Investigating the identity and role of the collecting duct intermediate cell

The classical model of the collecting duct (CD) defines two functionally distinct epithelial cell types - principal cells (PC) and intercalated cells (IC). A third, intermediate cell type, which expresses both PC and IC markers, has also been described in the literature but its exact role is yet to be determined. Plasticity within the CD has also been noted, both in vivo and in mCCDcl1 cells leading to questions regarding the origin of these cells and the mechanism underlying their differentiation. This work details the investigation of the intermediate cell type in the mCCDcl1 and mpkCCDcl4 cell lines. Using immunocytochemistry, RT-PCR and scRNA-Seq, both cell lines were determined to widely express classical PC, IC and recently identified intermediate cell markers. Heterogeneity of mpkCCDcl4 cell populations was sustained through single cell cloning indicating plasticity within the cell line. In line with previous work on the mCCDcl1 cell line, it is concluded that the mpkCCDcl4 cells is heterogenous and not a PC cell line as once thought. As these cell lines mirror what is observed in vivo, this suggests that the mCCDcl1 and mpkCCDcl4 cell lines are appropriate for studying CD cellular composition and plasticity, in particular the intermediate cell type that is still poorly understood. Despite this, functional differences were noted in the mCCDcl1 and mpkCCDcl4 cells lines in terms of sodium transport, with scRNA-Seq data leading to the hypothesis that up- and down-regulation of certain genes upstream of the epithelial sodium channel (ENaC) and those related to intracellular cholesterol storage, may be in part, be responsible. Understanding the mechanisms of plasticity within the CD will improve understanding of the kidney both under physiological and pathophysiological conditions. This work reports on one such condition where CD plasticity is observed – in mouse and rat models of the syndrome of apparent mineralocorticoid excess (SAME), conferred by an HSD11B2 knockout. A shift in cell fate from PC to intermediate cell was observed in knockout animals which was not recovered at maturity. Similar studies in mice on a high salt diet and under the ACTH-induced model of Cushing’s syndrome displayed no alteration in cell type composition. The results in the SAME study indicate the importance of cell plasticity activation under certain genetic diseases and pose further questions about the role and function of the intermediate cell type

Abnormal neonatal sodium handling in skin precedes hypertension in the SAME rat

We discovered high Na(+) and water content in the skin of newborn Sprague-Dawley rats, which reduced ~ 2.5-fold by 7 days of age, indicating rapid changes in extracellular volume (ECV). Equivalent changes in ECV post birth were also observed in C57Bl/6 J mice, with a fourfold reduction over 7 days, to approximately adult levels. This established the generality of increased ECV at birth. We investigated early sodium and water handling in neonates from a second rat strain, Fischer, and an Hsd11b2-knockout rat modelling the syndrome of apparent mineralocorticoid excess (SAME). Despite Hsd11b2(-/-) animals exhibiting lower skin Na(+) and water levels than controls at birth, they retained ~ 30% higher Na(+) content in their pelts at the expense of K(+) thereafter. Hsd11b2(-/-) neonates exhibited incipient hypokalaemia from 15 days of age and became increasingly polydipsic and polyuric from weaning. As with adults, they excreted a high proportion of ingested Na(+) through the kidney, (56.15 ± 8.21% versus control 34.15 ± 8.23%; n = 4; P < 0.0001), suggesting that changes in nephron electrolyte transporters identified in adults, by RNA-seq analysis, occur by 4 weeks of age. Our data reveal that Na(+) imbalance in the Hsd11b2(-/-) neonate leads to excess Na(+) storage in skin and incipient hypokalaemia, which, together with increased, glucocorticoid-induced Na(+) uptake in the kidney, then contribute to progressive, volume contracted, salt-sensitive hypertension. Skin Na(+) plays an important role in the development of SAME but, equally, may play a key physiological role at birth, supporting post-natal growth, as an innate barrier to infection or as a rudimentary kidney

Oxygen levels determine the ability of glucocorticoids to influence neutrophil survival in inflammatory environments

GCs are highly effective in treating a wide range of inflammatory diseases but are limited in their ability to control neutrophilic lung inflammation in conditions such as COPD. Neutrophil apoptosis, a central feature of inflammation resolution, is delayed in response to microenvironmental cues, such as hypoxia and inflammatory cytokines, present at inflamed sites. GCs delay neutrophil apoptosis in vitro, and this may therefore limit the ability of GCs to control neutrophilic inflammation. This study assesses the effect GCs have on hypoxia- and inflammatory cytokine-induced neutrophil survival. Human neutrophils were treated with GCs in the presence or absence of GM-CSF or inflammatory macrophage-CM at a range of oxygen concentrations (21–1% oxygen). Neutrophil apoptosis and survival were assessed by flow cytometry and morphological analysis and neutrophil function, by stimulus-induced shape change and respiratory burst. Dexamethasone promoted neutrophil survival at 21%, 10%, and 5% oxygen but not at 1% oxygen. Interestingly, GM-CSF and inflammatory CM increased neutrophil survival significantly, even at 1% oxygen, with cells remaining functionally active at 96 h. Dexamethasone was able to reduce the prosurvival effect of GM-CSF and inflammatory CM in a hypoxic environment. In conclusion, we found that GCs do not augment neutrophil survival in the presence of severe hypoxia or proinflammatory mediators. This suggests that GCs would not promote neutrophil survival at sites of inflammation under these conditions

Structure-Guided Design of Highly Selective and Potent Covalent Inhibitors of ERK1/2

The RAS/RAF/MEK/ERK signaling pathway has been targeted with a number of small molecule inhibitors in oncology clinical development across multiple disease indications. Importantly, cell lines with acquired resistance to B-RAF and MEK inhibitors have been shown to maintain sensitivity to ERK1/2 inhibition by small molecule inhibitors. There are a number of selective, noncovalent ERK1/2 inhibitors reported along with the promiscuous hypothemycin (and related analogues) that act via a covalent mechanism of action. This article reports the identification of multiple series of highly selective covalent ERK1/2 inhibitors informed by structure-based drug design (SBDD). As a starting point for these covalent inhibitors, reported ERK1/2 inhibitors and a chemical series identified via high-throughput screening were exploited. These approaches resulted in the identification of selective covalent tool compounds for potential <i>in vitro</i> and <i>in vivo</i> studies to assess the risks and or benefits of targeting this pathway through such a mechanism of action