Duct Cell Types Research Articles

Identifying Molecular Regulators of Kidney Collecting Duct Cell Type Plasticity

Identifying Molecular Regulators of Kidney Collecting Duct Cell Type Plasticity

Galanin System in the Human Bile Duct and Perihilar Cholangiocarcinoma.

Perihilar cholangiocarcinoma (pCCA) is characterised by poor outcomes. Early diagnosis is essential for patient survival. The peptide galanin (GAL) and its receptors GAL1-3 are expressed in various tumours. Detailed characterisation of the GAL system in pCCA is lacking. Our study sought to characterise GAL and GAL1-3 receptor (GAL1-3-R) expression in the healthy human bile duct, in cholestasis and pCCA. Immunohistochemical staining was performed in healthy controls (n = 5) and in the peritumoural tissues (with and without cholestasis) (n = 20) and tumour tissues of pCCA patients (n = 33) using validated antibodies. The score values of GAL and GAL1-3-R expression were calculated and statistically evaluated. GAL and GAL1-R were expressed in various bile duct cell types. GAL2-R was only slightly but still expressed in almost all the examined tissues, and GAL3-R specifically in cholangiocytes and capillaries. In a small pCCA patient cohort (n = 18), high GAL expression correlated with good survival, whereas high GAL3-R correlated with poor survival. Our in-depth characterisation of the GAL system in the healthy human biliary duct and pCCA in a small patient cohort revealed that GAL and GAL3-R expression in tumour cells of pCCA patients could potentially represent suitable biomarkers for survival.

Human Ureteric Bud Organoids Derived From Pluripotent Stem Cells Recapitulate Embryonic Development and Differentiate Into Collecting Duct Cell Types With Functional Ion Transport

Human Ureteric Bud Organoids Derived From Pluripotent Stem Cells Recapitulate Embryonic Development and Differentiate Into Collecting Duct Cell Types With Functional Ion Transport

Human ureteric bud organoids recapitulate branching morphogenesis and differentiate into functional collecting duct cell types.

Directed differentiation of human pluripotent stem cells (hPSCs) into functional ureteric and collecting duct (CD) epithelia is essential to kidney regenerative medicine. Here we describe highly efficient, serum-free differentiation of hPSCs into ureteric bud (UB) organoids and functional CD cells. The hPSCs are first induced into pronephric progenitor cells at 90% efficiency and then aggregated into spheres with a molecular signature similar to the nephric duct. In a three-dimensional matrix, the spheres form UB organoids that exhibit branching morphogenesis similar to the fetal UB and correct distal tip localization of RET expression. Organoid-derived cells incorporate into the UB tips of the progenitor niche in chimeric fetal kidney explant culture. At later stages, the UB organoids differentiate into CD organoids, which contain >95% CD cell types as estimated by single-cell RNA sequencing. The CD epithelia demonstrate renal electrophysiologic functions, with ENaC-mediated vectorial sodium transport by principal cells and V-type ATPase proton pump activity by FOXI1-induced intercalated cells.

Human Pluripotent Stem Cell-Derived Kidney Organoids with Improved Collecting Duct Maturation and Injury Modeling

Maximizing the potential of human kidney organoids for drug testing and regenerative medicine and to model development and disease requires addressing cell immaturity, the lack of a mature collecting system, and off-target cell types. By independently generating two kidney progenitor cell populations-metanephric mesenchyme and ureteric bud (UB)-like cells-we could generate kidney organoids with a collecting system. We also identify the hormones aldosterone and arginine vasopressin (AVP) as critical to promote differentiation of collecting duct cell types including both principal cells (PCs) and intercalated cells (ICs). The resulting PCs express aquaporin-2 (AQP2) protein, which undergoes translocation to the apical membrane after vasopressin or forskolin stimulation. By single-cell RNA sequencing (scRNA-seq), we demonstrate improved proximal tubule maturation and reduced off-target cell populations. We also show appropriate downregulation of progenitor cell types, improved modeling of tubular injury, the presence of urothelium (Uro), and the ability of Notch pathway modulation to regulate PC:IC ratios during organoid development.

Aldosterone‐Induced Transcripts Identified from Rapidly Isolated Collecting Duct Cells

In the distal nephron, the steroid hormone aldosterone regulates sodium reabsorption via the epithelial sodium channel ENaC. Previous attempts to identify the aldosterone‐induced proteins that mediate the action of aldosterone on ENaC have primarily used transcriptional profiling of cultured cells. Here, we present a method to rapidly isolate collecting duct cells from the mouse kidney for transcriptional profiling. Our objective is to use this method, in combination with dietary sodium manipulation, to identify transcripts that are differentially expressed in the collecting ducts of mouse kidney under conditions of high versus low plasma aldosterone.An antibody against the cell adhesion molecule L1‐CAM was previously used for fluorescence activated cell sorting (FACS) of collecting duct cells from human kidney. We verified L1‐CAM is also expressed in the collecting ducts of mouse kidney using immunofluorescence. We next developed a protocol for physical and enzymatic separation of mouse kidney into a single cell suspension. The kidney cells were labeled with L1‐CAM magnetic microbeads as well as antibodies against markers for endothelial cells (CD31) and immune cells (CD45). This allowed isolation of an L1‐CAM+ cell population using magnetic activated cell sorting (MACS), followed by separation of collecting duct cells from L1‐CAM+ endothelial and immune cells using FACS. RNA was purified from the FACS‐isolated collecting duct cells and, following poly(A) selection, used to generate Illumina TruSeq libraries. The libraries were sequenced on the Illumina HiSeq platform and the reads were aligned to the mouse genome using the Subread aligner. The aligned reads were mapped to transcripts using feature Counts and differentially expressed transcripts are identified using EdgeR.To identify transcripts that are differentially expressed in the collecting ducts of mouse kidney under conditions of high versus low plasma aldosterone, C57BL/6J mice were fed sodium‐deficient diet (to stimulate endogenous aldosterone) or high‐sodium diet (to suppress endogenous aldosterone). After 5 days of dietary sodium manipulation, the kidneys were harvested, collecting duct cells were isolated, and RNA was sequenced and analyzed according to the above protocol. Of the differentially expressed transcripts, 234 were up‐regulated by aldosterone (more highly expressed in the sodium‐deficient diet samples) while 276 were down‐regulated by aldosterone (more highly expressed in the high‐sodium diet samples); false discovery rate < 0.05, no log2FC or log2CPM threshold. The transcriptome data were then compared with recently published single‐cell RNA‐seq data for the major collecting duct cell types, specifically intercalated cells and principal cells. It was found that 16 of the up‐regulated transcripts and 32 of the down‐regulated transcripts were highly expressed in principal cells, thereby limiting the number of candidate transcripts for subsequent validation. Of the transcripts that were highly expressed in principal cells, Sgk1 was the most differentially expressed transcript induced by aldosterone.Support or Funding InformationU.S. Department of Veterans Affairs: I01BX002228This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Transcriptomes of major renal collecting duct cell types in mouse identified by single-cell RNA-seq

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 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, c-Kit. Single-cell RNA-seq using the IC- 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 aquaporin-2 and 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 cross-talk 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.

Elf5 is a principal cell lineage specific transcription factor in the kidney that contributes to Aqp2 and Avpr2 gene expression

The mammalian kidney collecting ducts are critical for water, electrolyte and acid-base homeostasis and develop as a branched network of tubular structures composed of principal cells intermingled with intercalated cells. The intermingled nature of the different collecting duct cell types has made it challenging to identify unique and critical factors that mark and/or regulate the development of the different collecting duct cell lineages. Here we report that the canonical Notch signaling pathway components, RBPJ and Presinilin1 and 2, are involved in patterning the mouse collecting duct cell fates by maintaining a balance between principal cell and intercalated cell fates. The relatively reduced number of principal cells in Notch-signaling-deficient kidneys offered a unique genetic leverage to identify critical principal cell-enriched factors by transcriptional profiling. Elf5, which codes for an ETS transcription factor, is one such gene that is down-regulated in kidneys with Notch-signaling-deficient collecting ducts. Additionally, Elf5 is among the earliest genes up regulated by ectopic expression of activated Notch1 in the developing collecting ducts. In the kidney, Elf5 is first expressed early within developing collecting ducts and remains on in mature principal cells. Lineage tracing of Elf5-expressing cells revealed that they are committed to the principal cell lineage by as early as E16.5. Over-expression of ETS Class IIa transcription factors, including Elf5, Elf3 and Ehf, increase the transcriptional activity of the proximal promoters of Aqp2 and Avpr2 in cultured ureteric duct cell lines. Conditional inactivation of Elf5 in the developing collecting ducts results in a small but significant reduction in the expression levels of Aqp2 and Avpr2 genes. We have identified Elf5 as an early maker of the principal cell lineage that contributes to the expression of principal cell specific genes.

Ultrastructural Characterization of Mammary Analogue Secretory Carcinoma of the Salivary Glands: A Distinct Entity from Acinic Cell Carcinoma?

Mammary analogue secretory carcinoma (MASC) of the salivary glands is a recently described neoplasm of the salivary glands with a characteristic morphology complemented by a specific cytogenetic translocation and gene rearrangements. Although immunophenotypic and cytogenetic differences allow for a more reliable distinction, ultrastructural features can also provide important information about the relationship between MASC, classic acinic cell carcinoma (AciCC), and AciCC intercalated duct cell-predominant variant. Following approval from the hospital's institutional review board, 7 cases of MASC, 8 cases of classic AciCC, and 4 cases of AciCC intercalated duct cell-predominant variant were retrieved from the pathology files of Massachusetts General Hospital from 2012 to 2015. Electron microscopy was performed using formalin-fixed, paraffin-embedded tissue. Ultrastructural features of all 19 neoplasms of the salivary glands were recorded. The predominant cell-types observed in MASCare those with intercalated/striated duct cell differentiation. These features include prominent invaginations of the cell surface studded with microvilli, and some intra- and intercellular lumina also with a microvillous surface. Classic AciCC dominant cell-type recapitulates acinar cell differentiation. These cells contain large intracytoplasmic zymogen-like granules. AciCC intercalated duct cell-predominant variant showed both cell populations in various proportions with the intercalated/striated duct cell type usually being the dominant one. MASC presents with distinctive ultrastructural features that allows its proper differentiation from classic AciCC. However, significant ultrastructural features overlaps between both AciCC intercalated duct cells-predominant and classic AciCC and MASC. These findings indicate a very close proximity between these tumors.

Ascl3 knockout and cell ablation models reveal complexity of salivary gland maintenance and regeneration

Expression of the transcription factor, Ascl3, marks a population of adult progenitor cells, which can give rise to both acinar and duct cell types in the murine salivary glands. Using a previously reported Ascl3EGFP-Cre/+ knock-in strain, we demonstrate that Ascl3-expressing cells represent a molecularly distinct, and proliferating population of progenitor cells located in salivary gland ducts. To investigate both the role of the Ascl3 transcription factor, and the role of the cells in which it is expressed, we generated knockout and cell-specific ablation models. Ascl3 knockout mice develop smaller salivary glands than wild type littermates, but secrete saliva normally. They display a lower level of cell proliferation, consistent with their smaller size. In the absence of Ascl3, the cells maintain their progenitor function and continue to generate both acinar and duct cells. To directly test the role of the progenitor cells, themselves, in salivary gland development and regeneration, we used Cre-activated expression of diphtheria toxin (DTA) in the Ascl3-expressing (Ascl3+) cell population, resulting in specific cell ablation of Ascl3+ cells. In the absence of the Ascl3+ progenitor cells, the mice developed morphologically normal, albeit smaller, salivary glands able to secrete saliva. Furthermore, in a ductal ligation model of salivary gland injury, the glands of these mice were able to regenerate acinar cells. Our results indicate that Ascl3+ cells are active proliferating progenitors, but they are not the only precursors for salivary gland development or regeneration. We conclude that maintenance of tissue homeostasis in the salivary gland must involve more than one progenitor cell population.

Impaired sodium excretion and increased blood pressure in mice with targeted deletion of renal epithelial insulin receptor

Renal tubule epithelial cells express the insulin receptor (IR); however, their value has not been firmly established. We generated mice with renal epithelial cell-specific knockout of the IR by Cre-recombinase-loxP recombination using a kidney-specific (Ksp) cadherin promoter. KO mice expressed significantly lower levels of IR mRNA and protein in kidney cortex (49-56% of the WT) and medulla (32-47%) homogenates. Immunofluorescence showed the greatest relative reduction in the thick ascending limb and collecting duct cell types. Body weight, kidney weight, and food and water intakes were not different from WT littermates. However, KO mice had significantly increased basal systolic blood pressure (BP, 15 mm Hg higher) as measured by radiotelemetry. In response to a volume load by gavage (20 ml/kg of body weight, 0.9% NaCl, 15% dextrose), KO mice had impaired natriuresis (37 +/- 10 versus 99 +/- 9 mmol of Na(+) per 2 h in WT). Furthermore, volume load led to a sustained increase in BP in KO mice only. In contrast, insulin administration i.p. (0.5 units/kg of body weight) resulted in a significant fall in BP in WT, but not in KO mice. To test the role of reduced renal nitric oxide (NO) production in these responses, basal urinary nitrates plus nitrites excretion (UNOx) was measured and found to be 61% lower in KO vs. WT mice. Furthermore, acute insulin increased UNOx by 202% in the WT, relative to a significantly blunted rise (67%) in KO animals. These results illuminate a previously uncharacterized role for renal IR to reduce BP and facilitate sodium and water excretion, possibly via NO production.

Notch signaling reveals developmental plasticity of Pax4+ pancreatic endocrine progenitors and shunts them to a duct fate

Relatively little is known about the developmental signals that specify the types and numbers of pancreatic cells. Previous studies suggested that Notch signaling in the pancreas inhibits differentiation and promotes the maintenance of progenitor cells, but it remains unclear whether Notch also controls cell fate choices as it does in other tissues. To study the impact of Notch in progenitors of the β cell lineage, we generated mice that express Cre-recombinase under control of the Pax4 promoter. Lineage analysis of Pax4 + cells demonstrates they are specified endocrine progenitors that contribute equally to four islet cell fates, contrary to expectations raised by the dispensable role of Pax4 in the specification of the α and PP subtypes. In addition, we show that activation of Notch in Pax4 + progenitors inhibits their differentiation into α and β endocrine cells and shunts them instead toward a duct fate. These observations reveal an unappreciated degree of developmental plasticity among early endocrine progenitors and raise the possibility that a bipotent duct-endocrine progenitor exists during development. Furthermore, the redirection of Pax4 + cells from α and β endocrine fates toward a duct cell type suggests a positive role for Notch signaling in duct specification and is consistent with the more widely defined role for Notch in cell fate determination.

Induction of Pancreatic Stem/Progenitor Cells into Insulin-Producing Cells by Adenoviral-Mediated Gene Transfer Technology

beta-Cell replacement therapy via islet transplantation is a promising possibility for the optimal treatment of type 1 diabetes; however, such an approach is severely limited by the shortage of donor organs. This problem could be overcome if it were possible to generate transplantable islets from stem cells. We showed previously that adult beta-cells might originate from duct or duct-associated cells. Ductal progenitor cells in the pancreas would become particularly useful for therapies that target beta-cell replacement in diabetic patients, because duct cell types are abundantly available in the pancreas of these patients and in donor organs. In this study, we examined which embryonic transcription factors in adult mouse and human duct cells could efficiently induce their differentiation into insulin-expressing cells. Infection with the adenovirus expressing PDX-1, Ngn3, NeuroD, or Pax4 induced the insulin gene expression. NeuroD was the most effective inducer of insulin expression in primary duct cells. Surprisingly, adenovirus Pax4 strongly induced Ngn3 expression, while Pax4 is considered the downstream target of Ngn3. These data suggest that the overexpression of transcription factors, especially NeuroD, facilitates pancreatic stem/progenitor cell differentiation into insulin-producing cells.

In vacuo isotope coded alkylation technique (IVICAT); an N‐terminal stable isotopic label for quantitative liquid chromatography/mass spectrometry proteomics

We present a new isotopic labeling strategy to modify the N-terminal amino group of peptides in a quantifiable reaction without the use of expensive reagents or solvents. The In Vacuo Isotope Coded Alkylation Technique (IVICAT) is a methylation reaction, carried out at low pressure (<100 mTorr), that results in a stable quaternary trimethylammonium group, thus adding a permanent positive charge at the N-terminus of peptides without modifying the epsilon-amino groups of lysine. The methylation reaction increases the signal intensity of modified peptides in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and liquid chromatography (LC)/MS and the isotopic peak pair differs by 9 mass units which can be easily resolved by either instrument. N-terminally trimethylated peptides exhibit collision-induced dissociation (CID) mass spectra that differ from their unmodified analogues by an enhanced b-ion series in MS2 spectra due to the fixed positive charge. Using LC/MS/MS with an LTQ mass analyzer for quantification, the experimentally determined ratios of H9- to D9-trimethyl-labeled peptides of beta-casein provided accurate estimates of the actual ratios with low % error. IVICAT labeling also accurately quantified proteins in rat kidney inner medullary collecting duct cell types, as judged by comparison with relative quantification by subsequent immunoblotting experiments. IVICAT labeling, when used in conjunction with the new proteomics software QUIL, can accurately report relative protein abundances and increase the sequence coverage of proteins of tissue proteomes.

Primary culture of collecting duct cell epithelium from neonate rabbit kidney in monolayer.

Cortical collecting duct cells from rabbit neonate kidney capsula fibrosa explants were grown in low (0.1%) serum medium on permeable culture supports. We found that over a period of 3-4 days in culture, the cells migrated from the explants, to form monolayers. The cells in monolayer displayed morphology similar to adult collecting duct cells. Approximately 24% of cells grown in monolayer were found to bind a specific beta-cell-probe, peanut agglutinin. Single cells were isolated by brief exposure to 0 Ca(2+)-Mg2+ solutions and the whole-cell clamp technique was used to measure macroscopic currents. The whole-cell conductance of 1.35 +/- 0.1 nS was reduced to 0.9 +/- 0.2 nS in the presence of 100 microM-amiloride. We conclude that these cells express cell membrane properties consistent with those of the collecting duct and that it may be a useful model for the study of cellular transport mechanisms and the factors controlling development of collecting duct cell types.

Endocytosis of native and glycosylated bovine serum albumin by duct cells of the rat parotid gland.

The ability of duct cells of the rat parotid gland to internalize bovine serum albumin (BSA) and several glycosylated albumins (glucosamide, galactosamide, fucosamide, lactosyl, p-aminophenyl-N-acetyl-D-glucosamide, p-aminophenyl-N-acetyl-D-mannopyranoside, p-aminophenyl-N-acetyl-D-galactosamide) was investigated. The various BSA preparations were infused into the gland via the main excretory duct, after which the tissues were fixed and prepared for light and electron microscopy. Immunolocalization of native BSA, as well as the glycosylated BSAs, was performed on thin sections, using an unlabeled antibody to BSA followed by protein A-colloidal gold. Gold particles were present over the lumina of both intercalated ducts and striated ducts, and over small endocytic structures and large vacuoles in the apical cytoplasm of both duct cell types. Endocytosis of the glycosylated BSAs by duct cells was greater than native BSA. Fucosylamide-BSA and mannopyranoside-BSA was taken up to a greater extent than the other glycosylated BSAs. Uptake by intercalated duct cells was greater than by striated duct cells, was independent of the concentration of the glycosylated BSA, and was reduced by an excess of the corresponding sugar. Striated duct cells showed some damage by the glycosylated BSAs that was concentration-dependent, and which was reduced in the presence of an excess of the corresponding sugar. These results suggest that endocytosis by salivary gland duct cells may involve specific recognition of carbohydrate residues and that the endocytosis of acinar secretory proteins observed in certain conditions may be due to increased and/or altered protein glycosylation.

Tumors of the exocrine pancreas in children and adolescents. A clinical and pathologic study of eight cases.

Eight patients with a tumor of the exocrine pancreas were treated or seen in consultation at the Children's Hospital Medical Center over the last 30 years. There were five girls and three boys ranging in age from 15 months to 18 years (average age: 8 years). Tumors were surgically resectable in six of eight cases--three by modified Whipple procedure and three by local resection. The average diameter of tumor was 13.6 cm (range: 3.8-20 cm). There were three adenocarcinomas of duct cell type, two acinar cell carcinomas, two acinar cell adenomas, and a papillary-cystic tumor of low-grade malignancy. One of the adenocarcinomas arose in a field of orthovoltage radiation and another occurred in a patient with tuberous sclerosis. Five of eight patients died, four with regional or distant metastases documented at autopsy. The patient with the papillary-cystic tumor experienced several local recurrences but was alive 14 years after initial surgery. Data from the literature indicate that acinar cell tumors are proportionately more common in children than in adults and have a somewhat better prognosis than tumors of duct cell type. Further reporting of cases with long-term follow-up is encouraged in order to gain clearer perspective of nonendocrine tumors of the pancreas in childhood.

Primary Carcinoma of the Liver (Cholangioma) with Hypoglycemic Convulsions

Summary A case of primary liver carcinoma of the bile duct cell type in a young negro has been presented. The outstanding manifestations were hypoglycemic convulsions. These were temporarily relieved by glucose but within a short time the convulsions came closer and lasted longer in spite of repeated concentrated infusion. Hepatic functional measurements showed abnormality but clinical manifestations of progressive hepatic failure exclusive of hypoglycemia were minimal.

Primary Liver Cell Carcinoma with Splenic Metastases

Primary liver-cell carcinoma is frequent among Malays and Mongols but relatively unusual in European races (Bonne). In Europe and the United States primary carcinoma of the liver, including both liver-cell and duct-cell types, is found in from 0.1 to 0.14 per cent of all necropsies (Herxheimer). Metastasis of carcinoma to the spleen is also generally considered to be of rare occurrence, though an accurate estimate of its frequency is difficult. Thus Yokohata found 6 cases of splenic metastases in a series of 29 carcinomas, Kurosu found 5 instances among 54 carcinomas, and Deelman 7 among 75. When, however, large numbers of unselected cases of carcinoma are systematically studied, the proportion of splenic metastases is found to be much smaller. Di Biasi encountered 50 examples in his own series of 2,422 cases (2.1 per cent) and 182 in 9,761 cases collected from other sources (1.9 per cent); Monti found splenic metastases in 28 of 649 cases of carcinoma and sarcoma; Warren and Hobson in 46 of 1,140 cases of carcinoma and sarcoma (4 per cent). Account must also be taken of differences in the morphologic criteria of splenic metastases. Those who find such metastases numerous generally include in their figures every case, without restriction, in which the presence of one or more cancer cells can be demonstrated in the spleen, even though these are free in the sinuses or blood vessels or occur only within the capsule. According to Krumbhaar and Scott, “cells in the blood stream do not constitute metastatic growth … tumor cells can be injected into or found in the circulation without necessarily causing tumor growth.” Braunstein pointed out that of Yokohata9s 6 cases only 2 were true splenic metastases, the others being either tumor emboli or growths in the capsule.