Urinary peptidome analyses for the diagnosis of chronic kidney disease in dogs

Chronic kidney disease (CKD) is clinically important in canine medicine. Current diagnostic tools lack sensitivity for detection of subclinical CKD. The aim of the present study was to evaluate urinary peptidome analysis for diagnosis of CKD in dogs. Capillary electrophoresis coupled to mass spectrometry analysis demonstrated presence of approximately 5400 peptides in dog urine. Comparison of urinary peptide abundance of dogs with and without CKD led to the identification of 133 differentially excreted peptides (adjusted P for each peptide <0.05). Sequence information was obtained for 35 of these peptides. This 35 peptide subset and the total group of 133 peptides were used to construct two predictive models of CKD which were subsequently validated by researchers masked to results in an independent cohort of 20 dogs. Both models diagnosed CKD with an area under the receiver operating characteristic (ROC) curve of 0.88 (95% confidence intervals [CI], 0.72–1.0). Most differentially excreted peptides represented fragments of collagen I, indicating possible association with fibrotic processes in CKD (similar to the equivalent human urinary peptide CKD model, CKD273). This first study of the urinary peptidome in dogs identified peptides that were associated with presence of CKD. Future studies are needed to validate the utility of this model for diagnosis and prediction of progression of canine CKD in a clinical setting

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

International audienceTwo 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

Cold- and Ouabain-resistance of Renal Na,K-ATPase in Cold-exposed and Hibernating Jerboas (Jaculus orientalis)

International audienceThe 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

Effect of Leptospira interrogans Endotoxin on Renal Tubular Na,K-ATPase and H,K-ATPase Activities

International audienc

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

International audienceBecause 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

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

International audience1. 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

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

International audienceClinical 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

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 µg 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 µg 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 µg 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 leptospirosi