Evaluation of cystine transport in cultured human kidney cells and establishment of cystinuria type I phenotype by antisense technology.
Cystinuria is a rare hereditary disease resulting in recurrent stone formation and the need for repeated invasive interventions. So far, two responsible genes have been identified which encode the two transporters, rBAT and b(0,+)AT forming a heterodimer to transport cystine in proximal tubular cells (PTC) and whose defect results in increased excretion of cystine. A human cell line mimicing the phenotype of cystinuria in vitro is yet to be developed. Human kidney (HK)-2 is a PTC line derived from normal HK. After determining the presence of rBAT gene by RT-PCR and Western blot analysis, radioactively labeled cystine (S(35)) was used to evaluate the functional presence of the amino acid transport in HK-2 cells when cultured in vitro. To achieve a cystinuria type I phenotype in HK-2 cells, the rBAT gene was silenced using antisense oligonucleotides complimentary to human rBAT mRNA. The reduced transport activity of cystine was then determined by radiolabeled cystine uptake measurements. RT-PCR and Western blot confirmed the expression of the rBAT gene in HK-2 cells. Considerable transport of the radio labeled cystine was observed in HK-2 cells and was linearly dependent on the incubation time with the amino acid. The cystine transport in rBAT knockdown cells after incubation with antisense oligonucleotides was significantly lower compared to control (0.76 vs. 0.98%; P=0.0008), proving a transient knock-down of the rBAT gene. This study demonstrates the presence of the b(0,+) amino acid transport system in human proximal tubular HK-2 cells when cultured in vitro. Inhibition of this transport system is possible by using antisense technology. A permanent inhibition of the cystine transport, based on our model, would be useful for the development and evaluation gene therapeutic approaches.
Wendt-Nordahl
Gunnar
G
Department of Urology, University Hospital Mannheim, Theodor-Kutzer-Ufer 1-3, 68135 Mannheim, Germany.
Sagi
Sreedhar
S
Bolenz
Christian
C
Alken
Peter
P
Michel
Maurice Stephan
MS
Knoll
Thomas
T
D026905:Amino Acid Transport Systems / Q000378:metabolism; D026923:Amino Acid Transport Systems, Basic / Q000235:genetics / Q000378:metabolism; D026921:Amino Acid Transport Systems, Neutral / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D003553:Cystine / Q000378:metabolism*; D003555:Cystinuria / Q000378:metabolism* / Q000473:pathology; D020868:Gene Silencing / Q000187:drug effects; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D010641:Phenotype; D012333:RNA, Messenger / Q000378:metabolism; D013462:Sulfur Radioisotopes
D026905:Amino Acid Transport Systems; D026923:Amino Acid Transport Systems, Basic; D026921:Amino Acid Transport Systems, Neutral; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; C489857:SLC3A1 protein, human; D013462:Sulfur Radioisotopes; D003553:Cystine