Inwardly rectifying potassium channels mediate polymyxin-induced nephrotoxicity.

Polymyxin antibiotics are often used as a last-line defense to treat life-threatening Gram-negative pathogens. However, polymyxin-induced kidney toxicity is a dose-limiting factor of paramount importance and can lead to suboptimal treatment. To elucidate the mechanism and develop effective strategies to overcome polymyxin toxicity, we employed a whole-genome CRISPR screen in human kidney tubular HK-2 cells and identified 86 significant genes that upon knock-out rescued polymyxin-induced toxicity. Specifically, we discovered that knockout of the inwardly rectifying potassium channels Kir4.2 and Kir5.1 (encoded by KCNJ15 and KCNJ16, respectively) rescued polymyxin-induced toxicity in HK-2 cells. Furthermore, we found that polymyxins induced cell depolarization via Kir4.2 and Kir5.1 and a significant cellular uptake of polymyxins was evident. All-atom molecular dynamics simulations revealed that polymyxin B1 spontaneously bound to Kir4.2, thereby increasing opening of the channel, resulting in a potassium influx, and changes of the membrane potential. Consistent with these findings, small molecule inhibitors (BaCl2 and VU0134992) of Kir potassium channels reduced polymyxin-induced toxicity in cell culture and mouse explant kidney tissue. Our findings provide critical mechanistic information that will help attenuate polymyxin-induced nephrotoxicity in patients and facilitate the design of novel, safer polymyxins.

PAX2 + Mesenchymal Origin of Gonadal Supporting Cells Is Conserved in Birds.

During embryonic gonadal development, the supporting cell lineage is the first cell type to differentiate, giving rise to Sertoli cells in the testis and pre-granulosa cells in the ovary. These cells are thought to direct other gonadal cell lineages down the testis or ovarian pathways, including the germline. Recent research has shown that, in contrast to mouse, chicken gonadal supporting cells derive from a PAX2/OSR1/DMRT1/WNT4 positive mesenchymal cell population. These cells colonize the undifferentiated genital ridge during early gonadogenesis, around the time that germ cells migrate into the gonad. During the process of somatic gonadal sex differentiation, PAX2 expression is down-regulated in embryonic chicken gonads just prior to up-regulation of testis- and ovary-specific markers and prior to germ cell differentiation. Most research on avian gonadal development has focused on the chicken model, and related species from the Galloanserae clade. There is a lack of knowledge on gonadal sex differentiation in other avian lineages. Comparative analysis in birds is required to fully understand the mechanisms of avian sex determination and gonadal differentiation. Here we report the first comparative molecular characterization of gonadal supporting cell differentiation in birds from each of the three main clades, Galloanserae (chicken and quail), Neoaves (zebra finch) and Palaeognathe (emu). Our analysis reveals conservation of PAX2+ expression and a mesenchymal origin of supporting cells in each clade. Moreover, down-regulation of PAX2 expression precisely defines the onset of gonadal sex differentiation in each species. Altogether, these results indicate that gonadal morphogenesis is conserved among the major bird clades.

Functional genomics and gene-environment interaction highlight the complexity of congenital heart disease caused by Notch pathway variants.

Congenital heart disease (CHD) is the most common birth defect and brings with it significant mortality and morbidity. The application of exome and genome sequencing has greatly improved the rate of genetic diagnosis for CHD but the cause in the majority of cases remains uncertain. It is clear that genetics, as well as environmental influences, play roles in the aetiology of CHD. Here we address both these aspects of causation with respect to the Notch signalling pathway. In our CHD cohort, variants in core Notch pathway genes account for 20% of those that cause disease, a rate that did not increase with the inclusion of genes of the broader Notch pathway and its regulators. This is reinforced by case-control burden analysis where variants in Notch pathway genes are enriched in CHD patients. This enrichment is due to variation in NOTCH1. Functional analysis of some novel missense NOTCH1 and DLL4 variants in cultured cells demonstrate reduced signalling activity, allowing variant reclassification. Although loss-of-function variants in DLL4 are known to cause Adams-Oliver syndrome, this is the first report of a hypomorphic DLL4 allele as a cause of isolated CHD. Finally, we demonstrate a gene-environment interaction in mouse embryos between Notch1 heterozygosity and low oxygen- or anti-arrhythmic drug-induced gestational hypoxia, resulting in an increased incidence of heart defects. This implies that exposure to environmental insults such as hypoxia could explain variable expressivity and penetrance of observed CHD in families carrying Notch pathway variants.

Gene-environment interaction impacts on heart development and embryo survival.

Congenital heart disease (CHD) is the most common type of birth defect. In recent years, research has focussed on identifying the genetic causes of CHD. However, only a minority of CHD cases can be attributed to single gene mutations. In addition, studies have identified different environmental stressors that promote CHD, but the additive effect of genetic susceptibility and environmental factors is poorly understood. In this context, we have investigated the effects of short-term gestational hypoxia on mouse embryos genetically predisposed to heart defects. Exposure of mouse embryos heterozygous for Tbx1 or Fgfr1/Fgfr2 to hypoxia in utero increased the incidence and severity of heart defects while Nkx2-5+/- embryos died within 2 days of hypoxic exposure. We identified the molecular consequences of the interaction between Nkx2-5 and short-term gestational hypoxia, which suggest that reduced Nkx2-5 expression and a prolonged hypoxia-inducible factor 1α response together precipitate embryo death. Our study provides insight into the causes of embryo loss and variable penetrance of monogenic CHD, and raises the possibility that cases of foetal death and CHD in humans could be caused by similar gene-environment interactions.

Gestational stress induces the unfolded protein response, resulting in heart defects.

Congenital heart disease (CHD) is an enigma. It is the most common human birth defect and yet, even with the application of modern genetic and genomic technologies, only a minority of cases can be explained genetically. This is because environmental stressors also cause CHD. Here we propose a plausible non-genetic mechanism for induction of CHD by environmental stressors. We show that exposure of mouse embryos to short-term gestational hypoxia induces the most common types of heart defect. This is mediated by the rapid induction of the unfolded protein response (UPR), which profoundly reduces FGF signaling in cardiac progenitor cells of the second heart field. Thus, UPR activation during human pregnancy might be a common cause of CHD. Our findings have far-reaching consequences because the UPR is activated by a myriad of environmental or pathophysiological conditions. Ultimately, our discovery could lead to preventative strategies to reduce the incidence of human CHD.

Cited2 is required in trophoblasts for correct placental capillary patterning.

CITED2 is a transcriptional co-factor with important roles in many organs of the developing mammalian embryo. Complete deletion of this gene causes severe malformation of the placenta, and results in significantly reduced embryonic growth and death from E14.5. The placenta is a complex organ originating from cells derived from three lineages: the maternal decidua, the trophectoderm, and the extra-embryonic mesoderm. Cited2 is expressed in many of these cell types, but its exact role in the formation of the placenta is unknown. Here we use a conditional deletion approach to remove Cited2 from overlapping subsets of trophectoderm and extra-embryonic mesoderm. We find that Cited2 in sinusoidal trophoblast giant cells and syncytiotrophoblasts is likely to have a non-cell autonomous role in patterning of the pericytes associated with the embryonic capillaries. This function is likely to be mediated by PDGF signaling. Furthermore, we also identify that loss of Cited2 in syncytiotrophoblasts results in the subcellular mislocalization of one of the major lactate transporters in the placenta, SLC16A3 (MCT4). We hypothesize that the embryonic growth retardation observed in Cited2 null embryos is due in part to a disorganized embryonic capillary network, and in part due to abnormalities of the nutrient transport functions of the feto-maternal interface.