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

View Article: PubMed Central - PubMed

ABSTRACT

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.

No MeSH data available.


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Absence of RIPK3 does not ameliorate NTS-induced nephritis in males or females.Male and female B6 and RIPK3 mice were injected i.p. with 8ul/g NTS. (A) 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. (B) 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.
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pone.0163611.g003: Absence of RIPK3 does not ameliorate NTS-induced nephritis in males or females.Male and female B6 and RIPK3 mice were injected i.p. with 8ul/g NTS. (A) 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. (B) 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.

Mentions: The above results indicate that RIPK3 is not required for the activation of the autoimmune response and the production of autoantibodies. To determine whether RIPK3 is important in the final phase of the nephritis, i.e. the tissue damage induced by antibodies and complement, we investigated the role of RIPK3 in the development of nephritis induced by administration of NTS. This is a serum from sheep immunized with extracts of mouse renal glomeruli that contains anti-glomeruli antibodies and triggers with a single injection a type II/III hypersensitivity, complement-dependent immune response. We found that both wild type and RIPK3-deficient mice, whether male or female, developed similarly high levels of blood urea nitrogen (BUN), indicating renal failure (Fig 3A). All mice stained positive for glomerular IgG and complement deposition with similar intensities (Fig 3B). In addition to BUN levels, pathology scoring of H&E sections per conventional means staining [22] demonstrated similar disease severity whether RIPK3 was present or not (Fig 3C). Our laboratory has previous shown that male mice lacking PARP1 develop less renal disease during NTS-nephritis than WT mice [11]. A direct relationship between the PARP1- and RIPK3-mediated death pathways has been controversial in the literature [29, 30]. Therefore, we crossed the B6.RIPK3-/- strain with a B6.PARP1-/- strain to generate a B6.RIP3-/-PARP1-/- double mutant mouse to investigate possible interactions between the RIP3- and PARP1—mediated necrotic pathways in the NTS model. RIPK3-/-PARP1-/- females develop similar levels of renal damage as the WT; however, the double mutant males develop reduced levels of renal damage but only in male mice (Fig 3A and 3C). These results are consistent with our previous work in B6.PARP1-/- mice and we conclude that reduced disease in males is due solely to the absence of PARP1 and is independent of RIPK3. In conclusion, our results demonstrate that RIPK3 is not critical for the development of nephritis not only in males but also in females, suggesting that the necrotic pathway in females is induced by an unknown mechanism, or by a redundant combination of PARP1 and RIPK3-driven pathways.


Immune-Mediated Nephropathy and Systemic Autoimmunity in Mice Does Not Require Receptor Interacting Protein Kinase 3 (RIPK3)
Absence of RIPK3 does not ameliorate NTS-induced nephritis in males or females.Male and female B6 and RIPK3 mice were injected i.p. with 8ul/g NTS. (A) 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. (B) 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.
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pone.0163611.g003: Absence of RIPK3 does not ameliorate NTS-induced nephritis in males or females.Male and female B6 and RIPK3 mice were injected i.p. with 8ul/g NTS. (A) 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. (B) 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.
Mentions: The above results indicate that RIPK3 is not required for the activation of the autoimmune response and the production of autoantibodies. To determine whether RIPK3 is important in the final phase of the nephritis, i.e. the tissue damage induced by antibodies and complement, we investigated the role of RIPK3 in the development of nephritis induced by administration of NTS. This is a serum from sheep immunized with extracts of mouse renal glomeruli that contains anti-glomeruli antibodies and triggers with a single injection a type II/III hypersensitivity, complement-dependent immune response. We found that both wild type and RIPK3-deficient mice, whether male or female, developed similarly high levels of blood urea nitrogen (BUN), indicating renal failure (Fig 3A). All mice stained positive for glomerular IgG and complement deposition with similar intensities (Fig 3B). In addition to BUN levels, pathology scoring of H&E sections per conventional means staining [22] demonstrated similar disease severity whether RIPK3 was present or not (Fig 3C). Our laboratory has previous shown that male mice lacking PARP1 develop less renal disease during NTS-nephritis than WT mice [11]. A direct relationship between the PARP1- and RIPK3-mediated death pathways has been controversial in the literature [29, 30]. Therefore, we crossed the B6.RIPK3-/- strain with a B6.PARP1-/- strain to generate a B6.RIP3-/-PARP1-/- double mutant mouse to investigate possible interactions between the RIP3- and PARP1—mediated necrotic pathways in the NTS model. RIPK3-/-PARP1-/- females develop similar levels of renal damage as the WT; however, the double mutant males develop reduced levels of renal damage but only in male mice (Fig 3A and 3C). These results are consistent with our previous work in B6.PARP1-/- mice and we conclude that reduced disease in males is due solely to the absence of PARP1 and is independent of RIPK3. In conclusion, our results demonstrate that RIPK3 is not critical for the development of nephritis not only in males but also in females, suggesting that the necrotic pathway in females is induced by an unknown mechanism, or by a redundant combination of PARP1 and RIPK3-driven pathways.

View Article: PubMed Central - PubMed

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus