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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 ubiquitination in TGF-β1 stimulated HPTC.(a) Lysates prepared from HPTC treated with 5 ng/ml TGF-β1 for 1, 3, 5 and 7 days were subjected to immunoblotting with the indicated antibodies. Cells treated with vehicle for 7 days was used as a control. (b) Immunoblotting of NLRP3 immunoprecipitates prepared from HPTC treated with TGF-β1 (5 ng/ml) for 1, 3, 5 and 7 days analyzed for ubiquitination. Untreated cells at 7 days was used as a control. Immunoblots are representative of 3 separate experiments.
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f7: NLRP3 ubiquitination in TGF-β1 stimulated HPTC.(a) Lysates prepared from HPTC treated with 5 ng/ml TGF-β1 for 1, 3, 5 and 7 days were subjected to immunoblotting with the indicated antibodies. Cells treated with vehicle for 7 days was used as a control. (b) Immunoblotting of NLRP3 immunoprecipitates prepared from HPTC treated with TGF-β1 (5 ng/ml) for 1, 3, 5 and 7 days analyzed for ubiquitination. Untreated cells at 7 days was used as a control. Immunoblots are representative of 3 separate experiments.

Mentions: Next we hypothesized that loss of NLRP3 signal at the mitochondria was a result of protein degradation mediated by either the ubiquitin-proteasome (UPS) or autophagy. The majority of intracellular proteins are degraded by the UPS pathway but aggregated proteins that are too large to enter the UPS pore are degraded by autophagy2829. To examine the involvement of autophagy in NLRP3 degradation by TGF-β1, immunoblotting was performed probing for the autophagy activated protein LC3 (microtubule-associated protein light chain 3b). The pattern of LC3-I expression followed that of NLRP3 with the highest level at 24 hours of TGF-β1 treatment (Fig. 7a). Beyond 24 hours, there was no significant conversion to LC3-II, a marker for autophagosome formation. Furthermore, levels of LAMP-1 decreased indicating that the lysosomal machinery was not upregulated and was unlikely to play a role in NLRP3 degradation in response to TGF-β1 stimulation (Fig. 7a). Interestingly, high molecular weight bands that migrated above ~180 kDa (in addition to the usual mobility of NLRP3 predicted at 118 kDa) were consistently observed when probing for NLRP3 by immunoblotting suggesting a role for ubiquitination in NLRP3 protein turnover. To determine if TGF-β1 stimulation increased NLRP3 ubiquitination, NLRP3 was immunoprecipitated from control and TGF-β1 treated HPTC followed by immunoblotting for ubiquitin. Immunoprecipitated NLRP3 demonstrated a similar pattern of expression over 7 days of TGF-β1 stimulation as previously observed in total cellular lysates with NLRP3 levels highest at day one followed by a decreasing signal over time (Fig. 7b). Indeed, prolonged treatment with TGF-β1 resulted in a progressive increase in high molecular weight ubiquitinated NLRP3 signal that paralleled the progressive loss of NLRP3 signal at the expected mobility of ~118 kDa (Fig. 7b). These results show that under conditions of prolonged stimulation with TGF-β1, HPTC lose their epithelial phenotype and reduced NLRP3 protein expression in part by ubiquitin mediated degradation.


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 ubiquitination in TGF-β1 stimulated HPTC.(a) Lysates prepared from HPTC treated with 5 ng/ml TGF-β1 for 1, 3, 5 and 7 days were subjected to immunoblotting with the indicated antibodies. Cells treated with vehicle for 7 days was used as a control. (b) Immunoblotting of NLRP3 immunoprecipitates prepared from HPTC treated with TGF-β1 (5 ng/ml) for 1, 3, 5 and 7 days analyzed for ubiquitination. Untreated cells at 7 days was used as a control. Immunoblots are representative of 3 separate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: NLRP3 ubiquitination in TGF-β1 stimulated HPTC.(a) Lysates prepared from HPTC treated with 5 ng/ml TGF-β1 for 1, 3, 5 and 7 days were subjected to immunoblotting with the indicated antibodies. Cells treated with vehicle for 7 days was used as a control. (b) Immunoblotting of NLRP3 immunoprecipitates prepared from HPTC treated with TGF-β1 (5 ng/ml) for 1, 3, 5 and 7 days analyzed for ubiquitination. Untreated cells at 7 days was used as a control. Immunoblots are representative of 3 separate experiments.
Mentions: Next we hypothesized that loss of NLRP3 signal at the mitochondria was a result of protein degradation mediated by either the ubiquitin-proteasome (UPS) or autophagy. The majority of intracellular proteins are degraded by the UPS pathway but aggregated proteins that are too large to enter the UPS pore are degraded by autophagy2829. To examine the involvement of autophagy in NLRP3 degradation by TGF-β1, immunoblotting was performed probing for the autophagy activated protein LC3 (microtubule-associated protein light chain 3b). The pattern of LC3-I expression followed that of NLRP3 with the highest level at 24 hours of TGF-β1 treatment (Fig. 7a). Beyond 24 hours, there was no significant conversion to LC3-II, a marker for autophagosome formation. Furthermore, levels of LAMP-1 decreased indicating that the lysosomal machinery was not upregulated and was unlikely to play a role in NLRP3 degradation in response to TGF-β1 stimulation (Fig. 7a). Interestingly, high molecular weight bands that migrated above ~180 kDa (in addition to the usual mobility of NLRP3 predicted at 118 kDa) were consistently observed when probing for NLRP3 by immunoblotting suggesting a role for ubiquitination in NLRP3 protein turnover. To determine if TGF-β1 stimulation increased NLRP3 ubiquitination, NLRP3 was immunoprecipitated from control and TGF-β1 treated HPTC followed by immunoblotting for ubiquitin. Immunoprecipitated NLRP3 demonstrated a similar pattern of expression over 7 days of TGF-β1 stimulation as previously observed in total cellular lysates with NLRP3 levels highest at day one followed by a decreasing signal over time (Fig. 7b). Indeed, prolonged treatment with TGF-β1 resulted in a progressive increase in high molecular weight ubiquitinated NLRP3 signal that paralleled the progressive loss of NLRP3 signal at the expected mobility of ~118 kDa (Fig. 7b). These results show that under conditions of prolonged stimulation with TGF-β1, HPTC lose their epithelial phenotype and reduced NLRP3 protein expression in part by ubiquitin mediated degradation.

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