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TNFα Amplifies DNaseI Expression in Renal Tubular Cells while IL-1β Promotes Nuclear DNaseI Translocation in an Endonuclease-Inactive Form.

Thiyagarajan D, Rekvig OP, Seredkina N - PLoS ONE (2015)

Bottom Line: TNFα-stimulation resulted in 3 distinct effects; increased DNaseI and IL-1β gene expression, and nuclear translocation of DNaseI.Tubular cells stimulated with TNFα and simultaneously transfected with IL-1β siRNA resulted in increased DNaseI expression but no nuclear translocation.Nuclear translocated DNaseI is shown to be enzymatically inactive, which may point at a new, yet unknown function of renal DNaseI.

View Article: PubMed Central - PubMed

Affiliation: RNA and Molecular Pathology Research Group, Department of Medical Biology, Faculty of Health Sciences, UiT The Arctic University of Norway, Tromsø, Norway.

ABSTRACT
We have demonstrated that the renal endonuclease DNaseI is up-regulated in mesangial nephritis while down-regulated during progression of the disease. To determine the basis for these reciprocal DNaseI expression profiles we analyse processes accounting for an early increase in renal DNaseI expression. Main hypotheses were that i. the mesangial inflammation and secreted pro-inflammatory cytokines directly increase DNaseI protein expression in tubular cells, ii. the anti-apoptotic protein tumor necrosis factor receptor-associated protein 1 (Trap 1) is down-regulated by increased expression of DNaseI due to transcriptional interference, and iii. pro-inflammatory cytokines promote nuclear translocation of a variant of DNaseI. The latter hypothesis emerges from the fact that anti-DNaseI antibodies stained tubular cell nuclei in murine and human lupus nephritis. The present study was performed on human tubular epithelial cells stimulated with pro-inflammatory cytokines. Expression of the DNaseI and Trap 1 genes was determined by qPCR, confocal microscopy, gel zymography, western blot and by immune electron microscopy. Results from in vitro cell culture experiments were analysed for biological relevance in kidneys from (NZBxNZW)F1 mice and human patients with lupus nephritis. Central data indicate that stimulating the tubular cells with TNFα promoted increased DNaseI and reduced Trap 1 expression, while TNFα and IL-1β stimulation induced nuclear translocation of the DNaseI. TNFα-stimulation resulted in 3 distinct effects; increased DNaseI and IL-1β gene expression, and nuclear translocation of DNaseI. IL-1β-stimulation solely induced nuclear DNaseI translocation. Tubular cells stimulated with TNFα and simultaneously transfected with IL-1β siRNA resulted in increased DNaseI expression but no nuclear translocation. This demonstrates that IL-1β promotes nuclear translocation of a cytoplasmic variant of DNaseI since translocation clearly was not dependent on DNaseI gene activation. Nuclear translocated DNaseI is shown to be enzymatically inactive, which may point at a new, yet unknown function of renal DNaseI.

No MeSH data available.


Related in: MedlinePlus

Renal expression of DNaseI and Trap 1 in (NZBxNZW)F1 mice during development of lupus nephritis.Mice were divided into 3 groups according to localization of electron dense structures (EDS) containing IgG (as traced by 5nm gold particles) in glomeruli as determined by immune electron microscopy (A). Pre-nephritic mice (Group 1) with no presence of EDS in glomeruli; mice with mesangial nephritis (Group 2) present EDS in the mesangium only; mice with membrano-proliferative nephritis (Group 3) have EDS both in the mesangium and in GBM (A). Expression patterns of DNaseI and Trap 1 were analysed by immune electron microscopy in Group 1–3 mice (B). DNaseI and Trap 1 were co-expressed in tubular cells in kidneys of Group 1 and Group 2 mice, while DNaseI staining was virtually absent in tubular cells in kidneys from Group 3 mice, while expression of Trap 1 was increased in these cells. The staining patterns were reflected by mRNA levels of renal DNaseI (C). In the left panel (C) average mRNA levels (±SD) are demonstrated in BW mice belonging to different groups (n = 5 per group). In the right panel (C), individual data on DNaseI mRNA levels in BW mice from Groups 1–3 (n = 3 per group) are demonstrated and compared with DNaseI gel zymography data (D). Renal DNaseI zymography data from all mice sacrificed every second week from 4–40 weeks are published in [25]. Significances: *<0.05; ***<0.0005.
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pone.0129485.g001: Renal expression of DNaseI and Trap 1 in (NZBxNZW)F1 mice during development of lupus nephritis.Mice were divided into 3 groups according to localization of electron dense structures (EDS) containing IgG (as traced by 5nm gold particles) in glomeruli as determined by immune electron microscopy (A). Pre-nephritic mice (Group 1) with no presence of EDS in glomeruli; mice with mesangial nephritis (Group 2) present EDS in the mesangium only; mice with membrano-proliferative nephritis (Group 3) have EDS both in the mesangium and in GBM (A). Expression patterns of DNaseI and Trap 1 were analysed by immune electron microscopy in Group 1–3 mice (B). DNaseI and Trap 1 were co-expressed in tubular cells in kidneys of Group 1 and Group 2 mice, while DNaseI staining was virtually absent in tubular cells in kidneys from Group 3 mice, while expression of Trap 1 was increased in these cells. The staining patterns were reflected by mRNA levels of renal DNaseI (C). In the left panel (C) average mRNA levels (±SD) are demonstrated in BW mice belonging to different groups (n = 5 per group). In the right panel (C), individual data on DNaseI mRNA levels in BW mice from Groups 1–3 (n = 3 per group) are demonstrated and compared with DNaseI gel zymography data (D). Renal DNaseI zymography data from all mice sacrificed every second week from 4–40 weeks are published in [25]. Significances: *<0.05; ***<0.0005.

Mentions: Baseline data for the BW mice included in this study have been published recently [25]. These mice are grouped according to morphology of the kidneys as determined by immune electron microscopy. Fig 1A demonstrates that Group 1 mice have no deposits of immune complexes in glomeruli; Group 2 mice have chromatin-IgG complex deposits, seen as electron dense structures (EDS) [5,25] in the mesangium; Group 3 mice have EDS in the mesangium and within the GBM. Individual mice from each group were further analysed for renal expression of DNaseI and Trap 1 by IEM. Fig 1B demonstrates that in Group 1 and Group 2 kidneys, DNaseI and Trap 1 are moderately expressed, while in Group 3 kidneys DNaseI expression could not be detected. Expression of Trap 1 in Group 3 mice was conversely strong compared with expression in Group 1 and Group 2 mice. As demonstrated in Fig 1C (left panel), renal DNaseI mRNA varied considerably in mice analyzed at various stages of the disease progression (n = 5 per group). Notably, renal DNaseI mRNA levels were particularly high in Group 2 compared with the levels in Group 1 and Group 3 BW mice. In Group 3, DNaseI mRNA levels were hardly detectable (Fig 1C, [25]). In the right panel in Fig 1C, DNaseI mRNA levels are demonstrated in individual mice from Groups 1–3 (n = 3 per group). Of particular interest is the observation of high to very high DNaseI levels in some Group 2 mice. Coherent results were obtained when comparing DNaseI mRNA levels and DNaseI enzyme activity (Fig 1C and 1D, respectively). This may mean that in Group 2 kidneys with mesangial nephritis, the inflammatory milieu is a candidate factor to explain early up-regulation of DNaseI. This hypothesis is subsequently tested in RPTEC by controlled in vitro experiments. Renal DNaseI zymography data from all mice sacrificed every second week from 4–40 weeks as part of a longitudinal study is recently published [25].


TNFα Amplifies DNaseI Expression in Renal Tubular Cells while IL-1β Promotes Nuclear DNaseI Translocation in an Endonuclease-Inactive Form.

Thiyagarajan D, Rekvig OP, Seredkina N - PLoS ONE (2015)

Renal expression of DNaseI and Trap 1 in (NZBxNZW)F1 mice during development of lupus nephritis.Mice were divided into 3 groups according to localization of electron dense structures (EDS) containing IgG (as traced by 5nm gold particles) in glomeruli as determined by immune electron microscopy (A). Pre-nephritic mice (Group 1) with no presence of EDS in glomeruli; mice with mesangial nephritis (Group 2) present EDS in the mesangium only; mice with membrano-proliferative nephritis (Group 3) have EDS both in the mesangium and in GBM (A). Expression patterns of DNaseI and Trap 1 were analysed by immune electron microscopy in Group 1–3 mice (B). DNaseI and Trap 1 were co-expressed in tubular cells in kidneys of Group 1 and Group 2 mice, while DNaseI staining was virtually absent in tubular cells in kidneys from Group 3 mice, while expression of Trap 1 was increased in these cells. The staining patterns were reflected by mRNA levels of renal DNaseI (C). In the left panel (C) average mRNA levels (±SD) are demonstrated in BW mice belonging to different groups (n = 5 per group). In the right panel (C), individual data on DNaseI mRNA levels in BW mice from Groups 1–3 (n = 3 per group) are demonstrated and compared with DNaseI gel zymography data (D). Renal DNaseI zymography data from all mice sacrificed every second week from 4–40 weeks are published in [25]. Significances: *<0.05; ***<0.0005.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4465975&req=5

pone.0129485.g001: Renal expression of DNaseI and Trap 1 in (NZBxNZW)F1 mice during development of lupus nephritis.Mice were divided into 3 groups according to localization of electron dense structures (EDS) containing IgG (as traced by 5nm gold particles) in glomeruli as determined by immune electron microscopy (A). Pre-nephritic mice (Group 1) with no presence of EDS in glomeruli; mice with mesangial nephritis (Group 2) present EDS in the mesangium only; mice with membrano-proliferative nephritis (Group 3) have EDS both in the mesangium and in GBM (A). Expression patterns of DNaseI and Trap 1 were analysed by immune electron microscopy in Group 1–3 mice (B). DNaseI and Trap 1 were co-expressed in tubular cells in kidneys of Group 1 and Group 2 mice, while DNaseI staining was virtually absent in tubular cells in kidneys from Group 3 mice, while expression of Trap 1 was increased in these cells. The staining patterns were reflected by mRNA levels of renal DNaseI (C). In the left panel (C) average mRNA levels (±SD) are demonstrated in BW mice belonging to different groups (n = 5 per group). In the right panel (C), individual data on DNaseI mRNA levels in BW mice from Groups 1–3 (n = 3 per group) are demonstrated and compared with DNaseI gel zymography data (D). Renal DNaseI zymography data from all mice sacrificed every second week from 4–40 weeks are published in [25]. Significances: *<0.05; ***<0.0005.
Mentions: Baseline data for the BW mice included in this study have been published recently [25]. These mice are grouped according to morphology of the kidneys as determined by immune electron microscopy. Fig 1A demonstrates that Group 1 mice have no deposits of immune complexes in glomeruli; Group 2 mice have chromatin-IgG complex deposits, seen as electron dense structures (EDS) [5,25] in the mesangium; Group 3 mice have EDS in the mesangium and within the GBM. Individual mice from each group were further analysed for renal expression of DNaseI and Trap 1 by IEM. Fig 1B demonstrates that in Group 1 and Group 2 kidneys, DNaseI and Trap 1 are moderately expressed, while in Group 3 kidneys DNaseI expression could not be detected. Expression of Trap 1 in Group 3 mice was conversely strong compared with expression in Group 1 and Group 2 mice. As demonstrated in Fig 1C (left panel), renal DNaseI mRNA varied considerably in mice analyzed at various stages of the disease progression (n = 5 per group). Notably, renal DNaseI mRNA levels were particularly high in Group 2 compared with the levels in Group 1 and Group 3 BW mice. In Group 3, DNaseI mRNA levels were hardly detectable (Fig 1C, [25]). In the right panel in Fig 1C, DNaseI mRNA levels are demonstrated in individual mice from Groups 1–3 (n = 3 per group). Of particular interest is the observation of high to very high DNaseI levels in some Group 2 mice. Coherent results were obtained when comparing DNaseI mRNA levels and DNaseI enzyme activity (Fig 1C and 1D, respectively). This may mean that in Group 2 kidneys with mesangial nephritis, the inflammatory milieu is a candidate factor to explain early up-regulation of DNaseI. This hypothesis is subsequently tested in RPTEC by controlled in vitro experiments. Renal DNaseI zymography data from all mice sacrificed every second week from 4–40 weeks as part of a longitudinal study is recently published [25].

Bottom Line: TNFα-stimulation resulted in 3 distinct effects; increased DNaseI and IL-1β gene expression, and nuclear translocation of DNaseI.Tubular cells stimulated with TNFα and simultaneously transfected with IL-1β siRNA resulted in increased DNaseI expression but no nuclear translocation.Nuclear translocated DNaseI is shown to be enzymatically inactive, which may point at a new, yet unknown function of renal DNaseI.

View Article: PubMed Central - PubMed

Affiliation: RNA and Molecular Pathology Research Group, Department of Medical Biology, Faculty of Health Sciences, UiT The Arctic University of Norway, Tromsø, Norway.

ABSTRACT
We have demonstrated that the renal endonuclease DNaseI is up-regulated in mesangial nephritis while down-regulated during progression of the disease. To determine the basis for these reciprocal DNaseI expression profiles we analyse processes accounting for an early increase in renal DNaseI expression. Main hypotheses were that i. the mesangial inflammation and secreted pro-inflammatory cytokines directly increase DNaseI protein expression in tubular cells, ii. the anti-apoptotic protein tumor necrosis factor receptor-associated protein 1 (Trap 1) is down-regulated by increased expression of DNaseI due to transcriptional interference, and iii. pro-inflammatory cytokines promote nuclear translocation of a variant of DNaseI. The latter hypothesis emerges from the fact that anti-DNaseI antibodies stained tubular cell nuclei in murine and human lupus nephritis. The present study was performed on human tubular epithelial cells stimulated with pro-inflammatory cytokines. Expression of the DNaseI and Trap 1 genes was determined by qPCR, confocal microscopy, gel zymography, western blot and by immune electron microscopy. Results from in vitro cell culture experiments were analysed for biological relevance in kidneys from (NZBxNZW)F1 mice and human patients with lupus nephritis. Central data indicate that stimulating the tubular cells with TNFα promoted increased DNaseI and reduced Trap 1 expression, while TNFα and IL-1β stimulation induced nuclear translocation of the DNaseI. TNFα-stimulation resulted in 3 distinct effects; increased DNaseI and IL-1β gene expression, and nuclear translocation of DNaseI. IL-1β-stimulation solely induced nuclear DNaseI translocation. Tubular cells stimulated with TNFα and simultaneously transfected with IL-1β siRNA resulted in increased DNaseI expression but no nuclear translocation. This demonstrates that IL-1β promotes nuclear translocation of a cytoplasmic variant of DNaseI since translocation clearly was not dependent on DNaseI gene activation. Nuclear translocated DNaseI is shown to be enzymatically inactive, which may point at a new, yet unknown function of renal DNaseI.

No MeSH data available.


Related in: MedlinePlus