Related Experiment Videos
Complement regulation in the rat glomerulus: Crry and CD59 regulate complement in glomerular mesangial and
R J Quigg1, B P Morgan, V M Holers
1Department of Medicine, University of Chicago, Illinois, USA.
Insights
Complement regulators Crry and CD59 were studied in rat kidney cells. Crry restricts the alternative complement pathway in glomerular endothelial and mesangial cells, while CD59 is active in endothelial cells.
Area of Science:
- Immunology
- Nephrology
- Cell Biology
Background:
- Complement regulators, including decay accelerating factor, membrane cofactor protein, and CD59, are found in human glomeruli.
- Crry serves as the rodent analog to decay accelerating factor and membrane cofactor protein.
- Understanding complement regulation in glomerular cells is crucial for kidney health.
Purpose of the Study:
- To investigate the presence and function of complement regulators Crry and CD59 in cultured rat glomerular endothelial cells (GEnC) and mesangial cells (MES).
- To elucidate the roles of Crry and CD59 in regulating both the classical and alternative complement pathways in these specific kidney cell types.
Main Methods:
- Immunoprecipitation and Western blotting were used to detect Crry and CD59 proteins in GEnC and MES.
- Northern analysis identified mRNA expression for Crry and CD59.
- Functional assays involved activating complement pathways (classical and alternative) and assessing cytotoxicity upon inhibition of Crry and CD59 using specific antibodies.
Main Results:
- Crry proteins (53, 65, 78 kD) and CD59 (20 kD) were detected in GEnC and MES, with varying expression levels.
- Crry mRNA was present in both cell types, and CD59 mRNA was found in GEnC and whole glomeruli.
- Inhibition of Crry function significantly increased cytotoxicity in both cell types, particularly via the alternative pathway. CD59 inhibition had a minor effect in MES but was functional in GEnC.
Conclusions:
- Crry is a key regulator of the alternative complement pathway in both rat glomerular endothelial and mesangial cells.
- Crry also plays a role in regulating the classical complement pathway in mesangial cells.
- CD59 is functionally active in glomerular endothelial cells but has a limited role in mesangial cells regarding complement regulation.
Abstract:
The complement regulators, decay accelerating factor, membrane cofactor protein, and CD59 are present in human glomeruli. Crry is the rodent analogue to the former two proteins. In this study, we examined complement regulation in cultured rat glomerular endothelial cells (GEnC) and mesangial cells (MES). Immunoprecipitation of 125I-labeled membrane proteins and Western blotting studies were performed with anti-Crry and anti-CD59. In both GEnC and MES, Crry was present as 53, 65, and 78 kD proteins. The 20 kD CD59 was apparent in GEnC. CD59 was also present in MES, but in relatively smaller quantities. By Northern analyses, 1.8 kb CD59 mRNA was present in GEnC as well as in RNA from isolated rat glomeruli. mRNA for Crry was present in both GEnC and MES as 2.2 kb species. The functional significance of these proteins was evaluated next. Anti-Thy 1.1 IgG was used to activate the complement classical pathway in MES. To inhibit the function of the complement regulators, anti-CD59 and/or anti-Crry F(ab')2 antibodies were added with anti-Thy 1.1. Inhibition of Crry function led to enhanced cytotoxicity, while there was no effect when CD59 function was inhibited. The complement alternative pathway was studied by adding complement in Mg-EGTA buffer. Inhibition of Crry led to productive alternative pathway activation, which was accentuated by anti-CD59 when Crry was incompletely inhibited. Alternative pathway regulation was also evaluated in GEnC. Inhibition of CD59 function alone had no effect in GEnC, while inhibition of Crry led to significant cytotoxicity from alternative pathway activation. Under conditions in which Crry was inactive, inhibition of CD59 further enhanced cytotoxicity. Therefore, Crry is present in both GEnC and MES and restricts the complement alternative pathway in both cell types. Crry also regulates the classical pathway in MES. CD59 is present and functionally active in GEnC, while it appears to have a minor role in MES.