bims-bicyki Biomed News
on Bicaudal-C1 and interactors in cystic kidney disease
Issue of 2021–04–04
thirty-two papers selected by
Céline Gagnieux, École Polytechnique Fédérale de Lausanne



  1. Clin Kidney J. 2021 Mar;14(3): 746-755
      For decades, researchers have been trying to decipher the complex pathophysiology of autosomal dominant polycystic kidney disease (ADPKD). So far these efforts have led to clinical trials with different candidate treatments, with tolvaptan being the only molecule that has gained approval for this indication. As end-stage kidney disease due to ADPKD has a substantial impact on health expenditures worldwide, it is likely that new drugs targeting kidney function will be developed. On the other hand, recent clinical observations and experimental data, including PKD knockout models in various cell types, have revealed unexpected involvement of many other organs and cell systems of variable severity. These novel non-cystic features, some of which, such as lymphopenia and an increased risk to develop infections, should be validated or further explored and might open new avenues for better risk stratification and a more tailored approach. New insights into the aberrant pathways involved with abnormal expression of PKD gene products polycystin-1 and -2 could, for instance, lead to a more directed approach towards early-onset endothelial dysfunction and subsequent cardiovascular disease. Furthermore, a better understanding of cellular pathways in PKD that can explain the propensity to develop certain types of cancer can guide post-transplant immunosuppressive and prophylactic strategies. In the following review article we will systematically discuss recently discovered non-cystic features of PKD and not well-established characteristics. Overall, this knowledge could enable us to improve the outcome of PKD patients apart from ongoing efforts to slow down cyst growth and attenuate kidney function decline.
    Keywords:  bronchiectasis; cancer; cardiovascular; endothelial; infections; inflammation; lymphopenia; polycystic kidney; polycystin
    DOI:  https://doi.org/10.1093/ckj/sfaa138
  2. Cells. 2021 Mar 03. pii: 532. [Epub ahead of print]10(3):
      The DNA damage response (DDR) pathway is upregulated in autosomal dominant polycystic kidney disease (ADPKD) but its functional role is not known. The ataxia-telangiectasia mutated (ATM) and AT and Rad3-related (ATR) protein kinases are key proximal transducers of the DDR. This study hypothesized that reducing either ATM or ATR attenuates kidney cyst formation and growth in experimental ADPKD. In vitro, pharmacological ATM inhibition by AZD0156 reduced three-dimensional cyst growth in MDCK and human ADPKD cells by up to 4.4- and 4.1-fold, respectively. In contrast, the ATR inhibitor, VE-821, reduced in vitro MDCK cyst growth but caused dysplastic changes. In vivo, treatment with AZD0156 by oral gavage for 10 days reduced renal cell proliferation and increased p53 expression in Pkd1RC/RC mice (a murine genetic ortholog of ADPKD). However, the progression of cystic kidney disease in Pkd1RC/RC mice was not altered by genetic ablation of ATM from birth, in either heterozygous (Pkd1RC/RC/Atm+/-) or homozygous (Pkd1RC/RC/Atm-/-) mutant mice at 3 months. In conclusion, despite short-term effects on reducing renal cell proliferation, chronic progression was not altered by reducing ATM in vivo, suggesting that this DDR kinase is dispensable for kidney cyst formation in ADPKD.
    Keywords:  ATM; ATR; DNA damage response; autosomal dominant polycystic kidney disease
    DOI:  https://doi.org/10.3390/cells10030532
  3. Can J Kidney Health Dis. 2021 ;8 20543581211000227
       Background: There is a perception that patients with autosomal dominant polycystic kidney disease (ADPKD) are more likely to develop kidney stones than the general population.
    Objective: To compare the rate of hospital encounter with kidney stones and the rate of stone interventions between patients with and without ADPKD.
    Design: Retrospective cohort study.
    Setting: Ontario, Canada.
    Patients: Patients with and without ADPKD who had a prior hospital encounter between 2002 and 2016.
    Measurements: Rate of hospital encounter with kidney stones and rate of stone intervention.
    Methods: We used inverse probability exposure weighting based on propensity scores to balance baseline indicators of health between patients with and without ADPKD. We followed each patient until death, emigration, outcomes, or March 31, 2017. We used a Cox proportional hazards model to compare event rates between the two groups.
    Results: Patients with ADPKD were at higher risk of hospital encounter with stones compared with patients without ADPKD (81 patients of 2094 with ADPKD [3.8%] vs 60 patients of 1902 without ADPKD [3.2%]; 8.9 vs 5.1 events per 1000 person-years; hazard ratio 1.6 [95% CI, 1.3-2.1]). ADPKD was not associated with a higher risk of stone intervention (49 of 2094 [2.3%] vs 47 of 1902 [2.4%]; 5.3 vs 3.9 events per 1000 person-years; hazard ratio 1.2 [95% CI = 0.9-1.3]).
    Limitations: We did not have information on kidney stone events outside of the hospital. There is a possibility of residual confounding.
    Conclusion: ADPKD was a significant risk factor for hospital encounters with kidney stones.
    Keywords:  administrative data; autosomal dominant polycystic kidney disease; epidemiology; kidney stones; population health research
    DOI:  https://doi.org/10.1177/20543581211000227
  4. Neurol Neurochir Pol. 2021 Mar 30.
       INTRODUCTION: Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic renal disorder. It leads to multiple extra-renal complications, with intracranial aneurysms (IA) among the most serious. Biological markers could become tools in identifying patients at risk of an IA. MicroRNAs 16 (miR-16) and 25 (miR-25) have been proposed as being markers of IAs in the general population. In the current study, we attempted to discover if they may also be considered markers of IAs in ADPKD.
    MATERIAL AND METHODS: 64 renal transplant recipients with ADPKD were included. After magnetic resonance angiography of the brain, they were divided into a case group (IA+, n = 13) and a control group (IA-, n = 51). Expression of miRNAs in plasma was analysed by qRT-PCR.
    RESULTS: The expression of miR-16 was higher in the control (IA-) group. There was no statistically significant difference between the groups in terms of miR-25 expression.
    CONCLUSIONS AND CLINICAL IMPLICATIONS: MicroRNA-16 is a potential marker of IAs in renal transplant recipients with ADPKD. It may become a tool to identify patients who should undergo screening for an IA.
    Keywords:  autosomal dominant polycystic kidney disease; biomarkers; intracranial aneurysm; kidney transplantation; micro RNA
    DOI:  https://doi.org/10.5603/PJNNS.a2021.0026
  5. Int J Mol Sci. 2021 Mar 16. pii: 3014. [Epub ahead of print]22(6):
      Single-gene defects have been revealed to be the etiologies of many kidney diseases with the recent advances in molecular genetics. Autosomal dominant polycystic kidney disease (ADPKD), as one of the most common inherited kidney diseases, is caused by mutations of PKD1 or PKD2 gene. Due to the complexity of pathophysiology of cyst formation and progression, limited therapeutic options are available. The roles of noncoding RNAs in development and disease have gained widespread attention in recent years. In particular, microRNAs in promoting PKD progression have been highlighted. The dysregulated microRNAs modulate cyst growth through suppressing the expression of PKD genes and regulating cystic renal epithelial cell proliferation, mitochondrial metabolism, apoptosis and autophagy. The antagonists of microRNAs have emerged as potential therapeutic drugs for the treatment of ADPKD. In addition, studies have also focused on microRNAs as potential biomarkers for ADPKD and other common hereditary kidney diseases, including HNF1β-associated kidney disease, Alport syndrome, congenital abnormalities of the kidney and urinary tract (CAKUT), von Hippel-Lindau (VHL) disease, and Fabry disease. This review assembles the current understanding of the non-coding RNAs, including microRNAs and long noncoding RNAs, in polycystic kidney disease and these common monogenic kidney diseases.
    Keywords:  Genetic kidney disease; PKD; microRNA; non-coding RNA
    DOI:  https://doi.org/10.3390/ijms22063014
  6. Ann Vasc Dis. 2021 Mar 25. 14(1): 68-70
      Autosomal dominant polycystic kidney disease (ADPKD) is the most common congenital kidney disease. However, reports on occasional cases of aortic dissection in PKD familial patients remain scarce. Herein, we describe rare aortic dissection cases in PKD familial patients (i.e., mother and daughter) and our successful treatment experience. The mother (84 years old) and daughter (53 years old) had a referral to us to treat type A acute aortic dissection. We performed emergency surgery and successfully treated the patients with an artificial graft. For comprehensive evaluation and treatment, ADPKD patients and their families should be screened for aortic diseases.
    Keywords:  ADPKD; aortic dissection
    DOI:  https://doi.org/10.3400/avd.cr.20-00149
  7. J Dev Biol. 2021 Mar 25. pii: 12. [Epub ahead of print]9(2):
      Primary cilia are ubiquitous microtubule-based organelles that serve as signaling hubs for numerous developmental pathways, most notably the Hedgehog (Hh) pathway. Defects in the structure or function of primary cilia result in a class of diseases called ciliopathies. It is well known that primary cilia participate in transducing a Hh signal, and as such ciliopathies frequently present with phenotypes indicative of aberrant Hh function. Interestingly, the exact mechanisms of cilia-dependent Hh signaling transduction are unclear as some ciliopathic animal models simultaneously present with gain-of-Hh phenotypes in one organ system and loss-of-Hh phenotypes in another. To better understand how Hh signaling is perturbed across different tissues in ciliopathic conditions, we examined four distinct Hh-dependent signaling centers in the naturally occurring avian ciliopathic mutant talpid2 (ta2). In addition to the well-known and previously reported limb and craniofacial malformations, we observed dorsal-ventral patterning defects in the neural tube, and a shortened gastrointestinal tract. Molecular analyses for elements of the Hh pathway revealed that the loss of cilia impact transduction of an Hh signal in a tissue-specific manner at variable levels of the pathway. These studies will provide increased knowledge into how impaired ciliogenesis differentially regulates Hh signaling across tissues and will provide potential avenues for future targeted therapeutic treatments.
    Keywords:  C2CD3; Hedgehog signaling; ciliopathies; craniofacial; hindgut; limb; neural tube; primary cilia; talpid2
    DOI:  https://doi.org/10.3390/jdb9020012
  8. Int J Mol Sci. 2021 Mar 11. pii: 2839. [Epub ahead of print]22(6):
      Diabetic kidney disease (DKD) is the leading cause of kidney failure. RhoA/Rho-associated protein kinase (ROCK) signaling is a recognized mediator of its pathogenesis, largely through mediating the profibrotic response. While RhoA activation is not feasible due to the central role it plays in normal physiology, ROCK inhibition has been found to be effective in attenuating DKD in preclinical models. However, this has not been evaluated in clinical studies as of yet. Alternate means of inhibiting RhoA/ROCK signaling involve the identification of disease-specific activators. This report presents evidence showing the activation of RhoA/ROCK signaling both in vitro in glomerular mesangial cells and in vivo in diabetic kidneys by two recently described novel pathogenic mediators of fibrosis in DKD, activins and cell-surface GRP78. Neither are present in normal kidneys. Activin inhibition with follistatin and neutralization of cell-surface GRP78 using a specific antibody blocked RhoA activation in mesangial cells and in diabetic kidneys. These data identify two novel RhoA/ROCK activators in diabetic kidneys that can be evaluated for their efficacy in inhibiting the progression of DKD.
    Keywords:  Rho-kinase; RhoA; activins; cell-surface GRP78; diabetic kidney disease; fibrosis
    DOI:  https://doi.org/10.3390/ijms22062839
  9. Stem Cell Res. 2021 Mar 22. pii: S1873-5061(21)00152-5. [Epub ahead of print]53 102306
      Autosomal dominant polycystic kidney disease (ADPKD) is one of the common genetic kidney disorders that are caused by mutations in PKD1 or PKD2 gene. In this report, the MUi026-A human induced pluripotent stem cell (hiPSC) line was established from the skin fibroblasts of a female ADPKD patient who had the PKD1 mutation with c.5878C > T. The iPSC line retained normal karyotype. The cells displayed embryonic stem cell-like characteristics with pluripotency marker expression and were able to differentiate into three germ layers.
    DOI:  https://doi.org/10.1016/j.scr.2021.102306
  10. J Nephrol. 2021 Apr 01.
      
    Keywords:  Autologous transplantation; Autosomal dominant polycystic kidney disease; Ectopic kidney
    DOI:  https://doi.org/10.1007/s40620-021-01008-y
  11. JCI Insight. 2021 Mar 30. pii: 141299. [Epub ahead of print]
      Despite the recent launch of Tolvaptan, the search for safer polycystic kidney disease (PKD) drugs continues. Ciclopirox (CPX) or its olamine salt (CPX-O) are contained in number of commercially available antifungal agents. CPX is also reported to possess anticancer activity. Several mechanisms of action have been proposed including chelation of iron and inhibition of iron dependent enzymes. Here, we show that CPX-O inhibited in vitro cystogenesis of primary human PKD cyst-lining epithelial cells cultured in a 3D collagen matrix. To assess in vivo role of CPX-O, we treated PKD mice with CPX-O. CPX-O reduced the kidney- to-body weight ratios of PKD mice. This was also associated with decreased cell proliferation, decreased cystic area and improved renal function. Ferritin levels were significantly elevated in cystic kidneys of PKD mice, and CPX-O treatment reduced renal ferritin levels. The reduction in ferritin was associated with increased ferritinophagy marker, NCOA4 which reversed upon CPX-O treatment in PKD mice. Interestingly, these effects on ferritin appeared independent of iron. These data suggest that CPX-O can induce ferritin degradation via ferritinophagy which is associated with decreased cyst growth progression in PKD mice. Most importantly these data indicate that CPX-O has the potential to treat autosomal dominant PKD.
    Keywords:  Chronic kidney disease; Nephrology
    DOI:  https://doi.org/10.1172/jci.insight.141299
  12. Curr Biol. 2021 Mar 24. pii: S0960-9822(21)00362-6. [Epub ahead of print]
      In animal cells, the functions of the microtubule cytoskeleton are coordinated by centriole-based centrosomes via γ-tubulin complexes embedded in the pericentriolar material or PCM.1 PCM assembly has been best studied in the context of mitosis, where centriolar SPD-2 recruits PLK-1, which in turn phosphorylates key scaffolding components like SPD-5 and CNN to promote expansion of the PCM polymer.2-4 To what extent these mechanisms apply to centrosomes in interphase or in differentiated cells remains unclear.5 Here, we examine a novel type of centrosome found at the ciliary base of C. elegans sensory neurons, which we show plays important roles in neuronal morphogenesis, cellular trafficking, and ciliogenesis. These centrosomes display similar dynamic behavior to canonical, mitotic centrosomes, with a stable PCM scaffold and dynamically localized client proteins. Unusually, however, they are not organized by centrioles, which degenerate early in terminal differentiation.6 Yet, PCM not only persists but continues to grow with key scaffolding proteins including SPD-5 expressed under control of the RFX transcription factor DAF-19. This assembly occurs in the absence of the mitotic regulators SPD-2, AIR-1 and PLK-1, but requires tethering by PCMD-1, a protein which also plays a role in the initial, interphase recruitment of PCM in early embryos.7 These results argue for distinct mechanisms for mitotic and non-mitotic PCM assembly, with only the former requiring PLK-1 phosphorylation to drive rapid expansion of the scaffold polymer.
    Keywords:  C. elegans; PCM; PCMD-1; PLK-1; SPD-5; centrioles; centrosomes; cilia; ciliogenesis
    DOI:  https://doi.org/10.1016/j.cub.2021.03.023
  13. Int J Mol Sci. 2021 Mar 16. pii: 3013. [Epub ahead of print]22(6):
      Motile cilia and homologous organelles, the flagella, are an early evolutionarily invention, enabling primitive eukaryotic cells to survive and reproduce. In animals, cilia have undergone functional and structural speciation giving raise to typical motile cilia, motile nodal cilia, and sensory immotile cilia. In contrast to other cilia types, typical motile cilia are able to beat in complex, two-phase movements. Moreover, they contain many additional structures, including central apparatus, composed of two single microtubules connected by a bridge-like structure and assembling numerous complexes called projections. A growing body of evidence supports the important role of the central apparatus in the generation and regulation of the motile cilia movement. Here we review data concerning the central apparatus structure, protein composition, and the significance of its components in ciliary beating regulation.
    Keywords:  Chlamydomonas; PCD; Trypanosoma; axoneme; central pair microtubules; male infertility
    DOI:  https://doi.org/10.3390/ijms22063013
  14. Hepatol Int. 2021 Apr 02.
       BACKGROUND: Most patients with autosomal-dominant polycystic kidney disease (ADPKD) develop liver cysts and polycystic liver disease as they age. To date, no simple clinical indicator has been confirmed to predict polycystic liver disease exacerbation. Furthermore, the effect of the type and location of mutation on disease progression of polycystic liver disease remains unclear. Here, we aimed to establish a simple liver cyst indicator for clinical practice and investigate whether gene mutations determined liver phenotype in patients with autosomal-dominant polycystic kidney disease.
    METHODS: In total, 129 patients with ADPKD were enrolled and liver cyst indicators were assessed based on mutation type (truncating mutation: nonsense, frameshift, and splicing mutation; non-truncating mutation: substitution) and mutation position. Liver cyst severity was determined using Gigot and Drenth classifications, based on their number, maximum diameter, and area ratio with the liver.
    RESULTS: We observed an overall prevalence of 62.8% for polycystic liver disease. Patients with PKD1 nonsense mutations, a type of PKD1 truncating mutation, exhibited more severe liver disease phenotypes than those without the mutation. We identified maximum diameter as a potential liver cyst indicator. Moreover, a subgroup analysis that included a PKD1 nonsense mutation cohort revealed that genetic mutations located closer to the 5' end of PKD1 were associated with a maximum diameter index value ≥ 6 cm.
    CONCLUSION: PKD1 nonsense mutations were associated with liver cyst severity, which along with maximum diameter index as a simple clinical indicator for liver cysts, may improve the treatment of polycystic liver disease associated with ADPKD.
    Keywords:  Cyst-liver area ratio; Drenth classification; Gigot classification; Liver cyst indicator; Maximum liver cyst diameter; Mutation type; Nonsense mutation; PKD1; Polycystic liver disease; Polycystin-1; Sex difference; Substitution mutation
    DOI:  https://doi.org/10.1007/s12072-021-10176-9
  15. Curr Biol. 2021 Mar 24. pii: S0960-9822(21)00361-4. [Epub ahead of print]
      During mitosis in animal cells, the centrosome acts as a microtubule organizing center (MTOC) to assemble the mitotic spindle. MTOC function at the centrosome is driven by proteins within the pericentriolar material (PCM), however the molecular complexity of the PCM makes it difficult to differentiate the proteins required for MTOC activity from other centrosomal functions. We used the natural spatial separation of PCM proteins during mitotic exit to identify a minimal module of proteins required for centrosomal MTOC function in C. elegans. Using tissue-specific degradation, we show that SPD-5, the functional homolog of CDK5RAP2, is essential for embryonic mitosis, while SPD-2/CEP192 and PCMD-1, which are essential in the one-cell embryo, are dispensable. Surprisingly, although the centriole is known to be degraded in the ciliated sensory neurons in C. elegans,1-3 we find evidence for "centriole-less PCM" at the base of cilia and use this structure as a minimal testbed to dissect centrosomal MTOC function. Super-resolution imaging revealed that this PCM inserts inside the lumen of the ciliary axoneme and directly nucleates the assembly of dendritic microtubules toward the cell body. Tissue-specific degradation in ciliated sensory neurons revealed a role for SPD-5 and the conserved microtubule nucleator γ-TuRC, but not SPD-2 or PCMD-1, in MTOC function at centriole-less PCM. This MTOC function was in the absence of regulation by mitotic kinases, highlighting the intrinsic ability of these proteins to drive microtubule growth and organization and further supporting a model that SPD-5 is the primary driver of MTOC function at the PCM.
    Keywords:  C. elegans; centrosome; cilia; microtubule; microtubule organizing center
    DOI:  https://doi.org/10.1016/j.cub.2021.03.022
  16. Am J Physiol Cell Physiol. 2021 Mar 31.
      Intraocular pressure (IOP) is dynamically regulated by the trabecular meshwork (TM), a mechanosensitive tissue that protects the eye from injury through dynamic regulation of aqueous humor outflow from the anterior chamber of the eye. In response to chronic IOP elevations, TM compensates for mechanical stress through increased actin polymerization, tissue stiffness and contractility. This process has been associated with open angle glaucoma, however, the mechanisms that link mechanical stress to pathological cytoskeletal remodeling downstream from the mechanotransducers remain poorly understood.We used fluorescence imaging and biochemical analyses to investigate cytoskeletal and focal adhesion remodeling in human TM cells stimulated with physiological strains. Mechanical stretch promoted F-actin polymerization, increased the number and size of focal adhesions, and stimulated the activation of the Rho-associated protein kinase (ROCK). Stretch-induced activation of the small GTPase RhoA, and tyrosine phosphorylations of focal adhesion proteins paxillin, focal adhesion kinase (FAK), vinculin and zyxin were time-dependently inhibited by ROCK inhibitor Y-27632, and by HC-067047, an antagonist of transient receptor potential vanilloid 4 (TRPV4) channels. Both TRPV4 and ROCK activation were required for zyxin translocation and increase in the number/size of focal adhesions in stretched cells. Y-27632 blocked actin polymerization without affecting calcium influx induced by membrane stretch and the TRPV4 agonist GSK1016790A. These results reveal that mechanical tuning of TM cells requires parallel activation of TRPV4, integrins and ROCK, with chronic stress leading to sustained remodeling of the cytoskeleton and focal complexes.
    Keywords:  TRPV4; cytoskeleton; focal adhesion; glaucoma; mechanotransduction
    DOI:  https://doi.org/10.1152/ajpcell.00599.2020
  17. HGG Adv. 2021 Jan 14. pii: 100016. [Epub ahead of print]2(1):
    University of Washington Center for Mendelian Genomics
      The Joubert-Meckel syndrome spectrum is a continuum of recessive ciliopathy conditions caused by primary cilium dysfunction. The primary cilium is a microtubule-based, antenna-like organelle that projects from the surface of most human cell types, allowing them to respond to extracellular signals. The cilium is partitioned from the cell body by the transition zone, a known hotspot for ciliopathy-related proteins. Despite years of Joubert syndrome (JBTS) gene discovery, the genetic cause cannot be identified in up to 30% of individuals with JBTS, depending on the cohort, sequencing method, and criteria for pathogenic variants. Using exome and targeted sequencing of 655 families with JBTS, we identified three individuals from two families harboring biallelic, rare, predicted-deleterious missense TMEM218 variants. Via MatchMaker Exchange, we identified biallelic TMEM218 variants in four additional families with ciliopathy phenotypes. Of note, four of the six families carry missense variants affecting the same highly conserved amino acid position 115. Clinical features included the molar tooth sign (N = 2), occipital encephalocele (N = 5, all fetuses), retinal dystrophy (N = 4, all living individuals), polycystic kidneys (N = 2), and polydactyly (N = 2), without liver involvement. Combined with existing functional data linking TMEM218 to ciliary transition zone function, our human genetic data make a strong case for TMEM218 dysfunction as a cause of ciliopathy phenotypes including JBTS with retinal dystrophy and Meckel syndrome. Identifying all genetic causes of the Joubert-Meckel spectrum enables diagnostic testing, prognostic and recurrence risk counseling, and medical monitoring, as well as work to delineate the underlying biological mechanisms and identify targets for future therapies.
    DOI:  https://doi.org/10.1016/j.xhgg.2020.100016
  18. Environ Toxicol. 2021 Mar 31.
      Exposure to carbon blacks (CBs) has been associated with the progression of pulmonary fibrosis, whereas the mechanism is still not clear. We therefore aimed to investigate the effect of RhoA/ROCK pathway on pulmonary fibrosis caused by CBs exposure. Western blot analysis indicated that CBs could promote the activation of RhoA/ROCK pathway and phosphorylation of p65 and IκBα in mice lung. However, ROCK inhibitor Y-27632 could attenuate phosphorylation levels of p65 and IκBα and restore histopathological changes of the lung tissue. Then, we evaluated the effect of RhoA/ROCK pathway on pulmonary fibrosis by detecting the expression levels of α-SMA, vimentin, and Collagen type-I (Col-I), which could be partly inhibited by Y-27632. It was assumed that inhibition of ROCK could be a promising therapeutic candidate for CBs-induced pulmonary fibrosis, which possibly through the blockage of RhoA/ROCK/NF-κB pathway.
    Keywords:  NF-kappa B; RhoA/rho kinase; Y-27632; carbon black; pulmonary fibrosis
    DOI:  https://doi.org/10.1002/tox.23135
  19. Cell Biol Int. 2021 Apr 03.
      Electromagnetic fields (EMFs) have emerged as a versatile means for osteoporosis treatment and prevention. However, its optimal application parameters are still elusive. Here we optimized the frequency parameter firstly by cell culture screening and then by animal experiment validation. Osteoblasts isolated from newborn rats (ROBs) were exposed 90 min/day to 1.8 mT SEMFs at different frequencies (ranging from 10 to 100 Hz, interval of 10 Hz). SEMFs of 1.8 mT inhibited ROB proliferation at 30, 40, 50, 60 Hz, but increased proliferation at 10, 70, 80 Hz. SEMFs of 10, 50 and 70 Hz promoted ROB osteogenic differentiation and mineralization as shown by alkaline phosphatase (ALP) activity, calcium content and osteogenesis-related molecule expression analyses, with 50 Hz showing greater effects than 10 and 70 Hz. Treatment of young rats with 1.8 mT SEMFs at 10, 50 or 100 Hz for 2 months significantly increased whole body bone mineral density (BMD) and femur micro-architecture, with the 50 Hz group showing the greatest effect. Furthermore, 1.8 mT SEMFs extended primary cilia lengths of ROBs and increased protein kinase A (PKA) activation also in a frequency-dependent manner, again with 50 Hz SEMFs showing the greatest effect. Pretreatment of ROBs with the PKA inhibitor KT5720 abolished the effects of SEMFs to increase primary cilia length and promote osteogenic differentiation/mineralization. These results indicate that 1.8 mT SEMFs have a frequency window effect in promoting osteogenic differentiation/mineralization in ROBs and bone formation in growing rats, which involve osteoblast primary cilia length extension and PKA activation. This article is protected by copyright. All rights reserved.
    Keywords:  Electromagnetic fields; Frequency; Osteoblasts; Osteoporosis; PKA; Peak bone mass; Primary cilia length; SEMFs
    DOI:  https://doi.org/10.1002/cbin.11606
  20. Int J Mol Sci. 2021 Mar 20. pii: 3176. [Epub ahead of print]22(6):
      The primary cilium plays a pivotal role during the embryonic development of vertebrates. It acts as a somatic signaling hub for specific pathways, such as Sonic Hedgehog signaling. In humans, mutations in genes that cause dysregulation of ciliogenesis or ciliary function lead to severe developmental disorders called ciliopathies. Beyond its role in early morphogenesis, growing evidence points towards an essential function of the primary cilium in neural circuit formation in the central nervous system. However, very little is known about a potential role in the formation of the peripheral nervous system. Here, we investigate the presence of the primary cilium in neural crest cells and their derivatives in the trunk of developing chicken embryos in vivo. We found that neural crest cells, sensory neurons, and boundary cap cells all bear a primary cilium during key stages of early peripheral nervous system formation. Moreover, we describe differences in the ciliation of neuronal cultures of different populations from the peripheral and central nervous systems. Our results offer a framework for further in vivo and in vitro investigations on specific roles that the primary cilium might play during peripheral nervous system formation.
    Keywords:  DRG; PNS; boundary cap cells; ciliogenesis; neural circuit formation; neural crest cells; primary cilium; sympathetic ganglia
    DOI:  https://doi.org/10.3390/ijms22063176
  21. Int J Mol Sci. 2021 Mar 23. pii: 3279. [Epub ahead of print]22(6):
      Numerous studies have assembled a complex picture, in which extracellular stimuli and intracellular signaling pathways modulate the chondrocyte phenotype. Because many diseases are mechanobiology-related, this review asked to what extent phenotype regulators control chondrocyte function through the cytoskeleton and cytoskeleton-regulating signaling processes. Such information would generate leverage for advanced articular cartilage repair. Serial passaging, pro-inflammatory cytokine signaling (TNF-α, IL-1α, IL-1β, IL-6, and IL-8), growth factors (TGF-α), and osteoarthritis not only induce dedifferentiation but also converge on RhoA/ROCK/Rac1/mDia1/mDia2/Cdc42 to promote actin polymerization/crosslinking for stress fiber (SF) formation. SF formation takes center stage in phenotype control, as both SF formation and SOX9 phosphorylation for COL2 expression are ROCK activity-dependent. Explaining how it is molecularly possible that dedifferentiation induces low COL2 expression but high SF formation, this review theorized that, in chondrocyte SOX9, phosphorylation by ROCK might effectively be sidelined in favor of other SF-promoting ROCK substrates, based on a differential ROCK affinity. In turn, actin depolymerization for redifferentiation would "free-up" ROCK to increase COL2 expression. Moreover, the actin cytoskeleton regulates COL1 expression, modulates COL2/aggrecan fragment generation, and mediates a fibrogenic/catabolic expression profile, highlighting that actin dynamics-regulating processes decisively control the chondrocyte phenotype. This suggests modulating the balance between actin polymerization/depolymerization for therapeutically controlling the chondrocyte phenotype.
    Keywords:  SOX9; actin depolymerization; actin polymerization; chondrocyte; collagen aggrecan fragments; cytoskeleton; fibrogenic expression profile; growth factor signaling; pro-inflammatory cytokine signaling; stress fiber
    DOI:  https://doi.org/10.3390/ijms22063279
  22. Naunyn Schmiedebergs Arch Pharmacol. 2021 Mar 29.
      The global incidence of myocardial infarction has been consistently high, and it is one of the main causes of poor cardiovascular prognosis. Dexmedetomidine (DEX) is a highly selective α2 receptor agonist. Recent studies have found that DEX has a protective effect on myocardial infarction, but its specific mechanism is still unclear. In this experiment, we permanently ligated the anterior descending branch of mice to explore the protective mechanism of DEX against myocardial infarction. Our study found that intraperitoneal injection of DEX for 7 days after myocardial infarction in mice can increase the reduction of ejection fraction (EF) and fractional shortening (FS) caused by myocardial infarction and significantly reduce the release of serum markers. The results of myocardial HE and Sirius red staining suggest that the changes in the myocardial structure of mice after using DEX are reduced. Immunohistochemistry shows that DEX reduces the expression of ROCK1 protein after myocardial infarction. TUNEL staining and the protein expression levels of cleaved caspase-3 and cleaved caspase-9 were used to detect cell apoptosis and results make clear that DEX can reduce the apoptosis caused by myocardial infarction. Western blot experiments showed that DEX can reduce the expression levels of ROCK1 and ROCK2 (Rho-kinase). At the same time, it was observed that DEX improved the Bcl-2/Bax ratio. The above results indicate that DEX reduces cardiomyocyte apoptosis and improves cardiac function likely through inhibiting the RhoA/ROCK signaling pathway. This study may provide new insights into the protective effect of DEX after myocardial infarction in mice.
    Keywords:  Apoptosis; Dexmedetomidine; Myocardial infarction; RhoA/ROCK
    DOI:  https://doi.org/10.1007/s00210-021-02082-6
  23. J Pharm Pharmacol. 2021 Mar 29. pii: rgab033. [Epub ahead of print]
       OBJECTIVES: The aim of this study was to evaluate the effects of fasudil on insulin resistance (IR) in HTR-8/SVneo cells.
    METHODS: HTR-8/SVneo cells were treated with insulin or/and fasudil. Cell proliferation, apoptosis, inflammation and related signalling pathways were assessed.
    KEY FINDINGS: Insulin treatment significantly enhanced the protein expressions of RhoA and Rho kinase (ROCK1 and ROCK2), but decreased glucose consumption. Administration of fasudil effectively promoted glucose uptake. Moreover, fasudil enhanced cell viability and the level of proliferating cell nuclear antigen (PCNA). Insulin-mediated cell apoptosis was inhibited by fasudil via the down-regulation of bax and cleaved-caspase-3, and the up-regulation of bcl-2. At the same time, fasudil led to the reduction of IL-1β, TNF-α, IL-6 and IL-8 mRNA levels in insulin-treated cells. In addition, RhoA, ROCK2 and phosphorylated myosin phosphatase target subunit-1 (p-MYPT-1) expressions were down-regulated by fasudil. Importantly, fasudil activated insulin receptor substrate-1 (IRS-1) through increasing p-IRS-1 (Tyr612) and p-Akt expressions. The nuclear NF-κB p65 and p-IκB-α levels were reduced via the administration of fasudil in insulin-treated cells.
    CONCLUSIONS: Fasudil mitigated IR by the promotion of cell proliferation, inhibition of apoptosis and inflammation and regulation of RhoA/ROCK/insulin/NF-κB signalling pathway through in vitro studies.
    Keywords:  HTR-8/SVneo cells; RhoA/Rho kinase/insulin/NF-κB signalling pathway; fasudil; insulin resistance
    DOI:  https://doi.org/10.1093/jpp/rgab033
  24. Eur Respir J. 2021 Apr 01. pii: 2000455. [Epub ahead of print]
      Development of therapeutic approaches for rare respiratory diseases is hampered by the lack of systems that allow medium-to-high-throughput screening of fully differentiated respiratory epithelium from affected patients. This is a particular problem for primary ciliary dyskinesia (PCD), a rare genetic disease caused by mutations in genes that adversely affect ciliary movement and consequently mucociliary transport. Primary cell culture of basal epithelial cells from nasal brush biopsies, followed by ciliated differentiation at air-liquid interface (ALI) has proven to be a useful tool in PCD diagnostics but the technique's broader utility, including in pre-clinical PCD research, has been restricted by the limited number of basal cells that it is possible to expand from such biopsies. Here, we describe an immunofluorescence screening method, enabled by extensive expansion of PCD patient basal cells and their culture into differentiated respiratory epithelium in miniaturised 96-well transwell format ALI cultures. Analyses of ciliary ultrastructure, beat pattern and beat frequency indicate that a range of different PCD defects can be retained in these cultures. As proof-of-principle, we performed a personalised investigation in a patient with a rare and severe form of PCD (reduced generation of motile cilia, RGMC), in this case caused by a homozygous nonsense mutation in the MCIDAS gene. The screening system allowed drugs that induce translational readthrough to be evaluated alone or in combination with nonsense-mediated decay inhibitors. Restoration of basal body formation in the patient's nasal epithelial cells was seen in vitro, suggesting a novel avenue for drug evaluation and development in PCD.
    DOI:  https://doi.org/10.1183/13993003.00455-2020
  25. Cells. 2021 Mar 16. pii: 663. [Epub ahead of print]10(3):
      The mechanical environment of cardiac cells changes continuously and undergoes major alterations during diseases. Most cardiac diseases, including atrial fibrillation, are accompanied by fibrosis which can impair both electrical and mechanical function of the heart. A key characteristic of fibrotic tissue is excessive accumulation of extracellular matrix, leading to increased tissue stiffness. Cells are known to respond to changes in their mechanical environment, but the molecular mechanisms underlying this ability are incompletely understood. We used cell culture systems and hydrogels with tunable stiffness, combined with advanced biophysical and imaging techniques, to elucidate the roles of the stretch-activated channel Piezo1 in human atrial fibroblast mechano-sensing. Changing the expression level of Piezo1 revealed that this mechano-sensor contributes to the organization of the cytoskeleton, affecting mechanical properties of human embryonic kidney cells and human atrial fibroblasts. Our results suggest that this response is independent of Piezo1-mediated ion conduction at the plasma membrane, and mediated in part by components of the integrin pathway. Further, we show that Piezo1 is instrumental for fibroblast adaptation to changes in matrix stiffness, and that Piezo1-induced cell stiffening is transmitted in a paracrine manner to other cells by a signaling mechanism requiring interleukin-6. Piezo1 may be a new candidate for targeted interference with cardiac fibroblast function.
    Keywords:  FAK; ROCK; Young’s modulus; actin; adhesion; calpain; cardiac fibrosis; cytoskeleton; heart; integrin
    DOI:  https://doi.org/10.3390/cells10030663
  26. Int J Mol Sci. 2021 Mar 03. pii: 2516. [Epub ahead of print]22(5):
      Monocyte to macrophage differentiation is characterized by the activation of various signal transduction pathways, which may be modulated by protein phosphorylation; however, the impact of protein kinases and phosphatases is not well understood yet. It has been demonstrated that actomyosin rearrangement during macrophage differentiation is dependent on Rho-associated protein kinase (ROCK). Myosin phosphatase (MP) target subunit-1 (MYPT1) is one of the major cellular substrates of ROCK, and MP is often a counter enzyme of ROCK; therefore, MP may also control macrophage differentiation. Changes in MP activity and the effects of MP activation were studied on PMA or l,25(OH)2D3-induced differentiation of monocytic THP-1 cells. During macrophage differentiation, phosphorylation of MYPT1 at Thr696 and Thr853 increased significantly, resulting in inhibition of MP. The ROCK inhibitor H1152 and the MP activator epigallocatechin-3-gallate (EGCG) attenuated MYPT1 phosphorylation and concomitantly decreased the extent of phosphorylation of 20 kDa myosin light chain. H1152 and EGCG pretreatment also suppressed the expression of CD11b and weakened the PMA-induced adherence of the cells. Our results indicate that MP activation/inhibition contributes to the efficacy of monocyte to macrophage differentiation, and this enzyme may be a target for pharmacological interventions in the control of disease states that are affected by excessive macrophage differentiation.
    Keywords:  EGCG; MYPT1; PMA; ROCK inhibitor; macrophage differentiation; myosin phosphatase
    DOI:  https://doi.org/10.3390/ijms22052516
  27. Front Cell Infect Microbiol. 2021 ;11 642271
      Trypanosomatids have a cytoskeleton arrangement that is simpler than what is found in most eukaryotic cells. However, it is precisely organized and constituted by stable microtubules. Such microtubules compose the mitotic spindle during mitosis, the basal body, the flagellar axoneme and the subpellicular microtubules, which are connected to each other and also to the plasma membrane forming a helical arrangement along the central axis of the parasite cell body. Subpellicular, mitotic and axonemal microtubules are extensively acetylated in Trypanosoma cruzi. Acetylation on lysine (K) 40 of α-tubulin is conserved from lower eukaryotes to mammals and is associated with microtubule stability. It is also known that K40 acetylation occurs significantly on flagella, centrioles, cilia, basal body and the mitotic spindle in eukaryotes. Several tubulin posttranslational modifications, including acetylation of K40, have been cataloged in trypanosomatids, but the functional importance of these modifications for microtubule dynamics and parasite biology remains largely undefined. The primary tubulin acetyltransferase was recently identified in several eukaryotes as Mec-17/ATAT, a Gcn5-related N-acetyltransferase. Here, we report that T. cruzi ATAT acetylates α-tubulin in vivo and is capable of auto-acetylation. TcATAT is located in the cytoskeleton and flagella of epimastigotes and colocalizes with acetylated α-tubulin in these structures. We have expressed TcATAT with an HA tag using the inducible vector pTcINDEX-GW in T. cruzi. Over-expression of TcATAT causes increased levels of the alpha tubulin acetylated species, induces morphological and ultrastructural defects, especially in the mitochondrion, and causes a halt in the cell cycle progression of epimastigotes, which is related to an impairment of the kinetoplast division. Finally, as a result of TcATAT over-expression we observed that parasites became more resistant to microtubule depolymerizing drugs. These results support the idea that α-tubulin acetylation levels are finely regulated for the normal progression of T. cruzi cell cycle.
    Keywords:  acetylation; cell division; cytoskeleton; flagella; tubulin (Microtubules)
    DOI:  https://doi.org/10.3389/fcimb.2021.642271
  28. Front Cell Neurosci. 2021 ;15 636017
      Rho-associated coiled-coil containing kinase isoform 2 (ROCK2) is a member of the AGC family of serine/threonine kinases and an extensively studied regulator of actin-mediated cytoskeleton contractility. Over the past decade, new evidence has emerged that suggests ROCK2 regulates autophagy. Recent studies indicate that dysregulation of autophagy contributes to the development of misfolded tau aggregates among entorhinal cortex (EC) excitatory neurons in early Alzheimer's disease (AD). While the accumulation of tau oligomers and fibrils is toxic to neurons, autophagy facilitates the degradation of these pathologic species and represents a major cellular pathway for tau disposal in neurons. ROCK2 is expressed in excitatory neurons and pharmacologic inhibition of ROCK2 can induce autophagy pathways. In this mini-review, we explore potential mechanisms by which ROCK2 mediates autophagy and actin dynamics and discuss how these pathways represent therapeutic avenues for Alzheimer's disease.
    Keywords:  Alzheimer’s disease; ROCK2; autophagy; dendritic spine; mTOR; tau
    DOI:  https://doi.org/10.3389/fncel.2021.636017
  29. J Diabetes Res. 2021 ;2021 9570405
       Objective: Abnormal signaling pathways play a crucial role in the mechanisms of podocyte injury in diabetic nephropathy. They also affect the recovery of podocytes after islet transplantation (IT). However, the specific signaling abnormalities that affect the therapeutic effect of IT on podocytes remains unclear. The purpose of this study was to assess whether the RhoA/ROCK/NF-κB signaling pathway is related to podocyte restoration after IT.
    Methods: A mouse model of diabetic nephropathy was established in vivo using streptozotocin. The mice were then subsequently reared for 4 weeks after islet transplantation to determine the effect of IT. Islet cells, CCG-1423 (RhoA Inhibitor), and fasudil (ROCK inhibitor) were then cocultured with podocytes in vitro to assess their protective effects on podocyte injury induced by high glucose (HG). Protein expression levels of RhoA, ROCK1, synaptopodin, IL-6, and MCP-1 in kidney tissues were then measured using immunohistochemistry and Western blotting techniques.
    Results: Islet transplantation reduced the expression levels of RhoA/ROCK1 and that of related inflammatory factors such as IL-6 and MCP-1 in the kidney podocytes of diabetic nephropathy. In the same line, islet cells reduced the expression of RhoA, ROCK1, and pp65 in immortalized podocytes under high glucose (35.0 mmol/L glucose) conditions.
    Conclusions: Islet transplantation can reverse podocyte injury in diabetes nephropathy by inhibiting the RhoA/ROCK1 signaling pathway. Islet cells have a strong protective effect on podocytes treated with high glucose (35.0 mmol/L glucose). Discovery of signaling pathways affecting podocyte recovery is helpful for individualized efficacy evaluation and targeted therapy of islet transplantation patients.
    DOI:  https://doi.org/10.1155/2021/9570405
  30. Clin Kidney J. 2021 Mar;14(3): 898-908
    REIN registry
       Background: Although end-stage renal disease (ESRD) is frequently used as an outcome marker for primary immunoglobulin A nephropathy (IgAN), the clinical course after reaching ESRD is not well documented. This study examined patients' characteristics and survival in ESRD-related biopsy-proven IgAN in France.
    Methods: French Renal Epidemiology and Information Network Registry data from 2010 to 2014 were used to analyse patients' survival and outcome in incident ESRD patients >16 years of age with biopsy-proven primary IgAN, in comparison with other primary and secondary glomerulonephritis (GN), adult polycystic kidney disease (ADPKD) or diabetes. Multivariable survival analysis was adjusted for age, sex, time on dialysis and comorbidities.
    Results: Among 17 138 incident dialysis patients with ESRD, IgAN (242.8/10 000 dialysis initiation) represents the most common GN related to ESRD during 2010. IgAN patients were the youngest, and had the fewest comorbidities and the highest use of peritoneal dialysis (PD) (17%). In comparison with the haemodialysis group, hazard ratios for death were not different in the preemptive transplantation group [0.46, 95% confidence interval (CI) 0.17-1.28] and in the PD group (0.77, 95% CI 0.44-1.33). Mortality rates in IgAN patients with preemptive transplantation and in those receiving dialysis waiting for transplantation were 2.9% (95% CI 0.0-5.6) and 6.7% (95% CI 0.9-12.3). Mortality rates of ADPKD patients receiving dialysis waiting for transplantation were higher (18%, 95% CI 3.1-30.6).
    Conclusion: IgAN has the best prognosis among primary and secondary GN. IgAN patients receiving dialysis waiting transplantation seem to have a more favourable prognosis than ADPKD patients, who usually comprise the reference population. The underlying reasons for the difference in access treatment modalities should be investigated to improve survival with respect to renal disease.
    Keywords:  IgA nephropathy end-stage renal disease; clinical epidemiology; dialysis modality; glomerulonephritis; mortality; outcomes
    DOI:  https://doi.org/10.1093/ckj/sfaa029
  31. Int J Mol Sci. 2021 Mar 22. pii: 3237. [Epub ahead of print]22(6):
      Currently, retinal pigment epithelium (RPE) transplantation includes sheet and single-cell transplantation, the latter of which includes cell death and may be highly immunogenic, and there are some issues to be improved in single-cell transplantation. Y-27632 is an inhibitor of Rho-associated protein kinase (ROCK), the downstream kinase of Rho. We herein investigated the effect of Y-27632 in vitro on retinal pigment epithelium derived from induced pluripotent stem cells (iPS-RPE cells), and also its effects in vivo on the transplantation of iPS-RPE cell suspensions. As a result, the addition of Y-27632 in vitro showed suppression of apoptosis, promotion of cell adhesion, and higher proliferation and pigmentation of iPS-RPE cells. Y-27632 also increased the viability of the transplant without showing obvious retinal toxicity in human iPS-RPE transplantation into monkey subretinal space in vivo. Therefore, it is possible that ROCK inhibitors can improve the engraftment of iPS-RPE cell suspensions after transplantation.
    Keywords:  ROCK inhibitor; iPS cells; retinal pigment epithelium; transplantation
    DOI:  https://doi.org/10.3390/ijms22063237