Virna Nowotny Carpio

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.

Noninvasive Tim-3 messenger RNA evaluation in renal transplant recipients with graft dysfunction.

Background. Renal biopsies are usually needed to elucidate graft dysfunction. In this study, T-cell immunoglobulin domain, mucin domain mRNA expression in the peripheral blood leukocytes (PBL) and urinary cells (UC) were studied as a noninvasive method for the diagnosis of acute rejection (AR) of kidney transplant patients with dysfunction. Methods. One hundred sixty biopsies were obtained from 115 patients. Blood and urine samples were collected immediately before the biopsies. Histopathologic diagnoses were acute tubular necrosis with superimposed AR or acute tubular necrosis in patients with delayed graft function (DGF), and (AR), or calcineurin inhibitor nephrotoxicity (CIN), or interstitial fibrosis and tubular atrophy in patients with acute graft dysfunction (AGD). Fifteen protocol biopsies of stable grafts were used as controls. mRNA relative quantification was performed by real-time polymerase chain reaction. Results. Gene expression in tissue, PBL, and UC was always higher in patients with AR than in patients with the other causes of graft dysfunction (P<0.001). Significant correlations of gene expression in different compartments were observed (P<0.001). The obtained diagnostic parameters were 100% accurate in the DGF group and, respectively, for blood and urine: sensitivity (87% and 84%); specificity (95% and 96%); positive predictive value (87% and 89%); negative predictive value (93% and 94%); and accuracy (91% and 93%) for the group of patients with AGD. Conclusion. T-cell immunoglobulin domain, mucin domain mRNA quantification by real-time polymerase chain reaction in PBL and UC of renal transplant patients undergoing DGF or AGD may become a useful tool for an accurate noninvasive diagnosis of AR.

Endothelin-1 and Endothelin A Receptor Immunoreactivity Is Increased in Patients with Diabetic Nephropathy

Background: Endothelin-1 (ET-1) is associated with progression of renal disease, acting as a vasoconstrictor and growth factor for mesangial cells. ET-1 and endothelin A receptor (ET-RA) might have a role in the development of diabetic nephropathy (DN). The aims of this study were to determine ET-1 and ET-RA expressions in patients with DN and to correlate these expressions with renal function and proteinuria. Materials and methods: This is a cross-sectional study comprising 13 patients with type 2 diabetes mellitus and DN, 10 patients with proteinuric IgA nephropathy, and 13 samples of normal kidney from tumor nephrectomies. Demographic and selected data were collected from medical charts. The distribution and intensity of ET-1 and ET-RA immunostaining in renal biopsies were determined by immunohistochemistry and these correlated with the estimated glomerular filtration rate (eGFR) and proteinuria. Results: Patients with DN and IgA nephropathy on biopsy had markedly increased staining for ET-1 in endothelial cells of glomerular and peritubular capillaries when compared with controls (p < 0.001). ET-RA staining was also more intense and more diffuse in DN and IgA nephropathy than in controls (p = 0.019) and was restricted to tubular epithelial cells. A positive correlation was observed between ET-1 expression and proteinuria (r = 0.634, p = 0.027), but both ET-1 and ET-RA expressions did not correlate with eGFR. Conclusion: In this preliminary report, the higher expressions of ET-1 and ET-RA found in both DN and IgA nephropathy suggest a potential role for the endothelin system in DN as well as in other nondiabetic glomerular diseases.

Kidney injury molecule-1 expression in human kidney transplants with interstitial fibrosis and tubular atrophy

BackgroundKidney injury molecule-1 (KIM-1) is expressed in tubular epithelial cells after injury and may have a role in the development of renal graft fibrosis. In this study we evaluated the molecular and protein expressions of KIM-1 in dysfunctional allografts and also mRNA KIM-1 expression in urine as potential biomarkers of graft fibrosis.MethodsProtein and mRNA levels in renal tissue and urinary sediment cells of 69 kidney transplant recipients that undertook for-cause graft biopsies were evaluated by immunohistochemistry and real-time polymerase chain reaction. The histopathology was classified according to the 2007 Banff schema.ResultsKIM-1 protein expression was increased in biopsies with interstitial fibrosis and tubular atrophy (IF/TA) compared with biopsies showing acute calcineurin inhibitor nephrotoxicity (CIN) (P <0.05). Kidney tissue KIM-1 mRNA signaling (in) was increased in biopsies with IF/TA compared with all other groups (P <0.05). In the urine cells KIM-1 mRNA was also increased in patients with IF/TA compared with patients with acute CIN (P <0.05). Significant correlations were found between KIM-1 protein and mRNA levels in tissue, between mRNA expressions in tissue and urine and between protein tissue expression and gene expression in the urine.ConclusionsKIM-1 seems to be a marker of kidney graft fibrosis. Urinary KIM-1 mRNA may become a useful non-invasive biomarker of the injuries that can trigger intra-graft fibrotic processes, such as interstitial fibrosis and tubular atrophy.

Analysis of FOXP3 gene and protein expressions in renal allograft biopsies and their association with graft outcomes

Background: The transcription factor FOXP3 is increased in acute renal rejection, but its influence on graft outcomes is unclear. This study correlated FOXP3 with dendritic cells and graft outcomes. Methods: We assessed 96 kidney transplants undergoing allograft biopsy for cause. FOXP3 mRNA was analyzed by real-time polymerase chain reaction (PCR) and FOXP3 protein and DCsCD83+ by immunohistochemistry. Graft function and survival were assessed at 5 years post-transplantation, as well as by independent predictors of graft loss. Results: Intragraft FOXP3 gene and protein expression were significantly correlated (r = 0.541, p < 0.001). Both FOXP3 mRNA and protein were increased in patients with acute rejection (AR). High expression of FOXP3 mRNA or protein in biopsies did not correlate with clinical variables, but there was a trend to higher positive variation in the glomerular filtration rate (GFR) from biopsy to last follow-up. Patients with FOXP3-mRNAhigh had more DCsCD83+ in biopsy, but these cells did not associate with AR. Five-year graft survival was not influenced by either FOXP3 mRNA or protein expressions. Conclusions: FOXP3 mRNA and protein had a good correlation in archival renal graft tissue. Increased FOXP3 expression was found in AR and FOXP3 associated with high numbers of DCs. However, both FOXP3 mRNA and protein was not associated with better allograft outcomes.

Correlações clinico-patológicas da marcação de C4d e sua influência na evolução de receptores de transplante renal

INTRODUCTION: C4d is a marker of antibody-mediated rejection (ABMR) in kidney allografts, although cellular rejection also have C4d deposits. OBJECTIVE: To correlate C4d expression with clinico-pathological parameters and graft outcomes at three years. METHODS: One hundred forty six renal transplantation recipients with graft biopsies by indication were included. C4d staining was performed by paraffin-immunohistochemistry. Graft function and survival were measured, and predictive variables of the outcome were determined by multivariate Cox regression. RESULTS: C4d staining was detected in 48 (31%) biopsies, of which 23 (14.7%) had diffuse and 25 (16%) focal distribution. Pre-transplantation panel reactive antibodies (%PRA) class I and II were significantly higher in C4d positive patients as compared to those C4d negative. Both glomerulitis and pericapillaritis were associated to C4d (p = 0.002 and p < 0.001, respectively). The presence of C4d in biopsies diagnosed as no rejection (NR), acute cellular rejection (ACR) or interstitial fibrosis/ tubular atrophy (IF/TA) did not impact graft function or survival. Compared to NR, ACR and IF/TA C4d-, patients with ABMR C4d+ had the worst graft survival over 3 years (p = 0.034), but there was no difference between ABMR versus NR, ACR and IF/TA that were C4d positive (p = 0.10). In Cox regression, graft function at biopsy and high %PRA levels were predictors of graft loss. CONCLUSIONS: This study confirmed that C4d staining in kidney graft biopsies is a clinically useful marker of ABMR, with well defined clinical and pathological correlations. The impact of C4d deposition in other histologic diagnoses deserves further investigation.