Hans-Peter Marti

Analysis of independent microarray datasets of renal biopsies identifies a robust transcript signature of acute allograft rejection

Transcriptomics could contribute significantly to the early and specific diagnosis of rejection episodes by defining ‘molecular Banff’ signatures. Recently, the description of pathogenesis‐based transcript sets offered a new opportunity for objective and quantitative diagnosis. Generating high‐quality transcript panels is thus critical to define high‐performance diagnostic classifier. In this study, a comparative analysis was performed across four different microarray datasets of heterogeneous sample collections from two published clinical datasets and two own datasets including biopsies for clinical indication, and samples from nonhuman primates. We characterized a common transcriptional profile of 70 genes, defined as acute rejection transcript set (ARTS). ARTS expression is significantly up‐regulated in all AR samples as compared with stable allografts or healthy kidneys, and strongly correlates with the severity of Banff AR types. Similarly, ARTS were tested as a classifier in a large collection of 143 independent biopsies recently published by the University of Alberta. Results demonstrate that the ‘in silico’ approach applied in this study is able to identify a robust and reliable molecular signature for AR, supporting a specific and sensitive molecular diagnostic approach for renal transplant monitoring.

Immunoliposome Targeting to Mesangial Cells: A Promising Strategy for Specific Drug Delivery to the Kidney

Mesangial cell-mediated nephropathies are a frequent cause of ESRD. Specific drug delivery to mesangial cells might be more effective and better tolerated than existing systemic treatments. Rat mesangial cells are characterized by Thy1.1 antigen expression. Therefore, OX7-coupled immunoliposomes (OX7-IL) were prepared by coupling liposomes with F(ab) fragments of OX7 mAb directed against Thy1.1 antigen. As the glomerular endothelium is fenestrated and no basement membrane separates glomerular capillaries from the mesangium, mesangial cells represent a particularly suitable target for drug delivery by OX7-IL. Therefore, the targeting efficacy of OX7-IL to mesangial cells was investigated. Specific targeting in vitro was obtained, and intravenous injection of OX-7-IL to rats showed a specific targeting of all mesangial cells in both kidneys. OX7-IL showed marked accumulation in the cytoplasm of rat mesangial cells, both in vitro and in vivo. This renal targeting was blocked when free OX7 F((ab)2) fragments were co-administered with OX7-IL. Rats that were given a single intravenous injection of low-dose doxorubicin encapsulated in OX7-IL showed extensive glomerular damage, whereas other parts of the kidney and other organs were spared. Free doxorubicin and the liposomal formulation of this agent had no effect. Thus, immunoliposomes are a very promising delivery system for the treatment of kidney diseases.

Design and methodology of the Swiss Transplant Cohort Study (STCS): a comprehensive prospective nationwide long-term follow-up cohort

In Switzerland, organ procurement is well organized at the national-level but transplant outcomes have not been systematically monitored so far. Therefore, a novel project, the Swiss Transplant Cohort Study (STCS), was established. The STCS is a prospective multicentre study, designed as a dynamic cohort, which enrolls all solid organ recipients at the national level. The features of the STCS are a flexible patient-case system that allows capturing all transplant scenarios and collection of patient-specific and allograft-specific data. Beyond comprehensive clinical data, specific focus is directed at psychosocial and behavioral factors, infectious disease development, and bio-banking. Between May 2008 and end of 2011, the six Swiss transplant centers recruited 1,677 patients involving 1,721 transplantations, and a total of 1,800 organs implanted in 15 different transplantation scenarios. 10xa0% of all patients underwent re-transplantation and 3% had a second transplantation, either in the past or during follow-up. 34% of all kidney allografts originated from living donation. Until the end of 2011 we observed 4,385 infection episodes in our patient population. The STCS showed operative capabilities to collect high-quality data and to adequately reflect the complexity of the post-transplantation process. The STCS represents a promising novel project for comparative effectiveness research in transplantation medicine.

Renal Allografts with IF/TA Display Distinct Expression Profiles of Metzincins and Related Genes

Chronic renal allograft injury is often reflected by interstitial fibrosis (IF) and tubular atrophy (TA) without evidence of specific etiology. In most instances, IF/TA remains an irreversible disorder, representing a major cause of long‐term allograft loss. As members of the protease family metzincins and functionally related genes are involved in fibrotic and sclerotic processes of the extracellular matrix (ECM), we hypothesized their deregulation in IF/TA. Gene expression and protein level analyses using allograft biopsies with and without Banff05 classified IF/TA illustrated their deregulation. Expression profiles of these genes differentiated IF/TA from Banff05 classified Normal biopsies in three independent microarray studies and demonstrated histological progression of IF/TA I to III. Significant upregulation of matrix metalloprotease‐7 (MMP‐7) and thrombospondin‐2 (THBS‐2) in IF/TA biopsies and sera was revealed in two independent patient sets. Furthermore, elevated THBS‐2, osteopontin (SPP1) and β‐catenin may play regulatory roles on MMP. Our findings further suggest that deregulated ECM remodeling and possibly epithelial to mesenchymal transition (EMT) are implicated in IF/TA of kidney transplants, and that metzincins and related genes play an important role in these processes. Profiling of these genes may be used to complement IF/TA diagnosis and to disclose IF/TA progression in kidney transplant recipients.

Effect of donor-specific transfusions on the outcome of renal allografts in the cyclosporine era

Despite the introduction of new immunosuppressive agents, a steady decline of functioning renal allografts after living donation is observed. Thus nonpharmacological strategies to prevent graft loss have to be reconsidered, including donor‐specific transfusions (DST). We introduced a cyclosporine‐based DST protocol for renal allograft recipients from living‐related/unrelated donation. From 1993 to 2003, 200u2003ml of whole blood, or the respective mononuclear cells from the potential living donor were administered twice to all of our 61 recipient candidates. The transplanted subjects were compared with three groups of patients without DST from the Collaborative Transplant Study (Heidelberg, Germany) during a 6‐year period. Six patients were sensitized without delay for a subsequent cadaveric kidney. DST patients had less often treatment for rejection and graft survival was superior compared with subjects from the other Swiss transplant centers (nu2003=u2003513) or from Western Europe (nu2003=u20037024). To diminish the probability that superior results reflect patient selection rather than effects of DST, a ‘matched‐pair’ analysis controlling for relevant factors of transplant outcome was performed. Again, this analysis indicated that recipients with DST had better outcome. Thus, our observation suggests that DST improve the outcome of living kidney transplants even when modern immunosuppressive drugs are prescribed.

Angiotensin-Converting Enzyme Inhibition but not Angiotensin II Receptor Blockade Regulates Matrix Metalloproteinase Activity in Patients with Glomerulonephritis

Equivalent long-term effects on the kidney are attributed to angiotensin-converting enzyme inhibitors (ACEI) and angiotensin II type 1 receptor blockers (ARB). Nevertheless, it is unknown to which degree effects of these compounds on individual inflammatory mediators, including matrix metalloproteinases (MMP), are comparable. On the basis of structural and functional differences, it was hypothesized that ACEI and ARB differentially regulate MMP activity. In a randomized, prospective crossover trial, the effect of an ACEI (fosinopril; 20 mg/d) and of an ARB (irbesartan; 150 mg/d) on MMP activity was evaluated. Ten hypertensive patients with glomerulonephritis and normal or mildly reduced creatinine clearance were studied. MMP activity and tissue inhibitors of metalloproteinase (TIMP) levels were analyzed in serum and urine: without therapy, with ACEI, with ARB, and with both agents combined. Treatment periods continued for 6 wk separated by periods of 4 wk each without therapy. Untreated patients with glomerulonephritis displayed distinctively higher serum levels of MMP-2 but much lower MMP-1/-8/-9 concentrations compared with healthy control subjects. Immunohistology of MMP-2 and MMP-9 in kidney biopsy specimen was accordingly. However, these patients excreted higher amounts of MMP-2 and MMP-9 in urine than healthy control subjects, possibly reflecting ongoing glomerular inflammation. In patients with glomerulonephritis, ACEI significantly reduced overall MMP serum activity to 25%, whereas ARB did not show any effect. Activities of MMP-1/-2/-8/-9 were also significantly inhibited by fosinopril but not by irbesartan. Levels of TIMP-1/-2 remained unaffected. In conclusion, ACEI and ARB differentially regulate MMP activity, which may ultimately have consequences in certain types of MMP-dependent glomerulonephritis.

Sirolimus and everolimus reduce albumin endocytosis in proximal tubule cells via an angiotensin II-dependent pathway

BACKGROUNDnThe proliferation signal inhibitors (PSIs) sirolimus (SRL) and everolimus (ERL) often induce proteinuria due to glomerular but also tubular dysfunction in transplant patients. The beneficial effect of angiotensin converting enzyme inhibitors (ACE-I) and angiotensin II (Ang II) type 1 receptor blockers (ARB) has been reported.nnnAIMnThis study aimed to investigate: (i) the role of an Ang II-dependent mechanism and Ang II type 1 receptor (AT(1)R) in the regulation of receptor-mediated albumin endocytosis on proximal tubular epithelial cells (PTEC) following PSI treatment; (ii) the specific roles of the albumin receptors cubilin and megalin in albumin binding and uptake in PTEC.nnnMETHODSnA human renal PTEC line (HK-2) was used in the study. The binding of Alexa 488 conjugated albumin was measured by flow cytometry. Albumin uptake and the expression of cubilin and megalin were determined by cellular ELISA.nnnRESULTSnThe administration of PSIs resulted in decreased albumin binding and uptake and downregulation of cubilin and megalin expression in PTEC. These effects were significantly reversed by the administration of an ACE-I (ramipril) or an ARB (losartan). Combined use of ramipril and losartan demonstrated additive effects on cubilin expression, but not on megalin expression or albumin binding and uptake.nnnCONCLUSIONSnOur findings suggest that decreased albumin endocytosis in PTECs following PSI treatment involve an Ang II-dependent pathway via AT(1)R. The albumin receptor megalin may play a more crucial role than cubulin, since its expression correlated directly with albumin uptake.

Metalloprotease Meprinβ in Rat Kidney: Glomerular Localization and Differential Expression in Glomerulonephritis

Meprin (EC 3.4.24.18) is an oligomeric metalloendopeptidase found in microvillar membranes of kidney proximal tubular epithelial cells. Here, we present the first report on the expression of meprinβ in rat glomerular epithelial cells and suggest a potential involvement in experimental glomerular disease. We detected meprinβ in glomeruli of immunostained rat kidney sections on the protein level and by quantitative RT-PCR of laser-capture microdissected glomeruli on the mRNA level. Using immuno-gold staining we identified the membrane of podocyte foot processes as the main site of meprinβ expression. The glomerular meprinβ expression pattern was altered in anti-Thy 1.1 and passive Heymann nephritis (PHN). In addition, the meprinβ staining pattern in the latter was reminiscent of immunostaining with the sheep anti-Fx1A antiserum, commonly used in PHN induction. Using Western blot and immunoprecipitation assays we demonstrated that meprinβ is recognized by Fx1A antiserum and may therefore represent an auto-antigen in PHN. In anti-Thy 1.1 glomerulonephritis we observed a striking redistribution of meprinβ in tubular epithelial cells from the apical to the basolateral side and the cytosol. This might point to an involvement of meprinβ in this form of glomerulonephritis.

Skin cancers in renal transplant recipients: A description of the renal transplant cohort in Bern

BACKGROUND/AIMSnSkin tumours, in particular squamous-cell carcinomas (SCC), are the most common malignant conditions developing in transplant recipients. The aim of this study is to investigate the frequency and type of skin cancer in patients receiving immunosuppressive therapy after organ transplantation.nnnMETHODSnMultivariate logistic regression analysis was performed on data of 243 renal transplant patients who attended the dermatology outpatient clinic for the first time after transplantation in the period January 2002-October 2005.nnnRESULTSnWe found an increased risk of actinic keratosis (AK) and SCC in renal transplant recipients with a basal cell carcinoma (BCC) / SCC ratio of 1:7. Older patients had AK more frequently (odds ratio [OR] 1.11, 95% confidence interval [CI] 1.06-1.15; p <0.0001) and SCC (OR 1.14, CI 1.07-1.22; p <0.0001) than younger patients. Men had AK (OR 0.19, CI 0.08-0.45; p = 0.0002) and SCC (OR 0.25, CI 0.07-0.89; p = 0.0332) more frequently than women. The duration of immunosuppressive therapy correlated significantly with the numbers of AKs (OR 1.15, CI 1.08-1.24; p <0.0001) and SCCs (OR 1.16, CI 1.05-1.28; p = 0.0025), and patients with fair skin had more AKs (OR 0.31, CI 0.14-1.24; p <0.0001) and SCCs (OR 0.11, CI 0.02-0.52; p = 0.0054) than darker skinned patients. We could not identify any specific immunosuppressive drug as a distinct risk factor for AK or non-melanoma skin cancer (NMSC).nnnCONCLUSIONnSkin cancers are increased in the renal transplant population. Main risk factors for skin cancers are fair skin type and long duration of immunosuppressive therapy. A follow-up programme is necessary for early detection of skin cancer and precancerous conditions. Preventive strategies should include specialist dermatological monitoring and self-examination.

Meta-Analyses Qualify Metzincins and Related Genes as Acute Rejection Markers in Renal Transplant Patients

Definition of acute renal allograft rejection (AR) markers remains clinically relevant. Features of T‐cell–mediated AR are tubulointerstitial and vascular inflammation associated with excessive extracellular matrix (ECM) remodeling, regulated by metzincins, including matrix metalloproteases (MMP). Our study focused on expression of metzincins (METS), and metzincins and related genes (MARGS) in renal allograft biopsies using four independent microarray data sets. Our own cases included normal histology (N, n = 20), borderline changes (BL, n = 4), AR (n = 10) and AR + IF/TA (n = 7). MARGS enriched in all data sets were further examined on mRNA and/or protein level in additional patients. METS and MARGS differentiated AR from BL, AR + IF/TA and N in a principal component analysis. Their expression changes correlated to Banff t‐ and i‐scores. Two AR classifiers, based on METS (including MMP7, TIMP1), or on MARGS were established in our own and validated in the three additional data sets. Thirteen MARGS were significantly enriched in AR patients of all data sets comprising MMP7, ‐9, TIMP1, ‐2, thrombospondin2 (THBS2) and fibrillin1. RT‐PCR using microdissected glomeruli/tubuli confirmed MMP7, ‐9 and THBS2 microarray results; immunohistochemistry showed augmentation of MMP2, ‐9 and TIMP1 in AR. TIMP1 and THBS2 were enriched in AR patient serum. Therefore, differentially expressed METS and MARGS especially TIMP1, MMP7/‐9 represent potential molecular AR markers.