Ingrid Lichtenstein

Filtration in surface glomeruli as regulated by flow rate through the loop of henle

SummaryA new pressure transducer microperfusion system has been used to measure quantitatively SN GFR, early proximal free flow pressure and stop flow pressure of varying flow rates through the loop of Henle in the range of 0 to 50 nl/min. Perfusing the loop with an isoosmolal artificial tubular fluid at physiological flow rates SN GFR was 19.9±1.1 and 27.7±1.0 nl/min in two different strains of antidiuretic rats. SN GFR increased when loop perfusion was interrupted. The question whether a feedback control mechanism of SN GFR is operative in the rat kidney was evaluated in further experiments in which early proximal tubular pressure was measured in functionally isolated proximal convolutions in vivo under conditions of constant flow as well as stopped flow. Experiments in which perfusion rate through the loop of Henle was varied demonstrated the existence of a feedback signal which originates downstream of the late proximal convolution and which affects filtration into individual early proximal segments. This feedback mechanism exhibited an asymmetrical behaviour: Elevation of loop perfusion above the control value resulted in an early proximal pressure drop, under simulated free flow conditions as well as under stop flow conditions. In contrast lowering of perfusion rate below the predetermined physiological value had no significant effect on early proximal pressures.

Metabolism of Dexamethasone: Sites and Activity in Mammalian Tissues

We have used in vitro techniques to study the metabolism of dexamethasone. Tissue slices, homogenates and microsomal fractions of various mammalian organs from rats and humans have been used. We focused particularly on the question of whether or not dexamethasone (Dexa) is oxidized at the C11-OH group by 11 beta-hydroxysteroid-dehydrogenase. High activities of this enzyme system for Dexa were localized in renal cortex and rectum. Material from both human and murine liver was ineffective. The main metabolite formed from Dexa in renal and intestinal systems was identified by different mass-spectrometric techniques including on line HPLC mass spectrometry as 11-dehydro-dexamethasone. This finding was corroborated by the observation that both corticosterone and glycyrrhetinic acid block the metabolic transformation of Dexa.

Endogenous inhibitors of 11β-OHSD: Existence and possible significance

Abstract Inhibition of 11β-hydroxysteroid dehydrogenase (11β-OHSD) by licorie-derived compounds and in cases of idiopathic impairment of this enzyme is known to result in hypermineralocorticoid syndromes, reflecting corticosteroid receptor activation by excess intracellular glucocorticoids. In this paper we address the question of whether or not endogenous inhibitors of 11β-OHSD exist that might cause pathological glucocorticoid metabolism. Using microsomal preparations we have demonstrated that bile acids are potent inhibitors of rat renal and human hepatic 11β-OHSD, with lithocholic acid exerting the strongest effect. The human renal enzyme is affected to a lesser extent. Serum of patients with cholestatic liver cirrhosis also inhibited 11β-OHSD activity, in parallel with total bile acid concentration. Cholesterol and its precursor lanosterol inhibited the enzymatic activity in microsomes from rat and human kidney cortex and human liver. We conclude that bile acids could contribute to the abnormalities of cortisol metabolism observed in cholestatic liver cirrhosis.

Corticosteroid metabolism in isolated rat kidney in vitro

Kidneys of male Sprague Dawley rats have been isolated and perfused in vitro in order to study the metabolism of corticosterone (B). B is the main endogenous corticosteroid in this species. Using3H-B and HPLC for the separation of steroid metabolites it has been possible to detect radioactive derivatives of B which have been denoted as met I, II and III. These substances were purified and compared with authentic reference hormones under different isocratic and gradient elution techniques. We observed chromatographic identity of met I with 11-dehydro-20-dihydro-B, of met II with 20-dihydro-B and of met III with 5α-H-4,5-dihydro-B. From the fact that conversion of B can be observed with normal (50 g · l−1 albumin in perfusate) and elevated (75 g · l−1) colloid osmotic pressure of the recirculating perfusate it can be concluded that B gets access to the metabolic site in renal tissue not solely by glomerular filtration and tubular reabsorption. The metabolites identified presently are excreted in the urine. Metopirone increased the concentration of met I and decreased the concentration of met II. This is compatible with the concept of a stimulatory effect of metopirone on a C-20-hydroxysteroid oxidoreductase and a C-11-hydroxysteroid dehydrogenase.

Renal metabolism of corticosteroid hormones

SummaryIK and STF from male and female rats have been used to study in vitro the renal metabolism of B. in male rat tissue four lipid soluble metabolites (I–IV) have been found, I+II being more polar and III+IV being less polar than B. I and II have been identified as 11-dehydro-20-hydroxy-B and 20-hydroxy-B. The structure of III and IV remains to be determined. Renal tissue from female rats produced predominantly III indicating sexual variations of steroid metabolism in kidneys. — The literature has been reviewed which documents that the kidneys in addition to B metabolize A, cortisol, progesterone and other corticosteroids.ZusammenfassungIn IK und STF von männlichen und weiblichen Ratten wurde der renale Stoffwechsel von B in vitro untersucht. Bei männlichen Ratten wurden vier lipidlösliche Metabolite (I–IV) gefunden. I und II waren polarer als B; die Strukturanalyse ergab für I: 11-dehydro-20-hydroxy-B und für II: 20-hydroxy-B. Die Struktur der beiden Metabolite III und IV, die weniger polar waren als B, konnte noch nicht aufgeklärt werden. Nierengewebe von weiblichen Ratten bildete aus B vorwiegend die weniger polaren Metabolite III und IV. Der renale Stoffwechsel von B ist somit geschlechtsabhängig. — Eine Übersicht über die Arbeiten in der Literatur belegt, daß die Nieren nicht nur B sondern auch A, Cortisol, Progesteron und andere CS metabolisieren.

Continuous Measurement of Renal Intratubular Pressures with a Combined Pressure Transducer Microperfusion System

SummaryA method has been developed which permits continuous recording of intratubular pressures and simultaneous infusion of test solution at rates of 0–50 nl/min through a single microcapillary. Design and operation of the system is described which consists of an ultraminiature pressure transducer, a micro-perfusion pump and the probing microcapillary. Sealing of the latter was achieved by a screw collar which permits rapid replacement of capillaries without deleterious pressure spikes. At optimal supply voltage the recorded signal was independent of input voltage changes (up to ± 100 mV) and directly proportional to a) pressure applied to the microcapillary tip and b) flow rate through the capillary. — As example the pressure increments following local infusion into the proximal convolution of indigocarmine stained saline or reabsorption inhibitors at a rate of 10 nl/min was measured. During the infusion of 10% mannitol intratubular pressure rose to a plateau and remained above control levels. Application of potassium cyanide (1.5×10−10 M/min) resulted in a gradual pressure increase starting at the onset of the intratubular perfusion. A qualitative similar rise was observed during administration of furosemide (2.2×10−11 M/min), however the effect exhibited a time delay of several minutes.ZusammenfassungEs wurde eine Methode entwickelt, die eine kontinuierliche Druckmessung in Rattennierentubuli bei gleichzeitiger Infusion von Testlösungen in einer Infusionsrate von 0–50 nl/min ermöglicht. Aufbau und Arbeitsweise des Systems, welches aus einem Ultraminiatur-Druckaufnehmer, einer Mikroperfusionspumpe und einer Mikropunktionscapillare besteht, werden beschrieben. Die Dichtung der Punktionscapillaren erfolgt über einen Schlitzkonus, der die einfache und sichere Auswechslung erlaubt und die Gefahr von kritischen Druckentwicklungen während der Capillarmontage ausschaltet. Im Bereich optimaler Betriebs-spannung war das Registriersignal unabhängig von Spannungsschwankungen (um±100 mV); ferner war es direkt proportional a) dem Druckanstieg an der Capillarspitze durch Gegendruck und b) der Strömungsrate durch die Capillarspitze. — Als Anwendungsbeispiele werden Druckregistrierungen bei direkter Infusion von Indigocarmin-gefärbter Kochsalzlösung und von Resorptionshemmern in das proximale Konvolut (10 nl/ml) mitgeteilt. 10% Mannit bedingt einen Anstieg des intratubularen Druckes über das Kontrollplateau. K-Cyanide (1,5×10−10 M/min) führt zu einer kontinuierlichen Druckzunahme, ebenso Furosemid (2,2×10−11 M/min), letzteres jedoch mit einer zeitlichen Verzögerung von mehreren Minuten.

Isoelectric focusing analysis of detergent extracted renal 11β-hydroxysteroid dehydrogenase

11 beta-hydroxysteroid dehydrogenase (11-HSD, EC 1.1.1.146) from rat renal cortex microsomes was solubilized using several detergents, the most effective being Zwittergent 3-10 and Triton X-100. The activity ratio oxidation/reduction of the reversible reaction corticosterone in equilibrium 11-dehydrocoticosterone varied depending on the detergent used. We attribute this variation to direct effects of different detergents on enzyme kinetics. In contrast, comparable results obtained with liver 11-HSD have been attributed to the possibility of spatially separated 11-oxidase and 11-reductase activities. In order to test whether renal 11-HSD represents a uniform oxido-reductase as generally assumed, or a dual enzyme system as has been recently proposed an attempt was made to characterize 11-HSD solubilized from renal microsomal fractions using isoelectric focusing (IEF). When 11-HSD was extracted with 1% Triton X-100 (= partially solubilized fraction) a heterogenous peak pattern was obtained. In contrast, IEF of 11-HSD extracted with 10% Triton X-100 (= delipidated fraction) resulted in a single peak at about pH 5.9 with both oxidative and reductive activity at practically identical positions within the gels. From this observation we conclude that the degree of detergent solubilization of a membrane bound protein affects its amphoteric properties and that removal of membranous lipids is a prerequisite for the analysis of its behaviour. Since the more delipidated fraction of 11-HSD revealed only one activity peak the data are compatible with the uniform enzyme concept since oxidative and reductive activities of renal cortical 11-HSD could not be separated.

Renal epithelial cell lines (BSC-1, MDCK, LLC-PK1) express 11β-hydroxysteroid dehydrogenase activity

Renal tissue of several species has been shown to express considerable 11 beta-hydroxysteroid dehydrogenase (11-HSD, EC 1.1.1.146) activity. However, it is uncertain as to which renal cell types exhibit 11-HSD activity. In the present study, we investigated corticosterone metabolism in BSC-1 cells, a continuous renal epithelial cell line derived from the African green monkey (Cercopithecus aethiops). In incubation experiments using 3H-labelled corticosterone and HPLC, we have demonstrated oxidative 11-HSD activity in intact monolayers of BSC-1 cells as well as in BSC-1 cell homogenates. 11-HSD activity in cell homogenates could be stimulated 7-9-fold by the addition of exogenous NADP+ (1 mM). In contrast, no reductive 11-HSD could be detected either in intact cells or in cell homogenates under various experimental conditions which were designed to favor reductive 11-HSD activity. Pilot experiments were performed in cell homogenates from two other renal epithelial cell lines derived from canine (MDCK) and porcine (LLC-PK1) kidney. They also revealed oxidative but no reductive 11-HSD activity. The data provide evidence for an epithelial localization of renal oxidative 11-HSD activity.

Does corticosteroid metabolism in target organs affect the cardiovascular system

The target organ metabolism of corticosteroids has been measured with biochemical and immunohistochemical techniques. Attention was focused on the key enzyme system 11 beta-hydroxysteroid oxidoreductase (11 beta-HSOR, EC 1.1.1.146). Several organs of rats, including kidney, colon, testis, pancreas, liver, lung and heart, express oxidative, as well as reductive activity, albeit with different ratios. The specific co-substrate preference in different organs points to the presence of isoforms of the enzyme, which can be inhibited by steroid compounds. In kidney tubules longitudinal heterogeneity of the enzyme distribution pattern has been reported. Inhibition of 11 beta-HSOR in transporting epithelia such as those of kidney and colon by liquorice, glycyrrhetinic acid and others leads to aldosterone-like effects of glucocorticosteroids. The mechanism of this effect is breakdown of the specific 11 beta-HSOR barrier for glucocorticosteroids which subsequently bind to mineralocorticosteroid receptors. Other possible mechanisms of interaction of the corticosteroid metabolism and the cardiovascular system are discussed.