FREE LIGHT CHAIN-INDUCED PROXIMAL TUBULE INJURY IS MEDIATED VIA - - PowerPoint PPT Presentation

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International Kidney and Monoclonal Gammopathy Research Group Fourth International IKMG Meeting

FREE LIGHT CHAIN-INDUCED PROXIMAL TUBULE INJURY IS MEDIATED VIA TOLL-LIKE RECEPTORS 2/4/6

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Vecihi Batuman, MD FASN Huberwald Professor of Medicine Tulane Unvisersity School of Medicine

Tulane University

DISCLOSURES

◼ I do not have a relationship with a for-profit organization to

disclose

◼ This research is supported by a grant from the Paul Teschan

Research Foundation (VB); and a Merit Review grant from the Veterans Affairs Department (PWS).

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Background

➢ Multiple myeloma (MM): malignancy of terminally differentiated plasma cells ➢ Renal involvement in MM is around 50% but often recognized late and leads to worse prognosis ➢ Inflammatory pathways triggered by the endocytosis of FLCs in the proximal tubule cells play a

significant role in the pathophysiology of FLC-associated kidney injury (KI).

➢ The role of innate immunity mediated by Toll-like receptors (TLR) in FLC-associated KI has not

been studied

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Cytokines TLRs (TLR2/3/4/6/9) Kidney Injury in MM Patients PROXIMAL TUBULE ?

complex Cubilin-megalin

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➢ Excessive endocytosis of FLCs cause proximal tubule injury by inducing production of inflammatory cytokines leading to a cascade of inflammatory pathways. ➢ The FLC-induced inflammation is mediated by activation

• f TLRs through generation of damage-associated

molecular patterns (DAMPs) released from the injured proximal tubule cells.

Hypotheses

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Methods

Blocking FLC endocytosis and TLR signaling molecules in PTCs exposed with different FLCs to confirm causal role of a specific TLR Chart review and urine collection from MM patients with renal involvement Isolation, purification and identification of urinary FLCs (Six different FLCs collected to date) Investigation of the cytotoxic effects of FLCs on kidney proximal tubule cells (PTCs) (Human cell lines RPTECs and HK2 were used for in-vitro studies) Screen kidney injury biomarkers (NGAL, KIM1, LCN and IL18) and TLRs protein and gene expression in PTCs exposed FLCs

Inclusion criteria: Age:18+ years, Male/ female with Myeloma kidney and from Tulane University Hospital, New Orleans, LA and Memorial Sloan-Kettering Cancer Center, New York, NY. Exclusion criteria: Ongoing dialysis. Vulnerable subjects e.g.: children, prisoners, and cognitively impaired subjects. Sample Size: 10-20

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Results

❖ To explore the FLCs induced Kidney Injury (KI) biomarkers; we exposed Human Proximal tubule cells (PTCs) to FLCs obtained from the urine of MM patients to assay for molecular signatures K and λ FLC exposure to Human proximal tubule cells causes cellular injury evident by the increased expression of known KI marker LCN2 (DAPI LCN2 ) Untreated (control) Proximal tubule cells (A) and RPTECs exposed to FLCs: Κ (B) and λ (C). A B C

Effect of FLCs on kidney proximal tubule cells

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Screening of candidate gene expressions for Kidney Injury in PTC exposed with 6 different FLCs (N=5)

Up-regulated genes: TNFα TLR4 MYD88 IL6 LCN2 CUBN TLR2 IL1-β Down-regulated genes: TP53 BCL2 κ1 κ2 κ3 λ1 λ2 λ3 No LC 25µM

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FLCs significantly upregulated expression of TLRs 2, 4, 6 and their downstream adaptor protein molecules MYD88 and TRIF

TLR4 gene expression

TLR4 Gene expression (Fold Change)

None 1  2  3  1  2  3  1 2 3 4

* * * * * *

TLR2 Gene expression (Fold Change)

None 1  2  3  1  2  3  0.0 0.5 1.0 1.5 2.0 2.5

TLR 2 gene expression

* * *

TLR 6 expression

TLR6 Gene expression (Fold Change)

None 1  2  3  1  2  3  0.0 0.5 1.0 1.5 2.0

* *

MYD88 expression

MYD88 Gene expression (Fold Change)

N

• n

e 1   2   3   1   2   3   0.0 0.5 1.0 1.5 2.0 2.5

* * * * *

TRIF expression

TRIF gene expression (Fold Change)

None 1  2  3  1  2  3  0.0 0.5 1.0 1.5 2.0

* *

None κ1 κ2 κ3 λ1 λ2 λ3

TLR4 β-Actin TLR2 TLR6

N=5; *P<0.05 (one-way ANOVA followed by Tukey’s multiple comparisons test)

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Release of HMGB1 into the medium from cells exposed to the FLCs. *p < 0.05

How do FLCs activate TLRs? The role of Damage Associated Molecular Patterns (DAMPs)– HMGB1

Vehicle λ1 λ2 λ3 κ1 κ2 κ3 BSA

* * * * * *

Role of HMGB1 in FLCs induced TLR2/4/6 expression

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Extracellular HMGB1 enhanced TLR2/4/6 expression and HMGB1 inhibitor (siRNA) decreased FLC-induced TLR2/4/6 expression

TLR6/-Actin

C 25  25  1 A A  A  0.0 0.5 1.0 1.5 2.0 2.5

* *

# #

K-LC λ-LC EC HMGB1 Control siRNA HMGB1siRNA

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K-LC λ-LC EC HMGB1 Control siRNA HMGB1siRNA

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TLR4 gene expression (FC) C 2 5 2 5 C E C C h h O 2 4 6 8

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K-LC λ-LC EC HMGB1 Control siRNA HMGB1siRNA

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TLR2/-Actin

C K 1 L 1 1 A A A 1 2 3 4

* # *

C 1 2 1 C 1 2 0.0 0.5 1.0 1.5 2.0 2.5

* * *

# # TLR4/-Actin

N=4; *P<0.05; #P<0.05; *Compared with No LC;

#Compared with FLC (k or λ)

(one-way ANOVA followed by Tukey’s multiple comparisons test)

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TLR inhibitor GIT-27 and pooled TLR2/4/6-siRNA reduced FLC-induced TNFα release

TLR2/-Actin

C _ 2 5 2 5  2 7 2 7 2 7  0.0 0.5 1.0 1.5

# #

* *

C _ 2 5 2 5  2 7 2 7 2 7  0.0 0.5 1.0 1.5 2.0 2.5

* *

# # TLR6/-Actin TLR4/-Actin

C _ 2 5 2 5  2 7 2 7 2 7  0.0 0.5 1.0 1.5 2.0

* *

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K-LC λ-LC GIT27

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TLR4 β-Actin TLR2 TLR6 β-Actin β-Actin

TNFa Gene expression (FC)

C C 2 4 6 4/6 0.0 0.5 1.0 1.5 2.0 2.5

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K-LC Control siRNA TLR2 siRNA TLR4siRNA TLR6 siRNA

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TNF-α (pg/ml)

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N=3; *P<0.05; #P<0.05;***P<0.001 *Compared with No LC; #Compared with FLC (k or λ) (one-way ANOVA followed by Tukey’s multiple comparisons test)

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Blocking endocytosis with hypertonic sucrose (0.25M) and Bafilomycin A1 inhibited FLC-induced TNFα release

N=3; *P<0.05; #P<0.05; *Compared with No LC; #Compared with FLC (k or λ) (one-way ANOVA followed by Tukey’s multiple comparisons test) * * *

# # # #

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Silencing Megalin and Cubilin in PTCs ameliorates FLC effects on TLR4 and TNFa gene expressions

K-LC Control siRNA Megalin-siRNA Cubilin-siRNA

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n e 2 5 A 2 5 5 5 0.0 0.5 1.0 1.5 2.0 2.5

TLR4 Gene expression (FC)

ne 25 NA 25 25 25 1 2 3 4

TNFα Gene expression (FC)

K-LC Control siRNA Megalin-siRNA Cubilin-siRNA

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N=3; *P<0.05; #P<0.05; *Compared with No LC; #Compared with FLC (k or λ) (one-way ANOVA followed by Tukey’s multiple comparisons test) *

# # # #

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Summary

NF-kB

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❖FLCs are toxic to PTCS and they induce production of inflammatory cytokines ❖FLCs upregulate expressions of TLRs 2, 4 and 6 and their adaptor protein molecules MYD88 and TRIF. ❖HMGB1 is the major DAMP responsible for FLC induced stimulation of TLRs. ❖Blocking endocytosis mitigates FLC toxicity on PTCs. ❖Innate immunity appears to play an important role in FLC-mediated tubule injury in myeloma.

Conclusions

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Acknowledgement

GRANT SUPPORT: Supported by Paul Teschan Research Fund (VB), VA Merit Review (PWS) RESEARCH TEAM: Rohit Upadhyay: Post-doctoral fellow, Nephrology Section, Tulane University School of Medicine Paul W Sanders: Dept. of Medicine, Division of Nephrology, University of Alabama- School of Medicine. Edgar A. Jaimes: Department of Medicine, Renal Service, Memorial Sloan Kettering Cancer Center, New York, NY Hana Safah: Department of Medicine (Hematology/Oncology),Tulane University Medical Center, New Orleans, LA, USA

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Thank You