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Context Cucumber (Cucumis sativus Linn. [Cucurbitaceae]) is widely known for its purgative, antidiabetic, antioxidant, and anticancer therapeutic potential. However, its effect on gastrointestinal (GI) disease is unrecognised. Objective This study investigated the effect of C. sativus fruit extract (CCE) on intestinal chloride secretion, motility, and motor function, and the role of TMEM16A chloride channels. Materials and methods CCE extracts were obtained from commercially available cucumber. Active fractions were then purified by HPLC and analysed by high resolution mass spectrometry. The effect of CCE on intestinal chloride secretion was investigated in human colonic T84 cells, ex vivo mouse intestinal tissue using an Ussing chamber, and the two-electrode voltage-clamp technique to record calcium sensitive TMEM16A chloride currents in Xenopus laevis oocytes. In vivo, intestinal motility was investigated using the loperamide-induced C57BL/6 constipation mouse model. Ex vivo contractility of mouse colonic smooth muscles was assessed by isometric force measurements. Results CCE increased the short-circuit current (ΔIsc 34.47 ± µA/cm2) and apical membrane chloride conductance (ΔICl 95 ± 8.1 µA/cm2) in intestinal epithelial cells. The effect was dose-dependent, with an EC50 value of 0.06 µg/mL. CCE stimulated the endogenous TMEM16A-induced Cl- current in Xenopus laevis oocytes. Moreover, CCE increased the contractility of smooth muscle in mouse colonic tissue and enhanced small bowel transit in CCE treated mice compared to loperamide controls. Mass spectrometry suggested a cucurbitacin-like analogue with a mass of 512.07 g/mol underlying the bioactivity of CCE. Conclusion A cucurbitacin-like analog present in CCE activates TMEM16A channels, which may have therapeutic potential in cystic fibrosis and intestinal hypodynamic disorders.

The pathophysiological nature of the common ABCG2 gout and hyperuricemia associated variant Q141K (rs2231142) remains undefined. Here, we use a human interventional cohort study (ACTRN12615001302549) to understand the physiological role of ABCG2 and find that participants with the Q141K ABCG2 variant display elevated serum urate, unaltered FEUA, and significant evidence of reduced extra-renal urate excretion. We explore mechanisms by generating a mouse model of the orthologous Q140K Abcg2 variant and find male mice have significant hyperuricemia and metabolic alterations, but only subtle alterations of renal urate excretion and ABCG2 abundance. By contrast, these mice display a severe defect in ABCG2 abundance and function in the intestinal tract. These results suggest a tissue specific pathobiology of the Q141K variant, support an important role for ABCG2 in urate excretion in both the human kidney and intestinal tract, and provide insight into the importance of intestinal urate excretion for serum urate homeostasis.

Autosomal dominant polycystic kidney disease (ADPKD) is the leading genetic cause of end stage renal disease characterized by progressive expansion of kidney cysts. To better understand the cell types and states driving ADPKD progression, we analyze eight ADPKD and five healthy human kidney samples, generating single cell multiomic atlas consisting of ~100,000 single nucleus transcriptomes and ~50,000 single nucleus epigenomes. Activation of proinflammatory, profibrotic signaling pathways are driven by proximal tubular cells with a failed repair transcriptomic signature, proinflammatory fibroblasts and collecting duct cells. We identify GPRC5A as a marker for cyst-lining collecting duct cells that exhibits increased transcription factor binding motif availability for NF-κB, TEAD, CREB and retinoic acid receptors. We identify and validate a distal enhancer regulating GPRC5A expression containing these motifs. This single cell multiomic analysis of human ADPKD reveals previously unrecognized cellular heterogeneity and provides a foundation to develop better diagnostic and therapeutic approaches.

Tetracycline-inducible gene expression systems have been used successfully to study gene function in vivo and in vitro renal epithelial models but the effects of the common inducing agent, doxycycline (DOX), on gene expression are not well appreciated. Here, we evaluated the DOX effects on the transcriptome of a widely used renal epithelial cell model, mIMCD3 cells, to establish a reference. Cells were grown on permeable filter supports in the absence and presence of DOX (3 or 6 days), and genome-wide transcriptome profiles were assessed using RNA-Seq. We found DOX significantly altered the transcriptome profile, changing the abundance of 1,549 transcripts at 3 days and 2,643 transcripts at 6 days. Within 3 days of treatment, DOX significantly decreased the expression of multiple signaling pathways (ERK, cAMP, and Notch) that are associated with cell proliferation and differentiation. Genes associated with cell cycle progression were subsequently downregulated in cells treated with DOX for 6 days, as were genes involved in cellular immune response processes and several cytokines and chemokines, correlating with a remarkable repression of genes encoding cell proliferation markers. The results provide new insight into responses of renal epithelial cells to DOX and a establish a resource for DOX-mediated gene expression systems.

Circulating urate levels are determined by the balance between urate production and excretion, a homeostasis regulated by the function of urate transporters in key epithelial tissues and cell types. Our understanding of these physiological processes and identification of the genes encoding the urate transporters has advanced significantly, leading to a greater ability to predict risk for urate associated diseases and identify new therapeutics that directly target urate transport. Here we review the identified urate transporters and their organization and function in the renal tubule, the intestinal enterocytes, and other important cell types to provide a fuller understanding of the complicated process of urate homeostasis and its role in human disease. Further, we provide a review of the genetic tools that have provided the unbiased catalyst for transporter identification as well as a discussion of the role of transporters in determining the observed significant sex differences in urate associated disease risk.

The role of mechanical forces driving kidney epithelial fluid transport and morphogenesis in kidney diseases is unclear. Here, using a microfluidic platform to recapitulate fluid transport activity of kidney cells, we report that renal epithelial cells can actively generate hydraulic pressure gradients across the epithelium. The fluidic flux declines with increasing hydraulic pressure until a stall pressure, in a manner similar to mechanical fluid pumps. For normal human kidney cells, the fluidic flux is from apical to basal, and the pressure is higher on the basal side. For human Autosomal Dominant Polycystic Kidney Disease cells, the fluidic flux is reversed from basal to apical. Molecular and proteomic studies reveal that renal epithelial cells are sensitive to hydraulic pressure gradients, changing gene expression profiles and spatial arrangements of ion exchangers and the cytoskeleton in different pressure conditions. These results implicate mechanical force and hydraulic pressure as important variables during kidney function and morphological change, and provide insights into pathophysiological mechanisms underlying the development and transduction of hydraulic pressure gradients.

Background High body mass index (BMI) is strongly associated with hyperuricaemia. It is unknown whether overweight and obesity influences serum urate primarily through increased urate production or reduced renal clearance of uric acid. The aim of this study was to determine the influence of BMI on the response to inosine, a purine nucleoside that functions as an intermediate in the purine salvage and degradation pathways. Methods Following an overnight fast, 100 healthy participants without gout attended a study visit. Blood and urine samples were taken prior to and over 180 min after 1.5 g oral inosine. Serum urate and fractional excretion of uric acid (FEUA) were analysed according to high BMI (≥ 25 kg/m2) and low/normal BMI (2) groups, and according to BMI as a continuous variable. Results Participants in the high BMI group (n = 52, mean BMI 30.8 kg/m2) had higher serum urate concentrations at baseline (P = 0.002) compared to those with low/normal BMI (mean BMI 21.8 kg/m2). However, the high BMI group had a smaller increase in serum urate following the inosine load (P = 0.0012). The two BMI groups had a similar FEUA at baseline (P = 0.995), but those in the high BMI group had a smaller increase in FEUA following the inosine (P = 0.0003). Similar findings were observed when analysing BMI as a continuous variable. Those with high BMI had a smaller increase in FEUA per increase in serum urate, compared to those with low BMI (P = 0.005). Conclusions In a fasting state, people with high BMI have elevated serum urate levels but similar FEUA values compared with those with low/normal BMI. Following a purine load, those with high BMI have an attenuated renal excretion of uric acid. These data, using an experimental method to dynamically assess human urate handling, suggest that people with high BMI have a higher renal capacity for uric acid reabsorption when fasted and following a dietary purine intake have reduced renal clearance. Trial registration Australia and New Zealand Clinical Trials Registry, ACTRN12615001302549, date of registration 30 November 2015.

Cystogenesis is a morphological consequence of numerous genetic diseases of the epithelium. In the kidney, the pathogenic mechanisms underlying the program of altered cell and tubule morphology are obscured by secondary effects of cyst expansion. Here, we developed a new 3D tubuloid system to isolate the rapid changes in protein localization and gene expression that correlate with altered cell and tubule morphology during cyst initiation. Mouse renal tubule fragments were pulsed with a cell differentiation cocktail including glial-derived neurotrophic factor (GDNF) to yield collecting duct-like tubuloid structures with appropriate polarity, primary cilia, and gene expression. Using the 3D tubuloid model with an inducible Pkd2 knockout system allowed the tracking of morphological, protein, and genetic changes during cyst formation. Within hours of inactivation of Pkd2 and loss of polycystin-2, we observed significant progression in tubuloid to cyst morphology that correlated with 35 differentially expressed genes, many related to cell junctions, matrix interactions, and cell morphology previously implicated in cystogenesis. This article has an associated First Person interview with the first author of the paper., Summary: A new three-dimensional in vitro model drives tubule morphogenesis with the addition of GDNF and demonstrates methods for interrogating the initiation of ADPKD cystogenesis.

Hyperuricemia, or elevated serum urate, causes urate kidney stones and gout and also increases the incidence of many other conditions including renal disease, cardiovascular disease, and metabolic syndrome. As we gain mechanistic insight into how urate contributes to human disease, a clear sex difference has emerged in the physiological regulation of urate homeostasis. This review summarizes our current understanding of urate as a disease risk factor and how being of the female sex appears protective. Further, we review the mechanisms of renal handling of urate and the significant contributions from powerful genome-wide association studies of serum urate. We also explore the role of sex in the regulation of specific renal urate transporters and the power of new animal models of hyperuricemia to inform on the role of sex and hyperuricemia in disease pathogenesis. Finally, we advocate the use of sex differences in urate handling as a potent tool in gaining a further understanding of physiological regulation of urate homeostasis and for presenting new avenues for treating the constellation of urate related pathologies.

Cystogenesis is a morphological consequence of numerous genetic diseases of the epithelium. In the kidney, the pathogenic mechanisms underlying the program of altered cell and tubule morphology are obscured by secondary effects of cyst expansion. Here, we developed a new 3D tubuloid system to isolate the rapid changes in protein localization and gene expression that correlate with altered cell and tubule morphology during cyst initiation. Mouse renal tubule fragments were pulsed with a cell differentiation cocktail including glial derived neurotrophic factor (GDNF) to yield collecting duct-like tubuloid structures with appropriate polarity, primary cilium, and gene expression. Using the 3D tubuloid model with an inducible Pkd2 knockout system allowed the tracking of morphological, protein, and genetic changes during cyst formation. Within hours of inactivation of Pkd2 and loss of polycystin-2, we observed significant progression in tubuloid to cyst morphology that correlated with 35 differentially expressed genes, many related to cell junctions, matrix interactions, and cell morphology previously implicated in cystogenesis.

Filipino Americans (FAs) are the third-largest Asian American subgroup in the United States (US). Some studies showed that FAs experience more cardiometabolic diseases (CMDs) than other Asian subgroups and non-Hispanic Whites. The increased prevalence of CMD observed in FAs could be due to genetics and social/dietary lifestyles. While FAs are ascribed as an Asian group, they have higher burdens of CMD, and adverse social determinants of health compared to other Asian subgroups. Therefore, studies to elucidate how FAs might develop CMD and respond to medications used to manage CMD are warranted. The ultimate goals of this study are to identify potential mechanisms for reducing CMD burden in FAs and to optimize therapeutic drug selection. Collectively, these investigations could reduce the cardiovascular health disparities among FAs.This is a cross-sectional epidemiological design to enroll 300 self-identified Filipino age 18 yrs. or older without a history of cancer and/or organ transplant from Virginia, Washington DC, and Maryland. Once consented, a health questionnaire and disease checklist are administered to participants, and anthropometric data and other vital signs are collected. When accessible, we collect blood samples to measure basic blood biochemistry, lipids, kidney, and liver functions. We also extract DNA from the blood or saliva for genetic and pharmacogenetic analyses. CMD prevalence in FAs will be compared to the US population. Finally, we will conduct multivariate analyses to ascertain the role of genetic and non-genetic factors in developing CMD in FAs. Virginia Commonwealth University IRB approved all study materials (Protocol HM20018500).This is the first community-based study to involve FAs in genomics research. The study is actively recruiting participants. Participant enrollment is ongoing. At the time of this publication, the study has enrolled 97 participants. This ongoing study is expected to inform future research to reduce cardiovascular health disparities among FAs.

The pathophysiological nature of the common ABCG2 gout and hyperuricemia associated variant Q141K (rs2231142) remains undefined. Here, we use a human interventional cohort study (ACTRN12615001302549) to understand the physiological role of ABCG2 and find that participants with the Q141K ABCG2 variant display elevated serum urate, unaltered FEUA, and significant evidence of reduced extra-renal urate excretion. We explore mechanisms by generating a mouse model of the orthologous Q140K Abcg2 variant and find male mice have significant hyperuricemia and metabolic alterations, but only subtle alterations of renal urate excretion and ABCG2 abundance. By contrast, these mice display a severe defect in ABCG2 abundance and function in the intestinal tract. These results suggest a tissue specific pathobiology of the Q141K variant, support an important role for ABCG2 in urate excretion in both the human kidney and intestinal tract, and provide insight into the importance of intestinal urate excretion for serum urate homeostasis.

TMEM16A (Transmembrane protein 16A or Anoctamin1) is a calcium-activated chloride channel. (CaCC),that exerts critical roles in epithelial secretion. However, its localization, function, and regulation in intestinal chloride (Cl−) secretion remain obscure. Here, we show that TMEM16A protein abundance correlates with Cl− secretion in different regions of native intestine activated by the Ca2+-elevating muscarinic agonist carbachol (CCH). Basal, as well as both cAMP- and CCH-stimulated Isc, was largely reduced in Ano1 ± mouse intestine. We found CCH was not able to increase Isc in the presence of apical to serosal Cl− gradient, strongly supporting TMEM16A as primarily a luminal Cl− channel. Immunostaining demonstrated apical localization of TMEM16A where it colocalized with NHERF1 in mouse colonic tissue. Cellular depletion of NHERF1 in human colonic T84 cells caused a significant reduction of both cAMP- and CCH-stimulated Isc. Immunoprecipitation experiments revealed that NHERF1 forms a complex with TMEM16A through a PDZ-based interaction. We conclude that TMEM16A is a luminal Cl− channel in the intestine that functionally interacts with CFTR via PDZ-based interaction of NHERF1 for efficient and specific cholinergic stimulation of intestinal Cl− secretion., Highlights • TMEM16A express apically and operate Cl− secretion in mouse intestinal tissue. • TMEM16A potentially interacts with NHERF1 via its C-terminal PDZ binding motif. • TMEM16A-NHERF1 complex is requisite for cAMP and Ca2+ mediated apical Cl− secretion.

Elevated serum urate levels can cause gout, an excruciating disease with suboptimal treatment. Previous GWAS identified common variants with modest effects on serum urate. Here we report large-scale whole-exome sequencing association studies of serum urate and kidney function among ≤19,517 European ancestry and African-American individuals. We identify aggregate associations of low-frequency damaging variants in the urate transporters SLC22A12 (URAT1; p = 1.3 × 10−56) and SLC2A9 (p = 4.5 × 10−7). Gout risk in rare SLC22A12 variant carriers is halved (OR = 0.5, p = 4.9 × 10−3). Selected rare variants in SLC22A12 are validated in transport studies, confirming three as loss-of-function (R325W, R405C, and T467M) and illustrating the therapeutic potential of the new URAT1-blocker lesinurad. In SLC2A9, mapping of rare variants of large effects onto the predicted protein structure reveals new residues that may affect urate binding. These findings provide new insights into the genetic architecture of serum urate, and highlight molecular targets in SLC22A12 and SLC2A9 for lowering serum urate and preventing gout., Elevated serum urate levels are a risk factor for gout. Here, Tin et al. perform whole-exome sequencing in 19,517 individuals and detect low-frequency genetic variants in urate transporter genes, SLC22A12 and SLC2A9, associated with serum urate levels and confirm their damaging nature in vitro and in silico.

Novel technologies, new understanding of the basement membrane composition, and better comprehension of the embryonic development of the mammalian kidney have led to explosive growth in the use of three-dimensional in vitro models to study a range of human disease pathologies (Clevers H. Cell 165: 1586–1597, 2016; Shamir ER, Ewald AJ. Nat Rev Mol Cell Biol 15: 647–664, 2014). The development of these effective model systems represents a new tool to study the progressive cystogenesis of autosomal dominant polycystic kidney disease (ADPKD). ADPKD is a prevalent and complex monogenetic disease, characterized by the pathological formation of fluid fill cysts in renal tissue (Grantham JJ, Mulamalla S, Swenson-Fields KI. Nat Rev Nephrol 7: 556–566, 2011; Takiar V, Caplan MJ. Biochim Biophys Acta 1812: 1337–1343, 2011). ADPKD cystogenesis is attributed to loss of function mutations in either PKD1 or PKD2, which encode for two transmembrane proteins, polycystin-1 and polycystin-2, and progresses with loss of both copies of either gene through a proposed two-hit mechanism with secondary somatic mutations (Delmas P, Padilla F, Osorio N, Coste B, Raoux M, Crest M. Biochem Biophys Res Commun 322: 1374–1383, 2004; Pei Y, Watnick T, He N, Wang K, Liang Y, Parfrey P, Germino G, St George-Hyslop P. Am Soc Nephrol 10: 1524–1529, 1999; Wu G, D’Agati V, Cai Y, Markowitz G, Park JH, Reynolds DM, Maeda Y, Le TC, Hou H Jr, Kucherlapati R, Edelmann W, Somlo S. Cell 93: 177–188, 1998). The exaggerated consequences of large fluid filled cysts result in fibrosis and nephron injury, leading initially to functional compensation but ultimately to dysfunction (Grantham JJ. Am J Kidney Dis 28: 788–803, 1996; Norman J. Biochim Biophys Acta 1812: 1327–1336, 2011; Song CJ, Zimmerman KA, Henke SJ, Yoder BK. Results Probl Cell Differ 60: 323–344, 2017). The complicated disease progression has scattered focus and resources across the spectrum of ADPKD research.

MC Cleophas,1,2 LA Joosten,1–3 LK Stamp,4 N Dalbeth,5 OM Woodward,6 Tony R Merriman7 1Department of Internal Medicine, 2Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, the Netherlands; 3Department of Medical Genetics, Iuliu Haţieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania; 4Department of Medicine, University of Otago Christchurch, Christchurch, 5Department of Medicine, University of Auckland, Auckland, New Zealand; 6Department of Physiology, University of Maryland School of Medicine, Baltimore, MD, USA; 7Department of Biochemistry, University of Otago, Dunedin, New Zealand Abstract: As a result of the association of a common polymorphism (rs2231142, Q141K) in the ATP-binding cassette G2 (ABCG2) transporter with serum urate concentration in a genome-wide association study, it was revealed that ABCG2 is an important uric acid transporter. This review discusses the relevance of ABCG2 polymorphisms in gout, possible etiological mechanisms, and treatment approaches. The 141K ABCG2 urate-increasing variant causes instability in the nucleotide-binding domain, leading to decreased surface expression and function. Trafficking of the protein to the cell membrane is altered, and instead, there is an increased ubiquitin-mediated proteasomal degradation of the variant protein as well as sequestration into aggresomes. In humans, this leads to decreased uric acid excretion through both the kidney and the gut with the potential for a subsequent compensatory increase in renal urinary excretion. Not only does the 141K polymorphism in ABCG2 lead to hyperuricemia through renal overload and renal underexcretion, but emerging evidence indicates that it also increases the risk of acute gout in the presence of hyperuricemia, early onset of gout, tophi formation, and a poor response to allopurinol. In addition, there is some evidence that ABCG2 dysfunction may promote renal dysfunction in chronic kidney disease patients, increase systemic inflammatory responses, and decrease cellular autophagic responses to stress. These results suggest multiple benefits in restoring ABCG2 function. It has been shown that decreased ABCG2 141K surface expression and function can be restored with colchicine and other small molecule correctors. However, caution should be exercised in any application of these approaches given the role of surface ABCG2 in drug resistance. Keywords: ABCG2, BCRP, gout, urate, uric acid, polymorphism, allopurinol

Diarrhea remains a continuous threat to human health worldwide. Scaling up the best practices for diarrhea prevention requires improved therapies. Diarrhea results from dysregulation of normal intestinal ion transport functions. Host-microbe contact is a key determinant of this response. Underlying mechanisms in the disease state are regulated by intracellular signals that modulate the activity of individual transport proteins responsible for ion transport and barrier function. Similarly, virulence factors of pathogens and their complex interaction with the host has shed light on the mechanism of enteric infection. Great advances in our understanding of the pathophysiologic mechanisms of epithelial transport, and host-microbe interaction have been made in recent years. Application of these new advances may represent strategies to decrease pathogen attachment, enhance intestinal cation absorption, decrease anion secretion and repair barrier function. This review highlights the new advances and better understanding in the pathophysiology of diarrheal diseases and their impact on therapy.

Serum urate concentrations are highly heritable and elevated serum urate is a key risk factor for gout. Genome-wide association studies (GWAS) of serum urate in African American (AA) populations are lacking. We conducted a meta-analysis of GWAS of serum urate levels and gout among 5820 AA and a large candidate gene study among 6890 AA and 21 708 participants of European ancestry (EA) within the Candidate Gene Association Resource Consortium. Findings were tested for replication among 1996 independent AA individuals, and evaluated for their association among 28 283 EA participants of the CHARGE Consortium. Functional studies were conducted using (14)C-urate transport assays in mammalian Chinese hamster ovary cells. In the discovery GWAS of serum urate, three loci achieved genome-wide significance (P< 5.0 × 10(-8)): a novel locus near SGK1/SLC2A12 on chromosome 6 (rs9321453, P= 1.0 × 10(-9)), and two loci previously identified in EA participants, SLC2A9 (P= 3.8 × 10(-32)) and SLC22A12 (P= 2.1 × 10(-10)). A novel rare non-synonymous variant of large effect size in SLC22A12, rs12800450 (minor allele frequency 0.01, G65W), was identified and replicated (beta -1.19 mg/dl, P= 2.7 × 10(-16)). (14)C-urate transport assays showed reduced urate transport for the G65W URAT1 mutant. Finally, in analyses of 11 loci previously associated with serum urate in EA individuals, 10 of 11 lead single-nucleotide polymorphisms showed direction-consistent association with urate among AA. In summary, we identified and replicated one novel locus in association with serum urate levels and experimentally characterize the novel G65W variant in URAT1 as a functional allele. Our data support the importance of multi-ethnic GWAS in the identification of novel risk loci as well as functional variants.

ATP-binding cassette (ABC) transporters form a large family of transmembrane proteins that facilitate the transport of specific substrates across membranes in an ATP-dependent manner. Transported substrates include lipids, lipopolysaccharides, amino acids, peptides, proteins, inorganic ions, sugars and xenobiotics. Despite this broad array of substrates, the physiological substrate of many ABC transporters has remained elusive. ABC transporters are divided into seven subfamilies, A-G, based on sequence similarity and domain organization. Here we review the role of members of the ABCG subfamily in human disease and how the identification of disease genes helped to determine physiological substrates for specific ABC transporters. We focus on the recent discovery of mutations in ABCG2 causing hyperuricemia and gout, which has led to the identification of urate as a physiological substrate for ABCG2.

Mammalian sweet, bitter, and umami taste is mediated by a single transduction pathway that includes a phospholipase C (PLC)β and one cation channel, TRPM5. However, in insects such as the fruit fly, Drosophila melanogaster , it is unclear whether different tastants, such as bitter compounds, are sensed in gustatory receptor neurons (GRNs) through one or multiple ion channels, as the cation channels required in insect GRNs are unknown. Here, we set out to explore additional sensory roles for the Drosophila TRPA1 channel, which was known to function in thermosensation. We found that TRPA1 was expressed in GRNs that respond to aversive compounds. Elimination of TRPA1 had no impact on the responses to nearly all bitter compounds tested, including caffeine, quinine, and strychnine. Rather, we found that TRPA1 was required in a subset of avoidance GRNs for the behavioral and electrophysiological responses to aristolochic acid. TRPA1 did not appear to be activated or inhibited directly by aristolochic acid. We found that elimination of the same PLC that leads to activation of TRPA1 in thermosensory neurons was also required in the TRPA1-expressing GRNs for avoiding aristolochic acid. Given that mammalian TRPA1 is required for responding to noxious chemicals, many of which cause pain and injury, our analysis underscores the evolutionarily conserved role for TRPA1 channels in chemical avoidance.

Genome-wide association studies (GWAS) have successfully identified common single nucleotide polymorphisms (SNPs) associated with a wide variety of complex diseases, but do not address gene function or establish causality of disease-associated SNPs. We recently used GWAS to identify SNPs in a genomic region on chromosome 4 that associate with serum urate levels and gout, a consequence of elevated urate levels. Here we show using functional assays that human ATP-binding cassette, subfamily G, 2 (ABCG2), encoded by the ABCG2 gene contained in this region, is a hitherto unknown urate efflux transporter. We further show that native ABCG2 is located in the brush border membrane of kidney proximal tubule cells, where it mediates renal urate secretion. Introduction of the mutation Q141K encoded by the common SNP rs2231142 by site-directed mutagenesis resulted in 53% reduced urate transport rates compared to wild-type ABCG2 ( P < 0.001). Data from a population-based study of 14,783 individuals support rs2231142 as the causal variant in the region and show highly significant associations with urate levels [whites: P = 10 −30 , minor allele frequency (MAF) 0.11; blacks P = 10 −4 , MAF 0.03] and gout (adjusted odds ratio 1.68 per risk allele, both races). Our data indicate that at least 10% of all gout cases in whites are attributable to this causal variant. With approximately 3 million US individuals suffering from often insufficiently treated gout, ABCG2 represents an attractive drug target. Our study completes the chain of evidence from association to causation and supports the common disease-common variant hypothesis in the etiology of gout.

The human propensity for high levels of serum uric acid (SUA) is a trait that has defied explanation. Is it beneficial? Is it pathogenic? Its role in the human diseases like gout and kidney stones was discovered over a century ago [Richette P, Bardin T. Lancet 375: 318–328, 2010; Rivard C, Thomas J, Lanaspa MA, Johnson RJ. Rheumatology (Oxford) 52: 421–426, 2013], but today emerging new genetic and epidemiological techniques have revived an age-old debate over whether high uric acid levels (hyperuricemia) independently increase risk for diseases like hypertension and chronic kidney disease [Feig DI. J Clin Hypertens (Greenwich) 14: 346–352, 2012; Feig DI, Madero M, Jalal DI, Sanchez-Lozada LG, Johnson RJ. J Pediatr 162: 896–902, 2013; Feig DI, Soletsky B, Johnson RJ. JAMA 300: 924–932, 2008; Wang J, Qin T, Chen J, Li Y, Wang L, Huang H, Li J. PLoS One 9: e114259, 2014; Zhu P, Liu Y, Han L, Xu G, Ran JM. PLoS One 9: e100801, 2014]. Part of the mystery of the role uric acid plays in human health stems from our lack of understanding of how humans regulate uric acid homeostasis, an understanding that could shed light on the historic role of uric acid in human adaptation and its present role in human pathogenesis. This review will highlight the recent work to identify the first important human uric acid secretory transporter, ABCG2, and the identification of a common causal ABCG2 variant, Q141K, for hyperuricemia and gout.

SUMMARYIn vertebrates, motile cilia line airways, oviducts and ventricles. Invertebrate cilia often control feeding, swimming and crawling, or gliding. Yet control and coordination of ciliary beating remains poorly understood. Evidence from the nudibranch mollusc, Tritonia diomedea, suggests that locomotory ciliated epithelial cells may be under direct electrical control. Here we report that depolarization of ciliated pedal epithelial (CPE)cells increases ciliary beating frequency (CBF), and elicits CBF increases similar to those caused by dopamine and the neuropeptide, TPep-NLS. Further,four CBF stimulants (zero external Cl-, depolarization, dopamine and TPep-NLS) depend on a common mode of action, viz. Ca2+influx, possibly through voltage-gated Ca2+ channels, and can be blocked by nifedipine. Ca2+ influx alone, however, does not provide all the internal Ca2+ necessary for CBF change. Ryanodine receptor(RyR) channel-gated internal stores are also necessary for CBF excitation. Caffeine can stimulate CBF and is sensitive to the presence of the RyR blocker dantrolene. Dantrolene also reduces CBF excitation induced by dopamine and TPep-NLS. Finally, W-7 and calmidazolium both block CBF excitation by caffeine and dopamine, and W-7 is effective at blocking TPep-NLS excitation. The effects of calmidazolium and W-7 suggest a role for Ca2+-calmodulin in regulating CBF, either directly or via Ca2+-calmodulin dependent kinases or phosphodiesterases. From these results we hypothesize dopamine and TPep-NLS induce depolarization-driven Ca2+ influx and Ca2+ release from internal stores that activates Ca2+-calmodulin, thereby increasing CBF.

SUMMARY The physiological mechanisms controlling ciliary beating remain largely unknown. Evidence exists supporting both hormonal control of ciliary beating and control via direct innervation. In the present study we investigated nervous control of cilia based locomotion in the nudibranch mollusc, Tritonia diomedea. Ciliated pedal epithelial (CPE) cells acting as locomotory effectors may be electrically excitable. To explore this possibility we characterized the cells' electrical properties, and found that CPE cells have large voltage dependent whole cell currents with two components. First, there is a fast activating outward Cl- current that is both voltage and Ca2+ influx dependent(ICl(Ca)). ICl(Ca) is sensitive to DIDS and 9-AC, and resembles currents of Ca2+-activated Cl- channels (CaCC). Ca2+ dependence also suggests the presence of voltage-gated Ca2+ channels; however, we were unable to detect these currents. The second current, a voltage dependent proton current(IH), activates very slowly and is sensitive to both Zn2+ and changes in pH. In addition we identify a new cilio-excitatory substance in Tritonia, viz., dopamine. Dopamine, in the 10 μmol l-1-1 mmol l-1 range, significantly increases ciliary beat frequency (CBF). We also found dopamine and Tritonia Pedal Peptide(TPep-NLS) selectively suppress ICl(Ca) in CPE cells,demonstrating a link between CBF excitation and ICl(Ca). It appears that dopamine and TPep-NLS inhibit ICl(Ca) not through changing [Ca2+]in, but directly by an unknown mechanism. Coupling of ICl(Ca) and CBF is further supported by our finding that DIDS and zero [Cl-]outboth increase CBF, mimicking dopamine and TPep-NLS excitation. These results suggest that dopamine and TPep-NLS act to inhibit ICl(Ca),initiating and prolonging Ca2+ influx, and activating CBF excitation.

Mutations in KLHL3 and CUL3, components of an E3 ligase complex, cause a familial disease of hyperkalemia and hypertension (PHAII). Here, we explore how KLHL3/CUL3 normally regulates the ROMK channel for potassium balance. We previously reported that ROMK is targeted for endocytosis by a specific clathrin adaptor molecule, ARH (Fang et al, JCI ’09). Because KLHL3/CUL3 targets ARH for proteasomal degradation (Fang et al, EB ’13), we wondered if ARH is a direct substrate of KLHL3/CUL3 ubiquitination. In vitro protein-protein interaction studies with purified ARH, KLHL3, CUL3 revealed that ARH directly interacts with KLHL3. We found separate interaction domains allow KLHL3 to simultaneously recruit CUL3 and the E3 ligase, ROC1, into a complex. As measured in ubiquitination assays in vitro with purified components, the direct interaction results in an increase in ARH ubiquitination, indicating that ARH is a direct substrate of KLHL3/CUL3. Importantly, KLHL3 localizes to the late DCT and CNT, overlapping with ...

Boid and Crotaline snakes use both their eyes and infrared-imaging facial pit organs to target homeothermic prey. These snakes can target in complete darkness, but the eyes can also effectively direct predatory strikes. We investigated the behavioral correlates of boid snakes’ simultaneous use of two imaging systems by testing whether congenital unilateral visual deprivation affects targeting performance. Normally sighted Burmese pythons exhibited average targeting angle of zero (on the midline axis of the head), but three unilaterally anophthalmic Burmese pythons targeted preferentially on the sighted side. A unilaterally anophthalmic amethystine python also targeted on the sighted side, and a unilaterally anophthalmic Brazilian rainbow boa tended to target on the sighted side, though its mean targeting angle was not significantly different from zero. When unilaterally anophthalmic Burmese pythons were temporarily blinded, mean strike angle changed to that of normally sighted snakes. These results show that while infrared-imaging snakes can shift between visual and infrared information under acute experimental conditions, loss of part of the visual field during development results in abnormal predatory targeting behavior. In contrast, normally sighted snakes subjected to temporary unilateral blinding do not target preferentially on the sighted side. Therefore, while loss of part of the visual field may be compensated for by infrared input in normal snakes, partial absence of visual input during development may alter central organization of visual information. Conversely, absence of half the visual field during development does not alter targeting performance based upon infrared input alone, suggesting that organization of the central infrared map does not depend upon normal organization of visual input.

Inhaled gene carriers must penetrate the highly viscoelastic and adhesive mucus barrier in the airway in order to overcome rapid mucociliary clearance and reach the underlying epithelium; however, even the most widely used viral gene carriers are unable to efficiently do so. We developed two polymeric gene carriers that compact plasmid DNA into small and highly stable nanoparticles with dense polyethylene glycol (PEG) surface coatings. These highly compacted, densely PEG-coated DNA nanoparticles rapidly penetrate human cystic fibrosis (CF) mucus ex vivo and mouse airway mucus ex situ. Intranasal administration of the mucus penetrating DNA nanoparticles greatly enhanced particle distribution, retention and gene transfer in the mouse lung airways compared to conventional gene carriers. Successful delivery of a full-length plasmid encoding the cystic fibrosis transmembrane conductance regulator protein was achieved in the mouse lungs and airway cells, including a primary culture of mucus-covered human airway epithelium grown at air–liquid interface, without causing acute inflammation or toxicity. Highly compacted mucus penetrating DNA nanoparticles hold promise for lung gene therapy.

The multidrug ATP-binding cassette, subfamily G, 2 (ABCG2) transporter was recently identified as an important human urate transporter, and a common mutation, a Gln to Lys substitution at position 141 (Q141K), was shown to cause hyperuricemia and gout. The nature of the Q141K defect, however, remains undefined. Here we explore the Q141K ABCG2 mutation using a comparative approach, contrasting it with another disease-causing mutation in an ABC transporter, the deletion of Phe-508 (ΔF508) in the cystic fibrosis transmembrane conductance regulator (CFTR). We found, much like in ΔF508 CFTR, that the Q141K mutation leads to instability in the nucleotide-binding domain (NBD), a defect that translates to significantly decreased protein expression. However, unlike the CFTR mutant, the Q141K mutation does not interfere with the nucleotide-binding domain/intracellular loop interactions. This investigation has also led to the identification of critical residues involved in the protein–protein interactions necessary for the dimerization of ABCG2: Lys-473 (K473) and Phe-142 (F142). Finally, we have demonstrated the utility of using small molecules to correct the Q141K defect in expression and function as a possible therapeutic approach for hyperuricemia and gout.

We previously showed that a truncation mutant of CFTR missing the first four transmembrane segments of TMD1, Δ264 CFTR, binds to key elements in the ER quality control mechanism to increase the amounts of the mature C band of both wt and ΔF508 CFTR through transcomplementation. Here, we created a new construct, Δ27–264 CFTR. Even though Δ27–264 CFTR is rapidly degraded in the proteasome, steady state protein can be detected by Western blot. Δ27–264 CFTR can also increase the amounts of the mature C band of both wt and ΔF508 CFTR through transcomplementation. Electrophysiology experiments show that Δ27–264 CFTR can restore chloride channel currents. Further experiments with the conduction mutant S341A show conclusively that currents are indeed generated by rescued channel function of ΔF508 CFTR. Immunoprecipitation studies show that Δ27–264 binds to ΔF508-CFTR, suggesting a bimolecular interaction. Thus the adeno-associated viral vector, rAAV-Δ27–264 CFTR, is a highly promising CF gene therapy vector, because it increases the amount of mature band C protein both from wt and ΔF508 CFTR, and rescues channel activity of ΔF508 CFTR.

Elevated serum urate concentrations can cause gout, a prevalent and painful inflammatory arthritis. By combining data from >140,000 individuals of European ancestry within the Global Urate Genetics Consortium (GUGC), we identified and replicated 28 genome-wide significant loci in association with serum urate concentrations (18 new regions in or near TRIM46, INHBB, SFMBT1, TMEM171, VEGFA, BAZ1B, PRKAG2, STC1, HNF4G, A1CF, ATXN2, UBE2Q2, IGF1R, NFAT5, MAF, HLF, ACVR1B-ACVRL1 and B3GNT4). Associations for many of the loci were of similar magnitude in individuals of non-European ancestry. We further characterized these loci for associations with gout, transcript expression and the fractional excretion of urate. Network analyses implicate the inhibins-activins signaling pathways and glucose metabolism in systemic urate control. New candidate genes for serum urate concentration highlight the importance of metabolic control of urate production and excretion, which may have implications for the treatment and prevention of gout. © 2013 Nature America, Inc. All rights reserved.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a genetic disorder resulting in large kidney cysts and eventual kidney failure. Mutations in either the PKD1 or PKD2/TRPP2 genes and their respective protein products, polycystin-1 (PC1) and polycystin-2 (PC2) result in ADPKD. PC2 is known to function as a non-selective cation channel, but PC1's function and the function of PC1 cleavage products are not well understood. Here we identify an endogenous PC1 cleavage product, P100, a 100 kDa fragment found in both wild type and epitope tagged PKD1 knock-in mice. Expression of full length human PC1 (FL PC1) and the resulting P100 and C-Terminal Fragment (CTF) cleavage products in both MDCK and CHO cells significantly reduces the store operated Ca(2+) entry (SOCE) resulting from thapsigargin induced store depletion. Exploration into the roles of P100 and CTF in SOCE inhibition reveal that P100, when expressed in Xenopus laevis oocytes, directly inhibits the SOCE currents but CTF does not, nor does P100 when containing the disease causing R4227X mutation. Interestingly, we also found that in PC1 expressing MDCK cells, translocation of the ER Ca(2+) sensor protein STIM1 to the cell periphery was significantly altered. In addition, P100 Co-immunoprecipitates with STIM1 but CTF does not. The expression of P100 in CHO cells recapitulates the STIM1 translocation inhibition seen with FL PC1. These data describe a novel polycystin-1 cleavage product, P100, which functions to reduce SOCE via direct inhibition of STIM1 translocation; a function with consequences for ADPKD.