Dominik Kentrup

FGF23 induces left ventricular hypertrophy

Chronic kidney disease (CKD) is a public health epidemic that increases risk of death due to cardiovascular disease. Left ventricular hypertrophy (LVH) is an important mechanism of cardiovascular disease in individuals with CKD. Elevated levels of FGF23 have been linked to greater risks of LVH and mortality in patients with CKD, but whether these risks represent causal effects of FGF23 is unknown. Here, we report that elevated FGF23 levels are independently associated with LVH in a large, racially diverse CKD cohort. FGF23 caused pathological hypertrophy of isolated rat cardiomyocytes via FGF receptor-dependent activation of the calcineurin-NFAT signaling pathway, but this effect was independent of klotho, the coreceptor for FGF23 in the kidney and parathyroid glands. Intramyocardial or intravenous injection of FGF23 in wild-type mice resulted in LVH, and klotho-deficient mice demonstrated elevated FGF23 levels and LVH. In an established animal model of CKD, treatment with an FGF-receptor blocker attenuated LVH, although no change in blood pressure was observed. These results unveil a klotho-independent, causal role for FGF23 in the pathogenesis of LVH and suggest that chronically elevated FGF23 levels contribute directly to high rates of LVH and mortality in individuals with CKD.

Damage of the endothelial glycocalyx in chronic kidney disease

BACKGROUND AND OBJECTIVES The 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. METHODS & RESULTS Shedding 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. CONCLUSION Our 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.

Nanomechanics of the endothelial glycocalyx in experimental sepsis.

The endothelial glycocalyx (eGC), a carbohydrate-rich layer lining the luminal side of the endothelium, regulates vascular adhesiveness and permeability. Although central to the pathophysiology of vascular barrier dysfunction in sepsis, glycocalyx damage has been generally understudied, in part because of the aberrancy of in vitro preparations and its degradation during tissue handling. The aim of this study was to analyze inflammation-induced damage of the eGC on living endothelial cells by atomic-force microscopy (AFM) nanoindentation technique. AFM revealed the existence of a mature eGC on the luminal endothelial surface of freshly isolated rodent aorta preparations ex vivo, as well as on cultured human pulmonary microvascular endothelial cells (HPMEC) in vitro. AFM detected a marked reduction in glycocalyx thickness (266 ± 12 vs. 137 ± 17 nm, P<0.0001) and stiffness (0.34 ± 0.03 vs. 0.21 ± 0.01 pN/mn, P<0.0001) in septic mice (1 mg E. coli lipopolysaccharides (LPS)/kg BW i.p.) compared to controls. Corresponding in vitro experiments revealed that sepsis-associated mediators, such as thrombin, LPS or Tumor Necrosis Factor-α alone were sufficient to rapidly decrease eGC thickness (-50%, all P<0.0001) and stiffness (-20% P<0.0001) on HPMEC. In summary, AFM nanoindentation is a promising novel approach to uncover mechanisms involved in deterioration and refurbishment of the eGC in sepsis.

Treatment of established left ventricular hypertrophy with fibroblast growth factor receptor blockade in an animal model of CKD

BACKGROUND Activation of fibroblast growth factor receptor (FGFR)-dependent signalling by FGF23 may contribute to the complex pathogenesis of left ventricular hypertrophy (LVH) in chronic kidney disease (CKD). Pan FGFR blockade by PD173074 prevented development of LVH in the 5/6 nephrectomy rat model of CKD, but its ability to treat and reverse established LVH is unknown. METHODS CKD was induced in rats by 5/6 nephrectomy. Two weeks later, rats began treatment with vehicle (0.9% NaCl) or PD173074, 1 mg/kg once-daily for 3 weeks. Renal function was determined by urine and blood analyses. Left ventricular (LV) structure and function were determined by echocardiography, histopathology, staining for myocardial fibrosis (Sirius-Red) and investigating cardiac gene expression profiles by real-time PCR. RESULTS Two weeks after inducing CKD by 5/6 nephrectomy, rats manifested higher (mean ± SEM) systolic blood pressure (208 ± 4 versus 139 ± 3 mmHg; P < 0.01), serum FGF23 levels (1023 ± 225 versus 199 ± 9 pg/mL; P < 0.01) and LV mass (292 ± 9 versus 220 ± 3 mg; P < 0.01) when compared with sham-operated animals. Thereafter, 3 weeks of treatment with PD173074 compared with vehicle did not significantly change blood pressure, kidney function or metabolic parameters, but significantly reduced LV mass (230 ± 14 versus 341 ± 33 mg; P < 0.01), myocardial fibrosis (2.5 ± 0.7 versus 5.4 ± 0.95% staining/field; P < 0.01) and cardiac expression of genes associated with pathological LVH, while significantly increasing ejection fraction (18 versus 2.5% post-treatment increase; P < 0.05). CONCLUSIONS FGFR blockade improved cardiac structure and function in 5/6 nephrectomy rats with previously established LVH. These data support FGFR activation as a potentially modifiable, blood pressure-independent molecular mechanism of LVH in CKD.

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

AIMS Chronic 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. METHODS AND RESULTS Male 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. CONCLUSION We 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.

Potential of Noninvasive Serial Assessment of Acute Renal Allograft Rejection by 18F-FDG PET to Monitor Treatment Efficiency

We propose 18F-FDG PET as a method to monitor acute rejection of allogeneic renal transplants in a rat model. Methods: Allogeneically transplanted (aTX) rats (binephrectomized Lewis–brown Norway to Lewis) served as the renal transplant model. aTX rats treated with cyclosporine A (CSA) served as a therapy monitoring group. Healthy control rats, rats with acute CSA nephrotoxicity, rats with acute tubular necrosis, syngeneically transplanted (sTX) rats, and aTX rats treated with CSA since postoperative day 0 served as controls. After surgery, renal glucose metabolism was assessed in vivo serially up to postoperative day 7 by performing small-animal PET 3 h after intravenous injection of 30 MBq of 18F-FDG. Mean radioactivity (cps/mm3 of tissue) was measured and the percentage injected dose calculated. Results were confirmed by histologic, functional, and autoradiographic analysis. Results: Renal 18F-FDG uptake was significantly elevated at postoperative day 4 in aTX rats, when compared with control, sTX, acute tubular necrosis, or CSA-treated rats (P < 0.05). In vivo 18F-FDG uptake correlated with the results of autoradiography and with inflammatory infiltrates observed on histologic examination. Notably, 18F-FDG PET assessed the response to therapy 48 h earlier than the time at which serum creatinine decreased and when histologic examination still showed signs of allograft rejection. In aTX rats, the CSA-susceptible graft infiltrate was dominated by activated cytotoxic T cells and monocytes/macrophages. Conclusion: 18F-FDG PET is an option to noninvasively assess early response to therapy in rat renal allograft rejection.

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.