New Strategies for Non-invasive Detection of Chronic Allograft Nephropathy
Condition: Chronic Rejection
Study type: Observational

Summary: 
      The purpose of this study is to help researchers find out more about a condition called
      "Chronic Allograft Nephropathy" (CAN). CAN is a complication that sometimes occurs after
      kidney transplantation and affects the function of the transplanted kidney. It is hoped that
      by studying blood, urine, and tissue samples of kidney transplant patients, new ways of
      diagnosing and treating CAN may be found.
    

Detailed description: 
      We hypothesize that patients with CAN will manifest different patterns of gene expression
      profiling (compared with non-CAN controls), detectable in peripheral blood mononuclear cells
      (PBMCs) and urine cells. By studying differences that exist in peripheral blood or in the
      graft and its local environment (urine samples), informative biomarkers may be discovered to
      improve treatment programs.

      Specific Aims:

      AIM 1: To investigate the genes involved in the molecular mechanism that characterize CAN in
      kidney transplant recipients, we will study gene expression in kidney biopsy samples from
      patients with CAN. For comparison analysis, we will study gene expression profiling in normal
      kidney biopsies (Control Group 1).

      AIM 2: To determine whether there is a role for assessing peripheral blood mononuclear cell
      (PBMC) and urine cell gene expression patterns as a means of measuring the immunologic and
      clinical status of kidney transplant recipients, we will compare gene expression profiles
      among PBMC, urine and biopsy samples.

      AIM 3: To establish the clinical utility of the set of markers resulting from AIMS 1 and 2 in
      the diagnostic of CAN, we will use "real time"(RT)-PCR in PBMC and urine samples employing a
      limited panel of markers in an independent group of patients.

      AIM 4: To study the role of the pre-established panel of molecular markers in the early
      detection of CAN in patients receiving mycophenolate mofetil (MMF) as immunosuppressive
      treatment, we will study urine and PBMC samples from patients more than 12 months
      post-transplantation, with normal creatinine, and proteinuria.

      Background and Significance:

      Chronic allograft nephropathy (CAN) is a devastating complication of kidney transplantation
      that is responsible for a significant proportion of graft loss (1-5). Although currently
      available immunosuppressive therapy is increasingly effective against acute rejection of a
      transplanted organ, chronic rejection (CR) remains difficult to treat. Nevertheless, a
      relevant proportion of patients with CR of a kidney transplant maintain satisfactory organ
      function for a long time. CAN is characterized by a progressive decline in kidney function,
      which is not attributable to a specific cause (6-8). CAN is a poorly known complex process
      resulting from as yet undefined etiology with both immune and non-immune components (8).
      Alloantigen-dependent causes of CAN include acute rejection episodes, particularly multiple,
      late, or vascular acute rejection episodes (9-13). With the use of a new immunosuppressive
      regimen, the frequency of acute rejection episodes has become reduced, which may possibly
      have an influence on future development of CAN. However, donor antigen-independent factors
      also play a role in the occurrence and progression of CAN. This includes donor-related
      factors such as donor age and number of mismatch, which have an impact on late graft outcome
      (13). Donation-related circumstances, such as prolonged cold ischemia time, are associated
      with delayed graft function and related to poor late renal transplant results.

      Recipient related factors may also act as co-factors contributing to an accelerated
      deterioration of the functional units in the kidney, including vascular, of renal arteries
      and a subsequent relative hypoxia of the kidney, frequently leading to interstitial fibrosis.
      Interestingly, many of the pathways involved in chronic injury and fibrosis are regulated
      very early in the course of the injury, when the downstream effects of these alterations are
      still not evident by pathology.

      The histopathologic features of CAN can be characterized by proliferative changes of the
      vascular wall, in which smooth muscle cells and parenchymal cells proliferate, leading to a
      narrowing of renal arteries, which may contribute to a relative hypoxia of the tissue.
      Subsequently, fibroblasts will contribute to fibrotic scarring in the vascular wall. CAN is
      also characterized by the occurrence of fibrosis in the interstitial tissue along with
      tubular atrophy (14). The glomeruli are also often affected by various degrees of
      glomerulosclerosis. An inflammatory infiltrate is frequently found and consists of invading
      lymphocytes and monocytes-macrophages into virtually all affected compartments of the kidney.
      The inflammatory infiltrate observed in CAN can be induced by both antigen-dependent and
      antigen-independent factors. These histopathologic changes can be graded according to the
      Banff system (14), which gives a score of the degree of CAN. These histopathologic changes
      act in concert, contributing to a gradual progression of renal allograft dysfunction.

      Fundamental studies in solid organ transplantation have characterized specific functional
      roles for individual genes in the immunological cascade leading to organ rejection.
      Nevertheless, significant redundancy in the immune system is suggested by clinical
      variability in acute rejection outcomes, in the progression of chronic rejection, and by the
      potential to acquire graft tolerance. Underlying all of these complex systems are concordant
      expression and co-regulation of multiple genes in known and novel molecular pathways.
      Ascertaining their individual functions in adapting the effector response is now possible
      with gene expression profiling using microarrays. Although cDNA microarrays (large numbers of
      genes represented by complementary DNAs or oligonucleotides immobilized onto microscope
      slides or silicon chips) were successfully used to profile gene expression in cancer
      (15,16,17,18,19), their impact in transplantation research has so far been relatively modest.

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      There is accumulating evidence that non-invasive immune monitoring may be useful in the early
      period after renal transplant (20,21), particularly with regard to predicting the presence of
      acute rejection. It is less clear whether chronic allograft nephropathy (CAN) is also
      associated with consistent changes in peripheral blood or urine cells.

      Moreover, current methods of diagnosing allograft dysfunction are inadequate for detecting
      the significant organ damage that occurs prior to the establishment of clinical
      manifestations. The development of assays or novel technologies that will allow detection of
      allograft dysfunction/rejection, monitor responses to therapy, and predict long-term outcomes
      is vital for the success of transplantation.

      In this project we propose to establish the molecular markers that characterize CAN using
      gene expression profiling in kidney biopsies of patients with CAN and to relate these
      findings with the profiles in PBMC and urine samples from the same patients. In addition, we
      will establish and validate (in a independent group of patients) the clinical utility of a
      limited set of molecular markers, resulting of the microarray analysis, using RT-PCR.

      V. Statistical Analysis:

      Data Security Plan Kidney tissue, PBMC and urine from kidney transplant patients with or
      without CAN will be studied. A Secure Data Base containing all the demographic information
      for the patients will be kept at the Virginia Commonwealth University, but patient
      identifiers will be removed and a study number assigned to each patient. The tissues required
      will include kidney biopsies only from patients with CAN.

      Human Subjects:

      A. Description and Research Materials Kidney tissues will be obtained with informed consent
      from patients with clinical suspicion of CAN (at least 15 patients). Tissue samples (needle
      biopsies) will be snap-frozen in liquid nitrogen within 5 minutes after excision and shipped
      under appropriate conditions, on dry ice, to maintain specimen integrity. All samples will be
      submitted for pathological evaluation, and a pathology report and clinical summary will be
      sent with the samples in all cases in a manner insuring patient confidentiality by removing
      patient identifiers and labeling with the assigned patient number.

      Peripheral blood and urine samples (1-2 tbs) will be obtained from patients at the biopsy
      time. Kidney biopsies will be obtained with informed consent from normal kidneys at
      transplantation (needle biopsy from donor kidney, N=10).

      Peripheral blood and urine samples will be collected monthly during 1 year with informed
      consent from kidney transplant patients with diagnostic of CAN confirmed by biopsy. As
      Control Group, stable long-term transplant patients (more than 12 months post-TX) with
      creatinine levels <1.6 mg/dL, and proteinuria less than 500mg/24hs, will be studied. (You may
      want to give an estimated number of patients to be included in the Control Group) All
      practices dealing with human subjects will be performed according to the ethical guidelines
      of the Institutional Review Board for the Protection of Human Subjects of the Virginia
      Commonwealth University.

      Recruitment Plan Patients seen at VCU Hume-Lee Transplant Clinic who qualify according to the
      inclusion/exclusion criteria will be asked to participate in this study.

      B. Potential Risks When blood is drawn there is a risk of bruising, bleeding, swelling or
      infection at the site where the blood is drawn.

      Kidney biopsies can be painful where the needle enters the body, and a bruise can form.
      Bleeding can occur where the needle is inserted in to the kidney. Blood can appear in the
      urine.

      Every effort will be made to maintain confidentiality. All identifying information will be
      kept in a secure data bank with access limited to research personnel.

      C. Risk Reduction The tissue being obtained upon tissue biopsy is part of the sample that
      would be obtained for diagnostic purposes and not specifically for this protocol. The blood
      samples will be obtained at the same time as venous access is already in place. A urine
      sample will be collected prior to the biopsy.

      All staff members handling tissue, PBMC and urine samples have been trained and are qualified
      to safely handle HCV infected tissue.

      D. Risk/Benefit Because this is not a treatment study, there are no specific risks to the
      study subjects. CAN will be diagnosed and treated in the same way, whether or not they
      participate in the study. There is no guarantee that patients will benefit from participation
      in this study. The knowledge gained may be invaluable in the future particularly for
      diagnosing of CAN.

      D. Compensation Plan Subjects will not be compensated for their participation in this study.
      E. Consent Issues Patients who meet study requirements will be consented.
    

Observational model: Cohort
Time perspective: Prospective
Sampling method: Probability Sample
Study population: 
        Patients Diagnosis of Chronic Allograft Nephropathy at VCUHS Clinic
      
Biospecimen description: 
      Blood, Urine
    
Keywords: Renal Transplantation; Immunosuppression; Chronic Rejection