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Volume: 12 Issue: 5 October 2014


Expression Patterns of B Cells in Acute Kidney Transplant Rejection

Objectives: To evaluate B-cell expression patterns and association with function and survival in dysfunctional kidney allografts.

Materials and Methods: There were 110 kidney transplant recipients included who had for-cause biopsies. Demographic and transplant data were collected. Immunostaining for B cells, plasma cells, and C4d was performed by the immunoperoxidase technique in paraffin-embedded samples. Cir-culating antihuman leukocyte antigen donor-specific antibodies were detected in a single-antigen assay at biopsy. The main outcomes were kidney graft survival and function.

The patients were evaluated in 3 groups according to the Banff classification: no rejection (40 patients), T-cell–mediated rejection (50 patients), and antibody-mediated rejection (20 patients).

Results: The CD138-positive plasma cell-rich infiltrates predominated in antibody-mediated rejection and were associated with stronger reactivity against panel antibodies (r = 0.41; P ≤ .001) and positive donor-specific antibodies (r = 0.32; P ≤ .006). The CD20-positive lymphocytes were associated with T-cell–mediated rejection, increased human leukocyte antigen mismatch, and frequency of retransplant. The CD138-positive cell infiltrates also were significantly greater in patients who had late than early rejection. There was no correlation between cellular CD20 and CD138 expression, and neither CD20 nor CD138 predicted worse graft function or survival. Other markers of antibody-mediated rejection such as C4d and donor-specific antibodies were associated with worse graft function and survival at 4 years after transplant. In multivariate analysis, C4d was the only risk factor associated with graft loss.

Conclusions: After kidney transplant, CD20-positive B-cell infiltrates were associated with T-cell–mediated rejection, and CD138-positive plasma cells were associated with antibody-mediated rejection. Graft loss was associated with the presence of C4d.

Key words : C4d, CD20, CD138, Donor-specific antibodies, Lymphocytes, Plasma cells


The incidence of acute cellular kidney graft rejection has been decreased with the use of potent immuno-suppressive drugs. However, grafts still are lost because of rejection. Antibody-mediated rejection (AMR) is associated with higher frequency of early and late renal allograft loss.1,2 In one-third of patients who have steroid-resistant acute rejection, AMR is demonstrated by C4d deposition in the peritubular capillaries and high antihuman leukocyte antigen (HLA) donor-specific antibody (DSA) titers.3 The presence of both C4d and DSA predicts worse graft dysfunction.4-7

In contrast with T-cell–mediated cellular rejection (TCMR), the function of B cells in organ transplant is unclear. The B cells at different stages of maturation, including immature CD20-positive cells, CD38-positive plasmablasts, and CD138-positive plasma cells, are present in clusters or diffuse infiltrates in grafts during acute rejection and are functionally active.8-10 In acute cellular rejection, the association between B cells, corticosteroid resistance, and poorer graft survival has been shown in some studies,10-12 but these data have not been confirmed in other studies.8,9,13 The participation of B cells in the humoral immune response also has been investigated.14,15 In kidneys undergoing rejection, strong correlations have been observed between infiltrating CD20-positive B cells, CD38-positive plasmablasts, plasma cells, circulating DSA and, to a lesser degree, C4d.16

The purpose of the present study was to evaluate B-cell expression patterns in biopsies of dysfunctional kidney allografts and to determine the correlation of these patterns with C4d deposition in peritubular capillaries, circulating DSA levels, and graft function and survival.

Materials and Methods

There were 110 patients included in this study who had kidney transplant between March 2008 and March 2012 at the Hospital de Clínicas de Porto Alegre and had available for-cause kidney biopsies with paraffin-embedded tissue. The study protocol conformed with the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Research Ethics Committee of Hospital de Clínicas de Porto Alegre before the study began. All patients signed a written informed consent form.

Clinical data
Demographic and transplant data were collected retrospectively, including pretransplant class I and class II panel-reactive antibody levels and HLA-A, -B, and -DR mismatches. Graft function was evaluated from serum creatinine levels at the time of biopsy and follow-up (mean, 44 mo). Class I and class II donor anti-HLA antibody levels, measured after transplant at the time of biopsy, were detected with flow cytometry (Luminex, Austin, TX, USA; LABScreen Single Antigen kit, One Lambda, Los Angeles, CA, USA). Mean fluorescence intensity > 500 indicated the presence of class I or class II anti-HLA antibodies.

The outcomes were graft survival and function, evaluated on the date of the most recent observation. Graft loss was defined as either return to dialysis or death with a functioning kidney.

The immunosuppression protocol included induction with basiliximab or anti-thymocyte globulin (Thymoglobulin, Sanofi-Aventis, Paris, France), followed by prednisone, mycophenolate mofetil or sodium, and a calcineurin inhibitor (cyclosporine or tacrolimus). The diagnosis of acute rejection was based on clinical signs, histopathology, and response to specific treatment. Acute cellular rejection was treated initially with intravenous methylprednisolone. Patients who had steroid-resistant rejection or a Banff 2B or higher classification were treated with anti-thymocyte globulin or muromonab-CD3. Patients who had acute AMR were treated with plasmapheresis and intravenous immunoglobulin.

Histopathologic diagnosis
Single kidney allograft biopsies were obtained from 110 patients. Histopathologic interpretation was performed according to the 2007 Banff classification by a pathologist who was blinded to the clinical data.17 Patients were grouped into 3 histologic categories of biopsies: no rejection (NR), TCMR, or AMR. The patients who had NR had with normal kidneys, acute tubular necrosis, calcineurin inhibitor nephrotoxicity, borderline changes, or nonspecific interstitial fibrosis and tubular atrophy. The diagnosis of AMR was made when C4d-positive staining showed > 25% peritubular capillaries and histopathology was compatible with the Banff criteria.17

For C4d detection, samples were incubated overnight at 4ºC in a 1:50 dilution of primary rabbit polyclonal anti-C4d antibody (American Research Products, Palo Alto, CA, USA) in antibody diluent (Renaissance, Biocare Medical, Concord, CA, USA), incubated with horseradish peroxidase (Biocare Medical), and stained with permanent 3-amino-9-ethyl-carbazole (Romulin, Biocare Medical). The criteria for C4d positivity were the linear and circumferential staining of 25% to 50% peritubular capillaries (focal) or > 50% peritubular capillaries (diffuse) in a cortical location. Samples that had < 25% peritubular capillaries stained were rated C4d-negative.17

For CD20, samples were incubated overnight at 4ºC in a 1:40 dilution of mouse monoclonal anti-CD20 antibody (Dako, Carpinteria, CA, USA) in 1% bovine serum albumin in phosphate-buffered saline and incubated with streptavidin-peroxidase (Vector Laboratories, Burlington, CA, USA). Quantification of CD20-positive cells was performed with digital analysis (Image-Pro Plus software, version 4.5, Media Cybernetics, Bethesda, MD, USA). The counts of positive events were performed with a microscope (original magnification ×400) and a resolution of 2560 × 1920 pixels (1 mm = 5900 pixels). Adjusted scores were generated for the fragment surface area (mm2).18

For CD138 (92 biopsies), samples were incubated overnight with a mouse monoclonal anti-CD138 antibody (Dako, Glostrup, Denmark), using a polymer detection system (Novolink Compact, Reagents Novocastra, Newcastle, UK). For detection, slides were incubated with a solution of 3-amino-9-ethyl-carbazole and examined with a microscope (original magnification ×100); positive cells were stained brown. For quantification, the numbers of positively stained cells were counted in 25 consecutive microscopic fields (original magnification ×400) corrected by a previously established factor of 5.096,19 and the results were expressed as the mean number of stained cells per mm2.

Statistical analyses
Data were analyzed with statistical software (SPSS for Windows, version 18.9, SPSS Inc., Armonk, NY, USA). Descriptive statistics were reported as number (%), mean ± standard deviation (SD), or median (range). Comparisons were made with Pearson chi-square test, analysis of variance with Bonferroni adjustment, or Kruskal-Wallis test. Correlations were evaluated with Pearson product moment or Spearman rank correlation. The cutoff points for CD20-positive and CD138-positive cells and the diagnostic performance of mean fluorescence intensity for development of AMR were determined using receiver operating characteristic curves. The effects of the immunologic markers on graft function were evaluated with the generalized estimating equation and gamma distribution with logarithmic function, and the effects on graft survival were analyzed using Kaplan-Meier method. The risk of graft loss was estimated using Cox proportional hazards regression model. The level of statistical significance was defined by P ≤ .05.


Clinical data
The 110 kidney transplant recipients had mean age 42 ± 12 years (range, 15-64 y) and were mostly male (68%) and white (96%). There were 64 patients (58%) who received a graft from deceased donors, and there were 10 retransplants (9%). Delayed graft function occurred in 58 patients (53%). Regarding immunosuppression, 69 patients (63%) received induction therapy with anti-thymocyte globulin (n = 19, 27%), basiliximab (n = 18, 26%), or muro-monab-CD3 (n = 7, 10%); 105 patients (95%) received a calcineurin inhibitor; and 104 patients (94%) received a mycophenolic acid derivative. Mean patient follow-up was 44 ± 19 months.

There were no significant differences between the groups in clinical variables except the NR group had the longest median time from transplant to biopsy (Table 1). The HLA-A, -B, and -DR mismatches were significantly more frequent in the TCMR than NR group (Table 1). The frequency of patients who had a last panel reactive antibody level > 20% and a median pretransplant anti-HLA class II peak panel reactive antibody level were greater in the AMR than other groups (Table 1).

Correlation between Banff categories and immuno-logical rejection markers
Immunohistochemical staining showed that C4d was present in 55 biopsies (50%), and 38 patients (35%) had circulating DSA (Table 2 and Figure 1). Class I DSA was detected in 45% AMR cases (comparison with other categories, P = .006), but the ratios of class II DSA were similar between the groups. All patients who had AMR were C4d-positive, and most patients with AMR were positive for C4d and class I or II circulating DSA (Table 2). The prevalence of DSAs increased with the intensity of C4d staining. The DSAs were present in 13 (23%) C4d-negative biopsies, 18 (32%) biopsies with focal C4d staining, and 30 (55%) biopsies with diffuse C4d deposition (P ≤ .02).

The median infiltrating CD20-positive cell density was significantly greater in the TCMR than NR or AMR groups (Table 2). The median absolute number of CD138-positive plasma cells was greater in patients with AMR than NR but similar to patients from the TCMR group (Table 2). In the subgroup of patients who had NR and interstitial fibrosis and tubular atrophy (16 patients), the number of CD20-positive cells (median, 9.5 cells/mm2; range, 5.8-18.0 cells/mm2) was higher than observed in patients who had other histologic types without evidence of rejection in the biopsies (P ≤ .05). In the 16 patients who had interstitial fibrosis and tubular atrophy, 13 patients had CD20 count ≥ 5 cells/mm2.

The effect of induction therapy on clinical and immunologic parameters was investigated. Patients who received anti-thymocyte globulin had a higher prevalence of C4d in the biopsy (anti-thymocyte globulin, 70%; no anti-thymocyte globulin, 28%; P ≤ .002) but the proportion of DSA did not differ (anti-thymocyte globulin, 33%; no anti-thymocyte globulin, 29%; not significant). The median (range) number of plasma cells was higher in patients who received anti-thymocyte globulin (17 [28-46] cells/m2) than patients who received basiliximab (3.5 [1.0-8.8] cells/m2; P ≤ .009), but the number of CD20-positive cells did not differ (anti-thymocyte globulin, 3.2 [0.5-17.4] cells/m2; basiliximab, 1.5 [0-5.4] cells/m2; not significant). Graft function and survival did not differ between the 2 groups at last follow-up.

Correlations between B cells, C4d, and donor-specific antibodies
The cutoffs to determine low or high infiltrating CD20-positive cells for diagnosis of TCMR, and CD138-positive plasma cell densities for diagnosis of AMR, were established with receiver operating characteristic curves. The area under the curve for CD20 was 0.77, and the cutoff was 5 cells/mm2 (sensitivity, 70%; specificity, 79%). For CD138, the area under the curve was 0.82 and the cutoff was 5 cells/mm2 (sensitivity, 72%; specificity, 90%).

Patients who had high or low densities of CD20-positive or CD138-positive cells in their biopsies did not differ in age, sex, ethnicity, or prevalence of delayed graft function. However, the group with a high CD20-positive cell density had a greater prevalence of deceased donors (high CD20-positive, 61%; low CD20-positive, 54%; P ≤ .05) and prior transplant (high CD20-positive, 15%; low CD20-positive, 2%; P ≤ .03) and a greater number of HLA mismatches (high CD20-positive, 4 ± 1; low CD20-positive, 3 ± 1; P ≤ .005). Patients with high CD138-positive cell densities had a greater prevalence of pregraft panel reactive antibody level > 20% (high CD138-positive, 36%; low CD138-positive, 14%; P ≤ .01) and class I or class II DSA-positive status (high CD138-positive, 52%; low CD138-positive, 24%; P ≤ .007). There were no associations between CD20-positive cells and class I or II DSA. The mean fluorescence intensity for class I or II anti-HLA antibody detection (500-1000, 1000-5000, or > 5000) did not correlate with either the CD20-positive or CD138-positive cell density.

In an analysis of the density of infiltrating B cells and plasma cells in patients who had TCMR or AMR (70 patients) according to posttransplant period and presence of early rejection (≤ 3 mo; 62 patients) or late rejection (> 3 mo; 8 patients), there was no significant difference in the number of CD20-positive cells in early rejection cases (early, 17.5 [1-84] cells/mm2; late, 2 [0.3-5] cells/mm2; not significant). However, the number of CD138-positive cells was significantly greater in late rejection cases (early, 3.4 [0-16] cells/mm2; late, 21 [18-59] cells/mm2; P ≤ .02).

In patients who had either TCMR or AMR, the presence of C4d in the graft biopsies did not correlate with the CD20-positive (r = 0.09; not significant) or CD138-positive cell density (r = 0.01; not significant) but did correlate with the presence of circulating DSA (r = 0.31; P ≤ .02). Positive DSA did not correlate with CD20-positive cell infiltration (r = -0.157; not significant), but did correlate with the CD138-positive plasma cell density (r = 0.42; P ≤ .006). There was no correlation between the CD20-positive and CD138-positive cell densities in the graft biopsy cellular infiltrates (r = 0.003; not significant).

Analysis of the Banff morphologic findings showed that tubulitis (any grade) was observed in 42 patients (63%) with ≥ 5 CD20-positive cells/mm2 and in 25 patients (37%) with < 5 CD20-positive cells/mm2 (P ≤ .04), but there were no differences in the other histologic parameters. Neutrophil margination in peritubular capillaries was observed in 14 patients (70%) with ≥ 5 CD138-positive cells/mm2 but only 6 patients (30%) with < 5 CD138-positive cells/mm2 (P ≤ .001). Acute glomerulitis was identified in 11 patients (73%) with ≥ 5 CD138-positive cells/mm2 but only 4 patients (27%) with < 5 CD138-positive cells/mm2 (P ≤ .001).

Effect of immunologic markers on graft outcomes
This analysis included only cases with TCMR or AMR in the graft biopsy. The mean serum creatinine level in the C4d-positive group was significantly higher at both the time of biopsy and at the last follow-up, compared with that in the C4d-negative group (time of biopsy: C4d-positive, 442.0 ± 265.2 μmol/L C4d-negative, 353.6 ± 176.8 μmol/L; P ≤ .03) (last follow-up: C4d-positive, 353.6 ± 265.2 μmol/L; C4d-negative, 265.2 ± 176.8 μmol/L; P ≤ .02). There were no significant differences in the evolution of graft function at any time (Figure 2). The evolution of serum creatinine until the end of follow-up was worse in C4d-positive patients, regardless of when it was measured (C4d-positive vs C4d-negative, P ≤ .006) (time, not significant). When comparing DSA-positive and DSA-negative patients, no differences were observed in the analyzed periods, but the combined analysis of C4d-positive/DSA-positive showed that graft function worsened regardless of the time of measurement (C4d-positive /DSA-positive vs C4d-negative /DSA-negative, P ≤ .03) (time, not significant).

Evaluation of the effect of immunologic markers on graft survival at almost 4 years after transplant showed that graft survival was significantly worse in C4d-positive patients (C4d-positive, 73%; C4d-negative, 87%; P ≤ .03) and in patients with DSAs (DSA-positive, 66%; DSA-negative, 82%; P ≤ .04) (Figure 3). The infiltrating CD20-positive B lymphocyte or CD138-positive plasma cell density had no association with graft survival (CD20-positive, 78%; CD20-negative, 81%; not significant) (CD138-positive, 81%; CD138-negative, 83%, not significant) (Figure 3).

The nonadjusted Cox proportional hazards regression model showed that presence of C4d or DSA was predictive of graft loss (Table 3). The mean fluorescence intensity for either class I or II anti-HLA antibodies (500-1000, 1000-5000, or > 5000) did not predict AMR or graft loss up to the end of follow-up.

A higher glomerular filtration rate at the time of biopsy, estimated with the Chronic Kidney Disease Epidemiology Collaboration CKD-EPI equation, was protective against graft loss in both the univariate and multivariate analyses (Table 3). In multivariate analysis, after adjusting for graft function at the time of biopsy, only positive C4d was an independent predictor of graft loss (Table 3).


The central role of T cells in acute cellular rejection has been well established. However, the occurrence of severe forms of rejection suggests that antibody-mediated mechanisms also might be involved.2-5,6,20,21 More recently, the function of B cells in immune allograft responses has been the focus of more intensive research.8,12,15,16,22-25

The primary objective of this study was to evaluate the associations of CD20-positive cells and CD138-positive plasma cells with graft outcomes in patients who had TCMR or AMR. A greater CD20-positive cell density was observed in the biopsies of patients who had TCMR, and a greater density of CD138-positive cells was observed in patients with AMR. This suggests that 2 recipient-derived B-cell compartments were identified. First, there was a cluster of CD27-positive memory cells and CD79-positive and CD20-positive cells that presented donor HLA antigens and sustained a continued T-cell response during ongoing rejection; however, these cells did not correlate with C4d or DSA, suggesting the involvement of a cellular mechanism of graft injury. A second group comprised late-strain B cells, CD138-positive mature plasma cells, and CD38-positive plasmablasts, which correlated strongly with circulating DSAs and, to a lesser extent, C4d and were not depleted by immunosuppressive therapy.14,16,24 Plasmapheresis, intravenous immunoglobulin, and rituximab reduced the number of these cells and the concentration of DSAs; however, these therapies did not affect long-term graft survival, perhaps because they were ineffective in blocking the production of anti-HLA antibodies by long-lasting and functionally active plasma cells.14 In the present study, induction therapy with anti-thymocyte globulin was associated with C4d and plasmocyte cell number in biopsy. However, receiving anti-thymocyte globulin or basiliximab had no effect on the prevalence of DSA, graft function, or survival.

Studies on the function of CD20-positive B cells in kidney graft rejection have reported conflicting results. In dysfunctional grafts, the CD20-positive cell density in acute rejection correlated with corticosteroid resistance and decreased graft survival.22 Worse outcomes also were reported for grafts with acute rejection with strong and diffuse CD20 expression.10 A correlation was reported with ≥ 3 CD20-positive cells/field with cellular rejection and worse graft survival but not an AMR.25 A correlation also was reported between nodular CD20-positive B cell infiltration and worse graft function in acute rejection; a similar correlation was not found with diffuse infiltration.19 However, other studies showed no associations between CD20-positive cell-rich infiltrates and acute treatment-resistant rejection or unfavorable graft outcomes.8,9,13 In the present study, CD20-positive cell-enriched infiltrates correlated with immunologic risk factors such as retransplant, higher HLA mismatch, and TCMR, but not with AMR, C4d, DSA, or the risk of graft loss. Factors such as the heterogeneity of the patient population, intensity of immunosuppression, time of post-transplant biopsy, and different methods of defining CD20-positive cells in biopsies might explain the discrepancies between these studies.

It is possible that in the present study, the threshold for CD20-positive B cells contributed to these negative findings, unlike other studies that showed a correlation with poorer graft outcomes.11,22 However, the established cutoff was similar to that of a previous study.25 We digitally analyzed the images to quantify the CD20-positive cell infiltrates and account for the patchy distribution of the positive cells. Therefore, the absolute number of CD20-positive cells was corrected by the area of cortical tissue to produce a final score with a lower magnitude. In addition, the type of interstitial CD20-positive cell distribution (nodular or diffuse infiltrate) might have prognostic importance,10,19,22 and the characterization of these distributions might have more accurately determined the graft outcomes.

In the present study, many CD20-positive B cells were observed in the infiltrates of a subset of biopsies without rejection, but with predominant nonspecific chronic damage (interstitial fibrosis and tubular atrophy). Resident B cells in the graft may remain capable of antigen presentation,24 maintaining a cellular immune and/or humoral response that might be harmful to the graft. In a separate analysis of this subgroup, no correlations were observed with C4d deposits or circulating DSA, possibly implying the presence of a persistent type of cellular response that may cause chronic graft damage.

Plasma cell infiltration during rejection of a transplanted kidney is uncommon, but the presence of such infiltration is associated with worse graft prognosis.12,16,23,26,27 The plasma cell density is significantly greater and associated with C4d and anti-HLA specific antibodies in patients with progressive functional losses than functionally stable patients.23 However, the cutoff used in the present study for high intragraft CD138-positive cell density might have been insufficient to detect an association with worse graft outcomes, and it is unclear whether plasma cell infiltration causes injury, irreversible damage, and long-term graft loss.

The lack of improvement in long-term kidney allograft survival might be associated with poor control of the immune response.28 New immuno-suppressive therapy targets should be developed and tested because different strains of B cells may initiate, participate in, or perpetuate immune responses in rejection. The B-cell-depleting agents and donor-specific anti-HLA antibodies such as rituximab and bortezomib have been tested previously.14 However, the prognostic value of these markers and the effect of these biological agents on long-term graft survival is unknown.

The time of biopsy after transplant might correlate with the extent and density of B-cell infiltration. In a small, retrospective study, the infiltrating CD20 cell density was greater in biopsies with TCMR that were performed from 2 weeks to 6 months than > 6 months after transplant, and this had a negative effect on graft outcome.29 In addition, the CD138-positive cell density and the estimated glomerular filtration rate at clinical rejection were predictive of graft loss, but the effects of CD20-positive cells on this outcome were minimal.30 The severity of graft dysfunction, which reflects the extent of tissue damage, is a known prognostic factor, and this also was demonstrated in the present study. However, after adjusting for estimated glomerular filtration rate in the multivariate model, we did not find CD20-positive or CD138-positive staining to be predictive of graft loss.

Limitations of the present study include the retrospective design, small number of patients with rejection, and short follow-up. The diagnosis of AMR was considered probable because circulating DSAs were not measured in all patients. We could not determine DSA against HLAs other than HLA-A, -B, and -DR because tests for other HLA antigens were not done in our institution. The absence of a standardized method for determining positive B-cell infiltrates in graft biopsies makes the analysis difficult. Although confirmation in studies with larger samples is necessary, the finding of higher plasma cell infiltration in late rejection cases suggests that a humoral component may be activated later, especially in patients who have poor compliance with immunosuppressive drugs and its relation with AMR, which was not evaluated in this study.31

Unlike C4d and donor-specific anti-HLA antibodies, B cells were not associated with worse function or graft loss during 4 years after transplant. In this study, C4d was the only consistent prognostic marker, even after adjusting for graft function and the presence of circulating DSA.


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Volume : 12
Issue : 5
Pages : 405 - 414
DOI : 10.6002/ect.2014.0049

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From the 1Postgraduate Program in Medicine/Medical Sciences, Federal University of Rio Grande do Sul, Division of Nephrology and Laboratory of Molecular Biology Applied to Nephrology, Hospital de Clínicas de Porto Alegre, Porto Alegre, Rio Grande do Sul; 2Laboratory of Genetic, Cellular, and Molecular Nephrology, Nephrology Division, University of São Paulo, São Paulo, São Paulo; and 3Division of Immunology, Hospital de Clínicas de Porto Alegre, Porto Alegre, Rio Grande do Sul, Brazil
Acknowledgements: This study received financial support from the Research Incentive Fund (Fundo de Incentivo à Pesquisa) of the Hospital das Clínicas de Porto Alegre (FIPE-HCPA) and the Program for Promotion of Postgraduate Studies (PROF) of the Coordination for the Improvement of Higher Education Personnel (CAPES). Virna Carpio received a PhD scholarship from CAPES. The authors have no competing financial interests.
Corresponding author: Francisco Veríssimo Veronese, MD, PhD, Division of Nephrology, Room 2030, Hospital de Clínicas de Porto Alegre. Ramiro Barcelos 2350, Porto Alegre, Rio Grande do Sul, 90035-003, Brazil
Phone: +55 51 3359 8295
Fax: +55 51 3359 8121