Cellular adenosine triphosphate production and consumption in the descending thin limb of Henle's loop in the rat.

To characterize the properties of cellular adenosine triphosphate (ATP) production and consumption in the descending thin limb of Henle's loop (DTL) in rats, we measured the cellular ATP content by applying the microchemiluminescence method. For ATP production, isolated DTL from the short- and long-loop nephron (SDL and LDL, respectively) was incubated with several substrates such as alanine, glucose, glutamine, beta-hydroxybutyrate (beta-HBA), lactate, and pyruvate; for ATP production from each substrate indicated that glucose and pyruvate were the preferred substrates to maintain cellular ATP in both SDL and LDL. ATP production from glutamine, beta-HBA, and lactate was substantial in LDL. In SDL, glutamine was the preferred substrate, and beta-HBA and lactate were minimal. The utilization of alanine was minimal in both LDL and SDL. The cellular ATP was significantly restored by ouabain at concentrations of 1.5 x 10(-5) and 1.5 x 10(-3) M in LDL, but in SDL only at 1.5 x 10(-3) M. In both segments, the cellular ATP level was significantly decreased by monensin above the concentrations of 1 micrograms/ml in LDL and 5 micrograms/ml in SDL. From these observations, we conclude that there exist clear differences between LDL and SDL in ATP-supplying substrate specificities and ATP-consuming properties.     

The thin limbs of Henle's loop in the rabbit

Summary The thin limbs of short and long loops of Henle of the rabbit kidney were studied by freeze fracture techniques. According to TEM studies of thin sections four segments are discernible: descending thin limbs of short loops, descending thin limbs of long loops, subdivided into an upper and a lower part, and ascending thin limbs (Kaissling and Kriz 1979). This division is supported by findings obtained with the freeze fracture technique and based on differences in the organization of the junctional complexes as well as on differences in the internal morphology of the cell membranes. The descending thin limbs of short loops have junctional complexes established by several closely arranged junctional strands and numerous desmosomes. The upper parts of the long descending thin limbs have tight junctions consisting of a variable number of strands; their outstanding characteristic after freeze fracture is a high density of intramembrane particles in both luminal and baso-lateral membranes. The tight junctions of the lower part of the long descending thin limbs consist of several anastomosing junctional strands, which are, in contrast, loosely arranged; the cell membranes contain only a sparse population of intramembrane particles. The ascending thin limbs are characterized by shallow tight junctions (frequently consisting of only one single junctional strand). Moreover, the epithelial cells of this segment are heavily interdigitated; thereby the tight junctions are correspondingly lengthened.In addition, this study presents further evidence that remarkable species differences occur among thin limb epithelia. The junctional complexes of the long descending thin limbs of the rabbit are organized quite differently from those of small rodents (e.g., rat, Psammomys).The data of this study support the concept that the tight junctions are the main determinant of ionic conductances of the paracellular pathway. However, with reference to recent findings from microperfusion studies, it becomes obvious that a correlation of the junctional morphology with the transepithelial water permeability is lacking, at least for the thin limbs.This investigation was supported by the Deutsche Forschungsgemeinschaft; project Kr 546 Henlesche Schleife     

Tamm-Horsfall protein-mRNA synthesis is localized to the thick ascending limb of Henle's loop in rat kidney

Tamm-Horsfall protein (THP) has been previously detected in cells of the thick ascending limb of Henle's loop (TAL) of different mammalian species using immunocytochemical methods. A nearly complete identity between THP and uromodulin, an immunosuppressive glycoprotein present in the urine of pregnant females, has been established recently. This paper describes the cellular location of THP mRNA by high-resolution in situ hybridization using a [35S]-labeled human uromodulin cRNA (antisense-) probe of a length of 665 base pairs. Control experiments were performed using an mRNA (sense-) probe of the same length. The probe was hybridized to frozen sections of the rat kidney. THP mRNA distribution in the kidney was found to be homologous to the immunocytochemical labeling pattern: Autoradiographic signal was present along the entire length of the TAL including the post-macula segment which leads to the distal convoluted tubule. Tubular cells of the macula densa were negative. Labeling intensity of the TAL epithelium was found to increase from the origin of the TAL at the transition between inner and outer medulla to its end beyond the macula densa. Labeling of the medullary segment in the inner stripe was weak, whereas outer medullary and cortical segments very strongly expressed THP mRNA. The glomerulus, the portions of the nephron proximal to the TAL, the distal convoluted tubule as well as the collecting duct system were negative.     

Tamm-Horsfall protein-mRNA synthesis is localized to the thick ascending limb of Henle's loop in rat kidney

Summary Tamm-Horsfall protein (THP) has been previously detected in cells of the thick ascending limb of Henle's loop (TAL) of different mammalian species using immunocytochemical methods. A nearly complete identity between THP and uromodulin, an immunosuppressive glycoprotein present in the urine of pregnant females, has been established recently. This paper describes the cellular location of THP mRNA by high-resolution in situ hybridization using a [³⁵S]-labeled human uromodulin cRNA (antisense-) probe of a length of 665 base pairs. Control experiments were performed using an mRNA (sense-) probe of the same length. The probe was hybridized to frozen sections of the rat kidney. THP mRNA distribution in the kidney was found to be homologous to the immunocytochemical labeling pattern: Autoradiographic signal was present along the entire length of the TAL including the post-macula segment which leads to the distal convoluted tubule. Tubular cells of the macula densa were negative. Labeling intensity of the TAL epithelium was found to increase from the origin of the TAL at the transition between inner and outer medulla to its end beyond the macula densa. Labeling of the medullary segment in the inner stripe was weak, whereas outer medullary and cortical segments very strongly expressed THP mRNA. The glomerulus, the portions of the nephron proximal to the TAL, the distal convoluted tubule as well as the collecting duct system were negative.     

Ion channels in the thick ascending limb of Henle's loop.

The thick ascending limb of Henle's loop (TAL) is polarized with respect to its conductances. The luminal membrane contains a K+ conductance which is made up by the synchronous operation of 60- to 80-pS K+ channels. The basolateral membrane contains a chloride conductance. This conductance corresponds most likely to a 30- to 60-pS Cl- channel present in this membrane. Our knowledge on the properties of the K+ channels of these cells has been increased rapidly by patch clamp studies: these K+ channels are inwardly rectifying. They are highly selective for K+ over Na+, Li+ and many other cations. They do not conduct Rb+, Cs+, NH+4 or other larger cations. In fact, all these three cations as well as choline, tetraethylammonium, lidocaine, verapamil, diltiazem, quinine, quinidine and Ba2+ inhibit these K+ channels. As apparent from kinetic studies the mechanisms of inhibition are different for the various blockers. The TAL K+ channels are downregulated by increasing cytosolic Ca2+ activity. Cytosolic adenosine trisphosphate (ATP) has a similar effect. This ATP inhibition is Ca2+ dependent. The affinity to ATP is augmented by increasing Ca2+. Cytosolic alkalinity increases the open probability of these channels, and cytosolic acidification has the opposite effect. This pH dependence is very marked. A change by 0.2 pH units leads to a more than twofold change in the open-channel probability. The basolateral chloride conductance reflects the properties of an outwardly rectifying 30- to 60-pS Cl- channel. This channel behaves, in many respects, like the Cl- channels of a multitude of Cl- transporting epithelia. It is characterized by two open and two closed states. It is highly selective for Cl- as compared with larger anions, and it is inhibited reversibly by Cl- channel blockers such as 5-nitro-2-(3-phenylpropylamino)-benzoate.     

A mathematical model of rabbit cortical thick ascending limb of the Henle's loop

A mathematical version of the cell model of the cortical thick ascending limb of the rabbit proposed by Greger and Schlatter ((1983) Pfügers Arch. 396, 325-334) is described. Available data are sufficient to compute the most important parameters. Simulations of experiments with perfused tubules in which the transepithelial voltage and conductance, and the intracellular electrical potential were measured in the course of ionic substitutions in the perfusing baths or treatment with ouabain or furosemide are in good agreement with the experimental results with the exception of those relating to dilution potential experiments. The model can be used in the analysis and planning of experiments and is capable of predicting the instantaneous values of ionic fluxes and intracellular concentrations and of cell volume.     

Short-term stimulation of cellular autophagy by furosemide in the thick ascending limb of Henle's loop in the rat kidney

Summary In an effort to investigate the functional relationship between cell-specific work and intracellular degradative processes, the effect of furosemide on cellular autophagy was investigated in two different portions of the nephron, namely, the thick ascending limb of Henle's loop (TAL), which is a main target of this drug, and the proximal convoluted tubule (PCT) as a reference structure. Eight male adult rats were treated with furosemide (60 mg/kg body weight, s.c.). Eight control animals received physiological saline. 1 to 4 h after the injections the animals were killed by perfusion fixation. Small specimens of kidney tissue from the inner stripe of the outer medulla and from the outer cortex were processed for electron microscopy; they were investigated morphometrically for volume fraction and numerical density of autophagic vacuoles (AVs). A significant increase of both parameters (volume fraction: 0.42 × 10^-4 to 1.09 × 10^-4; numerical density: 4.2 × 10⁵/mm³ to 15.5 × 10⁵/mm³) was seen under the influence of furosemide in TAL cells, whereas PCT cells did not show a significant increase in volume fraction or any increase in numerical density of AVs. These data suggest that the functional unloading of TAL, via blocking of the Na⁺- 2Cl^- — K⁺ co-transport by furosemide, results in adaptative structural unloading, i.e., an increased sequestration of cytoplasmic components into AVs, within a short-time interval.     

Effect of tubular inhomogeneities on filter properties of thick ascending limb of Henle's loop

We used a simple mathematical model of rat thick ascending limb (TAL) of the loop of Henle to predict the impact of spatially inhomogeneous NaCl permeability, spatially inhomogeneous NaCl active transport, and spatially inhomogeneous tubular radius on luminal NaCl concentration when sustained, sinusoidal perturbations were superimposed on steady-state TAL flow. A mathematical model previously devised by us that used homogeneous TAL transport and fixed TAL radius predicted that such perturbations result in TAL luminal fluid NaCl concentration profiles that are standing waves. That study also predicted that nodes in NaCl concentration occur at the end of the TAL when the tubular fluid transit time equals the period of a periodic perturbation, and that, for non-nodal periods, sinusoidal perturbations generate non-sinusoidal oscillations (and thus a series of harmonics) in NaCl concentration at the TAL end. In the present study we find that the inhomogeneities transform the standing waves and their associated nodes into approximate standing waves and approximate nodes. The impact of inhomogeneous NaCl permeability is small. However, for inhomogeneous active transport or inhomogeneous radius, the oscillations for non-nodal periods tend to be less sinusoidal and more distorted than in the homogeneous case and to thus have stronger harmonics. Both the homogeneous and non-homogeneous cases predict that the TAL, in its transduction of flow oscillations into concentration oscillations, acts as a low-pass filter, but the inhomogeneities result in a less effective filter that has accentuated non-linearities.     

Countercurrent exchange in the inner renal medulla: Vasa recta-descending limb system

A model of countercurrent exchange has been developed to simulate transport of salt, urea and water among vasa recta and descending limbs of the loop of Henle in the inner medulla. These vessels are abstracted as three concentric cylinders: the inne one represents descending vasa recta, the middle one represents ascending vasa recta and the outer one represents descending limbs. The capillary plexus, which connects the ascending and descending vasa recta, is modeled as a series of well-mixe compartments. Multicomponent transport equations for the sytem are derived from steady state mass balances and simple passive flux relations. The resulting set of nonlinear equations are solved numerically by an iterative Gauss-Seidel algorithm with under-relaxation. Simulations yield the salt and urea concentrations as well as volume flow rates in all tubes and compartments. The simulations indicate that solute concentrations can increase monotonically toward the papillae even if all transport processes within the exchanger are passive and source fluxes decrease monotonically toward the papillae.     

Separation of renal medullary cells: isolation of cells from the thick ascending limb of Henle's loop

A homogeneous population of single cells from the thick ascending limb of Henle's loop (TALH) has been isolated from the rabbit kidney medulla. A total medullary cell suspension was prepared by a series of collagenase, hyaluronidase, and trypsin digestions and separated on a Ficoll gradient (2.6-30.7% wt/wt). Morphologically, the cells isolated from the TALH were homogeneous and showed polarity within their plasma membrane structure, with a few blunt microvilli on their apical surface and deep infoldings of the basal-lateral membrane. Biochemically, the TALH cells were highly enriched in calcitonin-sensitive adenylate cyclase and Na, K-ATPase. Alkaline phosphatase and arginine vasopressin- sensitive adenylate cyclase, highly concentrated in proximal tubule and collecting duct, were present only in low concentrations in the TALH cells. Additionally, furosemide, a diuretic inhibiting sodium chloride transport in the TALH in vivo, inhibited oxygen consumption of the TALH cells in a dose-dependent manner. The TALH cells were viable, as judged by morphological appearance, trypan blue exclusion, the response of oxygen consumption to 2,4-dinitrophenol, succinate and ouabain, and the cellular Na, K and ATP levels.     

The transition of the thick ascending limb of Henle's loop into the distal convoluted tubule in the nephron of the rat kidney

Summary Examination of serial semithin sections of rat kidney cortex and a subsequent electron microscopic study of selected areas revealed that the characteristic epithelium of the cortical part of the thick ascending limb of Henle extends for a varying distance beyond the macula densa. The transition from the relatively thin epithelium of the thick ascending limb at this site to the three -or even four-fold thicker epithelium of the convoluted part of the distal tubule is sharply defined and occurs without the interposition of an intermediate cell type.The position of the macula densa at the end but still clearly within the ascending limb of Henle's loop is functionally interpreted to guarantee the separation of the sensor point macula densa from disturbing influences which might arise from the secretory activity of the subsequent tubular portion.Investigations supported by the Deutsche Forschungsgemeinschaft. The skillful technical assistance of Mrs. Saliha Sabanovic is gratefully acknowledged     

Phospholipid metabolism in the rat renal inner medulla

In view of the importance of phospholipids as a source of precursor fatty acids for the high prostaglandin synthesis in the renal inner medulla, we studied pathways of phospholipid esterification and degradation in the rat inner medulla. De novo acylation of [14C]arachidonate occurred predominantly in position 2 of phosphatidylcholine in the microsomal fraction. This newly esterified [14C]arachidonate was accessible to deacylation by a microsomal phospholipase A2 (EC 3.1.1.4) with alkaline optimum which was Ca2+-dependent and resistant to 0.1% deoxycholate. No phospholipase A1 (EC 3.1.1.32) activity against endogenous labeled phosphatidylcholine could be demonstrated in the microsomal fraction. When exogenous phosphatidylcholine labeled at position 2 was deacylated by renomedullary homogenates, labeled free fatty acid but no labeled lysophosphatidylcholine was recovered in the reaction products. This could be attributed to further degradation of generated lysophosphatidylcholine by a cytosolic lysophospholipase (EC 3.1.1.5). Sodium deoxycholate at a concentration of 0.1% or higher inhibited the lysophospholipase and allowed the demonstration of both A2 and A1 alkaline phospholipase activities in the homogenate. The major in vitro pathway of lysophosphatidylcholine disposition is further degradation by a cytosolic lysophospholipase, while reutilization for phosphatidylcholine synthesis through the action of a predominantly microsomal acyltransferase appears to be a minor pathway. In the presence of several acyl-CoAs, reutilization of lysophosphatidylcholine is significantly increased by an acyl-CoA:lysophosphatidylcholine acyltransferase (EC 2.3.1.23) but there is no preferential transfer of arachidonyl-CoA compared to other acyl-CoAs.     

Lack of inhibition of vasopressin-stimulated NA+K+ATPase by atrial natriuretic factor in the rat renal medullary thick ascending limb of Henle's loop

The anti-diuretic hormone, arginine vasopressin (AVP) stimulates the activity of Na+K+ATPase in the rat renal medullary thick ascending limb of Henle's loop (mTAL). Atrial natriuretic factor (ANF) has been suggested to exert a tubular effect on the mammalian nephron, perhaps in part, by interacting with other hormones. In the present study, we investigated the effect of rat ANF with and without AVP upon mTAL Na+K+ATPase activity using cytochemical methods. ANF alone failed to inhibit or stimulate Na+K+ATPase activity in mTAL at any of the concentrations tested (10 nmol-0.1 pmol l-1). Unlike the rat hypothalamic digitalis-like factor, ANF (10 nmol-10 fmol l-1) did not inhibit Na+K+ATPase activity after stimulation with AVP (1 fmol l-1) for either 4 or 10 min. The results suggest that ANF does not exert an effect on mTAL, either alone or in conjunction with AVP.     

Proteomic analysis of cellular response to osmotic stress in thick ascending limb of Henle's loop (TALH) cells

Epithelial cells of the thick ascending limb of Henle's loop (TALH cells) play a major role in the urinary concentrating mechanism. They are normally exposed to variable and often very high osmotic stress, which is particularly due to high sodium and chloride reabsorption and very low water permeability of the luminal membrane. It is already established that elevation of the activity of aldose reductase and hence an increase in intracellular sorbitol are indispensable for the osmotic adaptation and stability of the TALH cells. To identify new molecular factors potentially associated with the osmotic stress-resistant phenotype in kidney cells, TALH cells exhibiting low or high levels of resistance to osmotic stress were characterized using proteomic tools. Two-dimensional gel analysis showed a total number of 40 proteins that were differentially expressed in TALH cells under osmotic stress. Twenty-five proteins were overexpressed, whereas 15 proteins showed a down-regulation. Besides the sorbitol pathway enzyme aldose reductase, whose expression was 15 times increased, many other metabolic enzymes like glutathione S-transferase, malate dehydrogenase, lactate dehydrogenase, alpha enolase, glyceraldehyde-3-phosphate dehydrogenase, and triose-phosphate isomerase were up-regulated. Among the cytoskeleton proteins and cytoskeleton-associated proteins vimentin, cytokeratin, tropomyosin 4, and annexins I, II, and V were up-regulated, whereas tubulin and tropomyosins 1, 2, and 3 were down-regulated. The heat shock proteins alpha-crystallin chain B, HSP70, and HSP90 were found to be overexpressed. In contrast to the results in oxidative stress the endoplasmic reticulum stress proteins like glucose-regulated proteins (GRP78, GRP94, and GRP96), calreticulin, and protein-disulfide isomerase were down-regulated under hypertonic stress.     

Active chloride transport in rabbit thick ascending limb of Henle's loop and elasmobranch rectal gland: chloride fluxes in isolated plasma membranes

Summary To investigate directly whether a sodium-potassium-chloride cotransport system is operating in the mammalian thick ascending limb of Henle's loop (TALH) and in the elasmobranch rectal gland, plasma membrane vesicles were prepared from TALH cells isolated from rabbit kidney outer medulla and from rectal glands ofSqualus acanthias, and chloride uptake was measured by a rapid filtration technique. Chloride uptake into TALH vesicles in the presence of a 25 mM Na₂SO₄, 25 mM K₂SO₄ gradient reached 70% of equilibrium at 2.5 min. In the presence of both sodium and potassium, the 15 s chloride uptake was inhibited 35% by 1 mM bumetanide. When either sodium or potassium was removed from the incubation medium, chloride uptake decreased to the level observed in the presence of 1 mM bumetanide. 0.5 mM SITS had no effect on chloride uptake by the plasma membrane vesicles. This sodium and potassium dependent, bumetanide sensitive chloride uptake was also observed under tracer exchange conditions. Chloride uptake into rectal gland plasma membrane vesicles in the presence of a 50 mM Na₂SO₄, 50 mM K₂SO₄ gradient reached 80% of equilibrium at 2.5 min. 1 mM bumetanide inhibited the 15 s uptake of chloride by 34% and removal of either sodium or potassium from the incubation medium reduced chloride uptake to the level observed in the presence of bumetanide under both gradient and tracer exchange conditions. These studies provide additional support for the hypothesis that a sodium-potassium-chloride cotransport system is operating in these epithelia.Abbreviations SITS 4-acetamido-4-isothiocyanato-stilbene-2,2-disulfonic acid - TALH thick ascending limb of Henle's loop