Associations between outdoor temperature and bright sunlight with metabolites in two population-based European cohorts.

BACKGROUND AND AIMS: Outdoor temperature and bright sunlight may directly and/or indirectly modulate systemic metabolism. We assessed the associations between outdoor temperature and bright sunlight duration with metabolomics. METHODS AND RESULTS: Cross-sectional analyses were undertaken in non-diabetic individuals from the Oxford BioBank (OBB; N = 6368; mean age 47.0 years, males 44%) and the Netherlands Epidemiology of Obesity (NEO; N = 5916; mean age 55.6 years, males 43%) study. Data on mean outdoor bright sunlight and temperature were collected from local weather stations in the week prior to blood sampling. Fasting serum levels of 148 metabolites, including 14 lipoprotein subclasses, were measured using NMR spectroscopy. Linear regression analyses were performed to assess the associations between mean outdoor temperature and bright sunlight duration with metabolomics adjusted for age, sex, body mass index, season and either outdoor temperature or bright sunlight. A higher mean outdoor temperature was associated with increased serum concentrations of lipoprotein (sub)particles (ß (SE) = 0.064 (0.018) SD per 5 °C, p = 5.03e-4) and certain amino acids such as phenylalanine (0.066 (0.016) SD, p = 6.44e-05) and leucine (0.111 (0.018) SD, p = 1.25e-09). In contrast, longer duration of bright sunlight was specifically associated with lower concentrations of very low-density lipoprotein (sub)particles (e.g., VLDL cholesterol (-0.024 (0.005) SD per 1-h bright sunlight, p = 8.06e-6)). The direction of effects was generally consistent between the OBB and NEO, although effect sizes were generally larger in the OBB. CONCLUSIONS: Increased bright sunlight duration is associated with an improved metabolic profile whilst higher outdoor temperature may adversely impact cardiometabolic health.

Investigating the impact of metabolic syndrome traits on telomere length: a Mendelian randomization study.

OBJECTIVE: Observational studies have reported bidirectional associations between metabolic syndrome (MetS) traits and short leukocyte telomere length (LTL), a TL marker in somatic tissues and a proposed risk factor for age-related degenerative diseases. However, in Mendelian randomization studies, longer LTL has been paradoxically associated with higher MetS risk. This study investigated the hypothesis that shorter LTL might be a consequence of metabolic dysfunction. METHODS: This study undertook univariable and multivariable Mendelian randomization. As instrumental variables for MetS traits, all of the genome-wide significant independent signals identified in genome-wide association studies for anthropometric, glycemic, lipid, and blood pressure traits conducted in European individuals were used. Summary-level data for LTL were obtained from a genome-wide association study conducted in the UK Biobank. RESULTS: Higher BMI was associated with shorter LTL (ß = -0.039, 95% CI: -0.058 to -0.020, p = 5 × 10-5 ) equivalent to 1.70 years of age-related LTL change. In contrast, higher low-density lipoprotein cholesterol was associated with longer LTL (ß = 0.022, 95% CI: 0.007 to 0.037, p = 0.003) equivalent to 0.96 years of age-related LTL change. Mechanistically, increased low-grade systemic inflammation, as measured by circulating C-reactive protein, and lower circulating linoleic acid levels might link higher BMI to shorter LTL. CONCLUSIONS: Overweight and obesity might promote the development of aging-related degenerative diseases by accelerating telomere shortening.

Obesity, Fat Distribution and Risk of Cancer in Women and Men: A Mendelian Randomisation Study.

Obesity and upper-body fat distribution are independent, cardiometabolic risk factors but whether they also display comparable associations with cancer risk is unknown. We investigated the causal relationships between body mass index (BMI) and BMI-adjusted waist-to-hip ratio (WHRadjBMI) and cancer risk and searched for potential drivers linking these traits to carcinogenesis using two-sample and multivariable Mendelian randomisation. In women, genetically instrumented higher BMI was associated with lower breast (OR = 0.87, 95% CI 0.81-0.93) and higher endometrial (OR = 1.75, 95% CI 1.55-1.96) cancer risk whilst WHRadjBMI was associated with higher colon cancer risk (OR = 1.22, 95% CI 1.07-1.42). In men, elevated BMI was associated with lower prostate cancer risk (OR = 0.91, 95% CI 0.85-0.98). Mechanistically, testosterone and insulin mediated 21% and 35%, respectively of the total, genetically determined association of BMI with endometrial cancer risk whilst HDL cholesterol and IGF-1 mediated 40% and 22%, respectively of the association between BMI and breast cancer risk. In men, testosterone mediated 21% of the association between BMI and prostate cancer risk. Colon cancer aside, the total amount of body fat might be more important than its location in modulating cancer susceptibility due to differential effects of obesity and fat distribution on adiposity-associated cancer drivers.

Sex hormones, adiposity, and metabolic traits in men and women: a Mendelian randomisation study.

OBJECTIVE: Epidemiological and clinical studies have highlighted important roles for sex hormones in the regulation of fat distribution and systemic metabolism. We investigated the bidirectional associations between bioavailable serum testosterone (BioT) in both sexes and oestradiol (E2) in men and adiposity and metabolic traits using Mendelian randomisation (MR). DESIGN AND METHODS: As genetic instruments for sex hormones, we selected all the genome-wide significant, independent signals from a genome-wide association studies (GWAS) in up to 425 097 European ancestry UK Biobank participants. European population-specific, summary-level data for adiposity, metabolic, and blood pressure traits were obtained from the largest publicly available GWAS. Sex-specific, two-sample MR analyses were used to estimate the associations of sex hormones with these traits and vice versa. RESULTS: In women, higher BioT was associated with obesity, upper-body fat distribution, and low HDL-cholesterol although, based on analyses modelling the sex hormone-binding globulin-independent effects of BioT, the last two associations might be indirect. Conversely, obesity and android fat distribution were associated with elevated serum BioT. In men, higher BioT was associated with lower hip circumference and lower fasting glucose. Reciprocally, obesity was associated with lower BioT and higher E2, while upper-body fat distribution and raised triglycerides were associated with lower E2. CONCLUSIONS: Adipose tissue and metabolic dysfunction are associated with deranged sex hormone levels in both sexes. In women, elevated BioT might be a cause of obesity. Conversely, in men, higher BioT appears to have beneficial effects on adiposity and glucose metabolism.

TCF7L2 plays a complex role in human adipose progenitor biology, which might contribute to genetic susceptibility to type 2 diabetes.

INTRODUCTION: Non-coding genetic variation at TCF7L2 is the strongest genetic determinant of type 2 diabetes (T2D) risk in humans. TCF7L2 encodes a transcription factor mediating the nuclear effects of WNT signaling in adipose tissue (AT). In vivo studies in transgenic mice have highlighted important roles for TCF7L2 in adipose tissue biology and systemic metabolism. OBJECTIVE: To map the expression of TCF7L2 in human AT, examine its role in human adipose cell biology in vitro, and investigate the effects of the fine-mapped T2D-risk allele at rs7903146 on AT morphology and TCF7L2 expression. METHODS: Ex vivo gene expression studies of TCF7L2 in whole and fractionated human AT. In vitro TCF7L2 gain- and/or loss-of-function studies in primary and immortalized human adipose progenitor cells (APCs) and mature adipocytes (mADs). AT phenotyping of rs7903146 T2D-risk variant carriers and matched controls. RESULTS: Adipose progenitors (APs) exhibited the highest TCF7L2 mRNA abundance compared to mature adipocytes and adipose-derived endothelial cells. Obesity was associated with reduced TCF7L2 transcript levels in whole subcutaneous abdominal AT but paradoxically increased expression in APs. In functional studies, TCF7L2 knockdown (KD) in abdominal APs led to dose-dependent activation of WNT/ß-catenin signaling, impaired proliferation and dose-dependent effects on adipogenesis. Whilst partial KD enhanced adipocyte differentiation, near-total KD impaired lipid accumulation and adipogenic gene expression. Over-expression of TCF7L2 accelerated adipogenesis. In contrast, TCF7L2-KD in gluteal APs dose-dependently enhanced lipid accumulation. Transcriptome-wide profiling revealed that TCF7L2 might modulate multiple aspects of AP biology including extracellular matrix secretion, immune signaling and apoptosis. The T2D-risk allele at rs7903146 was associated with reduced AP TCF7L2 expression and enhanced AT insulin sensitivity. CONCLUSIONS: TCF7L2 plays a complex role in AP biology and has both dose- and depot-dependent effects on adipogenesis. In addition to regulating pancreatic insulin secretion, genetic variation at TCF7L2 might also influence T2D risk by modulating AP function.

HOTAIR interacts with PRC2 complex regulating the regional preadipocyte transcriptome and human fat distribution.

Mechanisms governing regional human adipose tissue (AT) development remain undefined. Here, we show that the long non-coding RNA HOTAIR (HOX transcript antisense RNA) is exclusively expressed in gluteofemoral AT, where it is essential for adipocyte development. We find that HOTAIR interacts with polycomb repressive complex 2 (PRC2) and we identify core HOTAIR-PRC2 target genes involved in adipocyte lineage determination. Repression of target genes coincides with PRC2 promoter occupancy and H3K27 trimethylation. HOTAIR is also involved in modifying the gluteal adipocyte transcriptome through alternative splicing. Gluteal-specific expression of HOTAIR is maintained by defined regions of open chromatin across the HOTAIR promoter. HOTAIR expression levels can be modified by hormonal (estrogen, glucocorticoids) and genetic variation (rs1443512 is a HOTAIR eQTL associated with reduced gynoid fat mass). These data identify HOTAIR as a dynamic regulator of the gluteal adipocyte transcriptome and epigenome with functional importance for human regional AT development.

Telomere length and metabolic syndrome traits: A Mendelian randomisation study.

Observational studies have revealed associations between short leucocyte telomere length (LTL), a TL marker in somatic tissues and multiple Metabolic Syndrome (MetS) traits. Animal studies have supported these findings by showing that increased telomere attrition leads to adipose tissue dysfunction and insulin resistance. We investigated the associations between genetically instrumented LTL and MetS traits using Mendelian Randomisation (MR). Fifty-two independent variants identified at FDR<0.05 from a genome-wide association study (GWAS) including 78,592 Europeans and collectively accounting for 2.93% of LTL variance were selected as genetic instruments for LTL. Summary-level data for MetS traits and for the MetS as a binary phenotype were obtained from the largest publicly available GWAS and two-sample MR analyses were used to estimate the associations of LTL with these traits. The combined effect of the genetic instruments was modelled using inverse variance weighted regression and sensitivity analyses with MR-Egger, weighted-median and MR-PRESSO were performed to test for and correct horizonal pleiotropy. Genetically instrumented longer LTL was associated with higher waist-to-hip ratio adjusted for body mass index (ß = 0.045 SD, SE = 0.018, p = 0.01), raised systolic (ß = 1.529 mmHg, SE = 0.332, p = 4x10-6 ) and diastolic (ß = 0.633 mmHg, SE = 0.222, p = 0.004) blood pressure, and increased MetS risk (OR = 1.133, 95% CI 1.057-1.215). Consistent results were obtained in sensitivity analyses, which provided no evidence of unbalanced horizontal pleiotropy. Telomere shortening might not be a major driver of cellular senescence and dysfunction in human adipose tissue. Future experimental studies should examine the mechanistic bases for the links between longer LTL and increased upper-body fat distribution and raised blood pressure.

The Relation Between Adult Weight Gain, Adipocyte Volume, and the Metabolic Profile at Middle Age.

CONTEXT: Weight gain during adulthood increases cardiometabolic disease risk, possibly through adipocyte hypertrophy. OBJECTIVE: We aimed to study the specific metabolomic profile of adult weight gain, and to examine its association with adipocyte volume. METHODS: Nuclear magnetic resonance-based metabolomics were measured in the Netherlands Epidemiology of Obesity (NEO) study (n = 6347, discovery) and Oxford Biobank (n = 6317, replication). Adult weight gain was calculated as the absolute difference between body mass index (BMI) at middle age and recalled BMI at age 20 years. We performed linear regression analyses with both exposures BMI at age 20 years and weight gain, and separately with BMI at middle age in relation to 149 serum metabolomic measures, adjusted for age, sex, and multiple testing. Additionally, subcutaneous abdominal adipocyte biopsies were collected in a subset of the Oxford Biobank (n = 114) to estimate adipocyte volume. RESULTS: Mean (SD) weight gain was 4.5 (3.7) kg/m2 in the NEO study and 3.6 (3.7) kg/m2 in the Oxford Biobank. Weight gain, and not BMI at age 20 nor middle age, was associated with concentrations of 7 metabolomic measures after successful replication, which included polyunsaturated fatty acids, small to medium low-density lipoproteins, and total intermediate-density lipoprotein. One SD weight gain was associated with 386 µm3 (95% CI, 143-629) higher median adipocyte volume. Adipocyte volume was associated with lipoprotein particles specific for adult weight gain. CONCLUSION: Adult weight gain is associated with specific metabolomic alterations of which the higher lipoprotein concentrations were likely contributed by larger adipocyte volumes, presumably linking weight gain to cardiometabolic disease.

CLC-5 and KIF3B interact to facilitate CLC-5 plasma membrane expression, endocytosis, and microtubular transport: relevance to pathophysiology of Dent's disease.

Renal tubular reabsorption is important for extracellular fluid homeostasis and much of this occurs via the receptor-mediated endocytic pathway. This pathway is disrupted in Dent's disease, an X-linked renal tubular disorder that is characterized by low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, and renal failure. Dent's disease is due to mutations of CLC-5, a chloride/proton antiporter, expressed in endosomes and apical membranes of renal tubules. Loss of CLC-5 function alters receptor-mediated endocytosis and trafficking of megalin and cubilin, although the underlying mechanisms remain to be elucidated. Here, we report that CLC-5 interacts with kinesin family member 3B (KIF3B), a heterotrimeric motor protein that facilitates fast anterograde translocation of membranous organelles. Using yeast two-hybrid, glutathione-S-transferase pull-down and coimmunoprecipitation assays, the COOH terminus of CLC-5 and the coiled-coil and globular domains of KIF3B were shown to interact. This was confirmed in vivo by endogenous coimmunoprecipitation of CLC-5 and KIF3B and codistribution with endosomal markers in mouse kidney fractions. Confocal live cell imaging in kidney cells further demonstrated association of CLC-5 and KIF3B, and transport of CLC-5-containing vesicles along KIF3B microtubules. KIF3B overexpression and underexpression, using siRNA, had reciprocal effects on whole cell chloride current amplitudes, CLC-5 cell surface expression, and endocytosis of albumin and transferrin. Clcn5(Y/-) mouse kidneys and isolated proximal tubular polarized cells showed increased KIF3B expression, whose effects on albumin endocytosis were dependent on CLC-5 expression. Thus, the CLC-5 and KIF3B interaction is important for CLC-5 plasma membrane expression and for facilitating endocytosis and microtubular transport in the kidney.

RSPO3 impacts body fat distribution and regulates adipose cell biology in vitro.

Fat distribution is an independent cardiometabolic risk factor. However, its molecular and cellular underpinnings remain obscure. Here we demonstrate that two independent GWAS signals at RSPO3, which are associated with increased body mass index-adjusted waist-to-hip ratio, act to specifically increase RSPO3 expression in subcutaneous adipocytes. These variants are also associated with reduced lower-body fat, enlarged gluteal adipocytes and insulin resistance. Based on human cellular studies RSPO3 may limit gluteofemoral adipose tissue (AT) expansion by suppressing adipogenesis and increasing gluteal adipocyte susceptibility to apoptosis. RSPO3 may also promote upper-body fat distribution by stimulating abdominal adipose progenitor (AP) proliferation. The distinct biological responses elicited by RSPO3 in abdominal versus gluteal APs in vitro are associated with differential changes in WNT signalling. Zebrafish carrying a nonsense rspo3 mutation display altered fat distribution. Our study identifies RSPO3 as an important determinant of peripheral AT storage capacity.

Genetic causes of hypercalciuric nephrolithiasis.

Renal stone disease (nephrolithiasis) affects 3-5% of the population and is often associated with hypercalciuria. Hypercalciuric nephrolithiasis is a familial disorder in over 35% of patients and may occur as a monogenic disorder that is more likely to manifest itself in childhood. Studies of these monogenic forms of hypercalciuric nephrolithiasis in humans, e.g. Bartter syndrome, Dent's disease, autosomal dominant hypocalcemic hypercalciuria (ADHH), hypercalciuric nephrolithiasis with hypophosphatemia, and familial hypomagnesemia with hypercalciuria have helped to identify a number of transporters, channels and receptors that are involved in regulating the renal tubular reabsorption of calcium. Thus, Bartter syndrome, an autosomal disease, is caused by mutations of the bumetanide-sensitive Na-K-Cl (NKCC2) co-transporter, the renal outer-medullary potassium (ROMK) channel, the voltage-gated chloride channel, CLC-Kb, the CLC-Kb beta subunit, barttin, or the calcium-sensing receptor (CaSR). Dent's disease, an X-linked disorder characterized by low molecular weight proteinuria, hypercalciuria and nephrolithiasis, is due to mutations of the chloride/proton antiporter 5, CLC-5; ADHH is associated with activating mutations of the CaSR, which is a G-protein-coupled receptor; hypophosphatemic hypercalciuric nephrolithiasis associated with rickets is due to mutations in the type 2c sodium-phosphate co-transporter (NPT2c); and familial hypomagnesemia with hypercalciuria is due to mutations of paracellin-1, which is a member of the claudin family of membrane proteins that form the intercellular tight junction barrier in a variety of epithelia. These studies have provided valuable insights into the renal tubular pathways that regulate calcium reabsorption and predispose to hypercalciuria and nephrolithiasis.

Mutational analysis of CLC-5, cofilin and CLC-4 in patients with Dent's disease.

BACKGROUND/AIMS: Dent's disease is caused by mutations in the chloride/proton antiporter, CLC-5, or oculo-cerebro-renal-syndrome-of-Lowe (OCRL1) genes. METHODS: Eighteen probands with Dent's disease were investigated for mutations in CLC-5 and two of its interacting proteins, CLC-4 and cofilin. Wild-type and mutant CLC-5s were assessed in kidney cells. Urinary calcium excretion following an oral calcium challenge was studied in one family. RESULTS: Seven different CLC-5 mutations consisting of two nonsense mutations (Arg347Stop and Arg718Stop), two missense mutations (Ser244Leu and Arg516Trp), one intron 3 donor splice site mutation, one deletion-insertion (nt930delTCinsA) and an in-frame deletion (523delVal) were identified in 8 patients. In the remaining 10 patients, DNA sequence abnormalities were not detected in the coding regions of CLC-4 or cofilin, and were independently excluded for OCRL1. Patients with CLC-5 mutations were phenotypically similar to those without. The donor splice site CLC-5 mutation resulted in exon 3 skipping. Electrophysiology demonstrated that the 523delVal CLC-5 mutation abolished CLC-5-mediated chloride conductance. Sixty percent of women with the CLC-5 deletion-insertion had nephrolithiasis, although calcium excretion before and after oral calcium challenge was similar to that in unaffected females. CONCLUSIONS: Three novel CLC-5 mutations were identified, and mutations in OCRL1, CLC-4 and cofilin excluded in causing Dent's disease in this patient cohort.

Associations of Outdoor Temperature, Bright Sunlight, and Cardiometabolic Traits in Two European Population-Based Cohorts.

CONTEXT: Seasonal variation in cold and light exposure may influence metabolic health. OBJECTIVE: We assessed the associations of bright sunlight and outdoor temperature with measures of glucose and lipid metabolism in two populations of middle-aged European subjects. DESIGN: Cross-sectional study. SETTING: Two population-based European cohorts. PARTICIPANTS: Middle-aged nondiabetic subjects from the Oxford Biobank (OBB; N = 4327; mean age, 41.4 years) and the Netherlands Epidemiology of Obesity (NEO) study (N = 5899; mean age, 55.6 years). INTERVENTIONS: Data on outdoor bright sunlight and temperature collected from local weather stations. MAIN OUTCOME MEASURES: Insulin resistance and fasting lipid levels. Multivariable regression analyses were adjusted for age, sex, percentage body fat, season, and either outdoor temperature or bright sunlight. RESULTS: In the OBB cohort, increased bright sunlight exposure was associated with lower fasting insulin [-1.27% (95% CI, -2.09 to -0.47%) per extra hour of bright sunlight], lower homeostatic model assessment for insulin resistance (-1.36%; 95% CI, -2.23 to -0.50), lower homeostatic model assessment for ß-cell function (-0.80%; 95% CI, -1.31 to -0.30), and lower triglyceride (-1.28%; 95% CI, -2.07 to -0.50) levels. In the NEO cohort generally unidirectional but weaker associations were observed. No associations between outdoor temperature and measures of glucose or lipid metabolism were detected following adjustment for bright sunlight. CONCLUSIONS: Bright sunlight, but not outdoor temperature, might be associated with increased insulin sensitivity and lower triglyceride levels.

An N-Ethyl-N-Nitrosourea (ENU)-Induced Tyr265Stop Mutation of the DNA Polymerase Accessory Subunit Gamma 2 (Polg2) Is Associated With Renal Calcification in Mice.

Renal calcification (RCALC) resulting in nephrolithiasis and nephrocalcinosis, which affects ∼10% of adults by 70 years of age, involves environmental and genetic etiologies. Thus, nephrolithiasis and nephrocalcinosis occurs as an inherited disorder in ∼65% of patients, and may be associated with endocrine and metabolic disorders including: primary hyperparathyroidism, hypercalciuria, renal tubular acidosis, cystinuria, and hyperoxaluria. Investigations of families with nephrolithiasis and nephrocalcinosis have identified some causative genes, but further progress is limited as large families are unavailable for genetic studies. We therefore embarked on establishing mouse models for hereditary nephrolithiasis and nephrocalcinosis by performing abdominal X-rays to identify renal opacities in N-ethyl-N-nitrosourea (ENU)-mutagenized mice. This identified a mouse with RCALC inherited as an autosomal dominant trait, designated RCALC type 2 (RCALC2). Genomewide mapping located the Rcalc2 locus to a ∼16-Mbp region on chromosome 11D-E2 and whole-exome sequence analysis identified a heterozygous mutation in the DNA polymerase gamma-2, accessory subunit (Polg2) resulting in a nonsense mutation, Tyr265Stop (Y265X), which co-segregated with RCALC2. Kidneys of mutant mice (Polg2+/Y265X ) had lower POLG2 mRNA and protein expression, compared to wild-type littermates (Polg2+/+ ). The Polg2+/Y265X and Polg2+/+ mice had similar plasma concentrations of sodium, potassium, calcium, phosphate, chloride, urea, creatinine, glucose, and alkaline phosphatase activity; and similar urinary fractional excretion of calcium, phosphate, oxalate, and protein. Polg2 encodes the minor subunit of the mitochondrial DNA (mtDNA) polymerase and the mtDNA content in Polg2+/Y265X kidneys was reduced compared to Polg2+/+ mice, and cDNA expression profiling revealed differential expression of 26 genes involved in several biological processes including mitochondrial DNA function, apoptosis, and ubiquitination, the complement pathway, and inflammatory pathways. In addition, plasma of Polg2+/Y265X mice, compared to Polg2+/+ littermates had higher levels of reactive oxygen species. Thus, our studies have identified a mutant mouse model for inherited renal calcification associated with a Polg2 nonsense mutation. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc.

Mice with a Brd4 Mutation Represent a New Model of Nephrocalcinosis.

Nephrolithiasis (NL) and nephrocalcinosis (NC), which comprise renal calcification of the collecting system and parenchyma, respectively, have a multifactorial etiology with environmental and genetic determinants and affect ∼10% of adults by age 70 years. Studies of families with hereditary NL and NC have identified >30 causative genes that have increased our understanding of extracellular calcium homeostasis and renal tubular transport of calcium. However, these account for <20% of the likely genes that are involved, and to identify novel genes for renal calcification disorders, we investigated 1745 12-month-old progeny from a male mouse that had been treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU) for radiological renal opacities. This identified a male mouse with renal calcification that was inherited as an autosomal dominant trait with >80% penetrance in 152 progeny. The calcification consisted of calcium phosphate deposits in the renal papillae and was associated with the presence of the urinary macromolecules osteopontin and Tamm-Horsfall protein, which are features found in Randall's plaques of patients with NC. Genome-wide mapping located the disease locus to a ∼30 Mbp region on chromosome 17A3.3-B3 and whole-exome sequence analysis identified a heterozygous mutation, resulting in a missense substitution (Met149Thr, M149T), in the bromodomain-containing protein 4 (BRD4). The mutant heterozygous (Brd4+/M149T ) mice, when compared with wild-type (Brd4+/+ ) mice, were normocalcemic and normophosphatemic, with normal urinary excretions of calcium and phosphate, and had normal bone turnover markers. BRD4 plays a critical role in histone modification and gene transcription, and cDNA expression profiling, using kidneys from Brd4+/M149T and Brd4+/+ mice, revealed differential expression of genes involved in vitamin D metabolism, cell differentiation, and apoptosis. Kidneys from Brd4+/M149T mice also had increased apoptosis at sites of calcification within the renal papillae. Thus, our studies have established a mouse model, due to a Brd4 Met149Thr mutation, for inherited NC. © 2019 American Society for Bone and Mineral Research.

Genetics of hypercalciuric nephrolithiasis: renal stone disease.

Renal stone disease (nephrolithiasis) affects 5% of adults and is often associated with hypercalciuria. Hypercalciuric nephrolithiasis is a familial disorder in more than 35% of patients, and may occur as a monogenic disorder, or as a polygenic trait involving 3 to 5 susceptibility loci in man and rat, respectively. Studies of monogenic forms of hypercalciuric nephrolithiasis in man, for example, Bartter syndrome, Dent's disease, autosomal dominant hypocalcemic hypercalciuria (ADHH), hypercalciuric nephrolithiasis with hypophosphatemia, and familial hypomagnesemia with hypercalciuria have helped to identify a number of transporters, channels, and receptors that are involved in regulating the renal tubular reabsorption of calcium. Thus, Bartter syndrome, an autosomal recessive disease, is caused by mutations of the bumetanide-sensitive Na-K-Cl (NKCC2) cotransporter, the renal outer-medullary potassium channel (ROMK), the voltage-gated chloride channel, CLC-Kb, or in its beta subunit, Barttin. Dent's disease, an X-linked disorder characterized by low molecular weight proteinuria, hypercalciuria, and nephrolithiasis, is due to mutations of the chloride/proton antiporter, CLC-5; ADHH is associated with activating mutations of the calcium-sensing receptor, which is a G protein-coupled receptor; hypophosphatemic hypercalciuric nephrolithiasis associated with rickets is due to mutations in the type 2c sodium-phosphate cotransporter (NPT2c); and familial hypomagnesemia with hypercalciuria is due to mutations of paracellin-1, which is a member of the claudin family of membrane proteins that form the intercellular tight junction barrier in a variety of epithelia. These studies have provided valuable insights into the renal tubular pathways that regulate calcium reabsorption and predispose to kidney stones and bone disease.

Mice with an N-Ethyl-N-Nitrosourea (ENU) Induced Tyr209Asn Mutation in Natriuretic Peptide Receptor 3 (NPR3) Provide a Model for Kyphosis Associated with Activation of the MAPK Signaling Pathway.

Non-syndromic kyphosis is a common disorder that is associated with significant morbidity and has a strong genetic involvement; however, the causative genes remain to be identified, as such studies are hampered by genetic heterogeneity, small families and various modes of inheritance. To overcome these limitations, we investigated 12 week old progeny of mice treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU) using phenotypic assessments including dysmorphology, radiography, and dual-energy X-ray absorptiometry. This identified a mouse with autosomal recessive kyphosis (KYLB). KYLB mice, when compared to unaffected littermates, had: thoraco-lumbar kyphosis, larger vertebrae, and increased body length and increased bone area. In addition, female KYLB mice had increases in bone mineral content and plasma alkaline phosphatase activity. Recombination mapping localized the Kylb locus to a 5.5Mb region on chromosome 15A1, which contained 51 genes, including the natriuretic peptide receptor 3 (Npr3) gene. DNA sequence analysis of Npr3 identified a missense mutation, Tyr209Asn, which introduced an N-linked glycosylation consensus sequence. Expression of wild-type NPR3 and the KYLB-associated Tyr209Asn NPR3 mutant in COS-7 cells demonstrated the mutant to be associated with abnormal N-linked glycosylation and retention in the endoplasmic reticulum that resulted in its absence from the plasma membrane. NPR3 is a decoy receptor for C-type natriuretic peptide (CNP), which also binds to NPR2 and stimulates mitogen-activated protein kinase (MAPK) signaling, thereby increasing the number and size of hypertrophic chondrocytes. Histomorphometric analysis of KYLB vertebrae and tibiae showed delayed endochondral ossification and expansion of the hypertrophic zones of the growth plates, and immunohistochemistry revealed increased p38 MAPK phosphorylation throughout the growth plates of KYLB vertebrae. Thus, we established a model of kyphosis due to a novel NPR3 mutation, in which loss of plasma membrane NPR3 expression results in increased MAPK pathway activation, causing elongation of the vertebrae and resulting in kyphosis.

LRP5 regulates human body fat distribution by modulating adipose progenitor biology in a dose- and depot-specific fashion.

Common variants in WNT pathway genes have been associated with bone mass and fat distribution, the latter predicting diabetes and cardiovascular disease risk. Rare mutations in the WNT co-receptors LRP5 and LRP6 are similarly associated with bone and cardiometabolic disorders. We investigated the role of LRP5 in human adipose tissue. Subjects with gain-of-function LRP5 mutations and high bone mass had enhanced lower-body fat accumulation. Reciprocally, a low bone mineral density-associated common LRP5 allele correlated with increased abdominal adiposity. Ex vivo LRP5 expression was higher in abdominal versus gluteal adipocyte progenitors. Equivalent knockdown of LRP5 in both progenitor types dose-dependently impaired ß-catenin signaling and led to distinct biological outcomes: diminished gluteal and enhanced abdominal adipogenesis. These data highlight how depot differences in WNT/ß-catenin pathway activity modulate human fat distribution via effects on adipocyte progenitor biology. They also identify LRP5 as a potential pharmacologic target for the treatment of cardiometabolic disorders.

Autosomal dominant hypercalciuria in a mouse model due to a mutation of the epithelial calcium channel, TRPV5.

Hypercalciuria is a major cause of nephrolithiasis, and is a common and complex disorder involving genetic and environmental factors. Identification of genetic factors for monogenic forms of hypercalciuria is hampered by the limited availability of large families, and to facilitate such studies, we screened for hypercalciuria in mice from an N-ethyl-N-nitrosourea mutagenesis programme. We identified a mouse with autosomal dominant hypercalciuria (HCALC1). Linkage studies mapped the Hcalc1 locus to a 11.94 Mb region on chromosome 6 containing the transient receptor potential cation channel, subfamily V, members 5 (Trpv5) and 6 (Trpv6) genes. DNA sequence analysis of coding regions, intron-exon boundaries and promoters of Trpv5 and Trpv6 identified a novel T to C transition in codon 682 of TRPV5, mutating a conserved serine to a proline (S682P). Compared to wild-type littermates, heterozygous (Trpv5(682P/+)) and homozygous (Trpv5(682P/682P)) mutant mice had hypercalciuria, polyuria, hyperphosphaturia and a more acidic urine, and ∼10% of males developed tubulointerstitial nephritis. Trpv5(682P/682P) mice also had normal plasma parathyroid hormone but increased 1,25-dihydroxyvitamin D(3) concentrations without increased bone resorption, consistent with a renal defect for the hypercalciuria. Expression of the S682P mutation in human embryonic kidney cells revealed that TRPV5-S682P-expressing cells had a lower baseline intracellular calcium concentration than wild-type TRPV5-expressing cells, suggesting an altered calcium permeability. Immunohistological studies revealed a selective decrease in TRPV5-expression from the renal distal convoluted tubules of Trpv5(682P/+) and Trpv5(682P/682P) mice consistent with a trafficking defect. In addition, Trpv5(682P/682P) mice had a reduction in renal expression of the intracellular calcium-binding protein, calbindin-D(28K), consistent with a specific defect in TRPV5-mediated renal calcium reabsorption. Thus, our findings indicate that the TRPV5 S682P mutant is functionally significant and study of HCALC1, a novel model for autosomal dominant hypercalciuria, may help further our understanding of renal calcium reabsorption and hypercalciuria.

A mouse model of early-onset renal failure due to a xanthine dehydrogenase nonsense mutation.

Chronic kidney disease (CKD) is characterized by renal fibrosis that can lead to end-stage renal failure, and studies have supported a strong genetic influence on the risk of developing CKD. However, investigations of the underlying molecular mechanisms are hampered by the lack of suitable hereditary models in animals. We therefore sought to establish hereditary mouse models for CKD and renal fibrosis by investigating mice treated with the chemical mutagen N-ethyl-N-nitrosourea, and identified a mouse with autosomal recessive renal failure, designated RENF. Three-week old RENF mice were smaller than their littermates, whereas at birth they had been of similar size. RENF mice, at 4-weeks of age, had elevated concentrations of plasma urea and creatinine, indicating renal failure, which was associated with small and irregularly shaped kidneys. Genetic studies using DNA from 10 affected mice and 91 single nucleotide polymorphisms mapped the Renf locus to a 5.8 Mbp region on chromosome 17E1.3. DNA sequencing of the xanthine dehydrogenase (Xdh) gene revealed a nonsense mutation at codon 26 that co-segregated with affected RENF mice. The Xdh mutation resulted in loss of hepatic XDH and renal Cyclooxygenase-2 (COX-2) expression. XDH mutations in man cause xanthinuria with undetectable plasma uric acid levels and three RENF mice had plasma uric acid levels below the limit of detection. Histological analysis of RENF kidney sections revealed abnormal arrangement of glomeruli, intratubular casts, cellular infiltration in the interstitial space, and interstitial fibrosis. TUNEL analysis of RENF kidney sections showed extensive apoptosis predominantly affecting the tubules. Thus, we have established a mouse model for autosomal recessive early-onset renal failure due to a nonsense mutation in Xdh that is a model for xanthinuria in man. This mouse model could help to increase our understanding of the molecular mechanisms associated with renal fibrosis and the specific roles of XDH and uric acid.