RESUMO
Our understanding of the complex pathophysiology of Heart failure with preserved ejection fraction (HFpEF) is limited by the lack of a robust in vivo model. Existing in-vivo models attempt to reproduce the four main phenotypes of HFpEF; ageing, obesity, diabetes mellitus and hypertension. To date, there is no in vivo model that represents all the haemodynamic characteristics of HFpEF, and only a few have proven to be reliable for the preclinical evaluation of potentially new therapeutic targets. HFpEF accounts for 50% of all the heart failure cases and its incidence is on the rise, posing a huge economic burden on the health system. Patients with HFpEF have limited therapeutic options available. The inadequate effectiveness of current pharmaceutical therapeutics for HFpEF has prompted the development of device-based treatments that target the hemodynamic changes to reduce the symptoms of HFpEF. However, despite the potential of device-based solutions to treat HFpEF, most of these therapies are still in the developmental stage and a relevant HFpEF in vivo model will surely expedite their development process. This review article outlines the major limitations of the current large in-vivo models in use while discussing how these designs have helped in the development of therapy devices for the treatment of HFpEF.
Assuntos
Diabetes Mellitus , Insuficiência Cardíaca , Hipertensão , Animais , Humanos , Volume Sistólico/fisiologia , Modelos AnimaisRESUMO
SIGNIFICANCE STATEMENT: CKD is accompanied by abnormal inflammation, which contributes to progressive loss of functional renal tissue and accelerated cardiovascular disease. Although studies have documented that dysregulation of monocyte maturation and function is associated with CKD and its complications, it is not well characterized. This study reveals that a distinctive human monocyte subtype with high propensity for releasing proinflammatory mediators and activating endothelial cells is increased in adults with CKD compared with adults with high cardiovascular risk and normal kidney function. It also demonstrates that human monocyte adhesion to endothelial layers and responses to specific inflammatory migration signals are enhanced in CKD. These findings offer insights into the mechanisms of CKD-associated intravascular and localized inflammation and may suggest potential targets for therapeutic interventions. BACKGROUND: Cardiovascular disease (CVD) in patients with CKD is associated with increased circulating intermediate monocytes (IMs). Dysregulation of monocyte maturation and function is associated with CKD and its complications, but it is incompletely characterized. METHODS: To explore monocyte repertoire abnormalities in CKD, we studied properties of monocyte subpopulations, including IM subpopulations distinguished by HLA-DR expression level, in individuals with or without CKD. Using flow cytometry, we profiled monocyte populations in blood samples from adults with CKD, healthy volunteers (HVs), and patient controls (PCs) with high CVD risk. Monocyte subpopulations were also derived from single-cell RNA-sequencing profiles of paired blood and biopsy samples from kidney transplant recipients. We quantified intracellular cytokine production, migration, and endothelial adhesion in ex vivo assays of PBMCs. RESULTS: Of four predefined blood monocyte subpopulations, only HLA-DR hi IMs were increased in individuals with CKD compared with HVs and PCs. In HVs and patients with CKD, LPS-stimulated HLA-DR hi IMs isolated from blood produced higher amounts of TNF and IL-1 ß than other monocyte populations. Single-cell analysis revealed four monocyte clusters common to blood and kidneys, including an HLA-DR hi IM-like cluster that was enriched in kidneys versus blood. Migration toward CCL5 and CX3CL1 and adhesion to primary endothelial cell layers were increased in monocyte subpopulations in individuals with CKD compared with HVs. Monocyte adhesion to endothelial cells was partly dependent on CX3CR1/CX3CL1 interaction. CONCLUSIONS: CKD is associated with an increased number of a distinctive proinflammatory IM subpopulation and abnormalities of monocyte migration and endothelial adhesion. Dysregulated monocyte maturation and function may represent targetable factors contributing to accelerated CVD in CKD.
Assuntos
Doenças Cardiovasculares , Insuficiência Renal Crônica , Adulto , Humanos , Monócitos , Células Endoteliais/metabolismo , Doenças Cardiovasculares/etiologia , Antígenos HLA-DR , Inflamação/patologiaRESUMO
Mechanical circulatory support (MCS) devices, such as left ventricular assist devices (LVADs) are very useful in improving outcomes in patients with advanced-stage heart failure. Despite recent advances in LVAD development, pump thrombosis is one of the most severe adverse events caused by LVADs. The contact of blood with artificial materials of LVAD pumps and cannulas triggers the coagulation cascade. Heat spots, for example, produced by mechanical bearings are often subjected to thrombus build-up when low-flow situations impair washout and thus the necessary cooling does not happen. The formation of thrombus in an LVAD may compromise its function, causing a drop in flow and pumping power leading to failure of the LVAD, if left unattended. If a clot becomes dislodged and circulates in the bloodstream, it may disturb the flow or occlude the blood vessels in vital organs and cause internal damage that could be fatal, for example, ischemic stroke. That is why patients with LVADs are on anti-coagulant medication. However, the anti-coagulants can cause a set of issues for the patient-an example of gastrointestinal (GI) bleeding is given in illustration. On account of this, these devices are only used as a last resort in clinical practice. It is, therefore, necessary to develop devices with better mechanics of blood flow, performance and hemocompatibility. This paper discusses the development of LVADs through landmark clinical trials in detail and describes the evolution of device design to reduce the risk of pump thrombosis and achieve better hemocompatibility. Whilst driveline infection, right heart failure and arrhythmias have been recognised as LVAD-related complications, this paper focuses on complications related to pump thrombosis, especially blood coagulopathy in detail and potential strategies to mitigate this complication. Furthermore, it also discusses the LVAD implantation techniques and their anatomical challenges.
Assuntos
Insuficiência Cardíaca , Coração Auxiliar , Trombose , Humanos , Coração Auxiliar/efeitos adversos , Trombose/etiologia , Trombose/prevenção & controle , Insuficiência Cardíaca/terapiaRESUMO
BACKGROUND: Cytomegalovirus (CMV) and BK polyoma virus (BKV) infection following kidney transplantation have been associated with allograft dysfunction and allograft loss. Reduction in immunosuppression is a mainstay of management yet has been associated with increased risk of rejection. According to international consensus guidelines, one approach to management of these viral infections is to discontinue the antimetabolite. Little is known surrounding long-term outcomes in these patients, and it remains unclear if consideration should be given to resuming the antimetabolite as variable re-escalation strategies have been reported. The objective was to describe episodes of rejection and identify risk factors for rejection following antimetabolite withdrawal after CMV or BKV DNAemia in kidney transplant recipients. METHODS: This single-center, retrospective review evaluated adult kidney transplant recipients with a serum CMV or BKV DNA PCR ≥500 copies/ml who underwent antimetabolite discontinuation. The primary outcome assessed was the incidence of biopsy-proven acute rejection (BPAR). RESULTS: One hundred fifty-nine patients were included. Overall, 14 patients (8.8%) experienced BPAR at a median of 1.6 years after antimetabolite discontinuation. Compared to CMV, discontinuation after BKV DNAemia was associated with a higher incidence of BPAR. Characteristics observed more frequently in patients with BPAR included younger age, female sex, higher initial viral load, and development of de novo donor-specific antibody (DSA). CONCLUSION: These findings suggest that antimetabolite discontinuation after CMV or BKV DNAemia in kidney transplant recipients is a reasonable and safe approach. Further prospective studies investigating optimal immunosuppression management following CMV or BKV DNAemia in kidney transplant recipients are warranted.
Assuntos
Vírus BK , Infecções por Citomegalovirus , Transplante de Rim , Infecções por Polyomavirus , Infecções Tumorais por Vírus , Adulto , Humanos , Feminino , Citomegalovirus/genética , Transplante de Rim/efeitos adversos , Infecções por Citomegalovirus/tratamento farmacológico , Infecções por Citomegalovirus/epidemiologia , Infecções por Citomegalovirus/etiologia , Estudos Prospectivos , Antimetabólitos , Imunossupressores/efeitos adversos , Infecções Tumorais por Vírus/complicações , TransplantadosRESUMO
Chimeric antigen receptor T cells (CAR-T) are genetically modified T cells with a chimeric antigen receptor directed against a specific tumor-associated antigen like CD19 in lymphoma. CAR-T cells have shown encouraging activity against recurrent and refractory diffuse large B cell lymphomas (DLBCL). However concurrent use of immunosuppressive agents was prohibited in most CAR-T trials effectively excluding patients with prior solid organ transplantation (SOT) and posttransplant lymphoproliferative disorders (PTLD). We report the outcomes for three patients with PTLD refractory to immunochemotherapy 10-20 years after SOT who received CAR-T therapy between January 2018 and December 2019. One patient had an orthotopic heart transplant, the second had a deceased donor kidney transplant, and the third had a pancreas after kidney transplant (PAK). All patients developed complications of CAR-T therapy such as cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome, and acute kidney injury requiring renal replacement therapy in the two out of three patients. All patients expired after withdrawal of care due to lack of response to CAR-T therapy. In addition, the PAK patient developed acute pancreatitis after CAR-T therapy. This case series identifies the challenges of using CAR-T therapy to manage refractory PTLD in SOT recipients and its possible complications.
Assuntos
Transtornos Linfoproliferativos , Transplante de Órgãos , Pancreatite , Receptores de Antígenos Quiméricos , Doença Aguda , Humanos , Transtornos Linfoproliferativos/etiologia , Transtornos Linfoproliferativos/terapia , Transplante de Órgãos/efeitos adversosRESUMO
BACKGROUND: In solid organ transplantation, donor-derived immune cells are assumed to decline with time after surgery. Whether donor leukocytes persist within kidney transplants or play any role in rejection is unknown, however, in part because of limited techniques for distinguishing recipient from donor cells. METHODS: Whole-exome sequencing of donor and recipient DNA and single-cell RNA sequencing (scRNA-seq) of five human kidney transplant biopsy cores distinguished immune cell contributions from both participants. DNA-sequence comparisons used single nucleotide variants (SNVs) identified in the exome sequences across all samples. RESULTS: Analysis of expressed SNVs in the scRNA-seq data set distinguished recipient versus donor origin for all 81,139 cells examined. The leukocyte donor/recipient ratio varied with rejection status for macrophages and with time post-transplant for lymphocytes. Recipient macrophages displayed inflammatory activation whereas donor macrophages demonstrated antigen presentation and complement signaling. Recipient-origin T cells expressed cytotoxic and proinflammatory genes consistent with an effector cell phenotype, whereas donor-origin T cells appeared quiescent, expressing oxidative phosphorylation genes. Finally, both donor and recipient T cell clones within the rejecting kidney suggested lymphoid aggregation. The results indicate that donor-origin macrophages and T cells have distinct transcriptional profiles compared with their recipient counterparts, and that donor macrophages can persist for years post-transplantation. CONCLUSIONS: Analysis of single nucleotide variants and their expression in single cells provides a powerful novel approach to accurately define leukocyte chimerism in a complex organ such as a transplanted kidney, coupled with the ability to examine transcriptional profiles at single-cell resolution.
Assuntos
Rejeição de Enxerto/imunologia , Transplante de Rim/efeitos adversos , Subpopulações de Linfócitos/imunologia , Análise de Sequência de RNA , Análise de Célula Única , Quimerismo , Variação Genética , Humanos , Doadores de Tecidos , Transplante HomólogoRESUMO
Malignancy after solid organ transplant is a common occurrence that is associated with increased morbidity and mortality. Literature in the general diabetic population has identified possible antineoplastic properties of metformin. This retrospective study aims to determine if metformin results in a malignancy risk reduction in a cohort of diabetic kidney, liver, and heart transplant recipients. The population included transplant recipients without use of metformin at any time point (DMO arm, n = 147) and those with use of metformin (DMM arm, n = 172); the two arms were matched based on organ type and transplant date prior to application of exclusion criteria. Recipients with prior malignancy, malignancy before diabetes diagnosis, and metformin duration <30 days were excluded. The primary endpoint of malignancy first occurrence post-transplant was not found to be statistically significant at 1, 5, 10, and 15 years. In the subgroup of heart transplant recipients, there was a significant reduction in malignancy at 15 years post-transplant. Older age and Caucasian race were identified as significant risk factors for malignancy, while never smoker was a protective factor. Metformin use in this solid organ transplant cohort was not found to significantly reduce malignancy risk compared to use of other anti-diabetic agents.
Assuntos
Metformina , Neoplasias , Transplante de Órgãos , Idoso , Humanos , Metformina/uso terapêutico , Neoplasias/epidemiologia , Neoplasias/etiologia , Transplante de Órgãos/efeitos adversos , Estudos Retrospectivos , Fatores de Risco , TransplantadosRESUMO
Microvascular injury is associated with accelerated kidney transplant dysfunction and allograft failure. Molecular pathology can identify new mechanisms of microvascular injury while improving on the diagnostic and prognostic capabilities of traditional histology. We conducted a case-control study of archived kidney biopsy specimens stored up to 10 years with microvascular injury (n = 50) compared with biopsy specimens without histologic injury (n = 45) from patients of similar age, race, and sex. We measured WNT gene expression with a multiplex quantification platform by using digital barcoding, given the importance of WNT reactivation to the response to wounding in the kidney microvasculature and other compartments. Of 210 genes from a commercial WNT panel, 71 were associated with microvascular injury and 79 were associated with allograft failure, with considerable overlap of genes between each set. Molecular pathology identified 46 biopsy specimens with molecular evidence of microvascular injury; 18 (39%) were either C4d negative, donor-specific antibody negative, or had no microvascular injury by histology. The majority of cases with molecular evidence of microvascular injury had poor long-term outcomes. We identified novel WNT pathway genes associated with microvascular injury and allograft failure in residual clinical biopsy specimens obtained up to 10 years earlier. Further mechanistic studies may identify the WNT pathway as a new diagnostic and therapeutic target.
Assuntos
Rejeição de Enxerto/diagnóstico , Isoanticorpos/efeitos adversos , Falência Renal Crônica/cirurgia , Transplante de Rim/efeitos adversos , Microvasos/patologia , Complicações Pós-Operatórias/diagnóstico , Via de Sinalização Wnt , Biomarcadores/metabolismo , Estudos de Casos e Controles , Estudos Transversais , Feminino , Seguimentos , Rejeição de Enxerto/etiologia , Rejeição de Enxerto/metabolismo , Sobrevivência de Enxerto , Humanos , Estudos Longitudinais , Masculino , Microvasos/lesões , Microvasos/metabolismo , Pessoa de Meia-Idade , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/metabolismo , Prognóstico , Fatores de RiscoRESUMO
Background Single-cell genomics techniques are revolutionizing our ability to characterize complex tissues. By contrast, the techniques used to analyze renal biopsy specimens have changed little over several decades. We tested the hypothesis that single-cell RNA-sequencing can comprehensively describe cell types and states in a human kidney biopsy specimen.Methods We generated 8746 single-cell transcriptomes from a healthy adult kidney and a single kidney transplant biopsy core by single-cell RNA-sequencing. Unsupervised clustering analysis of the biopsy specimen was performed to identify 16 distinct cell types, including all of the major immune cell types and most native kidney cell types, in this biopsy specimen, for which the histologic read was mixed rejection.Results Monocytes formed two subclusters representing a nonclassical CD16+ group and a classic CD16- group expressing dendritic cell maturation markers. The presence of both monocyte cell subtypes was validated by staining of independent transplant biopsy specimens. Comparison of healthy kidney epithelial transcriptomes with biopsy specimen counterparts identified novel segment-specific proinflammatory responses in rejection. Endothelial cells formed three distinct subclusters: resting cells and two activated endothelial cell groups. One activated endothelial cell group expressed Fc receptor pathway activation and Ig internalization genes, consistent with the pathologic diagnosis of antibody-mediated rejection. We mapped previously defined genes that associate with rejection outcomes to single cell types and generated a searchable online gene expression database.Conclusions We present the first step toward incorporation of single-cell transcriptomics into kidney biopsy specimen interpretation, describe a heterogeneous immune response in mixed rejection, and provide a searchable resource for the scientific community.
Assuntos
Falência Renal Crônica/genética , Transplante de Rim/métodos , Rim/citologia , Rim/patologia , Transcriptoma/genética , Aloenxertos , Biomarcadores/análise , Biópsia por Agulha , Comunicação Celular , Análise por Conglomerados , Perfilação da Expressão Gênica/métodos , Rejeição de Enxerto/genética , Rejeição de Enxerto/imunologia , Humanos , Imuno-Histoquímica , Falência Renal Crônica/patologia , Falência Renal Crônica/cirurgia , Masculino , Valores de Referência , Análise de Sequência de RNA , Adulto JovemRESUMO
Pierson syndrome is a congenital nephrotic syndrome with eye and neurologic defects caused by mutations in laminin ß2 (LAMB2), a major component of the glomerular basement membrane (GBM). Pathogenic missense mutations in human LAMB2 cluster in or near the laminin amino-terminal (LN) domain, a domain required for extracellular polymerization of laminin trimers and basement membrane scaffolding. Here, we investigated an LN domain missense mutation, LAMB2-S80R, which was discovered in a patient with Pierson syndrome and unusually late onset of proteinuria. Biochemical data indicated that this mutation impairs laminin polymerization, which we hypothesized to be the cause of the patient's nephrotic syndrome. Testing this hypothesis in genetically altered mice showed that the corresponding amino acid change (LAMB2-S83R) alone is not pathogenic. However, expression of LAMB2-S83R significantly increased the rate of progression to kidney failure in a Col4a3-/- mouse model of autosomal recessive Alport syndrome and increased proteinuria in Col4a5+/- females that exhibit a mild form of X-linked Alport syndrome due to mosaic deposition of collagen α3α4α5(IV) in the GBM. Collectively, these data show the pathogenicity of LAMB2-S80R and provide the first evidence of genetic modification of Alport phenotypes by variation in another GBM component. This finding could help explain the wide range of Alport syndrome onset and severity observed in patients with Alport syndrome, even for family members who share the same COL4 mutation. Our results also show the complexities of using model organisms to investigate genetic variants suspected of being pathogenic in humans.
Assuntos
Anormalidades Múltiplas/genética , Anormalidades do Olho/genética , Falência Renal Crônica/genética , Laminina/genética , Nefrite Hereditária/genética , Nefrite Hereditária/metabolismo , Síndrome Nefrótica/genética , Proteinúria/genética , Distúrbios Pupilares/genética , Animais , Autoantígenos/genética , Colágeno Tipo IV/genética , Modelos Animais de Doenças , Progressão da Doença , Anormalidades do Olho/complicações , Feminino , Membrana Basal Glomerular/metabolismo , Humanos , Laminina/metabolismo , Camundongos , Camundongos Transgênicos , Mutação de Sentido Incorreto , Síndromes Miastênicas Congênitas , Nefrite Hereditária/patologia , Síndrome Nefrótica/complicações , Fenótipo , Polimerização , Distúrbios Pupilares/complicaçõesRESUMO
Detecting acute rejection in kidney transplantation has been traditionally done using histological analysis of invasive allograft biopsies, but this method carries a risk and is not perfect. Transplant professionals have been working to develop more accurate or less invasive biomarkers that can predict acute rejection or subsequent worse allograft survival. These biomarkers can use tissue, blood or urine as a source. They can comprise individual molecules or panels, singly or in combination, across different components or pathways of the immune system. This review highlights the most recent evidence for biomarker efficacy, especially from multicenter trials.
Assuntos
Rejeição de Enxerto/diagnóstico , Falência Renal Crônica/cirurgia , Transplante de Rim/efeitos adversos , Aloenxertos/patologia , Biomarcadores/análise , Biópsia/efeitos adversos , Ensaios Clínicos como Assunto , Reações Falso-Negativas , Reações Falso-Positivas , Rejeição de Enxerto/sangue , Rejeição de Enxerto/patologia , Rejeição de Enxerto/urina , Sobrevivência de Enxerto , Humanos , Rim/patologia , Valor Preditivo dos Testes , Transplante Homólogo/efeitos adversosRESUMO
BACKGROUND: Many COL4A5 splice region variants have been described in patients with X-linked Alport syndrome, but few have been confirmed by functional analysis to actually cause defective splicing. We sought to demonstrate that a novel COL4A5 splice region variant in a family with Alport syndrome is pathogenic using functional studies. We also describe an alternative method of diagnosis. METHODS: Targeted next-generation sequencing results of an individual with Alport syndrome were analyzed and the results confirmed by Sanger sequencing in family members. A splicing reporter minigene assay was used to examine the variant's effect on splicing in transfected cells. Plucked hair follicles from patients and controls were examined for collagen IV proteins using immunofluorescence microscopy. RESULTS: A novel splice region mutation in COL4A5, c.1780-6T>G, was identified and segregated with disease in this family. This variant caused frequent skipping of exon 25, resulting in a frameshift and truncation of collagen α5(IV) protein. We also developed and validated a new approach to characterize the expression of collagen α5(IV) protein in the basement membranes of plucked hair follicles. Using this approach we demonstrated reduced collagen α5(IV) protein in affected male and female individuals in this family, supporting frequent failure of normal splicing. CONCLUSIONS: Differing normal to abnormal transcript ratios in affected individuals carrying splice region variants may contribute to variable disease severity observed in Alport families. Examination of plucked hair follicles in suspected X-linked Alport syndrome patients may offer a less invasive alternative method of diagnosis and serve as a pathogenicity test for COL4A5 variants of uncertain significance.
Assuntos
Colágeno Tipo IV/genética , Folículo Piloso/patologia , Nefrite Hereditária/diagnóstico , Nefrite Hereditária/genética , Adolescente , Membrana Basal/patologia , Criança , Éxons/genética , Feminino , Testes Genéticos , Taxa de Filtração Glomerular , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Microscopia de Fluorescência , Mutação , Linhagem , Fenótipo , Isoformas de Proteínas/genética , Splicing de RNA/genética , Análise de Sequência de RNA , Adulto JovemRESUMO
FSGS is a clinical disorder characterized by focal scarring of the glomerular capillary tuft, podocyte injury, and nephrotic syndrome. Although idiopathic forms of FSGS predominate, recent insights into the molecular and genetic causes of FSGS have enhanced our understanding of disease pathogenesis. Here, we report a novel missense mutation of the transcriptional regulator Wilms' Tumor 1 (WT1) as the cause of nonsyndromic, autosomal dominant FSGS in two Northern European kindreds from the United States. We performed sequential genome-wide linkage analysis and whole-exome sequencing to evaluate participants from family DUK6524. Subsequently, whole-exome sequencing and direct sequencing were performed on proband DNA from family DUK6975. We identified multiple suggestive loci on chromosomes 6, 11, and 13 in family DUK6524 and identified a segregating missense mutation (R458Q) in WT1 isoform D as the cause of FSGS in this family. The identical mutation was found in family DUK6975. The R458Q mutation was not found in 1600 control chromosomes and was predicted as damaging by in silico simulation. We depleted wt1a in zebrafish embryos and observed glomerular injury and filtration defects, both of which were rescued with wild-type but not mutant human WT1D mRNA. Finally, we explored the subcellular mechanism of the mutation in vitro. WT1(R458Q) overexpression significantly downregulated nephrin and synaptopodin expression, promoted apoptosis in HEK293 cells and impaired focal contact formation in podocytes. Taken together, these data suggest that the WT1(R458Q) mutation alters the regulation of podocyte homeostasis and causes nonsyndromic FSGS.
Assuntos
Glomerulosclerose Segmentar e Focal/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas WT1/genética , Adolescente , Adulto , Animais , Movimento Celular , Sobrevivência Celular , Exoma , Feminino , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Ligação Genética , Glomerulosclerose Segmentar e Focal/metabolismo , Células HEK293 , Humanos , Masculino , Mutação de Sentido Incorreto , Nefrose/etiologia , Nefrose/metabolismo , Podócitos/fisiologia , Análise de Sequência de DNA , Proteínas WT1/deficiência , Adulto Jovem , Peixe-Zebra , Proteínas de Peixe-Zebra/deficiênciaRESUMO
The emerging role of the transient receptor potential cation channel isotype 6 (TRPC6) as a central contributor to various pathological processes affecting podocytes has generated interest in the development of therapeutics to modulate its function. Recent insights into the regulation of TRPC6 have revealed PKG as a potent negative modulator of TRPC6 conductance and associated signaling via its phosphorylation at two highly conserved amino acid residues: Thr(69)/Thr(70) (Thr(69) in mice and Thr(70) in humans) and Ser(321)/Ser(322) (Ser(321) in mice and Ser(322) in humans). Here, we tested the role of PKG in modulating TRPC6-dependent responses in primary and conditionally immortalized mouse podocytes. TRPC6 was phosphorylated at Thr(69) in nonstimulated podocytes, but this declined upon ANG II stimulation or overexpression of constitutively active calcineurin phosphatase. ANG II induced podocyte motility in an in vitro wound assay, and this was reduced 30-60% in cells overexpressing a phosphomimetic mutant TRPC6 (TRPC6T70E/S322E) or activated PKG (P < 0.05). Pretreatment of podocytes with the PKG agonists S-nitroso-N-acetyl-dl-penicillamine (nitric oxide donor), 8-bromo-cGMP, Bay 41-2772 (soluble guanylate cyclase activator), or phosphodiesterase 5 (PDE5) inhibitor 4-{[3',4'-(methylenedioxy)benzyl]amino}[7]-6-methoxyquinazoline attenuated ANG II-induced Thr(69) dephosphorylation and also inhibited TRPC6-dependent podocyte motility by 30-60%. These data reveal that PKG activation strategies, including PDE5 inhibition, ameliorate ANG II-induced podocyte dysmotility by targeting TRPC6 in podocytes, highlighting the potential therapeutic utility of these approaches to treat hyperactive TRPC6-dependent glomerular disease.
Assuntos
Angiotensina II/farmacologia , Movimento Celular/efeitos dos fármacos , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Regulação para Baixo/efeitos dos fármacos , Inibidores da Fosfodiesterase 5/farmacologia , Podócitos/metabolismo , Canais de Cátion TRPC/metabolismo , Animais , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Células Cultivadas , Regulação para Baixo/fisiologia , Células HEK293 , Humanos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos , Modelos Animais , Fatores de Transcrição NFATC/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Podócitos/citologia , Podócitos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Canal de Cátion TRPC6RESUMO
Focal segmental glomerulosclerosis (FSGS) is a histological lesion with many causes, including inherited genetic defects, with significant proteinuria being the predominant clinical finding at presentation. Mutations in COL4A3 and COL4A4 are known to cause Alport syndrome (AS), thin basement membrane nephropathy, and to result in pathognomonic glomerular basement membrane (GBM) findings. Secondary FSGS is known to develop in classic AS at later stages of the disease. Here, we present seven families with rare or novel variants in COL4A3 or COL4A4 (six with single and one with two heterozygous variants) from a cohort of 70 families with a diagnosis of hereditary FSGS. The predominant clinical finding at diagnosis was proteinuria associated with hematuria. In all seven families, there were individuals with nephrotic-range proteinuria with histologic features of FSGS by light microscopy. In one family, electron microscopy showed thin GBM, but four other families had variable findings inconsistent with classical Alport nephritis. There was no recurrence of disease after kidney transplantation. Families with COL4A3 and COL4A4 variants that segregated with disease represent 10% of our cohort. Thus, COL4A3 and COL4A4 variants should be considered in the interpretation of next-generation sequencing data from such patients. Furthermore, this study illustrates the power of molecular genetic diagnostics in the clarification of renal phenotypes.
Assuntos
Autoantígenos/genética , Colágeno Tipo IV/genética , Glomerulosclerose Segmentar e Focal/genética , Adolescente , Adulto , Criança , Análise Mutacional de DNA , Exoma , Feminino , Testes Genéticos , Genótipo , Membrana Basal Glomerular/ultraestrutura , Glomerulosclerose Segmentar e Focal/complicações , Glomerulosclerose Segmentar e Focal/patologia , Perda Auditiva/genética , Hematúria/etiologia , Humanos , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Fenótipo , Podócitos/ultraestrutura , Proteinúria/etiologia , Adulto JovemAssuntos
Rejeição de Enxerto/prevenção & controle , Transplante de Rim , Polímeros/administração & dosagem , Tacrolimo/administração & dosagem , Adulto , Cátions , Função Retardada do Enxerto/prevenção & controle , Nefropatias Diabéticas/complicações , Nefropatias Diabéticas/cirurgia , Feminino , Glomerulonefrite por IGA/complicações , Glomerulonefrite por IGA/cirurgia , Humanos , Hiperpotassemia/tratamento farmacológico , Imunossupressores , Falência Renal Crônica/cirurgia , Masculino , Pessoa de Meia-Idade , Segurança do Paciente , Transplantados , Resultado do TratamentoRESUMO
BACKGROUND: Rejection requires cell-cell contact involving immune cells. Inferring the transcriptional programs of cell-cell interactions from single-cell RNA-sequencing (scRNA-seq) data is challenging as spatial information is lost. METHODS: We combined a CD45 pos enrichment strategy with Cellular Indexing of Transcriptomes and Epitopes by sequencing based quantification of leukocyte surface proteins to analyze cell-cell interactions in 11 human kidney transplant biopsies encompassing a spectrum of rejection diagnoses. scRNA-seq was performed using the 10X Genomics platform. We applied the sequencing physically interacting cells computational method to deconvolute the transcriptional profiles of heterotypic physically interacting cells. RESULTS: The 11 human allograft biopsies generated 31 203 high-quality single-cell libraries. Clustering was further refined by combining Cellular Indexing of Transcriptomes and Epitopes by sequencing data from 6 different leukocyte-specific surface proteins. Three of 6 doublet clusters were identified as physically interacting cell complexes; macrophages or dendritic cells bound to B cells or plasma cells; natural killer (NK) or T cells bound to macrophages or dendritic cells and NK or T cells bound to endothelial cells. Myeloid-lymphocyte physically interacting cell complexes expressed activated and proinflammatory genes. Lymphocytes physically interacting with endothelial cells were enriched for NK and CD4 T cells. NK cell-endothelial cell contact caused increased expression of endothelial proinflammatory genes CXCL9 and CXCL10 and NK cell proinflammatory genes CCL3 , CCL4 , and GNLY . CONCLUSIONS: The transcriptional profiles of physically interacting cells from human kidney transplant biopsies can be inferred from scRNA-seq data using the sequencing physically interacting cells method. This approach complements previous methods that estimate cell-cell physical contact from scRNA-seq data.
Assuntos
Células Endoteliais , Rejeição de Enxerto , Humanos , Rim/patologia , Transcriptoma , Aloenxertos , Proteínas de Membrana/genética , Epitopos , Análise de Célula ÚnicaRESUMO
Cardiovascular pathology is the leading cause of death and disability in the Western world, and current diagnostic testing usually evaluates the anatomy of the vessel to determine if the vessel contains blockages and plaques. However, there is a growing school of thought that other measures, such as wall shear stress, provide more useful information for earlier diagnosis and prediction of atherosclerotic related disease compared to pulsed-wave Doppler ultrasound, magnetic resonance angiography, or computed tomography angiography. A novel algorithm for quantifying wall shear stress (WSS) in atherosclerotic plaque using diagnostic ultrasound imaging, called Multifrequency ultrafast Doppler spectral analysis (MFUDSA), is presented. The development of this algorithm is presented, in addition to its optimisation using simulation studies and in-vitro experiments with flow phantoms approximating the early stages of cardiovascular disease. The presented algorithm is compared with commonly used WSS assessment methods, such as standard PW Doppler, Ultrafast Doppler, and Parabolic Doppler, as well as plane-wave Doppler. Compared to an equivalent processing architecture with one-dimensional Fourier analysis, the MFUDSA algorithm provided an increase in signal-to-noise ratio (SNR) by a factor of 4-8 and an increase in velocity resolution by a factor of 1.10-1.35. The results indicated that MFUDSA outperformed the others, with significant differences detected between the typical WSS values of moderate disease progression (p = 0.003) and severe disease progression (p = 0.001). The algorithm demonstrated an improved performance for the assessment of WSS and has potential to provide an earlier diagnosis of cardiovascular disease than current techniques allow.