Mouse Model Of Glomerulonephritis Research Articles

Noninvasive Detection of iC3b/C3d Deposits in the Kidney Using a Novel Bioluminescent Imaging Probe.

Histologic quantification of complement C3 deposits in kidney biopsies provides prognostic information in patients with glomerulonephritis. Unfortunately, kidney biopsies are invasive procedures that cannot be performed regularly and only provide a snapshot of a small portion of one kidney at the time of sampling. We have developed a method to noninvasively detect specific C3 fragment deposition throughout both kidneys, using a monoclonal antibody targeting tissue-bound iC3b/C3d linked to a bioluminescent resonance energy transfer construct that emits near-infrared light. In a mouse model of glomerulonephritis, the probe detected iC3b/C3d in kidneys of live mice by bioluminescent imaging. This demonstrates that noninvasive imaging with an anti-iC3b/C3d probe can be used to monitor inflammation in the kidneys.

Axl regulated survival/proliferation network and its therapeutic intervention in mouse models of glomerulonephritis

BackgroundLupus nephritis (LN) is the most common and serious complication of systemic lupus erythematosus (SLE). LN pathogenesis is not fully understood. Axl receptor tyrosine kinase is upregulated and contributes to the pathogenic progress in LN. We have reported that Axl disruption attenuates nephritis development in mice.MethodsIn this study, we analyzed the gene expression profiles with RNA-seq using renal cortical samples from nephritic mice. Axl-KO mice were bred onto a B6.lpr spontaneous lupus background, and renal disease development was followed and compared to the Axl-sufficient B6.lpr mice. Finally, anti-glomerular basement membrane (anti-GBM) Ab-induced nephritic mice were treated with Axl small molecule inhibitor, R428, at different stages of nephritis development. Blood urine nitrogen levels and renal pathologies were evaluated.ResultsTranscriptome analysis revealed that renal Axl activation contributed to cell proliferation, survival, and motility through regulation of the Akt, c-Jun, and actin pathways. Spontaneous lupus-prone B6.lpr mice with Axl deficiency showed significantly reduced kidney damages and decreased T cell infiltration compared to the renal damage and T cell infiltration in Axl-sufficient B6.lpr mice. The improved kidney function was independent of autoAb production. Moreover, R428 significantly reduced anti-GBM glomerulonephritis at different stages of GN development compared to the untreated nephritic control mice. R428 administration reduced inflammatory cytokine (IL-6) production, T cell infiltration, and nephritis disease activity.ConclusionsResults from this study emphasize the important role of Axl signaling in LN and highlight Axl as an attractive target in LN.

Conventional NK Cells and Type 1 Innate Lymphoid Cells Do Not Influence Pathogenesis of Experimental Glomerulonephritis.

Innate lymphoid cells (ILCs) that express NK cell receptors (NCRs) and the transcription factor T-bet populate nonlymphoid tissues and are crucial in immune responses against viral infections and malignancies. Recent studies highlighted the heterogeneity of this ILC population and extended their functional spectrum to include important roles in tissue homeostasis and autoimmunity. In this article, we provide detailed profiling of NCR+T-bet+ ILC populations in the murine kidney, identifying conventional NK (cNK) cells and type 1 ILCs (ILC1s) as the two major subsets. Induction of renal inflammation in a mouse model of glomerulonephritis did not substantially influence abundance or phenotype of cNK cells or ILC1s in the kidney. For functional analyses in this model, widely used depletion strategies for total NCR+ ILCs (anti-NK1.1 Ab application) and cNK cells (anti-asialoGM1 serum application) were unreliable tools, because they were accompanied by significant off-target depletion of kidney NKT cells and CD8+ T cells, respectively. However, neither depletion of cNK cells and ILC1s in NKT cell-deficient mice nor specific genetic deletion of cNK cells in Ncr1 Cre/wt × Eomes fl/fl mice altered the clinical course of experimental glomerulonephritis. In summary, we show in this article that cNK cells and ILC1s are dispensable for initiation and progression of immune-mediated glomerular disease and advise caution in the use of standard Ab depletion methods to study NCR+ ILC function in mouse models.

Response Gene to Complement -32 facilitates local recruitment of IL-17-producing cells in immune complex mediated glomerulonephritis through the CCR6/CCL20 axis

Abstract Response Gene to Complement (RGC)-32 is a cell cycle regulator induced by complement activation, growth factors and cytokines. RGC-32 mediates TGF-β dependent profibrotic pathways, while in immune cells promotes the differentiation of Th17 cells. In the nephrotoxic nephritis (NTN) mouse model of glomerulonephritis (GN), RGC-32 promotes organ damage through proinflammatory and profibrotic effects. To define the mechanisms underlying the role of RGC-32 in immune complex GN, we induced NTN in WT and RGC-32−/− mice. Proteinuria, blood urea nitrogen and kidney histopathology were determined to assess kidney damage. Single cell suspension of infiltrating kidneys and spleens cells were analyzed by flow cytometry and PCR for expression of IL-17A, CCR6, CCL20, CXCL1, CXCL2 and CXCL5. Induction of NTN in RGC-32−/− mice resulted in decreased expression of IL-17A and CCR6 mRNA and decreased proportion of renal IL-17A+ CD4 T cells. The decrease in IL-17A+ CD4 cells was paralleled by decreased mRNA expression of CCL20 in both the renal tissue and infiltrating kidney cells of RGC-32−/− mice. In vitro, CD4+ T cells from spleens of RGC-32−/− mice cultured under Th17 conditions displayed decreased expression of CCL20 mRNA. These data suggest a role for RGC-32 in the positive feedback loop that enhances Th17 cell recruitment. The decrease in renal IL-17A+ cells was associated with decreased expression of IL-17 induced chemokines CXCL1, CXCL2 and CXCL5 and decreased local infiltration of neutrophils. These results suggest that RGC-32 promotes the proinflammatory pathway in immune complex mediated GN by enhancing local recruitment of IL-17A+ cells through the CCR6/CCL20 axis. RGC-32 is a potential novel therapeutic target in the treatment of LN.

Crucial Roles of the Protein Kinases MK2 and MK3 in a Mouse Model of Glomerulonephritis

Elevated mitogen-activated protein kinase p38 (p38 MAPK) signaling has been implicated in various experimental and human glomerulopathies, and its inhibition has proven beneficial in animal models of these diseases. p38 MAPK signaling is partially mediated through MK2 and MK3, two phylogenetically related protein kinases that are its direct substrates. The current study was designed to determine the specific roles of MK2 and MK3 in a mouse model of acute proliferative glomerulonephritis, using mice with disrupted MK2 and/or MK3 genes. We found that the absence of MK3 alone worsened the disease course and increased mortality slightly compared to wild-type mice, whereas the absence of MK2 alone exhibited no significant effect. However, in an MK3-free background, the disease course depended on the presence of MK2 in a gene dosage-dependent manner, with double knock-out mice being most susceptible to disease induction. Histological and renal functional analyses confirmed kidney damage following disease induction. Because the renal stress response plays a crucial role in kidney physiology and disease, we analyzed the stress response pattern in this disease model. We found that renal cortices of diseased mice exhibited a pronounced and specific pattern of expression and/or phosphorylation of stress proteins and other indicators of the stress response (HSPB1, HSPB6, HSPB8, CHOP, eIF2α), partially in a MK2/MK3 genotype-specific manner, and without induction of a general stress response. Similarly, the expression and activation patterns of other protein kinases downstream of p38 MAPK (MNK1, MSK1) depended partially on the MK2/MK3 genotype in this disease model. In conclusion, MK2 and MK3 together play crucial roles in the regulation of the renal stress response and in the development of glomerulonephritis, which can potentially be exploited to develop novel therapeutic approaches to treat glomerular disease.

SiRNA-Based Therapy Ameliorates Glomerulonephritis

RNA interference by short interfering RNAs (siRNAs) holds promise as a therapeutic strategy, but use of siRNAs in vivo remains limited. Here, we developed a system to target delivery of siRNAs to glomeruli via poly(ethylene glycol)-poly(l-lysine)-based vehicles. The siRNA/nanocarrier complex was approximately 10 to 20 nm in diameter, a size that would allow it to move across the fenestrated endothelium to access to the mesangium. After intraperitoneal injection of fluorescence-labeled siRNA/nanocarrier complexes, we detected siRNAs in the blood circulation for a prolonged time. Repeated intraperitoneal administration of a mitogen-activated protein kinase 1 (MAPK1) siRNA/nanocarrier complex suppressed glomerular MAPK1 mRNA and protein expression in a mouse model of glomerulonephritis; this improved kidney function, reduced proteinuria, and ameliorated glomerular sclerosis. Furthermore, this therapy reduced the expression of the profibrotic markers TGF-beta1, plasminogen activator inhibitor-1, and fibronectin. In conclusion, we successfully silenced intraglomerular genes with siRNA using nanocarriers. This technique could aid the investigation of molecular mechanisms of renal disease and has potential as a molecular therapy of glomerular diseases.

Streptococcal pyrogenic exotoxin B antibodies in a mouse model of glomerulonephritis

Streptococcal pyrogenic exotoxin B is an extracellular cysteine protease. Only nephritis-associated strains of group A streptococci secrete this protease and this may be involved in the pathogenesis of post-streptococcal glomerulonephritis. Mice were actively immunized with a recombinant protease inactive exotoxin B mutant or passively immunized with exotoxin B antibody. Characteristics of glomerulonephritis were measured using histology, immunoglobulin deposition, complement activation, cell infiltration, and proteinuria. None of the mice given bovine serum albumin or exotoxin A as controls showed any marked changes. Immunoglobulin deposition, complement activation, and leukocyte infiltration occurred only in the glomeruli of exotoxin B-hyperimmunized mice. One particular anti-exotoxin B monoclonal antibody, 10G, was cross-reactive with kidney endothelial cells and it caused kidney injury and proteinuria when infused into mice. This cross-reactivity may be involved in the pathogenesis of glomerulonephritis following group A streptococcal infection.

Heterogeneity of antigen expression explains controversy over glomerular macrophage accumulation in mouse glomerulonephritis.

Many antibody labelling studies have suggested that there are few or no glomerular macrophages in mouse models of glomerulonephritis, despite the presence of a prominent interstitial macrophage infiltrate. These findings conflict with studies of human and rat glomerulonephritis. Therefore, we examined whether heterogeneity of macrophage antigen expression could explain this apparent discrepancy. Kidneys were collected from normal mice and mice killed at 2 and 10 days after induction of accelerated anti-glomerular basement membrane (GBM) glomerulonephritis. Following fixation, macrophages were detected by immunoperoxidase staining in serial kidney sections using antibodies recognising CD11b, F4/80 and CD68. Induction of anti-GBM nephritis caused a progressive increase in glomerular and interstitial leukocytes. At days 2 and 10, there were more CD68+ macrophages in glomeruli than macrophages expressing CD11b or F4/80. At day 10, CD68+ macrophages accounted for almost all glomerular CD45+ total leukocytes. In contrast, CD11b+ and F4/80+ macrophages at day 10 accounted for only 65 and 13% of glomerular leukocytes, respectively. However, in the interstitium the number of macrophages expressing CD68, CD11b and F4/80 were not different. Antibody detection of mouse CD68 identifies all glomerular macrophages in mouse anti-GBM nephritis, indicating a similar infiltrate to that seen in human and rat anti-GBM nephritis. Our finding of substantial heterogeneity in glomerular macrophage antigen expression in this model suggests that previous studies of mouse glomerulonephritis may have underestimated glomerular macrophages and their role in glomerular injury.

Immunopathology of adoptive immunization in mice chronically infected with lymphocytic choriomeningitis virus

Adoptive immunization (AI) of mice chronically infected with lymphocytic choriomeningitis (LCM) virus results in a marked reduction in serum virus titer and elevation in serum anti-LCM antibody titer. The histopathology associated with these serological parameters was examined using light and immunofluorescence microscopy. Liver tissue was cleared of LCM antigen within 21 days of cell transfer, thus providing a simple histologic marker for AI in this mouse model of glomerulonephritis. Cytoplasmic LCM antigen in renal tubules and brain was not cleared after AI. Cytoplasmic deposits of mouse immunoglobulin similar to deposits of LCM antigen were noted in several tissues, suggesting a role for antibody in adoptive immunization.