An in vitro model to study cystinuria
BAUS ePoster online library. Rhodes H. 06/30/16; 132025; P11-10
Ms. Hannah Rhodes
Ms. Hannah Rhodes
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Abstract
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P11-10

Introduction

Cystinuria is an inherited renal stone disease caused by mutations in the amino acid exchanger system b0,+ found on the proximal tubular epithelial cells (PTEC) of the kidney. Defects in either subunit of the transporter, rBAT or b0,+AT, lead to the formation of recurrent cystine stones and significant renal impairment. There are no effective treatments available, and no recent therapeutic advances. Mutation analysis has recently been made available in the UK. This work aims to establish a functional in vitro model to investigate the cellular effects of the common British mutations in rBAT and b0,+AT to enable the identification of new therapeutic targets at the molecular level.  

Materials and Methods

Epitope-tagged rBAT and b0,+AT were expressed and characterised in human conditionally immortalised PTEC, and cystine uptake was quantified using radio-labelled cystine assays.  

Results

Immunoprecipitation studies confirmed that expressed rBAT and b0,+AT formed dimers in PTEC in vitro. Biotinylation studies confirmed correct trafficking of the dimerised transporter to the plasma membrane. Radiolabelled cystine uptake more than doubled in cells co-expressing both rBAT and b0,+AT compared to untransfected PTEC, or PTEC expressing rBAT or b0,+AT alone.

Conclusions

We have established a functional in vitro model that can be used to further interrogate system b0,+ in the cell type specifically affected in cystinuria. This model can be manipulated to investigate known and discovered cystinuria mutations with the aim of facilitating development of novel disease therapies. 

P11-10

Introduction

Cystinuria is an inherited renal stone disease caused by mutations in the amino acid exchanger system b0,+ found on the proximal tubular epithelial cells (PTEC) of the kidney. Defects in either subunit of the transporter, rBAT or b0,+AT, lead to the formation of recurrent cystine stones and significant renal impairment. There are no effective treatments available, and no recent therapeutic advances. Mutation analysis has recently been made available in the UK. This work aims to establish a functional in vitro model to investigate the cellular effects of the common British mutations in rBAT and b0,+AT to enable the identification of new therapeutic targets at the molecular level.  

Materials and Methods

Epitope-tagged rBAT and b0,+AT were expressed and characterised in human conditionally immortalised PTEC, and cystine uptake was quantified using radio-labelled cystine assays.  

Results

Immunoprecipitation studies confirmed that expressed rBAT and b0,+AT formed dimers in PTEC in vitro. Biotinylation studies confirmed correct trafficking of the dimerised transporter to the plasma membrane. Radiolabelled cystine uptake more than doubled in cells co-expressing both rBAT and b0,+AT compared to untransfected PTEC, or PTEC expressing rBAT or b0,+AT alone.

Conclusions

We have established a functional in vitro model that can be used to further interrogate system b0,+ in the cell type specifically affected in cystinuria. This model can be manipulated to investigate known and discovered cystinuria mutations with the aim of facilitating development of novel disease therapies. 

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