Retrospective data analysis of animal poisoning events in Liguria.

Intentional poisoning represents a serious risk to domestic and wild animals, and it can be an environmental and human health issue as well . This paper is a retrospective study, which covers a decade, based on animal poisoning cases and poisoned baits that were submitted for diagnostic examinations to the Veterinary Medical Research Institute for Piedmont, Liguria and the Aosta Valley (IZS-PLVA) in Liguria region. All data were collected through a passive surveillance system introduced in Italy by a decree of the Ministry of Health in January 2009. 43.2% of the animal poisoning cases were confirmed by toxicological analysis, whereas toxic agents were detected in 31.1% of the baits. The most affected animal species were dogs and cats, followed by synanthropic birds,. Only 4% of the total poisoning events analysed involved wild animals and cases of livestock poisoning were minimal. An increased number of cases in January, March, April and August was noticed, but no seasonal trend was detected. The most affected areas were the ones with the highest level of urbanization and population density. The major cause of the poisonings and the most common substances detected in the examined baits were anticoagulants whereas cholinesterase inhibitors, organochlorine pesticides and carbamates were detected in a minor number of cases. This study raises concerns about deliberate animal poisoning in ligurian region and highlights the necessity to fight this phenomenon as it endangers animals, humans and environment.

Chloride channels in basolateral TAL membranes. XVIII. Phenylglyoxal induces functional mcCIC-Ka activity in basolateral MTAL membranes.

Cultured mouse MTAL cells contain more mRNA encoding the Cl- channel mcCIC-Ka, which mediates CTAL Cl- absorption, than mRNA encoding the Cl- channel mmCIC-Ka, which mediates MTAL Cl- absorption. mmCIC-Ka and mcCIC-Ka have three functional differences: 1) mmCIC-Ka open time probability, Po, increases with increasing cytosolic Cl-, but variations in cytosolic Cl- do not affect Po in mcCIC-Ka; 2) mmCIC-Ka is gated by (ATP + PKA), while (ATP + PKA) have no effect on Po in mcCIC-Ka; and 3) mmCIC-Ka channels have single-ion occupancy, while mcCIC-Ka channels have multi-ion occupancy. Using basolateral vesicles from MTAL cells fused into bilayers, we evaluated the effects of 1 mM cytosolic phenylglyoxal (PGO), which binds covalently to lysine or arginine, on Cl- channels. With PGO pretreatment, Cl- channels were uniformly not gated either with increases in cytosolic-face Cl- or with (ATP + PKA) at 2 mM cytosolic-face Cl-; and they exhibited multi-ion occupancy kinetics typical for mcCIC-Ka channels. Thus, in basolateral MTAL membranes, blockade of Cl- access to arginine or lysine residues on mmCIC-Ka by PGO results in Cl- channels having the functional characteristics of mcCIC-Ka channels.

Cl- channels in basolateral TAL membranes. XIX. Cytosolic Cl- regulates mmCIC-Ka and mcCIC-Ka channels.

We evaluated the effects of culturing mouse MTAL cells under conditions that suppressed steady-state cytosolic Cl- on chloride channels fused into bilayers from basolateral vesicles of cultured MTAL cells. We used two agents to suppress Cl- entry: 10(-6) M PGE2 and 10(-4) M bumetanide. Basolateral Cl- channels from control cultured MTAL cells exhibited the signature characteristics of mmCIC-Ka channels: increased open-time probability (Po) either by raising cytosolic-face [Cl-] or, at 2 mM cytosolic Cl-, by adding (ATP + PKA), and first-order conductance kinetics. Either 10(-6) M PGE2 or 10(-4) M bumetanide in culture media reduced steady-state MTAL cytosolic Cl-. Chloride channels from these cells exhibited characteristics unique to CTAL mcCIC-Ka channels, namely: no augmentation of Po either by raising cytosolic Cl- or with cytosolic (ATP + PKA), and multi-ion occupancy. Semi-quantitative RT-PCR and real-time quantitative PCR showed that culturing MTAL cells with 10(-6) M PGE2 or 10(-4) M bumetanide reduced mRNA levels encoding mmCIC-Ka but not mRNA levels encoding mcCIC-Ka. However, when MTAL cells were cultured under control conditions, and then pre-incubated for 60 minutes with 10(-4) M bumetanide, cytosolic Cl- fell acutely but Cl- channels exhibited characteristics of mmCIC-Ka channels. Thus PGE2 and bumetanide, both of which lower steady-state MTAL cytosolic Cl- concentrations, inhibit either the transcriptional and/or the translational processes for mmCIC-Ka synthesis.

Cl channels in basolateral TAL membranes. XVII. Kinetic properties of mcClC-Ka, a basolateral CTAL Cl- channel.

This paper describes the kinetics of Cl- flux through mcClC-Ka Cl- channels from basolateral membranes of mouse CTAL cells. We have cloned two separate but highly homologous Cl- channels, mmClC-Ka from cultured mouse MTAL cells and mcClC-Ka from cultured mouse CTAL cells. The mmClC-Ka and mcClC-Ka channels appear to mediate net Cl- absorption in the MTAL and CTAL, respectively. The kinetics of Cl- permeation through mmClC-Ka channels exhibit traditional criteria for a first-order process, including saturation kinetics. Thus mmClC-Ka channels operate functionally as if the channels were occupied by a single Cl- ion at any given time. In the present studies, we examined conductance-concentration relations in mcClC-Ka channels, and compared both mole-fraction effects and ion selectivity characteristics in mmClC-Ka and mcClC-Ka channels. In mcClC-Ka channels, we observed both self-block at high external Cl- concentrations and, at constant ionic strength, an anomalous mole-fraction effect using external solutions containing varying F-/Cl- concentrations. Neither effect was obtained in mmClC-Ka channels. These data are consistent with the possibility that Cl- permeation through mcClC-Ka channels involved multi-ion occupancy channels that expressed single-file behavior.

Chloride channels in the loop of Henle.

Cl- transport in the loop of Henle is responsible for reclamation of 25-40% of the filtered NaCl load and for the formation of dilute urine. Our understanding of the physiologic and molecular mechanisms responsible for Cl- reabsorption in both the thin ascending limb and thick ascending limb of Henle's loop has increased greatly over the last decade. Plasma membrane Cl- channels are known to play an integral role in transcellular Cl- transport in both the thin and thick ascending limbs. This review focuses on the functional characteristics and molecular identities of these Cl- channels, as well as the role of these channels in the pathophysiology of disease.

Cl(-) channels in basolateral TAL membranes XV. Molecular heterogeneity between cortical and medullary channels.

We have isolated two new and highly homologous cDNAs, mmClC-Ka from mouse outer medulla and mcClC-Ka from mouse cortex. In both cases, mRNA was obtained from the indicated region and subjected to RT-PCR using primers from the nucleotide sequence of rbClC-Ka, which encodes basolateral Cl(-) channels (termed rbClC-Ka) in rabbit MTAL. The predicted protein products of mmClC-Ka and mcClC-Ka, mmClC-Ka and mcClC-Ka, respectively, were 85% homologous and had predicted molecular weights of 75 kDa. The predicted protein sequences for mmClC-Ka and rbClC-Ka had three cytosolic sites-threonine 185, threonine 187 and serine 270-which were absent in mcClC-Ka. These three moieties represent potential sites for phosphorylation of mmClC-Ka and rbClC-Ka, but not of mcClC-Ka, and may account for the failure of (ATP + PKA) to increase the open time probability P(o) in basolateral CTAL Cl(-) channels. We prepared antisense oligonucleotides specific for nonhomologous regions of these two cDNAs, mmAntisense for mmClC-Ka and mcAntisense for mcClC-Ka. Using anti-rbClC-Ka, a polyclonal antibody to rbClC-Ka, we found that, when transfected into cultured mouse MTAL and CTAL cells, mmAntisense suppressed the appearance of the 75 kDa band by 50% in vesicles from MTAL but not CTAL cells, while transfection of MTAL and CTAL cells with mcAntisense suppressed appearance of the 75 kDa band in vesicles from CTAL but not MTAL cells. mmAntisense transfection also prolonged the half-time (T(1/2), sec) for (36)Cl(-) efflux in cultured MTAL cells from 82.4 +/- 6.8 sec (sem) to 187.8 +/- 9.5 sec (n = 5; P = 0.0001) while mcAntisense transfection had no such effect. Conversely, in cultured CTAL cells, mcAntisense transfection prolonged the T(1/2) for (36)Cl(-) efflux from 80.9 +/- 6.3 sec to 191.8 +/- 6.5 sec (n = 5; P = 0.00005), while mmAntisense had no such effect. We conclude that mmClC-Ka and mcClC-Ka may encode the basolateral Cl(-) channels mediating net Cl(-) absorption in mouse MTAL and CTAL, respectively.

Cl- channels in basolateral TAL membranes. XIV. Kinetic properties of a basolateral MTAL Cl- channel.

BACKGROUND: This article reports studies on the kinetics of chloride (Cl-) conductance in Cl- channels fused into bilayers from basolaterally enriched vesicles from rabbit outer medulla. A considerable body of evidence indicates that these channels represent rbClC-Ka, a 77 kDa kidney-specific protein of the ClC family of Cl- channels. rbClC-Ka, a candidate channel for mediating net Cl- absorption in the medullary thick ascending limb (MTAL), has been cloned from rabbit outer medulla and localized by immunofluorescence to basolateral membranes of the MTAL. Thus, this is the first account, to our knowledge, of the kinetics of ion permeation through a renal Cl- channel mediating net basolateral Cl- absorption in the thick ascending limb of Henle (TALH), and this channel may represent rbClC-Ka. METHODS: The electrophysiological properties of these channels were studied by fusing basolaterally enriched MTAL vesicles into planar bilayer membranes. RESULTS: Cl- conductance through these channels was concentration dependent and saturable. The relationship between gCl (pS) and symmetrical aqueous Cl- concentrations could be expressed in terms of the Michaelis equation with a limiting conductance (GClmax, pS) of 114 pS at infinitely high aqueous Cl- concentrations and a K1/2 of 163 mM Cl-. A log-log plot of the conductance-Cl- concentration relations, in the nonsaturating Cl- concentration range, had a slope of 0.91, that is, virtually unity. The relatively impermeant anion I- produced a voltage-dependent conductance blockade that could be overcome at high electric field strengths. CONCLUSIONS: The experimental data described earlier here fulfill the traditional criteria for a first-order process with a single Cl- ion occupying these channels at a given time. Although the channels may contain multiple ion binding sites, the latter function, in integral kinetic terms, as a single rate-limiting locus.

Pathogenesis of renal sodium retention in congestive heart failure.

This article summarizes briefly some factors responsible for edema in chronic congestive heart failure. It is now generally thought that so-called 'backward failure' is a manifestation of diastolic dysfunction, while systolic 'pump failure' is a disease that depends on two key factors: an inadequate cardiac output, and renal salt and water retention. The key elements involved in what might be termed the 'integrated volume response' are hemodynamic and renal factors. The hemodynamic factors include vasoconstriction, tachycardia and a reduced venous capacitance. These responses occur within minutes, while salt and water retention occurs over days to weeks. The key renal elements modulating sodium retention in congestive heart failure include, at a minimum, four variables. First, there is a reduction in renal blood flow produced by the almost simultaneous operation of alpha- and beta-catecholamines, antidiuretic hormone, the endothelins, and angiotensin II. Second, activation of the tubuloglomerular feedback system enhances intrarenal angiotensin II release, which augments proximal sodium absorption. In addition, beta-catechols also enhance proximal sodium absorption. A third key element involved in renal sodium retention is activation of apical sodium channels, ENaC, of principal cells in the cortical collecting tubule by aldosterone and by vasopressin. Finally, the inner medullary collecting duct becomes resistant to the action of atrial natriuretic peptide, thus adding a final dimension to the syndrome of sodium retention in underfilling.

Cl- channels in basolateral TAL membranes: XIII. Heterogeneity between basolateral MTAL and CTAL Cl- channels.

BACKGROUND: Antidiuretic hormone (ADH) or adenosine 3', 5'-cyclic phosphate (cAMP) analogues augment net NaCl absorption in microperfused mouse medullary thick ascending limb (MTAL) segments but not in cortical thick ascending limb (CTAL) segments. This ADH-dependent MTAL effect is due to increased apical Na+/K+/2Cl- admittance and apical K+ recycling accompanied by a rise in calculated intracellular Cl- concentrations and by a threefold rise in basolateral Cl- conductance. rbClC-Ka, a 75.2 member of the ClC family of Cl- channels, mediates net Cl- absorption in the MTAL. The gating characteristics of rbClC-Ka channels from their intracellular surfaces are, to our knowledge, unique among Cl- channels. The channels are activated by small increases in intracellular Cl- (K1/2 = 10 mM Cl-). Adenosine triphosphate plus the catalytic subunit of protein kinase A (ATP + PKA) gate rbClC-Ka when cytosolic Cl- concentrations are 25 mM. Thus, in mouse MTAL segments, ADH-dependent rises in cytosolic Cl- are primarily responsible for basolateral Cl- conductance increases. METHODS: These experiments compared the properties of Cl- channels fused into bilayers from basolaterally enriched vesicles from cultured mouse CTAL cells with rbClC-Ka channels. RESULTS: The key findings were that anti-rbClC-Ka, antibody that recognizes and blocks rbClC-Ka, recognized and blocked basolateral Cl- channels in CTAL cells, that the extracellular faces of the CTAL channels were, like rbClC-Ka, substrate gated with a K1/2 of approximately 170 mM Cl-, and that, unlike rbClC-Ka channels, cytosolic faces of basolateral CTAL Cl- channels were not gated by either increasing cytosolic Cl- concentrations or cytosolic (ATP + PKA). This failure of activation of basolateral CTAL Cl- channels was confirmed using excised patch clamp studies. Finally, on Western blots, anti-rbClC-Ka recognized a 74 kDa band on basolateral CTAL vesicles. CONCLUSIONS: Basolateral CTAL Cl- channels probably share a high degree of structural homology and possibly molecular mass with rbClC-Ka channels. However, significant differences between rbClC-Ka channels and CTAL Cl- channels account for the inability of increasing either cytosolic Cl- or (PKA + ATP) to raise Po in CTAL basolateral Cl- channels.

An overview of salt absorption by the nephron.

The purpose of this brief commentary is to provide a description of how renal physiology, and more particularly, renal tubular physiology, has evolved over the past thirty years, since the occasion, obviously, is a celebration of the Thirtieth Course on Advances in Nephrology and Dialysis. My arguments will begin by quoting from Homer Smith's book, The Kidney, and merging Smith's observations, which were integrative in the sense that they did not specify detail, with detailed incursions into tubular physiology, first at a cellular level and then at a molecular level. For convenience, the nephron is divided into four functional segments: the proximal nephron; the ascending limb; the distal nephron; and the collecting duct. Each of these carries out a specific function. The proximal nephron absorbs about two-thirds of filtered sodium, without dissociating salt and water absorption. The thick ascending limb absorbs 25% of filtered Na+, but no water. The distal nephron absorbs 10% of filtered Na+ in close relation with K+ and, to some extent, H+ secretion. Finally, the collecting duct includes three kinds of cells: the cortical collecting duct, which is responsible not only for Na+ absorption and K+ secretion, but also for the bulk of the absorption of free water; the outer medullary collecting duct (OMCD), which is largely responsible for the final steep drop in urine pH which occurs between cortex and papilla; and the inner medullary collecting duct (IMCD), whose major function is the final absorption of approximately 5% of filtered Na+.

Mechanisms of tubular sodium chloride transport.

Extracellular fluid volume is determined by sodium and its accompanying anions. There are control mechanisms which regulate sodium balance in the body. These include high and low pressure baroreceptors, intrarenal baroreceptors, renal autoregulation, tubuloglomerular feedback, aldosterone, and numerous other physical and hormonal factors. Sodium transport by the nephron involves active and passive processes which occur in several different nephron segments. Mechanisms of cotransport, Na(+)-H+ exchange, antiporters and ion-specific channels are all utilized by the nephron to maintain sodium balance. These regulatory factors and transport mechanisms for sodium in the kidney will he discussed in detail.