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1.
Br J Clin Pharmacol ; 90(6): 1503-1513, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38504437

RESUMO

AIMS: The aim of this study was to characterize the population pharmacokinetics of AZD8233, an antisense oligonucleotide (ASO) that targets the PCSK9 transcript to reduce hepatocyte PCSK9 protein production and plasma levels. AZD8233 utilizes generation 2.5 S-constrained ethyl motif (cET) chemistry and is conjugated to a triantennary N-acetylgalactosamine (GalNAc3) ligand for targeted hepatocyte uptake. METHODS: A non-linear mixed-effect modelling approach utilizing NONMEM software was applied to AZD8233 concentration-time data from 3416 samples in 219 participants from four phase 1-2 studies, one in healthy volunteers (NCT03593785) and three in patients with dyslipidaemia (NCT04155645, NCT04641299 and NCT04823611). RESULTS: The final model described the AZD8233 plasma concentration-time profile from four phase 1-2 studies in healthy volunteers or participants with dyslipidaemia, covering a dose range of 4 to 120 mg. The pharmacokinetics of AZD8233 were adequately described by a two-compartment model with first-order absorption. The supra-proportional increase in maximum plasma concentration (Cmax) across the observed dose range was described by non-linear Michaelis-Menten elimination (maximum elimination rate, 9.9 mg/h [12% relative standard error]; concentration yielding half-maximal elimination rate, 4.8 mg/L [18% relative standard error]). Body weight, sex, estimated glomerular filtration rate and disease status (healthy participant vs. patient with dyslipidaemia) were identified as factors affecting exposure to AZD8233. CONCLUSIONS: Covariate analysis showed body weight to be the main factor affecting exposure to AZD8233, which largely explained the higher Cmax observed in the Asian population relative to non-Asians.


Assuntos
Dislipidemias , Oligonucleotídeos Antissenso , Pró-Proteína Convertase 9 , Humanos , Masculino , Feminino , Pessoa de Meia-Idade , Adulto , Dislipidemias/tratamento farmacológico , Dislipidemias/genética , Dislipidemias/sangue , Oligonucleotídeos Antissenso/farmacocinética , Oligonucleotídeos Antissenso/administração & dosagem , Pró-Proteína Convertase 9/genética , Adulto Jovem , Voluntários Saudáveis , Modelos Biológicos , Idoso , Relação Dose-Resposta a Droga , Adolescente
2.
EMBO J ; 37(15)2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29925518

RESUMO

Polycystic kidney disease (PKD) and other renal ciliopathies are characterized by cysts, inflammation, and fibrosis. Cilia function as signaling centers, but a molecular link to inflammation in the kidney has not been established. Here, we show that cilia in renal epithelia activate chemokine signaling to recruit inflammatory cells. We identify a complex of the ciliary kinase LKB1 and several ciliopathy-related proteins including NPHP1 and PKD1. At homeostasis, this ciliary module suppresses expression of the chemokine CCL2 in tubular epithelial cells. Deletion of LKB1 or PKD1 in mouse renal tubules elevates CCL2 expression in a cell-autonomous manner and results in peritubular accumulation of CCR2+ mononuclear phagocytes, promoting a ciliopathy phenotype. Our findings establish an epithelial organelle, the cilium, as a gatekeeper of tissue immune cell numbers. This represents an unexpected disease mechanism for renal ciliopathies and establishes a new model for how epithelial cells regulate immune cells to affect tissue homeostasis.


Assuntos
Quimiocina CCL2/metabolismo , Cílios/patologia , Doenças Renais Císticas/congênito , Rim Policístico Autossômico Dominante/patologia , Proteína Quinase C/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Quinases Ativadas por AMP , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular , Proteínas do Citoesqueleto , Cães , Células Epiteliais/metabolismo , Feminino , Células HEK293 , Humanos , Doenças Renais Císticas/patologia , Túbulos Renais/citologia , Túbulos Renais/patologia , Macrófagos/metabolismo , Células Madin Darby de Rim Canino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagocitose/fisiologia , Rim Policístico Autossômico Dominante/genética , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Peixe-Zebra
3.
PLoS Pathog ; 16(9): e1008870, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32991634

RESUMO

The two T-box transcription factors T-bet and Eomesodermin (Eomes) are important regulators of cytotoxic lymphocytes (CTLs), such as activated CD8 T cells, which are essential in the fight against intracellular pathogens and tumors. Both transcription factors share a great degree of homology based on sequence analysis and as a result exert partial functional redundancy during viral infection. However, the actual degree of redundancy between T-bet and Eomes remains a matter of debate and is further confounded by their distinct spatiotemporal expression pattern in activated CD8 T cells. To directly investigate the functional overlap of these transcription factors, we generated a new mouse model in which Eomes expression is under the transcriptional control of the endogenous Tbx21 (encoding for T-bet) locus. Applying this model, we demonstrate that the induction of Eomes in lieu of T-bet cannot rescue T-bet deficiency in CD8 T cells during acute lymphocytic choriomeningitis virus (LCMV) infection. We found that the expression of Eomes instead of T-bet was not sufficient for early cell expansion or effector cell differentiation. Finally, we show that imposed expression of Eomes after acute viral infection promotes some features of exhaustion but must act in concert with other factors during chronic viral infection to establish all hallmarks of exhaustion. In summary, our results clearly underline the importance of T-bet in guiding canonical CTL development during acute viral infections.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular/fisiologia , Proteínas com Domínio T/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Proteínas Fetais/metabolismo , Regulação da Expressão Gênica/fisiologia , Interferon gama/metabolismo , Camundongos Transgênicos
4.
BMC Nephrol ; 23(1): 208, 2022 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-35698028

RESUMO

Diabetic kidney disease (DKD) is the leading cause of kidney failure worldwide. Mortality and morbidity associated with DKD are increasing with the global prevalence of type 2 diabetes. Chronic, sub-clinical, non-resolving inflammation contributes to the pathophysiology of renal and cardiovascular disease associated with diabetes. Inflammatory biomarkers correlate with poor renal outcomes and mortality in patients with DKD. Targeting chronic inflammation may therefore offer a route to novel therapeutics for DKD. However, the DKD patient population is highly heterogeneous, with varying etiology, presentation and disease progression. This heterogeneity is a challenge for clinical trials of novel anti-inflammatory therapies. Here, we present a conceptual model of how chronic inflammation affects kidney function in five compartments: immune cell recruitment and activation; filtration; resorption and secretion; extracellular matrix regulation; and perfusion. We believe that the rigorous alignment of pathophysiological insights, appropriate animal models and pathology-specific biomarkers may facilitate a mechanism-based shift from recruiting 'all comers' with DKD to stratification of patients based on the principal compartments of inflammatory disease activity.


Assuntos
Diabetes Mellitus Tipo 2 , Nefropatias Diabéticas , Animais , Biomarcadores , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/tratamento farmacológico , Nefropatias Diabéticas/etiologia , Humanos , Inflamação/complicações , Inflamação/tratamento farmacológico , Rim
5.
PLoS Biol ; 16(8): e2005651, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30080851

RESUMO

Cilia are organelles specialized in movement and signal transduction. The ciliary transient receptor potential ion channel polycystin-2 (TRPP2) controls elementary cilia-mediated physiological functions ranging from male fertility and kidney development to left-right patterning. However, the molecular components translating TRPP2 channel-mediated Ca2+ signals into respective physiological functions are unknown. Here, we show that the Ca2+-regulated mitochondrial ATP-Mg/Pi solute carrier 25 A 25 (SLC25A25) acts downstream of TRPP2 in an evolutionarily conserved metabolic signaling pathway. We identify SLC25A25 as an essential component in this cilia-dependent pathway using a genome-wide forward genetic screen in Drosophila melanogaster, followed by a targeted analysis of SLC25A25 function in zebrafish left-right patterning. Our data suggest that TRPP2 ion channels regulate mitochondrial SLC25A25 transporters via Ca2+ establishing an evolutionarily conserved molecular link between ciliary signaling and mitochondrial metabolism.


Assuntos
Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Cílios/metabolismo , Canais de Cátion TRPP/metabolismo , Animais , Antiporters/metabolismo , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Drosophila melanogaster/metabolismo , Heterozigoto , Humanos , Canais Iônicos/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/metabolismo , Transdução de Sinais , Peixe-Zebra
6.
Kidney Int ; 94(5): 849-851, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30348299

RESUMO

Progressive cyst expansion in autosomal-dominant polycystic kidney disease has been shown to cause regional hypoxia in cystic and pericystic tissue. This results in up-regulation of hypoxia-inducible transcription factor (HIF) 1α in cyst epithelial cells. However, the functional impact of HIF-1α in cystic kidney disease was unknown. A new study has shown that HIF-1α promotes cyst progression in a mouse model of autosomal-dominant polycystic kidney disease.


Assuntos
Doenças Renais Policísticas , Rim Policístico Autossômico Dominante , Animais , Cistos , Células Epiteliais , Subunidade alfa do Fator 1 Induzível por Hipóxia , Camundongos
7.
J Am Soc Nephrol ; 28(8): 2529-2539, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28373276

RESUMO

Hyperinsulinemic hypoglycemia (HI) and congenital polycystic kidney disease (PKD) are rare, genetically heterogeneous disorders. The co-occurrence of these disorders (HIPKD) in 17 children from 11 unrelated families suggested an unrecognized genetic disorder. Whole-genome linkage analysis in five informative families identified a single significant locus on chromosome 16p13.2 (logarithm of odds score 6.5). Sequencing of the coding regions of all linked genes failed to identify biallelic mutations. Instead, we found in all patients a promoter mutation (c.-167G>T) in the phosphomannomutase 2 gene (PMM2), either homozygous or in trans with PMM2 coding mutations. PMM2 encodes a key enzyme in N-glycosylation. Abnormal glycosylation has been associated with PKD, and we found that deglycosylation in cultured pancreatic ß cells altered insulin secretion. Recessive coding mutations in PMM2 cause congenital disorder of glycosylation type 1a (CDG1A), a devastating multisystem disorder with prominent neurologic involvement. Yet our patients did not exhibit the typical clinical or diagnostic features of CDG1A. In vitro, the PMM2 promoter mutation associated with decreased transcriptional activity in patient kidney cells and impaired binding of the transcription factor ZNF143. In silico analysis suggested an important role of ZNF143 for the formation of a chromatin loop including PMM2 We propose that the PMM2 promoter mutation alters tissue-specific chromatin loop formation, with consequent organ-specific deficiency of PMM2 leading to the restricted phenotype of HIPKD. Our findings extend the spectrum of genetic causes for both HI and PKD and provide insights into gene regulation and PMM2 pleiotropy.


Assuntos
Hiperinsulinismo Congênito/complicações , Hiperinsulinismo Congênito/genética , Mutação , Fosfotransferases (Fosfomutases)/genética , Doenças Renais Policísticas/complicações , Doenças Renais Policísticas/genética , Regiões Promotoras Genéticas/genética , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino
8.
Pflugers Arch ; 469(2): 303-311, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27987038

RESUMO

Recent advances in genome editing technologies have enabled the rapid and precise manipulation of genomes, including the targeted introduction, alteration, and removal of genomic sequences. However, respective methods have been described mainly in non-differentiated or haploid cell types. Genome editing of well-differentiated renal epithelial cells has been hampered by a range of technological issues, including optimal design, efficient expression of multiple genome editing constructs, attainable mutation rates, and best screening strategies. Here, we present an easily implementable workflow for the rapid generation of targeted heterozygous and homozygous genomic sequence alterations in renal cells using transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeat (CRISPR) system. We demonstrate the versatility of established protocols by generating novel cellular models for studying autosomal dominant polycystic kidney disease (ADPKD). Furthermore, we show that cell culture-validated genetic modifications can be readily applied to mouse embryonic stem cells (mESCs) for the generation of corresponding mouse models. The described procedure for efficient genome editing can be applied to any cell type to study physiological and pathophysiological functions in the context of precisely engineered genotypes.


Assuntos
Diferenciação Celular/genética , Células Epiteliais/metabolismo , Genoma/genética , Rim/metabolismo , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA Complementar/genética , Células-Tronco Embrionárias/metabolismo , Edição de Genes/métodos , Genótipo , Humanos , Camundongos , Doenças Renais Policísticas/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética
9.
Kidney Int ; 89(4): 949-55, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26924047

RESUMO

Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic cause of end-stage renal disease. The molecular pathogenesis of ADPKD is not completely known, and there is no approved therapy. To date, there is limited knowledge concerning the molecular consequences of specific disease-causing mutations. Here we show that the ADPKD missense variant TRPP2(D511V) greatly reduces TRPP2 protein stability, and that TRPP2(D511V) function can be rescued in vivo by small molecules targeting the TRPP2 degradation pathway. Expression of the TRPP2(D511V) protein was significantly reduced compared to wild-type TRPP2. Inhibition of lysosomal degradation of TRPP2(D511V) by the US Food and Drug Administration (FDA)-approved drug chloroquine strongly increased TRPP2 protein levels in vitro. The validation of these results in vivo requires appropriate animal models. However, there are currently no mouse models harboring human PKD2 missense mutations, and screening for chemical rescue of patient mutations in rodent models is time-consuming and expensive. Therefore, we developed a Drosophila melanogaster model expressing the ortholog of TRPP2(D511V) to test chemical rescue of mutant TRPP2 in vivo. Notably, chloroquine was sufficient to improve the phenotype of flies expressing mutant TRPP2. Thus, this proof-of-concept study highlights the potential of directed therapeutic approaches for ADPKD, and provides a rapid-throughput experimental model to screen PKD2 patient mutations and small molecules in vivo.


Assuntos
Antirreumáticos/uso terapêutico , Cloroquina/uso terapêutico , Rim Policístico Autossômico Dominante/genética , Canais de Cátion TRPP/genética , Animais , Antirreumáticos/farmacologia , Cloroquina/farmacologia , Drosophila melanogaster , Avaliação Pré-Clínica de Medicamentos , Feminino , Células HEK293 , Células HeLa , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Masculino , Mutação de Sentido Incorreto , Rim Policístico Autossômico Dominante/tratamento farmacológico , Rim Policístico Autossômico Dominante/metabolismo , Estabilidade Proteica , Canais de Cátion TRPP/metabolismo
10.
J Biol Chem ; 289(21): 14854-67, 2014 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-24719335

RESUMO

Glycosylation plays a critical role in the biogenesis and function of membrane proteins. Transient receptor potential channel TRPP2 is a nonselective cation channel that is mutated in autosomal dominant polycystic kidney disease. TRPP2 has been shown to be heavily N-glycosylated, but the glycosylation sites and the biological role of N-linked glycosylation have not been investigated. Here we show, using a combination of mass spectrometry and biochemical approaches, that native TRPP2 is glycosylated at five asparagines in the first extracellular loop. Glycosylation is required for the efficient biogenesis of TRPP2 because mutations of the glycosylated asparagines result in strongly decreased protein expression of the ion channel. Wild-type and N-glycosylation-deficient TRPP2 is degraded in lysosomes, as shown by increased TRPP2 protein levels upon chemical inhibition of lysosomal degradation. In addition, using pharmacological and genetic approaches, we demonstrate that glucosidase II (GII) mediates glycan trimming of TRPP2. The non-catalytic ß subunit of glucosidase II (GIIß) is encoded by PRKCSH, one of the genes causing autosomal dominant polycystic liver disease (ADPLD). The impaired GIIß-dependent glucose trimming of TRPP2 glycosylation in ADPLD may explain the decreased TRPP2 protein expression in Prkcsh(-/-) mice and the genetic interaction observed between TRPP2 and PRKCSH in ADPLD. These results highlight the biological importance of N-linked glycosylation and GII-mediated glycan trimming in the control of biogenesis and stability of TRPP2.


Assuntos
Asparagina/metabolismo , Lisossomos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Asparagina/genética , Sítios de Ligação/genética , Western Blotting , Linhagem Celular , Células Cultivadas , Glucosidases/genética , Glucosidases/metabolismo , Glicosilação , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Espectrometria de Massas , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Mutação , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteólise , Piruvato Desidrogenase Quinase de Transferência de Acetil
11.
Handb Exp Pharmacol ; 222: 675-711, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24756726

RESUMO

It has been exciting times since the identification of polycystic kidney disease 1 (PKD1) and PKD2 as the genes mutated in autosomal dominant polycystic kidney disease (ADPKD). Biological roles of the encoded proteins polycystin-1 and TRPP2 have been deduced from phenotypes in ADPKD patients, but recent insights from vertebrate and invertebrate model organisms have significantly expanded our understanding of the physiological functions of these proteins. The identification of additional TRPP (TRPP3 and TRPP5) and polycystin-1-like proteins (PKD1L1, PKD1L2, PKD1L3, and PKDREJ) has added yet another layer of complexity to these fascinating cellular signalling units. TRPP proteins assemble with polycystin-1 family members to form receptor-channel complexes. These protein modules have important biological roles ranging from tubular morphogenesis to determination of left-right asymmetry. The founding members of the polycystin family, TRPP2 and polycystin-1, are a prime example of how studying human disease genes can provide insights into fundamental biological mechanisms using a so-called "reverse translational" approach (from bedside to bench). Here, we discuss the current literature on TRPP ion channels and polycystin-1 family proteins including expression, structure, physical interactions, physiology, and lessons from animal model systems and human disease.


Assuntos
Canais de Cátion TRPP/metabolismo , Animais , Permeabilidade da Membrana Celular , Regulação da Expressão Gênica , Predisposição Genética para Doença , Humanos , Ativação do Canal Iônico , Potenciais da Membrana , Camundongos , Camundongos Knockout , Fenótipo , Conformação Proteica , Transdução de Sinais , Relação Estrutura-Atividade , Canais de Cátion TRPP/química , Canais de Cátion TRPP/deficiência , Canais de Cátion TRPP/genética
12.
Kidney Int Rep ; 9(6): 1876-1891, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38899206

RESUMO

Introduction: Inflammation is a significant contributor to cardiorenal morbidity and mortality in diabetic kidney disease (DKD). The pathophysiological mechanisms linking systemic, subacute inflammation and local, kidney injury-initiated immune maladaptation is partially understood. Methods: Here, we explored the expression of proinflammatory cytokines in patients with DKD; investigated mouse models of type 1 and type 2 diabetes (T2D); evaluated glomerular signaling in vitro; performed post hoc analyses of systemic and urinary markers of inflammation; and initiated a phase 2b clinical study (FRONTIER-1; NCT04170543). Results: Transcriptomic profiling of kidney biopsies from patients with DKD revealed significant glomerular upregulation of interleukin-33 (IL-33). Inhibition of IL-33 signaling reduced glomerular damage and albuminuria in the uninephrectomized db/db mouse model (T2D/DKD). On a cellular level, inhibiting IL-33 improved glomerular endothelial health by decreasing cellular inflammation and reducing release of proinflammatory cytokines. Therefore, FRONTIER-1 was designed to test the safety and efficacy of the IL-33-targeted monoclonal antibody tozorakimab in patients with DKD. So far, 578 patients are enrolled in FRONTIER-1. The baseline inflammation status of participants (N > 146) was assessed in blood and urine. Comparison to independent reference cohorts (N > 200) validated the distribution of urinary tumor necrosis factor receptor 1 (TNFR1) and C-C motif chemokine ligand 2 (CCL2). Treatment with dapagliflozin for 6 weeks did not alter these biomarkers significantly. Conclusion: We show that blocking the IL-33 pathway may mitigate glomerular endothelial inflammation in DKD. The findings from the FRONTIER-1 study will provide valuable insights into the therapeutic potential of IL-33 inhibition in DKD.

13.
Adv Ther ; 41(3): 1168-1183, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38280066

RESUMO

INTRODUCTION: Anemia of chronic kidney disease (CKD) has a high incidence and is associated with many disease conditions. Iron dysmetabolism is an important contributor to anemia in CKD patients. METHODS: ALTAI, a randomized, active-controlled, phase 4 trial, investigated the efficacy of roxadustat versus recombinant human erythropoietin (rHuEPO) on gastrointestinal iron absorption in patients with anemia of CKD (stage 4/5). The primary endpoint was change from baseline to day 15 in gastrointestinal iron absorption (serum iron area under the concentration-time curve; AUC0-3h) following single-dose oral iron. RESULTS: Twenty-five patients with a mean age of 55.1 years were randomized 1:1 to roxadustat (n = 13) or rHuEPO (n = 12). Baseline iron profiles were similar between treatment groups. Change from baseline to day 15 in serum iron AUC0-3h was not statistically significantly different between the roxadustat and rHuEPO groups. Mean (SD) change from baseline in serum iron AUC0-3h was 11.3 (28.2) g × 3 h/dl in the roxadustat group and - 0.3 (9.7) g × 3 h/dl in the rHuEPO group. Roxadustat treatment was associated with decreased hepcidin and also increased transferrin, soluble transferrin receptor, and total iron-binding capacity (TIBC), with nominal significance. The proportion of patients experiencing one or more adverse events was 38.5% when treated with roxadustat and 16.7% with rHuEPO. CONCLUSIONS: The study showed no significant difference between roxadustat and rHuEPO in iron absorption but was underpowered because of recruitment challenges. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT04655027.


Assuntos
Anemia , Insuficiência Renal Crônica , Humanos , Pessoa de Meia-Idade , Anemia/tratamento farmacológico , Anemia/etiologia , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/terapia , Ferro/uso terapêutico , Glicina/efeitos adversos , Isoquinolinas/efeitos adversos , China , Hemoglobinas/análise
14.
Biology (Basel) ; 11(5)2022 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-35625437

RESUMO

Patients with autosomal dominant polycystic kidney disease (ADPKD) and tuberous sclerosis complex (TSC) are born with normal or near-normal kidneys that later develop cysts and prematurely lose function. Both renal cystic diseases appear to be mediated, at least in part, by disease-promoting extracellular vesicles (EVs) that induce genetically intact cells to participate in the renal disease process. We used centrifugation and size exclusion chromatography to isolate the EVs for study. We characterized the EVs using tunable resistive pulse sensing, dynamic light scattering, transmission electron microscopy, and Western blot analysis. We performed EV trafficking studies using a dye approach in both tissue culture and in vivo studies. We have previously reported that loss of the Tsc2 gene significantly increased EV production and here demonstrate that the loss of the Pkd1 gene also significantly increases EV production. Using a cell culture system, we also show that loss of either the Tsc2 or Pkd1 gene results in EVs that exhibit an enhanced uptake by renal epithelial cells and a prolonged half-life. Loss of the primary cilia significantly reduces EV production in renal collecting duct cells. Cells that have a disrupted Pkd1 gene produce EVs that have altered kinetics and a prolonged half-life, possibly impacting the duration of the EV cargo effect on the recipient cell. These results demonstrate the interplay between primary cilia and EVs and support a role for EVs in polycystic kidney disease pathogenesis.

15.
Sci Rep ; 12(1): 18211, 2022 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-36307508

RESUMO

Genome editing tools such as CRISPR/Cas9 enable the rapid and precise manipulation of genomes. CRISPR-based genome editing has greatly simplified the study of gene function in cell lines, but its widespread use has also highlighted challenges of reproducibility. Phenotypic variability among different knockout clones of the same gene is a common problem confounding the establishment of robust genotype-phenotype correlations. Optimized genome editing protocols to enhance reproducibility include measures to reduce off-target effects. However, even if current state-of-the-art protocols are applied phenotypic variability is frequently observed. Here we identify heterogeneity of wild-type cells as an important and often neglected confounding factor in genome-editing experiments. We demonstrate that isolation of individual wild-type clones from an apparently homogenous stable cell line uncovers significant phenotypic differences between clones. Strikingly, we observe hundreds of differentially regulated transcripts (477 up- and 306 downregulated) when comparing two populations of wild-type cells. Furthermore, we show a variety of cellular and biochemical alterations in different wild-type clones in a range that is commonly interpreted as biologically relevant in genome-edited cells. Heterogeneity of wild-type cells thus contributes to variability in genome-edited cells when these are generated through isolation of clones. We show that the generation of monoclonal isogenic wild-type cells prior to genomic manipulation reduces phenotypic variability. We therefore propose to generate matched isogenic control cells prior to genome editing to increase reproducibility.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Sistemas CRISPR-Cas/genética , Reprodutibilidade dos Testes , Edição de Genes/métodos , Linhagem Celular , Células Cultivadas
16.
CPT Pharmacometrics Syst Pharmacol ; 11(12): 1569-1577, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36126230

RESUMO

Here, we show model-informed drug development (MIDD) of a novel antisense oligonucleotide, targeting PCSK9 for treatment of hypocholesteremia. The case study exemplifies use of MIDD to analyze emerging data from an ongoing first-in-human study, utility of the US Food and Drug Administration MIDD pilot program to accelerate timelines, innovative use of competitor data to set biomarker targets, and use of MIDD to optimize sample size and dose selection, as well as to accelerate and de-risk a phase IIb study. The focus of the case-study is on the cross-functional collaboration and other key MIDD enablers that are critical to maximize the value of MIDD, rather than the technical application of MIDD.


Assuntos
Oligonucleotídeos Antissenso , Pró-Proteína Convertase 9 , Humanos , Pró-Proteína Convertase 9/genética , Preparações Farmacêuticas , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Antissenso/uso terapêutico , Desenvolvimento de Medicamentos
17.
Adv Exp Med Biol ; 704: 287-313, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21290302

RESUMO

The founding member of the TRPP family, TRPP2, was identified as one of the disease genes causing autosomal dominant polycystic kidney disease (ADPKD). ADPKD is the most prevalent, potentially lethal, monogenic disorder in humans, with an average incidence of one in 400 to one in 1,000 individuals worldwide. Here we give an overview of TRPP ion channels and Polycystin-1 receptor proteins focusing on more recent studies. We include the Polycystin-1 family since these proteins are functionally linked to TRPP channels.


Assuntos
Canais de Cátion TRPP/fisiologia , Animais , Humanos , Rim Policístico Autossômico Dominante/fisiopatologia
18.
Autophagy ; 17(9): 2384-2400, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-32967521

RESUMO

Mutations in the PKD1 gene result in autosomal dominant polycystic kidney disease (ADPKD), the most common monogenetic cause of end-stage renal disease (ESRD) in humans. Previous reports suggested that PKD1, together with PKD2/polycystin-2, may function as a receptor-cation channel complex at cilia and on intracellular membranes and participate in various signaling pathways to regulate cell survival, proliferation and macroautophagy/autophagy. However, the exact molecular function of PKD1 and PKD2 has remained enigmatic. Here we used Pkd1-deficient mouse inner medullary collecting duct cells (mIMCD3) genetically deleted for Pkd1, and tubular epithelial cells isolated from nephrons of doxycycline-inducible conditional pkd1fl/fl;Pax8rtTA;TetOCre+ knockout mice to show that the lack of Pkd1 caused diminished lysosomal acidification, LAMP degradation and reduced CTSB/cathepsin B processing and activity. This led to an impairment of autophagosomal-lysosomal fusion, a lower delivery of ubiquitinated cargo from multivesicular bodies (MVB)/exosomes to lysosomes and an enhanced secretion of unprocessed CTSB into the extracellular space. The TFEB-dependent lysosomal biogenesis pathway was however unaffected. Pkd1-deficient cells exhibited increased activity of the calcium-dependent CAPN (calpain) proteases, probably due to a higher calcium influx. Consistent with this notion CAPN inhibitors restored lysosomal function, CTSB processing/activity and autophagosomal-lysosomal fusion, and blocked CTSB secretion and LAMP degradation in pkd1 knockout cells. Our data reveal for the first time a lysosomal function of PKD1 which keeps CAPN activity in check and ensures lysosomal integrity and a correct autophagic flux.Abbreviations: acCal: acetyl-calpastatin peptide; ADPKD: autosomal dominant polycystic kidney disease; CI-1: calpain inhibitor-1; CQ: chloroquine; Dox: doxycycline; EV: extracellular vesicles; EXO: exosomes; LAMP1/2: lysosomal-associated membrane protein 1/2; LGALS1/GAL1/galectin-1: lectin, galactose binding, soluble 1; LMP: lysosomal membrane permeabilization; mIMCD3: mouse inner medullary collecting duct cells; MV: microvesicles; MVB: multivesicular bodies; PAX8: paired box 8; PKD1/polycystin-1: polycystin 1, transient receptor potential channel interacting; PKD2/polycystin-2: polycystin 2, transient receptor potential cation channel; Tet: tetracycline; TFEB: transcription factor EB; VFM: vesicle-free medium; WT: wild-type.


Assuntos
Calpaína , Canais de Cátion TRPP , Animais , Autofagia , Calpaína/metabolismo , Lisossomos/metabolismo , Camundongos , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/metabolismo
19.
Sci Rep ; 11(1): 15139, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301992

RESUMO

Autosomal dominant polycystic kidney disease (ADPKD) affects more than 12 million people worldwide. Mutations in PKD1 and PKD2 cause cyst formation through unknown mechanisms. To unravel the pathogenic mechanisms in ADPKD, multiple studies have investigated transcriptional mis-regulation in cystic kidneys from patients and mouse models, and numerous dysregulated genes and pathways have been described. Yet, the concordance between studies has been rather limited. Furthermore, the cellular and genetic diversity in cystic kidneys has hampered the identification of mis-expressed genes in kidney epithelial cells with homozygous PKD mutations, which are critical to identify polycystin-dependent pathways. Here we performed transcriptomic analyses of Pkd1- and Pkd2-deficient mIMCD3 kidney epithelial cells followed by a meta-analysis to integrate all published ADPKD transcriptomic data sets. Based on the hypothesis that Pkd1 and Pkd2 operate in a common pathway, we first determined transcripts that are differentially regulated by both genes. RNA sequencing of genome-edited ADPKD kidney epithelial cells identified 178 genes that are concordantly regulated by Pkd1 and Pkd2. Subsequent integration of existing transcriptomic studies confirmed 31 previously described genes and identified 61 novel genes regulated by Pkd1 and Pkd2. Cluster analyses then linked Pkd1 and Pkd2 to mRNA splicing, specific factors of epithelial mesenchymal transition, post-translational protein modification and epithelial cell differentiation, including CD34, CDH2, CSF2RA, DLX5, HOXC9, PIK3R1, PLCB1 and TLR6. Taken together, this model-based integrative analysis of transcriptomic alterations in ADPKD annotated a conserved core transcriptomic profile and identified novel candidate genes for further experimental studies.


Assuntos
Células Epiteliais/patologia , Epitélio/patologia , Rim Policístico Autossômico Dominante/genética , Transcrição Gênica/genética , Animais , Células Cultivadas , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/genética , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , Doenças Renais Policísticas/genética , Doenças Renais Policísticas/patologia , Rim Policístico Autossômico Dominante/patologia , Transdução de Sinais/genética , Canais de Cátion TRPP/genética
20.
Sci Rep ; 11(1): 16532, 2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34400685

RESUMO

Recent advances in induced pluripotent stem cells (iPSCs), genome editing technologies and 3D organoid model systems highlight opportunities to develop new in vitro human disease models to serve drug discovery programs. An ideal disease model would accurately recapitulate the relevant disease phenotype and provide a scalable platform for drug and genetic screening studies. Kidney organoids offer a high cellular complexity that may provide greater insights than conventional single-cell type cell culture models. However, genetic manipulation of the kidney organoids requires prior generation of genetically modified clonal lines, which is a time and labor consuming procedure. Here, we present a methodology for direct differentiation of the CRISPR-targeted cell pools, using a doxycycline-inducible Cas9 expressing hiPSC line for high efficiency editing to eliminate the laborious clonal line generation steps. We demonstrate the versatile use of genetically engineered kidney organoids by targeting the autosomal dominant polycystic kidney disease (ADPKD) genes: PKD1 and PKD2. Direct differentiation of the respective knockout pool populations into kidney organoids resulted in the formation of cyst-like structures in the tubular compartment. Our findings demonstrated that we can achieve > 80% editing efficiency in the iPSC pool population which resulted in a reliable 3D organoid model of ADPKD. The described methodology may provide a platform for rapid target validation in the context of disease modeling.


Assuntos
Sistemas CRISPR-Cas , Descoberta de Drogas/métodos , Edição de Genes/métodos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Terapia de Alvo Molecular , Rim Policístico Autossômico Dominante/genética , Células A549 , Animais , Diferenciação Celular , Células Cultivadas , Doxiciclina/farmacologia , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Rim/citologia , Organoides/efeitos dos fármacos , Rim Policístico Autossômico Dominante/tratamento farmacológico , RNA Guia de Cinetoplastídeos/genética , Suínos , Canais de Cátion TRPP/genética
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