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Division of Nephrology/Department of Emergency and Transplantation, University of Bari, Italy.
Correspondence to Dr. Giuseppe Grandaliano, Division of Nephrology/Department of Emergency and Transplantation, University of Bari, Polyclinic, Piazza Giulio Cesare, 11, 70124 Bari, Italy. Phone: +39 080 5592787; Fax: +39 080 5575710; E-mail: g.grandaliano{at}nephro.uniba.it
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The activation of the coagulation cascade leads to the partial proteolysis of prothrombin with subsequent local thrombin generation (10). This serine protease may then be accumulated in its active form within the fibrin clots and released locally over a prolonged period of time (11). Thrombin, besides its physiologic action in the clotting cascade, is known to modulate a variety of cell functions through the interaction with specific cell surface receptors (12,13). All of the known thrombin receptors belong to the protease-activated receptor (PAR) family and are characterized by a peculiar proteolytic mechanism of activation (14,15,16). Indeed, receptor activation occurs when thrombin cleaves the extracellular domain of the receptor exposing a tethered ligand (14). Among the receptors of the PAR family, thrombin can interact specifically with PAR-1, -3, and -4 (14,15,16). Of these three signaling receptor proteins, however, only PAR-1 has been shown to be expressed in the kidney (14,17). Although proximal tubular cells (PTC) may represent a potential target for thrombin, no information is available on the presence of PAR-1 on their surface as well as on their activation by thrombin.
Tubular damage and monocyte infiltration are two of the histopathologic hallmarks of acute and chronic tubulointerstitial injury (18,19). Tubular necrosis and atrophy, especially in the setting of acute tubulointerstitial damage, are potentially reversible, although the mechanisms responsible for the regenerative response are still poorly understood (18). Monocyte interstitial infiltration is a key step in the pathogenesis of tubulointerstitial damage and monocyte chemotactic peptide (MCP-1), expressed by tubular cells, may be involved in the monocyte influx into the interstitial space (19,20).
In the present study, PAR-1 expression by human PTC was investigated in vivo and in vitro. Moreover, the thrombin effect on human PTC mitogenesis and MCP-1 gene and protein expression was evaluated.
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Materials and Methods |
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Cell Isolation and Culture
HK2, an immortalized PTC line from normal adult human kidney
(21), was obtained from
American Type Culture Collection (Manassas, VA). Cells were grown to
confluence in DMEM/F12 medium supplemented with 5% FBS, 100 U/ml penicillin,
100 µg/ml streptomycin, 2 mM L-glutamine, 5 µg/ml insulin, 5 µg/ml
transferrin, 5 ng/ml sodium selenite, 5 pg/ml T3, 5 ng/ml hydrocortisone, 5
pg/ml prostaglandin E1, and 10 ng/ml EGF. For passage, confluent cells were
washed with phosphate-buffered saline (PBS), removed with 0.05% trypsin/0.02%
ethylenediaminetetra-acetic acid in PBS, and plated in DMEM/F12 medium.
Immunohistochemistry and Immunocytochemistry
Apparently normal kidney portions from 10 patients undergoing nephrectomy
for renal cell carcinoma and cultured HK2 cells plated on 8-well multitest
slides (ICN, Aurora, OH) were used to investigate PAR-1 protein expression.
The detection of this thrombin receptor was performed on frozen 4-µm-thick
kidney sections and on subconfluent HK2, fixed in 4% paraformaldehyde, using
specific mouse monoclonal anti-human PAR-1 antibody directed against an
epitope (corresponding to PAR-1 residues 51 to 64) in the N terminus of the
receptor that is retained after PAR-1 cleavage at 1:200 dilution
(17). HK2 cells were
serum-starved overnight and incubated in serum-free medium with or without
thrombin (5 U/ml) for 15 min, before fixation. The anti-PAR-1 antibody
recognizes the extracellular domain of the inactive as well as of active
PAR-1. Immobilized mouse antibodies were detected by the immunoalkaline
phosphatase (APAAP) method with affinity-purified rabbit anti-mouse IgG (Dako,
Glostrup, Denmark) and APAAP complex (1:50 dilution; Dako), following a
two-step technique as described previously
(19). Alkaline phosphatase was
developed with New Fuchsin (Sigma). Negative controls were performed by
omitting the primary or secondary antibodies, and using nonimmune mouse or
rabbit antiserum as first layer.
3H-Thymidine Incorporation
DNA synthesis was measured as the amount of
[methyl-3H]-thymidine incorporated into TCA-precipitable material,
as described previously (22).
Briefly, HK2 were plated in 24-well dishes at a density of 4 x
104 cells/well, grown to confluence, and made quiescent by being
placed in serum-free medium for 48 h. The cell monolayer was then incubated
with thrombin at the indicated concentrations for 24 h at 37°C. In
separate sets of experiments, cells were preincubated with genistein (25
µM), daidzein (25 µM), PP1 (25 µM), and PP3 (25 µM) for 18 h
before adding thrombin. At the end of the incubation period, cells were pulsed
for 4 h with 1.0 µCi/ml 3H-thymidine. The medium was then
removed, the cells were washed twice in ice-cold 5% TCA, and then incubated in
5% TCA for 5 min. The monolayer was solubilized by adding 0.75 ml of 0.25N
NaOH in 0.1% sodium dodecyl sulfate (SDS). Half-milliliter aliquots were then
neutralized and counted in scintillation fluid using a beta counter.
In parallel experiments, cell proliferation was determined by direct cell counting after 24 and 48 h of incubation with thrombin (5 U/ml), as described previously (23).
RNA Isolation and Northern Blot Analysis
HK2 cells were plated in 75-mm2 flasks and cultured as described
above. After reaching confluence, cells were serum-starved for 48 h and then
incubated for the indicated time periods with thrombin (5 U/ml). In separate
sets of experiments, cells were preincubated with genistein (25 µM),
daidzein (25 µM), PP1 (25 µM), and PP3 (25 µM) for 18 h before adding
thrombin. At the end of incubation, cells were lysed with 4 M guanidinium
isothiocyanate containing 25 mM sodium citrate, pH 7.0, 0.5% sarcosyl, and 0.1
mM ß-mercaptoethanol. Total RNA was isolated by the single-step method,
using phenol and chloroform/isoamyl alcohol
(24).
MCP-1 gene expression was studied by Northern blotting, as described previously (24). Briefly, electrophoresis of 20 µg of total RNA from each experimental condition was carried out in 1% agarose gel with 2.2 M formaldehyde. The RNA was then transferred overnight onto a nylon membrane (Schleicher & Schuell, Dassel, Germany). The membrane was stained with ethidium bromide to evaluate the 28S and 18S ribosomal bands and prehybridized at 42°C for 2 h in 50% formamide, 0.5% SDS, 5x SSC, and 0.1 mg/ml salmon sperm DNA. A 0.7-kb fragment of the baboon MCP-1 cDNA was used as a probe (25). The DNA fragment was labeled by random priming using a commercially available kit (Amersham) and [32P]dCTP (specific activity, 3000 Ci/mmol). The probe (106 cpm/ml) was added to 10 ml of prehybridization solution, and the blots were hybridized for 16 h at 42°C. The membranes were then washed once in 2x SSC, 0.1% SDS at room temperature for 5 min, once in the same buffer at 55°C for 30 min, and in 1x SSC, 0.1% SDS at 55°C for an additional 30 min. After drying, membranes were exposed to a Kodak X-OMAT film with intensifying screens at -70°C.
Enzyme-Linked Immunosorbent Assay
HK2 cells plated in 6-well dishes and grown to 70% confluence were
serum-starved for 24 h and then incubated for 24 h in serum-free medium with
or without thrombin (5 U/ml). At the end of the incubation, the supernatant
was harvested, centrifuged for 10 min at 1000 x g to remove the
cell debris, and stored at -80°C until used. MCP-1 measurement in the
supernatant was performed using a commercial human MCP-1 enzyme-linked
immunosorbent assay (ELISA) kit (Quantikine; R&D Systems, Abingdon, United
Kingdom). This is a multiple sandwich solid-phase enzyme immunoassay that uses
a specific monoclonal antibody raised against human MCP-1. The sensitivity of
the ELISA is 5 pg/ml. The MCP-1 concentration of the unknown samples was
determined by interpolation into a standard curve developed with known amounts
of recombinant human MCP-1 protein. MCP-1 protein concentration was normalized
to cell counts.
Western Blot
HK2 were plated in 60-mm2 Petri dishes and grown to confluence
in DMEM/F12 medium supplemented with 10% FBS. The cells were incubated for 48
h in serum-free medium and then exposed to thrombin (5 U/ml) for the indicated
time periods. At the end of the treatment, the cell monolayer was rapidly
rinsed twice with ice-cold PBS and lysed in 100 µl of RIPA buffer (1 mM
phenylmethylsulfonyl fluoride, 5 mM ethylenediaminetetra-acetic acid, 1 mM
sodium orthovanadate, 150 mM sodium chloride, 8 µg/ml leupeptin, 1.5%
Nonidet P-40, and 20 mM Tris-HCl, pH 7.4). The lysates were set on ice for 30
min and centrifuged at 10,000 x g at 4°C for 5 min. The
supernatants were collected and stored at -80°C until used. Aliquots
containing 7.5 µg of proteins from each lysate were subjected to
SDS-polyacrylamide gel electrophoresis on a 7.5% gel under reducing conditions
and then electrotransferred onto nitrocellulose membrane (HybondTM C;
Amersham). The filter was blocked overnight at room temperature with 2% bovine
serum albumin in PBS containing 0.1% Tween 20 (TBS) and incubated with
monoclonal anti-phosphotyrosine antibody at room temperature for 4 h. The
membranes were washed twice in TBS and incubated for 2 h at room temperature
with horseradish peroxidase-conjugated sheep anti-mouse IgG at 1:1500 dilution
in TBS. The membranes were washed three times at room temperature in TBS and
then once with 0.1% SDS in PBS. The ECL enhanced chemiluminescence system
(Amersham) was used for detection.
Immunoprecipitation
Confluent HK2 cells in 60-mm2 culture dishes were placed in
serum-free medium for 48 h. Thrombin (5 U/ml) was then added for the indicated
time periods. Cells were washed twice with ice-cold PBS and lysed in
situ with RIPA buffer for 30 min at 4°C. The cell lysate was
centrifuged at 10,000 x g for 30 min at 4°C. One hundred
micrograms of protein from the supernatant was first incubated with
anti-phosphotyrosine antibodies for 2 h on a rocking platform at 4°C and
then with agarose-linked protein A for 2 h at 4°C. The immunobeads were
washed twice with RIPA buffer and twice with 0.5 mM LiCl, 0.1 mM Tris-HCl, pH
7.5, 1 mM sodium orthovanadate. The beads were then resuspended in sample
buffer and boiled. The immunoprecipitated proteins were separated by
electrophoresis on a 7.5% polyacrylamide gel and transferred onto a
nitrocellulose membrane. The membrane was blocked as described previously and
incubated with rabbit anti-src antibody (1:1000) for 4 h at room temperature,
washed, and incubated with horseradish peroxidase-conjugated mouse anti-rabbit
IgG (1:1500). The ECL system was used for detection of the horseradish
peroxidase-coupled antibodies.
Statistical Analyses
Data are presented as mean ± SD and compared by ANOVA. P
< 0.05 was considered significant.
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Results |
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Thrombin is a powerful mitogen for several cell types in culture (26,27,30). It is conceivable that thrombin, frequently activated in the setting of acute and potentially reversible tubular damage, may represent a regenerative stimulus for hPTC. To support this hypothesis, the effect of thrombin on hPTC DNA synthesis was investigated. As shown in Figure 3, the serine protease induced a dose-dependent increase in tritiated thymidine uptake that reached statistical significance at 0.05 U/ml and peaked at 5 U/ml. The proliferative effect of thrombin was further confirmed by direct cell counting. Indeed, incubation with this protease, at the concentration of 5 U/ml, caused a statistically significant increase in PTC number after 24 and 48 h (Figure 4).
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Interstitial monocyte infiltration is a histopathologic hall-mark of acute and chronic tubulointerstitial disease (19). MCP-1 is a specific and powerful chemotactic factor for monocytes, and its expression is strikingly upregulated during the development of tubulointerstitial damage (20,31). The expression of this chemokine has been demonstrated to be induced by thrombin in vascular smooth muscle and in endothelial and mesangial cells, but not in epithelial cells (25,32,33). Thus, the effect of thrombin on MCP-1 gene expression was investigated in cultured hPTC. Thrombin at the dose that maximally stimulated DNA synthesis caused a marked upregulation of MCP-1 mRNA abundance that was evident already at 3 h and was still present after 24 h (Figure 5). To determine whether the increased MCP-1 mRNA levels correlated with an increased translation, the MCP-1 protein concentration was evaluated, by ELISA, in the supernatant of serum-starved hPTC after 24 h of incubation in serum-free medium in the presence and in the absence of thrombin (5 U/ml). As shown in Figure 6, thrombin induced a statistically significant increase in the MCP-1 protein released into cell supernatant.
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Tyrosine phosphorylation of growth factor receptors plays an important role in their mitogenic and cell-activating effect (34). In the past few years, different G protein-coupled receptor agonists, including thrombin, have been shown to induce tyrosine phosphorylation of several cellular proteins (13,34). To investigate the effect of thrombin on tyrosine phosphorylation of cellular proteins in hPTC, equal amounts of protein from unstimulated and thrombin-stimulated cells were separated by SDS-polyacrylamide gel electrophoresis and analyzed by immunoblotting, using a specific anti-phosphotyrosine monoclonal antibody. Thrombin, at the dose that maximally stimulated DNA synthesis, caused a transient increase in the cellular levels of tyrosine-phosphorylated proteins, with the most prominent phosphorylated bands of 60, 70, and 90 kD (Figure 7). This early cellular effect of thrombin has been shown in different in vitro systems to be dependent on the activation of cytoplasmic and/or receptor tyrosine kinases. In platelets, thrombin stimulation induced a strong and rapid activation of c-src, a ubiquitous cytoplasmic tyrosine kinase, whereas in fibroblasts, keratinocytes, and COS-7 cells, thrombin has been shown to cross-activate the EGF receptor, a transmembrane tyrosine kinase (35,36). To investigate whether thrombin activates c-src in hPTC, the state of tyrosine phosphorylation of this enzyme was investigated as indirect evidence of its activation. For this purpose, cell lysates from unstimulated and stimulated PTC were immunoprecipitated with anti-phosphotyrosine antibodies and blotted with anti-src antibody. As shown in Figure 8, thrombin induced a time-dependent increase in tyrosine-phosphorylated c-src that peaked at 30 min. PTC express in vivo and in vitro the EGF receptor (37). Thus, the phosphorylation of this receptor in response to thrombin was also evaluated, but no EGF receptor tyrosine phosphorylation was observed upon thrombin stimulation (data not shown).
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To determine the role of the early tyrosine kinase activation in thrombin-induced DNA synthesis and MCP-1 gene expression, the effect of a specific tyrosine kinase inhibitor, genistein, on these two cellular responses was evaluated. Genistein at a concentration of 25 µM, a dose that completely blocks tyrosine phosphorylation, abolished completely thrombin-induced DNA synthesis as well as MCP-1 expression, whereas its inactive analogue daidzein was unable to influence both thrombin effects (Figures 9 and 10). The central role of tyrosine kinase activation in thrombin-elicited DNA synthesis was further confirmed using a second specific tyrosine kinase inhibitor, herbimycin A, with a mechanism of action different from genistein (Figures 9 and 10). Herbimycin A inhibited thymidine incorporation and MCP-1 expression induced by thrombin to the same extent as genistein. To better define the role of src activation in thrombin-induced DNA synthesis, the effect of a specific src inhibitor, PP1, was investigated. Preincubation of PTC with PP1 at the concentration of 25 µM significantly inhibited the increase in DNA synthesis caused by thrombin, whereas PP3, the inactive analogue of PP1, at the same molar concentration, was unable to influence the thrombin proliferative effect (Figure 11).
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Discussion |
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The present study demonstrated for the first time that PTC express both in vivo and in vitro PAR-1, and, therefore, may be considered potential targets for the modulatory action of thrombin. Interestingly, the thrombin receptor was localized mainly at the basolateral level. This localization, facing the interstitial space, may be pathogenically relevant. Indeed, fibrin deposits, the potential sites of thrombin accumulation, are mainly described in the peritubular capillaries and along the tubular basement membrane (1,2,3,4,5,6). In addition, in only a few tubular sections was luminal expression of PAR-1 observed. Although there are no reports on the presence of thrombin within the intratubular protein casts in proteinuric glomerular diseases, we hypothesize that thrombin generated within the glomerular tuft may reach the urinary space and activate the PTC by interacting with PAR-1 present at the luminal level.
The interaction of thrombin with the PAR-1 present on PTC leads to an increase in DNA synthesis and MCP-1 gene and protein expression. Both of these responses may be relevant in the setting of acute as well as chronic tubulointerstitial damage. Most of the acute conditions associated with interstitial fibrin deposition are characterized by potentially reversible tubular damage (2,4,6). Acute renal ischemia was the first pathologic condition in which this association was demonstrated (2). The ligation of the renal artery causes tubular necrosis and the intrarenal activation of the coagulation system with the subsequent extensive deposition of fibrin within the interstitial space (2,38). In this scenario, the ability of thrombin to act as a "growth factor" for PTC and stimulate a regenerative response may represent the first step toward the potential recovery, once the ischemic injury has been removed.
The ability of thrombin to induce MCP-1 gene and protein expression could represent a key event in the development of acute allograft rejection and in the progression of chronic rejection. Indeed, both acute and chronic renal graft rejection are characterized by a diffuse monocytic infiltrate (39). Monocytes, once recruited within the interstitial space, may represent a reservoir of cytokines and growth factors that can prime and maintain the activation of resident cells (40). Although monocytes may play a pivotal role in the pathogenesis of interstitial damage, the mechanisms underlying their influx into the interstitial space are still largely undefined. The local release of chemokines may represent the initial step in this event (19). In the growing chemokine family, MCP-1 represents the most specific and powerful chemotactic and activating factor for monocytes (41). We have recently demonstrated an increased MCP-1 expression at the tubular level in acute transplant rejection that was significantly correlated with monocyte infiltration (31). The infiltrating monocytes represent a major source of tissue factor, and thus powerful inducers of clotting cascade activation (42). The subsequent release of thrombin and its induction of MCP-1 production by PTC may stimulate a further influx and activation of circulating monocytes, closing a positive feedback loop and amplifying the phenomenon. Indeed, in human renal allograft rejection, activated interstitial macrophages are closely associated with fibrin deposits (43). Moreover, the hypothesis of a relevant role for thrombin in the setting of acute dysfunction of the renal allograft is further supported by the recent observation that proximal tubular cells express and produce anti-thrombin III, and the depletion of tubular anti-thrombin in the donor kidney is correlated with the degree of allograft function at 3 d after transplantation (44).
The mechanisms underlying thrombin-induced cell activation are still poorly understood. All of the known thrombin receptors, including PAR-1, belong to the G protein receptor superfamily (14,15,16). This class of receptors signal inside the cells through the interaction with one or more heterotrimeric G protein(s), leading to the activation of the phospholipase C-protein kinase C pathway on one side and to the modulation of adenylcyclase on the other (12,13,34). Although signaling receptors have always been rigidly divided in tyrosine kinase and G protein-coupled receptors, cross-talk between these two systems frequently occurs in rapidly induced cellular responses (34). In the past 10 yr, an increasing body of evidence suggested the activation of different tyrosine kinases in response to thrombin and the relevance of this phenomenon in thrombin-induced cell activation (45,46,47). Interestingly, both cellular responses described in the present study relied on the activation of the same signaling pathway involving protein-tyrosine phosphorylation. Indeed, thrombin stimulated the tyrosine phosphorylation of an array of cellular proteins in cultured hPTC. Although the precise identity of these phosphoproteins remains to be determined, the 60-kD protein most likely represents one of the cytoplasmic tyrosine kinases of the c-src family. In platelets, thrombin has been shown to activate different tyrosine kinases of this family, including c-src, fyn, yes, and lyn (45). These observations were, at least partially, reproduced in other cell types (45). However, recently it has been demonstrated that this serine protease can cross-activate the EGF receptor in several cell lines (36,47). PTC express in vivo and in vitro both c-src and EGF receptor at high levels and both of these kinases play a key role in different physiologic processes (37,48). In the present study, it was demonstrated that c-src, but not the EGF receptor, is strikingly autophosphorylated in response to thrombin in human PTC with a time course closely resembling the one observed for protein-tyrosine phosphorylation. This observation indirectly suggests the activation of c-src in response to thrombin and its potential role in protein tyrosine phosphorylation induced by this protease.
The mechanisms of c-src activation upon thrombin stimulation and its role in thrombin-induced cellular effects are still controversial. Indeed, both Chen et al. and Luttrell et al. demonstrated that the activation of this cytoplasmic tyrosine kinase is, at least partially, G protein-dependent (46,49). Moreover, Luttrell et al. reported that src activation may link G protein-coupled receptors to the ras-mitogen-activated protein kinase pathway, the main mitogenic pathway for thrombin (49). On the other hand, Kranenburg et al. demonstrated that src activation is G protein-independent and is not necessary for G protein-induced mitogen-activated protein kinase activation (50). In the present study, however, the mitogenic effect of thrombin seems to be completely dependent on src activation, because the specific src inhibitor PP1 significantly inhibited thrombin-induced DNA synthesis. On the other hand, we cannot definitively describe the role of src in thrombin-elicited MCP-1 expression because both PP1 and its inactive analogue PP3 equally inhibited this thrombin effect (data not shown).
In conclusion, PTC express in vivo and in vitro PAR-1 and represent a potential target for thrombin. This serine protease, interacting with PAR-1 and activating c-src, might represent a regenerative and proinflammatory stimulus for PTC in acute and chronic tubulointerstitial damage.
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Footnotes |
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References |
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-thrombin by surface-bound fibrin. J Lab
Clin Med 97:403
-411, 1980
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