Structural bases for Na+-Cl- cotransporter inhibition by thiazide diuretic drugs and activation by kinases.

The Na+-Cl- cotransporter (NCC) drives salt reabsorption in the kidney and plays a decisive role in balancing electrolytes and blood pressure. Thiazide and thiazide-like diuretics inhibit NCC-mediated renal salt retention and have been cornerstones for treating hypertension and edema since the 1950s. Here we determine NCC co-structures individually complexed with the thiazide drug hydrochlorothiazide, and two thiazide-like drugs chlorthalidone and indapamide, revealing that they fit into an orthosteric site and occlude the NCC ion translocation pathway. Aberrant NCC activation by the WNKs-SPAK kinase cascade underlies Familial Hyperkalemic Hypertension, but it remains unknown whether/how phosphorylation transforms the NCC structure to accelerate ion translocation. We show that an intracellular amino-terminal motif of NCC, once phosphorylated, associates with the carboxyl-terminal domain, and together, they interact with the transmembrane domain. These interactions suggest a phosphorylation-dependent allosteric network that directly influences NCC ion translocation.

From Fish Physiology to Human Disease: The Discovery of the NCC, NKCC2, and the Cation-Coupled Chloride Cotransporters.

The renal Na-K-2Cl and Na-Cl cotransporters are the major salt reabsorption pathways in the thick ascending limb of Henle loop and the distal convoluted tubule, respectively. These transporters are the target of the loop and thiazide type diuretics extensively used in the world for the treatment of edematous states and arterial hypertension. The diuretics appeared in the market many years before the salt transport systems were discovered. The evolving of the knowledge and the cloning of the genes encoding the Na-K-2Cl and Na-Cl cotransporters were possible thanks to the study of marine species. This work presents the history of how we came to know the mechanisms for the loop and thiazide type diuretics actions, the use of marine species in the cloning process of these cotransporters and therefore in the whole solute carrier cotransproters 12 (SLC12) family of electroneutral cation chloride cotransporters, and the disease associated with each member of the family.

The V-type H+-ATPase is targeted in antidiuretic hormone control of the Malpighian "renal" tubules.

Like other insects, secretion by mosquito Malpighian tubules (MTs) is driven by the V-type H+-ATPase (VA) localized in the apical membrane of principal cells. In Aedes aegypti, the antidiuretic neurohormone CAPA inhibits secretion by MTs stimulated by select diuretic hormones; however, the cellular effectors of this inhibitory signaling cascade remain unclear. Herein, we demonstrate that the VA inhibitor bafilomycin selectively inhibits serotonin (5HT)- and calcitonin-related diuretic hormone (DH31)-stimulated secretion. VA activity increases in DH31-treated MTs, whereas CAPA abolishes this increase through a NOS/cGMP/PKG signaling pathway. A critical feature of VA activation involves the reversible association of the cytosolic (V1) and membrane (Vo) complexes. Indeed, higher V1 protein abundance was found in membrane fractions of DH31-treated MTs, whereas CAPA significantly decreased V1 abundance in membrane fractions while increasing it in cytosolic fractions. V1 immunolocalization was observed strictly in the apical membrane of DH31-treated MTs, whereas immunoreactivity was dispersed following CAPA treatment. VA complexes colocalized apically in female MTs shortly after a blood meal consistent with the peak and postpeak phases of diuresis. Comparatively, V1 immunoreactivity in MTs was more dispersed and did not colocalize with the Vo complex in the apical membrane at 3 h post blood meal, representing a time point after the late phase of diuresis has concluded. Therefore, CAPA inhibition of MTs involves reducing VA activity and promotes complex dissociation hindering secretion. Collectively, these findings reveal a key target in hormone-mediated inhibition of MTs countering diuresis that provides a deeper understanding of this critical physiological process necessary for hydromineral balance.

Corticotropin-releasing factor-like diuretic hormone acts as a gonad-inhibiting hormone in adult female, Rhodnius prolixus.

Within insects, corticotropin-releasing factor/diuretic hormones (CRF/DHs) are responsible for the modulation of a range of physiological and behavioural processes such as feeding, diuresis, and reproduction. Rhopr-CRF/DH plays a key role in feeding and diuresis in Rhodnius prolixus, a blood-gorging insect and a vector for human Chagas disease. Here, we extend our understanding on the role of this neurohormone in reproduction in adult female R. prolixus. Double-label immunohistochemistry displays co-localized staining of CRF-like and the glycoprotein hormone (GPA2/GPB5) subunit GPB5-like immunoreactivity in the same neurosecretory cells (NSCs) in the mesothoracic ganglionic mass (MTGM) and in their neurohemal sites in adult female R. prolixus, suggesting these peptides could work together to regulate physiological processes. qPCR analysis reveals that the transcript for Rhopr-CRF/DH receptor 2 (Rhopr-CRF/DH-R2) is expressed in reproductive tissues and fat body (FB) in adult female R. prolixus, and its expression increases post blood meal (PBM), a stimulus that triggers diuresis and reproduction. Using RNA interference, transcript expression of Rhopr-CRF/DH-R2 was knocked down, and egg production monitored by examining the major yolk protein, vitellogenin (Vg), the number and quality of eggs laid, and their hatching ratio. Injection of dsCRFR2 into adult females reduces Rhopr-CRF/DH-R2 transcript expression, accelerates oogenesis, increases the number of eggs produced, and reduces hatching rate in female R. prolixus. Downregulation of Rhopr-CRF/DH-R2 leads to an increase in the transcript expression of RhoprVg1 in the fat body and ovaries, and increases the transcript level for the Vg receptor, RhoprVgR, in the ovaries. A significant increase in Vg content in the fat body and in the hemolymph is also observed. Incubation of isolated tissues with Rhopr-CRF/DH leads to a significant decrease in transcript expression of RhoprVg1 in the fat body and RhoprVg1 in the ovaries. In addition, Rhopr-CRF/DH reduces transcript expression of the ecdysteroid biosynthetic enzymes and reduces ecdysteroid titer in the culture medium containing isolated ovaries. These results suggest the involvement of the CRF-signaling pathway in reproduction, and that Rhopr-CRF/DH acts as a gonad-inhibiting hormone in the adult female R. prolixus, as previously shown for the colocalized glycoprotein, GPA2/GPB5.

Diuretic hormone 31 activates two G protein-coupled receptors with differential second messengers for diuresis in Drosophila suzukii.

Diuretic hormones (DHs) bind to G protein-coupled receptors (GPCRs), regulating water and ion balance to maintain homeostasis in animals. Two distinct DHs are known in insects: calcitonin (CT)-like DH31 and corticotropin-releasing factor (CRF)-like DH44. In this study, we identified and characterized DH31 and two DH31 GPCR variants, DH31-Ra and DH31-Rb, from spotted-wing drosophila, Drosophila suzukii, a globally prevalent vinegar fly causing severe damage to small fruits. Both GPCRs are active, but DH31-Ra is the dominant receptor based on gene expression analyses and DH31 peptide binding affinities. A notable difference between the two variants lies in 1) the GPCR structures of their C-termini and 2) the utilization of second messengers, and the amino acid sequences of the two variants are identical. DH31-Ra contains 12 additional amino acids, providing different intracellular C-terminal configurations. DH31-Ra utilizes both cAMP and Ca2+ as second messengers, whereas DH31-Rb utilizes only cAMP; this is the first time reported for an insect CT-like DH31 peptide. DH31 stimulated fluid secretion in D. suzukii adults, and secretion increased in a dose-dependent manner. However, when the fly was injected with a mixture of DH31 and CAPA, an anti-diuretic hormone, fluid secretion was suppressed. Here, we discuss the structures of the DH31 receptors and the differential signaling pathways, including second messengers, involved in fly diuresis. These findings provide fundamental insights into the characterization of D. suzukii DH31 and DH31-Rs, and facilitate the identification of potential biological targets for D. suzukii management.

Neuropeptide diuretic hormone 31 mediates memory and sleep via distinct neural pathways in Drosophila.

Memory formation and sleep regulation are critical for brain functions in animals from invertebrates to humans. Neuropeptides play a pivotal role in regulating physiological behaviors, including memory formation and sleep. However, the detailed mechanisms by which neuropeptides regulate these physiological behaviors remains unclear. Herein, we report that neuropeptide diuretic hormone 31 (DH31) positively regulates memory formation and sleep in Drosophila melanogaster. The expression of DH31 in the dorsal and ventral fan-shaped body (dFB and vFB) neurons of the central complex and ventral lateral clock neurons (LNvs) in the brain was responsive to sleep regulation. In addition, the expression of membrane-tethered DH31 in dFB neurons rescued sleep defects in Dh31 mutants, suggesting that DH31 secreted from dFB, vFB, and LNvs acts on the DH31 receptor in the dFB to regulate sleep partly in an autoregulatory feedback loop. Moreover, the expression of DH31 in octopaminergic neurons, but not in the dFB neurons, is involved in forming intermediate-term memory. Our results suggest that DH31 regulates memory formation and sleep through distinct neural pathways.

Periviscerokinin (Cap2b; CAPA) receptor silencing in females of Rhipicephalus microplus reduces survival, weight and reproductive output.

BACKGROUND: The cattle fever tick, Rhipicephalus (Boophilus) microplus, is a vector of pathogens causative of babesiosis and anaplasmosis, both highly lethal bovine diseases that affect cattle worldwide. In Ecdysozoa, neuropeptides and their G-protein-coupled receptors play a critical integrative role in the regulation of all physiological processes. However, the physiological activity of many neuropeptides is still unknown in ticks. Periviscerokinins (CAP2b/PVKs) are neuropeptides associated with myotropic and diuretic activities in insects. These peptides have been identified only in a few tick species, such as Ixodes ricinus, Ixodes scapularis and R. microplus, and their cognate receptor only characterized for the last two. METHODS: Expression of the periviscerokinin receptor (Rhimi-CAP2bR) was investigated throughout the developmental stages of R. microplus and silenced by RNA interference (RNAi) in the females. In a first experiment, three double-stranded (ds) RNAs, named ds680-805, ds956-1109 and ds1102-1200, respectively, were tested in vivo. All three caused phenotypic effects, but only the last one was chosen for subsequent experiments. Resulting RNAi phenotypic variables were compared to those of negative controls, both non-injected and dsRNA beta-lactamase-injected ticks, and to positive controls injected with beta-actin dsRNA. Rhimi-CAP2bR silencing was verified by quantitative reverse-transcriptase PCR in whole females and dissected tissues. RESULTS: Rhimi-CAP2bR transcript expression was detected throughout all developmental stages. Rhimi-CAP2bR silencing was associated with increased female mortality, decreased weight of surviving females and of egg masses, a delayed egg incubation period and decreased egg hatching (P < 0.05). CONCLUSIONS: CAP2b/PVKs appear to be associated with the regulation of female feeding, reproduction and survival. Since the Rhimi-CAP2bR loss of function was detrimental to females, the discovery of antagonistic molecules of the CAP2b/PVK signaling system should cause similar effects. Our results point to this signaling system as a promising target for tick control.

Functional characterization of a diuretic hormone receptor associated with desiccation, starvation and temperature tolerance in gypsy moth, Lymantria dispar.

Variety of diuretic hormone neuropeptides is known to regulate water and ion balance in invertebrates. By activating their specific neuropeptide, diuretic hormone receptor (DHR) transmits extracellular signals into the cell, and then produces functional cell activity, which plays an important role in regulating physiology and behavior. However, little is known about the function of DHR gene in Lymantria dispar. DHR gene was firstly identified in L. dispar and its physiological functions were investigated using RNA interference (RNAi) technology. The results showed that except for the 6th instar larvae, the expression levels of DHR gene in the larval stages are higher than that in the egg, pupal and adult stages. The DHR gene is highly expressed in hindgut and midgut tissues. The L. dispar larvae significantly increased their water content and high temperature tolerance after the DHR was silenced, while decreasing excretion and feeding behavior. The physiological function of DHR is associated with desiccation, high temperature and starvation resistance. DHR could contribute to future development of novel insecticide to manage this global forest pest population.

Biochemical and molecular effects of a commercial diuretic with herbal extract on experimentally induced urolithiasis in chickens.

This study evaluated the diuretic, antioxidant, anti-inflammatory, and immunological effects of a commercial diuretic (CD) (composed of ammonium chloride, potassium citrate, sodium chloride, ascorbic acid, biotin, halfa bar extract, and hexamine) on chickens with induced urolithiasis. A total of 100 one-day-old white Hy-Line chicks were fed a basal diet containing 20% crude protein (CP) and 1% Ca until they reached 48 days of age. Then, the birds were divided into five groups (G1-G5). G1 was fed a basal diet and kept as a negative control, G2 was fed a high protein (HP) diet containing 25% crude protein, G3 was fed high calcium (HC) diet containing 5% Ca, G4 was fed HP diet supplemented with CD, and G5 was fed HC diet supplemented with CD. The CD was supplemented with drinking water (at a dose of 0.5 ml/ liter) for 1 week. The experiment was held for 78 days. Clinical signs, postmortem lesions, and mortality rates were observed. Biochemical analytes, redox status biomarkers, and expression of interleukin-6 (IL-6) and interferon-gamma (IFN-γ) were measured. Tissue samples were taken for histopathological examination. No signs of CD toxicity were observed during the toxicity test prior to the experiment. Compared to all groups, birds in G2 and G3 showed impaired renal function and alterations in biochemical, redox status, lipid peroxidation, post-mortem, and histopathological lesions along with upregulation of IL-6 and IFN-γ in the kidney and spleen. In conclusion, commercial diuretic supplementation for one week improves renal function, redox status, immune and anti-inflammatory responses in chickens with induced urolithiasis.

Fluid Secretion by Malpighian Tubules of Rhodnius prolixus: Neuroendocrine Control With New Insights From a Transcriptome Analysis.

Rhodnius prolixus (the kissing bug and a major vector of Chagas disease) is an obligate blood feeder that in the case of the fifth instar consumes up to 10 times its unfed body weight in a single 20-minute feed. A post-prandial diuresis is initiated, within minutes of the start of gorging, in order to lower the mass and concentrate the nutrients of the meal. Thus, R. prolixus rapidly excretes a fluid that is high in NaCl content and hypo-osmotic to the hemolymph, thereby eliminating 50% of the volume of the blood meal within 3 hours of gorging. In R. prolixus, as with other insects, the Malpighian tubules play a critical role in diuresis. Malpighian tubules are not innervated, and their fine control comes under the influence of the neuroendocrine system that releases amines and neuropeptides as diuretic or antidiuretic hormones. These hormones act upon the Malpighian tubules via a variety of G protein-coupled receptors linked to second messenger systems that influence ion transporters and aquaporins; thereby regulating fluid secretion. Much has been discovered about the control of diuresis in R. prolixus, and other model insects, using classical endocrinological studies. The post-genomic era, however, has brought new insights, identifying novel diuretic and antidiuretic hormone-signaling pathways whilst also validating many of the classical discoveries. This paper will focus on recent discoveries into the neuroendocrine control of the rapid post-prandial diuresis in R. prolixus, in order to emphasize new insights from a transcriptome analysis of Malpighian tubules taken from unfed and fed bugs.

Discovery of MK-8153, a Potent and Selective ROMK Inhibitor and Novel Diuretic/Natriuretic.

A renal outer medullary potassium channel (ROMK, Kir1.1) is a putative drug target for a novel class of diuretics with potential for treating hypertension and heart failure. Our first disclosed clinical ROMK compound, 2 (MK-7145), demonstrated robust diuresis, natriuresis, and blood pressure lowering in preclinical models, with reduced urinary potassium excretion compared to the standard of care diuretics. However, 2 projected to a short human half-life (∼5 h) that could necessitate more frequent than once a day dosing. In addition, a short half-life would confer a high peak-to-trough ratio which could evoke an excessive peak diuretic effect, a common liability associated with loop diuretics such as furosemide. This report describes the discovery of a new ROMK inhibitor 22e (MK-8153), with a longer projected human half-life (∼14 h), which should lead to a reduced peak-to-trough ratio, potentially extrapolating to more extended and better tolerated diuretic effects.

Loop Diuretics: Clinical Application Information for Nephrology Nurses and Practitioners.

Loop diuretic medications work by inhibiting sodium reabsorption in the renal tubules. The net effect is increasing in urinary sodium and water excretion. Loop diuretics are routinely used for many clinical indications, and nephrology practitioners are well informed in the management of their use in daily practice. This article highlights key information on the most commonly used loop diuretics (e.g., furosemide and torsemide) and provides important clinical features related to pharmacokinetics properties, dosing consideration, route of administration, side effects, and other considerations for practitioners.

Distal convoluted tubule sexual dimorphism revealed by advanced 3D imaging.

The thiazide-sensitive Na+-Cl- cotransporter (NCC) is more abundant in kidneys of female subjects than of male subjects. Because morphological remodeling of the distal convoluted tubule (DCT) is dependent on NCC activity, it has been generally assumed that there is a corresponding sexual dimorphism in the structure of the DCT, leading to a larger female DCT. Until now, this has never been directly examined. Here, optical clearing techniques were combined with antibody labeling of DCT segment markers, state-of-the-art high-speed volumetric imaging, and analysis tools to visualize and quantify DCT morphology in male and female mice and study the DCT remodeling response to furosemide. We found an unexpected sex difference in the structure of the DCT. Compared with the male mice, female mice had a shorter DCT, a higher cellular density of NCC, and a greater capacity to elongate in response to loop diuretics. Our study revealed a sexual dimorphism of the DCT. Female mice expressed a greater density of NCC transporters in a shorter structure to protect Na+ balance in the face of greater basal distal Na+ delivery yet have a larger reserve and structural remodeling capacity to adapt to unique physiological stresses. These observations provide insight into mechanisms that may drive sex differences in the therapeutic responses to diuretics.

Diuretic Therapy for Patients With Heart Failure: JACC State-of-the-Art Review.

Expansion of extracellular fluid volume is central to the pathophysiology of heart failure. Increased extracellular fluid leads to elevated intracardiac filling pressures, resulting in a constellation of signs and symptoms of heart failure referred to as congestion. Loop diuretics are one of the cornerstones of treatments for heart failure, but in contrast to other therapies, robust clinical trial evidence to guide the use of diuretics is sparse. A nuanced understanding of renal physiology and diuretic pharmacokinetics is essential for skillful use of diuretics in the management of heart failure in both the inpatient and outpatient settings. Diuretic resistance, defined as an inadequate quantity of natriuresis despite an adequate diuretic regimen, is a major clinical challenge that generally portends a poor prognosis. In this review, the authors discuss the fundamental mechanisms and physiological principles that underlie the use of diuretic therapy and the available data on the optimal use of diuretics.

Renal secretion of hydrochlorothiazide involves organic anion transporter 1/3, organic cation transporter 2, and multidrug and toxin extrusion protein 2-K.

Hydrochlorothiazide (HCTZ) is the most widely used thiazide diuretic for the treatment of hypertension either alone or in combination with other antihypertensives. HCTZ is mainly cleared by the kidney via tubular secretion, but the underlying molecular mechanisms are unclear. Using cells stably expressing major renal organic anion and cation transporters [human organic anion transporter 1 (hOAT1), human organic anion transporter 3 (hOAT3), human organic cation transporter 2 (hOCT2), human multidrug and toxin extrusion 1 (hMATE1), and human multidrug and toxin extrusion 2-K (hMATE2-K)], we found that HCTZ interacted with both organic cation and anion transporters. Uptake experiments further showed that HCTZ is transported by hOAT1, hOAT3, hOCT2, and hMATE2-K but not by hMATE1. Detailed kinetic analysis coupled with quantification of membrane transporter proteins by targeted proteomics revealed that HCTZ is an excellent substrate for hOAT1 and hOAT3. The apparent affinities (Km) for hOAT1 and hOAT3 were 112 ± 8 and 134 ± 13 µM, respectively, and the calculated turnover numbers (kcat) were 2.48 and 0.79 s-1, respectively. On the other hand, hOCT2 and hMATE2-K showed much lower affinity for HCTZ. The calculated transport efficiency (kcat/Km) at the single transporter level followed the rank order of hOAT1> hOAT3 > hOCT2 and hMATE2-K, suggesting a major role of organic anion transporters in tubular secretion of HCTZ. In vitro inhibition experiments further suggested that HCTZ is not a clinically relevant inhibitor for hOAT1 or hOAT3. However, strong in vivo inhibitors of hOAT1/3 may alter renal secretion of HCTZ. Together, our study elucidated the molecular mechanisms underlying renal handling of HCTZ and revealed potential pathways involved in the disposition and drug-drug interactions for this important antihypertensive drug in the kidney.

Systematic analysis and identification of unexpected interactions from the neuroprotein drug interactome in hydrocephalus pharmacological intervention.

Hydrocephalus is a neurological condition caused by an abnormal accumulation of cerebrospinal fluid; pharmacological intervention of the disease has been found to elicit a variety of adverse drug reactions (ADRs) in central nervous system (CNS) by unexpectedly targeting certain functional neuroproteins. Here, a systematic neuroprotein drug interactome (SNDI) is created for 11 hydrocephalus drugs/metabolites plus 20 control drugs across 518 druggable pockets on the surface of 472 CNS neuroproteins via a large-scale molecular docking approach. Heuristic clustering analysis of the SNDI profile divides the 31 investigated drug ligands into a distinct panel and a background panel; the former consists of two hydrocephalus drugs (Furosemide and Triamterene) and their respective metabolites (Furosemide glucuronide and Hydroxytriamterene) that are inferred to have generally high affinity towards the whole array of neuroprotein pockets. A total of 13 neuroproteins are enriched in gene ontology semantic mining as putative unexpected targets of the distinct panel, and their intermolecular interactions with hydrocephalus drugs/metabolites are investigated in detail using dynamics simulation and energetics analysis. We also perform kinase assay and viability test to substantiate the interactome analysis. It is found that the Furosemide and Triamterene have significant cytotoxic effects on normal human astrocytes, in which the Triamterene can inhibit the neurokinase ROCK2, a representative of putative unexpected targets, with a high activity, which is comparable with the sophisticated ROCK2 inhibitor Fasudil.

First identification of a diuretic, hydrochlorothiazide, in hair: Application to a doping case and interpretation of the results.

An athlete contested an adverse analytical finding involving hydrochlorothiazide, and requested our laboratory for testing his hair. As there is no reference in the literature about identification of hydrochlorothiazide in hair, several volunteers were first enrolled (4 after a single 25 mg administration and 10 with daily therapeutic treatment). A specific method was developed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS. Hair samples were decontaminated with dichloromethane and 30 mg were incubated in buffer at pH 7.0 for 15 hours at 50°C. Then, 5 mL ethyl acetate was added for extraction. Linearity was observed for hydrochlorothiazide concentrations ranging from 5 to 2000 pg/mg. The limit of quantification was 5 pg/mg. The coefficients of variation (CVs) of repeatability and matrix effect were lower than 20%. Analysis of the 0-2-cm segment of the 4 volunteers having received a single dose, collected 1 month after administration, was negative at the limit of quantification. The hair of the 10 patients (proximal 2 cm) on daily treatment was positive with concentrations ranging from 12 to 1845 pg/mg, with no correlation between daily dose and concentration. The athlete's hair tested positive for hydrochlorothiazide at 36 pg/mg in the segment corresponding to the period of the urinary control. Since a single exposure to hydrochlorothiazide is not detectable in hair and based on the results of the patients on daily treatment, the concentration found in the athlete has been interpreted as corresponding to repeated exposures, where it was not possible to establish the dosage and the frequency.

Chronobiology in nephrology: the influence of circadian rhythms on renal handling of drugs and renal disease treatment.

INTRODUCTION: Chronobiology studies the phenomenon of rhythmicity in living organisms. The circadian rhythms are genetically determined and regulated by external synchronizers (the daylight cycle). Several biological processes involved in the pharmacokinetics and pharmacodynamics of drugs are subjected to circadian variations. Chronopharmacology studies how biological rhythms influence pharmacokinetics, pharmacodynamics, and toxicity, and determines whether time-of-day administration modifies the pharmacological characteristics of the drug. Chronotherapy applies chronopharmacological studies to clinical treatments, determining the best biological time for dosing: when the beneficial effects are maximal and the incidence and/or intensity of related side effects and toxicity are minimal. Most water-soluble drugs or drug metabolites are eliminated by urine through the kidney. The rate of drug clearance in the urine depends on several intrinsic variables related to renal function including renal blood flow, glomerular filtration rate, the ability of the kidney to reabsorb or to secrete drugs, urine flow, and urine pH, which influences the degree of urine acidification. Curiously, all these variables present a circadian behavior in different mammalian models. CONCLUSION: The circadian rhythms have influence in the renal physiology, pathophysiology, and pharmacology, and these data should be taken into account in clinical nephrology practice.