Tris DBA ameliorates IgA nephropathy by blunting the activating signal of NLRP3 inflammasome through SIRT1‐ and SIRT3‐mediated autophagy induction

Abstract Tris (dibenzylideneacetone) dipalladium (Tris DBA), a small‐molecule palladium complex, can inhibit cell growth and proliferation in pancreatic cancer, lymphocytic leukaemia and multiple myeloma. Given that this compound is particularly active against B‐cell malignancies, we have been suggested that it can alleviate immune complexes (ICs)–mediated conditions, especially IgA nephropathy (IgAN). The therapeutic effects of Tris DBA on glomerular cell proliferation and renal inflammation and mechanism of action were examined in a mouse model of IgAN. Treatment of IgAN mice with Tris DBA resulted in markedly improved renal function, albuminuria and renal pathology, including glomerular cell proliferation, neutrophil infiltration, sclerosis and periglomerular inflammation in the renal interstitium, together with (Clin J Am Soc Nephrol. 2011, 6, 1301‐1307) reduced mitochondrial ROS generation; (Am J Physiol‐Renal Physiol. 2011. 301, F1218‐F1230) differentially regulated autophagy and NLRP3 inflammasome; (Clin J Am Soc Nephrol. 2012, 7, 427‐436) inhibited phosphorylation of JNK, ERK and p38 MAPK signalling pathways, and priming signal of the NLRP3 inflammasome; and (Free Radic Biol Med. 2013, 61, 285‐297) blunted NLRP3 inflammasome activation through SIRT1‐ and SIRT3‐mediated autophagy induction, in renal tissues or cultured macrophages. In conclusion, Tris DBA effectively ameliorated the mouse IgAN model and targeted signalling pathways downstream of ICs‐mediated interaction, which is a novel immunomodulatory strategy. Further development of Tris DBA as a therapeutic candidate for IgAN is warranted.

the mouse IgAN model and targeted signalling pathways downstream of ICs-mediated interaction, which is a novel immunomodulatory strategy. Further development of Tris DBA as a therapeutic candidate for IgAN is warranted.

| INTRODUC TI ON
IgA nephropathy (IgAN) is the most prevalent primary glomerulonephritis worldwide, featuring mesangial cells and mononuclear leucocyte infiltration in the glomerulus and renal interstitial tissues. [1][2][3] The renal disorder has been shown to progress to uraemia in 20%-40% of the patients. 4,5 Clinically, current treatment for IgAN and its progression of disease is still insufficient, because the benefits of these non-disease targeted drugs are modest. Most current therapies target production of antibodies non-selectively, rather than the effect of immune complexes (ICs) directly. Therefore, novel therapeutics are urgently needed for the treatment of this renal disorder. Recently, we demonstrated that overproduction of reactive oxygen species (ROS) and mononuclear leucocyte infiltration into the kidney is involved in the development and progression of IgAN. [6][7][8] Furthermore, we 9,10 and others 11 have shown that NLRP3 inflammasome are implicated in the development and deterioration of IgAN. On the other hand, we showed that activation of MAPK signalling pathways in mesangial cells and macrophages and T cells infiltrated in the glomerulus may be involved in the pathogenesis of IgAN. 12 Activation of NLRP3 inflammasome results in the production of IL-1β and IL-18, involving a priming signal from pathogen-associated molecular patterns and an activation signal. [13][14][15] In this regard, ICs can initiate the activation of the NLRP3 inflammasome in macrophages in IgAN. [6][7][8] Recently, we have shown overproduction of IL-1β or IL-18 in mouse IgAN models, pointing to NLRP3 inflammasome and related signalling pathways as a major mechanism underlying the progression and deterioration of IgAN. [6][7][8] Mammalian SIRT1 deacetylates various target proteins relevant to apoptosis, cell cycle, circadian rhythms, mitochondrial function and metabolism. Growing evidence suggests the beneficial effects of sirtuin (SIRT) 1 and 3, which are NAD-dependent deacetylases. SIRT1 and SIRT3 are able to inhibit IL-1β expression and the production of reactive oxygen species (ROS), 16,17 and therefore, it is implicated in autophagy induction. 18 Activation of SIRT1 can inhibit NLRP3 inflammasome activation and subsequent IL-β secretion, and SIRT1 knockdown can enhance the activation of NLRP3 inflammasome in cultured endothelial cells. 19,20 In addition, SIRT3 is renoprotective by inhibiting mitochondrial ROS production and NLRP3 inflammasome activation. 21 Activation of SIRT1 can inhibit NLRP3 inflammasome activation and subsequent IL-β secretion, and SIRT1 knockdown can enhance the activation of NLRP3 inflammasome in cultured endothelial cells. 19,20 In addition, SIRT3 is renoprotective by inhibiting mitochondrial ROS production and NLRP3 inflammasome activation. 21 Autophagy is being regulated precisely and plays a vital role in maintaining cell homeostasis. It is a typical protective, pro-survival response at the initiation of damages. 22 Autophagy is involved in the inhibition on IL-1β secretion 23,24 and degrading pro-IL-1β expression in macrophages. 25,26 On the other hand, SIRT1 and SIRT3 are implicated in mTOR nutrient-sensing pathways and regulating autophagy machinery depending on ATG5 and LC3B in autophagy induction in macrophages and liver cells. 27 In addition, autophagy inhibits NLRP3 inflammasome and innate immune response and inflammation, 28,29 from which evidence supports that autophagy plays a negative regulator in the activation of NLRP3 inflammasome for the restoration of tissue homeostasis after damage in immune-mediated diseases, including IgAN. 10,11,30 Tris (Dibenzylideneacetone) dipalladium (Tris DBA) is a small-molecule palladium complex, and it has been shown to suppress cell growth and proliferation of pancreatic cancer, lymphocytic leukaemia and multiple myeloma. 31,32 Tris DBA can reduce Src/NMT-1 complex in melanoma cells and inhibit its downstream signalling, such as MAPK. 33 However, the effects of Tris DBA on inflammatory disease have yet to be determined. Given that IgAN is mediated in large part by the proliferation and activation of B cells, and Tris DBA has been shown to be effective against B-cell malignancy, we evaluated it against IgAN. This is the first study to evaluate the efficacy of this agent against an ICs-mediated chronic kidney disease.
In the present study, we showed the therapeutic effect of Tris DBA on IgAN in a mouse model and the mechanism of action of the compound, involving inhibition of a MAPK-mediated priming signal of NLRP3 inflammasome and enhancement of SIRT1 and SIRT3-mediated induction of autophagy and subsequent NLRP3 inflammasome suppression. This compound targets ICs-macrophage interactions as a novel immunomodulatory event. These results suggest that the pure compound be a drug candidate for treating IgAN.

| Tris DBA and optimal dose selection
Tris DBA palladium was purchased from Sigma-Aldrich and dissolved in KATIMIN (China Chemical & Pharmaceutical Co.) for in vivo studies or in DMSO for in vitro studies.

| IgAN mouse model and experimental protocol
Reference1 two normal control groups were included in this study as follows: 8-week-old C57BL/6 mice were injected with saline and served as saline control, while those injected with vehicle (KATIMIN) were served as vehicle control 2 ; groups for IgAN and IgAN with treatment were as follows: IgAN was induced by consecutive 28 daily injections of purified IgA anti-phosphorylcholine antibodies and pneumococcal C-polysaccharide antigen (PnC) as described previously. 8

| Urine albumin and renal function measurement
Serum samples were collected to measure the levels of BUN and Cr Urine samples were collected weekly, and albuminuria was determined by the ratio of urine albumin to urine Cr, as described previously. 9,10,34

| Renal pathology and IHC
For histopathological assessment, renal cortical tissue samples were fixed in 10% buffered formalin and embedded in paraffin for haematoxylin and eosin staining. Fifty glomeruli were examined in at least two renal tissue fields of view per slide under a light microscope at a magnification of 400×. Cell proliferation, glomerular sclerosis, neutrophil infiltration and periglomerular mononuclear leucocyte infiltration were recorded as described previously. 4 For IHC, F4/80 (Serotec, CA, USA) and CD3 (Dako, Glostrup, Denmark Dako) were used in formalin-fixed and paraffin-embedded renal sections, followed by incubation with biotinylated secondary antibodies and avidin-biotin-peroxidase complexes (both from Dako) as described previously. 4,9,10 An image analysis software (Pax-it; Paxcam) (https://www.paxit. com) was used to detect and sort objects or areas, and to quantify the number of positive cells for CD3 + and F4/80 + expression in twenty randomly selected fields of the glomeruli and periglomerular interstitial compartments, respectively, at a magnification of 400× by light microscopy. 35

| Real-time PCR analysis
RNA was extracted using REzol (Protech Technology, Taipei, Taiwan), and real-time PCRs were performed using SYBR Green RT-PCR Reagents Kit (Applied Biosystems) prepared as described previously. 9 The specific primer pairs used for realtime PCR analysis were as follows: mouse NLRP3 forward: 5′ CTGTG TGTGG GACTG AAGCA C-3′; mouse NLRP3 reverse:

| ROS detection
At ROS levels in renal tissues, the samples were incubated at room temperature with Krebs-Hepes buffer containing 1.25 mmol/L lucigenin (Sigma) as substrate immediately after the mice were killed, and luminescence counts were determined in duplicate at multilabel microplate reader (Hidex), as described previously. 36 ROS activity was expressed as relative luminescence units (RLU) per 15 minutes per milligram of the dry weight of the renal tissue (ie RLU/15min/mg) or as RLU/15 min/mL. For the 10 μm of frozen section slides, in situ superoxide anion production was determined in the mouse kidneys by DHE labelling dye (30 μmol/L). Renal tissues were incubated the dye for five min in a dark chamber on an orbital shaker at room temperature, followed by observing under a fluorescence microscope system (Olympus). For the quantified by counting the percentage of the total nuclei that were positive per kidney cross section as described previously. 9,10 Mitochondrial ROS levels in BMDMs, isolated from C57BL/6, measured by MitoSOXbased flow cytometry (Thermo) after Tris DBA treatment, priming with IgA-ICs for 5.5 hours and stimulation with ATP. The data are expressed as the means ± SEM for three separate experiments.

| GPx and NF-κB activity assay
GPx activity in the renal tissue was measured using a commercial glutathione peroxidase assay kit (Cayman, Michigan, USA) according to the manufacturer's instructions as described previously, 4 and nuclear NF-κB p65 activation was quantified using an ELISA-based TransAM NF-κB kit (Active Motif) according to manufacturer's instructions.

| Cell models
Murine macrophage cell line J774A.1 was purchased from the American Type Culture Collection (Rockville, MD). The cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated foetal calf serum and 2 mL l-glutamine (Life Technologies) at 37°C in a 5% CO 2 incubator. Briefly, 1 × 10 6 cells/mL was incubated for 30 minutes with or without Tris DBA before them primed with IgA immune complexes (IgA-ICs) prepared with the purified IgA anti-phosphorylcholine (5 ng/mL) and PnC (100 ng/mL) was used to stimulate of macrophages for 5.5-hour serves as NLRP3 inflammasome priming signal including NLRP3 and pro-IL-1β activation), and then for 30 minutes with 5 mmol/L ATP serves as NLRP3 inflammasome activation signal including IL-1β production and caspase-1 activation. Bone marrow-derived macrophages (BMDMs) were used for ex vivo experiments to measure their levels of mitochondrial ROS production as reported previously. 29,37  were then fixed in 4% paraformaldehyde for 30 minutes followed by washed in PBS. DAPI for nuclei staining. Image analysis was performed using the confocal microscope (FV1000-IX81, Olympus).

| Data analysis
The data are presented as the mean ± standard error of the mean (SEM), and comparisons between two groups were performed using Student's t test. The data from in vitro and ex vivo experiments were analysed using one-way ANOVA and subsequent Scheffe's test. A P value < .05 was considered statistically significant for each of the experiments.

| Renal function, proteinuria and renal pathology
To validate the potential therapeutic effects of Tris DBA on IgAN mice, first, we performed clinical assessment and renal pathology. To assess renal function, serum levels of BUN and Cr were determined. As shown in Figure 1A

| NLRP3 inflammasome activation in renal tissues
In the past, we showed that NLRP3 inflammasome is activated in the mouse IgAN model. 4,9,10 We examined whether Tris DBA could inhibit renal NLRP3 inflammasome activation in Tris    Table 1). Similarly, the Tris DBA + IgAN mice showed significantly reduced renal protein levels of all these proteins compared with Vehicle + IgAN mice, although the latter exhibited greatly increased expression levels of all the proteins in relevance to saline control mice ( Figure 2B,C, Table 1). Autophagy can regulate the activation of NLRP3 inflammasome in renal injury. [23][24][25][26] As shown in Figure 2D,E, renal levels of LC3B I/II protein were markedly

| ROS production in renal tissues and bone marrow-derived macrophages (BMDMs)
Both IgAN mice treated with saline (saline + IgAN) and Vehicle + IgAN mice had significantly higher levels of ROS in renal tissues, but this effect was significantly decreased in Tris DBA + IgAN mice ( Figure 3A,B, Table 1). Similarly, although the percentage of the DHE-positive cells was significantly increased in both saline + IgAN and Vehicle + IgAN mice compared with saline control or normal control mice treated with vehicle (vehicle control mice), this effect was markedly suppressed in Tris DBA + IgAN mice. In parallel, the levels of the NADPH oxidase p47phox (NADPH p47) and NQO1 were measured in renal tissues. As shown in Figure 3C, renal levels of NADPH p47 were significantly increased in both saline + IgAN and Vehicle + IgAN mice compared with those of saline control or vehicle control mice, but this effect was inhibited in Tris DBA + IgAN mice.
Adequate activation of antioxidant pathway is a key mechanism in cellular defence against ROS production. 38,39 In contrast, greatly increased renal NQO1 was observed in Tris DBA + IgAN mice compared with that of saline + IgAN or Vehicle + IgAN mice ( Figure 3C, Table 1). In consistence with these findings, Tris DBA + IgAN mice showed significantly elevated GPx compared with saline + IgAN or Vehicle + IgAN mice ( Figure 3D, Table 1). Mitochondrial ROS plays an essential role in the process of NLRP3 inflammasome activation. 40,41 The cells were first treated Tris DBA for 1 hour and then co-incubated with IgA-ICs (Tris DBA + IgA-ICs BMDMs). As shown in Figure 3E, mitochondrial ROS production was greatly reduced in

| Tris DBA reduced NLRP3 inflammasome activation in cultured macrophages
Recently, we found that full activation of the NLRP3 inflammasome requires both a priming signal such as IgA-ICs and activation signal such as ATP to produce the mature IL-1β. 10 ATP, as an inducer for the activation signal of NLRP3 inflammasome activation, is required for IgA-ICs-mediated IL-1β secretion. 10 Next, the activation signal for NLRP3 inflammation activation was examined by determining the levels of caspase-1 and IL-1β using cultured macrophages. As shown in Figure 4, upon treatment with ATP (NLRP3 inflammasome activator), increased secretion levels of mature IL-1β ( Figure 4A,B) and caspase-1 ( Figure 4C) were observed in the Vehicle + IgA-ICs macrophages compared to saline, but this effect was markedly inhibited in Tris DBA + IgA-ICs macrophages.
The potential mechanism of action for Tris DBA to exert its inhibitory effect on NLRP3 inflammasome activation was evaluated in

| Autophagy-mediated inhibition of NLRP3 inflammasome
Autophagy can inhibit priming and activation signals of NLRP3 inflammasome. [23][24][25][26] First, we examined whether Tris DBA could enhance autophagy induction in macrophages without IgA-ICs stimulation. As shown in Figure 5A SIRT1 and SIRT3 have been reported to enhance the autophagy. 27 As shown in Figure 6A, treatment with Tris DBA significantly increased the protein expression of SIRT1 starting from 6 hours and persisted until 24 hours, and SIRT3 at 24 hours in J774A.1 macrophages in a dose-dependent manner. Next, the cells were first treated with EX-527, an inhibitor of SIRT1, followed by the treatment with Tris DBA or shSIRT3 macrophages to elucidate whether SIRT1 or SIRT3 was involved in autophagy induction in the macrophages treated with Tris DBA. As shown in Figure 6B,C, Tris DBA induced significantly increased expression of LC3B I/II and p62 in the cells. Moreover, this effect was abolished in the macrophages deficient in SIRT1 and SIRT3. In addition, although reduction of IL-1β secretion and caspase-1 was observed in Tris DBA + IgA-ICs macrophages, this effect was reversed by the administration of EX-527 and shSIRT3 ( Figure 6D-G), respectively.

| D ISCUSS I ON
In the present study, we show the therapeutic effect of Tris DBA on IgAN in mice, the mechanism of action involving inhibition of were treated with Tris DBA in the study (Figure 2).
In our previous study, we demonstrate that ATP contributed the NLRP3 inflammasome-mediated IL-1β production after treated with IgA-ICs in antigen presenting cells by dose-dependent manner. 10 In this regard, we found that ATP, as an inducer for the activation signal for NLRP3 inflammasome activation, is required for IgA-ICs-mediated IL-1β secretion in macrophages. In addition, it should be noted that IgA1 deposition may induce NLRP3 expression and macrophage transdifferentiation of podocyte in IgAN. 11 Thus, the activation of NLRP3 inflammasome might contribute by both ATP and IgA1 and trigger the renal inflammation through renal cells and macrophages in IgAN. In addition, infiltration of macrophages into the kidney is a hallmark of IgAN, 7,9,10  IgA-ICs, IgA immune complexes; ROS, reactive oxygen species; SIRT1, sirtuin 1; SIRT3, sirtuin 3, mtROS, mitochondrial ROS p62, another autophagy marker, is regulated by the balance between transcriptional regulation (incoming flux) and post-translational autophagic degradation (outgoing flux) of the protein. 48 In the current study, Tris DBA may exert its effect at the level of both the synthesis and degradation of autophagy in macrophages.
It is established that ATG5 plays a crucial role in the membrane elongation and dissociate from the membrane upon completion of autophagosome formation. 49 The isoform LC3B I is synthesized in the cytosol, and after conjugation to phosphatidylethanolamine, the activated isoform LC3B II is located within the autophagosomal membrane, followed by its disruption after autolysosome formation. 50 Consistent with this notion, in the present study, increased expression of LC3B II was observed at days 1, 3 and 7, but not at day 28 in the mice treated with Tris DBA (Figure 2C), suggesting that LC3B II was produced more than disrupted at early stage, while the production of LC3B II was reduced due to degradation through lysosomal hydroplanes at day 28. Besides, the ATG5 expression was opposite to the LC3B II in the mice. Therefore, the underlying mechanism deserves further investigation.
As illustrated in Figure 7, it is suggested that Tris DBA was able to effectively ameliorate the mouse IgAN model. This beneficial effect might involve blunting of mitochondrial ROS production, a MAPK (ERK, JNK)-mediated priming signal of the NLRP3 inflammasome and differentially regulating the autophagy/NLRP3 inflammasome axis through SIRT1 and SIRT3. Thus, further development of Tris DBA as a therapeutic candidate for IgAN is warranted. from the Tri-Service General Hospital, Taipei, Taiwan.

CO N FLI C T O F I NTE R E S T
All the authors have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.