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'Special k' and a loss of cell-to-cell adhesion in proximal tubule-derived epithelial cells: modulation of the adherens junction complex by ketamine.

Hills CE, Jin T, Siamantouras E, Liu IK, Jefferson KP, Squires PE - PLoS ONE (2013)

Bottom Line: These physical changes matched the concentration-dependent (0.1-1 mg/mL) cytotoxic effect of Ketamine and reflect a loss in expression of the key adherens junction proteins epithelial (E)- and neural (N)-cadherin and β-catenin.Down-regulation of protein expression does not involve the pro-fibrotic cytokine TGFβ, nor is it regulated by the usual increase in expression of Slug or Snail, the transcriptional regulators for E-cadherin.Understanding Ketamine-induced renal pathology may identify targets for future therapeutic intervention.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, University of Warwick, Coventry, United Kingdom. C.Hills@warwick.ac.uk

ABSTRACT
Ketamine, a mild hallucinogenic class C drug, is the fastest growing 'party drug' used by 16-24 year olds in the UK. As the recreational use of Ketamine increases we are beginning to see the signs of major renal and bladder complications. To date however, we know nothing of a role for Ketamine in modulating both structure and function of the human renal proximal tubule. In the current study we have used an established model cell line for human epithelial cells of the proximal tubule (HK2) to demonstrate that Ketamine evokes early changes in expression of proteins central to the adherens junction complex. Furthermore we use AFM single-cell force spectroscopy to assess if these changes functionally uncouple cells of the proximal tubule ahead of any overt loss in epithelial cell function. Our data suggests that Ketamine (24-48 hrs) produces gross changes in cell morphology and cytoskeletal architecture towards a fibrotic phenotype. These physical changes matched the concentration-dependent (0.1-1 mg/mL) cytotoxic effect of Ketamine and reflect a loss in expression of the key adherens junction proteins epithelial (E)- and neural (N)-cadherin and β-catenin. Down-regulation of protein expression does not involve the pro-fibrotic cytokine TGFβ, nor is it regulated by the usual increase in expression of Slug or Snail, the transcriptional regulators for E-cadherin. However, the loss in E-cadherin can be partially rescued pharmacologically by blocking p38 MAPK using SB203580. These data provide compelling evidence that Ketamine alters epithelial cell-to-cell adhesion and cell-coupling in the proximal kidney via a non-classical pro-fibrotic mechanism and the data provides the first indication that this illicit substance can have major implications on renal function. Understanding Ketamine-induced renal pathology may identify targets for future therapeutic intervention.

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Related in: MedlinePlus

Ketamine evoked changes in AJ-protein expression in HK2-cells.To assess the effect of Ketamine on expression of key AJ proteins, HK2 cells were cultured in 5 mM glucose containing media for 48 hrs prior to overnight serum starvation. Cells were stimulated for 24 hrs with Ketamine (0.1–1 mg/mL) under serum-free conditions and morphological and phenotypic changes assessed. Phase contrast microscopy (panel A) and TRITC conjugated phalloidin (panel B) confirmed the dose dependent effects of Ketamine on cell morphology and cytoskeletal reorganization respectively. Whole cell expression of E-cadherin and its transcriptional co-repressor Snail were determined by western blotting. Ketamine decreased expression of E-cadherin (panel C) Snail (panel F) and Slug (panel G). Compartmental localisation of E-cadherin, Snail and Slug expression were determined for membrane (M), cytosol (C), nuclear (N) and cytoskeletal (CK) fractions +/− Ketamine (1 mg/mL). Ketamine altered the cellular localization of all three proteins, compared to control (panels Hi, Hiv and Hv respectively). Ketamine also down-regulated expression of N-cadherin (panel D) and β-catenin (panel E), with a loss in expression being apparent throughout the cell (panel Hii and Hiii respectively). Upper panels show representative blots for each protein and re-probed for α-tubulin as a loading control. Lower panels show mean (±SEM) densitometry data, normalised against the non-stimulated low glucose control (100%), from 3 or more separate experiments. Each lane in the representative blot corresponds to the associated bar in the graph. Key significances are shown, *P<0.05, *P<0.01, ***P<0.001.
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pone-0071819-g002: Ketamine evoked changes in AJ-protein expression in HK2-cells.To assess the effect of Ketamine on expression of key AJ proteins, HK2 cells were cultured in 5 mM glucose containing media for 48 hrs prior to overnight serum starvation. Cells were stimulated for 24 hrs with Ketamine (0.1–1 mg/mL) under serum-free conditions and morphological and phenotypic changes assessed. Phase contrast microscopy (panel A) and TRITC conjugated phalloidin (panel B) confirmed the dose dependent effects of Ketamine on cell morphology and cytoskeletal reorganization respectively. Whole cell expression of E-cadherin and its transcriptional co-repressor Snail were determined by western blotting. Ketamine decreased expression of E-cadherin (panel C) Snail (panel F) and Slug (panel G). Compartmental localisation of E-cadherin, Snail and Slug expression were determined for membrane (M), cytosol (C), nuclear (N) and cytoskeletal (CK) fractions +/− Ketamine (1 mg/mL). Ketamine altered the cellular localization of all three proteins, compared to control (panels Hi, Hiv and Hv respectively). Ketamine also down-regulated expression of N-cadherin (panel D) and β-catenin (panel E), with a loss in expression being apparent throughout the cell (panel Hii and Hiii respectively). Upper panels show representative blots for each protein and re-probed for α-tubulin as a loading control. Lower panels show mean (±SEM) densitometry data, normalised against the non-stimulated low glucose control (100%), from 3 or more separate experiments. Each lane in the representative blot corresponds to the associated bar in the graph. Key significances are shown, *P<0.05, *P<0.01, ***P<0.001.

Mentions: The transition of tubular epithelial cells from a typical, cobblestone morphology to a fibrotic phenotype is commonly associated with reorganisation of cell architecture and alterations in the expression of epithelial proteins involved in adherens and tight junction formation [12]–[13]. HK2 cells were cultured in 5 mM glucose containing media for 48 hrs prior to overnight serum starvation. Cells were stimulated for 24 hrs with Ketamine (0.1–1 mg/mL) under serum-free conditions and morphological and phenotypic changes assessed. At 24 hrs, Ketamine induced a concentration-dependent change in cell morphology towards an elongated fibroblast-like phenotype (Fig. 2A). These gross morphological changes were accompanied by re-organisation of the actin cytoskeleton from a diffuse transcellular network of F-actin filaments that spanned the cytosol, into more dense peripheral stress fibres (Fig. 2B).


'Special k' and a loss of cell-to-cell adhesion in proximal tubule-derived epithelial cells: modulation of the adherens junction complex by ketamine.

Hills CE, Jin T, Siamantouras E, Liu IK, Jefferson KP, Squires PE - PLoS ONE (2013)

Ketamine evoked changes in AJ-protein expression in HK2-cells.To assess the effect of Ketamine on expression of key AJ proteins, HK2 cells were cultured in 5 mM glucose containing media for 48 hrs prior to overnight serum starvation. Cells were stimulated for 24 hrs with Ketamine (0.1–1 mg/mL) under serum-free conditions and morphological and phenotypic changes assessed. Phase contrast microscopy (panel A) and TRITC conjugated phalloidin (panel B) confirmed the dose dependent effects of Ketamine on cell morphology and cytoskeletal reorganization respectively. Whole cell expression of E-cadherin and its transcriptional co-repressor Snail were determined by western blotting. Ketamine decreased expression of E-cadherin (panel C) Snail (panel F) and Slug (panel G). Compartmental localisation of E-cadherin, Snail and Slug expression were determined for membrane (M), cytosol (C), nuclear (N) and cytoskeletal (CK) fractions +/− Ketamine (1 mg/mL). Ketamine altered the cellular localization of all three proteins, compared to control (panels Hi, Hiv and Hv respectively). Ketamine also down-regulated expression of N-cadherin (panel D) and β-catenin (panel E), with a loss in expression being apparent throughout the cell (panel Hii and Hiii respectively). Upper panels show representative blots for each protein and re-probed for α-tubulin as a loading control. Lower panels show mean (±SEM) densitometry data, normalised against the non-stimulated low glucose control (100%), from 3 or more separate experiments. Each lane in the representative blot corresponds to the associated bar in the graph. Key significances are shown, *P<0.05, *P<0.01, ***P<0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3756955&req=5

pone-0071819-g002: Ketamine evoked changes in AJ-protein expression in HK2-cells.To assess the effect of Ketamine on expression of key AJ proteins, HK2 cells were cultured in 5 mM glucose containing media for 48 hrs prior to overnight serum starvation. Cells were stimulated for 24 hrs with Ketamine (0.1–1 mg/mL) under serum-free conditions and morphological and phenotypic changes assessed. Phase contrast microscopy (panel A) and TRITC conjugated phalloidin (panel B) confirmed the dose dependent effects of Ketamine on cell morphology and cytoskeletal reorganization respectively. Whole cell expression of E-cadherin and its transcriptional co-repressor Snail were determined by western blotting. Ketamine decreased expression of E-cadherin (panel C) Snail (panel F) and Slug (panel G). Compartmental localisation of E-cadherin, Snail and Slug expression were determined for membrane (M), cytosol (C), nuclear (N) and cytoskeletal (CK) fractions +/− Ketamine (1 mg/mL). Ketamine altered the cellular localization of all three proteins, compared to control (panels Hi, Hiv and Hv respectively). Ketamine also down-regulated expression of N-cadherin (panel D) and β-catenin (panel E), with a loss in expression being apparent throughout the cell (panel Hii and Hiii respectively). Upper panels show representative blots for each protein and re-probed for α-tubulin as a loading control. Lower panels show mean (±SEM) densitometry data, normalised against the non-stimulated low glucose control (100%), from 3 or more separate experiments. Each lane in the representative blot corresponds to the associated bar in the graph. Key significances are shown, *P<0.05, *P<0.01, ***P<0.001.
Mentions: The transition of tubular epithelial cells from a typical, cobblestone morphology to a fibrotic phenotype is commonly associated with reorganisation of cell architecture and alterations in the expression of epithelial proteins involved in adherens and tight junction formation [12]–[13]. HK2 cells were cultured in 5 mM glucose containing media for 48 hrs prior to overnight serum starvation. Cells were stimulated for 24 hrs with Ketamine (0.1–1 mg/mL) under serum-free conditions and morphological and phenotypic changes assessed. At 24 hrs, Ketamine induced a concentration-dependent change in cell morphology towards an elongated fibroblast-like phenotype (Fig. 2A). These gross morphological changes were accompanied by re-organisation of the actin cytoskeleton from a diffuse transcellular network of F-actin filaments that spanned the cytosol, into more dense peripheral stress fibres (Fig. 2B).

Bottom Line: These physical changes matched the concentration-dependent (0.1-1 mg/mL) cytotoxic effect of Ketamine and reflect a loss in expression of the key adherens junction proteins epithelial (E)- and neural (N)-cadherin and β-catenin.Down-regulation of protein expression does not involve the pro-fibrotic cytokine TGFβ, nor is it regulated by the usual increase in expression of Slug or Snail, the transcriptional regulators for E-cadherin.Understanding Ketamine-induced renal pathology may identify targets for future therapeutic intervention.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, University of Warwick, Coventry, United Kingdom. C.Hills@warwick.ac.uk

ABSTRACT
Ketamine, a mild hallucinogenic class C drug, is the fastest growing 'party drug' used by 16-24 year olds in the UK. As the recreational use of Ketamine increases we are beginning to see the signs of major renal and bladder complications. To date however, we know nothing of a role for Ketamine in modulating both structure and function of the human renal proximal tubule. In the current study we have used an established model cell line for human epithelial cells of the proximal tubule (HK2) to demonstrate that Ketamine evokes early changes in expression of proteins central to the adherens junction complex. Furthermore we use AFM single-cell force spectroscopy to assess if these changes functionally uncouple cells of the proximal tubule ahead of any overt loss in epithelial cell function. Our data suggests that Ketamine (24-48 hrs) produces gross changes in cell morphology and cytoskeletal architecture towards a fibrotic phenotype. These physical changes matched the concentration-dependent (0.1-1 mg/mL) cytotoxic effect of Ketamine and reflect a loss in expression of the key adherens junction proteins epithelial (E)- and neural (N)-cadherin and β-catenin. Down-regulation of protein expression does not involve the pro-fibrotic cytokine TGFβ, nor is it regulated by the usual increase in expression of Slug or Snail, the transcriptional regulators for E-cadherin. However, the loss in E-cadherin can be partially rescued pharmacologically by blocking p38 MAPK using SB203580. These data provide compelling evidence that Ketamine alters epithelial cell-to-cell adhesion and cell-coupling in the proximal kidney via a non-classical pro-fibrotic mechanism and the data provides the first indication that this illicit substance can have major implications on renal function. Understanding Ketamine-induced renal pathology may identify targets for future therapeutic intervention.

Show MeSH
Related in: MedlinePlus