1 Conjoint Renal Laboratory, Queensland Health Pathology Service and 2 Department of Renal Medicine, Royal Brisbane and Women's Hospital, Herston 4029, Brisbane, Australia
Correspondence and offprint requests to: Dr Helen Healy, Department of Renal Medicine, Royal Brisbane Hospital, Herston 4029, Brisbane, Australia. E-mail: Helen_Healy{at}health.qld.gov.au
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Abstract |
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Methods. Plasma was collected from healthy volunteers and fractionated into albumin-rich (30100 kDa), high molecular weight (100440 kDa) and combined (30440 kDa) fractions. PTECs were exposed to 10 mg/ml of the protein fractions for 24, 48 and 72 h. Apoptosis was measured using fluorescein isothiocyanate (FITC)annexinV and TUNEL. Necrosis was measured using propidium iodide, metabolic activity by MTT and cell proliferation by bromodeoxyuridine incorporation. Fas and FasL expression was analysed by western blotting.
Results. Exposure to the 100440 and 30440 kDa fractions produced significant increases in apoptosis at all time points, whereas PTECs exposed to the 30100 kDa fraction were not significantly different from control cells. There were no changes in the rates of necrosis as a result of protein loading. A significant reduction in metabolic activity was observed in PTECs exposed to the 100440 and 30440 kDa fractions, but not to the 30100 kDa fraction. Cell proliferation was significantly reduced by 24 h in cells exposed to the 100440 and 30440 kDa fractions. By 48 and 72 h, all the three fractions had inhibited cell proliferation. PTECs exposed to the 100440 and the 30440 kDa fractions showed a significant upregulation in the expression of Fas and FasL. Overall, the high molecular weight fraction was more toxic than the albumin-rich or combined fraction.
Conclusion. Increased apoptosis and decreased cell proliferation are the major mechanisms of cell death in human PTECs in response to protein overload. These effects may be mediated at least in part by overexpression of the Fas/FasL system. The severity of such changes is largely determined by the high molecular weight fraction (100440 kDa) rather than the albumin-rich fraction.
Keywords: apoptosis; cell proliferation; Fas; necrosis; protein overload; proximal tubular epithelial cells
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Introduction |
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Emerging evidence suggests that, irrespective of the initial insult, apoptosis may be the final mechanism of cell death in many renal diseases [4]. Although several reports have documented apoptosis in various renal diseases [4], the role of apoptosis in proteinuric conditions has not been well established. Some studies have suggested that high concentrations of albumin are pro-apoptotic for proximal tubular epithelial cells (PTECs) [57], while others have reported that albumin is not pro-apoptotic but a survival factor for PTECs [8,9].
Discrepancies over the role of apoptosis in response to protein overload can, at least in part, be attributed to differences in experimental design. Previous studies have generally used a single component (albumin) in very high concentrations to study the effect of protein loading on PTECs [57]. We reasoned that as the glomerular ultrafiltrate contains a mixture of proteins (either in normal or disease conditions), the use of a wide range of proteins rather than any single component might resemble more closely the in vivo situation and serve as a model to study the pathological changes in both selective and non-selective proteinuric renal diseases.
The mechanism(s) linking cellular insults such as protein loading to cell death are poorly understood. Recently, the Fas/Fas ligand (FasL) pathway has attracted interest as a mediator of kidney damage [10,11]. Fas (Apo-1, CD95) is a type I transmembrane protein belonging to the tumour necrosis factor receptor superfamily [10,11]. FasL, a type II transmembrane protein, belongs to the same family of death factors [10,11]. By binding to its receptor (Fas), FasL initiates a cascade of events leading to cell death [10,11]. Although the Fas/FasL system has been suggested to play a crucial role in mediating apoptosis in renal disease [10,11], their role in mediating cellular injury in response to protein overload in primary cultures of human PTECs has not been reported hitherto.
This study was undertaken to assess the effect of plasma-derived proteins on primary cultures of human PTECs, and the possible involvement of the Fas/FasL system in protein overload-induced apoptosis. Plasma proteins were isolated from healthy volunteers, and various fractions prepared. The effects of a 30100 kDa fraction (representative of selective proteinuria), 100440 kDa fraction (high molecular weight fraction) and 30440 kDa fraction (representative of non-selective proteinuria) on apoptosis, necrosis and proliferation of PTECs were examined.
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Subjects and methods |
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Chemicals
The chemicals were obtained either from Sigma (St Louis, MO), Gibco (Invitrogen Corporation, CA) or Roche (Roche Diagnostics, IN) unless otherwise stated.
Cell culture
PTECs were isolated and cultured as previously described [12]. In brief, renal cortical tissues from the normal pole of nephrectomy samples were collected in pre-cooled Hanks balanced salt solution (HBSS) containing penicillin (50 U/ml), streptomycin (50 µg/ml) and amphoterecin B (0.125 µg/ml). After removing the capsule, the extreme cortex was cut into small pieces and centrifuged at 200 g for 5 min at 4°C. After discarding the supernatant, the tissue fragments were suspended in type II collagenase (1 mg/ml in HBSS) and incubated for 1 h at 37°C. The digest was passed through a 100 µm sieve followed by a 40 µm sieve (Beckton Dickinson, NJ). The sieved cells were centrifuged (200 g, 5 min, 4°C) and seeded into 75 cm2 tissue culture flasks that had been coated with bovine collagen S and fetal calf serum. The cells were grown in defined medium, a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 containing 15 mM HEPES buffer, -glutamine and pyridoxine hydrochloride. The medium was supplemented with epidermal growth factor (10 ng/ml), insulin (10 µg/ml), transferrin (5 µg/ml), selenium (5 ng/ml), hydrocortisone (36 ng/ml), triiodothyronine (4 pg/ml), penicillin (50 U/ml), streptomycin (50 µg/ml) and amphoterecin B (0.125 µg/ml). Morphological, biochemical and transport characteristics [12] confirmed the cells to be of proximal tubular origin.
Plasma fractionation
Blood was collected in heparinized tubes from healthy volunteers and the plasma was separated (3000 r.p.m., 12 min, 4°C). Plasma (69 ml) was diluted with an equal volume of 0.02 M phosphate-buffered saline (PBS) and the proteins fractionated by gel filtration using a K26/100 column (26 mm i.d., bed height 94 cm) packed with Sephacryl S-300 superfine gel (Pharmacia Biotech, Uppsala, Sweden). A flow rate of 2.25 ml/cm2/h at 6°C was used. Eluant was collected in 5 ml vials using an FC 204 fraction collector (Gilson Medical Electronics, Middleton, WI). Plasma proteins were separated into three broad peaks comprising the lipoproteins, high molecular weight proteins and an albumin-rich peak (Figure 1a). Molecular weights were estimated by native polyacrylamide gradient gel electrophoresis (PAGGE, 160 min at 150 V, 4°C; Figure 1b) using 216% TBE gels (Alamo Gels, Alamo, TX). SDS gels are not suitable for this procedure, because of the loss of quaternary protein structure and conversion of oligomeric proteins into their constituent polypeptides. Proteins were visualized by staining with Coomassie blue (Gradipure, Gradipore Ltd, Sydney, Australia) and destained in 6% acetic acid. Based on the column elution profile (Figure 1a) and the electrophoresis gel (Figure 1b), individual fractions were identified and pooled for the preparation of protein fractions with particular molecular weight ranges: 30100 kDa (albumin-rich fraction), 100440 kDa (high molecular weight proteins) and 30440 kDa (combined fraction which includes albumin and high molecular weight proteins). The 30100 kDa fraction was prepared by pooling proteins in the albumin peak range, followed by ultrafiltration (2630 g, 23 min, 20°C) using an Amicon Ultra-15 filter device with a 100 kDa nominal molecular weight cut-off (Millipore Corporation, Bedford, MA) to remove contaminating high molecular weight proteins. The filtrate was then concentrated by ultrafiltration (2630 g, 23 min, 20°C) with a 30 kDa filter (Millipore Corporation). The 100440 kDa fraction was prepared by ultrafiltering pooled proteins in the high molecular weight range (Figure 1a) using a 100 kDa filter. The retentate was used in cell experiments. The 30440 kDa fraction was prepared by pooling proteins with molecular weights 440 kDa and concentrating the mixture by ultrafiltration using a 30 kDa filter. Molecular weight ranges were confirmed by native PAGE (180 V, 60 min) using 12% Tris-glycine gels (Bio-Rad Laboratories, Hercules, CA). Protein concentrations were determined using the BCA assay (Pierce, Rockford, IL). The concentrated fractions were frozen in aliquots at 20°C until further use.
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Experimental protocol
PTECs (5 x 104 cells) were cultured in non-collagen-coated 24-well culture plates in defined medium for 5 days to 9095% confluence. For other culture plates, the cell numbers were adjusted accordingly. Cells were then washed and incubated in basic medium for 24 h. Basic medium, which was devoid of growth factors except for apo-transferrin (5 µ/ml) and antibiotics, was used for all experiments. After 24 h, the cells were incubated with 10 mg/ml of the 30100, 100440 and 30440 kDa fractions for 24, 48 and 72 h. The proteins were dissolved in basic medium to a final concentration of 10 mg/ml. The volume of culture medium in each well was 1 ml. PBS (0.02 M) collected in heparinized tubes and subjected to appropriate dilutions was used as vehicle treatment (PBS group). The experiments were conducted using passage 2 and 3 cells.
Apoptosis and necrosis determination by flow cytometry
Apoptosis and necrosis were determined by flow cytometry using a commercially available apoptosis detection kit (Annexin-V-FLUOS Staining Kit, Roche). Briefly, after the appropriate treatment periods, non-adherent cells were pelleted and added to trypsinized and pelleted adherent cells. The cells were resuspended in 100 µl of binding buffer containing fluorescein isothiocyanate (FITC)annexinV and propidium iodide and incubated at room temperature for 15 min. After the incubation period, 300 µl of binding buffer was added and the cells were analysed in a FACS Calibur (Beckton Dickinson). Ten thousand events were recorded from each treatment group.
Apoptosis assay using in situ end labelling (TUNEL)
We used a commercially available ApopTag Plus Peroxidase in situ Apoptosis Detection Kit (Intergen Company, Purchase, NY), which is based on the TUNEL method (TdT-mediated dUTP nick end labelling). Briefly, PTECs were grown on Thermanox coverslips (Nalge Nunc International, NY) in 24-well plates. After the appropriate treatment periods, the cells were processed following the manufacturer's instructions. The colour was developed with 3',3'-diaminobenzidine and counterstained with haematoxylin, washed in ethanol, cleared in xylene and mounted in DePex. Five different areas were centralized under a 40x objective and the cells that fell within the 100 squares of the eyepiece graticule were counted. The number of apoptotic cells was expressed as the percentage of the total cells counted.
Metabolic activity assay using MTT
Metabolic activity was measured using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). In brief, cells were grown in 96-well culture plates. The culture volume was 100 µl. Ninety minutes before the end of the appropriate treatment periods, 10 µl of MTT (5 mg/ml in PBS) was added to each culture well. After incubation at 37°C, the culture medium was removed and the purple crystals formed were dissolved in 150 µl of 0.1 M HCl in isopropanol. The absorbance was measured using a microplate reader at 570 nm with a background correction of 690 nm.
Cell proliferation assay using BrdU incorporation assay
Cell proliferation was measured using a commercially available kit (Cell proliferation ELISA BrdU colorimetric, Roche). In brief, PTECs cultured in 96-well plates were treated with 10 mg/ml of the protein preparations for 24, 48 and 72 h. The volume of the culture medium was 100 µl. Ninety minutes before the pre-determined time periods, cells were incubated with 10 µl of bromodeoxyuridine (BrdU) labelling solution. After incubating at 37°C for 90 min, the culture medium was removed and the cells were treated with Fixdenat for 15 min. The Fixdenat was removed and the cells were incubated with anti-POD labelling solution and incubated at room temperature for 60 min. Appropriate negative controls were included. After three washes using the BrdU wash solution, the colour was developed using the substrate and read using a microplate reader at a wavelength of 370 nm with a reference wavelength of 492 nm.
Western blotting for Fas and FasL
After appropriate time periods, the culture medium was removed and the cells washed twice in PBS. Cells were lysed in RIPA buffer (1x PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) and centrifuged for 10 min at 13 000 r.p.m. at 4°C. The supernatant was collected and the protein content measured using BCA protein assay reagent (Pierce, Rockford, IL). The lysates were aliquoted and stored at 80°C until further use. The cell lysates were mixed with equal volume of non-reducing electrophoresis sample buffer (1 ml of glycerol, 3 ml of 10% SDS, 1.25 ml of 1 M TrisHCl, 1 mg of bromophenol blue) and boiled for 5 min. The proteins were resolved on a 12% TrisHCl gel (Biorad, Hercules, CA) and electro-transferred (100 V, 60 min) into Hybond-C Extra nitrocellulose membrane (Amersham Biosciences). After blocking with western blocking buffer (WBB) (10% non-fat powdered milk in PBS, 0.05% Tween-20), the membranes were incubated with primary antibodies (Santa Cruz Biotechnology, CA) against Fas (rabbit polyclonal IgG, catalogue no. sc-714, 1:200 in WBB) or FasL (rabbit polyclonal IgG, catalogue no. sc-6237, 1:200 in WBB) for 1 h at room temperature. After washing in PBST (PBS containing 0.05% Tween-20), the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit IgG, sc-2004, 1:10 000 in WBB) for 1 h. After three washes in PBST followed by two washes in PBS, the band intensity was detected by SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) and exposure to Fuji medical X-ray film.
Statistical analysis
The experiments were repeated three times in duplicate using cells obtained from three different nephrectomy samples. The results are expressed as the mean±SE. Comparisons between groups were analysed using analysis of variance. Pair-wise multiple comparisons employed the Tukey test. Analyses were performed using Graphpad Instat software (San Diego, CA). P<0.05 was considered significant. As the results in the PBS groups did not produce any significant difference from those of untreated cells, these are not included in the results.
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Results |
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Effect of protein treatment on apoptosis and necrosis
Flow cytometry for annexin binding showed that exposure of PTECs to proteins in the 30100 kDa range did not induce any significant changes in apoptosis when compared with the control (untreated) group (Figure 2). In contrast, PTECs exposed to the 100440 and 30440 kDa fractions resulted in a 2- to 3-fold increase in apoptosis at all time points (Figure 2).
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Discussion |
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This study aimed to mimic, as closely as possible, the conditions encountered in protein overload proteinuria in vivo. We used a wide range of plasma proteins rather than a single protein species to reflect the in vivo condition closely. In selective proteinuria, proteins in the range of 40100 kDa are filtered, whereas in non-selective proteinuria, the range can be up to 440 kDa [3,13] or even 1000 kDa in cases of severe nephrosis [3]. However, in general, any fraction >440 kDa would only be minimally filtered even in severe glomerular damage [13]. During the preparation of the plasma protein fraction, lipoproteins and proteins >440 kDa were specifically excluded. Also, the final ultrafiltration and concentration step using the centrifugal filters with a 30 kDa nominal molecular weight cut-off resulted in most of the <30 kDa proteins being lost in the filtrate, and proteins of 3066 kDa were just detectable in our protein gels (Figure 1c).
The physiological concentration of proteins in proximal tubular ultrafiltrate in humans is not clear. Micropuncture studies in rat kidneys suggest that the normal ultrafiltrate protein concentration of 0.020.03 mg/ml may increase 100-fold in proteinuric renal diseases [14]. In humans with severe nephrosis, urinary protein excretion may exceed 4000 mg/24 h. Assuming a urine volume of 12004000 ml, the urine protein concentration would therefore be 13.3 mg/ml. This is the excreted amount, and the concentrations of protein in proximal tubular ultrafiltrate should be considerably higher. The protein concentration used in the present study (10 mg/ml) is therefore comparable with levels which might be expected in the proximal tubular ultrafiltrate, at least in nephrotic syndrome.
Controversy exists over the species of filtered protein responsible for the pathological changes in proteinuric renal diseases. The predominance of albumin in proteinuric urine of humans and animals [3,15,16] suggests that albumin plays a key role in the pathophysiology of tubulointerstitial injury. However, other studies have shown that albumin may not be the mediator of progressive renal insufficiency. For example, in minimal change nephrosis, there may be heavy albuminuria but progressive renal insufficiency is rare [2]. Furthermore, the adriamycin nephrosis [17] and subtotal nephrectomy [18] models of progressive renal insufficiency in the Nagase analbuminaemic rat produce high molecular weight proteinuria without significant albuminuria. Albumin, therefore, cannot be implicated in the pathology in these animal models.
In vitro studies have also been controversial. Erkan et al. [6] showed that treatment of LLC-PK1 cells with 20 mg/ml of delipidated bovine serum albumin (BSA) produced a dose- and duration-dependent induction of apoptosis and suggested that the pro-apoptotic effect was specific to albumin and not shared by the fatty acid moiety of BSA. However, very high concentrations of fatty acid-bearing albumin (3050 mg/ml) also induced apoptosis of human PTECs [7]. Arici et al. attributed the pro-apoptotic effect of albumin to the fatty acid moiety of the protein [7]. On the other hand, Lee et al. [9] showed that treatment of human PTECs with 1 mg/ml of delipidated BSA for 48 h did not induce apoptosis. The induction of apoptosis by albumin in previous studies appears to be associated with the use of very high concentrations of the protein (whether delipidated or non-delipidated). These studies [6,7] have used a concentration of albumin in the 2050 mg/ml range, at least 25 times higher than the approximate concentration of albumin in our 30100 kDa fraction (estimated at 9.5 mg/ml).
Our findings are compatible with previous studies [2,17,18] which suggested that albumin may not be the major factor responsible for tubular cell deletion in proteinuric conditions. These observations also support recent clinical findings that show a correlation between the high molecular weight proteins (>100 kDa) and the severity of pathological changes observed in proteinuric conditions [1922]. In our study, although some pathological changes were induced by the 30100 kDa fraction, these were minimal (Figures 24 and 6). Major effects on the other hand were induced by exposure to the 100440 kDa fraction, which by comparison was more toxic than the other fractions studied. As the entry of the high molecular weight fraction alone into the tubular lumen is not possible in vivo, a 30440 kDa fraction was also included in this study. This latter fraction also induced significant apoptosis, but the changes were relatively less than those observed with the 100440 kDa fraction. The attenuated effect may reflect the lower concentration of proteins in the 100440 kDa range present in the combined fraction. This observation suggests that the presence or absence of the high molecular weight fraction (100440 kDa) in the glomerular ultrafiltrate might be of primary pathophysiological importance in eventually determining the severity and progression of tubular atrophy in proteinuric conditions. The model described here mimics the pathological changes observed in selective and non-selective proteinuria: in selective proteinuria (where proteins in the range of 30100 kDa are filtered), the tubulointerstitial changes are minimal, whereas in non-selective proteinuria (proteins in the range of 30440 kDa are filtered) the changes are severe.
In order to explore the mechanism(s) behind the protein overload-induced changes, attention was focused on the Fas/FasL system. The constitutive expression of the Fas/FasL system in proximal tubular cells is controversial, with some studies supporting a constitutive expression [10,23,24] while others report a negative finding [25]. The differences have been attributed largely to the methodology used in each study. In our study, Fas and FasL were undetectable in untreated cells and barely detectable in cells exposed to the albumin-rich (30100 kDa) fraction (Figure 8). However, western blotting revealed a significant and abundant increase in Fas and FasL expression in PTECs exposed to the 100440 and 30440 kDa protein fractions (Figure 8). Interestingly, Fas and FasL expression was induced by the two fractions that induced significant apoptosis and inhibited metabolic activity (100440 and 30440 kDa fractions), suggesting that the toxicity of a particular protein fraction is dependent upon or related to its ability to induce the expression of the Fas/FasL system. In LLC-PK1 cells, albumin-induced apoptosis has been shown to be associated with a dose-dependent overexpression of Fas [6].
The upregulation of Fas and FasL in response to protein overload can promote apoptosis in two ways. First, the upregulation increases the probability of binding of FasL to its receptor (Fas) [23], thereby initiating a cascade of substrate-specific pro-apoptotic proteases called caspases [10,11]. Secondly, Fas and FasL can also initiate the apoptotic process independently of each other [23]. Fas upregulation may precipitate spontaneous oligomerization of intracellular Fas domains, leading to FasL-independent apoptosis in a fashion similar to Fas-overexpressing cells [23]. Similarly, FasL itself can activate intracellular signalling pathways leading to cell cycle arrest resulting in apoptosis [11,23].
In conclusion, we have identified a fraction (100440 kDa proteins) of normal human plasma, prepared from freshly drawn blood, which may explain the accelerated tubulointerstitial damage observed in non-selective proteinuria in vivo. Our findings linking the high molecular weight fraction with the severity of changes observed in this study are supported by the recent clinical studies which found that proteinuria defined by the >100 kDa fraction was the best predictor of renal disease progression [1922]. Maintenance of tissue mass is dependent upon the balance between the rate of cell death and proliferation. In proteinuric conditions, this equilibrium may shift in favour of cell deletion due to increased apoptosis and decreased cell proliferation. The present study has demonstrated both processes in protein-treated human PTECs, that necrosis is not a significant factor in this system, and that these processes are at least in part mediated by the Fas/FasL system. The presence of high molecular weight proteins in the glomerular ultrafiltrate may be critical in determining the severity of the tubular atrophy in proteinuric conditions in vivo.
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Acknowledgments |
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Conflict of interest statement. None declared.
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References |
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