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Biomedical subjects

M Horster

Publications and source records attributed to M Horster.

At least 19 recordsLinked to original sources

Embryonic epithelial membrane transporters.

Embryonic epithelial membrane transporters are organized into transporter families that are functional in several epithelial organs, namely, in kidney, lung, pancreas, intestine, and salivary gland. Family members (subtypes) are developmentally expressed in plasma membranes in temporospatial patterns that are 1) similar for one subtype within different organs, like aquaporin-1 (AQP1) in lung and kidney; 2) different between subtypes within the same organ, like the amiloride-sensitive epithelial sodium channel (ENaC) in lung; and 3) apparently matched among members of different transporter families, as alpha-ENaC with AQP1 and -4 in lung and with AQP2 in kidney. Finally, comparison of temporal expression patterns in early embryonic development of transporters from different families [e.g., cystic fibrosis transmembrane conductance regulator (CFTR), ENaC, and outer medullary potassium channel] suggests regulatory activating or inactivating interactions in defined morphogenic periods. This review focuses on embryonic patterns, at the mRNA and immunoprotein level, of the following transporter entities expressed in epithelial cell plasma membranes: ENaC; the chloride transporters CFTR, ClC-2, bumetanide-sensitive Na-K-Cl cotransporter, Cl/OH, and Cl/HCO(3); the sodium glucose transporter-glucose transporter; the sodium/hydrogen exchanger; the sodium-phosphate cotransporter; the ATPases; and AQP. The purpose of this article is to relate temporal and spatial expression patterns in embryonic and in early postnatal epithelia to developmental changes in organ structure and function.

Animals↗

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.

Alternative Splicing↗

Analysis of mouse glomerular podocyte mRNA by single-cell reverse transcription-polymerase chain reaction.

Selective investigation of glomerular podocytes is not possible using conventional methods in vivo. Analysis of glomerular epithelium-derived cells in culture yields dubious results because of the rapid dedifferentiation of podocytes. We developed a modification of the polymerase chain reaction (PCR) method previously used to analyze cultured neurons. Podocytes harvested from freshly dissected glomeruli are ideal target cells for this modified, single cell reverse transcription-PCR method to reproducibly identify specific mRNA species from resident intact podocytes in vivo.

Actins↗

Chloride channels ClC-2 and ICln mRNA expression differs in renal epithelial ontogeny.

Development-dependent mRNA expression of the chloride channels ClC-2 and ICln was studied by quantitative reverse transcriptase-polymerase chain reaction in rat ureteric bud and cortical collecting duct primary monolayer cultures. Abundance of ClC-2 mRNA increased in ureteric bud cells between embryonic day 15 (E15) and E17, peaked at postnatal day 3 (P3), and was down-regulated at P7 when morphogenesis is complete, suggesting a specific embryonic function. Expression of ICln mRNA, in contrast, up-regulated continuously with development.

Animals↗

Detection of multiple vascular endothelial growth factor splice isoforms in single glomerular podocytes.

Glomerular podocytes are major determinants of filtration permselectivity in the glomerulus. Although the molecular mechanisms determining the characteristics of the glomerular filtration unit are incompletely understood, vascular endothelial growth factor (VEGF) has been implicated. To analyze this process in situ, we established a method that allows exploration of in vivo mRNA expression of podocytes using single-cell reverse transcriptase-polymerase chain reaction (RT-PCR). Microdissected mouse glomeruli were held in a patch-clamp apparatus, and single podocytes were harvested by aspiration. After lysis, the cells were reverse transcribed, and PCR was performed (45 cycles). The podocyte nature of the material was confirmed by detection of podocyte-specific mRNA (glomerular epithelial protein 1 and Wilms' tumor protein 1). Using specific oligonucleotide primers, VEGF was detected in mRNA obtained from renal cortex, single microdissected glomeruli, cultured murine podocytes, and single podocytes in situ. All cells examined expressed three VEGF isoforms (121, 165, and 189). These differ in their capacity for binding to extracellular matrix and could have different potencies regulating glomerular endothelial permeability. Our approach should allow a semiquantitative, isoform-specific evaluation of VEGF mRNA expression in podocytes during nephrogenesis and in glomerular disease.

Animals↗

Single cell RT-PCR analysis of ClC-2 mRNA expression in ureteric bud tip.

Embryonic epithelia at the tip of the ureteric bud (UB) face the interspace between epithelial and mesenchymal cells and are fundamentally involved in reciprocal signaling during early nephrogenesis. To characterize their membrane conductive proteins, patch-clamp and single cell RT-PCR techniques were applied to embryonic rat UBs [embryonic day 17 (day E17)] microdissected from the outer cortex. Cells at the UB tip had a high whole cell conductance (14 +/- 2 nS/10 pF, n = 8). The main fractional conductance resembled that of Ca-activated Cl channels in nonepithelial cells, with its time-dependent activation at depolarizing and inactivation at hyperpolarizing voltages. A second Cl-selective current fraction, by contrast, activated slowly during strong hyperpolarization, suggestive of a ClC-2-mediated conductance. To determine the origin of this current, cytoplasm was harvested into the patch pipette, RNA was reverse transcribed, and cDNA encoding the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeper gene or the ClC-2 Cl channel was amplified by polymerase chain reaction (PCR). GAPDH and ClC-2 PCR products were identified in 23 and 8 (out of a total of 57) single cell cDNA samples, respectively. ClC-2 PCR products with two different lengths were obtained, which might be due to two alternatively spliced ClC-2 mRNA isoforms. This first and combined approach by patch-clamp and single cell RT-PCR techniques to embryonic epithelia indicates that 1) cells at the UB tip express a phenotype remarkably different from that of postembryonic collecting duct principal cells and that 2) ClC-2 is likely to have a key role in early nephrogenesis.

Animals↗

Renal function and renotropic effects of secretin in cystic fibrosis.

In ten cystic fibrosis patients and nine age-matched controls, renal function was determined before and after infusion of secretin. Under baseline conditions creatinine excretion and clearance were significantly elevated, exclusively due to those patients who were homozygous for the DF508 mutation (153 vs 132 ml/min*1.73m2), whereas the glomerular filtration rate, measured by inulin clearance showed no difference. Renal plasma flow and the fractional reabsorption rates of electrolytes were similar in patients and controls. During secretin infusion renal plasma flow increased and the fractional reabsorption rates of electrolytes decreased in both groups. The patients had a increased metabolic clearance (2900 vs 1660 ml/min*m2) and endogenous production rate (9,9 vs 2,5 pmol/min*m2) of of secretin. In conclusion global renal function and electrolyte handling, in particular chloride permeability, are unchanged in cystic fibrosis. Individuals expressing the DF508 genotype showed a selective elevation of creatinine excretion and clearance. The secretion and metabolic clearance of secretin are increased in cystic fibrosis.

Adolescent↗

Epithelial nephrogenesis.

The metanephric, or definitive, kidney forms as a result of inductive processes between tissues of two distinct embryologic origins, the metanephric mesenchymal blastema and the ureteric bud. After inductive signalling between these primordial tissues, mesenchymal cells aggregate next to the branching ureteric bud tip, convert to epithelial cells and differentiate into the diverse cell populations of the nephron. The ureteric bud enters branching morphogenesis and gives rise to the collecting duct system. Nephrogenesis has become a target system by which to study developmental processes including embryonic inductive interactions, mesenchyme-to-epithelium conversion, cell lineage pathways, epithelial cell polarization, branching morphogenesis and spatio-temporal expression of transcription factors. This review summarizes data on cellular and extracellular events during epithelial metanephrogenesis.

Animals↗

Colonic-crypt-derived epithelia express induced ion transport differentiation in monolayer cultures on permeable matrix substrata.

The processes of transport differentiation from stem cell to the terminally differentiated cell in intact colonic crypts are difficult to study because access to the lumen is limited. Colonocytes were isolated from the lower two-thirds of rat distal colon crypts and grown to confluence on reconstituted basement membranes and permeable support in primary culture. Crypt and surface cells were distinguished by the uptake of [3H]thymidine and [3H]leucine and by brushborder fluorescence binding. Ion concentrations in apical and basolateral compartments of filter monolayer cultures after 48 h of incubation on days 16-18 were (in mM): apical, Na+ 116 +/- 4 (n = 48) and K+ 6 +/- 1 (n = 48); basolateral, Na+ 151 +/- 3 and K+ 3.7 +/- 0.5, respectively (mean +/- SE). Aldosterone (10(-8) M), added to the basolateral compartment from days 10-18, changed apical Na+ to 72 +/- 6 mM and apical K+ to 13 +/- 4 mM (n = 23). Dexamethasone (10(-8) M) changed apical Na+ to 84 +/- 7 mM but did not influence apical K+ (n = 22). Transmonolayer electrical potential difference (VtM; control medium; days 8-10) was 5 +/- 1 mV (n = 16; apical compartment negative); electrical resistance (RtM) was 217 +/- 21 omega.cm2 and short circuit current (ISC) was 21 +/- 5 microA.cm-2. Amiloride (0.1 mM; n = 12) in the apical medium decreased VtM to 2 +/- 1 mV and ISC to 11 +/- 4 microA.cm-2. Aldosterone (10(-8) M) after 1 week in the basolateral compartment (n = 21) changed VtM to 12.3 +/- 3 mV, RtM to 92 +/- 9 omega.cm2, and ISC to 138 +/- 23 microA.cm-2. Apical amiloride (0.1 mM; n = 9) decreased the induced VtM to -3 +/- 1 mV and ISC to -13 +/- 7 microA.cm-2. Colonic-crypt-derived epithelial cells proliferate and differentiate in primary culture, when grown on reconstituted basement membrane substratum and in supplemented medium, to form monolayers that express net Na+ absorption and net K+ secretion after 1 week. Na+ and K+ vectorial transport differentiation is primarily regulated by aldosterone, which specifically induces apical conductive Na+ transfer. Mineralocorticoid and glucocorticoid hormones appear to have differing actions on ion transport in functionally surface-type colonocytes derived in culture from isolated crypt-type cells.

Aldosterone↗

Peptide-dependent regulation of epithelial nephron functions.

It has become evident that the nephron is an important target organ of many of the regulatory peptides; this brief overview will not attempt to consider the vast amount of work on peptide-dependent kidney functions; instead, it will emphasize recent work directed towards understanding intracellular signal pathways between peptide ligand-receptor interaction and expression of physiological transport responses in renal epithelial cells. The awareness that peptide hormones of differing origin, e.g., intestinal and cardiac, share at least some of the signal steps in nephron cells, has stimulated work on nephron segmental analysis of receptor binding, of second messengers, of membrane G proteins, of protein phosphorylation, and of final membrane transport responses, such as peptide-dependent ion channel regulation. Peptides involved in cell growth and differentiation, e.g., growth factors, appear to act through part of the signal pathway shared by other peptides. The peptides selected for the purpose of this review, then, are those that have been linked, by experimental evidence, to intracellular messenger systems in nephron epithelia.

Animals↗

Bleb formation during anoxia is not a prerequisite for eventual cell death in renal tubule cells.

Membrane deformations, generally termed blebs, are a hallmark of cells subjected to noxious stimuli or injurious conditions. Although not lethal itself, bleb formation has been used as an indicator of the stage of cell injury. Whether or not bleb formation is an integral part of the sequence of events that lead to irreversible injury, or occurs in all cells within a group subjected to the same conditions is not known. We demonstrate here that renal epithelial cells in culture produce blebs when subjected to sustained periods of anoxia and substrate deprivation. However, the occurrence of blebs does not correlate with eventual cell death. The data suggest that bleb formation may be associated with prolonged survival.

Animals↗

Hormonal regulation of electrolyte and water transport in the colon.

The colon participates in water and electrolyte homeostasis by the absorption of sodium (Na) and water as well as by potassium (K) secretion. The primary step of colonic transport is the active Na transport via a transcellular route. Steroidal hormones considerably increase Na absorption by utilizing two mechanisms: (1) passive Na entry into the cells in enhanced by an increased membrane permeability; (2) active transport capacity is increased by a stimulation of ATPase synthesis. Mineralocorticoid versus glucocorticoid actions of steroids have not yet been clearly differentiated; parallel influences are possible. Active chloride (Cl) secretion is found in the colon under certain pathological conditions and is induced by a number of factors, e.g., hormones produced by pancreas tumors. Cellular events involve a rise of intracellular cAMP and calcium (Ca) concentrations, and altered Cl permeabilities. Functional changes of colonic epithelial cells caused by hormones assume a significant role in the etiology of diarrhea, as well as in compensatory processes by which an intestinal loss of electrolytes and water is prevented.

Adrenal Cortex Hormones↗

Effects of nicotine on epithelial nephron cells in culture.

Long-term effects of nicotine on cultured cells derived from defined segments of the nephron were studied in vitro. Nicotine was added during a 10-day incubation period to a defined nephron culture medium (NCM) which had been supplemented with one of the following: fetal calf serum (FCS), dexamethasone (D), aldosterone (A), or epidermal growth factor (EGF). Thymidine incorporation (10(-15) mmol/10(3) cells) into cultured cells of the cortical thick ascending loop of Henle (TAL) and of the cortical collecting tubule (CT) was inhibited by nicotine (5 X 10(1) ng/ml) when incubated in NCM only; the presence of FCS or of D (10(-8) M) or of D and EGF (25 ng/ml) prevented this inhibitory effect. Nicotine at a concentration of 5 X 10(2) ng/ml was inhibitory in TAL and CT even in the presence of FCS. Na-K-ATPase activity (pmol/10(3) cells X min-1) was stimulated significantly by D when compared with NCM; nicotine (5 X 10(2) ng/ml) prevented this inductive effect. Transepithelial voltage was 27.1 +/- 6 (mV) after incubation with A (10(-9) M), and 7.2 +/- 4.1 when incubated with A and nicotine (5 X 10(2) ng/ml). These data indicate that nicotine may interfere with factors stimulating cell proliferation, sodium pump enzyme activity, and ion flux (voltage). An effect of nicotine on the cellular Na-entry step would be consistent with the data.

Animals↗

Renal countercurrent system: role of collecting duct convergence and pelvic urea predicted from a mathematical model.

A differential equation model of the renal countercurrent system has been developed and physiological data from nephron segments were incorporated together with recently suggested urea recycling from renal pelvis to inner medulla and, particularly, an exponential reduction in the number of collecting tubules towards the renal papilla. The role of these features for the countercurrent concentrating mechanism has been studied by simulation runs. The computations, using the multiple shooting method, provide predictions about concentration profiles for salt and urea in tubes (nephron segments) and in the central core along the entire medullary countercurrent system. The results indicate that this model, without active salt or urea transport in the inner medulla, yields concentration gradients along the medullary axis compatible with those measured in the tissue.

Animals↗

Aldosterone on sodium transport of rat distal colon in long-term adrenalectomy during acute and chronic substitution.

1. The influence of aldosterone upon water and sodium transport properties of the distal colon was studied in long-term adrenalectomy (11-29 days).2. Six groups of rats were used: I, normal (control); II, adrenalectomized; III, adrenalectomized, acutely substituted with aldosterone (200 mug/kg 4 h); IV, adrenalectomized rats receiving aldosterone simultaneously with the specific inhibitor spironolactone (40 mg/kg within 4 h); V, adrenalectomized, substituted chronically with aldosterone (2 x 75 mug/kg day); VI, adrenalectomized, substituted chronically with dexamethasone (120 mug/kg day).3. Distal colon segments were perfused in vivo with isotonic Ringer solution. In addition, a hypotonic electrolyte solution (Na(+) 111 mM) was used in groups I and II.4. In adrenalectomy (group II), net water absorption (J(v)) was significantly decreased from (normal) 54.4 mul/h cm(2)+/-10.5 (n = 9) to 41.2 mul/h cm(2)+/-7.3 (n = 4), and net Na(+) absorption (J(Na)) was decreased from 13.6 mumol/h cm(2)+3.5 to 8.5 mumol/h cm(2)+/-0.9 (isotonic perfusate). Similarly, J(v) was decreased from 54.0 mul/h cm(2)+/-8.3 (n = 4) to 37.3 mul/h cm(2)+/-4.2 (n = 7), and J(Na) from 8.6 mumol/h cm(2)+/-2.1 to 4.2 mumol/h cm(2)+/-2.1 (hypotonic perfusate).5. Acute aldosterone substitution in adrenalectomy (III) had no effect upon J(v) (37.1 mul/h cm(2)+/-10.3; n = 5) but increased J(Na) to 10.3 mumol/h cm(2)+/-0.3.6. The luminal Na(+) steady-state concentration was higher in group II (11.2 mmol l(-1)+/-3.6; n = 6) than in group I (3.3 mmol l(-1)+/-1.4; n = 29). Acute aldosterone substitution restored this value to normal (3.0 mmol l(-1)+/-1.2; n = 4). The aldosterone effect was partly blocked by spironolactone: the Na(+) steady-state concentration was 6.4 mmol/l+/-0.6 (n = 3) in group IV.7. At the steady-state luminal Na(+) concentration, the osmotically driven net water fluxes were not different in groups I and II, indicating that the hydraulic permeability coefficient is not altered in adrenalectomy.8. In group V, J(v) (54.9 mul/h cm(2)+/-10.9; n = 7) and J(Na) (11.9 mumol/h cm(2)+/-1.7; n = 6) were not significantly different from normal.9. In group VI, J(v) (37.3 mul/h cm(2)+/-6.0; n = 5) and J(Na) (8.0 mumol/h cm(2)+/-1.4) were not significantly different from group II.10. The mineralocorticoid effects of aldosterone in long-term adrenalectomy appear to represent the principal determining factors of colonic J(v) and J(Na).

Adrenalectomy↗

Hydraulic permeability coefficient and sodium steady-state luminal concentration of the in vivo perfused rat distal colon.

The distal colon (rat) was perfused in vivo at low rates (1-2 ml/h). Dialyzed polyethyleneglycol 4,000 (PEG) was used to vary the luminal osmotic activity (pi eff). Perfusate sodium concentration of 3.7 mmol 1-1 +/- 0.9 (SD) resulted in an effluent Na+ of 3.3 mmol 1-1 +/- 1.4. Potassium concentrations of 15 or 25 mmol 1-1 remained unaltered. At this cationic steady-state, transmural net water flux (Jv) was linearly correlated (r = -0.96, n = 24) to the logarithm of pi eff. Jv was zero at 634 mosm 1-1. Lp (microliters h-1 cm-2 mosm-1 l) was 0.195 at pi eff 85 mosm 1-1 and 0.046 at 1,050 mosm 1-1. These data characterize the distal colon as an epithelium with high capacity for salt conservation.

Animals↗

Tissue culture in nephrology: potential and limits for the study of renal disease.

Kidney cells, when isolated and cultivated in vitro, retain differentiated renal properties. Glomerular epithelial and mesangial cells from animal and human kidneys express their normal ultrastructure and the ability for basement membrane biosynthesis. Mesangial cells in culture have been utilized particularly for the study of hormonal tissue receptors, of prostaglandin production, and of their contractile response to various hormonal stimuli. Cells of tubule origin have been a valuable tool for the study of transport mechanism which, as a consequence of the heterogeneity of nephron functions, can not be assessed in vivo. Ion transport and its structural basis, as well as transport regulation by hormones has been studied in established epithelial cell lines. Induction of ion transport and enzyme activities, and the control of cell proliferation and differentiation has also been succesfully evaluated in cultured epithelia derived from the kidney. Future work will attempt to prepare cell lines from defined nephron segments to study chemical and physical phenomena of renal disease.

Biological Transport↗