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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

The effect of purified IgG from healthy controls or patients with lupus nephritis on tyrosine phosphorylation in podocytes at two different IgG concentrations. Podocytes were grown in medium containing purified IgG from patients with LN, or normal controls. Lysates were blotted with antiphosphotyrosine antibody and equal loading of protein confirmed with GAPDH. The groups were compared using the Mann–Whitney test for significance. (A) Medium containing 100 μg/mL IgG, with representative western blots. No difference was seen between normal control and lupus nephritis IgG-exposed cells. (B) Medium containing 1.5 mg/mL IgG, with representative western blots. Exposure to this concentration of IgG from patients with lupus nephritis led to a relative reduction in protein tyrosine phosphorylation compared with healthy control IgG.
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LUPUS2014000013F4: The effect of purified IgG from healthy controls or patients with lupus nephritis on tyrosine phosphorylation in podocytes at two different IgG concentrations. Podocytes were grown in medium containing purified IgG from patients with LN, or normal controls. Lysates were blotted with antiphosphotyrosine antibody and equal loading of protein confirmed with GAPDH. The groups were compared using the Mann–Whitney test for significance. (A) Medium containing 100 μg/mL IgG, with representative western blots. No difference was seen between normal control and lupus nephritis IgG-exposed cells. (B) Medium containing 1.5 mg/mL IgG, with representative western blots. Exposure to this concentration of IgG from patients with lupus nephritis led to a relative reduction in protein tyrosine phosphorylation compared with healthy control IgG.

Mentions: In order to prove that antibodies, rather than any other plasma component, were responsible for influencing protein tyrosine phosphorylation, IgG was purified from LN and healthy control plasma samples. Podocytes were grown in medium containing purified IgG at two concentrations: 100 μg/mL, a concentration typically used in experiments with monoclonal antibodies,25 and 1.5 mg/mL to mirror the amount of IgG present in the plasma exposure experiments. Exposing podocytes to purified IgG from patients with LN at the higher (1.5 mg/mL) (figure 4B), but not at the lower (100 µg/mL) concentration (figure 4A), led to a relative decrease in tyrosine phosphorylation compared with IgG from healthy donors, thus recapitulating the initial plasma exposure experiments (p=0.001).


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)

The effect of purified IgG from healthy controls or patients with lupus nephritis on tyrosine phosphorylation in podocytes at two different IgG concentrations. Podocytes were grown in medium containing purified IgG from patients with LN, or normal controls. Lysates were blotted with antiphosphotyrosine antibody and equal loading of protein confirmed with GAPDH. The groups were compared using the Mann–Whitney test for significance. (A) Medium containing 100 μg/mL IgG, with representative western blots. No difference was seen between normal control and lupus nephritis IgG-exposed cells. (B) Medium containing 1.5 mg/mL IgG, with representative western blots. Exposure to this concentration of IgG from patients with lupus nephritis led to a relative reduction in protein tyrosine phosphorylation compared with healthy control IgG.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

LUPUS2014000013F4: The effect of purified IgG from healthy controls or patients with lupus nephritis on tyrosine phosphorylation in podocytes at two different IgG concentrations. Podocytes were grown in medium containing purified IgG from patients with LN, or normal controls. Lysates were blotted with antiphosphotyrosine antibody and equal loading of protein confirmed with GAPDH. The groups were compared using the Mann–Whitney test for significance. (A) Medium containing 100 μg/mL IgG, with representative western blots. No difference was seen between normal control and lupus nephritis IgG-exposed cells. (B) Medium containing 1.5 mg/mL IgG, with representative western blots. Exposure to this concentration of IgG from patients with lupus nephritis led to a relative reduction in protein tyrosine phosphorylation compared with healthy control IgG.
Mentions: In order to prove that antibodies, rather than any other plasma component, were responsible for influencing protein tyrosine phosphorylation, IgG was purified from LN and healthy control plasma samples. Podocytes were grown in medium containing purified IgG at two concentrations: 100 μg/mL, a concentration typically used in experiments with monoclonal antibodies,25 and 1.5 mg/mL to mirror the amount of IgG present in the plasma exposure experiments. Exposing podocytes to purified IgG from patients with LN at the higher (1.5 mg/mL) (figure 4B), but not at the lower (100 µg/mL) concentration (figure 4A), led to a relative decrease in tyrosine phosphorylation compared with IgG from healthy donors, thus recapitulating the initial plasma exposure experiments (p=0.001).

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