Salivary potassium measured by genetically encoded potassium ion indicators as a surrogate for plasma potassium levels in hemodialysis patients-a proof-of-concept study.

BACKGROUND: Hyperkalemia is a common complication in cardiorenal patients treated with agents interfering with renal potassium (K+) excretion. It frequently leads to discontinuation of potentially life-saving medication, which has increased the importance of K+ monitoring. Non-invasive means to detect hyperkalemia are currently unavailable, but would be of potential use for therapy guidance. The aim of the present study was to assess the analytical performance of genetically encoded potassium-ion indicators (GEPIIs) in measuring salivary [K+] ([K+]Saliva) and to determine whether changes of [K+]Saliva depict those of [K+]Plasma. METHODS: We conducted this proof-of-concept study: saliva samples from 20 healthy volunteers as well as plasma and saliva from 29 patients on hemodialysis (HD) before and after three consecutive HD treatments were collected. We compared [K+]Saliva as assessed by the gold standard ion-selective electrode (ISE) with GEPII measurements. RESULTS: The Bland-Altmann analysis showed a strong agreement (bias 0.71; 95% limits of agreement from -2.79 to 4.40) between GEPII and ISE. Before treatment, patients on HD showed significantly higher [K+]Saliva compared with healthy controls [median 37.7 (30.85; 48.46) vs 23.8 (21.63; 25.23) mmol/L; P < .05]. [K+]Plasma in HD patients decreased significantly after dialysis. This was paralleled by a significant decrease in [K+]Saliva, and both parameters increased until the subsequent HD session. Despite similar kinetics, we found weak or no correlation between [K+]Plasma and [K+]Saliva. CONCLUSION: GEPIIs have shown an excellent performance in determining [K+]Saliva. [K+]Plasma and [K+]Saliva exhibited similar kinetics. To determine whether saliva could be a suitable sample type to monitor [K+]Plasma, further testing in future studies are required.

Impact of cardiovascular risk stratification strategies in kidney transplantation over time.

BACKGROUND: Kidney transplant recipients exhibit a dramatically increased cardiovascular (CV) risk. In 2007, Austrian centres implemented a consensus of comprehensive CV screening programme prior to kidney transplantation (KT). The consensus placed a particular emphasis on screening for coronary artery disease (CAD) with cardiac computed tomography (CT) or coronary angiography (CAG) in patients with diabetes mellitus, known CAD or those having multiple conventional CV risk factors. Here, we investigate if this affected risk stratification and post-transplant CV outcomes. METHODS: In a retrospective chart review, we evaluated 551 KTs performed from 2003 to 2015 in our centre. Patients were categorized into three groups: KT before (2003-07), directly after (2008-11) and 5 years after (2012-15) implementation of the consensus. We analysed clinical characteristics, the rate of cardiac CTs and CAGs prior to KT as well as major adverse cardiac events (MACEs) during a 2-year follow-up after KT. RESULTS: The three study groups showed a homogeneous distribution of comorbidities and age. Significantly more cardiac CTs (13.6% versus 10.2% versus 44.8%; P = 0.002) and CAGs (39.6% versus 43.9% versus 56.2%; P = 0.003) were performed after the consensus. Coronary interventions were performed during 42 out of 260 CAGs (16.2%), the cumulative 2-year MACE incidence was 8.7%. Regarding MACE occurrence, no significant difference between the three groups was found. CONCLUSION: CV risk stratification has become more rigorous and invasive after the implementation of the consensus; however, this was not associated with an improvement in CV outcome.

IP3-mediated STIM1 oligomerization requires intact mitochondrial Ca2+ uptake.

Mitochondria contribute to cell signaling by controlling store-operated Ca(2+) entry (SOCE). SOCE is activated by Ca(2+) release from the endoplasmic reticulum (ER), whereupon stromal interacting molecule 1 (STIM1) forms oligomers, redistributes to ER-plasma-membrane junctions and opens plasma membrane Ca(2+) channels. The mechanisms by which mitochondria interfere with the complex process of SOCE are insufficiently clarified. In this study, we used an shRNA approach to investigate the direct involvement of mitochondrial Ca(2+) buffering in SOCE. We demonstrate that knockdown of either of two proteins that are essential for mitochondrial Ca(2+) uptake, the mitochondrial calcium uniporter (MCU) or uncoupling protein 2 (UCP2), results in decelerated STIM1 oligomerization and impaired SOCE following cell stimulation with an inositol-1,4,5-trisphosphate (IP3)-generating agonist. Upon artificially augmented cytosolic Ca(2+) buffering or ER Ca(2+) depletion by sarcoplasmic or endoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitors, STIM1 oligomerization did not rely on intact mitochondrial Ca(2+) uptake. However, MCU-dependent mitochondrial sequestration of Ca(2+) entering through the SOCE pathway was essential to prevent slow deactivation of SOCE. Our findings show a stimulus-specific contribution of mitochondrial Ca(2+) uptake to the SOCE machinery, likely through a role in shaping cytosolic Ca(2+) micro-domains.

The endocannabinoid N-arachidonoyl glycine (NAGly) inhibits store-operated Ca2+ entry by preventing STIM1-Orai1 interaction.

The endocannabiniod anandamide (AEA) and its derivate N-arachidonoyl glycine (NAGly) have a broad spectrum of physiological effects, which are induced by both binding to receptors and receptor-independent modulations of ion channels and transporters. The impact of AEA and NAGly on store-operated Ca(2+) entry (SOCE), a ubiquitous Ca(2+) entry pathway regulating many cellular functions, is unknown. Here we show that NAGly, but not AEA reversibly hinders SOCE in a time- and concentration-dependent manner. The inhibitory effect of NAGly on SOCE was found in the human endothelial cell line EA.hy926, the rat pancreatic ß-cell line INS-1 832/13, and the rat basophilic leukemia cell line RBL-2H3. NAGly diminished SOCE independently from the mode of Ca(2+) depletion of the endoplasmic reticulum, whereas it had no effect on Ca(2+) entry through L-type voltage-gated Ca(2+) channels. Enhanced Ca(2+) entry was effectively hampered by NAGly in cells overexpressing the key molecular constituents of SOCE, stromal interacting molecule 1 (STIM1) and the pore-forming subunit of SOCE channels, Orai1. Fluorescence microscopy revealed that NAGly did not affect STIM1 oligomerization, STIM1 clustering, or the colocalization of STIM1 with Orai1, which were induced by Ca(2+) depletion of the endoplasmic reticulum. In contrast, independently from its slow depolarizing effect on mitochondria, NAGly instantly and strongly diminished the interaction of STIM1 with Orai1, indicating that NAGly inhibits SOCE primarily by uncoupling STIM1 from Orai1. In summary, our findings revealed the STIM1-Orai1-mediated SOCE machinery as a molecular target of NAGly, which might have many implications in cell physiology.

Generation of Red-Shifted Cameleons for Imaging Ca²âº Dynamics of the Endoplasmic Reticulum.

Cameleons are sophisticated genetically encoded fluorescent probes that allow quantifying cellular Ca2+ signals. The probes are based on Förster resonance energy transfer (FRET) between terminally located fluorescent proteins (FPs), which move together upon binding of Ca2+ to the central calmodulin myosin light chain kinase M13 domain. Most of the available cameleons consist of cyan and yellow FPs (CFP and YFP) as the FRET pair. However, red-shifted versions with green and orange or red FPs (GFP, OFP, RFP) have some advantages such as less phototoxicity and minimal spectral overlay with autofluorescence of cells and fura-2, a prominent chemical Ca2+ indicator. While GFP/OFP- or GFP/RFP-based cameleons have been successfully used to study cytosolic and mitochondrial Ca2+ signals, red-shifted cameleons to visualize Ca2+ dynamics of the endoplasmic reticulum (ER) have not been developed so far. In this study, we generated and tested several ER targeted red-shifted cameleons. Our results show that GFP/OFP-based cameleons due to miss-targeting and their high Ca2+ binding affinity are inappropriate to record ER Ca2+ signals. However, ER targeted GFP/RFP-based probes were suitable to sense ER Ca2+ in a reliable manner. With this study we increased the palette of cameleons for visualizing Ca2+ dynamics within the main intracellular Ca2+ store.

Molecularly distinct routes of mitochondrial Ca2+ uptake are activated depending on the activity of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA).

The transfer of Ca(2+) across the inner mitochondrial membrane is an important physiological process linked to the regulation of metabolism, signal transduction, and cell death. While the definite molecular composition of mitochondrial Ca(2+) uptake sites remains unknown, several proteins of the inner mitochondrial membrane, that are likely to accomplish mitochondrial Ca(2+) fluxes, have been described: the novel uncoupling proteins 2 and 3, the leucine zipper-EF-hand containing transmembrane protein 1 and the mitochondrial calcium uniporter. It is unclear whether these proteins contribute to one unique mitochondrial Ca(2+) uptake pathway or establish distinct routes for mitochondrial Ca(2+) sequestration. In this study, we show that a modulation of Ca(2+) release from the endoplasmic reticulum by inhibition of the sarco/endoplasmatic reticulum ATPase modifies cytosolic Ca(2+) signals and consequently switches mitochondrial Ca(2+) uptake from an uncoupling protein 3- and mitochondrial calcium uniporter-dependent, but leucine zipper-EF-hand containing transmembrane protein 1-independent to a leucine zipper-EF-hand containing transmembrane protein 1- and mitochondrial calcium uniporter-mediated, but uncoupling protein 3-independent pathway. Thus, the activity of sarco/endoplasmatic reticulum ATPase is significant for the mode of mitochondrial Ca(2+) sequestration and determines which mitochondrial proteins might actually accomplish the transfer of Ca(2+) across the inner mitochondrial membrane. Moreover, our findings herein support the existence of distinct mitochondrial Ca(2+) uptake routes that might be essential to ensure an efficient ion transfer into mitochondria despite heterogeneous cytosolic Ca(2+) rises.

Assessment of Mitochondrial Ca2+ Uptake.

Mitochondrial Ca2+ uptake regulates mitochondrial function and contributes to cell signaling. Accordingly, quantifying mitochondrial Ca2+ signals and elaborating the mechanisms that accomplish mitochondrial Ca2+ uptake are essential to gain our understanding of cell biology. Here, we describe the benefits and drawbacks of various established old and new techniques to assess dynamic changes of mitochondrial Ca2+ concentration ([Ca2+]mito) in a wide range of applications.

Hypomagnesemia Is a Risk Factor for Infections after Kidney Transplantation: A Retrospective Cohort Analysis.

INTRODUCTION: Magnesium (Mg2+) deficiency is a common finding in the early phase after kidney transplantation (KT) and has been linked to immune dysfunction and infections. Data on the association of hypomagnesemia and the rate of infections in kidney transplant recipients (KTRs) are sparse. METHODS: We conducted a single-center retrospective cohort study of KTRs transplanted between 2005 and 2015. Laboratory data, including serum Mg2+ (median time of the Mg2+ measurement from KT: 29 days), rate of infections including mainly urinary tract infections (UTI), and common transplant-related viral infections (CMV, polyoma, EBV) in the early phase after KT were recorded. The primary outcome was the incidence of infections within one year after KT, while secondary outcomes were hospitalization due to infection, incidence rates of long-term (up to two years) infections, and all-cause mortality. RESULTS: We enrolled 376 KTRs of whom 229 patients (60.9%) suffered from Mg2+ deficiency defined as a serum Mg2+ < 0.7 mmol/L. A significantly higher incidence rate of UTIs and viral infections was observed in patients with versus without Mg2+ deficiency during the first year after KT (58.5% vs. 47.6%, p = 0.039 and 69.9% vs. 51.7%, p < 0.001). After adjustment for potential confounders, serum Mg2+ deficiency remained an independent predictor of both UTIs and viral infections (odds ratio (OR): 1.73, 95% CI: 1.04-2.86, p = 0.035 and OR: 2.05, 95% CI: 1.23-3.41, p = 0.006). No group differences according to Mg2+ status in hospitalizations due to infections and infection incidence rates in the 12-24 months post-transplant were observed. In the Cox regression analysis, Mg2+ deficiency was not significantly associated with all-cause mortality (HR: 1.15, 95% CI: 0.70-1.89, p = 0.577). CONCLUSIONS: KTRs suffering from Mg2+ deficiency are at increased risk of UTIs and viral infections in the first year after KT. Interventional studies investigating the effect of Mg2+ supplementation on Mg2+ deficiency and viral infections in KTRs are needed.

Impact of Timely Public Health Measures on Kidney Transplantation in Austria during the SARS-CoV-2 Outbreak-A Nationwide Analysis.

SARS-CoV-2 led to considerable morbidity/mortality worldwide and tremendously impacted on daily life. Strict lockdown measures were implemented early to contain the viral outbreak in Austria. Massive changes in organizational structures of healthcare facilities followed with unclear implications on the care of non-COVID-19-affected patients. We studied the nationwide impact of COVID-19 on kidney transplantation in Austria during the first six months of 2020. Concurrent with general lockdown measures, all kidney transplant activity was suspended from 13 March to 9 April. Nevertheless, between January and June, total transplant (p = 0.48) and procured donor organ numbers (p = 0.6) did not differ significantly from earlier years. Ten (0.18%) of 5512 prevalent Austrian kidney transplant recipients were diagnosed with SARS-CoV-2. The case fatality rate (one death; 10%) in renal transplant patients was less than in other countries but higher than in Austria's general population (2.4%). We conclude that early and strict general lockdown measures imposed by the government allowed an early, however cautious, re-opening of Austrian transplant programs and played a crucial role for the favorable outcomes of SARS-CoV-2 in Austrian kidney transplant patients. Even though it may be uncertain whether similar results may be obtainable in other countries, the findings may support early intervention strategies during similar episodes in the future.

High-Resolution Imaging of STIM/Orai Subcellular Localization Using Array Confocal Laser Scanning Microscopy.

The expression of chimeras that consist of a fluorescent protein (FP) conjugated with a protein of interest provides the ability to visualize, track, and quantify the subcellular localization and dynamics of specific proteins in biological samples. Array confocal laser scanning microscopy is an eminently suitable technique for live-cell imaging of FP-tagged fusion proteins. Here, we describe real-time monitoring of the subcellular dynamics of the stromal-interacting molecule 1 (STIM1) and Orai1, the key protagonists of store-operated Ca2+ entry (SOCE) under resting conditions, and upon Ca2+ mobilization from the endoplasmic reticulum (ER).

Novel genetically encoded fluorescent probes enable real-time detection of potassium in vitro and in vivo.

Changes in intra- and extracellular potassium ion (K+) concentrations control many important cellular processes and related biological functions. However, our current understanding of the spatiotemporal patterns of physiological and pathological K+ changes is severely limited by the lack of practicable detection methods. We developed K+-sensitive genetically encoded, Förster resonance energy transfer-(FRET) based probes, called GEPIIs, which enable quantitative real-time imaging of K+ dynamics. GEPIIs as purified biosensors are suitable to directly and precisely quantify K+ levels in different body fluids and cell growth media. GEPIIs expressed in cells enable time-lapse and real-time recordings of global and local intracellular K+ signals. Hitherto unknown Ca2+-triggered, organelle-specific K+ changes were detected in pancreatic beta cells. Recombinant GEPIIs also enabled visualization of extracellular K+ fluctuations in vivo with 2-photon microscopy. Therefore, GEPIIs are relevant for diverse K+ assays and open new avenues for live-cell K+ imaging.

Anemia management in cancer patients with chronic kidney disease.

Anemia is a common complication of cancer and chronic kidney disease (CKD) associated with decreased physical performance as well as poor prognosis for life expectancy. Renal and cancer-induced anemia share common features regarding pathogenesis and therapeutic strategies. It is typically treated with iron substitution, erythropoiesis-stimulating agents (ESA) and in refractory cases with red blood cell transfusions. However, studies of the past few years unveiled numerous setbacks in the use of ESAs. These included a higher risk of cerebrovascular events and increased mortality without the improvement of cardiovascular outcomes in patients with CKD. Moreover, particularly negative results were observed in patients with previous cancer history under ESA therapy. These unfavorable findings have forced the clinicians to reevaluate the management of renal anemia. This led to decrease of ESA usage, while iron substitution and alternative therapeutic options gained more significance. Iron supplementation is also accompanied with certain risks ranging from gastrointestinal complications to severe allergic reactions and increased rate of infections. Furthermore, the evaluation of the long-term safety of excessive iron therapy is still lacking, especially in CKD patients with cancer. In the absence of these clinical studies, this review aims to summarize the currently available therapeutic strategies in anemia management of CKD patients with concomitant cancer.

Assessment of mitochondrial Ca²âº uptake.

Mitochondrial Ca(2+) uptake regulates mitochondrial function and contributes to cell signaling. Accordingly, quantifying mitochondrial Ca(2+) signals and elaborating the mechanisms that accomplish mitochondrial Ca(2+) uptake are essential to gain our understanding of cell biology. Here, we describe the benefits and drawbacks of various established old and new techniques to assess dynamic changes of mitochondrial Ca(2+) concentration ([Ca(2+)]mito) in a wide range of applications.

N-Arachidonoyl glycine suppresses Na⁺/Ca²âº exchanger-mediated Ca²âº entry into endothelial cells and activates BK(Ca) channels independently of GPCRs.

BACKGROUND AND PURPOSE: N-Arachidonoyl glycine (NAGly) is a lipoamino acid with vasorelaxant properties. We aimed to explore the mechanisms of NAGly's action on unstimulated and agonist-stimulated endothelial cells. EXPERIMENTAL APPROACH: The effects of NAGly on endothelial electrical signalling were studied in combination with vascular reactivity. KEY RESULTS: In EA.hy926 cells, the sustained hyperpolarization to histamine was inhibited by the non-selective Na⁺/Ca²âº exchanger (NCX) inhibitor bepridil and by an inhibitor of reversed mode NCX, KB-R7943. In cells dialysed with Cs⁺-based Na⁺-containing solution, the outwardly rectifying current with typical characteristics of NCX was augmented following histamine exposure, further increased upon external Na⁺ withdrawal and inhibited by bepridil. NAGly (0.3-30 µM) suppressed NCX currents in a URB597- and guanosine 5'-O-(2-thiodiphosphate) (GDPßS)-insensitive manner, [Ca²âº]i elevation evoked by Na⁺ removal and the hyperpolarization to histamine. In rat aorta, NAGly opposed the endothelial hyperpolarization and relaxation response to ACh. In unstimulated EA.hy926 cells, NAGly potentiated the whole-cell current attributable to large-conductance Ca²âº-activated K⁺ (BK(Ca)) channels in a GDPßS-insensitive, paxilline-sensitive manner and produced a sustained hyperpolarization. In cell-free inside-out patches, NAGly stimulated single BK(Ca) channel activity. CONCLUSION AND IMPLICATIONS: Our data showed that NCX is a Ca²âº entry pathway in endothelial cells and that NAGly is a potent G-protein-independent modulator of endothelial electrical signalling and has a dual effect on endothelial electrical responses. In agonist pre-stimulated cells, NAGly opposes hyperpolarization and relaxation via inhibition of NCX-mediated Ca²âº entry, while in unstimulated cells, it promotes hyperpolarization via receptor-independent activation of BK(Ca) channels.

Spatiotemporal correlations between cytosolic and mitochondrial Ca(2+) signals using a novel red-shifted mitochondrial targeted cameleon.

The transfer of Ca(2+) from the cytosol into the lumen of mitochondria is a crucial process that impacts cell signaling in multiple ways. Cytosolic Ca(2+) ([Ca(2+)](cyto)) can be excellently quantified with the ratiometric Ca(2+) probe fura-2, while genetically encoded Förster resonance energy transfer (FRET)-based fluorescent Ca(2+) sensors, the cameleons, are efficiently used to specifically measure Ca(2+) within organelles. However, because of a significant overlap of the fura-2 emission with the spectra of the cyan and yellow fluorescent protein of most of the existing cameleons, the measurement of fura-2 and cameleons within one given cell is a complex task. In this study, we introduce a novel approach to simultaneously assess [Ca(2+)](cyto) and mitochondrial Ca(2+) ([Ca(2+)](mito)) signals at the single cell level. In order to eliminate the spectral overlap we developed a novel red-shifted cameleon, D1GO-Cam, in which the green and orange fluorescent proteins were used as the FRET pair. This ratiometric Ca(2+) probe could be successfully targeted to mitochondria and was suitable to be used simultaneously with fura-2 to correlate [Ca(2+)](cyto) and [Ca(2+)](mito) within same individual cells. Our data indicate that depending on the kinetics of [Ca(2+)](cyto) rises there is a significant lag between onset of [Ca(2+)](cyto) and [Ca(2+)](mito) signals, pointing to a certain threshold of [Ca(2+)](cyto) necessary to activate mitochondrial Ca(2+) uptake. The temporal correlation between [Ca(2+)](mito) and [Ca(2+)](cyto) as well as the efficiency of the transfer of Ca(2+) from the cytosol into mitochondria varies between different cell types. Moreover, slow mitochondrial Ca(2+) extrusion and a desensitization of mitochondrial Ca(2+) uptake cause a clear difference in patterns of mitochondrial and cytosolic Ca(2+) oscillations of pancreatic beta-cells in response to D-glucose.