Immune-Mediated Nephropathy and Systemic Autoimmunity in Mice Does Not Require Receptor Interacting Protein Kinase 3 (RIPK3)

<div><p>Immune mediated nephropathy is one of the most serious manifestations of lupus and is characterized by severe inflammation and necrosis that, if untreated, eventually leads to renal failure. Although lupus has a higher incidence in women, both sexes can develop lupus glomerulonephritis; nephritis in men develops earlier and is more severe than in women. It is therefore important to understand the cellular and molecular mechanisms mediating nephritis in each sex. Previous work by our lab found that the absence or pharmacological inhibition of Poly [ADP-ribose] polymerase 1 (PARP-1), an enzyme involved in DNA repair and necrotic cell death, affects only male mice and results in milder nephritis, with less in situ inflammation, and diminished incidence of necrotic lesions, allowing for higher survival rates. A second pathway mediating necrosis involves Receptor-Interacting Serine-Threonine Kinase 3 (RIPK3); in this study we sought to investigate the impact of RIPK3 on the development of lupus and nephritis in both sexes. To this end, we used two inducible murine models of lupus: chronic graft versus host disease (cGvHD) and pristane-induced lupus; and nephrotoxic serum (NTS)-induced nephritis as a model of immune mediated nephropathy. We found that the absence of RIPK3 has neither positive nor negative impact on the disease development or progression of lupus and nephritis in all three models, and in both male and female mice. We conclude that RIPK3 is dispensable for the pathogenesis of lupus and immune mediated nephropathy as to accelerate, worsen or ameliorate the disease.</p></div

Pristane-induced systemic autoimmunity is not dependent on RIPK3.

<p><i>(A)</i> Pristane treatment induced similar ANA patterns between WT and RIPK3-/- mice. Experiments were performed with 14 mice per strain, p>0.05 using Chi Square analysis. <i>(B)</i> After 6 months and <i>(C)</i> after 2 weeks from pristane injection, peritoneal immune cell infiltrates were characterized by flow cytometry. There were similar percentages of B cells, T cells, CD11b+ monocytes, and CD11b+/CD11c+ conventional dendritic cells (cDCs) present in the peritoneal cavity during pristine-induced systemic autoimmunity. 5 mice per strain were used for the acute, 14 mice per strain were used for the chronic (6 month) experiments. Data are represented by ± SEM and population differences (p>0.05) measured using Ttest.</p

Absence of RIPK3 does not ameliorate NTS-induced nephritis in males or females.

<p>Male and female B6 and RIPK3 mice were injected i.p. with 8ul/g NTS. <i>(A)</i> BUN levels were measured using Azostix throughout the experiment to monitor kidney disease. Male and female mice lacking RIPK3 did not have significantly decreased renal damage with NTS treatment. The data shown have 5–8 (pooled experiments) mice and p>0.05 as measured by Wilcoxon-Rank Sum analysis. <i>(B)</i> Kidney sections from NTS-treated and control mice were stained to detect IgG and C3 deposition. (C) Kidneys from the NTS-treated mice were sectioned and stained with H&E (size bars = 200um). The sections were scored for severity of glomerulonephritis, interstitial nephritis, and vessel damage. N = 5–8 mice per strain and significance was, measured by Mann-Whitney U test, * = p ≥ 0.05. Representative images for histology grading (10x magnification) are located below the scoring graphs. Data shown are pooled from three sets of experiments, except the RIP3-/-PARP1-/- experiments which were two sets. Data represented as Mean ± SD.</p

Absence of RIPK3 does not affect the development of autoimmunity induced by cGvHD or pristane.

<p><i>(A)</i> After induction of cGvHD, anti-dsDNA and anti-chromatin levels were similar between B6 and RIPK3-/- mice in both males and females. Data are represented as Mean ± SEM of 10 mice per group (pooled from two experiments) and all p-values were >0.05 (T-test). Autoantibody levels of PBS-treated mice are represented by the dotted line. <i>(B)</i> After cGVHD induction, total IgG levels were similar between B6 and RIPK3-/- mice in both males and females. Data are represented as Mean ± SEM of 10 mice per group and all p-values were >0.05 (T-test). Total IgG levels were increased at 3 and 6 weeks compared to pre-bleed (T-test). Pre-bleed vs. 3 weeks: * p≤0.05, ** p≤ 0.01. Pre-bleed vs. 6 weeks: # p≤0.05.</p

Absence of RIPK3 does not cause a reduction in necrotic cell death within the kidney during NTS-nephritis.

<p>Percentage of apoptotic and necrotic TUNEL+ cells were determined by staining for TUNEL (red), active Caspase 3 (green), and nuclei (DAPI-blue). 10x size bar = 200μm, 40x size bar = 50μm. At least 50 TUNEL+ cells were recorded for each mouse, N = 5 mice per strain. Necrotic nuclei were characterized by only TUNEL+ staining, while apoptotic cells were positive for both TUNEL and active-caspase 3. Percentages of positive cells between B6 and RIPK3-/-, in each sex were not statistically significant (p≥0.05) as determined by T-test. Data is represented as Mean ± SEM of 2–5 mice per group. Experiments were repeated at least twice.</p