Albumin induces endoplasmic reticulum stress and apoptosis in renal proximal tubular cells
2006; Elsevier BV; Volume: 70; Issue: 8 Linguagem: Inglês
10.1038/sj.ki.5001704
ISSN1523-1755
AutoresTakamoto Ohse, Reiko Inagi, Tetsuhiro Tanaka, Takashi Ota, Toshio Miyata, Ichiro Kojima, Julie R. Ingelfinger, Satoshi Ogawa, Toshiro Fujita, Masaomi Nangaku,
Tópico(s)Cannabis and Cannabinoid Research
ResumoChronic proteinuria appears to be a key factor in tubulointerstitial damage. Recent studies have emphasized a pathogenic role of endoplasmic reticulum (ER) stress which is induced by the accumulation of misfolded proteins in ER, extracellular stress, etc. In the present study, we investigated ER stress and ER stress-induced apoptosis in proximal tubular cells (PTCs). Immortalized rat PTCs (IRPTCs) were cultured with bovine serum albumin (BSA). The viability of IRPTCs decreased proportionately with BSA overload in a time-dependent manner. Quantitative real-time polymerase chain reaction analysis revealed that 40 mg/ml BSA increases mRNA of ER stress markers by 7.7- and 4.6-fold (glucose-regulated protein 78 (GRP78) and oxygen-regulated protein 150 (ORP150), respectively) as compared to control. The increased expression of ORP150 and GRP78 in IRPTCs with albumin overload was detected by Western blot and immunofluorescence study. These in vitro observations were supported by in vivo studies, which demonstrated that ER stress proteins were upregulated at PTCs in experimental proteinuric rats. Furthermore, increased ER stress-induced apoptosis and activation of caspase-12 were observed in IRPTCs with albumin overload and kidneys of experimental proteinuric rats. We confirmed that apoptotic cell death was attenuated by co-incubation with caspase-3 inhibitor or calpain inhibitors. These results indicate that the ER stress-induced apoptosis pathway contributed to the insult of tubular cells by proteinuria. In conclusion, renal tubular cells exposed to high protein load suffer from ER stress. ER stress may subsequently lead to tubular damage by activation of caspase-12. Chronic proteinuria appears to be a key factor in tubulointerstitial damage. Recent studies have emphasized a pathogenic role of endoplasmic reticulum (ER) stress which is induced by the accumulation of misfolded proteins in ER, extracellular stress, etc. In the present study, we investigated ER stress and ER stress-induced apoptosis in proximal tubular cells (PTCs). Immortalized rat PTCs (IRPTCs) were cultured with bovine serum albumin (BSA). The viability of IRPTCs decreased proportionately with BSA overload in a time-dependent manner. Quantitative real-time polymerase chain reaction analysis revealed that 40 mg/ml BSA increases mRNA of ER stress markers by 7.7- and 4.6-fold (glucose-regulated protein 78 (GRP78) and oxygen-regulated protein 150 (ORP150), respectively) as compared to control. The increased expression of ORP150 and GRP78 in IRPTCs with albumin overload was detected by Western blot and immunofluorescence study. These in vitro observations were supported by in vivo studies, which demonstrated that ER stress proteins were upregulated at PTCs in experimental proteinuric rats. Furthermore, increased ER stress-induced apoptosis and activation of caspase-12 were observed in IRPTCs with albumin overload and kidneys of experimental proteinuric rats. We confirmed that apoptotic cell death was attenuated by co-incubation with caspase-3 inhibitor or calpain inhibitors. These results indicate that the ER stress-induced apoptosis pathway contributed to the insult of tubular cells by proteinuria. In conclusion, renal tubular cells exposed to high protein load suffer from ER stress. ER stress may subsequently lead to tubular damage by activation of caspase-12. Chronic proteinuria is now considered to play an essential role in progression of tubulointerstitial damage which is considered as a final common pathway to end-stage renal disease.1.Remuzzi G. Bertani T. 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The present study was designed to delineate how ER stress induces apoptosis in PTCs exposed to protein overload. In order to mimic the in vivo conditions of PTCs exposed to proteinuria, we employed albumin overload. To evaluate that albumin overload damages immortalized rat PTCs (IRPTCs), we performed lactate dehydrogenase (LDH) release assays to evaluate cell viability after exposure to high albumin concentrations. Indeed, cell viability was decreased in a dose-dependent manner when cells were incubated with bovine serum albumin (BSA) (Figure 1a). Subsequently, IRPTCs were incubated with 40 mg/ml BSA for various incubation times. The ratio of LDH release increased in a time-dependent manner (Figure 1b). Similar results were observed by utilizing albumin derived from human serum instead of BSA (data not shown). To determine whether albumin overload induces ER stress in IRPTCs, we evaluated changes in mRNA levels of representative ER stress proteins using quantitative real-time polymerase chain reaction (PCR). Upregulation of mRNAs of two of these proteins, oxygen-regulated protein (ORP150) and glucose-regulated protein (GRP78), occurred in a time-dependent manner (Figure 2). IRPTCs were also incubated with various concentrations of albumin (0, 10, 20, 40 mg/ml) for 24 h, and a significant increase in GRP78 and ORP150 mRNA was observed in cells exposed to 40 mg/ml BSA for 24 h (7.7±0.92-fold (P<0.005) and 4.6±1.2-fold increase (P<0.05), respectively) as compared to cells cultured in serum-free media at the same time point (data of other doses are not shown). We evaluated the expression of the representative ER stress proteins at the protein level (Figure 3a–d). Immunofluorescence studies revealed constitutive GRP78 expression in control cells (Figure 3a), which was increased by albumin exposure (Figure 3b). Baseline expression of ORP150 (Figure 3c) was also increased by albumin overload (Figure 3d). Furthermore, the subcellular distribution of GRP78 or ORP150 was expanded by BSA exposure (Figure 3b and d). The expression of GRP78 was colocalized with that of ER resident protein, calnexin (Figure 3e). Western blot analysis followed by densitometry revealed that albumin overload significantly upregulated expression levels of GRP78 and ORP150 (Figure 3f and g). Next, we examined ER stress in an animal model of massive proteinuria, puromycin aminonucleoside (PAN) nephropathy,28.Tanaka T. Miyata T. Inagi R. et al.Hypoxia in renal disease with proteinuria and/or glomerular hypertension.Am J Pathol. 2004; 165: 1979-1992Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar and compared expression of various ER stress proteins in the kidneys of these animals to those of normal control rats. As shown in Figure 4a, GRP78 was expressed in normal kidneys; immunohistochemical analysis using tubule-specific markers identified that expression of GRP78 was limited in distal tubules under baseline conditions. On the other hand, ORP150 was not detected in normal kidneys (Figure 4d). In PAN nephropathy, expression of GRP78 was not only observed in distal tubules but in proximal tubules, and the expression of ORP150 was now present in distal and proximal tubules (Figure 4g–l). These results suggest that ER stress was induced in proximal tubules in association with the massive proteinuria induced in this model. To explore the pathophysiology of cell death, we examined whether apoptosis occurred in IRPTCs incubated with BSA. Staining with Hoechst33342 revealed nuclear shrinkage and condensation, which suggest that albumin overload induced apoptosis in IRPTCs (Figure 5a). These condensed nuclei were also stained with propidium iodide (PI). Apoptosis was confirmed by Annexin V assay as described below, and the percentage of apoptotic cell death in IRPTCs treated with BSA was higher than that in untreated-IRPTCs (33.9±3.0 vs 20.5±2.7%, P<0.001). To clarify the association of caspase-3, we utilized a cell-permeable and irreversible caspase-3 inhibitor (z-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-CH2F (z-DEVD-fmk)) and examined LDH release. The result suggested that caspase-3 is involved in apoptosis as induced by albumin overload in IRPTC. Thus, the caspase-3 inhibitor suppressed cell death, but not entirely to baseline levels (Figure 5b). To investigate a role of calpain in cell death associated with ER stress induced by albumin overload, we employed calpain inhibitors, such as N-acetyl-Leu-Leu-Nle-CHO (ALLN) (calpain inhibitor I), PD150606, and calpeptin. ALLN appeared remarkably effective in suppressing cell death in a dose-dependent manner (Figure 6). Annexin V assay revealed that apoptosis of IRPTCs induced by albumin overload in IRPTCs was significantly suppressed by ALLN (Figure 7).Figure 7The ratio of apoptosis was examined by flow cytometric analysis using Annexin V assay. IRPTCs, which were preincubated with 60 μM ALLN, significantly improved their viability (*P<0.01).View Large Image Figure ViewerDownload (PPT) Caspase-12 has been reported to play an important role as an essential mediator in apoptosis induced by ER stress. Therefore, we examined whether caspase-12 was upregulated or activated by BSA overload. In IRPTCs with BSA overload, immunofluorescence studies revealed that the total protein levels of caspase-12 were upregulated (Figure 8a and b). In addition, Western blot analysis followed by densitometry demonstrated that the active form of caspase-12 was increased by BSA overload, while it was attenuated by ALLN (Figure 8c and d). Activation of caspase-12 was also observed in in vivo studies utilizing PAN nephropathy rats: intensity of the band of active caspase-12 increased in kidneys of PAN nephropathy rats as compared to normal rats (827.7±72.5 vs 407.5±167.6 pixel2, P<0.05) (Figure 8e). Furthermore, to confirm contribution of calpain, other calpain inhibitors, such as PD150606, which has been considered a cell-permeable, selective non-peptide calpain inhibitor, and calpeptin, which is considered an inhibitor for calpain and papain, were employed. Both of these compounds also suppressed cell death (Figure 9). These results indicated that calpain activation was associated with ER stress induced by albumin overload, contributing to subsequent apoptosis. In the present study, we demonstrated that PTCs exposed to albumin underwent the state of ER stress. Why does albumin overload induce ER stress in PTCs? PTCs internalize proteins by a process of endolysosomal phagocytosis.29.Park C.H. Maack T. Albumin absorption and catabolism by isolated perfused proximal convoluted tubules of the rabbit.J Clin Invest. 1984; 73: 767-777Crossref PubMed Scopus (174) Google Scholar Endocytic vesicles of the lysosomal-processing pathway normally carry the reabsorbed protein toward the degenerative lysosomal compartment. A high-capacity reabsorption system would deal with filtered proteins that largely exceed the normal concentration at the apical surface of PTCs. Filtered albumin and other proteins that accumulate within intracellular compartments of PTCs may perturb cell functions by several mechanisms. 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Previous papers reported various protein concentrations in proximal tubules in normal animals by means of micropuncture techniques.36.Oken D.E. Cotes S.C. Mende C.W. Micropuncture study of tubular transport of albumin in rats with aminonucleoside nephrosis.Kidney Int. 1972; 1: 3-11Abstract Full Text PDF PubMed Scopus (61) Google Scholar, 37.Lewy J.E. Pesce A. Micropuncture study of albumin transfer in aminonucleoside nephrosis in the rat.Pediatr Res. 1973; 7: 553-559Crossref PubMed Scopus (47) Google Scholar, 38.Brunskill N. Mechanisms of albumin uptake by proximal tubular cells.Am J Kidney Dis. 2001; 37: S17-S20Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar In addition, the concentration of albuminuria reaching proximal tubular lumen right after filtration from glomeruli in nephrotic syndrome is not yet determined. In the present studies, we employed the albumin concentrations based on previous studies of the model of albumin overload.5.Zoja C. Donadelli R. 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Zoja C. et al.Protein overload-induced NF-kappaB activation in proximal tubular cells requires H(2)O(2) through a PKC-dependent pathway.J Am Soc Nephrol. 2002; 13: 1179-1189PubMed Google Scholar We observed that protein overload induced ORP150 and GRP78 in this study. In addition, cell death was detected after 12 h exposure to protein, and it seems to precede the increase of ORP150 and GRP78 expression. To date, several pathways of ER stress were reported,42.Kaufman R.J. Orchestrating the unfolded protein response in health and disease.J Clin Invest. 2002; 110 (Review): 1389-1398Crossref PubMed Scopus (1032) Google Scholar and ORP150 and GRP78 are considered as protective agents. Our findings suggest that death signal pathways overwhelmed activation of protective pathways, or that activation of protective pathways may be activated following death signal pathways. Although apoptosis in PTCs exposed to high concentration of albumin in vitro43.Amore P. Cirina A. Iavarone G.C. et al.Proteinuria induces apoptosis in cultured tubular cells.J Am Soc Nephrol. 1996; 7: 1691Google Scholar and in vivo44.Fernandez L. Romero M. Soto H. et al.Increased apoptosis in acute puromycin aminonucleoside nephrosis.Exp Nephrol. 2001; 9: 99-108Crossref PubMed Scopus (17) Google Scholar was reported previously, a precise mechanism of this apoptosis remains unknown. Our studies demonstrated that ER stress induced by albumin overload was associated with apoptosis of PTCs. The mechanism by which ER stress is coupled to an apoptotic response had been a mystery until caspase-12 was characterized by Nakagawa and Yuan.24.Nakagawa T. Yuan J. Cross-talk between two cysteine protease families. Activation of caspase-12 by calpain in apoptosis.J Cell Biol. 2000; 150: 887-894Crossref PubMed Scopus (1006) Google Scholar Caspase-12 is ubiquitously and constitutively expressed, but unlike other caspases, caspase-12 is specifically activated by the insults that elicit ER stress and is not activated by other death stimuli. Although previous reports showed dose- and duration-dependent upregulation of the Fas-FADD-caspase 8 pathway in PTCs exposed to albumin,40.Erkan E. De Leon M. Devarajan P. Albumin overload induces apoptosis in LLC-PK(1) cells.Am J Physiol Renal Physiol. 2001; 280: F1107-F1114PubMed Google Scholar we demonstrated proteolytic activation of caspase-12, indicating association of caspase-12 in ER stress-induced apoptosis of PTCs with albumin overload. The activated form of caspase-12 was detected to some extent even in normal samples in our Western blot and it may reflect activation of this enzyme during sample preparation of cell extract as pointed out by Nakagawa et al.21.Nakagawa T. Zhu H. Morishima N. et al.Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta.Nature. 2000; 403: 98-103Crossref PubMed Scopus (2832) Google Scholar and Van de Craen M et al.45.Van de Craen M. Vandenabeele P. Declercq W. et al.Characterization of seven murine caspase family members.FEBS Lett. 1997; 403: 61-69Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar To support this notion, we could see the same phenomenon in the datasheet provided by the manufacturer (Calbiochem). Following its activation at the ER, caspase-12 can directly process downstream caspases in the cytosol. Caspase-12 directly cleaves procaspase-9, leading to caspase-9-dependent activation of caspase-3.46.Morishima N. 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Couser W.G. et al.Mechanisms and consequences of proteinuria.Lab Invest. 1986; 54: 479-498PubMed Google Scholar Other researchers investigating the impact of albumin on PTCs have employed a similar range of protein concentration.5.Zoja C. Donadelli R. Colleoni S. et al.Protein overload stimulates RANTES production by proximal tubular cells depending on NF-kappa B activation.Kidney Int. 1998; 53: 1608-1615Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar, 6.Wang Y. Rangan G.K. Tay Y.C. et al.Induction of monocyte chemoattractant protein-1 by albumin is mediated by nuclear factor kappaB in proximal tubule cells.J Am Soc Nephrol. 1999; 10: 1204-1213PubMed Google Scholar, 30.Tang S. Leung J.C. Abe K. et al.Albumin stimulates interleukin-8 expression in proximal tubular epithelial cells in vitro and in vivo.J Clin Invest. 2003; 111: 515-527Crossref PubMed Scopus (217) Google Scholar To investigate association of protein load and ER stress-induced cell death in vivo, we employed PAN nephropathy, which causes with massive proteinuria. Fernandez et al.44.Fernandez L. Romero M. Soto H. et al.Increased apoptosis in acute puromycin aminonucleoside nephrosis.Exp Nephrol. 2001; 9: 99-108Crossref PubMed Scopus (17) Google Scholar previously reported apoptosis of PTCs in kidneys of PAN nephropathy, but a precise mechanism of the apoptosis remained to be determined. The present study demonstrates that GRP78 is constitutively expressed mainly in distal tubules of the normal kidney, new information. Furthermore, GRP78 was upregulated in the PTCs of the nephrotic kidney and caspase-12 was activated in these kidneys. These results suggested association of protein load-induced ER stress and apoptosis of PTCs in PAN nephropathy kidneys, supporting the observations of our in vitro experiments. Our experiments also revealed an essential role of calpain in ER stress-induced apoptosis in PTCs exposed to albumin. Nakagawa et al.24.Nakagawa T. Yuan J. Cross-talk between two cysteine protease families. Activation of caspase-12 by calpain in apoptosis.J Cell Biol. 2000; 150: 887-894Crossref PubMed Scopus (1006) Google Scholar reported that caspase-12 is cleaved by calpain and activated when exposed to the insult of ER stress. In our experiments, we explored a role of calpain in PTCs exposed to a high concentration of albumin utilizing three kinds of calpain inhibitors, all of which showed remarkable effects on amelioration of tubular injury. In addition, ALLN decreased ER stress-induced apoptosis of the cells with albumin overload. Our results clarified a role of calpain in the pathway of apoptosis induced by a high concentration of albumin, and raised a therapeutic possibility of calpain inhibitors against tubulointerstitial injury by chronic massive proteinuria. In conclusion, we demonstrated that albumin overload leads to ER stress-induced apoptosis in PTCs. Calpain and caspase-12 might be involved in this apoptosis pathway, and may serve as promising therapeutic targets in the future under proteinuric conditions. We employed lipid- and endotoxin-free BSA (Sigma, St Louis, MO, USA) for avoiding the infl
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