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NLRP3 Localizes to the Tubular Epithelium in Human Kidney and Correlates With Outcome in IgA Nephropathy.

Chun J, Chung H, Wang X, Barry R, Taheri ZM, Platnich JM, Ahmed SB, Trpkov K, Hemmelgarn B, Benediktsson H, James MT, Muruve DA - Sci Rep (2016)

Bottom Line: While NLRP3 expression in IgAN was detected in glomeruli, it remained largely confined to the tubular epithelial compartment.In vitro NLRP3 mRNA and protein expression were transiently induced in HPTC by TGF-β1 but subsequently diminished over time as cells lost their epithelial phenotype in a process regulated by transcription and ubiquitin-mediated degradation.Consistent with the in vitro data, low NLRP3 mRNA expression in kidney biopsies was associated with a linear trend of higher risk of composite endpoint of doubling serum creatinine and end stage renal disease in patients with IgAN.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada.

ABSTRACT
Nod-like receptor pyrin domain-containing-3 (NLRP3) has been implicated in the pathogenesis of experimental renal injury, yet its characterization in human kidney disease remains largely unexplored. NLRP3 expression was evaluated in human kidney biopsies, primary renal tubular cells (HPTC) and correlated to disease outcomes in patients with IgA nephropathy (IgAN). NLRP3 localized to renal tubules in normal human kidney tissue and to mitochondria within HPTC by immunohistochemistry and immunofluorescence microscopy. Compared to control kidneys, NLRP3 gene expression was increased in biopsies of patients with IgAN. While NLRP3 expression in IgAN was detected in glomeruli, it remained largely confined to the tubular epithelial compartment. In vitro NLRP3 mRNA and protein expression were transiently induced in HPTC by TGF-β1 but subsequently diminished over time as cells lost their epithelial phenotype in a process regulated by transcription and ubiquitin-mediated degradation. Consistent with the in vitro data, low NLRP3 mRNA expression in kidney biopsies was associated with a linear trend of higher risk of composite endpoint of doubling serum creatinine and end stage renal disease in patients with IgAN. Taken together, these data show that NLRP3 is primarily a kidney tubule-expressed protein that decreases in abundance in progressive IgAN.

No MeSH data available.


Related in: MedlinePlus

NLRP3 signal at mitochondria in TGF-β1 stimulated HPTC.Indirect immunofluorescence probing for NLRP3 (green) and GM130 (red) or mitochondria (Mitotracker Red) in HPTC untreated or treated with TGF-β1 (5 ng/ml) for 7 days. Bottom rows show magnified images of the outlined areas for single and merged images acquired from the red and green channels with yellow indicating colocalization. Scale bars represent 20 μm for full images and 10 μm for insets. Images are representative of experiments performed 3 independent times.
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f6: NLRP3 signal at mitochondria in TGF-β1 stimulated HPTC.Indirect immunofluorescence probing for NLRP3 (green) and GM130 (red) or mitochondria (Mitotracker Red) in HPTC untreated or treated with TGF-β1 (5 ng/ml) for 7 days. Bottom rows show magnified images of the outlined areas for single and merged images acquired from the red and green channels with yellow indicating colocalization. Scale bars represent 20 μm for full images and 10 μm for insets. Images are representative of experiments performed 3 independent times.

Mentions: To further examine the biology of NLRP3 in the context of human tubular injury and fibrosis, we stimulated HPTC for up to seven days with TGF-β1 to model chronic epithelial cell injury and fibrosis in vitro. Following TGF-β1 stimulation, the HPTC gradually acquired a fibroblastic phenotype with an obvious progressive change in morphology most notable following seven days of treatment (Fig. 5a). Both NLRP3 protein (Fig. 5b,c) and mRNA (Fig. 5d) expression in HPTC peaked after treatment with TGF-β1 for three days. Beyond three days, the level of NLRP3 protein and mRNA expression decreased concomitantly with progression of TGF-β1 mediated phenotypic change that included increased α-SMA and reduced levels of E-cadherin (Fig. 5b–d). Consistent with the loss of epithelial cell phenotype induced by prolonged TGF-β1 stimulation in HPTC, the epithelial cell injury marker KIM-1 also decreased significantly over time (Fig. 5b,c). Following seven days of TGF-β1 stimulation, Smad 2/3 remained in the nucleus suggesting persistent activation of the Smad-dependent signaling pathway and clear loss of Zo-1 membrane staining suggesting loss of epithelial morphology (Fig. 5e,f). Interestingly, NLRP3 signal diminished at the mitochondria with preservation of the mitochondrial architecture suggesting reduced levels of NLRP3 at the mitochondria rather than structural compromise of the organelle itself (Fig. 6). Taken together, these data show that there is an initial rise in NLRP3 expression induced by TGF-β1, but ongoing stimulation and loss of epithelial phenotype in HPTC is associated with decreased NLRP3 protein and mRNA expression.


NLRP3 Localizes to the Tubular Epithelium in Human Kidney and Correlates With Outcome in IgA Nephropathy.

Chun J, Chung H, Wang X, Barry R, Taheri ZM, Platnich JM, Ahmed SB, Trpkov K, Hemmelgarn B, Benediktsson H, James MT, Muruve DA - Sci Rep (2016)

NLRP3 signal at mitochondria in TGF-β1 stimulated HPTC.Indirect immunofluorescence probing for NLRP3 (green) and GM130 (red) or mitochondria (Mitotracker Red) in HPTC untreated or treated with TGF-β1 (5 ng/ml) for 7 days. Bottom rows show magnified images of the outlined areas for single and merged images acquired from the red and green channels with yellow indicating colocalization. Scale bars represent 20 μm for full images and 10 μm for insets. Images are representative of experiments performed 3 independent times.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4837396&req=5

f6: NLRP3 signal at mitochondria in TGF-β1 stimulated HPTC.Indirect immunofluorescence probing for NLRP3 (green) and GM130 (red) or mitochondria (Mitotracker Red) in HPTC untreated or treated with TGF-β1 (5 ng/ml) for 7 days. Bottom rows show magnified images of the outlined areas for single and merged images acquired from the red and green channels with yellow indicating colocalization. Scale bars represent 20 μm for full images and 10 μm for insets. Images are representative of experiments performed 3 independent times.
Mentions: To further examine the biology of NLRP3 in the context of human tubular injury and fibrosis, we stimulated HPTC for up to seven days with TGF-β1 to model chronic epithelial cell injury and fibrosis in vitro. Following TGF-β1 stimulation, the HPTC gradually acquired a fibroblastic phenotype with an obvious progressive change in morphology most notable following seven days of treatment (Fig. 5a). Both NLRP3 protein (Fig. 5b,c) and mRNA (Fig. 5d) expression in HPTC peaked after treatment with TGF-β1 for three days. Beyond three days, the level of NLRP3 protein and mRNA expression decreased concomitantly with progression of TGF-β1 mediated phenotypic change that included increased α-SMA and reduced levels of E-cadherin (Fig. 5b–d). Consistent with the loss of epithelial cell phenotype induced by prolonged TGF-β1 stimulation in HPTC, the epithelial cell injury marker KIM-1 also decreased significantly over time (Fig. 5b,c). Following seven days of TGF-β1 stimulation, Smad 2/3 remained in the nucleus suggesting persistent activation of the Smad-dependent signaling pathway and clear loss of Zo-1 membrane staining suggesting loss of epithelial morphology (Fig. 5e,f). Interestingly, NLRP3 signal diminished at the mitochondria with preservation of the mitochondrial architecture suggesting reduced levels of NLRP3 at the mitochondria rather than structural compromise of the organelle itself (Fig. 6). Taken together, these data show that there is an initial rise in NLRP3 expression induced by TGF-β1, but ongoing stimulation and loss of epithelial phenotype in HPTC is associated with decreased NLRP3 protein and mRNA expression.

Bottom Line: While NLRP3 expression in IgAN was detected in glomeruli, it remained largely confined to the tubular epithelial compartment.In vitro NLRP3 mRNA and protein expression were transiently induced in HPTC by TGF-β1 but subsequently diminished over time as cells lost their epithelial phenotype in a process regulated by transcription and ubiquitin-mediated degradation.Consistent with the in vitro data, low NLRP3 mRNA expression in kidney biopsies was associated with a linear trend of higher risk of composite endpoint of doubling serum creatinine and end stage renal disease in patients with IgAN.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada.

ABSTRACT
Nod-like receptor pyrin domain-containing-3 (NLRP3) has been implicated in the pathogenesis of experimental renal injury, yet its characterization in human kidney disease remains largely unexplored. NLRP3 expression was evaluated in human kidney biopsies, primary renal tubular cells (HPTC) and correlated to disease outcomes in patients with IgA nephropathy (IgAN). NLRP3 localized to renal tubules in normal human kidney tissue and to mitochondria within HPTC by immunohistochemistry and immunofluorescence microscopy. Compared to control kidneys, NLRP3 gene expression was increased in biopsies of patients with IgAN. While NLRP3 expression in IgAN was detected in glomeruli, it remained largely confined to the tubular epithelial compartment. In vitro NLRP3 mRNA and protein expression were transiently induced in HPTC by TGF-β1 but subsequently diminished over time as cells lost their epithelial phenotype in a process regulated by transcription and ubiquitin-mediated degradation. Consistent with the in vitro data, low NLRP3 mRNA expression in kidney biopsies was associated with a linear trend of higher risk of composite endpoint of doubling serum creatinine and end stage renal disease in patients with IgAN. Taken together, these data show that NLRP3 is primarily a kidney tubule-expressed protein that decreases in abundance in progressive IgAN.

No MeSH data available.


Related in: MedlinePlus