Screening for health-related quality of life and its determinants in Fabry disease: A cross-sectional multicenter study.

BACKGROUND: Fabry disease (FD) is a rare X-linked lysosomal storage disorder caused by α-galactosidase A (α-Gal A) deficiency. The progressive accumulation of globotriaosylceramide results in life-threatening complications, including renal, cardiac, and cerebrovascular diseases. In order to improve health care of FD-patients, knowledge of its predictors is important. The aim of our study was to evaluate health-related quality of life (HrQol) in FD and to identify its independent determinants by exploring a wide range of demographic, social and clinical parameters. RESULTS: In this cross-sectional multicenter study, 135 adult patients with FD were recruited at three specialized European centers in Germany and Switzerland. Demographics, social status and clinical parameters as well as data on HrQol (EQ5D, EQ VAS) and depression were collected by means of self-reporting questionnaires and confirmed by medical records. HrQol and its predictors were evaluated by univariate and multivariate regression analyses. The study population consisted of 78 female and 57 male FD patients (median age 48 yrs) of whom 80.7% (N = 109) were on enzyme replacement therapy (ERT) and 10.4% (N = 14) were on chaperone treatment. Univariate analysis revealed various factors reducing HrQol such as age > 40 years, classic phenotype, organ involvement (kidney and heart disease, stroke/transient ischemic attack (TIA), gastrointestinal disturbances), depression, and burning limb pain. However, only the following factors were identified as independent predictors of decreased HrQol: classic phenotype, kidney and heart disease, stroke/TIA, depression, and burning limb pain. ERT and chaperone therapy were independent determinants of increased HrQol. CONCLUSIONS: Modifiable factors, such as burning limb pain and depression, identified as independent predictors of HrQol-deterioration should be addressed in programs aiming to improve HrQol in FD. A multidisciplinary approach is essential in FD-patients since diverse organ involvement prominently compromises HrQol in affected patients. Our findings showed that the classic phenotype is a strong predictor of worsening HrQol.

Treatment switch in Fabry disease- a matter of dose?

BACKGROUND: Patients with Fabry disease (FD) on reduced dose of agalsidase-beta or after switch to agalsidase-alfa show a decline in chronic kidney disease epidemiology collaboration-based estimated glomerular filtration rate (eGFR) and a worsened plasma lyso-Gb3 decrease. Hence, the most effective dose is still a matter of debate. METHODS: In this prospective observational study, we assessed end-organ damage and clinical symptoms in 78 patients who had received agalsidase-beta (1.0 mg/kg) for >1 year, which were assigned to continue this treatment (agalsidase-beta, regular-dose group, n=17); received a reduced dose of agalsidase-beta and subsequent switch to agalsidase-alfa (0.2 mg/kg) or a direct switch to 0.2 mg/kg agalsidase-alfa (switch group, n=22); or were re-switched to agalsidase-beta after receiving agalsidase-alfa for 12 months (re-switch group, n=39) with a follow-up of 88±25 months. RESULTS: No differences for clinical events were observed for all groups. Patients within the re-switch group started with the worst eGFR values at baseline (p=0.0217). Overall, eGFR values remained stable in the regular-dose group (p=0.1052) and decreased significantly in the re-switch and switch groups (p<0.0001 and p=0.0052, respectively). However, in all groups males presented with an annual loss of eGFR by -2.9, -2.5 and -3.9 mL/min/1.73 m² (regular-dose, re-switch, switch groups, all p<0.05). In females, eGFR decreased significantly only in the re-switch group by -2.9 mL/min/1.73 m² per year (p<0.01). Lyso-Gb3 decreased in the re-switch group after a change back to agalsidase-beta (p<0.05). CONCLUSIONS: Our data suggest that a re-switch to high dosage of agalsidase results in a better biochemical response, but not in a significant renal amelioration especially in classical males.

Precision medicine in Fabry disease.

Fabry disease (FD) is a rare X-linked lysosomal storage disorder caused by mutations in the α-galactosidase A (GLA) gene, leading to a deficiency in α-galactosidase A. The lysosomal accumulation of glycosphingolipids, primarily globotriaosylceramide (Gb3) and its deacylated form, globotriaosylsphingosine (lyso-Gb3), results in progressive renal failure, cardiomyopathy associated with cardiac arrhythmia and recurrent cerebrovascular events, significantly limiting life expectancy in affected patients. In male patients, a definitive diagnosis of FD involves demonstrating a GLA deficiency in leucocytes. In females, because of the potential high residual enzymatic activity, the diagnostic gold standard requires molecular genetic analyses. The current treatment options for FD include recombinant enzyme replacement therapies (ERTs) with intravenous agalsidase-α (0.2 mg/kg body weight) or agalsidase-ß (1 mg/kg body weight) every 2 weeks as well as an oral pharmacological chaperone (migalastat 123 mg every other day) that selectively and reversibly binds to the active sites of amenable mutant forms of the GLA enzyme. These therapies facilitate cellular Gb3 clearance and an overall improvement of disease burden. However, ERT can lead to infusion-associated reactions, as well as the formation of neutralizing anti-drug antibodies in ∼40% of all ERT-treated males, leading to an attenuation of therapy efficacy. This article reviews the clinical presentation, diagnosis and interdisciplinary clinical management of FD and discusses the therapeutic options, with a special focus on precision medicine, accounting for individual variability in genetic mutations, Gb3 and lyso-Gb3 levels, allowing physicians to predict more accurately which prevention and treatment strategy is best for which patient.

Generation and Characterization of a Polyclonal Human Reference Antibody to Measure Anti-Drug Antibody Titers in Patients with Fabry Disease.

Male patients with Fabry disease (FD) are at high risk for the formation of antibodies to recombinant α-galactosidase A (AGAL), used for enzyme replacement therapy. Due to the rapid disease progression, the identification of patients at risk is highly warranted. However, currently suitable references and standardized protocols for anti-drug antibodies (ADA) determination do not exist. Here we generate a comprehensive patient-derived antibody mixture as a reference, allowing ELISA-based quantification of antibody titers from individual blood samples. Serum samples of 22 male patients with FD and ADAs against AGAL were pooled and purified by immune adsorption. ADA-affinities against agalsidase-α, agalsidase-ß and Moss-AGAL were measured by quartz crystal microbalance with dissipation monitoring (QCM-D). AGAL-specific immune adsorption generated a polyclonal ADA mixture showing a concentration-dependent binding and inhibition of AGAL. Titers in raw sera and from purified total IgGs (r2 = 0.9063 and r2 = 0.8952, both p < 0.0001) correlated with the individual inhibitory capacities of ADAs. QCM-D measurements demonstrated comparable affinities of the reference antibody for agalsidase-α, agalsidase-ß and Moss-AGAL (KD: 1.94 ± 0.11 µM, 2.46 ± 0.21 µM, and 1.33 ± 0.09 µM, respectively). The reference antibody allows the ELISA-based ADA titer determination and quantification of absolute concentrations. Furthermore, ADAs from patients with FD have comparable affinities to agalsidase-α, agalsidase-ß and Moss-AGAL.

α-Galactosidase a Deficiency in Fabry Disease Leads to Extensive Dysregulated Cellular Signaling Pathways in Human Podocytes.

Fabry disease (FD) is caused by mutations in the α-galactosidase A (GLA) gene encoding the lysosomal AGAL enzyme. Loss of enzymatic AGAL activity and cellular accumulation of sphingolipids (mainly globotriaosylcermide) may lead to podocyturia and renal loss of function with increased cardiovascular morbidity and mortality in affected patients. To identify dysregulated cellular pathways in FD, we established a stable AGAL-deficient podocyte cell line to perform a comprehensive proteome analysis. Imbalanced protein expression and function were analyzed in additional FD cell lines including endothelial, epithelial kidney, patient-derived urinary cells and kidney biopsies. AGAL-deficient podocytes showed dysregulated proteins involved in thermogenesis, lysosomal trafficking and function, metabolic activity, cell-cell interactions and cell cycle. Proteins associated with neurological diseases were upregulated in AGAL-deficient podocytes. Rescues with inducible AGAL expression only partially normalized protein expression. A disturbed protein expression was confirmed in endothelial, epithelial and patient-specific cells, pointing toward fundamental pathway disturbances rather than to cell type-specific alterations in FD. We conclude that a loss of AGAL function results in profound changes of cellular pathways, which are ubiquitously in different cell types. Due to these profound alterations, current approved FD-specific therapies may not be sufficient to completely reverse all dysregulated pathways.

FAbry STabilization indEX (FASTEX): Clinical evaluation of disease progression in Fabry patients.

BACKGROUND: Two established scores, the Mainz Severity Score Index (MSSI) and Fabry Disease Severity Scoring System (DS3), quantify the disease burden in Fabry disease (FD), while the recent developed FAbry STabilization indEX (FASTEX) aims to detect disease progression. OBJECTIVE: MSSI, DS3 and FASTEX were compared to evaluate disease stability or progression in a prospective cohort of Fabry patients under enzyme replacement therapy (ERT). METHODS: Disease load of 62 patients (28 [45%] females) treated with ERT (26 [42%] under agalsidase-alfa) was assessed using the current scores and re-assessed after 12 months of treatment. Fifteen (24%) patients were ERT-naïve at baseline. RESULTS: All scores showed a correlation with each other, while MSSI and DS3 showed the strongest (Pearson r: 0.81, p < .0001). Plasma lyso-Gb3 levels in naïve patients correlated with increasing DS3 and MSSI scores (Pearson r: 0.60, p < .05; Pearson r: 0.64, p < .01; respectively), but not with the total weighted FASTEX score. Longitudinal analysis suggested a stable disease course using DS3 and MSSI. Only males long-term-treated with agalsidase-alfa presented with a slight increase of the general MSSI score (p = .0084). By contrast, the FASTEX score demonstrates that only 21 patients (33.9%) were stable, all other patients presented a disease progression. Patients with an unstable FASTEX mainly suffered from a significant loss of renal function (eGFRcreat: -2.7 ± 7.3 ml/min/1.73 m2, p = .0298). CONCLUSION: We conclude that the FASTEX seems to be a simple and user friendly, valuable tool to assess early changes in disease progression even in smaller patient cohorts and short term surveillance.

Detailed epitope mapping of neutralizing anti-drug antibodies against recombinant α-galactosidase A in patients with Fabry disease.

BACKGROUND: Fabry disease (FD) is a lysosomal storage disease, treatable by enzyme replacement therapy (ERT) that substitutes deficient α-galactosidase A (AGAL). The formation of neutralizing anti-drug antibodies (ADA) inhibiting AGAL activity is associated with disease progression in affected male patients. In the current study, we performed a detailed epitope mapping of ADAs from antibody-positive males against infused AGAL. METHODS: A detailed epitope mapping for 34 male FD patients with neutralizing ADAs against AGAL was performed. Based on this data, in silico analyses were used to identify potential epitope clusters and mapped surface-located or buried epitopes. ELISA-based assays against α-galactosidase B (NAGA) were performed to identify ADAs that potentially recognize shared epitopes of AGAL and NAGA. A subset of 20 patients was analyzed to assess if NAGA-recognizing ADAs against AGAL might affect long-term outcomes under ERT. RESULTS: Thirty percent of the AGAL active site was recognized by patients' ADAs. No differences between buried and surface-located epitopes were observed. Dependent on the epitopes, ADAs against AGAL were also able to recognize human NAGA. Patients with NAGA recognizing anti-AGAL antibodies presented with lower plasma NAGA activities. The presence of NAGA-recognizing ADAs had no effect on disease progression. CONCLUSION: In conclusion, our current data underline previous reports demonstrating a large variation of antibody epitopes against AGAL. Detailed epitope mapping in affected patients might be the first step for the generation of patient-specific blocking peptides and/or immune adsorption columns for an individually tailored anti-antibody strategy.

Soluble adenylyl cyclase (sAC) regulates calcium signaling in the vascular endothelium.

The vascular endothelium acts as a selective barrier between the bloodstream and extravascular tissues. Intracellular [Ca2+]i signaling is essential for vasoactive agonist-induced stimulation of endothelial cells (ECs), typically including Ca2+ release from the endoplasmic reticulum (ER). Although it is known that interactions of Ca2+ and cAMP as ubiquitous messengers are involved in this process, the individual contribution of cAMP-generating adenylyl cyclases (ACs), including the only soluble AC (sAC; ADCY10), remains less clear. Using life-cell microscopy and plate reader-based [Ca2+]i measurements, we found that human immortalized ECs, primary aortic and cardiac microvascular ECs, and primary vascular smooth muscle cells treated with sAC-specific inhibitor KH7 or anti-sAC-small interfering RNA did not show endogenous or exogenous ATP-induced [Ca2+]i elevation. Of note, a transmembrane AC (tmAC) inhibitor did not prevent ATP-induced [Ca2+]i elevation in ECs. Moreover, l-phenylephrine-dependent constriction of ex vivo mouse aortic ring segments was also reduced by KH7. Analysis of the inositol-1,4,5-trisphosphate (IP3) pathway revealed reduced IP3 receptor phosphorylation after KH7 application, which also prevented [Ca2+]i elevation induced by IP3 receptor agonist adenophostin A. Our results suggest that sAC rather than tmAC controls the agonist-induced ER-dependent Ca2+ response in ECs and may represent a treatment target in arterial hypertension and heart failure.-Mewes, M., Lenders, M., Stappers, F., Scharnetzki, D., Nedele, J., Fels, J., Wedlich-Söldner, R., Brand, S.-M., Schmitz, B., Brand, E. Soluble adenylyl cyclase (sAC) regulates calcium signaling in the vascular endothelium.

Neutralising anti-drug antibodies in Fabry disease can inhibit endothelial enzyme uptake and activity.

Fabry disease (FD) is a lysosomal storage disease, treatable by enzyme replacement therapy (ERT) that substitutes deficient α-galactosidase A (AGAL). The formation of neutralising anti-drug antibodies (ADA) inhibiting AGAL activity during infusion is associated with disease progression in affected male patients. In this study we analysed if ADAs also inhibit endothelial enzyme uptake as well as intracellular enzyme activity. Therefore, fluorescence-labelled AGAL in combination with ADA-positive sera from FD patients (n = 8) was used to analyse enzyme uptake in endothelial and FD-specific cells. Furthermore, immune adsorption and a comprehensive ADA epitope mapping were performed. Pre-incubation of AGAL with ADAs significantly inhibited intracellular enzyme activity, which was rescued by immune adsorption (both P < .01). ADAs from some patients also inhibited enzyme uptake. ADA epitope mapping identified an epitope at position 121 to 140 aa potentially responsible for uptake inhibition for these patients. Further analyses revealed the presence of stable AGAL/ADA-immune complexes at pH 4.5 and decreased intracellular enzyme activity in endothelial cells (P < .001). Finally, the pre-incubation of AGAL with ADAs resulted in a reduced depletion of intracellular globotriaosylceramide in patient-derived AGAL-deficient cells, demonstrating a direct negative impact of ADAs on intracellular clearance. Neutralising ADAs may not only inhibit infused AGAL activity, but according to their epitopes can also inhibit endothelial AGAL uptake. Indeed, internalised AGAL/ADA-complexes may not dissociate, underlining the importance of novel therapeutic approaches for ADA reduction and prevention to increase therapy efficiency in affected patients.

Mutation-specific Fabry disease patient-derived cell model to evaluate the amenability to chaperone therapy.

BACKGROUND: Patients with Fabry disease (FD) and amenable mutations can be treated with the chaperone migalastat to restore endogenous α-galactosidase A (AGAL) activity. However, certain amenable mutations do not respond biochemically in vivo as expected. Here, we aimed to establish a patient-specific and mutation-specific cell model to evaluate the amenability to chaperone therapy in FD. METHODS: Since current tests to determine amenability are limited to heterologous mutation expression in HEK293T cells with endogenous AGAL activity, we generated CRISPR/Cas9-mediated AGAL-deficient HEK293T cells as a basis for mutant overexpression. Furthermore, primary urinary cells from patients were isolated and immortalised as a patient-specific cell model system to evaluate the amenability to chaperone therapy. RESULTS: Under treatment (>13 months), carriers of p.N215S (n=6) showed a significant reduction of plasma lyso-Gb3 (p<0.05). Lyso-Gb3 levels in carriers of p.L294S increased (p<0.05) and two patients developed severe albuminuria. Both missense mutations were amenable in wild-type HEK293T cells (p<0.05), but presented different responses in CRISPR/Cas9-mediated AGAL knockouts and immortalised urinary cells. Chaperone incubation resulted in increased AGAL activity (p<0.0001) and intracellular globotriaosylceramide (Gb3) reduction (p<0.05) in immortalised p.N215S cells but not in p.L294S and IVS2+1 G>A cells. CONCLUSION: We conclude that repeated AGAL activity measurements in patients' white blood cells are mandatory to assess the in vivo amenability to migalastat. Plasma lyso-Gb3 might be an appropriate tool to measure the biochemical response to migalastat. Patients with low AGAL activities and increasing lyso-Gb3 levels despite in vitro amenability might not benefit sufficiently from chaperone treatment.

Predicting the Development of Anti-Drug Antibodies against Recombinant alpha-Galactosidase A in Male Patients with Classical Fabry Disease.

Fabry Disease (FD) is a rare, X-linked, lysosomal storage disease that mainly causes renal, cardiac and cerebral complications. Enzyme replacement therapy (ERT) with recombinant alpha-galactosidase A is available, but approximately 50% of male patients with classical FD develop inhibiting anti-drug antibodies (iADAs) that lead to reduced biochemical responses and an accelerated loss of renal function. Once immunization has occurred, iADAs tend to persist and tolerization is hard to achieve. Here we developed a pre-treatment prediction model for iADA development in FD using existing data from 120 classical male FD patients from three European centers, treated with ERT. We found that nonsense and frameshift mutations in the α-galactosidase A gene (p = 0.05), higher plasma lysoGb3 at baseline (p < 0.001) and agalsidase beta as first treatment (p = 0.006) were significantly associated with iADA development. Prediction performance of a Random Forest model, using multiple variables (AUC-ROC: 0.77) was compared to a logistic regression (LR) model using the three significantly associated variables (AUC-ROC: 0.77). The LR model can be used to determine iADA risk in individual FD patients prior to treatment initiation. This helps to determine in which patients adjusted treatment and/or immunomodulatory regimes may be considered to minimize iADA development risk.

Effects of high-intensity interval training on microvascular glycocalyx and associated microRNAs.

High-intensity interval training (HIIT) has been proposed to exert vasculoprotective effects. This study aimed to evaluate whether HIIT affects the microvasculature, including the endothelial glycocalyx barrier, and to identify associated microRNAs (miRNAs). Fifty healthy participants (23.1 ± 3.0 yr) performed a 4-wk 4 × 30-s all-out running HIIT. Sidestream dark-field imaging was performed at baseline and follow-up to detect changes of the sublingual microvasculature including the endothelial glycocalyx. Exercise parameters were determined by continuous running field test and documentation of high-intensity runs. miRNAs potentially associated with glycocalyx thickness were selected by structured literature search and blood samples for miRNA, and lactate measurements were drawn at baseline and follow-up HIIT. At baseline, a correlation between maximal exercise performance capacity and glycocalyx thickness (determined by perfused boundary region) was detected (P = 0.045, r = 0.303). Increased exercise performance at follow-up also correlated with glycocalyx thickness (P = 0.031, r = 0.416), and increased high-intensity sprinting speed was associated with an increased number of perfused vessels (P = 0.0129, r = 0.449). Literature search identified miR-143, -96-5p, and -24, which were upregulated by HIIT already at baseline and showed an association with peak blood lactate levels after sprints (all P < 0.05). Moreover, increased baseline miR-143 levels predicted increased glycocalyx thickness at follow-up (AUCmiR-143 = 0.92, 95% confidence interval, 0.81-1.0, P = 0.0008). Elevated resting miR-126 levels after the intervention were associated with cell-free versican mRNA levels. We conclude that HIIT induces changes in the endothelial glycocalyx of the microvasculature. Associated miRNAs such as miR-143 may represent a tool for monitoring early vasculoprotective adaptations to physical activity. NEW & NOTEWORTHY High-intensity interval training is known to improve health-related fitness in general and in lifestyle-induced chronic diseases. To visualize microvasculature structure and to detect exercise-induced changes, sublingual sidestream dark-field imaging microscopy was used, and circulating miRNAs were measured. This study shows that exercise-induced changes correlate with associated circulating miRNA, which might be useful for monitoring vasculoprotective effects. Furthermore, sidestream dark-field imaging may represent a sensitive tool for the early detection of exercise-induced systemic vascular changes.

Dose-Dependent Effect of Enzyme Replacement Therapy on Neutralizing Antidrug Antibody Titers and Clinical Outcome in Patients with Fabry Disease.

BACKGROUND: Use of enzyme replacement therapy (ERT) to treat Fabry disease, caused by deficient lysosomal α-galactosidase A activity, can lead to formation of neutralizing antidrug antibodies (ADAs). These antibodies are associated with increased accumulation of plasma globotriaosylceramide (Gb3) and disease progression. Because agalsidase ERT can saturate ADA-binding sites during infusions (achieving agalsidase/antibody equilibrium), we investigated in this open cohort study whether saturated patients (who have excess agalsidase after infusions) experience better clinical outcomes compared with not saturated patients (who have excess ADAs after infusions). METHODS: We isolated ADAs from sera of 26 men with Fabry disease receiving ERT (for a median of 94 months) and determined the amount of agalsidase necessary for antibody saturation. Clinical and biochemical outcomes included measurements of eGFR, interventricular septum thickness, and lyso-Gb3. RESULTS: ADA titers decreased significantly in all patients during infusion. Agalsidase-α and agalsidase-ß had similar ADA-binding capacity and comparable ADA saturation frequency. Fourteen patients with saturated ADAs presented with mild (but significant) loss of eGFR, stable septum thickness, and significantly decreased lyso-Gb3 levels. The 12 not saturated patients had a more pronounced and significant loss of eGFR, increased septum thickness, and a smaller, nonsignificant reduction in lyso-Gb3, over time. In three patients, dose escalation resulted in partially elevated ADA titers, but importantly, also in reduced lyso-Gb3 levels. CONCLUSIONS: A not saturated ADA status during infusion is associated with progressive loss of eGFR and ongoing cardiac hypertrophy. Dose escalation can result in saturation of ADAs and decreasing lyso-Gb3 levels, but may lead to increased ADA titers.

Fabry disease under enzyme replacement therapy-new insights in efficacy of different dosages.

Background: Fabry patients on reduced dose of agalsidase-beta or after switch to agalsidase-alfa show a decline in estimated glomerular filtration rate (eGFR) and an increase of the Mainz Severity Score Index. Methods: In this prospective observational study, we assessed end-organ damage and clinical symptoms in 112 patients who had received agalsidase-beta (1.0 mg/kg) for >1 year, who were (i) non-randomly assigned to continue this treatment regime (regular-dose group, n = 37); (ii) received a reduced dose of agalsidase-beta and subsequent switch to agalsidase-alfa (0.2 mg/kg) or a direct switch to 0.2 mg/kg agalsidase-alfa (switch group, n = 38); or (iii) were re-switched to agalsidase-beta after receiving agalsidase-alfa for at least 12 months (re-switch group, n = 37) with a median follow-up of 53 (38-57) months. Results: eGFR of patients in the regular-dose group remained stable. Patients in the switch group showed an annual eGFR loss of - 4.6 ± 9.1 mL/min/1.73 m2 (P < 0.05). Patients in the re-switch group also had an eGFR loss of - 2.2 ± 4.4 mL/min/1.73 m2 after re-switch to agalsidase-beta, but to a lower degree compared with the switch group (P < 0.05). Patients in the re-switch group suffered less frequently from diarrhoea (relative risk 0.42; 95% confidence interval 0.19-0.93; P = 0.02). Lyso-Gb3 remained stable in the switch (P = 0.97) and the regular-dose (P = 0.48) groups, but decreased in the re-switch group after change of the therapy regimen (P < 0.05). Conclusions: After switch to agalsidase-alfa, Fabry patients experienced a continuous decline in eGFR, while this decline was attenuated in patients who were re-switched to agalsidase-beta. Decreasing lyso-Gb3 levels may indicate a better treatment response in the latter group.

Salt-induced Na+/K+-ATPase-α/ß expression involves soluble adenylyl cyclase in endothelial cells.

High dietary salt intake may lead to vascular stiffness, which predicts cardiovascular diseases such as heart failure, and myocardial and cerebral infarctions as well as renal impairment. The vascular endothelium is a primary target for deleterious salt effects leading to dysfunction and endothelial stiffness. We hypothesize that the Ca2+- and bicarbonate-activated soluble adenylyl cyclase (sAC) contributes to Na+/K+-ATPase expression regulation in vascular endothelial cells and is an important regulator of endothelial stiffness. In vitro stimulation of vascular endothelial cells with high sodium (150 mM Na+)-induced Na+/K+-ATPase-α and Na+/K+-ATPase-ß protein expression determined by western blot. Promoter analyses revealed increased cAMP response element (CRE)-mediated Na+/K+-ATPase-α transcriptional activity under high sodium concentrations. Inhibition of sAC by the specific inhibitor KH7 or siRNA reduced the sodium effects. Flame photometry revealed increased intracellular sodium concentrations in response to high sodium stimulations, which were paralleled by elevated ATP levels. Using atomic force microscopy, a nano-technique that measures cellular stiffness and deformability, we detected significant endothelial stiffening under increased sodium concentrations, which was prevented by inhibition of sAC using KH7 and Na+/K+-ATPase using ouabain. Furthermore, analysis of primary aortic endothelial cells in an in vitro aging model revealed an impaired Na+/K+-ATPase-α sodium response and elevated intracellular sodium levels with cellular aging. We conclude that sAC mediates sodium-induced Na+/K+-ATPase expression in vascular endothelium and is an important regulator of endothelial stiffness. The reactivity of Na+/K+-ATPase-α expression regulation in response to high sodium seems to be impaired in aging endothelial cells and might be a component of endothelial dysfunction.

Renal function predicts long-term outcome on enzyme replacement therapy in patients with Fabry disease.

BACKGROUND: Renal and cardiac involvement is responsible for substantial morbidity and mortality in Fabry disease (FD). We analysed the incidence of FD-related renal, cardiac and neurologic end points in patients with FD on long-term enzyme replacement therapy (ERT). METHODS: A retrospective analysis of prospectively collected data from two German FD centres was performed. The impact of renal and cardiac function at ERT-naïve baseline on end point development despite ERT was analysed. RESULTS: Fifty-four patients (28 females) receiving ERT (mean 81 ± 21 months) were investigated. Forty per cent of patients were diagnosed with clinical end points before ERT initiation and 50% of patients on ERT developed new clinical end points. In patients initially diagnosed with an end point before ERT initiation, the risk for an additional end point on ERT was increased {hazard ratio [HR] 3.83 [95% confidence interval (CI) 1.61-9.08]; P = 0.0023}. A decreased glomerular filtration rate (eGFR) ≤75 mL/min/1.73 m2 in ERT-naïve patients at baseline was associated with an increased risk for cardiovascular end points [HR 3.59 (95% CI 1.15-11.18); P = 0.0273] as well as for combined renal, cardiac and neurologic end points on ERT [HR 4.77 (95% CI 1.93-11.81); P = 0.0007]. In patients with normal kidney function, left ventricular hypertrophy at baseline predicted a decreased end point-free survival [HR 6.90 (95% CI 2.04-23.27); P = 0.0018]. The risk to develop an end point was independent of sex. CONCLUSIONS: In addition to age, even moderately impaired renal function determines FD progression on ERT. In patients with FD, renal and cardiac protection is warranted to prevent patients from deleterious manifestations of the disease.

Serum-Mediated Inhibition of Enzyme Replacement Therapy in Fabry Disease.

Fabry disease (FD) is a progressive multisystemic disorder, treatable with recombinant enzyme replacement therapy (agalsidase). However, recent studies suggest an endogenous inhibition of agalsidase in patients with FD, as reported for other lysosomal storage diseases. To assess the clinical consequences of serum-mediated agalsidase inhibition in affected patients, we determined the agalsidase inhibition status of 168 patients (68 male) with FD and compared outcomes of inhibition-positive patients with those of inhibition-negative patients. The assessment included clinical events during time on agalsidase, determination of renal and cardiac function, and evaluation of FD-related symptoms. The frequency of serum-mediated agalsidase inhibition was 40% in agalsidase-treated males. Inhibition did not depend on the compound initially used (agalsidase-α or -ß). Agalsidase inhibition was associated with higher lyso-globotriaosylceramide levels and worse disease severity scores in patients. Compared with agalsidase inhibition-negative men, agalsidase inhibition-positive men showed greater left ventricular mass (P=0.02) and substantially lower renal function (difference in eGFR of about -30 ml/min per 1.73 m(2); P=0.04), which was confirmed by a longitudinal 5-year retrospective analysis. Additionally, affected patients presented more often with FD-typical symptoms, such as diarrhea, fatigue, and neuropathic pain, among others. Therefore, patients with poor clinical outcome on agalsidase should be tested for agalsidase inhibition. Future studies are warranted to determine if affected patients with FD benefit from acute reduction of anti-agalsidase antibodies or long-term immune modulation therapies to suppress agalsidase inhibition and to identify mechanisms that minimize antibody generation against agalsidase.

Patients with Fabry Disease after Enzyme Replacement Therapy Dose Reduction and Switch-2-Year Follow-Up.

Because of the shortage of agalsidase-ß supply between 2009 and 2012, patients with Fabry disease either were treated with reduced doses or were switched to agalsidase-α. In this observational study, we assessed end organ damage and clinical symptoms with special focus on renal outcome after 2 years of dose-reduction and/or switch to agalsidase-α. A total of 89 adult patients with Fabry disease who had received agalsidase-ß (1.0 mg/kg body wt) for >1 year were nonrandomly assigned to continue this treatment regimen (regular-dose group, n=24), to receive a reduced dose of 0.3-0.5 mg/kg and a subsequent switch to 0.2 mg/kg agalsidase-α (dose-reduction-switch group, n=28), or to directly switch to 0.2 mg/kg agalsidase-α (switch group, n=37) and were followed-up for 2 years. We assessed clinical events (death, myocardial infarction, severe arrhythmia, stroke, progression to ESRD), changes in cardiac and renal function, Fabry-related symptoms (pain, hypohidrosis, diarrhea), and disease severity scores. Determination of renal function by creatinine and cystatin C-based eGFR revealed decreasing eGFRs in the dose-reduction-switch group and the switch group. The Mainz Severity Score Index increased significantly in these two groups (P=0.02 and P<0.001, respectively), and higher frequencies of gastrointestinal pain occurred during follow-up. In conclusion, after 2 years of observation, all groups showed a stable clinical disease course with respect to serious clinical events. However, patients under agalsidase-ß dose-reduction and switch or a direct switch to agalsidase-α showed a decline of renal function independent of the eGFR formula used.