7 research outputs found
mCCDcl1 cells exhibit a transitional phenotype: implications for collecting duct plasticity
The cortical collecting duct of the mammalian kidney plays a critical role in the
regulation of body volume, sodium pH and osmolarity and is composed of two distinct
cells types, principal cells and intercalated cells. Each cell type is detectable in the kidney
by the localization of specific transport proteins such as Aqp2 and ENaC in principal cells
and V-ATPase B1 and Cx30 in intercalated cells. mCCDcl1 cells have been widely used
as a mouse principal cell line on the basis of their physiological characteristics. In this
study, the mCCDcl1 parental cell line and three sub-lines cloned from isolated single cells
(Ed1, Ed2, and Ed3) were grown on filters to assess their transepithelial resistance,
transepithelial voltage, equivalent short circuit current and expression of the cell-specific
markers Aqp2, ENaC, V-ATPaseB1 and Cx30. The parental mCCDcl1 cell line presented
amiloride-sensitive electrogenic sodium transport indicative of principal cell function,
however immunocytochemistry and RT-PCR showed that some cells expressed the
intercalated cell-specific markers V-ATPase B1 and Cx30, including a subset of cells also
positive for Aqp2 and ENaC. The three subclonal lines contained cells that were positive
for both intercalated and principal cell-specific markers. The vertical transmission of both
principal and intercalated cell characteristics via single cell cloning, reveals the plasticity
of mCCDcl1 cells, and a direct lineage relationship between these two physiologically
important cell types, and is consistent with mCCDcl1 cells being precursor cells. For
observation of live mCCDcl1 in an environment closer to in vivo conditions, a model of
collecting duct was designed and developed using 3D printing of porous polymers.
mCCDcl1 were cultured successfully and demonstrated improved characteristics compared
to classic culture such as improved lifespan, different morphology and increased protein
expression, and retained their phenotypic plasticity
Dissociation of sodium-chloride cotransporter expression and blood pressure during chronic high dietary potassium supplementation
Dietary potassium (K+) supplementation is associated with a lowering effect in blood pressure (BP), but not all studies agree. Here, we examined the effects of short- and long-term K+ supplementation on BP in mice, whether differences depend on the accompanying anion or the sodium (Na+) intake and molecular alterations in the kidney that may underlie BP changes. Relative to the control diet, BP was higher in mice fed a high NaCl (1.57% Na+) diet for 7 weeks or fed a K+free diet for 2 weeks. BP was highest on a K+-free/high NaCl diet. Commensurate with increased abundance and phosphorylation of the thiazide sensitive sodium-chloride-cotransporter (NCC) on the K+-free/high NaCl diet, BP returned to normal with thiazides. Three weeks of a high K+ diet (5% K+) increased BP (predominantly during the night) independently of dietary Na+ or anion intake. Conversely, 4 days of KCl feeding reduced BP. Both feeding periods resulted in lower NCC levels but in increased levels of cleaved (active) α and γ subunits of the epithelial Na+ channel ENaC. The elevated BP after chronic K+ feeding was reduced by amiloride but not thiazide. Our results suggest that dietary K+ has an optimal threshold where it may be most effective for cardiovascular health
Comparative Studies of Renin-Null Zebrafish and Mice Provide New Functional Insights
Background: The renin-angiotensin system is highly conserved across vertebrates, including zebrafish, which possess orthologous genes coding for renin-angiotensin system proteins, and specialized mural cells of the kidney arterioles, capable of synthesising and secreting renin. Methods: We generated zebrafish with CRISPR-Cas9-targeted knockout of renin ( ren −/− ) to investigate renin function in a low blood pressure environment. We used single-cell (10×) RNA sequencing analysis to compare the transcriptome profiles of renin lineage cells from mesonephric kidneys of ren −/− with ren +/+ zebrafish and with the metanephric kidneys of Ren1 c −/− and Ren1 c +/+ mice. Results: The ren −/− larvae exhibited delays in larval growth, glomerular fusion and appearance of a swim bladder, but were viable and withstood low salinity during early larval stages. Optogenetic ablation of renin-expressing cells, located at the anterior mesenteric artery of 3-day-old larvae, caused a loss of tone, due to diminished contractility. The ren −/− mesonephric kidney exhibited vacuolated cells in the proximal tubule, which were also observed in Ren1 c −/− mouse kidney. Fluorescent reporters for renin and smooth muscle actin ( Tg(ren:LifeAct-RFP; acta2:EGFP )), revealed a dramatic recruitment of renin lineage cells along the renal vasculature of adult ren −/− fish, suggesting a continued requirement for renin, in the absence of detectable angiotensin metabolites, as seen in the Ren1 YFP Ren1 c −/− mouse. Both phenotypes were rescued by alleles lacking the potential for glycosylation at exon 2, suggesting that glycosylation is not essential for normal physiological function. Conclusions: Phenotypic similarities and transcriptional variations between mouse and zebrafish renin knockouts suggests evolution of renin cell function with terrestrial survival
Characterisation of a Mouse Kidney Cortical Collecting Duct Cell Line Suggests Plasticity Between Cell Types:Experimental Biology 2017
The kidney collecting duct is composed of principal (PC) and intercalated (IC) cells responsible for the reabsorption of water and sodium, and the acid-base balance, respectively. The immortalized mCCDcl1 cell line is widely used as a model for PCs since cells respond to glucocorticoid (MR) and mineralocorticoid (MR) hormones, such as aldosterone, and give reproducible electrophysiological responses.The effect of culture media on sodium transport in the mCCDcl1 cell line was tested by electrophysiological methods and immunostaining. The cells were cultured on filters in two media: the original medium published by Gaeggeler et al.(2005), and an alternative medium using a different source of sodium selenite supplement (Gibco). The cells transepithelial voltage and resistance across monolayers of cells was measured using an epithelial volt-ohm-meter, allowing calculation of the equivalent short circuit current by Ohm’s law. Basal currents recorded from cells cultured in the original medium measured −9.97μA/cm2, were amiloride-sensitive and stimulated by ~2.6 fold following aldosterone treatment (−26.78μA/cm2). Cells cultured in the alternative medium exhibited negligible current and no response to aldosterone was observed.Immunostaining was performed in the alternative media on the mCCDcl1 cells and on isolated subclones using antibodies against PC and IC markers: Aquaporin 2 (Aqp2) and the alpha subunit of the epithelial sodium channel (α-ENaC) for PC, Connexin 30 (Cx30) and vacuolar H+ ATPase (V-ATPase) for IC. Both the original cell line and the clonal sub-lines exhibited varied expression of Aqp2 and Cx30, with evidence of co-expression in sub-populations of cells. 41.8% of cells from the parental sub-line express both PC and IC markers whilst 23.8 to 58.7% cells in the clonal sub-lines express both PC and IC markers. Expression of both PC and IC markers was confirmed by PCR analyses, and RNA sequencing. Immunostaining for α-ENaC revealed staining across the apical membrane of the cells when cultured in the original medium and perinuclear staining when cells were cultured in the alternative medium.These results show that the provenance of the culture media supplements has a direct consequence on the phenotype of the cells and correlates with their capacity for sodium transport. Secondly, mCCDcl1 cells can present characteristics of both principal and intercalated cells and may have the capacity to switch between the two phenotypes.Support or Funding InformationBritish Heart Foundation Center of Research Excellence PhD Studentshi
Loss of Adam10 disrupts ion transport in immortalised kidney collecting duct cells
The kidney cortical collecting duct (CCD) comprises of principal cells (PC), intercalated cells (IC) and the recently discovered intermediate cell type. Kidney pathology in a mouse model of the syndrome of apparent aldosterone excess (SAME) revealed plasticity of the cortical collecting duct (CCD), with altered principal cell (PC): intermediate cell: intercalated cell (IC) ratio. The self-immortalized mouse CCD cell line, mCCDcl1, 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 mCCDcl1 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 (gamma-ENaC) and Hsd11b2 (11ßHSD2), 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 gamma-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 polarisation.Mullins, John; Assmus, Adrienne; Mullins, Linda. (2021). Loss of Adam10 disrupts ion transport in immortalised kidney collecting duct cells, [dataset]. University of Edinburgh; BHF Centre for Cardiovascular Science. https://doi.org/10.7488/ds/3022
Dissociation of sodium-chloride cotransporter expression and blood pressure during chronic high dietary potassium supplementation
Dietary potassium (K+) supplementation is associated with a lowering effect in blood pressure (BP), but not all studies agree. Here, we examined the effects of short- and long-term K+ supplementation on BP in mice, whether differences depend on the accompanying anion or the sodium (Na+) intake and molecular alterations in the kidney that may underlie BP changes. Relative to the control diet, BP was higher in mice fed a high NaCl (1.57% Na+) diet for 7 weeks or fed a K+free diet for 2 weeks. BP was highest on a K+-free/high NaCl diet. Commensurate with increased abundance and phosphorylation of the thiazide sensitive sodium-chloride-cotransporter (NCC) on the K+-free/high NaCl diet, BP returned to normal with thiazides. Three weeks of a high K+ diet (5% K+) increased BP (predominantly during the night) independently of dietary Na+ or anion intake. Conversely, 4 days of KCl feeding reduced BP. Both feeding periods resulted in lower NCC levels but in increased levels of cleaved (active) α and γ subunits of the epithelial Na+ channel ENaC. The elevated BP after chronic K+ feeding was reduced by amiloride but not thiazide. Our results suggest that dietary K+ has an optimal threshold where it may be most effective for cardiovascular health