Aims The inherited stone-forming condition cystinuria is most commonly caused by mutations in the SLC3A1 or SLC7A9 genes. These encode the two subunits, rBAT and b0,+AT, of the heterodimeric cystine transporter, which is located on the apical membrane of proximal tubular cells (PTC). Defects in this transporter cause multiple, bilateral and recurrent cystine stones. Trafficking of the rBAT/b0,+AT transporter has been shown to be disrupted by mutations of SLC3A1 and SLC7A9 in other cell lines, but this has not yet been demonstrated in a relevant cell type. Our aim is to interrogate cystine transport in vitro to identify new therapeutic targets at the molecular level.
Methods PTC from urine of healthy patients were conditionally immortalised using a temperature sensitive SV40 construct. Fluorescent tags for wild type and mutated SLC3A1 and SLC7A9 sequences were generated, and used to study the trafficking of the rBAT/b0,+AT heterodimer, and quantify cystine uptake using radio-labelled cystine assays.
Results Constructs for wild type and mutated rBAT and b0,+AT were transfected into human conditionally immortalised PTC, and imaged in real time to demonstrate transporter trafficking. Functional assays of cystine transport are underway to quantify the effects of clinically relevant cystinuria mutations.
Conclusion We have established an in vitro study of the cystine heterodimer rBAT/b0,+AT in human PTC which can be used to investigate known and discovered cystinuria mutations, and ultimately facilitate development of novel therapies for this disease.