Long-term renal graft outcome after parathyroidectomy - a retrospective single centre study.

BACKGROUND: Surgical correction of hyperparathyroidism after kidney transplantation has been associated with significant graft function decline. We examined the effects of parathyroidectomy on short- and long-term graft function and its potential predictors. METHODS: For this retrospective, monocentric study we identified 48 (5.5%) out of 892 patients from our protocol biopsy program who received renal transplantation between 2000 and 2007, with parathyroidectomy after transplantation. Data from up to three years after parathyroidectomy was collected and analyzed with multivariable linear regression analyses. RESULTS: Main indications for parathyroidectomy were hypercalcemia and graft calcifications. Parathyroidectomy was successful in 47 patients, with a median drop in serum intact parathormone (iPTH) from 394 to 21 pg/ml. Mean estimated glomerular fitration rate (eGFR) before parathyroidectomy was 60 ± 26 ml/min. At three months after parathyroidectomy, the eGFR was 46 ± 18 ml/min (p < 0.001) but remained stable at one and three years (50 ± 20; 49 ± 20 ml/min). The median annual eGFR change was - 0.5 ml/min before and + 1.0 ml/min after parathyroidectomy. Multivariable modeling identified high iPTH levels and higher eGFR before parathyroidectomy as predictors of the eGFR drop after parathyroidectomy. Lower graft function twelve months after parathyroidectomy was predicted by the eGFR before and the iPTH drop after surgery. CONCLUSIONS: These results indicate that the extent of parathyroidectomy is critical and too much lowering of iPTH should be avoided by timely parathyroidectomy, before reaching extreme high iPTH values. In view of the observed loss of eGFR, parathyroidectomy can be considered safe in patients with an eGFR above 30 ml/min.

Soluble neprilysin, NT-proBNP, and growth differentiation factor-15 as biomarkers for heart failure in dialysis patients (SONGBIRD).

BACKGROUND: Dialysis patients are at increased risk of HF. However, diagnostic utility of NT-proBNP as a biomarker is decreased in patients on dialysis. GDF-15 and cNEP are biomarkers of distinct mechanisms that may contribute to HF pathophysiology in such cohorts. The aim of this study was to determine whether growth differentiation factor-15 (GDF-15) and circulating neprilysin (cNEP) improve the diagnosis of congestive heart failure (HF) in patients on dialysis. METHODS AND RESULTS: We compared circulating concentrations of NT-proBNP, GDF-15, and cNEP along with cNEP activity in patients on chronic dialysis without (n = 80) and with HF (n = 73), as diagnosed by clinical parameters and post-dialysis echocardiography. We used correlation, linear and logistic regression as well as receiver operating characteristic (ROC) analyses. Compared to controls, patients with HF had higher median values of NT-proBNP (16,216 [interquartile range, IQR = 27739] vs. 2883 [5866] pg/mL, p < 0.001), GDF-15 (7512 [7084] vs. 6005 [4892] pg/mL, p = 0.014), but not cNEP (315 [107] vs. 318 [124] pg/mL, p = 0.818). Median cNEP activity was significantly lower in HF vs. controls (0.189 [0.223] vs. 0.257 [0.166] nmol/mL/min, p < 0.001). In ROC analyses, a multi-marker model combining clinical covariates, NT-proBNP, GDF-15, and cNEP activity demonstrated best discrimination of HF from controls (AUC = 0.902, 95% CI 0.857-0.947, p < 0.001 vs. base model AUC = 0.785). CONCLUSION: We present novel comparative data on physiologically distinct circulating biomarkers for HF in patients on dialysis. cNEP activity but not concentration and GDF-15 provided incremental diagnostic information over clinical covariates and NT-proBNP and may aid in diagnosing HF in dialysis patients.

Patient Perspectives on Renal Replacement Therapy Modality Choice: A Multicenter Questionnaire Study on Bioethical Dimensions.

Background:Peritoneal dialysis (PD) incidence and prevalence in Germany are low compared with hemodialysis (HD), an underachievement with multifactorial causes. Patient perspectives on renal replacement therapy (RRT) choice play a growing role in research. To date, and to the best of our knowledge, the importance of bioethical dimensions in the context of RRT choice has not been analyzed. The aim of this multicenter questionnaire study was to delineate differences in patient perspectives of PD vs HD in terms of bioethical dimensions, thus helping nephrologists target potential PD candidates more efficiently.Methods:A total of 121 stable outpatients from 2 tertiary care hospitals and 4 dialysis clinics were surveyed for bioethical dimensions ("autonomy," "beneficence," "non-maleficence," "justice," and "trust") with ranking and Likert scale items. Inclusion criteria were RRT > 3 months, age ≥ 18 years, and sufficient cognitive and language skills.Results:A surprisingly high percentage of patients felt excluded from the RRT choice process. Peritoneal dialysis patients were more critical of RRT. They used more versatile information sources on RRT, whereas HD patients were mainly informed by their nephrologist. Peritoneal dialysis patients felt more often dissatisfied with RRT than HD patients and had less trust in their co-patients. However, PD patients felt less autonomy impairment regarding body integrity, fluid balance, and dialysis in general.Conclusions:Our study demonstrates that PD patients showed more scrutiny of their situation as patients, especially their co-patients. Their treatment empowered them toward feeling more autonomous than HD patients. These new insights into patient perspectives on RRT choice might facilitate modality choice for nephrologists.

oxLDL inhibits differentiation of mesenchymal stem cells into osteoblasts via the CD36 mediated suppression of Wnt signaling pathway.

Bone abnormalities as a consequence of osteoblast deregulation are associated with several diseases such as diabetes and chronic kidney disease. Important role for oxidized low density lipoproteins (oxLDL) in the pathophysiology of bone disorders has been reported. However, little is known about the effects and mechanisms of oxLDL on the process of osteoblastogenesis in human mesenchymal stem cells (MSCs). We show that oxLDL concentrations of ~ 10-25 µg protein (0.43-1.0 µM MDA/mg protein) inhibited the differentiation of MSCs to osteoblasts. We demonstrate that the underlying mechanism entails the suppression of the Wnt signaling through the down-regulation of ß-catenin. Further, we show the association of scavenger receptor CD36 with the receptors LRP5/6 and Frizzled in mediating the oxLDL effects on the differentiation of MSCs to pre-osteoblasts. Inhibiting CD36 restored osteoblasts differentiation in the presence of oxLDL. Our findings suggest that oxLDL interferes with the canonical Wnt signaling pathway in a CD36 dependent manner leading to an inhibition of osteoblastogenesis.

oxLDL inhibits differentiation and functional activity of osteoclasts via scavenger receptor-A mediated autophagy and cathepsin K secretion.

Resorptive activity of osteoclasts is important for maintaining bone homeostasis. Endogenous compounds such as oxidized low density lipoprotein (oxLDL) have been shown to disturb this activity. While some studies have investigated the effects of oxLDL on the process of osteoclastogenesis, the underlying mechanism are not fully understood. We show here that oxLDL concentrations of ~10-25 µg protein (0.43-1.0 µM MDA/mg protein) completely blocked the formation of functional osteoclasts. The underlying mechanism implies an inhibition of autophagy that in turn leads to a decreased fusion of cathepsin K (CatK)-loaded lysosomal vesicles with the ruffled border membrane. As result, a lower secretion of CatK and impaired protonation of the resorption lacunae by vacuolar-ATPase (v-ATPase) is observed in the presence of oxLDL. We demonstrate that scavenger receptor A (SR-A) mediates oxLDL effects on osteoclastogenesis and repressing this receptor partially rescued oxLDL effects. Collectively, our data provides an insight into the possible mechanism of oxLDL on osteoclastogenesis suggesting that it does not perturb the packaging of CatK and v-ATPase (V-a3) in the secretory lysosome, but inhibits the fusion of these lysosomes to the ruffled border. The relevance of our findings suggests a distinct link between oxLDL, autophagy and osteoclastogenesis.

A case report of severe calciphylaxis - suggested approach for diagnosis and treatment.

BACKGROUND: Calciphylaxis is a serious complication in patients with chronic kidney disease associated mineral and bone disorder. It can occur in conditions with low and high bone turnover. So far, there are no definite diagnostic and therapeutic guidelines which may prevent the devastating outcome in many calciphylaxis patients. We report a case which clearly illustrates that knowledge of the underlying bone disorder is essential for a directed treatment. Based on this experience we discuss a systematic diagnostic and therapeutic approach in patients with calciphylaxis. CASE PRESENTATION: We report a patient with severe calciphylaxis. Initial evaluation showed an elevated serum parathormone concentration and a bone-specific alkaline phosphatase activity in the upper normal range; however, the bone biopsy clearly showed adynamic bone disease. Extended dialysis with low calcium dialysate concentration and citrate anticoagulation, and administration of teriparatide led to a further increase in bone-specific alkaline phosphatase activity and most importantly, resulted in an activated bone turnover as confirmed by a second bone biopsy 11 weeks later. CONCLUSIONS: This case illustrates that laboratory tests cannot reliably differentiate between high and low bone turnover in calciphylaxis patients. More importantly, this case highlights the fact that specific therapies that alter bone metabolism cannot be applied without knowledge of the bone status. On this background, we suggest that bone biopsies should be an integral part in the diagnosis and therapeutic decision in these patients and should be evaluated in further studies.

A comparative study of bone biopsies from the iliac crest, the tibial bone, and the lumbar spine.

BACKGROUND: Patients with an impaired renal function show a high incidence of bone and mineral disturbances. These 'chronic kidney disease - mineral and bone disorders' (CKD-MBD) range from high turnover osteoporosis to adynamic bone disease. Currently, the histomorphometric analysis of a bone biopsy taken from the iliac crest is viewed as the gold standard for CKD-MBD subtype differentiation. However, the clinical relevance of such a biopsy is questionable since iliac crest fractures are an extremely rare finding. Therefore, we aimed to elucidate if the histomorphometric parameter 'trabecular bone volume (BV/TV)' from the iliac crest is representative for other biopsy locations. We chose two skeletal sites of higher fracture risk for testing, namely, the tibial bone and the lumbar spine, to examine if the current gold standard of bone biopsy is indeed golden. METHODS: Bone biopsies were taken from 12 embalmed body donors at the iliac crest, the proximal tibia, and the lumbar vertebral body, respectively. Masson-Goldner stained sections of methyl methacrylate embedded biopsies were used for trabecular bone volume calculation. Furthermore, exemplary µ-computed tomography (XtremeCT) scans with subsequent analysis were performed. RESULTS: Median values of trabecular bone volume were comparable between all body donors with median (interquartile range, IQR) 18.3% (10.9-22.9%) at the iliac crest, 21.5% (9.5-40.1%) at the proximal tibia, and 16.3% (11.4-25.0%) at the lumbar spine. However, single values showed extensive intra-individual variation, which were also confirmed by XtremeCT imaging. CONCLUSIONS: Distinct intra-individual heterogeneity of trabecular bone volume elucidate why a bone biopsy from one site does not necessarily predict patient relevant endpoints like hip or spine fractures. Physicians interpreting bone biopsy results should know this limitation of the current gold standard for CKD-MBD diagnostic, especially, when systemic therapeutic decisions should be based on it.

Urokinase receptor mediates osteoclastogenesis via M-CSF release from osteoblasts and the c-Fms/PI3K/Akt/NF-κB pathway in osteoclasts.

Bone remodeling is a dynamic process based on a fine-tuned balance between formation and degradation of bone. Osteoblasts (OBLs) are responsible for bone formation and bone resorption is mediated by osteoclasts (OCLs). The mechanisms regulating the OBL-OCL balance are critical in health and disease; however, they are still far from being understood. We reported recently that the multifunctional urokinase receptor (uPAR) mediates osteogenic differentiation of mesenchymal stem cells (MSCs) to OBLs and vascular calcification in atherosclerosis. Here, we address the question of whether uPAR may also be engaged in regulation of osteoclastogenesis. We show that uPAR mediates this process in a dual fashion. Thus, uPAR affected OBL-OCL interplay. We observed that osteoclastogenesis was significantly impaired in co-culture of monocyte-derived OCLs and in OBLs derived from MSCs lacking uPAR. We show that expression and release, from OBLs, of macrophage colony-stimulating factor (M-CSF), which is indispensable for OCL differentiation, was inhibited by uPAR loss. We further found that uPAR, on the other hand, controlled formation, differentiation, and functional properties of macrophage-derived OCLs. Expression of osteoclastogenic markers, such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K, was impaired in OCLs derived from uPAR-deficient macrophages. The requirement of uPAR for osteoclastogenesis was further confirmed by immunocytochemistry and in bone resorption assay. We provide evidence that the underlying signaling mechanisms involve uPAR association with the M-CSF binding receptor c-Fms followed by c-Fms phosphorylation and activation of the PI3K/Akt/NF-κB pathway in OCLs. We further show that uPAR uses this pathway to regulate a balance between OCL differentiation, apoptosis, and cell proliferation. Our study identified uPAR as an important and multifaceted regulator of OBL-OCL molecular interplay that may serve as an attractive target in bone disease and ectopic calcification.

Urokinase receptor mediates osteogenic differentiation of mesenchymal stem cells and vascular calcification via the complement C5a receptor.

Vascular calcification is a severe consequence of several pathological processes with a lack of effective therapy. Recent studies suggest that circulating and resident mesenchymal stem cells (MSC) contribute to the osteogenic program of vascular calcification. Molecular mechanisms underlying MSC osteogenic potential and differentiation remain, however, sparsely explored. We investigated a role for the complement receptor C5aR in these processes. We found that expression of C5aR was upregulated upon differentiation of human MSC to osteoblasts. C5aR inhibition by silencing and specific antagonist impaired osteogenic differentiation. We demonstrate that C5aR expression upon MSC differentiation was regulated by the multifunctional urokinase receptor (uPAR). uPAR targeting by siRNA resulted in complete abrogation of C5aR expression and consequently in the inhibition of MSC-osteoblast differentiation. We elucidated the NFκB pathway as the mechanism utilized by the uPAR-C5aR axis. MSC treatment with the NFκB inhibitor completely blocked the differentiation process. Nuclear translocation of the p65 RelA component of the NFκB complex was induced under osteogenic conditions and impaired by the inhibition of uPAR or C5aR. Dual-luciferase reporter assays demonstrated enhanced NFκB signaling upon MSC differentiation, whereas uPAR and C5aR downregulation lead to inhibition of the NFκB activity. We show involvement of the Erk1/2 kinase in this cascade. In vivo studies in a uPAR/LDLR double knockout mouse model of diet-induced atherosclerosis revealed impaired C5aR expression and calcification in aortic sinus plaques in uPAR(-/-)/LDLR(-/-) versus uPAR(+/+)/LDLR(-/-) control animals. These results suggest that uPAR-C5aR axis via the underlying NFκB transcriptional program controls osteogenic differentiation with functional impact on vascular calcification in vivo.

Invariant natural killer T cells are depleted in renal impairment and recover after kidney transplantation.

BACKGROUND: Altered immune function in patients with renal failure results in both susceptibility to infection and increased inflammatory response. Invariant natural killer T (iNKT) cells are a conserved, immunoregulatory T lymphocyte subset that responds to lipid antigens with near-immediate cytokine production and cytotoxicity. iNKT cells are required for the antibacterial host response. Whether renal failure and renal replacement therapy alter iNKT cell abundance or phenotype has not been investigated. METHODS: iNKT cells were studied by flow cytometry in the peripheral blood of patients with acute renal failure, chronic haemo- and peritoneal dialysis (PD), chronic kidney disease and after renal transplantation. RESULTS: A very marked reduction in iNKT lymphocytes was found in acute renal failure before the first haemodialysis (HD) session. iNKT cells were depleted in end-stage renal disease patients receiving either HD or PD. iNKT cell depletion was accentuated after an HD session. Lesser degrees were observed in patients with non-dialysis-dependent chronic kidney disease. CD56 and CD161 NK cell marker expression was decreased in renal impairment. CD56(+) and CD161(+) iNKT cells produced more interferon-γ than negative cells of the same donor. Within the first year after kidney transplantation, the decrease in iNKT cells and their NK cell markers was reverted. CONCLUSIONS: We describe for the first time that iNKT lymphocytes are reduced in end-stage renal disease and further depleted by HD. iNKT cells are important for early host response including activation of other immune cells and their depletion may contribute to immune dysfunction in renal disease.

Transgastrically placed endoscopic vacuum-assisted closure system as an addition to transgastric necrosectomy in necrotizing pancreatitis (with video).

BACKGROUND: Endoscopic transluminal débridement of infected pancreatic necrosis has been proved to be an important alternative to surgical débridement. Recently, endoscopic vacuum-assisted closure (EVAC) has been described as a new effective treatment option in upper intestinal anastomotic leaks. OBJECTIVE: To test whether the EVAC can be applied to transgastrically accessible infected cavities. DESIGN: Single-center case study. SETTING: Academic medical center. PATIENTS: Two patients with necrotizing pancreatitis. MAIN OUTCOME MEASUREMENT: Successful closure of leak. RESULTS: We successfully applied EVAC to treat transgastrically accessible necrotic cavities. LIMITATIONS: Small case number. CONCLUSIONS: EVAC might be an important additional endoscopic treatment option for infected pancreatic necrosis, especially if established endoscopic treatment options fail.

The tight junction protein ZO-2 and Janus kinase 1 mediate intercellular communications in vascular smooth muscle cells.

Recent evidence points to a multifunctional role of ZO-2, the tight junction protein of the MAGUK (membrane-associated guanylate kinase-like) family. Though ZO-2 has been found in cell types lacking tight junction structures, such as vascular smooth muscle cells (VSMC), little is known about ZO-2 function in these cells. We provide evidence that ZO-2 mediates specific homotypic cell-to-cell contacts between VSMC. Using mass spectrometry we found that ZO-2 is associated with the non-receptor tyrosine kinase Jak1. By generating specific ZO-2 constructs we further found that the N-terminal fragment of ZO-2 molecule is responsible for this interaction. Adenovirus-based expression of Jak1 inactive mutant demonstrated that Jak1 mediates ZO-2 tyrosine phosphorylation. By means of RNA silencing, expression of Jak1 mutant form and fluorescently labeled ZO-2 fusion protein we further specified that active Jak1, but not Jak1 inactive mutant, mediates ZO-2 localization to the sites of intercellular contacts. We identified the urokinase receptor uPAR as a pre-requisite for these cellular events. Functional requirement of the revealed signaling complex for VSMC network formation was confirmed in experiments using Matrigel and in contraction assay. Our findings imply involvement of the ZO-2 tight junction independent signaling complex containing Jak1 and uPAR in VSMC intercellular communications. This mechanism may contribute to vascular remodeling in occlusive cardiovascular diseases and in arteriogenesis.

The tight junction protein ZO-2 mediates proliferation of vascular smooth muscle cells via regulation of Stat1.

AIMS: Recent evidence suggests that the zonula occludens protein 2 (ZO-2) might have additional cellular functions, beyond regulation of paracellular permeability of epithelial and endothelial cells. Deregulation of ZO-2 in response to ischaemia, hypertensive stress, and vascular injury implies its involvement in cardiovascular disorders, most likely via regulating the functional behaviour of vascular smooth muscle cells (VSMC). However, a role of ZO-2 in VSMC biology has yet to be established. Our study was designed to understand the specific functions of ZO-2 in human VSMC. METHODS AND RESULTS: The expression of ZO-2 and Stat1 upon vascular injury was studied using ex vivo organ culture of coronary arteries combined with immunohistochemistry. ZO-2 silencing in human primary VSMC was achieved by means of lentiviral gene transfer. Cell proliferation was assessed by analysing DNA synthesis and by cell counting. Stat1 expression was examined using immunoblotting, immunocytochemistry, TaqMan, and fluorescence activated cell sorting (FACS) analysis. Functional relevance of Stat1 up-regulation was studied using a Stat1 promoter-luciferase reporter assay and intracellular microinjections of a Stat1 specific antibody. ZO-2 was highly expressed in the media and neointima of dilated but not of control arteries, whereas expression of the transcription factor Stat1 was inversely regulated upon injury. Analysis of VSMC with down-regulated ZO-2 revealed increased expression of Stat1 in these cells, whereas Stat1 phosphorylation was not affected. Stat1 up-regulation in VSMC with ZO-2 silencing resulted in a coordinate activation of Stat1-specific genes and consequently led to inhibition of cell proliferation. This effect was restored by microinjection of a Stat1 neutralising antibody. CONCLUSION: Our data suggest that the tight junction protein ZO-2 is involved in regulation of VSMC growth control upon vascular injury that is mediated by the transcription factor Stat1. Our findings point to a novel function of ZO-2 in VSMC and implicate ZO-2 as a novel important molecular target in pathological states of vascular remodelling in cardiovascular diseases.

Tyk2 mediates effects of urokinase on human vascular smooth muscle cell growth.

The urokinase (uPA)/uPA receptor (uPAR) system plays a role in the response of the vessel wall to injury, presumably by modulating vascular smooth muscle cell (VSMC) functional behaviour. The Jak/Stat signaling pathway has been implicated to mediate the uPA/uPAR-directed cell migration and proliferation in VSMC. We have therefore investigated the underlying molecular mechanisms, which remained not completely understood. In particular, we aimed at identification of the kinase involved in the signaling cascade leading to Stat1 phosphorylation by uPA and its impact on VSMC growth. We performed expression in VSMC of kinase-deficient mutant forms of the Janus kinases Jak1 and Tyk2 and used different cell culture models imitating the response to vascular injury. We provide evidence that Tyk2, but not Jak1, mediates uPA-induced Stat1 phosphorylation and VSMC growth inhibition and suggest a novel function for Tyk2 as an important modulator of the uPA-directed VSMC functional behaviour at the place of injury.