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1.
Proc Natl Acad Sci U S A ; 119(25): e2203179119, 2022 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-35696569

RESUMO

Recent advances in single-cell sequencing provide a unique opportunity to gain novel insights into the diversity, lineage, and functions of cell types constituting a tissue/organ. Here, we performed a single-nucleus study of the adult Drosophila renal system, consisting of Malpighian tubules and nephrocytes, which shares similarities with the mammalian kidney. We identified 11 distinct clusters representing renal stem cells, stellate cells, regionally specific principal cells, garland nephrocyte cells, and pericardial nephrocytes. Characterization of the transcription factors specific to each cluster identified fruitless (fru) as playing a role in stem cell regeneration and Hepatocyte nuclear factor 4 (Hnf4) in regulating glycogen and triglyceride metabolism. In addition, we identified a number of genes, including Rho guanine nucleotide exchange factor at 64C (RhoGEF64c), Frequenin 2 (Frq2), Prip, and CG1093 that are involved in regulating the unusual star shape of stellate cells. Importantly, the single-nucleus dataset allows visualization of the expression at the organ level of genes involved in ion transport and junctional permeability, providing a systems-level view of the organization and physiological roles of the tubules. Finally, a cross-species analysis allowed us to match the fly kidney cell types to mouse kidney cell types and planarian protonephridia, knowledge that will help the generation of kidney disease models. Altogether, our study provides a comprehensive resource for studying the fly kidney.


Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Fator 4 Nuclear de Hepatócito , Túbulos de Malpighi , Proteínas do Tecido Nervoso , Fatores de Transcrição , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Fator 4 Nuclear de Hepatócito/genética , Rim/citologia , Rim/fisiologia , Túbulos de Malpighi/citologia , Túbulos de Malpighi/fisiologia , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Regeneração , Análise de Sequência de RNA/métodos , Análise de Célula Única , Células-Tronco/metabolismo , Células-Tronco/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
2.
Cell Rep ; 39(11): 110933, 2022 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-35705028

RESUMO

Generation of new kidneys can be useful in various research fields, including organ transplantation. However, generating renal stroma, an important component tissue for structural support, endocrine function, and kidney development, remains difficult. Organ generation using an animal developmental niche can provide an appropriate in vivo environment for renal stroma differentiation. Here, we generate rat renal stroma with endocrine capacity by removing mouse stromal progenitor cells (SPCs) from the host developmental niche and transplanting rat SPCs. Furthermore, we develop a method to replace both nephron progenitor cells (NPCs) and SPCs, called the interspecies dual replacement of the progenitor (i-DROP) system, and successfully generate functional chimeric kidneys containing rat nephrons and stroma. This method can generate renal tissue from progenitors and reduce xenotransplant rejection. Moreover, it is a safe method, as donor cells do not stray into nontarget organs, thus accelerating research on stem cells, chimeras, and xenotransplantation.


Assuntos
Rim , Néfrons , Nicho de Células-Tronco , Células-Tronco , Animais , Diferenciação Celular , Quimera , Rim/citologia , Camundongos , Néfrons/citologia , Ratos , Células-Tronco/citologia
3.
Gene ; 822: 146324, 2022 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-35182681

RESUMO

The advent of RNA sequencing technology provides insight into the dynamic nature of tremendous transcripts within Crandell-Reese feline kidney (CRFK) cells in response to canine parvovirus (CPV-2c) infection. A total of 1,603 genes displayed differentially expressed genes (DEGs), including 789 up-regulated genes and 814 downregulated genes in the infected cells. Gene expression profiles have shown a subtle pattern of defense mechanism and immune response to CPV through significant DEGs when extensively examined via Gene Ontology (GO) and pathway analysis. Prospective GO analysis was performed and identified several enriched GO biological process terms with significant participating roles in the immune system process and defense response to virus pathway. A Gene network was constructed using the 22 most significantly enriched genes of particular interests in defense response to virus pathways to illustrate the key pathways. Eleven genes (C1QBP, CD40, HYAL2, IFNB1, IFNG, IL12B, IL6, IRF3, LSM14A, MAVS, NLRC5) were identified, which are directly related to the defense response to the virus. Results of transcriptome profiling permit us to understand the heterogeneity of DEGs during in vitro experimental study of CPV infection, reflecting a unique transcriptome signature for the CPV virus. Our findings also demonstrate a distinct scenario of enhanced CPV responses in CRFK cells for viral clearance that involved multistep and perplexity of biological processes. Collectively, our data have given a fundamental role in anti-viral immunity as our highlights of this study, thus providing outlooks on future research priorities to be important in studying CPV.


Assuntos
Perfilação da Expressão Gênica/veterinária , Redes Reguladoras de Genes , Rim/citologia , Parvovirus Canino/patogenicidade , Animais , Linhagem Celular , Cães , Regulação da Expressão Gênica , Ontologia Genética , Rim/química , Rim/virologia , Modelos Biológicos , RNA-Seq
4.
Biomed Res Int ; 2022: 5239255, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35132377

RESUMO

Endothelial cells are heterogeneous, stemming from multiple organs, but there is still little known about the connection between the brain and kidney endothelial cells, especially in homeostasis. In this study, scRNA-seq results were obtained to compare genetic profiles and biological features of tissue-specific endothelial cells. On this basis, seven endothelial cell subpopulations were identified, two of which were upregulated genes in pathways related to stroke and/or depression, as characterized by neuroinflammation. This study revealed the similarities and distinctions between brain and kidney endothelial cells, providing baseline information needed to fully understand the relationship between renal diseases and neuroinflammation, such as stroke and depression.


Assuntos
Encéfalo/citologia , Células Epiteliais/fisiologia , Homeostase/fisiologia , Rim/citologia , Transcriptoma , Biologia Computacional , Homeostase/genética , Humanos
5.
Nat Commun ; 13(1): 611, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-35105870

RESUMO

Organs consist of the parenchyma and stroma, the latter of which coordinates the generation of organotypic structures. Despite recent advances in organoid technology, induction of organ-specific stroma and recapitulation of complex organ configurations from pluripotent stem cells (PSCs) have remained challenging. By elucidating the in vivo molecular features of the renal stromal lineage at a single-cell resolution level, we herein establish an in vitro induction protocol for stromal progenitors (SPs) from mouse PSCs. When the induced SPs are assembled with two differentially induced parenchymal progenitors (nephron progenitors and ureteric buds), the completely PSC-derived organoids reproduce the complex kidney structure, with multiple types of stromal cells distributed along differentiating nephrons and branching ureteric buds. Thus, integration of PSC-derived lineage-specific stroma into parenchymal organoids will pave the way toward recapitulation of the organotypic architecture and functions.


Assuntos
Rim/citologia , Rim/fisiologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia , Animais , Diferenciação Celular , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Néfrons , Organogênese/genética , Organogênese/fisiologia , Organoides/citologia , Transcriptoma
6.
Toxins (Basel) ; 14(2)2022 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-35202147

RESUMO

Pseudorabies, caused by pseudorabies virus (PRV), is the main highly infectious disease that severely affects the pig industry globally. T-2 toxin (T2), a significant mycotoxin, is widely spread in food and feeds and shows high toxicity to mammals. The potential mechanism of the interaction between viruses and toxins is of great research value because revealing this mechanism may provide new ideas for their joint prevention and control. In this study, we investigated the effect of T2 on PRV replication and the mechanism of action. The results showed that at a low dose (10 nM), T2 had no significant effect on porcine kidney 15 (PK15) cell viability. However, this T2 concentration alleviated PRV-induced cell injury and increased cell survival time. Additionally, the number of PK15 cells infected with PRV significantly reduced by T2 treatment. Similarly, T2 significantly decreased the copy number of PRV. Investigation of the mechanism revealed that 10 nM T2 significantly inhibits PRV replication and leads to downregulation of oxidative stress- and apoptosis-related genes. These results suggest that oxidative stress and apoptosis are involved in the inhibition of PRV replication in PK15 cells by low-concentration T2. Taken together, we demonstrated the protective effects of T2 against PRV infection. A low T2 concentration inhibited the replication of PRV in PK15 cells, and this process was accompanied by downregulation of the oxidative stress and apoptosis signaling pathways. Our findings partly explain the interaction mechanism between T2 and PRV, relating to oxidative stress and apoptosis, though further research is required.


Assuntos
Células Epiteliais/efeitos dos fármacos , Herpesvirus Suídeo 1/efeitos dos fármacos , Toxina T-2/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Células Epiteliais/virologia , Herpesvirus Suídeo 1/fisiologia , Rim/citologia , Estresse Oxidativo/efeitos dos fármacos , Suínos
7.
Cell Mol Life Sci ; 79(2): 97, 2022 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-35084555

RESUMO

Tau is a cytoskeletal protein that is expressed mainly in neurons and is involved in several cellular processes, such as microtubule stabilization, axonal maintenance, and transport. Altered tau metabolism is related to different tauopathies being the most important Alzheimer's disease where aberrant hyperphosphorylated and aggregated tau is found in the central nervous system. Here, we have analyzed that function in kidney by using tau knockout mice generated by integrating GFP-encoding cDNA into exon 1 of MAPT (here referred to as TauGFP/GFP). IVIS Lumina from PerkinElmer demonstrated GFP expression in the kidney. We then demonstrated by qPCR that the main tau isoform in the kidney is Tau4R. The GFP reporter allowed us to demonstrate that tau is found in the glomeruli of the renal cortex, and specifically in podocytes. This was further confirmed by immunohistochemistry. TauGFP/GFP mice present a podocyte cytoskeleton more dynamic as they contain higher levels of detyrosinated tubulin than wild-type mice. In addition, transmission electron microscopy studies demonstrated glomerular damage with a decrease in urinary creatinine. Our results prove that tau has an important role in kidney metabolism under normal physiological conditions.


Assuntos
Rim/metabolismo , Microtúbulos/metabolismo , Podócitos/metabolismo , Tauopatias/metabolismo , Proteínas tau/metabolismo , Animais , Citoesqueleto/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Rim/citologia , Rim/ultraestrutura , Glomérulos Renais/metabolismo , Glomérulos Renais/ultraestrutura , Camundongos da Linhagem 129 , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Microscopia Imunoeletrônica , Tauopatias/genética , Proteínas tau/genética
8.
Mol Immunol ; 143: 27-40, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35016116

RESUMO

CXC chemokine receptor 4 (CXCR4), a member of the G-protein-coupled receptor family, plays an important role in host immune responses. Within the teleost lineage, there are two paralogs of CXCR4; however, the role of CXCR4 in teleost B cells is poorly understood. In this study, we determined the cDNA sequences of the two CXCR4 paralogs from the Japanese sea bass (Lateolabrax japonica; LjCXCR4a and LjCXCR4b). Sequence and phylogenetic tree analyses revealed that LjCXCR4a and LjCXCR4b are most closely related to CXCR4a and CXCR4b, respectively, in the large yellow croaker (Larimichthys crocea). CXCR4 transcripts were mainly expressed in the gills, and their expression in different tissues was altered upon infection with Vibrio harveyi. LjCXCR4a and LjCXCR4b protein levels were upregulated in infected B cells. Knockdown of LjCXCR4a and LjCXCR4b in B cells by RNA interference, the phagocytic activity of B cells was not affected. Furthermore, knockdown of LjCXCR4a, not of LjCXCR4b, was observed to inhibit LjIgM expression in lipopolysaccharide-stimulated B cells. In addition, knockdown of LjCXCR4a, not of LjCXCR4b, was found to reduce reactive oxygen species levels in B cells. Our results indicate that LjCXCR4a and LjCXCR4b modulate the immune response of Japanese sea bass B cells against bacterial infection, albeit via different pathways.


Assuntos
Linfócitos B/imunologia , Bass/imunologia , Imunidade , Receptores CXCR4/química , Receptores CXCR4/metabolismo , Homologia de Sequência de Aminoácidos , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos/imunologia , Bass/sangue , Bass/genética , Citocinas/genética , Citocinas/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Imunoglobulina M/metabolismo , Rim/citologia , Macrófagos/metabolismo , Fagocitose , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores CXCR4/genética , Vibrio/fisiologia
9.
Sci Rep ; 12(1): 1443, 2022 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-35087095

RESUMO

Extracellular vesicles (EVs) are membrane enclosures released by eukaryotic cells that carry bioactive molecules and serve to modulate biological responses in recipient cells. Both increased EV release and altered EV composition are associated with the development and progression of many pathologies including cancer. Hypoxia, a feature of rapidly growing solid tumours, increases the release of EVs. However, the molecular mechanisms remain unknown. The hypoxia inducible factors (HIFs) are transcription factors that act as major regulators of the cellular adaptations to hypoxia. Here, we investigated the requirement of HIF pathway activation for EV release in Human Embryonic Kidney Cells (HEK293). Time course experiments showed that EV release increased concomitantly with sustained HIF1α and HIF2α activation following the onset of hypoxia. shRNA mediated knock-down of HIF1α but not HIF2α abrogated the effect of hypoxia on EV release, suggesting HIF1α is involved in this process. However, stabilization of HIF proteins in normoxic conditions through: (i) heterologous expression of oxygen insensitive HIF1α or HIF2α mutants in normoxic cells or (ii) chemical inhibition of the prolyl hydroxylase 2 (PHD2) repressor protein, did not increase EV release, suggesting HIF activation alone is not sufficient for this process. Our findings suggest HIF1α plays an important role in the regulation of EV release during hypoxia in HEK293 cells, however other hypoxia triggered mechanisms likely contribute as stabilization of HIF1α alone in normoxia is not sufficient for EV release.


Assuntos
Hipóxia Celular , Células Epiteliais/metabolismo , Vesículas Extracelulares/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Embrião de Mamíferos , Células Epiteliais/citologia , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Rim/citologia
10.
Lab Invest ; 102(3): 227-235, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34244616

RESUMO

Single-cell RNA sequencing (scRNA-seq) data has been widely used to profile cellular heterogeneities with a high-resolution picture. Clustering analysis is a crucial step of scRNA-seq data analysis because it provides a chance to identify and uncover undiscovered cell types. Most methods for clustering scRNA-seq data use an unsupervised learning strategy. Since the clustering step is separated from the cell annotation and labeling step, it is not uncommon for a totally exotic clustering with poor biological interpretability to be generated-a result generally undesired by biologists. To solve this problem, we proposed an active learning (AL) framework for clustering scRNA-seq data. The AL model employed a learning algorithm that can actively query biologists for labels, and this manual labeling is expected to be applied to only a subset of cells. To develop an optimal active learning approach, we explored several key parameters of the AL model in the experiments with four real scRNA-seq datasets. We demonstrate that the proposed AL model outperformed state-of-the-art unsupervised clustering methods with less than 1000 labeled cells. Therefore, we conclude that AL model is a promising tool for clustering scRNA-seq data that allows us to achieve a superior performance effectively and efficiently.


Assuntos
Algoritmos , Perfilação da Expressão Gênica/métodos , RNA-Seq/métodos , Análise de Célula Única/métodos , Aprendizado de Máquina não Supervisionado , Animais , Células Cultivadas , Análise por Conglomerados , Humanos , Rim/citologia , Rim/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Reprodutibilidade dos Testes , Bexiga Urinária/citologia , Bexiga Urinária/metabolismo
11.
Life Sci ; 295: 120167, 2022 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-34822795

RESUMO

AIMS: Regeneration of discarded human kidneys has been considered as an ideal approach to overcome organ shortage for the end-stage renal diseases (ESRDs). The aim of this study was to develop an effective method for preparation of kidney scaffolds that retain the matrix structure required for proliferation and importantly, differentiation of human adipose-derived mesenchymal stem cells (hAd-MSCs) into renal cells. MAIN METHODS: We first compared two different methods using triton X-100 and sodium dodecyl sulfate (SDS) for human kidney decellularization; followed by characterization of the prepared human renal extracellular matrix (ECM) scaffolds. Then, hAd-MSCs were seeded on the scaffolds and cultured for up to 3 weeks. Next, viability, proliferation, and migration of seeded hAd-MSCs underwent histological and scanning electron microscopy (SEM) assessments. Moreover, differentiation of hAd-MSCs into kidney-specific cell types was examined using immunohistochemistry (IHC) staining and qRT-PCR. KEY FINDINGS: Our results indicated that triton X-100 was a more effective detergent for decellularization of human kidneys compared with SDS. Moreover, attachment and proliferation of hAd-MSCs within the recellularized human kidney scaffolds, were confirmed. Seeded cells expressed epithelial and endothelial differentiation markers, and qRT-PCR results indicated increased expression of platelet and endothelial cell adhesion molecule 1 (PECAM-1), paired box 2 (PAX2), and E-cadherine (E-CDH) as markers of differentiation into epithelial and endothelial cells. SIGNIFICANCE: These observations indicate the effectiveness of decellularization with triton X-100 to generate suitable human ECM renal scaffolds, which supported adhesion and proliferation of hAd-MSCs and could induce their differentiation towards a renal lineage.


Assuntos
Rim/citologia , Octoxinol/farmacologia , Engenharia Tecidual/métodos , Bioengenharia/métodos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Detergentes/química , Células Endoteliais/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Humanos , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/metabolismo , Octoxinol/análise , Octoxinol/química , Dodecilsulfato de Sódio/química , Tecidos Suporte/química
12.
Physiol Genomics ; 54(2): 45-57, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-34890513

RESUMO

Recent studies have identified at least 20 different kidney cell types based upon chromatin structure and gene expression. Histone deacetylases (HDACs) are epigenetic transcriptional repressors via deacetylation of histone lysines resulting in inaccessible chromatin. We reported that kidney epithelial HDAC1 and HDAC2 activity is critical for maintaining a healthy kidney and preventing fluid-electrolyte abnormalities. However, to what extent does Hdac1/Hdac2 knockdown affect chromatin structure and subsequent transcript expression in the kidney? To answer this question, we used single nucleus assay for transposase-accessible chromatin-sequencing (snATAC-seq) and snRNA-seq to profile kidney nuclei from male and female, control, and littermate kidney epithelial Hdac1/Hdac2 knockdown mice. Hdac1/Hdac2 knockdown resulted in significant changes in the chromatin structure predominantly within the promoter region of gene loci involved in fluid-electrolyte balance such as the aquaporins, with both increased and decreased accessibility captured. Moreover, Hdac1/Hdac2 knockdown resulted different gene loci being accessible with a corresponding increased transcript number in the kidney, but among all mice only 24%-30% of chromatin accessibility agreed with transcript expression (e.g., open chromatin and increased transcript). To conclude, although chromatin structure does affect transcription, ∼70% of the differentially expressed genes cannot be explained by changes in chromatin accessibility and HDAC1/HDAC2 had a minimal effect on these global patterns. Yet, the genes that are targets of HDAC1 and HDAC2 are critically important for maintaining kidney function.


Assuntos
Cromatina/genética , Células Epiteliais/metabolismo , Histona Desacetilase 1/genética , Histona Desacetilase 2/genética , Rim/metabolismo , Transcriptoma/genética , Animais , Aquaporina 1/genética , Aquaporina 1/metabolismo , Aquaporina 2/genética , Aquaporina 2/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cromatina/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação/métodos , Feminino , Perfilação da Expressão Gênica/métodos , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Rim/citologia , Masculino , Camundongos Knockout , RNA-Seq/métodos
13.
Development ; 149(1)2022 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-34878095

RESUMO

Expansion of interstitial cells in the adult kidney is a hallmark of chronic disease, whereas their proliferation during fetal development is necessary for organ formation. An intriguing difference between adult and neonatal kidneys is that the neonatal kidney has the capacity to control interstitial cell proliferation when the target number has been reached. In this study, we define the consequences of inactivating the TGFß/Smad response in the mouse interstitial cell lineage. We find that pathway inactivation through loss of Smad4 leads to overproliferation of interstitial cells regionally in the kidney medulla. Analysis of markers for BMP and TGFß pathway activation reveals that loss of Smad4 primarily reduces TGFß signaling in the interstitium. Whereas TGFß signaling is reduced in these cells, marker analysis shows that Wnt/ß-catenin signaling is increased. Our analysis supports a model in which Wnt/ß-catenin-mediated proliferation is attenuated by TGFß/Smad to ensure that proliferation ceases when the target number of interstitial cells has been reached in the neonatal medulla.


Assuntos
Proliferação de Células , Rim/metabolismo , Proteína Smad4/metabolismo , Animais , Células Cultivadas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Rim/citologia , Rim/crescimento & desenvolvimento , Células Mesangiais/citologia , Células Mesangiais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína Smad4/genética , Fator de Crescimento Transformador beta/metabolismo , Via de Sinalização Wnt
14.
Tissue Cell ; 74: 101699, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34891081

RESUMO

Patients with end-stage renal disease often need dialysis to maintain their lives because of donor organ shortage. The creation of a transplantable graft to permanently replace kidney function would overcome the organ shortage problem and the morbidity associated with immunosuppression. In the present study, we decellularized rat kidneys by the perfusion of detergent, yielding acellular scaffolds with the vascular, uretic, as well as cortical and medullary architecture. To regenerate the functional organ, we seeded tubular epithelial cells and mouse kidney progenitor cells from the ureter together with endothelial cells and mouse kidney progenitor cells from the renal artery. The renal constructs from seeded cells were cultured in a whole-organ bioreactor. After 3 months of organ culture, the seeded cells formed renal tubules, grew in the glomeruli, and some mouse kidney progenitor cells were also scattered in the interstitium. We tested the function of the bioengineered kidney with standardized perfusate in vitro. The bioengineered kidney not only produced urine but also reabsorbed albumin, glucose, and calcium. We conclude that seeded cell-based bioengineering of kidneys with physiological secreting and reabsorbing properties is possible and holds therapeutic promise.


Assuntos
Reatores Biológicos , Rim/química , Rim/metabolismo , Células-Tronco/metabolismo , Engenharia Tecidual , Tecidos Suporte/química , Animais , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Humanos , Rim/citologia , Camundongos , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-Dawley , Células-Tronco/citologia
15.
Adv Sci (Weinh) ; 9(2): e2102358, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34747142

RESUMO

Rapid and high-resolution histological imaging with minimal tissue preparation has long been a challenging and yet captivating medical pursuit. Here, the authors propose a promising and transformative histological imaging method, termed computational high-throughput autofluorescence microscopy by pattern illumination (CHAMP). With the assistance of computational microscopy, CHAMP enables high-throughput and label-free imaging of thick and unprocessed tissues with large surface irregularity at an acquisition speed of 10 mm2 /10 s with 1.1-µm lateral resolution. Moreover, the CHAMP image can be transformed into a virtually stained histological image (Deep-CHAMP) through unsupervised learning within 15 s, where significant cellular features are quantitatively extracted with high accuracy. The versatility of CHAMP is experimentally demonstrated using mouse brain/kidney and human lung tissues prepared with various clinical protocols, which enables a rapid and accurate intraoperative/postoperative pathological examination without tissue processing or staining, demonstrating its great potential as an assistive imaging platform for surgeons and pathologists to provide optimal adjuvant treatment.


Assuntos
Encéfalo/citologia , Técnicas Histológicas/métodos , Rim/citologia , Pulmão/citologia , Microscopia/métodos , Aprendizado de Máquina não Supervisionado , Animais , Humanos , Camundongos , Modelos Animais
16.
J Ethnopharmacol ; 284: 114733, 2022 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-34644589

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Kidney stones is one of the common diseases of the urinary system. The primary cause of kidney stone formation is the thermodynamic supersaturation of lithogenic solutes in urine, which desaturates by nucleation, crystal growth and aggregation of minerals and salts, mainly Calcium oxalate (CaOx). One of the potential therapies is to develop drug molecules to inhibit or prevent CaOx crystallization in urine. Traditional Chinese medicines (TCMs) provided an efficient approach for the treatment of kidney stones with a specialized-designed recipe of medicinal herbs. But the action details of these herbs were poorly understood due to their complex components, and whether the effective constituents of herbs have an inhibitory effect on the process of stone formation has not been evaluated. AIM OF THE STUDY: This study aims to develop and identify inhibitor substitutes from a library of kidney stone prescriptions in traditional Chinese medicines to prevent pathological kidney stone formation. MATERIALS AND METHODS: As many as twenty Chinese medicines were extracted and separated into five different polar extracts, the inhibition performance of which on CaOx crystallization was explored by recording and comparing crystallization kinetics. The potential inhibitor molecules in the inhibitory extracts were confirmed by HPLC and their retardation efficacy was evaluated by quantifying nucleation and growth kinetics using colorimetry. Then the inhibitor-COM crystal interactions and specificity were examined by morphology evolution and surface structure analysis. In vitro inhibition performance of inhibitors on crystal growth and attachment of CaOx crystals to human renal epithelial cells were further evaluated by recording the nucleation and adhesive crystal numbers. RESULTS AND CONCLUSION: Water- and n-butanol- soluble extracts from 20 kinds of herbs show almost 100% inhibition percentage, and the n-butanol extracts was found better than commercial drug citrate. Twenty-one molecule substitutes were identified from these extracts, and among them polyphenols display the best inhibition efficacy to retard CaOx crystallization. The high-throughput colorimetric assay and morphology examinations reveals thirteen out of 21 molecules show inhibition potential and disrupt growth of CaOx monohydrate crystals by interacting with exposed Ca2+ and C2O42- on the (100) and (010) surfaces. Moreover, these inhibitors also display pronounced performance in protecting renal epithelial cells by inhibiting nucleation and adhesion of CaOx crystals to cells, thus reducing stone formation. The structure-performance correlation among 19 screened molecules that inhibitors having pKa<3.5, logD (pH = 6) <0, H-number>0.1 mmol are the best in suppressing CaOx crystallization. Our findings provide a novel solution to design and manufacture inhibitor drugs from Chinese medicines for preventing pathological kidney stones formation.


Assuntos
Oxalato de Cálcio/urina , Medicamentos de Ervas Chinesas/farmacologia , Células Epiteliais/efeitos dos fármacos , Cálculos Renais/prevenção & controle , Cristalização , Ensaios de Triagem em Larga Escala , Humanos , Técnicas In Vitro , Rim/citologia , Rim/efeitos dos fármacos , Medicina Tradicional Chinesa/métodos , Plantas Medicinais/química
17.
Int J Mol Sci ; 22(24)2021 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-34948355

RESUMO

Cellular senescence of renal tubular cells is associated with chronic diseases and age-related kidney disorders. Therapies to antagonize senescence are, therefore, explored as novel approaches in nephropathy. Exosomes derived from human mesenchymal stroma-/stem-like cells (MSC) entail the transfer of multiple bioactive molecules, exhibiting profound regenerative potential in various tissues, including therapeutic effects in kidney diseases. Here, we first demonstrate that exosomes promote proliferation and reduce senescence in aged MSC cultures. For potential therapeutic perspectives in organ rejuvenation, we used MSC-derived exosomes to antagonize senescence in murine kidney primary tubular epithelial cells (PTEC). Exosome treatment efficiently reduced senescence while diminishing the transcription of senescence markers and senescence-associated secretory phenotype (SASP) factors. Concomitantly, we observed less DNA damage foci and more proliferating cells. These data provide new information regarding the therapeutic property of MSC exosomes in the development of renal senescence, suggesting a contribution to a new chapter of regenerative vehicles in senotherapy.


Assuntos
Senescência Celular , Células Epiteliais/citologia , Exossomos/metabolismo , Rim/citologia , Células-Tronco Mesenquimais/citologia , Animais , Células Cultivadas , Células Epiteliais/metabolismo , Exossomos/transplante , Humanos , Rim/metabolismo , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos C57BL
18.
STAR Protoc ; 2(4): 100998, 2021 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-34950883

RESUMO

A key facet of epithelial differentiation is the assembly of actin-based protrusions known as microvilli, which amplify apical membrane surface area for various cell functions. To probe mechanisms of microvillus assembly, we developed a protocol using spinning disk confocal microscopy to directly visualize microvillus biogenesis on the surface of cultured porcine kidney epithelial cell monolayers engineered to express fluorescent proteins. This protocol offers access to the molecular details of individual protrusion growth events at high spatiotemporal resolution. For complete details on the use and execution of this protocol, please refer to Gaeta et al. (2021).


Assuntos
Células Epiteliais/citologia , Rim/citologia , Microscopia Confocal/métodos , Microvilosidades , Animais , Técnicas de Cultura de Células , Células Cultivadas , Microvilosidades/química , Microvilosidades/metabolismo , Suínos
19.
Cell Mol Life Sci ; 79(1): 53, 2021 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-34950960

RESUMO

SIRT6 is an NAD+ dependent deacetylase that belongs to the mammalian sirtuin family. SIRT6 is mainly located in the nucleus and regulates chromatin remodeling, genome stability, and gene transcription. SIRT6 extensively participates in various physiological activities such as DNA repair, energy metabolism, oxidative stress, inflammation, and fibrosis. In recent years, the role of epigenetics such as acetylation modification in renal disease has gradually received widespread attention. SIRT6 reduces oxidative stress, inflammation, and renal fibrosis, which is of great importance in maintaining cellular homeostasis and delaying the chronic progression of kidney disease. Here, we review the structure and biological function of SIRT6 and summarize the regulatory mechanisms of SIRT6 in kidney disease. Moreover, the role of SIRT6 as a potential therapeutic target for the progression of kidney disease will be discussed. SIRT6 plays an important role in kidney disease. SIRT6 regulates mitochondrial dynamics and mitochondrial biogenesis, induces G2/M cycle arrest, and plays an antioxidant role in nephrotoxicity, IR, obstructive nephropathy, and sepsis-induced AKI. SIRT6 prevents and delays progressive CKD induced by hyperglycemia, kidney senescence, hypertension, and lipid accumulation by regulating mitochondrial biogenesis, and has antioxidant, anti-inflammatory, and antifibrosis effects. Additionally, hypoxia, inflammation, and fibrosis are the main mechanisms of the AKI-to-CKD transition. SIRT6 plays a critical role in the AKI-to-CKD transition and kidney repair through anti-inflammatory, antifibrotic, and mitochondrial quality control mechanisms. AKI Acute kidney injury, CKD Chronic kidney disease.


Assuntos
Nefropatias/metabolismo , Rim/metabolismo , Sirtuínas , Animais , Epigênese Genética , Humanos , Rim/citologia , Rim/patologia , Camundongos , Mitocôndrias/metabolismo , Sirtuínas/química , Sirtuínas/fisiologia
20.
Dev Cell ; 56(22): 3042-3051, 2021 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-34813766

RESUMO

Adult organs are vascularized by specialized blood vessels. In addition to inter-organ vascular heterogeneity, each organ is arborized by structurally and functionally diversified populations of endothelial cells (ECs). The molecular pathways that are induced to orchestrate inter- and intra- organ vascular heterogeneity and zonation are shaped during development and fully specified postnatally. Notably, intra-organ specialization of ECs is associated with induction of angiocrine factors that guide cross-talk between ECs and parenchymal cells, establishing co-zonated vascular regions within each organ. In this review, we describe how microenvironmental tissue-specific biophysical, biochemical, immune, and inflammatory cues dictate the specialization of ECs with zonated functions. We delineate how physiological and biophysical stressors in the developing liver, lung, and kidney vasculature induce specialization of capillary beds. Deciphering mechanisms by which vascular microvasculature diversity is attained could set the stage for treating regenerative disorders and promote healing of organs without provoking fibrosis.


Assuntos
Células Endoteliais/citologia , Rim/irrigação sanguínea , Microvasos/citologia , Neovascularização Fisiológica/fisiologia , Regeneração/fisiologia , Animais , Diferenciação Celular/fisiologia , Humanos , Rim/citologia
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