Tulane University 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 Vecihi Batuman, MD FASN Huberwald Professor of Medicine Tulane Unvisersity School of Medicine 1 1
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). 2
Background Tulane University ➢ 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 PROXIMAL TUBULE ? TLRs (TLR2/3/4/6/9) Kidney Injury in Cytokines MM Cubilin-megalin Patients complex 3
Tulane University Hypotheses ➢ 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 of TLRs through generation of damage-associated molecular patterns (DAMPs) released from the injured proximal tubule cells. 4
Methods Tulane University Chart review and urine collection from MM patients with renal involvement Inclusion criteria: Age:18+ years, Male/ female with Myeloma kidney Exclusion criteria: Ongoing dialysis. Vulnerable subjects and from Tulane University Hospital, New Orleans, LA and Memorial e.g.: children, prisoners, and cognitively impaired subjects. Sloan-Kettering Cancer Center, New York, NY. Sample Size: 10-20 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 Blocking FLC endocytosis and TLR signaling molecules in PTCs exposed with different FLCs to confirm causal role of a specific TLR 5
Results Tulane University Effect of FLCs on kidney proximal tubule cells ❖ 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 A B C 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). 6
Screening of candidate gene expressions for Kidney Injury Tulane University in PTC exposed with 6 different FLCs (N=5) Up-regulated genes: TNF α TLR4 MYD88 IL6 LCN2 CUBN TLR2 IL1- β Down-regulated genes: TP53 BCL2 κ 1 κ 3 λ 1 λ 2 λ 3 κ 2 No LC 25µM 7
FLCs significantly upregulated expression of TLRs 2, 4, 6 and their downstream Tulane University adaptor protein molecules MYD88 and TRIF TLR2 Gene expression (Fold Change) TLR6 Gene expression (Fold Change) TLR4 Gene expression (Fold Change) TLR 6 expression TLR4 gene expression TLR 2 gene expression 2.5 2.0 4 * * * * * 2.0 * 1.5 3 * * * 1.5 * * 1.0 2 1.0 0.5 1 0.5 0.0 0.0 0 None 1 2 3 1 2 3 None 1 2 3 1 2 3 None 1 2 3 1 2 3 None κ 1 κ 2 κ 3 λ 1 λ 2 λ 3 MYD88 Gene expression (Fold Change) TRIF gene expression (Fold Change) TRIF expression MYD88 expression 2.0 2.5 TLR4 * 2.0 * * 1.5 * * * * TLR2 1.5 1.0 1.0 TLR6 0.5 0.5 β -Actin 0.0 0.0 e 1 2 3 1 2 3 n None 1 2 3 1 2 3 o N 8 N=5; *P<0.05 (one- way ANOVA followed by Tukey’s multiple comparisons test)
Role of HMGB1 in FLCs induced TLR2/4/6 expression Tulane University How do FLCs activate TLRs? The role of Damage Associated Molecular Patterns (DAMPs) – HMGB1 * * * * * * Vehicle λ 1 λ 2 λ3 κ1 κ2 κ3 BSA Release of HMGB1 into the medium from cells exposed to the FLCs. *p < 0.05 9
Extracellular HMGB1 enhanced TLR2/4/6 expression and HMGB1 inhibitor Tulane University (siRNA) decreased FLC-induced TLR2/4/6 expression 4 2.5 * * 2.0 3 * TLR2/ -Actin TLR6/ -Actin * 1.5 2 # 1.0 8 # # TLR4 gene expression (FC) 1 0.5 * 6 * 0 0.0 K-LC - + - - - + - C 1 1 1 A A A C 25 25 A A 1 A K L 2.5 4 λ -LC * - - + - - - + * * * 2.0 - - - + - - - EC HMGB1 TLR4/ -Actin 2 - - - - + - - # Control siRNA # 1.5 # - - - - - + + # HMGB1siRNA 0 1.0 C 5 5 C C h h O C 2 2 E K-LC - + - - + - - + - 0.5 λ -LC - - + - - + - - + N=4; *P<0.05; # P<0.05; EC HMGB1 0.0 - - - + + + - - - K-LC *Compared with No LC; - + - - - + - C 1 2 1 2 1 C - - - - - - + - - Control siRNA λ -LC # Compared with FLC (k or λ ) - - + - - - + - - - - - - - + + HMGB1siRNA - - - + - - - (one-way ANOVA followed by EC HMGB1 - - - - + - - Tukey’s multiple comparisons Control siRNA - - - - - + + test) HMGB1siRNA 10
TLR inhibitor GIT-27 and pooled TLR2/4/6-siRNA reduced FLC-induced TNF α release Tulane University TNFa Gene expression (FC) 2.5 TLR2 TLR6 TLR4 * 2.0 β -Actin β -Actin β -Actin 1.5 # 1.5 2.0 2.5 1.0 * # * # * * * * 2.0 0.5 1.5 TLR2/ -Actin TLR4/ -Actin TLR6/ -Actin 1.0 # 1.5 0.0 # # 1.0 # - + + + + + K-LC C C 2 4 6 4/6 # 1.0 # 0.5 - + - - - - Control siRNA 0.5 0.5 - - + - - + TLR2 siRNA 0.0 0.0 0.0 - - - + - + TLR4siRNA - + - - + - K-LC - + - - + - - + - - + - C 5 5 7 7 C 5 5 C 5 5 7 7 7 7 7 7 7 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 _ _ _ - - - - + + TLR6 siRNA - - + - - + - - + - - + - - + - - + λ -LC - - - + + + - - - + + + - - - + + + GIT27 TNF- α (pg/ml) 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) *** *** *** *** 11
Blocking endocytosis with hypertonic sucrose (0.25M) and Bafilomycin A1 Tulane University inhibited FLC-induced TNF α 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) 12
Silencing Megalin and Cubilin in PTCs ameliorates FLC effects on TLR4 and Tulane University TNFa gene expressions TNF α Gene expression (FC) TLR4 Gene expression (FC) 4 2.5 * 2.0 3 * 1.5 2 1.0 # # 1 # # 0.5 # 0.0 0 K-LC K-LC - + - + + + - + - + + + ne 25 e 5 A 5 NA 25 25 5 5 25 n 2 2 - - + - - - - - + - - - Control siRNA Control siRNA - - - + - + - - - + - + Megalin-siRNA Megalin-siRNA - - - - + + - - - - + + Cubilin-siRNA Cubilin-siRNA 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) 13
Summary Tulane University NF-kB 14
Tulane University Conclusions ❖ 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. 15
Tulane University 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 16
Tulane University Thank You 17
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