PS-B08-13: PLASMIN INHIBITION ALLEVIATES HYPERTENSION AND PODOCYTE INJURY IN DAHL SALT-SENSITIVE RATS
Qinyuan DengYutaka KakizoeYasunobu IwataTerumasa NakagawaYoshikazu MiyasatoMiyuki NakagawaKayo NishiguchiYu NagayoshiMasataka AdachiYuki NaritaYuichiro IzumiTakashige KuwabaraYuko TsudaMasashi Mukoyama
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Objective: The epithelial sodium channel (ENaC) in the kidney plays pivotal roles in blood pressure regulation, and the γ subunit is activated by extracellular serine proteases. In proteinuric renal diseases, plasmin filtered through injured glomeruli proteolytically activates γENaC. In addition, filtered plasmin directly causes podocyte injury. We previously reported that Dahl salt-sensitive (DS) rats fed a high-salt (HS) diet developed severe hypertension and proteinuria together with γENaC activation, and that a synthetic serine protease inhibitor, camostat mesilate, mitigated these changes. However, the role of plasmin in DS rats remains unclear. In this study, we evaluated the relationship between plasmin and hypertension as well as glomerular injury and the effects of plasmin inhibitors in DS rats. Design and method: Five-week-old male DS rats were divided into normal-salt (NS) diet, HS diet, and HS + plasmin inhibitors [tranexamic acid (TA, 2 mg/mL TA in drinking water) and synthetic plasmin inhibitor YO-2 (4 mg/kg/day, intraperitoneal injection)] groups. After systolic blood pressure measurement and 24-h urine collection over time for 5 weeks, the rats were sacrificed for biochemical examination. Results: The HS group displayed severe hypertension and proteinuria together with activation of plasmin in urine and γENaC in the kidney, which were not attenuated by TA. On the other hand, YO-2 mitigated both hypertension and proteinuria [SBP (mmHg): NS, 130.8 ± 7.5; HS, 209.0 ± 14.2; HS+YO-2, 183.8 ± 8.4; Urinary protein (mg/day): NS, 15.4 ± 3.2; HS 259.0 ± 129.3; HS+YO-2, 94.5 ± 31.5]. YO-2 inhibited the attachment of plasmin(ogen) to podocytes and alleviated podocyte injury by reducing glomerular apoptotic cells. Furthermore, YO-2 suppressed the upregulation of protease-activated receptor-1 and phosphorylated ERK1/2 as well as mRNA expression of inflammatory and pro-fibrotic cytokines in the kidney. Conclusion: These results indicate that plasmin plays important roles in the development of salt-sensitive hypertension and glomerular injuries in a rat model of hypertension, and suggest that plasmin inhibition could be a potential therapeutic strategy against salt-sensitive hypertension.Keywords:
Epithelial sodium channel
We previously reported that Dot1a‐AF9 complex inhibits the transcription of α ‐ ENaC by hypermethylating histone H3 K79 at its promoter. Aldosterone downregulates this complex through various mechanisms. AF17 upregulates transcription of all ENaC genes and their key regulators by competitively binding to and promoting nuclear export of Dot1a. However, the significance of these transcriptional mechanisms in regulating Na + transport has not been fully defined. To address this question, mouse inner medullary collecting duct cell line‐3 (mIMCD3) and mouse cortical collecting duct M1 cells were used to determine ENaC activity as measured by benzamil‐sensitive intracellular Na + concentration ([Na + ] i ) using fluorescence imaging or benzamil‐sensitive equivalent short circuit current (I sc ) using an EVOM ohm/volt meter. In both cell lines, addition of benzamil significantly decreased the basal [Na + ] i and I sc values. Benzamil‐sensitive [Na + ] i or I sc values were significantly decreased by overexpression of Dot1a or AF9, but increased by AF17 overexpression. The effect of aldosterone on benzamil‐sensitive [Na + ] i and I sc was impaired by blocking either transcription or translation. Our data suggest that ENaC is largely responsible for Na + uptake in these cells, and that transcriptional changes in ENaC genes mediated by Dot1a, AF9, AF17 or aldosterone treatment are translated into changes in ENaC‐mediated Na + transport.
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Background: The amiloride-sensitive Epithelial Sodium Channel (ENaC) is critical in maintaining Na + balance, extracellular fluid volume and long term blood pressure control.ENaC is composed of three main subunits α, β, & γ.While α ENaC is critical for channel functionality, β & γ ENaC maximize channel function.To date, there are four alternatively spliced forms of the α subunit of ENaC (α ENaC-a, -b, -c, & -d) that have been published in rats, in addition to the major α ENaC transcript.While α ENaC-a, -c & -d transcripts are low abundance transcripts compared to fulllength α ENaC, α ENaC-b is a higher abundance and salt-sensitive transcript compared to full-length α ENaC.Presentation of the hypothesis: α ENaC-b protein, which is preferentially produced in Dahl R rats, to a greater extent on high salt diet, exerts a dominant negative effect on full-length α ENaC subunit by physically binding to and trapping full-length α ENaC subunit in the endoplasmic reticulum, and finally accelerating full-length α ENaC proteolytic degradation in a dose-dependent manner.Testing the hypothesis: 1) To examine the mRNA and protein abundance of α ENaC-b relative to α ENaC full-length in kidney, lung, and taste tissues of Dahl rats.2) To compare the expression (mRNA and protein) of α ENaC-b in kidneys of Dahl S and R rats on regular and high salt diet.3) To examine the putative binding of α ENaC-b proteins to full-length α ENaC in vitro and to determine the impact of such binding on full-length α ENaC expression in vitro.Implications of the hypothesis: Our studies will be the first to demonstrate the over-expression of salt-sensitive α ENaC-b spliced form in kidney tissues of Dahl R rats at the expense of full-length α ENaC.The current proposal will provide highly novel insights into the putative mechanisms leading to ENaC hypoactivity in high-salt-fed Dahl R rats.Finally, findings from the present proposal will uncover a new mechanism by which alternative splicing may control the regulation of ENaC expression/function.
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Stabilized, active plasmin is a novel thrombolytic for direct delivery to clots. Although it is known that protease inhibitors in plasma inhibit plasmin, the amount of plasmin that can be added to plasma/blood before free plasmin is observed is not clear. Determination of free plasmin activity in plasma using chromogenic substrates represents a challenge due to false-positive signals from plasmin entrapped by alpha2-macroglobulin. Size-exclusion chromatography was used to separate the plasmin-alpha2-macroglobulin complex from uninhibited, free plasmin. In this in-vitro study, exogenous plasmin is effectively inhibited up to 2.4 micromol/l after 5-min incubation with plasma at 37 degrees C. Initially, plasmin was consumed predominantly by alpha2-antiplasmin up to 1.2 micromol/l plasmin. Following exhaustion of alpha2-antiplasmin, plasmin was further consumed by alpha2-macroglobulin up to 2.4 micromol/l plasmin added to human plasma. Whole human blood was found to have an increased inhibitory capacity over that of plasma; free plasmin activity could be measured only above 3.8 micromol/l added plasmin. In conclusion, several mechanisms exist that control plasmin activity in human blood; in addition to alpha2-antiplasmin and alpha2-macroglobulin, blood cells contribute to the inhibition of exogenously administered plasmin. These in-vitro results indicate that doses of plasmin up to approximately 12 mg/kg in humans can be completely inactivated by blood.
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The epithelial sodium channel (ENaC) plays an important role in controlling Na + homeostasis, extracellular fluid volume, and blood pressure. Copper metabolism Murr1 domain-containing protein 1 (COMMD1) interacts with ENaC and downregulates ENaC. COMMD1 belongs to the COMMD family consisting of COMMD1–10, and all COMMD family members share a C-terminal COMM domain. Here, we report that COMMD2–10 also interacts with ENaC, and COMMD3 and COMMD9 were selected for further study. Amiloride-sensitive current in mammalian epithelia expressing ENaC was significantly reduced by COMMD3 or COMMD9, and ENaC expression at the cell surface was significantly decreased in the presence of COMMD3 or COMMD9. COMMD3 and COMMD9 retained their ability to reduce current when COMMD1 was knocked down. COMMD3 and COMMD9 were widely expressed in kidney and were colocalized with ENaC in renal collecting duct cells. These data suggest that COMMD3 and COMMD9 may be endogenous regulators of ENaC to regulate Na + transport through altering ENaC cell surface expression.
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The paper describes properties of proteases A and B isolated from the biological insecticide bitoxibacillin by sulphate precipitation and Sephadex G-75 gel filtration. Proteases A and B of bitoxibacillin belong to the neutral bacterial proteases. pH optimum was found to be 6.0 and 7.5 for detection of proteolytic activity of protease A and protease B, respectively. Thermal stability of proteases A and B was similar and increased by 25% upon addition of CaCl2. Both proteases were inhibited with EDTA. The molecular weight of proteases A and B was estimated to be 57,000 and 47,000, respectively.
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ANALYSIS OF PROTEASE KINDS AND ACTIVITIES IN SEVEN KINDS OF TISSUES AND ORGANS OF ANDRIAS DAVIDIANUS
Objective Finding out the kinds and activities in protease of brain,heart,lung,kidney,eyes,skin and muscle of Andrias davidianus. Methods Using protease returned electrophoresis technique(G-PAGE). Results 1.Activity of proteases in brain was weak at pH 4.5,and showed no activity at pH 7.0 and pH 9.5;2.Proteases of heart and lung had activities at pH 4.5 and pH 7.0;3.Activity of kidney proteases was strong at pH 4.5 and stronger at pH 7.0,and kinds of proteases were more at pH 7.0 than at pH 4.5;4.Proteases in eyes had no activity at pH 4.5 and pH 9.5;5.Activity of proteases in muscle was very strong at pH 7.0 and pH 9.5,and kinds of proteases were more at pH 7.0 than at pH 9.5;6.Activity of skin proteases was similar to that of muscle,but the former was weaker than the later. Conclusions Among the seven kinds of tissues and organs of Andrias davidianus,kinds and activities of kidney and muscle proteases were stronger and more than others;and the eyes showed almost no protease activity.The proteases-activity optimal pH of brain was acidic;The proteases-activity optimal pH of heart,lung and kidney was acid of neutral;and the proteases-activity optimal pH of skin and muscle was neutral.
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Podocytes play a pivotal role in maintaining glomerular filtration function through their interdigitated foot processes. However, the mechanisms that govern the podocyte cytoskeletal rearrangement remain unclear. Through analyzing the transcriptional profile of renal biopsy specimens from patients with diabetic nephropathy (DN) and control donors, we identify SLIT-ROBO ρGTPase-activating protein 2a (SRGAP2a) as one of the main hub genes strongly associated with proteinuria and glomerular filtration in type 2 DN. Immunofluorescence staining and Western blot analysis revealed that human and mouse SRGAP2a is primarily localized at podocytes and largely colocalized with synaptopodin. Moreover, podocyte SRGAP2a is downregulated in patients with DN and db/db mice at both the mRNA and the protein level. SRGAP2a reduction is observed in cultured podocytes treated with tumor growth factor-β or high concentrations of glucose. Functional and mechanistic studies show that SRGAP2a suppresses podocyte motility through inactivating RhoA/Cdc42 but not Rac1. The protective role of SRGAP2a in podocyte function also is confirmed in zebrafish, in which knockdown of SRGAP2a, a SRGAP2 ortholog in zebrafish, recapitulates podocyte foot process effacement. Finally, increasing podocyte SRGAP2a levels in db/db mice through administration of adenovirus-expressing SRGAP2a significantly mitigates podocyte injury and proteinuria. The results demonstrate that SRGAP2a protects podocytes by suppressing podocyte migration.
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Abstract Proteases are fundamental for a plethora of biological processes, including signalling and tissue remodelling, and dysregulated proteolytic activity can result in pathogenesis. In this review, we focus on a subclass of membrane‐bound and soluble proteases that are defined as channel‐activating proteases (CAPs), since they induce Na + ion transport through an autocrine mechanism when co‐expressed with the highly amiloride‐sensitive epithelial sodium channel (ENaC) in Xenopus oocytes. These experiments first identified CAP1 (channel‐activating protease 1, prostasin) followed by CAP2 (channel‐activating protease 2, TMPRSS4) and CAP3 (channel‐activating protease 3, matriptase) as in vitro mediators of ENaC current. Since then, more serine‐, cysteine‐ and metalloproteases were confirmed as in vitro CAPs that potentially cleave and regulate ENaC, and thus this nomenclature was not further followed, but is accepted as functional term or alias. The precise mechanism of ENaC modulation by proteases has not been fully elucidated. Studies in organ‐specific protease knockout models revealed evidence for their role in increasing ENaC activity, although the proteases responsible for ENaC activation are yet to be identified. We summarize recent findings in animal models of these CAPs with respect to their implication in ENaC activation. We discuss the consequences of dysregulated CAPs underlying epithelial phenotypes in pathophysiological conditions, and the role of selected protease inhibitors. We believe that these proteases may present interesting therapeutic targets for diseases with aberrant sodium homoeostasis.
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