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Journal of the American Society of... Apr 2024Proteinuria predicts accelerated decline in kidney function in CKD. The pathologic mechanisms are not well known, but aberrantly filtered proteins with enzymatic...
SIGNIFICANCE STATEMENT
Proteinuria predicts accelerated decline in kidney function in CKD. The pathologic mechanisms are not well known, but aberrantly filtered proteins with enzymatic activity might be involved. The urokinase-type plasminogen activator (uPA)-plasminogen cascade activates complement and generates C3a and C5a in vitro / ex vivo in urine from healthy persons when exogenous, inactive, plasminogen, and complement factors are added. Amiloride inhibits uPA and attenuates complement activation in vitro and in vivo . In conditional podocin knockout (KO) mice with severe proteinuria, blocking of uPA with monoclonal antibodies significantly reduces the urine excretion of C3a and C5a and lowers tissue NLRP3-inflammasome protein without major changes in early fibrosis markers. This mechanism provides a link to proinflammatory signaling in proteinuria with possible long-term consequences for kidney function.
BACKGROUND
Persistent proteinuria is associated with tubular interstitial inflammation and predicts progressive kidney injury. In proteinuria, plasminogen is aberrantly filtered and activated by urokinase-type plasminogen activator (uPA), which promotes kidney fibrosis. We hypothesized that plasmin activates filtered complement factors C3 and C5 directly in tubular fluid, generating anaphylatoxins, and that this is attenuated by amiloride, an off-target uPA inhibitor.
METHODS
Purified C3, C5, plasminogen, urokinase, and urine from healthy humans were used for in vitro / ex vivo studies. Complement activation was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and ELISA. Urine and plasma from patients with diabetic nephropathy treated with high-dose amiloride and from mice with proteinuria (podocin knockout [KO]) treated with amiloride or inhibitory anti-uPA antibodies were analyzed.
RESULTS
The combination of uPA and plasminogen generated anaphylatoxins C3a and C5a from intact C3 and C5 and was inhibited by amiloride. Addition of exogenous plasminogen was sufficient for urine from healthy humans to activate complement. Conditional podocin KO in mice led to severe proteinuria and C3a and C5a urine excretion, which was attenuated reversibly by amiloride treatment for 4 days and reduced by >50% by inhibitory anti-uPA antibodies without altering proteinuria. NOD-, LRR- and pyrin domain-containing protein 3-inflammasome protein was reduced with no concomitant effect on fibrosis. In patients with diabetic nephropathy, amiloride reduced urinary excretion of C3dg and sC5b-9 significantly.
CONCLUSIONS
In conditions with proteinuria, uPA-plasmin generates anaphylatoxins in tubular fluid and promotes downstream complement activation sensitive to amiloride. This mechanism links proteinuria to intratubular proinflammatory signaling. In perspective, amiloride could exert reno-protective effects beyond natriuresis and BP reduction.
CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER
Increased Activity of a Renal Salt Transporter (ENaC) in Diabetic Kidney Disease, NCT01918488 and Increased Activity of ENaC in Proteinuric Kidney Transplant Recipients, NCT03036748 .
Topics: Humans; Mice; Animals; Urokinase-Type Plasminogen Activator; Plasminogen; Amiloride; Fibrinolysin; Diabetic Nephropathies; Inflammasomes; Mice, Inbred NOD; Proteinuria; Complement Activation; Anaphylatoxins; Fibrosis
PubMed: 38254266
DOI: 10.1681/ASN.0000000000000312 -
International Journal of Molecular... Oct 2023In proteinuric renal diseases, the serine protease (SP) plasmin activates the epithelial sodium channel (ENaC) by cleaving its γ subunit. We previously demonstrated...
In proteinuric renal diseases, the serine protease (SP) plasmin activates the epithelial sodium channel (ENaC) by cleaving its γ subunit. We previously demonstrated that a high-salt (HS) diet provoked hypertension and proteinuria in Dahl salt-sensitive (DS) rats, accompanied by γENaC activation, which were attenuated by camostat mesilate (CM), an SP inhibitor. However, the effects of CM on plasmin activity in DS rats remain unclear. In this study, we investigated the effects of CM on plasmin activity, ENaC activation, and podocyte injury in DS rats. The DS rats were divided into the control diet, HS diet (8.0% NaCl), and HS+CM diet (0.1% CM) groups. After weekly blood pressure measurement and 24-h urine collection, the rats were sacrificed at 5 weeks. The HS group exhibited hypertension, massive proteinuria, increased urinary plasmin, and γENaC activation; CM treatment suppressed these changes. CM prevented plasmin(ogen) attachment to podocytes and mitigated podocyte injury by reducing the number of apoptotic glomerular cells, inhibiting protease-activated receptor-1 activation, and suppressing inflammatory and fibrotic cytokine expression. Our findings highlight the detrimental role of urinary plasmin in the pathogenesis of salt-sensitive hypertension and glomerular injury. Targeting plasmin with SP inhibitors, such as CM, may be a promising therapeutic approach for these conditions.
Topics: Rats; Animals; Serine Proteinase Inhibitors; Fibrinolysin; Podocytes; Rats, Inbred Dahl; Hypertension; Serpins; Sodium Chloride, Dietary; Proteinuria; Blood Pressure; Kidney
PubMed: 37958726
DOI: 10.3390/ijms242115743 -
Seminars in Thrombosis and Hemostasis Jul 2024Fibrinolytic agents catalyze the conversion of the inactive proenzyme plasminogen into the active protease plasmin, degrading fibrin within the thrombus and recanalizing... (Review)
Review
Fibrinolytic agents catalyze the conversion of the inactive proenzyme plasminogen into the active protease plasmin, degrading fibrin within the thrombus and recanalizing occluded vessels. The history of these medications dates to the discovery of the first fibrinolytic compound, streptokinase, from bacterial cultures in 1933. Over time, researchers identified two other plasminogen activators in human samples, namely urokinase and tissue plasminogen activator (tPA). Subsequently, tPA was cloned using recombinant DNA methods to produce alteplase. Several additional derivatives of tPA, such as tenecteplase and reteplase, were developed to extend the plasma half-life of tPA. Over the past decades, fibrinolytic medications have been widely used to manage patients with venous and arterial thromboembolic events. Currently, alteplase is approved by the U.S. Food and Drug Administration (FDA) for use in patients with pulmonary embolism with hemodynamic compromise, ST-segment elevation myocardial infarction (STEMI), acute ischemic stroke, and central venous access device occlusion. Reteplase and tenecteplase have also received FDA approval for treating patients with STEMI. This review provides an overview of the historical background related to fibrinolytic agents and briefly summarizes their approved indications across various thromboembolic diseases.
Topics: Humans; Fibrinolytic Agents; Thromboembolism; History, 20th Century
PubMed: 38428841
DOI: 10.1055/s-0044-1781451 -
International Journal of Molecular... Sep 2023The fibrinolytic system is a key player in keeping the haemostatic balance, and changes in fibrinolytic capacity can lead to both bleeding-related and thrombosis-related... (Review)
Review
The fibrinolytic system is a key player in keeping the haemostatic balance, and changes in fibrinolytic capacity can lead to both bleeding-related and thrombosis-related disorders. Our knowledge of the fibrinolytic system has expanded immensely during the last 75 years. From the first successful use of thrombolysis in myocardial infarction in the 1960s, thrombolytic therapy is now widely implemented and has reformed treatment in vascular medicine, especially ischemic stroke, while antifibrinolytic agents are used routinely in the prevention and treatment of major bleeding worldwide. Despite this, this research field still holds unanswered questions. Accurate and timely laboratory diagnosis of disturbed fibrinolysis in the clinical setting remains a challenge. Furthermore, despite growing evidence that hypofibrinolysis plays a central role in, e.g., sepsis-related coagulopathy, coronary artery disease, and venous thromboembolism, there is currently no approved treatment of hypofibrinolysis in these settings. The present review provides an overview of the fibrinolytic system and history of its discovery; measurement methods; clinical relevance of the fibrinolytic system in diagnosis and treatment; and points to future directions for research.
Topics: Humans; Antifibrinolytic Agents; Clinical Relevance; Coronary Artery Disease; Fibrin Clot Lysis Time; Fibrinolysis
PubMed: 37762481
DOI: 10.3390/ijms241814179 -
The Journal of Investigative Dermatology May 2024The skin is the first host tissue that the tick mouthparts, tick saliva, and a tick-borne pathogen contact during feeding. Tick salivary glands have evolved a complex...
The skin is the first host tissue that the tick mouthparts, tick saliva, and a tick-borne pathogen contact during feeding. Tick salivary glands have evolved a complex and sophisticated pharmacological arsenal, consisting of bioactive molecules, to assist blood feeding and pathogen transmission. In this work, persulcatin, a multifunctional molecule that targets keratinocyte function and hemostasis, was identified from Ixodes persulcatus female ticks. The recombinant persulcatin was expressed and purified and is a 25-kDa acidic protein with 2 Kunitz-type domains. Persulcatin is a classical tight-binding competitive inhibitor of proteases, targeting plasmin (K: 28 nM) and thrombin (K: 115 nM). It blocks plasmin generation on keratinocytes and inhibits their migration and matrix protein degradation; downregulates matrix metalloproteinase 2 and matrix metalloproteinase 9; and causes a delay in blood coagulation, endothelial cell activation, and thrombin-induced fibrinocoagulation. It interacts with exosite I of thrombin and reduces thrombin-induced endothelial cell permeability by inhibiting vascular endothelial-cadherin disruption. The multifaceted roles of persulcatin as an inhibitor and modulator within the plasminogen-plasmin system and thrombin not only unveil further insights into the intricate mechanisms governing wound healing but also provide a fresh perspective on the intricate interactions between ticks and their host organisms.
Topics: Ixodes; Animals; Fibrinolysin; Keratinocytes; Thrombin; Cell Movement; Humans; Blood Coagulation; Female; Endothelial Cells; Wound Healing
PubMed: 37996063
DOI: 10.1016/j.jid.2023.10.026 -
International Journal of Gynaecology... Sep 2023To evaluate the hemostatic effects of tranexamic acid (TXA) ex vivo in women with pre-eclampsia.
OBJECTIVE
To evaluate the hemostatic effects of tranexamic acid (TXA) ex vivo in women with pre-eclampsia.
METHODS
This was an ex vivo study involving 45 normal pregnant women and 45 women with pre-eclampsia (nine with mild and 36 with severe features) matched for age, gestational age, and body mass index. Blood samples were collected and divided into two parts. The first served as the pre-TXA sample, while the second was spiked with TXA and served as the post-TXA sample. Plasma levels of D-dimer and plasmin-antiplasmin complex (PAP) were determined using enzyme-linked immunosorbent assay.
RESULTS
The mean D-dimer and PAP values in the pre-TXA samples differed significantly between groups. Following spiking with TXA, the mean D-dimer and PAP levels did not differ significantly in the pre-TXA and post-TXA samples (P = 0.560 and P = 0.500, respectively) in the pre-eclampsia cohort. In normal pregnancy, the mean D-dimer and PAP levels in the post-TXA samples did not differ significantly (P = 0.070 and P = 0.050, respectively) from the pre-TXA samples following TXA spiking.
CONCLUSION
TXA did not significantly affect D-dimer and PAP levels in pre-eclampsia, suggesting that TXA may not increase the thrombotic risks in patients with pre-eclampsia.
Topics: Pregnancy; Female; Humans; Hemostatics; Tranexamic Acid; Pre-Eclampsia; Body Mass Index
PubMed: 37067045
DOI: 10.1002/ijgo.14779 -
The Journal of Biological Chemistry Oct 2023Most serine proteases are synthesized as inactive zymogens that are activated by cleavage by another protease in a tightly regulated mechanism. The urokinase-type...
Most serine proteases are synthesized as inactive zymogens that are activated by cleavage by another protease in a tightly regulated mechanism. The urokinase-type plasminogen activator (uPA) and plasmin cleave and activate each other, constituting a positive feedback loop. How this mutual activation cycle begins has remained a mystery. We used hydrogen deuterium exchange mass spectrometry to characterize the dynamic differences between the inactive single-chain uPA (scuPA) and its active form two-chain uPA (tcuPA). The results show that the C-terminal β-barrel and the area around the new N terminus have significantly reduced dynamics in tcuPA as compared with scuPA. We also show that the zymogen scuPA is inactive but can, upon storage, become active in the absence of external proteases. In addition to plasmin, the tcuPA can activate scuPA by cleavage at K158, a process called autoactivation. Unexpectedly, tcuPA can cleave at position 158 even when this site is mutated. TcuPA can also cleave scuPA after K135 or K136 in the disordered linker, which generates the soluble protease domain of uPA. Plasmin cleaves scuPA exclusively after K158 and at a faster rate than tcuPA. We propose a mechanism by which the uPA receptor dimerization could promote autoactivation of scuPA on cell surfaces. These results resolve long-standing controversies in the literature surrounding the mechanism of uPA activation.
PubMed: 37607618
DOI: 10.1016/j.jbc.2023.105179 -
Thrombosis Research Oct 2023Cranial and extra-cranial vascular events are among the major determinants of morbidity and mortality in Giant Cell Arteritis (GCA). Vascular events seem mostly of...
BACKGROUND
Cranial and extra-cranial vascular events are among the major determinants of morbidity and mortality in Giant Cell Arteritis (GCA). Vascular events seem mostly of inflammatory nature, although the precise pathogenetic mechanisms are still unclear. We investigated the role of oxidation-induced structural and functional fibrinogen modifications in GCA. The effects of the anti-IL6R tocilizumab in counteracting these mechanisms were also assessed.
MATERIALS AND METHODS
A cross-sectional study was conducted on 65 GCA patients and 65 matched controls. Leucocyte reactive oxygen species (ROS) production, redox state, and fibrinogen structural and functional features were compared between patients and controls. In 19 patients receiving tocilizumab, pre vs post treatment variations were assessed.
RESULTS
GCA patients displayed enhanced blood lymphocyte, monocyte and neutrophil ROS production compared to controls, with an increased plasma lipid peroxidation and a reduced total antioxidant capacity. This oxidative impairment resulted in a sustained fibrinogen oxidation (i.e. dityrosine content 320 (204-410) vs 136 (120-176) Relative Fluorescence Units (RFU), p < 0.0001), with marked alterations in fibrinogen secondary and tertiary structure [intrinsic fluorescence: 134 (101-227) vs 400 (366-433) RFU, p < 0.001]. Structural alterations paralleled a remarkable fibrinogen functional impairment, with a reduced ability to polymerize into fibrin and a lower fibrin susceptibility to plasmin-induced lysis. In patients receiving tocilizumab, a significant improvement in redox status was observed, accompanied by a significant improvement in fibrinogen structural and functional features (p < 0.001).
CONCLUSIONS
An impaired redox status accounts for structural and functional fibrinogen modifications in GCA, suggesting a potential role of tocilizumab for cardiovascular prevention in GCA.
Topics: Humans; Giant Cell Arteritis; Interleukin-6; Reactive Oxygen Species; Fibrinogen; Cross-Sectional Studies; Hemostatics; Fibrin
PubMed: 37598635
DOI: 10.1016/j.thromres.2023.08.011 -
Biomedicine & Pharmacotherapy =... Oct 2023The chronic disease psoriasis is associated with severe inflammation and abnormal keratinocyte propagation in the skin. Tranexamic acid (TXA), a plasmin inhibitor, is...
The chronic disease psoriasis is associated with severe inflammation and abnormal keratinocyte propagation in the skin. Tranexamic acid (TXA), a plasmin inhibitor, is used to cure serious bleeding. We investigated whether TXA ointment mitigated Imiquimod (IMQ)-induced psoriasis-like inflammation. Furthermore, this study investigated the effect of noncytotoxic concentrations of TXA on IL-17-induced human keratinocyte (HaCaT) cells to determine the status of proliferative psoriatic keratinocytes. We found that TXA reduced IMQ-induced psoriasis-like erythema, thickness, scaling, and cumulative scores (erythema plus thickness plus scaling) on the back skin of BALB/c mice. Additionally, TXA decreased ear thickness and suppressed hyperkeratosis, hyperplasia, and inflammation of the ear epidermis in IMQ-induced BALB/c mice. Furthermore, TXA inhibited IMQ-induced splenomegaly in BALB/c mouse models. In IL-17-induced HaCaT cells, TXA inhibited ROS production and IL-8 secretion. Interestingly, TXA suppressed the IL-17-induced NFκB signaling pathway via IKK-mediated IκB degradation. TXA inhibited IL-17-induced activation of the NLRP3 inflammasome through caspase-1 and IL1β expression. TXA inhibited IL-17-induced NLRP3 inflammasome activation by enhancing autophagy, as indicated by LC3-II accumulation, p62/SQSTM1 expression, ATG4B inhibition, and Beclin-1/Bcl-2 dysregulation. Notably, TXA suppressed IL-17-induced Nrf2-mediated keratin 17 expression. N-acetylcysteine pretreatment reversed the effects of TXA on NFκB, NLRP3 inflammasomes, and the Nrf2-mediated keratin 17 pathway in IL-17-induced HaCaT cells. Results further confirmed that in the ear skin of IMQ-induced mice, psoriasis biomarkers such as NLRP3, IL1β, Nrf2, and keratin 17 expression were downregulated by TXA treatment. TXA improves IMQ-induced psoriasis-like inflammation in vivo and psoriatic keratinocytes in vitro. Tranexamic acid is a promising future treatment for psoriasis.
Topics: Humans; Animals; Mice; Interleukin-17; Tranexamic Acid; Inflammasomes; NLR Family, Pyrin Domain-Containing 3 Protein; Keratin-17; NF-E2-Related Factor 2; Psoriasis; Dermatitis; Skin; Keratinocytes; Inflammation; Imiquimod; NF-kappa B; Mice, Inbred BALB C; Disease Models, Animal
PubMed: 37573659
DOI: 10.1016/j.biopha.2023.115307 -
PLoS Pathogens Jul 2023Leptospirosis, a zoonosis with worldwide distribution, is caused by pathogenic spirochetes belonging to the genus Leptospira. Bacterial outer membrane proteins (OMPs),...
Leptospirosis, a zoonosis with worldwide distribution, is caused by pathogenic spirochetes belonging to the genus Leptospira. Bacterial outer membrane proteins (OMPs), particularly those with surface-exposed regions, play crucial roles in pathogen dissemination and virulence mechanisms. Here we characterized the leptospiral Membrane Protein L36 (MPL36), a rare lipoprotein A (RlpA) homolog with a C-terminal Sporulation related (SPOR) domain, as an important virulence factor in pathogenic Leptospira. Our results confirmed that MPL36 is surface exposed and expressed during infection. Using recombinant MPL36 (rMPL36) we also confirmed previous findings of its high plasminogen (PLG)-binding ability determined by lysine residues of the C-terminal region of the protein, with ability to convert bound-PLG to active plasmin. Using Koch's molecular postulates, we determined that a mutant of mpl36 has a reduced PLG-binding ability, leading to a decreased capacity to adhere and translocate MDCK cell monolayers. Using recombinant protein and mutant strains, we determined that the MPL36-bound plasmin (PLA) can degrade fibrinogen. Finally, our mpl36 mutant had a significant attenuated phenotype in the hamster model for acute leptospirosis. Our data indicates that MPL36 is the major PLG binding protein in pathogenic Leptospira, and crucial to the pathogen's ability to attach and interact with host tissues during infection. The MPL36 characterization contributes to the expanding field of bacterial pathogens that explore PLG for their virulence, advancing the goal to close the knowledge gap regarding leptospiral pathogenesis while offering a novel potential candidate to improve diagnostic and prevention of this important zoonotic neglected disease.
Topics: Cricetinae; Animals; Leptospira; Plasminogen; Fibrinolysin; Leptospira interrogans; Protein Binding; Leptospirosis; Bacterial Outer Membrane Proteins; Recombinant Proteins
PubMed: 37486929
DOI: 10.1371/journal.ppat.1011313