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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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The role of PI3-K, ERK and p38 MAPK in mediating the Ketamine response.HK2 cells were cultured in 5 mM glucose containing media for 48 hrs prior to overnight serum starvation. Cells were stimulated with Ketamine (1 mg/mL) for 24 hrs in the presence or absence of Wortmannin (2 µM), PD98059 (10 µM), and SB203580 (1 µM) under serum-free conditions. Phase contrast microscopy (panel A) and TRITC conjugated phalloidin (panel B) were used to confirm if blockade of PI3-K, ERK or p38 MAPK was able to negate Ketamine-induced changes in cell morphology and cytoskeletal reorganization. HK2 cells were cultured in 5 mM glucose containing media for 48 hrs prior to overnight serum starvation. Cells were stimulated with Ketamine (1 mg/mL) for 24 hrs in the presence or absence of Wortmannin (2 µM), PD98059 (10 µM), and SB203580 (1 µM) under serum-free conditions and the expression levels of E-cadherin (panel C), N-cadherin (panel D), β-catenin (panel E), Snail (panel F) and Slug (panel G) determined by immuno-blotting. 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-g005: The role of PI3-K, ERK and p38 MAPK in mediating the Ketamine response.HK2 cells were cultured in 5 mM glucose containing media for 48 hrs prior to overnight serum starvation. Cells were stimulated with Ketamine (1 mg/mL) for 24 hrs in the presence or absence of Wortmannin (2 µM), PD98059 (10 µM), and SB203580 (1 µM) under serum-free conditions. Phase contrast microscopy (panel A) and TRITC conjugated phalloidin (panel B) were used to confirm if blockade of PI3-K, ERK or p38 MAPK was able to negate Ketamine-induced changes in cell morphology and cytoskeletal reorganization. HK2 cells were cultured in 5 mM glucose containing media for 48 hrs prior to overnight serum starvation. Cells were stimulated with Ketamine (1 mg/mL) for 24 hrs in the presence or absence of Wortmannin (2 µM), PD98059 (10 µM), and SB203580 (1 µM) under serum-free conditions and the expression levels of E-cadherin (panel C), N-cadherin (panel D), β-catenin (panel E), Snail (panel F) and Slug (panel G) determined by immuno-blotting. 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: Having confirmed that Ketamine does not depend on TGF-β dependent signalling and that it can evoke activation of MAPK signalling, we examined whether the morphological and phenotypic effects of Ketamine could be arrested or reversed by pharmacological intervention with downstream inhibitors of the MAPK pathway. Cells were cultured in 5 mM glucose containing media for 48 hrs prior to an overnight serum starvation. Cells were stimulated for 24 hrs with the highest, non-lethal concentration of Ketamine (1 mg/mL) in the presence or absence of wortmannin (2 µM), PD98059 (10 µM), and SB203580 (1 µM) under serum-free conditions. Phase contrast morphology confirmed that incubation with wortmannin, PD98059, or SB203580 partly reversed gross Ketamine induced changes in cell morphology (See Fig. 5A). Qualitatively, SB203580 treated cells most closely resembled the cobblestone appearance observed under control conditions, and suggest that p38 MAPK may partially mediate the Ketamine response. Restoration of a normal cellular architecture may reflect the partial reclamation of E-cadherin expression following SB203580 treatment (see Fig. 5C). Contradictory to this, TRITC-conjugated phalloidin, suggested that the pattern of filamentous F-actin staining seen in control cells, was partially restored following treatment with the ERK inhibitor PD98059 and not SB203580 (Fig. 5B).


'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)

The role of PI3-K, ERK and p38 MAPK in mediating the Ketamine response.HK2 cells were cultured in 5 mM glucose containing media for 48 hrs prior to overnight serum starvation. Cells were stimulated with Ketamine (1 mg/mL) for 24 hrs in the presence or absence of Wortmannin (2 µM), PD98059 (10 µM), and SB203580 (1 µM) under serum-free conditions. Phase contrast microscopy (panel A) and TRITC conjugated phalloidin (panel B) were used to confirm if blockade of PI3-K, ERK or p38 MAPK was able to negate Ketamine-induced changes in cell morphology and cytoskeletal reorganization. HK2 cells were cultured in 5 mM glucose containing media for 48 hrs prior to overnight serum starvation. Cells were stimulated with Ketamine (1 mg/mL) for 24 hrs in the presence or absence of Wortmannin (2 µM), PD98059 (10 µM), and SB203580 (1 µM) under serum-free conditions and the expression levels of E-cadherin (panel C), N-cadherin (panel D), β-catenin (panel E), Snail (panel F) and Slug (panel G) determined by immuno-blotting. 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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3756955&req=5

pone-0071819-g005: The role of PI3-K, ERK and p38 MAPK in mediating the Ketamine response.HK2 cells were cultured in 5 mM glucose containing media for 48 hrs prior to overnight serum starvation. Cells were stimulated with Ketamine (1 mg/mL) for 24 hrs in the presence or absence of Wortmannin (2 µM), PD98059 (10 µM), and SB203580 (1 µM) under serum-free conditions. Phase contrast microscopy (panel A) and TRITC conjugated phalloidin (panel B) were used to confirm if blockade of PI3-K, ERK or p38 MAPK was able to negate Ketamine-induced changes in cell morphology and cytoskeletal reorganization. HK2 cells were cultured in 5 mM glucose containing media for 48 hrs prior to overnight serum starvation. Cells were stimulated with Ketamine (1 mg/mL) for 24 hrs in the presence or absence of Wortmannin (2 µM), PD98059 (10 µM), and SB203580 (1 µM) under serum-free conditions and the expression levels of E-cadherin (panel C), N-cadherin (panel D), β-catenin (panel E), Snail (panel F) and Slug (panel G) determined by immuno-blotting. 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: Having confirmed that Ketamine does not depend on TGF-β dependent signalling and that it can evoke activation of MAPK signalling, we examined whether the morphological and phenotypic effects of Ketamine could be arrested or reversed by pharmacological intervention with downstream inhibitors of the MAPK pathway. Cells were cultured in 5 mM glucose containing media for 48 hrs prior to an overnight serum starvation. Cells were stimulated for 24 hrs with the highest, non-lethal concentration of Ketamine (1 mg/mL) in the presence or absence of wortmannin (2 µM), PD98059 (10 µM), and SB203580 (1 µM) under serum-free conditions. Phase contrast morphology confirmed that incubation with wortmannin, PD98059, or SB203580 partly reversed gross Ketamine induced changes in cell morphology (See Fig. 5A). Qualitatively, SB203580 treated cells most closely resembled the cobblestone appearance observed under control conditions, and suggest that p38 MAPK may partially mediate the Ketamine response. Restoration of a normal cellular architecture may reflect the partial reclamation of E-cadherin expression following SB203580 treatment (see Fig. 5C). Contradictory to this, TRITC-conjugated phalloidin, suggested that the pattern of filamentous F-actin staining seen in control cells, was partially restored following treatment with the ERK inhibitor PD98059 and not SB203580 (Fig. 5B).

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