Animal Models of Lupus Nephritis: The Past, Present and a Future Outlook

2.1. MRL/lpr

The Murphy-Ross Large (MRL) strain was developed by crossing several strains, including C3H/Di, LG/J, C57BL/6, and AKR/J [24,25]. One of the MRL sub-strains carries a spontaneous mutation named lpr for lymphoproliferation and develops a severe SLE-like phenotype characterized at the cellular level by the accumulation of double negative (DN: CD4−CD8−) B220+ T cells. Like in SLE patients [26], the DN T cells are autoreactive and pathogenic [27], making this model relevant to SLE pathogenesis. Lpr corresponds to non-functional transcripts of the Fas gene, a key regulator of apoptosis in immune cells [28]. In this strain, male and female mice are affected (though females show a more pronounced phenotype) and produce autoantibodies against dsDNA and Sm, leading to large amounts of IC that induce renal and skin pathology [29]. Unlike SLE patients, these mice develop a massive lymphadenopathy. The data generated from the MRL/lpr strain has led to, among others, a better understanding of the role of TRL7 and TLR9 in lupus [30], differences in TLR activation in B cells and dendritic cells [31], and were used for the first time to report the critical role of the extrafollicular pathway for the development of autoreactive B cells [32]. Additionally, these mice have shown that the kidney infiltrating T cells in LN have an exhausted metabolic and functional phenotype [33]. Complement plays a critical role in nephritis in these mice, as a deficiency in the regulatory factor H accelerates kidney pathology [34,35]. MRL/lpr mice become azotemic, leading to 70% mortality by 14 weeks of age. Unlike other lupus models (e.g., NZB/NZW F1 discussed below), nephritis in MRLlpr mice is independent of Type I interferons and FcγRs, which are two major drivers of SLE pathogenesis in general, including LN. This represents clear limitations to the relevance of the MRL/lpr model to human LN, in addition to the confounding massive lymphoproliferation [36]. C57BL/6-Fas^lpr mice expressing the lpr mutation on a non-autoimmune C57BL/6 (B6) background develop systemic autoimmunity without clinical pathology and reduced lymphadenopathy [37]. These B6.lpr mice have been used to investigate the involvement of IL-17 in lupus [38,39]. Finally, the MRL/lpr model has the major advantage of being the most rapid, with the highest incidence and severity of disease amongst the spontaneous models. Hence, it has been extensively used as a pre-clinical model of LN to test novel therapeutics [23,40]. For example, the MRL/lpr mice expressing human CD20 on their B cells were used to demonstrate the superiority of obinutuzumab, a type II anti-CD20 monoclonal antibody over rituximab [41]. These mice were also utilized to highlight the efficacy of idebenone, an antioxidant that bypasses complex I activity and enhances ATP synthesis [42].

2.2. The BXSB model

The BXSB strain is a recombinant inbred strain developed in the 1970s by crossing of C57BL/6 and SB/Le mice ((B6 X SB/Le) F1 to SB/Le, hence BXSB) [43,44]. The BXSB male mice develop an accelerated disease compared to females due to the Y chromosome-linked autoimmune accelerator (Yaa) [44,45], which has been identified as the translocation of a telomeric region of the X chromosome to the Y chromosome, resulting in the duplication of the toll-like receptor 7 (Tlr7) gene [46,47]. This increased TLR7 expression activates the Type I interferon cascade, which is critical in SLE pathogenesis [48]. BXSB.Yaa mice have a relatively low titer of autoantibodies reactive to DNA and histones but produce a higher titer of anti-ribonucleoproteins (RNP) IgG with typical speckled patterns [49]. Male BXSB mice develop exudative, proliferative nephritis with neutrophilic infiltrates and severe proteinuria by three months of age [29,50]. The kidneys show prominent IgG and C3 deposits in the glomerular mesangium and along the capillary walls, with 50% of the mice developing fatal nephritis starting around five months and 90% at around eight months of age [29,49]. Female mice develop a weaker disease phenotype with around 50% mortality around 15 months of age. Therefore, despite its predominant presentation in males, the BXSB/Yaa strain models the consequences of an overreactive TLR7/Type 1 IFN pathway and associated renal failure.

Hybrid (NZW x BXSB)F1 male mice carry the Yaa-associated two copies of Tlr7 and develop accelerated lupus with antiphospholipid syndrome (APS) [51] as well as proliferative glomerulonephritis [46]. Female (NZW x BXSB)F1 mice with only one active copy of Tlr7 develop late-onset glomerulonephritis but not APS [51]. In addition to inflammatory glomerulonephritis, the males develop immune-mediated thrombocytopenia and a thrombotic vasculopathy with myocardial infarcts, myocardial fibrosis, and dilated cardiomyopathy [52,53]. Given the importance of antiphospholipid antibodies in lupus [54], this model is a valuable tool to dissect the mechanisms and interventions in SLE-associated antiphospholipid syndrome [53].

2.3. The NZB/NZW F1 model

The NZB/NZW F1 hybrid (NZB/W F1) is a spontaneous model of SLE and LN [55,56] and it is phenotypically a close approximation of human SLE and LN. As in humans, female mice are more severely affected, and anti-dsDNA antibodies are detected at around three months of age [57]. Female mice develop splenomegaly and chronic polyclonal B cell activation [29,50]. The kidney pathology presents as sclerosis with proteinaceous deposits in the mesangium, diffuse proliferation of glomerular cells, and tubular casts. As in LN patients, prominent glomerular crescents are observed, and large periglomerular and interstitial inflammatory infiltrate [29,55]. A few critical contributions of the NZB/W F1 model to the field include the identification of the therapeutic effect of BAFF inhibition [58], the role of type 1 IFN [59], and the identification of LN biomarkers [60]. We have shown that early treatment with a combination of metformin and CTLA4Ig decreases the development of LN in this model without attenuation of autoantibody production [61].

The serendipitous breeding between B/W and NZW mice led to the development of a panel of inbred New Zealand Mixed (NZM) mouse strains, several of which showed a highly penetrant SLE and LN resembling disease in parental NZB/W F1mice [56,62]. Pioneering studies on identifying the SLE susceptibility loci in murine SLE were conducted with the NZM2410 strain [62,63]. Identifying three lupus susceptibility loci (Sle1, Sle2, and Sle3 on chromosomes 1, 4, and 7, respectively) in the NZM2410 strain has also led to the development of congenic strains with unique advantages. Intercrossing the three Sle1, Sle2, and Sle3 susceptibility loci was sufficient to develop a lupus phenotype on the non-autoimmune B6 genetically resistant strain [63,64]. The B6.NZM2410.Sle1.Sle2.Sle3 is unique as it shares 95% of its genome with B6 and has provided original insight into the metabolic control of the disease [65–67]. The corresponding single (Sle1) and bi-congenic (Sle1.Sle3) are also well suited to breed with B6-based gene knockouts. For instance, deletion of the plasmacytoid dendritic cells (pDC)-specific transcription factor Tcf4 in B6.Sle1.Sle3 mice provided genetic evidence that pDCs are critically involved in developing SLE [68]. Furthermore, the transfer of bone marrow cells from B6.Sle1 mice into irradiated B6 recipients was sufficient to recapitulate the autoimmune phenotypes of B6.Sle1 mice, demonstrating that they are entirely accounted for by hematopoietic cells [69]. Bone marrow transfer from these NZM2410-derived congenics into mice with specific gene knockout (e.g., in the kidney) provides a powerful tool for studying end-organ resistance or susceptibility to lupus.

A notable mention in the NZM sub-strains is NZM2328 [70,71], which develops nephritis in two stages: acute glomerulonephritis followed by more chronic nephritis [72,73]. These mice were vital in showing that autoantibodies, especially anti-dsDNA antibodies, are not required to develop renal disease in SLE [74,75].

3.1. Pristane-induced lupus

Initially used for priming mice to obtain ascite fluid to produce monoclonal antibodies, an intraperitoneal injection of pristane (2,6,10,14 tetramethylpentadecane, a major component of mineral oil) leads to the development of anti-RNP, anti-DNA and anti-histone antibodies in BALB/c mice [76,77]. In this inducible model, the BALB/c and SJL strains present glomerular IgG and complement deposits with cellular proliferation and develop proteinuria, whereas B6 mice develop milder (Class II like) disease [78]. In contrast to most spontaneous models of SLE, the anti-RNP antibodies class switches to IgG, while anti-DNA antibodies are almost entirely IgM [77]. These mice gradually develop nephritis six months after injection with focal to diffuse proliferative glomerulonephritis and moderate proteinuria [77]. Pristane-treated BALB/c mice also develop lung hemorrhagic, capillary damage-associated pathology, and TNFα-driven arthritis that may resemble arthralgia and capillaritis occurring in SLE patients [79–81]. In the SJL strain, pristane-induced lupus is more severe in females than males [82]. Compared to males, the kidneys of pristane-injected SJL female mice present with significantly more glomerular hypercellularity and necrosis [82]. Pristane-induced lupus is driven by type 1 IFN [83] and thus, it is a valuable tool for investigating type 1 IFN-induced pathology, a critical pathogenic pathway in human lupus. This model was used to define the protective role of TLR9 in lupus as BALB/c mice with TLR9 deficiency developed more severe lupus than their WT TLR9-sufficient mice [84]. Pristane-induced lupus is an excellent example of how an environmental factor can induce lupus-like disease in a non-autoimmune background.

3.2. Nephrotoxic Serum Nephritis

The nephrotoxic serum nephritis (NTN), also known as anti-glomerular basement nephritis, is an inducible IC-mediated model of glomerulonephritis that resembles human Goodpasture’s Disease [85,86]. The antigen (anti-mouse glomerular basement membrane antibodies raised commonly in rabbits or sheep (donor) after immunization with mouse glomerular preparation) binds the mouse glomerular basement membrane (GBM), leading to IC deposition and inflammatory response. Most nephrotoxic serum preparations are crude as they contain immunoglobulins against other structural elements of the kidney. Administration of the heterologous donor anti-GBM antiserum (NTS) leads to complement-dependent, transient renal injury (day 0–4) [87], followed by the development of mouse anti–rabbit or sheep antibodies in the later, autologous stage (day 7–14) during which IC deposits are observed in the glomeruli, resulting in glomerulonephritis with renal failure [88]. A modified accelerated model of NTS has also been developed wherein mice are pre-sensitized to the heterologous anti-GBM serum by preimmunization with normal, donor heterologous serum a few days (Day −4) before the introduction of the heterologous anti-GBM serum (Day 0), thus initiating the autologous phase of disease concomitant with the introduction of the anti-GBM serum [88,89].

The binding of polyclonal antibodies to the glomerular endothelial wall and GBM antigens triggers local immune activation and the secretion of proinflammatory chemokines and cytokines. In the heterologous phase, glomerular injury depends largely on complement activation and neutrophil-dominated inflammation [90–92]. During the autologous phase, the renal dendritic cells promote nephritogenic T-cell responses [93]. At this stage, a Th1 response stimulates the macrophages to mediate renal damage in a classical type IV hypersensitivity reaction [94], though a pathogenic Th17 response has also been reported [95]. Using this model, we have recently shown the importance of iron-mediated tubulointerstitial injury in the pathogenesis of LN [96]. Since the phenotype of NTN involves changes in the renal functional parameters, glomerular and secondary tubulointerstitial injury, renal leukocyte infiltration, and adaptive cellular and humoral immune responses [96], it serves as an all-encompassing tool to tease out delicate mechanisms associated with LN-like chronic IC-induced GN. The NTN model allows investigations into the pathogenesis of IC-mediated glomerulonephritis independent of genetic background and gender and is highly synchronized.

3.3. Chronic graft-versus-host model of systemic lupus erythematosus

Graft versus host disease (cGVHD) models are generated by transferring major histocompatibility complex (MHC) mismatched immune cells into a recipient/host. Compared to the acute GVDH model [97], the chronic GVHD (cGVHD) model, where I-A^bm12B6(C)-H2-Ab1^bm12/KhEgJ (bm12) splenocytes are transferred into B6 mice produce symptoms closely resembling SLE. In this model, the adoptive transfer of immune cells from bm12 mice, a strain identical to B6 except for three amino acid substitutions on the MHC class II _I-A chain, into I-A^b B6 mice leads to alloactivation of donor CD4+ T cells by recipient APCs [98]. The activated alloreactive donor CD4+ T cells then provide help to host self-reactive B cells [99,100]. Some of the key features include the expansion of donor-derived T follicular helper(Tfh) cells, recipient-derived germinal center (GC) B cells and plasma cells, and the production of anti-dsDNA, anti-ssDNA, anti-chromatin, and anti-RBC antibodies [98]. During the later phase, the recipient mice develop glomerulonephritis associated with IgG deposits in the kidneys’ glomerular, interstitial, and vascular segments [101]. Host CD4+ T cells do not participate in the cGVHD induction, but their presence is essential for nurturing B cells through IL-4 and/or CD40 stimulation [101,102].

Like the NTN model, there are several advantages of this model. Genetically modified B6 mice can serve as either donors or recipients. This allows dissecting the contribution of specific genes and modified cells to disease progression. Furthermore, disease development in the bm12 model requires only two weeks for ANA production, compared to several months for most spontaneous models [103]. Furthermore, unlike spontaneous models that develop disease at different time points, the disease onset and progression in this model is highly synchronized. This permits the use of appropriately sized cohorts to test novel therapeutics during different stages of SLE/LN.

3.4. Humanized Models of Lupus

Though mouse models provide insights on SLE/LN pathogenesis, they do not fully recapitulate the complexities of human SLE. There are major differences between the mouse and human immune system [104,105] and genetics [106,107] pose challenges in understanding the complexities of SLE and evaluation of therapeutic protocols [105]. There have been some attempts to bridge the differences between the murine and human immune systems by using humanized mice as models of SLE. [108]. Immune cell-mediated lupus-like disease can be initiated by engrafting immunodeficient mice with either peripheral blood leukocytes from SLE patients or healthy hematopoietic stem cells, followed by lupus induction with pristane [108]. Proteinuria, a marker of kidney injury in LN, was observed using both approaches [109,110]. The first approach was used to evaluate the therapeutic potential of targeting the Kv1.3 channel on T cells with nanoparticles [109]. However, the injury induced by human immune cells to murine renal parenchyma may not provide a reliable model for in-depth mechanistic studies or hold clinical relevance.