Abstract:Labarca M, Nizar JM, Walczak EM, Dong W, Pao AC, Bhalla V. Harvest and primary culture of the murine aldosterone-sensitive distal nephron. Am J Physiol Renal Physiol 308: F1306 -F1315, 2015. First published March 25, 2015 doi:10.1152/ajprenal.00668.2014The aldosterone-sensitive distal nephron (ASDN) exhibits axial heterogeneity in structure and function from the distal convoluted tubule to the medullary collecting duct. Ion and water transport is primarily divided between the cortex and medulla of the ASDN, r… Show more
“…DBA-positive staining of differentiated tubuloids in our system reinforced the upregulation of principal cell-specific transcription factors ( Fig. 3 C) ( Chen et al, 2017 ; Labarca et al, 2015 ; Murata et al, 1983 ). Fig.…”
Cystogenesis is a morphological consequence of numerous genetic diseases of the epithelium. In the kidney, the pathogenic mechanisms underlying the program of altered cell and tubule morphology are obscured by secondary effects of cyst expansion. Here, we developed a new 3D tubuloid system to isolate the rapid changes in protein localization and gene expression that correlate with altered cell and tubule morphology during cyst initiation. Mouse renal tubule fragments were pulsed with a cell differentiation cocktail including glial-derived neurotrophic factor (GDNF) to yield collecting duct-like tubuloid structures with appropriate polarity, primary cilia, and gene expression. Using the 3D tubuloid model with an inducible Pkd2 knockout system allowed the tracking of morphological, protein, and genetic changes during cyst formation. Within hours of inactivation of Pkd2 and loss of polycystin-2, we observed significant progression in tubuloid to cyst morphology that correlated with 35 differentially expressed genes, many related to cell junctions, matrix interactions, and cell morphology previously implicated in cystogenesis.This article has an associated First Person interview with the first author of the paper.
“…DBA-positive staining of differentiated tubuloids in our system reinforced the upregulation of principal cell-specific transcription factors ( Fig. 3 C) ( Chen et al, 2017 ; Labarca et al, 2015 ; Murata et al, 1983 ). Fig.…”
Cystogenesis is a morphological consequence of numerous genetic diseases of the epithelium. In the kidney, the pathogenic mechanisms underlying the program of altered cell and tubule morphology are obscured by secondary effects of cyst expansion. Here, we developed a new 3D tubuloid system to isolate the rapid changes in protein localization and gene expression that correlate with altered cell and tubule morphology during cyst initiation. Mouse renal tubule fragments were pulsed with a cell differentiation cocktail including glial-derived neurotrophic factor (GDNF) to yield collecting duct-like tubuloid structures with appropriate polarity, primary cilia, and gene expression. Using the 3D tubuloid model with an inducible Pkd2 knockout system allowed the tracking of morphological, protein, and genetic changes during cyst formation. Within hours of inactivation of Pkd2 and loss of polycystin-2, we observed significant progression in tubuloid to cyst morphology that correlated with 35 differentially expressed genes, many related to cell junctions, matrix interactions, and cell morphology previously implicated in cystogenesis.This article has an associated First Person interview with the first author of the paper.
“…S1 B .) We chose fluorophore-labeled Dolichos biflorus agglutinin (DBA), a lectin, to decorate principal cells (PCs) (9, 10). Initially, we carried out RNA-Seq analysis of pooled flow-sorted c-Kit + cells, comparing them to DBA + cells.…”
Prior RNA sequencing (RNA-Seq) studies have identified complete transcriptomes for most renal epithelial cell types. The exceptions are the cell types that make up the renal collecting duct, namely intercalated cells (ICs) and principal cells (PCs), which account for only a small fraction of the kidney mass, but play critical physiological roles in the regulation of blood pressure, extracellular fluid volume and extracellular fluid composition. To enrich these cell types, we used fluorescence-activated cell sorting (FACS) that employed well established lectin cell surface markers for PCs and type B ICs, as well as a newly identified cell surface marker for type A ICs, viz. c-Kit. Single-cell RNA-Seq using the 1C- and PC-enriched populations as input enabled identification of complete transcriptomes of A-ICs, B-ICs and PCs. The data were used to create a freely-accessible online gene-expression database for collecting duct cells. This database allowed identification of genes that are selectively expressed in each cell type including cell-surface receptors, transcription factors, transporters and secreted proteins. The analysis also identified a small fraction of hybrid cells expressing both aquapor¡n-2 and either anion exchanger 1 or pendrin transcripts. In many cases, mRNAs for receptors and their ligands were identified in different cells (e.g. Notch2 chiefly in PCs vs Jag1 chiefly in ICs) suggesting signaling crosstalk among the three cell types. The identified patterns of gene expression among the three types of collecting duct cells provide a foundation for understanding physiological regulation and pathophysiology in the renal collecting duct.SIGNIFICANCE STATEMENTA long-term goal in mammalian biology is to identify the genes expressed in every cell type of the body. In kidney, the expressed genes (“transcriptome”) of all epithelial cell types have already been identified with the exception of the cells that make up the renal collecting duct, responsible for regulation of blood pressure and body fluid composition. Here, a technique called "single-cell RNA-Seq" was used in mouse to identify transcriptomes for the major collecting-duct cell types: type A intercalated cells, type B intercalated cells and principal cells. The information was used to create a publicly-accessible online resource. The data allowed identification of genes that are selectively expressed in each cell type, informative for cell-level understanding of physiology and pathophysiology.
“…We isolated kidney tubular cells from mice treated with vehicle or furosemide for 3 weeks. Once isolated and re-immersed in PBS buffer, we incubated samples at 1:200 dilutions with LTL-FITC to detect proximal tubular cells and Dolichos Biflorus Agglutinin–rhodamine (DBA-Rh) to detect principal cells 101,102 . During this incubation, we slowly rotated samples at room temperature before placing on ice.…”
Section: Methodsmentioning
confidence: 99%
“…After spin down for 2 minutes at 1000 g, we resuspended pellets in mRING containing 0.2% collagenase and 0.2% hyaluronidase then shook at 2,600 rpm for 45 minutes at 37 o C. We disrupted tubular fragments by passing the pellet gently through an 18-gauge needle 3x before 30-minute incubation with DNAse at 37 o C. We further washed and centrifuged pellets at 1,000 rpm for 2 min multiple times until solution was clear in order to remove debris. For flow cytometry experiments, we re-suspended pellets in PBS, passed 2X through a 40 µm strainer and then 2X through a 20 µm strainer, spun one time at 1,000 g for 10 minutes, incubated with fluorophoreconjugated lectin and/or DAPI for 30 min at RT, and then placed on ice 102…”
Furosemide, a widely prescribed diuretic for edema-forming states, inhibits sodium reabsorption in the thick ascending limb of the nephron. Tubular adaptation to diuretics has been observed, but the range of mechanisms along the nephron has not been fully explored. Using morphometry, we show that furosemide induces renal tubular epithelial hyperplasia selectively in distal nephron segments. By comparison, we find progressive cellular hypertrophy in proximal and distal nephron segments. We next utilize single cell RNA sequencing of vehicle- and furosemide-treated mice to define potential mechanisms of diuretic resistance. Consistent with distal tubular cell hyperplasia, we detect a net increase in DCT cell number and Birc5, an anti-apoptotic and pro-growth gene, in a subset of DCT cells, as the most prominently up-regulated gene across the nephron. We also map a gradient of cell-specific transcriptional changes congruent with enhanced distal sodium transport. Furosemide stimulates expression of the mitogen IGF-1. Thus, we developed a mouse model of inducible deletion of renal tubular IGF-1 receptor and show reduced kidney growth and proximal, but not distal, tubular hypertrophy by furosemide. Moreover, genes that promote enhanced bioavailability of IGF-1 including Igfbp1 and Igfbp5 are significantly and differentially expressed in proximal tubular segments and correspond to IGF-1R-dependent hypertrophy. In contrast, downstream PI3-kinase signaling genes including Pdk1, Akt1, Foxo3, FKBP4, Eif2BP4, and Spp1 are significantly and differentially expressed in distal nephron segments and correspond to IGF-1R-independent hypertrophy. These findings highlight novel mechanisms of tubular remodeling and diuretic resistance, provide a repository of transcriptional responses to a common drug, and expand the implications of long-term loop diuretic use for human disease.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
