| 1. |
- Bergwall, Lovisa, et al.
(författare)
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Amplification of the Melanocortin-1 Receptor in Nephrotic Syndrome Identifies a Target for Podocyte Cytoskeleton Stabilization
- 2018
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- The melanocortin-1 receptor (MC1R) in podocytes has been suggested as the mediator of the ACTH renoprotective effect in patients with nephrotic syndrome with the mechanism of action beeing stabilization of the podocyte actin cytoskeleton. To understand how melanocortin receptors are regulated in nephrotic syndrome and how they are involved in restoration of filtration barrier function, melanocortin receptor expression was evaluated in patients and a rat model of nephrotic syndrome in combination with cell culture analysis. Phosphoproteomics was applied and identified MC1R pathways confirmed using biochemical analysis. We found that glomerular MC1R expression was increased in nephrotic syndrome, both in humans and in a rat model. A MC1R agonist protected podocytes from protamine sulfate induced stress fiber loss with the top ranked phoshoproteomic MC1R activated pathway beeing actin cytoskeleton signaling. Actin stabilization through the MC1R consisted of ERK1/2 dependent phosphorylation and inactivation of EGFR signaling with stabilization of synaptopodin and stressfibers in podocytes. These results further explain how patients with nephrotic syndrome show responsiveness to MC1R receptor activation by decreasing EGFR signaling and as a consequence restore filtration barrier function by stabilizing the podocyte actin cytoskeleton.
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| 2. |
- Boi, Roberto, et al.
(författare)
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Podocyte Geranylgeranyl Transferase Type-I Is Essential for Maintenance of the Glomerular Filtration Barrier
- 2023
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Ingår i: Journal of the American Society of Nephrology. - : Ovid Technologies (Wolters Kluwer Health). - 1046-6673 .- 1533-3450. ; 34:4, s. 641-655
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Tidskriftsartikel (refereegranskat)abstract
- Significance StatementA tightly regulated actin cytoskeleton attained through balanced activity of RhoGTPases is crucial to maintaining podocyte function. However, how RhoGTPases are regulated by geranylgeranylation, a post-translational modification, has been unexplored. The authors found that loss of the geranylgeranylation enzyme geranylgeranyl transferase type-I (GGTase-I) in podocytes led to progressive albuminuria and foot process effacement in podocyte-specific GGTase-I knockout mice. In cultured podocytes, the absence of geranylgeranylation resulted in altered activity of its downstream substrates Rac1, RhoA, Cdc42, and Rap1, leading to alterations of & beta;1-integrins and actin cytoskeleton structural changes. These findings highlight the importance of geranylgeranylation in the dynamic management of RhoGTPases and Rap1 to control podocyte function, providing new knowledge about podocyte biology and glomerular filtration barrier function.BackgroundImpairment of the glomerular filtration barrier is in part attributed to podocyte foot process effacement (FPE), entailing disruption of the actin cytoskeleton and the slit diaphragm. Maintenance of the actin cytoskeleton, which contains a complex signaling network through its connections to slit diaphragm and focal adhesion proteins, is thus considered crucial to preserving podocyte structure and function. A dynamic yet tightly regulated cytoskeleton is attained through balanced activity of RhoGTPases. Most RhoGTPases are post-translationally modified by the enzyme geranylgeranyl transferase type-I (GGTase-I). Although geranylgeranylation has been shown to regulate activities of RhoGTPases and RasGTPase Rap1, its significance in podocytes is unknown.MethodsWe used immunofluorescence to localize GGTase-I, which was expressed mainly by podocytes in the glomeruli. To define geranylgeranylation's role in podocytes, we generated podocyte-specific GGTase-I knockout mice. We used transmission electron microscopy to evaluate FPE and measurements of urinary albumin excretion to analyze filtration barrier function. Geranylgeranylation's effects on RhoGTPases and Rap1 function were studied in vitro by knockdown or inhibition of GGTase-I. We used immunocytochemistry to study structural modifications of the actin cytoskeleton and & beta;1 integrins.ResultsDepletion of GGTase-I in podocytes in vivo resulted in FPE and concomitant early-onset progressive albuminuria. A reduction of GGTase-I activity in cultured podocytes disrupted RhoGTPase balance by markedly increasing activity of RhoA, Rac1, and Cdc42 together with Rap1, resulting in dysregulation of the actin cytoskeleton and altered distribution of & beta;1 integrins.ConclusionsThese findings indicate that geranylgeranylation is of crucial importance for the maintenance of the delicate equilibrium of RhoGTPases and Rap1 in podocytes and consequently for the maintenance of glomerular integrity and function. A tightly regulated actin cytoskeleton attained through balanced activity of RhoGTPases is crucial to maintaining podocyte function. However, how RhoGTPases are regulated by geranylgeranylation, a post-translational modification, has been unexplored. The authors found that loss of the geranylgeranylation enzyme geranylgeranyl transferase type-I (GGTase-I) in podocytes led to progressive albuminuria and foot process effacement in podocyte-specific GGTase-I knockout mice. In cultured podocytes, the absence of geranylgeranylation resulted in altered activity of its downstream substrates Rac1, RhoA, Cdc42, and Rap1, leading to alterations of & beta;1-integrins and actin cytoskeleton structural changes. These findings highlight the importance of geranylgeranylation in the dynamic management of RhoGTPases and Rap1 to control podocyte function, providing new knowledge about podocyte biology and glomerular filtration barrier function.
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| 3. |
- Buvall, Lisa, 1976, et al.
(författare)
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Antibodies against the beta1-adrenergic receptor induce progressive development of cardiomyopathy
- 2007
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Ingår i: J Mol Cell Cardiol. - : Elsevier BV. - 0022-2828. ; 42:5, s. 1001-7
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Tidskriftsartikel (refereegranskat)abstract
- Different immune disturbances have been found among patients with dilated cardiomyopathy (DCM), including antibodies directed against different cardiac antigens, such as the second extracellular loop of the beta(1)-adrenergic receptor. The aim of our study was to investigate antibodies directed against the second extracellular loop of the beta(1)-adrenergic receptor effect on cardiac functions at an early and late stage during DCM development. This was made in a mouse model, in which DCM was induced by immunization with the second extracellular loop of the beta(1)-adrenergic receptor. Mice were immunized for 14 or 25 weeks respectively with the second extracellular loop of the beta(1)-adrenergic receptor. At 14 weeks, there was no decreased heart function reviled by echocardiography at rest, but when dobutamine stress echocardiography was used, a lower cardiac reserve was shown in the mice with antibodies against the second extracellular loop of the beta(1)-adrenergic receptor. By 25 weeks, decreased heart function, dilatation of the left ventricle and thinner left ventricular posterior wall were observed. Further biochemical analyses at 25 weeks showed increased mRNA expressions for beta(1)-adrenergic receptor kinase, monocyte chemoattractant protein-1 and the brain natriuretic peptide as well as increased concentrations of complement factor 3 in sera in the immunized animals. Our data suggest a cardiotoxic effect of antibodies directed against the second extracellular loop of the beta(1)-adrenergic receptor and a capacity to induce DCM with progressive remodeling, decreased cardiac function, altered beta(1)AR signaling and upregulation of proinflammatory components.
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| 4. |
- Buvall, Lisa, 1976, et al.
(författare)
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Orellanine specifically targets renal clear cell carcinoma
- 2017
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Ingår i: Oncotarget. - : Impact Journals, LLC. - 1949-2553. ; 8:53, s. 91085-91098
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Tidskriftsartikel (refereegranskat)abstract
- Renal cell carcinoma (RCC), arising from the proximal tubule in the kidney, accounts for approximately 85% of kidney cancers and causes over 140,000 annual deaths worldwide. In the last decade, several new therapies have been identified for treatment of metastatic RCC. Although these therapies increase survival time compared to standard care, none of them has curative properties. The nephrotoxin orellanine specifically targets proximal tubular epithelial cells, leaving other organs unaffected. We therefore hypothesized that the selective toxicity of orellanine extends to clear cell RCC (ccRCC) cells since they emanate from proximal tubular cells. Orellanine would thus target both primary and metastatic ccRCC in vitro and in vivo. We found that orellanine induces dose-dependent cell death in proximal tubular cells and in all ccRCC cells tested, both primary and cell lines, with no toxicity detected in control cells. The toxic action of orellanine involve decreased protein synthesis, disrupted cell metabolism and induction of apoptosis. In nude rats carrying human ccRCC xenografts, brief orellanine treatment eliminated more than 90% of viable tumor mass compared to control rats. This identifies orellanine as a potential treatment concept for ccRCC patients on dialysis, due to its unique selective toxicity towards ccRCC.
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| 5. |
- Buvall, Lisa, 1976
(författare)
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Pathophysiological importance of Beta1-Adrenergic receptor autoantibodies in the development of cardiomyopathy and heart failure. Clinically and experimentally
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Idiopathic Dilated Cardiomyopathy (DCM) is a heart muscle disease of unknown originthat is characterized by ventricular dilation and the impaired contraction of the left orboth ventricles, resulting in progressive heart failure and sudden cardiac death. Antibodiesagainst different cardiac proteins have been found in DCM; they include antibodiesagainst the â1-adrenergic receptor (â1AR). The second extracellular loop of the â1AR(â1AR ECII) has been shown to be the most immunogenic target of this receptor inDCM patients. Recently, new immunomodulatory treatments which improve heartfunction, such as intravenous immunoglobulin treatment (IVIG) and immunoadsorption(IA), have been tested in DCM patients.The aim of our study was to elucidate the underlying mechanism regarding: 1) therole of â1AR antibodies in the beneficial effect following IVIG or IA treatment and 2) theimportance of â1AR antibodies and their interplay with inflammation in the developmentof cardiomyopathy at both early and late stages of the disease.IVIG treatment was not shown to involve the neutralization of â1AR antibodies,since the â1AR ECII antibody titer was not lowered following IVIG treatment. On theother hand, the removal of antibodies in IA treatment plays an important role in cardiacimprovement among DCM patients by removing antibodies which have cardiotoxiceffects in terms of chronotropic action, complement-dependent cytotoxicity andapoptosis. Our in vivo models (rat and mouse), which were immunized with a peptidecorresponding to the â1AR ECII, showed signs of adverse cardiac remodeling and cardiacdysfunction. The transcription of G-coupled receptor kinase 2 (GRK2) was upregulatedin both animal models. The mice displayed a reduction in cardiac reserve at an early stage,followed by more pronounced cardiomyopathic changes at a later stage, with increasedheart weight, left ventricular dilation and thinner left ventricular posterior walls. The laterstage in the mice was also correlated to an increase in inflammatory molecules, such ascomplement component 3 in plasma, and increased transcription of monocytechemotactant protein 1 in the heart.These results reinforce the pathological importance of â1AR ECII antibodies in thedevelopment of cardiomyopathy and heart failure and promote specific immunologicaltreatment in the management of DCM.
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| 6. |
- Buvall, Lisa, 1976, et al.
(författare)
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Phenotype of early cardiomyopathic changes induced by active immunization of rats with a synthetic peptide corresponding to the second extracellular loop of the human beta-adrenergic receptor
- 2006
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Ingår i: Clin Exp Immunol. - : Oxford University Press (OUP). - 0009-9104. ; 143:2, s. 209-15
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Tidskriftsartikel (refereegranskat)abstract
- In the failing human heart, due to idiopathic dilated cardiomyopathy, it has been suggested that the beta1-adrenergic receptor (beta1AR) is a potential pathogenic autoantigen. The aim of the present study was to investigate whether immunization of rats with a synthetic peptide corresponding to the second extracellular loop of the beta1AR (beta1AR EC(II)) was able to induce the early stage of cardiomyopathy and also to investigate immunological and receptor functional parameters at a transcriptional level to permit insights into the autoimmune mechanism in cardiomyopathy. Eleven Whistar Fur rats were immunized with a beta1AR EC(II) peptide (H26R) once a month during 12 months and seven control rats were injected with vehicle according to the same procedure used for the immunized group. Cardiac function, beta1AR autoantibodies and their functional effects on cardiomyocytes were analysed. beta1AR receptor signalling, immunological and cardiomyocyte stretch markers were determined on transcriptional level. In H26R immunized rats, beta1AR autoantibodies were shown to be present and functionally active, cardiac functions in terms of fractional shortening were decreased and beta1-adrenergic receptor kinase (GRK2) mRNA were increased compared with the control group. These data have shown that immunization of rats with a putative antigenic peptide was able to induce an early stage phenotype of cardiomyopathy in the form of cardiac dysfunction and up-regulation of GRK2 as the first step in the desensitization process of the beta1AR, implying the pathological importance of the beta1AR autoantibody.
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| 7. |
- Buvall, Lisa, 1976, et al.
(författare)
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Synaptopodin Is a Coincidence Detector of Tyrosine versus Serine/Threonine Phosphorylation for the Modulation of Rho Protein Crosstalk in Podocytes.
- 2017
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Ingår i: Journal of the American Society of Nephrology : JASN. - 1533-3450. ; 28:3, s. 837-851
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Tidskriftsartikel (refereegranskat)abstract
- Tyrosine and serine/threonine signal-transduction pathways influence many aspects of cell behavior, including the spatial and temporal regulation of the actin cytoskeleton. However, little is known about how input from diverse tyrosine and serine/threonine kinases is integrated to control Rho protein crosstalk and actin remodeling, which are critically important in podocyte health and disease. Here we unveil the proteolytically-regulated, actin organizing protein synaptopodin as a coincidence detector of tyrosine versus serine/threonine phosphorylation. We show that serine/threonine and tyrosine kinases duel for synaptopodin stability versus degradation. EGFR/Src-mediated tyrosine phosphorylation of synaptopodin in podocytes promotes binding to the serine/threonine phosphatase calcineurin. This leads to the loss of 14-3-3 binding, resulting in synaptopodin degradation, Vav2 activation, enhanced Rac1 signaling, and ultimate loss of stress fibers. Our studies reveal how synaptopodin, a single proteolytically-controlled protein, integrates antagonistic tyrosine versus serine/threonine phosphorylation events for the dynamic control of the actin cytoskeleton in podocytes.
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| 8. |
- Elvin, Johannes, et al.
(författare)
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Melanocortin 1 Receptor Agonist Protects Podocytes Through Catalase and RhoA Activation.
- 2016
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Ingår i: American journal of physiology. Renal physiology. - : American Physiological Society. - 1522-1466 .- 1931-857X. ; 310:9
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Tidskriftsartikel (refereegranskat)abstract
- Drugs containing adrenocorticotropic hormone (ACTH) have been used as therapy for patients with nephrotic syndrome. We have previously shown that ACTH and a selective agonist for the melanocortin 1 receptor (MC1R) exert beneficial actions in experimental membranous nephropathy with reduced proteinuria, reduced oxidative stress, improved glomerular morphology and function. Our hypothesis is that MC1R activation in podocytes elicits beneficial effects by promoting stress fibers and maintaining podocyte viability. To test the hypothesis, we cultured podocytes and used highly specific agonists for the MC1R. The podocytes were subjected to the nephrotic-inducing agent puromycin aminonucleoside and downstream effects of MC1R activation on podocyte survival; antioxidant defense and cytoskeleton dynamics were studied. To increase the response and enhance the intracellular signals, podocytes were transduced to overexpress MC1R. We show that puromycin promotes MC1R expression in podocytes and that activation of the MC1R promotes an increase of catalase activity and reduces oxidative stress, which results in dephosphorylation of p190RhoGAP and formation of stress fibers through RhoA. In addition, MC1R agonists protects against apoptosis. Together, these mechanisms protect the podocyte against puromycin. Our findings strongly support the hypothesis that selective MC1R activating agonists protect podocytes and may therefore be useful to treat patients with nephrotic syndromes commonly considered as podocytopathies.
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| 9. |
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| 10. |
- Matsui, Shinobu, et al.
(författare)
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Specific removal of beta1-adrenoceptor autoantibodies by immunoabsorption in rabbits with autoimmune cardiomyopathy improved cardiac structure and function.
- 2006
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Ingår i: Journal of molecular and cellular cardiology. - : Elsevier BV. - 0022-2828. ; 41:1, s. 78-85
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Tidskriftsartikel (refereegranskat)abstract
- Growing evidence suggests that the beta1-adrenoceptor-directed autoimmune mechanism may play an important role in the pathogenesis of idiopathic dilated cardiomyopathy. The aim of this study is to further study the effect of specific immunoabsorption of anti-beta1-adrenoceptor autoantibodies on cardiac structure and function in autoimmune cardiomyopathy in rabbits. Twenty-four male rabbits were divided into 2 groups: (1) one immunized with beta1-adrenoceptor peptide (beta1 group, n=16), and (2) the other receiving saline injection as a control (control group, n=8). Immunization was performed once a month for 8 months. A high concentration of anti-beta1-adrenoceptor autoantibodies was exhibited throughout the immunization period. Rabbits in the beta1 group showed increased left ventricular end-diastolic diameter (LVDd), decreased left ventricular ejection fraction (LVEF) and increased LV mass/body weight ratio after the 8th month. Immunoabsorption with beta1-adrenoceptor peptide column was able to remove up to 35% of anti-beta1-adrenoceptor autoantibodies in 2 h, resulting in decreased LVDd and increased LVEF 3 months after. Specific removal of anti-beta1-adrenoceptor autoantibodies improved cardiac structure and function in experimental autoimmune cardiomyopathy. These results suggest that anti-beta1-adrenoceptor autoantibodies are of pathogenic importance in the induction of cardiomyopathy, and that specific immunoabsorption as an emerging therapy may be considered when anti-beta1-adrenoceptor autoantibodies are pathophysiologically involved.
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