A gain-of-function filamin A mutation in mouse platelets induces thrombus instability.

BACKGROUND: Filaminopathies A are rare disorders affecting the brain, intestine, or skeleton, characterized by dominant X-linked filamin A (FLNA) gene mutations. Macrothrombocytopenia with functionally defective platelets is frequent. We have described a filaminopathy A male patient, exhibiting a C-terminal frame-shift FLNa mutation (Berrou et al., Arterioscler Thromb Vasc Biol. 2017;37:1087-1097). Contrasting with female patients, this male patient exhibited gain of platelet functions, including increased platelet aggregation, integrin αIIbß3 activation, and secretion at low agonist concentration, raising the issue of thrombosis risk. OBJECTIVES: Our goal is to assess the thrombotic potential of the patient FLNa mutation in an in vivo model. METHODS: We have established a mutant FlnA knock-in mouse model. RESULTS: The mutant FlnA mouse platelets phenocopied patient platelets, showing normal platelet count, lower expression level of mutant FlnA, and gain of platelet functions: increased platelet aggregation, secretion, and αIIbß3 activation, as well as increased spreading and clot retraction. Surprisingly, mutant FlnA mice exhibited a normal bleeding time, but with increased re-bleeding (77%) compared to wild type (WT) FlnA mice (27%), reflecting hemostatic plug instability. Again, in an in vivo thrombosis model, the occlusion time was not altered by the FlnA mutation, but arteriolar embolies were increased (7-fold more frequent in mutant FlnA mice versus WT mice), confirming thrombus instability. CONCLUSIONS: This study shows that the FlnA mutation found in the male patient induced gain of platelet functions in vitro, but thrombus instability in vivo. Implications for the role of FLNa in physiology of thrombus formation are discussed.

New insights into regulation of αIIbß3 integrin signaling by filamin A.

Background: Filamin (FLN) regulates many cell functions through its scaffolding activity cross-linking cytoskeleton and integrins. FLN was shown to inhibit integrin activity, but the exact mechanism remains unclear. Objectives: The aim of this study was to evaluate the role of filamin A (FLNa) subdomains on the regulation of integrin αIIbß3 signaling. Methods: Three FLNa deletion mutants were overexpressed in the erythro-megakaryocytic leukemic cell line HEL: Del1, which lacks the N-terminal CH1-CH2 domains mediating the FLNa-actin interaction; Del2, lacking the Ig-like repeat 21, which mediates the FLNa-ß3 interaction; and Del3, lacking the C-terminal Ig repeat 24, responsible for FLNa dimerization and interaction with the small Rho guanosine triphosphatase involved in actin cytoskeleton reorganisation. Fibrinogen binding to HEL cells in suspension and talin-ß3 proximity in cells adherent to immobilized fibrinogen were assessed before and after αIIbß3 activation by the protein kinase C agonist phorbol 12-myristate 13-acetate. Results: Our results show that FLNa-actin and FLNa-ß3 interactions negatively regulate αIIbß3 activation. Moreover, FLNa-actin interaction represses Rac activation, contributing to the negative regulation of αIIbß3 activation. In contrast, the FLNa dimerization domain, which maintains Rho inactive, was found to negatively regulate αIIbß3 outside-in signaling. Conclusion: We conclude that FLNa negatively controls αIIbß3 activation by regulating actin polymerization and restraining activation of Rac, as well as outside-in signaling by repressing Rho.

Relevance of platelet desialylation and thrombocytopenia in type 2B von Willebrand disease: preclinical and clinical evidence.

Patients with type 2B von Willebrand disease (vWD) (caused by gain-of-function mutations in the gene coding for von Willebrand factor) display bleeding to a variable extent and, in some cases, thrombocytopenia. There are several underlying causes of thrombocytopenia in type 2B vWD. It was recently suggested that desialylation-mediated platelet clearance leads to thrombocytopenia in this disease. However, this hypothesis has not been tested in vivo The relationship between platelet desialylation and the platelet count was probed in 36 patients with type 2B von Willebrand disease (p.R1306Q, p.R1341Q, and p.V1316M mutations) and in a mouse model carrying the severe p.V1316M mutation (the 2B mouse). We observed abnormally high elevated levels of platelet desialylation in both patients with the p.V1316M mutation and the 2B mice. In vitro, we demonstrated that 2B p.V1316M/von Willebrand factor induced more desialylation of normal platelets than wild-type von Willebrand factor did. Furthermore, we found that N-glycans were desialylated and we identified αIIb and ß3 as desialylation targets. Treatment of 2B mice with sialidase inhibitors (which correct platelet desialylation) was not associated with the recovery of a normal platelet count. Lastly, we demonstrated that a critical platelet desialylation threshold (not achieved in either 2B patients or 2B mice) was required to induce thrombocytopenia in vivo In conclusion, in type 2B vWD, platelet desialylation has a minor role and is not sufficient to mediate thrombocytopenia.

A mutation of the human EPHB2 gene leads to a major platelet functional defect.

The ephrin transmembrane receptor family of tyrosine kinases is involved in platelet function. We report the first EPHB2 variant affecting platelets in 2 siblings (P1 and P2) from a consanguineous family with recurrent bleeding and normal platelet counts. Whole-exome sequencing identified a c.2233C>T variant (missense p.R745C) of the EPHB2 gene. P1 and P2 were homozygous for this variant, while their asymptomatic parents were heterozygous. The p.R745C variant within the tyrosine kinase domain was associated with defects in platelet aggregation, αIIbß3 activation, and granule secretion induced by G-protein-coupled receptor (GPCR) agonists and convulxin, as well as in thrombus formation on collagen under flow. In contrast, clot retraction, flow-dependent platelet adhesion, and spreading on fibrinogen were only mildly affected, indicating limited effects on αIIbß3 outside-in signaling. Most importantly, Lyn, Syk, and FcRγ phosphorylation, the initial steps in glycoprotein VI (GPVI) platelet signaling were drastically impaired in the absence of platelet-platelet contact, indicating a positive role for EPHB2 in GPVI activation. Likewise platelet activation by PAR4-AP showed defective Src activation, as opposed to normal protein kinase C activity and Ca2+ mobilization. Overexpression of wild-type and R745C EPHB2 variant in RBL-2H3 (rat basophilic leukemia) cells stably expressing human GPVI confirmed that EPHB2 R745C mutation impaired EPHB2 autophosphorylation but had no effect on ephrin ligand-induced EPHB2 clustering, suggesting it did not interfere with EPHB2-ephrin-mediated cell-to-cell contact. In conclusion, this novel inherited platelet disorder affecting EPHB2 demonstrates this tyrosine kinase receptor plays an important role in platelet function through crosstalk with GPVI and GPCR signaling.

A mouse model of pseudohypoaldosteronism type II reveals a novel mechanism of renal tubular acidosis.

Pseudohypoaldosteronism type II (PHAII) is a genetic disease characterized by association of hyperkalemia, hyperchloremic metabolic acidosis, hypertension, low renin, and high sensitivity to thiazide diuretics. It is caused by mutations in the WNK1, WNK4, KLHL3 or CUL3 gene. There is strong evidence that excessive sodium chloride reabsorption by the sodium chloride cotransporter NCC in the distal convoluted tubule is involved. WNK4 is expressed not only in distal convoluted tubule cells but also in ß-intercalated cells of the cortical collecting duct. These latter cells exchange intracellular bicarbonate for external chloride through pendrin, and therefore, account for renal base excretion. However, these cells can also mediate thiazide-sensitive sodium chloride absorption when the pendrin-dependent apical chloride influx is coupled to apical sodium influx by the sodium-driven chloride/bicarbonate exchanger. Here we determine whether this system is involved in the pathogenesis of PHAII. Renal pendrin activity was markedly increased in a mouse model carrying a WNK4 missense mutation (Q562E) previously identified in patients with PHAII. The upregulation of pendrin led to an increase in thiazide-sensitive sodium chloride absorption by the cortical collecting duct, and it caused metabolic acidosis. The function of apical potassium channels was altered in this model, and hyperkalemia was fully corrected by pendrin genetic ablation. Thus, we demonstrate an important contribution of pendrin in renal regulation of sodium chloride, potassium and acid-base homeostasis and in the pathophysiology of PHAII. Furthermore, we identify renal distal bicarbonate secretion as a novel mechanism of renal tubular acidosis.

Consequences of SPAK inactivation on Hyperkalemic Hypertension caused by WNK1 mutations: evidence for differential roles of WNK1 and WNK4.

Mutations of the gene encoding WNK1 [With No lysine (K) kinase 1] or WNK4 cause Familial Hyperkalemic Hypertension (FHHt). Previous studies have shown that the activation of SPAK (Ste20-related Proline/Alanine-rich Kinase) plays a dominant role in the development of FHHt caused by WNK4 mutations. The implication of SPAK in FHHt caused by WNK1 mutation has never been investigated. To clarify this issue, we crossed WNK1+/FHHt mice with SPAK knock-in mice in which the T-loop Thr243 residue was mutated to alanine to prevent activation by WNK kinases. We show that WNK1+/FHHT:SPAK 243A/243A mice display an intermediate phenotype, between that of control and SPAK 243A/243A mice, with normal blood pressure but hypochloremic metabolic alkalosis. NCC abundance and phosphorylation levels also decrease below the wild-type level in the double-mutant mice but remain higher than in SPAK 243A/243A mice. This is different from what was observed in WNK4-FHHt mice in which SPAK inactivation completely restored the phenotype and NCC expression to wild-type levels. Although these results confirm that FHHt caused by WNK1 mutations is dependent on the activation of SPAK, they suggest that WNK1 and WNK4 play different roles in the distal nephron.

Acute genetic ablation of pendrin lowers blood pressure in mice.

BACKGROUND: Pendrin, the chloride/bicarbonate exchanger of ß-intercalated cells of the renal connecting tubule and the collecting duct, plays a key role in NaCl reabsorption by the distal nephron. Therefore, pendrin may be important for the control of extracellular fluid volume and blood pressure. METHODS: Here, we have used a genetic mouse model in which the expression of pendrin can be switched-on in vivo by the administration of doxycycline. Pendrin can also be rapidly removed when doxycycline administration is discontinued. Therefore, our genetic strategy allows us to test selectively the acute effects of loss of pendrin function. RESULTS: We show that acute loss of pendrin leads to a significant decrease of blood pressure. In addition, acute ablation of pendrin did not alter significantly the acid-base status or blood K + concentration. CONCLUSION: By using a transgenic mouse model, avoiding off-target effects related to pharmacological compounds, this study suggests that pendrin could be a novel target to treat hypertension.

LIM kinase/cofilin dysregulation promotes macrothrombocytopenia in severe von Willebrand disease-type 2B.

von Willebrand disease type 2B (VWD-type 2B) is characterized by gain-of-function mutations of von Willebrand factor (vWF) that enhance its binding to platelet glycoprotein Ibα and alter the protein's multimeric structure. Patients with VWD-type 2B display variable extents of bleeding associated with macrothrombocytopenia and sometimes with thrombopathy. Here, we addressed the molecular mechanism underlying the severe macrothrombocytopenia both in a knockin murine model for VWD-type 2B by introducing the p.V1316M mutation in the murine Vwf gene and in a patient bearing this mutation. We provide evidence of a profound defect in megakaryocyte (MK) function since: (a) the extent of proplatelet formation was drastically decreased in 2B MKs, with thick proplatelet extensions and large swellings; and (b) 2B MKs presented actin disorganization that was controlled by upregulation of the RhoA/LIM kinase (LIMK)/cofilin pathway. In vitro and in vivo inhibition of the LIMK/cofilin signaling pathway rescued actin turnover and restored normal proplatelet formation, platelet count, and platelet size. These data indicate, to our knowledge for the first time, that the severe macrothrombocytopenia in VWD-type 2B p.V1316M is due to an MK dysfunction that originates from a constitutive activation of the RhoA/LIMK/cofilin pathway and actin disorganization. This suggests a potentially new function of vWF during platelet formation that involves regulation of actin dynamics.

A fate-mapping approach reveals the composite origin of the connecting tubule and alerts on "single-cell"-specific KO model of the distal nephron.

The distal nephron is a heterogeneous part of the nephron composed by six different cell types, forming the epithelium of the distal convoluted (DCT), connecting, and collecting duct. To dissect the function of these cells, knockout models specific for their unique cell marker have been created. However, since this part of the nephron develops at the border between the ureteric bud and the metanephric mesenchyme, the specificity of the single cell markers has been recently questioned. Here, by mapping the fate of the aquaporin 2 (AQP2) and Na+-Cl- cotransporter (NCC)-positive cells using transgenic mouse lines expressing the yellow fluorescent protein fluorescent marker, we showed that the origin of the distal nephron is extremely composite. Indeed, AQP2-expressing precursor results give rise not only to the principal cells, but also to some of the A- and B-type intercalated cells and even to cells of the DCT. On the other hand, some principal cells and B-type intercalated cells can develop from NCC-expressing precursors. In conclusion, these results demonstrate that the origin of different cell types in the distal nephron is not as clearly defined as originally thought. Importantly, they highlight the fact that knocking out a gene encoding for a selective functional marker in the adult does not guarantee cell specificity during the overall kidney development. Tools allowing not only cell-specific but also time-controlled recombination will be useful in this sense.

WNK-SPAK-NCC cascade revisited: WNK1 stimulates the activity of the Na-Cl cotransporter via SPAK, an effect antagonized by WNK4.

The with-no-lysine (K) kinases, WNK1 and WNK4, are key regulators of blood pressure. Their mutations lead to familial hyperkalemic hypertension (FHHt), associated with an activation of the Na-Cl cotransporter (NCC). Although it is clear that WNK4 mutants activate NCC via Ste20 proline-alanine-rich kinase, the mechanisms responsible for WNK1-related FHHt and alterations in NCC activity are not as clear. We tested whether WNK1 modulates NCC through WNK4, as predicted by some models, by crossing our recently developed WNK1-FHHt mice (WNK1(+/FHHt)) with WNK4(-/-) mice. Surprisingly, the activated NCC, hypertension, and hyperkalemia of WNK1(+/FHHt) mice remain in the absence of WNK4. We demonstrate that WNK1 powerfully stimulates NCC in a WNK4-independent and Ste20 proline-alanine-rich kinase-dependent manner. Moreover, WNK4 decreases the WNK1 and WNK3-mediated activation of NCC. Finally, the formation of oligomers of WNK kinases through their C-terminal coiled-coil domain is essential for their activity toward NCC. In conclusion, WNK kinases form a network in which WNK4 associates with WNK1 and WNK3 to regulate NCC.

WNK1-related Familial Hyperkalemic Hypertension results from an increased expression of L-WNK1 specifically in the distal nephron.

Large deletions in the first intron of the With No lysine (K) 1 (WNK1) gene are responsible for Familial Hyperkalemic Hypertension (FHHt), a rare form of human hypertension associated with hyperkalemia and hyperchloremic metabolic acidosis. We generated a mouse model of WNK1-associated FHHt to explore the consequences of this intronic deletion. WNK1(+/FHHt) mice display all clinical and biological signs of FHHt. This phenotype results from increased expression of long WNK1 (L-WNK1), the ubiquitous kinase isoform of WNK1, in the distal convoluted tubule, which in turn, stimulates the activity of the Na-Cl cotransporter. We also show that the activity of the epithelial sodium channel is not altered in FHHt mice, suggesting that other mechanisms are responsible for the hyperkalemia and acidosis in this model. Finally, we observe a decreased expression of the renal outer medullary potassium channel in the late distal convoluted tubule of WNK1(+/FHHt) mice, which could contribute to the hyperkalemia. In summary, our study provides insights into the in vivo mechanisms underlying the pathogenesis of WNK1-mediated FHHt and further corroborates the importance of WNK1 in ion homeostasis and blood pressure.

KLHL3 mutations cause familial hyperkalemic hypertension by impairing ion transport in the distal nephron.

Familial hyperkalemic hypertension (FHHt) is a Mendelian form of arterial hypertension that is partially explained by mutations in WNK1 and WNK4 that lead to increased activity of the Na(+)-Cl(-) cotransporter (NCC) in the distal nephron. Using combined linkage analysis and whole-exome sequencing in two families, we identified KLHL3 as a third gene responsible for FHHt. Direct sequencing of 43 other affected individuals revealed 11 additional missense mutations that were associated with heterogeneous phenotypes and diverse modes of inheritance. Polymorphisms at KLHL3 were not associated with blood pressure. The KLHL3 protein belongs to the BTB-BACK-kelch family of actin-binding proteins that recruit substrates for Cullin3-based ubiquitin ligase complexes. KLHL3 is coexpressed with NCC and downregulates NCC expression at the cell surface. Our study establishes a role for KLHL3 as a new member of the complex signaling pathway regulating ion homeostasis in the distal nephron and indirectly blood pressure.

Decreased ENaC expression compensates the increased NCC activity following inactivation of the kidney-specific isoform of WNK1 and prevents hypertension.

Mutations in WNK1 and WNK4 lead to familial hyperkalemic hypertension (FHHt). Because FHHt associates net positive Na(+) balance together with K(+) and H(+) renal retention, the identification of WNK1 and WNK4 led to a new paradigm to explain how aldosterone can promote either Na(+) reabsorption or K(+) secretion in a hypovolemic or hyperkalemic state, respectively. WNK1 gives rise to L-WNK1, an ubiquitous kinase, and KS-WNK1, a kinase-defective isoform expressed in the distal convoluted tubule. By inactivating KS-WNK1 in mice, we show here that this isoform is an important regulator of sodium transport. KS-WNK1(-/-) mice display an increased activity of the Na-Cl cotransporter NCC, expressed specifically in the distal convoluted tubule, where it participates in the fine tuning of sodium reabsorption. Moreover, the expression of the ROMK and BKCa potassium channels was modified in KS-WNK1(-/-) mice, indicating that KS-WNK1 is also a regulator of potassium transport in the distal nephron. Finally, we provide an alternative model for FHHt. Previous studies suggested that the activation of NCC plays a central role in the development of hypertension and hyperkalemia. Even though the increase in NCC activity in KS-WNK1(-/-) mice was less pronounced than in mice overexpressing a mutant form of WNK4, our study suggests that the activation of Na-Cl cotransporter is not sufficient by itself to induce a hyperkalemic hypertension and that the deregulation of other channels, such as the Epithelial Na(+) channel (ENaC), is probably required.

Cross-talk between mineralocorticoid and angiotensin II signaling for cardiac remodeling.

Experimental and clinical studies show that aldosterone/mineralocorticoid receptor (MR) activation has deleterious effects in the cardiovascular system that may cross-talk with those of angiotensin II (Ang II). This study, using a transgenic mouse model with conditional and cardiomyocyte-restricted overexpression of the human MR, was designed to assess the cardiac consequences of Ang II treatment and cardiomyocyte MR activation. Two-month-old MHCtTA/tetO-hMR double transgenic males (DTg) with conditional, cardiomyocyte-specific human MR expression, and their control littermates were infused with Ang II (200 ng/kg per minute) or vehicle via osmotic minipump. Ang II induced similar increases in systolic blood pressure in control and DTg mice but a greater increase in left ventricle mass/body weight in DTg than in control mice. In DTg mice, Ang II-induced left ventricle hypertrophy and diastolic dysfunction without affecting systolic function, as assessed by echography. These effects were associated with an increase in the expression of collagens and fibronectin, matrix metalloproteinase 2 and matrix metalloproteinase 9 activities, and histological fibrosis. Ang II treatment of DTg mice did not affect inflammation markers, but oxidative stress was substantially increased, as indicated by gp91 expression, apocynin-inhibitable NADPH oxidase activity, and protein carbonylation. These molecular and functional alterations were prevented by pharmacological MR antagonism. Our findings indicate that the effects of Ang II and MR activation in the heart are additive. This observation may be relevant to the clinical use of MR or of combined Ang II type 1 receptor-MR antagonists for hypertrophic cardiomyopathies or for heart failure, particularly when diastolic dysfunction is associated with preserved systolic function.

Conditional glucocorticoid receptor expression in the heart induces atrio-ventricular block.

Corticosteroid hormones (aldosterone and glucocorticoids) and their receptors are now recognized as major modulators of cardiovascular pathophysiology, but their specific roles remain elusive. Glucocorticoid hormones (GCs), which are widely used to treat acute and chronic diseases, often have adverse cardiovascular effects such as heart failure, hypertension, atherosclerosis, or metabolic alterations. The direct effects of GC on the heart are difficult to evaluate, as changes in plasma GC concentrations have multiple consequences due to the ubiquitous expression of the glucocorticoid receptor (GR), resulting in secondary effects on cardiac function. We evaluated the effects of GR on the heart in a conditional mouse model in which the GR was overexpressed solely in cardiomyocytes. The transgenic mice displayed electrocardiogram (ECG) abnormalities: a long PQ interval, increased QRS and QTc duration as well as chronic atrio-ventricular block, without cardiac hypertrophy or fibrosis. The ECG alterations were reversible on GR expression shutoff. Isolated ventricular cardiomyocytes showed major ion channel remodeling, with decreases in I(Na), I(to), and I(Kslow) activity and changes in cell calcium homeostasis (increase in C(al), in Ca2+ transients and in sarcoplasmic reticulum Ca2+ load). This phenotype differs from that observed in mice overexpressing the mineralocorticoid receptor in the heart, which displayed ventricular arrhythmia. Our mouse model highlights novel effects of GR activation in the heart indicating that GR has direct and specific cardiac effects in the mouse.

Conditional mineralocorticoid receptor expression in the heart leads to life-threatening arrhythmias.

BACKGROUND: Life-threatening cardiac arrhythmia is a major source of mortality worldwide. Besides rare inherited monogenic diseases such as long-QT or Brugada syndromes, which reflect abnormalities in ion fluxes across cardiac ion channels as a final common pathway, arrhythmias are most frequently acquired and associated with heart disease. The mineralocorticoid hormone aldosterone is an important contributor to morbidity and mortality in heart failure, but its mechanisms of action are incompletely understood. METHODS AND RESULTS: To specifically assess the role of the mineralocorticoid receptor (MR) in the heart, in the absence of changes in aldosteronemia, we generated a transgenic mouse model with conditional cardiac-specific overexpression of the human MR. Mice exhibit a high rate of death prevented by spironolactone, an MR antagonist used in human therapy. Cardiac MR overexpression led to ion channel remodeling, resulting in prolonged ventricular repolarization at both the cellular and integrated levels and in severe ventricular arrhythmias. CONCLUSIONS: Our results indicate that cardiac MR triggers cardiac arrhythmias, suggesting novel opportunities for prevention of arrhythmia-related sudden death.