Galectin 3 protects from cisplatin-induced acute kidney injury by promoting TLR-2-dependent activation of IDO1/Kynurenine pathway in renal DCs

Abstract

Strategies targeting cross-talk between immunosuppressive renal dendritic cells (DCs) and T regulatory cells (Tregs) may be effective in treating cisplatin (CDDP)-induced acute kidney injury (AKI). Galectin 3 (Gal-3), expressed on renal DCs, is known as a crucial regulator of immune response in the kidneys. In this study, we investigated the role of Gal-3 for DCs-mediated expansion of Tregs in the attenuation of CDDP-induced AKI. Methods: AKI was induced in CDDP-treated wild type (WT) C57BL/6 and Gal-3 deficient (Gal-3^-/-) mice. Biochemical, histological analysis, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, real-time PCR, magnetic cell sorting, flow cytometry and intracellular staining of renal-infiltrated immune cells were used to determine the differences between CDDP-treated WT and Gal-3^-/- mice. Newly synthesized selective inhibitor of Gal-3 (Davanat) was used for pharmacological inhibition of Gal-3. Recombinant Gal-3 was used to demonstrate the effects of exogenously administered soluble Gal-3 on AKI progression. Pam3CSK4 was used for activation of Toll-like receptor (TLR)-2 in DCs. Cyclophosphamide or anti-CD25 antibody were used for the depletion of Tregs. 1-Methyl Tryptophan (1-MT) was used for pharmacological inhibition of Indoleamine 2,3-dioxygenase-1 (IDO1) in TLR-2-primed DCs which were afterwards used in passive transfer experiments. Results: CDDP-induced nephrotoxicity was significantly more aggravated in Gal-3^-/- mice. Significantly reduced number of immunosuppressive TLR-2 and IDO1-expressing renal DCs, lower serum levels of KYN, decreased presence of IL-10-producing Tregs and significantly higher number of inflammatory IFN-γ and IL-17-producing neutrophils, Th1 and Th17 cells were observed in the CDDP-injured kidneys of Gal-3^-/- mice. Pharmacological inhibitor of Gal-3 aggravated CDDP-induced AKI in WT animals while recombinant Gal-3 attenuated renal injury and inflammation in CDDP-treated Gal-3^-/- mice. CDDP-induced apoptosis, driven by Bax and caspase-3, was aggravated in Gal-3^-/- animals and in WT mice that received Gal-3 inhibitor (CDDP+Davanat-treated mice). Recombinant Gal-3 managed to completely attenuate CDDP-induced apoptosis in CDDP-injured kidneys of Gal-3^-/- mice. Genetic deletion as well as pharmacological inhibition of Gal-3 in renal DCs remarkably reduced TLR-2-dependent activation of IDO1/KYN pathway in these cells diminishing their capacity to prevent transdifferentiation of Tregs in inflammatory Th1 and Th17 cells. Additionally, Tregs generated by Gal-3 deficient DCs were not able to suppress production of IFN-γ and IL-17 in activated neutrophils. TLR-2-primed DCs significantly enhanced capacity of Tregs for attenuation of CDDP-induced AKI and inflammation and expression of Gal-3 on TLR-2-primed DCs was crucially important for their capacity to enhance nephroprotective and immunosuppressive properties of Tregs. Adoptive transfer of TLR-2-primed WTDCs significantly expanded Tregs in the kidneys of CDDP-treated WT and Gal-3^-/- recipients resulting in the suppression of IFN-γ and IL-17-driven inflammation and alleviation of AKI. Importantly, this phenomenon was not observed in CDDP-treated WT and Gal-3^-/- recipients of TLR-2-primed Gal-3^-/-DCs. Gal-3-dependent nephroprotective and immunosuppressive effects of renal DCs was due to the IDO1-induced expansion of renal Tregs since either inhibition of IDO1 activity in TLR-2-primed DCs or depletion of Tregs completely diminished DCs-mediated attenuation of CDDP-induced AKI. Conclusions: Gal-3 protects from CDDP-induced AKI by promoting TLR-2-dependent activation of IDO1/KYN pathway in renal DCs resulting in increased expansion of immunosuppressive Tregs in injured kidneys. Activation of Gal-3:TLR-2:IDO1 pathway in renal DCs should be further explored as new therapeutic approach for DC-based immunosuppression of inflammatory renal diseases.

Keywords: 3-dioxygenase-1; Galectin 3; Indoleamine 2; Toll-like receptor-2; cisplatin-induced acute kidney injury; renal dendritic cells.

Figure 1

Genetic deletion of Gal-3 significantly aggravates CDDP-induced AKI, apoptosis and inflammation. WT and Gal-3^-/- mice were injected with a single, intraperitoneal dose of CDDP (16 mg/kg body weight). Acute renal failure was aggravated in Gal-3^-/- mice 72 h after CDDP treatment, as evidenced by increased concentration of urea and creatinine (A), higher histological score (B) and more severe tubular epithelial cell injury, tubular dilation, and intra-tubular cast formation in the cortex of CDDP-injured kidneys (C). Genetic deletion of Gal-3 significantly increased apoptosis in CDDP-injured kidneys as evidenced by increased expression of Bax and caspase-3 and decreased expression of Bcl-2 (D). CDDP treatment significantly up-regulated expression of Gal-3 (E) and TLR-2 (F). Gal-3 deletion significantly attenuated CDDP-induced increase of TLR-2 in the kidneys and significantly increased expression of AGEs (G). Significantly increased serum levels of Th1 and Th17-promoting inflammatory cytokines (IL-12, IL-1β, IL-6 and IL-23) (H) and decreased levels of immunosuppressive IL-10 (I) and KYN (J) were observed in CDDP-treated Gal-3^-/- animals. Data from two individual experiments with 8 mice per group are shown as Mean ± SEM; *p<0.05, **p<0.01,***p<0.001.

Figure 2

Davanat significantly aggravated CDDP-induced AKI in WT animals while rGal-3 attenuated renal injury and inflammation in CDDP-treated Gal-3^-/- mice. Davanat was intraperitoneally injected in CDDP-treated WT animals (100 μg/day), for three consecutive days before CDDP administration (16 mg/kg body weight). Gal-3^-/- mice received single intravenous injection of rGal-3 (5 μg), 24 hours before CDDP administration. Davanat significantly enhanced CDDP-induced AKI in WT mice, as evidenced by elevated serum levels of urea and creatinine (A) and significantly increased histological score (B). Histological analysis revealed more severe tubular epithelial cell injury in the cortex of the kidneys of CDDP+Davanat-treated WT mice (C). Expression of pro-apoptotic Bax and caspase-3 were significantly higher and expression of anti-apoptotic Bcl-2 was significantly lower in the kidneys of CDDP+Davanat-treated WT mice compared to CDDP-only-treated WT animals (D).Serum levels of urea and creatinine (E), histological score (F), injury of tubular epithelial cells (G) and expression of pro-apoptotic Bax and caspase-3 (H) were significantly lower, while expression of anti-apoptotic Bcl-2 was significantly higher (H) in the kidneys of CDDP+rGal-3-treated Gal-3^-/- mice. Individual data points with Mean, obtained in one experiment with 6 mice per group; *p<0.05, **p<0.01,***p<0.001.

Figure 3

Gal-3 deficiency promoted development of inflammatory phenotype in renal-infiltrated neutrophils and macrophages. Bar graphs (left panels) and representative density plots obtained by flow cytometry analysis and intracellular staining of renal-infiltrated neutrophils and macrophages derived from saline or CDDP-treated WT and Gal-3^-/- mice, 72 h after CDDP administration (16 mg/kg body weight). There was no significant difference in total number of CD45+Gr-1+neutrophils (gated as CD45+Gr-1+cells in population of renal infiltrated cells) between CDDP-injured kidneys of WT and Gal-3^-/- animals (A). Significantly higher number of inflammatory, IFN-γ (B) and IL-17-producing neutrophils (C), and notably lower total number of IL-4-producing (D) and IL-10 producing neutrophils (E) were noticed in the kidneys of CDDP-treated Gal-3^-/- mice. Representative density plots show percentages of IFN-y-, IL-17-, IL-4- and IL-10-producing neutrophils (Fig.2B-E), gated in the population of CD45+Gr-1+ renal infiltrated cells. Strong positive correlation was observed between histological score and total number of renal-infiltrated macrophages (F) which was found in significantly higher number in the kidneys of CDDP-treated Gal-3^-/- mice (G). Representative density plots showing higher percentage of macrophages in renal infiltrated cells of CDDP-treated Gal-3^-/- mice compared to similarly treated WT animals (G, right panel). Macrophages are determined as F4/80+cells, gated in population of CD45+CD11c- renal infiltrated cells. Significantly higher number of IL-12-producing M1 macrophages (H, left panel), but lower number of IL-10-producing M2 macrophages (I, right panel) were noticed in the kidneys of CDDP-treated Gal-3^-/- mice. Positive correlation was observed between histological score and total number of IL-12-producing renal-infiltrated M1 macrophages (H, middle panel) and negative correlation was noticed between histological score and total number of IL-10-producing renal-infiltrated M2 macrophages (I, middle panel). Representative density plots show percentages of IL-12-producing (H, right panel) and IL-10-producing macrophages (I, right panel), gated in the population of F4/80+ renal infiltrated cells of CDDP-treated WT and Gal-3^-/- animals. Data from two individual experiments with 8 mice per group are shown as Mean ± SEM; *p<0.05, **p<0.01,***p<0.001.

Figure 4

Gal-3 deficiency significantly increased total number of inflammatory Th1 and Th17 cells and reduced presence of regulatory T cells in CDDP-injured kidneys. Bar graphs (left panels) and representative density plots obtained by flow cytometry analysis and intracellular staining of renal-infiltrated CD4+T cells derived from saline or CDDP-treated WT and Gal-3^-/- mice, 72 h after CDDP administration (16 mg/kg body weight). Gal-3 deficiency resulted with increased presence of CD4+T cells in the CDDP-injured kidneys (A, left panel). Representative density plots showing CD4+ T lymphocytes, gated as CD4+ cells in population of renal infiltrated cells (A, right panel). Significantly higher total number of T-bet-expressing (B) and IFN-γ-producing (C) CD4+Th1 cells and RORγT-expressing (D) and IL-17-producing (E) CD4+Th17 cells, but reduced presence of immunosuppressive IL-10-producing CD4+T cells (F), CD4+FoxP3+ Tregs (G) and STAT-3-expressing Treg (H) were noticed in the kidneys of CDDP-treated Gal-3^-/- mice. Representative density plots showing percentages of T-bet-expressing, IFN-γ-producing, RORγT-expressing, IL-17-producing, IL-10-producing, FoxP3-expressing cells, gated in the population of CD4+Tcells (B-G, right panels) and percentage of STAT-3-expressing cells in gated population of CD4+FoxP3+ Tregs (H, right panel), isolated from the kidneys of CDDP-treated WT and Gal-3-/- mice, 72h after CDDP administration. Real-time PCR gene analysis showing significantly higher mRNA expression of T-bet, IFN-γ, RORγT, IL-17 and lower mRNA expression of FoxP3 in CDDP-injured kidneys of Gal-3-/- mice compared to similarly treated WT animals (I). Data from two individual experiments with 8 mice per group are shown as Mean ± SEM; *p<0.05, **p<0.01,***p<0.001.

Figure 5

Genetic deletion of Gal-3 did not alter migratory capacity and immunosuppressive properties of Tregs. Tregs were fluorescence-labeled using pre-incubation with carboxyfluorescein diacetate succin-imidyl ester (CFSE). CSFE-labeled WT and Gal-3^-/-Tregs were intravenously injected (1x10⁶ Tregs/mouse) in CDDP-treated WT mice and their number and function were evaluated by flow cytometry. Representative dotplots showing similar percentage and total number of CSFE-labeled WT and Gal-3^-/-Tregs in the kidneys of CDDP-treated WT recipients. Engrafted WT and Gal-3^-/- Tregs managed to significantly attenuate CDDP-induced AKI, as evidenced by significantly lower serum levels of urea and creatinine (B) and reduced histological score (C), but there was no significant difference in the extent of AKI between CDDP-treated mice that received WT and Gal-3^-/-Tregs, which was confirmed by representative kidney tissue sections. WT and Gal-3^-/- Tregs significantly reduced total number of IFN-γ and IL-17-producing neutrophils (5D-E) and CD4+ T cells (5F-G) but there was no significant difference in the total number of these cells between CDDP-treated mice that received WT and Gal-3^-/-Tregs. Individual data points with Mean, obtained in one experiment with 6 mice per group; *p<0.05, **p<0.01,***p<0.001.

Figure 6

Target disruption of Gal-3 significantly attenuated immunosuppressive capacity of renal-infiltrated DCs and enhanced their potential to generate detrimental Th1 and Th17 immune response in CDDP-injured kidneys. Bar graphs (left panels) and representative density plots obtained by flow cytometry analysis and intracellular staining of renal-infiltrated DCs derived from saline or CDDP-treated WT and Gal-3^-/- mice, 72 h after CDDP administration (16 mg/kg body weight). Gal-3 deficiency resulted with increased presence of F4/80-CD11+DCs in the CDDP-injured kidneys (A). Representative density plots showing higher percentage of DCs in renal infiltrated cells of CDDP-treated Gal-3^-/- mice compared to similarly treated WT animals (A, right panel). DCs are determined as F4/80- CD11c+ renal infiltrated cells. Significantly lower number of IL-10-producing (B) and IDO1-expressing F4/80-CD11+DCs, but remarkably higher number of CD80-expressing (D), CD86-expressing (E), I-A-expressing (F), IL-12-producing (G) and IL-23-producing F4/80-CD11+DCs were noticed in the kidneys of CDDP-treated Gal-3^-/- mice. Representative density plots showing percentages of IL-10-producing, IDO1-, CD80-, CD86-, I-A-expressing, IL-12 and IL-23-producing cells, gated in the population of F4/80-CD11c+ renal DCs isolated from the kidneys of CDDP-treated WT and Gal-3^-/- mice, 72 h after CDDP injection (B-G, right panels). Data from two individual experiments with 8 mice per group are shown as Mean ± SEM; *p<0.05, **p<0.01.

Figure 7

Gal-3 is required for TLR-2-dependent activation of IDO1/KYN pathway in renal DCs and consequent generation of immunosuppressive Tregs. There was strong positive correlation between percentages of Gal-3+CD11c+DCs and TLR-2+CD11c+DCs in the kidneys (A). Representative flow cytometry dot plots showing expression of Gal-3 in TLR-2+ cells previously gated in the population of F4/80-CD11c+ renal DCs (B). Significantly lower number of TLR-2-expressing CD11c+DCs was observed in the kidneys of CDDP-treated Gal-3^-/- mice compared to CDDP-treated WT animals, 72 h after CDDP injection (16 mg/kg body weight). There was strong positive correlation between percentages of IDO1+CD11c+DCs and TLR-2+CD11c+DCs in the kidneys (D). Representative flow cytometry dot plots showing expression of IDO1 in TLR-2+ cells previously gated in the population of F4/80-CD11c+ renal DCs (E). Representative dot plots showing that majority of TLR-2+Gal-3+IDO1+DCs express IL-10 (F). Significantly lower concentrations of KYN were noticed in supernatants of Gal-3^-/-DCs^Pam3CSK4 and WTDCs^{PAM3CSK4+Davanat} compared to WTDCs^Pam3CSK4 (G). Real-time PCR gene analysis (H), ELISA (I) and flow cytometry (J) results showing expression and production of inflammatory IFN-γ, IL-17, and anti-inflammatory IL-10 in Tregs (H, J) and appropriate supernatants (I) before and after culturing with WTDCs^Pam3CSK4, Gal-3-/-DCs^Pam3CSK4 or WT DCs^{PAM3CSK4+Davanat} in contact-independent manner within transwell system (ratio between DCs and Tregs was 1:10). Real-time PCR gene analysis (K) and ELISA (L) results showing expression and production of inflammatory IFN-γ, IL-17 and anti-inflammatory IL-10 in LPS-activated neutrophils that were cultured with Tregs previously stimulated by WTDCs^Pam3CSK4, Gal-3^-/-DCs^Pam3CSK4 or WT DCs^{PAM3CSK4+Davanat}. Transwell systems were used to separate neutrophils and Tregs and ratio between neutrophils and Tregs was 10:1. Data from two individual experiments with 8 mice per group are shown as Mean ± SEM; *p<0.05, **p<0.01;***p<0.001.

Figure 8

Transfer of WTDCs^Pam3CSK4 significantly attenuated CDDP-induced AKI in WT recipients by promoting expansion of immunosuppressive Tregs in IDO1/KYN-dependent manner. TLR-2-primed DCs, isolated from the kidneys of untreated WT and Gal‐3^-/- mice (WTDCs^Pam3CSK4 and Gal‐3^-/-DCs^Pam3CSK4), were intravenously injected (5×10⁵cells/mouse) in CDDP-treated WT recipients (WT^{WTDCsPam3CSK4} and WT^{Gal‐3-/-DCsPam3CSK4}) two days prior CDDP administration (16 mg/kg body weight). IDO1 was inhibited in TLR-2-primed renal DCs (WTDCs^{Pam3CSK4+1-MT}) and Gal-3^-/-DCs (Gal-3^-/-DCs^{Pam3CSK4+1-MT}) by using 1-methyl tryptophan (1-MT; 2 mM). Transfer of WTDCs^Pam3CSK4 managed to attenuate CDDP-injured AKI and inflammation in CDDP-treated WT recipients, as evidenced by notably lower serum levels of urea (A), creatinine (B), decreased histological score (C), reduced extent of renal injury in the cortex of CDDP-injured kidneys (D), decreased serum levels of inflammatory, Th1/Th17-related cytokines and increased serum levels of immunosuppressive IL-10 and KYN (E), significantly higher presence of Tregs (F), and IL-10-producing neutrophils (G) and CD4+T cells (H), but notably decreased of IFN-γ (I) and IL-17-producing neutrophils (J) as well as IFN-γ (K) and IL-17-producing CD4+T cells (L). Transfer of WTDCs^{Pam3CSK4+1-MT} or Gal-3^-/-DCs^Pam3CSK4 did not expand immunosuppressive Tregs (F), IL-10-producing neutrophils (G) and CD4+Tcells (H) in CDDP-injured kidneys, but promoted polarization of renal-infiltrating neutrophils (I-J) and CD4+T cells (K-L) towards IFN-γ-producing (I, K) and IL-17-producing cells (J, L), resulting in significant aggravation of CDDP-induced AKI in WT recipients (A-D). There was no significant difference between CDDP-treated WT recipients of Gal-3^-/-DCs^{Pam3CSK4+1-MT} and Gal-3^-/-DCs^Pam3CSK4 (A-L). Data from two individual experiments with 8 mice per group are shown as Mean ± SEM; *p<0.05, **p<0.01;***p<0.001.

Figure 9

Transfer of WTDCs^Pam3CSK4 significantly attenuated CDDP-induced AKI in Gal-3^-/- recipients by promoting expansion of immunosuppressive Tregs in IDO1/KYN-dependent manner. TLR-2-primed DCs, isolated from the kidneys of untreated WT and Gal‐3^-/- mice (WTDCs^Pam3CSK4 and Gal‐3^-/-DCs^Pam3CSK4), were intravenously injected (5×10⁵cells/mouse) in CDDP-treated Gal-3^-/- recipients (Gal-3^{-/-WTDCsPam3CSK4} and Gal-3^{-/-Gal‐3-/-DCsPam3CSK4}) two days prior CDDP administration (16 mg/kg body weight). IDO1 was inhibited in TLR-2-primed renal DCs (WTDCs^{Pam3CSK4+1-MT}) and Gal-3^-/-DCs (Gal-3^-/-DCs^{Pam3CSK4+1-MT}) by using 1-methyl tryptophan (1-MT; 2 mM). Intravenous injection of WTDCs^Pam3CSK4 attenuated CDDP-injured AKI and inflammation in CDDP-treated Gal-3^-/- recipients, as evidenced by notably lower serum levels of urea (A), creatinine (B), decreased histological score (C), reduced extent of renal injury in the cortex of CDDP-injured kidneys (D), decreased serum levels of inflammatory, Th1/Th17-related cytokines and increased serum levels of immunosuppressive IL-10 and KYN (E), significantly reduced total number of IFN-γ (F) and IL-17-producing neutrophils (G), IFN-γ-(H) and IL-17-producing CD4+T cells (J), but remarkably increased number of immunosuppressive Tregs (J), IL-10-producing neutrophils (K) and CD4+T cells (L). Transfer of WTDCs^{Pam3CSK4+1-MT} or Gal-3^-/-DCs^Pam3CSK4 did not expand immunosuppressive Tregs (J), IL-10-producing neutrophils (K) and CD4+Tcells (L) in CDDP-injured kidneys, but promoted polarization of renal-infiltrating neutrophils (F-G) and CD4+T cells (H-I) towards IFN-γ-producing (F, H) and IL-17-producing cells (G, I), resulting in significant aggravation of CDDP-induced AKI in Gal-3^-/- recipients (A-D). There was no significant difference between CDDP-treated WT recipients of Gal-3^-/-DCs^{Pam3CSK4+1-MT} and Gal-3^-/-DCs^Pam3CSK4 (A-L). Data from two individual experiments with 8 mice per group are shown as Mean ± SEM; *p<0.05, **p<0.01;***p<0.001.

Figure 10

Expression of Gal-3 on TLR-2-primed DCs is crucially important for their capacity to enhance nephroprotective and immunosuppressive properties of Tregs. For the depletion of Tregs, anti-CD25 monoclonal antibody (250 μg/mouse) was intraperitoneally given to mice 3 days before CDDP administration (16 mg/kg body weight). For transfer experiments non-primed Tregs, Tregs primed with WTDC^Pam3CSK4, WTDC^{Pam3CSK4+Davanat} or Gal-3^-/- DC^Pam3CSK4 (1x10⁶ Tregs/ mouse) were intravenously injected in CDDP-treated animals 18 h before induction of AKI. Significantly lower serum levels of urea and creatinine (A) and reduced histological score accompanied with attenuated renal injury, observed in the cortex of CDDP-injured kidneys (B) were observed in Treg-depleted CDDP-treated mice that received WTDC^Pam3CSK4-primed Tregs compared to the Treg-depleted CDDP-injured animals that received non-primed Tregs. There was significantly lower number of T-bet-expressing, IFN-γ-, IL-17- and TNF-α-producing neutrophils (C-F) and remarkably higher number of IL-10-producing neutrophils (G) in the kidneys of Treg-depleted CDDP-treated mice that received WTDC^Pam3CSK4-primed Tregs. Significantly higher serum concentrations of urea and creatinine (A), more severe injury of proximal tubular epithelial cells and increased histological score (B), significantly higher number of inflammatory neutrophils (C-F) and significantly reduced number of immunosuppressive, IL-10-producing neutrophils (G) in the kidneys of Treg-depleted CDDP-treated mice that received Gal-3^-/-DC^Pam3CSK4 or WTDC^{Pam3CSK4+Davanat} -primed Tregs compared to Treg-depleted CDDP-treated mice that received WTDC^Pam3CSK4-primed Tregs. Individual data points with Mean, obtained in one experiment with 6 mice per group; *p<0.05, **p<0.01,***p<0.001.

Figure 11

Depletion of Tregs completely diminished Gal-3-dependent capacity of TLR-primed renal DCs to suppress IFN-γ and IL-17 driven inflammation in CDDP-injured kidneys. For the depletion of Tregs, CDDP-treated WT^WTDCPam3CSK4 and Gal-3^{-/-WTDCPam3CSK4} mice received either cyclophosphamide (CY; 10 mg/kg) 3 days before CDDP administration (16 mg/kg body weight) or anti-CD25 (P61) monoclonal antibody (250 μg/mouse). Significantly elevated serum levels of urea (A) and creatinine (B), increased histological score (C), more severe tubular epithelial cell injury (D), elevated serum levels of IFN-γ and IL-17 (E), increased presence of IFN-γ- and IL-17-producing neutrophils (F-G), and IFN-γ- and IL-17-producing CD4+T cells (H-I) were noticed in the kidneys of CY+CDDP- and anti-CD25+CDDP-treated WT and Gal-3^-/- recipients of WTDCs^Pam3CSK4 compared to CDDP-treated WT^{WTDCsPam3CSK4} and Gal-3^{-/-WTDCsPam3CSK4} that were not depleted from Tregs (J). Data from two individual experiments with 8 mice per group are shown as Mean ± SEM; *p<0.05, **p<0.01;***p<0.001.

Figure 12

Expression of Gal-3 in renal DCs is necessary for optimal TLR-2-dependent activation of IDO1/KYN pathway and for consequent expansion of renal-infiltrating Tregs. Gal-3, expressed on renal DCs, serves as receptor for AGEs which promotes TLR-2-dependent activation of IDO1/KYN pathway and protects against CDDP-caused AKI by inducing expansion of Tregs which, in turn, promote generation of immunosuppressive, IL-10-producing phenotype in renal-infiltrated neutrophils and prevent inflammation driven by IFN-γ- and IL-17-producing neutrophils, Th1 and Th17 cells (upper panel). Gal-3 deficiency resulted in reduced uptake of AGEs, attenuated activation of TLR-2-dependent IDO1/KYN pathway in renal DCs, decreased production of KYN, reduced presence of immunosuppressive Tregs and enhanced expansion of inflammatory IFN-γ- and IL-17-producing neutrophils, Th1 and Th17 cells in the kidneys, resulting in significant aggravation of CDDP-induced AKI (lower panel).