/ mpdcgi?rtn=docs/home). Non-IBD segments were identified by filtering for polymorphic SNP between FVB/NJ and CAST/eiJ strains. Gene annotation was performed using the PANTHER database (http://www.pantherdb.org/).
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DISCLOSURE
All the authors declared no competing interests. ACKNOWLEDGMENTS
This work is supported by NIH 1PO1 DK56492-01. AGG is supported by the Columbia Center for Glomerular diseases and the Emerald Foundation. SUPPLEMENTARY MATERIAL Supplementary Data Table S. Positional candidates within the 95th percent confidence interval in F2 cross. Table S1. Positional candidates with nonsynonymlous variants.
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Supplementary material is linked to the online version of the paper at http://www.nature.com/ki
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