Dataset Information

Cisplatin-induced downregulation of OCTN2 affects carnitine wasting.

ABSTRACT: Carnitine is an essential cofactor for mitochondrial fatty acid oxidation that is actively reabsorbed by the luminal transporter Octn2 (Slc22a5). Because the nephrotoxic agent cisplatin causes urinary loss of carnitine in humans, we hypothesized that cisplatin may affect Octn2 function.Excretion of carnitine and acetylcarnitine was measured in urine collected from mice with or without cisplatin administration. The transport of carnitine was assessed in cells that were transfected with OCT1 or OCT2. The effect of cisplatin treatment on gene expression was analyzed using a mouse GeneChip array and validated using quantitative reverse transcriptase-PCR.In wild-type mice, urinary carnitine excretion at baseline was ?3-fold higher than in mice lacking the basolateral cisplatin transporters Oct1 and Oct2 [Oct1/2(-/-) mice], indicating that carnitine itself undergoes basolateral uptake into the kidney. Transport of carnitine by OCT2, but not OCT1, was confirmed in transfected cells. We also found that cisplatin caused an increase in the urinary excretion of carnitine and acetylcarnitine in wild-type mice but not in Oct1/2(-/-) mice, suggesting that tubular transport of cisplatin is a prerequisite for this phenomenon. Cisplatin did not directly inhibit the transport of carnitine by Octn2 but downregulated multiple target genes of the transcription factor peroxisome proliferator activated receptor ?, including Slc22a5, in the kidney of wild-type mice that were absent in Oct1/2(-/-) mice.Our study shows a pivotal role of Oct1 and Oct2 in cisplatin-related disturbances in carnitine homeostasis. We postulate that this phenomenon is triggered by deactivation of peroxisome proliferator activated receptor ? and leads to deregulation of carnitine-shuttle genes.

SUBMITTER: Lancaster CS

PROVIDER: S-EPMC3531239 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Cisplatin-induced downregulation of OCTN2 affects carnitine wasting.

Lancaster Cynthia S CS Hu Chaoxin C Franke Ryan M RM Filipski Kelly K KK Orwick Shelley J SJ Chen Zhaoyuan Z Zuo Zhili Z Loos Walter J WJ Sparreboom Alex A

Clinical cancer research : an official journal of the American Association for Cancer Research 20100921 19

<h4>Purpose</h4>Carnitine is an essential cofactor for mitochondrial fatty acid oxidation that is actively reabsorbed by the luminal transporter Octn2 (Slc22a5). Because the nephrotoxic agent cisplatin causes urinary loss of carnitine in humans, we hypothesized that cisplatin may affect Octn2 function.<h4>Experimental design</h4>Excretion of carnitine and acetylcarnitine was measured in urine collected from mice with or without cisplatin administration. The transport of carnitine was assessed in ...[more]

PMID: 20858838

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Project description:Primary carnitine deficiency is caused by impaired activity of the Na(+)-dependent OCTN2 carnitine/organic cation transporter. Carnitine is essential for entry of long-chain fatty acids into mitochondria and its deficiency impairs fatty acid oxidation. Most missense mutations identified in patients with primary carnitine deficiency affect putative transmembrane or intracellular domains of the transporter. Exceptions are the substitutions P46S and R83L located in an extracellular loop close to putative glycosylation sites (N57, N64, and N91) of OCTN2. P46S and R83L impaired glycosylation and maturation of OCTN2 transporters to the plasma membrane. We tested whether glycosylation was essential for the maturation of OCTN2 transporters to the plasma membrane. Substitution of each of the three asparagine (N) glycosylation sites with glutamine (Q) decreased carnitine transport. Substitution of two sites at a time caused a further decline in carnitine transport that was fully abolished when all three glycosylation sites were substituted by glutamine (N57Q/N64Q/N91Q). Kinetic analysis of carnitine and sodium-stimulated carnitine transport indicated that all substitutions decreased the Vmax for carnitine transport, but N64Q/N91Q also significantly increased the Km toward carnitine, indicating that these two substitutions affected regions of the transporter important for substrate recognition. Western blot analysis confirmed increased mobility of OCTN2 transporters with progressive substitutions of asparagines 57, 64 and/or 91 with glutamine. Confocal microscopy indicated that glutamine substitutions caused progressive retention of OCTN2 transporters in the cytoplasm, up to full retention (such as that observed with R83L) when all three glycosylation sites were substituted. Tunicamycin prevented OCTN2 glycosylation, but it did not impair maturation to the plasma membrane. These results indicate that OCTN2 is physiologically glycosylated and that the P46S and R83L substitutions impair this process. Glycosylation does not affect maturation of OCTN2 transporters to the plasma membrane, but the 3 asparagines that are normally glycosylated are located in a region important for substrate recognition and turnover rate.

Dataset Information

Cisplatin-induced downregulation of OCTN2 affects carnitine wasting.

Publications

Cisplatin-induced downregulation of OCTN2 affects carnitine wasting.

Similar Datasets

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