*Department of Internal Medicine and Therapeutics, Osaka University Graduate School of Medicine, Osaka, Center of Tsukuba Advanced Research Alliance, Institute of Applied Biochemistry, University of Tsukuba, Ibaraki, and Graduate School of Life Science, Kobe Women’s University, Kobe, Japan.
Correspondence to: Dr. Masaru Takenaka, Department of Internal Medicine and Therapeutics (Box A8), Osaka University Graduate School of Medicine, Osaka, 565-0871, Japan. Phone: +81787314416; Fax: +81787325161; E-mail: masaru{at}suma.kobe-wu.ac.jp
ABSTRACT. Renal proximal tubular cells activated by reabsorptionof protein are thought to play significant roles in the progressionof kidney diseases. It was hypothesized that the signal transducerand activator of transcription (STAT) proteins may be activatedby proteinuria in proximal tubular cells. To test this hypothesis,murine proximal tubular cells were treated with albumin (30mg/ml medium) for various lengths of time. The results showedthat albumin could activate Stat1 and Stat5 within 15 min inproximal tubular cells. The activation of STATs was mediatedmostly by Jak2 and required no protein synthesis. In addition,activation of Stat1 occurred even after neutralization of IFN-.The activation of STATs was inhibited by N-acetyl-L-cysteine,a precursor of glutathione and a reactive oxygen species (ROS)scavenger, and fluorescence-activated cell sorter analysis showedupregulation of intracellular ROS after albumin overloading,suggesting that albumin per se could generate ROS in proximaltubular cells. The activation of STATs occurred by way of theROS generating system, and especially through the membrane-boundNADPH oxidase system. Reduced activities of glutathione peroxidaseand catalase could also be responsible for the accumulationof intracellular ROS. Hence, not only the ROS generating system,but also the ROS scavenging system may contribute to the inductionof ROS by albumin. These findings support the hypothesis thatproximal tubular cells are activated and generate ROS by reabsorptionof abundant urinary proteins filtered through the glomerularcapillaries, and as a consequence, various IFN-–inducibleproteins are synthesized through IFN-–independent activationof STAT signaling.
Janus kinase (JAK) and the signal transducer and activator oftranscription (STAT) proteins were originally defined largelyin the context of interferon (IFN) signaling (1–3). TheJAK/STAT signaling pathway was also first defined in the samecontext. A total of seven different STAT family members (Stat1,Stat2, Stat3, Stat4, Stat5a, Stat5b, and Stat6) have now beenidentified in mammalian cells (4), and a large number of cytokines,growth factors, reactive oxygen species (ROS), and others arenow known to trigger STAT activation (5–7). The fundamentalroles of STATs in highly diverse biologic processes have beenidentified by using STAT knockout mice and/or by tissue-specificdeletions (8,9). These processes include innate and adaptiveimmune function, embryonic development, cell differentiation,cell proliferation, survival, and apoptosis (10–14), andthe STAT family has become a therapeutic target in human cancer(15).
Renal proximal tubular cells play central roles in various kidneydiseases by producing chemokines such as regulated upon activation,normal T cell expressed and secreted (RANTES) (16), and thisis more pronounced in the presence of IFN- than IL-1 or TNF-(17). Moreover, the level of proteinuria, which is independentof mean arterial BP, is reportedly one of the best predictorsfor disease progression toward end-stage renal failure (18,19).Microalbuminuria is known as an important early sign of diabeticnephropathy (20,21) and of progressive loss of renal functionin the nondiabetic population (22). Recent studies have shownthat proximal tubular cells are activated by reabsorption ofabundant urinary proteins filtered through the glomerular capillaries,producing various chemokines that lead to kidney disease progression(23). Identification of the signal transduction pathway in activatedproximal tubular cells is, however, still incomplete.
To study changes in in vivo gene expression in proximal tubularcells caused by proteinuria, we constructed an expression profileof proximal tubular cells isolated from an albumin-overloadedproteinuria mouse model by use of the body map procedure (24–29).These data showed that the expression patterns in proximal tubularcells were changed dramatically by proteinuria. In view of therenal damage caused by proteinuria, it is of considerable interestthat several immunity-related genes, including interferon regulatoryfactor–1, major histocompatibility complex (MHC) classI, MHC class II, and monocyte chemoattractant protein–1(MCP-1), were found to be upregulated (26). These proteinuria-inducedgenes are likely to participate in kidney disease progression,and it is noteworthy that almost all of them are also inducedby IFN- (30).
Although IFN- is essentially a cytokine with direct antiviralactivity, its properties also include regulation of the immuneresponse, antigen presentation of phagocytes through the MHCclass I and II pathways, and orchestration of leukocyte-endotheliuminteractions through an intermediary in the JAK/STAT signalingpathway (30). Hence, we were interested in the relationshipbetween the STAT family and proteinuria-initiated gene expressionin proximal tubular cells.
In previous in vitro experiments, protein-overloaded proximaltubular cells were found to activate transcription of a numberof genes encoding inflammatory molecules (31,32). However, thesignaling pathways involved have not been analyzed in detail.Because it is likely that many inflammatory cytokines have variouseffects on the later stages of these pathways, we have focusedmainly on the early stage to avoid such complications.
In this report, we present our results showing that albuminper se can generate ROS in proximal tubular cells, resultingin the activation of the STAT signaling pathway. We suggestthat the membrane-bound NADPH oxidase system is important asan ROS generating system. Albumin-induced activation of Stat1and Stat5 in murine proximal tubular cells (mProx24) cells wasfound to be mediated mostly by Jak2, without the need for proteinsynthesis.
Antibodies
The Stat1, Stat3, Stat5, phospho-Stat1 (Y701), phospho-Stat3(Y705) and phospho-Stat5 (Y694) antibodies were purchased fromCell Signaling (Beverly, MA). The Jak1, Jak2, and phospho-Jak2(Y1007/Y1008) antibodies were obtained from Upstate Biotechnology(Lake Placid, NY), and the phospho-Jak1 (Y1022/Y1023) antibodyfrom Affinity BioReagents (Golden, CO). The Tyk2 and phospho-Tyk2(Y1054/Y1055) antibodies were obtained from Santa Cruz Biotechnology(Santa Cruz, CA), and anti-murine IFN- antibody from Peprotech(Princeton, NJ).
Chemicals
Cycloheximide (CHX) and AG490 were purchased from Calbiochem(La Jolla, CA). Protein A agarose beads, nylon membranes (hybond-P)for Western blot test and, Hyperfilm ECL films were obtainedfrom Amersham Pharmacia Biotech (Buckinghamshire, UK) and nylonmembranes (Biodyne B) for electrophoretic mobility shift assaysfrom Pall (East Hills, NY). The Pierce Supersignal substratechemiluminescence detection kit and the BCA Protein Assay Reagentkit were purchased from Pierce Biotechnology (Rockford, IL).5- (and 6-) Chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate(CM-H2DCFDA) was purchased from Molecular Probes (Eugene, OR),FCS and L-glutamine from InVitrogen (Carlsbad, CA), and CompleteMini from Boehringer Mannheim (Mannheim, Germany). CompleteMini tablets inhibit a broad spectrum of serine, cysteine, andmetalloproteases as well as calpains. BSA (Fraction V, IgG free,low endotoxin), apo-transferrin (apoTf), N-acetyl-L-cysteine(NAC), diphenylene iodonium chloride (DPI), and all other chemicalswere purchased from Sigma Chemical (St. Louis, MO).
Cells and Cell Culture
The murine proximal tubular cells (mProx24 cells derivative;patent WO9927363, JP, US, EU) (33,34) were cultured in DMEM/F-12medium containing 10% FCS. The mProx24 cells were examined beforehandto determine whether they could produce a valid activation ofSTATs in response to IFN- and IL-6. We used FITC-labeled BSAto confirm their ability to generate albumin endocytosis (datanot shown). In addition, we verified the upregulation of somegenes induced by IFN- when albumin was added to the medium (datanot shown).
Confluent mProx24 cells were rinsed three times with serum-freemedium and starved in the serum-free medium for 24 h to eliminatethe effect of FCS. mProx24 cells were then treated with albumin(30 mg/ml medium). Albumin mediums were filtered with a 0.22-µmfilter unit (Millipore, Carrigtwohill, County Cork, Ireland)and polymyxin B was added suppress the influence of lipopolysaccharide.When drugs or antibodies were added, mProx24 cells were pretreatedwith the drugs or antibodies for 1 h before exposure to albumin.The concentration (0.05 ng/ml) of anti–IFN- antibody wasdetermined according to the manufacturer’s instructions.This concentration resulted in a valid inhibition of Stat1-activationby IFN- (data not shown).
Trypan Blue Uptake Test
To evaluate cell viability by trypan blue dye exclusion, mProx24cells were treated with the antioxidants, NAC (20 mM), rotenone(10 µM), or DPI (10 µM) 1 h before and during the30-min incubation with albumin (30 mg/ml). At the end of thestimulation, the cells were rinsed three times with ice-coldPBS, detached with trypsin, and resuspended in medium diluted1:20 with trypan blue solution. Live cells and stained deadcells were then counted by a hemocytometer.
Immunoprecipitation, SDS-PAGE, and Western Blot Analysis
At the end of the treatment with albumin, the cells were rinsedthree times with ice-cold PBS with 1 mM Na3VO4, solubilizedin ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl,1% NP40, 0.25% sodium deoxycholate, 1 mM EDTA, 1 mM PMSF, 1mM Na3VO4, 1 mM NaF, Complete Mini), and rotated for 1 h at4°C. The total cell lysates were centrifuged at 14,000 xg for 10 min at 4°C. They were then incubated with the relevantantibody on ice for 2 h, and the antibody complexes were collectedon protein A agarose beads during a 1-h incubation at 4°C.The beads were washed three times with the lysis buffer andboiled for 20 min in SDS-PAGE sample buffer. A total of 50 µgof each sample was separated by 7% SDS-PAGE and transferredto a nylon membrane (Hybond-P), where it was blocked by a 30-minincubation at room temperature in TPBS (20 mM Tris-HCl, pH 7.6,137 mM NaCl, 0.05% Tween-20) plus 5% BSA and incubated overnightat 4°C with the relevant primary antibody. Subsequently,the membranes were washed three times for 5 min each with TPBSand incubated for 30 min with HRP-conjugated goat anti-rabbitIgG. After extensive washing, the bound antibody was visualizedon Hyperfilm ECL film. Membranes were then incubated at 55°Cfor 30 min in stripping buffer (100 mM 2-ME, 2% SDS, 62.5 mMTris-HCl, pH 6.8) to prepare them for a second round of immunoblotting.
Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assay (EMSA)
After the indicated treatment, the cells were rinsed three timeswith ice-cold PBS containing 1 mM Na3VO4, scraped from the dishand pelleted at 14,000 x g for 1 min at 4°C. Cells werethen resuspended in the same buffer and pelleted as describedabove. The resultant pellets were resuspended in 400 µlof ice-cold buffer A (10 mM HEPES-KOH pH 7.8, 10 mM KCl, 0.1mM EDTA pH 8.0, 0.1% NP-40, 1 mM DTT, 0.5 mM PMSF, 1 mM Na3VO4,Complete Mini). The lysate was vortexed vigorously for 20 s,and the nuclei were pelleted at 14,000 x g for 1 min at 4°C,followed by resuspension of the nuclear pellets in 100 µlof buffer C (50 mM HEPES-KOH pH 7.8, 420 mM KCl, 0.1 mM EDTApH 8.0, 5 mM MgCl2, 20% glycerol, 1 mM DTT, 0.5 mM PMSF, 1 mMNa3VO4, Complete Mini) and rotation for 1 h at 4°C. Theextracted proteins were separated from the residual nuclei at14,000 x g for 15 min at 4°C, and the supernatant fractionswere used as nuclear extracts. EMSA was performed with the Panomics’EMSA Kit (Panomics, Redwood City, CA) according to the manufacturer’sinstructions.
Assessment of Intracellular ROS
Intracellular ROS generation was assessed in mProx24 cells bymeans of an oxidant-sensitive dye, CM-H2DCFDA. Suspensions ofthe cells (106 cells) were incubated with 5 µM CM-H2DCFDAfor 15 min at 37°C in the serum-free medium. After centrifugationand washing to remove unincorporated probe, cells were treatedwith several concentrations of albumin medium for 15 min at37°C and placed on ice. Mean fluorescence intensity of DCFin the cells was measured by a flow cytometer (FACSCalibur;Becton Dickinson Biosciences, Franklin Lakes, NJ).
Measurement of SOD, Glutathione Peroxidase, and Catalase Activities, and the Total Reduced Form of Glutathione
The SOD activity was measured with a Superoxide Dismutase AssayKit (Trevigen, Gaithersburg, MD). The cellular glutathione peroxidase(GPx) activity was determined with a Bioxytech GPx-340 (OxisResearch,Portland, OR). The catalase activity was measured with an AmplexRed Catalase Assay Kit (Molecular Probes, Eugene, OR). The totalreduced form of glutathione (GSH) was determined with a BioxytechGSH-400 (OxisResearch). Each of the kits was used accordingto the manufacturer’s instructions.
Activation of STATs in mProx24 Cells by Albumin
We first investigated whether members of the STAT family wereactivated by albumin. mProx24 cells were treated with albumin(30 mg/ml medium) for various lengths of time. Western blotanalyses showed that Stat1 and Stat5 were rapidly activatedwithin 15 min of exposure to albumin, with Stat5 in particularshowing early and obvious activation. Stat3, however, demonstratedno detectable activation after 15 min (Figure 1A). To confirmStat1 and Stat5 activation, nuclear extracts were prepared fromthe albumin-treated cells and analyzed by EMSA. This approachalso showed rapid activation of Stat1 and Stat5 within 15 minof exposure to albumin. These results were finally confirmedby competition and supershift analyses (Figure 1B). To verifythe later activation of Stat3 after albumin overloading, mProx24cells were treated with albumin for longer lengths of time.Western blot analyses showed that Stat3 activation occurredonly after a 4-h exposure to albumin (Figure 1C).
Figure 1. Western blot analysis and electrophoretic mobility shift assays (EMSA) of activated Stat1 and Stat5 in mouse renal proximal tubular cells (mProx24 cells) treated with albumin. mProx24 cells were treated with albumin for various time periods. (A) Western blot analyses detected phosphorylated Stat1 and Stat5 15 min after addition of albumin, indicating that albumin could induce signal transducer and activator of transcription (STAT) signaling pathways. Stat3 showed no significant changes. Western blot analyses of Stat1, Stat3, and Stat5 are shown as controls. (B) EMSA confirmed rapid activation of Stat1 and Stat5 within 15 min of exposure to albumin. These results were confirmed by competition (Comp) and supershift (SS) analyses by using control samples. Band shifts are indicated by arrows. (C) Western blot detection of phosphorylated Stat3 4 h after addition of albumin. Western blot analysis of Stat3 are shown as controls. Three independent experiments were performed, and results are shown for one representative experiment.
Activation of Albumin-Induced STATs through Jak2
The rapid phosphorylation of Stat1 and Stat5 indicated thatalbumin is, like cytokines, a direct stimulant of the JAK family.We examined the time courses of the phosphorylation of Jak1,Jak2, and Tyk2 in albumin-overloaded mouse mProx24 cells. Westernblot analyses showed that rapid activation of Jak2 took placewithin 15 min of exposure to albumin, compared with no obviousactivation of Jak1 or Tyk2 (Figure 2A). To confirm that theactivation of Stat1 and Stat5 was under the control of Jak2,we tested the effect of AG490 (20 µM), a specific inhibitorof Jak2. The results in Figure 2B indicate that AG490 inhibitedthe activation of Jak2 and prevented the activation of Stat1and Stat5, compared with the control. Given the strong evidencethat JAK kinases are in vivo STAT kinases (35), our resultssuggest that the albumin-induced activation of Stat1 and Stat5in mProx24 cells was mediated mostly by Jak2.
Figure 2. Activation of Jak2 in mouse renal proximal tubular (mProx24) cells treated with albumin mediates the activation of Stat1 and Stat5. (A) Phosphorylated Jak2 was detected 15 min after addition of albumin, demonstrating that albumin induced JAK signaling pathways. Jak1 and Tyk2 showed no significant changes. Western blot results for Jak2, Jak1, and Tyk2 are shown as controls. (B) mProx24 cells were transferred to a medium containing albumin (30 mg/ml medium) in the presence of AG490 (20 µM). AG490 inhibited the activation of Jak2 (only control and 30-min data shown) and prevented the activation of Stat1 and Stat5 compared with the control, indicating that albumin-induced activation of Stat1 and Stat5 was mediated mostly by Jak2. Western blot analyses for Stat1 and Stat5 are shown as controls. Three independent experiments were performed, and results are shown for one representative experiment.
Moreover, to investigate whether protein synthesis was involvedin the activation of Stat1 and Stat5, we tested the effect ofCHX (2 µg/ml), a protein synthesis inhibitor. The resultsin Figure 3A indicate that CHX did not affect the albumin-inducedTyr-phosphorylation of STATs within 15 min, thus demonstratingthat the albumin-induced activation of Stat1 and Stat5 occurredwithout protein synthesis.
Figure 3. Effects of cycloheximide or anti–IFN- antibody on albumin-induced Stat1 and Stat5 activation. Mouse renal proximal tubular (mProx24) cells were transferred to a medium containing albumin (30 mg/ml medium) in the presence of cycloheximide (2 µg/ml) or anti–IFN- antibody (0.05 ng/ml). (A) Cycloheximide could not inhibit albumin-induced activation of Stat1 and Stat5. (B) Anti–IFN- antibody could not inhibit albumin-induced activation of Stat1. Three independent experiments were performed, and results are shown for one representative experiment.
Although the JAK/STAT signaling pathway appeared to be activatedby albumin per se, some cytokines and/or growth factors mayhave been involved in this phenomenon. Hence, the effect ofan inhibitory antibody on the JAK/STAT signaling pathway wasexamined. It is well known that Jak2 fulfills an essential functionin response to IFN- (36). We therefore used an anti–IFN-antibody (0.05 ng/ml) as a representative cytokine-neutralizationantibody to check whether albumin could induce IFN- secretionfrom mProx24 cells. mProx24 cells were pretreated with anti-IFN-antibody for 1 h before exposure to albumin. The results indicatedthat anti–IFN- antibody did not prevent the albumin-inducedTyr-phosphorylation of Stat1, suggesting that the albumin-inducedactivation of Stat1 was not a direct effect of IFN- (Figure 3B).
Activation of STATs in mProx24 Cells Was Not Specific to Albumin
To investigate whether the activation of the STATs was specificto albumin, mProx24 cells were treated with apoTf (30 mg/mlmedium) for 15 min. ApoTf is one of the major components ofplasma proteins and consists of transferrin that is not saturatedwith iron. Western blot analyses showed that there was alsorapid activation of Stat1 and Stat5 within 15 min of exposureto apoTf (Figure 4), indicating that the activation of the STATswas not albumin specific.
Figure 4. Western blot analyses of activated Stat1 and Stat5 in mouse renal proximal tubular (mProx24) cells treated with addition of apoTf. Phosphorylated Stat1 and Stat5 were detected 15 min after addition of apoTf, indicating that apoTf induced signal transducer and activator of transcription (STAT) signaling pathways. Western blot analyses for Stat1 and Stat5 are shown as controls. Three independent experiments were performed, and results are shown for one representative experiment.
Assessment of ROS Generation by Albumin and apoTf
Recently, it was reported that albumin upregulated ROS generationin proximal tubular cells (37,38). Therefore, to directly assesswhether albumin could induce oxidative stress in mProx24 cells,we used the CM-H2DCFDA method and fluorescence-activated cellsorter (FACS) analysis. After CM-H2DCFDA incubation, mProx24cells were treated with albumin for 15 min. Accumulation ofDCF in mProx24 cells was measured with a flow cytometer by monitoringthe fluorescence at 526 nm (39). ROS generation was found tobe upregulated compared with the control within 15 min aftereither albumin or apoTf had been added to the medium (Figure 5).Dose-dependent upregulation of intracellular ROS generationwas observed, as previously reported (37). The result was notchanged by addition of DMSO, which was used as the solvent forAG490, rotenone, and DPI (data not shown).
Figure 5. Assessment of reactive oxygen species (ROS) by fluorescence-activated cell sorter analysis. After CM-H2DCFDA incubation, mouse renal proximal tubular (mProx24) cells were treated with albumin for 15 min. Accumulation of DCF was measured with a flow cytometer by monitoring fluorescence at 526 nm. The increase was assumed to be proportional to the concentration of superoxide anions and hydrogen peroxide in the mProx24 cells. (A) Results are shown for one representative experiment. ROS generation was observed even in the control mProx24 cells (black line). The ROS generation was upregulated within 15 min after albumin (yellow line; 3 mg/ml, blue line; 30 mg/ml) overloading, but NAC (green line), a precursor of glutathione (GSH) and a ROS scavenger, blocked ROS generation. (B) Intracellular ROS formation was expressed as a ratio of the mean fluorescence intensity of control cells incubated in an albumin-free medium. Results are the means ± SD of triplicate experiments. $P < 0.01 versus control cells. *P < 0.005 versus control cells. **P < 0.001 versus control cells.
To inhibit ROS generation by albumin, we checked the effectof NAC (20 mM), a precursor of GSH and a ROS scavenger, rotenone(10 µM), an inhibitor of complex I of the mitochondrialrespiratory chain, and DPI (10 µM), an inhibitor of membraneNADPH oxidase. All three were able to block ROS generation byalbumin (Figure 5). These results supported our finding thatalbumin could induce oxidative stress in mProx24 cells. ROSgeneration was observed even in the control mProx24 cells afterstarvation, compared with the findings with antioxidants.
Activation of Albumin-Induced STATs through ROS
FACS analyses showed that albumin-induced ROS generation couldbe blocked by the three antioxidants. To determine whether theactivation of Stat1 and Stat5 occurred by way of oxidative stress,we first tested the effect of NAC (20 mM). The results presentedin Figure 6 indicate that NAC prevented albumin-induced Tyr-phosphorylationof Stat1 and Stat5 within 15 min (Figure 6, A and B). Moreover,even the activation of Stat1 and Stat5 in the control cellswas suppressed by pretreatment with NAC (Figure 6A). This suggeststhat the activation of Stat1 and Stat5 could take place throughthe upregulation of ROS generation.
Figure 6. Effects of N-acetyl-L-cysteine (NAC) on albumin-induced Stat1 and Stat5 activation. Mouse renal proximal tubular (mProx24) cells were transferred to a medium containing albumin (30 mg/ml medium) in the presence of NAC (20 mM). (A) Western blot analyses showed that activation of Stat1 and Stat5 was inhibited by NAC, indicating that the activation of Stat1 and Stat5 is mediated by reactive oxygen species (ROS). Western blot analyses for Stat1 and Stat5 are shown as controls. (B) Electrophoretic mobility shift assay (EMSA) also confirmed the inhibition of Stat1 and Stat5 by NAC. Three independent experiments were performed, and results are shown for one representative experiment.
The mitochondrial respiratory chain is one of the major intracellularsources of ROS and indispensable to cell function. We investigatedthe involvement of mitochondria in the albumin-induced ROS withan inhibitor of the mitochondrial respiratory chain, rotenone(10 µM). The results showed that rotenone prevented thealbumin-induced Tyr-phosphorylation of Stat1 and Stat5 within15 min, compared with the positive control (Figure 7, A and B).Pretreatment with rotenone also suppressed the activationof Stat1 and Stat5 in the control cells (Figure 7B). This indicatedthat inhibition of the mitochondrial respiratory chain couldsuppress ROS generation by albumin or by starvation.
Figure 7. Effects of rotenone or diphenylene iodonium chloride (DPI) on albumin-induced Stat1 and Stat5 activation. Mouse renal proximal tubular (mProx24) cells were transferred to a medium containing albumin (30 mg/ml medium) in the presence of rotenone (10 µM) or DPI (10 µM). Western blot analyses showed that activation of Stat1 and Stat5 was inhibited by either rotenone (B) or DPI (C), compared with the positive control (A), indicating that the activation of Stat1 and Stat5 was mediated by reactive oxygen species (ROS). Western blot analyses for Stat1 and Stat5 are shown as controls. Three independent experiments were performed, and results are shown for one representative experiment.
Intracellular generation of ROS in response to ligands is oftenmediated by the activity of membrane-bound NADPH oxidase (40,41).DPI (10 µM) also prevented the albumin-induced Tyr-phosphorylationof Stat1 and Stat5 (Figure 7, A and C), but did not seem tosuppress the activation of Stat1 and Stat5 in the control cells(Figure 7C). This suggests that ROS generated by albumin aremainly derived from the membrane-bound NADPH oxidase system.
Because pretreatment with NAC and rotenone even suppressed theactivation of Stat1 and Stat5 in the control cells, we evaluatedthe cell viability in the presence of NAC, rotenone and DPIby trypan blue dye exclusion. The antioxidants did not havemuch effect on cell viability, at least during the 30-min incubationwith albumin (30 mg/ml) (Table 1).
Table 1. Viable cell count by trypan blue dye exclusion
Measurement of SOD, GPx, and Catalase Activities
The membrane-bound NADPH oxidase system produces superoxideanions in cells, and intracellular SOD is the main enzyme thatmetabolizes these anions. Because interference with the SODactivity could induce accumulation of superoxide anions, wemeasured the SOD activity after mProx24 cells had been treatedwith albumin for 10 min. No marked change was observed (Figure 8A).These results indicate that altered SOD activity was notresponsible for the accumulation of intracellular ROS afterexposure of mProx24 cells to albumin.
Figure 8. Measurement of SOD, glutathione peroxidase (GPx), and catalase activity, and the total reduced form of Glutathione. (A) SOD activity was measured after 10-min albumin treatment of mouse renal proximal tubular (mProx24) cells, but showed no significant change. (B) GPx and catalase activities were measured after 10-min albumin treatment of mProx24 cells. Both GPx and catalase activities were reduced after albumin overloading. (C) Quantity of total glutathione (GSH) was determined after 10-min albumin treatment of mProx24 cells. The quantity increased after starvation but showed no significant change after treatment with albumin compared with control. The histograms represent the means of three independent experiments (means ± SD).
SOD produces hydrogen peroxide in cells, and intracellular GPxand catalase are the main enzymes that convert hydrogen peroxideinto water to prevent production of hydroxyl radicals. Becausereduction of GPx and catalase activities could induce accumulationof hydrogen peroxide, we measured the GPx and catalase activitiesafter mProx24 cells had been treated with albumin for 10 min.Figure 8B showed that both enzymatic activities were reducedby albumin. These results indicate that changes in GPx and catalaseactivities could be responsible for the accumulation of intracellularROS after exposure of mProx24 cells to albumin.
Measurement of the Total Reduced Form of Glutathione
Generally, intracellular proteins do not contain disulfide bondsbecause the high cytosolic concentration of free sulfhydryl(-SH) reducing agents breaks such bonds (42). We hypothesizedthat a large amount of proteins with disulfide bonds, such asalbumin and apoTf, might lead to oxidative stress when ingestedinto cells. To address this, we measured the shift in intracellularGSH levels. GSH is a naturally occurring tripeptide whose nucleophilicand reducing properties play a central role in the antioxidantsystem of most aerobic cells. GSH is crucial to the maintenanceof the -SH level of proteins in cells (43). Hence, we determinedthe quantity of the total reduced form of GSH under variousconditions of albumin loading. The quantity of GSH increasedafter albumin starvation, but did not show any marked changecompared with control cells after treatment with albumin for10 min (Figure 8C). Our findings suggest that the increase inGSH was induced by long-term upregulation of intracellular ROS,and that GSH was not consumed for at least 10 min after thecells were exposed to albumin.
We have previously constructed gene expression profiles by meansof direct sequencing procedures (26,29), and identified severalimmunity-related genes, such as MHC class I and II, by comparingthe profiles of control and disease model proximal tubular cells.Renal MHC proteins are expressed in response to inflammationand renal injury, and their expression is thought to be regulatedsolely by IFN- (44). Because the IFN- pathway usually requiresthe activation of Stat1 (45,46), we hypothesized that proteinuriacould activate proximal tubular cells directly by activationof the STAT signaling pathway. Our results indicate that albuminactivates proximal tubular cells in an IFN-–independentmanner after ROS-mediated STAT activation.
Our data also demonstrate that Stat1/5 and Stat3 show differentresponses to albumin. The activation of Stat1 and Stat5 wasclearly identified within 15 min. In contrast, no manifest activationof Stat3 could be identified after a 1-h exposure to albumin,and activation of Stat3 was observed only after a 4-h exposure.The mechanisms responsible for these different responses arestill obscure, but the differences may depend on the cell type.In macrophages, Stat1 has been reported to induce sterilizingactivity and the production of various complements, chemokines,and adhesion molecules (3). Stat5, which was originally identifiedas a mammary gland factor regulated by prolactin, is also activatedby multiple cytokines, including IL-2, IL-3, and GM-CSF, whichare macrophage-activating factors. In contrast, IL-10–inducedStat3 activation suppresses macrophage activity, and mice lackingStat3 show abnormally enhanced macrophage activity and developchronic inflammation (47). The different responses of Stat1/5and Stat3 in the renal proximal tubular cell line seem to besimilar to those in macrophages, suggesting that albumin overloadingcan induce proinflammatory processes. Stat3 is activated byvarious cytokines, such as IL-6, that are also induced by IFN-(30). Therefore, the activation of Stat3 at a late stage afteralbumin overloading might be due to a secondary response afterStat1 activation by albumin.
ROS generation was even observed in the control mProx24 cellsafter starvation, leading to a slight activation of Stat1 andStat5. This is a previously reported result that supports ourfinding that serum deprivation can induce elevated ROS and anoxidative state in cells (48). Nuclear factor kappa B (NF-B)is also induced by ROS (38) and is one of the key componentsthat cause immune reactions in kidneys (49). A recent reportof a new and interesting Stat5 signal transduction mechanismsuggests that Stat5 could be a potential activator of NF-B insome types of hematopoietic cells (50). In addition, many IFN-–induciblegenes, such as RANTES and MCP-1, were found to be induced synergisticallyby IFN- and TNF- (51). This synergism is reportedly involvedin the interaction between activated Stat1 and NF-B (11,30).Hence, the activation of Stat1 and Stat5 by albumin may be importantin the induction of transcripts that cause kidney disease progression.
Recent reports have demonstrated rapid and significant increasesin intracellular ROS after growth factor or cytokine stimulation.These types of ROS appear to be essential for a host of downstreamsignaling events, including cell proliferation, apoptosis, andinflammation, and thus contribute to the development of diseases(52). Simon et al. (7) reported that members of the STAT familyof transcription factors were activated in fibroblasts and A-431carcinoma cells in response to H2O2. This activation occurredwithin 5 min, could be inhibited by antioxidants, and did notrequire protein synthesis. These findings hence indicate thatthe JAK/STAT pathway responds to intracellular ROS.
Our results indicate that the membrane-bound NADPH oxidase systemseems to be relevant in the induction of ROS by albumin. Ithas been found that the activity of a membrane-bound NADPH oxidaseoften mediates intracellular generation of ROS in response toligands (40,41). The ROS in macrophages are also produced byNADPH oxidase after phagocytosis (53). Hence, it is particularlyinteresting that the endocytic uptake of albumin in renal proximaltubular cells seems also to be responsible for ROS generation.Further investigation is needed, however, to clarify the precisemechanisms of ROS generation by NADPH oxidase after albuminoverloading. Our results also indicate that the GPx and catalaseactivities changed within 10 min after albumin loading, suggestingthat albumin could interfere with GPx and catalase activitiesby way of glia maturation factor–beta (54). The low activitiesof GPx and catalase could be responsible for the accumulationof the intracellular ROS after albumin ingestion into mProx24cells. Hence, it seems that not only the ROS generating systembut also the ROS-scavenging system may contribute to the inductionof ROS by albumin.
We next focus on the amino acid sequences of albumin and apoTf.The amino acid sequences of human, bovine, and rodent albuminare highly homologous. Albumin itself contains a high percentageof cysteine that is organized into a characteristic repeatingdisulfide pattern (55). Generally, intracellular proteins donot contain disulfide bonds because the high cytosolic concentrationof free sulfhydryl (-SH) reducing agents breaks such bonds (42).We hypothesized that a large amount of proteins with disulfidebonds, such as albumin and apoTf, might cause oxidative stresswhen ingested into the cells. However, our data indicate thatthe amount of GSH was not changed for at least 10 min afteralbumin overloading, suggesting that disulfide bonds are nota primary cause of ROS induction.
Several mechanisms have been proposed by which ROS might activateintracellular kinases such as Jak2. One of these mechanismsis inactivation of protein tyrosine phosphatases (PTPs) thatmay be susceptible to oxidation (56). Tyrosine phosphorylationof proteins is dependent on the balance between kinases andPTPs within a cell. Therefore, it is feasible that inactivationof PTPs could induce tyrosine phosphorylation of Jak2. Anotherpossible mechanism involves Fyn, a member of the src family,which can regulate the activation of Jak2 by H2O2 in fibroblasts(57). These proposed mechanisms need to be verified in futurestudies.
The fact that almost all cytokines activate the JAK/STAT pathwaysuggests that STATs play important roles in the inflammatoryprocess. Our results suggest that albumin, a major componentof proteinuria, exerts cytokine-like effects to activate proximaltubular cells after ROS-mediated STAT activation. The activatedSTAT signaling could then result in cell proliferation, productionof several cytokines/growth factors, and induction of immuneresponses that are presumably involved in the progression ofkidney diseases. The results reported here could thus providean insight into the intrinsic toxicity of proteinuria and mayprovide further clues regarding therapeutic approaches to inhibitionof the signal transduction pathways involved in proteinuria.
Acknowledgments
This study was supported in part by a Grant-in-Aid for ScientificResearch from the Ministry of Education, Science, Sports andCulture, Japan, and in part by Renal Discoveries, the BaxterExtramural Grant Program, USA. We thank Dr. Toshio Hirano (Departmentof Molecular Oncology, Graduate School of Medicine, Osaka University)for his valuable suggestion about STAT signaling pathways andNaoko Horimoto for her technical assistance.
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Received for publication March 28, 2003.
Accepted for publication October 31, 2003.
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Top
Abstract
Introduction
. The activation of STATs was inhibited by N-acetyl-L-cysteine, a precursor of glutathione and a reactive oxygen species (ROS) scavenger, and fluorescence-activated cell sorter analysis showed upregulation of intracellular ROS after albumin overloading, suggesting that albumin per se could generate ROS in proximal tubular cells. The activation of STATs occurred by way of the ROS generating system, and especially through the membrane-bound NADPH oxidase system. Reduced activities of glutathione peroxidase and catalase could also be responsible for the accumulation of intracellular ROS. Hence, not only the ROS generating system, but also the ROS scavenging system may contribute to the induction of ROS by albumin. These findings support the hypothesis that proximal tubular cells are activated and generate ROS by reabsorption of abundant urinary proteins filtered through the glomerular capillaries, and as a consequence, various IFN-
(17). Moreover, the level of proteinuria, which is independent of mean arterial BP, is reportedly one of the best predictors for disease progression toward end-stage renal failure (18,19). Microalbuminuria is known as an important early sign of diabetic nephropathy (20,21) and of progressive loss of renal function in the nondiabetic population (22). Recent studies have shown that proximal tubular cells are activated by reabsorption of abundant urinary proteins filtered through the glomerular capillaries, producing various chemokines that lead to kidney disease progression (23). Identification of the signal transduction pathway in activated proximal tubular cells is, however, still incomplete. 
B) is also induced by ROS (38) and is one of the key components that cause immune reactions in kidneys (49). A recent report of a new and interesting Stat5 signal transduction mechanism suggests that Stat5 could be a potential activator of NF-
