Parallel generation of extensive vascular networks with application to an archetypal human kidney model.

Given the relevance of the inextricable coupling between microcirculation and physiology, and the relation to organ function and disease progression, the construction of synthetic vascular networks for mathematical modelling and computer simulation is becoming an increasingly broad field of research. Building vascular networks that mimic in vivo morphometry is feasible through algorithms such as constrained constructive optimization (CCO) and variations. Nevertheless, these methods are limited by the maximum number of vessels to be generated due to the whole network update required at each vessel addition. In this work, we propose a CCO-based approach endowed with a domain decomposition strategy to concurrently create vascular networks. The performance of this approach is evaluated by analysing the agreement with the sequentially generated networks and studying the scalability when building vascular networks up to 200 000 vascular segments. Finally, we apply our method to vascularize a highly complex geometry corresponding to the cortex of a prototypical human kidney. The technique presented in this work enables the automatic generation of extensive vascular networks, removing the limitation from previous works. Thus, we can extend vascular networks (e.g. obtained from medical images) to pre-arteriolar level, yielding patient-specific whole-organ vascular models with an unprecedented level of detail.

Leptospira and inflammation.

Leptospirosis is an important zoonosis and has a worldwide impact on public health. This paper will discuss both the role of immunogenic and pathogenic molecules during leptospirosis infection and possible new targets for immunotherapy against leptospira components. Leptospira, possess a wide variety of mechanisms that allow them to evade the host immune system and cause infection. Many molecules contribute to the ability of Leptospira to adhere, invade, and colonize. The recent sequencing of the Leptospira genome has increased our knowledge about this pathogen. Although the virulence factors, molecular targets, mechanisms of inflammation, and signaling pathways triggered by leptospiral antigens have been studied, some questions are still unanswered. Toll-like receptors (TLRs) are the primary sensors of invading pathogens. TLRs recognize conserved microbial pattern molecules and activate signaling pathways that are pivotal to innate and adaptive immune responses. Recently, a new molecular target has emerged--the Na/K-ATPase--which may contribute to inflammatory and metabolic alteration in this syndrome. Na/K-ATPase is a target for specific fatty acids of host origin and for bacterial components such as the glycolipoprotein fraction (GLP) that may lead to inflammasome activation. We propose that in addition to TLRs, Na/K-ATPase may play a role in the innate response to leptospirosis infection.

Regulation of Na+, K(+)-ATPase in the rat outer medullary collecting duct during potassium depletion.

Because in outer medullary collecting ducts (OMCD) of K(+)-depleted rats, K+ secretion is abolished, whereas Na+, K(+)-ATPase, which energizes this secretion, is markedly stimulated, it has been proposed that Na+, K(+)-ATPase was mislocated to the apical cell membrane and energized K+ reabsorption. This hypothesis has been supported by paradoxical effects of ouabain in K(+)-depleted compared with normal rats. However, we have recently shown that ouabain inhibits not only Na+, K(+)-ATPase but also apical H+, K(+)-ATPase in the OMCD of K(+)-depleted rats. Therefore, this study was designed to evaluate whether previous observations were accounted for by Na+, K(+)-ATPase or by ouabain-sensitive H+, K(+)-ATPase. Na+, K(+)-ATPase was distinguished from H+, K(+)-ATPase by its insensitivity to Sch-28080. Results indicate that the hydrolytic and transport activities of Na+, K(+)-ATPase, the number of its functional units, and the expression of mRNA of its alpha 1 and beta 1 subunits were increased threefold or more in the OMCD of rats fed a K(+)-depleted diet for 2 wk. By immunofluorescence, Na+, K(+)-ATPase staining was strongly increased in K(+)-depleted rats but remained localized to the basolateral pole of OMCD principal cells. In conclusion, K+ depletion is associated with marked induction of functional Na+, K+ pumps at the basolateral pole of rat OMCD. Therefore, reduced K+ secretion might result from inhibition of apical K+ conductances and stimulation of basolateral K+ recycling. It is proposed that increased Na+, K(+)-ATPase participates in the increased Na+ reabsorption prevailing in collecting ducts of K(+)-depleted rats.

Cold- and ouabain-resistance of renal Na,K-ATPase in cold-exposed and hibernating jerboas (Jaculus orientalis).

The temperature dependence and the ouabain sensitivity of Na,K-ATPase was examined in the nephron of normal, cold-exposed, and hibernating jerboas. The transport and hydrolytic activity of renal Na,K-ATPase displayed similar temperature dependence in rats and normal jerboas. Cold-resistance of Na,K-ATPase appeared in cold-exposed jerboas and further increased during hibernation. Three subpopulations of Na,K-ATPase displaying very high (Ki approximately 10(-13) M), high (Ki approximately 10(-9) M) and low sensitivity to ouabain (Ki approximately 10(-6) M) were detected in the thick ascending limb and collecting duct of jerboas. In thick ascending limbs, the subpopulation of very high sensitivity to ouabain disappeared in cold-exposed animals, which accounted for the previously reported decrease in Na,K-ATPase activity. In collecting ducts of cold-exposed animals, the subpopulation of very high sensitivity to ouabain also disappeared, but the resulting decrease in activity was overbalanced by the appearance of the subpopulation of high sensitivity.

Purification and characterization of a Na+, K+ ATPase inhibitor found in an endotoxin of Leptospira interrogans.

We showed previously that the glycolipoprotein fraction prepared from Leptospira interrogans inhibited the Na+,K+ ATPase enzyme purified from brain or kidney and in isolated nephron segments (M. Younes-Ibrahim, P. Burth, M. V. Castro Faria, B. Buffin-Meyer, S. Marsy, C. Barlet-Bas, L. Cheval, and A. Doucet, C. R. Acad. Sci. Paris Ser. III 318:619-625, 1995). In the present communication, we have demonstrated that unsaturated fatty acids such as oleic and palmitoleic acids, which are adsorbed to this fraction, are effective inhibitors of the enzyme.

Na,K-ATPase: a molecular target for Leptospira interrogans endotoxin.

On the basis of our report that a glycolipoprotein fraction (GLP) extracted from Leptospira interrogans contains a potent inhibitor of renal Na,K-ATPase, we proposed that GLP-induced inhibition of Na,K-ATPase might be the primary cellular defect in the physiopathology of leptospirosis. The present study was designed to test this hypothesis by determining whether or not 1). GLP inhibits all the isoforms of Na,K-ATPase which are expressed in the tissues affected by leptospirosis, 2) Na,K-ATPase from leptospirosis-resistant species, such as the rat, is sensitive to GLP, 3) GLP inhibits Na,K-ATPase from intact cells, and 4) GLP inhibits ouabain-sensitive H,K-ATPase. The results indicate that in the rabbit, a leptospirosis-sensitive species, GLP inhibits with similar efficiency (apparent IC50: 120-220 micrograms protein GLP/ml) all isoforms of Na,K-ATPase known to be expressed in target tissues for the disease. Na,K-ATPase from rat kidney displays a sensitivity to GLP similar to that of the rabbit kidney enzyme (apparent IC50: 25-80 and 50-150 micrograms protein GLP/ml for rat and rabbit, respectively), indicating that resistance to the disease does not result from the resistance of Na,K-ATPase to GLP. GLP also reduces ouabain-sensitive rubidium uptake in rat thick ascending limbs (pmol mm-1 min-1 +/- SEM; control: 23.8 +/- 1.8; GLP, 88 micrograms protein/ml: 8.2 +/- 0.9), demonstrating that it is active in intact cells. Finally, GLP had no demonstrable effect on renal H,K-ATPase activity, even on the ouabain-sensitive form, indicating that the active principle of GLP is more specific for Na,K-ATPase than ouabain itself. Although the hypothesis remains to be demonstrated in vivo, the present findings are compatible with the putative role of GLP-induced inhibition of Na,K-ATPase as an initial mechanism in the physiopathology of leptospirosis.

Na, K-ATPase: a molecular target for Leptospira interrogans endotoxin

On the basis of our report that a glycolipoprotein fraction (GLP) extracted from Leptospira interrogans contains a potent inhibitor of renal Na,K-ATPase, we proposed that GLP-induced inhibition of Na,K-ATPase might be the primary cellular defect in the physiopathology of leptospirosis. The present study was designed to test this hypothesis by determining whether or not 1) GLP inhibits all the isoforms of Na,K-ATPase which are expressed in the tissues affected by leptospirosis, 2) Na,K-ATPase from leptospirosis-resistant species, such as the rat, is sensitive to GLP, 3) GLP inhibits Na,K-ATPase from intact cells, and 4) GLP inhibits ouabain-sensitive H,K-ATPase. The results indicate that in the rabbit, a leptospirosis-sensitive species, GLP inhibits with similar efficiency (apparent IC5O: 120-220 mug protein GLP/ml) all isoforms of Na,K-ATPase known to be expressed in target tissues for the disease. Na,K-ATPase from rat kidney displays a sensitivity to GLP similar to that of the rabbit kidney enzyme (apparent IC50: 25-80 and 50-150 mug protein GLP/ml for rat and rabbit, respectively), indicating that resistance to the disease does not result from the resistance of Na,K-ATPase to GLP. GLP also reduces ouabain-sensitive rubidium uptake in rat thick ascending limbs (pmol mm-1 min-1 ñ SEM; control: 23.8 ñ 1.8; GLP, 88 mug protein/ml: 8.2 ñ 0.9), demonstrating that it is active in intact cells. Finally, GLP had no demonstrable effect on renal H,K-ATPase activity, even on the ouabain-sensitive form, indicating that the active principle of GLP is more specific for Na,K-ATPase than ouabain itself. Although the hypothesis remains to be demonstrated in vivo, the present findings are compatible with the putative role of GLP-induced inhibition of Na,K-ATPase as an initial mechanism in the physiopathology of leptospirosis.

K depletion modifies the properties of Sch-28080-sensitive K-ATPase in rat collecting duct.

Two distinct Sch-28080-sensitive K-adenosine triphosphatases (K-ATPases) were previously described in the rat nephron: a ouabain-resistant K-ATPase (type I) present in collecting ducts (CD) and a ouabain-sensitive from (type II) located in proximal tubules (PT) and thick ascending limbs (TAL). In K-depleted rats, K-ATPase activity is increased in CD, whereas it is reduced in PT and TAL. Because expression of colonic H-K-ATPase is restricted to the CD of K-depleted rats, we hypothesized that K-ATPase from the CD of K-depleted rats might be different from types I and II. Indeed, type III K-ATPase displays higher sensitivities to ouabain and to Sch-28080 than type II, a lower sensitivity to Sch-28080 than type I, and, conversely to types I and II, it can be stimulated by Na+. Pharmacological differences between types II and III K-ATPases were confirmed by [3H]ouabain binding experiments. Thus the rat kidney expresses three K-ATPases that differ by their pharmacological and kinetic properties, their distribution profile along the nephron and their behavior during K depletion.

Na-K-ATPase along rat nephron after subtotal nephrectomy: effect of enalapril.

Tubular overwork is thought to be a promoter of the tubular hypertrophy and renal failure that occur in response to renal mass reduction. Because Na-K-adenosinetriphosphatase (Na-K-ATPase) is an index of tubular work, we evaluated the effects of subtotal nephrectomy and of enalapril therapy, which delays the evolution of renal lesions, on tubular hypertrophy and Na-K-ATPase activity along the rat nephron. Within 6 wk, 70% reduction of renal mass engendered hypertrophy of the proximal convoluted tubule (PCT), thick ascending limb (TAL), and collecting duct (CD), as well as parallel increments in Na-K-ATPase activity per millimeter tubule length (Na-K-ATPase activity per unit surface area was not modified by subtotal nephrectomy). Chronic enalapril therapy prevented part of the hypertrophy (but not Na-K-ATPase stimulation) of the PCT and the whole stimulation of Na-K-ATPase (but not hypertrophy) in the CD, whereas it had no effect on the TAL. Enalapril effect on Na-K-ATPase in CD might result from reduced bradykinin metabolism, as the reduction in urinary excretion of bradykinin observed in subtotally nephrectomized rats was prevented by enalapril therapy.

Regulation of renal Na+,K(+)-ATPase in rat thick ascending limb during K+ depletion: evidence for modulation of Na+ affinity.

1. NaCl reabsorption along the loop of Henle is reduced in K(+)-depleted rats. Because Na+,K(+)-ATPase energizes this transport and because K+ depletion is known to induce an upregulation of Na+,K(+)-ATPase in most tissues, the regulation of this enzyme was investigated at the level of single thick ascending limbs of the loop of Henle freshly microdissected from rats fed either a normal (control rats) or a low-K+ diet (LK rats). 2. Within 2 weeks of K+ depletion, Na+,K(+)-ATPase activity and [3H]ouabain binding were increased by 30-50% in the medullary portion of the thick ascending limb (MTAL). 3. Despite this increase in the number of Na+,K(+)-ATPase units, the transport capacity of the Na+,K+ pump, determined by ouabain-sensitive Rb+ uptake in the presence of an extracellular concentration of Rb+ mimicking the kalaemia determined in control (4.0 mM Rb+) and LK rats (2.3 mM Rb+), was reduced in MTAL from LK rats. 4. Inhibition of the Na+,K+ pump was not accounted for by changes in either extracellular K+ or intracellular Na+ concentrations, but by a decrease in the pump affinity for Na+. 5. Because this change in the apparent affinity of the Na+,K+ pump for Na+ was detectable in intact but not in permeabilized MTAL cells, it is probably induced by a rapidly reversible cytosolic factor.

Ouabain-sensitive and -insensitive K-ATPases in rat nephron: effect of K depletion.

Because a ouabain-sensitive H-K-adenosinetriphosphatase (H-K-ATPase) has been identified recently in the amphibian bladder, we evaluated whether such an ATPase might exist also in the mammalian kidney, along with the ouabain-insensitive H-K-ATPase previously described in the collecting duct. For this purpose, we searched for an Na-independent, K-stimulated, ouabain- and Sch-28080-inhibitable ATPase activity in single segments of rat nephron. Ouabain-sensitive K-stimulated ATPase activity was detected in the absence of Na+ in rat proximal convoluted and straight tubules and in medullary and cortical thick ascending limbs of Henle's loop but not in collecting ducts. This K-ATPase differs from Na-K-ATPase by 1) its absence of requirement for Na, 2) its sensitivity to Sch-28080, 3) its higher sensitivity to ouabain, and 4) its absence in the collecting duct. It differs from the collecting duct H-K-ATPase by 1) its distribution along the nephron, 2) its sensitivity to ouabain, and 3) its lower sensitivity to Sch-28080. Furthermore, in rats fed a K-depleted diet for 2 wk, ouabain-sensitive K-ATPase activity was markedly reduced in both proximal tubules and thick ascending limbs, whereas collecting duct H-K-ATPase was upregulated.

Inhibition of Na,K-ATPase by an endotoxin extracted from Leptospira interrogans: a possible mechanism for the physiopathology of leptospirosis.

Clinical manifestations of leptospirosis include disorders of the electrolytical balance which might be related to inhibition of Na,K-ATPase. Although the physiopathological cellular mechanism of leptospirosis remains unknown, a bacterial endotoxin has been incriminated. Therefore, we evaluated whether a glycolipoprotein fraction extracted from Leptospira interrogans and known to be cytotoxic might inhibit Na,K-ATPase. This glycolipoprotein fraction (GLP) inhibited Na,K-ATPase activity in rabbit kidney epithelial cells as well as Na,K-ATPase purified from rabbit kidney medulla. Inhibition was dose-dependent, and at maximum it almost abolished Na,K-ATPase activity whereas it had no effect on other enzymes. The GLP did not change the apparent affinity of Na,K-ATPase for potassium whereas it increased that for sodium, revealing a mechanism of inhibition different from that of ouabain. Finally, the inhibitory principle present in the GLP preparation was thermostable and was curtailed by the presence of albumin. In conclusion, a glycolipoproteic fraction extracted from Leptospira interrogans contains a specific inhibitor of Na,K-ATPase. This glycolipoproteic fraction which is present in diseased tissues might induce, through this inhibitor, cellular dysfunctions responsible for the symptoms, in particular those associated with electrolytical disorders such as disturbances of renal electrolyte handling, cardiac arrhythmia or diarrhoea.