Hypertonicity-induced mitochondrial membrane permeability in renal medullary interstitial cells: protective role of osmolytes

Abstract

Background: Hyperosmotic stress causes cell death through activation of apoptotic pathways if the protective osmolyte response is impaired. In this study we attempt to elucidate the molecular mechanisms of hypertonicity-induced apoptosis and the effect of major organic osmolytes upon those.

Methods: Hypertonicity-induced changes in Bcl2-family protein abundance and the presence of cytochrome c and apoptosis inducing factor (AIF) in the cytoplasm, were measured using western blot and immunofluorescence labeling. To determine dissipation of mitochondrial membrane potential (Delta Psi) though the permeability transition pore (PTP), the lipophilic cationic carbocyanine fluorescence probe JC-1 and TMRM fluorescence probes were used.

Results: Hypertonic culture conditions increase the abundance of proapoptotic Bax and the concentration of cytochrome c and apoptosis inducing factor (AIF) in the cytoplasm. These changes are associated with a dissipation of Delta Psi and increased permeability of the PTP. We further show that organic osmolytes stabilize the Delta Psi and decrease the concentration of cytochrome c and AIF in the cytoplasm.

Conclusion: Our study shows that organic osmolytes prevent hypertonicity-induced apoptosis by preventing dissipation of Delta Psi through stabilization of the PTP. These findings further support the important role of organic osmolytes in preventing hypertonicity-mediated cell death in medullary kidney cells.

Fig. 1

Cytochrome c levels in cytosolic and mitochondrial fractions of medullary interstitial cells (MICs) following exposure to 600 mosmol/L for 3, 6, 12 and 24 hours. Cytosolic cytochrome c abundance was markedly increased at 6 and 12 hours. Cytochrome c levels were determined using western blot; iso=isotonic medium (295 mosol/L), h= hours, HT=hypertonic culture medium. All values are mean ± SEM of n=4. *p<0.05 compared to isotonic control at 0 hours.

Fig. 2

Immunofluorescence labeling (Texas red) of Apoptosis Inducing Factor (AIF) in medullary interstitial cells (MICs) under isotonic and hypertonic conditions (400 and 600 mosmol/L for 12 hours). Preincubation of MICs with 1 mmol/L betaine followed by exposure to 600 mosmol/L for 12 hours markedly decreased AIF staining (right lower quadrant). AIF/β-actin ratio was determined by western blot in MICs exposed to 400 and 600 mosmol/L for 12 and 24 hours respectively. All values are mean ± SEM of n=4; *p<0.05 compared to isotonic controls.

Fig. 3

A: Medullary interstitial cell (MIC) mitochondria were labeled with TMRM (5 μM, red) and calcein AM (1 μM, green) under isotonic conditions, followed by exposure to 600 mosmol/L medium tonicity for 6 hours (middle column) and 12 hours (right column). Hypertonicity induced rapid loss of TMRM and calcein fluorescence over the course of 12 hours (top row, right image). Preincubation of MICs with 1 mM/L betaine preserved mitochondrial potential as shown by persistent strong TMRM stain over 12 hours. Knock down of Bax (insert) using silencing RNA also increased TMRM staining over 12 hours of hypertonic exposure (bottom row, right image). TMRM= tetramethylrhodamine methyl ester, iso=isotonic conditions. B: Cytoplasmic Bax levels increased in MICs that were exposed to 600 mosmol/L for 6 (middle column) and 12 hours (right column). After 12 hours of hypertonic medium, Bax fluorescence (green, top row, right image) was strongest in mitochondria (co-labeled with MitoTracker, red, second row from top, right image). To the contrary Bcl-2 fluorescence (green, second row from bottom) was markedly decreased at 6 and 12 hours of exposure to 600 mosmol/L (middle and right image). Bcl-2 primarily located in mitochondrial regions of the cytoplasm as shown by the Mitotracker stained organelles (red, bottom row) under isotonic conditions. Exposure to 600 mosmol/L medium showed persistent mitotracker staining of mitochondria for 6 and 12 hours (bottom row). Images are representative of four experiments.

Fig. 4

Bcl-2/Bax ratio was determined by western blot in MICs under isotonic conditions (0 hour) and after exposure to 600 mosmol/L medium tonicity for 6, 12 and 24 hours. Compared to isotonic controls, the Bcl-2/Bax ratio markedly decreased at 6, 12 and 24 hours following exposure to hypertonic culture conditions. Preincubation of MICs with 1 mmol/L betaine markedly inhibited Bax expression at 6, 12 and 24 hours of hypertonicity and reversed the ratio in favor of Bcl-2. Western blot is representative of four individual experiments. Values in graph blot are mean±SEM of n=4. *p<0.05 compared to identical time point without betaine treatment.

Fig. 5

Isolated mitochondria (1mg/ml) from medullary interstitial cells were suspended in isotonic and hypertonic (600 mosmol/L) PBS buffer for 30 or 60 minutes and changes in mitochondrial membrane potential (Δψ) were measured by the JC-1 fluorescence ratio of red to green (see methods). CCCP (50 μM) was used as positive control. The osmolytes betaine, inositol or sorbitol were added to hypertonic buffer (600 msmol/L, PBS) at 1 mmol/L. Hypertonicity dissipated Δψ to a similar degree as CCCP. Addition of betaine, inositol and sorbitol significantly diminished Δψ dissipation. *p<0.05 compared to samples at identical time points and 600 mosmol/L medium tonicity without osmolyte supplementation.

Fig. 6

Medullary interstitial cells (MICs) were loaded with TMRM and exposed to 600 mosmol/L for 12 hours with or without Cyclosporine A (CsA). CCCP (50 μM) was used as positive control to dissipate the mitochondrial membrane potential both under isotonic and hypertonic conditions (top and middle row, middle images). Hypertonic medium caused significant loss of membrane potential, which was prevented by CsA (middle row, left and right images). Preloading MICs with organic osmolytes (1 mmol/L medium concentration), prior to exposure to 600 mosmol/L for 12 hours, preserved mitochondrial as shown by persistent TMRM fluorescence.