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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

Subcellular localization of NLRP3 in HPTC and human podocyte cell cultures.(a) Indirect immunofluorescence in HPTC and immortalized, differentiated human podocytes probing for NLRP3 (green) in the presence of DAPI (blue) for merged images. Scale bar represent 20 μm (b) Immunoblotting for NLRP3, podocin and tubulin in lysates prepared from THP-1 cells (positive control), HPTC, or immortalized human podocytes. Representative of experiments performed at least 3 independent times.
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f3: Subcellular localization of NLRP3 in HPTC and human podocyte cell cultures.(a) Indirect immunofluorescence in HPTC and immortalized, differentiated human podocytes probing for NLRP3 (green) in the presence of DAPI (blue) for merged images. Scale bar represent 20 μm (b) Immunoblotting for NLRP3, podocin and tubulin in lysates prepared from THP-1 cells (positive control), HPTC, or immortalized human podocytes. Representative of experiments performed at least 3 independent times.

Mentions: Since NLRP3 is mainly expressed in tubules, in vitro studies were next performed to characterize intracellular NLRP3 expression and localization by immunocytochemistry. Consistent with our immunohistochemistry (IHC) data, NLRP3 expression was clearly detectable in primary human proximal tubular cells (HPTC) but not in a differentiated human podocyte cell line by immunofluorescence or immunoblotting (Fig. 3a,b). NLRP3 expression in HPTC with epithelial characteristics was confirmed by indirect IF microscopy co-staining for NLRP3 and the tight junction protein Zo-1 (Fig. 4a). At the cellular level, NLRP3 expression was confirmed in Zo-1 positive epithelial cells, but did not co-localize with Zo-1 per se. Isotype controls were negative (data not shown). NLRP3 has been proposed to localize to cytoplasm, mitochondrial-associated endoplasmic reticulum membranes (MAMs) and mitochondria in macrophages72526 and to mitochondria in cardiac fibroblasts27. NLRP3 expression closely resembled the pattern of mitochondria in HPTC and significantly colocalized with mitochondrial-specific protein cytochrome C (Fig. 4d) and the mitochondrial dye, Mitotracker Red (Fig. 4e). NLRP3 also colocalized to a lesser extent with the MAMs marker calreticulin, but not to GM130, a Golgi complex protein (Fig. 4b,c). These data are consistent with prior studies and indicate that NLRP3 is a tubular epithelial expressed protein confined primarily to mitochondria and also to MAMs.


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)

Subcellular localization of NLRP3 in HPTC and human podocyte cell cultures.(a) Indirect immunofluorescence in HPTC and immortalized, differentiated human podocytes probing for NLRP3 (green) in the presence of DAPI (blue) for merged images. Scale bar represent 20 μm (b) Immunoblotting for NLRP3, podocin and tubulin in lysates prepared from THP-1 cells (positive control), HPTC, or immortalized human podocytes. 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

f3: Subcellular localization of NLRP3 in HPTC and human podocyte cell cultures.(a) Indirect immunofluorescence in HPTC and immortalized, differentiated human podocytes probing for NLRP3 (green) in the presence of DAPI (blue) for merged images. Scale bar represent 20 μm (b) Immunoblotting for NLRP3, podocin and tubulin in lysates prepared from THP-1 cells (positive control), HPTC, or immortalized human podocytes. Representative of experiments performed at least 3 independent times.
Mentions: Since NLRP3 is mainly expressed in tubules, in vitro studies were next performed to characterize intracellular NLRP3 expression and localization by immunocytochemistry. Consistent with our immunohistochemistry (IHC) data, NLRP3 expression was clearly detectable in primary human proximal tubular cells (HPTC) but not in a differentiated human podocyte cell line by immunofluorescence or immunoblotting (Fig. 3a,b). NLRP3 expression in HPTC with epithelial characteristics was confirmed by indirect IF microscopy co-staining for NLRP3 and the tight junction protein Zo-1 (Fig. 4a). At the cellular level, NLRP3 expression was confirmed in Zo-1 positive epithelial cells, but did not co-localize with Zo-1 per se. Isotype controls were negative (data not shown). NLRP3 has been proposed to localize to cytoplasm, mitochondrial-associated endoplasmic reticulum membranes (MAMs) and mitochondria in macrophages72526 and to mitochondria in cardiac fibroblasts27. NLRP3 expression closely resembled the pattern of mitochondria in HPTC and significantly colocalized with mitochondrial-specific protein cytochrome C (Fig. 4d) and the mitochondrial dye, Mitotracker Red (Fig. 4e). NLRP3 also colocalized to a lesser extent with the MAMs marker calreticulin, but not to GM130, a Golgi complex protein (Fig. 4b,c). These data are consistent with prior studies and indicate that NLRP3 is a tubular epithelial expressed protein confined primarily to mitochondria and also to MAMs.

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