Michael Steinhausen

Eine Methode zur Differenzierung proximaler und distaler Tubuli der Nierenrinde von Ratten in vivo und ihre Anwendung zur Bestimmung tubulärer Strömungsgeschwindigkeiten

Zur Differertzierung proximaler und distaler Harnkanälchen von Rat ten in vivo wird vielfach die von Wlnz beschriebene Methode der intratubulärert Indigocarmin-Injektion verwendet. Eine Differenzierung proximaler und distaler Tubuli ohne Mikropunktion und Färbung ist trotz Größenund Fcrmnnterschieden auch dem Geübten keinesfalls immer möglich (Witz). Bei der Suche nach einem weniger aufwendigen Verfahren prüften wir das Bild der Nierenrinde in vivo nach der sehr viel einfacheren i.v. Injektion einer Reihe von Farbstoffen. Dabei zeigte sich, daß Lisse~mingrün (Summenformel: C~7tt3409S~Na2), ein Triphenylmethan-Farbstoff, welcher bisher in der Elektrophoresetechnik (BRACKENI%IDG:E; I)ANGERFIELD U. SMITE; DISCOMBE; JONES u. Wrt¢STANLE¥ U. a.), bei Zellund Tumoruntersuchungen als Vitalfarbstoff (GoLI)ACRE U. SYLVEN; HOLlViBERG) sowie bei Untersuchungen über die Capillarpermeabilität der Froschschwimmhaut nach UV-Bestrahlung (MÜr,L~~) benutzt wurde, nach i.v. Stoßinjektion die gewünschte I)ifferenzierung leic:ht ermöglicht. Darüber hinaus erlaubt die Methode auch Abschätzungen intratubulärer Strömungsgeschwindigkeiten, welche kürzlich erstmalig von THv~AV u. I)~~TJEZ~ nach intratubulärer Indigocarmin-Injektion gemessen wurden.

Renovascular Effects of Adenosine Receptor Agonists

The purpose of the present experiments was to determine the renovascular effects of the adenosine agonists N-ethyl-carboxamide adenosine (NECA), N6-cyclohexyl adenosine (CHA) and 2-chloro adenosine (2-CLA). The diameter of pre- and postglomerular vessels in the split hydronephrotic kidney of Inactin-anesthetized rats was measured by in vivo television microscopy. All metabolically stable adenosine agonists were topically applied into the renal tissue bath. NECA, a preferential A2 adenosine receptor agonist, induced dose-dependent marked pre- and slight postglomerular vasodilation except for a small constrictory effect on the afferent arteriole near the glomerulus. Application of CHA, a selective A1 adenosine receptor agonist, led to a vasoconstriction of all preglomerular vessels, the extent of which was greatest at the most distal segment of the afferent arteriole. 2-CLA, a nonselective agonist, produced a small decrease in diameter in all preglomerular vessels, a marked constriction of the afferent arteriole at sites near the glomerulus, and a slight dilation of postglomerular vessels. Glomerular blood flow (GBF) was increased by NECA, and decreased by CHA and 2-CLA. The effects of CHA in reducing GBF were greater than those of 2-CLA. From these experiments it is concluded that vascular A1 and A2 adenosine receptors are present in the kidney and that activation of A1 receptors is associated with preglomerular vasoconstriction only, whereas activation of A2 receptors mediates pre- and postglomerular vasodilation with a lack of vasodilatory response of the distal afferent arteriole. Furthermore, these data indicate that nonselective occupation of both receptor subclasses is associated with marked vasoconstriction of the afferent arteriole and little vasodilation of the efferent arteriole.

Rho-Kinase Inhibition Blunts Renal Vasoconstriction Induced by Distinct Signaling Pathways In Vivo

In addition to intracellular calcium, which activates myosin light chain (MLC) kinase, MLC phosphorylation and hence contraction is importantly regulated by MLC phosphatase (MLCP). Recent evidence suggests that distinct signaling cascades of vasoactive hormones interact with the Rho/Rho kinase (ROK) pathway, affecting the activity of MLCP. The present study measured the impact of ROK inhibition on vascular F-actin distribution and on vasoconstriction induced by activation/inhibition of distinct signaling pathways in vivo in the microcirculation of the split hydronephrotic rat kidney. Local application of the ROK inhibitors Y-27632 or HA-1077 induced marked dilation of pre- and postglomerular vessels. Activation of phospholipase C with the endothelin ET B agonist IRL 1620, inhibition of soluble guanylyl cyclase with 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), or inhibition of adenylyl cyclase with the adenosine A1 agonist N6-cyclopentyladenosine (CPA) reduced glomerular blood flow (GBF) by about 50% through vasoconstriction at different vascular levels. ROK inhibition with Y-27632 or HA-1077, but not protein kinase C inhibition with Ro 31-8220, blunted ET B-induced vasoconstriction. Furthermore, the reduction of GBF and of vascular diameters in response to ODQ or CPA were abolished by pretreatment with Y-27632. ROK inhibitors prevented constriction of preglomerular vessels and of efferent arterioles with equal effectiveness. Confocal microscopy demonstrated that Y-27632 did not change F-actin content and distribution in renal vessels. The results suggest that ROK inhibition might be considered as a potent treatment of renal vasoconstriction, because it interferes with constriction induced by distinct signaling pathways in renal vessels without affecting F-actin structure.

Micropuncture studies after temporary ischemia of rat kidneys

SummaryMicropuncture experiments were carried out on rat kidneys subjected to temporary ischemia (TI). Unilateral TI was performed by clamping the blood supply to the left kidney for 60 min. Time period between TI and investigating the kidney ranged from 1 h to 13 days. In another series of experiments blood supply to the left kidney was severely diminished for 24 h by a clip.-39% of animals developed anuria of the experimental kidney; in the other animals, urine flow rate was markedly decreased after TI (P<0.02). Employing the tracer microinjection technique considerable amounts (51%-oliguric kidneys; 17%-nonoliguric kidneys) of14C-labelled inulin injected into a proximal tubule of the damaged kidney were found in the urine of the contralateral kidney indicating backdiffusion of inulin through the damaged tubular epithelium. The amount of inulin recovered from both kidneys was decreased. Backdiffusion of inulin was detectable up to seven days after TI. Under control conditions 2% of inulin injected was found on the contralateral side, inulin recovered from both kidneys being approx. 100%. Dye intensity of Lissamine-green passing through the nephron was decreased noticeably after TI. Proximal tubular diameter and proximal free-flow pressure showed a large variability, the means being not significantly differnt from controls.—The results indicate that the concurrence of at least two factors are involved in the pathogenesis of acute renal damage induced by TI: Backdiffusion of tubular fluid through the damaged tubular epithelium and a decrease of glomerular filtration rate. The measurement of the clearance of inulin in kidneys damaged by ischemia does not represent the actual GFR, but underestimates this value proportionately to the degree of backdiffusion of inulin.

Effect of intrarenally infused parathyroid hormone‐related protein on renal blood flow and glomerular filtration rate in the anaesthetized rat

1 Parathyroid hormone‐related protein (PTHrP) is expressed in the kidney and acts on vascular PTH/PTHrP receptors to vasodilate the isolated kidney and to stimulate renin release. However, effects of PTHrP on renal blood flow (RBF) and glomerular filtration rate (GFR) in vivo have not been assessed in the absence of its cardiac, peripheral and central effects. We investigated the renal effects of PTH and PTHrP infused into the left renal artery of anaesthetized rats. 2 Intrarenal infusions, adjusted to generate increasing concentrations of human PTHrP(1–34) and rat PTH(1–34) in renal plasma (2 × 10−11 to 6 × 10−9 m) produced a comparable dose‐dependent increase in RBF. The rise was 4% at the lowest and 34% at the highest concentrations of peptides. Up to a concentration of 2 × 10−9 m, mean arterial pressure (MAP) and heart rate were not affected, but at 6 × 10−9 m, intrarenally infused peptides reached the peripheral circulation, and caused a fall in MAP within a few minutes. While MAP returned to basal value after the last peptide infusion, RBF remained more than 10% above control for at least 30 min. 3 Two competitive PTH/PTHrP receptor antagonists, [Nle8,18, Tyr34]‐bPTH(3–34)amide and [Leu11, D‐Trp12]‐hPTHrP(7–34)amide (2 × 10−8 m) were devoid of agonist activity, but markedly antagonized the dose‐dependent increase in RBF elicited by PTHrP. 4 GFR and urine flow were measured in left PTHrP‐infused experimental kidney and right control kidney. Renal PTHrP concentration of 10−10 m elevated left RBF by 10%, and GFR by 20% without significantly increasing filtration fraction, and increased urine flow by 57%. In the right control kidney GFR and diuresis did not change. 5 The results indicate that PTHrP has similar renal haemodynamic effects as PTH and increases RBF, GFR and diuresis in anaesthetized rats.

Microcirculation of the epimyocardial layer of the heart

To study microcirculation of the heart in vivo, we developed a new technique of epi-illumination of the beating rat ventricular myocardium. Five tiny needles connected to the operation table were horizontally inserted into the ventricular myocardium just beneath the epicardium in order to restrict cardiac movement during contraction and to enhance the amount of light reflected from the structures under study. The in vivo light and fluorescent microscopy were performed with the Ultropak and the Fluopak systems (Leitz) combined with a highly sensitive television camera and recording system.In 142 individual in vivo measurements (6 animals) the mean capillary diameter amounted to 6.0±1.0 μ. In the same experiments, the mean intercapillary distance was 18.7±1.7 μ. In agreement with the literature, the capillaries of the epimyocardial layer of the rat heart demonstrate a mixed countercurrent flow pattern. In contrast to other authors, we observed no recruitment of resting capillaries after hypoxia.

Localization of endothelin ETA and ETB receptor‐mediated constriction in the renal microcirculation of rats.

1. The aim of the study was to visualize endothelin‐1 (ET‐1)‐mediated constriction in renal vessels of cortical and juxtamedullary glomeruli in the split hydronephrotic rat kidney in vivo and to functionally characterize the ET receptor subtypes involved. 2. ET‐1 (10(‐9) M) constricted preglomerular vessels (by 6‐18%) and efferent arterioles (by 11‐13%), and decreased glomerular blood flow (GBF, by 55%) of cortical and juxtamedullary glomeruli. 3. The ETA antagonist BQ‐123 (10(‐6) M), as well as the ETB antagonist BQ‐788 (2 x 10(‐7) M) and IRL 1038 (10(‐6) M), shifted the concentration‐response curve of GBF for ET‐1 to the right by one order of magnitude. While BQ‐123 antagonized ET‐1 constriction only in preglomerular vessels, BQ‐788 and IRL 1038 were effective both in preglomerular vessels and efferent arterioles. 4. The ETB agonist IRL 1620 (10(‐8) M) reduced GBF by 50% and constricted efferent arterioles (by 20‐33%) about two times more than preglomerular vessels (by 6‐14%). 5. Our results suggest that in renal cortical and juxtamedullary vessels of rats, ET‐1‐induced preglomerular vasoconstriction is mediated by ETA and ETB receptors, while efferent vasoconstriction is predominantly mediated by ETB receptors, which might have important consequences for the regulation of glomerular filtration pressure by ET.

Concentration of lissamine green in proximal tubules of antidiuretic and mercury poisoned rats and the permeability of these tubules

ZusammenfassungEs gelang mit Hilfe von Sudanrot-gefärbtem Ricinusöl die Splitdrop-Methode so zu modifizieren, daß Lissamingrün (LG)-Konzentrationen in einzelnen proximalen Tubulusschlingen der Rattenniere mikrophotometrisch bestimmt werden konnten. Im Verlauf des Splitdrop-Versuches kam es zu einer Konzentrationszunahme von LG, welche jedoch nur bei Testlängen über 100 μ der Abnahme des Lösungsmittelvolumens (berechnet aus intratubulärem Durchmesser und Testtropfenlänge) entsprach. Dies wurde im wesentlichen auf eine unvollständige Ölblockade im Grenzbereich zwischen Ölblock und Testtropfen bezogen.Anwendung fand die modifizierte Splitdrop-Methode zur Bestimmung der tubulären Permeabilität nach Sublimat-Intoxikation. Im Gegensatz zum Kontrollversuch kam es hier während des Splitdrop-Versuches zu einer Entfärbung von LG. Dies wurde zu einem kleinen Teil auf die Bildung einer LG-Leuko-Verbindung bezogen, welche LG mit geschädigtem Zelleiweiß eingeht. Darüber hinaus muß es aber auch zu einer erheblichen Permeabilitätssteigerung der proximalen Tubuluswand gekommen sein, da in Mikroperfusionsversuchen gezeigt werden konnte, daß proximal injiziertes, C14-markiertes Inulin nach Sublimat-Intoxikation in großen Mengen über die andere — nicht punktierte — Niere ausgeschieden wurde.SummaryBy coloring castor oil with Sudan-red we were able to modify the split droplet method so that concentrations of Lissamine green (LG) could be determined by microphotometry in individual proximal tubular loops of rat kidneys. In the course of the split droplet experiments an increase in the concentration of LG occured, which corresponded to the decrease in volume of the solvent (calculated from the intratubular diameter and the length of the test droplet) only with test lengths of over 100 μ. The change in concentration at shorter lengths of test droplets may have resulted from an incomplete blockage between the oil block and the test droplet.The modified split droplet method was applied to determine tubular permeability after mercury poisoning. In contrast to the control experiment, the test droplet stained with LG became decolorized during the split droplet experiments, a change which in small part may have been caused by the binding of LG with protein to form an uncolored compound. The protein resulted by the damage of the tubular cells after mercury poisoning. Furthermore, a considerable increase in the permeability of the proximal tubular wall must have occured, since we could show in microperfusion experiments that proximally injected C14-labelled Inulin was excreted in large quantities through the other (unpunctured) kidney after mercury poisoning.

Interaction between adenosine and angiotensin II in renal microcirculation

In order to examine the possibility of an interaction between adenosine and angiotensin II (A II) in the control of the renal microcirculation, we studied the effects of agonists and antagonists of both substances by means of in vivo microscopy in the split hydronephrotic rat kidney. In a first series of experiments (n = 6), local application of the A II receptor antagonist saralasin (10(-6) mol.liter-1 abolished the vasoconstriction and the reduction of glomerular blood flow induced by the A1-adenosine receptor agonist N6-cyclohexyladenosine (CHA, local concentration 10(-7) mol.liter-1). Without saralasin (second series, n = 6), CHA reduced glomerular blood flow and decreased vessel diameters as previously reported from our laboratory. In a third series of experiments (n = 6), A II significantly reduced vessel diameters and glomerular blood flow both alone and during blockage of the A1-adenosine receptor by the selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 10(-5) mol.liter-1). In additional experiments, we excluded nonspecific receptor effects of saralasin and confirmed the inhibitory action of DPCPX on the adenosine-induced vasoconstriction. We suppose that adenosine needs a functioning A II receptor system for its vasoconstrictor action, whereas A II can induce a nonadenosine-dependent vasoconstriction.

INCIDENCE LIGHT MICROSCOPY AND HISTOLOGY OF RENAL TUBULE SIZE UNDER VARIABLE CONDITIONS OF DIURESIS

An narkotisierten Ratten und Katzen wurde die Nierenoberflache mit dem Auflichtmikroskop photographiert und so der Lumendurchmesser und der Gesamtdurchmesser proximaler und distaler Tubuli bestimmt. BeiAntidiurese betragt der proximale Lumendurchmesser von 70 g schweren Ratten in vivo 16,4±1,8μ, bei 300 bis 330 g schweren Ratten 22,8±0,7μ, und bei Katzen 17,2±0,7μ. Nach Unterbrechung der glomerularen Filtration (Aortenabklemmung oder Hilusdurchtrennung) kommt es innerhalb von 20 bis 120 sec zum Kollaps der proximalen Tubuli, wahrend die distalen Tubuli offen bleiben; dieser Kollaps proximaler Tubuli bei Antidiurese wird mit einer auch postmortal noch ablaufenden Ruckresorption von Tubulusurin erklart.