Autosomal Dominant Tubulointerstitial Kidney Disease (ADTKD) is caused by mutations in one of at least five genes and leads to kidney failure usually in mid adulthood. Throughout the literature, variable numbers of families have been reported, where no mutation can be found and therefore termed ADTKD-not otherwise specified. Here, we aim to clarify the genetic cause of their diseases in our ADTKD registry. Sequencing for all known ADTKD genes was performed, followed by SNaPshot minisequencing for the dupC (an additional cytosine within a stretch of seven cytosines) mutation of MUC1. A virtual panel containing 560 genes reported in the context of kidney disease (nephrome) and exome sequencing were then analyzed sequentially. Variants were validated and tested for segregation. In 29 of the 45 registry families, mutations in known ADTKD genes were found, mostly in MUC1. Sixteen families could then be termed ADTKD-not otherwise specified, of which nine showed diagnostic variants in the nephrome (four in COL4A5, two in INF2 and one each in COL4A4, PAX2, SALL1 and PKD2). In the other seven families, exome sequencing analysis yielded potential disease associated variants in novel candidate genes for ADTKD; evaluated by database analyses and genome-wide association studies. For the great majority of our ADTKD registry we were able to reach a molecular genetic diagnosis. However, a small number of families are indeed affected by diseases classically described as a glomerular entity. Thus, incomplete clinical phenotyping and atypical clinical presentation may have led to the classification of ADTKD. The identified novel candidate genes by exome sequencing will require further functional validation.

Background: High sensitivity assays for the determination of cardiac troponin I (cTnI) are able to reliably measure cTnI far below the 99th percentile of healthy persons (hs-cTnI) and display sex-specific differences. There is uncertainty regarding the clinical utility of hs-cTnI in asymptomatic hemodialysis (HD) patients and if sex-specific differences also apply in this cohort. Methods: In this multicenter study we measured hs-cTnI and sensitive cTnI (s-TnI) concentrations (both on Siemens Centaur) in 215 HD patients from a predialytic sample to determine the prevalence of elevated concentrations above the 99th percentile, the association with baseline characteristics, prognostic accuracy for death, and sex-specific differences. Results: Hs-cTnI and s-cTnI concentrations were below the 99th percentile in 93% and 85% of patients with a median concentration of 12 ng/L (interquartile range 7-66) and 19 ng/L (12; 31, p < 0.0001). Hs-cTnI and s-cTnI concentrations were independently associated with age (p < 0.05) and ischemic cardiac disease (p < 0.05), but not with residual renal function. Both hs-cTnI and s-cTnI were predictors of death after median follow-up of 2.6 years with an AUC of 0.733 and 0.744, respectively (both p < 0.0001). Important sex-differences emerged for hs-cTnI, but not for s-cTnI: first, women had significantly lower hs-cTnI concentrations than men (p = 0.03); second, hs-cTnI had significantly higher prognostic accuracy for death in women than for men (AUC 0.824 vs. 0.674, p = 0.04). Conclusions: The majority of HD patients have (h)s-cTnI concentrations below the 99th percentile. High normal values are predictive of death. Hs-cTnI allows to elucidate important sex-differences in HD patients with lower concentrations and higher prognostic accuracy in women.

Background/Aims: Cardiovascular calcification contributes to the increased mortality in hemodialysis patients. Sclerostin was identified as an antianabolic bone factor causing soft tissue calcification. Data on prospective large-scale studies associating sclerostin with mortality in hemodialysis patients are so far inconsistent. Methods: In our multicenter prospective longitudinal study following hemodialysis patients, we assessed the associations of sclerostin and bone remodeling markers with long-term mortality. We evaluated the relationship between circulating sclerostin, Fibroblast growth factor 23 (FGF23) and traditional bone remodeling markers. Sclerostin levels in hemodialysis patients were compared with healthy controls. Results: We enrolled 239 hemodialysis patients with a median follow up of 1461 days. In Cox regression analysis, FGF23 (HR 1.40;95%CI 1.11-1.76), parathyroid hormone (PTH) (HR 1.80;95%CI 1.44-2.26) and alkaline phosphatase (AP) (HR 1.50;95%CI 1.10-2.04) per SD, 25(OH)vitamin D (HR 0.42;95%CI 0.23-0.76) per natural log but not sclerostin (HR 1.02;95%CI 0.75-1.38) per SD increase were associated with mortality. FGF23, PTH and AP were negatively associated with sclerostin. Among control and hemodialysis females, sclerostin levels were lower than in men. Conclusion: Higher FGF23, PTH, AP and lower 25(OH)vitamin D but not sclerostin predict long-term mortality. Sclerostin was negatively associated with FGF23, PTH and AP and lower in females than in males.

Vasopressin, endothelin and adrenomedullin are vasoactive peptides that regulate vascular tone and might play a role in hypertensive diseases. Recently, laboratory assays have been developed to measure stable fragments of vasopressin, endothelin and adrenomedullin. Little is known about their diagnostic and prognostic value in hemodialysis patients. In this study, we measured the plasma concentration of copeptin, mid-regional-pro-adrenomedullin (MR-pro-ADM) and C-terminal pro-endothelin 1 (CT-pro-ET1) in stable ambulatory hemodialysis patients (n = 239) and investigated their associations with clinical factors and mortality. In all patients enrolled, the plasma concentrations of copeptin, MR-pro-ADM and CT-pro-ET1 were largely elevated with a median concentration of 132 pmol/L (interquartile range [IQR] 78-192) for copeptin, 1.26 nmol/L (IQR 1.02-1.80) for MR-pro-ADM and 149 pmol/L (IQR 121-181) for CT-pro-ET1. The plasma concentrations of all vasoactive peptide fragments correlated with time on dialysis and plasma β2-microglobulin concentration and were negatively correlated to residual diuresis. The plasma concentration of MR-pro-ADM was a strong predictor of all-cause (univariate hazard ratio for a 10-fold increase 9.94 [3.14;32], p<0.0001) and cardiovascular mortality (hazard ratio 34.87 [5.58;217], p = 0.0001) within a 3.8-year follow-up. The associations remained stable in models adjusted for dialysis specific factors and were attenuated in a full model adjusted for all prognostic factors. Plasma copeptin concentration was weakly associated with cardiovascular mortality (only in univariate analysis) and CT-pro-ET1 was not associated with mortality at all. In conclusion, vasoactive peptide fragments are elevated in hemodialysis patients because of accumulation and, most likely, increased release. Increased concentrations of MR-pro-ADM are predictive of mortality.

Deranged calcium-phosphate metabolism contributes to the burden of morbidity and mortality in dialysis patients. This study aimed to assess the association of the phosphaturic hormone fibroblast growth factor 23 (FGF23) and soluble Klotho with all-cause mortality. We measured soluble Klotho and FGF23 levels at enrolment and two weeks later in 239 prevalent hemodialysis patients. The primary hypothesis was that low Klotho and high FGF23 are associated with increased mortality. The association between Klotho and atrial fibrillation (AF) at baseline was explored as secondary outcome. AF was defined as presence of paroxysmal, persistent or permanent AF. During a median follow-up of 924 days, 59 (25%) patients died from any cause. Lower Klotho levels were not associated with mortality in a multivariable adjusted analysis when examined either on a continuous scale (HR 1.25 per SD increase, 95% CI 0.84-1.86) or in tertiles, with tertile 1 as the reference category (HR for tertile two 0.65, 95% CI 0.26-1.64; HR for tertile three 2.18, 95% CI 0.91-2.23). Higher Klotho levels were associated with the absence of AF in a muItivariable logistic regression analysis (OR 0.66 per SD increase, 95% CI 0.41-1.00). Higher FGF23 levels were associated with mortality risk in a multivariable adjusted analysis when examined either on a continuous scale (HR 1.45 per SD increase, 95% CI 1.05-1.99) or in tertiles, with the tertile 1 as the reference category (HR for tertile two 1.63, 95% CI 0.64-4.14; HR for tertile three 3.91, 95% CI 1.28-12.20). FGF23 but not Klotho levels are associated with mortality in hemodialysis patients. Klotho may be protective against AF.

Background/Aims: Determination of peptide biomarkers such as troponins, natriuretic peptides or the recently reported FGF23 can be useful to identify hemodialysis patients with a high risk of mortality. However, it is desirable to focus on few robust parameters to warrant their routine application. Methods: In a prospective cohort study with 239 prevalent hemodialysis patients we studied the prognostic significance of 10 simultaneously determined modern peptide biomarkers (high sensitive troponin I and T, NT-pro-BNP, BNP, MR-pro-ANP, MR-pro-ADM, CT-pro-ET1, copeptin, FGF23 and a-Klotho) and compared them with parameters traditionally associated with mortality (PTH, Ca, Pi, albumin, CRP, cholesterol, AP). Results: After a follow-up of 4 years, plasma concentration of troponins, natriuretic peptides, MR-pro-ADM, FGF23 as well as PTH, CRP, AP were significantly higher in deceased patients (n=95). Hazard ratios from cox regression on a continuous scale (doubling of plasma concentration) or relative in tertiles were highest for high sensitive troponins, followed by natriuretic peptides and MR-pro-ADM (1.6-2.0 and 2.3-5.5, resp.). C-indices were also highest for troponins (0.708-0.746), followed by natriuretic peptides (0.706-0.731). Traditional parameters had low c-indices (0.598-0.655). Stepwise cox regression revealed that among all parameters troponin I, NT-pro-BNP, PTH and CRP remained independent predictors of mortality and a composite score had the highest c-index (0.799 [0.740-0.849]). Conclusions: Among peptide biomarkers high sensitive troponins and to a lesser extent natriuretic peptides are strong predictors of mortality in asymptomatic hemodialysis patients, followed by markers of mineral-bone disease and inflammation.

Aims/hypothesis In steroid diabetes insulin secretion does not adequately compensate for enhanced hepatic gluconeogenesis and peripheral insulin resistance. Previous studies suggest that activation of the transcription factor forkhead box O1 (FOXO1) contributes to glucocorticoid-induced beta cell death. This study examines the role and regulation of FOXO1 in insulin-secreting cells. Methods INS-1E cells and mouse islet cells were cultured in the presence of dexamethasone. Signalling pathways were modified pharmacologically or by small interfering (si)RNA-mediated inhibition of protein synthesis. Changes in protein abundance and phosphorylation were analysed by western blotting, and subcellular localisation was assessed using confocal microscopy. Transcript levels were examined by RT-PCR. Results Surprisingly, downregulation of FOXO1 by siRNA did not affect dexamethasone-induced apoptosis or Bim expression, but it prevented the effects of the pan protein kinase B (AKT) inhibitor (Akti-1/2). Indeed, dexamethasone and Akti-1/2 synergistically increased beta cell death and Bim expression. Akti-1/2 triggered dephosphorylation and nuclear translocation of FOXO1. Glucocorticoid-receptor activation stimulated Foxo1 transcription, but FOXO1 phosphorylation was unchanged and the cytosolic concentration of FOXO1 remained high in relation to its nuclear concentration. However, subcellular fractionation revealed a significant increase in both cytosolic and nuclear FOXO1 compared with untreated cells. Dexamethasone diminished Pdx1 mRNA level, an effect which was not reversed by siRNA against Foxo1 . Downregulation of AKT isoforms and serum/glucocorticoid-regulated kinase 1 (SGK1) suggests that only sustained suppression of all three AKT isoforms caused dephosphorylation and nuclear accumulation of FOXO1. Conclusions/interpretation This study reveals that FOXO1 is not the main mediator of glucocorticoid-receptor-induced beta cell apoptosis, but rather that it escalates beta cell death when AKT activity is inhibited by distinct pathways.

Background: In hemodialysis patients, elevated plasma troponin concentrations are a common finding that has even increased with the advent of newly developed sensitive assays. However, the interpretation and relevance of this is still under debate.Methods: In this cross-sectional study, we analyzed plasma concentrations of sensitive troponin I (TnI) and troponin T (TnT) in stable ambulatory hemodialysis patients (n = 239) and investigated their associations with clinical factors and mortality.Results: In all of the enrolled patients, plasma TnI or TnT was detectable at a median concentration of 14 pg/ml (interquartile range: 7-29) using the Siemens TnI ultra assay and 49 pg/ml (31-74) using the Roche Elecsys high sensitive TnT assay. Markedly more patients exceeded the 99th percentile for TnT than for TnI (95% vs. 14%, p<0.0001). In a multivariate linear regression model, TnT was independently associated with age, gender, systolic dysfunction, time on dialysis, residual diuresis and systolic blood pressure, whereas TnI was independently associated with age, systolic dysfunction, pulse pressure, time on dialysis and duration of a HD session. During a follow-up period of nearly two years, TnT concentration above 38 pg/mL was associated with a 5-fold risk of death, whereas elevation of TnI had a gradual association to mortality.Conclusion: In hemodialysis patients, elevations of plasma troponin concentrations are explained by cardiac function and dialysis-related parameters, which contribute to cardiac strain. Both are highly predictive of increased risk of death.

Background: Plasma concentrations of natriuretic peptides are often elevated in chronic hemodialysis patients and difficult to interpret due to accumulation, high incidence of cardiac disease and changes in volume status. Mid-regional pro-ANP is a newly developed assay whereas BNP and its fragment NT-pro-BNP are available for a longer time. In this cross-sectional study, we compared the plasma concentration of MR-pro-ANP, BNP and NT-pro-BNP in stable ambulatory hemodialysis patients (n = 239) and investigated their associations with clinical factors such as residual diuresis, cardiac status and interdialytic weight gain and with mortality. Methods and Results: In all patients enrolled, the plasma concentration of all natriuretic peptides were largely elevated with a median concentration of 337 pg/ml (interquartile range 146-684) for BNP, 4435 pg/ml (1687-16228) for NT-proBNP and 907 pmol/L (650-1298) for MR-pro-ANP. Plasma concentration of all natriuretic peptides correlated independently with age, degree of systolic dysfunction and negatively with residual diuresis. Dependency on residual renal clearance was strongest for the fragments MR-pro-ANP and NT-pro-BNP. The plasma concentration of all natriuretic peptides was associated with mortality within 2 years of follow-up. Receiver-operated curves revealed a low sensitivity (32-45%), but high specificity for all natriuretic peptides (85-93%) resulting in a high negative predictive (82-87%). Best cut-off values obtained from were 18 611 pg/ml for NT-pro-BNP, 958 pg/ml for BNP and 1684 pmol/L for MR-pro-ANP. Conclusions: In hemodialysis patients, the fragments NTproBNP and MR-pro-ANP are largely elevated compared to BNP which is explained by accumulation. The prognostic performance of MR-pro-ANP is similar to that of NT-pro-BNP or BNP.

Human JPCs (jaw periosteal cells) are a promising source for the engineering of cell-based osteoinductive grafts in oral surgery. For this purpose, cell characteristics of this stem cell source should be elucidated in detail. Analysis of gene expression profiles may help us to evaluate key factors and cellular targets of JPC osteogenesis. Because little is known about the interplay of osteogenic-related components, we analysed the expression of different collagen types reflecting important players for extracellular matrix assembly and of TIMPs (tissue inhibitors of metalloproteinases) responsible for the inhibition of matrix degradation. Gene expression analyses using microarrays and quantitative RT—PCR (reverse transcription—PCR) during JPC osteogenesis revealed the induction of several collagen types' expression (VII, VIII, XI and XII), and some of them (types I, VIII and XI) seemed to be susceptible to BMP-2 (bone morphogenetic protein-2) that is known to be a potent osteogenic inducer of periosteal cells. Among the TIMPs, only TIMP-4 and RECK (reversion-inducing cysteine-rich protein with Kazal motifs) expressions were strongly up-regulated during JPC osteogenesis. Proteome profiler analysis of supernatants from untreated and differentiated JPCs confirmed the gene expression data in terms of TIMP expression. In summary, we identified new collagen types and TIMPs that seem to play important roles during the osteogenesis of jaw periosteal progenitor cells.

The second messenger cAMP plays an important role in the regulation of renin gene expression. Nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) is known to stimulate renin gene transcription acting through PPARγ-binding sequences in renin promoter. We show now that activation of PPARγ by unsaturated fatty acids or thiazolidinediones drastically augments the cAMP-dependent increase of renin mRNA in the human renin-producing cell line Calu-6. The underlying mechanism involves potentiation of agonist-induced cAMP increase and up-regulation of adenylate cyclase 6 (AC6) gene expression. We identified a palindromic element with a 3-bp spacer (Pal3) in AC6 intron 1 (AC6Pal3). AC6Pal3 bound PPARγ and mediated trans-activation by PPARγ agonist. AC6 knockdown decreased basal renin mRNA level and attenuated the maximal PPARγ-dependent stimulation of the cAMP-induced renin gene expression. AC6Pal3 decoy oligonucleotide abrogated the PPARγ-dependent potentiation of cAMP-induced renin gene expression. Treatment of mice with PPARγ agonist increased AC6 mRNA kidney levels. Our data suggest that in addition to its direct effect on renin gene transcription, PPARγ "sensitizes" renin gene to cAMP via trans-activation of AC6 gene. AC6 has been identified as PPARγ target gene with a functional Pal3 sequence.

Human jaw periosteum-derived cells (JPCs) represent an alternative cell source to bone marrow-derived mesenchymal stem cells for tissue engineering applications in the oral and maxillofacial surgery. In this study we investigated how far the presence or expression of human mesenchymal stem cell antigen-1/tissue non-specific alkaline phosphatase (MSCA-1/TNAP) and LNGFR (CD271) can be utilized to select and enrich the osteogenic progenitor cell fraction from the entire JPC population. Depending on their mineralization capacity, we classified the human isolated JPCs into mineralizing (mJPCs) and non-mineralizing JPCs (nmJPCs). Flow cytometric analyses revealed that undifferentiated mJPCs expressed MSCA-1/TNAP at significant higher levels than nmJPCs at day 5 and 10 of osteogenesis. Western blot analyses showed increased MSCA-1/TNAP expression levels in mJPCs during osteogenesis, whereas in nmJPCs MSCA-1/TNAP expression remained undetectable. Using the MSCA-1 and LNGFR specific antibodies, we separated the positive and negative fractions from the entire mJPC population. In order to analyse the mineralization capacity of the MSCA-1+ and LNGFR+ cell subsets, we quantified the calcium deposition in both subpopulations in comparison to the respective negative subpopulations. The MSCA-1+/TNAP+ cell fraction showed a significant higher osteogenic capacity compared to the MSCA-1-/TNAP- cell fraction whereas the LNGFR+/- cell fractions did not differ in their osteogenic potential. Our findings suggest that MSCA-1 may represent a promising osteogenic marker for mJPC.

Neben dem Diabetes mellitus kommt der arteriellen Hypertonie eine wesentliche Bedeutung für die Entwicklung arteriosklerotischer Erkrankungen zu. Bei Hypertoniepatienten spielt die Kontrolle des Blutdruckes eine entscheidende Rolle zur Verbesserung des kardiovaskulären Outcomes und der Vermeidung mikrovaskulärer Komplikationen. Dies gilt umso mehr in Hochrisikokollektiven wie bei Patienten mit Diabetes mellitus. Bedingt durch die drastische Zunahme von Typ-2-Diabetikern in den letzten Jahrzehnten ist auch eine Zunahme der diabetischen Nephropathie zu verzeichnen – heutzutage stellt diese Krankheitsentität die häufigste Ursache für eine dialysepflichtige terminale Niereninsuffizienz in den westlichen Industrieländern dar. Zusätzlich bedeutet bereits eine geringe Einschränkung der glomerulären Filtrationsrate einen weiteren kardiovaskulären Risikofaktor. Beiden Erkrankungen gemein ist die Albuminurie als Screeningmarker für einen renalen Endorganschaden.