Mutations in DSTYK and dominant urinary tract malformations.

BACKGROUND: Congenital abnormalities of the kidney and the urinary tract are the most common cause of pediatric kidney failure. These disorders are highly heterogeneous, and the etiologic factors are poorly understood. METHODS: We performed genomewide linkage analysis and whole-exome sequencing in a family with an autosomal dominant form of congenital abnormalities of the kidney or urinary tract (seven affected family members). We also performed a sequence analysis in 311 unrelated patients, as well as histologic and functional studies. RESULTS: Linkage analysis identified five regions of the genome that were shared among all affected family members. Exome sequencing identified a single, rare, deleterious variant within these linkage intervals, a heterozygous splice-site mutation in the dual serine-threonine and tyrosine protein kinase gene (DSTYK). This variant, which resulted in aberrant splicing of messenger RNA, was present in all affected family members. Additional, independent DSTYK mutations, including nonsense and splice-site mutations, were detected in 7 of 311 unrelated patients. DSTYK is highly expressed in the maturing epithelia of all major organs, localizing to cell membranes. Knockdown in zebrafish resulted in developmental defects in multiple organs, which suggested loss of fibroblast growth factor (FGF) signaling. Consistent with this finding is the observation that DSTYK colocalizes with FGF receptors in the ureteric bud and metanephric mesenchyme. DSTYK knockdown in human embryonic kidney cells inhibited FGF-stimulated phosphorylation of extracellular-signal-regulated kinase (ERK), the principal signal downstream of receptor tyrosine kinases. CONCLUSIONS: We detected independent DSTYK mutations in 2.3% of patients with congenital abnormalities of the kidney or urinary tract, a finding that suggests that DSTYK is a major determinant of human urinary tract development, downstream of FGF signaling. (Funded by the National Institutes of Health and others.).

A retrotransposon insertion in the 5' regulatory domain of Ptf1a results in ectopic gene expression and multiple congenital defects in Danforth's short tail mouse.

Danforth's short tail mutant (Sd) mouse, first described in 1930, is a classic spontaneous mutant exhibiting defects of the axial skeleton, hindgut, and urogenital system. We used meiotic mapping in 1,497 segregants to localize the mutation to a 42.8-kb intergenic segment on chromosome 2. Resequencing of this region identified an 8.5-kb early retrotransposon (ETn) insertion within the highly conserved regulatory sequences upstream of Pancreas Specific Transcription Factor, 1a (Ptf1a). This mutation resulted in up to tenfold increased expression of Ptf1a as compared to wild-type embryos at E9.5 but no detectable changes in the expression levels of other neighboring genes. At E9.5, Sd mutants exhibit ectopic Ptf1a expression in embryonic progenitors of every organ that will manifest a developmental defect: the notochord, the hindgut, and the mesonephric ducts. Moreover, at E 8.5, Sd mutant mice exhibit ectopic Ptf1a expression in the lateral plate mesoderm, tail bud mesenchyme, and in the notochord, preceding the onset of visible defects such as notochord degeneration. The Sd heterozygote phenotype was not ameliorated by Ptf1a haploinsufficiency, further suggesting that the developmental defects result from ectopic expression of Ptf1a. These data identify disruption of the spatio-temporal pattern of Ptf1a expression as the unifying mechanism underlying the multiple congenital defects in Danforth's short tail mouse. This striking example of an enhancer mutation resulting in profound developmental defects suggests that disruption of conserved regulatory elements may also contribute to human malformation syndromes.

Identification of the nephropathy-susceptibility locus HIVAN4.

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 genes. 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 chromosome 6 G3-F3, with BALB alleles conferring a twofold reduction in severity (peak LOD score = 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 gene products are enriched in the glomerulus and interact with HIV-1 proteins. 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.

APOL1 variants increase risk for FSGS and HIVAN but not IgA nephropathy.

A chromosome 22q13 locus strongly associates with increased risk for idiopathic focal segmental glomerulosclerosis (FSGS), HIV-1-associated nephropathy (HIVAN), and hypertensive ESRD among individuals of African descent. Although initial studies implicated MYH9, more recent analyses localized the strongest association within the neighboring APOL1 gene. In this replication study, we examined the six top-most associated variants in APOL1 and MYH9 in an independent cohort of African Americans with various nephropathies (44 with FSGS, 21 with HIVAN, 32 with IgA nephropathy, and 74 healthy controls). All six variants associated with FSGS and HIVAN (additive ORs, 1.8 to 3.0; P values 3 × 10(-2) to 5 × 10(-5)) but not with IgA nephropathy. In conditional and haplotype analyses, two APOL1 haplotypes accounted for virtually all of the association with FSGS and HIVAN on chromosome 22q13 (haplotype P value = 5.6 × 10(-8)). To assess the role of MYH9 deficiency in nephropathy, we crossbred Myh9-haploinsufficient mice (Myh9(+/-)) with HIV-1 transgenic mice. Myh9(+/-) mice were healthy and did not demonstrate overt proteinuria or nephropathy, irrespective of the presence of the HIV-1 transgene. These data further support the strong association of genetic variants in APOL1 with susceptibility to FSGS and HIVAN among African Americans.

Accelerated development of collapsing glomerulopathy in mice congenic for the HIVAN1 locus.

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-HIVAN1(CAST) 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=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.

Long-term survival of transplanted allogeneic cells engineered to express a T cell chemorepellent.

BACKGROUND: Alloantigen specific T cells have been shown to be required for allograft rejection. The chemokine, stromal cell derived factor-1 (SDF-1) at high concentration, has been shown to act as a T-cell chemorepellent and abrogate T-cell infiltration into a site of antigen challenge in vivo via a mechanism termed fugetaxis or chemorepulsion. We postulated that this mechanism could be exploited therapeutically and that allogeneic cells engineered to express a chemorepellent protein would not be rejected. METHODS: Allogeneic murine insulinoma beta-TC3 cells and primary islets from BALB/C mice were engineered to constitutively secrete differential levels of SDF-1 and transplanted into allogeneic diabetic C57BL/6 mice. Rejection was defined as the permanent return of hyperglycemia and was correlated with the level of T-cell infiltration. The migratory response of T-cells to SDF-1 was also analyzed by transwell migration assay and time-lapse videomicroscopy. The cytotoxicity of cytotoxic T cell (CTLs) against beta-TC3 cells expressing high levels of SDF-1 was measured in standard and modified chromium-release assays in order to determine the effect of CTL migration on killing efficacy. RESULTS: Control animals rejected allogeneic cells and remained diabetic. In contrast, high level SDF-1 production by transplanted cells resulted in increased survival of the allograft and a significant reduction in blood glucose levels and T-cell infiltration into the transplanted tissue. CONCLUSIONS: This is the first demonstration of a novel approach that exploits T-cell chemorepulsion to induce site specific immune isolation and thereby overcomes allograft rejection without the use of systemic immunosuppression.

Refinement of the HIVAN1 Susceptibility Locus on Chr. 3A1-A3 via Generation of Sub-Congenic Strains.

HIV-1 transgenic mice on the FVB/NJ background (TgFVB) represent a validated model of HIV-associated nephropathy (HIVAN). A major susceptibility locus, HIVAN1, was previously mapped to chromosome 3A1-A3 in a cross between TgFVB and CAST/EiJ (CAST) strains, and introgression of a 51.9 Mb segment encompassing HIVAN1 from CAST into TgFVB resulted in accelerated development of nephropathy. We generated three sub-congenic strains carrying CAST alleles in the proximal or distal regions of the HIVAN1 locus (Sub-II, 3.02-38.93 Mb; Sub-III, 38.45-55.1 Mb and Sub-IV, 47.7-55.1 Mb, build 38). At 5-10 weeks of age, histologic injury and proteinuria did not differ between HIV-1 transgenic Sub-II and TgFVB mice. In contrast, HIV-1 transgenic Sub-III and Sub-IV mice displayed up to 4.4 fold more histopathologic injury and 6-fold more albuminuria compared to TgFVB mice, similar in severity to the full-length congenic mice. The Sub-IV segment defines a maximal 7.4 Mb interval for HIVAN1, and encodes 31 protein coding genes: 15 genes have missense variants differentiating CAST from FVB, and 14 genes show differential renal expression. Of these, Frem1, Foxo1, and Setd7 have been implicated in the pathogenesis of nephropathy. HIVAN1 congenic kidneys are histologically normal without the HIV-1 transgene, yet their global transcriptome is enriched for molecular signatures of apoptosis, adenoviral infection, as well as genes repressed by histone H3 lysine 27 trimethylation, a histone modification associated with HIV-1 life cycle. These data refine HIVAN1to 7.4 Mb and identify latent molecular derangements that may predispose to nephropathy upon exposure to HIV-1.

The molecular pathogenesis of HIV-1 associated nephropathy: recent advances.

HIV-1-associated nephropathy (HIVAN) is a major complication of HIV-1 infection, frequently resulting in kidney failure. HIVAN arises due to HIV-1-induced dysregulation of podocytes, the glomerular epithelial cells that establish and maintain the kidney filtration barrier. Host genetic factors are important for the development of HIVAN. The risk of HIVAN is greatest in populations of African ancestry, and is attributable to a genetic variation at the APOL1 locus on chromosome 22. Mouse models of HIVAN enable delineation of dysregulated pathways underlying disease. Identification of HIVAN susceptibility loci in a mouse model, combined with expression quantitative trait locus mapping, has demonstrated that murine HIVAN loci transregulate podocyte gene expression. HIV-1 induces perturbations in podocyte expression response, suggesting that HIV-1 potentially interferes with compensatory pathways that normally restore cellular homeostasis in the face of genetic mutations. These findings present a framework for identification of podocyte transregulators and reconstruction of the molecular networks connecting susceptibility genes to the development of nephropathy.

Reversible cell-cycle entry in adult kidney podocytes through regulated control of telomerase and Wnt signaling.

Mechanisms of epithelial cell renewal remain poorly understood in the mammalian kidney, particularly in the glomerulus, a site of cellular damage in chronic kidney disease. Within the glomerulus, podocytes--differentiated epithelial cells crucial for filtration--are thought to lack substantial capacity for regeneration. Here we show that podocytes rapidly lose differentiation markers and enter the cell cycle in adult mice in which the telomerase protein component TERT is conditionally expressed. Transgenic TERT expression in mice induces marked upregulation of Wnt signaling and disrupts glomerular structure, resulting in a collapsing glomerulopathy resembling those in human disease, including HIV-associated nephropathy (HIVAN). Human and mouse HIVAN kidneys show increased expression of TERT and activation of Wnt signaling, indicating that these are general features of collapsing glomerulopathies. Silencing transgenic TERT expression or inhibiting Wnt signaling through systemic expression of the Wnt inhibitor Dkk1 in either TERT transgenic mice or in a mouse model of HIVAN results in marked normalization of podocytes, including rapid cell-cycle exit, re-expression of differentiation markers and improved filtration barrier function. These data reveal an unexpected capacity of podocytes to reversibly enter the cell cycle, suggest that podocyte renewal may contribute to glomerular homeostasis and implicate the telomerase and Wnt-ß-catenin pathways in podocyte proliferation and disease.

Prkdc participates in mitochondrial genome maintenance and prevents Adriamycin-induced nephropathy in mice.

Adriamycin (ADR) is a commonly used chemotherapeutic agent that also produces significant tissue damage. Mutations to mitochondrial DNA (mtDNA) and reductions in mtDNA copy number have been identified as contributors to ADR-induced injury. ADR nephropathy only occurs among specific mouse inbred strains, and this selective susceptibility to kidney injury maps as a recessive trait to chromosome 16A1-B1. Here, we found that sensitivity to ADR nephropathy in mice was produced by a mutation in the Prkdc gene, which encodes a critical nuclear DNA double-stranded break repair protein. This finding was confirmed in mice with independent Prkdc mutations. Overexpression of Prkdc in cultured mouse podocytes significantly improved cell survival after ADR treatment. While Prkdc protein was not detected in mitochondria, mice with Prkdc mutations showed marked mtDNA depletion in renal tissue upon ADR treatment. To determine whether Prkdc participates in mtDNA regulation, we tested its genetic interaction with Mpv17, which encodes a mitochondrial protein mutated in human mtDNA depletion syndromes (MDDSs). While single mutant mice were asymptomatic, Prkdc/Mpv17 double-mutant mice developed mtDNA depletion and recapitulated many MDDS and ADR injury phenotypes. These findings implicate mtDNA damage in the development of ADR toxicity and identify Prkdc as a MDDS modifier gene and a component of the mitochondrial genome maintenance pathway.

Susceptibility loci for murine HIV-associated nephropathy encode trans-regulators of podocyte gene expression.

Multiple studies have linked podocyte gene variants to diverse sporadic nephropathies, including HIV-1-associated nephropathy (HIVAN). We previously used linkage analysis to identify a major HIVAN susceptibility locus in mouse, HIVAN1. We performed expression quantitative trait locus (eQTL) analysis of podocyte genes in HIV-1 transgenic mice to gain further insight into genetic susceptibility to HIVAN. In 2 independent crosses, we found that transcript levels of the podocyte gene nephrosis 2 homolog (Nphs2), were heritable and controlled by an ancestral cis-eQTL that conferred a 3-fold variation in expression and produced reactive changes in other podocyte genes. In addition, Nphs2 expression was controlled by 2 trans-eQTLs that localized to the nephropathy susceptibility intervals HIVAN1 and HIVAN2. Transregulation of podocyte genes was observed in the absence of HIV-1 or glomerulosclerosis, indicating that nephropathy susceptibility alleles induce latent perturbations in the podocyte expression network. Presence of the HIV-1 transgene interfered with transregulation, demonstrating effects of gene-environment interactions on disease. These data demonstrate that transcript levels of Nphs2 and related podocyte-expressed genes are networked and suggest that the genetic lesions introduced by HIVAN susceptibility alleles perturb this regulatory pathway and transcriptional responses to HIV-1, increasing susceptibility to nephropathy.

Murine B16 melanomas expressing high levels of the chemokine stromal-derived factor-1/CXCL12 induce tumor-specific T cell chemorepulsion and escape from immune control.

The chemokine, stromal-derived factor-1/CXCL12, is expressed by normal and neoplastic tissues and is involved in tumor growth, metastasis, and modulation of tumor immunity. T cell-mediated tumor immunity depends on the migration and colocalization of CTL with tumor cells, a process regulated by chemokines and adhesion molecules. It has been demonstrated that T cells are repelled by high concentrations of the chemokine CXCL12 via a concentration-dependent and CXCR4 receptor-mediated mechanism, termed chemorepulsion or fugetaxis. We proposed that repulsion of tumor Ag-specific T cells from a tumor expressing high levels of CXCL12 allows the tumor to evade immune control. Murine B16/OVA melanoma cells (H2b) were engineered to constitutively express CXCL12. Immunization of C57BL/6 mice with B16/OVA cells lead to destruction of B16/OVA tumors expressing no or low levels of CXCL12 but not tumors expressing high levels of the chemokine. Early recruitment of adoptively transferred OVA-specific CTL into B16/OVA tumors expressing high levels of CXCL12 was significantly reduced in comparison to B16/OVA tumors, and this reduction was reversed when tumor-specific CTLs were pretreated with the specific CXCR4 antagonist, AMD3100. Memory OVA-specific CD8+ T cells demonstrated antitumor activity against B16/OVA tumors but not B16/OVA.CXCL12-high tumors. Expression of high levels of CXCL12 by B16/OVA cells significantly reduced CTL colocalization with and killing of target cells in vitro in a CXCR4-dependent manner. The repulsion of tumor Ag-specific T cells away from melanomas expressing CXCL12 confirms the chemorepellent activity of high concentrations of CXCL12 and may represent a novel mechanism by which certain tumors evade the immune system.