Bettina Jung

Kidney injury molecule-1 and N-acetyl-ß-d-glucosaminidase in chronic heart failure: possible biomarkers of cardiorenal syndrome

Patients with chronic heart failure are often characterized by impaired renal function, also referred to as cardiorenal syndrome (CRS). The aim of this study was to assess whether novel markers of kidney injury are elevated in chronic heart failure and CRS.

Toll-Like Receptor 4 in Experimental Kidney Transplantation: Early Mediator of Endogenous Danger Signals

The role of toll-like receptors (TLRs) has been described in the pathogenesis of renal ischemia/reperfusion injury, but data on the expression and function of TLR4 during renal allograft damage are still scarce. We analyzed the expression of TLR4 in an experimental rat model 6 and 28 days after allogeneic kidney transplantation in comparison to control rats and rats after syngeneic transplantation. On day 6, a significant induction in TLR4 expression – restricted to the glomerular compartment – was found in acute rejecting allografts only. TLR4 expression strongly correlated with renal function, and TLR4 induction was accompanied by a significant increase in CC chemokine expression within the graft as well as in urinary CC chemokine excretion. TLR4 induction may be caused by an influx of macrophages as well as TLR4-expressing intrinsic renal cells. Fibrinogen deposition in renal allografts correlated with renal TLR4 expression and may act as a potent stimulator of chemokine release via TLR4 activation. This study provides, for the first time, data about the precise intrarenal localization and TLR4 induction after experimental kidney transplantation. It supports the hypothesis that local TLR4 activation by endogenous ligands may be one pathological link from unspecific primary allograft damage to subsequent chemokine release, infiltration and activation of immune cells leading to deterioration of renal function and induction of renal fibrosis.

Infiltration of Macrophages Correlates with Severity of Allograft Rejection and Outcome in Human Kidney Transplantation

Objective Despite substantial progress in recent years, graft survival beyond the first year still requires improvement. Since modern immunosuppression addresses mainly T-cell activation and proliferation, we studied macrophage infiltration into the allografts of 103 kidney transplant recipients during acute antibody and T-cell mediated rejection. Macrophage infiltration was correlated with both graft function and graft survival until month 36 after transplantation. Results Macrophage infiltration was significantly elevated in antibody-mediated and T-cell mediated rejection, but not in kidneys with established IFTA. Treatment of rejection with steroids was less successful in patients with more prominent macrophage infiltration into the allografts. Macrophage infiltration was accompanied by increased cell proliferation as well as antigen presentation. With regard to the compartmental distribution severity of T-cell-mediated rejection was correlated to the amount of CD68+ cells especially in the peritubular and perivascular compartment, whereas biopsies with ABMR showed mainly peritubular CD68 infiltration. Furthermore, severity of macrophage infiltration was a valid predictor of resulting creatinine values two weeks as well as two and three years after renal transplantation as illustrated by multivariate analysis. Additionally performed ROC curve analysis showed that magnitude of macrophage infiltration (below vs. above the median) was a valid predictor for the necessity to restart dialysis. Having additionally stratified biopsies in accordance to the magnitude of macrophage infiltration, differential CD68+ cell infiltration was reflected by striking differences in overall graft survival. Conclusion The differences in acute allograft rejection have not only been reflected by different magnitudes of macrophage infiltration, but also by compartment-specific infiltration pattern and subsequent impact on resulting allograft function as well as need for dialysis initiation. There is a robust relationship between macrophage infiltration, accompanying antigen-presentation and resulting allograft function.

Biomarker-guided Intervention to Prevent Acute Kidney Injury After Major Surgery: The Prospective Randomized Bigpak Study

Objective:To determine the impact of renal biomarker–guided implementation of the Kidney Disease Improving Global Outcomes (KDIGO) care bundle on the incidence of acute kidney injury (AKI) after major noncardiac surgery in a single-center unblinded randomized clinical trial. Background:Early optimization of volume status and discontinuation of nephrotoxic medication before the occurrence of AKI may be the crucial step to reduce preventable AKI. Methods:The urinary biomarker−triggered KDIGO care bundle (early optimization of fluid status, maintenance of perfusion pressure, discontinuation of nephrotoxic agents) was compared to standard intensive care unit (ICU) care in 121 patients with an increased AKI risk after major abdominal surgery that was determined by urinary biomarker (inhibitor of metalloproteinase-2 × insulin-like growth factor–binding protein 7) >0.3. Incidence of overall AKI, severity of AKI, length of stay, major kidney events at discharge, and cost effectiveness were evaluated. Results:The overall stages of AKI were not statistically different between the 2 groups, but in patients with inhibitor of metalloproteinase-2 × insulin-like growth factor–binding protein 7 values of 0.3 to 2.0 a subgroup analysis demonstrated a significantly reduced incidence of AKI 13/48 (27.1%) in the intervention group compared to control 24/50 (48.0%, P = 0.03). Incidence of moderate and severe AKI (P = 0.04), incidence of creatinine increase >25% of baseline value (P = 0.01), length of ICU, and hospital stay (P = 0.04) were significantly lower in the intervention group. Intervention was associated with cost reduction. There were no significant differences regarding renal replacement therapy, in-hospital mortality, or major kidney events at hospital discharge. Conclusions:Early biomarker-based prediction of imminent AKI followed by implementation of KDIGO care bundle reduced AKI severity, postoperative creatinine increase, length of ICU, and hospital stay in patients after major noncardiac surgery.

Impact of Toll-like receptor 2 expression in renal allograft rejection

BACKGROUND An important role of TLR2 has been shown in various experimental models of renal ischaemia/reperfusion injury. To study the expression of TLR2 in renal allograft rejection systematically, we established an experimental rat transplantation model. METHODS TLR2 expression was analysed in 99 human renal allograft biopsies, and in rat allografts at Day 6 and 28 after experimental renal transplantation. To discriminate whether regulation of TLR2 was following immunological processes after allogeneic transplantation or was a consequence from ischaemia/reperfusion injury, control animals subjected to syngeneic transplantation or to ischaemia/reperfusion damage were also investigated. RESULTS TLR2 mRNA was significantly elevated in rat allografts with acute rejection on Day 6 and decreased spontaneously towards Day 28. TLR2 induction correlated with renal function and TLR2 excretion in the urine of transplanted rats. TLR2 staining was also significantly increased in human allografts with acute rejection. TLR2 protein could be localized in tubular epithelial cells and vascular endothelial cells, and in CD68- and CD4-positive infiltrating cells. CONCLUSIONS TLR2 is markedly up-regulated in both experimental and human acute renal allograft rejection. Our data suggest a role for TLR2 during allogen-dependent graft damage after renal transplantation.

Traditional and Nontraditional Cardiovascular Risk Factors and Estimated Risk for Coronary Artery Disease in Renal Transplant Recipients: A Single-Center Experience

Background/Aims: The prevalence of cardiovascular disease in renal transplant recipients is markedly higher than in the general population due to the high prevalence of traditional cardiovascular risk factors, renal transplant function impairment and treatment with immunosuppressive drugs that affect blood pressure, cholesterol and blood glucose levels. Methods: Cross-sectional analysis using our renal transplant clinic cohort investigating (1) the cardiovascular risk factors present in this cohort, and (2) estimating their impact on the risk of coronary artery disease (CAD) by using the Framingham algorithm. Results: Control of modifiable cardiovascular risk factors in 231 renal transplant recipients is suboptimal, i.e. 47.2% of patients are hypertensive, 10.3% actively smoke, 39.4% have serum cholesterol concentrations >200 mg/dl, and 19.7% have diabetes mellitus. Blood pressure, age, hyperlipidemia, smoking and diabetes modulate the estimated CAD risk in males and females. Furthermore, a short time period (less than 1 year) since transplantation and increased serum creatinine levels negatively influenced the CAD risk in this patient population. Conclusion: According to current guidelines, the control of modifiable cardiovascular risk factors in renal transplant recipients is suboptimal. The decreasing CAD risk over time after transplantation may be due to the reduction of immunosuppressive drugs with time and survival bias.

B-cell activating factor BAFF reflects patients' immunological risk profile after kidney transplantation

The B-cell activating factor BAFF plays an important role in the development and maturation of B-lymphocytes, which can contribute to the generation of donor-specific antibodies and thus may influence graft function and graft survival. Inconsistent data on the role of BAFF levels after renal transplantation for the formation of donor-specific antibodies and the contribution for allograft rejection exist. The aim of the current study was to determine to what extent the degree of pre-immunization is reflected by each patients BAFF levels before transplantation and in the follow-up. Furthermore, the impact of BAFF on allograft rejection frequency as well as severity and resulting allograft function over time was analyzed. Additionally, the impact of viral infections on BAFF levels after transplantation - as a potential confounder - was examined. For this purpose, a group of pre-sensitized patients (PRA>0%, (52±24% on average), n=40) was compared with non-sensitized patients (PRA=0%, n=62) and in a subsequent analysis stratification in accordance to the detected BAFF level was performed. Pre-sensitized patients had significantly higher BAFF levels before transplantation and suffered significantly more often from early steroid-resistant, mainly antibody-mediated rejections. A result which was confirmed also in highly sensitized patients with PRA levels >50%. Additionally, in the follow-up patients with either rising BAFF levels over time or BAFF levels above the median also had significantly more often antibody mediated rejections. Additionally, patients with BAFF levels above detected median even displayed impaired creatinine values as well as an induced eGFR slope up to month 48 after transplantation. The occurrence of viral infections (CMV, BKV) was only an additional influencing factor in the absence of concomitant allograft rejections. Therefore, the B-cell proliferation factor BAFF appears not only to reflect the immunological risk profile of patients in the context of kidney transplantation, it may possibly be further developed as a predictor of patients with an increased risk profile for subsequent allograft rejection and impaired allograft function.

Comprehensive morphometric analysis of mononuclear cell infiltration during experimental renal allograft rejection

The role of specific subtypes of infiltrating cells in acute kidney allograft rejection is still not clear and was so far not examined by different analyzing methods under standardized conditions of an experimental kidney transplantation model. Immunohistochemical staining of CD3, CD20 and CD68 was performed in rat allografts, in syngeneically transplanted rats and in control rats with a test duration of 6 and 28 days. The detailed expression and localization of infiltrating cells were analyzed manually in different kidney compartments under light microscope and by the two different morphometric software programs. Data were correlated with the corresponding kidney function as well as with histopathological classification. The information provided by the morphometric software programs on the infiltration of the specific cell types after renal transplantation was in accordance with the manual analysis. Morphometric methods were solid to analyze reliably the induction of cellular infiltrates after renal transplantation. By manual analysis we could clearly demonstrate the detailed localization of the specific cell infiltrates in the different kidney compartments. Besides infiltration of CD3 and CD68 infiltrating cells, a robust infiltration of CD20 B-cells in allogeneically transplanted rats, even at early time points after transplantation was detected. Additionally an MHC class I expression could reliable be seen in allogeneically transplanted rats. The infiltration of B-cells and the reliable antigen presentation might act as a silent subclinical trigger for subsequent chronic rejection and premature graft loss.

Hypertonic Stress Promotes the Upregulation and Phosphorylation of Zonula Occludens 1

Tight junction molecules form a barrier between adjacent cells and mediate the cells’ ability to develop membranes that constitute boundaries of different compartments within the body. Membranes with selective ion and water passage are important for the electrolyte and water homeostasis in the kidney. Due to their role in the urinary concentration process, renal medullary cells are exposed to hyperosmotic stress. Therefore, we were interested in the question of how mouse inner medullary collecting duct cells (mIMCD3) manage to maintain their cell-cell contacts, despite hypertonicity-induced cell shrinkage. Employing mRNA expression analysis, we found that the zonula occludens type 1 (Zo-1), multi-PDZ domain protein 1 (MUPP1) and cortactin mRNA levels were upregulated in a tonicity-dependent manner. Using Western blot analysis, immunoprecipitation and immunofluorescence, we show that the Zo-1 protein is upregulated, phosphorylated and linked to the actin cytoskeleton in response to hypertonic stress. After cell exposure to hypertonicity, rearrangement of the actin cytoskeleton resulted in a stronger colocalization of actin fibres with Zo-1. Urea, which generates hyperosmolality, but no transcellular gradient, did not induce changes in Zo-1 protein expression or actin rearrangement. This data indicates that Zo-1 is a response protein to inner medullary tonicity and that extracellular stressors can promote Zo-1 protein expression, tyrosine phosphorylation and cytoskeleton association.

Development of Organized Intra-Graft Lymphocyte Clusters – Role of B Cell Activating Factor BAFF.

Background Chronic allograft rejection is a major cause of graft loss in solid organ transplantation. Local intra-graft B and T lymphocytes form organized clusters resembling lymphoid follicles, which have been associated with worsened graft outcome. The B cell activating factor BAFF is an important B cell survival factor and may influence the development of tertiary lymphoid organs (TLO). We have studied the expression of TLO-promoting factors and BAFF and their correlation with TLO in a rat model of renal transplantation (RTx). In parallel, we are examining the relationship of intra-graft BAFF levels and TLO formation in renal transplant patients. Methods MHC-mismatched rats (Brown Norway ->Lewis) were used in this model of RTx. Rats were subjected to chronic underimmunosuppression (CyA 5mg/KG/every 2 days) and sacrificed on d28 and d56 post-Tx. Expression of TLO-promoting factors (CCL19, CCL21, CXCL13, Lymphotoxin-ß) and BAFF were measured by qPCR, immunohistochemistry or flow cytometry. Intra-graft leukocyte and B cell subsets were assessed by flow cytometry (CD11b/c, CD163, CD3, CD45R, IgM, CD38, CD27) and intra-renal spacial distribution was assessed by immunofluorescence. Cell proliferation and formation of germinal centres was assessed using Ki67-staining. Specific molecules of T/B cell interaction (CD40/CD40L, ICOS/ICOSL), and B and T cell transcription factors (Bcl-6, Blimp-1) were assessed by qPCR. An interventional anti-BAFF antibody is also being tested in this model. In parallel, renal transplant biopsies from our patient cohort were assessed for BAFF and the occurrence of lymphoid follicles by immunohistochemistry (CD3, CD20). Results and Discussion We demonstrated the induction of TLO-promoting factors (CCL19/CCL21, CXCL13, Lymphotoxin-ß, and BAFF) and the development of organized lymphocyte clusters after prolonged underimmunosuppression in a rat model of RTx. We have characterized microanatomical and functional features of these lymphocyte clusters, including T and B cell zones, germinal centres, and expression of costimulatory molecules, B cell differentiation markers and transcription factors related to effector functions of humoral alloreactivity (e.g. Ig class switching, antibody production). The effect of an interventional anti-BAFF antibody on intra-graft lymphocytes is being tested in this rat RTx model. Furthermore, intra-graft BAFF expression and the occurrence of lymphoid aggregates and TLO is correlated in our renal transplant patient collective. Conclusion Under prolonged conditions of suboptimal immunosuppression TLO-promoting factors and BAFF are upregulated and organized intra-graft lymphocyte clusters are induced. This may augment local cellular and humoral alloreactivity and thereby contribute to chronic allograft injury. BAFF may act as a pivotal factor for the intra-renal B cell axis and subsequently for TLO formation.