Limits...
'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.

Show MeSH

Related in: MedlinePlus

Ketamine stimulates phosphorylation of ERK and p38 MAPK.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 0–30 minutes under serum free conditions and expression of p-ERK and p-P38 assessed by immunoblotting. Upper panels show representative blots for each protein and re-probed for Total ERK and Total p38 respectively 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<0.01, *** P<0.001.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3756955&req=5

pone-0071819-g004: Ketamine stimulates phosphorylation of ERK and p38 MAPK.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 0–30 minutes under serum free conditions and expression of p-ERK and p-P38 assessed by immunoblotting. Upper panels show representative blots for each protein and re-probed for Total ERK and Total p38 respectively 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<0.01, *** P<0.001.

Mentions: HK2 cells were cultured in 5 mM glucose containing media for 48 hrs prior to overnight serum starvation. Cells were stimulated for 0–30 minutes with Ketamine (1 mg/mL) and expression levels of p-P38 and p-P42/44 assessed by immunoblotting (Fig. 4). P-ERK expression levels increased by 166±11% (P<0.01), 222±27% (P<0.01) and 259±30% (P<0.01), at 1, 5 and 10 minutes respectively as compared to control (Fig. 4A, n = 3). p-P38 expression levels were also altered in response to Ketamine with levels increasing by 63±4% (P<0.01), and 98±25% (P<0.001), at 10 and 30 minutes respectively as compared to control (Fig. 4B, n = 3).


'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 stimulates phosphorylation of ERK and p38 MAPK.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 0–30 minutes under serum free conditions and expression of p-ERK and p-P38 assessed by immunoblotting. Upper panels show representative blots for each protein and re-probed for Total ERK and Total p38 respectively 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<0.01, *** P<0.001.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0071819-g004: Ketamine stimulates phosphorylation of ERK and p38 MAPK.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 0–30 minutes under serum free conditions and expression of p-ERK and p-P38 assessed by immunoblotting. Upper panels show representative blots for each protein and re-probed for Total ERK and Total p38 respectively 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<0.01, *** P<0.001.
Mentions: HK2 cells were cultured in 5 mM glucose containing media for 48 hrs prior to overnight serum starvation. Cells were stimulated for 0–30 minutes with Ketamine (1 mg/mL) and expression levels of p-P38 and p-P42/44 assessed by immunoblotting (Fig. 4). P-ERK expression levels increased by 166±11% (P<0.01), 222±27% (P<0.01) and 259±30% (P<0.01), at 1, 5 and 10 minutes respectively as compared to control (Fig. 4A, n = 3). p-P38 expression levels were also altered in response to Ketamine with levels increasing by 63±4% (P<0.01), and 98±25% (P<0.001), at 10 and 30 minutes respectively as compared to control (Fig. 4B, n = 3).

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