Chapter2: The Role Of Inflammasomes in Glomerulonephritis

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7. Inflammasome Involvement in Autoimmune Kidney Diseases

A summary of the main publications related to inflammasome investigations in glomerulonephritis is shown in Table 1.

LDG, low-density granulocytes; NETs, neutrophils extracellular traps; dsDNA, anti-double-stranded DNA; SLE, systemic lupus erythematosus; LN, lupus nephritis; NFkB, nuclear factor kappa B; TNF-c, tumor necrosis factor α; siRNA, small interfering RNA; INF, interferon; HMGB1, high-mobility group box 1 protein; LPS, Lipopolysaccharide; ATP, adenosine triphosphate; CASP1, caspase-1; ANCA, Anti-Neutrophilic Cytoplasmic Autoantibody; NE, neutrophil elastase; PR3, proteinase 3; MPO, myeloperoxidase; NCGN, necrotizing crescentic glomerulonephritis; Phox, NADPH oxidase; PR3, proteinase 3; IgAN, IgA nephropathy; mRNA, messenger RNA; shRNA, short hairpin RNA; Il-1ra, interleukin-1 receptor antagonist; anti-GBM, anti-glomerular basement membrane; PMN, polymorphonuclear; ICAM-1, intercellular adhesion molecule-1; IL-1RI, IL1 type 1 receptor.

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7.1.Lupus Nephritis

SLE is a chronic disease that frequently affects the kidney. Lupus nephritis (LN) is the most common renal disease, involving approximately 50% of patients with SLE. This autoimmune disease mostly affects women of the reproductive age. In men, the disorder could be more aggressive. Patients usually have LN at an early age, and it usually presents itself in the initial stages of the disease. Patients with this renal impairment have an increased mortality rate. In total, 10-30% of patients with LN progress to renal failure requiring kidney replacement therapy.

Irregularities in innate and adaptive immunity contribute to the pathogenesis of SLE. LN occurs when the transcription of genes associated with neutrophils increases. The rise in IFN precedes the activation of neutrophils. The increment of IFN causes the differentiation of B cells into plasmablasts and produces inflammation of specific tissues through neutrophils and active myeloid cells. When these neutrophils die, extracellular neutrophil traps(NETs) appear. NETs are meshing-chromatin fibers combined with granules derived from antimicrobial peptides and enzymes that play an important defense role [74]. These meshes help to maintain antigen-specific autoantibody production.

The formation of immune complexes that are deposited in the glomerulus is derived from the production of antibodies against nuclear and cellular antigens. Immune complexes can activate complement and cause kidney damage, especially through the alternative pathway. Plasma interstitial cells generated by B and T cells aggregate in the renal tubulointerstitium also generating the production of autoantibodies [73].

An increase in the inflammasome's components was observed in biopsies of patients with LN as PICARD(ASC), caspase-1, and IL-18, indicating their contribution to the disease 75]. Furthermore, the increased transcription of IL-18 in the tubulointerstitial and glomerular compartments correlates with the severity of LN and the onset of proteinuria. 

7.1.1.In Vitro Model

The activation of the inflammasome in cells of innate immunity could trigger or amplify an autoimmune response. After exposure to an inflammatory stimulus such as LPS, isolated fresh monocytes increase the activation of the inflammasome characterized by the rise in caspase-1, IL-1β, and IL-18. A caspase-1 inhibitor added to in vitro cultures reduces IL-18 production.

As mentioned previously, the NETosis mechanism contributes to the death of neutrophils in SLE patients. Evidence from groups of researchers suggests that SLE patients are characterized by an imbalance between the development and clearance of NETs, which produces tissue damage. Specifically, low-density proinflammatory granulocytes that occur in the bloodstream of SLE patients allow a much greater capacity to produce NETs[79].

Kahlenbertg and her coworkers [80] firstly demonstrated that NETs are, partly, activators of the inflammasome through the externalization of LL-37. NETs externalize various antimicrobial peptides. Specifically, cathelicidin LL-37 is a peptide synthesized by neutrophils, monocytes, and macrophages, among others, with activity against several pathogens. LL-37 externalization in NETs has been identified in neutrophils from SLE patients [79].In this study [80], the authors purified and isolated human and murine macrophages. Results showed that LL-37 activated the NLRP3 inflammasome in macrophages and that SLE patients were more likely to activate the inflammasome in response to LL-37 and NETs compared to macrophages from control patients. This stimulation perpetuates the increase in IL-1β and IL-18, which, in turn, will promote NETosis resulting in disease flares or organ damage, mainly kidneys, skin, and brain. Furthermore, their data suggested that the NLRP3 inflammasome is required for caspase-1 activation by LL-37. 

7.1.2.Animal Model

It is noteworthy to mention that in various murine studies, NLRP3 has been associated with LN. Kahlenberg et al. [81], studied the role of caspase-1 in the induction of murine lupus. Wild-type mouse models were exposed to pristane-developed lupus-related autoantibodies and active response to INF type I. Following pristane exposure, caspase-1 -/- mice did not have increased levels of IL-1β or IL-18, suggesting that caspase-1 played a role in the transcription of these cytokines. In addition, caspase-1-/-mice showed less development of autoantibodies and immune complexes related to glomerulonephritis, contrasting with wild-type mice. P2X7, an extracellular ATP-gated ion channel receptor, has also been shown to play a role in NLRP3 activation and LN development. The use of a selective P2X7 antagonist in brilliant blue Gin MLR/Ipr mice produced a downregulation of the NLRP3/ASC/Caspase-1 complex and therefore a suppression of IL-β. This reduced LN severity, proteinuria, and blood urea nitrogen levels in mice. Likewise, P2X7/NLRP3 inhibition decreases the Th17:Treg ratio, decreasing anti-double-stranded DNA antibodies(anti-dsDNA). NZM2328 mice that were injected intravenously with adenovirus-expressing interferon-c particles confirmed these results [82]. Increased expression of P2X7 has also been observed in the kidney tissue of patients with SLE [83]. A neutralizing monocle antibody to high-mobility box 1 protein, a ubiquitin nuclear protein that binds to DNA, has also been shown to decrease IL-β, IL-6, IL-17, and IL-18 levels and caspase-l in kidneys of BXSB mice. In addition, this model also attenuated proteinuria, glomerulonephritis, renal immune complex deposits, and circulating anti-dsDNA.

Another inflammatory pathway that affects LN is NF-kB. NF-KB is a transcription factor that participates in innate and adaptive immunity [85].In human studies, a correlation has been described between the activation of NF-kB and the histological and renal function impairment [86]. Zhao et al. [87] studied whether the inhibition of both pathways decreased LN progression in lupus-prone MRL/LPR mice. This interest was triggered by the renal pathophysiological role played by NF-kB. Inhibition of NF-kB and NLRP3 by Bay11-7082 prevented their formation and activation, respectively, resulting in an improvement in established kidney damage in LN. Moreover, Bayl1-7082 decreased renal immune complex deposits and serum anti-dsDNA levels.

AIM2 has also been implicated in the pathogenesis of SLE. However, its inhibition has been shown to be two-edged in relation to the pathophysiology of LN. Zhang and coworkers analyzed the correlation between the severity of LN and AIM2 in SLE patients and lupus mice. AIM2 expression was elevated in PBMCs from SLE patients. In addition, AIM2 correlated with macrophage activation and was augmented in macrophages induced by lymphocyte-derived apoptotic DNA. The inhibition of AIM2 by siRNA decreased the infiltration of macrophages in renal tissue and produced an improvement in nephritis [88]However, other researchers found that the inhibition of AIM2 generated susceptibility to developing SLE. p202 negatively regulates AIM2 in some mouse strains, increasing INF and predisposition to SLE.

7.1.3. Human Model

The role of the inflammasome in autoimmune diseases has been widely described. Studies with different SNPs related to inflammasome in patients with SLE have been reported in the literature. Pontillo et al.[90] analyzed 14 SNPs in 7 inflammasome genes, such as NLRP1, NLRP3, NLRC4, AIM2, CARD8, CASP1, and IL1B. The study showed, for the first time, an association between SNPs and SLE in a population from southern Brazil. The NLRP1 rs2670660 SNP, especially when combined with the NLRP1 variant rs12150220, confers an increased risk of SLE and developing nephritis, arthritis, and rash. Other SNPs also described in autoimmune diseases, such as celiac disease [91] and diabetes [21], were not associated with SLE disease in their population [90]. They also found no association with SLE with respect to AIM2 or IL1B polymorphisms [90]. Furthermore, results from da Cruz et al. found a gain of function in the NLRP3 rs10754558 variant in patients with LN.

7.2.ANCA Glomerulonephritis

ANCA associated with vasculitis (AAV)is a life-threatening autoimmune disease characterized by antibody-mediated glomerulonephritis and necrotizing vasculitis. AAV affects small and medium vessels, especially organs such as the kidney and lung. Pauci-immune and necrotizing glomerulonephritis are frequently associated in patients with vasculitis being more prevalent in men over 50 years of age. ANCA vasculitis is usually associated with ANCA-myeloperoxidase (MPO), ANCA-proteinase 3(PR3), or ANCA-negative serotype positivity. This pathology is classified into different clinical variants such as microscopic polyangiitis, granulomatosis with polyangiitis(Wegener), and Eosinophilic granulomatosis with polyangiitis(Churg-Strauss), or vasculitis limited to renal tissue [93]. Both innate and adaptive immunity participate in the development of AAV, although the exact mechanisms remain to be elucidated.

Neutrophils play a fundamental role in the pathogenesis of AAV inflicting tissue damage after degranulation induced by ANCA antibodies. Apart from antibodies, T cells are also involved in disease pathogenesis. Neutrophils secrete cytokines that recruit more neutrophils and other inflammatory cells. Infiltration of T cells is also part of the granulomas. The benefit of anti-T cell therapies demonstrates the involvement of this cell in AAV. The Th1 and Th17 phenotypes are involved in the acute phase. An increase in C5a and, therefore, a participation of the alternative complement pathway has been reported.

7.2.1.In Vitro Model

Inflammasome components such as cytokines are important mediators in AAV. Both Il-1β and Il-18 have been related to the pathogenesis of AAV, thus the implication of the inflammasome in the inflammation cascade of this disease is expected.

l-18 plays a role in neutrophil chemoattraction independent of TNFa priming [95], contrary to what was reported in other studies. Hewins et al. indicated that, in the presence of an anti-TNFα antibody, ANCA-induced superoxide production was not decreased. This would explain the persistence of tissue damage in the presence of anti-TNFα treatment. Furthermore, Hewins and colleagues demonstrated renal Il-18 expression in patients with ANCA vasculitis.

7.2.2.Animal Model

Several studies establish ANCA necrotizing crescentic GN(NCGN) in animal models [97]. Dipeptidyl peptidase (DPPI) is a cysteine protease responsible for activating neutrophil serine proteases (NPS)such as cathepsin G(CG), neutrophil elastase (NE), and PR3. These enzymes, responsible for modulating inflammation, have been related to the pathophysiology of ANCA vasculitis. In an anti-MPO antibody-induced experimental model of NCGN, a protective role of DPPI in kidney disease was demonstrated with a local decrease in inflammatory cytokines, especially IL-1β [98]. In fact, the elevation and processing of Il-1β by PR3 and NE have been linked [99]. The group of Scheiber et al.[98]demonstrated that active PR3 or active PR3/NE causes an increase in cytokines and anti-MPO antibodies, generating NCGN. They produced NE-/PR3-mice that were protected from NCGN. This demonstrated the role of NSP in ANCA nephropathy. In addition, they noticed that treatment with anakinra, an IL-1 receptor antagonist, downregulates the inflammatory cascade and protects against NCGN.

Another different pathway that induces Il-1β production and causes NCGN is related to phagocyte NADPH oxidase (Phox). Phox is a heme protein heterodimer from pg91phox and p22phox responsible for generating ROS producing tissue damage [100]. Though, some studies explain that ROS is also involved in the knockdown of inflammation [101]. Another study from the German group of Schreiber et al. [100] discovered the role of Phoxin in limiting the inflammasome by downregulating its components such as caspase-1 and thus IL-1β. Those authors created an antibody-mediated anti-MPO model. Transplanted mice with gp91phox-deficient or p47phox-deficient bone marrow showed greater histological involvement with more inflammation and necrosis, compared to mice with wild-type bone marrow. Additionally, they also generated pg91phox/caspase-1-deficient bone marrow transplant mice. In this case, mice with double deficiency improved NCGN compared to mice with only pg91phox- deficiency. These results hypothesize that Phox limits the activity of caspase-1 and therefore the role of the inflammasome.

7.2.3.Human Model

Elevated serum levels of the cascade of the inflammasome components have also been found in patients with ANCA vasculitis. IL-18 levels have been seen in patients with ANCA vasculitis regardless of MPO or PR3 values. It has been generally accepted that renal interstitial damage had to be associated with glomerular damage, but there are case reports of patients with ANCA vasculitis with only interstitial injury [103,104]. Tashiro et al. demonstrated no correlation between glomerular damage and interstitial damage in biopsies from patients with ANCA vasculitis. They glimpsed the correlation of IL-1β levels with the severity of tubulointerstitial damage. Moreover, infiltrating macrophages showed positive staining for NLRP3 at the tubulointerstitium without detecting this positivity in the glomerulus. On the contrary, Hewins et al. [95] who found upregulated IL-18 in renal biopsies, reported that positivity in the glomerulus has been found in podocytes while in the tubulointerstitium IL-18-positive has been observed in infiltrating macrophages, myofibroblasts, and tubular epithelial cells.

The activation of NOD-like receptors in patients with the active stage of ANCA vasculitis is from Wang et al. [106]. The mean optical densities of NOD2, NLRP3, and NLRC5, both in the glomerulus and in the tubulointerstitium, were significantly higher in patients with ANCA vasculitis than in healthy controls, in patients with minimal change disease, and in patients with type IV LN. NLRs were mainly expressed in podocytes and in infiltrating monocytes and macrophages but hardly expressed in glomeruli, results similar to those of Tashiro et al. The expression of NOD2 and NLRC5 correlated with clinicopathological involvement, while NLRP3 did not [106]. Unlike these researchers, Tashiro and colleagues did correlate NLRP3 with the severity of kidney damage.

7.3.IgA Nephropathy

IgA nephropathy(IgAN) is the main cause of renal failure due to glomerulonephritis in the world. Components of innate immunity are also involved in this nephropathy. The deposition of IgA aggregates or IgA immune complexes and subsequent activation of T cells causing inflammation is considered the main cause of the disease. The lgA subclass deposited in the glomerulus is the IgA1, which plays a central role in the pathophysiology of the disease. Mesangial cell proliferation is the typical histological finding of IgAN.Mesangial cells undergo proliferation under the action of IL-1, among other cytokines. The contribution of cytokines involved in the inflammasome cascade suggests a role for this inflammatory component in IgAN.

The alternative complement pathway and lectin pathways are also involved in the development of the disease since C3, C4, C4d, properdin, C5b-C9, and mannose-binding lectin are usually detected in renal biopsy.

7.3.1.Animal Model

Researchers have demonstrated IL-1 expression in kidney tissue affected with IgAN [110,111]. Chen et al., using an animal model of IgAN with ddY mice found decreased mesangial proliferation in mice treated with IL-1 receptor antagonist (IL-1ra). These results suggested that IL-1 is enrolled in IgAN development, evidencing a potential role of the inflammasome cascade in IgAN.

7.3.2.Human/In Vitro Model

The role of NLRP3 in lgAN remains to be discovered. The Canadian team of Chun et al. was the first to evaluate in vivo and in vitro the expression of NLRP3 in the kidney of patients with IgAN and the progression of the disease. They found that NLRP3 was expressed mainly in the tubules with no staining in the normal glomerulus. However, in patients with IgAN, NLRP3 expression was detected in the glomerulus, although it was more increased in the tubules. Both in human kidney biopsies and in low passage human cells, they established a decrease in NLRP3 during tubular damage. Equally the immunostaining results and the NLRP3 mRNA expression corroborated the presence of NLRP3 and its subsequent loss after renal injury. These discoveries suggest that NLRP3 may be a biomarker of tubular integrity. In addition, NLRP3 plays its role in the early stages of kidney disease being implicated in chronic kidney disease. However, due to study limitations, the researchers were unable to report on the functional role of the inflammasome.

Other researchers were able to glimpse the role of NLRP3 in the pathophysiology of lgAN.IgA immune complexes elicited the activation of the NLRP3 inflammasome in macrophages. This, in turn, stimulated the production of ROS by the mitochondria. They performed a mouse model with IgAN knockout for NLRP3. The generation of IgA immune complexes was inhibited by knockout mice. A regaining in renal function was described in NLRP3 knockout and in the kidney-targeting delivery of shRNA of NLRP3. Finally, the researchers clarify that they cannot exclude the role of other inflammasomes in lgAN and that the use of shNLRP3 could be a treatment to improve or prevent the disease.

7.4. Anti-Glomerular Basement Membrane Glomerulonephritis

Anti-glomerular basement membrane (anti-GBM) is an infrequent autoimmune dis-ease that affects the small vessels of the kidneys and lungs. Patients develop antibodies against the non-collagenous domain of the α3 chain of type IV collagen present in the basal membrane of those organs[115]. Although humoral immunity plays a central role, the participation of cellular immunity has also been reported. Thus, the lgG1 and IgG3 subclasses have been clearly related to the severity of the disease. The deposition of antibodies in kidney vessels can originate inflammation by activating complement and the Fc receptor. On the other hand, the increase in CD4+ T cells has been correlated with the severity of the disease. Peripheral CD4+ progress in the presence of c3 (IV) NC1.In addition, in animal models, CD4+ has been shown to be a trigger for the development of anti-GBM antibodies.

Animal Model

The major cytokines derived from the inflammasome cascade, IL-1, and IL-18, have been shown to have a pathophysiological role in patients with anti-GBM disease. In a mouse model of anti-GBM, Il-1β-/-and IL1 type 1 receptor(IL-1R)-/-mice, the role of IL-1 isoforms， IL-1α and IL-1β， in anti-GMB GN was studied. IL-1ß mice showed less development of crescentic formations, and recruitment of macrophages and T cells, while IL-1R1 -/-mice appeared to have a role in the immune response since they had fewer antibodies in serum. Furthermore, other animal models have demonstrated the proinflammatory role of IL-18 in renal inflammation.

Glomerular infiltration by macrophages is probably one of the major sources of IL-1 cytokine production. Several studies have analyzed the chemoattractant role of this cytokine and have implemented treatment with antagonists IL-1ra in a rat model with anti-GBM. Both the group of Lan et al. and Tang et al. demonstrated that by using IL-1ra there was a decrease in the infiltration of glomerular macrophages and an improvement in proteinuria. Lan et al. also revealed a stoppage of renal function worsening and prevented histological progression such as the formation of glomerular crescents. Tang et al. obtained a decreased expression of ICAM-1 after treatment with IL-1ra, which was also associated with a decline in the infiltration of polymorphonuclear (PMN) cells and monocytes.

However, following the findings found by Timoshenko et al. [117] on the contribution of IL-1β in nephritis in anti-GBM, other authors concluded, using a murine model, that only dendritic cells that reside mainly in the tubulointerstitium express pro-IL-1β and therefore they activate NLRP3 and caspase-1 secreting mature IL-1. They showed that the inflammasome axis does not contribute to glomerular inflammation since glomerular cells could not produce IL-1 during sterile inflammation.

8. Final Remarks

As comprehensively detailed, many studies have demonstrated the role of the inflammasome in glomerulonephritis. However, further insights into the pathophysiological mechanism research focus on inflammasome and autoimmune diseases are needed.

On the other hand, the participation of the inflammasome in immunity encourages the need for new therapeutic weapons aimed at its modulation. Recently, antagonists of IL-1ra are already approved to treat non-renal diseases such as rheumatoid arthritis, CAPS, Familial Mediterranean fever, and Still's a disease. Additionally, for now, there is development for treating autoimmune diseases in patients that are non-responsive or over time are refractory to treatment with TNF-α antagonists and/or T-cell co-stimulation antagonists with an IL-18 antagonist. Given that the IL-1 blockade or IL-18 antagonist has been successful in non-human models of renal diseases modulating inflammasome activation. Perhaps, we are ready to introduce these targets in the nephrological clinics. However, the blockade of a single cytokine could be not enough to downregulate the activation of the inflammasome, then polytherapy could be considered. To our knowledge, while there are developed clinical trials about autoimmune and inflammatory diseases, there are no ongoing clinical trials involving glomerulonephritis and inflammasomes. Thus, further efforts in the exploration into how treatments affect the activity of the inflammasome axis could be a promising therapy.