Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved. e90 Abstracts Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved M O N D A Y O R A L S ORAL SESSION ORAL SESSION 7C GENETICS, GENOMICS, PROTEOMICS, METABOLOMICS OP.7C.01 UROMODULIN-NACL INTERACTION ON MRNA EXPRESSION OF ION TRANSPORTERS IN ISOLATED TUBULES OF UMOD+/+ AND UMOD-/- MICE L. Graham, D. Graham, M.W. McBride, A.F. Dominiczak, S. Padmanabhan. University of Glasgow, Glasgow, UNITED KINGDOM Objective: UMOD –/– mice have low blood pressure and decreased salt-sensi- tivity indicating interactions between UMOD and sodium transport in the TAL. However, it’s unclear whether the effect of UMOD is solely through interaction with cognate molecules in the TAL or whether the perturbations in physiol- ogy are more complex with involvement of both TAL and other parts of the nephron. We propose to answer this by profiling mRNA expression levels of the main ion transporters and signalling molecules in tubules from UMOD +/+ and UMOD –/– mice. Design and method: Male mice of 12 weeks of age were treated ± 2% NaCl ad libitum in the drinking water for 6 weeks (n = 5 per group). Following the stimula- tion period mice were sacrificed, kidneys excised, and tubule isolations prepared for mRNA expression analysis utilising qRT-PCR. Results: The following transporters were differentially expressed in the UMOD –/– mice: Proximal convoluted tubule; NHE3 (down regulated during steady state and further attenuated during salt loading) and SLC26A4 (up regulated during steady state and remained unchanged during salt loading); Thick ascending limb/ Distal convoluted tubule; NKCC2, ROMK, ClC-KB, Barttin, NCC, and NHE3, were all down regulated during steady state and further attenuated during salt loading, whereas SLC26A4, and Nedd4 were up regulated during steady state and remained unchanged during salt loading. TNFa was up regulated at steady state and further increased during salt loading; Macula densa; ACE2 and COX2 were up regulated during steady state and remained unchanged during salt load- ing, AT1 and A1AR were down regulated during steady state and remained un- changed with salt loading, and NOS1 was attenuated during steady state and remained down regulated during salt loading; finally, the Collecting ducts; SL- C26A4 was up regulated during steady state and remained unchanged during salt loading and ENaC down regulated during steady state and remained unchanged with salt loading. Conclusions: This study demonstrates the impact of the absence of UMOD gene on renal transporter gene expression across the length of the nephron. Addition- ally, genes involved in tubuloglomerular feedback, revealing a key role of UMOD in sodium homeostasis which should lead to new therapies for hypertension by targeting this gene. OP.7C.02 BLOOD PRESSURE-ASSOCIATED POLYMORPHISMS IN SLC4A7 (SODIUM/ BICARBONATE CO-TRANSPORTER NBCN1) ARE LINKED WITH GENE EXPRESSION AND INTRACELLULAR PH REGULATION F. Ng 1 , E. Bødtkjer 2 , M.J. Caulfield 1 , S. Ye 1,3 . 1 Department of Clinical Pharmacology, William Harvey Research Institute, London, UNITED KINGDOM, 2 Department of Biomedicine, Aarhus University, Aarhus, DENMARK, 3 Depart- ment of Cardiovascular Sciences, University of Leicester, Leicester, UNITED KINGDOM Objective: Genomewide-association studies have identified a number of loci influencing blood pressure, one of which is located near SLC4A7 (lead SNP rs13082711). SLC4A7 encodes an electroneutral Na + , HCO3 – co-transporter, NBCn1, which contributes to intracellular pH (pHi) regulation in a range of tissues, including vascular smooth muscle and endothelium. Additionally, the NBCn1 knockout mouse demonstrates an altered blood pressure phenotype. We investigated the potential influence of SLC4A7 genotype on DNA-protein interac- tion, gene expression, and pHi regulation. Design and method: Primary cultures of human umbilical vein endothelial cells and artery smooth muscle cells of different SLC4A7 genotypes were used. DNA-protein interactions were identified by formaldehyde-associated isolation of regulatory elements (FAIRE), electrophoretic mobility shift assays (EMSA), and DNA-pulldown assays. Gene expression was quantified by allelic imbal- ance analyses and protein immunoblots. Intracellular acidification was induced by the ammonium-prepulse method. NBCn1 protein function was evaluated as Na+,HCO3– co-transport activity, based on the rate of dimethyl-amiloride-insen- sitive, Na+- and HCO3–-dependent pHi recovery. A10 rat aortic smooth muscle cells transfected with either 326E or 326K SLC4A7 variants were used to verify primary cultures results. Results: FAIRE studies showed that the chromatin of the risk allele was preferen- tially open, allowing increased transcription. EMSAs demonstrated allele-depen- dent interactions with nuclear proteins. Protein sequencing from DNA-pulldown assays failed to identify the nuclear proteins involved. Consistent with allele-de- pendent DNA-protein interactions, the risk allele was associated with increased NBCn1 mRNA and protein expression. Furthermore, increased NBCn1 expres- sion in risk allele carriers was associated with increased in vitro protein function, as demonstrated by greater Na+,HCO3– co-transport activity during intracellular acidosis and higher baseline steady-state pHi compared to protective allele homo- zygotes. A10 cells overexpressing either 326E or 326K NBCn1 variants demon- strated equivalent increases in Na+,HCO3– co-transport activity, suggesting the amino acid change is less important than expression levels. Conclusions: We have outlined a potential mechanism for the SLC4A7 locus in- fluencing blood pressure via NBCn1 gene transcription, protein expression, and pHi regulation of human vascular smooth muscle and endothelial cells. This effect appears independent of the associated E326K amino acid change. OP.7C.03 KLOTHO GENE POLYMORPHISM INTERACTS WITH ADDUCIN-ENDOGENOUS OUABAIN- NA + -K + ATPASE SYSTEM IN SALT SENSITIVE HYPERTENSION S. Tentori, C. Lanzani, L. Citterio, E. Brioni, M. Simonini, L. Zagato, E. Messaggio, P. Manunta. IRCCS San Raffaele Scientific Institute, Università Vita Salute San Raffaele, Milan, ITALY Objective: The genetic basis of salt sensitive (SSH) hypertension consists in a very complex network of dynamic interaction among genetic-environmental fac- tors that may change with aging. The existence of a regulatory genetic network (RGN) as triggering mechanism of SSH has been already postulated for Adducin- Endogenous Ouabain-Na+-K+ ATPase (ADD-EO-NKA) system. The observation that Klotho (KL) is a newly discovered aging suppressor gene can directly affect the Na+, K+-ATPase activity opening new possible interaction with the RGN. Aim of this study is to evaluate the interactions between Add-EO-NKA and KL genes on pressure-natriuresis relationship in SSH. Design and method: A large cohort of 655 naive hypertensive patients underwent acute Na Load test for phenotyping the pressure natriuresis relationship (PNat). Results: Univariate analysis showed significant interactions between Klotho G/T SNP and RGN network (SNPs in loci of ADDs, EO synthesis, metabolism, and activity) with PNat slope. Furthermore, those patients carrying together mutate KL and NCX1 (SLC8A1) variants showed a rightward shit of PNat curve than in wild type condition. Simi- lar differences are present in delta systolic BP (8.92 vs 2.26 mmHg), with lower fractional excretion of Na (FE Na 1.48 vs 2.40%), and increased circulating EO (251.41 vs 190.75 pM) after Na load.