PMID- 18074125
OWN - NLM
STAT- MEDLINE
DCOM- 20080701
LR  - 20211020
IS  - 0300-5623 (Print)
IS  - 0300-5623 (Linking)
VI  - 36
IP  - 1
DP  - 2008 Feb
TI  - Evaluation of cystine transport in cultured human kidney cells and establishment 
      of cystinuria type I phenotype by antisense technology.
PG  - 25-9
AB  - 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.
FAU - Wendt-Nordahl, Gunnar
AU  - Wendt-Nordahl G
AD  - Department of Urology, University Hospital Mannheim, Theodor-Kutzer-Ufer 1-3, 
      68135 Mannheim, Germany.
FAU - Sagi, Sreedhar
AU  - Sagi S
FAU - Bolenz, Christian
AU  - Bolenz C
FAU - Alken, Peter
AU  - Alken P
FAU - Michel, Maurice Stephan
AU  - Michel MS
FAU - Knoll, Thomas
AU  - Knoll T
LA  - eng
PT  - Journal Article
DEP - 20071207
PL  - Germany
TA  - Urol Res
JT  - Urological research
JID - 0364311
RN  - 0 (Amino Acid Transport Systems)
RN  - 0 (Amino Acid Transport Systems, Basic)
RN  - 0 (Amino Acid Transport Systems, Neutral)
RN  - 0 (Oligonucleotides, Antisense)
RN  - 0 (RNA, Messenger)
RN  - 0 (SLC3A1 protein, human)
RN  - 0 (Sulfur Radioisotopes)
RN  - 48TCX9A1VT (Cystine)
SB  - IM
MH  - Amino Acid Transport Systems/metabolism
MH  - Amino Acid Transport Systems, Basic/genetics/metabolism
MH  - Amino Acid Transport Systems, Neutral/genetics/metabolism
MH  - Biological Transport/drug effects
MH  - Cell Line
MH  - Cystine/*metabolism
MH  - Cystinuria/*metabolism/pathology
MH  - Gene Silencing/drug effects
MH  - Humans
MH  - Kidney Tubules, Proximal/drug effects/*metabolism/pathology
MH  - Oligonucleotides, Antisense/*pharmacology
MH  - Phenotype
MH  - RNA, Messenger/metabolism
MH  - Sulfur Radioisotopes
EDAT- 2007/12/13 09:00
MHDA- 2008/07/02 09:00
CRDT- 2007/12/13 09:00
PHST- 2007/07/18 00:00 [received]
PHST- 2007/11/21 00:00 [accepted]
PHST- 2007/12/13 09:00 [pubmed]
PHST- 2008/07/02 09:00 [medline]
PHST- 2007/12/13 09:00 [entrez]
AID - 10.1007/s00240-007-0127-z [doi]
PST - ppublish
SO  - Urol Res. 2008 Feb;36(1):25-9. doi: 10.1007/s00240-007-0127-z. Epub 2007 Dec 7.