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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

Comparison of the effects of growing podocytes in plasma from patients with lupus nephritis (LN), rheumatoid arthritis or non-renal lupus. Representative antiphosphotyrosine blots from experiments comparing the effect of growing podocytes for 24 h in normal control (NC) plasma versus plasma from patients with LN (A), rheumatoid arthritis (RA, B) or non-renal systemic lupus erythematosus (NR-SLE, C). Cells were lysed and protein tyrosine phosphorylation assessed by western blot. The graphs display densitometry, with the median and IQR for each group. Medians were compared using the Mann–Whitney test. As already shown, LN plasma leads to a reduction in protein tyrosine phosphorylation, whereas the effect of plasma from patients with RA or NR-SLE is no different to NC. Equal loading was ensured by blotting for either actin or GAPDH.
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LUPUS2014000013F3: Comparison of the effects of growing podocytes in plasma from patients with lupus nephritis (LN), rheumatoid arthritis or non-renal lupus. Representative antiphosphotyrosine blots from experiments comparing the effect of growing podocytes for 24 h in normal control (NC) plasma versus plasma from patients with LN (A), rheumatoid arthritis (RA, B) or non-renal systemic lupus erythematosus (NR-SLE, C). Cells were lysed and protein tyrosine phosphorylation assessed by western blot. The graphs display densitometry, with the median and IQR for each group. Medians were compared using the Mann–Whitney test. As already shown, LN plasma leads to a reduction in protein tyrosine phosphorylation, whereas the effect of plasma from patients with RA or NR-SLE is no different to NC. Equal loading was ensured by blotting for either actin or GAPDH.

Mentions: This observation was specific to LN patients since similar changes were not observed when podocytes were exposed to plasma from patients with rheumatoid arthritis (a disease where podocyte pathology is not observed) or from patients with SLE, but with no history of renal involvement (figure 3A–C). In addition, this effect appeared to be specific to podocytes since no changes in protein tyrosine phosphorylation were observed when cells from an irrelevant epithelial line (HeLa cells) were cultured in medium containing plasma from LN patients (data not shown).


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)

Comparison of the effects of growing podocytes in plasma from patients with lupus nephritis (LN), rheumatoid arthritis or non-renal lupus. Representative antiphosphotyrosine blots from experiments comparing the effect of growing podocytes for 24 h in normal control (NC) plasma versus plasma from patients with LN (A), rheumatoid arthritis (RA, B) or non-renal systemic lupus erythematosus (NR-SLE, C). Cells were lysed and protein tyrosine phosphorylation assessed by western blot. The graphs display densitometry, with the median and IQR for each group. Medians were compared using the Mann–Whitney test. As already shown, LN plasma leads to a reduction in protein tyrosine phosphorylation, whereas the effect of plasma from patients with RA or NR-SLE is no different to NC. Equal loading was ensured by blotting for either actin or GAPDH.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

LUPUS2014000013F3: Comparison of the effects of growing podocytes in plasma from patients with lupus nephritis (LN), rheumatoid arthritis or non-renal lupus. Representative antiphosphotyrosine blots from experiments comparing the effect of growing podocytes for 24 h in normal control (NC) plasma versus plasma from patients with LN (A), rheumatoid arthritis (RA, B) or non-renal systemic lupus erythematosus (NR-SLE, C). Cells were lysed and protein tyrosine phosphorylation assessed by western blot. The graphs display densitometry, with the median and IQR for each group. Medians were compared using the Mann–Whitney test. As already shown, LN plasma leads to a reduction in protein tyrosine phosphorylation, whereas the effect of plasma from patients with RA or NR-SLE is no different to NC. Equal loading was ensured by blotting for either actin or GAPDH.
Mentions: This observation was specific to LN patients since similar changes were not observed when podocytes were exposed to plasma from patients with rheumatoid arthritis (a disease where podocyte pathology is not observed) or from patients with SLE, but with no history of renal involvement (figure 3A–C). In addition, this effect appeared to be specific to podocytes since no changes in protein tyrosine phosphorylation were observed when cells from an irrelevant epithelial line (HeLa cells) were cultured in medium containing plasma from LN patients (data not shown).

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