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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 expression in normal human kidney.(a) Immunoperoxidase staining in frozen sections of normal kidney tissue (obtained from a representative human nephrectomy sample) using mouse monoclonal NLRP3 or rabbit polyclonal platelet derived growth factor receptor-β (PDGFR-β) antibodies. Scale bar 100 μm. (b) Indirect immunofluorescence for NLRP3 (Cryo2) in (i) frozen unfixed, (ii) methanol-acetone fixed or (iii) paraffin embedded normal kidney tissue. An antibody to NLRP3 (Sigma) was unable to detect NLRP3 in paraffin embedded samples (iv). Scale bar represents 50 μm. (c) Immunofluorescence using dual-labeling confocal microscopy probing for NLRP3 (green), PDGFR-β (red) and nuclei (DAPI: blue, in merged images) in paraffin embedded or frozen sections of normal kidney tissue. Scale bar represents 100 μm. Images are representative of experiments performed at least 3 independent times.
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f1: NLRP3 expression in normal human kidney.(a) Immunoperoxidase staining in frozen sections of normal kidney tissue (obtained from a representative human nephrectomy sample) using mouse monoclonal NLRP3 or rabbit polyclonal platelet derived growth factor receptor-β (PDGFR-β) antibodies. Scale bar 100 μm. (b) Indirect immunofluorescence for NLRP3 (Cryo2) in (i) frozen unfixed, (ii) methanol-acetone fixed or (iii) paraffin embedded normal kidney tissue. An antibody to NLRP3 (Sigma) was unable to detect NLRP3 in paraffin embedded samples (iv). Scale bar represents 50 μm. (c) Immunofluorescence using dual-labeling confocal microscopy probing for NLRP3 (green), PDGFR-β (red) and nuclei (DAPI: blue, in merged images) in paraffin embedded or frozen sections of normal kidney tissue. Scale bar represents 100 μm. Images are representative of experiments performed at least 3 independent times.

Mentions: NLRP3 localization in the human kidney has been inconclusive with reported localization at podocytes and tubular epithelial cells7102021. To clarify NLRP3 localization in the human kidney, cryosections and paraffin embedded sections of histologically normal tissue obtained from human kidney nephrectomies were stained with immunoperoxidase or processed for indirect immunofluorescence (IF) and confocal microscopy. In normal kidney tissue stained with immunoperoxidase, NLRP3 localized primarily to tubules whereas platelet derived growth factor receptor-beta (PDGFR-β), a well-established marker of mesangial cells2324, localized to glomeruli as expected (Fig. 1a). NLRP3 localization to tubules was verified in cryosections and paraffin embedded sections by IF (Fig. 1b,c). NLRP3 (Cryo-2; Adipogen) consistently and specifically detected NLRP3 at tubules in frozen or paraffin sections but an antibody to NLRP3 (Sigma) was unable to detect NLRP3 in paraffin embedded samples. Isotype controls were negative in all cases (Supplementary Fig. 1). Taken together, these results suggest that NLRP3 primarily localizes to the tubular epithelium in normal human kidney with virtually no glomerular localization.


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 expression in normal human kidney.(a) Immunoperoxidase staining in frozen sections of normal kidney tissue (obtained from a representative human nephrectomy sample) using mouse monoclonal NLRP3 or rabbit polyclonal platelet derived growth factor receptor-β (PDGFR-β) antibodies. Scale bar 100 μm. (b) Indirect immunofluorescence for NLRP3 (Cryo2) in (i) frozen unfixed, (ii) methanol-acetone fixed or (iii) paraffin embedded normal kidney tissue. An antibody to NLRP3 (Sigma) was unable to detect NLRP3 in paraffin embedded samples (iv). Scale bar represents 50 μm. (c) Immunofluorescence using dual-labeling confocal microscopy probing for NLRP3 (green), PDGFR-β (red) and nuclei (DAPI: blue, in merged images) in paraffin embedded or frozen sections of normal kidney tissue. Scale bar represents 100 μm. Images are representative of experiments performed at least 3 independent times.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: NLRP3 expression in normal human kidney.(a) Immunoperoxidase staining in frozen sections of normal kidney tissue (obtained from a representative human nephrectomy sample) using mouse monoclonal NLRP3 or rabbit polyclonal platelet derived growth factor receptor-β (PDGFR-β) antibodies. Scale bar 100 μm. (b) Indirect immunofluorescence for NLRP3 (Cryo2) in (i) frozen unfixed, (ii) methanol-acetone fixed or (iii) paraffin embedded normal kidney tissue. An antibody to NLRP3 (Sigma) was unable to detect NLRP3 in paraffin embedded samples (iv). Scale bar represents 50 μm. (c) Immunofluorescence using dual-labeling confocal microscopy probing for NLRP3 (green), PDGFR-β (red) and nuclei (DAPI: blue, in merged images) in paraffin embedded or frozen sections of normal kidney tissue. Scale bar represents 100 μm. Images are representative of experiments performed at least 3 independent times.
Mentions: NLRP3 localization in the human kidney has been inconclusive with reported localization at podocytes and tubular epithelial cells7102021. To clarify NLRP3 localization in the human kidney, cryosections and paraffin embedded sections of histologically normal tissue obtained from human kidney nephrectomies were stained with immunoperoxidase or processed for indirect immunofluorescence (IF) and confocal microscopy. In normal kidney tissue stained with immunoperoxidase, NLRP3 localized primarily to tubules whereas platelet derived growth factor receptor-beta (PDGFR-β), a well-established marker of mesangial cells2324, localized to glomeruli as expected (Fig. 1a). NLRP3 localization to tubules was verified in cryosections and paraffin embedded sections by IF (Fig. 1b,c). NLRP3 (Cryo-2; Adipogen) consistently and specifically detected NLRP3 at tubules in frozen or paraffin sections but an antibody to NLRP3 (Sigma) was unable to detect NLRP3 in paraffin embedded samples. Isotype controls were negative in all cases (Supplementary Fig. 1). Taken together, these results suggest that NLRP3 primarily localizes to the tubular epithelium in normal human kidney with virtually no glomerular localization.

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