Nov 4, 1996 - hibitors phlorizin and phloretin were used to in- vestigate the role of speciflc 1-glucose transport proteins. Application of either basolateral phlori ...
American journal of Patholog,I V'ol. 150, No. 3, March 1997 Copyright t American Society for Investigative Patholog
Polarity of Stimulation and Secretion of Transforming Growth Factor-f31 by Cultured Proximal Tubular Cells
Aled 0. Phillips, Robert Steadman, Kimberley Morrisey, and John D. Williams From the Institute of Nephrology, University of Wales College
of Medicine, Cardiff Royal Infirmary, Cardiff, United Kingdom
Proximal tubular epithelial ceUls are the most abundant cells in the renal cortex, and recent studies suggest that they may play an important role in initiating pathological changes in renal disease. Transforming growthfactor (TGF)-(31 has been implicated as a majorfactor controling the development and progression of renal flbrosis in numerous diseases, including diabetic nephropathy. We have recently demonstrated that human proximal tubular epitheial cells synthesize and secrete TGF-f41 after the sequential addition of both 25 mmol/L -glucose and platelet-derived growth factor (PDGF). The present study examines the control of this synthesis and in particular the polar requirements of the stimulation and the direction of release of the protein. A proximal tubular ceUl line (LLC-PK1) was cultured on porous tissue culture inserts. Confluent ceUls were exposed to 25 mmol/L 1-glucose on either their apical or basolateral aspect. TGF- 41 mRNA induction (reverse transcriptase polynerase chain reaction) occurred only after basolateral exposure. Similarly, TGF- (3 synthesis and secretion was induced only by the subsequent addition of PDGF to the basolateral aspect of the cells. In contrast, TGF-(31 protein secretion was detected equally in the apical and basolateral compartments. This effect was maximal after 12-hour PDGF stimulation and represented a threefold increase over controlsfor TGF- 31 in both the apical and basolateral compartments (n = 3, P < 0.05 versus control). The glucose transporter inhibitors phlorizin and phloretin were used to investigate the role of speciflc 1-glucose transport proteins. Application of either basolateral phlorizin or phloretin at the time of addition of 25
mmol/L D-glucose to the same compartment inhibited TGF-,B1 synthesis in response to PDGF. Maximal inhibition was achieved at 0.5 mmol/L ofeither inhibitor (phlorizin percent inhibition of apical TGF-181, 75%, P = 0.015, and of basolateral TGF-(31, 78%/, P = 0.015; phloretin percent inhibition of apical TGF-(81, 680%, P = 0.03, and of basolateral TGF,31, 79%, P = 0.001, n = 5, P versus control). No inhibition was seen with apical application of either inhibitor. These data demonstrate that the priming ofproximal tubular cels for TGF-(81 synthesis occurs only after basolateral exposure of the cells to 25 mmol/L 1-glucose. This mechanism is dependent on the activity of the basolateral Dglucose transporter GLUT-1. In another series of experiments, TGF-,31 synthesis in response to the addition of basolateral PDGF was also induced after basolateral pretreatment with D-galactose but not 2-deoxy--glucose. This priming effect demonstrates the dependence of this response on glucose metabolism by the cells, not simply the activity of the GLUT-1 transporter, as both 2-deoxy-D-glucose and D-galactose are transported by GLUT-1, although only the latter is metabolized. The extrapolation of these results to diabetic nephropathy would suggest that it is changes in the interstitial concentration of glucose rather than the urinary glucose level that likely modulate the synthesis of the profibrotic cytokine TGF-(31 and thereby influence the progression ofinterstitialflbrosis. (Amj Pathol 1997, 150:1101-1111) Renal failure is a common complication of diabetes mellitus, occurring in approximately 30% of patients A. 0. Phillips is a recipient of a training fellowship from the National Kidney Research Fund. R. Steadman is supported by the Kidney Research Unit for Wales Foundation, and K. Morrisey is supported by the Medical Research Council. Accepted for publication November 4, 1996. Address reprint requests to Dr. A. 0. Phillips, Institute of Nephrology, Cardiff Royal Infirmary, Newport Road, Cardiff, CF2 1SZ UK.
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with insulin-dependent diabetes mellitus and 10 to 20% of patients with non-insulin-dependent diabetes.1, 2 Diabetic nephropathy has, in the past, been considered to be a primary glomerular disease. There is now strong evidence, however, that a progressive decline in renal function is also closely correlated with the degree of renal interstitial fibrosis.3' 4 As a result, several studies have focused on the mechanisms of induction of these interstitial changes and in particular the role of cytokines and growth factors. Among the molecules that have been widely studied is transforming growth factor-131 (TGF-,13). This cytokine appears to have a central role in promoting the synthesis and the accumulation of different extracellular matrix components.5-9 Furthermore, there is now in vivo evidence that has directly linked TGF-p1 to the pathogenesis of diabetic nephropathy, both in animal models and human disease.10-13 Recently, epidemiological evidence has indicated that complications of diabetes are related to poor glycemic control.14 15 As only 30% of diabetic patients develop nephropathy, however, hyperglycemia alone may be insufficient to initiate pathological changes. We have previously demonstrated that exposure of cultured human renal proximal tubular epithelial cells to elevated D-glucose concentrations induced TGF-31 mRNA synthesis without any associated change in TGF-f31 protein synthesis.16 Application of platelet-derived growth factor (PDGF) as a second stimulus, however, triggered TGF-,B1 protein secretion in D-glucose-primed cells by a post-transcriptional mechanism.17 In diabetes, proximal tubular cells may be exposed to conditions of elevated glucose concentration either apically as a result of glycosuria or basely as a result of elevated interstitial tissue concentrations of glucose. In vivo, proximal tubular cells have an asymmetric distribution of transport proteins that facilitate the reabsorption of water, glucose, and electrolytes from the glomerular filtrate. Sugar transport consists of two sequential processes. Movement from lumen to cell across the brush border is by sodium-glucose symporter activity (SGLT), and its exit from the cell occurs by facultative GLUT-1 activity. Similarly, systemic or local inflammation may result in increased local levels of cytokines and growth factors that may synergize with hyperglycemia to initiate TGF-31 protein synthesis. Such changes in cytokine activity may occur at either the apical or basolateral side of the cell. This then raises the question of the polarity of stimulation as well as the polarity of secretion of TGF-,B1. In the current study, we have examined the polar requirements of proximal tubular cells for D-glucose
priming and of TGF-,B1 secretion using the porcine LLC-PK1 tubular cell line, grown in polarized culture on tissue culture inserts. As alteration of glucose transport has been proposed as a possible mechanism of the complications associated with diabetes mellitus,18-20 we have examined the dependence of the priming effect of D-glucose on the function of both the apical sodium-glucose co-transporter (SGLT) and on the basolateral facultative glucose transporter (GLUT-1). In addition, using D-galactose and the nonmetabolized hexose, 2-deoxy-D-glucose (2dglc), we have examined the contributions of glucose transport and metabolism to its priming effect. The data demonstrate that exposure of the cells on their basolateral but not their apical aspect induced TGF-,1 gene transcription. Neither apical not basolateral D-glucose exposure led to the secretion of TGF-,31 protein. The application of PDGF to Dglucose-pretreated cells initiated TGF-,31 protein synthesis but only when applied to the basolateral aspect of the cell. Secretion of TGF-j3l under these conditions was detected equally in the apical and basolateral compartments. The priming effect of Dglucose was abolished by the addition of either phlorizin or phloretin to the basolateral aspect of the cells at the time of addition of basolateral D-glucose but not when added to the apical compartment. Furthermore, the priming effect of D-glucose could be mimicked by D-galactose but not by 2dglc, suggesting that the D-glucose-dependent induction the TGF-,B1 mRNA involved transport of D-glucose by the facultative glucose transporter GLUT 1 and its subsequent metabolism by the cell.
Materials and Methods Cell Culture Experiments to study the polarity of stimulation and secretion of TGF-,31 and the control of TGF-,B1 gene expression were performed using LLC-PK1 cells, which are a widely used porcine cell line of proximal tubular origin. Growth medium for cell culture comprised M199 with 10% fetal cult serum (Life Technologies, Paisley, UK). Cells were grown at 370C in 5% C02 and 95% air. For the determination of polarity of the effects of D-glucose, cells were seeded onto porous tissue culture inserts (0.4-gm pore size; Becton Dickinson, Oxford, UK). Reassembly of tight junctions and integrity of the monolayer was demonstrated by serial measurements of transepithelial resistance using a commercially available system (Millicell-ERS Resistance System, Millipore, Watford, UK) as previously described.21 Confirmation that
Polarity of Stimulation and Secretion of TGF-j31
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formed under serum-free conditions after a growth arrest period of 72 hours in serum-free medium. Porcine PDGF was obtained from R&D Systems (125-PD-005, Abingdon, UK). D-Glucose, D-galactose, and glucose transport inhibitors (phlorizin and phloretin) were obtained from Sigma Chemicals (Poole, UK). 2dgic was obtained from Fluka Chemicals (Gillingham, UK).
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125I Albumin Diffusion (% control) Figure 1. Correlationi of transepithelial resistance with phenol red (A) radiolabeled albumin (B) diffusion across LLC-PK1 cells grown on tissue culture inserts. Aftersubculture, cells were plated onto permeable tissue cuilture inserts. A: Phenol red flux across the cell layer uwas measured by replacing the culture medium in the basolateral conipartment with phenol-red-free M199. Standard M199 was added to the apical compartment. Samples were collectedfrom the basolateral compartment at timed intervals. Phenol redflux was calculated by measutring absorbance at 540 nm, and this was correlated to the transepithelial resistance measurement takeni at the same time point. Conttrol measurements wvere made using diffusion ofphenol red across a tissue culture insert to uhich no cells were added. Phenol red diffusion was expressed as a percentage of conitrol. B: Radiolabeled albumin permeability was assessed by replacing the culture medium in the apical compartment with M199 containing 1 ,ul of 1251-labeled albuminl. After 60 minutes, 200 ,u l of medium was removedfrom the basolateral compartment and radioactivity was counted. Inserts without cells were used to derive control values. Diffusion of albumin was expressed as a percentage of control, and this was correlated to transepithelial resistance measuremetnts taken at the same time points. or
peak increases in transepithelial resistance were a reliable indicator of the formation of a confluent monolayer was demonstrated by the correlation of transepithelial resistance measurements to diffusion of both phenol red (Figure 1A) and 1251-labeled albumin (Figure 1 B) across the monolayer. Monolayer integrity was also demonstrated by electron and light microscopy (Figure 2). All experiments were per-
RNA Isolation, Reverse Transcription (RT), and Polymerase Chain Reaction (PCR) Amplification The effect of elevated D-glucose concentration on TGF-,B1, SGLT, and GLUT-1 gene expression was examined by RT-PCR. Control experiments were performed with L-glucose. At the defined time periods, total cellular RNA was extracted from both control and stimulated cells after lysis with 4 mol/L guanidine isothiocyanate and centrifugation through 5.7 mol/L cesium chloride in 0.1 mol/L EDTA.22 Total RNA was reverse transcribed to cDNA with M-MLV reverse transcriptase (Gibco Life Technologies, Paisley, UK), using the random hexamers method as previously described.23 Briefly, the reaction mixture contained 1 ,ul of random hexamers (100 ,umol/L; Pharmacia Biosystems, Milton Keynes, UK), 5 Al of NTPs (2.5 mmol/L, Gibco/BRL Life Technologies), 2 ,ul of 1OX PCR buffer (100 mmol/L Tris/HCI, 500 mmol/L KCI, 15 mmol/L MgCI2, and 0.01% w/v gelatin), 2 ,l] of dithiothreitol (0.1 mol/L, Gibco/BRL Life Technologies), 1 Al of RNAse inhibitor (Promega, Southampton, UK), 1 ,ul of M-MLV Superscript reverse transcriptase (200 U; Gibco/BRL Life Technologies), and 1 ,ug of total RNA. PCR amplification was performed in a total volume of 50 ,ul (2 ,lI of RT product and 48 ,tl of master mix (36.25 ,ul of H20, 1.25 ,ul of 5' primer (20 ,umol/L), 1.25 ,ll of 3' primer (20 ,umol/L), 4 ,tl of NTPs, 5 ,ul of 1OX PCR buffer, and 0.25 ,tl of Taq polymerase (2.5 U; Amplitaq, ILS, London, UK) using a Perkin Elmer 480 thermocycler (Applied Biosystems, Warrington, UK). The PCR protocol for TGF-,31 mRNA and a-actin was as follows: 1 st cycle 72°C for 10 minutes; 2nd cycle, 94°C for 3 minutes, 55°C for 1 minute, and 720C for 1 minute; 3rd through 38th cycles, 94°C for 40 seconds, 55°C for 1 minute, and 72°C for 1 minute. The final cycle was 94°C for 1 minute and 600C for 10 minutes. PCR was performed for 35 cycles for TGF-,B1 and a-actin and 30 cycles for SGLT and GLUT-1. One-tenth of the PCR reaction from both test and control (a-actin) product were mixed and separated
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Figure 2. Morphology of LLC-PK1 grown on tissue culture inserts. Cells were seeded onto 0.4-gtm porous tissue culture inserts. Once peak resistance uwas attained, cells were fixed for light (A) and electron (B) microscopy.
by flat-bed electrophoresis in 3% w/v NuSieve GTG agarose gels (Flowgen Instruments, Sittingbourne, UK), stained with ethidium bromide (Sigma), and photographed. The negatives were scanned using a densitometer (model 620 video densitometer, BioRad Laboratories, Hemelhempstead, UK), and the density of the bands was compared with those of the housekeeping gene. Results were expressed as ratios of the gene of interest to a-actin, normalized to the first time point of each experiment. The sequences of the amplification primers were as follows: TGF-,31, 5'-ACCGGCCCTTCCTGCTCCTCA-3' and 5'-CGCCCGGGTTGTGCTGGTTGT-3'
can be measured indirectly after acid activation of samples. All supernatant samples were assayed before and after acid activation to determine the amount of active or latent TGF-,31 secretion, respectively. Inter-assay precision ranged from 4.3 to 9.6% (coefficient of variation), and intra-assay precision ranged from 2.8 to 8.6% (coefficient of variation). There was no detectable cross-reactivity with a range of other cytokines and growth factors, including TGF-,32 and TGF-33.
(288 bp)24; SGLT, 5'-CCTGGCTGGACGAAGTATGGTG-3' and 5'-GGCCCCCTGTGATTGTGTAAAG-3' (413 bp)25; GLUT-1, 5'-GGGGGCATGATTGGCTCCTTCTCT-3' and 5'-ACTCTTGGCCCGGTTCTCCTCGTT-3' and 5'-ACTCTTGGCCCGGTTCTCCTCGTT-3' (456 bp)26; a-actin, 5'-CCTTCCTGGGCATGGAGTCCT-3' and 5'-GGAGCAATGATCTTGATCTT-3' (204 bp).27
For each individual experiment, the mean of duplicate determinations was calculated. The data are presented as means ± SEM of n experiments. Statistical analysis between two groups was performed using the paired Student's t-test, with a value of P < 0.05 considered to represent a significant difference.
Determination of TGF-,B1 Protein Secretion TGF-31 protein generation was quantitated by specific antibody capture enzyme-linked immunoassay (ELISA) as previously described.28 The assay is sensitive to 0.2 ng/ml with a range up to 100 ng/ml. The assay detects the active 25-kd human TGF-31. Active TGF-,31 is measured directly, and latent TGF-l31
Statistics
Results Effects of D-Glucose on TGF-,B1 Gene Expression Confluent monolayers of growth-arrested LLC-PK1 cells on porous tissue culture inserts were exposed to 25 mmol/L D-glucose on either their apical aspect or their basolateral aspect for up to 72 hours. Apical
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exposure of the cells did not alter TGF-[31 mRNA expression. In contrast, basolateral exposure led to an increase in TGF-,[1 mRNA first detectable after 3 hours (Figure 3). Maximal induction was reached at 12 hours, at which time there was a 4.5-fold increase in the TGF-,[1/a-actin mRNA ratio over control. Exposure of cells to 25 mmol/L L-glucose on either their apical or basolateral aspects did not induce TGF-[1
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cretion was maximal 12 hours after application of PDGF, at which time there was a 2.5-fold increase in apical (P = 0.02, n = 5) and a 3-fold increase in basolateral (P = 0.01, n = 5) TGF-,/1 secretion over control (Figure 4). This TGF-,B1 was detected only after transient acidification of the supernatant samples, indicating that TGF-/1 was secreted in its latent form.29 Pretreatment of cells with 25 mmol/L L-glucose either
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TGF-/31 Secretion In contrast to the increases in gene expression seen to basolateral stimulation with D-glucose, under the same conditions, there was no detectable increase in the secretion of active or latent TGF-31 as assessed by specific ELISA of cell culture supernatants. We have previously demonstrated that exposure of 25 mmol/L D-glucose-pretreated cells to PDGF induced TGF-,/1 synthesis and secretion by proximal tubular epithelial cells.16 In the current study, LLCPK1 cells were pretreated with either apical or basolateral 25 mmol/L D-glucose for 48 hours before the application of porcine PDGF (50 ng/ml) to either compartment. TGF-,B1 protein was quantified over the subsequent 48 hours by ELISA. Increased TGF-[31 protein in the cell culture supernatant was detected only when cells were pre-exposed to 25 mmol/L D-glucose on their basolateral aspect and subsequently stimulated by porcine PDGF also on their basolateral aspect. Under these conditions, secretion of TGF-,/1 occurred equally into both the apical and basolateral compartments. TGF-,B1 sein response
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