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【摘要】 Proteinuria contributes to chronic kidney disease by stimulating renal tubular epithelial cells to produce cytokines such as monocyte chemoattractant protein-1 (MCP-1). The present study determined whether cellular overexpression of heme oxygenase-1 (HO-1) can influence albumin-stimulated MCP-1 production. In response to bovine serum albumin, NRK-52E cells constitutively overexpressing HO-1 (HO-1 OE cells) exhibit less induction of MCP-1 mRNA and less production of MCP-1 protein compared with similarly treated, control NRK-52E cells (CON cells). In wild-type NRK-52E cells, and under these conditions, we demonstrate that the induction of MCP-1 is critically dependent on intact NF- B binding sites in the MCP-1 promoter. In response to albumin, CON cells exhibit activation of NF- B, and this is reduced in HO-1 OE cells. Albumin also activates ERK1/2 and increases ERK activity, both of which are exaggerated in HO-1 OE cells. Studies with an inhibitor of MAPK/ERK kinase (U0126) demonstrate that the inhibitory effects of U0126 on MCP-1 production are attenuated in HO-1 OE cells. We conclude that HO-1 overexpression in the proximal tubule reduces MCP-1 production in response to albumin, and this occurs, at least in part, by inhibiting an ERK-dependent, NF- B-dependent pathway at a site that is distal to the activation of ERK. These findings suggest that the induction of HO-1 in the proximal tubule, as occurs in chronic kidney disease, may be a countervailing response that reduces albumin-stimulated production of cytokines such as MCP-1.
【关键词】 proteinuria extracellular signalregulated kinase / tubulointerstitial disease cytoprotection
EMPLOYED AS A DIAGNOSTIC INDEX for intrinsic kidney disease, proteinuria is also a prognostic index of considerable reliability: the severity of proteinuria commonly predicts the risk for progressive kidney disease and the rate at which kidney function is lost ( 4, 11, 15, 25, 32, 42, 58 ). This faithfulness of proteinuria as a prognostic index for kidney disease reflects, at least in part, pathogenetic pathways that are predictably entrained when abnormal amounts of protein leak across the glomerular filtration barrier and into the urinary space ( 4, 11, 15, 25, 32, 42, 58 ). Such appearance of albumin and other proteins in the urinary space leads to the incorporation of these proteins by the proximal tubule, and, once within proximal tubular epithelial cells, albumin and other proteins can instigate intracellular signaling and other pathways that culminate in the elaboration of inflammatory and fibrogenic cytokines ( 4, 11, 15, 25, 32, 42, 58 ). Intracellular uptake of filtered proteins by the proximal tubule, for example, can impose oxidative stress, recruit the MAPK, JAK/STAT, and other signaling pathways, and activate transcription factors such as NF- B and activator protein-1, with the attendant elicitation of damaging cytokines, chemokines, and other species ( 16, 26, 30, 42, 46, 47, 53, 54, 58 ).
One such chemokine is monocyte chemoattractant protein-1 (MCP-1), the latter present in increased amounts in the kidney and urine in diverse types of kidney disease ( 10, 13, 27, 40, 52, 57 ). MCP-1 is one of the most potent chemoattractants ever described for monocytes and is also an inducer of T cells and natural killer cells ( 5, 6, 8 ). Independent of its chemoattracting effects on leukocytes, MCP-1 can induce transforming growth factor- 1, a potent fibrogenic cytokine, in resident kidney cells ( 56 ), and MCP-1 is itself directly proinflammatory on the proximal tubule, in part, by activating NF- B ( 48, 49 ). Indeed, in assorted types of chronic kidney disease, the expression of MCP-1 in the kidney or the amounts of MCP-1 in the urine predict the severity of tubulointerstitial disease and the risk for loss of kidney function ( 10, 13, 27, 40, 52, 57 ).
Our laboratory?s prior studies noted that MCP-1 is significantly upregulated under unstressed and stressed states in the kidney in mutant mice unable to express heme oxygenase-1 (HO-1) ( 36, 39 ). HO-1 is the inducible isozyme of the major heme-degrading enzyme, HO. This isozyme is induced by diverse stressors and insults to the kidney and other tissues, and HO-1 often confers a protective response in injured tissue by interrupting inflammatory, ischemic, immune-mediated, and other pathways of injury (1-3, 18, 19, 31, 33, 45). In assorted types of human kidney disease and in diverse experimental models of kidney injury, HO-1 is upregulated in the diseased kidney, localizing mainly in the proximal and distal tubules (1-3, 14, 18, 19, 23, 28, 29, 31, 33, 37, 38, 45, 50). Such expression of HO-1 in the proximal tubule in proteinuric human kidney disease correlates with the severity of proteinuria, hematuria, and tubulointerstitial disease ( 28, 37, 38, 44 ). However, to date, the functional significance of such expression of HO-1 in the proximal tubule in proteinuric kidney disease is unknown: no data are available that address whether such upregulation of HO-1 by the proximal tubule is a response that vitiates, exacerbates, or bears little if any functional relevance to the injurious effects of proteinuria on the proximal tubule.
The present study was initiated in an attempt to address this issue. This study utilized an established model system whereby the damaging effects of protein, such as albumin, on the proximal tubule can be analyzed. For example, as previously shown by others and as recently demonstrated by us, the exposure of the proximal tubular epithelial cells to bovine serum albumin (BSA) leads to a robust tubular induction of MCP-1 ( 38, 46, 53, 54 ). To simulate the sustained upregulation of HO-1 in the proximal tubule, as observed in human kidney disease and in experimental models of kidney disease, we engineered an established proximal tubular epithelial cell line to constitutively overexpress HO-1 (HO-1 OE cells). We then examined the response of these HO-1 OE cells to albumin, using the attendant expression of MCP-1 as a representative, clinically relevant, inflammatory response.
METHODS
Cell culture. NRK-52E cells (ATCC, Manassas, VA) were cultured at 37°C in 95% air-5% CO 2 in DMEM (Invitrogen, Carlsbad, CA) containing 4.5 g/l glucose, 10% heat-inactivated FBS (Atlanta Biologicals, Lawrenceville, GA), and the following supplements (Mediatech, Herndon, VA): 20 mM HEPES, 1 mM sodium pyruvate, 100 U/ml of penicillin, and 100 µg/ml streptomycin. Similarly, HO-1 overexpressing NRK-52E cells (HO-1 OE cells) and corresponding control cells (CON cells) were cultured in the medium above with the addition of 100 µg/ml G418 (Mediatech). Cells were grown to confluence, after which the serum-containing medium was removed. Cells were then exposed to the BSA-containing medium, and cells or conditioned media were studied after the duration of exposure, as indicated for each protocol. For incubation with BSA (15 mg/ml, A8806, Sigma-Aldrich, St. Louis, MO) and other treatments, serum-free DMEM with 20 mM HEPES (test medium) was used. In some studies, the MAPK/ERK kinase (MEK) inhibitor U0126 ( 46 ) (Promega, Madison, WI) was employed, with an initial concentrated stock solution dissolved in DMSO, followed by dilution to final concentration with test medium.
HO-1 overexpression in NRK-52E cells. Plasmid pSFFV/HO-1 was constructed by insertion of the rat HO-1 cDNA downstream of the Friend spleen focus-forming virus 5' long-terminal repeat in the expression vector pSFFV/neo. NRK 52E cells were stably transfected with pSFFV/neo (CON cells) or pSFFV/HO-1 (HO-1 OE cells), by methods previously described in detail ( 21 ). Transfectants were selected in G418, and individual clones were selected by limited dilution. Western analysis was used to confirm HO-1 expression, and HO activity was determined in microsomal preparations from CON and HO-1 OE cells by determining the rate of generation of bilirubin, as previously described ( 22 ).
Northern analysis for MCP-1 mRNA expression. CON and HO-1 OE cells were washed with PBS, and total RNA was extracted using the TRIzol method (Invitrogen). Ten micrograms of total RNA were separated on an agarose gel and transferred to a nylon membrane. Membranes were hybridized overnight with a 32 P-labeled rat MCP-1 cDNA probe. Autoradiograms were evaluated for loading and transfer by assessing the density of the 18S rRNA on an ethidium bromide-stained membrane, as previously described ( 34 ).
ELISA for MCP-1 protein expression. Production of MCP-1 protein by CON and HO-1 OE cells in response to BSA was measured in conditioned media using an ELISA kit (BD Pharmingen, San Diego, CA), according to the manufacturer?s protocol. Results were standardized against total cellular protein determined using a Lowry-based assay kit (BioRad Laboratories, Hercules, CA).
Western analysis. Cells were washed with cold PBS and lysed in a buffer consisting of PBS, pH 7.4, 1% Igepal CA-630, 0.1% SDS, 0.5% deoxycholic acid, 1 mM sodium orthovanadate, protease inhibitor cocktail (Complete, Roche, Indianapolis, IN), and phosphatase inhibitor cocktail (Sigma-Aldrich). Lysates were collected by scraping, sonicated for 10 s, and centrifuged at 18,000 g to pellet the insoluble debris. Western analysis was performed on the resulting supernatants, as previously described in detail ( 17, 35 ). Briefly, 25 µg of protein were separated on 10% Tris·HCl gels (BioRad) and transferred to polyvinylidene difluoride membrane. Polyclonal antibodies, obtained from Cell Signaling Technology (Danvers, MA), were employed as primary antibodies for the analysis of the levels of p42/44 (no. 9102), phospho-p42/44 (no. 9101), JNK/SAPK (no. 9252), phospho-JNK/SAPK (no. 9251), p38 (no. 9212), phospho-p38 (no. 9211), STAT1 (no. 9172), phospho-STAT1 (no. 9171), phosho-STAT5 (no. 9351), and phospho-JAK2 (no. 3771). Additionally, a monoclonal antibody for STAT5 (no. 610192, BD Pharmingen) and polyclonal antibodies for HO-1 (SPA-895, StessGen, Ann Arbor, MI), JAK2 (no. 06-255, Upstate, Lake Placid, NY), and BSA (BioVision, Mountain View, CA) were also used. Horseradish peroxidase-conjugated goat anti-rabbit and goat anti-mouse polyclonal antibodies were used as secondary antibodies, as appropriate, and detection was achieved using a chemiluminescence method (enhanced chemiluminescence, Amersham Pharmacia Biotech, Piscataway, NJ).
ERK kinase assay. The ERK kinase assay was performed in CON and HO-1 OE cells exposed to BSA using a previously employed method ( 55 ). After incubation for the indicated times at 37°C, the cells were lysed, and postnuclear supernatants were incubated with 15 µl packed protein A-Sepharose beads and 2 µl anti-ERK2 antibody for 1 h at 4°C, and the immunoprecipitates were washed, followed by resuspension in kinase buffer (30 mM Tris·HCl, 0.1 mM EGTA, 10 mM magnesium acetate, 1 mM -glycerophosphate, pH 7.4, containing 1 mM DTT, and 5 mM n -octyl - D -glucopyranoside). ERK kinase reactions were initiated with 20 µl of kinase buffer containing 10 µM ATP, 5 µg myelin basic protein, and 10 µCi [ - 32 P]ATP. After 10 min at 30°C, the reactions were terminated with 8 µl 4 x SDS sample buffer and heated at 100°C for 5 min. The reaction products were separated by SDS-PAGE through a 15% polyacrylamide gel. The proteins were transferred to Immobilon P, and incorporation of radioactivity into myelin basic protein was assessed by autoradiography.
MCP-1 promotor analysis with mutant NF- B sites. The MCP-1 promoter and flanking region were isolated from rat genomic DNA as described ( 7 ). Primers were based on GenBank accession no. AF079313. The forward primer was 5'-TGTGAGAGCTGCTTGGCTGTAAC-3' (nucleotide 1220-1242), and the reverse primer was 5'-TCTGGCTTCAGTGAGAGTTGGC-3' (nucleotide 3635-3614). The genomic PCR product was sequenced and subcloned into pGL3-Basic, a promoter-less firefly luciferase reporter (Promega). A truncated MCP-1 construct with deletion of the two NF- B binding sites in the distal enhancer region (AF079313 base pairs 1293-1302 and 1319-1328) was produced by amplification of base pair 1335-3635 using the forward primer 5'-TTATCCTACTCTGCCTCTG-3' and the reverse primer described above. The PCR products were sequenced and subcloned into pGL3-Basic. NRK-52E cells were cotransfected with the MCP-1 promoter construct (350 ng) or the NF- B deletion MCP-1 promoter construct (350 ng), and phRG-Basic (45 ng) to control for transfection efficiency. Transfections were performed using FuGENE 6 Transfection Reagent (Roche Molecular Biochemical, Indianapolis, IN), according to the manufacturer?s instructions. Eighteen hours after transfection, BSA was added, 24 h after which cells were rinsed and lysed. Luciferase activity was assessed using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI).
Electrophoretic mobility shift assay. Activation of NF- B was assessed by electrophoretic mobility shift assay (EMSA), as previously employed, and currently modified by our laboratory ( 24, 36, 39 ). Following incubations, cells were washed, collected by centrifugation, and resuspended in 400-µl buffer consisting of 10 mM HEPES, pH 7.9, 10 mM KCl, 2 mM MgCl 2, and 0.1 mM EDTA, and containing the following protease inhibitors: 0.5 mM DTT, 10 µM pepstatin, 1 µM PMSF, 0.05 µg/ml leupeptin, and 10 µM aprotinin. Cells were lysed with the addition of 50 µl 10% Nonidet P-40, mixed by inversion, and incubated on ice for 5 min. Nuclei were resuspended in an extraction buffer (50 mM HEPES, pH 7.9, 300 mM NaCl, 50 mM KCl, 10% glycerol, and protease inhibitors as above) and mixed vigorously, after which the nuclei were pelleted. The extracted nuclear protein was quantified using a Bradford-based reagent (Bio-Rad) and used for EMSA. Five micrograms of nuclear extract were added to binding reactions containing the following reagents: 20 mM HEPES, pH 8.0, 100 mM NaCl, 1 mM MgCl 2, 0.05 mM EDTA, 0.5 mM DTT, 3% glycerol, 0.2 mg/ml poly(dI+dC), and 50,000 counts/min of [ - 32 P]ATP-labeled NF- B probe (catalog no. E3291, Promega) and incubated at room temperature for 30 min. The binding reactions were analyzed by electrophoresis on a 5% non-denaturing polyacrylamide gel and visualized by autoradiography. Supershift analysis was performed using a concentrated anti-p65 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, catalog no. sc-109): 1 µl of antibody was added to 5 µg of nuclear extract and allowed to incubate on ice for 15 min before the binding reaction.
Assessment of apoptosis. The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay was used to evaluate apoptosis in CON and HO-1 OE cells exposed to BSA for 18 h. The assay was performed employing the Apoptag Plus Peroxidase In Situ Apoptosis Detection Kit (Chemicon International, Temecula, CA), as previously described in detail ( 12, 17, 38 ).
Statistics. Results are expressed as means ± SE and are considered statistically significant for P < 0.05. For comparison between unpaired groups, the Student t -test or the Mann-Whitney test was employed as appropriate.
RESULTS
We first confirmed that the HO-1 OE cells exhibit increased expression of HO-1. As demonstrated in Fig. 1, expression of HO-1 protein along with HO activity were markedly increased in these cells compared with CON cells.
Fig. 1. Characterization of heme oxygenase-1 (HO-1) expression/HO activity in control (CON) and HO-1 overexpressing (HO-1 OE) cells. A : Western analysis for the expression of HO-1 in CON and HO-1 OE cells as increasing amounts of protein are loaded onto the gel. Equivalency of loading is confirmed by -actin. B : HO activity as determined by the rate of bilirubin generation by microsomes extracted from CON and HO-1 OE cells, n = 4 in each group. * P < 0.05 vs. CON.
In our studies, we established equivalency of loading of albumin by CON and HO-1 OE cells. As demonstrated in Fig. 2, comparable amounts of albumin were incorporated and detected in these cells, as assessed at three different time points.
Fig. 2. Assessment of uptake and content of albumin in CON and HO-1 OE cells exposed to bovine serum albumin (BSA). Western analysis for the expression of BSA was performed in cells exposed to BSA-free and BSA-containing media for 5, 15, and 30 min.
Albumin-driven induction of MCP-1 differed in CON and HO-1 OE cells ( Fig. 3 ). Albumin markedly stimulated MCP-1 mRNA expression in CON cells, whereas MCP-1 mRNA expression in HO-1 OE cells following exposure to albumin was reduced by 63%. This stimulation of intracellular MCP-1 mRNA in CON cells was accompanied by markedly increased amounts of extracellular MCP-1 protein; the latter was also significantly reduced in albumin-stimulated HO-1 OE cells ( Fig. 4 ). In these studies, we assessed cell viability by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay; apoptosis was not induced by albumin, either in the CON or HO-1 OE cells (data not shown).
Fig. 3. Northern analysis for the expression of monocyte chemoattractant protein-1 (MCP-1). CON and HO-1 OE cells were exposed to BSA-containing or BSA-free media for 8 h, after which total RNA was extracted and Northern analysis performed for the expression of MCP-1. The numbers below the Northern analysis are the mean standardized densitometric values for each group. Equivalency of loading is assessed by 18S rRNA.
Fig. 4. Production of MCP-1 protein by CON and HO-1 OE cells exposed to BSA-containing (+BSA) or BSA-free (-BSA) media. MCP-1 was measured in conditioned media after 18 h of exposure to either BSA-containing (+BSA) or BSA-free (-BSA) media. Data for CON and HO-1 OE cells are shown in open and solid bars, respectively; n = 4 in each group. * P < 0.05, BSA-treated CON cells vs. BSA-treated HO-1 OE cells.
We next sought to determine the mechanisms underlying the basis for such reduction of MCP-1 production. Depending on the experimental setting and stimulus, a number of transcription factors are incriminated in inducing expression of the MCP-1 gene. We first determined the extent to which NF- B activation determines MCP-1 expression in our model system. Studies were undertaken in which the wild-type MCP-1 promoter or the MCP-1 promoter with mutated NF- B binding sites were transfected into wild-type NRK-52E cells and exposed to albumin. As a positive control, we assessed the effect of the cytokine, TNF-, on MCP-1 expression. As demonstrated in Fig. 5, the upregulation of the wild-type MCP-1 promoter by TNF- was completely dependent on NF- B. Albumin also elicited upregulation of MCP-1 that was dominantly dependent on activation of NF- B: the MCP-1 promoter with mutated NF- B binding sites evinced little, if any, response to albumin.
Fig. 5. Activity of wild-type MCP-1 promoter and MCP-1 promoter with mutated NF- B binding sites (MCP-1 NF- B) following exposure to vehicle, TNF-, or BSA. Luciferase activity in wild-type NRK-52E cells, transfected with either the wild-type or the mutated MCP-1 promoter, was determined under conditions described in the text. n = 3 in each condition.
Since NF- B is critically required for the induction of MCP-1 in this model system, we examined, using EMSA, the binding capacity of NF- B in these cells following exposure to albumin. As shown in Fig. 6, albumin induced a time-dependent increase in NF- B binding in CON cells, most notably after 60 min of exposure, and such binding was reduced in HO-1 OE cells. To confirm the presence of this transcription factor in the shifted band, an anti-p65 antibody was added to the extracts before the binding reaction; in both CON and HO-1 OE cells, this band was supershifted in the presence of the anti-p65 antibody.
Fig. 6. Electrophoretic mobility shift assay (EMSA) for the expression of NF- B. EMSA was performed using extracts from CON and HO-1 OE cells treated with BSA-free media, and following exposure to BSA for 15, 30, and 60 min. Supershifting was conducted using a p65 antibody. ns, Nonspecific band.
Recent studies have incriminated the activation of ERK1/2 (p42/44) and the JAK/STAT pathways in driving cytokine expression in response to albumin ( 9, 41, 46 ). Our initial studies demonstrated that, in our model system, activation of ERK1/2 occurred in response to albumin, whereas activation of JAK/STAT did not (data not shown); albumin also failed to upregulate p38 and p46 (data not shown). We thus focused on ERK1/2 and determined whether a differential response with regards to ERK1/2 occurred in CON and HO-1 OE cells. As shown in Fig. 7, in CON cells, albumin provoked an early and transient induction of phospho-ERK1/2 (p-ERK1/2), the latter subsiding at subsequent time points. HO-1 OE cells demonstrated a higher baseline expression of p-ERK1/2, and this expression appeared more sustained following the exposure to albumin. Given these unexpected findings, we also undertook measurements of ERK activity so as to corroborate or refute these findings observed on Western analysis. The ERK activity assay, shown in Fig. 8, demonstrated greater augmentation in ERK activity at 5 min and greater persistence of activity at 1 h in HO-1 OE cells following exposure to albumin. Thus these data demonstrate that the reduced expression of MCP-1 in response to albumin in HO-1 OE cells cannot be ascribed to blunted activation of ERK1/2.
Fig. 7. Western analysis for the expression of ERK1/2. Top : expression of phospho-ERK1/2 (p-ERK1/2); bottom : total ERK1/2 in CON and HO-1 OE cells in the absence of BSA and following exposure to BSA for 5, 15, and 30 min.
Fig. 8. ERK activity assay following exposure to BSA. ERK activity was determined in CON and HO-1 OE cells under basal conditions and following exposure to BSA for 5 min or 1 h.
To determine the functional contribution of ERK1/2 in inducing MCP-1 in CON and HO-1 OE cells, we examined the effect of an inhibitor of MEK, U0126, on albumin-induced MCP-1 production in these cells. U0126 reduced MCP-1 production by 60% in CON cells ( Fig. 9 ). Production of MCP-1 in vehicle-treated, albumin-stimulated HO-1 OE cells was reduced compared with vehicle-treated, albumin-stimulated CON cells. The inhibitory effect of U0126 on MCP-1 production in albumin-stimulated HO-1 OE cells was blunted compared with its effect in albumin-stimulated CON cells: the mean reductions in MCP-1 achieved by U0126 in albumin-stimulated CON and HO-1 OE cells were 19.2 ± 0.4 vs. 10.3 ± 0.5 ng/mg protein, P < 0.001. That the inhibition of MCP-1 by U0126 is attenuated when HO-1 is overexpressed, in conjunction with findings regarding ERK expression and activity ( Figs. 7 and 8 ), in aggregate, suggest that the inhibitory effects of HO-1 overexpression reside distal to ERK activation in the ERK-dependent pathway for MCP-1 production.
Fig. 9. Effect of U0126 or vehicle (Veh) on MCP-1 production by CON and HO-1 OE cells exposed to BSA-containing media. MCP-1 concentration was measured in the conditioned media; n = 4 in each group. * P < 0.05 vs. vehicle-treated, BSA-exposed CON cells; P < 0.05 vs. vehicle-treated, BSA-exposed HO-1 OE cells.
DISCUSSION
A vigorous chemoattractant for monocytes/macrophages, MCP-1 is a critical participant in the pathogenesis of assorted nephropathies, vasculopathies, and inflammatory conditions ( 5, 6, 8, 49 ). This broad involvement of MCP-1 in kidney and other diseases reflects numerous other effects of MCP-1, in addition to its capacity to recruit monocytes/macrophages, and these include the following: recruitment of memory T cells and natural killer cells; stimulation of cytokine production by inflammatory and other cells; induction of tissue factor and other procoagulant effects; activation and migration of endothelial cells; and proliferation and migration of smooth muscle cells ( 5, 6, 8, 49 ). MCP-1 has direct proinflammatory effects on resident renal cells, inducing, for example, NF- B and other proinflammatory pathways in proximal tubular epithelial cells ( 48, 49 ). MCP-1 participates in a number of reverberating loops that could sustain tissue injury. For example, MCP-1 is induced by oxidant stress, the MAPK system, and by NF- B, and, in turn, in certain cells, MCP-1 itself induces oxidative stress, the MAPK system, and NF- B ( 5, 6, 8, 49 ).
A substantial body of literature attests to the role of increased expression of MCP-1 in the kidney as a contributor to chronic kidney disease ( 10, 13, 27, 40, 52, 57 ). For example, in assorted nephropathies, upregulation of MCP-1 occurs in the kidney, appearing mainly in the renal tubular epithelium and interstitium, and urinary excretion of MCP-1 is also increased in chronic kidney disease. Renal expression of MCP-1 correlates with renal histological injury and recruitment of macrophages, while urinary excretion of MCP-1 correlates with albuminuria, numbers of infiltrating macrophages, renal histological injury, and renal survival ( 10, 13, 27, 40, 52, 57 ). In experimental kidney injury, maneuvers that interrupt receptor-mediated actions of MCP-1 markedly reduce renal injury. For example, the administration of a mutant MCP-1 gene ( 7ND ), which abrogates the effect of MCP-1 on its receptor ( CCR2 ), reduces macrophage recruitment and interstitial fibrosis following urinary tract obstruction ( 51 ); CCR2 -/- mice exhibit reduced fibrosis following urinary tract obstruction ( 20 ), and the administration of 7ND significantly reduces the expression of MCP-1, macrophage infiltration, tubular injury, and collagen and transforming growth factor- 1 expression in mice subjected to the protein-overload model of tubulointerstitial damage ( 43 ).
In view of these considerations, we thus employed MCP-1 as a representative cytokine in examining the effect of cellular overexpression of HO-1 on the proinflammatory influence of albumin on the renal proximal tubule. Our model system recapitulated previously described findings demonstrating marked expression of MCP-1 mRNA in proximal tubular epithelial cells exposed to albumin, and we also confirmed that such expression is associated with increased production of MCP-1 protein in the extracellular fluid. HO-1 OE cells exhibited reduced expression of MCP-1 mRNA, along with decreased amounts of MCP-1 protein, following exposure to albumin. These findings thus demonstrate that overexpression of HO-1 in the proximal tubule can interrupt the inflammatory response engendered in albumin-exposed renal proximal tubules. However, as shown by our laboratory?s prior findings, upregulation of HO-1, assessed either by HO activity, HO-1 mRNA, or HO-1 protein, does not occur in renal proximal epithelial cells exposed to albumin ( 38 ). The observed induction of HO-1 in the proximal tubule in chronic kidney disease thus originates from some other stimulus-i.e., cytokines or other proinflammatory species, oxidized lipid, transition metal-bearing proteins, glycated or other chemically altered albumin, among other possibilities-rather than albumin per se acting on the proximal tubule. Nonetheless, the induction of HO-1 in the proximal tubule can mitigate the inflammatory effects of albumin on this nephron segment.
To examine the basis for this attenuation of MCP-1 production in HO-1 OE cells, we considered whether reduced activation of NF- B occurred. NF- B is a transcription factor that instigates inflammatory cascades by stimulating a number of cytokines and chemokines. In our model system, we first examined the extent to which NF- B is involved in inducing MCP-1 expression in wild-type NRK-52E cells exposed to albumin. Using a MCP-1 promoter with mutated NF- B binding sites, we observed that the induction of MCP-1 by albumin was markedly reduced in such mutants, thereby indicating a critical and dominant role for NF- B in effecting albumin-driven MCP-1 expression in our model. Subsequent studies demonstrated that NF- B was activated in a time-dependent fashion in CON cells exposed to albumin, and that such activation was significantly attenuated in HO-1 OE cells. From these studies, we conclude that the reduced MCP-1 expression may result from a diminution in the extent to which NF- B is activated by albumin in HO-1 OE cells.
Recent studies have called attention to activation of ERK1/2 in inducing NF- B and, in turn, MCP-1 expression in renal tubular epithelial cells exposed to albumin ( 46 ). Other signaling pathways that are incriminated in mediating the intracellular effects of albumin involve the JAK/STAT pathway ( 30 ). In our model system, we saw no evidence of activation of JAK/STAT, whereas upregulation of ERK1/2 occurred in CON cells exposed to albumin; we thus explored this pathway. Phosphorylation of ERK1/2 in HO-1 OE cells exposed to albumin was surprising: basal phosphorylation of ERK1/2 was increased in HO-1 OE cells, and such phosphorylation persisted for a longer period in HO-1 OE cells upon exposure to albumin. To examine this further, we undertook measurements of ERK activity, and indeed our studies are the first to demonstrate that, in this model system, increased phosphorylation of ERK1/2, as assessed by Western analysis, is attended by increased ERK activity. Assessment of ERK activity in HO-1 OE cells corroborated the findings on Western analysis, that is, heightened activity along with greater persistence of such activity in HO-1 OE cells after exposure to albumin. Thus the suppressed production of MCP-1 in HO-1 OE cells exposed to albumin cannot be ascribed to lesser induction of ERK1/2 compared with that observed in albumin-exposed CON cells.
Studies were then undertaken using an inhibitor of MEK. The elaboration of MCP-1 in albumin-treated CON cells was reduced by 60% following exposure to the MEK inhibitor, findings that corroborate the previously described dependency of albumin-stimulated MCP-1 expression on p-ERK1/2 ( 46 ). In HO-1 OE cells, the effect of the inhibitor of MEK on the production of MCP-1 was blunted. That the inhibition of MCP-1 production by U0126 is attenuated when HO-1 is overexpressed, in conjunction with findings regarding ERK expression and activity, in aggregate, suggests that the inhibitory effects of HO-1 overexpression reside distal to ERK activation in the ERK-dependent pathway for MCP-1 production. Based on prior studies ( 46 ) and our present findings, we suggest that the inhibitory effects of HO-1 on albumin-induced MCP-1 production reside, at least in part, at the site in the following pathogenetic cascade marked by the asterisk: albumin p-ERK * NF- B MCP-1. It is also possible, however, that the inhibitory effects of overexpression of HO-1 may involve ERK-independent mechanisms.
Activation of ERK1/2 is a recognized cellular response to stress. The increased upregulation of p-ERK1/2 in the vehicle-treated HO-1 OE cells under basal conditions, and the more sustained expression of p-ERK1/2 in response to albumin, were quite unanticipated; indeed, this was the reason for pursuing additional analysis, as provided by the ERK activity assay. While ERK1/2 is involved in upregulating HO-1 in response to certain stimuli ( 3, 45 ), we are unaware of any other study that has addressed the effect of altered expression of HO-1 on p-ERK1/2 expression. The basis for heightened expression of p-ERK1/2 under unstressed and stressed conditions in HO-1 OE cells merits additional exploration, but is beyond the scope of the present study. Whatever the underlying mechanism, these data underscore the capacity of overexpression of HO-1 to dampen the stimulatory effect of ERK1/2 on MCP-1 expression, even when activation of ERK1/2 is increased, as occurs in HO-1 OE cells.
The present findings are complementary to our laboratory?s prior studies demonstrating that there is heightened activation of MCP-1 in HO-1-deficient states ( 36, 39 ). For example, studies utilizing HO-1 -/- mice demonstrate that an exaggerated expression of MCP-1 occurs in response to heme proteins, ischemia-reperfusion injury, and aging, and in these instances, this exaggerated expression of MCP-1 is attended by heightened activation of NF- B. The present findings, demonstrating that overexpression of HO-1 inhibits albumin-driven activation of NF- B and induction of MCP-1, provide additional evidence that one of the mechanisms whereby HO-1 inhibits inflammatory responses may involve the suppression of NF- B and NF- B-dependent genes such as MCP-1. However, we wish to emphasize that this mechanism would not be restricted to MCP-1, and other NF- B-dependent genes may be similarly suppressed. An assessment of such genes is beyond the scope of the present study.
In conclusion, we demonstrate that constitutive overexpression of HO-1 by proximal tubular epithelial cells reduces production of MCP-1 by these cells in response to albumin. We conclude that this suppressive effect involves mechanisms that are distal to the activation of ERK1/2 and are associated with a suppressive effect on NF- B activation. Additionally, as proteins other than albumin, such as IgG, can also activate NF- B (and presumably NF- B-dependent genes such as MCP-1) in proximal tubules ( 26 ), these suppressive effects of HO-1, conceivably, may also interrupt the inflammatory transformation of the proximal tubule caused by these other proteins. Based on the present findings, we suggest that the sustained induction of HO-1 in the proximal tubule, as observed in human chronic kidney disease, represents a countervailing response that mitigates the inflammatory effects of albumin on the proximal tubule.
GRANTS
These studies were funded by the following National Institutes of Health Grants: DK47060 (K. A. Nath), DK16105 (J. P. Grande), and AI62261 and AI40384 (A. D. Badley).
ACKNOWLEDGMENTS
We gratefully acknowledge the secretarial expertise of Sharon Heppelmann in the preparation of this work.
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作者单位：1 Division of Nephrology and Hypertension, 2 Department of Laboratory Medicine and Pathology, 3 Department of Pediatrics and Adolescent Medicine, 4 Department of Immunology, 5 Division of Infectious Diseases, 6 Program in Translational Immunovirology and Biodefense, Mayo Clinic College of Medicine,