Michelle Goldsworthy1,2, Asha Bayliss2, Anna Gluck2, Akira Wiberg3, Benjamin Turney1, DominicFurniss3, Rajesh Thakker2, Sarah Howles1,2
 1Nuffield Department of Surgical Sciences, University of Oxford, United Kingdom.2Academic Endocrine Unit, Radcliffe Department of Medicine, University of Oxford, UnitedKingdom.3Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Universityof Oxford, United Kingdom.
 Nephrolithiasis is a major health burden with a poorly understood pathogenesis. We conducteda genome-wide association study in British and Japanese populations identifying twentynephrolithiasis-associated loci. Mutations in the calcium-sensing receptor (CaSR) causedisorders of calcium homeostasis and five identified loci (DGKD, DGKH, WDR72, GPIC1 and BCR)were predicted to influence CaSR-signalling.
 In a validation population, we demonstrated that genotype at the DGKD-associated locuscorrelated with urinary calcium excretion but not serum calcium concentration. In vitro studiesdemonstrated that knockdown and overexpression of DGKD resulted in biased CaSR-signalling.Thus, treatment of CaSR-expressing HEK cells with DGKD-targeted siRNA (DGKD-KD), resulted indecreased MAPK responses to alterations in extracellular calcium concentration [Ca2+]e, asassessed by SRE-reporter and ERK-phosphorylation (pERK) assays, when compared to cellstreated with scrambled siRNA (WT) but without alteration in intracellular calcium responses[Ca2+]i as assessed by NFAT-reporter and Fluo-4 calcium assays (SRE maximal response DGKD-KD=5.28 fold change vs. WT=7.20 p=0.0065, pERK maximal response DGKD-KD=24.77, vs. WT=39.46 fold change, p=0.0056). Conversely, DGKD overexpression (DGKD-OE) increased MAPKresponses but suppressed [Ca2+]i responses to alterations in [Ca2+]e (SRE maximal responseDGKD-OE =14.13 fold change vs. WT=9.06 fold change, p=0.01; NFAT maximal response DGKDOE=13.67 fold change vs WT=59.16 fold change, p=0.0001).
 Our results demonstrate that alterations in DGKD expression cause biased CaSR-signalling. Thisbiased signalling may provide an explanation for the correlation of genotype at the DGKDassociatedlocus with urinary calcium excretion but not serum calcium concentration. Ourfindings suggest that biased CaSR-signalling may be a common cause of nephrolithiasis.
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