K+-induced HSP-72 expression is mediated via rapid Ca2+ influx in renal epithelial cells

Oliver Eickelberg, John Geibel, Frank Seebach, Gerhard Giebisch, and Michael Kashgarian

Departments of Pathology, Surgery, and Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520-8023


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Pathophysiological stimuli, including hypoxia, lead to K+ efflux from the intracellular lumen to the extracellular space, thereby increasing local tissue K+ concentrations and depolarizing resident cells. In this study, we investigated the effects of increased extracellular K+ concentrations ([K+]e) on heat shock protein (HSP) expression in the porcine proximal tubule epithelial cell line LLC-PK1. We analyzed HSP-25, HSP-72, HSC-73, and HSP-90 protein expression by Western blot analyses and HSP-72 promoter activity by luciferase reporter gene assays using the proximal 1,440 bp of the HSP-72 promoter. Elevating [K+]e from 20 to 50 mM increased HSP-72 protein expression and promoter activity but did not affect HSP-25, HSC-73, or HSP-90 levels. Addition of identical concentrations of sodium chloride did not increase HSP-72 expression to a similar amount. The Ca2+ channel blocker diltiazem and the Ca2+-specific chelator EGTA-AM abolished high [K+]e-induced HSP-72 expression by 69.7 and 75.2%, respectively, indicating that the transcriptional induction of HSP-72 involves Ca2+ influx. As measured by confocal microscopy using the Ca2+ dye fluo 3-AM, we also observed a rapid increase of intracellular Ca2+ concentration as early as 30 s after placing LLC-PK1 cells in high [K+]e. We further analyzed whether Ca2+ influx was necessary for induction of HSP-72 expression by high [K+]e using Ca2+-free medium. Here, induction of HSP-72 in response to high [K+]e was completely abolished. Our data thus demonstrate activation of a protective cellular response to ionic stress, e.g., elevated K+ concentrations, by specifically increasing protein levels of HSP-72.

ischemia; heat shock proteins; LLC-PK1; stress response; calcium ion; potassium ion


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

LIVING ORGANISMS share a common molecular response to stress characterized by rapid induction of stress-specific transcription factors and a change in the pattern of gene expression (9, 24, 34). The stress response mediates adaption and survival of an organism/cell after an initial insult. Heat shock proteins (HSP) are the major class of proteins presumed to confer cytoprotection against injury. HSP constitute a family of evolutionary highly conserved proteins that is categorized into subfamilies according to molecular size and amino acid sequence homology. HSP are found throughout the cell and, under cellular stress, the amount of all HSP isoforms can constitute up to 5% of total cellular proteins (4, 28, 40).

HSP subfamilies comprise both constitutive and inducible isoforms. Constitutive isoforms (HSC-73 and HSP-90) assist in proper protein folding and membrane translocation processes during protein translation (24, 30). In contrast, inducible isoforms, most notably HSP-72, are believed to be the main contributors to cytoprotection against injuries, such as heat, ionic stress, or hypoxia (2, 9, 10). In this regard, HSP-72 has been shown to protect intracellular proteins from precipitation and aggregation during injury (4, 19, 20). A relevant in vivo example is the prior induction of HSP by nonlethal injuries that protect transplanted pig kidneys from ischemic injury (35) or rabbit hearts against ischemic-reperfusion injury (6).

Hypoxic injury of the kidney, often observed in diseases such as arteriosclerosis, shock, or renal cancer, leads to a significant loss of organ function (22, 25, 44). The kidney is a highly perfused organ and is very susceptible to hypoxic insults. Interestingly, susceptibility to hypoxia among different compartments within the kidney varies widely. Most susceptible to hypoxia are proximal tubule cells. Here, oxygen or substrate deprivation leads to membrane depolarization, K+ efflux, and, depending on the severity of the injury, cell death by necrosis or apoptosis (5, 14, 27). Whereas apoptosis has little effects on neighboring cells, necrotic cells release their intracellular contents into the interstitium and thereby increase local tissue K+ concentrations [extracellular K+ concentration ([K+]e); see Ref. 22]. Increased [K+]e, as high as 60 mM, have been found in the brain after injury or hypoxia (17, 33). In the kidney, [K+]e up to 40 mM are measured in the papillae, even under physiological conditions (3, 29, 39). Interestingly, [K+]e also increases when intracellular ATP is reduced below the levels needed to maintain Na-K-ATPase activity (21, 41). As such, increased tissue [K+]e is also observed in conditions of sublethal injury without evidence for necrosis.

The aim of this study was to define the effects of increased [K+]e on HSP expression in LLC-PK1 cells at elevated levels of [K+]e or extracellular Na+ concentration ([Na+]e). HSP expression was assessed by Western blot analyses and HSP-72 promoter activity by luciferase reporter gene analyses using a fragment of the HSP-72 promoter. We demonstrate that high [K+]e is a potent and specific activator of HSP-72 expression. Increased protein expression of HSP-72 is a result of activation of its promoter and is best explained by rapid Ca2+ influx via voltage-gated Ca2+ channels. Induction of HSP-72 could therefore represent a possible mechanism accounting for autoprotection of cells against ionic stress, such as increased local K+ concentrations.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. LLC-PK1 cells were obtained from ATCC (Manassas, VA) and were cultured in alpha -MEM supplemented with 10% FBS, 8 mM L-glutamine, and 20 mM HEPES (media and supplements from GIBCO-BRL Life Technologies, Grand Island, NY). Cells were grown until 80% confluent, incubated for 24 h in FBS-free alpha -MEM, and subjected to treatment as indicated. No antibiotics or antimycotics were added to the culture conditions at any time.

Western blot analysis of HSP expression. For Western blot analysis, cells were seeded on 60-mm cell culture dishes and allowed to reach 80% confluence. After 24 h of serum starvation, cultures were stimulated by increasing extracellular K+ to the indicated values, and whole cell extracts were prepared. In brief, cells were washed two times with ice-cold PBS and harvested by scraping in 1 ml of PBS. Samples were centrifuged for 30 s at 6,000 g, and cell pellets were resuspended in 400 µl reducing sample buffer [62.5 mM Tris · HCl (pH 6.8), 2% wt/vol SDS, 10% glycerol, 50 mM dithiothreitol, and 0.1% wt/vol bromphenol blue supplemented with Complete (Boehringer Mannheim, Indianapolis, IN), a set of proteinase inhibitors]. Samples were heated to 95°C for 10 min and centrifuged at 13,000 g (5 min), and the supernatants were taken as whole cell extracts. Expression of HSP-25, HSP-72, HSP-90, or HSC-73 was determined by Western blot analysis using 7.5% SDS-PAGE. Aliquots of cell extracts were applied to the gels and run at 25 mA constant current for 3 h at room temperature. After electrophoresis, proteins were blotted on nitrocellulose transfer membranes overnight with 1 mA/cm2 at room temperature. Membranes were blocked in 5% skim milk in TBS-Tween for 1 h at room temperature (TBS-Tween is 10 mM Tris, 150 mM NaCl, and 0.05% Tween 20, pH 8.0) to reduce unspecific binding and were incubated with antibodies against HSP-25, HSP-72, HSP-90, or HSC-73 (StressGen, Victoria, BC) at room temperature for 1 h. Membranes were washed three times with TBS-Tween and incubated with the secondary, peroxidase-coupled antibody (Amersham Life Science, Buckinghamshire, UK) at a dilution of 1:10,000 for 1 h at room temperature. Specific bands were visualized using the enhanced chemiluminescence system from Amersham Life Science according to the manufacturer's instructions, as previously described (7).

Luciferase reporter gene assays. A 1.44-kb promoter fragment of the HSP-72 gene immediately upstream of the transcriptional start site (obtained from StressGen) was subcloned in correct orientation in front of a luciferase reporter gene (pGL3 Basic; Promega, Madison, WI). LLC-PK1 cells were transfected with this construct by lipofection, and luciferase reporter gene assays were carried out as described earlier (8). In brief, cells were seeded in 48-well plates (1 × 104 cells/well) and serum deprived for 24 h. Cells were subjected to liposomal transfection using the cationic lipid Tfx-50 (Promega) at a DNA-to-lipid ratio of 1:3 (using 0.3 µg plasmid/well). Transfections were carried out in the absence of FBS for 2 h at 37°C in humidified atmosphere. After transfection, cells were overlaid with specific media, as indicated. After 16 h, cells were washed two times with ice-cold PBS and lysed, and equal amounts of lysates were analyzed for firefly luciferase expression. In brief, 10-µl aliquots of cell lysates were mixed with 50 µl of luciferase reagent buffer, and luminescence of the samples was integrated over a time period of 10 s in a luminometer. To control for transfection efficiency and unspecific inductions, identical experiments were carried out in parallel using reporter gene vector without the HSP-72 promoter sequence (pGL3 control; Promega).

Measurements of intracellular Ca2+ concentration in LLC-PK1 cells. LLC-PK1 cells were cultured to confluence on glass coverslips. Before microscopic analysis, cells were incubated for 20 min in medium containing the Ca2+-sensing dye fluo 3-AM ester (Molecular Probes, Eugene, OR) at 1 µmol/l to allow dye uptake and ester hydrolysis. Cells were washed with medium alone to remove excess dye. Coverslips were placed on the microscope, background fluorescence was measured in regular medium with excitation at 488 nm, and emission was monitored at 535 nm. Cells were then placed in medium containing 30 mM K+, and changes in Ca2+ were monitored, as described earlier (43). Fluorescence intensity was determined by measuring pixel values over each cell of interest. Images (512 × 512 × 12-bit deep) were measured over selected areas before stimulation and then every 30 s over a time frame of 30 min during this study. Sequential frames were acquired at 2-s intervals, with each image comprising an average of eight frames with ~30 s between images. Each protocol lasted for 30 min, and images were stored on hard disc. At least five LLC-PK1 cells were analyzed in each experiment per cover slip studied. Data were expressed in arbitrary fluorescence intensity units, summarized as means ± SE, and analyzed by pairing measurements obtained during baseline and experimental maneuvers. A paired Student's t-test was used, with significance taken at P < 0.05.

Statistical analysis. All experiments were performed at least in triplicate using two independent sets of experiments. Luciferase assays were done in quadruplicates for each stimulation. Homogeneity of groups was analyzed by two-tailed Student's t-test for each time point or concentration.

Chemicals. Chemicals used in this study were diltiazem, EGTA-AM, thapsigargin, 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), and ionomycin (all from Sigma, St. Louis, MO).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

High [K+]e specifically upregulates HSP-72 expression in LLC-PK1 cells. Increasing [K+]e imposes ionic stress on cells and leads to membrane depolarization. We therefore analyzed HSP expression in LLC-PK1 cells under control conditions and under conditions of elevated [K+]e, ranging from 20 to 50 mM. Control cultures were maintained in regular culture media containing 5.5 mM [K+]e. HSP expression was assessed by Western blot analyses of whole cell extracts after placing the cells in elevated [K+]e for the indicated time periods. Figure 1A represents characteristic Western blots demonstrating rapid upregulation of HSP-72 protein as early as 6 h after placing the cells in culture media containing 50 mM KCl. As shown, LLC-PK1 cells exhibit constitutive expression levels of HSP-72 under physiological [K+]e. These levels of HSP-72 are rapidly upregulated by high [K+]e, and increased levels of HSP-72 are still observed after 24 h of exposure to high [K+]e (Fig. 1). In contrast, protein levels of HSP-90, HSC-73, and HSP-25 are not affected by high [K+]e (Fig. 1A), indicating that the induction is specific for HSP-72.


View larger version (59K):
[in this window]
[in a new window]
 
Fig. 1.   Effect of 50 mM extracellular K+ concentration ([K+]e) on the expression of heat shock protein (HSP) gene expression in LLC-PK1 cells. LLC-PK1 cells were cultured to subconfluence and placed in media containing 50 mM [K+]e, and total proteins were extracted at the indicated times. Equal aliquots (10 µg) were applied to 7.5% SDS-PAGE, separated by gel electrophoresis, and blotted on nitrocellulose membranes. Blots were hybridized with antibodies specific for HSP-25, HSP-72, HSP-90, or HSC-73 (all obtained from StressGen Biotechnologies), as indicated in A. B: densitometric analyses of HSP-72 induction by 50 mM [K+]e representative of 3 independent experiments with LLC-PK1 cells. Rel units, relative units. *Significance of P < 0.005.

These experiments were performed by adding defined concentrations of KCl to the culture media. As such, the trigger for HSP-72 induction could be 1) higher concentrations of K+, 2) higher concentrations of Cl-, or 3) increased osmolarity of the culture media. To address this issue, we analyzed the effects of elevating osmolarity or Cl- concentrations by adding identical concentrations of NaCl to the culture media. Figure 2A depicts a representative Western Blot demonstrating that the induction of HSP-72 is primarily mediated by the high concentrations of K+. Incubation of cells in elevated [K+]e (50 mM) led to increased expression of HSP-72, whereas identical concentrations of extracellular NaCl concentration had no apparent effects on HSP-72 protein levels in our model (Fig. 2B).


View larger version (41K):
[in this window]
[in a new window]
 
Fig. 2.   High [K+]e, but not high extracellular Na+ concentration ([Na+]e) or osmolarity, is the trigger for increased HSP-72 expression in LLC-PK1 cells. Cells were prepared as described and placed in media containing either 30 or 50 mM [K+]e or [Na+]e, respectively, as indicated for 12 h. Total proteins were extracted, separated by SDS-PAGE, and transferred to nitrocellulose membranes. Membranes were then blotted with an antibody specific for HSP-72. B: densitometric analyses representative of 3 independent experiments with LLC-PK1 cells. *Significance of P < 0.005.

High [K+]e activates the HSP-72 promoter. To investigate the molecular mechanism of K+-induced HSP-72 induction, we tested whether high [K+]e induces HSP-72 protein expression via direct activation of the HSP-72 promoter. A luciferase reporter gene vector containing the proximal 1,440 bp of the human HSP-72 promoter, termed pHSP-72luc, was generated. LLC-PK1 cells were transfected with pHSP-72luc and placed in regular media ([K+]e = 5.5 mM) or media with elevated [K+]e ranging from 10 to 100 mM. Control experiments were performed by addition of NaCl instead of KCl, as described for Western blot analyses. Figure 3 summarizes luciferase activities in cells placed in either high [K+]e or [Na+]e over a time course of 18 h. Consistent with the results of Western Blot analyses, elevated [K+]e from 30 to 100 mM clearly upregulates HSP-72 promoter activity. As shown in Fig. 3, LLC-PK1 cells placed in media containing [K+]e of 10, 30, 50, and 100 mM exhibited increased HSP-72 promoter activity of 136, 266, 477, and 947%, respectively. In contrast, identical concentrations of [Na+]e led to significantly smaller inductions of HSP-72 promoter activity than respective [K+]e. Here, media containing [Na+]e of 30, 50, and 100 mM led to induction of HSP-72 promoter activities to 130, 172, and 305% of control, respectively (Fig. 3). It is of interest to note that luciferase assays, as described above, exhibited a higher sensitivity than the Western blot assays. Addition of 30 mM [K+]e led to a significant induction of pHSP-72luc promoter activity (Fig. 3), whereas the induction of HSP-72 protein by 30 mM [K+]e was not significant (Fig. 2).


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of high [K+]e or [Na+]e on promoter activity of the HSP-72 gene promoter. A reporter construct including 1,440 bp of the HSP-72 gene promoter was fused to the coding sequence of firefly luciferase (pHSP-72luc), as described in MATERIALS AND METHODS. LLC-PK1 cells were transiently transfected with pHSP-72luc by lipofection and were placed in media containing either [K+]e or [Na+]e at the indicated concentrations. After 24 h, cells were lysed, and luciferase activity was measured as described under MATERIALS AND METHODS. A separate set of experiments was performed using the native luciferase vector without the HSP-72 promoter sequence as a control. RLU, relative light units. Values are means of quadruplicates; *significance of P < 0.001. Data are representative of 5 independent sets of experiments.

HSP-72 induction by high [K+]e is controlled by Ca2+ influx. Increasing [K+]e leads to depolarization of the membrane potential. In excitable cell types such as neurons, this induces rapid Ca2+ influx from the extracellular space via voltage-gated Ca2+ channels (23). We investigated whether this mechanism could also account for HSP-72 induction by high [K+]e in LLC-PK1 cells. We therefore analyzed pHSP-72luc expression in response to high [K+]e under cotreatment with several Ca2+-modifying agents. We used 1) diltiazem, an L-type Ca2+ channel blocker, 2) EGTA-AM, a membrane-permeable form of the Ca2+-specific chelator EGTA, 3) TMB-8, an inhibitor of intracellular Ca2+ release, and 4) thapsigargin, an inhibitor of the endoplasmic Ca2+-ATPase. These compounds were used to analyze the specific contributions of Ca2+ fluxes derived from distinct subcellular compartments.

As depicted in Fig. 4, diltiazem and EGTA-AM significantly inhibited high [K+]e-induced HSP-72 promoter activity. Diltiazem led to a significant reduction of HSP-72 promoter activity over a concentration range from 1 nM to 10 µM (inhibition of 62.1, 64.8, and 69.7% at concentrations of 0.01, 0.1, and 1 µM, respectively; Fig. 4A). High [K+]e-induced HSP-72 promoter activity was even more effectively inhibited by EGTA-AM. EGTA-AM (at concentrations of 0.01-1 µM) led to inhibition of HSP-72 promoter activity of 72.0, 68.2, and 75.2% (Fig. 4B). In contrast, no significant inhibition of HSP-72 promoter activity was observed when TMB-8 was used (Fig. 4C). Concentrations of TMB-8 ranging from 0.1 nM to 1 µM did not significantly alter HSP-72 induction by high [K+]e.


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of Ca2+ modification on high [K+]e-induced HSP-72 promoter activity. LLC-PK1 cells were transfected with pHSP-72luc and then incubated in media containing 50 mM [K+]e and diltiazem (A), EGTA-AM (B), 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8; C), or thapsigargin (D) at the indicated concentrations. After 24 h, cells were lysed, and luciferase activity was measured as described under MATERIALS AND METHODS. Values are means of quadruplicates; *significance of P < 0.001. Data are representative of 3 independent sets of experiments.

In general, enhanced Ca2+ influx is rapidly sequestered in intracellular stores. By using thapsigargin, we investigated whether this mechanism also controlled HSP-72 expression. As summarized in Fig. 4D, thapsigargin alone led to significant induction of HSP-72 promoter activity, to 384.6 and 165.9% of control values at concentrations of 0.1 and 0.01 µM, respectively. In addition, HSP-72 promoter activity in response to high [K+]e was amplified in the presence of thapsigargin. Thapsigargin, at 0.1 µM, increased HSP-72 promoter activity to 853.8% (compared with 281.5% by high [K+]e alone). Cotreatment with 0.01 µM thapsigargin still increased activity to 616.7%, whereas 1 nM was not effective any more (Fig. 4D).

High [K+]e induces Ca2+ influx. The results described above strongly suggested that exposing LLC-PK1 cells to high [K+]e results in Ca2+ influx from the extracellular space. To further address this, LLC-PK1 cells were loaded with the Ca2+-sensing dye fluo 3-AM and challenged with 30 mM [K+]e, and Ca2+ levels were monitored by confocal fluorescent microscopy. As shown in Fig. 5, an increase in cytosolic Ca2+ was detected as early as 30 s after exposure to 30 mM [K+]e. Fluorescence peaked at 90 s (to 346% of control levels) and returned thereafter to baseline levels by 240 s (Fig. 5A). A summary is presented in Fig. 5B. Interestingly, increases in intracellular Ca2+ concentration ([Ca2+]i) were specifically seen in the cytosolic compartment (Fig. 5, C and D) but were absent in nuclei. Nucleoli exhibited background staining for Ca2+ before stimulation, but no additional Ca2+ spots were observed in nuclei of LLC-PK1 cells after stimulation with 30 mM [K+]e.


View larger version (62K):
[in this window]
[in a new window]
 
Fig. 5.   Effects of elevated [K+]e on intracellular Ca2+ levels in LLC-PK1 cells. Cells were plated on coverslips and grown to subconfluence. Coverslips were then placed in perfusion chambers, and the cells were loaded with fluo 3-AM and monitored by fluorescence confocal microscopy. Baseline Ca2+ levels were analyzed in regular medium containing 5.5 mM [K+]e. Medium was then changed to 30 mM [K+]e, and changes in free intracellular Ca2+ were monitored using fluo 3-AM at the indicated time points. A: characteristic time course of changes in free Ca2+. T, time. B: summary statistics. C and D: representative images obtained at time 0 and t = 90 s after placing the cells in 30 mM [K+]e, respectively.

Ca2+ influx is required for high [K+]e-induced HSP-72 induction. To test whether this Ca2+ influx was required for high [K+]e-induced HSP-72 induction, we transiently transfected LLC-PK1 cells with pHSP-72luc. Cells were then stimulated with 50 mM [K+]e, either in culture media containing regular Ca2+ concentration of 1.8 mM or in Ca2+-free media. As depicted in Fig. 6, induction of HSP-72 promoter activity by high [K+]e in regular media was 792.8% (48,852 ± 8,690 vs. 5,279 ± 1,495 relative light units). However, HSP-72 induction was almost completely abolished when cells were stimulated with high [K+]e in Ca2+-free media. High [K+]e of 50 mM only slightly increased HSP-72 promoter activity to 115.1% (18,876 ± 1,882 vs. 16,402 ± 772 relative light units) in the absence of extracellular Ca2+ (Fig. 6). This lack of HSP-72 promoter induction did not reflect a general unresponsiveness of the HSP-72 promoter in Ca2+-free culture conditions because promoter activity was still stimulated by the proteasome inhibitors MG-132 and PPI, both known to induce HSP-72 expression (data not shown). With the use of these substances, HSP-72 promoter induction was similar in control (Ca2+ concentration = 1.8 mM) or Ca2+-free culture media. This indicates that, in the setting of our experiments, Ca2+ influx is specifically required for high [K+]e-induced HSP-72 expression.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 6.   Ca2+ influx is required for HSP-72 promoter activation by high [K+]e. Cells were transfected with pHSP-72luc and then incubated in media containing 50 mM [K+]e, either under conditions of regular extracellular Ca2+ (1.8 mM) or under conditions of no extracellular Ca2+, as indicated. Cells were lysed after 24 h, and luciferase activity was measured as described under MATERIALS AND METHODS. Experiments were performed in quadruplicates; *significance of P < 0.001. Data are representative of 3 independent sets of experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our experiments demonstrate that a major cytoprotective protein, the inducible HSP-72, is rapidly and specifically induced by elevations in [K+]e. HSP-72 induction is the result of direct activation of the HSP-72 promoter, which required enhanced Ca2+ influx in response to high [K+]e.

Few studies have addressed the effects of elevated [K+]e on gene expression in nonexcitable cells, such as renal epithelial cells, although they are frequently exposed to high [K+]e, under physiological and pathophysiological conditions. In vivo, the highest K+ concentrations are measured in the renal papilla where [K+]e of 50 mM are frequently encountered (39). Under pathophysiological conditions associated with ischemia, ATP depletion, or cell death, tissue K+ concentration can rapidly rise to [K+]e of 40 mM because of the massive K+ release of necrotic cells (3, 21, 41). In traumatic brain injury, [K+]e increases from 4 mM to 20-50 mM in the cerebral cortex and to 20-30 mM in brain stem cells (33). Corresponding observations have also been made in acute myocardial ischemia (38). It is therefore reasonable to assume that, during acute tubular necrosis or hypoxic kidney injury, interstitial [K+]e may reach levels between 40 and 60 mM. In this respect, upregulation of HSP-72 in renal epithelial cells may reflect an autoprotective process of adjacent cells to preserve their biological and structural functionality. Upregulation of HSP-72 by 50 mM [K+]e in our investigation was rapid and could thus possibly indicate a self-preserving mechanism of cells in response to ionic stress such as high [K+]e resulting from cellular necrosis.

With respect to the effect of increased HSP expression before injury, most investigations have found that increases in HSP protein levels protect most organs from subsequent injuries (1, 4, 20, 31, 36, 40). Representative examples include studies of reperfusion injury in the heart, in which high [K+]e solutions reduced the severity of injury, compared with solutions containing a regular [K+]e of 5.9 mM. Solutions with [K+]e of 20 mM significantly improved regained heart function if used during and after reperfusion injury (15, 42). In organ transplantation, high [K+]e solutions are used to preserve many organ transplants ex vivo during transplantation procedures. Although the role of HSP has yet to be investigated in this context, it is tempting to assume that changes in HSP expression levels by high [K+]e may contribute to preservation of organ functions during transplantation procedures.

A rapid and transient Ca2+ influx was required for HSP-72 induction by [K+]e in LLC-PK1 cells. As shown by use of specific Ca2+ channel blockers, this Ca2+ influx is mediated by activation of voltage-gated Ca2+ channels in the cell membrane. Interestingly, the existence of voltage-gated Ca2+ channels in distal tubules and mesangial cells in humans has recently been demonstrated (11, 45). Analogously, cells that lack voltage-gated Ca2+ channels do not respond with Ca2+ influx during depolarization with high [K+]e, as shown recently in NIE-115 neuroblastoma cells and embryonic kidney cells (HEK) (12). Although both diltiazem and EGTA-AM significantly inhibited high [K+]e-induced HSP-72 promoter activity, the inhibition was not complete. This may indicate that 1) alternate signal transduction mechanisms independent of Ca2+ influx may contribute to the induction, or 2) Ca2+ channels not affected by L-type-specific channel blockers may be present in the proximal tubule. However, because most of the induction is inhibited by EGTA-AM and diltiazem, we propose that Ca2+ influx via L-type Ca2+ channels represents the major signal transduction mechanism in our setting.

Interestingly, baseline HSP-72 promoter activity was significantly higher in Ca2+-free media (Fig. 6). This may be a result of the fact that Ca2+ depletion represents a major stress for LLC-PK1 cells. The activities of many protein kinases and phosphatases necessary for normal cellular functions depend on Ca2+ (13, 26, 37). As such, increases in HSP-72 levels under Ca2+ depletion may be one mechanism for preserving their functions because of its well-documented chaperon activity.

Which molecules could be the immediate targets of this transient Ca2+ influx? Sustained influx of Ca2+ is thought to initiate cell necrosis and apoptosis rather than specific signaling cascades (22). In the present study, stimulation of Ca2+ influx in response to high [K+]e was transient and did not lead to sustained increases in [Ca2+]i. LLC-PK1 cells, left in solutions containing [K+]e of 30-50 mM for 24 h, did not exhibit increased cell death compared with cells left in normal [K+]e (data not shown). In this respect, the transient Ca2+ influx represents a trigger to initiate a specific change in HSP gene expression rather than to instigate the cell death machinery. Furthermore, the transient Ca2+ influx was required for increased HSP-72 expression. It would therefore be intriguing to investigate the immediate upstream effector molecules/kinases that control increased HSP-72 expression in response to elevated [K+]e and [Ca2+]i.

It has been observed before that Ca2+ is necessary for HSP-72 induction. In rat proximal tubule cells, heat shock increases both HSP-72 mRNA and [Ca2+]i (46). Similar to our experiments, HSP-72 induction was abrogated in the absence of Ca2+, lending further support to the notion that a transient increase in [Ca2+]i is a major regulator of increased HSP-72 synthesis. In fact, many stimuli leading to increased HSP-72 expression induce a transient increase in [Ca2+]i. In addition to heat shock, maneuvers that enhance Ca2+ uptake stimulate HSP-72 expression (16, 18, 46). Increased Ca2+ uptake therefore seems to play a major role in increasing HSP-72 expression, although other mechanisms could contribute to HSP-72 induction by high [K+]e.

In the present study, we have demonstrated that high [K+]e was required for HSP-72 induction, as similar increases in HSP-72 were not observed after addition of high [Na+]e or extracellular Cl- concentration. High [K+]e depolarizes the membrane potential and leads to changes in cell volume and pHi. It is possible that either depolarization or an increase in cell volume is the immediate trigger for increased HSP-72 expression in our model. In Madin-Darby canine kidney (MDCK) cells, elevations of K+ only slightly increase HSP-72 levels (32). In these experiments by Neuhofer et al. (32), HSP-72 induction was highest when the cells were subjected to high sodium and acidification. These differences could possibly be the result of different origins of MDCK and LLC-PK1 cells and associated adaptive mechanisms (proximal tubule vs. collecting duct). In contrast, Kiang et al. (18) have described Ca2+-dependent increases in HSP-72 in response to heat shock that were apparently independent of decreases in pHi, as analyzed by acid loading of A 431 cells. Increases in HSP-72 expression therefore seem to be highly cell specific and determined by the actual trigger and/or set of signal-transducing elements. Further studies need to address the immediate upstream mechanism that mediates the observed increase in HSP-72 in LLC-PK1 cells.

In summary, we report that high [K+]e potently induces HSP-72 expression. Upregulation of inducible chaperones such as HSP-72 may contribute importantly to a cell's attempt to preserve essential intracellular structures and thereby facilitate its survival. These mechanisms may be encountered in many pathophysiological conditions coinciding with cell death and subsequent increases in [K+]e, such as ischemic injuries, tissue hypoxia, or tumorigenesis.


    ACKNOWLEDGEMENTS

We are indebted to A. Mann and Dr. M. Stankewich for generous help and advice.


    FOOTNOTES

O. Eickelberg is a Feodor-Lynen Fellow supported by the Alexander von Humboldt-Foundation.

Address for reprint requests and other correspondence: O. Eickelberg, Yale Univ. School of Medicine, Dept. of Pathology, 310 Cedar St., New Haven, CT 06520-8023 (E-mail: oliver.eickelberg{at}yale.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 24 July 2000; accepted in final form 12 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Angelidis, CE, Lazaridis I, and Pagoulatos GN. Constitutive expression of heat-shock protein 70 in mammalian cells confers thermoresistance. Eur J Biochem 199: 35-39, 1991[Abstract].

2.   Beck, FX, Neuhofer W, and Muller E. Molecular chaperones in the kidney: distribution, putative roles, and regulation. Am J Physiol Renal Physiol 279: F203-F215, 2000[Abstract/Free Full Text].

3.   Beck, FX, Ohno A, Dorge A, and Thurau K. Ischemia-induced changes in cell element composition and osmolyte contents of outer medulla. Kidney Int 48: 449-457, 1995[ISI][Medline].

4.   Benjamin, IJ, and McMillan DR. Stress (heat shock) proteins: molecular chaperones in cardiovascular biology and disease. Circ Res 83: 117-132, 1998[Abstract/Free Full Text].

5.   Brezis, M, and Epstein FH. Cellular mechanisms of acute ischemic injury in the kidney. Annu Rev Med 44: 27-37, 1993[ISI][Medline].

6.   Currie, RW, Tanguay RM, and Kingma JG, Jr. Heat-shock response and limitation of tissue necrosis during occlusion/reperfusion in rabbit hearts. Circulation 87: 963-971, 1993[Abstract].

7.   Eickelberg, O, Kohler E, Reichenberger F, Bertschin S, Woodtli T, Erne P, Perruchoud AP, and Roth M. Extracellular matrix deposition by primary human lung fibroblasts in response to TGF-beta1 and TGF-beta3. Am J Physiol Lung Cell Mol Physiol 276: L814-L824, 1999[Abstract/Free Full Text].

8.   Eickelberg, O, Pansky A, Koehler E, Bihl M, Tamm M, Hildebrand P, Perruchoud AP, Kashgarian M, and Roth M. Molecular mechanisms of TGF-beta antagonism by interferon gamma  and cyclosporine A in lung fibroblasts. FASEB J 15: 797-806, 2001[Abstract/Free Full Text].

9.   Feder, ME, and Hofmann GE. Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology. Annu Rev Physiol 61: 243-282, 1999[ISI][Medline].

10.   Feige, U, and Polla BS. Hsp70---a multi-gene, multi-structure, multi-function family with potential clinical applications. Experientia 50: 979-986, 1994[ISI][Medline].

11.   Hall, DA, Carmines PK, and Sansom SC. Dihydropyridine-sensitive Ca2+ channels in human glomerular mesangial cells. Am J Physiol Renal Physiol 278: F97-F103, 2000[Abstract/Free Full Text].

12.   Hargreaves, AC, Lummis SC, and Taylor CW. Ca2+ permeability of cloned and native 5-hydroxytryptamine type 3 receptors. Mol Pharmacol 46: 1120-1128, 1994[Abstract].

13.   Hubbard, SR, and Till JH. Protein tyrosine kinase structure and function. Annu Rev Biochem 69: 373-398, 2000[ISI][Medline].

14.   Jones, DP. Renal metabolism during normoxia, hypoxia, and ischemic injury. Annu Rev Physiol 48: 33-50, 1986[ISI][Medline].

15.  Ju H. Protective effects of high potassium administered after ischemic arrest against reperfusion injury in isolated rat hearts. Chung Hua Hsin Hsueh Kuan Ping Tsa Chih 20: 182-184, 196-197, 1992.

16.   Kantengwa, S, Capponi AM, Bonventre JV, and Polla BS. Calcium and the heat-shock response in the human monocytic line U-937. Am J Physiol Cell Physiol 259: C77-C83, 1990[Abstract/Free Full Text].

17.   Katayama, Y, Becker DP, Tamura T, and Hovda DA. Massive increases in extracellular potassium and the indiscriminate release of glutamate following concussive brain injury. J Neurosurg 73: 889-900, 1990[ISI][Medline].

18.   Kiang, JG, Carr FE, Burns MR, and McClain DE. HSP-72 synthesis is promoted by increase in [Ca2+]i or activation of G proteins but not pHi or cAMP. Am J Physiol Cell Physiol 267: C104-C114, 1994[Abstract/Free Full Text].

19.   Kiang, JG, and Tsokos GC. Heat shock protein 70 kDa: molecular biology, biochemistry, and physiology. Pharmacol Ther 80: 183-201, 1998[ISI][Medline].

20.   Komatsuda, A, Wakui H, Oyama Y, Imai H, Miura AB, Itoh H, and Tashima Y. Overexpression of the human 72 kDa heat shock protein in renal tubular cells confers resistance against oxidative injury and cisplatin toxicity. Nephrol Dial Transplant 14: 1385-1390, 1999[Abstract].

21.   LeFurgey, A, Spencer AJ, Jacobs WR, Ingram P, and Mandel LJ. Elemental microanalysis of organelles in proximal tubules. I. Alterations in transport and metabolism. J Am Soc Nephrol 1: 1305-1320, 1991[Abstract].

22.   Lieberthal, W, and Levine JS. Mechanisms of apoptosis and its potential role in renal tubular epithelial cell injury. Am J Physiol Renal Fluid Electrolyte Physiol 271: F477-F488, 1996[Abstract/Free Full Text].

23.   Macias, W, Carlson R, Rajadhyaksha A, Barczak A, and Konradi C. Potassium chloride depolarization mediates CREB phosphorylation in striatal neurons in an NMDA receptor-dependent manner. Brain Res 890: 222-232, 2001[ISI][Medline].

24.   Mathew, A, and Morimoto RI. Role of the heat-shock response in the life and death of proteins. Ann NY Acad Sci 851: 99-111, 1998[Free Full Text].

25.   Meyrier, A, Hill GS, and Simon P. Ischemic renal diseases: new insights into old entities. Kidney Int 54: 2-13, 1998[ISI][Medline].

26.   Millward, TA, Zolnierowicz S, and Hemmings BA. Regulation of protein kinase cascades by protein phosphatase 2A. Trends Biochem Sci 24: 186-191, 1999[ISI][Medline].

27.   Mohaupt, M, and Kramer HJ. Acute ischemic renal failure: review of experimental studies on pathophysiology and potential protective interventions. Ren Fail 11: 177-185, 1989[ISI][Medline].

28.   Morimoto, RI. Regulation of the heat shock transcriptional response: cross talk between a family of heat shock factors, molecular chaperones, and negative regulators. Genes Dev 12: 3788-3796, 1998[Free Full Text].

29.   Muller, E, Neuhofer W, Burger-Kentischer A, Ohno A, Thurau K, and Beck F. Effects of long-term changes in medullary osmolality on heat shock proteins HSp25, HSP60, HSP72 and HSP73 in the rat kidney. Pflügers Arch 435: 705-712, 1998[ISI][Medline].

30.   Netzer, WJ, and Hartl FU. Protein folding in the cytosol: chaperonin-dependent and -independent mechanisms. Trends Biochem Sci 23: 68-73, 1998[ISI][Medline].

31.   Neuhofer, W, Muller E, Burger-Kentischer A, Fraek ML, Thurau K, and Beck F. Pretreatment with hypertonic NaCl protects MDCK cells against high urea concentrations. Pflügers Arch 435: 407-414, 1998[ISI][Medline].

32.   Neuhofer, W, Muller E, Grunbein R, Thurau K, and Beck FX. Influence of NaCl, urea, potassium and pH on HSP72 expression in MDCK cells. Pflügers Arch 439: 195-200, 1999[ISI][Medline].

33.   Nilsson, P, Hillered L, Olsson Y, Sheardown MJ, and Hansen AJ. Regional changes in interstitial K+ and Ca2+ levels following cortical compression contusion trauma in rats. J Cereb Blood Flow Metab 13: 183-192, 1993[ISI][Medline].

34.   Nover, L, and Scharf KD. Heat stress proteins and transcription factors. Cell Mol Life Sci 53: 80-103, 1997[ISI][Medline].

35.   Perdrizet, GA, Kaneko H, Buckley TM, Fishman MA, and Schweizer RT. Heat shock protects pig kidneys against warm ischemic injury. Transplant Proc 22: 460-461, 1990[ISI][Medline].

36.   Saad, S, Kanai M, Awane M, Yamamoto Y, Morimoto T, Isselhard W, Minor T, Troidl H, Ozawa K, and Yamaoka Y. Protective effect of heat shock pretreatment with heat shock protein induction before hepatic warm ischemic injury caused by Pringle's maneuver. Surgery 118: 510-516, 1995[ISI][Medline].

37.   Sallese, M, Iacovelli L, Cumashi A, Capobianco L, Cuomo L, and De Blasi A. Regulation of G protein-coupled receptor kinase subtypes by calcium sensor proteins. Biochim Biophys Acta 1498: 112-121, 2000[ISI][Medline].

38.   Sato, R, Yamazaki J, and Nagao T. Temporal differences in actions of calcium channel blockers on K+ accumulation, cardiac function, and high-energy phosphate levels in ischemic guinea pig hearts. J Pharmacol Exp Ther 289: 831-839, 1999[Abstract/Free Full Text].

39.   Seldin, DW, and Giebisch GH. The Kidney: Physiology and Pathophysiology. New York: Raven, 1992.

40.   Smith, DF, Whitesell L, and Katsanis E. Molecular chaperones: biology and prospects for pharmacological intervention. Pharmacol Rev 50: 493-514, 1998[Abstract/Free Full Text].

41.   Spencer, AJ, LeFurgey A, Ingram P, and Mandel LJ. Elemental microanalysis of organelles in proximal tubules. II. Effects of oxygen deprivation. J Am Soc Nephrol 1: 1321-1333, 1991[Abstract].

42.   Tani, M, and Neely JR. Mechanisms of reduced reperfusion injury by low Ca2+ and/or high K+. Am J Physiol Heart Circ Physiol 258: H1025-H1031, 1990[Abstract/Free Full Text].

43.   Van Why, SK, Kim S, Geibel J, Seebach FA, Kashgarian M, and Siegel NJ. Thresholds for cellular disruption and activation of the stress response in renal epithelia. Am J Physiol Renal Physiol 277: F227-F234, 1999[Abstract/Free Full Text].

44.   Van Why, SK, and Siegel NJ. Heat shock proteins in renal injury and recovery. Curr Opin Nephrol Hypertens 7: 407-412, 1998[ISI][Medline].

45.   Weiergraber, M, Pereverzev A, Vajna R, Henry M, Schramm M, Nastainczyk W, Grabsch H, and Schneider T. Immunodetection of alpha1E voltage-gated Ca(2+) channel in chromogranin-positive muscle cells of rat heart, and in distal tubules of human kidney. J Histochem Cytochem 48: 807-819, 2000[Abstract/Free Full Text].

46.   Yamamoto, N, Smith MW, Maki A, Berezesky IK, and Trump BF. Role of cytosolic Ca2+ and protein kinases in the induction of the hsp70 gene. Kidney Int 45: 1093-1104, 1994[ISI][Medline].


Am J Physiol Renal Fluid Electrolyte Physiol 281(2):F280-F287
0363-6127/01 $5.00 Copyright © 2001 the American Physiological Society