Immunosuppressive drugs modes of action.

The innate and adaptive immune systems work as a complex interplay between different cell types, involving cytokines and chemokines mediating extracellular and paracrine effects. At the intracellular level, the inflammatory cascade is mediated by multifaceted processes that have been better described in the last 10 years. Immunosuppressive agents available in clinical practice act at different points of those cascades at the intracellular or extracellular level. Those drugs can mediate their effects on one or more cell types finally limiting inflammation and immune responses to antigens. Every immunosuppressive agent is characterized by intrinsic toxicity and side effects that may be due to the same therapeutic pathways or to off-target secondary effect of each molecule. We will here review the mechanisms of action of the most widely used immunosuppressive agents in the field of solid organ transplantation and autoimmune disorders, describing the mechanisms underlying both the therapeutic and secondary effects.

In vivo CD40 Silencing by siRNA Infusion in Rodents and Evaluation by Kidney Immunostaining.

The co-stimulatory molecule CD40 and its ligand CD40L play a key role in the regulation of immunological processes and are involved in the pathophysiology of autoimmune and inflammatory diseases. Inhibition of the CD40-CD40L axis is a promising therapy, and a number of strategies and techniques have been designed to hinder its functionality. Our group has broad experience in silencing CD40 using RNAi technology, and here we summarize protocols for the systemic administration of a specific anti-CD40 siRNA in different rodents models, in addition to the subsequent quantification of CD40 expression in murine kidneys by immunostaining. The use of RNAi technology with specific siRNAs to silence genes is becoming an essential method to investigate gene functions and is rapidly emerging as a therapeutic tool. Graphic abstract: CD40 siRNA mechanism.

The gene silencing of IRF5 and BLYSS effectively modulates the outcome of experimental lupus nephritis.

Systemic lupus erythematosus is a highly complex and heterogeneous autoimmune disease mostly mediated by B cells. It is characterized by circulating self-reactive antibodies that deposit and form immune complexes in kidney, leading to irreparable tissue damage and resulting in lupus nephritis. In a New Zealand Black X New Zealand White F1 mouse model, we tested two different small interfering RNA (siRNA) silencing treatments against interferon regulatory factor 5 (IRF5) and B cell-activating factor (BLYSS) expression and their combination in a second set of animals. The administration of these two siRNAs separately prevented the progression of proteinuria and albuminuria at similar levels to that in cyclophosphamide animals. These treatments effectively resulted in a reduction of serum anti-double-stranded DNA (dsDNA) antibodies and histopathological renal score compared with non-treated group. Treated groups showed macrophage, T cell, and B cell infiltrate reduction in renal tissue. Moreover, kidney gene expression analysis revealed that siRNA treatments modulated very few pathways in contrast to cyclophosphamide, despite showing similar therapeutic effects. Additionally, the combined therapy tested in a second set of animals, in which the disease appeared more virulent, exhibited better results than monotherapies in the disease progression, delaying the disease onset and ameliorating the disease outcome. Herein, we provide the potential therapeutic effect of both selective IRF5 and BLYSS silencing as an effective and potential treatment, particularly in early phases of the disease.

Influence of the Circadian Timing System on Tacrolimus Pharmacokinetics and Pharmacodynamics After Kidney Transplantation.

Introduction: Tacrolimus is the backbone immunosuppressant after solid organ transplantation. Tacrolimus has a narrow therapeutic window with large intra- and inter-patient pharmacokinetic variability leading to frequent over- and under-immunosuppression. While routine therapeutic drug monitoring (TDM) remains the standard of care, tacrolimus pharmacokinetic variability may be influenced by circadian rhythms. Our aim was to analyze tacrolimus pharmacokinetic/pharmacodynamic profiles on circadian rhythms comparing morning and night doses of a twice-daily tacrolimus formulation. Methods: This is a post-hoc analysis from a clinical trial to study the area under curve (AUC) and the area under effect (AUE) profiles of calcineurin inhibition after tacrolimus administration in twenty-five renal transplant patients. Over a period of 24 h, an intensive sampling (0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 12.5, 13, 13.5, 14, 15, 20, and 24 h) was carried out. Whole blood and intracellular tacrolimus concentrations and calcineurin activity were measured by UHPLC-MS/MS. Results: Whole blood and intracellular AUC12-24 h and Cmax achieved after tacrolimus night dose was significantly lower than after morning dose administration (AUC0-12 h) (p < 0.001 for both compartments). AUE0-12 h and AUE12-24 h were not statistically different after morning and night doses. Total tacrolimus daily exposure (AUC0-24 h), in whole blood and intracellular compartments, was over-estimated when assessed by doubling the morning AUC0-12 h data. Conclusion: The lower whole blood and intracellular tacrolimus concentrations after night dose might be influenced by a distinct circadian clock. This significantly lower tacrolimus exposure after night dose was not translated into a significant reduction of the pharmacodynamic effect. Our study may provide conceptual bases for better understanding the TDM of twice-daily tacrolimus formulation.

Donor/Recipient HLA Molecular Mismatch Scores Predict Primary Humoral and Cellular Alloimmunity in Kidney Transplantation.

Donor/recipient molecular human leukocyte antigen (HLA) mismatch predicts primary B-cell alloimmune activation, yet the impact on de novo donor-specific T-cell alloimmunity (dnDST) remains undetermined. The hypothesis of our study is that donor/recipient HLA mismatches assessed at the molecular level may also influence a higher susceptibility to the development of posttransplant primary T-cell alloimmunity. In this prospective observational study, 169 consecutive kidney transplant recipients without preformed donor-specific antibodies (DSA) and with high resolution donor/recipient HLA typing were evaluated for HLA molecular mismatch scores using different informatic algorithms [amino acid mismatch, eplet MM, and Predicted Indirectly Recognizable HLA Epitopes (PIRCHE-II)]. Primary donor-specific alloimmune activation over the first 2 years posttransplantation was assessed by means of both dnDSA and dnDST using single antigen bead (SAB) and IFN-γ ELISPOT assays, respectively. Also, the predominant alloantigen presenting pathway priming DST alloimmunity and the contribution of main alloreactive T-cell subsets were further characterized in vitro. Pretransplantation, 78/169 (46%) were DST+ whereas 91/169 (54%) DST-. At 2 years, 54/169 (32%) patients showed detectable DST responses: 23/54 (42%) dnDST and 31/54 (57%) persistently positive (persistDST+). 24/169 (14%) patients developed dnDSA. A strong correlation was observed between the three distinct molecular mismatch scores and they all accurately predicted dnDSA formation, in particular at the DQ locus. Likewise, HLA molecular incompatibility predicted the advent of dnDST, especially when assessed by PIRCHE-II score (OR 1.014 95% CI 1.001-1.03, p=0.04). While pretransplant DST predicted the development of posttransplant BPAR (OR 5.18, 95% CI=1.64-16.34, p=0.005) and particularly T cell mediated rejection (OR 5.33, 95% CI=1.45-19.66, p=0.012), patients developing dnDST were at significantly higher risk of subsequent dnDSA formation (HR 2.64, 95% CI=1.08-6.45, p=0.03). In vitro experiments showed that unlike preformed DST that is predominantly primed by CD8+ direct pathway T cells, posttransplant DST may also be activated by the indirect pathway of alloantigen presentation, and predominantly driven by CD4+ alloreactive T cells in an important proportion of patients. De novo donor-specific cellular alloreactivity seems to precede subsequent humoral alloimmune activation and is influenced by a poor donor/recipient HLA molecular matching.

Anonymity of organ donors under discussion.

The World Health Organization declares in its Guiding Principles on Human Cell, Tissue and Organ Transplantation that organizations must ensure that the personal anonymity and privacy of donors and recipients are always protected. Hence, most of the European transplantation programs forbid any form of direct contact between the family of the deceased donor and the transplant recipient. The anonymity is guarded to protect both parties against potential abuse, manipulation or financial pressure. However, legislation on anonymity of organ donation has come under discussion in the last few years, especially in Italy where the National Committee for Bioethics has recently positioned in favor of allowing organs donor families and transplant recipients to meet, if both parties wish to. Most donor families need to "complete" the biography of the deceased and make peace with their decision to consent to the donation; on the other hand, a considerable proportion of recipients wish to acknowledge the role that the donor and their family played in saving their lives and make peace with having a part of the donor sustaining their life, while not feeling guilty. Thus, a resilient model built on confidentiality, autonomy and freedom to make informed choice should be considered in those countries where a change in the transplantation law is currently debated.

Macrophage Overexpressing NGAL Ameliorated Kidney Fibrosis in the UUO Mice Model.

BACKGROUND/AIMS: Alternatively activated macrophages (AAM) have regenerative and anti-inflammatory characteristics. Here, we sought to evaluate whether AAM cell therapy reduces renal inflammation and fibrosis in the unilateral ureteral obstruction (UUO) mice model. METHODS: We stabilized macrophages by adenoviral vector NGAL (Neutrophil gelatinase-associated lipocalin-2) and infused them into UUO mice. To ascertain whether macrophages were capable of reaching the obstructed kidney, macrophages were stained and detected by in vivo cell tracking. RESULTS: We demonstrated that some infused macrophages reached the obstructed kidney and that infusion of macrophages overexpressing NGAL was associated with reduced kidney interstitial fibrosis and inflammation. This therapeutic effect was mainly associated with the phenotype and function preservation of the transferred macrophages isolated from the obstructed kidney Conclusions: Macrophage plasticity is a major hurdle for achieving macrophage therapy success in chronic nephropathies and could be overcome by transferring lipocalin-2.

Effector Antitumor and Regulatory T Cell Responses Influence the Development of Nonmelanoma Skin Cancer in Kidney Transplant Patients.

BACKGROUND: Chronic immunosuppression promotes nonmelanocytic squamous cell carcinoma (SCC) after kidney transplantation. Adaptive and innate immunity play a key role controlling tumor growth and are influenced by different immunosuppressive agents. We hypothesized that functional impairment of tumor-specific T cell responses due to calcineurin inhibitors (CNI) could contribute to SCC development, whereas conversion to mammalian target of rapamycin inhibitors (mTOR-i) could recover this protective immune response. METHODS: Peripheral tumor-specific T cell responses against main SCC-derived antigens using the IFN-γ enzyme-linked immunospot assay and intratumor (IT) and circulating immune phenotypes (CD4 + T, CD8 + T, CD20 + B, CD56 + NK, FOXP3 + regulatory T [Treg] cells) were explored in a cross-sectional analysis in 59 kidney transplant patients with SCC on CNI (KT-CNI-SCC) or mTOR-i (KT-mTORi-SCC), 25 nontransplants developing SCC (NoKT-SCC) and 6 healthy controls. Moreover, 25 KT-CNI-SCC were switched to mTOR-i and evaluated after 12 months. RESULTS: Kidney transplant patients showed lower IT infiltrates and tumor-specific T cell responses than NoKT-SCC, and intratumoral and circulating FOXP3 + Treg cells were higher in KT-mTORi-SCC (P < 0.05). Tumor-specific T cell responses were significantly lower in KT-CNI-SCC than KT-mTORi-SCC and NoKT-SCC and predicted SCC relapses (area under the curve = 0.837; P < 0.05). One-year after mTOR-i conversion, a significant increase in FOXP3 + Treg cell numbers and tumor-specific T cell responses were observed, reaching similar levels than KT-mTORi-SCC and NoKT-SCC patients. CONCLUSIONS: Tumor-specific T cell responses are strongly impaired in CNI-treated patients but recover after mTOR-i conversion, reducing SCC relapses.

Molecular and Functional Noninvasive Immune Monitoring in the ESCAPE Study for Prediction of Subclinical Renal Allograft Rejection.

BACKGROUND: Subclinical acute rejection (sc-AR) is a main cause for functional decline and kidney graft loss and may only be assessed through surveillance biopsies. METHODS: The predictive capacity of 2 novel noninvasive blood biomarkers, the transcriptional kidney Solid Organ Response Test (kSORT), and the IFN-γ enzyme-linked immunosorbent spot assay (ELISPOT) assay were assessed in the Evaluation of Sub-Clinical Acute rejection PrEdiction (ESCAPE) Study in 75 consecutive kidney transplants who received 6-month protocol biopsies. Both assays were run individually and in combination to optimize the use of these techniques to predict sc-AR risk. RESULTS: Subclinical acute rejection was observed in 22 (29.3%) patients (17 T cell-mediated subclinical rejection [sc-TCMR], 5 antibody-mediated subclinical rejection [sc-ABMR]), whereas 53 (70.7%) showed a noninjured, preserved (stable [STA]) parenchyma. High-risk (HR), low-risk, and indeterminate-risk kSORT scores were observed in 15 (20%), 50 (66.7%), and 10 (13.3%) patients, respectively. The ELISPOT assay was positive in 31 (41%) and negative in 44 (58.7%) patients. The kSORT assay showed high accuracy predicting sc-AR (specificity, 98%; positive predictive value 93%) (all sc-ABMR and 58% sc-TCMR showed HR-kSORT), whereas the ELISPOT showed high precision ruling out sc-TCMR (specificity = 70%, negative predictive value = 92.5%), but could not predict sc-ABMR, unlike kSORT. The predictive probabilities for sc-AR, sc-TCMR, and sc-ABMR were significantly higher when combining both biomarkers (area under the curve > 0.85, P < 0.001) and independently predicted the risk of 6-month sc-AR in a multivariate regression analysis. CONCLUSIONS: Combining a molecular and immune cell functional assay may help to identify HR patients for sc-AR, distinguishing between different driving alloimmune effector mechanisms.

Clinical approach to kidney disease in kidney recipients in Spain.

BACKGROUND AND OBJECTIVES: In the present study, clinical criteria used by Spanish nephrologists when approaching chronic kidney disease (CKD) in kidney recipients, as well as their level of maintenance and control of renal function, were evaluated. METHODS: An epidemiological, observational, multicenter, nation-wide, prospective study was carried out, with a 6-month follow-up period. Three hundred and sixty-eight adult patients with stage3 kidney disease after a 24-month or longer post-transplantation follow-up period were included. Visits schedule included a retrospective visit, a baseline visit, an optional mid-term visit, and a final visit at month6. RESULTS: Mean time since kidney transplantation was 8.2±5.4years. Most common pre-transplant cardiovascular risk factors were high blood pressure (80.2%), followed by high cholesterol levels (61.7%). Serum creatinine levels showed a statistically significant decrease from baseline visit to 6-month visit (0.06±0.22; P<.0001), and glomerular filtration rate (GFR) reduction was -1.03±6.14 (P=0.0014). Significant independent prognostic factors for GFR worsening were: higher 24-hour proteinuria (OR=1.001 per mg; P=.020), longer time since transplantation (OR=1.009 per month; P=.017), and lower hemoglobin levels (OR=1.261 per g/dl; P=.038). Donor age also had some negative influence (OR=1.021 per year; P=.106). Biopsies were obtained in only 8% of kidney transplant recipients with stage 3 CKD with an intervention being carried out in 25.4% of cases. CONCLUSIONS: Secondary markers and factors resulting in CKD progression, particularly anemia, are still frequently uncontrolled after kidney transplantation. Only about 2% of patients benefit from a therapeutic intervention based on a biopsy. Clinical perception differs from objective measures, which results in an obvious clinical inertia regarding risk factor control in such patients.

Actitud clínica frente a la disfunción renal en receptores de un trasplante renal en España / Clinical approach to kidney disease in kidney recipients in Spain

Antecedentes y objetivos: El presente estudio ha evaluado el criterio clínico que utilizan los nefrólogos españoles frente a la disfunción renal crónica (DRC) en receptores de trasplante renal (TR), y el grado de mantenimiento y control de la disfunción renal. Métodos: Estudio observacional, epidemiológico, multicéntrico, nacional y prospectivo, con un período de seguimiento de 6 meses. Se incluyeron 368 pacientes adultos con disfunción renal de grado3 con un período mínimo de evolución posterior al trasplante de 24meses. La programación de las visitas incluyó una visita retrospectiva, una visita inicial, una visita intermedia opcional y una visita final al sexto mes. Resultados: El tiempo medio desde el TR fue de 8,2±5,4años. La hipertensión (80,2%), seguida por la hipercolesterolemia (61,7%), fueron los factores de riesgo cardiovascular previos al trasplante más frecuentes. Las concentraciones de creatinina sérica entre la visita inicial y la visita de los 6meses mostraron una diferencia estadísticamente significativa de 0,06±0,22 (p<0,0001), y la diferencia del filtrado glomerular (FG) fue de −1,03±6,14 (p=0,0014). Los factores pronósticos independientes significativos del empeoramiento del FG fueron: proteinuria a 24h más alta (OR=1,001 por cada mg; p=0,020), más tiempo desde el trasplante (OR=1,009 por cada mes; p=0,017) y concentraciones bajas de hemoglobina (OR=1,261 por cada g/dl; p=0,038). También se observó cierta influencia negativa de la edad del donante (OR=1,021 por cada año; p=0,106). Solo se realizó biopsia en el 8% de los casos de receptores de TR con DRC de grado 3, suponiendo alguna intervención en el 25,4% de los casos. Conclusiones: Con frecuencia los marcadores secundarios y los factores de progresión de la DRC siguen sin estar controlados después del TR, principalmente la anemia. Solo aproximadamente el 2% de pacientes se benefician de una intervención terapéutica basada en una biopsia. Existe una disparidad entre la percepción clínica y los parámetros objetivos, que conduce a una clara inercia clínica del control de los factores de riesgo de estos pacientes (AU)

Background and objectives: In the present study, clinical criteria used by Spanish nephrologists when approaching chronic kidney disease (CKD) in kidney recipients, as well as their level of maintenance and control of renal function, were evaluated. Methods: An epidemiological, observational, multicenter, nation-wide, prospective study was carried out, with a 6-month follow-up period. Three hundred and sixty-eight adult patients with stage3 kidney disease after a 24-month or longer post-transplantation follow-up period were included. Visits schedule included a retrospective visit, a baseline visit, an optional mid-term visit, and a final visit at month6. Results: Mean time since kidney transplantation was 8.2±5.4years. Most common pre-transplant cardiovascular risk factors were high blood pressure (80.2%), followed by high cholesterol levels (61.7%). Serum creatinine levels showed a statistically significant decrease from baseline visit to 6-month visit (0.06±0.22; P<.0001), and glomerular filtration rate (GFR) reduction was −1.03±6.14 (P=0.0014). Significant independent prognostic factors for GFR worsening were: higher 24-hour proteinuria (OR=1.001 per mg; P=.020), longer time since transplantation (OR=1.009 per month; P=.017), and lower hemoglobin levels (OR=1.261 per g/dl; P=.038). Donor age also had some negative influence (OR=1.021 per year; P=.106). Biopsies were obtained in only 8% of kidney transplant recipients with stage 3 CKD with an intervention being carried out in 25.4% of cases. Conclusions: Secondary markers and factors resulting in CKD progression, particularly anemia, are still frequently uncontrolled after kidney transplantation. Only about 2% of patients benefit from a therapeutic intervention based on a biopsy. Clinical perception differs from objective measures, which results in an obvious clinical inertia regarding risk factor control in such patients (AU)

Measurement of ganciclovir concentration in human plasma by ultra-performance liquid chromatography-tandem mass spectrometry.

BACKGROUND: Ganciclovir/valganciclovir plays an important role in the treatment and prevention of cytomegalovirus disease after organ transplantation. MATERIAL AND METHODS: We developed and validated a simple chromatographic method by ultra-performance liquid chromatography tandem mass spectrometry to measure plasma concentration of ganciclovir in human plasma. Chromatographic separation was achieved using an Acquity(®) UPLC(®) BEH™ (2.1×50mm id, 1.7µm) reverse-phase C18 column, with a water/methanol linear gradient containing ammonium acetate/formic acid at a 0.4mL/min flow rate. Ganciclovir and its internal standard (acyclovir) were detected by electrospray ionization mass spectrometry in positive ion multiple reaction monitoring mode. RESULTS: The limit of detection and quantification were 0.03 and 0.06mg/L, respectively, and linearity was observed between 0.06 and 30.0mg/L. Intra-day and day-to-day coefficients of variation and relative biases ranged from 3.6 to 5.4%, 4.2 to 6.2%, -2.6 to -1.1% and -4.0 to -2.8%, respectively. Recovery values were greater than 81.9%. Evaluation of the matrix effect showed ion suppression for ganciclovir and acyclovir. No carry-over was observed. CONCLUSIONS: The validated method is useful for both therapeutic drug monitoring and pharmacokinetic studies. It could be applied to the daily clinical laboratory practice to measure the concentration of ganciclovir in human plasma.

A randomized trial of basiliximab with three different patterns of cyclosporin A initiation in renal transplant from expanded criteria donors and at high risk of delayed graft function.

This study assays therapy with basiliximab and different patterns of cyclosporin A (CsA) initiation in renal transplant (RT) recipients from expanded criteria donors (ECD) and at high risk of delayed graft function (DGF). A multicentre six-month open-label randomized trial with three parallel groups treated with basiliximab plus steroids, mycophenolate mofetil and different patterns of CsA initiation: early within 24 h post-RT at 3 mg/kg/d (Group 1; n = 38), and at 5 mg/kg/d (Group 2; n = 40), or delayed after 7-10 d at 5 mg/kg/d (Group 3; n = 36). There were no differences among groups in six months GFR (43.1 +/- 12, 48.0 +/- 14 and 47.2 +/- 17 mL/min, respectively), DGF (Group 1: 31%, Group 2: 37%, Group 3: 42%), nor biopsy-proven acute rejection, although clinically treated and biopsy-proven acute rejection was significantly higher in Group 3 (25%) vs. Group 1 (5.3%, p < 0.05). At six months no differences were observed in death-censored graft survival or patient survival. Induction therapy with basiliximab and three CsA-ME initiation patterns in RT recipients from ECD and at high risk of DGF presented good renal function and graft survival at six months. Late onset group did not achieve improvement in DGF rate and showed a higher incidence of clinically treated and biopsy-proven acute rejection.

Evaluation of pre-implantation kidney biopsies: comparison of Banff criteria to a morphometric approach.

BACKGROUND: Donor glomerulosclerosis, interstitial fibrosis, and fibrous intimal thickening correlate with graft outcome. We evaluate chronic lesions in donor biopsies according to Banff criteria and with a morphometric technique to ascertain their predictive value on graft outcome. METHODS: We evaluated 77 cadaveric donor biopsies according to Banff criteria. Glomerulosclerosis was expressed as the percentage of global sclerotic glomeruli. The following morphometric parameters were obtained: cortical interstitial volume fraction (Vvint/c), cortical glomerular volume fraction (Vvglom/c), mean glomerular volume (Vg), mean and maximal intimal arterial volume fraction (Vvintima/art), and Vvintima/art of the largest artery. We evaluated the correlation of histologic lesions with delayed graft function, 3 months' glomerular filtration rate (GFR), and death-censored graft survival. RESULTS: Multivariate logistic regression showed that delayed graft function was associated with cv score [relative risk (RR) 4.2 and 95% CI 1.1 to 16.0) and glomerulosclerosis (RR 1.06 and 95% CI 1.01 to 1.13). Stepwise regression showed that Vvint/c and glomerulosclerosis were independent predictors of 3 months' GFR (R= 0.62, P= 0.0001). Repeated analysis not considering morphometric parameters showed that glomerulosclerosis, cv score and ci score were independent predictors of 3 months' GFR (R= 0.64, P= 0.0001). A donor chronic damage score was generated considering glomerulosclerosis, cv score and ci score. This score after adjusting for clinical variables was associated with 3 months' GFR (R= 0.71, P < 0.0001) and death-censored graft survival (RR 2.2 and 95% CI 1.3 to 3.7). CONCLUSION: Combined evaluation of donor glomerulosclerosis, chronic vascular and interstitial damage according to Banff criteria allows a precise prediction of graft outcome. Morphometric evaluation of donor biopsies does not improve the predictive value of semiquantitative grading.

Pilot randomized study of early tacrolimus withdrawal from a regimen with sirolimus plus tacrolimus in kidney transplantation.

We performed a randomized trial to compare two regimens of low-risk kidney allograft recipients in the first year after transplantation. Both regimens initially included sirolimus, tacrolimus and steroids; one with long-term maintenance with these drugs vs. tacrolimus withdrawal. Group I: sirolimus levels of 4-8 ng/mL, plus tacrolimus 8-12 ng/mL for 3 months, and 5-10 ng/mL after month 3. Group II: sirolimus concentration of 8-16 ng/mL, plus tacrolimus 3-8 ng/mL with tacrolimus elimination from month 3 onwards. Owing to difficulties in achieving target levels, the protocol was amended to increase the doses. Eighty-seven patients were recruited. In the intention-to-treat analysis, glomerular filtration rate (GFR) at 12 months, adjusted to zero for graft loss, was similar in both groups (58.8 and 59.9 mL/min). Analysis of patients remaining on protocol showed that GFR was higher in group II only in the patients postamendment (58.4 and 72.9 mL/min, p = 0.03). Rates of biopsy-confirmed rejection (BCAR) were 9.3% and 22.7% in groups I and II, respectively (p = NS). After amendment, BCAR rates were 10.3% and 11.1% (p = NS). Diastolic blood pressure was significantly lower in patients who eliminated tacrolimus (80.4 vs. 75.6 mmHg) (p = 0.03). Combining sirolimus and tacrolimus with adequate loading doses was associated with a low incidence of BCAR, and allowed tacrolimus elimination in a high proportion of patients, which may be followed by amelioration in renal function and blood pressure.

Angiotensin converting enzyme genotype and chronic allograft nephropathy in protocol biopsies.

Genotype DD of the angiotensin-converting enzyme (ACE) is not associated with an increased incidence of native renal diseases, although it could modulate progression to renal failure in patients who already display chronic lesions. Because its role in renal allograft degeneration is not well characterized, whether ACE genotype was associated with the prevalence of chronic allograft nephropathy (CAN) was studied, in a group of protocol biopsies from 180 patients, or with the incidence of CAN in 152 patients with at least two sequential biopsies. As a control group, ACE genotype was also studied in 41 donors and 72 healthy subjects. For analyzing the influence of ACE genotype in graft survival, patients were grouped into six categories (II-normal biopsy, ID-normal, DD-normal, II-CAN, ID-CAN and DD-CAN). Finally, relative renal ACE mRNA levels were measured in 67 cases by real-time PCR using the delta threshold cycle method. ACE-DD genotype was more frequent in patients who received a transplant than in control subjects (43.3% versus 30.1%, P = 0.026), but prevalence (DD = 42.7% versus non-DD = 42.2%) or incidence (DD = 24.6% versus non-DD = 29.9%) of CAN was not different regarding recipient ACE genotype. Furthermore, patients with the ACE-DD genotype and CAN had the poorest graft survival (II-normal = 100%, ID-normal = 91%, DD-normal = 84%, II-CAN = 100%, ID-CAN = 66%, and DD-CAN = 36%; P = 0.034) and higher ACE mRNA levels than non-DD and CAN (DD = -3.36 +/- 2.35 versus non-DD = -5.65 +/- 1.72-fold in ACE copies; P = 0.012). It is concluded that ACE-DD genotype is not associated with an increased prevalence or incidence of CAN but is actually associated with higher ACE mRNA levels and poorer graft survival in patients who already display CAN.

Splicing alterations in human renal allografts: detection of a new splice variant of protein kinase Par1/Emk1 whose expression is associated with an increase of inflammation in protocol biopsies of transplanted patients.

Protein kinase Emk1/Par1 (GenBank accession no. X97630) has been identified as a regulator of the immune system homeostasis. Since immunological factors are critical for the development of chronic allograft nephropathy (CAN), we reasoned that expression of Par1/Emk1 could be altered in kidney allografts undergoing CAN. In this paper, we have analysed the association among renal allograft lesions and expression of Par1/Emk1, studied by RT-PCR on total RNA from 51 protocol biopsies of transplanted kidneys, five normal kidneys, and five dysfunctional allografts. The most significant result obtained has been the detection of alterations in the normal pattern of alternative splicing of the Par1/Emk1 transcript, alterations that included loss of expression of constitutively expressed isoforms, and the inclusion of a cryptic exon to generate a new Emk1 isoform (Emk1C). Expression of Emk1C was associated with an increase in the extension of the interstitial infiltrate (0.88+/-0.33 in Emk1C([+]) vs. 0.41+/-0.50 in Emk1C([-]); P<0.011), and with a trend to display higher interstitial scarring (0.66+/-0.70 vs. 0.29+/-0.52; P=0.09) in protocol biopsies when evaluated according to the Banff schema. Moreover, a higher mean arterial pressure (MAP) was also observed (110+/-11 vs. 99+/-11 mm Hg; P=0.012). From these results we propose that Par1/Emk1 could have a role in the development of CAN in kidney allografts.