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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

Intracellular position of podocyte proteins after exposure to healthy or lupus nephritis plasma. Human podocytes were incubated for 48 h with heat-inactivated plasma from six lupus nephritis (LN) patients and six healthy donors. Cells were then fixed and stained for α-actinin and CD2AP (CD2-associated protein) using immunofluorescence staining (see ‘Methods”). Cells were examined by confocal microscopy, by two independent assessors and the number of cells with membrane staining was noted. Representative images showing α-actinin staining after culture with LN (A) and healthy (B) plasma. The percentage of cells with membrane staining was compared between the two groups using a t test (C). Representative images showing CD2AP staining after culture with LN (D) and healthy (E) plasma. The percentage of cells with membrane staining was compared between the two groups using a t test (F). No significant differences in distribution of α-actinin or CD2AP were found.
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LUPUS2014000013F1: Intracellular position of podocyte proteins after exposure to healthy or lupus nephritis plasma. Human podocytes were incubated for 48 h with heat-inactivated plasma from six lupus nephritis (LN) patients and six healthy donors. Cells were then fixed and stained for α-actinin and CD2AP (CD2-associated protein) using immunofluorescence staining (see ‘Methods”). Cells were examined by confocal microscopy, by two independent assessors and the number of cells with membrane staining was noted. Representative images showing α-actinin staining after culture with LN (A) and healthy (B) plasma. The percentage of cells with membrane staining was compared between the two groups using a t test (C). Representative images showing CD2AP staining after culture with LN (D) and healthy (E) plasma. The percentage of cells with membrane staining was compared between the two groups using a t test (F). No significant differences in distribution of α-actinin or CD2AP were found.

Mentions: Plasma obtained from six patients with LN and six healthy donors was heat inactivated to remove viable cytokines and complement but retain immunoglobulins. The six patients whose samples were used in this part of the study were LN4, LN8, LN9, LN10, LN11 and LN17. They were chosen because they were among the earliest samples available in this study and not on the basis of particular clinical characteristics. The cellular distributions of α-actinin-4 and CD2AP, both proteins important for podocyte function, were examined by immunofluorescence following 48 h culture with HI plasma from patients and controls (figure 1A,B,D and E show representative immunofluorescence images). The characteristic peripheral pattern of staining for CD2AP and α-actinin-419 was assessed; however, quantitative analysis revealed no significant difference between podocytes exposed to plasma from LN patients compared with healthy donors (figure 1C,F).


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)

Intracellular position of podocyte proteins after exposure to healthy or lupus nephritis plasma. Human podocytes were incubated for 48 h with heat-inactivated plasma from six lupus nephritis (LN) patients and six healthy donors. Cells were then fixed and stained for α-actinin and CD2AP (CD2-associated protein) using immunofluorescence staining (see ‘Methods”). Cells were examined by confocal microscopy, by two independent assessors and the number of cells with membrane staining was noted. Representative images showing α-actinin staining after culture with LN (A) and healthy (B) plasma. The percentage of cells with membrane staining was compared between the two groups using a t test (C). Representative images showing CD2AP staining after culture with LN (D) and healthy (E) plasma. The percentage of cells with membrane staining was compared between the two groups using a t test (F). No significant differences in distribution of α-actinin or CD2AP were found.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

LUPUS2014000013F1: Intracellular position of podocyte proteins after exposure to healthy or lupus nephritis plasma. Human podocytes were incubated for 48 h with heat-inactivated plasma from six lupus nephritis (LN) patients and six healthy donors. Cells were then fixed and stained for α-actinin and CD2AP (CD2-associated protein) using immunofluorescence staining (see ‘Methods”). Cells were examined by confocal microscopy, by two independent assessors and the number of cells with membrane staining was noted. Representative images showing α-actinin staining after culture with LN (A) and healthy (B) plasma. The percentage of cells with membrane staining was compared between the two groups using a t test (C). Representative images showing CD2AP staining after culture with LN (D) and healthy (E) plasma. The percentage of cells with membrane staining was compared between the two groups using a t test (F). No significant differences in distribution of α-actinin or CD2AP were found.
Mentions: Plasma obtained from six patients with LN and six healthy donors was heat inactivated to remove viable cytokines and complement but retain immunoglobulins. The six patients whose samples were used in this part of the study were LN4, LN8, LN9, LN10, LN11 and LN17. They were chosen because they were among the earliest samples available in this study and not on the basis of particular clinical characteristics. The cellular distributions of α-actinin-4 and CD2AP, both proteins important for podocyte function, were examined by immunofluorescence following 48 h culture with HI plasma from patients and controls (figure 1A,B,D and E show representative immunofluorescence images). The characteristic peripheral pattern of staining for CD2AP and α-actinin-419 was assessed; however, quantitative analysis revealed no significant difference between podocytes exposed to plasma from LN patients compared with healthy donors (figure 1C,F).

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