Exploring the role of TRPM4 in calcium-dependent triggered activity and cardiac arrhythmias.

Cardiac arrhythmias pose a major threat to a patient's health, yet prove to be often difficult to predict, prevent and treat. A key mechanism in the occurrence of arrhythmias is disturbed Ca2+ homeostasis in cardiac muscle cells. As a Ca2+-activated non-selective cation channel, TRPM4 has been linked to Ca2+-induced arrhythmias, potentially contributing to translating an increase in intracellular Ca2+ concentration into membrane depolarisation and an increase in cellular excitability. Indeed, evidence from genetically modified mice, analysis of mutations in human patients and the identification of a TRPM4 blocking compound that can be applied in vivo further underscore this hypothesis. Here, we provide an overview of these data in the context of our current understanding of Ca2+-dependent arrhythmias.

Leukopenia in autosomal dominant polycystic kidney disease: a single-center cohort of kidney transplant candidates with post-transplantation follow-up.

Background: Autosomal dominant polycystic kidney disease (ADPKD) has occasionally been associated with lower peripheral white blood cell (WBC) counts. This study aimed to investigate the peripheral blood cell counts in a large cohort of kidney transplant recipients before and after kidney transplantation and its potential impact on post-transplant outcomes. Methods: This was a retrospective study with long-term follow-up data of 2090 patients who underwent a first kidney transplantation in the Leuven University Hospitals, of whom 392 had ADPKD. Results: In total, 2090 patients who underwent a first kidney transplantation in the Leuven University Hospitals were included, of whom 392 had ADPKD. Both pre- and post-transplantation, ADPKD patients had significantly lower total WBC counts, and more specifically lower neutrophil, lymphocyte and eosinophil counts compared with the non-ADPKD patients. This observation was independent of potential confounders such as level of inflammation, smoking habit, vitamins and pre-transplant medication. Overall survival and kidney transplant survival were significantly better in ADPKD vs non-ADPKD transplant recipients and a longer time to first infection was observed. However, no association between blood cell counts and outcome differences was found. Conclusions: In conclusion, this large single-center study reports a strong and independent association between ADPKD and lower peripheral WBC counts both before and after kidney transplantation. Considering the role of inflammation in disease progression, further investigation into the role of WBC in ADPKD is needed.

TRPM4 regulates hilar mossy cell loss in temporal lobe epilepsy.

BACKGROUND: Mossy cells comprise a large fraction of excitatory neurons in the hippocampal dentate gyrus, and their loss is one of the major hallmarks of temporal lobe epilepsy (TLE). The vulnerability of mossy cells in TLE is well known in animal models as well as in patients; however, the mechanisms leading to cellular death is unclear. RESULTS: Transient receptor potential melastatin 4 (TRPM4) is a Ca2+-activated non-selective cation channel regulating diverse physiological functions of excitable cells. Here, we identified that TRPM4 is present in hilar mossy cells and regulates their intrinsic electrophysiological properties including spontaneous activity and action potential dynamics. Furthermore, we showed that TRPM4 contributes to mossy cells death following status epilepticus and therefore modulates seizure susceptibility and epilepsy-related memory deficits. CONCLUSIONS: Our results provide evidence for the role of TRPM4 in MC excitability both in physiological and pathological conditions.

Low agreement between various eGFR formulae in pediatric and young adult ADPKD patients.

BACKGROUND: Young autosomal dominant polycystic kidney disease (ADPKD) patients are becoming the new target population for the development of new treatment options. Determination of a reliable equation for estimated glomerular filtration rate (eGFR) from early stages is needed with the promising potential interventional therapies. METHODS: Prospective and longitudinal study on a cohort of 68 genotyped ADPKD patients (age range 0-23 years) with long-term follow-up. Commonly used equations for eGFR were compared for their relative performance. RESULTS: The revised Schwartz formula (CKiD) showed a highly significant decline in eGFR with aging (- 3.31 mL/min/1.73 m2/year, P < 0.0001). The recently updated equation by the Schwartz group (CKiDU25) showed a smaller (- 0.90 mL/min/1.73 m2/year) but significant (P = 0.001) decline in eGFR with aging and also showed a significant sex difference (P < 0.0001), not observed by the other equations. In contrast, the full age spectrum (FAS) equations (FAS-SCr, FAS-CysC, and the combined) showed no age and sex dependency. The prevalence of hyperfiltration is highly dependent on the formula used, and the highest prevalence was observed with the CKiD Equation (35%). CONCLUSIONS: The most widely used methods to calculate eGFR in ADPKD children (CKiD and CKiDU25 equations) were associated with unexpected age or sex differences. The FAS equations were age- and sex-independent in our cohort. Hence, the switch from the CKiD to CKD-EPI equation at the transition from pediatric to adult care causes implausible jumps in eGFR, which could be misinterpreted. Having reliable methods to calculate eGFR is indispensable for clinical follow-up and clinical trials. A higher resolution version of the Graphical abstract is available as Supplementary information.

Risk Severity Model for Pediatric Autosomal Dominant Polycystic Kidney Disease Using 3D Ultrasound Volumetry.

BACKGROUND: Height-adjusted total kidney volume (htTKV) measured by imaging defined as Mayo Imaging Class (MIC) is a validated prognostic measure for autosomal dominant polycystic kidney disease (ADPKD) in adults to predict and stratify disease progression. However, no stratification tool is currently available in pediatric ADPKD. Because magnetic resonance imaging and computed tomography in children are difficult, we propose a novel 3D ultrasound-based pediatric Leuven Imaging Classification to complement the MIC. METHODS: A prospective study cohort of 74 patients with genotyped ADPKD (37 female) was followed longitudinally with ultrasound, including 3D ultrasound, and they underwent in total 247 3D ultrasound assessments, with patients' median age (interquartile range [IQR]) at diagnosis of 3 (IQR, 0-9) years and at first 3D ultrasound evaluation of 10 (5-14) years. First, data matching was done to the published MIC classification, followed by subsequent optimization of parameters and model type. RESULTS: PKD1 was confirmed in 70 patients (95%), PKD2 in three (4%), and glucosidase IIα unit only once (1%). Over these 247 evaluations, the median height was 143 (IQR, 122-166) cm and total kidney volume was 236 (IQR, 144-344) ml, leading to an htTKV of 161 (IQR, 117-208) ml/m. Applying the adult Mayo classification in children younger than 15 years strongly underestimated ADPKD severity, even with correction for height. We therefore optimized the model with our pediatric data and eventually validated it with data of young patients from Mayo Clinic and the Consortium for Radiologic Imaging Studies of Polycystic Kidney Disease used to establish the MIC. CONCLUSIONS: We proposed a five-level Leuven Imaging Classification ADPKD pediatric model as a novel classification tool on the basis of patients' age and 3D ultrasound-htTKV for reliable discrimination of childhood ADPKD severity.

Pharmacokinetics of Oral Rebaudioside A in Patients with Type 2 Diabetes Mellitus and Its Effects on Glucose Homeostasis: A Placebo-Controlled Crossover Trial.

BACKGROUND AND OBJECTIVES: Rebaudioside A, a steviol glycoside, is deglycosylated by intestinal microflora prior to the absorption of steviol and conjugation to steviol glucuronide. While glucose-lowering properties are observed for rebaudioside A in mice, they have been attributed to the metabolites steviol and steviol glucuronide. We aimed to characterize the pharmacokinetic and pharmacodynamic properties of rebaudioside A and its metabolites in patients with early-onset type 2 diabetes mellitus (T2DM). METHODS: This randomized, placebo-controlled, open-label, two-way crossover trial was performed in subjects with T2DM on metformin or no therapy at the University Hospitals Leuven, Belgium. Following oral rebaudioside A (3 g), plasma concentrations of rebaudioside A, steviol and steviol glucuronide were determined. The effect on glucose homeostasis was examined by an oral glucose tolerance test (OGTT) performed 19 h following rebaudioside A administration, i.e. the presumed time of maximal steviol and steviol glucuronide concentrations. The primary pharmacodynamic endpoint was the difference in area under the blood glucose concentration-time curve during the first 2 h of the OGTT (AUCGlucose(0-2h)) for rebaudioside A vs. placebo. RESULTS: In total, 30 subjects [63.5 (57.8-69.0) years of age, 86.7% male] completed the trial. Rebaudioside A was detected as early as 1 h after administration in nearly all subjects. As expected, steviol and steviol glucuronide reached their maximal concentrations at 19.5 h following rebaudioside A administration. Rebaudioside A did not lower the AUCGlucose(0-2h) compared to placebo (- 0.7 (95% CI - 22.3; 20.9) h·mg/dL, P = 0.95). Insulin and C-peptide concentrations were also comparable between both conditions (P > 0.05). CONCLUSION: Rebaudioside A is readily absorbed after oral administration and metabolized to steviol and steviol glucuronide. However, no effect on glucose nor insulin or C-peptide excursion was observed during the OGTT at the time of maximal metabolite concentrations. Thus, no antidiabetic properties of rebaudioside A could be observed in patients with T2DM after single oral use. CLINICAL TRIAL REGISTRATION: Registered on ClinicalTrials.gov (NCT03510624).

Acute inhibition of transient receptor potential vanilloid-type 4 cation channel halts cytoskeletal dynamism in microglia.

Microglia, the resident macrophages of the central nervous system, are highly motile cells that support brain development, provision neuronal signaling, and protect brain cells against damage. Proper microglial functioning requires constant cell movement and morphological changes. Interestingly, the transient receptor potential vanilloid 4 (TRPV4) channel, a calcium-permeable channel, is involved in hypoosmotic morphological changes of retinal microglia and regulates temperature-dependent movement of microglial cells both in vitro and in vivo. Despite the broad functions of TRPV4 and the recent findings stating a role for TRPV4 in microglial movement, little is known about how TRPV4 modulates cytoskeletal remodeling to promote changes of microglial motility. Here we show that acute inhibition of TRPV4, but not its constitutive absence in the Trpv4 KO cells, affects the morphology and motility of microglia in vitro. Using high-end confocal imaging techniques, we show a decrease in actin-rich filopodia and tubulin dynamics upon acute inhibition of TRPV4 in vitro. Furthermore, using acute brain slices we demonstrate that Trpv4 knockout microglia display lower ramification complexity, slower process extension speed and consequently smaller surveyed area. We conclude that TRPV4 inhibition triggers a shift in cytoskeleton remodeling of microglia influencing their migration and morphology.

TRPM4 inhibition by meclofenamate suppresses Ca2+-dependent triggered arrhythmias.

AIMS: Cardiac arrhythmias are a major factor in the occurrence of morbidity and sudden death in patients with cardiovascular disease. Disturbances of Ca2+ homeostasis in the heart contribute to the initiation and maintenance of cardiac arrhythmias. Extrasystolic increases in intracellular Ca2+ lead to delayed afterdepolarizations and triggered activity, which can result in heart rhythm abnormalities. It is being suggested that the Ca2+-activated nonselective cation channel TRPM4 is involved in the aetiology of triggered activity, but the exact contribution and in vivo significance are still unclear. METHODS AND RESULTS: In vitro electrophysiological and calcium imaging technique as well as in vivo intracardiac and telemetric electrocardiogram measurements in physiological and pathophysiological conditions were performed. In two distinct Ca2+-dependent proarrhythmic models, freely moving Trpm4-/- mice displayed a reduced burden of cardiac arrhythmias. Looking further into the specific contribution of TRPM4 to the cellular mechanism of arrhythmias, TRPM4 was found to contribute to a long-lasting Ca2+ overload-induced background current, thereby regulating cell excitability in Ca2+ overload conditions. To expand these results, a compound screening revealed meclofenamate as a potent antagonist of TRPM4. In line with the findings from Trpm4-/- mice, 10 µM meclofenamate inhibited the Ca2+ overload-induced background current in ventricular cardiomyocytes and 15 mg/kg meclofenamate suppressed catecholaminergic polymorphic ventricular tachycardia-associated arrhythmias in a TRPM4-dependent manner. CONCLUSION: The presented data establish that TRPM4 represents a novel target in the prevention and treatment of Ca2+-dependent triggered arrhythmias.

On Methods for the Measurement of the Apelin Receptor Ligand Apelin.

Apelin exists in many isoforms, both in the circulation and in specific tissues. Apelin peptides have a short half-life but preservation before measurement is scarcely studied. Reproducible mass spectrometry methods to specifically measure a broad range of apelinergic peptide isoforms are currently lacking. A sample protocol to conserve apelinergic peptides in the preanalytical phase and a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method to measure apelinergic isoforms was developed. Apelin was measured in plasma. For validation, human embryonic kidney (HEK) cells transfected with cDNA for preproapelin were used. Results were compared with a validated radioimmunoassay (RIA) method. Acidifying plasma to pH 2.5 improves post-sampling stability of apelin. HPLC-MS/MS was unable to detect apelin isoforms in plasma of healthy volunteers (n = 16) and chronic kidney disease patients (n = 4). RIA could detect apelin in concentrations between 71 and 263 fmol/l in 10 healthy volunteers. An optimized preanalytical protocol was developed. A sensitive and specific HPLC-MS/MS method failed to detect apelin in human plasma. Apelin-36 was detected in HEK cells transfected with cDNA for preproapelin. Currently, RIA with relatively selective antibodies is the best alternative for the measurement of apelin but novel sensitive and specific methods are needed.

Is autosomal dominant polycystic kidney disease an early sweet disease?

The clinical course of autosomal dominant polycystic kidney disease (ADPKD) starts in childhood. Evidence of the beneficial impact of early nephron-protective strategies and lifestyle modifications on ADPKD prognosis is accumulating. Recent studies have described the association of overweight and obesity with rapid disease progression in adults with ADPKD. Moreover, defective glucose metabolism and metabolic reprogramming have been reported in distinct ADPKD models highlighting these pathways as potential therapeutic targets in ADPKD. Several "metabolic" approaches are currently under evaluation in adults, including ketogenic diet, food restriction, and metformin therapy. No data are available on the impact of these approaches in childhood thus far. Yet, according to World Health Organization (WHO), we are currently facing a childhood obesity crisis with an increased prevalence of overweight/obesity in the pediatric population associated with a cardio-metabolic risk profile. The present review summarizes the knowledge about the role of glucose metabolism in the pathophysiology of ADPKD and underscores the possible harm of overweight and obesity in ADPKD especially in terms of long-term cardiovascular outcomes and renal prognosis.

The emerging role of the apelinergic system in kidney physiology and disease.

The apelinergic system (AS) is a novel pleiotropic system with an essential role in renal and cardiovascular physiology and disease, including water homeostasis and blood pressure regulation. It consists of two highly conserved peptide ligands, apelin and apela, and a G-protein-coupled apelin receptor. The two ligands have many isoforms and a short half-life and exert both similar and divergent effects. Vasopressin, apelin and their receptors colocalize in hypothalamic regions essential for body fluid homeostasis and interact at the central and renal levels to regulate water homeostasis and diuresis in inverse directions. In addition, the AS and renin-angiotensin system interact both systemically and in the kidney, with implications for the cardiovascular system. A role for the AS in diverse pathological states, including disorders of sodium and water balance, hypertension, heart failure, pre-eclampsia, acute kidney injury, sepsis and diabetic nephropathy, has recently been reported. Furthermore, several metabolically stable apelin analogues have been developed, with potential applications in diverse diseases. We review here what is currently known about the physiological functions of the AS, focusing on renal, cardiovascular and metabolic homeostasis, and the role of the AS in associated diseases. We also describe several hurdles and research opportunities worthy of the attention of the nephrology community.

Interdependent Regulation of Polycystin Expression Influences Starvation-Induced Autophagy and Cell Death.

Autosomal dominant polycystic kidney disease (ADPKD) is mainly caused by deficiency of polycystin-1 (PC1) or polycystin-2 (PC2). Altered autophagy has recently been implicated in ADPKD progression, but its exact regulation by PC1 and PC2 remains unclear. We therefore investigated cell death and survival during nutritional stress in mouse inner medullary collecting duct cells (mIMCDs), either wild-type (WT) or lacking PC1 (PC1KO) or PC2 (PC2KO), and human urine-derived proximal tubular epithelial cells (PTEC) from early-stage ADPKD patients with PC1 mutations versus healthy individuals. Basal autophagy was enhanced in PC1-deficient cells. Similarly, following starvation, autophagy was enhanced and cell death reduced when PC1 was reduced. Autophagy inhibition reduced cell death resistance in PC1KO mIMCDs to the WT level, implying that PC1 promotes autophagic cell survival. Although PC2 expression was increased in PC1KO mIMCDs, PC2 knockdown did not result in reduced autophagy. PC2KO mIMCDs displayed lower basal autophagy, but more autophagy and less cell death following chronic starvation. This could be reversed by overexpression of PC1 in PC2KO. Together, these findings indicate that PC1 levels are partially coupled to PC2 expression, and determine the transition from renal cell survival to death, leading to enhanced survival of ADPKD cells during nutritional stress.

TRPM4 links calcium signaling to membrane potential in pancreatic acinar cells.

Transient receptor potential cation channel subfamily M member 4 (TRPM4) is a Ca2+-activated nonselective cation channel that mediates membrane depolarization. Although, a current with the hallmarks of a TRPM4-mediated current has been previously reported in pancreatic acinar cells (PACs), the role of TRPM4 in the regulation of acinar cell function has not yet been explored. In the present study, we identify this TRPM4 current and describe its role in context of Ca2+ signaling of PACs using pharmacological tools and TRPM4-deficient mice. We found a significant Ca2+-activated cation current in PACs that was sensitive to the TRPM4 inhibitors 9-phenanthrol and 4-chloro-2-[[2-(2-chlorophenoxy)acetyl]amino]benzoic acid (CBA). We demonstrated that the CBA-sensitive current was responsible for a Ca2+-dependent depolarization of PACs from a resting membrane potential of -44.4 ± 2.9 to -27.7 ± 3 mV. Furthermore, we showed that Ca2+ influx was higher in the TRPM4 KO- and CBA-treated PACs than in control cells. As hormone-induced repetitive Ca2+ transients partially rely on Ca2+ influx in PACs, the role of TRPM4 was also assessed on Ca2+ oscillations elicited by physiologically relevant concentrations of the cholecystokinin analog cerulein. These data show that the amplitude of Ca2+ signals was significantly higher in TRPM4 KO than in control PACs. Our results suggest that PACs are depolarized by TRPM4 currents to an extent that results in a significant reduction of the inward driving force for Ca2+. In conclusion, TRPM4 links intracellular Ca2+ signaling to membrane potential as a negative feedback regulator of Ca2+ entry in PACs.

The Ca2+-activated cation channel TRPM4 is a positive regulator of pressure overload-induced cardiac hypertrophy.

Pathological left ventricular hypertrophy (LVH) occurs in response to pressure overload and remains the single most important clinical predictor of cardiac mortality. The molecular pathways in the induction of pressure overload LVH are potential targets for therapeutic intervention. Current treatments aim to remove the pressure overload stimulus for LVH, but do not completely reverse adverse cardiac remodelling. Although numerous molecular signalling steps in the induction of LVH have been identified, the initial step by which mechanical stretch associated with cardiac pressure overload is converted into a chemical signal that initiates hypertrophic signalling remains unresolved. In this study, we show that selective deletion of transient receptor potential melastatin 4 (TRPM4) channels in mouse cardiomyocytes results in an approximately 50% reduction in the LVH induced by transverse aortic constriction. Our results suggest that TRPM4 channel is an important component of the mechanosensory signalling pathway that induces LVH in response to pressure overload and represents a potential novel therapeutic target for the prevention of pathological LVH.

Enhanced MCP-1 Release in Early Autosomal Dominant Polycystic Kidney Disease.

INTRODUCTION: Autosomal dominant polycystic kidney disease (ADPKD) causes kidney failure typically in adulthood, but the disease starts in utero. Copeptin, epidermal growth factor (EGF), and monocyte chemoattractant protein-1 (MCP-1) are associated with severity and hold prognostic value in adults but remain unstudied in the early disease stage. Kidneys from adults with ADPKD exhibit macrophage infiltration, and a prominent role of MCP-1 secretion by tubular epithelial cells is suggested from rodent models. METHODS: In a cross-sectional study, plasma copeptin, urinary EGF, and urinary MCP-1 were evaluated in a pediatric ADPKD cohort and compared with age-, sex-, and body mass index (BMI)-matched healthy controls. MCP-1 was studied in mouse collecting duct cells, human proximal tubular cells, and fetal kidney tissue. RESULTS: Fifty-three genotyped ADPKD patients and 53 controls were included. The mean (SD) age was 10.4 (5.9) versus 10.5 (6.1) years (P = 0.543), and the estimated glomerular filtration rate (eGFR) was 122.7 (39.8) versus 114.5 (23.1) ml/min per 1.73 m2 (P = 0.177) in patients versus controls, respectively. Plasma copeptin and EGF secretion were comparable between groups. The median (interquartile range) urinary MCP-1 (pg/mg creatinine) was significantly higher in ADPKD patients (185.4 [213.8]) compared with controls (154.7 [98.0], P = 0.010). Human proximal tubular cells with a heterozygous PKD1 mutation and mouse collecting duct cells with a PKD1 knockout exhibited increased MCP-1 secretion. Human fetal ADPKD kidneys displayed prominent MCP-1 immunoreactivity and M2 macrophage infiltration. CONCLUSION: An increase in tubular MCP-1 secretion is an early event in ADPKD. MCP-1 is an early disease severity marker and a potential treatment target.

Continuous glucose monitoring during pregnancy in healthy mice.

During pregnancy, metabolic adaptations occur to maintain the balance between maternal and foetal growth, including increased insulin secretion and decreased insulin sensitivity. When the body fails to adjust, gestational diabetes mellitus develops. To gain insight in the pregnancy-induced adaptations, we applied continuous glucose monitoring via telemetric transmitters. We show that continuous glucose monitoring in conscious, non-stressed, freely moving mice throughout the full pregnancy is feasible, accurate and safe. We show that healthy mice during a full pregnancy develop adaptations in glucose homeostasis reminiscent of those in pregnant women. Furthermore, continuous glucose monitoring allows the complete analysis of all aspects of glucose excursions associated with spontaneous feeding episodes, and the thorough analysis of glycaemic variability. In conclusion, continuous glucose monitoring allows a detailed description of the glycaemic status during pregnancy, which will help to unravel specific mechanisms for gestational diabetes mellitus.

Putting the pressure on endocytosis in the kidney.

In Science Signaling, Gualdani and colleagues provide evidence that the ion channel TRPV4 functions as a mechanosensor in the renal proximal tubules and show that TRPV4 activity modulates protein reabsorption.

Cytopenia in autosomal dominant polycystic kidney disease (ADPKD): merely an association or a disease-related feature with prognostic implications?

Autosomal dominant polycystic kidney disease (ADPKD) is associated with distinct cytopenias in observational studies; the most consistent and strongest association is seen with alternations in the lymphocytic lineages. Although the underlying mechanism of these associations is unclear, it has been hypothesized to be secondary to sequestration of white blood cells in cystic organs, or related to the uremic environment in chronic kidney disease (CKD). However, since mutations in PKD1 or -2 affect several immunomodulating pathways, cytopenia may well be an unrecognized extrarenal manifestation of ADPKD. Furthermore, many important questions on the clinical implications of this finding and the effect on the disease course in these patients are unanswered. In this review article, we provide an overview of the current evidence on cytopenia in ADPKD and explore the underlying mechanisms of this association and its potential prognostic implications. Based on the current literature, we hypothesize that polycystin deficiency can disturb immune cell homeostasis and that cytopenia is thus an intrinsic feature of ADPKD, related to genetic factors. Taken together, these findings warrant further investigation to establish the exact etiology and role of cytopenia in patients with ADPKD.