Luis F. Quintana

Application of Label-free Quantitative Peptidomics for the Identification of Urinary Biomarkers of Kidney Chronic Allograft Dysfunction

The advent of quantitative proteomics opens new opportunities in biomedical and clinical research. Although quantitative proteomics methods based on stable isotope labeling are in general preferred for biomolecular research, biomarker discovery is a case example of a biomedical problem that may be better addressed by using label-free MS techniques. As a proof of concept of this paradigm, we report the use of label-free quantitative LC-MS to profile the urinary peptidome of kidney chronic allograft dysfunction (CAD). The aim was to identify predictive biomarkers that could be used to personalize immunosuppressive therapies for kidney transplant patients. We detected (by LC-M/MS) and quantified (by LC-MS) 6000 polypeptide ions in undigested urine specimens across 39 CAD patients and 32 control individuals. Although unsupervised hierarchical clustering differentiated between the groups when including all the identified peptides, specific peptides derived from uromodulin and kininogen were found to be significantly more abundant in control than in CAD patients and correctly identified the two groups. These peptides are therefore potential biomarkers that might be used for the diagnosis of CAD. In addition, ions at m/z 645.59 and m/z 642.61 were able to differentiate between patients with different forms of CAD with specificities and sensitivities of 90% in a training set and, significantly, of ∼70% in an independent validation set of samples. Interestingly low expression of uromodulin at m/z 638.03 coupled with high expression of m/z 642.61 diagnosed CAD in virtually all cases. Multiple reaction monitoring experiments further validated the results, illustrating the power of our label-free quantitative LC-MS approach for obtaining quantitative profiles of urinary polypeptides in a rapid, comprehensive, and precise fashion and for biomarker discovery.

Urine Proteomics to Detect Biomarkers for Chronic Allograft Dysfunction

Despite optimal immunosuppressive therapy, more than 50% of kidney transplants fail because of chronic allograft dysfunction. A noninvasive means to diagnose chronic allograft dysfunction may allow earlier interventions that could improve graft half-life. In this proof-of-concept study, we used mass spectrometry to analyze differences in the urinary polypeptide patterns of 32 patients with chronic allograft dysfunction (14 with pure interstitial fibrosis and tubular atrophy and 18 with chronic active antibody-mediated rejection) and 18 control subjects (eight stable recipients and 10 healthy control subjects). Unsupervised hierarchical clustering showed good segregation of samples in groups corresponding mainly to the four biomedical conditions. Moreover, the composition of the proteome of the pure interstitial fibrosis and tubular atrophy group differed from that of the chronic active antibody-mediated rejection group, and an independent validation set confirmed these results. The 14 protein ions that best discriminated between these two groups correctly identified 100% of the patients with pure interstitial fibrosis and tubular atrophy and 100% of the patients with chronic active antibody-mediated rejection. In summary, this study establishes a pattern for two histologic lesions associated with distinct graft outcomes and constitutes a first step to designing a specific, noninvasive diagnostic tool for chronic allograft dysfunction.

Effect of mTOR inhibitor on body weight: from an experimental rat model to human transplant patients

The aim was to study the influence of sirolimus (SRL) on body weight in a rat model and in kidney transplant patients. Wistar rats (15 weeks old) were either treated with vehicle (VEH; n = 8) or SRL (n = 7) 1.0 mg/kg three times per week for 12 weeks. Body mass and food intake were measured weekly. Adipocyte diameter was determined in hematoxylin–eosin stains. The body mass index (BMI) obtained from clinical kidney transplant trials comparing SRL‐based with cyclosporine‐based therapy was analyzed. Animals: SRL produced a decrease of the weight gain curve. At the end of the study, mean body weight in the SRL group was lower than in the VEH group (356 vs. 507 g, P < 0.01) in spite of comparable food intake normalized for body weight was not different. Mean adipocyte diameter was 36 μm in VEH and 25 μm in SRL rats (P = 0.009). Mean SRL blood trough concentration was 38 ng/ml. Kidney transplant patients: Two years after transplantation, BMI was significantly lower in the SRL‐based treatment arm compared to cyclosporine (24.17 ± 2.99 vs. 25.97 ± 5.01 kg/m2, P = 0.031). SRL treatment leads to less body mass. Adipocyte cell diameter was reduced in SRL‐treated animals. A possible explanation may be the effects of SRL on metabolic regulation and cell growth.

Two-dimensional Difference Gel Electrophoresis Urinary Proteomic Profile in the Search of Nonimmune Chronic Allograft Dysfunction Biomarkers

Introduction. Despite advances in therapeutics, graft loss associated with chronic allograft dysfunction (CAD) remains high. Urinary proteomic analysis is a noninvasive method that could be used to detect and evaluate CAD in renal transplant recipients. This study was aimed to establish the normal proteome map of stable transplant patients and to validate the utility of two-dimensional difference gel electrophoresis (2DE-DIGE) in identifying new candidates as urinary biomarkers of CAD. Methods. Morning spot urine samples that were collected from kidney transplant recipients with biopsy-proven interstitial fibrosis and tubular atrophy (IFTA) stages 0-I-II/III (n=8/group) under immunosuppressive treatment with tacrolimus plus mycophenolate with or without prednisone. 2DE silver staining and mass spectrometry analyses were used to establish the normal proteome map, and 2DE-DIGE and mass spectrometry were used to identify proteins exhibiting differential abundance. Results and Conclusions. This study defines the normal proteome of stable renal transplant patients, which is composed of several plasma proteins, as well as of immunologic proteins that are probably specific to transplant recipients. The 2DE-DIGE study showed 19 proteins with differential concentrations, depending on the IFTA histologic score. These 19 proteins could be used as urinary biomarkers of the severity of IFTA in renal transplant recipients.

B-cell depleting agents for ANCA vasculitides: a new therapeutic approach.

Vasculitides associated with anti-neutrophil cytoplasmic antibody (ANCA) serum positivity affecting small and medium-sized vessels are defined as ANCA-associated vasculitis (AAV). Glomerulonephritis in AAV is characterized by focal necrosis, crescent formation, and few or no immunoglobulin deposits. In vitro and animal evidence suggests that ANCA play a pathogenic role in AAV. Specific gene expression signatures are reported to predict long-term prognosis in AAV, suggesting the possibility of individualizing therapy and identifying new therapeutic targets. Although immunosuppressants and glucocorticoids are the cornerstone of AAV therapy, results from two recent randomized controlled trials have shown the non-inferiority of rituximab, compared with cyclophosphamide, for the induction of remission in patients with severe AAV. In fact, in April 2011, the US Food and Drug Administration (FDA) approved rituximab, combined with glucocorticoids, as a front-line therapy for adult patients with granulomatosis with polyangiitis (GPA; Wegeners granulomatosis) or microscopic polyangiitis. This new indication for rituximab provided the first ever FDA-approved therapy for these two diseases and the first alternative to cyclophosphamide for the treatment of severe disease in almost 40years. However, issues regarding the use of maintenance therapy after rituximab, the concurrent use of cyclophosphamide and the toxicity of rituximab remain unanswered and should be clarified in ongoing and future randomized controlled trials.

Efficacy of plasma exchange and immunoadsorption in systemic lupus erythematosus and antiphospholipid syndrome: A systematic review

Extracorporeal treatments have been used since the 1970s in the management of systemic lupus erythematosus (SLE). A randomised controlled trial comparing the efficacy of standard of care (SOC) combined with plasma exchange against SOC alone in patients with lupus nephritis revealed no difference in terms of renal outcome. Subsequently, initial expectations have been dampened and further experience with plasma exchange is mainly limited to observational studies and single case reports. Beneficial effects have been reported in patients with refractory disease course or in pregnancy with prior complications due to SLE and antiphospholipid syndrome. A more specific form of extracorporeal treatment, immunoadsorption (IAS), has emerged as a valuable option in the treatment of SLE. In line with the plasma exchange experience, IAS seems to have beneficial effects in patients with refractory disease, contraindications to standard immunosuppression or during pregnancy. The mechanism IAS relates to autoantibody removal but for plasma exchange removal of activated complement components, coagulation factors, cytokines and microparticles may also be relevant. Both treatment forms have good safety profiles although reactions to blood product replacement in plasma exchange and procedure related complications such as bleeding or catheter-related infections have occurred. There is a need to more clearly define the clinical utility of plasma exchange and IAS in refractory lupus and APS subgroups.

Antiphospholipase A2 Receptor Antibody Levels Predict the Risk of Posttransplantation Recurrence of Membranous Nephropathy.

Background Secretory phospholipase A2 receptor (PLA2R) is the target antigen of the auto-antibodies produced in most (∼70%) patients with primary membranous nephropathy (pMN). The applicability of anti-PLA2R1 antibody monitoring for the prediction of MN recurrence in kidney transplant recipients still is a matter of debate. Methods We sought to characterize the presence and concentration of anti-PLA2R antibodies by enzyme-linked immunosorbent assay (ELISA) in a cohort of 21 patients with pMN before and after transplantation to evaluate whether anti-PLA2R concentrations could predict pMN recurrence. Results The presence of pMN recurrence was significantly correlated with the existence of a positive ELISA assay at graft biopsy or with high level of anti-PLA2R1 activity before transplantation (P = 0.03). In the receiver operating characteristic analysis, anti-PLA2R levels (cut-off of 45 U/mL) during the pretransplantation period accurately predicted pMN recurrence, with a sensitivity of 85.3%, specificity of 85.1%, negative predictive value of 92%, and an area under the curve of 90.8%. This finding supports the hypothesis that anti-PLA2R cause pMN recurrence in humans and indicates the need to prove in an experimental model. Furthermore, 6 of 7 patients with recurrence were carriers of HLA DQA1* 05:01/05 and DQB1* 02:01, confirming these DQ alleles as those associated with higher anti-PLA2R levels. Conclusions This study is the first to demonstrate pretransplantation circulating anti-PLA2R antibodies in a cohort of renal transplant recipients who prospectively developed recurrent disease. Currently, anti-PLA2R levels measured by ELISA may be a rational tool to establish the risk of MN recurrence in renal allograft recipients.

Elevated factor H–related protein 1 and factor H pathogenic variants decrease complement regulation in IgA nephropathy

IgA nephropathy (IgAN), a frequent cause of chronic kidney disease worldwide, is characterized by mesangial deposition of galactose-deficient IgA1-containing immune complexes. Complement involvement in IgAN pathogenesis is suggested by the glomerular deposition of complement components and the strong protection from IgAN development conferred by the deletion of the CFHR3 and CFHR1 genes (ΔCFHR3-CFHR1). Here we searched for correlations between clinical progression and levels of factor H (FH) and FH-related protein 1 (FHR-1) using well-characterized patient cohorts consisting of 112 patients with IgAN, 46 with non-complement-related autosomal dominant polycystic kidney disease (ADPKD), and 76 control individuals. Patients with either IgAN or ADPKD presented normal FH but abnormally elevated FHR-1 levels and FHR-1/FH ratios compared to control individuals. Highest FHR-1 levels and FHR-1/FH ratios are found in patients with IgAN with disease progression and in patients with ADPKD who have reached chronic kidney disease, suggesting that renal function impairment elevates the FHR-1/FH ratio, which may increase FHR-1/FH competition for activated C3 fragments. Interestingly, ΔCFHR3-CFHR1 homozygotes are protected from IgAN, but not from ADPKD, and we found five IgAN patients with low FH carrying CFH or CFI pathogenic variants. These data support a decreased FH activity in IgAN due to increased FHR-1/FH competition or pathogenic CFH variants. They also suggest that alternative pathway complement activation in patients with IgAN, initially triggered by galactose-deficient IgA1-containing immune complexes, may exacerbate in a vicious circle as renal function deterioration increase FHR-1 levels. Thus, a role of FHR-1 in IgAN pathogenesis is to compete with complement regulation by FH.

Eculizumab in pregnancy-associated atypical hemolytic uremic syndrome: insights for optimizing management

Pregnancy-associated atypical hemolytic uremic syndrome is a systemic disease associated with high morbidity and mortality rates, caused by dysregulation of the alternative complement pathway, leading to uncontrolled complement activation resulting in thrombotic microangiopathy. This condition can be effectively treated by anti-C5 therapy, which controls complement activation. Treatment can be safely discontinued after complete remission and resolution of the precipitating cause, especially in patients with a low-risk genetic profile.

Urine Proteomics Biomarkers in Renal Transplantation: An Overview

A major goal of clinical proteomics was to identify biomarkers that can aid in the diagnosis and prognosis of different conditions. These biomarkers will not only assist the clinician in the diagnosis of a disease but they will also give directions as to which therapy may be more appropriate for each patient, thus contributing to the development of personalized medicine. This review discusses the current concepts in urine proteomics aimed at identifying predictive biomarkers that could detect the presence of acute rejection or chronic allograft dysfunction early on and for instance be used to personalize immunosuppressive therapies for kidney transplant patients.