Dataset Information

Loss of Adam10 Disrupts Ion Transport in Immortalized Kidney Collecting Duct Cells.

ABSTRACT: The kidney cortical collecting duct (CCD) comprises principal cells (PCs), intercalated cells (IC), and the recently discovered intermediate cell type. Kidney pathology in a mouse model of the syndrome of apparent aldosterone excess revealed plasticity of the CCD, with altered PC:intermediate cell:IC ratio. The self-immortalized mouse CCD cell line, mCCD_cl1, shows functional characteristics of PCs, but displays a range of cell types, including intermediate cells, making it ideal to study plasticity. We knocked out Adam10, a key component of the Notch pathway, in mCCD_cl1 cells, using CRISPR-Cas9 technology, and isolated independent clones, which exhibited severely affected sodium transport capacity and loss of aldosterone response. Single-cell RNA sequencing revealed significantly reduced expression of major PC-specific markers, such as Scnn1g (γ-ENaC) and Hsd11b2 (11βHSD2), but no significant changes in transcription of components of the Notch pathway were observed. Immunostaining in the knockout clone confirmed the decrease in expression of γ-ENaC and importantly, showed an altered, diffuse distribution of PC and IC markers, suggesting altered trafficking in the Adam10 knockout clone as an explanation for the loss of polarization.

SUBMITTER: Assmus A

PROVIDER: S-EPMC8187228 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Loss of Adam10 Disrupts Ion Transport in Immortalized Kidney Collecting Duct Cells.

Assmus Adrienne A Mullins Linda L Ward Mairi M Dobie Ross R Hunter Robert R Henderson Neil C NC Mullins John J JJ

Function (Oxford, England) 20210510 4

The kidney cortical collecting duct (CCD) comprises principal cells (PCs), intercalated cells (IC), and the recently discovered intermediate cell type. Kidney pathology in a mouse model of the syndrome of apparent aldosterone excess revealed plasticity of the CCD, with altered PC:intermediate cell:IC ratio. The self-immortalized mouse CCD cell line, mCCD<sub>cl1</sub>, shows functional characteristics of PCs, but displays a range of cell types, including intermediate cells, making it ideal to st ...[more]

PMID: 34131651

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Project description:Kidneys are essential for acid-base homeostasis, especially when organisms cope with changes in acid or base dietary intake. Because collecting ducts constitute the final site for regulating urine acid-base balance, we undertook to identify the gene network involved in acid-base transport and regulation in the mouse outer medullary collecting duct (OMCD). For this purpose, we combined kidney functional studies and quantitative analysis of gene expression in OMCDs, by transcriptome and candidate gene approaches, during metabolic acidosis. Furthermore, to better delineate the set of genes concerned with acid-base disturbance, the OMCD transcriptome of acidotic mice was both compared to that of normal mice and mice undergoing an adaptative response through potassium depletion. Metabolic acidosis, achieved through a NH4Cl-supplemented diet for 3 days, not only induced acid secretion, but also stimulated the aldosterone and vasopressin systems and triggered cell proliferation. Accordingly, metabolic acidosis increased the expression of genes involved in acid-base transport, sodium transport, water transport and cell proliferation. In particular, more than 25 transcripts encoding proteins involved in urine acidification (subunits of H-ATPase, kidney anion exchanger, chloride channel Clcka, carbonic anhydrase 2, aldolase) were co-regulated during acidosis. We propose that these trancripts cooperating to a same function and co-regulated during acidosis constitute a functional regulon. Keywords: SAGE; Kidney collecting duct; Mouse; Metabolic acidosis; V-ATPase Approximately 650 OMCDs were microdissected, as previously described from collagenase-treated kidneys of 6 mice fed the NH4Cl-supplemented diet for 3 days. 20960 tags were sequenced in the acidosis SAGE library. After withdrawing duplicate ditags and linker sequences, the 19146 tags remaining corresponded to 8847 different tags.This acidosis library is similar in size and diversity to the normal mouse OMCD libraries previously generated (GEO accession number : GSM23478). The tag abundance in the two library was compared and discussed.

Dataset Information

Loss of Adam10 Disrupts Ion Transport in Immortalized Kidney Collecting Duct Cells.

Publications

Loss of Adam10 Disrupts Ion Transport in Immortalized Kidney Collecting Duct Cells.

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