Role of renal expression of CD68 in the long-term prognosis of proliferative lupus nephritis
|Cristiane B. Dias1 • Patrıcia´||Malafronte1 • Jin Lee2 • Aline Resende1 •|
|Lectıcia´||Jorge1 • Cilene C. Pinheiro1 • Denise Malheiros2 • Viktoria Woronik1|
Introduction Renal histology of proliferative lupus nephritis (LN) shows increased macrophage infiltration, but its association with renal outcome is a matter of debate. Here, we investigate the potential relationship that mac-rophage expression has with renal prognosis in patients with proliferative LN.
Methods Fifty patients newly diagnosed with prolifera-tive LN were followed for a median of 8 years. Laboratory testing was conducted at diagnosis, after induction therapy and at the final follow-up evaluation. Renal biopsies were obtained at diagnosis and underwent immunohistochemical analysis with anti-CD68 and monocyte chemoattractant protein 1 monoclonal antibodies. Patients were stratified at final follow-up evaluation into glomerular filtration rate (GFR) [60 ml/min/1.73 m2 (non-progressor group;
- = 24) and GFR B60 ml/min/1.73 m2 (progressor group; n = 26). All patients were treated with prednisone and six pulses of cyclophosphamide on induction therapy. Con-ventional maintenance therapy was administered in both groups.
Results Compared to progressors, the non-progressor group showed a lower chronicity index (p = 0.01) and fewer CD68-positive cells in the renal tubules (p = 0.01) and particularly in the renal interstitium (p = 0.0003).
- Cristiane B. Dias firstname.lastname@example.org; email@example.com
- Nephrology Department, University of Sa˜o Paulo School of Medicine Hospital das Clı´nicas, Rua Dr. Ovı´dio Pires de Campos, 225, Cerqueira Ce´sar, Sa˜o Paulo, SP 05403-010, Brazil
- Department of Pathology, University of Sa˜o Paulo School of Medicine Hospital das Clı´nicas, Sa˜o Paulo, Brazil
Baseline and final serum creatinine correlated positively with the chronicity index (r = 0.3, p = 0.01 and r = 0.3, p = 0.04, respectively), and final serum creatinine corre-lated positively with interstitial expression of CD68 (r = 0.4, p = 0.0006).
Conclusion Renal expression of CD68 and the chronicity index are associated with progression to chronic kidney disease in patients with proliferative LN.
Keywords Lupus nephritis Macrophage inflammatory proteins Tissue macrophage Prognosis
Lupus nephritis (LN) is a kidney inflammation disease caused by systemic lupus erythematosus (SLE). Studies have shown that LN is associated with substantial mor-bidity and mortality, as well as with progression to the final stages of chronic kidney disease (CKD), which occurs in up to 30 % of LN patients [1, 2]. At LN diagnosis, clinical features and certain aspects of the renal histology are known prognostic factors for CKD progression [3, 4].
In a study involving African-American patients with LN, hypertension and a reduction in creatinine clearance at diagnosis were shown to be predictors of poor renal out-comes and proved superior to renal histological findings as prognostic factors . In another study, involving LN patents from various ethnic groups, multivariate analysis revealed that lower creatinine clearance at diagnosis, hypertension, and non-remission after treatment were associated with poor renal outcomes . A clinical study of 50 LN patients followed for 9 years showed that the intensity of tubulointerstitial nephritis at diagnosis was the only histopathological parameter associated with poor
renal outcomes . In addition, Giannico and Fogo  highlighted studies of LN in which a higher chronicity index in tissue sample of renal biopsy at diagnosis was associated with poor renal outcomes, as were vascular lesions of immunological or atherosclerotic etiology .
Although inflammatory mononuclear cells are often seen in renal biopsies of patients with various glomeru-lopathies, including the proliferative forms of LN , there is a lack of studies evaluating their role as prognostic markers of renal outcomes. In lupus-prone NZB/W F1 mice, onset of proteinuria has been shown to occur con-currently with increased renal expression of inflammatory proteins produced by macrophages such as monocyte chemoattractant protein 1 (MCP-1) . In MRL-Faslpr mice, which exhibit autoimmune disease similar to human lupus, increased renal expression of macrophages in the early stages of disease has been associated with poor renal survival .
In clinical settings, the effects of inflammatory cells in the renal tissue of LN patients are controversial. In a ret-rospective study of adults with proliferative forms of LN, tissue expression of macrophages (CD68) and platelets (CD61) was not found to correlate with renal function at baseline . However, Hill et al.  demonstrated that the presence of macrophages in glomeruli correlates with baseline serum creatinine and proteinuria, while macro-phages in renal tubules correlate with progression. In children and adolescents with proliferative forms of LN, Marks et al.  showed that glomerular expression of CD68 and MCP-1 correlated with proteinuria and poor renal prognosis.
The role of macrophages in the inflammatory process has been extensively discussed in the literature. According to Anders and Ryu , there are four different macro-phage phenotypes: the pro-inflammatory M1 macrophages, expressing CD68, CD14, and CCR2; anti-inflammatory M2c macrophages, expressing interleukin 10R; profibrotic M2a macrophages, expressing major histocompatibility complex class II; and fibrolytic M2b macrophages, expressing CD68. Therefore, at sites of inflammation, there should be a balance between classically activated M1 macrophages, which promote tissue injury, and alterna-tively activated M2 macrophages, which promote tissue repair . The inflammatory environment, cytokines, and other products could drive macrophage polarization toward a profibrotic phenotype that contributes to growth factor secretion mediators of fibrotic products, resulting in renal fibrosis and progression to CKD . However, there is much controversy regarding the immunoregulatory pro-cesses involved in macrophage phenotype differentiation, mainly because there is still a need to identify surface markers that would allow precise phenotyping . Within this context, we undertook the present study in order to
determine whether tissue macrophage expression, analyzed in renal biopsy samples obtained at diagnosis, are associ-ated with renal prognosis in patients with LN.
Patients and methods
Inclusion of patients
We enrolled 50 consecutive adult patients undergoing regular follow-up in the Nephrology Department of the University of Sa˜o Paulo School of Medicine Hospital das Clı´nicas, in Sa˜o Paulo, Brazil, between 2006 and 2010, that met at least four of the American Rheumatism Association criteria for SLE , and had a renal-biopsy confirmed diagnosis of proliferative LN categorized as class III or IV according to the International Society of Nephrology/Renal Pathology Society (ISN/RPS) classification . The patients were followed for a median of 8 years (in-terquartile range 4–9 years). Patients who had taken non-steroidal anti-inflammatory drugs were excluded, as were those with diabetes, hepatitis B, hepatitis C, or human immunodeficiency virus (HIV) infection, as well as those who had been under follow-up treatment for less than 6 months.
Overall disease activity was assessed using the SLE Disease Activity Index (SLEDAI); renal disease activity at diagnosis of LN was assessed by the renal SLEDAI score ; and race was assessed by a physician-assessed clas-sification of skin color, based solely on a visual and sub-jective estimation of the ancestry of the patient .
At diagnosis, after induction therapy, and at the final fol-low-up evaluation, we performed the following laboratory tests: serum creatinine; glomerular filtration rate (GFR), as determined by the Modification of Diet in Renal Disease (MDRD) equation; C3 and C4 complement; antinuclear antibody (ANA) and anti-double-stranded DNA (anti-dsDNA); blood cell counts; urinary protein and creatinine; and urinalysis. We determined antinuclear antibody by immunofluorescence in HEp-2 cells and anti-dsDNA by immunofluorescence using Crithidia luciliae as the sub-strate, complemented with radial immunodiffusion.
In a subgroup of 20 patients, we collected urine samples for the determination of MCP-1 expression. The samples, which were obtained at diagnosis and after 6 months of induction therapy, were placed in sterile containers and centrifuged at 3009g for 5 min at 4 LC to remove cells and precipitates, after which they were stored at -80 LC and thawed only on the day of the analytical procedure. To analyze the samples, we used enzyme-linked
immunosorbent assay (R&D Systems, Minneapolis, MN, USA), and MCP-1 levels were expressed in pg/ml cor-rected for urinary creatinine.
Renal biopsy and immunohistochemistry
Renal biopsies were performed at diagnosis of LN. Biopsy samples were analyzed using optical microscopy and immunofluorescence. Only samples containing ten or more glomeruli were considered acceptable for analysis. Activity and chronicity indices for class III and IV pro-liferative LN were determined as recommended in the ISN/RPS guidelines . Active lesions were defined as follows: endocapillary hypercellularity with or without leukocyte infiltration and with substantial luminal reduc-tion; karyorrhexis; fibrinoid necrosis; glomerular base-ment membrane rupture; cellular or fibrocellular crescents; subendothelial deposits identifiable by light microscopy (wire loops); and intraluminal immune aggregates (hyaline thrombi). We defined chronic lesions as follows: glomerular sclerosis (segmental, global); fibrous adhesions; and fibrous crescents. Each lesion received a score of 3 points. Vascular lesions, regardless of type, were categorized as absent or present. Immuno-histochemical study was performed with anti-CD68 (DAKO, Glostrup, Denmark) and MCP-1 monoclonal antibody (R&D Systems). Positive cells were counted in the glomerular, tubular, and interstitial compartments. The results are expressed as cells/glomerulus and cells/ microscopic field.
Patients were stratified into two groups by renal outcome: GFR B60 ml/min/1.73 m2 (progressor group); and [60 ml/min/1.73 m2 (non-progressor group).
All patients were treated with prednisone (1 mg/kg per day) for 8 weeks and cyclophosphamide in monthly pulses for 6 months as induction therapy. The maintenance therapy was prednisone in tapered doses, combined with azathio-prine or mycophenolate mofetil (conventional treatment). There was no statistical difference between the two groups in terms of the use of these immunosuppressive drugs. Renin-angiotensin system blockers were used in 85 % of the patients, and statin therapy was administered in 45 %, also with no statistical difference between the groups.
Continuous data are presented as mean ± standard devia-tion or as median and interquartile range (IQR), and cate-gorical data are presented as percentages. In comparisons between the two groups, we analyzed continuous data using the unpaired t test or the Mann–Whitney test, as appropriate, and categorical data using the Chi square test. The strength of correlations was determined with Pearson’s or Spearman’s correlation coefficient. The level of statis-tical significance was set at p \ 0.05.
Table 1 shows the clinical and biochemical characteristics of patients at diagnosis of LN. Of the 50 patients evaluated, 47 (94 %) were female, 25 (50 %) were white and their mean age was 27 ± 10 years.
Patients were stratified into two groups according to renal outcome. At the end of follow-up of 8 (4–9) years, there were 24 patients in the GFR [60 ml/min/1.73 m2 (non-progressor group) and 26 patients in the GFR
Table 1 Baseline clinical and biochemical characteristics of patients
|Parameter||n = 50|
|Age (years), mean ± SD||27 ± 10|
|White race, n (%)||25 (50)|
|Serum creatinine (mg/dl), mean ± SD||2.3 ± 1.7|
|MDRD-determined GFR (ml/min/1.73 m2), mean ± SD||43.8 ± 32.8|
|Serum albumin (g/dl), mean ± SD||2.6 ± 0.8|
|Proteinuria (g/day), mean ± SD||4.2 ± 2.6|
|C3 complement (mg/dl), mean ± SD||52.6 ± 23.2|
|C4 complement (mg/dl), mean ± SD||7.3 ± 3.9|
|Overall SLEDAI score, mean ± SD||25.2 ± 6.0a|
|Renal SLEDAI score, mean ± SD||11.8 ± 2.6a|
SD standard deviation, GFR glomerular filtration rate, MDRD modification of diet in renal disease (equation), SLEDAI Systemic Lupus Erythematosus Disease Activity Index
- Overall and renal SLEDAI scores were calculated in 34 patient
B60 ml/min/1.73 m2 (progressor group). Non-progressors compared to progressors showed lower baseline C4 levels (p = 0.01), higher renal SLEDAI scores (p = 0.02), lower chronicity index on histology of renal biopsy samples (p = 0.01), and lower numbers of CD68-positive cells in the renal tubules and particularly in the renal interstitium (p = 0.01 and p = 0.0003, respectively; Table 2).
After 6 months of LN diagnosis (Table 3), patients in the non-progressor group showed lower serum creatinine levels (p = 0.001) and higher serum albumin levels (p = 0.02) than those in the progressor group.
At the final follow-up evaluation, the two groups dif-fered significantly only concerning renal function param-eters, serum creatinine and MDRD-determined GFR,
according to the protocol criteria used to delineate the groups (Table 4).
Correlations between clinical parameters and CD68 tissue expression were studied. As can be seen in Table 5, baseline and final serum creatinine correlated positively with the chronicity index (r = 0.3, p = 0.01 and r = 0.3, p = 0.04, respectively). Final serum creatinine also cor-related positively with interstitial expression of CD68 (r = 0.4, p = 0.0036). Although baseline proteinuria did not correlate with any of the parameters studied, protein-uria after 6 months of treatment correlated positively with interstitial expression of CD68 (r = 0.4, p = 0.006). The severity of SLE assessed by overall SLEDAI score corre-lated negatively with the chronicity index (r = – 0.3,
Table 2 Non-progressor and progressor group characteristics at baseline
|[60 ml/min/1.73 m2||B60 ml/min/1.73 m2|
|(n = 24)||(n = 26)|
|Age (years), mean ± SD||26.3||± 9.0||27.6||± 11.0||ns|
|Female gender, n (%)||22 (91.6)||25 (96.1)||ns|
|Serum creatinine (mg/dl), mean ± SD||2.0 ± 1.5||2.6 ± 1.8||ns|
|MDRD-determined GFR (ml/min/1.73 m2), median (IQR)||39.0||(25.8–68.0)||25.2||(16.5–55.0)||ns|
|Serum albumin (g/dl), mean ± SD||2.5 ± 0.9||2.6 ± 0.9||ns|
|Proteinuria (g/day), mean ± SD||4.1||± 2.7||4.2 ± 2.7||ns|
|C3 complement (mg/dl), mean ± SD||46.4||± 30.2||56.1||± 18.3||ns|
|C4 complement (mg/dl), mean ± SD||5.7 ± 3.4||8.7 ± 3.7||0.01|
|Overall SLEDAI, mean ± SD||24.0||± 6.7a||25.1||± 7.1b||ns|
|Renal SLEDAI, mean ± SD||13.0||± 2.6a||10.7||± 2.4b||0.02|
|Activity index, mean ± SD||5.0 ± 1.7||3.9 ± 2.2||ns|
|Chronicity index, mean ± SD||2.3 ± 2.1||4.1 ± 2.3||0.01|
|Vascular lesion(s), n (%)||5 (20.8)||7 (26.9)||ns|
|CD68? cells/tubule field, median (IQR)||2.0||(1.0–3.0)||6.0 (1.3–10.1)||0.01|
|CD68? cells/interstitial field, median (IQR)||5.3||(3.3–10.0)||38.2||(8.5–101.8)||0.0003|
|CD68? cells/glomerulus, median (IQR)||5.0||(2.9–10.2)||9.0 (2.5–15.5)||ns|
GFR glomerular filtration rate, SD standard deviation, IQR interquartile range, ns not significant, MDRD Modification of Diet in Renal Disease (equation), SLEDAI Systemic Lupus Erythematosus Disease Activity Index
- Calculated in 18 patients
- Calculated in 16 patients
Table 3 Non-progressor and progressor group characteristics after 6 months
|[60 ml/min/1.73 m2||B60 ml/min/1.73 m2|
|(n = 24)||(n = 26)|
|Serum creatinine (mg/dl), median (IQR)||0.9||(0.8–1.1)||1.6||(0.9–3.6)||0.001|
|Serum albumin (g/dl), median (IQR)||4.1||(3.7–4.3)||3.6||(3.1–4.0)||0.02|
|Proteinuria (g/day), median (IQR)||0.4||(0.2–1.7)||1.0||(0.7–3.5)||ns|
|C3 complement (mg/dl), mean ± SD||105.9 ± 29.0||94.2 ± 36.6||ns|
|C4 complement (mg/dl), median (IQR)||18 (15–23)||18 (14–30)||ns|
GFR glomerular filtration rate, IQR interquartile range, SD standard deviation
Table 4 Non-progressor and progressor group characteristics at the end of follow-up
|[60 ml/min/1.73 m2||B60 ml/min/1.73 m2|
|(n = 24)||(n = 26)|
|Serum creatinine (mg/dl), mean ± SD||0.8 ± 0.1||5.4 ± 3.2||\0.0001|
|MDRD-determined GFR (ml/min/1.73 m2), median (IQR)||93.0 (78.0–116.5)||10.0 (5.8–34.7)||\0.0001|
|Serum albumin (g/dl), median (IQR)||4.2 (4.0–4.4)||4.1 (3.3–4.4)||ns|
|Proteinuria (g/day), median (IQR)||0.5 (0.2–1.1)||0.9 (0.2–2.9)||ns|
|C3 complement (mg/dl), median (IQR)||102.0 (84.0–123.5)||95.0 (59.0–120.0)||ns|
|C4 complement (mg/dl), median (IQR)||17.0 (12.0–27.0)||16.5 (11.2–24.0)||ns|
|Follow-up (years), mean ± SD||8.1 ± 2.8||7.2 ± 3.0||ns|
|GFR glomerular filtration rate, SD standard deviation, IQR interquartile range|
|Table 5 Expression of CD68|
|Parameter||Interstitial CD68||Glomerular CD68||Chronicity index|
|and the chronicity index, in|
|relation to laboratory tests and||R||p||r||P||R||p|
|Baseline serum creatinine||0.3||0.01|
|Final serum creatinine||0.4||0.0036||0.3||0.04|
|Proteinuria at 6 months||0.4||0.006|
|Overall SLEDAI score||-0.3||0.04|
|Renal SLEDAI score||0.4||0.0006||0.3||0.04|
SLEDAI Systemic Lupus Erythematosus Disease Activity Index
p = 0.04), whereas the renal SLEDAI score correlated positively with glomerular and interstitial expression of CD68 (r = 0.3, p = 0.04 and r = 0.4, p = 0.0006, respectively) (Table 5). The immune activation status, as assessed by baseline C3 levels, correlated positively with the chronicity index (r = 0.3, p = 0.02).
In the subgroup of 20 patients, MCP1 involvement and correlations with renal parameters and disease progression were studied. Urinary levels of MCP-1 were measured prospectively at baseline, median 1918 pg/mg creatinine (IQR 1264–4387), and at the end of induction therapy, median 717.5 pg/mg creatinine (IQR 378–1200) and as expected after treatment there was a significant decrease (p = 0.0008) in this inflammatory mediator. Considering the sample as a whole, median MCP-1 expression in the renal biopsy sample was 1 cell/microscopic field (IQR 0–18) and 93 cells/microscopic field (IQR 15–271) in the tubules and interstitium, respectively.
Baseline urinary MCP-1 correlated negatively with final serum creatinine (r = -0.4, p = 0.03). Baseline interstitial MCP-1 expression correlated positively with proteinuria after 6 months of induction therapy (r = 0.3, p = 0.03), as well as with CD68 expression in all tissue compartments: glomerular (r = 0.4, p = 0.0009); interstitial (r = 0.6, p \ 0.0001); and tubular (r = 0.32, p = 0.02).
Local induction of inflammatory markers, primarily glomerular and interstitial accumulation of macrophages, is a characteristic feature of proliferative LN. Such cells, together with dendritic cells and resident T cells, constitute the major source of inflammatory cytokines. Macrophages are important cells in inflammatory, allergic, and immune processes. They are currently broadly classified as M1 or M2 macrophages, the latter category being subdivided into M2a, M2b, and M2c phenotypes . Whereas M1 macrophages are pro-inflammatory, M2a macrophages are pro-fibrotic , M2b macrophages are fibrolytic, and M2c macro-phages are anti-inflammatory, possibly attenuating the fibrotic process . However, the idea that there are ‘‘good’’ and ‘‘bad’’ macrophages has not been fully established. The plasticity of macrophages and their differentiation into the M1 and M2 functional phenotypes might represent the extremes along a continuum of differential pathways. The presence of different cytokines in the inflammatory milieu could drive macrophage differentiation, as shown by Chen et al.  who demonstrated that LN activity in an experi-mental model was related to the glycosylated protein gran-ulin, described as an autocrine growth factor that polarizes macrophages toward the M2b phenotype, which they found
In the present study, we corroborated the findings of previous studies associating the histological chronicity index with clinical disease progression, as assessed by the final serum creatinine values. In addition, the chronicity index correlated positively with baseline serum creatinine and negatively with systemic LN activity (overall SLEDAI score), as shown in the literature. Alexopoulos et al.  had already demonstrated that the chronicity index and intersti-tial infiltrate of lymphocytes and macrophages at renal tissue biopsy of patients with LN correlated with declining renal function. In our study, increased interstitial expression of CD68 was observed mainly in the LN patients categorized as progressors on the basis of their final GFR. We also found that glomerular CD68 expression correlated positively with the renal SLEDAI score and did not correlate with serum creatinine, suggesting that the interstitial process is more important than the glomerular process in the determination of renal outcomes of patients with LN.
Macrophages are considered transient cells that are recruited to intraglomerular or interstitial areas in order to modulate immune responses . However, the initial process of wound healing can evolve to chronic fibrosis and progressive CKD. The chemokine MCP-1, produced by M1 macrophages infiltrating kidney tissue or resident in the kidney, plays an important role in the immunopatho-genesis of LN. Singh et al.  reported that urinary MCP-1 levels were high during disease activity in all LN patients, as well as in patients who did not respond to treatment. Rosa et al.  showed that, in patients with SLE, urinary MCP-1 correlated with LN activity and with the overall SLEDAI score.
Rovin et al.  demonstrated that urinary MCP-1 is a sensitive predictor of renal SLE flare and appears to increase 2–4 months before flare . The authors found that, after appropriate treatment, the majority of clinically improved patients showed urinary MCP-1 levels comparable to those of the controls, whereas urinary MCP-1 remained elevated in
- % despite clinical remission, as well as in all of the patients who showed no improvement, raising the possibility that high urinary MCP-1 levels indicate ongoing subclinical inflam-mation in that group . In the present study, we found that urinary MCP-1 levels decreased with immunosuppressive therapy, and the levels at baseline correlated negatively with final serum creatinine values. Therefore, we can speculate that MCP-1 is mainly a product of the M1 macrophage response, which is attenuated by immunosuppressive therapy. We also found that renal tissue expression of MCP-1, in any com-partment, did not correlate with urinary MCP-1, although interstitial MCP-1 expression correlated positively with pro-teinuria after 6 months and with CD68 renal tissue expression. Our more significant results point to CD68 tissue expression,
which in humans is associated with the M1 macrophage phenotype, and a classical Th1 activation over-expressing various pro-inflammatory cytokines including MCP-1 . Therefore, as expected, tissue expression of CD68 and MCP-1 were correlated showing the interaction affinity with CCR2 receptor expressed in activated monocytes downstream CCL2/MCP1 stimulation.
Increased interstitial over glomerular and tubular expression of CD68 was observed mainly in patients that progressed to low GFR, 38.2 (8.5–101.8) vs. 5.3 (3.3–10.0), p = 0.0003. Considering correlations, glomerular CD68 correlated positively with renal SLEDAI score and did not correlate with serum creatinine, sug-gesting that renal function is regulated by multiple mech-anisms besides glomerular inflammation. In addition, CD68 interstitial expression showed important and useful clinical correlations with follow-up creatinine levels, pro-teinuria at 6 months, and renal SLEDAI score.
Gonzalo et al.  studying active LN patients obtained results similar to ours, reporting a significant increase in the numbers of CD68-positive glomerular and extra-glomerular macrophages, as well as showing that the glomerular infiltration correlated with the overall SLEDAI score, anti-dsDNA, serum complement, proteinuria, and hematuria, whereas the extraglomerular infiltration corre-lated with the overall SLEDAI score and proteinuria. However, those authors found no correlation between macrophage infiltration and renal function at biopsy, probably because their study sample did not include patients followed up to CKD. In addition, Zhao et al. , studying patients with vasculitis, showed that serum crea-tinine levels at biopsy correlated with glomerular and interstitial expression of CD68.
Complement activation is thought to be involved in the tissue damage associated with LN activity. Studies attempting to determine whether changes in plasma levels of complement can serve as biomarkers of LN flare or pro-gression have reported conflicting results . Birmingham et al.  showed decreased complement values at renal SLE flare, although they found that those values showed low sensitivity and specificity for detecting such flares . There have been few studies evaluating the relationship between complement activation and progression to CKD. In another study of LN patients, Colares et al.  showed that low baseline complement C3 values were associated with progression to CKD . In contrast, we did not find a logical association between complement and progression to CKD. Clearly, complement activation and LN progression is a complex question that merits further study.
Our results show that patients with proliferative LN exhibit intense inflammatory activity, mainly tubulointer-stitial, characterized by infiltration of CD68 macrophages with an M1 phenotype and a Th1 cytokine inflammatory
profile marked by MCP-1 activation. Nevertheless, M2 macrophages can be present simultaneously in those active lesions. On this issue, Zhao et al.  concluded that it cannot be determined whether these are the same macro-phages expressing each of those markers, representing the plasticity of macrophage differentiation.
Our patients were treated with immunosuppression therapy, as is recommended in LN, and some progressed to CKD. It is well known that insufficient macrophage deac-tivation and over-expression of transforming growth factor b (TGFb) are linked to renal fibrosis. In a study of biopsy samples obtained from patients with LN, Hill et al.  found that the persistence of glomerular and extra-glomerular macrophage infiltrates after treatment (in a second biopsy) correlated with long-term renal outcomes.
In summary, our most significant result points to CD68 renal over-expression in active LN and its correlation with CKD progression. In clinical settings of LN patients, it is an important achievement. However, a weakness of our study was insufficient macrophage phenotype characteri-zation (M2) and analysis of its role in inflammatory deactivation or chronic activation.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of interest
Ethical statement The procedures involved in this study have been performed in accordance with the ethical. The Research Ethics Committee of the University of Sa˜o Paulo School of Medicine Hospital das Clı´nicas approved the study protocol
Informed consent Informed consent was obtained from all indi-vidual participants included in the study.
- Mok CC (2012) Understanding lupus nephritis: diagnosis, man-agement, and treatment options. Int J Women’s Health 4:213–222
- Davidson A, Aranow C (2006) Pathogenesis and treatment of systemic lupus erythematosus nephritis. Curr Opin Rheumatol 18:468–475
- Carlos Franco C, Yoo W, Franco D, Xu Z (2010) Predictors of end stage renal disease in African Americans with lupus nephritis. Bull NYU Hosp Jt Dis 68(4):251–256
- Oe Ayodele, Ig Okpechi, Cr Swanepoel (2010) Predictors of poor renal outcome in patients with biopsy-proven lupus nephritis. Nephrology 15:482–490
- Nived O, Hallengren CS, Alm P, Jo¨nsen A, Sturfelt G, Bengtsson AA (2013) An observational study of outcome in SLE patients with biopsy-verified glomerulonephritis between 1986 and 2004 in a defined area of Southern Sweden: the clinical utility of the ACR renal response criteria and predictors for renal outcome. Scand J Rheumatol 42:383–389
- Giannico G, Fogo AB (2013) Lupus nephritis: is the kidney biopsy currently necessary in the management of lupus nephritis? Clin J Am Soc Nephrol 8:138–145
- Wu LH, Yu F, Tan Y, Qu Z, Chen MH, Wang SX, Liu G, Zhao MH (2013) Inclusion of renal vascular lesions in the 2003 ISN/ RPS system for classifying lupus nephritis improves renal out-come predictions. Kidney Int 83:715–723
- Yang N, Isbel NM, Nikolic-Paterson DJ, Li Y, Ye R, Atkins RC, Lan HY (1998) Local Macrophage proliferation in human glomerulonephritis. Kidney Int 54:143–151
- Schiffer L, Bethunaickan LR, Ramanujam M, Huang W, Schiffer M, Tao H, Madaio MM, Bottinger EP, Davidson A (2008) Activated renal macrophages are markers of disease onset and disease remission in lupus nephritis. J Immunol 1(3):1938–1947
- Menke J, Rabacal WA, Byrne KT, Iwata Y, Schwartz MM, Stanley ER, Schwarting A, Kelley VR (2009) Circulating CSF-1 promotes monocyte and macrophage phenotypes that enhance lupus nephritis. J Am Soc Nephrol 20:2581–2592
- Gonzalo E, Toldos O, Martı´nez-Vidal MP, Ordon˜ez MC, Santi-ago B, Ferna´ndez-Nebro A, Loza E, Garcı´a I, Leo´n M, Pablos JL, Galindo M (2012) Clinicopathologic correlations of renal microthrombosis and inflammatory markers in proliferative lupus nephritis. Arthritis Res Ther 14:1–8
- Hill GS, Delahousse M, Nochy D, Re´my P et al (2001) Predictive power of second renal biopsy in lupus nephritis: significance of macrophages. Kidney Int 59:304–316
- Marks SD, Williams SJ, Tullus K, Sebire N (2008) Glomerular expression of monocyte chemoattractant protein-1 is predictive of poor renal prognosis in pediatric lupus nephritis. Nephrol Dial Transplant 23:3521–3526
- Anders HJ, Ryu M (2011) Renal microenvironments and mac-rophage phenotypes determine progression or resolution of renal inflammation and fibrosis. Kidney Int 80:915–925
- Ricardo SD, van Goor H, Eddy AA (2008) Macrophage diversity in renal injury and repair. J Clin Invest 118:3522–3530
- Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, Rothfield NF, Schaller JG, Talal N, Winchester RJ (1982) The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 25:1271–1277
- Weening JJ, D’Agati V, Schwartz MM, Seshan SV, Alpers CE, Appel GB, Balow JE, Bruijn JA, Cook T, Ferrario F, Fogo AG, Ginzler EM, Hebert L, Hill G, Hill P, Jennette JC, Kong NC, Lesavre P, Lockshin M, Looi LM, Makino H, Moura L, Nagatam M, on behalf of the International Society of Nephrology and Renal Pathology Society working group on the classification of lupus nephritis (2004) The Classification of Glomerulonephritis in Systemic Lupus Erythematosus Revisited. J Am Soc Nephrol 15:241–250
- Bombardier C, Gladmann DD, Urowitz MB, Caron D, Chang CH (1992) Derivation of the SLEDAI: a disease activity index for lupus patients. Arthritis Rheum 35:630–640
- Sa´nchez E, Rasmussen A, Riba L, Acevedo-Vasquez E, Kelly JA et al (2012) Impact of genetic ancestry and sociodemographic status on the clinical expression of systemic lupus erythematosus in American Indian-European populations. Arthritis Rheum 64:3687–3694
- Chen X, Wen Z, Xu W, Xiong S (2013) Granulin exacerbates lupus nephritis via enhancing macrophage M2b polarization. PLoS One 8:1–12
- Alexopoulos E, Seron D, Hartley RB, Cameron JS (1990) Lupus nephritis: correlation of interstitial cells with glomerular function. Kidney Int 37(1):100–109
- Singh RG, Usha, Rathore SS, Behura SK, Singh NK (2012) Urinary MCP-1 as diagnostic and prognostic marker in patients with lupus nephritis flare. Lupus 21:1214–1218
- Rosa RF, Takei K, Araujo NC, Loduca SMA, Szajubok JCM, Chahade WH (2012) Monocyte chemoattractant-1 as a urinary
biomarker for the diagnosis of activity of lupus nephritis in Brazilian patients. J Rheumatol 39(10):1948–1954
- Rovin BH, Song H, Birmingham DJ, Hebert LA, Yu CI, Nagaraja HN (2005) Urine chemokines as biomarkers of human systemic lupus erythematosus activity. J Am Soc Nephrol 16(12):467–473
- Zhao L, David MZ, Hyjek E, Chang A, Meehan SM (2015) M2 macrophage infiltrates in the early stages of ANCA-associated pauci-immune necrotizing GN. Clin J Am Soc 10:54–62
- Tsokos GC (2004) Exploring complement activation to develop biomarkers for systemic lupus erythematosus. Arthritis Reum 50(11):3404–3407
- Birmingham DJ, Irshaid F, Nagaraja HN, Zou X, Tsao BP, Wu H, Yu CY, Hebert LA, Rovin BH (2010) The complex nature of serum C3 and C4 as biomarkers of lupus renal flare. Lupus 19(11):1272–1280
- Colares VS, Titan SMO, Pereira AC, Malafronte P, Cardena MM, Santos S, Santos PC, Fridman C, Barros RT, Woronik V (2014) MYH9 and APOL1 gene polymorphisms and the risk of CKD in patients with lupus nephritis from an admixture population. PLoS One 9(3):1–8