Alexander Grabner

Damage of the endothelial glycocalyx in chronic kidney disease

BACKGROUND AND OBJECTIVESnThe endothelial glycocalyx (eGC), a mesh of anionic biopolymers covering the luminal surface of endothelial cells, is considered as an intravascular compartment that protects the vessel wall against pathogenic insults in cardiovascular disease. We hypothesized that chronic kidney disease (CKD) is associated with reduced eGC integrity and subsequent endothelial dysfunction.nnnMETHODS & RESULTSnShedding of two major components of the eGC, namely syndecan-1 (Syn-1) and hyaluronan (HA), was measured by ELISA in 95 patients with CKD (stages 3-5) and 31 apparently healthy controls. Plasma levels of Syn-1 and HA increased steadily across CKD stages (5- and 5.5-fold, respectively P < 0.001) and were independently associated with impaired renal function after multivariate adjustment. Furthermore, Syn-1 and HA correlated tightly with plasma markers of endothelial dysfunction such as soluble fms-like tyrosine kinase-1 (sFlt-1), soluble vascular adhesion molecule-1 (sVCAM-1), von-Willebrand-Factor (vWF) and angiopoietin-2 (P < 0.001). Experimentally, excessive shedding of the eGC, evidenced by 11-fold increased Syn-1 plasma levels, was also observed in an established rat model of CKD, the 5/6-nephrectomized rats. Consistently, an atomic force microscopy-based approach evidenced a significant decrease in eGC thickness (360 ± 79 vs. 157 ± 29 nm, P = 0.001) and stiffness (0.33 ± 0.02 vs. 0.22 ± 0.01 pN/nm, P < 0.001) of aorta endothelial cell explants isolated from CKD rats.nnnCONCLUSIONnOur findings provide evidence for damage of the atheroprotective eGC as a consequence of CKD and potentially open a new avenue to pathophysiology and treatment of cardiovascular disease in renal patients.

The cysteines of the extracellular loop are crucial for trafficking of human organic cation transporter 2 to the plasma membrane and are involved in oligomerization

Human organic cation transporter 2 (hOCT2) is involved in transport of many endogenous and exogenous organic cations, mainly in kidney and brain cells. Because the quaternary structure of transmembrane proteins plays an essential role for their cellular trafficking and function, we investigated whether hOCT2 forms oligomeric complexes, and if so, which part of the transporter is involved in the oligomerization. A yeast 2‐hybrid mating‐based split‐ubiquitin system (mbSUS), fluorescence resonance energy transfer, Western blot analysis, crosslinking experiments, immunofluorescence, and uptake measurements of the fluorescent organic cation 4‐(4‐(dimethylamino) styryl)‐N‐methylpyridinium were applied to human embryonic kidney 293 (HEK293) cells transfected with hOCT2 and partly also to freshly isolated human proximal tubules. The role of cysteines for oligomerization and trafficking of the transporter to the plasma membranes was investigated in cysteine mutants of hOCT2. hOCT2 formed oligomers both in the HEK293 expression system and in native human kidneys. The cysteines of the large extracellular loop are important to enable correct folding, oligomeric assembly, and plasma membrane insertion of hOCT2. Mutation of the first and the last cysteines of the loop at positions 51 and 143 abolished oligomer formation. Thus, the cysteines of the extracellular loop are important for correct trafficking of the transporter to the plasma membrane and for its oligomerization.—Brast, S., Grabner, A., Sucic, S., Sitte, H. H., Hermann, E., Pavenstädt, H., Schlatter, E., and Ciarimboli, G. The cysteines of the extracellular loop are crucial for trafficking of human organic cation transporter 2 to the plasma membrane and are involved in oligomerization. FASEB J. 26, 976‐986 (2012). www.fasebj.org

Cardioprotective effect of calcineurin inhibition in an animal model of renal disease

AIMSnChronic kidney disease is directly associated with cardiovascular complications. Heart remodelling, including fibrosis, hypertrophy, and decreased vascularization, is frequently present in renal diseases. Our objective was to investigate the impact of calcineurin inhibitors (CNI) on cardiac remodelling and function in a rat model of renal disease.nnnMETHODS AND RESULTSnMale Sprague Dawley rats were divided into six groups: sham-operated rats, 5/6 nephrectomized rats (Nx) treated with vehicle, CNI (cyclosporine A 5.0 or 7.5, or tacrolimus 0.5 mg/kg/day) or hydralazine (20 mg/kg twice a day) for 14 days, starting on the day of surgery. Creatinine clearance was significantly lower and blood pressure significantly higher in Nx rats when compared with controls. Morphological and echocardiographic analyses revealed increased left ventricular hypertrophy and decreased number of capillaries in Nx rats. Treatment with CNI affected neither the renal function nor the blood pressure, but prevented the development of cardiac hypertrophy and improved vascularization. In addition, regional blood volume improved as confirmed by contrast agent-based echocardiography. Hydralazine treatment did not avoid heart remodelling in this model. Gene expression analysis verified a decrease in hypertrophic genes in the heart of CNI-treated rats, while pro-angiogenic and stem cell-related genes were upregulated. Moreover, mobilization of stem/progenitor cells was increased through manipulation of the CD26/SDF-1 system.nnnCONCLUSIONnWe conclude from our studies that CNI-treatment significantly prevented cardiac remodelling and improved heart function in Nx rats without affecting renal function and blood pressure. This sheds new light on possible therapeutic strategies for renal patients at high cardiovascular risk.

Hydroxyfasudil-Mediated Inhibition of ROCK1 and ROCK2 Improves Kidney Function in Rat Renal Acute Ischemia-Reperfusion Injury

Renal ischemia-reperfusion (IR) injury (IRI) is a common and important trigger of acute renal injury (AKI). It is inevitably linked to transplantation. Involving both, the innate and the adaptive immune response, IRI causes subsequent sterile inflammation. Attraction to and transmigration of immune cells into the interstitium is associated with increased vascular permeability and loss of endothelial and tubular epithelial cell integrity. Considering the important role of cytoskeletal reorganization, mainly regulated by RhoGTPases, in the development of IRI we hypothesized that a preventive, selective inhibition of the Rho effector Rho-associated coiled coil containing protein kinase (ROCK) by hydroxyfasudil may improve renal IRI outcome. Using an IRI-based animal model of AKI in male Sprague Dawley rats, animals treated with hydroxyfasudil showed reduced proteinuria and polyuria as well as increased urine osmolarity when compared with sham-treated animals. In addition, renal perfusion (as assessed by 18F-fluoride Positron Emission Tomography (PET)), creatinine- and urea-clearances improved significantly. Moreover, endothelial leakage and renal inflammation was significantly reduced as determined by histology, 18F-fluordesoxyglucose-microautoradiography, Evans Blue, and real-time PCR analysis. We conclude from our study that ROCK-inhibition by hydroxyfasudil significantly improves kidney function in a rat model of acute renal IRI and is therefore a potential new therapeutic option in humans.

Circulating Endothelial Progenitor Cells in Kidney Transplant Patients

Background Kidney transplantation (RTx) leads to amelioration of endothelial function in patients with advanced renal failure. Endothelial progenitor cells (EPCs) may play a key role in this repair process. The aim of this study was to determine the impact of RTx and immunosuppressive therapy on the number of circulating EPCs. Methods We analyzed 52 RTx patients (58±13 years; 33 males, mean ± SD) and 16 age- and gender-matched subjects with normal kidney function (57±17; 10 males). RTx patients received a calcineurin inhibitor (CNI)-based (65%) or a CNI-free therapy (35%) and steroids. EPC number was determined by double positive staining for CD133/VEGFR2 and CD34/VEGFR2 by flow cytometry. Stromal cell-derived factor 1 alpha (SDF-1) levels were assessed by ELISA. Experimentally, to dissociate the impact of RTx from the impact of immunosuppressants, we used the 5/6 nephrectomy model. The animals were treated with a CNI-based or a CNI-free therapy, and EPCs (Sca+cKit+) and CD26+ cells were determined by flow cytometry. Results Compared to controls, circulating number of CD34+/VEGFR2+ and CD133+/VEGFR2+ EPCs increased in RTx patients. There were no correlations between EPC levels and statin, erythropoietin or use of renin angiotensin system blockers in our study. Indeed, multivariate analysis showed that SDF-1 – a cytokine responsible for EPC mobilization – is independently associated with the EPC number. 5/6 rats presented decreased EPC counts in comparison to control animals. Immunosuppressive therapy was able to restore normal EPC values in 5/6 rats. These effects on EPC number were associated with reduced number of CD26+ cells, which might be related to consequent accumulation of SDF-1. Conclusions We conclude that kidney transplantation and its associated use of immunosuppressive drugs increases the number of circulating EPCs via the manipulation of the CD26/SDF-1 axis. Increased EPC count may be associated to endothelial repair and function in these patients.

PET with 18F-FDG–Labeled T Lymphocytes for Diagnosis of Acute Rat Renal Allograft Rejection

We proposed small-animal PET with 18F-FDG–labeled T lymphocytes as a new method for image-based diagnosis of acute allogeneic renal transplant rejection (AR) established in a rat model. Methods: One and 2 h after tail vein injection of 30 × 106 ex vivo 18F-FDG–labeled human T cells into male 10-wk-old uninephrectomized, allogeneically transplanted rats (aTX; Lewis–brown Norway [LBN] to Lewis), whole-body radioactivity distribution was assessed in vivo by small-animal PET (postoperative day 4), and percentage injected dose (%ID) as a parameter of T-cell infiltration was assessed and compared between graft and native kidney. In vivo results were confirmed by autoradiography and staining of human CD3 after postmortem dissection. Syngeneically transplanted rats (sTX) (LBN to LBN), rats with ischemia–reperfusion injury (IRI) (45-min warm ischemia), and rats subjected to acute cyclosporine A (CSA) toxicity (50 mg/kg for 2 d intraperitoneally) served as controls. Results: The accumulation of labeled cells was significantly elevated in allografts with AR (1.07 ± 0.28 %ID), compared with native control kidneys (0.49 ± 0.18 %ID) (P < 0.0001). No differences were found among native controls, sTX, CSA toxicity, and kidneys with IRI. In vivo uptake of 18F-FDG cells measured in the PET scanner correlated with results obtained by autoradiography, histologic evaluation, and polymerase chain reaction. Conclusion: We proposed graft PET imaging using 18F-FDG–labeled T cells as a new option to detect rat renal AR with a low dose of 18F-FDG in a noninvasive, fast, and specific manner in rats.

SPECT- and PET-Based Approaches for Noninvasive Diagnosis of Acute Renal Allograft Rejection

Molecular imaging techniques such as single photon emission computed tomography (SPECT) or positron emission tomography are promising tools for noninvasive diagnosis of acute allograft rejection (AR). Given the importance of renal transplantation and the limitation of available donors, detailed analysis of factors that affect transplant survival is important. Episodes of acute allograft rejection are a negative prognostic factor for long-term graft survival. Invasive core needle biopsies are still the “goldstandard” in rejection diagnostics. Nevertheless, they are cumbersome to the patient and carry the risk of significant graft injury. Notably, they cannot be performed on patients taking anticoagulant drugs. Therefore, a noninvasive tool assessing the whole organ for specific and fast detection of acute allograft rejection is desirable. We herein review SPECT- and PET-based approaches for noninvasive molecular imaging-based diagnostics of acute transplant rejection.

Renal Contrast‐Enhanced Sonography Findings in a Model of Acute Cellular Allograft Rejection

Noninvasive methods to diagnose and differentiate acute cellular rejection from acute tubular necrosis or acute calcineurin inhibitor toxicity are still missing. Because T lymphocytes play a decisive role in early states of rejection, we investigated the suitability and feasibility of antibody‐mediated contrast‐enhanced ultrasound by using microbubbles targeted to CD3+, CD4+, or CD8+ T cells in different models of renal disease. In an established rat renal transplantation model, CD3‐mediated ultrasound allows the detection of acute rejection as early as on postoperative day 2. Ultrasound signal intensities increased with the severity of inflammation. Further, an early response to therapy could be monitored by using contrast‐enhanced sonography. Notably, acute tubular necrosis occurring after ischemia–reperfusion injury as well as acute calcineurin inhibitor toxicity could easily be differentiated. Finally, the quantified ultrasound signal correlated significantly with the number of infiltrating T cells obtained by histology and with CD3 mRNA levels, as well as with chemokine CXCL9, CXCL11, and CCL19 mRNA but not with KIM‐1 mRNA expression, thereby representing the severity of graft inflammation but not the degree of kidney injury. In summary, we demonstrate that antibody‐mediated contrast‐enhanced ultrasound targeting T lymphocytes could be a promising tool for an easy and reproducible assessment of acute rejection after renal transplantation.

Non-Invasive Diagnosis of Acute Renal Allograft Rejection − Special Focus on Gamma Scintigraphy and Positron Emission Tomography

The number of patients treated for end-stage renal failure continuously increases. Because treatment alternatives are limited and transplants are often the first therapeutic choice, the numbers of patients joining the waiting lists in countries world-wide rises. At present trans‐ plantation medicine is one of the most progressive fields of medicine. Gradually the “half-life” of renal transplants improved and the five years survival rate ranges now above 80% [1;2]. Despite of the advances made within the last decades, acute rejection (AR) is still a risk for graft survival. The incidence of rejection episodes depends on several factors, e.g., the organ (status), co-morbidities, medication and compliance. Thus, in different situations the incidence of AR varies between 13-53% in the first year after transplantation [3], and, in most cases, cellular and humoral immunity mediated rejections can be distinguished. Usually, AR pro‐ ceeds substantially as an acute cellular rejection whereas humoral rejection comprises only a smaller proportion of AR [4]. Every single episode of an AR is a negative prognostic factor, increasing the risk for development of chronic allograft deterioration and worsening long-term graft survival [5;6]. Interestingly, the impact of AR on chronic renal allograft failure as the main cause for death-censored graft-loss after kidney transplantation increases, whereas the severity of the episode itself is an independent risk factor [7-9]. Therefore, early detection and rapid and effective treatment of AR are essential to preserve graft`s function. Clinically established screening methods such as elevated serum creatinine, occurrence or aggravation of proteinu‐ ria, oliguria, hypertension, graft tenderness, or peripheral edema, often lack the desired sen‐ sitivity and specificity for early diagnoses of AR. Hence,a compelling need for high sensitive

Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using 18F-FDG PET

The number of patients with end-stage renal disease, and the number of kidney allograft recipients continuously increases. Episodes of acute cellular allograft rejection (AR) are a negative prognostic factor for long-term allograft survival, and its timely diagnosis is crucial for allograft function (1). At present, AR can only be definitely diagnosed by core-needle biopsy, which, as an invasive method, bares significant risk of graft injury or even loss. Moreover, biopsies are not feasible in patients taking anticoagulant drugs and the limited sampling site of this technique may result in false negative results if the AR is focal or patchy. As a consequence, this gave rise to an ongoing search for new AR detection methods, which often has to be done in animals including the use of various transplantation models. Since the early 60s rat renal transplantation is a well-established experimental method for the examination and analysis of AR (2). We herein present in addition small animal positron emission tomography (PET) using (18)F-fluorodeoxyglucose (FDG) to assess AR in an allogeneic uninephrectomized rat renal transplantation model and propose graft FDG-PET imaging as a new option for a non-invasive, specific and early diagnosis of AR also for the human situation (3). Further, this method can be applied for follow-up to improve monitoring of transplant rejection (4).