H. Haller

Protocol biopsies in renal transplantation: Insights into patient management and pathogenesis

A 1‐day symposium on the application of protocol biopsies in renal transplantation was held in Boston, 21 July 2006. Representatives from centers with extensive experience in the use of protocol biopsies for routine patient care and research reported results on the pathological findings and their value in patient management. The consensus was that protocol biopsies, in experienced hands, are a safe and valuable means of detecting subclinical disease that can benefit from modification of therapy. Furthermore, molecular studies reveal evidence of activity or progression not readily appreciated by histological techniques. Wider application is expected in multicenter clinical trials to predict and validate outcomes. The principal barrier to wider use of protocol biopsies is knowledge of the benefits of intervention.

Lymphatic Neoangiogenesis in Human Renal Allografts: Results from Sequential Protocol Biopsies

Neoangiogenesis of lymphatic vessels may be important for the cellular immune response in renal transplants. To determine the prevalence and chronology of lymph vessel proliferation and its relation to cellular infiltrates and allograft function, we analyzed sequential protocol biopsies (n = 162), taken at 6, 12 and 26 weeks after transplantation. Biopsies were stained with an antibody against podoplanin and lymphatic vessel density was quantified per square millimeter.

The Molecular Phenotype of 6-Week Protocol Biopsies from Human Renal Allografts: Reflections of Prior Injury but Not Future Course

We assessed the molecular phenotype of 107 6‐week protocol biopsies from human renal allografts, using Affymetrix microarrays. Transcript changes were summarized as nonoverlapping pathogenesis‐based transcript sets (PBTs) reflecting inflammation (T cells, macrophages, IFNG effects) and the injury–repair response of the parenchyma, stroma and microcirculation‐increased (‘injury‐up’) and decreased (‘injury‐down’) transcripts. The molecular changes were highly correlated with each other, even when all rejection and borderline cases were excluded. Inflammation and injury‐down PBTs correlated with histologic inflammation and tubulitis, and the inflammation transcripts were greater in kidneys diagnosed as T cell‐mediated or borderline rejection. Injury‐up PBTs did not correlate with histopathology but did correlate with kidney function: thus functional disturbances are represented in transcript changes but not in histopathology. PBT changes correlated with prior delayed graft function. However, there was little difference between live donor kidneys and deceased donor kidneys that had not shown delayed graft function. Molecular changes did not predict future biopsies for clinical indications, rejection episodes, functional deterioration or allograft loss. Thus while detecting T cell‐mediated inflammation, the molecular phenotype of early protocol biopsies mostly reflects the injury–repair response to implantation stresses, and has little relationship to future events and outcomes.

Acute Tubular Injury in Protocol Biopsies of Renal Grafts: Prevalence, Associated Factors and Effect on Long-Term Function

Acute tubular injury (ATI) is commonly observed in renal allografts, especially early after transplantation. This study analyzes prevalence and associated clinical conditions of ATI in serial protocol biopsies (pBx) and indication biopsies (iBx), and its impact on long‐term graft function.

Course and Relevance of Arteriovenous Fistulas After Renal Transplant Biopsies

Arteriovenous fistulas (AVFs) after renal transplant biopsy are considered harmless. However, verification of the clinical course has not been thoroughly documented. We evaluated the data of our outpatient renal transplant biopsy program regarding the clinical course of AVFs after 2824 biopsies since 2000. We also reviewed all selective renal transplant embolizations. AVFs were the most frequent biopsy complications (8.3%). Seventy‐seven percent of AVFs disappeared spontaneously. Renal function in patients with AVFs was not different compared to those without during 2 years of observation. There were no differences in AVFs comparing protocol or indication biopsies, needle size, the time after transplantation, the use of acetylic salicylic acid or serum‐creatinine at biopsy. Living or younger donors were less likely to get postbiopsy AVFs. Ten embolizations were performed. Only one patient was from our outpatient biopsy program. Nine others were biopsied as inpatients in the course of complications during 6 weeks after transplant. Six of nine successfully embolized patients profited with improvement of renal function. Large AVFs occur most commonly shortly after transplantation in patients with poor graft function. There is no established test predicting which patient will benefit from embolization; however, Doppler‐determined resistive index may help in this regard.

CXCL13 as a Novel Marker for Diagnosis and Disease Monitoring in Pediatric PTLD

Posttransplant lymphoproliferative disease (PTLD) is a severe complication of immunosuppressive treatment in organ‐grafted children. Early diagnosis of PTLD is hampered by both unspecific clinical symptoms and lack of easy accessible markers. The homeostatic chemokine CXCL13, which plays a crucial role in B‐cell homing and lymphoid organ development, is expressed in some lymphomatous diseases. This study aims to investigate whether serum CXCL13 (sCXCL13) levels correlate with occurrence and regression of PTLD in pediatric solid‐organ graft recipients. Serum samples from PTLD patients (n = 21), patients with Epstein–Barr virus (EBV) reactivation (n = 18), and healthy age‐matched controls (n = 19) were tested for CXCL13 using a commercially available ELISA kit. sCXCL13 levels were significantly higher in PTLD patients than in healthy children. PTLD patients had also higher sCXCL13 values than pediatric solid‐organ recipients with EBV reactivation. An increase in sCXCL13 levels was observed from EBV reactivation to PTLD diagnosis in most cases. Elevated sCXCL13 levels were detected up to 2 years prior to PTLD diagnosis and correlated well with response to cytoreductive treatment in individual patients. sCXCL13, thus, may be a readily available surrogate marker for the diagnosis of PTLD and for monitoring of response to treatment in patients with initially elevated sCXCL13 levels.

Expression of pro- and antifibrotic genes in protocol biopsies from renal allografts with interstitial fibrosis and tubular atrophy.

AIM Better understanding of early onset of interstitial fibrosis and tubular atrophy (IF/TA), as the morphological surrogate of renal allograft deterioration might improve outcome after renal transplantation. MATERIAL AND METHODS We quantified mRNA expression of 3 profibrotic (transforming growth factor-beta (TGF-beta), tissue transglutaminase (tTG), tissue inhibitor of matrix metalloproteases (TIMP-1)) and 1 antifibrotic (matrix metalloprotease-2 (MMP-2)) molecule in protocol biopsies from renal allografts. From 107 transplants, two sequential protocol biopsies (6 weeks and 6 months) were analyzed. We evaluated a control group showing no IF/TA in both biopsies (n = 65) and a IF/TA group developing IF/TA at 6 months (n = 42). Expression data were correlated with clinical and histological risk factors for IF/TA and allograft function. RESULTS The expression of the genes correlated strongly with each other, particularly the profibrotic genes and in patients who developed IF/TA. Analyzing protocol biopsies from stable grafts, not all patients in both groups showed increased gene expression. In patients with increased gene expression a significantly higher tTG expression (matrix stabilization) at 6 weeks and a significantly lower MMP-2 expression (failure in matrix degradation) at 6 months were observed in the IFTA group compared to controls. Multivariate logistic regression revealed donor age positively and TIMP-1 expression at 6 weeks inversely correlated with IF/TA at 6 months. CONCLUSIONS We conclude that a disturbance in the equilibrium of pro- and antifibrotic pathways is decisive for early onset of IF/TA in renal allografts: insufficient degradation of exaggerated matrix production apparently changes the balance in the direction of IF/TA.

Compartment-specific quantitative gene expression analysis after laser microdissection from archival renal allograft biopsies.

BACKGROUND Various immunological and non-immunological pathomechanisms are responsible for the cellular damage in renal allografts. Since the kidney is an anatomically complex organ with functional and morphological heterogeneous compartments (interstitium, tubuli, vessels, glomeruli), the local response to injury maybe variable, therefore, the identification of local pathomechanisms is important. AIM To elucidate any discrepancies in quantitative mRNA expression profiles between a total specimen analysis and a cell-specific evaluation after laser microdissection. METHODS Real-time RT-PCR was performed for complement component C3 and heme oxygenase-1 (HO-1) genes compared to the housekeeping gene beta-actin using whole section RNA extracted from formalin-fixed and paraffin-embedded archival material of 16 explanted, rejected renal allografts. Ten non-transplant nephrectomies served as controls. For five cases from each group, five different compartments of the organs (interstitium, proximal tubuli, distal tubuli, vessels, glomeruli) were microdissected and quantitative analysis for C3 and HO-1 was performed identically. RESULTS Whole section mRNA expression analysis: the data showed a constant expression of the housekeeping gene beta-actin, a 7-fold increased expression of C3 and a 3-fold decreased expression of HO-1 in the allograft group as compared to the control group. mRNA expression results from microdissected compartments: in the control group, C3 and HO-1 expression could only be detected in the proximal tubuli of all cases whereas all five compartments analyzed from the rejecting kidneys showed expression of the two genes. In the allografts, expression levels of the investigated genes varied considerably not only among the different compartments but between individual cases as well. CONCLUSION Laser microdissection combined with real-time RT-PCR is a feasible approach for retrospective quantitative gene expression analysis in formalin-fixed and paraffin-embedded renal allograft specimens. As shown for C3 and HO-1, cell-specific expression patterns ofpathogenetically relevant genes vary considerably between individual cases. A close correlation of morphology and cell-specific gene expression analysis will contribute to the elucidation of the complex pathogenesis of chronic renal allograft nephropathy.

Authors Reply to Budde et al.: ‘No Evidence for Relationship Between Infiltrates in Renal Protocol Biopsies and Outcome’

We thank Budde and colleagues (1) for their interest in our article (2). We agree with Budde that the GFR at 2 years is only weakly, but significantly (p = 0.001) associated with the sum of infiltrates. We previously showed that other pathologies, that is calcification and chronic changes in our series also have effects on long-term allograft function and allograft nephropathy (3,4). Budde’s anticipation, that multivariate analysis including those and other clinical factors might define long-term outcome better, is well taken. However, it was never the sole scope of the study to develop a model to predict allograft outcome. As stated in the introduction and shown in the results, major aims were to describe prevalence and localization of different infiltrates in protocol and indication biopsies, and to determine how these infiltrates correlate with the current Banff schema and secondarily with allograft function. We agree with the comment from Budde et al. that infiltrates in a single biopsy do not predict outcome. However, it is incorrect, as stated in the first paragraph of Budde’s letter, that we found persistent inflammation within the first 6 months only in protocol biopsies to be associated with later allograft function. In fact, we included all biopsies (protocol and indication) in this analysis taken within the first year after transplantation. To exclude bias due to lack of biopsy results, thus missing diagnosis of infiltrates, the analysis was done in a subgroup, which was defined to have at least all three protocol biopsies (See the Results, ‘Infiltrate number and allograft outcome’, page 362 in Ref. 2).