Albumin-bound fatty acids induce mitochondrial oxidant stress and impair antioxidant responses in proximal tubular cells
2006; Elsevier BV; Volume: 70; Issue: 4 Linguagem: Inglês
10.1038/sj.ki.5001629
ISSN1523-1755
AutoresDavid Ishola, Jan A. Post, Mirjan M. van Timmeren, Stephan J. L. Bakker, R. Goldschmeding, H A Koomans, Branko Braam, Jaap A. Joles,
Tópico(s)Effects of Radiation Exposure
ResumoAlbumin induces oxidative stress and cytokine production in proximal tubular cells (PTECs). Albumin-bound fatty acids (FAs) enhance tubulopathic effects of albumin in vivo. We proposed that FA aggravation of albumin-induced oxidative stress in PTECs might be involved. We hypothesized that mitochondria could be a source of such stress. Using a fluorescent probe, we compared reactive oxygen species (ROS) production after exposure of PTECs to bovine serum albumin (BSA) alone or loaded with oleic acid (OA-BSA) (3–30 g/l for 2 h). There was no difference in cellular albumin uptake, but OA-BSA dose-dependently induced more ROS than BSA alone (P<0.001). OA-BSA-induced ROS was significantly alleviated by mitochondrial inhibition, but not by inhibitors of nicotinamide adenine dinucleotide phosphate hydrogenase (NADPH) oxidase, xanthine oxidase, or nitric oxide synthase. Gene expression analysis showed that neither the NADPH oxidase component p22phox nor xanthine oxidase was induced by BSA or OA-BSA. OA-BSA, in contrast to BSA, failed to induce mitochondrial manganese superoxide dismutase 2 (SOD2) expression. OA-BSA showed a greater capacity than BSA to downregulate heme oxygenase-1 mRNA expression and accentuate inflammatory cytokine mRNA and protein. Supplementation of SOD activity with EUK-8 reduced ROS, and interleukin-6 protein expression was suppressed by both mitochondrial inhibition and SOD augmentation. Thus, in PTECs, FAs accentuate albumin-induced oxidative stress and inflammatory cytokine expression via increased mitochondrial ROS, while frustrating protective antioxidant responses. Albumin induces oxidative stress and cytokine production in proximal tubular cells (PTECs). Albumin-bound fatty acids (FAs) enhance tubulopathic effects of albumin in vivo. We proposed that FA aggravation of albumin-induced oxidative stress in PTECs might be involved. We hypothesized that mitochondria could be a source of such stress. Using a fluorescent probe, we compared reactive oxygen species (ROS) production after exposure of PTECs to bovine serum albumin (BSA) alone or loaded with oleic acid (OA-BSA) (3–30 g/l for 2 h). There was no difference in cellular albumin uptake, but OA-BSA dose-dependently induced more ROS than BSA alone (P<0.001). OA-BSA-induced ROS was significantly alleviated by mitochondrial inhibition, but not by inhibitors of nicotinamide adenine dinucleotide phosphate hydrogenase (NADPH) oxidase, xanthine oxidase, or nitric oxide synthase. Gene expression analysis showed that neither the NADPH oxidase component p22phox nor xanthine oxidase was induced by BSA or OA-BSA. OA-BSA, in contrast to BSA, failed to induce mitochondrial manganese superoxide dismutase 2 (SOD2) expression. OA-BSA showed a greater capacity than BSA to downregulate heme oxygenase-1 mRNA expression and accentuate inflammatory cytokine mRNA and protein. Supplementation of SOD activity with EUK-8 reduced ROS, and interleukin-6 protein expression was suppressed by both mitochondrial inhibition and SOD augmentation. Thus, in PTECs, FAs accentuate albumin-induced oxidative stress and inflammatory cytokine expression via increased mitochondrial ROS, while frustrating protective antioxidant responses. Proximal tubular epithelial cells (PTECs) treated with excess albumin produce numerous pro-oxidant and proinflammatory substances.1.Eddy A.A. Proteinuria and interstitial injury.Nephrol Dial Transplant. 2004; 19: 277-281Crossref PubMed Scopus (135) Google Scholar, 2.Imai E. Nakajima H. Kaimori J.Y. Albumin turns on a vicious spiral of oxidative stress in renal proximal tubules.Kidney Int. 2004; 66: 2085-2087Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 3.Zoja C. Benigni A. Remuzzi G. Cellular responses to protein overload: key event in renal disease progression.Curr Opin Nephrol Hypertens. 2004; 13: 31-37Crossref PubMed Scopus (115) Google Scholar, 4.Zandi-Nejad K. Eddy A.A. Glassock R.J. et al.Why is proteinuria an ominous biomarker of progressive kidney disease?.Kidney Int. 2004; 66: S76-S89Abstract Full Text Full Text PDF Scopus (131) Google Scholar, 5.Zoja C. Morigi M. Remuzzi G. Proteinuria and phenotypic change of proximal tubular cells.J Am Soc Nephrol. 2003; 14: S36-S41Crossref PubMed Google Scholar, 6.Brunskill N.J. Albumin signals the coming of age of proteinuric nephropathy.J Am Soc Nephrol. 2004; 15: 504-505Crossref PubMed Scopus (34) Google Scholar There is evidence that such albumin-stimulated PTEC activity contributes to tubulointerstitial inflammation in vivo. For example, in human nephrotic kidneys, interleukin 8 (IL-8) mRNA and protein localized mainly to tubular epithelial cells, and the distribution of infiltrating interstitial leukocytes approximated with IL-8-expressing tubules.7.Tang S. Leung J.C. Abe K. et al.Albumin stimulates interleukin-8 expression in proximal tubular epithelial cells in vitro and in vivo.J Clin Invest. 2003; 111: 515-527Crossref PubMed Scopus (217) Google Scholar Similarly, in proteinuric rats, osteopontin was upregulated in proximal tubules with adjacent interstitial inflammatory infiltrates.8.Abbate M. Zoja C. Corna D. et al.In progressive nephropathies, overload of tubular cells with filtered proteins translates glomerular permeability dysfunction into cellular signals of interstitial inflammation.J Am Soc Nephrol. 1998; 9: 1213-1224PubMed Google Scholar These findings indicate that PTECs are not merely structural elements but are active players in pathophysiologic processes. Oxidative stress plays a key role in these events. In PTECs exposed to albumin, increased intracellular production of reactive oxygen species (ROS) was required for activation of nuclear factor kappaB and expression of IL-87.Tang S. Leung J.C. Abe K. et al.Albumin stimulates interleukin-8 expression in proximal tubular epithelial cells in vitro and in vivo.J Clin Invest. 2003; 111: 515-527Crossref PubMed Scopus (217) Google Scholar and macrophage chemoattractant protein 1.9.Morigi M. Macconi D. Zoja C. et al.Protein overload-induced NF-kappaB activation in proximal tubular cells requires H(2)O(2) through a PKC-dependent pathway.J Am Soc Nephrol. 2002; 13: 1179-1189PubMed Google Scholar Some effects of albumin on PTECs are not owing to the molecule itself, but rather to fatty acids (FAs) bound to it. Excess PTEC fibronectin production was induced by oleic acid-complexed albumin but not by pure albumin.10.Arici M. Brown J. Williams M. et al.Fatty acids carried on albumin modulate proximal tubular cell fibronectin production: a role for protein kinase C.Nephrol Dial Transplant. 2002; 17: 1751-1757Crossref PubMed Scopus (50) Google Scholar FA presented to PTECs via albumin is efficiently taken up, leading to such effects as altered cellular growth,11.Thomas M.E. Schreiner G.F. Contribution of proteinuria to progressive renal injury: consequences of tubular uptake of fatty acid bearing albumin.Am J Nephrol. 1993; 13: 385-398Crossref PubMed Scopus (103) Google Scholar disturbed metabolism,11.Thomas M.E. Schreiner G.F. Contribution of proteinuria to progressive renal injury: consequences of tubular uptake of fatty acid bearing albumin.Am J Nephrol. 1993; 13: 385-398Crossref PubMed Scopus (103) Google Scholar,12.Thomas M.E. Morrison A.R. Schreiner G.F. Metabolic effects of fatty acid-bearing albumin on a proximal tubule cell line.Am J Physiol. 1995; 268: F1177-F1184PubMed Google Scholar increased apoptosis,13.Arici M. Chana R. Lewington A. et al.Stimulation of proximal tubular cell apoptosis by albumin-bound fatty acids mediated by peroxisome proliferator activated receptor-gamma.J Am Soc Nephrol. 2003; 14: 17-27Crossref PubMed Scopus (116) Google Scholar and the release of lipid metabolites with immunologic14.Kees-Folts D. Sadow J.L. Schreiner G.F. Tubular catabolism of albumin is associated with the release of an inflammatory lipid.Kidney Int. 1994; 45: 1697-1709Abstract Full Text PDF PubMed Scopus (138) Google Scholar and biochemical15.Lindner A. Hinds T.R. Joly A. et al.Neutral lipid from proteinuric rat urine is a novel inhibitor of the red blood cell calcium pump.J Am Soc Nephrol. 1999; 10: 1170-1178PubMed Google Scholar properties that may be pathologically important. The concept of albumin-bound FA toxicity is supported by in vivo evidence. In rodents, we16.van Timmeren M.M. Bakker S.J. Stegeman C.A. et al.Addition of oleic acid to delipidated bovine serum albumin aggravates renal damage in experimental protein-overload nephrosis.Nephrol Dial Transplant. 2005; 20: 2349-2357Crossref PubMed Scopus (28) Google Scholar and others17.Thomas M.E. Harris K.P. Walls J. et al.Fatty acids exacerbate tubulointerstitial injury in protein-overload proteinuria.Am J Physiol Renal Physiol. 2002; 283: F640-F647Crossref PubMed Scopus (112) Google Scholar,18.Kamijo A. Kimura K. Sugaya T. et al.Urinary free fatty acids bound to albumin aggravate tubulointerstitial damage.Kidney Int. 2002; 62: 1628-1637Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar have found that albumin-bound FAs aggravate albumin-induced nephropathic effects, including renal cortical apoptosis,17.Thomas M.E. Harris K.P. Walls J. et al.Fatty acids exacerbate tubulointerstitial injury in protein-overload proteinuria.Am J Physiol Renal Physiol. 2002; 283: F640-F647Crossref PubMed Scopus (112) Google Scholar tubulointerstitial inflammation,16.van Timmeren M.M. Bakker S.J. Stegeman C.A. et al.Addition of oleic acid to delipidated bovine serum albumin aggravates renal damage in experimental protein-overload nephrosis.Nephrol Dial Transplant. 2005; 20: 2349-2357Crossref PubMed Scopus (28) Google Scholar, 17.Thomas M.E. Harris K.P. Walls J. et al.Fatty acids exacerbate tubulointerstitial injury in protein-overload proteinuria.Am J Physiol Renal Physiol. 2002; 283: F640-F647Crossref PubMed Scopus (112) Google Scholar, 18.Kamijo A. Kimura K. Sugaya T. et al.Urinary free fatty acids bound to albumin aggravate tubulointerstitial damage.Kidney Int. 2002; 62: 1628-1637Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar and glomerular injury.16.van Timmeren M.M. Bakker S.J. Stegeman C.A. et al.Addition of oleic acid to delipidated bovine serum albumin aggravates renal damage in experimental protein-overload nephrosis.Nephrol Dial Transplant. 2005; 20: 2349-2357Crossref PubMed Scopus (28) Google Scholar Moreover, nephrotic patients with the relatively benign minimal change disease (MCD) have more than threefold lower urinary albumin FA content than individuals with non-MCD varieties of nephrosis that tend to run a more aggressive course.19.Ghiggeri G.M. Ginevri F. Candiano G. et al.Characterization of cationic albumin in minimal change nephropathy.Kidney Int. 1987; 32: 547-553Abstract Full Text PDF PubMed Scopus (63) Google Scholar These observations together indicate that albumin-bound FA exerts extra deleterious effects on PTECs, on top of those of albumin alone. However, information about these processes remains limited, and in particular it is not known whether albumin-bound FA can increase oxidative stress. We employed an in vitro protein overload model of cultured PTECs to explore two questions relating to the idea that albumin-bound FAs exaggerate oxidant effects of albumin on PTECs. First, we postulated that FAs increase the ROS-inducing capacity of albumin. We tested this hypothesis by comparing ROS production after exposure to bovine serum albumin (BSA) either alone or loaded with oleic acid (OA-BSA). We found that cells exposed to OA-BSA produced up to 2.5-fold more ROS than those treated with BSA alone. Second, we explored the source of OA-BSA-induced ROS in these cells. Evidence from another cell type suggests that FAs stimulate ROS production from the mitochondria.20.Koshkin V. Wang X. Scherer P.E. et al.Mitochondrial functional state in clonal pancreatic beta-cells exposed to free fatty acids.J Biol Chem. 2003; 278: 19709-19715Crossref PubMed Scopus (99) Google Scholar In vascular endothelial cells exposed to lipids, there is an important mitochondrial contribution to ROS.21.Zmijewski J.W. Moellering D.R. Le Goffe C. et al.Oxidized LDL induces mitochondrially associated reactive oxygen/nitrogen species formation in endothelial cells.Am J Physiol Heart Circ Physiol. 2005; 289: H852-H861Crossref PubMed Scopus (118) Google Scholar Thus, we hypothesized that mitochondria may also be a major source in PTECs upon treatment with albumin-bound FAs. Our findings supported this hypothesis. We went further to assess the responses of cellular antioxidant defence mechanisms to the FA-induced disturbance of redox balance, and to explore whether the resultant excess oxidant stress is of functional consequence in terms of cellular proinflammatory transcriptional and translational activity. The present study reveals a failure in the mitochondrial antioxidant response as a mechanism by which FAs bound to albumin can induce oxidative and inflammatory stresses in PTECs. Electro-immunodiffusion assays (Table 1) showed similar albumin uptake by HK-2 cells after BSA or OA-BSA treatment for 2 h, in a dose-dependent manner. Similar uptake was also visualized by immunohistochemistry (not shown). The results are comparable with previous findings in similar cells.22.Brunskill N.J. Stuart J. Tobin A.B. et al.Receptor-mediated endocytosis of albumin by kidney proximal tubule cells is regulated by phosphatidylinositide 3-kinase.J Clin Invest. 1998; 101: 2140-2150Crossref PubMed Scopus (76) Google Scholar, 23.Choi J.S. Kim K.R. Ahn D.W. et al.Cadmium inhibits albumin endocytosis in opossum kidney epithelial cells.Toxicol Appl Pharmacol. 1999; 161: 146-152Crossref PubMed Scopus (19) Google Scholar, 24.Takaya K. Koya D. Isono M. et al.Involvement of ERK pathway in albumin-induced MCP-1 expression in mouse proximal tubular cells.Am J Physiol Renal Physiol. 2003; 284: F1037-F1045Crossref PubMed Scopus (102) Google ScholarTable 1Albumin uptake after exposure to albumin with or without bound OATreatment: albumin concentration (g/l)Cellular albumin content after 2 h (μg BSA/mg total cellular protein)Control00BSA312.3 (2.7)*P<0.001 vs control,1532.4 (2.3)*P<0.001 vs control,P<0.05, ## P<0.01 vs 3g/l.3045.2 (2.4)*P<0.001 vs control,P<0.05, ## P<0.01 vs 3g/l.OA-BSA313.6 (1.8)*P<0.001 vs control,1524.8 (2.5)*P<0.001 vs control,P<0.05, ## P<0.01 vs 3g/l.3042.0 (6.9)*P<0.001 vs control,P<0.05, ## P<0.01 vs 3g/l.BSA, bovine serum albumin; OA, oleic acid.Mean (s.e.m.) of three independent experiments (μg albumin/mg total cellular protein).There were no significant treatment or time differences.* P<0.001 vs control,# P<0.05, ## P<0.01 vs 3 g/l. Open table in a new tab BSA, bovine serum albumin; OA, oleic acid. Mean (s.e.m.) of three independent experiments (μg albumin/mg total cellular protein). There were no significant treatment or time differences. Incubation of HK-2 cells with OA-BSA for 2 h resulted in markedly and significantly greater ROS production than BSA alone, at every concentration tested. OA-BSA induced ROS dose-dependently (up to fourfold increase from control level), whereas BSA induced moderate (up to 1.5-fold) ROS increase, which attained significance only at the highest concentration (Figure 1a). Cell viability was not impaired by 2-h BSA or OA-BSA exposure (3-[4,5-dimethylthiazol]-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) release assays; Figure 1b). The property of albumin as an excellent carrier for many hydrophobic substances (besides FAs) raises the concern that it could acquire unknown contaminants in the course of commercial preparation and purification processes. To confirm that the effects we observed were not influenced by such contamination, we repeated some experiments using a different formulation of low endotoxin (0–2.0 EU/mg), low FA (0–0.2 mg/g) BSA (SERVA Electrophoresis GmbH, Heidelberg, Germany). As shown in Table 2, the pattern of ROS production induced by the SERVA product was similar to that of the Sigma product described in the above paragraph.Table 2ROS measurement after treatment of HK-2 cells with a different albumin formulationAlbumin content (g/l)BSAOA-BSA 31.5 (0.2)2.4 (0.2)**P<0.001 vs control.#P<0.01, BSA vs OA-BSA.151.9 (0.2)**P<0.001 vs control.3.3 (0.4)**P<0.001 vs control.P<0.05,302.3 (0.4)*P<0.01,3.6 (0.5)**P<0.001 vs control.#P<0.01, BSA vs OA-BSA.0 (control)1.0 (0.03)BSA, bovine serum albumin; HK-2, human kidney-2; OA, oleic acid; ROS, reactive oxygen species.Mean (s.e.m.) of three independent experiments.* P<0.01,** P<0.001 vs control.# P<0.05,## P<0.01, BSA vs OA-BSA. Open table in a new tab BSA, bovine serum albumin; HK-2, human kidney-2; OA, oleic acid; ROS, reactive oxygen species. Mean (s.e.m.) of three independent experiments. In further experiments, cells were exposed to OA-BSA (30 g/l) for 2 h. In intervention studies to probe possible sources of ROS (Figure 2), OA-BSA-induced increase was significantly attenuated by the mitochondrial respiratory chain blockers rotenone (ROT) (complex I inhibitor) and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (respiratory uncoupling agent), but not by 2-thenoyltrifuoroacetone (complex II inhibitor). The specific nicotinamide adenine dinucleotide phosphate hydrogenase (NADPH) oxidase inhibitor apocynin (APO) had no significant effect. Nω-nitro-L-arginine methyl ester and allopurinol also had no effect, excluding uncoupled nitric oxide synthase or xanthine oxidase as ROS sources. To probe whether BSA or OA-BSA induce pro-oxidant transcriptional changes that could influence cellular ROS sources, we performed reverse transcription-polymerase chain reaction (PCR) studies on the NADPH components p22phox (cytosolic component) and rac1 and rac2 (membrane-bound components). We also tested the xanthine oxidase gene. Although rac1 and rac2 were both induced by either treatment, the vital enzyme component p22phox was not. There was no change in xanthine oxidase expression. Illustrative PCR bands and semiquantitative densitometry of gene expression are shown (Figure 3a). We assessed the transcriptional response of major cellular antioxidant enzymes (Figure 3b). In cells treated with BSA alone, there was notable upregulation of manganese superoxide dismutase (SOD2) despite the comparatively mild ROS increase. In marked contrast to BSA, OA-BSA failed to induce SOD2. Expression of other redox enzyme genes, copper–zinc superoxide dismutase (SOD1) and glutathione peroxidase, was not significantly regulated by either treatment. To explore the functional implication of the antioxidant gene expression pattern noted above, we tested whether supplementation of SOD might alleviate ROS induced by OA-BSA. We used the saleno-manganese SOD mimetic agent EUK-8, which indeed markedly alleviated ROS (Figure 4). For an insight into whether bound FAs might also influence albumin-induced inflammatory effects, we studied the gene expression of cytokines IL-6 and IL-8. Both treatments similarly increased cytokine expression. However, OA-BSA had a greater tendency to suppress the expression of heme oxygenase-1 (HO-1) (Figure 3c), a molecule with important renal antioxidant and anti-inflammatory effects.25.Chung S.W. Perrella M.A. Role of HO-1 in renoprotection: location, location, location.Kidney Int. 2004; 65: 1968-1969Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar,26.Yang Y. Ohta K. Shimizu M. et al.Selective protection of renal tubular epithelial cells by heme oxygenase (HO)-1 during stress-induced injury.Kidney Int. 2003; 64: 1302-1309Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar To assess whether the observed effects in gene expression are reflected at protein level, we measured IL-6 in cells treated with BSA or OA-BSA. OA-BSA induced significantly greater cellular expression of IL-6 protein than BSA (Table 3), despite their similar effect at the level of gene expression. We then tested whether antioxidant intervention would ameliorate the effect of OA-BSA. The saleno-manganese complex and SOD mimetic agent EUK-8, and the mitochondrial blockers rotenone and CCCP, all reduced IL-6 protein expression to baseline (control) levels. The intervention agents acting alone did not significantly alter baseline levels (not shown).Table 3IL-6 protein expression in HK-2 cells after treatment with BSA or OA-BSAIL-6 (ng/mg total cellular protein)Control0.96 (0.1)BSA1.65 (0.2)*P<0.05 vs control.OA-BSA2.38 (0.4)**P=0.005 vs control.OA-BSA+EUK-81.16 (0.2)OA-BSA+rotenone0.90 (0.4)OA-BSA+CCCP1.10 (0.4)CCCP, carbonyl cyanide 3-chlorophenylhydrazone; BSA, bovine serum albumin; IL, interleukin; OA, oleic acid.Mean (s.e.m.) of up to eight independent experiments.* P<0.05 vs control.** P=0.005 vs control. Open table in a new tab CCCP, carbonyl cyanide 3-chlorophenylhydrazone; BSA, bovine serum albumin; IL, interleukin; OA, oleic acid. Mean (s.e.m.) of up to eight independent experiments. The present study provides observations suggesting that albumin-bound FAs augment the oxidative capacity of albumin on PTECs via excess mitochondrial ROS, in the face of an impaired mitochondrial ROS-scavenging response. A complex of albumin with OA (OA-BSA) significantly and dose-dependently increased intracellular ROS much more than albumin alone (BSA). OA-BSA stimulation experiments in the presence of various inhibitors implicated mitochondria and not NADPH oxidase, xanthine oxidase, or nitric oxide synthase as the main source of ROS. A manganese SOD mimetic agent, EUK-8, alleviated the OA-BSA-induced ROS increase. Not only did OA-BSA markedly increase mitochondrial ROS, but also adversely affected gene expression of the key mitochondrial antioxidant system, the manganese-containing SOD2. Whereas BSA induced protective SOD2 gene expression, this response was completely absent with OA-BSA. Parallel to increased oxidative stress, OA-BSA aggravated the protein expression of the inflammatory cytokine IL-6 beyond the level of BSA alone. This effect was redressed by both SOD supplementation and mitochondrial inhibition, suggesting that the dual-mechanism oxidant effect of albumin-bound FAs promotes proinflammatory PTEC activity. There is as yet no broad consensus regarding the specific sites of ROS production on the mitochondrial electron transport chain. Complex I is thought to be important, but complexes II and III are also known to be capable of ROS production, and there are variations between tissues.27.Brand M.D. Affourtit C. Esteves T.C. et al.Mitochondrial superoxide: production, biological effects, and activation of uncoupling proteins.Free Radic Biol Med. 2004; 37: 755-767Crossref PubMed Scopus (774) Google Scholar,28.Degli Esposti M. Measuring mitochondrial reactive oxygen species.Methods. 2002; 26: 335-340Crossref PubMed Scopus (135) Google Scholar We probed possible ROS contribution from complexes I and II, and from the respiratory coupling step. Only the complex II inhibitor 2-thenoyltrifuoroacetone failed to substantially reduce OA-BSA-induced ROS, suggesting complex I as the key location of ROS production within PTEC mitochondria. In experiments probing other possible ROS sources apart from mitochondria, APO, a specific inhibitor of NADPH oxidase complex assembly,29.Stolk J. Hiltermann T.J. Dijkman J.H. et al.Characteristics of the inhibition of NADPH oxidase activation in neutrophils by apocynin, a methoxy-substituted catechol.Am J Respir Cell Mol Biol. 1994; 11: 95-102Crossref PubMed Scopus (557) Google Scholar failed to attenuate ROS. Moreover, gene expression studies indicated that the crucial NADPH oxidase component p22phox was not induced by OA-BSA. p22phox plays an essential role in NADPH oxidase function by facilitating transfer of the cytosolic elements to the membrane, enabling catalytic activation.30.Babior B.M. NADPH oxidase.Curr Opin Immunol. 2004; 16: 42-47Crossref PubMed Scopus (615) Google Scholar Interestingly, we found that PTECs robustly express both of the low molecular weight G-protein NADPH oxidase complex components rac1 and rac2. Both molecules tended to be upregulated after OA-BSA treatment, but apparently without functional significance in the absence of p22phox induction. In further inhibition studies of cellular ROS-producing enzymes, neither nitric oxide synthase inhibition with Nω-nitro-L-arginine methyl ester nor blockade of xanthine oxidase with allopurinol yielded significant reductions in ROS. Marked reduction of OA-BSA-induced ROS was observed with EUK-8, a manganese–salen compound that possesses efficient catalytic SOD antioxidant activity.31.Doctrow S.R. Huffman K. Marcus C.B. et al.Salen-manganese complexes as catalytic scavengers of hydrogen peroxide and cytoprotective agents: structure–activity relationship studies.J Med Chem. 2002; 45: 4549-4558Crossref PubMed Scopus (226) Google Scholar,32.Kachadourian R. Menzeleev R. Agha B. et al.High-performance liquid chromatography with spectrophotometric and electrochemical detection of a series of manganese(III) cationic porphyrins.J Chromatogr B Analyt Technol Biomed Life Sci. 2002; 767: 61-67Crossref PubMed Scopus (24) Google Scholar Crucially, EUK-8 also has potent catalase activity, precluding the risk of SOD-driven H2O2 accumulation. Thus, an SOD/catalase enzymatic axis (driving the reaction sequence O2•− → H2O2 → H2O+O2) appears to be the pivotal redox pathway in this OA-BSA model of PTEC protein overload (Figure 5). Gene expression of other cellular antioxidant enzymes, SOD1 and glutathione peroxidase, was not regulated by either BSA or OA-BSA. Mitochondria produce ROS during the course of normal cellular metabolism, and the local scavenging action of mitochondrial SOD (SOD2) is a major protector of the organelle, and indeed the whole cell, from auto-oxidation (via ROS diffusion into the cytoplasm).33.Melov S. Mitochondrial oxidative stress. Physiologic consequences and potential for a role in aging.Ann NY Acad Sci. 2000; 908: 219-225Crossref PubMed Scopus (140) Google Scholar Excess mitochondrial ROS can have devastating effects, as illustrated by severe lifespan reductions in SOD2 null mice.27.Brand M.D. Affourtit C. Esteves T.C. et al.Mitochondrial superoxide: production, biological effects, and activation of uncoupling proteins.Free Radic Biol Med. 2004; 37: 755-767Crossref PubMed Scopus (774) Google Scholar,33.Melov S. Mitochondrial oxidative stress. Physiologic consequences and potential for a role in aging.Ann NY Acad Sci. 2000; 908: 219-225Crossref PubMed Scopus (140) Google Scholar This contrasts with remarkably milder phenotypes in mice lacking other SOD isoforms. In the present study, OA-BSA increased mitochondrial ROS and suppressed the protective gene induction of SOD2 seen with BSA alone. To further assess the cellular defensive response to increased oxidative stress, we examined the gene expression of HO-1, a widely distributed enzyme that plays an important role in maintaining cellular redox balance. HO-1 induction is seen as a defensive response to injurious stimuli25.Chung S.W. Perrella M.A. Role of HO-1 in renoprotection: location, location, location.Kidney Int. 2004; 65: 1968-1969Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar and is specifically protective of PTECs against various types of oxidant agents.26.Yang Y. Ohta K. Shimizu M. et al.Selective protection of renal tubular epithelial cells by heme oxygenase (HO)-1 during stress-induced injury.Kidney Int. 2003; 64: 1302-1309Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar Contrary to our expectation of increased expression as an attempt to counter oxidant stress, we surprisingly observed that HO-1 was downregulated in PTECs exposed to BSA, an effect that was enhanced by OA-BSA. Thus it appears that, apart from aggravating albumin-induced oxidative stress, albumin-borne FAs also further depress the HO-1 antioxidant protection. However, in contrast to our finding of suppressed HO-1 expression in protein-overloaded human PTECs, it was recently reported that no changes occurred in HO-1 expression in kidneys of rats that received a brief course of albumin injections (7 days), or in cultured rat PTECs that were exposed to albumin.34.Pedraza-Chaverri J. Murali N.S. Croatt A.J. et al.Proteinuria as a determinant of renal expression of heme oxygenase-1: studies in models of glomerular and tubular proteinuria in the rat.Am J Physiol Renal Physiol. 2006; 290: F196-F204Crossref PubMed Scopus (23) Google Scholar This contrast may be a further example of differences in renal transcriptional HO-1 reaction between humans and other species. Marked differences are known between human and murine renal HO-1 gene responses to various stimuli, including heat shock, hypoxia, and hyperosmolarity.35.Sikorski E.M. Hock T. Hill-Kapturczak N. et al.The story so far: molecular regulation of the heme oxygenase-1 gene in renal injury.Am J Physiol Renal Physiol. 2004; 286: F425-F441Crossref PubMed Scopus (202) Google Scholar OA-BSA significantly escalated cellular IL-6 protein mass beyond the level induced by BSA alone. As IL-6 gene expression was increased to a similar degree by the two treatments, these data suggest that the critical effect of albumin-bound FAs in the promotion of PTEC proinflammatory capacity may be at the level of gene translation rather than transcription. OA-BSA-induced IL-6 was suppressed by CCCP and ROT, confirming the key role of mitochondria-derived oxidant stress, as well as by EUK-8, emphasizing the importance of SOD. FAs may play a crucial role in the translational control of IL-6 expression, and albumin-bound lipids could escalate proinflammatory tendencies within PTECs by driving the gene translation of IL-6 and possibly other cytokines, via ROS-dependent mechanisms. Such effects may at least partly explain
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