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Kidney organoids cultured on adherent matrices in the presence of superfusate flow generate vascular networks and exhibit more mature podocyte and tubular compartments compared with static controls (Homan KA, Gupta N, Kroll KT, Kolesky DB, Skylar-Scott M, Miyoshi T, Mau D, Valerius MT, Ferrante T, Bonventre JV, Lewis JA, Morizane R. Nat Methods 16: 255-262, 2019; Takasato M, Er PX, Chiu HS, Maier B, Baillie GJ, Ferguson C, Parton RG, Wolvetang EJ, Roost MS, Chuva de Sousa Lopes SM, Little MH. Nature 526: 564-568, 2015.). However, their physiological function has yet to be systematically investigated. Here, we measured mechano-induced changes in intracellular Ca2+ concentration ([Ca2+]i) in tubules isolated from organoids cultured for 21-64 days, microperfused in vitro or affixed to the base of a specimen chamber, and loaded with fura-2 to measure [Ca2+]i. A rapid >2.5-fold increase in [Ca2+]i from a baseline of 195.0 ± 22.1 nM (n = 9; P ≤ 0.001) was observed when microperfused tubules from organoids >40 days in culture were subjected to luminal flow. In contrast, no response was detected in tubules isolated from organoids <30 days in culture. Nonperfused tubules (41 days) subjected to a 10-fold increase in bath flow rate also exhibited a threefold increase in [Ca2+]i from baseline (P < 0.001). Mechanosensitive PIEZO1 channels contribute to the flowinduced [Ca2+]i response in mouse distal tubule (Carrisoza-Gaytan R, Dalghi MG, Apodaca GL, Kleyman TR, Satlin LM. The FASEB J 33: 824.25, 2019.). Immunodetectable apical and basolateral PIEZO1 was identified in tubular structures by 21 days in culture. Basolateral PIEZO1 appeared to be functional as basolateral exposure of nonperfused tubules to the PIEZO1 activator Yoda 1 increased [Ca2+]i (P ≤ 0.001) in segments from organoids cultured for >30 days, with peak [Ca2+]i increasing with advancing days in culture. These results are consistent with a maturational increase in number and/or activity of flow/stretch-sensitive Ca2+ channels, including PIEZO1, in tubules of static organoids in culture. [ABSTRACT FROM AUTHOR]

The water channel aquaporin-1 (AQP1) is the principal water pathway for isotonic water reabsorption in the kidney proximal tubule (PT). We investigated flow-mediated fluid (Jv) and HCO- 3 (JHCO3 ) reabsorption in PTs of the mouse kidney by microperfusion in wild-type (WT) and AQP1 knockout (KO) mice. Experiments were simulated in an adaptation of a mathematical model of the rat PT. An increase in perfusion rate from 5 to 20 nL/min increased Jv and JHCO3 in PTs of WT mice. AQP1 KO mice significantly decreased Jv at low and high flow rates compared with control. In contrast, JHCO3 was not reduced at either low or high flow rates. Cell volume showed no significant difference between WT and AQP1 KO mice. Renal clearance experiments showed significantly higher urine flow in AQP1 KO mice, but there was no significant difference in either Na+ and K+ or HCO-3 excretion. Acid-base parameters of blood pH, PCO2, HCO-3, and urine pH were the same in both WT and KO mice. In model calculations, tubules whose tight junction (TJ) water permeability (Pf) was that assigned to the rat TJ, showed no difference in Jv between WT and KO, whereas TJ Pf set to 25% of the rat predicted Jv concordant with our observations from AQP1 KO. These results affirm the dominance of AQP1 in mediating isotonic water reabsorption by the mouse PT and demonstrate that flowstimulated HCO3 reabsorption is intact and independent of AQP1. With reference to the model, the findings also suggest that TJ water flux in the Pf is less prominent in the mouse than in the rat kidney. NEW & NOTEWORTHY We found an absence of flow-dependent modulation of fluid absorption but no effect on either proximal tubule (Pf) HCO-3 absorption or acid-base parameters in the aquaporin 1 (AQP1) knockout mouse. We affirmed the dominance of the water channel AQP1 in mediating isotonic water reabsorption by the mouse PT and demonstrated that flow-stimulated HCO-3 reabsorption is independent of AQP1. With reference to the model, the findings also suggest that tight junctional water flux in the PT is less prominent in the mouse than rat kidney. [ABSTRACT FROM AUTHOR]

HCO3− secretion in distal airways is critical for airway mucosal defense. HCO3−/H+ transport across the apical membrane of airway surface epithelial cells was studied by measuring intracellular pH in luminally microperfused freshly dissected mice bronchioles. Functional studies demonstrated that CFTR, ENaC, Cl−–HCO3− exchange, Na+-H+ exchange, and Na+–HCO3− cotransport are involved in apical HCO3−/H+ transport. RT-PCR of isolated bronchioles detected fragments from Cftr, α, β, γ subunits of ENaC, Ae2, Ae3, NBCe1, NBCe2, NBCn1, NDCBE, NBCn2, Nhe1, Nhe2, Nhe4, Nhe5, Slc26a4, Slc26a6, and Slc26a9. We assume that continuous decline of intracellular pH following alkaline load demonstrates time course of HCO3− secretion into the lumen which is perfused with a HCO3−-free solution. Forskolin-stimulated HCO3− secretion was substantially inhibited by luminal application of CFTRinh-172 (5 μM), H2DIDS (200 μM), and amiloride (1 μM). In bronchioles from a cystic fibrosis mouse model, basal and acetylcholine-stimulated HCO3− secretion was substantially impaired, but forskolin transiently accelerated HCO3− secretion of which the magnitude was comparable to wild-type bronchioles. In conclusion, we have characterized apical HCO3−/H+ transport in native bronchioles. We have demonstrated that cAMP-mediated and Ca2+-mediated pathways are involved in HCO3− secretion and that apical HCO3− secretion is largely mediated by CFTR and H2DIDS-sensitive Cl−–HCO3− exchanger, most likely Slc26a9. The impairment of HCO3− secretion in bronchioles from a cystic fibrosis mouse model may be related to the pathogenesis of early lung disease in cystic fibrosis. [ABSTRACT FROM AUTHOR]

BACKGROUND: Negative pressure wound therapy (NPWT) has been established over years for treatment of chronic and complex wounds. OBJECTIVE: Aim of this study was to investigate the effect NPWT on the microperfusion. METHODS: Prospective single centre analysis of patients treated with NPWT due to acute (ACUTE) wounds after fasciotomy or patients with chronic wounds (CHRONIC) due to a chronic limb threatening ischemia was performed. NPWT was conducted through a three days sequence with a negative pressure of –120 mmHg. Before after and during the entire period of therapy the microperfusion was assessed (O2C™, LEA Medizintechnik). RESULTS: Comparison of the perfusion values of 28 patients (CHRONIC/ACUTE 5/23, women/men 8/20) before and after the NPWT sequence showed a non-significant improvement in the CHRONIC group (supine position: p = 0.144, elevated position p = 0.068) and a significant decrease in the ACUTE group (supine position p = 0.012, elevated position p = 0.034). This effect could also been demonstrated during the NPWT over time (CHRONIC: supine position: p = 0.320, elevated position: p = 0.053, ACUTE: supine position: p = 0.021, elevated position: p = 0.012). CONCLUSION: Microperfusion measurements showed alterations and differences in wound bed perfusion of acute and chronic wounds; acute wounds tended to a decrease of blood flow, whereas this effect was not seen in chronic wounds in peripheral artery disease. [ABSTRACT FROM AUTHOR]

Assessment and monitoring of changes in microcirculatory perfusion, perfusion dynamic, vessel structure and oxygenation is crucial in management of arterial hypertension. Constant search for non-invasive methods has led the clinical focus towards the vasculature of the retina, which offers a large opportunity to detect the early phase of the functional and structural changes in the arterial hypertension and can reflect changes in brain vasculature. We review all the available methods of retinal microcirculation measurements including angiography, oximetry, retinal vasculature assessment software, Optical Coherence Tomography Angiography, Adaptive Optics and Scanning Laser Doppler Flowmetry and their application in clinical research. To further analyse the applicability of described methods in hypertension research we performed a systematic search of the PubMed electronic database (April 2020). In our analysis, we included 111 articles in which at least one of described methods was used for assessment of microcirculation of the retina in hypertensive individuals. Up to this point, the methods most commonly published in studies of retinal microcirculation in arterial hypertension were Scanning Laser Doppler Flowmetry followed shortly by Optical Coherence Tomography Angiography and retinal vasculature assessment software. While none of described methods enables the simultaneous measurement of all microcirculatory parameters, certain techniques are widely used in arterial hypertension research, while others gain popularity in screening. [ABSTRACT FROM AUTHOR]

Background: Aim of this prospective experimental study was to assess effects of systemic hypoxemia and hypovolemia on global and gastrointestinal oxygenation and perfusion in anesthetized horses. Therefore, we anesthetized twelve systemically healthy warmblood horses using either xylazine or dexmedetomidine for premedication and midazolam and ketamine for induction. Anesthesia was maintained using isoflurane in oxygen with either xylazine or dexmedetomidine and horses were ventilated to normocapnia. During part A arterial oxygen saturation (SaO2) was reduced by reducing inspiratory oxygen fraction in steps of 5%. In part B hypovolemia was induced by controlled arterial exsanguination via roller pump (rate: 38 ml/ kg/h). Mean arterial blood pressure (MAP), heart rate, pulmonary artery pressure, arterial and central venous blood gases and cardiac output were measured, cardiac index (CI) was calculated. Intestinal microperfusion and oxygenation were measured using laser Doppler flowmetry and white-light spectrophotometry. Surface probes were placed via median laparotomy on the stomach, jejunum and colon. Results: Part A: Reduction in arterial oxygenation resulted in a sigmoid decrease in central venous oxygen partial pressure. At SaO2 < 80% no further decrease in central venous oxygen partial pressure occurred. Intestinal oxygenation remained unchanged until SaO2 of 80% and then decreased. Heart rate and pulmonary artery pressure increased significantly during hypoxemia. Part B: Progressive reduction in circulating blood volume resulted in a linear decrease in MAP and CI. Intestinal perfusion was preserved until blood loss resulted in MAP and CI lower 51 ± 5 mmHg and 40 ± 3 mL/kg/min, respectively, and then decreased rapidly. Conclusions: Under isoflurane, intestinal tissue oxygenation remained at baseline when arterial oxygenation exceeded 80% and intestinal perfusion remained at baseline when MAP exceeded 51 mmHg and CI exceeded 40 mL/kg/min in this group of horses. [ABSTRACT FROM AUTHOR]

Key points The digestive tract of larval and adult Drosophila is an excellent analogue of the mammalian gut. Enterocytes of the posterior midgut are separated by septa, with no paracellular path, and therefore perform both immune and transport functions., Using microperfusion electrophysiology, we show that larvae emerging from the embryo into sterile medium have symmetrical apical and basal membrane conductances while larvae emerging into non-sterile medium have apical membranes fivefold more conductive than basal membranes. The channels inserted into the apical membranes could originate in microbiata or host and mediate recognition of microbes., Entomopathogenic cyclic peptide toxins deplete intracellular ions reversibly, forming transient ion channels that do not conduct water, unlike an ionophore like nystatin that depletes ions irreversibly., We show the feasibility of studying the interaction of a single microbial species, or tractable combinatorials of microbial species, with only enterocytes in the primary epithelial barrier., Abstract Microbiota colonizing exposed epithelial surfaces are vital for sustenance of metazoan life, but communication between microbiota, epithelial cells and the host immune system is only beginning to be understood. We address this issue in the posterior midgut epithelium of Drosophila larvae where nutrient transport and immune functions are exclusively transcellular. We showed that larvae emerging into a sterile post-embryonic environment have symmetrical apical and basal membranes. In contrast, larvae emerging into non-sterile media, the source of microbiota, have markedly asymmetrical membranes, with apical membrane conductance more than fivefold higher than the basal membrane. As an example of pathogen action, we showed that the entomopathogenic fungal toxin destruxin A (Dx) depleted intracellular ions. Reversibility of action of Dx was verified by bilayer reconstitution in forming transient non-specific channels that conduct ions but not water. Dx was also less effective from the apical side as compared to the basal side of the epithelium. We also showed that intercellular septa are not conductive in non-sterile cells, even though most cells are isopotential. Luminal microbiota therefore impart asymmetry to the epithelium, by activation of apical membrane conductance, enhancing inter-enterocyte communication, separated by insulating septa, via the gut lumen. These results also open the possibility of studying the basis of bidirectional molecular conversation specifically between enterocytes and microbiota that enables discrimination between commensals and pathogens, establishment of the former, and elimination of the latter. [ABSTRACT FROM AUTHOR]

The thick ascending limb (TAL) of Henle’s loop drives paracellular Na+, Ca2+, and Mg2+ reabsorption via the tight junction (TJ). The TJ is composed of claudins that consist of four transmembrane segments, two extracellular segments (ECS1 and -2), and one intracellular loop. Claudins interact within the same (cis) and opposing (trans) plasma membranes. The claudins Cldn10b, -16, and -19 facilitate cation reabsorption in the TAL, and their absence leads to a severe disturbance of renal ion homeostasis. We combined electrophysiological measurements on microperfused mouse TAL segments with subsequent analysis of claudin expression by immunostaining and confocal microscopy. Claudin interaction properties were examined using heterologous expression in the TJ-free cell line HEK 293, live-cell imaging, and Förster/FRET. To reveal determinants of interaction properties, a set of TAL claudin protein chimeras was created and analyzed. Our main findings are that (i) TAL TJs show a mosaic expression pattern of either cldn10b or cldn3/cldn16/cldn19 in a complex; (ii) TJs dominated by cldn10b prefer Na+ over Mg2+, whereas TJs dominated by cldn16 favor Mg2+ over Na+; (iii) cldn10b does not interact with other TAL claudins, whereas cldn3 and cldn16 can interact with cldn19 to form joint strands; and (iv) further claudin segments in addition to ECS2 are crucial for trans interaction. We suggest the existence of at least two spatially distinct types of paracellular channels in TAL: a cldn10b-based channel for monovalent cations such as Na+ and a spatially distinct site for reabsorption of divalent cations such as Ca2+ and Mg2+. [ABSTRACT FROM AUTHOR]

Key points The cortical collecting duct (CCD) plays an essential role in sodium homeostasis by fine-tuning the amount of sodium that is excreted in the urine., Ex vivo, the microperfused CCD reabsorbs sodium in the absence of lumen-to-bath concentration gradients., In the present study, we show that, in the presence of physiological lumen-to-bath concentration gradients, and in the absence of endocrine, paracrine and neural regulation, the mouse CCD secretes sodium, which represents a paradigm shift., This secretion occurs via the paracellular route, as well as a transcellular pathway that is energized by apical H+/K+-ATPase type 2 pumps operating as Na+/K+ exchangers., The newly identified transcellular secretory pathway represents a physiological target for the regulation of sodium handling and for anti-hypertensive therapeutic agents., Abstract In vitro microperfusion experiments have demonstrated that cortical collecting ducts (CCDs) reabsorb sodium via principal and type B intercalated cells under sodium-depleted conditions and thereby contribute to sodium and blood pressure homeostasis. However, these experiments were performed in the absence of the transepithelial ion concentration gradients that prevail in vivo and determine paracellular transport. The present study aimed to characterize Na+, K+ and Cl− fluxes in the mouse CCD in the presence of physiological transepithelial concentration gradients. For this purpose, we combined in vitro measurements of ion fluxes across microperfused CCDs of sodium-depleted mice with the predictions of a mathematical model. When NaCl transport was inhibited in all cells, CCDs secreted Na+ and reabsorbed K+; Cl− transport was negligible. Removing inhibitors of type A and B intercalated cells increased Na+ secretion in wild-type (WT) mice but not in H+/K+-ATPase type 2 (HKA2) knockout mice. Further inhibition of basolateral NaCl entry via the Na+-K+-2Cl− cotransporter in type A intercalated cells reduced Na+ secretion in WT mice to the levels observed in HKA2−/− mice. With no inhibitors, WT mouse CCDs still secreted Na+ and reabsorbed K+. In vivo, HKA2−/− mice excreted less Na+ than WT mice after switching to a high-salt diet. Taken together, our results indicate that type A intercalated cells secrete Na+ via basolateral Na+-K+-2Cl− cotransporters in tandem with apical HKA2 pumps. They also suggest that the CCD can mediate overall Na+ secretion, and that its ability to reabsorb NaCl in vivo depends on the presence of acute regulatory factors. [ABSTRACT FROM AUTHOR]

Angiotensin II (ANG II) is secreted by the proximal tubule resulting in a luminal concentration that is 100- to 1,000-fold greater than that in the blood. Luminal ANG II has been shown to stimulate sodium transport in the proximal tubule and distal nephron. Surprisingly, luminal ANG II inhibits NaCl transport in the medullary thick ascending limb (mTAL), a nephron segment responsible for a significant amount of NaCl absorption from the glomerular ultrafiltrate. We confirmed that addition of 10-8 M ANG II to the lumen inhibited mTAL chloride transport (220 ± 19 to 165 ± 25 pmol·mm-1·min-1, P < 0.01) and examined whether an interaction with basolateral norepinephrine existed to simulate the in vivo condition of an innervated tubule. We found that in the presence of a 10-6 Mnorepinephrine bath, luminal ANG II stimulated mTAL chloride transport from 298 ± 18 to 364 ± 42 pmol·mm-1·min-1 (P < 0.05). Stimulation of chloride transport by luminal ANG II was also observed with 10-3 M bath dibutyryl cAMP in the bathing solution and bath isoproterenol. A bath of 10-5 H-89 blocked the stimulation of chloride transport by norepinephrine and prevented the effect of luminal ANG II to either stimulate or inhibit chloride transport. Bath phentolamine, an α-adrenergic agonist, also prevented the decrease in mTAL chloride transport by luminal ANG II. Thus luminal ANG II increases chloride transport with basolateral norepinephrine; an effect likely mediated by stimulation of cAMP. Alpha-1 adrenergic stimulation prevents the inhibition of chloride transport by luminal ANG II. [ABSTRACT FROM AUTHOR]

The thick ascending limb of Henle's loop (TAL) drives an important part of the reabsorption of divalent cations. This reabsorption occurs via the paracellular pathway formed by the tight junction (TJ), which in the TAL shows cation selectivity. Claudins, a family of TJ proteins, determine the permeability and selectivity of this pathway. Mice were fed with normal or high-Ca diet, and effects on the reabsorptive properties of cortical and medullary TAL segments were analysed by tubule microdissection and microperfusion. Claudin expression was investigated by immunostaining and quantitative PCR. We show that the TAL adapted to high Ca load in a sub-segment-specific manner. In medullary TAL, transcellular NaCl transport was attenuated. The transepithelial voltage decreased from 10.9 ± 0.6 mV at control diet to 8.3 ± 0.5 mV at high Ca load, thereby reducing the driving force for Ca and Mg uptake. Cortical TAL showed a reduction in paracellular Ca and Mg permeabilities from 8.2 ± 0.7 to 6.2 ± 0.5 ∙ 10 cm/s and from 4.8 ± 0.5 to 3.0 ± 0.2 · 10 cm/s at control and high-Ca diet, respectively. Expression, localisation and regulation of claudins 10, 14, 16 and 19 differed along the corticomedullary axis: Towards the cortex, the main site of divalent cation reabsorption in TAL, high-Ca intake led to a strong upregulation of claudin-14 within TAL TJs while claudin-16 and -19 were unaltered. Towards the inner medulla, only claudin-10 was present in TAL TJ strands. In summary, high-Ca diet induced a reduction of divalent cation reabsorption via a diminution of NaCl transport and driving force in mTAL and via decreased paracellular permeabilities in cTAL. We reveal an important regulatory pattern along the corticomedullary axis and improve the understanding how the kidney disposes of detrimental excess Ca. [ABSTRACT FROM AUTHOR]

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Objectives: Aim of this clinical study was to evaluate the angiosome concept with regard to the microcirculation of the foot in patients with CLI and to evaluate its relevance by means of combined laser Doppler flowmetrie and white-light tissue spectrophotometry. Methods: Twenty-eight patients who underwent leg revascularization in the stage of CLI were prospectively examined. The microperfusion was assessed by light guided spectrophotometry. The measuring points were set according to the angiosome concept into direct and indirect revascularized areas of the foot. Investigations were performed pre and postinterventionally and after 4 and 12 weeks in baseline-position as well as in an elevated position of the leg. Results: Microcirculation parameters (oxygen saturation, blood flow, velocity) of the revascularized leg showed a significant increase in elevation and baseline position compared to the preoperative values in most analyses. No significant differences between the direct and indirect revascularized angiosome were apparent. Conclusion: The light-guided spectrophotometry measurement proved to be feasible in terms of measuring changes in the microcirculation after leg revascularization. However, our data do not support the value of the "angiosome concept" concerning the individual changes in microperfusion of the foot in patients with CLI. [ABSTRACT FROM AUTHOR]

Complications rates after free flap transplantation still amount up to 5%. Consequently a reliable monitoring system is of high importance in plastic and reconstructive surgery. The following guidelines provide an overviewof the current opportunities for free flap planning and monitoring with ultrasound and in particular with contrast enhanced ultrasound. [ABSTRACT FROM AUTHOR]

Caffeine is one of the most commonly consumed pharmacologically active ingredients in the Western world. It is postulated to cause peripheral vasoconstriction and decreased digital blood flow. As a result, many hand surgeons forbid caffeine consumption post-operatively by patients undergoing replantation surgery for fear of compromising healing. We hypothesized that caffeine has no effect on digital microvascular perfusion. Healthy volunteers were recruited and digital microperfusion was assessed using laser Doppler probes attached to the finger pulp, both before and after ingestion of 100 mg of caffeine. A total of 34 patients were included in the final study. The mean flow before the consumption of caffeine was 226.15 PU. The mean flow following the consumption of caffeine was 197.7 PU. This decrease was not statistically significant. This study revealed no decrease in digital blood flow following the ingestion of 100 mg of caffeine by healthy volunteers, as measured by laser Doppler flow monitoring.Level of Evidence: 3 [ABSTRACT FROM PUBLISHER]

To define the stoichiometry and molecular identity of the Cl-/HCO3- exchanger in the apical membrane of pancreatic duct cells, changes in luminal pH and volume were measured simultaneously in interlobular pancreatic ducts isolated from wild-type and Slc26a6-null mice. Transepithelial fluxes of HCO3- and Cl- were measured in the presence of anion gradients favoring rapid exchange of intracellular HCO3- with luminal Cl- in cAMP-stimulated ducts. The flux ratio of Cl- absorption/ HCO3- secretion was∼0.7 in wild-type ducts and∼1.4 in Slc26a6-/- ducts where a different Cl-/HCO3 - exchanger, most likely SLC26A3, was found to be active. Interactions between Cl-/HCO3- exchange and cystic fibrosis transmembrane conductance regulator (CFTR) in cAMP-stimulated ducts were examined by measuring the recovery of intracellular pH after alkali-loading by acetate prepulse. Hyperpolarization induced by luminal application of CFTRinh-172 enhanced HCO3- efflux across the apical membrane via SLC26A6 in wild-type ducts but significantly reduced HCO3- efflux in Slc26a6-/- ducts. In microperfused wild-type ducts, removal of luminal Cl-, or luminal application of dihydro-4,4=-diisothiocyanatostilbene-2,2=-disulphonic acid to inhibit SLC26A6, caused membrane hyperpolarization, which was abolished in Slc26a6-/- ducts. In conclusion, we have demonstrated that deletion of Slc26a6 alters the apparent stoichiometry of apical Cl-/ HCO3 - exchange in native pancreatic duct. Our results are consistent with SLC26A6 mediating 1:2 Cl-/HCO3 - exchange, and the exchanger upregulated in its absence, most probably SLC26A3, mediating 2:1 exchange. [ABSTRACT FROM AUTHOR]

Objective: Clinical hepatic diffusion weighted imaging (DWI) generally relies on mono-exponential diffusion. The aim was to demonstrate that mono-exponential diffusion in the liver is contaminated by microperfusion and that the bi-exponential model is required. Methods: Nineteen fasting healthy volunteers were examined with DWI (seven b-values) using fat suppression and respiratory triggering (1.5 T). Five different regions in the liver were analysed regarding the mono-exponentially fitted apparent diffusion coefficient (ADC), and the bi-exponential model: molecular diffusion ( D), microperfusion ( D) and the respective fractions ( f). Data were compared using ANOVA and Kruskal-Wallis tests. Simulations were performed by repeating our data analyses, using just the DWI series acquired with b-values approximating those of previous studies. Results: Median mono-exponentially fitted ADCs varied significantly ( P < 0.001) between 1.107 and 1.423 × 10 mm/s for the five regions. Bi-exponential fitted D varied between 0.923 and 1.062 × 10 mm/s without significant differences ( P = 0.140). D varied significantly, between 17.8 and 46.8 × 10 mm/s ( P < 0.001). F-tests showed that the diffusion data fitted the bi-exponential model significantly better than the mono-exponential model ( F > 21.4, P < 0.010). These results were confirmed by the simulations. Conclusion: ADCs of normal liver tissue are significantly dependent on the measurement location because of substantial microperfusion contamination; therefore the bi-exponential model should be used. Key Points: [ABSTRACT FROM AUTHOR]

Prenatal insults have been shown to lead to elevated blood pressure in offspring when they are studied as adults. Prenatal administration of dexamethasone and dietary protein deprivation have demonstrated that there is an increase in transporter abundance for a number of nephron segments but not the subunits of the epithelial sodium channel (ENaC) in the cortical collecting duct. Recent studies have shown that aldosterone is elevated in offspring of protein-deprived mothers when studied as adults, but the physiological importance of the increase in serum aldosterone is unknown. As an indirect measure of ENaC activity, we compared the natriuretic response to benzamil in offspring of mothers who ate a low-protein diet (6%) with those who ate a normal diet (20%) for the last half of pregnancy. The natriuretic response to benzamil was greater in the 6% group (821.1 ± 161.0 μmol/24 h) compared with the 20% group (279.1 ± 137.0 μmol/24 h), consistent with greater ENaC activity in vivo (P < 0.05). In this study, we also directly studied cortical collecting tubule function from adult rats using in vitro microperfusion. There was no difference in basal or vasopressin-stimulated osmotic water permeability. However, while cortical collecting ducts of adult offspring whose mothers ate a 20% protein diet had no sodium transport (-1.9 ± 3.1 pmol.mm-1.min-1), the offspring of rats that ate a 6% protein diet during the last half of pregnancy had a net sodium flux of 10.7 ± 2.6 pmol.mm-1.min-1 (P = 0.01) in tubules perfused in vitro. Sodium transport was measured using ion-selective electrodes, a novel technique allowing measurement of sodium in nanoliter quantities of fluid. Thus we directly demonstrate that there is prenatal programming of cortical collecting duct sodium transport. [ABSTRACT FROM AUTHOR]

Barttin, a gene product of BSND, is one of four genes responsible for Bartter syndrome. Coexpression of barttin with ClC-K chloride channels dramatically induces the expression of ClC-K current via insertion of ClC-K-barttin complexes into plasma membranes. We previously showed that stably expressed R8L barttin, a disease-causing missense mutant, is retained in the endoplasmic reticulum (ER) of Madin-Darby canine kidney (MDCK) cells, with the barttin β-subunit remaining bound to ClC-K α-subunits (Hayama A, Rai T, Sasaki S, Uchida S. Histochem Cell Biol 119: 485-493, 2003). However, transient expression of R8L barttin in MDCK cells was reported to impair ClC-K channel function without affecting its subcellular localization. To investigate the pathogenesis in vivo, we generated a knockin mouse model of Bartter syndrome that carries the R8L mutation. These mice display disease-like phenotypes (hypokalemia, metabolic alkalosis, and decreased NaCl reabsorption in distal tubules) under a low-salt diet. Immunofluorescence and immunoelectron microscopy revealed that the plasma membrane localization of both R8L barttin and the ClC-K channel was impaired in these mice, and transepithelial chloride transport in the thin ascending limb of Henle's loop (tAL) as well as thiazide-sensitive chloride clearance were significantly reduced. This reduction in transepithelial chloride transport in tAL, which is totally dependent on ClC-K1/barttin, correlated well with the reduction in the amount of R8L barttin localized to plasma membranes. These results suggest that the major cause of Bartter syndrome type IV caused by R8L barttin mutation is its aberrant intracellular localization. [ABSTRACT FROM AUTHOR]

Purpose Anastomotic leakage is associated with increased morbidity and mortality. However, there is no accurate tool to predict its occurrence. We evaluated the predictive value of visible light spectroscopy (VLS), a novel method to measure tissue oxygenation [saturated O2 (StO2)], for anastomotic leakage of the colon and the rectum. Method Oxygen saturation in the bowel was measured in 77 colorectal resections. The anastomosis was between 2 and 30 cm (mean 13 cm) from the anal verge. The oxygen saturation was measured in the colon and rectum before and after anastomosis construction. This was compared with a reference measurement in the caecum. Data on postoperative complications were prospectively collected. Results Anastomotic leakage occurred in 14 (18%) patients. When compared with a leaking anastomosis, normal anastomoses showed rising O2 values during the operation (mean StO2 72.1 ± 9.0–76.7 ± 8.0 vs 73.9 ± 7.9–73.1 ± 7.4) ( P ≤ 0.05). There were also higher StO2 values in the caecum compared with those which ultimately leaked (73.6 ± 5.7 normal anastomoses , 69.6 ± 5.6 anastomotic leaks) ( P ≤ 0.05). Both StO2 values were predictive of anastomotic leakage. Conclusion Tissue oxygenation O2 appears to be a potentially useful means of predicting anastomotic leakage after colorectal anastomosis. [ABSTRACT FROM AUTHOR]

Objectives: The evaluation of subepithelial tumors of the stomach is normally the domain of gastroscopy and endoscopic ultrasound. We investigated these rare tumors using transabdominal B-mode ultrasound and performed perfusion analysis of these tumors with contrast enhanced ultrasound. Methods: Patients with gastrointestinal stromal tumors (GIST, n = 3), leiomyoma (n = 1) and schwannoma (n = 1) were routinely examined using conventional B-mode-ultrasound, colour Doppler ultrasound and contrast-enhanced ultrasound (contrast media: Sonovue®; ultrasound device: Siemens Acuson Sequoia™ 512). Gastroscopy, endosonography with puncture of the subepithelial tumor and computed tomography were also performed in all patients. After surgery, the resected stomach tumors were correlated with the preoperative imaging findings. Results: All calculated tumor sizes using any imaging modalities showed a good correlation with the macroscopic tumor sizes ex-vivo. Histologically increased tumor size of the GISTs was correlated with large, central avascular areas. The GISTs and the leiomyoma presented with mixed echogenicity in B-mode-ultrasound. Colour Doppler ultrasound was able to detect some vessels in the periphery of the tumor only. Using contrast-enhanced ultrasound the GISTs and the leiomyoma presented hypervascular. The contrast pattern of these lesions was from the periphery to the centre or diffuse or a progressive centrifugal fill in during the arterial phase. We also registered slowly progressive washout starting at the end of the arterial phase and increasing into the late phase. The contrast media behaviour in the schwannoma was different from that describt above within the GISTs: it was noted to have a diffuse intralesional pattern at the start of the arterial phase followed by an early, rapidly progressing washout-phenomenon. Conclusion: In our pilot study B-mode transabdominal ultrasound was able to visualise gastric subepithelial tumors larger than three centimetre. Contrast-enhanced ultrasound is a proven method in clinical practice for the perfusion analysis of gastric subepithelial tumors. It can also be used for the planning of ultrasound-guided biopsies to avoid punctures of necrotic tumor parts. [ABSTRACT FROM AUTHOR]

An elevation in angiotensin II (Ang II) levels is a common occurrence in spontaneously hypertensive rats (SHRs). Infusions of Ang II and a high salt diet increase the activity of NADPH oxidase that stimulates superoxide anion (O−2) generation and increases the expression of certain subunits of NADPH oxidase. Apocynin, an NADPH oxidase inhibitor with antihypertensive effects, is able to inhibit the release of superoxide anion by inhibiting NADPH oxidase activity and blocking the migration of p47 phox to the mitochondrial membrane. The aim of our study was to evaluate the antihypertensive effects of apocynin in SHRs and Wistar rats (WKYs) using a micropuncture technique. After microperfusion of both the proximal and distal tubules, we found that SHRs treated with apocynin showed a decrease in the free-flow collection of the proximal tubule (PT), which was not affected in WKYs. Moreover, significant differences were not demonstrated in the distal tubule (DT), probably due a mechanism of compensation that occurs in the loop of Henle. In conclusion, it is possible that the mechanisms of reabsorption in the PT are controlled by the interactions of O−2 and nitric oxide (NO). These data could suggest a higher activity of NADPH oxidase and increase in reactive oxygen species (ROS) production in the PT during hypertension. [ABSTRACT FROM AUTHOR]

In the collecting duct (CD), H-K-ATPases function in cation reabsorption and H secretion. This study evaluated H-K-ATPase-mediated H secretion along the mouse CD, measured as EIPA and luminal bafilomycin A1-insensitive intracellular pH (pHi) recovery from acute H loading (NH4) using BCECF. pHi recovery was measured in 1) microperfused cortical, outer medullary, and inner medullary CDs (CCD, OMCD, and IMCD) from C57BL/6J mice fed a normal diet and 2) common murine CD cell lines. H-K-ATPase activity along the native, microperfused CD was greatest in the CCD, less in the OMCD, and least in the IMCD (0.10 ± 0.02, 0.04 ± 0.01, and 0.01 ± 0.002 U/mm, respectively). H-K-ATPase activity was 0.30 ± 0.03 and 0.26 ± 0.03 in Aand B-type ICs, respectively, and was sensitive to Sch-28080 or ouabain. pHi recovery was greatest in the OMCDi cell line (0.25 ± 0.01) and less in mpkCCDc14 (0.17 ± 0.01), mIMCD-K2 (0.12 ± 0.01), and mIMCD-3 (0.05 ± 0.01) cells. EIPA inhibited the majority of pH recovery in these cells (100%, 64%, 75%, and 80% in mpkCCDc14, OMCD1, mIMCD-K2, and mIMCD-3, respectively). In OMCD1 cells, where EIPA-insensitive pH, recovery was greatest, H-K-ATPase activity was 0.10 ± 0.01 and was significantly inhibited (80%) by Sch-28080. We conclude that 1) H-K-ATPase-mediated H secretion in the native mouse CD is greatest in the ICs of the CCD, 2) Aand B-type ICs possess HKa1 and HKcr2 H-K-ATPase activity, and 3) the OMCD1 cell line best exhibits H-K-ATPase. [ABSTRACT FROM AUTHOR]

Dagan A, Habib S, Gattineni J, Dwarakanath V, Baum M. Prenatal programming of rat thick ascending limb chloride transport by low-protein diet and dexamethasone. Am J Physiol Re gut Integr Comp Physiol 297: R93-R99, 2009. First published April 29, 2009; doi: 10.1152/ajpregu.9 1006.2008.-Prenatal administration of dexamethasone and a low-protein diet has been shown to result in hypertension in the offspring when they are adults. The cause for the hypertension is unknown. The purpose of this study was to examine whether there was prenatal programming of thick ascending limb transport. Rats were administered either dexamethasone for 4 days (0.2 mg/kg body wt) by intraperitoneal injection daily between the 15th and 18th day of gestation, or they were fed a low-protein diet (6% protein) or an isocaloric normal protein diet (20% protein) from day 12 gestation until birth. The offspring were studied as adults. Prenatal dexamethasone and dietary protein deprivation resulted in an increase in blood pressure. Offspring of mothers fed a low-protein diet had an increase in medullaiy but not cortical bumetanide-sensitive Na-K-2C1 cotransporter (NKCC2) protein abundance (P < 0.01). There was not a statistically significant increase in medullary NKCC2 by prenatal dexamethasone (P = 0.07). Both prenatal administration of dexamethasone and a low-protein diet resulted in an increase in medullary thick ascending limb chloride transport compared with control (298 ± 33 pmoles∙mm[sup-1]∙min[sup-1], 280 ± 26 pmoles∙mm[sup-1]∙min[sup-1], and 191 ± 21 pmoles∙mm[sup-1]∙min[sup-1], respectively P < 0.05). There was a higher lumen-positive transepithelial potential difference in the prenatal dexamethasone and low-protein group compared with control as well. Administration of furosemide for 24 h resulted in a decrease in blood pressure in the low-protein group but not the control group. This study demonstrates that insults administered to the fetus can program altered sodium transport. Increased tubular sodium transport is a likely cause for the hypertension by prenatal programming. [ABSTRACT FROM AUTHOR]

There is a resurgence of interest in the Drosophila midgut on account of its potential value in understanding the structure, development and function of digestive organs and related epithelia. The recent identification of regenerative or stem cells in the adult gut of Drosophila has opened up new avenues for understanding development and turnover of cells in insect and mammalian gastrointestinal tracts. Conversely, the physiology of the Drosophila gut is less well understood as it is a difficult epithelial preparation to study under controlled conditions. Recent progress in microperfusion of individual segments of the Drosophila midgut, in both larval and adult forms, has enabled ultrastructural and electrophysiological study and preliminary characterization of cellular transport processes in the epithelium. As larvae are more active feeders, the transport rates are higher than in adults. The larval midgut has at least three segments: an anterior neutral zone, a short and narrow acid-secreting middle segment and a long and wider posterior segment (which is the best studied) that secretes base (probably HCO3-) into the lumen. The posterior midgut has a lumen-negative transepithelial potential (35-45mV) and a high resistance (800-1400 Ω.cm²) that correlates with little or no lateral intercellular volume. The primary transport system driving base secretion into the lumen appears to be a bafilomycin-A1-sensitive, electrogenic H+ V-ATPase located on the basal membrane, which extrudes acid into the haemolymph, as inferred from the extracellular pH gradients detected adjacent to the basal membrane. The adult midgut is also segmented (as inferred from longitudinal gradients of pH dye-indicators in the lumen) into anterior, middle and posterior regions. The anterior segment is probably absorptive. The middle midgut secretes acid (pH<4.0), a process dependent on a carbonic- anhydrase-catalysed W pool. Cells of the middle segment are alternately absorptive (apically amplified by ≈9-fold, basally amplified by >90-fold) and secretory (apically amplified by >90-fold and basally by ≈10-fold). Posterior segment cells have an extensively dilated basal extracellular labyrinth, with a volume larger than that of anterior segment cells, indicating more fluid reabsorption in the posterior segment. The luminal pH of anterior and posterior adult midgut is 7-9. These findings in the larval and adult midgut open up the possibility of determining the role of plasma membrane transporters and channels involved in driving not only H+ fluxes but also secondary fluxes of other solutes and water in Drosophila. [ABSTRACT FROM AUTHOR]

Adenosine mediates Na+ reabsorption in the proximal tubule (PT) and other segments by activating adenosine type 1 receptors (A1-AR). We tested the hypothesis that A1-AR in the PT is regulated by salt intake and participates in the kidney adaptation to changes in salt intake. Absolute fluid reabsorption (Jv) was measured by direct in vivo microperfusion and recollection in rats maintained on low (LS; 0.03% Na, wt/wt)-, normal (NS; 0.3% Na)-, and high-salt (HS; 3.0% Na) diets for 1 wk. The effect of microperfusion of BG97 19 a highly selective inhibitor of A1-ARs or adenosine deaminase (AD), which metabolizes adenosine, was measured in each group. Jv was higher in PT from LS rats (LA: 2.8 ± 0.2 vs. NS: 2.1 ± 0.2 nl·min-1·mm-1, P < 0.001). Jv, in HS rats was not different from NS. BG9719 reduced Jv in LS rats by 66 ± 6% (LS: 2.8 ± 0.2 vs LS+CVT: 1.3 ± 0.3 nl·min-1·mm-1, P < 0.001), which was greater than its effect in NS (45 ± 4%) or HS (41 ± 4%) rats. AD reduced Jv similarly, suggesting that A1-ARs are activated by local production of adenosine. Expression of A1-AR mRNA and protein was higher (P < 0.01) in micro-dissected PTs in LS rats compared with NS and HS. We conclude that A1-ARs in the PT are increased by low salt intake and that A1-AR participates in the increased PT reabsorption of solute and fluid in response to low salt intake. [ABSTRACT FROM AUTHOR]

Two classes of H pumps, H-K-ATPase and H-ATPase, contribute to luminal acidification and HCO3 transport in the collecting duct (CD). At least two H-K-ATPase h-subunits are expressed in the CD: HKα1 and HKα2. Both exhibit K dependence but have different inhibitor sensitivities. The HKα1 H-K-ATPase is Sch-28080 sensitive, whereas the pharmacological profile of the HKα2 H-K-ATPase is not completely understood. The present study used a nonpharmacological, genetic approach to determine the contribution of HKα1 and HKα2 to cortical CD (CCD) intercalated cell (IC) proton transport in mice fed a normal diet. intracellular pH (pHi) recovery was determined in ICs using in vitro microperfusion of CCD after an acute intracellular acid load in wild-type mice and mice of the same strain lacking expression of HKα1, HKα2, or both H-K-ATPases (HKα1,2). A-type and B-type ICs were differentiated by luminal loading with BCECF-AM and peritubular chloride removal from CO2/HCO3-buffered solutions to identify the membrane locations of Cl/HCO3 exchange activity. H-ATPase- and Na/H exchange-mediated H transport were inhibited with bafilomycin A1 (100 nM) and EIPA (10 μM), respectively. Here, we report 1) initial pHi and buffering capacity were not significantly altered in the ICs of HKα-deficient mice, 2) either HKα1 or HKα2 deficiency resulted in slower acid extrusion, and 3) A-type ICs from HKα1,2-deficient mice had significantly slower acid extrusion compared with A-type ICs from HKα1-deficient mice alone. These studies are the first nonpharmacological demonstration that both HKα1 and HKα2 contribute to H secretion in both A-type and B-type ICs in animals fed a normal diet. [ABSTRACT FROM AUTHOR]

Copyright of Canadian Journal of Physiology & Pharmacology is the property of Canadian Science Publishing and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

A method for microperfusion of isolated segments of the midgut epithelium of Drosophila larvae has been developed to characterize cellular transport pathways and membrane transporters. Stereological ultrastructural morphometry shows that this epithelium has unusually long tight junctions, with little or no lateral intercellular volume normally found in most epithelia. Amplification of the apical and basal aspects of the cells, by ≈ 17-fold and ≈ 7-fold, respectively, predicts an almost exclusively transcellular transport system for solutes. This correlates with the high lumen-negative transepithelial potential ( V t) of 38 to 45 mV and high resistance ( R t) of 800 to 1400 Ω • cm2 measured by terminated cable analysis, in contrast to other microperfused epithelia like the renal proximal tubule. Several blockers (amiloride 10−4 M, ouabain 10−4 M, bumetanide 10−4 M), K+-free solutions, or organic solutes such as D-glucose 10 m M or DL-alanine 0.5 m M failed to affect V t or R t. Bafilomycin-A1 (3 to 5 μ M) decreased V t by ≈ 40% and short-circuit current ( I sc) by ≈ 50%, and decreased intracellular pH when applied from the basal side only, consistent with an inhibition of an electrogenic V-H+-ATPase located in the basal membrane. Gradients of H+ were detected by pH microelectrodes close to the basal aspect of the cells or within the basal extracellular labyrinth. The apical membrane is more conductive than the basal membrane, facilitating secretion of base (presumably HCO3 −), driven by the basal V-H+-ATPase. [ABSTRACT FROM AUTHOR]

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In previous studies examining the role of glucocorticoids and thyroid hormone on the maturation of the Na2/H2 antiporter (NHE3), we found attenuation in the maturational increase in proximal tubule apical Na2 /H2 antiporter activity but no change in NHE3 mRNA abundance in either glucocorticoid-deficient or hypothyroid rats. In addition, prevention of the maturational increase in either hormone failed to totally prevent the maturational increase in Na2/H2 antiporter activity. We hypothesized that one hormone played a compensatory role when the other was deficient. The present study examined whether combined deficiency of thyroid and glucocorticoid hormones would completely prevent the maturation of the Na 2/H2 antiporter. Adrenalectomy was performed in 9-day- old hypothyroid Sprague-Dawley rats, a time before the normal postnatal maturational increase in these hormones occurs. Nine- and 30-day-old adrenalectomized (ADX), hypothyroid rats had comparable NHE3 mRNA abundance, which was 5- to 10-fold less than 30-day-old ADX, hypothyroid rats that received corticosterone-thyroxine replacement and 30-day-old sham control rats (P < 0.05). Brush-border membrane NHE3 protein abundance was comparable in 9- and 30day-old ADX, hypothyroid groups and ∼20-fold lower than both the 30-day replacement and 30-day sham groups (P < 0.05). Similarly, the replacement and sham groups had higher sodium-dependent proton secretion than 9- and 30- day-old ADX, hypothyroid groups (P < 0.05). We conclude that combined deficiency of both hormones totally prevents the maturational increase in NHE3 mRNA and protein abundance and Na2/H2 antiporter activity. [ABSTRACT FROM AUTHOR]

The proximal tubule contains an autonomous renin-angiotensin system that regulates transport independently of circulating angiotensin II. Androgens are known to increase expression of angiotensinogen, but the effect of androgens on proximal tubule transport is unknown. In this in vivo microperfusion study, we examined the effect of androgens on proximal tubule transport. The volume reabsorptive rate in Sprague-Dawley rats given dihydrotestosterone (DHT) injections was significantly higher than in control rats given vehicle injections (4.57 ± 0.31 vs. 3.31 ± 0.23 nl·min-1·mm -1, P < 0.01). Luminally perfusing with either enalaprilat (10-4 M) to inhibit production of angiotensin II or losartan (10-8 M) to block the angiotensin receptor decreased the proximal tubule volume reabsorptive rate in DHT treated rats to a significantly greater degree than in control vehicle injected rats. The renal expression of angiotensinogen was shown to be higher in the DHT-treated animals, using Northern blot analysis. The expression of angiotensin receptors, determined by specific binding of angiotensin II, was not different in the two groups of animals. Brush-border membrane protein abundance of the Na/H exchanger, a membrane transport protein under angiotensin II regulation, was also higher in DHT-treated rats vs. control rats. Rats that received DHT had higher blood pressures than the control rats but had no change in their glomerular filtration rate. In addition, serum angiotensin II levels were lower in DHT- treated vs. control rats. These results suggest that androgens may directly upregulate the proximal tubule renin-angiotensin system, increase the volume reabsorptive rate, and thereby increase extracellular volume and blood pressure and secondarily decrease serum angiotensin II levels. [ABSTRACT FROM AUTHOR]

The balance of vascular tone between afferent (Af-) and efferent arterioles (Ef-Arts) is a crucial determinant of glomerular hemodynamics. Thus, to understand renal physiology and pathophysiology it is important to study the mechanisms that control their vascular resistance. In order to directly study these mechanisms, we have developed several in vitro microperfusion preparations of these arterioles, which have the advantage of allowing us to observe the arteriolar diameter directly in the absence of systemic hemodynamic and hormonal influences. Using these preparations, we have found that angiotensin II (Ang II) causes much stronger constriction in Ef- than in Af-Arts and that this difference is mediated by nitric oxide (NO)- and prostaglandin (PG)-induced modulation of Ang II action in the Af-Art. We have also found that the vasoconstrictor effect of Ang II on Ef-Arts is modulated by PG produced by the upstream glomerulus. Thus, this may be a mechanism whereby the glomerulus controls its own capillary pressure by releasing PG and thereby adjusting the resistance of the downstream Ef-Art. In addition, we have found that in these arterioles activation of the Ang II type 2 (AT2) receptor causes endothelium-dependent vasodilation, which modulates the vasoconstrictor action mediated by its type 1 (AT1) receptors. Such modulator mechanisms that regulate Af- and Ef-Art tone may play an important role in the precise control of glomerular hemodynamics, and their alterations may play a role in the pathophysiology of renal diseases, including hypertension. Indeed, we have demonstrated that the vasoconstrictor action of Ang II on the Af-Art is exaggerated in spontaneously hypertensive rats, an animal model of human essential hypertension, due to an impaired function of the AT2 receptor before the development of hypertension. Because such exaggerated vasoconstriction leads to the elevation of renal vascular resistance (an important pathogenic factor for essential hypertension), our findings suggest that impaired function of the AT2 receptor in Af-Arts may play a role in the pathophysiology of essential hypertension. [ABSTRACT FROM AUTHOR]

The balance of the vascular tone between afferent (Af-Arts) and efferent arterioles (Ef-Arts) is a crucial determinant of glomerular hemodynamics. In order to directly study the mechanisms that regulate their vascular tone, we have developed several in-vitro microperfusion preparations of these arterioles, which allow us to observe the arteriolar diameter in the absence of systemic hemodynamic and hormonal influences. In the Af-Art, but not Ef-Art, we have demonstrated the presence of two intrinsic mechanisms, myogenic response and macula densa-mediated tubuloglomerular feedback, both of which play an important role in the control of vascular tone. We also found that both of these mechanisms are modulated by endogenous nitric oxide (NO). In addition, several humoral factors (such as angiotensin II [Ang II] and prostaglandins [PGs]) play an important role in the control of the tone of these arterioles although their actions are not identical in Af- and Ef-Arts. We found that Ang II caused much stronger constriction in Ef- than in Af-Arts, and that this difference was mediated by the NO-induced modulation of Ang II action in the Af-Art. We have also found that the vasoconstrictor effect of Ang II on Ef-Arts is modulated by PGs produced by the upstream glomerulus. Thus, this may be a mechanism whereby the glomerulus controls its own capillary pressure by releasing PGs, thereby adjusting the resistance of the downstream Ef-Art. Such different mechanisms that regulate Af- and Ef-Art tone play an important role in the precise control of glomerular hemodynamics. [ABSTRACT FROM AUTHOR]

Mice aged 4 or 8 weeks were fed with a low-Mg2+ diet for 1, 2, 3 or 4 days. After 1 day of diet, the urinary excretion of Mg2+ and Ca2+ was strongly reduced in both animal groups (4 and 8 weeks), accompanied by a significant fall in plasma Mg2+ concentration and an increase in urinary volume. This profile persisted after 2, 3 or 4 days of dietary Mg2+ restriction. After 1 day of diet, transepithelial ion net fluxes of Na+, Cl–, Ca2+ and Mg2+ (JNa, JCl, JCa, JMg) measured in vitro from isolated perfused cortical thick ascending limbs (CTALs) of these animals remained unchanged. After 2 days of diet, measurements of JCa and JMg in isolated perfused CTALs showed that transepithelial Mg2+ and Ca2+ reabsorption were enhanced in CTALs from Mg2+-depleted, 8-week-old animals, whereas transepithelial Mg2+ and Ca2+ transport were not altered in 4-week-old mice. JNa and JCl and the transepithelial potential (PDte) were not modified in CTALs from either animal group. Our results suggest that a low-Mg2+ diet leads to urinary retention of Mg2+ and Ca2+ which is most likely due to increased Mg2+ and Ca2+ transport in the CTAL. Furthermore, in response to dietary Mg2+ restriction, the reabsorption of divalent cations in the CTAL of adult, but not of young, mice undergoes cellular adaptation. [ABSTRACT FROM AUTHOR]

Renal vasoconstrictor action of angiotensin II (Ang II) is exaggerated in the spontaneously hypertensive rat (SHR) before development of hypertension. We have recently demonstrated that in the rabbit afferent arteriole (Af-Art) activation of the AT2 receptor causes vasodilation, which modulates the vasoconstrictor action of Ang II mediated by the AT1 receptor. In this study, we tested the hypothesis that vasoconstrictor action of Ang II is exaggerated in SHR Af-Arts due to an impaired function of the AT2 receptor before development of hypertension. Af-Arts were microdissected from the superficial cortex of 4- to 5-week-old SHR or age-matched Wistar Kyoto rats (WKY), and perfused at 60 mm Hg in vitro. Ang II (10[sup –11] to 10[sup –8] M) decreased the luminal diameter of Af-Arts of both strains in a dose-dependent manner. However, the constriction was stronger in SHR; at 10[sup –10] M, the diameter decreased by 34 ± 4% in SHR (n = 6) compared to 18 ± 3% in WKY (n = 6; p < 0.01). Pretreatment with PD123319 (PD), an AT2 receptor antagonist, significantly augmented Ang II-induced constriction in WKY but not SHR Af-Arts; at 10[sup –10] M, the diameter now decreased by 41 ± 5 and 37 ± 1% in SHR (n = 6) and WKY (n = 6), respectively. Thus, blockade of the AT2 receptor abolished the difference in Ang II action on Af-Arts between strains. Moreover, with the AT1 receptor blockade Ang II caused dose-dependent dilation of preconstricted Af-Arts only in WKY (27 ± 5% at 10[sup –8] M, n = 5), and the dilation was abolished by simultaneous treatment with PD. In contrast, no such dilation was observed in SHR Af-Arts. These results suggest that activation of the AT2 receptor modulates AT1 receptor vasoconstriction in WKY Af-Arts, while impaired modulatory function of AT2 receptor may play a role in the exaggerated vasoconstrictor action of Ang II on the Af-Art of prehypertensive SHR. [ABSTRACT FROM AUTHOR]

As luminal fluid moves from the seminiferous tubule and enters the rete testis, its protein concentration declines from approximately 6 mg/ml to 1 mg/ml. It was therefore suggested that protein is either 1) utilized by the spermatozoa, 2) transported across the epithelium of the terminal segment of the seminiferous tubule, the tubuli recti or rete testis, or 3) absorbed and degraded by the epithelium. Horseradish peroxidase (HRP), a protein marker, was microperfused into single seminiferous tubules or perfused directly into the rete. After fixation, the HRP was localized histochemically and the tissue observed under the light- and electron microscope. HRP was taken up via pinocytotic vesicles into the cytoplasm of the Sertoli cells and germ cells but did not permeate extracellularly beyond the tight junctions. Similar results were obtained in the cells lining the terminal segment and the tubuli recti. The rete epithelium showed uptake of HRP into coated and noncoated vesicles, while some cells additionally revealed diffuse cytoplasmic distribution of HRP. The terminal segment, tubuli recti, and rete testis may be important routes by which proteins may leave the testicular fluid either to be degraded or to enter the blood. [ABSTRACT FROM AUTHOR]

We examined microlocalization of mRNA coding for adrenomedullin (AM), using reverse transcription- polymerase chain reaction (RT-PCR), and the effects of AM on adenosine 3',5'-cyclic monophosphate (cAMP) generation and water transport in microdissected rat nephron segments. We also examined intraglomerular site of the expression of AM and AM-stimulated cAMP generation in cultured rat mesangial cells (MC). RT-PCR demonstrated the signals for AM mRNA in glomerulus (Glm), cortical collecting duct (CCD), outer medullary collecting duct (OMCD), and inner medullary collecting duct (IMCD) but not in proximal convoluted tubule (PCT) or medullary thick ascending limb (MTAL). AM (10-7 M) stimulated cAMP generation in Glm » CCD - IMCD > OMCD but not in PCT or MTAL, which corresponded to the results of the expression of AM mRNA. AM (10-8 M) slightly increased osmotic water permeability by 24% in perfused terminal IMCD. Northern blot analysis revealed high expression of AM mRNA in MC. AM (10-7 M) stimulated cAMP generation in MC both in the presence and absence of fetal calf serum, suggesting that AM-dependent cAMP generation was evident both in cycling MC and in quiescent MC. AM may work as a diuretic peptide mainly by increasing glomerular filtration rate via cAMP in MC. [ABSTRACT FROM AUTHOR]

Arginine vasopressin (AVP) can selectively decrease blood flow in the renal medulla, but the sites of vasoconstriction are uncertain. We have examined the effects of vasopressin-receptor agonists and antagonists on the diameters of outer medullary descending vasa recta (OMDVR), isolated and perfused in vitro. AVP can constrict OMDVR, apparently via V1a receptors. Ablumenal AVP (10-10-10-6 M) or the selective V1a-receptor agonist [Phe²,Ile³,Orn8] - vasopressin (PO-VT; 10-8 M) constricted OMDVR focally and (at higher AVP concentrations) transiently. The V1a agonist [deamino-Cys¹,D-3-(pyridyl)Ala²,Arg8]vasopressin (DP-VP; 10-8 M) and the V2 agonist [deamino-Cys¹,D-Arg8]vasopressin (DDAVP; 10-8 M) did not constrict OMDVR. The V1a antagonist [d(CH2)5¹,O-Me-Tyr²,Arg8]vasopressin (CTM-VP; 10-10 or 10-8 M) inhibited vasoconstriction by AVP (10-9 M), whereas the V2 antagonist [d(CH2)5¹,D-Ile²,Ile4,Arg8]vasopressin (II-VP) at low concentration (10-10 M) did not. V2 stimulation seems to inhibit V1a constriction of OMDVR. DDAVP prevented constriction by PO-VT (10-8 M) applied at the same time and dilated OMDVR preconstricted with PO-VT. [ABSTRACT FROM AUTHOR]

Simultaneous capillary and luminal microperfusion studies were performed in the rat proximal tubule to determine the effects of the beta agonist isoproterenol and the alpha agonist phenylephrine on water absorption. Capillary and luminal perfusion solutions were composed such that organic solutes were not present, no bicarbonate was present in the lumen, and no chloride gradient was imposed. Under such conditions, water absorption (Jv) averaged 0.36 +/- 0.11 nl . min-1 . mm-1. the addition of isoproterenol to the capillary solution in concentrations of 10(-6) and 10(-4) M resulted in significantly higher Jv's of 0.68 +/- 0.10 and 0.71 +/- 0.11 nl . min-1 . mm-1, respectively. The enhancing effect of isoproterenol was inhibited by the beta blocker propranolol (10(-4) M), but not by the alpha blocker phentolamine (10(-7) M). The addition of phenylephrine (10(-6) M) to the capillary perfusion solution also resulted in a significantly higher Jv of 0.84 +/- 0.14 nl . min-1, an effect inhibited by phentolamine (10(-7) M), but not by propranolol (10(-4) M). Neither phentolamine nor propranolol alone in the concentrations indicated had an effect on water absorption. These experiments indicate that both alpha and beta agonists stimulate water absorption in the superficial proximal tubule of the rat. This effect appears to be relatively specific for each class of agonist, as demonstrated by the effects of the specific antagonists. [ABSTRACT FROM AUTHOR]

Clearance and micropuncture experiments were performed in rats to study the renal handling of oxalate. The C-oxalate to H-inulin clearance ratio (C/C) was 1.36±0.04 and was lowered by probenecid (200 mg/kg) to 1.11±0.03 (±S.E., n=6, P<0.005). an attempt was made to localize the assumed secretion of oxalate in three different micropuncture protocols. In free flow micropuncture experiments single nephron clearance of oxalate was not different when obtained from proximal or distal tubular puncture sites. The fractional delivery of oxalate averaged 0.84 ±0.03 regardless of the puncture site from midproximal to late distal. This finding indicates a net outflux of oxalate in an early proximal loop since oxalate is freely ultrafilterable. In microperfusion experiments the mean recovery of oxalate ranged from 79-90%. The outflux of oxalate correlated linearly with the tubular load ( r=0.95). The results suggest that no net secretion occurs in superficial nephron segments accessible for micropuncture. Since whole kidney clearances of oxalate always exceeded glomerular filtration rate, it is concluded that net addition of oxalate into the tubular fluid can occur at sites beyond the superficial late distal tubules or is due to higher delivery of oxalate by deep cortical nephrons. [ABSTRACT FROM AUTHOR]

The permselectivity of the upper portion of long descending limb of Henle (LDLu) was investigated with electrophysiological methods in the isolated perfused tubule preparation of hamster kidney. The diffusion potential ( V) was determined in three different protocols. In protocol 1, the tubules were initially perfused with modified Krebs Ringer's solution on the both sides of the epithelium. Then the bath solution was exchanged consecutively with another solution in which 50 mmol/l NaCl replaced by 50 mmol/l KCl, RbCl, NHCl, CsCl, LiCl, NaBr, NaNO, NaI, Na acetate or 75 mmol/l NaCl replaced by mannitol. The permeabilities for these ions relative to chloride were calculated by Goldman's constant field equation. The segment was found to be cation selective, with all cations being 5-9 times more permeable than all anions. The sequence of permeability was K>Rb>Li>NH=Cs≧ Na≫Cl≧Br≧NO≧I>Acetate. In protocol 2, pure 150 mM NaCl was used for the basal solution to avoid interference by other ions. The bathing solution was exchanged by other solutions which contained 150 mmol/l KCl, NHCl, CsCl, RbCl, LiCl, NaI, NaBr, NaNO, Na acetate or 75 mmol/l NaCl with mannitol. Thus simple biionic substitution was performed. Again, the segment was found to be cation selective, with all cations being 4-10 times more permeable than all anions. The sequence of ion permeability was NH>K>Rb>Na>Cs> Li≫NO≧Cl≧I≧Br≧Acetate. In protocol 3,75 mmol/l of NaCl with mannitol was applied either in the bath or in the lumen and the potential difference was compared on the same tubule. The diffusion potential across the epithelium was found to be completely symmetrical, suggesting that it reflects mainly, if not entirely, the permselectivity of the paracellular pathway. It is concluded that the upper portion of descending limb of Henle of long-loop nephron is highly permselective for cations. [ABSTRACT FROM AUTHOR]

The unidirectional resorption rates of l-glutamate (initial concentrations of 0.07, 0.66, 2.0 or 20.0 mmol·l, d-glutamate (0.66 mmol·l in the presence or absence of 20 mmol·l l-glutamate), and of l-aspartate (0.073, 0.3, 0.66, 2.0 or 5.0 mmol·l) were determined in the rat proximal convolution. l-Glutamate resorption was saturable. A permeability coefficient (P) of <−20 μm·s, and a maximum resorption rate ( J) of 0.15±0.015 (SEM) nmol ·s·m at a K of 0.17±0.025 (SEM) mmol·l was obtained for l-glutamate. For l-aspartate, J was 0.13 ±0.005 at a K of 0.1±0.013. A free flow glutamate concentration profile along the proximal convolution was (I) predicted from these constants and (II) actually measured by means of free flow micropuncture. The data agree very well and show that more than 90% of the filtered load is resorbed within the first third of the proximal convolution. The late proximal and early distal free flow recoveries of l-glutamate amounted to 5.3±1.7% (SEM) and 6.6±1.4% of the filtered load, respectively. In contrast to this, unidirectional resorption during the microperfusion of the same tubule section was high: fractional resorption amounted to ca. 96% at 2 mmol·l initial l-glutamate. It fell to 35 or 33% respectively if the initial l-glutamate concentration was 20 mmol·l or if the resorption of 0.66 mmol·l d-glutamate in presence of 20 mmol·l l-glutamate was measured. The fractional excretion of endogenous l-glutamate in the final urine amounted to 0.13±0.012% of the filtered load. It is concluded that l-glutamate and l-aspartate are quickly resorbed in early parts of the proximal convolution (low K). Saturation already occurs when there is a small increase in the filtered load (low J) The nephron section between the late proximal and early distal nephron sites also reabsorbs 'acidic' amino acids. Normally, however, the back leak cancels this out, and net flux becomes zero. Deep nephrons seem to handle amino acids somewhat differently than superficial nephrons do. [ABSTRACT FROM AUTHOR]

Single sections of superficial proximal convolutions of rat kidney were microperfused in vivo and in situ. The perfusion fluids contained radioactively labelled l- or d-aspartate, l-glutamate, l-pyroglutamate, or N-methyl- d-aspartate. l-γ-Carboxyglutamate as well as the other amino acids were added in the unlabelled from. Results. l- and d-Aspartate (0.073 mmol·1) are quickly resorbed at about the same rate. d-Aspartate resorption was blocked by l-aspartate (5 mmol·1) but not by β-alanine (5 mmol·1). l-Aspartate resorption was inhibited by l-glutamate (2 mmol·1) but not by d-glutamate, l-asparagine, l-phenylalanine or by succinate (2 mmol·1, each). The fast resorption of l-glutamate (0.073 mmol·1) was blocked by d-aspartate, l-cysteate (2 mmol·1), but not by 3-mercaptopicolinic acid (0.15 mmol·1), l-glutamine, 2-oxoglutarate, taurine, N-methyl- l-glutamate or kainic acid (2 mmol·1, each). l-γ-Carboxyglutamate (0.66 mmol·1) and N-methyl- d-aspartate (2μmol·1) were found to be resorbed only at an extremely small rate. l-pyroglutamate (0.076 mmol·1) resorption was not influenced by l-glutamate (1 mmol·1). Fractional excretion of γ-carboxyglutamate was 7-25% ( l-from) or 45-70% ( d-form) at an artificially elevated plasma level of 12μmol·1. It is concluded that l- and d-aspartate, l-glutamate, l-cysteate and, to a much smaller extent, l-γ-carboxyglutamate, are accepted by the tubular resorption mechanism highly specific for 'acidic' amino acids. N-Substitution, the amidation of the β- or γ-carboxyl group, or the removal of the α-amino moiety almost completely abolish the ability of such compounds to be resorbed via this carrier; N-methylated or γ-carboxylated derivatives of 'acidic' amino acids are not resorbed at all from the proximal tubule. The resorption of glutamate, but not of aspartate, is highly stereospecific. [ABSTRACT FROM AUTHOR]

In microperfusion experiments renal tubular reabsorption ofS- andC-labelled l-cysteine (=cys), l-cystine (= cys-cys), and l-cystathionine was measured in vivo et situ at different initial concentrations. The interactions of cys and cys-cys with several neutral amino acids were investigated. The cys reabsorption mechanism was found to be saturable and has a high capacity and a low affinity for cys. An J-value of 3.22±0.88 nmol · m · s and a K-value of 7.5±0.7 mmol · l were estimated. A saturation of cys-cys reabsorption could not be demonstrated. The fractional reabsorption rate (=FRR) of cys-cys was about 85% at initial concentrations of 0.01, 0.08, and 0.4 mmol · l after a perfusion distance of 2 mm. The FRR of l-cystathionine at an initial concentration of 0.115 mmol · l was only 30% under the same conditions. After perfusion of tubule segments between late proximal and early distal loops the recovery of cys, cys-cys, and cystathionine was smaller than 10%. The FRR of cys was decreased only by l-methionine. Six other neutral amino acids had no effect. On the other hand the FRR of cys-cys was reduced significantly by any of the tested neutral amino acids. The inhibitory effect increased in the order l-alanine < l-methionine < l-citrulline < α-aminoisobutyric acid < l-phenylalanine < cycloleucine. The FRR of l-methionine and l-phenylalanine was slightly reduced in the presence of cys. It is concluded from these results that cys-cys shares a transport system with other neutral amino acids which is not identical with the reabsorption mechanism for cys. Reabsorption of cys, cys-cys, and cystathionine occurs also in a tubular section between late proximal and early distal sites. [ABSTRACT FROM AUTHOR]

Interactions of l-cysteine (=cys) and l-cystine (=cys-cys), and dibasic amino acids were investigated during tubular reabsorption by microperfusion experiments in rat kidney. The following results were obtained: The dibasic amino acids l-ornithine and l-canavanine were strong inhibitors of cys-cys reabsorption. The arginine analogue agmatine and the lysine analogue 2,6-diaminopimelic acid had no effect. The oxidizing agent azodicarboxylic acid bis-dimethylamide (=diamide) decreased the fractional reabsorption rate (=FRR) of cys-cys (0.08 mmol·l) from 84% to 60% when present in the perfusion fluid in a concentration of 10 mmol·l. Diamide did not affect the reabsorption of a dibasic amino acid ( l-arginine) nor of a neutral amino acid ( l-phenylalanine). The FRR of l-arginine and l-ornithine could not be decreased by adding cys-cys to the perfusion fluid. Cys had just as little effect on the reabsorption of l-arginine like agmatine. In the presence of α-aminoisobutyric acid a slight reduction of the FRR of l-arginine could be observed. The dibasic amino acids l-arginine and l-canavanine had no influence on the FRR of cys when dithioerythritol was added to the perfusion fluid. Conclusions: More than one site exists for tubular reabsorption of cys-cys. One of these may be shared by dibasic amino acids. Cys is reabsorbed by a separate and specific transport system. A reduction of cys-cys to cys takes place rather in the tubular cell than in the lumen. [ABSTRACT FROM AUTHOR]

The present study was designed to define the prerequisites of carbonic anhydrase independent bicarbonate reabsorption. In free flow experiments during systemic application of carbonic anhydrase inhibitor benzolamide (50 mg/kg B. W.) bicarbonate recovery in % of filtered load was found to be 74±8% in late proximal convoluted tubules, 39±6% in distal convoluted tubules and 32±4% in urine, indicating that most of carbonic anhydrase independent bicarbonate reabsorption occurs in tubule segments prior to distal convoluted tubules. In vivo continuous microperfusion experiments in proximal convoluted tubules demonstrated that luminal benzolamide (0.5 mmol/l) virtually abolishes net bicarbonate fluxes, when bicarbonate concentration in the luminal perfusate (25 mmol/l) is close to peritubular plasma concentration (24.4 mmol/l). In contrast, a significant downhill reabsorptive flux occurs, when perfusate bicarbonate concentration is 75 mmol/l and a significant downhill secretory flux is observed, when the perfusate is initially free of bicarbonate. The corresponding apparent permeabilities are 1.0±0.1·10 cm/s for influx and 1.6±0.4·10 cm/s for efflux of bicarbonate. Clearance studies reveal that carbonic anhydrase dependent and independent bicarbonate reabsorption are not saturable but depend on the rate of volume reabsorption in the kidney. In conclusion, passive movements of bicarbonate do occur in proximal convoluted tubules and most likely contribute to carbonic anhydrase independent bicarbonate reabsorption. [ABSTRACT FROM AUTHOR]

The effects of three electrolyte transport inhibitors on the secretory and absorptive function of the human eccrine sweat gland were examined. Amiloride and furosemide at 10 mol/l had no detectable effect on secretion while ouabain at 10 mol/l completely inhibited secretion. In contrast, when amiloride, ouabain and furosemide were applied at 10 mol/l to the luminal surface of the sweat duct by microperfusion, only amiloride blocked Na reabsorption. When the same inhibitors were added to the bathing solution at the same concentration, only ouabain inhibited Na reabsorption detectably. Present findings suggest that the secretory epithelium is permeable to solute and water movement while the duct epithelium is probably impermeable with respect to fluid movement across it. [ABSTRACT FROM AUTHOR]

Together the two rat kidneys accumulated a total of 31.7±1.6% of the intravenously injected amount of 7 nmoles egg-white-lysozyme (measured as iodine 125-lysozyme) within 10 min. The low molecular weight protein lysozyme and other basic substances were injected simultaneously in order to evaluate whether these basic substances can inhibit the tenal lysozyme accumulation. The inhibitory effect of various basic compounds was dose-dependent with a maximal reduction of lysozyme accumulation to 11.7±0.8%. The basic substances could be divided into three groups depending upon the micromolar amount injected at which a 50% inhibition was achieved (0.3-1.2 μmoles: cytochrome C, ribonuclease; 10.9 μmoles: spermine; 501-688 μmoles: l-arginine, l-lysine). The neutral myoglobin had no effect on renal lysozyme accumulation. The inhibitory potency appeared to increase with increasing molecular weight and pI value of the substances tested. Microperfusion experiments of proximal convoluted tubules of rat kidney revealed that luminal reabsorption of the basie lysozyme can be inhibited by the basic protein cytochrome C in a dose-dependent fashion. In these experiments the perfusion solution contained 57 μmol·l lysozyme, an intratubular lysozyme concentration at which the tubular lysozyme reabsorption was found to be about 80% saturated. A 50% inhibition of the tubular endocytic lysozyme reabsorption was achieved at a cytochrome C concentration of 102 μmol·l. [ABSTRACT FROM AUTHOR]