Urinary CD80 Excretion Increases in Idiopathic Minimal-Change Disease
Eduardo H. Garin*,
Leila N. Diaz*,
Wei Mu,
Clive Wasserfall,
Carlos Araya*,
Mark Segal and
Richard J. Johnson
Divisions of * Pediatric Nephrology and Nephrology, Hypertension and Transplantation and Department of Immunology, University of Florida, Gainesville, Florida
Correspondence: Dr. Eduardo H. Garin, University of Florida, Box 100296, Gainesville, FL 32610. Phone: 352-392-4434; Fax: 352-392-7107; E-mail: garineh{at}peds.ufl.edu
Received for publication August 1, 2007.
Accepted for publication August 11, 2008.
CD80 is expressed on all antigen-presenting cells and is presenton podocytes in a number of experimental models of nephroticsyndrome. We tested whether urinary soluble CD80 increased withidiopathic minimal-change disease (MCD). We collected urineand serum samples from patients with MCD in relapse and in remission,patients with nephrotic syndrome resulting from other glomerulardiseases (FSGS, membranoproliferative glomerulonephritis, IgAnephropathy, and membranous nephropathy), patients with systemiclupus erythematosus, and normal control subjects. Urinary concentrationsof soluble CD80 in patients with relapsed MCD were significantlyhigher compared with those observed in patients with MCD inremission, other glomerular diseases, and systemic lupus erythematosuswith and without proteinuria and healthy control subjects. Urinaryconcentrations of soluble CTLA-4, which is a negative regulatorof CD80, were not statistically different in patients with relapsedMCD compared with those in remission. The urinary soluble CD80/CTLA-4ratio was >100-fold higher in patients with relapsed MCDcompared with those in remission (P < 0.008). In contrast,serum concentrations of soluble CD80 and CTLA-4 did not distinguishpatients with MCD in relapse and in remission. In conclusion,urinary soluble CD80 is elevated in idiopathic MCD, which couldbe relevant to both diagnosis and pathogenesis.
Idiopathic minimal-change disease (MCD) is the most common nephroticsyndrome in children and adolescents.1 The disease is considereda disorder of T cell function.2,3 Although the mechanism ofthe proteinuria is unknown, a circulating cytokine has beenpostulated as the link between proteinuria and the T cell dysfunction.2–4
Podocytes are specialized and highly differentiated epithelialcells that form a layer between the glomerular basement membraneand the urinary space. In a series of experiments, Reiser etal.5 found that these cells could acquire dendritic cell–likefunctions, in which they can be induced to express CD80, a transmembraneprotein that provides a co-stimulatory signal for T cell activation.Mice administered LPS developed podocyte CD80 expression andproteinuria; when LPS was administered to the CD80–/–knockout mouse, no increase in urinary protein excretion wasobserved.5 The authors further showed that podocyte CD80 activationby LPS could occur in SCID mice that lack T cells.5 Furthermore,CD80 expression could be shown in both experimental models ofnephrotic syndrome (e.g., aminonucleoside nephrosis) and humanlupus nephritis.5 These findings led the authors to proposethat podocyte CD80 expression might be involved in the pathogenesisof MCD.6
Some podocyte antigens are known to be shed, where they canbe found in the urine.7 This led us to hypothesize that solubleCD80 (sCD80) might be detected in the urine in patients withMCD. Furthermore, T regulatory cells are known to secrete solubleCTLA-4 (sCTLA-4), which can bind CD80 and block the co-stimulatoryactivation of T cells.8–10 If T cells are releasing acytokine that can induce MCD, such as IL-13,11 then one mightpostulate that an inadequate release of sCTLA-4 might lead tocontinued activation of the T cells and possibly persistentCD80 expression by dendritic cells. We therefore measured urinarysCD80 and urinary sCTLA-4 levels in children with MCD and comparedthe findings with healthy children and children with other glomerulardiseases.
Clinical characteristics of the patients with MCD and controlgroups are shown in Table 1.
We measured urinary sCD80 excretion adjusted for urinary creatininein patients with MCD (n = 19), patients with other glomerulardiseases (n = 11), and normal control subjects (n = 9; Figure 1A).Urinary sCD80 excretion was elevated in patients with MCD inrelapse compared with patients with MCD in remission (P <0.001), patients with other glomerular diseases (P < 0.001),and healthy control subjects (P < 0.01). When we studiedeight patients with MCD during relapse off immunosuppressivetherapy and 1 mo after remission was induced, the differenceswere still marked (P < 0.01; Figure 1B). In four patientswith MCD, we measured urine CD80 excretion during various stagesof their nephrotic syndrome. In these patients, changes in proteinuriawere paralleled by changes in urinary sCD80 excretion (Figure 1C).Urinary sCD80 was not significantly elevated in healthy controlsubjects or in patients with nephrotic syndrome and other glomerulardiseases when compared with patients with MCD in remission (Figure 1A).In patients with MCD in relapse, there was no correlation betweensCD80 and proteinuria (Figure 2).
Figure 1. (A) sCD80 urinary excretion (ng/g creatinine) in patients with MCD and control subjects. Comparisons: P < 0.0001 between patients with MCD in relapse and MCD in remission; P < 0.001 between patients with MCD in relapse and other glomerular diseases; P < 0.001 between patients with MCD in relapse and healthy control subjects; P = NS among patients with MCD in remission, patients with nephrotic syndrome and other glomerular diseases, and normal control subjects. (B) Serial sCD80 urinary concentration (ng/g creatinine) in eight patients with MCD during relapse and remission. P < 0.01 in patients with MCD with relapse versus remission. (C) Serial sCD80 urinary concentrations (solid line) and concomitant urine protein/creatinine ratio (bars) in patients with MCD.
In contrast with the urinary findings, serum sCD80 concentrationswere not different among patients with MCD in relapse, patientswith MCD in remission, and normal control subjects (307 ±78, 297 ± 58, and 374 ± 28, pg/ml, mean ±SEM, respectively).
In a separate study, we examined concomitant urine sCD80 andsCTLA-4 concentrations during relapse and during early remissionon therapy in patients with MCD. Results are presented in Figure 3A.As in our previous study, urinary sCD80 was significantly increasedduring relapse when compared with values measured during remission(P < 0.009). Urinary sCTLA-4 concentrations in patients inrelapse were not statistically significant to those observedin patients in remission (P = 0.25). When the ratio of urinarysCD80 to sCTLA-4 was evaluated in patients with MCD, the ratiochanged from 10.80 ± 6.10 during relapse to 0.10 ±0.05 during remission (mean ± SEM; P < 0.008; Figure 3B).In contrast, the same ratio in patients with FSGS and proliferativeglomerulonephritis and nephrotic syndrome was 0.66 ±0.28 (mean ± SEM).
Figure 3. (A) Urinary excretion of CD80 (ng/g creatinine; mean ± SEM) and sCTLA-4 in patients with MCD in relapse and in remission. (B) Urinary ratio of CD80 (ng/g creatinine) and sCTLA-4 (ng/g creatinine) in patients with MCD in relapse and in remission.
Serum sCTLA-4 levels in patients with MCD in relapse (376 ±174 pg/ml, mean ± SEM; n = 8) were not statisticallydifferent from the values observed for patients in remission(229 ± 86 pg/ml; n = 9). No significant difference inserum sCD80/sCTLA-4 ratio between relapse and remission wasobserved (1.8 ± 0.6 [n = 8] and 6.1 ± 5.1 [n =7], mean ± SEM, respectively).
Urinary CD80 levels in patients with systemic lupus erythematosus(SLE) without proteinuria (urinary protein-creatinine ratio<0.5; 111 ± 34 ng/mg creatinine, mean ± SEM;n = 13) were not statistically different from those observedfor patients with proteinuria (136 ± 65 ng/mg creatinine,mean ± SEM; n = 8; P = 0.96). Urinary CD80 values includingall patients with SLE (127 ± 31 ng/mg creatinine, mean± SEM) were significantly lower than those observed forpatients with MCD in relapse (P = 0.0001) but not significantlydifferent from those seen in patients with MCD in remissionand healthy control subjects.
CD80 is a dendritic-associated receptor that mediates co-stimulatorysignaling of the T cell. Expression of CD80 on podocytes wasdemonstrated by Reiser et al.5,6 in several experimental modelsof glomerular disease associated with nephrotic syndrome. Similarly,CD80 was been reported to be expressed on podocytes in humanlupus nephritis.5
In this study, we demonstrated that urinary sCD80 was elevatedin MCD during relapse with levels returning to the normal rangein remission. Furthermore, serial studies showed that the urinarysCD80 excretion paralleled changes in proteinuria in four ofthe patients. The increase in urinary sCD80 was not simply areflection of proteinuria per se, because it was not observedin patients who had proteinuria and/or nephrotic syndrome andhad other glomerular diseases. In addition, serum sCD80 levelswere not different for patients with MCD in relapse from patientswith MCD in remission and normal control subjects, documentingthat the increased urinary excretion could not be accountedfor by higher serum levels.
Patients with lupus nephritis and proteinuria have been reportedto have CD80 expression on their podocytes.5 Despite this observation,we did not find urinary sCD80 levels to be increased in patientswith SLE and proteinuria. Indeed, levels of sCD80 in the urinewere not different from those observed in healthy control subjectsand in patients with MCD in remission. In addition, sCD80 urinaryexcretion in patients with SLE was significantly lower thanthat seen in patients with MCD in relapse; however, some ofour patients with SLE and with or without concomitant proteinuriadid have increased urinary sCD80 excretion. Urinary sCD80 levelsare difficult to interpret in patients with SLE because thesepatients have been reported to have elevated serum sCD80 levelsunrelated to the activity of the disease.12 Because sCD80 hasa molecular weight of 23 kD, the increased urinary sCD80 seenin some of our patients with SLE could be due to higher serumsCD80 values.
The co-stimulatory activation of T cells can be blocked by sCTLA-4released by T regulatory cells,8–10 and sCTLA-4 may alsonegatively regulate the CD80-bearing dendritic cell.9 This ledus to hypothesize that sCTLA-4 might be reduced in patientswith MCD in relapse. Although we detected no statistically significantdifferences in serum or urinary CTLA-4 between patients withMCD in relapse and in remission, we did observe that the ratioof urinary sCD80 to sCTLA-4 was markedly higher in patientswith MCD in relapse compared with remission. The relative decreasein urinary sCTLA-4 to sCD80 during relapse could be due to theinability of the T regulatory cells from patients with MCD torelease sCTLA-4. We recently demonstrated that T regulatorycells are normal in number but severely dysfunctional in patientswith MCD in relapse.13 More studies are needed to confirm ourhypothesis that this defect could result in uncontrolled CD80podocyte activation with persistent proteinuria.
This pilot study raises several interesting questions. First,it raises the possibility that urinary sCD80 levels may be helpfulin the diagnosis of patients with MCD. Although larger patientpopulations will be necessary to study before concluding thatthis test may be specific for MCD, it is interesting that noneof the four patients with FSGS and nephrotic-range proteinuriahad elevated sCD80 levels. Second, it raises the question ofthe source of the CD80. Theoretically, a number of cells withinthe kidney could produce CD80, including podocytes, macrophages,dendritic cells, and tubular cells.5,14,15 If the podocyte isthe source expressing CD80, then it could be relevant to thepathogenesis of the disease. In this regard, we were recentlyable to demonstrate CD80 expression in a few biopsies of patientswith MCD in relapse (unpublished observations).
In conclusion, urinary sCD80 is increased in patients with MCDin relapse and seems to correlate with the activity of the disease.Further studies to examine the role of urinary sCD80 as a diagnostic,pathogenic, and prognostic marker are indicated as well as todetermine the role, if any, that sCTLA-4 has in this process.
Patients
The study was approved by the institutional review board ofthe University of Florida, and written informed consent wasobtained before participation. Children and adolescents withbiopsy-proven MCD (n = 19), defined according to the InternationalStudy for Kidney Diseases in Children, were studied.16 The majorityof patients with MCD during relapse and during remission werereceiving variable amounts of immunosuppressive therapy. Controlsubjects (n = 9) included a group of age- and gender-matchedhealthy children and adolescents. None of the normal controlsubjects included in the study had any underlying immunologicdisorder. In addition, we examined patients with FSGS (n = 4),membranoproliferative glomerulonephritis (n = 2), membranousnephropathy (n = 3), IgA nephropathy (n = 2), and SLE (n = 21).Definitions of glomerular diseases were based on establishedcriteria according to the International Study for Kidney Diseasesin Children.16
Methods
Relapse of the nephrotic syndrome was defined as the presenceof proteinuria of 3+ using the tetrabromophenol-citrate buffercolorimetric qualitative dipstick test, confirmed by a urineprotein/creatinine ratio >2.0, and a concomitant serum albuminof <3.5 g/L during the course of the episode. Complete remissionwas defined as no proteinuria using the colorimetric qualitativetest and a urinary protein/creatinine ratio of <0.2 on arandom urine sample.
We measured sCD80 in blood and urine using a commercially availableELISA kit (Bender MedSystems, Burlingame, CA). We measured sCTLA-4in blood and urine according to Oaks and Hallet10 with minormodifications. We measured urinary creatinine and proteinuriausing an autoanalyzer.
Statistical Analysis
We conducted statistical analysis using nonparametric ANOVA(Kruskal-Wallis test). We determined differences between meanswith the Mann-Whitney U test or the Wilcoxon signed rank testwhen applicable. We calculated the correlation between urinarysCD80 and urinary protein using Spearman's correlation coefficienttest.
Support for this study was provided by startup funds from theDepartment of Pediatrics, National Institutes of Health grantsDK-52121 and HL-68607, Gatorade funds from the Division of AdultNephrology, and a T32 training grant for L.D. (DK-07518).
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
R.J.J.'s current affiliation is Division of Renal Diseases andHypertension, University of Colorado Denver, Aurora, CO.
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Abstract
Introduction
3+ using the tetrabromophenol-citrate buffer colorimetric qualitative dipstick test, confirmed by a urine protein/creatinine ratio >2.0, and a concomitant serum albumin of <3.5 g/L during the course of the episode. Complete remission was defined as no proteinuria using the colorimetric qualitative test and a urinary protein/creatinine ratio of <0.2 on a random urine sample. 
