Single-Cell Chromatin and Gene-Regulatory Dynamics of Mouse Nephron Progenitors.

BACKGROUND: We reasoned that unraveling the dynamic changes in accessibility of genomic regulatory elements and gene expression at single-cell resolution will inform the basic mechanisms of nephrogenesis. METHODS: We performed single-cell ATAC-seq and RNA-seq both individually (singleomes; Six2GFP cells) and jointly in the same cells (multiomes; kidneys) to generate integrated chromatin and transcriptional maps in mouse embryonic and neonatal nephron progenitor cells. RESULTS: We demonstrate that singleomes and multiomes are comparable in assigning most cell states, identification of new cell type markers, and defining the transcription factors driving cell identity. However, multiomes are more precise in defining the progenitor population. Multiomes identified a "pioneer" bHLH/Fox motif signature in nephron progenitor cells. Moreover, we identified a subset of Fox factors exhibiting high chromatin activity in podocytes. One of these Fox factors, Foxp1, is important for nephrogenesis. Key nephrogenic factors are distinguished by strong correlation between linked gene regulatory elements and gene expression. CONCLUSION: Mapping the regulatory landscape at single-cell resolution informs the regulatory hierarchy of nephrogenesis. Paired single-cell epigenomes and transcriptomes of nephron progenitors should provide a foundation to understand prenatal programming, regeneration after injury, and ex vivo nephrogenesis.

Mutations in PRDM15 Are a Novel Cause of Galloway-Mowat Syndrome.

BACKGROUND: Galloway-Mowat syndrome (GAMOS) is characterized by neurodevelopmental defects and a progressive nephropathy, which typically manifests as steroid-resistant nephrotic syndrome. The prognosis of GAMOS is poor, and the majority of children progress to renal failure. The discovery of monogenic causes of GAMOS has uncovered molecular pathways involved in the pathogenesis of disease. METHODS: Homozygosity mapping, whole-exome sequencing, and linkage analysis were used to identify mutations in four families with a GAMOS-like phenotype, and high-throughput PCR technology was applied to 91 individuals with GAMOS and 816 individuals with isolated nephrotic syndrome. In vitro and in vivo studies determined the functional significance of the mutations identified. RESULTS: Three biallelic variants of the transcriptional regulator PRDM15 were detected in six families with proteinuric kidney disease. Four families with a variant in the protein's zinc-finger (ZNF) domain have additional GAMOS-like features, including brain anomalies, cardiac defects, and skeletal defects. All variants destabilize the PRDM15 protein, and the ZNF variant additionally interferes with transcriptional activation. Morpholino oligonucleotide-mediated knockdown of Prdm15 in Xenopus embryos disrupted pronephric development. Human wild-type PRDM15 RNA rescued the disruption, but the three PRDM15 variants did not. Finally, CRISPR-mediated knockout of PRDM15 in human podocytes led to dysregulation of several renal developmental genes. CONCLUSIONS: Variants in PRDM15 can cause either isolated nephrotic syndrome or a GAMOS-type syndrome on an allelic basis. PRDM15 regulates multiple developmental kidney genes, and is likely to play an essential role in renal development in humans.

Biallelic Pathogenic GFRA1 Variants Cause Autosomal Recessive Bilateral Renal Agenesis.

BACKGROUND: Congenital anomalies of the kidney and urinary tract (CAKUT) are one of the most common malformations identified in the fetal stage. Bilateral renal agenesis (BRA) represents the most severe and fatal form of CAKUT. Only three genes have been confirmed to have a causal role in humans (ITGA8, GREB1L, and FGF20). METHODS: Genome sequencing within a diagnostic setting and combined data repository analysis identified a novel gene. RESULTS: Two patients presented with BRA, detected during the prenatal period, without additional recognizable malformations. They had parental consanguinity and similarly affected, deceased siblings, suggesting autosomal recessive inheritance. Evaluation of homozygous regions in patient 1 identified a novel, nonsense variant in GFRA1 (NM_001348097.1:c.676C>T, p.[Arg226*]). We identified 184 patients in our repository with renal agenesis and analyzed their exome/genome data. Of these 184 samples, 36 were from patients who presented with isolated renal agenesis. Two of them had loss-of-function variants in GFRA1. The second patient was homozygous for a frameshift variant (NM_001348097.1:c.1294delA, p.[Thr432Profs*13]). The GFRA1 gene encodes a receptor on the Wolffian duct that regulates ureteric bud outgrowth in the development of a functional renal system, and has a putative role in the pathogenesis of Hirschsprung disease. CONCLUSIONS: These findings strongly support the causal role of GFRA1-inactivating variants for an autosomal recessive, nonsyndromic form of BRA. This knowledge will enable early genetic diagnosis and better genetic counseling for families with BRA.

Uncovering Modifier Genes of X-Linked Alport Syndrome Using a Novel Multiparent Mouse Model.

BACKGROUND: Mutations in COL4A5 are responsible for 80% of cases of X-linked Alport Syndrome (XLAS). Although genes that cause AS are well characterized, people with AS who have similar genetic mutations present with a wide variation in the extent of kidney impairment and age of onset, suggesting the activities of modifier genes. METHODS: We created a cohort of genetically diverse XLAS male and female mice using the Diversity Outbred mouse resource and measured albuminuria, GFR, and gene expression. Using a quantitative trait locus approach, we mapped modifier genes that can best explain the underlying phenotypic variation measured in our diverse population. RESULTS: Genetic analysis identified several loci associated with the variation in albuminuria and GFR, including a locus on the X chromosome associated with X inactivation and a locus on chromosome 2 containing Fmn1. Subsequent analysis of genetically reduced Fmn1 expression in Col4a5 knockout mice showed a decrease in albuminuria, podocyte effacement, and podocyte protrusions in the glomerular basement membrane, which support the candidacy of Fmn1 as a modifier gene for AS. CONCLUSION: With this novel approach, we emulated the variability in the severity of kidney phenotypes found in human patients with Alport Syndrome through albuminuria and GFR measurements. This approach can identify modifier genes in kidney disease that can be used as novel therapeutic targets.

Copy Number Variant Analysis and Genome-wide Association Study Identify Loci with Large Effect for Vesicoureteral Reflux.

BACKGROUND: Vesicoureteral reflux (VUR) is a common, familial genitourinary disorder, and a major cause of pediatric urinary tract infection (UTI) and kidney failure. The genetic basis of VUR is not well understood. METHODS: A diagnostic analysis sought rare, pathogenic copy number variant (CNV) disorders among 1737 patients with VUR. A GWAS was performed in 1395 patients and 5366 controls, of European ancestry. RESULTS: Altogether, 3% of VUR patients harbored an undiagnosed rare CNV disorder, such as the 1q21.1, 16p11.2, 22q11.21, and triple X syndromes ((OR, 3.12; 95% CI, 2.10 to 4.54; P=6.35×10-8) The GWAS identified three study-wide significant and five suggestive loci with large effects (ORs, 1.41-6.9), containing canonical developmental genes expressed in the developing urinary tract (WDPCP, OTX1, BMP5, VANGL1, and WNT5A). In particular, 3.3% of VUR patients were homozygous for an intronic variant in WDPCP (rs13013890; OR, 3.65; 95% CI, 2.39 to 5.56; P=1.86×10-9). This locus was associated with multiple genitourinary phenotypes in the UK Biobank and eMERGE studies. Analysis of Wnt5a mutant mice confirmed the role of Wnt5a signaling in bladder and ureteric morphogenesis. CONCLUSIONS: These data demonstrate the genetic heterogeneity of VUR. Altogether, 6% of patients with VUR harbored a rare CNV or a common variant genotype conferring an OR >3. Identification of these genetic risk factors has multiple implications for clinical care and for analysis of outcomes in VUR.

mtor Haploinsufficiency Ameliorates Renal Cysts and Cilia Abnormality in Adult Zebrafish tmem67 Mutants.

BACKGROUND: Although zebrafish embryos have been used to study ciliogenesis and model polycystic kidney disease (PKD), adult zebrafish remain unexplored. METHODS: Transcription activator-like effector nucleases (TALEN) technology was used to generate mutant for tmem67, the homolog of the mammalian causative gene for Meckel syndrome type 3 (MKS3). Classic 2D and optical-clearing 3D imaging of an isolated adult zebrafish kidney were used to examine cystic and ciliary phenotypes. A hypomorphic mtor strain or rapamycin was used to inhibit mTOR activity. RESULTS: Adult tmem67 zebrafish developed progressive mesonephric cysts that share conserved features of mammalian cystogenesis, including a switch of cyst origin with age and an increase in proliferation of cyst-lining epithelial cells. The mutants had shorter and fewer distal single cilia and greater numbers of multiciliated cells (MCCs). Absence of a single cilium preceded cystogenesis, and expansion of MCCs occurred after pronephric cyst formation and was inversely correlated with the severity of renal cysts in young adult zebrafish, suggesting a primary defect and an adaptive action, respectively. Finally, the mutants exhibited hyperactive mTOR signaling. mTOR inhibition ameliorated renal cysts in both the embryonic and adult zebrafish models; however, it only rescued ciliary abnormalities in the adult mutants. CONCLUSIONS: Adult zebrafish tmem67 mutants offer a new vertebrate model for renal cystic diseases, in which cilia morphology can be analyzed at a single-nephron resolution and mTOR inhibition proves to be a candidate therapeutic strategy.

Integration of GWAS Summary Statistics and Gene Expression Reveals Target Cell Types Underlying Kidney Function Traits.

BACKGROUND: Genetic variants identified in genome-wide association studies (GWAS) are often not specific enough to reveal complex underlying physiology. By integrating RNA-seq data and GWAS summary statistics, novel computational methods allow unbiased identification of trait-relevant tissues and cell types. METHODS: The CKDGen consortium provided GWAS summary data for eGFR, urinary albumin-creatinine ratio (UACR), BUN, and serum urate. Genotype-Tissue Expression Project (GTEx) RNA-seq data were used to construct the top 10% specifically expressed genes for each of 53 tissues followed by linkage disequilibrium (LD) score-based enrichment testing for each trait. Similar procedures were performed for five kidney single-cell RNA-seq datasets from humans and mice and for a microdissected tubule RNA-seq dataset from rat. Gene set enrichment analyses were also conducted for genes implicated in Mendelian kidney diseases. RESULTS: Across 53 tissues, genes in kidney function-associated GWAS loci were enriched in kidney (P=9.1E-8 for eGFR; P=1.2E-5 for urate) and liver (P=6.8·10-5 for eGFR). In the kidney, proximal tubule was enriched in humans (P=8.5E-5 for eGFR; P=7.8E-6 for urate) and mice (P=0.0003 for eGFR; P=0.0002 for urate) and confirmed as the primary cell type in microdissected tubules and organoids. Gene set enrichment analysis supported this and showed enrichment of genes implicated in monogenic glomerular diseases in podocytes. A systematic approach generated a comprehensive list of GWAS genes prioritized by cell type-specific expression. CONCLUSIONS: Integration of GWAS statistics of kidney function traits and gene expression data identified relevant tissues and cell types, as a basis for further mechanistic studies to understand GWAS loci.

Pax2 and Pax8 Proteins Regulate Urea Transporters and Aquaporins to Control Urine Concentration in the Adult Kidney.

BACKGROUND: As the glomerular filtrate passes through the nephron and into the renal medulla, electrolytes, water, and urea are reabsorbed through the concerted actions of solute carrier channels and aquaporins at various positions along the nephron and in the outer and inner medulla. Proliferating stem cells expressing the nuclear transcription factor Pax2 give rise to renal epithelial cells. Pax2 expression ends once the epithelial cells differentiate into mature proximal and distal tubules, whereas expression of the related Pax8 protein continues. The collecting tubules and renal medulla are derived from Pax2-positive ureteric bud epithelia that continue to express Pax2 and Pax8 in adult kidneys. Despite the crucial role of Pax2 in renal development, functions for Pax2 or Pax8 in adult renal epithelia have not been established. METHODS: To examine the roles of Pax2 and Pax8 in the adult mouse kidney, we deleted either Pax2, Pax8, or both genes in adult mice and examined the resulting phenotypes and changes in gene expression patterns. We also explored the mechanism of Pax8-mediated activation of potential target genes in inner medullary collecting duct cells. RESULTS: Mice with induced deletions of both Pax2 and Pax8 exhibit severe polyuria that can be attributed to significant changes in the expression of solute carriers, such as the urea transporters encoded by Slc14a2, as well as aquaporins within the inner and outer medulla. Furthermore, Pax8 expression is induced by high-salt levels in collecting duct cells and activates the Slc14a2 gene by recruiting a histone methyltransferase complex to the promoter. CONCLUSIONS: These data reveal novel functions for Pax proteins in adult renal epithelia that are essential for retaining water and concentrating urine.

Novel Risk Loci Identified in a Genome-Wide Association Study of Urolithiasis in a Japanese Population.

BACKGROUND: A family history of urolithiasis is associated with a more than doubling of urolithiasis risk, and a twin study estimating 56% heritability of the condition suggests a pivotal role for host genetic factors. However, previous genome-wide association studies (GWAS) have identified only six risk-related loci. METHODS: To identify novel urolithiasis-related loci in the Japanese population, we performed a large-scale GWAS of 11,130 cases and 187,639 controls, followed by a replication analysis of 2289 cases and 3817 controls. Diagnosis of urolithiasis was confirmed either by a clinician or using medical records or self-report. We also assessed the association of urolithiasis loci with 16 quantitative traits, including metabolic, kidney-related, and electrolyte traits (such as body mass index, lipid storage, eGFR, serum uric acid, and serum calcium), using up to 160,000 samples from BioBank Japan. RESULTS: The analysis identified 14 significant loci, including nine novel loci. Ten regions showed a significant association with at least one quantitative trait, including metabolic, kidney-related, and electrolyte traits, suggesting a common genetic basis for urolithiasis and these quantitative traits. Four novel loci were related to metabolic traits, obesity, hypertriglyceridemia, or hyperuricemia. The remaining ten loci were associated with kidney- or electrolyte-related traits; these may affect crystallization. Weighted genetic risk score analysis indicated that the highest risk group (top 20%) showed an odds ratio of 1.71 (95% confidence interval, 1.42 to 2.06) - 2.13 (95% confidence interval, 2.00 to 2.27) compared with the reference group (bottom 20%). CONCLUSIONS: Our findings provide evidence that host genetic factors related to regulation of metabolic and crystallization pathways contribute to the development of urolithiasis.

Synergistic Genetic Interactions between Pkhd1 and Pkd1 Result in an ARPKD-Like Phenotype in Murine Models.

BACKGROUND: Autosomal recessive polycystic kidney disease (ARPKD) and autosomal dominant polycystic kidney disease (ADPKD) are genetically distinct, with ADPKD usually caused by the genes PKD1 or PKD2 (encoding polycystin-1 and polycystin-2, respectively) and ARPKD caused by PKHD1 (encoding fibrocystin/polyductin [FPC]). Primary cilia have been considered central to PKD pathogenesis due to protein localization and common cystic phenotypes in syndromic ciliopathies, but their relevance is questioned in the simple PKDs. ARPKD's mild phenotype in murine models versus in humans has hampered investigating its pathogenesis. METHODS: To study the interaction between Pkhd1 and Pkd1, including dosage effects on the phenotype, we generated digenic mouse and rat models and characterized and compared digenic, monogenic, and wild-type phenotypes. RESULTS: The genetic interaction was synergistic in both species, with digenic animals exhibiting phenotypes of rapidly progressive PKD and early lethality resembling classic ARPKD. Genetic interaction between Pkhd1 and Pkd1 depended on dosage in the digenic murine models, with no significant enhancement of the monogenic phenotype until a threshold of reduced expression at the second locus was breached. Pkhd1 loss did not alter expression, maturation, or localization of the ADPKD polycystin proteins, with no interaction detected between the ARPKD FPC protein and polycystins. RNA-seq analysis in the digenic and monogenic mouse models highlighted the ciliary compartment as a common dysregulated target, with enhanced ciliary expression and length changes in the digenic models. CONCLUSIONS: These data indicate that FPC and the polycystins work independently, with separate disease-causing thresholds; however, a combined protein threshold triggers the synergistic, cystogenic response because of enhanced dysregulation of primary cilia. These insights into pathogenesis highlight possible common therapeutic targets.

Identification of Acer2 as a First Susceptibility Gene for Lithium-Induced Nephrogenic Diabetes Insipidus in Mice.

BACKGROUND: Lithium, mainstay treatment for bipolar disorder, causes nephrogenic diabetes insipidus and hypercalcemia in about 20% and 10% of patients, respectively, and may lead to acidosis. These adverse effects develop in only a subset of patients treated with lithium, suggesting genetic factors play a role. METHODS: To identify susceptibility genes for lithium-induced adverse effects, we performed a genome-wide association study in mice, which develop such effects faster than humans. On day 8 and 10 after assigning female mice from 29 different inbred strains to normal chow or lithium diet (40 mmol/kg), we housed the animals for 48 hours in metabolic cages for urine collection. We also collected blood samples. RESULTS: In 17 strains, lithium treatment significantly elevated urine production, whereas the other 12 strains were not affected. Increased urine production strongly correlated with lower urine osmolality and elevated water intake. Lithium caused acidosis only in one mouse strain, whereas hypercalcemia was found in four strains. Lithium effects on blood pH or ionized calcium did not correlate with effects on urine production. Using genome-wide association analyses, we identified eight gene-containing loci, including a locus containing Acer2, which encodes a ceramidase and is specifically expressed in the collecting duct. Knockout of Acer2 led to increased susceptibility for lithium-induced diabetes insipidus development. CONCLUSIONS: We demonstrate that genome-wide association studies in mice can be used successfully to identify susceptibility genes for development of lithium-induced adverse effects. We identified Acer2 as a first susceptibility gene for lithium-induced diabetes insipidus in mice.

Early B Cell Factor 1 (EBF1) Regulates Glomerular Development by Controlling Mesangial Maturation and Consequently COX-2 Expression.

BACKGROUND: We recently showed the transcription factor Early B cell factor 1 (EBF1) is essential for the last stages of metanephric development, and that mice globally deficient in EBF1 display impaired maturation of peripheral glomeruli. EBF1 is present within multiple glomerular cell types, including the glomerular mesangium and podocytes. METHODS: To identify which cell type is driving the glomerular developmental defects in the global EBF1 knockout mice, we deleted EBF1 from the mesangium/pericytes (Foxd1-cre) or podocytes (Podocin-cre) in mice. RESULTS: Deletion of EBF1 from Foxd1 lineage cells resulted in hypoplastic kidneys, poorly differentiated peripheral glomeruli, and decreased proximal tubular mass in the outer cortex. Renal insufficiency was apparent at P21 when proteinuria presents, fibrosis of both the glomeruli and interstitium rapidly progresses, microthrombi appear, and hematuria develops. Approximately half of the Foxd1+, Ebf1fl/fl mice die before they are 3 months old. Mice with podocyte-targeted deletion of EBF1 exhibited no developmental abnormalities. Mice with Ebf1 deficiency in Foxd1 lineage cells shared characteristics with Ptgs2/COX-2-insufficient models, and mechanistic investigation revealed impaired calcineurin/NFATc1 activation and decreased COX-2 expression. Deletion of COX-2 from the interstitial/mesangial lineage displayed a less severe phenotype than EBF1 deficiency in mice. Overexpressing COX-2 in the EBF1-deficient mice, however, partially restored glomerular development. CONCLUSIONS: The results suggest that EBF1 regulates metanephric development at the last stages of glomerular maturation through its actions in the stromal progenitor (Foxd1+) lineage where it mediates proper regulation of calcineurin/NFAT signaling and COX-2 expression.

Sprouty1 Controls Genitourinary Development via its N-Terminal Tyrosine.

BACKGROUND: Studies in mice suggest that perturbations of the GDNF-Ret signaling pathway are a major genetic cause of congenital anomalies of the kidney and urinary tract (CAKUT). Mutations in Sprouty1, an intracellular Ret inhibitor, results in supernumerary kidneys, megaureters, and hydronephrosis in mice. But the underlying molecular mechanisms involved and which structural domains are essential for Sprouty1 function are a matter of controversy, partly because studies have so far relied on ectopic overexpression of the gene in cell lines. A conserved N-terminal tyrosine has been frequently, but not always, identified as critical for the function of Sprouty1 in vitro. METHODS: We generated Sprouty1 knockin mice bearing a tyrosine-to-alanine substitution in position 53, corresponding to the conserved N-terminal tyrosine of Sprouty1. We characterized the development of the genitourinary systems in these mice via different methods, including the use of reporter mice expressing EGFP from the Ret locus, and whole-mount cytokeratin staining. RESULTS: Mice lacking this tyrosine grow ectopic ureteric buds that will ultimately form supernumerary kidneys, a phenotype indistinguishable to that of Sprouty1 knockout mice. Sprouty1 knockin mice also present megaureters and vesicoureteral reflux, caused by failure of ureters to separate from Wolffian ducts and migrate to their definitive position. CONCLUSIONS: Tyrosine 53 is absolutely necessary for Sprouty1 function during genitourinary development in mice.

Protein Kinase 2ß Is Expressed in Neural Crest-Derived Urinary Pacemaker Cells and Required for Pyeloureteric Contraction.

Nonobstructive hydronephrosis, defined as dilatation of the renal pelvis with or without dilatation of the ureter, is the most common antenatal abnormality detected by fetal ultrasound. Yet, the etiology of nonobstructive hydronephrosis is poorly defined. We previously demonstrated that defective development of urinary tract pacemaker cells (utPMCs) expressing hyperpolarization-activated cyclic nucleotide-gated channel 3 (HCN3) and the stem cell marker cKIT causes abnormal ureteric peristalsis and nonobstructive hydronephrosis. However, further investigation of utPMC development and function is limited by lack of knowledge regarding the embryonic derivation, development, and molecular apparatus of these cells. Here, we used lineage tracing in mice to identify cells that give rise to utPMCs. Neural crest cells (NCCs) indelibly labeled with tdTomato expressed HCN3 and cKIT. Furthermore, purified HCN3+ and cKIT+ utPMCs were enriched in Sox10 and Tfap-2α, markers of NCCs. Sequencing of purified RNA from HCN3+ cells revealed enrichment of a small subset of RNAs, including RNA encoding protein kinase 2ß (PTK2ß), a Ca2+-dependent tyrosine kinase that regulates ion channel activity in neurons. Immunofluorescence analysis in situ revealed PTK2ß expression in NCCs as early as embryonic day 12.5 and in HCN3+ and cKIT+ utPMCs as early as embryonic day 15.5, with sustained expression in HCN3+ utPMCs until postnatal week 8. Pharmacologic inhibition of PTK2ß in murine pyeloureteral tissue explants inhibited contraction frequency. Together, these results demonstrate that utPMCs are derived from NCCs, identify new markers of utPMCs, and demonstrate a functional contribution of PTK2ß to utPMC function.

MYH9 E1841K Mutation Augments Proteinuria and Podocyte Injury and Migration.

Intronic variants of the MYH9 gene that encodes the nonmuscle myosin heavy chain IIA are associated with diabetic nephropathy in European Americans and with sickle cell disease-associated nephropathy. However, the causal functional variants of MYH9 have remained elusive. Rare missense mutations in MYH9 cause macrothrombocytopenia and are occasionally associated with development of nephropathy. The E1841K mutation is among the common MYH9 missense mutations and has been associated with nephropathy in some carriers. To determine the contribution of the E1841K mutation in kidney disease, we studied the effects of the E1841K mutation in mice subjected to high salt or angiotensin II (Ang II) as models of hypertension and in mice subjected to renal mass reduction as a model of CKD. Despite similar levels of BP among wild-type (MYH9+/+ ) mice and mice heterozygous (MYH9+/E1841K ) and homozygous (MYH9E1841K/E1841K ) for the mutation in each model, MYH9E1841K/E1841K mice exhibited mildly increased albuminuria in response to high salt; severe albuminuria, nephrinuria, FSGS, and podocyte foot effacement in Ang II-induced hypertension; and early mortality in the renal mass reduction model. Treatment with candesartan during Ang II-induced hypertension attenuated kidney disease development in MYH9E1841K/E1841K mice. In vitro, isolated primary podocytes from MYH9E1841K/E1841K mice exhibited increased lamellipodia formation and reorganization of F-actin stress fibers. Wound healing assays revealed that MYH9+/+ podocytes had the lowest migration rate, followed by MYH9+/E1841K then MYH9E1841K/E1841K podocytes. In conclusion, the MYH9 E1841K variant alters podocyte cytoskeletal structure and renders podocytes more susceptible to injury after a damaging stimulus.

Targeted Exome Sequencing Identifies PBX1 as Involved in Monogenic Congenital Anomalies of the Kidney and Urinary Tract.

Congenital anomalies of the kidney and urinary tract (CAKUT) occur in three to six of 1000 live births, represent about 20% of the prenatally detected anomalies, and constitute the main cause of CKD in children. These disorders are phenotypically and genetically heterogeneous. Monogenic causes of CAKUT in humans and mice have been identified. However, despite high-throughput sequencing studies, the cause of the disease remains unknown in most patients, and several studies support more complex inheritance and the role of environmental factors and/or epigenetics in the pathophysiology of CAKUT. Here, we report the targeted exome sequencing of 330 genes, including genes known to be involved in CAKUT and candidate genes, in a cohort of 204 unrelated patients with CAKUT; 45% of the patients were severe fetal cases. We identified pathogenic mutations in 36 of 204 (17.6%) patients. These mutations included five de novo heterozygous loss of function mutations/deletions in the PBX homeobox 1 gene (PBX1), a gene known to have a crucial role in kidney development. In contrast, the frequency of SOX17 and DSTYK variants recently reported as pathogenic in CAKUT did not indicate causality. These findings suggest that PBX1 is involved in monogenic CAKUT in humans and call into question the role of some gene variants recently reported as pathogenic in CAKUT. Targeted exome sequencing also proved to be an efficient and cost-effective strategy to identify pathogenic mutations and deletions in known CAKUT genes.

The Genetic Landscape of Renal Complications in Type 1 Diabetes.

Diabetes is the leading cause of ESRD. Despite evidence for a substantial heritability of diabetic kidney disease, efforts to identify genetic susceptibility variants have had limited success. We extended previous efforts in three dimensions, examining a more comprehensive set of genetic variants in larger numbers of subjects with type 1 diabetes characterized for a wider range of cross-sectional diabetic kidney disease phenotypes. In 2843 subjects, we estimated that the heritability of diabetic kidney disease was 35% (P=6.4×10-3). Genome-wide association analysis and replication in 12,540 individuals identified no single variants reaching stringent levels of significance and, despite excellent power, provided little independent confirmation of previously published associated variants. Whole-exome sequencing in 997 subjects failed to identify any large-effect coding alleles of lower frequency influencing the risk of diabetic kidney disease. However, sets of alleles increasing body mass index (P=2.2×10-5) and the risk of type 2 diabetes (P=6.1×10-4) associated with the risk of diabetic kidney disease. We also found genome-wide genetic correlation between diabetic kidney disease and failure at smoking cessation (P=1.1×10-4). Pathway analysis implicated ascorbate and aldarate metabolism (P=9.0×10-6), and pentose and glucuronate interconversions (P=3.0×10-6) in pathogenesis of diabetic kidney disease. These data provide further evidence for the role of genetic factors influencing diabetic kidney disease in those with type 1 diabetes and highlight some key pathways that may be responsible. Altogether these results reveal important biology behind the major cause of kidney disease.

Diversification of Cell Lineages in Ureter Development.

The mammalian ureter consists of a mesenchymal wall composed of smooth muscle cells and surrounding fibrocytes of the tunica adventitia and the lamina propria and an inner epithelial lining composed of layers of basal, intermediate, and superficial cells. How these cell types arise from multipotent progenitors is poorly understood. Here, we performed marker analysis, cell proliferation assays, and genetic lineage tracing to define the lineage relations and restrictions of the mesenchymal and epithelial cell types in the developing and mature mouse ureter. At embryonic day (E) 12.5, the mesenchymal precursor pool began to subdivide into an inner and outer compartment that began to express markers of smooth muscle precursors and adventitial fibrocytes, respectively, by E13.5. Smooth muscle precursors further diversified into lamina propria cells directly adjacent to the ureteric epithelium and differentiated smooth muscle cells from E16.5 onwards. Uncommitted epithelial progenitors of the ureter differentiated into intermediate cells at E14.5. After stratification into two layers at E15.5 and three cell layers at E18.5, intermediate cells differentiated into basal cells and superficial cells. In homeostasis, proliferation of all epithelial and mesenchymal cell types remained low but intermediate cells still gave rise to basal cells, whereas basal cells divided only into basal cells. These studies provide a framework to further determine the molecular mechanisms of cell differentiation in the tissues of the developing ureter.

Polymorphisms in α-Defensin-Encoding DEFA1A3 Associate with Urinary Tract Infection Risk in Children with Vesicoureteral Reflux.

The contribution of genetic variation to urinary tract infection (UTI) risk in children with vesicoureteral reflux is largely unknown. The innate immune system, which includes antimicrobial peptides, such as the α-defensins, encoded by DEFA1A3, is important in preventing UTIs but has not been investigated in the vesicoureteral reflux population. We used quantitative real-time PCR to determine DEFA1A3 DNA copy numbers in 298 individuals with confirmed UTIs and vesicoureteral reflux from the Randomized Intervention for Children with Vesicoureteral Reflux (RIVUR) Study and 295 controls, and we correlated copy numbers with outcomes. Outcomes studied included reflux grade, UTIs during the study on placebo or antibiotics, bowel and bladder dysfunction, and renal scarring. Overall, 29% of patients and 16% of controls had less than or equal to five copies of DEFA1A3 (odds ratio, 2.09; 95% confidence interval, 1.40 to 3.11; P<0.001). For each additional copy of DEFA1A3, the odds of recurrent UTI in patients receiving antibiotic prophylaxis decreased by 47% when adjusting for vesicoureteral reflux grade and bowel and bladder dysfunction. In patients receiving placebo, DEFA1A3 copy number did not associate with risk of recurrent UTI. Notably, we found that DEFA1A3 is expressed in renal epithelium and not restricted to myeloid-derived cells, such as neutrophils. In conclusion, low DEFA1A3 copy number associated with recurrent UTIs in subjects in the RIVUR Study randomized to prophylactic antibiotics, providing evidence that copy number polymorphisms in an antimicrobial peptide associate with UTI risk.

Refining Genotype-Phenotype Correlation in Autosomal Dominant Polycystic Kidney Disease.

Renal disease variability in autosomal dominant polycystic kidney disease (ADPKD) is strongly influenced by the gene locus (PKD1 versus PKD2). Recent studies identified nontruncating PKD1 mutations in approximately 30% of patients who underwent comprehensive mutation screening, but the clinical significance of these mutations is not well defined. We examined the genotype-renal function correlation in a prospective cohort of 220 unrelated ADPKD families ascertained through probands with serum creatinine ≤1.4 mg/dl at recruitment. We screened these families for PKD1 and PKD2 mutations and reviewed the clinical outcomes of the probands and affected family members. Height-adjusted total kidney volume (htTKV) was obtained in 161 affected subjects. Multivariate Cox proportional hazard modeling for renal and patient survival was performed in 707 affected probands and family members. Overall, we identified pathogenic mutations in 84.5% of our families, in which the prevalence of PKD1 truncating, PKD1 in-frame insertion/deletion, PKD1 nontruncating, and PKD2 mutations was 38.3%, 4.3%, 27.1%, and 30.3%, respectively. Compared with patients with PKD1 truncating mutations, patients with PKD1 in-frame insertion/deletion, PKD1 nontruncating, or PKD2 mutations have smaller htTKV and reduced risks (hazard ratio [95% confidence interval]) of ESRD (0.35 [0.14 to 0.91], 0.10 [0.05 to 0.18], and 0.03 [0.01 to 0.05], respectively) and death (0.31 [0.11 to 0.87], 0.20 [0.11 to 0.38], and 0.18 [0.11 to 0.31], respectively). Refined genotype-renal disease correlation coupled with targeted next generation sequencing of PKD1 and PKD2 may provide useful clinical prognostication for ADPKD.