RESUMO
The advent of personalized medicine has driven the development of novel approaches for obtaining detailed cellular and molecular information from clinical tissue samples. Tissue cytometry is a promising new technique that can be used to enumerate and characterize each cell in a tissue and, unlike flow cytometry and other single-cell techniques, does so in the context of the intact tissue, preserving spatial information that is frequently crucial to understanding a cell's physiology, function, and behavior. However, the wide-scale adoption of tissue cytometry as a research tool has been limited by the fact that published examples utilize specialized techniques that are beyond the capabilities of most laboratories. Here we describe a complete and accessible pipeline, including methods of sample preparation, microscopy, image analysis, and data analysis for large-scale three-dimensional tissue cytometry of human kidney tissues. In this workflow, multiphoton microscopy of unlabeled tissue is first conducted to collect autofluorescence and second-harmonic images. The tissue is then labeled with eight fluorescent probes, and imaged using spectral confocal microscopy. The raw 16-channel images are spectrally deconvolved into 8-channel images, and analyzed using the Volumetric Tissue Exploration and Analysis (VTEA) software developed by our group. We applied this workflow to analyze millimeter-scale tissue samples obtained from human nephrectomies and from renal biopsies from individuals diagnosed with diabetic nephropathy, generating a quantitative census of tens of thousands of cells in each. Such analyses can provide useful insights that can be linked to the biology or pathology of kidney disease. The approach utilizes common laboratory techniques, is compatible with most commercially-available confocal microscope systems and all image and data analysis is conducted using the VTEA image analysis software, which is available as a plug-in for ImageJ.
Assuntos
Técnicas Citológicas , Imageamento Tridimensional , Rim/citologia , Microscopia de Fluorescência por Excitação Multifotônica , Software , Corantes Fluorescentes , Humanos , Microscopia ConfocalRESUMO
Fibroblast Growth Factor 23 (FGF23) is a bone-derived hormone that reduces kidney phosphate reabsorption and 1,25(OH)2 vitamin D synthesis via its required co-receptor alpha-Klotho. To identify novel genes that could serve as targets to control FGF23-mediated mineral metabolism, gene array and single-cell RNA sequencing were performed in wild type mouse kidneys. Gene array demonstrated that heparin-binding EGF-like growth factor (HBEGF) was significantly up-regulated following one-hour FGF23 treatment of wild type mice. Mice injected with HBEGF had phenotypes consistent with partial FGF23-mimetic activity including robust induction of Egr1, and increased Cyp24a1 mRNAs. Single cell RNA sequencing showed overlapping HBEGF and EGF-receptor expression mostly in the proximal tubule, and alpha-Klotho expression in proximal and distal tubule segments. In alpha-Klotho-null mice devoid of canonical FGF23 signaling, HBEGF injections significantly increased Egr1 and Cyp24a1 with correction of basally elevated Cyp27b1. Additionally, mice placed on a phosphate deficient diet to suppress FGF23 had endogenously increased Cyp27b1 mRNA, which was rescued in mice receiving HBEGF. In HEK293 cells with stable alpha-Klotho expression, FGF23 and HBEGF increased CYP24A1 mRNA expression. HBEGF, but not FGF23 bioactivity was blocked with EGF-receptor inhibition. Thus, our findings support that the paracrine/autocrine factor HBEGF could play novel roles in controlling genes downstream of FGF23 via targeting common signaling pathways.
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Fatores de Crescimento de Fibroblastos , Vitamina D , Animais , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/genética , Glucuronidase/genética , Células HEK293 , Humanos , Rim , Camundongos , Minerais , FosfatosRESUMO
BACKGROUND: Idiopathic nodular mesangial sclerosis, also called idiopathic nodular glomerulosclerosis (ING), is a rare clinical entity with an unclear pathogenesis. The hallmark of this disease is the presence of nodular mesangial sclerosis on histology without clinical evidence of diabetes mellitus or other predisposing diagnoses. To achieve insights into its pathogenesis, we queried the clinical, histopathologic and transcriptomic features of ING and nodular diabetic nephropathy (DN). METHODS: All renal biopsy reports accessioned at Indiana University Health from 2001 to 2016 were reviewed to identify 48 ING cases. Clinical and histopathologic features were compared between individuals with ING and DN (n = 751). Glomeruli of ING (n = 5), DN (n = 18) and reference (REF) nephrectomy (n = 9) samples were isolated by laser microdissection and RNA was sequenced. Immunohistochemistry of proline-rich 36 (PRR36) protein was performed. RESULTS: ING subjects were frequently hypertensive (95.8%) with a smoking history (66.7%). ING subjects were older, had lower proteinuria and had less hyaline arteriolosclerosis than DN subjects. Butanoate metabolism was an enriched pathway in ING samples compared with either REF or DN samples. The top differentially expressed gene, PRR36, had increased expression in glomeruli 248-fold [false discovery rate (FDR) P = 5.93 × 10-6] compared with the REF and increased 109-fold (FDR P = 1.85 × 10-6) compared with DN samples. Immunohistochemistry revealed a reduced proportion of cells with perinuclear reaction in ING samples as compared to DN. CONCLUSIONS: Despite similar clinical and histopathologic characteristics in ING and DN, the uncovered transcriptomic signature suggests that ING has distinct molecular features from nodular DN. Further study is warranted to understand these relationships.
Assuntos
Diabetes Mellitus , Nefropatias Diabéticas , Síndrome Nefrótica , Diabetes Mellitus/patologia , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Humanos , Glomérulos Renais/patologia , Síndrome Nefrótica/patologia , Proteinúria/patologia , Esclerose/patologiaRESUMO
Mouse double minute 2 (Mdm2) is a multifaceted oncoprotein that is highly regulated with distinct domains capable of cellular transformation. Loss of Mdm2 is embryonically lethal, making it difficult to study in a mouse model without additional genetic alterations. Global overexpression through increased Mdm2 gene copy number (Mdm2Tg ) results in the development of hematopoietic neoplasms and sarcomas in adult animals. In these mice, we found an increase in osteoblastogenesis, differentiation, and a high bone mass phenotype. Since it was difficult to discern the cell lineage that generated this phenotype, we generated osteoblast-specific Mdm2 overexpressing (Mdm2TgOb ) mice in 2 different strains, C57BL/6 and DBA. These mice did not develop malignancies; however, these animals and the MG63 human osteosarcoma cell line with high levels of Mdm2 showed an increase in bone mineralization. Importantly, overexpression of Mdm2 corrected age-related bone loss in mice, providing a role for the proto-oncogenic activity of Mdm2 in bone health of adult animals.
Assuntos
Calcificação Fisiológica/fisiologia , Osteossarcoma/patologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proto-Oncogenes/fisiologia , Análise de Variância , Animais , Densidade Óssea/fisiologia , Remodelação Óssea/fisiologia , Osso Esponjoso/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese/fisiologia , Proto-Oncogene MasRESUMO
The Lnk adapter protein negatively regulates the signaling of thrombopoietin (TPO), the main megakaryocyte (MK) growth factor. Lnk-deficient (-/-) mice have increased TPO signaling and increased MK number. Interestingly, several mouse models exist in which increased MK number leads to a high bone mass phenotype. Here we report the bone phenotype of these mice. MicroCT and static histomorphometric analyses at 20 weeks showed the distal femur of Lnk-/- mice to have significantly higher bone volume fraction and trabecular number compared to wild-type (WT) mice. Notably, despite a significant increase in the number of osteoclasts (OC), and decreased bone formation rate in Lnk-/- mice compared to WT mice, Lnk-/- mice demonstrated a 2.5-fold greater BV/TV suggesting impaired OC function in vivo. Additionally, Lnk-/- mouse femurs exhibited non-significant increases in mid-shaft cross-sectional area, yet increased periosteal BFR compared to WT femurs was observed. Lnk-/- femurs also had non-significant increases in polar moment of inertia and decreased cortical bone area and thickness, resulting in reduced bone stiffness, modulus, and strength compared to WT femurs. Of note, Lnk is expressed by OC lineage cells and when Lnk-/- OC progenitors are cultured in the presence of TPO, significantly more OC are observed than in WT cultures. Lnk is also expressed in osteoblast (OB) cells and in vitro reduced alkaline phosphatase activity was observed in Lnk-/- cultures. These data suggest that both direct effects on OB and OC as well as indirect effects of MK in regulating OB contributes to the observed high bone mass. J. Cell. Biochem. 118: 2231-2240, 2017. © 2017 Wiley Periodicals, Inc.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Trombopoetina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Western Blotting , Células da Medula Óssea/metabolismo , Ciclo Celular/genética , Ciclo Celular/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Feminino , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Megacariócitos/metabolismo , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese/genética , Osteogênese/fisiologia , Células RAW 264.7 , Trombopoetina/genética , Microtomografia por Raio-XRESUMO
Colistin sulfate (polymixin E) is an antibiotic prescribed with increasing frequency for severe Gram-negative bacterial infections. As nephrotoxicity is a common side effect, the discovery of pharmacogenomic markers associated with toxicity would benefit the utility of this drug. Our objective was to identify genetic markers of colistin cytotoxicity that were also associated with expression of key proteins using an unbiased, whole genome approach and further evaluate the functional significance in renal cell lines. To this end, we employed International HapMap lymphoblastoid cell lines (LCLs) of Yoruban ancestry with known genetic information to perform a genome-wide association study (GWAS) with cellular sensitivity to colistin. Further association studies revealed that single nucleotide polymorphisms (SNPs) associated with gene expression and protein expression were significantly enriched in SNPs associated with cytotoxicity (p ≤ 0.001 for gene and p = 0.015 for protein expression). The most highly associated SNP, chr18:3417240 (p = 6.49 × 10-8), was nominally a cis-expression quantitative trait locus (eQTL) of the gene TGIF1 (transforming growth factor ß (TGFß)-induced factor-1; p = 0.021) and was associated with expression of the protein HOXD10 (homeobox protein D10; p = 7.17 × 10-5). To demonstrate functional relevance in a murine colistin nephrotoxicity model, HOXD10 immunohistochemistry revealed upregulated protein expression independent of mRNA expression in response to colistin administration. Knockdown of TGIF1 resulted in decreased protein expression of HOXD10 and increased resistance to colistin cytotoxicity. Furthermore, knockdown of HOXD10 in renal cells also resulted in increased resistance to colistin cytotoxicity, supporting the physiological relevance of the initial genomic associations.
Assuntos
Antibacterianos/farmacologia , Colistina/farmacologia , Proteínas de Homeodomínio/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Antibacterianos/efeitos adversos , Antibacterianos/toxicidade , Linhagem Celular , Linhagem Celular Tumoral , Colistina/efeitos adversos , Colistina/toxicidade , Resistência a Medicamentos/genética , Estudo de Associação Genômica Ampla , Humanos , Polimorfismo de Nucleotídeo Único , Locos de Características QuantitativasRESUMO
C-Mpl is the receptor for thrombopoietin (TPO), the main megakaryocyte (MK) growth factor, and c-Mpl is believed to be expressed on cells of the hematopoietic lineage. As MKs have been shown to enhance bone formation, it may be expected that mice in which c-Mpl was globally knocked out (c-Mpl(-/-) mice) would have decreased bone mass because they have fewer MKs. Instead, c-Mpl(-/-) mice have a higher bone mass than WT controls. Using c-Mpl(-/-) mice we investigated the basis for this discrepancy and discovered that c-Mpl is expressed on both osteoblasts (OBs) and osteoclasts (OCs), an unexpected finding that prompted us to examine further how c-Mpl regulates bone. Static and dynamic bone histomorphometry parameters suggest that c-Mpl deficiency results in a net gain in bone volume with increases in OBs and OCs. In vitro, a higher percentage of c-Mpl(-/-) OBs were in active phases of the cell cycle, leading to an increased number of OBs. No difference in OB differentiation was observed in vitro as examined by real-time PCR and functional assays. In co-culture systems, which allow for the interaction between OBs and OC progenitors, c-Mpl(-/-) OBs enhanced osteoclastogenesis. Two of the major signaling pathways by which OBs regulate osteoclastogenesis, MCSF/OPG/RANKL and EphrinB2-EphB2/B4, were unaffected in c-Mpl(-/-) OBs. These data provide new findings for the role of MKs and c-Mpl expression in bone and may provide insight into the homeostatic regulation of bone mass as well as bone loss diseases such as osteoporosis.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese/genética , Receptores de Trombopoetina/genética , Trombopoetina/genética , Animais , Animais Recém-Nascidos , Densidade Óssea , Contagem de Células , Diferenciação Celular , Divisão Celular , Efrina-B2/genética , Efrina-B2/metabolismo , Homeostase/genética , Fator Estimulador de Colônias de Macrófagos/genética , Fator Estimulador de Colônias de Macrófagos/metabolismo , Megacariócitos/citologia , Megacariócitos/metabolismo , Camundongos , Camundongos Knockout , Osteoblastos/citologia , Osteoclastos/citologia , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Ligante RANK/genética , Ligante RANK/metabolismo , Receptor EphB2/genética , Receptor EphB2/metabolismo , Receptor EphB4/genética , Receptor EphB4/metabolismo , Receptores de Trombopoetina/deficiência , Transdução de Sinais , Crânio/citologia , Crânio/metabolismo , Trombopoetina/metabolismoRESUMO
Osteoblast differentiation and migration are necessary for bone formation during bone remodeling. Mice lacking the proline-rich tyrosine kinase Pyk2 (Pyk2-KO) have increased bone mass, in part due to increased osteoblast proliferation. Megakaryocytes (MKs), the platelet-producing cells, also promote osteoblast proliferation in vitro and bone-formation in vivo via a pathway that involves Pyk2. In the current study, we examined the mechanism of action of Pyk2, and the role of MKs, on osteoblast differentiation and migration. We found that Pyk2-KO osteoblasts express elevated alkaline phosphatase (ALP), type I collagen and osteocalcin mRNA levels as well as increased ALP activity, and mineralization, confirming that Pyk2 negatively regulates osteoblast function. Since Pyk2 Y402 phosphorylation is important for its catalytic activity and for its protein-scaffolding functions, we expressed the phosphorylation-mutant (Pyk2(Y402F) ) and kinase-mutant (Pyk2(K457A) ) in Pyk2-KO osteoblasts. Both Pyk2(Y402F) and Pyk2(K457A) reduced ALP activity, whereas only kinase-inactive Pyk2(K457A) inhibited Pyk2-KO osteoblast migration. Consistent with a role for Pyk2 on ALP activity, co-culture of MKs with osteoblasts led to a decrease in the level of phosphorylated Pyk2 (pY402) as well as a decrease in ALP activity. Although, Pyk2-KO osteoblasts exhibited increased migration compared to wild-type osteoblasts, Pyk2 expression was not required necessary for the ability of MKs to stimulate osteoblast migration. Together, these data suggest that osteoblast differentiation and migration are inversely regulated by MKs via distinct Pyk2-dependent and independent signaling pathways. Novel drugs that distinguish between the kinase-dependent or protein-scaffolding functions of Pyk2 may provide therapeutic specificity for the control of bone-related diseases.
Assuntos
Quinase 2 de Adesão Focal/genética , Quinase 2 de Adesão Focal/metabolismo , Megacariócitos/citologia , Osteoblastos/citologia , Animais , Diferenciação Celular , Movimento Celular , Células Cultivadas , Técnicas de Cocultura , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Megacariócitos/metabolismo , Camundongos , Osteoblastos/metabolismo , Fosforilação , Transdução de SinaisRESUMO
Emerging data suggest that megakaryocytes (MKs) play a significant role in skeletal homeostasis. Indeed, osteosclerosis observed in several MK-related disorders may be a result of increased numbers of MKs. In support of this idea, we have previously demonstrated that MKs increase osteoblast (OB) proliferation by a direct cell-cell contact mechanism and that MKs also inhibit osteoclast (OC) formation. As MKs and OCs are derived from the same hematopoietic precursor, in these osteoclastogenesis studies we examined the role of the main MK growth factor, thrombopoietin (TPO) on OC formation and bone resorption. Here we show that TPO directly increases OC formation and differentiation in vitro. Specifically, we demonstrate the TPO receptor (c-mpl or CD110) is expressed on cells of the OC lineage, c-mpl is required for TPO to enhance OC formation in vitro, and TPO activates the mitogen-activated protein kinases, Janus kinase/signal transducer and activator of transcription, and nuclear factor-kappaB signaling pathways, but does not activate the PI3K/AKT pathway. Further, we found TPO enhances OC resorption in CD14+CD110+ human OC progenitors derived from peripheral blood mononuclear cells, and further separating OC progenitors based on CD110 expression enriches for mature OC development. The regulation of OCs by TPO highlights a novel therapeutic target for bone loss diseases and may be important to consider in the numerous hematologic disorders associated with alterations in TPO/c-mpl signaling as well as in patients suffering from bone disorders.
Assuntos
Diferenciação Celular/genética , Proliferação de Células/genética , Osteoclastos/metabolismo , Proteínas Recombinantes/administração & dosagem , Trombopoetina/administração & dosagem , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula , Proliferação de Células/efeitos dos fármacos , Hematopoese/genética , Humanos , Megacariócitos/metabolismo , Megacariócitos/patologia , Camundongos , Camundongos Knockout , Osteoclastos/patologia , Receptores de Trombopoetina/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Células-Tronco/efeitos dos fármacos , Trombopoetina/metabolismoRESUMO
GATA-1(low/low) mice have an increase in megakaryocytes (MKs) and trabecular bone. The latter is thought to result from MKs directly stimulating osteoblastic bone formation while simultaneously inhibiting osteoclastogenesis. Osteoprotegerin (OPG) is known to inhibit osteoclastogenesis and OPG(-/-) mice have reduced trabecular and cortical bone due to increased osteoclastogenesis. Interestingly, GATA-1(low/low) mice have increased OPG levels. Here, we sought to determine whether GATA-1 knockdown in OPG(-/-) mice could rescue the observed osteoporotic bone phenotype. GATA-1(low/low) mice were bred with OPG(-/-) mice and bone phenotype assessed. GATA-1(low/low) × OPG(-/-) mice have increased cortical bone porosity, similar to OPG(-/-) mice. Both OPG(-/-) and GATA-1(low/low) × OPG(-/-) mice, were found to have increased osteoclasts localized to cortical bone, possibly producing the observed elevated porosity. Biomechanical assessment indicates that OPG(-/-) and GATA-1(low/low) × OPG(-/-) femurs are weaker and less stiff than C57BL/6 or GATA-1(low/low) femurs. Notably, GATA-1(low/low) × OPG(-/-) mice had trabecular bone parameters that were not different from C57BL/6 values, suggesting that GATA-1 deficiency can partially rescue the trabecular bone loss observed with OPG deficiency. The fact that GATA-1 deficiency appears to be able to partially rescue the trabecular, but not the cortical bone phenotype suggests that MKs can locally enhance trabecular bone volume, but that MK secreted factors cannot access cortical bone sufficiently to inhibit osteoclastogenesis or that OPG itself is required to inhibit osteoclastogenesis in cortical bone.
Assuntos
Fator de Transcrição GATA1/deficiência , Megacariócitos/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteoprotegerina/metabolismo , Animais , Reabsorção Óssea/genética , Fêmur/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Osteogênese/genética , Osteoprotegerina/deficiênciaRESUMO
Recent studies suggest that megakaryocytes (MKs) may play a significant role in skeletal homeostasis, as evident by the occurrence of osteosclerosis in multiple MK related diseases (Lennert et al., 1975; Thiele et al., 1999; Chagraoui et al., 2006). We previously reported a novel interaction whereby MKs enhanced proliferation of osteoblast lineage/osteoprogenitor cells (OBs) by a mechanism requiring direct cell-cell contact. However, the signal transduction pathways and the downstream effector molecules involved in this process have not been characterized. Here we show that MKs contact with OBs, via beta1 integrin, activate the p38/MAPKAPK2/p90RSK kinase cascade in the bone cells, which causes Mdm2 to neutralizes p53/Rb-mediated check point and allows progression through the G1/S. Interestingly, activation of MAPK (ERK1/2) and AKT, collateral pathways that regulate the cell cycle, remained unchanged with MK stimulation of OBs. The MK-to-OB signaling ultimately results in significant increases in the expression of c-fos and cyclin A, necessary for sustaining the OB proliferation. Overall, our findings show that OBs respond to the presence of MKs, in part, via an integrin-mediated signaling mechanism, activating a novel response axis that de-represses cell cycle activity. Understanding the mechanisms by which MKs enhance OB proliferation will facilitate the development of novel anabolic therapies to treat bone loss associated with osteoporosis and other bone-related diseases.
Assuntos
Diferenciação Celular/genética , Megacariócitos/citologia , Osteoblastos/citologia , Transdução de Sinais/genética , Ciclo Celular/genética , Linhagem da Célula , Proliferação de Células/genética , Células Cultivadas , Humanos , Sistema de Sinalização das MAP Quinases/genética , Megacariócitos/metabolismo , Osteoblastos/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismoRESUMO
Chronic kidney disease (CKD) is associated with renal metabolic disturbances, including impaired fatty acid oxidation (FAO). Nicotinamide adenine dinucleotide (NAD + ) is a small molecule that participates in hundreds of metabolism-related reactions. NAD + levels are decreased in CKD, and NAD + supplementation is protective. However, both the mechanism of how NAD + supplementation protects from CKD, as well as the cell types most responsible, are poorly understood. Using a mouse model of Alport syndrome, we show that nicotinamide riboside (NR), an NAD + precursor, stimulates renal peroxisome proliferator-activated receptor α signaling and restores FAO in the proximal tubules, thereby protecting from CKD in both sexes. Bulk RNA-sequencing shows that renal metabolic pathways are impaired in Alport mice and dramatically activated by NR in both sexes. These transcriptional changes are confirmed by orthogonal imaging techniques and biochemical assays. Single nuclei RNA-sequencing and spatial transcriptomics, both the first of their kind from Alport mice, show that NAD + supplementation restores FAO in the proximal tubules with minimal effects on the podocytes. Finally, we also report, for the first time, sex differences at the transcriptional level in this Alport model. Male Alport mice had more severe inflammation and fibrosis than female mice at the transcriptional level. In summary, the data herein identify both the protective mechanism and location of NAD + supplementation in this model of CKD.
RESUMO
Spatial -OMICS technologies facilitate the interrogation of molecular profiles in the context of the underlying histopathology and tissue microenvironment. Paired analysis of histopathology and molecular data can provide pathologists with otherwise unobtainable insights into biological mechanisms. To connect the disparate molecular and histopathologic features into a single workspace, we developed FUSION (Functional Unit State IdentificatiON in WSIs [Whole Slide Images]), a web-based tool that provides users with a broad array of visualization and analytical tools including deep learning-based algorithms for in-depth interrogation of spatial -OMICS datasets and their associated high-resolution histology images. FUSION enables end-to-end analysis of functional tissue units (FTUs), automatically aggregating underlying molecular data to provide a histopathology-based medium for analyzing healthy and altered cell states and driving new discoveries using "pathomic" features. We demonstrate FUSION using 10x Visium spatial transcriptomics (ST) data from both formalin-fixed paraffin embedded (FFPE) and frozen prepared datasets consisting of healthy and diseased tissue. Through several use-cases, we demonstrate how users can identify spatial linkages between quantitative pathomics, qualitative image characteristics, and spatial --omics.
RESUMO
There is a need to define regions of gene activation or repression that control human kidney cells in states of health, injury, and repair to understand the molecular pathogenesis of kidney disease and design therapeutic strategies. Comprehensive integration of gene expression with epigenetic features that define regulatory elements remains a significant challenge. We measure dual single nucleus RNA expression and chromatin accessibility, DNA methylation, and H3K27ac, H3K4me1, H3K4me3, and H3K27me3 histone modifications to decipher the chromatin landscape and gene regulation of the kidney in reference and adaptive injury states. We establish a spatially-anchored epigenomic atlas to define the kidney's active, silent, and regulatory accessible chromatin regions across the genome. Using this atlas, we note distinct control of adaptive injury in different epithelial cell types. A proximal tubule cell transcription factor network of ELF3, KLF6, and KLF10 regulates the transition between health and injury, while in thick ascending limb cells this transition is regulated by NR2F1. Further, combined perturbation of ELF3, KLF6, and KLF10 distinguishes two adaptive proximal tubular cell subtypes, one of which manifested a repair trajectory after knockout. This atlas will serve as a foundation to facilitate targeted cell-specific therapeutics by reprogramming gene regulatory networks.
Assuntos
Cromatina , Rim , Humanos , Cromatina/genética , Túbulos Renais Proximais , Nível de Saúde , Contagem de CélulasRESUMO
The proliferation and differentiation of osteoblast (OB) precursors are essential for elaborating the bone-forming activity of mature OBs. However, the mechanisms regulating OB proliferation and function are largely unknown. We reported that OB proliferation is enhanced by megakaryocytes (MKs) via a process that is regulated in part by integrin signaling. The tyrosine kinase Pyk2 has been shown to regulate cell proliferation and survival in a variety of cells. Pyk2 is also activated by integrin signaling and regulates actin remodeling in bone-resorbing osteoclasts. In this study, we examined the role of Pyk2 and actin in the MK-mediated increase in OB proliferation. Calvarial OBs were cultured in the presence of MKs for various times, and Pyk2 signaling cascades in OBs were examined by Western blotting, subcellular fractionation, and microscopy. We found that MKs regulate the temporal expression of Pyk2 and its subcellular localization. We also found that MKs regulate the expression of two alternatively spliced isoforms of Pyk2 in OBs, which may regulate OB differentiation and proliferation. MKs also induced cytoskeletal reorganization in OBs, which was associated with the caspase-mediated cleavage of actin, an increase in focal adhesions, and the formation of apical membrane ruffles. Moreover, BrdU incorporation in MK-stimulated OBs was blocked by the actin-polymerizing agent, jasplakinolide. Collectively, our studies reveal that Pyk2 and actin play an important role in MK-regulated signaling cascades that control OB proliferation and may be important for therapeutic interventions aimed at increasing bone formation in metabolic diseases of the skeleton.
Assuntos
Actinas/metabolismo , Caspases/metabolismo , Quinase 2 de Adesão Focal/biossíntese , Regulação Enzimológica da Expressão Gênica/fisiologia , Megacariócitos/metabolismo , Osteoblastos/metabolismo , Processamento Alternativo , Animais , Diferenciação Celular/fisiologia , Proliferação de Células , Células Cultivadas , Adesões Focais/metabolismo , Isoenzimas/biossíntese , Megacariócitos/citologia , Camundongos , Osteoblastos/citologiaRESUMO
Kidney stone disease causes significant morbidity and increases health care utilization. In this work, we decipher the cellular and molecular niche of the human renal papilla in patients with calcium oxalate (CaOx) stone disease and healthy subjects. In addition to identifying cell types important in papillary physiology, we characterize collecting duct cell subtypes and an undifferentiated epithelial cell type that was more prevalent in stone patients. Despite the focal nature of mineral deposition in nephrolithiasis, we uncover a global injury signature characterized by immune activation, oxidative stress and extracellular matrix remodeling. We also identify the association of MMP7 and MMP9 expression with stone disease and mineral deposition, respectively. MMP7 and MMP9 are significantly increased in the urine of patients with CaOx stone disease, and their levels correlate with disease activity. Our results define the spatial molecular landscape and specific pathways contributing to stone-mediated injury in the human papilla and identify associated urinary biomarkers.
Assuntos
Cálculos Renais , Medula Renal , Humanos , Medula Renal/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinase 7 da Matriz , Oxalato de Cálcio/metabolismo , Transcriptoma , Cálculos Renais/genética , Cálculos Renais/metabolismoRESUMO
There is a need to define regions of gene activation or repression that control human kidney cells in states of health, injury, and repair to understand the molecular pathogenesis of kidney disease and design therapeutic strategies. However, comprehensive integration of gene expression with epigenetic features that define regulatory elements remains a significant challenge. We measured dual single nucleus RNA expression and chromatin accessibility, DNA methylation, and H3K27ac, H3K4me1, H3K4me3, and H3K27me3 histone modifications to decipher the chromatin landscape and gene regulation of the kidney in reference and adaptive injury states. We established a comprehensive and spatially-anchored epigenomic atlas to define the kidney's active, silent, and regulatory accessible chromatin regions across the genome. Using this atlas, we noted distinct control of adaptive injury in different epithelial cell types. A proximal tubule cell transcription factor network of ELF3 , KLF6 , and KLF10 regulated the transition between health and injury, while in thick ascending limb cells this transition was regulated by NR2F1 . Further, combined perturbation of ELF3 , KLF6 , and KLF10 distinguished two adaptive proximal tubular cell subtypes, one of which manifested a repair trajectory after knockout. This atlas will serve as a foundation to facilitate targeted cell-specific therapeutics by reprogramming gene regulatory networks.
RESUMO
Hematopoietic stem (HSC) and progenitor (HPC) cell fate is governed by intrinsic and extrinsic parameters. We examined the impact of hematopoietic niche elements on HSC and HPC function by analyzing the combined effect of osteoblasts (OBs) and stromal cells (SCs) on Lineage(-)Sca-1(+)CD117(+) (LSK) cells. CFU expansion and marrow repopulating potential of cultured Lineage(-)Sca-1(+)CD117(+) cells were significantly higher in OB compared with SC cultures, thus corroborating the importance of OBs in the competence of the hematopoietic niche. OB-mediated enhancement of HSC and HPC function was reduced in cocultures of OBs and SCs, suggesting that SCs suppressed the OB-mediated hematopoiesis-enhancing activity. Although the suppressive effect of SC was mediated by adipocytes, probably through up-regulation of neuropilin-1, the OB-mediated enhanced hematopoiesis function was elaborated through Notch signaling. Expression of Notch 2, Jagged 1 and 2, Delta 1 and 4, Hes 1 and 5, and Deltex was increased in OB cultures and suppressed in SC and OB/SC cultures. Phenotypic fractionation of OBs did not segregate the hematopoiesis-enhancing activity but demonstrated that this function is common to OBs from different anatomic sites. These data illustrate that OBs promote in vitro maintenance of hematopoietic functions, including repopulating potential by up-regulating Notch-mediated signaling between HSCs and OBs.
Assuntos
Medula Óssea/metabolismo , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Osteoblastos/citologia , Transdução de Sinais/fisiologia , Nicho de Células-Tronco/fisiologia , Animais , Comunicação Celular , Diferenciação Celular/fisiologia , Proliferação de Células , Separação Celular , Células Cultivadas , Técnicas de Cocultura , Citometria de Fluxo , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoblastos/metabolismo , Receptores Notch/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Estromais/citologia , Células Estromais/metabolismoRESUMO
Diabetic kidney disease (DKD) remains the leading cause of end-stage kidney disease despite decades of study. Alterations in the glomerulus and kidney tubules both contribute to the pathogenesis of DKD although the majority of investigative efforts have focused on the glomerulus. We sought to examine the differential expression signature of human DKD in the glomerulus and proximal tubule and corroborate our findings in the db/db mouse model of diabetes. A transcriptogram network analysis of RNAseq data from laser microdissected (LMD) human glomerulus and proximal tubule of DKD and reference nephrectomy samples revealed enriched pathways including rhodopsin-like receptors, olfactory signaling, and ribosome (protein translation) in the proximal tubule of human DKD biopsy samples. The translation pathway was also enriched in the glomerulus. Increased translation in diabetic kidneys was validated using polyribosomal profiling in the db/db mouse model of diabetes. Using single nuclear RNA sequencing (snRNAseq) of kidneys from db/db mice, we prioritized additional pathways identified in human DKD. The top overlapping pathway identified in the murine snRNAseq proximal tubule clusters and the human LMD proximal tubule compartment was carboxylic acid catabolism. Using ultra-performance liquid chromatography-mass spectrometry, the fatty acid catabolism pathway was also found to be dysregulated in the db/db mouse model. The Acetyl-CoA metabolite was down-regulated in db/db mice, aligning with the human differential expression of the genes ACOX1 and ACACB. In summary, our findings demonstrate that proximal tubular alterations in protein translation and carboxylic acid catabolism are key features in both human and murine DKD.
Assuntos
Diabetes Mellitus , Nefropatias Diabéticas , Animais , Ácidos Carboxílicos/metabolismo , Diabetes Mellitus/metabolismo , Nefropatias Diabéticas/metabolismo , Rim/patologia , Glomérulos Renais/patologia , Camundongos , Biossíntese de ProteínasRESUMO
Sepsis is a dynamic state that progresses at variable rates and has life-threatening consequences. Staging patients along the sepsis timeline requires a thorough knowledge of the evolution of cellular and molecular events at the tissue level. Here, we investigated the kidney, an organ central to the pathophysiology of sepsis. Single-cell RNA-sequencing in a murine endotoxemia model revealed the involvement of various cell populations to be temporally organized and highly orchestrated. Endothelial and stromal cells were the first responders. At later time points, epithelial cells upregulated immune-related pathways while concomitantly downregulating physiological functions such as solute homeostasis. Sixteen hours after endotoxin, there was global cell-cell communication failure and organ shutdown. Despite this apparent organ paralysis, upstream regulatory analysis showed significant activity in pathways involved in healing and recovery. This rigorous spatial and temporal definition of murine endotoxemia will uncover precise biomarkers and targets that can help stage and treat human sepsis.