96 results on '"Lipschutz JH"'
Search Results
2. Electronic health records in hospitals.
- Author
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Lipschutz JH and Lipschutz, Josh H
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- 2009
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3. PAR1-mediated Non-periodical Synchronized Calcium Oscillations in Human Mesangial Cells.
- Author
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Stefanenko M, Fedoriuk M, Mamenko M, Semenikhina M, Nowling TK, Lipschutz JH, Maximyuk O, Staruschenko A, and Palygin O
- Subjects
- Humans, Calcium metabolism, Cells, Cultured, Cell Proliferation, Receptors, Thrombin metabolism, Receptor, PAR-1 metabolism, Mesangial Cells metabolism, Calcium Signaling drug effects, Thrombin metabolism, Thrombin pharmacology
- Abstract
Mesangial cells offer structural support to the glomerular tuft and regulate glomerular capillary flow through their contractile capabilities. These cells undergo phenotypic changes, such as proliferation and mesangial expansion, resulting in abnormal glomerular tuft formation and reduced capillary loops. Such adaptation to the changing environment is commonly associated with various glomerular diseases, including diabetic nephropathy and glomerulonephritis. Thrombin-induced mesangial remodeling was found in diabetic patients, and expression of the corresponding protease-activated receptors (PARs) in the renal mesangium was reported. However, the functional PAR-mediated signaling in mesangial cells was not examined. This study investigated protease-activated mechanisms regulating mesangial cell calcium waves that may play an essential role in the mesangial proliferation or constriction of the arteriolar cells. Our results indicate that coagulation proteases such as thrombin induce synchronized oscillations in cytoplasmic Ca2+ concentration of mesangial cells. The oscillations required PAR1 G-protein coupled receptors-related activation, but not a PAR4, and were further mediated presumably through store-operated calcium entry and transient receptor potential canonical 3 (TRPC3) channel activity. Understanding thrombin signaling pathways and their relation to mesangial cells, contractile or synthetic (proliferative) phenotype may play a role in the development of chronic kidney disease and requires further investigation., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Physiological Society.)
- Published
- 2024
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4. Cilia-deficient renal tubule cells are primed for injury with mitochondrial defects and aberrant tryptophan metabolism.
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Zuo X, Winkler B, Lerner K, Ilatovskaya DV, Zamaro AS, Dang Y, Su Y, Deng P, Fitzgibbon W, Hartman J, Park KM, and Lipschutz JH
- Subjects
- Animals, Dogs, Madin Darby Canine Kidney Cells, Reactive Oxygen Species metabolism, Kidney Tubules metabolism, Kidney Tubules pathology, Mice, Tumor Suppressor Proteins metabolism, Tumor Suppressor Proteins genetics, Tumor Suppressor Proteins deficiency, Mice, Knockout, Cilia metabolism, Cilia pathology, Mitochondria metabolism, Mitochondria pathology, Tryptophan metabolism, Acute Kidney Injury metabolism, Acute Kidney Injury pathology, Acute Kidney Injury genetics
- Abstract
The exocyst and Ift88 are necessary for primary ciliogenesis. Overexpression of Exoc5 (OE), a central exocyst component, resulted in longer cilia and enhanced injury recovery. Mitochondria are involved in acute kidney injury (AKI). To investigate cilia and mitochondria, basal respiration and mitochondrial maximal and spare respiratory capacity were measured in Exoc5 OE, Exoc5 knockdown (KD), Exoc5 ciliary targeting sequence mutant (CTS-mut), control Madin-Darby canine kidney (MDCK), Ift88 knockout (KO), and Ift88 rescue cells. In Exoc5 KD, Exoc5 CTS-mut, and Ift88 KO cells, these parameters were decreased. In Exoc5 OE and Ift88 rescue cells they were increased. Reactive oxygen species were higher in Exoc5 KD, Exoc5 CTS-mut, and Ift88 KO cells compared with Exoc5 OE, control, and Ift88 rescue cells. By electron microscopy, mitochondria appeared abnormal in Exoc5 KD, Exoc5 CTS-mut, and Ift88 KO cells. A metabolomics screen of control, Exoc5 KD, Exoc5 CTS-mut, Exoc5 OE, Ift88 KO, and Ift88 rescue cells showed a marked increase in tryptophan levels in Exoc5 CTS-mut (113-fold) and Exoc5 KD (58-fold) compared with control cells. A 21% increase was seen in Ift88 KO compared with rescue cells. In Exoc5 OE compared with control cells, tryptophan was decreased 59%. To determine the effects of ciliary loss on AKI, we generated proximal tubule-specific Exoc5 and Ift88 KO mice. These mice had loss of primary cilia, decreased mitochondrial ATP synthase, and increased tryptophan in proximal tubules with greater injury following ischemia-reperfusion. These data indicate that cilia-deficient renal tubule cells are primed for injury with mitochondrial defects in tryptophan metabolism. NEW & NOTEWORTHY Mitochondria are centrally involved in acute kidney injury (AKI). Here, we show that cilia-deficient renal tubule cells both in vitro in cell culture and in vivo in mice are primed for injury with mitochondrial defects and aberrant tryptophan metabolism. These data suggest therapeutic strategies such as enhancing ciliogenesis or improving mitochondrial function to protect patients at risk for AKI.
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- 2024
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5. Role of the β 2 -adrenergic receptor in podocyte injury and recovery.
- Author
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Arif E, Solanki AK, Rahman B, Wolf B, Schnellmann RG, Nihalani D, and Lipschutz JH
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- Animals, Mice, Acute Kidney Injury metabolism, Acute Kidney Injury pathology, Adrenergic beta-2 Receptor Agonists pharmacology, Albuminuria metabolism, Mice, Inbred C57BL, Doxorubicin pharmacology, Doxorubicin toxicity, Formoterol Fumarate pharmacology, Mice, Knockout, Podocytes metabolism, Podocytes drug effects, Podocytes pathology, Receptors, Adrenergic, beta-2 metabolism
- Abstract
Background: Podocytes have a remarkable ability to recover from injury; however, little is known about the recovery mechanisms involved in this process. We recently showed that formoterol, a long-acting β
2 -adrenergic receptor (β2 -AR) agonist, induced mitochondrial biogenesis (MB) in podocytes and led to renoprotection in mice. However, it is not clear whether this effect was mediated by formoterol acting through the β2 -AR or if it occurred through "off-target" effects., Methods: We genetically deleted the β2 -AR specifically in murine podocytes and used these mice to determine whether formoterol acting through the podocyte β2 -AR alone is sufficient for recovery of renal filtration function following injury. The podocyte-specific β2 -AR knockout mice (β2 -ARfl/fl /PodCre) were generated by crossing β2 -AR floxed mice with podocin Cre (B6.Cg-Tg(NPHS2-cre)295Lbh/J) mice. These mice were then subjected to both acute and chronic glomerular injury using nephrotoxic serum (NTS) and adriamycin (ADR), respectively. The extent of injury was evaluated by measuring albuminuria and histological and immunostaining analysis of the murine kidney sections., Results: A similar level of injury was observed in β2 -AR knockout and control mice; however, the β2 -ARfl/fl /PodCre mice failed to recover in response to formoterol. Functional evaluation of the β2 -ARfl/fl /PodCre mice following injury plus formoterol showed similar albuminuria and glomerular injury to control mice that were not treated with formoterol., Conclusions: These results indicate that the podocyte β2 -AR is a critical component of the recovery mechanism and may serve as a novel therapeutic target for treating podocytopathies., (© 2024. The Author(s).)- Published
- 2024
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6. High water intake induces primary cilium elongation in renal tubular cells.
- Author
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Kong MJ, Han SJ, Seu SY, Han KH, Lipschutz JH, and Park KM
- Abstract
Background: The primary cilium protrudes from the cell surface and functions as a mechanosensor. Recently, we found that water intake restriction shortens the primary cilia of renal tubular cells, and a blockage of the shortening disturbs the ability of the kidneys to concentrate urine. Here, we investigate whether high water intake (HWI) alters primary cilia length, and if so, what is its underlying mechanism and its role on kidney urine production., Methods: Experimental mice were given free access to normal water (normal water intake) or 3% sucrose-containing water for HWI for 2 days. Some mice were administered with U0126 (10 mg/kg body weight), an inhibitor of MEK kinase, from 2 days before HWI, daily. The primary cilium length and urine amount and osmolality were investigated., Results: HWI-induced diluted urine production and primary cilium elongation in renal tubular cells. HWI increased the expression of α-tubulin acetyltransferase 1 (αTAT1), leading to the acetylation of α-tubulins, a core protein of the primary cilia. HWI also increased phosphorylated ERK1/2 (p-ERK1/2) and exocyst complex component 5 (Exoc5) expression in the kidneys. U0126 blocked HWI-induced increases in αTAT1, p-ERK1/2, and Exoc5 expression. U0126 inhibited HWI-induced α-tubulin acetylation, primary cilium elongation, urine amount increase, and urine osmolality decrease., Conclusion: These results show that increased water intake elongates the primary cilia via ERK1/2 activation and that ERK inhibition prevents primary cilium elongation and diluted urine production. These data suggest that the elongation of primary cilium length is associated with the production of diluted urine.
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- 2024
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7. β 2 -Adrenergic receptor agonists as a treatment for diabetic kidney disease.
- Author
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Arif E, Medunjanin D, Solanki A, Zuo X, Su Y, Dang Y, Winkler B, Lerner K, Kamal AI, Palygin O, Cornier MA, Wolf BJ, Hunt KJ, and Lipschutz JH
- Subjects
- Humans, Animals, Mice, Adrenergic beta-2 Receptor Agonists therapeutic use, Streptozocin, Formoterol Fumarate therapeutic use, Receptors, Adrenergic therapeutic use, Diabetic Nephropathies drug therapy, Diabetes Mellitus, Type 1 drug therapy, Diabetes Mellitus, Type 2 complications, Diabetes Mellitus, Type 2 drug therapy, Pulmonary Disease, Chronic Obstructive drug therapy, Kidney Failure, Chronic drug therapy, Kidney Failure, Chronic etiology
- Abstract
We have previously shown that the long-acting β
2 -adrenergic receptor (β2 -AR) agonist formoterol induced recovery from acute kidney injury in mice. To determine whether formoterol protected against diabetic nephropathy, the most common cause of end-stage kidney disease (ESKD), we used a high-fat diet (HFD), a murine type 2 diabetes model, and streptozotocin, a murine type 1 diabetes model. Following formoterol treatment, there was a marked recovery from and reversal of diabetic nephropathy in HFD mice compared with those treated with vehicle alone at the ultrastructural, histological, and functional levels. Similar results were seen after formoterol treatment in mice receiving streptozotocin. To investigate effects in humans, we performed a competing risk regression analysis with death as a competing risk to examine the association between Veterans with chronic kidney disease (CKD) and chronic obstructive pulmonary disease (COPD), who use β2 -AR agonists, and Veterans with CKD but no COPD, and progression to ESKD in a large national cohort of Veterans with stage 4 CKD between 2011 and 2013. Veterans were followed until 2016 or death. ESKD was defined as the initiation of dialysis and/or receipt of kidney transplant. We found that COPD was associated with a 25.6% reduction in progression from stage 4 CKD to ESKD compared with no COPD after adjusting for age, diabetes, sex, race-ethnicity, comorbidities, and medication use. Sensitivity analysis showed a 33.2% reduction in ESKD in Veterans with COPD taking long-acting formoterol and a 20.8% reduction in ESKD in Veterans taking other β2 -AR agonists compared with those with no COPD. These data indicate that β2 -AR agonists, especially formoterol, could be a treatment for diabetic nephropathy and perhaps other forms of CKD. NEW & NOTEWORTHY Diabetic nephropathy is the most common cause of ESKD. Formoterol, a long-acting β2 -adrenergic receptor (β2 -AR) agonist, reversed diabetic nephropathy in murine models of type 1 and 2 diabetes. In humans, there was an association with protection from progression of CKD in patients with COPD, by means of β2 -AR agonist intake, compared with those without COPD. These data indicate that β2 -AR agonists, especially formoterol, could be a new treatment for diabetic nephropathy and other forms of CKD.- Published
- 2024
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8. β-Arrestin pathway activation by selective ATR1 agonism promotes calcium influx in podocytes, leading to glomerular damage.
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Semenikhina M, Fedoriuk M, Stefanenko M, Klemens CA, Cherezova A, Marshall B, Hall G, Levchenko V, Solanki AK, Lipschutz JH, Ilatovskaya DV, Staruschenko A, and Palygin O
- Subjects
- Rats, Animals, Humans, TRPC6 Cation Channel metabolism, Calcium metabolism, beta-Arrestins metabolism, Angiotensin Receptor Antagonists pharmacology, Rats, Inbred Dahl, Angiotensin-Converting Enzyme Inhibitors pharmacology, TRPC Cation Channels metabolism, TRPC Cation Channels pharmacology, Podocytes metabolism, Kidney Diseases metabolism, Hypertension metabolism
- Abstract
Angiotensin receptor blockers (ARBs) are the first-line treatment for hypertension; they act by inhibiting signaling through the angiotensin 1 receptor (AT1R). Recently, a novel biased AT1R agonist, TRV120027 (TRV), which selectively activates the β-arrestin cascade and blocks the G-protein-coupled receptor pathway has been proposed as a potential blood pressure medication. Here, we explored the effects of TRV and associated β-arrestin signaling in podocytes, essential cells of the kidney filter. We used human podocyte cell lines to determine β-arrestin's involvement in calcium signaling and cytoskeletal reorganization and Dahl SS rats to investigate the chronic effects of TRV administration on glomerular health. Our experiments indicate that the TRV-activated β-arrestin pathway promotes the rapid elevation of intracellular Ca2+ in a dose-dependent manner. Interestingly, the amplitude of β-arrestin-mediated Ca2+ influx was significantly higher than the response to similar Ang II concentrations. Single-channel analyses show rapid activation of transient receptor potential canonical (TRPC) channels following acute TRV application. Furthermore, the pharmacological blockade of TRPC6 significantly attenuated the β-arrestin-mediated Ca2+ influx. Additionally, prolonged activation of the β-arrestin pathway in podocytes resulted in pathological actin cytoskeleton rearrangements, higher apoptotic cell markers, and augmented glomerular damage. TRV-activated β-arrestin signaling in podocytes may promote TRPC6 channel-mediated Ca2+ influx, foot process effacement, and apoptosis, possibly leading to severe defects in glomerular filtration barrier integrity and kidney health. Under these circumstances, the potential therapeutic application of TRV for hypertension treatment requires further investigation to assess the balance of the benefits versus possible deleterious effects and off-target damage., (© 2023 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)
- Published
- 2023
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9. Breaking New Ground: The Crucial Role of Animal Research in the Advancement of Rhabdomyolysis-Induced AKI Treatment and Prevention.
- Author
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Semenikhina M, Lipschutz JH, and Palygin O
- Subjects
- Animals, Endothelin-1 adverse effects, Kidney, Animal Experimentation, Rhabdomyolysis chemically induced, Acute Kidney Injury etiology
- Abstract
Competing Interests: None declared.
- Published
- 2023
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10. Shortening of primary cilia length is associated with urine concentration in the kidneys.
- Author
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Kong MJ, Han SJ, Seu SY, Han KH, Lipschutz JH, and Park KM
- Abstract
Background: The primary cilium, a microtubule-based cellular organelle present in certain kidney cells, functions as a mechano-sensor to monitor fluid flow in addition to various other biological functions. In kidneys, the primary cilia protrude into the tubular lumen and are directly exposed to pro-urine flow and components. However, their effects on urine concentration remain to be defined. Here, we investigated the association between primary cilia and urine concentration., Methods: Mice either had free access to water (normal water intake, NWI) or were not allowed access to water (water deprivation, WD). Some mice received tubastatin, an inhibitor of histone deacetylase 6 (HDAC6), which regulates the acetylation of α-tubulin, a core protein of microtubules., Results: WD decreased urine output and increased urine osmolality, concomitant with apical plasma membrane localization of aquaporin 2 (AQP2) in the kidney. After WD, compared with after NWI, the lengths of primary cilia in renal tubular epithelial cells were shortened and HDAC6 activity increased. WD induced deacetylation of α-tubulin without altering α-tubulin levels in the kidney. Tubastatin prevented the shortening of cilia through increasing HDAC6 activity and consequently increasing acetylated α-tubulin expression. Furthermore, tubastatin prevented the WD-induced reduction of urine output, urine osmolality increase, and apical plasma membrane localization of AQP2., Conclusions: WD shortens primary cilia length through HDAC6 activation and α-tubulin deacetylation, while HDAC6 inhibition blocks the WD-induced changes in cilia length and urine output. This suggests that cilia length alterations are involved, at least in part, in the regulation of body water balance and urine concentration.
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- 2023
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11. Hydrogen sulfide, a gaseous signaling molecule, elongates primary cilia on kidney tubular epithelial cells by activating extracellular signal-regulated kinase.
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Han SJ, Kim JI, Lipschutz JH, and Park KM
- Abstract
Primary cilia on kidney tubular cells play crucial roles in maintaining structure and physiological function. Emerging evidence indicates that the absence of primary cilia, and their length, are associated with kidney diseases. The length of primary cilia in kidney tubular epithelial cells depends, at least in part, on oxidative stress and extracellular signal-regulated kinase 1/2 (ERK) activation. Hydrogen sulfide (H
2 S) is involved in antioxidant systems and the ERK signaling pathway. Therefore, in this study, we investigated the role of H2 S in primary cilia elongation and the downstream pathway. In cultured Madin-Darby Canine Kidney cells, the length of primary cilia gradually increased up to 4 days after the cells were grown to confluent monolayers. In addition, the expression of H2 S-producing enzyme increased concomitantly with primary cilia length. Treatment with NaHS, an exogenous H2 S donor, accelerated the elongation of primary cilia whereas DL-propargylglycine (a cystathionine γ-lyase inhibitor) and hydroxylamine (a cystathionine-β-synthase inhibitor) delayed their elongation. NaHS treatment increased ERK activation and Sec10 and Arl13b protein expression, both of which are involved in cilia formation and elongation. Treatment with U0126, an ERK inhibitor, delayed elongation of primary cilia and blocked the effect of NaHS-mediated primary cilia elongation and Sec10 and Arl13b upregulation. Finally, we also found that H2 S accelerated primary cilia elongation after ischemic kidney injury. These results indicate that H2 S lengthens primary cilia through ERK activation and a consequent increase in Sec10 and Arl13b expression, suggesting that H2 S and its downstream targets could be novel molecular targets for regulating primary cilia.- Published
- 2021
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12. Phosphorylation of slit diaphragm proteins NEPHRIN and NEPH1 upon binding of HGF promotes podocyte repair.
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Solanki AK, Arif E, Srivastava P, Furcht CM, Rahman B, Wen P, Singh A, Holzman LB, Fitzgibbon WR, Budisavljevic MN, Lobo GP, Kwon SH, Han Z, Lazzara MJ, Lipschutz JH, and Nihalani D
- Subjects
- Animals, Cell Line, Glomerular Filtration Rate physiology, Hepatocyte Growth Factor physiology, Humans, Intercellular Junctions metabolism, Kidney pathology, Kidney Glomerulus metabolism, Membrane Proteins genetics, Mice, Peptides metabolism, Phosphorylation, Podocytes metabolism, Protein Binding physiology, Signal Transduction physiology, Hepatocyte Growth Factor metabolism, Membrane Proteins metabolism
- Abstract
Phosphorylation (activation) and dephosphorylation (deactivation) of the slit diaphragm proteins NEPHRIN and NEPH1 are critical for maintaining the kidney epithelial podocyte actin cytoskeleton and, therefore, proper glomerular filtration. However, the mechanisms underlying these events remain largely unknown. Here we show that NEPHRIN and NEPH1 are novel receptor proteins for hepatocyte growth factor (HGF) and can be phosphorylated independently of the mesenchymal epithelial transition receptor in a ligand-dependent fashion through engagement of their extracellular domains by HGF. Furthermore, we demonstrate SH2 domain-containing protein tyrosine phosphatase-2-dependent dephosphorylation of these proteins. To establish HGF as a ligand, purified baculovirus-expressed NEPHRIN and NEPH1 recombinant proteins were used in surface plasma resonance binding experiments. We report high-affinity interactions of NEPHRIN and NEPH1 with HGF, although NEPHRIN binding was 20-fold higher than that of NEPH1. In addition, using molecular modeling we constructed peptides that were used to map specific HGF-binding regions in the extracellular domains of NEPHRIN and NEPH1. Finally, using an in vitro model of cultured podocytes and an ex vivo model of Drosophila nephrocytes, as well as chemically induced injury models, we demonstrated that HGF-induced phosphorylation of NEPHRIN and NEPH1 is centrally involved in podocyte repair. Taken together, this is the first study demonstrating a receptor-based function for NEPHRIN and NEPH1. This has important biological and clinical implications for the repair of injured podocytes and the maintenance of podocyte integrity., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
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13. Targeting myosin 1c inhibits murine hepatic fibrogenesis.
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Arif E, Wang C, Swiderska-Syn MK, Solanki AK, Rahman B, Manka PP, Coombes JD, Canbay A, Papa S, Nihalani D, Aspichueta P, Lipschutz JH, and Syn WK
- Subjects
- Animals, Collagen Type I, alpha 1 Chain, Fibroblasts pathology, Hepatic Stellate Cells metabolism, Hepatic Stellate Cells pathology, Liver pathology, Liver Cirrhosis genetics, Liver Cirrhosis pathology, Mice, Myosin Type I genetics, Phosphorylation, Signal Transduction physiology, Transforming Growth Factor beta metabolism, Fibroblasts metabolism, Liver metabolism, Liver Cirrhosis metabolism, Myosin Type I metabolism
- Abstract
Myosin 1c (Myo1c) is an unconventional myosin that modulates signaling pathways involved in tissue injury and repair. In this study, we observed that Myo1c expression is significantly upregulated in human chronic liver disease such as nonalcoholic steatohepatitis (NASH) and in animal models of liver fibrosis. High throughput data from the GEO-database identified similar Myo1c upregulation in mice and human liver fibrosis. Notably, transforming growth factor-β1 (TGF-β1) stimulation to hepatic stellate cells (HSCs), the liver pericyte and key cell type responsible for the deposition of extracellular matrix, upregulates Myo1c expression, whereas genetic depletion or pharmacological inhibition of Myo1c blunted TGF-β-induced fibrogenic responses, resulting in repression of α-smooth muscle actin (α-SMA) and collagen type I α 1 chain (Col1α1) mRNA. Myo1c deletion also decreased fibrogenic processes such as cell proliferation, wound healing response, and contractility when compared with vehicle-treated HSCs. Importantly, phosphorylation of mothers against decapentaplegic homolog 2 (SMAD2) and mothers against decapentaplegic homolog 3 (SMAD3) were significantly blunted upon Myo1c inhibition in GRX cells as well as Myo1c knockout (Myo1c-KO) mouse embryonic fibroblasts (MEFs) upon TGF-β stimulation. Using the genetic Myo1c-KO mice, we confirmed that Myo1c is critical for fibrogenesis, as Myo1c-KO mice were resistant to carbon tetrachloride (CCl4)-induced liver fibrosis. Histological and immunostaining analysis of liver sections showed that deposition of collagen fibers and α-SMA expression were significantly reduced in Myo1c-KO mice upon liver injury. Collectively, these results demonstrate that Myo1c mediates hepatic fibrogenesis by modulating TGF-β signaling and suggest that inhibiting this process may have clinical application in treating liver fibrosis. NEW & NOTEWORTHY The incidences of liver fibrosis are growing at a rapid pace and have become one of the leading causes of end-stage liver disease. Although TGF-β1 is known to play a prominent role in transforming cells to produce excessive extracellular matrix that lead to hepatic fibrosis, the therapies targeting TGF-β1 have achieved very limited clinical impact. This study highlights motor protein myosin-1c-mediated mechanisms that serve as novel regulators of TGF-β1 signaling and fibrosis.
- Published
- 2021
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14. Conditional Loss of the Exocyst Component Exoc5 in Retinal Pigment Epithelium (RPE) Results in RPE Dysfunction, Photoreceptor Cell Degeneration, and Decreased Visual Function.
- Author
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Rohrer B, Biswal MR, Obert E, Dang Y, Su Y, Zuo X, Fogelgren B, Kondkar AA, Lobo GP, and Lipschutz JH
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- Animals, Mice, Mice, Inbred C57BL, Mice, Knockout, Photoreceptor Cells metabolism, Retinal Pigment Epithelium metabolism, Vision Disorders metabolism, Zebrafish, Photoreceptor Cells pathology, Retinal Degeneration, Retinal Pigment Epithelium pathology, Vesicular Transport Proteins physiology, Vision Disorders pathology
- Abstract
To characterize the mechanisms by which the highly conserved exocyst trafficking complex regulates eye physiology in zebrafish and mice, we focused on Exoc5 (also known as sec10 ), a central exocyst component. We analyzed both exoc5 zebrafish mutants and retinal pigmented epithelium (RPE)-specific Exoc5 knockout mice. Exoc5 is present in both the non-pigmented epithelium of the ciliary body and in the RPE. In this study, we set out to establish an animal model to study the mechanisms underlying the ocular phenotype and to establish if loss of visual function is induced by postnatal RPE Exoc5-deficiency. Exoc5
-/- zebrafish had smaller eyes, with decreased number of melanocytes in the RPE and shorter photoreceptor outer segments. At 3.5 days post-fertilization, loss of rod and cone opsins were observed in zebrafish exoc5 mutants. Mice with postnatal RPE-specific loss of Exoc5 showed retinal thinning associated with compromised visual function and loss of visual photoreceptor pigments. Abnormal levels of RPE65 together with a reduced c-wave amplitude indicate a dysfunctional RPE. The retinal phenotype in Exoc5-/- mice was present at 20 weeks, but was more pronounced at 27 weeks, indicating progressive disease phenotype. We previously showed that the exocyst is necessary for photoreceptor ciliogenesis and retinal development. Here, we report that exoc5 mutant zebrafish and mice with RPE-specific genetic ablation of Exoc5 develop abnormal RPE pigmentation, resulting in retinal cell dystrophy and loss of visual pigments associated with compromised vision. Together, these data suggest that exocyst-mediated signaling in the RPE is required for RPE structure and function, indirectly leading to photoreceptor degeneration.- Published
- 2021
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15. Desert hedgehog-primary cilia cross talk shapes mitral valve tissue by organizing smooth muscle actin.
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Fulmer D, Toomer KA, Glover J, Guo L, Moore K, Moore R, Stairley R, Gensemer C, Abrol S, Rumph MK, Emetu F, Lipschutz JH, McDowell C, Bian J, Wang C, Beck T, Wessels A, Renault MA, and Norris RA
- Subjects
- Actins metabolism, Animals, Extracellular Matrix metabolism, Heart Valve Diseases, Hedgehog Proteins physiology, Mice, Mitral Valve Prolapse genetics, Mitral Valve Prolapse metabolism, Muscle, Smooth metabolism, Muscle, Smooth physiology, Myocytes, Smooth Muscle metabolism, Neuropeptides metabolism, Phenotype, Signal Transduction, Transcription Factors metabolism, rac1 GTP-Binding Protein metabolism, Cilia metabolism, Hedgehog Proteins metabolism, Mitral Valve embryology
- Abstract
Non-syndromic mitral valve prolapse (MVP) is the most common heart valve disease affecting 2.4% of the population. Recent studies have identified genetic defects in primary cilia as causative to MVP, although the mechanism of their action is currently unknown. Using a series of gene inactivation approaches, we define a paracrine mechanism by which endocardially-expressed Desert Hedgehog (DHH) activates primary cilia signaling on neighboring valve interstitial cells. High-resolution imaging and functional assays show that DHH de-represses smoothened at the primary cilia, resulting in kinase activation of RAC1 through the RAC1-GEF, TIAM1. Activation of this non-canonical hedgehog pathway stimulates α-smooth actin organization and ECM remodeling. Genetic or pharmacological perturbation of this pathway results in enlarged valves that progress to a myxomatous phenotype, similar to valves seen in MVP patients. These data identify a potential molecular origin for MVP as well as establish a paracrine DHH-primary cilium cross-talk mechanism that is likely applicable across developmental tissue types., Competing Interests: Declaration of competing interest None., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2020
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16. A Functional Binding Domain in the Rbpr2 Receptor Is Required for Vitamin A Transport, Ocular Retinoid Homeostasis, and Photoreceptor Cell Survival in Zebrafish.
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Solanki AK, Kondkar AA, Fogerty J, Su Y, Kim SH, Lipschutz JH, Nihalani D, Perkins BD, and Lobo GP
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- Animals, Carrier Proteins metabolism, Liver metabolism, Membrane Proteins metabolism, Photoreceptor Cells metabolism, Zebrafish, Zebrafish Proteins metabolism, Cell Survival physiology, Eye metabolism, Homeostasis physiology, Retinoids metabolism, Vitamin A blood
- Abstract
Dietary vitamin A/all- trans retinol/ROL plays a critical role in human vision. ROL circulates bound to the plasma retinol-binding protein (RBP4) as RBP4-ROL. In the eye, the STRA6 membrane receptor binds to circulatory RBP4 and internalizes ROL. STRA6 is, however, not expressed in systemic tissues, where there is high affinity RBP4 binding and ROL uptake. We tested the hypothesis that the second retinol binding protein 4 receptor 2 (Rbpr2), which is highly expressed in systemic tissues of zebrafish and mouse, contains a functional RBP4 binding domain, critical for ROL transport. As for STRA6, modeling and docking studies confirmed three conserved RBP4 binding residues in zebrafish Rbpr2. In cell culture studies, disruption of the RBP4 binding residues on Rbpr2 almost completely abolished uptake of exogenous vitamin A. CRISPR-generated rbpr2- RBP4 domain zebrafish mutants showed microphthalmia, shorter photoreceptor outer segments, and decreased opsins, which were attributed to impaired ocular retinoid content. Injection of WT-Rbpr2 mRNA into rbpr2 mutant or all- trans retinoic acid treatment rescued the mutant eye phenotypes. In conclusion, zebrafish Rbpr2 contains a putative extracellular RBP4-ROL ligand-binding domain, critical for yolk vitamin A transport to the eye for ocular retinoid production and homeostasis, for photoreceptor cell survival.
- Published
- 2020
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17. The Use of High-Throughput Transcriptomics to Identify Pathways with Therapeutic Significance in Podocytes.
- Author
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Solanki AK, Srivastava P, Rahman B, Lipschutz JH, Nihalani D, and Arif E
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- Animals, Doxorubicin pharmacology, Humans, Kidney Diseases chemically induced, Kidney Diseases pathology, Mice, Phosphorylation drug effects, Podocytes pathology, Puromycin Aminonucleoside pharmacology, Doxorubicin adverse effects, Gene Expression Regulation drug effects, Kidney Diseases metabolism, Podocytes metabolism, Puromycin Aminonucleoside adverse effects, Signal Transduction drug effects, Transcriptome drug effects
- Abstract
Podocytes have a unique structure that supports glomerular filtration function, and many glomerular diseases result in loss of this structure, leading to podocyte dysfunction and ESRD (end stage renal disease). These structural and functional changes involve a complex set of molecular and cellular mechanisms that remain poorly understood. To understand the molecular signature of podocyte injury, we performed transcriptome analysis of cultured human podocytes injured either with PAN (puromycin aminonucleoside) or doxorubicin/adriamycin (ADR). The pathway analysis through DE (differential expression) and gene-enrichment analysis of the injured podocytes showed Tumor protein p53 (P53) as one of the major signaling pathways that was significantly upregulated upon podocyte injury. Accordingly, P53 expression was also up-regulated in the glomeruli of nephrotoxic serum (NTS) and ADR-injured mice. To further confirm these observations, cultured podocytes were treated with the P53 inhibitor pifithrin-α, which showed significant protection from ADR-induced actin cytoskeleton damage. In conclusion, signaling pathways that are involved in podocyte pathogenesis and can be therapeutically targeted were identified by high-throughput transcriptomic analysis of injured podocytes.
- Published
- 2019
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18. Primary cilia and the exocyst are linked to urinary extracellular vesicle production and content.
- Author
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Zuo X, Kwon SH, Janech MG, Dang Y, Lauzon SD, Fogelgren B, Polgar N, and Lipschutz JH
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- ADP-Ribosylation Factor 6, Animals, Cells, Cultured, Dogs, Humans, Madin Darby Canine Kidney Cells metabolism, Mice, Mice, Knockout, Vesicular Transport Proteins deficiency, Cilia metabolism, Exocytosis, Extracellular Vesicles metabolism, Kidney metabolism, Vesicular Transport Proteins metabolism
- Abstract
The recently proposed idea of "urocrine signaling" hypothesizes that small secreted extracellular vesicles (EVs) contain proteins that transmit signals to distant cells. However, the role of renal primary cilia in EV production and content is unclear. We previously showed that the exocyst, a highly conserved trafficking complex, is necessary for ciliogenesis; that it is present in human urinary EVs; that knockdown (KD) of exocyst complex component 5 (EXOC5), a central exocyst component, results in very short or absent cilia; and that human EXOC5 overexpression results in longer cilia. Here, we show that compared with control Madin-Darby canine kidney (MDCK) cells, EXOC5 overexpression increases and KD decreases EV numbers. Proteomic analyses of isolated EVs from EXOC5 control, KD, and EXOC5-overexpressing MDCK cells revealed significant alterations in protein composition. Using immunoblotting to specifically examine the expression levels of ADP-ribosylation factor 6 (ARF6) and EPS8-like 2 (EPS8L2) in EVs, we found that EXOC5 KD increases ARF6 levels and decreases EPS8L2 levels, and that EXOC5 overexpression increases EPS8L2. Knockout of intraflagellar transport 88 (IFT88) confirmed that the changes in EV number/content were due to cilia loss: similar to EXOC5, the IFT88 loss resulted in very short or absent cilia, decreased EV numbers, increased EV ARF6 levels, and decreased Eps8L2 levels compared with IFT88-rescued EVs. Compared with control animals, urine from proximal tubule-specific EXOC5-KO mice contained fewer EVs and had increased ARF6 levels. These results indicate that perturbations in exocyst and primary cilia affect EV number and protein content.
- Published
- 2019
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19. Defects in the Exocyst-Cilia Machinery Cause Bicuspid Aortic Valve Disease and Aortic Stenosis.
- Author
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Fulmer D, Toomer K, Guo L, Moore K, Glover J, Moore R, Stairley R, Lobo G, Zuo X, Dang Y, Su Y, Fogelgren B, Gerard P, Chung D, Heydarpour M, Mukherjee R, Body SC, Norris RA, and Lipschutz JH
- Subjects
- Animals, Aortic Valve metabolism, Aortic Valve pathology, Aortic Valve Stenosis genetics, Bicuspid Aortic Valve Disease, Case-Control Studies, Cilia pathology, Gene Frequency, Genome-Wide Association Study, Genotype, Heart Defects, Congenital genetics, Heart Valve Diseases genetics, Heart Valve Diseases metabolism, Humans, Mice, Mice, Knockout, Polymorphism, Single Nucleotide, Vesicular Transport Proteins genetics, Vesicular Transport Proteins metabolism, Zebrafish, Zebrafish Proteins genetics, Zebrafish Proteins metabolism, Aortic Valve abnormalities, Aortic Valve Stenosis pathology, Cilia physiology, Heart Defects, Congenital pathology, Heart Valve Diseases pathology
- Abstract
Background: Bicuspid aortic valve (BAV) disease is a congenital defect that affects 0.5% to 1.2% of the population and is associated with comorbidities including ascending aortic dilation and calcific aortic valve stenosis. To date, although a few causal genes have been identified, the genetic basis for the vast majority of BAV cases remains unknown, likely pointing to complex genetic heterogeneity underlying this phenotype. Identifying genetic pathways versus individual gene variants may provide an avenue for uncovering additional BAV causes and consequent comorbidities., Methods: We performed genome-wide association Discovery and Replication Studies using cohorts of 2131 patients with BAV and 2728 control patients, respectively, which identified primary cilia genes as associated with the BAV phenotype. Genome-wide association study hits were prioritized based on P value and validated through in vivo loss of function and rescue experiments, 3-dimensional immunohistochemistry, histology, and morphometric analyses during aortic valve morphogenesis and in aged animals in multiple species. Consequences of these genetic perturbations on cilia-dependent pathways were analyzed by Western and immunohistochemistry analyses, and assessment of aortic valve and cardiac function were determined by echocardiography., Results: Genome-wide association study hits revealed an association between BAV and genetic variation in human primary cilia. The most associated single-nucleotide polymorphisms were identified in or near genes that are important in regulating ciliogenesis through the exocyst, a shuttling complex that chaperones cilia cargo to the membrane. Genetic dismantling of the exocyst resulted in impaired ciliogenesis, disrupted ciliogenic signaling and a spectrum of cardiac defects in zebrafish, and aortic valve defects including BAV, valvular stenosis, and valvular calcification in murine models., Conclusions: These data support the exocyst as required for normal ciliogenesis during aortic valve morphogenesis and implicate disruption of ciliogenesis and its downstream pathways as contributory to BAV and associated comorbidities in humans.
- Published
- 2019
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20. The role of the exocyst in renal ciliogenesis, cystogenesis, tubulogenesis, and development.
- Author
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Lipschutz JH
- Abstract
The exocyst is a highly conserved eight-subunit protein complex (EXOC1-8) involved in the targeting and docking of exocytic vesicles translocating from the trans-Golgi network to various sites in renal cells. EXOC5 is a central exocyst component because it connects EXOC6, bound to the vesicles exiting the trans-Golgi network via the small GTPase RAB8, to the rest of the exocyst complex at the plasma membrane. In the kidney, the exocyst complex is involved in primary ciliognesis, cystogenesis, and tubulogenesis. The exocyst, and its regulators, have also been found in urinary extracellular vesicles, and may be centrally involved in urocrine signaling and repair following acute kidney injury. The exocyst is centrally involved in the development of other organs, including the eye, ear, and heart. The exocyst is regulated by many different small GTPases of the RHO, RAL, RAB, and ARF families. The small GTPases, and their guanine nucleotide exchange factors and GTPase-activating proteins, likely give the exocyst specificity of function. The recent development of a floxed Exoc5 mouse line will aid researchers in studying the role of the exocyst in multiple cells and organ types by allowing for tissue-specific knockout, in conjunction with Cre-driver mouse lines.
- Published
- 2019
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21. The motor protein Myo1c regulates transforming growth factor-β-signaling and fibrosis in podocytes.
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Arif E, Solanki AK, Srivastava P, Rahman B, Tash BR, Holzman LB, Janech MG, Martin R, Knölker HJ, Fitzgibbon WR, Deng P, Budisavljevic MN, Syn WK, Wang C, Lipschutz JH, Kwon SH, and Nihalani D
- Subjects
- Animals, Disease Models, Animal, Doxorubicin toxicity, Female, Fibrosis, Gene Expression Regulation, Humans, Kidney Diseases chemically induced, Male, Mice, Mice, Knockout, Myosin Type I genetics, Podocytes drug effects, Promoter Regions, Genetic, Transcription, Genetic, Growth Differentiation Factor 15 genetics, Kidney Diseases pathology, Myosin Type I metabolism, Podocytes pathology, Signal Transduction genetics, Transforming Growth Factor beta metabolism
- Abstract
Transforming growth factor-β (TGF-β) is known to play a critical role in the pathogenesis of many progressive podocyte diseases. However, the molecular mechanisms regulating TGF-β signaling in podocytes remain unclear. Using a podocyte-specific myosin (Myo)1c knockout, we demonstrate whether Myo1c is critical for TGF-β-signaling in podocyte disease pathogenesis. Specifically, podocyte-specific Myo1c knockout mice were resistant to fibrotic injury induced by Adriamycin or nephrotoxic serum. Further, loss of Myo1c also protected from injury in the TGF-β-dependent unilateral ureteral obstruction mouse model of renal interstitial fibrosis. Mechanistic analyses showed that loss of Myo1c significantly blunted TGF-β signaling through downregulation of canonical and non-canonical TGF-β pathways. Interestingly, nuclear rather than the cytoplasmic Myo1c was found to play a central role in controlling TGF-β signaling through transcriptional regulation. Differential expression analysis of nuclear Myo1c-associated gene promoters showed that nuclear Myo1c targeted the TGF-β responsive gene growth differentiation factor (GDF)-15 and directly bound to the GDF-15 promoter. Importantly, GDF15 was found to be involved in podocyte pathogenesis, where GDF15 was upregulated in glomeruli of patients with focal segmental glomerulosclerosis. Thus, Myo1c-mediated regulation of TGF-β-responsive genes is central to the pathogenesis of podocyte injury. Hence, inhibiting this process may have clinical application in treating podocytopathies., (Copyright © 2019 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)
- Published
- 2019
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22. Disruption of the exocyst induces podocyte loss and dysfunction.
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Nihalani D, Solanki AK, Arif E, Srivastava P, Rahman B, Zuo X, Dang Y, Fogelgren B, Fermin D, Gillies CE, Sampson MG, and Lipschutz JH
- Subjects
- Animals, Apoptosis, Cell Movement, Exocytosis, Humans, Kidney Glomerulus metabolism, Membrane Proteins genetics, Membrane Proteins metabolism, Mice, Mice, Inbred C57BL, Mice, Knockout, Nephrotic Syndrome genetics, Phosphorylation, Podocytes metabolism, Protein Transport, Proteinuria etiology, Proteinuria pathology, Signal Transduction, Gene Deletion, Kidney Glomerulus pathology, Nephrotic Syndrome pathology, Podocytes pathology, Vesicular Transport Proteins physiology
- Abstract
Although the slit diaphragm proteins in podocytes are uniquely organized to maintain glomerular filtration assembly and function, little is known about the underlying mechanisms that participate in trafficking these proteins to the correct location for development and homeostasis. Identifying these mechanisms will likely provide novel targets for therapeutic intervention to preserve podocyte function following glomerular injury. Analysis of structural variation in cases of human nephrotic syndrome identified rare heterozygous deletions of EXOC4 in two patients. This suggested that disruption of the highly-conserved eight-protein exocyst trafficking complex could have a role in podocyte dysfunction. Indeed, mRNA profiling of injured podocytes identified significant exocyst down-regulation. To test the hypothesis that the exocyst is centrally involved in podocyte development/function, we generated homozygous podocyte-specific Exoc5 (a central exocyst component that interacts with Exoc4) knockout mice that showed massive proteinuria and died within 4 weeks of birth. Histological and ultrastructural analysis of these mice showed severe glomerular defects with increased fibrosis, proteinaceous casts, effaced podocytes, and loss of the slit diaphragm. Immunofluorescence analysis revealed that Neph1 and Nephrin, major slit diaphragm constituents, were mislocalized and/or lost. mRNA profiling of Exoc5 knockdown podocytes showed that vesicular trafficking was the most affected cellular event. Mapping of signaling pathways and Western blot analysis revealed significant up-regulation of the mitogen-activated protein kinase and transforming growth factor-β pathways in Exoc5 knockdown podocytes and in the glomeruli of podocyte-specific Exoc5 KO mice. Based on these data, we propose that exocyst-based mechanisms regulate Neph1 and Nephrin signaling and trafficking, and thus podocyte development and function.
- Published
- 2019
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23. Primary cilia defects causing mitral valve prolapse.
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Toomer KA, Yu M, Fulmer D, Guo L, Moore KS, Moore R, Drayton KD, Glover J, Peterson N, Ramos-Ortiz S, Drohan A, Catching BJ, Stairley R, Wessels A, Lipschutz JH, Delling FN, Jeunemaitre X, Dina C, Collins RL, Brand H, Talkowski ME, Del Monte F, Mukherjee R, Awgulewitsch A, Body S, Hardiman G, Hazard ES, da Silveira WA, Wang B, Leyne M, Durst R, Markwald RR, Le Scouarnec S, Hagege A, Le Tourneau T, Kohl P, Rog-Zielinska EA, Ellinor PT, Levine RA, Milan DJ, Schott JJ, Bouatia-Naji N, Slaugenhaupt SA, and Norris RA
- Subjects
- Adaptor Proteins, Signal Transducing genetics, Adaptor Proteins, Signal Transducing metabolism, Animals, Base Sequence, Extracellular Matrix metabolism, Female, Genome-Wide Association Study, Heart Valves diagnostic imaging, Heart Valves growth & development, Humans, Male, Mice, Knockout, Mitral Valve Prolapse diagnostic imaging, Mitral Valve Prolapse genetics, Morphogenesis, Pedigree, Time Factors, Tumor Suppressor Proteins metabolism, Cilia pathology, Mitral Valve Prolapse etiology
- Abstract
Mitral valve prolapse (MVP) affects 1 in 40 people and is the most common indication for mitral valve surgery. MVP can cause arrhythmias, heart failure, and sudden cardiac death, and to date, the causes of this disease are poorly understood. We now demonstrate that defects in primary cilia genes and their regulated pathways can cause MVP in familial and sporadic nonsyndromic MVP cases. Our expression studies and genetic ablation experiments confirmed a role for primary cilia in regulating ECM deposition during cardiac development. Loss of primary cilia during development resulted in progressive myxomatous degeneration and profound mitral valve pathology in the adult setting. Analysis of a large family with inherited, autosomal dominant nonsyndromic MVP identified a deleterious missense mutation in a cilia gene, DZIP1 A mouse model harboring this variant confirmed the pathogenicity of this mutation and revealed impaired ciliogenesis during development, which progressed to adult myxomatous valve disease and functional MVP. Relevance of primary cilia in common forms of MVP was tested using pathway enrichment in a large population of patients with MVP and controls from previously generated genome-wide association studies (GWAS), which confirmed the involvement of primary cilia genes in MVP. Together, our studies establish a developmental basis for MVP through altered cilia-dependent regulation of ECM and suggest that defects in primary cilia genes can be causative to disease phenotype in some patients with MVP., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)
- Published
- 2019
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24. The exocyst acting through the primary cilium is necessary for renal ciliogenesis, cystogenesis, and tubulogenesis.
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Zuo X, Lobo G, Fulmer D, Guo L, Dang Y, Su Y, Ilatovskaya DV, Nihalani D, Rohrer B, Body SC, Norris RA, and Lipschutz JH
- Subjects
- Animals, DNA, Complementary genetics, Dogs, Gene Knockdown Techniques, Humans, Kidney Diseases pathology, Madin Darby Canine Kidney Cells, Mutagenesis, Site-Directed, Protein Binding, Protein Transport, RNA, Messenger metabolism, Vesicular Transport Proteins genetics, Zebrafish, Cilia physiology, Cysts pathology, Kidney metabolism, Kidney Tubules growth & development, Vesicular Transport Proteins metabolism
- Abstract
The exocyst is a highly conserved protein complex found in most eukaryotic cells and is associated with many functions, including protein translocation in the endoplasmic reticulum, vesicular basolateral targeting, and ciliogenesis in the kidney. To investigate the exocyst functions, here we exchanged proline for alanine in the highly conserved V X P X ciliary targeting motif of EXOC5 (exocyst complex component 5), a central exocyst gene/protein, and generated stable EXOC5 ciliary targeting sequence-mutated (EXOC5CTS-m) Madin-Darby canine kidney (MDCK) cells. The EXOC5CTS-m protein was stable and could bind other members of the exocyst complex. Culturing stable control, EXOC5-overexpressing (OE), Exoc5-knockdown (KD), and EXOC5CTS-m MDCK cells on Transwell filters, we found that primary ciliogenesis is increased in EXOC5 OE cells and inhibited in Exoc5-KD and EXOC5CTS-m cells. Growing cells in collagen gels until the cyst stage, we noted that EXOC5-OE cells form mature cysts with single lumens more rapidly than control cysts, whereas Exoc5-KD and EXOC5CTS-m MDCK cells failed to form mature cysts. Adding hepatocyte growth factor to induce tubulogenesis, we observed that EXOC5-OE cell cysts form tubules more efficiently than control MDCK cell cysts, EXOC5CTS-m MDCK cell cysts form significantly fewer tubules than control cell cysts, and Exoc5-KD cysts did not undergo tubulogenesis. Finally, we show that EXOC5 mRNA almost completely rescues the ciliary phenotypes in exoc5- mutant zebrafish, unlike the EXOC5CTS-m mRNA, which could not efficiently rescue the phenotypes. Taken together, these results indicate that the exocyst, acting through the primary cilium, is necessary for renal ciliogenesis, cystogenesis, and tubulogenesis.
- Published
- 2019
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25. Correction to: Exocyst Complex Member EXOC5 Is Required for Survival of Hair Cells and Spiral Ganglion Neurons and Maintenance of Hearing.
- Author
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Lee B, Baek JI, Min H, Bae SH, Moon K, Kim MA, Kim YR, Fogelgren B, Lipschutz JH, Lee KY, Bok J, and Kim UK
- Abstract
The original article contains an error for a grant number in the Acknowledgements section.
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- 2019
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26. Exocyst Complex Member EXOC5 Is Required for Survival of Hair Cells and Spiral Ganglion Neurons and Maintenance of Hearing.
- Author
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Lee B, Baek JI, Min H, Bae SH, Moon K, Kim MA, Kim YR, Fogelgren B, Lipschutz JH, Lee KY, Bok J, and Kim UK
- Subjects
- Animals, Apoptosis, Cell Survival, DNA-Binding Proteins metabolism, Dependovirus metabolism, Epithelium pathology, Hair Cells, Auditory ultrastructure, Hearing Loss metabolism, Hearing Loss pathology, Integrases metabolism, Mice, Inbred C57BL, Nerve Degeneration pathology, Neurites metabolism, Neurons metabolism, Organ of Corti metabolism, Organ of Corti ultrastructure, Stereocilia metabolism, Stereocilia ultrastructure, Transcription Factors metabolism, Vesicular Transport Proteins deficiency, Hair Cells, Auditory metabolism, Hair Cells, Auditory pathology, Hearing, Neurons pathology, Spiral Ganglion pathology, Vesicular Transport Proteins metabolism
- Abstract
The exocyst, an octameric protein complex consisting of Exoc1 through Exoc8, was first determined to regulate exocytosis by targeting vesicles to the plasma membrane in yeast to mice. In addition to this fundamental role, the exocyst complex has been implicated in other cellular processes. In this study, we investigated the role of the exocyst in cochlear development and hearing by targeting EXOC5, a central exocyst component. Deleting Exoc5 in the otic epithelium with widely used Cre lines resulted in early lethality. Thus, we generated two different inner ear-specific Exoc5 knockout models by crossing Gfi1
Cre mice with Exoc5f/f mice for hair cell-specific deletion (Gfi1Cre/+ ;Exoc5f/f ) and by in utero delivery of rAAV-iCre into the otocyst of embryonic day 12.5 for deletion throughout the otic epithelium (rAAV2/1-iCre;Exoc5f/f ). Gfi1Cre/+ ;Exoc5f/f mice showed relatively normal hair cell morphology until postnatal day 20, after which hair cells underwent apoptosis accompanied by disorganization of stereociliary bundles, resulting in progressive hearing loss. rAAV2/1-iCre;Exoc5f/f mice exhibited abnormal neurite morphology, followed by apoptotic degeneration of spiral ganglion neurons (SGNs) and hair cells, which led to profound and early-onset hearing loss. These results demonstrate that Exoc5 is essential for the normal development and survival of cochlear hair cells and SGNs, as well as the functional maintenance of hearing.- Published
- 2018
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27. Downregulation of exocyst Sec10 accelerates kidney tubule cell recovery through enhanced cell migration.
- Author
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Noh MR, Jang HS, Song DK, Lee SR, Lipschutz JH, Park KM, and Kim JI
- Subjects
- Animals, Biological Assay, Cell Line, Cell Movement drug effects, Diacylglycerol Kinase genetics, Diacylglycerol Kinase metabolism, Dogs, Enzyme Inhibitors pharmacology, Exocytosis, Gene Expression Regulation, Kidney Tubules pathology, Madin Darby Canine Kidney Cells, Male, Mice, Mice, Inbred C57BL, Piperidines pharmacology, Quinazolinones pharmacology, RNA, Small Interfering genetics, RNA, Small Interfering metabolism, Reperfusion Injury genetics, Reperfusion Injury metabolism, Reperfusion Injury pathology, Vesicular Transport Proteins agonists, Vesicular Transport Proteins antagonists & inhibitors, Vesicular Transport Proteins metabolism, Wound Healing physiology, Diacylglycerol Kinase antagonists & inhibitors, Kidney Tubules metabolism, Reperfusion Injury prevention & control, Vesicular Transport Proteins genetics
- Abstract
Migration of surviving kidney tubule cells after sub-lethal injury, for example ischemia/reperfusion (I/R), plays a critical role in recovery. Exocytosis is known to be involved in cell migration, and a key component in exocytosis is the highly-conserved eight-protein exocyst complex. We investigated the expression of a central exocyst complex member, Sec10, in kidneys following I/R injury, as well as the role of Sec10 in wound healing following scratch injury of cultured Madin-Darby canine kidney (MDCK) cells. Sec10 overexpression and knockdown (KD) in MDCK cells were used to investigate the speed of wound healing and the mechanisms underlying recovery. In mice, Sec10 decreased after I/R injury, and increased during the recovery period. In cell culture, Sec10 OE inhibited ruffle formation and wound healing, while Sec10 KD accelerated it. Sec10 OE cells had higher amounts of diacylglycerol kinase (DGK) gamma at the leading edge than did control cells. A DGK inhibitor reversed the inhibition of wound healing and ruffle formation in Sec10 OE cells. Conclusively, downregulation of Sec10 following I/R injury appears to accelerate recovery of kidney tubule cells through activated ruffle formation and enhanced cell migration., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2018
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28. The Retinol-Binding Protein Receptor 2 (Rbpr2) Is Required for Photoreceptor Survival and Visual Function in the Zebrafish.
- Author
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Lobo GP, Pauer G, Lipschutz JH, and Hagstrom SA
- Subjects
- 3T3 Cells, Animals, Cell Survival, Humans, Intestinal Absorption physiology, Intestinal Mucosa metabolism, Larva, Liver metabolism, Mice, Pancreas metabolism, Photoreceptor Cells, Vertebrate pathology, Photoreceptor Cells, Vertebrate physiology, RNA, Messenger biosynthesis, RNA, Messenger genetics, Recombinant Proteins metabolism, Retinol-Binding Proteins, Plasma genetics, Retinol-Binding Proteins, Plasma physiology, Transfection, Vision Disorders etiology, Vision Disorders metabolism, Vision Disorders pathology, Zebrafish, Zebrafish Proteins deficiency, Zebrafish Proteins genetics, Photoreceptor Cells, Vertebrate cytology, Vitamin A pharmacokinetics, Zebrafish Proteins physiology
- Abstract
Vitamin A/retinol (ROL) and its metabolites (retinoids) play critical roles in eye development and photoreception. Short-term dietary vitamin A deficiency (VAD) manifests clinically as night blindness, while prolonged VAD is known to cause retinal pigment epithelium (RPE) and photoreceptor degeneration. Therefore, sustained uptake of dietary vitamin A, for ocular retinoid production, is essential for photoreceptor health and visual function. The mechanisms influencing the uptake, storage, and supply of dietary vitamin A, for ocular retinoid production, however, are not fully understood. We investigated, in zebrafish, the physiological role of the retinol-binding protein receptor 2 (Rbpr2), for the uptake of dietary ROL, which is necessary for vision. NIH3T3 cells expressing zebrafish Rbpr2 showed plasma membrane localization patterns and were capable of ROL uptake from its bound form. Using whole-mount in situ hybridization, Rbpr2 was found to be expressed exclusively in the liver, intestine, and pancreas, of staged zebrafish larvae. At 5.5 days post fertilization, TALEN-generated rbpr2 mutants (rbpr2
-/- ) had smaller eyes and shorter OS lengths and showed loss of PNA (cones) and rhodopsin (rods) by immunofluorescence staining. Finally, tests for visual function using optokinetic response (OKR) showed no consistent OKR in rbpr2-/- larval zebrafish. Our analysis, therefore, suggests that Rbpr2 is capable of ROL uptake and loss of this membrane receptor in zebrafish results in photoreceptor defects that adversely affect visual function.- Published
- 2018
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29. Zebrafish as models to study ciliopathies of the eye and kidney.
- Author
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Shi Y, Su Y, Lipschutz JH, and Lobo GP
- Abstract
Cilia are highly-conserved organelles projecting from the cell surface of nearly every cell type in vertebrates. Ciliary proteins have essential functions in human physiology, particularly in signaling and organ development. As cilia are a component of almost all vertebrate cells, cilia dysfunction can manifest as a constellation of features that characteristically include, retinal degeneration, renal disease and cerebral anomalies. The terminology "Ciliopathies" refers to inherited human disorders caused by genetic mutations in ciliary genes, leading to cilia dysfunctions that form an important and ever expanding multi-organ disease spectrum. Ciliopathies are a diverse class of congenital diseases, with twenty-four recognized syndromes caused by mutations in at least ninety different genes. In order to start to dissect the phenotypes of each disease associated with ciliary dysfunction it is necessary to understand the mechanisms underlying the phenotype using suitable animal models. Here, we review the advantages of the zebrafish as a vertebrate model for human ciliopathies, with a focus on ciliopathies affecting the eye and the kidney.
- Published
- 2017
30. Adaptor protein CD2AP and L-type lectin LMAN2 regulate exosome cargo protein trafficking through the Golgi complex.
- Author
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Kwon SH, Oh S, Nacke M, Mostov KE, and Lipschutz JH
- Published
- 2017
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31. Targeting Neph1 and ZO-1 protein-protein interaction in podocytes prevents podocyte injury and preserves glomerular filtration function.
- Author
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Sagar A, Arif E, Solanki AK, Srivastava P, Janech MG, Kim SH, Lipschutz JH, Kwon SH, Ashish, and Nihalani D
- Subjects
- Animals, Cells, Cultured, Glomerular Filtration Rate drug effects, Humans, Kidney drug effects, Kidney metabolism, Kidney physiopathology, Membrane Proteins chemistry, Membrane Proteins genetics, Mice, Molecular Docking Simulation, Podocytes metabolism, Protein Binding drug effects, Protein Domains, Zebrafish, Zonula Occludens-1 Protein chemistry, Zonula Occludens-1 Protein genetics, Isodesmosine pharmacology, Membrane Proteins metabolism, Podocytes drug effects, Zonula Occludens-1 Protein metabolism
- Abstract
Targeting protein-protein interaction (PPI) is rapidly becoming an attractive alternative for drug development. While drug development commonly involves inhibiting a PPI, in this study, we show that stabilizing PPI may also be therapeutically beneficial. Junctional proteins Neph1 and ZO-1 and their interaction is an important determinant of the structural integrity of slit diaphragm, which is a critical component of kidney's filtration system. Since injury induces loss of this interaction, we hypothesized that strengthening this interaction may protect kidney's filtration barrier and preserve kidney function. In this study, Neph1-ZO-1 structural complex was screened for the presence of small druggable pockets formed from contributions from both proteins. One such pocket was identified and screened using a small molecule library. Isodesmosine (ISD) a rare naturally occurring amino acid and a biomarker for pulmonary arterial hypertension was selected as the best candidate and to establish the proof of concept, its ability to enhance Neph1-CD and ZO-1 binding was tested. Results from biochemical binding analysis showed that ISD enhanced Neph1 and ZO-1 interaction under in vitro and in vivo conditions. Importantly, ISD treated podocytes were resistant to injury-induced loss of transepithelial permeability. Finally, mouse and zebrafish studies show that ISD protects from injury-induced renal damage.
- Published
- 2017
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32. The exocyst is required for photoreceptor ciliogenesis and retinal development.
- Author
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Lobo GP, Fulmer D, Guo L, Zuo X, Dang Y, Kim SH, Su Y, George K, Obert E, Fogelgren B, Nihalani D, Norris RA, Rohrer B, and Lipschutz JH
- Subjects
- Animals, Mice, Mice, Inbred C57BL, Mutation, Photoreceptor Cells, Vertebrate pathology, Retina pathology, Vesicular Transport Proteins deficiency, Vesicular Transport Proteins metabolism, Zebrafish, Cilia metabolism, Exocytosis, Photoreceptor Cells, Vertebrate metabolism, Retina metabolism
- Abstract
We previously have shown that the highly conserved eight-protein exocyst trafficking complex is required for ciliogenesis in kidney tubule cells. We hypothesized here that ciliogenic programs are conserved across organs and species. To determine whether renal primary ciliogenic programs are conserved in the eye, and to characterize the function and mechanisms by which the exocyst regulates eye development in zebrafish, we focused on exoc5 , a central component of the exocyst complex, by analyzing both exoc5 zebrafish mutants, and photoreceptor-specific Exoc5 knock-out mice. Two separate exoc5 mutant zebrafish lines phenocopied exoc5 morphants and, strikingly, exhibited a virtual absence of photoreceptors, along with abnormal retinal development and cell death. Because the zebrafish mutant was a global knockout, we also observed defects in several ciliated organs, including the brain (hydrocephalus), heart (cardiac edema), and kidney (disordered and shorter cilia). exoc5 knockout increased phosphorylation of the regulatory protein Mob1, consistent with Hippo pathway activation. exoc5 mutant zebrafish rescue with human EXOC5 mRNA completely reversed the mutant phenotype. We accomplished photoreceptor-specific knockout of Exoc5 with our Exoc5 fl/fl mouse line crossed with a rhodopsin-Cre driver line. In Exoc5 photoreceptor-specific knock-out mice, the photoreceptor outer segment structure was severely impaired at 4 weeks of age, although a full-field electroretinogram indicated a visual response was still present. However, by 6 weeks, visual responses were eliminated. In summary, we show that ciliogenesis programs are conserved in the kidneys and eyes of zebrafish and mice and that the exocyst is necessary for photoreceptor ciliogenesis and retinal development, most likely by trafficking cilia and outer-segment proteins.
- Published
- 2017
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33. A role for primary cilia in aortic valve development and disease.
- Author
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Toomer KA, Fulmer D, Guo L, Drohan A, Peterson N, Swanson P, Brooks B, Mukherjee R, Body S, Lipschutz JH, Wessels A, and Norris RA
- Subjects
- Animals, Aortic Valve growth & development, Bicuspid Aortic Valve Disease, Cell Differentiation genetics, Cell Differentiation physiology, Cell Proliferation genetics, Cell Proliferation physiology, Extracellular Matrix metabolism, Female, Gene Regulatory Networks genetics, Gene Regulatory Networks physiology, Hedgehog Proteins genetics, Hedgehog Proteins metabolism, Humans, Immunohistochemistry, Male, Mice, Tumor Suppressor Proteins genetics, Tumor Suppressor Proteins metabolism, Aortic Valve abnormalities, Aortic Valve metabolism, Cilia metabolism, Cilia physiology, Heart Valve Diseases metabolism
- Abstract
Background: Bicuspid aortic valve (BAV) disease is the most common congenital heart defect, affecting 0.5-1.2% of the population and causing significant morbidity and mortality. Only a few genes have been identified in pedigrees, and no single gene model explains BAV inheritance, thus supporting a complex genetic network of interacting genes. However, patients with rare syndromic diseases that stem from alterations in the structure and function of primary cilia ("ciliopathies") exhibit BAV as a frequent cardiovascular finding, suggesting primary cilia may factor broadly in disease etiology., Results: Our data are the first to demonstrate that primary cilia are expressed on aortic valve mesenchymal cells during embryonic development and are lost as these cells differentiate into collagen-secreting fibroblastic-like cells. The function of primary cilia was tested by genetically ablating the critical ciliogenic gene Ift88. Loss of Ift88 resulted in abrogation of primary cilia and increased fibrogenic extracellular matrix (ECM) production. Consequentially, stratification of ECM boundaries normally present in the aortic valve were lost and a highly penetrant BAV phenotype was evident at birth., Conclusions: Our data support cilia as a novel cellular mechanism for restraining ECM production during aortic valve development and broadly implicate these structures in the etiology of BAV disease in humans. Developmental Dynamics 246:625-634, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)
- Published
- 2017
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34. Adaptor Protein CD2AP and L-type Lectin LMAN2 Regulate Exosome Cargo Protein Trafficking through the Golgi Complex.
- Author
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Kwon SH, Oh S, Nacke M, Mostov KE, and Lipschutz JH
- Subjects
- Adaptor Proteins, Signal Transducing genetics, Adaptor Proteins, Vesicular Transport genetics, Adaptor Proteins, Vesicular Transport metabolism, Animals, Biological Transport, Active physiology, Cytoskeletal Proteins genetics, Dogs, Exosomes genetics, Golgi Apparatus genetics, HEK293 Cells, Humans, Mannose-Binding Lectins genetics, Membrane Transport Proteins genetics, Receptors, G-Protein-Coupled genetics, Receptors, G-Protein-Coupled metabolism, Adaptor Proteins, Signal Transducing metabolism, Cytoskeletal Proteins metabolism, Exosomes metabolism, Golgi Apparatus metabolism, Mannose-Binding Lectins metabolism, Membrane Transport Proteins metabolism
- Abstract
Exosomes, 40-150-nm extracellular vesicles, transport biological macromolecules that mediate intercellular communications. Although exosomes are known to originate from maturation of endosomes into multivesicular endosomes (also known as multivesicular bodies) with subsequent fusion of the multivesicular endosomes with the plasma membrane, it remains unclear how cargos are selected for exosomal release. Using an inducible expression system for the exosome cargo protein GPRC5B and following its trafficking trajectory, we show here that newly synthesized GPRC5B protein accumulates in the Golgi complex prior to its release into exosomes. The L-type lectin LMAN2 (also known as VIP36) appears to be specifically required for the accumulation of GPRC5B in the Golgi complex and restriction of GPRC5B transport along the exosomal pathway. This may occur due to interference with the adaptor protein GGA1-mediated trans Golgi network-to-endosome transport of GPRC5B. The adaptor protein CD2AP-mediated internalization following cell surface delivery appears to contribute to the Golgi accumulation of GPRC5B, possibly in parallel with biosynthetic/secretory trafficking from the endoplasmic reticulum. Our data thus reveal a Golgi-traversing pathway for exosomal release of the cargo protein GPRC5B in which CD2AP facilitates the entry and LMAN2 impedes the exit of the flux, respectively., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)
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- 2016
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35. Dynamin Binding Protein (Tuba) Deficiency Inhibits Ciliogenesis and Nephrogenesis in Vitro and in Vivo.
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Baek JI, Kwon SH, Zuo X, Choi SY, Kim SH, and Lipschutz JH
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- Animals, Cilia genetics, Cilia metabolism, Cytoskeletal Proteins genetics, Dogs, Gene Knockdown Techniques, Madin Darby Canine Kidney Cells, Mice, Zebrafish, Zebrafish Proteins genetics, cdc42 GTP-Binding Protein genetics, cdc42 GTP-Binding Protein metabolism, Cytoskeletal Proteins metabolism, Kidney embryology, Organogenesis physiology, Zebrafish Proteins metabolism
- Abstract
Dysfunction of renal primary cilia leads to polycystic kidney disease. We previously showed that the exocyst, a protein trafficking complex, is essential for ciliogenesis and regulated by multiple Rho and Rab family GTPases, such as Cdc42. Cdc42 deficiency resulted in a disruption of renal ciliogenesis and a polycystic kidney disease phenotype in zebrafish and mice. Here we investigate the role of Dynamin binding protein (also known as Tuba), a Cdc42-specific guanine nucleotide exchange factor, in ciliogenesis and nephrogenesis using Tuba knockdown Madin-Darby canine kidney cells and tuba knockdown in zebrafish. Tuba depletion resulted in an absence of cilia, with impaired apical polarization and inhibition of hepatocyte growth factor-induced tubulogenesis in Tuba knockdown Madin-Darby canine kidney cell cysts cultured in a collagen gel. In zebrafish, tuba was expressed in multiple ciliated organs, and, accordingly, tuba start and splice site morphants showed various ciliary mutant phenotypes in these organs. Co-injection of tuba and cdc42 morpholinos at low doses, which alone had no effect, resulted in genetic synergy and led to abnormal kidney development with highly disorganized pronephric duct cilia. Morpholinos targeting two other guanine nucleotide exchange factors not known to be in the Cdc42/ciliogenesis pathway and a scrambled control morpholino showed no phenotypic effect. Given the molecular nature of Cdc42 and Tuba, our data strongly suggest that tuba and cdc42 act in the same ciliogenesis pathway. Our study demonstrates that Tuba deficiency causes an abnormal renal ciliary and morphogenetic phenotype. Tuba most likely plays a critical role in ciliogenesis and nephrogenesis by regulating Cdc42 activity., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)
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- 2016
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36. Unilateral nephrectomy elongates primary cilia in the remaining kidney via reactive oxygen species.
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Han SJ, Jang HS, Kim JI, Lipschutz JH, and Park KM
- Subjects
- Animals, Blood Pressure drug effects, Carrier Proteins genetics, Carrier Proteins metabolism, Cell Line, Cyclin-Dependent Kinase Inhibitor p21 genetics, Cyclin-Dependent Kinase Inhibitor p21 metabolism, Epithelial Cells cytology, Epithelial Cells drug effects, Epithelial Cells metabolism, Extracellular Signal-Regulated MAP Kinases metabolism, Gene Expression, Hydrogen Peroxide metabolism, Kidney drug effects, Kidney Tubules cytology, Kidney Tubules drug effects, Kidney Tubules metabolism, Membrane Proteins, Metalloporphyrins pharmacology, Mice, Organ Size, Vesicular Transport Proteins genetics, Vesicular Transport Proteins metabolism, Cilia drug effects, Cilia metabolism, Kidney cytology, Kidney metabolism, Nephrectomy, Reactive Oxygen Species metabolism
- Abstract
The length of primary cilia is associated with normal cell and organ function. In the kidney, the change of functional cilia length/mass is associated with various diseases such as ischemia/reperfusion injury, polycystic kidney disease, and congenital solitary kidney. Here, we investigate whether renal mass reduction affects primary cilia length and function. To induce renal mass reduction, mice were subjected to unilateral nephrectomy (UNx). UNx increased kidney weight and superoxide formation in the remaining kidney. Primary cilia were elongated in proximal tubule cells, collecting duct cells and parietal cells of the remaining kidney. Mn(III) Tetrakis (1-methyl-4-pyridyl) porphyrin (MnTMPyP), an antioxidant, reduced superoxide formation in UNx-mice and prevented the elongation of primary cilia. UNx increased the expression of phosphorylated ERK, p21, and exocyst complex members Sec8 and Sec10, in the remaining kidney, and these increases were prevented by MnTMPyP. In MDCK, a kidney tubular epithelial cell line, cells, low concentrations of H2O2 treatment elongated primary cilia. This H2O2-induced elongation of primary cilia was also prevented by MnTMPyP treatment. Taken together, these data demonstrate that kidney compensation, induced by a reduction of renal mass, results in primary cilia elongation, and this elongation is associated with an increased production of reactive oxygen species (ROS).
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- 2016
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37. Arl13b and the exocyst interact synergistically in ciliogenesis.
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Seixas C, Choi SY, Polgar N, Umberger NL, East MP, Zuo X, Moreiras H, Ghossoub R, Benmerah A, Kahn RA, Fogelgren B, Caspary T, Lipschutz JH, and Barral DC
- Subjects
- ADP-Ribosylation Factors genetics, Abnormalities, Multiple, Animals, Cerebellum abnormalities, Eye Abnormalities, Genetic Association Studies, HeLa Cells, Humans, Kidney metabolism, Kidney Diseases, Cystic, Mice, Mice, Knockout, Microtubules metabolism, Mutation, NIH 3T3 Cells, Retina abnormalities, Vesicular Transport Proteins metabolism, Zebrafish, Zebrafish Proteins genetics, Zebrafish Proteins metabolism, ADP-Ribosylation Factors metabolism, Cilia metabolism
- Abstract
Arl13b belongs to the ADP-ribosylation factor family within the Ras superfamily of regulatory GTPases. Mutations in Arl13b cause Joubert syndrome, which is characterized by congenital cerebellar ataxia, hypotonia, oculomotor apraxia, and mental retardation. Arl13b is highly enriched in cilia and is required for ciliogenesis in multiple organs. Nevertheless, the precise role of Arl13b remains elusive. Here we report that the exocyst subunits Sec8, Exo70, and Sec5 bind preferentially to the GTP-bound form of Arl13b, consistent with the exocyst being an effector of Arl13b. Moreover, we show that Arl13b binds directly to Sec8 and Sec5. In zebrafish, depletion of arl13b or the exocyst subunit sec10 causes phenotypes characteristic of defective cilia, such as curly tail up, edema, and abnormal pronephric kidney development. We explored this further and found a synergistic genetic interaction between arl13b and sec10 morphants in cilia-dependent phenotypes. Through conditional deletion of Arl13b or Sec10 in mice, we found kidney cysts and decreased ciliogenesis in cells surrounding the cysts. Moreover, we observed a decrease in Arl13b expression in the kidneys from Sec10 conditional knockout mice. Taken together, our results indicate that Arl13b and the exocyst function together in the same pathway leading to functional cilia., (© 2016 Seixas et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)
- Published
- 2016
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38. Total Kidney Volume in Autosomal Dominant Polycystic Kidney Disease: A Biomarker of Disease Progression and Therapeutic Efficacy.
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Alam A, Dahl NK, Lipschutz JH, Rossetti S, Smith P, Sapir D, Weinstein J, McFarlane P, and Bichet DG
- Subjects
- Biomarkers blood, Disease Progression, Female, Humans, Kidney Function Tests, Male, Organ Size physiology, Polycystic Kidney, Autosomal Dominant genetics, Prognosis, Risk Assessment, Severity of Illness Index, Glomerular Filtration Rate physiology, Polycystic Kidney, Autosomal Dominant pathology, Polycystic Kidney, Autosomal Dominant therapy
- Abstract
Autosomal dominant polycystic kidney disease (ADPKD) is the most common potentially life-threatening monogenic disorder in humans, characterized by progressive development and expansion of fluid-filled cysts in the kidneys and other organs. Ongoing cyst growth leads to progressive kidney enlargement, whereas kidney function remains stable for decades as a result of hyperfiltration and compensation by unaffected nephrons. Kidney function irreversibly declines only in the late stages of the disease, when most of the parenchyma is lost to cystic and fibrotic tissue and the remaining compensatory capacity is overwhelmed. Hence, conventional kidney function measures, such as glomerular filtration rate, do not adequately assess disease progression in ADPKD, especially in its early stages. Given the recent development of potential targeted therapies in ADPKD, it has become critically important to identify relevant biomarkers that can be used to determine the degree of disease progression and evaluate the effects of therapeutic interventions on the course of the disease. We review the current evidence to provide an informed perspective on whether total kidney volume (TKV) is a suitable biomarker for disease progression and whether TKV can be used as an efficacy end point in clinical trials. We conclude that because cystogenesis is the central factor leading to kidney enlargement, TKV appears to be an appropriate biomarker and is gaining wider acceptance. Several studies have identified TKV as a relevant imaging biomarker for monitoring and predicting disease progression and support its use as a prognostic end point in clinical trials., (Copyright © 2015 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.)
- Published
- 2015
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39. Urothelial Defects from Targeted Inactivation of Exocyst Sec10 in Mice Cause Ureteropelvic Junction Obstructions.
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Fogelgren B, Polgar N, Lui VH, Lee AJ, Tamashiro KK, Napoli JA, Walton CB, Zuo X, and Lipschutz JH
- Subjects
- Animals, Animals, Newborn, Anuria genetics, Anuria metabolism, Blotting, Western, Disease Models, Animal, Gene Expression Regulation, Developmental, Humans, Hydronephrosis genetics, Hydronephrosis metabolism, Kidney Pelvis embryology, Kidney Pelvis pathology, Mice, Knockout, Mice, Transgenic, Microscopy, Fluorescence, Reverse Transcriptase Polymerase Chain Reaction, Time Factors, Ureteral Obstruction metabolism, Urothelium embryology, Urothelium pathology, Vesicular Transport Proteins metabolism, Kidney Pelvis metabolism, Ureteral Obstruction genetics, Urothelium metabolism, Vesicular Transport Proteins genetics
- Abstract
Most cases of congenital obstructive nephropathy are the result of ureteropelvic junction obstructions, and despite their high prevalence, we have a poor understanding of their etiology and scarcity of genetic models. The eight-protein exocyst complex regulates polarized exocytosis of intracellular vesicles in a large variety of cell types. Here we report generation of a conditional knockout mouse for Sec10, a central component of the exocyst, which is the first conditional allele for any exocyst gene. Inactivation of Sec10 in ureteric bud-derived cells using Ksp1.3-Cre mice resulted in severe bilateral hydronephrosis and complete anuria in newborns, with death occurring 6-14 hours after birth. Sec10 FL/FL;Ksp-Cre embryos developed ureteropelvic junction obstructions between E17.5 and E18.5 as a result of degeneration of the urothelium and subsequent overgrowth by surrounding mesenchymal cells. The urothelial cell layer that lines the urinary tract must maintain a hydrophobic luminal barrier again urine while remaining highly stretchable. This barrier is largely established by production of uroplakin proteins that are transported to the apical surface to establish large plaques. By E16.5, Sec10 FL/FL;Ksp-Cre ureter and pelvic urothelium showed decreased uroplakin-3 protein at the luminal surface, and complete absence of uroplakin-3 by E17.5. Affected urothelium at the UPJ showed irregular barriers that exposed the smooth muscle layer to urine, suggesting this may trigger the surrounding mesenchymal cells to overgrow the lumen. Findings from this novel mouse model show Sec10 is critical for the development of the urothelium in ureters, and provides experimental evidence that failure of this urothelial barrier may contribute to human congenital urinary tract obstructions.
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- 2015
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40. A post-developmental genetic screen for zebrafish models of inherited liver disease.
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Kim SH, Wu SY, Baek JI, Choi SY, Su Y, Flynn CR, Gamse JT, Ess KC, Hardiman G, Lipschutz JH, Abumrad NN, and Rockey DC
- Subjects
- Animals, Disease Models, Animal, Ethylnitrosourea toxicity, Genetic Predisposition to Disease, Humans, Liver pathology, Non-alcoholic Fatty Liver Disease genetics, Zebrafish genetics, Genetic Testing methods, Mutation, Non-alcoholic Fatty Liver Disease pathology, Zebrafish growth & development
- Abstract
Nonalcoholic fatty liver disease (NAFLD) is one of the most common causes of chronic liver disease such as simple steatosis, nonalcoholic steatohepatitis (NASH), cirrhosis and fibrosis. However, the molecular pathogenesis and genetic variations causing NAFLD are poorly understood. The high prevalence and incidence of NAFLD suggests that genetic variations on a large number of genes might be involved in NAFLD. To identify genetic variants causing inherited liver disease, we used zebrafish as a model system for a large-scale mutant screen, and adopted a whole genome sequencing approach for rapid identification of mutated genes found in our screen. Here, we report on a forward genetic screen of ENU mutagenized zebrafish. From 250 F2 lines of ENU mutagenized zebrafish during post-developmental stages (5 to 8 days post fertilization), we identified 19 unique mutant zebrafish lines displaying visual evidence of hepatomegaly and/or steatosis with no developmental defects. Histological analysis of mutants revealed several specific phenotypes, including common steatosis, micro/macrovesicular steatosis, hepatomegaly, ballooning, and acute hepatocellular necrosis. This work has identified multiple post-developmental mutants and establishes zebrafish as a novel animal model for post-developmental inherited liver disease.
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- 2015
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41. Cdc42 and sec10 Are Required for Normal Retinal Development in Zebrafish.
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Choi SY, Baek JI, Zuo X, Kim SH, Dunaief JL, and Lipschutz JH
- Subjects
- Animals, Eye Proteins genetics, Gene Silencing physiology, Organ Size, Retinal Degeneration physiopathology, Vesicular Transport Proteins genetics, Zebrafish embryology, Zebrafish Proteins genetics, cdc42 GTP-Binding Protein genetics, Eye Proteins physiology, Retina embryology, Vesicular Transport Proteins physiology, Zebrafish Proteins physiology, cdc42 GTP-Binding Protein physiology
- Abstract
Purpose: To characterize the function and mechanisms of cdc42 and sec10 in eye development in zebrafish., Methods: Knockdown of zebrafish cdc42 and sec10 was carried out using antisense morpholino injection. The phenotype of morphants was characterized by histology, immunohistology, and transmission electron microscopy (TEM). To investigate a synergistic genetic interaction between cdc42 and sec10, we titrated suboptimal doses of cdc42 and sec10 morpholinos, and coinjected both morpholinos. To study trafficking, a melanosome transport assay was performed using epinephrine., Results: Cdc42 and sec10 knockdown in zebrafish resulted in both abnormal eye development and increased retinal cell death. Cdc42 morphants had a relatively normal retinal structure, aside from the absence of most connecting cilia and outer segments, whereas in sec10 morphants, much of the outer nuclear layer, which is composed of the photoreceptor nuclei, was missing and RPE cell thickness was markedly irregular. Knockdown of cdc42 and sec10 also resulted in an intracellular transport defect affecting retrograde melanosome transport. Furthermore, there was a synergistic genetic interaction between zebrafish cdc42 and sec10, suggesting that cdc42 and sec10 act in the same pathway in retinal development., Conclusions: We propose a model whereby sec10 and cdc42 play a central role in development of the outer segment of the retinal photoreceptor cell by trafficking proteins necessary for ciliogenesis.
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- 2015
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42. Exocyst Sec10 protects renal tubule cells from injury by EGFR/MAPK activation and effects on endocytosis.
- Author
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Fogelgren B, Zuo X, Buonato JM, Vasilyev A, Baek JI, Choi SY, Chacon-Heszele MF, Palmyre A, Polgar N, Drummond I, Park KM, Lazzara MJ, and Lipschutz JH
- Subjects
- Acute Kidney Injury enzymology, Acute Kidney Injury genetics, Acute Kidney Injury pathology, Animals, Animals, Genetically Modified, Disease Models, Animal, Dogs, Enzyme Activation, ErbB Receptors antagonists & inhibitors, ErbB Receptors genetics, Gene Knockdown Techniques, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Kidney Tubules drug effects, Kidney Tubules pathology, Madin Darby Canine Kidney Cells, Oxidative Stress, Phosphorylation, Protein Binding, Protein Kinase Inhibitors pharmacology, Signal Transduction, Time Factors, Transfection, Vesicular Transport Proteins genetics, Zebrafish genetics, Zebrafish Proteins genetics, Acute Kidney Injury prevention & control, Endocytosis drug effects, ErbB Receptors metabolism, Kidney Tubules enzymology, Mitogen-Activated Protein Kinases metabolism, Vesicular Transport Proteins metabolism, Zebrafish Proteins metabolism
- Abstract
Acute kidney injury is common and has a high mortality rate, and no effective treatment exists other than supportive care. Using cell culture models, we previously demonstrated that exocyst Sec10 overexpression reduced damage to renal tubule cells and speeded recovery and that the protective effect was mediated by higher basal levels of mitogen-activated protein kinase (MAPK) signaling. The exocyst, a highly-conserved eight-protein complex, is known for regulating protein trafficking. Here we show that the exocyst biochemically interacts with the epidermal growth factor receptor (EGFR), which is upstream of MAPK, and Sec10-overexpressing cells express greater levels of phosphorylated (active) ERK, the final step in the MAPK pathway, in response to EGF stimulation. EGFR endocytosis, which has been linked to activation of the MAPK pathway, increases in Sec10-overexpressing cells, and gefitinib, a specific EGFR inhibitor, and Dynasore, a dynamin inhibitor, both reduce EGFR endocytosis. In turn, inhibition of the MAPK pathway reduces ligand-mediated EGFR endocytosis, suggesting a potential feedback of elevated ERK activity on EGFR endocytosis. Gefitinib also decreases MAPK signaling in Sec10-overexpressing cells to levels seen in control cells and, demonstrating a causal role for EGFR, reverses the protective effect of Sec10 overexpression following cell injury in vitro. Finally, using an in vivo zebrafish model of acute kidney injury, morpholino-induced knockdown of sec10 increases renal tubule cell susceptibility to injury. Taken together, these results suggest that the exocyst, acting through EGFR, endocytosis, and the MAPK pathway is a candidate therapeutic target for acute kidney injury.
- Published
- 2014
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43. A possible zebrafish model of polycystic kidney disease: knockdown of wnt5a causes cysts in zebrafish kidneys.
- Author
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Huang L, Xiao A, Wecker A, McBride DA, Choi SY, Zhou W, and Lipschutz JH
- Subjects
- Animals, Animals, Genetically Modified, Gene Knockdown Techniques, Green Fluorescent Proteins genetics, Oligonucleotides, Antisense genetics, Polycystic Kidney Diseases metabolism, Wnt Proteins deficiency, Wnt Proteins metabolism, Wnt-5a Protein, Zebrafish, Disease Models, Animal, Polycystic Kidney Diseases genetics, Wnt Proteins genetics
- Abstract
Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to "off-target" effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.
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- 2014
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44. The exocyst and regulatory GTPases in urinary exosomes.
- Author
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Chacon-Heszele MF, Choi SY, Zuo X, Baek JI, Ward C, and Lipschutz JH
- Abstract
Cilia, organelles that function as cellular antennae, are central to the pathogenesis of "ciliopathies", including various forms of polycystic kidney disease (PKD). To date, however, the molecular mechanisms controlling ciliogenesis and ciliary function remain incompletely understood. A recently proposed model of cell-cell communication, called "urocrine signaling", hypothesizes that a subset of membrane bound vesicles that are secreted into the urinary stream (termed exosome-like vesicles, or ELVs), carry cilia-specific proteins as cargo, interact with primary cilia, and affect downstream cellular functions. This study was undertaken to determine the role of the exocyst, a highly conserved eight-protein trafficking complex, in the secretion and/or retrieval of ELVs. We used Madin-Darby canine kidney (MDCK) cells expressing either Sec10-myc (a central component of the exocyst complex) or Smoothened-YFP (a ciliary protein found in ELVs) in experiments utilizing electron gold microscopy and live fluorescent microscopy, respectively. Additionally, human urinary exosomes were isolated via ultracentrifugation and subjected to mass-spectrometry-based proteomics analysis to determine the composition of ELVs. We found, as determined by EM, that the exocyst localizes to primary cilia, and is present in vesicles attached to the cilium. Furthermore, the entire exocyst complex, as well as most of its known regulatory GTPases, are present in human urinary ELVs. Finally, in living MDCK cells, ELVs appear to interact with primary cilia using spinning disc confocal microscopy. These data suggest that the exocyst complex, in addition to its role in ciliogenesis, is centrally involved in the secretion and/or retrieval of urinary ELVs., (© 2014 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.)
- Published
- 2014
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45. Cilia and polycystic kidney disease, kith and kin.
- Author
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Huang L and Lipschutz JH
- Subjects
- Animals, Cilia physiology, Disease Models, Animal, Humans, Kidney cytology, Kidney pathology, Morphogenesis, Cilia pathology, Polycystic Kidney, Autosomal Dominant genetics, Polycystic Kidney, Autosomal Dominant pathology
- Abstract
In the past decade, cilia have been found to play important roles in renal cystogenesis. Many genes, such as PKD1 and PKD2 which, when mutated, cause autosomal dominant polycystic kidney disease (ADPKD), have been found to localize to primary cilia. The cilium functions as a sensor to transmit extracellular signals into the cell. Abnormal cilia structure and function are associated with the development of polyscystic kidney disease (PKD). Cilia assembly includes centriole migration to the apical surface of the cell, ciliary vesicle docking and fusion with the cell membrane at the intended site of cilium outgrowth, and microtubule growth from the basal body. This review summarizes the most recent advances in cilia and PKD research, with special emphasis on the mechanisms of cytoplasmic and intraciliary protein transport during ciliogenesis., (Copyright © 2014 Wiley Periodicals, Inc.)
- Published
- 2014
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46. Novel MAPK-dependent and -independent tubulogenes identified via microarray analysis of 3D-cultured Madin-Darby canine kidney cells.
- Author
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Chacon-Heszele MF, Zuo X, Hellman NE, McKenna S, Choi SY, Huang L, Tobias JW, Park KM, and Lipschutz JH
- Subjects
- Animals, Cell Polarity, Dogs, Gene Expression Regulation, Developmental, Gene Expression Regulation, Enzymologic, Gene Knockdown Techniques, Gene Regulatory Networks, Hepatocyte Growth Factor metabolism, Kidney Tubules growth & development, Kidney Tubules pathology, Madin Darby Canine Kidney Cells, Matrix Metalloproteinase 1 genetics, Matrix Metalloproteinase 1 metabolism, Organogenesis, RNA Interference, RNA, Messenger metabolism, Real-Time Polymerase Chain Reaction, Reproducibility of Results, Gene Expression Profiling methods, Kidney Tubules enzymology, MAP Kinase Signaling System genetics, Mitogen-Activated Protein Kinases genetics, Mitogen-Activated Protein Kinases metabolism, Oligonucleotide Array Sequence Analysis, Tissue Culture Techniques
- Abstract
Cystogenesis and tubulogenesis are basic building blocks for many epithelial organs, including the kidney. Most researchers have used two-dimensional (2D) cell culture to investigate signaling pathways downstream of hepatocyte growth factor (HGF). We hypothesize that three-dimensional (3D) collagen-grown Madin-Darby canine kidney (MDCK) cells, which form cysts and then tubulate in response to HGF, are a much more in vivo-like system for the identification of novel tubulogenes. With the use of a canine microarray containing over 20,000 genes, 2,417 genes were identified as potential tubulogenes that were differentially regulated, exclusively in 3D-grown MDCK cells. Among these, 840 were dependent on MAPK signaling. Importantly, this work shows that many putative tubulogenes, previously identified via microarray analysis of 2D cultures, including by us, do not change in 3D culture and vice versa. The use of a 3D-culture system allowed for the identification of novel MAPK-dependent and -independent genes that regulate early renal tubulogenesis in vitro, e.g., matrix metalloproteinase 1 (MMP1). Knockdown of MMP1 led to defects in cystogenesis and tubulogenesis in 3D-grown MDCK cells, most likely due to problems establishing normal polarity. We suggest that data obtained from 2D cultures, even those using MDCK cells treated with HGF, should not be automatically extrapolated to factors important for cystogenesis and tubulogenesis. Instead, 3D culture, which more closely replicates the biological environment and is therefore a more accurate model for identifying tubulogenes, is preferred. Results from the present analysis will be used to build a more accurate model of the signaling pathways that control cystogenesis and tubulogenesis.
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- 2014
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47. Expression of Drosophila forkhead transcription factors during kidney development.
- Author
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Baek JI, Choi SY, Chacon-Heszele MF, Zuo X, and Lipschutz JH
- Subjects
- Amino Acid Sequence, Animals, Animals, Newborn, Base Sequence, DNA, Complementary genetics, Gene Expression Regulation, Developmental, Mice, Molecular Sequence Data, Rats, Forkhead Transcription Factors genetics, Kidney growth & development, Kidney metabolism, Nuclear Proteins genetics, Transcription Factors genetics
- Abstract
The Drosophila forkhead (Dfkh) family of transcription factors has over 40 family members. One Dfkh family member, BF2 (aka FoxD1), has been shown, by targeted disruption, to be essential for kidney development. In order to determine if other Dfkh family members were involved in kidney development and to search for new members of this family, reverse transcriptase polymerase chain reaction (RT-PCR) was performed using degenerate primers of the consensus sequence of the DNA binding domain of this family and developing rat kidney RNA. The RT-PCR product was used to probe RNA from a developing rat kidney (neonatal), from a 20-day old kidney, and from an adult kidney. The RT-PCR product hybridized only to a developing kidney RNA transcript of ∼2.3 kb (the size of BF2). A lambda gt10 mouse neonatal kidney library was then screened, using the above-described RT-PCR product as a probe. Three lambda phage clones were isolated that strongly hybridized to the RT-PCR probe. Sequencing of the RT-PCR product and the lambda phage clones isolated from the developing kidney library revealed Dfkh BF2. In summary, only Dfkh family member BF2, which has already been shown to be essential for nephrogenesis, was identified in our screen and no other candidate Dfkh family members were identified., (Published by Elsevier Inc.)
- Published
- 2014
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48. Wnt5a is necessary for normal kidney development in zebrafish and mice.
- Author
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Huang L, Xiao A, Choi SY, Kan Q, Zhou W, Chacon-Heszele MF, Ryu YK, McKenna S, Zuo X, Kuruvilla R, and Lipschutz JH
- Subjects
- Animals, Disease Models, Animal, Female, Gene Knockout Techniques, Incidence, Kidney abnormalities, Kidney Diseases, Cystic epidemiology, Kidney Diseases, Cystic etiology, Kidney Diseases, Cystic physiopathology, Male, Mice, Mice, Knockout, Models, Animal, Wnt Proteins deficiency, Wnt Proteins genetics, Wnt-5a Protein, Zebrafish, Zebrafish Proteins deficiency, Zebrafish Proteins genetics, Kidney embryology, Kidney physiology, Wnt Proteins physiology, Zebrafish Proteins physiology
- Abstract
Background: Wnt5a is important for the development of various organs and postnatal cellular function. Little is known, however, about the role of Wnt5a in kidney development, although WNT5A mutations were identified in patients with Robinow syndrome, a genetic disease which includes developmental defects in kidneys. Our goal in this study was to determine the role of Wnt5a in kidney development., Methods: Whole-mount in situ hybridization was used to establish the expression pattern of Wnt5a during kidney development. Zebrafish with wnt5a knockdown and Wnt5a global knockout mice were used to identify kidney phenotypes., Results: In zebrafish, wnt5a knockdown resulted in glomerular cyst formation and dilated renal tubules. In mice, Wnt5a global knockout resulted in pleiotropic, but severe, kidney phenotypes, including agenesis, fused kidney, hydronephrosis and duplex kidney/ureter., Conclusions: Our data demonstrated the important role of Wnt5a in kidney development. Disrupted Wnt5a resulted in kidney cysts in zebrafish and pleiotropic abnormal kidney development in mice., (© 2014 S. Karger AG, Basel.)
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- 2014
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49. Activation of ERK accelerates repair of renal tubular epithelial cells, whereas it inhibits progression of fibrosis following ischemia/reperfusion injury.
- Author
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Jang HS, Han SJ, Kim JI, Lee S, Lipschutz JH, and Park KM
- Subjects
- Animals, Blotting, Western, Butadienes pharmacology, Cilia metabolism, Cilia pathology, Creatinine blood, Enzyme Activation, Enzyme Inhibitors pharmacology, Epithelial Cells metabolism, Extracellular Matrix metabolism, Fibrosis etiology, Fibrosis metabolism, Fluorescent Antibody Technique, Immunoenzyme Techniques, Kidney Tubules metabolism, MAP Kinase Kinase 1 antagonists & inhibitors, MAP Kinase Kinase 1 metabolism, Male, Mice, Mice, Inbred C57BL, NF-kappa B metabolism, Nitriles pharmacology, Phosphorylation, Reactive Oxygen Species metabolism, Reperfusion Injury metabolism, Reperfusion Injury pathology, Signal Transduction, Transforming Growth Factor beta1 metabolism, Epithelial Cells cytology, Extracellular Signal-Regulated MAP Kinases metabolism, Fibrosis prevention & control, Kidney Tubules cytology, Reperfusion Injury complications
- Abstract
Extracellular signal-regulated kinase (ERK) signals play important roles in cell death and survival. However, the role of ERK in the repair process after injury remains to be defined in the kidney. Here, we investigated the role of ERK in proliferation and differentiation of tubular epithelial cells, and proliferation of interstitial cells following ischemia/reperfusion (I/R) injury in the mouse kidney. Mice were subjected to 30min of renal ischemia. Some mice were administered with U0126, a specific upstream inhibitor of ERK, daily during the recovery phase, beginning at 1day after ischemia until sacrifice. I/R caused severe tubular cell damage and functional loss in the kidney. Nine days after ischemia, the kidney was restored functionally with a partial restoration of damaged tubules and expansion of fibrotic lesions. ERK was activated by I/R and the activated ERK was sustained for 9days. U0126 inhibited the proliferation, basolateral relocalization of Na,K-ATPase and lengthening of primary cilia in tubular epithelial cells, whereas it enhanced the proliferation of interstitial cells and accumulation of extracellular matrix. Furthermore, U0126 elevated the expression of cell cycle arrest-related proteins, p21 and phospholylated-chk2 in the post-ischemic kidney. U0126 mitigated the post-I/R increase of Sec10 which is a crucial component of exocyst complex and an important factor in ciliogenesis and tubulogenesis. U0126 also enhanced the expression of fibrosis-related proteins, TGF-β1 and phosphorylated NF-κB after ischemia. Our findings demonstrate that activation of ERK is required for both the restoration of damaged tubular epithelial cells and the inhibition of fibrosis progression following injury., (© 2013.)
- Published
- 2013
- Full Text
- View/download PDF
50. Cdc42 deficiency causes ciliary abnormalities and cystic kidneys.
- Author
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Choi SY, Chacon-Heszele MF, Huang L, McKenna S, Wilson FP, Zuo X, and Lipschutz JH
- Subjects
- Animals, Apoptosis, Cell Proliferation, Disease Models, Animal, Fibrosis, In Vitro Techniques, Kidney Diseases, Cystic metabolism, Kidney Tubules, Collecting metabolism, Kidney Tubules, Collecting pathology, Kidney Tubules, Collecting physiopathology, Kidney Tubules, Distal metabolism, Kidney Tubules, Distal pathology, Kidney Tubules, Distal physiopathology, Mice, Mice, Knockout, Mice, Transgenic, Mitogen-Activated Protein Kinase Kinases physiology, Signal Transduction physiology, Vesicular Transport Proteins deficiency, Vesicular Transport Proteins genetics, Vesicular Transport Proteins metabolism, Zebrafish, Zebrafish Proteins deficiency, Zebrafish Proteins genetics, Zebrafish Proteins metabolism, cdc42 GTP-Binding Protein genetics, cdc42 GTP-Binding Protein metabolism, Cilia metabolism, Cilia pathology, Kidney Diseases, Cystic pathology, Kidney Diseases, Cystic physiopathology, Phenotype, cdc42 GTP-Binding Protein deficiency
- Abstract
Ciliogenesis and cystogenesis require the exocyst, a conserved eight-protein trafficking complex that traffics ciliary proteins. In culture, the small GTPase Cdc42 co-localizes with the exocyst at primary cilia and interacts with the exocyst component Sec10. The role of Cdc42 in vivo, however, is not well understood. Here, knockdown of cdc42 in zebrafish produced a phenotype similar to sec10 knockdown, including tail curvature, glomerular expansion, and mitogen-activated protein kinase (MAPK) activation, suggesting that cdc42 and sec10 cooperate in ciliogenesis. In addition, cdc42 knockdown led to hydrocephalus and loss of photoreceptor cilia. Furthermore, there was a synergistic genetic interaction between zebrafish cdc42 and sec10, suggesting that cdc42 and sec10 function in the same pathway. Mice lacking Cdc42 specifically in kidney tubular epithelial cells died of renal failure within weeks of birth. Histology revealed cystogenesis in distal tubules and collecting ducts, decreased ciliogenesis in cyst cells, increased tubular cell proliferation, increased apoptosis, increased fibrosis, and led to MAPK activation, all of which are features of polycystic kidney disease, especially nephronophthisis. Taken together, these results suggest that Cdc42 localizes the exocyst to primary cilia, whereupon the exocyst targets and docks vesicles carrying ciliary proteins. Abnormalities in this pathway result in deranged ciliogenesis and polycystic kidney disease.
- Published
- 2013
- Full Text
- View/download PDF
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