Biased calcium-sensing receptor signalling in the pathogenesis of kidney stone disease
BAUS ePoster online library. Howles S. 06/21/21; 319100; p6-2
Disclosure(s): I have no disclosures
Dr. Sarah Howles
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Abstract
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Introduction
Nephrolithiasis is a major clinical and economic health burden with a poorly understood pathogenesis. We conducted a genome-wide association study in British and Japanese populations identifying twenty nephrolithiasis-associated loci. Mutations in the calcium-sensing receptor (CaSR) cause disorders of calcium homeostasis and five loci identified at GWAS (DGKD, DGKH, WDR72, GPIC1 and BCR) were predicted to influence CaSR signalling. We demonstrated that genotype at the DGKD-associated locus correlated with urinary calcium excretion but not serum calcium concentration in a cohort of stone forming patients.
Methods
To define the effects of altered DGKD-expression on CaSR-signalling, in vitro studies were undertaken using HEK293 cells stably transfected with the CaSR.
Results
Transfection of HEK293-CaSR cells with DGKD-targetted siRNA resulted in decreased intracellular responses to alterations in extracellular calcium concentration as assessed by SRE-reporter and ERK-phosphorylation (pERK) assays when compared to cells transfected with scrambled siRNA. However, no alterations in intracellular responses as assessed by NFAT-reporter and Fluo-4 calcium assays were detected. In contrast, DGKD-overexpression increased intracellular responses as assessed by SRE-reporter assays and decreased intracellular responses as assessed by NFAT-reporter assays but without alteration in intracellular responses as assessed by pERK and Fluo-4 calcium assays.
Conclusions
Our results demonstrate that alterations in DGKD expression cause biased CaSR-signalling. This biased signalling may provide an explanation for the correlation of genotype at the DGKD-associated locus with urinary calcium excretion but not serum calcium concentration. Our findings suggest that biased CaSR-signalling may be a common cause of nephrolithiasis that represents a potential target for novel therapeutics.
Nephrolithiasis is a major clinical and economic health burden with a poorly understood pathogenesis. We conducted a genome-wide association study in British and Japanese populations identifying twenty nephrolithiasis-associated loci. Mutations in the calcium-sensing receptor (CaSR) cause disorders of calcium homeostasis and five loci identified at GWAS (DGKD, DGKH, WDR72, GPIC1 and BCR) were predicted to influence CaSR signalling. We demonstrated that genotype at the DGKD-associated locus correlated with urinary calcium excretion but not serum calcium concentration in a cohort of stone forming patients.
Methods
To define the effects of altered DGKD-expression on CaSR-signalling, in vitro studies were undertaken using HEK293 cells stably transfected with the CaSR.
Results
Transfection of HEK293-CaSR cells with DGKD-targetted siRNA resulted in decreased intracellular responses to alterations in extracellular calcium concentration as assessed by SRE-reporter and ERK-phosphorylation (pERK) assays when compared to cells transfected with scrambled siRNA. However, no alterations in intracellular responses as assessed by NFAT-reporter and Fluo-4 calcium assays were detected. In contrast, DGKD-overexpression increased intracellular responses as assessed by SRE-reporter assays and decreased intracellular responses as assessed by NFAT-reporter assays but without alteration in intracellular responses as assessed by pERK and Fluo-4 calcium assays.
Conclusions
Our results demonstrate that alterations in DGKD expression cause biased CaSR-signalling. This biased signalling may provide an explanation for the correlation of genotype at the DGKD-associated locus with urinary calcium excretion but not serum calcium concentration. Our findings suggest that biased CaSR-signalling may be a common cause of nephrolithiasis that represents a potential target for novel therapeutics.
Introduction
Nephrolithiasis is a major clinical and economic health burden with a poorly understood pathogenesis. We conducted a genome-wide association study in British and Japanese populations identifying twenty nephrolithiasis-associated loci. Mutations in the calcium-sensing receptor (CaSR) cause disorders of calcium homeostasis and five loci identified at GWAS (DGKD, DGKH, WDR72, GPIC1 and BCR) were predicted to influence CaSR signalling. We demonstrated that genotype at the DGKD-associated locus correlated with urinary calcium excretion but not serum calcium concentration in a cohort of stone forming patients.
Methods
To define the effects of altered DGKD-expression on CaSR-signalling, in vitro studies were undertaken using HEK293 cells stably transfected with the CaSR.
Results
Transfection of HEK293-CaSR cells with DGKD-targetted siRNA resulted in decreased intracellular responses to alterations in extracellular calcium concentration as assessed by SRE-reporter and ERK-phosphorylation (pERK) assays when compared to cells transfected with scrambled siRNA. However, no alterations in intracellular responses as assessed by NFAT-reporter and Fluo-4 calcium assays were detected. In contrast, DGKD-overexpression increased intracellular responses as assessed by SRE-reporter assays and decreased intracellular responses as assessed by NFAT-reporter assays but without alteration in intracellular responses as assessed by pERK and Fluo-4 calcium assays.
Conclusions
Our results demonstrate that alterations in DGKD expression cause biased CaSR-signalling. This biased signalling may provide an explanation for the correlation of genotype at the DGKD-associated locus with urinary calcium excretion but not serum calcium concentration. Our findings suggest that biased CaSR-signalling may be a common cause of nephrolithiasis that represents a potential target for novel therapeutics.
Nephrolithiasis is a major clinical and economic health burden with a poorly understood pathogenesis. We conducted a genome-wide association study in British and Japanese populations identifying twenty nephrolithiasis-associated loci. Mutations in the calcium-sensing receptor (CaSR) cause disorders of calcium homeostasis and five loci identified at GWAS (DGKD, DGKH, WDR72, GPIC1 and BCR) were predicted to influence CaSR signalling. We demonstrated that genotype at the DGKD-associated locus correlated with urinary calcium excretion but not serum calcium concentration in a cohort of stone forming patients.
Methods
To define the effects of altered DGKD-expression on CaSR-signalling, in vitro studies were undertaken using HEK293 cells stably transfected with the CaSR.
Results
Transfection of HEK293-CaSR cells with DGKD-targetted siRNA resulted in decreased intracellular responses to alterations in extracellular calcium concentration as assessed by SRE-reporter and ERK-phosphorylation (pERK) assays when compared to cells transfected with scrambled siRNA. However, no alterations in intracellular responses as assessed by NFAT-reporter and Fluo-4 calcium assays were detected. In contrast, DGKD-overexpression increased intracellular responses as assessed by SRE-reporter assays and decreased intracellular responses as assessed by NFAT-reporter assays but without alteration in intracellular responses as assessed by pERK and Fluo-4 calcium assays.
Conclusions
Our results demonstrate that alterations in DGKD expression cause biased CaSR-signalling. This biased signalling may provide an explanation for the correlation of genotype at the DGKD-associated locus with urinary calcium excretion but not serum calcium concentration. Our findings suggest that biased CaSR-signalling may be a common cause of nephrolithiasis that represents a potential target for novel therapeutics.
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