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., chiefly in PCs vs. 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.

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One hallmark of trivalent N-acetylgalactosamine (GalNAc)-conjugated siRNAs is the remarkable durability of silencing that can persist for months in preclinical species and humans. Here, we investigated the underlying biology supporting this extended duration of pharmacological activity. We found that siRNA accumulation and stability in acidic intracellular compartments is critical for long-term activity. We show that functional siRNA can be liberated from these compartments and loaded into newly generated Argonaute 2 protein complexes weeks after dosing, enabling continuous RNAi activity over time. Identical siRNAs delivered in lipid nanoparticles or as GalNAc conjugates were dose-adjusted to achieve similar knockdown, but only GalNAc–siRNAs supported an extended duration of activity, illustrating the importance of receptor-mediated siRNA trafficking in the process. Taken together, we provide several lines of evidence that acidic intracellular compartments serve as a long-term depot for GalNAc–siRNA conjugates and are the major contributor to the extended duration of activity observed in vivo.

Hypomagnesemia affects insulin resistance and is a risk factor for diabetes mellitus type 2 (DM2) and gestational diabetes mellitus (GDM). Two single nucleotide polymorphisms (SNPs) in the epithelial magnesium channel TRPM6 (V 1393 I, K 1584 E) were predicted to confer susceptibility for DM2. Here, we show using patch clamp analysis and total internal reflection fluorescence microscopy, that insulin stimulates TRPM6 activity via a phosphoinositide 3-kinase and Rac1-mediated elevation of cell surface expression of TRPM6. Interestingly, insulin failed to activate the genetic variants TRPM6 (V 1393 I) and TRPM6(K 1584 E), which is likely due to the inability of the insulin signaling pathway to phosphorylate TRPM6(T 1391 ) and TRPM6(S 1583 ). Moreover, by measuring total glycosylated hemoglobin (TGH) in 997 pregnant women as a measure of glucose control, we demonstrate that TRPM6(V 1393 I) and TRPM6(K 1584 E) are associated with higher TGH and confer a higher likelihood of developing GDM. The impaired response of TRPM6(V 1393 I) and TRPM6(K 1584 E) to insulin represents a unique molecular pathway leading to GDM where the defect is located in TRPM6. G estational diabetes mellitus (GDM) is a condition in which women without previously diagnosed diabetes exhibit high blood glucose levels during pregnancy. Babies born to mothers with GDM are typically at increased risk of large for gestational age (LGA), low blood sugar, and jaundice (1). Women with GDM are at a higher risk for preeclampsia and Caesarean section (1) as well as developing diabetes mellitus type 2 (DM2) later in life (2). GDM affects 3-10% of pregnancies, depending on the population studied. No specific cause has been identified, but it is believed that in particular sex hormones (i.e., estrogen, progesterone, prolactin) produced during pregnancy increases a woman's resistance to insulin, resulting in impaired glucose tolerance (1, 3). Moreover, pregnant women are prone to lose magnesium (Mg 2+ ). Bardicef et al. (4) demonstrated that pregnancy itself is a condition of intracellular Mg 2+ depletion. This depletion was more pronounced in women affected by GDM. The elevation in female hormones as well as Mg 2+ deficiency during pregnancy impairs insulin sensitivity and these disturbances may even act synergistically.There is growing evidence suggesting that Mg 2+ deficiency is a significant risk factor for the development of insulin resistance and subsequently hypertension and DM2 (5-8), but the underlying molecular mechanism is unknown. The first evidence suggesting a direct connection between Mg 2+ deficiency and the occurrence of metabolic diseases came from the identification of a monogenic disease primarily characterized by significant hypomagnesemia that was caused by a mutation in a mitochondrial tRNA (9). Moreover, in a recent genome-wide association (GWA) study, it was demonstrated that certain SNPs nominally associated with hypomagnesemia also correlate with fasting glucose levels, again supporting the hypothesis of a direct link between Mg 2+ a...

Highlights d Gene expression profiling mapped systemic macrophage responses to sepsis, MI, and stroke d The tissue microenvironment determined phenotypic adaptions following remote injury d Local proliferation dominated over recruitment in expanding tissue macrophage numbers d Alveolar macrophage priming post MI increased resilience against subsequent pneumonia

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Epithelial cells line the lumen of tubular organs and are key players in their respective functions. They establish a unique luminal environment by providing a protective barrier and by performing vectorial transport of ions, nutrients, solutes, proteins, and water. Complex intercellular communication networks, specific for each organ, ensure their interaction with adjacent epithelial and nonepithelial cells, allowing them to respond to and modulate their immediate environment. In the epididymis, several epithelial cell types work in a concerted manner to establish a luminal acidic milieu that is essential for the post-testicular maturation and storage of spermatozoa. The epididymis also prevents autoimmune responses against auto-antigenic spermatozoa, while ensuring protection against ascending and blood pathogens. This is achieved by a network of immune cells that are in close contact and interact with epithelial cells. This review highlights the coordinated interactions between spermatozoa, basal cells, principal cells, narrow cells, clear cells, and immune cells that contribute to the maturation, protection, selection, and storage of spermatozoa in the lumen of the epididymis. Figure 1 Visualization of narrow cells (NCs) and clear cells (CCs) in the epididymis of transgenic mice expressing EGFP under the control of the promoter of the V-ATPase B1 subunit (B1-EGFP). EGFP + NCs (green) are located in the initial segment (IS), and EGFP + CCs (green) are located in the caput (A), corpus (C), and cauda (D) regions. (B) In the IS, NCs have a 'champagne glass' appearance and their nuclei are located in the apical region of the epithelium, compared to adjacent principal cells (PCs). A dense network of filamentous actin is seen in the apical stereocilia of PCs (labeled in red using phalloidin). Nuclei are labeled in blue using DAPI. Bars: A, C, D = 500 lm, B = 5 lm. ANDROLOGY

Recent experiments have demonstrated that dynein motors exhibit catch bonding behavior, in which the unbinding rate of a single dynein decreases with increasing force, for a certain range of force. Motivated by these experiments, we study the effect of catch bonding on unidirectional transport properties of cellular cargo carried by multiple dynein motors. We introduce a threshold force bond deformation (TFBD) model, consistent with the experiments, wherein catch bonding sets in beyond a critical applied load force. We find catch bonding can result in dramatic changes in the transport properties, which are in sharp contrast to kinesin-driven unidirectional transport, where catch bonding is absent. We predict that under certain conditions, the average velocity of the cellular cargo can actually increase as applied load is increased. We characterize the transport properties in terms of a velocity profile plot in the parameter space of the catch bond strength and the stall force of the motor. This plot yields predictions that may be experimentally accessed by suitable modifications of motor transport and binding properties.