Gerald S. Braun

CFTR mRNA and its truncated splice variant (TRN-CFTR) are differentially expressed during collecting duct ontogeny.

The collecting duct epithelium originates from the embryonic ureter by branching morphogenesis. Ontogeny‐dependent changes of CFTR mRNA expression were assessed by quantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR) in primary monolayer cultures of rat ureteric buds (UB) and cortical collecting ducts, microdissected at different embryonic and postnatal developmental stages. The amount of wild‐type CFTR‐specific PCR product in UB declined to 20% of the initial value between embryonic gestational day E15 and postnatal day P1. After birth the CFTR product increased transiently between P1 and P7 by a factor of 10 and decreased towards day P14. PCR products specific for TRN‐CFTR, a truncated splice variant, however, were low in early embryonic cells, increased markedly between day E17 and P2, and reached a plateau postnatally. Therefore, mRNA encoding TRN‐CFTR does not appear to have a specific embryonic‐morphogenetic function. By contrast, such function is suggested for wild‐type CFTR mRNA as its abundance was high in early embryonic nephrogenesis, as well as during a postnatal period shortly before branching morphogenesis is completed.

Expression of the epithelial sodium channel (ENaC) during ontogenic differentiation of the renal cortical collecting duct epithelium.

Abstract The NaCl-reabsorbing collecting duct epithelium develops by budding and branching of the embryonic ureter. The expression of Na+ channels during this branching morphogenesis was studied in the outermost branches of rat ureteric buds (UB; embryonic day E15 to postnatal day P6) and in cortical collecting ducts (CCD; days P7–P28) in primary monolayer culture. Expression of both Na+ channel mRNA and of Na+-selective membrane conductance were estimated by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and by patch-clamp recording, respectively. UB and CCD uniformly represented a principal-like cell type in culture. Messenger RNA encoding the α-ENaC subunit was detected in oligo-dT primed cDNA (5 ng) of embryonic UB cells (E15–17) after 30 PCR cycles. The abundance of α-ENaC mRNA, when normalized by reference to β-actin, was higher by a factor of 2 in postnatal (P1–6) UB and by a factor of 5 in CCD cells (P7–14) compared with the embryonic stage. Highly Na+-selective, low-conductance channels were identified in apical patches from both UB and CCD monolayers, but only CCD cells exhibited macroscopic, amiloride-sensitive Na+ currents in whole-cell patch-clamp recordings. We conclude that α-ENaC mRNA and functional Na+ channel protein are expressed already before morphogenesis of the CCD is completed and prior to the onset of epithelial NaCl reabsorption.

Mycoplasma pneumoniae: Usual suspect and unsecured Diagnosis in the acute setting

Abstract Mycoplasma pneumoniae is one of the most common known bacterial pathogens of the respiratory tract, especially in patients between 5 and 30 years of age. It may be encountered at a relatively high rate in the non-life-threatened fraction of Emergency Department (ED) patients presenting with upper respiratory symptoms or cough. Yet its hallmarks are very non-specific, including a great variety of presentations from mild pharyngitis to potentially life-threatening complications such as the Stevens-Johnson Syndrome. Here, we describe a typical case of pneumonia due to Mycoplasma pneumoniae in a young adult with mild pharyngitis as the leading symptom. Disease presentation, complications, diagnostic means, therapeutic options, and suspicious clinical settings are discussed to provide a review on the clinical aspects of the disease that are important in the ED setting.

Ontogeny of purinergic receptor-regulated Ca2+ signaling in mouse cortical collecting duct epithelium.

Changes in ATP-induced increase in [Ca²⁺]_i during collecting duct ontogeny were studied in primary monolayer cultures of mouse ureteric bud (UB) and cortical collecting duct (CCD) cells by Fura-PE3 fluorescence ratio imaging. In UB (embryonic day E14 and postnatal day P1) the ATP-stimulated increase (EC₅₀ ≈ 1 µM) in fluorescence ratio (ΔR_ATP) was independent of extracellular Ca²⁺ and insensitive to the P2 purinoceptor-antagonist suramin (1 mM). From day P7 onward when CCD morphogenesis had been completed ΔR_ATP increased and became dependent on extracellular Ca²⁺. This ATP-stimulated Ca²⁺ entry into CCD cells was non-capacitative and suramin (1 mM)-insensitive, but sensitive to nifedipine (30 µM) and enhanced by Bay K8644 (15 µM), a blocker and an agonist of L-type Ca²⁺ channels, respectively. Quantitative RT-PCR demonstrated similar mRNA expression of L-type Ca²⁺ channel α1-subunit, P2Y₁, P2Y₂, and P2X_4b purinoceptors in UB and CCD monolayers while the abundance of P2X₄ mRNA increased with CCD morphogenesis. In conclusion, both embryonic and postnatal cells express probably P2Y₂-stimulated Ca²⁺ release from intracellular stores. With development, the CCD epithelium acquires ATP-stimulated Ca²⁺ entry via L-type Ca²⁺ channels. This pathway might by mediated by the increasing expression of P2X₄-receptors resulting in an increasing ATP-dependent membrane depolarization and activation of L-type Ca²⁺ channels.

Bradykinin-stimulated Cl– secretion in T84 cells. Role of Ca2+-activated hSK4-like K+ channels

Abstract Bradykinin (BK)-stimulated colonic Cl– secretion was studied in T84 colonic adenocarcinoma cells by measuring BK (50 nM)-evoked changes in cytosolic free [Ca2+] ([Ca2+]i), membrane conductance and transepithelial ion transport. In T84 cells grown on impermeable supports, BK stimulated a transient increase in [Ca2+]i as assessed by fura-2 ratio imaging. In cell-attached, patch-clamp recordings, BK transiently activated low-conductance K channels. These channels were activated/inactivated reversibly in inside-out patches by switching [Ca2+]i in the bath between 30 nM and 100 nM. Excised channels recorded with 160 mM [K+] in bath and pipette exhibited an inwardly rectifying current/voltage-relation, conductances of 10±1 pS and 34±4 pS (n=10) at positive and negative voltages, respectively, and a 15-fold lower permeability for Na+ than for K+. The mean open probability of these channels did not depend on voltage but increased with increasing [Ca2+]i with an apparent concentration for a half-maximal response (EC50) of 110 nM, resembling that of hSK4 K+ channels. Application of the reverse transcriptase-polymerase chain reaction technique showed hSK4 messenger ribonucleic acid (mRNA) to be expressed in T84 cells. Macroscopic currents in T84 cells showed a similar dependence on [Ca2+]i. Whole cell conductance (in nS/10pF) increased from 0.5±0.1 (n=6) at 10 nM [Ca2+]i in the pipette solution to 1.5±0.2 (n=7) at 100 nM, and to 2.0±0.5 (n=7) at 1 µM due to activation of a K+ conductance. In Ussing-chambered T84 monolayers grown on filters, BK did not evoke a short-circuit current (Isc). When, however, the monolayers were pre-stimulated by forskolin (1 µM), BK further enhanced Cl–secretion (ΔIsc=21±5 µA/cm2, n=10) transiently and biphasically. In conclusion, BK enhances cyclic adenosine monophosphate-stimulated Cl– secretion in T84 cells, probably via basolateral, Ca2+-liganded activation of low-conductance hSK4-type K+ channels.

Development of renal function.

The mammalian metanephric kidney develops following a general principle of organogenesis of epithelial organs, i.e., along the tree-like structure of an arborizing ductal system (the ureteric bud and cortical collecting duct). In parallel, the proximal portions of the uriniferous tubule develop by mesenchymal-to-epithelial transition of the neighbouring mesenchyme. On one hand, vectorial transport systems in nephrogenesis should be functional at the onset of glomerular filtration in any of the newly formed nephron generations to prevent loss of salt, water and metabolites. On the other hand, developing nephron epithelia must serve the needs of organ-formation such as cell proliferation and fluid-secretion for morphogenic purposes. This review intends to summarize current data and concepts on the development of renal epithelial functions with an emphasis on ion channels. Current model systems are introduced, such as ureteric bud cell monolayer culture, in vitro nephron culture, HEK293 cell culture, and the dissection of tubular cells for direct analysis. The current data on the developmental expression and functions of ENaC Na(+) channels, the CFTR, ClC-2 Cl(ndash;) channels, L-type Ca(2+) channels, P2 purinoceptors, and the Kir6.1/SUR2, ROMK (Kir1.1), and Kv K(+) channels are presented.