Limits...
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

Tyrosine phosphorylation of proteins in podocyte lysates following exposure to healthy or lupus nephritis plasma. Human podocytes were incubated with heat-inactivated (HI) plasma from five lupus nephritis (LN) patients and five healthy donors. Cells were lysed following 24 (i) or 48 (ii) h exposure, and protein tyrosine phosphorylation assessed by western blot. This demonstrated globally reduced tyrosine phosphorylation at 24 h in the lupus nephritis plasma exposed-cells, which had returned to normal levels by 48 h. Representative western blots (A) showing tyrosine phosphorylation of podocyte proteins following exposure to plasma from LN patients or from normal controls (NCs). Actin was used to check equal loading of protein. Results were quantified using densitometry and the median and IQR from 5 NCs and 5 patients are shown (B). Data from the two groups were compared using the Mann–Whitney test for significance.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4225730&req=5

LUPUS2014000013F2: Tyrosine phosphorylation of proteins in podocyte lysates following exposure to healthy or lupus nephritis plasma. Human podocytes were incubated with heat-inactivated (HI) plasma from five lupus nephritis (LN) patients and five healthy donors. Cells were lysed following 24 (i) or 48 (ii) h exposure, and protein tyrosine phosphorylation assessed by western blot. This demonstrated globally reduced tyrosine phosphorylation at 24 h in the lupus nephritis plasma exposed-cells, which had returned to normal levels by 48 h. Representative western blots (A) showing tyrosine phosphorylation of podocyte proteins following exposure to plasma from LN patients or from normal controls (NCs). Actin was used to check equal loading of protein. Results were quantified using densitometry and the median and IQR from 5 NCs and 5 patients are shown (B). Data from the two groups were compared using the Mann–Whitney test for significance.

Mentions: However, a significant reduction in global protein tyrosine phosphorylation was observed in podocytes exposed to plasma from LN patients compared with the effect of plasma from healthy controls after 24 h culture (p=0.0008) (figure 2). By 48 h, differences between the groups were no longer seen (figure 2).


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)

Tyrosine phosphorylation of proteins in podocyte lysates following exposure to healthy or lupus nephritis plasma. Human podocytes were incubated with heat-inactivated (HI) plasma from five lupus nephritis (LN) patients and five healthy donors. Cells were lysed following 24 (i) or 48 (ii) h exposure, and protein tyrosine phosphorylation assessed by western blot. This demonstrated globally reduced tyrosine phosphorylation at 24 h in the lupus nephritis plasma exposed-cells, which had returned to normal levels by 48 h. Representative western blots (A) showing tyrosine phosphorylation of podocyte proteins following exposure to plasma from LN patients or from normal controls (NCs). Actin was used to check equal loading of protein. Results were quantified using densitometry and the median and IQR from 5 NCs and 5 patients are shown (B). Data from the two groups were compared using the Mann–Whitney test for significance.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

LUPUS2014000013F2: Tyrosine phosphorylation of proteins in podocyte lysates following exposure to healthy or lupus nephritis plasma. Human podocytes were incubated with heat-inactivated (HI) plasma from five lupus nephritis (LN) patients and five healthy donors. Cells were lysed following 24 (i) or 48 (ii) h exposure, and protein tyrosine phosphorylation assessed by western blot. This demonstrated globally reduced tyrosine phosphorylation at 24 h in the lupus nephritis plasma exposed-cells, which had returned to normal levels by 48 h. Representative western blots (A) showing tyrosine phosphorylation of podocyte proteins following exposure to plasma from LN patients or from normal controls (NCs). Actin was used to check equal loading of protein. Results were quantified using densitometry and the median and IQR from 5 NCs and 5 patients are shown (B). Data from the two groups were compared using the Mann–Whitney test for significance.
Mentions: However, a significant reduction in global protein tyrosine phosphorylation was observed in podocytes exposed to plasma from LN patients compared with the effect of plasma from healthy controls after 24 h culture (p=0.0008) (figure 2). By 48 h, differences between the groups were no longer seen (figure 2).

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