Effect of Taurine-Conjugated Ursodeoxycholic Acid on Endoplasmic Reticulum Stress and Apoptosis Induced by Advanced Glycation End Products in Cultured Mouse Podocytes

Diabetic nephropathy is the leading cause of end-stage renal disease and is clinically characterized by proteinuria and progressive renal insufficiency. None of the currently available pharmacologic interventions for diabetic nephropathy can completely forestall or reverse the progression of the disease.The podocyte is a terminally differentiated cell with a limited capacity to proliferate and regenerate after injury, so this loss cannot be compensated. Any disease process that causes podocyte loss, either from detachment or apoptosis, will lead to a decrease in the density of podocytes. Reduction in podocyte density is an important determinant of progressive diabetic nephropathy [1] and precedes the development of renal dysfunction and albuminuria in diabetic patients and animal models of diabetes mellitus [1,2,3]. Recently, Chuang et al.[ 4] demonstrated that advanced glycation end products (AGEs) induced apoptosis in podocytes during the early stage of diabetic nephropathy. However, the mechanism for AGEs to trigger apoptosis of podocytes remains to be elucidated.Apoptotic pathways are activated by a diverse array of extrinsic and intrinsic signals. Early studies suggested that mitochondria played a central role in apoptosis [5]. Recent studies challenge the central role of mitochondria in apoptosis and suggest that some apoptotic signals may bypass mitochondria to directly activate caspases [6]. A recent study showed that endoplasmic reticulum (ER) stress triggered apoptosis without involvement of mitochondria [7]. The ER is a cellular organelle where secretory and membrane proteins are synthesized and modified and is also a major intracellular Ca2+ storage compartment inside the cell. The ER plays a critical role in protein biosynthesis, folding and trafficking, and maintenance of intracellular calcium homeostasis. A threat to equilibrium between the load of nascent client proteins synthesized and the capacity of ER is referred to as ER stress [8]. A variety of agents, including calcium ionophores, toxins, and oxidative stress can induce ER stress. Both Ca2+ overload and depletion of the ER Ca2+ storage can result in changes in protein folding and in ER stress. The mammalian ER stress response can be separated into two phases, adaptation and apoptosis. Cells initially adapt to the accumulation of unfolded proteins by inducing ER-resident chaperones such as glucose-regulated protein 78 (GRP78) and glucose-regulated protein 94 (GRP94). However, if this adaptation does not prove sufficient, the apoptotic response is initiated. The induction of GRP78, a calcium-dependent chaperon of ER lumen, has been used widely as an indicator of ER stress [9]. Previous studies have shown that ER stress conditions have been observed in numerous diseases including Alzheimer disease [10], Parkinson disease [11], Huntington disease [12] as well as cardiovascular diseases [13], indicating that ER stress-induced apoptosis is an important factor in pathophysiological conditions. Furthermore, ER stress induces apoptosis of pancreatic cells and the subsequent development of diabetes mellitus [14,15]. However, a role of ER stress in AGEs-induced podocytes apoptosis remains to be elucidated. The present study was designed to delineate how ER stress induces apoptosis in podocytes exposed to AGEs and the role of the Ca2+ signals in this event.Tauroursodeoxycholic acid (TUDCA) is a hydrophilic bile acid that is normally produced endogenously in humans at very low levels. TUDCA is formed in the conjugation pathway of ursodeoxycholic acid, which is commonly used as a bile acid replacement therapy for the treatment of certain cholestatic syndromes [16]. Recent reports have shown that TUDCA can modulate ER functionin vitro and in vivo [17, 18]. Özcan et al.[ 18] demonstrated that TUDCA enhanced the adaptive capacity of the ER and acted as potent antidiabetic modalities with potential application in the treatment of type 2 diabetes. In this study, we investigated whether TUDCA could alleviate the increased ER stress seen in AGEs-induced apoptosis of murine podocytes.Endotoxin-free AGE-bovine serum albumin (BSA) was prepared from endotoxin-free, lyophilized BSA (fraction IV; Sigma-Aldrich, Inc., USA), as described previously [19]. BSA was incubated under sterile conditions with D-glucose at 37°C for 8 weeks. Unincorporated sugars were then removed by dialysis against phosphate-buffered saline. Control nonglycated BSA was incubated under the same conditions with the exception of the absence of reducing sugars. Before the in vitro study, reagents were tested for endotoxin levels by the Limulus amebocyte assay and were found to contain less than 0.2 ng/ml of endotoxin.A thermosensitive immortalized mouse podocyte cell line, kindly provided by Professor Peter Mundel (Albert Einstein College of Medicine, New York, N.Y., USA), was used for this study. This cell line has been conditionally immortalized from normal mouse podocytes with a temperature-sensitive mutant of immortalized SV40 T antigen. These cells have been previously characterized in detail elsewhere [20]. All cells were grown on dishes coated with type I collagen (Sigma-Aldrich, Inc., USA) and in RPMI media supplemented with heat-inactivated 10% fetal calf serum (HyClone, USA), 100 U/ml penicillin, and 100 U/ml streptomycin. At the ‘permissive’ temperature of 33°C, the cells were treated with 10–15 U/ml of mouse recombinant γ-interferon (R&D, USA) and the SV40 T antigen is active and allows the cells to proliferate rapidly. To silence the transgene and induce the differentiation of podocytes, the cells were maintained at 37°C in the same medium without γ-interferon under nonpermissive conditions for 14 days before the experiments were conducted.Protein levels of GRP78 in podocytes were examined by Western blotting. Podocytes following different treatments were lysed with sodium dodecyl sulfate (SDS) sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM dithiothreitol, and 0.1% bromphenol blue). Samples were heated at 100°C for 5–10 min before loading and separated by 12% SDS polyacrylamide gel electrophoresis. Protein was transferred electrophoretically to polyvinylidene fluoride membranes (Immun-Blot PVDF membrane, 0.2 µm; Bio-Rad, USA) in transfer buffer containing 48 mmol/l Tris-HCl, 39 mmol/l glycine, 0.037% SDS, and 20% methanol, and blocked with 5% BSA for 4 h at room temperature. The immunoblots were incubated with mouse anti-human GRP78 polyclonal antibody (BD Biosciences, San Jose, Calif., USA) overnight at 4°C. The membrane was incubated with a horseradish peroxidase-conjugated secondary antibody (Sigma-Aldrich, Inc., USA) for 2 h at room temperature followed by three 15-min washings. After washing again, the immunoblots were detected by enhanced chemiluminescence reagent according to the manufacturer’s instructions. Equivalent protein loading and transfer efficiency were verified by staining for β-actin (Sigma-Aldrich, Inc., USA). The bands were analyzed with a Bio-Rad Gel Imaging System (Bio-Rad ChemiDoc XRS, USA) and Quantity One 1-D Analysis Software (Bio-Rad, USA).Apoptotic cells were quantified by measuring externalized phosphatidylserine assessed by annexin V-FITC and by propidium iodide (PI) uptake. Annexin V is a Ca2+-dependent phospholipid-binding protein that has a high affinity for phosphatidylserine. Because externalization of phosphatidylserine occurs in the early stages of apoptosis, annexin V staining can identify apoptotic cells early. Therefore, staining with annexin V in conjunction with vital dye such as PI allows to identify early apoptotic cells (annexin positive, PI negative) and nonapoptotic non-necrotic cells (annexin negative, PI negative).Detection of early apoptosis in podocytes was performed according to the manufacturer’s instructions (Nanjing Keygen Biotech. Co., Ltd., China). In brief, fresh podocytes (105 cells/ml) were harvested and centrifuged at 450 g for 5 min at 4°C. After washing twice with PBS, the cell pellet was resuspended in 100 µl of ice-cold diluted binding buffer and incubated with 5 µl annexin V and 5 µl PI for 10 min at room temperature in the dark. Next, the cell sample was diluted with 400 µl of binding buffer. Finally, the cell pellet was analyzed with FACScan flow cytometer (BD Biosciences, San Jose, Calif., USA). Negative control was carried out for each sample (incubation with binding buffer alone).Intracellular free Ca2+ was determined with the Ca2+-sensitive fluorescent dye Fluo-3/AM (Molecular Probes, Eugene, Oreg., USA). Podocytes grown on coverslips were rinsed with a phosphate-buffered Ringer-like solution (145 mM NaCl, 1.6 mM K2HPO4, 0.4 mM KH2PO4, 1.3 mM CaCl2, 1.03 mM MgCl2, and 5 mM D-glucose, pH 7.4) and were incubated with 5 µM Fluo-3/AM in a phosphate-buffered Ringer-like solution at 37°C. After loaded with Fluo-3/AM for 30 min, cells were washed three times with phosphate-buffered Ringer-like solution. Fluo-3-loaded cells were transferred to the recording chamber (approximately 0.5 ml in volume) mounted on the stage of a laser confocal microscope (Zeiss Axiovert LSM510) linked to a CCD camera (Orca-ER; Hamamatsu, Hamamatsu City, Japan). Fluo-3 fluorescence images were acquired continuously at rates of 0.33 Hz. Each experiment monitored 15–20 cells. The fluorescent images of cells were collected every 0.5–20 s for fast signals and analyzed frame by frame with a confocal microscope. The Ca2+ level was expressed as a pseudo-ratio value (F/F₀) of the actual fluorescence intensity (F) divided by the basal intensity of the [Ca2+]i at rest (F₀).The data are presented as mean ± SE for the number of experiments indicated. One-way analysis of variance was used to determine significant differences among groups. All statistics were generated using SPSS 11.0 software, and all figures were drawn using Origin 75 software.p value of <0.05 was considered to indicate statistical significance.To study the effect of AGEs on podocytes apoptosis in vitro, AGEs or control BSA were incubated with podocytes. Increase of the AGEs concentration from 10 to 40 µM enhanced apoptosis of podocytes cultured for 24 h in a concentration-dependent manner (fig. 1). Figure 1a shows that only 4.56% of podocytes cultured in medium supplemented without AGEs (normal control) for 24 h were annexin V-positive/PI-negative podocytes representing apoptotic cells (lower right quadrangle). However, when podocytes were cultured in medium with 40 µM AGEs, 23.01% podocytes underwent apoptosis, as compared with normal control (p < 0.05; fig. 1b). The time dependency of apoptosis by 40 µM AGEs was analyzed for incubation times of 6, 12, 24 and 48 h (fig. 2). AGEs-induced apoptosis increased in a time-dependent manner, which was maximal after 48 h, but not BSA. Quantitative analysis indicated that AGEs cultured for 24 h stimulated a 4.4-fold increase in apoptotic cells compared with normal control (time 0) or BSA control (time 0) (p < 0.05, fig. 2b).We investigated whether the ER stress was involved in AGEs-induced apoptosis of podocytes. Western blot analysis followed by densitometry (fig. 3a) revealed that AGEs in the concentration range between 10 and 40 µM dose-dependently enhanced GRP78 expression when applied for 24 h. As shown in figure 3, AGEs (40 µM), but not BSA (40 µM), significantly upregulated the expression of GRP78 at the protein level in a time-dependent manner (fig. 3b), which could be detected as early as 6 h after AGEs treatment and became more evident after 24 and 48 h. These results together indicated that AGEs caused ER stress in mouse podocytes.Since disturbance of the [Ca2+]i homeostasis has been reported to cause ER stress, we speculated that AGEs might have some impact on the [Ca2+]i. To examine this possibility, podocytes were loaded with the Ca2+ indicator dye Fluo-3 and the [Ca2+]i was monitored upon exposure to 40 µM AGEs (fig. 4a). In the presence of extracellular Ca2+, AGEs rapidly (within 20 s) increased the [Ca2+]i, which reached the maximum within 40 s and lasted for about 3 min and declined to the baseline by 10 min (fig. 4a, b). When extracellular Ca2+ was chelated with 5 mM EGTA, AGEs could still evoke an increase in the [Ca2+]i and last for a shorter time, although to a lesser extent (fig. 4c). These results suggest that AGEs may cause an increase in the [Ca2+]i through influx of extracellular Ca2+. Further, podocytes were pretreated with 2 µM thapsigargin before exposure to AGEs. Thapsigargin is a sesquiterpene lactone of guaianolide type isolated from the Mediterranean plant Thapsia garganica L. It is widely used experimentally as a potent and selective inhibitor of ER Ca2+-ATPase and commonly employed to deplete ER Ca2+ content [21]. In the absence of ER Ca2+, AGEs increased the [Ca2+]i of the thapsigargin-treated cells, but reached the maximum to a lesser extent (fig. 4c). These results together support our notion that the AGEs-induced increase in the [Ca2+]i is through both release of ER stores and influx of extracellular Ca2+.TUDCA appeared remarkably effective in suppressing AGEs-induced apoptosis. Incubation of cells with TUDCA alone did not induce apoptosis. Annexin V assay revealed that apoptosis of podocytes induced by AGEs was suppressed by TUDCA (10, 25, 50 and 100 µM) in a dose-dependent manner. However, coincubation with TUDCA (100 µM) significantly reduced AGE-induced cell apoptosis 24 h after preparation, compared with AGEs (40 µM)-treated podocytes (fig. 5).To investigate a role of ER stress in cell apoptosis induced by AGEs, we evaluated whether TUDCA could alleviate the increased ER stress seen in AGEs-treated mouse podocytes. Administration of TUDCA (10, 25, 50 and 100 µM) on podocytes treated with 40 µM AGEs abrogated GRP78 expression in a concentration- dependent manner (fig. 6). TUDCA (50 and 100 µM) pretreatment significantly rescued podocytes from AGEs-induced ER stress (fig. 6).Podocyte depletion has been proposed as a hallmark of both proteinuria and glomerulosclerosis for many years and is now considered a central problem in progression of renal diseases [22]. Here we show that AGEs induced apoptosis in podocytes through the ER stress pathway, providing a novel molecular mechanism for podocyte depletion in diabetic nephropathy. Our data demonstrated for the first time that the role of ER stress in apoptosis of murine podocytes when exposed to AGEs and the ability of TUDCA to prevent ER stress-induced apoptosis.Recent studies suggested that apoptosis could be induced by an ER stress pathway independent from mitochondria. ER stress has been studied for a long time, but mainly in neuropathology, such as Parkinson disease and Alzheimer disease [10, 11]. Recently, it was reported that ER stress may play an important role in several renal diseases, including the renal tubular injury caused by the analgesic and antipyretic drug paracetamol (acetaminophen) [23], glomerular epithelial cell injury induced by complement attack [24], podocyte injury induced by excessive accumulation of secretory proteins [25], and renal ischemia reperfusion injury [24]. However, scanty information is available about ER stress and diabetic nephropathy. Our studies demonstrated that GRP78 expression, an indicator of ER stress, induced by AGEs was associated with apoptosis of podocytes.The glucose-regulated proteins (GRPs), a family of molecular chaperones and Ca2+-binding stress proteins located in the ER, are also induced by stress [26]. Induction of GRPs by ER stress protects cells against a variety of toxic insults including Ca2+ ionophores, oxidative stress, topoisomerase inhibitors, and cytotoxic T cells. Thus, multiple stress proteins may be important in the cellular tolerance response. In addition to being a sensor of ER stress, GRP78 also serves as a gatekeeper to the activation of the stress transducers. Under normal conditions, most of the GRP78 is bound to three ER transmembrane receptors: pancreatic ER kinase-like ER kinase, activating transcription factor 6 and inositol-requiring enzyme to keep them inactive, whereas free GRP78 is present at a low level. When ER stress happens, the misfolded/unfolded proteins complex with free GRP78. The depletion of free GRP78 is a cue for the dissociation of GRP78 from its transducer clients, thus leading to their activation. A top priority is to contain the damage by decreasing general protein synthesis and increasing the production of GRP78. If the rescue effort is insufficient to mend the injury, the decision to commit to apoptosis may be triggered, although the mechanism behind this is not yet fully understood [27]. Our experiments found that AGEs induced GRP78 expression to rescue cells in a dose- and time-dependent manner. But the effort was insufficient to mend the injury and apoptosis was triggered.Storage, release, and uptake of calcium are regulated by proteins in the ER. Alterations in Ca2+ homeostasis cause ER stress. Recent studies suggested that cellular Ca2+ overload or ER Ca2+ load, or perturbation of intracellular Ca2+ compartmentalization, can cause cytotoxicity and trigger apoptosis [28]. Therefore, the high sensitivity of the podocytes to AGEs-induced apoptosis may be because of their high ER Ca2+ load. In fact, we showed in this study that AGEs had an impact on the Ca2+ homeostasis of podocytes. It evoked a rapid rise in the [Ca2+]i through both the release of ER Ca2+ stores and the influx of extracellular Ca2+, suggesting a role for AGEs in activating Ca2+-release channels and/or inhibiting Ca2+-ATPases. More importantly, we demonstrated that the AGEs-induced increase in [Ca2+]i might subsequently cause ER stress, resulting in apoptosis.Chemical enhancement of ER function to cope with cell apoptosis provides a unique approach to managing diabetes nephropathy. Several lines of evidence suggest that chemical chaperones enhance ER functional capacity [29]. Chemical or pharmaceutical chaperones, such as 4-phenyl butyric acid, trimethylamine N-oxide dihydrate, are a group of low-molecular-weight compounds known to stabilize protein conformation, improve ER folding capacity, and facilitate the trafficking of mutant proteins. TUDCA, a hydrophilic bile acid derivative, diminishes ER stress responses in cultured liver cells [17, 18]. Özcan et al.[ 18] demonstrated that TUDCA could alleviate ER stress, established normoglycemia, and rescued insulin action in mice with type 2 diabetes. In this study, we found that TUDCA could alleviate the increased ER stress and apoptosis exposed to AGEs in murine podocytes in a dose-dependent manner. The ability of TUDCA to alleviate AGEs-induced ER stress and apoptosis provides strong support to the ER stress-based mechanism in diabetic nephropathy and demonstrates the feasibility of targeting ER function for therapeutic gain. At this moment, the protective mechanisms of the TUDCA against AGEs-induced apoptosis in podocytes deserve further investigation.In summary, AGEs was shown to trigger apoptosis in mouse podocytes in vitro. We found that the ability of AGEs to upregulate ER stress and apoptosis was significantly alleviated in TUDCA-treated cells. Our experiments with TUDCA reinforce the idea that AGEs acts through ER stress, at least in part, to induce apoptosis. This study suggested that chemical chaperones in general, and TUDCA in particular, might warrant clinical investigation as treatments for diabetic nephropathy.