Potassium-Switch Signaling Pathway Dictates Acute Blood Pressure Response to Dietary Potassium.

BACKGROUND: Potassium (K+)-deficient diets, typical of modern processed foods, increase blood pressure (BP) and NaCl sensitivity. A K+-dependent signaling pathway in the kidney distal convoluted tubule, coined the K+ switch, that couples extracellular K+ sensing to activation of the thiazide-sensitive NaCl cotransporter (NCC) and NaCl retention has been implicated, but causality has not been established. METHODS: To test the hypothesis that small, physiological changes in plasma K+ (PK+) are translated to BP through the switch pathway, a genetic approach was used to activate the downstream switch kinase, SPAK (SPS1-related proline/alanine-rich kinase), within the distal convoluted tubule. The CA-SPAK (constitutively active SPS1-related proline/alanine-rich kinase mice) were compared with control mice over a 4-day PK+ titration (3.8-5.1 mmol) induced by changes in dietary K+. Arterial BP was monitored using radiotelemetry, and renal function measurements, NCC abundance, phosphorylation, and activity were made. RESULTS: As PK+ decreased in control mice, BP progressively increased and became sensitive to dietary NaCl and hydrochlorothiazide, coincident with increased NCC phosphorylation and urinary sodium retention. By contrast, BP in CA-SPAK mice was elevated, resistant to the PK+ titration, and sensitive to hydrochlorothiazide and salt at all PK+ levels, concomitant with sustained and elevated urinary sodium retention and NCC phosphorylation and activity. Thus, genetically locking the switch on drives NaCl sensitivity and prevents the response of BP to potassium. CONCLUSIONS: Low K+, common in modern ultraprocessed diets, presses the K+-switch pathway to turn on NCC activity, increasing sodium retention, BP, and salt sensitivity.

Dietary potassium stimulates Ppp1Ca-Ppp1r1a dephosphorylation of kidney NaCl cotransporter and reduces blood pressure.

Consumption of low dietary potassium, common with ultraprocessed foods, activates the thiazide-sensitive sodium chloride cotransporter (NCC) via the with no (K) lysine kinase/STE20/SPS1-related proline-alanine-rich protein kinase (WNK/SPAK) pathway to induce salt retention and elevate blood pressure (BP). However, it remains unclear how high-potassium "DASH-like" diets (dietary approaches to stop hypertension) inactivate the cotransporter and whether this decreases BP. A transcriptomics screen identified Ppp1Ca, encoding PP1A, as a potassium-upregulated gene, and its negative regulator Ppp1r1a, as a potassium-suppressed gene in the kidney. PP1A directly binds to and dephosphorylates NCC when extracellular potassium is elevated. Using mice genetically engineered to constitutively activate the NCC-regulatory kinase SPAK and thereby eliminate the effects of the WNK/SPAK kinase cascade, we confirmed that PP1A dephosphorylated NCC directly in a potassium-regulated manner. Prior adaptation to a high-potassium diet was required to maximally dephosphorylate NCC and lower BP in constitutively active SPAK mice, and this was associated with potassium-dependent suppression of Ppp1r1a and dephosphorylation of its cognate protein, inhibitory subunit 1 (I1). In conclusion, potassium-dependent activation of PP1A and inhibition of I1 drove NCC dephosphorylation, providing a mechanism to explain how high dietary K+ lowers BP. Shifting signaling of PP1A in favor of activation of WNK/SPAK may provide an improved therapeutic approach for treating salt-sensitive hypertension.

Dietary anions control potassium excretion: it is more than a poorly absorbable anion effect.

The urinary potassium (K+) excretion machinery is upregulated with increasing dietary K+, but the role of accompanying dietary anions remains inadequately characterized. Poorly absorbable anions, including [Formula: see text], are thought to increase K+ secretion through a transepithelial voltage effect. Here, we tested if they also influence the K+ secretion machinery. Wild-type mice, aldosterone synthase (AS) knockout (KO) mice, or pendrin KO mice were randomized to control, high-KCl, or high-KHCO3 diets. The K+ secretory capacity was assessed in balance experiments. Protein abundance, modification, and localization of K+-secretory transporters were evaluated by Western blot analysis and confocal microscopy. Feeding the high-KHCO3 diet increased urinary K+ excretion and the transtubular K+ gradient significantly more than the high-KCl diet, coincident with more pronounced upregulation of epithelial Na+ channels (ENaC) and renal outer medullary K+ (ROMK) channels and apical localization in the distal nephron. Experiments in AS KO mice revealed that the enhanced effects of [Formula: see text] were aldosterone independent. The high-KHCO3 diet also uniquely increased the large-conductance Ca2+-activated K+ (BK) channel ß4-subunit, stabilizing BKα on the apical membrane, the Cl-/[Formula: see text] exchanger, pendrin, and the apical KCl cotransporter (KCC3a), all of which are expressed specifically in pendrin-positive intercalated cells. Experiments in pendrin KO mice revealed that pendrin was required to increase K+ excretion with the high-KHCO3 diet. In summary, [Formula: see text] stimulates K+ excretion beyond a poorly absorbable anion effect, upregulating ENaC and ROMK in principal cells and BK, pendrin, and KCC3a in pendrin-positive intercalated cells. The adaptive mechanism prevents hyperkalemia and alkalosis with the consumption of alkaline ash-rich diets but may drive K+ wasting and hypokalemia in alkalosis.NEW & NOTEWORTHY Dietary anions profoundly impact K+ homeostasis. Here, we found that a K+-rich diet, containing [Formula: see text] as the counteranion, enhances the electrogenic K+ excretory machinery, epithelial Na+ channels, and renal outer medullary K+ channels, much more than a high-KCl diet. It also uniquely induces KCC3a and pendrin, in B-intercalated cells, providing an electroneutral KHCO3 secretion pathway. These findings reveal new K+ balance mechanisms that drive adaption to alkaline and K+-rich foods, which should guide new treatment strategies for K+ disorders.

A New Understanding of Potassium's Influence Upon Human Health and Renal Physiology.

Potassium channels are expressed in virtually all cell types, and their activity is the dominant determinant of cellular membrane potential. As such, potassium flux is a key regulator of many cellular processes including the regulation of action potentials in excitable cells. Subtle changes in extracellular potassium can initiate signaling processes vital for survival (insulin signaling) while more extreme and chronic changes may lead to pathological states (acid-base disturbances and cardiac arrhythmia). While many factors acutely influence extracellular potassium levels, it is principally the role of the kidneys to maintain potassium balance by matching urinary excretion with dietary intake. When this balance is disrupted, human health is negatively impacted. In this review, we discuss evolving views of dietary potassium intake as means of preventing and mitigating diseases. We also provide an update on a molecular pathway called the potassium switch, a mechanism by which extracellular potassium regulates distal nephron sodium reabsorption. Finally, we review recent literature describing how several popular therapeutics influence potassium homeostasis.

Three-dimensional echo decorrelation monitoring of radiofrequency ablation in ex vivo bovine liver.

Three-dimensional (3D) echo decorrelation imaging was investigated for monitoring radiofrequency ablation (RFA) in ex vivo bovine liver. RFA experiments (N = 14) were imaged by 3D ultrasound using a matrix array, with in-phase and quadrature complex echo volumes acquired about every 11 s. Tissue specimens were then frozen at -80 °C, sectioned, and semi-automatically segmented. Receiver operating characteristic (ROC) curves were constructed for assessing ablation prediction performance of 3D echo decorrelation with three potential normalization approaches, as well as 3D integrated backscatter (IBS). ROC analysis indicated that 3D echo decorrelation imaging is potentially a good predictor of local RFA, with the best prediction performance observed for globally normalized decorrelation. Tissue temperatures, recorded by four thermocouples integrated into the RFA probe, showed good correspondence with spatially averaged decorrelation and statistically significant but weak correlation with measured echo decorrelation at the same spatial locations. In tests predicting ablation zones using a weighted K-means clustering approach, echo decorrelation performed better than IBS, with smaller root mean square volume errors and higher Dice coefficients relative to measured ablation zones. These results suggest that 3D echo decorrelation and IBS imaging are capable of real-time monitoring of thermal ablation, with potential application to clinical treatment of liver tumors.

Rapid development of vasopressin resistance in dietary K+ deficiency.

The association between diabetes insipidus (DI) and chronic dietary K+ deprivation is well known, but it remains uncertain how the disorder develops and whether it is influenced by the sexual dimorphism in K+ handling. Here, we determined the plasma K+ (PK) threshold for DI in male and female mice and ascertained if DI is initiated by polydipsia or by a central or nephrogenic defect. C57BL6J mice were randomized to a control diet or to graded reductions in dietary K+ for 8 days, and kidney function and transporters involved in water balance were characterized. We found that male and female mice develop polyuria and secondary polydipsia. Altered water balance coincided with a decrease in aquaporin-2 (AQP2) phosphorylation and apical localization despite increased levels of the vasopressin surrogate marker copeptin. No change in the protein abundance of urea transporter-A1 was observed. The Na+-K+-2Cl- cotransporter decreased only in males. Desmopressin treatment failed to reverse water diuresis in K+-restricted mice. These findings indicate that even a small fall in PK is associated with nephrogenic DI (NDI), coincident with the development of altered AQP2 regulation, implicating low PK as a causal trigger of NDI. We found that PK decreased more in females, and, consequently, females were more prone to develop NDI. Together, these data indicate that AQP2 regulation is disrupted by a small decrease in PK and that the response is influenced by sexual dimorphism in K+ handling. These findings provide new insights into the mechanisms linking water and K+ balances and support defining the disorder as "potassium-dependent NDI."NEW & NOTEWORTHY This study shows that aquaporin-2 regulation is disrupted by a small fall in plasma potassium levels and the response is influenced by sexual dimorphism in renal potassium handling. The findings provided new insights into the mechanisms by which water balance is altered in dietary potassium deficiency and support defining the disorder as "potassium-dependent nephrogenic diabetes insipidus."

Detection of specific Treponema species and Dichelobacter nodosus from digital dermatitis (Mortellaro's disease) lesions in Swiss cattle.

INTRODUCTION: The aim of this study was to determine the prevalence of the three Treponema species as well as D. nodosus in Digital dermatitis (DD) and slurry of Swiss cattle using PCR. A total of 86 specimens from 24 farms were enrolled in the study. Slurry samples from 21 DD-affected and one unaffected farm were collected to assess the potential of environmental transmission. Nested and real-time PCR were performed from the specimens to detect Treponema species and D. nodosus, respectively. The DD-stages were positive for at least one or more of the DD-associated Treponema species in 50 of 61 cases (82.0%) and in 9 of 25 cases (36.0%) in unaffected animals. Infected animals with small focal active lesions showed a significantly lower prevalence (14.8%) compared to the other DD stages (67.2%; P=0.011). Most prevalent was T. phagedenis (65.1%). D. nodosus was detected in 51.8% of clinical DD lesions and 24.1% in unaffected cases, but its presence was not significantly associated with the various DD-stages. All samples positive for D. nodosus contained the acid protease gene aprB2 but were negative for aprV2, the latter associated with virulence in sheep foot rot. Control farms were negative for all DD-associated Treponema species while positive for aprB2 and negative for aprV2. The presence of aprB2 suggests it is ubiquitous in the animal environment. With respect to the slurry samples, three out of 21 specimens (14.3%) were positive for one or more of the DD-associated Treponema species and eleven out of 21 specimens (52.4%) were positive for aprB2 and negative for aprV2 of D. nodosus. In conclusion, an association was found between the presence of clinical DD and specific Treponema species, while for D. nodosus no such link with DD lesions could be observed.

INTRODUCTION: La Dermatite digitée (DD) chez les bovins est une maladie infectieuse podale multifactorielle, qui est devenue un problème émergent pour le bien-être animal et pour l'économie au niveau mondial. Trois espèces de Treponema, T. pedis, T. medium et T. phagedenis, sont associées avec la DD. Cependant, leur prévalence est inconnue en Suisse. Il a également été rapporté que Dichelobacter nodosus pouvait contribuer au développement de la DD. Le but de cette étude a été de déterminer la prévalence des trois espèces de Treponema ainsi que de D. nodosus dans des lésions de DD et du lisier de vaches suisses, en utilisant des techniques basées sur la PCR. Vingt-deux exploitations avec de la DD clinique et deux exploitations sans signes cliniques de DD ont été inclues dans l'étude. Un total de 86 échantillons de cas de DD ont été prélevés (M1, n=15; M2, n=19; M3, n=9; M4, n=2, M4.1, n=16 and M5, n=25) en utilisant des coton-tiges secs et stériles. De plus, afin d'évaluer le potentiel de transmission par l'environnement, des échantillons de lisier ont été prélevés sur des exploitations atteintes de DD (n=21) et sur une exploitation à stabulation libre exempte de DD. La PCR nichée et la PCR en temps réel ont ensuite été utilisées sur l'ADN extrait des échantillons afin de détecter les espèces de Treponema et D. nodosus, respectivement. Les associations entre la présence d'espèces de Treponema et de D. nodosus avec le statut DD des animaux ont été évaluées avec le test Pearson's Chi-Square. Les stades de DD (M1 à M4.1) et M5 (peau saine) étaient positifs pour au moins une ou plusieurs des espèces de Treponema associées à la DD dans 50 de 61 cas (82.0%) et 9 de 25 cas (36.0%), respectivement. Les lésions M1 ont montré une prévalence nettement inférieure (14.8%) comparé aux autres stades de DD (M2, M3, M4 et M4.1; 67.2%; P=0.011). T. phagedenis était prédominant (65.1%). D. nodosus a été détecté dans 51.8% des lésions cliniques de DD (M1 à M4) et 24.1% des échantillons M5, mais sa présence n'était pas associée significativement avec les divers stades de DD. Tous les échantillons positifs pour D. nodosus contenaient le gène de la protéase acide aprB2, mais étaient négatifs pour aprV2, un gène associé à la virulence dans le piétin des moutons. Les exploitations de contrôle étaient négatives pour toutes les espèces de Treponema associées à la DD, mais positives pour aprB2 et négatives pour aprV2. La présence du gène aprB2 suggère qu'il est ubiquitaire dans l'environnement des animaux et n'est pas une association en soi avec le piétin des moutons. En ce qui concerne les échantillons de lisier, trois des 21 échantillons (14.3%) étaient positifs pour au moins une ou plusieurs espèces de Treponema associées à la DD et onze des 21 échantillons (52.4%) étaient positifs pour aprB2 et négatifs pour aprV2 de D. nodosus. En conclusion, une association a été trouvée entre la présence de DD clinique et des espèces de Treponema spécifiques, alors que pour D. nodosus aucun lien avec des lésions de DD n'a pu être observé. Cette étude démontre la présence des trois espèces de Tréponèmes chez les bovins suisses et facilite la compréhension de l'implication de Treponema spécifiques dans les lésions de DD.

Mechanism of Hyperkalemia-Induced Metabolic Acidosis.

Background Hyperkalemia in association with metabolic acidosis that are out of proportion to changes in glomerular filtration rate defines type 4 renal tubular acidosis (RTA), the most common RTA observed, but the molecular mechanisms underlying the associated metabolic acidosis are incompletely understood. We sought to determine whether hyperkalemia directly causes metabolic acidosis and, if so, the mechanisms through which this occurs.Methods We studied a genetic model of hyperkalemia that results from early distal convoluted tubule (DCT)-specific overexpression of constitutively active Ste20/SPS1-related proline-alanine-rich kinase (DCT-CA-SPAK).Results DCT-CA-SPAK mice developed hyperkalemia in association with metabolic acidosis and suppressed ammonia excretion; however, titratable acid excretion and urine pH were unchanged compared with those in wild-type mice. Abnormal ammonia excretion in DCT-CA-SPAK mice associated with decreased proximal tubule expression of the ammonia-generating enzymes phosphate-dependent glutaminase and phosphoenolpyruvate carboxykinase and overexpression of the ammonia-recycling enzyme glutamine synthetase. These mice also had decreased expression of the ammonia transporter family member Rhcg and decreased apical polarization of H+-ATPase in the inner stripe of the outer medullary collecting duct. Correcting the hyperkalemia by treatment with hydrochlorothiazide corrected the metabolic acidosis, increased ammonia excretion, and normalized ammoniagenic enzyme and Rhcg expression in DCT-CA-SPAK mice. In wild-type mice, induction of hyperkalemia by administration of the epithelial sodium channel blocker benzamil caused hyperkalemia and suppressed ammonia excretion.Conclusions Hyperkalemia decreases proximal tubule ammonia generation and collecting duct ammonia transport, leading to impaired ammonia excretion that causes metabolic acidosis.

Constitutively Active SPAK Causes Hyperkalemia by Activating NCC and Remodeling Distal Tubules.

Aberrant activation of with no lysine (WNK) kinases causes familial hyperkalemic hypertension (FHHt). Thiazide diuretics treat the disease, fostering the view that hyperactivation of the thiazide-sensitive sodium-chloride cotransporter (NCC) in the distal convoluted tubule (DCT) is solely responsible. However, aberrant signaling in the aldosterone-sensitive distal nephron (ASDN) and inhibition of the potassium-excretory renal outer medullary potassium (ROMK) channel have also been implicated. To test these ideas, we introduced kinase-activating mutations after Lox-P sites in the mouse Stk39 gene, which encodes the terminal kinase in the WNK signaling pathway, Ste20-related proline-alanine-rich kinase (SPAK). Renal expression of the constitutively active (CA)-SPAK mutant was specifically targeted to the early DCT using a DCT-driven Cre recombinase. CA-SPAK mice displayed thiazide-treatable hypertension and hyperkalemia, concurrent with NCC hyperphosphorylation. However, thiazide-mediated inhibition of NCC and consequent restoration of sodium excretion did not immediately restore urinary potassium excretion in CA-SPAK mice. Notably, CA-SPAK mice exhibited ASDN remodeling, involving a reduction in connecting tubule mass and attenuation of epithelial sodium channel (ENaC) and ROMK expression and apical localization. Blocking hyperactive NCC in the DCT gradually restored ASDN structure and ENaC and ROMK expression, concurrent with the restoration of urinary potassium excretion. These findings verify that NCC hyperactivity underlies FHHt but also reveal that NCC-dependent changes in the driving force for potassium secretion are not sufficient to explain hyperkalemia. Instead, a DCT-ASDN coupling process controls potassium balance in health and becomes aberrantly activated in FHHt.

American Brachytherapy Society Task Group Report: Use of androgen deprivation therapy with prostate brachytherapy-A systematic literature review.

PURPOSE: Prostate brachytherapy (PB) has well-documented excellent long-term outcomes in all risk groups. There are significant uncertainties regarding the role of androgen deprivation therapy (ADT) with brachytherapy. The purpose of this report was to review systemically the published literature and summarize present knowledge regarding the impact of ADT on biochemical progression-free survival (bPFS), cause-specific survival (CSS), and overall survival (OS). METHODS AND MATERIALS: A literature search was conducted in Medline and Embase covering the years 1996-2016. Selected were articles with >100 patients, minimum followup 3 years, defined risk stratification, and directly examining the role and impact of ADT on bPFS, CSS, and OS. The studies were grouped to reflect disease risk stratification. We also reviewed the impact of ADT on OS, cardiovascular morbidity, mortality, and on-going brachytherapy randomized controlled trials (RCTs). RESULTS: Fifty-two selected studies (43,303 patients) were included in this review; 7 high-dose rate and 45 low-dose rate; 25 studies were multi-institutional and 27 single institution (retrospective review or prospective data collection) and 2 were RCTs. The studies were heterogeneous in patient population, risk categories, risk factors, followup time, and treatment administered, including ADT administration and duration (median, 3-12 months);71% of the studies reported a lack of benefit, whereas 28% showed improvement in bPFS with addition of ADT to PB. The lack of benefit was seen in low-risk and favorable intermediate-risk (IR) disease and most high-dose rate studies. A bPFS benefit of up to 15% was seen with ADT use in patients with suboptimal dosimetry, those with multiple adverse risk factors (unfavorable IR [uIR]), and most high-risk (HR) studies. Four studies reported very small benefit to CSS (2%). None of the studies showed OS advantage; however, three studies reported an absolute 5-20% OS detriment with ADT. Literature suggests that OS detriment is more likely in older patients or those with pre-existing cardiovascular disease. Four RCTs with an adequate number of patients and well-defined risk stratification are in progress. One RCT will answer the question regarding the role of ADT with PB in favorable IR patients and the other three RCTs will focus on optimal duration of ADT in the uIR and favorable HR population. CONCLUSIONS: Patients treated with brachytherapy have excellent long-term disease outcomes. Existing evidence shows no benefit of adding ADT to PB in low-risk and favorable IR patients. UIR and HR patients and those with suboptimal dosimetry may have up to 15% improvement in bPFS with addition of 3-12 months of ADT, with uncertain impact on CSS and a potential detriment on OS. To minimize morbidity, one should exercise caution in prescribing ADT together with PB, in particular to older men and those with existing cardiovascular disease. Due to the retrospective nature of this evidence, significant selection, and treatment bias, no definitive conclusions are possible. RCT is urgently needed to define the potential role and optimal duration of ADT in uIR and favorable HR disease.

Faecal parameters as biomarkers of the equine hindgut microbial ecosystem under dietary change.

Faeces could be used for evaluating the balance of the equine hindgut microbial ecosystem, which would offer a practical method for assessing gut health and how this relates to disease. However, previous studies concluded that faeces microbial ecosystem was not representative of the proximal hindgut (caecum and ventral colon). This study aimed to evaluate if variations of the faecal microbial ecosystem were similar to those observed in the proximal hindgut. Six horses, fistulated in the caecum and right ventral (RV) colon, were subjected to a gradual change of diet, from a 100% hay (high fibre) diet (2.2 DM kg/day per 100 kg BW) to a 57% hay+43% barley (high starch) diet (0.8 DM kg/day per 100 kg BW hay and 0.6 DM kg/day per 100 kg BW barley). The two diets were iso-energetic and fed over a 3-week trial period. Samples of digesta from the caecum, RV colon and faeces were collected two times on the 10th and 20th day of the trial, for each diet to assess the microbial ecosystem parameters by both classical culture technics and biochemical methods. The variations observed in the caecal and colonic bacterial composition (increase in total anaerobic, amylolytic and lactate-utilizing and decrease in cellulolytic bacteria concentrations) and microbial activity (changes in volatile fatty acids concentrations and increase in lactate concentrations) demonstrated that the hay+barley diet caused changes in the hindgut microbial ecosystem. Similar variations were observed in the faecal microbial ecosystem. Feeding the hay+barley diet resulted in higher concentrations of faecal lipopolysaccharides. The functional bacterial group concentrations (cellulolytics, amylolytics and lactate utilizers) were significantly correlated between caecum and faeces and between colon and faeces. From analyses of the metabolites produced from microbial activity, only valerate concentration in the caecum and the proportion of propionate were significantly correlated with the same parameters in the faeces. Results of the principal component analysis performed between all the caecal/faecal and colonic/faecal parameters revealed that the total anaerobic and cellulolytic bacteria concentrations, as well as valerate, l-lactate and lipopolysaccharide concentrations were strongly correlated with several microbial parameters in the caecum (P|0.45|) and in the colon (P|0.50|). This demonstrated that faecal samples and their bacterial analyses could be used to represent caecum and RV colon hindgut microbial ecosystem in terms of variations during a change from a high-fibre to a high-starch diet, and thus could be markers of particular interest to diagnostic proximal hindgut microbial disturbances.

Relationship Among Viremia/Viral Infection, Alloimmunity, and Nutritional Parameters in the First Year After Pediatric Kidney Transplantation.

The Immune Development in Pediatric Transplantation (IMPACT) study was conducted to evaluate relationships among alloimmunity, protective immunity, immune development, physical parameters, and clinical outcome in children undergoing kidney transplantation. We prospectively evaluated biopsy-proven acute rejection (BPAR), de novo donor-specific antibody (dnDSA) formation, viremia, viral infection, T cell immunophenotyping, and body mass index (BMI)/weight Z scores in the first year posttransplantation in 106 pediatric kidney transplant recipients. Outcomes were excellent with no deaths and 98% graft survival. Rejection and dnDSAs occurred in 24% and 22%, respectively. Pretransplant cytomegalovirus (CMV) and Epstein-Barr virus (EBV) serologies and subsequent viremia were unrelated to BPAR or dnDSA. Viremia occurred in 73% of children (EBV, 34%; CMV, 23%; BMK viremia, 23%; and JC virus, 21%). Memory lymphocyte phenotype at baseline was not predictive of alloimmune complications. Patients who developed viral infection had lower weight (-2.1) (p = 0.028) and BMI (-1.2) (p = 0.048) Z scores at transplantation. The weight difference persisted to 12 months compared with patients without infection (p = 0.038). These data indicate that there is a high prevalence of viral disease after pediatric kidney transplantation, and underweight status at transplantation appears to be a risk factor for subsequent viral infection. The occurrence of viremia/viral infection is not associated with alloimmune events.

HORSE SPECIES SYMPOSIUM: The microbiome of the horse hindgut: History and current knowledge.

In the early 1990s, the equine hindgut microbial ecosystem looked like a "black box." Its vital role in hydrolyzing and fermenting fiber, thus providing energy to the host, was recognized. Although there was a critical lack of information on the hindgut microbes, their role in preventing intestinal diseases was suggested. Traditionally, the microbes of the horse hindgut were studied using culture-dependent techniques. More recently, culture-independent methods have been used and provided further insight. This review presents the history and updated knowledge regarding the microbes that live inside the different intestinal ecosystems and which collective genomes compose the hindgut microbiome. In the first section, the quantification and diversity are described for each microbial community as well as the implication of plant fiber degradation and their crucial role for an herbivore host. The microbial communities are presented in chronological order of discovery: due to their large size, protozoa were brought to light as early as 1843 in the horse cecum; in 1897, bacteria were described in the horse intestine; as early as 1910, monoflagellated eukaryotic organisms resembling protozoa were observed in the horse cecum; since then, they have been identified to be zoospores of anaerobic fungi; in 1970, bacteriophage-like particles were recognized in the cecum and colon of pony and horse; and finally, in 1996, archaea were identified in the horse cecum. The second section discusses the variations that can occur between digestive segments or between individuals. The representativeness of the fecal microbiota to the hindgut one is debated, especially as the majority of recent studies conducted on the horse hindgut are in fact focused on the feces, rather than the cecum or colon. Also, the representation of microbiota between individuals is questioned. It has long been suggested in the literature that some ponies or horses that were more susceptible to intestinal diseases may harbor a specific intestinal microbiota. Alternatively, some new studies aim at identifying a core microbiome between all individual equine. A deeper knowledge of the microbiome and its core may allow improvement of nutrition and health, understanding of the onset of digestive diseases, and the development tools for health monitoring and disease prevention.

Integrated compensatory network is activated in the absence of NCC phosphorylation.

Thiazide diuretics are used to treat hypertension; however, compensatory processes in the kidney can limit antihypertensive responses to this class of drugs. Here, we evaluated compensatory pathways in SPAK kinase-deficient mice, which are unable to activate the thiazide-sensitive sodium chloride cotransporter NCC (encoded by Slc12a3). Global transcriptional profiling, combined with biochemical, cell biological, and physiological phenotyping, identified the gene expression signature of the response and revealed how it establishes an adaptive physiology. Salt reabsorption pathways were created by the coordinate induction of a multigene transport system, involving solute carriers (encoded by Slc26a4, Slc4a8, and Slc4a9), carbonic anhydrase isoforms, and V-type H⁺-ATPase subunits in pendrin-positive intercalated cells (PP-ICs) and ENaC subunits in principal cells (PCs). A distal nephron remodeling process and induction of jagged 1/NOTCH signaling, which expands the cortical connecting tubule with PCs and replaces acid-secreting α-ICs with PP-ICs, were partly responsible for the compensation. Salt reabsorption was also activated by induction of an α-ketoglutarate (α-KG) paracrine signaling system. Coordinate regulation of a multigene α-KG synthesis and transport pathway resulted in α-KG secretion into pro-urine, as the α-KG-activated GPCR (Oxgr1) increased on the PP-IC apical surface, allowing paracrine delivery of α-KG to stimulate salt transport. Identification of the integrated compensatory NaCl reabsorption mechanisms provides insight into thiazide diuretic efficacy.

SPAK-mediated NCC regulation in response to low-K+ diet.

The NaCl cotransporter (NCC) of the renal distal convoluted tubule is stimulated by low-K(+) diet by an unknown mechanism. Since recent work has shown that the STE20/SPS-1-related proline-alanine-rich protein kinase (SPAK) can function to stimulate NCC by phosphorylation of specific N-terminal sites, we investigated whether the NCC response to low-K(+) diet is mediated by SPAK. Using phospho-specific antibodies in Western blot and immunolocalization studies of wild-type and SPAK knockout (SPAK(-/-)) mice fed a low-K(+) or control diet for 4 days, we found that low-K(+) diet strongly increased total NCC expression and phosphorylation of NCC. This was associated with an increase in total SPAK expression in cortical homogenates and an increase in phosphorylation of SPAK at the S383 activation site. The increased pNCC in response to low-K(+) diet was blunted but not completely inhibited in SPAK(-/-) mice. These findings reveal that SPAK is an important mediator of the increased NCC activation by phosphorylation that occurs in the distal convoluted tubule in response to a low-K(+) diet, but other low-potassium-activated kinases are likely to be involved.

Haemodialysis is an effective treatment in acute metabolic decompensation of maple syrup urine disease.

Acute metabolic decompensation in maple syrup urine disease can occur during intercurrent illness and is a medical emergency. A handful of reports in the medical literature describe the use of peritoneal dialysis and haemodialysis as therapeutic inventions. We report the only patient from our centre to have haemodialysis performed in this setting. Combined with dietary BCAA restriction and calorific support, haemodialysis allows rapid reduction in plasma leucine concentrations considerably faster than conservative methods.

Immune cell function assay does not identify biopsy-proven pediatric renal allograft rejection or infection.

Management of pediatric renal transplant patients involves multifactorial monitoring modalities to ensure allograft survival and prevent opportunistic infection secondary to immunosuppression. An ICFA, which utilizes CD4 T-cell production of ATP to assess immune system status, has been used to monitor transplant recipients and predict susceptibility of patients to rejection or infection. However, the validity of this assay to reflect immune status remains unanswered. In a two-yr retrospective study that included 31 pediatric renal transplant recipients, 42 patient blood samples were analyzed for immune cell function levels, creatinine, WBC (white blood cell) count, immunosuppressive drug levels, and viremia, concurrent with renal biopsy. T-cell ATP production as assessed by ICFA levels did not correlate with allograft rejection or with the presence or absence of viremia. ICFA levels did not correlate with serum creatinine or immunosuppressive drug levels, but did correlate with WBC count. The ICFA is unreliable in its ability to reflect immune system status in pediatric renal transplantation. Further investigation is necessary to develop methods that will accurately predict susceptibility of pediatric renal transplant recipients to allograft rejection and infection.

NEU1 sialidase regulates the sialylation state of CD31 and disrupts CD31-driven capillary-like tube formation in human lung microvascular endothelia.

The highly sialylated vascular endothelial surface undergoes changes in sialylation upon adopting the migratory/angiogenic phenotype. We recently established endothelial cell (EC) expression of NEU1 sialidase (Cross, A. S., Hyun, S. W., Miranda-Ribera, A., Feng, C., Liu, A., Nguyen, C., Zhang, L., Luzina, I. G., Atamas, S. P., Twaddell, W. S., Guang, W., Lillehoj, E. P., Puché, A. C., Huang, W., Wang, L. X., Passaniti, A., and Goldblum, S. E. (2012) NEU1 and NEU3 sialidase activity expressed in human lung microvascular endothelia. NEU1 restrains endothelial cell migration whereas NEU3 does not. J. Biol. Chem. 287, 15966-15980). We asked whether NEU1 might regulate EC capillary-like tube formation on a Matrigel substrate. In human pulmonary microvascular ECs (HPMECs), prior silencing of NEU1 did not alter tube formation. Infection of HPMECs with increasing multiplicities of infection of an adenovirus encoding for catalytically active WT NEU1 dose-dependently impaired tube formation, whereas overexpression of either a catalytically dead NEU1 mutant, NEU1-G68V, or another human sialidase, NEU3, did not. NEU1 overexpression also diminished EC adhesion to the Matrigel substrate and restrained EC migration in a wounding assay. In HPMECs, the adhesion molecule, CD31, also known as platelet endothelial cell adhesion molecule-1, was sialylated via α2,6-linkages, as shown by Sambucus nigra agglutinin lectin blotting. NEU1 overexpression increased CD31 binding to Arachis hypogaea or peanut agglutinin lectin, indicating CD31 desialylation. In the postconfluent state, when CD31 ectodomains are homophilically engaged, NEU1 was recruited to and desialylated CD31. In postconfluent ECs, CD31 was desialylated compared with subconfluent cells, and prior NEU1 silencing completely protected against CD31 desialylation. Prior CD31 silencing and the use of CD31-null ECs each abrogated the NEU1 inhibitory effect on EC tube formation. Sialyltransferase 6 GAL-I overexpression increased α2,6-linked CD31 sialylation and dose-dependently counteracted NEU1-mediated inhibition of EC tube formation. These combined data indicate that catalytically active NEU1 inhibits in vitro angiogenesis through desialylation of its substrate, CD31.

SPAK isoforms and OSR1 regulate sodium-chloride co-transporters in a nephron-specific manner.

STE20/SPS-1-related proline-alanine-rich protein kinase (SPAK) and oxidative stress-related kinase (OSR1) activate the potassium-dependent sodium-chloride co-transporter, NKCC2, and thiazide-sensitive sodium-chloride cotransporter, NCC, in vitro, and both co-localize with a kinase regulatory molecule, Cab39/MO25α, at the apical membrane of the thick ascending limb (TAL) and distal convoluted tubule (DCT). Yet genetic ablation of SPAK in mice causes a selective loss of NCC function, whereas NKCC2 becomes hyperphosphorylated. Here, we explore the underlying mechanisms in wild-type and SPAK-null mice. Unlike in the DCT, OSR1 remains at the TAL apical membrane of KO mice where it is accompanied by an increase in the active, phosphorylated form of AMP-activated kinase. We found an alterative SPAK isoform (putative SPAK2 form), which modestly inhibits co-transporter activity in vitro, is more abundant in the medulla than the cortex. Thus, enhanced NKCC2 phosphorylation in the SPAK knock-out may be explained by removal of inhibitory SPAK2, sustained activity of OSR1, and activation of other kinases. By contrast, the OSR1/SPAK/M025α signaling apparatus is disrupted in the DCT. OSR1 becomes largely inactive and displaced from M025α and NCC at the apical membrane, and redistributes to dense punctate structures, containing WNK1, within the cytoplasm. These changes are paralleled by a decrease in NCC phosphorylation and a decrease in the mass of the distal convoluted tubule, exclusive to DCT1. As a result of the dependent nature of OSR1 on SPAK in the DCT, NCC is unable to be activated. Consequently, SPAK(-/-) mice are highly sensitive to dietary salt restriction, displaying prolonged negative sodium balance and hypotension.

Role of BK channels in hypertension and potassium secretion.

PURPOSE OF REVIEW: This review summarizes recent studies of hypertension associated with a defect in renal K excretion due to genetic deletions of various components of the large, Ca-activated K channel (BK), and describes new evidence and theories regarding K secretory roles of BK in intercalated cells. RECENT FINDINGS: Isolated perfused tubule methods have revealed the importance of BK in flow-induced K secretion. Subsequently, mice with genetically deleted BK subunits revealed the complexities of BK-mediated K secretion. Deletion of BKα results in extreme aldosteronism, hypertension, and an absence of flow-induced K secretion. Deletion of the BKß1 ancillary subunit results in decreased handling of a K load, increased plasma K, mild aldosteronism and hypertension that is exacerbated by a high K diet. Deletion of BKß4 (ß4KO) leads to insufficient K handling, high plasma K, fluid retention, but with milder hypertension. Fluid retention in ß4KO may be the result of insufficient flow-induced secretion of adenosine triphosphate (ATP), which normally inhibits epithelial Na channels (ENaCs). SUMMARY: Classical physiological analysis of electrolyte handling in knockout mice has enlightened our understanding of the mechanism of handling K loads by renal K channels. Studies have focused on the different roles of BK-α/ß1 and BK-α/ß4 in the kidney. BKß1 hypertension may be a 'three-hit' hypertension, involving a K secretory defect, elevated production of aldosterone, and increased vascular tone. The disorders observed in BK knockout mice have shed new insights on the importance of proper renal K handling for maintaining volume balance and blood pressure.