Kapil Chaudhary HHS Public Access 1 Rahul Shinde1 Haiyun ......Amino acid metabolism inhibits antibody-driven kidney injury by inducing autophagy Kapil Chaudhary1, Rahul Shinde1, Haiyun

Amino acid metabolism inhibits antibody-driven kidney injury by inducing autophagy

Kapil Chaudhary1, Rahul Shinde1, Haiyun Liu1, Jaya P. Gnana-Prakasam2, Rajalakshmi Veeranan-Karmegam2, Lei Huang1,3, Buvana Ravishankar1,7, Jillian Bradley1, Nino Kvirkvelia4, Malgorzata McManamin4, Wei Xiao1, Daniel Kleven5, Andrew L. Mellor1,4, Michael P. Madaio4, and Tracy L. McGaha1,4,6

1Cancer Immunology, Inflammation, and Tolerance Program, GRU Cancer Center

2Signaling and Angiogenesis Program, GRU Cancer Center

3Department of Radiology, Medical College of Georgia, Georgia Regents University

4Department of Medicine, Medical College of Georgia, Georgia Regents University

5Department of Pathology, Medical College of Georgia, Georgia Regents University

Abstract

Inflammatory kidney disease is a major clinical problem that can result in end-stage renal failure.

Here we show that antibody mediated inflammatory kidney injury and renal disease in a mouse

nephrotoxic serum nephritis (NTN) model was inhibited by amino acid metabolism and a

protective autophagic response. The metabolic signal was driven by IFN-γ-mediated induction of

indoleamine 2,3 dioxygenase 1 (Ido1) enzyme activity with subsequent activation of a stress

response dependent on the eIF2α kinase general control nonderepressible 2 (GCN2). Activation of

GCN2 suppressed proinflammatory cytokine production in glomeruli and reduced macrophage

recruitment to the kidney during the incipient stage of antibody induced glomerular inflammation.

Further, inhibition of autophagy or genetic ablation of Ido1 or Gcn2 converted antibody-induced,

self-limiting nephritis to fatal end-stage renal disease. Conversely, increasing kidney IDO1

activity or treating mice with a GCN2 agonist induced autophagy and protected mice from

nephritic kidney damage. Finally kidney tissue from patients with antibody-driven nephropathy

showed increased IDO1 abundance, GCN2 activation and autophagy induction. Thus, these

findings support the hypothesis that the IDO-GCN2 pathway in glomerular stromal cells is a

critical negative feedback mechanism that limits inflammatory renal pathology by inducing

autophagy.

6Address correspondence to: Dr. Tracy McGaha, PhD, Department of Medicine, Medical College of Georgia, CN4143, 1120 15th

Street, Augusta, GA, 30912, USA, [email protected] address: Department of Cancer Immunology, Genentech, 1 DNA Way South, San Francisco, CA, 94080, USA

Author contributions. RS, HL, JPG-N, R V-K, LH, BR, JB, NK, and MM contributed to experimental design and execution. DK provided human materials and critical review of the manuscript. ALM and MPM were involved in experimental planning and execution, critical assessment of data, and reviewing the manuscript. KC contributed to experimental design and execution, analysis of data, writing and review of the manuscript. TLM was responsible for experimental design, data analysis and interpretation and wrote the manuscript.

Competing interests. The authors declare that they have no competing interests.

HHS Public AccessAuthor manuscriptJ Immunol. Author manuscript; available in PMC 2016 June 15.

Published in final edited form as:J Immunol. 2015 June 15; 194(12): 5713–5724. doi:10.4049/jimmunol.1500277.

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Introduction

In humans and experimental models of glomerular disease loss of the specialized epithelial

cells in the glomerulus (podocytes) has been directly linked to accelerated

glomerulosclerosis, a substantial cause of decreased glomerular filtration rate and end stage

renal disease (ESRD) (1). Since podocytes have a limited ability to proliferate and

regenerate after injury, podocyte responses to inflammation-driven damage play a key

mechanistic role in the outcome of glomerular disease. Autophagy is a critical response

downstream of diverse stress signals (such as cytokine-driven activation, nutrient depletion,

and so on) (2-4). Moreover, autophagy is required for podocyte survival under basal

conditions as mice with a podocyte-specific defect in autophagy show age-dependent signs

of advanced glomerulosclerosis and decline in kidney function (5). Stress is detected at the

cellular level by multiple homeostatic sensing systems. The integrated stress response is

comprised of four of Ser/Thr kinases that target the α subunit of eukaryotic initiation factor

2 (eIF2α). These kinases are activated by multiple environmental signals including amino

acid deprivation, endoplasmic reticulum stress, heme limitation, and DNA damage (6). The

activation of integrated stress response kinases and subsequent eIF2α phosphorylation alters

cellular transcription and translation driving expression of stress response genes including

those encoding autophagy factors and cytokines (7-9).

There has been long-standing clinical interest in the impact of diet and nutritional stress on

kidney disease progression. While it is not understood on a mechanistic level, there is a

reason to believe that nutrient availability in the microenvironment is a critical determinant

factor in disease outcome. For example, dietary protein or amino acid restriction stress

reduced pathology in clinical and experimental glomerulonephritis and renal ischemia

(10-14). However, the reported effects of a low protein diet in human renal disease have

been more variable and thus the efficacy of dietary protein or amino acid restriction on

chronic kidney disease pathophysiology is not clear (15, 16).

Active enzymatic metabolism of amino acids and the subsequent stress it elicits is

recognized as a regulatory feed back mechanism that alters disease outcomes (8, 17, 18).

Indoleamine 2, 3 dioxygenase 1 (IDO1) is an intracellular tryptophan-metabolizing enzyme

that is expressed by monocytic, epithelial, and endothelial-lineage cells (18-21). Our lab and

others have shown that IDO1-driven tryptophan depletion has a significant impact on

immune cell behavior regulating inflammatory cytokine production in macrophages and

dendritic cells, inducing proliferative arrest in naïve T cells, and promoting tolerogenic

FoxP3+ regulatory T cell maturation and activation (7, 8, 22-24). Thus is it perhaps not

surprising that IDO1 regulates immunity and pathology in a wide array of inflammatory

conditions including infection, autoimmunity, and cancer (19). Interferons are the principle

mechanistic drivers of IDO1 induction, although both transforming growth factor β (TGF-β)

and toll like receptor (TLR)-dependent mechanisms can stimulate IDO1 gene activity (25,

26). IDO1 may act as a negative feed back mechanism protecting the kidney from antibody

mediated inflammatory injury (27); however, data demonstrating IDO1 expression in

inflamed kidney is not definitive and mechanisms underlying this protective effect are not

known.

Chaudhary et al. Page 2

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Amino acid deficiencies are detected by the integrated stress response kinase general control

nonderepressible 2 (GCN2) (28). GCN2 kinase signals are activated by accumulation of any

deacylated (namely, uncharged) tRNA and are required and sufficient for transduction of

intracellular amino acid limitation signals (29, 30). Phosphorylation of eIF2α substantially

reduces global protein synthesis, but translation from relatively rare mRNA species, such as

activating transcription factor 4 (ATF4), are selectively derepressed (31). ATF4, a basic

leucine zipper transcription factor, is a nodal regulator of integrated stress response-driven

transcription and manifestation of the stress response. An ATF4 target gene product, C/EBP-

β homologous protein-10 (CHOP10, CHOP), functionally cooperates with ATF4 to regulate

expression of amino acid transporters, aminoacyl tRNA synthetases, and genes involved in

autophagy suggesting a direct link between GCN2 activation and autophagy responses (32).

In this study we hypothesized inflammatory induction of IDO1 with subsequent tryptophan

depletion-driven GCN2 activation constitutes a metabolic regulatory circuit limiting

pathology in a mouse model of anti-glomerular basement membrane (GBM) disease. The

data presented below show IDO1 and GCN2-dependent regulation is a key mechanism

restricting inflammatory kidney damage and manipulation of IDO1 or GCN2 activity may

have therapeutic benefit.

Materials and methods

Mice

Female 8-10 week old C57BL6/J (B6), B6.Ido1−/−, B6.Eif2ak4tm1.2Dron (Gcn2 KO),

B6.Ifnar1−/−, and B6.Ifnγr1−/− mice were obtained from The Jackson Laboratories and

maintained under specific-pathogen-free conditions in the Georgia Regents University

animal facilities in accordance with Institutional Animal Care and Use Committee

guidelines.

Human Kidney

For patients P7 to P18: tissue was obtained from core needle biopsy taken with ultrasound

guidance. The tissue was separated and the portion of renal parenchyma reserved for

immunofluorescence studies was snap frozen in OCT compound (Tissue Tek) after

collection and stored at −70°C. All experiments were conducted with residual tissue

collected for clinical diagnostic purposes under studies and protocols approved by the

Georgia Regents University institutional review board.

For patients P1 to P5 kidney cortex samples were obtained from the national disease

research interchange (NDRI) national rare disease biospecimen resource. All kidney cortices

were collected in accordance with institutional review board guidelines and regulations.

Patient information is described in table 1.

Nephrotoxic Serum Nephritis (NTN)

Non-accelerated NTN was induced by passive transfer of preabsorbed sheep nephrotoxic

serum (NTS) intravenously in 8-10-week-old mice. Serum from the same batch was used for

all experiments. NTS dose was titrated in B6 mice to induce mild (8 μl/gm, low-dose) and



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severe (20 μl/gm, high-dose) pathology. Functional quantification of kidney function was

done by urine albumin/creatinine ratio and BUN estimation (33). Urinary albumin was

measured by mouse albumin specific ELISA (Bethyl Labs). BUN and creatinine

concentrations were measured by enzymatic colorimetric estimation (Stanbio).

Pathology and Immunofluorescence

To assess target organ pathology, kidneys were fixed with 10% formalin in PBS for 48 h and

embedded in paraffin. Kidney sections were stained with Periodic acid-Schiff (PAS) and

hematoxylin reagent. Stained kidney sections were scored for renal damage, as previously

described (34, 35). To assay for immune deposits, kidneys were embedded in optimal

cutting temperature compound (Tissue-Tek) and snap-frozen. Sections (5 μm) were air-

dried, fixed with ice cold methanol, blocked with 3% non-fat milk and stained with rabbit

anti-mouse IDO1 (kindly provided by Dr. David Munn), rabbit anti-mouse LC3 (Cell

signaling Technology), anti-synaptopodin (Santa Cruz Biotechnology), rabbit anti-mouse

cleaved caspase3 (Cell signaling Technology), or FITC conjugated rat anti-mouse CD31

(PCAM1). For immunocytochemistry, 30k to 50k podocytes were cultured in chamber slides

(BD Falcon) and fixed for 3 minutes in ice-cold methanol. To stain for actin filaments

Alexafluor-488 conjugated phalloidin (Thermo-Fisher) was used. In some experiments

TUNEL staining was done on frozen sections using commercially available kits

(Invitrogen). Fluorescent images were captured using Zeiss LSM 510 Meta confocal

microscope.

Quantification of infiltrated F4/80+, CD11c+ and CD11c+ cells in kidney tissue sections

was carried out by immunofluorescence as previously described (36). In the same

experiment, IDO1 expression by these cells was also analyzed by co-localizing IDO1

staining with the cell specific staining. In brief, kidney sections from day 5 post-NTN mice

were co-labeled with IDO1, DAPI and either of F4/80 (eBioscience), CD11c or CD11b

antibodies (BD Pharmingen). Positive cell were identified by presence of staining

surrounding the nuclei staining (DAPI). Cells were counted manually in 5 randomly selected

non-overlapping fields at 25× magnification covering minimum 2-3 glomeruli per field for

each mouse. Microscope magnification, selected area and other imaging parameters were

kept constant between the samples.

Glomerular purification

Mice were anaesthetized and perfused with 8 × 106 magnetic Dynabeads™ (Lifescience

Technologies) diluted in 20 ml of Hanks balanced salt solution (HBSS) through the left

ventricle. After perfusion, kidneys were removed, minced into approximately 1 mm3 pieces

and digested in Collagenase (1mg/ml Collagenase A [Worthington], 100 U/ml

deoxyribonuclease-I [Sigma] in RPMI media without fetal calf serum) at 37°C for 30

minutes with gentle agitation. Collagenase-digested tissue was pressed through a 100 μm

nylon cell strainer (Thermo-Fisher). The filtered was again passed through a 100 μm cell

strainer without pressing. The filtrate was then centrifuged at 200 × g for 5 min at 4°C. The

pellet was resuspended in 2 ml of HBSS. Glomeruli containing Dynabeads™ were isolated

by a magnetic particle concentrator (magnet) and isolated glomeruli were washed at least

three times with HBSS. During the entire procedure kidney tissues were kept at 4°C, except



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for the collagenase digestion at 37°C. From purified glomeruli, mRNA and protein were

extracted for quantitative PCR and western blot.

Cell culture

Conditionally immortalized mouse podocytes (37), were cultured and maintained in

collagen-I coated flasks in RPMI-1640 media supplemented with 10% FCS, 100 units/ml

penicillin/streptomycin, 10U/ml mouse recombinant IFN-γ. Confluent cells were passaged

and kept at 37°C without IFNγ to differentiate for 2 weeks in Dulbecco’s modified Eagle’s

medium containing 5% FCS, 5.5 mM glucose, and antibiotics. Podocyte differentiation was

defined by synaptopodin expression confirmed by immunocytochemistry.

Reagents

DNPs were freshly prepared before every injection by adding polyethylenimine (PEI)

(InvivoGen) with CpG−free plasmid DNA (pGiant, InvivoGen) according to the

manufacturers directions. Mice were injected i/v with 30 μg pDNA (N/P ratio of 10:1) in

200μl of 5% glucose solution. 1-Methyl-D-tryptophan (D1MT), 1-Methyl-L-tryptophan

(L1MT), or a racemic mixture of the D and L isomers (Sigma) was prepared as a 20 mM

stock and used at a final concentration of 20 μM to treat podocytes for in vitro experiments

(38). Halofuginone hydrobromide (HF) (Sigma) stored as 2 mg/ml stock solution in

methanol at −20°C was used at a final concentration of 40 ng/ml to treat podocytes for in-

vitro experiments. For in-vivo experiments, HF was resuspended in phosphate buffered

saline (PBS) (4μg/gm) and given i/v as described. 3-methyl adenine (3-MA) (Sigma)

dissolved in 1N NaOH at 10 mg/ml and diluted to 10mM in phosphate buffered saline for

in-vitro experiments and 30 mg/kg for in-vivo studies.

siRNA interference

Transient knock down of GCN2 was done using Lipofectamine™ 2000 (Invitrogen). Briefly,

for each sample in a 6 well plate, 500pmol of siRNA against Gcn2 or scrambled controls

(Santa Cruz Biotechnology) in 250 μl of Opti-MEM™ (Thermo-Fisher Scientific) media was

mixed with Lipofectamine™ 2000, and after 20 minutes the mixture was added to the

podocyte culture. After 6-8 hours of incubation, media was changed to normal media used

for differentiated podocyte culture. Inhibition of GCN2 expression was confirmed by real

time PCR and Western blot after 14-20 hours before using cells for the experiments.

Kynurenine and Trp measurements and IDO enzymatic activity

High-performance liquid chromatography (HPLC) analysis was performed as previously

described (7). Briefly, 100 μl of culture medium was diluted with 10 μl (1/10 volume) of 150

mM sodium acetate (NaAc), pH 4.0, and incubated at room temperature for 2 min. Fifty

microliters (1/4 volume) of 30% trichloroacetic acid (TCA) was then added, and sample

mixtures were incubated on ice for 5 min before being spun in a precooled (4°C) centrifuge

at 2000 × g. For HPLC, 50 μl of sample was loaded by autosampler (Beckman Coulter

model 508) and separated on a C18 (Shimadzu) column using the following conditions:

mobile phase A, 2.5% acetonitrile in 15 mM NaAc, pH 4.0; mobile phase B, 100%

acetonitrile; and flow rate, 1.2 ml/min. The mobile phase was delivered with a Beckman



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Coulter model 126 solvent module at a flow rate of 1.2 ml per minute. Kynurenine was

detected with a Beckman Coulter model 166 detector at 360 nm, and tryptophan was

detected using a Jasco FP-1520 fluorescent detector at an excitation wavelength of 285 nm

and an emission wavelength of 365 nm.

IDO activity was measured as previously described (39). Briefly, tissues were homogenized

with a Polytron homogenizer (Kinematica) in 1.5 volumes of ice-cold 0.14 M KCl-20 mM

potassium phosphate buffer (pH 7). The homogenate samples were centrifuged at 7000 × g

and 4°C for 10 min. An aliquot of supernatant was taken for the measurement of IDO

activity. The reaction mixture contained 50 μl enzyme preparation and 50 μl substrate

solution (100mM potassium phosphate buffer pH 6.5, 50μM methylene blue, 20μg catalase,

50 mM ascorbate, and 0.4 mM L-TRP). After incubation at 37°C, samples were acidified

with 3% perchloric acid and centrifuged at 7000×g, 4°C, 10 min. The concentrations of the

enzymatic products were measured by HPLC. IDO activity was calculated as picomoles

kynurenine per hour per milligram tissue.

Gene expression quantification

RNA from cells or tissue were purified using RNeasy RNA purification kits (Qiagen), and

250 ng of RNA was reverse transcribed using a random-hexamer cDNA reverse

transcription kit (Clontech). For the PCR, 1 μl of cDNA was amplified and PCR was done

using IQ Sybr green super mix (Bio-Rad) on an iQ5 real-time PCR detection system (Bio-

Rad). Results were analyzed with the accompanying software according to the

manufacturer’s instructions. PCR for mouse (or human as indicated) β-actin (i.e. Actb), 18S

ribosomal RNA, Ido1, Chop, Gadd45b, Atg3, Atg5, Il6, Ccl2, and Tnfα was done using

previously described primers (19, 22).

Immunoblotting

Western blot was done according to previously published methodology (40). Specific

antibodies against mouse Wilms’ tumor-1 and podocin were purchased from Abcam while

antibodies against LC3, cleaved caspase 3, and β-actin were purchased from Cell Signaling

Technology.

Elisa

To measure kidney cortical cytokines, renal cortices were physically separated from medulla

5 days after NTS injection, weighed, and ground with a pestle and mortar containing 5g

sterile sand (Sigma) and 100 μl of PBS+protease inhibitor cocktail (Sigma)/10mg of tissue.

The material was centrifuged to sediment the sand/ground tissue and supernatant was

collected for assessment of cytokines or chemokines indicated. All measurements were done

using commercially available reagents and done as per manufactures instructions

(eBioscience).

Quantification of LC3 puncta in vitro

Autophagosome formation was quantified by counting LC3 puncta manually. Briefly,

differentiated podocytes were plated on chamber slide and treated. At the experimental

endpoint, cells were gently washed once with PBS and fixed with methanol at −20°C for 3



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minutes. Cells were stained for LC3 and LC3+ punctate structures were counted manually in

50-80 cells. Size of the punctate was variable.

Flow cytometry

Kidney cortices were collected and minced finely with a blade. The kidneys were then

incubated with 500 U of collagenase (Sigma) 30 min at 37°C. From the digest, a single-cell

suspension was washed 3 times in PBS+1% BSA and processed for staining. To stain

hematopoietic cells in kidney, anti-CD45, anti-Ly6G, anti-F4/80, anti-CD4, anti-CD8 (all

eBioscience), anti-CD11b, anti-MHCII, and anti-CD86 (BD Pharmingen) were used.

Stained cells were formalin fixed and subjected to flow cytometry on a FACS Canto flow

cytometer (BD Bioscience). Data were analyzed with FlowJo software (Tree Star).

Statistical analysis

Means, SDs, and unpaired student t test results were used to analyze the data. When

comparing two groups, a P value of ≤ 0.05 was considered to be significant. Animal survival

data were analyzed with Kaplan–Meier survival plots followed by the log-rank test.

Results

IDO activity is protective in antibody-mediated glomerulonephritis

To elucidate the functional role of IDO1 in the nephrotoxic serum nephritis (NTN) model of

anti-GBM disease, Ido1 knockout (Ido1 KO) and C57BL/6 (B6) mice were challenged with

sheep anti-mouse nephrotoxic serum (NTS) at a dose that induced mild, self-limiting

glomerular inflammation in B6 mice (35). As expected, B6 mice developed albuminuria that

peaked on or about day 5 and then returned to baseline levels by day 21. At this dose blood

urea nitrogen (BUN) concentrations remained normal (Fig 1A) and the histology remained

normal or minimally altered (Fig 1B and IC). By contrast, Ido1 KO mice developed

fulminant nephritis evidenced by increased albuminuria and BUN concentrations compared

to NTS-injected B6 mice on day 5 and day 21 (Fig 1A). These outcomes were associated

with increased pathology in Ido1 KO mice that showed features of severe

glomerulonephritis including peri-glomerular and interstitial infiltration, fibrocellular

crescent formation, glomerular capillary thrombosis, thickened glomerular basement

membrane, and tubular damage (Fig 1B and 1C). There was progressive and fatal end-stage

renal disease by day 45 after-NTS administration in Ido1 KO mice; in contrast, all wild-type

mice survived (Fig 1D). Similarly, when mice were treated with a lethal dose of NTS, the

Ido1 KO mice had accelerated mortality, with all animals succumbing by day 15 (Fig 1D).

In the NTN model of anti-GBM disease, inflammation and pathology during the first 5 to 7

days post-injury is driven by innate inflammation mediated primarily by cellular responses

to antibody deposition precipitating infiltration of neutrophils and macrophages (41-43).

Thus the increase in proteinuria and BUN in Ido1 KO mice compared to controls at day 5

raised the possibility that IDO1 activity was induced early during the disease process

limiting NTS-driven pathology. Immunofluorescence microscopy showed IDO1 protein was

significantly increased in inflamed kidney and appeared to be primarily in glomeruli (Fig

1E). IDO1+ staining patterns in the glomeruli overlapped with staining for the podocyte



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marker synaptopodin suggesting podocytes were producing IDO1 in response to NTS-driven

inflammation (Fig 1E). In contrast, there was no overlap of IDO1 staining with the

endothelial cell marker CD31 suggesting glomerular endothelium did not express IDO1 after

NTS administration (Fig 1E). There were a few IDO1+ cells in the interstitium which co-

stained with the macrophage marker F4/80 suggesting other cell types were expressing

IDO1 in the inflamed renal parenchyma (Fig 2). To test if NTS administration induced Ido1

expression, mRNA from renal cortices and purified glomeruli was measured. We observed

that NTS administration induced a 27-fold increase in Ido1 mRNA in lysate from total renal

cortices (not shown); however, purified glomeruli showed a 10-fold enrichment in Ido1

message induction relative to whole kidney tissues (i.e. a 230-fold induction in Ido1 mRNA

in NTS-treated versus control glomeruli, Fig 1F). Thus while we cannot completely rule out

a role for monocyte-derived IDO1 in regulating the pathogenic antibody-driven renal

response, it is likely that the majority of IDO activity is contributed by glomerular stroma.

Since Ido1 gene activity can be induced by both type I and type II interferons (IFN), we

tested whether IFNs are required for Ido1 induction by NTS. Mice with a genetic disruption

of the gene coding for the IFNα receptor 1 protein fail to respond to either IFNα or IFNβ.

However, lack of type I IFN responsiveness had no effect on Ido1 mRNA induction in

glomeruli after NTS injection (Fig 1F). In contrast, glomeruli from mice lacking IFNγ

responsiveness (Ifnγr1 knockout) showed a 3-fold reduction in Ido1 mRNA compared to

NTS-injected B6 glomeruli (Fig 1F). Thus the data suggest IFNγ responsiveness is a key

mechanism driving Ido1 gene activity in NTN.

By enzymatically catabolizing tryptophan, IDO drives GCN2 activation and subsequent

elicitation of the integrated stress response. This pathway modulates inflammatory potential

and cytokine production in sterile and septic inflammatory settings (7, 8, 22). Thus we

postulated that GCN2 was required to limit antibody-driven kidney pathology.

Phenocopying the observations in Ido1 KO mice, Gcn2 knockout (Gcn2 KO) mice showed

an exaggerated response to low dose NTS with significantly increased albuminuria and

BUN concentrations at both 5 and 21 days post-injection compared to B6 mice (Fig 3A).

Gcn2 KO mice also showed marked glomerular pathology analogous to that seen in Ido1

KO mice (Fig 3B and 3C). Ultimately NTS-driven kidney inflammation progressed to

ESRD in Gcn2 KO mice with death in all members of the group in response to low dose

NTS and accelerated death when exposed to high-dose NTS (Fig 3D). This result suggests

that GCN2 is critical to limit pathology during NTN.

NTN induces rapid production of inflammatory cytokines and chemokines. Since IDO1

inhibits cytokine production in inflammatory conditions(22), we reasoned in the absence of

IDO1 mediated activation of GCN2 there would be increased inflammatory cytokine

responses in NTN. Consistent with this hypothesis, Ido1 KO and Gcn2 KO glomeruli

exhibited a significant increase in Tnfα and Il6 mRNA expression compared to controls 5

days after low dose NTS injection (Fig 4A). Accordingly, there was a 2-fold increase for

both IL-6 and TNFα protein detectible in kidney cortex homogenate as measured by ELISA

(Fig 4B). In contrast, NTS-driven induction of Tgfβ1 expression in glomeruli was lost in

Ido1 KO and Gcn2 KO glomeruli (Fig 4A). In agreement with the expression data, when

cortical TGFβ1 protein was measured there was a 2-fold increase in the NTS treated group,



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an effect that was lost in Ido1 KO animals (Fig 4B). Yet, there was not a significant

difference in TGFβ1 in Gcn2 KO cortical samples (Fig 4B). Overall induction of TGFβ1

protein was relatively low in the cortex at this time-point (i.e. 5 days after NTS injection)

likely contributing to the lack of correlation between mRNA and protein. When we

measured other cytokines, we found Ido1 and Gcn2 deletion had no impact on NTS-driven

production of IL-1β, IL-17A, or IFNγ protein in inflamed renal cortices suggesting that

IDO1 and GCN2 have a selective impact on inflammatory cytokine production in the kidney

(supplemental Fig 1).

We observed that NTS-treated Ido1 KO and Gcn2 KO glomeruli showed a significant

increase in expression and overall cortical protein abundance of the macrophage-recruiting

chemokine C-C motif ligand 2 (CCL2, also known as monocyte chemotactic protein 1,

MCP-1) (Fig 4A and 4B). Thus we examined inflamed kidney for the impact Ido1 and Gcn2

deletion had on macrophage infiltration. Both Ido1 KO and Gcn2 KO mice showed a 2-fold

increase in the presence of cells expressing the common leukocyte marker CD45 relative to

controls suggesting an increase in hematopoietic cell infiltration (Fig 5A). Neither Ido1 or

Gcn2 deficiency increased NTS driven T cell infiltration in the inflamed kidney

(supplemental Fig 2); however, there was a significant increase in CD11b+F4/80+

macrophages in Ido1 KO and Gcn2 KO kidneys compared to NTS-injected controls (Fig

5B). FACS analyses showed the infiltrating macrophages had higher surface abundance of

MHCII and CD86, indicating the cells were in a more activated state compared to controls

(Fig 5B). Taken together these data indicate that IDO1 and GCN2 are required to limit

myeloid infiltration and activation in response to renal inflammation.

GCN2 mediates the IDO1 driven amino acid stress response and autophagy in podocytes

Since our data show that IDO1 is induced by NTS primarily in the glomeruli in association

with synaptopodin+ cells (Fig 1E) and genetic deletion of the Ifnγr1 significantly reduced

Ido1 mRNA induction after NTS injection (Fig 1F), we hypothesized that IFN induced by

NTS deposition in the kidney may drive IDO1-GCN2 signaling in podocytes. We first

evaluated the impact of IFN stimulation on the induction of IDO1-dependent stress signals

in podocytes in culture. Both IFN-γ and IFN-β induced prominent Ido1 expression in

podocytes (supplemental Fig 3A and Fig 6A) and IFNγ drove significant reduction of

tryptophan concentrations in podocyte cultures, an effect that was reversible by addition of a

pharmacologic inhibitor of IDO, 1-methyl tryptophan (supplemental Fig 3B). Thus IFN-

stimulation directly induces enzymatically active IDO in podocytes. Moreover, IFNγ

induced a prominent stress response in podocytes with expression of Chop and Gadd45b

increasing 10-fold relative to baseline (Fig 6A). The stress response was dependent on IDO

and GCN2 activity since either adding D-1-methyl tryptophan (D1MT) to the cultures or

knocking down Gcn2 with siRNA significantly reduced induction of both Chop and

Gadd45b (Fig 6A). Asparagine synthetase (ASNS) is induced by amino acid deficiency in a

GCN2 and ATF4 dependent manner (44). Mirroring the pattern observed for Chop and

Gadd45b, Asns was induced by IFN-γ, and induction was abrogated by inhibition of IDO or

reduction of Gcn2 mRNA (Fig 6A). Since this podocyte cell line is maintained in an

immature state by culture at 33°C in the presence of IFNγ (37), there was the possibility that

differences in gene expression were due to altered maturation state of the podocytes after



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IFNγ simulation. However, overnight culture with IFN-γ did not change expression of the

differentiated podocyte markers Wilms’ tumor-1 (WT-1) or podocin indicating that in our

experimental system podocyte maturation status was not the driving factor in observed gene

expression patterns (supplemental Fig 4A). Thus, these data support for hypothesis that

podocytes respond to IFN-stimulation by rapid induction of IDO1 and the downstream

GCN2-dependent stress program.

IDO-GCN2 signals activate a protective autophagy response in podocytes

Integrated stress response activation initiates autophagy which reduces energy expenditure

and promotes replenishment of cellular amino acid pools (4). Moreover, in terminally

differentiated, post-mitotic cells (e.g. podocytes) autophagy is a key mechanism for cell

survival in homeostatic conditions (45) and promoters for the autophagy genes ATG3,

ATG5, BECN1, and LC3 contain motifs that can interact with the GCN2-induced proteins

ATF4 and CHOP (32). Thus, we tested the hypothesis that IDO1 activity during glomerular

inflammation drives autophagy through activation of the GCN2-driven integrated stress

response signal in podocytes. When podocytes were stimulated with IFNγ, Atg3 and Atg5

expression were significantly increased and inhibition of IDO by addition of D1MT or

supplementing culture media with excess L-tryptophan significantly attenuated both Atg3

and Atg5 mRNA induction (Fig 6B). Likewise, siRNA knockdown of Gcn2 reduced

expression of Atg3 and Atg5 suggesting that IFNγ-driven activation of podocytes induces

autophagic stress responses in podocytes by IDO-driven activation of the GCN2 pathway.

Consistent with this, stimulation with IFNγ resulted in punctate staining patterns for

microtubule-associated protein 1A/1B-light chain 3 (LC3), a characteristic feature of

autophagosome formation (Fig 6C). LC3 is cytosolic in the absence of autophagy; however,

it is rapidly modified during autophagy by the addition of phosphatidylethanolamine

generating the LC3II isoform and promoting association with autolysosomes (46, 47). When

we examined podocyte cultures by Western blot we only observed the presence of the LC3II

isoform (supplemental Fig 4B). This is consistent with Tomino and colleagues who reported

differentiated podocytes show high basal LC3II (48). Simulation of podocytes with IFNγ

resulted in loss of LC3 detectible by Western blot, an effect that was reversible by inhibition

of lysosome acidification by addition chloroquine (supplemental Fig 4B). Inhibition of IDO

by D1MT treatment, or reduction of Gcn2 expression by siRNA-mediated knock down

substantially reduced punctate LC3 staining (Fig 6C), suggesting a role for IDO and GCN2

for IFN-driven autophagy in podocytes. Halofuginone activates GCN2 by inhibiting prolyl-

tRNA sythetase (49, 50). Treating podocytes with halofuginone phenocopied the IDO-

dependent effects of IFNγ on podocytes inducing expression of Atg5 in a GCN2 dependent

manner and driving LC3 punctate staining in podocytes (supplemental Fig 4C to 4D). These

data suggest that IFNγ induces rapid autophagic responses in podocytes primarily by an IDO

and GCN2-driven pathway.

Autophagy protects podocytes from endoplasmic reticulum stress and mice lacking this

activity exhibit progressive podocyte apoptosis and severe glomerular disease. We

hypothesized GCN2-driven autophagy in response to IFNγ may be a feedback mechanism

protecting podocytes from apoptosis in the inflamed kidney. Treatment of podocytes in vitro

with the antibiotic puromycin causes time and dose dependent apoptosis via reactive oxygen



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species generation (51). Thus we tested if GCN2 activation would protect podocytes from

puromycin-driven apoptotic death. Podocyte culture with puromycin resulted in a 5-fold

increase in apoptosis; however, addition of halofuginone reduced podocyte apoptosis 2-fold

compared to controls suggesting that GCN2 signaling was protective (Fig 6E); Further

supporting a role for autophagy in prevention of apoptosis, pharmacologic inhibition of

autophagy by addition of 3-methyladenine (3-MA) (52) completely abrogated the protective

effect of halofuginone (Fig 6E). These data suggest that GCN2 dependent autophagy inhibits

podocyte apoptosis and may be protective in inflammatory settings.

To test this mechanism in vivo we injected mice with low dose NTS and examined stress

response gene and autophagy induction. Consistent with our in vitro data, low dose NTS

induced significant expression of Chop, Gadd45b, and Asns in purified glomeruli 5 days

after injection (Fig 7A). Moreover, stress gene induction was dependent on IDO1 and GCN2

as Ido1 KO and Gcn2 KO mice showed abrogated induction for all stress genes examined.

Similarly, NTS administration induced significant Ido1 and Gcn2-dependent expression of

Atg3 and Atg5 suggesting autophagy was stimulated by IDO1-mediated activation of GCN2

(Fig 7A). Consistent with this finding, LC3 immuno-staining in kidney sections showed

punctate staining specifically in podocytes in NTS-injected mice (Fig 7B), and increased

LC3I and LC3II in purified glomeruli from the NTS treatment group (Fig 7C). In contrast,

neither Ido1 or Gcn2 KO mice exhibited substantial punctate LC3 staining in

immunofluorescence imaging (Fig 7B) or increases in LC3I or LC3II detectible by Western

blot (Fig 7C). Thus, this collectively suggests a failure of inflammation-driven autophagy

induction. Next, we examined glomerular activation of the executioner caspase, caspase3,

and apoptosis 5 days after low dose NTS administration. The glomeruli of B6 mice after low

dose NTS showed little caspase3 cleavage (Fig 7E and supplemental Fig 4E) and few

TUNEL+ cells (Fig 7D) suggesting minimal apoptosis. In contrast, we observed increased

TUNEL+ cells in the glomeruli of NTS-injected Ido1 and Gcn2 KO mice compared to

controls (Fig 7D). Similarly, we observed increased cleaved caspase3 in purified glomerular

lysates (supplemental Fig 4E) and cleaved caspase3+ podocytes in renal tissue sections (Fig

7E). Thus these data suggest GCN2 and IDO1 are required to limit apoptosis associated with

inflammatory, antibody-driven glomerular disease.

The data support the hypothesis that IDO1 and GCN2 provide signals via induction of

autophagy protecting the kidney from antibody driven inflammation. Based on this, we

reasoned inhibition of autophagy would increase the pathogenicity of low dose NTS and

phenocopy genetic ablation of Ido1 and Gcn2. To test this we treated mice with 3-MA at the

time of low-dose NTS administration. Similar to Ido1 and Gcn2 KO mice, we found

inhibition of autophagy by 3-MA during the induction of NTS-driven nephritis significantly

increased proteinuria and BUN (Fig 7F). Control mice receiving low dose NTS alone

showed a non-significant elevation in proteinuria and BUN concentrations 5 days after NTS

injection that returned to baseline by day 21. In contrast, mice that had received 3-MA in

combination with low-dose NTS showed progressive, chronic proteinuria and elevated BUN

concentrations (Fig 7F). Histological alterations in 3-MA-treated mice with low dose NTS at

day 21 were similar to Ido1 and Gcn2 KO mice with fibrocellular crescents affecting 80% of

glomeruli and advanced tubular pathology (Fig 8). These findings suggest that autophagy



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induction early during NTS-driven nephritis is a critical regulatory mechanism to limit

pathology.

Manipulation of IDO1 or GCN2 activity protects the kidney from antibody-mediated injury

We next tested the hypothesis that exogenous manipulation of either IDO1 or GCN2 activity

would provide therapeutic benefit in NTS driven nephritis. Deoxyribonucleic acid (DNA)

nanoparticles (DNPs) made of polyethylenimine complexed with unmethylated cytosine-

phosphodiester-guanine motif free (CpGfree) DNA stimulate IDO activity via the stimulator

of IFN genes (STING) induced IFN-β pathway (38, 39). Because a single dose of DNPs

containing CpG-free DNA induced prominent IDO activity in the kidney (Fig 9A), we tested

their ability to limit nephritis in response to high dose NTS exposure. High-dose NTS

caused proteinuria and elevated BUN concentrations (Fig 9B) and glomerular and tubular

pathology (Fig 9C and 9D). However, treatment with DNPs significantly improved kidney

function (Fig 9B) and reduced overall renal pathology (Fig 9C and 9D). Neutrophil

gelatinase-associated lipocalin (NGAL) expression is upregulated in several models of renal

injury and may be a biomarker of chronic kidney disease (53-56). Consistent with this, NTS

injected mice showed a 27-fold increase in Ngal expression over control mice; and Ngal

mRNA induction was significantly reduced in DNP-treated mice (Fig 9E). DNP-mediated

protection required IDO1, as DNPs did not improve renal function (Fig 9B) or pathology

measurements (Fig 9C to 9E) in Ido1 KO mice. We then tested the ability of the GCN2

agonist halofuginone to protect the kidney from NTS-mediated injury. Similar to DNP

treatment, mice receiving halofuginone with high dose NTS showed marked reduction in

albuminuria and BUN concentrations relative to controls (Fig 9F). In contrast to DNPs

however, halofuginone treatment improved renal function in Ido1 KO mice suggesting

activation of GCN2 circumvents the need of IDO1 activity for protection from kidney

pathology. Moreover, we found halofuginone did not improve disease outcomes in Gcn2

KO after high dose NTS injection (Fig 9F). Thus activation of IDO1 or GCN2 can reduce

renal decline associated with inflammatory kidney disease.

IDO1 and GCN2 activity are increased in immune-mediated glomerulonephritis in human kidney

Antibody and immune complex driven glomerular inflammation is associated with IFN

production in the renal parenchyma. Since IDO1 is induced by IFNs, we hypothesized that

IDO1 induction and downstream GCN2 pathway activation is a common feature of this

disease etiology. Thus we quantified IDO1 expression and stress signal induction in a panel

of tissues isolated from renal cortices of patients diagnosed with either Goodpasture’s

syndrome (GPS, P2) or lupus (P3 to P6, for patient characteristics see table 1), both

antibody-driven glomerular nephropathies. All samples showed increases in expression of

IDO1, CHOP and ASNS relative to normal kidney suggestive of IDO1 dependent activation

of the GCN2-driven ISR (Fig 10A).

We then we examined renal biopsy tissue from patients diagnosed with GPS, lupus

nephritis, or membranous nephritis for the presence of IDO1 and autophagy activity (LC3

punctate staining, for patient information see table I). IDO1 staining was weak in healthy

human kidney tissue and there was very little LC3 staining suggesting low autophagy (Fig



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10B, P1). IDO1 staining was variable in disease tissue likely reflecting the variable

pathology assessment and disease activity at the time of collection; however several patients

showed IDO1 staining (Fig 10B). In particular, renal tissue from patients P2, P9 to P12, P15,

and P17 showed prominent diffuse presence of IDO1 in the glomeruli (Fig 10B). Moreover,

the intensity of IDO1 and LC3 staining loosely correlated such that samples that stained

prominently for IDO1 generally exhibited strong LC3II staining as well (Fig 10B). Thus the

data suggest IDO activity is induced in antibody-driven nephritic disease may drive both

stress signals and autophagy responses in the glomerulus.

Discussion

Our findings identify IFN-induced, IDO1-dependent tryptophan catabolism as a critical

mechanism that protects renal tissues from antibody mediated tissue injury. Protection was

mediated through activation of a GCN2 kinase signal that triggers a downstream metabolic

stress response inducting autophagy in podocytes. Moreover, we show that treatments to

induce IDO1 or activate GCN2 directly elicited effective protective responses to NTS,

substantiating the importance of the protection pathway.

Podocytes were crucial to the benefit observed. Antibody deposition in renal tissues incites

local inflammation that causes protein oxidation, DNA damage, and apoptosis leading to

podocyte death. Thus podocytes must engage regulatory mechanisms to compensate for

injury accompanying immune nephritis. Our report demonstrates that induction of IDO

activity during the early phases of NTN is a critical step limiting glomerular apoptosis and

inflammatory pathology. IDO is an intracellular enzyme that metabolizes both cellular and

micro-environmental tryptophan stores to suppress inflammation and regulate innate and

adaptive immunity (19). The Ido1 gene promoter contains several interferon-stimulated

response elements (ISREs) and gamma activated sequence (GAS) motifs and is thus

sensitive to interferon type I and II driven expression and enzymatic activity (57, 58). We

observed IFNγ protein is induced by day 5 after NTS administration (as seen in

supplemental Fig 3) suggesting that early IFNγ production may drive Ido1 gene activity in

podocytes. The hypothesis of IFNγ-driven IDO1 induction is supported by the finding that

Ifnγr1 KO mice showed a significant reduction in Ido1 mRNA induction after injury.

However, the extent of Ido1 message ablation was only 50% as compared to controls. This

suggests other factors are involved in IDO1 induction. One possibility is that type I IFN may

compensate for the lack of IFNγ responsiveness. Supporting this hypothesis we observed

that treatment of podocytes with type I IFN in vitro induced prominent Ido1 expression,

although the induction was 20-fold lower than induction after IFN stimulation (as shown in

sFig 3A). Similarly, systemic administration of DNPs induces IDO1 by driving type I IFN

production in vivo (38). Since we observed protection conveyed by DNPs in an IDO1-

dependent manner the data strongly suggests IDO1 induction after antibody-driven kidney

injury is dependent on upstream interferon release in response to NTS challenge.

IDO1 regulates immunity via two primary mechanisms: i) tryptophan catabolism activates

the amino acid starvation response transmitted by the GCN2 arm of the integrated stress

response, ii) some tryptophan metabolites released by cells expressing IDO (e.g.

kynurenine) activate the aryl hydrocarbon receptor to regulate T cell and monocytic cell



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function. Our data suggest that IDO1 modulated NTN via GCN2 activation since outcomes

in Gcn2 KO mice phenocopied outcomes in Ido1 KO mice, as evidenced by exaggerated

NTS-induced renal pathology, cytokine expression profiles in renal cortices, and induction

of stress and autophagy genes. Our results reveal that GCN2 provides a molecular signal

critical for limiting inflammatory pathology induced by NTS deposition in the glomeruli

demonstrating endogenous GCN2-activation, as a consequence of endogenous inflammatory

responses, drives IDO1 activity in the glomeruli playing a dominant role in limiting NTS-

mediated inflammatory pathology.

IDO1 is a potent regulator of adaptive immunity. Thus, it is likely that effects of IDO1 on

glomerulonephritic disease progression involve suppression of stromal pathology and

downstream T and B cell responses to NTS deposition. However, our data show that Ido1

gene activity is induced in the kidney at a time when adaptive responses have not yet

manifest (i.e. 5 days). Moreover functional differences in Ido1 and Gcn2 KO mice as

compared to wild type controls are evident early in the disease process indicating IDO1

driven induction of GCN2 signals are critical for renal responses to antibody injury.

In terminally differentiated podocytes stress response mechanisms play a key function in

cell survival within inflammatory environments. Podocytes exhibit a higher rate of basal as

well as inflammation-driven autophagy and mice deficient in autophagy related gene 5

(Atg5) specifically in podocytes show increased onset of relatively severe, age-related

glomerulosclerosis (5). Evidence from mouse models of non-immune glomerular disease

also supports the notion that autophagy is cytoprotective for podocytes (5, 59-62); however,

the role of autophagy in inflammatory renal disease has never been tested. Our data suggests

basal autophagy activity in podocytes is significantly increased early during the course of

NTS-driven nephritis and inhibition of autophagy drastically increases inflammatory renal

dysfunction. The GCN2 signal has broad effects on cellular phenotypes regulating

expression of genes involved in amino acid synthesis and transport, redox potential, fatty

acid production and inflammation (19). Recently, it was reported that GCN2 could drive

amino acid starvation-induced autophagy in vitro by induction of ATF4 and CHOP (32).

Moreover, stimulation of renal epithelial cells with IFNγ induced IDO dependent autophagy

in vitro (6), suggesting autophagy genes are an biologic target of the GCN2 signal. Yet these

results were derived from artificial restriction of protein/ amino acids in the diet or from in

vitro culture experiments, thus the role of autophagy in renal disease is not clear. Our results

strongly suggest autophagy induction in podocytes is a requisite regulatory feedback

mechanism in renal inflammation; moreover, we show that IDO1 induced GCN2 signaling

is a obligatory driver of autophagy in response to antibody injury as genetic ablation of IDO

or GCN2 activity led to loss of the autophagic response, causing relatively mild

inflammation to convert into severe end-stage renal disease.

In summary, this study shows IDO1-driven activation of GCN2-dependent signals is a

required for induction of autophagy and ultimately podocyte survival and suppression of

inflammatory pathology associated with glomerulonephritis. Moreover we demonstrate that

induction of IDO or GCN2 activity improves kidney function after antibody injury

suggesting therapeutic targeting to enhance this regulatory circuit is possible. In the future it

will be critical to determine the full extent to which IDO and GCN2 modulate inflammatory



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pathology in human disease. Nevertheless, given the cytoprotective effect of GCN2

activation it is likely manipulation of the GCN2-driven metabolic signal will show efficacy

in a range of inflammatory renal diseases.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgements

The authors would like to thank Dr. David Munn for comments and advice during the development of this research report.

Funding. This work is supported by NIAID grants AI092213, AI099043, and AI105500 (TLM) and NIDDK grant DK081140 (MPM).

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Figure 1. Ido1 induction is a key feedback mechanism limiting anti-glomerular basement membrane antibody driven nephritisMild nephrotoxic serum nephritis was induced in wild type and Ido1 KO mice by injection

of low-dose NTS. (A) Functional status of kidney was assessed by albuminuria (albumin/

creatinine ratio, ACR) and blood urea nitrogen (BUN) measurement in urine and blood

samples. (B) Paraffin embedded kidney sections stained with periodic acid–Schiff reagent

and hematoxylin. Images are representative of 5 mice per group. Bar= 100 μm. (C) Glomerular and tubular pathology of mice from A was scored in a blinded manner. For % of

glomeruli with crescent determination at least 100 glomeruli were examined. (D) Survival of

wild type and Ido1 KO mice challenged with low and high dose NTS. (E) Kidney

cryosections from mice 5 days after low-dose NTS injection were stained for IDO1 (red)

and the podocyte antigen synaptopodin (synp, green) or the endothelial antigen CD31

(green). Bar= 100 μm. (F) Ido1 mRNA from purified glomeruli was measured by rtPCR 5

days after low dose NTS injection from B6 mice with the genotypes indicated. For A, C, and

F *=p<0.05 and **=p<0.01 as determined by student t test. For A, C, and F n=5 mice per

group and the error bars indicate standard deviation (SD). For D, n=8 to 12 mice per group.

Each experiment shown was repeated three times with similar results.



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Figure 2. Ido1 deficiency increases renal macrophage and dendritic cell accumulation in response to low-dose NTSMice of the genotype indicated were injected with low-dose NTS. 5 days later kidney was

collected and frozen sections were examined by immunofluorescence analysis for the

presence of CD11c+ dendritic cells, F4/80+ macrophages and CD11b+ myeloid cells. The

number of cells positive for each marker was determined as described in Materials and

Methods. Images are representative for 5 mice per group. Bars to the right are the mean

values for 5 mice +/− the standard deviation. **= p<0.01 as determined by the student T-

test. Bars= 100 μm. Experiment was repeated three times with similar results.



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Figure 3. GCN2 is required to restrict NTS-driven glomerulopathy(A) Mice of the indicated genotype were injected with low-dose NTS and proteinuria and

blood urea nitrogen were assessed as described in Fig 1. (B) Paraffin embedded kidney

sections stained with periodic acid–Schiff reagent and hematoxylin. Images are

representative of 5 mice per group. Bar= 100 μm. (C) Glomerular and tubular pathology of

mice from A was scored in blinded manner as described in methods. (D) Survival of wild

type and GCN2KO mice challenged with low and high dose NTS, determined over a period

of 20 to 60 days. For A to C *=p<0.05 and **=p<0.01 as determined by student t test. For

graphs n=5 mice per group and the error bars indicate standard deviation (SD). For D, n=8

mice per group. Each experiment shown was repeated at least three times with similar

results.



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Figure 4. IDO1-GCN2 signaling restricts cytokine production during glomerular inflammation(A) Expression of CCL-2, TNF-α, and IL-6 mRNA was quantified in isolated glomeruli 5

days after low dose NTS injection. Results are normalized for β-actin expression as

previously described (22). (B) Kidney cortical tissue homogenates from mice treated as

described in A were analyzed for the indicated proteins by ELISA. *=p<0.05 and **=p<0.01

as determined by student t test. For graphs n=5 mice per group and the error bars indicate

standard deviation (SD).

Experiments were repeated three times with similar results.



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Figure 5. IDO1 and GCN2 restrict macrophage infiltration and activation in the kidney after antibody-driven injuryMice of the indicated genotype were injected with low-dose NTS and 5 days later kidney

was collected for FACS analysis of infiltrates. (A) Samples were gated based on size and

granularity and the extent of CD45+ hematopoietic cell infiltrate was determined by FACS

analysis. (B) Representative dot plot gated on the CD45+Ly6G− cell population stained for

the macrophage marker F4/80 and CD11b. MFI-mean fluorescence intensity. *=p<0.05 and

**=p<0.01 as determined by student t test. For graphs n=5 mice per group and the error bars

indicate standard deviation (SD). The experiment was repeated three times with similar

results. MFI- mean fluorescence intensity. Cell # is the number of cells detected per kidney.



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Figure 6. IDO1-dependent activation of GCN2 is required for IFN-driven induction of podocyte autophagy in vitro(A) Podocytes cultures were treated with IFN-γ in the presence or absence of D1MT or

siRNA against Gcn2 and mRNA for indicated genes was measured by rtPCR. (B) Podocyte

cultures treated as A were assessed for Atg3 and Atg5 mRNA expression. In some groups L-

Trp was added to offset the effects of IDO activity. (C) Podocytes were treated as described

in A and autophagosome formation was assessed by immunofluorescence staining for the

autophagy marker LC3 (red, punctate staining) and counterstained for actin (green) and

nuclear DAPI staining. Some groups were treated with D1MT or siRNA against GCN2.

Quantification of LC3 puncta was done as described in Methods. (D) Podocyte cultures

were treated with the GCN2 agonist halofuginone (HF) and Atg3 mRNA expression was

determined by rtPCR. Gcn2 knockdown with siRNA or control scrambled siRNA treatment

was done as in A. (E) Podocyte apoptosis was induced by treatment with puromycin +/−HF

or 3-MA. Apoptosis was quantified by annexin-V staining through FACS analysis. For

mRNA analysis all samples were normalized against β-actin. For graphs n=5 biologic

samples per group and the error bars indicate standard deviation. Experiments were least



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three times with similar results. *=p<0.05, **= p<0.01, ns= not significant as determined by

the student t test. Experiments were repeated four times with similar results.



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Figure 7. Antibody mediated nephritis induces an IDO1 and GCN2 dependent stress response and autophagy in vivo(A) mRNA expression was measured by rtPCR in purified glomeruli from mice of the

indicated genotype 5 days after injection with low-dose NTS. Values were normalized for β-

actin expression. Graphs show means from n=5 mice per group and the error bars indicate

standard deviation (SD). (B, D, and E) Frozen kidney sections collected from mice treated

as in A were stained for markers indicated and examined by immunofluorescence

microscopy. Images are representative of 5 mice per group. Bar= 25 μm (C) Glomeruli from

mice treated as in A were isolated and lysates were tested for LC3 and β-actin by Western

blot. For each group 2 representative samples are shown out of 5 mice per group. (F) B6

mice were injected with 3-MA at the same time as injection with low-dose NTS. Functional

status of kidney was then assessed by albuminuria (albumin/creatinine ratio, ACR) and

blood urea nitrogen (BUN) in urine and blood samples. Graphs show means from n=5 mice

per group and the error bars indicate standard deviation (SD). *=p<0.05, **= p<0.01, ns=

not significant as determined by the student t test. All experiments were repeated three or

more times with similar results. Synp= synaptopodin.



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Figure 8. Autophagy inhibition increases kidney pathology after low-dose NTS administrationB6 mice were injected with 3-MA (30mg/kg i/p) at day −2, −1, 0, +1, and +2 relative to i/v

injection of low-dose NTS. Kidneys were collected at day 21 post-NTS administration and

paraffin embedded kidney sections were stained with periodic acid–Schiff reagent and

hematoxylin. The tissue from mice injected with 3-MA and NTS exhibited extensive

nephritis with crescent formation and numerous tubular casts associated with tubule dilation

and necrosis. Images are representative for 5 mice per group. Bars= 100 μm. Experiment

was repeated three times with similar results.



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Figure 9. IDO or GCN2 induction decreases severity of renal pathology in antibody-mediated inflammation(A) IDO enzymatic activity in kidney was measured ex vivo 24 hours after administration of

DNA-nanoparticles conjugates (DNP). (B) Mice of the indicated genotype were injected

with DNPs and high dose NTS as described in methods. Functional status of kidney was

assessed by albuminuria (albumin/creatinine ratio, ACR) and blood urea nitrogen (BUN)

measurement in urine and blood samples. (C) Paraffin embedded kidney sections stained

with periodic acid–Schiff reagent and hematoxylin. Images are representative of 5 mice per

group. Bar= 100 μm. (D) Glomerular and tubular pathology of mice from B was scored in

blinded manner. (E) The expression of neutrophil gelatinase-associated lipocalin (NGAL)

was determined by rtPCR in mRNA from renal cortices of mice after injection of high-dose

NTS and treatment with DNPs as in A. Values are relative to baseline NGAL expression in

B6 controls and normalized against β-actin. (F) Mice of the indicated genotype were

injected with halofuginone (HF) on before, at the same time, or after injection of high dose

NTS. Functional status of kidney was assessed by albuminuria (albumin/creatinine ratio,

ACR) and blood urea nitrogen (BUN) measurement in urine and blood samples. Bars and

line graph data points show mean values from 5 mice per group and error bars indicate SD.



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*=p<0.05, **=p<0.01, ns= not significant as determined by the student t test. Experiments

were repeated three times with similar results.



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Figure 10. IDO1 expression, stress signals, and autophagy are increased in human antibody mediated

renal disease. (A) mRNA expression was measured by rtPCR in renal cortices of kidneys

collected post mortem from patients with lupus or Goodpasture’s syndrome and normalized

against 18S RNA. Bars are relative value from individual tissue samples measured in 3

separate experiments. (B) Frozen renal sections from biopsies obtained from patients

diagnosed with lupus nephritis, membranous glomerulonephritis, or Goodpasture’s

syndrome were stained for IDO1 and the autophagy marker LC3. Bars= 100 μm. For patient

information see table 1.



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Table IDemographic information and disease diagnosis of patients. AA-African American

PatientID

Sex Race AGE Disease Diagnosis

P1 Female Caucasian 72 Healthy control

P2 Male Caucasian 37 Goodpasture’s syndrome

P3 Female Caucasian 82 Lupus




P7 Female AA 11 Lupus nephritis class II and class V

P8 Female AA 50 Lupus nephritis class III (A) and class V

P9 Male AA 11 Lupus nephritis class II

P10 Female AA 29 Lupus nephritis class III (A/C)

P11 Female AA 25 Lupus nephritis class IV-G (A/C)

P12 Female AA 52 Lupus nephritis IV-S (C)

P13 Male AA 33 Lupus nephritis class III (A/C)

P14 Female AA 28 Lupus nephritis class IV-S (A)

P15 Male AA 28 Membranous glomerulonephritis

P16 Female AA 65 Membranous glomerulonephritis

P17 Female AA 22 Membranous glomerulonephritis

P18 Male AA 49 Membranous glomerulonephritis


Documents

Kapil Chaudhary HHS Public Access 1 Rahul Shinde1 Haiyun ......Amino acid metabolism inhibits antibody-driven kidney injury by inducing autophagy Kapil Chaudhary1, Rahul Shinde1, Haiyun