Biology subjects

Ellison, D. H.

Publications and source records attributed to Ellison, D. H..

6 recordsLinked to original sources

bioRxiv2026

Differential Assembly of Native ENaC Complexes Across Mouse Epithelial Tissues

The epithelial sodium channel (ENaC) governs sodium and fluid absorption in the lung, kidney, and colon, but the organization of native ENaC complexes has remained difficult to define because of their low abundance and biochemical instability. To enable direct analysis of native assemblies, we generated a knock-in mouse in which the endogenous {gamma} subunit is fused at its C terminus to mVenus, a 3C protease cleavage site, and a 3xFLAG epitope (ENaC{gamma}-VF). The tag preserves physiological ENaC function, as ENaC{gamma}-VF mice display normal electrolyte handling, benzamil affinity, and amiloride-sensitive Na:K responses indistinguishable from wild-type animals. Using fluorescence-detection size-exclusion chromatography and single-molecule pull-down, we directly monitor intact native ENaC complexes from lung, kidney, and colon and uncover marked tissue-to-tissue differences in channel abundance and apparent complex size. Dual-color analysis with a fluorescent Fab against ENaC marks fully assembled {beta}{gamma} channels, while {gamma}-based fluorescence reports the broader population of {gamma}-containing assemblies. In combination, the ENaC{gamma}-VF line provides a biochemical anchor for identifying regulatory and trafficking proteins that co-purify with native ENaC complexes. These data show that ENaC architecture in vivo is heterogeneous, and establish ENaC{gamma}-VF mice as a platform for dissecting how epithelial environments shape ENaC assembly, composition, and regulation.

biochemistry↗

bioRxiv2025

Kidney kallikrein-1 contributes to cleavage of gamma-ENaC in vivo

The epithelial sodium channel (ENaC) is essential for sodium reabsorption and potassium homeostasis in the distal nephron, where its activity is controlled by mineralocorticoid signaling and downstream proteolytic processing of channel subunits. While cleavage of the {gamma}-ENaC subunit has been implicated in aldosterone-mediated sodium transport, the identity of mineralocorticoid receptor (MR)-regulated proteases responsible for this process remains uncertain. Here, we investigated the role of kallikrein-1 (encoded by Klk1), a serine protease expressed in the connecting tubule and cortical collecting duct (CNT/CCD), as a mediator of ENaC activation. Using CRISPR/Cas9, we generated a conditional Klk1-floxed allele and established mice with CNT/CCD-specific deletion of Klk1 by crossing with Calb1-Cre (CNT-Klk1-/-). On a low sodium, high potassium diet, CNT-Klk1-/- mice exhibited [~]85% less renal kallikrein-1 expression, yet maintained normal serum electrolytes, urinary potassium excretion, and aldosterone responses. Western blot analysis revealed significantly less cleavage of {gamma}-ENaC and -ENaC in CNT-Klk1-/- kidneys, accompanied by more total NCC abundance. Despite impaired ENaC proteolysis, amiloride-sensitive sodium excretion was preserved, indicating intact ENaC function. These findings identify renal kallikrein-1 as a protease that contributes to ENaC subunit processing in vivo. However, the absence of overt sodium or potassium handling defects in CNT-Klk1-/- mice suggests that kallikrein-1 deficiency is not sufficient to disrupt overall ENaC function, likely due to compensatory mechanisms from redundant proteolytic or non-proteolytic pathways. Together, our results refine the role of kallikrein-1 as a modulator, rather than a sole determinant, of ENaC activation and highlight the complexity of aldosterone-dependent sodium transport in the distal nephron. New & NoteworthyUsing a novel connecting tubule / cortical collecting duct specific kallikrein-1 knockout model, we show that {gamma}- and -ENaC cleavage is impaired by loss of renal kallikrein-1 without major disturbances in sodium or potassium handling. These findings highlight redundancy among ENaC regulatory pathways and suggest that proteolytic cleavage, while biochemically evident, may not be an accurate marker of ENaC-mediated sodium transport under physiological stress.

physiology↗

bioRxiv2025

Distinct cell types along thick ascending limb express pathways for monovalent and divalent cation transport

Kidney thick ascending limb cells reabsorb sodium, potassium, calcium, and magnesium and contribute to urinary concentration. These cells are typically viewed as of a single type that recycles potassium across the apical membrane and generates a lumen-positive transepithelial voltage driving calcium and magnesium reabsorption, although variability in potassium channel expression has been reported. Additionally, recent transcriptomic analyses suggest that different cell types exist along this segment, but classifications have varied and have not led to a new consensus model. We used immunolocalization, electrophysiology and enriched single nucleus RNA-Seq to identify thick ascending limb cell types in rat, mouse and human. We identified three major TAL cell types defined by expression of potassium channels and claudins. One has apical potassium channels, low basolateral potassium conductance, and is bordered by a sodium-permeable claudin. A second lacks apical potassium channels, has high basolateral potassium conductance and is bordered by calcium- and magnesium-permeable claudins. A third type also lacks apical potassium channels and has a high basolateral potassium conductance, but these cells are ringed by sodium-permeable claudins. The recognition of diverse cell types resolves longstanding questions about how solute transport can be modulated selectively and how disruption of these cells leads to human disease.

physiology↗

bioRxiv2024

NRBP1 and TSC22D proteins impact distal convoluted tubule physiology through modulation of the WNK pathway

The With No lysine (WNK) kinases regulate processes such as cell volume and epithelial ion transport through the modulation of Cation Chloride Cotransporters such as the NaCl cotransporter, NCC, present in the distal convoluted tubule (DCT) of the kidney. Recently, the interaction of WNKs with Nuclear Receptor Binding Protein 1 (NRBP1) and Transforming Growth Factor {beta}-Stimulated Clone 22 Domain (TSC22D) proteins was reported. Here we explored the effect of NRBP1 and TSC22Ds on WNK signaling in vitro and in the DCT. TSC22D1.1, TSC22D2, and NRBP1 are localized in DCT WNK bodies, which are cytoplasmic biomolecular condensates associated with WNK activation. In HEK293 cells, long TSC22D isoforms and NRBP1 increase WNK4 activity. DCT-specific NRBP1 knockout mice have reduced NCC phosphorylation and activate a compensatory response. Thus, NRBP1 and long TSC22D proteins are positive modulators of WNK signaling and modulate Na+ reabsorption in the kidney. NRBP1 and TSC22Ds likely influence WNK signaling in other tissues, impacting various physiological processes. TeaserThe pseudokinase NRBP1 and its associated TSC22D proteins modulate WNK kinases to regulate sodium reabsorption in the kidney.

physiology↗

bioRxiv2023

Enriched Single-Nucleus RNA-Sequencing reveals unique attributes of distal convoluted tubule cells

BackgroundThe distal convoluted tubule (DCT) comprises two subsegments, DCT1 and DCT2, with different functional and molecular characteristics. The functional and molecular distinction between these segments, however, has been controversial. MethodsTo understand the heterogeneity within the DCT population with better clarity, we enriched for DCT nuclei by using a mouse line combining "Isolation of Nuclei TAgged in specific Cell Types" and NCC (sodium chloride cotransporter)-driven inducible Cre recombinase. We sorted the fluorescently labeled DCT nuclei using Fluorescence-Activated Nucleus Sorting, and performed single nucleus transcriptomics. ResultsAmong 25,183 DCT cells, 70% were from DCT1 and 30% from DCT2. Additionally, there was a small population (<1%) enriched in proliferation-related genes, such asTop2a, Cenpp, and Mki67. Both DCT1 and DCT2 express NCC, magnesium transport genes are more abundant along DCT1; whereas calcium, electrogenic sodium and potassium transport genes are more abundant along DCT2. The transition between these two segments are gradual with a transitional zone where DCT1 and DCT2 cells are interspersed. The expression of the homeobox genes is not consistent between all DCT cells, suggesting that they develop along different trajectories. ConclusionTranscriptomics analysis of an enriched rare cell population using genetically targeted approach offers better clarification of the function and classification. The DCT segment is short, yet, can be separated into two sub-types that serve distinct functions, and are speculated to derive from different origins during development. Significance StatementHigh-resolution snRNAseq data indicate a clear separation between primary sites of calcium and magnesium handling within DCT. Both DCT1 and DCT2 express Slc12a3, but these subsegments serve distinctive functions, with more abundant magnesium handling genes along DCT1 and more calcium handling genes along DCT2. The data also provides insight into the plasticity of the distal nephron-collecting duct junction, formed from cells of separate embryonic origins. By focusing/changing gradients of gene expression, the DCT can morph into different physiological cell states on demand.

physiology↗

bioRxiv2021

Potassium effects on NCC are attenuated during inhibition of Cullin E3-ubiquitin ligases

The thiazide sensitive sodium-chloride co-transporter (NCC) plays a vital role in maintaining sodium (Na+) and potassium (K+) homeostasis. NCC activity is modulated by the with-no-lysine kinases 1 and 4 (WNK1 and WNK4), the abundance of which are controlled by the RING-type E3 ligase Cullin 3 (Cul3) and its substrate adapter Kelch-like protein 3. Dietary K+ intake has an inverse correlation with NCC activity, but the mechanism underlying this phenomenon remains to be fully elucidated. Here, we investigated the involvement of other members of the Cullin family in mediating K+ effects on NCC phosphorylation (active form) and abundance. In kidneys from mice fed diets varying in K+ content, there were negative correlations between NCC (phosphorylated and total) and active (neddylated) forms of Cullins (Cul1, 3, 4 and 5). High dietary K+ effects on phosphorylated NCC were attenuated in Cul3 mutant mice (CUL3-Het/{Delta}9). Short-term (30 min) and long-term (24 h) alterations in the extracellular K+ concentration did not affect Cullin neddylation levels in ex vivo renal tubules. Short-term, the ability of high extracellular K+ to decrease NCC phosphorylation was preserved in the presence of MLN4924 (pan Cullin inhibitor), but the response to low extracellular K+ was absent. Long-term, MLN4924 attenuated the effects of high extracellular K+ on NCC phosphorylation and responses to low extracellular K+ were absent. Our data suggest that in addition to Cul3, other Cullins are involved in mediating the effects of K+ on NCC phosphorylation and abundance.

physiology↗