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Pathogenic autoantibodies from patients with lupus nephritis cause reduced tyrosine phosphorylation of podocyte proteins, including tubulin.

Manson JJ, Mills K, Jury E, Mason L, D'Cruz DP, Ni L, Saleem M, Mathieson P, Isenberg D, Rahman A - Lupus Sci Med (2014)

Bottom Line: HI LN plasma did not alter expression or cellular distribution of podocyte-specific proteins but caused a significant reduction in podocyte protein tyrosine phosphorylation compared with plasma from healthy controls (p=0.0008).This result was replicated using purified IgG but was not seen with plasma from rheumatoid arthritis or non-renal lupus patients.Since tubulin is an important component of podocyte major processes, these results suggest that autoantibodies from LN patients may exert an important pathogenic effect by dephosphorylation of this protein in podocytes.

View Article: PubMed Central - PubMed

Affiliation: Division of Medicine , Centre for Rheumatology Research, University College London, Rayne Institute , London , UK.

ABSTRACT

Introduction: The tertiary structure of normal podocytes prevents protein from leaking into urine. Patients with lupus nephritis (LN) develop proteinuria, and kidney biopsies from these patients display a number of podocyte abnormalities including retraction of podocyte processes. Autoantibodies have been shown to deposit in the kidneys of patients and mice with LN and are believed to play a key role in causing renal inflammation and dysfunction. The objective of this research was to study the effects of IgG antibodies from patients with LN on cultured human podocytes.

Methods: We exposed a human podocyte cell line to heat-inactivated (HI) plasma and purified polyclonal IgG from the following groups of subjects; patients with LN, patients with lupus without nephritis, patients with rheumatoid arthritis and healthy controls. We measured expression and intracellular distribution of podocyte-specific proteins and global phosphorylation of tyrosine. We then used mass spectrometry to identify the major protein targets of this phosphorylation.

Results: HI LN plasma did not alter expression or cellular distribution of podocyte-specific proteins but caused a significant reduction in podocyte protein tyrosine phosphorylation compared with plasma from healthy controls (p=0.0008). This result was replicated using purified IgG but was not seen with plasma from rheumatoid arthritis or non-renal lupus patients. The dominant tyrosine phosphorylated protein in podocytes was 55 kDa in size and was identified as tubulin.

Conclusions: Since tubulin is an important component of podocyte major processes, these results suggest that autoantibodies from LN patients may exert an important pathogenic effect by dephosphorylation of this protein in podocytes.

No MeSH data available.


Related in: MedlinePlus

Silver-stained gel showing the major tyrosine-phosphorylated proteins in podocytes. In order to identify the key tyrosine phosphorylated proteins, podocyte lysates were subjected to immunoprecipitation with mouse antiphosphotyrosine antibody. The products of the immunoprecipitation were run on polyacrylamide gels and visualised by silver staining. The unbound proteins are shown in lane A, and the bound (ie, tyrosine phosphorylated) proteins in lane B. Proteins bands 1–5 were excised from the polyacrylamide gel and subjected to in-gel proteolytic digestion before their analysis and identification using mass spectrometry.
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LUPUS2014000013F5: Silver-stained gel showing the major tyrosine-phosphorylated proteins in podocytes. In order to identify the key tyrosine phosphorylated proteins, podocyte lysates were subjected to immunoprecipitation with mouse antiphosphotyrosine antibody. The products of the immunoprecipitation were run on polyacrylamide gels and visualised by silver staining. The unbound proteins are shown in lane A, and the bound (ie, tyrosine phosphorylated) proteins in lane B. Proteins bands 1–5 were excised from the polyacrylamide gel and subjected to in-gel proteolytic digestion before their analysis and identification using mass spectrometry.

Mentions: Examination of the western blots shown in figure 2A revealed dominant tyrosine phosphorylated protein bands at approximately 55  and 80 kDa. In order to understand the functional implications of the observed dephosphorylation, the tyrosine phosphorylated proteins were isolated by immunoprecipitation using an antiphosphotyrosine antibody before separation on polyacrylamide gels, and the protein bands visualised by silver staining (figure 5). The proteins of interest, and a number of control bands, were excised from the polyacrylamide gel and subjected to in-gel proteolytic digestion before analysis and identification using electrospray QTOF mass spectrometry.


Pathogenic autoantibodies from patients with lupus nephritis cause reduced tyrosine phosphorylation of podocyte proteins, including tubulin.

Manson JJ, Mills K, Jury E, Mason L, D'Cruz DP, Ni L, Saleem M, Mathieson P, Isenberg D, Rahman A - Lupus Sci Med (2014)

Silver-stained gel showing the major tyrosine-phosphorylated proteins in podocytes. In order to identify the key tyrosine phosphorylated proteins, podocyte lysates were subjected to immunoprecipitation with mouse antiphosphotyrosine antibody. The products of the immunoprecipitation were run on polyacrylamide gels and visualised by silver staining. The unbound proteins are shown in lane A, and the bound (ie, tyrosine phosphorylated) proteins in lane B. Proteins bands 1–5 were excised from the polyacrylamide gel and subjected to in-gel proteolytic digestion before their analysis and identification using mass spectrometry.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4225730&req=5

LUPUS2014000013F5: Silver-stained gel showing the major tyrosine-phosphorylated proteins in podocytes. In order to identify the key tyrosine phosphorylated proteins, podocyte lysates were subjected to immunoprecipitation with mouse antiphosphotyrosine antibody. The products of the immunoprecipitation were run on polyacrylamide gels and visualised by silver staining. The unbound proteins are shown in lane A, and the bound (ie, tyrosine phosphorylated) proteins in lane B. Proteins bands 1–5 were excised from the polyacrylamide gel and subjected to in-gel proteolytic digestion before their analysis and identification using mass spectrometry.
Mentions: Examination of the western blots shown in figure 2A revealed dominant tyrosine phosphorylated protein bands at approximately 55  and 80 kDa. In order to understand the functional implications of the observed dephosphorylation, the tyrosine phosphorylated proteins were isolated by immunoprecipitation using an antiphosphotyrosine antibody before separation on polyacrylamide gels, and the protein bands visualised by silver staining (figure 5). The proteins of interest, and a number of control bands, were excised from the polyacrylamide gel and subjected to in-gel proteolytic digestion before analysis and identification using electrospray QTOF mass spectrometry.

Bottom Line: HI LN plasma did not alter expression or cellular distribution of podocyte-specific proteins but caused a significant reduction in podocyte protein tyrosine phosphorylation compared with plasma from healthy controls (p=0.0008).This result was replicated using purified IgG but was not seen with plasma from rheumatoid arthritis or non-renal lupus patients.Since tubulin is an important component of podocyte major processes, these results suggest that autoantibodies from LN patients may exert an important pathogenic effect by dephosphorylation of this protein in podocytes.

View Article: PubMed Central - PubMed

Affiliation: Division of Medicine , Centre for Rheumatology Research, University College London, Rayne Institute , London , UK.

ABSTRACT

Introduction: The tertiary structure of normal podocytes prevents protein from leaking into urine. Patients with lupus nephritis (LN) develop proteinuria, and kidney biopsies from these patients display a number of podocyte abnormalities including retraction of podocyte processes. Autoantibodies have been shown to deposit in the kidneys of patients and mice with LN and are believed to play a key role in causing renal inflammation and dysfunction. The objective of this research was to study the effects of IgG antibodies from patients with LN on cultured human podocytes.

Methods: We exposed a human podocyte cell line to heat-inactivated (HI) plasma and purified polyclonal IgG from the following groups of subjects; patients with LN, patients with lupus without nephritis, patients with rheumatoid arthritis and healthy controls. We measured expression and intracellular distribution of podocyte-specific proteins and global phosphorylation of tyrosine. We then used mass spectrometry to identify the major protein targets of this phosphorylation.

Results: HI LN plasma did not alter expression or cellular distribution of podocyte-specific proteins but caused a significant reduction in podocyte protein tyrosine phosphorylation compared with plasma from healthy controls (p=0.0008). This result was replicated using purified IgG but was not seen with plasma from rheumatoid arthritis or non-renal lupus patients. The dominant tyrosine phosphorylated protein in podocytes was 55 kDa in size and was identified as tubulin.

Conclusions: Since tubulin is an important component of podocyte major processes, these results suggest that autoantibodies from LN patients may exert an important pathogenic effect by dephosphorylation of this protein in podocytes.

No MeSH data available.


Related in: MedlinePlus