Profiling at mRNA, protein, and metabolite levels reveals alterations in renal amino acid handling and glutathione metabolism in kidney tissue of Pept2-/- mice.
PEPT2 is an integral membrane protein in the apical membrane of renal epithelial cells that operates as a rheogenic transporter for di- and tripeptides and structurally related drugs. Its prime role is thought to be the reabsorption of filtered di- and tripeptides contributing to amino acid homeostasis. To elucidate the role of PEPT2 in renal amino acid metabolism we submitted kidney tissues of wild-type and a Pept2(-/-) mouse line to a comprehensive transcriptome, proteome and metabolome profiling and analyzed urinary amino acids and dipeptides. cDNA microarray analysis identified 147 differentially expressed transcripts in transporter-deficient animals, and proteome analysis by 2D-PAGE and MALDI-TOF-MS identified 37 differentially expressed proteins. Metabolite profiling by GC-MS revealed predominantly altered concentrations of amino acids and derivatives. Urinary excretion of amino acids demonstrated increased glycine and cysteine/cystine concentrations and dipeptides in urine were assessed by amino acid analysis of urine samples before and after in vitro dipeptidase digestion. Dipeptides constituted a noticeable fraction of urinary amino acids in Pept2(-/-) animals, only, and dipeptide-bound glycine and cystine were selectively increased in Pept2(-/-) urine samples. These findings were confirmed by a drastically increased excretion of cysteinyl-glycine (cys-gly). Urinary loss of cys-gly together with lower concentrations of cysteine, glycine, and oxoproline in kidney tissue and altered expression of mRNA and proteins involved in glutathione (GSH) metabolism suggests that PEPT2 is predominantly a system for reabsorption of cys-gly originating from GSH break-down, thus contributing to resynthesis of GSH.
Frey
Isabelle M
IM
Molecular Nutrition Unit, Technical University of Munich, Freising, Germany.
Rubio-Aliaga
Isabel
I
Siewert
Anne
A
Sailer
Daniela
D
Drobyshev
Aleksey
A
Beckers
Johannes
J
de Angelis
Martin Hrabé
MH
Aubert
Julie
J
Bar Hen
Avner
A
Fiehn
Oliver
O
Eichinger
Hans M
HM
Daniel
Hannelore
H
D000596:Amino Acids / Q000378:metabolism / Q000652:urine*; D000818:Animals; D004151:Dipeptides / Q000378:metabolism / Q000652:urine; D015180:Electrophoresis, Gel, Two-Dimensional; D020869:Gene Expression Profiling; D005978:Glutathione / Q000378:metabolism* / Q000652:urine; D007668:Kidney / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008954:Models, Biological; D020411:Oligonucleotide Array Sequence Analysis; D040901:Proteomics; D012333:RNA, Messenger / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D027981:Symporters / Q000235:genetics / Q000502:physiology*
D000596:Amino Acids; D004151:Dipeptides; D012333:RNA, Messenger; D027981:Symporters; C093694:hydrogen-coupled oligopeptide transporter PepT2; C028505:cysteinylglycine; D005978:Glutathione