ABSTRACT
SIGNIFICANCE STATEMENT: 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 ß1-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. BACKGROUND: Impairment 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. METHODS: We 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 ß1 integrins. RESULTS: Depletion 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 ß1 integrins. CONCLUSIONS: These 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.
Subject(s)
Kidney Diseases , Podocytes , Mice , Animals , Podocytes/metabolism , Glomerular Filtration Barrier , Albuminuria/metabolism , Kidney Diseases/metabolism , Mice, Knockout , Transferases/metabolism , Integrins/metabolismABSTRACT
Deciphering the pathophysiological mechanisms of primary podocytopathies that can lead to end-stage renal disease and increased mortality is an unmet need. Studying how microRNAs (miRs) interfere with various signaling pathways enables identification of pathomechanisms, novel biomarkers and potential therapeutic options. We investigated the expression of miR-200c in urine from patients with different renal diseases as a potential candidate involved in podocytopathies. The role of miR-200c for the glomerulus and its potential targets were studied in cultured human podocytes, human glomerular endothelial cells and in the zebrafish model. miR-200c was upregulated in urine from patients with minimal change disease, membranous glomerulonephritis and focal segmental glomerulosclerosis and also in transforming growth factor beta (TGF-ß) stressed glomerular endothelial cells, but not in podocytes. In zebrafish, miR-200c overexpression caused proteinuria, edema, podocyte foot process effacement and glomerular endotheliosis. Although zinc finger E-Box binding homeobox 1/2 (ZEB1/2), important in epithelial to mesenchymal transition (EMT), are prominent targets of miR-200c, their downregulation did not explain our zebrafish phenotype. We detected decreased vegfaa/bb in zebrafish overexpressing miR-200c and could further prove that miR-200c decreased VEGF-A expression and secretion in cultured human podocytes. We hypothesize that miR-200c is released from glomerular endothelial cells during cell stress and acts in a paracrine, autocrine, as well as context-dependent manner in the glomerulus. MiR-200c can cause glomerular damage most likely due to the reduction of podocyte VEGF-A. In contrast, miR-200c might also influence ZEB expression and therefore EMT, which might be important in other conditions. Therefore, we propose that miR-200c-mediated effects in the glomerulus are context-sensitive.
Subject(s)
Endothelial Cells , MicroRNAs , Animals , Humans , Endothelial Cells/metabolism , Epithelial-Mesenchymal Transition , MicroRNAs/genetics , MicroRNAs/metabolism , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism , Zebrafish/genetics , Zebrafish/metabolismABSTRACT
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.
Subject(s)
Microfilament Proteins/physiology , Podocytes/physiology , Serine/metabolism , Threonine/metabolism , Tyrosine/metabolism , rhoA GTP-Binding Protein/physiology , Animals , Calcineurin/metabolism , Cells, Cultured , Mice , Phosphorylation , Receptor Cross-Talk , Signal TransductionABSTRACT
Drugs containing adrenocorticotropic hormone have been used as therapy for patients with nephrotic syndrome. We have previously shown that adrenocorticotropic hormone and a selective agonist for the melanocortin 1 receptor (MC1R) exert beneficial actions in experimental membranous nephropathy with reduced proteinuria, reduced oxidative stress, and 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 MC1R. 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 intracellular signals, podocytes were transduced to overexpress MC1R. We showed that puromycin promotes MC1R expression in podocytes and that activation of MC1R promotes an increase of catalase activity and reduces oxidative stress, which results in the dephosphorylation of p190RhoGAP and formation of stress fibers through RhoA. In addition, MC1R agonists protect 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.
Subject(s)
Catalase/metabolism , Podocytes/drug effects , Receptor, Melanocortin, Type 1/agonists , rhoA GTP-Binding Protein/metabolism , Animals , Antimetabolites , Cells, Cultured , Enzyme Activation , Mice , Nephrosis/chemically induced , Nephrosis/metabolism , Oxidative Stress/drug effects , Puromycin Aminonucleoside , Reactive Oxygen Species/metabolism , Receptor, Melanocortin, Type 1/genetics , Stress Fibers/drug effectsABSTRACT
Podocyte injury and resulting albuminuria are hallmarks of diabetic nephropathy, but targeted therapies to halt or prevent these complications are currently not available. Here, we show that the immune-related molecule B7-1/CD80 is a critical mediator of podocyte injury in type 2 diabetic nephropathy. We report the induction of podocyte B7-1 in kidney biopsy specimens from patients with type 2 diabetes. Genetic and epidemiologic studies revealed the association of two single nucleotide polymorphisms at the B7-1 gene with diabetic nephropathy. Furthermore, increased levels of the soluble isoform of the B7-1 ligand CD28 correlated with the progression to ESRD in individuals with type 2 diabetes. In vitro, high glucose conditions prompted the phosphatidylinositol 3 kinase-dependent upregulation of B7-1 in podocytes, and the ectopic expression of B7-1 in podocytes increased apoptosis and induced disruption of the cytoskeleton that were reversed by the B7-1 inhibitor CTLA4-Ig. Podocyte expression of B7-1 was also induced in vivo in two murine models of diabetic nephropathy, and treatment with CTLA4-Ig prevented increased urinary albumin excretion and improved kidney pathology in these animals. Taken together, these results identify B7-1 inhibition as a potential therapeutic strategy for the prevention or treatment of diabetic nephropathy.
Subject(s)
B7-1 Antigen/physiology , Diabetes Mellitus, Type 1/complications , Diabetic Nephropathies/etiology , Podocytes , Adult , Aged , Animals , Female , Humans , Male , Mice , Mice, Inbred C57BL , Middle Aged , Up-RegulationABSTRACT
Apolipoprotein L1 (APOL1) high-risk genotypes are associated with increased risk of chronic kidney disease (CKD) in people of West African ancestry. Given the importance of endothelial cells (ECs) in CKD, we hypothesized that APOL1 high-risk genotypes may contribute to disease via EC-intrinsic activation and dysfunction. Single cell RNA sequencing (scRNA-seq) analysis of the Kidney Precision Medicine Project dataset revealed APOL1 expression in ECs from various renal vascular compartments. Utilizing two public transcriptomic datasets of kidney tissue from African Americans with CKD and a dataset of APOL1-expressing transgenic mice, we identified an EC activation signature; specifically, increased intercellular adhesion molecule 1 (ICAM-1) expression and enrichment in leukocyte migration pathways. In vitro, APOL1 expression in ECs derived from genetically modified human induced pluripotent stem cells and glomerular ECs triggered changes in ICAM-1 and platelet endothelial cell adhesion molecule 1 (PECAM-1) leading to an increase in monocyte attachment. Overall, our data suggest the involvement of APOL1 as an inducer of EC activation in multiple renal vascular beds with potential effects beyond the glomerular vasculature.
ABSTRACT
Kidney disease is a complex disease with several different etiologies and underlying associated pathophysiology. This is reflected by the lack of effective treatment therapies in chronic kidney disease (CKD) that stop disease progression. However, novel strategies, recent scientific breakthroughs, and technological advances have revealed new possibilities for finding novel disease drivers in CKD. This review describes some of the latest advances in the field and brings them together in a more holistic framework as applied to identification and validation of disease drivers in CKD. It uses high-resolution 'patient-centric' omics data sets, advanced in silico tools (systems biology, connectivity mapping, and machine learning) and 'state-of-the-art' experimental systems (complex 3D systems in vitro, CRISPR gene editing, and various model biological systems in vivo). Application of such a framework is expected to increase the likelihood of successful identification of novel drug candidates based on strong human target validation and a better scientific understanding of underlying mechanisms.
ABSTRACT
Confusion of various nephrotoxic Cortinarius species with edible mushrooms occurs every year throughout Europe and North America. The toxin, orellanine (OR), accumulates selectively in renal tubular epithelium with ensuing renal failure after several days as the only clinical manifestation. This study was performed to clarify the mechanisms behind the kidney damage. Sprague-Dawley rats, 100 g bw, received various doses of purified OR ip (0-5 mg/kg bw). One week later, renal function (GFR) was determined (51Cr-EDTA), ascorbyl radicals in venous blood were analyzed using electron spin resonance, and oxidative protein damage was evaluated immunohistochemically. One OR-treated group (3.5 mg/kg) simultaneously received superoxide dismutase (SOD) targeted to tubular epithelium (HC-SOD; 10 mg/kg ip daily for 5 days). RT-PCR was used for analysis of mRNA expression of genes related to oxidative stress. OR caused a dose-dependent decrease in GFR, paralleled by increased levels of ascorbyl radicals and oxidative protein damage. Antioxidant treatment with HC-SOD decreased renal function even more and also increased tissue damage and mortality. Renal mRNA levels for key components in the antioxidative defense were strongly decreased, whereas those for several cytokines were increased. The data strongly suggest that OR nephrotoxicity in vivo is mediated by oxidative stress, including a virtual shutdown of important antioxidative enzymes. We interpret the unexpected effect of HC-SOD in terms of unbalanced SOD and catalase levels in the presence of OR, leading to massive generation of *OH and cell death.
Subject(s)
2,2'-Dipyridyl/analogs & derivatives , Oxidative Stress/drug effects , Renal Insufficiency/physiopathology , 2,2'-Dipyridyl/toxicity , Agaricales , Animals , Female , Glomerular Filtration Rate/drug effects , Glutathione Peroxidase/biosynthesis , Kidney Tubules, Proximal/metabolism , Kidney Tubules, Proximal/physiopathology , Mushroom Poisoning/complications , Mushroom Poisoning/metabolism , Mushroom Poisoning/physiopathology , Rats , Rats, Sprague-Dawley , Renal Insufficiency/etiology , Renal Insufficiency/metabolism , Superoxide Dismutase/metabolismABSTRACT
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.
Subject(s)
Actin Cytoskeleton/metabolism , Nephrotic Syndrome/metabolism , Podocytes/ultrastructure , Receptor, Melanocortin, Type 1/analysis , Animals , Cells, Cultured , ErbB Receptors/metabolism , Glomerular Filtration Barrier , Humans , Phosphorylation , Proteomics/methods , Rats , Receptor, Melanocortin, Type 1/agonists , Receptor, Melanocortin, Type 1/metabolismABSTRACT
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.
ABSTRACT
The ubiquitously expressed adapter proteins Nck1/2 interact with a multitude of effector molecules to regulate diverse cellular functions including cytoskeletal dynamics. Here we show that Nck1, but not Nck2, is a substrate of c-Cbl-mediated ubiquitination. We uncover lysine 178 in Nck1 as the evolutionarily conserved ubiquitin acceptor site. We previously reported that synaptopodin, a proline-rich actin-binding protein, induces stress fibres by blocking the Smurf1-mediated ubiquitination of RhoA. We now find that synaptopodin competes with c-Cbl for binding to Nck1, which prevents the ubiquitination of Nck1 by c-Cbl. Gene silencing of c-Cbl restores Nck1 protein abundance and stress fibres in synaptopodin knockdown cells. Similarly, expression of c-Cbl-resistant Nck1(K178R) or Nck2 containing the SH3 domain 2 of Nck1 restores stress fibres in synaptopodin-depleted podocytes through activation of RhoA signalling. These findings reveal proteasomal regulation as a key factor in the distinct and non-redundant effects of Nck on RhoA-mediated actin dynamics.
Subject(s)
Actins/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Oncogene Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , rhoA GTP-Binding Protein/metabolism , Adaptor Proteins, Signal Transducing/genetics , Cytoskeleton/genetics , Cytoskeleton/metabolism , Enzyme Activation , HEK293 Cells , Humans , Oncogene Proteins/genetics , Protein Binding , Proteolysis , Proto-Oncogene Proteins c-cbl/genetics , Proto-Oncogene Proteins c-cbl/metabolism , Ubiquitination , rhoA GTP-Binding Protein/geneticsABSTRACT
An intact kidney filter is vital to retention of essential proteins in the blood and removal of waste from the body. Damage to the filtration barrier results in albumin loss in the urine, a hallmark of cardiovascular disease and kidney failure. Here we found that the ion channel TRPC5 mediates filtration barrier injury. Using Trpc5-KO mice, a small-molecule inhibitor of TRPC5, Ca2+ imaging in isolated kidney glomeruli, and live imagining of podocyte actin dynamics, we determined that loss of TRPC5 or its inhibition abrogates podocyte cytoskeletal remodeling. Inhibition or loss of TRPC5 prevented activation of the small GTP-binding protein Rac1 and stabilized synaptopodin. Importantly, genetic deletion or pharmacologic inhibition of TRPC5 protected mice from albuminuria. These data reveal that the Ca2+-permeable channel TRPC5 is an important determinant of albuminuria and identify TRPC5 inhibition as a therapeutic strategy for the prevention or treatment of proteinuric kidney disease.
Subject(s)
Albuminuria/prevention & control , Glomerular Filtration Barrier/physiology , TRPC Cation Channels/physiology , Albuminuria/chemically induced , Albuminuria/genetics , Amino Acid Sequence , Animals , Calcium Signaling , HEK293 Cells , Humans , Kidney Glomerulus/metabolism , Kidney Glomerulus/ultrastructure , Lipopolysaccharides/toxicity , Male , Mice , Mice, Knockout , Molecular Sequence Data , Neuropeptides/metabolism , Podocytes/ultrastructure , Protamines/toxicity , Rats , TRPC Cation Channels/analysis , TRPC Cation Channels/antagonists & inhibitors , TRPC Cation Channels/genetics , rac1 GTP-Binding Protein/metabolismABSTRACT
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.