O49
HUMAN PROXIMAL TUBULAR CELL MODELS OF CYSTINURIA
Rhodes HL1, Woodward MN2, Smithson S3, Tomson CR4, Williams M5, Welsh GI1,
Coward RJ1
1. Academic Renal Unit, School of Clinical Sciences, University of Bristol
2. Department of Paediatric Urology, Bristol Royal Hospital for Children
3. Clinical Genetics, University Hospitals Bristol NHS Foundation Trust
4. Department of Renal Medicine, Southmead Hospital, Bristol
5. Bristol Genetics Laboratory, Southmead Hospital
INTRODUCTION: Cystinuria causes recurrent cystine stone formation in the urinary tract. In
approximately 90% of cystinuria patients mutations in the cystinuria genes, SLC3A1 and
SLC7A9, lead to a reduction of cystine and dibasic amino acid reabsorption from the proximal
tubule. The cystine is poorly soluble in urine and precipitates to form stones. Current treatments
aim to solubilise cystine in urine by urine dilution, optimising pH or chelating cystine. However
the medications are poorly tolerated, have severe side effects and even compliant patients can
continue to form and pass painful kidney stones.
Our aim is to develop novel therapies for cystinuria that specifically target the proximal tubular
cells affected by this disease. Initial genotyping of 26 patients in our cohort revealed a broad
variety and combination of cystinuric mutations in these patients, such that studying the cell
biology of cystinuria for a single genotype is inadequate. In order to overcome this we are
establishing proximal tubular epithelial cell lines (PTEC) from cystinuria patients with a variety
of mutations to interrogate their cell biology and investigate ways of manipulating the cells to
increase cystine reabsorption.
METHODS: Ethical approval was obtained for patients with cystinuria (cystine stones
confirmed by stone analysis) to provide a venous blood sample and fresh urine specimen. DNA
extracted from blood samples was analysed using High Throughput sequencing of the SLC3A1
and SLC7A9 genes and Multiplex Ligation-dependent Probe Amplification (MLPA) to identify
larger gene rearrangements.
Exfoliated cells from urine of cystinuria patients were isolated and conditionally immortalised
using a temperature sensitive SV40 construct. PTEC were identified and subcloned, then
assessed for markers of PTEC on immunofluorescence.
RESULTS: At present PTEC have been conditionally immortalised from 4 cystinuria patients.
These all have different cystinuric genotypes; 1 is a compound heterozygote and 3 are
homozygous. Cultured cells demonstrate PTEC markers aminopeptidase N (CD13) and
aquaporin-1 on immunofluorescence. Further characterisation is underway along with functional
assays of cystine transport to quantify the effects of the differing cystinuria mutations.
CONCLUSION: We are establishing natural human mutant cell lines for a variety of cystinuria
genetic variants. We aim to utilise these cell lines to interrogate the effects of differing
cystinuria mutations on the cystine transporter (rBAT/b0,+AT). This in vitro model will enable
us to investigate ways of manipulating the cystine transporter and identifying novel therapeutic
targets at the molecular level to develop new treatments that patients find easier to tolerate with
fewer or no side effects.