Cellular Antioxidant Mechanisms Control Immunoglobulin Light Chain-Mediated Proximal Tubule Injury

Abstract

A major cause of morbidity and mortality in multiple myeloma is kidney injury from overproduction of monoclonal immunoglobulin light chains (FLC). FLC can induce damage through the production of hydrogen peroxide, which activates pro-inflammatory and pro-apoptotic pathways. The present study focused on catalase, a highly conserved antioxidant enzyme that degrades hydrogen peroxide. Initial findings were that FLC increased hydrogen peroxide levels but also decreased catalase levels and activity in proximal tubule epithelium. In order to clarify, we showed that the phosphatidylinositol 3-kinase inhibitor, LY294002, inhibited FLC-induced Akt-mediated deactivation of Forkhead box O class 3a (FoxO3a) and increased catalase activity in proximal tubule cells. Augmented catalase activity decreased FLC-mediated production of hydrogen peroxide as well as the associated increase in High Mobility Group Box 1 (HMGB1) protein release and caspase-3 activity. Coincubation of cells with FLC and an allosteric activator of Sirtuin 1 (SIRT1) was also sufficient to increase catalase activity and promote similar cytoprotective effects. Our studies confirmed that the mechanism of downregulation of catalase by FLC involved deactivation of FoxO3a and inhibition of SIRT1. Mechanistic understanding of catalase regulation allows for future treatments that target pathways that increase catalase in the setting of proximal tubule injury from FLC.

Graphical Abstract

Introduction

A major function of the kidney proximal tubule is to reclaim low molecular weight proteins that are filtered from the bloodstream by the glomerulus and appear in the tubular fluid. Included among these proteins are immunoglobulin free light chains (FLC), which are readily reabsorbed by the proximal tubule, where they typically undergo hydrolysis [1]. In plasma cell dyscrasias such as multiple myeloma, the production of monoclonal FLC can dramatically increase, requiring the proximal tubules to metabolize significant amounts of these proteins [2]. Proximal tubule absorption of some monoclonal FLC, however, generates hydrogen peroxide (H₂O₂) [3, 4] in amounts sufficient to activate redox-sensitive signaling pathways that include mitogen-activated protein kinase 5, also known as apoptosis signal-regulating kinase 1 (ASK1) [5], and Signal Transducer and Activator of Transcription 1 (STAT1), High Mobility Group protein B1 (HMGB1) and Toll-like Receptor 4 (TLR4) [4, 6]. These pro-apoptotic and pro-inflammatory pathways facilitate the progressive kidney injury observed in the setting of monoclonal FLC production.

In addition to intrinsic physicochemical features that determined the degree of oxidative stress produced by the FLC [4], we hypothesized that host antioxidant defense mechanisms, particularly catalase activity in the kidney, play an important modulating role in this process. Regulation of catalase activity occurs primarily at the transcriptional level and involves several nuclear factors [7], although there are also post-translational events that determine the cellular location of catalase and the subsequent contribution to anti-oxidant defense [8]. Forkhead box O class 3a (FoxO3a), a nuclear member of the subgroup of the Forkhead family of transcription factors, responds to cellular stress by transcriptionally activating critical gene responses that include catalase [9, 10]. A major counterregulatory factor of nuclear FoxO3a activity is protein kinase B (Akt), which phosphorylates a serine residue within the nuclear localization domain of FoxO3a, resulting in loss of function by extrusion of FoxO3a from the nucleus into the cytoplasm [11]. FoxO3a-mediated transcription of catalase requires Peroxisome proliferator-activated receptor Gamma Coactivator 1-alpha (PGC-1α) to serve as a co-activator [12]. Importantly, Sirtuin1 (SIRT1), a NAD⁺-dependent deacetylase that is typically localized to the nucleus, also regulates antioxidant gene expression specifically through both FoxO3a and PGC-1α [13]. Thus, the interactions among FoxO3a, Akt, PGC-1α, and SIRT1 determine cellular catalase activity.

In this paper, a series of studies used in vivo and in vitro models of monoclonal FLC-mediated proximal tubule injury to determine if signaling pathways that control endogenous catalase activity governed the cellular responses to FLC. The mechanisms by which catalase activity was regulated in the proximal tubule and the responses to monoclonal FLC were explored, with focus particularly on the role of oxidative stress in regulation of FoxO3a and SIRT1.

Materials and Methods

Protein reagents

Two unique human monoclonal immunoglobulin free light chains (FLC) - one κ and one λ - were purified using standard methods from the urine of patients with multiple myeloma, light chain proteinuria, and evidence of significant kidney injury clinically determined to be of tubular origin [4, 6, 14, 15]. Briefly, the protein precipitate developed from addition of ammonium sulfate, 70% saturation was harvested, dissolved in deionized water, and extensively dialyzed against deionized water and then 0.01 M sodium phosphate buffer, pH 7.6. The FLC were then purified using ion exchange chromatography. Following dialysis against distilled water, the FLC were lyophilized and stored at −20° C until use. The purity and identity of FLCs were confirmed by SDS-PAGE and western blotting [4, 6, 14, 15]. These proteins were arbitrarily labeled κ2 and λ3, which were not indications of the light chain subtype. These FLC readily dissolved in phosphate-buffered saline (PBS), which served as the vehicle control in the in vivo studies. Human β₂ microglobulin (B2M) was obtained commercially (Cat# M4890, MilliporeSigma, Saint Louis, MO). B2M was used as a control for the in vitro studies, because it is a circulating low molecular weight protein that has a three-dimensional structure that resembles an immunoglobulin constant domain [16] and is handled by the kidney similarly to FLC, appearing in the glomerular ultrafiltrate and metabolized by the proximal tubule [17].

Commercial Reagents

LY294002 (Cat# 15447-36-6, Selleckchem, Houston, TX), a pharmacological inhibitor of phosphatidylinositol 3-kinase (PI3K) [18], was dissolved in 0.001% DMSO final and used at a concentration of 50 μM. SRT1720 (Cat# 1001645-58-4, Selleckchem), an allosteric activator of Sirtuin 1 (SIRT1) [19, 20], was dissolved in 0.001% DMSO final and used at a concentration of 10 μM. Sirtinol (Cat# 410536-97-9, Selleckchem), a selective inhibitor of SIRT1 activity [21], was dissolved in 0.004% DMSO and used at a final concentration of 40 μM. The solvents alone served as the corresponding vehicle controls. Antibodies directed against total STAT1 (Cat# 9172); pSTAT1(Tyr701) (D4A7, Cat# 7649); total Akt (pan) (C67E7, Cat# 4691); p-Akt(Ser473) (D9E, Cat# 9271); total FoxO3a (75D8, Cat# 2497); p-FoxO3a(Ser253) (D18H8, Cat# 13129); total SIRT1 (D1D7, Cat# 9475); catalase (D4P7B, Cat# 12980) were all obtained commercially (Cell Signaling Technology Inc., Danvers, MA). GAPDH (Cat# Ab8245) served as a loading normalization control and was obtained commercially (Abcam Inc., Cambridge, MA). Secondary antibodies used for western analyses included Alexa Fluor 680-conjugated goat anti-rabbit antibody (Cat# 712-625-150) or goat anti-mouse IgG (Cat# 115—625-146); and Alexa Fluor 790-conjugated goat anti-rabbit antibody (Cat# 111-655-144) or goat anti-mouse IgG (Cat#[ISP]115-655-146); all were obtained commercially (Jackson ImmunoResearch Laboratores Inc., West Grove, PA).

Animal and Tissue Preparation

Colonies of Stat1-knockout mice with a C57BL/6J genetic background (termed Stat1^−/− mice) and littermate controls (termed Stat1^+/+ mice) were established, confirmed using PCR-based genotyping, and maintained in a gnotobiotic facility [4]. Studies were conducted using 16 Stat1^+/+ and 16 Stat1^−/− mice. Stat1^−/− mice grew normally and were phenotypically normal under these conditions. At the start of the experiment, 8-week-old male Stat1^+/+ and Stat1^−/− mice (n = 8/group) received daily intraperitoneal injections of either vehicle (PBS) (Gibco, Thermo Fisher Scientific) or with κ2 FLC (0.165 mg/g BW) in PBS. The experiments were concluded on day 10, when the mice were anesthetized using isoflurane and sacrificed. Total RNA was isolated from right kidney cortex as described [4]. RNA was sequenced on NextSeq500 System and the library was prepared with the Agilent SureSelect Stranded mRNA kit. Raw sequencing (fastq) files were subjected to quality control analysis using FastQC (v0.11.5) [http://www.bioinformatics.babraham.ac.uk/projects/fastqc/]. Cleaned sequence reads were aligned to mouse genome (GRCm38) using TopHat v2.1.0 [22] or STAR v2.5.3.a [23]. Differential expression analysis was performed using DESeq2 [24], following standard protocol [https://bioconductor.org/packages/release/bioc/vignettes/DESeq2/inst/doc/DESeq2.html]. The left kidney cortex was homogenized in RIPA buffer (25 mM Tris, pH 7.4; 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) or buffer from assay kits. Total soluble protein concentration in lysates was determined using a bicinchoninic acid assay (BCA) kit (cat# 23227, BCA Protein Assay Reagent Kit, Thermo Fisher Scientific Pierce Protein Research Products, Rockford, IL).

Cell Culture Studies

Human proximal tubular epithelial cells (HK-2 cells), which have been well characterized [25], were obtained from the American Type Culture Collection (ATCC, CRL-2190). Monolayers of HK-2 cells were grown on 6-well plates (Corning-Costar, Corning Life Sciences) that were precoated with 5 μg/cm² collagen type 1 (Rat Tail Collagen Type 1, cat# A1048301, Gibco, Thermo Fisher Scientific) and incubated at 37°C with 5% CO₂ and 95% air in keratinocyte serum-free medium (K-SFM) (Cat# 10724-011, Gibco, Thermo Fisher Scientific) supplemented with recombinant human epidermal growth factor (5 ng/ml) and bovine pituitary extract (50 μg/ml). Medium was exchanged at 48-hour intervals, and the cells were not used beyond 25 to 30 passages.

At the start of the experiment, following incubation of confluent HK-2 cells, the medium was exchanged for keratinocyte serum-free medium (K-SFM) that contained one of the FLC (1 mg/ml) or B2M (1 mg/ml). The FLC concentration (1 mg/ml) was within the expected concentration range to which proximal tubule cells are exposed based on the serum levels found in patients with multiple myeloma, and the estimated glomerular sieving coefficients for these low-molecular-weight proteins [4–6, 26]. In some experiments, 50 μM LY94002 (cat# s1105 Selleckchem), 40 μM Sirtinol (cat# s2804 Selleckchem), or 10 μM SRT1720 (cat# s1129 Selleckchem) were added to the medium just prior to the addition of the low molecular weight proteins. Following incubation for 24 hours at 37°C, the medium was harvested promptly for assays and cells were collected and lysed in radioimmunoprecipitation (RIPA) buffer or buffer from commercial assay kits. The lysis buffer contained a protease inhibitor cocktail (cat# 1861284, Pierce Halt Protease and Phosphatase inhibitor, Thermo Scientific, Rockford, IL). Cell supernatants and cell or tissue lysates were clarified by centrifugation and stored at −70°C until assayed.

Quantification of H₂O₂ in medium

Levels of H₂O₂ by HK-2 cells were quantified using a kit (Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit, Molecular Probes), following the protocol provided by the manufacturer. Treated medium samples and standards were mixed with Amplex Red working solution and incubated at room temperature for 30 minutes, protected from light. Fluorescence was excited at 535 nm and emission detected at 590 nm (Spectramax M2e Microplate Reader; Molecular Devices, Sunnyvale, CA). All samples were tested in duplicate and averaged.

Western Blot Analyses

Total protein concentration was determined using a bicinchoninic acid (BCA) assay (cat# 23227, BCA Protein Assay Reagent Kit, Thermo Fisher Scientific Pierce Protein Research Products, Rockford, IL). Tissue and cell lysates (20-60 μg total protein) were boiled for 3 min in Laemmli buffer and separated using 7-12% SDS-PAGE (Bio-Rad Laboratories, Hercules, CA), before electrophoretic transfer onto PVDF membranes. The membranes were blocked in 5% nonfat milk and then probed with an antibody (diluted 1:1000) that recognized specifically catalase (D4P7B, Cat# 12980), STAT1 (Cat# 9172) and pSTAT1(Y701) (D4A7 Cat# 7649), FoxO3a (75D8 Cat#2497) and p-FoxO3a(S253) (D18H8 Cat# 13129), Akt and p-Akt(S473) (C67E7 Cat# 4691), PP2A (clone 1D6, Cat# 05-421, Millipore), and GAPDH (Cat# Ab8245), diluted 1:5000. After washes, the blots were incubated for 1 h at room temperature with Alexa Fluor 680- or 790-conjugated AffiniPure secondary antibody (1:10,000 dilution). The bands were detected using the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE), and densitometric analysis was performed using Image Studio Software (LI-COR Biosciences, Lincoln, NE).

Quantification of High Mobility Group Box 1 (HMGB1) Protein

Medium levels of HMGB1 were determined with a HMGB1 ELISA kit (cat# st51011 IBL International GMBH, Hamburg, Germany), following the protocol provided by the manufacturer. Collected cell culture medium, 50 μl, and 50 μl of diluent buffer were added to an antibody-coated 96-well plate and incubated at 37°C for 20 hours. After washing, 100 μl of enzyme conjugate were added into each well, and incubated at room temperature for 2 hours, followed by substrate and stop solution. HMGB1 levels were quantified using a colorimetric plate reader (Spectramax M2e Microplate Reader; Molecular Devices, Sunnyvale, CA).

Determination of Catalase Activity

Catalase activity was quantified using commercially available kits (OxiSelect Catalase Activity Assay Kit, cat# STA-339 and cat# STA-341, Cell Biolabs, Inc., San Diego, CA). Cells at 2 x 10⁶were resuspended in 1X assay buffer and homogenized on ice by sonication. Following the protocol provided by the manufacturer, the reaction included hydrogen peroxide and detection reagent. Fluorescence was detected in the 590 nm range during excitation in the 545 nm range (Spectramax M2e Microplate Reader; Molecular Devices, Sunnyvale, CA). All samples were tested in duplicate and averaged. Cell viability was determined using a fluorescence microplate assay and resazurin (alamarBlue™ HS Cell Viability Reagent, Cat# A50101, Thermo Fisher Scientific, Waltham MA).

Protein Phosphatase 2 (PP2a) Activity Assay

PP2a activity was assayed using a kit (PP2A Immunoprecipitation Phosphatase Assay Kit, cat#17-313, Millipore, Temecula, CA), following the manufacturer’s protocol. Treated HK-2 cells at 2 X10⁶ were resuspend in 0.3 ml of phosphatase extraction buffer, containing 20 mM imidazole HCl, 2 mM EGTA, pH 7.0, with 10 μg/mL each of aprotinin, leupeptin, and pepstatin, and 1 mM benzamidine and 1 mM PMSF. The cells were homogenized, and centrifuged at 2000xg for 5 min. Total protein concentration of the supernatants was determined using a BCA Protein Assay Reagent Kit (cat# 23227, Pierce Protein Research Products, Thermo Fisher Scientific), and 250 μg of lysates were used for each phosphatase activity assay. Cellular lysates were immunoprecipitated with 4 μg of anti-PP2a, C subunit antibody (clone 1D6, cat# 05-421, Millipore) and 40 μL of Protein A agarose slurry for 2 h at 4°C. Following washes, phosphatase substrate was added. After a 10-min development time, absorbance was read at 650 nm using a microplate reader (Spectramax M2e Microplate Reader; Molecular Devices, Sunnyvale, CA). Absorbance was compared to the standard curve to determine PP2a activity. Along with determination of PP2a activity, western analysis of the immunoprecipitates was performed using the same antibody that was directed against the catalytic subunit of anti-PP2a.

Determination of Cytoplasmic Caspase-3 Activity

Cytoplasmic caspase-3 activity was quantified using a colorimetric assay (cat# ab39401, Caspase-3 Assay Kit, Abcam Inc., Cambridge, MA), using the labeled substrate DEVD-p-NA and following the protocol provided by the manufacturer. Cytoplasmic lysates were collected from pelleted cells using lysis buffer with protease inhibitors (Cat# 1861284, Complete Protease Inhibitor tablets; Roche Diagnostics GmbH, Mannheim, Germany). Caspase-3 activity was quantified using a microplate reader (Spectramax M2e Microplate Reader; Molecular Devices, Sunnyvale, CA).

Determination of Sirtuin 1 (SIRT1) Activity

SIRT1 activity was detected in cell lysates using a commercial kit (SIRT1 Activity Assay Kit, cat# ab156065 Abcam Inc., Cambrdge, MA). Following the protocol provided by the manufacturer, SIRT1 activity was quantified using a fluorescence-labeled acetylated peptide. When the rate of the reaction was constant, a stop solution was added to each well, and fluorescence intensity was detected using a microplate fluorescence reader (SpectraMax M2e Microplate Reader, Molecular Devices). Assays were performed in duplicate and averaged.

Statistics

All data including those represented graphically were expressed as mean ± SEM. For multiple group comparisons, either one-way analysis of variance (ANOVA) or two-way ANOVA, followed by Tukey’s multiple comparisons test, was performed using Prism, version 8.4.3. Two-way ANOVA partitioned the overall variance of the outcome variable into three components, plus a residual (or error) term. It computed P values that tested three null hypotheses: an interaction P value for the null hypothesis that there was no interaction between the two factors on the response, as well as P values for the null hypotheses that each factor had no effect on the response. P values as well as the significance demonstrated through subsequent post-hoc testing were provided in the figures and figure legends, where appropriate. P<0.05 was considered statistically significant, except in the RNA analysis studies, where genes altered by absolute fold change with adjusted P value <0.01 were considered to be significantly differentially expressed.

Study Approval

This study was carried out in strict accordance with the recommendations in the NIH’s Guide for the Care and Use of Laboratory Animals. The IACUC of the University of Alabama at Birmingham approved the project. The IRB of the Birmingham Department of Veterans Affairs provided annual continuing oversight and approval of this research activity.

Results

Human immunoglobulin free light chains (FLC) decreased catalase activity in vivo and in vitro.

Examination of antioxidant gene expression in kidney cortex using Kidney Cell Explorer [27] demonstrated predominance in proximal tubule epithelium. Gene expression in kidney cortex was determined in colonies of Stat1^+/+ mice that were injected intraperitoneally daily for 10 days with vehicle (PBS) or with a nephrotoxic monoclonal κ2 FLC, 0.165 mg/g BW. By this time, Stat1^+/+ mice that received the FLC demonstrated oxidant-induced kidney injury [4]. Expression of the major antioxidant genes tested did not increase, but specifically catalase showed a significant reduction with the administration of FLC, when compared with mice given the vehicle control (Table 1). With this observation, the focus became catalase. Catalase activity in kidney cortex was then determined in both Stat1^+/+ and Stat1^−/− mice following treatment with kappa2 FLC. Kidney cortical lysates from Stat1^−/− mice treated with vehicle had higher amounts of catalase and catalase activity (15.3±0.21 versus 13.9±0.22 U per mg total protein; P=0.0001) than corresponding lysates from Stat1^+/+ mice treated with vehicle. Parenteral administration of FLC reduced catalase and catalase activity in lysates of kidney cortex of both Stat1^+/+ and Stat1^−/− mice, compared with lysates from vehicle-treated mice (Figure 1). These findings indicated that the inhibitory effect of the FLC on catalase occurred independently of STAT1. Subsequent studies incubating human kidney epithelial cells (HK-2) overnight in medium containing human κ2 and λ3 FLC, 1 mg/ml, confirmed the inhibitory effect of both monoclonal FLC on catalase activity (Figure 2A). In these and subsequent experiments, human β₂ microglobulin (B2M), 1 mg/ml, was used as the control, since this protein was another low molecular weight protein that was metabolized by the kidney proximal tubule [17] and was not toxic in this model [4]. Additional experiments confirmed that the production of H₂O₂ and catalase activity did not differ between cells incubated in medium containing vehicle (PBS) or B2M (Supplemental Figure 1). Associated with the decrease in catalase activity were increases in FLC-induced medium H₂O₂ levels detected using Amplex Red (Figure 2B), caspase-3 activity (Figure 2C), and medium HMGB1 levels (Figure 2D). Addition of both FLC, but not B2M, produced a time-dependent reduction in cell viability (Supplemental Figure 2).

Table 1.

Relative expression of antioxidant genes in kidney cortex from mice (n=4 in each group) treated with either a monoclonal FLC or vehicle control (PBS) for 10 days.

Gene Description	PBS-treated mice	FLC-treated mice	P value	Padj*
catalase [Source: MGI Symbol;Acc:MGI:88271]	39608±1603	32064±1120	0.0005	0.0067
glutathione peroxidase 1 [Source: MGI Symbol;Acc:MGI:104887]	58809±3434	64237±4099	0.2759	0.5677
glutathione peroxidase 4 [Source: MGI Symbol;Acc:MGI:104767]	12032±564	10643±328	0.0491	0.2071
superoxide dismutase 1, soluble [Source: MGI Symbol;Acc:MGI:98351]	15809±988	13630±458	0.0372	0.1742
superoxide dismutase 2, mitochondrial [Source: MGI Symbol;Acc:MGI:98352]	11414±527	11653±224	0.7196	0.8855
glutaredoxin [Source:MGI Symbol;Acc:MGI:2135625]	1005±76	1088±77	0.3939	0.6789
glutaredoxin 2 (thioltransferase) [Source:MGI Symbol;Acc:MGI:1916617]	1088±27	1206±27	0.0672	0.2525
thioredoxin 1 [Source: MGI Symbol;Acc:MGI:98874]	8932±400	8725±481	0.7436	0.8946
thioredoxin 2 [Source:MGI Symbol;Acc:MGI:1929468]	5325±308	5507±229	0.6386	0.8434

^*Padj, P value adjusted for multiple tests of significance

Figure 1. Levels of catalase as well as catalase activity decreased in kidney cortex of mice injected daily for 10 days with the human FLC (kappa2), 0.165 mg/g body weight.

A, Compared with the vehicle (PBS)-treated control groups of mice, lysates of kidney cortex of both Stat1^−/− and wild type mice injected with the kappa2 FLC demonstrated reduced amounts of catalase. (n=4 mice in each group; *P<0.05; **P<0.002; ****P <0.0001; 2-way ANOVA) B, Compared with the vehicle-treated control groups of mice, catalase activity in the kidney cortex of wild type mice declined with treatment with the kappa2 FLC. The absence of Stat1 enhanced levels of catalase and catalase activity, compared with data from the wild type mice, demonstrating a beneficial effect of loss of Stat1 on endogenous catalase activity. However, irrespective of the presence or absence of Stat1, parenteral administration of the kappa2 FLC reduced catalase and catalase activity in kidney cortex, indicating an effect that was independent of Stat1. (n=8 mice in each group; ***P=0.0001; ****P <0.0001; 2-way ANOVA)

Figure 2. Overnight incubation of human kidney epithelial cells (HK-2) in medium containing human kappa2 and lambda3 FLC (both 1 mg/ml) reduced catalase activity and increased H₂O₂ levels, caspase-3 activity, and medium High Mobility Group Box 1 (HMGB1) protein.

A, Compared with data from HK-2 cells incubated in medium containing human beta-2 microglobulin (B2M, 1 mg/ml), catalase activity declined in HK2 cells treated with both of the FLC (n=8 in each group; ****P <0.0001; ANOVA). Compared with HK-2 cells incubated in medium containing B2M, cultured cells incubated with both of the FLC also produced (B) higher levels of H₂O₂ in the medium (n=8 in each group; ****P <0.0001; ANOVA); (C) higher cellular activity levels of caspase-3 (n=8 in each group; ****P <0.0001; ANOVA), which is the final enzyme in the pathway that promotes apoptosis; and (D) higher levels of HMGB1 (n=8 in each group; ****P <0.0001; ANOVA), a key damage-associated molecular pattern (DAMP) that is known to activate TLR4 signaling.

Both κ2 and λ3 FLC reduced activity of Protein Phosphatase 2 (PP2a) and activated the protein kinase B (Akt) pathway.

Because protein serine/threonine phosphatases, including PP2a, have been shown to be sensitive to H₂O₂ [28, 29], PP2a activity was quantified. Compared with immunoprecipitates of lysates from cells incubated with B2M, PP2a activity decreased in immunoprecipitates from cells incubated with both FLC; notably, the catalytic subunit of PP2a in the samples did not change (Figure 3A). The decline in PP2a activity prompted an analysis of changes in Akt activity, which has been shown to be regulated by PP2a in a breast cancer model [30]. Compared with cells that were incubated with B2M, cells incubated in medium containing both FLC showed increased activation of Akt, indicated by phosphorylation of Akt at S473 (Figure 3B), which stabilizes phosphorylation of T308 and the activation state of Akt [31].

Figure 3. The activities of the ubiquitously expressed Protein Phosphatase 2 (PP2a) and Protein Kinase B (Akt) were determined in the model.

A, Following overnight incubation of HK-2 cells in medium containing the kappa and lambda FLC, cellular PP2a activity (left panel) declined (n=8 in each group; ****P <0.0001; ANOVA). However, despite the reduced activity, western analysis of samples (right panel) of the immunoprecipitates (n=5 in each group) showed levels of the catalytic (C) subunit PP2a did not differ (P=0.3993; ANOVA) among the three groups. Ig, immunoglobulin B, Western analyses of cell lysates following incubation in medium containing the kappa2 (left panel) or the lambda3 (right panel) FLC showed an increase in the activity of Akt, indicated by phosphorylation of Akt at amino acid residue S473 (n=6 in each group; ****P <0.0001; ANOVA).

Inhibition of the PI3K/Akt pathway during incubation with FLC inhibited phosphorylation of Forkhead box O class 3a (FoxO3a).

Initial experiments again demonstrated FLC-mediated increase in p-Akt(S473). In addition, the PI3K inhibitor, LY294002 (LY), inhibited the activation of Akt in HK-2 cells incubated in medium containing both FLC (Figure 4A). The introduction of LY294002 also caused a modest decrease in p-Akt in the B2M controls. Because FoxO3a transcriptionally activated catalase [9, 10] and was a substrate of Akt, which phosphorylates and inhibits FoxO3a [11], the activity of this enzyme was then determined. FLC-treated cells exhibited increased amounts of p-FoxO3a(S253), compared with cells incubated with FLC alone, and co-treatment with LY294002 inhibited this response (Figure 4B).

Figure 4. An integral role of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in the regulation of Forkhead box O class 3a (FoxO3a) was shown during incubation of HK-2 cells with the kappa2 and lambda3 FLC.

A, Western analyses of HK-2 cell lysates following incubation in medium containing the kappa2 (left panel) or the lambda3 (right panel) FLC, 1 mg/ml, again showed the increase in Akt activity and inhibition when the cells were co-incubated with LY294002 (n=4 in each group; ****P <0.0001; 2-way ANOVA). B, Western analyses of cell lysates following incubation in medium containing the kappa2 (left panel) or the lambda3 (right panel) FLC showed the increase in inhibition of FoxO3a (indicated by phosphorylation at S253) and prevention of inhibition when the cells were co-incubated with LY294002 (n=4 in each group; ***P =0.001; ****P <0.0001; 2-way ANOVA).

Inhibition of PI3K upregulated catalase and catalase activity, and decreased H₂O₂ in FLC-treated HK-2 cells.

In order to clarify further the correlation of PI3K/Akt activation with catalase, catalase activity and H₂O₂, HK-2 cells incubated in medium containing FLC or B2M were studied in the presence or absence of LY29400. Western analyses showed a decrease in relative catalase levels in FLC-incubated cells compared to B2M control. Co-incubation with LY29400 resulted in increases in the levels of catalase for both B2M and FLC treatment groups; however, the increase was significantly pronounced in FLC-treated groups (Figure 5A). Changes in catalase activity were similar to catalase levels and increased with the co-incubation of the cells with LY294002 (Figure 5B). Addition of LY294002 reduced medium H₂O₂ levels. Taken together, these findings demonstrated the PI3K/Akt pathway as a regulator of catalase and FLC-mediated H₂O₂ production.

Figure 5. The PI3K/Akt pathway regulated catalase, catalase activity, and the amount of H₂O₂ during incubation of HK-2 cells with the kappa2 and lambda3 FLC.

A, Compared with data from HK-2 cells incubated in medium containing B2M, 1 mg/ml, western analyses again showed the decline in catalase levels in HK-2 cells following incubation in medium containing the kappa2 (left panel) or the lambda3 (right panel) FLC, 1 mg/ml. Co-incubation with LY294002 reversed the decrease in catalase (n=4 in each group; **P <0.005; ***P <0.001; ****P <0.0001; 2-way ANOVA). B, The pattern of changes in catalase activities was similar to catalase levels and increased with the co-incubation of the cells with LY294002 (n=8 in each group; ****P <0.0001; 2-way ANOVA). Mean medium H₂O₂ levels increased when the HK-2 cells were co-incubated in medium containing the FLC and LY294002 (n=8 in each group; *P <0.05 ****P <0.0001; 2-way ANOVA).

FLC-induced increases in STAT1 and caspase-3 activities were prevented by inhibiting the PI3K/Akt axis.

Our prior studies showing monoclonal FLC-induced activation of STAT1 and release of HMGB1 [4, 6] and activation of caspase-3 [5] were recreated in HK-2 cells treated with both FLC (Figure 6A–C). Co-incubation with LY294002 resulted in reductions in pSTAT1(Y701) (Figure 6A), medium HMGB1 (Figure 6B), and caspase-3 activity (Figure 6C), when compared with cells incubated with the FLC alone and B2M controls. The role of catalase levels in determining catalase activity and H₂O₂ production was also examined in these experiments (Figure 7A). Mean catalase activity correlated (r²=0.7269; P<0.0001) directly with catalase determined using western analyses. In addition, mean medium H₂O₂ levels inversely correlated (r² = 0.8234; P < 0.0001) with mean cell catalase levels.

Figure 6. The PI3K/Akt pathway regulated activation of the STAT1 pathway, release of HMGB1 into the medium, and caspase-3 activity during incubation of HK-2 cells with the kappa2 and lambda3 FLC.

A, Activation of STAT1, indicated by tyrosine phosphorylation at residue 701, increased during incubation of HK-2 cells in medium containing either of the FLC, 1 mg/ml. Co-incubation with LY294002 inhibited activation of STAT1 (n=4 in each group; ****P <0.0001; 2-way ANOVA). Compared with data from the B2M-treated control group, medium HMGB1 levels (B) and caspase-3 activity (C) increased when HK-2 cells were incubated in medium containing the kappa2 and lambda3 FLC. Co-incubation with LY294002 returned both values toward control levels (n=8 in each group; ****P <0.0001; 2-way ANOVA).

Figure 7. Production of catalase correlated with activity and inversely with H₂O₂.

A, Levels of catalase determined using western analyses correlated directly (r²=0.7269; P<0.0001) with catalase activity and inversely (r² = 0.8234; P < 0.0001) with mean medium H₂O₂ levels. B, Because of the importance of catalase production in this model, the activity of sirtuin 1 (SIRT1) was determined. Compared with data obtained from HK-2 cells incubated in medium containing B2M, cells incubated in medium containing either of the FLC, 1 mg/ml, showed decreased cellular SIRT1 activity (n=8 in each group; ****P <0.0001; ANOVA).

The level of Sirtuin1 (SIRT1) activity regulated catalase, H₂O₂, activation of STAT1/HMGB1, and caspase-3 activity.

Because SIRT1 has been shown to regulate catalase transcription [13], the effect of FLC administration on SIRT1 activity was determined in our model. Following overnight incubation of HK-2 cells in medium containing the κ2 and λ3 FLC, SIRT1 activity in cell lysates decreased in comparison to lysates from HK-2 cells incubated in medium that contained B2M (Figure 7B). To demonstrate the effect of SIRT1 on catalase activity, H₂O₂ production, HMGB1 release into the medium, and caspase-3 activity, we co-incubated HK-2 cells with sirtinol, an inhibitor of SIRT1 activity [21], and SRT1720, an allosteric activator of SIRT1 [19, 20]. In these studies, the 40-μM dose of sirtinol used by Hasegawa, et al. [21] provided sufficient inhibitory effects on SIRT1. The dose of SRT1720 used in this study was based upon dose-response experiments published by Funk, et al [19]. Experiments demonstrated the inhibitory effects of sirtinol, 40 μM, and augmenting effects of SRT1720, 10 μM, on SIRT1 activity in the setting of both the FLC and B2M (Figure 8, left column). When HK-2 cells incubated in medium containing FLC or B2M were treated with sirtinol, catalase activity fell and medium H₂O₂ and HMGB1 increased; caspase-3 activity increased further in cells co-incubated with the FLC. In contrast, co-incubation with SRT1720 increased catalase activity and reduced medium H₂O₂ and HMGB1; caspase-3 activity decreased further in cells co-incubated with the FLC (Figure 8). Similar augmentation and inhibitory effects on STAT1 activation were demonstrated with co-incubation with sirtinol and SRT1720, respectively (Supplemental Figure 3).

Figure 8. SIRT1 activity regulated activation of the STAT1 pathway, release of HMGB1 into the medium, and caspase-3 activity during incubation of HK-2 cells with the kappa2 and lambda3 FLC.

SIRT1 activity decreased with incubation of HK-2 cells in medium containing either of the FLC, 1 mg/ml. These studies examined if altering human SIRT1 activity using an inhibitor (Sirtinol, 40 μM) or a selective activator (SRT1720, 10 μM) modified outcomes. The top row represented data using the control protein, B2M (1 mg/ml), while the second and third rows contained data from HK-2 cells incubated in medium containing kappa2 (1 mg/ml) and lambda3 (1 mg/ml) FLC, respectively. The first column showed the anticipated decrease and increase in SIRT1 activity with the addition of Sirtinol and SRT1720, respectively. Sirtinol further decreased catalase activity, increased medium H₂O₂ and HMGB1, and increased caspase-3 activity during incubation in medium containing the FLC. In contrast, SRT1720 promoted the opposite effects (n=8 in each group; *P <0.05; **P <0.005; ***P <0.001; ****P <0.0001; ANOVA).

Discussion

The proximal tubule is the site of metabolism of low molecular weight proteins, but in clinical settings that overproduce monoclonal FLC, this epithelium can be injured by oxidative stress that develops during the uptake and catabolism of these molecules [1, 3–6, 14, 26, 32–35]. The purpose of this series of studies was to explore the potential functional role of responses of proximal tubule epithelium to monoclonal FLC-induced oxidative stress and to provide potential interventions that mitigate this proximal tubulopathic process. The initial finding was a decrease in catalase expression and activity in the kidney cortex in vivo when mice were administered a monoclonal FLC from a patient with clinical kidney injury. This unexpected finding prompted a series of subsequent mechanistic studies using cultured human proximal tubule epithelial cells. These studies showed that catalase activity was reduced in cells incubated in medium containing FLC. In turn, catalase activity determined the amount of FLC-mediated production of H₂O₂ as well as the associated upregulation of pro-inflammatory and pro-apoptotic pathways. Critical determinants of catalase activity included inhibition of the redox-sensitive PP2a, a ubiquitously expressed phosphatase that regulates a variety of signal transduction events including the PI3K/Akt pathway [28–30, 36]. Because PP2a activity declined but western analysis did not show changes in the amount of the catalytic unit of PP2a in the samples, the findings were consistent with the known reversible inhibition of PP2a activity by H₂O₂ [28, 29]. In our studies, FLC-induced activation of Akt downregulated the activity of FoxO3, which in turn decreased catalase activity. Altogether, the data supported a direct influence of oxidative stress on the regulation of PI3K/Akt activity, which itself influenced the degree of oxidative injury generated by the FLC.

In addition to these signal transduction pathways, SIRT1 activity has also been shown to participate in catalase production [13, 21]. Catalase expression by SIRT1 involves the deacetylation of FoxO3a and PGC-1α, permitting their interaction and catalase transcription [7, 13]. In our in vitro model, incubation of HK-2 cells with both FLC reduced SIRT1 activity, likely through the known effect of H₂O₂ on depletion of NAD⁺, which limited SIRT1 activity [37]. Thus, in these specific models of kidney injury from monoclonal FLC, the interactions among several redox-sensitive signaling pathways determined the amount of intracellular catalase activity available to regulate oxidative stress in the proximal tubule (Figure 9). A primary focus of the present studies was the mechanism of catalase expression and the findings demonstrated significant correlations between relative catalase levels and catalase activity. However, prevention of movement of catalase from the peroxisome to the cytosol was recently shown to be an important response to oxidative stress. In particular, H₂O₂ promoted the phosphorylation of Peroxisomal Biogenesis Factor 14 (Pex14), preventing transport of catalase into peroxisomes [8]. One limitation of our findings related to the untested possibility that the production of H₂O₂ was sufficient to activate this signal transduction mechanism in addition to those signaling events identified and characterized in this paper. Future studies are therefore indicated to detect these events as well as test the potential involvement of the other identified signaling events in the determination of the intracellular location of catalase in this model of oxidative stress.

Figure 9. Summary of the findings of the present studies.

The data in this paper fit a paradigm that was initiated by endocytosis of the monoclonal FLC into proximal tubule epithelium. Generation of H₂O₂ inhibited PP2a activity, which has been shown to facilitate the activation of the PI3K/Akt pathway. Activated Akt phosphorylated and inactivated Forkhead box O class 3a (FoxO3a), inhibiting catalase production, which further promoted redox-mediated downstream activation of the STAT1/HMGB1/TLR axis and caspase-3 by activating Apoptosis Signal Kinase 1 (ASK1). SIRT1 activity decreased in the presence of the FLC, but modulating SIRT1 activity regulated catalase production, likely through the known regulation of the complex formed by FoxO3a and Peroxisome proliferator-activated receptor Gamma Coactivator 1-alpha (PGC-1α).

Our prior studies showed the benefit of reducing agents in the prevention of cytotoxicity from FLC [4, 35]. Importantly, the present findings uncovered additional targets that modulated the activities of either Akt or SIRT1 in FLC-mediated cell injury. By preventing the activation of the PI3K/Akt pathway, the inhibition of FoxO3a through Akt-mediated phosphorylation was also prevented. The result was an increase in catalase, which limited the activation of the STAT1/HMGB1 pathway as well as caspase-3 activation. Despite the FLC-mediated inhibition of SIRT1 activity, the efficacy of agents that enhanced (or decreased) SIRT1 activity was not impaired. As a result, inhibition of SIRT1 activity worsened outcomes, while allosteric activation of SIRT1 improved outcomes. This latter observation has translational potential since activators of SIRT1, such as SRT1720, or agents that increase cellular NAD⁺ levels are in a planning phase or presently in clinical trials for a variety of diseases and are overall well tolerated [38].

Conclusions

Taken together, our findings provided a mechanistic link for how FLC induced injury in proximal renal tubules. Regulation of catalase in proximal tubule epithelial cells was mediated by PI3K/Akt, FoxO3a and SIRT1 and, although not directly examined in these studies, likely also Peroxisome proliferator-activated receptor Gamma Coactivator 1-alpha (PGC-1α) [7]. H₂O₂ is considered a functionally important metabolite, but at higher doses can saturate basal redox proteins and require catalase to limit toxicity [39]. The apparent paradox of reduced catalase activity by monoclonal FLC was caused primarily by activation of signal transduction pathways that promoted cell survival but also inhibited FoxO3a (Figure 9) [11]. Inhibition of PI3K prevented activation of Akt and restored FoxO3a function, resulting in activation of an antioxidant program that increased catalase, decreased H₂O₂ levels, and reduced activation of pro-inflammatory and apoptotic pathways. These data were also consistent with the role of FoxO3a activation in preventing cardiac hypertrophy specifically through catalase expression [10]. SIRT1 activity also fell with exposure to FLC, but the addition of allosteric activators of SIRT1 was sufficient to increase catalase activity and promote cytoprotection. Our research demonstrated that the level of catalase activity was modifiable and provided a framework for further research by clarifying how epithelial cells reacted to FLC. The involvement of SIRT1 in catalase regulation holds promise for the as yet unfulfilled potential of SIRT1 activators that increase FoxO3a activity and catalase in the clinical setting.

Highlights.

• Immunoglobulin light chains (FLC) reduced catalase in proximal tubule epithelium.

• FLC decreased Protein Phosphatase 2a (PP2a) activity and increased Akt activity.

• Akt deactivated FoxO3a and decreased catalase in proximal tubule cells.

• Sirtuin 1 (SIRT1) activity fell with exposure to FLC.

• Allosteric activators of SIRT 1 increased catalase activity and cycenterrotection.

• Catalase downregulation by FLC involved inhibition of FoxO3a and SIRT1.