Francisco José Veríssimo Veronese

FOXP3+ regulatory T cells: From suppression of rejection to induction of renal allograft tolerance☆

Naturally occurring and induced regulatory T cells (Tregs) can become hyporesponsive and anergic to antigen stimulation in autoimmune diseases and allograft rejection. The mechanisms of suppression of effector T cells by Tregs remain unclear, but there are in vitro and in vivo evidences showing that these cells are able to suppress antigen-specific responses via direct cell-to-cell contact, secrete anti-inflammatory cytokines such as TGF-β and IL-10, and inhibit the generation of memory T cells, among others. The transcription factor FOXP3 is a specific marker of Tregs and its deficiency is associated with autoimmune diseases and inflammation. During acute rejection of kidney allografts, an augmented FOXP3 gene expression as well as increased CD4(+)CD25(+)FOXP3(+) and other cell populations are observed in graft biopsies. However, it is not clear whether Tregs migrate into the graft and are retained there to suppress the inflammatory process, or whether they are directly associated with more complex mechanisms to induce immune tolerance. FOXP3(+) Tregs may direct the immune response toward a graft acceptance program, potentially affecting the long-term survival of transplanted organs and tissues. Immunosuppressive drugs modulate the number and function of circulating Tregs and FOXP3 expression. Experimental and clinical studies have shown that mTOR inhibitors have positive and calcineurin inhibitors negative effects on Tregs, but it is difficult to set apart the effect of multiple other factors known to be associated with short- and long-term renal graft outcomes. This review aimed to describe the functions of Tregs and its transcription factor FOXP3 in suppression of immune response during rejection and in induction of kidney graft tolerance, as well as to review the individual effects of immunosuppressive drugs on Tregs.

Reproducibility of the Banff classification in subclinical kidney transplant rejection.

Abstract:  The Banff classification for kidney allograft pathology has proved to be reproducible, but its inter and intraobserver agreement can vary substantially among centres. The aim of this study was to evaluate Banff reproducibility of surveillance renal allograft biopsies among renal pathologists from different transplant centres. This study included 32 renal transplant patients with stable graft function. Biopsies were performed 2 and 12 months post‐transplant. Histology was interpreted according to the Banff schema by three renal pathologists, and inter and intraobserver agreement were measured. The best reproducibility was obtained for the presence or absence of acute rejection (AR), with kappa values ranging from moderate (κ = 0.47; p = 0.006) to good (κ = 0.72; p = 0.0001). However, the agreement for ‘suspicious for AR’ category was poor between all observers. For scoring and grading interstitial inflammation and intimal arteritis the agreement were poor and moderate, respectively. Reproducibility for the presence or absence of chronic allograft nephropathy (CAN) was heterogeneous, ranging from poor (κ = 0.13; p = NS) to moderate (κ = 0.56; p = 0.007). Scoring chronic changes such as fibrous intimal thickening gave a reasonable interobserver agreement. Intraobserver reproducibility was good for presence or absence of AR, but was poor for the diagnosis of CAN. In conclusion, histologic analysis of stable renal allografts based on Banff criteria showed a good agreement for the diagnosis of AR and a reasonable kappa for CAN, but reproducibility for scoring and grading showed a substantial interobserver variation.

Diagnostic accuracy of the protein/creatinine ratio in urine samples to estimate 24-h proteinuria in patients with primary glomerulopathies: a longitudinal study

BACKGROUND The protein/creatinine (P/C) ratio in urine samples has been used in the clinical management of patients with glomerular diseases. The aim of this study is to perform a prospective evaluation of the P/C ratio accuracy in determining critical levels of proteinuria in patients with glomerulopathies. METHODS This is a longitudinal study of 41 adult patients with primary glomerulopathies treated with immunosuppressive drugs or angiotensin-converting enzyme inhibitors in a 6-month follow-up. Correlation and agreement level between P24 and the P/C ratio were evaluated. Kappa statistic was employed to evaluate concordance between the two methods taking into account clinically relevant categories of proteinuria. ANOVA for repeated measures was employed. Diagnostic accuracy of the P/C ratio was evaluated by receiver-operator curves (ROC). RESULTS There was a significant correlation between P24 and the P/C ratio during the 6-month period (P < 0.001 in all time points). Mean differences between P24 and P/C ratios at baseline and from the first to the sixth month were 2.00, 1.88, 1.22, 1.07, 0.65, 0.34 and 0.57 respectively. In spite of the lower agreement between P24 and the P/C ratio for higher levels of proteinuria, we found substantial Kappa values for categories of proteinuria in all periods. ROC considering the cut-off levels of 0.20 g and 3.5 g for P24 showed that the P/C ratio had a very good accuracy, with areas under the curve of 0.99 (95% CI: 0.97-1.00) and 0.99 (95% CI: 0.99-1.00), respectively. CONCLUSION This longitudinal analysis corroborates the findings of previous cross-sectional studies, supporting the use of the P/C ratio as an accurate test to define critical levels of proteinuria.

Effect of Peripheral and Respiratory Muscle Training on the Functional Capacity of Hemodialysis Patients

Patients on hemodialysis (HD) show changes in muscle structure and function reducing their functional capacity. This study was conduted to assess the effects of respiratory muscle training (RMT) and peripheral muscle training (PMT) during dialysis on functional parameters, inflammatory state, and quality of life (QoL) in patients on HD. Randomized controlled trial included 39 patients on HD, and they were divided into three groups: RMT (n = 11), PMT (n = 14), and controls (C, n = 14). Training was performed during the HD session for 10 weeks. Maximal inspiratory pressure (PImax), maximal expiratory pressure (PEmax), forced vital capacity (FVC), six-minute walk test (6MWT), Kt/Vsp, biochemical parameters, and inflammatory state (i.e., level of high sensitivity C-reactive protein) were evaluated. Variation from baseline was calculated by Analysis of Covariance (ANCOVA). The ΔPImax was 22.5 ± 3.2, 9.1 ± 2.9, and −4.9 ± 2.8 cmH2O in the RMT, PMT and C, respectively (p < 0.001); ΔPEmax was 10.8 ± 6.6, 3.7 ± 5.9, and −15.6 ± 5.9 cmH2O respectively (p = 0.014). The Δ6MWT was significantly greater in RMT and PMT (65.5 ± 9; 30.8 ± 8 m) than in C (−0.5 ± 8.1 m), p < 0.001. Although biochemical parameters decreased after training, Kt/V remained unchanged. CRP decreased only in the RMT and PMT groups. There was a significant increase in QoL scores in the training groups (vs. C) in energy/fatigue (p = 0.002), sleep (p < 0.001), pain (p < 0.001), and list of symptoms/problems (p = 0.014). A short period of RMT or PMT during HD significantly improved functional capacity, with RMT showing greater effect than PMT. Muscle training improved biochemical and inflammatory markers, but a direct cause and effect relationship could not be established by this study.

Prevalence and immunohistochemical findings of subclinical kidney allograft rejection and its association with graft outcome

Abstract:  Subclinical acute rejection (SAR) occurs in about 30% of stable renal transplant patients and may be a risk factor for a poor allograft outcome. In the present study, the prevalence and clinical features of subclinical rejection, and the expression of immune activation markers in surveillance graft biopsies were assessed and correlated with late graft outcomes. Protocol biopsies were obtained at 2 and 12 months post‐transplant in 32 and 26 patients, respectively, with stable renal function. The Banff 1997 criteria were used for histological diagnosis. Graft function and survival and proteinuria were assessed during the 36 months of follow‐up. Immunohistochemical evaluation of cell subpopulations and immunoactivation markers were performed on protocol biopsies. The prevalence of SAR at 2 months and of chronic allograft nephropathy (CAN) at 12 months in representative biopsies was 55 and 50%, respectively. Patients with SAR presented mononuclear cell infiltration with an increased expression of CD3, CD4, CD68, IL‐2R and granzyme B. Kidney graft function was significantly worse in patients with SAR at 2 months who had chronic rejection on biopsy at 12 months, but SAR was not associated with a worse graft function, greater proteinuria or a lower graft survival in 3 yr of follow‐up. In conclusion, we found an elevated prevalence of SAR at 2 months after transplantation with an increased expression of activation markers. Although an association of SAR with poor graft outcome was not observed, our results suggest that SAR is an immunologically active process and underscore the importance of protocol biopsies in the surveillance of transplanted kidneys.

Noninvasive Analyses of Kidney Injury Molecule-1 Messenger RNA in Kidney Transplant Recipients With Graft Dysfunction

BACKGROUND Kidney graft fibrosis is a major factor related to chronic loss of kidney function. At present, the finding of fibrosis depends on the analysis of tissue in the renal biopsy, which has important limitations. In this study, we evaluated the messenger mRNA transcription and gene expression of kidney injury molecule-1 (KIM-1) in kidney tissue and in urinary sediment cells of kidney transplant patients with graft dysfunction aiming at the development of techniques that may allow the noninvasive diagnosis of interstitral fibrosis/tubular atrophy (IF/TA). PATIENTS AND METHODS RNA extracted from cells in tissue and urine of 77 renal transplant patients whose biopsies were classified according to the Banff scheme-2007. Four diagnostic groups were established: (1) acute tubular necrosis (n = 9); (2) acute rejection (n = 49); (3) acute calcineurin inhibitors nephrotoxicity (n = 10); and (4) interstitial fibrosis and tubular atrophy (IFTA, n = 29). Tissue and urine cell RNA was amplified and quantification were made by real-time polymerase chain reactron. Data from the quantification of gene expression are presented as median and 25th to 75th percentiles. RESULTS Messenger RNA levels of the KIM-1 gene were higher in the biopsies (26.17; 3.38-294.53) and urinary sediment cells (0.09; 0-5.81) of the patients classified as having IF/TA as compared with all others groups. A significant correlation between gene expression in samples of urine and tissue cells was found (P < .01). CONCLUSION These initial data suggests that KIM-1 gene mRNA quantification can be used as a noninvasive biomarker of IF/TA.

Doença renal crônica, inflamação e aterosclerose: novos conceitos de um velho problema

Chronic kidney disease (CKD) has reached epidemic proportions in the last few years, generating an emergent public health problem. Common risk factors for CKD and cardiovascular disease (CVD) are now well known resulting in a high prevalence rate of cardiovascular events which are the main cause of death in CKD patients. Development of accelerated atherosclerosis is related to traditional risk factors such as diabetes mellitus, arterial hypertension, dislipidemia and smoking, but recently other non traditional factors were found to be significantly associated with cardiovascular mortality, including inflammation, oxidative stress, endothelial dysfunction and uremia, even at early stages of CKD. Inflammatory markers such as C-reactive protein, interleukin 6 and fibrinogen are all correlated with cardiovascular death. The MIA syndrome is characterized by the association between inflammation, malnutrition and accelerated atherosclerosis, a condition commonly found in uremic patients, which is related to the genesis of CVD. Other important factors are the high level of oxidative stress, expressed by oxidized lipids, proteins and carbohydrates (AGES) (Advanced Glycation End Products), which cause tissue damage and endothelial dysfunction, that is aggraveted by the uremic environment and other factors. These alterations are the basis for the pathogenic process of atherosclerosis and CVD in CKD patients, contributing to their high morbidity/ mortality. This article is an updated review of the mechanisms of inflammation and oxidative stress and their relation to atherosclerosis in CKD.

Molecular markers in subclinical acute rejection of renal transplants

Abstract:  In this study, we evaluated the expression of molecular markers of acute rejection in protocol biopsies of patients with and without subclinical acute rejection (SAR). Protocol biopsies were performed at 2 months (n = 21) and 12 months (n = 14) after kidney transplantation in patients with stable allograft function. After biopsy tissue RNA isolation, reverse transcription and polymerase chain reaction (RT‐PCR) for the glyceraldehyde 3‐phospate dehydrogenase (GAPDH), perforin, granzyme B and Fas ligand genes were performed. The Banff 97 classification was used for histological diagnosis. Creatinine concentrations at 2 months were significantly higher in patients with SAR (1.46 ± 0.27 × 1.18 ± 0.24; p < 0.02). Perforin transcripts were found in 15 biopsy specimens, 10 of which had histological signs of SAR (p = 0.06). Granzyme B expression was found in 10 specimens, nine of which had SAR (p < 0.01). Fas ligand was expressed in seven specimens, and six of them were classified as SAR (p < 0.01). Perforin expression had the highest sensitivity (81%) for the diagnosis of SAR. Granzyme B and Fas ligand had specificity of 90%. At 12 months, there was no significant difference in creatinine concentrations for patients with and without previous SAR (1.63 ± 0.57 × 1.28 ± 0.31; p = 0.10). Molecular analysis revealed that there was no statistically significant difference in the expression of perforin and granzyme B in patients with and without SAR. Fas ligand expression was observed in five samples, four of which had histological signs of SAR (p = 0.03). At 12 months, perforin expression had the highest sensitivity (83%), and Fas ligand, the highest specificity (88%) for the diagnosis of SAR. We concluded that the expression of genes that encode proteins involved in the cytolytic attack against the allograft is increased in kidneys with SAR. These findings support the understanding that SAR is an active immune process potentially deleterious to renal allografts.

Messenger RNA levels of podocyte-associated proteins in subjects with different degrees of glucose tolerance with or without nephropathy

BackgroundTo investigate gene expression of podocyte-specific proteins in urine of diabetes and prediabetes subjects and the association of these proteins with albuminuria.MethodsFifteen controls, 19 prediabetes, and 67 diabetes subjects were included. Messenger RNA of nephrin, podocin, podocalyxin, synaptopodin, TRPC6, alpha-actinin-4, and TGF-β1 were measured using RT-PCR. Podocyte marker expression was correlated with albuminuria, glycemic control, and renal function. The diagnostic performance of the genes used to detect increased albuminuria was assessed using ROC curves and Poisson regressions.ResultsPodocyte marker expression was significantly higher in diabetic subjects. Urinary nephrin was correlated with increasing levels of albuminuria; risk of albuminuria increased by 20% for every one-unit increase in the log10 of nephrin mRNA. Nephrinuria was found in 53%, 71%, and 90% of normo-, micro-, and macroalbuminuric diabetes subjects, respectively (p = 0.023). Urinary nephrin, podocalyxin, TRPC6, podocin, and alpha actinin-4 were correlated with glycemic control and albuminuria but not with renal function.ConclusionsDiabetes subjects had higher urinary mRNA levels of podocyte proteins than nondiabetic subjects, even the normoalbuminuric patients. Nephrinuria was correlated with diabetic nephrophathy stage and predicted pathological albuminuria. Urinary mRNA levels of podocyte markers of prediabetic subjects did not differ from controls.

Performance of CKD-EPI equation to estimate glomerular filtration rate as compared to MDRD equation in South Brazilian individuals in each stage of renal function

Abstract Background: The Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation seems to correct the overdiagnosis of chronic kidney disease (CKD) provided by Modification of Diet in Renal Disease (MDRD) equation. However, this point has not been tested in some ethnic groups. This study investigated the performance of MDRD and CKD-EPI equations in South Brazilian individuals. Methods: This cross-sectional study included 354 individuals including healthy volunteers, diabetic and non-diabetic individuals with or without CKD. Glomerular filtration rate (GFR) was measured by the 51Cr-EDTA single-injection method (51Cr-GFR). Accuracy (P30), bias, and Bland-Altman agreement plots were evaluated. Results: In the group as a whole, 51Cr-GFR was 87±37 (6-187), CKD-EPI eGFR, 82±30 (6-152), and MDRD eGFR, 77±28 (6-156) mL/min/1.73 m2 (p<0.001 for all comparisons). Analyzing the subset of individuals with 51Cr-GFR <60 mL/min/1.73 m2, P30 values were, respectively, 76% and 84% for MDRD and for CKD-EPI (p<0.001) while for 51Cr-GFR ≥60 mL/min/1.73 m2, P30 values were 57.5% for both equations (p=1.000). For MDRD and CKD-EPI, mean bias were negative for GFRs <60 (–11 vs. –12, p=0.221) and positive for values >60 (16 vs. 9, p<0.001). In multivariate analysis, absolute bias was unfavorably influenced by measured GFR >60 (for MDRD) and being diabetic or younger (for CKD-EPI). Conclusions: CKD-EPI reduces GFR underestimation in individuals with GFRs >60, but still presents a quite low accuracy at this GFR range. Moreover, it tends to overestimate GFR in subjects with GFRs <60 mL/min/1.73 m2. CKD stages 1 and 2, diabetes and young age had a negative influence on the performance of the equations.