Heat shock protein 27 expression in human proximal tubule cells exposed to lethal and sublethal concentrations of CdCl2.

The expression of hsp 27 mRNA and protein was determined in cultured human proximal tubule (HPT) cells exposed to lethal and sublethal concentrations of Cd2+ under both acute and extended conditions. Initial procedures demonstrated that HPT cells display the classic stress response following physical and chemical stress. Heat stress (42.5 degrees C for 1 hr) caused an increase in both hsp 27 mRNA and protein as well as a shift in the protein to a more phosphorylated state. Results were similar when the cells were subjected to chemical stress (exposure to 100 microM sodium arsenite for 4 hr). Acute exposure to 53 microM CdCl2 for 4 hr also resulted in an increase in hsp 27 mRNA and protein and a shift to the more phosphorylated protein isoform. Extended Cd2+ exposure involved continuous treatment with Cd2+ at both lethal and sublethal levels over a 16-day time course. The results of this treatment showed that chronic exposure to Cd2+ failed to increase either hsp 27 mRNA or protein expression in HPT cells, even at lethal Cd2+ concentrations. In fact, hsp 27 protein levels decreased as compared to controls at both lethal and sub-lethal exposure to Cd2+. These findings imply that hsp 27 expression in human proximal tubule cells may have two distinct modes depending on the nature (acute vs. chronic) of the stress. ImagesFigure 1Figure 2Figure 3Figure 4Figure 5Figure 6Figure 7

The expression of hsp 27 mRNA and protein was determined in cultured human proximal tubule (HPT) cells exposed to lethal and sublethal concentrations of Cd2+ under both acute and extended conditions. Initial procedures demonstrated that HPT cells display the classic stress response following physical and chemical stess. Heat stres (42.5`C for 1 hr) caused an increase in both hsp 27 mRNA and protein as well as a shf in the protein to a more phosphorylated state. Results were similar when the edls were subjected to chemical s (exposure to 100 pM sodium arsenite for 4 hr). Acute exposure to 53 pM CdCI2 for 4 hr also resulted in an increase in hsp 27 mRNA and protein and a shift to the more phosphorylated protein isoform. Extnded Cd2+ exposure involved continuous treatment with +C.2 at both lethal and sublethal level over a 16-day ime coure. The results of this treatment showed that chronic exposure to Cd2* failed to increase either hsp 27 mRNA or protein expression in HPT cell, even at lethal C2 concentrations. In fict, hisp 27 protein levels decreased as compared to contros at both lethal and sublethal eposure to C&. These findings imply that hp 27 expression in hum an proximal tubule cells may have two distinct modes depending on the natur (acute vs. chronic) of the stress. Key uorda cadmium, gene expression, heat shock, heavy metals, hp 27, proximal tubule, sodium arsenite. Envin Heaut Perpe 107:545-552 (1999). [Online 2 June 1999] http://ehpnetl.niehs.nih.gov/docs/I999/107p545-552somji/absraatMmnl The kidney, and in particular the proximal tubule, is critically affected by chronic exposure to the environmental pollutant Cd2+ in both animals and humans (1,2). Nephrotoxicity results from a slow accumulation of CdP in the proximal tubules of the kidney.
The earliest markers of chronic Cd2+ nephrotoxicity are disorders of proximal tubule transport characterized by low-molecularweight proteinuria, increased high-molecularweight protein excretion, and a variety of other proximal tubule ion transport disorders (1,(3)(4)(5)(6). Selective and direct absorption of Cd2+ by proximal tubules has been demonstrated using microinjection techniques (7). In an effort to define the fundamental mechanistic processes underlying human Cd2+_ induced nephrotoxicity, we used a cell culture model of the human proximal tubule (HPT). The HPT cell culture model retains important features of proximal tubule cell differentiation (8)(9)(10). These retained features are stable with cell passage and include a consistent enzyme histochemical profile, sodiumdependent glucose transport, parathyroid hormone stimulation of cAMP, generation of an apical-negative potential difference, and the presence of gap junctions. When these cells are exposed to sublethal concentrations of Cd2+, they exhibit the transport and ultrastructural alterations expected from in vivo knowledge of Cd-induced nephrotoxicity (11)(12)(13). Also in agreement with in vivo findings is the fact that at lethal CdP concentrations, the cells undergo necrotic cell death (11). This model system is currently being used to define the roles and interactions of the stress response superfamily of proteins in protection and recovery from Cd2+ exposure. Initial examinations centered on the metallothionein gene family because these proteins are known for their ability to bind and sequester heavy metals (14)(15)(16). The current study focuses on the role of the stress response protein, hsp 27, as a possible mediator of Cd2+-induced nephrotoxicity. This examination was motivated by recent studies demonstrating that enhanced hsp 27 expression has a role in the protection and recovery of the proximal tubule cell during and after brief periods of renal ischemia (17)(18)(19). This finding suggested that hsp 27 expression may also have a role in the protection of the renal proximal tubule from the cytotoxic effects of the environmental pollutant, cadmium.
Hsp 27 is a member of a large superfamily of proteins with molecular weights ranging from 8 to 170 kD and collectively referred to as the heat shock (hsp) or stress response proteins (20,21). In humans, hsp 27 is encoded by a single active gene located on chromosome 9 (22). Cell lines that overexpress hsp 27 protein exhibit an enhanced ability to survive and recover from heat stress (23)(24)(25)(26)(27)(28)(29). Increasing hsp 27 expression by transiently or stably transfecting cell lines confers increased cellular resistance to a variety of toxicants including doxorubicin, daunorubicin, actinomycin D, vincristine, colchicine, arsenite, hydrogen peroxide, and tumor necrosis factor (23)(24)(25)27,30). Hsp 27 appears to exert its effects on cell survival, at least in part, through a chaperone action that stabilizes microfilament dynamics. Hsp 27 regulates actin dynamics, and hsp 27 overexpression prevents microfilament disruption and enhances mitogen-stimulated actin polymerization (23)(24)(25)31,32). Hsp 27 also demonstrates actin capping activity (25,31). Hsp 27 is phosphorylated at serine residues in response to heat shock or mitotic stimuli, suggesting a role in the regulation of signal transduction pathways. Recent studies also indicate that hsp 27 is involved in the regulation of programmed cell death in several cell lines (33)(34)(35).

Materials and Methods
Cell culture. Stock cultures of HPT cells were grown in 75-cm2 T-flasks using procedures previously described by this laboratory (8,X2. The growth medium was a serum-free formulation consisting of a 1:1 mixture of Dulbecco's modified Eagles' medium and Ham's F-12 growth medium supplemented with selenium (5 ng/mL), insulin (5 pg/mL), transferrin (5 pg/mL), hydrocortisone (36 ng/mL), triiodothyronine (4 pg/mL), and epidermal growth factor (10 ng/mL). The growth surface was treated with a collagen matrix to promote cell attachment and subculture. The cells were fed fresh growth medium every 3 days, and at confluence (normally 3-6 days post subculture) were subcultured using trypsin-EDTA (0.05%, 0.02%). For use in experimental protocols, the cells were subcultured in six-well plates at a 1:2 ratio and allowed to reach confluence (6 days after subculture) before initiation of experimental protocols. The cells were fed every 3 days. HPT cells between passages 5 and 7 were used in the present study. The three isolates of HPT cells were derived from normal cortical tissue obtained from kidneys removed for renal cell carcinoma. The kidneys were from a 72-year-old female, a 63year-old male, and a 58-year-old female.
Cell viability. The effect of the various treatments on the viability of confluent cell monolayers was determined by counting cell nuclei of viable cells remaining attached to the culture surface using the nuclear stain DAPI (4',6-diamidino-2-phenylindole) and Kontron KS400 image analysis software (Zeiss, Thornwood, NY), as described previously (15). At the indicated time points, wells containing the cell monolayers were fixed for 15 min in 70% ethanol, rehydrated with phosphate-buffered saline (PBS), and stained with 10 pL DAPI (10 pg/mL in distilled water). Each well was examined under epifluorescent illumination at 40 x magnification on a Zeiss Axiovert 35 (Zeiss) linked to the computer with an Optronics DEI 470 CCD camera (Optronics, Goleta, CA). For each time point, a minimum of 20 fields per well and three wells per data point were determined. Both nuclear counts and total nuclear area were obtained from the program and yielded equivalent results.
Isolation of total RNA, RT-PCR, and Northern analysis. Total RNA was isolated according to the protocol supplied with TRI REAGENT (Molecular Research Center, Inc., Cincinnati, OH) as described previously by this laboratory (15). The concentration and purity of the RNA samples were determined using spectrophotometer scan in the ultraviolet (UV) region and ethidium bromide (EtBr) visualization of 12   7.2) 1 mM EDTA 1.0% SDS for 15 min at 50°C. Blots were wrapped in plastic and exposed overnight at -76°C for autoradiography. Radioactive probes were removed from membranes by immersion in boiling 0.1% SDS. Stripped blots were reprobed with radiolabeled cDNA complementary to human glyceraldehyde 3-phosphate dehydrogenase mRNA (Clontech, Palo Alto, CA) for loading correction. Western analysis. Cells were washed twice with PBS and lysed directly in the flask by addition of 400 pL (85°C) 1 x SDS buffer (2% SDS, 100 mM dithiothreitol, and 50 mM Tris-HCI, pH 6.8). The cell lysate was heated in a boiling water bath for 10 min. DNA was sheared by repeated passage through a 23-gauge needle. The samples were centrifuged at 10,000g for 10 min at room temperature and the supernatant transferred to a new tube. The concentration of protein in the samples was determined by the bicinchoninic acid protein assay (Pierce Chemical Co., Rockford, IL). Equal amounts of total cellular protein were separated on 12% SDS-containing polyacrylamide gels and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad). Membranes were blocked with 10% (w/v) nonfat milk in PBS, incubated with a mouse monoclonal antibody specific for human hsp 27 10.0 mM NaF, 2% ampholines, 5% f-mercaptoethanol, and 2% Triton X-100. The proteins were focused on 4% polyacrylamide capillary tube gels containing 9 M urea, 1.5% 5/7 Biolyte, and 0.5% 3/10 Biolyte ampholines (Bio-Rad). Capillary tube gels containing focused proteins were placed at the top of 12% polyacrylamide slab minigels, followed by separation of proteins in the second dimension. Resolved proteins were electrotransferred onto PVDF membranes (Bio-Rad). Hsp 27 phosphoisoforms were detected using procedures identical to those described for Western analysis.
Integrated optical density (IOD). IOD values were determined using an image analysis work station configured with Kontron KS 400 software. For IOD evaluations of EtBr-stained gels, inverted images were used. In the heat-shock, acute CdCI2, and sodium arsenite protocols, IOD

Results
Hsp 27 expression in HPT cells exposed to heat shock. The classic method used to examine the response of cultured mammalian cells to physical stress is exposure to elevated temperature, usually 42-440C, followed by a recovery period at normal temperature. To determine the effect of heat shock on hsp 27 expression in HPT cells, confluent cells from three independent cell isolates were exposed to an elevated temperature of 42.5°C for 1 hr followed by a recovery period of 48 hr at 370C. Exposure to heat shock clearly resulted in an increase in the amount of hsp 27 mRNA for all three HPT cell isolates as determined by Northern analysis (Figure IA, B). Hsp 27 mRNA was increased at the end of the 1-hr heat shock period, continued to increase during the initial hour of the recovery period, and remained elevated for the next 12-16 hr before returning to control values 24 hr postheat shock. Hsp 27 mRNA levels were also examined on the same total RNA samples using an RT-PCR assay to determine if results would be equivalent to those found with Northern analysis ( Figure IC, D). This is important because RT-PCR analysis consumes a much smaller amount of total RNA than Northern analysis. The relative amounts of hsp 27 mRNA were similar for both Northern and RT-PCR assay methods. The expression of hsp 27 protein was also determined at selected time points of the heat shock protocol by Western analysis (Figure 2A, B). Hsp 27 protein was expressed under control conditions and did not increase during the initial 1-hr period at 42.5°C. Hsp 27 protein levels increased in the recovery period, reaching peak values by [8][9][10][11][12] hr. The phosphorylation state of hsp 27 protein was also determined as a function of heat shock and recovery ( Figure 2C, D). In the control condition, hsp 27 protein was present in the unphosphorylated and mono-phosphorylated state. There was an increase in phosphorylation during the 1-hr heat shock, as noted by a faint additional immunoreactive spot representing the di-phosphorylated state. There was no loss of cell viability during the treatment and recovery periods (data not shown).
Hsp 27 expression in HPTcellsfollowing acute exposure to arsenite and CdClI The classic method to evaluate the response of cultured cells to chemical stress is exposure to sodium arsenite followed by a recovery period that involves the removal of the chemical stress through a change of the culture medium and renewed incubation at 37°C. This method was used to determine if acute exposure to Cd2+ induces hsp 27 expression in HPT cells. Confluent cells were exposed to 53 pM Cd2+ for 4 hr, followed by a 48-hr recovery period in Cd2+-free growth media. The Cd2+ concentration used was established in preliminary experiments to be an exposure level resulting in the death of 15-30% of the cells by the end of the recovery period. The effect of this level of exposure was confirmed by monitoring HPT cell viability over the total time course of exposure and recovery ( Figure 3A). Exposure to 53 iM Cd2+ clearly resulted in an increase in the amount of hsp 27 mRNA, as determined by RT-PCR analysis ( Figure 3B, C). The increase in hsp 27 mRNA was rapid, 5-10-fold over control, occurred largely within the first 4 hr of Cd> exposure, and was not dependent on a Cd2P-free recovery period. This elevated level of hsp 27 mRNA was maintained 4-8 hr into the recovery period and returned to control values by 48 hr of recovery in Cd_ free growth medium.
The level of hsp 27 protein was also increased by acute Cd2+ exposure ( Figure   3D, E). Hsp 27 protein was maximally elevated following 1 hr of Cd2+ exposure, remained elevated 8-12 hr into the recovery period, and returned to control values following 24 hr of recovery in Cd2+-free growth medium. Exposure to 53 j1M Cd2+ resulted in an immediate and prolonged shift of the hsp 27 isoform pattern to an enhanced phosphorylation state ( Figure 3F). The shift in the phosphoisoform pattern occurred within the first hr of Cd2+ exposure and was retained in the recovery period.
For comparison, confluent HPT cells were also exposed to 100 pM sodium arsenite for 4 hr, followed by a 48-hr recovery period ( Figure 4). Exposure to 100 pM sodium arsenite resulted in an increase in the amount of hsp 27 mRNA ( Figure 4B,  C). The level of hsp 27 mRNA was relatively constant during the initial hours of sodium arsenite exposure and began to increase after 4 hr. Four hours into the recovery period, there was a large increase in the level of hsp 27 mRNA. The increased level of hsp 27 mRNA was sustained for 12 hr of recovery and thereafter rapidly returned to control values. Hsp 27 protein also increased as a consequence of sodium arsenite treatment ( Figure 4D, E). Hsp 27 protein levels increased following 1 hr of sodium arsenite treatment and remained elevated 24 hr into the recovery period. The phosphorylation state of hsp 27 protein was also evaluated for HPT cells exposed to sodium arsenite. The unphosphorylated and mono-phosphorylated forms of hsp 27 were evident in the control condition. Sodium arsenite exposure resulted in a shift in the isoform pattern of hsp 27 to a more phosphorylated state following 4 hr of exposure ( Figure 4F). Hsp Figure 6B). The level of hsp 27 mRNA expression was also not altered as compared to control for any of the Cd2+ treatment groups regardless of Cd2P dose or Cd2+-induced cell lethality ( Figure 6). The level of hsp 27 protein expression was also relatively constant over the 16-day time course for control cells ( Figure 7B). In contrast, the level of hsp 27 protein was reduced as compared to control at each concentration of Cd2+ (Figure 7). The pattern of hsp 27 phosphoisoforms was evaluated after 24 hr of exposure to each Cd2+ concentration and was identical to control cells (data not shown).

Discussion
The first goal of the present study was to determine if acute exposure to Cd2+ evokes the hsp 27  Although studies in the renal system are limited, a role for the hsp 27 response in protection of the proximal tubule cell from acute Cd2+ exposure can be inferred from recent studies on renal ischemia. In studies using the rodent model, evidence shows that induction of the hsp 27 stress response can attenuate the effects of acute renal ischemia (17)(18)(19). The expression and intracellular distribution of hsp 25 (the rodent homologue to human hsp 27) was evaluated in rat renal cortex following 45 min of renal ischemia with subsequent reflow (17). Cortical hsp 25 was induced within 2 hr of reflow, peak values were reached by 6 hr, and elevated levels were maintained after 24 hr of reflow. The shift in hsp 25 between the detergent soluble and insoluble cytoskeletal fractions and the localization of hsp 25 within the proximal tubule cell as a function of ischemia and recovery both suggested specific interactions between hsp 25 and actin during the early postischemic reorganization of the cytoskeleton. The suggestion that hsp 25 provides assistance in the reorganization of the actin cytoskeleton following renal ischemia is consistent with one of the known functions of hsp 27 Figure 5. HPT cell continuous exposure to CdCI2. Abbreviations: DAPI, 4',6-diamidino-2-phenylindole; HPT, human proximal tubule; IOD, integrated optical density.Three HPT cell isolates were exposed to 9, 27, and 45 pM CdCI2 for a period of 16 days. Computer-assisted cell counts are shown for one HPT cell isolate. DAPI-stained nuclei in 20 fields for each triplicate well were counted and results are expressed as percentage of control.
known to occur in ischemia. A series of studies demonstrated that the loss of proximal tubule cell structure and transport function associated with renal ischemia involves alterations in the actin cytoskeleton [reviewed by Molitoris (36)]. The induction of hsp 27 in HPT cells by acute Cd2+ exposure would likewise be expected to stabilize the actin filament network and help preserve proximal tubule transport function. Recent studies in other systems have demonstrated that expression ofhsp 27 regulates apoptosis (33)(34)(35). In studies using L929 cells that constitutively express the Fas receptor, expression of human hsp 27 inhibits apoptosis mediated by stimulation of the Fas receptor (34). In addition, hsp 27 expression interfered with apoptotic cell death mediated by staurosporine (33,34). A similar finding for a protective effect of the small hsp against apoptosis was also observed in U937 and Wehi-s cells exposed to actinomycin D, camptothecin, and etoposide (35). HPT cells express Fas under normal growth conditions and increased Fas expression occurs after treatment with interferon-y (37). These observations suggest that the rapid induction of hsp 27 protein and phosphorylation early in the time course of acute exposure to Cd2+ may protect the proximal tubule cell against programmed cell death.
The dassic methods used to examine the stress response in cultured cells evaluate the presence or absence of the response after a short duration of agent exposure. The results discussed for hsp 27 expression in Cd2+-exposed HPT cells can then be categorized as early events within the initial 4 hr of Cd2+ exposure. Although this illustrates the hsp 27 stress response following acute exposure to Cd2 , it is likely that actual exposure in many instances is more prolonged and involves lower concentrations. An additional   (15). Within the first 24 hr of exposure to 9 pM Cd2+, HPT cells showed a 10to 20-fold increase in MT protein levels and this increase continued to the end of the time course where MT represented 7 to 10% of total cell protein.
Because of the high binding affinity of MT for Cd2+, it would be expected that Cd2+ is in the unsequestered state for only a brief period following the initial exposure of the cells before complexing with MT. Assuming that hsp 27 induction occurs in response to Cd2+ in the unsequestered state, then hsp 27 induction would only be expected to occur immediately after Cd2+ exposure-the brief interval before MT protein induction and complexation with MT. This would provide a mechanism to explain the current finding that hsp 27 expression is limited to an early transient induction in Cd2+-exposed HPT cells.
Because the Cdsequestering MT protein level continues to increase with time, this explanation is also consistent with the finding that hsp 27 expression is not increased later in the time course. Although the induction of MT may explain the lack of a sustained or long-term induction of hsp 27 expression by Cd2+, it does not explain why the levels of hsp 27 protein are decreased by prolonged exposure to lethal and sublethal concentrations of Cd2+. Although there is no explanation for this finding at present, it does not appear to be due to a nonspecific overall decrease in protein synthesis because MT protein is increased at the same time hsp 27 protein is decreased.
Two distinct roles can be proposed for hsp 27 expression when the HPT cell is exposed to Cd2+. Induction early in the time course of Cd21 exposure may protect the cell while the Cd2+-binding protein MT is being synthesized. This would serve to stabilize the actin filament network of the cell in a fashion similar to that proposed to occur during renal ischemia and subsequent reflow (17)(18)(19). Direct evidence that a transient elevation of hsp 27 expression can provide cellular resistance to Cd2+ toxicity comes from recent studies with mouse embryonic stem cells transfected with sense or antisense hsp 27 cDNA (39). In these studies, the level of hsp 27 expression was directly correlated with cellular resistance to the toxicity of CdCl2, HgCl2, cis-platinum (11)-diamine dichloride, or sodium arsenite within a 12-hr exposure period. Protection against programmed cell death provided by the early induction of hsp 27 is speculative based solely on hsp 27 involvement in programmed cell death decision in other cell systems (33)(34)(35). In contrast, the inability to sustain constitutive levels of hsp 27 protein may potentially have deleterious effects, as was demonstrated when HPT cells were subjected to a longer period of sustained Cd2+ exposure. Whereas the induction of hsp 27 protein has been proposed to stabilize actin filament dynamics, the loss of hsp 27 protein would be expected to render the actin filaments susceptible to damage. The demonstration that constitutive levels of hsp 27 protein are not maintained in HPT cells during a chronic course of Cd2+ exposure suggests that the cytoskeleton might be a site particularly susceptible to damage in cadmium-induced nephropathy.