CD80 and Angiopoietin-like 4 in glomerulopathies Gabriel Cara-Fuentes M.D.; Alfons Segarra† M.D.; Changli Wei‡ M.D.; Heiman Wang B.S.; Eduardo H. Garin M.D. Division of Pediatric Nephrology - University of Florida – Gainesville, Fl. †Department of Nephrology – Vall D’Hebron Hospital - Barcelona, Spain ‡ Department of Internal Medicine – Rush University- Chicago, IL Abstract Results Background: Minimal change disease (MCD) is considered a podocytopathy. We have shown that in MCD there is an increased podocyte expression of CD80 that may mediate proteinuria. Recently, the hyposialylated form of angiopoietin-like 4 (Angptl4) secreted by podocytes has been also suggested to play a role in proteinuria in MCD in humans and in several animal models of focal segmental Glomerulosclerosis-FSGS- and membranous nephropathy-MN-. Objective: To determine the role of CD80 and Angptl4 as mediators of proteinuria in MCD and other glomerulopathies in humans. Methods: Patients. 31, 31 and 32 patients with biopsy-proven MCD, FSGS and MN respectively and 18 control subjects were included. Urinary and serum CD80 and Angptl4 were measured by Elisa. In vitro studies. Human podocytes were maintained in RPMI 1640 containing 10% fetal bovine serum (FBS), 1% insulin-transferrin-selenium-A supplement (Invitrogen, Carlsbad, CA), penicillin (100 U/mL) and streptomycin (100 μg/mL). Cells were grown at 33°C in 95% air, 5% CO2 and then converted to differentiated cells by incubating at 37°C for 10 to 14 days. Differentiated cells were kept in 1% FBS growth media for 16 h and then incubated with 15% of serum from MCD patients in relapse, MCD in remission and 1% FBS growth media. Podocyte expression of CD80 and Angptl4 were measured by Western-Blot analysis. 2-D gel electrophoresis. pI of Angptl4 was determined in urine of a MCD patient in relapse. Immunofluorescence (IF). Frozen tissue sections were stained for CD80 (anti-human polyclonal CD80 goat antibody, R&D system) and Angptl4 (XXX). Statistical analysis using Mann-Whitney U test to compare two groups and Wilcoxon matched-pairs signed rank test for comparison of paired groups. Correlation between proteinuria and CD80 and Angptl4 were calculated using Spearman correlation. Results: Urinary CD80 was increased in MCD patients during relapse compared to those in remission and to patients with active FSGS, MN and to control subjects. Serum CD80 was lower in patients with glomerulopathies during relapse compared to normal controls. Urinary Angptl4 was increased in patients with glomerulopathies compared to normal controls, but no differences were found amount different types of nephrotic syndrome. Serum Angptl4 was higher in normal controls compared to patients with glomerulopathies. Urinary CD80 correlated with proteinuria in MCD, FSGS and MN whereas urinary Angptl4 correlated with proteinuria in FSGS and MN but not in MCD. Angptl4 detected in urine of a MCD patient had a pI of 5.4. Human podocytes exposed to sera from MCD patients in relapse showed a significant increase in CD80 expression, but not Angtpl4, compared to those exposed to sera from MCD patients in remission. Glomerular Angptl4 expression by IF was increased in kidney tissue from MCD patients in remission compared to MCD in relapse and controls. Glomerular CD80 expression by IF was increased in MCD in relapse compared to controls. Urinary CD80 in different types of glomerulopathies Serum CD80 and Angptl4 in different types of glomerulopathies Urinary CD80 is increased in MCD patients during relapse compared to remission, and to those with active FSGS, MN and control subjects Serum CD80 was decreased in patients with nephrotic syndrome compared to controls, this difference was statistically significant in the FSGS group compared to controls Serum Angptl4 was decreased in patients with nephrotic syndrome compared to controls. This difference reached statistically significance in the MN group compared to controls Urinary Angptl4 in different types of glomerulopathies Immunofluorescences studies: CD80 and Angptl-4 in MCD in relapse and controls Urinary Angptl4 is increased in nephrotic patients with MCD, FSGS and MN during relapse compared to controls. No difference was found among patients with different types of nephrotic syndrome Angptl4 expression in kidney tissue (Angptl4: green; DAPI: blue) CD80 expression in kidney tissue (CD80: green; DAPI: blue) MCD MCD MCD MCD Control Control Control Control In vitro experiments – Human podocyte cultures 2-D gel electrophoresis CD80 expression, but not Angplt4, is increased in podocytes cultured with sera from MCD patients in relapse Urinary Angptl4 has a pI of 5.4 in MCD in relapse Conclusions Urinary CD80 excretion in MCD in relapse is likely due to CD80 overexpression by podocytes that seems to be related to the presence of a circulating factor. Urinary CD80 is increased in MCD during relapse compared to MCD during remission and to patients with FSGS and MN during relapse and control subjects, suggesting that CD80 may play a role in proteinuria in MCD. Urinary Angptl4 is increased in patients with MCD, FSGS and MN during relapse compared to same patients during remission of proteinuria and to control subjects. Increased urinary Angptl4 in MCD in relapse is likely due to increased glomerular filtration of serum angptll4. The source of urinary Angptl4 is unlikely to be the podocyte since a) podocytes do not show increased Angtpl4 expression when cultured with sera from MCD patients in relapse and b) glomerular Angptl4 expression is similar in MCD in relapse compared to controls. Urinary Angptl4 in MCD in relapse has a pI of 5.4. Therefore it seems unlikely to have any role on neutralizing negative charges at the glomerular basement membrane. While CD80 seems to play a role in proteinuria in MCD, Angptl4 detected in urine in MCD and other glomerulopathies appears to be the result, rather than the cause, of a leaky glomerular filtration barrier. # pI MW 40 5.48 66 45 5.43 59