Clonal hematopoiesis of indeterminate potential (CHIP) is a common form of age-related somatic mosaicism that is associated with significant morbidity and mortality. CHIP mutations can be identified in peripheral blood samples sequenced using approaches that cover the whole genome, whole exome or targeted genetic regions; however, differentiating true CHIP mutations from sequencing artifacts and germline variants is a considerable bioinformatic challenge. We present a stepwise method that combines filtering based on sequencing metrics, variant annotation, and population-based associations to increase the accuracy of CHIP calls. We apply this approach to ascertain CHIP in ~550,000 individuals in the UK Biobank complete whole exome cohort and the All of Us Research Program initial whole genome release cohort. CHIP ascertainment on this scale unmasks recurrent artifactual variants and highlights the importance of specialized filtering approaches for several genes including TET2 and ASXL1. We show how small changes in filtering parameters can considerably increase CHIP misclassification and reduce the effect size of epidemiological associations. Our high-fidelity call set refines prior population-based associations of CHIP with incident outcomes. For example, the annualized incidence of myeloid malignancy in individuals with small CHIP clones is 0.03%/year, which increases to 0.5%/year amongst individuals with very large CHIP clones. We also find a significantly lower prevalence of CHIP in individuals of self-reported Latino or Hispanic ethnicity in All of Us, highlighting the importance of including diverse populations. The standardization of CHIP calling will increase the fidelity of CHIP epidemiological work and is required for clinical CHIP diagnostic assays.

BACKGROUND AND PURPOSEEndothelin-1 (ET-1) is increased in patients with sickle cell disease and may contribute to the development of sickle cell nephropathy. The current study was designed to determine whether ET-1 acting via the ET A receptor contributes to renal injury in a mouse model of sickle cell disease. EXPERIMENTAL APPROACHAdult, humanized HbSS (homozygous for sickle Hb) mice had increased ET-1 mRNA expression in both the cortex and the glomeruli compared with mice heterozygous for sickle and Hb A (HbAS controls). In the renal cortex, ET A receptor mRNA expression was also elevated in HbSS (sickle) mice although ET B receptor mRNA expression was unchanged. Ligand binding assays confirmed that sickle mice had increased ET A receptors in the renal vascular tissue when compared with control mice. KEY RESULTSIn response to PKC stimulation, reactive oxygen species production by isolated glomeruli from HbSS sickle mice was increased compared with that from HbSA controls, an effect that was prevented by 1 week in vivo treatment with the selective ET A antagonist, ABT-627. Protein and nephrin excretion were both elevated in sickle mice, effects that were also significantly attenuated by ABT-627. Finally, ET A receptor antagonism caused a significant reduction in mRNA expression of NADPH oxidase subunits, which may contribute to nephropathy in sickle cell disease. CONCLUSIONS AND IMPLICATIONSThese data support a novel role for ET-1 in the progression of sickle nephropathy, specifically via the ET A receptor, and suggest a potential role for ET A receptor antagonism in a treatment strategy. AbbreviationsET-1, endothelin-1; HbAS, heterozygous for sickle and Hb A; HbSS, homozygous for sickle Hb; ROS, reactive oxygen species; SAD, HbS Antillies and D-Punjab; SCD, sickle cell disease; SCN, sickle cell nephropathy BJP British Journal of Pharmacology

Speed JS, Hyndman KA, Roth K, Heimlich JB, Kasztan M, Fox BM, Johnston JG, Becker BK, Jin C, Gamble KL, Young ME, Pollock JS, Pollock DM. High dietary sodium causes dyssynchrony of the renal molecular clock in rats. Am J Physiol Renal Physiol 314: F89-F98, 2018. First published September 27, 2017; doi:10.1152/ajprenal.00028.2017.-Dyssynchrony of circadian rhythms is associated with various disorders, including cardiovascular and metabolic diseases. The cell autonomous molecular clock maintains circadian control; however, environmental factors that may cause circadian dyssynchrony either within or between organ systems are poorly understood. Our laboratory recently reported that the endothelin (ET-1) B (ET) receptor functions to facilitate Na excretion in a time of day-dependent manner. Therefore, the present study was designed to determine whether high salt (HS) intake leads to circadian dyssynchrony within the kidney and whether the renal endothelin system contributes to control of the renal molecular clock. We observed that HS feeding led to region-specific alterations in circadian clock components within the kidney. For instance, HS caused a significant 5.5-h phase delay in the peak expression of Bmal1 and suppressed Cry1 and Per2 expression in the renal inner medulla, but not the renal cortex, of control rats. The phase delay in Bmal1 expression appears to be mediated by ET-1 because this phenomenon was not observed in the ET-deficient rat. In cultured inner medullary collecting duct cells, ET-1 suppressed Bmal1 mRNA expression. Furthermore, Bmal1 knockdown in these cells reduced epithelial Na channel expression. These data reveal that HS feeding leads to intrarenal circadian dyssynchrony mediated, in part, through activation of ET receptors within the renal inner medulla.

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Aim The current study was designed to determine whether ET-1 derived from endothelial cells contributes to oxidative stress in the glomerulus of mice subjected to a high salt diet and/or hypoxia. Methods C57BL6/J control mice or vascular endothelial cell ET-1 knockout (VEET KO) mice were subjected to three-hour exposure to hypoxia (8% O2) and or 2 weeks of high salt diet (4% NaCl) prior to metabolic cage assessment of renal function and isolation of glomeruli for determination of reactive oxygen species (ROS). Results In control mice, hypoxia significantly increased urinary protein excretion during the initial 24 hrs, but only in animals on a high salt diet. Hypoxia increased glomerular ET-1 mRNA expression in control, but not in vascular endothelial cell ET-1 knockout (VEET KO) mice. Under normoxic conditions, mice on a high salt diet had approximately 150% higher glomerular ET-1 mRNA expression compared to a normal salt diet (p<0.05). High salt diet administration significantly increased glomerular ROS production in flox control, but not in glomeruli isolated from VEET KO mice. In C57BL6/J mice, the ETA receptor selective antagonist, ABT-627, significantly attenuated the increase in glomerular ROS production produced by high salt diet. In addition, chronic infusion of C57BL6/J mice with a sub-pressor dose of ET-1 (osmotic pumps) significantly increased levels of glomerular ROS that were prevented by ETA antagonist treatment. Conclusion These data suggest that both hypoxia and a high salt diet increases glomerular ROS production via endothelial derived ET-1-ETA receptor activation and provide a potential mechanism for ET-1 induced nephropathy.